We Have Updated Our Rapid Micro Methods News Page!

Great news for those of you who follow our rapid micro methods news page! We have just competed the migration of all of our news articles into a more user friendly format, which is very similar to our current RMM blog. As of today, all of our 2012, 2011 and 2010 news articles and press releases are now available for your review. Using this new format, there are a number of novel features that you can now utilize:

• Review a list of the most popular-read news articles
• Send articles to other social networks
• Provide your comments
• And in a few weeks (after the new pages are indexed), you will be able to search the entire news archive 

Additionally, all of our future news articles will now be automatically uploaded to our Twitter and Facebook pages. If you haven't already done so, I encourage you to follow us on Twitter and/or Facebook for a "rapid" way to stay in touch with what's happening in the world of RMMs.

Please visit our updated News Page now by clicking on the link at the top of this page!

Bacteria Will Prevent the End of the World

It's December 21, 2012. The sun is shining in Florida and there are no earthquakes, asteroids, solar storms, colliding planets or planetary alignments. However, the temperature did dip into the 40's last night, and for some Floridians, that meant the end of the world as we know it.

During this year I have blogged on the advancement of rapid microbiological methods (RMM) and the introduction of novel technologies for the rapid detection of organisms in a variety of industry sectors, including pharmaceutical, environmental, clinical and food. For the most part, these systems are utilized to detect the microorganisms that can cause us harm and where they are coming from. And in light of today's supposedly end of the world prophecies, here are a few bad-bug scenarios worth noting:

1. Nickel-eating bacteria may have worsened the world's worst mass die-off by producing huge amounts of methane, a new study suggests. The study is the latest attempt to explain how most of the world's ocean species died off in just a few hundred thousand years at the end of the Permian era, about 250 million years ago. The researchers presented their findings earlier this month at the annual meeting of the American Geophysical Union. The study proposes that a series of steps caused the mass extinction, but that bacteria played a key role. First, massive volcanic activity in Siberia released nickel into the atmosphere, which somehow reached the ocean. As a result, populations of ocean-dwelling bacteria that use nickel in their metabolic pathway exploded, releasing huge amounts of methane into the atmosphere and depleting ocean oxygen levels as a byproduct of that metabolism. Because methane is a greenhouse gas, the catastrophic gas release trapped heat in the atmosphere and caused the mass extinction by making the climate uninhabitable.

2. As bacteria evolve to evade antibiotics, common infections could become deadly, according to Dr. Margaret Chan, director general of the World Health Organization. Speaking at a conference in Copenhagen earlier this year, Chan said antibiotic resistance could bring about “the end of modern medicine as we know it.”

“We are losing our first-line antimicrobials,” she said Wednesday in her keynote address at the conference on combating antimicrobial resistance. “Replacement treatments are more costly, more toxic, need much longer durations of treatment, and may require treatment in intensive care units.” Chan said hospitals have become “hotbeds for highly-resistant pathogens” like methicillin-resistant Staphylococcus aureus, “increasing the risk that hospitalization kills instead of cures.”

Indeed, diseases that were once curable, such as tuberculosis, are becoming harder and more expensive to treat. Chan said treatment of  multidrug resistant tuberculosis was “extremely complicated, typically requiring two years of medication with toxic and expensive medicines, some of which are in constant short supply. Even with the best of care, only slightly more than 50 percent of these patients will be cured.” Antibiotic-resistant strains of salmonella, E. coli, and gonorrhea have also been discovered.

“Some experts say we are moving back to the pre-antibiotic era. No. This will be a post-antibiotic era. In terms of new replacement antibiotics, the pipeline is virtually dry,” said Chan. “A post-antibiotic era means, in effect, an end to modern medicine as we know it. Things as common as strep throat or a child’s scratched knee could once again kill.”

The dearth of effective antibiotics could also make surgical procedures and certain cancer treatments risky or even impossible, Chan said. “Some sophisticated interventions, like hip replacements, organ transplants, cancer chemotherapy and care of preterm infants, would become far more difficult or even too dangerous to undertake,” she said.

All things considered, it looks like microorganisms can play a role in armageddon. Not so fast my friends. The folks at Cracked.com have put things into perspective. Here is an overview of their thoughts on how bacteria can actually prevent the apocalypse from happening....

Not all bacteria is of the flesh-eating, "kill it before it kills you" variety. Some of it is actually good for you. Maybe you've seen the commercials where it helps people's gastrointestinal flow, for instance.

But even more beneficial than that, there are some strains of bacteria that not only can perform massive, superhero-level feats, but are probably going to be what stands between us and an apocalyptic future. Here are six examples.

#1. Controlling the Weather

If you've ever been unlucky enough to get caught in a hailstorm then you probably know that it's pretty painful. And while at the time you may have been rhetorically asking the heavens why they were so insistent firing tiny balls of ice at your face, now you have an actual answer: Nearly 85 percent of all hailstones have bacteria at the center.

The bacteria is called Pseudomonas syringae and when it's kicked up into the air, it collects condensation fury, forming water droplets (or in the case of hail, painful pellets). Generally the moisture in clouds needs something to cling around in order to create precipitation, and the bacteria provide the perfect nucleus.

So how does this information help us? Well it could allow us to weaponize water, destroying the windshields of our most hated enemies. Or we could use it to stop droughts.

We know that this particular bacterium causes water to freeze about 7 degrees Fahrenheit higher than usual, which means it can create snow and ice in slightly warmer temperatures. For that reason, mountain resorts have been using Pseudomonas syringae to make fake snow since the late '80s. But for anyone who's not jetsetting to Aspen this winter, there are more practical applications as well. Scientists say it's possible that planting crops already infected with these bacteria may help overcome droughts by inducing rain.

