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1.
PLoS Negl Trop Dis ; 14(7): e0007871, 2020 07.
Artículo en Inglés | MEDLINE | ID: mdl-32628669

RESUMEN

Leprosy, caused by Mycobacterium leprae, has plagued humanity for thousands of years and continues to cause morbidity, disability and stigmatization in two to three million people today. Although effective treatment is available, the disease incidence has remained approximately constant for decades so new approaches, such as vaccine or new drugs, are urgently needed for control. Research is however hampered by the pathogen's obligate intracellular lifestyle and the fact that it has never been grown in vitro. Consequently, despite the availability of its complete genome sequence, fundamental questions regarding the biology of the pathogen, such as its metabolism, remain largely unexplored. In order to explore the metabolism of the leprosy bacillus with a long-term aim of developing a medium to grow the pathogen in vitro, we reconstructed an in silico genome scale metabolic model of the bacillus, GSMN-ML. The model was used to explore the growth and biomass production capabilities of the pathogen with a range of nutrient sources, such as amino acids, glucose, glycerol and metabolic intermediates. We also used the model to analyze RNA-seq data from M. leprae grown in mouse foot pads, and performed Differential Producibility Analysis to identify metabolic pathways that appear to be active during intracellular growth of the pathogen, which included pathways for central carbon metabolism, co-factor, lipids, amino acids, nucleotides and cell wall synthesis. The GSMN-ML model is thereby a useful in silico tool that can be used to explore the metabolism of the leprosy bacillus, analyze functional genomic experimental data, generate predictions of nutrients required for growth of the bacillus in vitro and identify novel drug targets.


Asunto(s)
Genoma Bacteriano , Lepra/microbiología , Redes y Vías Metabólicas , Mycobacterium leprae/genética , Mycobacterium leprae/metabolismo , Animales , Humanos , Ratones , Ratones Desnudos , Mycobacterium leprae/crecimiento & desarrollo
2.
Int J Infect Dis ; 98: 6-13, 2020 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-32553715

RESUMEN

OBJECTIVES: Understanding the nature of Mycobacterium leprae transmission is vital to implement better control strategies for leprosy elimination. The present study expands the knowledge of county-level strain diversity, distribution, and transmission patterns of leprosy in endemic provinces of China. METHODS: We genetically characterized 290 clinical isolates of M. leprae from four endemic provinces using variable number tandem repeats (VNTR) and single nucleotide polymorphisms (SNPs). Attained genetic profiles and cluster consequences were contrasted with geographical and migration features of leprosy at county levels. RESULTS: Considering the allelic variability of 17 VNTR loci by the discriminatory index, (GTA)9, (AT)17, (AT)15, (TA)18, (TTC)21, and (TA)10 are reported to be more highly polymorphic than other loci. The VNTR profile generated the low-density clustering pattern in the counties of Sichuan and Yunnan, whereas clusters have been observed from the isolates from Huayuan (N = 6), Yongding (N = 3), Zixing (N = 3), Chenxi (N = 2) and Zhongfang (N = 2) counties of Hunan, and Zhijin (N = 3), Anlong (N = 2), Zhenning (N = 2), and Xixiu (N = 2) counties of Guizhou. In some clusters, people's social relations have been observed between villages. From the 290 clinical isolates, the most predominantly reported SNP was 3K (278, 95.8%), followed by SNP 1D (10, 3.4%), which are typically observed to be predominant in China. We also detected the novel SNP 3J (2, 0.8%), which has not yet been reported in China. CONCLUSION: The clustering pattern of M. leprae indicates the transmission of leprosy still persists at county levels, suggesting that there is a need to implement better approaches for tracing the close contacts of leprosy patients.


Asunto(s)
Lepra/microbiología , Mycobacterium leprae/aislamiento & purificación , Alelos , China/epidemiología , Análisis por Conglomerados , ADN Bacteriano/genética , Genotipo , Geografía , Humanos , Lepra/epidemiología , Lepra/transmisión , Repeticiones de Minisatélite , Epidemiología Molecular , Mycobacterium leprae/clasificación , Mycobacterium leprae/genética , Filogenia , Polimorfismo de Nucleótido Simple
3.
Int J Infect Dis ; 96: 172-179, 2020 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-32371193

