A T-cell diagnostic test for cystic echinococcosis based on Antigen B peptides.

Cystic echinococcosis (CE) immunodiagnosis is still imperfect. We recently set-up a whole-blood test based on the interleukin (IL)-4 response to the native Antigen B (AgB) of Echinococcus granulosus. However, AgB is encoded by a multigene family coding for five putative subunits. Therefore, the aims of this study were to analyse the IL-4 response to peptides spanning the immunodominant regions of the five AgB subunits and to evaluate the accuracy of this assay for CE diagnosis. Peptides corresponding to each subunit were combined into five pools. A pool containing all peptides was also used (total pool). IL-4 evaluated by enzyme-linked immunosorbent assay was significantly higher in patients with CE compared to those without (NO-CE subjects) when whole-blood was stimulated with AgB1 and with the total pool. Moreover, IL-4 levels in response to the total pool were significantly increased in patients with active cysts. Receiver Operator Curve analysis identified a cut-off point of 0.59 pg/mL predicting active cysts diagnosis with 71% sensitivity and 82% specificity in serology-positive CE patients. These data, if confirmed in a larger cohort, offer the opportunity to develop new diagnostic tools for CE based on a standardized source of AgB as the peptides.

Evaluation of a multi-antigen test based on B-cell epitope peptides for the serodiagnosis of pulmonary tuberculosis.

SETTING: Two sample panels: 1) 20 pulmonary tuberculosis (PTB) patients and 10 healthy subjects from a country with a low incidence of TB (Italy); and 2) 47 PTB patients and 26 healthy subjects from a country with a high incidence of TB (Morocco). OBJECTIVE: To identify a combination of Mycobacterium tuberculosis peptides useful for the serodiagnosis of active PTB. METHODS: Fifty-seven B-cell epitope peptides of M. tuberculosis were evaluated by immunoenzymatic assay and the data were analysed using logistic regression analysis and the random forest method. RESULTS: The best discriminating peptide between PTB patients and healthy subjects from the sample of the low TB incidence country was the 23 amino acid peptide of the Rv3878 protein. The sensitivity and specificity were respectively 65% and 100%. The same peptide had a sensitivity and specificity of respectively 47% and 100% for the sample from the high TB incidence country. The best combination of peptides was a pool of nine peptides which had a sensitivity of 70.2% and a specificity of 100% in the high TB incidence country. CONCLUSIONS: The 9-peptide pool can be useful in identifying patients with active PTB.

M. avium binding to HLA-DR expressed alleles in silico: a model of phenotypic susceptibility to sarcoidosis.

Sarcoidosis is a systemic granulomatosis disease of unknown origin where a number of microbes, in particular M. tuberculosis and non-tuberculous mycobacteria, have been hypothesized to play a role in disease pathogenesis, possibly through bacterial antigen-driven hypersensitivity. To test this concept, we used bioinformatic tools allowing the identification of antigenic peptides in whole microbial genomes to analyze the interaction between the expressed HLA-DR gene allelic variants and the HLA-DR immunome of all pathogenic bacteria in a population of 149 sarcoidosis affected subjects and 447 controls, all HLA-typed at high resolution. We show here that patients with the Löfgren's syndrome, express HLA-DR alleles that recognize in silico a significantly higher number of bacterial antigen epitopes compared to the control population (18,496+9,114 vs 17,954+8,742; p<0.00001), and the chronic sarcoidosis affected population (17,954+8,742; p<0.00001 vs Löfgren's and controls). Further, the analysis of the ability of the HLA-DR allele combinations expressed by the Löfgren's and the chronic sarcoidosis affected subjects to recognize M. avium epitopes demonstrates that a significantly larger number of Löfgren's are capable of top affinity recognition, compared to chronic sarcoidosis (45% vs 17%, p<0.0037). Finally, both Löfgren's and chronic sarcoidosis subjects expressed HLA-DR allele combinations capable of M. tuberculosis and M. avium epitope recognition at higher affinity than tuberculosis affected subjects (p<0.01 all comparisons). In conclusion, we propose that - at least in a subgroup of affected subjects - sarcoidosis might be part of a spectrum of granulomatous responses to several agents where the Löfgren's syndrome represents the hyper-reactive end of the spectrum while pulmonary tuberculosis and atypical mycobacterial infections might represent the opposite end.

A model of phenotypic susceptibility to tuberculosis: deficient in silico selection of Mycobacterium tuberculosis epitopes by HLA alleles.

