Circulating Biomarkers, Fraction of Exhaled Nitric Oxide, and Lung Function in Patients With Human Immunodeficiency Virus and Tuberculosis.

BACKGROUND: Identification of proinflammatory factors responding to Mycobacterium tuberculosis is important to reduce long-term sequelae of pulmonary tuberculosis (TB). METHODS: We examined the association between plasma biomarkers, the fraction of exhaled nitric oxide (FeNO), and lung function among a prospective cohort of 105 adults newly diagnosed with TB/human immunodeficiency virus (HIV) in South Africa. Participants were followed for 48 weeks from antiretroviral therapy (ART) initiation with serial assessments of plasma biomarkers, FeNO, lung function, and respiratory symptoms. Linear regression and generalized estimating equations were used to examine the associations at baseline and over the course of TB treatment, respectively. RESULTS: At baseline, higher FeNO levels were associated with preserved lung function, whereas greater respiratory symptoms and higher interleukin (IL)-6 plasma levels were associated with worse lung function. After ART and TB treatment initiation, improvements in lung function were associated with increases in FeNO (rate ratio [RR] = 86 mL, 95% confidence interval [CI] = 34-139) and decreases in IL-6 (RR = -118 mL, 95% CI = -193 to -43) and vascular endothelial growth factor ([VEGF] RR = -178 mL, 95% CI = -314 to -43). CONCLUSIONS: Circulating IL-6, VEGF, and FeNO are associated with lung function in adults being treated for TB/HIV. These biomarkers may help identify individuals at higher risk for post-TB lung disease and elucidate targetable pathways to modify the risk of chronic lung impairment among TB survivors.

Qualification of the differential leukocyte count and immunophenotyping in cryopreserved ex vivo whole blood assay.

We developed a flow cytometry-based assay, termed Differential Leukocyte Counting and Immunophenotyping in Cryopreserved Ex vivo whole blood (DLC-ICE), that allows quantification of absolute counts and frequencies of leukocyte subsets and measures expression of activation, phenotypic and functional markers. We evaluated the performance of the DLC-ICE assay by determining inter-operator variability for processing fresh whole blood (WB) from healthy donors collected at multiple clinical sites. In addition, we assessed inter-operator variability for staining of fixed cells and robustness across different anticoagulants. Accuracy was evaluated by comparing DLC-ICE measurements to real-time cell enumeration using an accredited hematology analyzer. Finally, we developed and tested the performance of a 27-colour immunophenotyping panel on cryopreserved fixed WB and compared results to matched fresh WB. Overall, we observed <20% variability in absolute counts and frequencies of granulocytes, monocytes and lymphocytes (T, B and NK cells) when fresh WB was collected in different anti-coagulant tubes, processed or stained by independent operators. Absolute cell counts measured across operators and anti-coagulants using the DLC-ICE method exhibited excellent correlation with the reference method, complete blood count (CBC) with differential, measured using a hematology analyzer (r2 > 0.9 for majority of measurements). A comparison of leukocyte immunophenotyping on fresh WB versus DLC-ICE processed blood yielded equivalent and linear results over a wide dynamic range (r2 = 0.94 over 10-104 cells/µL). These results demonstrate low variability across trained operators, high robustness, linearity and accuracy, supporting utility of the DLC-ICE assay for large cohort studies involving multiple clinical research sites.

Pulmonary restriction predicts long-term pulmonary impairment in people with HIV and tuberculosis.

