Establishing the extent, geographical distribution and mechanisms of insecticide resistance in malaria vectors is a prerequisite for resistance management. Here, we report a widespread distribution of insecticide resistance in the major malaria vector An. funestus across Uganda and western Kenya under the control of metabolic resistance mechanisms.
Female An. funestus collected throughout Uganda and western Kenya exhibited a Plasmodium infection rate between 4.2 to 10.4%. Widespread resistance against both type I (permethrin) and II (deltamethrin) pyrethroids and DDT was observed across Uganda and western Kenya. All populations remain highly susceptible to carbamate, organophosphate and dieldrin insecticides. Knockdown resistance plays no role in the pyrethroid and DDT resistance as no kdr mutation associated with resistance was detected despite the presence of a F1021C replacement. Additionally, no signature of selection was observed on the sodium channel gene. Synergist assays and qRT-PCR indicated that metabolic resistance plays a major role notably through elevated expression of cytochrome P450s. DDT resistance mechanisms differ from West Africa as the L119F-GSTe2 mutation only explains a small proportion of the genetic variance to DDT resistance.
The extensive distribution of pyrethroid and DDT resistance in East African An. funestus populations represents a challenge to the control of this vector. However, the observed carbamate and organophosphate susceptibility offers alternative solutions for resistance management.
Background. Defining the parameters that modulate vaccine responses in African populations will be imperative to design effective vaccines for protection against HIV, malaria, tuberculosis, and dengue virus infections. This study aimed to evaluate the contribution of the patient-specific immune microenvironment to the response to the licensed yellow fever vaccine 17D (YF-17D) in an African cohort.
Methods. We compared responses to YF-17D in 50 volunteers in Entebbe, Uganda, and 50 volunteers in Lausanne, Switzerland. We measured the CD8+ T cell and B cell responses induced by YF-17D and correlated them with immune parameters analyzed by flow cytometry prior to vaccination.
Results. We showed that YF-17D–induced CD8+ T cell and B cell responses were substantially lower in immunized individuals from Entebbe compared with immunized individuals from Lausanne. The impaired vaccine response in the Entebbe cohort associated with reduced YF-17D replication. Prior to vaccination, we observed higher frequencies of exhausted and activated NK cells, differentiated T and B cell subsets and proinflammatory monocytes, suggesting an activated immune microenvironment in the Entebbe volunteers. Interestingly, activation of CD8+ T cells and B cells as well as proinflammatory monocytes at baseline negatively correlated with YF-17D–neutralizing antibody titers after vaccination. Additionally, memory T and B cell responses in preimmunized volunteers exhibited reduced persistence in the Entebbe cohort but were boosted by a second vaccination.
Conclusion. Together, these results demonstrate that an activated immune microenvironment prior to vaccination impedes efficacy of the YF-17D vaccine in an African cohort and suggest that vaccine regimens may need to be boosted in African populations to achieve efficient immunity.
Trial registration. Registration is not required for observational studies.
Funding. This study was funded by Canada’s Global Health Research Initiative, Defense Threat Reduction Agency, National Institute of Allergy and Infectious Diseases, Bill & Melinda Gates Foundation, and United States Agency for International Development.
Access to timely and accurate diagnostic tests has a significant impact in the management of diseases of global concern such as malaria. While molecular diagnostics satisfy this need effectively in developed countries, barriers in technology, reagent storage, cost and expertise have hampered the introduction of these methods in developing countries. In this study a simple, lab-on-chip PCR diagnostic was created for malaria that overcomes these challenges.
The platform consists of a disposable plastic chip and a low-cost, portable, real-time PCR machine. The chip contains a desiccated hydrogel with reagents needed for Plasmodium specific PCR. Chips can be stored at room temperature and used on demand by rehydrating the gel with unprocessed blood, avoiding the need for sample preparation. These chips were run on a custom-built instrument containing a Peltier element for thermal cycling and a laser/camera setup for amplicon detection.
This diagnostic was capable of detecting all Plasmodium species with a limit of detection for Plasmodium falciparum of 2 parasites/μL of blood. This exceeds the sensitivity of microscopy, the current standard for diagnosis in the field, by ten to fifty-fold. In a blind panel of 188 patient samples from a hyper-endemic region of malaria transmission in Uganda, the diagnostic had high sensitivity (97.4%) and specificity (93.8%) versus conventional real-time PCR. The test also distinguished the two most prevalent malaria species in mixed infections, P. falciparum and Plasmodium vivax. A second blind panel of 38 patient samples was tested on a streamlined instrument with LED-based excitation, achieving a sensitivity of 96.7% and a specificity of 100%.
