Evaluation of HIV testing strategies and monitoring of immune responses in HIV-vaccinated individuals in Tanzania

This thesis describes studies on the evaluation of human immunodeficiency virus (HIV) enzyme-linked immunosorbent assays (ELISAs) and simple rapid HIV assays for use in HIV testing strategies in resource-limited settings and studies of HIV vaccine-induced immune responses. Peripheral blood mononuclear cell (PBMC) preparation techniques were also studied in preparation for use in the HIV vaccine trials. The performance of two antibody ELISAs (Vironostika Uni-Form II plus O and Enzygnost anti-HIV-1/2 Plus) and two new diagnostic HIV antigen/antibody combination ELISAs (Murex and Vironostika HIV Uni-Form II antigen/antibody) was evaluated using 1380 serum samples from Tanzanian individuals (paper I). The sensitivity at initial testing was 100% for all assays except Vironostika Uni-Form II plus O which showed one false negative sample at initial testing but 100% sensitivity after repeat testing. The initial specificity was 99.8% for Enzygnost, 98.9% for each of the antigen/antibody ELISAs and 97.0% for Vironostika Plus O ELISA. An alternative confirmatory HIV testing strategy based on initial testing on any of the two antigen/antibody assays followed by testing of reactive samples on the Enzygnost anti-HIV-1/2 Plus assay gave 100% specificity (95% CI; 99.7-100%). The performance of five simple rapid HIV antibody assays was evaluated using 1433 whole blood samples (paper II). The sensitivity at initial testing of Determine, SD Bioline and Uni-Gold was 100% while First Response and Stat-Pak had a sensitivity of 99.5% and 97.7%, respectively, which increased to 100% on repeat testing. The initial specificity of the Uni-Gold assay was 100% while the specificities were 99.6%, 99.4%, 99.6% and 99.8% for Determine, SD Bioline, First Response and Stat-Pak assays, respectively. An alternative confirmatory HIV testing strategy based on initial testing on SD Bioline followed by testing of reactive samples on the Determine gave 100% sensitivity (95% CI; 99.1-100) and 100% specificity (95% CI; 96-99.1) with Uni-Gold as tiebreaker for discordant results and was adopted as a national algorithm in Tanzania. Standard Ficoll-Paque gradient (FIP) centrifugation, BD vacutainer cell preparation tube (CPT) and Greiner Bio-One LeucoSep tube techniques for PBMC preparation were evaluated (paper III). No differences in mean recovery or mean viability of fresh PBMCs were observed between FIP centrifugation and CPT techniques used in Stockholm. In Dar es Salaam, recovery and viability of PBMCs isolated by FIP technique was higher compared to CPT purified cells. LeucoSep cell separation gave a higher yield and viability than FIP cell separation. The cells purified by the different techniques at the two sites performed equally well in interferon-gamma (IFN-γ) enzyme-linked immunospot (ELISpot) assays. In a phase 1 HIV-1 DNA prime MVA boost vaccine trial in Sweden (HIVIS01/02), HIV-specific lymphoproliferative responses were tested by a [3H]-thymidine uptake assay and a flow-cytometric assay using whole blood (FASCIA-WB) (paper IV). A FASCIA using PBMC (FASCIA-PBMC) was also employed (n=14).Two weeks after the HIV-MVA boost 35 of 38 (92%) vaccinees were reactive by the thymidine uptake assay. Thirty-two of 38 (84%) vaccinees were reactive by the CD4+ T-cell FASCIA-WB, and 7 of 38 (18%) also exhibited CD8+ T-cell responses. There was strong correlation between the proliferative responses measured by the thymidine uptake assay and CD4+ T-cell FASCIA-WB (r=0.68; P < 0.01). Fourteen vaccinees were analyzed using all three assays. Ten of 14 (71%) and 11/14 (79%) demonstrated CD4+ T-cell responses in FASCIA-WB and FASCIA-PBMC, respectively. CD8+ T-cell reactivity was observed in 3/14 (21%) and 7/14 (50%) using the FASCIA-WB and FASCIA-PBMC, respectively. All 14 were reactive by the thymidine uptake assay. A FASCIA-PBMC, which allows simultaneous phenotyping, may be an option to the [3H] thymidine uptake assay for assessment of vaccine-induced T-cell proliferation, especially in isotope-restricted settings. In the HIVIS03 phase I/II HIV vaccine trial in Tanzania, sixty HIV-uninfected volunteers randomised to three groups of 20, received DNA plasmid vaccine 1 mg intradermally (id) or 3.8 mg intramuscularly (im) or placebo using a needle-free injection device (paper V). DNA plasmids vectoring HIV-1 genes gp160 subtypes A, B, C; rev B; p17/p24 gag A, B and Rtmut B were given at weeks 0, 4 and 12. Recombinant MVA (108 pfu) expressing HIV-1 Env, Gag, Pol of CRF01_AE or placebo was administered im at month 9 and 21. The vaccines were well tolerated. Two weeks after the first HIV-MVA boost 35/35 (100%) vaccinees had IFN-γ ELISpot responses; 35 (100%) to Gag and 31 (89%) to Env. Two to four weeks after the second HIV-MVA boost, 28/29 (97%) vaccinees had IFN-γ responses. The id -primed recipients had significantly higher responses to Env than im recipients after HIV-MVA boost. Intracellular cytokine staining for Gag-specific IFN-γ/IL-2 production showed both CD8+ and CD4+ T-cell responses. All vaccinees had HIV-specific lymphoproliferative responses. All vaccinees reacted in diagnostic HIV serological tests and 26/29 (90%) had antibodies against gp160 after the second HIV-MVA boost. A high neutralizing antibody response rate (31-83% depending on the clade B or AE virus tested) was demonstrated using a PBMC assay. In conclusion, this vaccine approach was safe and highly immunogenic

