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.

Profiling of Intracellular Metabolites: An Approach to Understanding the Characteristic Physiology of Mycobacterium leprae.

Mycobacterium leprae is the causative agent of leprosy and also known to possess unique features such as inability to proliferate in vitro. Among the cellular components of M. leprae, various glycolipids present on the cell envelope are well characterized and some of them are identified to be pathogenic factors responsible for intracellular survival in host cells, while other intracellular metabolites, assumed to be associated with basic physiological feature, remain largely unknown. In the present study, to elucidate the comprehensive profile of intracellular metabolites, we performed the capillary electrophoresis-mass spectrometry (CE-MS) analysis on M. leprae and compared to that of M. bovis BCG. Interestingly, comparison of these two profiles showed that, in M. leprae, amino acids and their derivatives are significantly accumulated, but most of intermediates related to central carbon metabolism markedly decreased, implying that M. leprae possess unique metabolic features. The present study is the first report demonstrating the unique profiles of M. leprae metabolites and these insights might contribute to understanding undefined metabolism of M. leprae as well as pathogenic characteristics related to the manifestation of the disease.

Mutations in the drug resistance-determining region of Mycobacterium lepromatosis isolated from leprosy patients in Mexico.

Mycobacterium lepromatosis, an independent species from Mycobacterium leprae, has been found to be a causative agent for diffuse lepromatous leprosy (DLL) in Mexico, but remains poorly studied. Here, the drug resistance-determining regions (DRDR) of folP1, rpoB and gyrA (conferring resistance to dapsone, rifampicin and quinolone, respectively) in M. lepromatosis from leprosy patients in Mexico were characterized. No mutations or silent mutations were found at previously characterized major sites in DRDR of M. lepromatosis. However, a non-synonymous mutation was found in codon 54 between two major sites of the folP1 DRDR in M. lepromatosis sequences. All M. lepromatosis isolates showed CAG sequence in codon 54 of folP1. Because the next codons 53 and 55 are known as major mutation sites for drug resistance, more detailed analysis using more samples is needed to determine whether it influences susceptibility to dapsone and/or efficiency of folate biosynthesis in M. lepromatosis or not.

[Rapid detection of mutations related to Mycobacterium leprae drug resistance by using Hp-rPCR (hairpin primer- real time PCR) method].

Rapid and simple detection method of drug resistance bacteria is required. In the present study, Hp-rPCR (hairpin primer-real time PCR) was applied to Mycobacterium leprae genes to detect mutations. Target sites of the method were as follows: first base and second base on 53rd codon and second base on 55th codon infolP1 gene for dapsone resistance, first base on 441st codon and 451st codon and second base on 456th and 458th codon in rpoB gene for rifampicin resistance, and first base on 89th codon and second base on 91st codon in gyrA gene for quinolone resistance which were common mutation sites in clinical reports. The total number of the target sites was 9. Mycobacterium leprae, Thai-53, Zensho-2 and Zensho-4 were used as reference bacteria in the present study and clear, reliable results were obtained. Double-blind study was conducted using 15 samples. The number of target sites was calculated as 135 in total by 9 sites in 15 samples. There was only one misreading in the blind samples and the sensitivity was more than 99%.

[Guidelines for the treatment of Hansen's disease in Japan (third edition)].

ad hoc committee of Japanese Leprosy Association recommends revised standard treatment protocol of leprosy in Japan, which is a modification of World Health Organization's multidrug therapy (WHO/MDT, 2010). For paucibacillary (PB) leprosy, 6 months treatment by rifampicin and dapsone (MDT/PB) is enough. However, for high bacterial load multibacillary (MB) leprosy, 12 months treatment seems insufficient. Thus, (A) For MB with bacterial index (BI) > 3 before treatment, 2 years treatment by rifampicin, dapsone and clofazimine (MDT/MB) is necessary. When BI becomes negative and active lesion is lost within 2 years, no maintenance therapy is necessary. When BI is still positive, one year of MDT/MB is added (3 years in total), followed by maintenance therapy by dapsone and clofazimine until BI negativity and loss of active lesions. (B) For MB with BI < 3 or fresh MB (less than 6 months after the onset of the disease) with BI > 3, 1 year treatment by MDT/MB is necessary. When BI becomes negative and active lesion is lost within one year, no maintenance therapy is necessary. When BI is still positive or active lesion is remaining, additional therapy with MDT/MB for one more year is recommended. Brief summary of diagnosis, purpose of therapy, character of drugs, and prevention of deformity is also described.

