C.diff cases may be twice as high as tests miss infection
The number of cases of the hospital bug C.diff could be twice as high as previously thought as current tests used by the NHS are failing to pick up the infection, experts have claimed. >
By Rebecca Smith, Medical Editor
Published: 7:15AM GMT 04 Nov 2009
There are around 3,000 cases of Clostridium difficile reported in hospitals in England each month but the true figure could be much higher as the current testing methods miss between 20 per cent and 80 per cent of cases, according to microbiologists.
Prof Gary French, consultant microbiologist at Kings College London and Guy's and St Thomas' Foundation Trust, has carried out research showing that the current test only correctly identifies around 38 per cent of cases of C.diff. Other trials have suggested about half of cases are found.
The research, which is due to be published in the Journal of Hospital Infection, suggests new methods using two-stages, one to screen for the presence of C.diff and if positive another more sophisticated test to establish if the bug is producing the toxins which cause symptoms or is lying dormant.
C.diff is a bacterium that can be found naturally in the gut of some people without causing problems but in others it can produce toxins which trigger diarrhoea. Vulnerable elderly patients and those who have been on antibiotics in hospital are more susceptible and the disease can prove fatal in some cases.
The results from current tests used in NHS hospitals can take several days.
Prof David Persing, consulting professor of pathology at Standford University in California and chief medical officer of Cepheid, a company which produces pathology tests, said his research has shown that the tests only found about 50 per cent of positive C.diff cases.
"The data was quite surprising. The tests are not doing anywhere near as well as we thought. For some strains they are only picking up about 20 per cent of the infections that are there. The vasst majority of patients who are infrected with some of these strains are being missed and are not being put in isolation. There is no attempt to even treat the patient if the test comes back negative," he said.
He said for strain 106, which is common in Britain, the current test only detected around 20 per cent of cases but for the more virulent 027 strain the tests found 80 per cent of cases.
The new tests are more accurate and faster, he said, but they can be three time more expensive. However, by finding positive cases faster, NHS hospitals could cut their infection rates and reduce duplicate tests to save money in the long run.
Prof French also found that the current tests are 'inadequate' and called for new methods to be employed. But he added that patients with symptoms are isolated and treated accordingly without waiting for test results and they may continue to be treated even if the result is negative.
He said: "From our paper and other studies, we show that the present tests are not as reliable as they should be… I think that these tests are really not good enough. We need recommendations to move to a new system and this should be debated."
Prof French said that although new tests may find more cases it should not be taken as a rise in C.diff prevalence but would be the result of more accurate testing. He said the downward trend in C.diff cases shown by mandatory reporting of cases to the Health Protection Agency is probably still the case.
He said: "The hygiene improvements across the board, shown by the dramatic reduction in MRSA rates, means I am convincted taht the reduction in C.diff rates is a true phenomenon.
"If we go to a new test that is more sensitive we are bound to see an increase in reported cases. That will not be a result of poor practice but the result of better testing."
The latest C. Difficile figures show that there were 6,855 cases reported in patients aged two years and over during the April to June 2009. This is a drop of 37 per cent on the same period last year.
Dr Christine McCartney, Executive Director for the Health Protection Agency's Healthcare Associated Infection and Anti-Microbial Resistance Programme, said: “Studies have suggested that different tests have better efficacy rates depending on the strain of Clostridium difficile.
“The Agency works closely with the Department of Health to ensure all infection control guidelines take any new strains of health care associated infection into account to ensure health care settings have the most up to date evidence and research possible to help combat healthcare associated infections.”
Brian Duerden, Inspector of Microbiology and Infection Control at the Department of Health said: "Accurate test results are essential for good patient care. In March this year we issued advice on a two-stage testing approach and reminded trusts that patients should be isolated and treated as Clostridium difficile patients if they were symptomatic, even if they tested negative.
"We will be evaluating the results of this study, along with other studies currently underway, as part of a review into healthcare acquired infection testing. We will continue to work tirelessly to tackle these infections."
Testing methods will be discussed at a symposium at Guy's and St Thomas' Hospital.
New, more expensive tests should be brought in, but hospital managers would need reassurance that any apparent rise in cases found by the new methods should not be seen as poor patient care, they said.
By Ed Susman
SAN FRANCISCO -- September 15, 2009 -- Researchers said that hospital-acquired infections with methicillin-resistant Staphylococcus aureus (MRSA) can be largely prevented by identifying carriers of the organism when patients are admitted to the hospital and then initiating aggressive isolation procedures, even if patients are not experiencing disease symptoms but are colonised by the bacteria.
Lance Peterson, MD, Microbiology and Infectious Diseases Research, NorthShore University HealthSystem, Chicago, Illinois, said if hospital staff is not aggressive in conducting isolation programs with persons carrying MRSA, the infection control program is doomed to fail.
The aggressive program initiated at his hospitals in 2005 resulted in a 70% reduction in MRSA infections rates, and even though the costs associated with the program added to the hospital budget, the resulting savings in preventing infections was triple the cost, Dr. Peterson said here on September 12 at the 49th Annual Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC).
The study at the 3 NorthShore system hospitals in Evanston, Illinois, revealed that about 7 of every 100 individuals who had MRSA colonisation developed a symptomatic infection. "The biggest risk factor for MRSA infection is to be colonised," he said. The 3 hospitals used real-time polymerase chain reaction testing to screen incoming patients for colonisation with MRSA.
Upon presentation at the hospital, patients had their nostrils swabbed for pathogens. If MRSA was detected the patients were isolated and treated with mupirocin for 5 days. Staff treating these patients wore gowns and gloves for contact precautions, he said. Patients were also given 2 antiseptic baths.
The polymerase chain reaction test is more expensive than traditional tests, and additional nursing care precautions also added costs to the program, Dr. Peterson said.
"The bottom line for us is that if you want to have a successful MRSA reduction program," he said, "you have to have a very aggressive, very rapid, and broad MRSA screening program."
Dr. Peterson and colleagues studied 37,179 consecutive patients who underwent MRSA-testing at admission. The prevalence of MRSA was 8.5%, but two-thirds of the individuals were not aware they were carriers of MRSA.
"Successful control of any epidemic relies on detection of those harbouring the pathogen and limiting further spread," he said. "The approach to control of the MRSA pandemic is no different. Most persons harbouring MRSA do not exhibit signs of infection and thus to detect all spreaders some surveillance must be done."
[Presentation title: Debate: Is Molecular Screening for MRSA Colonization Worth the Cost? Case in Favour. Abstract 408]
Source - ( Sep 15, 2009 )
Sepsis is a major healthcare problem. There are approximately 750,000 cases of sepsis each year in the U.S. And the number is growing.
Sepsis results from complex interactions between infecting microorganisms and host immune, inflammatory, and coagulation responses. Severe sepsis is defined as sepsis with organ dysfunction. Severe sepsis with hypotension, despite adequate fluid resuscitation, is septic shock. Septic shock and multiorgan dysfunction are the most common causes of death in sepsis patients. Mortality associated with severe sepsis remains unacceptably high—20 to 50%. When shock is present, mortality is even higher—40 to 60%.
Distinguishing patients with localized infections or a systemic inflammatory response (SIRS) from those with sepsis is challenging. SIRS is not specific to sepsis and can result from other conditions such as acute pancreatitis and immunodeficiencies. This makes a quick diagnosis, difficult.
Today, blood culture and culture techniques are the gold standard for detection of infection. Physicians order cultures as soon as two or more SIRS criteria are identified. The turnaround time for culture/blood culture is lengthy, ranging from 48 to 72 hours. As a result, antimicrobial therapy administration usually begins before culture results are available.
The choice of appropriate broad-spectrum antimicrobial therapy is tricky because of the rising prevalence of resistant pathogens. Today, this choice is left up to the physician’s intuition. Patients with severe sepsis or septic shock, however, have little margin for error in what therapy they receive. Consequently, there is a huge unmet need for fast turnaround tests that enable early administration of antimicrobial therapy/antibiotics by rapid identification of infection and facilitate choice of antimicrobial therapy through rapid identification and detection of causative pathogen.
Future sepsis diagnostic options include single-analyte immunoassays and molecular identification, for which opportunities exist at both the front and back ends.
Among all single-analyte biomarkers, detection of a protein biomarker called procalcitonin (PCT) in serum holds the most promise. Serum levels of PCT have been shown to increase in patients with an infection; high values will be seen in cases of severe sepsis and septic shock.
Widespread use, however, is lacking. This is partly due to the lack of clinical trials with proper negative controls that differentiate septic from sepsis-like patients. Another difficulty lies with the riskiness of using a cut-off threshold of PCT to rule-out sepsis. Scientia’s research indicates that the possibility of false negatives is daunting and subsequent denial of antibiotic could increase the probability of mortality.
Another potential application of PCT is in therapy monitoring. PCT kinetics can be used to assess the effectiveness of treatment. If PCT levels do not begin to decline in a patient after four days of antibiotic treatment, the physician should consider changing the antibiotic regimen.
B.R.A.H.M.S. owns the IP on PCT and offers the test today along with bioMerieux. Several other immunoassay providers, including Roche Diagnostics and Siemens, have licensed PCT from B.R.A.H.M.S. And are offering it or intend to offer it on their immunoassay platforms.
Molecular identification of sepsis-causing pathogens might prove extremely helpful for rapid identification of pathogens and major antimicrobial resistance determinants. Rapid detection of hard-to-grow pathogens and antibiotic-resistant pathogens such as MRSA and VRE can significantly improve clinical outcome, enabling a narrowing of the spectrum of antibiotic coverage.
SeptiFast, a molecular sepsis test, has been launched by Roche Molecular Diagnostics in Europe. Adoption of this test has been disappointing as a result of its limited coverage of pathogens, automation issues, contamination problem, and cost.
Given the substantial barriers, many molecular diagnostic companies do not plan on taking up the daunting task of introducing a front-end molecular identification test and have resorted to the back end of identification of organisms directly in positive blood cultures instead.
The simplest of these back-end molecular identification tests is the PNA FISH (peptide nucleic acid fluorescent in situ hybridization) test. PNA FISH has the advantage of rapid hybridization kinetics and offers a sensitive way to identify S. Aureus, Enterococcus faecalis, or Candida albicans in positive blood culture samples. Identification is typically done within a few hours after a positive blood culture result is obtained. PNA FISH is available today and is offered by AdvanDx.
Another emerging methodology for identification of organisms in positive blood cultures is real-time PCR. Several companies such as Cepheid (with bioMerieux) are working toward developing such a test.
Sepsis is a complex and life-threatening disease. Significant improvements in the care, management, and treatment of sepsis patients have been realized recently. While early diagnosis remains a challenge and a rapid, sensitive, and specific diagnostic test is still lacking, these new approaches open up a whole new dimension in early diagnosis of sepsis and are likely to evolve into solutions for this major unmet need.
Source - ( Jul 16, 2009 )
Tuberculosis (TB) has taken a backseat over the last few decades as it has not received the funding appropriate to such a major health threat to the developing world. The World Health Organisation (WHO) published statistics last month showing that worldwide TB rates are stabilising at approximately 14 million cases, comprising one-third of the world’s population.
There are, however, a few countries where TB rates are increasing. The Health Protection Agency (HPA) has released statistics showing that the UK has seen a 2% increase (the only European country where TB is actually on the rise) and in African countries where HIV/TB co-infection is rife, TB deaths have quadrupled over the last 15 years. Clearly, tuberculosis is making a comeback - the disease is stronger now than ever before, and is rapidly becoming more drug-resistant, usually by gene mutations. In fact, much of the antibiotic resistance observed among TB strains is multi-drug resistance (MDR), showing resistance toward at least two first-line drugs. TB is second only to HIV as the world’s deadliest infectious disease, claiming a life every 20 seconds.
One of the key obstacles in controlling TB infection is the lack of fast and accurate diagnostic methods, particularly in areas of the world with the greatest disease burden. This article will examine those current diagnostics and reveal details of a new test that is set to revolutionise the way that TB is diagnosed and, therefore, controlled throughout the world.
Over the last year, the World Health Organisation has expressed concern that they may not be able to reach the targets for TB control as defined in the UN’s Millennium Development Goals (MDG’s). Those targets for 2015 included minimum requirements for cases detected and treated and required falling rates of TB incidence. Additionally, as defined in 2006 by the Stop TB Partnership, a global consortium of public health, non-government organisations, World Bank, pharmaceutical and other corporate stakeholders, governments, and academic institutions, prevalence and mortality rates should be decreased to one-half of the 1990 rates. This would essentially mean that 50 million people would receive treatment and 14 million lives would be saved.
As we know, early detection and proper treatment are essential to combat TB. UN Secretary-General, Ban Ki-Moon, argued that although “the epidemic is continuing its decline, the [global] rate of decline is far too slow, Unless we accelerate action, the numbers of those falling ill will continue to grow.” A 2008 report stated that “the detection rate increased only marginally to 61%, short of the 65% benchmark for 2006 contained in the Stop TB Partnership Global Plan and the ultimate target of 70%.”
One-third of TB cases come from Sub-Saharan Africa and Southeast Asia, where the main attributable factors are widespread poverty and a lack of adequate public healthcare.
People who have TB, or live in poverty, can often become mired in a vicious circle of continuous proliferation of both. TB most commonly attacks people in their working years crippling their ability to earn a living. Additionally, the costs associated with healthcare for TB can put families of sufferers under tremendous financial strain. This has a particularly significant impact on children who often become malnourished and have to leave school to take care of their family. In some cases, these children ultimately become orphans.
In turn, people living in poverty who cannot afford or access healthcare often share living space with many people, some of whom are already infected with TB. Spread of infection becomes much more likely in such settings.
There is a strong connection between HIV and TB. A third of all HIV/AIDS sufferers are co-infected with TB. The WHO Global TB Report stated that approximately 1.37 million HIV/AIDS sufferers were diagnosed with TB in 2008 - indeed TB is the leading cause of death amongst HIV sufferers. Shockingly, new data show that 25% of all TB deaths are HIV-related, double the amount previously thought.
Optimal TB management requires rapid detection, treatment, and prevention - but current technologies are outdated and impractical. The most commonly used methods have not changed in more than 125 years. According to the WHO, “only 2% of multi-drug resistant TB (MDR-TB) cases worldwide are being diagnosed and treated appropriately.”
The current testing method begins with spreading the patient’s sputum on a glass slide, treating the material with a special stain, and observing the slide under a microscope to detect the stained bacteria. The stain does not wash off the bacteria on the slide even when an acid solution is used, hence the organisms are called acid-fast bacilli (AFB). But this method, as practiced in high-burden parts of the world, fails to detect half of all active cases; a failure rate that is even higher in those co-infected with HIV. Tests that are more accurate exact a price, both monetarily and in the prolonged length of time it takes to achieve accurate detection of the TB bacteria by cultivating them in the laboratory. Due to the slow growth rate of the TB organism (the bacterium has a doubling time of 24 hours), growing the organism in culture can take weeks before results are available. It then takes even longer to determine if there is drug resistance, because the positive culture requires a second cultivation in the presence and absence of antibiotics in order to determine which antimicrobial agents are effective in inhibiting its growth. Employing all these procedures currently required to diagnose drug-resistant TB means that results may not be available for months.
Unfortunately for many patients in the developing world, this amounts to a post-mortem diagnosis, particularly as they are often co-infected with both TB and HIV - a rapidly fatal combination. Additionally, because diagnosis takes so long, these patients are sent back into the community, rather than isolated and treated appropriately, allowing for further transmission of the organism within the community in the local area.
In most of the developed world, isolation of patients with TB is implemented to prevent transmission to surrounding family and communities. However, even in the UK there are currently no guidelines advocating isolation during the time that the laboratory testing is being conducted, making a rapid diagnosis even more necessary.
Until now, if a clinician were to request a quicker TB diagnostic method than culture, accuracy would suffer. Sputum smear testing alone can theoretically produce results within a day. However 30% or more of patients whose sputum is smear-negative ultimately reveal positive cultures in follow-up tests, proving this diagnostic method to be highly insensitive and dangerously inaccurate. Missed diagnoses via smear testing are estimated to contribute to as much as 20% of newly transmitted TB infections. And in an unexpected twist of fate, patients with HIV/TB co-infection are actually more likely to be smear-negative but to progress rapidly to a fatal outcome.
As World TB Day arrived at the end of last month, Ki-Moon called for faster action to combat TB on a global scale. He highlighted the need for infection prevention, early detection, and worldwide availability of treatment, whilst calling for more effort to combat MDR-TB, XDR-TB (extensively drug-resistant TB), and the TB/HIV co-epidemic.
With this in mind, he called for organisations to come together and develop diagnostic solutions that will prevent the spread of the disease. Specifically, the WHO has called for faster and more accurate diagnostics based on the detection of specific DNA patterns in a gene called rpoB, found in all TB bacteria, that is considered to be a surrogate marker for MDR-TB.
In line with these announcements, a number of organisations have collaborated to develop a new diagnostic test for TB. The new test detects TB in less than two hours, is highly accurate, and has recently become available in Europe. The test is run on Cepheid’s GeneXpert System, a leading molecular testing platform, and was developed jointly by Cepheid, the Foundation for Innovative New Diagnostics (FIND), the University of Medicine and Dentistry of New Jersey (UMDNJ), and the US-run National Institute of Allergy and Infectious Diseases (NIAID).
Not only is the advanced technology able to detect the TB organism directly from sputum, it can also simultaneously detect rifampicin resistant strains of the infection via the presence of mutations in the rpoB gene, which is generally used as a surrogate marker for multi-drug resistance. And the test boasts unprecedented levels of sensitivity for detection of both smear-positive and smear-negative TB cases. Especially for HIV co-infected patients, the latter feature could be a life-saver.
This new test is one of the most important diagnostic developments to have occurred in many years. It is the most technologically advanced test for TB ever developed, yet it is simple enough to perform in all corners of the world, including resource-limited settings in which it is most needed.
When an individual is suspected of having a TB infection, the first step is often a chest X-ray. If the chest X-ray is suspicious, the patient is asked to cough and produce a sputum specimen, which is mixed with a solution that kills the TB organisms and placed into a small plastic GeneXpert cartridge. The GeneXpert System carries out steps that would have, until recently, required a dedicated laboratory facility and highly trained molecular diagnostic specialists. Within the cartridge, specimen processing and reagent mixing involving dozens of micro-pipetting steps are used to carry out nested real-time polymerase chain reaction (PCR), a method to multiply DNA in a test tube dramatically faster than occurs naturally in cultures. This reaction amplifies a DNA signature sequence that is specific to TB complex bacteria. DNA amplification and detection occur simultaneously within about 90 minutes. Because the test procedure is so simple, it is possible to run a STAT test on demand for patients with X-ray findings suspicious for TB, thus maximising the medical value of the test. At this time, all other PCR-based tests require multiple hands-on processing steps that necessitate performing the tests in batches.
Building on an ingenious protocol developed by Dr David Alland, M.D. And his colleagues at UMDNJ, the team engineered a protocol inside the GeneXpert cartridge that detects the presence of 5 different mutations in the core of the rpoB gene, now proven to detect virtually all rifampicin resistant strains of TB. Because rifampicin resistance is almost always present when there is resistance to other drugs, this result can be used to predict MDR-TB. Still more important is that the presence of rifampicin resistance predicts failure of first-line therapy, guiding the clinician to the most effective choice of second-line treatment and potentially avoiding costly treatment failures that lead to progressive disease and continued spread to others.
Tuberculosis is not an infection that is going to vanish overnight and, contrary to popular belief, it is not a disease from the 19th century that we no longer need to worry about, although it should be. It is a tragedy that we have not yet managed to eradicate this devastating infection from the world, both in developing countries and in our own supposedly developed country. Its close links with poverty and the HIV virus make it a much more widespread and serious issue in Africa and Southeast Asia, but it is also a concern in the UK and Europe. With over 8,000 new cases of TB reported every year in the UK, we are seeing the number of cases increase, bucking the global trend. It is my sincere hope that this new diagnostic test, which will soon be available throughout the world, will mark the tipping point when we finally start to win the battle against TB.
Source - ( Jul 24, 2009 )
By Geri Aston
When MultiCare Health System started rapid screening for methicillin-resistant Staphylococcus aureus (MRSA) in late 2007, officials paid close attention to one intensive care unit because of its history as an infection source. In the first quarter of 2008, before the screening protocol was consistently adopted, the medical-surgical ICU in question had three cases of health care-associated MRSA, says Marcia Patrick, R.N., MSN, infection control director for MultiCare Health System, Tacoma, Wash. In the second through fourth quarters, one patient was colonized with the bacteria after admission, but no patients became infected in any of the system’s five ICUs. “Early identification means we can get positive patients into isolation quickly,” Patrick says. “With good use of gowns, gloves and hand hygiene, we reduce the risk to other patients.”
Because of heightened attention to prevention of health care-associated infections (HAI) and advances in testing technology, many hospitals are considering making the move to rapid tests. But the decision is a complicated one that depends on an individual hospital’s circumstances, say hospital officials, infection control experts and test makers.
Rapid tests fall into two broad categories: screening (for the presence of an infection) and diagnostic (to determine the source). Major developments have occurred in both categories in recent years that have expanded hospitals’ options.
When it comes to rapid screening tests, most attention is on MRSA, says Patrick, who is an Association for Professionals in Infection Control and Epidemiology board member. “That’s probably the biggest thing health care facilities are struggling with, in terms of do we or don’t we want to go the rapid route.”
When making the decision, it’s important not to look at the tests in isolation, says Susan A. Levine, Ph.D., vice president of health technology research and consulting for Hayes Inc., a health technology assessment company in Lansdale, Pa. “These tests are very clever, but it’s really more important to have a system in place to support them and even to figure out if you need them.”
Hospitals should make sure they have a good infection control program in place before deciding whether to move to a rapid test, Levine says. Instituting proper hand hygiene, wound care and environmental cleanliness standards might solve most of the problem.
If a hospital has a strong infection control system and low HAI rates, it might not make sense to bring in a rapid MRSA screening test and its associated costs, says Daniel Diekema, M.D., an epidemiologist at the Iowa City VA Medical Center and a professor of internal medicine and pathology at the University of Iowa Carver College of Medicine. “You have to work with your clinicians and laboratorians up front to decide if it’s justified to bring a test on board,” he says. “Will it make a clinical impact?”
If all the infection control protocols are in place and a problem persists, a hospital might want to bring in a rapid screening test, says Diekema, who chairs the Society for Healthcare Epidemiology of America’s scientific meeting planning committee.
The question then becomes which patients to test and when. This decision, too, depends on the hospital’s circumstances. The options include screening all admitted patients or just those admitted to specific areas. The Veterans Health Administration has required screening of almost all inpatients since 2007. MultiCare tests all ICU patients, says Patrick. The system also began screening certain surgical patients, such as those getting total joint replacement or spine surgery, because infection in those cases can be so medically devastating.
Some hospitals opt only to screen patients with risk factors for carrying or getting MRSA in the hospital, such as patients transferred from nursing homes or immunocompromised patients, Levine notes. However, not all at-risk patients are easy to identify, especially as MRSA has spread into the community, says David Persing, M.D., Ph.D., chief medical and technology officer for test-maker Cepheid, Sunnyvale, Calif., (www.cepheid.com).
Hospitals have several options from which to pick once they’ve made the decision to go with a rapid screening test. Results from time-honored standard cultures take about 48 hours. But newer technology allows labs to grow cultures in 18 to 24 hours using chromogenic media. Hospitals also could go with real-time polymerase chain reaction (PCR) tests, which rely on gene amplification and provide results in two hours or less.
Sizing up needs
In evaluating their choices, hospitals should look at a number of factors, including test performance, ease of use and cost. Other questions to ask are whether the test can be run with current lab personnel, given their numbers, training, lab space and certification; how often tests would be run; how much room is available to isolate positive patients; and what kind of technical support and training the test manufacturer provides.
The two available PCR screening tests perform comparably well, several experts say, so other issues come into play. One is frequency of testing. Cepheid tests can be done on demand on individual patients, while Becton, Dickinson and Co. (BD, www.bd.com) tests are run in batches, so they’re done less frequently. For example, MultiCare uses the BD test, which the lab runs once a day at 11 a.m., with results coming back between 1:30 p.m. And 2 p.m., Patrick notes.