BACKGROUND: While tuberculosis is considered a risk factor for chronic obstructive pulmonary disease, a restrictive pattern of pulmonary impairment may actually be more common among tuberculosis survivors. We aimed to determine the nature of pulmonary impairment before and after treatment among people with HIV and tuberculosis and identify risk factors for long-term impairment. METHODS: In this prospective cohort study conducted in South Africa, we enrolled adults newly diagnosed with HIV and tuberculosis who were initiating antiretroviral therapy and tuberculosis treatment. We measured lung function and symptoms at baseline, 6, and 12 months. We compared participants with and without pulmonary impairment and constructed logistic regression models to identify characteristics associated with pulmonary impairment. RESULTS: Among 134 participants with a median CD4 count of 110 cells/µl, 112 (83%) completed baseline spirometry at which time 32 (29%) had restriction, 13 (12%) had obstruction, and 9 (7%) had a mixed pattern. Lung function was dynamic over time and 30 (33%) participants had impaired lung function at 12 months. Baseline restriction was associated with greater symptoms and with long-term pulmonary impairment (adjusted odds ratio 5.44, 95% confidence interval 1.16-25.45), while baseline obstruction was not (adjusted odds ratio 1.95, 95% confidence interval 0.28-13.78). CONCLUSIONS: In this cohort of people with HIV and tuberculosis, restriction was the most common, symptomatic, and persistent pattern of pulmonary impairment. These data can help to raise awareness among clinicians about the heterogeneity of post-tuberculosis pulmonary impairment, and highlight the need for further research into mediators of lung injury in this vulnerable population.

Declines in Lung Function After Antiretroviral Therapy Initiation in Adults With Human Immunodeficiency Virus and Tuberculosis: A Potential Manifestation of Respiratory Immune Reconstitution Inflammatory Syndrome.

End-organ impairment has received relatively little research attention as a possible manifestation of tuberculosis immune reconstitution inflammatory syndrome (TB-IRIS). In this prospective cohort study, one-half of adults with human immunodeficiency virus and pulmonary tuberculosis experienced meaningful declines in lung function on antiretroviral therapy, suggesting a role for lung function in TB-IRIS definitions.

A Common NLRC4 Gene Variant Associates With Inflammation and Pulmonary Function in Human Immunodeficiency Virus and Tuberculosis.

BACKGROUND: Inflammasomes mediate inflammation in adults living with both human immunodeficiency virus (HIV) and tuberculosis (TB), but the relevance of inflammasome gene polymorphisms in TB-associated pulmonary damage is unknown. We hypothesized that functional single-nucleotide polymorphisms (SNPs) in inflammasome pathway genes modify systemic and pulmonary inflammation, contributing to respiratory impairment in adults living with HIV/pulmonary TB. METHODS: This was a prospective cohort study set in South Africa following individuals living with HIV/TB up to 48 weeks post-antiretroviral therapy (ART) initiation. Ten functional SNPs in 5 inflammasome pathway genes were related to circulating inflammatory biomarkers and lung function assessed by spirometry pre- and post-ART initiation. Analyses used 2-sided t tests, Wilcoxon rank sum tests, Spearman correlation coefficients, linear regression, and generalized estimating equation models. RESULTS: Among 102 patients with baseline samples, the minor allele (T) in NLRC4 rs385076 was independently associated with lower levels of interleukin (IL)-18 and IL-6 before and up to 12 weeks post-ART initiation (Benjamini-Hochberg corrected P values < .02). Patients with the CT/TT genotypes also had improved lung function vs CC patients up to 48 weeks post-ART initiation (forced vital capacity, 206 mL higher; 95% confidence interval [CI], 67-345 mL; P = .004 and forced expiratory volume in 1 second, 143 mL higher; 95% CI, 11-274 mL; P = .034). CONCLUSIONS: A common SNP in the NLRC4 inflammasome may modify TB-associated inflammation in clinically relevant ways. This SNP may identify high-risk groups for lung damage in TB. Inhibition of NLRC4 activity may be an important approach for TB host-directed therapy.

Lung Injury on Antiretroviral Therapy in Adults With Human Immunodeficiency Virus/Tuberculosis.

