Synergistic antigen combinations for the development of interferon gamma release assays for paucibacillary leprosy.

The development of immunodiagnostic tests for paucibacillary leprosy (PB) is based on Mycobacterium leprae specific-cell mediated immunity (CMI)/IFN-γ production. Recently, novel M. leprae protein antigens that stimulate CMI have been described. This study evaluated different M. leprae antigen combinations in whole blood assay (WBA). Five study groups were tested (20 per group): newly diagnosed, untreated PB patients and multibacillary leprosy patients (MB); household contacts of MB patients (HHC); healthy endemic controls (EC); pulmonary tuberculosis patients (TB). WBA (heparinized, 24 h 37 °C 5% CO2) were stimulated with: 10 µg/ml of each individual M. leprae recombinant protein (rML) and five combinations of rML (46f + LID-1, ML0276 + LID-1, ML2055 + ML1632 + ML2044, ML0276 + 46f, ML2055 + LID-1)-M. leprae cell sonicate (MLCS, 10 µg/ml), PHA (1 µg/ml), and PBS alone. Human IFN-γ ELISA (QuantiFERON-TB Gold/QFT-G, Cellestis) was performed using stimulated plasma (arbitrary cut-off = 50 pg/ml). Three out of five antigen combinations (46f + LID-1, ML0276 + LID-1, ML2055 + ML1632 + ML2044) were able to increase the levels of IFN-γ production in WBA in a larger number of responders among both PB leprosy and contacts. However, the magnitude of IFN-γ responses was higher among contacts. The antigen combination (46f + ML0276) stimulated IFN-γ only in symptomatic PB leprosy patients and not in asymptomatic contacts. Few controls (EC, TB) responded to combinations (0-15%), indicating the specificity of the response in an endemic area with high BCG coverage. The synergistic effect of new combinations of M. leprae proteins upon IFN-γ production in WBA indicates their potential use for the development of an interferon gamma release assay/IGRA for the diagnosis of PB leprosy.

Evaluation of various cytokines elicited during antigen-specific recall as potential risk indicators for the differential development of leprosy.

Leprosy is a dermato-neurological disease caused by Mycobacterium leprae infection that manifests across a wide range of clinical and immunological outcomes. Diagnosis is still currently based on clinical manifestations and simple tests are needed. This study investigated whether biomarkers induced by defined M. leprae proteins in 24-h whole blood assays (WBA) could discriminate active leprosy patients from at-risk contacts. Newly diagnosed, untreated paucibacillary (PB; tuberculoid leprosy/borderline tuberculoid [TT/BT]) and multibacillary (MB; borderline lepromatous/lepromatous leprosy [BL/LL]) leprosy patients, as well as healthy household contacts (HHC) of MB patients, were recruited in central western Brazil (Goiânia/Goiás). Cell-based responses to the ML0276, ML1623, ML0405, ML1632, 92f, and ML1011 antigens were measured by Luminex 14-plex assays detecting eotaxin, IFNγ, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-15, IL-17A, IL-23, IL-31, IP-10, and TNFα. Our data reinforce that IFNγ is currently the best indicator of the antigen-specific cellular immune response of TT/BT leprosy and demonstrate that the same antigens promote the secretion of IL-4 in blood from BL/LL leprosy patients. While none of the biomarkers tested could discriminate leprosy patients from HHC, our data indicate that, although most HHC antigen-specific responses are qualitatively similar to TT/BT patients, some HHC can respond similarly to BL/LL patients.

Human immunodeficiency virus type 1 and hepatitis C virus co-infection and viral subtypes at an HIV testing center in Brazil.

