Cytotoxicity responses to selected ESAT-6 and CFP-10 peptides in tuberculosis

Cytotoxicity responses were studied for the ESAT-6 peptides Esp1, Esp6, Esp7, Esp8, and CFP-10 peptides, Cfp6, Cfp7, Cfp8, Cfp9 (synthetic 20-mer peptides) and the recombinant ESAT-6, CFP-10 proteins. Cytolytic molecules perforin, granzymes A and B, granulysin responses in healthy household contacts (HHC) and pulmonary tuberculosis patients (PTB), were studied by intracellular flow cytometry. Functional cytotoxicity was studied in both the groups for the peptides Esp6 and Cfp8 by an enzyme (lactate dehydrogenase) based assay. The results revealed that cytolytic molecule positive CD4+ and CD8+ T cells were increased in HHC in response to Esp1, Esp6, Cfp8 and Cfp9 immunogenic peptides compared to PTB. Functional cytotoxicity results showed higher cytotoxicity (not statistically significant) to be exhibited by the peptide Esp6 than Cfp8 in the HHC.     

Improved humoral immunity against tuberculosis ESAT-6 antigen by chimeric DNA prime and protein boost strategy

Ag85A and ESAT-6 proteins of Mycobacterium tuberculosis (M.TB) are important protective antigens. The 32-kDa Ag85A is a strong immunogen in both small and large animals. However, the 6-kDa ESAT-6 has relatively low inherent immunogenicity, especially in large animals. To improve the immunogenicity of ESAT-6 in animals, we made chimeric DNA vaccines, HG856K and HG856A, by inserting the esat-6 gene into the Kpn I or Acc I endonuclease restriction site of the ag85a gene, respectively. BALB/c mice were injected intramuscularly three times with the 10-microg singular DNA vaccine (HG85 encoding for Ag85A or HG6 encoding for ESAT-6) or chimeric DNA vaccine (HG856K or HG856A) followed by electroporation (EP). Ten days after the last DNA vaccination, mice received a booster immunization intraperitoneally with 50-microg pure recombinant protein Ag85A or ESAT-6 without adjuvant. Additional groups of mice immunized with chimeric DNA vaccines were boosted with two mixed proteins (Ag85A/ESAT-6) at the same time. The results showed that the immunogenicity of M.TB ESAT-6 antigen was not improved by priming with the HG6 DNA vaccine. However, the humoral immunity against the ESAT-6 antigen was significantly increased in the mice primed with chimeric DNA vaccines, HG856K or HG856A, followed by boosting with ESAT-6 or ESAT-6/Ag85A mixed proteins.     

Synthetic Peptides are Better Than Native Antigens for Development of ELISA Assay for Diagnosis of Tuberculosis

Immunodiagnosis of both pulmonary tuberculosis (PTB) and extrapulmonary tuberculosis has remained challenging. In the present work, in-house developed synthetic peptide based antibody detection assay was assessed and validated with antigen based assay for effective diagnosis of tuberculosis (TB). The study population included both tuberculous meningitis (TBM) (n = 60) admitted to Neurology IPD wards of our Institute hospital and PTB cases (n = 57) recruited from high TB endemic zones. Peptides of five highly immunogenic Mycobacterium tuberculosis (MTB) proteins (Ag85B, 45 kDa, HSP-16, CFP-10 and ESAT-6) were designed and synthesized. The designed peptides were evaluated in samples of both TBM and PTB cases, respectively, using in-house developed antibody detection method. The developed tests were further compared and validated with MTB native proteins based antibody detection ELISA. Sensitivity and specificity of peptide assay were significantly higher or almost similar (p < 0.05) in TBM and PTB as compared to native proteins based ELISA. Among all peptides, diagnostic reliability of Ag 85B peptide A1 was higher for both forms of TB. Peptide-based antibody assay is cost effective, simple and may be interchangeable with conventional Antigen based ELISA assays for effective diagnosis of TB in the developing world.     

