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1.
PLoS Negl Trop Dis ; 14(5): e0008325, 2020 05.
Artículo en Inglés | MEDLINE | ID: mdl-32453754

RESUMEN

Leprosy urgently needs a precise and early diagnostic tool. The sensitivity of the direct (bacilli staining, Mycobacterium leprae DNA) and indirect (antibody levels, T cell assays) diagnostics methods vary based on the clinical form. Recently, PCR-based M. leprae DNA detection has been shown to differentially diagnose leprosy from other dermatological conditions. However, accuracy can still be improved, especially for use with less invasive clinical samples. We tested different commercial DNA extraction kits: DNeasy Blood & Tissue, QIAamp DNA Microbiome, Maxwell 16 DNA Purification, PowerSoil DNA Isolation; as well as in-house phenol-chloroform and Trizol/FastPrep methods. Extraction was performed on M. leprae-infected mouse footpads and different clinical samples of leprosy patients (skin biopsies and scrapings, lesion, oral and nasal swabs, body hair, blood on FTA cards, peripheral whole blood). We observed that the Microbiome kit was able to enrich for mycobacterial DNA, most likely due the enzymatic digestion cocktail along with mechanical disruption involved in this method. Consequently, we had a significant increase in sensitivity in skin biopsies from paucibacillary leprosy patients using a duplex qPCR targeting 16S rRNA (M. leprae) and 18S rRNA (mammal) in the StepOnePlus system. Our data showed that the presence of M. leprae DNA was best detected in skin biopsies and skin scrapings, independent of the extraction method or the clinical form. For multibacillary patients, detection of M. leprae DNA in nasal swabs indicates the possibility of having a much less invasive sample that can be used for the purposes of DNA sequencing for relapse analysis and drug resistance monitoring. Overall, DNA extracted with the Microbiome kit presented the best bacilli detection rate for paucibacillary cases, indicating that investments in extraction methods with mechanical and DNA digestion should be made.


Asunto(s)
ADN Bacteriano/aislamiento & purificación , Mycobacterium leprae/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Animales , ADN Bacteriano/genética , Humanos , Ratones , Mycobacterium leprae/genética , ARN Bacteriano/genética , ARN Ribosómico 16S/genética , Sensibilidad y Especificidad
2.
PLoS Negl Trop Dis ; 14(3): e0008127, 2020 03.
Artículo en Inglés | MEDLINE | ID: mdl-32203502

RESUMEN

Understanding the prevalence of M. leprae infection in armadillos is important because of evidence from Brazil and other countries of an association between contact with armadillos and the development of Hansen's Disease (leprosy). Our aim was to characterize studies which have investigated natural M. leprae infection in wild armadillos in Brazil, and to quantify and explore variability in the reported prevalence of infection. We conducted a systematic review (PROSPERO CRD42019155277) of publications in MEDLINE, EMBASE, Global Health, Scopus, LILACS, Biblioteca Digital Brasileira de Teses e Dissertações, Catálogo de Teses e Dissertações de CAPES, and Biblioteca Virtual em Saúde up to 10/2019 using Mesh and text search terms (in English, Portuguese, Spanish, and French). The 10 included studies represented a total sample of 302 armadillos comprising 207 (69%) Dasypus novemcinctus, 67 (22%) Euphractus sexcinctus, 16 (5%) Priodontes maximus, 10 (3%) Cabassous unicinctus, and 2 (1%) Cabassous tatouay from 7 different states. Methods used included histopathology (4 studies), PGL-1 and LID-1 antigen detection (4 studies) and examination for clinical signs of disease (4 studies). Eight studies used PCR of which 7 targeted the RLEP repetitive element and 3 tested for inhibitory substances. M. leprae prevalence by PCR ranged from 0% (in 3 studies) to 100% in one study, with a summary estimate of 9.4% (95% CI 0.4% to 73.1%) and a predictive interval of 0-100%. The average prevalence is equivalent to 1 in 10 armadillos in Brazil being infected with M. leprae, but wide variation in sample estimates means that the prevalence in any similar study would be entirely unpredictable. We propose instead that future studies aim to investigate transmission and persistence of M. leprae within and between armadillo populations, meanwhile adopting the precautionary principle to protect human health and an endangered species in Brazil.


Asunto(s)
Armadillos/microbiología , Lepra/epidemiología , Lepra/veterinaria , Mycobacterium leprae/aislamiento & purificación , Animales , Animales Salvajes/microbiología , Brasil/epidemiología , ADN Bacteriano/análisis , Bases de Datos Factuales , Mapeo Geográfico , Mycobacterium leprae/genética , Reacción en Cadena de la Polimerasa , Prevalencia , Secuencias Repetitivas de Ácidos Nucleicos , Zoonosis/epidemiología , Zoonosis/microbiología
3.
Am J Trop Med Hyg ; 102(3): 547-552, 2020 03.
Artículo en Inglés | MEDLINE | ID: mdl-31933458

