Healthy Endemic Controls Research Articles

Evaluation of Plasmodium vivax ELISA for the blood screen

Plasmodium vivax malaria is the indigenous strain in the Republic of Korea (ROK). Plasmodium vivax can be transmitted through the transfusions of various blood components, which became a severe problem with the safety of blood transfusions and blood-related products in ROK. We evaluated a P.vivax-specific enzyme-linked immunosorbent assay (Genedia Malaria Ab ELISA 2.0, Green Cross, ROK) with blood samples from four groups: 251 samples from P.vivax-infected patients, 39 samples from post-treatment patients upon follow-up, 200 samples from healthy volunteers and 421 samples from domestic travellers to and from high endemic areas of ROK. The positive cases from the ELISA test were confirmed by both Giemsa microscopic and polymerase chain reaction methods. The clinical sensitivity and specificity of detecting P.vivax with ELISA test were 94.4% and 99.0%, respectively. Thirteen of 421 domestic travellers (3.0%) to endemic areas tested positive. The results indicate the effectiveness of detecting antibodies against P.vivax in blood with Genedia Malaria Ab ELISA 2.0 test in a large blood screen setting.

Detection of LD body in Peripheral Blood Buffy-coat from Suspected Kala-azar Cases of Bangladesh

The present study has been carried out in an attempt to observe the usefulness of peripheral blood buffy-coat microscopic examination for detection of LD-body from clinically suspected Kala-azar cases. Total 127 individuals are included in this study as cases and controls. Among them 67 are clinically suspected Kala-azar cases included from one of the Kala-azar endemic areas of Bangladesh and 60 are taken as endemic and nonendemic healthy controls. Peripheral venous blood and bone marrow aspirate collected and tested for LD bodies from clinically suspected Kala-azar cases in Rajshahi Medical College Hospital, during the period of July 2006 to June 2007. Serum from all cases and controls were tested by rK39 ICT for serological identification of visceral leishmaniasis. Laboratory examinations were performed in the Department of Microbiology and Immunology, BSMMU, Dhaka. Out of 67 study cases bone marrow smear microscopy for LD bodies positive in 44 (66.67%) and buffy-coat smear microscopy positive for 21 (31.34%); diagnostic sensitivity and specificity of buffy-coat smear microscopy was 47.72% and 100% in comparison with bone marrow examinations. Buffy-coat smear made form the 23 splenomegalic patients, microscopy revealed significant number 17 (73.91%) of the cases were positive for LD-body and all of the four hepato-splenomegalic patients were positive (p-value >0.05, reached from 2 test). Results of rK39 ICT for detection of visceral leishmaniasis cases show 100% sensitivity and 90% specificity. Peripheral blood buffy coat examination for LD body in splenomegalic cases of Kala-azar may replace bone marrow examinations.Bangladesh J Med Microbiol 2010; 03 (02): 27-32

Sensitivity and specificity of the Leishmania OligoC‐TesT and NASBA‐oligochromatography for diagnosis of visceral leishmaniasis in Kenya

To estimate the sensitivity and specificity of the OligoC-TesT and nucleic acid sequence-based amplification coupled to oligochromatography (NASBA-OC) for molecular detection of Leishmania in blood from patients with confirmed visceral leishmaniasis (VL) and healthy endemic controls from Kenya. Blood specimens of 84 patients with confirmed VL and 98 endemic healthy controls from Baringo district in Kenya were submitted to both assays. The Leishmania OligoC-TesT showed a sensitivity of 96.4% (95% confidence interval [CI]: 90-98.8%) and a specificity of 88.8% (95% CI: 81-93.6%), while the sensitivity and specificity of the NASBA-OC were 79.8% (95% CI: 67-87%) and 100% (95% CI: 96.3-100%), respectively. Our findings indicate high sensitivity of the Leishmania OligoC-TesT on blood while the NASBA-OC is a better marker for active disease.

Evaluation of Ex Vivo Human Immune Response against Candidate Antigens for a Visceral Leishmaniasis Vaccine

People cured from visceral leishmaniasis (VL) develop protection mediated by Th1-type cellular responses against new infections. We evaluated cytokine responses against 6 defined candidate vaccine antigens in 15 cured VL subjects and 5 healthy endemic controls with no evidence of previous exposure to Leishmania parasites. Of the 6 cytokines examined, only interferon-gamma (IFN-gamma) differentiated cured VL patients from non-exposed individuals, with cured patients mounting a significantly higher IFN-gamma response to a crude parasite antigen preparation. Among candidate vaccine antigens tested, the largest number of cured subjects recognized cysteine proteinase B, leading to heightened IFN-gamma responses, followed by sterol 24-c-methyltransferase. These two antigens were the most immunogenic and protective antigens in a murine VL model, indicating a relationship between T cell recall responses of humans cured from VL and protective efficacy in an experimental model. Further studies may help prioritize antigens for clinical development of a subunit vaccine against VL.

