Seroprevalence of Brucella Infection in a Cohort of HIV-Positive Malawian Pregnant Women Living in Urban Areas.

Background: The seroprevalence of Brucella infection in sub-Saharan regions is high, and no recent data are available for Malawi, a country in which >60% of the population is involved in agropastoral activity. Aim: To evaluated the seroprevalence of Brucella in a cohort of HIV-positive pregnant women, living in an urban setting in Malawi. Methods: Sera of 201 pregnant women were tested for Brucella IgG. The Rose Bengal Plate Test and Serum Agglutination Tube test were used to determine antibody titer. Results: Five out of 201 (2.48%) women show positivity to Brucella, consistent with a past exposition to the infection. All five women delivered healthy infants, but two of them reported previous abortion/stillbirths, with a higher rate than those of the rest of the cohort (40% vs. 21.5%). Conclusions: This is one of the first reports of exposure of pregnant women to Brucella infection in Malawi, providing evidence of Brucella occurrence in an urban setting. Control programs should be introduced to reduce its impact on animal and human health.

HIV-exposed infants with EBV infection have a reduced persistence of the immune response to the HBV vaccine.

BACKGROUND: In sub-Saharan African countries Epstein Barr virus (EBV) infection occurs in early childhood. We aim to investigate the factors associated with EBV acquisition and the impact of EBV infection on the humoral response to HBV vaccination in infants born from HIV-positive, antiretroviral-treated mothers in Malawi. METHODS: A total of 149 HIV-exposed infants were included in this longitudinal study. EBV anti-VCA IgG were measured using an ELISA assay. The EBV seroconversion was correlated with the maternal viro-immunological conditions, with infant growth and immunological vulnerability, and with the humoral response to the HBV vaccine. RESULTS: No infant was EBV-positive at 6 months (n. 52 tested). More than a third of infants (49/115 or 42.6 %) on study beyond 6 months seroconverted at 12 months. At 24 months, out of 66 tested infants, only 13 remained EBV-uninfected, while 53 (80.3 %) acquired EBV infection, rising the total proportion of EBV seroconversion to 88.7 % (102/115 infants). EBV seroconversion was significantly associated with a low maternal educational status but had no impact on infant growth or vulnerability to infections. Reduced HBsAb levels and accelerated waning of antibodies were associated with early EBV seroconversion. CONCLUSIONS: We found a heterogeneous timing of acquisition of EBV with the majority of infants born from HIV + mothers acquiring infection after 6 months. Anti-HBs levels were lower and appeared to wane faster in infants acquiring EBV infection.

Dried blood spots for the quantitative evaluation of IgG isotypes and correlation with serum samples in HIV-exposed uninfected (HEU) infants.

BACKGROUND: The determination of IgG levels and their subclasses can provide clinically relevant information on the status of the immune system. Here we determined the sensitivity and reproducibility of the quantification of IgG subclasses from Dried Blood Spots (DBS) in Malawian uninfected infants exposed to HIV (HEU). METHODS: Sixty paired samples of serum and DBS from HEU infants were used. Samples were collected from 1, 6, and 24-month old infants. IgGs concentrations from both serum and DBS were analyzed by BN ProSpec Siemens assay, using a different setting for sample dilutions. The reproducibility of the DBS method was tested on 10 samples run twice, starting from the DBS extraction process. To assess the systematic, proportional, and random differences, we computed the Passing-Bablok regression, and the Bland-Altman analysis to estimate the total mean bias between the two tests. RESULTS: The IgG isotypes concentrations from serum and DBS showed significant differences in all the comparisons. Generally, the DBS method underestimated IgG subclasses' values showing a recovery range between 51.2% and 77.6%. Passing Bablok regression on age-based groups showed agreement for IgG, IgG1, and IgG2, but not for IgG3 and IgG4. The mean bias obtained with the Bland Altman test varied largely depending on IgG isotypes (-0.02-2.21 g/l) Coefficient of variation <7.0% was found in the repeated tests for IgG, IgG1, IgG3, and IgG4, while it was 12.4% for IgG2. CONCLUSIONS: Varying degrees of differences were seen in the IgGs measurement in the two different matrices. In IgGs analysis, the DBS method offers promise for population-based research, but the results should be carefully evaluated and considered as a relative value since they are not equivalent to the serum concentrations.

Social determinants of male partner attendance in women's prevention-of mother-to-child transmission program in Malawi.

