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.

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.

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.

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.

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.

Impact of Extended Combination Antiretroviral Therapy on the Decline of HIV Prevalence in Pregnant Women in Malawi.

Combination antiretroviral therapy has been shown to reduce HIV transmission and incident infections. In recent years, Malawi has significantly increased the number of individuals on combination antiretroviral drugs through more inclusive treatment policies. Using a retrospective observational cohort design, records with HIV test results were reviewed for pregnant women attending a referral hospital in Malawi over a 5-year period, with viral load measurements recorded. HIV prevalence over time was determined, and results correlated with population viral load. A total of 11 052 women were included in this analysis, with 440 (4.1%) HIV infections identified. HIV prevalence rates in pregnant women in Malawi halved from 6.4% to 3.0% over 5 years. Mean viral loads of adult patients decreased from 120 000 copies/mL to less than 20 000 copies/mL. Results suggest that community viral load has an effect on HIV incidence rates in the population, which in turn correlates with reduced HIV prevalence rates in pregnant women.

Antibodies against pneumococcal capsular polysaccharide in Malawian HIV-positive mothers and their HIV-exposed uninfected children.

Background HIV-exposed uninfected children have a higher risk of infection and mortality compared to HIV-unexposed children and the reasons for this vulnerability are still under investigation. AIM: This study aimed to evaluate the influence of maternal HIV parameters on the passive transfer of anti-pneumococcal capsular polysaccharide (PCP) IgG and to determine whether the concentrations of specific IgG might be related to the morbidity and mortality in HIV-exposed uninfected children. Methods One hundred and twenty-six Malawian HIV-infected pregnant women and their uninfected children were studied. Antiretroviral treatment-naive women started a nevirapine-based triple combination regimen from the third trimester of pregnancy until at least 6 months of exclusive breastfeeding. Mother/child pairs were followed until 2 years after delivery. Plasma anti-PCP IgG titers (in mothers at 26 weeks of gestation and in infants at 1 and 6 months) were determined by an enzyme-linked immunosorbent assay. None of these women and children had received any vaccination against pneumococcal polysaccharides. Results Maternal anti-PCP IgG concentration was independent from viral load (p = 0.848), CD4 count (p = 0.740) and WHO stage (p = 0.450). However, the child/mother ratio of anti-PCP IgG measured at 1 month among infants was significantly reduced in pairs whose mothers had HIV-RNA > 10 000 copies/ml (p = 0.043) and CD4 < 350 cells/µl (p = 0.090) before antiretroviral therapy (ART). No clear associations between anti-PCP IgG and respiratory-related deaths were found, but respiratory infection episodes were more frequent among children with lower anti-PCP IgG ratio (p = 0.046). Conclusion This study indicates that HIV pre-ART conditions in mothers may influence the rate of specific immunoglobulins transfer, increasing infants vulnerability to respiratory infections.

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.

Viral sequence analysis of HIV-positive women and their infected children: insight on the timing of infection and on the transmission network.

We used high-resolution phylogenetic methods in the context of mother-to-child transmission to obtain information on the timing of the infection and on the transmission network. A total of 33 pol sequences (from maternal peripheral blood, from breast milk, and from plasma of children) belonging to five cases of HIV infant transmission were studied. Using time-scaled phylogeny we were able to estimate that in two cases the transmission occurred after the recommended duration of breastfeeding, supporting a longer, not reported, duration of breastfeeding as a significant factor associated with HIV infant acquisition in this cohort. Among the postnatal infections we were also able to demonstrate that the cell-free virus in breast milk was the most likely population associated with the event of transmission. Our study showed that a coalescent-based model within a Bayesian statistical framework can provide important information that can contribute to optimizing preventive strategies.

The impact of HBV or HCV infection in a cohort of HIV-infected pregnant women receiving a nevirapine-based antiretroviral regimen in Malawi.

