Comparative Analysis of Leptospirosis And Scrub Typhus: Clinical, Molecular And Metabolomic Approach

Objective 1: The retrospective data of the hospitalized leptospirosis and scrub typhus patients from January 2022 to December 2023 either by IgM ELISA or PCR will be enrolled and the data will be collected on a structured proforma from the Medical Records Department of Kasturba Hospital, Manipal. Data collection: The clinical, demographic, and laboratory parameters will be collected in the patient proforma of both infections after obtaining IEC. Statistical analysis: The collected data of the patient will be initially recorded in a Microsoft Excel spreadsheet. The analysis will be conducted using the Statistical Package for Social Sciences for Windows (SPSS). Univariate analysis and multivariate logistic regression analysis will be performed to distinguish the two groups based on significant differences between binary and continuous variables. Significant variables identified through regression analysis will be used to calculate adjusted odds ratios and generate the scoring system. The ROC-AUC will be used to determine the optimal cut-off for the scoring system, with sensitivity and specificity calculated accordingly.

Objective 2: Patient screening and enrolling: Patients with IgM ELISA or PCR confirmed cases of leptospirosis and scrub typhus, who are hospitalized in Kasturba Hospital, Manipal, will be screened and enrolled. Consent forms will be signed by each study participant who fulfills the inclusion and exclusion criteria and is willing to be part of the study. Data collection: The clinical, demographic, and laboratory parameters will be collected in the patient proforma of both infections after obtaining IEC. The clinical scores will be calculated after obtaining the variables. The patients will be presumptively diagnosed as having either leptospirosis or scrub typhus. After confirmation of the diagnosis based on serological tests or PCR, the patients will be finally classified as having either leptospirosis or scrub typhus. Statistical analysis: SPSS software for Windows will be used. Continuous variables will be presented as mean (SD) or median (range) depending on the distribution of data and will be compared using t-test or Mann-Whitney test. Chi-square test or Fischer’s test will be used to compare dichotomous variables. A two-sided P <0.05 will be considered as statistically significant. Sensitivity, specificity, positive predictive value, and negative predictive value will be calculated.

Objective 3: Patient screening and enrolling: Patients admitted to Kasturba Hospital, Manipal, and suspected to have leptospirosis and scrub typhus during the study period (1.5 years) will be screened by RDT test and enrolled into the study by confirming with IgM ELISA after obtaining written informed consent. Screening and sample collection: All patients fitting into eligible criteria will be screened by Rapid Diagnostic Test for leptospirosis and scrub typhus, and the positive patients will be enrolled in the study based on positive IgM ELISA. A 5 mL sterile EDTA and a plain blood sample will be collected each in the 1st week of illness, and serum will be separated and transferred into cryovials for storage at -20℃. Approximately 10 mL of urine samples from leptospirosis-positive patients in the 2nd week of illness will be collected in a sterile container and stored at 4°C. Eschar will be collected into a sterile container from the scrub typhus-suspected patients presented with eschar in the 2nd week of illness and transported to the laboratory for storage at 2-8℃ (described by CDC – Eschar-associated Rickettsioses). All the samples will be collected from the study participants who fulfill the inclusion criteria. DNA extraction: DNA extraction will be employed by the salting-out method from buffy-coat samples of patients. From collected urine samples DNA will be extracted using a method described by WHO guidelines (Human leptospirosis: guidance for diagnosis, surveillance, and control). From eschar samples, DNA will be extracted by the kit-based method. The extracted DNA will be quantified and eluted using 50 μL of TE buffer into 1.5 mL cryovials and stored at -20 °C until further use. Conventional PCR: Primer designing and PCR optimization: The PCR will be standardized using primers for the LipL32 gene, as described by Shukla S et al., and for the 56-kDa tsa gene, as described by Janardhanan J et al. Using certain primers, conventional PCR for LipL32 and tsa56 genes will be carried out, and the outcomes will be recorded. Agarose Gel Electrophoresis: The PCR products that have been amplified will undergo electrophoretic separation on a 1.5% agarose gel that contains TBE buffer containing Ethidium Bromide and a molecular marker. The gel can then be observed with the aid of a UV transilluminator. Purification of PCR products and DNA sequencing: PCR amplicons will be isolated using a PCR purification kit following the manufacturer’s instructions. The obtained PCR amplicons will be confirmed by sequence analysis. The determination of prevailing genotypes and serovars after performing the Microscopic Agglutination Test and their association with the disease severity will be determined for both infections by using the criteria for the severity of leptospirosis and scrub typhus based on organ involvement and laboratory parameters.

Objective 4: Screening and sample collection: All patients fitting into eligible criteria will be screened by Rapid Diagnostic Test for leptospirosis and scrub typhus, and the patients positive for IgM ELISA will be enrolled in the study. The sterile plain blood sample will be collected, and serum will be separated and transferred into cryovials for storage at -80℃. Approximately 50 mL of urine samples from leptospirosis and scrub typhus-positive patients will be collected in a sterile container and stored at -20°C. All the samples will be collected from the study participants who fulfill the inclusion criteria. 3 mL of sterile plain blood and 50 mL of urine samples from 102 healthy individuals will be used as controls for the metabolomic study. Metabolomic Analysis: Confirmed 168 leptospirosis and 135 scrub typhus serum and urine samples will be transported for targeted metabolomic analysis, and the collected 10 serum and urine samples from Scrub typhus, Leptospirosis, and age-sex-matched healthy Individuals will be transported to an outside laboratory for untargeted metabolomic analysis, which will determine the alteration of metabolites in these samples during the infection. Target panel analysis will be performed across all the samples, and a clinically significant data set will be decided based on global metabolomics. The metabolites will be analyzed and quantified from the collected samples to determine the alteration and to identify a better biomarker for the early detection of leptospirosis and scrub typhus infection.