HLA-DR allelic variants have been associated with tuberculosis (TB) susceptibility in different populations with risk ratios of 3.7 to 7.2. We hypothesized that the genetic susceptibility to TB depends upon the reduced capability of HLA-class II alleles of TB patients to bind and select peptide antigen from the Mycobacterium tuberculosis (MTB) expressed genome. To test this hypothesis, we developed a software that can predict HLA-DR restricted epitopes within the whole MTB genome based on quantitative peptide binding matrices. We analyzed the number of MTB epitopes recognized in two previously described populations of TB patients and matched controls and in a control population comprised of individuals affected by a sarcoid-like granuloma induced by beryllium and by healthy exposed controls. The number of putative epitopes within the whole MTB genome which could be bound by any HLA-DR allele (HLA-DR immunome of MTB) was 405,422 out of 1,304,277 possible 9-mers i.e., 31.08% of the global capability, instead of the expected 35%. When tested at an affinity level equivalent of the 1% of the best binder peptides, the HLA-DR alleles (HLA-DRB1*0801, *0802, *1401, *1501 and *1502) associated with TB susceptibility recognized a significantly lower mean number of MTB-epitopes (7,862 +/- 4,258) than the MTB-epitopes recognized by HLA-DR alleles (HLA-DRB1*0301, *0701, *1101, *1102, *1301 and *1302) negatively associated with TB (11,376 +/- 1,984, p<0.032). The number of epitopes bound at high affinity out of the whole MTB genome by the combination of the two HLA-DR alleles carried by each individual was lower in TB patients [TB-population 1: 11,341 +/- 908 (mean+SEM); TB-population 2: 15,303 +/- 657] than in matched healthy controls (CTR-population 1: 13,587 +/- 605, p<0.03 vs TB-population 1; CTR-population 2: 1,6841 +/- 555, p<0.04 vs TB-population 2). No difference was seen in individuals with the sarcoid-like granuloma induced by beryllium compared to the exposed healthy (beryllium-hypersensitivity: 17,593 +/- 447; controls 18,014 +/- 421; p=0.57). The data suggest that HLA-DR alleles associated with susceptibility to tuberculosis may be endowed with a reduced capability to bind at high affinity T-cell epitopes and select them for antigen presentation. The same alleles may contribute to determine the reaction to mycobacteria in non tuberculous granulomatous disorders.

Neonatal protection and preterm birth reduction following maternal group B streptococcus vaccination in a mouse model.

PURPOSE: Evaluate effects of maternal immunization in a mouse model of Group B Streptococcus (GBS) vaginal colonization using clinical isolates. MATERIALS AND METHODS: Female pregnant mice were immunized with heat-killed GBS 21 days before pregnancy and were inoculated intravaginally with GBS cultures (5 × 107 CFU twice a day for three days) from the 16th day of pregnancy. Gestation period and mice survival were monitored. Maternal anti-GBS IgG levels have been determined by ELISA analysis in vaccinated, unvaccinated mothers and newborns. RESULTS: Maternal immunization before pregnancy provided protection to newborns for three of the four GBS strains used. Evaluation of the immunogenicity showed that this vaccination induced higher levels of IgG in vaccinated compared to unvaccinated dams and the presence of antibodies in the offspring at embryonic and postnatal age, and a Th1 response and high levels of IgG2a subclass antibody and IFN-γ were detected. A significant reduction of preterm births was observed in vaccinated mothers (p< 0.05). CONCLUSIONS: Our finding suggest that vaccinated mothers could protect their progeny from GBS infection and preterm birth through passive immunization. The proposed mouse model may represent a noninvasive and effective tool to investigate pathogenetic mechanisms of GBS ascending infection and for vaccine protection studies.

Accuracy of an immune diagnostic assay based on RD1 selected epitopes for active tuberculosis in a clinical setting: a pilot study.

A previous case-control study reported that an in-vitro interferon (IFN)-gamma response to early secreted antigenic target (ESAT)-6 selected peptides was associated with active tuberculosis (A-TB). The objective of the present pilot study was to evaluate the diagnostic accuracy of this assay for TB disease in a clinical setting. An IFN-gamma ELISPOT assay was performed on samples from patients with suspected A-TB using two peptides selected from ESAT-6 protein and three peptides selected from culture filtrate 10 (CFP-10) proteins. The results were compared with those obtained by two commercially available assays approved for diagnosis of TB infection (T SPOT-TB and QuantiFERON-TB Gold) which use ESAT-6/CFP-10 (RD1) overlapping peptides. Sensitivity to the RD1 selected peptides was 70% (positive for 16 of 23 patients with microbiologically diagnosed A-TB) and specificity was 91% (positive for three of 32 controls). In contrast, the sensitivity and specificity were 91% and 59%, respectively, for T SPOT-TB, and were 83% and 59%, respectively, for QuantiFERON-TB Gold. The RD1 selected peptides assay had the highest diagnostic odds ratio for A-TB. Thus, the results suggest that an assay based on RD1 selected peptides has a higher diagnostic accuracy for A-TB in a clinical setting compared with commercially available assays based on RD1 overlapping peptides.

