Saturday, December 14, 2013

WHO: Countries Seek Improvements in Malaria Tests

Manufactures and implementers of rapid diagnostic tests (RDTs), for checking malaria parasites, have agreed on a set of required features in an effort to scale up use.

The consensus was reached at a meeting in Antwerp earlier this week jointly organised by the Roll Back Malaria Partnership and the Institute of Tropical Medicine in the Belgian city, according to a statement made available to The Guardian in Dar es Salaam.

The statement said the Antwerp meeting generated a prioritised list of requirements to meet country needs after assessing the interchangeability and user-friendliness of nearly 60 different RDTs on the market.

Over the past one and a half years, an international group of researchers coordinated by Prof Jan Jacobs of the Institute of Tropical Medicine in Antwerp assessed similarities and differences between commercially available RDTs. The research was commissioned by the Roll Back Malaria Partnership.

“We found that many of the shortcomings, such as confusing names, unclear and non-consistent labeling and terminology, variations in design and test procedures can be overcome in the short term,” said Prof Jacobs.

“Field observations show that even small things like the readability of the test instructions can make the difference between a correct and a failed test,” he noted.

“Harmonisation will also benefit manufacturers. History shows that harmonisation boosts business and market, while a level playing field protects producers from unfair competition from cheaper but substandard products,” added the professor.

The efforts to harmonise RDTs are widely expected to accelerate the implementation of universal diagnostic testing in the public and private sectors, reduce the global requirements for antimalarial treatment and advance progress towards the Roll Back Malaria Partnership goal of near-zero malaria deaths by 2015.

“This latest move to harmonise rapid diagnostic tests will help all countries to implement the WHO recommendations to ‘test, treat and track’ malaria – and build the trust of health workers in the accuracy of the malaria test results,” said RBM Executive Director Dr Fatoumata Nafo, who addressed the gathering.

“By bringing together manufacturers, technical experts and end-users, we can improve these lifesaving tools, improve market flexibility and advance the scale up of malaria diagnosis and treatment,” added Dr Nafo.

John Oluoch Nyamuni of Kenya’s Health ministry said: “With easily identifiable, interchangeable and user-friendly RDTs, we can dramatically increase the testing of malaria – which will result in more rational administration of antimalarial medicines. Harmonisation will also help countries to buy a quality product.” Currently, RDTs vary in diagnostic performance, manner of use, and price.

Countries prefer to stay with the same RDT rather than go through the costly exercise of retraining thousands of health workers, or run the risk of error in the manipulation of the test, which can detect malaria parasites in a tiny drop of blood.

According to the World Malaria Report launched earlier this week by WHO, the numbers of procured rapid diagnostic tests is increasing, as is the reported rate of diagnostic testing in the public sector in the African Region, which increased from 37 per cent in 2010 to 61 per cent last year, and from 44 per cent to 64 per cent globally.

Most of the increase in testing in the Africa Region is due to increased use of RDTs, which accounted for 40 per cent of all cases tested in the region last year.

However, millions of people with suspected malaria still do not receive a diagnostic test, and many with confirmed infections do not receive appropriate malaria treatments.

Globally, an estimated 3.4 billion people are at risk of malaria, with 80 per cent of cases and 90 per cent of deaths estimated to occur in Sub-Saharan Africa.

Children under five years of age and pregnant women are most severely affected.

Between 2000 and 2012 the world has seen tremendous progress against malaria, with global deaths decreasing by about 45 per cent and deaths in Africa decreasing by 49 per cent.

Meanwhile global efforts to curb malaria have saved the lives of 3.3 million people since 2000, the World Health Organisation said on Wednesday.

It said this translates into cutting global death rates from the mosquito-borne disease by 45 per cent and by half in children aged under five.

WHO said in its World Malaria Report 2013 that expanded prevention and control measures helped produce declines in malaria deaths and illness. Of the 3.3 million lives saved, most were in the ten countries with the highest malaria burden and among children under age five, the group most afflicted by the disease.

“Investments in malaria control, mostly since 2007, have paid off tremendously,” said Ray Chambers, the United Nations Secretary General’s special envoy for malaria.

According to the WHO report, child deaths fell to fewer than 500,000 last year. The report includes information from 102 countries with malaria transmission and shows that, overall, there were an estimated 207 million cases of malaria last year, which caused some 627,000 deaths.

That compared with an estimated 219 million cases and 660,000 deaths in 2010, the most recent year for which numbers are available.

“This remarkable progress is no cause for complacency: absolute numbers of malaria cases and deaths are not going down as fast as they could," WHO Director General Dr Margaret Chan said in a statement accompanying release of the report. "The fact that so many people are infected and dying from mosquito bites is one of the greatest tragedies of the 21st century."

Malaria is endemic in more than 100 countries worldwide but can be prevented by the use of bed nets and indoor spraying to keep the mosquitoes that carry the disease at bay. The mosquito-borne parasitic disease kills hundreds of thousands of people a year, mainly babies in the poorest parts of sub-Saharan Africa.

An estimated 3.4 billion people continue to be at risk for malaria, mostly in southeast Asia and in Africa where around 80 percent of cases occur.

Chambers said progress against malaria has been threatened by funding cuts in 2011-2012, which translated into a flattening in the curve of the decline. The WHO report noted significant drops in delivery of insecticide-treated bed nets in its 2013 report.

But that could begin to ease. Last month, the Global Fund to Fight AIDS, Tuberculosis and Malaria, UNICEF, the UK's Department for International Development and the U.S. President's Malaria Initiative agreed to provide over 200 million nets in the next 12 to 18 months, which will replace 120 million existing bed nets and provide 80 million new ones.

WHO also continues to track emerging parasite resistance to artemisinin, the core component of malaria drugs known as artemisinin-based combination therapies, or ACTs, and mosquito resistance to insecticides.

Four countries in southeast Asia reported artemisinin resistance in 2013, and 64 countries found evidence of insecticide resistance, suggesting recent gains against malaria "are still fragile," Dr Robert Newman, director of the WHO Global Malaria Programme, said in a statement.