On-demand tests give hospitals more flexibility, but they pose a lab staffing question, she says. “Do you have the manpower for someone to step away from their routine work and go set one of these up to get an immediate result?”
Many hospitals avoid the big capital expenditure with “reagent rental” agreements, says Barbara Kalavik, spokeswoman for BD, Franklin Lakes, N.J. Under this approach, the hospital finances use of the PCR machine by committing to the manufacturer’s reagents and paying somewhat more for them.
Two other considerations are test volume and the lab’s turnaround time. Depending on those factors, batched PCR test results could potentially have the same turnaround time as the less-expensive chromogenic plates, Patrick notes.
Hospitals also have to address whether they plan to isolate patients while awaiting MRSA screening test results and if they have space to do so, Levine says. If the hospital has plenty of single-bed rooms in which patients are essentially isolated, a slower test might be a viable option. Other hospitals may not have the space to pre-emptively isolate patients for 18 or more hours, in which case the more rapid PCR test might be the way to go.
“A lot of hospitals have at least considered the [PCR] test because it really does speed up the process,” Levine says. “It becomes reasonable when someone is being admitted for surgery to have the test results before surgery.”
Most U.S. Hospitals do not isolate patients pre-emptively, Persing says. Instead, they make the bed assignment, wait for the test results and then react. In hospitals where patients typically share rooms, that practice poses an exposure risk when a patient tests positive for carrying MRSA, he says. Although it’s more expensive up front, getting the test results before bed assignment can save money later in bed management, Persing argues.
The two PCR test manufacturers only have U.S. Approval to sell rapid screening products for MRSA. However, last year both BD and Cepheid introduced products in Europe that screen for vancomycin-resistant enterococci. Cepheid plans on submitting its test for Food and Drug Administration (FDA) approval, Persing says. BD’s product currently is under FDA review, Kalavik says.
An advantage of the PCR machines is that they can be used to detect other bacteria as tests for those organisms become available, Patrick says. For example, MultiCare plans to use BD’s Clostridium difficile PCR diagnostic test. The company announced in January that it had FDA clearance for U.S. Marketing.
Rapid diagnostic tests for HAIs are quickly evolving. The technologies allow for quick identification of the organism causing a patient’s bloodstream infection. This means clinicians can prescribe the right antibiotic more quickly, which benefits the patient and helps prevent antibiotic resistance, several experts say.
Last year, BD and Cepheid received FDA approval for their rapid PCR diagnostic tests for methicillin-sensitive S. Aureus (MSSA) and MRSA. The tests detect the organisms in blood culture bottles showing gram-positive cocci. The products yield results in two hours or less, depending on the maker, as compared with the several days traditional tests take. Knowing whether the patient is infected with resistant or nonresistant staph allows the clinician to tailor antibiotic therapy accordingly, Persing says.
Another rapid diagnostic testing option is peptide nucleic acid fluorescence in situ hybridization (PNA FISH). The technology uses fluorescent molecular gene probes that target specific microbes to identify which organism is causing a patient’s bloodstream infection. Like PCR tests, PNA FISH is run using a sample from a positive blood culture. Results take about two and a half hours.
Beyond MRSA testing
The technology can detect a number of bacteria, most of which can cause bloodstream infections in hospitalized patients, says Graeme Forrest, M.D., assistant professor of medicine in the Oregon Health and Science University division of infectious diseases. These include S. Aureus, enterococci, streptococci, pseudomonas and Escherichia coli. PNA FISH also tests for yeast and fungal infections.
PNA FISH products by test manufacturer AdvanDx (www.advandx.com ) cover 95 percent of organisms causing bloodstream infections, and the Woburn, Mass., company has plans to develop tests for the remaining 5 percent, says marketing director Joen T. Johansen.
When evaluating PCR and PNA FISH technology, hospitals should consider a number of factors, Forrest says. They include: “what works within the resources of your center, what’s practical, what can be achieved and what can fit in the space you have. It also depends on your tech people—what their qualifications are.”
The technologies have some major differences. One is cost. PNA FISH requires less investment up front, about $4,000 to $4,500, Johansen says. After that, the reagents cost about the same as PCR tests, Forrest notes.
The two types of tests have advantages and disadvantages in terms of which organisms they detect. A major limitation of the PNA FISH staph test is that it does not show whether the bacteria is methicillin resistant, Forrest says. On the plus side, PNA FISH products are available for a wider range of bacteria.
The PNA FISH enterococci test differentiates between E. Faecalis and E. Faecium, which is often vancomycin resistant (VRE). According to the Centers for Disease Control and Prevention, enterococci cause about one out of every eight infections in hospitals, and about 30 percent of those are VRE.
The PCR diagnostic tests’ main disadvantage is that its U.S. Use is limited to only staph in the case of Cepheid, and staph and C. Difficile for BD. However, that could change soon. Cepheid’s rapid diagnostic test for C. Difficile is available in Europe. The company is submitting its product, which yields results in 45 minutes and can identify the strain responsible for hospital epidemics, to the FDA, Persing says. He expects approval before the year is out.
Cepheid also has released in Europe a rapid diagnostic test for tuberculosis. The sputum test not only determines the presence of the bacteria but identifies whether it is drug resistant. It takes about 45 minutes, rather than between four and six weeks for the traditional technique, Persing says. He anticipates that the company will begin the U.S. Regulatory approval process next year.
When it comes to diagnosis of bloodstream infections, Forrest believes hospitals should move to rapid products for the clinical benefit. “The microbiology labs in some small centers are still doing the same things we’ve been doing for the past 80 to 90 years.”
The main barrier is the price. “Unfortunately with hospitals, it’s a cost to the lab. They’re really tightening up any costs at the moment,” Forrest says.
He urges hospitals to take a long view. “If we can shorten lengths of stay, shorten the use of unnecessary antibiotics, have less ICU stays because [patients] get the right drug, it’s going to save a lot of money on the other end.”
Hospitals interested in investing in rapid tests should keep an eye on the horizon, Levine says. Some interesting products could be hitting the market in the coming years. One is a five-hour MRSA screening test that uses bacteriophages, viruses that infect bacteria.
MicroPhage Inc. (www.microphage.com ), the test’s maker, announced in January that it had completed its first multicenter clinical trial. The company, located in Longmont, Colo., predicts the product will be available to U.S. Hospitals later this year.
A diagnostic MRSA test under development uses metal ions to identify the bacteria’s genes without the need for the gene amplification technique on which PCR tests rely. If approved, this metalized-DNA approach could be used by labs that lack the sophistication to run PCR tests, notes Adnavance (www.adnavance.com ), the San Diego company developing the test.
Rapid tests for HAIs are still a new phenomenon, Levine says. The first quick-screening test for MRSA was approved in 2004 and the second in 2007, she notes. In the last quarter of 2008, 447 Cepheid GeneXpert Systems were in place in the United States, says spokeswoman Darwa Peterson. About 100 U.S. Labs are using AdvanDx’s PNA FISH products, Johansen says.
“What we’re seeing is just the start of these rapid tests,” Levine says. “I think they’ll become more user-friendly and easier to use at the point of care, and that would all be good.”
Source - ( Jul 23, 2009)
Survivors Network, the Chicago-based nonprofit and the official organization that launched World MRSA Day earlier this year is building momentum in the US, the UK and worldwide in its humanitarian grass-roots effort to raise awareness of the MRSA epidemic.
"As governments and world agencies continue to put their focus on swine flu and prepare to spend billions of dollars on it as they did with Avian flu; the true epidemic/pandemic, MRSA continues to be virtually ignored", states Jeanine Thomas, president of MRSA Survivors Network and the National Spokesperson for MRSA. "It is unconscionable what has been allowed to happen and MRSA has been swept under the carpet in healthcare facilities for decades and the purpose of World MRSA Day is to raise awareness."
Healthcare industry companies are stepping up and sponsoring World MRSA Day in the US and in the UK to raise awareness and save lives. In the U.S.; 3M, Tec Labs, Cepheid, Pfizer and others have lent their support and all have become true heroes in this movement to save lives.
The inaugural pre-launch kick-off event in the U.S. Will be held Oct. 1st at Loyola University in Chicago with an international press conference, followed by the event. MRSA Action UK, who shares and alliance with MRSA Survivors Network are planning their event for Oct. 2nd. MRSA activists are organizing and holding events in their communities on Oct. 2nd and throughout October, MRSA Awareness Month. Activists can list their event on the official site- www.worldmrsaday.org.
MRSA Survivors Network and fellow activists are pushing for immediate action, legislation, funding, more stringent and pro-active infection control measures and enforcement by the World Health Organization, the Centers for Disease Control and governments to stop the massive loss of life and human suffering from preventable infections. World MRSA Day is embracing all survivors and their families who have suffered or lost a loved one from any multi-drug resistant infection.
Jeanine Thomas of MRSA Survivors Network was the first advocate in the U.S. To raise the alarm about MRSA and other healthcare-acquired infections and began her crusade in 2003. Ms. Thomas is a survivor of MRSA, sepsis, osteomyelitis and C. Diff and became critically ill and nearly died from the results of ankle surgery. She now lives with chronic MRSA infections.
MRSA Survivors Network
<p><a href="http://www.medicalnewstoday.com/articles/154878.php" target="_blank"><b>Source - ( Jun 01, 2009 )</b></a></p>
MRSA Survivors Network, the Chicago-based nonprofit and the official organization that launched World MRSA Day earlier this year is building momentum in the US, the UK and worldwide in its humanitarian grass-roots effort to raise awareness of the MRSA epidemic.
"As governments and world agencies continue to put their focus on swine flu and prepare to spend billions of dollars on it as they did with Avian flu; the true epidemic/pandemic, MRSA continues to be virtually ignored", states Jeanine Thomas, president of MRSA Survivors Network and the National Spokesperson for MRSA. "It is unconscionable what has been allowed to happen and MRSA has been swept under the carpet in healthcare facilities for decades and the purpose of World MRSA Day is to raise awareness."
Healthcare industry companies are stepping up and sponsoring World MRSA Day in the US and in the UK to raise awareness and save lives. In the U.S.; 3M, Tec Labs, Cepheid, Pfizer and others have lent their support and all have become true heroes in this movement to save lives.
The inaugural pre-launch kick-off event in the U.S. Will be held Oct. 1st at Loyola University in Chicago with an international press conference, followed by the event. MRSA Action UK, who shares and alliance with MRSA Survivors Network are planning their event for Oct. 2nd. MRSA activists are organizing and holding events in their communities on Oct. 2nd and throughout October, MRSA Awareness Month. Activists can list their event on the official site- www.worldmrsaday.org.
MRSA Survivors Network and fellow activists are pushing for immediate action, legislation, funding, more stringent and pro-active infection control measures and enforcement by the World Health Organization, the Centers for Disease Control and governments to stop the massive loss of life and human suffering from preventable infections. World MRSA Day is embracing all survivors and their families who have suffered or lost a loved one from any multi-drug resistant infection.
Jeanine Thomas of MRSA Survivors Network was the first advocate in the U.S. To raise the alarm about MRSA and other healthcare-acquired infections and began her crusade in 2003. Ms. Thomas is a survivor of MRSA, sepsis, osteomyelitis and C. Diff and became critically ill and nearly died from the results of ankle surgery. She now lives with chronic MRSA infections.
MRSA Survivors Network
“Fast and accurate diagnosis of MDR TB is critical if the increase in this especially deadly form of TB is to be slowed,” says David Alland, M.D., of the University of Medicine and Dentistry of New Jersey, in Newark. “It is important to distinguish patients who have drug-susceptible disease and can be started on a routine regimen of one or more TB drugs from those whose infections are caused by drug-resistant strains of M. Tb. If the latter can be identified as soon as they come to the clinic for treatment they can be safely separated from the rest of the hospital patients and will not spread MDR TB to others.”
Dr. Alland and his collaborators at Cepheid, Inc., of Sunnyvale, Calif., have had success identifying MDR TB using a test that they say is well suited to conditions in countries where health care resources are limited and where TB is widespread. Dr. Alland explains that the testing method they have developed using a special assay is essentially a biotech lab in a small plastic cartridge. The test quickly, automatically, and accurately tells doctors whether a sample of patient sputum (material expelled from the lungs and throat by coughing) contains drug-resistant or drug-susceptible M. Tb. The test is contained in a sealed cartridge to protect the patient sample and prevent contamination of the sample. A contaminated sample could result in inaccurate or false test results.
One early hurdle for the researchers was finding a simple way to extract M. Tb DNA from the thick, sticky sputum. Ordinarily, processing sputum is labor-intensive and requires expensive equipment, such as centrifuges, which are not always available in resource-limited settings. After some effort, the scientists devised a way to liquefy sputum with chemicals that can be added directly into the sputum collection cup. The sputum can then be easily transferred into the sample processing cartridge using a simple plastic pipette, eliminating the need to spin samples in a centrifuge.
After the M. Tb DNA is extracted from the sputum sample it is amplified for testing, through a technique called real-time polymerase chain reaction. When the sample is large enough, five kinds of lab-made fluorescent molecular tags, called beacons, are applied simultaneously. Each beacon glows in a different color when it attaches to M. Tb DNA. In the presence of drug-susceptible TB, all five colors are visible. If any one of the colors is absent at the end of the test, it means the sample contains drug-resistant M. Tb. Dr. Alland and his colleagues reported that their molecular beacon assay took less than 2 hours and correctly identified rifampin-resistant TB in almost all of the samples tested.
In 2004, the scientists used the molecular beacon assay to correctly distinguish rifampin-resistant and rifampin-susceptible strains of M. Tb in sputum samples taken from patients in areas of high TB incidence in north India and Mexico. Rifampin is one of the first line drugs used to treat TB. It is also one of the drugs to which strains of M. Tb have developed resistance, and importantly, resistance to this drug has been shown to be a reliable marker for MDR TB.
Along with Dr. Alland and his collaborators from Cepheid, Inc., the partnership now includes Geneva, Switzerland-based Foundation for Innovative New Diagnostics. The group has initiated field tests of their all-in-one diagnostic cartridge in Azerbaijan, India, Germany, Peru, South Africa and Uganda.
El-Hajj, H. H. Et al. Detection of rifampin resistance in Mycobacterium tuberculosis in a single tube with molecular beacons. J Clin Microbiol. 2001 Nov;39(11):4131-7.
Varma-Basil, M. Et al. Rapid detection of rifampin resistance in Mycobacterium tuberculosis isolates from India and Mexico by a molecular beacon assay. J Clin Microbiol. 2004 Dec;42(12):5512-6.
Thirteen years after Roche launched the industry with a test for HIV load based on transcript abundance, molecular diagnostics is in full flower. A $3.3 billion market growing at 17 percent annually, the field includes assays for disease predisposition, screening, diagnosis, prognosis, monitoring, and predicting treatment efficacy, using markers ranging from SNPs to methylcytosine, messenger RNA to microRNA. Bringing digital power to traditional analog medicine, the field "is absolutely going to revolutionize health care," says Harry Glorikian, managing partner at Scientia Advisors. "I believe it with every fiber in my being."
By Jeffrey M. Perkel
Inclusion of companies in this article does not indicate endorsement by either AAAS or Science, nor is it meant to imply that their products or services are superior to those of other companies.
About a year ago, 48-year-old Jeff Gulcher's doctor called to say Jeff had cancer. His prostate was teeming with cells scored at Gleason-6, the high end of intermediate grade. The recommendation: radical prostatectomy.
The diagnosis was shocking. Gulcher wasn't at risk as far as he knew: he had no family history of early prostate cancer, and he had no symptoms. What he did have was a suspicious genotype.
Gulcher had paid $985 for the deCODEme test, a genome-scale single nucleotide polymorphism screening service from deCODE Genetics. The test uses Illumina microarrays to profile about a million loci, reporting back on the relatively small number of conditions that can be tied to the results. Scattered across 10 chromosomes, the test's 13 prostate cancer–related markers suggested that Gulcher's lifetime risk for the disease is about twice the average, or 32 percent. That's not a diagnosis, but a warning, like elevated cholesterol. It means, pay attention.
Gulcher's physician recommended a prostate specific antigen (PSA) test. Such screening typically doesn't begin until age 50. In this case, the test came back at 2.0 ng/mL, "in sort of the mid-normal range," Gulcher says. Normally harmless, that value plus the genotype data prompted his urologist to refer Jeff for a biopsy.
"I'm thinking, there's no way I have cancer," Gulcher recalls. "I'll go through the motions, have the biopsy. But just having this higher risk doesn't mean I'm going to get prostate cancer by any stretch of the imagination."
Fortunately, Gulcher didn't rely on his intuition, and the surgeon excised his tumor intact; postoperatively, it was reassessed at high grade. PSA score down to zero, Gulcher appears to have dodged a bullet, all thanks to a molecular diagnostic that he actually helped develop. Gulcher is deCODE's co-founder and chief scientific officer.
"This genetic test is reclassifying people who are thought to have average risk into somebody who really is at higher risk, and may benefit from extra surveillance," Gulcher says.
That, in a nutshell, is the promise of molecular diagnostics (MDx). Whether stratifying patients for heightened or diminished scrutiny, advising doctors on treatment decisions, or diagnosing disease, the field is redefining medicine.
Every diagnostic is molecular, whether measuring cholesterol for heart disease, radiolabeled glucose for brain imaging, or mass spectrometric peaks for ovarian cancer. So just what, exactly, is a "molecular" diagnostic?
According to Harry Glorikian of Scientia Advisors, the term generally applies to assays that detect nucleic acids, whether single nucleotide polymorphisms, mutations, or RNAs, with a sprinkling of "certain very high-quality, high-value protein assays," as well.
They come in a variety of flavors. Besides predisposition tests like deCODEme, there are assays for diagnosis, prognosis, prediction of drug response, and disease monitoring. Such tests, says Glorikian, represent a fundamental break from what he calls the traditional "gestalt" diagnostic approach. Blood pressure, temperature, and blood chemistry "are not digital indicators but analog indicators," Glorikian says, "where [the doctor] uses the computer between his/her ears, looks at all this stuff, bins it together, and says aha! This is what I think."
By contrast many MDx tests provide quantitized, digital data, as well as actionable results.
Take Down syndrome. Traditionally, a woman's likelihood of having a baby with trisomy-21 is first assessed by noninvasive screens based on maternal serum proteins and ultrasound. Yet these assays provide risk scores rather than absolute diagnoses, and must often be supplemented with more accurate, but also invasive tests such as amniocentesis, which literally count fetal chromosomes.
Sequenom's trisomy-21 MDx, set for launch later this year, blends these two approaches. Using mass spectrometry to count fetal chromosome 21–derived transcripts in maternal blood, the test is both noninvasive and quantitative—though not, per se, digital; the test measures the ratio of maternal and paternal alleles from chromosome 21.
"It's an absolute result," says Harry Stylli, Sequenom's president and CEO. "You're not dealing with a risk score." As a result, high-risk pregnancies may be properly managed, while the number of pregnancies unnecessarily subjected to invasive procedures—currently about 140,000 per year, Stylli estimates—"will dramatically decrease." [Editor's Note: It came to our attention shortly after publication of this article that the release of Sequenom's test has been delayed due to questions about some of their test data.]
Perhaps no genes more starkly illustrate the actionable power of MDx than BRCA1 and BRCA2. Mutations in these genes carry such high risk of breast and ovarian cancer (about 82 percent) that some carriers opt for prophylactic radical mastectomy, using Myriad Genetics' BRACAnalysis blood test to help guide their decision.
Based on full-length Sanger dideoxy gene sequencing, BRACAnalysis is an unusual test; sequencing is more typically suited to research labs. Yet unlike proteins or transcripts, Gulcher notes, DNA "is the only stable molecule in your body." It enables Myriad to pick up novel mutations. "Unfortunately there are a very large number of mutations in the genes that we look at, so it is not a mutation-specific test, it is a full DNA sequence test," explains Pete Meldrum, Myriad's president and CEO.
Myriad offers the test as a lab-developed "home brew" assay available through the company's Clinical Laboratory Improvement Amendments (CLIA)–certified facility in Salt Lake City, Utah. Not yet vetted by the US Food and Drug Administration (FDA), such tests can only be offered by the lab that develops them.
In this case, results come back in about 10 days. That's not generally a problem for predisposition tests. But sometimes, and especially when confronting acute medical issues, patients don't have that kind of time.
"In general, the faster the results become available, the more likely it is that they are going to be actionable," says David Persing, executive vice president and chief medical and technology officer at Cepheid.
When speed is required, real-time or quantitative PCR (qPCR) delivers. Most labs have a PCR machine, and most lab techs are competent to run them.
"Real-time PCR is quite a forgiving technique," says Stephen Little, CEO of DxS. "You can put different levels and quantities of samples into the test and it will still give a good result."
Even so, clinical labs often delay crucial tests because they must process them in batches. Cepheid's GeneXpert system was built to circumvent this problem, says Persing. A modular, independently addressable real-time PCR system, the GeneXpert is "the first sample-in, answer-out machine for real-time PCR to hit the market," says Glorikian.
Anyone can use it, Persing says. In one study, nurses running the company's Group B Streptococcus test in the labor and delivery suite "delivered results that were highly accurate, and were as reliable and as good as the ones delivered by the lab."
In part, that's because there's no sample prep; the system processes raw samples straight to analysis. Results can arrive in as little as 31 minutes, fast enough to catch patients before they leave the doctor's office.
Cepheid's new Xpert MTB/RIF assay reports in less than two hours both if an individual has tuberculosis and whether it is resistant to rifampicin. Traditional culture methods take weeks to make that assessment, says Persing, during which time patients may unwittingly spread the infection.
Cepheid's assays are mostly based on Applied Biosystems' TaqMan reagents, modified oligonucleotides that contain a fluorescent dye on one end and a quencher on the other. During PCR, this oligo binds its target DNA between the two amplification primers, where it is chewed up by the polymerase's 5' to 3' exonuclease activity, separating dye from quencher in a burst of fluorescence.
DxS uses an alternative detection reagent, called a "scorpion" probe, for its TheraScreen assays.
A scorpion, says Little, is "a self-quenching molecule." A hairpin with a PCR primer at its 3' end, the scorpion serves as a 5' PCR primer. The hairpin is complementary to part of the amplified sequence, and like a TaqMan probe bears both a fluorophore and a quencher. In the absence of amplification, the closed hairpin keeps the two dyes in close proximity, dousing fluorescence. But after amplification, the probe can denature, unfold like a scorpion's tail, and hybridize to the amplified segment. In the process, the fluorophore and quencher dissociate.
"That's the signal that the reaction has taken place and we use it to signal the presence or absence of the mutations that we look for," says Little.
In the case of DxS's K-RAS and EGFR assays, the resulting data can guide treatment. Knowledge of a K-RAS mutation helps oncologists choose between cetuximab and panitumumab in colorectal cancer, while different mutations in EGFR affect which works better for nonsmall-cell lung cancer (NSCLC), gefitinib or erlotinib.
Though many MDx probe single nucleic acids, a small but growing number profile panels of genes simultaneously.
"As our understanding of genetics increases, we are going to learn that most diseases are multigenic in nature," says Amit Kumar, president and CEO of Combimatrix, which develops MDx based on array comparative genomic hybridization; "It's not just one gene that's causing a particular illness or disease; it's a combination of multiple genes functioning together."
Three such tests have been cleared under the FDA's "in vitro diagnostics–multivariate index assay" guidelines, all based on gene expression. XDx's 20-gene AlloMap test uses reverse transcriptase (RT) PCR to monitor heart transplant rejection; Agendia's 70-gene MammaPrint microarray assay predicts whether breast cancer patients are likely to relapse and benefit from chemotherapy; and Pathwork Diagnostics' Tissue of Origin test uses a 1,500-gene array to determine the source of a primary tumor in patients that present only with metastases.
"In order to effectively treat a metastasis, you have to treat it in the same way that you treat the primary tumor," explains Ronen Tamir, chief commercialization officer at Rosetta Genomics, whose "miRview mets" test addresses the same problem using a 48-gene RT-PCR assay. In other words, a breast tumor that metastasizes to the colon should be treated as breast cancer.
Such tests may be less digital, perhaps, than the Down syndrome or BRCA tests—they sometimes provide probabilities rather than certainties—yet they still can help doctors make smarter treatment choices.
