Genetic diversity of Mycobacterium leprae: Need to move towards genome-wide approaches.

ABSTRACT: Leprosy, an ancient disease, continues to be a public health concern as it remains endemic in several countries. After reaching the elimination target (1/10,000) as a public health problem in 2005 in India, around 1.2 lakh cases have been detected every year over the last decade indicating active transmission of leprosy bacillus (Mycobacterium leprae). Single-nucleotide polymorphisms (SNPs), genomic insertions/deletions and variable-number tandem repeats (VNTRs) have been identified as genetic markers for tracking M. leprae transmission. As the leprosy bacilli cannot be cultured in vitro, molecular testing of M. leprae genotypes is done by polymerase chain reaction-based sequencing which provides a practical alternative for the identification of strains as well as drug resistance-associated mutations. Whole-genome sequencing (WGS) of M. leprae directly from clinical samples has also proven to be an effective tool for identifying genetic variations which can further help refine the molecular epidemiological schemes based on SNPs and VNTRs. However, the WGS data of M. leprae strains from India are scarce, being responsible for a gross under-representation of the genetic diversity of M. leprae strains present in India and need to be addressed suitably. Molecular studies of leprosy can provide better insight into phylogeographic markers to monitor the transmission dynamics and emergence of antimicrobial resistance. An improved understanding of M. leprae transmission is essential to guide efficient leprosy control strategies. Therefore, this review compiles and discusses the current status of molecular epidemiology, genotyping and the potential of genome-wide analysis of M. leprae strains in the Indian context.

Novel mutations found in Mycobacterium leprae DNA repair gene nth from central India.

INTRODUCTION: The importance of DNA repair enzymes in maintaining genomic integrity is highlighted by the hypothesis that DNA damage by reactive oxygen/nitrogen species produced inside the host cell is essential for the mutagenesis process. Endonuclease III (Nth), formamidopyrimide (Fpg) and endonuclease VIII (Nei) DNA glycosylases are essential components of the bacterial base excision repair process. Mycobacterium leprae lost both fpg/nei genes during the reductive evolution event and only has the nth (ML2301) gene. This study aims to characterize the mutations in the nth gene of M. leprae strains and explore its correlation with drug-resistance. METHOD: A total of 91 M. leprae positive DNA samples extracted from skin biopsy samples of newly diagnosed leprosy patients from NSCB Hospital Jabalpur were assessed for the nth gene as well as drug resistance-associated loci of the rpoB, gyrA and folP1 genes through PCR followed by Sanger sequencing. RESULTS: Of these 91 patients, a total of two insertion frameshift mutations, two synonymous and seven nonsynonymous mutations were found in nth in seven samples. Sixteen samples were found to be resistant to ofloxacin and one was found to be dapsone resistant as per the known DRDR mutations. No mutations were found in the rpoB region. Interestingly, none of the nth mutations were identified in the drug-resistant associated samples. CONCLUSION: The in-silico structural analysis of the non-synonymous mutations in the Nth predicted five of them were to be deleterious. Our results suggest that the mutations in the nth gene may be potential markers for phylogenetic and epidemiological studies.

Multiplex PCR-based RFLP assay for early identification of prevalent Mycobacterium leprae genotypes.

Mycobacterium leprae is classified into four SNP genotypes and 16 subtypes (from 1A to 4P) that exhibit phylogeographical association reported from around the world. Among them, genotypes 1D and 3I represent more than 60% of M. leprae strains. Here, we report a new method for M. leprae genotyping which identifies the genotypes 1D and 3I by combining multiplex PCR amplification and restriction fragment length polymorphism (RFLP) of a M. leprae DNA amplicons using AgeI restriction enzyme. Agarose gel electrophoresis showed a deletion of 11 bp only among 3I genotypes by electrophoresis. When this multiplex PCR reaction is subjected to AgeI digestion, successful restriction digestion shows three bands for all the genotypes except 1D where only two bands were observed due to loss of restriction site. This method gives us the advantage of 1-step identification of the two most prevalent strains of M. leprae without using specialized equipments such as the Sanger sequencing system or quantitative PCR.