Human immunodeficiency virus (HIV) testing sites have been recognized recently as potential settings for hepatitis C virus (HCV) screening since both viruses share common routes of transmission. HIV and HCV prevalence, predictors, co-infection rates, and viral subtypes were studied in 592 attendants at an anonymous HIV Counseling and Testing Center in central Brazil. Anti-HIV-1 and -HCV antibodies were screened by ELISA, and Western blots were used to confirm HIV infection. Among HIV-seropositive samples, reverse transcriptase-polymerase chain reaction (RT-PCR) and nested-PCR were used to subtype HIV-1 by the Heteroduplex Mobility Analysis (HMA) and HCV by the line probe assay (INNO-LiPA). HIV and HCV seroprevalence was 3.2% (95% CI 2.0-4.9) and 2.5% (95% CI 1.5-4.0), respectively. Intravenous drug use was the risk factor most strongly associated with both HIV and HCV infections, even in a population with few intravenous drug users (n = 6); incarceration was also associated with HCV. HIV/AIDS-positive sexual partner and homosexual/bisexual behaviors were associated independently with HIV-1. The prevalence of HCV infection among HIV-positive persons was 42% (95% CI 20-66), higher than in HIV-negative persons (1.2%; 95% CI 0.5-2.5). HIV-1 subtype B was identified in the env and gag regions of the genome. HCV subtype 3a predominated among co-infected persons and one HCV subtype 1a was detected. Overall, a similar prevalence of HIV and HCV infections and a higher prevalence of HCV among HIV-positive persons were observed. Integrated HIV and HCV screening at HIV testing sites may represent a unique opportunity to provide diagnosis and prevention strategies at a single visit.

Distinct histopathological patterns in single lesion leprosy patients treated with single dose therapy (ROM) in the Brazilian Multicentric Study.

This paper aims to describe the histomorphologic features of skin biopsies of single lesion leprosy patients recruited at outpatient clinics in four Brazilian states in the Northeast (Amazonas and Rondonia), Southeast (Rio de Janeiro) and Center-West (Goiás) between October 1997 and December 1998. Patients clinically diagnosed as single skin lesion paucibacillary (SSL-PB) leprosy had a standard 4-mm punch biopsy taken from the lesion before rifampin, ofloxacin, minocycline (ROM) therapy. The features of the cellular inflammatory infiltrates, the presence of nerve involvement and acid-fast bacilli (AFB) were used to categorize SSL-PB biopsies into different histopathological groups. Two-hundred-seventy-eight (93.0%) out of 299 patients had a skin biopsy available. Seven single lesion patients were diagnosed as BL or LL leprosy types (MB) by the histopathological exams and 12 cases were excluded due to other skin diseases. Therefore, 259 patients had skin lesions with histomorphological features compatible with PB leprosy categorized as follows: 33.6% (N = 87) of the biopsies represented well-circumscribed epithelioid cell granuloma (Group 1); 21.6% (N = 56) less-circumscribed epithelioid cell granuloma (Group 2); 12.0% (N = 31) were described as mononuclear inflammatory infiltrate permeated with epithelioid cells (Group 3), and 29.7% (N = 77) had perivascular/periadnexal mononuclear inflammatory infiltrate (Group 4). Minimal/no morphological alteration in the skin was detected in only 8 (3.1%) SSL-PB patients categorized as Group 5, who were considered to have leprosy by clinical parameters. SSL-PB leprosy patients recruited in a multicentric study presented histomorphology readings comprising the whole PB leprosy spectrum but also a few MB cases. These results indicate heterogeneity among SSL-PB patients, with a predominance of well-circumscribed and less-circumscribed epithelioid cell granulomas (Groups 1 and 2) in the sites studied and the heterogeneity of local cellular immune response.

Single lesion paucibacillary leprosy: baseline profile of the Brazilian Multicenter Cohort Study.

