Nonzwakazi Bangani

Screening for Cryptococcal Antigenemia in Patients Accessing an Antiretroviral Treatment Program in South Africa

BACKGROUND Cryptococcal meningitis is a leading cause of death in patients with acquired immunodeficiency syndrome and contributes substantially to the high early mortality in antiretroviral treatment (ART) programs in low-resource settings. Screening for cryptococcal antigen in patients who enroll in ART programs may identify those at risk of cryptococcal meningitis and permit targeted use of preemptive therapy. METHODS In this retrospective study, cryptococcal antigen was measured in stored plasma samples obtained from patients when they enrolled in a well-characterized ART cohort in South Africa. The predictive value of screening for cryptococcal antigen before initiation of ART for development of microbiologically confirmed cryptococcal meningitis or death during the first year of follow-up was determined. RESULTS Of 707 participants with a baseline median CD4 cell count of 97 cells/microL (interquartile range, 46-157 cells/microL), 46 (7%) were positive for cryptococcal antigen. Antigenemia was 100% sensitive for predicting development of cryptococcal meningitis during the first year of ART, and in multivariate analysis, it was an independent predictor of mortality (adjusted hazard ratio, 3.2; 95% confidence interval, 1.5-6.6). Most cases (92%) of cryptococcal meningitis developed in patients with a CD4 cell count <or= 100 cells/microL. In this subset of patients, a cryptococcal antigen titer >or 1:8 was 100% sensitive and 96% specific for predicting incident cryptococcal meningitis during the first year of ART in those with no history of the disease. CONCLUSIONS Cryptococcal antigen screening before initiation of ART in patients with a CD4 cell count <or=100 cells/microL is highly effective for identifying those at risk of cryptococcal meningitis and death and might permit implementation of a targeted preemptive treatment strategy.

Reciprocal seasonal variation in vitamin D status and tuberculosis notifications in Cape Town, South Africa

Vitamin D deficiency is associated with susceptibility to tuberculosis (TB) in HIV-uninfected people in Europe, but it is not known whether such an association exists among HIV-infected people in subtropical Africa. We conducted a cross-sectional study to determine whether vitamin D deficiency was associated with susceptibility to active TB in HIV-uninfected (n = 196) and HIV-infected (n = 174) black Africans in Cape Town, South Africa. We also investigated whether there was evidence of seasonal variation in vitamin D status and TB notifications in this setting over an 8-y period. Vitamin D deficiency (serum 25-hydroxyvitamin D [25(OH)D] <50 nmol/L) was present in 232 (62.7%) of 370 participants and was associated with active TB in both HIV-uninfected (odds ratio = 5.2, 95% confidence interval: 2.8–9.7; P < 0.001) and HIV-infected (odds ratio = 5.6, 95% confidence interval: 2.7–11.6; P < 0.001) people. Vitamin D status varied according to season: The mean serum 25(OH)D concentration was highest in January through March and lowest in July through September (56.8 vs. 30.7 nmol/L, respectively; P < 0.001). Reciprocal seasonal variation in TB notifications was observed: The mean number of TB notifications per quarter for Cape Town in 2003 to 2010 was lowest in April through June and highest in October through December (4,222 vs. 5,080; P < 0.001). Vitamin D deficiency is highly prevalent among black Africans in Cape Town and is associated with susceptibility to active TB both in the presence and absence of HIV infection. Reciprocal seasonal variation in serum 25(OH)D concentration and TB notifications suggests that seasonal variations in vitamin D status and TB incidence in this setting are causally related.

Utility of interferon-γ ELISPOT assay responses in highly tuberculosis-exposed patients with advanced HIV infection in South Africa