The bacteria is whipped up into the air the same way the pollen of plants would be, except once it climbs high in the atmosphere, the Pseudomonas syringae encourages rain in the area. Even if you're not on board with the idea of genetically engineering plants to be infected with bacteria, researchers think that just planting crops that encourage the bacteria would have a similar effect. And likewise, planting crops that the bacteria doesn't like might encourage droughts. So there might come a day when solving the world's water problems is just a matter of ordering up some manipulative microbes.

#2. Colonizing Other Planets

An unspoken rule among party-goers is that anyone left at the end of the night helps to clean up a little, so naturally everybody tries to leave early to avoid dealing with the mess. Well what if we could apply that same lack of accountability to Earth? If we never solve the energy crisis and don't get a handle on greenhouses gases, it may be possible in the future to just leave for another planet. Surely we won't mess that one up, right?

Apparently, a bacteria called Deinococcus radiodurans is going to help us along the way. This organism, which is nicknamed "Conan the Bacterium" is famous for its ability to not die. It shrugs off an astronomical amount of radioactivity, up to 1.5 million Rads, which is equivalent to 1.5 million Teenage Mutant Ninja Turtles, or 3,000 times more radiation than it takes to kill a human. It's also about 750,000 times more than the maximum daily measured radiation on Mars. See where we're going with this?

You may know that "terraforming" is the process of making another planet, like Mars, more Earth-like before we even get there. Obviously that's difficult if not impossible if the work involves millions of humans in bulky suits and hundreds of ships taking them back and forth. But microbes like Deinococcus radiodurans open up the possibility of sending a bunch of them spilling out onto the Martian surface and letting them do the work for us. For instance, even if Mars had our atmosphere, we couldn't grow plants there because the soil is toxic. But we have decoded the genome of Deinococcus radiodurans, and therefore could one day engineer a version that would change the composition of the Martian soil, making it plant-friendly.

But there are broader applications; just examining the way the microbe resists damage from radiation tells us a lot about how to engineer other things to do the same (radiation is normally harmful because it damages DNA, and Deinococcus radiodurans has a mechanism for rapidly repairing that damage). We could theoretically do everything from engineering goods to survive the long trip through space to genetically altering the astronauts themselves to be impervious supermen or superwomen.

#3. Eating Battleships

The biggest problem with ships is that when they're old and unusable, there's nowhere to retire them other than the bottom of the ocean. The U.N. estimates that over 3 million ships are located on the ocean floor, with fewer than a thousand that anyone has any plans to clean up. And that's not even covering all the defunct oil rigs down there. As cool as it might be to go exploring sunken battleships on the bottom of the sea in search of treasures and corpses, what we really need is a way to clean up the mess.

Enter Halomonas titanicai, a bacterium that loves eating metal and could do all the cleaning up for us. The Titanic sank in 1912, where it sat undisturbed for over 70 years. Well, "undisturbed" isn't entirely accurate. During that time, a bacterium sprouted colonies all over the vessel and they are eating the ship. This bacterium adheres to metal, then create rust knobs which appear to be slowly devouring the ship. It's good news for anyone who is mildly interested in ocean health, but sadly, bad news for anyone who was substituting an old cruise ship for an actual relationship.

Because of the great work the bacteria is doing on the Titanic, researchers don't see any reason we can't use the same cultures to clean up other oil rigs and ships. Or, conversely, knowing exactly how the bacteria eats away at metal can inform how we build boats in the future so that they are stronger. By finding a way to prevent this bacteria from colonizing, we can ensure that oil rigs stay structurally sound for a lot longer. Then again, if one of them collapses and it's resistant to the bacteria, then we're right back where we started with steel trash on the floor of the ocean.

#4. Fixing Obesity

Your intestines play host to about 500 different species of bacteria, and that's a good thing because bacteria break down and absorb the food our bodies can't digest alone. The last thing anyone wants in their belly is all the food you ever ate ever, right?

Recently, scientists have found that when we eat a high-fat, sugar rich diet, we not only pack in the calories, we also encourage the growth of bacteria called Firmicutes in our intestines, which happen to love Pringles and Twix bars. They love fatty foods so much that they devour it, breaking the compound down until it is sure to be absorbed by the body.

In a study that changed the diet of lab mice from low-fat, plant based meals to fatty foods, the mice picked up a new set of bad bacteria overnight and started packing on the pounds. They even stayed fat after switching back to low-fat foods. So that's the bad news. Bad bacteria makes you fat. Here's the good news: Good bacteria makes you skinny! Surprise!

Daily intake of a unique lactic acid bacteria was shown to keep the fat-loving bacteria away, which is great news for people who despise the idea of working out. Scientists tested the effectiveness of Lactobacillus plantarum HEAL19 by feeding it to baby rats every day, even before they were born. Then those rats were fed some high fat, fast-foodish diet and despite enjoying fatty foods, the rats with the lactic acid bacteria living inside their gut stayed leaner.

So, the theory goes that by intentionally ingesting something Lactobacillus regularly and from an early age, you can allow this stuff to colonize inside of you like tiny but stern diet camp councilors that constantly keep obesity in check.

#5. Cleaning up Oil Spills

Thanks to recent oil spill tragedies, you might have already known that there's a bacterium that eats oil. What you probably didn't know was that the bacterium is called Alcanivorax borkumensis. It is pretty rare in unpolluted waters, but once an oil spill occurs, it shows up like a superhero. We'd equate it to Aquaman, but technically the bacteria is just a little bit better at its job. The microbes start multiplying quickly in the event of a spill, fattening themselves on oil ... but only to a certain point. The BP oil spill overwhelmed the existing oil-eating bacteria and they weren't nearly as helpful as they were for the Exxon Valdez spill in 1989. But some people think they have the answer to that problem. With fertilizer. Specifically, nitrogen and phosphorous. The same stuff that they're putting on the (nonorganic) crops that are feeding the world could also fertilize the bacteria that loves oil. Adding fertilizer to the water increases the size and number of the bacteria so there are more out there to cut through all that oil.