RESUMEN

Resistance to anti-leprosy drugs is on the rise. Several studies have documented resistance to rifampicin, dapsone, and ofloxacin in patients with leprosy. We looked for point mutations within the folP1, rpoB, and gyrA gene regions of the Mycobacterium leprae genome predominantly in the neural form of leprosy. DNA samples from 77 nerve tissue samples were polymerase chain reaction (PCR)-amplified for M leprae DNA and sequenced for drug resistance-determining regions of genes rpoB, folP1, and gyrA. The mean age at presentation and onset was 38.2 ± 13.4 (range 14-71) years and 34.9 ± 12.6 years (range 10-63) years, respectively. The majority had borderline tuberculoid leprosy (53 [68.8%]). Mutations associated with resistance were identified in 6/77 (7.8%) specimens. Mutations seen were those associated with resistance to rifampicin, ofloxacin, and dapsone. All the six patients were drug-naive. The clinical and pathological manifestations in this group did not differ from the drug-sensitive group. This study highlights the occurrence of resistance to the standard multidrug therapy and ofloxacin in leprosy. Among the entire cohort, 1/77 (1.3%) showed resistance to rifampicin, 2/77 (2.6%) to dapsone, and 5/77 (6.4%) to ofloxacin. Six new patients showing infection by mutant strains indicated the emergence of primary resistance. Resistance to ofloxacin could be due to frequent use of quinolones for many bacterial infections. The results of the study indicate the need for development of a robust and strict surveillance system for detecting drug resistance in leprosy in India.


Asunto(s)
Leprostáticos/uso terapéutico , Lepra/tratamiento farmacológico , Lepra/microbiología , Mycobacterium leprae/efectos de los fármacos , Adolescente , Adulto , Anciano , Niño , Preescolar , Estudios de Cohortes , Estudios Transversales , Farmacorresistencia Bacteriana , Quimioterapia Combinada , Femenino , Humanos , India/epidemiología , Lactante , Leprostáticos/farmacología , Lepra/epidemiología , Masculino , Pruebas de Sensibilidad Microbiana , Persona de Mediana Edad , Mutación , Mycobacterium leprae/genética , Adulto Joven
4.
PLoS Negl Trop Dis ; 13(12): e0007946, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31881061

RESUMEN

BACKGROUND: Although leprosy is efficiently treated by multidrug therapy, resistance to first-line (dapsone, rifampin) and second-line (fluoroquinolones) drugs has been described worldwide. However, the characteristics of drug resistance in Southwest China remain unknown. Furthermore, the sensitivity of polymerase chain reaction (PCR)/sequencing for resistance detection is limited, especially for paucibacillary (PB) leprosy patients. The current study aimed to develop a nested PCR/sequencing and TaqMan SNP Genotyping Assay to increase the sensitivity of the method used to detect drug resistance in Mycobacterium leprae and to reveal the nature of M. leprae drug resistance in Southwest China. METHODOLOGY/PRINCIPAL FINDINGS: Seventy-six specimens, including skin biopsy (n = 64), formalin-fixed paraffin-embedded (FFPE) (n = 11) and skin-slit smear (SSS) (n = 1) samples from multibacillary (MB, n = 70) and PB (n = 6) leprosy patients from Southwest China, were included in this study. The presence of mutations in drug resistance-determining regions (DRDRs) of the rpoB, folP1, and gyrA genes, which are associated with rifampicin, dapsone, and quinolone resistance, respectively, was detected by PCR/sequencing, as recommended by the WHO, and the nested PCR and TaqMan SNP Genotyping Assay developed in this study. Mutations in the folP gene were detected in 19 (25.00%) samples, indicating dapsone-resistant M. leprae, with one (1.31%) sample showing mutations in two genes, folP and gyrA, reflecting multidrug-resistant strains to dapsone and ofloxacin. However, no rpoB mutation was detected. Compared with PCR/sequencing, nested PCR increased the sensitivity of detecting rpoB (from 51.39% to 78.94% for leprosy patients and from 0.00% to 50.00% for PB), gyrA (from 75.00% to 80.26% for leprosy patients and from 50.00% to 66.67% for PB), and folP1 (from 5.26% to 84.21% for leprosy patients and from 0.00% to 66.67% for PB). Moreover, the TaqMan SNP Genotyping Assay showed greater sensitivity for folP1 detection (from 5.26% to 78.94-86.84% for leprosy patients and from 0.00% to 33.33%-83.33% for PB patients) than the PCR/sequencing method. In addition, the latter method was able to more easily distinguish heterozygous genotypes and mutant homozygous genotypes from homozygous genotypes. CONCLUSIONS/SIGNIFICANCE: Nested PCR/sequencing and the TaqMan SNP Genotyping Assay are rapid and highly sensitive methods for detecting drug resistance in leprosy cases. The current study revealed that diamino-diphenylsulfone (DDS; also known as dapsone) resistance in M. leprae, as indicated by folP1 gene detection, is still the most concerning form of drug resistance in leprosy patients from Southwest China.


Asunto(s)
Farmacorresistencia Bacteriana , Técnicas de Genotipaje/métodos , Lepra/microbiología , Pruebas de Sensibilidad Microbiana/métodos , Mycobacterium leprae/efectos de los fármacos , Mycobacterium leprae/genética , Reacción en Cadena de la Polimerasa/métodos , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , China , Femenino , Genes Bacterianos , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Mycobacterium leprae/aislamiento & purificación , Sensibilidad y Especificidad , Análisis de Secuencia de ADN/métodos , Adulto Joven
5.
Emerg Microbes Infect ; 8(1): 1479-1489, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31621517