BACKGROUND: Male partners are rarely present during PMTCT (Prevention-Mother-To-Child-Transmission) services in Sub-Saharan Africa (SSA). Male involvement is increasingly recognised as an important element of women's access to care. This study aims to identify the socio-demographic characteristics, HIV-Knowledge, Attitude and Practice (KAP) among women accompanied and not accompanied by their male partners. METHODS: We included pregnant women enrolled in PMTCT programme between August 2018 and November 2019 in the Southern Region of Malawi. Eligible women were aged 18 years or older, living with a male partner, enrolled for the first time in one of the four selected facilities. We provided a KAP survey to women and their partners attending the facilities. Our primary objective was to assess and analyse the proportion of women who were accompanied by their partner at least once. We applied descriptive statistics and logistic regressions to study the association between being accompanied and explanatory variables. RESULTS: We enrolled 128 HIV-positive women: 82 (64.1%) were accompanied by their male partners and 46 (35.9%) were alone. In the multivariable model, women's unemployment and owning a means of transport are negatively associated with male attendance (respectively adjusted OR 0.32 [95% CI, 0.11-0.82] and 0.23 [95% CI, 0.07-0.77]), whereas, in the univariable model, high women's level of knowledge of HIV is positively associated with male attendance (OR 2.17 [95% CI, 1.03-4.58]). Level of attitude and practice toward HIV were not significantly associated to our study variable. CONCLUSIONS: Our study shows a high male attendance in Malawi compared to other studies performed in SSA. This study highlights that women's level of knowledge on HIV and their economic condition (employment and owning a means of transport) affects male attendance. Moreover, the study points out that gender power relationships and stringent gender norms play a crucial role thus they should be considered to enhance male involvement.

Dynamics of immunoglobulin G subclasses during the first two years of life in Malawian infants born to HIV-positive mothers.

BACKGROUND: Maternal antibodies are key components of the protective responses of infants who are unable to produce their own IgG until 6 months of life. There is evidence that HIV-exposed uninfected children (HEU) have IgG levels abnormalities, that can be partially responsible for the higher vulnerability to infections in the first 2 years of the life of this population. This retrospective study aimed to characterize the dynamics in plasma levels of total IgG and their isotypes during the first 2 years of life in HEU infants exclusively breastfed through 6 months of age. METHODS: Total IgG, IgG1, IgG2, IgG3 and IgG4 isotypes, and IgM and IgA plasma concentrations were determined by nephelometric methods in 30 Malawian infants born to HIV-positive women at month 1, 6 and 24 of life. RESULTS: At 1-month infants had a median concentration of total IgG of 8.48 g/l, (IQR 7.57-9.15), with an overrepresentation of the IgG1 isotype (89.0% of total) and low levels of IgG2 (0.52 g/l, IQR, 0.46-0.65). Total IgG and IgG1 concentrations were lower at 6 months (- 2.1 and - 1.12 g/dl, respectively) reflecting disappearance of maternal antibodies, but at 24 months their levels were higher with respect to the reported reference values for age-matched pairs. Abnormal isotype distribution was still present at 24 months with IgG2 remaining strongly underrepresented (0.87 g/l, 7.5% of total IgG). CONCLUSION: HIV exposure during pregnancy and breastfeeding seems to influence the IgG maturation and isotype distribution that persist in 2-year old infants.

IgG abnormalities in HIV-positive Malawian women initiating antiretroviral therapy during pregnancy persist after 24 months of treatment.

OBJECTIVES: Hypergammaglobulinemia and anomalies in the IgG subclass distribution are common in HIV-infected individuals and persist even after many years of antiretroviral therapy (ART). The aim of this study was to investigate the IgG profile and dynamics in pregnant HIV-infected Malawian women in the Option B era. METHODS: Thirty-seven treatment-naive women received ART from the third trimester of pregnancy to 6 months post delivery (end of the breastfeeding period). ART continuation (group C) or interruption (group I) was then decided on the basis of the CD4+ cell count at enrolment (>350 or ≤350/µl). Total IgG and IgG subclasses were determined in maternal serum using a nephelometric assay at baseline and at 6 and 24 months postpartum. RESULTS: At enrolment, 36/37 women had IgG levels >15g/l and there was a predominance of the IgG1 isotype (more than 90%) in parallel with underrepresentation of IgG2 (5.0%). After 6 months of ART, both groups showed a significant median decrease in total IgG (-3.1g/l in group I, -3.5g/l in group C) and in IgG1 (-4.0g/l and -3.6g/l, respectively), but only a modest recovery in IgG2 levels (+0.16 in group I, +0.14g/l in group C). At month 24, hypergammaglobulinemia was still present in 73.7% of women in group C, although a significant reduction was observed in total IgG level and in IgG1 and IgG3 subclasses (p<0.0001 in all cases). IgG2 levels did not show any significant change. In group I at 24 months, total IgG and IgG subclasses had returned to levels comparable to those at baseline. CONCLUSIONS: The beneficial effects of 24 months of ART appear to be limited in the B-cell compartment, with an incomplete reduction of total IgG levels and no recovery of IgG2 depletion. A short ART period did not have significant effects on IgG abnormalities in women who interrupted treatment.

