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) 

Monday, August 5, 2013

Breathalyzer Detects Infections in Mice; May Replace Blood Testing in Humans

Breath analysis may prove to be an accurate, noninvasive way to quickly determine the severity of bacterial and other infections, according to a UC Irvine study appearing online today in the open-access journal PLOS ONE.

Employing a chemical analysis method developed for air pollution testing, UC Irvine microbiologists and chemists were able to correlate inflammation levels in laboratory mice to the amount of naturally produced carbon monoxide and other gases in breath samples.

The findings point to human applications of this technology in emergency rooms and intensive care units, potentially augmenting or replacing blood tests.

"Breath analysis has been showing promise as a diagnostic tool in a number of chronic diseases," said Dr. Alan Barbour, professor of microbiology & molecular genetics and medicine. "This study provides the first evidence … that it can be used for rapid clinical assessment of infections, which can lead to prompt institution of effective treatments."

Barbour collaborated with UC Irvine chemist Donald Blake, utilizing a gas analysis method devised for the Rowland-Blake lab's atmospheric chemistry research, which measures the level of trace gases that contribute to local and regional air pollution. It's one of the few research groups in the world recognized for its ability to gauge precisely at the parts-per-trillion level. Previous breath sampling work by the Rowland-Blake lab has involved diabetes, cystic fibrosis and kidney failure.

Barbour believed that breath analysis could additionally be used on infections, which elicit strong inflammatory responses in the body. Several compounds, or "biomarkers," are by-products of these responses. They can be identified in blood but also detected in exhaled breath.

Studying mice with bacterial blood infections, the researchers found that increases in the severity of infection elicited significantly higher amounts of carbon monoxide in relation to carbon dioxide in breath samples, making carbon monoxide a reliable biomarker for the presence and intensity of infection. Importantly, the carbon monoxide returned to normal levels soon after an antibiotic was given.

"Using a breath analysis method like this could help physicians in the emergency room and ICU make critical decisions about serious infections more quickly than if they had to wait for blood tests to come back from the lab," Barbour said.

He and Blake will next expand their research to human breath samples. Their diagnostic method is currently under patent review.

Charlotte Hirsch, Arash Ghalyanchi Langeroudi, Simone Meinardi, Eric Lewis and Azadeh Shojaee Estabragh of UC Irvine also contributed to the study, which was funded by a National Institute of Allergy & Infectious Diseases grant to the Pacific Southwest Regional Center of Excellence for Biodefense & Emerging Infectious Diseases (AI-065359).


Alan G. Barbour, Charlotte M. Hirsch, Arash Ghalyanchi Langeroudi, Simone Meinardi, Eric R. G. Lewis, Azadeh Shojaee Estabragh, Donald R. Blake. Elevated Carbon Monoxide in the Exhaled Breath of Mice during a Systemic Bacterial Infection. PLoS ONE, 2013; 8 (7): e69802 DOI: 10.1371/journal.pone.0069802


Blood is the specimen of choice for most laboratory tests for diagnosis and disease monitoring. Sampling exhaled breath is a noninvasive alternative to phlebotomy and has the potential for real-time monitoring at the bedside. Improved instrumentation has advanced breath analysis for several gaseous compounds from humans. However, application to small animal models of diseases and physiology has been limited. To extend breath analysis to mice, we crafted a means for collecting nose-only breath samples from groups and individual animals who were awake. Samples were subjected to gas chromatography and mass spectrometry procedures developed for highly sensitive analysis of trace volatile organic compounds (VOCs) in the atmosphere. We evaluated the system with experimental systemic infections of severe combined immunodeficiency Mus musculus with the bacterium Borrelia hermsii. Infected mice developed bacterial densities of ~107 per ml of blood by day 4 or 5 and in comparison to uninfected controls had hepatosplenomegaly and elevations of both inflammatory and anti-inflammatory cytokines. While 12 samples from individual infected mice on days 4 and 5 and 6 samples from uninfected mice did not significantly differ for 72 different VOCs, carbon monoxide (CO) was elevated in samples from infected mice, with a mean (95% confidence limits) effect size of 4.2 (2.8–5.6), when differences in CO2 in the breath were taken into account. Normalized CO values declined to the uninfected range after one day of treatment with the antibiotic ceftriaxone. Strongly correlated with CO in the breath were levels of heme oxygenase-1 protein in serum and HMOX1 transcripts in whole blood. These results (i) provide further evidence of the informativeness of CO concentration in the exhaled breath during systemic infection and inflammation, and (ii) encourage evaluation of this noninvasive analytic approach in other various other rodent models of infection and for utility in clinical management.

Source: PLOS ONE and Science Daily