Performance of molecular methods for the detection of Salmonella in human stool specimens [version 1; peer review: awaiting peer review]

Background: The relationship between asymptomatic Salmonella exposure within the gastrointestinal tract and Salmonella 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 Salmonella in stool. The study aimed to determine a reliable molecular diagnostic test for Salmonella in stool specimens. Methods: We optimized an in-house monoplex real time polymerase chain reaction (PCR) for the detection of Salmonella TTR and InvA genes in stool by including a selenite broth pre-culture step for Salmonella 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 TTR and InvA 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. Results: TTR and InvA primers were both able to detect all the different Salmonella 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 Open Peer Review Reviewer Status AWAITING PEER REVIEW Any reports and responses or comments on the article can be found at the end of the article. Page 1 of 11 Wellcome Open Research 2020, 5:237 Last updated: 18 NOV 2020


Introduction
Salmonellae cause a huge global burden of morbidity and mortality. They are globally estimated to be responsible for 300,000 deaths [1][2][3][4] . Salmonella enterica serovars Typhi and Paratyphi A are the predominant cause of invasive Salmonella infections in south and southeast Asia, and cause between 129,000 to 223,000 global deaths per year 1,3,5 . In contrast, non-typhoidal Salmonella (NTS) serovars, principally S. Typhimurium and S. Enteritidis, are a common cause of invasive disease in sub-Saharan Africa (sSA) 4,6 . In 2017, NTS caused an estimated 535,000 cases with sSA having the highest incidence 7 . Risk factors for invasive NTS (iNTS) disease include young age, recent malaria, and advanced HIV disease. Case fatality rates for iNTS in young children, people infected with HIV, and living in the sSA region were estimated at 13.5%, 41.8% and 15.8% respectively 4 . This is in marked contrast to the presentation of Salmonella disease in high-income countries, where NTS typically cause a self-limiting diarrhoeal disease in healthy individuals, while bloodstream or focal infections are rare and mainly occur in individuals with specific risk factors such as diabetes, neoplastic and autoimmune disease, or immunosuppressive therapy 8 . It is notable, however, in both settings, that invasive NTS disease in adults and children are not always associated with diarrhoea 9 .
We previously described in under-five year-old children the sequential development of cellular and humoral immunity against the Salmonella serovars causing iNTS disease, and that acquisition of this immunity is associated with decreasing incidence of disease 10,11 , suggesting that this immunity is protective. Previous studies have reported that healthy young children experience transient asymptomatic episodes of gastrointestinal infection with non-typhoidal Salmonella 12,13 , and we therefore hypothesise that episodes of asymptomatic Salmonella exposure in the healthy gastrointestinal tract during early childhood may facilitate the development of protective immunity. Balanced against this beneficial effect of exposure, diarrhoeal disease results from enteric Salmonella exposure, and invasive NTS disease also follows episodes of asymptomatic gastrointestinal exposure in susceptible children including those with malaria or malnutrition, or immunocompromised individuals.
Elucidating the relationship between Salmonella exposure events within the gastrointestinal tract and resultant Salmonella immunity or Salmonella disease is thus critical for understanding the pathogenesis of iNTS disease. Lack of affordable and rapid diagnostic tools for the detection of bloodstream and intestinal Salmonella disease hampers our understanding of Salmonella disease epidemiology and pathogenesis. Blood culture is considered the gold standard diagnostic test for Salmonella bacteremia and is highly specific but has a number of drawbacks; poor turn-around time of between 2 to 7 days, and low sensitivity of about 20% -30% for samples collected 7 days post-infection [14][15][16] . Molecular detection of Salmonella in blood also has limited apparent sensitivity, and different assays are in development 13,17 .
Stool culture is similarly considered the gold standard test for the detection of Salmonella in the intestinal tract. However, stool culture, even for diarrhoeal disease when the bacterial load is likely to be high, has poor sensitivity (<50%), and is labour and time consuming 18 . Real-time PCR has a short turnaround time and is potentially highly sensitive compared to standard culture, and has the capacity for automation and testing for multiple targets 19 . However, stool PCR test performance is hindered by PCR inhibitors and a large number of genetically closely related enteric bacteria. These pose a challenge in the generation of highly specific and sensitive primers for real-time PCR (qPCR) for Salmonella. Furthermore, a lower infective load of Salmonella colonization during asymptomatic infection may further limit detection by PCR.
With this background, we validated an in-house monoplex qPCR method for the detection of Salmonella in stool specimens, and compared them with a validated multiplex based qPCR and standard stool culture. Both qPCR assays used primers and probes based on the Salmonella tetrathionate respiration gene (TTR), and the Salmonella invasion gene A (InvA). Stool specimens were collected from healthy, mainly asymptomatic healthy Malawian children aged 6-18 months. Assessing the performance of a diagnostic test is challenging when the existing "gold standard" test being used has known low sensitivity or specificity. Statistical methods, such as the Latent Marcov model, are used to assess the performance of diagnostic tests without assigning a gold standard test. Since the current "gold standard" is known to lack sensitivity, we employed a latent Markov model, in order to estimate the specificities and sensitivities of PCR methods without assigning a gold standard.