Immunogenicity and protective efficacy study using combination of four tuberculosis DNA vaccines

Immune response and protective efficacy for the combination of four tuberculosis DNA vaccines were evaluated in this study. We obtained 1:200 antibody titers against Ag85B 21d after mice were vaccinated for the first time by four recombinant eukaryotic expression vectors containing coding sequences for Ag85B, MPT-64, MPT-63 and ESAT-6. The titers of Ag85B were elevated to 1:102400 after the second injection and decreased to 1:12800 after the third injection. Antibody titers for MPT-64 and MPT-63 reached 1:25600 21 d after the first vaccination, and were then decreased following the second and third injections. No antigen-specific antibody titer against ESAT-6 was detected in sera harvested from immunized mice at any time. These DNA vaccines evoked specific IFN-λ responses in the spleens of vaccinated mice as well. When challenged with M. tuberculosis H37Rv, we found that the lungs of the vaccinated mice produced 99.8% less bacterial counts than that of the empty-vector control group and the bacterial counts were also significantly less than that of the BCG group. Histopathological analyses showed that the lungs of vaccinated mice produced no obvious caseation while over 50%-70% of the pulmonary parenchyma tissue produced central caseation in the vector control group. Our results indicated that the combination of four tuberculosis DNA vaccines may generate high levels of immune responses and result in better animal protection.     

Immunogenicity and protective efficacy study using combination of four tuberculosis DNA vaccines

Tuberculosis is an ancient scourge of mankind. According to statistics, there are more than 10 million new cases of tuberculosis each year and the annual death toll for tuberculosis exceeds three millions. The current available BCG is of questionable efficacy, and its protection ranges from 0 to 85%. Therefore, developing a safe and effective vaccine against this scourge is very important. Previous studies have shown that the secreted proteins of Mycobacterium tuberculosis (M. TB) can induc…     

Identification of early secretory antigen target-6 epitopes for the immunodiagnosis of active tuberculosis

The early secretory antigenic target (ESAT)-6 purified protein and peptides from Mycobacterium tuberculosis were evaluated as antigens for the immunodiagnosis of tuberculosis (TB). Because the control of TB requires improved diagnostic procedures, efforts have increased to identify Mycobacterium tuberculosis-specific epitopes for the immunodiagnosis of active TB and to discriminate between active and latent states of infection. Two multiepitopic peptides from ESAT-6 protein were selected by computational analysis. Patients with active TB (7 HIV(+) and 20 HIV(-)) and control patients (17 HIV(+) and 28 HIV(-)) were enrolled. Enzyme-linked immunospot assay analysis for interferon-g expression by peripheral blood mononuclear cells was quantified after stimulation with selected ESAT-6 peptides, purified protein derivative, or the intact ESAT-6 protein. During active TB, 20 of 27 patients responded in vitro to ESAT-6 peptides and 23 of 27 patients to purified protein derivative. None of the controls without active TB, including individuals with latent TB infection, recognized ESAT-6 peptides. By contrast, latently infected individuals did respond in vitro to both intact ESAT-6 protein and purified protein derivative. Thus, high T-cell response frequencies to ESAT-6 peptides are present only during active TB and can be used to discriminate between active and latent forms of infection.     

Recombinant BCG coexpressing Ag85B, ESAT-6 and mouse-IFN-gamma confers effective protection against Mycobacterium tuberculosis in C57BL/6 mice

In this study, the protective efficacy of a novel recombinant bacille Calmette Géurin (BCG) strain (rBCG-AEI) expressing fusion protein the antigen 85B (Ag85B)- the 6-kDa early secreted antigen target (ESAT-6)-IFN-gamma against Mycobacterium tuberculosis H37Rv in mice was evaluated. The immunogenicity study showed that rBCG-AEI could induce higher specific antibody titers and significantly increase cellular immune response than BCG, or rBCG-A strain (expressing Ag85B), or rBCG-AE strain (expressing fusion protein Ag85B-ESAT-6). The protective experiment demonstrated that rBCG-AEI could confer similar or even better protective efficacy against M. tuberculosis infection compared with others in organ bacterial loads, lung histopathology and net weight gain or loss. The results suggested that rBCG-AEI is a potential candidate for further study.     

