Preventing False- Positive Results in Pathogen Testing

Issue 3

Photo: Sergey Nivens

A magazine of Romer Labs®

Preventing FalsePositive Results in Pathogen Testing Eight Myths of Pathogen Testing Rapid Screening System Contributes to Reduction of U.S. Egg-borne SE

Photo: Marco G Faria

Content

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Preventing False-Positive Results in Pathogen Testing Costly and tedious to follow up on, false-positive results in pathogen detection can nonetheless be minimized.

By Stefan Widmann, Product Manager Romer Labs®

Eight Myths of Pathogen Testing What stands in the way of an effective pathogen testing program for your company? Spot-On puts a lid on the myths surrounding pathogen detection.

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By Meredith Sutzko and Stefan Widmann, Product Managers Romer Labs®

Spot On is a quarterly publication of Romer Labs Division Holding GmbH, distributed free-of-charge.

ISSN: 2414-2042

Contributors: Simon M. Shane, Mark Muldoon, Meredith Sutzko, Stefan Widmann Graphic: Reinhold Gallbrunner Research: Kurt Brunner

Publisher: Romer Labs Division Holding GmbH Erber Campus 1 3131 Getzersdorf, Austria Tel: +43 2782 803 0 www.romerlabs.com

©Copyright 2023, Romer Labs® All rights reserved. No part of this publication may be reproduced in any material form for commercial purposes without the written permission of the copyright holder except in accordance with the provisions of the Copyright, Designs and Patents Act 1998.

Photo: Kartinkin77

Editors: Cristian Ilea, Simone Schreiter

Rapid Screening System Contributes to Reduction of U.S. Egg-borne SE

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While total biosecurity programs for the entire layer and egg value chain are crucial for the control of Salmonella Enteritidis, highly sensitive and specific lateral flow immunoassays, such as RapidChek® SELECT™, can drastically reduce the costs associated with compliance through highly accurate screening results.

By Simon M. Shane, FRCVS, BVSc, PhD, MBL, ACPV

All photos herein are the property of Romer Labs or used with license.

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Spot On Issue 3

Editorial Improving Food Testing: Viruses to the Rescue!

According to a recent World Health Organization (WHO) report, foodborne diseases caused by the ingestion of various bacteria, viruses, parasites, toxins and chemicals result in an estimated 600 million illnesses worldwide per year (1 in 10 persons), and over 420,000 deaths. Thirty percent of these occur in young children less than 5 years of age. Pathogenic bacteria such as E. coli O157:H7, non-typhoidal Salmonella, and Listeria monocytogenes, are responsible for most of the diseases found in that category.

In many geographical regions, the food industry has adapted a farm-to-fork approach to monitoring bacterial pathogens as part of their hazard prevention programs. This consists of testing both the farm production and food processing environments as well as live and final food products for pathogens.

This increased testing has led to the need for increasingly rapid and yet highlyreliable test methods that can be used to ensure that a healthy and nutritious food product makes it to the consumer’s plate in an efficient manner. However, as rapid methods are increasingly implemented worldwide for foodborne pathogen monitoring (approximately 50% of all tests), innovative means to ensure their accuracy and reliability are required.

In this issue of Spot On, we explore several key aspects of food pathogen testing in the food industry. In the featured technology article, we describe how bacteriophage, bacterial viruses also called “phage”, are used as a replacement of antibiotics to control the growth of competitive nonpathogenic bacteria often found in food samples and by doing so, safer, faster, and more accurate detection of the target pathogenic bacteria can be achieved. In a subsequent article, Dr. Simon Shane, a key opinion leader in the egg and poultry industry, shares his views of how rapid test methods can be used in direct support of critical food safety regulations. And in yet another article, insight is provided into how rapid methods can be implemented as onsite tools for food pathogen monitoring in a production facility.

So, sit back and enjoy this new issue of Spot On describing yet more of Romer Labs’ innovations that address key challenges in food safety testing.

Mark Muldoon Director of Research and Development, Romer Labs Technology, Inc.

A magazine of Romer Labs®

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Photo: Sergey Nivens

Preventing FalsePositive Results in Pathogen Testing

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Spot On Issue 3

Costly and tedious to follow up on, false-positive results in pathogen detection can be mini­ mized with phages that reduce the occurrence of cross-reacting bacteria in pathogen tests. Cross-reacting bacteria can cause false positive results and inhibit the growth of the pathogen in question.