BACKGROUND: Immune restoration on antiretroviral therapy (ART) can drive inflammation in people living with human immunodeficiency virus (HIV) who have pulmonary tuberculosis (TB), but its effects on the lungs have not been assessed. We evaluated associations between pulmonary inflammation, recovery of pathogen-specific CD4 T-cell function, and lung injury prior to and after ART initiation in adults with HIV and pulmonary TB. METHODS: This was a prospective cohort study in South Africa, following adults with HIV and pulmonary TB prior to and up to 48 weeks after ART initiation. Pulmonary-specific inflammation was defined as total glycolytic activity (TGA) on [18]F-fluorodeoxyglucose (FDG) positron emission tomography-computed tomography (PET-CT) at baseline and 4 weeks after ART initiation. Spirometry, respiratory symptom tests, and flow cytometry were performed at the same times to assess lung involvement and the frequency of mycobacteria-specific CD4 T-cells. In addition, we evaluated lung function longitudinally up to 48 weeks after ART initiation. RESULTS: Greater lung TGA on FDG PET-CT was associated with worse lung function and respiratory symptoms prior to ART initiation, and nearly half of subjects experienced worsening lung inflammation and lung function at Week 4 of ART. Worsening Week 4 lung inflammation and pulmonary function were both associated with greater increases in pathogen-specific functional CD4 T-cell responses on ART, and early decreases in lung function were independently associated with persistently lower lung function months after TB treatment completion. CONCLUSIONS: Increases in pulmonary inflammation and decreases in lung function are common on ART, relate to greater ART-mediated CD4 T-cell restoration, and are associated with the persistent impairment of lung function in individuals with HIV/TB.

Predictors of male condom use among sexually active heterosexual young women in South Africa, 2012.

BACKGROUND: In South Africa, young women are at disproportionate risk of HIV infection with about 2363 new infections per week in 2015. Proper condom use is one of the most effective HIV/AIDS prevention strategies among sexually active persons. Understanding factors associated with male condom use in this key population group is important to curb the spread of HIV. This study determined practices and predictors of male condom use among sexually active young women in South Africa. METHODS: The 2012 National HIV Communication Survey measured the extent of exposure to communication activities for HIV prevention among men and women aged 16-55 years in South Africa. We performed a secondary data analysis on a subset of this survey, focussing on 1031 women aged 16-24 years who reported having had sex in the past 12 months. We determined predictors of male condom use using the unconditional multivariable logistic regression model. RESULTS: Of the 1031 young women, 595 (57.8%) reported using a male condom at last sex, 68.4% in women aged 16-19 years and 54.5% in women aged 20-24 years (p < 0.001). Delayed sexual debut [20 years or above] (Adjusted Odds Ratio [aOR] 2.1, 95% CI: 1.2 to 3.7, p = 0.006); being a student (aOR 1.6, 95% CI: 1.2 to 2.3, p = 0.005); and exposure to HIV communication programmes (aOR 3.1, 95% CI: 1.2 to 8.6, p = 0.025) were significantly associated with male condom use at last sex. CONCLUSION: Male condom use was a common practice among young women and was associated with delayed sexual debut and exposure to HIV communication programmes. Behavioral interventions and HIV communication programmes should therefore encourage young women to delay initiation of sex and promote usage of male condoms.

Risk factors for tuberculosis smear non-conversion in Eden district, Western Cape, South Africa, 2007-2013: a retrospective cohort study.

BACKGROUND: Tuberculosis (TB) continues to be a major global health problem. While progress has been made to improve TB cure rates, South Africa's 76 % smear-positive pulmonary TB (PTB) case cure rate remains below the WHO target of 85 %. We report on the trends of TB smear non-conversion and their predictors at the end of an intensive phase of treatment, and how this impacted on treatment outcomes of smear-positive PTB cases in Eden District, Western Cape Province, South Africa. METHODS: Routinely collected, retrospective data of smear-positive PTB cases from the electronic TB register in Eden District between 2007 and 2013 was extracted. Non-conversion was defined as persistent sputum smear-positive PTB cases at the end of the two or three month intensive phase of treatment. Chi-square test for linear trend and simple linear regression analysis were used to analyse the change in percentages and slope of TB smear non-conversion rates over time. Risk factors for TB non-conversion, and their impact on treatment outcomes, were evaluated using logistic regression models. RESULTS: Of 12,742 total smear-positive PTB cases included in our study, 12.8 % (n = 1627) did not sputum smear convert; 13.3 % (1411 of 10,574) of new cases and 9.9 % (216 of 2168) of re-treatment cases. Although not statistically significant in either new or re-treatment cases, between 2007 and 2013, smear non-conversion decreased from 16.4 to 12.7 % (slope = -0.60; 95 % CI: -1.49 to 0.29; p = 0.142) in new cases, and from 11.3 to 10.8 % in re-treatment cases (slope = -0.29; 95 % CI: -1.06 to 0.48; p = 0.376). Male gender, HIV co-infection and a >2+ acid fast bacilli (AFB) smear grading at the start of TB treatment were independent risk factors for non-conversion (p < 0.001). Age was a risk factor for non-conversion in new cases, but not for re-treatment cases. Non-conversion was also associated with unsuccessful treatment outcomes (p < 0.01), including treatment default and treatment failure. CONCLUSIONS: Smear-positive PTB cases, especially men and those with identified risk factors for non-conversion, should be closely monitored throughout their treatment period. The South African TB control program should invest in patient adherence counselling and education to mitigate TB non-conversion risk factors, and to improve conversion and TB cure rates.

