Optimising the yield from bronchoalveolar lavage on human participants in infectious disease immunology research.

Bronchoalveolar lavage (BAL) is becoming a common procedure for research into infectious disease immunology. Little is known about the clinical factors which influence the main outcomes of the procedure. In research participants who underwent BAL according to guidelines, the BAL volume yield, and cell yield, concentration, viability, pellet colour and differential count were analysed for association with important participant characteristics such as active tuberculosis (TB) disease, TB exposure, HIV infection and recent SARS-CoV-2 infection. In 337 participants, BAL volume and BAL cell count were correlated in those with active TB disease, and current smokers. The right middle lobe yielded the highest volume. BAL cell and volume yields were lower in older participants, who also had more neutrophils. Current smokers yielded lower volumes and higher numbers of all cell types, and usually had a black pellet. Active TB disease was associated with higher cell yields, and higher proportions of granulocytes, but this declined at the end of treatment. HIV infection was associated with lower cell yields and more bloody pellets, and recent SARS-CoV-2 infection with a higher proportion of lymphocytes. These results allow researchers to optimise their participant and end assay selection for projects involving lung immune cells.

Sildenafil, a Type-5 Phosphodiesterase Inhibitor, Fails to Reverse Myeloid-Derived Suppressor Cell-Mediated T Cell Suppression in Cells Isolated From Tuberculosis Patients.

Successful TB treatment is hampered by increasing resistance to the two most effective first-line anti-TB drugs, namely isoniazid and rifampicin, thus innovative therapies focused on host processes, termed host-directed therapies (HDTs), are promising novel approaches for increasing treatment efficacy without inducing drug resistance. We assessed the ability of Sildenafil, a type-5 phosphodiesterase inhibitor, as a repurposed compound, to serve as HDT target, by counteracting the suppressive effects of myeloid-derived suppressor cells (MDSC) obtained from active TB cases on T-cell responsiveness. We confirm that MDSC suppress non-specific T-cell activation. We also show that Sildenafil treatment fails to reverse the MDSC-mediated suppression of T-cell functions measured here, namely activation and proliferation. The impact of Sildenafil treatment on improved immunity, using the concentration tested here, is likely to be minimal, but further identification and development of MDSC-targeting TB host-directed therapies are warranted.

Longitudinal Dynamics of a Blood Transcriptomic Signature of Tuberculosis.

Rationale: Performance of blood transcriptomic tuberculosis (TB) signatures in longitudinal studies and effects of TB-preventive therapy and coinfection with HIV or respiratory organisms on transcriptomic signatures has not been systematically studied. Objectives: We evaluated longitudinal kinetics of an 11-gene blood transcriptomic TB signature, RISK11, and effects of TB-preventive therapy (TPT) and respiratory organisms on RISK11 signature score, in HIV-uninfected and HIV-infected individuals. Methods: RISK11 was measured in a longitudinal study of RISK11-guided TPT in HIV-uninfected adults, a cross-sectional respiratory organisms cohort, or a longitudinal study in people living with HIV (PLHIV). HIV-uninfected RISK11+ participants were randomized to TPT or no TPT; RISK11- participants received no TPT. PLHIV received standard-of-care antiretroviral therapy and TPT. In the cross-sectional respiratory organisms cohort, viruses and bacteria in nasopharyngeal and oropharyngeal swabs were quantified by real-time quantitative PCR. Measurements and Main Results: RISK11+ status was transient in most of the 128 HIV-negative participants with longitudinal samples; more than 70% of RISK11+ participants reverted to RISK11- by 3 months, irrespective of TPT. By comparison, reversion from a RISK11+ state was less common in 645 PLHIV (42.1%). Non-HIV viral and nontuberculous bacterial organisms were detected in 7.2% and 38.9% of the 1,000 respiratory organisms cohort participants, respectively, and among those investigated for TB, 3.8% had prevalent disease. Median RISK11 scores (%) were higher in participants with viral organisms alone (46.7%), viral and bacterial organisms (42.8%), or prevalent TB (85.7%) than those with bacterial organisms other than TB (13.4%) or no organisms (14.2%). RISK11 could not discriminate between prevalent TB and viral organisms. Conclusions: Positive RISK11 signature status is often transient, possibly due to intercurrent viral infection, highlighting potentially important challenges for implementation of these biomarkers as new tools for TB control.

Establishment of a Patient-Derived, Magnetic Levitation-Based, Three-Dimensional Spheroid Granuloma Model for Human Tuberculosis.