Description of study participants and specimens
Stool specimens collected from a longitudinal cohort of children aged 6 -18 months who were recruited from Zingwangwa Health Centre (ZHC) in Blantyre, Malawi, were used to compare the performance of molecular and standard culture for detection of Salmonella in stool. The main study started recruitment in August 2013 and follow up was concluded in December 2014 17 . Group sensitization of the study, by well-trained study nurses, was done to parents or guardians of six month old children attending a vaccination clinic at ZHC. Individual sensitization was also done to parents or guardian that were interested in joining the study. Children who met the inclusion criteria of being healthy were recruited into the study after obtaining consent. Children born preterm (less than 38 weeks' gestation), HIV positive or HIV exposed, and those with fever >38°C or any acute illness were excluded from the study 20 .
Stool samples were collected monthly until they were aged 18 months. Stool specimens were collected in sterile and clean containers and transported to the laboratory on the same day. From 60 children who were recruited at 6 months of age, 10 children withdrew from the study, and 600 stool specimens were collected and tested by culture, on the day of sample collection at the College of Medicine and Malawi Liverpool Wellcome Laboratory. Molecular tests were done on frozen samples that were available at the time the tests were done.

Salmonella stool culture
A matchstick head-size sample of stool was inoculated in selenite F broth (Oxoid, UK, catalog number: 2300631) and aerobically incubated overnight at 37 °C for 18-24 hours. The top layer (1 ml) of an overnight culture was spun at 20,000 g for 5 minutes and the pellet was sub-cultured on Xylose Lysine Deoxycholate (XLD) agar (Oxoid, UK, catalog number: 2547703). An aliquot of the selenite broth was also frozen for molecular detection (below). Presumptive Salmonella colonies were cultured onto sheep blood agar (Oxoid, UK, catalog number: 2910831) and MacConkey agar plates (Oxoid, UK, catalog number: 2529552) and incubated aerobically at 37°C for 18-24 hours. Salmonella colonies were then distinguished from other enteric bacteria (i.e. Citrobacter and Serratia) using triple sugar iron agar (Oxoid, UK, catalog number: 1882283) and Urea agar (Oxoid, UK, catalog number: 1779617) biochemical tests. Further Salmonella identification was determined using API® 10S (bioMérieux, France, catalog number: 1007181060) according to the manufacturer's instructions.

Monoplex-qPCR TTR and InvA assay Validation of the monoplex-qPCR TTR and InvA assay.
For the monoplex-qPCR, the TTR primers and probe were designed and validated by Federal Institute for Risk Assessment, Berlin, Germany, according to the published DNA sequence of the S. enterica serotype Typhimurium ttr locus for Salmonella detection (GenBank accession no. AF282268) which had demonstrated high specificity and sensitivity rates when used on food samples 21 . The InvA DNA primers and probe used were previously designed in-house in Malawi for blood samples 17 . Both primers required optimisation for use in stool specimens. The DNA sequence of all the primers and probes used in this study are listed in Table 1.

Specificity of TTR and InvA primer/probe set for
Salmonella compared to other local pathogens To determine the specificity of the primers for Salmonella compared to other local pathogens, 9 different locally isolated and whole genome sequenced Salmonella strains and 26 pure isolates of non-Salmonella bacterial strains locally isolated from blood culture were tested using TTR and InvA primer/probe sets ( Table 2). These strains were chosen because they are genetically closely related to Salmonella or because their growing conditions are similar to Salmonella. These strains were collected from MLW bacterial blood culture repository. Overnight cultures of the frozen samples were made on SBA or LB agar. One colony was then cultured in liquid media. After reaching stationary growth phase, a known and matched concentration of about 10 6 CFU was used for DNA extraction using QIAamp Fast DNA Stool Mini Kit (QIAGEN, Netherlands, catalog number: 51604) but without the bead beating step. Miles and Misra technique was used for bacteria quantification.

