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

The relationship between asymptomatic exposure within the gastrointestinal tract and 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 in stool. The study aimed to determine a reliable molecular diagnostic test for in stool specimens. We optimized an in-house monoplex real time polymerase chain reaction (PCR) for the detection of and genes in stool by including a selenite broth pre-culture step for 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 and 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 a period of 12 months. We employed a latent Markov model to estimate the specificities and sensitivities of PCR methods. : TTR and InvA primers were both able to detect all the different serovars tested, and had superior limits of detection if DNA was extracted after selenite pre-culture. TTR sensitivity and specificity for monoplex-PCR were (99.53%, 95.46%) and for multiplex-PCR (90.30%, 99.30%) respectively. InvA 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 mono-TTR had the highest NPV (99.67%). Test methods demonstrated high concordance although stool culture and monoplexed TTR primers had superior specificity and sensitivity respectively. The use of selenite pre-enrichment step increased detection rate. Taken together, molecular detection methods used here could be used to reveal the true extent of both asymptomatic and symptomatic exposure events.