[Detection of Mycobacterium leprae DNA in nasal swab]. / Deteccção do DNA de Mycobacterium leprae em secreção nasal.

Studies have demonstrated high sensibility of the polimerase chain reaction (PCR) technique in the identification of the Mycobacterium leprae DNA . This study aimed to evalue the PCR sensibility at the detection of the M. leprae DNA in nasal swab of leprosy patients and to compare the results with the bacilloscopy and multibacillary (MBs) and paucibacilares (PBs) forms. Nasal secretion samples of 24 leprosy patients were collected, and were preserved in one and two lise's solution. The PCR results were highly significant (p <0.0000) and they revealed grater sensibility than bacilloscopy, in several clinical forms. Nevertheless, still different studies are necessary, testing new markers and preservatives, with the purpose of lifting up the sensibility of this technique, in nasal secretion samples.

Recombinant Mycobacterium leprae protein associated with entry into mammalian cells of respiratory and skin components.

BACKGROUND: The transmission of Mycobacterium leprae, the causative pathogen of leprosy, has been postulated to occur mainly through upper respiratory route rather than skin-to-skin contact via minor injuries. The M. leprae genome contains mce1A gene, which encodes a putative mammalian cell entry protein. However, to date, there have been no functional analyses of the M. leprae mce1A gene product. OBJECTIVE: The aim of this study was to elucidate a possible relationship between this transmission mechanism and the mce1A gene product. METHODS: We analyzed the cell uptake activity in vitro of polystyrene latex beads coated with a purified recombinant (r-) protein expressed by a 849-bp locus within the mce1A gene. RESULTS: The r-protein promoted uptake of the beads into human nasal epithelial cells derived from nasal polyps, human bronchial epithelial cell line, normal human dermal fibroblasts, normal human microvascular endothelial cells and normal human keratinocytes cultured at 0.01 mM extracellular calcium concentration [Ca]; no uptake occurred with keratinocytes cultured at 1.2mM [Ca]. CONCLUSION: These results suggest that the mce1A gene product can mediate M. leprae entry into respiratory epithelial cells as their natural target cells, which may be the main mode of transmission. Endothelial cells, on the other hand, may serve as the reservoir of the bacilli for long-term infection. The M. leprae Mce1A protein has potential important implications for mode of transmission and pathogenesis of leprosy.

Detection of Mycobacterium leprae DNA by polymerase chain reaction in the blood and nasal secretion of Brazilian household contacts.

DNA samples from blood and nasal swabs of 125 healthy household contacts was submitted to amplification by polymerase chain reaction (PCR) using a Mycobacterium leprae-specific sequence as a target for the detection of subclinical infection with M. leprae. All samples were submitted to hybridization analysis in order to exclude any false positive or negative results. Two positive samples were confirmed from blood out of 119 (1.7%) and two positive samples from nasal secretion out of 120 (1.7%). The analysis of the families with positive individuals showed that 2.5% (n = 3) of the contacts were relatives of multibacilary patients while 0.8% of the cases (n = 1) had a paucibacilary as an index case. All positive contacts were followed up and after one year none of them presented clinical signs of the disease. In spite of the PCR sensitivity to detect the presence of the M. leprae in a subclinical stage, this molecular approach did not seem to be a valuable tool to screen household contacts, since we determined a spurious association of the PCR positivity and further development of leprosy.

Characterization of leprosy based on the nasal lipid profile.

While extracting the M. leprae from the nasal flushings of leprosy patients it was found that these organisms were trapped in the waxy layer, between the aqueous and the chloroform layers. Thin layer chromotography (TLC) analysis of this layer, using chloroform-methanol-water system, revealed different spots when sprayed with acid alcohol and heated at 160 degrees C. The TLC profile of lipids of lepromatous and borderline (MB according to the WHO terminology) leprosy patients was distinctly different from that of tuberculoid leprosy patients and normal human volunteers. A simple, economical and fast procedure to characterize patients belonging to different spectra has been developed.

Disposition of rifampicin following intranasal and oral administration.

