Optimization of immunodot blot assay for diagnosis of Typhoid and Paratyphoid fever using the TPTest for comparison

Abstract

Typhoid and paratyphoid (enteric) fever are responsible for morbidity and mortality worldwide. Bangladesh is an endemic zone of enteric fever where sanitation and hygienic conditions are poor. Rapid, accurate diagnosis of enteric fever and early treatment of the patients with appropriate antimicrobials are essential for recovery and for prevention of complications and death. Bone marrow culture is the gold standard for diagnosis of enteric fever but it is an invasive procedure and blood culture method and widal test have low sensitivity and specificity respectively. Thus development of an early and reliable diagnostic method is necessary. Previous studies reported that a novel diagnostic method, TPTest is very useful for diagnosis of typhoid and paratyphoid fever and it gives a preliminary result within 24 hours and a confirmatory result after 48 hours. The sensitivity and specificity of the TPTest are 100% and 78-97% respectively. The TPTest detects Salmonella enterica serotype Typhi specific antibodies in peripheral blood antibodies (PBA) specimen by ELISA. The PBA specimen is prepared by isolating the lymphocytes form whole blood using ficoll density gradient centrifugation method and culturing the cells at 37oC with 5% CO2 supplementation. For implication of the TPTest in laboratories with less facilities and expertise immunodot blot assay was used to detect the response in PBA specimen. The specimen was prepared by isolating the blood cells using RBC lysis and culturing the cells at 37oC with no 5% CO2 supply. In this study, optimization of the simplified methods of the TPTest was done at different stages and the results were evaluated with the results of blood culture and with the standard TPTest method. The findings of this study suggest that the optimized immunodot blot assay and the simplified cell separation methods will be helpful for diagnosis of typhoid and paratyphoid fever.