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.

Associations of HLA class II alleles with pulmonary tuberculosis in Thais.

Tuberculosis is an important infectious disease in Thailand. Susceptibility to tuberculosis is influenced not only by the environment but also by host genetic factors. In this study, we investigated HLA alleles in 82 patients with tuberculosis from Bangkok and in 160 normal controls. HLA-DRB1, DQA1 and DQB1 genotyping was performed by the PCR-SSO method. The frequency of HLA-DQB1*0502 was increased in tuberculosis patients compared to the normal controls (P = 0.01, OR = 2.06). In contrast, the frequencies of DQA1*0601 and DQB1*0301 were decreased in tuberculosis patients compared to the controls (P = 0.02 and P = 0.01, respectively). Our results suggest that HLA-DQB1*0502 may be involved in the development of pulmonary tuberculosis, whereas HLA-DQA1*0601 and DQB1*0301 may be associated with protection against tuberculosis.

Antibody detection for the diagnosis of tuberculous pleuritis.

SETTING: University Hospital, Bangkok, Thailand. OBJECTIVE: To evaluate the diagnostic value of antibody detection in serum and in pleural effusion as a marker of tuberculous pleuritis (TBP). DESIGN: Cross-sectional study. MATERIALS AND METHODS: One hundred and fifty-five patients with pleural effusion who underwent diagnostic evaluation at Siriraj Hospital between March 1999 and May 2000 were recruited. Samples of pleural fluid were examined biochemically, cytologically and microbiologically. Pathological examination of pleural tissue was also performed. The diagnosis of TBP or other diagnosis was made by either pathological finding or culture result. Immunochromatographic tuberculosis (ICT-TB) tests for antibody detection were then performed using the stored serum samples and effusions from those patients with a final definite diagnosis. This test detects antibodies to five secreted antigens of Mycobacterium tuberculosis, including the 38 kDa antigen. RESULTS: We investigated 67 patients with TBP, 44 with malignant pleural effusions, seven with transudates and one with cryptococcal pleuritis. The combined ICT-TB serum and effusion tests were positive in 34/67 TBP and 22/52 non-TBP patients. The sensitivity, specificity, positive predictive value and negative predictive value of the ICT-TB test were 50.7, 57.7, 60.7 and 47.6%, respectively. In 11 TBP patients with human immunodeficiency virus (HIV) co-infection, the sensitivity of the ICT-TB test was 45.6%. There was no correlation between the test positivity and culture result or duration of disease. CONCLUSIONS: The diagnostic value of antibody detection in TBP is modest in an area with intermediate prevalence of tuberculosis, independently of HIV serological status.

Detection of Mycobacterium tuberculosis from sputum specimens using one-tube nested PCR.

One-tube nested PCR was developed for diagnosis of pulmonary tuberculosis using sequences based on thel6SrRNA gene. The usage of primers 16SOL, 16SOR, 16SIL and 16SIR with optimized conditions could detect 555 bp DNA band from 21 species, 41 strains of mycobacteria and one isolate of Nocardia asteroides. It also revealed a specific 306 bp DNA band from 59 strains of M. tuberculosis complex. Cross amplification was observed in M. marinum, M. ulcerans and a few isolates of M. fortuitum complex. The developed method could detect as little as 100 fg of M. tuberculosis DNA. The PCR mixtures could be stored at 0 degrees C for 2 months or at -20 degrees C for at least 20 months without decrease in sensitivity. Using one-tube nested PCR for detection of M. tuberculosis compared with acid fast staining and culture results from 153 sputum specimens revealed 88.6% sensitivity and 89.2% specificity in smear positive specimens and 93.2% sensitivity and 85.0% specificity in culture positive specimens.

Intrapulmonary pharmacokinetics of ofloxacin in drug-resistant tuberculosis.

OBJECTIVE: To determine the lung epithelial lining fluid (ELF) and serum levels of ofloxacin in drug-resistant tuberculosis patients during treatment. DESIGN: Ten drug-resistant tuberculosis patients treated with ofloxacin containing regimens for at least 2 weeks were enrolled in the study. Subjects ingested ofloxacin 10 mg/kg and other anti-tuberculosis agents after overnight fasting. RESULTS: Serum and bronchoalveolar lavage fluid were collected at 4 hours after treatment and assayed by high performance liquid chromatography. The mean concentrations of ofloxacin in serum and ELF were 5.889 +/- 1.096 and 16.583 +/- 8.697 mg/L, respectively. The mean ratio of ELF-to-serum ofloxacin concentration was 2.825 +/- 1.275. CONCLUSION: Ofloxacin can penetrate well into the intra-alveolar fluid of patients treated for drug-resistant tuberculosis. The lung ELF concentrations were consistently higher than the minimal inhibitory concentrations of Mycobacterium tuberculosis as determined in vitro.

Can serial qualitative polymerase chain reaction monitoring predict outcome of pulmonary tuberculosis treatment?