RESUMEN

Reports on antimicrobial resistance (AMR) of Mycobacterium leprae, relationship with bacteriological index (BI), and transmission in China are limited. We investigated the emergence of AMR mutations, the relationship between BI and AMR in complete, moderate and lack of BI decline cases, and molecular epidemiological features of AMR cases by enrolling 290 leprosy cases from four endemic provinces. Seven (2.41%), one (0.34%), five (1.72%), one (0.34%), and one (0.34%) strains had single mutations in folP1, rpoC, gyrA, gyrB, and 23S rRNA, respectively. Double mutations in folP1 and gyrA, rpoB and gyrA, and gyrA and 23S rRNA were observed in one (0.34%) strain each. Mutated strains occurred in three out of 81 (95% CI-0.005-0.079, p = 0.083) cases with complete BI decline, in seven out of 103 (95% CI 0.018-0.117, p = 0.008) cases with moderate BI decline, and in four out of 34 (95% CI 0.003-0.231, p = 0.044) cases with lack of BI decline. Most of these mutated strains were geographically separated and diverged genotypically. AMR mutations may not be the main cause of the lack of BI decline. The low transmission of AMR strains at the county level indicates an ongoing transmission at close contact levels.


Asunto(s)
Farmacorresistencia Bacteriana , Leprostáticos/farmacología , Lepra/microbiología , Mycobacterium leprae/efectos de los fármacos , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , China/epidemiología , Femenino , Humanos , Lepra/epidemiología , Masculino , Pruebas de Sensibilidad Microbiana , Persona de Mediana Edad , Mutación , Mycobacterium leprae/clasificación , Mycobacterium leprae/genética , Mycobacterium leprae/aislamiento & purificación , Filogenia , Adulto Joven
6.
PLoS Negl Trop Dis ; 13(10): e0007731, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-31577795

RESUMEN

BACKGROUND: Detection and pathology analysis of Mycobacterium leprae using skin biopsy tissues are essential for leprosy diagnosis and monitoring response to treatment. Although formalin fixation of patient tissues may not be ideal for molecular studies, biopsy samples are the most accessible material from suspected cases. Therefore, clinical molecular laboratories must be able to utilize formalin-fixed, paraffin-embedded (FFPE) material. OBJECTIVE: To determine the best molecular method for diagnosing and monitoring leprosy in FFPE specimens, we developed a single-tube nested PCR (STNPCR) (131 bp) and SYBRGreen PCR (101 bp) assay using primers for the M. leprae-specific repetitive element (RLEP) gene and evaluated the results compared to those using previously established RLEP primers (372 bp). METHODS: FFPE biopsy samples obtained from 145 leprosy patients (during or after multidrug therapy (MDT)) and patients with 29 other confounding dermatoses were examined by the bacteria index (BI) and by simple PCR, STNPCR, and SYBRGreen PCR using primers amplifying a 372-bp, 131-bp or 101-bp fragment of RLEP, respectively. RESULTS: In leprosy patients receiving MDT, STNPCR showed a highest specificity of 100% and a positive predictive value (PPV) of 100%. For multibacillary (MB), paucibacillary (PB) and all leprosy patients, the highest sensitivities were 91.42%, 39.13%, and 67.92%, negative predictive values (NPVs) were 8.57%, 60.36%, and 32.07%, and the highest accuracies were 93.93%, 62.67%, and 74.81%, respectively, higher than the results of SYBRGreen PCR and simple PCR. For post-MDT leprosy patients, SYBRGreen PCR showed the highest sensitivity of 50.0%, highest specificity of 100%, a PPV of 100%, an NPV of 100% and the highest accuracy of 83.72% for MB patients, which were higher than those of STNPCR and simple PCR. STNPCR showed the highest sensitivity of 26.66% and 34.48%, highest specificity of 100% and 100%, a PPV of 100% and 100%, NPV of 72.50% and 60.21%, and highest accuracy of 75.00% and 67.24% for PB and leprosy patients, respectively, higher than those of SYBRGreen PCR and simple PCR. CONCLUSIONS: These findings suggest that STNPCR or SYBRGreen PCR (131-bp and 101-bp fragment amplification, respectively) for RLEP using FFPE specimens performs better as a diagnostic test and for monitoring response to MDT than does simple PCR based on 372-bp fragment amplification. Additionally, STNPCR showed increased sensitivity for PB diagnosis using FFPE specimens, which can be transferred remotely or retrieved from previous leprosy patients.


Asunto(s)
Formaldehído , Lepra/diagnóstico , Mycobacterium leprae/aislamiento & purificación , Adhesión en Parafina/métodos , Reacción en Cadena de la Polimerasa/métodos , Biopsia/métodos , China , Cartilla de ADN , ADN Bacteriano/genética , Quimioterapia Combinada , Humanos , Leprostáticos/uso terapéutico , Lepra/tratamiento farmacológico , Mycobacterium leprae/genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , Sensibilidad y Especificidad , Piel/microbiología
7.
J Med Microbiol ; 68(11): 1629-1640, 2019 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-31553301

