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1.  Determinants of hypertension in a young adult Ugandan population in epidemiological transition—the MEPI-CVD survey 
BMC Public Health  2015;15:830.
High blood pressure is the principal risk factor for stroke, heart failure and kidney failure in the young population in Africa. Control of hypertension is associated with a larger reduction in morbidity and mortality in younger populations compared with the elderly; however, blood pressure control efforts in the young are hampered by scarcity of data on prevalence and factors influencing awareness, treatment and control of hypertension. We aimed to describe the prevalence of prehypertension and hypertension among young adults in a peri-urban district of Uganda and the factors associated with occurrence of hypertension in this population.
This cross-sectional study was conducted between August, 2012 and May 2013 in Wakiso district, a suburban district that that encircles Kampala, Uganda’s capital city. We collected data on socio-demographic characteristics and hypertension status using a modified STEPs questionnaire from 3685 subjects aged 18–40 years selected by multistage cluster sampling. Blood pressure and anthropometric measurements were performed using standardized protocols. Fasting blood sugar and HIV status were determined using a venous blood sample. Association between hypertension status and various biosocial factors was assessed using logistic regression.
The overall prevalence of hypertension was 15 % (95 % CI 14.2 – 19.6) and 40 % were pre-hypertensive. Among the 553 hypertensive participants, 76 (13.7 %) were aware of their diagnosis and all these participants had initiated therapy with target blood pressure control attained in 20 % of treated subjects. Hypertension was significantly associated with the older age-group, male sex and obesity. There was a significantly lower prevalence of hypertension among participants with HIV OR 0.6 (95 % CI 0.4–0.8, P = 0.007).
There is a high prevalence of high blood pressure in this young periurban population of Uganda with sub-optimal diagnosis and control. There is previously undocumented high rate of treatment, a unique finding that may be exploited to drive efforts to control hypertension. Specific programs for early diagnosis and treatment of hypertension among the young should be developed to improve control of hypertension. The relationship between HIV infection and blood pressure requires further clarification by longitudinal studies.
Electronic supplementary material
The online version of this article (doi:10.1186/s12889-015-2146-y) contains supplementary material, which is available to authorized users.
PMCID: PMC4552375  PMID: 26315787
2.  Predictors for MTB Culture-Positivity among HIV-Infected Smear-Negative Presumptive Tuberculosis Patients in Uganda: Application of New Tuberculosis Diagnostic Technology 
PLoS ONE  2015;10(7):e0133756.
The existing World Health Organization diagnostic algorithms for smear-negative TB perform poorly in HIV-infected individuals. New TB diagnostics such as urine TB lipoarabinomannan (LAM) could improve the accuracy and reduce delays in TB diagnosis in HIV-infected smear-negative presumptive TB. We sought to determine predictors for MTB culture-positivity among these patients.
This study was nested into a prospective evaluation of HIV-infected outpatients and inpatients clinically suspected to have TB who were screened by smear-microscopy on two spot sputum samples. Data on socio-demographics, clinical symptoms, antiretroviral therapy, CXR, CD4 count, mycobacterial sputum and blood cultures and TB-LAM were collected. Logistic regression and conditional inference tree analysis were used to determine the most predictive indicators for MTB culture-positivity.
Of the 418 smear-negative participants [female, 64%; median age (IQR) 32 (28-39) years, median CD4 106 (IQR 22 - 298) cells/mm3], 96/418 (23%) were sputum and/ or blood culture-positive for Mycobacterium tuberculosis (MTB) complex. Abnormal CXR (aOR 3.68, 95% CI 1.76- 7.71, p=0.001) and positive urine TB-LAM (aOR 6.21, 95% CI 3.14-12.27, p< 0.001) were significantly associated with MTB culture-positivity. Previous TB treatment (aOR 0.41, 95% CI 0.17-0.99, p=0.049) reduced the likelihood of a positive MTB culture. A conditional inference tree analysis showed that positive urine TB-LAM and abnormal CXR were the most predictive indicators of MTB culture-positivity. A combination of urine TB-LAM test and CXR had sensitivity and specificity of 50% and 86.1% respectively overall, and 70.8% and 84.1% respectively among those with CD4<100 cells/mm3.
A positive urine TB-LAM test and an abnormal CXR significantly predict MTB culture-positivity among smear-negative HIV-infected presumptive TB patients while previous TB treatment reduces the likelihood of a positive MTB culture. Validation studies to assess the performance of diagnostic algorithms that include urine TB-LAM in the diagnosis of smear-negative TB in HIV-infected individuals are warranted.
PMCID: PMC4519276  PMID: 26222142
3.  Immunoadjuvant Prednisolone Therapy for HIV-Associated Tuberculosis: A Phase 2 Clinical Trial in Uganda 
The Journal of infectious diseases  2005;191(6):856-865.
Human immunodeficiency virus (HIV)–infected patients with tuberculosis (TB) respond to effective antituberculous therapy, but their prognosis remains poor. Mounting evidence from clinical studies supports the concept of copathogenesis in which immune activation that is triggered by TB and mediated by cytokines stimulates viral replication and worsens HIV infection, especially when immune function is preserved.
We performed a phase 2, randomized, double-blind, placebo-controlled clinical trial in Kampala, Uganda, to determine whether immunoadjuvant prednisolone therapy in HIV-infected patients with TB who have CD4+ T cell counts ≥200 cells/μL is safe and effective at increasing CD4+ T cell counts.
Short-term prednisolone therapy reduced levels of immune activation and tended to produce higher CD4+ T cell counts. Although prednisolone therapy was associated with a more rapid clearance of Mycobacterium tuberculosis from the sputum, it was also associated with a transient increase in HIV RNA levels, which receded when prednisolone therapy was discontinued. The intervention worsened underlying hypertension and caused fluid retention and hyperglycemia.