BackgroundInterferon-gamma (IFN-γ) ELISPOT assays incorporating Mycobacterium tuberculosis-specific antigens are useful in the diagnosis of tuberculosis (TB) or latent infection. However, their utility in patients with advanced HIV is unknown. We studied determinants of ELISPOT responses among patients with advanced HIV infection (but without active TB) living in a South African community with very high TB notification rates.MethodsIFN-γ responses to ESAT-6 and CFP-10 in overnight ELISPOT assays and in 7-day whole blood assays (WBA) were compared in HIV-infected patients (HIV+, n = 40) and healthy HIV-negative controls (HIV-, n = 30) without active TB. Tuberculin skin tests (TSTs) were also done.ResultsELISPOTs, WBAs and TSTs were each positive in >70% of HIV- controls, reflecting very high community exposure to M. tuberculosis. Among HIV+ patients, quantitative WBA responses and TSTs (but not the proportion of positive ELISPOT responses) were significantly impaired in those with CD4 cell counts <100 cells/μl compared to those with higher counts. In contrast, ELISPOT responses (but not WBA or TST) were strongly related to history of TB treatment; a much lower proportion of HIV+ patients who had recently completed treatment for TB (n = 19) had positive responses compared to those who had not been treated (11% versus 62%, respectively; P < 0.001). Multivariate analysis confirmed that ELISPOT responses had a strong inverse association with a history of recent TB treatment (adjusted OR = 0.06, 95%CI = 0.10–0.40, P < 0.01) and that they were independent of CD4 cell count and viral load. Among HIV+ individuals who had not received TB treatment both the magnitude and proportion of positive ELISPOT responses (but not TST or WBA) were similar to those of HIV-negative controls.ConclusionThe proportion of positive ELISPOT responses in patients with advanced HIV infection was independent of CD4 cell count but had a strong inverse association with history of TB treatment. This concurs with the previously documented low TB risk among patients in this cohort with a history of recent treatment for TB. These data suggest ELISPOT assays may be useful for patient assessment and as an immuno-epidemiological research tool among patients with advanced HIV and warrant larger scale prospective evaluation.

High prevalence of subclinical tuberculosis in HIV-1-infected persons without advanced immunodeficiency: implications for TB screening

Background The prevalence of asymptomatic tuberculosis (TB) in recently diagnosed HIV-1-infected persons attending pre-antiretroviral therapy (ART) clinics is not well described. In addition, it is unclear if the detection of Mycobacterium tuberculosis in these patients clearly represents an early asymptomatic phase leading to progressive disease or transient excretion of bacilli. Objective To describe the prevalence and outcome of subclinical TB disease in HIV-1-infected persons not eligible for ART. Methods The study was conducted in 274 asymptomatic ART-naïve HIV-1-infected persons in Khayelitsha Day Hospital, Cape Town, South Africa. All participants were screened for TB using a symptom screen and spoligotyping was performed to determine genotypes. Results The prevalence of subclinical TB disease was 8.5% (95% CI 5.1% to 13.0%) (n=18; median days to culture positivity 17 days), with 22% of patients being smear-positive. Spoligotyping showed a diverse variety of genotypes with all paired isolates being of the same spoligotype, effectively excluding cross-contamination. 56% of patients followed up developed symptoms 3 days to 2 months later. All were well and still in care 6–12 months after TB diagnosis; 60% were started on ART. A positive tuberculin skin test (OR 4.96, p=0.064), low CD4 count (OR 0.996, p=0.06) and number of years since HIV diagnosis (OR 1.006, p=0.056) showed trends towards predicting TB disease. Conclusion This study found a high prevalence but good outcome (retained in care) of subclinical TB disease in HIV-1-infected persons. The results suggest that, in high HIV/TB endemic settings, a positive HIV-1 test should prompt TB screening by sputum culture irrespective of symptoms, particularly in those with a positive tuberculin skin test, longer history of HIV infection and low CD4 count. Operational difficulties in resource-constrained settings with respect to screening with TB culture highlight the need for rapid and affordable point-of-care tests to identify persons with clinical and subclinical TB disease.

Plasma levels of soluble urokinase-type plasminogen activator receptor (suPAR) and early mortality risk among patients enrolling for antiretroviral treatment in South Africa

