Evaluation of antibody detection against the NDO-BSA, LID-1 and NDO-LID antigens as confirmatory tests to support the diagnosis of leprosy in Yunnan province, southwest China.

Although multidrug therapy (MDT) has been widely used for the treatment of leprosy for nearly 40 y, the disease remains a public health concern in some areas. The early detection of leprosy cases is vital to interrupt Mycobacterium leprae transmission, but currently diagnosis is typically achieved during the recognition of clinical symptoms by professional staff performing physical examinations in conjunction with microbiological assessment of slit skin smears (SSSs) and histopathology. In the last 10 y, serum antibody detection tests have emerged to aid leprosy diagnosis. Here we evaluated the ability of antigens NDO-BSA and LID-1 (ML0405 and ML2331) and the conjugate of these, NDO-LID, to detect antibodies in the sera of 113 leprosy patients and 166 control individuals in Yunnan province in southwest China. We found that each antigen was readily detected by sera from multibacillary (MB) patients, with sensitivities of 97.3%, 97.3% and 98.6% for NDO-BSA, LID-1 and NDO-LID, respectively. Even among paucibacillary (PB) patients the antigens detected antibodies in 74.4%, 56.4% and 69.2% of serum samples, respectively. Receiver operating characteristics (ROC) curve analysis indicated that, irrespective of the leprosy case classification as MB or PB, the detection efficiency obtained with NDO-LID was better than that obtained with the other two antigens (with LID-1 being a slightly better than NDO-BSA). Our results indicate the utility of NDO-LID in assisting in the diagnosis of PB and MB leprosy patients and that these antibody detection assays represent powerful diagnostic tools. We suggest that could be implemented into the procedures of local health centres in leprosy-endemic regions to assist in earlier diagnosis.

Correlates of immune exacerbations in leprosy.

Leprosy is still a considerable health threat in pockets of several low and middle income countries worldwide where intense transmission is witnessed, and often results in irreversible disabilities and deformities due to delayed- or misdiagnosis. Early detection of leprosy represents a substantial hurdle in present-day leprosy health care. The dearth of timely diagnosis has, however, particularly severe consequences in the case of inflammatory episodes, designated leprosy reactions, which represent the major cause of leprosy-associated irreversible neuropathy. There is currently no accurate, routine diagnostic test to reliably detect leprosy reactions, or to predict which patients will develop these immunological exacerbations. Identification of host biomarkers for leprosy reactions, particularly if correlating with early onset prior to development of clinical symptoms, will allow timely interventions that contribute to decreased morbidity. Development of a point-of-care (POC) test based on such correlates would be a definite game changer in leprosy health care. In this review, proteomic-, transcriptomic and metabolomic research strategies aiming at identification of host biomarker-based correlates of leprosy reactions are discussed, next to external factors associated with occurrence of these episodes. The vast diversity in research strategies combined with the variability in patient- and control cohorts argues for harmonisation of biomarker discovery studies with geographically overarching study sites. This will improve identification of specific correlates associated with risk of these damaging inflammatory episodes in leprosy and subsequent application to rapid field tests.

The oral cavity in leprosy: what clinicians need to know.

Leprosy is a chronic infectious disease caused by Mycobacterium leprae, a bacillus that has a tropism for skin and peripheral nerves. Leprosy treatment is based on a multidrug therapy established by the World Health Organization in 1982 and, despite its widespread use, Brazil ranks second worldwide in numbers of cases. Oral involvement in leprosy has been poorly described in the literature, and few studies have shown that although the bacillus is found in mucosa, specific leprosy lesions are rare and affect patients with advanced stages of the disease. This review aimed to assess the literature on oral manifestations in leprosy and the aspects involving oral cavity in leprosy pathogenesis.

Colonization by Helicobacter pylori of leprosy patients in Spain: immunomodulation to low molecular weight antigens of H. pylori.

We studied the prevalence of Helicobacter pylori in patients with leprosy and the effects of co-infection on the immune response to Helicobacter antigens in the polar groups of leprosy (lepromatous and tuberculoid). We showed that there is no difference in the prevalence of H. pylori in patients with leprosy as compared to a non-leprosy population. We also demonstrated that the immune response to low molecular weight H. pylori antigens (35, 26 and 19 kDa) differs in patients with lepromatous as compared to those with tuberculoid leprosy. In lepromatous leprosy, we show that there is a higher prevalence of the 35 and 26 kDa antigens, but a lower prevalence of the 19 kDa antigen. These immunological results are consistent with previous histopathological studies illustrating a more severe gastrointestinal inflammation in lepromatous patients; importantly, a response to the 35 kDa antigen is recognized as a marker for the development of ulcerative disease.

