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We determined the impact of the COVID-19 pandemic on mycobacterial diagnostic services. 40 laboratories from 22 countries completed an online questionnaire covering the redeployment of the laboratory infrastructure and/or staff for SARS-CoV-2 testing, staff shortages and supply chain disruptions. 28 laboratories reported monthly numbers of samples processed for mycobacterial investigations and monthly numbers of M. tuberculosis complex (MTBC) PCRs performed between October 1st 2018 and October 31st 2020. More than half (23/40) of the participating TB laboratories reported having performed COVID-19 diagnostics in the early phase of the pandemic, in part with negative impact on the mycobacterial service activities. All participating laboratories reported shortages of consumables and laboratory equipment due to supply chain issues. Average monthly sample numbers decreased by 24% between January 2020 and October 2020 compared to pre-pandemic averages. At the end of the study period, most participating laboratories had not returned to pre-pandemic average MTBC PCR throughput. [ABSTRACT FROM AUTHOR]

Background: Cystic echinococcosis (CE), caused by Echinococcus granulosus sensu lato, is a neglected zoonosis. Its diagnosis relies on imaging, supported by serology, while only imaging is useful for staging and follow-up. Since diagnostic tools and expertise are not widely available, new accurate and easily implementable assays for the diagnosis and follow-up of CE are highly needed. Methodology/Principal Findings: We aimed to identify new E. granulosus antigens through a bioinformatics selection applied to the parasite genome, followed by peptide microarray screening and validation in ELISA, using independent panels of sera from patients with hepatic CE and clinically relevant controls. From 950 proteins selected in silico, 2,379 peptides were evaluated by microarray for IgG reactivity and eight candidates selected for validation. Reactivity to one peptide was significantly higher in the CE group (p = 0.044), but had suboptimal diagnostic accuracy. Conclusions/Significance: Here we performed bioinformatics analysis and peptide microarray for antigen discovery, useful for the diagnosis of CE. Eight candidates were selected and validated. Reactivity to one peptide associated to CE but had suboptimal diagnostic accuracy. Importantly, the database developed in this study may be used to identify other antigenic candidates for CE diagnosis and follow-up. Author summary: Cystic echinococcosis (CE) is a complex, neglected parasitic zoonosis. Tools and expertise in the diagnosis of CE are not widely available and the development of accurate and easily implementable diagnostic assays for CE has been highlighted as a priority also by the WHO. Antigen discovery for the immunodiagnosis of CE has relied so far on classical methods, which are impractical and poorly sensitive. Here, starting from the Echinococcus granulosus sensu stricto genome, we aimed to identify antigenic peptides having a potential for CE diagnosis based on a multiple-criteria strategy involving the bioinformatics selection of proteins, followed by screening by peptide microarray and validation by ELISA, using a panel of well-characterized sera from patients with hepatic CE in all development stages, and from clinically relevant controls. This methodology has virtually never been applied in the field of CE. Importantly, the developed database can be used to identify other E. granulosus s.s. antigenic candidates. [ABSTRACT FROM AUTHOR]

Background: Cystic echinococcosis (CE) is a complex disease for which clear understanding of clinical manifestations is needed to avoid misdiagnosis, inappropriate treatment, and severe complications. We evaluated the accuracy of a whole-blood stimulation test based on Interleukin (IL)-4 detection in response to Antigen B (AgB) of Echinococcus granulosus sensu lato to discriminate cyst viability and detect cyst reactivation in patients with hepatic CE. Methodology/Principal findings: Thirty patients with CE3b cysts and 37 patients with spontaneously-inactivated CE4-CE5 cysts were recruited (T0). After enrollment, 5 patients with CE3b cysts received albendazole, resulting in cyst solidification (CE4) in 4/5. Within a two-year follow-up, the whole-blood test was repeated at two time-points, in ≥14 (T1) and in ≥4 (T2) patients per group. IL-4 and a panel of other soluble factors were measured in the stimulated plasma. Baseline IL-4 levels were significantly higher in patients with CE3b compared to those with CE4 cysts (p = 0.006). Test accuracy for CE3b diagnosis had a sensitivity of 33–60% and a specificity of 76–95%, depending on the cut-off applied. Overall, IL-4 levels did not change significantly over time in either group; however, patients within the CE3b group showed a significant decrease of IL-1ra, IL-6, IL-8, G-CSF, IFN-γ, IP-10, MCP-1, MIP-1α, FGF at T1 compared to T0 (p≤0.042). Conclusions/Significance: Whole-blood IL-4-response to AgB is significantly higher in patients with active compared to inactive CE but apparently not modulated over time after treatment. On the contrary, the levels of IL-1ra, IL-6, IL-8, G-CSF, IFN-γ, IP-10, MCP-1, MIP-1α, FGF significantly decreased in active CE during follow-up. Additional studies are needed to understand whether these findings might have a clinical significance for patients' follow-up. Author summary: Cystic echinococcosis (CE) is a complex disease for which a clear understanding of clinical manifestations is needed to avoid misdiagnosis, inappropriate treatment, and severe complications. The development of a laboratory test able to assess CE cyst viability and reactivation over time would facilitate the classification of patients and cysts, their rational assignment to a clinical management option and the implementation of a regular follow-up. Here, we evaluated the accuracy of a cytokine-based whole-blood test to detect the reactivation and the viability of hepatic CE cysts. The results showed that IL-4-response to AgB is not modulated over-time after treatment, while, IL-1ra, IL-6, IL-8, G-CSF, IFN-γ, IP-10, MCP-1, MIP-1α, FGF levels significantly decreased in active CE during follow-up. Additional studies are needed to understand whether these findings might have a clinical significance for patients' follow-up. [ABSTRACT FROM AUTHOR]

