Factors responsible for asthma and rhinitis among Kuwaiti schoolchildren.

OBJECTIVE: The aim of this study was to assess the prevalence of asthma among schoolchildren and to determine the level house dust mites in Kuwaiti homes and its role in asthma and rhinitis. SUBJECTS AND METHOD: The International Study of Asthma and Allergy in Children questionnaire was distributed to schoolchildren in the age group of 5-7 years, after random sampling from all the five governorates of Kuwait. The questionnaires were completed and initiated by parents with the help of the investigator and parents. House dust was collected from the bedroom floor of 549 houses in the same geographical areas where the schoolchildren were sampled, extracted and assayed for mite Der p 1 by ELISA method. RESULTS: The estimated prevalence of asthma was 22.4% and that of rhinitis was 23%. House dust collected from the bedroom floor was found to contain low levels of Der p 1. There was no significant difference (p = 0.969) in the level of Der p 1 between areas in Kuwait. The highest levels of Der p 1 ranged from 0.02 to 0.10 mg/g in 3.5% of the total samples examined. CONCLUSIONS: There is a high prevalence of asthma and rhinitis among the schoolchildren in Kuwait. However, the level of dust mitogens investigated in this study was below the level of concern, thus undermining their role in increasing asthma cases in Kuwait. Therefore, further studies are needed to understand the role of other mite allergens and other factors that contribute to the increased prevalence of allergic diseases in Kuwaiti children.

Effect of cigarette smoking on sputum smear conversion in adults with active pulmonary tuberculosis.

Although factors influencing sputum smear conversion in tuberculosis have been studied well, the effect of smoking is largely unknown. Excluding those with incomplete history or drug resistant isolates, 339 patients out of the 526 sputum positive patients registered between 1998 and 2000 were studied. At the end of 2 months, smokers and non-smokers converted with almost the same frequency to a negative sputum status {P=0.065, OR (95%CI) 0.47 (0.21-1.06)}. Although gender or age had no effect on sputum conversion with respect to smoking status, expatriate smokers as a whole showed a significant difference. (P=0.039). On applying logistic regression model, smokers with far advanced radiographic abnormalities (P<0.038) or with 3+ smear status (P=0.011), were found to have a less chance of an early smear conversion. In conclusion smoking did not influence sputum smear conversion in tuberculosis. However, as expatriate smokers and smokers with advanced disease showed a delay in smear conversion, smoking should be discouraged in patients with pulmonary tuberculosis.

In vitro cellular immune responses to complex and newly defined recombinant antigens of Mycobacterium tuberculosis.

The immunological diagnosis and development of new antituberculosis vaccines require the characterization of Mycobacterium tuberculosis antigens inducing cell-mediated immune responses. In this study, we have tested peripheral blood mononuclear cells (PBMC) from tuberculosis (TB) patients (n = 43) and Bacille Calmette-Guérin (BCG)-vaccinated healthy subjects (n = 24) for in vitro cellular immune responses, as indicated by antigen-induced proliferation and interferon (IFN)-gamma secretion, in response to a panel of complex (culture filtrate and cell wall preparations) and single recombinant antigens (Mtb8.4, Mtb9.8, Mtb9.9, Mtb32A, Mtb39A, Mtb40, Mtb41 and Ag85B) of M. tuberculosis. The results of cellular responses showed that the majority (ranging from 70 to 98%) of TB patients and healthy donors responded to the complex antigens in antigen-induced proliferation and IFN-gamma secretion assays. However, when PBMC from the same groups of patients and healthy donors were tested with the recombinant antigens, TB patients showed strong recognition (>50% responders) of Mtb9.8 and Mtb39A in proliferation assays (median SI = 6.2 and 6.4, respectively) and of Mtb9.8, Mtb39A, Mtb40 and Ag85B in IFN-gamma assays (median delta IFN-gamma= 15.5, 10.8, 7.8 and 8.1 U/ml, respectively). BCG-vaccinated healthy donors showed weak (<30% responders) to moderate (31-50% responders) responses to all of the recombinant antigens in both assays. When PBMC of a subset of TB patients (n = 11) were tested for secretion of protective Th1 cytokines [IFN-gamma, tumour necrosis factor (TNF)-alpha and interleukin (IL)-12] and the immunosuppressive cytokine IL-10, the complex CF and CW antigens as well as the recombinant Mtb9.8, Mtb9.9, Mtb40 and Ag85B induced the secretion of both types of cytokines. On the other hand, Mtb41 induced only IL-10, while Mtb8.4, Mtb32Aand Mtb39A induced the secretion of one or more of Th1 cytokines, but not IL-10. In conclusion, the recombinant antigens inducing the secretion of Th1 cytokines could be useful as subunit vaccine candidates against TB.