The benefits of prednisolone therapy on immune activation and CD4+ T cell counts do not outweigh the risks of adverse events in HIV-infected patients with TB and preserved immune function.
PMCID: PMC4515766  PMID: 15717259
4.  Lower Pre-Treatment T Cell Activation in Early- and Late-Onset Tuberculosis-Associated Immune Reconstitution Inflammatory Syndrome 
PLoS ONE  2015;10(7):e0133924.
Tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) is an inflammatory complication in HIV-TB co-infected patients receiving antiretroviral therapy (ART). The role of disturbed T cell reconstitution in TB-IRIS is not well understood. We investigated T cell activation and maturation profiles in patients who developed TB-IRIS at different intervals during ART.
Twenty-two HIV-TB patients who developed early-onset TB-IRIS and 10 who developed late-onset TB-IRIS were matched for age, sex and CD4 count to equal numbers of HIV-TB patients who did not develop TB-IRIS. Flow cytometry analysis was performed on fresh blood, drawn before and after ART initiation and during TB-IRIS events. T cell activation and maturation was measured on CD4+ and CD8+ T cells using CD45RO, CD38, HLA-DR, CCR7 and CD27 antibodies.
CD8+ T cell activation before ART was decreased in both early-onset (77% vs. 82%, p = 0.014) and late-onset (71% vs. 83%, p = 0.012) TB-IRIS patients compared to non-IRIS controls. After ART initiation, the observed differences in T cell activation disappeared. During late-onset, but not early-onset TB-IRIS, we observed a skewing from memory to terminal effector CD4+ and CD8+ T cell populations (p≤0.028).
Our data provide evidence of reduced CD8+ T cell activation before ART as a common predisposing factor of early- and late-onset TB-IRIS. The occurrence of TB-IRIS itself was not marked by an over-activated CD8+ T cell compartment. Late- but not early-onset TB-IRIS was characterized by a more terminally differentiated T cell phenotype.
PMCID: PMC4514632  PMID: 26208109
5.  Short Communication: Circulating Plasma HIV-1 Viral Protein R in Dual HIV-1/Tuberculosis Infection 
Circulating free HIV-1 viral protein R (Vpr) is found in up to one third of subjects with HIV-1 infection. Free Vpr presumably shares some of the immunopathogenic effects of cell-associated Vpr. Here we assessed Vpr in plasma and pleural fluid from HIV/tuberculosis (TB) dually infected subjects with pleural TB and from plasma of patients with pulmonary HIV/TB. Vpr was assessed by western blot analysis. In plasma from HIV/TB subjects with pulmonary TB free Vpr could be detected in 47%. Only one subject, among 26 tested, with HIV monoinfection showed plasma Vpr activity. The majority (87.5%) of patients with pleural HIV/TB demonstrated free Vpr reactivity in their plasma. However, no Vpr activity was found in autologous pleural fluid samples from pleural HIV/TB patients. Standard (s) Vpr reactivity was reduced markedly by the addition of sVpr to pleural fluid from HIV-uninfected subjects. A high incidence of plasma Vpr reactivity in HIV/TB patients implies heightened processing and release of this HIV-1 accessory protein during HIV/TB coinfection. The contribution of free Vpr to HIV-1 immunopathogenesis during HIV/TB needs to be studied.
PMCID: PMC4076988  PMID: 24798102
6.  FCRL5 Delineates Functionally Impaired Memory B Cells Associated with Plasmodium falciparum Exposure 
PLoS Pathogens  2015;11(5):e1004894.
Exposure to Plasmodium falciparum is associated with circulating “atypical” memory B cells (atMBCs), which appear similar to dysfunctional B cells found in HIV-infected individuals. Functional analysis of atMBCs has been limited, with one report suggesting these cells are not dysfunctional but produce protective antibodies. To better understand the function of malaria-associated atMBCs, we performed global transcriptome analysis of these cells, obtained from individuals living in an area of high malaria endemicity in Uganda. Comparison of gene expression data suggested down-modulation of B cell receptor signaling and apoptosis in atMBCs compared to classical MBCs. Additionally, in contrast to previous reports, we found upregulation of Fc receptor-like 5 (FCRL5), but not FCRL4, on atMBCs. Atypical MBCs were poor spontaneous producers of antibody ex vivo, and higher surface expression of FCRL5 defined a distinct subset of atMBCs compromised in its ability to produce antibody upon stimulation. Moreover, higher levels of P. falciparum exposure were associated with increased frequencies of FCRL5+ atMBCs. Together, our findings suggest that FCLR5+ identifies a functionally distinct, and perhaps dysfunctional, subset of MBCs in individuals exposed to P. falciparum.
Author Summary
A subset of “atypical” memory B cells found in individuals with high exposure to P. falciparum has been hypothesized to be dysfunctional, based on phenotypic similarities to analogous cells found in HIV-infected individuals. However, the functional capabilities of these cells have been poorly characterized in the setting of malaria exposure, and previous reports have been controversial regarding whether these cells produce antibody. In our study, we analyze the molecular programming of atypical memory B cells, find that they are dysfunctional in a manner similar to that observed in B cells from HIV-infected individuals, and present data that may reconcile previously conflicting studies. By delineating the transcriptional landscape of atMBCs and identifying expression of FCRL5 as a key marker of dysfunction, we provide a foundation for improving our understanding of the role of these cells in immunity to malaria.
PMCID: PMC4438005  PMID: 25993340
7.  Cellular Immune Activation in Cerebrospinal Fluid From Ugandans With Cryptococcal Meningitis and Immune Reconstitution Inflammatory Syndrome 
The Journal of infectious diseases  2014;211(10):1597-1606.
Human immunodeficiency virus (HIV)-associated cryptococcal meningitis (CM) is characterized by high fungal burden and limited leukocyte trafficking to cerebrospinal fluid (CSF). The immunopathogenesis of CM immune reconstitution inflammatory syndrome (IRIS) after initiation of antiretroviral therapy at the site of infection is poorly understood.
