Stefanie Theuring

Male Involvement in PMTCT Services in Mbeya Region, Tanzania

Throughout all stages of programmes for the prevention of mother-to-child-transmission of HIV (PMTCT), high dropout rates are common. Increased male involvement and couples’ joint HIV counselling/testing during antenatal care (ANC) seem crucial for improving PMTCT outcomes. Our study assessed male attitudes regarding partner involvement into ANC/PMTCT services in Mbeya Region, Tanzania, conducting 124 individual interviews and six focus group discussions. Almost all respondents generally supported PMTCT interventions. Mentioned barriers to ANC/PMTCT attendance included lacking information/knowledge, no time, neglected importance, the services representing a female responsibility, or fear of HIV-test results. Only few perceived couple HIV counselling/testing as disadvantageous. Among fathers who had refused previous ANC/PMTCT attendance, most had done so even though they were not perceiving a disadvantage about couple counselling/testing. The contradiction between men’s beneficial attitudes towards their involvement and low participation rates suggests that external barriers play a large role in this decision-making process and that partner’s needs should be more specifically addressed in ANC/PMTCT services.

Adherence to Combination Prophylaxis for Prevention of Mother-to-Child-Transmission of HIV in Tanzania

Background Since 2008, Tanzanian guidelines for prevention of mother-to-child-transmission of HIV (PMTCT) recommend combination regimen for mother and infant starting in gestational week 28. Combination prophylaxis is assumed to be more effective and less prone to resistance formation compared to single-drug interventions, but the required continuous collection and intake of drugs might pose a challenge on adherence especially in peripheral resource-limited settings. This study aimed at analyzing adherence to combination prophylaxis under field conditions in a rural health facility in Kyela, Tanzania. Methods and Findings A cohort of 122 pregnant women willing to start combination prophylaxis in Kyela District Hospital was enrolled in an observational study. Risk factors for decline of prophylaxis were determined, and adherence levels before, during and after delivery were calculated. In multivariate analysis, identified risk factors for declining pre-delivery prophylaxis included maternal age below 24 years, no income-generating activity, and enrolment before 24.5 gestational weeks, with odds ratios of 5.8 (P = 0.002), 4.4 (P = 0.015) and 7.8 (P = 0.001), respectively. Women who stated to have disclosed their HIV status were significantly more adherent in the pre-delivery period than women who did not (P = 0.004). In the intra- and postpartum period, rather low drug adherence rates during hospitalization indicated unsatisfactory staff performance. Only ten mother-child pairs were at least 80% adherent during all intervention phases; one single mother-child pair met a 95% adherence threshold. Conclusions Achieving adherence to combination prophylaxis has shown to be challenging in this rural study setting. Our findings underline the need for additional supervision for PMTCT staff as well as for clients, especially by encouraging them to seek social support through status disclosure. Prophylaxis uptake might be improved by preponing drug intake to an earlier gestational age. Limited structural conditions of a healthcare setting should be taken into serious account when implementing PMTCT combination prophylaxis.

Emergence of minor drug-resistant HIV-1 variants after triple antiretroviral prophylaxis for prevention of vertical HIV-1 transmission.

Background WHO-guidelines for prevention of mother-to-child transmission of HIV-1 in resource-limited settings recommend complex maternal antiretroviral prophylaxis comprising antenatal zidovudine (AZT), nevirapine single-dose (NVP-SD) at labor onset and AZT/lamivudine (3TC) during labor and one week postpartum. Data on resistance development selected by this regimen is not available. We therefore analyzed the emergence of minor drug-resistant HIV-1 variants in Tanzanian women following complex prophylaxis. Method 1395 pregnant women were tested for HIV-1 at Kyela District Hospital, Tanzania. 87/202 HIV-positive women started complex prophylaxis. Blood samples were collected before start of prophylaxis, at birth and 1–2, 4–6 and 12–16 weeks postpartum. Allele-specific real-time PCR assays specific for HIV-1 subtypes A, C and D were developed and applied on samples of mothers and their vertically infected infants to quantify key resistance mutations of AZT (K70R/T215Y/T215F), NVP (K103N/Y181C) and 3TC (M184V) at detection limits of <1%. Results 50/87 HIV-infected women having started complex prophylaxis were eligible for the study. All women took AZT with a median duration of 53 days (IQR 39–64); all women ingested NVP-SD, 86% took 3TC. HIV-1 resistance mutations were detected in 20/50 (40%) women, of which 70% displayed minority species. Variants with AZT-resistance mutations were found in 11/50 (22%), NVP-resistant variants in 9/50 (18%) and 3TC-resistant variants in 4/50 women (8%). Three women harbored resistant HIV-1 against more than one drug. 49/50 infants, including the seven vertically HIV-infected were breastfed, 3/7 infants exhibited drug-resistant virus. Conclusion Complex prophylaxis resulted in lower levels of NVP-selected resistance as compared to NVP-SD, but AZT-resistant HIV-1 emerged in a substantial proportion of women. Starting AZT in pregnancy week 14 instead of 28 as recommended by the current WHO-guidelines may further increase the frequency of AZT-resistance mutations. Given its impact on HIV-transmission rate and drug-resistance development, HAART for all HIV-positive pregnant women should be considered.

