Laboratory methods and surveillance

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Overview

Identification of a specific pathogen usually begins with a physician ordering a stool sample for a patient. Once the pathogen is identified, the isolate may be transferred to provincial or federal public health laboratories for additional typing and testing such as serotyping, phage typing, pulsed-field gel electrophoresis (PFGE), multiple loci VNTR analysis (MLVA), anti-microbial resistance (AMR) profiling, and whole genome sequencing (WGS). These additional tests are an important component of investigations as isolates with matching genetic fingerprints or profiles are more likely to have originated from a common source, or have an epidemiological relationship, than those with different profiles.  If pathogens are identified in a food sample, their genetic profiles are compared to those isolated from human cases that are part of the outbreak investigation.  Determining if the isolates “match” provides critical evidence for an outbreak investigation.

There are a number of different types of laboratory surveillance programs in Canada that are used to monitor enteric disease trends over time, detect clusters and outbreaks, and inform outbreak response through source attribution data. When analyzed separately, each surveillance program has its own unique purpose; but when linked to other surveillance programs, they can help to inform outbreak investigations and detect clusters that may otherwise go unnoticed.  The earlier a cluster is detected, the greater the likelihood a source is identified which ultimately helps to reduce the number of people who may become ill.

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Laboratory methods

Serotyping

Serotyping is a method used to further differentiate between the various bacterial strains based on the presence or absence of particular antigens (i.e., receptors on the external surface of the cell).  For example, there are over 2000 different strains of the Salmonella bacteria, the most common being Salmonella enterica serovar Enteritidis. The Kauffman and White classification scheme is a system that classifies various Salmonella strains into serotypes, based on surface antigens. Serotyping is often done at provincial public health laboratories. If a serotype is common, additional sub-typing tests/methods are often performed.

Phage typing 

Phage typing (PT) is a subtyping method that tests a bacterium’s vulnerability to bacterial viruses. This can be used in conjunction with other subtyping methods to increase the discriminatory power. By adding phage typing, analyses can be focused to include only cases that have the predominant phage type (e.g., Salmonella Enteritidis with PT 8). Phage typing was phased out in Canada in 2017 and is no longer being performed at the National Microbiology Laboratory.

Anti-microbial Resistance (AMR)

Pathogens can be tested for their resistance to particular antimicrobials. Isolates with similar resistance categories are grouped together. AMR profiling is often done at hospital and private laboratories, as it is important for patient care (i.e., to avoid treating patients with antibiotics that the pathogens causing the disease are resistant to, thereby prolonging illness).

Pulsed-Field Gel Electrophoresis (PFGE)

In Canada, pulsed-field gel electrophoresis (PFGE) is performed at provincial and federal public health laboratories. PFGE is a technique that uses enzymes to cut bacterial DNA strands into fragments. These fragments are then separated based on size using an electric field, which results in the creation of a PFGE pattern (i.e., “DNA fingerprint”). Once these PFGE patterns are generated, they are entered into an electronic database of DNA fingerprints at the National Microbiology Laboratory (NML). This enables the rapid comparison of patterns allowing for the detection of geographically dispersed outbreaks of foodborne bacterial disease at an earlier stage compared to traditional laboratory surveillance. This allows for more timely interventions to outbreaks and reduced impact on public health. PFGE’s discriminatory power can be increased by using more than one enzyme, and the enzymes used varies by pathogen. Following the transition to whole genome sequencing (WGS) in Canada in 2017, PFGE was phased out at the national level for multiple pathogens including Listeria, Salmonella, E.coli and Shigella.

Multi-Locus Variable number of tandem repeat Analysis (MLVA) 

Multiple-locus variable number tandem repeat analysis (MLVA) is a subtyping method that characterizes bacteria by counting the number of tandem repeats (i.e., naturally occurring repetitive DNA sequences) at specific points on the bacterial genome. It is typically used as a complementary technique to PFGE and is currently only performed by a limited number of laboratories in Canada. As of December 2014, in Canada MLVA is only used routinely for E. coli isolates.

Whole Genome Sequencing (WGS)

Whole genome sequencing (WGS) is a laboratory process that determines the complete genetic (DNA or RNA) sequence of an organism (e.g., bacterium or virus). Different analytical (bioinformatics) methods will then be employed to determine the relatedness between different isolates using these sequences. Within the context of an outbreak investigation, isolates that are more closely related to each other may have come from the same source (e.g., contaminated food product), while isolates that are not closely related to each other are less likely to have originated from the same source. In 2017, the National Microbiology Laboratory (NML) initiated a transition to WGS, the current “gold standard” method for comparing the genetic profiles of most foodborne bacterial pathogens in Canada. At the national level, WGS is currently being performed routinely for all isolates of Listeria, Salmonella, E.coli and Shigella.

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Laboratory surveillance programs

National Enteric Surveillance Program (NESP)

The National Enteric Surveillance Program (NESP) is a program administered by the Enteric Diseases Program at the National Microbiology Laboratory and the Centre of Foodborne, Environmental, and Zoonotic Infectious Diseases, Public Health Agency of Canada (PHAC). Through the NESP, each provincial public health laboratory provides the NML with weekly totals of new laboratory-confirmed enteric illnesses in their respective jurisdictions. These numbers are summarized into a weekly report summarizing trends in illness at the national and provincial levels, which highlights pathogens for which cases are reported above baseline levels. The weekly surveillance reports, as well as online data, are available to provincial and federal public health professionals. 

