Prevalence of Diabetes in Tuberculosis Patients in Kathmandu Valley, Nepal.

In this descriptive cross-sectional study, the data on the prevalence of diabetes mellitus (DM) among tuberculosis (TB) patients at the Urban Directly Observed Treatment Centers in the Kathmandu, Bhaktapur, and Lalitpur districts of Nepal were collected. The prevalence of DM was assessed in 67 previously treated TB (PTTB) and 214 new TB patients. DM was diagnosed in 8 PTTB and 20 new TB patients. Clinical interviews identified 14 patients with DM, rapid blood glucose test was used to diagnose DM in 4 patients, and oral glucose tolerance test was used to diagnose DM in another 4 patients. Impaired glucose tolerance and impaired fasting glycemia were observed in 8 and 5 patients, respectively. The 18-24-year age group had the largest number of new TB patients (82, 38.3%). However, the incidence of DM among TB patients was higher in the >35-year age group. Moreover, DM was diagnosed in 24.2% of PTTB patients and in 23.1% of new TB patients. To determine the impact of DM screening in TB patients, a larger number of samples should be analyzed. DM screening for patients with TB is expected to start in developing countries. This should be initiated by conducting clinical interviews about DM and glucose tests using rapid kits.

Coordinated In Vitro Release of Granulysin, Perforin and IFN-γ in TB and HIV/TB Co-Infection Associated with Clinical Outcomes before and after Anti-TB Treatment.

Granule-associated killing molecules released from cytotoxic T lymphocytes participate as a crucial step in immunity against tuberculosis (TB), but the role of coordinated production remains controversial. Coordinated release of effector molecules in vitro after stimulating peripheral blood mononuclear cells (PBMCs) of active TB or HIV/TB coinfection patients with PPD, purified protein derivative of tuberculin and avirulent Mtb, H37Ra, an attenuated strain were investigated in association with clinical outcomes. Perforin, granzyme-B, granulysin and IFN-γ were measured using ELISA. Before anti-TB treatment, PBMCs of TB stimulated with PPD or H37Ra released higher perforin, granzyme-B, and granulysin levels than in HIV/TB and released significantly higher IFN-γ (p = 0.045, p = 0.022). Granulysin positively correlated with perforin in TB (p = 0.042, r = 0.385), HIV/TB coinfection (p = 0.003, r = 0.941) after PPD stimulation, and after H37Ra stimulation in TB (p = 0.005, r = 0.549), but negatively correlated with granzyme B in TB (p = 0.042, r = -0.386), HIV/TB coinfection (p = 0.042, r = 0.754) were noted. After anti-TB treatment, increased levels of perforin, granulysin and IFN-γ in TB or HIV/TB upon PPD or H37Ra stimulation, and decreased granzyme-B levels after PPD (p = 0.003) or H37Ra (p = 0.028) stimulation in TB were observed. These results suggest that granulysin may act synergistic with perforin and IFN-γ in TB, indicating its crucial function in host immunity to tuberculosis. Future studies with larger numbers of patients ought to be conducted in the future.

Circulating granulysin levels in healthcare workers and latent tuberculosis infection estimated using interferon-gamma release assays.

BACKGROUND: Granulysin (GNLY) is produced by human lymphocyte subpopulations and exhibits antimicrobial activity against Mycobacterium tuberculosis. We examined the association between GNLY levels in blood and latent tuberculosis (TB) infection. METHODS: Latency of TB infection among Vietnamese healthcare workers was estimated using interferon-gamma release assays (IGRA), and serum GNLY concentrations were measured using enzyme-linked immunosorbent assays. The levels of GNLY expression in whole blood and the presence of GNLY alleles with the exon-4 polymorphism rs11127 were also determined using PCR-based methods. RESULTS: Among 109 study participants, 41 (37.6 %) were IGRA positive and had significantly lower serum GNLY concentrations compared with IGRA-negative participants (adjusted mean, 95 % confidence interval; 2.03, 1.72-2.44 vs. 2.48, 2.10-2.92 ng/ml, P = 0.0127; analysis of covariance). Serum GNLY concentrations and TB antigen-stimulated interferon-gamma values were weakly inversely correlated (r = -0.20, P = 0.0333). Serum GNLY concentrations varied with GNLY genotypes even after adjustment for gender and age (adjusted P = 0.0015) and were moderately correlated with GNLY expression in blood cells (r = 0.40, P < 0.0001). In subsequent analyses, low serum GNLY concentrations were significantly associated with IGRA status (adjusted odds ratio and 95 % confidence interval, 0.55 and 0.31-0.98, respectively), although GNLY genotype and mRNA levels were not. CONCLUSIONS: Decreased GNLY, presumably at the protein level, is linked to the immunological condition of latent TB infection.