Distinct kinetics of Gag-specific CD4+ and CD8+ T cell responses during acute HIV-1 infection.

HIV infection is characterized by a gradual deterioration of immune function, mainly in the CD4 compartment. To better understand the dynamics of HIV-specific T cells, we analyzed the kinetics and polyfunctional profiles of Gag-specific CD4(+) and CD8(+) T cell responses in 12 subtype C-infected individuals with different disease-progression profiles, ranging from acute to chronic HIV infection. The frequencies of Gag-responsive CD4(+) and CD8(+) T cells showed distinct temporal kinetics. The peak frequency of Gag-responsive IFN-γ(+)CD4(+) T cells was observed at a median of 28 d (interquartile range: 21-81 d) post-Fiebig I/II staging, whereas Gag-specific IFN-γ(+)CD8(+) T cell responses peaked at a median of 253 d (interquartile range: 136-401 d) and showed a significant biphasic expansion. The proportion of TNF-α-expressing cells within the IFN-γ(+)CD4(+) T cell population increased (p = 0.001) over time, whereas TNF-α-expressing cells within IFN-γ(+)CD8(+) T cells declined (p = 0.005). Both Gag-responsive CD4(+) and CD8(+) T cells showed decreased Ki67 expression within the first 120 d post-Fiebig I/II staging. Prior to the disappearance of Gag-responsive Ki67(+)CD4(+) T cells, these cells positively correlated (p = 0.00038) with viremia, indicating that early Gag-responsive CD4 events are shaped by viral burden. No such associations were observed in the Gag-specific CD8(+) T cell compartment. Overall, these observations indicated that circulating Gag-responsive CD4(+) and CD8(+) T cell frequencies and functions are not synchronous, and properties change rapidly at different tempos during early HIV infection.

Fluidity of HIV-1-specific T-cell responses during acute and early subtype C HIV-1 infection and associations with early disease progression.

Deciphering immune events during early stages of human immunodeficiency virus type 1 (HIV-1) infection is critical for understanding the course of disease. We characterized the hierarchy of HIV-1-specific T-cell gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) assay responses during acute subtype C infection in 53 individuals and associated temporal patterns of responses with disease progression in the first 12 months. There was a diverse pattern of T-cell recognition across the proteome, with the recognition of Nef being immunodominant as early as 3 weeks postinfection. Over the first 6 months, we found that there was a 23% chance of an increased response to Nef for every week postinfection (P = 0.0024), followed by a nonsignificant increase to Pol (4.6%) and Gag (3.2%). Responses to Env and regulatory proteins appeared to remain stable. Three temporal patterns of HIV-specific T-cell responses could be distinguished: persistent, lost, or new. The proportion of persistent T-cell responses was significantly lower (P = 0.0037) in individuals defined as rapid progressors than in those progressing slowly and who controlled viremia. Almost 90% of lost T-cell responses were coincidental with autologous viral epitope escape. Regression analysis between the time to fixed viral escape and lost T-cell responses (r = 0.61; P = 0.019) showed a mean delay of 14 weeks after viral escape. Collectively, T-cell epitope recognition is not a static event, and temporal patterns of IFN-γ-based responses exist. This is due partly to viral sequence variation but also to the recognition of invariant viral epitopes that leads to waves of persistent T-cell immunity, which appears to associate with slower disease progression in the first year of infection.