By Stefan Widmann, Product Manager, Romer Labs

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alse-positive results cause extra costs for food producers, in addition to the extra time needed to confirm the results. According to ISO/FDA/USDA standards, the process of confirming test results begins with streaking selective agar plates from the presumptive positive sample. These agar plates must be incubated for two days to prove whether it is a real positive result sample, and incubated at a different temperature from that of routine samples. If a lab does not have an additional incubator, the main incubator must be set aside and adjusted to the required temperature, which means downtime as the incubator cannot be used for enrichments.

Another down side of the confirmation step is that the selective agar plates cost money. As they have a very limited shelf-life, it is hard to keep them in stock.

Overall, false-positive tests demand unnecessary time and money.

The false-positive issue

False-positive results may occur with each pathogen detection method.

False-positive results may occur with every pathogen detection method. It does not matter if it is a DNA, a biochemical or an immunological based technology.

They are all dealing with the same issue of higher sensitivity versus higher selectivity. A good approach is to eliminate cross reacting bacteria before detection, and antibiotics are used for this purpose. But for most applications, antibiotics lack selectivity.

Overcoming the selectivity challenge Bacteriophages (or phages) are the most abundant organisms in our environment and are present in high numbers in water, food and various other sources. They were discovered by the Canadian biologist Félix Hubert d’Hérelle in 1917. Their name means “bacteria eaters”, which somewhat defines what they do.

Bacteriophages have high host specificity, attaching themselves to bacteria. The big advantage of using phages isthat they are harmless to humans, animals and plants. In fact, humans are routinely exposed to phages through food or water consumption with no negative effects.

Phages can be called “the natural enemies” of the bacteria to which they are specific to. In processed meat and meat products, there are about 108 phages per gram.

High numbers of phages are also present in the human gastrointestinal tract. Before antibiotics became widely available, phages were used to treat bacterial infections.

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Cross-reacting bacteria can be eliminated with the help of phages.

Used for decades The so-called phage therapy was developed in the USA and the former USSR. Even as antibiotics were being developed in the western world, the former Soviet bloc continued its research on phage therapy. In the post-cold war era, research activities on phages were continued in Georgia where they are still being carried out today.

Phages have very high specificity to their host bacteria, a characteristic that presents potentially interesting applications for food and feed, as well as the biotech industry. Today, phages are used as additives to eliminate pathogenic organisms in food, as an instrument to detect bacteria, or as supplement in the Romer Labs SELECT™ media system, to reduce or even eliminate competitive bacterial flora, enabling target pathogenic bacteria species to grow to a detectable level.

They are more selective than antibiotics, and microbes do not develop any resistance to them.

Identifying cross-reactive bacteria To identify phages, which can be used in selective enrichment media or other applications to inhibit or even kill competitive bacteria, the key question is: How can I even begin to find them?

The best way to start is by looking at food or feed samples, because these test materials contain bacteria which should be inhibited during enrichment. Bacte-

ria strains that are distantly related, to the pathogen searched for, are not the target of the phages, but strains that are closely related to the target pathogen could cause false-positive results.

Transgenic? Not at all!

After the cross-reacting background bacteria are identified, the next step is to search for a phage that can act against it. Phages can be found everywhere, such as in raw sewage, surface water, food and the agricultural environment.

The easiest way to extract them, if it is a solid matrix, is centrifugation. In this way, the phages will stay in the supernatant, while other components such as bacteria will form a pellet at the bottom of the container. This supernatant contains a heterogeneous mixture of different phages.

Next the different phages are isolated and the bacterial host they infect will have to be determined.

This could be done by the so-called “soft agar overlay technique or by simple spotting”. In this technique, a lawn of bacteria is overlaid on a traditional agar plate and the phage supernatants are spotted on the bacterial lawn.

After an overnight incubation, the phages create plaques (or zones of clearing), which are visible to the naked eye. Plaques are zones where the phages have killed the bacteria by a lytic reaction. Now, the phage clones can be isolated from the agar, which means that they are all copies of the same phage strain.

How phages help

Photo: Marco G Faria

It is now time to find the best phages for killing cross-reacting bacteria in pathogen enrichments. This is achieved by testing phages against the cross-reacting bacteria as well as the target pathogen. A microtiter format is preferred to minimize costs and speed up the tests.

A visible lytic reaction in a well indicates that the phage is effective against the bacteria. After several lab trials, suitable phages can finally be identified – these are phages that inhibit the growth of closely related bacterial strains while at the same time do not affect the growth of the target pathogen.

These phages can then be used to eliminate cross reacting bacteria in pathogen detection test systems, reducing the incidence of false-positive signals in a pathogen monitoring program and thus save a lot of money and time.