Dynamics of immune parameters during the treatment of active tuberculosis showing negative interferon gamma response at the time of diagnosis.

OBJECTIVES: In the performance of interferon gamma release assays (IGRA) for the diagnosis of tuberculosis (TB) infection, false-negative results are a major obstacle. In active TB patients, treatment-dependent changes of the negative test results remain unknown. METHODS: The treatment course of 19 smear-positive/culture-confirmed TB patients who had IGRA-negative results by QuantiFERON-TB in-tube (QFT-IT) method at the time of diagnosis (month 0) in a previous study, were monitored in the present study. Blood was further collected at months 2 and 7, and the concentrations of 27 immune molecules were measured in the plasma supernatants remaining after performing the IGRA, using a suspension array system. RESULTS: After initiating treatment, eight of the 19 QFT-IT-negative patients showed positive conversion, whereas the remaining 11 (58%) did not; the interferon gamma (IFN-γ) response was restored to levels higher than 1 IU/ml in only three of the eight patients with positive conversion. Plasma concentrations of interleukin 1 receptor antagonist, interleukin 2, and interferon gamma-induced protein 10 remained low after Mycobacterium tuberculosis-specific antigen stimulation at months 2 and 7 in the continuously QFT-IT-negative group, whereas the parameters were elevated only in the transiently QFT-IT-negative group. CONCLUSIONS: It was demonstrated that a majority of active TB patients showing negative IGRA results did not regain sufficient levels of immune responsiveness despite successful treatment.

Mycobacterium tuberculosis strains spreading in Hanoi, Vietnam: Beijing sublineages, genotypes, drug susceptibility patterns, and host factors.

Beijing genotype strains are divided into two major sublineages, ancient (atypical) and modern (typical) types, but their phenotypic variations remain largely unknown. Mycobacterium tuberculosis (MTB) isolates from Hanoi, Vietnam, were analyzed by single-nucleotide polymorphisms and spoligotyping. Patient information and drug susceptibility patterns were obtained. Genetic clustering was assessed by variable number of tandem repeat (VNTR) locus sets. Multivariate analysis was also performed to investigate factors possibly associated with these sublineages. Of the 465 strains tested, 175 (37.6%) belonged to the ancient Beijing sublineage and 97 (20.9%) were of the modern Beijing sublineage. Patients with the Beijing genotype were significantly younger and more undernourished than those with non-Beijing genotype. The proportion of clustered strains calculated from 15 locus-optimized mycobacterial interspersed repetitive units [optimized-(MIRU)15]-, optimized-MIRU24-, optimized-MIRU28-, Japan Anti-Tuberculosis Association (JATA)15-, and JATA18-VNTRs were 55.7%, 49.2%, 33.8%, 44.5%, and 32.0%, respectively. Ancient and modern Beijing genotype strains were more frequently clustered than non-Beijing genotype strains, even when using VNTR sets with high discriminatory power. Isoniazid and streptomycin resistance tended to be more frequently observed in ancient Beijing strains than in modern Beijing strains and others. Our findings may provide insight into area-dependent differences in Beijing family strain characteristics.

Association between tuberculosis recurrence and interferon-γ response during treatment.