Association of HIV-specific and total CD8+ T memory phenotypes in subtype C HIV-1 infection with viral set point.

Understanding early immunological events during HIV-1 infection that may set the course of disease progression is important for identifying correlates of viral control. This study explores the association of differentiation profiles of HIV-specific and total memory CD8(+) T cells with viral set point. A cohort of 47 HIV-1-infected individuals, with differing viral set points at 12 mo, were recruited during acute infection. We identified that the magnitude of IFN-gamma(+) T cell responses at 6 mo postinfection did not associate with viral set point at 12 mo. A subset of 16 individuals was further studied to characterize CD8(+) T cells for expression patterns of markers for memory differentiation, survival (CD127), senescence (CD57), and negative regulation (programmed death-1). We show that viral control and the predicted tempo of HIV disease progression in the first year of infection was associated with a synchronous differentiation of HIV-specific and total CD8(+) memory subpopulations. At 6-9 mo postinfection, those with low viral set points had a significantly higher proportion of early differentiated HIV-specific and total memory CD8(+) cells of a central memory (CD45RO(+)CD27(+)CCR7(+)) and intermediate memory (CD45RO(-)CD27(+)CCR7(-)) phenotype. Those with high viral set points possessed significantly larger frequencies of effector memory (CD45RO(+)CD27(-)CCR7(-)) cells. The proportions of memory subsets significantly correlated with CD38(+)CD8(+) T cells. Thus, it is likely that a high Ag burden resulting in generalized immune activation may drive differentiation of HIV-specific and total memory CD8(+) T cells.

Human immunodeficiency virus-specific gamma interferon enzyme-linked immunospot assay responses targeting specific regions of the proteome during primary subtype C infection are poor predictors of the course of viremia and set point.

It is unknown whether patterns of human immunodeficiency virus (HIV)-specific T-cell responses during acute infection may influence the viral set point and the course of disease. We wished to establish whether the magnitude and breadth of HIV type 1 (HIV-1)-specific T-cell responses at 3 months postinfection were correlated with the viral-load set point at 12 months and hypothesized that the magnitude and breadth of HIV-specific T-cell responses during primary infection would predict the set point. Gamma interferon (IFN-gamma) enzyme-linked immunospot (ELISPOT) assay responses across the complete proteome were measured in 47 subtype C HIV-1-infected participants at a median of 12 weeks postinfection. When corrected for amino acid length and individuals responding to each region, the order of recognition was as follows: Nef > Gag > Pol > Rev > Vpr > Env > Vpu > Vif > Tat. Nef responses were significantly (P < 0.05) dominant, targeted six epitopic regions, and were unrelated to the course of viremia. There was no significant difference in the magnitude and breadth of responses for each protein region with disease progression, although there was a trend of increased breadth (mean, four to seven pools) in rapid progressors. Correlation of the magnitude and breadth of IFN-gamma responses with the viral set point at 12 months revealed almost zero association for each protein region. Taken together, these data demonstrate that the magnitude and breadth of IFN-gamma ELISPOT assay responses at 3 months postinfection are unrelated to the course of disease in the first year of infection and are not associated with, and have low predictive power for, the viral set point at 12 months.

Transmission of HIV-1 CTL escape variants provides HLA-mismatched recipients with a survival advantage.

One of the most important genetic factors known to affect the rate of disease progression in HIV-infected individuals is the genotype at the Class I Human Leukocyte Antigen (HLA) locus, which determines the HIV peptides targeted by cytotoxic T-lymphocytes (CTLs). Individuals with HLA-B*57 or B*5801 alleles, for example, target functionally important parts of the Gag protein. Mutants that escape these CTL responses may have lower fitness than the wild-type and can be associated with slower disease progression. Transmission of the escape variant to individuals without these HLA alleles is associated with rapid reversion to wild-type. However, the question of whether infection with an escape mutant offers an advantage to newly infected hosts has not been addressed. Here we investigate the relationship between the genotypes of transmitted viruses and prognostic markers of disease progression and show that infection with HLA-B*57/B*5801 escape mutants is associated with lower viral load and higher CD4+ counts.

