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Abstract Background In Africa, tuberculous meningitis (TBM) is an important opportunistic infection in HIV-positive patients. Current diagnostic tools for TBM perform sub-optimally. In particular, the rapid diagnosis of TBM is challenging because smear microscopy has a low yield and PCR is not widely available in resource-poor settings. Methods We evaluated the performance outcome of a novel standardized lipoarabinomannan (LAM) antigen-detection assay, using archived cerebrospinal fluid samples, in 50 African TBM suspects of whom 68% were HIV-positive. Results Of the 50 participants 14, 23 and 13 patients had definite, probable and non-TBM, respectively. In the non-TB group there were 5 HIV positive patients who were lost to follow-up and in whom concomitant infection with Mycobacterium tuberculosis could not be definitively excluded. The test sensitivities and specificities were as follows: LAM assay 64% and 69% (cut-point 0.22), smear microscopy 0% and 100% and PCR 93% and 77%, respectively. Conclusion In this preliminary proof-of-concept study, a rapid diagnosis of TBM could be achieved using LAM antigen detection. Although specificity was sub-optimal, the estimates provided here may be unreliable because of a classification bias inherent in the study design where it was not possible to exclude TBM in the presumed non-TBM cases owing to a lack of clinical follow-up. As PCR is largely unavailable, the LAM assay may well prove to be a useful adjunct for the rapid diagnosis of TBM in high HIV-incidence settings. These preliminary results justify further enquiry and prospective studies are now required to definitively establish the place of this technology for the diagnosis of TBM.

Background Tuberculous meningitis (TBM) mortality is high and current diagnostics perform suboptimally. We evaluated the diagnostic performance of a DNA-based assay (GeneXpert Ultra) against a new same-day immunodiagnostic assay that detects unstimulated interferon-gamma (IRISA-TB). Methods In a stage 1 evaluation, IRISA-TB was evaluated in biobanked samples from Zambia (n = 82; tuberculosis [TB] and non-TBM), and specificity in a South African biobank (n = 291; non-TBM only). Given encouraging results, a stage 2 evaluation was performed in suspected TBM patients from Zimbabwe and Malawi (n = 668). Patients were classified as having definite, probable or possible TBM, or non-TBM based on their microbiological results, cerebrospinal fluid (CSF) chemistry, and whether they received treatment. Results In the stage 1 evaluation, sensitivity and specificity of IRISA-TB were 75% and 87% in the Zambian samples, and specificity was 100% in the South African samples. In the stage 2 validation, IRISA-TB sensitivity (95% confidence interval [CI]) was significantly higher than Xpert Ultra (76.2% [55.0%–89.4%] vs 25% [8.9%–53.3%]; P =.0048) when trace readouts were considered negative. Specificity (95% CI) was similar for both assays (91.4% [88.8%–93.4%] vs 86.9% [83.4%–89.8%]). When the Xpert Ultra polymerase chain reaction product was verified by sequencing, the positive predictive value of trace readouts in CSF was 27.8%. Sensitivity of IRISA-TB was higher in human immunodeficiency virus (HIV)–infected versus uninfected participants (85.8% vs 66.7%). Conclusions As a same-day rule-in test, IRISA-TB had significantly better sensitivity than Xpert Ultra in a TB/HIV-endemic setting. An immunodiagnostic approach to TBM is promising, and further studies are warranted. [ABSTRACT FROM AUTHOR]

Rationale: In the upper respiratory tract, replicating (culturable) severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is recoverable for ∼4–8 days after symptom onset, but there is a paucity of data about the frequency and duration of replicating virus in the lower respiratory tract (i.e., the human lung). Objectives: We undertook lung tissue sampling (needle biopsy) shortly after death in 42 mechanically ventilated decedents during the Beta and Delta waves. An independent group of 18 ambulatory patients served as a control group. Methods: Lung biopsy cores from decedents underwent viral culture, histopathological analysis, electron microscopy, transcriptomic profiling, and immunohistochemistry. Measurements and Main Results: Thirty-eight percent (16 of 42) of mechanically ventilated decedents had culturable virus in the lung for a median of 15 days (persisting for up to 4 wk) after symptom onset. Lung viral culture positivity was not associated with comorbidities or steroid use. Delta but not Beta variant lung culture positivity was associated with accelerated death and secondary bacterial infection (P < 0.05). Nasopharyngeal culture was negative in 23.1% (6 of 26) of decedents despite lung culture positivity. This hitherto undescribed biophenotype of lung-specific persisting viral replication was associated with an enhanced transcriptomic pulmonary proinflammatory response but with concurrent viral culture positivity. Conclusions: Concurrent rather than sequential active viral replication continues to drive a heightened proinflammatory response in the human lung beyond the second week of illness and was associated with variant-specific increased mortality and morbidity. These findings have potential implications for the design of interventional strategies and clinical management of patients with severe coronavirus disease (COVID-19). [ABSTRACT FROM AUTHOR]