Monday, November 25, 2013

PCR: 30 Years Young and Still Going Strong

The polymerase chain reaction (PCR) is a molecular biology method that exponentially amplifies very small quantities of DNA (e.g., one or a few copies). The result is millions of copies of a specific DNA sequence that can be used to detect the presence of a specific microorganism or pathogen. The method was direst developed 30 years ago (1983) by Kary Mullis. I came across a nice review detailing the advancements of PCR over the past 30 years in Genetic Engineering & Biotechnology News. Below is an excerpt from this article.

Novel techniques, automated machines, sophisticated synthetic and bioprospected polymerases, and developments in symbiotic sequencing technologies have transformed PCR into a multifaceted, adaptable molecular tool.

Such advancements, however, have tethered PCR to expensive laboratories, requiring ever more specialized equipment and technicians. Looking to the future, improvements in PCR technologies will simplify and speed up the process, reduce costs, and enable miniaturization of PCR devices, taking diagnostics out of the lab and placing it firmly at the point of care (POC).

PCR is increasingly used to interrogate the genomes of humans, animals, bacteria, and viruses in different ways. Though the process has long been routinely used for detection of infectious diseases such as malaria and flu, quantitative PCR (qPCR) and digital PCR (dPCR) have significantly increased the sensitivity and precision of such tests.

The ability to identify single nucleotide polymorphisms (SNPs) or regions of DNA associated with infectious disease drug resistance is crucial to effectively treating diseases, and thereby preventing further spread within the population, or overprescribing therapeutics that will have no benefit.

The development of dPCR has seen advances in the precision and sensitivity of PCR for absolute quantification of nucleic acid targets, key capabilities for the detection and treatment of food-borne pathogens such as salmonella and E. coli, as well as rare mutations such as those seen in cancerous cells.

Neoplastic cells have been successfully detected in the sputum, urine, and stool of patients with lung, bladder, and colon cancer respectively, and circulating tumor cells have been shown to be a useful biomarker for metastatic breast cancer.

In addition, the development of dPCR as a noninvasive prenatal testing (NIPT) diagnostic using maternal blood is a promising focus of current research.

High-Quality Diagnostics

Though PCR is now used for a multitude of diagnostic applications, from infectious diseases to NIPT, these tests are costly, and it can take weeks to return results to the patient. As technology moves forward, and more complex automated devices allow for real-time results with higher specificity and resolution, affordability and accessibility are expected to improve. But such gains have yet to materialize.

As the cost of standard polymerases falls, new, high-priced PCR machines, which require costly reagents and polymerases, are incrementally introduced to maintain the market share of nextgen technologies.

Rather than allowing more accessibility, these new PCR machines require increasingly well-stocked labs and well-trained staff. The infrastructure and skill levels required prohibit the use of such tests outside the developed world. Reversing this trend requires new thinking and disruptive, game-changing technology.

Recent advances in microfluidics now allow for POC PCR. This technology will make PCR-based diagnostic testing accessible in various settings worldwide, especially in remote, underserved communities where the detection of drug resistance markers for infectious diseases could revolutionize healthcare provision.

Microfluidics and PCR

Microfluidic techniques have developed from the advent of dPCR, which relies upon the ability to generate small-reaction volumes in an accurate and repeatable manner. Single-molecule isolation technologies have shown promise of late, with Quanterix utilizing single-molecule array technology to produce an extremely sensitive assay.

Innovative researchers have been applying microfluidics techniques to PCR, with the recent production of thermal cyclers utilizing microwells, capillaries, and microdroplet formation. Companies such as RainDance Technologies and QuantaLife provide extremely high levels of sensitivity with their microdroplet PCR devices. Further microfluidic advancement is pivotal to future advances in amplification techniques and nucleic acid detection.

Microfluidics technology increases the speed of PCR by orders of magnitude. Speed is only theoretically limited by the processivity of the polymerase and the length of the region to be amplified.

In reality, the limiting factor is often the time it takes to cycle the reaction mixture through the reaction temperatures, so-called ramping. Ramping in conventional PCR machines is time- and energy-intensive due to the thermal mass of the block and is not readily made portable.

Improvements in buffer systems and engineered enzymes have increased the speed of standard benchtop thermal cyclers. Typically, however, these methods still take over 90 minutes, with 40 minutes being considered fast. This is far too long to be practical in a clinical POC setting.

Very few commercially available devices are capable of truly rapid PCR, none of which can be considered portable. BJS Technologies, though it doesn’t utilize true microfluidics technology, has developed the xxpress benchtop microwell thermal cycler. This device is capable of 40 cycles in just 10 minutes because it has a ramp rate of 10°C per second.

Microfluidic PCR systems fall into two categories—microwell reactors and continuous flow. Microwell thermal cyclers are essentially miniaturized versions of traditional PCR machines, reducing the input and chamber volumes such that the time it takes to heat and cool the reaction mix is likewise reduced.

Roche’s LightCycler holds PCR mix within capillaries, thereby increasing the surface-to-volume ratio and reducing the cycle time. Despite a runtime of approximately 30 minutes, the LightCycler technology has been commercially successful.

Continuous flow devices, on the other hand, require heating zones of different temperatures, over or between which the reaction mixture is passed in microfluidic channels. This lab-on-chip approach greatly reduces energy consumption, as once heat has been added to the system it only has to be maintained and not ramped. Provided there is good thermal contact between the flowing reaction mixture and the heaters, extremely rapid PCR can be achieved.

With microfluidic channels, surface area is increased and the time to reach thermal equilibrium at any given temperature zone becomes fractions of seconds. This equates to rapid ramping. An additional benefit of continuous flow devices is that they utilize disposable plastic microfluidics cassettes in lieu of large batch processing machines, lowering the cost per test and eliminating the possibility of cross-contamination between samples.

The increased surface-to-volume ratio in microfluidic PCR is not without its challenges. Polymerase can become denatured on the walls of microfluidic channels, and primer concentration often has to be increased for the same reasons. Inhibition can be overcome by careful choice of materials and utilizing surface chemistry to increase hydrophobicity.

Alternatively, active or passive passivation layers can be used to decrease the high adsorption rate of reagents by the materials. Pumping mechanisms and channel dimensions also require careful consideration because of their effect on laminar flow and dispersion. Research teams worldwide are optimizing continuous flow PCR, with advances in materials, heating and cooling systems, and connectors, all required to provide more efficient integrated systems.