Leprosy is transmitted by dissemination of M.leprae which are lodged in the nose of the patients suffering from multibacillary (MB) type of the disease. Rifampicin, a potent bactericidal antileprotic drug is given orally to the patients with a view to make the infective cases non-infective. Earlier work by us has shown that intranasal administration of rifampicin helps in reducing the M.leprae load in the nose much faster than after conventional oral administration. In the present study, rifampicin concentrations in plasma/urine/nasal wash of healthy volunteers following oral and intranasal administration were determined. Following intranasal administration, rifampicin was not detectable in plasma and high concentrations were measured in the nasal wash. Following oral administration, rifampicin was not detectable in the nasal wash indicating that enough antibiotic levels are not available for clearing M.leprae from nose.

Development of a mucosal challenge test for leprosy using leprosin A.

There is little information about the mucosal immune response in leprosy. We have developed a nasal provocation test with leprosin A which will be used to investigate mucosal immunity to Mycobacterium leprae. Initial studies were performed with increasing doses of leprosin A (1.0 pg/ml-10 micrograms/ml) to determine the optimal safe dose of leprosin A. Anti-M. leprae IgA antibody and normal IgA concentrations were measured in the saliva of leprosy contacts and controls before and after instillation of leprosin A. Nasal leprosin A was well tolerated up to a concentration of 10 micrograms/ml without side effects. None of the six subjects who had not been exposed to leprosy had salivary IgA against whole M. leprae, whereas IgA was detected from 64 h to 140 h following instillation of leprosin A in all of the leprosy hospital workers and in 15 out of 18 healthy household contacts tested. There was no correlation between serum and salivary anti-M. leprae IgA levels before and after testing. Salivary IgA anti-lipoarabinomannan responses were seen in 12 out of 20 household contacts. Normal salivary IgA concentrations varied from 8 to 240 mg/l. The leprosin A nasal provocation test appears to be a safe method for the investigation of the role of mucosal immunity in the pathogenesis of leprosy.

Evaluation of PCR mediated DNA amplification in non-invasive biological specimens for subclinical detection of Mycobacterium leprae.

DNA from Mycobacterium leprae, present in non-invasive clinical samples from leprosy patients, such as nasal secretion and hair bulbs, was submitted to amplification by the polymerase chain reaction using a M. leprae-specific repetitive sequence as a target. After optimization of sample processing and of the PCR conditions, we were able to detect DNA from M. leprae in both types of clinical samples, even from paucibacillary leprosy patients. The use of hair bulbs and nasal secretion as clinical samples for screening of household contacts and for the evaluation of a risk population, or for the follow-up of patients under chemotherapy, and monitoring of bacterial load is discussed.

Loss of viability of Mycobacterium leprae isolated from nasal secretions of lepromatous leprosy patients following daily rifampicin and DDS therapy.

Excreta from blowing their noses was collected from 4 previously untreated multibacillary (LL) patients in the ALERT hospital, immediately before and during daily treatment with 600 mg rifampicin and 100 mg dapsone (DDS). The Mycobacterium leprae recovered from the nasal secretions were enumerated and inoculated into the footpads of normal mice. Bacilli recovered from 2 of the patients failed to infect mice after 1 day's treatment, and all infectivity of the bacilli from the other 2 patients was lost after 2 days' treatment. These findings demonstrate the rapidity with which rifampicin-containing multidrug treatment is likely to reduce a patient's level of infection to their contacts.

Joint chemotherapy trials in lepromatous leprosy conducted in Thailand, the Philippines, and Korea.

Chemotherapy trials in lepromatous leprosy using various combinations of existing antileprosy drugs were conducted jointly by Korea, The Philippines, and Thailand. The general objective of these trials was to determine the most effective and practicable regimen or regimens for field application. Lepromatous patients were divided into two groups: Group I was comprised of new, untreated patients infected with dapsone-sensitive Mycobacterium leprae and Group II consisted of relapsed patients with dapsone-resistant disease. Four different regimens were administered to each group for 5 years. Comparison among the regimens was based on antileprotic efficacy, drug safety, acceptability, field practicability, and economic feasibility. No significant differences were noted among the various regimens as judged by the reduction in the bacterial index (BI), clinical response, and change in biopsy index. Toxicity was seen only in the regimens containing prothionamide and rifampin. The regimens were acceptable to the patients and all were found practical for field use. Clofazimine, even in low doses, was found to suppress the frequency and severity of erythema nodosum leprosum. A multidrug regimen effective against both new and relapsed cases of lepromatous leprosy, whether dapsone sensitive or dapsone resistant, is recommended for field use. Given priority, the cost of the regimens is affordable in the three countries.