We characterized the lineage and activation status of mononuclear cells in blood and CSF of HIV-infected patients with noncryptococcal meningitis (NCM) (n = 10), those with CM at day 0 (n = 40) or day 14 (n = 21) of antifungal therapy, and those with CM-IRIS (n = 10).
At diagnosis, highly activated CD8+ T cells predominated in CSF in both CM and NCM. CM-IRIS was associated with an increasing frequency of CSF CD4+ T cells (increased from 2.2% to 23%; P = .06), a shift in monocyte phenotype from classic to an intermediate/proinflammatory, and increased programmed death ligand 1 expression on natural killer cells (increased from 11.9% to 61.6%, P = .03). CSF cellular responses were distinct from responses in peripheral blood.
After CM, T cells in CSF tend to evolve with the development of IRIS, with increasing proportions of activated CD4+ T cells, migration of intermediate monocytes to the CSF, and declining fungal burden. These changes provide insight into IRIS pathogenesis and could be exploited to more effectively treat CM and prevent CM-IRIS.
PMCID: PMC4407762  PMID: 25492918
cryptococcal meningitis; cryptococcus; HIV; cerebrospinal fluid; immune responses; cell activation
8.  How we determined the most reliable solid medium for studying treatment of tuberculosis 
Phase 2 clinical trials for tuberculosis (TB) treatment require reliable culture methods to determine presence or absence of Mycobacterium tuberculosis (Mtb) over the course of therapy, as these trials are based primarily on bacteriological endpoints. We evaluate which of 5 solid media is most reliable: Lowenstein-Jensen (LJ) egg-based medium and 4 Middlebrook agar media (nonselective 7H10 and 7H11 and selective 7H10 and 7H11). We analyze 393 specimens from 50 HIV-negative Ugandan adults with newly-diagnosed, pulmonary TB and high acid-fast bacillus smear grade. Specimens were collected every 2–4 weeks during the first 12 weeks of therapy. We compare the results for each culture to 2 composite reference standards—one that was deemed positive if any solid culture was positive for Mtb and another based on latent class analysis. Both reference standards established that the 2 selective Middlebrook media most reliably determine the presence or absence of Mtb (P<0.003), largely because of their lower contamination rates. We also showed that results on Middlebrook media were similar to each other, while LJ was most frequently discordant. Contaminated results appeared more likely to be truly negative than to harbor undetected Mtb.
PMCID: PMC4070601  PMID: 24661816
Middlebrook agar culture media; Lowenstein-Jensen culture medium; composite reference standard; latent class model
9.  The Effect of a Bidirectional Exchange on Faculty and Institutional Development in a Global Health Collaboration 
PLoS ONE  2015;10(3):e0119798.
The MUYU Collaboration is a partnership between Mulago Hospital-Makerere University College of Health Sciences (M-MakCHS), in Kampala, Uganda, and the Yale University School of Medicine. The program allows Ugandan junior faculty to receive up to 1 year of subspecialty training within the Yale hospital system. The authors performed a qualitative study to assess the effects of this program on participants, as well as on M-MakCHS as an institution.
Data was collected via semi-structured interviews with exchange participants. Eight participants (67% of those eligible as of 4/2012) completed interviews. Study authors performed data analysis using standard qualitative data analysis techniques.
Analysis revealed themes addressing the benefits, difficulties, and opportunities for improvement of the program. Interviewees described the main benefit of the program as its effect on their fund of knowledge. Participants also described positive effects on their clinical practice and on medical education at M-MakCHS. Most respondents cited financial issues as the primary difficulty of participation. Post-participation difficulties included resource limitations and confronting longstanding institutional and cultural habits. Suggestions for programmatic improvement included expansion of the program, ensuring appropriate management of pre-departure expectations, and refinement of program mentoring structures. Participants also voiced interest in expanding post-exchange programming to ensure both the use of and the maintenance of new capacity.
The MUYU Collaboration has benefitted both program participants and M-MakCHS, though these benefits remain difficult to quantify. This study supports the assertion that resource-poor to resource-rich exchanges have the potential to provide significant benefits to the resource-poor partner.
PMCID: PMC4370667  PMID: 25799567
10.  Predictors and outcomes of mycobacteremia among HIV-infected smear- negative presumptive tuberculosis patients in Uganda 
Sputum smear microscopy for tuberculosis (TB) diagnosis lacks sensitivity in HIV-infected symptomatic patients and increases the likelihood that mycobacterial infections particularly disseminated TB will be missed; delays in diagnosis can be fatal. Given the duration for MTB growth in blood culture, clinical predictors of MTB bacteremia may improve early diagnosis of mycobacteremia. We describe the predictors and mortality outcome of mycobacteremia among HIV-infected sputum smear-negative presumptive TB patients in a high prevalence HIV/TB setting.
Between January and November 2011, all consenting HIV-infected adults suspected to have TB (presumptive TB) were consecutively enrolled. Diagnostic assessment included sputum smear microscopy, urine Determine TB lipoarabinomannan (LAM) antigen test, mycobacterial sputum and blood cultures, chest X-ray, and CD4 cell counts in addition to clinical and socio-demographic data. Patients were followed for 12 months post-enrolment.
Of 394 sputum smear-negative participants [female, 63.7%; median age (IQR) 32 (28–39) years], 41/394 (10.4%) had positive mycobacterial blood cultures (mycobacteremia); all isolates were M. tuberculosis (MTB). The median CD4 cell count was significantly lower among patients with mycobacteremia when compared with those without (CD4 31 versus 122 cells/μL, p < 0.001). In a multivariate analysis, male gender [OR 3.4, 95%CI (1.4-7.6), p = 0.005], CD4 count <100 cells/μL [OR 3.1, 95% CI (1.1-8.6), p = 0.030] and a positive lateral flow urine TB LAM antigen test [OR 15.3, 95%CI (5.7-41.1), p < 0.001] were significantly associated with mycobacteremia. At 12 months of follow-up, a trend towards increased mortality was observed in patients that were MTB blood culture positive (35.3%) compared with those that were MTB blood culture negative (23.3%) (p = 0.065).
