Scrub typhus in Puducherry, India: Application of nested PCR targeting three different genes – 56 kDa, 47 kDa and groEL of Orientia tsutsugamushi and comparison with ST IgM ELISA

BACKGROUND & OBJECTIVES: Scrub typhus (ST), an important zoonosis caused by Orientia tsutsugamushi, is now prevalent throughout India. While demonstration of IgM antibody by Indirect Immunofluorescence Assay (IFA) is the gold standard serological test, IgM ELISA is an alternative. Demonstration of O. tsutsugamushi DNA in the blood or eschar confirms infection in the early febrile period. METHODS: Scrub typhus nested PCR (n-PCR) for 56 kDa, 47 kDa and groEL genes and ST IgM ELISA were performed for 210 clinically suspected ST patients. As healthy controls, 70 voluntary blood donors were included. Statistical analysis was performed for laboratory parameters using Fisher exact test/chi-square test. Ninety-five PCR products of n-PCR positive samples were purified and submitted for gene sequencing. RESULTS: PCR was positive for one or more gene targets in 75.71% of IgM ELISA positive patients and 10% of antibody negative patients. All voluntary blood donors were negative for both antibodies and DNA. Gene sequences of 95 n-PCR positive products confirmed the presence of Orientia tsutsugamushi DNA in the samples and NCBI database accession numbers MG601875 to MG601969 were obtained. INTERPRETATION & CONCLUSION: Compared to IgM ELISA, sensitivity of three PCRs was 30, 51.43 and 61.43% for 56 kDa, 47 kDa and groEL targets, respectively. Since IgM ELISA positivity can persist up to one year, PCR confirms ST diagnosis in the acute phase of the illness, in the presence of IgM and even before IgM appears. Inclusion of all three genes - 56 kDa, 47 kDa and groEL, instead of a single 56 kDa target, identifies and confirms maximum number of ST patients.