RESUMEN

BACKGROUND: Human-to-human transmission of Mycobacterium leprae among household contacts of active leprosy cases is significant, and surveillance of household contacts is vital to interrupting the transmission chain for this disease. This study was conducted to identify similarities in M. leprae strains, based on genomic single nucleotide polymorphisms (SNPs), among cases and their household contacts and in multicase families in order to decipher possible associations, transmission links, various clinical conditions of index cases that enhance person-to-person transmission, and timelines for transmission patterns. METHODS: PCR for M. leprae DNA detection (amplification of the Rlep gene) and SNP subtyping of M. leprae strains was performed for 61 index cases and one of their household contacts. Additionally, we studied six families with multiple cases of leprosy, to understand timelines of infectivity and its relation to severity of the disease in the index cases. RESULTS: Index cases with lepromatous (LL) and borderline lepromatous (BL) leprosy, together with a positive bacteriological index (BI) for M. leprae, result in a higher percentage of their contacts subclinically infected with M. leprae, with odds ratios (OR) of 6.6 (95% confidence interval (CI) 1.6-27.6) for BL and LL, and 7.07 (CI 1.41-35.41) for BI-positive index cases. 75% of the case-contact pairs had a similar SNP subtype of M. leprae. The timeline of infection in multicase families revealed that contacts were infected during the BI-positive period of the index case. CONCLUSION: Using molecular methods, we determined that positivity for M. leprae DNA in contacts of index leprosy cases was attributed to clinical characteristics of leprosy in the index cases. LL and BL forms of leprosy, together with positive BI, contributed to dissemination of infection to household contacts. In conclusion, we found a relationship between SNP subtypes within index case-contact pairs. This method can help decipher the transmission patterns and identify individuals at risk of contracting leprosy.


Asunto(s)
Lepra/epidemiología , Mycobacterium leprae/genética , Adolescente , Adulto , Composición Familiar , Femenino , Humanos , Lepra/microbiología , Lepra/transmisión , Masculino , Persona de Mediana Edad , Epidemiología Molecular , Mycobacterium leprae/clasificación , Mycobacterium leprae/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo de Nucleótido Simple , Adulto Joven
4.
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
5.
Am J Trop Med Hyg ; 103(1): 209-213, 2020 07.
Artículo en Inglés | MEDLINE | ID: mdl-32285768

RESUMEN

Identification of Mycobacterium leprae DNA by polymerase chain reaction (PCR) is a reliable and an affordable method to confirm leprosy. DNA from 87 nerve samples (61 from paraffin blocks and 26 fresh samples) was extracted. Mycobacterium leprae DNA was amplified by PCR from 80/87 (92%) specimens. Patients were seen over a period of 11 years (2007-2019), and leprosy was diagnosed based on clinical and characteristic histopathology findings. The clinical diagnostic possibilities were as follows: leprous neuropathy in 73/80 (91.3%), mononeuritis multiplex of unknown etiology in four (5.0%), vasculitic neuropathy in two (2.5%), and distal symmetric sensory motor neuropathy in one (1.3%). The biopsied nerves were as follows: superficial radial = 34 (42.6%), dorsal cutaneous branch of ulnar = 19 (23.8%), sural = 18 (22.5%), and superficial peroneal = 9 (11.3%), and corresponding neurological deficits were recorded in 77 (96.3%) cases. The histopathological diagnoses in total group were as follows: (borderline tuberculoid (BT) = 52, tuberculoid (TT) = 8, borderline lepromatous (BL) = 8, borderline borderline (BB) = 3, nonspecific inflammation = 3, healed/fibrosed = 4, and axonopathy = 2). Acid fast bacilli (AFB) was demonstrated in 11 (13.7%) samples. For comparison, 31 clinically and histopathologically defined non-leprous disease control nerves (inherited neuropathy = 20, vasculitis = 8, and nutritional neuropathy = 3) subjected to PCR were negative for M. leprae DNA. In most instances, there are multiple thickened peripheral nerves in suspected cases of leprosy, but neurological deficits pertaining to the thickened nerve are not as widespread. The current findings emphasize the importance of selecting the most appropriate nerve for biopsy to obtain a positive PCR result. We infer that clinical, histopathological, and PCR tests complement each other to help achieve a definitive diagnosis of leprosy particularly in pure neuritic leprosy and in leprous neuropathy with negative skin smears/biopsy.