BACKGROUND: Coinfection with the hepatitis viruses is common in the HIV population in sub-Saharan Africa. The aim of this study was to assess, in a cohort of HIV-infected pregnant women receiving antiretroviral drugs (ARVs), the prevalence of HBV and HCV infections and to determine the impact of these infections on the occurrence of liver toxicity and on the viro-immunological response. METHODS: Women were screened for HBsAg and HCV-RNA before starting, at week 25 of gestational age, an antiretroviral regimen consisting of lamivudine and nevirapine plus either stavudine or zidovudine. Women with CD4+ < 350/mm3 continued ARVs indefinitely, while the other women interrupted treatment 6 months postpartum (end of breastfeeding period). Both groups were followed for 2 years after delivery. Liver function was monitored by alanine aminotransferase (ALT) measurement. The Cox proportional hazards model was used to identify factors associated with the emergence of liver toxicity. RESULTS: A total of 28 women out of the 309 enrolled in the study (9.1%) were coinfected with HBV (n. 27), or HCV (n. 1). During follow-up 125 women (40.4%) developed a grade ≥ 1 ALT elevation, 28 (9.1%) a grade ≥ 2 and 6 (1.9%) an elevation defining grade 3 toxicity. In a multivariate model including age, baseline CD4+ count and hemoglobin level, the presence of either HBV or HCV infection was significantly associated with the development of an ALT increase of any grade (P = 0.035). Moderate or severe liver laboratory toxicity (grade ≥ 2) was more frequent among women with baseline CD4+ > 250/mm3 (P = 0.030). In HBV-infected women a baseline HBV-DNA level above 10,000 IU/ml was significantly associated to the development of liver toxicity of grade ≥ 1 (P = 0.040). Coinfections had no impact on the immunological and virological response to antiretroviral drugs up to 2 years after delivery. CONCLUSIONS: In this cohort of nevirapine-treated women the presence of HBV or HCV was associated only to the development of mild liver toxicity, while the occurrence of moderate or severe hepatoxicity was correlated to a baseline CD4+ count > 250/mm3. No statistically significant effect of the coinfections was observed on the efficacy of antiretroviral therapy.

Viro-immunological response and emergence of resistance in HIV-infected women receiving combination antiretroviral regimens for the prevention of mother-to-child transmission in Malawi.

OBJECTIVES: To identify factors associated with detectable viral load and the emergence of drug resistance in a cohort of HIV-infected pregnant women in Malawi receiving antiretroviral combination regimens for the prevention of mother-to-infant transmission. METHODS: The study included 260 treatment-naive women who had received a three-drug nevirapine-based regimen from week 25 of gestational age until 6 months after delivery. HIV RNA was determined at month 6 and drug resistance was assessed if viral load was >50 copies/mL. Attendance at the scheduled follow-up visits was used as an indirect measure of treatment adherence. RESULTS: The rate of detectable HIV RNA at 6 months was 9.6% (25/260). The only significant predictor of this occurrence was the presence of ≥1 missed visit during follow-up (P = 0.012). Resistance was assessed in 19 of these women: 7 (37%) had a wild-type virus and the other 12 (63%) had resistance-associated mutations (nucleoside reverse transcriptase inhibitor, 7/12; non-nucleoside reverse transcriptase inhibitor, 11/12). Three of 12 cases (25%) in which mutations were detected had a viral load <1000 copies/mL. The emergence of resistance was not correlated with the presence of baseline mutations in either plasma or archived DNA. CONCLUSIONS: In this cohort of women, detectable HIV RNA 6 months post-partum was infrequent and associated with low adherence to the treatment programme. Mutations were present in 63% of the women with detectable viral load at 6 months who had samples available for resistance testing. The impact of resistance on treatment re-initiation in women discontinuing drugs after the risk of transmission has ceased can be limited.

Reduction of maternal mortality with highly active antiretroviral therapy in a large cohort of HIV-infected pregnant women in Malawi and Mozambique.

BACKGROUND: HIV infection is a major contributor to maternal mortality in resource-limited settings. The Drug Resource Enhancement Against AIDS and Malnutrition Programme has been promoting HAART use during pregnancy and postpartum for Prevention-of-mother-to-child-HIV transmission (PMTCT) irrespective of maternal CD4 cell counts since 2002. METHODS: Records for all HIV+ pregnancies followed in Mozambique and Malawi from 6/2002 to 6/2010 were reviewed. The cohort was comprised by pregnancies where women were referred for PMTCT and started HAART during prenatal care (n = 8172, group 1) and pregnancies where women were referred on established HAART (n = 1978, group 2). RESULTS: 10,150 pregnancies were followed. Median (IQR) baseline values were age 26 years (IQR:23-30), CD4 count 392 cells/mm(3) (IQR:258-563), Viral Load log10 3.9 (IQR:3.2-4.4), BMI 23.4 (IQR:21.5-25.7), Hemoglobin 10.0 (IQR: 9.0-11.0). 101 maternal deaths (0.99%) occurred during pregnancy to 6 weeks postpartum: 87 (1.1%) in group 1 and 14 (0.7%) in group 2. Mortality was 1.3% in women with

Maternal antiretroviral therapy for the prevention of mother-to-child transmission of HIV in Malawi: maternal and infant outcomes two years after delivery.

