Clinical and immunological data about HIV in older adults from low and middle income countries is scarce. We aimed to describe differences between younger and older adults with HIV starting antiretroviral therapy in two low–income African countries.
Setting: HIV clinics in Uganda and Zimbabwe.
Design: Secondary exploratory cross-sectional analysis of the DART randomized controlled trial.
Outcome Measures: Clinical and laboratory characteristics were compared between adults aged 18-49 years (younger) and ≥ 50 years (older), using two exploratory multivariable logistic regression models, one with HIV viral load (measured in a subset pre-ART) and one without.
A total of 3316 eligible participants enrolled in DART were available for analysis; 219 (7%) were ≥ 50 years and 1160 (35%) were male. Across the two adjusted regression models, older adults had significantly higher systolic blood pressure, lower creatinine clearance and were consistently less likely to be females compared to younger adults with HIV. Paradoxically, the models separately suggested that older adults had statistically significant (but not clinically important) higher CD4+ cell counts and higher plasma HIV–1 viral copies at initiation. Crude associations between older age and higher baseline hemoglobin, body mass index, diastolic blood pressure and lower WHO clinical stage were not sustained in the adjusted analysis.
Our study found clinical and immunological differences between younger and older adults, in a cohort of Africans starting antiretroviral therapy. Further investigations should explore how these differences could be used to ensure equity in service delivery and affect outcomes of antiretroviral therapy.
Tuberculosis (TB) remains a global health threat with 9 million new cases and 1.4 million deaths per year. In order to develop a protective vaccine, we need to define the antigens expressed by Mycobacterium tuberculosis (Mtb), which are relevant to protective immunity in high-endemic areas.
We analysed responses to 23 Mtb antigens in a total of 1247 subjects with different HIV and TB status across 5 geographically diverse sites in Africa (South Africa, The Gambia, Ethiopia, Malawi and Uganda). We used a 7-day whole blood assay followed by IFN-γ ELISA on the supernatants. Antigens included PPD, ESAT-6 and Ag85B (dominant antigens) together with novel resuscitation-promoting factors (rpf), reactivation proteins, latency (Mtb DosR regulon-encoded) antigens, starvation-induced antigens and secreted antigens.
There was variation between sites in responses to the antigens, presumably due to underlying genetic and environmental differences. When results from all sites were combined, HIV- subjects with active TB showed significantly lower responses compared to both TST- and TST+ contacts to latency antigens (Rv0569, Rv1733, Rv1735, Rv1737) and the rpf Rv0867; whilst responses to ESAT-6/CFP-10 fusion protein (EC), PPD, Rv2029, TB10.3, and TB10.4 were significantly higher in TST+ contacts (LTBI) compared to TB and TST- contacts fewer differences were seen in subjects with HIV co-infection, with responses to the mitogen PHA significantly lower in subjects with active TB compared to those with LTBI and no difference with any antigen.
Our multi-site study design for testing novel Mtb antigens revealed promising antigens for future vaccine development. The IFN-γ ELISA is a cheap and useful tool for screening potential antigenicity in subjects with different ethnic backgrounds and across a spectrum of TB and HIV infection states. Analysis of cytokines other than IFN-γ is currently on-going to determine correlates of protection, which may be useful for vaccine efficacy trials.
When manifested as Mycobacterium tuberculosis (MTB) bacteremia, disseminated MTB infection clinically mimics other serious blood stream infections often hindering early diagnosis and initiation of potentially life-saving anti-tuberculosis therapy. In a cohort of hospitalized HIV-infected Ugandan patients with severe sepsis, we report the frequency, management and outcomes of patients with MTB bacteremia and propose a risk score based on clinical predictors of MTB bacteremia.
We prospectively enrolled adult patients with severe sepsis at two Ugandan hospitals and obtained blood cultures for MTB identification. Multivariable logistic regression modeling was used to determine predictors of MTB bacteremia and to inform the stratification of patients into MTB bacteremia risk categories based on relevant patient characteristics.
Among 368 HIV-infected patients with a syndrome of severe sepsis, eighty-six (23%) had MTB bacteremia. Patients with MTB bacteremia had a significantly lower median CD4 count (17 vs 64 lymphocytes/mm3, p<0.001) and a higher 30-day mortality (53% vs 32%, p = 0.001) than patients without MTB bacteremia. A minority of patients with MTB bacteremia underwent standard MTB diagnostic testing (24%) or received empiric anti-tuberculosis therapy (15%). Independent factors associated with MTB bacteremia included male sex, increased heart rate, low CD4 count, absence of highly active anti-retroviral therapy, chief complaint of fever, low serum sodium and low hemoglobin. A risk score derived from a model containing these independent predictors had good predictive accuracy [area under the curve = 0.85, 95% CI 0.80–0.89].