CCL2 Responses to Mycobacterium tuberculosis Are Associated with Disease Severity in Tuberculosis

BackgroundLeucocyte activating chemokines such as CCL2, CCL3, and CXCL8 together with proinflammatory IFNγ, TNFα and downmodulatory IL10 play a central role in the restriction of M. tuberculosis infections, but is unclear whether these markers are indicative of tuberculosis disease severity.MethodologyWe investigated live M. tuberculosis- and M. bovis BCG- induced peripheral blood mononuclear cell responses in patients with tuberculosis (TB) and healthy endemic controls (ECs, n = 36). TB patients comprised pulmonary (PTB, n = 34) and extrapulmonary groups, subdivided into those with less severe localized extrapulmonary TB (L-ETB, n = 16) or severe disseminated ETB (D-ETB, n = 16). Secretion of CCL2, IFNγ, IL10 and CCL3, and mRNA expression of CCL2, TNFα, CCL3 and CXCL8 were determined.Results M. tuberculosis- and BCG- induced CCL2 secretion was significantly increased in both PTB and D-ETB (p<0.05, p<0.01) as compared with L-ETB patients. CCL2 secretion in response to M. tuberculosis was significantly greater than to BCG in the PTB and D-ETB groups. M. tuberculosis-induced CCL2 mRNA transcription was greater in PTB than L-ETB (p = 0.023), while CCL2 was reduced in L-ETB as compared with D-ETB (p = 0.005) patients. M. tuberculosis –induced IFNγ was greater in L-ETB than PTB (p = 0.04), while BCG-induced IFNγ was greater in L-ETB as compared with D-ETB patients (p = 0.036). TNFα mRNA expression was raised in PTB as compared with L-ETB group in response to M. tuberculosis (p = 0.02) and BCG (p = 0.03). Mycobacterium-induced CCL3 and CXCL8 was comparable between TB groups.ConclusionsThe increased CCL2 and TNFα in PTB patients may support effective leucocyte recruitment and M. tuberculosis localization. CCL2 alone is associated with severity of TB, possibly due to increased systemic inflammation found in severe disseminated TB or due to increased monocyte infiltration to lung parenchyma in pulmonary disease.

ESAT6-induced IFNgamma and CXCL9 can differentiate severity of tuberculosis.

BackgroundProtective responses against Mycobacterium tuberculosis are dependent on appropriate T cell and macrophage activation. Mycobacterial antigen six kDa early secreted antigenic target (ESAT6) and culture filtrate protein 10 (CFP10) can detect M. tuberculosis specific IFNγ responses. However, most studies have been performed in non-endemic regions and to study pulmonary tuberculosis (PTB). We have studied ESAT6 and CFP10 induced cytokine and chemokines responses in PTB and extrapulmonary (EPul) TB.MethodologyIFNγ, IL10, CXCL9 and CCL2 responses were determined using an ex vivo whole blood assay system in PTB (n = 30) and EPulTB patients with limited (LNTB, n = 24) or severe (SevTB, n = 22) disease, and in healthy endemic controls (ECs). Responses to bacterial LPS were also determined.Principal FindingsESAT6- and CFP10-induced IFNγ was comparable between ECs and TB patients. Both ESAT6- and CFP10-induced IFNγ secretion was greater in LNTB than PTB. ESAT6-induced CXCL9 was greater in EPulTB as compared with PTB, with an increase in SevTB as compared with LNTB. CFP10-induced CCL2 was higher in PTB than LNTB patients. LPS-stimulated CXCL9 was greatest in SevTB and LPS-induced CCL2 was increased in PTB as compared with LNTB patients. A positive correlation between ESAT6-induced IFNγ and CXCL9 was present in all TB patients, but IFNγ and CCL2 was only correlated in LNTB. ESAT-induced CCL2 and CXCL9 were significantly associated in LNTB while correlation in response to LPS was only present in SevTB.ConclusionsESAT6 induced IFNγ and CXCL9 can differentiate between limited and severe TB infections.

