Salmonella enterica serovar typhi (S. typhi) is the Gram-negative bacterium leading cause of community-acquired bloodstream infections in many low- and middle-income countries[1, 2] and responsible for typhoid fever, a systemic infection characterized by prolonged fever, headache, and abdominal symptoms[2]. 

Salmonella enterica serovars typhi, paratyphi A, paratyphi B, and paratyphi C are collectively known as typhoidal Salmonella, which are human host-restricted bacteria responsible for typhoid and paratyphoid (enteric) fever. All other serovars are grouped as nontyphoidal Salmonella (NTS), which may infect a wide range of animals or be host-adapted to specific nonhuman species[3]. 

Accurate and rapid diagnosis of typhoid fever is crucial for effective treatment and to prevent the spread of the disease, especially in endemic regions. While traditional methods like blood culture remain the gold standard, they are often time-consuming and can have limited sensitivity, particularly if antibiotics have been initiated.

Molecular identification techniques, especially those based on the Polymerase Chain Reaction (PCR), have revolutionized S. typhi detection by offering superior speed, sensitivity, and specificity. PCR-based methods amplify specific DNA sequences unique to the pathogen, allowing for its direct detection in clinical samples[4].

The success of PCR for S. typhi identification hinges on the careful selection of target genes that are both highly conserved within S. typhi and absent or significantly different in other Salmonella serovars and common commensal bacteria. Several genes have been explored as PCR targets, each with its own advantages and limitations.

The tviA gene (or tviB) within the viaB operon is frequently targeted due to its strong association with S. typhi and its essential role in Vi antigen production. PCR assays targeting tviA have shown high specificity for S. typhi[5, 6]. However, it's important to note that S. paratyphi C and some S. dublin strains also possess the Vi capsule, which can lead to false positives for S. typhi if not combined with other targets in a multiplex PCR. While fliC-d is characteristic of S. typhi, it is also found in some other Salmonella serovars (e.g., S. muenchen). Therefore, using fliC-d alone for S. typhi identification lacks absolute specificity. Similarly, the tyv gene is specific for S. typhi, while prt is found in S. paratyphi A. These can be useful for differentiating between typhoidal serovars[7]. However, like fliC-d, they may not be exclusively specific to S. typhi when considered in isolation from all other Salmonella serovars.

With the availability of whole-genome sequences, bioinformatic approaches have identified chromosomal genes that are highly conserved in S. typhi but absent or significantly divergent in other Salmonella serovars and non-Salmonella bacteria[8, 9]. These genes represent potentially more specific targets. Studies have reported several such genes (e.g., STY0307, STY0322, STY0326, STY2020, STY2021, STY0201, stoD) demonstrated 100% sensitivity and specificity when tested against panels of S. typhi, non-typhi Salmonella, and other bacterial isolates. These targets aim to overcome the limitations of previously used genes that might share homology with other serovars[10]. These target genes are reported in detecting S. typhi but their specificity needs to be evaluated with more clinical strains.

These newer, highly specific targets are increasingly being incorporated into single-gene PCR or multiplex PCR assays for robust and definitive S. typhi identification. In the present investigation, we aimed to analysed single-gene target PCR assays (STY0201, STY0307, STY0322, STY0326, STY2020, STY2021) for the reliable molecular identification of Salmonella typhi strains recovered from blood cultures of patients presenting with typhoid fever.

Screening and identification of Salmonella typhi

Salmonella typhi, the test organism, was isolated from blood samples obtained from patients clinically diagnosed with typhoid fever at Adesh Hospital, Bathinda. Isolation was conducted using Xylose Lysine Deoxycholate (XLD, Hi-media) selective agar. Briefly, 100 µl of blood was aseptically inoculated into 10 ml of nutrient broth and incubated at 37°C for 24 hours for enrichment. Subsequently, 100 µl of the enriched broth culture was spread onto XLD selective agar plates, which were incubated at 37°C for 24 hours or until bacterial colonies developed. All isolates used in this study were confirmed as S. typhi through conventional culture techniques, biochemical characterization, and 16S rRNA gene analysis.

