Four-Month Rifapentine Regimens with or without Moxifloxacin for Tuberculosis.

BACKGROUND: Rifapentine-based regimens have potent antimycobacterial activity that may allow for a shorter course in patients with drug-susceptible pulmonary tuberculosis. METHODS: In an open-label, phase 3, randomized, controlled trial involving persons with newly diagnosed pulmonary tuberculosis from 13 countries, we compared two 4-month rifapentine-based regimens with a standard 6-month regimen consisting of rifampin, isoniazid, pyrazinamide, and ethambutol (control) using a noninferiority margin of 6.6 percentage points. In one 4-month regimen, rifampin was replaced with rifapentine; in the other, rifampin was replaced with rifapentine and ethambutol with moxifloxacin. The primary efficacy outcome was survival free of tuberculosis at 12 months. RESULTS: Among 2516 participants who had undergone randomization, 2343 had a culture positive for Mycobacterium tuberculosis that was not resistant to isoniazid, rifampin, or fluoroquinolones (microbiologically eligible population; 768 in the control group, 791 in the rifapentine-moxifloxacin group, and 784 in the rifapentine group), of whom 194 were coinfected with human immunodeficiency virus and 1703 had cavitation on chest radiography. A total of 2234 participants could be assessed for the primary outcome (assessable population; 726 in the control group, 756 in the rifapentine-moxifloxacin group, and 752 in the rifapentine group). Rifapentine with moxifloxacin was noninferior to the control in the microbiologically eligible population (15.5% vs. 14.6% had an unfavorable outcome; difference, 1.0 percentage point; 95% confidence interval [CI], -2.6 to 4.5) and in the assessable population (11.6% vs. 9.6%; difference, 2.0 percentage points; 95% CI, -1.1 to 5.1). Noninferiority was shown in the secondary and sensitivity analyses. Rifapentine without moxifloxacin was not shown to be noninferior to the control in either population (17.7% vs. 14.6% with an unfavorable outcome in the microbiologically eligible population; difference, 3.0 percentage points [95% CI, -0.6 to 6.6]; and 14.2% vs. 9.6% in the assessable population; difference, 4.4 percentage points [95% CI, 1.2 to 7.7]). Adverse events of grade 3 or higher occurred during the on-treatment period in 19.3% of participants in the control group, 18.8% in the rifapentine-moxifloxacin group, and 14.3% in the rifapentine group. CONCLUSIONS: The efficacy of a 4-month rifapentine-based regimen containing moxifloxacin was noninferior to the standard 6-month regimen in the treatment of tuberculosis. (Funded by the Centers for Disease Control and Prevention and others; Study 31/A5349 ClinicalTrials.gov number, NCT02410772.).

A Standardized Approach for Collection of Objective Data to Support Outcome Determination for Late-Phase Tuberculosis Clinical Trials.

Rationale: We developed a standardized method, possible poor treatment response (PPTR), to help ascertain efficacy endpoints in Study S31/A5349 (NCT02410772), an open-label trial comparing two 4-month rifapentine-based regimens with a standard 6-month regimen for the treatment of pulmonary tuberculosis (TB). Objectives: We describe the use of the PPTR process and evaluate whether the goals of minimizing bias in efficacy endpoint assessment and attainment of relevant data to determine outcomes for all participants were achieved. Methods: A PPTR event was defined as the occurrence of one or more prespecified triggers. Each PPTR required initiation of a standardized evaluation process that included obtaining multiple sputum samples for microbiology. Measurements and Main Results: Among 2,343 participants with culture-confirmed drug-susceptible TB, 454 individuals (19.4%) had a total of 534 individual PPTR events, of which 76.6% were microbiological (positive smear or culture at or after 17 wk). At least one PPTR event was experienced by 92.4% (133 of 144) of participants with TB-related unfavorable outcome and between 13.8% and 14.7% of participants with favorable and not-assessable outcomes. A total of 75% of participants with TB-related unfavorable outcomes had microbiological confirmation of failure to achieve a disease-free cure. Conclusions: Standardized methodologies, such as our PPTR approach, could facilitate unbiased efficacy outcome determinations, improve discrimination between outcomes that are related and unrelated to regimen efficacy, and enhance the ability to conduct pooled analyses of contemporary trials.