Relationship between Circulating Levels of IFN‐γ, IL‐10, CXCL9 and CCL2 in Pulmonary and Extrapulmonary Tuberculosis is Dependent on Disease Severity

Protection against Mycobacterium tuberculosis infection is dependent on T cell and macrophage activation regulated by cytokines. Cytokines and chemokines produced at disease sites may be released into circulation. Data available on circulating cytokines in tuberculosis (TB) is mostly on pulmonary TB (PTB) with limited information on extrapulmonary disease (EPul-TB). We measured interferon-gamma (IFN-gamma), interkeukin-10 (IL-10), CXCL9 and CCL2 in sera of patients (n = 80) including; PTB (n = 42), EPul-TB (n = 38) and BCG vaccinated healthy endemic controls (EC, n = 42). EPul-TB patients comprised those with less severe (LNTB) or severe (SevTB) disease. Serum IFN-gamma, IL-10 and CXCL9 levels were significantly greater while CCL2 was reduced in TB patients as compared with EC. IFN-gamma was significantly greater in PTB as compared with LNTB (P = 0.002) and SevTB (P = 0.029). CXCL9 was greater in PTB as compared with LNTB (P = 0.009). In contrast, CCL2 levels were reduced in PTB as compared with LNTB (P = 0.021) and SevTB (P = 0.024). A Spearman's rank correlation analysis determined a positive association between IFN-gamma and IL-10 (rho = 0.473, P = 0.002) and IFN-gamma and CXCL9 (rho = 0.403, P = 0.008) in the PTB group. However, in SevTB, only IFN-gamma and CXCL9 were positively associated (rho = 0.529, P = 0.016). Systemic levels of cytokines are reflective of local responses at disease sites. Therefore, our data suggests that in PTB increased IFN-gamma and CXCL9 balanced by IL-10 may result in a more effective cell mediated response in the host. However, elevated inflammatory chemokines CXCL9 and CCL2 in severe EPul-TB without concomitant down modulatory cytokines may exacerbate disease related pathology and hamper restriction of M. tuberculosis infection.

The significance of blood levels of IgM, IgA, IgG and IgG subclasses in Sudanese visceral leishmaniasis patients.

We developed an ELISA test using leishmania antigenic extracts to detect antigen-specific antibody responses, including subclass and isotype analysis, in visceral leishmaniasis (VL) patients from the Sudan. A total of 92 parasitologically proven patients were compared with cutaneous leishmaniasis, schistosomiasis, malaria, onchocerciasis and tuberculosis patients, as well as with healthy endemic and non-endemic controls. Some VL patients were examined before and after chemotherapy. VL patients showed significantly higher IgG responses compared with all other groups (93.4% sensitivity, 93.7% specificity), and higher (but not significantly) IgM responses. All groups showed low IgA levels. All IgG subclasses, IgG1, 2, 3, and 4, showed higher levels in patients than all other groups, with IgG1 and IgG3 levels being significantly reduced following treatment. The rank order for specificity and sensitivity for IgG subclasses was IgG3 > IgG1 > IgG2 > IgG4.

Comparative evaluation of parasitology and serological tests in the diagnosis of visceral leishmaniasis in India: a phase III diagnostic accuracy study

In this phase III trial for diagnostics for visceral leishmaniasis (VL) in India, we compared parasitological diagnosis with several serological tests: direct agglutination test (freeze dried; DAT-FD), rK-39 strip test, rK-26 strip test and a latex agglutination test for antigen detection in urine (KAtex) in 452 subjects from the endemic regions of Bihar, India. The subjects were segregated into four categories: 230 confirmed patients, 52 probable cases, 70 non-cases and 100 healthy endemic controls. The first two groups were used for estimating sensitivity, the latter two for specificity. Sensitivity of DAT-FD was 98.9%, rK-39: 98.9%, KAtex: 67.0% and rK-26: 21.3%. Sensitivity of DAT-FD on blood taken on filter paper (DAT-FDF) was 99.3%, which was comparable with that using serum. Specificity of serological tests was comparable and high (DAT-FD and DAT-FDF: 94%, rK-39 strip test: 97%, KAtex: 99% and rK-26 strip test: 100%). The classical 'gold standard' parasitological demonstration in splenic smear performed poorly as it missed 18.4% of cases that benefited from VL treatment. Reproducibility of the serological tests between field and central laboratories was excellent (kappa = 1.0, 0.99, 0.96 and 0.94 respectively for microscopy, DAT-FD, rK-39 strip test and rK-26 strip test). A high degree of agreement was observed between DAT-FD and rK-39 strip test (kappa = 0.986). Although DAT-FD and rK-39 strip test were highly sensitive with excellent specificity, the ease of use of the latter makes it most suitable for the diagnosis of VL in the field conditions.