[Berylliosis in Italy: a case of "sarcoidosis" under the threshold limit value]. / La berilliosi in Italia: una caso di "sarcoidosi" sotto la soglia di esposizione.

BACKGROUND: Berylliosis is caused by a chronic immune reaction to beryllium; in Italy the first case of beryllium exposure-related disease was described in 1935 by Fabroni-Marradi and two additional cases of beryllium disease were subsequently described by Ambrosi and co-workers in 1968. No case has since been recognized using the standardized criteria including immunological testing. OBJECTIVES: To describe a case report of clinically significant berylliosis that occurred in a man exposed to beryllium for ten years in the workplace at concentrations below the permitted threshold limit value. METHODS: The man complained of dyspnoea, dry cough, weakness and weight loss for the past year and was at first diagnosed as suffering from sarcoidosis because of increased angiotensin converting enzyme levels, alteration of hepatic and renal functional indexes, the presence of diffused reticulo-nodular lung abnormalities with high resolution computed tomography that also showed enlarged mediastinal lymph nodes, abnormal lung physiology with reduced diffusion capacity and a bronchial biopsy showing granulomatous lesions. Because of the occupational history immunological testing and high resolution HLA class II typing were performed. RESULTS: The high response to beryllium in the lymphocytes proliferation test and the HLA typing which revealed the presence of the two susceptibility markers HLA-DPGlu69 and HLA-DRPhe47 led to a diagnosis of berylliosis. CONCLUSIONS: The importance is stressed of suspecting a diagnosis of berylliosis in the proper occupational contexts and encouraging the use of immunological tests for diagnosis, and also the need for critical revision of the permitted threshold limit values.

Beryllium binding to HLA-DP molecule carrying the marker of susceptibility to berylliosis glutamate beta 69.

Berylliosis is a chronic granulomatous disorder caused by inhalation of Be dusts that is driven by the accumulation of Be-specific CD4+ Th1-cells at disease sites. Susceptibility to berylliosis has been associated with the supratypic variant of HLA-DP gene coding for glutamate at position beta69 (HLA-DPbetaGlu69). The aim of this study was to test the hypothesis that the HLA-DPbetaGlu69 residue plays a role in the interaction with Be. To this end, soluble HLA-DP2 molecule (carrying betaGlu69) and its mutated form carrying lysine at position beta69 (HLA-DP2Lys69) were produced in Drosophila melanogaster and then used in a Be binding assays. BeSO4 (1-1000 microM) was used to compete for the binding of the biotinilated invariant chain-derived peptide CLIP (50 microM). BeSO4 was capable of compete out biotin-CLIP binding from the HLA-DP2 (IC50%: 4.5 microM of BeSO4 at pH 5.0 and 5.5 microM of BeSO4 at pH 7.5), but not from the HLA-DP2Lys69 molecule (IC50%: 480 microM of BeSO4 at pH 5.0 and 220 microM of BeSO4 at pH 7.5). Moreover, the binding of NFLD.M60, a MoAb recognizing an epitope in the HLA-DP peptide binding region, to the HLA-DP2, but not to the HLA-DP2Lys69 soluble molecules was inhibited BeSO4. NFLD.M60 binding to HLA-DP2, but not to HLA-DP2Lys69 stably transfected murine cells was also inhibited by Be both at pH 5.0 and at pH 7.5. The data indicate a direct interaction of Be with the HLA-DPGlu69 molecule, in the absence of antigen processing.

Expression of CCR5 is increased in human monocyte-derived macrophages and alveolar macrophages in the course of in vivo and in vitro Mycobacterium tuberculosis infection.