Implementation of genetic screening test to reduce the incidence of dapsone hypersensitivity syndrome among patients with leprosy in Papua, Indonesia: a study protocol.

INTRODUCTION: The mainstay of leprosy treatment is multidrug treatment (MDT), which contains rifampicin, dapsone and clofazimine. The occurrence of dapsone hypersensitivity syndrome (DHS), a sudden, potentially fatal and traumatic adverse reaction due to dapsone, may affect treatment adherence and may result in fatality if untreated. Before MDT administration, screening for HLA-B*13:01 in patients with leprosy can potentially reduce DHS risk. The study aims to assess the effectiveness of using a screening test for HLA-B*13:01 in reducing the incidence of DHS and to evaluate the feasibility of using the quantitative PCR-based screening tool as DHS predictors before dapsone administration using individual patient testing in a referral centralised-lab model. METHODS AND ANALYSIS: A total of 310 newly diagnosed patients with leprosy will be recruited from health centres in two highly endemic districts in Indonesia. Dried blood will be taken on filter paper as the specimen receptacle to collect DNA from the patients and transported at room temperature to the leprosy referral laboratory before MDT administration. Checking for HLA-B*13:01 from human DNA is performed using the Nala PGx 1301 V.1 kit. The results will be shared with the leprosy health workers on the site via phone call and courier. Patients with a positive test result will be treated with MDT without dapsone, and patients with a negative result will be treated with complete MDT. Physical examination (weight, height, skin, muscle and nerve function examination), complete blood tests (including renal function test) will be carried out at baseline. Follow-up will be performed at the fourth and eighth weeks to observe any development of adverse drug reactions. ETHICS AND DISSEMINATION: The ethical approval for the study was issued by the Ethical Committee of the National Institute of Health Research and Development, Ministry of Health, Indonesia. Written informed consent will be sought from all participants.

Role of TlyA in the Biology of Uncultivable Mycobacteria.

TlyA proteins are related to distinct functions in a diverse spectrum of bacterial pathogens, including mycobacterial spp. There are several annotated proteins that function as hemolysin or pore-forming molecules that play an important role in the virulence of pathogenic organisms. Many studies reported the dual activity of mycobacterial TlyA as 'hemolysin' and 'Sadenosylmethionine dependent rRNA methylase'. To act as a hemolysin, a sequence must have a signal sequence and transmembrane segment, which helps the protein enter the extracellular environment. Interestingly, the mycobacterial tlyA has neither traditional signal sequences of general/ sec/tat pathways nor any transmembrane segments. Still, it can reach the extracellular milieu with the help of non-classical signal mechanisms. Also, retention of tlyA in cultivable mycobacterial pathogens (such as Mycobacterium tuberculosis and M. marinum) as well as uncultivated mycobacterial pathogens despite their extreme reductive evolution (such as M. leprae, M. lepromatosis and M. uberis) suggests its crucial role in the evolutionary biology of pathogenic mycobacteria. Numerous virulence factors have been characterised by the uncultivable mycobacteria, but the information of TlyA protein is still limited in terms of molecular and structural characterisation. The genomic insights offered by comparative analysis of TlyA sequences and their conserved domains reveal pore-forming activity, which further confirms its role as a virulence protein, particularly in uncultivable mycobacteria. Therefore, this review presents a comparative analysis of the mycobacterial TlyA family by sequence homology and alignment to improve our understanding of this unconventional hemolysin and RNA methyltransferase TlyA of uncultivable mycobacteria.

Repurposing Drugs to Combat Drug Resistance in Leprosy: A Review of Opportunities.