Evaluation of ID-PaGIA syphilis antibody test.

BACKGROUND: Laboratory diagnosis of syphilis is usually accomplished by serology. There are currently a large number of different commercial treponemal tests available that vary in format, sensitivity and specificity. AIM: To evaluate the ID-PaGIA Syphilis Antibody Test as an alternative to other specific treponemal tests for primary screening or confirmation of diagnosis. METHODS: Serum samples from healthy adults (n = 100) were used for detection of specificity of ID-PaGIA. To evaluate sensitivity of ID-PaGIA serum samples (n = 101) from patients with confirmed or suspected syphilis were tested for syphilis antibodies with FTA-Abs IgM, ID-PaGIA, ELISA IgM and TPHA tests. RESULTS: No false-positive results were found with ID-PaGIA. Sensitivity of various treponemal tests was the following: FTA-Abs IgM: 95.5%, ID-PaGIA and ELISA IgM: 94%, and TPHA 75%. The positive and negative predictive values of ID-PaGIA were 100 and 89.5%, respectively. CONCLUSIONS: Compared with other treponemal tests ID-PaGIA has excellent sensitivity and specificity.

Immunogenicity and protective efficacy of "Mycobacterium w" against Mycobacterium tuberculosis in mice immunized with live versus heat-killed M. w by the aerosol or parenteral route.

As the disease caused by Mycobacterium tuberculosis continues to be a burden, there is a concerted effort to find new vaccines to combat this problem. One of the important vaccine strategies is whole bacterial vaccines. This approach relies on multiple antigens and built-in adjuvanticity. Other mycobacterial strains which share cross-reactive antigens with M. tuberculosis have been considered as alternatives to M. bovis for vaccine use. One such strain, "Mycobacterium w", had been evaluated for its immunomodulatory properties in leprosy. A vaccine against leprosy based on killed M. w is approved for human use, where it has resulted in clinical improvement, accelerated bacterial clearance, and increased immune responses to Mycobacterium leprae antigens. M. w shares antigens not only with M. leprae but also with M. tuberculosis, and initial studies have shown that vaccination with killed M. w induces protection against tuberculosis in Mycobacterium bovis BCG responder, as well as BCG nonresponder, strains of mice. Hence, we further studied the protective potential of M. w and the underlying immune responses in the mouse model of tuberculosis. We analyzed the protective efficacy of M. w immunization in both live and killed forms through the parenteral route and by aerosol immunization, compared with that of BCG. Our findings provide evidence that M. w has potential protective efficacy against M. tuberculosis. M. w activates macrophage activity, as well as lymphocytes. M. w immunization by both the parenteral route and aerosol administration gives higher protection than BCG given by the parenteral route in the mouse model of tuberculosis.

Study of cross-reactivity of Mycobacterium leprae reactive salivary IgA with other environmental mycobacteria.

Majority of the endemic population is exposed to Mycobacterium leprae but very few develop disease. Humoral mucosal immune response mediated through M. leprae reactive salivary antibodies has been suggested to be quite important in the protective immunity. As the endemic population is also exposed to many environmental mycobacteria, we tested saliva from 121 subjects for the cross-reactivity of the M. leprae reactive salivary antibodies to mycobacteria like M. smegmatis and M. phlei. Saliva samples were treated with these two mycobacteria prior to testing M. leprae reactive antibodies by ELISA. In 59 subjects (48.76%), original and cross-reacted saliva showed same absorbance values suggesting no cross-reactivity. 26 subjects (21.49%) showed less than 25% drop in the OD values whereas 21 subjects (17.4%) showed 25% to 50% drop after reacting saliva with the mycobacteria. 15 subjects (12.4%) showed more that 50% drop in OD. The data suggest that though in half of subjects antibodies did not cross-react with mycobacteria tested, there were subjects where antibodies showed cross-reactivity to mycobacteria suggesting that positive salivary M. leprae reactive IgA response could be to some extent due to exposure to environmental mycobacteria and it could also be protective against M. leprae.

Leprosy-specific B-cells within cellular infiltrates in active leprosy lesions.