BACKGROUND: The aim of this study was to assess the use of qualitative one-tube nested polymerase chain reaction (PCR) for monitoring the treatment response in smear-positive pulmonary tuberculosis, and the factors determining the negative conversion of sputum smear, culture, and PCR during treatment. METHODOLOGY: A total of 53 patients receiving a standard short course of chemotherapy with 24 months follow-up period after treatment cessation were included in the study. Sputum specimens were collected serially for smear, culture, and PCR until the treatment was complete. RESULTS: The conversion rate for sputum culture, smear, and PCR at 8 weeks after treatment were 84.9, 58.5, and 47.1%, and at 16 weeks of treatment were 100, 88.7, and 79.2%, respectively. At the end of the treatment period, there were four PCR persisters, one of whom had disease relapse. Only cavitary disease had an influence over the negative conversion of the smear and PCR at 8 weeks (RR 3.5, 95% CI 1.04-11.95, P=0.04 for smear; RR 5.06, 95% CI 1.196-21.42, P=0.03 for PCR). CONCLUSION: Qualitative PCR was not useful for monitoring therapy in smear-positive pulmonary tuberculosis. Mycobacterium DNA was cleared slowly in cavitary disease. The PCR may be performed at the time of treatment cessation to identify those with potential for disease relapse.

Reference spirometric values for healthy lifetime nonsmokers in Thailand.

The normal spirometric reference values for Thai people are still not yet available. The aim of this study was to establish standard spirometric equations for Thai people. Subjects 10 years of age and over were selected and their demographic distributions represented that of the population of the whole country. Inclusion criteria were strictly lifetime nonsmokers, no history of chronic cardiopulmonary disease (using a modified ATS--DLD 78 respiratory adult questionnaire), normal standard chest radiograph and unremarkable physical examination. They had to be without respiratory symptoms at the time of the study. Spirometric values were obtained by 5 turbine system 'Pony graphic' (Cosmed, Italy) spirometers which met ATS recommendations. A normal group of 2299 women and 1655 men were selected. Regression analyses using sex, height and age as independent variables were used to provide equations for predicted values. The results were: [table: see text] FVC and FEV1 from this study are close to the Chinese but are 8-20 per cent lower than the Caucasians. These predicted equations are recommended to be used for future reference values in the Thai population.

Diagnostic value of bronchoalveolar lavage and postbronchoscopic sputum cytology in peripheral lung cancer.

The objective of this study was to evaluate the value of bronchoalveolar lavage (BAL) and postbronchoscopic sputum cytology in diagnosing peripheral lung cancer. We performed a prospective study in 55 patients with lesions on chest radiographs who were suspected of having lung cancer and had non-endoscopically visible lesions on fiberoptic bronchoscopy. The sequence of procedures in all cases was BAL and transbronchial forceps biopsy. The final diagnosis of these patients were primary lung cancer in 30 patients, metatastic lung cancer in five and benign diseases in 20. In the primary lung cancer group, BAL was positive for malignant cells in 14 of the 30 patients (46.7%). In seven (50%) of these patients, the cell type diagnosed by BAL agreed with the final diagnosis. The diagnostic yield of BAL was influenced by the size and segmental location of the lesion. Bronchoalveolar lavage provided a higher diagnostic yield (46.7%) than transbronchial biopsy (16.7%). In five patients with metastatic lung cancer and 20 patients with benign disease, BAL gave negative results in all. Postbronchoscopic sputum cytology was positive in only two of the 26 patients (7.7%) from whom samples could be obtained. Bronchoalveolar lavage cytology proved to be a valuable diagnostic tool in detecting peripheral, primary lung cancer. Postbronchoscopic sputum cytology provided no significant additional information.

Hemothorax from gestational trophoblastic disease: a case report.

A 19-year-old woman who presented with right-sided hemothorax was diagnosed to have gestational trophoblastic disease with pleural metastasis. A CT scan of the chest revealed a pleural mass and serum Beta-subunit of HCG was high. After treatment the hormonal level became normal and the patient remains asymptomatic.

Peripheral gangrene in nonfatal pediatric cerebral malaria: a report of two cases.

Two Thai girls aged 10 and 13 years from the same rural area were admitted to Paholpolpayuhasena Hospital, Kanchanaburi, Thailand during the rainy season of 1989 with cerebral malaria. After several days of conventional treatment, both developed gangrene involving the feet and toes, but the lesions healed and no other complications were seen. In the absence of convincing clinical and laboratory evidence of vasculitis or coagulopathy, it seems likely that host factors (dehydration, sluggish peripheral circulation, platelet activation, subclinical intravascular coagulation) combined with strain-specific parasite factors (tissue sequestration of mature forms, rosette formation) may predispose to peripheral microvascular occlusion sufficient to produce infarction of tissue in susceptible children. However, despite the apparently ominous appearance of such lesions in a comatose child, the prognosis seems good.