Application of BactTiter-Glo ATP bioluminescence assay for Mycobacterium tuberculosis detection.

BACKGROUND: Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), remains a global health threat, necessitating faster and more accessible diagnostic methods. This study investigates critical parameters in the application of a commercial ATP bioluminescence assay for the detection of MTB. METHOD: Our objective was to optimize the ATP bioluminescence protocol using BacTiter-Glo™ for MTB, investigating the impact of varying volumes of MTB suspension and reagent on assay sensitivity, evaluating ATP extraction methods, establishing calibration curves, and elucidating strain-specific responses to antimicrobial agents. RESULTS: ATP extraction methods showed no significant improvement over controls. Calibration curves revealed a linear correlation between relative light units (RLU) and colony-forming units (CFU/mL), establishing low detection limits. Antimicrobial testing demonstrated strain-specific responses aligning with susceptibility and resistance patterns. CONCLUSION: Our findings contribute to refining ATP bioluminescence protocols for enhanced MTB detection and susceptibility testing. Further refinements and validation efforts are warranted, holding promise for more efficient diagnostic platforms in the future.

Early alveolar macrophage response and IL-1R-dependent T cell priming determine transmissibility of Mycobacterium tuberculosis strains.

Mechanisms underlying variability in transmission of Mycobacterium tuberculosis strains remain undefined. By characterizing high and low transmission strains of M.tuberculosis in mice, we show here that high transmission M.tuberculosis strain induce rapid IL-1R-dependent alveolar macrophage migration from the alveolar space into the interstitium and that this action is key to subsequent temporal events of early dissemination of bacteria to the lymph nodes, Th1 priming, granulomatous response and bacterial control. In contrast, IL-1R-dependent alveolar macrophage migration and early dissemination of bacteria to lymph nodes is significantly impeded in infection with low transmission M.tuberculosis strain; these events promote the development of Th17 immunity, fostering neutrophilic inflammation and increased bacterial replication. Our results suggest that by inducing granulomas with the potential to develop into cavitary lesions that aids bacterial escape into the airways, high transmission M.tuberculosis strain is poised for greater transmissibility. These findings implicate bacterial heterogeneity as an important modifier of TB disease manifestations and transmission.

Silk Fibroin/Poly(vinyl Alcohol) Microneedles as Carriers for the Delivery of Singlet Oxygen Photosensitizers.

Photodynamic therapy (PDT) is a medical treatment in which a combination of a photosensitizing drug and visible light produces highly cytotoxic reactive oxygen species (ROS) that leads to cell death. One of the main drawbacks of PDT for topical treatments is the limited skin penetration of some photosensitizers commonly used in this therapy. In this study, we propose the use of polymeric microneedles (MNs) prepared from silk fibroin and poly(vinyl alcohol) (PVA) to increase the penetration efficiency of porphyrin as possible applications in photodynamic therapy. The microneedle arrays were fabricated from mixtures in different proportions (1:0, 7:3, 1:1, 3:7, and 0:1) of silk fibroin and PVA solutions (7%); the polymer solutions were cast in polydimethylsiloxane (PDMS) molds and dried overnight. Patches containing grids of 10 × 10 microneedles with a square-based pyramidal shape were successfully produced through this approach. The polymer microneedle arrays showed good mechanical strength under compression force and sufficient insertion depth in both Parafilm M and excised porcine skin at different application forces (5, 20, 30, and 40 N) using a commercial applicator. We observe an increase in the cumulative permeation of 5-[4-(2-carboxyethanoyl) aminophenyl]-10,15,20-tris-(4-sulphonatophenyl) porphyrin trisodium through porcine skin treated with the polymer microneedles after 24 h. MNs may be a promising carrier for the transdermal delivery of photosensitizers for PDT, improving the permeation of photosensitizer molecules through the skin, thus improving the efficiency of this therapy for topical applications.

Increase of CD4+CD25highFoxP3+ cells impairs in vitro human microbicidal activity against Mycobacterium tuberculosis during latent and acute pulmonary tuberculosis.