Hematological Changes in Women and Infants Exposed to an AZT-Containing Regimen for Prevention of Mother-to-Child-Transmission of HIV in Tanzania

Introduction Tanzanian guidelines for prevention of mother-to-child-transmission of HIV (PMTCT) recommend an antiretroviral combination regimen involving zidovudine (AZT) during pregnancy, single-dosed nevirapine at labor onset, AZT plus Lamivudine (3TC) during delivery, and AZT/3TC for 1–4 weeks postpartum. As drug toxicities are a relevant concern, we assessed hematological alterations in AZT-exposed women and their infants. Methods and Materials A cohort of HIV-positive women, either with AZT intake (n = 82, group 1) or without AZT intake (n = 62, group 2) for PMTCT during pregnancy, was established at Kyela District Hospital, Tanzania. The cohort also included the infants of group 1 with an in-utero AZT exposure ≥4 weeks, receiving AZT for 1 week postpartum (n = 41), and infants of group 2 without in-utero AZT exposure, receiving a prolonged 4-week AZT tail (n = 58). Complete blood counts were evaluated during pregnancy, birth, weeks 4–6 and 12. Results For women of group 1 with antenatal AZT intake, we found a statistically significant decrease in hemoglobin level, red blood cells, white blood cells, granulocytes, as well as an increase in red cell distribution width and platelet count. At delivery, the median red blood cell count was significantly lower and the median platelet count was significantly higher in women of group 1 compared to group 2. At birth, infants from group 1 showed a lower median hemoglobin level and granulocyte count and a higher frequency of anemia and granulocytopenia. At 4–6 weeks postpartum, the mean neutrophil granulocyte count was significantly lower and neutropenia was significantly more frequent in infants of group 2. Conclusions AZT exposure during pregnancy as well as after birth resulted in significant hematological alterations for women and their newborns, although these changes were mostly mild and transient in nature. Research involving larger cohorts is needed to further analyze the impact of AZT-containing regimens on maternal and infant health.

Antiretroviral Therapy Programme Retention and Outcomes after 12 Months in a Retrospective Patient Cohort in Fort Portal, Uganda: The Ongoing Challenge of Male ART Performance

Background: Antiretroviral therapy (ART) programmes have been extensively scaled-up in countries like Uganda. While success of these programmes largely depends on lasting patient retention, attrition rates are often especially high in the first year after treatment initiation. Our study aimed at analysing recent data of a Ugandan ART patient cohort regarding 12 months ART outcomes and programme retention. Methods: Virika Hospital in Fort Portal, Western Uganda, is offering ART services according to national treatment standards. A routinely collected patient monitoring database was used for retrospective analysis, following-up 369 patients for 12 months after enrolment. Primary outcome indicator was 12 months retention (being alive and active in programme). We assessed clinical ART progress, attrition, mortality and influencing factors, particularly gender differences. Results: From 369 ART patients, two-thirds were female. Overall ART outcomes and adherence in this cohort were satisfying, but in men, improvement of CD4-cell counts and weight gain after 12 months were significantly lower than in female patients. In total, one in eight patients (12.2%) was subject to all-cause attrition after 12 months. We identified low CD4-cell count at ART enrolment as the only independent risk factor for attrition after 12 months (p=0.037), while male patients were at highest risk for this, showing significantly lower CD4-cell counts at ART initiation (p=0.008). Conclusion: We found overall outcomes of this ART programme encouraging, however, attention must be paid particularly to male ART patients. Men were underepresented in our cohort, enrolled in the ART programme at later disease stage, and showed worse ART outcomes after 12 months. Our data suggests that the known challenge of male ART performance is persisting, and that it has not sufficiently been addressed in the past years. Especially the problem of male late-presenters in ART programmes should lead to action in health services planning and implementation, for example by offering more HIV testing opportunities to men.