BACKGROUND: Optimized preventive strategies are needed to reach the objective of eliminating pediatric AIDS. This study aimed to define the determinants of residual HIV transmission in the context of maternal antiretroviral therapy (ART) administration to pregnant women, to assess infant safety of this strategy, and to evaluate its impact on maternal disease. METHODOLOGY/PRINCIPAL FINDINGS: A total of 311 HIV-infected pregnant women were enrolled in Malawi in an observational study and received a nevirapine-based regimen from week 25 of gestation until 6 months after delivery (end of breastfeeding period) if their CD4+ count was > 350/mm(3) at baseline (n = 147), or indefinitely if they met the criteria for treatment (n. 164). Mother/child pairs were followed until 2 years after delivery. The Kaplan-Meier method was used to estimate HIV transmission, maternal disease progression, and survival at 24 months. The rate of HIV infant infection was 3.2% [95% confidence intervals (CI) 1.0-5.4]. Six of the 8 transmissions occurred among mothers with baseline CD4+ count > 350/mm(3). HIV-free survival of children was 85.8% (95% CI 81.4-90.1). Children born to mothers with baseline CD4+ count < 350/mm(3) were at increased risk of death (hazard ratio 2.6, 95% CI 1.1-6.1). Among women who had stopped treatment the risk of progression to CD4+ count < 350/mm(3) was 20.6% (95% CI 9.2-31.9) by 18 months of drug discontinuation. CONCLUSIONS: HIV transmission in this cohort was rare however, it occurred in a significative proportion among women with high CD4+ counts. Strategies to improve treatment adherence should be implemented to further reduce HIV transmission. Mortality in the uninfected exposed children was the major determinant of HIV-free survival and was associated to maternal disease stage. Given the considerable proportion of women reaching the criteria for treatment within 18 months of drug discontinuation, life-long ART administration to HIV-infected women should be considered.

Emergence of lamivudine resistance hepatitis B virus mutations in pregnant women infected with HBV and HIV receiving antiretroviral prophylaxis for the prevention of mother-to-infant transmission in Malawi.

HIV/HBV co-infection is highly prevalent in sub-Saharan Africa. The aim of this study was to determine if the use of triple combination lamivudine-containing prophylaxis for the prevention of mother-to-infant HIV transmission was associated with the emergence of lamivudine HBV mutations. The study included 21 pregnant co-infected women in Malawi who received either zidovudine or stavudine plus lamivudine and nevirapine from week 25 of gestation until 6 months after delivery or indefinitely if they met the criteria for treatment (CD4+ <350/mm(3)). HBV-DNA was determined using the Roche COBAS assay. Resistance mutations were assessed by the Trugene assay (Siemens Diagnostics). At baseline 33% of the women were HBeAg positive and had HBV-DNA > 10(4) IU/ml. Median CD4 count was 237 cells/mm(3) and median HIV-RNA was 3.8 log(10) copies/ml. After a median of 259 days of treatment, HBV-DNA was detectable in 9 out of 21 patients (42.8%). In three cases the HBV-DNA level was >10(4) IU/ml. Resistance mutations (M204I in five cases and L180M + M204I/V in one case) were present in 6 (28.6%) patients. Women with a resistant virus had significantly higher baseline HBV-DNA levels than those not developing resistance (1.1 × 10(7) IU/ml vs. 20.8 IU/ml, P = 0.022). Levels of ALT and AST were higher in women with resistant viruses compared to those retaining a wild-type virus. A high rate of lamivudine resistance was seen in this cohort of pregnant women. Follow-up of these patients will clarify if the presence of resistance has a significant impact on liver disease.

Antiretroviral prophylaxis for breastfeeding transmission in Malawi: drug concentrations, virological efficacy and safety.

BACKGROUND: Limited information is available on antiretroviral concentrations in women/infant pairs receiving prophylaxis for breastfeeding transmission of HIV and on the relationship between drug levels and the virological and haematochemistry parameters. METHODS: Patient population included HIV-positive pregnant women receiving antiretroviral prophylaxis from gestational week 25 until 6 months after delivery and their breastfed infants. Blood and breast milk samples were collected at delivery, and at months 1, 3 and 6 postpartum. Drug concentrations were measured by liquid chromatography-mass spectrometry. RESULTS: Overall, 66 women were studied: 29 received zidovudine (ZDV), lamivudine (3TC) and nevirapine (NVP), 28 stavudine (d4T), 3TC and NVP, and 9 ZDV, 3TC and lopinavir/ritonavir (LPV/r). Women who received >9 weeks of pre-partum prophylaxis were significantly more likely to have an undetectable viral load both in plasma and in breast milk at delivery. No emergence of resistance mutations was observed in breast milk. Breast milk/plasma concentration ratios were 0.6 for ZDV, 3TC and NVP, 1.0 for d4T and 0.4 for LPV/r. Only NVP reached significant levels in the infants. No correlation with any adverse events, including infant anaemia, was observed with drug concentrations. Two infants who acquired HIV infection had non-nucleoside reverse transcriptase inhibitor mutations at month 6. CONCLUSIONS: Maternal administration of these three regimens up to 6 months postpartum was effective and safe for both mothers and infants. No significant correlation was found between drug concentrations and infant haematological parameters, supporting the hypothesis that other factors may contribute to the development of anaemia in these settings.