Serodiagnosis And Molecular Characterization Of Rickettsia In And Around Vijayapura North Karnataka, India.

Abstract

Background Rickettsiosis is the most covert, re-emerging vector borne bacterial disease of public health importance distributed in many parts of the world. The disease is mostly go unnoticed or misdiagnosed due to low manifestation and non availability of specific diagnostic tests at all levels of healthcare setup. Failure of timely diagnosis and treatment leads to significant morbidity and mortality. Aim & Objectives: The present study aimed to demonstrate the presence of rickettsial infections in and around Vijayapur district using both serological and molecular diagnostic tests. Methods: In the present study, 572 blood samples from clinically suspected patients were screened for the presence of antibodies against rickettsial infections by routinely used Weil felix test. For the serological confirmation, all the serum samples were tested further by more sensitive and specific diagnostic assay, R.conorii and Scrub typhus specific IgM ELISA. To detect the etiological agent of the rickettsial infection, blood clot of ELISA positive samples were tested by nested PCR using the primers specific to the gene encoding different antigens of Rickettsia and Orientia. Sanger sequencing was performed to confirm the amplification of the gene. Phylogenetic tree was constructed to compare the sequences with other strains. Results: Out of 572 cases, 213 (37.23%) samples have shown titre of 1:160 and above with Weil felix OX antigens suggestive of seropositivity for rickettsial infection. Among 213 WF positive cases, 180 (31.46) samples have shown agglutination with OX2 antigen suggestive of SFG Rickettsial infection, and 63 (11.01%) cases shown agglutination with OXK antigen suggestive of Scrub typhus infection. In R. conorii IgM ELISA, 56 sampleswere positive out of 572. In Scrub typhus IgM ELISA, out of 432 samples 23 samples proved to be scrub typhus infection serologically. In nPCR for Scrub typhus, two samples have amplified successfully with the primers specific to 483bp segment of gene encoding 56kDa antigen of Orientia tsutsugumushi. Both the samples were sequenced by using Sanger sequencing technique. Sequences of the study sample have shown closest homology with many other strains reported from various parts of India and Southeast Asia. Phylogenetic analysis O. tsutsugumushi with sequences of the study have shown close relatedness with the strain reported from Korea. Several attempts were made to standardize the nested PCR for the detection of Rickettsia genus specific citrate synthase gene (gltA) and SFGR specific outer membrane protein gene (ompA) using specific primers. However, in spite of multiple attempts we could not standardize the PCR. Conclusion: Findings of our study demonstrated that Rickettsial infection is also circulating and causing acute febrile illness in and around Vijayapura, North Karnataka region. Significant seropositivity was observed for SFGR than Scrub typhus; however molecular detection of scrub typhus has proved its existence. More seropositivity was observed during the cooler months (August to January). Clinical diagnosis of rickettsial infection is really challenging as most of the common symptoms are non specific, and leads to misdiagnosis if not thoroughly examined. As the routinely used Weil felix test is lacking both sensitivity and specificity, inclusion of more specific tests like IgM ELISA and/or nPCR in routine diagnostic course would be highly beneficial for accurate diagnosis and patient management.