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Spot On Issue 3

MYTHS

of Pathogen Testing

What

What stands in the way of an effective pathogen testing program for your company? Spot On puts a lid on the myths surrounding pathogen detection in food products and identifies some common gaps in testing procedures.

By Meredith Sutzko and Stefan Widmann, Product Managers Romer Labs®

Myth 1 Environmental pathogen testing isn’t improving my food safety program Most regulations for pathogens require end-product testing before a food production lot can be safely released for distribution and retail. However, there are several challenges associated with the testing of end products.

Firstly, variables such as background microflora, inhibitory characteristics, pH level and salt concentration make pathogen detection difficult. Secondly, sampling methods and sample heterogeneity can lead to inaccuracies, especially if levels of contamination are very low. How can food producers be confident that the number of samples tested is sufficient to ensure that the lot is entirely free of dangerous pathogens?

Environmental testing has proven to be a very useful tool in many food safety programs and adapted as part of preventive controls under the FDA Food Safety Modernization Act (FSDA). Environmental monitoring programs (or EMPs) allow a food producer to find growth niches in production areas and take action to eliminate any risk associated with food contamination.

Areas with a high risk of pathogen contamination should be sampled, as the purpose of environmental testing is A magazine of Romer Labs®

to find positives. The goal of monitoring is to eradicate potential pathogens from the facility and production line before actual contamination takes place, and give food producers an early indication of any contamination risk/ problem.

Lateral flow strip tests do not need any extra equipment.

Myth 2 In-house pathogen testing requires substantial investments Service labs often tell customers that it is too expensive and laborious to test for food pathogens in their own facility. That might be true if standard procedures (FDA BAM, ISO) are used, as these generally require a lot of hands-on time. Other methods such as ELISA and real-time PCR require expensive equipment.

In fact, the only equipment you need for in-house tests are an incubator for the enrichment phase and a scale to weigh samples. An autoclave is optional, and only if you wish to sterilize your own media or for waste disposal. There may also be a need for a separate room for enrichment and testing.

When using lateral flow strips, there is no need for any extra equipment to interpret the results and therefore, no substantial investment is necessary.

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Myth 3 Pathogen testing requires well-trained staff

Small companies can test for pathogens on

Rapid methods for pathogens have been streamlined from the days when only highly trained microbiologists could administer such tests, along with countless tubes, plates and a discerning eye. A variety of skill sets are still required, depending on the method but many test kits can be run effectively by trained staff. Not all test methods require highly educated staff.

In many companies, lab staff may have been transferred from the production plant and may not have a scientific degree. Test methods should therefore be simple and robust, with minimal steps to help streamline workflow in the lab and minimize the chance of errors.

A lab must also be able to demonstrate that their staff have been trained in the prevailing test method. Some companies also participate in proficiency test programs to ensure that their lab staff can work independently to obtain accurate test results.

their own.

Myth 4 Lab safety requirements deter small companies from conducting their own tests Food pathogens in the so-called risk group 2, such as Salmonella, Listeria and E.coli O157, can be handled in rooms with some minor adjustments. Pathogens in this risk group are not considered hazardous to laboratory workers and the community in general.

According to the EN 12128 (European Standard), Bio-

Table 1. Selective agar plates for various pathogens Pathogen

USDA MLG

FDA BAM

ISO

E. coli O157

Rainbow® agar

CT-SMAC Second selective agar

Listeria monocytogenes

MOX agar

CT-SMAC Rainbow® agar or R&F ® E. coli O157:H7 agar

Salmonella species

XLT4 BGS

Source: USDA MLG, FDA BAM, ISO

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Esculin-based agar (OXA, MOX, Palcam) Chromogenic selective agar (Chromagar Listeria, Agar Listeria according to Ottaviani and Agosti)

XLD HE BS

Oxford agar Palcam

safety in Microbiological and Biomedical Laboratories (US Centers for Disease Control and Prevention) and the World Health Organization (WHO) Biosafety Manual, such rooms should be marked with the biohazard symbol and have access restricted. Only those working with the pathogens should have access to this lab room.

The surfaces in the room need to be resistant to chemicals and therefore also to disinfectants. These lab safety requirements can be easily managed by small food producers.

Myth 5 I cannot dispose of contaminated test material Yes you can. Here are three options for safe disposal: 1) Use an autoclave and set the temperature at 121 °C for at least 20 minutes. The material can now be safely disposed of as residual waste.

2) Use a microwave disinfection system. The material can now be safely disposed of as residual waste.

3) Delegate a waste company for that task. They will order special containers to collect the contaminated waste and discard it for you.