RESUMEN

Introduction. ML1899 is conserved in all mycobacterium sp. and is a middle member of mle-ML1898 operon involved in mycolic acid modification.Aim. In the present study attempts were made to characterize ML1899 in detail.Methodology. Bioinformatics tools were used for prediction of active-site residues, antigenic epitopes and a three-dimensional model of protein. The gene was cloned, expressed and purified as His-tagged protein in Escherichia coli for biophysical/biochemical characterization. Recombinant protein was used to treat THP-1 cells to study change in production of nitric oxide (NO), reactive oxygen species (ROS), cytokines and chemokines using flowcytometry/ELISA.Results. In silico analysis predicted ML1899 as a member of α/ß hydrolase family with GXSXG-motif and Ser126, His282, Asp254 as active-site residues that were confirmed by site-directed mutagensis. ML1899 exhibited esterase activity. It hydrolysed pNP-butyrate as optimum substrate at pH 8.0 and 50 °C with 5.56 µM-1 min-1 catalytic efficiency. The enzyme exhibited stability up to 60 °C temperature and between pH 6.0 to 9.0. K m, V max and specific activity of ML1899 were calculated to be 400 µM, 40 µmoles min-1 ml-1 and 27 U mg- 1, respectively. ML1899 also exhibited phospholipase activity. The protein affected the survival of macrophages when treated at higher concentration. ML1899 enhanced ROS/NO production and up-regulated pro-inflammatory cytokines and chemokine including TNF-α, IFN-γ, IL-6 and IL-8 in macrophages. ML1899 was also observed to elicit humoral response in 69 % of leprosy patients.Conclusion. These results suggested that ML1899, an esterase could up-regulate the immune responses in favour of macrophages at a low concentration but kills the THP-1 macrophages cells at a higher concentration.


Asunto(s)
Proteínas Bacterianas/inmunología , Esterasas/inmunología , Lepra/microbiología , Mycobacterium leprae/enzimología , Secuencia de Aminoácidos , Anticuerpos Antibacterianos/inmunología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Citocinas/genética , Citocinas/inmunología , Estabilidad de Enzimas , Esterasas/química , Esterasas/genética , Femenino , Humanos , Concentración de Iones de Hidrógeno , Cinética , Lepra/inmunología , Macrófagos/inmunología , Macrófagos/microbiología , Masculino , Mycobacterium leprae/química , Mycobacterium leprae/genética , Mycobacterium leprae/inmunología , Óxido Nítrico/inmunología , Especies Reactivas de Oxígeno/inmunología , Alineación de Secuencia
8.
Homo ; 70(2): 105-118, 2019 Oct 24.
Artículo en Inglés | MEDLINE | ID: mdl-31486822

RESUMEN

Orosháza site no. 10 (Southeast Hungary) contains the partially excavated archaeological remains of an 11-13th century CE Muslim merchant village and its cemetery located in close proximity to Christian villages of the same era. The skeleton of a young woman (grave no. 16) from the last phase of the cemetery use was identified with rhinomaxillary lesions associated with lepromatous leprosy. The right parietal bone also exhibited signs of cranial trauma, possibly caused by symbolic trepanation, a well-known ritual practice in the 9-11th century CE Carpathian Basin. The retrospective diagnosis of the disease was supported by ancient DNA analysis, as the samples were positive for Mycobacterium leprae aDNA, shown to be of genotype 3. Contrary to the general practice of the era, the body of the young female with severe signs of leprosy was interred among the regular graves of the Muslim cemetery in Orosháza, which may reflect the unique cultural background of the community.


Asunto(s)
Cementerios/historia , Islamismo/historia , Lepra/historia , Adulto , Huesos/microbiología , Huesos/patología , ADN Antiguo/análisis , ADN Bacteriano/análisis , ADN Bacteriano/genética , Femenino , Historia Medieval , Humanos , Hungría , Lepra/microbiología , Masculino , Mycobacterium leprae/genética , Paleopatología , Adulto Joven
9.
BMC Infect Dis ; 19(1): 753, 2019 Aug 28.
Artículo en Inglés | MEDLINE | ID: mdl-31462296

RESUMEN

BACKGROUND: Leprosy continues to be a health problem in endemic areas. More than 200,000 new cases of leprosy per year suggest that transmission of the disease is still ongoing, presumably as airborne infection through nasal droplets. Late diagnosis supports continued transmission and increases the individual risk for functional disabilities. Laboratory tools are considered beneficial to facilitate early detection and clinical assessment of cases. The aim of this study was to validate molecular tools allowing detection, quantification and assessment of viability of M. leprae from nasal swab samples which are easy to obtain without the need of any invasive procedures. METHODS: Validation of two real-time PCRs detecting M. leprae DNA (RLEP qPCR) and RNA (16S rRNA RT qPCR) was conducted on "must not detect"/"must detect" samples and 160 pre-treatment nasal swab samples from 20 clinically diagnosed multibacillary (MB) leprosy patients from Togo. RESULTS: Both assays were 100% M. leprae specific and showed analytical sensitivities of three templates each. Out of 20 clinically diagnosed MB leprosy patients, 15 (75.0%) had a positive RLEP qPCR result from nasal swab samples. The 16S rRNA RT qPCR detected viable bacilli in nasal swab samples of ten out of these 15 RLEP positive patients (66.7%). CONCLUSION: The combined RLEP/16S rRNA (RT) qPCR assay provides a sensitive and specific tool to determine the bacterial load and viability of M. leprae from nasal swab samples and is applicable for early diagnosis, monitoring treatment response and investigating the role of nasal carriage of M. leprae in human-to-human transmission through aerosol infection.