These results describe the development of a lab-on-chip PCR diagnostic from initial concept to ready-for-manufacture design. This platform will be useful in front-line malaria diagnosis, elimination programmes, and clinical trials. Furthermore, test chips can be adapted to detect other pathogens for a differential diagnosis in the field. The flexibility, reliability, and robustness of this technology hold much promise for its use as a novel molecular diagnostic platform in developing countries.
Human immunodeficiency virus type 1 (HIV-1) prevalence and incidence in the fishing communities on Lake Victoria in Uganda are high. This population may play a role in driving the HIV epidemic in Uganda including the spread of transmitted drug resistance (TDR). We report data on TDR in this population among antiretroviral (ARV)-naive, recently infected individuals about 5 years after ARV scaling-up in Uganda. We identified phylogenetic transmission clusters and combined these with volunteer life histories in order to understand the sexual networks within this population. From a prospective cohort of 1,000 HIV-negative individuals recruited from five communities, 51 seroconverters were identified over a period of 2 years. From these, whole blood was collected and population sequencing of the HIV-1 pol gene (protease/reverse transcriptase) was performed from plasma. Drug resistance mutations (DRMs) were scored using the 2009 WHO list for surveillance of TDR. TDR prevalence categories were estimated using the WHO recommended truncated sampling technique for the surveillance of TDR for use in resource-limited settings (RLS). Of the samples 92% (47/51) were successfully genotyped. HIV-1 subtype frequencies were 15/47 (32%) A1, 20/47 (43%) D, 1/47 (2%) C, 1/47 (2%) G, and 10/47 (21%) unique recombinant forms. Nonnucleoside reverse transcriptase inhibitor (NNRTI) drug resistance mutation K103N was identified in two individuals and V106A in one (6%) suggesting that the level of TDR was moderate in this population. No nucleoside/tide reverse transcriptase inhibitor (NRTI) or protease inhibitor (PI) DRMs were detected. In this study, we identified five transmission clusters supported by high bootstrap values and low genetic distances. Of these, one pair included the two individuals with K103N. Two of the genotypic clusters corresponded with reported sexual partnerships as detected through prior in-depth interviews. The level of TDR to NNRTIs in these ARV-naive individuals was moderate by WHO threshold survey categorization. The transmission clusters suggest a high degree of sexual partner mixing between members of these communities.
Although the An. funestus group conceals one of the major malaria vectors in Africa, little is known about the dynamics of members of this group across the continent. Here, we investigated the species composition, infection rate and susceptibility to insecticides of this species group in Uganda.
Indoor resting blood-fed Anopheles adult female mosquitoes were collected from 3 districts in Uganda. Mosquitoes morphologically belonging to the An. funestus group were identified to species by PCR. The sporozoite infection rates were determined by TaqMan and a nested PCR. Susceptibility to major insecticides was assessed using WHO bioassays. The potential role of four candidate resistance genes was assessed using qRT-PCR.
An. funestus s.s. and An. parensis, were the only members of the An. funestus group identified. Both species were sympatric in Masindi (North-West), whereas only An. parensis was present in Mityana (Central) and Ntungamo (South-West). The Plasmodium falciparum infection detected in An. parensis (4.2%) by TaqMan could not be confirmed by nested PCR, whereas the 5.3% infection in An. funestus s.s. was confirmed. An. parensis was susceptible to most insecticides, however, a moderate resistance was observed against deltamethrin and DDT. In the sympatric population of Masindi, resistance was observed to pyrethroids (permethrin and deltamethrin) and DDT, but all the resistant mosquitoes belonged to An. funestus s.s. No significant over-expression was observed for the four P450 candidate genes CYP6M7, CYP9K1, CYP6P9 and CYP6AA4 between deltamethrin resistant and control An. parensis. However, when compared with the susceptible FANG An. funestus s.s strain, the CYP9K1 is significantly over-expressed in An. parensis (15-fold change; P < 0.001), suggesting it could play a role in the deltamethrin resistance.
The contrasting infection rates and insecticide susceptibility profiles of both species highlights the importance of accurate species identification for successful vector control programs.
An. parensis; An. funestus; Malaria; Insecticide resistance; Vector control; Uganda
We report on the adherence experience of a group of people living with HIV on ART over six years in Uganda.
Between 2005 and 2009, we followed up 41 participants who were also part of a clinical trial comparing home and facility based delivery of ART in Jinja, eastern Uganda. We conducted qualitative in-depth interviews at enrolment, 3, 6, 18 and 30 months to capture experiences with adherence over time. In 2011 we returned to these participants to find out how they were fairing with long term adherence. We managed to retrace 24 participants and interviewed them about their experience. We thematically analysed the data and compared findings over time.