Myth 6 There is no need to further analyze samples after a positive test result All commercially available rapid methods are screening tests and require cultural confirmation after a “presumptive positive” test result. Confirmation requires streaking the enriched test portion to selective agars in order to isolate a typical colony based on morphological and biochemical characteristics. This is especially true when testing end products or foodstuffs.

For environmental samples, cultural confirmation is seldom performed as added sanitation practices are often put in place. There are several options for selective agar plates, with the most common ones found in the USDA Microbiology Laboratory Guidebook (MLG), FDA Bacteriological Analytical Manual (BAM), or ISO reference guides (see Table 1).

Myth 7 There is no reason to test for Listeria species when L. monocytogenes is the adulterant XLD Second selective agar

Listeria monocytogenes (L’mono) may be the only regulated Listeria strain but it is not the only pathogenic one. Listeria ivanovii is another pathogenic strain that may occur in your facility.

Another reason for testing Listeria spp. (all Listeria Spot On Issue 3

strains), especially in the processing environment, is to track growth niches. This is because L’mono can potentially grow where other Listeria strains thrive. It is a matter of probability whether L’mono is present or not.

If a lot of non-pathogenic Listeria strains (such as Listeria innocua) are present in a sample, they might “overgrow” L’ mono in the enrichment process and potentially cause a false negative result. It is not possible to grow only one Listeria species (i.e., L’mono) during selective enrichment.

Myth 8 All commercially available methods are the same There are a plethora of rapid methods available on the market today and choosing between them can be daunting. How does a food producer determine which method is best for them? All commercially available rapid methods consist of two parts – an enrichment followed by a detection step. Different media are used during the enrichment phase, using either a conventional or proprietary broth.

The main difference lies in the detection step. Immunoassay methods detect proteins while PCR methods detect DNA. The following evaluation criteria should be considered: inclusivity, exclusivity, sensitivity, specificity, detection limit, reproducibility, repeatability, and certification (see Table 2).

Rapid methods differ significantly with regard to workflow, ease of use, and throughput. Several other

Table 3. Pros and cons of different test methods

Lateral Flow

Table 2. Typical Criteria for Qualifying Pathogen Detection Methods Evaluation criteria

Definition

Inclusivity

Ability to detect different strains of the target pathogen

Sensitivity

Validated to detect a low concentration of target pathogen (i.e., few false negatives)

Exclusivity

Ability to not detect non-target organisms

Specificity

Validated on naturally contaminated samples (i.e., few false positives)

Reproducibility

Ability to yield the same results in different labs with different personnel

Detection limit

Repeatability

Compatibility with existing and standard enrichment media Availability of positive control strains for enrichment Certification

Concentration of cells required for a positive test result (cfu/g or cfu/mL) Ability to yield the same results in the same lab with the same equipment and personnel Method should employ the use of a unique strain that can be differentiated from wild-type strains to assess potential lab contamination Preferably AOAC or AFNOR approved

features of a test method that should be considered include capital equipment expenditure, training, time to result, and cost. All of these attributes must be taken into consideration when implementing a rapid method.

There is no single method that will fit all food producers (see Table 3).

Automated ELISA

DNA Detection

Accuracy Sensitivity Specificity Ease of Use Capital Equipment Training Time to Result Cost Throughput

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Rapid Screening System Contributes to Reduction of U.S. Egg-borne SE While total biosecurity programs for the entire layer and egg value chain are crucial for the control of Salmonella Enteritidis, highly sensitive and specific lateral flow immunoassays can drastically reduce the costs associated with compliance through highly accurate screening results.

By Simon M. Shane, FRCVS, BVSc, PhD, MBL, ACPV

E

gg-borne Salmonella Enteritidis* (SE) infection has been drastically reduced in the U.S. commercial egg industry. Following cases in the EU in the early 1970s, the index outbreak occurred in 1979 in New England. This was followed by extension through the Mid-Atlantic States in the early 1980s and ultimately emergence in the Southwest and Pacific regions in the 1990s.

Total SE cases in the U.S. peaked at 10,200 in 1995 but had declined to 5,000 by 2005. Incidence rates in the Mid-Atlantic States, clearly the worst affected region, attained 9.1 per 100,000 in 1994. By 2007, the level was lower at 2.5 per 100,000 population.

The US Centers for Disease Control and Prevention projected that the 5,333 confirmed cases of SE actually *

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Salmonella enterica Serotype Enteritidis

represented 170,000 cases nationwide, of which 108,000 were attributed to consumption of infected eggs. Irrespective of the validity of these projections, based on a series of contested assumptions, it was evident that a problem existed, stimulating Federal intervention.