Seroprevalence of hepatitis B virus infection among antenatal clinic attendees at a tertiary hospital in Dar es Salaam, Tanzania

Hepatitis B virus (HBV) infection is a serious public health problem in sub-Saharan Africa. The risk of vertical transmission increases if the mother is hepatitis B surface antigen (HBsAg) positive and more so when she is also hepatitis B envelope antigen (HBeAg) positive. Since 2000, the World Health Organization recommends screening of pregnant women for HBV infection. However, this is not currently practiced in Tanzania. The objective of this study was to determine seroprevalence and associated factors of HBV infection among pregnant women attending antenatal clinic at the Muhimbili National Hospital, in Dar es Salaam, Tanzania. This cross-sectional study was conducted from August-September 2010. Data on socio-demographic characteristics, obstetric and risk factors such as history of blood transfusion, and risky sexual practices was collected.  Blood samples were tested for HBsAg, HBeAg, antibodies to HBsAg (Anti-HBs), IgM antibodies to hepatitis B core antigen (Anti-HBc) and anti-HIV antibodies. A total of 310 pregnant women (28.5±5.4 years) were enrolled in the study. Thirty-one percent (96) of the women were primigravidae. Most of studied women were of low educational status and married. The seroprevalence of HBsAg was 3.9% (12/310) and none had detectable anti-HBs. None had IgM HBcAb, thus excluding acute HBV infection. All women tested negative for HBeAg. The overall seroprevalence of HIV infection was 9.7%. Three of 12 (25%) women had HBV-HIV co-infection. None of the assessed associated factors were significantly related to HBV infection. In conclusion, the seroprevalence of HBsAg among women attending antenatal care at Muhimbili National Hospital is moderate. It is recommended to introduce routine antenatal screening for HBV and “at birth dose” vaccination to new born babies of mothers found to be HBsAg positive

Predictors of Bacterial Vaginosis among Pregnant Women Attending Antenatal Clinic at Tertiary Care Hospital in Tanzania: A Cross Sectional Study