Nearly 1 in 4 adult HIV-infected patients hospitalized with severe sepsis in 2 Ugandan hospitals had MTB bacteremia. Among patients in whom MTB was suspected, standard tests for diagnosing pulmonary MTB were inaccurate for correctly classifying patients with or without bloodstream MTB infection. A MTB bacteremia risk score can improve early diagnosis of MTB bacteremia particularly in settings with increased HIV and MTB co-infection.
Dysglycemia during sepsis is associated with poor outcomes in resource-rich settings. In resource-limited settings, hypoglycemia is often diagnosed clinically without the benefit of laboratory support. We studied the utility of point-of-care glucose monitoring to predict mortality in severely septic patients in Uganda.
Prospective observational study.
One national and two regional referral hospitals in Uganda.
We enrolled 532 patients with sepsis at three hospitals in Uganda. The analysis included 418 patients from the three sites with inhospital mortality data, a documented admission blood glucose concentration, and evidence of organ dysfunction at admission (systolic blood pressure ≤100 mm Hg, lactate > 4 mmol/L, platelet number <100,000/µL, or altered mental status).
Measurements and Main Results
We evaluated the association between admission point-of-care blood glucose concentration and inhospital mortality. We also assessed the accuracy of altered mental status as a predictor of hypoglycemia. Euglycemia occurred in 33.5% (140 of 418) of patients, whereas 16.3% (68 of 418) of patients were hypoglycemic and 50.2% (210 of 418) were hyperglycemic. Univariate Cox regression analyses comparing inhospital mortality among hypoglycemic (35.3% [24 of 68], hazard ratio 2.0, 95% confidence interval 1.2–3.6, p = .013) and hyperglycemic (29.5% [62 of 210], hazard ratio 1.5, 95% confidence interval 0.96–2.4, p = .08) patients to euglycemic (19.3% [27 of 140]) patients showed statistically significantly higher rates of inhospital mortality for patients with hypoglycemia. Hypoglycemia (adjusted hazard ratio 1.9, 95% confidence interval 1.1–3.3, p = .03) remained significantly and independently associated with inhospital mortality in the multivariate model. The sensitivity and specificity of altered mental status for hypoglycemia were 25% and 86%, respectively.
Hypoglycemia is an independent risk factor for inhospital mortality in patients with severe sepsis and cannot be adequately assessed by clinical examination. Correction of hypoglycemia may improve outcomes of critically ill patients in resource-limited settings.
Africa; hypoglycemia; mortality; outcomes; severe sepsis; Uganda
The world health organization (WHO) declared tuberculosis (TB) a global emergency, mainly affecting people in sub-Saharan Africa. However there is little data about the burden of TB among adolescents. We estimated the prevalence and incidence of TB and assessed factors associated with TB among adolescents aged 12–18 years in a rural population in Uganda in order to prepare the site for phase III clinical trials with novel TB vaccines among adolescents.
In a prospective cohort study, we recruited 5000 adolescents and followed them actively, every 6 months, for 1–2 years. Participants suspected of having TB were those who had any of; TB signs and symptoms, history of TB contact or a positive tuberculin skin test (TST) of ≥10 mm. Laboratory investigations included sputum smear microscopy and culture.
Of the 5000 participants, eight culture confirmed cases of TB were found at baseline: a prevalence of 160/100,000 (95% confidence interval (CI), 69–315). There were 13 incident TB cases detected in an average of 1.1 person years: an incidence of 235/100,000 person years (95% CI, 125–402). None of the confirmed TB cases were HIV infected. Predictors for prevalent TB disease were: a history of TB contact and a cough ≥ 2 weeks at baseline and being out of school, while the only predictor for incident TB was a positive TST during follow-up.
The TB incidence among adolescents in this rural part of Uganda seemed too low for a phase III TB vaccine trial. However, the study site demonstrated capability to handle a large number of participants with minimal loss to follow-up and its suitability for future clinical trials. Improved contact tracing in TB program activities is likely to increase TB case detection among adolescents. Future studies should explore possible pockets of higher TB incidence in urban areas and among out of school youth.
Vitamin D deficiency has been reported among patients with tuberculosis in Africa despite abundant sunshine. Vitamin D plays a fundamental role in improving anti tuberculosis immunity, reducing progression and severity of TB in humans.
In this descriptive cross sectional study, 260 hospitalized adults with a confirmed diagnosis of TB were enrolled into the study from the pulmonology wards of Mulago national referral and teaching hospital, Uganda. The serum concentrations of 25-hydroxyvitamin D or 25 (OH) D were determined by an electrochemilumniscence immunoassay. Vitamin D deficiency, vitamin D insufficiency, severe and very severe vitamin D deficiency were defined as serum 25(OH) D concentrations of ≤ 20 ng/ml, 21–29 ng/ml, < 10 ng/ml and <5 ng/ml respectively.