Initially there were few barriers to adherence and many followed the adherence guidance closely. By year six, relaxation of these rules was noticeable although self-reported adherence continued to be high. Alcohol consumption was more common than before. Some relatives of the participants who had died claimed that some deaths were a result of alcohol. While participants reported that ART had allowed them to reclaim independence and return to work the changes in work and social routines created new challenges for adherence. Side effects like lipodystrophy were not only causing some stigma but for some tested their faith in the drugs. Many participants reported resumption of sexual lives but apart from those who selected same status partners, disclosure to new partners was minimal.
Good adherence practice to ART wanes over the long-term, and people who may have disclosed at initiation find it difficult to do so to new partners once they are healthy. Further adherence interventions and support with disclosure over the course of therapy may need to be considered. (Words: 283)
Few women in Uganda access intermittent preventive treatment of malaria in pregnancy (IPTp) with sulfadoxine-pyrimethamine (SP). Previous studies have shown that high costs, frequent stock-out of drugs, supplies and poor quality of care are the greatest hindrance for women to access health services. In order to increase adherence to IPTp, we conceptualised an intervention that offset delivery care costs through providing a mama kit, created awareness on health benefits of IPTp and built trust between the provider and the client.
The new strategy was conceived along four constructs namely: 1) creating awareness by training midwives to explain the benefits of SP and the importance of adhering to the two doses of SP as IPTp to all pregnant women who attended ANC and consented to the study. Midwives were trained for two days in customer care and to provide a friendly environment. The pregnant women were also informed of the benefits of attending ANC and delivering at health facilities. 2) Each woman was promised a mama kit during ANC; 3) trust was built by showing the mama kit to each woman and branding it with her name; 4) keeping the promise by providing the mama kit when women came to deliver. The strategy to increase adherence to two doses of SP and encourage women to deliver at health facilities was implemented at two health facilities in Mukono district (Kawolo hospital and Mukono health centre IV). The inclusion criteria were women who: i) consented to the study and ii) were in the second trimester of pregnancy. All pregnant women in the second trimester (4-6 months gestation) who attended ANC and consented to participate in the study were informed of the benefits of SP, the importance of delivering at health facilities, were advised to attend the scheduled visits, promised a mama kit and ensured the kit was available at delivery. The primary outcome was the proportion of pregnant women adhering to a two dose SP regimen.
A total of 2,276 women received the first dose of SP and 1,656 (72.8%) came back for the second dose. 1,069 women were involved in the evaluation (384 had participated in the intervention while 685 had not). The main reasons that enabled those who participated in the intervention to adhere to the two doses of IPTp and deliver at the study facilities were: an explanation provided on the benefits of IPTp and delivering at health facilities (25.1%), availability of a mama kit at delivery (24.6%), kind midwives (19.8%) and fearing complications of pregnancy (8.5%). Overall, 78.0% of these women reported that they were influenced to adhere to IPTp by the intervention. In a multivariable regression, nearby facility, P = 0. 007, promising a mama kit, P = 0.002, kind midwives, P = 0.0001 and husbands’ encouragement, P = 0.0001 were the significant factors influencing adherence to IPTp with SP.
The new strategy was a good incentive for women to attend scheduled ANC visits, adhere to IPTp and deliver at the study facilities. Policy implications include the urgent need for developing a motivation package based on the Health-Trust Model to increase access and adherence to IPTp.
Malaria in pregnancy; IPTp; Adherence; Mama kit; Health-Trust Model; Uganda
Stigma is a barrier to HIV prevention and treatment. There is a limited understanding of the types of stigma facing people living with HIV (PLHIV) on antiretroviral therapy (ART). We describe the stigma trajectories of PLHIV over a 5-year period from the time they started ART.
Longitudinal qualitative in-depth interviews were conducted with 41 members of The AIDS Support Organisation (TASO) from 2005 to 2008 in Jinja, Uganda, who were part of a pragmatic cluster-randomised trial comparing two different modes of ART delivery (facility and home). Participants were stratified by gender, ART delivery arm and HIV stage (early or advanced) and interviewed at enrolment on to ART and then after 3, 6, 18 and 30 months. Interviews focused on stigma and ART experiences. In 2011, follow-up interviews were conducted with 24 of the participants who could be traced. Transcribed texts were translated, coded and analyzed thematically.
Stigma was reported to be very high prior to starting ART, explained by visible signs of long-term illnesses and experiences of discrimination and abuse. Early coping strategies included: withdrawal from public life, leaving work due to ill health and moving in with relatives. Starting ART led to a steady decline in stigma and allowed the participants to take control of their illness and manage their social lives. Better health led to resumption of work and having sex but led to reduced disclosure to employers, colleagues and new sexual partners. Some participants mentioned sero-sorting in order to avoid questions around HIV sero-status. A rise in stigma levels during the 18 and 30 month interviews may be correlated with decreased disclosure. By 2011, ART-related stigma was even more pronounced particularly among those who had started new sexual relationships, gained employment and those who had bodily signs from ART side-effects.