Background: Bacterial vaginosis (BV) is one of the most common genital tract infections in pregnancy associated with an increased risk of pregnancy losses, maternal and perinatal morbidity and mortality. Different social behavioural and obstetric factors can contribute to the development of BV. Determining the predictors of BV could be the best way of identifying women at high risk of developing the disease.Methods: This was a cross-sectional study conducted between December 2017 and February 2018 to determine the prevalence and&nbsp; predictors of BV among pregnant women attending antenatal Clinic (ANC) at Muhimbili National Hospital (MNH), Tanzania. Participants&nbsp; were recruited using systematic random sampling. For each consented participant, a pretested questionnaire was filled, a pelvic&nbsp; examination was done and a sample was collected. BV was diagnosed using Nugent’s score. Data was analysed using Statistical Package for Social Scientists (SPSS) version 23.0. Bivariate and multivariate logistic regression analysis was done to determine factors that were independently associated with BV.Results: 178 (26.7%) pregnant women out of 667 were diagnosed positive for BV. In the bivariate analysis (Table 3), age (COR 1.71; 95% CI, 1.16-2.52), level of education (COR 4.08, 95% CI, 2.84-5.84), gravidity (COR, 1.52, 95% CI; 1.04-2.23), parity (COR 1.69, 95% CI; 1.18-2.42), vaginal douching (COR 2.89, 95% CI; 1.96-4.27), HIV status (COR 9.37, 95%CI; 4.12-21.28), history of STI (COR 2.49 95% CI; 1.46-4.25), LTSP (COR 2.76, 95% CI; 1.68-4.54) and age of first coitus (COR 3.19, 95% CI; 2.24-4.56) were significantly associated with BV. After adjusting for confounders in multivariate analysis, the following risk factors remained significantly associated with BV; age of 21- 29 years (AOR, 2.22, 95%CI; 1.45-3.49), primary education level (AOR 3.97, 95% CI; 2.63-5.98), vaginal douching (AOR 3.68, 95% CI; 2.35-5.76), HIV status (AOR 6.44, 95% CI; 2.62-15.88), STI infection (AOR 2.34, 95% CI; 1.25-4.37), more than one LTSP (AOR 2.69, 95% CI; 1.53-4.74) and age of less than 18 years of first coitus (AOR 2.16, 95% CI; 1.42-3.30).Conclusion: The prevalence of BV in pregnant women was found to be high. Age of less than 30 years, primary education level and below, vaginal douching, HIV infection, STI, more than one lifetime sexual partners and early age of sexual debut were found to be significant predictors of BV. The high prevalence of BV in our population should necessitate policy makers to include screening and treatment of BV in the future policy of antenatal care package, as BV is associated with significant maternal and neonatal morbidity and mortality. Women should also be educated on harmful effects of certain behavioural practices such as vaginal douching that predispose to BV. In addition symptoms of BV such as abnormal vaginal discharge during pregnancy are inconsistent, under reported and often overlooked. Therefore, a high-risk approach can be used for screening and treatment of asymptomatic women

Vitamin D Status and TB Treatment Outcomes in Adult Patients in Tanzania: A Cohort Study.

Vitamin D is an immunomodulator and can alter response to tuberculosis (TB) treatment, though randomised trials have been inconclusive to date. We present one of the first comprehensive analysis of the associations between vitamin D status and TB treatment, T-cell counts and nutritional outcomes by HIV status. Cohort study. Outpatient clinics in Tanzania. 25-hydroxyvitamin D levels were assessed in a cohort of 677 patients with TB (344 HIV infected) initiating anti-TB treatment at enrolment in a multivitamin supplementation (excluding vitamin D) trial (Clinicaltrials.gov identifier: NCT00197704). Information on treatment outcomes such as failure and relapse, HIV disease progression, T-cell counts and anthropometry was collected routinely, with a median follow-up of 52 and 30 months for HIV-uninfected and HIV-infected patients, respectively. Cox and binomial regression, and generalised estimating equations were used to assess the association of vitamin D status with these outcomes. Mean 25-hydroxyvitamin D concentrations at enrolment were 69.8 (±21.5) nmol/L (27.9 (±8.6) ng/mL). Vitamin D insufficiency (<75 nmol/L) was associated with a 66% higher risk of relapse (95% CI 4% to 164%; 133% higher risk in HIV-uninfected patients). Each unit higher 25-hydroxyvitamin D levels at baseline were associated with a decrease of 3 (p=0.004) CD8 and 3 (p=0.01) CD3 T-cells/µL during follow-up in patients with HIV infection. Vitamin D insufficiency was also associated with a greater decrease of body mass index (BMI; -0.21 kg/m(2); 95% CI -0.39 to -0.02), during the first 8 months of follow-up. No association was observed for vitamin D status with mortality or HIV disease progression. Adequate vitamin D status is associated with a lower risk of relapse and with improved nutritional indicators such as BMI in patients with TB, with or without HIV infection. Further research is needed to determine the optimal dose of vitamin D and effectiveness of daily vitamin D supplementation among patients with TB