BACKGROUND: Regulatory T cells (Tregs) play a critical role during Mycobacterium tuberculosis (Mtb) infection, modulating host responses while neutralizing excessive inflammation. However, their impact on regulating host protective immunity is not completely understood. Here, we demonstrate that Treg cells abrogate the in vitro microbicidal activity against Mtb. METHODS: We evaluated the in vitro microbicidal activity of peripheral blood mononuclear cells (PBMCs) from patients with active tuberculosis (TB), individuals with latent tuberculosis infection (LTBI, TST+/IGRA+) and healthy control (HC, TST-/IGRA-) volunteers. PBMCs, depleted or not of CD4+CD25+ T-cells, were analyzed to determine frequency and influence on microbicidal activity during in vitro Mtb infection with four clinical isolates (S1, S5, R3, and R6) and one reference strain (H37Rv). RESULTS: The frequency of CD4+CD25highFoxP3+ cells were significantly higher in Mtb infected whole blood cultures from both TB patients and LTBI individuals when compared to HC. Data from CD4+CD25+ T-cells depletion demonstrate that increase of CD4+CD25highFoxP3+ is associated with an impairment of Th-1 responses and a diminished in vitro microbicidal activity of LTBI and TB groups. CONCLUSIONS: Tregs restrict host anti-mycobacterial immunity during active disease and latent infection and thereby may contribute to both disease progression and pathogen persistence.

Mycobacterium tuberculosis progresses through two phases of latent infection in humans.

Little is known about the physiology of latent Mycobacterium tuberculosis infection. We studied the mutational rates of 24 index tuberculosis (TB) cases and their latently infected household contacts who developed active TB up to 5.25 years later, as an indication of bacterial physiological state and possible generation times during latent TB infection in humans. Here we report that the rate of new mutations in the M. tuberculosis genome decline dramatically after two years of latent infection (two-sided p < 0.001, assuming an 18 h generation time equal to log phase M. tuberculosis, with latency period modeled as a continuous variable). Alternatively, assuming a fixed mutation rate, the generation time increases over the latency duration. Mutations indicative of oxidative stress do not increase with increasing latency duration suggesting a lack of host or bacterial derived mutational stress. These results suggest that M. tuberculosis enters a quiescent state during latency, decreasing the risk for mutational drug resistance and increasing generation time, but potentially increasing bacterial tolerance to drugs that target actively growing bacteria.

Differentially culturable tubercle bacteria dynamics during standard anti-tuberculosis treatment: A prospective cohort study.

This study aimed to evaluate the dynamics of culture filtrate dependent subpopulations of Mycobacterium tuberculosis in a prospective cohort study following 17 patients through a standard 6-month anti-tuberculosis regimen, performing monthly sputum collection. We performed the limiting dilution method with culture filtrate supplementation of liquid media in pre- and post-treatment sputum samples to assess the bacillary load and to evaluate the Mycobacterium tuberculosis subpopulation dynamics within the 6-months standard anti-tuberculosis regimen. We found that supplementation increased the bacillary load by 30% in pre-treatment samples (p = 0.0005) and 35% in samples after one month of treatment (p = 0.0977). We found a weak linear correlation between the decrease of Mycobacterium tuberculosis growth in liquid media with and without culture filtrate supplementation (ρ = 0.54; p = 0.026). None of the patients had bacilli recovery after two months of treatment. Our study constitutes the first follow-up regarding Mycobacterium tuberculosis subpopulation dynamics throughout a standard 6-month anti-tuberculosis treatment and also supports the use of culture filtrate to increase bacillary load in liquid media. Moreover, it highlights that any new treatment regimens should test the efficacy of the drugs in all Mycobacterium tuberculosis subpopulations.

Cross-validation of existing signatures and derivation of a novel 29-gene transcriptomic signature predictive of progression to TB in a Brazilian cohort of household contacts of pulmonary TB.