结核分枝杆菌重要诊断用抗原研究进展

血清学试验是结核诊断的重要依据。随着科学技术的进步,新的结核诊断用抗原不断被发现。我们简要综述了结核菌素蛋白衍生物、抗原85复合体、38kDa磷酸盐转运蛋白、6kDa早期分泌性蛋白、10kDa培养滤液蛋白、免疫性蛋白MPT64、主要分泌性免疫蛋白MPB70、表面脂蛋白MPB83等8种结核分枝杆菌重要抗原作为结核诊断用抗原的研究进展。     

Highly focused T cell responses in latent human pulmonary Mycobacterium tuberculosis infection

The elucidation of the molecular and immunological mechanisms mediating maintenance of latency in human tuberculosis aids to develop more effective vaccines and to define biologically meaningful markers for immune protection. We analyzed granuloma-associated lymphocytes (GALs) from human lung biopsies of five patients with latent Mycobacterium tuberculosis (MTB) infection. MTB CD4+ and CD8+ T cell response was highly focused in the lung, distinct from PBL, as assessed by TCR-CDR3 spectratyping coupled with a quantitative analysis of TCR VB frequencies. GALs produced IFN-gamma in response to autologous macrophages infected with MTB and to defined MTB-derived HLA-A2-presented peptides Ag85a242-250, Ag85b199-207, early secreted antigenic target 6 (ESAT-6)28-36, 19-kDa Ag88-97, or the HLA-DR-presented ESAT-6(1-20) epitope. Immune recognition of naturally processed and presented MTB epitopes or the peptide ESAT-6(1-20) could be linked to specific TCR VB families, and in two patients to unique T cell clones that constituted 19 and 27%, respectively, of the CD4+ and 17% of the CD8+ GAL population. In situ examination of MTB-reactive GALs by tetramer in situ staining and confocal laser-scanning microscopy consolidates the presence of MHC class I-restricted CD8+ T cells in MTB granuloma lesions and supports the notion that clonally expanded T cells are crucial in immune surveillance against MTB.     

Interferon gamma response to combinations 38 kDa/CFP-10, 38 kDa/MPT-64, ESAT-6/MPT-64 and ESAT-6/CFP-10, each related to a single recombinant protein of Mycobacterium tuberculosis in individuals from tuberculosis endemic areas

Several antigens of Mycobacterium tuberculosis have been identified and specificity to one or multiple antigens could determine the distinction between protective and pathogenic host reaction. Therefore T cell immune response to combinations 38 kDa/CFP-10, 38 kDa/MPT-64, ESAT-6/MPT-64 and ESAT-6/CFP-10 (each related to a single protein of Mycobacterium tuberculosis) in individuals from tuberculosis endemic areas have been examined. ELISA was used to detect IFN-gamma production in PBMC priming with single proteins and combinations in a panel of 105 individuals: 38 tuberculosis patients (6 untreated and 32 treated) and 67 healthy controls with tuberculin skin test positive or negative (TST). Brazilian TB patients highly recognized ESAT-6 (66%), but combinations improved response in the following order: ESAT-6/MPT-64 (89%) > ESAT-6/CFP-10 (73%) > 38 kDa/CFP-10 (70%), the last combination showing the highest specificity (TST(/) = 42% and TST(-) = 83%). Average IFN-gamma production in TB patients was signifi-cantly higher for 38 kDa/CFP-10 (P = 0.012) and 38 kDa/MPT-64 (P <0.035), when compared to single antigens. None of the combinations was able to discriminate TB patients from TST(+) controls; however, 38 kDa/CFP-10 displayed a borderline significance (P = 0.053). Similar to the ESAT-6/CFP-10 combination, IFN-gamma response to 38 kDa/CFP-10 showed an increased tendency in treated patients, although not signifi-cant (P = 0.16). We demonstrated for the first time that 38 kDa/CFP-10 had prediction sensitivity for TB patients similar to the ESAT-6/CFP-10 combination and also significant response improvement related to the single proteins with more selective reactivity among TST-positive individuals, which could be of potential interest for diagnostic evaluation for tuberculosis infection.     