Rationale: A human model to better understand tuberculosis immunopathogenesis and facilitate vaccine development is urgently needed.Objectives: We evaluated the feasibility, safety, and immunogenicity of live bacillus Calmette-Guérin (BCG) in a lung-oriented controlled human infection model.Methods: We recruited 106 healthy South African participants with varying degrees of tuberculosis susceptibility. Live BCG, sterile PPD, and saline were bronchoscopically instilled into separate lung segments (n = 65). A control group (n = 34) underwent a single bronchoscopy without challenge. The primary outcome was safety. Cellular and antibody immune signatures were identified in BAL before and 3 days after challenge using flow cytometry, ELISA, RNA sequencing, and mass spectrometry.Measurements and Main Results: The frequency of adverse events was low (9.4%; n = 10), similar in the challenge versus control groups (P = 0.8), and all adverse events were mild and managed conservatively in an outpatient setting. The optimal PPD and BCG dose was 0.5 TU and 104 cfu, respectively, based on changes in BAL cellular profiles (P = 0.02) and antibody responses (P = 0.01) at incremental doses before versus after challenge. At 104 versus 103 cfu BCG, there was a significant increase in number of differentially expressed genes (367 vs. 3; P < 0.001) and dysregulated proteins (64 vs. 0; P < 0.001). Immune responses were highly setting specific (in vitro vs. in vivo) and compartment specific (BAL vs. blood) and localized to the challenged lung segments.Conclusions: A lung-oriented mycobacterial controlled human infection model using live BCG and PPD is feasible and safe. These data inform the study of tuberculosis immunopathogenesis and strategies for evaluation and development of tuberculosis vaccine candidates. [ABSTRACT FROM AUTHOR]

The article focuses on human lung challenge model to evaluate the safety and immunogenicity of purified protein derivative (PPD) and live Bacille-Calmette-Guerin (BCG) culture. It mentions PPD and BCG dilutions were prepared from GMP-grade pharmaceutical stocks in a Class II bio-safety cabinet under sterile conditions. It also mentions bronchoscopies were performed by the study pulmonologist and laryngeal mask (LMA) was used to protect the airway.

Background: The effects of the widely used progestin-only injectable contraceptives, medroxyprogesterone acetate (MPA) and norethisterone acetate (NET-A), on host susceptibility to Mycobacterium tuberculosis (Mtb) are unknown.Methods: We recruited human immunodeficiency virus-uninfected females, not taking any contraceptives, from Cape Town, South Africa, to evaluate the effect of MPA, NET-A, and dexamethasone on Mtb containment in monocyte-derived macrophages co-incubated with purified protein derivative (PPD)-driven peripheral blood-derived effector cells.Results: MPA (P < .005) and dexamethasone (P < .01), but not NET-A, significantly attenuated Mtb containment in Mtb-infected macrophages co-cultured with PPD-driven effector cells at physiologically relevant concentrations and in a dose-dependent manner. Antagonizing the glucocorticoid receptor with mifepristone (RU486) abrogated the reduction in Mtb containment. In PPD-stimulated peripheral blood mononuclear cells, MPA and dexamethasone, but not NET-A, upregulated (median [interquartile range]) regulatory T cells (5.3% [3.1%-18.2%]; P < .05), reduced CD4+ T-cell interferon-γ (21% [0.5%-28%]; P < .05) and granzyme B production (12.6% [7%-13.5%]; P < .05), and reduced CD8+ perforin activity (2.2% [0.1%-7%]; P < .05). RU486 reversed regulatory T-cell up-regulation and the inhibitory effect on Th1 and granzyme/perforin-related pathways.Conclusions: MPA, but not NET-A, subverts mycobacterial containment in vitro and downregulates pathways associated with protective CD8+- and CD4+-related host immunity via the glucocorticoid receptor. These data potentially inform the selection and use of injectable contraceptives in tuberculosis-endemic countries. [ABSTRACT FROM AUTHOR]