Restoration of mycobacterial antigen-induced proliferation and interferon-gamma responses in peripheral blood mononuclear cells of tuberculosis patients upon effective chemotherapy.

Peripheral blood mononuclear cells (PBMC) were obtained from culture-proven tuberculosis (TB) patients before and after 2 and 6 months of chemotherapy with a multi-drug regimen. PBMC were tested for cellular responses in antigen-induced proliferation and interferon-gamma (IFN-gamma) assays in response to complex mycobacterial antigens (whole cell Mycobacterium bovis BCG and M. tuberculosis, cell walls and short-term culture filtrate [ST-CF] of M. tuberculosis), fractionated ST-CF antigens (fractions F1-F10) and ESAT-6. The responses in TB patients before anti-TB treatment were low (median stimulation index (SI)=1-7, median delta IFN-gamma=0-12 U ml(-1), and percent responders=13-67%) to all the antigenic preparations. Following the administration of anti-TB chemotherapy for 2 months, there were significant (P<0.05) improvements in the cellular responses (median SI=9-76, median delta IFN-gamma=3-70 U ml(-1), and percent responders=33-100%) to most of the antigenic preparations tested. However, concanavalin A-induced proliferation responses of PBMC from the same patients before and after 2 months of chemotherapy were high and comparable (median SI=101 and 114, respectively, P>0.05, 100% responders). A further increase in IFN-gamma responses (median delta IFN-gamma=14-250 U ml(-1) and percent responders=43-100%) to mycobacterial antigens was observed in patients receiving chemotherapy for 6 months. Among the ST-CF fractions, F1 and F2 containing low molecular mass proteins resulted in the highest responses, whereas ESAT-6 showed responses comparable to these fractions only in a minority of the patients. HLA-DR typing of these patients showed heterogeneity in the expression of molecules encoded by HLA-DRB genes. These results show that effective chemotherapy restores cellular responses of TB patients to a large number of M. tuberculosis antigens, which could be useful in monitoring the efficacy of anti-TB treatment.

Influence of smoking on cutaneous delayed-type hyper sensitivity reaction by tuberculin skin test.

Tuberculin test (purified protein derivative) is currently accepted as a standard investigation used in the diagnosis of tuberculosis (TB). Although the sensitivity of the test is reliable, a substantial number of those subjected to screening for TB by such test are cigarette smokers. This study is designed to investigate the effect of smoking on cell-mediated delayed-type cellular hypersensitivity (DTH) reaction by PPD. Prospective, case-control study was conducted at the Chest and TB unit of Chest Hospital Kuwait. The study population consisted of 357 healthy volunteers serving as controls and 200 in-patients under direct medical supervision and treatment for tuberculosis as cases. The mean age was 33.69 +/- 8.6 SD; 286 were current smokers and 271 were lifetime non-smokers. PPD test was done using 2TU RT23 SSI-Denmark on all subjects. Median PPD was significant among the cases (P=0.03) between smokers and non-smokers and was highly significant among the healthy controls (P<0.001). No significant difference was seen between median pack years of smoking and PPD levels among the patient group (P=0.264) but the difference was significant among the control group (P<0.001). Univariate analysis of variance (ANOVA) on PPD, taking into account age, pack years of smoking, ethnic groups and BCG scar showed sufficient response but was not statistically significant to all these factors. Smoking habit does not appear to influence the cutaneous delayed type hypersensitivity reaction by tuberculin skin test.

Human Th1 cell lines recognize the Mycobacterium tuberculosis ESAT-6 antigen and its peptides in association with frequently expressed HLA class II molecules.

