Utility of immunoglobulin isotypes against LID-1 and NDO-LID for, particularly IgG1, confirming the diagnosis of multibacillary leprosy

BACKGROUND Leprosy remains a health problem in many countries, with difficulties in diagnosis resulting in delayed treatment and more severe disabilities. Antibodies against several Mycobacterium leprae antigens have, however, shown value as diagnostic and/or prognostic markers. OBJECTIVES The objective of this study was to evaluate serum immunoglobulin (Ig) IgM and IgG subclass reactivity against three M. leprae specific antigens: NDO-HSA, a conjugate formed by natural octyl disaccharide bound to human serum albumin; LID-1, the fusion protein product of the ml0405 and ml2331 genes; and NDO-LID, a combination of LID-1 and NDO. METHODS Sera from healthy controls, paucibacillary (PB) and multibacillary (MB) leprosy patients, and their respective household contacts, were evaluated for the presence of antigen-specific IgM, IgG, and IgG subclass antibodies by enzyme-linked immunosorbent assay (ELISA). The sensitivity and specificity of each ELISA were evaluated by receiver operating characteristic (ROC) curve analysis. FINDINGS Our data confirm that serum IgM antibodies against NDO-HSA and IgG antibodies against LID-1, as well as IgG/M antibodies against NDO-LID, are markedly increased in MB patients. For the first time, our data reveal a selective increase in IgG1 and IgG3 antibodies against LID-1 and NDO-LID in MB patients, demonstrating that these antibody isotypes are suitable for differentiation between MB and PB patients. ROC curve analysis indicates an improved capacity for diagnosing MB leprosy patients using the detection of IgG antibodies, particularly the IgG1 isotype, specific to LID-1 and NDO-LID over the performance levels attained with NDO-HSA. CONCLUSIONS Our findings indicate that serological tests based on the detection of antigen-specific IgG1 antibodies are a useful tool to differentiate MB from PB patients, and indicate the enhanced performance of the LID-1 and NDO-LID antigens in the serodiagnosis of leprosy.

Unveiling healthy carriers and subclinical infections among household contacts of leprosy patients who play potential roles in the disease chain of transmission

Leprosy transmission still occurs despite the availability of highly effective treatment. The next step towards successfully eliminating leprosy is interrupting the chain of transmission of the aetiological agent, Mycobacterium leprae. In this investigation, we provide evidence that household contacts (HHCs) of leprosy patients might not only have subclinical infections, but may also be actively involved in bacilli transmission. We studied 444 patients and 1,352 contacts using anti-phenolic glycolipid-I (PGL-I) serology and quantitative polymerase chain reaction (qPCR) to test for M. leprae DNA in nasal swabs. We classified the patients according to the clinical form of their disease and the contacts according to the characteristics of their index case. Overall, 63.3% and 34.2% of patients tested positive by ELISA and PCR, respectively. For HHCs, 13.3% had a positive ELISA test result and 4.7% had a positive PCR test result. The presence of circulating anti-PGL-I among healthy contacts (with or without a positive PCR test result from nasal swabs) was considered to indicate a subclinical infection. DNA detected in nasal swabs also indicates the presence of bacilli at the site of transmission and bacterial entrance. We suggest that the concomitant use of both assays may allow us to detect subclinical infection in HHCs and to identify possible bacilli carriers who may transmit and disseminate disease in endemic regions. Chemoprophylaxis of these contacts is suggested.

Mycobacterium leprae virulence-associated peptides are indicators of exposure to M: leprae in Brazil, Ethiopia and Nepal