The goal of this study was to identify individuals at risk of progression and reactivation among household contacts (HHC) of pulmonary TB cases in Vitoria, Brazil. We first evaluated the predictive performance of six published signatures on the transcriptional dataset obtained from peripheral blood mononuclear cell samples from HHC that either progressed to TB disease or not (non-progressors) during a five-year follow-up. The area under the curve (AUC) values for the six signatures ranged from 0.670 to 0.461, and the PPVs did not reach the WHO published target product profiles (TPPs). We therefore used as training cohort the earliest time-point samples from the African cohort of adolescents (GSE79362) and applied an ensemble feature selection pipeline to derive a novel 29-gene signature (PREDICT29). PREDICT29 was tested on 16 progressors and 21 non-progressors. PREDICT29 performed better in segregating progressors from non-progressors in the Brazil cohort with the area under the curve (AUC) value of 0.911 and PPV of 20%. This proof of concept study demonstrates that PREDICT29 can predict risk of progression/reactivation to clinical TB disease in recently exposed individuals at least 5 years prior to disease development. Upon validation in larger and geographically diverse cohorts, PREDICT29 can be used to risk-stratify recently infected for targeted therapy.

Host Determinants of Infectiousness in Smear-Positive Patients With Pulmonary Tuberculosis.

BACKGROUND: Epidemiologic data suggests that only a minority of tuberculosis (TB) patients are infectious. Cough aerosol sampling is a novel quantitative method to measure TB infectiousness. METHODS: We analyzed data from three studies conducted in Uganda and Brazil over a 13-year period. We included sputum acid fast bacilli (AFB) and culture positive pulmonary TB patients and used a cough aerosol sampling system (CASS) to measure the number of colony-forming units (CFU) of Mycobacterium tuberculosis in cough-generated aerosols as a measure for infectiousness. Aerosol data was categorized as: aerosol negative (CFU = 0) and aerosol positive (CFU > 0). Logistic regression models were built to identify factors associated with aerosol positivity. RESULTS: M. tuberculosis was isolated by culture from cough aerosols in 100/233 (43%) TB patients. In an unadjusted analysis, aerosol positivity was associated with fewer days of antituberculous therapy before CASS sampling (p = .0001), higher sputum AFB smear grade (p = .01), shorter days to positivity in liquid culture media (p = .02), and larger sputum volume (p = .03). In an adjusted analysis, only fewer days of TB treatment (OR 1.47 per 1 day of therapy, 95% CI 1.16-1.89; p = .001) was associated with aerosol positivity. CONCLUSION: Cough generated aerosols containing viable M. tuberculosis, the infectious moiety in TB, are detected in a minority of TB patients and rapidly become non-culturable after initiation of antituberculous treatment. Mechanistic studies are needed to further elucidate these findings.

Further evidence of Mycobacterium tuberculosis in the sputum of culture-negative pulmonary tuberculosis suspects using an ultrasensitive molecular assay.

BACKGROUND: Rapid diagnosis of pulmonary tuberculosis (TB) is critical to TB control. However, many patients with paucibacillary TB disease remain undiagnosed. Current TB elimination goals require new tools to diagnose early disease. We evaluated performance of the Totally Optimized PCR (TOP) TB assay, a novel ultrasensitive molecular test. METHODS: We assessed analytical specificity against nontuberculous mycobacteria (NTM), and estimated the diagnostic accuracy of TOP in a pilot study in Brazil (n = 46) and a cross-sectional study in Boston (n = 60). We compared TOP results to culture and a composite reference standard (CRS). RESULTS: TOP exhibited no cross-reactivity against NTM. We tested 132 respiratory specimens from 106 patients with suspected pulmonary TB. The pilot demonstrated feasibility and 100% (95% CI 85-100) sensitivity in predominantly smear-positive specimens; TOP's specificity against solid media culture was low (58%, 37-77) but improved against a CRS (93%, 68-100). Similarly, when using the CRS in the Boston study, TOP (88%, 1-99) had greater sensitivity than solid or liquid media culture (25%, 3-65) and similar specificity (both 100%, 93-100). CONCLUSIONS: The TOP assay enables detection of M. tuberculosis in culture-negative paucibacillary disease. While the use of TOP for the diagnosis of paucibacillary disease will require further clinical validation, its high sensitivity indicate a more immediate utility as a rule out TB test.