Detection of lepromatous leprosy patients shedding M. leprae in nasal droppings by enzyme immunoassay.

Enzyme immunoassays (EIAs) for detection of lepromatous leprosy (LL) patients harbouring M. leprae in nasal mucosa are described. One EIA measures IgM antibodies against the synthetic disaccharide (ND-BSA) residue of phenolic glycolipid I of M. leprae, whereas the other titrates primarily IgG antibodies against sonicate supernatant antigens of Mycobacterium w. (M.w.). Fifty coded leprosy sera were analysed by EIAs under a double blind code. Amongst the 20 LL patients with positive nasal smear, 18 (90%) were positive in EIA based on ND-BSA, in comparison to 19 (95%) in EIA using M.w. antigens. The assays can be performed on fresh serum samples or on blood samples collected on filter paper discs. These assays can be useful for leprosy control programmes.

"Nose-blow" smears in multibacillary leprosy patients.

332 nose-blow specimens have been examined from 73 untreated multibacillary patients of leprosy before and periodically after they were put on a maximal, minimal or an intermediate multi-drug regimen. 80% of these specimens were found to be positive for acid fast bacilli initially. Bacillary positivity rate was more in samples containing pus or blood. Bacilli were seen in LL, LI as well as BL patients. Nearly half of the cases became negative for AFB in their nose-blow specimens within 3 months of initiation of treatment whereas none of these patients has become negative in skin smears. However, a few cases have continued to discharge bacilli in their nasal secretions even after 12 months of multi-drug regimen therapy.

Rifampin in the treatment of leprosy.

The minimal inhibitory concentration of rifampin for Mycobacterium leprae is less than 1 microgram/ml. Therapy with rifampin has proved efficacious both in mice experimentally infected with M. leprae and in humans with leprosy. Rifampin kills M. leprae more rapidly than do other antileprosy drugs currently available. Consequently, M. leprae bacilli from patients with lepromatous disease are rendered noninfectious within three weeks after the institution of rifampin therapy, as determined in the mouse footpad test system. Administration of this antibiotic substantially reduces the quantities of M. leprae discharged in the nasal secretions of lepromatous patients within three weeks, thus rapidly decreasing the potential infectivity of these individuals. Intermittent rifampin therapy for leprosy has been successful, with a low incidence of adverse reactions to the drug. Worldwide, the prevalence of primary and secondary resistance of M. leprae to dapsone has increased markedly. Therefore, the World Health Organization recommends a multidrug regimen that includes intermittent administration of rifampin for the treatment of leprosy.

The nose in lepromatous leprosy; bacteriological and histopathological studies of patients treated with dapsone monotherapy for varying periods of time.

A clinical, bacteriological and histopathological investigation of 62 patients with lepromatous leprosy attending a hospital in South India is reported, with particular emphasis on the activity of the disease in the nose. Twelve of the patients were from a group of 34 new patients who had been originally examined 5 years previously, and subsequently treated with dapsone (DDS) monotherapy. A further 50 lepromatous patients were also examined, who had been treated for periods ranging from 3 months to 10 years. With a few exceptions, there was good correlation between the clinical and histopathological findings in the skin and nose. Evidence of disease activity was demonstrated among three-quarters of the patients who had been treated for over a year. Failure to achieve quiescence was explained in most of the patients by failure to collect their dapsone treatment or to ingest it regularly as demonstrated by the determination of DDS/creatinine ratios on urine samples collected at the time of their visit to the clinic. Although the compliance of most patients was relatively satisfactory during the first 12 months of treatment, thereafter it deteriorated markedly. In contrast to the clinical, bacteriological, and histopathological evidence of disease activity in the skin and nose of most patients, in only one of the patients treated for more than a year was a positive nose-blow encountered. This suggests that the infectivity of DDS-treated lepromatous patients within this time and this diminished infectivity often persists despite poor drug compliance and continuing disease activity.

Importance of nasal lesions in early lepromatous leprosy.

There are some 20 million people in the world with leprosy. In the lepromatous form of the illness the nose becomes infected very early in the disease process. The nasal discharge which occurs is heavily bacillated and is the most potent source of exit of Mycobacterium leprae from the body. The necessity for early diagnosis and treatment of leprosy in the absence of an effective vaccine is discussed and the pathological changes that occur in the nose are outlined. The roles which the leprologist and the rhinologist are able to play are mentioned.