Leprosy is caused by extremely slow-growing and uncultivated mycobacterial pathogens, namely Mycobacterium leprae and M. lepromatosis. Nearly 95% of the new cases of leprosy recorded globally are found in India, Brazil, and 20 other priority countries (WHO, 2019), of which nearly two-third of the cases are reported in India alone. Currently, leprosy is treated with dapsone, rifampicin, and clofazimine, also known as multi-drug therapy (MDT), as per the recommendations of WHO since 1981. Still, the number of new leprosy cases recorded globally has remained constant in last one-decade, and resistance to multiple drugs has been documented in various parts of the world, even though relapses are rare in patients treated with MDT. Antimicrobial resistance testing against M. leprae or the evaluation of the anti-leprosy activity of new drugs remains a challenge as leprosy bacilli cannot grow in vitro. Besides, developing a new drug against leprosy through conventional drug development process is not economically attractive or viable for pharma companies. Therefore, a promising alternative is the repurposing of existing drugs/approved medications or their derivatives for assessing their anti-leprosy potential. It is an efficient method to identify novel medicinal and therapeutic properties of approved drug molecules. Any combinatorial chemotherapy that combines these repurposed drugs with the existing first-line (MDT) and second-line drugs could improve the bactericidal and synergistic effects against these notorious bacteria and can help in achieving the much-cherished goal of "leprosy-free world". This review highlights novel opportunities for drug repurposing to combat resistance to current therapeutic approaches.

Epidemiological scenario of leprosy in marginalized communities of India: Focus on scheduled tribes.

The Scheduled Tribes (STs) are designated among the most disadvantaged social groups in India. Until the year 2005 (pre-elimination era of leprosy in India), several leprosy-specific control field programmes were implemented, which have been discontinued subsequently. Since then, leprosy diagnosis and treatment have been integrated with General Health Services. Thereafter, specialized expertise for the early diagnosis of leprosy has been gradually diminishing, especially at the peripheral clinics in remote areas. Hence, leprosy cases usually remain undetected for a long time and persist as endemic reservoirs. The tribal population of India accounts for just 8.6 per cent of the overall population. However, 18.5 per cent of the new leprosy cases were detected within the tribal community in the year 2020, indicating a disproportionately high burden of leprosy among the tribal population. Recent data suggest that these health disparities can be mainly related to the increased marginalization of STs as compared to other communities. This shows the need to further explore the current situation of leprosy in STs so that suitable interventions can address the contributing factors, leading to health inequalities in disadvantaged socio-economic groups. Therefore, this review aims to present the current distribution of leprosy in marginalized communities with a special emphasis on STs. Further, this review discusses how resources might be mobilized for such communities to find and treat undetected leprosy patients in STs to enable effective control of leprosy through early detection and timely treatment.

Simultaneous detection and differentiation between Mycobacterium leprae and Mycobacterium lepromatosis using novel polymerase chain reaction primers.

We have developed a polymerase chain reaction-based method to detect and distinguish Mycobacterium leprae and Mycobacterium lepromatosis using a single set of primers based on a 45-bp difference in the amplicon size of their rpoT gene. This method can also help in detecting the cases of co-infection in a single experiment.

Mycobacterium lepromatosis MLPM_5000 is a potential heme chaperone protein HemW and mis-annotation of its orthologues in mycobacteria.

