Samples
A prospective study was conducted among hospitalized acute febrile illness patients with suspected scrub typhus in central Nepal for a period of one year, April 2017 to March 2018. Cases of uncharacterized acute fever for more than 4 days were included in the study after excluding the confirmed cases of other febrile illness caused by malaria, dengue, leptospirosis, and enteric fever. Single blood sample were collected from this subset of acute febrile illness patients that were hospitalized. The blood samples were centrifuged at 3000 rpm for 5 min to separate the serum. Serum samples were stored at − 80 °C until they were analyzed.
Scrub typhus detect™ IgM ELISA
IgM antibody to Orientia tsutsugamushi was detected by using Scrub Typhus Detect™ Kit, InBios International, USA containing the recombinant p56kDa type specific antigens of Orientia tsutsugamushi Karp, Kato, Gilliam and TA 716 strains according to the manufacturer’s instruction. Optical density was measured by HumaReader HS, ELISA reader, optical densities (ODs) > 0.50 was considered positive. The cut-off was calculated following recommendations for determining the endemic cut-off titre in the kit protocol. The cut-off calculated from healthy volunteer was mean OD (0.23) + 3 standard deviation (0.09) =0.50. We proposed a cut-off OD value of > 0.50 for chitwan and surrounding region based on our finding.
IgM immunofluorescence assay
Antibodies against Scrub Typhus Group were tested using Orientia tsutsugamushi (Gilliam, Karp, Kato) strains and Orientia chuto antigens. The antigens were prepared in the Australian Rickettsial Reference Laboratory, Geelong, Australia by culturing the organism in L929 cell line and RPMI media (Invitrogen) supplemented with 5% fetal bovine serum. Individual antigens were coated onto rickettsial screening slides containing 40 individual wells, air dried and fixed in acetone. Serum samples were diluted 1:128 in 2% casein buffer and spotted onto the slide in duplicate and incubated at 37 °C for 40 min in a moist chamber to allow for the binding of antigen and antibody. With each slide tested, positive and negative controls were included. Slides were washed 3 times in PBS and dried. An anti-human FITC labeled IgM conjugate was then added and slides incubated at 37 °C for 40 min in a moist chamber. Slides were washed once more, air dried, mounted and observed under the fluorescent microscope. Positive results are indicated when fluorescence intensity was equal to or greater than the positive control. The diagnostic cutoff > 1:128 was considered positive which was derived after testing the serum samples of healthy controls from that particular region. Negative results were reported when the sera didn’t fluoresce at a dilution of 1:128. Positive serum samples were serially titrated 1:128, 1:256, 1:512, 1:1024, 1:2048, 1:4096, 1:8192, 1:16384 etc. to end point titers with individual antigens.
Quality control
Positive and negative controls were included with each slide that if either failed in a screening or titration slide, tests were repeated. In instances of continuation of assay failure both the antibody and antigen controls were re-titrated to see if there had been a shift in the antibody endpoint or if the antigen had lost its reactivity. Whenever necessary fresh controls and antigens were optimised prior to repeating of the assay with the specimens.
Statistical analysis
The collected data were entered in Epi info 3.5 from CDC and exported to IBM SPSS version 16.0 (SPSS Inc. Chicago, USA). The sensitivities, specificities, positive predictive values, negative predictive values of the serological tests were calculated using MedCalc for windows, version 18.11.3 (MedCalc, Software, Ostend, Belgium). STARD 2015 guidelines for reporting diagnostic accuracy studies was strictly followed [8].