This study has shown that while ART comes with health benefits which help individuals to get rid of previously stigmatising visible signs, an increase in stigma may be noticed after about five years on ART, leading to reduced disclosure. ART adherence counselling should reflect changing causes and manifestations of stigma over time.
Stigma; Antiretroviral therapy; Disclosure; Uganda; Side effects; Sero-sorting
Malaria is a public health problem in Uganda; affecting mainly women and children. Effective treatment has been hampered by over-diagnosis and over-treatment with anti-malarial drugs among patients presenting with fever. In order to understand the effect of drug pressure on sulfadoxine-pyrimethamine (SP) resistance in pregnancy, a sample of pregnant women presenting with fever in out–patient clinics was studied. The main objective was to assess prescription patterns and drug use in pregnancy especially SP; and draw implications on the efficacy of SP for intermittent preventive treatment of malaria in pregnancy (IPTp).
A total of 998 pregnant women with a history of fever were interviewed and blood samples taken for diagnosis of malaria and HIV infections. Data were captured on the drugs prescribed for the current febrile episode and previous use of drugs especially SP, anti-retroviral drugs (ARVs) and cotrimoxazole.
Few pregnant women, 128 (12.8%) were parasitaemic for P.falciparum; and of these, 72 (56.3%) received first-line treatment with Artemether-lumefantrine (Coartem®) 14 (10.9%) SP and 33 (25.8%) quinine. Of the parasite negative patients (non-malarial fevers), 186 (21.4%) received Coartem, 423 (48.6%) SP and 19 (2.1%) cotrimoxazole. Overall, malaria was appropriately treated in 35.5% of cases. Almost all febrile pregnant women, 91.1%, were sleeping under a mosquito net. The majority of them, 911 (91.3%), accepted to have an HIV test done and 92 (9.2%) were HIV positive. Of the HIV positive women, 23 (25.0%) were on ARVs, 10 (10.9%) on cotrimoxazole and 30 (32.6%) on SP. A significant proportion of women, 40 (43.5%), were on both SP and cotrimoxazole. Age and occupation were associated with diagnosis and treatment of malaria and HIV infections.
There is inappropriate treatment of malaria and non-malarial fevers among pregnant women in these facilities. This is due to non-adherence to the guidelines. Over-prescription and use of anti-malarial drugs, especially SP may have implications on resistance against SP for malaria prevention in pregnancy. The policy implications of these findings are to evaluate SP efficacy as IPTp; and the need to enforce adherence to the current clinical treatment guidelines.
Malaria; Pregnant women; Appropriate treatment; SP; Uganda
Although international guidelines recommend initiating antiretroviral therapy (ART) when a patient’s CD4 cell count is ≤350 cells/μL, most patients in resource-limited settings present with much lower CD4 cell counts. The lowest level that their CD4 cell count reaches, the nadir, may have long-term consequences in terms of mortality. We examined this health state in a large cohort of HIV+ patients in Uganda.
This was an observational study of HIV patients in Uganda aged 14 years or older, who were enrolled in 10 major clinics across Uganda.
We assessed the CD4 nadir of patients, using their CD4 cell count at initiation of ART, stratified into categories (,50, 50–99, 100–149, 150–249, 250+ cells/μL). We constructed Kaplan–Meier curves to assess the differences in survivorship for patients left-censored at 1 year and 2 years after treatment initiation. We used Cox proportional hazards regression to model the associations between CD4 nadir and mortality. We adjusted mortality for loss-to-follow-up.
Of 22,315 patients, 20,129 patients had greater than 1 year of treatment follow-up. Among these patients, 327 (1.6%) died and 444 (2.2%) were lost to follow-up. After left-censoring at one year, relative to lowest CD4 strata, patients with higher CD4 counts had significantly lower rates of mortality (CD4 150–249, hazard ratio [HR] 0.60, 95% confidence interval [CI]: 0.45–0.82, P = 0.001; 250+, HR 0.66, 95% CI, 0.44–1.00, P = −0.05). Male sex, older age, and duration of time on ART were independently associated with mortality. When left-censoring at 2 years, CD4 nadir was no longer statistically significantly associated with mortality.
After surviving for 1 year on ART, a CD4 nadir was strongly predictive of longer-term mortality among patients in Uganda. This should argue for efforts to increase engagement with patients to ensure a higher CD4 nadir at initiation of treatment.
antiretroviral therapy; ART; CD4; prognosis; sub-Saharan Africa
Ed Mills and colleagues argue that a more balanced approach to gender is needed so that both men and women are involved in HIV treatment and prevention.
Because men in Africa are less likely to access HIV/AIDS care than women, we aimed to determine if men have differing outcomes from women across a nationally representative sample of adult patients receiving combination antiretroviral therapy in Uganda.