Background: Urine LAM testing has been approved by the WHO for use in hospitalised patients with advanced immunosuppression. However, sensitivity remains suboptimal. We therefore examined the incremental diagnostic sensitivity of early morning urine (EMU) versus random urine sampling using the Determine® lateral flow lipoarabinomannan assay (LF-LAM) in HIV-TB co-infected patients.Methods: Consenting HIV-infected inpatients, screened as part of a larger prospective randomized controlled trial, that were treated for TB, and could donate matched random and EMU samples were included. Thus paired sample were collected from the same patient, LF-LAM was graded using the pre-January 2014, with grade 1 and 2 manufacturer-designated cut-points (the latter designated grade 1 after January 2014). Single sputum Xpert-MTB/RIF and/or TB culture positivity served as the reference standard (definite TB). Those treated for TB but not meeting this standard were designated probable TB.Results: 123 HIV-infected patients commenced anti-TB treatment and provided matched random and EMU samples. 33% (41/123) and 67% (82/123) had definite and probable TB, respectively. Amongst those with definite TB LF-LAM sensitivity (95%CI), using the grade 2 cut-point, increased from 12% (5-24; 5/43) to 39% (26-54; 16/41) with random versus EMU, respectively (p = 0.005). Similarly, amongst probable TB, LF-LAM sensitivity increased from 10% (5-17; 8/83) to 24% (16-34; 20/82) (p = 0.001). LF-LAM specificity was not determined.Conclusion: This proof of concept study indicates that EMU could improve the sensitivity of LF-LAM in hospitalised TB-HIV co-infected patients. These data have implications for clinical practice. [ABSTRACT FROM AUTHOR]

Increased risk of tuberculosis (TB) associated with HIV-1 infection is primarily attributed to deficient T helper (Th)1 immune responses, but most people with active TB have robust Th1 responses, indicating that these are not sufficient to protect against disease. Recent findings suggest that favourable outcomes following Mycobacterium tuberculosis infection arise from finely balanced inflammatory and regulatory pathways, achieving pathogen control without immunopathology. We hypothesised that HIV-1 and antiretroviral therapy (ART) exert widespread changes to cell mediated immunity, which may compromise the optimal host protective response to TB and provide novel insights into the correlates of immune protection and pathogenesis. We sought to define these effects in patients with active TB by transcriptional profiling of tuberculin skin tests (TST) to make comprehensive molecular level assessments of in vivo human immune responses at the site of a standardised mycobacterial challenge. We showed that the TST transcriptome accurately reflects the molecular pathology at the site of human pulmonary TB, and used this approach to investigate immune dysregulation in HIV-1/TB co-infected patients with distinct clinical phenotypes associated with TST reactivity or anergy and unmasking TB immune reconstitution inflammatory syndrome (IRIS) after initiation of ART. HIV-1 infected patients with positive TSTs exhibited preserved Th1 responses but deficient immunoregulatory IL10-inducible responses. Those with clinically negative TSTs revealed profound anergy of innate as well as adaptive immune responses, except for preservation of type 1 interferon activity, implicated in impaired anti-mycobacterial immunity. Patients with unmasking TB IRIS showed recovery of Th1 immunity to normal levels, but exaggerated Th2-associated responses specifically. These mechanisms of immune dysregulation were localised to the tissue microenvironment and not evident in peripheral blood. TST molecular profiling categorised different mechanisms of immunological dysfunction in HIV-1 infection beyond the effects on CD4 T cells, each associated with increased risk of TB disease and amenable to host-directed therapies. [ABSTRACT FROM AUTHOR]