Asunto(s)
Lepra/diagnóstico , Mycobacterium leprae/genética , Nervios Periféricos/microbiología , Enfermedades del Sistema Nervioso Periférico/microbiología , Reacción en Cadena de la Polimerasa , Adolescente , Adulto , Anciano , Niño , ADN Bacteriano/genética , Humanos , Lepra/complicaciones , Lepra/microbiología , Lepra/patología , Lepra Paucibacilar/complicaciones , Lepra Paucibacilar/diagnóstico , Lepra Paucibacilar/microbiología , Lepra Paucibacilar/patología , Lepra Tuberculoide/complicaciones , Lepra Tuberculoide/diagnóstico , Lepra Tuberculoide/microbiología , Lepra Tuberculoide/patología , Persona de Mediana Edad , Enfermedades del Sistema Nervioso Periférico/diagnóstico , Enfermedades del Sistema Nervioso Periférico/etiología , Enfermedades del Sistema Nervioso Periférico/patología , Reacción en Cadena de la Polimerasa/métodos , Adulto Joven
6.
Fontilles, Rev. leprol ; 32(4): 253-261, ene.-abr. 2020. ilus
Artículo en Español | IBECS | ID: ibc-193431

RESUMEN

La lepra en la infancia cursa con una diversidad de manifestaciones clínicas e histopatológicas que hacen necesario un minucioso examen cutáneo en todo niño, que presente lesiones dermatológicas sugestivas y una fuente infecciosa sospechosa. Para un oportuno diagnóstico es indispensable que el medico tenga siempre presente la enfermedad, así como la destreza al realizar el examen clínico, ya que muchas lesiones cutáneas suelen ser asintomáticas y con frecuencia simulan otros cuadros dermatológicos. El rango de edad en el cual la población infantil se encuentra más afectada está dentro de los 10 y 15 años. En la infancia la lepra refleja características clínicas del adulto, guardando ciertas particularidades; las formas paucibacilar son más comunes entre los dos y nueve años y las formas multibacilares entre los 10 a 14 años. En Cuba han sido reportados desde agosto de 1989 hasta diciembre 2016 un total de 135 casos de Lepra en pacientes en edad pediátrica de los cuales 44 (32,6 %), han sido atendidos en el Hospital Pediátrico Docente Juan Manuel Márquez. OBJETIVO: presentación clínica de tres casos de lepra infantil con formas clínicas diferentes. CONCLUSIONES: La lepra en la infancia cursa con una diversidad de manifestaciones clínicas e histopatológicas, que hacen necesario un minucioso examen cutáneo en todo nino, que presente lesiones dermatológicas sugestivas y una fuente infecciosa sospechosa. El mayor énfasis en la detección y vigilancia temprana de esta enfermedad se debe a que alguno de los niños que recientemente han sido diagnosticados ya mostraban signos de discapacidad


Leprosy in childhood presents a variety of clinical and histopathological manifestations that require a thorough skin examination in every child, presenting suggestive dermatological lesions and a suspicious infectious source. For a timely diagnosis it is essential that the doctor always keeps in mind the disease, as well as the knowledge for performing the clinical examination, since many skin lesions are usually asymptomatic and often simulate other dermatological conditions. The age range in which children are most affected is most affected is within 10 and 15 years. In childhood, leprosy reflects the clinical characteristics of the adult, keeping certain peculiarities; paucibacillary forms are more common between two and nine years old and multibacillary forms between 10 and 14 years old. In Cuba, a total of 135 cases of leprosy have been reported from August 1989 to December 2016 in pediatric patients, of which 44 (32.6%) have been treated at the Juan Manuel Marquez Teaching Pediatric Hospital. OBJECTIVE: clinical presentation of three cases of childhood leprosy with different clinical forms. CONCLUSIONS: Childhood leprosy has a variety of clinical and histopathological manifestations, which require a thorough skin examination in every child, presenting suggestive dermatological lesions and a suspicious infectious source. The greatest emphasis on the detection and early surveillance of this disease is due to the fact that some of the children who have recently been diagnosed already showed signs of disability


Asunto(s)
Humanos , Masculino , Femenino , Preescolar , Niño , Adolescente , Lepra Dimorfa/diagnóstico , Lepra Paucibacilar/diagnóstico , Portador Sano , Mycobacterium leprae/genética , Reacción en Cadena de la Polimerasa
7.
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
8.
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
9.
J Appl Microbiol ; 128(6): 1814-1819, 2020 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-31981442

