Abstract
Background: Typhoid fever a major public health problem in developing countries. Epidemiological investigations of Salmonella enterica serovar Typhi (Salmonella Typhi) infections using molecular sub-typing methods are challenging due to the highly homologous nature of Salmonella Typhi strains. In recent years, several approaches in using multiple-locus variable-number tandem-repeats analysis (MLVA) for molecular sub-typing of Salmonella Typhi have been made. To date, a standardized set of variable-number tandem-repeats (VNTR) loci for the typing of homologous Salmonella Typhi strains has not been established. The aim of our study was to develop and evaluate a MLVA assay consisting of 5 VNTR markers to analyse Salmonella Typhi strains from Sub-Saharan Africa (SSA). Methods & Materials: To develop and evaluate the MLVA assay, 50 Salmonella Typhi strains from humans were selected from the culture collection at the Centre for Enteric Diseases (CED) at the National Institute for Communicable Diseases (NICD) from a potential 1080 isolates. This strain panel represented the diverse Salmonella Typhi pulsed-field gel electrophoresis (PFGE) profiles in the CED database, specimen collection dates and geographic areas within the SSA region. The 50 Salmonella Typhi strains were used to evaluate 12 polymorphic VNTR loci that were previously published. These include TR1, TR2, TR3, TR4, TR5, Sal02, Sal06, Sal10, Sal16, Sal20, TR4500 and TR4600. The assay included PCR amplification of VNTR loci using fluorescently labelled primers and size determination of PCR products by capillary electrophoresis. Results: Of the 12 VNTR loci that were evaluated, 5 (TR3, TR4, TR5, Sal06, Sal10 and TR4500) were found unsuitable as they showed poor allele variation (between 1 and 3 alleles). The remaining 6 VNTR loci showed good allele variation and good diversity indices; with Sal20, Sal 16 and TR1 having 6, 9 and 11 alleles respectively; and Sal02, TR4699 and TR2 having 15, 20 and 23 alleles respectively. Conclusion: In summary, we have identified 6 polymorphic VNTR loci suitable for MLVA analysis of Salmonella Typhi strains. The five most diverse VNTR loci will be selected for the MLVA assay and will be used to analyse Salmonella Typhi strains from SSA. This work will assist in rapidly identifying strain relatedness and assist outbreak detection in typhoid fever outbreaks.
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