HIV incidence and predictors of inconsistent condom use among adult men enrolled into an HIV vaccine preparedness study, Rustenburg, South Africa.

INTRODUCTION: Understanding HIV incidence and risk behaviour among populations being considered for HIV vaccine studies is necessary for the appropriate design of trials. METHODS: Between May 2012 and June 2015, we recruited men aged 18-49 years from urban and peri-urban areas of Rustenburg, a mining town in the North West Province, South Africa. Men who reported HIV-risk behaviour were followed for nine to 12 months to determine HIV incidence and factors associated with condom use. RESULTS: A total of 400 HIV uninfected men were enrolled; 366 (91.5%) had at least one follow-up visit and were included in the analysis; 47.6% were under 25 years of age. HIV incidence was 1.9 per 100 person-years (95% CI: 0.79-4.56). Among heterosexual men (N = 339), 80.8% reported having vaginal intercourse with multiple partners in the past three months, among whom 74.1% reported inconsistent condom use. Sixty-eight percent reported vaginal intercourse with new female partners, of whom 40.6% reported inconsistent condom use. Over half (55.6%) of men who had sex with men (N = 27) reported anal intercourse with multiple male partners in the past three months, of whom 68.2% reported using condoms inconsistently. Men who had more than two female partners in the last three months (n = 121) were more likely to use condoms inconsistently (aOR 4.31, 95% CI: 1.34-13.8); in contrast, those with more than one new female sex partner (aOR 0.13, 94% CI 0.04-0.44), and whose sexual debut was after 19 years of age (aOR 0.39, 95% CI: 0.15-1.01) were less likely to use condoms inconsistently. CONCLUSION: HIV incidence was low and similar to other studies of heterosexual men in South Africa. To identify men at high risk for HIV for enrolment in prevention trials, future researchers may need to focus on those who report early sexual debut and who report having multiple sexual partners. Men in newer relationships appear to use condoms more frequently.

Evaluating the electronic tuberculosis register surveillance system in Eden District, Western Cape, South Africa, 2015.

BACKGROUND: Tuberculosis (TB) surveillance data are crucial to the effectiveness of National TB Control Programs. In South Africa, few surveillance system evaluations have been undertaken to provide a rigorous assessment of the platform from which the national and district health systems draws data to inform programs and policies. OBJECTIVE: Evaluate the attributes of Eden District's TB surveillance system, Western Cape Province, South Africa. METHODS: Data quality, sensitivity and positive predictive value were assessed using secondary data from 40,033 TB cases entered in Eden District's ETR.Net from 2007 to 2013, and 79 purposively selected TB Blue Cards (TBCs), a medical patient file and source document for data entered into ETR.Net. Simplicity, flexibility, acceptability, stability and usefulness of the ETR.Net were assessed qualitatively through interviews with TB nurses, information health officers, sub-district and district coordinators involved in the TB surveillance. RESULTS: TB surveillance system stakeholders report that Eden District's ETR.Net system was simple, acceptable, flexible and stable, and achieves its objective of informing TB control program, policies and activities. Data were less complete in the ETR.Net (66-100%) than in the TBCs (76-100%), and concordant for most variables except pre-treatment smear results, antiretroviral therapy (ART) and treatment outcome. The sensitivity of recorded variables in ETR.Net was 98% for gender, 97% for patient category, 93% for ART, 92% for treatment outcome and 90% for pre-treatment smear grading. CONCLUSIONS: Our results reveal that the system provides useful information to guide TB control program activities in Eden District. However, urgent attention is needed to address gaps in clinical recording on the TBC and data capturing into the ETR.Net system. We recommend continuous training and support of TB personnel involved with TB care, management and surveillance on TB data recording into the TBCs and ETR.Net as well as the implementation of a well-structured quality control and assurance system.