We have used a synthetic-peptide approach to map epitope regions of the Mycobacterium tuberculosis ESAT-6 antigen recognized by human T cells in relation to major histocompatibility complex (MHC) restriction. ESAT-6-specific CD4+ T-cell lines were established by stimulating peripheral blood mononuclear cells from 25 HLA-DR-typed tuberculosis patients with complete antigen in vitro. The established T-cell lines were then screened for proliferation and interferon-gamma (IFN-gamma) secretion in response to eight overlapping 20-mer peptides covering the ESAT-6 sequence. The response of the T-cell lines to ESAT-6 and peptides from a human leucocyte antigen (HLA)-heterogeneous group of donors suggested the presence of multiple epitopes and promiscuous recognition of the antigen. Analysis of antigen and peptide recognition in the presence of anti-HLA class I and class II antibodies suggested that the T-cell lines recognized ESAT-6 in association with HLA-DR and -DQ molecules. Furthermore, testing of selected T-cell lines with ESAT-6 and the peptides in the presence of autologous and allogeneic HLA-DR- and -DQ-typed antigen-presenting cells identified HLA-DR2, -DR52 and -DQ2 amongst the HLA molecules involved in the presentation of ESAT-6 and its peptides to human Th1 cells. In addition, the T-cell lines were cytotoxic for monocytes and macrophages pulsed with ESAT-6 and peptides. In conclusion, the recognition of ESAT-6 by IFN-gamma-secreting and cytotoxic CD4+ T cells in association with frequently expressed HLA class II molecules supports the application of this antigen to either specific diagnosis or subunit vaccine design.

Haemoptysis: aetiology, evaluation and outcome--a prospective study in a third-world country.

Haemoptysis is an alarming symptom, and the management depends upon the aetiology. Emergency management depends upon localization of the site of bleeding by roentgenogram, computerized chest tompgraphy and bronchoscopy. We prospectively evaluated 52 patients with haemoptysis admitted to the Chest Hospital, Kuwait for 1 year (January 1998 to December 1998) and followed them up for 1 year (January 1999 to December 1999). There were 42 males (80.8%) and 10 (19.2%) females, with a mean age of 42.2 (16-86) years. Of these, 26.9% were Kuwaiti nationals, 36.5% were Arab non-Kuwaiti nationals, 34.6% were Asians and 1.9% were other nationals. The aetiologies of haemoptysis were bronchiectasis (21.2%), old pulmonary tuberculosis with bronchiectasis (17.3%), active pulmonary tuberculosis (15.4%), bronchitis (5.8%), aspergilloma, rheumatic heart disease and carcinoid (1.9%). Aetiology could not be identified in 25% of patients. The site of bleeding in haemoptysis could not be localized by the consultants in 18 (32%) by roentgenogram. 16 patients (37%) by CT scan and 23 patients (50%) by Fibreoptic bronchoscopy. Sequential estimation of hemoglobin showed a mean of 13.56 (SD 1.9) and 13.31 (SD 1.8) after 24 h. The difference in mean was statistically significant (p<0.036). Conservative management was given in 80.8%, and embolotherapy or surgical intervention in 19.2% of patients. Only 12% of patients had recurrent haemoptysis at 1-year follow up. In conclusion, bronchiectasis and pulmonary tuberculosis were the major causes of haemoptysis in this study. Roentgenogram, CT scan and fibreoptic bronchoscopy are useful for localizing the site of bleeding. Sequential estimation of haemoglobin may be helpful in assessing the severity of haemoptysis, but larger studies are required to address this observation. The outcome of haemoptysis is generally good, with a low mortality and recurrence rate.

Identification and HLA restriction of naturally derived Th1-cell epitopes from the secreted Mycobacterium tuberculosis antigen 85B recognized by antigen-specific human CD4(+) T-cell lines.

Antigen 85B (Ag85B/MPT59) is a major secreted protein from Mycobacterium tuberculosis which is a promising candidate antigen for inclusion in novel subunit vaccines against tuberculosis (TB). The present study was undertaken to map naturally derived T-cell epitopes from M. tuberculosis Ag85B in relation to major histocompatibility complex (MHC) class II restriction. Antigen-specific CD4(+) T-cell lines were established from HLA-typed TB patients and Mycobacterium bovis BCG vaccinees by stimulation of peripheral blood mononuclear cells with purified Ag85B in vitro. The established T-cell lines were then tested for proliferation and gamma interferon (IFN-gamma) secretion in response to 31 overlapping synthetic peptides (18-mers) covering the entire sequence of the mature protein. The results showed that the epitopes recognized by T-cell lines from TB patients were scattered throughout the Ag85B sequence whereas the epitopes recognized by T-cell lines from BCG vaccinees were located toward the N-terminal part of the antigen. The T-cell epitopes represented by peptides p2 (amino acids [aa] 10 to 27), p3 (aa 19 to 36), and p11 (aa 91 to 108) were frequently recognized by antigen-specific T-cell lines from BCG vaccinees in both proliferation and IFN-gamma assays. MHC restriction analysis demonstrated that individual T-cell lines specifically recognized the complete Ag85B either in association with one of the self HLA-DRB1, DRB3, or DRB4 gene products or nonspecifically in a promiscuous manner. At the epitope level, panel studies showed that peptides p2, p3, and p11 were presented to T cells by HLA-DR-matched as well as mismatched allogeneic antigen-presenting cells, thus representing promiscuous epitopes. The identification of naturally derived peptide epitopes from the M. tuberculosis Ag85B presented to Th1 cells in the context of multiple HLA-DR molecules strongly supports the relevance of this antigen to subunit vaccine design.