Continuous flow PCR was born out of capillary electrophoresis in the early 1990s. It was pioneered by Andreas Manz, who has been central in the early development of microchip devices for chemical and biological analysis. However, during this time it was not commercialized.

More recently, academic labs, such as that of Niel Crews, have further developed the early iterations of continuous flow PCR, although the majority of these devices have only been used in the research setting. Thermal Gradient has produced a commercially available continuous flow PCR device capable of sub-10 minute runtime by pumping PCR reaction mix through a sandwich of two or three heating zones.

The single-use devices are intended for integration into POC devices. While rapid PCR would save time and money for researchers, it could also save lives if put into the hands of healthcare workers as part of a POC diagnostic test.

Continuous flow PCR is ideal for POC applications with amplification of DNA possible in just a few minutes, microfluidic PCR opens up the possibility for rapid diagnostic testing. When combined with automated sample preparation and DNA detection technologies, such as QuantuMDx’s Q-POC device, the development of which also benefits from microfluidics engineering, a portable, fully integrated POC diagnostic device could extend MDx to resource-limited settings.

Continuous flow PCR, with low power, sample size, and reagent volume requirements, is ideal for inclusion in a handheld device, which will be produced at much lower cost than traditional PCR machines due to the lack of robotics. Per-test costs also will be lower due to the much smaller volumes of reagents required.

Moreover, due to the speed of of amplification the speed of molecular analysis will be improved, and with assay times in the 10-minute range, this makes it relevant to in-field or in-clinic MDx testing.

The resulting sample-to-result device will not only be less costly than traditional PCR machines but significantly easier to operate, negating the need for well-stocked laboratories and highly skilled technicians. Such a technology leap could provide resource-scarce settings with access to the high-quality PCR-based diagnostic testing that is available in the developed world.

Providing diagnostic results in minutes rather than days, weeks, or months could revolutionize healthcare worldwide by surmounting many of the obstacles associated with traditional healthcare provision models in the developing world.

Lack of reliable electricity supply, clean water, transportation (particularly cold chain transportation for reagents), and highly skilled technicians are all hurdles that could be overcome by a simple POC test. Such a test would make complex diagnostics affordable and accessible, and could improve outcomes through early detection and treatment of disease.

Advancements in speed, accuracy, and cost control will open up developed and developing healthcare economies to the advantages of state-of-the-art PCR and related diagnostic technologies, including reduced test costs as healthcare costs skyrocket, reduced waiting times as hospitals overflow, and improved accuracy for priority diagnostics.

This will aid in diagnosis and treatment of patients worldwide, whether for a drug-resistant infectious disease, cancer, or a genetic condition. The development of such cutting-edge diagnostics is dependent on the creation of novel rapid microfluidic PCR methods, and the resulting technologies are now set to change the basis of genetic diagnosis.

Source: Genetic Engineering & Biotechnology News

Thursday, November 21, 2013

Rapid Detection of Superbugs

A new lab test that detects antibiotic resistance genes quickly could help doctors choose the right drugs to knock out superbugs.

Patients affected by a bacterial infection can usually be treated with an antibiotic. But sometimes a resistant bacterial strain is causing the infection. In a hospital setting, doctors ideally want to know if they are dealing with such bacteria and which drugs they should choose. But if the doctor runs a test it can take days to get a result. Now, a European project is paving the way for much more rapid tests using DNA biochips. The aim is to rapidly screen disease-causing bacteria using a microarray to spot which resistant genes are present in bacteria.

The scientists, as part of the Antiresdev project, developed an array that could test for 116 antibiotic resistant genes from one class of bacteria, and 90 resistant genes from the other class of bacteria. The "arrays" contain pits with a DNA probe that lights up if a specific gene is present.

It is important to search for many resistance genes because bacteria have a habit of exchanging bits of DNA that make for useful anti-drug defences, making the bacteria resistant or even untreatable. This is bad news for patients infected with a superbug. "A lot of these resistance genes are on mobile elements. They can transfer all sorts of different resistant genes if they are put under the right pressures," explains project scientist Peter Mullany, who is also a molecular microbiologist at University College London, UK. He adds: "There are lots of resistance genes too, with at least 30 for tetracycline [an antibiotic] alone."

Quick detection technologies could therefore provide a diagnostic without delay to help direct the therapeutic choice. "The chip arrays are rapid and will tell us if any of the resistance genes on the array are present in a particular environment. If resistance genes are present, which allow the bacteria to resist a clinically important antibiotic, clinicians may choose to use an alternative antibiotic to which no resistance genes are present," Mullany tells

The team also discovered a new genetic fragment that gives bacteria an ability to resist an antibiotic called minocycline and an antiseptic for wounds called cetrimide bromide. Identifying the fragment's presence could allow doctors to take steps preventing its spread to harmful bacteria.

Until now, the DNA biochips were used to investigate how various antibiotics influenced the kinds of resistant bacteria that were present. It also helped determine which persisted at various sites in the human body – mouth, skin and nose. This gave insight into how antibiotics affect 'friendly' microorganisms naturally present in our bodies. "There are a lot of unknowns in this area, as the majority of microorganisms can't be cultured," says Mullany. "But if you can get rapid knowledge about which antibiotics genes are present, you have a better chance that treatment will be successful."

Experts welcome this development. Microarray allows the simultaneous detection of a large number of genes in the bacterial cell in one test. "The result which is obtained is a complex pattern of spots or dots, representing the individual resistance genes, which is read by machine and tells us which genes are present," explains Chris Teale, head of antimicrobial resistance at the Animal Health and Veterinary Laboratories Agency in the UK, "This technique is a great benefit when screening isolates for resistance because so many different genes can be screened at the same time."

The threat of antibiotic resistant pathogens is global, notes Richard Goering professor of medical microbiology and immunology at Creighton University, Omaha, Nebraska, USA, whose research deals with antibiotic resistance genes in Methicillin-resistant Staphylococcus aureus (MRSA). "The issue in dealing with these is two-fold, diagnosis and treatment. There are long standing worldwide problems with slowdown in pharma research and development for new drugs but still the bottom line is that the sooner the diagnosis is made the better the potential therapeutic outcome," Goering explains.

