Comparison of 6-week PMTCT outcomes for HIV-exposed and HIV-unexposed infants in the era of lifelong ART: Results from an observational prospective cohort study.

BACKGROUND: Lifelong antiretroviral therapy (ART) reduces mother-to-child HIV transmission (MTCT) and improves maternal health. Data on the outcomes of HIV-exposed infants (HEI) compared to their unexposed counterparts in the era of universal ART is limited. We compared birth and 6-week outcomes among infants born to HIV-positive and HIV-negative women in Lesotho.
METHODS: 941 HIV-negative and 653 HIV-positive pregnant women were enrolled in an observational cohort to evaluate the effectiveness of prevention of mother-to-child HIV transmission (PMTCT) program after implementation of universal maternal ART in 14 health facilities. Pregnancy, delivery, birth, and 6-week data were collected through participant interviews and medical record review. DNA PCR testing for HEI was conducted within 2 weeks of birth and at around 6 weeks of age. Data were analysed to estimate the distribution of birth outcomes, mortality, HIV transmission and HIV-free survival at 6 weeks.
RESULTS: HIV-positive women were older (mean age of 28.7 vs. 24.4 years) and presented for antenatal care earlier (mean gestational age of 23.0 weeks vs 25.3 weeks) than HIV-negative women. Prematurity was more frequent among HEI, 7.8% vs. 3.6%. There was no difference in rates of congenital anomalies between HEI (1.0%) and HIV-unexposed infants (HUI) (0.6%). Cumulative HIV transmission was 0.9% (N = 4/431) (95% CI:0.25-2.36) at birth and 1.0% (N = 6/583) (95% CI:0.38-2.23) at 6 weeks. Overall mortality, including stillbirths, was 5.2% and 6.0% by 6 weeks for HUI and HEI respectively. Among liveborn infants, 6-week HIV-free survival for HEI was 95.6% (95% CI:93.7-97.1) compared to 96.8% (95% CI:95.4-97.9) survival for HUI.
CONCLUSIONS: Implementation of universal maternal ART lowers MTCT at 6 weeks of age with no differences in congenital anomalies or early mortality between HIV exposed Infants and HIV unexposed infants. However, HIV exposed infants continue to have high rates of prematurity despite improved maternal health on ART.

Introduction

Effective prevention of mother to child transmission (PMTCT) programs offering universal life-long antiretroviral therapy (ART) reduce HIV transmission to children from their mother and improve maternal health [1,2]. A critical question remains as to whether the reduction in MTCT rates and improvement in maternal health are coupled with improvement in birth outcomes and survival among HIV-exposed infants (HEI) to match those of HIV-unexposed infants (HUI).

There have been conflicting data reported on birth outcomes among HIV-positive women who are on lifelong ART compared to HIV negative women. Some studies found that adverse birth outcomes, such as increased preterm deliveries, stillbirths and low birth weight, occurred more frequently among HEI [3-8]. Other studies found no association between use of ART and adverse birth outcomes [9,10].

Intrauterine and perinatal HIV transmission measurement in the era of lifelong ART is limited. In Rwanda, a study measuring HIV-free survival in a cohort of HEI, found a 6-week Mother to child transmission rate of 0.5% (95% CI:0.2–1.6) demonstrating the effectiveness of lifelong ART for HIV-positive pregnant women in preventing perinatal HIV transmission [11]. UNAIDS spectrum data in Lesotho reported an estimated 6-week transmission of 6.9% in 2016 [1].

Lesotho is a mountainous country in southern Africa with one of the highest HIV prevalence documented in the world. The Lesotho Ministry of Health implemented universal ART for all HIV-positive pregnant women in 2013 using a once-daily fixed dose combination of tenofovir disoproxil fumarate (TDF), lamivudine (3TC) and efavirenz (EFZ) [12,13]. HIV-exposed infants are given nevirapine (NVP) for 6 weeks after birth.