Rifapentine With and Without Moxifloxacin for Pulmonary Tuberculosis in People With Human Immunodeficiency Virus (S31/A5349).

BACKGROUND: Tuberculosis (TB) Trials Consortium Study 31/AIDS Clinical Trials Group A5349, an international randomized open-label phase 3 noninferiority trial showed that a 4-month daily regimen substituting rifapentine for rifampin and moxifloxacin for ethambutol had noninferior efficacy and was safe for the treatment of drug-susceptible pulmonary TB (DS-PTB) compared with the standard 6-month regimen. We explored results among the prespecified subgroup of people with human immunodeficiency virus (HIV) (PWH). METHODS: PWH and CD4+ counts ≥100 cells/µL were eligible if they were receiving or about to initiate efavirenz-based antiretroviral therapy (ART). Primary endpoints of TB disease-free survival 12 months after randomization (efficacy) and ≥ grade 3 adverse events (AEs) on treatment (safety) were compared, using a 6.6% noninferiority margin for efficacy. Randomization was stratified by site, pulmonary cavitation, and HIV status. PWH were enrolled in a staged fashion to support cautious evaluation of drug-drug interactions between rifapentine and efavirenz. RESULTS: A total of 2516 participants from 13 countries in sub-Saharan Africa, Asia, and the Americas were enrolled. Among 194 (8%) microbiologically eligible PWH, the median CD4+ count was 344 cells/µL (interquartile range: 223-455). The rifapentine-moxifloxacin regimen was noninferior to control (absolute difference in unfavorable outcomes -7.4%; 95% confidence interval [CI] -20.8% to 6.0%); the rifapentine regimen was not noninferior to control (+7.5% [95% CI, -7.3% to +22.4%]). Fewer AEs were reported in rifapentine-based regimens (15%) than the control regimen (21%). CONCLUSIONS: In people with HIV-associated DS-PTB with CD4+ counts ≥100 cells/µL on efavirenz-based ART, the 4-month daily rifapentine-moxifloxacin regimen was noninferior to the 6-month control regimen and was safe. CLINICAL TRIALS REGISTRATION: NCT02410772.

Systemic DPP4/CD26 is associated with natural HIV-1 control: Implications for COVID-19 susceptibility.

The current intersection of the COVID-19 and HIV-1 pandemics, has raised concerns about the risk for poor COVID-19 outcomes particularly in regions like sub-Saharan Africa, disproportionally affected by HIV. DPP4/CD26 has been suggested to be a potential therapeutic target and a biomarker for risk in COVID-19 patients with high risk co-morbidities. We therefore evaluated soluble DPP4 (sDPP4) levels and activity in plasma of 131 HIV-infected and 20 HIV-uninfected South African individuals. Flow cytometry was performed to compare cell surface expression of DPP4/CD26 and activation markers on peripheral blood mononuclear cells of extreme clinical phenotypes. Progressors had lower specific DPP4 activity and lower frequency of CD3+ T-cells expressing CD26 than HIV-1 controllers, but more activated CD3+CD26+ T-cells. The frequency of CD26-expressing T-cells negatively correlated with HLA-DR+ and CD38+ T-cells. Divergent DPP4/CD26 expression between HIV-1 controllers and progressors may have implications for risk and treatment of COVID-19 in people living with HIV.

Comparing rates of mycobacterial clearance in sputum smear-negative and smear-positive adults living with HIV.