Mutation analysis of mycobacterial rpoB genes and rifampin resistance using recombinant Mycobacterium smegmatis.

Rifampin is a major drug used to treat leprosy and tuberculosis. The rifampin resistance of Mycobacterium leprae and Mycobacterium tuberculosis results from a mutation in the rpoB gene, encoding the ß subunit of RNA polymerase. A method for the molecular determination of rifampin resistance in these two mycobacteria would be clinically valuable, but the relationship between the mutations and susceptibility to rifampin must be clarified before its use. Analyses of mutations responsible for rifampin resistance using clinical isolates present some limitations. Each clinical isolate has its own genetic variations in some loci other than rpoB, which might affect rifampin susceptibility. For this study, we constructed recombinant strains of Mycobacterium smegmatis carrying the M. leprae or M. tuberculosis rpoB gene with or without mutation and disrupted their own rpoB genes on the chromosome. The rifampin and rifabutin susceptibilities of the recombinant bacteria were measured to examine the influence of the mutations. The results confirmed that several mutations detected in clinical isolates of these two pathogenic mycobacteria can confer rifampin resistance, but they also suggested that some mutations detected in M. leprae isolates or rifampin-resistant M. tuberculosis isolates are not involved in rifampin resistance.

Real-time PCR and high-resolution melt analysis for rapid detection of Mycobacterium leprae drug resistance mutations and strain types.

Drug resistance surveillance and strain typing of Mycobacterium leprae are necessary to investigate ongoing transmission of leprosy in regions of endemicity. To enable wider implementation of these molecular analyses, novel real-time PCR-high-resolution melt (RT-PCR-HRM) assays without allele-specific primers or probes and post-PCR sample handling were developed. For the detection of mutations within drug resistance-determining regions (DRDRs) of folP1, rpoB, and gyrA, targets for dapsone, rifampin, and fluoroquinolones, real-time PCR-HRM assays were developed. Wild-type and drug-resistant mouse footpad-derived strains that included three folP1, two rpoB, and one gyrA mutation types in a reference panel were tested. RT-PCR-HRM correctly distinguished the wild type from the mutant strains. In addition, RT-PCR-HRM analyses aided in recognizing samples with mixed or minor alleles and also a mislabeled sample. When tested in 121 sequence-characterized clinical strains, HRM identified all the folP1 mutants representing two mutation types, including one not within the reference panel. The false positives (<5%) could be attributed to low DNA concentration or PCR inhibition. A second set of RT-PCR-HRM assays for identification of three previously reported single nucleotide polymorphisms (SNPs) that have been used for strain typing were developed and validated in 22 reference and 25 clinical strains. Real-time PCR-HRM is a sensitive, simple, rapid, and high-throughput tool for routine screening known DRDR mutants in new and relapsed cases, SNP typing, and detection of minor mutant alleles in the wild-type background at lower costs than current methods and with the potential for quality control in leprosy investigations.

A lipopeptide facilitate induction of Mycobacterium leprae killing in host cells.

Little is known of the direct microbicidal activity of T cells in leprosy, so a lipopeptide consisting of the N-terminal 13 amino acids lipopeptide (LipoK) of a 33-kD lipoprotein of Mycobacterium leprae, was synthesized. LipoK activated M. leprae infected human dendritic cells (DCs) to induce the production of IL-12. These activated DCs stimulated autologous CD4+ or CD8+ T cells towards type 1 immune response by inducing interferon-gamma secretion. T cell proliferation was also evident from the CFSE labeling of target CD4+ or CD8+ T cells. The direct microbicidal activity of T cells in the control of M. leprae multiplication is not well understood. The present study showed significant production of granulysin, granzyme B and perforin from these activated CD4+ and CD8+ T cells when stimulated with LipoK activated, M. leprae infected DCs. Assessment of the viability of M. leprae in DCs indicated LipoK mediated T cell-dependent killing of M. leprae. Remarkably, granulysin as well as granzyme B could directly kill M. leprae in vitro. Our results provide evidence that LipoK could facilitate M. leprae killing through the production of effector molecules granulysin and granzyme B in T cells.