Limits of detection in different conditions
A well-characterized invasive S. Typhimurium ST313 strain (D23580), isolated from an HIV negative child in Malawi, and representative of our commonest invasive blood stream infections, was used as a reference strain for determining limits of detection in varying kinds of sample 22,23 . Three types of Salmonella sample were prepared for comparison using RT-PCR; 1) pure Salmonella Table 1. List of primers and probes sequences used in this study. Primers and probes sequences used in this study include inhouse designed InvA, TTR previously validated for Salmonella detection in food, and TAC-InvA and TAC-TTR used on a well validated TAC assay as pan Salmonella primers. isolates picked from a blood agar plate, 2) Salmonella cultured in selenite broth and 3) Salmonella spiked into stool. Salmonella stool spiking in stool was done to determine the inhibitory effect that stool may have on the assay which could then affect the limit of detection. For this, a stool sample was collected from healthy individual and confirmed Salmonella negative by culture. The stool sample was thereafter diluted with PBS (50% w/v) and then spiked with S. Typhimurium, D23580 at varying doses of viable bacteria. The viable dose of Salmonella was adjusted across a range from to 10 0 -10 6 CFU/ml, and quantified using Miles and Misra technique. DNA was extracted for RT-PCR, as above. All experiments were repeated three times on different days, by the same operator. A previously-optimised in-house PCR protocol was used 17 . Briefly, the master-mix for RT-PCR was prepared using predefined quantities. A total of 20μl master-mix for each sample was comprised of the following: 12.5μl Platinum® Quantitative PCR Super Mix-UDG (Life Technologies, USA, Catalog number: 11730025), 0.10μl specific forward primer, 0.10 specific reverse primer, 0.10 specific probe (all primers and probes at 200nM), 0.05μl ROX reference dye (Life Technologies, USA, Catalog number: 12223012) at 50nM final concentration, and 7.15μl nuclease-free water. This mixture was transferred to  To extract total nucleic acid (TNA) from the clinical samples for TAC assay, we used the same DNA extraction kit and protocol that were used to extract whole-stool DNA for the monoplex qPCR assay, with the addition of internal extraction positive controls. For TNA extraction, each sample was extracted together with internal positive controls, Phocine Herpesvirus (PHhv) and MS2. PHhv and MS2 were added to the inhib-itX buffer before being added to each sample, as previously described 24 . An assay was considered to have passed when both MS2 and PhHv internal positive (amplification crossing the threshold) and negative controls (no amplification crossing the threshold line) passed, and when the sample had sigmoid curve that crossed the threshold line. Only results for Salmonella are reported here.

Statistical analysis
Data were recorded and analysed in MS Excel (version 16.14.1 (18061302)). Sensitivities and specificities of the different PCR methods were estimated using a latent Markov model (LMM) 25 . We have previously described the LMM and various extensions that we considered for modeling longitudinal diagnostic test data 26 . We implemented the LMM within a Bayesian framework using R (version v3.5.1) and JAGS (version 4.3.0) via the rjags (version 4.6) R package 27 . LMMs have been extensively used for discrete-time longitudinal data in the absence of a gold standard diagnostic procedure 28,29 . We considered several LMMs, with and without mixed effects and with either time-homogeneous or time-heterogeneous transition matrices 26 . Convergence and identifiability of the LMM were checked by inspecting trace plots and computing Gelman-Rubin potential scale reduction factors 30 . The more complex models exhibited poor mixing or convergence of MCMC chains (most likely due to the sparse number of positive samples) and as a result the LMM we used for this dataset is a basic LMM with no random effects and a time-homogeneous transition matrix. To report positive predictive values (PPV) and negative predictive values (NPV), we calculated an estimate of the infection prevalence. For the Bayesian LMM, we report maximum a posteriori (MAP) parameter estimates together with 95% credible intervals (Crl), specifically the highest posterior density intervals (HDI) with 95% coverage. All other analyses report (frequentist) parameter estimates and corresponding 95% confidence estimates (CI).

Ethical considerations
Ethical approval for this work was granted by the University of Malawi, College of Medicine Research Ethics Committee (P.01/13/1327). Written informed consent was obtained from the parent or guardian of each participating child.

