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PLoS Neglected Tropical Diseases Apr 2014In leprosy, classic diagnostic tools based on bacillary counts and histopathology have been facing hurdles, especially in distinguishing latent infection from active... (Review)
Review
In leprosy, classic diagnostic tools based on bacillary counts and histopathology have been facing hurdles, especially in distinguishing latent infection from active disease and diagnosing paucibacillary clinical forms. Serological tests and IFN-gamma releasing assays (IGRA) that employ humoral and cellular immune parameters, respectively, are also being used, but recent results indicate that quantitative PCR (qPCR) is a key technique due to its higher sensitivity and specificity. In fact, advances concerning the structure and function of the Mycobacterium leprae genome led to the development of specific PCR-based gene amplification assays for leprosy diagnosis and monitoring of household contacts. Also, based on the validation of point-of-care technologies for M. tuberculosis DNA detection, it is clear that the same advantages of rapid DNA detection could be observed in respect to leprosy. So far, PCR has proven useful in the determination of transmission routes, M. leprae viability, and drug resistance in leprosy. However, PCR has been ascertained to be especially valuable in diagnosing difficult cases like pure neural leprosy (PNL), paucibacillary (PB), and patients with atypical clinical presentation and histopathological features compatible with leprosy. Also, the detection of M. leprae DNA in different samples of the household contacts of leprosy patients is very promising. Although a positive PCR result is not sufficient to establish a causal relationship with disease outcome, quantitation provided by qPCR is clearly capable of indicating increased risk of developing the disease and could alert clinicians to follow these contacts more closely or even define rules for chemoprophylaxis.
Topics: Humans; Leprosy; Molecular Diagnostic Techniques; Mycobacterium leprae; Real-Time Polymerase Chain Reaction
PubMed: 24722358
DOI: 10.1371/journal.pntd.0002655 -
Bulletin of the World Health... 1971Considerable developments have occurred in the application of the method for growing Mycobacterium leprae in the mouse foot-pad since it was first described about 10... (Review)
Review
Considerable developments have occurred in the application of the method for growing Mycobacterium leprae in the mouse foot-pad since it was first described about 10 years ago. The method has been used to study growth curves and histology in normal and in thymectomized irradiated mice, to identify supposed isolates of Myco. leprae that have been made in tissue-culture or in non-living media, to evaluate tests of experimental vaccines, to investigate applications to clinical investigations (the loss of infectivity during chemotherapy as a means of monitoring a drug trial, the demonstration of drug-resistance, and the clinical problem of the patient who responds poorly to therapy), and to study new drugs-e.g., dapsone, acedapsone, clofazimine, and rifampicin.
Topics: Animals; BCG Vaccine; Clofazimine; Dapsone; Disease Models, Animal; Drug Evaluation; Drug Evaluation, Preclinical; Drug Resistance, Microbial; Humans; Leprosy; Mice; Mycobacterium leprae; Radiation Effects; Rifampin; Thymectomy
PubMed: 4950564
DOI: No ID Found -
Antimicrobial Agents and Chemotherapy Jun 2000Two Mycobacterium leprae genes, folP1 and folP2, encoding putative dihydropteroate synthases (DHPS), were studied for enzymatic activity and for the presence of...
Two Mycobacterium leprae genes, folP1 and folP2, encoding putative dihydropteroate synthases (DHPS), were studied for enzymatic activity and for the presence of mutations associated with dapsone resistance. Each gene was cloned and expressed in a folP knockout mutant of Escherichia coli (C600DeltafolP::Km(r)). Expression of M. leprae folP1 in C600DeltafolP::Km(r) conferred growth on a folate-deficient medium, and bacterial lysates exhibited DHPS activity. This recombinant displayed a 256-fold-greater sensitivity to dapsone (measured by the MIC) than wild-type E. coli C600, and 50-fold less dapsone was required to block (expressed as the 50% inhibitory concentration [IC(50)]) the DHPS activity of this recombinant. When the folP1 genes of several dapsone-resistant M. leprae clinical isolates were sequenced, two missense mutations were identified. One mutation occurred at codon 53, substituting an isoleucine for a threonine residue (T53I) in the DHPS-1, and a second mutation occurred in codon 55, substituting an arginine for a proline residue (P55R). Transformation of the C600DeltafolP::Km(r) knockout with plasmids carrying either the T53I or the P55R mutant allele did not substantially alter the DHPS activity compared to levels produced by recombinants containing wild-type M. leprae folP1. However, both mutations increased dapsone resistance, with P55R having the greatest affect on dapsone resistance by increasing the MIC 64-fold and the IC(50) 68-fold. These results prove that the folP1 of M. leprae encodes a functional DHPS and that mutations within this gene are associated with the development of dapsone resistance in clinical isolates of M. leprae. Transformants created with M. leprae folP2 did not confer growth on the C600DeltafolP::Km(r) knockout strain, and DNA sequences of folP2 from dapsone-susceptible and -resistant M. leprae strains were identical, indicating that this gene does not encode a functional DHPS and is not involved in dapsone resistance in M. leprae.
