Impacts of Culture-Independent Diagnostic Practices on Public Health Surveillance for Bacterial Enteric Pathogens

Disease Control and Environmental Epidemiology Division, Colorado Department of Public Health and Environment, 4300 Cherry Creek Dr S, Denver, CO 80426, USA.
Clinical Infectious Diseases (Impact Factor: 8.89). 06/2012; 54 Suppl 5(suppl 5):S432-9. DOI: 10.1093/cid/cis267
Source: PubMed


For decades, culture has been the mainstay of diagnostic testing for bacterial enteric pathogens. This paradigm is changing
as clinical laboratories adopt culture-independent methods, such as antigen-based tests and nucleic acid–based assays. Public
health surveillance for enteric infections addresses 4 interrelated but distinct objectives: case investigation for localized
disease control; assessment of disease burden and trends to prioritize and assess impact of population-based control measures;
outbreak detection; and microbiologic characterization to improve understanding of pathogens, their virulence mechanisms,
and epidemiology. We summarize the challenges and opportunities that culture-independent tests present and suggest strategies,
such as validation studies and development of culture-independent tests compatible with subtyping, that could be adopted to
ensure that surveillance remains robust. Many of these approaches will require time and resources to implement, but they will
be necessary to maintain a strong surveillance system. Public health practitioners must clearly explain the value of surveillance,
especially how outbreak detection benefits the public, and collaborate with all stakeholders to develop solutions.