RESUMEN

AIMS: Diagnosis of leprosy, a chronic infection caused by Mycobacterium leprae, predominantly depends on clinical manifestations and histopathological analysis, hampering rapid and accurate diagnostics. Our aim was to increase accuracy of leprosy diagnosis by improving M. leprae's DNA detection based on polymerase chain reaction (PCR) technique using new specific primers for the RLEP repetitive sequence. METHODS AND RESULTS: The specific target region, RLEP, of M. leprae's genome was selected based on comparative genomics. After confirming the specificity of this region, using blastn analysis, primers were designed and tested for their in silico specificity. To evaluate the specificity and sensitivity of these primers in vitro, 184 blood samples from patients were used in qPCR. The new primer pair LYON1/LYON2 produced 91% positive samples, whereas the current primer pair LP1/LP2 produced 46%. Specificity and DNA detection limit test were carried out to compare the efficiency of the developed primer pair. The LYON1/LYON2 primer showed 100% specificity, whereas LP1/LP2 showed 64%. The DNA detection limit of LYON1/LYON2 was 10 copies of bacterial genomes per millilitre, whereas LP1/LP2 was 1000 copies of bacterial genomes per millilitre. CONCLUSIONS: In conclusion, the developed LYON1/LYON2 primer pair presented to be a specific and sensitive new molecular marker for the diagnosis of leprosy. SIGNIFICANCE AND IMPACT OF THE STUDY: The development of a specific primer pair for the detection of the M. leprae genome through qPCR technique contributes to a fast, sensitive and specific diagnosis, which is essential to prevent spreading and progression of this disease.


Asunto(s)
Lepra/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Mycobacterium leprae/aislamiento & purificación , ADN Bacteriano/genética , Femenino , Genoma Bacteriano/genética , Humanos , Secuencias Repetitivas Esparcidas/genética , Lepra/sangre , Lepra/microbiología , Mycobacterium leprae/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Sensibilidad y Especificidad
10.
Transbound Emerg Dis ; 67(2): 1032-1034, 2020 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-31733134

RESUMEN

Leprosy is a human infectious disease caused by Mycobacterium leprae or Mycobacterium lepromatosis that can also occur in animals and even manifest as zoonosis. Recently, both mycobacteria were detected in red squirrels (Sciurus vulgaris) from the British Isles. To further explore the presence of leprosy bacilli in North-West Europe, we screened Belgian and Dutch squirrels. Tissue samples from 115 animals tested by qPCR were negative for both pathogens. No molecular or pathological evidence was found of the presence of these zoonotic pathogens in North-West Europe.


Asunto(s)
Lepra/veterinaria , Mycobacterium leprae/aislamiento & purificación , Mycobacterium/aislamiento & purificación , Sciuridae/microbiología , Animales , Bélgica/epidemiología , Humanos , Lepra/microbiología , Mycobacterium/genética , Mycobacterium leprae/genética , Países Bajos/epidemiología , Reino Unido/epidemiología , Zoonosis
12.
Comp Immunol Microbiol Infect Dis ; 68: 101397, 2020 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-31775113

RESUMEN

Leprosy was recognized as a zoonotic disease, associated with nine-banded armadillos (Dasypus novemcinctus) in the Southern United States of America in 2011. In addition, there is growing evidence to support a role for armadillos in zoonotic leprosy in South America. The current study evaluated twenty specimens of the six-banded armadillo (Euphractus sexcinctus), collected from rural locations in the state of Rio Grande do Norte (RN), Brazil for evidence of infection with Mycobacterium leprae. Serum was examined using two "in-house" enzyme-linked immunosorbent assays (ELISAs) and via two commercially available (ML flow and NDO-LID®) immunochromatographic lateral flow (LF) tests, for detection of the PGL-I and/or LID-1 antigens of the bacterium. The presence of M. leprae DNA in liver tissue was examined using the multi-copy, M. leprae-specific repetitive element (RLEP), as target in conventional and nested PCR assays. Molecular and anti-PGL-I-ELISA data indicated that 20/20 (100 %) of the armadillos were infected with M. leprae. The corresponding detection levels recorded with the LF tests were 17/20 (85 %) and 16/20 (85 %), for the NDO-LID® and ML flow tests, respectively. Our results indicate that, in common with D. novemcinctus, six banded armadillos (a species hunted and reared as a food-source in some regions of Brazil, including RN), represent a potential reservoir of M. leprae and as such, their role in a possible zoonotic cycle of leprosy within Brazil warrants further investigation.


Asunto(s)
Armadillos/microbiología , Reservorios de Enfermedades/veterinaria , Lepra/veterinaria , Mycobacterium leprae/genética , Mycobacterium leprae/inmunología , Animales , Brasil/epidemiología , Reservorios de Enfermedades/microbiología , Ensayo de Inmunoadsorción Enzimática , Femenino , Lepra/epidemiología , Masculino , Reacción en Cadena de la Polimerasa , Zoonosis/epidemiología , Zoonosis/microbiología
13.
s.l; s.n; 2020. 15 p. ilus, graf, tab.
No convencional en Inglés | Sec. Est. Saúde SP, CONASS, HANSEN, SESSP-ILSLPROD, Sec. Est. Saúde SP, SESSP-ILSLACERVO, Sec. Est. Saúde SP | ID: biblio-1146399