Silent transmission of Mycobacterium leprae, as evidenced by stable leprosy incidence rates in various countries, remains a health challenge despite the implementation of multidrug therapy worldwide. Therefore, the development of tools for the early diagnosis of M. leprae infection should be emphasised in leprosy research. As part of the continuing effort to identify antigens that have diagnostic potential, unique M. leprae peptides derived from predicted virulence-associated proteins (group IV.A) were identified using advanced genome pattern programs and bioinformatics. Based on human leukocyte antigen (HLA)-binding motifs, we selected 21 peptides that were predicted to be promiscuous HLA-class I T-cell epitopes and eight peptides that were predicted to be HLA-class II restricted T-cell epitopes for field-testing in Brazil, Ethiopia and Nepal. High levels of interferon (IFN)-γ were induced when peripheral blood mononuclear cells (PBMCs) from tuberculoid/borderline tuberculoid leprosy patients located in Brazil and Ethiopia were stimulated with the ML2055 p35 peptide. PBMCs that were isolated from healthy endemic controls living in areas with high leprosy prevalence (EChigh) in Ethiopia also responded to the ML2055 p35 peptide. The Brazilian EChigh group recognised the ML1358 p20 and ML1358 p24 peptides. None of the peptides were recognised by PBMCs from healthy controls living in non-endemic region. In Nepal, mixtures of these peptides induced the production of IFN-γ by the PBMCs of leprosy patients and EChigh. Therefore, the M. leprae virulence-associated peptides identified in this study may be useful for identifying exposure to M. leprae in population with differing HLA polymorphisms.

Molecular typing of Mycobacterium leprae strains from northern India using short tandem repeats.

Background & objectives: Due to the inability to cultivate Mycobacterium leprae in vitro and most cases being paucibacillary, it has been difficult to apply classical genotyping methods to this organism. The objective of this study was therefore, to analyze the diversity among M. leprae strains from Uttar Pradesh, north India, by targeting ten short tandem repeats (STRs) as molecular markers. Methods: Ninety specimens including 20 biopsies and 70 slit scrappings were collected in TE buffer from leprosy patients, who attended the OPD of National JALMA Institute for Leprosy and Other Mycobacterial Diseases, Tajganj, Agra, and from villages of Model Rural Health Research Unit (MRHRU) at Ghatampur, Kanpur, Uttar Pradesh. DNA was extracted from these specimens and ten STRs loci were amplified by using published and in-house designed primers. The copy numbers were determined by electrophoretic mobility as well as sequence analysis. Phylogenetic analysis was done on variable number of tandem repeats (VNTRs) data sets using start software. Results: Diversity was observed in the cross-sectional survey of isolates obtained from 90 patients. Allelic index for different loci was found to vary from 0.7 to 0.8 except for rpoT for which allelic index was 0.186. Similarity in fingerprinting profiles observed in specimens from the cases from same house or nearby locations indicated a possible common source of infection. Such analysis was also found to be useful in discriminating the relapse from possible reinfection. Interpretation & conclusions: This study led to identification of STRs eliciting polymorphism in north Indian strains of M. leprae. The data suggest that these STRs can be used to study the sources and transmission chain in leprosy, which could be very important in monitoring of the disease dynamics in high endemic foci.

Considerações sobre o envolvimento da cavidade bucal na hanseníase / Considerations on oral cavity involvement in leprosy

As lesões causadas pela proliferação do Mycobacterium leprae (M. leprae) foram significantemente reduzidas nos últimos anos com a detecção precoce de casos novos.Consequentemente, as lesões maxilofaciais e da mucosa bucal passaram ser cada vez menos relatadas. A despeito de ser menos evidente, a infecção da cavidade bucal pelo M. leprae pode revelar detalhes importantes a respeito da transmissibilidade e imunopatogenia da hanseníase.A associação entre a infecção da mucosa bucal e a perda óssea alveolar, bem como a participação da resposta imune local na proteção contra a doença, ainda são tópicos de pesquisa não explorados totalmente. Infelizmente, o tratamento da hanseníase não possibilitou a prevenção total de suas seqüelas, nem impediu transmissão dessa doença. As razões acima justificam rever algumas perguntas não respondidas do passado.

Lesions secondary to Mycobacterium leprae (M. leprae) proliferation have been significantly abbreviated in recent years due to early detection of new cases of leprosy. Consequently, oral and maxillofacial lesions have been each time less reported. In spite of been less evident, the infection of oral cavity by M. leprae may reveal important details regarding the transmission and immunopathology of leprosy. The association between oral mucosal infection and alveolar bone loss, as well as the role of local immune response in protection against this pathogen, are still not fully explored. Unfortunately, the treatment of leprosy did not allow the full prevention of sequels neither hindered the transmission of this disease. The reasons above justify reviewing some unanswered questions of the past.