Transmission phenotype of Mycobacterium tuberculosis strains is mechanistically linked to induction of distinct pulmonary pathology.

In a study of household contacts (HHC), households were categorized into High (HT) and Low (LT) transmission groups based on the proportion of HHC with a positive tuberculin skin test. The Mycobacterium tuberculosis (Mtb) strains from HT and LT index cases of the households were designated Mtb-HT and Mtb-LT, respectively. We found that C3HeB/FeJ mice infected with Mtb-LT strains exhibited significantly higher bacterial burden compared to Mtb-HT strains and also developed diffused inflammatory lung pathology. In stark contrast, a significant number of mice infected with Mtb-HT strains developed caseating granulomas, a lesion type with high potential to cavitate. None of the Mtb-HT infected animals developed diffused inflammatory lung pathology. A link was observed between increased in vitro replication of Mtb-LT strains and their ability to induce significantly high lipid droplet formation in macrophages. These results support that distinct early interactions of Mtb-HT and Mtb-LT strains with macrophages and subsequent differential trajectories in pathological disease may be the mechanism underlying their transmission potential.

Cough-aerosol cultures of Mycobacterium tuberculosis in the prediction of outcomes after exposure. A household contact study in Brazil.

BACKGROUND: Mycobacterium tuberculosis cultures of cough-generated aerosols from patients with pulmonary tuberculosis (TB) are a quantitative method to measure infectiousness and to predict secondary outcomes in exposed contacts. However, their reproducibility has not been established. OBJECTIVE: To evaluate the predictive value of colony-forming units (CFU) of M. tuberculosis in cough aerosols on secondary infection and disease in household contacts in Brazil. METHODS: Adult sputum smear+ and culture+ pulmonary TB cases underwent a standard evaluation and were categorized according to aerosol CFU. We evaluated household contacts for infection at baseline and at 8 weeks with TST and IGRA, and secondary disease. RESULTS: We enrolled 48 index TB cases; 40% had negative aerosols, 27% low aerosols (<10 CFU) and 33% high aerosols (≥10 CFU). Of their 230 contacts, the proportion with a TST ≥10 mm at 8 weeks was 59%, 65% and 75%, respectively (p = 0.34). Contacts of high aerosol cases had greater IGRA readouts (median 4.6 IU/mL, IQR 0.02-10) when compared to those with low (0.8, 0.2-10) or no aerosol (0.1, 0-3.7; p = 0.08). IGRA readouts in TST converters of high aerosol cases (median 20 IU/mL, IQR 10-24) were larger than those from aerosol-negative (0.13, 0.04-3; p = o.o2). 8/9 (89%) culture+ secondary TB cases occurred in contacts of aerosol+ cases. CONCLUSION: Aerosol CFU predicts quantitatively IGRA readouts among household contacts of smear positive TB cases. Our results strengthen the argument of using cough aerosols to guide targeted preventive treatment strategies, a necessary component of current TB elimination projections.

Sputum sample collected over a period of 5 h: A reliable procedure for early bactericidal activity studies.

Our study was designed to test the hypothesis that an early morning sputum may be sufficient for calculation of early bactericidal activity (EBA). Patients underwent sputum collection randomly (spot, 5 h and 12 h) in consecutive days. The median CFU count in the spot samples group was 5.67 log10 CFU/mL compared to 6.17 log10 CFU/mL in 5 h and 6.23 log10 CFU/mL in 12 h samples. Inter-patient comparison showed low coefficient of variation for both 12 h (11%) and 5 h samples (10%). Intrapatient samples analysis demonstrated that the median bacillary load variation (0.037 log10 CFU/mL and 0.022 log10 CFU/mL for 5 and 12 h samples respectively) was comparable to the other EBA studies and did not vary significantly from one day of collection to another. We concluded that 5 h pooled sputum when collected appropriately in the morning can be sufficient for calculation of EBA.