In Brazil, there is little information about the clinical and epidemiological characteristics of paucibacillary, single skin lesion leprosy patients (SSL-PB). Only recently has the official notification system distinguished leprosy patients with a single lesion as a clinical entity, for whom the single-dose ROM (rifampin, ofloxacin and minocycline) regimen has been recommended. In this paper, we describe the baseline clinical features and the immunological background of a multicenter cohort of SSL-PB leprosy cases enrolled between December 1997-1998. Patients were recruited at health centers located in the following regions: Southeast = Rio de Janeiro; North = Amazon and Rondônia states and Center-West = Goiás state. Eligible cases were newly detected, untreated single-lesion leprosy patients without thickened nerve involvement, and were assessed by clinical, bacilloscopic and histopathological exams. The Mitsuda skin test and anti-PGL-I serology (ELISA) were also performed. Of the 299 SSL-PB leprosy patients, 259 (86.6%) fulfilled the criteria for single-dose ROM intervention. Our results showed that patients recruited from different sites had similar features, considering the clinical and immunological profiles. There was a predominance of adults (mean age 32.4; S.D. = 16.0), and a BCG scar was detected in 76.7% of the children (< or = 15 years old). Only 7 cases were diagnosed as the multibacillary type, representing less than 3% of the patients being misclassified. Our data indicate that in Brazil SSL-PB case ascertainment based on clinical and bacilloscopic criteria can be accurately defined under a routine control program; 75.0% of SSL-PB cases were Mitsuda positive (> or = 5 mm) and seropositivity for anti-PGL-I was detected in 17.3% of the patients. These data are compatible with effective cell-mediated immunity and low bacillary load, suggesting favorable clinical outcomes for most SSL-PB participants of this cohort.

Assessment of anti-PGL-I as a prognostic marker of leprosy reaction.

The anti-phenolic glycolipid-I (PGL-I) assay as currently applied for leprosy is conceived as an early marker of asymptomatic infection, early disease diagnosis and cure monitoring. Its use as a prognostic marker of reaction is still a matter of controversy. We conducted a case-control study to investigate whether IgM and IgG anti-PGL-I antibodies could discriminate patients at increased risk of developing reactions. Eligible cases were untreated leprosy patients at the onset of type 1 and type 2 reactions recruited from among 600 concurrent, newly detected, untreated leprosy patients attending an outpatient clinic in central Brazil. For the patients with reaction, approximately the same number of leprosy cases without reaction matched as to bacterial index (BI), age and gender were randomly selected. Individuals without clinical leprosy were evaluated as healthy controls. Sera from type 1 reaction (N = 43) and type 2 reaction (N = 26) patients were tested by an ELISA using PGL-I synthetic disaccharide-BSA antigen and 1:300 sera dilution (cut-off point > or = 0.2 OD). Antibody profiles were evaluated by exploratory data analysis and reverse cumulative distribution curves. The IgG anti-PGL-I response did not have a defined pattern, being detected only at low levels. Our results indicate that leprosy patients, independently of their reactional status, produce high levels of IgM anti-PGL-I, demonstrating a strong correlation between the magnitude of antibody response and the BI. Patients with a higher BI were at least 3.4 times more prone to produce an antibody response compared to healthy controls.

Trypanosoma cruzi stocks isolated from acute Chagas disease patients lead to lethal murine infection.

Ten Trypanosoma cruzi stocks recently isolated from patients in acute and chronic phases of Chagas disease were inoculated to susceptible (A/Sn) mice. The mice were inoculated with 10(4) trypomastigotes intraperitoneally and monitored for parasitaemia and mortality for up to 300 d. The results demonstrated that (i) T. cruzi stocks isolated from patients in the acute phase killed animals, while stocks from patients in the chronic phase did not; (ii) survival curves differed statistically among mice infected with lethal stocks, and (iii) parasite burden did not affect the mortality rate of mice.

Leishmania major-specific CD8+ T cells are inducers and targets of nitric oxide produced by parasitized macrophages.