Majority of the study participants were males (146, 56.2%) and < 35 years (154, 59.2%). The mean age ± SD was 34.7 ± 9.5 years. Two hundred eight (80%) patients were HIV co-infected with a median CD4 count of 68 cells/mm3 (IQR: 17–165). The prevalence of vitamin D deficiency, vitamin D insufficiency, severe and very severe vitamin D deficiency among the hospitalized adult tuberculosis patients was 44.2%, 23.5%, 13.5% and 4.2% respectively. The median (IQR) vitamin D concentration in ng/ml was 22.55 (14.59-33.31).
Vitamin D deficiency was more prevalent in patients with hypoalbuminemia (97.4%), anemia (86.1%), HIV co-infected patients with CD4 count <200cells/mm3 (83.2%) and hypocalcemia corrected for serum albumin levels (67%).
Vitamin D deficiency is very common among hospitalized adult tuberculosis patients in Uganda especially in patients with hypoalbuminemia, anemia, HIV co-infected patients with CD4 count <200cells/mm3 and hypocalcemia corrected for serum albumin levels.
Vitamin D deficiency; Tuberculosis; Uganda
In sub-Saharan Africa, sepsis is an important cause of mortality but optimal sepsis management including fluid resuscitation, early antibiotic administration and patient monitoring is limited by a lack of supplies and skilled health workers.
To evaluate whether early, monitored sepsis management provided by a study medical officer can improve survival among patients with severe sepsis admitted to two public hospitals in Uganda.
DESIGN, SETTING and PATIENTS
A prospective before and after study of an intervention cohort (n=426) with severe sepsis receiving early, monitored sepsis management compared to an observation cohort (n=245) of similarly ill patients with severe sepsis receiving standard management after admission to the medical wards of two Ugandan hospitals.
Early sepsis management provided by a dedicated study medical officer comprised of fluid resuscitation, early antibiotics and regular monitoring in the first 6 hours of hospitalization.
Kaplan-Meier survival and unadjusted and adjusted Cox proportional hazards analysis were used to compare the effect of early, monitored sepsis management on 30-day mortality between the intervention cohort (enrolled May 2008 to May 2009) and observation cohort (enrolled July 2006 to November 2006).
The majority (86%) of patients in both cohorts were HIV-infected. Median fluid volume provided in the first 6 hours of hospitalization was higher in intervention than observation cohort patients (3000 vs. 500 mL, p<0.001) and a greater proportion of intervention cohort patients received antibacterial therapy in less than one hour (67% vs 30.4%, p<0.001). Mortality at 30 days was significantly lower in the intervention cohort compared to the observation cohort (33.0% vs 45.7%, log-rank p=0.005). After adjustment for potential confounders, the hazard of 30-day mortality was 26% less in the intervention cohort compared to the observation cohort (adjusted HR=0.74, 95% CI=0.55–0.98). Mortality among the 13% of intervention patients who developed signs of respiratory distress was associated with baseline illness severity rather than fluid volume administered.
Early, monitored management of severely septic patients in Uganda improves survival and is feasible and safe even in a busy public referral hospital.
Sepsis; Management Bundle; Fluid Therapy; Uganda; Africa; Mortality; Pulmonary Edema
Most HIV-infected subjects exhibit a progressive rise in CD4 T-cell counts after initiation of highly active antiretroviral therapy (HAART). However, a subset of individuals exhibit very poor CD4 T-cell recovery despite effective control of HIV-RNA viraemia. We evaluated CD4 T-cell proliferation among suboptimal responders and its correlation with CD4 T-cell activation.
The magnitude of CD4 increase (difference between absolute CD4 counts at baseline and absolute CD4 counts at 4 years of ART) was grouped into 4 quartiles for the 211 patients with sustained HIV-RNA viral suppression. Cases of ‘Suboptimal immune responders’ included patients within the lowest quartile [Median CD4 increase 165 (Range −43-298) cells/μl; n=52] and a comparison group of ‘Optimal immune responders’ was defined as patients within the highest quartile of CD4 increase [Median CD4 increase 528 (Range 417–878) cells/μl; n=52]. Frozen PBMC were thawed and analysed from a convenient sample of 39 suboptimal responders and 48 optimal responders after 4 years of suppressive antiretroviral therapy. T-cell activation was measured by proportions of T-cells expressing surface marker CD38 and HLADR (CD4+CD38+HLA-DR+ and CD8+CD38+HLA-DR+ cells). T-cell proliferation was determined by the extent of carboxyfluorescein diacetate succinimidyl ester (CFSE) dye dilution on culture day 5 of PBMCs in the presence of antigen (SEB, PPD, CMVpp65, GagA and GagD). Samples were analyzed on a FACS Calibur flow cytometer and flow data was analyzed using FlowJo and GraphPad.
Overall, CD4 T-cell proliferation on stimulation with SEB, PPD, CMVpp65, Gag A and Gag D.antigens, was lower among suboptimal than optimal responders; this was significant for SEB (CD4+ p=0.003; CD8+ p=0.048) and PPD antigens (CD8+ p=0.038). Among suboptimal responders, T-cell proliferation decreased with increasing immune activation (Negative correlation; slope = −0.13±−0.11) but not among optimal responders.
