Molecular detection of scrub typhus in Tirupati, Andhra Pradesh, India.

caused by Orientia tsutsugamushi, a gram-negative intracellular coccobacillus. It is transmitted to humans and other vertebrates through the bite of the larval trombiculid mites (known as Chiggers) harbouring the etiological agent. The mites normally feed upon single vertebrate hosts, usually rodents. Febrile illness typically begins after the bite of an infected chigger and lasts for 7–10 days1. Headache, lymphadenopathy, hepatosplenomegaly, rash, and an eschar usually develop in the infected person. Scrub typhus have been reported from Southeast Asia, Japan, Malaysia, Kampuchea, Thailand, Vietnam, Southeastern Siberia, Sri Lanka, Indonesia, Philippines, Korea, Western Pacific Islands, Pakistan, Astrakhan, India, and northern Australia forming the so called “tsutsugamushi triangle” 2-4. In India, epidemics of scrub typhus have been reported from states of Andhra Pradesh and neighbouring states, as well as from the sub-Himalayan states5-8. The aim of the present study was to diagnose the clinically suspected scrub typhus patients serologically by WeilFelix (WF) test, IgM ELISA and nested-PCR targeting 56-kDa type-specific antigen gene. From January 2013 to July 2014, 113 blood samples of patients clinically suspected to have scrub typhus, attending Sri Venkateswara Institute of Medical Sciences (SVIMS), Tirupati, India were included in the study. Patients presented with intermittent fever and having five out of the following eight clinical features, namely headache, myalgia, malaise, cough, nausea, abdominal pain, lymphadenopathy, hepatomegaly, splenomegaly, rash and eschar were included in the study9. Two ml of blood samples were collected without anticoagulant before the administration of empirical treatment with antibiotics. The Institutional Ethical Clearance was taken from Institute Ethics Committee, SVIMS, Tirupati, India. Serum and blood clot were separated and preserved at –80°C until processing. Demographic details of patients, the clinical course of illness and complications of infection were reviewed from the medical records. The samples were tested serologically by two techniques. The WF test was performed with each sample by using in-house prepared Proteus mirabilis OX-K antigens. Detection of IgM antibodies against O. tsutsugamushi was performed by commercial ELISA kit (InBiOS International Inc., USA) as per manufacturer’s instructions. The IgM ELISA test was initially standardized using serum samples from healthy blood donors and the OD cut-off of 0.5 was taken 3±SD from the mean. Further, validation was done using known scrub typhus sera (confirmed by PCR) and sera from patients with other diseases like malaria and enteric fever and also healthy controls. A positive and negative control provided in the kit as well as in-house positive control for every run were used. Molecular analysis of the serologically positive samples were carried at Department of Virology, Sri Venkateswara University (SVU), Tirupati, India. DNA was extracted from blood clot of each sample by using the method as described by Furuya et al10. The 56-kDa type-specific antigen (TSA) gene was amplified by nestedPCR (N-PCR) using the following primers: