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. 2022 Aug 2;17(8):e0269835. doi: 10.1371/journal.pone.0269835

Prompt HIV diagnosis and antiretroviral treatment in postpartum women is crucial for prevention of mother to child transmission during breastfeeding: Survey results in a high HIV prevalence community in southern Mozambique after the implementation of Option B+

Sheila Fernández-Luis 1,2,*, Laura Fuente-Soro 1,2, Tacilta Nhampossa 1,3, Elisa Lopez-Varela 1,2, Orvalho Augusto 1, Ariel Nhacolo 1, Olalla Vazquez 4, Anna Saura-Lázaro 1,2, Helga Guambe 5, Kwalila Tibana 5, Bernadette Ngeno 6, Adelino José Chingore Juga 7, Jessica Greenberg Cowan 7, Marilena Urso 7, Denise Naniche 1,2
Editor: Rena C Patel8
PMCID: PMC9345360  PMID: 35917332

Abstract

Objective

World Health Organization recommends promoting breastfeeding without restricting its duration among HIV-positive women on lifelong antiretroviral treatment (ART). There is little data on breastfeeding duration and mother to child transmission (MTCT) beyond 24 months. We compared the duration of breastfeeding in HIV-exposed and HIV-unexposed children and we identified factors associated with postpartum-MTCT in a semi-rural population of Mozambique.

Methods

This cross-sectional assessment was conducted from October-2017 to April-2018. Mothers who had given birth within the previous 48-months in the Manhiça district were randomly selected to be surveyed and to receive an HIV-test along with their children. Postpartum MTCT was defined as children with an initial HIV positive result beyond 6 weeks of life who initiated breastfeeding if they had a first negative PCR result during the first 6 weeks of life or whose mother had an estimated date of infection after the child’s birth. Cumulative incidence accounting for right-censoring was used to compare breastfeeding duration in HIV-exposed and unexposed children. Fine-Gray regression was used to assess factors associated with postpartum-MTCT.

Results

Among the 5000 mother-child pairs selected, 69.7% (3486/5000) were located and enrolled. Among those, 27.7% (967/3486) children were HIV-exposed, 62.2% (2169/3486) were HIV-unexposed and for 10.0% (350/3486) HIV-exposure was unknown. Median duration of breastfeeding was 13.0 (95%CI:12.0–14.0) and 20.0 (95%CI:19.0–20.0) months among HIV-exposed and HIV-unexposed children, respectively (p<0.001). Of the 967 HIV-exposed children, 5.3% (51/967) were HIV-positive at the time of the survey. We estimated that 27.5% (14/51) of the MTCT occurred during pregnancy and delivery, 49.0% (2551) postpartum-MTCT and the period of MTCT remained unknown for 23.5% (12/51) of children. In multivariable analysis, mothers’ ART initiation after the date of childbirth was associated (aSHR:9.39 [95%CI:1.75–50.31], p = 0.001), however breastfeeding duration was not associated with postpartum-MTCT (aSHR:0.99 [95%CI:0.96–1.03], p = 0.707).

Conclusion

The risk for postpartum MTCT was nearly tenfold higher in women newly diagnosed and/or initiating ART postpartum. This highlights the importance of sustained HIV screening and prompt ART initiation in postpartum women in Sub-Saharan African countries. Under conditions where HIV-exposed infants born to mothers on ART receive adequate PMTCT, extending breastfeeding duration may be recommended.

Introduction

Globally in 2019 there were 1.7 million children living with HIV [1]. Children predominantly acquire HIV infection through mother-to-child transmission (MTCT), either during pregnancy, delivery, or breastfeeding. Without preventive interventions, the risk of MTCT is 30–40%, and breastfeeding is responsible for one-third to one-half of these transmissions [2]. However, specific preventive interventions such as antiretroviral treatment (ART) to the mother and antiretroviral prophylaxis to the child can reduce MTCT to less than 5%, even in high HIV burden settings [3, 4].

In order to eliminate MTCT, the Joint United Nations Programme on HIV/AIDS (UNAIDS) and United States President’s Emergency Plan for AIDS Relief (PEPFAR) launched the Start Free Stay Free AIDS Free strategy in 2016 [5]. It focuses on the 23 countries with the highest burden of pregnant women and children living with HIV. In 2018, these 23 countries had a rate of MTCT during pregnancy and delivery, (calculated as first 6-week MTCT-rate), of 6.3% [95%CI: 4.9–9.1%] and a rate of MTCT at the end of breastfeeding of 11.8% [95%CI: 9.8–15.2%] [5]. The number of new infections was unevenly distributed; of 130 000 children who acquired HIV in 2018, half came from just six countries (Kenya, Mozambique, Nigeria, South Africa, Uganda and the United Republic of Tanzania) [5].

Breastfeeding contributes substantially to the health, development and survival of young children, particularly in settings with high mortality from diarrhea, pneumonia and malnutrition among children under five years of age [6]. Among HIV-exposed children, breastfeeding has been shown to increase the HIV-free survival at 9 and 18 months of life compared with formula-feeding [7, 8]. Breastfeeding practices of women living with HIV are influenced by the social circumstances in which mothers make decisions, the fear of transmission, and also the attitudes, knowledge, and updating of health care workers about current guidelines [9–11].

World Health Organization (WHO) adapted its guidelines over time, aiming to balance the benefits of breastfeeding with the risks of HIV transmission. In 2007, WHO recommended exclusive breastfeeding for six months unless replacement feeding was feasible, sustainable and safe for the HIV-positive mothers and their infants [12]. In 2010, the recommendation was extended to 12 months, while antiretrovirals were provided either to the mother or the infant throughout breastfeeding to reduce the risk of postnatal HIV transmission [13]. By 2016, the B+ strategy was brought to scale globally, ensuring that the HIV-positive pregnant and breastfeeding women were offered lifelong ART regardless of their CD4 count. At the same time, enhanced post-natal prophylaxis with daily zidovudine (ZDV) and nevirapine (NVP) for a total of 12 weeks was recommended among HIV-exposed children at high risk of acquiring HIV who were breastfeeding [14]. With the expansion of durable ART access for lactating women, WHO again updated its recommendations, advising that HIV+ women should continue to breastfeed for at least 12 months, but ideally for up to 24 months or longer while remaining fully ART adherent [15]. However, this revised guidance promoting extended breastfeeding was based on low to moderate quality evidence [15].

Mozambique adopted the B+ strategy in 2013 [16] and the “test and treat” strategy, in which ART is initiated for all people living with HIV as soon as possible after diagnosis, in 2016 [17]. Nevertheless, in 2018, although more than 95% of HIV-positive pregnant women in Mozambique received ART, the rate of MTCT at the end of breastfeeding was 15.0% [95% CI: 11.8–19.0%]:7% during the first 6-weeks postpartum and 8% during breastfeeding [5], accounting for 10% of global MTCT infections among the Start Free Stay Free AIDS Free priority countries [5]. In 2011, the median duration of total breastfeeding in the general population of Mozambique was 20.8 months [18]. However, there are no updated data on total duration of breastfeeding disaggregated by mother’s HIV-serostatus or on the impact of breastfeeding duration on mother-to-child transmission in Mozambique. Although there are no data about ART adherence and viral load during breastfeeding period at national level, a controlled clinical trial performed in central Mozambique between 2014–2015 among pregnant and breastfeeding women in B+ showed that without intervention, 52.3%, 46.1% and 38.3% of them returned for 30-day, 60-day and 90-day ART refills, respectively [19]. In the other hand, PEPFAR data showed viral load coverage in pregnant women was approximately 60% and viral suppression 80% at national level in 2020 [20]. Until September 2019, postnatal prophylaxis consisted of 6 weeks NVP to all infants [21, 22] and thereafter enhanced postnatal prophylaxis has been recommended to all HIV exposed infants in Mozambique [23].

We compared the duration of breastfeeding in HIV-exposed and HIV-unexposed children and we identified sociodemographic and HIV-care factors associated with postpartum MTCT, through a cross-sectional household survey in a semi-rural population of southern Mozambique with a high HIV community prevalence [24].

Methods

Study setting

The study was conducted within the Health and Demographic Surveillance System (HDSS) run by the Manhiça Research Health Center since 1996, which is located in Maputo Province, southern Mozambique [25]. The HDSS platform currently extends over the entire district of Manhiça, which has an area of 2,380 square kilometers and covers 46,441 households and 201,383 inhabitants, each one with a unique identification number. Every household is visited twice a year to collect data on vital events such as births, deaths, pregnancies and migrations [25]. Verbal autopsies are used to attribute a cause of death to all recorded death events, including those that occurred in the community, in accordance with WHO Verbal Autopsies Instrument Form 2016 [26].

The Manhiça District is served by fifteen health centers, one rural hospital and one referral district hospital. All public health facilities offer free access to HIV care and treatment. Routine patient-level HIV clinical data is recorded by providers in a paper-based system and prospectively entered into an electronic patient tracking system.

At the time of the study, the B+ strategy was already implemented in all the health facilities which provided free ART to HIV-positive pregnant or breastfeeding mothers and 6 week Nevirapine prophylaxis for HIV-exposed children, regardless of both the feeding method and whether the mother’s diagnosis and ART initiation occurred during pregnancy or breastfeeding period [21, 27]. The ART regimen that most pregnant and lactating women received during the time period of this study was Tenofovir Disoproxil Fumarate/Lamivudine/Efavirenz (TDF+3TC+EFV) [21, 27].

Study design and study population

Between October 2017 and April 2018, 5000 of the total children born alive in the previous 48 months within the HDSS were randomly selected to participate in this cross-sectional household survey. After informed consent was obtained, the survey was conducted with mothers, or in case of a mother’s absence, migration or death, with the child’s primary caregiver. Study HIV counselors administered a specific questionnaire designed to capture sociodemographic characteristics, HIV testing history and ART, antenatal care and duration of breastfeeding.

For each individual mother and child, HIV-status was ascertained through documentation of previous testing, conducting age-appropriate testing with laboratory confirmation or verbal autopsy. Mothers who do not know their status or self-report being HIV-negative were tested at survey, as well as the HIV-exposed children. For children under 18 months of age, HIV diagnosis was determined with molecular testing through HIV DNA Polymerase Chain Reaction (PCR). Children 18 months or older and mothers were tested following the National HIV testing algorithm [21] which included two serial rapid diagnostic tests, Determine [28] and Unigold [29]. Documented known HIV-positive individuals were not re-tested, however for study purposes, all HIV positive participants (including those who were diagnosed prior to or during the study visit) underwent confirmatory testing through Geenius HIV-1/2 Confirmatory Assay [30]. Clinical documentation was also used to obtain information about gestational age and infant antiretroviral prophylaxis. Verbal autopsy from HDSS database was used to ascertain HIV status in children and mothers who had died before the survey. Hospitalizations and outpatients’ visits were also obtained through the HDSS database. Information about maternal viral load and CD4 was extracted from the routinely collected data in the electronic patient tracking system, a Microsoft access database [31] co-managed by the Ministry of Health and other stakeholders, where each participant living with HIV had a unique numeric identifier that allows follow-up through the continuum of care [32].

Definitions

HIV exposure was defined as follows: i) a child whose mother had a documented HIV-infection before birth or at the end of breastfeeding (confirmed exposure) and ii) a child born to a self-reported HIV-positive mother for whom the time of the mother’s infection could not be determined (probable exposure). Children born from HIV negative mothers were considered HIV-unexposed. If the mother was deceased and her HIV-status could not be confirmed, the child´s exposure was considered unknown and were excluded from the analysis.

Date of HIV infection in the mother was estimated as follows:

  1. In case of documentation of a previous HIV-negative test, date of infection was assumed to be equal to the midpoint between the last negative HIV test and the day of the survey for mothers of children who are less than 23 months of age. If the interval between the test and the survey was less than 24 months, the midpoint between last negative HIV test and first positive test was used. If the interval was greater than 24 months, the date of seroconversion was not estimated due to the larger uncertainty in the estimation.

  2. In case of documentation of a previous HIV-positive test, this was assumed to be the date of infection.

  3. In case of no previous documentation and a first positive test on the day of the survey, time of infection was defined as the midpoint between serosurvey and date of most recent delivery.

MTCT was assumed to occur during pregnancy and delivery if the child had a positive PCR result during the first 6 weeks of life [15, 33, 34]. Postpartum MTCT was defined for children with an initial HIV positive result beyond 6 weeks of life who initiated breastfeeding if 1) they had a first negative PCR during the first 6 weeks of life, or 2) did not have a prior negative PCR but whose mother had an estimated date of infection after the child’s birth. For children born to mothers with date of infection prior to child’s birth but without a DNA PCR by 6 weeks of age, the date of MTCT was considered unknown.

Breastfeeding included any type of breastfeeding (exclusive, mixed and any breastfeeding after the introduction of complementary feeding) since birth. The mother or caregiver self-reported the total duration of any breastfeeding in months at the time of survey.

Statistical analysis

Medians and interquartile ranges (IQR) were calculated to describe continuous variables and categorical variables were summarized using frequencies and its 95% confidence intervals. Comparisons between groups were made using Pearson chi-square or Fisher exact test and Kruskal Wallis tests, as applicable. In addition, we performed two analyses:

First, we estimated breastfeeding duration in HIV-exposed and HIV-unexposed children with cumulative incidence of breastfeeding cessation, accounting for right censoring. Children who had not initiated breastfeeding were excluded from this analysis. HIV-exposure was evaluated as a factor associated with breastfeeding duration through Fine-Gray regression, using mortality as competing risk and adjusted for age and sex in a multivariable model.

Second, among HIV-exposed children who had been breastfed at any time, we performed a Fine-Gray regression analysis to assess factors associated with postpartum MTCT, adjusting for age and sex and considering mortality a competing risk factor. Infants with MTCT during pregnancy and delivery and children in which it was not possible to establish whether MTCT was during pregnancy and delivery or postpartum were excluded from this analysis. A multivariable model was built including the variables with a p-value lower than 0.20 in the bivariate analysis and with less than 20% missing values. Time-varying covariates were handled by episode splitting. Variables age of the child, sex of the child, mother ART initiation and breastfeeding duration were forced-in covariates due to their clinical relevance. The variable ‘mother ART initiation’ was treated as a binary variable: ART initiation before delivery yes/no, and had more than 20% missing values. The missing data was addressed through multiple imputation using a logistic regression imputation method including our outcome variable and the other predictor variables. A total of 20 imputations were performed.

Data was analyzed using Stata statistical software version 16 (Stata Corp., College Station, Texas, USA) [35].

We conducted a sensitivity analysis considering the time of infection of the mother as random date selected from a uniform distribution, a point at the quarter of the interval between the two dates considered at definition and a point at the three-quarters of the interval between the two dates specified above in definitions section.

We conduct another two sensitivity analysis considering the 61 children HIV-exposed with unknown HIV serostatus as HIV-positive and the 12 children HIV-positive with no information on time of HIV acquisition as postpartum MTCT, respectively.

Ethics statement

This study was approved by the Mozambican National Bioethics Committee and the Barcelona Hospital Clinic Institutional Review Board. It was also reviewed in accordance with CDC human research protection procedures and was determined to be research, but CDC investigators did not interact with human subjects or have access to identifiable data or specimens for research purposes. Written informed consent was obtained from the mothers/caregivers of all children for the mothers/caregiver and children participation. In case of mothers between 14–16 years old, informed consent was provided by the legal representative of the young mother, after the mother’s consent.

Results

Among the 5000 mother/caregiver-child pairs randomly selected for participation, 4826 children were eligible (96.5%) and 174 children were older than 54 months at the time of the visit, and excluded of the study. A total of 1340 mother/child pairs were not located and 3486 were enrolled. Among those, 27.7% (967/3486) children were considered to be HIV-exposed (probable or confirmed), 62.2% (2169/3486) were HIV-unexposed and for 10.0% (350/3486) HIV-exposure was unknown (Fig 1). Taking into account the estimated date of infection of the mother according to assumptions in methods, 77.7% (751/967) were considered exposed to HIV during pregnancy and delivery, 13.2% (128/967) were exposed only in the postpartum period and 9.1% (88/967) were unknown. Among the children exposed only in the postpartum period, 47.6% (61/128) were characterized as HIV-exposed based on maternal HIV testing performed during the study visit and 14% (18/128) were still breastfeeding when their mothers started on ART.

Fig 1. Study profile among the 5000 mother-child pairs randomly selected for the study.

Fig 1

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Percentages are calculated over the previous parent box. MTCT = maternal to child transmission. MTCT during pregnancy and delivery was defined as having a positive PCR result during the first 6 weeks of life. Postpartum MTCT was defined for children with an HIV positive result beyond 6 weeks of life who initiated breastfeeding if 1) they had a first negative PCR during the first 6 weeks of life, or 2) did not have a prior negative PCR but whose mother had an estimated date of infection after the child’s birth.

Baseline characteristics differed between HIV-positive and HIV-negative mothers, and their respective children, with the exception of place of birth, child’s gender, gestational age and age of the child at the time of study visit (Table 1).

Table 1. Characteristics of HIV-exposed and HIV-unexposed children at the time of the survey (n = 3136).

The 350 children for whom HIV exposure could not be ascertained are not included.

HIV-UNEXPOSED (N = 2169) HIV-EXPOSED (N = 967) TOTAL (N = 3136)  
N % N % N % p value
MOTHER
Age of the mother at delivery in years (IQR) * 22.6 (18.8–29.3) 28.7 (23.4–33.4) 24.8 (19.7–31.2) <0.001
Mother located during the household survey ** Yes 2164 99.8% 871 90.1% 3035 96.8% <0.001
Absent or migrated 0 0.0% 85 8.8% 85 2.7%
Died 5 0.2% 11 1.1% 16 0.5%
    Education level** Illiteracy 295 13.6% 189 19.5% 484 15.4% <0.001
  Primary 1555 71.7% 674 69.7% 2229 71.1%
  Seconday or higher 317 14.6% 91 9.4% 408 13.0%
  Unknown 2 0.1% 13 1.3% 15 0.5%
    Marital status*** Single 265 12.2% 121 12.5% 386 12.3% <0.001
  Married 1737 80.1% 693 71.7% 2430 77.5%
  Divorced/Widowed 167 7.7% 153 15.8% 320 10.2%
    Main source of Income** Domestic 66 3.0% 10 1.0% 76 2.4% <0.001
  No fix salary/agriculture 974 44.9% 497 51.4% 1471 46.9%
  Fix Salary 1129 52.1% 458 47.4% 1587 50.6%
  Unknown 0 0.0% 2 0.2% 2 0.1%
    Parity** Primipara 723 33.3% 136 14.1% 859 27.4% <0.001
  Secundipara 458 21.1% 184 19.0% 642 20.5%
  Multipara 987 45.5% 647 66.9% 1634 52.1%
  Unknown 1 0.0% 0 0.0% 1 0.0%
    Antenatal clinic visit** No 152 7.0% 18 1.9% 170 5.4% <0.001
Yes 2017 93.0% 949 98.1% 2966 94.6%
CHILD
    Child found Yes 2107 97.1% 920 95.1% 3027 96.5% 0.018
Absent or migrated 23 1.1% 15 1.6% 38 1.2%
Died 39 1.8% 32 3.3% 71 2.3%
    Age at survey in months (IQR) 23.5 (14.5–35.3) 24.6 (15.7–36.4) 23.9 (14.9–35.7) 0.053
    Gender Female 1126 51.9% 479 49.5% 1605 51.2% 0.218
  Male 1043 48.1% 488 50.5% 1531 48.8%
    Born in Mozambique No 38 1.8% 24 2.5% 62 2.0% 0.175
  Yes 2131 98.2% 943 97.5% 3074 98.0%
    Gestational Age <37 weeks 153 7.1% 79 8.2% 232 7.4% 0.088
  ≥ 37 weeks 1243 57.3% 514 53.2% 1757 56.0%
  Unknown 773 35.6% 374 38.7% 1147 36.6%
    Birth order of baby 1–3 1524 70.3% 538 55.6% 2062 65.8% <0.001
  >3 644 29.7% 429 44.4% 1073 34.2%
  Unknown 1 0.0% 0 0.0% 1 0.0%
        Breastfeeding at any time No 11 0.5% 28 2,9% 39 1.2% <0.001
Yes 2158 99.5% 939 97,1% 3097 98.8%
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IQR: Interquartile Range.

*Kruskal Wallis test.

**Fisher exact test.

***Pearson chi-square

HIV positive mothers were significantly more absent from the household at the time of survey with the caregiver responding, as compared to HIV-negative mothers (8.8% vs 0.0%, p<0.001). HIV-positive mothers were significantly older, with a median age of 28.7 years (IQR: 23.4–33.4) compared with HIV-negative mothers whose median age at survey was 22.6 years (IQR: 18.8–29.3), p<0.001. Almost all the mothers had attended at least one antenatal visit, but the proportion was higher among the HIV-positive mothers (98.1% vs 93.0%, p<0.001).

Only 34.1% (330/967) of HIV-positive mothers had at least one viral load result at the time of survey. However, 75.8% (250/330) of the mothers with viral load results were virally suppressed.

Breastfeeding duration among HIV-exposed and HIV-unexposed children

In our cohort, the proportion of children who had never breastfed was significantly higher among HIV-exposed children (2.9%, 28/967), than among HIV-unexposed children (0.5%, 11/2169), p<0.001 (Table 1).

The median duration of breastfeeding was 13.0 (95%CI: 12.0–14.0) and 20.0 months (95%CI: 19.0–20.0) among HIV-exposed and HIV-unexposed children respectively, and the risk of discontinuing breastfeeding was almost two-fold higher among HIV-exposed children [adjusted Sub-Hazard Ratio (aSHR) 1.85 (95%CI: 1.67–2.05), p<0.001] (Fig 2). The cumulative incidence of HIV-exposed children breastfeeding dropped at 6 months as compared to unexposed children. At 12 months, only 56.4% (95%CI: 53.2%-59.5%) of HIV-exposed infants were breastfeeding as compared to 77.3% (75.4%-79.0%) of unexposed infants. The gap in breastfeeding between HIV exposed and unexposed children continued through to 18 months of age.

Fig 2. Breastfeeding duration among HIV-exposed and HIV-unexposed breastfed children.

Fig 2

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Cumulative incidence was expressed as the proportion of children breastfeeding in a given time period after birth accounting for right censoring and adjusted by sex and age of the child. Children who had not initiated breastfeeding were excluded from this analysis. N = 3097.

Postpartum MTCT and associated factors

Of the 967 HIV-exposed children, 5.3% (51/967) were HIV-positive, 88.4% (855/967) were HIV-negative and 6.2% (61/967) with unknown serostatus at the time of the survey. Among the HIV-positive, according to the definitions in methods, we estimated that 49.0% (25/51) of the MTCT occurred postpartum, 27.5% (14/51) during pregnancy and delivery and for 23.5% (12/51) of children the period of infection remained unknown.

Among the 61 children with unknown HIV serostatus, 62.3% (38/61) were female, 4 never breastfed and among the 57 who initiated breastfeeding, the median duration of breastfeeding was 12 (95%CI: 10.4–15.1) months. From 12 of children with unknown period of MTCT, 41.7% (5/12) were female, and all had initiated breastfeeding with the median duration being 15.0 months, (95%CI: 5.6–24.0).

The estimation of the period of MTCT among the 51 HIV-positive children remained the same regardless of the method of estimating the infection time of the mother (as random date selected from a uniform distribution, as a point at the quarter of the interval between the two dates considered at definition and as a point at the three-quarters of the interval between the two dates specified above in definitions section).

Table 2 shows the sociodemographic and clinical characteristics among children with postpartum MTCT compared to HIV-exposed uninfected children, after the exclusion of children with unknown HIV-status, children with MTCT during pregnancy or delivery, children with HIV infection without attribution to pregnancy, delivery or breastfeeding and children who never initiated breastfeeding.

Table 2. Mother and child clinical and sociodemographic characteristics of HIV-exposed uninfected children and HIV-exposed children who acquired HIV through postpartum MTCT.

N = 856. Children who never breastfed, children with HIV unknown status, children who acquired HIV through MTCT during pregnancy and delivery and HIV positive children with no information on time of HIV acquisition were excluded from this analysis.

HIV-exposed uninfected children (N = 831) HIV exposed children with postpartum MTCT (N = 25) TOTAL (N = 856)
 
Characteristics N % N % N % p value
MOTHER
    Age of the mother at delivery in years. Median (IQR)* 28.7 (23.4–33.4) 26.5 (22.4–33.0) 28.7 (23.4–33.3) 0.577
Mother located during the household survey ** yes 748 90.0% 23 92.0% 771 90.1% 1.000
absent or migrated 75 9.0% 2 8.0% 77 9.0%
died 8 1.0% 0 0.0% 8 0.9%
    Education level** Illiteracy 171 20.6% 3 12.0% 174 20.3% 0.403
  Primary 579 69.7% 18 72.0% 597 69.7%
  Seconday or higher 68 8.2% 4 16.0% 72 8.4%
  Unknown 13 1.6% 0 0.0% 13 1.5%
    Marital status** Single 112 13.5% 1 4.0% 113 13.2% 0.321
  Married 586 70.5% 21 84.0% 607 70.9%
  Divorced/Widowed 133 16.0% 3 12.0% 136 15.9%
    Main source of Income** Domestic 6 0.7% 0 0.0% 6 0.7% 0.456
  No fix salary/agriculture 428 51.5% 16 64.0% 444 51.9%
  Fix Salary 395 47.5% 9 36.0% 404 47.3%
  Unknown 2 0.2% 0 0.0% 2 0.2%
    Parity** Primipara 116 14.0% 4 16.0% 120 14.0% 0.906
  Secundipara 152 18.3% 4 16.0% 156 18.2%
  Multipara 563 67.7% 17 68.0% 580 67.8%
    Antenatal clinic visit** No 14 1.7% 1 4.0% 15 1.8% 0.361
Yes 817 98.3% 24 96.0% 841 98.2%
    Mother HIV diagnosis** Before the date of childbirth 653 78.6% 9 36.0% 662 77.3% <0.001
  After the date of childbirth 102 12.3% 15 60.0% 117 13.7%  
  Unknown 76 9.1% 1 4.0% 77 9.0%  
    Mother ART initiation*** Before the date of childbirth 513 61.7% 6 24.0% 519 60.6% <0.001
  After the date of childbirth 95 11.4% 13 52.0% 108 12.6%  
  Unknown 223 26.8% 6 24.0% 229 26.8%  
    Mother CD4 at childbirth** <200 cel/mm3 42 5.1% 4 16.0% 46 5.4% 0.083
  200–500 cel/mm3 175 21.1% 5 20.0% 180 21.0%  
  >500 cel/mm3 327 39.4% 6 24.0% 333 38.9%  
  Unknown 287 34.5% 10 40.0% 297 34.7%  
    Mother viral load at childbirth** <1000copies/ml 247 29.7% 7 28.0% 254 29.7% 0.352
  ≥1000copies/ml 45 5.4% 3 12.0% 48 5.6%  
  Unknown 539 64.9% 15 60.0% 554 64.7%  
CHILD
    Child located during the household survey** yes 813 97.8% 20 80.0% 833 97.3% <0.001
absent or migrated 2 0.2% 0 0.0% 2 0.2%
died 16 1.9% 5 20.0% 21 2.5%
    Age at survey in months. Median (IQR) 24.6 (15.6–35.6) 30.0 (20.9–34.2) 24.6 (15.7–36.5) 0.369
    Gender*** Female 420 50.5% 9 36.0% 429 50.1% 0.152
  Male 411 49.5% 16 64.0% 427 49.9%
    Born in Mozambique** No 19 2.3% 1 4.0% 20 2.3% 0.451
  Yes 812 97.7% 24 96.0% 836 97.7%
    Gestational Age** <37 weeks 67 8.1% 2 8.0% 69 8.1% 0.345
  ≥ 37 weeks 451 54.3% 10 40.0% 461 53.9%
  Unknown 313 37.7% 13 52.0% 326 38.1%
    Birth order of baby *** 1–3 463 55.7% 13 52.0% 476 55.6% 0.713
  >3 368 44.3% 12 48.0% 380 44.4%
    Number of Outpatient visits before survey*** 0 374 45.0% 5 20.0% 379 44.3% 0.021
1 92 11.1% 6 24.0% 98 11.5%  
≥2 365 43.9% 14 56.0% 379 44.3%  
    Number of Hospitalizations before survey** 0 783 94.2% 18 72.0% 801 93.6% 0.001
1 39 4.7% 5 20.0% 44 5.1%  
≥2 9 1.1% 2 8.0% 11 1.3%  
    Duration of breastfeeding in months. Median (IQR)   12.0 (8.0–17.0) 12.2 (12.0–18.0) 12.0 (8.0–17.0) 0.310
    Received Antiretroviral prophylaxis** Yes 688 82.8% 13 52.0% 132 15.4% <0.001
No 20 2.4% 3 12.0% 701 81.9%  
Unknown 123 14.8% 9 36.0% 23 2.7%  
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IQR: Interquartile Range.

*Kruskal Wallis test.

**Fisher exact test.

***Pearson chi-square

Sociodemographic data (Age, Eductional level, Marital status, Income, Parity), antenatal clinic visits and breastfeeding information were self-reported.

HIV data were obtained through medical documentation (Mother HIV diagnosis, Mother ART initiation) or HIV database (Mother cd4 at child birth, Mother viral load at child birth). Hospitalizations and outpatients’ visits were obtained through HDSS database.

Gestational Age and Infant Antiretroviral prophylaxis were obtained through medical documentation.

Mothers with postpartum MTCT were mostly diagnosed (60.0%) and initiated ART (52.0%) after the child was born, compared with the 12.3% and 11.4% of diagnosis and ART initiation after the birth among the mothers of HIV exposed uninfected children, p<0.001. Furthermore, only 52.0% (13/25) of children with postpartum MTCT had received antiretroviral prophylaxis at any time after birth compared to 82.8% (688/831) of HIV-exposed uninfected children, p<0.001.

A total of 80.0% (20/25) of children with postpartum-MTCT had attended at least one unscheduled outpatient visits before the survey and a total of 28.0% (7/25) had been hospitalized, compared with the 55.0% (457/831) and 5.8% (48/831) of the HIV exposed uninfected children p = 0.021 and p = 0.001, respectively.

Table 3 shows bivariable and multivariable analysis of factors associated with postpartum MTCT after adjusting for child’s sex and age at survey. Children with unknown HIV-status, children with MTCT during pregnancy or delivery and children who never initiated breastfeeding were excluded from this analysis.

Table 3. Mother and child clinical and sociodemographic risk factors associated with postpartum MTCT among HIV-exposed children.

N = 856. Fine-Gray subdistribution hazard regression with death as a competing risk was conducted. The same exclusion factors as those described in Table 2 were applied. The multivariable model was built of including the variables with a p-value lower than 0.20 in the bivariate analysis and with less than 20% missing values. Variables age of child, mother ART initiation and breastfeeding duration were forced-in covariates due to their clinical relevance. Multiple imputation was performed in mother ART initiation. Mother HIV diagnosis was excluded because of collinearity.

Factors Univariable Model* Multivariable Model**
SHR (95% Conf. Interval) p-value aSHR (95% Conf. Interval) p-value
Mother
    Age of the mother at delivery (in years) 0.99 0.93–1.05 0.627
    Education level (N = 843) No education 1   0.284      
  Basic 1.76 0.52–5.96      
  Medium/High 3.31 0.74–14.82      
    Marital status Single 1   0.333      
  Married 3.95 0.53–29.39      
  Divorced/Widowed 2.52 0.26–24.24      
    Parity Primipara 1   0.931      
  Secundipara 0.77 0.19–3.07      
  Multipara 0.87 0.29–2.60      
    Antenatal clinic visit No 1   0.398      
Yes 0.44 0.06–3.01      
    Mother HIV diagnosis1 (N = 779) before the date of childbirth 1   0.008      
  after the date of childbirth 4.20 1.46–12.06      
    Mother ART initiation1 before the date of childbirth 1   <0.001      
  after the date of childbirth 9.18 3.87–21.80 9.39 1.75–50.31 0.009
    Mother cd4 at childbirth (N = 559) <200 cel/mm3 1          
  200–500 cel/mm3 0.32 0.09–1.16 0.042      
  >500 cel/mm3 0.20 0.06–0.71        
    Mother viral load at childbirth (N = 302) <1000copies/ml 1          
  ≥1000copies/ml 2.30 0.60–8.82 0.226      
Children
    Age at survey in months   1.01 0.99–1.04 0.348 1.01 0.97–1.06 0.577
    Gender Male 1   0.160 1    
  Female 1.80 0.79–4.06 1.99 0.67–5.96 0.218
    Gestational Age (N = 530) <37 weeks 1   0.699      
  ≥ 37 weeks 0.74 0.16–3.40      
    Birth order of baby 1–3 1   0.722      
  >3 1.15 0.53–2.52      
    Born in Mozambique No 1   0.575      
  Yes 0.56 0.07–4.24      
    Median time of breastfeeding   1.01 0.98–1.05 0.557 0.99 0.96–1.03 0.707
    Received Antiretroviral prophylaxis (N = 724) Yes 1     1    
No 7.25 2.13–24.73 0.002 3.01 0.55–16.54 0.205
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*N = 856 unless otherwise specified

** N = 818

SHR: subhazard ratio.

aSHR:adjusted subhazard ratio.

1 Time-varying covariates. Time-varying covariates were handled by episode splitting

Our results show that the duration of breastfeeding was not associated with postpartum MTCT (aSHR: 0.99 [95%CI: 0.96–1.03], p = 0.707). By contrast, children born from mothers who initiated ART at any time after delivery were more likely to acquire HIV postpartum (aSHR: 9.39 [95%CI: 1.75–50.31], p = 0.009).

The estimations obtained in postpartum MTCT related factors were not impacted when we performed sensitivity analysis including the children with unknown HIV status (61 children) or the children with unknown period of HIV infection (12 children): breastfeeding was not associated with postpartum MTCT (aSHR: 0.99 [95%CI: 0.96–1.02] p = 0.547 and aSHR: 0.99 [95%CI: 0.96–1.03] p = 0.660, respectively) and children born from mothers who initiated ART at any time after delivery were more likely to acquire HIV postpartum (aSHR: 8.77 [95%CI: 1.61–47.77] p = 0.012 and aSHR: 8.67 [95%CI: 1.64–45.87] p = 0.011, respectively).

Discussion

In this study, HIV-exposed children breastfed for significantly less time and had a nearly two-fold higher risk of discontinuation of breastfeeding over 48 months compared with non-exposed children. Evidence of association between breastfeeding duration and postpartum MTCT was not observed. In contrast, mother ART initiation after the date of child birth was associated with a nearly ten-fold higher risk of postpartum MTCT (aSHR: 9.39 [95%CI: 1.75–50.31], p = 0.009).

Our results demonstrate that children born from mothers who initiated ART after childbirth had a higher risk of acquiring HIV during the breastfeeding period, as previously indicated by other studies [36]. At the time of the study, the B+ strategy was already implemented and lifelong ART was recommended to HIV-positive pregnant or breastfeeding mothers [21], [27]. However, success of the B+ strategy in reaching all pregnant and breastfeeding women is highly dependent on a sustained frequency of HIV testing not only during pregnancy but during the post-partum period. The reasons for not initiating ART could be multiple: others may not have initiated lack of awareness of HIV status, death, not willing or because of service delivery shortfalls or stockouts. A study conducted in southern Mozambique between 2008 to 2011 before B+ implementation, found an HIV incidence in women of 3.2/100 women-years (95%CI: 2.30–4.46) in breastfeeding women during the postpartum period. In absence of treatment, this was reflected by a postpartum-MTCT rate of 21% at 18-months of age among their children [37]. In Mozambique, at the time of the study, re-testing in all pregnant women every three months during pregnancy was recommended, however, delivery and the postpartum period were not targeted time points for re-testing [38, 39]. Our results suggest that establishing specific retesting times during the postpartum period in areas of high HIV incidence could reinforce the prevention of MTCT in LMIC, particularly in areas of high HIV prevalence where the risk of contracting HIV during the breastfeeding period is high. This would facilitate initiation of ART in breastfeeding mothers and antiretroviral prophylaxis in their HIV exposed infants. In addition to retesting, pre-exposure prophylaxis (PrEP) among women who remain at risk for HIV acquisition in the postpartum period may be an effective approach to reduce MTCT. In 2018 Mozambique began with the pilot implementation of PrEP in sero-discordant couples in Zambezia province and will expand PrEP nationally in 2022 to additional target groups, including pregnant and breastfeeding women at risk and key populations [40].

The vast majority (>97%) of the mothers in our study initiated breastfeeding. This is in agreement with data from Demographic and Health Surveys (2000–2013) for 57 countries which showed a consistently high percentage of children who had ever breastfed across all regions (weighted mean 98.2%, range of countries 87.8–99.8%) [41]. In terms of duration of breastfeeding, in a study conducted in 2019 in an urban population of South Africa, similar to our results, the duration of breastfeeding was also significantly lower among HIV-positive mothers as compared to HIV-negative mothers [3.9 months vs 9.0 months, respectively, p<0.001] [42]. The striking difference in the duration of breastfeeding between women in our study and those studies by Roux et al are likely due to shorter breastfeeding duration in urban compared to rural populations, compounded by social and contextual barriers, as previously described [43, 44]. Advice of health workers, influence of relatives, stigma, conflicting opinions about the risk for MTCT and poor dissemination of policies have been described as main reasons affecting breastfeeding among HIV-positive women in the past [45–47]. However, little is known about the barriers for prolonged breastfeeding after the implementation of the 2016 WHO feeding guidelines.

The higher risk of discontinuing breastfeeding in HIV-exposed children could have important health implications for this population. A clinical trial conducted in Uganda demonstrated higher rates of serious gastroenteritis among HIV-exposed uninfected infants with early breastfeeding cessation (8.0/1000 child-months) when compared to later breastfeeding cessation (3.1/1000 child-months; p<0.001) [48]. In addition, early cessation of breastfeeding is associated with a lower probability of HIV-free survival compared with longer breastfed infants who had lower overall mortality [44].

Our results suggest that in the context of B+ (lifelong ART to the mother and antiretroviral prophylaxis to the children), duration of breastfeeding is not associated with an increased risk of postpartum MTCT. Bispo et al (2017) in their systematic review found a pooled estimated rate of overall HIV transmission by age six months of 3.5% and a pooled postnatal transmission rate by six months of 1.1% in women who were on ART from early-mid pregnancy and breastfed for 6 months [4]. To our knowledge, no such study has been published after the expansion of B+ prevention MTCT programs. Thus, our results fill the gap in knowledge on the risk of MTCT associated with breastfeeding beyond 6 months of age in the context of B+ strategy that recommends lifelong antiretroviral treatment for all pregnant and breastfeeding women living with HIV.

Effective strategies to increase the duration of safe breastfeeding in HIV-exposed children could allow them to reap the benefits of breastfeeding through the second year of life such as decreased morbidity and mortality in comparison to HIV exposed infants who are weaned earlier [49, 50]. Highlighted strategies proposed by the literature to promote breastfeeding among HIV-exposed children include increased coverage of extended nevirapine prophylaxis to the infants, active support and breastfeeding counseling [51]. Other strategies such as widespread access to viral load testing could mitigate the fear of HIV transmission, reported by mothers and health workers as a barrier to breastfeeding [52]. In the absence of viral load testing, expansion of antiretroviral prophylaxis among HIV-exposed children until the end of breastfeeding i one strategy supported by literature [44].

This study has several limitations. First, 27% of the randomly selected mother/child pairs were not located and we do not have information about their HIV status. Second, breastfeeding duration was self-reported (in months) by mothers/caregivers for both HIV-exposed and HIV non-exposed children patients at the time of the survey. As we have analyzed children born up to 48 months prior to data collection, potential memory bias could affect the estimations of breastfeeding duration. Third, due to missing data, it was not possible to establish the HIV status of 6.2% (61/967) of HIV exposed children and it was not possible to establish the period of MTCT (during pregnancy and delivery or during breastfeeding) of 23.5% (12/51) of HIV infected children. We conducted a sensitivity analysis considering the 61 children with unknown HIV serostatus as HIV-positive and the 12 HIV-infected children with unknown period of MTCT as postpartum MTCT. It did not impact the estimates of postpartum-MTCT and associated factors, even if the 12 with had a median duration of breastfeeding of 15 months (95%CI: 5.6–24.0) Further studies with larger sample size and breastfeeding periods of 24 months or longer are needed. Fourth, the exact date of mothers’ seroconversion was unknown and was established according to the assumptions described in the methods section. A sensitivity analysis considering the time of infection as random date selected from a uniform distribution, a point at the quarter of the interval between the two dates and a point at the three-quarters of the interval between the two dates was robust and did not impact the classification of time of HIV acquisition among HIV-positive children. Fifth, service delivery shortfalls or stockouts of ART were not assessed during the study, which may have affected the ART initiation of among HIV-positive mothers and ARV prophylaxis among HIV-exposed infants. Finally, in Mozambique viral load became routinely available after 2016, and viral load coverage has slowly climbed over time. As a result, more than 60% of HIV infected mothers included in the study did not have any viral load results prior to the survey, thus no adjustments by viral suppression were possible in the multivariable model.

Conclusion

The risk for postpartum MTCT was nearly tenfold higher in women who were newly diagnosed or initiated ART during the postpartum period as compared to those who initiated ART prior to childbirth. HIV-exposed children breastfed for significantly less time compared with non-exposed children. Breastfeeding duration was not observed to be associated with an increased risk of MTCT in the postpartum period in women on ART. These results emphasize the importance of repeat HIV testing in the postpartum period, and support PrEP in breastfeeding women at high risk for HIV infection. Moreover, further public health messaging to encourage prolonged breastfeeding among HIV-exposed infants born to mothers adherent to ART in Sub-Saharan African countries could bring long term health benefits for children.

Supporting information

S1 Appendix. Study questionnaires in English.

(ZIP)

Click here for additional data file. (678.9KB, zip)
S2 Appendix. Study questionnaires in Portuguese.

(ZIP)

Click here for additional data file. (403.4KB, zip)

Acknowledgments

We acknowledge support from the Spanish Ministry of Science, Innovation and Universities through the “Centro de Excelencia Severo Ochoa 2019–2023” Program (CEX2018-000806-S), and support from the Generalitat de Catalunya through the CERCA Program. We want specially acknowledge Elisabeth Salvo for their contributions to this work. The authors gratefully acknowledge the Ministry of Health of Mozambique, our research team, collaborators, and especially all communities and participants involved.

Data Availability

Data cannot be shared publicly because of ethical restrictions. Data contain potentially sensitive information and national ethics committee (CNBS) does not authorize data sharing without a protocol request specifying the objectives and the researchers who will have access to the data. Data are available under request (contact via llorenc.quinto@isglobal.org) for researchers who meet the criteria for access to confidential data. We are sharing a copy, in both the original language and English of the questionnaire of the study as Supporting Information.

Funding Statement

This research has been supported by the President's Emergency Plan for AIDS Relief (PEPFAR) through the Center for Disease Control (CDC) under the terms of CoAg GH000479. The findings and conclusions in this report are those of the author(s) and do not necessarily represent the official position of the CDC. S.F.L. receives a pre-doctoral fellowship from the Secretariat of Universities and Research, Ministry of Enterprise and Knowledge of the Government of Catalonia and cofounded by European Social Fund. E.L.V. is supported by a Spanish Pediatrics Association (AEP) fellowship and a Ramon Areces Foundation fellowship. For the remaining authors none were declared.

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Decision Letter 0

Rena C Patel

19 Oct 2021

PONE-D-21-25758Prolonged breastfeeding is safe in HIV-exposed children: Survey results in a high HIV prevalence community in southern Mozambique after the implementation of Option B+PLOS ONE

Dear Dr. Fernandez,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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The authors have attempted to tackle an important question in the PMTCT world of how much transmission likely occurs at the various stages of the pregnancy/delivery/postpartum periods, and what associations exist with duration of breastfeeding with HIV transmissions. The work, however, as noted by the reviewers, has marked limitations, which dampens my overall enthusiasm.  Duration of breastfeeding is retrospectively self-reported more than 2 years back, if I understand the methods correctly.  Not enough details on the overall sample from which they randomly selected 5000 pairs to follow is not given, nor justification for why 5000.  I am a little confused by their methods and use of proportional hazards modeling when at times events/person time (i.e., Poisson modeling) and not time to event analysis is needed, though the journal may need formal biostatistical review for this point if the authors choose to stay with their current methods.  On a small note, small English formatting and copy editing aspects (e.g. tables split on multiple pages) simply makes it harder to the read this paper with joy.  Ultimately, as one reviewer put it, there is marked over-interpretation of their data.

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Reviewers' comments:

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Comments to the Author

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Reviewer #1: No

Reviewer #2: Partly

Reviewer #3: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: I Don't Know

Reviewer #3: Yes

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Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #2: Yes

Reviewer #3: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: General:

1. This study has the potential to make important contributions to the literature on this subject. With the success of PMTCT programs, vertical transmission rates have been declining; however, MTCT has not been eliminated, and the relative contribution of postpartum/breastfeeding MTCT to the vertical transmission events that continue to occur is not well understood. This study suggests that postpartum/breastfeeding MTCT most commonly occurs in the context of women newly acquiring HIV in the postpartum period and in the absence of infant ARV prophylaxis. The authors emphasize that duration of breastfeeding was not associated with postpartum MTCT, suggesting that prolonged breastfeeding can/should be supported in this context; however, this conclusion does not seem to logically follow from the results. Rather, it seems that the emphasis should be on prevention, diagnosis, and treatment of women who remain at risk for HIV acquisition in the postpartum period. It may be true that prolonged breastfeeding might be safe (assuming that the mother and baby are adherent to ART and ARV prophylaxis, respectively), but these data/findings do not strongly support this conclusion. That all said, the interpretation and messaging need to be modified. If these issues and the following comments are adequately addressed, then I think this article can reconsidered for publication.

Title:

2. I appreciate the sentiment, but to declare prolonged breastfeeding “safe” without further context is too strong. Furthermore, the context was a cross-sectional sample of mother-child pairs, of whom nearly 30% could not be found and therefore had unknown (and perhaps worse) outcomes. Also, among the subset of HIV-exposed infants who were located and tested, there was an overall MTCT rate of 5.3%, more than half of which was attributed to postpartum/breastfeeding. All said, a postpartum/breastfeeding MTCT rate of 2.7% (26/967) should not be celebrated as “safe”. Just because self-reported breastfeeding duration was not associated with post-partum MTCT in your multivariable model, it does not mean that the postpartum MTCT was not attributable to breastfeeding; in fact, it seems that breastfeeding in the absence of maternal HIV diagnosis, maternal ART, and infant ARV prophylaxis might have been the cause for these MTCT events. Consider tempering the title and revising the interpretation and messaging throughout the manuscript.

Abstract:

3. The study design should be more clearly stated. From what I can gather, this is a cross-sectional assessment of an observational cohort.

4. 69.7% (3486/5000) were “found and interviewed”. Were all of these women and their children also tested for HIV, as suggested in the Methods. If so, what was the median age [IQR] of children when they were tested, and were all tested after complete cessation of breastfeeding?

5. How can 10% of children have unknown HIV exposure status when/if all of the mothers were tested for HIV in the context of this study (see Comment #4, above)? Perhaps this is made clear later in the manuscript.

6. “P-value” does not need to be spelled out (e.g., p<0.001 should suffice).

7. For the same reasons outlined in Comment #2 (re: Title), I do not think the Conclusion is well supported by the Results. To me, the results suggest that more needs to be done to promote uptake of infant ARV prophylaxis, and that perhaps when mothers are virally suppressed AND infants are appropriately prophylaxed, then prolonged breastfeeding can and should be supported in this context.

Introduction:

8. In the last sentence of the 5th paragraph (line 52) of the Introduction, there is a typo. Presumably, “2014-1015” should be “2014-2015”.

9. In the 5th paragraph of the Introduction, the authors state that “there is no data about ART adherence and viral load during breastfeeding period at national level…”, so they instead cite Reference #18 to illustrate that there is substantial non-adherence to ART refills in the first 3 months postpartum. Firstly, it should be “there ARE no data…” (plural). Secondly, aren’t there PEPFAR data about retention and viral suppression that can be disaggregated for pregnancy/breastfeeding status (or at least for women of child bearing age) to approximate national statistics?

10. There is sufficient introduction to breastfeeding guidelines for HIV+ women, but there is insufficient background information about HIV-exposed infant ARV prophylaxis guidelines, which have also evolved over time. Since your main finding was that lack of infant ARV prophylaxis was associated with postpartum MTCT, it seems like more background on this needs to be presented (i.e., WHO and Mozambican guidelines).

Methods:

11. In the last paragraph of the Statistical Analysis section of the Methods (line 157), the authors state that there were “61 children with unknown HIV serostatus”, but in the Abstract it is noted that “for 10.1% (350/3486) HIV-exposure was unknown.” This distinction becomes clear once the reader gets to Figure 1, but please clarify this in the text as well.

Results:

12. In the Study Design and Study Population section of the Methods (line 84) it is stated that “5000 children born alive in the previous 48 months were randomly selected”, but in the first sentence of the Results section (line 174) it is stated that 174 children were ineligible/excluded because they were “older than 48 months”. How is this possible?

13. In the second sentence of the Results it is noted that “1340 [27%] mother/child pairs were not located”, but no further explanation is provided in the text. In Figure 1, it is noted that these were mostly “absent/migrant”, but this is not well characterized. Also in Table 1, among those who were included/located, it seems that all of those women who were “absent or migrated” were HIV+ (n=85; 8.8%) vs. HIV- (n=0), but the numbers are different for the HIV-exposed (n=15; 1.6%) vs unexposed infants (n=23; 1.1%). Whatever the case, this might suggest that these were not missing at random. Can the authors provide some additional details as to why mother-child pairs might not have been located, and whether the characteristics of those not located were similar/different to those who ultimately participated in the study? Is it possible that those who were not located were also more likely to be non-adherent to PMTCT care and therefore more likely to have postpartum MTCT? All said, I am concerned that the study findings might not be generalizable based on this limitation. This must be addressed or at least acknowledged as a severe limitation.

14. Very minor point, but in the Results (line 178) it is stated that for 10.0% HIV-exposure was unknown, while in the Abstract it says 10.1%. Please reconcile.

15. In line 181 it is stated that “13.4% (130/967) were exposed only in the postpartum period”. What proportion of these were characterized as such based on maternal HIV testing performed during the study visit? What proportion of these women were diagnosed with HIV and started on ART while the baby was still breastfeeding?

16. In lines 186-188 it is stated “HIV-positive mothers were significantly older, with a median age of 28.7 years (IQR: 23.4-187 33.4) and almost all (98.1%) had attended at least one antenatal visit, when compared with HIV-negative mothers.” Can you please include the statistics for the HIV-negative women for comparison, rather than requiring the reader to cross-reference Table 1.

17. In line 189, it is stated that, “Only 34.1% of HIV-positive mothers had at least one viral load result at the time of survey.” What proportion of these were virologically suppressed?

18. In the Postpartum MTCT and Associated Factors section of the Results (line 208), “6,2%” should be “6.2%”.

19. In the Postpartum MTCT and Associated Factors section of the Results, the sentence “Among the 61 children with unknown HIV serostatus, 62.3% (38/61) were female, 4 never breastfed and among the 57 who initiated breastfeeding, the median duration of breastfeeding was 12 (95%CI: 10.4 - 15.1) months and a total of 53 would meet the definition of postpartum MTCT” (lines 212-215) is quite confusing and needs to be re-written to enhance clarity.

20. In the Postpartum MTCT and Associated Factors section of the Results, the paragraph found in lines 227-232 is essential to contextualizing the findings of this study. The fact that, “Mothers with postpartum MTCT were mostly diagnosed (57.7%) and initiated ART (50.0%) after the child was born, compared with the 12.3% and 11.4% of diagnosis and ART initiation after the birth among the mothers of HIV exposed uninfected children, p-value<0.001” indicates a problem that heretofore has not been very well discussed. This frames the problem not only as postpartum MTCT among mother-child pairs breastfeeding in the context of known HIV status during the antepartum/peripartum period, but rather postpartum HIV diagnosis and ART initiation (and lack thereof) among women who were previously HIV-negative or HIV-unknown status. Similarly, that “only 50.0% (13/26) of children with postpartum MTCT had received antiretroviral prophylaxis at any time after birth compared to 82.8% (688/831) of HIV exposed uninfected children, p-value<0.001” should not come as a surprise when nearly two-thirds of their mothers were not diagnosed with HIV until after the child was born. This further emphasizes that the issue has less to do with prescription and adherence to infant ARV prophylaxis and more to do with mothers not knowing their HIV status and/or not benefiting from ART in the postpartum period and while breastfeeding.

21. In the Postpartum MTCT and Associated Factors section of the Results, the paragraph found in lines 233-236 states, “A total of 80.8% (21/26) of children with postpartum-MTCT had attended at least one unscheduled outpatient visits before the survey and a total of 26.9% (7/26) had been hospitalized, compared with the 55.0% (457/831) and 5.8% (48/831) of the HIV exposed uninfected children p-value=0.018 and p-value=0.001, respectively”, which suggests there may have been opportunities for postpartum infant/mother HIV diagnosis during this encounters. Do we know whether HIV-testing/diagnosis was done during these encounters?

Figure 1:

22. This entire figure could use some formatting/attention to detail. For example, some boxes have text aligned left while others are centered, some statements are incomplete, and some arrows are not well aligned.

23. The first exclusion (dashed arrow pointing to the right, just below the box that states “5000 randomly selected”) states “174 children >54 months at the”. This statement is incomplete, and it seems to be in conflict with what was reported in the text of the Results section (48 vs 54 months?).

24. The second exclusion (dashed arrow pointing to the left, just below the box that states “4826 children eligible”) states “17 houses not”. This statement is incomplete.

25. It’s still not clear to me why 350 (10%) of women and 61 (6.3%) of HIV-exposed infants have unknown HIV serostatus, when as HIV testing should have been performed per study protocol. Did these refuse testing? If so, this should be stated. If not, please provide some other explanation.

Table 1:

26. In the PDF, this table gets cut-off then continues on a second page. Might be easier to read/critique if re-formatted (landscape orientation, smaller font size, etc.).

27. At the top of the table, “Puesto como footnote” needs to be translated to English.

Table 2:

28. In the PDF, this table gets cut-off then continues onto three pages. Might be easier to read/critique if re-formatted (landscape orientation, smaller font size, etc.).

29. The p-value in the second row (“Mother located during the household survey”) is “1,000”. I’m assuming this is an error.

30. What is the source of the variables included in Table 2? This is not very well described in the Methods. In particular, was infant ARV prophylaxis based on self-report vs. medical records?

Table 3:

31. Again, in the PDF, this table gets cut-off then continues onto three pages. Might be easier to read/critique if re-formatted (landscape orientation, smaller font size, etc.).

32. Why weren’t “mother HIV diagnosis” and “mother ART initiation” included in the multivariable model. These were both strongly associated with postpartum MTCT in the univariate model. And, in terms of a priori assumptions, I would expect these to account for most of the MTCT risk. If the issue is missingness, then consider multiple imputation. This seems like a major flaw that needs to be addressed.

33. On the flip side of Comment #32, why was breastfeeding duration “forced” into multivariable model. It’s no surprise that breastfeeding duration wasn’t associated with MTCT, when the more important variables are timing of maternal HIV diagnosis and ART initiation; quantity is less important than quality (i.e., breastfeeding duration only becomes relevant when contextualized with timing of HIV diagnosis and ART initiation).

34. “Survey” is misspelled in “Age at survAy in months”.

Discussion:

35. The statement in the 4th paragraph of the Discussion (lines 290-292), “Our results suggest that in the context of B+ (lifelong ART to the mother and antiretroviral prophylaxis to the children), duration of breastfeeding is not associated with an increased risk of postpartum MTCT” is not entirely correct. It may in fact be true that duration of breastfeeding is not associated with an increased risk of postpartum MTCT when B+ guidelines are followed; but in this study the MTCT events seem to have occurred predominantly among women newly diagnosed with HIV and started on ART in the postpartum period. If you want to answer the question of impact of duration of breastfeeding in the context of B+, then you first have to control/adjust for timing of maternal HIV diagnosis and ART initiation (and ideally virologic control), both of which were strongly associated with MTCT in your univariable analysis but were not included in your multivariable analysis.

36. Likewise, the statement at the end of the 4th paragraph of the Discussion (lines 299-302) “Our findings reaffirm the 2016 guidelines on HIV-exposed infant feeding [14], which recommend that HIV-positive mothers who are well-controlled on ART should breastfeed their children for two years or more, as should HIV-negative mothers[14]” is an overstatement. Rather, than supporting WHO guidelines about prolonged breastfeeding (the veracity of which are not in question), this study instead highlights other very important issues, namely the need for ongoing HIV prevention, diagnosis, and ART initiation among women who remain at risk for HIV acquisition in the postpartum setting. I see this study as more of a justification for postpartum PREP and serially repeated HIV testing in postpartum period, rather than supporting prolonged breastfeeding. The authors start to touch on this in the 5th paragraph of the Discussion. All said, it seems as though the authors went into this analysis with a presupposition to prove (i.e., that prolonged breastfeeding is safe), rather than lettering the data speak for themselves. I strongly recommend reassessing the interpretation of the results and the messaging of the discussion and conclusions.

37. I have deferred further critiques of the discussion, until the Methodological issues and interpretation of the Results are addressed, as above.

Reviewer #2: Prolonged breastfeeding is safe in HIV-exposed children: Survey results in a high HIV prevalence community in southern Mozambique after implementation of Option B+

Fernandez-Luis et al.

This study looks at both duration of breastfeeding among HIV-exposed and HIV-unexposed infants as well as factors associated with postpartum transmission of HIV in Mozambique. HIV-exposed infants received six weeks of nevirapine, whether they were breastfeeding or not.

The authors conclude that women living with HIV breastfeed for a shorter median duration than women not living with HIV and that infants not receiving 6 weeks of nevirapine prophylaxis are at higher risk of HIV acquisition.

1) The study design is a bit confusing: women were chosen randomly to be interviewed but this was primarily a retrospective chart review combined with mother’s memories of what she did during pregnancy and postpartum.

2) What information do the authors have about maternal adherence to ART? In the era of U=U (undetectable equals untransmissible) in the world of sexual transmission, many obstetricians and pediatricians seek information on the effect of maternal adherence on transmission. Flynn et al have documented a 0.3% transmission rate at 6 months and 0.6% transmission rate at 12 months among women with undetectable viral loads. The Mozambique study addresses the “real world” of not having easy availability of viral loads but there is little said about maternal adherence. The authors cite a study by Pfeiffer 2017 that documented only 38% of women living with HIV still obtaining ART refills for themselves 90 days postpartum but do not comment on adherence among their study participants. Did their survey ask about adherence?

3) The actual risk of transmission appears to be 51% of 5.3%, or 2.7% (per Figure 1). That rate is significantly higher than the transmission rates in the PROMISE study (Flynn et al). Please comment.

4) I would also like some discussion of why the authors think that women living with HIV breastfed for a shorter period of time than women who did not have HIV. I can imagine that those with HIV may have been aware of some risk of transmission of HIV via breastmilk and, therefore, weaned their babies earlier. Did the surveys ask women why they stopped breastfeeding when they did? If not, what reasons do the authors think would explain early cessation?

5) If the standard of care in Mozambique is to give six weeks of infant prophylaxis, what were the reasons mothers gave for not giving it?

6) Of special interest to us in the U.S., Canada, and Europe, adding infant prophylaxis in this study was associated with a significant decrease in postpartum transmission. In high resource countries some pediatricians have opted to give nevirapine through cessation of breastfeeding in addition to continuing maternal ART. This study lends credence to that approach.

Although this study attempts to add important information to the literature, I am not sure that the study design allows us to confidently draw conclusions from it.

Reviewer #3: PLOS ONE

Please make objective more direct. "We aimed to compare" can be changed to "We compared...."

It is not clear what is meant by "Among the 5000 mother-child pairs selected, 69.7% (3486/5000) were found and

interviewed. " Please revise the terms "found" and "interviewed" to something like located, enrolled and surveyed.

Who were the initial 5000? All mothers who gave birth in the study interval? Please clarify.

The method used to estimate the mode of transmission should be specified in the methods of the abstract.

Line 16 - take out "In addition."

line 26 - updating does not need a hyphen

Line 56- change to just "We compared the duration..."

The introduction is excellently written and has great content.

Please explain how breastfeeding duration was obtained. Expand this section at is is the main dependent variable and currently not well explained in terms of how BF status and duration was obtained accurately. Reference a method please.

Mothers were surveyed, not interviewed. Or call this a structured interview. Survey is more appropriate since no qualitative data was obtained.

Results: do not have a sub-heading for sociodemographic characteristics. Make this frist first paragraph with no boldface.

Combine limitations in one paragraph.

Should the title be revised to include the dual object of the study which is to compare BF duration and MTCT transmission by ART prophylaxes status?

The conclusion is well written and supports the results. Well done!

**********

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PLoS One. 2022 Aug 2;17(8):e0269835. doi: 10.1371/journal.pone.0269835.r002

Author response to Decision Letter 0

17 Mar 2022

Reviewer #1: General:

1. This study has the potential to make important contributions to the literature on this subject. With the success of PMTCT programs, vertical transmission rates have been declining; however, MTCT has not been eliminated, and the relative contribution of postpartum/breastfeeding MTCT to the vertical transmission events that continue to occur is not well understood. This study suggests that postpartum/breastfeeding MTCT most commonly occurs in the context of women newly acquiring HIV in the postpartum period and in the absence of infant ARV prophylaxis. The authors emphasize that duration of breastfeeding was not associated with postpartum MTCT, suggesting that prolonged breastfeeding can/should be supported in this context; however, this conclusion does not seem to logically follow from the results. Rather, it seems that the emphasis should be on prevention, diagnosis, and treatment of women who remain at risk for HIV acquisition in the postpartum period. It may be true that prolonged breastfeeding might be safe (assuming that the mother and baby are adherent to ART and ARV prophylaxis, respectively), but these data/findings do not strongly support this conclusion. That all said, the interpretation and messaging need to be modified. If these issues and the following comments are adequately addressed, then I think this article can reconsidered for publication.

Answer: Thank you for general comments. It is true that the natural conclusion that logically flows from the results is on prevention, diagnosis and treatment of women who remain at-risk for HIV acquisition. Thus, we modified the interpretation and messaging putting more emphasis on prevention, diagnosis, and treatment of women who remain at risk for HIV acquisition in the postpartum period and on infant ARV prophylaxis. In addition, we have softened the messaging on supporting prolonged breastfeeding for those mothers who started breastfeeding in LMIC situations where mother and baby are adherent to ART and ARV prophylaxis, respectively.

Title:

2. I appreciate the sentiment, but to declare prolonged breastfeeding “safe” without further context is too strong. Furthermore, the context was a cross-sectional sample of mother-child pairs, of whom nearly 30% could not be found and therefore had unknown (and perhaps worse) outcomes. Also, among the subset of HIV-exposed infants who were located and tested, there was an overall MTCT rate of 5.3%, more than half of which was attributed to postpartum/breastfeeding. All said, a postpartum/breastfeeding MTCT rate of 2.7% (26/967) should not be celebrated as “safe”. Just because self-reported breastfeeding duration was not associated with post-partum MTCT in your multivariable model, it does not mean that the postpartum MTCT was not attributable to breastfeeding; in fact, it seems that breastfeeding in the absence of maternal HIV diagnosis, maternal ART, and infant ARV prophylaxis might have been the cause for these MTCT events. Consider tempering the title and revising the interpretation and messaging throughout the manuscript.

Answer: Thank you for the comment, the title has been modified eliminating the word “safe” and reinforcing the importance of MTCT prevention. We have also revised the messaging throughout the manuscript.

Abstract:

3. The study design should be more clearly stated. From what I can gather, this is a cross-sectional assessment of an observational cohort.

Answer: Thank you for your comment. We have specified that it is a cross sectional assessment.

4. 69.7% (3486/5000) were “found and interviewed”. Were all of these women and their children also tested for HIV, as suggested in the Methods. If so, what was the median age [IQR] of children when they were tested, and were all tested after complete cessation of breastfeeding?

Answer:

Thank you for your comment.

Mothers who do not know their status or self-report being HIV-negative were tested at survey, as well as the HIV-exposed children. For those children whose mother was not available and the main caregiver provided consent, age-appropriate testing was also offered. Documented known HIV-positive individuals were not re-tested, however a Geenius HIV-1/2 Confirmatory Assay was performed. This information has been added in the methods of the manuscript.

Median age [IQR] of children at survey was 23.9 (14.9 - 35.7) months as described in table 1.

5. How can 10% of children have unknown HIV exposure status when/if all of the mothers were tested for HIV in the context of this study (see Comment #4, above)? Perhaps this is made clear later in the manuscript.

Answer: We didn’t add this information in the abstract due to the limitations in the number of words. However, after your comment more information about these 350 mothers with unknown serostatus has been added in figure 1:

317 migration/absence (9.1%)

1 indeterminate (0.03%)

1 death (0.03%)

31 refusals (0.9%)

We have also included more information about the 61 children with Unknown HIV serostatus in figure 1:

15 migration/absence (1.5%)

4 death (0.4%)

21 refusals (2.2%)

21 no information (2.2%)

6. “P-value” does not need to be spelled out (e.g., p<0.001 should suffice).

Answer: thank you for your comment. We have changed P-value with p.

7. For the same reasons outlined in Comment #2 (re: Title), I do not think the Conclusion is well supported by the Results. To me, the results suggest that more needs to be done to promote uptake of infant ARV prophylaxis, and that perhaps when mothers are virally suppressed AND infants are appropriately prophylaxed, then prolonged breastfeeding can and should be supported in this context.

Answer: Thank you for your comment. As explained in previous responses, the conclusion has been revised.

Introduction:

8. In the last sentence of the 5th paragraph (line 52) of the Introduction, there is a typo. Presumably, “2014-1015” should be “2014-2015”.

Answer: Thank you for your comment. It has been corrected.

9. In the 5th paragraph of the Introduction, the authors state that “there is no data about ART adherence and viral load during breastfeeding period at national level…”, so they instead cite Reference #18 to illustrate that there is substantial non-adherence to ART refills in the first 3 months postpartum. Firstly, it should be “there ARE no data…” (plural). Secondly, aren’t there PEPFAR data about retention and viral suppression that can be disaggregated for pregnancy/breastfeeding status (or at least for women of child bearing age) to approximate national statistics?

Answer: typo has been corrected. We have added PEPFAR data about viral suppression in pregnancy.

10. There is sufficient introduction to breastfeeding guidelines for HIV+ women, but there is insufficient background information about HIV-exposed infant ARV prophylaxis guidelines, which have also evolved over time. Since your main finding was that lack of infant ARV prophylaxis was associated with postpartum MTCT, it seems like more background on this needs to be presented (i.e., WHO and Mozambican guidelines).

Answer: thank you for the recommendation. We have added more background on ARV prophylaxis guidelines

Methods:

11. In the last paragraph of the Statistical Analysis section of the Methods (line 157), the authors state that there were “61 children with unknown HIV serostatus”, but in the Abstract it is noted that “for 10.1% (350/3486) HIV-exposure was unknown.” This distinction becomes clear once the reader gets to Figure 1, but please clarify this in the text as well.

Answer: Thank you for your comment.

There are 350 children with HIV-exposure unknown due to the status of the mother was not possible to determine. These 350 children who we didn’t know if were or not exposed to HIV were excluded of the analysis. In addition, among the HIV-exposed children, we have 61 children HIV-exposed with unknown HIV serostatus as it was not possible to perform HIV test in these children.

We have specified that the 350 children with unknown HIV-exposures were excluded and we have also reworded the sentence about the 61 children with unknown HIV serostatus for more clarification. In addition, in figure 1, we have added information about the reasons for unknown HIV exposure and HIV status, respectively.

Results:

12. In the Study Design and Study Population section of the Methods (line 84) it is stated that “5000 children born alive in the previous 48 months were randomly selected”, but in the first sentence of the Results section (line 174) it is stated that 174 children were ineligible/excluded because they were “older than 48 months”. How is this possible?

Answer: Thank you for your comment. It was mainly to an error in the date of birth in the HDSS database. During the informed consent process, the field workers of the study verified the inclusion criteria including the date of birth and found that those 174 children were not eligible to participate in the study.

13. In the second sentence of the Results it is noted that “1340 [27%] mother/child pairs were not located”, but no further explanation is provided in the text. In Figure 1, it is noted that these were mostly “absent/migrant”, but this is not well characterized. Also in Table 1, among those who were included/located, it seems that all of those women who were “absent or migrated” were HIV+ (n=85; 8.8%) vs. HIV- (n=0), but the numbers are different for the HIV-exposed (n=15; 1.6%) vs unexposed infants (n=23; 1.1%). Whatever the case, this might suggest that these were not missing at random. Can the authors provide some additional details as to why mother-child pairs might not have been located, and whether the characteristics of those not located were similar/different to those who ultimately participated in the study? Is it possible that those who were not located were also more likely to be non-adherent to PMTCT care and therefore more likely to have postpartum MTCT? All said, I am concerned that the study findings might not be generalizable based on this limitation. This must be addressed or at least acknowledged as a severe limitation.

Answer: Thank you for the comment. We have added more information on the reasons that 27% of the mother/child pairs initially randomly selected were not located. We don’t have information about HIV serostatus of these mothers and we have added this as a limitation of the study.

Please note that Table 1 is not related with these 27% mother/child pairs not located. Table 1 includes children born from mothers with known HIV status (967 HIV positive and 2169 negative). This table describes whether the mother was located and surveyed or not (and this means that the survey was performed with the caregiver).

14. Very minor point, but in the Results (line 178) it is stated that for 10.0% HIV-exposure was unknown, while in the Abstract it says 10.1%. Please reconcile.

Answer: Thank you for your comment. It has been corrected

15. In line 181 it is stated that “13.4% (130/967) were exposed only in the postpartum period”. What proportion of these were characterized as such based on maternal HIV testing performed during the study visit? What proportion of these women were diagnosed with HIV and started on ART while the baby was still breastfeeding?

Answer:

Thank you for the comment. Among the children exposed only in the postpartum period 47.6% were characterized as HIV-exposed based on maternal HIV testing performed during the study visit and 14% were still breastfeeding when their mothers started on ART. We have added this information.

16. In lines 186-188 it is stated “HIV-positive mothers were significantly older, with a median age of 28.7 years (IQR: 23.4-187 33.4) and almost all (98.1%) had attended at least one antenatal visit, when compared with HIV-negative mothers.” Can you please include the statistics for the HIV-negative women for comparison, rather than requiring the reader to cross-reference Table 1.

Answer: Thank you for the suggestion. The statistics for HIV-negative women have been added.

17. In line 189, it is stated that, “Only 34.1% of HIV-positive mothers had at least one viral load result at the time of survey.” What proportion of these were virologically suppressed?

Answer: Thank you for your question. Only 34.1%% (330/967) of HIV-positive mothers had at least one viral load result at the time of survey. However, 75.8% (250/330) of the mothers with viral load result were virally suppressed. We have added this information.

18. In the Postpartum MTCT and Associated Factors section of the Results (line 208), “6,2%” should be “6.2%”.

Answer: Thank you for your comment. It has been corrected

19. In the Postpartum MTCT and Associated Factors section of the Results, the sentence “Among the 61 children with unknown HIV serostatus, 62.3% (38/61) were female, 4 never breastfed and among the 57 who initiated breastfeeding, the median duration of breastfeeding was 12 (95%CI: 10.4 - 15.1) months and a total of 53 would meet the definition of postpartum MTCT” (lines 212-215) is quite confusing and needs to be re-written to enhance clarity.

Answer: thank you for the comment. It has been corrected.

20. In the Postpartum MTCT and Associated Factors section of the Results, the paragraph found in lines 227-232 is essential to contextualizing the findings of this study. The fact that, “Mothers with postpartum MTCT were mostly diagnosed (57.7%) and initiated ART (50.0%) after the child was born, compared with the 12.3% and 11.4% of diagnosis and ART initiation after the birth among the mothers of HIV exposed uninfected children, p-value<0.001” indicates a problem that heretofore has not been very well discussed. This frames the problem not only as postpartum MTCT among mother-child pairs breastfeeding in the context of known HIV status during the antepartum/peripartum period, but rather postpartum HIV diagnosis and ART initiation (and lack thereof) among women who were previously HIV-negative or HIV-unknown status. Similarly, that “only 50.0% (13/26) of children with postpartum MTCT had received antiretroviral prophylaxis at any time after birth compared to 82.8% (688/831) of HIV exposed uninfected children, p-value<0.001” should not come as a surprise when nearly two-thirds of their mothers were not diagnosed with HIV until after the child was born. This further emphasizes that the issue has less to do with prescription and adherence to infant ARV prophylaxis and more to do with mothers not knowing their HIV status and/or not benefiting from ART in the postpartum period and while breastfeeding.

Answer: thank you for your comment. We agree with the reviewer and we have highlighted in the discussion that “Mothers may not have initiated ART because of their lack of awareness of their HIV status, death, not willing or because of service delivery shortfalls or stockouts.” We have included data on HIV incidence in breastfeeding women during the postpartum period in Mozambique and we have suggested that “Our results suggest that improving the reach of PrEP among women who remain at risk for HIV acquisition in the postpartum period, as well as establishing specific retesting times during the postpartum period in areas of high HIV incidence would reinforced the prevention of MTCT in LMIC and would facilitate initiation of ART in breastfeeding mothers and antiretroviral prophylaxis in their HIV exposed infants.”

21. In the Postpartum MTCT and Associated Factors section of the Results, the paragraph found in lines 233-236 states, “A total of 80.8% (21/26) of children with postpartum-MTCT had attended at least one unscheduled outpatient visits before the survey and a total of 26.9% (7/26) had been hospitalized, compared with the 55.0% (457/831) and 5.8% (48/831) of the HIV exposed uninfected children p-value=0.018 and p-value=0.001, respectively”, which suggests there may have been opportunities for postpartum infant/mother HIV diagnosis during this encounters. Do we know whether HIV-testing/diagnosis was done during these encounters?

Answer: Thank you for your comment. We agree with the reviewer that that information would be very interesting, however this information is unfortunately unavailable. We have added in the discussion that these are potential opportunities for HIV testing/Diagnosis.

Figure 1:

22. This entire figure could use some formatting/attention to detail. For example, some boxes have text aligned left while others are centered, some statements are incomplete, and some arrows are not well aligned.

Answer: Thank you for the observation. The figure has been corrected.

23. The first exclusion (dashed arrow pointing to the right, just below the box that states “5000 randomly selected”) states “174 children >54 months at the”. This statement is incomplete, and it seems to be in conflict with what was reported in the text of the Results section (48 vs 54 months?).

Answer: Thank you for the observation. The figure and the text of the results section have been corrected.

24. The second exclusion (dashed arrow pointing to the left, just below the box that states “4826 children eligible”) states “17 houses not”. This statement is incomplete.

Answer: Thank you for the observation. The figure has been corrected.

25. It’s still not clear to me why 350 (10%) of women and 61 (6.3%) of HIV-exposed infants have unknown HIV serostatus, when as HIV testing should have been performed per study protocol. Did these refuse testing? If so, this should be stated. If not, please provide some other explanation.

Answer: Thank you for the observation, the information has been added.

Table 1:

26. In the PDF, this table gets cut-off then continues on a second page. Might be easier to read/critique if re-formatted (landscape orientation, smaller font size, etc.).

Answer: Thank you for your comment. It has been sent in excel format.

27. At the top of the table, “Puesto como footnote” needs to be translated to English.

Answer: Thank you for the observation. It has been removed.

Table 2:

28. In the PDF, this table gets cut-off then continues onto three pages. Might be easier to read/critique if re-formatted (landscape orientation, smaller font size, etc.).

Answer: Thank you for your comment. It has been sent in excel format.

29. The p-value in the second row (“Mother located during the household survey”) is “1,000”. I’m assuming this is an error.

Answer: The p-value is 1.000, we have corrected it.

30. What is the source of the variables included in Table 2? This is not very well described in the Methods. In particular, was infant ARV prophylaxis based on self-report vs. medical records?

Answer: thank you for the question. Sociodemographic (Age, Educational level, Marital status, Income, Parity), antenatal care and breastfeeding information were self-reported during the survey. HIV data were obtained through medical documentation (Mother HIV diagnosis, Mother ART initiation) or HIV database (Mother cd4 at child birth, Mother viral load at child birth). Hospitalizations and outpatients’ visits were obtained through HDSS database. Gestational Age and Infant Antiretroviral prophylaxis were obtained through medical documentation. This information has been added in methods section.

Table 3:

31. Again, in the PDF, this table gets cut-off then continues onto three pages. Might be easier to read/critique if re-formatted (landscape orientation, smaller font size, etc.).

Answer: Thank you for your comment. It has been sent re-formatted.

32. Why weren’t “mother HIV diagnosis” and “mother ART initiation” included in the multivariable model. These were both strongly associated with postpartum MTCT in the univariate model. And, in terms of a priori assumptions, I would expect these to account for most of the MTCT risk. If the issue is missingness, then consider multiple imputation. This seems like a major flaw that needs to be addressed.

Answer: The initial reason for not including “mother ART initiation” was more than 20% missing values and because “mother HIV diagnosis” was eliminated in the forward stepwise selection. However, after your recommendation we have performed the regression without using the stepwise selection process and we have performed multiple imputation on the “mother ART initiation” variable treated as a binary variable: ART initiation before delivery yes/no. We have assumed that missing data are missing at random and used a logistic regression model as a method to impute. In the imputation model we have included our outcome variable and the other predictor variables included in the final multivariable model and which have p>0.2 in the univariable model and have less than 20% missing value. We have made a total of 20 imputations. Using these parameters for the additional analysis, we obtained similar results with no observation of evidence of association between breastfeeding duration and postpartum MTCT. In contrast, mother ART initiation after the date of childbirth was associated with postpartum MTCT. We have not added “mother HIV diagnosis” in the regression because of collinearity with “mother ART initiation.” We feel this additional step to our analysis has made our results more robust and thank the reviewer for the suggestion.