T-cell immune activation and exhaustion were associated with poor proliferation among suboptimal responders to HAART despite sustained viral suppression. We recommend studies to further understand the mechanisms leading to impaired T-cell function among suboptimal responders as well as the potential role of immune modulation in optimizing CD4 count and functional recovery after HAART.
T-cell proliferation; Immune activation; Suboptimal immune recovery; HAART immune responses; HIV/AIDS
We aimed to compare the steady-state pharmacokinetic parameters and tolerability of Triomune 40® (stavudine 40 mg, lamivudine 150 mg and nevirapine 200 mg) and branded formulations of these drugs in HIV-infected Ugandans.
This includes a randomized, open-label, cross-over study of HIV-infected patients stable on therapy for 1 month. Patients were randomized to generic or branded formulation. Plasma pharmacokinetics were assessed after 1 month. The following day, alternate formulation was administered, and 1 month later, drug pharmacokinetics were re-assessed. Plasma pharmacokinetics were determined using HPLC–UV detection. Similarity between steady-state pharmacokinetic parameters was assessed using the US Food and Drug Administration standards for bioequivalency testing. Tolerability was assessed using questionnaires.
Sixteen (10 females) patients completed the study. Median (IQR) age, weight and CD4 count were 37 (33.7–40) years, 65 (63.4–66) kg and 292 (220.7–344.5) cells/mm3, respectively. All patients received co-trimoxazole. The geometric mean ratio (90% CI) for stavudine, lamivudine and nevirapine was 0.92 (0.78–1.08), 1.11 (0.95–1.30) and 0.84 (0.64–1.11), respectively, for Cmax, and 0.83 (0.70–0.97), 1.06 (0.94–1.20) and 0.88 (0.71–1.10), respectively, for AUC. Stavudine plasma concentrations were significantly lower for the generic formulation. Pharmacokinetic parameter inter-individual variability ranged from 29% to 99%. There were no differences in tolerability for the two formulations.
Pharmacokinetic profiles of generic and branded drugs were similar. Differences particularly with regard to stavudine were demonstrated. Surveillance of the quality of generic antiretroviral drugs in the target populations is needed. Capacity building for pharmacokinetic research in resource-limited settings is a priority.
antiretroviral drugs; PK; Uganda
The HIV/AIDS epidemic has evolved with an increasing burden in older adults. We assessed for knowledge about aging and HIV/AIDS, among clinicians in Kampala district, Uganda.
A cross-sectional survey of 301 clinicians complemented by 9 key-informant interviews between May and October 2011. Data was analyzed by multivariable logistic regression for potential determinants of clinician knowledge about HIV/AIDS in older adults, estimating their adjusted Odds Ratios (aOR) and 95% confidence intervals (95% CI) using Stata 11.2 software.
Two-hundred and sixty-two questionnaires (87.7%) were returned. Respondents had a median age of 30 years (IQR 27–34) and 57.8% were general medical doctors. The mean knowledge score was 49% (range 8.8%–79.4%). Questions related to co-morbidities in HIV/AIDS (non-AIDS related cancers and systemic diseases) and chronic antiretroviral treatment toxicities (metabolic disorders) accounted for significantly lower scores (mean, 41.7%, 95% CI: 39.3%–44%) compared to HIV/AIDS epidemiology and prevention (mean, 65.7%, 95% CI: 63.7%–67.7%). Determinants of clinician knowledge in the multivariable analysis included (category, aOR, 95% CI): clinician age (30–39 years; 3.28∶1.65–9.75), number of persons with HIV/AIDS seen in the past year (less than 50; 0.34∶0.14–0.86) and clinical profession (clinical nurse practitioner; 0.31∶0.11–0.83). Having diploma level education had a marginal association with lower knowledge about HIV and aging (p = 0.09).
Our study identified gaps and determinants of knowledge about HIV/AIDS in older adults among clinicians in Kampala district, Uganda. Clinicians in low and middle income countries could benefit from targeted training in chronic care for older adults with HIV/AIDS and long-term complications of antiretroviral treatment.