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    • "The current practice of sending pure cultures from primary laboratories to the reference laboratories for FWD pathogens may undergo changes with the advent of new primary diagnostic and typing techniques, in particular new applications of PCR, mass spectrometry, WGS and metagenomics[3,4,5,6,7]. As a result, in some cases, and predominantly driven by cost reduction, there will no longer be a pure culture required for the primary laboratories' diagnostic testing that can then subsequently be sent for typing. "
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    ABSTRACT: Molecular typing, and typing in general, has a long history of application to surveillance of food- and waterborne diseases (FWD) for public health purposes. The advent of new microbial typing and detection techniques, in particular whole genome sequencing (WGS) and culture-independent diagnostic methods, brings about a fundamental change in the way diagnostics and typing are performed for foodborne infections. To respond to the strategic needs related to the introduction of WGS into public health microbiology and facilitate the further development of EU/EEA-wide molecular-typing-enhanced surveillance for FWD, ECDC sent a call for interest to public health experts to form an expert group on introduction of next-generation typing methods for surveillance of food- and waterborne diseases (FWD-NEXT Expert Group), composed of microbiologists, epidemiologists and bioinformaticians. This report, produced by the FWD-NEXT Expert Group, focuses on four pathogens: Salmonella, Listeria monocytogenes, verocytotoxin–producing Escherichia coli (VTEC) and Campylobacter. It is written from the country perspective and covers the entire process from sample provision and sequencing to data analysis, and finally data sharing and collaboration between different organisations. As such it serves to inform and support countries that are planning to, or are in the process of, implementing WGS for routine surveillance and outbreak investigation of FWD. The main audiences of the report are therefore national public health reference laboratories, national level epidemiologists and their immediate stakeholders. The report will also contribute to the ECDC strategy and roadmap for integration of molecular typing into European-level surveillance, response and epidemic preparedness, in particular for food- and waterborne diseases. In addition, IT departments of organisations that will be performing WGS on a routine basis may also find parts of the report relevant for their planning, especially on data storage and computing capacity requirements. It is expected that WGS will eventually become the sole standard method for genotyping of FWD pathogens for public health purposes, with additional phenotypic tests such as antimicrobial resistance only being performed in situations where the phenotype cannot be reliably predicted from the sequence. In the meantime, laboratories that already perform WGS should ideally also still perform the current typing techniques if necessary on selected isolates, such as outbreak-related ones, so that data remain comparable across organisations and can be used for surveillance purposes and further validation during the transition phase. Largely independent of the typing method, it would be very beneficial to have isolates from positive samples (from food, food processing plants, animals, feed and the environment) available for real-time typing as well, since this allows complementing the traditional route of epidemiological investigation that is often unable to identify a potential vehicle or does so when the corresponding batches are no longer available for sampling. A legal framework, as already in place in some countries, can be very helpful, even if it is not required, to ensure that national reference laboratories receive a sufficient selection of both human and positive food samples for further typing. A cost comparison between WGS and current typing methods indicates that already, on a per isolate basis, WGS can be less expensive than current typing methods for E. coli and Campylobacter. For Listeria, the cost is more or less the same, and for Salmonella, depending on the throughput, the cost can still be somewhat higher. The total time required for WGS is already comparable to that of current typing methods. As WGS technology is still evolving rapidly, the cost and total time can be expected to decrease further and become less expensive for all pathogens compared with current typing methods. Taking into account the higher accuracy of the method for delineating epidemiologically relevant clusters, the potential for preventing additional cases through earlier detection of clusters and outbreaks, as well as identification of the vehicle, is also higher than for current typing methods. The actual laboratory work using WGS as the standard genotyping method will become simpler as only one genotyping method needs to be used per pathogen. In addition, the differences between pathogens are often small and limited to the DNA extraction process rather than to the preparation of the sequence library that forms the input material for the sequencer. It is therefore also easier to pool typing capacity. At the same time however, protocols for DNA library preparation provided by the manufacturer often benefit from some optimisation and hence are not yet standardised. The inter- and intralaboratory reproducibility of WGS results also needs to be assessed better. WGS is different from current typing methods in the sense that in addition to the actual laboratory work also substantial subsequent data processing, storage and analysis is required in order to extract useful information from the large amount of generated data. The required storage capacity needs to be taken into account when planning the introduction of the method. It likely only becomes an important factor when reaching several thousands of isolates and consequently the terabyte range. Similarly, computing capacity also needs to be taken into account, but likely only becomes an important factor when processing more than one hundred isolates per week. The routine analyses of WGS data for public health purposes are not yet standardised. However, a model process for routine analysis is described, including nomenclature assignment, cluster detection, prediction of relevant phenotypes and use of prior epidemiological knowledge. Many of these steps involve the usage of collaborative resources or databases, which are each described separately. The WGS nomenclature database for a particular pathogen is the most important of these collaborative resources, enabling effective communication between organisations and standardised analyses. Since there is currently no agreed standard database for any pathogen, minimum requirements are proposed for any such database in order to be acceptable as a standard. In addition, two types of WGS nomenclature are described, one based on core genome multilocus sequence typing (cgMLST), and one that is a true hierarchical or taxonomical classification. Collaboration and data sharing between organisations and countries is required due to the international dimension of FWD pathogens and food trade in particular. National public health reference laboratories form the first line of such collaboration, as they are usually the first to have sufficient information, i.e. the microbiological typing data, to allow linkage of cases at national level and subsequent detection of human clusters or outbreaks. Ideally, as soon as these typing data are available, they should also be sent to an international database to allow timely detection of microbiological clusters at the international level. The typing data have to be accompanied by some descriptive data, in particular a relevant date such as the date of sampling. For WGS typing data, several options are possible for what can be sent through, from raw reads to assembled genomes to nomenclature only. The most realistic option at present seems first to submit nomenclature, and then when a cluster is detected based on that, actual sequence data can be sent through as well to allow more detailed analysis.
    Full-text · Technical Report · Oct 2015
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    • "Alternatives to culturing bacterial agents of disease use nucleic acid and antigenbased tests. While these are faster than culture methods, they are not without limitations making it worthwhile to invest energy into ameliorating current culture techniques (Cronquist et al., 2012; Jones and Gerner-Smidt, 2012). As discussed above, various VBNC-inducing conditions have been identified to date and these conditions are most likely just the tip of the iceberg. "

    Full-text · Article · Jun 2014 · Frontiers in Microbiology
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    • "Alternatives to culturing bacterial agents of disease use nucleic acid and antigenbased tests. While these are faster than culture methods, they are not without limitations making it worthwhile to invest energy into ameliorating current culture techniques (Cronquist et al., 2012; Jones and Gerner-Smidt, 2012). As discussed above, various VBNC-inducing conditions have been identified to date and these conditions are most likely just the tip of the iceberg. "
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    ABSTRACT: Many bacterial species have been found to exist in a viable but non-culturable (VBNC) state since its discovery in 1982. VBNC cells are characterized by a loss of culturability on routine agar, which impairs their detection by conventional plate count techniques. This leads to an underestimation of total viable cells in environmental or clinical samples, and thus poses a risk to public health. In this review, we present recent findings on the VBNC state of human bacterial pathogens. The characteristics of VBNC cells, including the similarities and differences to viable, culturable cells and dead cells, and different detection methods are discussed. Exposure to various stresses can induce the VBNC state, and VBNC cells may be resuscitated back to culturable cells under suitable stimuli. The conditions that trigger the induction of the VBNC state and resuscitation from it are summarized and the mechanisms underlying these two processes are discussed. Last but not least, the significance of VBNC cells and their potential influence on human health are also reviewed.
    Full-text · Article · Jun 2014 · Frontiers in Microbiology
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