According to Steve Shak, chief medical officer at Genomic Health, whose 21-gene RT-PCR–based Oncotype DX assay also predicts breast cancer relapse, the most common treatment for women with node-negative, estrogen receptor–positive breast cancer is chemotherapy. Yet only about 4 percent of patients benefit from that option; the rest would do just as well with surgery plus hormonal therapy alone.
Now, using Oncotype DX, about one-third of treatment decisions change, Shak says. "Some women with high recurrence scores who might have been treated with hormonal therapy alone are then given hormonal therapy plus chemotherapy," he says. Similarly, "there are many women who might have considered chemotherapy but based on the results will be confident in getting hormonal therapy alone." Agendia cites similar numbers for its MammaPrint diagnostic.
Yet when it comes to multivariate MDx, array-based assays like MammaPrint are relatively rare; frequently, developers use arrays for test development, reverting to RT-PCR once the hard work of biomarker identification is done. Such was the case for Oncotype DX and AlloMap.
"The thing with arrays is they are a little bit clunky to use, whereas RT-PCR, if you can use it, is a much easier technology to fit into a lab," says Little. "Ease of use and convenience are very important considerations when you move into diagnostics."
Ease of use and convenience are especially important when it comes to more esoteric markers, such as DNA methylation or microRNAs.
LabCorp's ColoSure test measures methylation of the vimentin gene in stool samples. Epigenomics is developing a blood-based assay for SEPT9 methylation. Both are early indicators of colon cancer, and both are based on qPCR detection of bisulfite-treated DNA (in which unmethylated cytosine is converted to uracil, while methyl-C remains unmodified).
"Having a very innovative test with a novel analyte [DNA methylation], we decided to be rather conservative when it comes to the assay technology," says Achim Plum, senior vice president for corporate development at Epigenomics.
Slated for European release this year, Epigenomics' assay won't be the first blood test–based colon cancer screen on the market; GeneNews beat them to the punch in 2008 with ColonSentry, based on microRNA expression.
Short, endogenous, regulatory RNAs that silence the translation of complementary mRNAs, microRNAs are relatively recent discoveries. Yet they have emerged as hot commodities in MDx, and Cepheid, Rosetta Genomics, Exiqon, and Combimatrix are all also pursuing them.
Exiqon's in-development assay for colorectal cancer prognosis is based on the detection of microRNAs with DNA analogs called Locked Nucleic Acids (LNA), because, says Søren Møller, Exiqon's vice president of research and development, standard nucleic acids just aren't specific enough.
"You don't have much sequence space," Møller explains. "microRNAs are 20–23 nucleotides long, and if you need to detect those in complex samples, you need very, very accurate detection techniques. LNA provides that advantage."
Rosetta's miRview squamous test uses RT-PCR of a single microRNA to help distinguish between squamous and nonsquamous NSCLC—an actionable result, says Tamir.
"Alimta is only labeled for nonsquamous NSCLC; Avastin is labeled for both," he says. But a "black box" warning on Avastin "warns against fatal hemorrhaging of 40 percent of the patients which are squamous."
A potentially life-saving decision based on a type of molecule researchers barely knew existed 10 years ago, it's just another example of putting the "personal" in personalized medicine. The MDx landscape continues to change as new technologies—like next-generation sequencing—come to the fore.
"There's a lot going on in this space, it's so exciting," says Glorikian. "Anybody who isn't in this space needs to ask themselves why."
Fred C. Tenover turned an early love of science into a successful microbiology career at the Centers for Disease Control and Prevention. He published 290 papers and became a noted specialist in drug resistant bacteria and moved into the private sector in 2008. Now 54, Dr. Tenover works at Cepheid, a molecular diagnostics company in Sunnyvale, Calif., where he's developing early warning tools to detect antibiotic resistant diseases.
Full name: Dr. Fred C. Tenover
Hometown: Kalamazoo, Mich.
Current position: Senior director, scientific affairs for Cepheid
First job: Phlebotomist
Favorite job: Current one
Education: B.S. From University of Dayton; M.S. And Ph.D. From University of Rochester
Years in the industry: 26
How I got to here in 10 words or less: I asked the right questions.
Q. You always had a love of science, but that wasn't what you planned to study when you arrived at college.
A. I had 16 years of Catholic education and had been interested in science since elementary school. So as a freshman in college, my intent was to go to the seminary and join a religious order where I could teach and still do scientific research.
Q. Did you ever have any problems reconciling those two halves of your life?
A. Not at all. I had read the work of a Jesuit priest named Pierre Teilhard de Chardin. He was also a serious scientist, a paleontologist, and the way he melded his science and faith became a model for me.
Q. But you ended up choosing science over theology. Why?
A. I did a three-month study tour at eight different universities throughout Europe and had a chance to talk to a lot of different people. That's when I decided science was more attractive to me than the theology side in terms of what I could do with my career. My faith still played a major role in my career.
Q. What did you plan to do once you actually decided to get a Ph.D?
A. I wanted to continue to do research particularly in antibiotic resistance, which was what much of my doctoral thesis focused on. I took a post doctoral fellowship at the University of Washington studying clinical microbiology in public health. I later became associate director of microbiology at the Seattle Veterans Administration. And I continued to teach at the University of Washington.
Q. Where did you go from there?
A. I was offered a job at the CDC in 1990 as the chief of the antibiotics investigation branch and later became director. It was an opportunity to focus on antimicrobial resistance and public health and lead a larger group of scientists. Plus, the CDC was the Mecca of infectious disease research.
Q. I understand you taught introductory microbiology to nonscience majors. Is there a secret to keeping nonscientists engaged when discussing technical topics?
A. Every lecture should have a lot of factual information and it should also be mildly entertaining. With infectious diseases as a topic, it's easy to get people to laugh in one moment and to scare the heck out of them the next. That's a great way to hold a group together, and you get great questions.
Q. What do you consider to be your most notable accomplishment while there?
A. One was the discovery of community MRSA [methicillin-resistant Staphylococcus aureus] in 1999. It's mostly spread person to person in hospitals by other patients or health-care workers that haven't washed their hands or worn their gloves.
Q. How did you discover it?
A. We saw a report of four kids from Minnesota and North Dakota, all from Indian reservations, that developed Staph. They were treated with antibiotics but died anyway. Shortly afterward, there were outbreaks in Pennsylvania, Mississippi, Georgia and Texas. This was a wake-up call since these are not strains of MRSA that leaked out (of a hospital). They developed in the community.
Q. One of your early investigations on MRSA involved professional football players. What did you find?
A. We investigated an outbreak with a professional football team in 2003. We found linemen had the most incidents of MRSA because -- we hypothesized -- they had the most skin-to-skin contact. I understand the early infections sidelined a few professional athletic careers including former Redskin defensive tackle Brandon Noble and college football player Ricky Lannetti. The CDC worked with the NFL and the NCAA to develop guidelines for wound management, prevention and care.
Q. Do you consider drug-resistant bacteria to be a bigger threat than flu?
A. Yes. There are more antibiotic resistant bacteria that threaten our health every day and will be for a long time to come.
Q. After years of public service, what prompted your move into the private sector?
A. I got a great job offer from Cepheid in 2008 to establish an external resource program with all of our customers, collecting organisms and characterizing them, looking for emerging resistance and changing strain types across the U.S. And internationally. It merges my love of microbial resistance and rapid diagnostics, something I saw the importance of when working in developing countries with the CDC and the World Health Organization.
Q. Was it a tough transition?
A. Not really, because I worked with a lot of diagnostic companies while at CDC. Helping them to improve products is something I already had in my portfolio.
Q. Your entire career has been dedicated to studying antimicrobial resistance. What's needed to control the problem?
A. We need better infection control, definitely need new antibiotics and new rapid diagnostics to find out where the patients are to find these infections.
Two new California laws are forcing hospitals to spend more to rein in “super bug” staph infections that employers blame for helping to push up their insurance rates.
An estimated $3 billion is spent annually to treat patients who are infected while in a hospital, a cost the facilities are increasingly being forced to bear themselves.
Medicare has stopped reimbursing for certain hospital-acquired infections, and there’s talk that HMOs and PPOs will follow suit, hospital officials report.
The first law, which began as Senate Bill 1058, requires hospital officials to publicly disclose their infection rates by 2011 and screen high-risk patients for methicillin-resistant staph aureus, or MRSAs. The other, Senate Bill 158, gives the Department of Health Services the authority to further investigate infection outbreaks and complaints about infection control. Both were passed by the Legislature and signed by Gov. Schwarzenegger in September 2008.
A Silicon Valley company that is seeing more business as a result of the increased testing is Cepheid Inc. Where CEO John Bishop said his company has seen increased interest from hospitals wanting to implement MRSA surveillance testing. The Sunnyvale-based company manufactures MRSA nasal swabs that deliver results within 72 minutes.
“During the 18 to 24 hours that traditional culture tests take to deliver results, an infected patient could be put in a semiprivate room and potentially sicken a noncolonized patient,” Bishop said.
The tests retail for $42 each, and the system that runs the molecular tests range from $27,000 to $400,000, based on the volume of testing a hospital does.
Bishop said business has ramped markedly — from $9 million in MRSA test sales in 2007 to $47 million in 2008.
Five states, including California, have mandated testing while 11 states have pending legislation to do so.
Suzanne Cistulli, director of infection prevention and control at O’Connor Hospital in San Jose, said the hospital is following guidelines set by the U.S. Centers for Disease Control and Prevention for a hand-hygiene program.
“There are hand sanitizers installed at all entries and at elevators,” Cistulli said. “We encourage visitors and staff to gel in and gel out.”
The hospital started using a stronger sanitizer a year ago to better fight the spread of infection.
“It’s four times the cost of what we were previously using,” Cistulli said.
In addition, O’Connor has also stepped up its screening of patients who might be infected with or be carriers of the MRSA bug.
O’Connor Hospital previously tested only patients in the intensive care unit but has extended its testing to patients who fall into the high-risk category. This includes anyone who has been hospitalized during the past 30 days or three times or more in the past year, patients from skilled nursing facilities and those who have a history of MRSA.
Every high-risk patient gets a nasal swab when admitted, which is cultured within 24 hours. A positive result means the patient is put in isolation.
The cost is $10 per culture, which is absorbed by the hospital. Cistulli estimates 1,300 patients have been screened so far this year. Of those patients, 12 percent test positive for MRSA.
“The target is to have zero infections, but that’s not realistic — there’s always going to be an incidence of infection in a hospital,” she said. “The goal is zero tolerance for noncompliance with best practices.”
Sequoia Hospital in Redwood City has increased its nasal swab testing in response to the new laws. As of Jan. 1, 30 percent to 40 percent of admitted patients are tested for MRSA.
Infection Control Coordinator Cheryl Flesner said the hospital’s infection rate last year was 0.5 per 1,000 patient days, compared with the national average of 2 per 1,000 patient days.
“Our goal is 0.4 for 2009,” she said.
The state’s recent decision to revise a corporate income tax formula is expected to encourage big pharmaceutical companies such as Genentech Inc. To stay in Silicon Valley.
For South San Francisco-based Genentech, which chose Oregon over California to build a commercial drug facility in 2006, the opportunity comes too late. But others will most likely benefit.
California had based its corporate income tax on a percentage of a company’s sales, payroll and property within the state. With this formula, companies were discouraged from adding jobs and facilities within California because doing so would increase their tax rate, said Matt Gardner, CEO of BayBio, a Northern California trade organization representing the life sciences.
The new rule, passed in the state budget, changes that. Corporations can now base their taxes solely on sales within the state, under the so-called single sales factor. Companies that have a lot of sales in California but few or no offices or employees can stay with the old formula if it’s more advantageous.
“It puts California in a better, more competitive situation with other states such as Oregon,” said Gardner, adding that BayBio has been lobbying on behalf of the formula since 2003. The incentive will go into effect Jan. 1, 2011.
Under the prior law, California’s tax structure penalized decisions to keep or expand Genentech’s operations in the state, said Genentech Executive Vice President and CFO David Ebersman.
“We ultimately made the decision to construct a $400 million facility in Hillsboro, Ore.,” Ebersman said. “Although we had land suitable for expansion at our existing California locations, California’s tax structure didn’t support in-state growth.”
The Oregon site is expected to be licensed and operational in 2010 and will employ about 300 people by 2015.
“We are very pleased with the adoption of single sales factor,” Ebersman said. “For Genentech, this puts California back into play as an option for locating our facilities.”
The revision affects major California companies, including biotech firms, that are already profitable. But companies approaching profitability and thinking about expansion have also been encouraged. Among those are Cepheid, a Sunnyvale molecular diagnostics company, and South San Francisco’s Monogram Biosciences Inc., which specializes in products to help guide and improve treatments of infectious diseases, cancer and other serious disorders.
Monogram CEO Bill Young said his company had revenue of about $63 million in 2008 and expects to break even by year-end, so executives are thinking about the financial implications of expansion.
Young said with the old formula, he would be encouraged to add facilities and employees outside the state.
“It’s very illogical,” Young said. “Now the system is where the actual sales are.”
Cepheid CEO John Bishop said his approximately 500-person company is well-positioned for growth, closing in on profitability, and has a number of diagnostics nearly ready for the market. He said it will now make sense to keep Cepheid’s growth in-state.
The company has grown from one facility to four in Sunnyvale. Bishop said it occupies about 120,000 to 130,000 square feet, but he believes it will need about a half million square feet over the next several years.
Source - ( Mar 09, 2009)
New law has S.J. hospitals turning up the fight against infections
STOCKTON - In the life-and-death war against germs, especially the superbug MRSA, a state law as of Jan. 1 requires hospitals to implement control measures and tests on patients in an effort to stop the spread of hospital-acquired infections.
There also is a public-disclosure element to the new law - Senate Bill 1058, known as Nile's Law - that requires hospitals to report their infection data to the California Department of Public Health. By 2011, that information will be posted on the department's Web site.
San Joaquin County's seven acute-care hospitals have developed stronger infection-control procedures, converted regular patient rooms to isolation rooms, and beefed up their staff training and patient-education programs.
The hospitals are required to screen certain high-risk patients for methicillin-resistant staph aureus within 24 hours of admittance to protect other patients. Those considered high-risk include patients admitted directly from nursing and care homes, other hospitals, correctional facilities and those with weakened immune systems.
The law requires hospitals to follow specific protocols in cleaning and disinfecting items including all televisions, telephones, bedding, elevators, meeting rooms, glucometers, gurneys, feeding pumps and cardiac monitors to stop the spread of MRSA.
Patients who have MRSA must be provided with information on how to recover and stop the spread of the infection.
Nationwide, the federal Centers for Disease Control and Prevention estimated that in 2005, more than 94,000 people developed a serious MRSA infection and 18,650 of them died during a hospital stay related to a serious MRSA infection.
For 2007, the state Department of Health Services estimated 240,000 patients each year develop an infection of some kind while in a California hospital, costing taxpayers about $3.1 billion.
But if all hospitals developed an intensive surveillance and prevention program, a "significant percentage" of those hospital-acquired infection cases could be eliminated, according to the health department.
Stockton's 188-bed Dameron Hospital already met or exceeded many of the new requirements long before Jan. 1.
"We were waiting for over a year for this law to work through the Legislature. We were ahead of the game," said Richard Wong, administrative director of Dameron's clinical laboratory that does all patient testing.
"Once that happened at the beginning of this year, all the hospitals were scrambling. Just the county hospital and ourselves were screening high-risk groups," Wong said.
Dameron has been testing high-risk patients since at least 1990, said Linda Knowles, a nurse epidemiologist and the head of Dameron's infection-control department.
A year ago, Wong said he persuaded Dameron administration to invest in new technology that allows individual test results for MRSA from a nasal or groin swab of a high-risk patient to be completed in less than two hours.
San Joaquin General Hospital has been testing all patients admitted to its intensive-care units for MRSA for two years and routinely has tested people transferred to the hospital from skilled nursing facilities, jails, board-and-care homes and other hospitals.
It added the testing of babies admitted to its intensive-care nursery and all immune-compromised surgery patients, county health director Ken Cohen said.
The new law did not come with any state funds attached, and because the testing is not ordered by physicians and therefore not a billable procedure, the hospitals must pay the costs.
Starting March 16, Lodi Memorial will have procedures in place to test all patients - not just those identified as high risk - within 20 hours of being admitted, costing the hospital an estimated $200,000 annually, spokeswoman Carol Farron said.
Source - ( Mar 05, 2009 )
By David Persing, MD, PhD, and Ellen Jo Baron, PhD
ost healthcare institutions in the United States have been watching an ominous trend of escalating proportions of methicillin-resistant Staphylococcus aureus infections. According to the Centers for Disease Control and Prevention, more people in the United States die annually from MRSA (estimated 18,650 deaths) than from AIDS (roughly 16,000 deaths).1
In addition to loss of life, MRSA costs the Amercan healthcare system over $ 2.5 billion in non-reimbursable costs.2 In terms of the healthcare setting, MRSA infection rates have increased over the past three decades â€” in 1974, MRSA accounted for 2% of staph infections; in 2004 the number rose to 63%.3
In healthcare environments, MRSA is spread from patients who already have an MRSA infection or who are colonized with the bacterium but do not have any symptoms. The harmful pathogen is passed to other patients through hand-to-hand contact or by touching contaminated surfaces such as bed rails
Many institutions have begun selective testing of some patients believed to be at higher risk. Several commercial chromogenic agar plates have been developed to culture nasal swabs for active surveillance of MRSA. Even with the most rapid culture turnaround time, however, results from cultures are not available for at least 18 hours after inoculation of the medium. Such results could reach the unit more than a day after a colonized patient has been sharing a room with a non-colonized patient.
Moving the non-colonized patient into another room at this point can be a social and public-relations nightmare for patients, their families, and hospital administrators. The alternative, instituting barrier precautions pre-emptively on all patients until their MRSA status is known, is costly and problematic; despite the success of this strategy in controlling MRSA in Denmark and the Netherlands, not all healthcare institutions in the United States have that capacity.
Selective testing of patients based on some risk assessment has been shown to detect less than 85% of the colonized patients in a hospital4; and the elaborate admission interview required to determine who might be at risk is counterproductive, disliked by nursing staff, and slows down admissions. Even less effective is passive surveillance, in which MRSA carrying patients are discovered and isolated only if cultures sent to the clinical laboratory yield MRSA. This approach fails to identify 70% of truly colonized patients.5
As MRSA outbreaks are widely reported in the news media, the publicâ€™s fears are increasing, putting pressure on policymakers to address the issue. Already five states have enacted legislation mandating surveillance of MRSA for high-risk units in a hospital, and 31 states have reporting requirements.
Many hospitals are now implementing systems that use fully integrated real-time PCR technology, in most cases for the purpose of implementing rapid MRSA surveillance as part of their overall infection-control program. Major advantages of such a system is a MRSA test available in cartridge form with (1) its ability to process and deliver results in less than one hour, enabling physicians to take the appropriate precautions before the pathogen has the opportunity to spread, and (2) its moderate complexity, allowing a non-clinical laboratory scientist to perform the test.
The new technology combines on-board sample preparation with real-time PCR amplification and detection functions for fully integrated and automated nucleic-acid analysis. Training to use a moderate complexity test is easy, and less experienced workers can perform such tests with minimal hands-on time. With a random-access system, a new sample can be added at any time. In many laboratories, the combination of ease of use and random access translates into 24/7 access to MRSA results.
Nearly 450 U.S. Hospitals (and over 900 worldwide) to date have chosen to deploy this technology, and laboratory technicians and technologists have voiced their satisfaction with the simplicity of the systemâ€™s workflow and its proven results.
Leaders at sites that have implemented the MRSA assay have been able to reduce infection rates and cut healthcare-associated costs while improving patient care.
"The patients with the highest risk of MRSA infection are those who undergo invasive surgeries making it a serious concern for our orthopedic hospital" says Maureen Spencer, RN, Med, CIC infection-control manager at New England Baptist Hospital in Boston. "Following invasive orthopedic surgical procedures, post-operative surgical wounds are vulnerable to infection â€” particularly bone infections which are among the toughest to treat.
"We were one of the first sites in the United States to implement a pre-screening program for all surgical patients for MRSA. When selecting a pre-screening program, time to result is a huge factor. It is critical that patients know their MRSA status before they leave their surgical consult in order to initiate a topical decolonization protocol and allow the surgeon to adjust the surgical antibiotic prophylaxis. Using the MRSA assay and the system, we proactively screen all surgical patients for MRSA at least two weeks prior to their procedure. If they are colonized with staph, doctors place the patients on a five-day decolonization protocol and conduct a second screening for MRSA before admission to the hospital for surgery. The goal is to reduce the introduction of MRSA into the hospital by ensuring patients are MRSA-free before they are admitted.
"Our data is showing that pre-screening surgical patients for MRSA lowers infection rates, creating a win-win situation for the hospital and patients. Since implementing the first PCR test in July of 2006 and moving to the MRSA test in early 2007, our hospital saw MRSA infection rates drop nearly 60%; from 0.46% to 0.18% in 2007 and decreasing further to 0.11% in 2008. This has helped the hospital decrease its orthopedic surgery hospital-acquired infection rates to 0.3% which is five times lower than the average national rate of 1.5%."
In Wooster, OH, Gail Woosley, manager of Lab Services for Wooster Community Hospital says, "The goal of our infection-control program is to lower healthcare-acquired infection rates and improve patient care by delivering physicians the test results they need as quickly as possible. In my 33 years in the field, it is not often that I am impressed by some of the new technology being made available, but this system really impressed me. Relative to other lab equipment, the new system technology is affordable, even for a small institution. If you have a device that can save you money in terms of patient time in the hospital, supplies used during their hospital stay, and the health of the patient, its value is not measurable.
"We find that technology to be very useful in three patient population groups.
In Stockton, CA, Richard Wong, administrative director of Pathology and Clinical Laboratory for Dameron Hospital, says, "We have been at the forefront in the San Joaquin Valley with regard to active surveillance on multidrug-resistant organisms since the 1987 implementation of its first screening program.
"Technology has allowed us to expand and improve on our infection- control methods as the nationwide threat of hospital-acquired infections grows. The MRSA test delivers the fastest time to result available today, making it an ideal technology for our critical initiative, allowing us to reduce hospital-acquired infection rates and provide the highest standards of patient safety.
On average, our hospital runs about 480 MRSA tests per month with plans to increase as we move towards universal screening of all patients who enter the site. Of importance, we have found the MRSA assay is 100% sensitive compared to culture-based tests."
More recently tests approved for the system we use include a combined test for S aureus (usually methicillin sensitive) and MRSA in blood cultures from patients with suspected sepsis, and a similar test for direct detection of both organisms in skin and soft-tissue infections. Both tests deliver results in less than one hour and their results can be used in real-time to guide optimal treatment or management decisions. Other tests in development that are relevant to infection control include a test for VRE, Clostridium difficile, and multidrug-resistant tuberculosis. It is becoming increasingly clear that the medical value of rapidly available, actionable results provided by the new technology can be an important ally in the "search-and-destroy" strategy being adopted by more and more hospitals.
David Persing, MD, PhD, is the executive VP and chief medical and technology officer for Cepheid. Ellen Jo Baron, PhD, is director of Medical Affairs for Cepheid. The systems and tests for which Cepheid is well-known are its GeneXpert System and its accompanying MRSA/S Aureus test.
Source - ( Feb 24, 2009 )
A recent study found that on any given day more than 7,000 patients are suffering from a C. Diff infection. An estimated 300 will die from it.
"This is an organism that's been around for three decades, but really, in just the last five or six years, has become epidemic," said Dr. Fred Tenover with Cepheid.
Tenover directed research into drug-resistant germs at the Centers for Disease Control and Prevention. He is now leading a team at Cepheid, a Sunnyvale company that developed a rapid DNA test to help hospitals spot the most toxic strains of C. Diff which attack the intestinal tract.
"This is an organism that can form very resistant spore, that is something that's resistant to disinfectants and to antibiotics," Tenover explained.
Since normal hospital cleaners will not kill the bacteria, it is often carried from room to room unknowingly by nurses and doctors.
Some hospitals have started isolating suspected patients and wiping their rooms and equipment with bleach, one of the few things that will kill C. Diff.
Destroying the bacteria in patients is also becoming increasingly difficult. The bug is showing increasing resistance to common antibiotics.