Results
TTR and InvA primers for Salmonella do not cross-react with closely related enteric micro-organisms We first validated the TTR and InvA primers that were used in the monoplex-qPCR assay, by assessing the sensitivity and specificity of the primers for Salmonella, using a standardized number of 10 0 -10 6 CFU/ml of 9 different locally-relevant Salmonella strains, and 26 non-Salmonella bacterial strains as indicated in Table 2. We included 17 strains of E. coli because of the close genomic relatedness of Salmonella and E. coli. Bacterial isolates that were either enriched in Selenite F broth (referred here as selenite sub-cultured) or not (referred here as direct culture) were used in this evaluation. We found that TTR and InvA assays both achieved 100% sensitivity and specificity either as direct isolates or selenite sub-cultured isolates. Table 2 demonstrates that all Salmonella strains tested positive with both monoplexed primer pairs, and all other bacterial strains were negative, confirming a lack of cross-reactivity.
Selenite broth culture enhances the detection of Salmonella in stool using either TTR or InvA primers The limits of detection (LOD) of qPCR for Salmonella were then determined using S. Typhimurium strain D23580 serially diluted, and tested as direct isolates, selenite broth cultured samples, or isolates spiked into a culture-negative stool specimen. We found that limits of detection for TTR were 1, 10 and 100 CFU/ml, and for InvA were 1, 100 and 100 CFU/ml for selenite sub-cultured broth, direct isolates and stoolspiked isolates respectively, with 98.5% qPCR efficiency for TTR and 97.2% qPCR efficiency for InvA. No statistically significant difference was observed in the LOD when TTR was compared with InvA in either direct isolates (p = 0.3212), selenite sub-cultured samples (P = 0.2534), or salmonella spiked stool samples (P = 0.2361). Importantly, we found that the TTR assay was significantly different when direct isolates (LOD = 10 CFU/ml) were compared with selenite sub-cultured samples (LOD = 1 CFU/ml) (p<0.0001), and when selenite sub-cultured isolates were compared to Salmonella spiked stool (p <0.0001), and there was no significant difference when direct isolates were compared to Salmonella spiked stool (p=0.2965).
Similarly, we found that detection in InvA qPCR assay direct isolates was significantly different compared to selenite broth cultures isolates (p < 0.0001), and selenite subculture isolates were also significantly different to Salmonella spiked stool (p < 0.0001), while no significant difference was found between direct isolates compared to Salmonella spiked stool samples (p = 0.2862). In summary, we found that selenite broth overnight liquid culture of stool samples enhanced the molecular detection of Salmonella using either TTR or InvA primers, even if culture of the broth remained negative.
TTR and InvA primers had both high specificity and sensitivity rates, whilst stool culture had high specificity but low sensitivity The samples from healthy children were used to determine the performance of stool culture, monoplex TTR, monoplex InvA, multiplex TAC TTR, and multiplex TAC InvA. Standard stool culture was performed on a total of 600 specimens at different time points. Molecular tests were used to detect Salmonella in the available 421 stool DNA specimens. We detected Salmonella in 23, 40, 29, 56, and 47 of 421 stool specimens, using standard stool culture, TTR, InvA, TAC-TTR, and TAC-InvA respectively. Of the 23 Salmonella stool culture-positive samples, 21 samples were also positive with either one or more molecular tests whilst 2 were negative with molecular tests.
Based on a time-homogeneous LMM without random effects (Table 3 and Figure 1A) we reported the specificities and sensitivities of the detection methods with their 95% credible intervals (Bayesian confidence intervals). The observed specificity rates from highest to lowest were for stool culture (99.99%), TAC-TTR (99.30%), TAC-InvA (98.00%), monoplex TTR (95.46%) and monoplex InvA (90.31%) respectively. The observed sensitivity rates from highest to lowest were monoplex TTR (99.53%), monoplex InvA (95.06%), TAC-TTR (90.30%), TAC-InvA (89.41%) and stool culture (62.88%) respectively (Table 3 and Figure 1A).  High negative and positive concordance for stool culture, monoplex TTR, monoplex InvA, Multiplex TTR, and multiplex InvA Next, we explored correlations between stool culture, monoplex TTR, monoplex InvA, Multiplex TTR, and multiplex InvA . In this exploration, we considered all test results, whether positive or negative. To account for both censored observations and the longitudinal nature of the data, we calculated repeated measures of correlation coefficients 31 using the ranks of observations for each test (akin to a repeated-measures Spearman correlation coefficient) for measuring the correlation between the Ct values for the four molecular tests and point biserial correlation coefficients based on ranks for measuring correlations between standard stool culture and each of the qPCR tests (Figure 2A). The correlation coefficients vary quite widely from 0.12 (monoplex InvA and TAC-InvA) to 0.8 (stool culture and TAC-TTR). Given that for truly negative samples, the Ct values are effectively randomly distributed near the threshold used to discriminate between positive and negative samples, and that most samples were negative in most tests, the somewhat weak correlations we observe can be driven by the random Ct values for negative samples. For this reason, using only the binary negative / positive outcomes for each test, we computed positive ( Figure 2B) and negative ( Figure 2C) concordance: for example, in Figure 2B, the intersection of the row labelled 'TTR' and the column labelled 'InvA' lists the proportion of positive test results for the TTR test that are also positive for the InvA test. Unexpectedly (given that most samples were negative), negative concordance ( Figure 2C) was very high, with the lowest negative concordance being 89%. Results for positive concordance ( Figure 2B) are also quite high, though there is more variation, ranging from 25% (for positive InvA results confirmed by positive stool cultures) to 100% (positive stool cultures confirmed by positive monoplex TTR or positive monoplex InvA).  Table 3 and Figure 1B.

