Comment on a Recent RMM Vendor Roundtable Discussion

In the most recent European Pharmaceutical Review (2013, Vol. 18, Issue 4), two articles provided discussion on rapid microbiological methods (RMM) in the pharmaceutical industry. The first article, “The rapid microbiological methods revolution,” written by Pfizer’s Emanuele Selvaggio, offers an overview of RMM implementation strategies including technology and cost considerations.

The second article describes a roundtable discussion that included responses from questions presented to four RMM vendors. Questions focused on perceived hurdles for RMM implementation, validation challenges, available guidance for statistical analysis and how the industry can accelerate RMM adoption and implementation.

Unfortunately, responses provided by some of the vendors demonstrated a general lack of understanding of current regulatory expectations and quality initiatives, as well as what existing validation guidance documents offer the industry.

For example, when discussing perceived hurdles for RMM implementation, Carrene Plummer of Azbil BioVigilant responded “the greatest hurdle is the lack of well-defined guidelines and regulatory expectations” and that “existing documents aren’t in step with RMMs evolution, either not considering RMMs or not addressing distinctions between them.” Plummer further stated “pharma companies express reluctance in making modifications without knowing if their validation approach will find regulatory acceptance...exacerbating the uncertainty is the ill-alignment of regulatory bodies in what is required for validation.” Jörg Stappert of Greiner Bio-One indicated “manufacturers of biologics are quite reluctant in establishing RMMs due to the lack of clear validation and/or acceptance criteria for these methods.”

Some of these responses are far from the truth and do nothing but continue to perpetuate myths and misconceptions associated with the validation and implementation of rapid methods within our industry. There are well-defined guidelines that address distinctions between methods, validation strategies and the use of statistics, and these are getting better (e.g., the revision to PDA Technical Report No. 33, due to be published within the next month). It should also be noted that for a number of years, companies have successfully validated and implemented RMMs using the guidance provided in TR33, USP 1223 and Ph. Eur. 5.1.6.  More importantly, the FDA, EMA and other worldwide regulators have put initiatives and policies in place that encourage the implementation of RMMs (e.g., EMA’s post-approval change management protocol, FDA’s strategic plan that includes the implementation of RMMs, and recent changes to the sterility testing of biologics as specified in the U.S. Code of Federal Regulations). Microbiologists representing these same regulatory authorities have also communicated the quality and technical advantages of RMMs over classical methods at professional meetings and in print. Also, the statement that biologics manufacturers are reluctant to establish RMMs is a gross distortion of the facts.  Biologics manufacturers have widely implemented RMMs for in-process in addition to finished product testing (e.g., sterility).

As for regulators accepting validation strategies and approaches, numerous firms have already implemented a variety of RMM technology platforms, such as ATP bioluminescence and solid phase cytometry, as alternatives to compendial methods such as the sterility test. And these same multinational firms have obtained approvals from multiple regulatory authorities, demonstrating that the “ill-alignment of regulatory bodies” may not be as apparent as this article suggests.

Next, the participants stated that existing guidance on the use of statistical methods is, in general, at a high-level and do not apply to specific RMM technologies. I don't disagree with this position; however, it is impossible for any single guidance document to address every existing or potential future technology and what statistical method may or may not be appropriate for use. Fortunately, the revision to PDA TR33 will provide enhanced guidance on the use of statistics during the validation of RMMs, and many of these recommendations originated from firms who have used these same strategies for methods that have been approved by worldwide regulatory authorities and are being used today.

Finally, the vendors commented that the industry should continue to work closely with regulators in developing a meaningful validation and implementation strategy. I agree. Open and honest dialogue between all parties is key to success. Darrick Niccum of TSI summed it up nicely when he stated, “only through open discussion and scientific analysis will appropriate applications for each RMM be identified and promulgated.”

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Doctors Misdiagnose Dengue as Cyst

Fever, along with excruciating abdominal pain, forced a 27-year-old woman to undergo a surgery for ovarian cyst. However, post-operation, she found out that the pain had nothing to do with the cyst, it was actually a manifestation of dengue.

The reason behind such wrong detection, say doctors, lies in the lack of uniformity in the diagnosis procedure of dengue.