At sites of Mycobacterium tuberculosis (MTB) infection, HIV-1 replication is increased during tuberculosis (TB). Here we investigated the role of positive transcription elongation factor (P-TEFb), comprised of CycT1 and CDK9, as the cellular cofactor of HIV-1 Tat protein in transcriptional activation of HIV-1 in mononuclear cells from HIV-1-infected patients with pleural TB. Expression of CycT1 in response to MTB was assessed in mononuclear cells from pleural fluid (PFMC) and blood (PBMC) from HIV/TB patients with pleural TB, and in blood monocytes (MN) from singly infected HIV-1-seropositive subjects. We then examined whether the CDK9 inhibitor, Indirubin 3′-monoxime (IM), was effective in inhibition of MTB-induced HIV-1 mRNA expression. We found higher expression of CycT1 mRNA in PFMCs as compared to PBMCs from HIV/TB-coinfected subjects. MTB induced the expression of CycT1 and HIV-1 gag/pol mRNA in both PFMCs from HIV/TB subjects and MN from HIV-1-infected subjects. CycT1 protein was also induced by MTB stimulation in PFMCs from HIV/TB patients, and both MN and in vitro-derived macrophages. Inhibition of CDK9 by IM in both PFMCs from HIV/TB and MN from HIV-1-infected subjects in response to MTB led to inhibition of HIV-1 mRNA expression. These data imply that IM may be useful as an adjunctive therapy in control of HIV-1 replication in HIV/TB dually infected subjects.
Rationale: The immunologic events surrounding primary Mycobacterium tuberculosis infection and development of tuberculosis remain controversial. Young children who develop tuberculosis do so quickly after first exposure, thus permitting study of immune response to primary infection and disease. We hypothesized that M. tuberculosis–specific CD8+ T cells are generated in response to high bacillary loads occurring during tuberculosis.
Objectives: To determine if M. tuberculosis–specific T cells are generated among healthy children exposed to M. tuberculosis and children with tuberculosis.
Methods: Enzyme-linked immunosorbent spot assays were used to measure IFN-γ production in response to M. tuberculosis–specific proteins ESAT-6/CFP-10 by peripheral blood mononuclear cells and CD8+ T cells isolated from Ugandan children hospitalized with tuberculosis (n = 96) or healthy tuberculosis contacts (n = 62).
Measurements and Main Results: The proportion of positive CD8+ T-cell assays and magnitude of CD8+ T-cell responses were significantly greater among young (<5 yr) tuberculosis cases compared with young contacts (P = 0.02, Fisher exact test, P = 0.01, Wilcoxon rank-sum, respectively). M. tuberculosis–specific T-cell responses measured in peripheral blood mononuclear cells were equivalent between groups.
Conclusions: Among young children, M. tuberculosis–specific CD8+ T cells develop in response to high bacillary loads, as occurs during tuberculosis, and are unlikely to be found after M. tuberculosis exposure. T-cell responses measured in peripheral blood mononuclear cells are generated after M. tuberculosis exposure alone, and thus cannot distinguish exposure from disease. In young children, IFN-γ–producing M. tuberculosis–specific CD8+ T cells provide an immunologic signature of primary M. tuberculosis infection resulting in disease.
Mycobacterium tuberculosis; infant; child; CD8-positive T lymphocytes; enzyme-linked immunosorbent spot
The World Health Organization (WHO) recommends collection of two sputum samples for tuberculosis (TB) diagnosis, with at least one being an early morning (EM) using smear microscopy. It remains unclear whether this is necessary even when sputum culture is employed. Here, we determined the diagnostic yield from spot and the incremental yield from the EM sputum sample cultures among TB-suspected adolescents from rural Uganda.
Sputum samples (both spot and early-morning) from 1862 adolescents were cultured by the Lowenstein-Jensen (LJ) and Mycobacterium Growth Indicator Tube (MGIT) methods. For spot samples, the diagnostic yields for TB were 19.0% and 57.1% with LJ and MGIT, respectively, whereas the incremental yields (not totals) of the early-morning sample were 9.5% and 42.9% (P < 0.001) with LJ and MGIT, respectively. Among TB-suspected adolescents in rural Uganda, the EM sputum culture has a high incremental diagnostic yield. Therefore, EM sputum in addition to spot sample culture is necessary for improved TB case detection.
Several population-wide HIV-1 subtype distribution studies in Uganda have evaluated relatively healthy clinic patients. Given the differences in HIV-1 disease progression based on subtype, we examined HIV-1 subtype distribution and disease outcomes among hospitalized patients with severe sepsis.
Patients with severe sepsis were enrolled at two hospitals in Uganda. Data collected included demographics, Karnofsky scores, highly active antiretroviral therapy (HAART) use, HIV-1 serostatus, CD4+ T cell concentration, whole blood lactate concentration, and blood cultures. HIV-1 subtypes were determined by sequencing parts of the gag and env genes, followed by phylogenetic analysis.
Of the 267 patients evaluated, 228 (85.4%) were HIV infected. The predominant HIV-1 subtypes were A (46%), D (17%), and AD recombinants (30%). HIV-1 subtypes B, C, and other recombinants were uncommon. Patients infected with HIV-1 subtypes A, D and AD viruses were similar in demographics, CD4+ T cell concentration, HAART use, Karnofsky scores, whole blood lactate concentration, and positive blood cultures. There was no difference in 30-day mortality from severe sepsis between the 3 groups (p = 0.99).