33. On the flip side of Comment #32, why was breastfeeding duration “forced” into multivariable model. It’s no surprise that breastfeeding duration wasn’t associated with MTCT, when the more important variables are timing of maternal HIV diagnosis and ART initiation; quantity is less important than quality (i.e., breastfeeding duration only becomes relevant when contextualized with timing of HIV diagnosis and ART initiation).

Answer: breastfeeding was forced because is the variables that we are specifically testing as part of this study.

34. “Survey” is misspelled in “Age at survAy in months”.

Answer: Thank you for your comment. It has been corrected

Discussion:

35. The statement in the 4th paragraph of the Discussion (lines 290-292), “Our results suggest that in the context of B+ (lifelong ART to the mother and antiretroviral prophylaxis to the children), duration of breastfeeding is not associated with an increased risk of postpartum MTCT” is not entirely correct. It may in fact be true that duration of breastfeeding is not associated with an increased risk of postpartum MTCT when B+ guidelines are followed; but in this study the MTCT events seem to have occurred predominantly among women newly diagnosed with HIV and started on ART in the postpartum period. If you want to answer the question of impact of duration of breastfeeding in the context of B+, then you first have to control/adjust for timing of maternal HIV diagnosis and ART initiation (and ideally virologic control), both of which were strongly associated with MTCT in your univariable analysis but were not included in your multivariable analysis.

Answer: Thank you for your suggestion. We have adjusted for maternal HIV diagnosis and ART initiation using multiple imputation in the regression analysis of postpartum MTCT. Due to >60% of women lacking a viral load result, we were unable to impute and adjust for viral load.

36. Likewise, the statement at the end of the 4th paragraph of the Discussion (lines 299-302) “Our findings reaffirm the 2016 guidelines on HIV-exposed infant feeding [14], which recommend that HIV-positive mothers who are well-controlled on ART should breastfeed their children for two years or more, as should HIV-negative mothers[14]” is an overstatement. Rather, than supporting WHO guidelines about prolonged breastfeeding (the veracity of which are not in question), this study instead highlights other very important issues, namely the need for ongoing HIV prevention, diagnosis, and ART initiation among women who remain at risk for HIV acquisition in the postpartum setting. I see this study as more of a justification for postpartum PREP and serially repeated HIV testing in postpartum period, rather than supporting prolonged breastfeeding. The authors start to touch on this in the 5th paragraph of the Discussion. All said, it seems as though the authors went into this analysis with a presupposition to prove (i.e., that prolonged breastfeeding is safe), rather than lettering the data speak for themselves. I strongly recommend reassessing the interpretation of the results and the messaging of the discussion and conclusions.

Answer: Thank you for your comment. We have reoriented the discussion and conclusions to highlight our results in the context of the need for ongoing HIV prevention, diagnosis, and ART initiation in the postpartum setting, including postpartum PREP.

37. I have deferred further critiques of the discussion, until the Methodological issues and interpretation of the Results are addressed, as above.

Answer: Thank you for the comment. We have addressed all the comments above.

 

Reviewer #2: Prolonged breastfeeding is safe in HIV-exposed children: Survey results in a high HIV prevalence community in southern Mozambique after implementation of Option B+

Fernandez-Luis et al.

This study looks at both duration of breastfeeding among HIV-exposed and HIV-unexposed infants as well as factors associated with postpartum transmission of HIV in Mozambique. HIV-exposed infants received six weeks of nevirapine, whether they were breastfeeding or not.

The authors conclude that women living with HIV breastfeed for a shorter median duration than women not living with HIV and that infants not receiving 6 weeks of nevirapine prophylaxis are at higher risk of HIV acquisition.

1) The study design is a bit confusing: women were chosen randomly to be interviewed but this was primarily a retrospective chart review combined with mother’s memories of what she did during pregnancy and postpartum.

Answer: thank you for tour comment. We have reworded methods section in order to facilitate the compression.

It this cross-sectional household survey conducted from October-2017 to April-2018. Mothers who had given birth within the previous 48-months in the Manhiça district were randomly selected to be surveyed and to receive an HIV-test along with their children. During the survey, study HIV counselors administered a specific questionnaire designed to capture sociodemographic characteristics, HIV testing history and ART, antenatal care and duration of breastfeeding. Clinical documentation was used to obtain information about HIV testing and HIV care during pregnancy and postpartum. The mother or caregiver self-reported the total duration of any breastfeeding in months at the time of survey.

2) What information do the authors have about maternal adherence to ART? In the era of U=U (undetectable equals untransmissible) in the world of sexual transmission, many obstetricians and pediatricians seek information on the effect of maternal adherence on transmission. Flynn et al have documented a 0.3% transmission rate at 6 months and 0.6% transmission rate at 12 months among women with undetectable viral loads. The Mozambique study addresses the “real world” of not having easy availability of viral loads but there is little said about maternal adherence. The authors cite a study by Pfeiffer 2017 that documented only 38% of women living with HIV still obtaining ART refills for themselves 90 days postpartum but do not comment on adherence among their study participants. Did their survey ask about adherence?

Answer: Unfortunately, adherence was not asked during the survey. We have highlighted more the importance of adherence in the discussion, according to your suggestion.

3) The actual risk of transmission appears to be 51% of 5.3%, or 2.7% (per Figure 1). That rate is significantly higher than the transmission rates in the PROMISE study (Flynn et al). Please comment.

Answer: thank you for your suggestion. We have added the comment in the discussion.

4) I would also like some discussion of why the authors think that women living with HIV breastfed for a shorter period of time than women who did not have HIV. I can imagine that those with HIV may have been aware of some risk of transmission of HIV via breastmilk and, therefore, weaned their babies earlier. Did the surveys ask women why they stopped breastfeeding when they did? If not, what reasons do the authors think would explain early cessation?

Answer: Thank you for your comment. The reason for stopped breastfeeding was not asked in the survey. We have added some reasons described in the literature as advice of health workers, influence of relatives, stigma, conflicting opinions about the risk for MTCT and poor dissemination of policies.

5) If the standard of care in Mozambique is to give six weeks of infant prophylaxis, what were the reasons mothers gave for not giving it?

Answer: Thank you for your comment. As explained in the discussion, we hypothesized that mothers may not have administered prophylaxis to their infants because of non-adherence, because of their lack of awareness of their HIV status or because of service delivery shortfalls or stockouts. We supported our hypothesis with a reference who showed high incidence of HIV in breastfeeding women during the postpartum period in Mozambique.

6) Of special interest to us in the U.S., Canada, and Europe, adding infant prophylaxis in this study was associated with a significant decrease in postpartum transmission. In high resource countries some pediatricians have opted to give nevirapine through cessation of breastfeeding in addition to continuing maternal ART. This study lends credence to that approach.

Answer: thank you for your suggestion. We agree. However, other reviewer asks for repeat the regression after multiple imputation of the variable mother ART initiation. After the inclusion of mother ART initiation in the regression, we have not found association between infant prophylaxis and MTCT and for this reason we have not added the information about infant prophylaxis in high income countries.

Although this study attempts to add important information to the literature, I am not sure that the study design allows us to confidently draw conclusions from it.

Answer: Thank you for the comment. We have reworded the conclusion.

 

Reviewer #3: PLOS ONE

Please make objective more direct. "We aimed to compare" can be changed to "We compared...."

Answer: thank you for your suggestion. It has been changed.

It is not clear what is meant by "Among the 5000 mother-child pairs selected, 69.7% (3486/5000) were found and interviewed.

" Please revise the terms "found" and "interviewed" to something like located, enrolled and surveyed.

Answer: thank you for your suggestion. We have used the words “located” and “enrolled” according to your suggestion.

Who were the initial 5000? All mothers who gave birth in the study interval? Please clarify.

Answer: Thank you for your comment. The initial 5000 mother-children pairs were randomly selected among all children born alive in the previous 48 months at the HDSS. The sentence has been reworded to clarify.

The method used to estimate the mode of transmission should be specified in the methods of the abstract.

Answer: Thank you for your suggestion. It has been added.

Line 16 - take out "In addition."

Answer: Thank you for your suggestion. It has been removed.

line 26 - updating does not need a hyphen

Answer: Thank you for your suggestion. It has been removed.

Line 56- change to just "We compared the duration..."

Answer: thank you for your suggestion. It has been changed.

The introduction is excellently written and has great content.

Answer: Thank you for your comment.

Please explain how breastfeeding duration was obtained. Expand this section at is is the main dependent variable and currently not well explained in terms of how BF status and duration was obtained accurately. Reference a method please.

Answer: thank you for your comment. The mother or caregiver self-reported the total duration of any breastfeeding at the time of survey. This information has been included in methods.

Mothers were surveyed, not interviewed. Or call this a structured interview. Survey is more appropriate since no qualitative data was obtained.

Answer: thank you for your comment. It has been corrected.

Results: do not have a sub-heading for sociodemographic characteristics. Make this frist first paragraph with no boldface.

Answer: sub-heading for sociodemographic characteristics has been deleted according your recommendation.

Combine limitations in one paragraph.

Answer: limitations have been combined in one paragraph, according to your suggestion.

Should the title be revised to include the dual object of the study which is to compare BF duration and MTCT transmission by ART prophylaxes status?

Answer: thank you for the comment, title has been modified.

The conclusion is well written and supports the results. Well done!

Answer: thank you for the comment.

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Decision Letter 1

Rena C Patel

30 May 2022

Prompt HIV diagnosis and treatment in postpartum women is crucial for prevention of mother to child transmission during breastfeeding: Survey results in a high HIV prevalence community in southern Mozambique after the implementation of Option B+.

PONE-D-21-25758R1

Dear Dr. Fernandez,

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Additional Editor Comments (optional):

Dear authors, I greatly appreciate that you have taken reviewer comments to heart in revising this manuscript, with significant changes in the methods (e.g., MI, modeling, etc.) and in reporting (write-up of methods and discussion). I still note some minor copy editing issues that can, hopefully, be corrected at that stage. I would like to personally apologize for the delay in reaching a decision of the work. Thank you, Rena

Reviewers' comments:

Acceptance letter

Rena C Patel

20 Jul 2022

PONE-D-21-25758R1

Prompt HIV diagnosis and treatment in postpartum women is crucial for prevention of mother to child transmission during breastfeeding: Survey results in a high HIV prevalence community in southern Mozambique after the implementation of Option B+.

Dear Dr. Fernandez:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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on behalf of

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Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Appendix. Study questionnaires in English.

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    S2 Appendix. Study questionnaires in Portuguese.

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    Data Availability Statement

    Data cannot be shared publicly because of ethical restrictions. Data contain potentially sensitive information and national ethics committee (CNBS) does not authorize data sharing without a protocol request specifying the objectives and the researchers who will have access to the data. Data are available under request (contact via llorenc.quinto@isglobal.org) for researchers who meet the criteria for access to confidential data. We are sharing a copy, in both the original language and English of the questionnaire of the study as Supporting Information.

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