The impact of ocular tuberculosis on vision after two months of intensive therapy

ABSTRACT Tuberculosis is an infectious disease of global importance with major economic and social burden accounting for 25% of all avoidable deaths in developing countries. Extrapulmonary involvement may occur either in association with clinically apparent pulmonary tuberculosis or in isolation. This cross-sectional descriptive study aimed to evaluate the impact of ocular tuberculosis in visual acuity at baseline and after two months of intensive anti-tuberculous therapy. A sample of 133 pulmonary tuberculosis patients, seven disseminated tuberculosis, and three pleural tuberculosis patients was evaluated. All patients underwent routine ophthalmic evaluation, including assessment of visual acuity, biomicroscopy, applanation tonometry, indirect ophthalmoscopy, and fluorescent angiography as appropriate. None of the patients had impaired visual acuity due to tuberculosis. A rate of 4.2% (6/143) of ocular involvement was found. None of the patients with ocular involvement were HIV-infected. Of the six patients with ocular involvement, five met the diagnostic criteria for probable and one for possible ocular lesions. As for the type of ocular lesions, two patients had bilateral findings: one had sclerouveitis and the second had choroidal nodules. The other four patients presented with unilateral lesions: peripheral retinal artery occlusion in the right eye (one case), choroidal nodules in the left eye (one case), and choroidal nodules in the right eye (two cases). Patients progressed favorably after two month of intensive therapy, with no significant reduction in vision.

The impact of ocular tuberculosis on vision after two months of intensive therapy.

Tuberculosis is an infectious disease of global importance with major economic and social burden accounting for 25% of all avoidable deaths in developing countries. Extrapulmonary involvement may occur either in association with clinically apparent pulmonary tuberculosis or in isolation. This cross-sectional descriptive study aimed to evaluate the impact of ocular tuberculosis in visual acuity at baseline and after two months of intensive anti-tuberculous therapy. A sample of 133 pulmonary tuberculosis patients, seven disseminated tuberculosis, and three pleural tuberculosis patients was evaluated. All patients underwent routine ophthalmic evaluation, including assessment of visual acuity, biomicroscopy, applanation tonometry, indirect ophthalmoscopy, and fluorescent angiography as appropriate. None of the patients had impaired visual acuity due to tuberculosis. A rate of 4.2% (6/143) of ocular involvement was found. None of the patients with ocular involvement were HIV-infected. Of the six patients with ocular involvement, five met the diagnostic criteria for probable and one for possible ocular lesions. As for the type of ocular lesions, two patients had bilateral findings: one had sclerouveitis and the second had choroidal nodules. The other four patients presented with unilateral lesions: peripheral retinal artery occlusion in the right eye (one case), choroidal nodules in the left eye (one case), and choroidal nodules in the right eye (two cases). Patients progressed favorably after two month of intensive therapy, with no significant reduction in vision.

Risk factors associated with cluster size of Mycobacterium tuberculosis (Mtb) of different RFLP lineages in Brazil.