Quick detection is therefore a positive step. "So, genomics and the ability to rapidly sequence bacterial chromosomes holds great promise in identifying bacterial genes associated with accurate detection of both the bug and what is it susceptible to treatment," Goering tells
However, more work lies ahead. "More such tests are needed especially for use in resource-poor regions where morbidity and mortality associated with infectious disease are always high," he adds, concluding that just because a gene is present, does not mean it is actively expressed, and therefore that molecular techniques will need to address this issue too.


Monday, November 18, 2013

Rapid Testing to Diagnose Influenza Leads to More Appropriate Care in the Emergency Department

When patients in the emergency department (ED) are diagnosed with influenza by means of a rapid test, they get fewer unnecessary antibiotics, are prescribed antiviral medications more frequently, and have fewer additional lab tests compared to patients diagnosed with influenza without testing, according to a new study. Published online in the Journal of the Pediatrics Infectious Diseases Society, the findings suggest that diagnosing influenza with a rapid diagnostic test leads to more appropriate, specific, and efficient care.

In the study, researchers used data from the National Hospital Ambulatory Medical Care Survey, a nationally representative sample of ED visits in the U.S. They identified children and adults across three influenza seasons (2007-2009) who were diagnosed with influenza in the ED. They looked at how the patients were diagnosed—either with the use of a rapid influenza test or without it—and the subsequent care they received.

Among patients diagnosed with influenza without rapid testing, 23 percent of the ED visits included a prescription for antibiotics, which are not effective in treating influenza, a viral infection. However, for patients who were diagnosed by rapid testing, only 11 percent of ED visits resulted in the patient getting antibiotics. Additional laboratory tests, including chest X-rays, blood tests, and urinalysis, were also ordered less frequently for patients whose influenza illness was diagnosed with a rapid test.

Notably, prescriptions for antiviral drugs, which can be effective in treating influenza when used early and appropriately, were more frequent (56 percent of ED visits) among patients diagnosed with influenza using a rapid test, compared to antiviral use among influenza patients diagnosed without testing (19 percent of ED visits).

"When results of influenza tests are available to physicians at the 'point of care,' they use this information to provide more appropriate patient management," said lead study author Anne J. Blaschke, MD, PhD, of the University of Utah School of Medicine. "While other studies have shown that physicians can accurately diagnose influenza without testing, our results suggest that using an influenza test increases diagnostic certainty and leads to the physician providing more specific and appropriate care."

The study suggests a significant impact from rapid influenza testing on physician decision making, patient care, and use of health care resources, the authors wrote, despite the limited sensitivity of currently available rapid tests, which miss a number of true cases of influenza. The development of more accurate and faster tests for influenza available at the bedside could further improve care for patients with influenza or other respiratory illness, they noted.

The researchers' findings build on previous studies by others, focused primarily on children, that found that rapid influenza testing can influence patient care in specific settings. This latest study breaks new ground, Dr. Blaschke said, by using nationwide data and by demonstrating that the findings apply to both adults and children, and across different practice types.

Source: Pediatric Infectious Diseases Society

Purdue U. Device Speeds Concentration Step in Food-Pathogen Detection

Researchers have developed a system that concentrates foodborne salmonella and other pathogens faster than conventional methods by using hollow thread-like fibers that filter out the cells, representing a potential new tool for speedier detection.

The machine, called a continuous cell concentration device, could make it possible to routinely analyze food or water samples to screen for pathogens within a single work shift at food processing plants.

"This approach begins to address the critical need for the food industry for detecting food pathogens within six hours or less," said Michael Ladisch, a distinguished professor of agricultural and biological engineering at Purdue University. "Ideally, you want to detect foodborne pathogens in one work shift, from start to finish, which means extracting the sample, concentrating the cells and detection."

A report from the Centers for Disease Control and Prevention (CDC) indicates a lack of recent progress in reducing foodborne infections and highlights the need for improved prevention. Although many foodborne illnesses have declined in the past 15 years, the number of laboratory-confirmed salmonella cases did not change significantly in 2012 compared with 2006 to 2008.

The first step in detecting foodborne pathogens is concentrating the number of cells in test samples. The new system enables researchers to carry out the concentration step within one hour, compared to a day for the standard method now in commercial use, said Ladisch, also a professor of biomedical engineering and director of Purdue's Laboratory of Renewable Resources Engineering (LORRE)

Findings are detailed in a research paper to appear in November in the journal Applied and Environmental Microbiology.

The paper was authored by doctoral student Xuan Li; LORRE research scientist Eduardo Ximenes; postdoctoral research associate Mary Anne Roshni Amalaradjou; undergraduate student Hunter B. Vibbert; senior research engineer Kirk Foster; engineering resources manager Jim Jones; microbiologist Xingya Liu; Arun K. Bhunia, a professor of food microbiology; and Ladisch.

Findings showed the system was able to concentrate inoculated salmonella by 500 to 1,000 times the original concentration in test samples. This level of concentration is required for accurate detection. Another finding showed the system recovered 70 percent of the living pathogen cells in samples, Ladisch said.

"This is important because if you filter microorganisms and kill them in the process that's self-defeating," he said. "The goal is to find out how many living microorganisms are present."

The machine was used to concentrate cells in a sample of chicken meat. The sample is first broken down into the consistency of a milkshake and chemically pretreated to prevent the filtering membranes from clogging. The fluid is then passed through 12 hollow-fiber filters about 300 microns in diameter that are contained in a tube about the size of a cocktail straw. The filtering process continues until pathogens if present are concentrated enough to be detected.

The technique, developed by researchers from Purdue's colleges of Engineering and Agriculture, could be performed during food processing or vegetable washing before the products are shipped.

The U.S. Department of Agriculture will test the system, which is not yet ready for commercialization.

One feature that could make the machine practical for commercial application is that it can be quickly cleaned between uses. The tubes are flushed with sodium hydroxide and alcohol.

Purdue has filed a patent application for the concept.

The research is funded by the U.S. Department of Agriculture, Purdue's Agricultural Research Programs and Center for Food Safety Engineering, and the Department of Agricultural and Biological Engineering.

Above Picture: Purdue University doctoral students, from left, Xuan Li and Seockmo Ku operate a new system that concentrates foodborne salmonella and other pathogens faster than conventional methods, representing a potential new tool for speedier detection. The research is led by Michael Ladisch, center, a distinguished professor of agricultural and biological engineering. (Purdue University photo/Steven Yang). Click on picture to enlarge.