OBJECTIVES: We investigated the relationship between tuberculosis recurrence and Mycobacterium tuberculosis antigen-stimulated interferon-gamma (IFN-γ) responses during treatment. METHODS: Plasma IFN-γ levels in active pulmonary tuberculosis patients (n = 407) were analyzed using QuantiFERON-TB Gold In-Tube™ (QFT-IT) at 0, 2, and 7 months of the 8-month treatment received from 2007 to 2009 and the patients were followed up for another 16 months after treatment. Risk factors for recurrence were assessed using the log-rank test and Cox proportional hazard models. Random coefficient models were used to compare longitudinal patterns of IFN-γ levels between groups. RESULTS: QFT-IT showed positive results in 95.6%, 86.2%, and 83.5% at 0, 2, and 7 months, respectively. The antigen-stimulated IFN-γ responses varied significantly during the treatment course (P < 0.0001). Unexpectedly, positive-to-negative conversion of QFT-IT results between 0 and 2 months was significantly associated with earlier recurrence (adjusted hazard ratio, 5.57; 95% confidence interval, 2.28-13.57). Time-dependent changes in IFN-γ levels were significantly different between the recurrence and nonrecurrence groups (P < 0.0001). CONCLUSIONS: Although the IGRA response varies individually, early response during the treatment course may provide an insight into host immune responses underlying tuberculosis recurrence.

Age-dependent association of mannose-binding lectin polymorphisms with the development of pulmonary tuberculosis in Viet Nam.

Mannose-binding lectin (MBL) binds to pathogens and induces complement-mediated opsonophagocytosis. Although the association between MBL2 polymorphisms and tuberculosis (TB) has been studied in various populations, the results are controversial. We explored the stages of TB associated with MBL2 polymorphisms. X/Y (rs7096206) and A/B (rs1800450) were genotyped in 765 new patients with active pulmonary TB without HIV infection and 556 controls in Hanoi, Viet Nam. The MBL2 nucleotide sequences were further analyzed, and plasma MBL levels were measured in 109 apparently healthy healthcare workers and 65 patients with TB. Latent TB infection (LTBI) was detected by interferon-gamma release assay (IGRA). The YA/YA diplotype, which exhibited high plasma MBL levels, was associated with protection against active TB in younger patients (mean age = 32)≦ 45 years old (odds ratio, 0.61; 95% confidence interval, 0.46-0.80). The resistant diplotype was less frequently found in the younger patients at diagnosis (P = 0.0021). MBL2 diplotype frequencies and plasma MBL levels were not significantly different between the IGRA-positive and -negative groups. MBL2 YA/YA exhibited a protective role against the development of TB in younger patients, whereas the MBL2 genotype and MBL levels were not associated with LTBI. High MBL levels may protect against the early development of pulmonary TB after infection.

Refusal of oral polio vaccine in northwestern Pakistan: a qualitative and quantitative study.

BACKGROUND: Refusal of the oral polio vaccine (OPV) is a difficulty faced by the Polio Eradication Initiative (PEI) in multiple endemic areas, including the Khyber Pakhtunkhwa Province (KPP), Pakistan. In 2007, we investigated community perceptions of the OPV and estimated the prevalence of OPV refusal in three districts in Swat Valley, KPP, a polio-endemic area. METHODS: Qualitative data concerning community perceptions were collected by focus group discussions among lady health workers (LHWs) and mothers with children <1 year old and by key informant interviews with local health managers and officials. Quantitative data collection followed using a questionnaire survey of 200 LHWs and a cluster sampling survey of 210 mothers (per district) with children <1 year old. RESULTS: The qualitative assessments identified the grounded theory of OPV refusal involving facts known by the residents that are related to the OPV (too frequent OPV campaigns, an OPV boycott in northern Nigeria in 2003 and that birth control is viewed as is against Islam), the local interpretations of these facts (perceptions that OPV contained birth control or pork, that OPV was a foreign/central plot against Muslims, and that the vaccination was against the Hadith and the fate determined by God) and different manifestations of OPV refusal. Among the three districts studied, the proportion of LHWs who encountered OPV refusal ranged from 0 to 33%, whereas among the districts, the proportions of mothers unwilling to give OPV to their children ranged from 0.5 to 5.7%. Refusal of other injectable vaccines was almost equally prevalent for reasons that were very similar. CONCLUSIONS: The PEI needs to reflect local value system in the path to polio eradication in the studied districts in the Swat Valley. The religious and cultural values as well as the interpretation of the international political situation are of particular importance.

Clonal expansion of Mycobacterium tuberculosis isolates and coexisting drug resistance in patients newly diagnosed with pulmonary tuberculosis in Hanoi, Vietnam.