The IL-17F sequence variant is associated with susceptibility to tuberculosis

The interleukin (IL)-17 gene plays a key role in host defence against infections from microbes, including Mycobacterium tuberculosis. Genetic factors contribute to host defence. However, whether genetic variation in IL-17 is associated with altered susceptibility to tuberculosis is unknown. A total of 596 pulmonary tuberculosis (PTB) patients, 176 extra-pulmonary tuberculosis (EPTB) patients, and 622 control patients from a Chinese Han population were recruited. Two single-nucleotide polymorphisms (SNPs) in IL-17F (rs1889570 and rs763780) and one SNP in IL-17A (rs2275913) were genotyped using the SNaPshot technique. Of the three SNPs in the IL-17 gene tested, there was an increased frequency of the rs1889570 G allele and the rs763780 C allele in the PTB patients and an increased frequency of the rs763780 C allele in the EPTB patients compared with the control patients. There were also significant differences in the distribution of the rs763780 genotype between the PTB and EPTB patients and the controls. The patients who had the CT/TT genotype of the rs763780 SNP were more susceptible to tuberculosis, compared to the CC genotype. There was no significant difference observed between the IL-17 SNPs when the PTB and EPTB patients were compared. Genetic variation in IL-17F is associated with altered susceptibility to tuberculosis and may provide valuable information in the development of tuberculosis.     

Rapid detection of Mycobacterium tuberculosis based on antigen 85B via real-time recombinase polymerase amplification

Tuberculosis (TB), as a common infectious disease, still remains a severe challenge to public health. Due to the unsatisfied clinical needs of currently available diagnostic vehicles, it is desired to establish a new approach for universally detecting Mycobacterium tuberculosis. Herein, we designed a real-time recombinase polymerase amplification (RPA) technology for identifying M. tuberculosis within 20 min at 39°C via custom-designed oligonucleotide primers and probe, which could specifically target antigen 85B (Ag85B). Particularly, the primers F4-R4 produced the fastest fluorescence signal with the probe among four pairs of designed primers in the RPA assays. The optimal primers/probe combination could effectively identify M. tuberculosis with the detection limit of 4·0 copies per μl, as it could not show a positive signal for the genomic DNA from other mycobacteria or pathogens. The Ag85B-based RPA could determine the genomic DNA extracted from M. tuberculosis with high reliability (100%, 22/22). More importantly, when testing clinical sputum samples, the real-time RPA displayed an admirable sensitivity (90%, 95% CI: 80·0-96·0%) and specificity (98%, 95% CI: 89·0-100·0%) compared to traditional smear microscopy, which was similar to the assay of Xpert MTB/RIF. This real-time RPA based Ag85B provides a promising strategy for the rapid and universal diagnosis of TB.     

Exchanging ESAT6 with TB10.4 in an Ag85B fusion molecule-based tuberculosis subunit vaccine: efficient protection and ESAT6-based sensitive monitoring of vaccine efficacy