Egg quality assurance programs During the late 1990s, individual states introduced the Egg Quality Assurance Programs requiring producers to conform to minimal standards of biosecurity, post-pack refrigeration, vaccination, assay of flocks for environmental contamination and diversion of eggs from infected flocks to breaking and pasteurization.

The elimination of vertically transmitted infection through successive generations, extending from pure lines to parent stock, was critical to the attainment of a meaningful reduction in the prevalence of SE in flocks.

Spot On Issue 3

Photo: Kartinkin77

The National Poultry Improvement Plan (NPIP) established in the early 1930s is responsible for the coordination of Federal, state and industry activities to eradicate vertically transmitted infections. The Plan mandates operating procedures, approves diagnostic tests and certifies hatcheries supplying day-old chicks and poults.

Originally focused on the eradication of Salmonella pullorum infection, the NPIP is now concerned with SE in laying strains, in addition to other pathogens in a wide range of avian species. Adherence to the requirements of the National Poultry Improvement Plan ensures a supply of commercial replacement pullet chicks from hatcheries certified free of SE. This is an essential foundation of industry efforts to prevent SE in commercial flocks.

In 2003, a nationally distributed brand of eggs introduced a rigorous SE prevention program involving vaccination and enhanced biosecurity required of all franchised egg producers. The use of lateral-flow immunoassay test kits was central to the successful implementation of the program. Since flocks were screened five times from the day of placement to the end of the second cycle of production, a rapid test with appropriate parameters was required in place of the conventional Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) microbiological procedure.

RapidChek® SELECT™ a highly sensitive test The Romer Labs RapidChek® SELECT™ lateral flow immunoassay has become the standard screening procedure to determine the presence of a Group D1 Salmonella in manure drag swabs or in egg pools. The procedure is regarded by the FDA as equivalent to the more laborious and expensive BAM methodology. Following the initial evaluation of the RapidChek® method, it was determined that the test demonstrated a sensitivity of 100 percent, critical for a screening test which should not provide false negatives.

Concurrently in the approval process assaying 141 Group D1 Salmonella isolates and 210 non-Group D1 Salmonella serotypes, the specificity of the test was determined to be 93 percent. This is an important parameter since a false positive assay from a 45-week old flock of 125,000 hens will incur substantial costs and disrupt operations.

Applying the BAM method a commercial laboratory examined 2,412 drag swab samples in 2010, yielding 1,477 “suspicious” colonies requiring further examination. From this complement only two SE positive samA magazine of Romer Labs®

ples were confirmed. The combined results from two mid-West state laboratories applying the RapidChek® assay in 2010 and 2011 yielded 106 “presumptive” positives from approximately 14,000 drag swabs examined.

Confirmatory bacteriologic assays identified 16 SE isolates from the routine samples submitted by producers.

Data from a Western state laboratory on 1,162 drag swabs compared the ability of the RapidChek® assay to detect Group D1 Salmonella against the conventional BAM microbiological assay. Eleven “presumptive” positives were identified using lateral-flow immunoassay compared to three SE positives applying the BAM procedure.

Method specificity is crucial from the perspective of financial losses.

Practical considerations The specificity of a screening assay is important from the perspective of potential financial loss. It is calculated that if a false positive environmental assay was obtained from a flock at the mandatory 40 to 45 weeks age period, withholding eggs from the market or diverting to breaking and pasteurization over a four-week period during which confirmatory test are performed could cost as much as $75,000 for a flock of 125,000 hens, depending on the prevailing market price of shell eggs and breaking stock.

In 2010, shortly after the introduction of the FDA Final Rule on Prevention of Salmonella, a widespread outbreak associated with one complex in Iowa resulted in an extensive recall ultimately numbering 500 million eggs. This case was an aberration and was due to gross mismanagement and did not reflect current practices in the industry.

In this 2010 outbreak, the initial round of assays derived from the audits conducted by the FDA applying the FDA BAM methodology showed an infection rate of 2.5 percent of flocks examined. This has since declined to negligible levels. There have not been any reported cases of SE attributed to eggs from 2011 onwards. This is due to diligent compliance with the principles of the FDA Final Rule and is based in large measure on the frequency and intensity of environmental monitoring.

This would not have been possible without the application of the RapidChek® SELECT™ SE test, which is used by commercial and state laboratories to conduct screening and compliance assays for the industry.

Epidemiologic evidence demonstrates that SE infection in the poultry industry is now associated with broiler meat and that SE isolates comprise an increasingly greater percentage of Salmonella serotypes recovered from carcass washes. Accordingly, programs of suppression and elimination are being applied, with the SE kit as the preferred screening technique for parent and growing flocks in the U.S. broiler industry.

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