Increased memory phenotypes of CD4+ and CD8+ T cells in children with sickle cell anaemia in Tanzania

Background: Infection is an important cause of morbidity in children with sickle cell anaemia (SCA). However, little is currently known regarding the spectrum of adaptive immune derangement in SCA, especially of populations in Sub-Saharan Africa. In this study, we investigated the phenotype and activation status of T and B lymphocytes among children with SCA in Tanzania.Methods: We compared 30 children with SCA aged 1–6 years in steady-state with 10 age-matched controls. We assessed white blood cell count, T and B lymphocyte phenotype and activation status using an automated haematology analyser and multiparameter Flow Cytometry.Results: In children with SCA, the absolute lymphocyte, monocyte and granulocyte counts were all increased. There was also an increase in proportion of central/transitional memory (42.4% vs. 33.3%, p = 0.0100), effector memory (7.8% vs. 5.4%, p = 0.0086) and terminally differentiated (2.3% vs. 1.3%, p = 0.0355) CD4+ T cells as well as effector memory CD8+ T cells (21.3% vs. 11.5%, p = 0.0060) in children with SCA. In contrast, there was no difference in naïve, classical memory, atypical memory and IgM memory B-cells between the two groups. The level of activation of both T and B cells were comparable between children with and without SCA. Furthermore, we observed a significant inverse correlation between frequency of the effector memory CD8+ T cells and haematocrit (Spearman rho = -0.3859, p = 0.0352). Conclusions: Children with SCA in Tanzania show an absolute increase in all leukocyte types, including lymphocytes, with skewing of both CD4+ and CD8+ T cells towards the memory phenotypes. These findings provide insights on the development of adaptive immunity which may have implications on vaccine responsiveness, allo-immunisation, auto-immune diseases and transplant immunology in children with SCA

Predominance of PVL-negative community-associated methicillin-resistant Staphylococcus aureus sequence type 8 in newly diagnosed HIV-infected adults, Tanzania

Difficult-to-treat infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are of concern in people living with HIV infection as they are more vulnerable to infection. We aimed to identify molecular characteristics of MRSA colonizing newly diagnosed HIV-infected adults in Tanzania. Individuals newly diagnosed with HIV infection were recruited in Dar es Salaam, Tanzania, from April 2017 to May 2018, as part of the randomized clinical trial CoTrimResist (ClinicalTrials.gov identifier: NCT03087890). Nasal/nasopharyngeal isolates of Staphylococcus aureus were susceptibility tested by disk diffusion method, and cefoxitin-resistant isolates were characterized by short-reads whole genome sequencing. Four percent (22/537) of patients carried MRSA in the nose/nasopharynx. MRSA isolates were frequently resistant towards gentamicin (95%), ciprofloxacin (91%), and erythromycin (82%) but less often towards trimethoprim-sulfamethoxazole (9%). Seventy-three percent had inducible clindamycin resistance. Erythromycin-resistant isolates harbored ermC (15/18) and LmrS (3/18) resistance genes. Ciprofloxacin resistance was mediated by mutations of the quinolone resistance-determining region (QRDR) sequence in the gyrA (S84L) and parC (S80Y) genes. All isolates belonged to the CC8 and ST8-SCCmecIV MRSA clone. Ninety-five percent of the MRSA isolates were spa-type t1476, and one exhibited spa-type t064. All isolates were negative for Panton-Valentine leucocidin (PVL) and arginine catabolic mobile element (ACME) type 1. All ST8-SCCmecIV-spa-t1476 MRSA clones from Tanzania were unrelated to the globally successful USA300 clone. Carriage of ST8 MRSA (non-USA300) was common among newly diagnosed HIV-infected adults in Tanzania. Frequent co-resistance to non-beta lactam antibiotics limits therapeutic options when infection occurs.publishedVersio