Increasing Partner Attendance in Antenatal Care and HIV Testing Services: Comparable Outcomes Using Written versus Verbal Invitations in an Urban Facility-Based Controlled Intervention Trial in Mbeya, Tanzania.

In many Sub-Saharan African settings male partner involvement in antenatal care (ANC) remains low, although great benefits for maternal and infant health outcomes have been long recognised, in particular regarding the prevention of HIV transmission. Yet there is paucity on evidence regarding the effectiveness of strategies to increase male partner involvement. This controlled intervention trial in Ruanda Health Centre in Mbeya, Tanzania, assessed the effectiveness of invitation letters for male involvement in ANC. Pregnant women approaching ANC without partners received official letters inviting the partner to attend ANC. A control group was instructed to verbally invite partners. Partner attendance was recorded at two subsequent ANC visits. Rates for male partner return, couple voluntary counselling and testing (CVCT), and influencing factors were analysed. From 199 ANC clients in total, 97 were assigned to the invitation letter group; 30 of these (30.9%) returned with their male partners for ANC. In the control group of 102 women, 28 (27.5%) returned with their partner. In both groups CVCT rates among jointly returning couples were 100%. Partner return/CVCT rate was not statistically different in intervention and control group (OR 1.2, p = 0.59). Former partner attendance at ANC during a previous pregnancy was the only factor found to be significantly linked with partner return (p = 0.03). Our study demonstrates that rather simple measures to increase male partner attendance in ANC and CVCT can be effective, with written and verbal invitations having comparable outcomes. In terms of practicability in Sub-Saharan African settings, we recommend systematic coaching of ANC clients on how to verbally invite male partners in the first instance, followed by written invitation letters for partners in case of their non-attendance. Further studies covering both urban and rural settings will be more informative for effective translation into policy.

Zidovudine Exposure in HIV-1 Infected Tanzanian Women Increases Mitochondrial DNA Levels in Placenta and Umbilical Cords

Background Zidovudine (AZT) constitutes part of the recommended regimens for prevention and treatment of HIV-1 infection. At the same time, AZT as well as HIV-1 infection itself may induce mitochondrial damage. In this study, we analyzed the impact of prenatal AZT-exposure on mitochondrial alterations in HIV-infected women and their infants. Methods Mitochondrial DNA (mtDNA) levels in placentas of HIV-1 infected Tanzanian women with and without prenatal AZT exposure, and in the umbilical cords of their AZT-exposed/unexposed infants were quantified using real-time PCR. Furthermore, we checked for the most common mitochondrial deletion in humans, the 4977 base pair deletion (dmtDNA4977) as a marker for mitochondrial stress. Results 83 women fulfilled the inclusion criteria. 30 women had been treated with AZT (median duration 56 days; IQR 43–70 days) while 53 women had not taken AZT during pregnancy. Baseline maternal characteristics in the two groups were similar. The median mtDNA levels in placentas and umbilical cords of women (311 copies/cell) and infants (190 copies/cell) exposed to AZT were significantly higher than in AZT-unexposed women (187 copies/cell; p = 0.021) and infants (127 copies/cell; p = 0.037). The dmtDNA4977 was found in placentas of one woman of each group and in 3 umbilical cords of AZT-unexposed infants but not in umbilical cords of AZT-exposed infants. Conclusions Antenatal AZT intake did not increase the risk for the common mitochondrial deletion dmtDNA4977. Our data suggests that AZT exposure elevates mtDNA levels in placentas and umbilical cords possibly by positively influencing the course of maternal HIV-1 infection.

Prevention of mother-to-child transmission of HIV: Postpartum adherence to Option B+ until 18 months in Western Uganda

Since 2012, the WHO recommends Option B+ for the prevention of mother-to-child transmission of HIV. This approach entails the initiation of lifelong antiretroviral therapy in all HIV-positive pregnant women, also implying protection during breastfeeding for 12 months or longer. Research on long-term adherence to Option B+ throughout breastfeeding is scarce to date. Therefore, we conducted a prospective observational cohort study in Fort Portal, Western Uganda, to assess adherence to Option B+ until 18 months postpartum. In 2013, we recruited 67 HIV-positive, Option B+ enrolled women six weeks after giving birth and scheduled them for follow-up study visits after six, twelve and 18 months. Two adherence measures, self-reported drug intake and amount of drug refill visits, were combined to define adherence, and were assessed together with feeding information at all study visits. At six months postpartum, 51% of the enrolled women were considered to be adherent. Until twelve and 18 months postpartum, adherence for the respective follow-up interval decreased to 19% and 20.5% respectively. No woman was completely adherent until 18 months. At the same time, 76.5% of the women breastfed for ≥12 months. Drug adherence was associated with younger age (p<0.01), lower travel costs (p = 0.02), and lower number of previous deliveries (p = 0.04). Long-term adherence to Option B+ seems to be challenging. Considering that in our cohort, prolonged breastfeeding until ≥12 months was widely applied while postpartum adherence until the end of breastfeeding was poor, a potential risk of postpartum vertical transmission needs to be taken seriously into account for Option B+ implementation.

