First isolate ofOrientia tsutsugamushifrom Vellore, South India

Abstract

AbstractBackgroundScrub typhus a common cause of acute febrile illness in India caused byOrientia tsutsugamushian obligate intracellular bacterium requiring cell culture for isolation. Cell lines like Vero and L929 are most suitable for isolating and maintaining this organism. This study was undertaken to isolate and characterize ofOrientia tsutsugamushifrom whole blood samples at a tertiary care centre in Southern India.MethodsThe PBMCs (peripheral blood mononuclear cells) collected from scrub typhus positive (47kDa qPCR positive) patients were inoculated into Vero and L929 cell line at 80% confluence for primary isolation. The inoculated flasks were incubated at 37°C with 5% CO2for 30 days and examined for presence ofOrientia tsutsugamushion the day 10, 15, 20 post-inoculation and everyday thereafter for a maximum of 30 days post inoculation. The scrapings were subjected to Giemsa staining, IFA, 47kDa qPCR and transmission electron microscopy (TEM). The isolates were passaged 3-4 times to ensure viability and then stored in DMEM with 10% FBS (-80°C). Genotyping of the isolates was performed by amplifying a 650 bp segment of the TSA 56 (type specific antigen 56) gene.ResultsAmongst the 50 samples inoculated, three were culture positive as confirmed by 47 kDa qPCR at 24thday of inoculation. This was further confirmed by Giemsa, IFA staining and TEM. The 650bp amplicons showed 99.5 to 100% homology withOrientia tsutsugamushiMW604716, MH003839, MW604718, MW604717, MH922787 and MH003838 strains. Phylogenetic analysis revealed that 2 isolates belong to TA763 genotype and one belongs to Gilliam genotype.ConclusionsWe have successfully isolated and characterised theOrientia tsutsugamushifor the first time at our centre from PBMCs. Based on the partial TSA56 gene sequence our isolates belongs to TA763 and Gilliam genotype. More number of samples are being processed for identifying further isolates followed by genomic analysis.