BackgroundSerum concentrations of soluble urokinase-type plasminogen activator receptor (suPAR) have a strong independent association with HIV-1-related mortality. The practical utility of plasma suPAR in assessing short-term all-cause mortality risk was evaluated in patients with advanced immunodeficiency enrolling in an antiretroviral treatment (ART) programme in South Africa.MethodsAn enzyme-linked immunosorbent assay (ELISA) was used to measure plasma concentrations of suPAR in patients at the time of enrolment to the ART programme. The association between plasma suPAR concentrations, baseline patient characteristics and cohort outcomes after 4 months of ART were determined.ResultsPatients (n = 293, 70% female) had a median age of 33 years and were followed up for a median of 5 months from enrolment. The median CD4 cell count was 47 cells/μl (IQR = 22–72) and 38% of patients had WHO stage 4 disease. 218 (74%) patients remained alive after 4 months of ART; 39 (13%) died and 36 (12%) were lost to the programme for other reasons. Patients who died had significantly higher plasma suPAR concentrations compared to those who either survived (P < 0.01) or left the programme for other reasons (P < 0.043). In multivariate analysis, higher log10 suPAR concentrations were significantly associated with lower CD4 cell counts, WHO clinical stage 4 disease and male sex. In multivariate analysis to identify factors associated with death, log10 suPAR concentration was the most strongly associated variable (P < 0.001). However, examination of sensitivity and specificity characteristics using receiver operating characteristic (ROC) analysis revealed that results from this assay did not have a discriminatory cut-point to provide clinically useful information.ConclusionPlasma suPAR concentration was the strongest independent predictor of short-term mortality risk among patients with advanced immunodeficiency enrolling in this ART programme. However, lack of a discriminatory threshold did not permit this marker to be used to triage patients according to short-term mortality risk.

Risk Factors Associated with Indeterminate Gamma Interferon Responses in the Assessment of Latent Tuberculosis Infection in a High-Incidence Environment

ABSTRACT The performance of gamma interferon (IFN-γ) release assays (IGRA) in the detection of latent tuberculosis (TB) infection is limited by the higher rates of indeterminate results in HIV-infected persons, who bear the brunt of TB disease in some high-burden settings. The objective of the study was to evaluate predictors of indeterminate IGRA results in the overall study population and in HIV-infected persons. The study setting is Khayelitsha, an informal township in the Western Cape of South Africa, with a high burden of TB and HIV infection. A total of 561 asymptomatic persons were recruited from the day hospital and youth centers. A questionnaire was used to collect demographic information, and blood tests, including CD4 counting and a 7-day in-house IGRA, were performed. The overall prevalence of indeterminate IGRA results was 8.6% (48/561), and this was higher in HIV-infected than in HIV-uninfected persons (11.5% [38/330] versus 4.3% [10/231], respectively; P = 0.003). In the overall study population, predictors of indeterminate IGRA results were the presence of HIV infection (odds ratio [OR], 2.36; 95% confidence interval [CI], 1.10 to 5.08) and the presence of a Mycobacterium bovis BCG scar (OR, 2.48; 95% CI, 1.23 to 5.01). Long-term township residents were significantly less likely to have indeterminate results than recent migrants (OR, 0.30; 95% CI, 0.11 to 0.80). Among HIV-infected persons, participants with CD4 counts of >200 cells/mm3 and long-term residents were significantly less likely to have indeterminate IGRA results (OR of 0.21 with a 95% CI of 0.09 to 0.48 and OR of 0.22 with a 95% CI of 0.07 to 0.68, respectively). We evaluated risk factors for indeterminate IGRA results and report a higher rate of indeterminate results among HIV-infected persons, particularly those with lower CD4 counts. Of note, a recent move to the township was associated with a higher risk of indeterminate IGRA results.

Smoking, BCG and Employment and the Risk of Tuberculosis Infection in HIV-Infected Persons in South Africa

Background The increased susceptibility to latent tuberculosis infection (LTBI) of HIV-1-infected persons represents a challenge in TB epidemic control. However few studies have evaluated LTBI predictors in a generalized HIV/TB epidemic setting. Methods The study recruited 335 HIV-infected participants from Khayelitsha, Cape Town between February 2008 and November 2010. Tuberculin skin tests and interferon-gamma release assays were performed on all participants and active TB excluded using a symptom screen, TB microscopy and culture. Results LTBI prevalence was 52.7% and 61.2% (TST and IGRA respectively). Being a recent TB contact (OR 2.07; 95% C.I. 1.15–3.69) was associated with TST positivity. Participants with a CD4>200 had a two-fold higher risk of IGRA positivity compared to those with CD4 counts <200 (OR 2.07; 95% C.I. 0.99–4.34). There was also a 19% increase in IGRA positivity risk for every additional year of schooling and a strong association between years of schooling and employment (p = 0.0004). A decreased risk of IGRA positivity was observed in persons with a BCG scar (OR 0.46; 95% C.I. 0.31–0.69) and in smokers (OR 0.47; 95% C.I. 0.23–0.96). Conclusion We report the novel findings of a decreased risk of IGRA positivity in HIV-infected smokers possibly due to decreased interferon production, and in the persons with a BCG scar suggesting a protective role for BCG in this population. We also found an increased risk of TST positivity in employed persons, possibly due to ongoing transmission in public modes of transport.

