Phylogenetic and immunological investigations of complete TSA56 ORF of Orientia tsutsugamushi present in acute encephalitis syndrome cases from eastern Uttar Pradesh, India.

Scrub typhus (ST) caused by Orientia tsutsugamushi (OT), has long been known to cause acute encephalitis syndrome (AES) and acute febrile illness (AFI). The immunodominant 56 kDa protein of OT, which is encoded by the 56 kDa gene (1600 bp encoding 516-541 amino acids) is a commonly studied antigen for genotype and serotype assignment. Previous studies from India have utilized partial type specific antigen (TSA) 56 kDa sequences for OT strain characterisation. On the other hand, understanding the antigenic diversity of current OT strains, is critical for developing specific diagnostic tests and vaccines against ST. As a result, the current study analyses antigenic variants using the entire TSA56 ORF of OT from AES cases. Phylogenetic investigation using complete TSA56 ORF sequences revealed Karp and Gilliam were the circulating predominant strains of OT. Furthermore, Immuno-informatical analysis demonstrated that the majority of high-binding affinity CD4 TCEs against the most prevalent Indian human leukocyte antigen alleles were present in the S-VDIII/IV and S-VDIV spacer regions of TSA56 ORF. TSA56 conserved spacer is crucial for OT immunological response investigations. Further, the pathophysiological effects of spacer domains in ST require further investigation. Furthermore, the characterization of the TSA56 spacer region of the OT from different parts of India is critical for developing region-specific ST diagnostic assays and vaccines.

Molecular Detection and Genetic Characterization of Orientia tsutsugamushi from Hospitalized Acute Encephalitis Syndrome Cases During Two Consecutive Outbreaks in Eastern Uttar Pradesh, India.

Background: Seasonal outbreaks of acute encephalitis syndrome (AES) have been reported especially in the pediatric population with a high case fatality rate in Eastern Uttar Pradesh, India. Orientia tsutsugamushi (OT) is a causative agent of scrub typhus that has been recently identified as a major cause of AES. However, the specific genotypes of OT responsible for AES cases of this region are not known. Therefore, the present study was undertaken to understand the molecular epidemiology of OT prevailing in the AES endemic Eastern Uttar Pradesh region of India. Methods: The study was conducted on 2529 hospitalized AES cases from August 2016 to December 2017. The presence of antibodies against OT from cerebrospinal fluid (CSF) and serum samples were tested using OT IgM enzyme-linked immunosorbent assay (ELISA), whereas OT DNA was tested from whole blood and CSF specimens targeting the partial gene of 56 kDa using nested PCR. Phylogenetic analysis was conducted with sequences (n = 241) generated in this study. Findings: Among the studied AES cases, 50% were found positive for antibodies against OT, whereas 37% of cases were positive for OT DNA. The genetic analysis study revealed that Gilliam (93.8%) is the prevailing genotype of OT followed by Karp (6.16%) genotype in AES cases. Furthermore, the Gilliam strains of this study showed they were >99% identical to earlier reported Gilliam strains from AES cases. Conclusion: We observed the presence of two main OT genotypes in AES cases, among which the majority of OT genotypes fall under the Gilliam clade. The understanding of predominant genotype will be beneficial for its future implications in vaccine development strategies and the development of rapid diagnostic tests.