Asunto(s)
Lepra/microbiología , Mycobacterium leprae/genética , Cavidad Nasal/microbiología , ARN Ribosómico 16S , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Preescolar , ADN Bacteriano/genética , Humanos , Lepra/diagnóstico , Lepra Multibacilar/diagnóstico , Lepra Multibacilar/microbiología , Persona de Mediana Edad , Mycobacterium leprae/aislamiento & purificación , Mycobacterium leprae/patogenicidad , ARN Ribosómico 16S/genética , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Togo , Adulto Joven
10.
Microbiol Spectr ; 7(4)2019 07.
Artículo en Inglés | MEDLINE | ID: mdl-31322104

RESUMEN

The mammalian nervous system is invaded by a number of intracellular bacterial pathogens which can establish and progress infection in susceptible individuals. Subsequent clinical manifestation is apparent with the impairment of the functional units of the nervous system, i.e., the neurons and the supporting glial cells that produce myelin sheaths around axons and provide trophic support to axons and neurons. Most of these neurotrophic bacteria display unique features, have coevolved with the functional sophistication of the nervous system cells, and have adapted remarkably to manipulate neural cell functions for their own advantage. Understanding how these bacterial pathogens establish intracellular adaptation by hijacking endogenous pathways in the nervous system, initiating myelin damage and axonal degeneration, and interfering with myelin maintenance provides new knowledge not only for developing strategies to combat neurodegenerative conditions induced by these pathogens but also for gaining novel insights into cellular and molecular pathways that regulate nervous system functions. Since the pathways hijacked by bacterial pathogens may also be associated with other neurodegenerative diseases, it is anticipated that detailing the mechanisms of bacterial manipulation of neural systems may shed light on common mechanisms, particularly of early disease events. This chapter details a classic example of neurodegeneration, that caused by Mycobacterium leprae, which primarily infects glial cells of the peripheral nervous system (Schwann cells), and how it targets and adapts intracellularly by reprogramming Schwann cells to stem cells/progenitor cells. We also discuss implications of this host cell reprogramming by leprosy bacilli as a model in a wider context.


Asunto(s)
Lepra/microbiología , Mycobacterium leprae/fisiología , Sistema Nervioso Periférico/microbiología , Adaptación Fisiológica , Animales , Humanos , Mycobacterium leprae/genética , Mycobacterium leprae/aislamiento & purificación , Células de Schwann/microbiología
11.
Diagn Microbiol Infect Dis ; 95(3): 114855, 2019 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-31285121

RESUMEN

Early diagnosis of leprosy is important for limiting the severity of disease, which may lead to disabilities and deformities if not treated timely. Multiplex PCR employing more than one gene, specific to target DNA, is more efficient detection tool. In the present study, slit skin scrapings, blood, nasal swabs and saliva from Paucibacillary (PB) and Multibacillary (MB) cases as well as household contacts of PB cases were tested by multiplex PCR using three different gene targets namely RLEP, 16SrRNA and sodA. We found an increase in overall diagnostic positivity for M. leprae DNA detection by M-PCR as compared to individual PCR. In case of nasal swabs using M-PCR the PPV, NPV were 0.5454, 0.8333 respectively. There is remarkable increase in PPV in SSS of PB cases and nasal swabs of HHCs using M-PCR. Conclusively, our finding suggests the utility of M-PCR for early diagnosis and household contact surveillance for leprosy.


Asunto(s)
Técnicas Bacteriológicas/métodos , Pruebas Diagnósticas de Rutina/métodos , Lepra/diagnóstico , Reacción en Cadena de la Polimerasa Multiplex , Mycobacterium leprae/aislamiento & purificación , Vigilancia de la Población/métodos , Diagnóstico Precoz , Humanos , Mycobacterium leprae/genética , Sensibilidad y Especificidad
12.
Acta Trop ; 197: 105041, 2019 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-31152726

RESUMEN

Leprosy is an ancient disease caused by the acid-fast bacillus Mycobacterium leprae, also known as Hansen's bacillus. M. leprae is an obligate intracellular microorganism with a marked Schwann cell tropism and is the only human pathogen capable of invading the superficial peripheral nerves. The transmission mechanism of M. leprae is not fully understood; however, the nasal mucosa is accepted as main route of M. leprae entry to the human host. The complete sequencing and the comparative genome analysis show that M. leprae underwent a genome reductive evolution process, as result of lifestyle change and adaptation to different environments; some of lost genes are homologous to those of host cells. Thus, M. leprae reduced its genome size to 3.3 Mbp, contributing to obtain the lowest GC content (approximately 58%) among mycobacteria. The M. leprae genome contains 1614 open reading frames coding for functional proteins, and 1310 pseudogenes corresponding to 41% of the genome, approximately. Comparative analyses to different microorganisms showed that M. leprae possesses the highest content of pseudogenes among pathogenic and non-pathogenic bacteria and archaea. The pathogen adaptation into host cells, as the Schwann cells, brought about the reduction of the genome and induced multiple gene inactivation. The present review highlights the characteristics of genome's reductive evolution that M. leprae experiences in the genetic aspects compared with other pathogens. The possible mechanisms of pseudogenes formation are discussed.