The impact of tuberculosis and of anti-tuberculosis therapy on composition and modification of human lung microbiota has been the object of several investigations. However, no clear outcome has been presented so far and the relationship between M. tuberculosis pulmonary infection and the resident lung microbiota remains vague. In this work we describe the results obtained from a multicenter study of the microbiota of sputum samples from patients with tuberculosis or unrelated lung diseases and healthy donors recruited in Switzerland, Italy and Bangladesh, with the ultimate goal of discovering a microbiota-based biomarker associated with tuberculosis. Bacterial 16S rDNA amplification, high-throughput sequencing and extensive bioinformatic analyses revealed patient-specific flora and high variability in taxon abundance. No common signature could be identified among the individuals enrolled except for minor differences which were not consistent among the different geographical settings. Moreover, anti-tuberculosis therapy did not cause any important variation in microbiota diversity, thus precluding its exploitation as a biomarker for the follow up of tuberculosis patients undergoing treatment. [ABSTRACT FROM AUTHOR]

Rationale: Treatment of multi-drug resistant Tuberculosis (MDR-TB) is challenging because it mostly relies on drugs with lower efficacy and greater toxicity than those used for drug-susceptible TB. Objectives: Aim of the study was to describe the frequency and type of adverse drug reactions in a cohort of MDR-TB patients and their potential impact on treatment outcome. Methods: We conducted a retrospective study in a cohort of MDR-TB patients enrolled at a tertiary referral hospital in Italy from January 2008 to December 2016. The records of patients were reviewed for epidemiological, clinical, microbiological and adverse drug reactions data. Results: Seventy-four MDR-TB patients (mean age 32 years, 58.1% males, 2 XDR, 12 pre-XDR TB) were extracted from the Institute data base and included in the retrospective study cohort in the evaluation period (January 2008—December 2016). Median length of treatment duration was 20 months (IQR 14–24). Treatment outcome was successful in 57 patients (77%; 51 cured, 6 treatment completed); one patient died and one failed (2.7% overall); 15 patients were lost to follow-up (20.3%). Sixty-six (89.2%) presented adverse drug reactions during the whole treatment period. Total number of adverse drug reactions registered was 409. Three hundred forty-six (84.6%) were classified as adverse events (AEs) and 63 (15.4%) were serious AEs (SAEs). One third of the total adverse drug reactions (134/409; 32.8%) was of gastrointestinal origin, followed by 47/409 (11.5%) ototoxic drug reactions, thirty-five (8.6%) regarded central nervous system and 33 (8.1%) affected the liver. All 63 SAEs required treatment suspension with 61 SAEs out of 63 (96.8%) occurring during the first six months of treatment. Factors associated with unsuccessful treatment outcome were smoking (p = 0.039), alcohol abuse (p = 0.005) and homeless condition (p = 0.044). Neither the number of antitubercular drugs used in different combinations nor the number of AEs showed significant impact on outcome. Patients who completed the treatment experienced a greater number of AEs and SAEs (p < 0.001) if compared to lost to follow-up patients. Conclusions: Our data demonstrate that, despite the high frequency of adverse drug reactions and long term therapy, the clinical management of MDR-TB patients in a referral center could reach successful treatment according to WHO target, by implementing active and systematic clinical and laboratory assessment to detect, report and manage suspected and confirmed adverse drug reactions. [ABSTRACT FROM AUTHOR]

Individuals exposed to Mycobacterium tuberculosis (Mtb) may be infected and remain for the entire life in this condition defined as latent tuberculosis infection (LTBI) or develop active tuberculosis (TB). Among the multiple factors governing the outcome of the infection, dendritic cells (DCs) play a major role in dictating antibacterial immunity. However, current knowledge on the role of the diverse components of human DCs in shaping specific T-cell response during Mtb infection is limited. In this study, we performed a comparative evaluation of peripheral blood circulating DC subsets as well as of monocyte-derived Interferon-α DCs (IFN-DCs) from patients with active TB, subjects with LTBI and healthy donors (HD). The proportion of circulating myeloid BDCA3+ DCs (mDC2) and plasmacytoid CD123+ DCs (pDCs) declined significantly in active TB patients compared to HD, whereas the same subsets displayed a remarkable activation in LTBI subjects. Simultaneously, the differentiation of IFN-DCs from active TB patients resulted profoundly impaired compared to those from LTBI and HD individuals. Importantly, the altered developmental trait of IFN-DCs from active TB patients was associated with down-modulation of IFN-linked genes, marked changes in molecular signaling conveying antigen (Ag) presentation and full inability to induce Ag-specific T cell response. Thus, these data reveal an important role of IFN-α in determining the induction of Mtb-specific immunity. [ABSTRACT FROM AUTHOR]