BACKGROUND: Pulmonary tuberculosis (TB) in people living with HIV (PLH) frequently presents as sputum smear-negative. However, clinical trials of TB in adults often use smear-positive individuals to ensure measurable bacterial responses following initiation of treatment, thereby excluding HIV-infected patients from trials. METHODS: In this prospective case cohort study, 118 HIV-seropositive TB patients were assessed prior to initiation of standard four-drug TB therapy and at several time points through 35 days. Sputum bacillary load, as a marker of treatment response, was determined serially by: smear microscopy, Xpert MTB/RIF, liquid culture, and colony counts on agar medium. RESULTS: By all four measures, patients who were baseline smear-positive had higher bacterial loads than those presenting as smear-negative, until day 35. However, most smear-negative PLH had significant bacillary load at enrolment and their mycobacteria were cleared more rapidly than smear-positive patients. Smear-negative patients' decline in bacillary load, determined by colony counts, was linear to day 7 suggesting measurable bactericidal activity. Moreover, the decrease in bacterial counts was comparable to smear-positive individuals. Increasing cycle threshold values (Ct) on the Xpert assay in smear-positive patients to day 14 implied decreasing bacterial load. CONCLUSION: Our data suggest that smear-negative PLH can be included in clinical trials of novel treatment regimens as they contain sufficient viable bacteria, but allowances for late exclusions would have to be made in sample size estimations. We also show that increases in Ct in smear-positive patients to day 14 reflect treatment responses and the Xpert MTB/RIF assay could be used as biomarker for early treatment response.

Isoniazid Preventive Therapy and Pregnancy Outcomes in Women Living With Human Immunodeficiency Virus in the Tshepiso Cohort.

BACKGROUND: Both pregnancy and human immunodeficiency virus (HIV) increase the risk of tuberculosis disease, which results in poor maternal, pregnancy, and infant outcomes. Isoniazid preventive therapy (IPT) reduces mortality among individuals living with HIV in high-burden settings but has recently been associated with adverse pregnancy outcomes when initiated during pregnancy. METHODS: In this secondary analysis, we used multivariable logistic regression to evaluate the association between IPT exposure and adverse pregnancy outcomes (fetal demise, prematurity, low birth weight, congenital anomaly) in pregnant women living with HIV enrolled as controls in the Tshepiso study, a prospective observational cohort of pregnant women living with HIV with and without tuberculosis disease in Soweto, South Africa, from 2011-2014. RESULTS: There were 151 women enrolled with known pregnancy outcomes; 69 (46%) reported IPT initiation during pregnancy. Of the 69 IPT-exposed women, 11 (16%) had an adverse pregnancy outcome compared with 23 (28%) IPT-unexposed women. The adjusted odds of having an adverse pregnancy outcome was 2.5 (95% confidence interval, 1.0-6.5; P = .048) times higher in IPT-unexposed women compared with IPT-exposed women after controlling for maternal age, CD4 count, viral load, antiretroviral regimen, body mass index, and anemia. CONCLUSIONS: IPT exposure during pregnancy was not negatively associated with pregnancy outcomes after controlling for demographic, clinical, and HIV-related factors. These results provide some reassurance that IPT can be safely used in the second or third trimester of pregnancy. Additional research is needed to evaluate the safety of IPT and new short-course tuberculosis preventive therapies during pregnancy.

Population Pharmacokinetics of Isoniazid, Pyrazinamide, and Ethambutol in Pregnant South African Women with Tuberculosis and HIV.