We estimated survival distributions for adult male and female patients using Kaplan-Meier, and constructed multivariable regressions to model associations of baseline variables with mortality. We assessed person-years of life lost up to age 55 by sex. To minimize the impact of patient attrition, we assumed a weighted 30% mortality rate among those lost to follow up.
We included data from 22,315 adults receiving antiretroviral therapy. At baseline, men tended to be older, had lower CD4 baseline values, more advanced disease, had pulmonary tuberculosis and had received less treatment follow up (all at p < 0.001). Loss to follow up differed between men and women (7.5 versus 5.9%, p < 0.001). Over the period of study, men had a significantly increased risk of death compared with female patients (adjusted hazard ratio 1.43, 95% CI 1.31-1.57, p < 0.001). The crude mortality rate for males differed importantly from females (43.9, 95% CI 40.7-47.0/1000 person-years versus 26.9, 95% CI 25.4-28.5/1000 person years, p < 0.001). The probability of survival was 91.2% among males and 94.1% among females at 12 months. Person-years of life lost was lower for females than males (689.7 versus 995.9 per 1000 person-years, respectively).
In order to maximize the benefits of antiretroviral therapy, treatment programmes need to be gender sensitive to the specific needs of both women and men. Particular efforts are needed to enroll men earlier into care.
As immune compromised HIV sero-positive people regain health after initiating antiretroviral treatment (ART), they may seek a return to an active 'normal' life, including sexual activity. The aim of the paper is to explore the changing sexual desires and behaviour of people on ART in Uganda over a 30 month period.
This study employed longitudinal qualitative interviews with forty people starting ART. The participants received their ART, adherence education and counselling support from The AIDS Support Organisation (TASO). The participants were selected sequentially as they started ART, stratified by sex, ART delivery mode (clinic or home-based) and HIV progression stage (early or advanced) and interviewed at enrolment, 3, 6, 18 and 30 months of their ART use.
Sexual desire changed over time with many reporting diminished desire at 3 and 6 months on ART compared to 18 and 30 months of use. The reasons for remaining abstinent included fear of superinfection or infecting others, fear that engaging in sex would awaken the virus and weaken them and a desire to adhere to the counsellors' health advice to remain abstinent. The motivations for resumption of sexual activity were: for companionship, to obtain material support, social norms around marriage, desire to bear children as well as to satisfy sexual desires. The challenges for most of the participants were using condoms consistently and finding a suitable sexual partner (preferably someone with a similar HIV serostatus) who could agree to have a sexual relationship with them and provide for their material needs.
These findings point to the importance of tailoring counselling messages to the changing realities of the ART users' cultural expectations around child bearing, marriage and sexual desire. People taking ART require support so they feel comfortable to disclose their HIV status to sexual partners.
Sexual desire; ART; HIV; Longitudinal; Sexual behaviour
Adolescents have been identified as a high-risk group for poor adherence to and defaulting from combination antiretroviral therapy (cART) care. However, data on outcomes for adolescents on cART in resource-limited settings remain scarce.
We developed an observational study of patients who started cART at The AIDS Service Organization (TASO) in Uganda between 2004 and 2009. Age was stratified into three groups: children (≤10 years), adolescents (11–19 years), and adults (≥20 years). Kaplan-Meier survival curves were generated to describe time to mortality and loss to follow-up, and Cox regression used to model associations between age and mortality and loss to follow-up. To address loss to follow up, we applied a weighted analysis that assumes 50% of lost patients had died.
A total of 23,367 patients were included in this analysis, including 810 (3.5%) children, 575 (2.5%) adolescents, and 21 982 (94.0%) adults. A lower percentage of children (5.4%) died during their cART treatment compared to adolescents (8.5%) and adults (10%). After adjusting for confounding, other features predicted mortality than age alone. Mortality was higher among males (p<0.001), patients with a low initial CD4 cell count (p<0.001), patients with advanced WHO clinical disease stage (p<0.001), and shorter duration of time receiving cART (p<0.001). The crude mortality rate was lower for children (22.8 per 1000 person-years; 95% CI: 16.1, 29.5), than adolescents (36.5 per 1000 person-years; 95% CI: 26.3, 46.8) and adults (37.5 per 1000 person-years; 95% CI: 35.9, 39.1).
This study is the largest assessment of adolescents receiving cART in Africa. Adolescents did not have cART mortality outcomes different from adults or children.