The roll out of lifelong ART for all pregnant women in Lesotho provided a unique opportunity to determine its effect on birth outcomes and survival of infants born to HIV-positive mothers compared with a similar cohort of HIV-negative mothers and their HIV-unexposed infants. The study also assessed the effectiveness of the Lesotho PMTCT program in reaching the goal of the virtual elimination of new pediatric HIV infections in selected sites in Lesotho.

Methods

Design

HIV-positive and HIV-negative pregnant women attending routine antenatal care (ANC) services were enrolled in a prospective observational cohort study from June 2014 to February 2016. Study personnel captured demographic, social, and medical information through participant interviews and extraction of medical record information during pregnancy, and at delivery, birth and 6 weeks postpartum.

Setting

The study was conducted in 3 districts in Lesotho–Botha-Bothe, Thaba-Tseka and Mohale’s Hoek. These districts were selected to include areas with varying levels of PMTCT service delivery coverage, and heterogeneity in health-seeking behavior due to variances in terrain (lowlands, foothills, mountains). We included all 5 hospitals in these districts and randomly selected 9 medium volume (100–200 ANC women/year) or high (>200 ANC women/year) health centers to be included in the study.

Population and enrollment

Eligible HIV-positive and HIV-negative pregnant women attending ANC in the study health facilities were recruited for this study. They were eligible for study enrollment if they resided in the area, planned to continue to receive services at the facility following delivery, were willing to have their infant co-enrolled after birth, and were willing to provide written informed consent. Population proportional sampling was used to estimate the enrollment target at each of the 14 study facilities. The sample size for HIV-positive women was calculated based on a 4% estimated HIV transmission at 6–8 weeks in Lesotho at the time of study initiation with a precision of 1.4% [12]. Consecutive consenting women were enrolled until the sample size was reached. HIV-negative women were enrolled into an HIV seroincidence cohort study with scheduled repeat HIV testing at 36 weeks gestation, delivery, and every 3–6 months postpartum until 24 months postpartum [14].

Data collection

Study data were collected by trained study nurses through structured interviews and by abstraction of data from clinical and laboratory records. Mother-infant pair information such as demographic variables, date of last menstrual cycle, HIV status of spouse, disclosure of HIV status was collected through structured interviews with the pregnant women. General health and clinical history, ARV use and toxicity, adherence, retention in care, family planning, infant feeding practices and infant growth were extracted from the women medical charts and clinic registers. Electronic tablets were used to enter data directly into a web-based database (SurveyCTO).

Gestational age at birth was estimated by the time between the date of last menstrual period given by the women at first ANC and the date of birth. Very preterm birth was defined as infant born at a gestational age of 28–32 weeks while preterm birth was infant born after 32 weeks but before 37 weeks [15]. In addition, miscarriage was defined as loss of pregnancy before the gestational age of 28 weeks and stillbirth was considered when the pregnancy was lost after 28 weeks [16].

Laboratory methods

Blood samples were collected from a subset of HEI at or within 2 weeks of birth and from all HEI at 6–8 weeks of age to determine their HIV infection status. Trained nurses obtained blood from infants by heel prick, which was spotted directly onto filter paper to create a dried blood spot for HIV DNA PCR testing as per the standard Ministry of Health (MOH) protocol. HIV testing was performed by the national reference laboratory using the Roche COBAS Ampliprep/COBAS TaqMan HIV-1 qualitative test (v2.0). Test results were obtained directly from the laboratory and entered into the study database.

Statistical analysis

Quantitative data analysis was performed using STATA version 15.1 (College Station, TX, USA). We summarized categorical variables using frequencies and percentages of participants and continuous variables using means (+/- standard deviation). Maternal baseline characteristics were stratified by HIV status at enrollment. We compared birth outcomes between HIV-exposed and HIV-unexposed infants. HIV-free survival was estimated as the proportion of children alive and HIV-negative among all exposed children. The precision around survival estimates was assessed by 95% confidence intervals. We used the Kaplan Meier curves to graphically display infant mortality, infection, and HIV free survival. We performed complete case analysis, and missing data were not imputed.

Ethical considerations

The study was approved by the Lesotho Ministry of Health Research and Ethics Committee, the Baylor College of Medicine Children’s Foundation Lesotho Institutional Review Board (IRB), and the George Washington University Committee on Human Research IRB. All women were informed of the study protocol, requirements, and risks and benefits, and provided written informed consent to participate in the study.

Results

Participant characteristics

A total of 1594 pregnant women (941 HIV-negative and 653 HIV-positive) were enrolled in the study with their infants (Fig 1). Eight HIV negative women seroconverted before delivery. Delivery information was available for 95.4% of HIV positive women (623/653 and 92.2% of HIV negative women (868/941). 623 HIV positive women gave birth to 631 HIV exposed infants (HEI) and 868 HIV negative women gave birth to 879 HIV unexposed infants (HUI). Six week-follow-up information was available for 577 and 831 HEI and HUI respectively.

Fig 1

Study enrollment for comparison of 6-week PMTCT outcomes for HEI and HUI in the era of lifelong ART.

Characteristics of HIV-positive and HIV-negative study women at enrollment are presented in Table 1. HIV-positive pregnant women were older than their HIV-negative counterparts with a mean age of 28.7 (+/- 5.5) compared to 24.4 (+/- 5.7) years. HIV-positive women also presented earlier for ANC at a mean gestational age of 23.0 (+/- 8.7) weeks compared to 25.3 (+/- 8.2) weeks. Overall, 83.5% of the women were married, and 59.3% had disclosed their HIV-status to their partner/husband. Concerning HIV status of spouses, 4.2% of HIV negative women had HIV positive partners while 29.6% of HIV positive women had an HIV negative partner.

Table 1

Characteristics of study women at enrollment.

	Maternal HIV Status		Total Mothers
	HIV-negative (N = 941)	HIV-positive (N = 653*)	Total (N = 1593)
	n (%)	n (%)	n (%)
Maternal age in years (mean +/- SD)	24.4 +/- 5.7	28.7 +/- 5.5	26.0 +/- 6
Gestational age at first ANC in weeks (mean, SD)	25.3 +/- 8.2	23.0 +/- 8.7	24.4 +/- 8.5
Marital Status: Married	801 (85.1)	528 (81.0)	1329 (83.4)
Mother disclosed HIV status to husband/partner	521 (55.4)	422 (64.9)	943 (59.3)
Missing Data	0	3	3
Maternal ARV Regimen at enrollment
TDF+3TC+EFV	N/A	548 (84.2)	548 (84.2)
TDF+3TC+NVP	N/A	25 (3.8)	25 (3.8)
AZT+3TC+EFV	N/A	28 (4.3)	28 (4.3)
AZT+3TC+NVP	N/A	29 (4.5)	29 (4.5)
ART-other regimens	N/A	7 (1.1)	7 (1.1)
None	N/A	14 (2.2)	14 (2.2)
Missing data	N/A	1	1
Husband/Partner’s HIV Status
Positive	19 (4.2)	230 (68.9)	249 (31.8)
Negative	422 (94.0)	99 (29.6)	521 (66.5)
Unknown	8 (1.8)	5 (1.5)	13 (1.7)
Not tested	492	318	810
Husband/Partner Taking ARVs
Yes	14 (73.7)	166 (72.2)	180 (72.3)
No	5 (26.3)	63 (27.4)	68 (27.3)
Unknown	0	1 (0.6)	1 (0.4)

*One woman was enrolled but excluded from analysis due to missing enrolment questionnaire data

Among HIV-positive women, 97.8% were receiving ART at enrollment with 84.2% receiving the TDF/3TC/EFV first-line regimen. Among the 619 women with data available on the timing of ART, 249 (40.2%) initiated ART before conception and 370 (59.8%) initiated ART after conception.