"Resistance is now becoming a problem to these two major antibiotics and the more we use it the more likely we're going to see resistance," said Dr. Jan Winetz at the Good Samaritan Hospital.
Winetz believes help could be on the way.
He is directing clinical trials of a new drug at Good Samaritan Hospital in San Jose called Par 101. It is a naturally-occurring compound that can attack the bacteria inside the intestines.
Dr. Winetz says, "It's specific for C. Difficile and actually more toxic to C. Difficile bacteria than the traditional antibiotics."
Double-blind clinical trials with Par 101 are taking place across the country and while the results are not yet available, Dr. Winetz says some intriguing patterns are emerging.
He also says finding an effective solution quickly is critical.
Epidemiologists believe C. Diff is about to become one of the most serious health threats to hit hospitals in decades.
Source - ( Feb 19, 2009 )
How fitting that the shorthand name of one of the wiliest health care-associated infections—C. Difficile—sounds so much like “c’est difficile,” French for “it is difficult.” With an increasing number of patients developing Clostridium difficile-related diarrhea, not to mention a hypervirulent strain on the loose, no one’s exactly murmuring a pleased “Ooh la la.” More like “Sacre bleu!”
“It looks like we haven’t reached the peak of the U.S. Epidemic as yet,” says C. Difficile epidemiology expert Dale Gerding, MD, who types C. Diff isolates sent to him by hospitals around the country. Dr. Gerding is associate chief of staff for research and development at Hines Veterans Affairs Hospital, Hines, Ill., and professor of medicine at Loyola University School of Medicine, Maywood, Ill. “We’re still battling the same problem.”
What problem is that? Take your pick: The C.-difficile-associated, severe, often recurring diarrhea in patients. The potential for toxic megacolon. The ability of spores to survive alcohol-based hand hygiene gels. At least one strain’s resistance to fluoroquinolone antibiotics. The insensitivity of some enzyme immunoassay tests commonly used in C. Difficile testing. Not to mention a recent national prevalence study by the Association for Professionals in Infection Control and Epidemiology that found that of 110,550 inpatients in 648 participating hospitals, 13 out of every 1,000 were either infected or colonized with C. Difficile—a number APIC calls “6.5 to 20 times greater than previous incidence estimates.” Oh, and Dr. Gerding’s statement that U.S. İnfection rates are increasing by roughly 20 percent each year. “It just keeps on going at almost a linear rate,” he says. Sort of like the Energizer Bunny, except much, much less adorable.
In other words, the picture isn’t much brighter than the last time CAP TODAY reported on the C. Difficile epidemic (“C. Difficile back with a vengeance,” May 2006). However, the news isn’t all bad. Several manufacturers are developing or introducing new, more sensitive tests to determine if a patient is infected with the bacteria. Potential new treatments are on the horizon. And with luck, the U.S. Department of Health and Human Services’ “HHS Action Plan to Prevent Healthcare-Associated Infections” (released last month), which calls for a 30 percent reduction in C. Difficile case rate per patient days and administrative/discharge data for ICD-9-CM-coded C. Difficile infections in the next five years, will spur more hospitals to action. Still, how much hope is there for stemming the C. Diff tide?
Certainly the continuing prevalence of the hypervirulent NAP1 strain isn’t soothing any worries. To refresh your memory: “This is a specific strain of C. Difficile that emerged first in North America, in Pennsylvania. This NAP1 strain has a genetic change that results in literally 16 to 23 times more toxin production in vitro,” explains William Jarvis, MD, principal investigator of the APIC study and president and co-founder of Jason and Jarvis Associates, a private consulting firm in health care epidemiology with offices in South Carolina, Oregon, and San Francisco.
“There’s no question that this strain is more virulent than other strains. It seems to be playing a big role” in the epidemic, says L. Clifford McDonald, MD, chief of the Prevention and Response Branch of the Centers for Disease Control and Prevention’s Division of Healthcare Quality Promotion. “We’re continuing to see it as the most common single strain in outbreaks in severe cases. That’s been better documented since 2006, with some studies showing that in humans who have more severe disease that that strain is responsible. But the exact reason for the hypervirulence has never been really determined.”
One ongoing question with regard to the NAP1 strain is how much significance to assign the fact that one of its characteristics is a deletion in the tcdC-negative regulator gene of toxin expression. One theory holds that this deletion is responsible for the strain’s increased virulence, and that that increased virulence causes more severe disease in humans. But, Dr. McDonald says: “It’s clear that it’s not just that deletion. Probably more important is an upstream point mutation. The point mutation does lead to early truncation of the protein and probable disregulation of toxin production.”
Unfortunately, the APIC survey was not able to determine the precise prevalence of the NAP1 strain. “The fact that [only] two percent [of the hospitals surveyed] are doing cultures means there’s no isolate” to send for testing, Dr. Jarvis points out. “So, not surprisingly, we had no facility report a NAP1 strain, even though we know from Dr. Gerding’s data that it has been seen in a very large number of U.S. States.”
“Just like the NAP1 strain emerged, there may be another C. Diff strain that emerges today, tomorrow, next year, 10 years from now, that spreads across the United States, the world,” he continues ominously, “and we’re going to be delayed in even recognizing that such a strain exists because no one has the capability of doing culture and identifying what it really is.”
Indeed, Dr. Gerding is already seeing new strains arise. “We’re still continuing to see this epidemic [NAP1] strain as the dominant strain, but we’re also starting to see other types come up now,” he says. “This is what we’ve been expecting.” Thus far most of those new strains appear to be toxinotype Os, that is, “they don’t have any major changes in their pathogenicity locus,” he says. “But none of them has undergone enough analysis to say much more than that.”
Another question: whether C. Difficile will become resistant to the antibiotics used to treat it—vancomycin and metronidazole—the way that the NAP1 strain has already become resistant to fluoroquinolones. At the moment, “there really isn’t any resistance to speak of,” Dr. Gerding says. That said, he adds, recent studies have shown that metronidazole is significantly less effective than vancomycin in treating patients with severe C. Diff infection. “But it has not been correlated with any resistance by the organism in vitro to the drug. It’s something else. It may have to do with the fact that we don’t have as much metronidazole staying in the gut as we do with vancomycin.”
By some accounts, the number of patients acquiring C. Difficile infections who do not have the typical risk factor—exposure to antibiotics—is growing. Ciarán Kelly, MD, associate professor of medicine at Harvard Medical School, chief of the Herrman L. Blumgart Internal Medicine Firm, and director of gastroenterology training at Beth Israel Deaconess Medical Center, Boston, goes so far as to say that previously C. Difficile acquisition without prior antibiotic exposure was considered a “freak occurrence.”
Dr. Gerding says, “I’m not too concerned. In the hospital, there seem to be almost no patients who haven’t had antibiotic exposure who get C. Diff. But in the community, we’re finding 30, 40 percent” who do not report having been exposed to antibiotics. “It may be that they don’t have any, or it may be that we just don’t know how to look for it, or how to interview the patients. There are more and more of those kinds of patients being reported. Whether this is a significant part of the C. Diff problem or not, I’m not certain. I still think the vast majority of cases are in the hospital, and they’re associated with antibiotics.”
Another still unknown aspect of C. Difficile is its incubation period. Previous studies have suggested that period is seven days or less, but, Dr. McDonald points out, “we certainly do see a lot of C. Difficile right after people are being discharged from the hospital. Whether that’s because they’re going out on antibiotics and getting it in the community, or actually they’re acquiring it in the hospital and developing disease in the community, we don’t know, but that makes us wonder whether the incubation period couldn’t be a little bit more prolonged.”
In addition, some studies have revealed the presence of C. Difficile in retail meats. “It’s been known since around 2000 that it was starting to show up in food-producing animals more—actually causing disease in neonatal pigs and being often found to colonize cattle,” Dr. McDonald says. “It’s now been noticed that some of the strains, predominantly one group of strains that were very prevalent in food-producing animals in the early part of this decade, are increasingly starting to be seen in humans. But at least as of this date, there is no conclusive evidence that you get C. Difficile from food.”
Then, too, the role of proton pump inhibitors, if any, in the spread of C. Diff infection has yet to be nailed down. Karen Carroll, MD, professor of pathology at Johns Hopkins University School of Medicine and director of the Division of Medical Microbiology at Johns Hopkins Hospital, says the topic “deserves a lot more study” and that “there’s some evolving literature to show that perhaps those are overprescribed and could be contributing to one’s risk, particularly in the community among healthy populations.”
But Dr. Gerding says the correlation, if any, is far from clear: “You’ll find about as many studies that indicate a risk for proton pump inhibitors of getting C. Diff as you will that find no risk.” And if PPIs do increase one’s risk of acquiring C. Diff infection, it’s not clear how. By neutralizing stomach acid, they might make it easier for the bacteria to survive in the body—“but the spores are pretty resistant to acid anyway,” he says. “The other possibility is that these drugs [the PPIs] have activity against some bacteria. So they could be acting like an antibiotic to upset the normal bacteria flora, and in the process of doing that, could make the patients susceptible to C. Diff infection just like an antibiotic does.”
Unfortunately, one C. Diff treatment that had looked especially promising—a toxin-binding agent from Genzyme Corp. Called tolevamer—has been taken off the table. “A lot of us in the field had significant hopes” for it, Dr. Kelly confesses. “Many of us believe that antibiotic treatment is intrinsically not the best way to approach the disease, and predisposes the patient to the most common complications, which is recurrence. A phase II study indicated that tolevamer was as effective as antibiotics and associated with lower rates of recurrence. We thought we were going to get the first new treatment for C. Diff in more than two decades.” The idea was that tolevamer would bind the toxins released by C. Difficile and remove them from the body. Instead, it ended up failing two large phase III studies. “It was very disappointing,” he says.
On the bright side, another investigational treatment, a narrow-spectrum antibiotic from Optimer Pharmaceuticals called fidaxomicin (also called OPT-80), remains promising. A recent phase III clinical trial showed that while its response rate was similar to that of vancomycin, fidaxomicin had significantly lower recurrence rates (15 percent compared with 25 percent for vancomycin). “The theory behind this antibiotic was that it would affect the healthy normal bacteria flora to a lesser extent,” Dr. Kelly says. “From what we know about that study so far, it seems as though that may be the case, but there isn’t a full peer-reviewed publication” yet.
In addition, Massachusetts Biologic Laboratory and Medarex Inc. Are collaborating on two injectable human monoclonal antibodies called CDA-1 and CDB-1, which, it’s hoped, will reduce relapse rates when given in conjunction with vancomycin or metronidazole. “That is a totally new approach, based on the observation that many people who become infected with C. Diff don’t develop the disease, but are symptomless carriers,” says Dr. Kelly. “The treatment is based on the idea that a faulty antibody response makes individuals susceptible.” A phase II study just yielded the report that the recurrence rates in study participants who received the active antibodies were 70 percent lower than those who received a placebo. So, Dr. Gerding says, between fidaxomicin and the new monoclonal antibodies, “it looks like we have at least two drugs or approaches that will result in lower relapse rates.”
Meanwhile, a more esoteric treatment, fecal transplant, has yet to be widely embraced. The idea is that by administering a filtered stool sample (usually donated by a family member) to a patient with multiple recurrences of C. Diff, normal gut flora can be reestablished. The “eww” factor aside, “all the case series that have been published at least have suggested almost 100 percent success rates,” Dr. McDonald says. “That really suggests that it works. But at the same time, they have not taken off, in part because a lot of people don’t feel comfortable doing them. There’s some sense of liability, the sense that it’s not standardized, you don’t really know what you’re giving them.” Besides, Dr. Gerding says: “I think it’s too desperate an approach. If we’re going to do something like this, we need to figure out how to construct a synthetic fecal transplant where we can control the constituents and make sure we don’t have anything in it that doesn’t belong there.”
On the prevention front, experts point to antibiotic stewardship programs in hospitals as a key way to control the spread of C. Difficile. Those programs might include such measures as automatic stop orders, so that, for example, a patient who receives antibiotic prophylaxis for a surgical procedure will receive only the appropriate number of doses. They might also include pharmacy formularies that restrict which antibiotics physicians can access, or online physician ordering systems that provide educational prompts to physicians ordering antibiotics. Just less than half (47 percent) of the institutions surveyed in the APIC study reported having implemented an antibiotic stewardship program, and “they were all doing a variety of different things” within their programs, Dr. Jarvis says. “Given that antibiotic controls and antibiotic stewardship are such a critical element of the comprehensive program for preventing C. Difficile-associated infection, we feel that more emphasis needs to be placed on that, and perhaps even having a person full or part time dedicated to that effort.”
With so many unknowns about the nature and future of the C. Difficile epidemic, what can laboratories do in the meantime? Says Dr. Gerding bluntly: “What they can do is better testing.”
If enzyme immunoassay testing for C. Difficile were a schoolchild, it would be the awkward kid with too few friends, rarely the first pick for the dream team—in this case of testing solutions. So why does just about everyone let it hang around? For one, it’s much faster than the much more reliable cytotoxin assays. It’s convenient and cost-effective. And though the sensitivities of some of the EIAs are reported to be only about 73 to 75 percent, there are those, like Meridian’s Premier and Immunocard, for which the sensitivity has been found to be higher (Planche T, et al. Diagnosis of Clostridium difficile infection by toxin detection kits: a systematic review. Lancet Infect Dis. 2008;8:777–784).
Still, sensitivity is a well-known problem, and the common physician practice of ordering an EIA multiple times on the same patient in an attempt to compensate for the problem can clog laboratory workflows and lead to unnecessary and costly patient isolation. However, “the biggest clinical consequence of the low sensitivity is the high level of falsely negative results, which of course can lead to delay in treatment,” Beth Israel’s Dr. Kelly says. “It’s a situation that none of us are content with, ordering a test and then ignoring the result because the test isn’t sensitive enough.”
“We’ve all gotten into the habit of testing two or three or four stool samples for C. Diff until we get a positive,” agrees Lance R. Peterson, MD, associate epidemiologist and director of microbiology and infectious diseases research in the Department of Pathology and Laboratory Medicine of north suburban Chicago’s NorthShore University HealthSystem, and professor of pathology and medicine at Northwestern University’s Feinberg School of Medicine, Chicago. And that’s not good. “When you’re using indirect assays such as enzyme immunoassays, there’s been no validation that repeating a test in a person on the same kind of sample within a few days that the test is even reliable to repeat. Even with the second test, your positive predictive value is less than 50 percent. So if you have a negative first test, and then a day or two later you do another stool sample and you have a positive, you have a 50–50 chance of having a true positive or a false positive.” Not only that, he adds, “if you have a 70-percent-sensitivity test and a 10 percent positive prevalence of disease in the samples you’re testing, it takes you five tests in order to detect all 100, or 10 percent of the 1,000 patients you’re sampling and by that time you have more false-positives than true positives.”
In the APIC study, the C. Difficile infection of nearly 95 percent of the patients reported was detected via EIA. “They [laboratories] are taking the easy way out, which is to buy a test off the shelf with poor sensitivity, but one that they can turn around quickly and without requiring a huge amount of technical skill,” Dr. Gerding says. “If they really wanted to get on top of this problem, they would back up their current enzyme immunoassay testing with something like culture and actually try to get the organism out of the stool. But most labs don’t have anaerobic culturing capability anymore, so that isn’t very practical. And they don’t want to do cell cytotoxin assays because that requires maintaining a cell line and also requires a fairly high skill level by the people who are looking at the cells to interpret the tests. It’s laborintensive, and, of course, they’ve had their budgets cut and they’re really struggling to be able to provide any kind of testing with the budgets they have.”
Taking EIA off the table, however, would leave nearly all laboratories without a fast, affordable way to detect C. Difficile. That’s where some new molecular tests come in. Several manufacturers, including Cepheid, Prodesse, and BD, are developing or introducing real-time PCR tests for the detection of C. Diff, while others are developing molecular tests that use other means.
“We think that molecular testing will provide the right technology at the right time for meeting the demands of the C. Diff diagnosis,” says Cepheid’s chief medical and technology officer, David Persing, MD, PhD. Cepheid’s Xpert PCR test for C. Difficile, which was released in Europe in November, detects the toxigenic form of the C. Diff genome and specifically identifies the NAP1 epidemic strain in less than an hour. Run on the company’s GeneXpert system, it detects three targets: toxin B (“the most reliable marker of toxigenic C. Diff,” Dr. Persing says); binary toxin, which has been implicated in some of the more virulent strains; and a single base deletion causing a frameshift mutation in the tcdC gene. “When you detect all three of those targets, what we found in our clinical trials is that it is an excellent indicator of the presence of the NAP1 epidemic strain,” says Dr. Persing. Specific detection of the NAP1 epidemic strain may be of benefit in tracking hospital outbreaks in real time, he says, thus aiding infection-control personnel in what he calls their “search-and-destroy mission.”
He’s particularly proud of the fact that the Xpert test, which generates results in about 45 minutes, doesn’t require batching and thus can be run on a stat basis. “All the other molecular tests out there run specimens in batches,” he says. “In fact, most of the laboratory-based enzyme immunoassays are run in batches as well. That limits their turnaround time.” In addition, he says, the Xpert’s sensitivity and specificity are about 96 percent compared with toxigenic culture. The company hopes to receive FDA approval for the test in the second half of this year.
Meanwhile, BD received FDA clearance in December for its real-time PCR test, the BD GeneOhm Cdiff assay, which targets the toxin B gene, detects the NAP1 strain, and produces results in about two hours. Medical affairs director Tobi Karchmer, MD, MS, says it is the first molecular C. Difficile assay cleared in the United States. In clinical trials, she says, the GeneOhm test was compared with cell culture cytotoxicity assay. “We had a set of data that came from fresh stool specimens, and in that case sensitivity was 93.8 percent compared to the cytotoxicity reference method, and the specificity was 95.5 percent. We had a second set of data from frozen stool specimens, and in that case the sensitivity was 100 percent and the specificity was 97.7 percent.”
One of BD’s clinical trial sites was Wishard Memorial Hospital, Indianapolis, which is affiliated with the Indiana University School of Medicine. “There hardly is a comparison” between the BD PCR test and the hospital’s existing C. Diff detection method, says Deanna Fuller, clinical research manager for the Department of Pathology and Laboratory Medicine. The clinical trial laboratory used a three-tier testing algorithm that called for common antigen and EIA; cytotoxin assays were then run on any positive results. In comparison to that method, the BD test achieved 95.8 percent sensitivity and 98.7 percent specificity, after discordant results were resolved, Fuller says.
Johns Hopkins Hospital also served as a clinical trial site for the GeneOhm assay, allowing Dr. Carroll to familiarize herself with it. Her laboratory has been using a two-step algorithm for detecting C. Diff—screening with an EIA antigen test for glutamate dehydrogenase, then testing antigen-positive samples with cell culture cytotoxin—but she was so impressed with the BD test that she plans to switch to it in the next six months or so. “While I don’t think it is as sensitive as toxigenic culture, I think it’s definitely better than cytotoxin testing and allows for same-day testing,” she says. “That said, PCR is expensive, and given the volume of testing that we do, what I’m thinking I might do is continue to screen with the antigen test and then confirm the positive antigens by PCR.”
Thomas Davis, MD, professor of pathology at Indiana University and director of Wishard’s clinical microbiology laboratory, also plans to institute the GeneOhm assay in his lab, but is similarly concerned about cost. “I don’t think there’s any way that this technology is going to be cheaper in the laboratory,” he says. “It’s probably going to cost twice or three times as much to do this technology as the EIA technology, but that’s not where you save the money. You save the money with patient management.”
Speaking of money, one potential barrier to going molecular for many laboratories is the need to purchase a thermal cycler, plus the costs associated with training workers to perform real-time PCR assays. That’s why Meridian Bioscience is touting its forthcoming Illumigene C. Difficile test—which, at CAP TODAY press time, was scheduled to begin clinical trials this month—as “molecular simplified.” The assay uses loop-mediated isothermal amplification technology, or LAMP, rather than polymerase chain reaction technology, Rich Connors, associate director of molecular marketing, explains. “It does with chemistry what PCR does with temperature. Is it any better or any worse [than PCR]? No, it’s just different. It’s a little bit quicker, it’s a little bit simpler, and it doesn’t require large amounts of capital equipment, because you don’t need a thermocycler.” He expects it to be the first FDA-approved assay based on LAMP technology.
And those much-maligned enzyme immunoassays? Connors points to the December 2008 Lancet Infectious Diseases article that reported median sensitivities for Meridian’s Premier and Immunocard assays for toxins A and B of 95 percent and 90 percent, respectively, based on a systematic review of the literature. Manufacturers’ instructions must be followed, he says. “EIAs are designed to evaluate samples from clearly symptomatic patients, and in these cases will give PPVs [positive predictive values] that are quite high. It’s also important to ensure that samples are collected and transported correctly.”
A new EIA is awaiting FDA clearance: Inverness Medical’s Techlab C. Diff Quik Chek Complete, which is a combined membrane EIA for glutamate dehydrogenase and the A and B toxins in a single assay. It can be completed in 30 minutes and does not require additional equipment or batching of specimens. “The beauty of the new GDH assay is that it detects most all of the negatives. It had a sensitivity of 91.7 percent compared to bacterial culture,” says clinical trials participant Wallace Greene, PhD, director of the diagnostic virology laboratory and associate professor of pathology, Penn State College of Medicine, Hershey. When compared with cytotoxic cell culture, all toxin-positive strains were detected by the GDH assay. However, the toxin EIA was only 95.2 percent sensitive. Specimens that are positive for GDH but negative for the toxins should be tested by more sensitive assays—either bacterial culture, cytotoxin, or molecular assay, if available, Dr. Greene says. “This allows a laboratory to report the negative specimens and most of the positive specimens in less than an hour.” Only a very few specimens would require further testing with the more expensive and labor-intensive molecular assays, he says.
With the C. Diff Quik Chek Complete, “instead of taking two or three days to report a negative result using the cytotoxin assay, thus allowing a patient to be removed from contact isolation, we can do it in less than an hour. In most cases, patients won’t even be placed in isolation. That’s a huge cost savings for the hospital.” Still, Dr. Greene wonders, can labs get away with a simple, rapid, and relatively cheap GDH assay to reliably detect negative specimens and report results essentially as they come into the lab, “or must we use a more expensive, time-consuming molecular assay that would only be run once or twice a day for all specimens to significantly improve sensitivity?
“Until we do a head-to-head comparison with our patient population, we just can’t answer that question,” he says.
Source - ( Feb 09, 2009 )
Birmingham Children’s Hospital NHS Foundation Trust has introduced a new way of detecting MRSA among its most critically ill young patients as part of its ongoing infection prevention and control programme. The Trust has already made significant progress in tackling healthcare-acquired infection, but is striving to improve its record through enhanced surveillance of patients admitted to the intensive care unit (ICU) and other key areas where patients are deemed to be vulnerable. The Healthcare Commission’s Hygiene Code Inspection Report on Birmingham Children’s Hospital NHS Foundation Trust, published in October 2008, observed that rates of MRSA infections have been below the national average for children’s Trusts for “most of the last seven years (and all of the last two), while the infection rate has decreased particularly significantly since 2006.” However, the Trust is taking its infection control and prevention strategy further by expanding its screening programme beyond the requirements outlined by the Government and by implementing more stringent, rapid testing. Despite having comparatively low levels of MRSA, the Trust was concerned that approximately half of MRSA cases were going undetected until the child had actually developed symptoms and fallen ill with the infection. It therefore took the decision to use a polymerase chain reaction (PCR) test, on “high risk” patients, to speed up results and the time taken to respond with appropriate interventions. The project, which initially focused on screening of admissions to the ICU, allows doctors to identify if a patient has MRSA within just one hour by using the GeneXpert test from Cepheid. Traditional culture-based testing, where the bacteria is grown in a petri dish in a microbiology lab, previously took up to three days to verify. Each result obtained using the molecular diagnostic test has been verified as accurate by a further culture test and the hospital has already identified cases of MRSA that they would not normally have picked up on. So far it has proven to be 100% accurate. Dr Jim Gray, consultant microbiologist at Birmingham Children’s Hospital commented: “It is a generally accepted belief in the medical profession that children are not really carriers of the MRSA bacterium. However, we are increasingly finding that this is not the case. “Obtaining an accurate and rapid result for MRSA is absolutely key at the emergency admission point in hospitals. For example, if a patient is critically ill and needs to be admitted to the ICU without delay, clinicians simply cannot afford to wait two to three days for a culture result to see whether the patient has MRSA. By then the patient will certainly have undergone several medical procedures that carry a risk of introducing MRSA into sites of the body where it can cause serious harm, such as the lungs and the bloodstream. Also, if the child is carrying the infection, by the time the results of MRSA cultures arrive back from the lab it may have spread to other children on the unit. “It made sense to start the screening with ICU patients as they are the most susceptible to contracting the MRSA infection due to the bacteria most commonly gaining access to the body through open wounds, injections, catheters and IV sites. It is vital to prevent MRSA from entering the ICU as it is extremely serious and potentially fatal for children who are already critically ill.” One of the advantages of the type of molecular testing being used is the fact that specimens do not have to be processed in batches, which are normally only performed once a day. Testing can be performed in a variety of locations by non-laboratory personnel, which opens up the possibility of providing screening on a 24/7 basis by staff who do not have expert diagnostic skills. It can be performed in non-standard locations in satellite laboratories, emergency department laboratories, or even in the patient admissions area. “In shared room settings, any delays in identifying carriers can cause anxiety for other patients and their carers who have been in close proximity to carriers,” said Dr David Persing, executive vice president and chief medical and technology officer of Cepheid: “They may witness patients who have tested positive being moved into isolation or may find they are moved themselves – in which case, you are presented with the problem of what to tell them. It also creates bed management challenges – faster turnaround times for results enable hospitals to make better decisions at the outset, thus avoiding the ‘bed shuffle’ two or three days after admission.”