[Practice of international collaborations in Southeast Asia].

Leprosy Research Center is engaged in the international collaborative research project since 1989. The project was consists of two parts. One part is the JICA training course which has been practiced since 1989. Another part is the international collaborative research which was started in Indonesia in the year 1991. Author has participated in this project since 1998. Then, we started collaboration with various organizations including Pakistan, Vietnam, and Myanmar. The contents of the collaborative research were mainly technical assistances for leprosy diagnosis and we have trained young doctors, staff to conduct serological diagnosis and molecular biological diagnosis of leprosy. The projects between the countries were succesful. Throughout the collaboration with foreign countries, author felt strongly that one of most important things in such collaboration was better communication and relation between people having different cultural background.

FTA card utility for PCR detection of Mycobacterium leprae.

The suitability of the FTA® elute card for the collection of slit skin smear (SSS) samples for PCR detection of Mycobacterium leprae was evaluated. A total of 192 SSS leprosy samples, of bacillary index (BI) 1 to 5, were collected from patients attending two skin clinics in Myanmar and preserved using both FTA® elute cards and 70% ethanol tubes. To compare the efficacy of PCR detection of DNA from each BI class, PCR was performed to amplify an M. leprae-specific repetitive element. Of the 192 samples, 116 FTA® elute card and 112 70% ethanol samples were PCR positive for M. leprae DNA. When correlated with BI, area under the curve (AUC) values of the respective receiver-operating characteristic curves were similar for the FTA® elute card and ethanol collection methods (AUC=0.6). Taken together, our results indicate that the FTA® elute card, which enables the collection, transport, and archiving of clinical samples, is an attractive alternative to ethanol preservation for the detection of M. leprae DNA.

Analysis of drug-resistant strains of Mycobacterium leprae in an endemic area of Vietnam.

BACKGROUND: Multidrug therapy has effectively reduced the number of leprosy cases in the world. However, the rate of reduction has decelerated over the years, giving early detection of Mycobacterium leprae and epidemiological study of relapse renewed relevance in attempts to eliminate the disease. METHODS: A molecular epidemiological survey for drug-resistant M. leprae was conducted in the central and highland regions of Vietnam. A total of 423 samples taken from patients, including 83 patients with new cases, 321 patients receiving treatment, and 19 patients with relapse, were studied for detection of M. leprae with mutations relating to drug resistance by sequencing the drug resistance determining region of the folP1, rpoB, and gyrA genes, which are responsible for dapsone, rifampicin, and ofloxacin resistance, respectively. RESULTS: Nineteen mutations were found in the folP1 gene samples, and no mutations relating to drug resistance were found in either the rpoB or gyrA genes. Samples from patients with relapse showed folP1 mutation rates as high as 57%, and the mutation rates in samples from new and recent cases were <10%. Patients with relapse who had histories of treatment with dapsone monotherapy showed high mutation rates (78%), compared with patients with relapse who had previously only received multidrug therapy (33%). CONCLUSIONS: Our study indicated high rates of dapsone resistance in patients with relapse, compared with patients with new and recent cases of leprosy. Moreover, it was observed that many of the patients with relapse who had dapsone-resistant mutations had histories of treatment with dapsone monotherapy.

Mutation analysis of the Mycobacterium leprae folP1 gene and dapsone resistance.