Topics: Amino Acid Sequence; Anti-Bacterial Agents; Dapsone; Dihydropteroate Synthase; Drug Resistance, Microbial; Gene Expression Regulation, Bacterial; Molecular Sequence Data; Mycobacterium leprae
PubMed: 10817704
DOI: 10.1128/AAC.44.6.1530-1537.2000 -
Antimicrobial Agents and Chemotherapy Oct 2014Mycobacterium leprae and Mycobacterium tuberculosis antimicrobial resistance has been followed with great concern during the last years, while the need for new drugs...
Mycobacterium leprae and Mycobacterium tuberculosis antimicrobial resistance has been followed with great concern during the last years, while the need for new drugs able to control leprosy and tuberculosis, mainly due to extensively drug-resistant tuberculosis (XDR-TB), is pressing. Our group recently showed that M. leprae is able to induce lipid body biogenesis and cholesterol accumulation in macrophages and Schwann cells, facilitating its viability and replication. Considering these previous results, we investigated the efficacies of two statins on the intracellular viability of mycobacteria within the macrophage, as well as the effect of atorvastatin on M. leprae infections in BALB/c mice. We observed that intracellular mycobacteria viability decreased markedly after incubation with both statins, but atorvastatin showed the best inhibitory effect when combined with rifampin. Using Shepard's model, we observed with atorvastatin an efficacy in controlling M. leprae and inflammatory infiltrate in the BALB/c footpad, in a serum cholesterol level-dependent way. We conclude that statins contribute to macrophage-bactericidal activity against Mycobacterium bovis, M. leprae, and M. tuberculosis. It is likely that the association of statins with the actual multidrug therapy effectively reduces mycobacterial viability and tissue lesion in leprosy and tuberculosis patients, although epidemiological studies are still needed for confirmation.
Topics: Animals; Antitubercular Agents; Atorvastatin; Cell Line; Drug Synergism; Heptanoic Acids; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Leprosy; Macrophages; Mice; Mice, Inbred BALB C; Mycobacterium leprae; Mycobacterium tuberculosis; Pyrroles; Rifampin; Simvastatin
PubMed: 25049257
DOI: 10.1128/AAC.01826-13 -
Journal of Clinical Microbiology Jun 2001Currently recommended control measures for treating leprosy with multidrug therapy should control the spread of drug-resistant strains; however, dapsone (DDS) resistance...
Currently recommended control measures for treating leprosy with multidrug therapy should control the spread of drug-resistant strains; however, dapsone (DDS) resistance continues to be reported. Comprehensive estimates of drug-resistant leprosy are difficult to obtain due to the cumbersome nature of the conventional drug susceptibility testing method using mouse footpad inoculation, which requires at least 6 months to obtain results. Recently, it has been determined that DDS-resistant strains contain missense mutations in codon 53 or 55 of the folP1 gene of Mycobacterium leprae, and definitive evidence linking these mutations with DDS resistance in M. leprae has been obtained. Based on these mutations, a heteroduplex DDS M. leprae (HD-DDS-ML) assay was developed for the simultaneous detection of M. leprae and of its susceptibility to DDS. The assay relies on the PCR amplification of an M. leprae-specific 231-bp fragment of folP1 containing codons 53 and 55. The PCR products are allowed to anneal to a universal heteroduplex generator, and the separation of the resultant DNA duplexes is accomplished by polyacrylamide gel electrophoresis. M. leprae was detected in crude cell lysates of skin biopsy specimen homogenates from eight leprosy patients and from M. leprae-infected mouse or armadillo tissues infected with 14 separate strains using the HD-DDS-ML assay. The assay was specific for M. leprae in a comparison with results obtained from 14 species of mycobacteria other than M. leprae and four bacterial species known to colonize human skin. The HD-DDS-ML assay detected as few as 100 M. leprae organisms present in homogenates of human skin and demonstrated a 93% correlation with DDS susceptibility as determined by both DNA sequencing of folP1 and mouse footpad susceptibility testing. The HD-DDS-ML assay provides a new tool for the simultaneous detection of M. leprae and of its susceptibility to DDS from a single specimen. The assay should prove useful for drug resistance surveillance in leprosy control programs when combined with similar molecular tests developed for other drug resistance markers.
