Isabela Maria Bernardes Goulart

Imunopatologia da hanseníase: a complexidade dos mecanismos da resposta imune do hospedeiro ao Mycobacterium leprae

Leprosy, whose etiologic agent Mycobacterium leprae, is an illness of ample clinical and immunopathological spectrum. Its clinical manifestations are correlated with distinct immunologic forms, varying from a vigorous immune response mediated by cells to M. leprae, with Th1 standard in the tuberculoid polar region, to an absence of specific cellular response to antigens of M. leprae in the lepromatous polar region, with predominance of Th2 response and exacerbation of humoral response. It is probable that different polymorphic genes determine susceptibility to M. leprae. Additional studies are necessary to clarify the complex interactions between cytokines and the role of the phenotypic diversity of cells network that contribute to the host defense. The comprehension of such mechanisms will provide new insights for the identification of agonists and/or antagonists for pro- or anti-inflammatory effects, and also will indicate possible situations for its appropriate use in immunologic and/or immunotherapeutic interventions.

Leprosy: diagnostic and control challenges for a worldwide disease.

Leprosy is a curable disease with well-defined etiology, but lacks better diagnostic tools, preventive and therapeutic strategies. The continued application of the Ridley–Jopling clinical classification that recognizes the natural diversity of the immune response has provided the basis for understanding leprosy, and this review proposes its implementation in all Reference Centers in order to standardize the diagnostic resources, aiming at the improvement of the disease control. Due to the broad bioepidemiological aspects of infection its eradication is difficult, and proper diagnosis of the disease and the correct clinical classification are required to ensure proper treatment. Tools and markers for diagnosis and prognosis, and the novel use of nanotechnology, as well as strategies for disease control and monitoring populations at higher risk are still continuous challenges, which will be specifically reviewed with additional insights. The use of the current diagnostic tools, such as ELISA and PCR has a very limited approach for leprosy that has been considered as a marginal disease; therefore, the current diagnostic tools must be applied extensively in the routine to accumulate clinical experience in order to improve their precise application, like what has been done in many other infectious diseases. Since a vaccine for leprosy presents an unpredictable future, the proposed chemoprophylaxis of contacts (healthy carriers and/or with subclinical infection) must also be employed in referral centers of endemic countries not only to evaluate its efficacy, but also because of the favorable cost–benefit ratio, given that there is no other available approach, besides the multi-drug therapy of patients. This strategy should readily be applied as a public health policy, and may lead to a substantial breakage of the transmission chain aiming a world without leprosy.

Leprosy pathogenetic background: a review and lessons from other mycobacterial diseases.

Leprosy is a disease caused by Mycobacterium leprae that initially affects the peripheral nervous system with patients exhibiting contrasting clinical, immunological, and pathological manifestations despite minimal genetic variation among bacilli isolates. Its clinical manifestations are related to M. leprae survival, innate and acquired immune responses, and interactions between host and bacterial proteins, preventing their invasion and infection, or promoting their development and pathogenesis. The complex molecular interactions in affected individuals influenced by the pathogenetic background will be explored in this review. However, the great genetic diversity imposes difficulty for understanding disease development, and it is likely that many factors and metabolic pathways regulating the immense and contrasting symptomatology will yet be revealed. Four pathways may play a central role in leprosy, including the TLR/LIR-7, VDR, TNF-α, and TGF-β1 for which a large amount of gene polymorphisms have been described that could potentially affect the clinical outcome. Cross-talk pathways may significantly change the course of the disease, depending on the specific disequilibrium of genic homeostasis, which is highly dependent on the environment, antigens that are presented to the host cell, and specific polymorphisms that interact with other genes, external factors, and pathogen survival, culminating in leprosy occurrence. Currently, the microarray-based genomic survey of gene polymorphisms, multiple gene expression analyses, and proteomic technologies, such as mass spectrometry and phage display applied in the discovery of antigens, represent a great potential for evaluating individual responses of leprosy patients and contacts to predict the outcome and progression of the disease. At present, none of the genes is good prognostic marker; however, in the near future we may use multiple targets to predict infection and leprosy development.