Diaminodiphenylsulfone (dapsone) has long been used as a first-line drug worldwide for the treatment of leprosy. Diagnosis for dapsone resistance of Mycobacterium leprae by DNA tests would be of great clinical value, but the relationship between the nucleotide substitutions and susceptibility to dapsone must be clarified before use. In this study, we constructed recombinant strains of cultivable Mycobacterium smegmatis carrying the M. leprae folP1 gene with or without a point mutation, disrupting their own folP gene on the chromosome. Dapsone susceptibilities of the recombinant bacteria were measured to examine influence of the mutations. Dapsone MICs for most of the strains with mutations at codon 53 or 55 of M. leprae folP1 were 2 to 16 times as high as the MIC for the strain with the wild-type folP1 sequence, but mutations that changed Thr to Ser at codon 53 showed somewhat lower MIC values than the wild-type sequence. Strains with mutations at codon 48 or 54 showed levels of susceptibility to dapsone comparable to the susceptibility of the strain with the wild-type sequence. This study confirmed that point mutations at codon 53 or 55 of the M. leprae folP1 gene result in dapsone resistance.

Immunostimulatory activity of major membrane protein II from Mycobacterium tuberculosis.

Previously, we observed that both major membrane protein II of Mycobacterium leprae (MMP-ML) and its fusion with M. bovis BCG (BCG)-derived heat shock protein 70 (HSP70) (Fusion-ML) are immunogenic and that recombinant BCG secreting either of these proteins effectively inhibits the multiplication of M. leprae in mice. Here, we purified M. tuberculosis-derived major membrane protein II (MMP-MTB) and its fusion with HSP70 (Fusion-MTB) in a lipopolysaccharide-free condition and evaluated their immunostimulatory abilities. Both MMP-MTB and Fusion-MTB activated monocyte-derived dendritic cells (DC) in terms of phenotype and interleukin-12 (IL-12) production, but Fusion-MTB more efficiently activated them than MMP-MTB did. The IL-12 production was a consequence of the ligation of those recombinant proteins with Toll-like receptor 2. The M. tuberculosis-derived and M. leprae-derived recombinant proteins activated naïve T cells of both CD4 and CD8 subsets, but M. tuberculosis-derived proteins were superior to M. leprae-derived proteins and fusion proteins were superior to MMP, regardless of the origin of the protein. Memory-type CD4(+) T cells obtained from BCG-vaccinated healthy individuals seem to be primed with MMP-MTB by the vaccination, and both M. tuberculosis-derived recombinant proteins produced perforin-producing CD8(+) T cells from memory-type CD8(+) T cells. Further, infection of DC and macrophages with M. tuberculosis H37Ra and H37Rv induced the expression of MMP on their surface. These results indicate that M. tuberculosis-derived MMP, as a sole protein or as part of a fusion protein, may be useful for developing new vaccinating agents against tuberculosis.

GM-CSF-mediated T-cell activation by macrophages infected with recombinant BCG that secretes major membrane protein-II of Mycobacterium leprae.

The potential of Mycobacterium bovis Bacillus Calmette-Guerin (BCG) needs to be augmented to efficiently activate CD4(+) T cells through macrophages. Mycobacterium leprae-derived recombinant major membrane protein (MMP)-II induced GM-CSF production from macrophages. A recombinant BCG-SM that secretes MMP-II more efficiently produced GM-CSF and activated interferon (IFN)-gamma-producing CD4(+) T cells than did vector control BCG when infected with macrophages. The T-cell activation by BCG-SM was dependent on the GM-CSF production by macrophages. Interleukin (IL)-10 production by macrophages stimulated with M. leprae was inhibited in a GM-CSF-dependent manner when the precursor monocytes were infected with BCG-SM. BCG inducing GM-CSF production was effective in macrophage-mediated T-cell activation partially through IL-10 inhibition.

Detection of serum antibodies to M. leprae major membrane protein-II in leprosy patients from Indonesia.