BACKGROUND: Newly diagnosed patients without anti-tuberculosis (TB) treatment histories have not often undergone drug susceptibility testing (DST), but have received the standard treatment regimen without information about their DST profiles in many countries with inadequate resources. METHODS: We collected 346 clinical isolates from previously untreated patients with smear-positive active TB in Hanoi, the capital of Vietnam. Of these, 339 were tested for susceptibility to four first-line anti-TB drugs, including isoniazid (INH), rifampicin (RMP), streptomycin (SM), and ethambutol (EMB), using the proportion method. A pyrazinamidase (PZase) test was used to assess pyrazinamide (PZA) resistance. Results of the culture-based drug susceptibility tests were confirmed by those from reverse hybridization-based line probe assays (LiPAs) that detected mutations associated with RMP, INH, PZA, and fluoroquinolone (FQ) resistance. To investigate a diversity of these strains, IS6110-probed restriction fragment length polymorphisms (RFLPs) were analyzed. Nucleotide sequences for furA-katG and fabG1-inhA operons, transcription units responsible for INH resistance, were also determined. RESULTS: Of the isolates tested, 127 (37.5%) were resistant to at least one of the four drugs, which included 93 (27.4%) isolates that were resistant to INH. RFLP analysis identified four clusters defined by similarity of the band patterns, which accounted for 46.1% of the tested isolates. Among the clustered isolates, 37.7% were resistant to INH, most of which (85.4%) carried a g944c mutation, which causes an S315T amino acid substitution, in the katG gene. CONCLUSIONS: Our results suggest that drug-resistant strains, particularly those with INH resistance characterized by a single mutation, S315T, are spreading in Hanoi, Vietnam. When RMP resistance is combined with this setting, patients are not easily cured by conventional short-term treatment. We will need to carefully monitor these trends and search for the origins and transmission routes of these strains.

Primary drug-resistant tuberculosis in Hanoi, Viet Nam: present status and risk factors.

INTRODUCTION: Resistance of Mycobacterium tuberculosis (MTB) to anti-tuberculosis (TB) drugs presents a serious challenge to TB control worldwide. We investigated the status of drug resistance, including multidrug-resistant (MDR) TB, and possible risk factors among newly diagnosed TB patients in Hanoi, the capital of Viet Nam. METHODS: Clinical and epidemiological information was collected from 506 newly diagnosed patients with sputum smear- and culture-positive TB, and 489 (96.6%) MTB isolates were subjected to conventional drug susceptibility testing, spoligotyping, and 15-locus variable numbers of tandem repeats typing. Adjusted odds ratios (aORs) were calculated to analyze the risk factors for primary drug resistance. RESULTS: Of 489 isolates, 298 (60.9%) were sensitive to all drugs tested. Resistance to isoniazid, rifampicin, streptomycin, ethambutol, and MDR accounted for 28.2%, 4.9%, 28.2%, 2.9%, and 4.5%, respectively. Of 24 isolates with rifampicin resistance, 22 (91.7%) were MDR and also resistant to streptomycin, except one case. Factors associated with isoniazid resistance included living in old urban areas, presence of the Beijing genotype, and clustered strains [aOR = 2.23, 95% confidence interval (CI) 1.15-4.35; 1.91, 1.18-3.10; and 1.69, 1.06-2.69, respectively). The Beijing genotype was also associated with streptomycin resistance (aOR = 2.10, 95% CI 1.29-3.40). Human immunodeficiency virus (HIV) coinfection was associated with rifampicin resistance and MDR (aOR = 5.42, 95% CI 2.07-14.14; 6.23, 2.34-16.58, respectively). CONCLUSION: Isoniazid and streptomycin resistance was observed in more than a quarter of TB patients without treatment history in Hanoi. Transmission of isoniazid-resistant TB among younger people should be carefully monitored in urban areas, where Beijing strains and HIV coinfection are prevalent. Choosing an optimal treatment regimen on the basis of the results of drug susceptibility tests and monitoring of treatment adherence would minimize further development of drug resistance strains.

Potential function of granulysin, other related effector molecules and lymphocyte subsets in patients with TB and HIV/TB coinfection.