Previously we have shown that Ag85B-ESAT-6 is a highly efficient vaccine against tuberculosis. However, because the ESAT-6 Ag is also an extremely valuable diagnostic reagent, finding a vaccine as effective as Ag85B-ESAT-6 that does not contain ESAT-6 is a high priority. Recently, we identified a novel protein expressed by Mycobacterium tuberculosis designated TB10.4. In most infected humans, TB10.4 is strongly recognized, raising interest in TB10.4 as a potential vaccine candidate and substitute for ESAT-6. We have now examined the vaccine potential of this protein and found that vaccination with TB10.4 induced a significant protection against tuberculosis. Fusing Ag85B to TB10.4 produced an even more effective vaccine, which induced protection against tuberculosis comparable to bacillus Calmette-Guerin vaccination and superior to the individual Ag components. Thus, Ag85B-TB10 represents a new promising vaccine candidate against tuberculosis. Furthermore, having now exchanged ESAT-6 for TB10.4, we show that ESAT-6, apart from being an excellent diagnostic reagent, can also be used as a reagent for monitoring vaccine efficacy. This may open a new way for monitoring vaccine efficacy in clinical trials.     

Performance of recombinant ESAT-6 antigen (ML0049) for detection of leprosy patients

AIMS: The study was aimed to evaluate the Mycobacterium leprae recombinant early secreted antigenic target-6 (rESAT-6) for its serological performance in leprosy patients. METHODS AND RESULTS: Employing enzyme-linked immunosorbent assay (ELISA), serum samples were tested for prevalence of immunoglobulin G antibodies against M. leprae rESAT-6. The results revealed that the sensitivity of the assay for smear-positive leprosy patients was 82.4% (14 of 17) while for smear-negative patients it was 19.4% (six of 31). Interestingly, the performance of ESAT-6-based assay was statistically comparable with anti-phenolic glycolipid-I antibody-detecting ELISA, a most widely studied serological assay in leprosy. Regarding specificity, none of the 48 controls was positive indicating that antibody response to ESAT-6 was highly specific. Moreover, a high concordance between bacterial index and anti-ESAT-6 antibody-detecting assay was noted. CONCLUSIONS: Recombinant ESAT-6 seems to be a potential serological reagent for detection of M. leprae infection. SIGNIFICANCE AND IMPACT OF THE STUDY: ESAT-6 serology may have utility for (i) early diagnosis, particularly, of highly infectious form (multibacillary, MB) of leprosy, (ii) monitoring the response in smear-positive leprosy patients during the course of the chemotherapy, (iii) classification of leprosy patients into MB and paucibacillary groups for treatment purpose. Hence, further research on these lines is warranted.     

Monokine induced by interferon gamma and IFN-gamma response to a fusion protein of Mycobacterium tuberculosis ESAT-6 and CFP-10 in Brazilian tuberculosis patients

IFN-gamma responses to Mycobacterium tuberculosis antigens ESAT-6 and CFP-10 have been proposed as specific markers of M. tuberculosis infection. Monokine induced by gamma interferon (MIG/CXCL9) has been shown to be expressed by IFN-gamma stimulated mononuclear cells and to attract activated T-cells through the chemokine receptor CXCR3. Since MIG is induced early in the response to IFN-gamma, measuring MIG may provide an interesting marker to assess downstream IFN-gamma induced responses, in contrast to assays that mainly focus on quantifying production of IFN-gamma per se. We, therefore, investigated MIG and IFN-gamma responses to a fusion protein of ESAT-6 and CFP-10, and compared responses to the conserved mycobacterial antigen 85B (Ag85B) and purified protein derivative (PPD) of M. tuberculosis, in 29 BCG vaccine controls and 24 TB patients. IFN-gamma secreting cells were determined by ELISPOT, and MIG production was measured by ELISA and flow cytometry. Production of MIG in response to ESAT-6/CFP-10, Ag85B and PPD correlated overall with increased numbers of IFN-gamma secreting cells (r=0.55, P<0.0001). A significant increase was noted among patients compared to controls in the secretion of IFN-gamma and MIG following stimulation with ESAT-6/CFP-10 or PPD (P<0.05). Moreover, MIG intracellular expression was higher in TB patients compared to BCG vaccines (P<0.05) in response to ESAT-6/CFP-10 or PPD. We conclude that MIG production correlates significantly with enhanced T-cell IFN-gamma production induced by M. tuberculosis-specific antigens ESAT-6/CFP-10. These results point to MIG as a potential novel biomarker that may be helpful in assessing downstream responses induced by IFN-gamma in TB.     