Prevalencia de lepra en Costa Rica, de 1998-2003 / Prevalence of leprosy in Costa Rica between 1998 and 2003

Se realizó un estudio de prevalencia de lepra durante el período 1998 al año 2003, con el objetivo de caracterizar la distribución de esta enfermedad en el país. Los casos fueron pacientes con un diagnóstico de lepra o enfermedad de Hansen confirmado por un médico, que estuviera consultando en los establecimientos de salud de la Caja Costarricense Seguro Social, CCSS, durante el período de estudio y pacientes que abandonaron el tratamiento sin curarse. Se excluyeron los casos curados, fallecidos y extranjeros no residentes en el país. Mediante búsqueda activa en la CCSS se identificaron los expedientes de los pacientes de 1998 al 2003 y los reportes de laboratorio de biopsia de piel y linfa cutánea confirmados por lepra. Del Ministerio de Salud, MS, se obtuvieron los casos notificados y del Instituto Nacional de Estadísticas y Censos los datos de población. Se elaboró un formulario que incluyó variables clínicas, epidemiológicas, de laboratorio y socioeconómicas. El análisis se realizó según provincia, cantón, grupos de edad y sexo. Se calculó la tasa de prevalencia, de detección de casos y porcentajes. Se identificaron, 114 expedientes en los servicios de salud pero sólo 89 casos, que representaron una prevalencia de 0,3 casos por diez mil habitantes. La enfermedad afectó a los mayores de 15 años, y a los hombres un 65 por ciento, 58. Puntarenas presentó la prevalencia por provincia más alta: 0,6X10000 habitantes, 23. Todos recibieron medicamentos para el tipo lepromatoso y en el Hospital San Juan de Dios se atendieron el 60 por ciento de ellos. El 28 por ciento reingresó a los servicios. Desde 1998, el MS no había realizado investigaciones epidemiológicas de los casos, ni seguimiento a sus contactos. El personal de los servicios de salud no cuenta con protocolos, ni normas de tratamiento clínico. Costa Rica eliminó la lepra como problema de salud pública, menos de 1 caso por diez mil habitantes, desde 1995...

A study of the prevalence of leprosy was conducted during the time period comprised between the years 1998 to 2003 with the purpose of characterizing the distribution of this illness in the country. Cases were those patients with a diagnosis of leprosy or Hansen´s disease confirmed by a physician which had consulted in the health establishments of the Costa Rican Social Security System (hereby, abbreviated as C.C.S.S.) during the study period as well as patients who abandoned treatment without having achieved a cure. Cases which were cured, who died or foreigners were excluded. By means of an active search in the CCSS, health records of patients between 1998 and 2003 were identified as well as their laboratory reports of skin biopsy and cutaneous lymph confirmatory of the diagnosis. Notified cases were obtained from the Ministry of Health and population data from the National Institute on Statistics and the Census. A form with clinical as well as epidemiological, laboratory and socio-economic variables was designed. Analysis was carried out by geographical area (both province as well as county), age groups and sex. A prevalence rate was calculated as well as a case-detection rate and the respective percentages. While 114 cases were identified in the health services, only 89 were included in our study which represents a prevalence of 0,3 cases per 100,000 inhabitants. The illness affected mainly those older than 15 years of age; 65 percent of the cases were male. The province of Puntarenas showed the highest prevalence with 0,6 per 10,000 inhabitants. All these patients received treatment for the lepromatous type of leprosy and the San Juan de Dios Hospital took care of 60% of them. 28% of the cases relapsed...

M. leprae typing by genomic diversity of clinical isolates in Korea / 나학회지

Recently, PCR-based typing would be of great value for Epidemiological investigation to identify infectious source of leprosy, understand transmission pattern, and distinguish between relapse & re-infection. Variable TTC DNA repeats in non-coding region and 6bp(GACATC) tandem repeats in rpoT gene revealed PCR products of different size may be useful to investigate the epidemiology of leprosy. Authors have typed clinical isolates of M. leprae in Korea using difference of TTC DNA repeats in non-coding region and 6bp(ACATCG) tandem repeats in rpoT gene. Of the sequence analysis of isolates(M. leprae) of 52 patients(44 Koreans, 8 foreigners; Bangladesh, Indonesia, Philippine, Sri Lanka, Thailand) M. leprae with 12 TTC repeats was showed most common(13 cases, 29.5%) in 44 Korean isolates and 42 Koreans(95.5% of Korean isolates) isolates demonstrated four copies of 6bp(ACATCG) tandem repeats in rpoT gene and the isolates with three copies were found in 2 Koreans and 8 foreigners.