People living with HIV who are taking antiretroviral therapy (ART) are increasingly involved in ‘positive prevention’ initiatives. These are generally oriented to promoting abstinence, ‘being faithful’ (partner reduction) and condom use (ABC). We conducted a longitudinal qualitative study with people living with HIV using ART, who were provided with adherence education and counselling support by a Ugandan nongovernmental organisation, The AIDS Support Organisation (TASO). Forty people were selected sequentially as they started ART, stratified by sex, ART delivery mode (clinic- or home-based) and HIV progression stage (early or advanced) and interviewed at enrolment and at 3, 6, 18 and 30 months. At initiation of ART, participants agreed to follow TASO's positive-living recommendations. Initially poor health prevented sexual activity. As health improved, participants prioritised resuming economic production and support for their children. With further improvements, sexual desire resurfaced and people in relationships cemented these via sex. The findings highlight the limitations of HIV prevention based on medical care/personal counselling. As ART leads to health improvements, social norms, economic needs and sexual desires increasingly influence sexual behaviour. Positive prevention interventions need to seek to modify normative and economic influences on sexual behaviour, as well as to provide alternatives to condoms.
HIV/AIDS; anti-retroviral therapy; sexual behaviour; Uganda
We examined the association between density of healthcare providers and patient outcomes using a large nationally representative cohort of patients receiving combination antiretroviral therapy (cART) in Uganda.
We obtained data from The AIDS Support Organization (TASO) in Uganda. Patients 18 years of age and older who initiated cART at TASO between 2004 and 2008 contributed to this analysis. The number of healthcare providers per 100 patients, the number of patients lost to follow-up per 100 person years and number of deaths per 100 person years were calculated. Spearman correlation was used to identify associations between patient loss to follow-up and mortality with the healthcare provider-patient ratios.
We found no significant associations between the number of patients lost to follow-up and physicians (p = 0.45), nurses (p = 0.93), clinical officers (p = 0.80), field officers (p = 0.56), and healthcare providers overall (p = 0.83). Similarly, no significant associations were observed between mortality and physicians (p = 0.65), nurses (p = 0.49), clinical officers (p = 0.73), field officers (p = 0.78), and healthcare providers overall (p = 0.73).
Patient outcomes, as measured by loss to follow-up and mortality, were not significantly associated with the number of doctors, nurses, clinical officers, field officers, or healthcare providers overall. This may suggest that that other factors, such as the presence of volunteer patient supporters or broader political or socioeconomic influences, may be more closely associated with outcomes of care among patients on cART in Uganda.
The Comprehensive T Cell Vaccine Immune Monitoring Consortium (CTC-VIMC) was created to provide standardized immunogenicity monitoring services for HIV vaccine trials. The ex vivo interferon-gamma (IFN-γ) ELISpot is used extensively as a primary immunogenicity assay to assess T cell-based vaccine candidates in trials for infectious diseases and cancer. Two independent, GCLP-accredited central laboratories of CTC-VIMC routinely use their own standard operating procedures (SOPs) for ELISpot within two major networks of HIV vaccine trials. Studies are imperatively needed to assess the comparability of ELISpot measurements across laboratories to benefit optimal advancement of vaccine candidates.
We describe an equivalence study of the two independently qualified IFN-g ELISpot SOPs. The study design, data collection and subsequent analysis were managed by independent statisticians to avoid subjectivity. The equivalence of both response rates and positivity calls to a given stimulus was assessed based on pre-specified acceptance criteria derived from a separate pilot study.
Detection of positive responses was found to be equivalent between both laboratories. The 95% C.I. on the difference in response rates, for CMV (−1.5%, 1.5%) and CEF (−0.4%, 7.8%) responses, were both contained in the pre-specified equivalence margin of interval [−15%, 15%]. The lower bound of the 95% C.I. on the proportion of concordant positivity calls for CMV (97.2%) and CEF (89.5%) were both greater than the pre-specified margin of 70%. A third CTC-VIMC central laboratory already using one of the two SOPs also showed comparability when tested in a smaller sub-study.
The described study procedure provides a prototypical example for the comparison of bioanalytical methods in HIV vaccine and other disease fields. This study also provides valuable and unprecedented information for future vaccine candidate evaluations on the comparison and pooling of ELISpot results generated by the CTC-VIMC central core laboratories.
The development of a rapid and efficient system to identify human immunodeficiency virus type 1 (HIV-1)-infected individuals with broad and potent HIV-1-specific neutralizing antibody responses is an important step toward the discovery of critical neutralization targets for rational AIDS vaccine design. In this study, samples from HIV-1-infected volunteers from diverse epidemiological regions were screened for neutralization responses using pseudovirus panels composed of clades A, B, C, and D and circulating recombinant forms (CRFs). Initially, 463 serum and plasma samples from Australia, Rwanda, Uganda, the United Kingdom, and Zambia were screened to explore neutralization patterns and selection ranking algorithms. Samples were identified that neutralized representative isolates from at least four clade/CRF groups with titers above prespecified thresholds and ranked based on a weighted average of their log-transformed neutralization titers. Linear regression methods selected a five-pseudovirus subset, representing clades A, B, and C and one CRF01_AE, that could identify top-ranking samples with 50% inhibitory concentration (IC50) neutralization titers of ≥100 to multiple isolates within at least four clade groups. This reduced panel was then used to screen 1,234 new samples from the Ivory Coast, Kenya, South Africa, Thailand, and the United States, and 1% were identified as elite neutralizers. Elite activity is defined as the ability to neutralize, on average, more than one pseudovirus at an IC50 titer of 300 within a clade group and across at least four clade groups. These elite neutralizers provide promising starting material for the isolation of broadly neutralizing monoclonal antibodies to assist in HIV-1 vaccine design.