Birth outcomes

Overall, 91.6% of study women delivered in a health facility and 96.8% (n = 1443) of infants were born alive (Table 2). There was no difference in the proportion of infant stillbirths, however, HIV-positive women were more likely to have had a macerated stillbirth (consistent with antepartum death), while HIV-negative women were more likely to have had an intrapartum death (fresh stillbirth or a liveborn infant who died within two hours of delivery). The risk of premature birth in HEI was more than double the risk of prematurity in HUI (7.8% vs 3.6%). The risk of very premature births (<32 weeks gestational age) was also substantially higher in HEI (2.2%) compared to HUI (0.4%).

Table 2

Birth outcomes by mother’s HIV status at delivery.

	Mother’s HIV Status at delivery		Total MothersN = 1491n (%)	P-value
	HIV-negativeN = 868n (%)	HIV-positiveN = 623n (%)
Miscarriage*	7 (0.8)	10 (1.6)	17 (1.1)	0.16
Mode of Delivery
Vaginal	748 (86.6)	525 (84.8)	1273(85.9)	0.11
Cesarean section	116 (13.4)	94 (15.2)	210(14.1)
Place of delivery
Health facility	786/858 (91.6)	560/620(90.3)	1346(91.1)
Home	67 (7.8)	53 (8.5)	120 (8.1)
Other	5 (0.5)	7 (1.1)	12 (0.8)
Missing data	10	2	12
Birth Outcome
Liveborn†	840 (96.8)	603 (96.8)	1443 (96.8)
Antepartum death	5 (0.6)	12 (1.9)	17 (1.1)	0.01
Intrapartum death	23 (2.6)	8 (1.3)	31 (2.1)
Newborn Maturity
Mature	835 (96.0)	574 (91.7)	1409 (94.2)	0.001
Premature	35 (4.0)	52 (8.3)	87 (4.8)
Very Premature delivery (<32 wks)	3 (0.4)	13 (2.2)	16 (1.1)	0.001
Premature delivery (<37 wks)	31 (3.6)	48 (7.8)	79 (1.6)	<0.01
Missing data	5	1	6
Newborn with congenital anomalies	9 (1.0)	4 (0.6)	13 (0.9	0.56
Birth Weight in kilograms
Normal weight	736 (90.8)	514(88.3)	1250(89.7)	0.15
Low Birth Weight (<2.5 kg)	75 (9.2)	68(11.7)	143(10.3)
Very Low Birth Weight (<1.5 kg)	7 (0.9)	8 (1.4)	15(1.1)	0.43

*There were no miscarriages or stillbirths recorded among the 8 women who seroconverted between enrollment and delivery

†This does not include babies who were born alive and died with two hours.

Of the 249 women who initiated ART pre-conception, 24 (9.6%) had premature babies compared to 27 (7.3%) among the 370 women who initiated ART post-conception. The rates of very low weight (<1.5kgs) and low birth weight (<2.5 kgs) among women who initiated ART before conception were 1.7% and 11.6% respectively compared to 1.2% and 11.9% among women who initiated ART after conception.

The rate of congenital anomalies was 0.6% and 1% among HEI and HUI respectively.

Infant survival

There were no substantial differences in the rates of infant survival at 6 weeks of age by infant HIV exposure status (Fig 2 and Table 3). When including all deaths (liveborn plus stillbirths), the estimated survival rates were 94.8% (95% CI: 93.1–96.1) among HUI and 94.0% (95% CI: 91.8–95.7) among HEI. Analysis of postnatal deaths only (excluding stillbirths), yielded estimated survival rates of 96.8% (95% CI: 95.4–97.9) and 96.7% (95% CI: 95.0–98.0) for HUI and HEI respectively. Adjusting for maternal mortality and gestational age at first ANC visit, infant HIV exposure status was not associated with early infant mortality (aOR = 1.06, 95% CI: 0.56–1.99).

Fig 2

Survival of HUI and HEI at six weeks of age including and excluding stillbirths.

Table 3

Survival of HUI and HEI at six weeks of age.

	HIV-Unexposed		HIV-Exposed
	Death(rate)	Survival(95% CI)	Death(rate)	Survival(95% CI)
Including stillbirths	46/877 (5.2%)	94.8% [93.1–96.1]	38/631 (6.0%)	94.0% [91.8–95.7]
Excluding stillbirths	27/858 (3.2%)	96.8% [95.4–97.9]	20/613 (3.3%)	96.7% [95.0–98.0]

Mortality and HIV-free survival among HEI

Six infants were diagnosed with HIV infection by 6 weeks of age, including 4 diagnosed at birth, and 2 diagnosed at 6 weeks of age (Table 4). The estimated HIV transmission rate among those tested at birth was 0.9% (95% CI: 0.25–2.36) and by 6 weeks the overall HIV transmission was 1.0% (95%CI: 0.38–2.23). The estimated HIV-free survival including stillbirths was 92.8% (95% CI: 90.5–94.8), and 95.6% (95% CI: 93.7–97.1) when stillbirths were excluded.

Table 4

HIV-free survival at six weeks of age.

	HIV-Exposed Infants
	Death(rate)	HIV infection(rate)	Infected/Death(rate)	HIV-Free Survival(95% CI)
Including stillbirths	38/627 (6.1%)	6/581 (1.0%)	44/613 (7.2%)	92.8% [90.4–94.7]
Excluding stillbirths	20/609(3.3%)	6/581 (1.0%)	26/595(4.4%)	95.6% [93.7–97.1]

Five of the 6 HIV-infected infants had mothers that were initiated on ART post-conception (Table 5). Five of the mothers of infected infants had records of viral load at delivery and of these, 4 women had viral loads above 100,000 copies/ml. All mothers were on a TDF/3TC/EFZ regimen as per the national guidelines.

Table 5

Timing of ART initiation and maternal viral load at delivery for HIV-infected infants.

Infant #	Maternal age (years)	Gestational age at first ANC(weeks)	Timing of ART initiation	Maternal duration on ART before delivery (months)	Infant maturity and weight (kg) at birth	Maternal ART regimen at enrollment+	Maternal VL at delivery (copies/ml)
1	23	32	Pre-conception	32.5	Mature—2.4	TLE	36,881
2	22	24	Post-conception	3.5	Mature—3.5	TLE	109,000
3	32	18	Post-conception	5.6	Mature—3.5	TLE	428,054
4	18	18	Post-conception	4.6	Mature—2.8	TLE	320,000
5	23	10	Post-conception	6.9	Mature—2.8	TLE	100,062
6	25	29	Post-conception	2.6	Mature—2.9	TLE	-

+All mothers were initiated on Tenofovir-Lamivudine-Efavirenz (TLE) after HIV diagnosis and remained on this regimen throughout the study.

At 6 weeks, the mortality rate was higher among premature babies 24.6% (17/69) compared to mature infants 2.1% (29/1391). There was also a difference in 6-8-week mortality between low birth weight infants compared to normal birth weight infants (7.0% vs. 1.9%). Very low birth weight infants had higher risk of death within 6–8 weeks compared to infants born weighing at least 1.5kgs or more (30% vs 2.2%).

Discussion

This is the first prospective cohort study in Lesotho comparing birth and 6-week outcomes between HEI and HUI in a large cohort of HIV-positive and negative women and their infants. We found that birth outcomes of HEI were similar to those of HUI except for the frequency of prematurity, which was found to be significantly higher among HEI. While prematurity among HEI has been reported in several other studies [17-19], there have been few studies conducted within routine health systems with large numbers of both HIV-positive and HIV-negative women in the era of lifelong ART for all pregnant women with TDF-based ART regimens. In our study, infant prematurity and low birth weight were not significantly different among women who initiated ART before conception compared to those who initiated ART after conception. This is consistent with other studies that reported no relationship between preconception ART and preterm delivery [17,18]. However, in a systematic review and meta-analysis of adverse pregnancy outcomes and timing of initiation of ART, Uthman et. al reported significantly higher risk of prematurity among HEI whose mothers initiated ART before conception compared to those who initiated ART after conception (pooled RR 1.20, 95%CI 1.01–1.44) [8].