Tuberculosis is an important cause of maternal morbidity, but little is known about the effects of pregnancy on antituberculosis drug concentrations. We developed population pharmacokinetic models to describe drug dispositions of isoniazid, pyrazinamide, and ethambutol in pregnant women with tuberculosis and HIV. HIV-positive pregnant women with tuberculosis receiving standard first-line tuberculosis treatment and participating in Tshepiso, a prospective cohort study in Soweto, South Africa, underwent sparse pharmacokinetic sampling at >36 weeks of gestation and 7 weeks postpartum. The effects of pregnancy on the pharmacokinetics of isoniazid, pyrazinamide, and ethambutol were investigated via population pharmacokinetic modeling. Isoniazid, pyrazinamide, and ethambutol concentrations were available for 29, 18, and 18 women, respectively. Their median weight was 66 kg while pregnant and 64 kg postpartum. No significant differences were observed in drug clearance, volume of distribution, or bioavailability during and after pregnancy. The model-estimated isoniazid, pyrazinamide, and ethambutol area under the concentration-time curve from 0 to 24 h (AUC0-24) medians were, respectively, 6.88, 419, and 16.5 mg · h/liter during pregnancy versus 5.01, 407, and 19.0 mg · h/liter postpartum. The model-estimated maximum concentration (Cmax) medians for isoniazid, pyrazinamide, and ethambutol were, respectively, 1.39, 35.9, and 1.82 mg/liter during pregnancy versus 1.43, 34.5, and 2.11 mg/liter postpartum. A posteriori power calculations determined that our analysis was powered 91.8%, 59.2%, and 90.1% at a P of <0.01 to detect a 40% decrease in the AUCs of isoniazid, pyrazinamide, and ethambutol, respectively. Pregnancy does not appear to cause relevant changes in the exposure to isoniazid, pyrazinamide, and ethambutol. Additional studies of antituberculosis drugs in pregnancy are needed.

Poor Obstetric and Infant Outcomes in Human Immunodeficiency Virus-Infected Pregnant Women With Tuberculosis in South Africa: The Tshepiso Study.

Background: Before the wide availability of antiretroviral therapy (ART), tuberculosis and human immunodeficiency virus (HIV) disease among pregnant women resulted in poor maternal and neonatal outcomes, including high rates of mother-to-child transmission of both HIV and tuberculosis. We aimed to describe the impact of tuberculosis among HIV-infected mothers on obstetric and infant outcomes in a population with access to ART. Methods: In this prospective cohort study, we followed up HIV-infected pregnant women with or without tuberculosis disease from January 2011 through January 2014 in Soweto, South Africa. Two controls were enrolled for each case patient, matched by enrollment time, maternal age, gestational age, and planned delivery clinic and followed up for 12 months after delivery. Results: We recruited 80 case patients and 155 controls, resulting in 224 live-born infants. Infants of mothers with HIV infection and tuberculosis disease had a higher risk of low birth weight (20.8% vs 10.7%; P = .04), prolonged hospitalization at birth (51% vs 16%; P < .001), infant death (68 vs 7 deaths per 1000 births; P < .001), and tuberculosis disease (12% vs 0%; P < .001) despite appropriate maternal therapy and infant tuberculosis preventive therapy. HIV transmission was higher among these infants (4.1% vs 1.3%; P = .20), though this difference was not statistically significant. Obstetric outcomes in coinfected women were also poorer with higher risks of maternal hospitalization (25% vs 11%; P = .005) and preeclampsia (5.5% vs 0.7%; P = .03). Conclusions: Tuberculosis in HIV coinfected pregnant women remains a significant threat to the health of both mothers and infants. Improving tuberculosis prevention and early diagnosis among pregnant women is critical.

Clinical Strains of Mycobacterium tuberculosis Representing Different Genotype Families Exhibit Distinct Propensities to Adopt the Differentially Culturable State.

Growing evidence points to the presence of differentially culturable tubercle bacteria (DCTB) in clinical specimens from individuals with active tuberculosis (TB) disease. These bacteria are unable to grow on solid media but can resuscitate in liquid media. Given the epidemiological success of certain clinical genotype families of Mycobacterium tuberculosis, we hypothesize that different strains may have distinct mechanisms of adaptation and tolerance. We used an in vitro carbon starvation model to determine the propensity of strains from lineages 2 and 4 that included the Beijing and LAM families respectively, to generate DCTB. Beijing strains were associated with a greater propensity to produce DCTB compared to LAM strains. Furthermore, LAM strains required culture filtrate (CF) for resuscitation whilst starved Beijing strains were not dependent on CF. Moreover, Beijing strains showed improved resuscitation with cognate CF, suggesting the presence of unique growth stimulatory molecules in this family. Analysis of starved Beijing and LAM strains showed longer cells, which with resuscitation were restored to a shorter length. Cell wall staining with fluorescent D-amino acids identified strain-specific incorporation patterns, indicating that cell surface remodeling during resuscitation was distinct between clinical strains. Collectively, our data demonstrate that M. tuberculosis clinical strains from different genotype lineages have differential propensities to generate DCTB, which may have implications for TB treatment success.