Mycobacteremia occurred in 10% of smear-negative patients and was associated with higher mortality compared with smear-negative patients without mycobacteremia. Advanced HIV disease (CD4 < 100 cells/mm3), male gender and positive lateral flow urine TB LAM test predicted mycobacteremia in HIV-infected smear-negative presumptive TB patients in this high prevalence TB/HIV setting.
PMCID: PMC4332438  PMID: 25888317
Predictors; Mortality; Mycobacterial infections; Bacteremia; Smear- negative; HIV; LAM; Sub-Saharan Africa
11.  Feasibility of establishing a biosafety level 3 tuberculosis culture laboratory of acceptable quality standards in a resource-limited setting: an experience from Uganda 
Despite the recent innovations in tuberculosis (TB) and multi-drug resistant TB (MDR-TB) diagnosis, culture remains vital for difficult-to-diagnose patients, baseline and end-point determination for novel vaccines and drug trials. Herein, we share our experience of establishing a BSL-3 culture facility in Uganda as well as 3-years performance indicators and post-TB vaccine trials (pioneer) and funding experience of sustaining such a facility.
Between September 2008 and April 2009, the laboratory was set-up with financial support from external partners. After an initial procedure validation phase in parallel with the National TB Reference Laboratory (NTRL) and legal approvals, the laboratory registered for external quality assessment (EQA) from the NTRL, WHO, National Health Laboratories Services (NHLS), and the College of American Pathologists (CAP). The laboratory also instituted a functional quality management system (QMS). Pioneer funding ended in 2012 and the laboratory remained in self-sustainability mode.
The laboratory achieved internationally acceptable standards in both structural and biosafety requirements. Of the 14 patient samples analyzed in the procedural validation phase, agreement for all tests with NTRL was 90% (P <0.01). It started full operations in October 2009 performing smear microscopy, culture, identification, and drug susceptibility testing (DST). The annual culture workload was 7,636, 10,242, and 2,712 inoculations for the years 2010, 2011, and 2012, respectively. Other performance indicators of TB culture laboratories were also monitored. Scores from EQA panels included smear microscopy >80% in all years from NTRL, CAP, and NHLS, and culture was 100% for CAP panels and above regional average scores for all years with NHLS. Quarterly DST scores from WHO-EQA ranged from 78% to 100% in 2010, 80% to 100% in 2011, and 90 to 100% in 2012.
From our experience, it is feasible to set-up a BSL-3 TB culture laboratory with acceptable quality performance standards in resource-limited countries. With the demonstrated quality of work, the laboratory attracted more research groups and post-pioneer funding, which helped to ensure sustainability. The high skilled experts in this research laboratory also continue to provide an excellent resource for the needed national discussion of the laboratory and quality management systems.
Electronic supplementary material
The online version of this article (doi:10.1186/1478-4505-13-4) contains supplementary material, which is available to authorized users.
PMCID: PMC4326287  PMID: 25589057
Acceptable quality standards; Biosafety level 3; Feasibility; Resource limited countries; TB culture
12.  Towards reframing health service delivery in Uganda: the Uganda Initiative for Integrated Management of Non-Communicable Diseases 
Global Health Action  2015;8:10.3402/gha.v8.26537.
The burden of non-communicable diseases (NCDs) in low- and middle-income countries (LMICs) is accelerating. Given that the capacity of health systems in LMICs is already strained by the weight of communicable diseases, these countries find themselves facing a double burden of disease. NCDs contribute significantly to morbidity and mortality, thereby playing a major role in the cycle of poverty, and impeding development.
Integrated approaches to health service delivery and healthcare worker (HCW) training will be necessary in order to successfully combat the great challenge posed by NCDs.
In 2013, we formed the Uganda Initiative for Integrated Management of NCDs (UINCD), a multidisciplinary research collaboration that aims to present a systems approach to integrated management of chronic disease prevention, care, and the training of HCWs.
Through broad-based stakeholder engagement, catalytic partnerships, and a collective vision, UINCD is working to reframe integrated health service delivery in Uganda.
PMCID: PMC4292588  PMID: 25563451
Non-communicable diseases; Health system strengthening; Integration; Multi-sectoral collaboration
13.  Global medical education partnerships to expand specialty expertise: a case report on building neurology clinical and research capacity 
Neurological disorders are a common cause of morbidity and mortality in sub-Saharan African, but resources for their management are scarce. Collaborations between training institutions in developed and resource-limited countries can be a successful model for supporting specialty medical education and increasing clinical and research capacity.
Case report
This report describes a US National Institutes of Health (NIH) funded Medical Education Partnership Initiative (MEPI) to enhance expertise in neurology, developed between Makerere University College of Health Sciences in Kampala, Uganda, and Case Western Reserve University School of Medicine in Cleveland, OH, USA.
This collaborative model is based on a successful medical education and research model that has been developed over the past two decades. The Ugandan and US teams have accumulated knowledge and 'lessons learned' that facilitate specialty expertise in neurological conditions, which are widespread and associated with substantial disability in resource-limited countries. Strengths of the model include a focus on community health care settings and a strong research component. Key elements include strong local leadership; use of remote technology, templates to standardize performance; shared exchanges; mechanisms to optimize sustainability and of dissemination activities that expand impact of the original initiative. Efficient collaborations are further enhanced by external and institutional support, and can be sequentially refined.
Models such as the Makerere University College of Health Sciences - Case Western Reserve University partnership may help other groups initiate collaborative education programmes and establish successful partnerships that may provide the opportunity to expand to other chronic diseases. A benefit of collaboration is that learning is two-directional, and interaction with other international medical education collaborators is likely to be of benefit to the larger global health community.