Tuberculous granulomas that develop in response to Mycobacterium tuberculosis (M. tuberculosis) infection are highly dynamic entities shaped by the host immune response and disease kinetics. Within this microenvironment, immune cell recruitment, polarization, and activation are driven not only by coexisting cell types and multicellular interactions but also by M. tuberculosis-mediated changes involving metabolic heterogeneity, epigenetic reprogramming, and rewiring of the transcriptional landscape of host cells. There is an increased appreciation of the in vivo complexity, versatility, and heterogeneity of the cellular compartment that constitutes the tuberculosis (TB) granuloma and the difficulty in translating findings from animal models to human disease. Here, we describe a novel biomimetic in vitro three-dimensional (3D) human lung spheroid granuloma model, resembling early "innate" and "adaptive" stages of the TB granuloma spectrum, and present results of histological architecture, host transcriptional characterization, mycobacteriological features, cytokine profiles, and spatial distribution of key immune cells. A range of manipulations of immune cell populations in these spheroid granulomas will allow the study of host/pathogen pathways involved in the outcome of infection, as well as pharmacological interventions. IMPORTANCE TB is a highly infectious disease, with granulomas as its hallmark. Granulomas play an important role in the control of M. tuberculosis infection and as such are crucial indicators for our understanding of host resistance to TB. Correlates of risk and protection to M. tuberculosis are still elusive, and the granuloma provides the perfect environment in which to study the immune response to infection and broaden our understanding thereof; however, human granulomas are difficult to obtain, and animal models are costly and do not always faithfully mimic human immunity. In fact, most TB research is conducted in vitro on immortalized or primary immune cells and cultured in two dimensions on flat, rigid plastic, which does not reflect in vivo characteristics. We have therefore conceived a 3D, human in vitro spheroid granuloma model which allows researchers to study features of granuloma-forming diseases in a 3D structural environment resembling in vivo granuloma architecture and cellular orientation.

Validation of a host blood transcriptomic biomarker for pulmonary tuberculosis in people living with HIV: a prospective diagnostic and prognostic accuracy study.

BACKGROUND: A rapid, blood-based triage test that allows targeted investigation for tuberculosis at the point of care could shorten the time to tuberculosis treatment and reduce mortality. We aimed to test the performance of a host blood transcriptomic signature (RISK11) in diagnosing tuberculosis and predicting progression to active pulmonary disease (prognosis) in people with HIV in a community setting. METHODS: In this prospective diagnostic and prognostic accuracy study, adults (aged 18-59 years) with HIV were recruited from five communities in South Africa. Individuals with a history of tuberculosis or household exposure to multidrug-resistant tuberculosis within the past 3 years, comorbid risk factors for tuberculosis, or any condition that would interfere with the study were excluded. RISK11 status was assessed at baseline by real-time PCR; participants and study staff were masked to the result. Participants underwent active surveillance for microbiologically confirmed tuberculosis by providing spontaneously expectorated sputum samples at baseline, if symptomatic during 15 months of follow-up, and at 15 months (the end of the study). The coprimary outcomes were the prevalence and cumulative incidence of tuberculosis disease confirmed by a positive Xpert MTB/RIF, Xpert Ultra, or Mycobacteria Growth Indicator Tube culture, or a combination of such, on at least two separate sputum samples collected within any 30-day period. FINDINGS: Between March 22, 2017, and May 15, 2018, 963 participants were assessed for eligibility and 861 were enrolled. Among 820 participants with valid RISK11 results, eight (1%) had prevalent tuberculosis at baseline: seven (2·5%; 95% CI 1·2-5·0) of 285 RISK11-positive participants and one (0·2%; 0·0-1·1) of 535 RISK11-negative participants. The relative risk (RR) of prevalent tuberculosis was 13·1 times (95% CI 2·1-81·6) greater in RISK11-positive participants than in RISK11-negative participants. RISK11 had a diagnostic area under the receiver operating characteristic curve (AUC) of 88·2% (95% CI 77·6-96·7), and a sensitivity of 87·5% (58·3-100·0) and specificity of 65·8% (62·5-69·0) at a predefined score threshold (60%). Of those with RISK11 results, eight had primary endpoint incident tuberculosis during 15 months of follow-up. Tuberculosis incidence was 2·5 per 100 person-years (95% CI 0·7-4·4) in the RISK11-positive group and 0·2 per 100 person-years (0·0-0·5) in the RISK11-negative group. The probability of primary endpoint incident tuberculosis was greater in the RISK11-positive group than in the RISK11-negative group (cumulative incidence ratio 16·0 [95% CI 2·0-129·5]). RISK11 had a prognostic AUC of 80·0% (95% CI 70·6-86·9), and a sensitivity of 88·6% (43·5-98·7) and a specificity of 68·9% (65·3-72·3) for incident tuberculosis at the 60% threshold. INTERPRETATION: RISK11 identified prevalent tuberculosis and predicted risk of progression to incident tuberculosis within 15 months in ambulant people living with HIV. RISK11's performance approached, but did not meet, WHO's target product profile benchmarks for screening and prognostic tests for tuberculosis. FUNDING: Bill & Melinda Gates Foundation and the South African Medical Research Council.