Human immunodeficiency virus (HIV) replicates more efficiently in Mycobacterium tuberculosis (MTB)-infected macrophages than in uninfected controls. We investigated whether this may be partly explained by changes in expression of CCR5 in the course of mycobacterial infection, as this molecule has been shown to be a coreceptor for HIV entry. Since the lung is the preferential organ of HIV replication in the course of tuberculosis, we preliminarily analyzed beta-chemokine receptor expression in alveolar macrophages from patients with active tuberculosis, using flow cytometry based on an MIP-1alpha ligand-biotin/avidin-FITC detection system. Increased MIP-1alpha receptor (MIP-1alphaR) expression in alveolar macrophages from infected patients was observed whereas no detectable expression could be revealed in uninfected controls. Since MIP-la can also bind CCR1 and CCR4, the presence of CCR5 mRNA was investigated in bronchoalveolar lavage (BAL) cells and detected in alveolar macrophages from tuberculosis patients only. The study was then extended to in vitro MTB-infected macrophages. Monocyte-derived macrophages (MDMs) were left to differentiate for 7 days before MTB H37Rv infection, and CCR5 expression was monitored, by using a specific monoclonal antibody, on days 1, 6, and 11 after infection. Increased CCR5 expression in MTB-infected macrophages was observed, with a peak on day 6 (64% in MTB-infected versus 33% in control cultures) and a decrease by day 11 (25% in MTB infected versus 13% in control cultures). These results show that CCR5 expression is enhanced in the course of in vitro MTB infection and during active pulmonary tuberculosis.

Use of discriminant analysis to assess disease activity in pulmonary tuberculosis with a panel of specific and nonspecific serum markers.

Several cell activation markers, acute phase reactants, enzymes, and antituberculous antibody serum levels have been proposed as possible markers to monitor disease activity in patients with tuberculosis. They have all shown limited sensitivity or specificity. The authors therefore attempted to generate a canonical variable using discriminant analysis, including sensitive and specific parameters, to be a reliable marker in classifying patients correctly during the course of pulmonary tuberculosis. The following parameters were selected: two soluble cell activation markers (soluble interleukin-2 receptor and sCD8); the levels of immunoglobulin (Ig) G and IgM antibodies against the A60 antigen complex; and the presence of specific antibodies directed to eight different A60 components, revealed by Western blot analysis. The tests were performed on sera from three groups of patients with pulmonary tuberculosis. The first group comprised 25 patients with onset tuberculosis, clinically active (OTCA), evaluated at the time of admission. The second group included 28 patients with chemotherapy-treated tuberculosis, clinically active (CTCA), 2 months after therapy had begun. The third group included 20 patients with tuberculosis, nonclinically active (TNCA), who had had at least 1 year of effective therapy. The authors obtained an 80.9% rate of correct classification for the three groups and a rate of 100% when OTCA and TNCA were compared. The patients with CTCA were scarcely differentiated and tended to be distributed into the two other groups. To improve the separation between patients with CTCA and those with OTCA, a second canonical variable was generated with a 91.7% rate of correct classification, as compared with 71% obtained using the sCD8 as the best single variable. The mean values of the last canonical variable were statistically different (Mann-Whitney test, P = .049) when stratified for acid fast bacilli-positive or negative CTCA patients (microscopic detection). Three patients, followed during the entire course their disease, were, as expected, correctly positioned with respect to the subsequent disease phases.

Evaluation of the BDProbeTec strand displacement amplification assay in comparison with the AMTD II direct test for rapid diagnosis of tuberculosis.

The BDProbeTec MTB assay for direct detection of Mycobacterium tuberculosis was evaluated in comparison with the AMTD-II assay on 94 samples from different patients with clinical suspicion of tuberculosis. Using a combination of culture on Lowenstein-Jensen medium (with or without preculture in BACTEC 9000) and clinical diagnosis as the standard, BDProbeTec MTB showed high sensitivity and specificity (96.1% and 100%, respectively), similar to AMTD-II (96.1% and 97.1%, respectively), with significantly higher sensitivity than the Ziehl-Neelsen stain for acid-fast bacilli (73%, p < 0.05).

Sensitivity and specificity of a multi-antigen ELISA test for the serological diagnosis of tuberculosis.

A new serological assay (DETECT-TB, BioChem ImmunoSystems), using three recombinant proteins and two synthetic peptides for the detection of the anti-Mycobacterium tuberculosis IgG, was evaluated using a panel of serum specimens collected from 100 tuberculosis (TB) patients and 270 controls, in comparison with a homemade ELISA test using purified protein derivative (PPD) as antigen. DETECT-TB presented a higher sensitivity (75%) compared to PPD-ELISA (56%; P < 0.01), while the specificity of each assay was similar (DETECT-TB 97%; PPD-ELISA 100%; P > 0.80). Receiver operating characteristics (ROC) analysis obtained with these data confirmed the higher level of performance of DETECT-TB in comparison with PPD-ELISA. Considering the rapidity, cost-effectiveness and simplicity of this assay, its use may provide useful clinical information aiding in the rapid diagnosis of difficult TB cases.