Optimization of blood culture PCR assay

To increase PCR sensitivity, it's crucial to first remove inhibitory human DNA from the sample. Additionally, a short pre-incubation of blood with culture broth boosts PCR amplification by increasing target DNA concentration. The 2 ml blood was added to 20 ml of culture media [3% (w/v) ox bile/tryptone soya broth] in 50 ml falcon tubes (Tarson) and incubated for 5 hrs at 37°C. Bacteria were concentrated by centrifugation at 8000 rpm for 10 min and the supernatant was removed. The human DNA removal by mixing thoroughly with an equal volume of mammalian cell lysis buffer (2 M Na2CO2 pH 9.8, 1% Triton-X100) for three minutes at ambient temperature to allow for complete fragmentation of human chromatin into DNA fragments. After the incubation step an equal volume of neutralization buffer (1.0 M Tris–HCl, pH 4.5) was added in order to prevent further cell lysis. The samples were then centrifuged at 5000 rpm for 10 min. The supernatants were discarded and bacterial pellets were resuscitated in 200 μl of 1x phosphate buffered saline (PBS) and used for isolation of bacterial DNA by a conventional method using the NaOH/SDS approach[11]. Similarly isolated colonies on XLD media also explored for the DNA isolation by NaOH/SDS approach and DNA processed for PCR amplification.

DNA Purity & integrity

The purity and concentration of the extracted DNA were assessed using a Cary UV-Visible Spectrophotometer (Agilent Technologies). DNA concentration was determined by measuring absorbance at 260 nm, while the A260/280 ratio was used to evaluate DNA purity. The extracted DNA was diluted with MilliQ water to a final concentration of 50 ng/µL and stored at −20 °C for subsequent analyses. Genomic DNA integrity was evaluated by electrophoresis on a 0.8% agarose gel at 80 V (BIO-RAD), followed by staining with ethidium bromide and visualization using a Gel Doc EZ imager (BIO-RAD).

Optimization of PCR

After S. typhi DNA extraction from the different patients and the amplification were done on BIO-RAD T100 Thermocycler with a set of six primers selected from previous study[10].

Each PCR assay was optimized to assess the effects and interactions of two main variables: S. typhi-specific gene primer concentrations and annealing temperatures, each tested at three levels (primer concentrations: 1.00, 1.50, and 2.00 µM; annealing temperatures: 50, 55, and 60°C). PCR amplification was performed in a total reaction volume of 20 µl, comprising 10 µl of 2× PCR master mix (SRL), 5 µl of DNA template, 2–4 µl of forward and reverse primers (combined), and MilliQ water to a final volume of 20 µl. The thermal cycling conditions were as follows: initial denaturation at 94°C for 2 minutes; 34 cycles of denaturation at 94°C for 45 seconds, annealing at 50–60°C for 45 seconds, and extension at 72°C for 45 seconds; with a final extension at 74°C for 5 minutes. PCR products were analyzed alongside a 3 kb molecular weight marker (SRL) on a 1.2% (w/v) agarose gel containing ethidium bromide and visualized using a Gel Doc EZ imager (BIO-RAD).

Sequencing

The purified PCR amplicons were submitted to Central University, Bathinda, for definitive identification through 16S rRNA region sequencing ([Table. 1]). The resulting nucleic acid sequences, derived from both forward and reverse cycle sequencing reactions, underwent bioinformatic analysis using DNA Baser software. This analysis involved sequence trimming and subsequent alignment with previously curated sequence data available in the GenBank database via the NCBI Basic Local Alignment Search Tool (BLAST).