The genome of a newly identified leprosy causing bacillus Mycobacterium lepromatosis was sequenced in 2015 wherein a gene MLPM_5000 was detected whose corresponding sequences are missing in its close relative Mycobacterium leprae, the well-known causal agent of leprosy. Thus MLPM_5000 is considered to be a specific genomic locus for differentiating M. lepromatosis from M. leprae. The locus was annotated as HemN (Coproporphyrinogen III oxidase) based on the available annotations in other mycobacterial species. However, we noticed that the MLPM_5000 and its orthologues in different mycobacterial species show a much higher degree of similarity with Escherichia coli HemW (378 aa) in comparison to the E. coli HemN (457 aa). Additionally, the fourth cysteine of the characteristic CX3CX2CXC motif of the E. coli HemN is replaced by a phenylalanine in the M. lepromatosis MLPM_5000 and its mycobacterial orthologues, which is a hallmark of heme chaperone protein HemW in E. coli and other species. Phylogenetic analysis of MLPM_5000 and its mycobacterial orthologues also showed that these proteins form a divergent phylogenetic clade with the HemW proteins of other species such as Escherichia coli and Lactococcus lactis. Further, Molecular Dynamics simulation studies also predicted that the residues of conserved HNXXYW motif of the MLPM_5000 may have a role in binding to heme part of the host hemoglobin, thereby suggesting it to be a HemW instead of HemN. Altogether, this work shows that MLPM_5000 and its mycobacterial orthologues are highly unlikely to be HemN. Therefore, the current annotations of mycobacterial HemN sequences should be corrected to heme chaperone 'HemW' in various protein databases. The study not only corrects the mis-annotation but also provides a new perspective in the context of evolutionary history of M. leprae and M. lepromatosis such as lack of HemW in M. leprae may explain some of the variations in the virulence between the two pathogens.

Comparison of target enrichment strategies for ancient pathogen DNA.

In ancient DNA research, the degraded nature of the samples generally results in poor yields of highly fragmented DNA; targeted DNA enrichment is thus required to maximize research outcomes. The three commonly used methods - array-based hybridization capture and in-solution capture using either RNA or DNA baits - have different characteristics that may influence the capture efficiency, specificity and reproducibility. Here we compare their performance in enriching pathogen DNA of Mycobacterium leprae and Treponema pallidum from 11 ancient and 19 modern samples. We find that in-solution approaches are the most effective method in ancient and modern samples of both pathogens and that RNA baits usually perform better than DNA baits.

Molecular epidemiology of leprosy: An update.

Molecular epidemiology investigations are notoriously challenging in the leprosy field mainly because the inherent characteristics of the disease as well as its yet uncultivated causative agents, Mycobacterium leprae and M. lepromatosis. Despite significant developments in understanding the biology of leprosy bacilli through genomic approaches, the exact mechanisms of transmission is still unclear and the factors underlying pathological variation of the disease in different patients remain as major gaps in our knowledge about leprosy. Despite these difficulties, the last two decades have seen the development of genotyping procedures based on PCR-sequencing of target loci as well as by the genome-wide analysis of an increasing number of geographically diverse isolates of leprosy bacilli. This has provided a foundation for molecular epidemiology studies that are bringing a better understanding of strain evolution associated with ancient human migrations, and phylogeographical insights about the spread of disease globally. This review discusses the advantages and drawbacks of the main tools available for molecular epidemiological investigations of leprosy and summarizes various methods ranging from PCR-based genotyping to genome-typing techniques. We also describe their main applications in analyzing the short-range and long-range transmission of the disease. Finally, we summarise the current gaps and challenges that remain in the field of molecular epidemiology of leprosy.

Population Genomics of Mycobacterium leprae Reveals a New Genotype in Madagascar and the Comoros.

Human settlement of Madagascar traces back to the beginning of the first millennium with the arrival of Austronesians from Southeast Asia, followed by migrations from Africa and the Middle East. Remains of these different cultural, genetic, and linguistic legacies are still present in Madagascar and other islands of the Indian Ocean. The close relationship between human migration and the introduction and spread of infectious diseases, a well-documented phenomenon, is particularly evident for the causative agent of leprosy, Mycobacterium leprae. In this study, we used whole-genome sequencing (WGS) and molecular dating to characterize the genetic background and retrace the origin of the M. leprae strains circulating in Madagascar (n = 30) and the Comoros (n = 3), two islands where leprosy is still considered a public health problem and monitored as part of a drug resistance surveillance program. Most M. leprae strains (97%) from Madagascar and Comoros belonged to a new genotype as part of branch 1, closely related to single nucleotide polymorphism (SNP) type 1D, named 1D-Malagasy. Other strains belonged to the genotype 1A (3%). We sequenced 39 strains from nine other countries, which, together with previously published genomes, amounted to 242 genomes that were used for molecular dating. Specific SNP markers for the new 1D-Malagasy genotype were used to screen samples from 11 countries and revealed this genotype to be restricted to Madagascar, with the sole exception being a strain from Malawi. The overall analysis thus ruled out a possible introduction of leprosy by the Austronesian settlers and suggests a later origin from East Africa, the Middle East, or South Asia.

