Research Projects

The present study addresses the urgent need for enhanced molecular surveillance of drug resistance in Mycobacterium leprae, specifically in areas like central India that are endemic and under-represented. The persistence of relapsed cases and rising antimicrobial resistance (AMR) highlight important gaps in diagnosis and treatment, even though leprosy is curable. The study aims to use SNP genotyping to molecularly distinguish relapse from reinfection and to concurrently identify primary and secondary drug resistance mutations in important genes (rpoB, folP1, gyrA/gyrB). The central hypothesis is that integrating SNP-based strain typing with resistance profiling can reveal the true origin of drug resistance, whether community-acquired (primary) or treatment-induced (secondary). This will be tested through nested PCR, multiplex PCR, and amplicon sequencing, followed by correlation with clinical history. For the proposed studymethodology,samples will be collected from the leprosy patients diagnosed as suspected relapse cases, based on the reappearance of symptoms following completion of multidrug therapy(MDT). Slit skin smear or tissue biopsies will be obtained from each patient, with a sample size of 100cases. Samples will be subjected to the presence of acid-fast bacilli(AFB), after which positive samples will be processed for DNA extraction using a standard commercial kit.Following extraction, SNP genotyping will be performed using a nested PCR approach. A multiplex PCR protocol will be optimised for simultaneous amplification of both target regions. The PCR products will be visualised on agarose gel, purified, and subjected to amplicon-based next-generation sequencing (NGS). The three main genes that determine drug resistance—folP1 (dapsone resistance), rpoB (rifampicin resistance), and gyrA/gyrB (fluoroquinolone resistance)—will be examined for alterations in all DNA-positive samples. Mutation hotspots within these genes will be amplified by PCR or nested PCR using approved or published primers. Amplicons will either be introduced to the sequencing after being visualised and purified.. Based on clinical history and SNP genotyping, mutations will be categorised as primary (in cases of reinfection) or secondary (in cases of relapse). Findings will be compared to clinical outcomes, drug exposure, and treatment history in order to comprehend the dynamics of resistance emergence in the population under study.A comparison analysis will be conducted to investigate the relationship between particular mutations and primary (transmitted) versus secondary (acquired) resistance after SNP genotyping and resistance mutation identification. The presence of resistance mutations in patients classified as reinfected (having a different strain genotype) will be interpreted as primary resistance, implying community-acquired drug-resistant strains. Relapse patients (same genotype) who have newly discovered or emerging resistance mutations, on the other hand, will be categorised as secondary resistance, suggesting that resistance may have arisen during or following therapy.For every case, the mutation patterns (gene involved, mutation type, codon location) will be recorded and examined. It will be determined which mutations are frequently associated with relapse and which are showing up in instances that are untreated or reinfected by analysing the frequency and distribution of these mutations. The project’s findings will offer a significant leap in understanding resistance dynamics, enabling patient-specific therapy, guiding national leprosy control strategies, and contributing to the global fight against AMR through region-specific, molecularly informed interventions.