Renal outcomes in patients initiated on tenofovir disoproxil fumarate-based antiretroviral therapy at a community health centre in Malawi.

Tenofovir-based antiretroviral therapy (TDF ART) is the first-line regimen for human immunodeficiency virus (HIV) in Africa. However, contemporary data on nephrotoxicity are lacking. We determined the renal outcomes of patients commenced on TDF ART in Malawi. ART-naïve patients initiated on TDF ART at a community health centre between 1 July 2013 and 31 December 2015 were included. The estimated glomerular filtration rate (eGFR, Cockcroft-Gault) was recorded at the initiation of therapy and over 18 months thereafter. The prevalence of renal impairment at ART initiation (eGFR < 60 ml/min) and the incidence of nephrotoxicity (eGFR < 50 ml/min) were determined. A total of 439 patients (median age: 32 years; 317 [72.2%] female) were included. Twenty-one (4.8%) patients had renal impairment at ART initiation; eGFR improved in all during follow-up. Nephrotoxicity occurred in 17 (4.0%) patients with eGFR > 50 ml/min at baseline, predominantly within the first six months of therapy. Increasing age and diastolic hypertension (>100 mmHg) were independent risk factors for nephrotoxicity development. The prevalence of kidney disease at ART initiation was 4.8% and nephrotoxicity occurred in 4.0%. Some eGFR decline may have been due to weight gain. Targeted monitoring of kidney function six months after TDF initiation should be considered in Malawi.

High CMV IgG antibody levels are associated to a lower CD4+ RESPONSE to antiretroviral therapy in HIV-infected women.

BACKGROUND: Virtually all HIV-infected women in sub-Saharan Africa have evidence of Cytomegalovirus (CMV) infection and levels of specific anti-CMV IgG have been suggested to represent more intense reactivation of subclinical infection. Studies have also shown direct influence of CMV on lymphocytes. OBJECTIVE: The aim of this study was to determine if levels of anti-CMV specific antibodies could impact on the immunological response to antiretroviral treatment (ART) in HIV-infected pregnant women. STUDY DESIGN: CMV-specific IgG were measured in HIV-infected pregnant women at 26 weeks of gestation (before ART initiation). Women received ART until 6 months postpartum or indefinitely according to local guidelines at the time of the study. Immunological and virological responses were assessed 6 months and 24 months after delivery. RESULTS: A total of 81 women were studied. At baseline high levels (above the median) of specific IgG were associated to a low CD4+ cell count (P<0.001), a high viral load (P=0.003), and to an older age (P=0.051). In a multivariate model adjusting for baseline CD4+ count, baseline viral load and age, the presence of low levels of CMV IgG was the only independent predictor of a a CD4+ count above 500/mm3 24 months after delivery among women on continuous therapy. CONCLUSIONS: In this cohort, levels of CVM IgG had a significant influence on the immunological response to ART, adding information to the known impact of CMV infection in the HIV-positive population, and underlining the need of new strategies to contain the infection.

CMV infection in a cohort of HIV-exposed infants born to mothers receiving antiretroviral therapy during pregnancy and breastfeeding.

Antiretroviral therapy has been shown to reduce rates of congenital CMV infection. Little information is available on the possible impact of antiretroviral therapy on postnatal breastfeeding-associated CMV infection acquisition. A cohort of 89 HIV-infected mothers and their children was studied. Women received antiretroviral therapy from week 25 of gestation until 6 months postpartum or indefinitely if meeting the criteria for treatment. All women were evaluated for CMV IgG presence and CMV DNA in breast milk. Children were tested for CMV infection by either the presence of IgM or the presence of CMV DNA in plasma at 1, 6 and 12 months and by the presence of IgG at 24 months. All mothers had high titers of CMV DNA in breast milk (5.7 log at Month 1 and 5.1 log at Month 6). Cumulative CMV infection rates were 60.3 % at Month 6, 69 % at Month 12 and 96.4 % at Month 24. There was a significant negative correlation between the duration of antiretroviral treatment during pregnancy and levels of CMV DNA in breast milk at Month 1 (P = 0.033). There was a trend for a correlation between high titers of CMV DNA in breast milk at 6 months and CMV infection at 6 months (P = 0.069). In this cohort, more than 95 % of the children had acquired CMV infection by 2 years of age. Besides breastfeeding, which played a major role, also horizontal transmission between 1 and 2 years was certainly relevant in determining CMV infection acquisition.