A Recent HIV Diagnosis Is Associated with Non-Completion of Isoniazid Preventive Therapy in an HIV-Infected Cohort in Cape Town

Introduction Despite high rates of successful treatment TB incidence in South Africa remains high, suggesting ongoing transmission and a large reservoir of latently infected persons. Isoniazid preventive therapy (IPT) is recommended as preventive therapy in HIV-infected persons. However, implementation has been slow, impeded by barriers and challenges including the fear of non-adherence. Objective and Methods The aim was to evaluate predictors of IPT non-completion. One hundred and sixty four antiretroviral therapy (ART)-naïve HIV-infected patients with tuberculin skin test ≥5 mm were recruited from Khayelitsha day hospital and followed up monthly. A questionnaire was used to collect demographic information. Results The overall completion rate was 69%. In multivariable analysis, there was a 29% decrease in risk of non-completion for every year after HIV diagnosis (OR 0.81; 95% C.I. 0.68–0.98). Self-reported alcohol drinkers (OR 4.05; 95% C.I. 1.89–9.06) also had a four-fold higher risk of non-completion, with a strong association between alcohol drinkers and smoking (χ2 27.08; p<0.001). Conclusion We identify patients with a recent HIV diagnosis, in addition to self-reported drinkers and smokers as being at higher risk of non-completion of IPT. The period of time since HIV diagnosis should therefore be taken into account when initiating IPT. Our results also suggest that smokers and alcohol drinkers should be identified and targeted for adherence interventions when implementing IPT on a wider scale.

A novel assay of antimycobacterial activity and phagocytosis by human neutrophils

Summary Despite abundant evidence that neutrophils arrive early at sites of mycobacterial disease and phagocytose organisms, techniques to assay phagocytosis or killing of mycobacteria by these cells are lacking. Existing assays for measuring the antimycobacterial activity of human leukocytes require cell lysis which introduces new bioactive substances and may be incomplete. They are also time-consuming and carry multiple risks of inaccuracy due to serial dilution and organism clumping. Flow cytometric techniques for measuring phagocytosis of mycobacteria by human cells have failed to adequately address the effects of organism clumping, quenching agents and culture conditions on readouts. Here we present a novel in-tube bioluminescence-based assay of antimycobacterial activity by human neutrophils. The assay yields intuitive results, with improving restriction of mycobacterial bioluminescence as the ratio of cells to organisms increases. We show that lysis of human cells is not required to measure luminescence accurately. We also present a phagocytosis assay in which we have minimised the impact of mycobacterial clumping, investigated the effect of various opsonisation techniques and established the correct usage of trypan blue to identify surface-bound organisms without counting dead cells. The same multiplicity of infection and serum conditions are optimal to demonstrate both internalisation and restriction of mycobacterial growth.

Brief Report: HIV-1 Infection Impairs CD16 and CD35 Mediated Opsonophagocytosis of Mycobacterium tuberculosis by Human Neutrophils.

Abstract:Using a flow cytometric assay, we investigated neutrophil–Mycobacterium tuberculosis opsonophagocytosis and the impact of HIV-1–infected serum on this process. The mean (±SD) percentage of neutrophils internalizing bacilli after 30 minutes incubation was significantly reduced by pretreatment with anti-CD16 (18.2% ± 8.1%, P < 0.001) or anti-CD35 antibody (23.2% ± 10.6%, P < 0.05) versus anti-CD4 controls (29.9% ± 8.1%). Blocking CD88 or CD11a did not affect internalization. Using heat-inactivated serum, maximal internalization was lower using HIV-1–infected serum versus HIV-1–uninfected. Using non–heat-inactivated serum, internalization decreased more rapidly with sequential dilutions of HIV-1–infected versus HIV-1–uninfected serum. CD16 and CD35 are important for neutrophil internalization of M. tuberculosis, whereas HIV-1 infection adversely affects opsonophagocytosis.