A week ago, Priya Kadam, a school teacher, started getting fever, along with severe pain in her abdomen. Her family took her to a nearby hospital, where she was admitted, but the doctors apparently could not detect anything wrong with her. But as the pain continued, they assumed that it was because of either appendicitis or an ovarian cyst.

"Her sonography reports showed everything normal except for a small cyst in her ovary," said Manisha, Priya's sister-in-law. "Priya is newly married. Doctors worried that there must have been some gynaecological problems with her. As the doctors could not diagnose what was causing the fever and the pain, they assumed that the cyst was the reason." Manisha is also a nurse by profession.

Within two days, doctors operated upon her and removed the cyst. After remaining in post-operative care for a day, Priya again started feeling uneasy. It was then that she was shifted to Kohinoor Hospital in Kurla and the doctors there diagnosed that the abdominal pain was a symptom for dengue.

Physician at Kohinoor Hospital Dr Amol Manekar, who treated Priya, said her condition was very bad when she was brought to them on Friday. "Her haemoglobin and white blood cell count had gone up, while her platelet count was down. Her urine output was low and her liver and kidneys had started to fail. But low platelet count is a sure presentation of dengue, and that is what we tested her for," he said.

But the result came negative for dengue in the rapid screening test. But based on clinical diagnosis, doctors still started treating her for dengue. "It is common for us to find atypical symptoms of dengue in patients at the fag end of monsoon. But not testing for dengue and instead, removing a cyst affected Priya's immunity system," said Dr Manekar.

Pointing out that no uniformity is followed in the diagnosis procedure of dengue, Dr Jayanti Shastri, head of microbiology department at civic-run Nair Hospital, said, "PCR test is conducted in the initial phase of the ailment. It is provided free of cost to civic patients. The antibody tests have limited value and certain kits detect both antigens and antibodies. But the problem is that diagnostic centres in the city use various test kits, which may give false positives or false negatives." "The rapid tests are quick-fix methods and cost less. But sometimes they can be inaccurate, and may show false negatives, even when the doctors can see that the person concerned is suffering from dengue. So it is always better to go through an ELISA test, a confirmatory procedure. But the rule has to be standardized," said Dr Manekar.

After being treated for dengue, Priya's condition has stabilized but she is still in the intensive care. "Had she not undergone the operation for cyst, she would have been strong enough to fight dengue on her own," said Manisha.

Source: The Times of India

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Join Us in the Rapid Micro Methods LinkedIn Group!

We have an active rapid microbiology discussion group on LinkedIn with more than 3,600 members! Many different industry sectors are represented, including pharmaceuticals, biotech, food and beverage, clinical and diagnostics, environmental, water and even homeland defense. We discuss existing and newly introduced rapid method technologies, applications, validation strategies, regulatory expectations and implementation success stories. You can join the Rapid Micro Methods discussion group by clicking on the following link: http://www.linkedin.com/groups?gid=3028275.

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Speedy Breedy Rapid Respirometer Added to Our RMM Matrix

Our rapid methods (RMM) Product Matrix page has just been updated to include a portable, rapid method that is based on measuring pressure changes as a result of growing microorganisms. The Bactest Speedy Breedy system detects pressure changes in a closed culture vessel when microorganisms respire. Applicable to aerobes, facultative anaerobes, anaerobes, microaerophilic bacteria and yeast, pressure changes are monitored continuously and data is presented in real-time. General media or selective media may be used, the latter for detecting specific microorganisms.

The RMM Product Matrix catalogues more than 60 qualitative, quantitative and microbial identification systems, detailing scientific principles, workflow, applications, time to result, throughput, sample size or type, sensitivity levels and organisms detected. The rapid methodologies discussed have been validated and implemented in a wide variety of industries, including pharmaceuticals, food and beverage, environmental, water, clinical and diagnostics, personal care and homeland defense.  

Our RMM Product Matrix may be accessed at http://rapidmicromethods.com/files/matrix.php.

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Dameron Hospital Uses MALDI-TOF Mass Spec for Rapid Diagnosis

Among the primary tasks for any physician is diagnosing what ails us. To do it properly, they need information - observing the patient, obtaining vital signs such as heart rate and blood pressure, and getting accurate results from lab tests.