Electronic supplementary material
The online version of this article (doi:10.1186/1478-4491-12-75) contains supplementary material, which is available to authorized users.
PMCID: PMC4531526  PMID: 25547168
Medical education; Neurology; Neurological disorders; Research education; Global health; Uganda; sub-Saharan Africa; Cardiovascular disease
14.  Contact Investigation for Active Tuberculosis Among Child Contacts in Uganda 
In a large prospective study in Kampala, Uganda, we found that case investigation can be effective (10% yield) in early detection of active tuberculosis in children <15 years old, with 71% culture confirmation.
Background. Tuberculosis is a large source of morbidity and mortality among children. However, limited studies characterize childhood tuberculosis disease, and contact investigation is rarely implemented in high-burden settings. In one of the largest pediatric tuberculosis contact investigation studies in a resource-limited setting, we assessed the yield of contact tracing on childhood tuberculosis and indicators for disease progression in Uganda.
Methods. Child contacts aged <15 years in Kampala, Uganda, were enrolled from July 2002 to June 2009 and evaluated for tuberculosis disease via clinical, radiographic, and laboratory methods for up to 24 months.
Results. Seven hundred sixty-one child contacts were included in the analysis. Prevalence of tuberculosis in our child population was 10%, of which 71% were culture-confirmed positive. There were no cases of disseminated tuberculosis, and 483 of 490 children (99%) started on isoniazid preventative therapy did not develop disease. Multivariable testing suggested risk factors including human immunodeficiency virus (HIV) status (odds ratio [OR], 7.90; P < .001), and baseline positive tuberculin skin test (OR, 2.21; P = .03); BCG vaccination was particularly protective, especially among children aged ≤5 years (OR, 0.23; P < .001). Adult index characteristics such as sex, HIV status, and extent or severity of disease were not associated with childhood disease.
Conclusions. Contact tracing for children in high-burden settings is able to identify a large percentage of culture-confirmed positive tuberculosis cases before dissemination of disease, while suggesting factors for disease progression to identify who may benefit from targeted screening.
PMCID: PMC3840405  PMID: 24077055
pediatric; child; tuberculosis; risk factors; contact tracing
15.  Cellular Immune Activation in Cerebrospinal Fluid From Ugandans With Cryptococcal Meningitis and Immune Reconstitution Inflammatory Syndrome 
The Journal of Infectious Diseases  2014;211(10):1597-1606.
Background. Human immunodeficiency virus (HIV)-associated cryptococcal meningitis (CM) is characterized by high fungal burden and limited leukocyte trafficking to cerebrospinal fluid (CSF). The immunopathogenesis of CM immune reconstitution inflammatory syndrome (IRIS) after initiation of antiretroviral therapy at the site of infection is poorly understood.
Methods. We characterized the lineage and activation status of mononuclear cells in blood and CSF of HIV-infected patients with noncryptococcal meningitis (NCM) (n = 10), those with CM at day 0 (n = 40) or day 14 (n = 21) of antifungal therapy, and those with CM-IRIS (n = 10).
Results. At diagnosis, highly activated CD8+ T cells predominated in CSF in both CM and NCM. CM-IRIS was associated with an increasing frequency of CSF CD4+ T cells (increased from 2.2% to 23%; P = .06), a shift in monocyte phenotype from classic to an intermediate/proinflammatory, and increased programmed death ligand 1 expression on natural killer cells (increased from 11.9% to 61.6%, P = .03). CSF cellular responses were distinct from responses in peripheral blood.
Conclusions. After CM, T cells in CSF tend to evolve with the development of IRIS, with increasing proportions of activated CD4+ T cells, migration of intermediate monocytes to the CSF, and declining fungal burden. These changes provide insight into IRIS pathogenesis and could be exploited to more effectively treat CM and prevent CM-IRIS.
PMCID: PMC4407762  PMID: 25492918
cryptococcal meningitis; cryptococcus; HIV; cerebrospinal fluid; immune responses; cell activation
16.  Antigen-Specific Interferon-Gamma Responses and Innate Cytokine Balance in TB-IRIS 
PLoS ONE  2014;9(11):e113101.
Tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) remains a poorly understood complication in HIV-TB patients receiving antiretroviral therapy (ART). TB-IRIS could be associated with an exaggerated immune response to TB-antigens. We compared the recovery of IFNγ responses to recall and TB-antigens and explored in vitro innate cytokine production in TB-IRIS patients.
In a prospective cohort study of HIV-TB co-infected patients treated for TB before ART initiation, we compared 18 patients who developed TB-IRIS with 18 non-IRIS controls matched for age, sex and CD4 count. We analyzed IFNγ ELISpot responses to CMV, influenza, TB and LPS before ART and during TB-IRIS. CMV and LPS stimulated ELISpot supernatants were subsequently evaluated for production of IL-12p70, IL-6, TNFα and IL-10 by Luminex.
Before ART, all responses were similar between TB-IRIS patients and non-IRIS controls. During TB-IRIS, IFNγ responses to TB and influenza antigens were comparable between TB-IRIS patients and non-IRIS controls, but responses to CMV and LPS remained significantly lower in TB-IRIS patients. Production of innate cytokines was similar between TB-IRIS patients and non-IRIS controls. However, upon LPS stimulation, IL-6/IL-10 and TNFα/IL-10 ratios were increased in TB-IRIS patients compared to non-IRIS controls.
TB-IRIS patients did not display excessive IFNγ responses to TB-antigens. In contrast, the reconstitution of CMV and LPS responses was delayed in the TB-IRIS group. For LPS, this was linked with a pro-inflammatory shift in the innate cytokine balance. These data are in support of a prominent role of the innate immune system in TB-IRIS.
