Application of Nested PCR and Loop-Mediated Isothermal Amplification (LAMP) to Target 56kDa gene in Scrub Typhus Patients and Phylogenetic Analysis to Identify Orientia tsutsugamushi Strains Circulating In and Around Puducherry

Abstract

Scrub typhus (ST) is a re-emerging zoonotic disease caused by Orientia tsutsugamushi and is transmitted by chiggers. Serological tests targeting IgM and/or IgG antibodies play a major role in the detection of ST cases. Orientia 56kDa genome is common target for the molecular diagosis of ST to identify the prevalence of specific serotypes of O. tsutsugamushi in and around Puducherry by targeting 56kDa gene with the application of phylogenetic analysis. This prospective laboratory-based study was conducted in a tertiary care teaching hospital, from November 2105 to March 2018. Blood samples were collected from out-patients/in-patients, and those tested positive for ST IgM ELISA (n=140) and an equal number of negative samples were archived and anonymized. Genomic DNA was extracted and analyzed by using Nested PCR and LAMP assay. The positive products were purified and sequenced. Phylogenetic tree was constructed based on the sequences. Among 280 samples, 45 (16.1%) N-PCR and 102 LAMP (36.43%) positivity was observed for 56kDa gene. Forty-one N-PCR positive products were sequenced and accession numbers were obtained (MG601875 to MG601917) for the isolates. Phylogenetic analysis was done by Maximum Likelihood methods and this study has showed that 32.3% are similar to the Karp isolates. Molecular diagnosis of Scrub typhus has become essential in case of doubtful serology and early acute phase of illness. Gene sequencing result indicates that most of them were different from the existing ones, which may belong to the newer strains. The identification of newer strains will be helpful in future for development of scrub typhus vaccine.