Oral mucosa as a source of Mycobacterium leprae infection and transmission, and implications of bacterial DNA detection and the immunological status

Leprosy is an important health problem in Brazil despite extensive use of multidrug therapy. The nasal mucosa is the preferential site of entry and exit of Mycobacterium leprae, and although lesions have been found in the oral mucosa, its potential involvement in the transmission of leprosy bacilli has never been investigated. We investigated the presence of the M. leprae DNA in buccal swabs of leprosy patients (334) and household contacts (1288) through polymerase chain reaction (PCR), and correlated this with clinical and laboratorial evaluations. The overall positivity for patients and contacts was 18.26% and 6.83%, respectively. Subclinical infection among contacts was considered when PCR and anti-PGL-1 ELISA presented positive results. This study provides evidence that the oral mucosa may be a secondary site of M. leprae transmission and infection, and contacts with bacillary DNA may be actively involved in transmission. We have also shown that bacilli DNA is more frequently found in the oral mucosa of PB patients. Our findings have great epidemiological relevance and indicate an additional strategy for leprosy control programmes and dental clinics.

Detection of Mycobacterium leprae DNA in skin lesions of leprosy patients by PCR may be affected by amplicon size

Despite the high sensitivity and specificity of PCR for infectious disease diagnostics, it has presented low sensitivity for Mycobacterium leprae DNA detection in the tuberculoid pole (TT and BT) of leprosy. In order to demonstrate the effect of amplicon size on the efficacy of PCR detection of M. leprae DNA in skin lesions of leprosy patients, two pairs of primers targeting the M. leprae genomic DNA, RLEP3 (X17153), were used to amplify fragments of 372 and 130-bp until their PCR end-points were reached after 40 reaction cycles. Skin biopsies of leprosy lesions in 110 non-treated patients were used for bacilloscopy index (BI) analysis and PCR tests. The 130-bp fragment was detected in 73.6% of samples (81/110), and classified as TT (40%), BT (55.5%), and 100% of BB, BL and LL. The 372-bp fragment was detected in 52.7% and classified as TT (13.3%), BT (33.3%), BB (64.7%), BL (83.3%), and LL (95.2%). The BI of biopsies was positive in 39.1% of samples, classified as TT (0%), BT (2.2%), BB (64.7%), BL (91.6%), and LL (95.2%). The shorter amplicon (130-bp) has improved diagnosis by 20.9 and 34.5% in relation to the 372-bp fragment and the BI, respectively, and has shown a superior sensitivity (73.6%), specificity (100%) and accuracy (86.2%). The 130-bp amplicon could not detect % of positive BI of biopsies in BT cases. Therefore, for confirmatory diagnosis, we propose the use of PCR detection of the 130-bp genomic target, especially when the tuberculoid pole forms are considered, which has reached 51.6% of positivity in this group.

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.

Susceptibility to leprosy may be conditioned by an interaction between the NRAMP1 promoter polymorphisms and the lepromin response.

Controversial results have been achieved by attempting to associate the NRAMP1 gene with Mycobacterium leprae susceptibility as well as with the Mitsuda reaction, which represents a specific immune response to M. leprae. This study evaluated this association as well as the interaction of the polymorphism (GT)(n) in the promoter region of the NRAMP1 gene with a specific immune response to M. leprae measured by the intradermal Mitsuda test in leprosy patients and in non-consanguineous household contacts. The study aimed to evaluate the association of this gene polymorphism with resistance or susceptibility to the disease, and/or with clinical forms of the disease, in a population in an endemic area served by the State Reference Center in Sanitary Dermatology and Leprosy, Federal University of Uberlandia, MG, Brazil. Leprosy patients (90) were diagnosed according to Ridley and Jopling criteria and they grouped into multibacillary (MB) and paucibacillary (PB) patients. The control group consisted of 61 non-consanguineous contacts. NRAMP1 promoter genotypes were obtained through amplification by the polymerase chain reaction (PCR) followed by the detection through the low ionic-strength single strand conformational polymorphism (LIS-SSCP) electrophoretic technique. There were no significant differences in the allelic and genotypic frequencies for alleles 2, 3, and 4 in relation to the Mitsuda test among patients and household contacts, nor between those with MB and PB forms. However, individuals with a negative lepromin response associated with genotypes 22 and 23 presented a 7- and 8-fold greater chance of developing leprosy, respectively. Therefore, the NRAMP1 gene promoter polymorphism exhibited an interaction with the lepromin response, suggesting that allele 2 of the NRAMP1 promoter is an independent genetic factor that predisposes cells to enable pathogen survival, probably due to its low efficiency in iron transport. However, establishment of the infection and disease development may be conditioned by other immunological and genetic factors.

