Evaluation of an SNP-Based Diagnostic Assay for Enteric Fever Detection in Resource-Limited Settings

The diagnosis of enteric fever has become difficult due to the nonspecific and overlapping clinical syndrome of typhoid and paratyphoid infections with other febrile illnesses. Moreover, the rapid emergence of fluoroquinolone-resistant typhoidal Salmonella and the lack of robust diagnostic methods highlight the urgent need for highly sensitive molecular techniques. Here, we evaluated the performance of a rapid, reliable, and cost-effective molecular diagnostic approach for detecting Salmonella Typhi, including the globally dominant haplotype H58 lineage (H58), and Salmonella Paratyphi A. An in-house-built conventional polymerase chain reaction (PCR) was performed on a collection of blood-culture-positive strains, and the sensitivity and specificity were compared with those of the standard blood culture results. H58 and non-H58 Typhi lineages with distinct resistance patterns were confirmed from the previously reported sequencing data. Our PCR result showed that target genes SSPA2308, STY2513, and STY0307 demonstrated 100% sensitivity and specificity for Salmonella Paratyphi A, Salmonella Typhi, and H58 Salmonella Typhi, respectively. The PCR assay reliably detected bacterial DNA at 5.2 × 104 colony-forming units (CFUs), with consistent amplification observed up to 10−1 dilution. This single-nucleotide polymorphism (SNP)-based diagnostic approach has added a new dimension to designing unique markers for multidrug-resistant (MDR)-associated H58 lineage detection and has the potential to inform local treatment algorithms.