Introduction: RD1-based Interferon-γ Release Assays (IGRAs) cannot distinguish latent from active tuberculosis (TB) disease. Conversely, a positive response to heparin-binding haemagglutinin (HBHA)-based IGRAs, among TB-infected subjects, correlates with Mycobacterium tuberculosis (Mtb) containment and low risk of TB progression. The aim of this study was to characterize HBHA-immune responses in HIV-infected and uninfected subjects with active TB or latent TB infection (LTBI). Methods: 49 subjects were prospectively enrolled: 22 HIV-uninfected (13 TB, 9 LTBI) and 27 HIV-infected (12 HIV-TB, 15 HIV-LTBI). Whole blood and peripheral blood mononuclear cells were stimulated with HBHA and RD1 antigens. Interferon (IFN)γ release was evaluated by ELISA whereas cytokine profile [IFNγ, tumor necrosis (TNF)α, interleukin (IL)2] and phenotype (CD45RA, CCR7) by flow cytometry. Results: Among LTBI individuals, HBHA stimulation induced IFNγ release in all the HIV-uninfected, while, only 4/15 HIV-infected responded. Within the active TB, only 5/13 HIV-uninfected and 1/12 HIV-TB patients responded. Interestingly, by cytometry we showed that CD4+ T-cells response to HBHA was significantly impaired in the HIV-infected subjects with TB or LTBI compared to the HIV-uninfected subjects. The phenotype of HBHA-specific CD4 T-cells showed a predominantly central memory (CM) and effector memory (EM) phenotype without differences among the groups. Differently, HBHA-specific CD8+ T-cells, showed mainly a CM and naïve phenotype in LTBI group while TB, HIV-LTBI and HIV-TB groups were characterized by EM or terminally differentiated phenotypes. Interestingly, differently than what observed for RD1, the cytokine profile of HBHA-specific T-cells evaluated by cytometry showed that the CD4+ T-cells were mostly monofunctional. Conversely, CD8-specific T-cells were mostly monofunctional for both HBHA and RD1 stimulations. Conclusions: These results characterize the impact of HIV infection in CD4- and CD8-specific response to HBHA in both LTBI and TB patients. HIV infection impairs the CD4 response to HBHA and likely this may lead to an impairment of TB control. [ABSTRACT FROM AUTHOR]

Background: During pulmonary tuberculosis (PTB) antibodies are generated to trehalose esters of mycolic acids which are cell wall lipids of Mycobacterium tuberculosis (Mtb). Attempts have been made to use these complex natural mixtures in serological tests for PTB diagnosis. Aim: The aim of this work was to determine whether a serological test based on a panel of defined individual trehalose esters of characteristic synthetic mycolic acids has improved diagnostic accuracy in distinguishing patients with culture positive PTB from individuals who were Mtb culture negative. Method: One hundred serum samples from well-characterized patients with presumptive tuberculosis, and diagnosed as having pulmonary smear and culture positive TB, or being culture and smear negative were evaluated by ELISA using different combinations of synthetic antigens and secondary antibodies. Using cut-off values determined from these samples, we validated this study blind in samples from a further 249 presumptive TB patients. Results: With the first 100 samples, detailed responses depended both on the precise structure of the antigen and on the secondary antibody. Using a single antigen, a sensitivity/specificity combination for smear and culture positive PTB detection of 85 and 88% respectively was achieved; this increased to 96% and 95% respectively by a statistical combination of the results with seven antigens. In the blind study a sensitivity/specificity of 87% and 83% was reached with a single antigen. With some synthetic antigens, the responses from all 349 samples were significantly better than those with the natural mixture. Combining the results for seven antigens allowed a distinction between culture positive and negative with a ROC AUC of 0.95. Conclusion: We have identified promising antigen candidates for serological assays that could be used to diagnose PTB and which could be the basis of a much-needed, simple, rapid diagnostic test that would bring care closer to communities. [ABSTRACT FROM AUTHOR]

Tuberculosis (TB) is one of the most important cause of morbidity and death among infectious diseases, and continuous efforts are needed to improve diagnostic tools and therapy. Previous published studies showed that the absolute cells number of monocytes or lymphocytes in peripheral blood or yet the ratio of monocytes to lymphocytes displayed the ability to predict the risk of active TB. In the present study we evaluated the ratio of monocytes to lymphocytes variation and we also analyzed the ex-vivo expression of CD64 on monocytes as tools to identify biomarkers for discriminating TB stages. Significant differences were found when the average ratio of monocytes to lymphocytes of active TB patients was compared with latent TB infection (LTBI) subjects, cured TB and healthy donors (HD). By the receiver operator characteristics (ROC) curve analysis the cut-off value of 0.285, allowed the discrimination of active TB from HD, with a sensitivity of 91.04% and a specificity of 93.55% (95% of confidence interval: 0.92–0.99). The ROC curve analysis comparing TB patients and LTBI groups, led to a sensitivity and the specificity of the assay of 85.07% and 85.71%, respectively (95% of confidence interval: 0.85 to 0.96). The upregulation of CD64 expression on circulating monocytes in active TB patients could represent an additional biomarker for diagnosis of active TB. In conclusion, we found that the ML ratio or monocyte absolute count or phenotypic measures show predictive value for active TB. [ABSTRACT FROM AUTHOR]