Differential immunogenicity and protective efficacy of DNA vaccines expressing proteins of Mycobacterium tuberculosis in a mouse model

BALB/c mice were vaccinated three times (2-week intervals) with plasmid DNA separately encoding antigen Ag85B, ESAT-6 or Ag85A from Mycobacterium tuberculosis. The protective efficacy of these DNA vaccines against intravenous M. tuberculosis H37Rv challenge infection was measured by counting bacterial loads in spleen and lung and recording changes in lung pathology. The splenocyte proliferative response to the corresponding antigens and antigen-specific interferon (IFN)-γ secreted by splenocytes of the vaccinated mice were also detected. We found a clear hierarchy of protective efficacies among the three DNA vaccines tested in this study. Plasmid DNA encoding Ag85A provided the strongest protection and showed the least change in lung pathology, followed by plasmid DNAs encoding Ag85B and ESAT-6. However, DNA-85B reduced comparative bacterial load in lung tissue, as did DNA-85A. Compared to the control group, protective efficacies conferred by different DNA vaccines were consistent with the lymphoproliferative responses to the corresponding antigens as well as the secretions of antigen-specific IFN-γ. Our study demonstrates that both Ag85A and Ag85B are the most promising of the candidate antigens tested for future TB vaccine development.     

Differential expression of CD64 in patients with Mycobacterium tuberculosis infection: A potential biomarker for clinical diagnosis and prognosis

To evaluate the clinical utility of neutrophil (n)CD64 index to diagnose pulmonary tuberculosis (PTB) and extrapulmonary TB (ePTB) and to predict the outcome of Mycobacterium tuberculosis infection. We recruited 189 patients with active TB and 140 controls and measured the differential expression of nCD64 index using flow cytometry. The receiver operating characteristics (ROC) curve analysis was performed to estimate the diagnostic performance of the nCD64 index and T‐SPOT.TB assay for the diagnosis of TB. Furthermore, we analysed whether the nCD64 index in patients with TB was correlated with inflammatory indicators. Finally, we assessed the prognosis of patients by following the dynamic changes of the nCD64 index once a week. The nCD64 index was significantly higher in active TB group (PTB and ePTB), than in the anti‐TB and healthy controls (HC) groups. The sensitivity and specificity of nCD64 index for the differential diagnosis of PTB and pneumonia (PN) patients were 68.33% and 77.55%, respectively. The sensitivity and specificity of nCD64 index for the diagnosis of tuberculous meningitis (TBM) were 53.85% and 100%, respectively. Furthermore, there was a weak correlation between the nCD64 index and inflammatory indicators. More importantly, with the improvement in patient condition, the nCD64 index started to decline in the first week of anti‐TB therapy and significantly decreased at 4 weeks after treatment. Our study demonstrated that the CD64 assay is a rapid, non‐invasive and stable method for clinical application, and the nCD64 index can serve as a potential biomarker for the diagnosis and prognosis of TB.     

结核分枝杆菌可视化抗体检测蛋白芯片的制备

目的：利用克隆表达的7种结核分枝杆菌优势表位抗原,建立可视化抗体检测蛋白芯片,用于结核病辅助诊断。方法：将7种结核分枝杆菌优势表位抗原,即38kD、ESAT-6、CFP10、MPT64、Mtb8、Mtb8.4和Mtb16.3点于修饰的基片上,制备可检测7种结核抗体的多靶点蛋白微阵列,建立免疫金银染色检测系统;使用该芯片对48例临床结核病患者血液样品进行检测,并与“金标准”痰涂片（48例）和痰培养（其中的29例）检测结果进行比较,分析其敏感性;对30名献血员血液样品进行检测,分析其特异性。结果：可视化抗体检测蛋白芯片的敏感性分别为98.5％和96.6％,而痰涂片和痰培养检测方法的敏感性分别为35.4％和48.3％;可视化抗体检测蛋白芯片的特异性为93.3％。结论：建立的结核分枝杆菌可视化抗体检测蛋白芯片检测敏感性显著高于痰涂片和痰培养方法,可用于结核病的临床辅助诊断,提高痰涂片和痰培养假阴性的检出率。     