Topics: Base Sequence; DNA, Bacterial; Dapsone; Drug Resistance, Microbial; Heteroduplex Analysis; Humans; Leprostatic Agents; Leprosy; Molecular Sequence Data; Mycobacterium leprae; Sensitivity and Specificity; Sequence Analysis, DNA
PubMed: 11376039
DOI: 10.1128/JCM.39.6.2083-2088.2001 -
PLoS Neglected Tropical Diseases Jan 2018Leprosy is caused by the bacterial pathogens Mycobacterium leprae and Mycobacterium lepromatosis. Apart from humans, animals such as nine-banded armadillos in the...
Leprosy is caused by the bacterial pathogens Mycobacterium leprae and Mycobacterium lepromatosis. Apart from humans, animals such as nine-banded armadillos in the Americas and red squirrels in the British Isles are naturally infected with M. leprae. Natural leprosy has also been reported in certain nonhuman primates, but it is not known whether these occurrences are due to incidental infections by human M. leprae strains or by M. leprae strains specific to nonhuman primates. In this study, complete M. leprae genomes from three naturally infected nonhuman primates (a chimpanzee from Sierra Leone, a sooty mangabey from West Africa, and a cynomolgus macaque from The Philippines) were sequenced. Phylogenetic analyses showed that the cynomolgus macaque M. leprae strain is most closely related to a human M. leprae strain from New Caledonia, whereas the chimpanzee and sooty mangabey M. leprae strains belong to a human M. leprae lineage commonly found in West Africa. Additionally, samples from ring-tailed lemurs from the Bezà Mahafaly Special Reserve, Madagascar, and chimpanzees from Ngogo, Kibale National Park, Uganda, were screened using quantitative PCR assays, to assess the prevalence of M. leprae in wild nonhuman primates. However, these samples did not show evidence of M. leprae infection. Overall, this study adds genomic data for nonhuman primate M. leprae strains to the existing M. leprae literature and finds that this pathogen can be transmitted from humans to nonhuman primates as well as between nonhuman primate species. While the prevalence of natural leprosy in nonhuman primates is likely low, nevertheless, future studies should continue to explore the prevalence of leprosy-causing pathogens in the wild.
Topics: Africa, Western; Animals; Cercocebus atys; Genetic Variation; Genome, Bacterial; Lemur; Leprosy; Macaca fascicularis; Mycobacterium leprae; Pan troglodytes; Philippines; Phylogeny; Primate Diseases
PubMed: 29381722
DOI: 10.1371/journal.pntd.0006190 -
Journal of Clinical Microbiology Aug 2019Many pathogens that caused devastating disease throughout human history, such as , , and , remain problematic today. Historical bacterial genomes represent a unique... (Review)
Review
Many pathogens that caused devastating disease throughout human history, such as , , and , remain problematic today. Historical bacterial genomes represent a unique source of genetic information and advancements in sequencing technologies have allowed unprecedented insights from this previously understudied resource. This minireview brings together example studies which have utilized ancient DNA, individual historical isolates (both extant and dead) and collections of historical isolates. The studies span human history and highlight the contribution that sequencing and analysis of historical bacterial genomes have made to a wide variety of fields. From providing retrospective diagnosis, to uncovering epidemiological pathways and characterizing genetic diversity, there is clear evidence for the utility of historical isolate studies in understanding disease today. Studies utilizing historical isolate collections, such as those from the National Collection of Type Cultures, the American Type Culture Collection, and the Institut Pasteur, offer enhanced insight since they typically span a wide time period encompassing important historical events and are useful for the investigating the phylodynamics of pathogens. Furthermore, historical sequencing studies are particularly useful for looking into the evolution of antimicrobial resistance, a major public health concern. In summary, although there are limitations to working with historical bacterial isolates, especially when utilizing ancient DNA, continued improvement in molecular and sequencing technologies and the resourcefulness of investigators mean this area of study will continue to expand and contribute to the understanding of pathogens.