Still, the process hasn't exactly been tested in deep waters yet, and one of the main consequences could be an overwhelming amount of algae (which also loves fertilizer). That kind of thick marine vegetation could overpower more fragile species. Still, it's been proven to work well near shores and can still do a whole lot of good.

#6. Turning Greenhouse Gases into Bricks

We don't need to explain why CO₂ is a problem for the environment right now -- it's why the nations of the world are scrambling to reduce emissions before we fry ourselves into a Venusian nightmare.

A lot of potential CO₂/global warming fixes are on the table, but sometimes just overcoming your enemy isn't enough. Sometimes you want to destroy them and use pieces of them in your buildings and roads just to remind yourself how superior you are. Well it turns out we can do just that with carbon dioxide.

There are two ways that nature deals with CO₂: either through photosynthesis in plants which turns out oxygen, or through bacteria which convert CO₂ into solid calcium bicarbonate. That kind of "air into stone" transformation sounds like alchemy, but about 40 percent of the chalk cliffs in the world are created by carbon dioxide absorbing microbes. Not only are oxygen and calcium bicarbonate less threatening for humanity and for the world, they are also completely useful to us.

A group of Indian scientists have discovered bacteria capable of creating building materials out of the carbon dioxide in the air, essentially creating a chemical reaction that turns a gas into a separate, solid compound. A rock-solid compound.

We can use calcium bicarbonate in building materials, agricultural lime, the purification of iron and even antacid tablets. The group of scientists speculates that modern factories could include bacteria chambers to convert the CO₂ before it leaves the building. This would allow a factory to produce massive stores of calcium bicarbonate while giving off only low levels of dangerous emissions, thus killing two birds with one chalky stone.

In Conclusion

So, there you have it. The world isn't coming to an end today, microorganisms may actually help us more than hurt us, and I have another year to blog about rapid methods!

Wishing you and yours a very enjoyable holiday season, and a very happy and healthy New Year!

Our Latest Newsletter is Now Available

Our final Newsletter of the year is now available for your review. The Newsletter will keep you informed of updates to our RMM News, Calendar of Events, Blog, Published References and RMM Technologies pages. 

To receive our Newsletter, please sign up by clicking on the following link: http://rapidmicromethods.com/newsletter/. We will then email the Newsletter directly to your inbox!

Approval for Rapid Diagnostic for Foodborne Pathogen has Ramifications for Healthcare

A Philadelphia life science startup with a pocket-sized diagnostic assay for detecting foodborne pathogens has received accreditation from the Association of Analytical Communities for a test for bacteria most commonly found in chickens - Campylobacter, according to a company statement. The test has ramifications not only for reducing healthcare costs, but also for how healthcare providers identify hospital-acquired infections.

The Campylobacter bacteria affects 2.5 million people each year in the US.

Invisible Sentinel’s Veriflow test uses a molecular detection system designed to comply with stricter food testing standards. Its designed to speed up the time it takes to identify whether samples are contaminated. It is also intended to make it easier to use and more easily transported. The assay utilizes a PCR detection method coupled with a rapid, visual, flow-based assay that develops in 3 minutes post PCR amplification and requires only 24 hours of non-specialized incubation for maximum sensitivity. The Veriflow™ Campylobacter system eliminates the need for microaerobic chambers, gel electrophoresis or fluorophore based detection of target amplifications, and does not require complex data analysis.

Once the AOAC greenlights a performance tested method, it is recognized by the US Department of Agriculture, the Food and Drug Administration, certain European Union counterparts and other global regulatory agencies.

Ben Pascal, the CEO, said its manufacturing facilities in the University City Science Center is up and running and the company is talks with distribution partners in the US and Europe. The Campylobacter assay is the first in a suite of tests that are expected to be rolled out out next year for listeria, E. coli and Salmonella. The company expects to more than double its staff of 12 next year, to support sales and marketing.

Pascal told MedCity News earlier this year that its diagnostic platform could be used to detect hospital acquired infections. Such a move would require securing 510(k) approval from the U.S. Food and Drug Administration, but Pascal said the design of its diagnostic platform means that when the company is ready to shift its attention to HAI, it could make such a submission in a relatively short timeframe.

Detecting Biothreats, Faster and Cheaper

Scientists at the Texas Biomedical Research Institute (TBRI) have created a fast and efficient way of developing tests for potential bioterror agents. The technique, published on November 5, 2012 in Scientific Reports, quickly identifies antibodies that recognize bacterial toxins or viral proteins in a few days, using simple equipment found in most facilities around the world.

This technique is “more suitable for resource limited laboratories” than traditional methods that require expensive equipment like chromatography systems,  said Kim Janda, a chemist from the Scripps Research Institute, who was not involved in the study. “I think it will find ample use in other laboratories in the future.” 

Currently, to find antibodies that recognize potential biological threats—a key step towards developing effective diagnostics—scientists start with a large panel of possible antibodies, and gradually isolate those that recognize a given target. It is a laborious process—each round of screening can identify hundreds of antibodies, which have to be individually purified using large cultures and expensive equipment like chromatography systems. The whole process can take months.

“I was faced with this dilemma of deconvoluting hundreds of antibodies, and didn’t want to spend a year purifying the darn things,” said TBRI’s Andrew Hayhurst , who studies ways to detect biothreats.