Higher SARS-CoV-2 seroprevalence in workers with lower socioeconomic status in Cape Town, South Africa.

BACKGROUND: Inequality is rife throughout South Africa. The first wave of COVID-19 may have affected people in lower socioeconomic groups worse than the affluent. The SARS-CoV-2 seroprevalence and the specificity of anti-SARS-CoV-2 antibody tests in South Africa is not known. METHODS: We tested 405 volunteers representing all socioeconomic strata from the workforce of a popular shopping and tourist complex in central Cape Town with the Abbott SARS-CoV-2 IgG assay. We assessed the association between antibody positivity and COVID-19 symptom status, medical history, and sociodemographic variables. We tested 137 serum samples from healthy controls collected in Cape Town prior to the COVID-19 pandemic, to confirm the specificity of the assay in the local population. RESULTS: Of the 405 volunteers tested one month after the first peak of the epidemic in Cape Town, 96(23.7%) were SARS-CoV-2 IgG positive. Of those who tested positive, 46(47.9%) reported no symptoms of COVID-19 in the previous 6 months. Seropositivity was significantly associated with living in informal housing, residing in a subdistrict with low income-per household, and having a low-earning occupation. The specificity of the assay was 98.54%(95%CI 94.82%-99.82%) in the pre-COVID controls. CONCLUSIONS: There is a high background seroprevalence in Cape Town, particularly in people of lower socioeconomic status. Almost half of cases are asymptomatic, and therefore undiagnosed by local testing strategies. These results cannot be explained by low assay specificity.

Biomarker-guided tuberculosis preventive therapy (CORTIS): a randomised controlled trial.

BACKGROUND: Targeted preventive therapy for individuals at highest risk of incident tuberculosis might impact the epidemic by interrupting transmission. We tested performance of a transcriptomic signature of tuberculosis (RISK11) and efficacy of signature-guided preventive therapy in parallel, using a hybrid three-group study design. METHODS: Adult volunteers aged 18-59 years were recruited at five geographically distinct communities in South Africa. Whole blood was sampled for RISK11 by quantitative RT-PCR assay from eligible volunteers without HIV, recent previous tuberculosis (ie, <3 years before screening), or comorbidities at screening. RISK11-positive participants were block randomised (1:2; block size 15) to once-weekly, directly-observed, open-label isoniazid and rifapentine for 12 weeks (ie, RISK11 positive and 3HP positive), or no treatment (ie, RISK11 positive and 3HP negative). A subset of eligible RISK11-negative volunteers were randomly assigned to no treatment (ie, RISK11 negative and 3HP negative). Diagnostic discrimination of prevalent tuberculosis was tested in all participants at baseline. Thereafter, prognostic discrimination of incident tuberculosis was tested in the untreated RISK11-positive versus RISK11-negative groups, and treatment efficacy in the 3HP-treated versus untreated RISK11-positive groups, during active surveillance through 15 months. The primary endpoint was microbiologically confirmed pulmonary tuberculosis. The primary outcome measures were risk ratio [RR] for tuberculosis of RISK11-positive to RISK11-negative participants, and treatment efficacy. This trial is registered with ClinicalTrials.gov, NCT02735590. FINDINGS: 20 207 volunteers were screened, and 2923 participants were enrolled, including RISK11-positive participants randomly assigned to 3HP (n=375) or no 3HP (n=764), and 1784 RISK11-negative participants. Cumulative probability of prevalent or incident tuberculosis disease was 0·066 (95% CI 0·049 to 0·084) in RISK11-positive (3HP negative) participants and 0·018 (0·011 to 0·025) in RISK11-negative participants (RR 3·69, 95% CI 2·25-6·05) over 15 months. Tuberculosis prevalence was 47 (4·1%) of 1139 versus 14 (0·78%) of 1984 in RISK11-positive compared with RISK11-negative participants, respectively (diagnostic RR 5·13, 95% CI 2·93 to 9·43). Tuberculosis incidence over 15 months was 2·09 (95% CI 0·97 to 3·19) vs 0·80 (0·30 to 1·30) per 100 person years in RISK11-positive (3HP-negative) participants compared with RISK11-negative participants (cumulative incidence ratio 2·6, 95% CI 1·2 to 5·9). Serious adverse events related to 3HP included one hospitalisation for seizures (unintentional isoniazid overdose) and one death of unknown cause (possibly temporally related). Tuberculosis incidence over 15 months was 1·94 (95% CI 0·35 to 3·50) versus 2·09 (95% CI 0·97 to 3·19) per 100 person-years in 3HP-treated RISK11-positive participants compared with untreated RISK11-positive participants (efficacy 7·0%, 95% CI -145 to 65). INTERPRETATION: The RISK11 signature discriminated between individuals with prevalent tuberculosis, or progression to incident tuberculosis, and individuals who remained healthy, but provision of 3HP to signature-positive individuals after exclusion of baseline disease did not reduce progression to tuberculosis over 15 months. FUNDING: Bill and Melinda Gates Foundation, South African Medical Research Council.