Multiple epitopes from the Mycobacterium tuberculosis ESAT-6 antigen are recognized by antigen-specific human T cell lines.

A synthetic-peptide approach was used to map epitope regions of the Mycobacterium tuberculosis 6-kDa early secreted antigen target (ESAT-6) by testing human CD4(+) T cell lines for secretion of IFN-gamma in response to recombinant ESAT-6 (rESAT-6) and overlapping 20-mer peptides covering the antigen sequence. The results demonstrate that all of the ESAT-6 peptides screened were able to induce IFN-gamma secretion from one or more of the T cell lines tested. Some of the individual T cell lines showed the capacity to respond to all peptides. Human leukocyte antigen (HLA-DR) typing of the donors showed that rESAT-6 was presented to T cells in association with multiple HLA-DR molecules. The results suggest that frequent recognition of the M. tuberculosis ESAT-6 antigen by T cells from patients with tuberculosis is due to the presence of multiple epitopes scattered throughout the ESAT-6 sequence.

Detection of Mycobacterium tuberculosis complex and non-tuberculous mycobacteria by multiplex polymerase chain reactions.

The ability of two-band and three-band multiplex polymerase chain reactions to detect and differentiate Mycobacterium tuberculosis complex from non-tuberculous mycobacteria was evaluated. The polymerase chain reactions differentiated between M. tuberculosis and non-tuberculous mycobacteria when standard strains and clinical isolates of mycobacteria were tested. The sensitivity of the two-band and three-band techniques to detect M. tuberculosis in clinical specimens, compared with smear and/or culture, was 88% and 75% respectively. Although both techniques showed 100% specificity, the superior sensitivity of the two-band technique suggests that it could be more useful in the diagnosis of tuberculosis and in differentiating M. tuberculosis complex from non-tuberculous mycobacteria.

Detection of Mycobacterium tuberculosis complex and non-tuberculous mycobacteria by multiplex polymerase chain reactions

The ability of two-band and three-band multiplex polymerase chain reactions to detect and differentiate Mycobacterium tuberculosis complex from non-tuberculous mycobacteria was evaluated. The polymerase chain reactions differentiated between M. tuberculosis and non-tuberculous mycobacteria when standard strains and clinical isolates of mycobacteria were tested. The sensitivity of the two-band and three-band techniques to detect M. tuberculosis in clinical specimens, compared with smear and/or culture, was 88% and 75% respectively. Although both techniques showed 100% specificity, the superior sensitivity of the two-band technique suggests that it could be more useful in the diagnosis of tuberculosis and in differentiating M. tuberculosis complex from non-tuberculous mycobacteria

Comparison of antigen-specific T-cell responses of tuberculosis patients using complex or single antigens of Mycobacterium tuberculosis.