So, together with TBRI colleague Laura Sherwood, he devised a solution. He starts by making extracts from different strains of E. coli, each engineered to produce a slightly different antibody. Each extract is loaded into a separate well on a large plate. The antibodies all have a molecule called biotin on their tail, which binds tightly to neutravidin, a protein that coats the wells.

Then, Hayhurst adds the target. It sticks to some of the immobilized antibodies, but not others. He adds another round of antibodies that bind to the immobilized targets. Finally, he applies another batch of neutravidin that sticks to the second layer of antibodies, this time labeled with a tag that can be made visible to the naked eye, such as a fluorescence marker. He rinses the plate to remove any loose tags, and simply looks for which wells are producing the right color, indicating the antibodies in that well successfully recognized the microbial target. 

Hayhurst and Sherwood used their method to identify pairs of antibodies against two potential bioterror weapons - botulinum toxin, one of the most potent known bacterial poisons, and Ebola virus. By rapidly finding antibodies that bind to such target, scientists could quickly develop tests for them. “It’s an environmental surveillance tool,” said Hayhurst.

The same method could also be used to develop new diagnostic tests for “virtually any target,” Hayhurst said. For the moment, he is focusing on known biothreats, but the technique could eventually be help create a rapid response to an unfamiliar threat used in an attack, he said. “The knowledge gained in targeting the known will be of use in smoothing the path to targeting the unknown.”

The system is incredibly simple, and takes very little equipment. All the E. coli strains can be grown in milliliter-sized table-top cultures. “Anyone in the world can just do this on their bench,” said Hayhurst. “It’s so simple, there’s no witchcraft involved. It’s about the cheapest way of doing this possible.”

When the cultures are ready, it takes just an hour to screen hundreds of antibodies at once. The technique immediately focuses an experimenter’s attention on the effective antibodies, bypassing the need to purify them. And once the right antibodies have been identified, Hayhurst can produce them en masse using the same engineered E. coli strains. “You can immediately start cranking out milligrams of antibodies,” he said.

“I am extremely excited by these innovations,” said Paul Gulig, a microbiologist from the University of Florida who was not involved in the study. “They streamlined many things.”

L. J. Sherwood & A. Hayhurst., “Hapten mediated display and pairing of recombinant antibodies accelerates assay assembly for biothreat countermeasures,” Scientific Reports, doi:10.1038/srep00807, 2012.

Source: http://www.the-scientist.com.

Veredus Introduces the Vere MTB Chip for Fast Diagnosis of TB

Tuberculosis (TB) is a common, and in many cases lethal infectious diseases caused by various strains of mycobacterium, usually Mycobacterium tuberculosis complex. It is spread through the air when people who have an active MTB infection cough, sneeze, or otherwise transmit their saliva through the air. Most infections in humans result in an asymptomatic, latent infection, and about one in ten latent infections eventually progress to active disease, which, if left untreated, kills more than 50% of those infected.

Global prospects for TB control are challenged by the emergence of drug-resistant strains, especially those that are multidrug resistant (MDR) and extensively drug resistant (XDR). Soon after anti-TB drugs became available in the 1940s came reports of drug resistance among patients undergoing treatment. The prevalence of TB resistance to a single drug was continuously on the rise in several parts of the world, and eventually in the early 1990s, multiple converging factors led to an explosive emergence of MDR-TB, defined as resistance to the two most effective first-line anti-TB agents, Isoniazid and Rifampicin. In 2010, there were an estimated 650 000 cases of MDR-TB.

The threat of tuberculosis has propelled the need of a new tuberculosis diagnostic test system that should be able to do fast and reliable detection and identification of Mycobacterium tuberculosis complex, with the capability to differentiate it from clinically relevant non-tuberculous mycobacterium species as well as detecting drug resistance especially multidrug resistance.

Singapore based Veredus Laboratories announced the launch of its VereMTB™ multiplexed lab-on-a-chip for the detection of various mutations of mycobacterium responsible for causing tuberculosis as well as nine other similar clinically interesting mycobacterium. The chip identifies the specific mycobacterium within three hours after being presented with a sample of coughed up direct sputum.

The technology doesn’t require culturing the bacteria, a slow process that can extend into days when rapid detection is key. The VereMTB is a nucleic acid-based, Lab-On-Chip (LOC) device which combines multiplex PCR and microarray hybridization assay to detect, differentiate and identify:10 different mycobacterium strains with special emphasis on Mycobacterium tuberculosis Complex (MTBC) and its Resistance to Rifampicin and/or Isoniazid from Pulmonary Clinical Specimens or Cultivated Samples (MDR-TB).

Based on STMicroelectronics’ technology, the VereMTB chip is currently undergoing evaluations by the Chinese Center for Disease Control and Prevention in Beijing, China as part of their ongoing program to assess new technologies for TB diagnostics. According to the 2012 World Health Organization report on TB, India and China combined have almost 40 percent of the world’s TB cases, and nearly 60% of multi-drug resistant cases in 2011 were in India, China, and the Russian Federation.

“At the main CDC National TB Reference Lab in Beijing, we have been evaluating VereMTB using samples, collected from across China with a special interest in detecting challenging multi-drug resistant strains that are difficult to detect using other methods,” said Professor Zhao Yanlin Director of National TB Reference Laboratory and Vice Director of the National Center for Tuberculosis Control and Prevention at the Chinese Center for Disease Control and Prevention. “The speed, accuracy and comprehensiveness of the results have been very promising. We look forward to continuing our collaboration with Veredus for new breakthroughs in diagnosing TB.”

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."

Source: http://news.yahoo.com/

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 http://spectroscopyNOW.com is an overview of their work.