This study aimed at the molecular identification of Salmonella typhi isolates obtained from the blood samples of patients diagnosed with typhoid fever. Three representative isolates were selected for 16S rRNA gene sequencing. The resulting sequences, submitted to GenBank with accession numbers MT065760, MT065761, and MT065762, were analysed using the NCBI BLASTn algorithm. The analysis confirmed their identity as S. typhi, showing 100% query coverage and sequence identity, with an E-value of 0.

To develop a rapid diagnostic tool, single gene-target PCR assays were optimized using six different primer sets, with an annealing temperature of 55 °C. These assays targeted the STY2020, STY2021, STY0201, STY0307, STY0322, and STY0326 genes, as previously described by Goay et al.[10] Clinical evaluation of the assays demonstrated successful detection of S. typhi by five targets: STY0307 (495 ± 5.0 bp), STY0326 (261 ± 1.0 bp), STY2020 (429 ± 4 bp), STY2021 (732 ± 2.0 bp), and STY0201 (1176 ± 6 bp). These assays accurately identified all S. typhi isolates without cross-reactivity or false positives among non-Salmonella isolates ([Fig. 1]; [Table. 2]), resulting in 100% sensitivity and 100% specificity. In contrast, the assay targeting STY0322 failed to detect any S. typhi strains.

Serial dilutions of S. typhi genomic DNA were performed to determine the detection limits of the optimized PCR assays. The assay targeting the STY0307 gene exhibited the highest sensitivity, with a detection limit of 5.0 pg. This was followed by the STY0201 and STY2021 assays, each with a detection limit of 10 pg. In contrast, the STY2020 and STY0326 assays demonstrated lower sensitivity, with detection limits of 50 pg and produced only faint bands during gel electrophoresis. These findings suggest that STY0307, STY2021, and STY0201 are more reliable and sensitive molecular markers for the detection of S. typhi compared to STY2020 and STY0326.

Notably, the primer targeting the STY0322 gene failed to produce any visible amplification bands during gel electrophoresis for all patient-derived S. typhi isolates. This contrasts with the findings of Goay et al.[10], who reported that STY0322, along with four other genes, was highly conserved among S. typhi strains.

Ngan et al.[12] and Pratap et al.[13] demonstrated that the STY0201 gene serves as an effective PCR target, with assays based on this gene exhibiting 100% sensitivity and specificity. The results of the present study are consistent with these findings. However, Goay et al.[10] reported that the STY0201 gene showed only 97.2% specificity, noting cross-reactivity with S. oslo and S. kissi. Additionally, Tracz et al.[14] found that the STY4220 locus was present in both Salmonella serovars Typhi and Paratyphi A.

Goay et al.[10] reported that the genes STY0307, STY0322, and STY0326 encode hypothetical proteins, while STY2020 and STY2021 encode putative bacteriophage proteins. Notably, STY0307, STY0322, and STY0326 are located within Salmonella Pathogenicity Island 6 (SPI-6), although their roles in bacterial virulence and pathogenicity remain uncharacterized. In the present study, the STY0322 gene was not detected among isolates from the local ethnic population, in contrast to its presence in Malaysian isolates as reported by Goay et al.[10], suggesting possible regional variation. Further studies are needed to substantiate this observation.

Additionally, the expression of STY2020 and STY0326 was found to be markedly low during typhoid infection in this study, compared to the findings of Goay et al.[10]. This highlights the need for further investigation into the expression profiles of these genes across different populations.

Overall, STY0307 and STY2021 emerged as the most specific and sensitive markers for the detection of S. typhi. These genes offer promising prospects for the development of novel point-of-care diagnostic assays that are cost-effective, straightforward, rapid, and accurate—particularly beneficial in resource-constrained environments. 

In conclusion, clinical testing in the present study revealed that three S. typhi-specific genes—STY0307, STY0201, and STY2021—demonstrated high sensitivity and specificity, outperforming STY2020 and STY0326. These genes represent promising diagnostic targets and could be effectively utilized in the development of DNA-based diagnostic tools for the accurate and sensitive clinical detection of typhoid fever.