Rapid Sample Processing for Detection of Food-Borne Pathogens via Cross-Flow Microfiltration  

Xuan Li,a,b Eduardo Ximenes,a,b Mary Anne Roshni Amalaradjou,d Hunter B. Vibbert,a,e* Kirk Foster,c Jim Jones,c Xingya Liu,a,b, Arun K. Bhunia,d,f Michael R. Ladisch a,b,c

Laboratory of Renewable Resources Engineering,a Department of Agricultural and Biological Engineering,b Weldon School of Biomedical Engineering,c Department of Food Science,d Department of Chemistry,e Department of Comparative Pathobiology,f  Purdue University

This paper reports an approach to enable rapid concentration and recovery of bacterial cells from aqueous chicken homogenates as a preanalytical step of detection. This approach includes biochemical pretreatment and prefiltration of food samples and development of an automated cell concentration instrument based on cross-flow microfiltration. A polysulfone hollow-fiber membrane module having a nominal pore size of 0.2 um constitutes the core of the cell concentration instrument. The aqueous chicken homogenate samples were circulated within the cross-flow system achieving 500- to 1,000-fold concentration of inoculated Salmonella enterica serovar Enteritidis and naturally occurring microbiota with 70% recovery of viable cells as determined by plate counting and quantitative PCR (qPCR) within 35 to 45 min. These steps enabled 10 CFU/ml microorganisms in chicken homogenates or 102 CFU/g chicken to be quantified. Cleaning and sterilizing the instrument and membrane module by stepwise hydraulic and chemical cleaning (sodium hydroxide and ethanol) enabled reuse of the membrane 15 times before replacement. This approach begins to address the critical need for the food industry for detecting food pathogens within 6 h or less.

Source: Purdue University

Thursday, October 31, 2013

Less than 1 Week Away to Learn About the NEW Rapid Micro Methods PDA TR33!

PDA TR33, "Evaluation, Validation and Implementation of Alternative and Rapid Microbiological Methods," has just been published and the new document provides enhanced guidance on equipment and method validation, regulatory expectations, return on investment, scientific principles and implementation strategies.

This comprehensive 90-minute live web seminar will provide attendees with an overview of the new TR33 and a practical understanding of how to apply the guidance when validating and implementing rapid methods (RMMs) in their own facilities. Attendees will also have an opportunity to ask questions about TR33 or any other topic associated with RMMs.

The webinar will take place on Wednesday, NOVEMBER 6 at two separate times to accommodate most geographic locations. Please register for this webinar at

Although not required, attendees may benefit from reading TR33 prior to the webinar. The new TR33 may be downloaded (free for existing PDA members until Nov. 30) or purchased at

Saturday, October 19, 2013

PDA Technical Report No. 33 is Now Available - Webinar on Nov. 6

As the Chairperson of the Technical Report No. 33 task force team, it is my pleasure to announce that the newly revised TR33, "Evaluation, Validation and Implementation of Alternative and Rapid Microbiological Methods," is now available for download by PDA members and the scientific community.

The revised TR significantly expands on the original 2000 document, providing enhanced guidance on validation, regulatory expectations, return on investment, scientific principles and implementation.

Please visit to access the TR.

If you are attending the PDA Global Conference on Pharmaceutical Microbiology in Bethesda next week, please join me on Tuesday from 11:00-11:30 am, where I will present an overview of the new TR.

If you cannot attend the PDA Micro meeting, I will be hosting a comprehensive 90-minute web seminar that will provide attendees with an overview of the new TR33 and a practical understanding of how to apply the guidance when validating and implementing rapid methods in their own facilities. Attendees will also have an opportunity to ask questions about TR33 or any other topic associated with RMMs. The webinar will take place on Wednesday, November 6 at two separate times to accommodate most geographic locations. Please register for this webinar at

Wednesday, October 9, 2013

Vote for the Encyclopedia of Rapid Microbiological Methods!

The 4th volume of the Encyclopedia of Rapid Microbiological Methods is up for an award! In recognition of the outstanding quality of their publications, PDA presents one distinguished Editor or Author with the PDA/DHI Award annually. This award is determined by readers and members.

If you enjoyed this volume and would like to vote for the Encyclopedia of Rapid Microbiological Methods, please visit and select Editor Michael Miller: "Encyclopedia of Rapid Microbiological Methods, Volume 4"

Friday, September 27, 2013

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.”

Wednesday, September 25, 2013

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

Monday, September 23, 2013

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:

Monday, September 16, 2013

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

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."

Tuesday, September 10, 2013

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

Thursday, September 5, 2013

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

Sunday, September 1, 2013

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

Monday, August 12, 2013

Free Rapid Method Web Seminar - Implementation Considerations and Case Study on the BioLumix System

On September 12, American Pharmaceutical Review will be hosting a free web seminar in which I will discuss the current state of rapid microbiological methods (RMM) and present a case study on the BioLumix System, which can be used for the detection of specified microorganisms and an estimation of cell count. This talk will be applicable to a number of industry sectors, including pharmaceutical, biotech, food and beverage. You can sign up for this webinar by visiting the registration page.

You can also review the system's workflow and characteristics on the Product Matrix page at

Tuesday, August 6, 2013

Rapid Methods and On-Farm Bacterial Testing of Foods

Each year, an estimated 47.8 million people in the U.S. will become ill from eating contaminated foods.[1] A study by the U.S. Centers for Disease Control and Prevention has recently concluded that leafy greens are responsible for almost half of these foodborne illnesses.[2] Foodborne outbreaks associated with produce have increased significantly, from 0.7 percent in the 1970s to 13 percent between 1990 and 2005.[3] From 1990 to 2005, there have been 713 recorded produce-related outbreaks and approximately 34,000 cases of illness associated with produce contamination.[3]

While demand has been growing for the consumption of fresh produce for better health and nutrition, at present, a pragmatic nonthermal process to reduce pathogenic risk in produce has not been put into practice. Food safety has continued to grow in importance, and the climate is changing to demand that stronger food safety programs are instituted throughout the food chain from farm to fork. Under the newly established Food Safety Modernization Act (FSMA), the U.S. Food and Drug Administration (FDA) will now have regulations for produce.[4] These regulations emphasize employee training, health and hygiene, agricultural water, biological soil amendments of animal origin, domesticated and wild animals, equipment, tools and buildings.[5]

Farmers will soon be responsible for validating the food safety of their on-farm water and soil. Part of the proposed ruling is to have agricultural water tested routinely to ensure that the water source is safe for its intended on-farm use. If the tested water fails the declared compliance, certain actions must be taken to make it safe (proposed sections 112.44 and 112.45). While these activities are intended to support the reduction of foodborne pathogens within produce, they will have a significant impact on many farmers’ methods of growing produce.