GPR183 Regulates Interferons, Autophagy, and Bacterial Growth During Mycobacterium tuberculosis Infection and Is Associated With TB Disease Severity.

Oxidized cholesterols have emerged as important signaling molecules of immune function, but little is known about the role of these oxysterols during mycobacterial infections. We found that expression of the oxysterol-receptor GPR183 was reduced in blood from patients with tuberculosis (TB) and type 2 diabetes (T2D) compared to TB patients without T2D and was associated with TB disease severity on chest x-ray. GPR183 activation by 7α,25-dihydroxycholesterol (7α,25-OHC) reduced growth of Mycobacterium tuberculosis (Mtb) and Mycobacterium bovis BCG in primary human monocytes, an effect abrogated by the GPR183 antagonist GSK682753. Growth inhibition was associated with reduced IFN-ß and IL-10 expression and enhanced autophagy. Mice lacking GPR183 had significantly increased lung Mtb burden and dysregulated IFNs during early infection. Together, our data demonstrate that GPR183 is an important regulator of intracellular mycobacterial growth and interferons during mycobacterial infection.

Prospective evaluation of host biomarkers other than interferon gamma in QuantiFERON Plus supernatants as candidates for the diagnosis of tuberculosis in symptomatic individuals.

BACKGROUND: There is an urgent need for new tools for the diagnosis of TB. We evaluated the usefulness recently described host biomarkers in supernatants from the newest generation of the QuantiFERON test (QuantiFERON Plus) as tools for the diagnosis of active TB. METHODS: We recruited individuals presenting at primary health care clinics in Cape Town, South Africa with symptoms requiring investigation for TB disease, prior to the establishment of a clinical diagnosis. Participants were later classified as TB or other respiratory diseases (ORD) based on the results of clinical and laboratory tests. Using a multiplex platform, we evaluated the concentrations of 37 host biomarkers in QuantiFERON Plus supernatants from study participants as tools for the diagnosis of TB. RESULTS: Out of 120 study participants, 35(29.2%) were diagnosed with active TB, 69(57.5%) with ORD whereas 16(13.3%) were excluded. 14(11.6%) of the study participants were HIV infected. Although individual host markers showed potential as diagnostic candidates, the main finding of the study was the identification of a six-marker biosignature in unstimulated supernatants (Apo-ACIII, CXCL1, CXCL9, CCL8, CCL-1, CD56) which diagnosed TB with sensitivity and specificity of 73.9%(95% CI; 51.6-87.8) and 87.6%(95% CI; 77.2-94.5), respectively, after leave-one-out cross validation. Combinations between TB-antigen specific biomarkers also showed potential (sensitivity of 77.3% and specificity of 69.2%, respectively), with multiple biomarkers being significantly different between TB patients, Quantiferon Plus Positive and Quantiferon Plus negative individuals with ORD, regardless of HIV status. CONCLUSIONS: Biomarkers detected in QuantiFERON Plus supernatants may contribute to adjunctive diagnosis of TB.