Comparison of rpoT gene of M. leprae strain from korean and foreign leprosy patients / 나학회지

The variance of tandem repeats in the rpoT gene of Mycobacterium leprae was recently demonstrated. The objects of this study was to examine the proportion and distributions of the genotypes of M. leprae in Korea and to compared it with genotypes of M. leprae form foreign leprosy patients using difference of the tandem repeats. Among 101 cases, 72 isolated from Korea and 4 cases from Japan (except Okinawa) demonstrated four copies of the 6 bp tandem repeats in the rpoT gene, and three copies were found in isolates from two korean, 2 cases of Okinawa in Japan, and those from Southeast Asian countries, Peru and Paraguay. These results reveal the genetic diversity of M. leprae and the related genotype-specific distribution in the world. In this study, a more detailed explanation can be also possible regarding the transmission route of M. leprae.

The upstream sequence of Mycobacterium leprae 18-kDa gene confers transcription repression activity in orientation-independent manner

In order to understand the role of the upstream region of the Mycobacterium leprae 18-kDa gene on the gene regulation, the region was divided into two at the -50 position from the first start codon of the gene and their effect on transcription was examined by using a LacZ transcriptional reporter gene assay. The presence of each of these two regions conferred transcription repression not only on its cognate M. lepraerae 18-kDa gene promoter, but also on a heterologous promoter such as the Mycobacterium bovis BCG hsp65 gene promoter. Moreover, it was found that these regions could confer transcription repression activity in both cases in an orientation-independent manner. Thus, these results indicate that the upstream region of the M. leprae 18-kDa gene harbors transcription repression responsive element(s) acting as an operator and can be further divided into two separately functional regions, suggesting a bipartite structure of the element(s). The identification of transcription repression activity of the upstream region in the M. leprae 18-kDa gene will contribute greatly for the understanding of the 18-kDa gene regulation mechanism, and provide also useful information for the manipulation of mycobacterium gene expression.

Using the technique of amplyfication of nucleic acide arrangement (NASBA) RNA to detect the bacilli of M.leprae in leprosy patient after treatment

The detection of structural ARN in ribosom-16sr RNA existing in living M.leprae has the value of valuating the efficacy of the treatment and make prognosis of the recurrence . NASBA is performed on 91 samples of biopsy of ear tissue of 2 groups of patients immediately after the treatment of the group I and 5 years after the treatment of the group II. In group I, the positive rate of NASBA is 1/29 and in group II is 3/62. All patients with positive NASBA are in the group of numerous bacilli (MB). In finishing the treatment, there is only 1 patient of MB group who has positive NASBA. Among 60 patients who finishes multichimiotherapy, after 5 years there are 3 patients with positive NASBA

Effects of Mycobacterium leprae , M . leprae Phenolic Glycolipid - 1 and Cytokines on the Nitric Oxide Generation of RAW 264 . 7 Macrophages / 대한피부과학회지

Background: Nitric oxide(NO) has been reproted to play an important role in macrophage-mediated microbicidal capacity for a variety of intracellular pathogens. NO generation is used as an indicator of microbicidal function of macrophages. OBJECTIVE: Our purpose is to investigate the production of NO rom macrophages phagocytized with Mycobacterium leprae or M. leprae phenolic glycolipid-1(PGL-1) for the purpose of elucidating the pathogenesis of leprosy. METHODS: We used a murine macrophage cell line, RAW 264.7. Macrophages were incubated with dead M. leprae or PGL-1, respectively and then treated with interfer n-gamma(IFN-r) and/or tumor necrosis factor-alpha(TNF-a). The release of NO was determined spectrophotometrically by measuring nitrite. RESULTS: M. Leprae and PGL-1 failed to stimulnte NO secretion execept at high bacteria-to-cell rations(50:1)and at the higheat concentrat,ion(100pg/ml) of PGL-1. IFN-r or IFN-r plus TNF-a markedly stimulated macrophages phagocyt,ized with M. leprae or PGL-1 to release NO . CONCLUSION: Defective IFN-r-dependent NO production of macrophages may be an important factor in the pathogenesis of leprosy.