BACKGROUND: Host effector mechanism against Mycobacterium tuberculosis (Mtb) infection is dependent on innate immune response by macrophages and neutrophils and the alterations in balanced adaptive immunity. Coordinated release of cytolytic effector molecules from NK cells and effector T cells and the subsequent granule-associated killing of infected cells have been documented; however, their role in clinical tuberculosis (TB) is still controversy. OBJECTIVE: To investigate whether circulating granulysin and other effector molecules are associated with the number of NK cells, iNKT cells, Vγ9(+)Vδ2(+) T cells, CD4(+) T cells and CD8(+) T cells, and such association influences the clinical outcome of the disease in patients with pulmonary TB and HIV/TB coinfection. METHODS: Circulating granulysin, perforin, granzyme-B and IFN-γ levels were determined by ELISA. The isoforms of granulysin were analyzed by Western blot analysis. The effector cells were analyzed by flow cytometry. RESULTS: Circulating granulysin and perforin levels in TB patients were lower than healthy controls, whereas the granulysin levels in HIV/TB coinfection were much higher than in any other groups, TB and HIV with or without receiving HAART, which corresponded to the number of CD8(+) T cells which kept high, but not with NK cells and other possible cellular sources of granulysin. In addition, the 17kDa, 15kDa and 9kDa isoforms of granulysin were recognized in plasma of HIV/TB coinfection. Increased granulysin and decreased IFN-γ levels in HIV/TB coinfection and TB after completion of anti-TB therapy were observed. CONCLUSION: The results suggested that the alteration of circulating granulysin has potential function in host immune response against TB and HIV/TB coinfection. This is the first demonstration so far of granulysin in HIV/TB coinfection.

Circulating levels of adiponectin, leptin, fetuin-A and retinol-binding protein in patients with tuberculosis: markers of metabolism and inflammation.

BACKGROUND: Wasting is known as a prominent feature of tuberculosis (TB). To monitor the disease state, markers of metabolism and inflammation are potentially useful. We thus analyzed two major adipokines, adiponectin and leptin, and two other metabolic markers, fetuin-A and retinol-binding protein 4 (RBP4). METHODS: The plasma levels of these markers were measured using enzyme-linked immunosorbent assays in 84 apparently healthy individuals (=no-symptom group) and 46 patients with active pulmonary TB around the time of treatment, including at the midpoint evaluation (=active-disease group) and compared them with body mass index (BMI), C-reactive protein (CRP), chest radiographs and TB-antigen specific response by interferon-γ release assay (IGRA). RESULTS: In the no-symptom group, adiponectin and leptin showed negative and positive correlation with BMI respectively. In the active-disease group, at the time of diagnosis, leptin, fetuin-A and RBP4 levels were lower than in the no-symptom group [adjusted means 2.01 versus 4.50 ng/ml, P<0.0001; 185.58 versus 252.27 µg/ml, P<0.0001; 23.88 versus 43.79 µg/ml, P<0.0001, respectively]. High adiponectin and low leptin levels were associated with large infiltrates on chest radiographs even after adjustment for BMI and other covariates (P=0.0033 and P=0.0020). During treatment, adiponectin levels increased further and then decreased. Leptin levels remained low. Initial low levels of fetuin-A and RBP4 almost returned to the normal reference range in concert with reduced CRP. CONCLUSIONS: Our data and recent literature suggest that low fat store and underlying inflammation may regulate these metabolic markers in TB in a different way. Decreased leptin, increased adiponectin, or this ratio may be a promising marker for severity of the disease independent of BMI. We should further investigate pathological roles of the balance between these adipokines.

Inter-rater agreement in the assessment of abnormal chest X-ray findings for tuberculosis between two Asian countries.

BACKGROUND: Inter-rater agreement in the interpretation of chest X-ray (CXR) films is crucial for clinical and epidemiological studies of tuberculosis. We compared the readings of CXR films used for a survey of tuberculosis between raters from two Asian countries. METHODS: Of the 11,624 people enrolled in a prevalence survey in Hanoi, Viet Nam, in 2003, we studied 258 individuals whose CXR films did not exclude the possibility of active tuberculosis. Follow-up films obtained from accessible individuals in 2006 were also analyzed. Two Japanese and two Vietnamese raters read the CXR films based on a coding system proposed by Den Boon et al. and another system newly developed in this study. Inter-rater agreement was evaluated by kappa statistics. Marginal homogeneity was evaluated by the generalized estimating equation (GEE). RESULTS: CXR findings suspected of tuberculosis differed between the four raters. The frequencies of infiltrates and fibrosis/scarring detected on the films significantly differed between the raters from the two countries (P < 0.0001 and P = 0.0082, respectively, by GEE). The definition of findings such as primary cavity, used in the coding systems also affected the degree of agreement. CONCLUSIONS: CXR findings were inconsistent between the raters with different backgrounds. High inter-rater agreement is a component necessary for an optimal CXR coding system, particularly in international studies. An analysis of reading results and a thorough discussion to achieve a consensus would be necessary to achieve further consistency and high quality of reading.