B-cells not only produce immunoglobulins and present antigens to T-cells, but also additional key roles in the immune system. Current knowledge on the role of B-cells in infections caused by intracellular bacteria is fragmentary and contradictory. We therefore analysed the phenotypical and functional properties of B-cells during infection and disease caused by Mycobacterium tuberculosis (Mtb), the bacillus causing tuberculosis (TB), and included individuals with latent TB infection (LTBI), active TB, individuals treated successfully for TB, and healthy controls. Patients with active or treated TB disease had an increased proportion of antibodies reactive with mycobacteria. Patients with active TB had reduced circulating B-cell frequencies, whereas only minor increases in B-cells were detected in the lungs of individuals deceased from TB. Both active TB patients and individuals with LTBI had increased relative fractions of B-cells with an atypical phenotype. Importantly, these B-cells displayed impaired proliferation, immunoglobulin- and cytokine- production. These defects disappeared upon successful treatment. Moreover, T-cell activity was strongest in individuals successfully treated for TB, compared to active TB patients and LTBI subjects, and was dependent on the presence of functionally competent B-cells as shown by cellular depletion experiments. Thus, our results reveal that general B-cell function is impaired during active TB and LTBI, and that this B-cell dysfunction compromises cellular host immunity during Mtb infection. These new insights may provide novel strategies for correcting Mtb infection-induced immune dysfunction towards restored protective immunity. [ABSTRACT FROM AUTHOR]

Cystic echinococcosis (CE) is a complex disease caused by Echinococcus granulosus (E. granulosus), and its immunophatogenesis is still not clearly defined. A peculiar feature of chronic CE is the coexistence of Th1 and Th2 responses. It has been suggested that Th1 cytokines are related to disease resistance, whereas Th2 cytokines are related to disease susceptibility and chronicity. The aim of this study was to evaluate, by multi-parametric flow cytometry (FACS), the presence of CE specific immune signatures. Methodology/Principal Findings We enrolled 54 subjects with suspected CE; 42 of them had a confirmed diagnosis, whereas 12 were classified as NO-CE. Based on the ultrasonography images, CE patients were further categorized as being in "active stages" (25) and "inactive stages" (17). The ability of CD4+ T-cells to produce IFN-γ, IL-2, TNF-α, Th2 cytokines or IL-10 was assessed by FACS on antigen-specific T-cells after overnight stimulation with Antigen B (AgB) of . . Cytokine profiles were evaluated in all the enrolled subjects. The results show that none of the NO-CE subjects had a detectable AgB-specific response. Among the CE patients, the frequency and proportions of AgB-specific CD4+ T-cells producing IL- 2+ TNF-α+ Th2+ or TNF-α+ Th2+ were significantly increased in the "active stages" group compared to the "inactive stages" group. Moreover, an increased proportion of the total polyfunctional subsets, as triple-and double-functional CD4 T-cells, was found in CE patients with active disease. The response to the mitogen, used as a control stimulus to evaluate the immune competence status, was characterized by the same cytokine subsets in all the subjects enrolled, independent of CE. Conclusions We demonstrate, for the first time to our knowledge, that polyfunctional T-cell subsets as IL-2+ TNF-α+ Th2+ triple-positive and TNF-α+ Th2+ double-positive specific T-cells associate with cyst biological activity. These results contribute to increase knowledge of CE immunophatogenesis and the disease outcome in terms of control and persistence. [ABSTRACT FROM AUTHOR]

Objectives: To evaluate the accuracy of the QuantiFERON-TB Gold assay (QFT-IT) in children with suspected active or latent TB infection (LTBI). Methods: A retrospective study was conducted on 621 children (0–14 years old) evaluated for TB infection or disease. Following clinical assessment, children were tested with the QFT-IT assay. Results: Among the 140 active TB suspects, we identified 19 cases of active disease. The overall sensitivity for active TB was 87.5%, ranging from 62.5% in children 25–36 months old to 100% in children older than 49 months. The overall specificity for active TB was 93.6%. Among the 481 children tested for LTBI screening, 38 scored positive and all but 2 had at least one risk factor for TB infection. Among the 26 children with indeterminate results, bacterial, viral or fungal pneumonia were later diagnosed in 11 (42.3%) cases and non-TB related extra-pulmonary infections in 12 (46.1%). Conclusions: Our results indicate that the children's response to QFT-IT associates to active TB and risk factors for LTBI. Moreover, we show that mitogen response is also found in children of 1 year of age, providing support for QFT-IT use also in young children. [ABSTRACT FROM AUTHOR]

Background: Interferon-release assays (IGRAs) for diagnosing active pulmonary tuberculosis (PTB) are not yet fully validated, particularly in high TB-endemic areas as the People's Republic of China (PRC). The aim of this report was to assess the performance of the QuantiFERON-TB Gold In-tube (QFT-GIT) and tuberculin skin test (TST), in addition to microbiological results, as contributors for diagnosing active PTB in the PRC. Methods/Principal Findings: A total of 300 PTB patients, 41 disease controls (DC) and 59 healthy community controls (HCC) were included prospectively between May 2010 and April 2011 from two provinces of the PRC (Heilongjiang and Zhejiang). The QFT-GIT and TST yielded an overall sensitivity for active TB of 80.9% and 86.2%, and a specificity of 36.6% and 26.8%, respectively. The province of origin and smear microscopy status did not significantly impact the diagnostic values for PTB. However, using the TST with a 10 mm cut-off point, a significantly higher proportion of LTBI was observed in the DC than the HCC (p=0.01). Discordant results between the QFT-GIT and TST were found among 1/3 of the PTB, HCC and DC. Two-thirds of the individuals presented TST-positive/QFT-GIT-negative discordant results. The TST-negative/QFT-GIT-positive result was not associated with age or bacillary load. Cumulative QFT-GIT and TST positive results increased the overall sensitivity (95.9%), but it was associated with a dramatic decrease of the overall specificity (24.8%) leading to a suboptimal PPV (80.1%) and a low NPV (61.1%). Conclusions/Significance: The usefulness of the QFT-GIT to diagnose active TB in high TB-endemic countries remains doubtful because like the TST, the QFT-GIT cannot distinguish between LTBI and active TB. Used as single stand-alone tests, both the QFT-GIT and TST have very limited roles in the diagnosis of active PTB. However, the combined use of SM, the TST and QFT-GIT may allow for the exclusion of ATB. [ABSTRACT FROM AUTHOR]

