Typhoid fever is a neglected re-emerging and potentially fatal infectious disease caused by S.Typhi. The disease is characterized by illness of multi-organ system and acquired through faecal-oral route by consumption of contaminated food and water. The disease is endemic in developing countries but alarming number of cases linked to domestic sources have been reported in industrialised countries. The clinical presentations of typhoid fever is highly variable and often overlap with other acute undifferentiated febrile illness (AUFI). Culture isolation is the gold standard for the diagnosis of typhoid. However, culture isolation is time-consuming, laborious with variable sensitivity and often limited to secondary or tertiary laboratory facilities. On the other hand, serological interference has been recognized as a major drawback in the reported diagnostic kits for typhoid fever. Hence, accurate, rapid and cost-effective diagnostic test are required for early life saving treatment and effective epidemiological intervention. This present study report the development of TYPHOIDYNE EIA using arrays of multi-antigen for definitive and differential diagnosis of typhoid fever. Multiple antigens were comprised of whole cell protein (WCP), cell surface protein(CSP) and surface depleted-whole cell protein(sdWCP) derived from differentially extracted whole cell bacterial proteins of S.Typhi and S.spp . Sensitivity and specificity of TYPHOIDYNE EIA were evaluated using panel of sera consisting of typhoid patient, typhoid vaccinated subject, healthy subject and subject with other diseases. The performance of the test was encouraging with sensitivity of 97.3% and specificity of 100%. The positive predictive value (PPV) and negative predictive value (NPV) of the assay were 100% and 97%, respectively. Multi-antigens derived from species-specific and genus conserved play an important role in profiling heterogenicity in typhoid immune response that lead to excellent synergistic effect in differential and definitive diagnosis of typhoid fever. This preliminary report showed that TYPHOIDYNE EIA successfully detected and differentiated typhoid cases, typhoid vaccinated individuals, typhoid carriers and healthy individuals. To our knowledge, this is the first report to describe the successful use of multi-antigens based microspot arrays enzyme immunoassay to characterize the heterogenicity of immune response and their application in diagnosis of typhoid fever.

Isolation, purification, and separation of complex mixtures is crucial in proteomic research. The conventional electrophoresis method for antigen characterization has limitations in separating low abundance components and selectively enriching important proteins with high degree of purity. Two-dimensional gel electrophoresis was introduced to overcome these limitations, but it also has inherent shortcomings in detecting hydrophobic proteins, low abundance proteins, or samples with proteins of various concentrations. Therefore, this study aims to develop an innovative approach for the enrichment and characterization of immunoreactive components found in differentially extracted whole cell bacterial protein derived from S.Typhi and S.spp. A modified liquid phase preparative isoelectric focusing (IEF) and SDS-PAGE method was used to purify and characterize the proteins. The modified liquid phase IEF efficiently fractionated the proteins into 20 fractions based on the pI value, providing a high-resolution power for protein separation, high throughput, and ease of performance. The fractionated proteins were then analysed by SDS-PAGE for their molecular weight, providing a simple and cost-efficient method for protein analysis. This innovative approach for the enrichment and characterization of immunoreactive components in differentially extracted whole cell bacterial protein derived from S.Typhi and S.spp has the potential to revolutionize the diagnosis and treatment of typhoid fever and other related diseases. By improving the sensitivity and accuracy of protein analysis, this study may lead to identification of exclusive disease biomarkers for early, accurate diagnosis of diseases, improved prognosis and treatment outcomes.

Typhoid fever is a multiorgan infectious disease caused by Salmonella typhi. It is transmitted through fecal oral route and can be fatal without proper treatment. Therefore, early diagnosis of typhoid fever is crucial. In the previous study, we have developed TYPHOIDYNE EIA, which showed excellent synergy between the genus conserved and species-specific antigens in the serodiagnosis of typhoid fever. TYPHOIDYNE EIA can effectively detect and differentiate typhoid patients, typhoid vaccinated subjects, healthy subjects, and subjects with other febrile illnesses. Following the successful development of TYPHOIDYNE EIA, in this report, we further characterize the antigenic components of differentially extracted S. typhi and S. spp recognized by IgM, IgG, and IgA antibody isotypes in typhoid patients and possible typhoid carrier by the western blot (WB) assay. The WB characterization revealed a dynamic pattern of recognition, with significant variations in the number of antigenic bands observed between the differentially extracted arrays of antigens. The reactivity of patient’s sera was divided into 3 regions, with region 1 (≥55 kDa) showing the strongest reactivity followed by region 2 (54 kDa–34 kDa) and region 3 (<34 kDa). Overall, the good synergy expressed in these bands suggests the potential role of these proteins in differentiating typhoid patients with possible typhoid carrier. The antigenic bands highlighted in this study are also identified as prospective biomarkers for diagnostic use and vaccine development.

Typhoid fever is an endemic disease that has been acknowledged as a major global health burden. Typhoid fever is a neglected re-emerging infectious disease that is transmitted through fecal oral route. Nevertheless, non-typhoidal Salmonella outbreak has been increasing globally with large number of cases involving immunocompromised individuals. The clinical diagnosis of typhoid is difficult due to the overlapping symptoms of typhoid fever, non-typhoidal Salmonella, and other associated febrile diseases, which causes to delayed treatment. Herein, this study aims to provide a reproducible, discriminative protein fingerprint of two different Salmonella serovars using a differential extraction procedure comprising of whole cell protein (WCP), cell surface protein (CSP) and surface-depleted whole cell protein (sdWCP) derived from whole cell bacterial protein of Salmonella Typhi (S.Typhi) and Salmonella spp (S.spp). In the present research, we perform comparative analysis to characterize protein profiles of two differentially extracted Salmonella serovars by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and dendrogram analysis. The findings showed that the proteins were reproducible in optimized concentration. Separation of protein into three different extractions revealed discriminative protein profiles with major and micro-heterogenicity. We observed the diverseness of differentially extracted proteins between two strains which provided an effective adjunct that can be used in identification of Salmonella strains. The results achieved when differential extraction procedure was applied, provides a promising, future opportunity for further immunoproteomic classifications.