BACKGROUND: Tuberculosis (TB) transmission is influenced by patient-related risk, environment and bacteriological factors. We determined the risk factors associated with cluster size of IS6110 RFLP based genotypes of Mycobacterium tuberculosis (Mtb) isolates from Vitoria, Espirito Santo, Brazil. METHODS: Cross-sectional study of new TB cases identified in the metropolitan area of Vitoria, Brazil between 2000 and 2010. Mtb isolates were genotyped by the IS6110 RFLP, spoligotyping and RDRio. The isolates were classified according to genotype cluster sizes by three genotyping methods and associated patient epidemiologic characteristics. Regression Model was performed to identify factors associated with cluster size. RESULTS: Among 959 Mtb isolates, 461 (48%) cases had an isolate that belonged to an RFLP cluster, and six clusters with ten or more isolates were identified. Of the isolates spoligotyped, 448 (52%) were classified as LAM and 412 (48%) as non-LAM. Our regression model found that 6-9 isolates/RFLP cluster were more likely belong to the LAM family, having the RDRio genotype and to be smear-positive (adjusted OR = 1.17, 95% CI 1.08-1.26; adjusted OR = 1.25, 95% CI 1.14-1.37; crude OR = 2.68, 95% IC 1.13-6.34; respectively) and living in a Serra city neighborhood decrease the risk of being in the 6-9 isolates/RFLP cluster (adjusted OR = 0.29, 95% CI, 0.10-0.84), than in the others groups. Individuals aged 21 to 30, 31 to 40 and > 50 years were less likely of belonging the 2-5 isolates/RFLP cluster than unique patterns compared to individuals < 20 years of age (adjusted OR = 0.49, 95% CI 0.28-0.85, OR = 0.43 95% CI 0.24-0.77and OR = 0. 49, 95% CI 0.26-0.91), respectively. The extrapulmonary disease was less likely to occur in those infected with strains in the 2-5 isolates/cluster group (adjustment OR = 0.45, 95% CI 0.24-0.85) than unique patterns. CONCLUSIONS: We found that a large proportion of new TB infections in Vitoria is caused by prevalent Mtb genotypes belonging to the LAM family and RDRio genotypes. Such information demonstrates that some genotypes are more likely to cause recent transmission. Targeting interventions such as screening in specific areas and social risk groups, should be a priority for reducing transmission.

Intensity of exposure to pulmonary tuberculosis determines risk of tuberculosis infection and disease.

Household contacts of pulmonary tuberculosis (TB) patients are at increased risk of TB infection and disease. However, their risk in relation to the intensity of exposure remains unknown.We studied smear-positive TB cases and their household contacts in Vitória, Brazil. We collected clinical, demographic and radiographic information from TB cases, and obtained tuberculin skin test (TST) and QuantiFERON-TB Gold (QFT) results from household contacts. We measured intensity of exposure using a proximity score and sleep location in relation to the TB index case and defined infection by TST ≥10 mm or QFT ≥0.35 UI·mL-1 We ascertained secondary TB cases by reviewing local and nationwide case registries.We included 160 TB index cases and 894 household contacts. 464 (65%) had TB infection and 23 (2.6%) developed TB disease. Risk of TB infection and disease increased with more intense exposures. In an adjusted analysis, the proximity score was associated with TB disease (OR 1.61, 95% CI 1.25-2.08; p<0.000); however, its diagnostic performance was only moderate.Intensity of exposure increased risk of TB infection and disease among household contacts; however, its diagnostic performance was still suboptimal. A biomarker to target preventive therapy is urgently needed in this at-risk population.

Genomic epidemiology of a national outbreak of post-surgical Mycobacterium abscessus wound infections in Brazil.

An epidemic of post-surgical wound infections, caused by a non-tuberculous mycobacterium, has been on-going in Brazil. It has been unclear whether one or multiple lineages are responsible and whether their wide geographical distribution across Brazil is due to spread from a single point source or is the result of human-mediated transmission. 188 isolates, collected from nine Brazilian states, were whole genome sequenced and analysed using phylogenetic and comparative genomic approaches. The isolates from Brazil formed a single clade, which was estimated to have emerged in 2003. We observed temporal and geographic structure within the lineage that enabled us to infer the movement of sub-lineages across Brazil. The genome size of the Brazilian lineage was reduced relative to most strains in the three subspecies of Mycobacterium abscessus and contained a novel plasmid, pMAB02, in addition to the previously described pMAB01 plasmid. One lineage, which emerged just prior to the initial outbreak, is responsible for the epidemic of post-surgical wound infections in Brazil. Phylogenetic analysis indicates that multiple transmission events led to its spread. The presence of a novel plasmid and the reduced genome size suggest that the lineage has undergone adaptation to the surgical niche.

Incident Mycobacterium tuberculosis infection in household contacts of infectious tuberculosis patients in Brazil.