PMCID: PMC4240578  PMID: 25415590
17.  Abbreviated HIV counselling and testing and enhanced referral to care in Uganda: a factorial randomised controlled trial 
The lancet global health  2013;1(3):e137-e145.
HIV counselling and testing and linkage to care are crucial for successful HIV prevention and treatment. Abbreviated counselling could save time; however, its effect on HIV risk is uncertain and methods to improve linkage to care have not been studied.
We did this factorial randomised controlled study at Mulago Hospital, Uganda. Participants were randomly assigned to abbreviated or traditional HIV counselling and testing; HIV-infected patients were randomly assigned to enhanced linkage to care or standard linkage to care. All study personnel except counsellors and the data officer were masked to study group assignment. Participants had structured interviews, given once every 3 months. We compared sexual risk behaviour by counselling strategy with a 6·5% non-inferiority margin. We used Cox proportional hazards analyses to compare HIV outcomes by linkage to care over 1 year and tested for interaction by sex. This trial is registered with (NCT00648232).
We enrolled 3415 participants; 1707 assigned to abbreviated counselling versus 1708 assigned to traditional. Unprotected sex with an HIV discordant or status unknown partner was similar in each group (232/823 [27·9%] vs 251/890 [28·2%], difference −0·3%, one-sided 95% CI 3·2). Loss to follow-up was lower for traditional counselling than for abbreviated counselling (adjusted hazard ratio [HR] 0·61, 95% CI 0·44–0·83). 1003 HIV-positive participants were assigned to enhanced linkage (n=504) or standard linkage to care (n=499). Linkage to care did not have a significant effect on mortality or receipt of co-trimoxazole. Time to treatment in men with CD4 cell counts of 250 cells per μL or fewer was lower for enhanced linkage versus standard linkage (adjusted HR 0·60, 95% CI 0·41–0·87) and time to HIV care was decreased among women (0·80, 0·66–0·96).
Abbreviated HIV counselling and testing did not adversely affect risk behaviour. Linkage to care interventions might decrease time to enrolment in HIV care and antiretroviral treatment and thus might affect secondary HIV transmission and improve treatment outcomes.
US National Institute of Mental Health.
PMCID: PMC4129546  PMID: 25104262
18.  Evaluation of Cepheid's Xpert MTB/RIF Test on Pleural Fluid in the Diagnosis of Pleural Tuberculosis in a High Prevalence HIV/TB Setting 
PLoS ONE  2014;9(7):e102702.
Diagnosis of pleural tuberculosis (TB) using routinely available diagnostic methods is challenging due to the paucibacillary nature of the disease. Histopathology and pleural tissue TB culture involves an invasive procedure which requires expertise and appropriate equipment, both often unavailable in many health units. Xpert MTB/Rif test has been widely evaluated in sputum specimens but data on its performance in pleural TB is scarce. We evaluated the accuracy of Cepheid's Xpert MTB/Rif test on pleural fluid in the diagnosis of pleural TB in Uganda.
Consenting adult patients with exudative pleural effusions underwent pleural biopsy and the tissue obtained subjected to Lowenstein-Jensen and mycobacterial growth indicator tube MTB cultures and histopathology. Pleural fluid for Xpert MTB/Rif testing was also collected. Data on socio-demographic characteristics, clinical symptoms, HIV status and CD4 count were also collected. Sensitivity, specificity, positive and negative predictive values of Xpert MTB/Rif test on pleural fluid in pleural TB diagnosis were calculated using pleural tissue MTB culture and/or histopathology as the reference standard.
Of the 116 participants [female 50%, mean age 34 (SD ±13], 87/116 (75%) had pleural TB confirmed on pleural tissue culture and/or histopathology. The Xpert MTB/Rif test identified 25 (28.7%) of the 87 confirmed pleural TB cases. The sensitivity and specificity of Xpert MTB/Rif test were 28.7% and 96.6% respectively while the positive and negative predictive values were 96.1% and 31.1% respectively.
Xpert MTB/Rif test on pleural fluid does not accurately diagnose pleural TB and therefore cannot be used as an initial evaluation test in patients with suspected pleural TB. New, rapid and accurate tests for the diagnosis of pleural TB are still warranted.
PMCID: PMC4106856  PMID: 25051491
19.  Genetic and Shared Environmental Influences on Interferon-γ Production in Response to Mycobacterium tuberculosis Antigens in a Ugandan Population 
Interferon-γ (IFN-γ) is a key cytokine in the immune response to Mycobacterium tuberculosis (Mtb). Many studies established IFN-γ responses are influenced by host genetics, however differed widely by the study design and heritability estimation method. We estimated heritability of IFN-γ responses to Mtb culture filtrate (CF), ESAT-6, and Antigen 85B (Ag85B) in 1,104 Ugandans from a household contact study. Our method separately evaluates shared environmental and genetic variance, therefore heritability estimates were not upwardly biased, ranging from 11.6% for Ag85B to 22.9% for CF. Subset analyses of individuals with latent Mtb infection or without human immunodeficiency virus infection yielded higher heritability estimates, suggesting 10–30% of variation in IFN-γ is caused by a shared environment. Immunosuppression does not negate the role of genetics on IFN-γ response. These estimates are remarkably close to those reported for components of the innate immune response. These findings have implications for the interpretation of IFN-γ response assays and vaccine studies.
PMCID: PMC3748477  PMID: 23629934
20.  Clinical and epidemiological characteristics of individuals resistant to M. tuberculosis infection in a longitudinal TB household contact study in Kampala, Uganda 
BMC Infectious Diseases  2014;14:352.
Despite sustained exposure to a person with pulmonary tuberculosis (TB), some M. tuberculosis (Mtb) exposed individuals maintain a negative tuberculin skin test (TST). Our objective was to characterize these persistently negative TST (PTST-) individuals and compare them to TST converters (TSTC) and individuals who are TST positive at study enrollment.