Targeted screening Although Birmingham Children’s Hospital has highlighted screening as having an important contribution to improving patient safety, the national focus has been on detection in adult populations. The Government has set the agenda for MRSA screening by stipulating that all elective admissions should be screened by March 2009 along with all emergency admissions “as soon as practicably possible”, but there are some exclusions, which includes groups within paediatrics. “Operational guidance was released in July 2008 which, for the first time, gave robust requirements around paediatrics, stating that we should be screening highrisk paediatric admissions, but it was no more specific than that,” Dr Gray commented. “At Birmingham Children’s Hospital, we have always had an interest in MRSA, but the national focus has been very much around bloodstream infections, which are only the tip of the iceberg. To put this into perspective, we typically see around one or two bloodstream infections per year at our hospital, but we probably see around 50 new patients with MRSA at one site or another. “For most of my career, I have kept detailed records of every patient with MRSA. When we looked back over our experience at the children’s hospital, over a period of 10 years, we found that there had been 410 patients with MRSA. Of these, 215 actually presented with infection. The remaining 195 were colonised but not symptomatic when we detected MRSA. This is a relatively small number, representing around 20 infections per year. However, we thought that if we could detect MRSA in patients before they became infected, we could put measures in place to prevent serious outcome.” When the Trust analysed the 215 cases presenting with infections, it found that the majority of cases were admissions to the intensive care, cardiac and surgery units. It therefore decided to target these high-risk patients for screening. Although they only accounted for around 20% of the patient population, this target group represented 80% of all MRSA cases – enabling the Trust to capture the large majority of infections in the most cost effective way. “We haven’t rolled out PCR beyond this targeted group, for the moment at least, because we needed to be sure that it would be cost effective. PCR is being piloted initially in the ICU to establish how common MRSA is in children and how accurate it is for this patient group,” Dr Gray continued.
Accuracy Commenting on the accuracy of the test, Dr Persing added: “The molecular technique is extremely sensitive and a number of users have come to us and said that they have 10% more ‘positives’ with this system. At first they queried the results, but when they re-analysed these cases and performed additional manipulations to enhance the sensitivity of the culture-based techniques, they found that in fact these were indeed positive cases. Molecular testing can apparently identify cases that would normally be missed, by usual surveillance techniques.” Dr Gray confirmed that the hospital had found a very good correlation between PCR and culture results, and concluded that the test had proven “very accurate”. Some of the Trust’s initial findings have now been revised, however, as increased data has been gathered. He observed: “In terms of prevalence of MRSA, when we started using PCR, it appeared that one in every 10 patients were testing positive. Following further testing of a greater number of patients (around 250-300) we now have more comprehensive statistical data and have found, in fact, that there is a prevalence of around 2-3% – reaffirming the belief that MRSA is much more prevalent in adults,” he commented. Dr Gray believes that the surveillance work the hospital is carrying out on MRSA is very important in building a better picture of the levels of MRSA colonisation within children: “We are learning a lot and asking ourselves many questions. For example, because of the nature of their condition, some of our haematology patients may be admitted as often as three or four times per month. Is it necessary and helpful to screen them every time they come back? “Hopefully the answers that we obtain will assist other UK health Trusts in developing a best practice policy regarding MRSA in child patients. As there is very little research being carried out on MRSA in paediatrics, we are learning as we go along.”
Response to screening Dr Gray observed that the screening programme has been well received by both staff and parents: “We have had a long standing interest in controlling MRSA and have historically performed some screening, so staff were generally comfortable with the implementation of the programme. When you start a screening programme, it is very important that staff understand why you are doing it and that tests are carried out within the desired timescale. It is important when setting up a system that everyone understands how it works and why it has been set up in a specific way. Moreover it needs to be monitored for compliance. “Other considerations include the need for a robust system for transmitting results and ensuring they are acted on. At Birmingham Children’s Hospital, once a positive results is obtained, an infection control nurse is notified, who will make sure the patient immediately receives the treatment required. We were concerned about how patients and their families would react to the screening programme, but there has been so much publicity surrounding the issues, that it has caused a lot less anxiety than we thought it would.” While surveys in adult hospitals show that MRSA is key concern for patients, it does not appear to raise the same level of concern in parents of children coming into hospital, he observed: “Not many people phone before their child is admitted and ask about the issue, and we considered whether screening might heighten anxiety around MRSA. However, we did not find this to be the case. We have a leaflet for those who want more information on the screening programme, but parents are not presenting with questions or concerns on the issue,” he commented. He added that as a relatively new children’s facility, the hospital is in a fortunate position of having ample isolation rooms, although the management of this requires a flexible approach. “While you may be isolating the patient, the parents are spending most of the day with the child and mixing with other parents at the hospital. You cannot stop that from happening. Although there is a theoretical risk of cross-infection, it has not proved to be a significant problem as we speak to parents about the risks and ask them to observe hand hygiene protocols. Children are either in hospital for a short time or are very ill, so isolation does not tend to be a problem from a psycho-social perspective. However, it is important to tailor the approach according to the individual.” The Trust now plans to expand the screening programme to include all elective surgical patients who require overnight admission, along with elective and emergency admissions to the liver and renal units, haematology and oncology wards.
Source - ( Jan 15, 2009 )
Staff photo/Tim Johnson - Kathie L. Rogers, PhD, clinical microbiology consultant at Jennie Edmundson Hospital, places a cartridge containing a DNA sample into a Cepheid GeneXpert machine that will make copies and yield test results in about an hour.
“This is a huge step forward for patients in this hospital, as well as infection control.”
Molecular testing, introduced at Jennie Edmundson Hospital in December, will produce faster lab results, reduce the risk of contamination and enable doctors to make quicker, better informed treatment decisions, said John Southard, M.D., pulmonologist at Jennie.
Jennie’s upgrade was aided by an advanced processing machine Southard called “the wave of the future.” The Cepheid GeneXpert allows technicians to test for specific bacteria and have results in about an hour.
“We can start the antibiotics that are most effective within an hour and a half,” he said.
Also important, doctors can avoid giving the patient a less effective antibiotic that might cause severe side effects.
Before molecular diagnostics, samples were used to grow lab cultures, which could take several days to produce results, Southard said. That meant doctors might have to begin treatment before knowing exactly what they were trying to fight.
“It’s kind of revolutionized infectious disease treatment,” he said.
“At the end of the day, it’s the patients who are going to benefit,” said Kathie Rogers, PhD, clinical microbiology consultant at Jennie.
The GeneXpert takes a DNA sample and sets off a chain reaction to quickly produce millions of copies of it. Chemical indicators glow brighter as the quantity increases, and the color helps the machine identify the bacteria. What once took three separate rooms is now accomplished in a small cartridge with several compartments. The cartridge reduces the chances of contamination and a ruined test. The machine can run four tests at a time, but that can be expanded to as many as 16 with the addition of more test modules.
Said Southard, “It’s a very complicated inner working, but it’s been made very simple for the user. It’s really a pretty amazing process that’s going on inside.”
The system was set up by Rogers, a longtime Jennie employee who completed her doctorate in microbiology in 2008. Rogers came to Jennie in 1985 as a medical technician. After 1 1/2 years, she went to New York. She returned to Jennie as a med tech in 1992 and, within a year, became technical supervisor of the lab, which she has been ever since.
“Without her, this wouldn’t have been done at all,” Southard said. “She’s just brought a whole new level to microbiology. She also works with me clinically seeing patients and deciding what the best treatment is.”
At this point, Jennie is using the GeneXpert to test for methicillin-resistant staphylococcus aureus, one of the most common causes of infections in hospitals. According to the Centers for Disease Control and Prevention, MRSA accounts for more than 60 percent of hospital-acquired S. Aureus infections in the U.S. More than 278,000 inpatients are infected by MRSA each year, and more than 17,000 MRSA-related hospitalizations end in death.
Jennie now conducts routine MRSA tests upon admission of certain high-risk patients, such as nursing home residents, patients with skin lesions and patients with a family history of infection, Rogers said.
“We’ve started selective surveillance,” she said. “All of the patients that have a predisposition to have MRSA are tested,” she said.
The hospital does not do blanket testing of all of its patients, Rogers said.
“There’s been no benefit shown for universal surveillance, at this point,” she said.
Jennie has 50 to 100 patients a month with MRSA, Southard said. Other targeted bacteria are not as common.
“I probably have, at any one time, five patients with MRSA involved in their infection,” he said.
The hospital may initially conduct about 100 tests a month, but that will increase as new tests are added and additional risk groups are added to the surveillance pool.
The hospital will also use the GeneXpert to test for Group B streptococcus in expectant mothers, vancomycin-resistant enterococci and clostridium difficile toxins. Group B strep can be passed to the baby during delivery and cause meningitis, sepsis or pneumonia.
Source - ( Dec 22, 2008 )
Lots of innovation is happening inside nondescript office buildings in Bothell, but this activity gets little attention because it takes place at branch operations of companies headquartered elsewhere. Cepheid, the maker of molecular diagnostic tools based in Sunnyvale, CA, is one of those companies.
So I jumped at an opportunity to interview CEO John Bishop a couple weeks ago to learn more about why a fast-growing company like Cepheid (NASDAQ: CPHD) made a strategic decision to build a 30-person chemistry team in Bothell, and why the venture is expected to grow.
First, though, a little background about the company. Cepheid’s revenues have boomed over the last six years, from about $15 million to a forecast of about $175 million for this year. One of the key drivers is a precise test for detecting MRSA bacteria—a common cause of staph infections. These bugs are growing fast, and represent about two-thirds of hospital acquired infections.
The Cepheid test tackles this problem by putting complex DNA amplification processes into a box that someone without expert lab training can operate. This is a big deal, because only about one-third of U.S. Healthcare facilities have labs certified and staffed to run the DNA amplification tests needed to identify MRSA, so they have to turn to contract labs that can take three or four days to get a result. Instead of fearing the worst and putting patients on potent antibiotics, even when it isn’t necessary, hospitals can shell out as much as $60,000 for a fully-equipped Cepheid machine that can give them the answer in about an hour.
More conventional diagnostic tests use antibodies to bind with proteins in the blood, but they can be less reliable and sometimes late to set off an alarm bell. This is why companies like Roche, Abbott Laboratories, Celera, and Becton Dickinson are all pushing to grab bigger stakes in the more precise world of molecular diagnostics. “With molecular, you can see what’s going on at a causative level,” Bishop says.
So Cepheid sees demand rising, particularly as it adapts the technology to hospitals that want to spot other worrisome bugs, like multi-drug resistant tuberculosis.
But how does the Bothell team fit into a company with 500 employees in North America and Europe? It all started in late 2003 when Alexander Gall, a chemist who formerly worked at Seattle Genetics and Epoch Pharamceuticals, told Bishop he was looking for a new job (right around the time when a lot of Seattle biotechs were laying off scientists). Bishop saw an opportunity to build something around Gall, so he hired him, and put him in charge of R&D.
Gall, and the people he oversees at the Bothell group, brought an important skill in their ability to modify the individual nucleic acid base chemicals—the building blocks of DNA—that make the Cepheid machines proprietary, Bishop says. “They are brilliant nucleic acid and organic chemists,” he says.
These modifications make the difference in the test’s reliability, and speed, Bishop says. This work is so integral to the company and its ability to make specialized probes and primers that Cepheid is planning to build up manufacturing capability near the chemistry group in Bothell. This work shouldn’t be outsourced, and will be kept close by the chemistry group, because “it’s high-value work for us, and we want to keep it with technically competent individuals,” Bishop says.
Bishop wouldn’t be specific about how many people it plans to hire here, but he was unequivocal about the fact that growth is coming here. “This will be a long-term growth program in Washington,” Bishop says.
Source - ( Dec 15, 2008 )
A Dorset hospital has begun to use a new kind of lab test that detects MRSA in hours rather than days.
Washington -- Use of real-time polymerase chain reaction to screen for group B Streptococcus in the delivery room could reduce the use of intrapartum antibiotics by more than half, compared with antenatal screening alone, the findings of a single-center study of 232 pregnant women suggest.
Current CDC guidelines call for vaginal and rectal swabs at 35-37 weeks' gestation and for all women with cultures positive for group B Streptococcus (GBS) to receive intravenous antibiotic prophylaxis during labor and delivery (MMWR 2002;51[RR1 l]:l-22). This practice has greatly reduced the rates of neonatal sepsis in the United States, but it is imperfect. Women whose status is unknown at the time of labor also must receive prophylaxis, resulting in overtreatment, while cultures can fail to detect GBS in women who are lightly colonized, resulting in failure to treat.
Rapid testing at the time of delivery using real-time polymerase chain reaction (RT-PCR) has the potential to solve these problems, Dr. Stefan Gerber and his associates said in a poster presentation at the jointly held annual Interscience Conference on Antimicrobial Agents and Chemotherapy and the annual meeting of the Infectious Diseases Society of America.
Of 232 women presenting for vaginal birth at University Center Hospital, Lausanne (Switzerland) in an 8-month span, 19% (44) had positive GBS cultures at 35-37 weeks, 65% (152) had negative cultures, and 16% (36) had unknown GBS status at the time of delivery Per the guidelines, 34% of the women (80) received prophylactic antibiotics during labor, but treatment was completed (at least two doses or at least 4 hours of intravenous antibiotics) in just 21% (17).
Lower vaginal and rectal swabs were obtained in all the women in the delivery room, and GBS detection was performed by both culture and RT-PCR, using Cepheid's Xpert GBS test, which runs on the Gen-eXpert System, a fully automated molecular testing system. Results were available in 75 minutes (compared with 2 days for cultures). By RT-PCR, just 15% (35) of the women were GBS positive, suggesting that 19% (45) of the women had received unnecessary prophylaxis, Dr. Gerber and his associates at the hospital reported.
Of the 35 PCR-positive women, 7 had negative cultures--presumably because they were only lightly colonized--and therefore would not have received antibiotics under the antenatal screening guidelines.
All 35 PCR-positive women received prophylaxis but just 7 (22%) completed it.
Since further work-up of the newborn is required when the mother doesn't receive complete antimicrobial prophylaxis, RT-PCR could potentially represent a significant cost saving. An ongoing study is investigating the cost-effectiveness of the approach, Dr. Gerber said in an interview.
Moreover, unlike cultures, which must be performed by technicians, the RT-PCR was performed by the midwives themselves. The technology could also be used by labor and delivery nurses or obstetricians, Dr. Gerber said in an interview.
Dr. Gerber stated that he had no financial ties to Cepheid. The company provided the equipment for the study, but no additional funding.
Most insurance plans cover the Xpert GBS test, which was approved by the Food and Drug Administration in 2006. It is categorized as "moderate complexity" by the FDA, meaning that non-laboratory health care professionals such as physicians and nurses can run the test, a Cepheid spokesman said in an interview.
The spokesman declined to say how many Xpert GBS tests are currently in use, but he did say that as of the last quarter of 2008, there were 848 GeneExpert Systems installed worldwide, capable of running the GBS test.
BY MIRIAM E. TUCKER
Source - ( Dec 04, 2008 )
This was discovered at Birmingham Children’s Hospital NHS Foundation Trust, after it introduced same-day MRSA screening for patients admitted to the PICU a month ago.
Previously, only high-risk cardiac patients had been screened, using traditional testing which can take up to three days to produce results.
However, the trust had become concerned that half of infections were not detected before symptoms appeared. There was one MRSA bacteraemia case last year and several surgical site infections.
Around 100 new patients on PICU have been screened using the new rapid polymerase chain reaction (PCR) test for MRSA – and 10% were carriers.
Natalie Edwards, senior sister in PICU, said: ‘We would have normally only known that these children carried MRSA once they had an infection. Now we know which ones are carriers we can quickly get them into treatment, with isolation and eradication. That way we can minimise cross-contamination.’
Children’s hospitals are not subject to Department of Health requirements to screen all elective admissions for MRSA from next March.
Birmingham Children's Hospital is the first in the country to use a new test from the US, that can detect the potentially fatal superbug MRSA.
The test means that the bug can be detected in an hour, whereas previously used tests to detect MRSA have taken around three days to establish whether a patient has contracted it or not.
Birmingham Children's Hospital is focusing on children in the ICU because they are most at risk of having the infection and doctors need a accurate and rapid result for MRSA.
Consultant microbiologist Dr Jim Gray said: "It made sense to start screening with ICU patients as they are the most susceptible to contracting the MRSA infection due to bacteria gaining access to the body through open wounds and
He went on: "It is vital to stop MRSA from entering the ICU as it is extremely serious and potentially fatal for children who are already critically ill.''
Source - ( Dec 03, 2008 )
James Netterwald, PhD, MT (ASCP)Senior Editor
Drug Discovery & Development - November 01, 2008
Collaborations bridge the gap between drug development and PCR-based companion diagnostic assays in the spirit of a more personalized approach.
Was Kerry Mullis insanely brilliant or brilliantly insane? That is the question. And polymerase chain reaction (PCR, for short) is the answer.
This little nucleic amplification method has turned the world upside-down and right-side-up again with sheer beauty, in the way that only a monumental scientific discovery like PCR can. It’s no wonder that Kerry Mullis received the Nobel Prize for his discovery.
PCR has been, and continues to be, the single most important tool in molecular biology. Enabling almost all molecular biology methods, PCR has been applied to basic biological research, drug discovery and development research, environmental research, forensics, and more.
“PCR is widely used to understand biology at the genomic level.” says Walter Koch, PhD, vice president and head of global research, Roche Molecular Diagnostics, Pleasanton, Calif. “As such, it is used as part of the target discovery effort including applications such as gene sequencing, for differential gene expression analysis, or as a preliminary step in areas such as microarray analysis.”
But PCR has gone beyond applications in basic and drug research and into the clinic, as the linchpin for building companion diagnostics. This article will discuss some of the ongoing research which has that aim in mind.
For clinical applications, HIV viral load testing—which first became available in the 1990s—is a prime example of the enduring value of a PCR-based molecular diagnostic assay. Commercialized first by Roche Molecular Diagnostics, the test was developed to augment the then-established biomarker for HIV infection, CD4-postive T-lymphocyte count, as a primary endpoint for HIV drug development and to monitor the effectiveness of HIV therapy in the clinical setting. “What has changed since the introduction of the first tests is that we also know that there are viral sequence mutations in HIV that are associated with drug-resistance,” says Koch. "By assessing the presence of these mutations in the protease gene and the reverse transcriptase gene, one can assess the likelihood of how well a patient will respond to a given drug combination treatment."
More recently, Roche Molecular Diagnostics has ventured into the realm of oncology. One project involves collaboration between Roche Molecular Diagnostics, Plexxikon (Berkeley, Calif.), and Roche Pharmaceuticals. Together, the teams are investigating the utility of the drug target, mutated b-Raf kinase, to select patients for clinical trials involving novel anti-Raf kinase compounds.
“In 70% of melanomas, perhaps 10% of colorectal cancers, as well as others, the b-Raf kinase is mutated, and this has become the target of a specific therapeutic designed to selectively inactivate the mutated kinase,” says Koch.
While Roche Pharmaceuticals is developing the anti-Raf drug, Roche Molecular Diagnostics is developing a real-time PCR-based companion diagnostic assay to test for the V600D mutation in the b-Raf gene. The test is designed for use in clinical trials, and if these are successful, to help physicians better identify patients who are candidates for this specific therapy. Initiating test development while the drug is still in clinical trials is intended to support the goal of launching both the drug and companion diagnostic simultaneously.
PCR at the start
A nother collaborative effort between Roche Pharmaceuticals and Roche Molecular Diagnostics is to work on developing a companion molecular diagnostic assay for MDM2 inhibitors, a novel class of anticancer agents developed by Roche called Nutlins. MDM2 is a protein that targets the p53 transcription factor for degradation, thus reducing its pro-apoptotic activity in normal human cells. Cancer chemotherapeutic agents that cause DNA damage activate the p53 pathway in cancer cells, thus resulting in their demise by apoptosis.
“If you could increase levels of p53 by blocking its interactions with MDM2, then that same program would be turned on and you would cause the cancer cells to commit suicide in the absence of any damage,” says Koch. By binding to the p53-binding cleft of MDM2, Nutlins do exactly that. Of course, biology never allows things to be THAT simple.
“In approximately 50% of all cancers, p53 is mutated. And when it is mutated, this approach is not going to work,” says Koch. To deal with this additional kink, Roche Molecular Diagnostics has designed an assay that first PCR-amplifies the p53 gene from DNA samples taken from each prospective clinical trial candidate, then uses a sequencing microarray to determine the p53 genotype. The idea is to identify patients who would or would not, based on p53 mutation status, be likely to respond to treatment with Nutlins. Currently, both the Nutlin and its companion diagnostic assay are still in development.
Let’s get clinical
David Persing, MD, PhD, executive vice president and chief medical and technology officer at Cepheid, Sunnyvale, Calif., knows a thing or two about designing molecular diagnostic assays. Rooted in second-generation PCR technology since its inception, Cepheid’s molecular diagnostic assays incorporate everything one needs to run the assays from sample prep to detection in a single, disposable cartridge. Each cartridge contains a single assay type and runs in a module in the instrumentation, which can run up to 48 independent modules, simultaneously, thereby eliminating the wait time associated with batch-mode processing.
"If you need a test result quickly and available on demand and around the clock in order to effect a treatment decision for a particular antibiotic, for instance, then batched testing is really not an option," says Persing. “So the kind of testing that we developed is really ideally suited for the pharmacodiagnostic applications that are being discussed in a lot of different contexts right now.” Cephid is now in discussions with multiple pharmaceutical companies interested in using one of their premiere technologies, the GeneXpert, to rapidly select clinical trial candidates for novel therapies against methicillin-resistant Staphylococcus aureus (MRSA).
All of Cepheid’s molecular diagnostic tests work by real-time PCR using fluorescent probes, which utilize many different fluorescent technologies including molecular beacons, scorpions, and TaqMan from Applied Biosystems. Cepheid’s instrumentation contains six different color channels that allow for simultaneous detection six independent reactions. After reserving one channel for an internal control, the other five channels can be used to detect amplification of five other targets from MRSA, for instance. “In the case of our MRSA SA assay, we detect two drug resistance markers and a species-identifying marker,” says Persing.
“Compared to other types of molecular methods, PCR is by far the most versatile,” says Persing. “PCR detects both DNA and RNA targets readily … can be multiplexed … is the technique that is going to be the mainstay of molecular diagnostics in the future.”
This article was published in Drug Discovery & Development magazine: Vol. 11, No. 11, November, 2008, pp. 30-34.
Source - ( Oct 31, 2008 )
A Sunnyvale company has developed a new test that can determine in just one hour if a patient has the superbug known as MRSA.
The traditional tests often took days before results were available.