RESUMEN

Leprosy is difficult to diagnose since it is caused by a bacterium that does not grow in vitro. Bacilli direct detection or the presence of specific antibodies can vary greatly depending on the clinical form. M. leprae direct DNA detection can aid clinical diagnosis, although invasive skin biopsies are still necessary to detect the pathogen or histological features consistent with leprosy. Here we show that a kit combining mechanical and chemical lysis efficiently removes host DNA and enriches for M. leprae DNA, allowing better detection of paucibacillary cases. We believe our findings can contribute to improving disease diagnosis, as well as early detection and that could help monitoring strategies(AU).


Asunto(s)
Humanos , Animales , Ratones , ADN Bacteriano/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Mycobacterium leprae/aislamiento & purificación , ADN Bacteriano/genética , ARN Bacteriano/genética , ARN Ribosómico 16S/genética , Sensibilidad y Especificidad , Mycobacterium leprae/genética
14.
Rev. Soc. Bras. Med. Trop ; 53: e20200197, 2020.
Artículo en Inglés | LILACS, Coleciona SUS, Sec. Est. Saúde SP | ID: biblio-1143857

RESUMEN

Abstract Slit skin smear and histopathological examinations are currently the main laboratory tools used to aid the diagnosis of leprosy. However, their sensitivity is low, and many cases are not detected. New methodologies have been studied to develop more accurate tests. This narrative review aims to raise attention to the results of molecular (polymerase chain reaction) and serological (Enzyme-Linked Immunosorbent Assay) tests applied to the diagnosis of leprosy, and to summarize the available information about the former. Original scientific articles published in indexed international journals, whose study involved aspects of the diagnosis and classification of leprosy cases or home contacts, were selected. The data were extracted independently using a standardized method that dictated the inclusion of the following information: diagnosis in Paucibacillary and Multibacillary cases and in household contacts; sample number; sample type; study design; studied variables; statistical analysis employed; main results; and limitations identified. In clinical practice, the results from molecular and serological tests are assessed separately, with moderate sensitivity and specificity. However, an integrated study of these methodologies has been suggested for greater accuracy in diagnosis.


Asunto(s)
Humanos , Lepra/diagnóstico , Mycobacterium leprae/genética , Ensayo de Inmunoadsorción Enzimática , Pruebas Serológicas , Reacción en Cadena de la Polimerasa , Sensibilidad y Especificidad
15.
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
16.
Sci Rep ; 9(1): 16675, 2019 11 13.
Artículo en Inglés | MEDLINE | ID: mdl-31723144

RESUMEN

Household contacts (HHC) of leprosy patients exhibit high-risk of developing leprosy and contact tracing is helpful for early diagnosis. From 2011 to 2018,2,437 HHC were examined in a clinic in Rio de Janeiro, Brazil and 16S qPCR was used for diagnosis and monitoring of contacts. Fifty-four HHCs were clinically diagnosed with leprosy at intake. Another 25 exhibited leprosy-like skin lesions at intake, 8 of which were confirmed as having leprosy (50% of which were qPCR positive) and 17 of which were diagnosed with other skin diseases (6% qPCR positive). In skin biopsies, qPCR presented a sensitivity of 0.50 and specificity of 0.94. Furthermore, 955 healthy HHCs were followed-up for at least 3 years and skin scrapings were collected from earlobes for qPCR detection. Positive qPCR indicated a non-significant relative risk of 2.52 of developing the disease. During follow-up, those who progressed towards leprosy exhibited 20% qPCR positivity, compared to 9% of those who remained healthy. Disease-free survival rates indicated that age had a significant impact on disease progression, where patients over 60 had a greater chance of developing leprosy [HR = 32.4 (3.6-290.3)]. Contact tracing combined with qPCR may assist in early diagnosis and age is a risk factor for leprosy progression.


Asunto(s)
Trazado de Contacto/métodos , ADN Bacteriano/análisis , ADN Ribosómico/análisis , Composición Familiar , Lepra/diagnóstico , Mycobacterium leprae/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Adolescente , Adulto , Brasil/epidemiología , Estudios de Casos y Controles , Niño , Preescolar , Estudios de Cohortes , Femenino , Estudios de Seguimiento , Humanos , Lactante , Lepra/epidemiología , Lepra/genética , Masculino , Persona de Mediana Edad , Técnicas de Diagnóstico Molecular , Mycobacterium leprae/genética , Factores de Tiempo , Adulto Joven
17.
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
18.
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
19.
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
20.
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
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