Leprosy is a spectral disease with polar lepromatous and tuberculoid forms correlating with enhanced humoral and cell-mediated immunity, respectively, against Mycobacterium leprae and the borderline forms, borderline lepromatous, midborderline, and borderline tuberculoid showing in-between clinical and immunological characteristics. Histopathologically, the cellular infiltrates of leprosy lesions show predominantly the presence of interacting T-cells and antigen presenting cells like macrophages, whereas the presence of B-cells has only been sporadically reported. The present study demonstrates by immunohistochemical techniques the presence of B-cells, including plasma cells, in active lesions from lepromatous leprosy, skin smear negative borderline lepromatous, and paucibacillary borderline tuberculoid leprosy. Furthermore, the study demonstrates the in situ production of M leprae-specific antibodies from BT lesions using an organotypic skin explant culture model. Finally, analysis of the cytokine release profile in supernatants of lesional organotypic skin cultures showed a microenvironment conducive to the differentiation and maturation of B-cells. The results demonstrate the presence of different functionally active B-cell stages within lesions of patients with leprosy, including borderline tuberculoid patients, which could secrete anti-M leprae-specific antibodies. However, their role in leprosy pathology remains to be elucidated.

B-cell immune responses in HIV positive and HIV negative patients with tuberculosis evaluated with an ELISA using a glycolipid antigen.

The diagnostic value of the PGL-Tb1 enzyme-linked immunosorbent assays (ELISA) was established following a survey study using sera from 220 Tuberculosis patients (including 69 HIV coinfected) and 324 controls. A higher percentage (76.8%) of the HIV-seropositive compared to the HIV-seronegative (58.9%) TB patients were ELISA positive (p=0.02) with a specificity of 94%. In HIV-positive TB patients, ELISA sensitivity was identical for all sites of disease and antibody levels were not affected by the CD4+ counts, PPD results, age or bacterial yield. Combining data for both the smear microscopy and ELISA maximized sensitivity. The kinetics of anti-PGL-Tb1 antibody was evaluated in cohort studies using sera collected before, during and after treatment for clinical TB for 79 TB patients (including 39 HIV coinfected). Statistically significant ELISA signals were observed in 51.3% of HIV-seropositive TB patients prior to the diagnosis of clinical TB and elevated antibody levels persisting 18 months after the end of antituberculous chemotherapy. Asymptomatic development of antibody also occurred in 22.7% of a cohort of 44 HIV-positive patients with a high risk of tuberculosis, but no correlation was found between persisting elevated antibody levels and progression to active disease. This antibody response in absence of disease, might reflect the control of an incipient tuberculosis infection by antituberculous prophylaxis or through an improved protective immune response associated with antiretroviral therapy.

Risk factors for developing leprosy--a population-based cohort study in Indonesia.

We identified risk factors associated with increased yearly incidence rates of leprosy in five island populations. Age, sex, household size and Mycobacterium leprae-specific antibodies as well as contact factors were studied. Of 94 index patients (patients diagnosed in 2000), 43 (46%) were classified as multibacillary (MB), 17 (19%) were seropositive for PGL-1 [corrected] antibodies and 6 (7%) had M. leprae DNA in nasal swabs as determined by polymerase chain reaction (PCR) testing. All PCR positive patients were also seropositive. Forty-four of 4903 initially symptom free persons developed leprosy within 4 years, giving an incidence rate of 298 per 1000 person-years. Men had a 22 times higher risk [95% confidence interval (CI): 1.2-4.1] of developing leprosy than women. People living in households with more than 7 members had a 3.1 times higher risk (95% CI: 1.3-7.3) than households of 1-4 members. Persons who were seropositive in 2000 had a 3.8 times higher risk (95% CI: 1.1-12.6) than seronegative persons. Household contacts of MB patients had an adjusted hazard ratio (aHR) of 4.6 (95% CI: 1.6-12.9) and household contacts of PCR positive patients an aHR of 9.36 (95% CI: 2.5-34.9) compared with non-contacts. Patients with PCR positive nasal swabs, suggesting nasal excretion of M. leprae, are probably the patients with the highest transmission potential. Since all index patients who were PCR positive were also seropositive, serology seems an adequate tool to identify these patients. Preventing seropositive persons from becoming seropositive and infectious patients might break the chain of transmission.

[Detection of antibodies to M. leprae in saliva of lepric patients].

Saliva of 116 lepric patients were scanned for antibodies in ELISA (with M. leprae cultivated in vitro used as antigen) in order to work out an invasion-free diagnostic tool for lepra. The ELISA findings for "saliva-serum" pairs from same patients showed an increased level of antibodies both in serum and saliva in 39.7% of cases, and matching of results (positive and negative ones) was observed in 73.3% of patients. The increasing level of specific antibodies as observed in intensification of the lepric process in blood serum occurred simultaneously with its increase in saliva. The invasion-free diagnostics of lepra is promising for examinations of contact persons and of population in endemic regions as well as for evaluating the efficiency of the antilepric therapy.