Association of IFNGR2 gene polymorphisms with pulmonary tuberculosis among the Vietnamese.

Interferon-γ (IFN-γ) is a key molecule of T helper 1 (Th1)-immune response against tuberculosis (TB), and rare genetic defects of IFN-γ receptors cause disseminated mycobacterial infection. The aim of the present study was to investigate whether genetic polymorphisms found in the Th1-immune response genes play a role in TB. In our study, DNA samples were collected from two series of cases including 832 patients with new smear-positive TB and 506 unrelated individuals with no history of TB in the general population of Hanoi, Vietnam. Alleles of eight microsatellite markers located around Th1-immune response-related genes and single nucleotide polymorphisms near the promising microsatellites were genotyped. A set of polymorphisms within the interferon gamma receptor 2 gene (IFNGR2) showed a significant association with protection against TB (P = 0.00054). Resistant alleles tend to be less frequently found in younger age at diagnosis (P = 0.011). Luciferase assays revealed high transcriptional activity of the promoter segment in linkage disequilibrium with resistant alleles. We conclude that the polymorphisms of IFNGR2 may confer resistance to the TB development of newly infected individuals. Contribution of the genetic factors to TB appeared to be different depending on age at diagnosis.

Analysis of factors lowering sensitivity of interferon-γ release assay for tuberculosis.

BACKGROUND: Imperfect sensitivity of interferon-γ release assay (IGRA) is a potential problem to detect tuberculosis. We made a thorough investigation of the factors that can lead to false negativity of IGRA. METHODS: We recruited 543 patients with new smear-positive pulmonary tuberculosis in Hanoi, Viet Nam. At diagnosis, peripheral blood was collected and IGRA (QuantiFERON-TB Gold In-Tube) was performed. Clinical and epidemiological information of the host and pathogen was collected. The test sensitivity was calculated and factors negatively influencing IGRA results were evaluated using a logistic regression model in 504 patients with culture-confirmed pulmonary tuberculosis. RESULTS: The overall sensitivity of IGRA was 92.3% (95% CI, 89.6%-94.4%). The proportions of IGRA-negative and -indeterminate results were 4.8% (95% CI, 3.1%-7.0%) and 3.0% (95% CI, 1.7%-4.9%). Age increased by year, body mass index <16.0, HIV co-infection and the increased number of HLA-DRB1*0701 allele that patients bear showed significant associations with IGRA negativity (OR = 1.04 [95% CI, 1.01-1.07], 5.42 [1.48-19.79], 6.38 [1.78-22.92] and 5.09 [2.31-11.22], respectively). HIV co-infection and the same HLA allele were also associated with indeterminate results (OR = 99.59 [95% CI, 15.58-625.61] and 4.25 [1.27-14.16]). CONCLUSIONS: Aging, emaciation, HIV co-infection and HLA genotype affected IGRA results. Assessment of these factors might contribute to a better understanding of the assay.

[Human genetic susceptibility to tuberculosis].

Similar to common diseases such as diabetes and hypertension, tuberculosis is a disease in which genetic predisposition is deeply involved. Linkage analysis, candidate gene association studies and animal models have been used to determine disease susceptibility genes to tuberculosis. Although many associated genes (NRAMP1, VDR, MBL and so on) have been reported thus far, the results are often inconsistent, partly because non-genetic host factors, environmental factors and virulence of pathogens also confer the risk and partly because systematic approaches have been adopted insufficiently. Genome wide association studies and large-scale international collaborative studies have recently been promoted, which are expected to identify high-risk individuals who develop the disease and to prevent tuberculosis effectively.

Decreased plasma granulysin and increased interferon-gamma concentrations in patients with newly diagnosed and relapsed tuberculosis.