Comparative evaluation of freeze‐dried and liquid antigens in the direct agglutination test for serodiagnosis of visceral leishmaniasis (ITMA‐DAT/VL)

The direct agglutination test (DAT) for visceral leishmaniasis (VL) with liquid (LQ) antigen is known to be only moderately reproducible because of inter-observer and batch-to-batch variability as well as its sensitivity to temperature and shaking during transport. We evaluated a DAT with freeze-dried (FD) antigen and compared it with the LQ antigen version. Blood samples of clinical VL suspects and healthy endemic controls were collected in Sudan, Nepal and India. Both test versions were performed in duplicate in the respective countries and in the reference laboratory. Interbatch variability and stability tests were conducted and agreement was examined within and between centres on a dichotomic scale by Cohen's kappa as well as on a continuous scale through Bland-Altman plots. The FD antigen remains fully active even after storage at 45 degrees C for 24 months. Using a cut-off titre of 1:6400, the agreement between the FD and the LQ formats was excellent. The major advantages of FD antigen are its better stability at higher temperatures and its longer shelf life, which make it much more suitable than the LQ version for use in the field.

Cloning, expression, and purification of a novel recombinant antigen from Leishmania donovani

Visceral leishmaniasis (VL) is a major health problem in the tropical and subtropical regions of the world. The conventional methods for diagnosis of Old World Visceral leishmaniasis are difficult, insensitive, and hazardous. There is no recombinant antigen from old world Leishmania species which can be commercially used for rapid diagnosis. There is an urgent need for a less invasive and accurate method. Here, we report a recombinant antigen from Indian Leishmania donovani for its diagnosis. The kinesin gene of a L. donovani clinical isolate (KE16) from India was PCR amplified for cloning and the immunodominant domain was expressed in Escherichia coli. This recombinant protein or Ld-rKE16 was evaluated for serodiagnosis of Indian kala-azar by ELISA. The recombinant antigen was found to be 100% sensitive and specific for Old World VL cases from India, Pakistan, China, and Turkey. The antigen showed no cross-reactivity with sera from other endemic diseases or healthy controls. The expressed Ld-rKE16 antigen is highly specific and sensitive for diagnosing visceral and post-kala-azar dermal leishmaniasis and is ready for commercialization.

Immune profiling of leprosy and tuberculosis patients to 15-mer peptides of Mycobacterium leprae and M. tuberculosis GroES in a BCG vaccinated area: implications for development of vaccine and diagnostic reagents.

Mycobacterium leprae (ML) GroES has been shown to induce strong T cell responses in tuberculoid as well as in exposed healthy contacts of leprosy patients, and therefore this antigen has been the focus of study as a potential vaccine candidate. Paradoxically, we have shown that ML GroES also induces extremely high titres of IgG1 antibody in leprosy patients across the disease spectrum, a response associated with disease progression. IgG1 antibodies in leprosy also show a negative association with interferon-gamma, a critical T cell cytokine responsible for macrophage activation and intracellular killing of mycobacteria. We therefore queried if antibody and T cell responses were being evoked by different epitopes in ML GroES proteins. To address the issue of epitope recognition in mycobacterial diseases, we have analysed 16 peptides (15-mer peptides) spanning the entire ML and M. tuberculosis GroES protein in leprosy (n = 19) and tuberculosis (n = 9) patients and healthy endemic controls (n = 8). Our analysis demonstrates clearly that the dominant peptides evokingT cell and IgG subclass antibodies were different. The target of both T and B cell responses were cross-reactive epitopes in all groups. Differences in disease and healthy states related to the strength (mean intensity) of the T cell and antibody response. IgG1 and IgG3 antibodies were associated with disseminated disease and IgG 2 and IgG4 with disease limitation. Such comprehensive immune profiling of antigen-specific responses is critical to understanding the disease pathogenesis and also if these reagents are to be exploited for either diagnostic or vaccine purposes.