Those lab tests often take time. That's time added to the patient's suffering and time that costs the doctor, clinic or hospital money. But that is changing for the better, thanks to new technology.

About a year ago, 202-bed Dameron Hospital in Stockton invested $250,000 to $300,000 in a mass spectrometer system called the Vitek MS, which helps the hospital's clinical laboratory scientists to detect in minutes unusual organisms that often took days to diagnose.

Dameron is one of the first hospitals in the nation and the only one with less than 500 beds to have this advanced system, according to the lab's administrative director, Richard Wong.

On Aug. 21, the U.S. Food and Drug Administration gave its approval for the Vitek MS to be marketed for automated identification of bacteria and yeasts that are known to cause serious illnesses and infections. It can identify 193 microorganisms and can perform up to 192 tests in a single automated series of testing, with each test taking about one minute.

The FDA's Alberto Gutierrez said "the ability for laboratories to use one device to identify almost 200 different microorganisms is a significant advance. ... Rapid identification of harmful microorganisms can improve the care of critically ill patients."

Prior to the FDA approval, Dameron conducted an in-house trial of the system, providing clinicians with presumptive test results while training four of its lab scientists in the highly precise techniques required to prepare specimens for testing.

"It's made a big difference in timeliness," said Michael Glasberg, Dameron's chief operating officer.

Abby Adesanya, Dameron's assistant pharmacy director, said that "studies have shown the sooner you can get the right medication to the patient, the better the outcome." In practical terms, the new system will cut by half the time it takes for the pharmacy to recommend the proper medication a patient should be taking for an infection, for example.

While the emphasis is on speedier results, accuracy is not being compromised, according to Dameron microbiology supervisor Joanne Gonsalves. Most test results are completed with 99.9 percent accuracy. "Anything less than that we verify with another method," Gonsalves said, noting that even with retesting, the system provides results much quicker than traditional testing.

Compared to other identification methods that require abundant organism growth for testing, mass spectrometry requires only a small amount of yeast or bacterial growth, so testing can start as soon as growth is visible, generally within 18 to 24 hours. Traditional methods can take up to five days to produce the same identification results.

The Vitek MS can identify yeasts and bacteria that are associated with skin infections, pneumonia, meningitis and bloodstream infections. People with compromised immune systems weakened by HIV/AIDS, cancer treatment or anti-rejection therapy following an organ transplant are particularly vulnerable to these infections.

The Vitek system uses a laser to break yeast and bacteria specimens into small particles that form a pattern unique to the microorganism. The Vitek MS then automatically compares the microorganism pattern to the 193 known yeasts and bacteria in the test system's database to identify the microorganism.

Glasberg said the hospital's goal in investing in the latest laboratory technology is about "getting the right medication to the right patient in a timely manner."

All hospitals, according to Wong, are seeing a "tremendous increase in the demand for fast, accurate lab testing. Like all hospitals, we're seeing more and more patients with multidrug-resistant infections, so it's critical to do all we can to identify these patients quickly so that the clinical staff can contain these infections and expedite treatment."

Wong said the cost to treat a single antibiotic resistant infection can run from $18,000 to $29,000 while the patient remains hospitalized for an additional one to two weeks.

"While some of this additional cost is reimbursed, much of it is not. If Vitek MS helps Dameron contain the spread of serious infections, it will quickly pay for itself. More importantly, this technology can help us treat patients more accurately and rapidly, avoiding unnecessary pain and suffering."

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Color-Changing Dots Detect Blood-Borne Bacteria

A team at the University of Illinois has developed a cheap disposable device containing a chemical sensing array (CSA) that can rapidly identify bacteria from the signature chemicals that they give off.

The new device consists of a plastic bottle, small enough to fit in the palm of a hand, filled with nutrient solution for bacteria to grow. Attached to the inside is an array of 36 pigment dots that change colour in response to chemicals released by bacteria. The device combines amplification of bacteria with detection and identification in a single sealed bottle.

A blood sample from a patient is injected into the bottle, which goes onto a simple shaker device to agitate the nutrient solution and encourage bacterial growth. Any bacteria present in the blood sample will grow and release a signature odour that changes the colours of the pigment dots. The results can be read in a pattern of colour changes unique to each strain of bacteria. The device was tested with nine microorganisms, including two strains of E. coli and two strains of E. faecalis.