Serological markers predicting tuberculosis in human immunodeficiency virus-infected patients.

SETTING: Human immunodeficiency virus (HIV)-positive patients retrospectively identified at the Hospital of Bari, Italy, with diagnosis of tuberculosis (TB) (n = 30) or non-tuberculous pneumonia (n = 29). Serum samples drawn at the time of diagnosis and one year before. Anti-purified protein derivative (PPD) and anti-diacyltrehalose (DAT) serum antibodies quantified by ELISA assay. OBJECTIVE: Since TB patients with HIV infection may present with elevated serum antibodies against Mycobacterium tuberculosis, we hypothesized that TB-specific antibody markers might predict TB in these subjects. DESIGN: A retrospective study was designed to assess the presence of M. tuberculosis-specific antibodies in HIV-positive patients developing TB. RESULTS: Of 30 HIV-positive TB patients, 24 (80%) had anti-PPD or anti-DAT antibodies at the time of TB diagnosis, and 20 (67%) one year before. In a sub-population of 16 of the 30 HIV-positive subjects, positivity for anti-PPD or anti-DAT antibodies one year before TB diagnosis was higher (11/16, 69%) than for the PPD skin test (4/16, 25%, P < 0.01). Antibody tests were specific for TB since positivity rates were lower both in patients with non-tuberculous pneumonia (P < 0.01) and in those with M. avium infection (P < 0.05). CONCLUSION: Antibody markers may predict TB in HIV-positive subjects, including those with negative PPD skin test.

Role of the HLA-DP Glu 69 and the TNF-alpha TNF-alpha 2 gene markers in susceptibility to beryllium hypersensitivity.

Berylliosis is an environmental chronic inflammatory disorder of the lung caused by inhalation of beryllium dusts, characterized by the accumulation of CD4+ T cells and macrophages in the lower respiratory tract. Beryllium presentation to CD4+ T cells from patients with berylliosis results in T cell activation and these Be-specific CD4+ T cells undergo clonal proliferation and Th1-type cytokine production such as interleukin-2, interferon-gamma and tumor necrosis factor-alpha. In exposed workers, genetic susceptibility to this granulomatous disorder is associated with major histocompatibility gene and the TNF-alpha gene. The HLA-DP glutamic 69 residue was shown to be the MHC genetic marker associated with disease susceptibility; furthermore the TNF-alpha TNFA-308*2 allele was found to be independently associated with HLA-DP Glu69 in the determination of berylliosis risk.

Different cytokine production and effector/memory dynamics of alpha beta+ or gamma delta+ T-cell subsets in the peripheral blood of patients with active pulmonary tuberculosis.

Immunity to M.tuberculosis (MTB) infection consists of interactions between various T-cell subsets that control the infection and prevent further reactivation. We analysed the effector/memory T-cell dynamics and cytokines production in the peripheral blood of patients with pulmonary tuberculosis (TB). We observed that the frequency of CD4+ T-cell effectors was significantly increased during active TB, confirming a major role of this T-cell subset in TB immunity. Pre-terminally differentiated CD8+ T-lymphocytes were increased in the peripheral blood as well. In contrast, we observed a reduced number of effector mycobacteria-reactive gammadelta+ T-lymphocytes with a specific defects in reacting to mycobacterial nonpeptidic antigens, suggesting that this innate response is rapidly lost during TB infection. Nevertheless, the frequency of gammadelta+ T-cells effectors in TB patients was higher than the alphabeta+ T-cell response to peptide from MTB-ESAT-6 protein and quantitatively similar to PPD reactivity. Thus, alphabeta+ and gammadelta+ T-cell differentiation and function are differently triggered by active TB infection.

Evaluation of a western blot test as a potential screening tool for occupational exposure to Mycobacterium tuberculosis in health care workers.