BACKGROUND: In household contact investigations of tuberculosis (TB), a second tuberculin skin test (TST) obtained several weeks after a first negative result consistently identifies individuals that undergo TST conversion. It remains unclear whether this delay in M. tuberculosis infection is related to differences in the infectious exposure, TST boosting, partial host resistance, or some other factor. METHODS: We conducted a household contact study Vitória, Brazil. Between 2008 and 2013, we identified culture-positive pulmonary TB patients and evaluated their household contacts with both a TST and interferon gamma release assay (IGRA), and identified TST converters at 8-12 weeks post study enrollment. Contacts were classified as TST-positive (≥10 mm) at baseline, TST converters, or persistently TST-negative. We compared TST converters to TST-positive and to TST-negative contacts separately, using generalized estimating equations. RESULTS: We enrolled 160 index patients and 838 contacts; 523 (62.4%) were TST+, 62 (7.4%) TST converters, and 253 (30.2%) TST-. TST converters were frequently IGRA- at 8-12 weeks. In adjusted analyses, characteristics distinguishing TST converters from TST+ contacts (no contact with another TB patient and residence ownership) were different than those differentiating them from TST- contacts (stronger cough in index patient and contact BCG scar). CONCLUSIONS: The individual risk and timing of M. tuberculosis infection within households is variable and dependent on index patient, contact and environmental factors within the household, and the surrounding community. Our findings suggest a threshold effect in the risk of infection in humans.

Strains of Mycobacterium tuberculosis transmitting infection in Brazilian households and those associated with community transmission of tuberculosis.

Molecular epidemiologic studies have shown that the dynamics of tuberculosis transmission varies geographically. We sought to determine which strains of Mycobacterium tuberculosis (MTB) were infecting household contacts (HHC), and which were causing clusters of tuberculosis (TB) disease in Vitoria-ES, Brazil. A total of 741 households contacts (445 TST +) and 139 index cases were characterized according to the proportion of contacts in each household that had a tuberculin skin test positive: low (LT) (≤40% TST+), high (HT) (≥70% TST+) and (40-70% TST+) intermediate (IT) transmission. IS6110-RFLP and spoligotyping analysis were performed only 139 MTB isolates from index cases and 841 community isolates. Clustering occurred in 45% of the entire study population. There was no statistically significant association between MTB household transmission category and clustering. Within the household study population, the proportion of clusters in HT and LT groups was similar (31% and 36%, respectively; p = 0.82). Among index cases isolates associated with households demonstrating TST conversion, the frequency of unique pattern genotypes was higher for index cases of the LT compared to HT households (p = 0.03). We concluded that clusters and lineages associated with MTB infection in HT households had no proclivity for increased transmission of TB in the community.

Mycobacterial Hsp65 antigen upregulates the cellular immune response of healthy individuals compared with tuberculosis patients.

Previously we showed that 65-kDa Mycobacterium leprae heat shock protein (Hsp65) is a target for the development of a tuberculosis vaccine. Here we evaluated peripheral blood mononuclear cells (PBMC) from healthy individuals or tuberculosis patients stimulated with two forms of Hsp65 antigen, recombinant DNA that encodes Hsp65 (DNA-HSP65) or recombinant Hsp65 protein (rHsp65) in attempting to mimic a prophylactic or therapeutic study in vitro, respectively. Proliferation and cytokine-producing CD4+ or CD8+ cell were assessed by flow cytometry. The CD4+ cell proliferation from healthy individuals was stimulated by DNA-HSP65 and rHsp65, while CD8+ cell proliferation from healthy individuals or tuberculosis patients was stimulated by rHSP65. DNA-HSP65 did not improve the frequency of IFN-gamma+ cells from healthy individuals or tuberculosis patients. Furthermore, we found an increase in the frequency of IL-10-producing cells in both groups. These findings show that Hsp65 antigen activates human lymphocytes and plays an immune regulatory role that should be addressed as an additional antigen for the development of antigen-combined therapies.