Isolation of Mycobacterium lepromatosis and Development of Molecular Diagnostic Assays to Distinguish Mycobacterium leprae and M. lepromatosis.

BACKGROUND: Mycobacterium leprae was thought to be the exclusive causative agent of leprosy until Mycobacterium lepromatosis was identified in a rare form of leprosy known as diffuse lepromatous leprosy (DLL). METHODS: We isolated M. lepromatosis from a patient with DLL and propagated it in athymic nude mouse footpads. Genomic analysis of this strain (NHDP-385) identified a unique repetitive element, RLPM, on which a specific real-time quantitative polymerase chain reaction assay was developed. The RLPM assay, and a previously developed RLEP quantitative polymerase chain reaction assay for M. leprae, were validated as clinical diagnostic assays according to Clinical Laboratory Improvement Amendments guidelines. We tested DNA from archived histological sections, patient specimens from the United States, Philippines, and Mexico, and US wild armadillos. RESULTS: The limit of detection for the RLEP and RLPM assays is 30 M. leprae per specimen (0.76 bacilli per reaction; coefficient of variation, 0.65%-2.44%) and 122 M. lepromatosis per specimen (3.05 bacilli per reaction; 0.84%-2.9%), respectively. In histological sections (n = 10), 1 lepromatous leprosy (LL), 1 DLL, and 3 Lucio reactions contained M. lepromatosis; 2 LL and 2 Lucio reactions contained M. leprae; and 1 LL reaction contained both species. M. lepromatosis was detected in 3 of 218 US biopsy specimens (1.38%). All Philippines specimens (n = 180) were M. lepromatosis negative and M. leprae positive. Conversely, 15 of 47 Mexican specimens (31.91%) were positive for M. lepromatosis, 19 of 47 (40.43%) were positive for M. leprae, and 2 of 47 (4.26%) contained both organisms. All armadillos were M. lepromatosis negative. CONCLUSIONS: The RLPM and RLEP assays will aid healthcare providers in the clinical diagnosis and surveillance of leprosy.

Ancient genomes reveal a high diversity of Mycobacterium leprae in medieval Europe.

Studying ancient DNA allows us to retrace the evolutionary history of human pathogens, such as Mycobacterium leprae, the main causative agent of leprosy. Leprosy is one of the oldest recorded and most stigmatizing diseases in human history. The disease was prevalent in Europe until the 16th century and is still endemic in many countries with over 200,000 new cases reported annually. Previous worldwide studies on modern and European medieval M. leprae genomes revealed that they cluster into several distinct branches of which two were present in medieval Northwestern Europe. In this study, we analyzed 10 new medieval M. leprae genomes including the so far oldest M. leprae genome from one of the earliest known cases of leprosy in the United Kingdom-a skeleton from the Great Chesterford cemetery with a calibrated age of 415-545 C.E. This dataset provides a genetic time transect of M. leprae diversity in Europe over the past 1500 years. We find M. leprae strains from four distinct branches to be present in the Early Medieval Period, and strains from three different branches were detected within a single cemetery from the High Medieval Period. Altogether these findings suggest a higher genetic diversity of M. leprae strains in medieval Europe at various time points than previously assumed. The resulting more complex picture of the past phylogeography of leprosy in Europe impacts current phylogeographical models of M. leprae dissemination. It suggests alternative models for the past spread of leprosy such as a wide spread prevalence of strains from different branches in Eurasia already in Antiquity or maybe even an origin in Western Eurasia. Furthermore, these results highlight how studying ancient M. leprae strains improves understanding the history of leprosy worldwide.