Cepheid's test looks at the DNA of the infecting organism and can even detect the strain of methicillin resistant
staphylococcus aureas. The quick diagnosis means patients can get the correct type of anitbiotic for their condition because its
possible they may have a less severe staph infection. Cepheid CEO John Bishop says the test may also be used on patients before
surgery to determine if they have MRSA, which may put them at higher risk of developing an infection during their procedure.
Source - ( Oct 28, 2008 )
A new method to detect the superbug MRSA that only takes a fraction of the time of previous tests has so far proved 100% accurate, according to the company that developed it.
Birmingham Children's Hospital has recently introduced the GeneXpert test for patients waiting to enter intensive care.
Molecular diagnostics firm Cepheid said not only does its test take just one hour to identify if a patient has MRSA, but every result has also been verified as accurate by later culture-based tests.
Detection of MRSA in patients heading for intensive care is crucial as they will likely undergo medical procedures that carry a risk of introducing MRSA into sites of the body where it can cause serious harm, such as the lungs and the bloodstream. It is also important in stopping the spread of the disease to other patients.
Previously, traditional tests � where the bacteria is grown in a Petri dish in a micro-biology lab � take up to three days to verify.
Consultant microbiologist Dr Jim Gray said: "It made sense to start the screening with ICU patients as they are the most susceptible to contracting the MRSA infection due to the bacteria most commonly gaining access to the body through open wounds, injections, catheters and IV sites."
Copyright PA Business 2008
Source - ( Oct 28, 2008 )
A SUPERBUG can be detected in more sick children after a new test was pioneered in Birmingham.
Doctors at Birmingham Children’s Hospital are now rewriting medical myths that children are not prone to carrying MRSA bacteria, with concerns that half of its cases were going undetected until too late.
The trust has become one of the first to use a 100 per cent accuracy Cepheid XpertMRSA test that produces results within an hour.
Lab workers using the advanced technology to screen all young patients in intensive care said half of MRSA cases were not being picked up until the child had actually developed symptoms and fallen ill.
Dr Jim Gray, consultant microbiologist, said two extra cases had been picked up by the test.
“Something that has become apparent is that levels of MRSA amongst children are actually higher than is commonly believed.
“It is a generally accepted belief in the medical profession that children are not really carriers of the MRSA bacterium, however we are increasingly finding that this is not the case.
“It made sense to start the screening with intensive care patients as they are the most susceptible to contracting MRSA, as it commonly gains access to the body through open wounds.
“It is vital to prevent MRSA from entering the this unit as it is extremely serious and potentially fatal for children who are already critically ill.”
The new test allows doctors to diagnose patients quickly with the only other lab test with the same accuracy taking three days, by which time the bug may have spread to more children.
Source - ( Oct 21, 2008 )
by Heather Buschman
PCR-based assays deliver speed and accuracy, and see increasing adoption in the clinical lab.
Rreal-time PCR (polymerase chain reaction) provides clinical diagnostic labs with the ability to specifically target a particular sequence of human or pathogen DNA or RNA and amplify it to a level that can be detected and measured. This type of test is sensitive enough to pick up small variations in genetic sequence and can be quantified to determine the relative amount of DNA in a sample. Before PCR, this level of specificity, sensitivity, and speed did not exist. In terms of infectious disease diagnostics, even miniscule amounts of pathogen-specific DNA or RNA can be artificially amplified, detected, and measured in just a few hours without the need to culture the specimen—a task that can be very difficult for some infectious agents.
"PCR is a fast, accurate, and highly sensitive method that can be used for a broad range of genetic tests that identify infectious organisms or influence cancer therapy," said David Persing, MD, PhD, executive VP and chief medical and technology officer at Cepheid, Sunnyvale, Calif.
Until recently, the use of PCR has been restricted to a small number of laboratories that have the dedicated space and technical skill to perform complicated molecular diagnostics procedures. Even then, specimens are often run in periodic batches, which can add several days of waiting time to a 1-hour PCR test. As the technology advances and costs drop, however, clinical labs are increasingly incorporating PCR techniques into their repertoire of diagnostic tests.
The polymerase chain reaction—usually known simply as PCR—is so named in part for the polymerase enzyme that uses primers bound to a DNA template to assemble a new strand of nucleic acids. The chain reaction occurs as the template is heated to strip apart the double helix, cooled to allow the polymerase to synthesize a new strand of DNA using one of the parent strands as a template, and then allowed to zip back up before the process starts again. As each new strand of DNA then becomes a template for subsequent rounds of replication, the amount of target sequence is dramatically amplified. RNA can also be detected by first applying a reverse transcriptase enzyme to generate a complementary DNA (cDNA) template before proceeding to PCR.
The recipe is simple: add the DNA template, a polymerase enzyme, nucleotides, and primers in a buffer, place the sample in a thermal cycler instrument, set the program to cycle through the necessary temperatures, and work on something else while it runs. During real-time PCR (RT-PCR), the concentration of DNA in the sample is detected and quantified in real time by an instrument measuring the amount of a dye that fluoresces when bound to double-stranded DNA.1 Fluorescence increases as the concentration of DNA in the sample increases.
At the end of a RT-PCR cycle, the sample temperature is raised to determine the melting point. Since melting points should be equal for samples amplified with the same pair of primers, plotting the decrease in fluorescence as strands separate will determine homogeneity and purity. Curve variations can indicate subtle sequence variations like a single base mutation, or problems such as contamination.
For many clinical labs, concerns over ease of use and standardization of assay design remain formidable barriers to the adoption of PCR-based diagnostics.2 Several companies are developing products specifically designed to address these concerns.
Molecular diagnostics can be applied to four areas of clinical testing: rapid identification, screening, classification, and monitoring. Each category has different clinical, operational, and economic needs required to maximize the benefits to patient care. Roche Diagnostics, Basel, Switzerland, markets its portfolio of instruments, which includes the COBAS TaqMan® and LightCycler® analyzers, to meet these distinct needs.
The COBAS TaqMan Analyzer is a RT-PCR system that automates amplification and detection of DNA or RNA for up to 96 samples and four assays at one time. This system can be paired with the COBAS AmpliPrep Instrument for a truly "sample in, result out" workflow. AmpliPrep automatically purifies nucleic acids from closed input tubes by lysing, washing, resuspending, and transferring samples directly to output tubes. When the two systems are docked together, the target template and a master mix of PCR reagents move directly to the COBAS TaqMan Analyzer for RT-PCR analysis. Without human intervention, the workflow is streamlined and the risk of contamination is minimized.
COBAS TaqMan is uniquely qualified to meet the needs for monitoring viral load. "A patient with HIV, for example, is tested regularly to confirm that the viral load is below a certain threshold," explains Harsha Mokashi, marketing manager at Roche Diagnostics. "Since any increase in viral load could mandate a change in treatment, measurements made over time must be consistent. The COBAS system delivers this continuity of care."
Roche's COBAS AmpliPrep/TaqMan system has been rigorously tested for its sensitivity and specificity in detecting pathogens such as HIV-1. Now, a TaqMan test for measuring Hepatitis B virus (HBV) DNA recently became the first of its kind approved in the United States.3 Tests for Hepatitis C virus (HCV) and cytomegalovirus (CMV) are currently in development.
To meet the needs of rapid identification, Roche also offers the LightCycler Analyzer, a carousel-based thermal cycler. With this instrument, RT-PCR can be performed in as little as 30 minutes in small batch sizes, eliminating the need to wait for enough samples to complete the batch before testing.
"Molecular diagnostics has redefined what it means to be timely. The LightCycler delivers speed to those tests where trimming off hours can deliver clinical impact," Mokashi says.
The GeneXpert Infinity-48 high-throughput system (Cepheid, Sunnyvale, Calif) automates the management of the entire molecular testing workflow. After a sample is loaded into a GeneXpert cartridge, the Infinity-48 system does the rest by managing the sample data, cartridge loading and unloading, and reporting of test results. It uses a robotic cartridge handling, managed by Cepheid's Xpertise™ software, to run up to 1,300 different tests during any 24-hour period.
"With each of the system's 48 testing modules managed as independent testing sites, the GeneXpert system can start test runs anytime samples are collected—24 hours per day, 365 days per year in under 2 hours," Persing says. "It's designed to improve workflow while accelerating time-to-result, enabling health care providers to make more informed patient-management and treatment decisions."
Industry trends in automation are moving toward high-throughput systems, but these platforms still rely on batch testing. "While batch testing may seem ideal for the select handful of national and regional reference laboratories looking for that level of throughput, it does not meet the needs of the average hospital," Persing says. "Cepheid's technology has democratized diagnostics, making it possible for almost anyone to perform a sophisticated molecular diagnostic test."
Currently, the Infinity-48 system is designed specifically for the detection of methicillin-resistant Staphylococcus aureus, enteroviral meningitis, and Group B Streptococcus. The platform also supports other assays for infectious diseases, genetic markers, and, soon, oncology.
Genetic heterogeneity, even within the same patient, can push clinical decisions to depend on analysis at the single-cell level. According to Frank Feist, executive director of Advalytix Business at Olympus America Inc, Concord, Mass, "Oncogene expression can vary from one cell to another in a single tumor, and immune cells can differ depending on their pathogen specificity, so clinical tests are increasingly looking to exploit the information contained in a single cell to inform diagnosis, prognosis, and treatment."
To simplify this process, Advalytix has developed the AmpliGrid and AmpliSpeed systems. AmpliGrid looks like a standard microscope slide, but is processed like a semiconductor chip to allow the surface to hold up to 48 1-µL samples safely in place. The drop can be placed by pipetting, laser capture microdissection, or flow cytometry, and visually verified under a microscope. Next, with the surface tension preventing any dripping or sliding, samples are covered with a mastermix of PCR reagents. Then, the whole slide drops right into the AmpliSpeed cycler, a miniature PCR thermal cycler customized for the AmpliGrid.
"The AmpliSpeed is so small you could take it home in your bag to run another cycle during dinner," Feist jokes. "It saves energy by relying on moving parts to create a temperature profiles, instead of bulky fans and heaters.
In just a couple of hours from start to finish, DNA from a single cell is amplified by the AmpliSpeed instrument and ready for standard RT-PCR analysis.
Accurate RT-PCR results rely on consistent loading of an appropriate concentration of template. The NanoDrop™ (Thermo Fisher Scientific Inc, Wilmington, Del) further streamlines molecular workflow by measuring the concentration of DNA or RNA in 10 seconds with only 1 µL.
Measuring the concentration of a nucleic acid sample usually requires sacrificing a substantial amount of sample to make dilutions, transferring it to a cuvette, and measuring the absorbancy with a standard spectrophotometer.
"The NanoDrop simply takes away the need for a container," says Philippe Desjardins, scientific marketing manager at Thermo Fisher Scientific Inc.
The NanoDrop brings speed and reproducibility to PCR-based diagnostics. The inherent surface tension of 1 µL of undiluted sample holds it in place as the drop is stretched between two points and a narrow optical beam is fired precisely in the middle, accurately reading a range of 2 to 3700 ng/µL of undiluted nucleic acid. The instrument is then simply wiped clean and ready for the next sample.
"The basic idea for the NanoDrop came from hearing molecular biologists complain about wasting their sample to take measurements," Desjardins says. "Waste is especially a shame when you have a limited amount, and that factor becomes even more important in clinical settings."
No one wants to wait 2 days for a result when a health care decision is in the balance, yet the cost of speedy RT-PCR instruments and reagents can be prohibitively expensive. Risk of contamination is also a huge issue, since results can easily be skewed by the unintentional amplification of just a few stray copies of DNA. Thus, clinical technicians must be well trained in RT-PCR techniques and assays must be standardized, whether by purchasing manufacturer-designed kits or developing in-house protocols. PCR experts encourage technicians to educate themselves by attending regional trade shows or talking to vendors that can properly demonstrate how to use these instruments.
In the end, the value of applying RT-PCR in the clinical laboratory environment comes down to a cost/benefit analysis. As Persing summarizes, "The technology comes at a premium price, but most users figure out very quickly that they can save money in labor and overhead, and most importantly by delivering the most effective health care."
Source - ( Oct 09, 2008 )
Cepheid (Nasdaq: CPHD) announced it received clearance from the U.S. Food & Drug Administration (FDA) to market its Xpert™ MRSA/SA Blood Culture (BC) test, which runs on the GeneXpert®System, for the detection of Methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus aureus (SA, typically methicillin susceptible) in blood culture bottles showing gram-positive cocci — in less than one hour.
Cepheid’s Xpert MRSA/SA BC test processes positive blood culture specimens to determine if a patient’s blood is infected with MRSA or SA, which are frequent causes of sepsis in hospitalized patients. This may enable physicians to quickly de-escalate from broad-spectrum antibiotic treatment to a more effective targeted therapy, thus reducing risk of resistance and improving patient outcomes. If the gram-positive cocci are neither MRSA nor SA, the physician may be able to stop antibiotics altogether.
“With the addition of our Xpert MRSA/SA Blood Culture diagnostic test, Cepheid is providing institutions with the most comprehensive product portfolio for rapid detection of MRSA and SA,” said John Bishop, Cepheid’s Chief Executive Officer. “The GeneXpert System is unique in its ability to combine the benefits of molecular testing with on-demand availability. In the case of sepsis, one of the ten leading causes of death in the United States, test results must be available quickly in order to have maximum impact.”
According to a 22-year study published in the New England Journal of Medicine, the incidence of sepsis is growing at 8.7 percent annually in the U.S, expected to reach more than one million by 2010, while cases of severe sepsis account for up to 11 percent of ICU admissions. The same study states that care of sepsis patients cost hospitals as much as $50,000 per patient, resulting in an annual healthcare burden of $17 billion.
Typically, physicians will order a set of blood culture bottles drawn from patients presenting with symptoms of systemic infections. Positive cultures can appear in as little as 6-24 hours, but then require further testing to identify the specific organism causing the infection. Currently, those additional tests — most notably to determine if the organism is methicillin-resistantormethicillin- susceptible Staphylococcus aureus — are done via slower culture testing methods.
“Same day diagnostic results for blood culture bottles growing gram-positive cocci will empower physicians to begin targeted therapy in septic patients far sooner than current culture-based methods,” said Dr. Ellen Jo Baron, Director of Clinical Microbiology Laboratories for Stanford University Hospital and Director of Medical Affairs at Cepheid. “These results, delivered 24-48 hours sooner, can potentially improve antibiotic stewardship and provide patients with the best, most effective therapeutic regimen.”
According to a Critical Care Medicine study, hospital costs associated with severe sepsis patients are 141 percent higher than those of other patients. The study also found that 72 percent of cases are under-reimbursed, and severe sepsis patients are five times as likely to be outliers.
Xpert MRSA/SA BC delivers on-demand test results to assist physicians in modifying patient treatment plans with targeted therapy to improve patient management, increase antimicrobial stewardship, and control treatment costs associated with the care of sepsis patients.
Source - ( Sep 26, 2008 )
Source - ( May 19, 2008 )
by Judy O'Rourke
An interview with David Persing, MD, PhD, executive VP and chief medical and technical officer
In the early 1990s, David Persing, MD, PhD, founded and oversaw the molecular microbiology lab at Mayo Clinic, Rochester, Minn, which pioneered many diagnostic techniques used in molecular pathology labs. He joined Cepheid, Sunnyvale, Calif, in 2004, where he leads efforts to fully automate the complex processes behind real-time molecular diagnostics so scientists, clinicians, and those with little training can tap the technology. The on-demand molecular diagnostics company develops and manufactures fully integrated systems and tests for genetic analysis. CLP recently caught up with Persing, who distilled his wide-ranging experiences into timely observations about where his company fits in the supply-and-demand food chain.
Q: How long were you director of Mayo Clinic's molecular microbiology lab?
A: For about 10 years. I was recruited in 1990 to set up molecular diagnostic testing for the infectious disease diagnostics activity. The microbiology laboratories at that time were just starting to get into molecular techniques.
Q: Describe some diagnostic techniques pioneered by the lab that play a key role in molecular pathology labs today.
A: We were early adopters of PCR technology for routine use in the laboratory. The big concern back then was that of contamination problems with PCR. PCR was viewed as incredibly powerful, but essentially a technique that could not be tamed for routine laboratory use—because of the fact that during the process of performing PCR you create billions or trillions of copies of your target sequence that you're amplifying to use to identify a particular organism. I spent the first couple of years at the Mayo Clinic figuring out ways to control the technique and make it usable as a routine laboratory method.
Q: You joined Cepheid as a director in May 2004, and became executive VP and chief medical and technology officer in August 2005. What is your role there?
A: When the opportunity came up to come in and reorient Cepheid from a biothreat company to a clinical diagnostics company, I jumped at the chance. What Cepheid has done is they've essentially taken my 4,000-square-foot laboratory at the Mayo Clinic with multiple rooms and separation between rooms and lab techs—all the stuff that we did within that PCR facility Cepheid accomplished inside of a little plastic cartridge that carries out all of the sample-processing steps. Nucleic acid extraction, purification, amplification by PCR is all carried out in a disposable cartridge that runs in a system called the GeneXpert, which is the automated instrument platform that all of our tests run on. Testing was so sophisticated it couldn't be done in your average hospital, and we thrived on this reference lab model. But I saw the Cepheid technology is essentially producing a trend in the opposite direction where you can take this very sophisticated technology with no compromise in performance—you could decentralize the whole process and make it possible for virtually any hospital anywhere at any time to run these tests.
Q: Have you snagged FDA approval?
A: All of our clinical diagnostics either have gone or will go through the FDA approval process. Right now we have three FDA-approved products on the market. We hope to see two more by the end of the year.
Q: Why did you align yourself with the company?
A: Because of the chance to really make a difference in diagnostics. One test that I think is going to have a huge impact is for MRSA testing. Our test is designed to identify a carrier of MRSA rapidly and to minimize exposure of that individual to other hospital personnel by virtue of instituting infection precautions—barrier precautions, contact isolation quickly.
The other area I'm really excited about is a program we're working on with a group called FIND (Foundation for Innovative New Diagnostics), which is funded by the Bill and Melinda Gates Foundation for the development of a rapid, extremely sensitive diagnostic cartridge for tuberculosis. Now, TB takes weeks or months to grow in the laboratory, and then if you really want to know if it's one of the bad TB strains, one of the multi-drug-resistant TB strains, you have to wait for a couple more weeks to be able to do drug-resistance testing.
We developed in the GeneXpert, with support from the NIAID (National Institutes of Allergy Infectious Disease) and the Gates foundation through FIND, a cartridge that goes straight from a sputum specimen to a definitive TB result within 90 minutes. It does that in a way that allows us to predict drug resistance as well, so it gives us two things—the identification of tuberculosis and the likelihood of drug resistance—very rapidly, so the patients can be identified at the clinic door as being carriers of TB, placed in the appropriate conditions for reducing the risk of transmission, and they can be given the right drugs without waiting for months for a drug-resistance result, which in current conditions often translates into a postmortem diagnosis.
Q: How are you working on improving microRNA identification and analysis by combining bioinformatic approaches and updating your database to detect all microRNA sequence and antisense targets with in-house-developed methodology?
A: About 2 years ago, we bought a company in France called Actigenics. It specialized in the development of bioinformatic techniques for predicting the presence of microRNA in the human genome, and we've been looking at microRNA as a unique diagnostic opportunity for developing novel diagnostic applications in areas like inflammation and oncology. We're focused heavily on discovering these microRNAs, characterizing them, and exploring their diagnostic value in panels of microRNAs that are measured by real-time PCR on our GeneXpert platform. The idea is to develop gene-expression profiles of microRNAs that are informative, that help make these decisions that I talked about relative to choice of therapy, whether to treat or not to treat, whether to use chemo or not to use chemo. These kinds of decisions are, we think, ultimately going to be resolved and facilitated by gene-expression profiling, and we think the microRNAs are going to have a big impact on the overall tests of the programs to stratify patients according to appropriate therapeutic options.
Q: Describe some industry trends.
A: I see the industry trends in automation moving toward larger systems. Higher-throughput systems, but still batch-based systems. That's great for the 12 laboratories in the universe that really need that level of throughput, but it doesn't really meet the needs of your average hospital of 150 beds that's 50 miles away from a major metro area. We have to get away from the central lab mentality, which is based on huge central-lab systems, because in order to get enough samples to justify having one of these instruments you have to transport samples across long distances in a reference lab model. There's a lot of interest now in improving turnaround time by decentralizing the technology.
Q: What is the future of the industry?
A: Molecular diagnostics is the largest growth area for diagnostics. We're going to see more drug companies building companion diagnostics into their products. We are going to see a lot more pharmacogenetics. The last thing a drug company wants is for their drug to be held hostage to a diagnostic test. Nobody wants to wait around for 2 days for a result when a prescribing decision is in the balance.
Source - ( Feb 15, 2008 )
BACKGROUND: Methicillin-resistant staphylococcus (MRSA) is a common cause of skin infections. It can also cause pneumonia, ear infections and sinusitis. MRSA bacteria are sometimes dubbed "superbugs" because they are highly resistant to common antibiotics like penicillin, making infections difficult to treat effectively. Bacteria are highly adaptive and over time, they naturally develop resistance, protecting them from incoming germs -- and antibiotics -- and making them harder to kill. If MRSA enters the body through the skin, it can cause irritating skin infections, but if it enters the lungs or bloodstream, it can cause serious blood infections, pneumonia, even death. According to the Centers for Disease Control and Prevention, about 90,000 hospital patients die each year as a result of contracting an infection while being treated for another condition. The estimated annual nationwide cost to treat infected patients is between $3.2 billion and $4.2 billion.
SPORTS ANTIMICROBIAL SYSTEM: The University of South Carolina is applying high-tech, proactive measures to combat the spread of bacterial infections throughout its athletic complex. One is the use of the SportsAide system -- a patented, non-leaching microtechnology that creates a durable, antimicrobial bond on sports surfaces and controls the growth of a wide array of bacteria, mold, fungi and algae. The antimicrobial substance is sprayed onto all surfaces, destroying bacteria on contact.
GENE X-PERT: Scientists at Cepheid have developed a simple new test called the GeneXpert to detect MRSA. The new test received FDA approval in April of last year. The GeneXpert can generate a result in two hours, enabling hospitals to rapidly identify infected carriers of MRSA and take timely control measures. Older tests took up to two days to reveal results. The ultimate goal is to lower the rate of hospital-acquired infections and improve overall patient care. The GeneXpert test simply asks users to place a patient sample in the cartridge and load it into the device. It's like having a complex molecular laboratory in a handheld box that can be used anywhere, any time.
AT HOME PREVENTION: There are several preventive measures that can be taken to stop the spread of MRSA at home. The CDC recommends you:
· Wash your hands with soap for as long as it takes you to recite the alphabet. When washing hands isn't possible, use an alcohol-based hand sanitizer
· Cover all cuts and scrapes with a clean bandage
· Don't ever touch another person's wounds or bandages
· Don't share personal items like towels or razors
· Dry clothes, sheets and towels in a dryer rather than hanging them out to dry
Source - ( Jan 02, 2008 )
I have written about the killer, super-bug staph infection MRSA, but new, proposed legislation in California is bound to make it a headline again -- and a big one!
The proposed law would give California, perhaps, the toughest MRSA reporting requirements -- it would require all high-risk patients be screened, such as people in hospital ICUs. California would also add MRSA to the list of communicable diseases that must be reported to state authorities. This would make California the fifth, and by far the most important, state to adopt these kinds of regulations.
Is this an opportunity for investors?
There are few plays, and the closest to being a pure play in 2008 is a small diagnostics company, Cepheid (CPHD). They have a new, state of the art test that is much faster than existing tests, for a variety of reasons, but the Department of Veterans Affairs is giving between 90% and 95% of its business to CPHD.
The company uses a razor and blades business model -- they sell hardware that can run any CPHD test, MRSA being one of the first and easily the most popular -- and make their real profits on test cartridges. The stock quadrupled from its low in 2007, in part due to new state and Medicare regs and the outbreak of community MRSA that has killed several non-patients. A CDC report saying at least 17,000 people a year die from MRSA also helped push the stock. I own shares in the company.
Another play is Swiss company Basilea (BSLN on the Zurich exchange). It is developing something called Ceftobiprole in Phase III trials for hospital acquired pneumonia but it also works on MRSA. The company believes it will be superior to the current gold standard for treatment, vancomycin, and in this world, all new antibiotics are welcome due to the inevitable problem with drug resistant strains. I do not own any Basilea stock.