Topics: Anti-Bacterial Agents; Bacteria; DNA, Ancient; Drug Resistance, Multiple, Bacterial; Evolution, Molecular; Genetic Variation; Genome, Bacterial; Humans; Mycobacterium leprae; Mycobacterium tuberculosis; Phylogeny; Sequence Analysis, DNA; Yersinia pestis
PubMed: 31092597
DOI: 10.1128/JCM.00100-19 -
Microbiology Spectrum Jun 2016The key question our work has sought to address has been, "What are the necessary and sufficient conditions that engender protection from intracellular pathogens in the... (Review)
Review
The key question our work has sought to address has been, "What are the necessary and sufficient conditions that engender protection from intracellular pathogens in the human host?" The origins of this work derive from a long-standing interest in the mechanisms of protection against two such paradigmatic intracellular pathogens, Mycobacterium tuberculosis and Mycobacterium leprae, that have brilliantly adapted to the human host. It was obvious that these pathogens, which cause chronic diseases and persist in macrophages, must have acquired subtle strategies to resist host microbicidal mechanisms, yet since the vast majority of individuals infected with M. tuberculosis do not develop disease, there must be some potent human antimicrobial mechanisms. What follows is not a comprehensive review of the vast literature on the role of human macrophages in protection against infectious disease, but a summary of the research in our two laboratories with collaborators that we hope has contributed to some understanding of mechanisms of resistance and pathogenesis. While mouse models revealed some necessary conditions for protection, e.g., innate immunity, Th1 cells and their cytokines, and major histocompatibility complex class I-restricted T cells, here we emphasize multiple antimicrobial mechanisms that exist in human macrophages that differ from those of most experimental animals. Prominent here is the vitamin D-dependent antimicrobial pathway common to human macrophages activated by innate and acquired immune responses, mediated by antimicrobial peptides, e.g., cathelicidin, through an interleukin-15- and interleukin-32-dependent common pathway that is necessary for macrophage killing of M. tuberculosis in vitro.
Topics: Animals; Disease Resistance; Host-Pathogen Interactions; Humans; Macrophages; Mycobacterium leprae; Mycobacterium tuberculosis
PubMed: 27337485
DOI: 10.1128/microbiolspec.MCHD-0006-2015 -
Philosophical Transactions of the Royal... Nov 2020As one of the oldest known human diseases, leprosy or Hansen's disease remains a public health concern around the world with over 200 000 new cases in 2018. Most human...
As one of the oldest known human diseases, leprosy or Hansen's disease remains a public health concern around the world with over 200 000 new cases in 2018. Most human leprosy cases are caused by , but a small number of cases are now known to be caused by , a sister taxon of . The global pattern of genomic variation in is not well defined. Particularly, in the Pacific Islands, the origins of leprosy are disputed. Historically, it has been argued that leprosy arrived on the islands during nineteenth century colonialism, but some oral traditions and palaeopathological evidence suggest an older introduction. To address this, as well as investigate patterns of pathogen exchange across the Pacific Islands, we extracted DNA from 39 formalin-fixed paraffin-embedded biopsy blocks dating to 1992-2016. Using whole-genome enrichment and next-generation sequencing, we produced nine genomes dating to 1998-2015 and ranging from 4-63× depth of coverage. Phylogenetic analyses indicate that these strains belong to basal lineages within the phylogeny, specifically falling in branches 0 and 5. The phylogeographical patterning and evolutionary dating analysis of these strains support a pre-modern introduction of into the Pacific Islands. This article is part of the theme issue 'Insights into health and disease from ancient biomolecules'.
Topics: Adolescent; Adult; Aged; American Samoa; Biological Evolution; Child; Evolution, Molecular; Female; Genome, Bacterial; Hawaii; Humans; Leprosy; Male; Micronesia; Middle Aged; Mycobacterium leprae; Pacific Islands; Phylogeography; Young Adult
PubMed: 33012236
DOI: 10.1098/rstb.2019.0582 -
Scientific Reports Jul 2019Leprosy, an important infectious disease in humans caused by Mycobacterium leprae (Mle), remains endemic in many countries. Notably, the pathogen cannot be cultured in...
Leprosy, an important infectious disease in humans caused by Mycobacterium leprae (Mle), remains endemic in many countries. Notably, the pathogen cannot be cultured in vitro, except in mouse footpads in vivo. The molecular basis of these characteristics and the mechanisms remain unknown. Consequently, analysis of Mle growth and survival is urgently needed to develop novel therapies against leprosy, including rapid, simple, and specific methods to detect infection. Here, we demonstrated the functional role and contribution of Mle-DNA gyrase, which regulates DNA topology, DNA replication, and chromosome segregation to promote bacterial growth and survival, in Mle growth and survival in vitro and in vivo. The optimum temperature for Mle-DNA gyrase activity was 30 °C. When the DNA gyrB-gyrA genes in Mycobacterium smegmatis were replaced with the Mle gyrase genes by allelic exchange, the recombinants could not grow at 37 °C. Moreover, using radiorespirometry analysis for viability of Mle bacilli, we found that Mle growth was more vigorous at 25-30 °C than at 37 °C, but was inhibited above 40 °C. These results propose that DNA gyrase is a crucial factor for Mle growth and survival and its sensitivity to temperature may be exploited in heat-based treatment of leprosy.
Topics: Cell Culture Techniques; DNA Gyrase; DNA Replication; DNA, Bacterial; Leprosy; Mycobacterium leprae
PubMed: 31346236
DOI: 10.1038/s41598-019-47364-5