BACKGROUND: Sero-diagnostic methods are the easiest way of diagnosing an infectious disease in developing countries. In leprosy, phenolic glycolipid-1 (PGL-I) based methods for the detection of leprosy are currently available, but the use of these methods has been hindered due to the inherent problems of sensitivity. We previously showed that antibodies to Major Membrane Protein-II (MMP-II) derived from Mycobacterium leprae could be used to diagnose leprosy in Japan. METHODS: Sera from patients and healthy individuals were collected with informed consent and the anti-MMP-II antibody levels of the sera were measured by enzyme-linked immunosorbent assay. The study was conducted at South Sulawesi and Bali, in Indonesia. The study population included 40 each of multibacillary leprosy and paucibacillary leprosy patients, 30 tuberculosis and 16 patients with typhoid. RESULTS: We evaluated the anti-MMP-II antibody levels in Indonesian individuals. The cut-off value was determined from receiver operator characteristic curve as 0.124 using the O.D. titers for patients with multibacillary leprosy, so that the sensitivity of the test was 97.5% and the specificity taking healthy individuals as controls was 984%. Using the determined cut-off values, 98% of multibacillary (MB) leprosy and 48% of paucibacillary (PB) leprosy patients had positive levels of anti-MMP-II antibodies, 13% of patients with typhoid and 22% of the household contacts of MB leprosy had positive levels of anti-MMP-II antibodies. CONCLUSIONS: Our results suggest that measuring anti-MMP-II antibody levels could facilitate the detection of leprosy in endemic countries.

Serological diagnosis of leprosy in patients in vietnam by enzyme-linked immunosorbent assay with Mycobacterium leprae-derived major membrane protein II.

A serological diagnostic test using phenolic glycolipid-I (PGL-I) developed in the 1980s is commercially available, but the method is still inefficient in detecting all forms of leprosy. Therefore, more-specific and -reliable serological methods have been sought. We have characterized major membrane protein II (MMP-II) as a candidate protein for a new serological antigen. In this study, we evaluated the effectiveness of the enzyme-linked immunosorbent assay (ELISA) using the MMP-II antigen (MMP-II ELISA) for detecting antibodies in leprosy patients and patients' contacts in the mid-region of Vietnam and compared to the results to those for the PGL-I method (PGL-I ELISA). The results showed that 85% of multibacillary patients and 48% of paucibacillary patients were positive by MMP-II ELISA. Comparison between the serological tests showed that positivity rates for leprosy patients were higher with MMP-II ELISA than with PGL-I ELISA. Household contacts (HHCs) showed low positivity rates, but medical staff members showed comparatively high positivity rates, with MMP-II ELISA. Furthermore, monitoring of results for leprosy patients and HHCs showed that MMP-II is a better index marker than PGL-I. Overall, the epidemiological study conducted in Vietnam suggests that serological testing with MMP-II would be beneficial in detecting leprosy.

Identification of trehalose dimycolate (cord factor) in Mycobacterium leprae.

Glycolipids of Mycobacterium leprae obtained from armadillo tissue nodules infected with the bacteria were analyzed. Mass spectrometric analysis of the glycolipids indicated the presence of trehalose 6,6'-dimycolate (TDM) together with trehalose 6-monomycolate (TMM) and phenolic glycolipid-I (PGL-I). The analysis showed that M. leprae-derived TDM and TMM possessed both alpha- and keto-mycolates centering at C78 in the former and at C81 or 83 in the latter subclasses, respectively. For the first time, MALDI-TOF mass analyses showed the presence of TDM in M. leprae.

Evaluation of major membrane protein-II as a tool for serodiagnosis of leprosy.

As serodiagnosis is the easiest way of diagnosing a disease, the utility of Mycobacterium leprae-derived major membrane protein-II (MMP-II), one of the immuno-dominant antigens, in the serodiagnosis of leprosy was examined. The percent positivity by an enzyme-linked immunosorbent assay for anti-MMP-II antibody was 82.4% for multi-bacillary leprosy, and the specificity of the test was 90.1%. For pauci-bacillary leprosy where cell-mediated immunity predominates, 39.0% showed positive results. These percentage values were significantly higher than these values obtained for existing phenolic glycolipid-I based methods, suggesting that MMP-II antibody detection would facilitate the diagnosis of leprosy.

[Current advances in leprosy research activities].

Due to the advent of multi-drug therapy (MDT) recommended by the WHO, for the treatment of leprosy, presently, leprosy is regarded as a "curable disease". The number of new cases in Japan is relatively very low, due to which the disease is likely to be neglected, but on scientific grounds, there is a necessity to perform in depth studies. Leprosy caused by M. leprae is still unclear on various aspects including transmission, immunology, nerve damage etc. Here we introduce the recent advances in the field of basic leprosy research.