First Study of NAT1 and NAT2 Polymorphisms in Bulgarian Patients with Balkan Endemic Nephropathy (Ben) and Healthy Controls

ABSTRACTN-acetyltransferases (NAT) are involved in the metabolic activation and deactivation of environmental carcinogens, such as arylamines. Several epidemiological studies evaluated the role of NAT1 and NAT2 polymorphisms in a number of cancers. The suspected role of polycyclic aromatic hydrocarbons in the etiology of Balkan Endemic Nephropathy (BEN), as well as the high risk of uroepithelial tumor development in BEN patients necessitates clarification of the role of xenobiotic metabolizing enzymes for BEN susceptibility. The aim of this study was to determine the frequency of different polymorphisms in N- acetyltransferases in patients with BEN and to define the role NAT1 and NAT2 as candidate modifiers of BEN susceptibility.96 Bulgarian BEN patients and 112 healthy Bulgarians as controls were genotyped for NAT1 (C559T, G560A, T640G, T1088A, C1095A) and NAT2 (C282T, T341C). Rapid cycle PCR followed by melting curve analysis was used for genotyping.The frequencies of NAT1 and NAT2 polymorphisms were similar in BEN patients and controls. The data did not reveal a significantly elevated risk for BEN with NAT1 alleles *3, *10 and *11. NAT2 slow acetylators, predicted by C282T and T341C, also did not show an increased risk for BEN.For the first time the frequencies of different polymorphisms in NAT1 and NAT2 in patients with BEN and in the healthy Bulgarian population were established. No evidence for a predisposing role of N- acetyltransferases in the development of BEN was revealed. This study needs conformation in other endemic regions of Balkan countries.

Erythema nodosum leprosum is associated with up-regulation of polyclonal IgG1 antibody synthesis.

Erythema nodosum leprosum (ENL) is a serious complication of lepromatous (L) disease in leprosy. We have previously shown that of the four IgG subclasses, IgG1 and IgG3 Mycobacterium leprae-specific antibodies are significantly lower in leprosy patients during ENL reaction compared with untreated L patients. To see if this decrease results from a down-regulation of antibody synthesis during ENL, the frequency of antibody-secreting B cells (ABSC) in the blood compartment was determined by ELISPOT and related to serum immunoglobulin concentrations (microgram/ABSC). Control groups consisted of 16 patients with stable L disease and 32 healthy endemic controls (EC). Paired samples were analysed during acute ENLS (n = 13) and after the reaction had subsided to identify changes associated with ENL. Polyclonal (PC) IgG1 was elevated in L patients compared with EC (325 micrograms versus 180 micrograms). Interestingly, patients during acute ENL showed concentrations higher than L patients (419 micrograms), which decreased after the reaction had subsided (260 micrograms), indicating the transient nature of the antibody response. IgG2 antibodies showed the reverse trend and were lower during ENL and increased after the reaction had subsided. The mean concentrations for PC IgG3 and IgG4 antibodies were similar during ENL and after the reaction had subsided. Thus, decrease in M. leprae-specific IgG1 and IgG3 antibodies is not related to down-regulation of B cell responses. Identification of factors which regulate PC IgG1 antibody synthesis may provide additional insights into determinants of ENL reactions.

Field evaluation of latex agglutination test for detecting urinary antigens in visceral leishmaniasis in Sudan

A latex agglutination test to detect urinary antigens for visceral leishmaniasis (VL) was studied. In 204 patients with suspected VL, KAtex had a sensitivity of 95.2% with good agreement with microscopy smears but poor agreement with 4 different serology tests. It was also positive in 2 confirmed VL cases co-infected with HIV. In all K4tex-positive confirmed cases actively followed up after treatment, the test became negative 1 month after completion of treatment. While IC4tex had a specificity of 100% in healthy endemic and non-endemic controls, the direct agglutination test (DAT) was positive in 14% of the KAtex-negative healthy endemic controls. KAtex is a simple addition to the diagnostics of VL particularly at field level and as a complementary test for the diagnosis of VL in smear-negative cases with positive DAT results.