Health care workers (HCWs) have a higher than average risk for contracting Mycobacterium tuberculosis (MTB) infection and tuberculosis (TB). No markers of MTB-exposure are available, and TB risk assessment is performed by tuberculin screening, identifying individuals with acquired MTB infection. This study evaluated a western blot (WB) anti-M. bovis A60 complex antibody as a MTB-exposure marker. WB reactivity was evaluated on 127 exposed and 28 non-exposed HCWs from four divisions of the Policlinico Hospital of Modena, and 140 non-exposed bacille Calmette-Guérin-vaccinated controls. Excess of occupational TB risk according to the Occupational Safety and Health Administration (OSHA) was calculated in each division. WB-positivity (%) was: (1) significantly higher in exposed HCWs compared with non-exposed (72% vs 25%, P < 0.00001), (2) highly related (r = 0.99) to OSHA risk excess in all divisions, (3) higher than non-exposed in HCWs with short (< 5 years) MTB-exposure (purified protein derivative [PPD], P > 0.18; WB, P < 0.04). PPD-positivity (%) was higher than controls only in HCWs with longer (> 5 years) MTB-exposure. The study suggests that the WB antibody might represent a more sensitive biological marker of MTB contact among exposed HCWs, related to the level of TB risk and detectable earlier than the PPD skin test, thus providing new tools for TB risk assessment in health care facilities.

Beryllium disease.

Berylliosis is an environmental chronic inflammatory disorder of the lung caused by inhalation of insoluble beryllium (Be) dusts and characterized by the accumulation of CD4+ T cells and macrophages in the lower respiratory tract. In response to Be inhalation, noncaseating granuloma formation and, eventually, fibrosis. The immunopathogenic process is maintained by Be-specific lung CD4+ T-lymphocytes. Consistent with the disease immunopathology, these Be-specific T cells have a T-helper 1 phenotype producing interleukin-2 and interferon-gamma, the macrophage-activating cytokine driving the granulomatous reaction. Previous studies have demonstrated that the glutamic acid in position 69 of the human leukocyte antigen class II b chain is strongly associated with increased susceptibility to Be in exposed workers, suggesting that human leukocyte antigen gene markers may be used as epidemiological probes to identify population groups at higher risk.

[Mycobacterium tuberculosis. From the gene to the diagnosis]. / Il microbatterio della tubercolosi. Dal gene alla diagnosi.

Pulmonary tuberculosis still in on the list of the world major health problems. Tuberculosis has not been eradicated yet, from developing countries. Furthermore, its incidence is increasing in the industrialized world, due to the human immunodeficiency virus (HIV) epidemic. In this regard, atypical clinical presentation of tuberculosis in individuals who have a deficient immune system, such as those at risk of tuberculosis because of HIV infection, makes the diagnostic process more difficult. Tuberculosis cases are often diagnosed later in HIV individuals compared to non-HIV individuals. The ensuing greater risk of contagion thus requires rapid and sensitive diagnostic protocols. In this context, several biotechnological tools have been developed that can be applied to the diagnosis of tuberculosis. M. tuberculosis genes have been cloned, monoclonal antibodies against pure proteins have been produced, thus enabling researchers to generate molecular and biochemical probes. As a consequence, DNA hybridization and DNA amplification techniques have been applied to the detection of mycobacteria, and ELISA kits of high sensitivity are been already made available. In regard to the latter, it is likely that monospecific and highly sensitive immunoassays will be developed that are directed against "active disease" immunodominant antigens. It may thus be expected that future new technologies will supplement the traditional tools for the diagnosis of tuberculosis and rapid diagnosis protocols will be available to chest clinicians in a foreseeable future.

HLA class II amino acid epitopes as susceptibility markers of sarcoidosis.

Sarcoidosis is a multisystemic disorder of unknown etiology, affecting primarily the lung and characterized by epithelioid granulomas. Disease association studies showed human leukocyte antigen (HLA) class II to be related to sarcoidosis. Initially, we studied the association of sarcoidosis with DQB1, and in the present study, we evaluated all amino acid variants of the HLA-DPB1, -DQB1, -DRB1, -DRB3, -DRB4 and -DRB5 genes to identify possible polymorphisms associated with the disease. Patients and controls were typed for class II genes to the allele level by sequence-based typing. Multiple logistic regression models showed DRAla71 and DQPhe9 to be independently associated with the disease. Subdivision of patients according to their radiographic stage resulted in identification of DRArg74 as independent associated residue in the RS I group, whereas DRAla71 and DQTyr30 were associated with RS II-IV groups. Polymorphic residues specifically associated with sarcoidosis shed new light on the characteristics of sarcoidosis-triggered peptides. Overall, pocket 9 of DQ and pocket 4 of DR seem to be the most important areas involved in the association with sarcoidosis.