As recommended for first-line treatment in Lesotho, 88% of HIV-positive women were on TDF-based ART regimens. In a systematic review and meta-analysis looking at safety of TDF-based regimens in pregnancy for HIV-positive women and their infants, the rates of prematurity and stillbirths were significantly lower among women on TDF-based ART compared to other ART regimens [20, 21]. However, even with the use of TDF regimens, our study showed that HEI still had a higher risk of prematurity compared to HUI.

We found that HEI were more likely to die antepartum (1.9% versus 0.6%), consistent with medical complications, while HUI were more likely to die during the intrapartum period (2.6% versus 1.3%), consistent with obstetrical complications. However, it may be important to note that these women were followed up only from their first ANC and we may have missed some of the antepartum deaths which occurred before women were enrolled in the study. A number of studies have explored causes of antepartum death among HEI. In South Africa and Botswana, maternal vascular malperfusion was more frequent among HIV-positive women and placenta insufficiency associated with hypertension accounted for most stillbirths [22,23]. In our study, postpartum infant mortality at 6 to 8 weeks was independently associated with gestational age, but not with HIV exposure status.

We found one of the lowest 6-week HIV transmission rates (1.0%) reported in Lesotho coupled with very high HIV-free survival among liveborn infants at 6 weeks of age. This is a significant improvement compared to the estimated 6-week transmission of 7% reported in Lesotho in 2016 and in other countries in the region [1, 24]. Two-thirds of the HIV-positive infants identified were infected in utero. Most women presented for their first ANC visit toward the end of the second trimester, contrary to the WHO recommendation for women to present during the first trimester. Early ANC visits are especially important for HIV-positive pregnant women because earlier ART initiation may further reduce MTCT in utero [12,25]. This was buttressed by the findings that almost all women who transmitted HIV to their infants had a high viral load despite being on ART. Therefore, we agree with Smith et. al that it is essential to maximize viral suppression for HIV-positive women on ART [25].

Our findings indicate that the current universal ART program within the setting of routine care is effective. Implementation of this program showed that high ANC utilization, and high uptake of ART during pregnancy, including a high proportion of facility-based deliveries, collectively led to improved pregnancy outcomes among HIV-positive women. Our study also demonstrates the importance of incorporating implementation research to document program effectiveness within routine, public health settings. Lesotho’s experience shows that when PMTCT programs are well implemented, routine program setting can achieve high effectiveness, comparable to more controlled research settings.

A limitation of our study is that it measured birth outcomes and 6-week HIV-free survival in a facility-based population and may have missed women and children who did not seek care in health facilities. We may have also missed women who lost pregnancies early. In addition, our study may have potential systematic errors arising from estimation of gestational age with the potential of misclassifying baby’s maturing at delivery. However, since ANC attendance in Lesotho is higher than in many African countries, we believe that the results are reflective of the Lesotho context [26, 27]. The successful reduction of perinatal HIV transmission that we report may not be comparable in areas with lower ANC attendance and low rates of facility-based deliveries. Additionally, study sites were purposively selected so the findings may not be generalizable to the whole country of Lesotho, especially to low volume facilities, which were not included in the study. However, the purposive selection of sites from the three geo-ecological settings in Lesotho (highlands, foothills, lowlands) does account for the variances in health-seeking behaviors.

Conclusion

Implementation of universal maternal ART was associated with low MTCT among infants at 6 weeks of age with no differences in congenital anomalies or early mortality between HEI and HUI. However, HEI continue to have increased rates of prematurity even in the era of lifelong combination ART.

S3_File.excel birth outcomes data set.

(XLSX)

Click here for additional data file.