A high proportion of HIV-1 subtypes A and AD recombinants was observed in this cohort of severely septic patients. The proportion of AD recombinants was higher in this cohort than in previous cohorts of Ugandan HIV-1 patients. No difference in baseline demographics, clinical factors or 30-day mortality was seen across HIV-subtypes.
Genetic epidemiological studies of complex diseases often rely on data from the International HapMap Consortium for identification of single nucleotide polymorphisms (SNPs), particularly those that tag haplotypes. However, little is known about the relevance of the African populations used to collect HapMap data for study populations conducted elsewhere in Africa. Toll-like receptor (TLR) genes play a key role in susceptibility to various infectious diseases, including tuberculosis. We conducted full-exon sequencing in samples obtained from Uganda (n = 48) and South Africa (n = 48), in four genes in the TLR pathway: TLR2, TLR4, TLR6, and TIRAP. We identified one novel TIRAP SNP (with minor allele frequency [MAF] 3.2%) and a novel TLR6 SNP (MAF 8%) in the Ugandan population, and a TLR6 SNP that is unique to the South African population (MAF 14%). These SNPs were also not present in the 1000 Genomes data. Genotype and haplotype frequencies and linkage disequilibrium patterns in Uganda and South Africa were similar to African populations in the HapMap datasets. Multidimensional scaling analysis of polymorphisms in all four genes suggested broad overlap of all of the examined African populations. Based on these data, we propose that there is enough similarity among African populations represented in the HapMap database to justify initial SNP selection for genetic epidemiological studies in Uganda and South Africa. We also discovered three novel polymorphisms that appear to be population-specific and would only be detected by sequencing efforts.
Nucleic acid amplification tests (NAATs) have offered hope for rapid diagnosis of tuberculosis (TB). However, their efficiency with smear-negative samples has not been widely studied in low income settings. Here, we evaluated in-house PCR assay for diagnosis of smear-negative TB using Lowenstein-Jensen (LJ) culture as the baseline test. Two hundred and five pulmonary TB (PTB) suspects with smear-negative sputum samples, admitted on a short stay emergency ward at Mulago Hospital in Kampala, Uganda, were enrolled. Two smear-negative sputum samples were obtained from each PTB suspect and processed simultaneously for identification of MTBC using in-house PCR and LJ culture.
Seventy two PTB suspects (35%, 72/205) were LJ culture positive while 128 (62.4%, 128/205) were PCR-positive. The sensitivity and specificity of in-house PCR for diagnosis of smear-negative PTB were 75% (95% CI 62.6-85.0) and 35.9% (95% CI 27.2-45.3), respectively. The positive and negative predictive values were 39% (95% CI 30.4-48.2) and 72.4% (95% CI 59.1-83.3), respectively, while the positive and negative likelihood ratios were 1.17 (95% CI 0.96-1.42) and 0.70 (95% CI 0.43-1.14), respectively. One hundred and seventeen LJ culture-negative suspects (75 PCR-positive and 42 PCR-negative) were enrolled for follow-up at 2 months. Of the PCR-positive suspects, 45 (60%, 45/75) were still alive, of whom 29 (64.4%, 29/45) returned for the follow-up visit; 15 (20%, 15/75) suspects died while another 15 (20%, 15/75) were lost to follow-up. Of the 42 PCR-negative suspects, 22 (52.4%, 22/42) were still alive, of whom 16 (72.7%, 16/22) returned for follow-up; 11 (26.2%, 11/42) died while nine (21.4%, 9/42) were lost to follow-up. Overall, more PCR-positive suspects were diagnosed with PTB during follow-up visits but the difference was not statistically significant (27.6%, 8/29 vs. 25%, 4/16, p = 0.9239). Furthermore, mortality was higher for the PCR-negative suspects but the difference was also not statistically significant (26.2% vs. 20% p = 0.7094).
In-house PCR correlates poorly with LJ culture for diagnosis of smear-negative PTB. Therefore, in-house PCR may not be adopted as an alternative to LJ culture.
Pulmonary tuberculosis; Smear-negative TB; HIV-infected; HIV-TB co-infection; CD4 cell counts; Nucleic acid amplification tests; In-house PCR; Lowenstein-Jensen culture; Sensitivity; Specificity; Resource limited settings
Introduction. While the introduction of highly active antiretroviral therapy decreased HIV-related morbidity and mortality rates in the sub-Saharan Africa, a subsequent increase in metabolic abnormalities has been observed. We sought to determine the prevalence of HIV-associated metabolic abnormalities among patients on first-line antiretroviral therapy (ART) in an ART clinic in Kampala, Uganda. Methods. Four hundred forty-two consecutive patients on first-line ART for at least 12 months were screened for eligibility in a cross-sectional study, and 423 were enrolled. Pre-ART patient characteristics were abstracted from medical charts, examinations included anthropometric measurement and physical assessment for lipodystrophy.