Summary Background There are few prospective data about the incidence and mortality associated with pulmonary tuberculosis in intensive care units (ICUs), and none on the accuracy and clinical effect of the Xpert-MTB/RIF assay in this setting. We aimed to measure the frequency of culture-positive tuberculosis in ICUs in Cape Town, South Africa and to assess the performance and effect on patient outcomes of Xpert MTB/RIF versus smear microscopy for diagnosis of tuberculosis. Methods We did a prospective burden of disease study with a randomised controlled substudy at the ICUs of four hospitals in Cape Town. Mechanically ventilated adults (≥18 years) with suspected pulmonary tuberculosis admitted between Aug 1, 2010, and July 31, 2013 (irrespective of the reason for admission), were prospectively investigated by culture, and by Xpert-MTB/RIF testing or smear microscopy, of tracheal aspirate samples. In the substudy, patients were randomly assigned (1:1), via a computer-generated allocation list, to smear microscopy or Xpert MTB/RIF. Participants, caregivers, and outcome assessors were not masked to group assignment. Only the laboratory staff were blinded to the clinical details of the participants. In November, 2012, Xpert MTB/RIF was adopted as the initial diagnostic test for respiratory samples in Western Cape province. Thereafter, patients received Xpert MTB/MIF and culture as standard of care. For the whole study cohort, the primary outcome was the frequency of bacteriologically confirmed tuberculosis. The primary endpoint of the randomised substudy was the proportion of culture-positive patients on treatment at 48 h after enrolment. The randomised substudy is registered with ClinicalTrials.gov , number NCT01530568 . Findings We investigated 341 patients for suspected pulmonary tuberculosis out of a total of 2309 ICU admissions. 46 (15%) of 317 patients included in the final analysis had a positive test for tuberculosis (Xpert MTB/RIF or culture). Culture-positive patients who failed to initiate treatment (adjusted HR 4·49, 95% CI 1·45–13·89) or who received inotropes (4·33, 1·49–12·60) were more likely to die. However, tuberculosis status was not associated with 28-day or 90-day mortality. In the substudy, we randomly assigned 115 patients to smear microscopy and 111 to Xpert MTB/RIF. Smear microscopy detected six (43%) of 14 culture-positive patients, and Xpert MTB/RIF detected 11 (100%) of 11 culture-positive patients (p=0·002). The proportion of culture-positive patients on treatment at 48 h was higher in the Xpert MTB/RIF group than in the smear microscopy group (11 [92%] of 12 vs nine [53%] of 17; p=0·043), although use of Xpert MTB/RIF had no effect on mortality or other patient outcomes. Interpretation Tuberculosis is fairly common in ICUs in high-burden settings, and clinicians should screen and test patients for tuberculosis with Xpert MTB/RIF where available. This test improves diagnostic yield and rates of treatment initiation, and reduces unnecessary treatment, but might not increase the total number of patients on treatment when empirical treatment is widely used. A suspected diagnosis of pulmonary tuberculosis should not exclude patients from ICU care in resource-limited settings because mortality is unaffected by the presence of this disease. Funding European and Developing Countries Clinical Trials Partnership, South African Medical Research Council, and the Discovery Foundation. [ABSTRACT FROM AUTHOR]

The determinants of Xpert MTB/RIF sensitivity, a widely used PCR test for the diagnosis of tuberculosis (TB) are poorly understood. We compared culture time-to-positivity (TTP; a surrogate of bacterial load), MTB/RIF TB-specific and internal positive control (IPC)-specific CT values, and clinical characteristics in patients with suspected TB who provided expectorated (n = 438) or induced sputum (n = 128), tracheal aspirates (n=71), bronchoalveolar lavage fluid (n=152), pleural fluid (n=76), cerebral spinal fluid (CSF; n=152), pericardial fluid (n=131), or urine (n=173) specimens. Median bacterial load (TTP in days) was the strongest associate of MTB/RIF positivity in each fluid. TTP correlated with CT values in pulmonary specimens but not extrapulmonary specimens (Spearman's coefficient 0.5043 versus 0.1437; p = 0.030). Inhibition affected a greater proportion of pulmonary specimens than extrapulmonary specimens (IPC CT > 34: 6% (47/731) versus 1% (4/381; p < 0.0001). Pulmonary specimens had greater load than extrapulmonary specimens [TTPs (interquartile range) of 11 (7–16) versus 22 (18–33.5) days; p < 0.0001]. HIV-infection was associated with a decreased likelihood of MTB/RIF-positivity in pulmonary specimens but an increased likelihood in extrapulmonary specimens. Mycobacterial load, which displays significant variation across different body compartments, is the main determinant of MTB/RIF-positivity rather than PCR inhibition. MTB/RIF CT is a poor surrogate of load in extrapulmonary specimens. [ABSTRACT FROM AUTHOR]