The new test produces results in 24 hours, compared to as much as 72 hours for current tests. It is also suitable for use in developing countries and other areas that lack expensive equipment in hospital labs.

“We have a solution to a major problem with the blood cultures that hospitals have used for more than 25 years to diagnose patients with blood-borne bacterial infections,” said Dr James Carey, who presented a report on the device at the 246th National Meeting & Exposition of the American Chemical Society (ACS). “The current technology involves incubating blood samples in containers for 24-48 hours just to see if bacteria are present. It takes another step and 24 hours or more to identify the kind of bacteria in order to select the right antibiotic to treat the patient. By then, the patient may be experiencing organ damage, or may be dead from sepsis.”

Sepsis, or blood poisoning, is a toxic response to blood-borne infections that kills more than 250,000 people each year in the United States alone. The domestic healthcare costs to treat sepsis exceed US 20 billion. In such a medical emergency, every minute counts, Carey explained, and giving patients the right antibiotics and other treatment can save lives.

Carey said the new device can identify eight of the most common disease-causing bacteria with almost 99% accuracy under clinically relevant conditions. Other microbes can cause sepsis, and the scientists are working to expand the test’s capabilities. But Carey said the device could make an impact now in reducing the toll of sepsis, especially in developing countries or other medically underserved areas.

“Our CSA blood culture bottle can be used almost anywhere in the world for a very low cost and minimal training,” Carey noted. “All you need is someone to draw a blood sample, an ordinary shaker, incubator, a desktop scanner and a computer.”

The group's research was also published in the Journal of the American Chemical Society. Clicking on the journal's link will download the paper.

Source: MTB europe

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Genome Sequencing Aids Hunt for Deadly Bugs

Public-health investigators were alarmed last year when they tried to solve an outbreak of a dangerous superbug at a large Denver hospital.

Eight patients had been infected with a strain of klebsiella pneumoniae bacteria that was resistant to nearly all antibiotics. To stop it, the disease detectives needed to know where and how it was spreading. But the patients had been all over the hospital, from operating rooms to intensive care. Standard lab tests showed the cases were related to one another, but offered no clues as to how the people had been infected.

So the disease detectives turned to a technology like the one used to decode the human genome. In a laboratory at the Centers for Disease Control and Prevention in Atlanta, scientists sequenced the bacteria samples' entire DNA. They found tiny mutations that the bacteria had made as they moved from patient to patient. That helped them divide the patients into smaller clusters and pinpoint transmission to two intensive-care and two other units of the hospital. New steps were taken to prevent the spread of infections in those units.

"We were able to clarify a lot that we could not otherwise," said Erin Epson, a CDC disease detective working at the Colorado Department of Public Health and Environment and a member of the investigative team.

Used for years now in research and academia, whole-genome sequencing has become faster and cheaper, allowing it to be deployed more widely. It is a powerful new weapon that public-health scientists have begun turning to in a bid to outflank deadly microbes emerging around the world.

Public-health leaders and scientists say decoding dangerous pathogens could revolutionize the fight against outbreaks.

"We can stop outbreaks quicker and figure out ways pathogens are spreading that we don't currently know," said Thomas Frieden, the director of the CDC, which is building its capacity to sequence and analyze pathogens.

President Barack Obama's administration is seeking $40 million in fiscal 2014 for an "advanced molecular detection" initiative to expand the CDC's capacity to sequence and analyze pathogens after a panel of experts determined the agency was behind with the technology. But it isn't clear whether Congress will approve the funding for the agency, whose budget was cut by $580 million to a total of $6.29 billion this fiscal year.

Sequencing a whole genome allows scientists to identify quickly how virulent a bug is and what drugs it is resistant to. They can see how it is mutating and evolving as it jumps from one person to another, allowing them to track—and, hopefully, stop—an outbreak in real time. "The accumulations of mutations in an organism are like its history," said Duncan MacCannell, senior adviser helping lead the CDC's advanced molecular-detection work.