The performance of tongue swabs for detection of pulmonary tuberculosis.

Introduction: Oral and/or tongue swabs have demonstrated ability to detect Mycobacterium tuberculosis (Mtb) in adults with pulmonary tuberculosis (TB). Swabs provide useful alternative specimens for diagnosis of TB using molecular assays however, the diagnostic pickup by culture requires further improvement and development. Several studies identified the presence of differentially culturable tubercle bacilli (DCTB) populations in a variety of clinical specimens. These organisms do not grow in routine laboratory media and require growth factors in the form of culture filtrate (CF) from logarithmic phase cultures of Mtb H37Rv. Methods: Herein, we compared the diagnostic performance of sputum and tongue swabs using Mycobacterial Growth Indicator Tube (MGIT) assays, Auramine smear, GeneXpert and DCTB assays supplemented with or without CF. Results: From 89 eligible participants, 83 (93%), 66 (74%) and 79 (89%) were sputum positive by MGIT, smear and GeneXpert, respectively. The corresponding tongue swabs displayed a lower sensitivity with 39 (44%), 2 (2.0%) and 18 (20%) participants respectively for the same tests. We aimed to improve the diagnostic yield by utilizing DCTB assays. Sputum samples were associated with a higher positivity rate for CF-augmented DCTB at 82/89 (92%) relative to tongue swabs at 36/89 (40%). Similarly, sputum samples had a higher positivity rate for DCTB populations that were CF-independent at 64/89 (72%) relative to tongue swabs at 26/89 (29%). DCTB positivity increased significantly, relative to MGIT culture, for tongue swabs taken from HIV-positive participants. We next tested whether the use of an alternative smear stain, DMN-Trehalose, would improve diagnostic yield but noted no substantial increase. Discussion: Collectively, our data show that while tongue swabs yield lower bacterial numbers for diagnostic testing, the use of growth supplementation may improve detection of TB particularly in HIV-positive people but this requires further interrogation in larger studies.

SMA-TB: study protocol for the phase 2b randomized double-blind, placebo-controlled trial to estimate the potential efficacy and safety of two repurposed drugs, acetylsalicylic acid and ibuprofen, for use as adjunct therapy added to, and compared with, the standard WHO recommended TB regimen.

BACKGROUND: The duration and regimen of tuberculosis (TB) treatment is currently based predominantly on whether the M. tuberculosis (Mtb) strain is drug-sensitive (DS) or multidrug-resistant (MDR) with doses adjusted by patients' weight only. The systematic stratification of patients for personalized treatment does not exist for TB. As each TB case is different, individualized treatment regimens should be applied to obtain better outcomes. In this scenario, novel therapeutic approaches are urgently needed to (1) improve outcomes and (2) shorten treatment duration, and host-directed therapies (HDT) might be the best solution. Within HDT, repurposed drugs represent a shortcut in drug development and can be implemented at the short term. As hyperinflammation is associated with worse outcomes, HDT with an anti-inflammatory effect might improve outcomes by reducing tissue damage and thus the risk of permanent sequelae. METHODS: SMA-TB is a multicentre randomized, phase IIB, placebo-controlled, three-arm, double-blinded clinical trial (CT) that has been designed in the context of the EC-funded SMA-TB Project ( www.smatb.eu ) in which we propose to use 2 common non-steroidal anti-inflammatory drugs (NSAID), acetylsalicylic acid (ASA) and ibuprofen (Ibu), as an HDT for use as adjunct therapy added to, and compared with, the standard of care (SoC) World Health Organization (WHO)-recommended TB regimen in TB patients. A total of 354 South African and Georgian adults diagnosed with confirmed pulmonary TB will be randomized into SoC TB treatment + placebo, SoC + acetylsalicylic acid or SoC + ibuprofen. DISCUSSION: SMA-TB will provide proof of concept of the HDT as a co-adjuvant treatment and identify the suitability of the intervention for different population groups (different epidemiological settings and drug susceptibility) in the reduction of tissue damage and risk of bad outcomes for TB patients. This regimen potentially will be more effective and targeted: organ saving, reducing tissue damage and thereby decreasing the length of treatment and sequelae, increasing cure rates and pathogen clearance and decreasing transmission rates. It will result in better clinical practice, care management and increased well-being of TB patients. TRIAL REGISTRATION: Clinicaltrials.gov NCT04575519. Registered on October 5, 2020.