Discussion
The burden of asymptomatic gastrointestinal exposure to Salmonella which could be linked to either the development of immunity, or conversely to blood-stream infection is not known, due to lack of robust Salmonella detection methods for stool specimens. This study aimed to optimize detection methods, and to validate and compare the performance of monoplex TTR and InvA qPCR assays (TTR and InvA), against TTR and InvA qPCR assays on a validated multiplex qPCR platform (TAC-TTR and TAC-InvA), and compare all molecular methods to standard Salmonella stool culture. Validation of the monoplex TTR and InvA primers showed that the primers do not cross react with other enteric pathogens, and LOD testing showed that selenite pre-culture promotes molecular detection, even when culture is negative. Stool culture demonstrated the highest specificity but low sensitivity than all the molecular tests. Stool culture, despite having low sensitivity, still Correlation coefficients for the four molecular tests (using Ct values) and stools culture using positive or negative (Figure 2A). Concordance coefficients for positive ( Figure 2B) and negative ( Figure 2C) diagnosis obtained using binary negative or positive outcomes for each test. For example, in Figure 2B, the intersection of the row labelled 'Culture' and the column labelled 'TTR' lists the proportion of positive test results for the Culture test that are also positive for the TTR test. Both the size and colour depth represent the magnitude of correlation.
remains important in Salmonella diagnosis. Culture allows for antimicrobial susceptibility testing and strain typing. TTR detected on the monoplex platform demonstrated superior sensitivity to stool culture, InvA, TAC-TTR, and TAC-InvA. All the test methods however displayed high concordance to each other.
Several studies have developed Salmonella detection methods based on antigen detection or nucleic acid amplification 16,18,32,33 . Both monoplex and multiplex nucleic acid amplificationbased detection methods have been developed [34][35][36][37] . Most of these have however focused on Salmonella detection in blood as opposed to stool specimens. Some multiplex qPCRs to specifically detect Salmonella and its serovars, or for the detection of multiple enteric pathogens in stool specimen (including Salmonella) have recently been developed 24,[38][39][40] . The advantage of multiplex qPCR is that it is fast in determining the main etiological agent in cases where the outcome is caused by multiple pathogens or different serovars, but it is expensive if one is interested in detecting only one particular pathogen. By contract, the advantage of a monoplex test is that it is economical. In this study, the same primer/ probe sets were tested using both the monoplex and multiplex qPCR platforms. The monoplex qPCR maximized sensitivity while the multiplex panel provided a balanced pay-off between sensitivity and specificity. The high sensitivities of the monoplex qPCR could be attributed to the use of selenite pre-cultured stool as opposed to extraction of DNA from neat stool samples which is used in the multiplex qPCR. Selenite sub-cultured stool samples were not be used on the multiplex platform because the manufacturer's protocol was followed. Other studies have, however, also demonstrated superior performance of monoplex qPCR when compared with multiplex qPCR. The monoplex qPCR is therefore ideal for studies that are only interested in determining the presence or absence of Salmonella whilst capitalizing on the sensitivity of the test while multiplex qPCR will have an added advantage if a study wants to detect multiple pathogens whilst having a pay-off between sensitivity and specificity.
The TTR primer/ probe set used in the monoplex qPCR was previously validated for use in food samples and required validation in stool specimens. Our in-house developed InvA primer/ probe set also required validation. Both assays demonstrated that they can detect all the different Salmonella strains including S. Enteritidis, S. Typhimurium, and S. Typhi strains which are the commonly isolated strains in Malawi and sSA 41 .
Comparing the limits of detection of different Salmonella isolate conditions demonstrated that selenite pre-culture achieves a significantly lower limit of detection (1 CFU/ml) as opposed to direct isolates (10 CFU/ml) and Salmonella-spiked stool (10 CFU/ml). Selenite F broth is a selective broth that suppresses the growth of fecal coliforms and streptococci in order to optimize Salmonella growth 42  We used an LMM to estimate the specificities and sensitivities of the 5 Salmonella detection methods. Stool culture demonstrated the highest specificity but had the lowest sensitivity. All molecular assays;TAC-TTR, TAC-InvA, TTR, and InvA, demonstrated high specificity and sensitivity rates. Compared to the other methods, the monoplex based qPCR TTR achieved the best sensitivity-specificity trade-off as it demonstrates