Federal and state officials are experimenting with whole-genome sequencing to get to the bottom of food-borne outbreaks and avoid fingering the wrong source. Using current lab methods to analyze salmonella, "it's hard to say it came from this lettuce and not that spinach," said Jill Taylor, interim director of the Wadsworth Center, New York state's public-health lab, which is working with the Food and Drug Administration to hone use of the technology. "With next-generation sequencing, you can really see the person has the same isolate [sample] as came from that lettuce. It's a much better tool to be able to say what the source was for an outbreak."

Last month, investigating an outbreak of listeria, CDC scientists compared sequenced bacteria from the suspected source, cheese, and found it to be "indistinguishable" from isolates from several suspected cases, said Peter Gerner-Smidt, branch chief of the agency's division of food-borne, waterborne and environmental diseases. They also sequenced isolates of two patients they weren't sure were part of the outbreak, he said. One was, he said.

Tuberculosis officials are working on ways to use next-generation sequencing to more rapidly identify drug-resistant forms of the disease, which are dangerous but often take weeks to confirm with current tests.

Meanwhile, scientists say the technology also helps them more deeply probe flu viruses. Life Technologies Corp., a maker of next-generation sequencing technology, set up a global influenza network earlier this year to sequence more samples once they are taken from patients, in the hope of detecting emerging strains earlier. "You're able to sample more, so you get a better idea of what is the most prevalent strain for the flu season," said a company spokesman.

Source: Wall Street Journal

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Rapid Test Aims for Quicker Notice of Beach Water Bacteria Levels

Each day, environmental health specialist Stan Sherman scoops water from several of 28 beaches in New Hanover County, places the samples on ice and transports them to a testing facility in Wilmington.

"You take part of the sample and empty it into a sterile vial, and add growth media," said Sherman, who works for the state Division of Marine Fisheries. "That's basically a mixture that's supposed to enhance the growth of bacteria. Then we incubate it at a certain temperature and look to see if there's been growth after 24 hours."

The test determines whether the water contains unsafe levels of enterococci, a fecal bacteria found in the intestines of warm-blooded animals that can indicate the presence of other disease-causing organisms. If the results indicate heightened bacteria levels, state officials will pose a swimming advisory at the affected beach - but because the test takes a full day to complete, those advisories are always based on yesterday's water samples.

"Basically, you've gone to the beach, gotten into the water, there was no sign, and then the next day you hear there's a beach advisory," said Rachel Noble, a professor of environmental biology at the University of North Carolina. "The sample you're hearing about the next morning was taken yesterday when you were swimming."

Noble's aiming to fix the lag using a method devised several years ago that returns water quality results in three to four hours, allowing state officials to post beach advisories on the same day the samples were taken. Using a process known as quantitative polymerase chain reaction, or qPCR, Noble's method also tests for enterococci. Because qPCR uses DNA markers to confirm the presence of the bacteria, results are available in two to three hours.

"That's not perfect. We'd love for it to take five to 10 minutes, but it's an improvement," she said. "Using the rapid method, you can take a sample at 7 or 8 a.m. and the results are posted at the beach by 11, by the time families and most people are putting their towels down."

The rapid testing method is in use at various places across the country, as far north as Racine, Wis., but implementing the system along the North Carolina coast is more complicated.

Water quality officials have been trained to use the process, but uncertain funding makes it difficult to standardize across the state.

"I think we would all like to do it. The problem is the money," said J.D. Potts, director of the state's recreational water quality program. "It costs a lot more, and your lab has to be equipped to run qPCR."

Purchasing the proper equipment and outfitting a lab would cost between $50,000 and $60,000, Noble said, a decrease from several years ago, when it would have run around $100,000.

"The cost of running individual samples is beginning to approach the same cost as the traditional methods, so that's one big advancement," she said. "We're getting to the point where, minus the capital cost, the price of the new and traditional methods are similar."

To implement the rapid method, the state would also need approval from the federal Environmental Protection Agency. At the beginning of this year, EPA recommended that states use rapid testing methods to measure enterococci, but the agency specifically endorses its own, similar process. To petition for statewide usage, officials would most likely have to present EPA with data showing that Noble's method is more user-friendly.

"If somebody takes our method and generates the data themselves and finds it's easier to use, then they'll begin to use it," Noble said. "At this point, adopting the method is really a matter of resources."

Source: StarNews Online

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