Identification of clinical, epidemiological and laboratory risk factors for leprosy reactions during and after multidrug therapy

This cross-sectional retrospective study evaluated 440 leprosy patients; 57% (251/440) had leprosy reactions during and/or after multidrug therapy, 80.5% (202/251) of whom presented with multibacillary leprosy. At diagnosis, positive bacterial index (BI) [odds ratio (OR) = 6.39; 95% confidence interval (CI): 4.1-10.1)] or polymerase chain reaction (PCR) (OR = 9.15; 95% CI: 5.4-15.5) in skin smears, anti-phenolic glycolipid-1 (anti-PGL-1) ELISA (OR = 4.77; 95% CI: 2.9-7.9), leucocytosis (OR = 9.97; 95% CI: 3.9-25.7), thrombocytopenia (OR = 5.72; 95% CI: 2.3-14.0) and elevated lactate dehydrogenase (OR = 2.38; 95% CI: 1.4-4.0) were potential markers for the development of reactions during treatment. After treatment, positive BI (OR = 8.47; 95% CI: 4.7-15.3) and PCR (OR = 6.46; 95% CI: 3.4-12.3) in skin smears, anti-PGL-1 ELISA (OR = 2.25; 95% CI: 1.3-3.9), anaemia (OR = 2.36; 95% CI: 1.2-4.5), leucocytosis (OR = 4.14; 95% CI: 1.5-11.6) and thrombocytopenia (OR = 3.70; 95% CI: 1.3-2.2) were risk factors for the occurrence of reactions during the study period. The identification of groups with an increased risk for developing reactions will allow for the timely development of a treatment plan to prevent nerve damage and, therefore, the appearance of the disabling sequelae associated with the stigma of leprosy.

Mycobacterium leprae DNA in peripheral blood may indicate a bacilli migration route and high-risk for leprosy onset

Leprosy epidemiological studies have been restricted to Mycobacterium leprae DNA detection in nasal and oral mucosa samples with scarce literature on peripheral blood. We present the largest study applying quantitative real-time PCR (qPCR) for the detection of M. leprae DNA in peripheral blood samples of 200 untreated leprosy patients and 826 household contacts, with results associated with clinical and laboratory parameters. To detect M. leprae DNA a TaqMan qPCR assay targeting the M. leprae ML0024 genomic region was performed. The ML0024 qPCR in blood samples detected the presence of bacillus DNA in 22.0% (44/200) of the leprosy patients: 23.2% (16/69) in paucibacillary (PB), and 21.4% (28/131) in multibacillary (MB) patients. Overall positivity among contacts was 1.2% (10/826), with similar percentages regardless of whether the index case was PB or MB. After a follow-up period of 7 years, 26 contacts have developed leprosy. Comparing the results of healthy contacts with those that become ill, ML0024 qPCR positivity at the time of diagnosis of their index case represented an impressive 14.78-fold greater risk for leprosy onset (95% CI 3.6-60.8; p <0.0001). In brief, contacts with positive PCR in blood at diagnosis of index cases are at higher risk of later leprosy onset and this marker might be combined with other prognostic markers for management of contacts, which requires further studies.

Antigen-specific assessment of the immunological status of various groups in a leprosy endemic region

BackgroundSerological tests can be important tools to assist in the diagnosis of leprosy and can contribute to an earlier diagnosis. The aim of this study was to evaluate the antibody responses against phenolic glycolipid-1 (PGL-1), natural disaccharide linked to human serum albumin via an octyl (NDO-HSA), Leprosy IDRI Diagnostic-1 (LID-1) and natural disaccharide octyl - Leprosy IDRI Diagnostic-1 (NDO-LID) in leprosy patients, household contacts of patients and the general population.MethodsEnzyme-linked immunosorbent assays were used to analyze the antigen-specific antibody responsesof 94 leprosy cases, 104 household contacts of cases and 2.494 individuals from the general population.ResultsA positive correlation was observed for the antibody responses to all antigens studied. A higher proportion of seropositivity for all antigens, along with stronger magnitude of response, was observed in multibacillary (MB) leprosy patients and household contacts of MB leprosy patients compared with the levels observed in paucibacillary (PB) leprosy patients and household contacts of PB leprosy patients. A substantial and significant positive correlation was found between seropositivity and the bacterial index for the leprosy patients. Anti-PGL-1 tests were more frequently positive than anti-NDO-HSA tests among patients with all clinical forms of leprosy and among the group of household contacts. The LID-1 and NDO-LID antigens showed a greater capacity to identify household contacts and individuals from the general population infected with M. leprae.ConclusionsTests that measure the antibody responses against LID-1, NDO-LID, NDO-HSA and PGL-1 were effective tools for the detection of patients with MB leprosy. Our data indicate that the anti-LID-1 and anti-NDO-LID responses were more effective than an anti-NDO-HSA response for the identification of individuals with subclinical infection.