12 Aug 2019

PONE-D-19-18206

Comparison of 6-week PMTCT outcomes for HIV-exposed and HIV-unexposed infants in the era of lifelong ART: results from an observational prospective cohort study

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Reviewer #1: Question 1: I answered "partly" for the following reasons

a) I would recommend the authors define the variables collected further in the Methods section. As an example, was marital status self-report? Same for HIV status of their partner and disclosure. Specifically, what variables were from interviews and which were from medical records. This information will allow readers to better assess the results.

b) On lines 245-247, the authors mention HIV-exposed infants being more likely to die antepartum. While I don't disagree with the point, follow-up of women would need to begin at conception to make this determination. Rather only some antenatal period is captured (time since first ANC visit).

Question 2: I would have answered partly, but it wasn't an option. There are two points I would like to raise.

a) I would encourage the authors to reconsider their usage of p-values and statistical significance regarding interpretations. See the recent issues of The American Statistician. For the interpretation of p-values used by the authors, it requires that all error is random error. However, there is likely systematic error (confounding), since this is observational data and potential confounders are unaccounted for. The presence of the systematic error invalidates the interpretation of p-values used. It may be better to describe the general trends without an appeal to statistical significance.

Related, the systematic errors of the study should be discussed further in the limitations. While selection bias is discussed, there remains measurement error (newborn maturity is difficult to measure), missing data (women/infants lost to follow-up), and confounding (differences in maturity between HIV-exposed and HIV-unexposed may be due to some common cause).

b) Have the authors considered using Kaplan-Meier or similar estimators to report a survival curve instead? This would convey more information than survival at a single time point. It would also clarify the time-scale of interest (time since birth or time since first ANC visit)

Some additional minor comments:

- I am confused by the numbers on Line 138-141. The second and third/fourth sentence of the paragraph seem to repeat the same information. Furthermore, both n=652 and n=631 are said to be the number of HIV-exposed infants. It is not clear to me why these numbers are different.

- I would encourage the authors to write out HIV-exposed and HIV-unexposed infants rather than use abbreviations.

- Line 178, the p-value does not match Table 2.

- Why were both means and standard deviation, and median and interquartile range used? Based on the Results, it seems that only mean and standard deviations were used, but both are mentioned in the Methods.

Reviewer #2: Great manuscript - well written.

Minor detail: table 4 - the author uses (*) in the table and (+) in the footnote

Reviewer #3: Thank you for the opportunity to review this manuscript that presents a prospective cohort study of women living with (N=653) and without (N=941) HIV and their infants up to 6 weeks of age in Lesotho receiving care within routine health services. The study compared birth outcomes (stillbirth, preterm birth, low birth weight, congenital anomalies) and survival at 6 weeks of age in HIV exposed and HIV unexposed infants and also described HIV acquisition by 6 weeks of age in HIV exposed infants. The study found substantially higher rates of preterm and very preterm birth in HEI compared to HUI and a remarkably low rate of HIV-acquisition of 1% in HEI at 6 weeks of age.

Overall it is a valuable paper documenting the experience in Lesotho. I recommend however some re-consideration of the interpretation of the results and analyses presented. Please see detailed comments below.

Major comments:

• I would be cautious to make any conclusions about difference (present or absent) in congenital anomalies – the proportions were 0.6% in HUI and 1.0% in HEI, and although this isn’t a statistically significant difference in this sample, these are rare outcomes with low rates and the study is underpowered to detect this difference, the point estimate of which is approximately a 50% increased prevalence in HEI compared to HUI.

• Similarly for very low birth weight, by the point estimates there is an approximately 50% increased risk for VLBW in HEI compared to HUI, but the proportions are very low and thus this sample under-powered to compare these outcomes.