Human immunodeficiency virus type 1 (HIV-1) and Mycobacterium leprae co-infection: HIV-1 subtypes and clinical, immunologic, and histopathologic profiles in a Brazilian cohort.

Co-infections with human immunodeficiency virus (HIV) and Mycobacterium leprae represent unique opportunities to investigate the interaction of both pathogens. We determined the immunologic, virologic, and histopathologic characteristics of 22 co-infected Brazilian patients (median age = 38 years, 81.8% males, 72.2% with paucibacillary leprosy, and 95.4% with acquired immunodeficiency syndrome). The HIV-1 subtypes B and BF predominated in envelope and gag heteroduplex mobility analysis. Borderline tuberculoid (BT), tuberculoid, lepromatous, and indeterminate morphology with CD3+, CD8+, and CD68+ cell distributions compatible with leprosy patients not infected with HIV were observed. Histologic evidence of nerve damage was observed in BT lesions. IgM antibody to M. leprae-specific phenolic glycolipid I was not detected. Two of six co-infected patients monitored during highly active antiretroviral therapy (HAART) developed a leprosy type 1 reaction after an increase in CD4+ cells, suggesting an immune restoration phenomenon. Clinical, immunologic, histopathologic, and virologic features among these HIV-leprosy co-infected patients indicate that each disease progressed as in single infection. However, HAART immune reconstitution may trigger potential adverse effects, such as leprosy acute inflammatory episodes.

Prevalence and incidence density of Mycobacterium leprae and Trypanosoma cruzi infections within a population of wild nine-banded armadillos.

A total of 415 wild 9-banded armadillos from the East Atchafalaya River Levee (Point Coupee, LA) were collected over 4 years to estimate the incidence and prevalence of Mycobacterium leprae and Trypanosoma cruzi and to discern any relationship between the 2 agents. M. leprae infections were maintained at a high steady prevalence rate year to year averaging 19%. T. cruzi antibody prevalence remained relatively low, averaging 3.9%, and varied markedly between years. Prevalence rates were independent, with only 3 armadillos coinfected with both agents. M. leprae incidence density ranged from 0.47 to 3.5 cases per 1,000 animal-days, depending on case definition, confirming active intense transmission of M. leprae among armadillos. No incident T. cruzi cases were found. These infections seem to occur independently and may be used in comparisons to understand better factors that may influence transmission of these agents.

Progress towards development of immunoassays for detection of Mycobacterium leprae infection, employing 35kDa antigen: an update.

The 35 kDa antigen of Mycobacterium leprae is a membrane component that contains both B and T-cell stimulating epitopes. Monoclonal antibodies, primarily specific to M. leprae, have been developed against this antigen. Moreover, this antigen has been genetically engineered. Using recombinant 35 kDa antigen and/or a monoclonal antibody against an epitope on 35 kDa, a variety of antibody/antigen detecting tests have been described for detection of M. leprae infection. 35 kDa protein also stimulates peripheral blood mononuclear cells (PBMCs) from the majority of paucibacillary (PB) patients. Approaches using combined antibody and T cell are discussed.

Prevalence and incidence density of Mycobacterium leprae and trypanosoma cruzi infections within a population of wild nine-banded armadillos

A total of 415 wild 9-banded armadillos from the East Atchafalaya River Levee (Point Coupee, LA) were collected over 4 years to estimate the incidence and prevalence of Mycobacterium leprae and Trypanosoma cruzi and to discern any relationship between the 2 agents. M. leprae infections were maintained at a high steady prevalence rate year to year averaging 19 per cent. T. cruzi antibody prevalence remained relatively low, averaging 3.9 per cent, and varied markedly between years. Prevalence rates were independent, with only 3 armadillos coinfected with both agents. M. leprae incidence density ranged from 0.47 to 3.5 cases per 1,000 animal-days, depending on case definition, confirming active intense transmission of M. leprae among armadillos. No incident T. cruzi cases were found. These infections seem to occur independently and may be used in comparisons to understand better factors that may influence transmission of these agents.

Large unilamellar vesicles as trehalose-stabilised vehicles for vaccines: storage time and in vivo studies.

Liposomes, as a pharmaceutical formulation must display a long shelf life. The recombinant heat-shock protein from Mycobacterium leprae (18-kDa hsp) or its N-acylated derivative, when entrapped within or externally associated with large unilamellar vesicles, acts as a T-epitope source. Freeze-fracture electron microscopy shows unequivocally that trehalose avoids aggregation and fusion of these vesicles. Formulations containing trehalose retained up to 98% of the entrapped protein. The highest antibody level is obtained with formulations containing trehalose. The adjuvant effect depends on the liposomal membrane integrity.