Current microbiological methods traditionally take between 24 and 72 hours to complete, plus the time it takes to send the sample to the laboratory for testing. In the operational process of produce, harvested product is shipped to distribution centers or sent directly to stores within 1 to 3 days to ensure the best quality and maintain that quality for at least 7 to 10 days. Adding more time to account for microbiological testing could be detrimental to overall product quality. Besides the time it takes to conduct testing, there are significant costs associated as well. The general cost to outsource a 100-mL water sample for an Escherichia coli/coliform assay to a microbiological lab ranges from $15 to $28 per sample. Due to the complexity of the test, currently this is the only option available for farmers.

During one of the most recent Q&A calls for the Produce Safety Alliance in regard to the Produce Safety Rules, many farmers expressed great concern about the proposed rulings for agricultural water. Farmers are very worried about how they will manage product if their water sampling results return out of regulatory compliance. One farmer pointed out that if he pulls a water sample on Monday, he would likely not get the results until Friday. The proposed rules require that you treat the water to ensure that the source is deemed safe; however, they don’t provide guidance on how to manage the produce that has been exposed to the contaminated water between Monday and Thursday. Although it is prudent that all contributors be involved in food safety, this issue illustrates a need for better on-farm tools to meet the upcoming food safety expectations.

While the current laws exempt small farms from mandated food safety plans such as a Good Agricultural Practices (GAP) certification, the climate is changing. Many wholesale and chain grocery buyers, such as Hannaford and Price Chopper, are requiring that their buyers have a GAP certification to reduce their business liability. Therefore, exempt farms that are not compliant to GAP certification may be at a significant competitive disadvantage if they do not initiate a food safety plan.

Testing on a Farm

A closer examination of the resources and requirements for on-farm testing reveals a significant difference from those of professional food labs. If rapid assays are to be used on a farm, either the on-farm resources would need to be upgraded or the assays would have to be modified to be amenable to this unique environment. Given the diversity in farm environments and cultures, the better approach would be improved design of the assays for use in nonlaboratory environments.

Rather than compare the testing environment on a farm with a processing plant or third-party testing laboratory, a more fitting model for diagnostic testing is in low-resource settings (LRS), such as rural India or sub-Saharan Africa. Scientists have been designing new strategies to aid in the diagnosis of chronic and infectious diseases in these challenging settings. The challenge is to develop a rapid assay for bacteria that does not include expensive external equipment, is low cost and requires little user training.[6] Given the resources and level of training on farms of all sizes, we could consider farms to be LRS.

In 2002, the U.S. Department of Agriculture’s Agricultural Research Service published an article entitled “On-Farm Testing for Pathogens on the Horizon.” The article outlines a detection method using fluorescent real-time polymerase chain reaction to detect pathogens. Although the detection was possible in 30 to 45 minutes, the instrumentation and cost make systems like this less practical for routine testing by a farmer. To design assays to be conducted on a farm, one needs to account for the farming environment and resources.

Diagnostic assays for on-farm use have different constraints compared with those used in traditional laboratory environment. These tools used on-farm must be rapid, low-cost, produce little waste and easy to use. In addition, pre-enrichment of pathogens should be avoided due to a lack of disposal options and increased risk of contamination. Currently, there are few pathogen diagnostic tools truly compatible with on-farm testing. The justification to provide on-farm testing seems to be growing.

Disposable, stand-alone, kit-based assays are ideal for testing in LRS. This type of system does not typically require expensive external diagnostic readers. The reliance on a single piece of equipment for testing increases maintenance requirements and often raises the initial cost of testing beyond what many farmers are willing to spend. Additionally, if the instrument breaks down and a service appointment is weeks away, the farmer must seek alternative means to test. The use of stand-alone kits therefore provides better reliability in these settings. As farmers cannot be expected to clean testing glassware on the farm, ideally all components of the kit would be disposable.

Nonprofit organizations such as Diagnostics for All, based in Cambridge, MA, and PATH in Seattle, WA, have been designing tests that can be used in LRS. Much of the funding for these projects has come from the Bill & Melinda Gates Foundation. Recently, Diagnostics for All established a group to aid small farmers in sub-Saharan Africa. These tests include bovine reproduction tests, milk spoilage tests and aflatoxin detection in maize. The goal of the projects is to allow farmers to maximize the price they receive for their commodities. The goals and requirements of these tests are clearly applicable to farms of all sizes in the United States as well.

Lateral Flow Shows Potential

Currently, several companies are manufacturing lateral flow assays to be used on the farm. These tests are easy to use, relatively inexpensive and very reliable. They are typically immunoassays utilizing colloidal gold for visual detection. Future requirements that include bacterial counts at low concentrations may require a new generation of rapid testing that is amenable to the farm environment.

Lateral flow tests are reliable enough that they have been used for in-home testing for decades (home pregnancy tests are probably the most familiar). FDA approval demonstrates confidence in this decades-old technology as a test that can be performed reliably outside of a laboratory setting. Until recently, lateral flow tests have been limited by relatively poor sensitivity and used almost strictly as a qualitative test. The increasing interest in diagnostics for LRS has encouraged research that addresses the current limitations of the traditional lateral flow assay. Although most of these research projects target infectious disease diagnostics in LRS, the technology can be seen as a potential benefit for farms everywhere.