Check out all the legislative and industry activity in this area. I believe all -- that’s right all -- patients will be screened for MRSA in the next 3-5 years, prompted in part by interest in the subject by tort attorneys. See if it belongs in your portfolio in some fashion.
Source - ( Jan 01, 2008 )
by Renee DiIulio
New developments continue to improve stat-testing turnaround.
Stat testing will never fall by the wayside, but its handling has undergone significant change. Stat-testing menus have expanded at the same time that some central laboratories have become so efficient that stat tests do not require special handling because all samples are turned around within stat-testing time guidelines. Other laboratories have implemented comprehensive point-of-care systems that provide urgently needed results at or near the patient's bedside within an even shorter period of time.
Stat originates with the Latin statim, which means immediately. According to Duane Morris, COO of Response Biomedical Corp, Burnaby, British Columbia, regulations often demand that stat-test results be delivered within 30 to 60 minutes.
Ron Berman, vice president of automation and systems at Beckman Coulter Inc, Fullerton, Calif, estimates that about 60% to 65% of an average hospital laboratory's testing is performed for outpatients, with the remainder performed for inpatients. "Typically, there are a lot of stats in that remaining 35%. How does the laboratorian prioritize?" Berman asks.
Guidelines, protocols, instrumentation, and software help meet the stat-testing demands of clinical laboratories. Manufacturers are constantly pushing to reduce turnaround times for their diagnostic products and to deliver more useful information that can help physicians make treatment decisions.
Beckman Coulter's recently released AutoMate 800 Sample Processing System permits users to place priority tubes in a special area, where they become the next tubes to be processed. "This allows the lab to have a process to manage tubes as they come in, but also a method to prioritize tubes that need special attention," Berman says.
Other automated features include sample loading and sorting; programmable, on-demand centrifugation; sample-volume detection (through the label); a decapper module; and intelligent aliquoting and tube labeling. Removing the manual factor from these steps helps to reduce errors and improve turnaround times.
Berman notes that all of Beckman Coulter's analyzers offer some method for handling stat samples, though these methods may differ from an engineering standpoint. The Power Processor automated sample processing system permits the operator to stop routine processing at the push of a button and manually initiate stat specimen processing. A chemistry analyzer pushes routine racks back to make room for a stat rack, also at the push of a button; hematology instruments can aspirate stat samples manually.
"There are all sorts of ways within software to be able to say, 'Pause what you are doing, finish the chemistries, and put this stat tube ahead for processing,'" Berman says. Automation, combined with process-management improvement, means that customers no longer need to process samples in batches, nor even to differentiate between stat and routine testing: turnaround is fast and consistent.
In addition, middleware can track stat specimens, alerting laboratories to their presence as soon as they are ordered. "The lab will know it's coming and can look for it," Berman says. The specimen is added to the system's running log, and an alert is sounded when time is running out or the results are ready. Beckman Coulter's DL2000 Data Manager features an instant-message window in which the alerts are displayed.
"There is a big focus on automation, but how does it work in combination with middleware and software to help with the physical management of tubes and data management?" Berman asks.
Data management can be even more challenging with point-of-care instruments if results are not stored and/or transferred to the laboratory information system (LIS) electronically. "Many point-of-care systems print the result on a piece of paper that disappears, so it is a challenge to know where those results are, who has run them, and if billing can match it to a patient or ordering physician," according to Bill Manchester, group product manager of WW marketing, critical care, Instrumentation Laboratory (IL), Lexington, Mass. IL features a system that offers LIS connectivity, whether the instrument is placed in the central laboratory or in patient care areas such as the intensive care unit or cardiovascular operating room.
The GEM Premier 4000 whole-blood analyzer is designed to work in the clinical laboratory or in a remote area where it will still deliver laboratory-quality results. Its moderate complexity means that technologists are not required to run the device; properly trained individuals may do so. The system monitors performance through Intelligent Quality Management (iQM), a real-time quality assurance program.
GEMweb Plus is the integrated operating software for the GEM Premier 4000. It enables all connected GEM instruments and computers to gain remote access to the system, permitting real-time supervision and automatic transfer of results. Orders can be downloaded from the LIS or hospital information system, samples can be validated, reports can be accessed, troubleshooting can be done remotely, and authorized users can be permitted access.
Training takes about 20 minutes, according to Manchester. Users can include nurses, respiratory care practitioners, and perfusionists, in addition to technologists. The system is so easy to use because the work is performed using a disposable cartridge, which minimizes the necessary manual intervention for sample or control analysis.
"The fluidics of the system, the sampler, pump tubing, distribution valve, waste container, and process control solutions—everything that would be a piece of a traditional blood gas analyzer is in the cartridge," Manchester says. He explains that the sample is inserted into the cartridge, where it is eventually run over a sensor card that performs the measurements. The unit also serves as the waste container and is designed for easy disposal. Manchester likens each replacement cartridge to a new analyzer.
Also, customers also can specify the test menu, so that resources will not be wasted on unneeded tests. The testing menu includes blood gases, CO-oximetry, electrolytes, glucose, lactate, metabolites, hematocrit, and pH. Xavier Nogue-Villa, IL's product manager for commercial strategy for the GEM Premier 4000, notes that the system takes about 90 seconds to produce a result (60 seconds for coagulation data). "There is a clinical benefit, particularly during surgery, to having the information immediately," Nogue-Villa says, comparing the turnaround time to an hour for results from a central laboratory.
Morris believes that additional point-of-care challenges include accuracy, precision, and compliance with regulatory requirements. "Accuracy relates to how close the result is to the true result, which is difficult for some analytes (such as troponin, where there is no true result)," Morris says. "The use of whole blood, a common point-of-care specimen, challenges instruments in terms of precision, and typically, it does not match that found on an analyzer."
Compliance is a third, and rather large, issue. "Nurses, physicians, and other hospital staff are not interested in running controls and providing documentation. They want self-maintaining equipment so they can concentrate on patient needs," Morris says.
Point-of-care devices, however, permit that same staff to focus more immediately on patient needs by providing information that helps to determine what those needs are, often more quickly than would be possible using the central laboratory. Sometimes, simply the elimination of transportation time makes a valuable difference.
Response Biomedical's RAMP system has unique features designed to address these limitations while offering a turnaround time of less than 20 minutes. The lateral-flow immunoassay system is composed of a portable fluorescent reader and single-use disposable test cartridges. The analysis incorporates the company's RAMP ratio, calculated using data from the detection and internal-control zones. "This corrects for a multitude of variability that could be present otherwise, improving the system's accuracy and precision over those that use absolute fluorescence," Morris says.
The device also features built-in controls (both positive and negative) that prevent samples from being run if the controls do not meet the standards programmed by the laboratory. Incorrect operator technique will also prevent sample processing; the system senses when the timing signals are off, whether due to the operator or to the device. "In either instance, the instrument produces a good result or no result at all," Morris says.
The result is also clear: a specific readout, rather than a line or color change. "No judgment is required on the part of the user," Morris says. This ease of use contributes to the moderate complexity rating, which means that the device can have a variety of users, including nurses and technologists. The device has been approved by the FDA for use with three cardiac markers: troponin I, creatine kinase MB, and myoglobin.
Point-of-care examinations are particularly valuable for super-stat specimens, which David Persing, PhD, MD, chief medical and technology officer for Cepheid, Sunnyvale, Calif, notes are more urgent than simple stat specimens. "Is the result needed before the patient leaves the ED? With super-stat specimens, time is of the essence and transport is a major limiting factor, so it makes sense to have the test run close to the patient. Regular stat testing may make better sense to run through a central lab for economies of scale and efficiency," Persing says.
Both models work, but Persing has noted a trend toward completing more urgent testing at or near the patient, even with tests as common as those used to detect methicillin-resistant Staphylococcus aureus (MRSA).
MRSA tests may not commonly be designated stat, but Persing notes that more MRSA testing can be expected to occur in different areas of patient care (such as admissions, where knowing the patient's MRSA status is sometimes necessary before assigning a bed). "That is a stat-testing indication," Persing says. Similarly, other tests also benefit from near-patient testing. The company's Smart GBS test for group B Streptococcus (GBS), which was approved for use on the SmartCycler system, can be run where patients in labor are admitted and in obstetrical clinics.
The Smart GBS test is the first polymerase chain reaction (PCR) test to have received a moderate complexity rating, according to Persing. Both Xpert tests run on the GeneXpert system, which accepts the cartridges in which the samples and reagents are combined. The instrument performs the sample preparation, PCR, and amplification in about 70 minutes and is not batch dependent. This rapid turnaround permits new testing protocols. For instance, GBS guidelines suggest that the test be performed around the 35th to 37th week of pregnancy. Some women, however, will miss the appointment and will not have the test performed at all; others will take it too early.
Persing has noticed a tendency to have the test run at an earlier stage. He says, "This can backfire, since there is less of a connection between the antepartum and intrapartum results. Studies show that the sensitivity of an antepartum culture for predicting intrapartum culture activity is roughly 50% to 60%. This means you are missing a lot of converted cases." Stat-testing demands are increasing because many facilities are implementing intrapartum testing, which is accomplished at or near the patient, to provide the results within the useful time range.
The company also offers an Xpert test for enteroviral meningitis and has products in development for vancomycin-resistant enterococci and Clostridium difficile. "We have a lot of involvement in hospital-acquired infections because that is our main focus," Persing says. The company aims to have PCR produce results in real time. What could be more stat than that?
Renee DiIulio is a contributing writer for CLP.
Advances in molecular diagnostics have transformed laboratory medicine. Robotic DNA and RNA extraction, DNA microarrays, multiplex protein detection, and gene-expression profiling are among the technologies making it possible to diagnose earlier and to create more precise treatment options for patients.
The Center for Molecular Medicine, or CMM, launched in Grand Rapids, Mich., a year ago, is a melding of research expertise and hospital and laboratory resources that is using these technologies to probe cancer, heart disease, mental illness, and other conditions at the DNA, RNA, and protein levels.
A $6 million joint venture between a hospital, Spectrum Health, and a research institute, the Van Andel Institute, the CMM aims, as one of the three pillars of its business model, to accelerate the tailoring of treatment based on the molecular makeup of a patient or a patient’s tumor, or both.
In a CAP TODAY interview with writer Anne Paxton, the center’s executive director, Daniel H. Farkas, PhD, talked about the new center, test platforms, molecular diagnostics’ effect on point-of-care testing, reimbursement and economic studies, IVDMIAs, gene patents, and more.
What is the Center for Molecular Medicine’s mission?
The center is really designed to do three things. One is to be a 21st century molecular diagnostics lab. Two, on a fee-for-service basis we plan to court not only classical clinical trials from pharmaceutical companies but also less classical trials of new content and new platforms from diagnostic and biotech companies. Three, the center will serve as an outlet for the molecular diagnostics content being developed at the research institute.
We’re a CLIA-certified, CAP-accredited clinical laboratory, and so we can bring those tests online in a regulatorily appropriate environment.
What led you to join the center?
I’ve established three hospital-based molecular diagnostics laboratories in the past, most recently at The Methodist Hospital in Houston, but I was interested in coming back to Michigan.
I found out about the center and they worked to recruit me for a long time. I joined in December 2006. We have five technologists now, an office manager, and an administrative support person, and can gear up quickly to add new people when new projects come in the door.
I see the center as an opportunity for me to use my skills in laboratory medicine and my skills in business development to bring genomics to a new level here in Grand Rapids. I think it’s a paradigm for a future path for molecular diagnostics, and I was excited to be part of a forward-looking, entrepreneurial community that was interested in blazing trails.
What kinds of testing are you conducting?
In my previous hospital-based molecular diagnostics jobs, my main mission was to add new items to the test menus for the sake of our patients and physicians. Here, that’s not my main goal. It’s an important part of what we are doing but ancillary to our main objective, so I’m intentionally keeping a light test menu of cutting-edge tests.
We’ve been open for about a year and we have three tests on the menu. Roche Diagnostics’ AmpliChip CYP450 test, a microarray-based test to examine patient genotypes, was the first test we brought online, in March 2007.
In October 2007 we began offering the CellSearch system from Veridex to identify and count CTCs, circulating tumor cells, in metastatic breast cancer patients. And on December 14, we became one of the first laboratories in the nation to offer the CellSearch system to test for CTCs and guide treatment of metastatic colorectal cancer. The CellSearch tests represent an important advance in how breast and colorectal cancer can be managed over the long term.
I don’t think I’m going to bring on that many tests very rapidly, because I want to support the clinical trials part of the business, and patients always come first. If you have too many tests on the laboratory test menu, then that tends to trump everything else.
Xceed Molecular just launched its automated Ziplex system to perform complex molecular assays, and the center is the first member of the Xceed Molecular collaborator program. What kind of work will you be doing with Xceed?
Our cooperation with Xceed Molecular fits into our overall strategic mission. Xceed is a perfect example of a biotech company that eventually would like to become a diagnostics company. They see their relationship with CMM—which sits across the street from a hospital and a research institute—as a great way for them to gain experience in the clinical laboratory and simultaneously develop content for their eventual molecular diagnostics test platform.
We’re doing two things with Xceed. First, in our laboratory we are demonstrating the equivalence of their system to the gold standard method for gene-expression profiling. And second, in collaboration with the Van Andel Institute, we’re helping Xceed move new array-based content toward commercialization.
As translational medicine moves from the research laboratory to the clinic, technology that is affordable, reliable, and automated will be critical. We believe the Ziplex system is very user-friendly for the clinical laboratory and therefore uniquely positioned to fulfill these needs.
Which molecular diagnostic test platforms do you envision being the most robust or most relevant over the next few years?
Clearly, real-time PCR, which has re-revolutionized molecular diagnostics yet again in this decade, will remain the most robust platform that will generate the highest volume for the foreseeable future. It’s just a great platform.
Gene-expression profiling is next. It is at the beginning, it is nascent, and we’re still gaining expertise as a laboratory medicine and medical care community in terms of how best to utilize gene-expression profiling. So that’s the future. But right now, the short answer to your question remains real-time PCR.
What impact do you think molecular diagnostics will have on point-of-care testing?
We’re actually working with a couple of companies to help them develop that technology.
Point-of-care molecular diagnostics used to be oxymoronic. But now, with real-time PCR and with advanced technologies that allow us to extract nucleic acids quickly and remotely, one can actually think about point-of-care molecular diagnostics. There are a good 10 or 12 companies I know about that, in various phases of development, are working to develop POC molecular diagnostics. They’re not ready for prime time—with the single exception of Cepheid and its platform called GeneXpert.
GeneXpert is a true sample-in, answer-out device. It’s not exactly point of care because it’s not portable, but it can be placed in a delivery room or emergency room or a surgical suite for the sorts of applications that make sense there. Two examples would be testing for group B strep in the delivery room and assessing surgical margins in tumor and lymph node excision in the OR—sort of a molecular frozen section.
Is the center involved in any studies related to reimbursement or economic issues?
CMM is working with two major payers, Priority Health and Blue Cross Blue Shield of Michigan. We’ve made more progress with the former, but I suspect we’ll have the same level of success with the Blues.
We’ve had a very active, very open dialog with Priority Health about the value of AmpliChip for certain subsets of patients. It’s a test that allows physicians to more rationally choose drugs and dosages to manage a patient. The disorder or disease that Priority Health, a local mental health facility, and CMM will be working on together includes depression and other mental illnesses for which drugs are prescribed. These drugs are metabolized by enzymes that are encoded by the genes interrogated on the AmpliChip.
So together we are designing a medico-economic study that will allow us to assess if the test has economic value. We already understand the medical value; we’re just trying to figure out where it fits in with the payment scheme of things.
Would there be a lot of demand for looking at metabolism of those drugs?
If you’re a depression patient, then yes. Any individual candidate for a drug metabolized by those enzymes which are interrogated indirectly by looking at the genes on the chip would be terribly interested in that.
I don’t want to bankrupt the system by suggesting every patient should be tested, because 25 percent of the drugs in any hospital’s pharmacopoeia are metabolized by the enzymes assessed by this chip. But we have to start somewhere, and we chose depression and other mental illnesses.
A lot of patients have unpredictable responses to antidepressants, and might have to try a whole series before finding one that works. Is that the kind of issue this study would address?
Exactly. So we’re going to try to do an economic study that finds real data to determine how many dollars did we waste in managing the patient because the drugs didn’t work and they’re on their third or fourth drug, how many days of work did they miss, what is the lost productivity—those sorts of economic questions.
And how far along is that project?
We are about a third of the way in. We hope that with 100 patients’ worth of data, medical economists at the insurance companies can draw some good conclusions.
How sophisticated are the economic models that would be needed to associate your research findings with payment?
The models are actually quite sophisticated. That’s exactly why I’m partnering with Priority Health and hopefully the Blues, because I know what I know and I know what I don’t know, and I certainly don’t know how to medically/economically model these sorts of questions. But I know from discussions with Priority Health that they have experts to do this sort of thing.
Would insurers have a strong incentive to pay more for the kinds of tests that could perform these kinds of studies?
There are two ways to answer that question. If economic data suggest that the test is beneficial at a certain price, then yes, it becomes easy to convince payers.
Even if the economic data don’t demonstrate that it’s financially advantageous, insurance companies do care about their patients, and if a $900 test, for example, saves $700 worth of drugs (in other words, there’s a net loss of $200), but their patient feels better sooner, I like to think that test is going to happen anyway. And I believe that it would.
Are there comparable types of studies going on throughout the country at payers or other research operations?
On the one hand, I hope so. On the other hand, from a selfish point of view, if we’re the first then that’s a feather in our cap. And if nobody else is conducting these studies, then I hope this article suggests to other parties/ insurers/laboratory partnerships to get on the stick.
How do you see CPT coding evolving to adapt to molecular diagnostics testing in the future?
That’s a hard question. The CPT code approach has to adapt to reimbursement for molecular diagnostic testing. The way by which dollar figures are associated with specific molecular testing CPT codes doesn’t work so well. Some tests are vastly over-reimbursed; some tests are wildly under-reimbursed.
I think the existence of array-based codes is a good step forward. The AMA, which of course puts out the codes, has demonstrated an understanding that arrays are a future part of laboratory medicine, specifically molecular diagnostics.
What has to change is the other half of the equation. Simply publishing a code without associated rational reimbursement is only half the battle. And if you don’t take it all the way across the goal line, then you haven’t scored a touchdown. We need rational, intelligent, data-derived discourse about what an appropriate dollar figure is for the CPT codes. There needs to be more dialog between those who peg a dollar figure to the codes and the practitioners of tests, namely medical professionals, so the right dollar figure—not too high, not too low—is set.
There has been a certain amount of critique about the Food and Drug Administration’s guidance on IVDMIAs (in vitro diagnostic multivariate index assays). Can you comment on that?
I think the FDA is appropriately concerned about new classes of tests coming into the marketplace. IVDMIAs are array-based tests, but the data goes through a “black box” software component so that I as a laboratory director don’t necessarily understand the algorithms or what’s going on with the data to generate the laboratory values that I then provide to my physicians.
And if one subscribes to the notion that the pathologist is the “doctor’s doctor,” that the pathologist acts as a consultant to the physician who orders the test, then I’m a little bit concerned about IVDMIAs, because I don’t feel I can give my physicians a complete story, a complete consult, the complete information.
I think that’s where the FDA is coming from. They want to put the tests through their paces to make sure they’re as robust as they should be.
Some bills on Capitol Hill right now, such as the Genomics and Personalized Medicine Act, address genetic discrimination. What impact might they have on your operations?
I have been involved in discussions on genetic information non-discrimination since 1997 when Rep. Louise Slaughter of New York introduced legislation. It’s 10 years later, and we still don’t have a bill that’s been signed into law to protect individuals against genetic information discrimination. So I’m not concerned [about impact on operations], because when these things become law, hopefully they will be well vetted and there will be lots of input from organizations like the CAP and the Association for Molecular Pathology, so that a bad law isn’t put into effect. In general, what I worry about are onerous, overly burdensome regulations being put on the laboratory when CLIA regulation, combined with CAP’s Laboratory Accreditation Program, provides excellent oversight already. I hope Congress doesn’t send anything up to the president that reinvents the wheel for laboratories that already know exactly what they’re doing and have been doing it for many, many years.
When those laws hit the books I will of course comply with them, but right now I’m interested in providing as much input as possible to make sure they do become the best possible laws.
Frankly, with 2008 being an election year, I can’t imagine you’d have to worry about it before 2009 at the very earliest.
The College is concerned about gene patents that restrict the types of tests pathologists can perform as diagnostic medical procedures. Can you comment on bills now pending in Congress that would prohibit patents from being obtained under certain circumstances?
It’s okay for folks to disagree, but it’s a complicated issue. Patents are legal; patents are the bedrock of our economy. It’s perfectly appropriate to prohibit patenting a gene sequence for all sorts of reasons, but if there is a particular polymorphism or signature or particular misspelling in the code or the gene-expression profile that a company has invested significant dollars and effort and resources into turning into a diagnostic test, I believe they have earned the right to enjoy patent protection.
Does the center have any plans for expansion?
We just doubled the size of the lab in anticipation of all the work we’ll be bringing in. So we have about 3,300 square feet of ultra-modern laboratory space.
We will see how we can do with five med techs and scientists supporting the work, but I expect that with the amount of business development I’ve been doing, and the amount of interest in the marketplace, we’ll have many more than the eight or nine projects I have right now, and the center is only going to be getting bigger.
What is the future of molecular testing?
Molecular diagnostics is poised for continued, excellent double-digit annual growth because of fields like pharmacogenomics, companion diagnostics, and personalized medicine.
I think molecular diagnosticians need to think about the future very carefully. There are a handful of cash cows on the molecular diagnostics laboratory test menu, including HIV viral load, Chlamydia/gonorrhea, cystic fibrosis, HPV, and maybe a couple of others. But beyond those, things move into low-volume, high-cost esoterica.
And if, selfishly, we’re not going to see our tests lost to the microbiology laboratory, then laboratories can look to CMM as something of a model for the future, whereby we are divesting ourselves of the higher-revenue, high-volume tests in exchange for relatively modest and light test menus. That way we can concentrate on developing new tests in conjunction with our academic colleagues, our research institute across the street, or with companies looking for opportunities to partner.
I would tell the readers of CAP TODAY that we’re being very, very successful at that at CMM, and if they want to copy the model, they can. I don’t mind because I think there’s plenty of business to go around, and if molecular laboratories think about those sorts of collaborative opportunities, then they’ll continue to thrive.
Source - ( Dec 01, 2007 )
It’s 2007, and at this point miRNA needs no introduction. Just in case you’re late to the party, though, miRNAs are small, single-stranded nucleotides, which were originally believed to have little impact on gene expression and protein formation.
In addition, miRNAs are accurate biomarkers for numerous diseases and are also involved in the expression of DNA. Scientists are still continuing to discover new roles for these nucleotides. How could we have possibly known that something so fascinating was to be found in the middle of absolutely nowhere?
Having established the importance of miRNA, the scientific community is now racing to identify regulatory miRNAs that control pathways involved in development and disease, and to fully characterize their regulatory behavior.
Because miRNAs are not mRNAs—they are smaller, not capped, and not polyadenylated—existing RNA-analysis tools have had to be adapted to work with miRNA. This includes nucleic acid isolation technology, miRNA detection and amplification technology, technologies for silencing and controlling miRNA signals, and computer software methods for profiling miRNA and analyzing the genome for miRNA sequences.
Qiagen’s (www.qiagen.com) contribution to the miRNA landscape has been in optimizing RNA-analysis tools for the smaller miRNAs—essentially creating lighter weight tackle for catching smaller fish. It has modified its industry-standard solid-phase mRNA purification system to create the miRNeasy system.
A related product, miRNeasy FFPE, enables purification of miRNAs from formalin-fixed, paraffin-embedded tissue samples. This is the form in which hospitals archive patient samples. These archives contain not only diseased tissue, but records pertaining to the treatment and outcome of the disease—a rich resource for mining miRNAs.
Following purification, Qiagen’s miScript system allows for the sensitive detection and precise quantification of hundreds of miRNAs from an RNA sample as small as 1 ng (which can be stored indefinitely for future access once converted to cDNA) via RT-PCR. Although microarrays can be used for analysis, RT-PCR has the advantage of being able to detect low-abundance miRNAs.