Canadian Laboratory Surveillance Network (CLSN) – PulseNet Canada

PulseNet Canada is the national molecular subtyping network for foodborne illness surveillance, providing a means for health officials to link cases in an outbreak investigation by identifying enteric illness clusters that are related by either pulsed-field gel electrophoresis (PFGE), multiple-locus variable number tandem repeat analysis (MLVA) or whole genome sequencing (WGS).  These techniques play an especially important role in identifying outbreaks when the numbers of cases are not above expected values.

The Canadian Laboratory Surveillance Network (CLSN) maintains an electronic discussion board, housed on the Canadian Network for Public Health Intelligence (CNPHI) platform, that allows laboratories in each province and territory, as well as NML and the Canadian Food Inspection Agency (CFIA), to post isolates from either cases or food that are genetically related. Follow-up is facilitated by the maintenance of a complex network of relationships with food safety partners at multiple levels, including Health Canada and CFIA, as well as provincial/territorial public health and agriculture authorities.

Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS)

The Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS) monitors trends in antimicrobial use and antimicrobial resistance in selected bacterial organisms from human, animal and food sources across Canada. The program is based on several representative and methodologically unified surveillance components which can be linked to examine the relationship between antimicrobials used in food-animals and humans and the associated health impacts.

FoodNet Canada

FoodNet Canada is a multi-partner initiative facilitated by the Public Health Agency of Canada. It supports activities that will reduce the burden of enteric disease through comprehensive sentinel site surveillance implemented through local public health units and provincial public health laboratories. FoodNet Canada is a preventive, multi-partner sentinel site system, facilitated by the Public Health Agency of Canada that identifies the sources of enteric illness in Canada. FoodNet Canada collects samples at the community level on human illness cases (i.e. exposures and behaviours) and along the farm to fork continuum (i.e. retail food, farm animals, and local water) to identify risks. FoodNet Canada’s core objectives are to i) detect changes in trends in human enteric disease and in levels of pathogen exposure from food, farm animal, and water sources (untreated) in a defined population; ii) strengthen source attribution efforts in Canada by determining significant exposures and risk factors for enteric illness; and iii) provide practical preventive information to prioritize risks, compare interventions and direct actions, and to assess the effectiveness of food safety programs and targeted public health interventions. 

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Limitations and challenges

There are many limitations and challenges to the various laboratory methodologies for enteric pathogens as well as the surveillance systems. In general, to be captured in a Canadian enteric illness surveillance system, an ill individual must: seek care; have a sample (e.g., stool, urine or blood) requested; and submit a sample for testing. In addition, the sample must be tested with a test capable of identifying the causative agent, and the positive test result must be reported to the surveillance system. Surveillance systems only capture a small portion of total illnesses given all these necessary steps (i.e., there is under-diagnosis and under-reporting taking place).

Even if cases are tested and captured through a surveillance system, it can be challenging to separate outbreak from sporadic cases. This may be addressed partially through additional testing, however, there is usually a limit to the discriminatory power of genetic typing, and it is not always feasible or practical to test all isolates. These limitations must be considered when analyzing and interpreting laboratory and surveillance data.

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Tools

National Enteric Surveillance Program (NESP)

  • This PDF document prepared by PHAC is a summary of data submitted to NESP by provinicial/territorial public health laboratories in 2012. The report includes reference tables with a complete list of species and serotype data reported to NESP as well as phage type data.

FoodNet Canada Reports and Publications

  • FoodNet Canada reports and publications provide information on the areas of greatest risk to human health to help direct food safety actions and programming as well as public health interventions, and to evaluate their effectiveness.

CIPARS Reports and Publications

  • The Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS) monitors trends in antimicrobial use and antimicrobial resistance in selected bacterial organisms from human, animal, and food sources across Canada.

PHAC Notifiable Diseases Online Database

  • The CNDSS on-line database provides data on national notifiable diseases including the number and rates of reported cases, data limitations, and disease descriptions.

National Collaborating Centre for Infectious Disease’s Notifiable Diseases Database

  • This searchable database contains the case definitions for all notifiable diseases in each of the provinces and territories.

Interpretation criteria for PFGE and MLVA

  • This reference document provides details on the strength of the evidence for interpreting PFGE and MLVA results. These criteria are used to assess matches among human cases, and to assess matches between food isolates and human cases.

CFIA: Canada’s 10 Least Wanted Foodborne Pathogens

  • This infographic prepared by the CFIA includes information on symptoms, onset time, transmission, potential sources, and preventative measures for ten foodborne pathogens.

FDA Bad Bug Book: Handbook of Foodborne Pathogenic Microorganisms and Natural Toxins

  • The second edition of the Bad Bug Book, published by the Center for Food Safety and Applied Nutrition, of the Food and Drug Administration (FDA), U.S. Department of Health and Human Services, provides current information about the major known agents that cause foodborne illness. The information provided in this handbook is not intended to be a comprehensive scientific or clinical reference, rather, it is abbreviated and general in nature, and is intended for practical use.

WHO Collaborating Centre for Reference and Research on Salmonella: Antigenic Formulae of the Salmonella Serovars (2007, 9th Edition)

  • This reference document prepared for the WHO Collaborating Centre for Reference and Research on Salmonella outlines the taxonomy and nomenclature of Salmonella and includes an alphabetic list of names given to serovars with their antigenic formulae

FDA Foodborne Illness-Causing Organisms Cheat Sheet

  • This PDF document provides a quick summary chart on foodborne illnesses, organisms involved, symptom onset times, signs and symptoms to expect, and food sources.

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