The familiar red or blue line in a lateral flow assay has proven ideal for situations requiring positive or negative results. Unfortunately, for situations where a quantitative result is required, such as FSMA’s generic E. coli standards for agricultural water, a simple positive or negative is insufficient. The ability to reliably quantitate color reactions on test paper has been proposed using smartphones.[7] Most smartphones and tablets now come equipped with powerful cameras that can be used to quantify colorimetric results. The popularity of these devices has placed potential analytical tools in the hands of many farmers. Test results could be instantly electronically logged with a date and location, allowing the farmer to maintain accurate records with minimal effort. Smartphones can even be used to quantify fluorescent or chemiluminescent assays.[8] The use of chemiluminescent and fluorescent detection in place of visual colorimetric assays can reduce the limit of detection that is orders of magnitude lower.[9, 10] As the limit of detection continues to decrease, we get closer to the ability to detect low numbers of bacteria without the need to pre-enrich or perform genetic amplification.

When developing assays to be used on-farm, below are some of the questions that should be considered:

•    What is the true cost of the test? This includes labor, initial equipment costs and space requirements.

•    Does the assay require a clean environment such as a biosafety cabinet?

•    Can the assay be run by someone who was trained in approximately an hour?

•    Does the assay result in any waste that requires special handling?

•    Does the assay require specialized storage beyond a standard refrigerator?

•    Can the results be easily interpreted?

•    What is the total assay time from sampling to results?

•    How does the test perform when compared with the current standard?

•    Does the assay require timed steps by the user? How critical is the timing?

•    How temperature sensitive is the assay?

•    Does the assay require pre-enrichment of pathogens?


While federal funds for this area of research are limited, it is possible that future testing requirements will create a market attractive enough for additional companies to look toward this technology. Many researchers have aimed for years at the ability to rapidly detect foodborne pathogens. Unfortunately, the instrumentation used in a food testing lab typically cannot be used on a farm where there is no laboratory. The solution must be pragmatic and low cost. A look at the research being performed to bring low-cost diagnostics to LRS such as sub-Saharan Africa suggests that on-farm applicable diagnostics may be on their way. For now, farmers can continue sending out samples and waiting for results. Hopefully, in the near future, technology may empower the farmer to conduct rapid microbiological testing on the farm to better ensure a safe product.

Amanda Kinchla, M.Sc., is an assistant professor and food safety specialist at the University of Massachusetts, Amherst. Professor Kinchla researches both pre- and postharvest food safety practices.

Sam Nugen, Ph.D., is an assistant professor at the University of Massachusetts, Amherst. He specializes in the development of low-cost diagnostic assays for low-resource settings.

1. Morris, J.G. Jr. 2011. How safe is our food? Emerg Infect Dis 17:126–128.
2. Painter, J.A., R.M. Hoekstra, T. Ayers, R.V. Tauxe, C.R. Braden, F.J. Angulo and P.M. Griffin. 2013. Attribution of foodborne illnesses, hospitalizations, and deaths to food commodities by using outbreak data, United States, 1998–2008. Emerg Infect Dis 19:407–415.
3. DeWaal, C.S. and F. Bhuiya. 2007. Outbreak alert! Closing the gaps in our federal food safety net. Washington, DC: Center for Science in the Public Interest.
5. LeBerre, V., E. Trevisiol, A. Dagkessamanskaia, S. Sokol, A.M. Caminade, J.P. Majoral, B. Meunier and J. Francois. 2003. Dendrimeric coating of glass slides for sensitive DNA microarrays analysis. Nucleic Acids Res 31:e88.
6. Yager, P., G.J. Domingo and J. Gerdes. 2008. Point-of-care diagnostics for global health. Annu Rev Biomed Eng 10:107–144.
7. Shen, L. J.A. Hagen and I. Papautsky. 2012. Point-of-care colorimetric detection with a smartphone. Lab Chip 12:4240–4243.
8. O’Driscoll, S., B.D. MacCraith and C.S. Burke. 2013. A novel camera phone-based platform for quantitative fluorescence sensing. Anal Methods 5:1904–1908.
9. Wang, Y. and S.R. Nugen. 2013. Development of fluorescent nanoparticle-labeled lateral flow assay for the detection of nucleic acids. Biomed Microdevices 10.1007/s10544-013-9760-1.
10. Wang, Y., C. Fill and S.R. Nugen. 2012. Development of chemiluminescent lateral flow assay for the detection of nucleic acids. Biosensors 2:32–42.

Source: Food Safety Magazine

Rapid Diagnostics of Infectious Diseases in Hospitals

Infection Control Today recently provided an overview of hospital-acquired infections and the need to utilize rapid methods to screen for pathogens such as MRSA and influenza...

Hospitals constantly struggle to combat healthcare-acquired infections (HAIs). In recent years, the development and use of rapid diagnostic testing for these infectious diseases has helped to alleviate some of the issue, allowing healthcare providers to screen patients at the point of admission and take necessary precautions for carriers. Hospitals also want to know this information as quickly as possible in order to document infections. They do not receive additional payments for conditions that were not present at the time of admission, according to the Center for Medicare and Medicaid Services (CMS) website.

Tests are now available for bloodstream infections such as streptococcus and enterococcus as well as methicillin-resistant Staphylococcus aureus (MRSA) and respiratory illnesses including influenza A and influenza B.

In the past, diagnostic testing for these infections were primarily culture-based, meaning healthcare providers collected a sample  from the patient, put it on an agar plate, incubated the sample and then waited to see if bacteria grew out of it, says Paul Schreckenberger, PhD, professor of pathology at the Stritch School of Medicine at Loyola University and the director of the clinical microbiology lab for Loyola University Hospital. The entire process took about 24 hours to detect bacteria, at which point the lab would begin various tests to determine what bacteria was actually present. These tests could take another 24 hours to complete.

"They're labor-intensive and they're slow," Schreckenberger says of the old, culture-based tests. Physicians often relied on their clinical reasoning to diagnose certain illnesses such as influenza instead of waiting for the test results, he adds.

New molecular tests allow labs to look at the DNA in a specimen and determine the infection.

"Now we have these molecular techniques that completely revolutionized how testing is done, where we can take specimens, like nasal secretions, look for the DNA specimen and know within the hour whether it's a bacteria or virus and what bacteria or virus is present," Schreckenberger says.