Background

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.

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.

PDA Microbiology Conference Update: Real Time Microbial Detection and Quality Control

Rapid microbiological methods (RMM) are utilized for a variety of applications, including in-process bioburden testing, environmental monitoring and finished product release for both sterile and non-sterile product. Recent advances in optical spectroscopic techniques now allow for the real-time detection, sizing and enumeration of microorganisms during volumetric air sampling. For the very first time, one of today’s sessions brought together three technology innovators that provide this type of instantaneous microbial detection capability. Following an introduction into the need for real-time monitoring that will support a QbD and PAT model for the parametric or real-time release of aseptically-filled, each innovator discussed the scientific and quality benefits, as well as contamination control opportunities that their technologies can provide.

I opened the session with a future vision for the real-time release of aseptically filled product. To realize this vision, I discussed the need for RMMs to be incorporated into the manufacturing process stream, providing real-time and continuous monitoring of in-process bioburden (e.g., pre- and post-filtration, and especially at the point of filling), in addition to real-time environmental monitoring. Additional considerations included the utilization of a robust manufacturing barrier system (e.g., an isolator or closed RABS), which would eliminate human-borne contaminants, as well as the incorporation of advanced aseptic filling technologies, such as blow-fill-seal and closed-vial filling technologies.

The next three presentations focused on real-time viable air monitoring technologies that are currently available to the pharmaceutical industry. Brief summaries and conclusions from each speaker are provided below.

IMD-A Systems for Instantaneous, Data-Rich Detection. Scott Morris, Applications Engineering Manager, BioVigilant

• A technical overview of two IMD-A instruments, validation data and potential applications were provided
• Continuous monitoring and technological sensitivity are a paradigm shift from traditional methods; these come with great benefits but will also require validation 
• Different applications and environments are unique; therefore, in-situ testing and qualification of optical spectroscopic/intrinsic fluorescence RMMs is key to success
• Data-rich feedback and software tools empower the end-user to extract relevant and actionable process knowledge, facilitating PAT and QbD, and allows the industry to move closer to real-time release

Real Time Viable Particle Detection: Key Capability and Application Considerations. Darrick Niccum, Global Product Manager-Biotechnology, TSI

• A technical review of TSI’s BioTrak Real Time Viable Particle Detector was provided
• This was followed by a review of laser induced fluorescence 
• Real time viable particle detection has great potential to improve pharmaceutical manufacturing process
• Some key performance parameters to consider include sample flow rate, aerosol efficiency, aerosol concentrator performance, an effective sampling rate, total particulate counting performance and discrimination capability

Application of Real-Time Microbial Monitoring in an Environmental Monitoring Program. Elizabeth Bennett, Microbiologist Application Scientist, Particle Measuring Systems

• A technical review of Particle Measuring System’s BioLaz Real-Time Microbial Monitor was provided
• Applications within filling lines, sterility test isolators, biosafety cabinets and during aseptic transfers was then discussed
• The benefits for using real-time environmental monitoring (EM) technologies include immediate notification for alarm response, the ability to partition finished product in the even of a microbial excursion (based on the timing of alarms), verification of acceptable biological levels prior to filling, faster batch release, reduced operator error and paperless data management
• Incorporation of this type of RMM into the existing EM program was further examined

PDA Microbiology Conference Update: Revision of USP 1223

USP Chapter 1223, Validation of Alternative Microbiological Methods, has been under revision for at least the past year. During today’s conference, Tony Cundell, Director, Analytical Sciences Microbiology, Merck Research Laboratories and Vice-Chair, USP General Chapters – Microbiology Expert Committee, provided an update to the chapter’s revision process and what we should see when the draft is published in a future issue of the Pharmacopeial Forum.

The revised chapter will most probably reduce or eliminate the prescriptive guidance that is currently found within the existing chapter. This will allow users of alternative or rapid technologies to have more flexibility in the validation and use of these novel systems. Here is an overview of the proposed changes:

• Will include wider discussions of instrument and method validation and address the relationship of alternate methods to the USP General Notices and other relevant chapters
• Address regulatory requirements
• Will introduce concepts of performance, results and equivalence to existing methods
• Considerations for QC product release assays versus referee tests
• Provide guidance on alternate methods for compendial microbial tests
• Will consider equipment selection and qualification with actual product
• User specifications
• Installation, operational and performance qualification considerations
• Better define specificity, limit of detection, ruggedness and robustness, and other validation criteria for qualitative and quantitative technologies
• The responsibility of end-users versus instrument suppliers
• Method suitability
• Statistical tools
• References

And don't look for recommendations on the best rapid method for a specific application, as there is no plan to include any additional discussions regarding scientific principles or proprietary technologies.

PDA Technical Report #33, Evaluation, Validation and Implementation of New Microbiological Testing Methods, which is also under revision, will fill in the gaps that the future USP 1223 will leave. For example, TR#33 will provide detailed guidance on every step of the validation and implementation process, regulatory guidance from the FDA. EMA and other agencies, business and return on investment considerations, as well as a review of the technical and scientific aspects of technologies that are currently available. The revised TR#33 is planned for completion in 2013.

Blogging from the PDA Microbiology Conference: Compounding Pharmacies and Fungal Meningitis

Another year has passed and once again, I am blogging from the PDA Global Conference on Pharmaceutical Microbiology. This is the 7th year for this signature scientific meeting, where microbiologists from around the world gather to learn about the latest technologies, industry best practices and regulatory expectations with regard to contamination control and microbiological considerations during the manufacture of drug product.