Levels of bone markers in a population of infants exposed in utero and during breastfeeding to tenofovir within an Option B+ programme in Malawi.

OBJECTIVES: No data are available on bone metabolism in infants exposed to tenofovir during breastfeeding. We investigated bone metabolism markers in the first year of life in infants from mothers who received tenofovir, lamivudine and efavirenz during pregnancy and 12 months of breastfeeding in a national Option B+ programme in Malawi. METHODS: Serum samples collected at 6 and 12 months in tenofovir-exposed infants and in a small sample of tenofovir-unexposed infants from the same clinical centre were analysed in batches for levels of bone-specific alkaline phosphatase (BAP; marker of bone formation) and of C-terminal telopeptide of type I collagen (CTX; marker of bone resorption). RESULTS: Overall, 136 tenofovir-exposed infants were evaluated. No infant had at either timepoint CTX values above the upper normal limit, while most of them had at 6 and 12 months levels of BAP above the upper normal limit for the age range. Levels of bone markers showed no differences by gender and no association with growth parameters. Tenofovir-unexposed and -exposed children had similar mean levels of bone markers at 6 months (CTX: 0.62 versus 0.55 ng/mL, P = 0.122; BAP: 384 versus 362 U/L, P = 0.631). CONCLUSIONS: No significant association between treatment with tenofovir and CTX or BAP levels was found. The high levels of BAP, coupled to the normal levels observed for CTX, might reflect primarily skeletal growth. Potential negative effects of prolonged exposure to tenofovir through breastfeeding cannot however be excluded and longitudinal studies that evaluate bone mineralization status in children enrolled in Option B+ programmes are warranted.

Virological Response and Drug Resistance 1 and 2 Years Post-Partum in HIV-Infected Women Initiated on Life-Long Antiretroviral Therapy in Malawi.

The objective of this study was to determine the virological response and the possible emergence of drug resistance at 1 and 2 years postpartum in HIV-positive pregnant women enrolled under the Option B approach and meeting the criteria for treatment. In the study, women with baseline CD4(+) <350/mm(3) received a combination of stavudine, lamivudine, and nevirapine during pregnancy (from week 25 of gestation) and continued it indefinitely after delivery. HIV-RNA was measured at 12 and 24 months postpartum. Drug resistance mutations were assessed in those with HIV-RNA >50 copies/ml. Baseline resistance mutations were assessed in the entire cohort. A total of 107 women were studied. At baseline, resistance mutations were seen in 6.6% of the women. At 12 months, 26.7% of the women had >50 copies/ml and among them 12.9% had virological failure (HIV-RNA >1,000 copies/ml). At 24 months, detectable HIV-RNA was seen in 28.3% of the women and virological failure in 10.1% of the women. Resistance mutations (mainly non-nucleoside reverse transcriptase inhibitors mutations) were seen in 40% of the women with detectable HIV-RNA. Baseline mutations did not correlate with virological failure or the emergence of resistance at later time points. Virological failure 2 years postpartum and emergence of resistance were rare in this cohort of HIV-infected women. These findings are reassuring in the light of the new strategies for the prevention of mother-to-child HIV transmission, recommending life-long antiretroviral therapy administration.

Growth indices in breastfed infants pre and postnatally exposed to tenofovir compared with tenofovir-unexposed infants.

We evaluated growth indices in two cohorts of Malawian infants exposed to tenofovir, lamivudine and efavirenz in utero and during 12 months of breastfeeding, and to stavudine/zidovudine, lamivudine and nevirapine in utero and during 6 months of breastfeeding. Growth indices were similar in the two cohorts at one and 6 months but were significantly better in the first group at 12 months, suggesting no negative effect of tenofovir and a significant benefit of extended breastfeeding.

Concentrations of tenofovir, lamivudine and efavirenz in mothers and children enrolled under the Option B-Plus approach in Malawi.