Background Tuberculous pericarditis (TBP) is associated with high morbidity and mortality, and is an important treatable cause of heart failure in developing countries. Tuberculous aetiology of pericarditis is difficult to diagnose promptly. The utility of the new quantitative PCR test (Xpert MTB/RIF) for the diagnosis of TBP is unknown. This study sought to evaluate the diagnostic accuracy of the Xpert MTB/RIF test compared to pericardial adenosine deaminase (ADA) and unstimulated interferon-gamma (uIFNγ) in suspected TBP. Methods From October 2009 through September 2012, 151 consecutive patients with suspected TBP were enrolled at a single centre in Cape Town, South Africa. Mycobacterium tuberculosis culture and/or pericardial histology served as the reference standard for definite TBP. Receiver-operating-characteristic curve analysis was used for selection of ADA and uIFNγ cut-points. Results Of the participants, 49% (74/151) were classified as definite TBP, 33% (50/151) as probable TBP and 18% (27/151) as non TBP. A total of 105 (74%) participants were human immunodeficiency virus (HIV) positive. Xpert-MTB/RIF had a sensitivity and specificity (95% confidence interval (CI)) of 63.8% (52.4% to 75.1%) and 100% (85.6% to 100%), respectively. Concentration of pericardial fluid by centrifugation and using standard sample processing did not improve Xpert MTB/RIF accuracy. ADA (≥35 IU/L) and uIFNγ (≥44 pg/ml) both had a sensitivity of 95.7% (88.1% to 98.5%) and a negative likelihood ratio of 0.05 (0.02 to 0.10). However, the specificity and positive likelihood ratio of uIFNγ was higher than ADA (96.3% (81.7% to 99.3%) and 25.8 (3.6 to 183.4) versus 84% (65.4% to 93.6%) and 6.0 (3.7 to 9.8); P = 0.03) at an estimated background prevalence of TB of 30%. The sensitivity and negative predictive value of both uIFNγ and ADA were higher than Xpert-MT/RIF (P < 0.001). Conclusions uIFNγ offers superior accuracy for the diagnosis of microbiologically confirmed TBP compared to the ADA assay and the Xpert MTB/RIF test. [ABSTRACT FROM AUTHOR]

Background The accuracy of currently available same-day diagnostic tools (smear microscopy and conventional nucleic acid amplification tests) for pleural tuberculosis (TB) is sub-optimal. Newer technologies may offer improved detection. Methods Smear-microscopy, adenosine deaminase (ADA), interferon gamma (IFN-γ), and Xpert MTB/RIF [using an unprocessed (1 ml) and centrifuged (~20 ml) sample] test accuracy was evaluated in pleural fluid from 103 consecutive patients with suspected pleural TB. Culture for M.tuberculosis and/or histopathology (pleural biopsy) served as the reference standard. Patients were followed prospectively to determine their diagnostic categorisation. Results Of 93 evaluable participants, 40 had definite-TB (reference positive), 5 probable-TB (not definite but treated for TB) and 48 non-TB (culture and histology negative, and not treated for TB). Xpert MTB/RIF sensitivity and specificity (95% CI) was 22.5% (12.4 - 37.6) and 98% (89.2 - 99.7), respectively, and centrifugation did not improve sensitivity (23.7%). The Xpert MTB/RIF internal positive control showed no evidence of inhibition. Biomarker specific sensitivity, specificity, PPV, and NPVs were: ADA (48.85 IU/L; rule-in cut-point) 55.3% (39.8 - 69.9), 95.2% (83.9 - 98.7), 91.4 (73.4 - 95.4), 69.7% (56.7 - 80.1); ADA (30 IU/L; clinically used cut-point) 79% (63.7 - 89), 92.7% (80.6 - 97.5), 91.0 (73.4 - 95.4), 82.7% (69.3 - 90.1); and IFN-γ (107.7 pg/ml; rule-in cut-point) 92.5% (80.2 - 97.5), 95.9% (86.1 - 98.9), 94.9% (83.2 - 98.6), 93.9% (83.5 - 97.9), respectively (IFN-γ sensitivity and NPV better than Xpert [p < 0.05] and rule-in ADA [p < 0.05]). Conclusion The usefulness of Xpert MTB/RIF to diagnose pleural TB is limited by its poor sensitivity. IFN-γ is an excellent rule-in test and, compared to ADA, has significantly better sensitivity and rule-out value in a TB-endemic setting. [ABSTRACT FROM AUTHOR]