The conference began with an outstanding first keynote address entitled Outbreaks Associated with Pharmaceutical Product: Steps for Prevention presented by Matthew J. Arduino, Dr. Ph., Lead Microbiologist, Chief Clinical and Environmental Microbiology Branch, Centers for Disease Control and Prevention.

During his presentation, Dr. Arduino reviewed the role of the CDC, key outbreaks that have occurred over the past few decades, where contamination has occurred, and how to prevent outbreaks and adverse events. It is interesting to note, that in light of very recent events associated with the fungal contamination of pharmacy compounded injectable product (see below), the number of products that have been contaminated by compounding pharmacies have increased since the 1990's. Microbial contaminants isolated from patients and products (contaminated at the compounding pharmacy level) have included Burkholderia cepacia, Pseudomonas aerugnosa, P. fluorescens, P. putida, Serratia marcescens, Exophiala dermatitidis, Elizabethkingae meningosepticum, Enterobacter cloacae, Fusarium spp, Bipolaris, Bullera spp and Rhodotorula.

Dr. Arduino concluded that outbreaks highlight the potential of certain products, manufacturing processes and infection control practices to cause high morbidity and mortality. There continues to be unsafe injection practices, and medication handling. Finally, we continue to see problems associated with compounding pharmacies.

Of course, the session wold not have been complete without a discussion of the fungal (Exserohilum rostratum) meningitis outbreak that has now affected patients in at least 17 states within the U.S. Nearly 14,000 patients may have received contaminated steroid injections since May 21, 2012, which have been produced by the New England Compounding Center (NECC) of Framingham, Mass. The potentially tainted drugs were sent to pain clinics and health care facilities in 23 states. As of today, infections related to the outbreak reached 308, with 23 deaths. That includes 304 cases of meningitis, stroke or other nervous system problems tied to epidural injections of contaminated steroids (methylprednisolone acetate), plus four infections in patients who received pain shots in joints such as the hip, knee shoulder or elbow. The death toll has held steady for a few days, but officials with the CDC said that doesn't necessarily mean that the outbreak is waning.

Dutch Bio-Hackers Mobilize Malaria Testing

Amplino may be the ultimate garage project. Three DIY bio-hackers have created a mobile malaria testing kit they claim can identify different strains of malaria with higher accuracy, and at lower levels of parasite concentrations than existing rapid diagnostic tests.

The testing device is connected via Bluetooth to a mobile phone, making it possible to track malaria outbreaks and the spread of particular strains. The team just won 40,000 EUR ($52,000) in the Vodafone Mobile for Good competition to further develop the kit.

Amplion’s young founders Jelmer Cnossen, Wouter Bruins, and Pieter van Boheemen have backgrounds in bio-informatics/mechanical engineering, cell biology, and functional genomics respectively. The group became fascinated by the DIYBio movement, which blends biology expertise with electronics, software development, and open source principles. Amplino’s team started to look at a technique called PCR, which copies a segment of DNA billions of times so that it can be analyzed. PCR is a well-established technique used in criminal DNA testing, disease diagnosis, and even testing whether food is halal.

A $600 “Build it yourself” OpenPCR machine already existed. The team decided to go one step further and develop a mobile device to do real-time PCR plus diagnosis of malaria. When you add a DNA binding dye to the multipled DNA mixture and shine light of a specific wavelength on it, the mixture will emit light when the malaria parasite is present.

In fact, the technology can detect any kind of pathogen, not just malaria, depending on the selection of a chemical component called a primer used in the device. A commercial real-time PCR setup can cost up to $30,000. Amplino built one for $60.

Malaria is a massive problem in the developing world. WHO estimates that up to 1 million people die each year from the disease, the majority of which are children in sub-Saharan Africa. The main methods of testing in the developing world are rapid diagnostic blood tests, which look like a pregnancy test and can be easily used in the field, and microscopy of a blood smear. The basic measure of accuracy of a malaria test is its sensitivity, in other words what percentage of infected people are correctly identified. Other factors in assessing a testing system include the concentration of parasites which need to be present in the blood for the test to correctly detect malaria, the number of false positives, storage life, or whether refrigeration,  clean water or trained medical staff are required.

Rapid diagnostic tests (RDTs) are less sensitive than lab tests like microscopy and cannot identify different types of malaria. Microscopy requires samples to be sent to a laboratory where trained health professionals review the blood samples. Incorrect diagnosis means that the limited supply of malaria drugs is not allocated optimally. However, testing at the point of care can still result in higher levels of correct patient treatment than more accurate, but slower, lab testing.  The World Health Organisation tests available RDTs each year to determine their sensitivity and PCR technology is used as the benchmark. So PCR can be regarded as the gold standard of malaria diagnostics.

Amplino’s test combines the accuracy of PCR with the mobility and ease of use of rapid diagnostic tests. It can be used by non-medical staff for immediate diagnosis of different types of malaria in the field. It can also detect malaria in pregnant women. During pregnancy, a woman’s immune system is suppressed, making her twice as likely to die from malaria. The parasites that cause the disease can hide in the placenta, making them much harder to detect. PCR can detect malaria at up to 10x lower concentrations of parasites in the blood than RDTs, making it suitable to identify re-infections as well as malaria in pregnancy.

Amplino estimates that the final mobile testing device will cost about $250, while the cartridge required per test costs between 50 cents and $1. Getting the device to market is still a long road. While the core PCR technology is not new, all medical devices need to be certified. This process, plus getting the prototype ready for manufacturing, could cost up to 1.5 million EUR over the next few years.

There are not many competing devices. InstantLabs makes a portable, real-time PCR system, but the company seems to focus mainly on the food safety industry. Lava Amp makes a $300 machine to run PCR but not to diagnose a particular disease.