Cytokine profiles using whole-blood assays can discriminate between tuberculosis patients and healthy endemic controls in a BCG-vaccinated population

Whole-blood assays (WB) provide a simple tool for assessing immune cytokine profiles which may be useful laboratory predictors of early disease, aiding the evaluation of new tuberculosis (TB) vaccines and offering insights into disease pathogenesis. Although BCG does not provide protection against pulmonary disease in TB endemic areas, it does modulate immune responses to mycobacterial antigens. It is important, therefore, to evaluate any new tool in an endemic setting in both BCG vaccinees and patients with tuberculosis. We have assessed the optimal conditions in terms of dose and kinetics of those cytokines which are released early (TNF-α, IL6 and TGF-β, IL10) or (interferon [IFN]-γ and IL5) in WB cultures stimulated with mitogens and mycobacterial antigens. Responses were studied in parallel in untreated TB patients and endemic control groups. Optimal responses to LPS (predominantly monocyte-derived) occurred on days 1–2, whereas for PHA (predominantly T-cell-derived), they were on days 3–5. Secreted Mycobacterium tuberculosis culture filtrate proteins (CFP) provided a stronger stimulus for monocyte-derived cytokines compared to PPD, but both antigens were comparable for induction of T-cell cytokines. Using unpaired Student's t-tests, pulmonary tuberculosis patients (P.TB; n=11), in response to CFP, showed higher monocyte-derived IL6 ( p=0.023) and IL10 ( p=0.042) compared to endemic controls (EC; n=13), and significantly suppressed T-cell-derived IFN-γ ( p=0.028) and IL5 ( p=0.012) secretion but increased IL10 ( p=0.047) on day 5, indicating that CFP is a strong stimulus for IL10 secretion in pulmonary TB patients. Extrapulmonary TB patients (E.TB; n=6) showed no elevation of early monocyte-derived cytokines to either PPD or CFP, but showed a marked suppression of the T-cell-derived cytokines IFN-γ (PPD, p=0.015; CFP, p=0.05) and IL5 (PPD, p=0.05; CFP, p=0.015). Cytokine analysis in WB cultures is, therefore, able to discriminate between active tuberculosis infection and nondiseased healthy controls.

Dominant recognition of a cross-reactive B-cell epitope in Mycobacterium leprae 10 K antigen by immunoglobulin G1 antibodies across the disease spectrum in leprosy.

Mycobacterium leprae-specific immunoglobulin G1 (IgG1) antibodies in patients with leprosy show a direct correlation with bacterial load (rho=0.748; P<0002) suggesting that IgG1 B-cell responses may be surrogate markers of disease progression. To investigate if this upregulation was a general feature of IgG1 responses to all M. leprae (ML) antigens, we analysed responses to several recombinant purified ML heat-shock proteins (HSP). Three recombinant HSPs (ML10 K, ML 18 K and ML 65 K) were tested for their ability to induce various IgG subclasses in patients with either the lepromatous (LL/BL, n=26) or tuberculoid form (BT/TT, n=39) of the disease as well as in healthy households (HC, n=14) and endemic controls (EC=19). Our major findings were: (1) selective augmentation of IgG1 antibody responses to ML10 K; (2) recognition of a restricted number of epitopes across the disease spectrum and healthy controls by IgG1 antibodies; (3) dominant recognition of cross-reactive epitopes which were common to both ML and MT 10 K. This response was not related to contamination with endotoxin. Epitope mapping using 15-mer overlapping peptides spanning the ML 10 000 MW revealed an immunodominant IgG1 binding peptide (aa41-55) in patients as well as healthy controls. This peptide is a shared epitope with M. tuberculosis 10 K suggesting that postswitched IgG1 B cells recognizing this epitope rather than naive B cells are being expanded.

Recognition of mycobacterial antigens by sera from patients with leprosy.

Mycobacterium leprae sonic extracts prepared from armadillo-derived bacteria were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting (immunoblotting) procedures and probed with serum or plasma samples from 20 patients with lepromatous leprosy and 14 healthy endemic controls. Five proteins of 33, 25, 18, 15, and 12 kilodaltons (kDa) were frequently recognized; the 33- and 15-kDa proteins were, respectively, recognized with high intensity by 16 and 13 of the 20 samples from patients with leprosy, whereas only one healthy donor had antibodies that recognized the 15-kDa protein. By the use of M. leprae-specific murine monoclonal antibodies it was demonstrated that the 33-, 25-, and 15-kDa antigens were different from those bound by the available murine monoclonal antibodies. The 18- and 12-kDa proteins detected had molecular masses similar to those detected by the corresponding murine monoclonal antibodies. The serum and plasma samples from patients with leprosy were also used to probe Western blots of a soluble extract of M. tuberculosis. They recognized, among others, antigens with molecular weights similar to those detected in the M. leprae antigenic preparations, although with less intensity and at a lower frequency.