Differentially culturable tubercle bacteria as a measure of tuberculosis treatment response.

Introduction: Routine efficacy assessments of new tuberculosis (TB) treatments include quantitative solid culture or routine liquid culture, which likely miss quantification of drug tolerant bacteria. To improve these assessments, comparative analyses using additional measures such as quantification of differentially culturable tubercle bacteria (DCTB) are required. Essential for enabling this is a comparative measure of TB treatment responses using routine solid and liquid culture with liquid limiting dilutions (LLDs) that detect DCTB in sputum. Methods: We recruited treatment-naïve TB patients, with and without HIV-infection, and serially quantified their sputum for DCTB over the course of treatment. Results: Serial sputum sampling in 73 individuals during their first 14 days of treatment demonstrated that clearance of DCTB was slower compared to routine solid culture. Treatment response appeared to be characterized by four patterns: (1) Classic bi-phasic bacterial clearance; (2) early non-responders with slower clearance; (3) paradoxical worsening with an increase in bacterial count upon treatment initiation; and (4) non-responders with no change in bacterial load. During treatment, LLDs displayed greater bacterial yield when compared with quantitative solid culture. Upon treatment completion, 74% [46/62] of specimens displayed residual DCTB and within this group, two recurrences were diagnosed. Residual DCTB upon treatment completion was associated with a higher proportion of MGIT culture, GeneXpert, and smear positivity at two months post treatment. No recurrences occurred in the group without residual DCTB. Discussion: These data indicate that DCTB assays detect distinct subpopulations of organisms in sputum that are missed by routine solid and liquid culture, and offer important alternatives for efficacy assessments of new TB treatments. The residual DCTB observed upon treatment completion suggests that TB treatment does not always eliminate all bacterial populations, a finding that should be investigated in larger cohorts.

Supplementation of sputum cultures with culture filtrate to detect tuberculosis in a cross-sectional study of HIV-infected individuals.

While some healthcare systems have shifted to molecular diagnostics, culture still remains the gold standard for tuberculosis diagnosis, but it is limited by its long duration to a positive result. Methods to reduce time to culture positivity (TTP) are urgently required. We determined if growth factor supplementation in the mycobacterial growth indicator tube (MGIT) culture system reduces TTP. MGITs were supplemented with fresh culture filtrate (CF) as a source of growth stimulatory molecules from axenic Mycobacterium tuberculosis culture. Different volumes of CF and media components were tested. The performance of these modified MGITs was assessed with sputum from HIV-TB co-infected individuals. Reducing the volume of MGIT cultures and removal of detergent from cultures grown to generate CF had a marginal but significant benefit on reducing TTP. In a subset of specimens, CF inhibited growth. Following optimization of methods, a reduced TTP occurred in specimens with low bacillary load as measured by GeneXpert, smear microscopy and colony forming units. Three specimens that were negative under standard conditions flagged positive following CF supplementation. Our data provide preliminary evidence that addition of CF to MGIT cultures can enhance detection of M. tuberculosis in HIV-TB co-infected patients with low sputum bacillary loads.

