Whole genome sequencing distinguishes between relapse and reinfection in recurrent leprosy cases

Background Since leprosy is both treated and controlled by multidrug therapy (MDT) it is important to monitor recurrent cases for drug resistance and to distinguish between relapse and reinfection as a means of assessing therapeutic efficacy. All three objectives can be reached with single nucleotide resolution using next generation sequencing and bioinformatics analysis of Mycobacterium leprae DNA present in human skin. Methodology DNA was isolated by means of optimized extraction and enrichment methods from samples from three recurrent cases in leprosy patients participating in an open-label, randomized, controlled clinical trial of uniform MDT in Brazil (U-MDT/CT-BR). Genome-wide sequencing of M. leprae was performed and the resultant sequence assemblies analyzed in silico. Principal findings In all three cases, no mutations responsible for resistance to rifampicin, dapsone and ofloxacin were found, thus eliminating drug resistance as a possible cause of disease recurrence. However, sequence differences were detected between the strains from the first and second disease episodes in all three patients. In one case, clear evidence was obtained for reinfection with an unrelated strain whereas in the other two cases, relapse appeared more probable. Conclusions/Significance This is the first report of using M. leprae whole genome sequencing to reveal that treated and cured leprosy patients who remain in endemic areas can be reinfected by another strain. Next generation sequencing can be applied reliably to M. leprae DNA extracted from biopsies to discriminate between cases of relapse and reinfection, thereby providing a powerful tool for evaluating different outcomes of therapeutic regimens and for following disease transmission.

Genomics: a Swiss army knife to fight leprosy

Charlotte Avanzi

Leprosy, a highly disabling and stigmatizing infectious disease, is caused by Mycobacterium leprae and the newly discovered agent, Mycobacterium lepromatosis. Though treatable with antibiotics, leprosy has still not been eradicated, and around 200,000 new cases are reported every year worldwide, mainly in India, Brazil, and Indonesia. Tipping the balance towards leprosy elimination begins with improving our understanding of the pathogenesis and the transmission of the disease, which remains poorly understood. Current research on leprosy is markedly hindered by our incapacity to cultivate the leprosy bacilli on artificial media, as well as by the variation of the clinical forms of the disease. The rise of genomics in the 2000s has helped to get around these problems by opening new ways of studying organisms. Tools were developed to recover enough genetic material for downstream genomic applications; however, none of them is yet suitable for high throughput purposes. In this thesis, we describe an optimized DNA extraction method from skin tissue that allows direct whole-genome sequencing, and enabled us to obtain ~ 250 genome sequences of M. leprae from different geographical locations throughout the world. Firstly, this dataset deepened our insight into the phylogeny of M. leprae, and points to the ancestral strain originating in East Asia and/or Europe. In addition, analysis of more than twenty drug-resistant strains revealed mutations in candidate genes potentially associated with new biological mechanisms such as drug resistance. Moreover, we analysed isolates from restricted geographic areas and from recurrent cases, and show that the distinction between relapse and reinfection with a closely related strain can be made but this remains challenging. The whole genome sequencing of M. lepromatosis was achieved in 2015, and the discovery and use of new specific molecular detection methods allowed us to identify M. lepromatosis in the red squirrel population in the British Isles. In parallel, M. leprae was also discovered in red squirrels on Brownsea Island in the south of England. Though the risk of transmission from animals to humans is not yet clear, the discovery of a new animal reservoir for leprosy bacilli in a non-endemic country raises the question about the existence of other such reservoirs, especially in endemic countries, which could contribute to ongoing transmission. Reliable and sensitive methods for detection of leprosy bacilli are crucial for early diagnosis and monitoring the disease. We show that efficient cell lysis during extraction increases the yield of genetic material recovered from leprosy bacilli and significantly improves the sensitivity of diagnosis by PCR for all leprosy forms. Overall, our results highlight the impact and efficiency of genomics and whole genome sequencing for uncovering new biological mechanisms in unculturable bacteria such as the leprosy bacilli. Our results generated new hypotheses that await testing, and underline the massive potential of omics and bioinformatics for better understanding and fighting the disease.

EPFL2018

Genomic Characterization ofMycobacterium lepraeto Explore Transmission Patterns Identifies New Subtype in Bangladesh

Charlotte Avanzi, Andrej Benjak, Stewart Cole

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. In 2001, the genome ofM. lepraewas first described and subsequently four genotypes (1-4) and 16 subtypes (A-P) were identified providing means to study global transmission patterns for leprosy. In order to understand the role of asymptomatic carriers we investigatedM. lepraecarriage 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, to studyM. lepraegenotype distribution in Bangladesh we explored strain diversity by whole genome sequencing (WGS) and Sanger sequencing. In the studied cohort in Bangladesh,M. lepraeDNA 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 theM. lepraestrains detected in this study belonged to genotype 1D (55%), followed by 1A (31%). Importantly, WGS allowed the identification of a newM. lepraegenotype, 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 theM. lepraestrains obtained including mutations in hypermutated genes, suggesting mixed colonization/infection or in-host evolution. In summary, we observed thatM. lepraeis 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 atM. leprae-infected or -colonized individuals.

FRONTIERS MEDIA SA2020

Genomics and the chemotherapy of leprosy

Stewart Cole

The information deduced from the genome sequence of Mycobacterium leprae is of immense value for the chemotherapy of leprosy. Knowing the complete set of genes, enzymes and proteins allows us to understand why some drugs are without effect whereas others are fully active. It may also enable better use to be made of existing drugs, such as beta-lactams, and opens new avenues for the development of novel compounds. M. leprae is relatively susceptible to a wide range of drugs, unlike the highly related tubercle bacillus, and several new multidrug regimens are in clinical trials. Genomics provides a number of possible explanations for this broader susceptibility as some of the genes encoding enzymes involved in antibiotic inactivation have decayed whereas the number of transporters available to contribute to drug efflux is considerably lower than in Mycobacterium tuberculosis. Several leads for new drug targets have been uncovered.