Background: Measures of bacillary load in patients with tuberculosis (TB) may be useful for predicting and monitoring response to treatment. The relationship between quantitative T-cell responses and mycobacterial load remains unclear. We hypothesised that, in a HIV-prevalent high burden setting, the magnitude of mycobacterial antigen-specific and nonspecific T-cell IFN-γ responses would correlate with (a) bacterial load and (b) culture conversion in patients undergoing treatment. Methods: We compared baseline (n = 147), 2 (n = 35) and 6 month (n = 13) purified-protein-derivative (PPD) and RD1-specific (TSPOT.TB and QFT-GIT) blood RD1-specific (TSPOT.TB; QFT-GIT) responses with associates of sputum bacillary load in patients with culture-confirmed TB in Cape Town, South Africa. Results: IFN-γ responses were not associated with liquid culture time-to-positivity, smear-grade, Xpert MTB/RIF-generated cycle threshold values or the presence of cavities on the chest radiograph in patients with culture-confirmed TB and irrespective of HIV-status. 2-month IGRA conversion rates (positive-to-negative) were negligible [<11% for TSPOT.TB (3/28) and QFT-GIT (1/29)] and lower compared to culture [60% (21/35); p<0.01]. Conclusions: In a high burden HIV-prevalent setting T-cell IFN-γ responses to M. tuberculosis-specific and non-specific antigens do not correlate with bacillary load, including Xpert MTB/RIF-generated CT values, and are therefore poorly suited for monitoring treatment and prognostication. [ABSTRACT FROM AUTHOR]

Introduction: Accurate quantification of mycobacterial load is important for the evaluation of patient infectiousness, disease severity and monitoring treatment response in human and in-vitro laboratory models of disease. We hypothesized that newer techniques would perform as well as solid media culture to quantify mycobacterial burden in laboratory specimens. Methods: We compared the turn-around-time, detection-threshold, dynamic range, reproducibility, relative discriminative ability, of 4 mycobacterial load determination techniques: automated liquid culture (BACTEC-MGIT-960), [3H]-uracil incorporation assays, luciferase-reporter construct bioluminescence, and quantitative PCR(Xpert -MTB/RIF) using serial dilutions of Mycobacterium bovis and Mycobacterium tuberculosis H37RV. Mycobacterial colony-forming-units(CFU) using 7H10-Middlebrook solid media served as the reference standard. Results: All 4 assays correlated well with the reference standard, however, bioluminescence and uracil assays had a detection threshold ≥1×103 organisms. By contrast, BACTEC-MGIT-960 liquid culture, although only providing results in days, was user-friendly, had the lowest detection threshold (<10 organisms), the greatest discriminative ability (1 vs. 10 organisms; p = 0.02), and the best reproducibility (coefficient of variance of 2% vs. 38% compared to uracil incorporation; p = 0.02). Xpert-MTB/RIF correlated well with mycobacterial load, had a rapid turn-around-time (<2 hours), was user friendly, but had a detection limit of ∼100 organisms. Conclusions: Choosing a technique to quantify mycobacterial burden for laboratory or clinical research depends on availability of resources and the question being addressed. Automated liquid culture has good discriminative ability and low detection threshold but results are only obtained in days. Xpert MTB/RIF provides rapid quantification of mycobacterial burden, but has a poorer discrimination and detection threshold. [ABSTRACT FROM AUTHOR]