We have screened peripheral blood mononuclear cells (PBMC) from tuberculosis (TB) patients for proliferative reactivity and interferon-gamma (IFN-gamma) secretion against a panel of purified recombinant (r) and natural (n) culture filtrate (rESAT-6, nMPT59, nMPT64 and nMPB70) and somatic-derived (rGroES, rPstS, rGroEL and rDnaK) antigens of Mycobacterium tuberculosis. The responses of PBMC to these defined antigens were compared with the corresponding results obtained with complex antigens, such as whole-cell M. tuberculosis, M. tuberculosis culture filtrate (MT-CF) and cell wall antigens, as well as the vaccine strain, Mycobacterium bovis bacillus Calmette-Guerin (BCG). In addition, M. tuberculosis and MT-CF-induced T-cell lines were tested in the same assays against the panel of purified and complex antigens. The compiled data from PBMC and T-cell lines tested for antigen-induced proliferation and IFN-gamma secretion showed that the most frequently recognized antigen was ESAT-6, followed by MPT59, GroES, MPB70, MPT64, DnaK, GroEL and PstS. The frequency of ESAT-6 responders, as measured both by proliferation (18/19) and secretion of IFN-gamma (16/19) was comparable to the results obtained with whole-cell M. tuberculosis, MT-CF and M. bovis BCG. We also observed that most of the high responders to complex antigens recognized all of the antigens tested (covariation), demonstrating that the repertoire of human T-cell specificities induced by natural infection is directed towards several unrelated culture filtrate as well as somatic-derived protein antigens. In conclusion, the results obtained suggest that the cellular immune response in humans is directed against several important target antigens of M. tuberculosis and that some antigens, such as ESAT-6, are recognized by a high number of individuals. Such antigens represent candidates to be used for development of specific diagnostic reagents or in subunit vaccines.

Polymerase chain reaction targeting of single- and multiple-copy genes of mycobacteria in the diagnosis of tuberculosis.

Tuberculosis is a worldwide health problem of major concern. Direct detection of Mycobacterium tuberculosis in clinical specimens is the best approach to identify the causative agent. Identification of M. tuberculosis by culture is the gold standard, but the results are delayed for days to weeks. Microscopic examination of smears is quite fast, but a sample must contain a large number of M. tuberculosis (> 7.5 x 10(3) organisms/ml) for smear positivity. To diagnose tuberculosis specifically within 1 d of receiving clinical specimens, we have established multiplex polymerase chain reaction (MPCR) assays by targeting DNA fragments in the genes present in single or multiple copies in the M. tuberculosis genome. The MPCR results are available within a few hours, and the detection limit for different targets ranges between 2 and 200 organisms. The targets selected in the MPCRs could differentiate between M. tuberculosis complex and other mycobacteria from culture-grown specimens. The MPCRs were compared with microscopic examination of smears and culture in the diagnosis of tuberculosis. Coded sputum samples from suspected tuberculosis patients were tested. The codes were broken at the end of the study and the results were compared. All the samples negative for smear and/or culture were also negative by multiple-copy gene MPCRs (specificity = 100%), whereas the single-copy gene MPCR showed 98% specificity. With respect to sensitivity, compared with culture, the single-copy gene MPCR showed a sensitivity of 92%, whereas the three- and two-band multiple-copy gene MPCRs exhibited sensitivities of 87% and 93%, respectively. These results suggest that the MPCRs could be helpful in early and specific diagnosis of pulmonary tuberculosis.

Establishment and evaluation of a multiplex polymerase chain reaction for detection of mycobacteria and specific identification of Mycobacterium tuberculosis complex.

OBJECTIVE: To establish a multiplex polymerase chain reaction for detection of mycobacteria and specific identification of Mycobacterium tuberculosis complex and to evaluate the test in the diagnosis of tuberculosis. DESIGN: Three sets of primers were used to amplify 383 bp, 240 bp and 131 bp DNA fragments from the genes encoding the 65 kDa, MPB64 and the 19 kDa proteins of M. tuberculosis in a single reaction tube. Reaction conditions were optimized with respect to the requirement of DMSO, concentration of MgCl2, annealing and denaturation temperatures and number of amplification cycles. Inhibitory activity in clinical samples was identified by amplifying a 500 bp DNA fragment of the phage lambda along with the mycobacterial targets within the same reaction tube. The multiplex PCR was evaluated in differentiating M. tuberculosis complex from other mycobacteria and in the diagnosis of tuberculosis by testing clinical specimens. RESULTS: Amplification of the 383 bp DNA fragments was specific to the genus Mycobacterium. The 240 bp DNA fragment was amplified from M. tuberculosis complex and M. fortuitum and the 131 bp DNA fragment was amplified from the mycobacteria of M. tuberculosis complex and M. scrofulaceum. All the three bands were amplified only from M. tuberculosis complex. Applicability of the multiplex PCR is demonstrated in differentiating M. tuberculosis complex from other mycobacteria by using standard strains and clinical isolates. The multiplex PCR was also useful in the detection of inhibitory activity and in the identification of M. tuberculosis complex directly in clinical samples. CONCLUSION: The multiplex PCR established in this study could differentiate M. tuberculosis complex from other mycobacteria. This test may also be helpful in the early and specific diagnosis of tuberculosis.