During a TB household contact study in Kampala, Uganda, PTST-, TSTC, and TST + individuals were identified. PTST- individuals maintained a negative TST over a 2 year observation period despite prolonged exposure to an infectious tuberculosis (TB) case. Epidemiological and clinical characteristics were compared, a risk score developed by another group to capture risk for Mtb infection was computed, and an ordinal regression was performed.
When analyzed independently, epidemiological risk factors increased in prevalence from PTST- to TSTC to TST+. An ordinal regression model suggested age (p < 0.01), number of windows (p < 0.01) and people (p = 0.07) in the home, and sleeping in the same room (p < 0.01) were associated with PTST- and TSTC. As these factors do not exist in isolation, we examined a risk score, which reflects an accumulation of risk factors. This compound exposure score did not differ significantly between PTST-, TSTC, and TST+, except for the 5–15 age group (p = 0.009).
Though many individual factors differed across all three groups, an exposure risk score reflecting a collection of risk factors did not differ for PTST-, TSTC and TST + young children and adults. This is the first study to rigorously characterize the epidemiologic risk profile of individuals with persistently negative TSTs despite close exposure to a person with TB. Additional studies are needed to characterize possible epidemiologic and host factors associated with this phenotype.
PMCID: PMC4091673  PMID: 24970328
Transmission risk factors; Latent Mtb infection; Exposure; Household characteristics; PPD test
21.  Systemic Immune Activation and Microbial Translocation in Dual HIV/Tuberculosis-Infected Subjects 
The Journal of Infectious Diseases  2013;207(12):1841-1849.
Background. Systemic immune activation is a strong predictor of progression of human immunodeficiency virus type 1 (HIV-1) disease and a prominent feature of infection with Mycobacterium tuberculosis.
Objective. To understand the role of systemic immune activation and microbial translocation in HIV/tuberculosis dually infected patients over the full spectrum of HIV-1 immunodeficiency, we studied circulating sCD14 and lipopolysaccharide (LPS) and their relationship to HIV-1 activity.
Methods. Two cohorts of HIV/tuberculosis subjects defined by CD4 T-cell count at time of diagnosis of tuberculosis were studied: those with low (<350/μL) and those with high (≥350/μL) CD4 T-cell count. Circulating soluble CD14 (sCD14) and LPS were assessed.
Results. Levels of sCD14 were higher in HIV/tuberculosis with high (≥350/μL) as compared to low CD4 T-cell count (P < .001). Whereas sCD14 levels remained elevated in HIV/tuberculosis subjects with lower CD4 T-cell counts despite treatment of tuberculosis, in HIV/tuberculosis patients with higher CD4 T-cell count (≥350/μL), levels declined regardless of whether highly active antiretroviral therapy (HAART) was included with the anti-tuberculosis regimen. Circulating LPS levels in HIV/tuberculosis patients with CD4 T-cell count ≥350/μL were unaffected by treatment of tuberculosis with or without HAART.
Conclusion. During HIV/tuberculosis, systemic immune activation is dissociated from microbial translocation. Changes in circulating sCD14 and LPS are dependent on CD4 T-cell count.
PMCID: PMC3654743  PMID: 23479321
HIV-1; tuberculosis; LPS; soluble CD14
22.  Tuberculin Skin Test Reversion following Isoniazid Preventive Therapy Reflects Diversity of Immune Response to Primary Mycobacterium tuberculosis Infection 
PLoS ONE  2014;9(5):e96613.
Healthy household contacts (HHC) of individuals with Tuberculosis (TB) with Tuberculin Skin Test (TST) conversions are considered to harbor latent Mycobacterium tuberculosis (Mtb), and at risk for TB. The immunologic, clinical, and public health implications of TST reversions that occur following Isoniazid preventive therapy (IPT) remain controversial.
To measure frequency of TST reversion following IPT, and variation in interferon-gamma (IFN-γ) responses to Mtb, in healthy Ugandan TB HHC with primary Mtb infection evidenced by TST conversion.
Prospective cohort study of healthy, HIV-uninfected, TST-negative TB HHC with TST conversions. Repeat TST was performed 12 months following conversion (3 months following completion of 9 month IPT course) to assess for stable conversion vs. reversion. Whole blood IFN-γ responses to Mtb antigen 85B (MtbA85B) and whole Mtb bacilli (wMtb) were measured in a subset (n = 27 and n = 42, respectively) at enrollment and TST conversion, prior to initiation of IPT.
Of 122 subjects, TST reversion was noted in 25 (20.5%). There were no significant differences in demographic, clinical, or exposure variables between reverters and stable converters. At conversion, reverters had significantly smaller TST compared to stable converters (13.7 mm vs 16.4 mm, respectively; p = 0.003). At enrollment, there were no significant differences in IFN-γ responses to MtbA85B or wMTB between groups. At conversion, stable converters demonstrated significant increases in IFN-γ responses to Ag85B and wMtb compared to enrollment (p = 0.001, p<0.001, respectively), while there were no significant changes among reverters.
TST reversion following IPT is common following primary Mtb infection and associated with unique patterns of Mtb-induced IFN-γ production. We have demonstrated that immune responses to primary Mtb infection are heterogeneous, and submit that prospective longitudinal studies of cell mediated immune responses to Mtb infection be prioritized to identify immune phenotypes protective against development of TB disease.
PMCID: PMC4010490  PMID: 24796677
23.  Two year mortality and associated factors in a cohort of children from rural Uganda 
BMC Public Health  2014;14:314.
As part of site development for clinical trials in novel TB vaccines, a cohort of infants was enrolled in eastern Uganda to estimate the incidence of tuberculosis. The study introduced several mortality reduction strategies, and evaluated the mortality among study participants at two years. The specific of objective of this sub-study was to estimate 2 year mortality and associated factors in this community-based cohort.
A community based cohort of 2500 infants was enrolled from birth up to 8 weeks of age and followed for 1–2 years. During follow up, several mortality reduction activities were implemented to enhance cohort survival and retention. The verbal autopsy process was used to assign causes of death.