Lines of Leishmania major-specific CD8+ T cells were derived from the lymph nodes and spleens of CBA mice, immune following resolution of a primary infection, 7 days after secondary challenge with viable L. major. Specific stimulation of these CD8+ T cells by bone marrow-derived macrophages infected with L. major led to the release of interferon-gamma by CD8+ T cells and nitric oxide by macrophages. Interestingly, the nitric oxide released by bone marrow-derived macrophages down-regulated the production of interferon-gamma by specifically activated CD8+ T cells. The proliferation and long-term maintenance of these parasite-specific CD8+ T cells was impaired by the nitric oxide produced by stimulating infected macrophages as a result of cytokines released by activated stimulating infected macrophages as a result of cytokines released by activated CD8+ T cells. Taken together, the results indicate that L. major-specific CD8+ T cells are sensitive to the toxic effect of the nitric oxide that they induce.

Leishmania major infection in BALB/c mice: protection or exacerbation by treatment with different doses of BCG.

The effect of live bacillus Calmette-Guérin (BCG), administered intraperitoneally to BALB/c mice, upon the development of lesions induced by subcutaneous infection with Leishmania major was examined. Lesions in mice given 10(7) BCG colony-forming units (CFU) 9 days before challenge with L. major were less severe and contained significantly fewer parasites than those of similarly infected control mice not given BCG. This effect of treatment with high doses of BCG upon the development of leishmanial lesions was observed using L. major promastigotes and amastigotes, whether or not 10(6) live BCG was included in the parasite inoculum. Lesions in mice given 5 x 10(4) BCG CFU 14 days before infection with L. major contained significantly fewer parasites than those of control mice not given BCG. Mice treated with low doses of BCG and infected with an L. major inoculum also comprising BCG exhibited larger lesions that contained more parasites. Interestingly, compared to naive mice infected with L. major, infection of naive mice with L. major mixed with live BCG consistently led to the development of more severe lesions that contained higher numbers of parasites. No correlation was found between the effect of BCG on the development of lesions induced by L. major and the amounts of IFN gamma, IL5 and TNF produced after in vitro antigenic challenge of either draining lymph node or spleen cells, the antigenic challenge being either live BCG or live L. major.

Trypanosoma cruzi and experimental Chagas' disease: characterization of a stock isolated from a patient with associated digestive and cardiac form

Um novo estoque de Trypanosoma cruzi isolado de paciente chagásico crônico, com a forma digestiva e cardiaca da doença, foi caracterizado através de infecçäo experimental em camundongos isogênicos A/Sn suscetíveis à infecçäo chagásica. As curvas de parasitemia mostraram picos de até 1,7x10**6 parasitas/ml näo se observando mortalidade até 300 dias após infecçäo. anticorpos da classe IgM foram encontrados na fase aguda até 40 dias e também na fase crônica e IgG foi detectada nas fases aguda e crônica. O exame histopatológico mostrou miotropismo para músculo liso do tubo digestivo e cardíaco

Trypanosoma cruzi and experimental Chagas' disease: characterization of a stock isolated from a patient with associated digestive and cardiac form.

A new Trypanosoma cruzi stock isolated from a patient in the chronic phase of Chagas' disease with the digestive and cardiac form of the disease was characterized by experimental infection in isogenic, susceptible, A/Sn strain mice. Parasitemia curves showed up to 1.7 x 10(6) parasites/ml and no mortality was observed up to 300 days post infection. Specific IgM was found in mice in the acute phase up to 40 days and also in the chronic phase. IgG antibodies were detected in the acute and chronic phase. Histopathology examination demonstrated myotropism to the digestive tract muscle layers and to the heart.

Isotype of antibodies responsible for immune lysis in Trypanosoma cruzi infected mice.

Serum obtained from mice infected with Trypanosoma cruzi have antibodies able to induce immune lysis in presence of complement. Gel filtration in Sephadex G-200 and affinity chromatography with rabbit anti-mouse IgG and protein A-Sepharose showed that the antibodies responsible for the immune serum lytic activity are exclusively located in Ig of IgG isotype, including IgG1, IgG2a, IgG2b and probably IgG3. It is suggested that antibodies responsible for protection against T. cruzi infection and antibodies responsible for immune lysis are the same.