Comparison of 454 Ultra-Deep Sequencing and Allele-Specific Real-Time PCR with Regard to the Detection of Emerging Drug-Resistant Minor HIV-1 Variants after Antiretroviral Prophylaxis for Vertical Transmission

Background Pregnant HIV-infected women were screened for the development of HIV-1 drug resistance after implementation of a triple-antiretroviral transmission prophylaxis as recommended by the WHO in 2006. The study offered the opportunity to compare amplicon-based 454 ultra-deep sequencing (UDS) and allele-specific real-time PCR (ASPCR) for the detection of drug-resistant minor variants in the HIV-1 reverse transcriptase (RT). Methods Plasma samples from 34 Tanzanian women were previously analysed by ASPCR for key resistance mutations in the viral RT selected by AZT, 3TC, and NVP (K70R, K103N, Y181C, M184V, T215Y/F). In this study, the RT region of the same samples was investigated by amplicon-based UDS for resistance mutations using the 454 GS FLX System. Results Drug-resistant HIV-variants were identified in 69% (20/29) of women by UDS and in 45% (13/29) by ASPCR. The absolute number of resistance mutations identified by UDS was twice that identified by ASPCR (45 vs 24). By UDS 14 of 24 ASPCR-detected resistance mutations were identified at the same position. The overall concordance between UDS and ASPCR was 61.0% (25/41). The proportions of variants quantified by UDS were approximately 2–3 times lower than by ASPCR. Amplicon generation from samples with viral loads below 20,000 copies/ml failed more frequently by UDS compared to ASPCR (limit of detection = 650 copies/ml), resulting in missing or insufficient sequence coverage. Conclusions Both methods can provide useful information about drug-resistant minor HIV-1 variants. ASPCR has a higher sensitivity than UDS, but is restricted to single resistance mutations. In contrast, UDS is limited by its requirement for high viral loads to achieve sufficient sequence coverage, but the sequence information reveals the complete resistance patterns within the genomic region analysed. Improvements to the UDS limit of detection are in progress, and UDS could then facilitate monitoring of drug-resistant minor variants in the HIV-1 quasispecies.

The challenge of referring HIV-positive pregnant women with treatment indication from PMTCT to ART services: a retrospective follow-up study in Mbeya, Tanzania

Providing full antiretroviral therapy (ART) to all HIV-positive, pregnant women with treatment indication could significantly reduce overall mother-to-child transmission. However, the effectiveness of referring HIV-positive antenatal care (ANC) clients with a treatment indication to ART services has rarely been assessed to date. We retrospectively followed-up data of a cohort of treatment-eligible ANC clients in Mbeya Region, Tanzania by retracing and merging registries of ANC, Care and Treatment Centers (CTC), and Infant Care. ART initiation and ART duration before delivery served as primary outcome indicators to assess referral effectiveness. We retraced data of 60 ANC clients with treatment indication: 39 (65%) started predelivery ART and 21 (35%) remained untreated during pregnancy. Eight (13.3%) did not initiate ART at all within the observation period. Women starting ART before delivery had significantly lower CD4-cell counts at enrollment than nonstarters (medians: 207.5 vs. 292 cells/µl; p = 0.013). Predelivery ART starters had experienced a significantly shorter duration between staff-declared “ART readiness” and actual ART start (medians: 0 vs. 28 days; p = 0.0004). The median ART duration prior to delivery was 57 days; only eight women (13.3%) accomplished ≥90 days ART intake during pregnancy. Early enrollment in ANC at ≤24 gestational weeks was associated with longer duration of predelivery ART. At maternity wards, 24.3% of treatment-eligible mothers and newborns with retraceable delivery data had received no or inadequate antiretrovirals. Within 6 months postdelivery, women attended on average 3.5 out of 6 requested CTC visits. Concluding, every third treatment-eligible woman in this cohort was not covered through ART before delivery, and predelivery ART duration was mostly suboptimal regarding vertical transmission prevention. HIV-positive women need to be encouraged to approach ANC early in pregnancy, and health services need to address unnecessary time gaps before ART initiation. In addition, inclusive ART services for HIV-positive ANC clients should be seriously discussed.