Background: The diagnosis of smear-negative or sputum-scarce tuberculosis (TB) is problematic as culture takes several weeks and representative biological samples are difficult to obtain. RD-1 antigen-specific interferon-c release assays (IGRAs) are sensitive and specific blood-based tests for the diagnosis of M. tuberculosis infection. The feasibility and diagnostic utility of this rapid immunodiagnostic assay, using cells from induced sputum, is unknown. Methodology/Principal Findings: Cells isolated from induced sputum were co-cultured with ESAT-6 and CFP-10 antigens using a standardized enzyme-linked immunospot (ELISPOT) assay (T-SPOT®.TB) in 101 consecutively recruited TB suspects or non-TB controls. An optimization phase using 28 samples was followed by a validation phase using samples from 73 participants (20 with definite or probable TB, and 48 with non-TB). Despite optimization of sputum processing 65/73 (89%) of the IGRAs in the validation phase were inconclusive. 44/73 (60%) tests failed due to sputum induction-related factors [sputum induction-related adverse events (n = 5), inadequate sputum volume (n = 8), non-homogenisable sputum (n = 7), and insufficient numbers of cells to perform the assay (n = 24)], whilst 20/73 (27%) tests failed due T-SPOTH.TB assay-related factors [excessive debris precluding reading of spots in the ELISPOT well (n = 6), failure of the positive control (n = 11), or high spot count in the negative control (n = 3)]. Only 8/73 (11%) of the available samples could therefore be correctly categorized (7 definite or probable TB, and 1 non-TB patient). Thus, 13/20 (65%) of the definite or probable TB cases remained undiagnosed. Conclusions/Significance: Rapid immunodiagnosis of pulmonary TB by antigen-specific IFN-γ ELISPOT responses, using cells from induced sputum, is possible. However, the test, in its current ELISPOT format, is not clinically useful because the majority of the assays are inconclusive. [ABSTRACT FROM AUTHOR]

Background: The accurate diagnosis of TB in HIV-infected patients, particularly with advanced immunosuppression, is difficult. Recent studies indicate that a lipoarabinomannan (LAM) assay (Clearview-TB®-ELISA) may have some utility for the diagnosis of TB in HIV-infected patients; however, the precise subgroup that may benefit from this technology requires clarification. The utility of LAM in sputum samples has, hitherto, not been evaluated. Methods: LAM was measured in sputum and urine samples obtained from 500 consecutively recruited ambulant patients, with suspected TB, from 2 primary care clinics in South Africa. Culture positivity for M. tuberculosis was used as the reference standard for TB diagnosis. Results: Of 440 evaluable patients 120/387 (31%) were HIV-infected. Urine-LAM positivity was associated with HIV positivity (p = 0.007) and test sensitivity, although low, was significantly higher in HIV-infected compared to uninfected patients (21% versus 6%; p<0.001), and also in HIV-infected participants with a CD4 <200 versus <200 cells/mm³ (37% versus 0%; p = 0.003). Urine-LAM remained highly specific in all 3 subgroups (95%-100%). 25% of smear-negative but culture-positive HIV-infected patients with a CD4 <200 cells/mm³ were positive for urine-LAM. Sputum-LAM had good sensitivity (86%) but poor specificity (15%) likely due to test cross-reactivity with several mouth-residing organisms including actinomycetes and nocardia species. Conclusions: These preliminary data indicate that in a high burden primary care setting the diagnostic usefulness of urine- LAM is limited, as a rule-in test, to a specific patient subgroup i.e. smear-negative HIV-infected TB patients with a CD4 count <200 cells/mm³, who would otherwise have required further investigation. However, even in this group sensitivity was modest. Future and adequately powered studies in a primary care setting should now specifically target patients with suspected TB who have advanced HIV infection. [ABSTRACT FROM AUTHOR]

Rationale: The optimal strategy for the diagnosis of latent tuberculosis infection is controversial. Adoption of a two-step strategy (tuberculin skin test [TST] followed by an IFN-gamma release assay [IGRA], compared with an IGRA alone), may be limited by TST-mediated boosting of subsequent IGRA responses. Assessment of within-subject IGRA variability will aid in establishing thresholds for conversions and reversions, and interpretation of serial testing results.Objectives: To determine short-term IGRA variability and the impact of TST on subsequent IGRA results.Methods: Within-subject variability and TST-mediated boosting of IGRA responses were evaluated in 26 South African participants with varying exposure risk. IGRAs (T-SPOT.TB, QuantiFERON-TB Gold In-Tube [QuantiFERON-TB-GIT], PPD, and heparin-binding hemagglutinin) were repeated four times over 21 days pre-TST, and on Days 3, 7, 28, and 84 post-TST administration.Measurements and Main Results: All participants showed within-subject IGRA variability. Changes of +/-3 spots (T-SPOT.TB) or +/-80% from the mean IFN-gamma response (QuantiFERON-TB-GIT) over 3 weeks explained 95% of the variability. Spontaneous conversions/reversions occurred in 7 of 26 subjects (27%) (6 for T-SPOT.TB and 1 for QuantiFERON-TB-GIT [P = 0.049]) during the within-patient variability studies (pre-TST). After the TST eight subjects (33%) boosted above the defined baseline variability. By Day 7 post-TST, but not Day 3, 2 (12.5%) initially IGRA-negative test subjects converted. By contrast, boosting of PPD and heparin-binding hemagglutinin occurred by Day 3 post-TST.Conclusions: When using a two-step screening strategy it appears safe to perform a QuantiFERON-TB-GIT or T-SPOT.TB IGRA within 3 days of performing the TST. A 3-spot or 80% IFN-gamma response variation, on either side of baseline values, explains 95% of the short-term variability and may be useful for interpreting conversions and reversions, and values close to the cut-point. [ABSTRACT FROM AUTHOR]