Tuberculosis (TB) is still the principal cause of death caused by a single infectious agent, and the balance between the bacillus and host defense mechanisms reflects the different manifestations of the pathology. The aim of this work was to study the role of myeloid-derived suppressor cells (MDSCs) during active pulmonary tuberculosis at the site of infection. We observed an expansion of MDSCs in the lung and blood of patients with active TB, which are correlated with an enhanced amount of nitric oxide in the plasma. We also found that these cells have the remarkable ability to suppress T-cell response, suggesting an important role in the modulation of the immune response against TB. Interestingly, a trend in the diminution of MDSCs was found after an efficacious anti-TB therapy, suggesting that these cells may be used as a potential biomarker for monitoring anti-TB therapy efficacy. [ABSTRACT FROM AUTHOR]

Several studies showed that assessing levels of specific circulating microRNAs (miRNAs) is a non-invasive, rapid, and accurate method for diagnosing diseases or detecting alterations in physiological conditions. We aimed to identify a serum miRNA signature to be used for the diagnosis of tuberculosis (TB). To account for variations due to the genetic makeup, we enrolled adults from two study settings in Europe and Africa. The following categories of subjects were considered: healthy (H), active pulmonary TB (PTB), active pulmonary TB, HIV co-infected (PTB/HIV), latent TB infection (LTBI), other pulmonary infections (OPI), and active extra-pulmonary TB (EPTB). Sera from 10 subjects of the same category were pooled and, after total RNA extraction, screened for miRNA levels by TaqMan low-density arrays. After identification of “relevant miRNAs”, we refined the serum miRNA signature discriminating between H and PTB on individual subjects. Signatures were analyzed for their diagnostic performances using a multivariate logistic model and a Relevance Vector Machine (RVM) model. A leave-one-out-cross-validation (LOOCV) approach was adopted for assessing how both models could perform in practice. The analysis on pooled specimens identified selected miRNAs as discriminatory for the categories analyzed. On individual serum samples, we showed that 15 miRNAs serve as signature for H and PTB categories with a diagnostic accuracy of 82% (CI 70.2–90.0), and 77% (CI 64.2–85.9) in a RVM and a logistic classification model, respectively. Considering the different ethnicity, by selecting the specific signature for the European group (10 miRNAs) the diagnostic accuracy increased up to 83% (CI 68.1–92.1), and 81% (65.0–90.3), respectively. The African-specific signature (12 miRNAs) increased the diagnostic accuracy up to 95% (CI 76.4–99.1), and 100% (83.9–100.0), respectively. Serum miRNA signatures represent an interesting source of biomarkers for TB disease with the potential to discriminate between PTB and LTBI, but also among the other categories. [ABSTRACT FROM AUTHOR]

Background: Although North American hospitals are switching from tuberculin testing (TST) to interferon-gamma release assays (IGRAs), data are limited on the association between occupational exposure and serial QuantiFERON-TB Gold In-Tube (QFT) results in healthcare workers (HCWs). Methods: In a cohort of Canadian HCWs, TST and QFT were performed at study enrolment (TST1 and QFT1) and 1 year later (TST2 and QFT2). Conversion and reversion rates were estimated, and correlation with TB exposure was assessed. Results: Among 258 HCWs, median age was 36.8 years, 188/258 (73%) were female and 183/258 (71%) were Canadian-born. In 245 subjects with a negative QFT1 we found a QFT conversion rate of 5.3% (13/245, 95% CI 2.9-8.9%). Using more stringent definitions, QFT conversion rates ranged from 2.0 to 5.3%. No TST conversions were found among the 241 HCWs with negative TST1, and no measure of recent TB exposure was associated with QFT conversions. In the 13 HCWs with a positive QFT1, 62% reverted. Conclusion: Using the conventional QFT conversion definition, we found a higher than expected rate of conversion. Recent occupational exposures were not associated with QFT conversions, and no TST conversions occurred in this cohort, suggesting the 'conversions' may not reflect new TB infection. [ABSTRACT FROM AUTHOR]

Tuberculosis (TB), caused by Mycobacterium tuberculosis (M.tb), remains the leading cause of mortality from a single infectious agent. Each year around 9 million individuals newly develop active TB disease, and over 2 billion individuals are latently infected with M.tb worldwide, thus being at risk of developing TB reactivation disease later in life. The underlying mechanisms and pathways of protection against TB in humans, as well as the dynamics of the host response to M.tb infection, are incompletely understood. We carried out whole-genome expression profiling on a cohort of TB patients longitudinally sampled along 3 time-points: during active infection, during treatment, and after completion of curative treatment. We identified molecular signatures involving the upregulation of type-1 interferon (α/β) mediated signaling and chronic inflammation during active TB disease in an Indonesian population, in line with results from two recent studies in ethnically and epidemiologically different populations in Europe and South Africa. Expression profiles were captured in neutrophil-depleted blood samples, indicating a major contribution of lymphocytes and myeloid cells. Expression of type-1 interferon (a/b) genes mediated was also upregulated in the lungs of M.tb infected mice and in infected human macrophages. In patients, the regulated gene expression-signature normalized during treatment, including the type-1 interferon mediated signaling and a concurrent opposite regulation of interferon-gamma. Further analysis revealed IL15RA, UBE2L6 and GBP4 as molecules involved in the type-I interferon response in all three experimental models. Our data is highly suggestive that the innate immune type-I interferon signaling cascade could be used as a quantitative tool for monitoring active TB disease, and provide evidence that components of the patient's blood gene expression signature bear similarities to the pulmonary and macrophage response to mycobacterial infection. [ABSTRACT FROM AUTHOR]