Asunto(s)
Aclimatación/genética , Evolución Molecular , Lepra/microbiología , Mycobacterium leprae/genética , Mycobacterium leprae/fisiología , ADN Bacteriano , Regulación Bacteriana de la Expresión Génica , Genoma Bacteriano , Humanos
13.
PLoS Negl Trop Dis ; 13(6): e0007400, 2019 06.
Artículo en Inglés | MEDLINE | ID: mdl-31181059

RESUMEN

BACKGROUND: Early detection of Mycobacterium leprae is a key strategy for disrupting the transmission chain of leprosy and preventing the potential onset of physical disabilities. Clinical diagnosis is essential, but some of the presented symptoms may go unnoticed, even by specialists. In areas of greater endemicity, serological and molecular tests have been performed and analyzed separately for the follow-up of household contacts, who are at high risk of developing the disease. The accuracy of these tests is still debated, and it is necessary to make them more reliable, especially for the identification of cases of leprosy between contacts. We proposed an integrated analysis of molecular and serological methods using artificial intelligence by the random forest (RF) algorithm to better diagnose and predict new cases of leprosy. METHODS: The study was developed in Governador Valadares, Brazil, a hyperendemic region for leprosy. A longitudinal study was performed, including new cases diagnosed in 2011 and their respective household contacts, who were followed in 2011, 2012, and 2016. All contacts were diligently evaluated by clinicians from Reference Center for Endemic Diseases (CREDEN-PES) before being classified as asymptomatic. Samples of slit skin smears (SSS) from the earlobe of the patients and household contacts were collected for quantitative polymerase chain reaction (qPCR) of 16S rRNA, and peripheral blood samples were collected for ELISA assays to detect LID-1 and ND-O-LID. RESULTS: The statistical analysis of the tests revealed sensitivity for anti-LID-1 (63.2%), anti-ND-O-LID (57.9%), qPCR SSS (36.8%), and smear microscopy (30.2%). However, the use of RF allowed for an expressive increase in sensitivity in the diagnosis of multibacillary leprosy (90.5%) and especially paucibacillary leprosy (70.6%). It is important to report that the specificity was 92.5%. CONCLUSION: The proposed model using RF allows for the diagnosis of leprosy with high sensitivity and specificity and the early identification of new cases among household contacts.


Asunto(s)
Ensayo de Inmunoadsorción Enzimática/métodos , Composición Familiar , Salud de la Familia , Lepra/diagnóstico , Mycobacterium leprae/genética , Mycobacterium leprae/inmunología , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Antibacterianos/sangre , Inteligencia Artificial , Brasil , Niño , Preescolar , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Femenino , Humanos , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Técnicas de Diagnóstico Molecular/métodos , ARN Ribosómico 16S/genética , Sensibilidad y Especificidad , Pruebas Serológicas/métodos , Adulto Joven
14.
Ann Hum Biol ; 46(2): 120-128, 2019 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-31137975

RESUMEN

Context: Tuberculosis and leprosy are readily recognised in human remains due to their typical palaeopathology. Both Mycobacterium tuberculosis (MTB) and Mycobacterium leprae (ML) are obligate pathogens and have been detected in ancient human populations. Objective: To demonstrate historical tuberculosis and leprosy cases in Europe and beyond using molecular methods, as human populations are associated with different mycobacterial genotypes. Methods: MTB and ML ancient DNA (aDNA) has been detected by DNA amplification using PCR, or by whole genome sequencing. Mycobacterial cell wall lipids also provide specific markers for identification. Results: In 18th century Hungary, the European indigenous MTB genotype 4 strains have been found. However, many individuals were co-infected with up to three MTB sub-genotypes. In 8th-14th century Europe significant differences in ML genotypes were found between northwest Europe compared with central, southern, or eastern Europe. In addition, several co-infections of MTB and ML were detected in historical samples. Conclusion: Both MTB and ML strain types differ between geographically separate populations. This is associated with ancient human migration after an evolutionary bottleneck and clonal expansion. The absence of indigenous leprosy in Europe today may be due to the greater mortality of tuberculosis in individuals who are co-infected with both organisms.


Asunto(s)
ADN Antiguo/análisis , Migración Humana/historia , Lepra/historia , Mycobacterium leprae/genética , Mycobacterium tuberculosis/genética , Tuberculosis/historia , Europa (Continente) , Genotipo , Historia del Siglo XVII , Historia del Siglo XVIII , Historia Medieval , Humanos , Lepra/microbiología , Paleopatología , Reacción en Cadena de la Polimerasa , Tuberculosis/microbiología , Secuenciación Completa del Genoma
15.
PLoS One ; 14(4): e0214051, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30947261

RESUMEN

BACKGROUND: Leprosy is a slow, chronic disorder caused by Mycobacterium leprae. India has achieved elimination of leprosy in December 2005 but new cases are being detected and continue to occur in some endemic pockets. The possible ways of transmission of leprosy is not fully understood and is believed that leprosy is transmitted from person to person in long term contact. Studying the transmission dynamics is further complicated by inability to grow M. leprae in culture medium and lack of animal models. More than one family members were found to be affected by leprosy in some highly endemic pockets. This study reported the transmission pattern of leprosy in a family having 4 patients. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the transmission of leprosy in a single family having 4 patients using microsatellite typing. DNA was isolated from slit skin smear samples taken from the patients and the isolated DNA were amplified using microsatellite loci TA11CA3. The amplified products were sequenced using Sanger's sequencing methods and the copy number variation in the microsatellite loci between strains were elucidated by multiple sequence alignment. The result showed that all the 4 members of the family acquired infection from 3 different strains of M. leprae from 3 different sources. The elder and middle daughters were infected by same types of strains having the repeat unit TA13CA3 and could have acquired the infection from social contacts of leprosy cases while the father and younger daughter were infected by strains with the repeat unit TA12CA3 and TA11CA3 and could have acquired infection from social contacts. CONCLUSIONS/SIGNIFICANCE: The study suggested that three family members viz, elder daughter, father and younger daughter could be infected by M. leprae from 3 different sources and the history of the disease and genetic analysis showed that the middle daughter acquired infection from her elder sister in due course of contact. This study implies that the transmission of leprosy not only occurred amongst the house hold members but also has been transmitted from social and neighborhood contacts in long term association with the them.


