Detection of new Mycobacterium leprae subtype in Bangladesh by genomic characterization to explore transmission patterns

Abstract

Mycobacterium leprae, the causative agent of leprosy, is an unculturable bacterium with a considerably reduced genome (3.27 Mb) compared to homologues mycobacteria from the same ancestry. M. leprae transmission is suggested to occur through aerosols but the exact mechanisms of infection remains unclear. In 2001, the genome of M. leprae was first described and subsequently four genotypes (1-4) and 16 subtypes (A-P) were identified providing means to study global transmission patterns for leprosy. We investigated M. leprae carriage as well as infection in leprosy patients (n=60) and healthy household contacts (HHC; n=250) from Bangladesh using molecular detection of the bacterial element RLEP in nasal swabs (NS) and slit skin smears (SSS). In parallel, we explored bacterial strain diversity by whole-genome sequencing (WGS) and Sanger sequencing. In the studied cohort in Bangladesh, M. leprae DNA was detected in 33.3% of NS and 22.2% of SSS of patients with bacillary index of 0 whilst in HHC 18.0% of NS and 12.3% of SSS were positive. The majority of the M. leprae strains detected in this study belonged to genotype 1D (55%), followed by 1A (31%). Importantly, WGS allowed the identification of a new M. leprae genotype, designated 1B-Bangladesh (14%), which clustered separately between the 1A and 1B strains. Moreover, we established that the genotype previously designated 1C, is not an independent subtype but clusters within the 1D genotype. Intraindividual differences were present between the M. leprae strains obtained including mutations in hypermutated genes, suggesting mixed colonization/infection or in-host evolution. In summary, we observed that M. leprae is present in asymptomatic contacts of leprosy patients fueling the concept that these individuals contribute to the current intensity of transmission. Our data therefore emphasize the importance of sensitive and specific tools allowing post-exposure prophylaxis targeted at M. leprae-infected or -colonized individuals.

Household Contacts of Leprosy Patients in Endemic Areas Display a Specific Innate Immunity Profile

“…The HCs diagnosed with leprosy at intake (DevLep) were not evenly distributed over the different households: in 14 households none of the HCs had developed leprosy, whereas in the other 17 households, 9-42% suffered from leprosy (Figure 1). Applying previous results on the presence of M. leprae DNA (25), indicated that in 10 households M. leprae DNA was not detected in any of the HCs in NS and in 13 households all HCs were negative in the SSS. Of the households where M. leprae DNA was detected, percentages of colonization varied from 7 to 100% (NS) and for infection from 10 to 66% (SSS; Figure 1).…”

“…The cohort used in this study originates from four districts in Bangladesh (Nilphamari, Rangpur, Panchagar, and Thakurgaon) and has been extensively described previously (25). The prevalence of leprosy in these districts was 0.9 per 10,000 and the new case detection rate 1.18 per 10,000 (Rural health program, the leprosy mission Bangladesh, yearly district activity report 2018).…”

“…Between July 2017 and May 2018, newly diagnosed leprosy patients (index case; n = 31) with BI ≥2 and between 3 and 15 HCs per index case (n = 279) were recruited (25). Leprosy was diagnosed based on clinical and bacteriological observations and classified as MB or PB as described by the WHO (5) and the BI was determined.…”

“…DNA isolated from the NS and SSS was used to perform RLEP PCR and qPCR as described previously (25). Presence of M. leprae DNA was considered if a sample was positive for RLEP qPCR with a Ct lower than 37.5 or was positive for RLEP PCR at least in two out of three independently performed PCRs to avoid false positives.…”

“…Previously, we analyzed nasal swabs (NS) and slit-skin smears (SSS) of household contacts (HCs) of MB leprosy patients with high bacillary loads for the presence of M. leprae DNA (25). Here we analyzed the same individuals to examine the correlation of the presence of M. leprae DNA with the levels of innate immune markers.…”