During the flu outbreak this fall in Chicago, the rapid diagnostic testing allowed Loyola to identify the specific strand of influenza spreading throughout the city and determine that the flu vaccine covered that specific strand. This helped the city alert people to the potential harm and encourage them to get their shot, Schreckenberger says. "The information we're able to get is so influential in many ways, not just for the individual patient but to inform the masses what's going on, why is everybody getting sick, what's causing it and what's the solution," Schreckenberger explains.

The test Loyola uses to determine respiratory illness screens for 17 viruses and three bacteria, considered the most common causes of respiratory illnesses, which account for about 95 percent of all respiratory illness causes, Schreckenberger says.

Screening Upon Admission

Even as hospitals work to prevent HAIs, certain infections like Clostridium difficile (C. difficile) actually increased among pediatric patients and patients older than 85 years, according to a 2011 paper published in Laboratory Medicine. Screening patients at admission acts as one way to help combat these growing numbers, the article adds.

Common HAIs such as MRSA, vancomycin-resistant Enterococci (VRE) and C. difficile can and should be screened for prior to or at the time of admission, the paper states. Testing for these bacteria and infections at the point of admission allows the hospital to efficiently implement isolation if needed. Longer wait times for results leads to patients either being unnecessarily isolated or not isolated at all, in which case they risk transmitting the bacteria elsewhere and possibly infecting other patients, the article explains.

Loyola University Hospital screens every patient, regardless of his or her reason for admission, for MRSA during admission using a polymerase-chain reaction (PCR) test. Nurses swab new patients' noses, where the staph is carried, and send the sample to the lab where they test to see if the DNA of the sample matches the known DNA of infections.  In order to detect potential infections, the lab heats the DNA sample to separate the double-helix. Agents that match the DNA of a possible infection such as MRSA are added to the sample , where it tries to find a complementary strand on the patient's DNA. If it finds a match, it will bind with it to form a double helix. At this time, it begins multiplying until there are about 10 million strands, at which time it is measurable. The entire PCR amplification process takes about one hour, far quicker than the tests of years past.

Most patients don't know they carry MRSA because they are asymptomatic, Schreckenberger says, and for that reason he adds, "You come into our hospital you are going to have your nose checked."

Years of testing suggests that about 7 percent of the population acts as a carrier for MRSA. Loyola admits approximately 100 patients a day, meaning that seven of those patients will likely be a carrier, he explains.

Necessary Precautions

Screening for MRSA helps the hospital take precautions against the spread of the infection or contamination of the patient's room. Patients likely have wounds and stitches that make them more susceptible to infections post-surgery, Schreckenberger says.

If a patient carries staph, he or she may touch his or her nose, at which time the staph is transferred to his or her hands and skin and ultimately makes contact with the wound, increasing the risk of infection. People tend to think that the hospital gives them the infection, but it can be transmitted from your own body, Schreckenberger says.

"It's going to be infected from you, whatever you are carrying on your body," Schreckenberger explains. "If you get a urinary tract infection, it's not because you stood too close to somebody. You get a urinary tract infection because your own flora gets in your urine and causes infection."

Identifying the patients who carry staph allows the hospitals to stay one step ahead and take necessary precautions to prevent the infection. First, the healthcare providers need to alert the patient to their status as a carrier and explain to them simple ways they can aid in prevention, including frequent handwashing, Schreckenberger says. Second, the hospital can put an antibacterial ointment in the patient's nose that eliminates the staph. They can also provide the patient with special baths using antiseptic soap to disinfect any of the staph that may be on his or her skin. The patient will be placed in contact isolation, so that any healthcare provider or visitor to the room must wear gowns and gloves. Finally, the room will be cleaned somewhat differently once the patient leaves the hospital, Schreckenberger concluded.

"Maybe the patient who is a carrier never gets an infection. That's possible, but they still have the staph in their nose, so that staph is on their sheets, on their bedrail, on their remote control and on everything they touch in that room," Schreckenberger says. "Just knowing that they carry that organism is enough to initiate all of these steps."

Healthcare providers also need to be cautious about what they touch when caring for a carrier. Even with gloves on, the healthcare provider may touch the bedrail or a remote control that the patient previously touched and then touch another surface further contaminating it.

"There's a lot of touching that goes on in the room, and we don't know what we're leaving behind when we touch all of these things," Schreckenberger says.

For this reason, hospitals should make sure to thoroughly clean  these rooms upon a patient's discharge. Loyola brings in isolation carts for patients in contact isolation. These carts contain a remote control, stethoscope, blood pressure cuff—important equipment that may come in contact with the patient and potential staph. After discharge, the hospital removes the cart from the room and either discards or sanitizes these instruments elsewhere, Schreckenberger says. All surfaces in the room are then thoroughly cleaned.

"We take a lot of precautions that would not normally be done every day on every patient who doesn't have an infection," he adds.

Impact of Rapid Diagnostic Testing

Thanks to the rapid screening tests, Loyola reduced the number of MRSA infections at their hospital.

"We used to get about 90 of these infections a year," Schreckenberger says. "Now we average about 30 a year, so we had a two-thirds reduction in the number of these by screening patients, letting them know they are carriers and taking all of these precautions."

In the past, the culture-based tests sometimes gave false negatives because there weren't enough bacteria present to properly detect and identify the issue. The new tests, however, will only be positive in the presence of the agent of interest, Schreckenberger says, adding that these tests are better and more accurate than any previous test. "Because they are DNA-based they are very specific for the target that we're looking for," he says.

When selecting the right rapid diagnostic test for these infections, consider four key elements—flexible throughput, turnaround time, cost and assay content, the article from Laboratory Medicine suggests. Can you run one or more tests at a time without compromising results? If only one test can be administered at a time, how many instruments are needed to ensure an efficient lab and how much space is needed for these instruments? Can the test be completed in approximately two hours or less? What will the test cost the hospital and what will it cost the patient? Are the results unambiguous?

If tests meet these requirements, screening procedures, like the MRSA screening at Loyola, can play a key role in the prevention of HAIs. They alert physicians and other healthcare providers what patients carry the bacteria or virus at the time of admission. Knowing from the start that a patient carries the staph can help the hospital take necessary precautions to ensure that the patient does not develop an infection and the room does not become contaminated.

Tara Boyd is a freelance writer for ICT.

Reference: Centers for Medicare and Medicaid Services

Source: Infection Control Today (ICT)