“Our ultimate goal would be to get money from the Gates foundation,” said Bruins. ”We need strategic, not purely financial investors.” The Vodafone prize will allow the team to produce a version of the prototype that can be manufactured on a large scale (they estimate this will take six months) and test it in the field in Burkina Faso.

Amplino is not seeking to patent the technology. “I actually have two patents pending (for other technologies), ” Bruins explained. “But is a patent really fit for our mission? Maybe we need to ditch the whole patent approach.”

Amplino was founded in 2012, is based in Leiden in the Netherlands, has three employees, and is privately funded.

Source: VentureBeat (http://venturebeat.com)

Rapid Methods in the Meat and Poultry Industries

Meat and poultry processors want faster, more accurate rapid-test results to protect their products, customers, consumers and their own companies from potential food-safety dangers. And rapid-test suppliers are endeavoring to satisfy this demand.

Stronger regulations from the US Dept. of Agriculture’s Food Safety and Inspection Service, Food and Drug Administration as well as the Food Safety Modernization Act are leading to increased rapid-test usage by industry. Growing food-safety awareness from the public and government also are playing a role in the demand for faster, more accurate tests, says Chris Lopez, technical sales and pricing analyst with San Antonio-based Food Safety Net Services. 

“Microbiological testing and high quality programs are a good starting point for a foundation to ensure the protocols of meat and poultry companies are protecting their products and their business,” he says. 
Various platforms of rapid testing use different scientific approaches to detect the microorganism of interest, Lopez says. These platforms usually fall under three main approaches: antibody-based, nucleic acid-based or enzymatic. 

Processors’ priorities

Many rapid-test providers tout their tests are user-friendly and that anyone from a trained molecular microbiologist to entry-level lab technician can conduct them. 

“When we chose our ATP [adenosine triphosphate] testing system, we looked for the system with accuracy, ease of use and understandable results,” says Darren Toczko, senior manager of food safety with Bar-S Foods Co., Phoenix, Ariz. 

“I want accuracy, sensitivity, reproducibility, costs, time-saving and ease of use,” adds Carl Zerr, director of international food safety and quality assurance with Rastelli Foods Group, Swedesboro, NJ. “The most important thing to me is accuracy.” 

Bar-S plants use ATP testing during pre-op inspections. At its corporate off-site laboratory, it uses a Listeria spp. test for processing environmental swabs. 

“If the surface is not clean, we know it within seconds,” Toczko says. “ATP testing does not replace Aerobic Plate Count/total plate count or Listeria testing. It is an indication of cleanliness, not of microbe level or type.” 

The Listeria spp. tests used in the Bar-S lab are fast, accurate and flexible to company sampling needs. This enables the processor to find and respond to any issues in a timely manner with confidence, he says. Bar-S’ Listeria spp. testing must be an AOAC method performed in an ISO 17025 laboratory. 

“Our company lab, which processes samples for four plants, has been ISO17025-accredited since 2006, with Listeria spp. on its scope of accreditation,” he adds. “Any Listeria method without an enrichment step to allow very low numbers of cells to grow would not be considered. Since Listeria is a slow grower, faster is not always better.” 

The test Bar-S uses requires a single-step enrichment and incubation for 40 to 48 hours, Toczko continues. “We stay away from methods which would lead to FSIS scrutiny of our results,” he adds. “It must also be flexible for increases in numbers of samples to be tested without excess increase in time, people or equipment,” he says. 

Rastelli Foods Group, a major national US foodservice distributor of beef, lamb, veal, pork, poultry and seafood products, incorporates rapid testing for E. coli O157 and Salmonella. It currently uses the FoodChek MICT system for the E. coli O157 test and upon AOAC certification, will also use the Listeria spp test. 

“I like the accuracy of the results, the ease of preparation and sample protocols to put everything together,” Zerr says of his E. coli O157 rapid test. “I didn’t need to hire a microbiologist to do the testing,” he adds. “The cost of the FoodChek Reader unit, test cassettes and pickup transportation savings I get by not sending every test to an independent lab is another factor.” 

The company conducts many in-house screening tests. Zerr still sends bi-weekly samples to an independent lab to confirm that his in-house test results are consistent, but he has reduced this practice by about 90 percent. 

Rastelli Foods now samples and tests products for the presence of E. coli O157 pathogens in its ground beef at 15-minute intervals. Before, tests may have been done two or three times a day. But over his first year despite increasing their testing, the company has saved around $14,000 with FoodChek’s system, Zerr says. 

Examine each technology 

A gap exists between current microbiology diagnostic products and technologies and what is ideally desired in rapid testing by plant QC managers, says William Hogan, president and CEO of FoodChek Systems Inc., Calgary, Alberta, Canada. Faster time-to-results (TTR) is the key requirement that enables better decision-making, improved economics and reduced risks, he adds. 

FoodChek’s patented MICT magnetic nanotechnology system eliminates human error by using a testing process that includes a bench-top electronic magnetic diagnostic reader, Hogan says. The FoodChek system is the only food-safety pathogen screening test that uses nanotechnology, which consistently produces accurate results in as little as eight hours, including the sample enrichment growth phase, he adds. 

FoodChek’s MICT Assay cassettes for E. coli O157 and Listeria spp provide very fast and accurate results in hours not days; are easy, five-step processes; and offer savings of up to 50 percent on the cost of pathogen testing, he continues. The company’s patent-pending Actero Enrichment Media also claim to have the fastest time-to-results in the bacteria growth phase; making the timeline to results up to 30 percent faster for E. coli and Listeria, and up to 70 percent faster for Salmonella. “Our MICT test cassettes give a quantitative result with a hard copy printout,” Hogan says. 