Identification of new ways to increase access to antiretroviral therapy in Africa is an urgent priority. We assessed whether home-based HIV care was as effective as was facility-based care.
We undertook a cluster-randomised equivalence trial in Jinja, Uganda. 44 geographical areas in nine strata, defined according to ratio of urban and rural participants and distance from the clinic, were randomised to home-based or facility-based care by drawing sealed cards from a box. The trial was integrated into normal service delivery. All patients with WHO stage IV or late stage III disease or CD4-cell counts fewer than 200 cells per μL who started antiretroviral therapy between Feb 15, 2005, and Dec 19, 2006, were eligible, apart from those living on islands. Follow-up continued until Jan 31, 2009. The primary endpoint was virological failure, defined as RNA more than 500 copies per mL after 6 months of treatment. The margin of equivalence was 9% (equivalence limits 0·69–1·45). Analyses were by intention to treat and adjusted for baseline CD4-cell count and study stratum. This trial is registered at http://isrctn.org, number ISRCTN 17184129.
859 patients (22 clusters) were randomly assigned to home and 594 (22 clusters) to facility care. During the first year, 93 (11%) receiving home care and 66 (11%) receiving facility care died, 29 (3%) receiving home and 36 (6%) receiving facility care withdrew, and 8 (1%) receiving home and 9 (2%) receiving facility care were lost to follow-up. 117 of 729 (16%) in home care had virological failure versus 80 of 483 (17%) in facility care: rates per 100 person-years were 8·19 (95% CI 6·84–9·82) for home and 8·67 (6·96–10·79) for facility care (rate ratio [RR] 1·04, 0·78–1·40; equivalence shown). Two patients from each group were immediately lost to follow-up. Mortality rates were similar between groups (0·95 [0·71–1·28]). 97 of 857 (11%) patients in home and 75 of 592 (13%) in facility care were admitted at least once (0·91, 0·64–1·28).
This home-based HIV-care strategy is as effective as is a clinic-based strategy, and therefore could enable improved and equitable access to HIV treatment, especially in areas with poor infrastructure and access to clinic care.
US Centers for Disease Control and Prevention and UK Medical Research Council.
In many HIV programmes in Africa, patients are assessed clinically and prepared for antiretroviral treatment over a period of 4–12 weeks. Mortality rates following initiation of ART are very high largely because patients present late with advanced disease. The rates of mortality and retention during the pre-treatment period are not well understood. We conducted an observational study to determine these rates.
HIV-infected subjects presenting at The AIDS Support Clinic in Jinja, SE Uganda, were assessed for antiretroviral therapy (ART). Eligible subjects were given information and counselling in 3 visits done over 4–6 weeks in preparation for treatment. Those who did not complete screening were followed-up at home. Survival analysis was done using poisson regression.
4321 HIV-infected subjects were screened of whom 2483 were eligible for ART on clinical or immunological grounds. Of these, 637 (26%) did not complete screening and did not start ART. Male sex and low CD4 count were associated independently with not completing screening. At follow-up at a median 351 days, 181 (28%) had died, 189 (30%) reported that they were on ART with a different provider, 158 (25%) were alive but said they were not on ART and 109 (17%) were lost to follow-up. Death rates (95% CI) per 100 person-years were 34 (22, 55) (n.18) within one month and 37 (29, 48) (n.33) within 3 months. 70/158 (44%) subjects seen at follow-up said they had not started ART because they could not afford transport.
About a quarter of subjects eligible for ART did not complete screening and pre-treatment mortality was very high even though patients in this setting were well informed. For many families, the high cost of transport is a major barrier preventing access to ART.
The gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) assay is used routinely to evaluate the potency of human immunodeficiency virus (HIV) vaccine candidates and other vaccine candidates. In order to compare candidates and pool data from multiple trial laboratories, validated standardized methods must be applied across laboratories. Proficiency panels are a key part of a comprehensive quality assurance program to monitor inter- and intralaboratory performance, as well as assay performance, over time. Seven International AIDS Vaccine Initiative-sponsored trial sites participated in the proficiency panels described in this study. At each laboratory, two operators independently processed identical sample sets consisting of frozen peripheral blood mononuclear cell (PBMC) samples from different donors by using four blind stimuli. PBMC recovery and viability after overnight resting and the IFN-γ ELISPOT assay performance were assessed. All sites demonstrated good performance in PBMC thawing and resting, with a median recovery of 78% and median viability of 95%. The laboratories were able to detect similar antigen-specific T-cell responses, ranging from 50 to >3,000 spot-forming cells per million PBMC. An approximate range of a half log in results from operators within or across sites was seen in comparisons of antigen-specific responses. Consistently low background responses were seen in all laboratories. The results of these proficiency panels demonstrate the ability of seven laboratories, located across three continents, to process PBMC samples and to rank volunteers with differential magnitudes of IFN-γ ELISPOT responses. These findings also illustrate the ability to standardize the IFN-γ ELISPOT assay across multiple laboratories when common training methods, reagents such as fetal calf serum, and standard operating procedures are adopted. These results are encouraging for laboratories that are using cell-based immunology assays to test HIV vaccines and other vaccines.
Clinical laboratory reference intervals have not been established in many African countries, and non-local intervals are commonly used in clinical trials to screen and monitor adverse events (AEs) among African participants. Using laboratory reference intervals derived from other populations excludes potential trial volunteers in Africa and makes AE assessment challenging. The objective of this study was to establish clinical laboratory reference intervals for 25 hematology, immunology and biochemistry values among healthy African adults typical of those who might join a clinical trial.
Methods and Findings
Equal proportions of men and women were invited to participate in a cross sectional study at seven clinical centers (Kigali, Rwanda; Masaka and Entebbe, Uganda; two in Nairobi and one in Kilifi, Kenya; and Lusaka, Zambia). All laboratories used hematology, immunology and biochemistry analyzers validated by an independent clinical laboratory. Clinical and Laboratory Standards Institute guidelines were followed to create study consensus intervals. For comparison, AE grading criteria published by the U.S. National Institute of Allergy and Infectious Diseases Division of AIDS (DAIDS) and other U.S. reference intervals were used. 2,990 potential volunteers were screened, and 2,105 (1,083 men and 1,022 women) were included in the analysis. While some significant gender and regional differences were observed, creating consensus African study intervals from the complete data was possible for 18 of the 25 analytes. Compared to reference intervals from the U.S., we found lower hematocrit and hemoglobin levels, particularly among women, lower white blood cell and neutrophil counts, and lower amylase. Both genders had elevated eosinophil counts, immunoglobulin G, total and direct bilirubin, lactate dehydrogenase and creatine phosphokinase, the latter being more pronounced among women. When graded against U.S.-derived DAIDS AE grading criteria, we observed 774 (35.3%) volunteers with grade one or higher results; 314 (14.9%) had elevated total bilirubin, and 201 (9.6%) had low neutrophil counts. These otherwise healthy volunteers would be excluded or would require special exemption to participate in many clinical trials.
To accelerate clinical trials in Africa, and to improve their scientific validity, locally appropriate reference ranges should be used. This study provides ranges that will inform inclusion criteria and evaluation of adverse events for studies in these regions of Africa.
An understanding of the health of potential volunteers in Africa is essential for the safe and efficient conduct of clinical trials, particularly for trials of preventive technologies such as vaccines that enroll healthy individuals. Clinical safety laboratory values used for screening, enrolment and follow-up of African clinical trial volunteers have largely been based on values derived from industrialized countries in Europe and North America. This report describes baseline morbidity during recruitment for a multi-center, African laboratory reference intervals study.
Asymptomatic persons, aged 18–60 years, were invited to participate in a cross-sectional study at seven sites (Kigali, Rwanda; Masaka and Entebbe, Uganda; Kangemi, Kenyatta National Hospital and Kilifi, Kenya; and Lusaka, Zambia). Gender equivalency was by design. Individuals who were acutely ill, pregnant, menstruating, or had significant clinical findings were not enrolled. Each volunteer provided blood for hematology, immunology, and biochemistry parameters and urine for urinalysis. Enrolled volunteers were excluded if found to be positive for HIV, syphilis or Hepatitis B and C. Laboratory assays were conducted under Good Clinical Laboratory Practices (GCLP).
Results and Conclusions
Of the 2990 volunteers who were screened, 2387 (80%) were enrolled, and 2107 (71%) were included in the analysis (52% men, 48% women). Major reasons for screening out volunteers included abnormal findings on physical examination (228/603, 38%), significant medical history (76, 13%) and inability to complete the informed consent process (73, 13%). Once enrolled, principle reasons for exclusion from analysis included detection of Hepatitis B surface antigen (106/280, 38%) and antibodies against Hepatitis C (95, 34%). This is the first large scale, multi-site study conducted to the standards of GCLP to describe African laboratory reference intervals applicable to potential volunteers in clinical trials. Approximately one-third of all potential volunteers screened were not eligible for analysis; the majority were excluded for medical reasons.