Asunto(s)
Lepra/microbiología , Repeticiones de Microsatélite/genética , Mycobacterium leprae/genética , Secuencia de Bases , Variaciones en el Número de Copia de ADN/genética , Familia , Femenino , Humanos , Masculino
16.
Emerg Microbes Infect ; 8(1): 109-118, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30866765

RESUMEN

Of the more than 190 distinct species of Mycobacterium genus, many are economically and clinically important pathogens of humans or animals. Among those mycobacteria that infect humans, three species namely Mycobacterium tuberculosis (causative agent of tuberculosis), Mycobacterium leprae (causative agent of leprosy) and Mycobacterium abscessus (causative agent of chronic pulmonary infections) pose concern to global public health. Although antibiotics have been successfully developed to combat each of these, the emergence of drug-resistant strains is an increasing challenge for treatment and drug discovery. Here we describe the impact of the rapid expansion of genome sequencing and genome/pathway annotations that have greatly improved the progress of structure-guided drug discovery. We focus on the applications of comparative genomics, metabolomics, evolutionary bioinformatics and structural proteomics to identify potential drug targets. The opportunities and challenges for the design of drugs for M. tuberculosis, M. leprae and M. abscessus to combat resistance are discussed.


Asunto(s)
Proteínas Bacterianas/química , Biología Computacional/métodos , Mycobacterium/genética , Análisis de Secuencia de ADN/métodos , Animales , Proteínas Bacterianas/metabolismo , Descubrimiento de Drogas , Farmacorresistencia Bacteriana , Genoma Bacteriano , Humanos , Anotación de Secuencia Molecular , Mycobacterium/metabolismo , Mycobacterium abscessus/genética , Mycobacterium abscessus/metabolismo , Mycobacterium leprae/genética , Mycobacterium leprae/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Conformación Proteica , Proteómica
17.
PLoS Negl Trop Dis ; 13(3): e0007284, 2019 03.
Artículo en Inglés | MEDLINE | ID: mdl-30883558

RESUMEN

BACKGROUND: The reduced amounts of Mycobacterium leprae (M. leprae) among paucibacillary (PB) patients reflect the need to further optimize methods for leprosy diagnosis. An increasing number of reports have shown that droplet digital polymerase chain reaction (ddPCR) is a promising tool for diagnosis of infectious disease among samples with low copy number. To date, no publications have investigated the utility of ddPCR in the detection of M. leprae. The aim of this study was to develop and evaluate a ddPCR assay for the diagnosis of PB leprosy. METHODOLOGY: The two most sensitive DNA targets for detection of M. leprae were selected from electronic databases for assessment of sensitivity and specificity by quantitative polymerase chain reaction (qPCR) and ddPCR. Control patients (n = 59) suffering from other dermatological diseases were used to define the cut-off of the duplex ddPCR assay. For comparative evaluation, qPCR and ddPCR assays were performed in 44 PB patients and 68 multibacillary (MB) patients. PRINCIPAL FINDINGS: M. leprae-specific repetitive element (RLEP) and groEL (encoding the 65 kDa molecular chaperone GroEL) were used to develop the ddPCR assay by systematically analyzing specificity and sensitivity. Based on the defined cut-off value, the ddPCR assay showed greater sensitivity in detecting M. leprae DNA in PB patients compared with qPCR (79.5% vs 36.4%), while both assays have a 100% sensitivity in MB patients. CONCLUSIONS/SIGNIFICANCE: We developed and evaluated a duplex ddPCR assay for leprosy diagnosis in skin biopsy samples from leprosy patients. While still costly, ddPCR might be a promising diagnostic tool for detection of PB leprosy.


Asunto(s)
Lepra Paucibacilar/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Mycobacterium leprae/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Piel/microbiología , Adolescente , Adulto , Anciano , Biopsia , Chaperonina 60/genética , Femenino , Humanos , Secuencias Repetitivas Esparcidas , Masculino , Persona de Mediana Edad , Mycobacterium leprae/genética , Sensibilidad y Especificidad , Adulto Joven
18.
PLoS Negl Trop Dis ; 13(3): e0007147, 2019 03.
Artículo en Inglés | MEDLINE | ID: mdl-30835722

RESUMEN

OBJECTIVE: The diagnosis of paucibacillary (PB) leprosy cases remains a challenge because of the absence of a confirmatory laboratory method. While quantitative polymerase chain reaction (qPCR) has been shown to provide reliable sensitivity and specificity in PB diagnoses, a thorough investigation of its efficacy in clinical practice has not yet been published. The present study evaluated patients with suspected leprosy skin lesions by using qPCR to identify PB individuals in the Leprosy Outpatient clinic at the Oswaldo Cruz Foundation in Rio de Janeiro, Brazil. METHODS: One hundred seventy-two suspected PB cases were included in the study. The patients were evaluated by a dermatologist at three different times. The clinical dermato-neurological examination and collected samples were performed on the first visit. On the second visit, the results of the histopathological analysis and PCR assay (DNA-based Mycobacterium leprae qPCR-targeting 16S gene) results were analyzed, and a decision regarding multi-drug therapy was made. A year later, the patients were re-examined, and the consensus diagnosis was established. RESULTS: In 58% (100/172) of cases, a conclusive diagnosis via histopathological analysis was not possible; however, 30% (30/100) of these cases had a positive PCR. One hundred ten patients (110/172) attended the third visit. The analysis showed that while the sensitivity of the histopathological test was very low (35%), a qPCR alone was more effective for identifying leprosy, with 57% sensitivity. CONCLUSION: The use of qPCR in suspected PB cases with an inconclusive histology improved the sensitivity of leprosy diagnoses.


