Monday, November 26, 2012

Outbreaks of Foodborne Illnesses Are Becoming Harder to Detect, Even Though Rapid Methods are Being Utilized

New diagnostic tests for common foodborne pathogens such as Salmonella, Campylobacter, and Escherichia coli may hinder the ability of public health officials to detect multistate outbreaks. The problem is an inability to trace contamination to its source.

In 2009 Alicia Cronquist, an epidemiologist with the Colorado Department of Public Health and Environment noticed that several rural clinics in her state had switched from traditional laboratory tests that relied on growing a culture to rapid nonculture tests. In the past, when patients were suspected of having certain foodborne illnesses, doctors routinely sent a stool sample to a laboratory, which detected a range of potential bacterial culprits. (Some foodborne infections, like Listeria, are diagnosed with blood tests.) An isolate, or sample of the bacterial colony at fault, would then be forwarded to local, state or federal officials, who had the DNA tested to determine the organism's specific strain. The telling DNA sequence, or "fingerprint," was entered into the PulseNet system so that public health officials could see if samples from other newly diagnosed patients matched the information in the database. Analysis of when and where people contracted an infection of that specific strain can help lead to the source of contamination, allowing investigators to remedy the situation.

But, Cronquist says, over the course of a year, a clear shift in the types of tests being run in local labs had resulted in much less information being shared with her department. "We saw our surveillance data changing, and by 2010, almost 15 percent of total case reports were using the nonculture tests," she says.

The new tests do have a lot going for them. They provide quicker results to the physician and patient. They are often less expensive and, in some cases, may not require a stool sample at all. What is more, some of them can spot pathogens that the culture-based tests do not and diagnose more infections.

Tennessee's state epidemiologist Timothy F. Jones notes, for instance, that culture tests for E. coli look for the 0157 strain, which is among the bacteria that produce the Shiga toxin; that strain infamously accounted for the outbreak of food poisoning from spinach this year. "With the new rapid tests," he says, "we can actually detect the whole class of Shiga-toxin producing bacteria. The rapid test is detecting additional bacteria we would have missed before."

But adoption of the new tests has meant that health officials, like Cronquist, are not always getting the isolate required to do the DNA fingerprinting that is needed to help identify a source of contamination, such as E. coli in lettuce or salmonella in raw spinach.

In that way, Jones says, "these rapid tests put us back where we were when we didn't have the ability to do [DNA] fingerprinting."

The trend is particularly worrisome because other ways of protecting the public from foodborne illnesses are also stumbling. According to the Centers for Disease Control and Prevention, one in six Americans (or 48 million) become sick from a foodborne disease each year and 3,000 die. A study released late last month by the U.S. Public Interest Research Group indicated the problem is not improving in part because laws like the Food Modernization Safety Act continue to languish in the White House's Office of Management and Budget. And a federal monitoring program—the U.S. Department of Agriculture's Microbiological Data Program, which tested produce for pathogens—began to be shut down on November 12. 

Jay M. Lieberman, medical director of infectious diseases for Quest Diagnostics, which serves approximately half the physicians and hospitals in the U.S., says that the wide adoption of the new tests means that public health officials will need to come up with new ways to monitor and respond to new outbreaks. For example, health officials will need to work with the labs to figure out how to get an isolate or find another way to characterize pathogens.

"By connecting cases, we can find problems in the food supply we might not have found," says John Besser, deputy chief of the CDC's Enteric Diseases Laboratory Branch. "The challenge for us is to develop a test that will provide all the information that we need without going to the isolate step. That's a significant challenge, but we all believe it's doable."

At present, no one is working on developing a test that can help public health officials trace outbreaks, though several companies continue to develop the new nonculture tests, including Abbott Laboratories, BD, Cepheid and Luminex. Besser says he expects laboratories to quickly adopt them when they become available. For the labs, the new tests mean quicker, cheaper results requiring fewer highly trained staff.

Although the problem of tracking pathogens related to foodborne illness is new, similar concerns were raised when nonculture tests were developed for detecting the sexually transmitted disease gonorrhea.

"When it comes to gonorrhea, almost all testing is done by nonculture techniques now," explains Quest's Lieberman. In response to that sea change, the CDC set up a surveillance project that allows it to monitor trends in a new way. At specified labs in 28 cities across the U.S., cultures are run and isolates taken from samples collected from the first 25 men found to have urethral gonorrhea each month. With that limited data set, public health officials can still track outbreaks while also allowing for broader use of the new nonculture tests.

Although that procedure has worked well for gonorrhea, whether it will also work for foodborne illnesses remains an open question.