High rate of antimicrobial resistance and multiple mutations in the dihydrofolate reductase gene among Streptococcus pneumoniae isolated from HIV-infected adults in a community setting in Tanzania

Objectives The aim of this study was to characterize molecular mechanisms of resistance to trimethoprim and other antibiotics in Streptococcus pneumoniae isolates from HIV-infected adults in Dar es Salaam, Tanzania. Methods A total of 1877 nasopharyngeal swabs were collected and screened for pneumococcal colonization from 537 newly diagnosed individuals with HIV at four clinic visits during a 1-year follow-up from 2017–2018 as part of the randomized clinical trial CoTrimResist (ClinicalTrials.gov ID: NCT03087890). Results A total of 76 pneumococcal isolates were obtained. Of the 70 isolates that could be serotyped, 42 (60.0%) were vaccine serotypes included in pneumococcal conjugate vaccine 23 (PCV23). The majority of isolates (73.7%; 56/76) were non-susceptible to penicillin (MICs of 0.06–2 μg/mL). Isolates were frequently resistant to co-trimoxazole (trimethoprim/sulfamethoxazole) (71.1%) but less so to azithromycin (22.4%), erythromycin (21.1%), chloramphenicol (18.4%), tetracycline (14.5%), clindamycin (10.5%) and levofloxacin (0%). Moreover, 26.3% were multidrug-resistant (resistant to ≥3 antibiotic classes). Vaccine-type pneumococci were resistant to more classes of antibiotics, were more frequently resistant to erythromycin, azithromycin, clindamycin and tetracycline, and had higher MICs to penicillin (median, 0.19 μg/mL; range, 0.002–1.5 μg/mL) compared with non-vaccine serotypes (median, 0.125 μg/mL; range, 0.012–0.25 μg/mL) (P = 0.003). Co-trimoxazole-resistant isolates carried from 1 to 11 different mutations in the dihydrofolate reductase (DHFR) gene, most commonly Ile100Leu (100%), Glu20Asp (91.8%), Glu94Asp (61.2%), Leu135Phe (57.1%), His26Tyr (53.1%), Asp92Ala (53.1%) and His120Gln (53.1%). Conclusion Streptococcus pneumoniae isolated from HIV-diagnosed patients were frequently non-susceptible to penicillin and co-trimoxazole. Most isolates carried multiple mutations in DHFR.publishedVersio

Low prevalence of detectable serum cardiac troponin I among healthy Tanzanian adults: observational study

Background: Cardiac troponin test is used in detecting various heart disorders. The objective of this study was to establish normal reference levels for serum cardiac Troponin I which could be utilized for selection of vaccines and determine any electrocardiogram (EKG) changes among healthy volunteers.Methods: A total of 263 healthy blood donors from Dar es Salaam, Tanzania were included in this sub-study. A thorough medical history and physical examination to rule out any major chronic disease like heart failure, chronic kidney diseases, diabetes mellitus and HIV was undertaken.  Ten mL of blood sample for the purpose of establishing normal reference values for Troponin I assessment and parallel EKG was performed to all participants. Results: Of the 263 subjects, males were156 (59.3%) and females were 107 (40.7%). Median (range) age was 34 years old. The manufacture’s reference level for serum Cardiac Troponin I was 0.00-0.39 µg/L. Serum Cardiac Troponin I was detected in two blood donors (0.76%). However, their Troponin I levels were within the manufacturer’s normal range (0.01-0.36 µg/L).  Clinically both subjects were healthy and their EKG tracing were unremarkable.Conclusions: Our study has shown that among healthy subjects, detectable serum cardiac Troponin I is a rare finding. The manufacturer’s range is applicable in our setting and can be used in the ongoing vaccine trial. The significance of minimally elevated serum cardiac Troponin I may represent a subclinical cardiac injury and have important clinical implications, a hypothesis that should be tested in future longitudinal outcome studies