Asunto(s)
Lepra Paucibacilar/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Mycobacterium leprae/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Adolescente , Adulto , Anciano , Brasil , ADN Bacteriano/genética , ADN Ribosómico/genética , Femenino , Histocitoquímica , Humanos , Masculino , Persona de Mediana Edad , Mycobacterium leprae/genética , Pacientes Ambulatorios , ARN Ribosómico 16S/genética , Sensibilidad y Especificidad , Adulto Joven
19.
PLoS Negl Trop Dis ; 13(3): e0006704, 2019 03.
Artículo en Inglés | MEDLINE | ID: mdl-30835734

RESUMEN

Leprosy is a chronic infection where the skin and peripheral nervous system is invaded by Mycobacterium leprae. The infection mechanism remains unknown in part because culture methods have not been established yet for M. leprae. Mce1A protein (442 aa) is coded by mce1A (1326 bp) of M. leprae. The Mce1A homolog in Mycobacterium tuberculosis is known to be associated with M. tuberculosis epithelial cell entry, and survival and multiplication within macrophages. Studies using recombinant proteins have indicated that Mce1A of M. leprae is also associated with epithelial cell entry. This study is aimed at identifying particular sequences within Mce1A associated with M. leprae epithelial cell entry. Recombinant proteins having N-terminus and C-terminus truncations of the Mce1A region of M. leprae were created in Escherichia coli. Entry activity of latex beads, coated with these truncated proteins (r-lep37 kDa and r-lep27 kDa), into HeLa cells was observed by electron microscopy. The entry activity was preserved even when 315 bp (105 aa) and 922 bp (308 aa) was truncated from the N-terminus and C-terminus, respectively. This 316-921 bp region was divided into three sub-regions: 316-531 bp (InvX), 532-753 bp (InvY), and 754-921 bp (InvZ). Each sub-region was cloned into an AIDA vector and expressed on the surface of E. coli. Entry of these E. coli into monolayer-cultured HeLa and RPMI2650 cells was observed by electron microscopy. Only E. coli harboring the InvX sub-region exhibited cell entry. InvX was further divided into 4 domains, InvXa-InvXd, containing sequences 1-24 aa, 25-46 aa, 47-57 aa, and 58-72 aa, respectively. Recombinant E. coli, expressing each of InvXa-InvXd on the surface, were treated with antibodies against these domains, then added to monolayer cultured RPMI cells. The effectiveness of these antibodies in preventing cell entry was studied by colony counting. Entry activity was suppressed by antibodies against InvXa, InvXb, and InvXd. This suggests that these three InvX domains of Mce1A are important for M. leprae invasion into nasal epithelial cells.


Asunto(s)
Proteínas Bacterianas/metabolismo , Lepra/microbiología , Mycobacterium leprae/patogenicidad , Tabique Nasal/microbiología , Proteínas Bacterianas/genética , Línea Celular , Recuento de Colonia Microbiana , ADN Bacteriano/genética , Escherichia coli/genética , Vectores Genéticos/genética , Células HeLa , Humanos , Microesferas , Mycobacterium leprae/genética , Mycobacterium leprae/crecimiento & desarrollo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
20.
Biochem Biophys Res Commun ; 509(3): 779-783, 2019 02 12.
Artículo en Inglés | MEDLINE | ID: mdl-30616886

RESUMEN

Repair of DNA alkylation damage is essential for maintaining genome integrity and Fe(II)/2-oxoglutarate(2OG)-dependent dioxygenase family of enzymes play crucial role in repairing some of the alkylation damages. Alkylation repair protein-B (AlkB) of Escherichia coli belongs to Fe(II)/2OG-dependent dioxygenase family and carries out DNA dealkylation repair. We report here identification of a hypothetical Mycobacterium leprae protein (accession no. ML0190) from the genomic database and show that this 615-bp open reading frame encodes a protein with sequence and structural similarity to Fe(II)/2OG-dependent dioxygenase AlkB. We identified mRNA transcript of this gene in the M. leprae infected clinical skin biopsy samples isolated from the leprosy patients. Heterologous expression of ML0190 in methyl methane sulfonate (MMS) sensitive and DNA repair deficient strain of Saccharomyces cerevisiae and Escherichia coli resulted in resistance to alkylating agent MM. The results of the present study imply that Mycobacterium leprae ML0190 is involved in protecting the bacterial genome from DNA alkylation damage.


Asunto(s)
Proteínas Bacterianas/genética , Escherichia coli/efectos de los fármacos , Metilmetanosulfonato/toxicidad , Mutágenos/toxicidad , Mycobacterium leprae/genética , Saccharomyces cerevisiae/efectos de los fármacos , Alquilación/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Escherichia coli/genética , Genes Bacterianos , Genoma Bacteriano/efectos de los fármacos , Humanos , Lepra/microbiología , Modelos Moleculares , Mycobacterium leprae/efectos de los fármacos , Saccharomyces cerevisiae/genética
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