Background: Current tools for the diagnosis of tuberculosis pleural effusions are sub-optimal. Data about the value of new diagnostic technologies are limited, particularly, in high burden settings. Preliminary case control studies have identified IFN-c-inducible-10kDa protein (IP-10) as a promising diagnostic marker; however, its diagnostic utility in a day-to-day clinical setting is unclear. Detection of LAM antigen has not previously been evaluated in pleural fluid. Methods: We investigated the comparative diagnostic utility of established (adenosine deaminase [ADA]), more recent (standardized nucleic-acid-amplification-test [NAAT]) and newer technologies (a standardized LAM mycobacterial antigendetection assay and IP-10 levels) for the evaluation of pleural effusions in 78 consecutively recruited South African tuberculosis suspects. All consenting participants underwent pleural biopsy unless contra-indicated or refused. The reference standard comprised culture positivity for M. tuberculosis or histology suggestive of tuberculosis. Principal Findings: Of 74 evaluable subjects 48, 7 and 19 had definite, probable and non-TB, respectively. IP-10 levels were significantly higher in TB vs non-TB participants (p<0.0001). The respective outcomes [sensitivity, specificity, PPV, NPV %] for the different diagnostic modalities were: ADA at the 30 IU/L cut-point [96; 69; 90; 85], NAAT [6; 93; 67; 28], IP-10 at the 28,170 pg/ml ROC-derived cut-point [80; 82; 91; 64], and IP-10 at the 4035 pg/ml cut-point [100; 53; 83; 100]. Thus IP-10, using the ROC-derived cut-point, missed ~20% of TB cases and mis-diagnosed ~20% of non-TB cases. By contrast, when a lower cut-point was used a negative test excluded TB. The NAAT had a poor sensitivity but high specificity. LAM antigendetection was not diagnostically useful. Conclusion: Although IP-10, like ADA, has sub-optimal specificity, it may be a clinically useful rule-out test for tuberculous pleural effusions. Larger multi-centric studies are now required to confirm our findings. [ABSTRACT FROM AUTHOR]

• In a high TB burden and HIV prevalence setting we show that different M.tb strains are associated with different host-specific IFN-γ responses. • We show that these strain-specific differences in host IFN-γ responses may be associated with mutations in the ESX-1 region. • Our data have implications for IGRA assay interpretation and provide another reason for variability in IGRA responses and help to explain the differences seen between different IGRA formats. Between-person variability in T-cell-specific interferon-gamma release assay (IGRA) responses and discordance between IGRA test formats are poorly understood. We evaluated the IFN-γ responses (QuantiFERON-TB Gold-In-Tube [QFT-GIT] and TSPOT-TB) stratified according to the Mycobacterium tuberculosis spoligotype of the culture isolate obtained from the same patients with confirmed active tuberculosis (n = 91). We further analysed differences within the RD-1-encoding ESX-1 region between the different strain types using whole genome sequencing. In HIV-uninfected patients, TSPOT.TB and QFT-GIT IFN-γ responses were 5-fold (p < 0.01) and 2-fold higher (p < 0.05) for those infected with family 33 compared to the LAM strain (additionally, TSPOT.TB responses were 5.6-fold [p < 0.05] and 2.6-fold higher [p < 0.05] for the patients infected with the family 33 versus the X strain and Beijing versus the LAM strain, respectively). Multivariate analysis revealed that strain type (determined by spoligotyping) was independently associated with the magnitude of the IGRA response (varied by IGRA test type) and this is likely explained by variability in the ESX-1 region of Mycobacteriumtuberculosis (determined by next-generation sequencing). These data have implications for the understanding of between-person heterogeneity in IGRA responses, Mycobateriumtuberculosis -specific host immunity, and the discordance between different IGRA test formats. [ABSTRACT FROM AUTHOR]