OBJECTIVES: To evaluate antiretroviral drug concentrations in mothers and infants enrolled under the Option B-Plus approach for the prevention of HIV mother-to-child transmission in Malawi and to assess the maternal virological response after 1 year of treatment. PATIENTS AND METHODS: Forty-seven women and 25 children were studied. Mothers were administered during pregnancy a combination of tenofovir, lamivudine and efavirenz and continued it during breastfeeding (up to 2 years) and thereafter. Drug concentrations were evaluated in mothers (plasma and breast milk) at 1 and 12 months post-partum and in infants (plasma) at 6 and 12 months of age. Drug concentrations were determined using an LC-MS/MS validated methodology. RESULTS: In breast milk, tenofovir concentrations were very low (breast milk/maternal plasma ratio = 0.08), while lamivudine was concentrated (breast milk/plasma ratio = 3) and efavirenz levels were 80% of those found in plasma. In infants, median levels at 6 months were 24 ng/mL tenofovir, 2.5 ng/mL lamivudine and 86.4 ng/mL efavirenz. At month 12, median levels were below the limit of quantification for the three drugs. No correlation was found between drug concentrations and laboratory parameters or indices of growth. HIV-RNA >1000 copies/mL was seen at month 1 in 15% of the women and at month 12 in 8.5%. Resistance was found in half of the women with detectable viral load. CONCLUSIONS: Breastfeeding infants under Option B-Plus are exposed to low concentrations of antiretroviral drugs. With this strategy, mothers had a good virological response 1 year after delivery.

Retention, transfer out and loss to follow-up two years after delivery in a cohort of HIV+ pregnant women in Malawi.

In this study, we analysed in a cohort of pregnant women followed for two years the proportion of women remaining at the same clinic, those who transferred to other clinics, and those lost to follow-up. The possible determinants of the loss to follow-up were also assessed in a setting of postpartum discontinuation based on CD4+ count. A total of 311 pregnant women received antiretroviral therapy from week 25 of gestational age until six months postpartum (end of breastfeeding period), or indefinitely if meeting the criteria for treatment (baseline CD4+ <350 cells/mm(3)). Twenty-four months after delivery, six women had died, 247 were in active follow-up, 21 had transferred to another antiretroviral therapy clinic and 37 were lost to follow-up (rate of loss to follow-up 13%, 95% CI 9.1-16.9%). The presence of a baseline CD4+ count above 350 cells/mm(3) was associated with a ten-fold higher risk of loss to follow-up after six months of delivery (hazard ratio: 9.8, 95% CI 2.2-42.7, for baseline CD4 >350 cells/mm(3) versus baseline CD4+ count below 350 cells/mm(3), p = 0.002). This finding suggests that discontinuation of drugs when the risk of transmission has ceased can have a negative impact on the retention in care of these women.

Drug resistance mutations 18 months after discontinuation of nevirapine-based ART for prevention of mother-to-child transmission of HIV in Malawi.

OBJECTIVES: The objective of this study was to determine the prevalence of drug resistance mutations among HIV-positive women in Malawi 18 months after discontinuing nevirapine-based ART for the prevention of mother-to-child transmission. PATIENTS AND METHODS: HIV-infected antiretroviral-naive (except for single-dose nevirapine) pregnant Malawian women receiving a nevirapine-based triple antiretroviral regimen from Week 25 of gestation until 6 months of breastfeeding were included in this analysis. Drug resistance was assessed in HIV-DNA 24 months post-partum and at baseline (before the initiation of treatment). In patients with resistance, the presence of mutations was also evaluated in the corresponding plasma samples. RESULTS: Seven out of 42 (16.7%) women studied had archived drug resistance at Month 24 [six cases had NNRTI-associated mutations and two cases the M184I mutation]. In four cases, resistance mutations were already present at baseline (all NNRTI mutations). In three cases, there was an emergence of 'new' resistance (also present in the plasma in one case). Of the 35 women without resistance mutations at Month 24, only one subject had resistance mutations at baseline. Baseline resistance was significantly more common among women with mutations at 24 months compared with those harbouring a WT virus (4/7 versus 1/35, P < 0.001). CONCLUSIONS: Among women who had discontinued drugs 6 months post-partum, only 3/42 (7.1%) had accumulated new resistance mutations in HIV-DNA 2 years after delivery. These findings are reassuring in terms of the safety of the Option B strategy for the prevention of HIV mother-to-child transmission.

Laboratory confirmation of clinically diagnosed malaria in a cohort of HIV-infected mothers and their children in Malawi.

To avoid overdiagnosis, accuracy in the identification of true malaria cases is of critical importance. Samples (either whole blood, dried blood spots or plasma/serum) collected at the time of clinically diagnosed malaria episodes in a cohort of Malawian HIV-infected mothers and their children were retrospectively tested with the enzyme-linked immunosorbent assay (ELISA) for HRP-2 (histidine-rich protein 2) detection. There were 55 and 56 clinically diagnosed cases of malaria in mothers and children, respectively, with samples available for testing. Rates of laboratory-confirmed episodes were 20% (11 of 55) in mothers and 16.1% (9 of 56) in children. Hemoglobin was lower in children with confirmed malaria compared to those with clinical malaria diagnosis. The results of our study support the widespread use of rapid diagnostic tests.