Role of microRNA (miRNA) has been highlighted in pathogen-host interactions recently. At present, their role in active pulmonary tuberculosis is unknown. The aim of the study was to delineate miRNA expression in sputum supernatant of patients with active pulmonary tuberculosis. Expression of miRNAs was evaluated by microarray analysis and differentially expressed miRNAs were validated by RT-qPCR. Secreted cytokines TNF-α and IL-6 were measured by ELISA. We found that 95 miRNAs were differentially expressed between tuberculosis group and controls. More miRNAs (52 out of 95 miRNAs) were underexpressed than overexpressed during tuberculosis infection. Overexpression of miR-3179, miR-147 and underexpression of miR-19b-2* in TB group compared with controls were confirmed in the validation cohort. TNF-α and IL-6 levels were not significantly altered between TB group and controls. For the first time, differential expression of miRNAs in sputum was found in active pulmonary tuberculosis. The study provides rationale for identifying the role of miRNAs in the pathogenesis of pulmonary tuberculosis and indicates potential for miRNA- based therapeutic strategies. [ABSTRACT FROM AUTHOR]

Objective: To determine the variation of IFN-c and IL-17 responses to M. tuberculosis antigens in healthy TST+ humans. Methods: We isolated peripheral blood mononuclear cells from 21 TST+ healthy adults, stimulated them with phytohemagglutinin (PHA), PPD, Ag85B, ESAT-6, and live M. bovis BCG, and assayed IFN-γ and IL-17 secretion by ELISA in supernatants after 24 or 72 hours of incubation respectively. Results: As in other studies, we found a wide range of IFN-γ responses to M. tuberculosis antigens; the variation significantly exceeded that observed in the same donors to the polyclonal T cell stimulus, phytohemagglutinin (PHA). In addition, we assayed IL-17 secretion in response to the same stimuli, and found less subject-to-subject variation. Analysis of the ratio of IFN-γ to IL-17 secretion on a subject-to-subject basis also revealed a wide range, with the majority of results distributed in a narrow range, and a minority with extreme results all of which were greater than that in the majority of subjects. The data suggest that study of exceptional responses to M. tuberculosis antigens may reveal immunologic correlates with specific outcomes of M. tuberculosis infection. Conclusion: Variation of IFNc and IFN-γ/IL-17 responses to mycobacterial antigens exceeds that of responses to the polyclonal stimulus, PHA, in TST positive healthy humans. This indicates a quantitative spectrum of human immune responses to infection with M. tuberculosis. Since the outcome of human infection with M. tuberculosis varies greatly, systematic study of multiple immune responses to multiple antigens is likely to reveal correlations between selected immune responses and the outcomes of infection. [ABSTRACT FROM AUTHOR]

Rationale: Due to the invasive nature of the procedures involved, most studies of Mycobacterium tuberculosis (Mtb)-specific immunity in humans have focused on the periphery rather than the site of active infection, the lung. Recently, antigens associated with Mtb-latency and -dormancy have been described using peripheral blood (PB) cells; however their response in the lung is unknown. The objective of this report was to evaluate, in patients prospectively enrolled with suspected active tuberculosis (TB), whether the latency antigen Rv2628 induces local-specific immune response in bronchoalveolar lavage (BAL) cells compared to PB cells. Material/Methods: Among the 41 subjects enrolled, 20 resulted with active TB. Among the 21 without active disease, 9 were defined as subjects with latent TB-infection (LTBI) [Quantiferon TB Gold In-tube positive]. Cytokine responses to Rv2628 were evaluated by enzyme linked immunospot (ELISPOT) assay and flow cytometric (FACS) analysis. RD1-secreted antigen stimulation was used as control. Results: There was a significantly higher frequency of Rv2628- and RD1-specific CD4+ T-cells in the BAL of active TB patients than in PB. However the trend of the response to Rv2628 in subjects with LTBI was higher than in active TB in both PB and BAL, although this difference was not significant. In active TB, Rv2628 and RD1 induced a cytokine-response profile mainly consisting of interferon (IFN)-γ-single-positive over double-IFN-γ/interleukin (IL)-2 T-cells in both PB and BAL. Finally, BALspecific CD4+ T-cells were mostly effector memory (EM), while peripheral T-cell phenotypes were distributed among naïve, central memory and terminally differentiated effector memory T-cells. Conclusions: In this observational study, we show that there is a high frequency of specific T-cells for Mtb-latency and RD1- secreted antigens (mostly IFN-γ-single-positive specific T-cells with an EM phenotype) in the BAL of active TB patients. These data may be important for better understanding the pathogenesis of TB in the lung. [ABSTRACT FROM AUTHOR]