An irony of all of this, says the CDC's Besser, is that the new tests for foodborne pathogens may be better than the old ones, but if they disrupt the public health systems, they "could result in a lot more people getting sick. That is the unintended consequence."


Monday, November 19, 2012

Ion/Molecule Reaction Mass Spectrometry Identifies Gram-Negative Bacteria

MALDI-TOF mass spectrometry is now being used to identify microorganisms, and this is based on an accurate molecular weight measurement and characterization of the organism's biomolecules, including proteins, peptides, polysaccharides and nucleic acids. Following microbial growth, approximately 105 cells are added to a target plate (usually stainless steel), and following the addition of an energy absorbing matrix, the mixture is ionized, and the ionized particles are accelerated in an electric field. The molecules are separated according to their mass to charge ratio, and the resulting mass spectrum is compared with an internal identification library or database.

Ion/molecule reaction mass spectrometry examines volatile or gas-phase compounds, and scientists in Germany and the UK are now using this technique for the rapid identification of Gram negative bacteria. The following excerpt from is an overview of their work.


Critically ill patients in hospitals can be severely affected by infections, which slow down their recovery or, in extreme cases, cause death. A recent international study found that the mortality rate in intensive care units rose from 11% in non-infected patients to 25% in those who had been infected. The majority of the infections (64%) were of respiratory origin and 62% of these were with Gram-negative bacteria.

It goes without saying that rapid identification of infection could be a lifesaver, helping the clinician to prescribe the appropriate antibiotics. However, the conventional microbiological tests can take several hours or days to complete, by which time any infection will have taken a stronger hold.

One alternative route being explored in recent years involves the volatile metabolites that are emitted from bacteria. There have been numerous reports outlining the use of GC/MS to distinguish between various microorganisms and these have led to direct mass spectrometry techniques like proton transfer reaction mass spectrometry which can produce results much quicker than GC/MS.

Now, a team of scientists in Europe has developed a method to discriminate between bacteria using ion/molecule reaction mass spectrometry (IMR MS) as part of a broader programme to investigate the use of volatile compounds for the diagnosis of infections in vivo. Their method has the advantage of providing results within three minutes and is suitable for automation, a big plus for clinical testing labs.

Michael Dolch and colleagues from the Ludwig-Maximilians-University of Munich, Germany, V&F Medical Development GmbH, Absam, Austria, and Avacta/Oxford Medical Diagnostics Ltd, Oxford, UK, published details of their new procedure in Journal of Applied Microbiology.

Ion/molecule reactions

The team concentrated on seven Gram-negative bacteria that were prevalent in patients with nocosomial pneumonia. They were grown on blood agar plates and their volatile metabolites were measured at various times by IMR MS using headspace analysis. The ion/molecule reactions were initiated using mercury ions which were allowed to react with the volatiles to produce positive ions for detection.

A set of 65 m/z values were selected for monitoring based on preliminary work. The identities of some of the peaks were confirmed by comparison with the IMR mass spectra of authentic compounds, including ammonia, hydrogen sulphide, propene, acetyl group, acetaldehyde, ethanol, methanethiol, acetone, propanal, toluene and indole.

The raw data signals were normalised against that of isoprene as a reference gas. Then peaks were marked as originating from the microorganisms if they exceeded a threshold intensity that was 100% different from the background noise of the broth solutions. These peaks were processed by principal components analysis to see if the bacterial species could be distinguished.

Positive identification of Gram-negative bacteria

No peaks reached the threshold value after 4 hours of incubation but all species showed significant increases for some masses after 8 hours and the peaks at m/z values of 105, 106 and 107 showed decreased abundances. These are likely to be benzyl derivatives but their structures were not elucidated.

After 24 hours of culture, the IMR mass spectra were sufficiently developed to allow the seven bacteria to be distinguished. Any combination of the first three components in the PCA of the signals allowed discrimination equally between Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, Proteus vulgaris and Serratia marcescens.

The clusters in the PCA plots for Enterobacter cloacae and Klebsiella oxytoca overlapped significantly. However, the introduction of minor spectral differences, namely the presence of peaks at m/z 29, 60 and 79 from K. oxytoca and their absence from E. cloacae, allowed their differentiation in a separate PCA analysis.

So, seven Gram-negative bacterial species can be detected after only 8 hours of growth and distinguished from each other after 24 hours of growth. Their early identification will allow timely intervention with a treatment program to combat their growth and protect the patients.

The rapid test is complete within three minutes using a process that is amenable to automation. “Currently no such diagnostic tool exists, but we certainly hope it will in the future allowing for species differentiation even outside the usual working hours, and thus shorten the time required for adaptions of antibiotic treatment in response to microbial testing results,” said Dolch.

