Development of molecular and immunological based diagnostic tools to detect Orientia tsutsugamushi (scrub typhus)...

Scrub typhus is a disease caused by a bacterial pathogen, Orientia tsutsugamushi which spreads through bites of infected chigger mite. Fever, headache, body aches and rash are common symptoms of scrub typhus. The absence of specific signs and symptoms of the disease makes diagnosis extremely difficult. In the present study, the blood samples from individuals with infections were obtained and analyzed for IgM and IgG antibodies using a commercial ELISA test kit. The present work was carried out to develop a molecular and immunological based technique which are more sensitive and accurate for the early detection of infected persons. The molecular diagnostics like PCR and nested PCR used for amplification of the 56 kDa type specific antigen (TSA) are more specific, sensitive and accurate. The PCR amplified partial 56 kDa TSA gene was cloned into the vector pET27b (+), and expressed in the BL21 host. The recombinant protein was purified by Ni-NTA column, and then the purified protein was used for production of polyclonal antibody in mice and rabbits. The antibody was used to develop immunological based diagnostic tools like ELISA, Western blot and dot blot for the early detection of scrub typhus infection. Totally, 141 infected samples were screened by PCR, nPCR, ELISA and western blot developed in the present study and the results were compared to commercial ELISA kit. Out of 141 samples analyzed, 58.8 % (83 samples) tested positive by PCR, 73 % (103 samples) by nPCR, and 86.5 % (122 samples) by ELISA. Western blot analysis of the 14 selected positive samples showed positive results (100 %). The newly developed immunological diagnostic tools and PCR techniques demonstrated high sensitivity and enhanced specificity, allowing for early detection of scrub typhus infection.