According to Eric Lader, director of R&D at Qiagen, no one knows whether extremely low abundance miRNAs are significant. “It might not be a great idea to ignore a whole class of miRNAs expressed at low levels.” RT-PCR picks up these low-abundance miRNAs. Microarray analysis, however, is still a valuable tool for large-scale miRNA profiling.
As Qiagen works to develop its toolbox for miRNA research, one area of development is the technology to identify true mRNA targets of miRNA. Current methods make use of miRNA mimics and inhibitors if there is already a predicted candidate for a miRNA target. However, it is difficult to link miRNA to its target without some prior association.
The biotech tool of RNA interference, or gene silencing, owes its existence to the miRNA pathway, revealed in 1998 by Andrew Fire and Craig Mello. They found that short pieces of double-stranded RNA could inhibit, or silence, genes. This discovery immediately instigated a push for therapeutics and tools to take advantage of the silencing effect. It is ironic, then, that gene silencing can actually be targeted at miRNA, basically turning off the cell’s natural turning-off device.
This was the plan of attack for Peng Jin, Ph.D., assistant professor, and his colleagues at Emory University. Dr. Jin’s team was drawn into the field of miRNA indirectly, via fragile X syndrome, a common cause of mental retardation.
One of the proteins lost in fragile X syndrome is FMRP, which is thought to regulate translation in neurons, and is known to associate with miRNAs as well as Dicer and Argonaute, key proteins in the miRNA pathway. Dr. Jin’s team wanted to know which genes were regulated by particular miRNAs, so they created an assay using siRNA to knock down miRNAs.
Using this method, they observed increases in target mRNA transcripts in response to knockdown of certain miRNAs. In addition to providing a valuable new method for analyzing miRNAs, this work has led to a potential breakthrough. “Basically, what we have taken is a chemical biology approach,” says Dr. Jin. “We have identified certain small molecules that modulate miRNA activity.” These results are not yet published.
The principle of miRNA knockdown can be used in the development of RNAi based therapeutics as well. Alnylam Pharmaceuticals (www.alnylam.com) entered into a joint venture with Isis Pharmaceuticals (www.isispharm.com) to form Regulus Therapeutics (www.regulusrx.com), dedicated to RNAi therapeutics targeted to miRNAs.
Regulus’ most advanced program targets mir122, which is expressed in hepatocytes and is required for replication of hepatitis C virus. “This whole approach opens a new frontier for pharmaceutical research. By antagonizing miRNAs, we can shut down an entire pathway of human disease. miRNAs control groups of genes that tend to be involved in disease. By targeting single miRNAs, we can have an impact on whole networks of genes involved in disease processes,” reports John Maraganore, Ph.D., president and CEO of Alnylam.
Third Wave Technologies (www.twt.com) is focused on nucleic acid testing and diagnostics. It has applied its technology to miRNA analysis with intriguing results. Third Wave’s Invader chemistry is based on a two-step reaction.
In the first step, an oligonucleotide binds to the target sequence, and then a probe also binds to it. In the case of a point mutation or other variation from the reference sequence, a single-base overlap is created. The cleavase enzyme then cleaves off the 5´ flap of the overlapping sequence. The released 5´ flap then binds to another probe, this time labeled with FRET molecules.
Subsequent cleavage of this complex results in a fluorescence signal that can be detected. Since both reactions happen over and over again, the signal from a single miRNA can be amplified repeatedly, leading to ten-million-fold amplification of a single signal sequence. Detection is carried out in real time, or as an endpoint.
“We actually detect the miRNA in the sample without altering the miRNA,” says Hatim Allawi, Ph.D., director of discovery and advanced technologies for Third Wave. Other advantages that Dr. Allawi cites for Invader assays are short assay times—as little as two hours, large (six log) quantitative dynamic range, and the ability to multiplex. Multiple targets can be assayed in the same tube using Invader chemistry.
“Our miRNA Invader assay comes in handy,” notes Dr. Allawi, “when screening through patient samples or clinical samples. Invader is highly quantitative and highly specific. The answer is not a false positive or a false negative.”
One of the most powerful tools of all for analyzing mRNA is the computer. Using specialized algorithms, researchers can analyze gene sequences for miRNAs and predict miRNA targets and pathways. Since unbiased miRNA analysis is difficult to impossible in vitro, a powerful software package to narrow down the hypotheticals is indispensible.
Actigenics (www.actigenics.com), now a division of Cepheid (www.cepheid.com), offers a comprehensive miRNA analysis platform (MiRgate).
Actigenics’ miRNA analysis begins with bioinformatic predicitions of thousands of novel miRNA candidates, followed by a custom microarray analysis to create a microarray expression profile for clinical samples. Then the MiRgate software suite correlates microarray results with significant pathways (using the company’s extensive database), producing a custom report for each sample. MiRgate will also analyze noncoding DNA for miRNAs, search for SNPs, and predict miRNA target sequences.
There is considerable affinity between Cepheid and Actigenics, explains David Persing, M.D., Ph.D., chief medical and technology officer and executive vice president for Cepheid. “We managed to acquire the expertise of a group that was extremely talented at being able to predict the presence of miRNA candidates within the human genome.
“Dr. Bernard Michot, formerly of INSERM, joined us as head of the group in the Cepheid France facility. He and his group of bioinformatics specialists developed a software package that is capable of producing accurate predictions of miRNA candidates and potential diagnostic markers not yet represented within the public domain data set.”
For Cepheid, this was an opportunity to access proprietary diagnostic targets for a broad array of diseases, and for Actigenics, it was an opportunity to commercialize a unique technology.
“We have reasons to believe miRNA markers might be among the most accurate for the diagnosis of cancer, inflammation, and autoimmune conditions such as systemic lupus, and there are a lot of areas yet to be explored by our system,” notes Dr. Persing. “What we expect to get out of it in practical terms is a short list of diagnostic markers that we can incorporate into tests on our GeneXpert diagnostic platform.”
In some cases, Cepheid has identified miRNA targets within regions of amplified and deleted chromosomes in cancers. For example, the region 17q12-22 is commonly amplified in breast cancer. It contains the gene for ErbB2, which is the target of Herceptin.
Cepheid predicted that two miRNAs from the same region would be overexpressed in breast cancer with potential regulatory roles apart from ErbB2 amplification. Overexpression of these two molecules was later demonstrated on microarrays. Interestingly, no miRNAs from the public domain data set were found to map to this region of the chromosome. These miRNAs may themselves be good diagnostic markers and they may in turn point to downstream pathways that may also be good candidates for new companion diagnostics in the mold of Herceptin/ErbB2.
Happily, the greatest obstacle to full exploitation of miRNA biology has been removed—that of simply not knowing it existed. miRNAs have begun to explain mysteries such as the mechanisms of embryonic development and the haywire genetics of cancer. And yet, the mind-boggling complexity of tiny regulatory elements that control hundreds of genes at a time may make us nostalgic for the days when noncoding DNA was junk and gene regulation consisted of a comfortably large protein stationed like a parking lot attendant on top of a very obvious promoter region.
But as with most nostalgia, we’d have to admit that the world is a more interesting place with miRNA in it, and as better tools become available for analyzing miRNA, those dizzying networks of interactions should slowly begin to make sense.
Source - ( Nov 16, 2007 )
A Sunnyvale company's stock and revenue has risen almost as fast as has the fear of the so-called "superbug" for which it's developed and marketed a highly sensitive diagnostic test.
In the six months since Cepheid Inc. Won FDA approval for its GeneXpert test, stock almost quadrupuled from a low of $7.40 to a high of $27.60, and third quarter revenue jumped to $36.3 million -- 53 percent above the same period the previous year.
"We're at the right place at the right time with the right solution," boasts CEO John Bishop. "And the primary driver, of course, is MRSA."
The rise of MRSA, or methicillin-resistant Staphylococcus aureus, has been one of the big medical panics of the year to date. It is now officially responsible for more than 94,000 serious infections and nearly 19,000 deaths a year, and according to a much-discussed article in the Journal of the American Medical Association, the death rate is expected to soon exceed that from HIV/AIDS.
Fear stems in part from the fact that the organism is a strain of a pervasive bacterium long associated with first-line antibiotics such as methicillin and amoxicillin. Though the most resistant strains have turned up in hospitals, a small but growing number are emerging in community settings.
Bishop says he himself acquired the infection at his Sunnyvale gym in late August.
"I evidently got it on my hands, took my shoes off at my domicile, didn't wash my hands first and got it on my foot. Within five days, it was looking pretty ugly."
After testing positive at company headquarters, the CEO went to a hospital emergency room where doctors listened to his account and then prescribed a combination of two powerful antibiotics -- Keflex and Bactrim. Even then, he says, the infection took 17 days to clear.
"It's important to get very fast and accurate diagnosis when you're dealing with MRSA," says Bishop. "The reason is because you need to treat patients appropriately, immediately. Which means we have to equip emergency rooms and get results within an hour; 24 or 48 hours later is not good enough."
Not quite so fast, says Dr. Henry Chambers, chief of infectious disease at San Francisco General Hospital, professor of medicine at UCSF, and a nationally recognized expert in the field.
It's not good enough to identify MRSA in a nasal swab, Chambers says, because, simply put, the mere presence of MRSA doesn't translate to infection.
Between 1 million and 10 million Americans, and 10 percent of Bay Area residents, walk around every day with MRSA in their nasal passages. But while they are colonized with MRSA, only a fraction are actually infected with MRSA. "There's an assumption that knowledge of nasal colonization is tremendously helpful," says Chambers, who laughingly refers to himself as "Cepheid's marketing nightmare."
"The question is what do you do with this information. You don't necessarily treat nasal colonization, because it's a normal condition that's present in a third of all people.
Yet that is precisely what the Veterans Affairs Healthcare System is doing, after infectious disease doctors in Pittsburgh discovered that they could slash MRSA rates by more than 70 percent after instituting a program to immediately identify and quarantine all MRSA patients.
Now, officials are rolling that program out at VA hospitals across the U.S. About 77 of them use Cepheid's GeneXpert, a molecular diagnostic test that analyzes genetic material to identify MRSA.
The equipment, which costs hospitals about $130,000, works like this: A nasal swab is dropped into a tube with a reagent, shaken up, placed in an eyedropper and then put into a cartridge that fits in a machine. The answer is delivered in about an hour.
"The beauty of GeneXpert is that it takes the technology and makes it transparent to the user," says Bishop. "Any medical professional can run the test, you can be fully trained to run this in 15 minutes."
In the Bay Area, MRSA has become so embedded that Chambers says he no longer waits to culture a blood sample before beginning presumptive treatment in patients with signs of infection.
"If you were sufficiently ill, there is no way I would withhold treatment," he says. Even, he says, if a nasal swab was negative. And that's because the organism can hide out in other areas of the body besides the nose.
Bishop takes exception to such presumptive therapy, arguing that it gives rise to even worse resistant organisms. "We're going to use the few antibiotics we have left if we use them on an indiscriminate basis," he says. "You can't just go out there and blast with a shotgun, you have to use a rifle and be very specific."
Source - ( Nov 01, 2007 )
by Mike May
In the late 1800s, Samuel Siegfried Karl Ritter von Basch invented the sphygmomanometer the analog device most medical professionals still use to measure blood pressure. Thus came the detection and quantitation of a significant, early biomarker. Over the last two centuries, many more biomarkers emerged, including today's sophisticated molecular biomarkers, a key ingredient of modern biotech and pharma. "Right now, biomarkers lie very close to the top of the value chain," says David Persing, chief medical and technology
officer at Cepheid in Sunnyvale, California. All companies use biomarkers. Hanno Langen, head of Roche's proteomics initiative in Basel, Switzerland, points out a wide range of possible applications, from risk assessment to diagnostics. "For example, a biomarker might tell you how a patient is likely to respond to a particular drug," he says. Moreover, biomarkers can be used early in drug development to predict toxic side-effects, and they can be used as diagnostics to assess a patient's condition.
In the October 2007 issue of Clinica Chimica Acta, for example, a team of scientists from McMaster University in Hamilton, Ontario, searched for biomarkers that would identify patients at a high risk of further problems, such as myocardial necrosis, after a visit to the emergency room with acute coronary syndrome. Such biomarkers could point out the patients who need the most aggressive treatment. These scientists assayed specimens from more than 200 patients, looking at 11 markers. One panel of potential biomarkers-interleuken-6, a version of brain natriuretic peptide (NT-proBNP), and E-selectin-picked out 60% of the patients with myocardial necrosis. As the authors conclude: "A biomarker panel analyzed early after pain onset can identify individuals at risk for both myocardial necrosis and the combined endpoint of death/myocardial infarction/heart failure." Nonetheless, the authors note that more research must be performed.
To be effective, though, biomarkers must be incorporated in a complete research program. As Persing says, biomarkers form the tip of a pyramid based on technologies needed to detect the biomarkers and the building blocks needed to use them, whether they are DNA, microRNA (miRNA), messenger RNA (mRNA), single nucleotide polymorphisms (SNPs), or the proteins encoded by these molecules. As a result, this field depends on increasingly sophisticated technology.
To identify proteins that could be biomarkers, scientists often turn to mass spectrometry (MS). Although one round of MS can reveal the components of a sample, two rounds of MS, or tandem MS, give an even more-complete view. In this so-called multiple reaction moni
In many cases, biomarkers mean proteins-such as prostate specific antigen (PSA), which has long been used as a market of prostate cancer-but many other types of molecules can be used. For example, scientists explore miRNA for biomarkers. These molecules participate in the regulation of gene expression, and they might sidestep some of the problems with using genes as biomarkers. "Thousands of genes change expression levels in cancer," says Persing. "It makes it very difficult to identify the genes that are most critical for distinguishing different types of cancer or determining the likelihood of progression." So Cepheid scientists focus on miRNA. "The data suggest that miRNAs are potentially less susceptible to individual variation," says Persing, "and they could also be more reliable as diagnostic biomarkers in general, in comparison to gene-based assays." Cepheid uses novel bioinformatic algorithms to mine the human genome for new miRNAs, and it is currently evaluating over 3,000 miRNA candidates. Among these, Cepheid scientists have identified hundreds of novel miRNAs that look like promising biomarkers for several forms of cancer, including breast, lung, and prostate.
Other targets are also gaining attention. At SGX Pharmaceuticals in San Diego, California, scientists see great promise in inhibiting MET, a receptor tyrosine kinase. It plays important roles in ordinary cell growth, proliferation, and motility, but it can also contribute to primary tumors and the onset of metastasis. "There are mutations of MET that are inherited in the germ line," says Stephen K. Burley, SGX's chief scientific officer and senior vice president, research. "This phenomenon gives rise to the hereditary form of papillary renal cell carcinoma." Even in the absence of germ line mutations, Burley adds that, "virtually all papillary renal cell tumors show evidence of MET activation."
MET also plays a role in subsets of other cancers. For example, about 20 percent of patients with non-small cell lung cancer show evidence of addiction to the MET oncoprotein (without that protein, the cancer dies). Consequently, a MET-related biomarker say, DNA, RNA, or a protein could be used as a diagnostic that determines which cancer patients should be treated with a MET inhibitor, such as SGX523, for which SGX plans to start Phase I studies in early 2008.
Source - ( Oct 28, 2007 )
Cepheid Inc. First made its name with anthrax-detection tests to guard the nation's post offices from bioterrorism attacks back in 2001.
Now, the Sunnyvale company's gene-based testing system is becoming part of an escalating nationwide defense against the deadly "superbug" called MRSA, which is often innocently spread by well-meaning health care workers.
Cepheid won Food and Drug Administration approval in April for its test for a life-threatening, antibiotic-resistant form of the common staph bacterium, a strain that now kills more people in the United States each year than the AIDS virus. The microbe, methicillin-resistant Staphylococcus aureus, or MRSA, can cause stubborn skin eruptions, pneumonia and bloodstream infections that can be fatal unless an effective antibiotic is given in time. Methicillin, penicillin and related drugs are powerless against the germ, so doctors must use alternative antibiotics such as vancomycin.
Just how widespread MRSA has become was revealed this month when scientists at the Centers for Disease Control and Prevention published results of a large-scale surveillance study based on samplings in the Bay Area and other regions. The researchers estimated that 94,360 cases of invasive MRSA infection occurred in the United States in 2005, resulting in 18,650 deaths.
Most testing for the aggressive staph germ takes 24 hours or longer, because technicians must wait for the microbe sample from a patient's nasal swab to grow in a culture dish. Cepheid reduced that time to 72 minutes with a test that recognizes the DNA signature of the resistant germ, said Cepheid Chief Executive Officer John Bishop. Patients who test positive can start treatment sooner with an antibiotic that still works against the bacteria, he said. The MRSA test is processed by the company's GeneXpert system, the automated analyzer also used with its anthrax test.
The scale of the demand for Cepheid's MRSA tests will depend on the outcome of an active debate among hospital and public health officials over the best methods to protect people from the dangerous microbe. Some experts say a significant portion of patients admitted to a hospital should have their noses swabbed for testing, said Dr. John Jernigan, a CDC medical epidemiologist and MRSA expert.
The germ, which spreads easily by contact with another person's skin or with contaminated objects such as bedrails and sheets, is a serious hazard for hospital patients. It can infiltrate the body through surgical wounds, catheters and other means.
The majority of MRSA cases originate in hospitals and health care facilities among patients with weakened immune systems and other risk factors. But the germ also can be brought into the hospital from outside. The recent CDC study found a significant incidence of MRSA exposure among healthy people who congregate in schools and other community centers.
Even so, some health care officials say mass testing at the point of hospital admission is not necessary if strict sterile procedures are used with all patients, Jernigan said. That includes old-fashioned measures such as frequent hand-washing and gown changes by health care staff.
Cepheid testing systems are in use under two contracts with the Department of Veterans Affairs, which just announced stringent new MRSA screening requirements at all 153 of its hospitals. All patients admitted to intensive-care units and other high-risk wards are tested, although the department does not mandate the use of rapid diagnostic systems such as Cepheid's.
In addition to its MRSA test, Cepheid sells two other clinical infectious disease tests for the GeneXpert system, and plans to introduce many more, Bishop said. But he said alarms over MRSA are already increasing demand for the instruments, and the company's manufacturing units are gearing up to meet it.
"MRSA will be the biggest driver of sales in the near term," he said. Cepheid's smallest GeneXpert analyzer costs $27,000. The price per MRSA test is $42.
Caris & Co. Analyst Zarak Khurshid estimates that Cepheid could reap $21 million in sales of the analyzer systems and nearly $33 million in MRSA test components in 2008.
"The business is growing extremely fast and could outstrip those estimates," Khurshid said. Caris & Co. Makes a market in Cepheid shares. In 2006, Cepheid's biothreat detection products contributed 58 percent of the company's $87.3 million in revenue.
The life-threatening form of the common "staph" bacterium is part of a larger class of drug-resistant infections created by the widespread use and overuse of antibiotics.
When such drugs are first introduced, they can stop the growth of nearly all the individual microbes causing an infection. But the antibiotic also gives a survival edge to mutated forms of the germ that can thrive despite the treatment. Over time, the antibiotic-resistant strains make up a larger and larger percentage of the bacterial population.
In 1974, methicillin-resistant Staphylococcus aureus infections accounted for only 2 percent of total staph infections, the CDC estimates. In 1995, that figure rose to 22 percent. Now, MRSAs account for more than 60 percent of staph infections.
It's natural to ask why doctors don't use alternative antibiotics such as vancomycin against all staph infections, given that patients with the resistant germ can worsen or die if their treatment begins with a drug that doesn't work, said Dr. Paul Akins, director of the neuro intensive-care unit at Kaiser Permanente in Sacramento.
But he said blanket use of alternative antibiotics would foster the development of germs resistant to those drugs as well, leaving doctors with fewer remaining weapons against MRSA. Vancomycin resistance is already showing up in staph cultures.
Several companies are trying to create a new generation of antibiotics against the microbe, including Theravance of South San Francisco. Last week, Theravance said the FDA has asked for further information about the clinical testing and manufacturing of its experimental antibiotic telavancin. The agency indicated it might approve the drug after reviewing the additional data.
Akins has been studying methods to reduce MRSA infections in his intensive-care unit, including screening all patients who are admitted to the unit.
He uses the old culture plate test, but he was intrigued by a description of Cepheid's rapid test. "It could be cost-effective," he said. Complications of MRSA can be pricey and dangerous to correct, he said.
On the other hand, the older lab tests have some advantages when it comes to choosing the right alternative antibiotic for each MRSA-infected patient, he said.
The culture dish includes small pieces of paper, each soaked in one of 10 or 15 different antibiotics. The microbes grow all over the papers containing drugs that won't work for that patient, he said. Akins chooses a drug that stops the microbe colony at the edge of the paper.
Source - ( Jan 14, 2007 )
Chris Somerville is energy agnostic.
That might sound odd for someone now heading the Energy Biosciences Institute, a research center supported by $500 million from oil giant BP plc. But while the EBI is starting its alternative energy work with a focus on biofuels, Somerville, a microbiologist by training, remains a strong believer in the potential of geothermal, wind and solar.
No one should discount the range of possibilities as researchers seek cleaner, more efficient and less costly ways of powering the world, he said, and the institute -- already the target of criticism because of its Big Oil connection -- shouldn't be pre-judged.
"We're not proposing a silver bullet. We're proposing magic buckshot," Somerville said. "We really need a lot of parallel approaches."
That, friends and colleagues said, is vintage Somerville -- focused but open to a range of possibilities and opportunities. And that, they said, is why Somerville has had uncustomary crossover success between the academic and business worlds.
Somerville is the first director of the EBI, based at the University of California, Berkeley. He has started three Bay Area companies and yet remains firmly planted in academia, formally moving from Stanford University to Berkeley this month with his EBI appointment.
Through it all, Somerville has focused on how plants can produce low-cost chemicals and other materials that can help the environment and people across the globe.
"There are very few people who on the one hand remain close to the science and at the other end have the interest or the capacity to develop the science in practical ways," said Chiron Corp. Co-founder Bill Rutter, who Somerville counts as a mentor.
Somerville, 60, may have been the best person for the EBI job, based only on his startup experience. LS9 Inc. is using synthetic biology to develop straight-to-the-tank biofuels; Poetic Genetics Inc. has outlicensed its technology for hemophilia treatments to Pain Therapeutics Inc.; and Mendel Biotechnology Inc. has won multimillion-dollar investments from BP and Monsanto Co. as it develops a seeds business for cellulosic biofuel crops.
Somerville has divested all but founder shares in the companies and has severed other ties to meet UC conflict of interest rules.
Somerville's interest in business came about accidentally more than a quarter century ago. A friend who operated a small, struggling chemical company casually mentioned his business' plight to Somerville and his wife, Shauna, an expert plant breeder. Shauna Somerville wrote a check that day, instantly making the Somervilles the fifth-largest shareholders in the company.
Raylo Chemicals ultimately became the largest privately held chemical company in Canada. The Somervilles held their stake for 10 years, until the company was sold in 1990 to Laporte Industries Ltd. Raylo was bought last year by Foster City's Gilead Sciences Inc.
That experience, learning the ups and downs of business, would come in handy as Somerville research evolved into his first company, Mendel.
Somerville in 1996 led a worldwide group that coordinated the sequencing of the genome of arabidopsis, a weedy plant that is a biological model for corn and wheat. The publicly released data was rich, and Somerville and a cadre of a half-dozen scientists seized in 1997 on parts of it to start Mendel.
After the company's first CEO failed to work out, Somerville filled the job part time for five years, eventually hiring and working with Neal Gutterson, Mendel's current CEO.
The company, now shifting gears from arabidopsis to cellulosic fuel feedstocks, this year attracted investments from BP and longtime partner Monsanto.
The initial arabidopsis research work had attracted top-line talent, including Samir Kaul, then a scientist at the J. Craig Venter Institute in Maryland.
Kaul, who headed the plant's genome sequencing work, didn't have a doctorate degree like other researchers. He remembered, however, that Somerville backed his leadership of the unit. "Chris said, 'He's delivering and performing ahead of schedule and ahead of budget,'" Kaul recalled.
Now a general partner at Khosla Ventures -- one of venture capital's biggest cleantech proponents -- Kaul a few years ago visited Somerville's offices at Stanford University, where Somerville led the Carnegie Institution's plant biology unit.
LS9 was born as a result.