A total of 152 children died over a median follow up period of 2.0 years. The overall crude mortality rate was 60.8/1000 or 32.9/1000 person years with 40 deaths per 1000 for children who died in their first year of life. Anaemia, malaria, diarrhoeal diseases and pneumonia were the top causes of death. There was no death directly attributed to tuberculosis. Significant factors associated with mortality were young age of a mother and child’s birth place not being a health facility.
The overall two year mortality in the study cohort was unacceptably high and tuberculosis disease was not identified as a cause of death. Interventions to reduce mortality of children enrolled in the cohort study did not have a significant impact. Clinical trials involving infants and young children in this setting will have to strengthen local maternal and child health services to reduce infant and child mortality.
PMCID: PMC4234345  PMID: 24708689
Mortality; Infants; Factors associated with mortality; Verbal autopsy
24.  High CD56++CD16- natural killer (NK) cells among suboptimal immune responders after four years of suppressive antiretroviral therapy in an African adult HIV treatment cohort 
BMC Immunology  2014;15:2.
Up to 40% of HIV-infected individuals receiving Highly Active Antiretroviral Therapy (HAART) have poor CD4+ T-cell recovery. The role of natural killer (NK) cells in immune recovery during HAART is not well understood. We described the profiles of NK cell subsets and their expression of activating receptor, NKG2D and cytotoxicity receptor NKp46 among suboptimal immune responders to despite four years of suppressive HAART.
A case control study utilized frozen peripheral blood mononuclear cells (PBMC) from a cohort of HIV-infected adults that initiated HAART in 2004/5, at CD4 < 200 cells/μl. Cases were ‘suboptimal’ responders; patients within the lowest quartile of CD4+ T-cell reconstitution, with a median CD4 count increase of 129 (-43-199) cells/μl (difference between CD4 count at baseline and after 4 years of HAART) and controls were ‘super-optimal’ responders; patients within the highest quartile of CD4 T-cell reconstitution with a median CD4 count increase of 528 (416-878) cells/μl). Expression of NK cell lineage markers (CD56+/-CD16+/-) and receptors NKG2D and NKp46, was measured among PBMC from 29 cases of ‘suboptimal’ responders’ and 23 controls of ‘super-optimal responders’, and compared among ‘suboptimal’ and ‘super-optimal’ responders. NK cell populations were compared using the Holm Sidak multiple comparison test and p values < 0.05 were considered statistically significant. Data was analyzed using FLOWJO and GraphPad Prism 6.
‘Suboptimal responders’ had a higher proportion of cytokine producing CD56++CD16+/- (CD56bri) NK cells than the ‘super-optimal responders’ p = 0.017, and CD56neg NK cells were lower among suboptimal than super-optimal responders (p = 0.007). The largest NK cell subset, CD56dim, was comparable among suboptimal responders and ‘super-optimal immune responders’. Expression of NKG2D and NKp46 receptors on NK cell subsets (CD56bri, CD56neg and CD56dim), was comparable among ‘suboptimal’ and ‘super-optimal’ immune responders.
The pro-inflammatory CD56++CD16-- NK cells were higher among ‘suboptimal’ responders relative to ‘super-optimal’ responders, despite four years of suppressive HAART. Alteration of NK cell populations could inhibit host immune responses to infections among suboptimal responders. We recommend further analysis of NK cell function among suboptimal immune responders in order to inform targeted interventions to optimize immune recovery among HAART-treated adults.
PMCID: PMC3915033  PMID: 24483185
Natural killer cells; Suppressive antiretroviral therapy; HAART; Suboptimal immune recovery; HAART; Sub-saharan Africa
25.  Doctoral training in Uganda: evaluation of mentoring best practices at Makerere university college of health sciences 
Good mentoring is a key variable for determining success in completing a doctoral program. We identified prevailing mentoring practices among doctoral students and their mentors, identified common challenges facing doctoral training, and proposed some solutions to enhance the quality of the doctoral training experience for both candidates and mentors at Makerere University College of Health Sciences (MakCHS).
This cross-sectional qualitative evaluation was part of the monitoring and evaluation program for doctoral training. All doctoral students and their mentors were invited for a half-day workshop through the MakCHS mailing list. Prevailing doctoral supervision and mentoring guidelines were summarised in a one-hour presentation. Participants were split into two homogenous students’ (mentees’) and mentors’ groups to discuss specific issues using a focus group discussion (FGD) guide, that highlighted four main themes in regard to the doctoral training experience; what was going well, what was not going well, proposed solutions to current challenges and perceived high priority areas for improvement. The two groups came together again and the note-takers from each group presented their data and discussions were recorded by a note-taker.
Twelve out of 36 invited mentors (33%) and 22 out of 40 invited mentees (55%) attended the workshop. Mentors and mentees noted increasing numbers of doctoral students and mentors, which provided opportunities for peer mentorship. Delays in procurement and research regulatory processes subsequently delayed students’ projects. Similarly, mentees mentioned challenges of limited; 1) infrastructure and mentors to support basic science research projects, 2) physical office space for doctoral students and their mentors, 3) skills in budgeting and finance management and 4) communication skills including conflict resolution. As solutions, the team proposed skills’ training, induction courses for doctoral students-mentor teams, and a Frequently Asked Questions’ document, to better inform mentors’, mentees’ expectations and experiences.
Systemic and infrastructural limitations affect the quality of the doctoral training experience at MaKCHS. Clinical and biomedical research infrastructure, in addition to training in research regulatory processes, procurement and finance management, communication skills and information technology, were highlighted as high priority areas for strategic interventions to improve mentoring within doctoral training of clinician scientists.
PMCID: PMC3897930  PMID: 24410984
Mentorship; Doctoral training; Supervision; Capacity building; Health care; Low and middle income countries; Uganda

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