Results. The prevalence of hyperglycemia and dyslipidemia was 16.3% (69/423) and 81.5% (345/423), respectively. Prevalence of dyslipidemia between stavudine- and zidovudine-based regimens (91% versus 72%; P < 0.001). Being on stavudine (aOR 4.79, 95%, 2.45–9.38) and peak body weight (aOR 1.44, 95% CI 1.05–1.97) were independent risk factors for dylipidemia. Stavudine (aOR 0.50, 95% CI 0.27–0.93) use was associated with lower risk for hyperglycemia while, and older age (aOR 1.31, 95% CI 1.11–1.56) and having a family history of DM (aOR 2.18, 95% CI 1.10–4.34) were independent risk factors for hyperglycemia. Conclusions. HIV-associated metabolic complications were prevalent among patients on thymidine analogue-containing ART regimens. Screening for lipid and glucose abnormalities should be considered in ART patients because of cardiovascular risks.
Co-administration of artemether/lumefantrine with antiretroviral therapy has potential for pharmacokinetic drug interactions. We investigated drug–drug interactions between artemether/lumefantrine and efavirenz or nevirapine.
We performed a cross-over study in which HIV-infected adults received standard six-dose artemether/lumefantrine 80/480 mg before and at efavirenz or nevirapine steady state. Artemether, dihydroartemisinin, lumefantrine, efavirenz and nevirapine plasma concentrations were measured and compared.
Efavirenz significantly reduced artemether maximum concentration (Cmax) and plasma AUC (median 29 versus 12 ng/mL, P < 0.01, and 119 versus 25 ng · h/mL, P < 0.01), dihydroartemisinin Cmax and AUC (median 120 versus 26 ng/mL, P < 0.01, and 341 versus 84 ng · h/mL, P < 0.01), and lumefantrine Cmax and AUC (median 8737 versus 6331 ng/mL, P = 0.03, and 280 370 versus 124 381 ng · h/mL, P < 0.01). Nevirapine significantly reduced artemether Cmax and AUC (median 28 versus 11 ng/mL, P < 0.01, and 123 versus 34 ng · h/mL, P < 0.01) and dihydroartemisinin Cmax and AUC (median 107 versus 59 ng/mL, P < 0.01, and 364 versus 228 ng · h/mL, P < 0.01). Lumefantrine Cmax and AUC were non-significantly reduced by nevirapine. Artemether/lumefantrine reduced nevirapine Cmax and AUC (median 8620 versus 4958 ng/mL, P < 0.01, and 66 329 versus 35 728 ng · h/mL, P < 0.01), but did not affect efavirenz exposure.
Co-administration of artemether/lumefantrine with efavirenz or nevirapine resulted in a reduction in artemether, dihydroartemisinin, lumefantrine and nevirapine exposure. These drug interactions may increase the risk of malaria treatment failure and development of resistance to artemether/lumefantrine and nevirapine. Clinical data from population pharmacokinetic and pharmacodynamic trials evaluating the impact of these drug interactions are urgently needed.
antimalarial; antiretroviral; malaria; drugs
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), causes 9 million new cases worldwide and 2 million deaths annually. Genetic linkage and association analyses have suggested several chromosomal regions and candidate genes involved in TB susceptibility. This study examines the association of TB disease susceptibility with a selection of biologically relevant genes on regions on chromosomes 7 (IL6 and CARD11) and 20 (CTSZ and MC3R), and fine mapping of the chromosome 7p22-p21 region, identified through our genome scan. We analyzed 565 individuals from Kampala, Uganda who were previously included in our genome-wide linkage scan. Association analyses were conducted for 1417 single-nucleotide polymorphisms (SNPs) that passed quality control. None of the candidate gene or fine mapping SNPs were found significantly associated with TB susceptibility (P > 0.10). When we restricted the analysis to HIV-negative individuals, two SNPs on chromosome 7 were significantly associated with TB susceptibility (P < 0.05). Haplotype analyses identified a significant risk haplotype in Cathepsin X (CTSZ) (p=0.0281, OR = 1.5493, 95% CI [1.039, 2.320]).
infectious disease; family study; TB genetics; fine map; immunogenetics
Severe malaria is a medical emergency with high mortality. Prompt achievement of therapeutic concentrations of highly effective anti-malarial drugs reduces the risk of death. The aim of this study was to assess the pharmacokinetics and pharmacodynamics of intravenous artesunate in Ugandan adults with severe malaria.
Fourteen adults with severe falciparum malaria requiring parenteral therapy were treated with 2.4 mg/kg intravenous artesunate. Blood samples were collected after the initial dose and plasma concentrations of artesunate and dihydroartemisinin measured by solid-phase extraction and liquid chromatography-tandem mass spectrometry. The study was approved by the Makerere University Faculty of Medicine Research and Ethics Committee (Ref2010-015) and Uganda National Council of Science and Technology (HS605) and registered with ClinicalTrials.gov (NCT01122134).
