Performance of molecular methods for the detection of Salmonella in human stool specimens

Abstract

<ns3:p> <ns3:bold>Background:</ns3:bold> The relationship between asymptomatic <ns3:italic>Salmonella</ns3:italic> exposure within the gastrointestinal tract and <ns3:italic>Salmonella</ns3:italic> bacteraemia is poorly understood, in part due to the low sensitivity of stool culture and the lack of validated molecular diagnostic tests for the detection of <ns3:italic>Salmonella</ns3:italic> in the stool. The study aimed to determine a reliable molecular diagnostic test for <ns3:italic>Salmonella</ns3:italic> in stool specimens. </ns3:p> <ns3:p> <ns3:bold>Methods:</ns3:bold> We optimised an in-house monoplex real-time polymerase chain reaction (PCR) for the detection of <ns3:italic>Salmonella</ns3:italic> <ns3:italic>ttr</ns3:italic> and <ns3:italic>InvA</ns3:italic> genes in stool by including a selenite broth pre-culture step for <ns3:italic>Salmonella</ns3:italic> before DNA extraction and validated their specificity against other local common pathogens. Then we assessed their performance against a well-validated multiplex PCR targeting the same <ns3:italic>ttr</ns3:italic> and <ns3:italic>InvA</ns3:italic> genes and against stool culture using clinical stool specimens collected from a cohort of 50 asymptomatic healthy Malawian children that were sampled at 1-month intervals over 12 months. We employed a latent Markov model to estimate the specificities and sensitivities of PCR methods. </ns3:p> <ns3:p> <ns3:bold>Results</ns3:bold> : <ns3:italic>Ttr</ns3:italic> and <ns3:italic>InvA</ns3:italic> primers were both able to detect all the different <ns3:italic>Salmonella</ns3:italic> serovars tested and had superior limits of detection when DNA was extracted after selenite pre-culture. T <ns3:italic>tr</ns3:italic> sensitivity and specificity for monoplex-PCR were (99.53%, 95.46%) and for multiplex-PCR (90.30%, 99.30%) respectively. <ns3:italic>InvA</ns3:italic> specificity and specificity for using monoplex-PCR was (95.06%, 90.31%) and multiplex-PCRs (89.41%, 98.00%) respectively. Sensitivity and specificity for standard stool culture were 62.88% and 99.99%, respectively. Culture showed the highest PPV (99.73%), and monoplex- <ns3:italic>ttr</ns3:italic> had the highest NPV (99.67%). </ns3:p> <ns3:p> <ns3:bold>Conclusion:</ns3:bold> Test methods demonstrated high concordance, although stool culture and monoplexed <ns3:italic>ttr</ns3:italic> primers had superior specificity and sensitivity, respectively. The use of selenite pre-enrichment step increased <ns3:italic>Salmonella</ns3:italic> detection rate. Taken together, molecular detection methods used here could be used to reveal the true extent of both asymptomatic and symptomatic <ns3:italic>Salmonella</ns3:italic> exposure events. </ns3:p>

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