Isolation of Orientia tsutsugamushi in Culture from Vellore, South India

Orientia tsutsugamushi, causative agent of scrub typhus is an obligate intracellular parasite. We present information on isolation of this pathogen at a tertiary care centre in Vellore, Southern India. PBMCs (peripheral blood mononuclear cells) collected from suspected scrub typhus patients were inoculated into Vero and L929 cell lines and incubated at 37°C with 5% CO2 for 30 days. They were examined for presence of Orientia tsutsugamushi on 10, 15, 20 days 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). Genotyping of the isolates was performed by amplifying a 650 bp segment of the TSA 56 (type specific antigen 56) gene. Amongst the 50 samples inoculated, three were culture positive as confirmed by 47 kDa qPCR on 24th day post inoculation. This was further confirmed by Giemsa, IFA staining and TEM. The 650bp amplicons showed 99.5 to 100% homology with Orientia tsutsugamushi MW604716, MH003839, MW604718, MW604717, MH922787 and MH003838 strains. Phylogenetic analysis revealed that 2 isolates belong to TA763 genotype and one belongs to Gilliam genotype. Orientia tsutsugamushi has been isolated for the first time at Vellore, South India from PBMCs. Complete genomic analysis will give more information.