Human leukocyte antigen associations with protection against tuberculosis infection and disease in human immunodeficiency virus-1 infected individuals, despite household tuberculosis exposure and immune suppression.

BACKGROUND: To determine the association of human leukocyte antigen (HLA) alleles as correlates of risk for and protection against tuberculin skin test (TST) positivity and active TB disease amongst HIV-infected adults. METHODS: Genomic DNA was extracted from 754 HIV-infected adults whole-blood. HLA-A, -B, -C and -DRB1 loci were genotyped by next generation sequencing methods. HLA alleles were analysed by the presence/absence of TST immune conversion and active TB disease and further stratified by exposure to a household TB contact, CD4+ T-cell count and, for active TB disease, TST-positivity. RESULTS: HLA-A*29:11 and - B*45:01/07 were associated with TST-positivity, while HLA-A*24:02, -A*29:02 and -B*15:16 with TST-negativity. In participants with a household TB contact, HLA-A*66:01, -A*68:02 and -B*49:01 were associated with TST-negativity. For TB disease, HLA-B*41:01, -C*06:02, -DRB1*04:01 and -DRB1*15:01 were associated with susceptibility, while HLA-B*07:02 and -DRB1*11:01 were protective, even for CD4+ T-cell count <350 cells/mm3. For initial TST-positivity and subsequent TB disease, HLA-A*01:01 and -DRB1*11:01 conveyed protection including for those with CD4+ T-cell count <350 cells/mm3. CONCLUSION: Several HLA alleles are noted as correlates of TB infection, risk and natural protection in HIV-infected individuals. HLA associations may enable risk stratification of those with HIV infection. Protective alleles may assist in future TB vaccine development.

Detection of differentially culturable tubercle bacteria in sputum using mycobacterial culture filtrates.

Rapid detection of tuberculosis (TB) infection is paramount to curb further transmission. The gold standard for this remains mycobacterial culture, however emerging evidence confirms the presence of differentially culturable tubercle bacteria (DCTB) in clinical specimens. These bacteria do not grow under standard culture conditions and require the presence of culture filtrate (CF), from axenic cultures of Mycobacterium tuberculosis (Mtb), to emerge. It has been hypothesized that molecules such as resuscitation promoting factors (Rpfs), fatty acids and cyclic-AMP (cAMP) present in CF are responsible for the growth stimulatory activity. Herein, we tested the ability of CF from the non-pathogenic bacterium Mycobacterium smegmatis (Msm) to stimulate the growth of DCTB, as this organism provides a more tractable source of CF. We also interrogated the role of Mtb Rpfs in stimulation of DCTB by creating recombinant strains of Msm that express Mtb rpf genes in various combinations. CF derived from this panel of strains was tested on sputum from individuals with drug susceptible TB prior to treatment. CF from wild type Msm did not enable detection of DCTB in a manner akin to Mtb CF preparations and whilst the addition of RpfABMtb and RpfABCDEMtb to an Msm mutant devoid of its native rpfs did improve detection of DCTB compared to the no CF control, it was not statistically different to the empty vector control. To further investigate the role of Rpfs, we compared the growth stimulatory activity of CF from Mtb, with and without Rpfs and found these to be equivalent. Next, we tested chemically diverse fatty acids and cAMP for growth stimulation and whilst some selective stimulatory effect was observed, this was not significantly higher than the media control and not comparable to CF. Together, these data indicate that the growth stimulatory effect observed with Mtb CF is most likely the result of a combination of factors. Future work aimed at identifying the nature of these growth stimulatory molecules may facilitate improvement of culture-based diagnostics for TB.

Performance of the Roche cobas MTB Assay for the Molecular Diagnosis of Pulmonary Tuberculosis in a High HIV Burden Setting.