Differential growth of Mycobacterium leprae strains (SNP genotypes) in armadillos.

Leprosy (Hansen's Disease) has occurred throughout human history, and persists today at a low prevalence in most populations. Caused by Mycobacterium leprae, the infection primarily involves the skin, mucosa and peripheral nerves. The susceptible host range for Mycobacterium leprae is quite narrow. Besides humans, nine banded armadillos (Dasypus novemcinctus) and red squirrels (Sciurus vulgaris) are the only other natural hosts for M. leprae, but only armadillos recapitulate the disease as seen in humans. Armadillos across the Southern United States harbor a single predominant genotypic strain (SNP Type-3I) of M. leprae, which is also implicated in the zoonotic transmission of leprosy. We investigated, whether the zoonotic strain (3I) has any notable growth advantages in armadillos over another genetically distant strain-type (SNP Type-4P) of M. leprae, and if M. leprae strains manifest any notably different pathology among armadillos. We co-infected armadillos (n = 6) with 2 × 109 highly viable M. leprae of both strains and assessed the relative growth and dissemination of each strain in the animals. We also analyzed 12 additional armadillos, 6 each individually infected with the same quantity of either strain. The infections were allowed to fulminate and the clinical manifestations of the disease were noted. Animals were humanely sacrificed at the terminal stage of infection and the number of bacilli per gram of liver, spleen and lymph node tissue were enumerated by Q-PCR assay. The growth of M. leprae strain 4P was significantly higher (P < 0.05) than 3I when each strain was propagated individually in armadillos. Significantly (P < 0.0001) higher growth of the 4P strain also was confirmed among animals co-infected with both 3I and 4P strain types using whole genome sequencing. Interestingly, the zoonotic strain does not exhibit any growth advantage in these non-human hosts, but the varied proliferation of the two M. leprae strains within armadillos suggest there are notable pathological variations between M. leprae strain-types.

Phylogenomics and antimicrobial resistance of the leprosy bacillus Mycobacterium leprae.

Leprosy is a chronic human disease caused by the yet-uncultured pathogen Mycobacterium leprae. Although readily curable with multidrug therapy (MDT), over 200,000 new cases are still reported annually. Here, we obtain M. leprae genome sequences from DNA extracted directly from patients' skin biopsies using a customized protocol. Comparative and phylogenetic analysis of 154 genomes from 25 countries provides insight into evolution and antimicrobial resistance, uncovering lineages and phylogeographic trends, with the most ancestral strains linked to the Far East. In addition to known MDT-resistance mutations, we detect other mutations associated with antibiotic resistance, and retrace a potential stepwise emergence of extensive drug resistance in the pre-MDT era. Some of the previously undescribed mutations occur in genes that are apparently subject to positive selection, and two of these (ribD, fadD9) are restricted to drug-resistant strains. Finally, nonsense mutations in the nth excision repair gene are associated with greater sequence diversity and drug resistance.

Phylogenomics and antimicrobial resistance of the leprosy bacillus Mycobacterium leprae

Leprosy is a chronic human disease caused by the yet-uncultured pathogen Mycobacterium leprae. Although readily curable with multidrug therapy (MDT), over 200,000 new cases are still reported annually. Here, we obtain M. leprae genome sequences from DNA extracted directly from patients' skin biopsies using a customized protocol. Comparative and phylogenetic analysis of 154 genomes from 25 countries provides insight into evolution and antimicrobial resistance, uncovering lineages and phylogeographic trends, with the most ancestral strains linked to the Far East. In addition to known MDT-resistance mutations, we detect other mutations associated with antibiotic resistance, and retrace a potential stepwise emergence of extensive drug resistance in the pre-MDT era. Some of the previously undescribed mutations occur in genes that are apparently subject to positive selection, and two of these (ribD, fadD9) are restricted to drug-resistant strains. Finally, nonsense mutations in the nth excision repair gene are associated with greater sequence diversity and drug resistance.

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.