Processors should use rapid tests as insurance to safeguard their business, Hogan insists. “I see [rapid tests] as a huge marketing tool,” he adds. “If you test more than your competitors, why would a consumer or retailer not buy from you? We’re creating the FoodChek symbol/brand for food safety to be like the Nike Swoosh. Rastelli Foods intends to be the first to put the FoodChek brand on its packaging.”

“[After the enrichment growth phase], our tests take 30 minutes to set up and 48 seconds to get results. We can find the results in the same production shift; nobody else can do this,” Hogan says. 

FoodChek’s E. coli O157 rapid test is AOAC-approved and USDA/FSIS comparable. A new AOAC-approved, USDA/FSIS comparable Listeria spp rapid test is due for release this October; its Campylobacter test is expected out in January 2013; and its Salmonella spp; and its new STECs (shiga-toxin producing E. coli) tests are expected to be released in the second quarter of 2012. 

More meat and poultry processors and testing labs are looking for technologies with faster turnaround times for their pathogen screening so decisions can be made earlier regarding releasing inventory or in-process product, concurs Christine Aleski, US pathogen specialist, 3M Food Safety Department, St. Paul Minn. 

“Time is money and this axiom rules in the quality control lab as well,” she adds. “The sooner the results are available and the more accurate those results, with fewer retests required, the better financial circumstances for the company.” 

Pathogen screening for Salmonella, E. coli O157(H7) and STECs are used most by 3M’s meat and poultry clients. 

Rapid molecular testing offers earlier release from test-and-hold on inventories; increased specificity and sensitivity to help reduce the number of repeat tests; streamlined workflow to increase lab efficiency and technician productivity; real-time results to help make critical decisions faster; robust hardware with minimal maintenance requirements and less downtime for the lab; and powerful software that is easy to use and operate, she says. 

3M’s Molecular Detection System is a simple, rapid, specific and cost-effective nucleic acid amplification and detection method, she adds. 

“3M combined two unique technologies – Isothermal DNA amplification and bioluminescence detection – to offer the specificity and sensitivity required in a pathogen test solution that is also fast, simple and cost-effective,” she adds. “Compared to other rapid-detection methods, the system improves efficiencies in the lab process by offering users only one preparation protocol across all assays and all matrices allowing for batch processing, easier training and less chance for human error.” 

Because the DNA amplification is detected via bioluminescence, the 3M Molecular Detection System offers the unique use of color-coded assay tubes to differentiate pathogen assays making it easier for technicians work with its system, she says. Bioluminescence provides real-time detection of the DNA amplification and simultaneous amplification and detection allows for detection of positive results before the end of the run (as early as 15 minutes). 

With a smaller footprint that a standard notebook computer, the 3M Molecular Detection Instrument requires significantly less space than the market leading rapid methods, she says. 

3M’s technology instrument is robust and portable with no need for recalibration, requires minimal maintenance and provides automatic diagnostics at start up. Isothermal DNA amplification proceeds at a constant temperature, removing the need for complicated instrumentation. Bioluminescence detection eliminates the need for high-cost excitation sources, fluorophores, fluorescent filters and detectors. Assays are available for testing Salmonella, Listeria and E. coli including O157 H7. 

Meat and poultry customers of Bio-Rad Laboratories Inc., Hercules, Calif., are using the company’s real-time PCR assays, says Wendy Lauer, senior product manager, food science division. These tests typically are for Salmonella and E. coli O157:H7. The company also offers an assay for STECs. 

However, its most popular assay is its Listeria assay for environmental samples. “That’s where they test the processing environment instead of the food to use Listeria as an indicator of hygiene,” Lauer says. 

Doing the culture method could be less expensive than a rapid method, but, regarding time-to-results – cost is a relative term. “If you have your food sitting in a warehouse waiting for test results, that’s an additional cost on top of your cost of testing,” Lauer says. “If you use a rapid test and get a result after 24 hrs. as opposed to 48 hrs., that’s one day more you have shelf-life on that product.” 

Another rapid-test benefit is the ability of high-throughput screening. If a company is processing 100, 200 or more samples at one time, it needs something that can fit into that workflow. Usually, rapid methods are designed for that, Lauer says. 

Sensitivity is key. “You want to have the right answer to your rapid testing [and not false negatives or positives],” Lauer says. “PCR methods detect DNA of the target, they’re extremely sensitive, which yield better results.” 

iQ-Check is the company’s line of real-time PCR kits. “We have a full menu of different tests,” Lauer says. “We have iQ-Check Salmonella; iQ-Check Listeria (species and monocytogenes); iQ-Check Campylobacter; iQ-Check O157; and iQ-Check-STEC. All of our tests can all be run at the same time because chances are people aren’t just running one pathogen. Most people are running Listeria for their environmentals and Salmonella for their products, and they’re possibly running an O157 as well. 

Enrichment times depend on the organism. Bio-Rad’s O157 test is validated for an eight hr. enrichment; STEC is 10 hours; Salmonella is 20 hrs. with no regrowth step; and Listeria 24 hrs. After the enrichment, the sample processing time is about 30-45 minutes and the run time is about an hour- and-a-half to two hrs. For the Listeria test, complete results are available between 26-27 hrs.; for O157 complete results are within 10-11 hrs.; Salmonella is less than 24 hrs. For all tests, the enrichment times are 24 hrs. or less.” 

‘Time is money’

Expect rapid testing to increase among US meat and poultry processors in the future, all sources agree. 

“Processors will be looking for ‘flawless information instantly,’” Hogan says. “It’s about gaining the fastest time-to-results. You’re going to see an entire movement towards technologies being the fastest because time is money.”

Source: MeatPoultry.com (http://www.meatpoultry.com)