Scrub Typhus Case Study

Molecular diagnosis of scrub typhus using nested PCR analysis in Chennai – A case report
Seethalakshmi S 1, Thangam Menon 1
1 Department of Microbiology, Dr. A L Mudaliar Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani, Chennai, India.

Abstract:
Scrub typhus is a mite borne rickettsial zoonosis caused by Orientia tsutsugamushi. We report a case of scrub typhus with ARDS and DKA in an adult diabetic male in Chennai. The diagnosis was made initially by serology and subsequently confirmed by PCR to detect the 56 kDa gene.

Introduction:
Scrub typhus is an acute febrile mite borne disease caused by Orientia tsutsugamushi which is under-diagnosed in India. Scrub typhus produced considerable morbidity and mortality

Hospital. The patient was presented with complaints of intermittent high grade fever for 10 days associated with vomiting, myalgia, head ache, body pain and burning micturition. He had a past history of malaria and treated in local hospital. He was a known patient of type II diabetes mellitus since two years. On physical examination no eschar formation or skin rash was observed. Laboratory results showed hemoglobin 12.9 g/dI, platelet count 1,00,000 cells/µL, mean corpuscular volume (MCV) 83 fL, mean corpuscular hemoglobin (MCH) 26.5pg, mean corpuscular haemoglobin concentration (MCHC) 32.0 g/dI, sodium 128 mEQ/L, potassium 4.2 mEQ/L, chlorine 98mEQ/L, urea 22 mg/dL and creatinine 0.9mg/dL . The patient was diagnosed with diabetic ketoacidosis. The smears for WIDAL and dengue serology were negative. The chest radiograph was suggestive features of ARDS. Abdominal ultrasonography showed hepatosplenomegaly with normal gall bladder, pancreas and

After transportation to the laboratory, the serum was separated from the blood sample and subjected to rapid card test (SD Bioline Tsutsugamushi test, SD Diagnostics, Korea), which is a solid phase immunochromatographic assay that detects IgG, IgM or IgA antibodies to O. tsutsugamushi. The IgM ELISA was also performed using InBios International Scrub typhus Detect IgM ELISA system as per manufacturer’s instructions. The raid card test and IgM ELISA showed the presence of antibodies against O. tsutsugamushi. The EDTA blood samples were subjected to DNA extraction using QIAamp DNA Mini Kit (Qiagen, GmBh, Germany) according to manufacturer’s instructions. The purified DNA samples were stored at −20°C. The 626 bp segment gene encoding the 56 kDa protein antigen of O. tsutsugamushi was amplified by nPCR using modified protocol of Izzard et al. The PCR products were visualized using the gel documentation system (Carestream Gel Logic 212 Pro, USA) and amplification of 626 bp was considered positive in the second round PCR. A BLAST search (http://www.ncbi.nlm.nih.gov/BLAST) of the amplified 56 kDa gene sequence was performed, which showed 99% homology with Orientia tsutsugamushi. The patient was treated with intravenous ceftriaxone (1g, twice a day) and doxycycline (100mg,