Background: A challenge in tuberculosis (TB) research is to develop a new immunological test that can help distinguish, among subjects responsive to QuantiFERON TB Gold In tube (QFT-IT), those who are able to control Mtb replication (remote LTBI, recent infection and past TB) from those who cannot (active TB disease). IFN-γ response to the Heparin-bindinghemagglutinin (HBHA) of Mtb has been associated with LTBI, but the cumbersome procedures of purifying the methylated and immunological active form of the protein from Mtb or M. bovis Bacillus Calmette et Guerin (BCG) have prevented its implementation in a diagnostic test. Therefore, the aim of the present study was to evaluate the IFN-γ response to methylated HBHA of Mtb produced in M. smegmatis (rHBHAms) in individuals at different stages of TB who scored positive to QFT-IT. Methodology/Principal Findings: 87 individuals at different stages of TB who scored positive to QFT-IT were selected. IFN-γ response to in vitro whole blood stimulation with rHBHAms was evaluated by short-term and long-term tests and detected by ELISA or flow cytometry. We demonstrated that the IFN-γ response to rHBHAms is mediated by CD4+ T-cells with an effector-memory phenotype. This response, evaluated by short-term-tests, is significantly lower in active TB than in remote LTBI (p = 0.0010) and past TB (p = 0.0152). These results were confirmed by long-term tests. The qualitative data confirmed that IFN-γ responses higher than the cut-off point identified by ROC analysis are associated with the status of non-active disease. Conclusions: In this study we show that the T-cell response to a recombinant and methylated HBHA of Mtb produced in M. smegmatis is useful to discriminate between active and non-active TB disease among those responsive to QFT-IT in a whole blood system. Further studies are needed to improve the accuracy of the assay. [ABSTRACT FROM AUTHOR]

Background: The suboptimal sensitivity of Interferon (IFN)-γ-based in-vitro assays, especially in immunocompromised individuals, emphasizes the need for alternative markers for diagnosing tuberculosis (TB). The objective of this study was to evaluate whether interferon-inducible protein (IP)-10, monocyte chemotactic protein (MCP)-2 and interleukin (IL)-2 can be useful biomarkers for evaluating a specific response to RD1 antigens associated to active TB disease in HIV-infected individuals. Methodology/Principal Findings: The study was carried out in India, the country with the highest TB burden in the world. Sixty-six HIV-infected individuals were prospectively enrolled, 28 with active-pulmonary-TB and 38 without. The whole blood assay based on RD1-selected peptides (experimental test) and QuantiFERON-TB Gold In tube (QFT-IT) was performed. Plasma was harvested at day-1-post-culture and soluble factors were evaluated by ELISA. The results indicate that by detecting IP-10, the sensitivity of the experimental test and QFT-antigen (75% and 85.7% respectively) for active TB was higher compared to the same assays based on IFN-γ (42.9% and 60.7% respectively) and was not influenced by the ability to respond to the mitogen. By detecting IP-10, the specificity of the experimental test and QFT-antigen (57.9% and 13.2% respectively) for active TB was lower than what was reported for the same assays using IFN-γ-detection (78.9% and 68.4% respectively). On the other side, in vitro IL-2 and MCP-2 responses were not significantly associated with active TB. Conclusions: HIV infection does not impair RD1-specific response detected by IP-10, while it significantly decreases IFN-γ-mediated responses. At the moment it is unclear whether higher detection is related to higher sensitivity or lower specificity of the assay. Further studies in high and low TB endemic countries are needed to elucidate this. [ABSTRACT FROM AUTHOR]

Background: The clinical application of IFN-c release assays (IGRAs) has recently improved the diagnosis of latent tuberculosis infection. In a multicenter study of the Tuberculosis Network European Trialsgroup (TBNET) we aimed to ascertain in routine clinical practice the accuracy of a novel assay using selected peptides encoded in the mycobacterial genomic region of difference (RD) 1 for the diagnosis of active tuberculosis in comparison with tuberculin skin test (TST), QuantiFERON-TB GOLD In-Tube (Cellestis Ltd., Carnegie, Australia) and T-SPOT.TB (Oxfordimmunotec, Abingdon, UK). Principal Findings: 425 individuals from 6 different European centres were prospectively enrolled. We found that sensitivity of the novel test, TST, QuantiFERON-TB GOLD In-Tube and T-SPOT.TB was respectively 73.1%, 85.3%, 78.1%, and 85.2%; specificity was respectively 70.6%, 48.0%, 61.9% and 44.3%; positive likelihood ratios were respectively 2.48, 1.64, 2.05, and 1.53; negative likelihood ratios were respectively 0.38, 0.31, 0.35, 0.33. Sensitivity of TST combined with the novel test, QuantiFERON-TB GOLD In-Tube and T-SPOT.TB increased up to 92.4%, 97.7% and 97.1%, respectively. The likelihood ratios of combined negative results of TST with, respectively, the novel test, QuantiFERON-TB GOLD In-Tube and T-SPOT.TB were 0.19, 0.07 and 0.10. Conclusions: The assay based on RD1 selected peptides has similar accuracy for active tuberculosis compared with TST and commercial IGRAs. Then, independently of the spectrum of antigens used in the assays to elicit mycobacterial specific immune responses, the novel test, IGRAs, and the TST do not allow an accurate identification of active tuberculosis in clinical practice. However, the combined use of the novel assay or commercial IGRAs with TST may allow exclusion of tuberculosis. [ABSTRACT FROM AUTHOR]