Granulysin and interferon-gamma (IFN-γ) have broad antimicrobial activity which controls Mycobacterium tuberculosis (M. tuberculosis) infection. Circulating granulysin and IFN-γ concentrations were measured and correlated with clinical disease in Thai patients with newly diagnosed, relapsed and chronic tuberculosis (TB). Compared to controls, patients with newly diagnosed, relapsed and chronic TB had lower circulating granulysin concentrations, these differences being significant only in newly diagnosed and relapsed TB (P < 0.001 and 0.004, respectively). Granulysin concentrations in patients with newly diagnosed and relapsed TB were significantly lower than in those with chronic TB (P= 0.003 and P= 0.022, respectively). In contrast, significantly higher circulating IFN-γ concentrations were found in patients with newly diagnosed and relapsed TB compared to controls (P < 0.001). The IFN-γ concentrations in newly diagnosed and relapsed patients were not significantly different from those of patients with chronic TB. However, in vitro stimulation of peripheral blood mononuclear cells (PBMCs) from patients with newly diagnosed, relapsed and chronic TB with purified protein derivative (PPD) or heat killed M. tuberculosis (H37Ra) enhanced production of granulysin by PBMCs. In vitro, stimulation of PBMCs of newly diagnosed TB patients with PPD produced greater amounts of IFN-γ than did controls, while those stimulated with H37Ra did not. The results demonstrate that patients with active pulmonary TB have low circulating granulysin but high IFN-γ concentrations, suggesting possible roles in host defense against M. tuberculosis for these agents.

Identification of tuberculosis-associated proteins in whole blood supernatant.

BACKGROUND: Biological parameters are useful tools for understanding and monitoring complicated disease processes. In this study, we attempted to identify proteins associated with active pulmonary tuberculosis (TB) using a proteomic approach. METHODS: To assess TB-associated changes in the composition of human proteins, whole blood supernatants were collected from patients with active TB and healthy control subjects. Two-dimensional difference gel electrophoresis (2D-DIGE) was performed to analyze proteins with high molecular weights (approximately >20 kDa). Baseline protein levels were initially compared between patients with active TB and control subjects. Possible changes of protein patterns in active TB were also compared ex vivo between whole blood samples incubated with Mycobacterium tuberculosis (Mtb)-specific antigens (stimulated condition) and under unstimulated conditions. Immunoblot and enzyme-linked immunosorbent assays (ELISA) were performed to confirm differences in identified proteins. RESULTS: Under the baseline condition, we found that the levels of retinol-binding protein 4 (RBP4), fetuin-A (also called α-HS-glycoprotein), and vitamin D-binding protein differed between patients with active TB and control subjects on 2D gels. Immunoblotting results confirmed differential expression of RBP4 and fetuin-A. ELISA results further confirmed significantly lower levels of these two proteins in samples from patients with active TB than in control subjects (P < 0.0001). Mtb-specific antigen stimulation ex vivo altered clusterin expression in whole blood samples collected from patients with active TB. CONCLUSIONS: We identified TB-associated proteins in whole blood supernatants. The dynamics of protein expression during disease progression may improve our understanding of the pathogenesis of TB.

Association between circulating full-length osteopontin and IFN-gamma with disease status of tuberculosis and response to successful treatment.

The T helper type 1 (Th1) immune response plays an important role in protective immunity, pathophysiology and development of tuberculosis (TB). To investigate whether osteopontin (OPN) and other Th1 response-related molecules are associated withTB disease status, including co-infection with HIV, and response to anti-TB treatment, circulating levels of full-length OPN (F-OPN), thrombin-cleaved N-terminal fragment of OPN (N-half OPN), IFN-gamma, IP-10, IL-18, IL-12/ IL-23 (p40), IL-10, IL-15 and C-reactive protein (CRP) were measured before and after anti-TB treatment. Patients with newly active pulmonary TB had significantly higher plasma levels of F-OPN, IFN-gamma and CRP than healthy controls (HC). F-OPN, N-half OPN, IFN-gamma, IP-10, IL-18 and IL-10 levels were higher in patients with extensive TB/HIV co-infection than in patients with a single disease of TB or HIV. Plasma levels of F-OPN correlated well with those of IP-10, IL-18 and N-half OPN among patients with active TB. The F-OPN, IFN-gamma, IP-10 and CRP levels decreased significantly after effective anti-TB treatment. These data suggest that circulating OPN and Th1 response-related molecules, including IFN-gamma, may be regulated in response to expansion of active TB and could serve as markers of disease activity before and during treatment.