Increased access to and improved sensitivities of methods for diagnosing Mycobacterium tuberculosis infection and detecting rifampicin and isoniazid resistance are needed. Herein, the performance of the new cobas MTB assay for use on cobas 6800/8800 Systems (Roche) was assessed and compared with two other commercial assays: RealTime MTB (Abbott), and Xpert MTB/RIF (Cepheid). Molecular PCR-based assays were conducted on sputum specimens from individuals with presumptive and confirmed tuberculosis (n = 294) from two clinical facilities in South Africa between December 2016 and October 2017. Liquid mycobacterial culture was the reference. Test sensitivities were 94.7% (95% CI, 88%-98%), 92.6% (95% CI, 85%-97%), and 91.6% (95% CI, 84%-96%) for cobas MTB, RealTime MTB, and Xpert MTB/RIF assays, respectively. cobas MTB sensitivity was unaffected by HIV coinfection (95.7%; 95% CI, 88%-99%; n = 176) and sediment testing (94.7%; 95% CI, 88%-98%). Sensitivities were 81.8% (95% CI, 60%-95%), 72.7% (95% CI, 50%-89%), and 72.7% (95% CI, 50%-89%) among smear-negative, culture-positive individuals (n = 221) for cobas MTB, RealTime MTB, and Xpert MTB/RIF assays, respectively. cobas MTB specificity was 95.7% (95% CI, 89%-99%) and 99% (95% CI, 94%-100%) among HIV coinfected and uninfected individuals, respectively. The cobas 6800/8800 system is already implemented in South Africa for high-throughput HIV viral load testing, making it suitable for integrated HIV/tuberculosis diagnostics.

Chronic HIV-1 Infection Alters the Cellular Distribution of FcγRIIIa and the Functional Consequence of the FcγRIIIa-F158V Variant.

Chronic HIV-infection modulates the expression of Fc gamma receptors (FcγRs) on immune cells and their antibody-dependent effector function capability. Given the increasingly recognized importance of antibody-dependent cellular cytotoxicity (ADCC) in HIV-specific immunity, we investigated the cellular distribution of FcγRIIIa on cytotoxic lymphocytes-natural killer cells and CD8+ T cells-and the effect of the FcγRIIIa-F158V variant on ADCC capacity in HIV-infected individuals (n = 23) and healthy controls (n = 23). Study participants were matched for F158V genotypes, carried two copies of the FCGR3A gene and were negative for FcγRIIb expression on NK cells. The distribution of CD56dimFcγRIIIabright and CD56negFcγRIIIabright NK cell subsets, but not FcγRIIIa surface expression, differed significantly between HIV-1 negative and HIV-1 positive donors. NK cell-mediated ADCC responses negatively correlated with the proportion of the immunoregulatory CD56brightFcγRIIIadim/neg cells and were lower in the HIV-1 positive group. Intriguingly, the FcγRIIIa-F158V variant differentially affected the NK-mediated ADCC responses for HIV-1 negative and HIV-1 positive donors. Healthy donors bearing at least one 158V allele had higher ADCC responses compared to those homozygous for the 158F allele (48.1 vs. 34.1%), whereas the opposite was observed for the HIV-infected group (26.4 vs. 34.6%), although not statistically significantly different. Furthermore, FcγRIIIa+CD8bright and FcγRIIIa+CD8dim T cell subsets were observed in both HIV-1 negative and HIV-1 positive donors, with median proportions that were significantly higher in HIV-1 positive donors compared to healthy controls (15.7 vs. 8.3%; P = 0.016 and 18.2 vs. 14.1%; P = 0.038, respectively). Using an HIV-1-specific GranToxiLux assay, we demonstrate that CD8+ T cells mediate ADCC through the delivery of granzyme B, which was overall lower compared to that of autologous NK cells. In conclusion, our findings demonstrate that in the presence of an HIV-1 infection, the cellular distribution of FcγRIIIa is altered and that the functional consequence of FcγRIIIa variant is affected. Importantly, it underscores the need to characterize FcγR expression, cellular distribution and functional consequences of FcγR genetic variants within a specific environment or disease state.