All study participants achieved prompt resolution of symptoms and complete parasite clearance with median (range) parasite clearance time of 17 (8–24) hours. Median (range) maximal artesunate concentration (Cmax) was 3260 (1020–164000) ng/mL, terminal elimination half-life (T1/2) was 0.25 (0.1-1.8) hours and total artesunate exposure (AUC) was 727 (290–111256) ng·h/mL. Median (range) dihydroartemisinin Cmax was 3140 (1670–9530) ng/mL, with Tmax of 0.14 (0.6 – 6.07) hours and T1/2 of 1.31 (0.8–2.8) hours. Dihydroartemisinin AUC was 3492 (2183–6338) ng·h/mL. None of the participants reported adverse events.
Plasma concentrations of artesunate and dihydroartemisinin were achieved rapidly with rapid and complete symptom resolution and parasite clearance with no adverse events.
Pharmacokinetics; Pharmacodynamics; Intravenous; Artesunate; Severe malaria
Enterocytozoon bieneusi is clinically the most significant microsporidian parasite associated with persistent diarrhea, wasting and cholangitis in 30-50% of individuals with HIV/AIDS, as well as in malnutritional children and in recipients of immunosuppressive therapy. However, the host immune responses to E. bieneusi have not been investigated until recently due to lack of sources of spores, cell culture system, and animal models. In this study, we purified spores from heavily infected human or monkey feces by serial salt-Percoll-sucrose-iodixanol centrifugation and the purity of spores was confirmed by FACS and scanning electron microscopy. Exposure of dendritic cells to E. bieneusi spores induced up-regulation of the surface markers and production of pro-inflammatory cytokines. The cytokine production was independent of Toll-like receptor 4, but MyD88-dependent, since dendritic cells from MyD88 knockout mice failed to secrete these pro-inflammatory cytokines, whereas dendritic cells from C3H/HeJ (a Toll-like receptor 4 mutant) were activated by E. bieneusi and secreted these cytokines. Furthermore, MyD88 deficient mice were susceptible to E. bieneusi infection, in contrast to wild type mice which resisted the infection. Collectively the data demonstrate innate recognition of E. bieneusi by dendritic cells and the importance of MyD88-dependent signaling in resisting infection in a murine challenge model.
Dendritic cells; Enterocytozoon bieneusi; innate immunity; MyD88; TLR
Background. Human immunodeficiency virus (HIV)–tuberculosis coinfection is associated with heightened immune activation, viral replication, and T cell dysfunction. We compared changes in T cell activation and function between patients receiving concurrent treatment for HIV-tuberculosis coinfection and those receiving treatment for tuberculosis alone.
Methods. HIV-infected adults with tuberculosis and CD4+ T cell counts >350 cells/mm3 were randomized to receive tuberculosis treatment alone (control arm; n = 36) or 6 months of antiretroviral therapy (ART) concurrent with tuberculosis treatment (intervention arm; n = 38). HIV viral load, T cell subsets, T cell activation, and cytokine production were measured at enrollment and every 3 months for 12 months.
Results. Differences in absolute CD4+ and CD8+ T cell counts were not observed between arms. Viral load was reduced while participants received ART; control patients maintained viral load at baseline levels. Both arms had significant reductions in T cell expression of CD38 and HLA-DR. Interferon-γ production in response to mitogen increased significantly in the intervention arm.
Conclusions. In HIV-infected adults with tuberculosis and CD4+ T cell counts >350 cells/mm3, both tuberculosis treatment and concurrent HIV-tuberculosis treatment reduce T cell activation and stabilize T cell counts. Concurrent ART with tuberculosis treatment does not provide additional, sustained reductions in T cell activation among individuals with preserved immunologic function.
Rapid HIV tests provide same-day results and are widely used in HIV testing programs in areas with limited personnel and laboratory infrastructure. The Uganda Ministry of Health currently recommends the serial rapid testing algorithm with Determine, STAT-PAK, and Uni-Gold for diagnosis of HIV infection. Using this algorithm, individuals who test positive on Determine, negative to STAT-PAK and positive to Uni-Gold are reported as HIV positive. We conducted further testing on this subgroup of samples using qualitative DNA PCR to assess the potential for false positive tests in this situation.
Of the 3388 individuals who were tested, 984 were HIV positive on two consecutive tests, and 29 were considered positive by a tiebreaker (positive on Determine, negative on STAT-PAK, and positive on Uni-Gold). However, when the 29 samples were further tested using qualitative DNA PCR, 14 (48.2%) were HIV negative.
Although this study was not primarily designed to assess the validity of rapid HIV tests and thus only a subset of the samples were retested, the findings show a potential for false positive HIV results in the subset of individuals who test positive when a tiebreaker test is used in serial testing. These findings highlight a need for confirmatory testing for this category of individuals.
False positive; HIV testing algorithm; Rapid diagnostic tests; Qualitative PCR testing