Background: The aim of this multi-centric prospective study in India was to assess the accuracy of a serological test as an additional tool for diagnosing active tuberculosis (ATB). In particular, an assay based on ELISA using a phenolic glycolipid (PGL-Tb1) or a fusion protein (ESAT-6/CFP10) was compared to the tuberculin skin test (TST) and the microbiological results according to HIV status., Methods: Individuals with and without ATB and HIV infection were enrolled. Serology and TST results were analyzed per se and in combination with the microbiological data., Results: Among the 778 ATB patients, 102 were HIV-infected, 316 HIV-uninfected and 360 had an HIV-unknown status. Of the 945 non-ATB subjects, 559 were at low risk (community adults) and 386 at high risk of M. tuberculosis exposure. Among those with ATB, the sensitivity of ELISA-PGL-Tb1 for ATB was higher than that of ELISA-ESAT-6/CFP10, both in HIV-infected (72.3% versus 63.7%, p = 0.29) and HIV-uninfected/HIV-unknown groups (40.5% versus 28.6%; p<0.0001), whereas the specificity was around 91% for both tests. Sensitivity for ATB increased when the results of the two ELISA were combined, reaching 75.5% in the HIV-infected and 50.9% in the group of HIV-uninfected/HIV-unknown ATB, with a significant decrease of the global specificity (83.9%). Analyzing the ELISA results with the microbiological results, we observed that the sensitivity of both serology tests was independent of the ATB patients' smear microscopy (SM) status and grade. Combining the results of SM with both ELISA, the detection of ATB patients significantly increased (p<0.0001), particularly in those with extrapulmonary TB (up to 45.1%) or HIV infection (up to 83.3%). No significant association was observed between TST and serology results., Conclusions: In this prospective multi-centric study, the combination of two rapid tests, such as SM and serology, might be useful in detecting ATB, especially in HIV-infected patients.

Background: The aim of this multicentric prospective study in India was to assess the performance of the QuantiFERON TB-Gold in tube (QFT-GIT), Tuberculin Skin Test (TST) and microbiological results as additional tools for diagnosing active tuberculosis (TB) and latent infection (LTBI) according to Human Immunodeficiency Virus (HIV) status., Methods: Individuals with and without active TB and HIV infection were enrolled between 2006-2008. QFT-GIT and TST results were analyzed per se and in combination with microbiological data., Results: Among the 276 individuals (96 active pulmonary TB and 180 no active TB) tested by QFT-GIT, 18 indeterminate results (6.5%) were found, more significantly numerous in the HIV-infected (15/92; 16.3%) than the HIV-uninfected (3/184; 1.6%)(p<0.0001). QFT-GIT sensitivity for active TB was 82.3% and 92.9% respectively after including or excluding indeterminate results. Clinical sensitivity was significantly lower in the HIV-infected (68.4%) than the HIV-uninfected (91.4%) patients (p = 0.0059). LTBI was detected in 49.3% of subjects without active TB but varied according to TB exposure. When the TST and QFT-GIT were concomitantly performed, the respective sensitivity for active TB diagnosis was 95.0% and 85.0% in the HIV-uninfected (p = 0.60), and 66.7% and 51.5% in the HIV-infected patients (p = 0.32). QFT-GIT and TST respective specificity for active TB in the HIV-uninfected was 25.0% and 57.1% (p = 0.028), and 64.8% and 83.3% in the HIV-infected (p = 0.047). In those with active TB, QFT-GIT results were not associated with microbiological parameters (smear grade, liquid culture status, time-to-positivity of culture) or clinical suspicion of active TB score (provided by the clinicians at enrollment). Combining microbiological tests with both immunological tests significantly increased sensitivity for active TB diagnosis (p = 0.0002), especially in the HIV-infected individuals (p = 0.0016)., Conclusion: QFT-GIT and TST have similar diagnostic value for active TB diagnosis. In HIV-infected patients, combining microbiological tests with both immunological tests significantly increases the sensitivity for active TB diagnosis.

Background: The aim of this multicentric prospective study in India was to assess the value of several microbiological tools that contribute to the diagnosis of tuberculosis (TB) according to HIV status., Methods: Standard microbiological tools on individual specimens were analyzed., Results: Among the 807 patients with active TB, 131 were HIV-infected, 316 HIV-uninfected and 360 had HIV-unknown status. Among the 980 non-active TB subjects, 559 were at low risk and 421 were at high risk of M. tuberculosis (Mtb) exposure. Sensitivity of smear microscopy (SM) was significantly lower in HIV-infected (42.2%) than HIV-uninfected (75.9%) (p = 0.0001) and HIV-unknown pulmonary TB patients (61.4%) (p = 0.004). Specificity was 94.5% in non-TB patients and 100% in health care workers (HCW) and healthy family contacts. Automated liquid culture has significantly higher diagnostic performances than solid culture, measured by sensitivity (74.7% vs. 55.9%) (p = 0.0001) and shorter median time to detection (TTD) (12.0 vs. 34.0 days) (p = 0.0001). Specificity was 100% in HCW and cured-TB patients, but was lower in non-TB patients (89%) due to isolation of Mycobacteria other than tuberculosis (MOTT). TTD by both methods was related to AFB score. Contamination rate was low (1.4%). AccuProbe hybridization technique detected Mtb in almost all culture-positive specimens, but MOTT were found in 4.7% with a significantly higher frequency in HIV-infected (15%) than HIV-uninfected TB patients (0.5%) (p = 0.0007). Pre-test classification significantly increased the diagnostic value of all microbiological tests in pulmonary TB patients (p<0.0001) but to a lesser degree in extrapulmonary TB patients., Conclusions: Conventional microbiological tools led to results similar to those already described in India special features for HIV-infected TB patients included lower detection by SM and culture. New microbiological assays, such as the automated liquid culture system, showed increased accuracy and speed of detection.