RESUMO
The number of registered leprosy patients world-wide has decreased dramatically after extensive application of WHO recommended Multiple Drug Therapy (MDT). The annual number of new cases has, however, been almost unchanged in several populations, indicating that the infection is still present at community level. Nasal carriage of Mycobacterium leprae DNA was studied in Lega Robi village in Ethiopia. MDT had been applied for more than ten years, and 718 residents over 5 years old were eligible for the study. During the first survey nasal swab samples were collected from 664 (92.5%) individuals. The results of a Peptide Nucleic Acid-ELISA test for M. leprae DNA interpreted by stringent statistical criteria were available for 589 (88.7%) subjects. Thirty-five (5.9%) individuals without clinical signs of leprosy were positive for M. leprae DNA. Seven PCR positive individuals lived in a household where one or two other members were also positive for M. leprae DNA. During a second survey 8 (46%) of 175 interpretable PNA-ELISA tests were positive. Of 137 individuals tested twice, only two were positive on both occasions whereas 10 were PCR positive only once. The study confirms the widespread distribution of M. leprae DNA in healthy individuals. The feasibility of curbing possible transmission of subclinical infection needs further consideration.
Assuntos
Feminino , Masculino , Adolescente , Adulto , Idoso , Criança , Humanos , Pessoa de Meia-Idade , DNA Bacteriano , Ensaio de Imunoadsorção Enzimática , Etiópia , Hanseníase , Mycobacterium leprae , Nariz , Reação em Cadeia da PolimeraseRESUMO
We report here a simplified method for the detection of nasal carriage of Mycobacterium leprae. DNA extracted from nasal swabs was analyzed by PCR, and M. leprae specific amplicons detected by means of a novel peptide-nucleic-acid-ELISA (PNA-ELISA) method. Parameters for the method were established using swabs taken from untreated lepromatous leprosy patients. We have developed this method to study nasal carriage in endemic populations. However, due to the sensitivity of PCR based techniques, we wished to assess the possibility of false positive samples arising in our method. We therefore examined samples taken from individuals in Norway, a country non-endemic for leprosy, using our technique. A total of 219 nasal swabs were collected and tested in our laboratory in London. All of these were found to be negative by our criteria. In order to corroborate our results, and also to assess the specificity of the method, a small number of these samples were randomly selected, and a known amount of M. leprae DNA added to them. All 219 samples were then retested using the same techniques under [quot ]double blind[quot ] conditions in our laboratory in India. All of the samples to which M. leprae DNA had been added were successfully identified by this method whereas all other swabs were negative. Taken together, these results suggest that the technique described here is simple, sensitive, and specific for use in large-scale epidemiological studies. This study, part of the larger MILEP 2 study, represents the first use of a PNA-PCR method for an epidemiological study of infection. The method using PNA-ELISA is significantly simpler and more rapid than gel based detection methods. The supply of laboratory consumables and overall detection procedure were simplified and standardized by use of PCR Ready-to-Go beads.
Assuntos
Humanos , Mycobacterium leprae/fisiologia , Mycobacterium leprae/genética , Mycobacterium leprae/imunologia , Reação em Cadeia da Polimerase/métodosAssuntos
Antígenos CD/metabolismo , Antígenos de Bactérias/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Apoptose , Glicoproteínas de Membrana/metabolismo , Granuloma de Corpo Estranho/imunologia , Granuloma de Corpo Estranho/patologia , Hanseníase/imunologia , Hanseníase/microbiologia , Hanseníase/patologia , Imuno-Histoquímica , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/patologia , Modelos Imunológicos , Mycobacterium leprae/imunologia , Mycobacterium tuberculosis/imunologia , Receptor fas/metabolismo , Tuberculose/imunologia , Tuberculose/microbiologia , Tuberculose/patologiaRESUMO
BACKGROUND: A third of the world's population has latent infection with Mycobacterium tuberculosis, and in areas of low endemicity, most cases of active tuberculosis arise as a result of reactivation of latent bacilli. We sought to establish the cellular location of these latent organisms to facilitate their elimination. METHODS: We applied in-situ PCR to sections of macroscopically normal lung tissue from 13 individuals from Ethiopia and 34 from Mexico who had died from causes other than tuberculosis. Sections of lung tissue from six Norwegian individuals (ie, individuals from a non-endemic population) acted as negative controls, and six Ethiopian tuberculosis cases acted as positive controls. FINDINGS: Control necropsy samples from the Norwegian individuals were all negative by in-situ PCR and conventional PCR, whereas all samples from known Ethiopian tuberculosis cases were positive by both methods. However, in macroscopically normal lung tissue from Ethiopian and Mexican individuals without tuberculous lesions, the in-situ PCR revealed five of 13 and ten of 34 positive individuals, respectively. These results were confirmed by conventional PCR with extracted DNA. Positive cells included alveolar and interstitial macrophages, type II pneumocytes, endothelial cells, and fibroblasts. INTERPRETATION: M. tuberculosis can persist intracellularly in lung tissue without histological evidence of tuberculous lesions. M. tuberculosis DNA is situated not only in macrophages but also in other non-professional phagocytic cells. These findings contradict the dominant view that latent organisms exist in old classic tuberculous lesions, and have important implications for strategies aimed at the elimination of latent and persistent bacilli.
Assuntos
DNA Bacteriano/isolamento & purificação , Pulmão/microbiologia , Mycobacterium tuberculosis/isolamento & purificação , Tuberculose Pulmonar/microbiologia , Adolescente , Adulto , Estudos de Casos e Controles , Criança , Endotélio Vascular/microbiologia , Feminino , Fibroblastos/microbiologia , Humanos , Pulmão/patologia , Macrófagos/microbiologia , Macrófagos Alveolares/microbiologia , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase/métodos , Marcação in Situ com Primers , Alvéolos Pulmonares/microbiologiaRESUMO
Peripheral blood lymphocytes from 105 subjects with different forms of leprosy and healthy contacts of leprosy patients weere stimulated in vitro different preparations of mycobacterial antugens alone or in combination with a suboptimal dose of phytohaemagglutinin (PHA). In nearly all individuals sonicated leprosy bacilli and PHA together gave a lower 3H-thymidine incorporation than didi the same dose of PHA alone. There was no difference in the degree of inhibition seen in the different patient groups or the healthy contacts. High doses of whole, washed Mycobacterium leprae, combined with PHA led to an increased thymidine incorporation in borderline tuberculoid leprosy patients who had experienced a reversal reaction, and in healthy contacts with less than 6 months exposure did not show an augmentation of the PHA-induced thymidine incorporation. The inhibition exerted by sonicated M. leprae was dose-dependent, seen even with very low doses of antigen, and was not due to direct Cytotoxicity, M. bovis, strain BCG, was weakly suppressive effect. There was no correlation between the suppressive effective of M. leprae antigens and the other mtcobacteria neither was there any correlation with the responses to the mycobacterial antigens alone. Many lepromatous leprosy patients showed significant suppression of background incorporation with addition of M. leprae antigens. This paper discusses wheter the apparent `non-responsiveness` in lepromatous leprosy could be due to active suppressor mechanisms operative in vitro.