Computational protocol: Rickettsial Illnesses as Important Causes of Febrile Illness in Chittagong, Bangladesh

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Protocol publication

[…] We conducted our study at Chittagong Medical College Hospital (CMCH), Chittagong, Bangladesh, during August 2014–September 2015. We enrolled into the study all consenting patients >12 years old who were admitted with a febrile illness to the medical wards and referred to the hospital’s malaria screening service with a history of fever for <3 weeks. Written informed consent was provided by all patients before inclusion in the study or by their relatives if the patient lacked capacity for providing consent. The study was approved by Chittagong Medical College ethics committee in Bangladesh and the Oxford Tropical Research Ethics Committee, United Kingdom.We collected admission blood samples and convalescent-phase blood samples (taken 7–14 days apart, where possible) into EDTA tubes and separated the samples into packed cells and plasma before storage at −30°C. We subjected packed cell admission samples of all patients to DNA extraction for real-time PCR screening by using the 47-kDa htra gene–based assay for Orientia spp. and the 17-kDa gene–based assay for Rickettsia spp. We subsequently confirmed positive results by nested PCR (nPCR) assays with product sequencing. For Orientia spp., we targeted the 56-kDa and 47-kDa gene targets. For Rickettsia spp., we targeted 17-kDa and performed ompB real-time PCR and gltA nPCR, as previously described (,). We aligned the resulting DNA sequences for construction of phylogenetic trees by using CLC Sequence Viewer 7.0.2 (http://www.clcbio.com). For serologic testing, all patient plasma samples underwent indirect immunofluorescence assays (IFA) by using slides from the Australian Rickettsial Reference Laboratory coated with O. tsutsugamushi (strains Karp, Kato, and Gilliam) for scrub typhus or R. typhi (Wilmington strain) for murine typhus. We used the following stringent diagnostic positivity criteria for scrub and murine typhus: a baseline IgM titer of >3,200 or a 4-fold rise to >3,200. In the absence of regional serologic positivity criteria for Bangladesh, we selected these cutoffs on the basis of experience from an area where scrub typhus is highly endemic (). In suspected cases of R. felis infection, we conducted IFA by using dedicated IFA slides from the Australian Rickettsial Reference Laboratory. We conducted statistical analyses by using Stata 14 (StataCorp LLC, College Station, TX, USA). […]

Pipeline specifications

Software tools CLC Sequence Viewer, CLC Assembly Cell
Application Phylogenetics
Organisms Homo sapiens, Mus musculus, Rickettsia felis, Orientia tsutsugamushi
Diseases Infection, Scrub Typhus