Risk Factors for Preterm Birth among HIV-Infected Tanzanian Women: A Prospective Study

Premature delivery, a significant cause of child mortality and morbidity worldwide, is particularly prevalent in the developing world. As HIV is highly prevalent in much of sub-Saharan Africa, it is important to determine risk factors for prematurity among HIV-positive pregnancies. The aims of this study were to identify risk factors of preterm (<37 weeks) and very preterm (<34 weeks) birth among a cohort of 927 HIV positive women living in Dar es Salaam, Tanzania, who enrolled in the Tanzania Vitamin and HIV Infection Trial between 1995 and 1997. Multivariable relative risk regression models were used to determine the association of potential maternal risk factors with premature and very premature delivery. High rates of preterm (24%) and very preterm birth (9%) were found. Risk factors (adjusted RR (95% CI)) for preterm birth were mother <20 years (1.46 (1.10, 1.95)), maternal illiteracy (1.54 (1.10, 2.16)), malaria (1.42 (1.11, 1.81)), Entamoeba coli (1.49 (1.04, 2.15)), no or low pregnancy weight gain, and HIV disease stage ≥2 (1.41 (1.12, 1.50)). Interventions to reduce pregnancies in women under 20, prevent and treat malaria, reduce Entamoeba coli infection, and promote weight gain in pregnant women may have a protective effect on prematurity

Antimicrobial resistance among producers and non-producers of extended spectrum beta-lactamases in urinary isolates at a tertiary Hospital in Tanzania

<p>Abstract</p> <p>Background</p> <p>Published data on the existence and magnitude of extended spectrum beta-lactamase (ESBL) production in urinary pathogens in local setting is limited. The aim of the present study was to determine the prevalence of antimicrobial resistance and ESBL production among <it>Escherichia coli </it>and <it>Klebsiella spp </it>from urine samples in a tertiary hospital. This was a cross sectional study conducted at Muhimbili National Hospital in Dar es Salaam, Tanzania.</p> <p>Findings</p> <p>A total of 270 <it>E.coli </it>and <it>Klebsiella spp </it>urinary pathogens from children and adults isolated from January to March 2010 were included in the study. <it>E. coli </it>and <it>Klebsiella spp </it>isolates were tested for antimicrobial susceptibility by the Clinical and Laboratory Standard Institute's disc diffusion method. These isolates were further screened for ESBL phenotype using cefotaxime and ceftazidime discs. Isolates with reduced sensitivity were confirmed using ESBL E-test strips. Of 270 isolates, 138 (51.1%) were <it>E. coli </it>and 132 (48.9%) were <it>Klebsiella spp</it>. ESBL was detected in 122 (45.2%) of all the isolates. ESBL- producing <it>E. coli </it>strains were significantly more resistance to cotrimoxazole (90.7%), ciprofloxacin (46.3%) and nalidixic acid (61.6%) than strains that did not produce ESBL (p < 0.05). Similarly, ESBL- producing <it>Klebsiella spp </it>strains were significantly more resistance to cotrimoxazole (92.6%), ciprofloxacin (25.0%), nalidixic acid (66.2%), and gentamicin (38.2%) than strains that did not produce ESBL (P < 0.05). Multi-drug resistance was found to be significantly (<it>P </it>< 0.05) more in ESBL producing isolates (90.5%) than non ESBL producers (68.9%). The occurrence of ESBL was significantly higher among isolates from inpatients than outpatients [95 (50.5%) vs. 27(32.9%)] (p = 0.008). The occurrence of ESBL was significantly higher among isolates from children than in adults [84 (54.9%) vs. 38(32.5%)] (p < 0.001).</p> <p>Conclusions</p> <p>High prevalence of ESBL-producing <it>E. coli </it>and <it>Klebsiella spp </it>strains was found among inpatients and children. Most of the ESBL- producing isolates were multi-drug resistant making available therapeutic choices limited. We recommend continued antibiotic surveillance as well comprehensive multi-center studies to address the emerging problem of ESBL-associated infections in order to preserve the continued usefulness of most antimicrobial drugs. Further more conducting molecular studies will help to evaluate the various ESBL types.</p