Rapid diagnostic testing for salmonella in clinical practice (Proceedings)

Salmonellaenterica is commonly associated with epidemic disease in veterinary hospitals and on-farm environmental contamination [1; 2].  As such it garners much of our attention.  Unfortunately, testing techniques for the detection of Salmonella in fecal and environmental samples is variable among laboratories and current testing methodology generally lacks in sensitivity.  Consequently, testing strategies generally test multiple samples, use lengthy enrichment steps, and may take 3-5 days to obtain results.  In that time, significant environmental contamination and disease transmission can occur, thus risk recognition and rapid identification are critical to effectively managing this agent in populations and their environments.

There are many methods available for the detection of S. enterica in samples relevant to veterinary medicine – including enriched culture, polymerase chain reaction (PCR), and lateral flow immunoassays (LFIs) – all  requiring varying levels of expertise and can impact time to detection, test results and interpretations, and ultimately disease control efforts [3-7].  The reliability of bacterial culture for S. enterica detection can be affected by the type of sample (feces, swab, or rectal biopsy), heterogeneity of target organism in the sample, sample weight, intermittent shedding, bacterial culture method, and laboratory proficiency. 

While experimentally, the analytic sensitivity of equine fecal culture has been found to be as few as 4 cfu/gram of feces when enriched in tetrathionate broth [8] and 100 cfu/gram of feces when enriched in selenite broth [9]; in practice, fecal culture is a relatively insensitive method.  This is likely due to intermittent shedding of relatively few organisms per gram of feces [10; 11], as well as the heterogeneous distribution of organisms within fecal samples [12].  Because of the need to use one or more enrichment steps, it can take up to 3 days to realize test results for a single fecal sample and the limited test sensitivity means that we typically need 3-5 cultures per animal, interpreting tests in parallel, to achieve reasonable sensitivity for the overall diagnostic process. 

Polymerase chain reaction (PCR) is generally considered to be a highly sensitive and specific method of Salmonella detection as most assays target highly conserved genes allowing for the detection of many different serotypes while minimizing cross-reaction with other common bacteria [13; 14].  The analytic sensitivity of PCR varies from 100 cfu/gram of equine feces with overnight enrichment to 1000 cfu/gram without enrichment; and PCR is generally more rapid than enriched culture, providing results within 1-2 days (versus 2-5 days) [9; 14-16].  Keep in mind that PCR does not replace the need for culture as generally we need more than a dichotomous (positive/negative) result to facilitate epidemiological investigations and on-going surveillance.     

Commercially-available lateral flow immunoassays (LIAs), developed for use in food safety microbiology, are a promising practical alternative to more traditional detection methods [8; 17].  In experimentally-inoculated equine fecal samples they have been shown to have an analytic sensitivity of 4 cfu/gram of feces after 18-hrs in selective broth culture [8].  The use of these tests does not require any specialized training or equipment – just need an incubator and pre-made media.  There is some variability in the ability of LFIs to detect different strains (serotypes) of Salmonella, however their low cost, ease of use, and reliability make them an appealing option for point-of-care testing in equine practice [18].  Follow-up culture of samples that are LFI-positive should be performed to characterize isolates and inform epidemiological investigations.

Detecting Salmonella in equine practice can be challenging – horses typically shed low numbers of organisms and tend do so intermittently [10; 11].  Regardless of the analytical sensitivity of test methods, this causes the overall detection system (i.e., sample type combined with sample processing and detection method) to have poorer epidemiological sensitivity (i.e., lower probability of detecting truly infected/shedding horses).  While testing larger sample volumes will improve upon test sensitivity up to a point [19], testing of multiple samples may also be utilized.  Interpreting multiple test results in parallel can improve the overall sensitivity of the testing strategy.  Case in point, a truly positive patient is more likely to culture-positive with increased number of samples tested [20].  In application this is generally accepted to be a minimum of 3-5 cultures obtained in a relatively short time frame – if all are negative then we can be reasonably sure that there is a low risk of Salmonella shedding [10].  Assuming independence of test results, this equates to an estimated sensitivity of 44% for a single culture, 66% for two cultures, 82% for three cultures, and 97% for 5 cultures [21]. 

There are many different Salmonella serotypes and their distribution can change over time [22].  Therefore, it is important to ensure that the test can detect the most commonly detected serotypes in a given geographic or practice location [6].  Finally, it is important to use tests that have been appropriately validated for use on veterinary relevant samples.

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