Tuesday, November 13, 2012

USP 1116: Points to Consider and the Role of Rapid Methods

At a recent PDA workshop on the revised USP Chapter 1116, Microbiological Evaluation of Clean Rooms and Other Controlled Environments, a number of speakers provided an overview of the changes but more importantly, the regulatory perspectives on the new informational monograph. With the introduction of real-time rapid microbiological method (RMM) technologies that are intended to detect, size and enumerate both total particulates and viable microorganisms in air, USP 1116 may be a welcome change, as these RMM’s are capable of continuous monitoring, single cell detection and enhanced trending of environmental monitoring (EM) data. Obviously, the ability of these systems to continuously trend non-zero results, especially in an ISO 5 or Grade A environment, may be worthwhile to the industry, and is relevant to the teachings of USP 1116.

One of the speakers at the workshop, Dr. David Hussong, Associate Director for New Drug Microbiology at FDA/CDER, stated that random detectable counts are to be expected even in the most controlled manufacturing areas, especially from operators, equipment, and possibly other sources. However, any excursions from expected EM specifications or acceptable levels are considered deviations that indicate a problem. Furthermore, operational data should establish a normal or expected baseline, and when low level or infrequent counts are expected, charting or trending of the data will certainly help to understand the overall control of the area over time. For example, this would help to visually demonstrate that three positive “hits” over three days is a worse trend than three positive “hits” over a month. This is where USP 1116 can come into play.

Dr. Hussong explained that the purpose of the new USP chapter is to deal with low numbers of recovery in cleanrooms. USP 1116 allows to trend and demonstrate process control, and to satisfy regulatory requirements that demonstrate process control, in addition to signaling operators when the process drifts away from established control parameters; however, it should be understood that the methods and acceptance criteria are to be established for each facility’s needs. Dr. Hussong stated that EM data:

  • Are NOT product release criteria
  • Are useful, but should be weighed against risks associated with the operations (e.g., a higher number of personnel or more manipulations)
  • Are needed to understand process control for aseptic filling operations, terminally sterilized product lines and non-sterile manufacturing
  • Should provide suitable alert and action level responses, and that excursions should have appropriately graded responses which are not necessarily an indication of an out-of-specification (OOS) finding
I had the opportunity to ask Dr. Hussong how the FDA views USP 1116 in light of existing regulatory expectations for using specific control levels for EM within controlled and classified manufacturing areas. Here is a brief summary of the Q&A exchange.

Q: Does the FDA currently expect firms to continue to meet existing levels for EM as specified in FDA’s aseptic processing guidance for industry?

A: Yes.

Q: Would this be applicable for all classified areas, including ISO 7 and 8?

A. Yes.

Q: Will the FDA allow companies to move toward trending (as described in USP 1116) and completely abolish the quantitative action/alert levels for EM?

A. Yes, but only with data to support this change.

Q. You represent the Review side of CDER. Does the Compliance side of CDER also accept the recommendations provided in USP 1116?

A. Grudgingly they do, but there are ongoing discussions between both sides.

Personally, I believe that trending in our cleanroom and controlled areas provides a more realistic understanding of environmental process control, and by implementing the continuous and real-time monitoring capabilities of laser induced fluorescence RMMs will help the industry in meeting the spirit of USP 1116. However, I caution the reader of this blog that the teachings of USP 1116 may not be widely accepted by all regulators, especially outside of the U.S. Additionally, there are some statements within the chapter that the industry should consider very carefully before implementing all of the recommendations as provided in USP 1116.

For example, USP 1116 states that “Excursions beyond approximately 15 cfu recovered from a single ISO 5 sample, whether from airborne, surface, or personnel sources, should happen very infrequently. When such ISO 5 excursions do occur, they may be indicative of a significant loss of control when they occur within the ISO 5 critical zone in close proximity to product and components. Thus, any ISO 5 excursion >15 cfu should prompt a careful and thorough investigation.” In all honesty, it is unclear to me how a level of 15 cfu was chosen to represent a loss of control in a critical manufacturing area. Why wouldn’t a level of 12, 10 or even 8 cfu be considered as indicative of loss of microbial control, especially when these levels are observed in a critical location? A very recent discussion I have had with a European regulator on this very subject confirms my notion that the chapter suggestions may not be universally accepted.

Therefore, firms should review and understand all of the chapter recommendations and their potential quality and regulatory impact(s) prior to removing their EM specifications or levels, and to include a trending strategy, in addition to their current methods, in order to accumulate the necessary data and scientific evidence that could allow them to follow the essence of USP 1116 at some point in the future.