Cytokine profiles in tuberculosis patients and healthy subjects in response to complex and single antigens of Mycobacterium tuberculosis

Peripheral blood mononuclear cells (PBMC) were obtained from tuberculosis (TB) patients and Mycobacterium bovis bacillus Calmette-Guerin vaccinated healthy subjects. PBMC were tested for secretion of tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), interleukin-5 (IL-5) and IL-10 in response to complex (whole cells, culture filtrate and cell walls), single secreted (Ag85B, ESAT6, MPT64, PstS and MPT70) and single cytosolic (DnaK, GroES and GroEL) antigens of Mycobacterium tuberculosis. In the absence of antigens, detectable concentrations of TNF-alpha, IFN-gamma and IL-10 were secreted by PBMC of both donor groups, but the concentrations of only IL-10 were significantly higher (P=0.015) in TB patients than in healthy subjects. In the presence of complex antigens, PBMC secreted IFN-gamma and TNF-alpha in response to all three preparations, whereas IL-10 was secreted in response to whole cells and cell walls only. In the presence of single antigens, IFN-gamma was secreted in response to Ag85B, ESAT6 and MPT64 in TB patients and ESAT6 in healthy donors. Except for GroEL and DnaK, single antigens did not induce TNF-alpha and IL-10 secretion from PBMC in either donor group. The secretion of IFN-gamma, but not IL-10, in the presence of Ag85B, ESAT6 and MPT64 supports their potential as subunit vaccine candidates against TB.     

Extrapulmonary Tuberculosis: Mycobacterium tuberculosis Strains and Host Risk Factors in a Large Urban Setting in Brazil

Background

Factors related to the development of extrapulmonary forms of tuberculosis (EPTB) are still poorly understood, particularly in high-endemic countries like Brazil. The objective of the paper is to determine host and Mycobacterium tuberculosis (MTB) strain-related factors associated with the development of EPTB in Espírito Santo state, Brazil.

Methods and Findings

We conducted a retrospective laboratory-based surveillance study of new tuberculosis (TB) cases diagnosed in Espírito Santo state, Brazil between 1998 and 2007. We genotyped 612 isolates of MTB from 606 TB patients using spoligotyping and IS6110-restriction fragment length polymorphism (RFLP) typing and compared sociodemographic and clinical characteristics of patients with pulmonary TB (PTB) and EPTB. Among 606 patients, 464 (77%) had PTB, 79 (13%) had EPTB, 51 (8%) had both, and 12 (2%) had miliary TB. The IS6110 RFLP analysis demonstrated that 250 (41%) isolates belonged to clustered RFLP patterns, 27 (11%) of which were from EPTB. We identified 73 clusters including 35 (48%) composed of 2 isolates each. By spoligotyping, 506 (83%) MTB isolates fell into known patterns and 106 (17%) fell into patterns with no family assignment; 297 (48%) isolates belonged to the Latin-American Mediterranean family. Higher school level (4-7 years OR: 0.16 95% CI 0.34-0.73 and > 8 years of education, OR 0.06 95% CI 0.009-0.50) white ethnicity (OR: 2.54 95% CI 1.03-6.25) and HIV infection (OR: 16.83 95% CI 5.23-54.18) were associated with EPTB. No specific strain lineage or percentage of clustering was associated with EPTB.

Conclusions

These results demonstrate that risk factors for EPTB are related more to host than to MTB strain lineage characteristics.