near-perfect sensitivity (99.53%) and still achieves high specificity (95.43%). All molecular test methods had significantly higher sensitivities than stool culture. High specificity and low sensitivity rate for culture have been widely reported 18 . Such low sensitivity rates should be taken into consideration when evaluating diagnostic tests. It is clear that a reference test with poor sensitivity is not adequate to evaluate alternative test methods. In such a situation alternative means of evaluating the assays should be used such as the LMM that has been used here. LMMs, and their counterpart for cross-sectional data, latent class models (LCMs), have been used to evaluate diagnostic tests for different pathogens including Salmonella 44 .
PPV and NPV vary depending on the prevalence of the condition being tested in any particular population. Our samples were collected from a population that was considered healthy and asymptomatic at the time of recruitment. Using the model-estimated stationary probability of being infected, we estimated the Salmonella infection prevalence of 5.25% in this population. With this prevalence estimate, stool culture demonstrated a high PPV when compared to molecular tests that had high NPVs. When prevalence is low, a small change in specificity will have significant effects on the PPV. Higher PPVs could be observed in a situation where prevalence is high such as when using a cohort of hospitalized diarrheal cases, or during a diarrhoeal outbreak.
Molecular methods had higher sensitivity but lower specificity, relative to stool culture. The loss in specificity is small compared to the gain in sensitivity and, in the case of Salmonella, the public health cost of false-negative results could be higher if the infection becomes potentially life-threatening due to withholding or delay of treatment. With the high sensitivity, molecular methods were able to detect asymptomatic Salmonella events, critical for the research questions we hoped to pose in this cohort. All the events that were detected here were asymptomatic in healthy children, which are potentially very important in transmission or the development of immunity. The detection of low bacterial burden events could also be relevant in settings like Malawi where unprescribed over-the-counter antibiotic procurement and use is common. Studies that have reported on risk factors of having a culturenegative result has indicated that antibiotic usage before sample collection is the main risk factor. Using molecular techniques such as PCR could overcome this challenge because it detects bacterial DNA regardless of the viability of the pathogen. This might increase the probability of identifying the infection and reduce sample processing time which could then with proper patient management and treatment if needed.
Our study has several limitations. One main limitation is the use of different sample types for the two qPCR platforms. The use of selenite sub-cultured stool samples in monoplex qPCR may have contributed to the superior performance when compared with the multiplex qPCR. We used neat stool samples for multiplex qPCR to comply with the manufacturer's protocol. Other studies have however demonstrated that testing primer/ probe sets in the monoplex platform perform better than in the multiplex qPCR platform. Clinical samples used to test the performance of the test are a limitation especially in determining the PPV and NPV. Clinical samples used in the study were collected from a cohort of children that were asymptomatic to Salmonella and remained healthy for most of the one-year study period. Using samples from participants with a clinical diagnosis of Salmonella or diarrhea would improve the PPV and NPV.

Conclusion
The data presented here demonstrate that the addition of selenite pre-enrichment step increases Salmonella detection in stool samples, and that TTR and InvA primer and probe sets used are able to detect different Salmonella strains. The ability of TTR to detect Salmonella with such high levels of specificity and sensitivity when tested using clinical samples collected from a cohort that was mostly healthy, make it a promising assay that could be used for research surveillance studies. The assays could be very useful in studying the transmission of Salmonella infections. This method may perform with different sensitivity and specificity in a chronic carriage, diarrhoeal or invasive Salmonella disease state, since the load and culturability of the pathogen within the stool may be different, and further validation studies would be needed We established that selenite pre-culture increased diagnostic yield for molecular detection and identified TTR primers as molecular tools that could best help to reveal the true extent of Salmonella exposure events within the gastrointestinal tract. This will allow us to understand their importance to diarrhoeal and invasive disease pathogenesis and epidemiology in the future.