• Methods

o Please can you add to the methods section how gestational age was determined and what the definitions for preterm and very preterm birth were (later in the results the definition of very premature birth is given as < 32 weeks). Would also suggest using the standard terminology of ‘preterm birth’ rather than ‘premature birth’.

o Please also add to methods how miscarriages and stillbirths were defined

• The rate of miscarriage is extremely low in both groups (up to 20% of pregnancies end in miscarriage) – this may be due to how miscarriages were defined or more likely due to under-ascertainment, with likely only late miscarriages documented in this sample of women who largely presented for antenatal care well after the first trimester. For these reasons I would be hesitant to present results on miscarriages at all.

• The results presented illustrate very well that there is a pathway between HIV exposure, preterm birth and early mortality – HIV exposure is associated with a higher rate of preterm birth and preterm birth is associated with early mortality. I was therefore confused by the very final sentence of the results briefly presenting the only multivariable model described in the paper, that evaluated the association between preterm birth and early mortality adjusted for HIV exposure. From the earlier results presented preterm birth is on the causal pathway between HIV exposure (HIV exposure � preterm birth � early infant mortality) and thus adjusting for either HIV exposure or preterm birth in the reciprocal association with mortality is inappropriate. Lines 250-251 in the discussion that lead from this, discount that HIV exposure is still problematic as this is the risk factor for the increased preterm birth that is leading to the increased mortality.

• From the descriptive results presented there are additional potential maternal confounders to take into consideration when evaluating HIV exposure and early infant mortality – particularly the differences in maternal age and gestation at first ANC. Were additional multivariable analyses conducted to take these differences into account.

• Was morbidity evaluated in any way – e.g. hospitalization before 6 weeks of age? There may be improving survival but with the higher rate of preterm birth there may still be a substantial hospitalization or other longer-term morbidity experienced by HEI (and the health care system) that is missing from this analysis. If not evaluated, this should be added as a limitation.

Minor comments:

• Would you consider using alternative terminology in place of “mother-to-child transmission”? Women with HIV have repeatedly expressed finding this term stigmatizing and prefer alternatives such as vertical HIV transmission or perinatal and postnatal HIV transmission. I understand that most country program names are still called PMTCT Programs, and this may be difficult to avoid, however in all other contexts alternatively terminology would be possible.

• Generally, it is also now appropriate to use people-first terminology and refer to women living with/without HIV rather than HIV positive/negative women.

Introduction

• If PMTCT is to be used, please write out in full when used the first time in line 44.

• Line 52-53 seems incomplete at the end “Some studies found that more adverse birth outcomes such as....[where more common/occurred more often?]”

Results

• Lines 140-142 (pg 8) Please can you explain the denominators for both the HEI and HUI groups. Why are the denominators in parentheses not the same as the number stated in the narrative text i.e Of the 631 HEI we had information on 95.4% (623/653) – why is the 653 different to the 631? Same for the HUI why is the 941 different to the 879 HUI?

• Thank you for the contribution of distinguishing macerated from fresh stillbirths. This additional classification of stillbirths has seldom been given in the HIV birth outcomes literature and this distinction helps to start to understand potential mechanistic pathways.

• Table 4 – column heading “Gestational Age” is unclear – gestational age when (at delivery, at ART initiation, at first presentation for ANC?)

**********

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Reviewer #2: No

Reviewer #3: Yes: Amy L. Slogrove

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27 Sep 2019

PONE-D-19-18206

Comparison of 6-week PMTCT outcomes for HIV-exposed and HIV-unexposed infants in the era of lifelong ART: results from an observational prospective cohort study

General Comments from the Academic Editor

We note that you have reported significance probabilities of 0 in places. Since p=0 is not strictly possible, please correct this to a more appropriate limit, eg 'p<0.0001'.

Thank for the observation, we have made the necessary correction throughout the manuscript.

We have heard back from reviewers. Both reviewers #1 and #3 provide very details suggestion on how to revise the manuscript and caution about the use of P-value, I particularly agree with Reviewer #1 suggestion to simply not use P-value.

Thank you for the observation, we have taken note and have adjusted the text accordingly.

________________________________________

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:

Comment 1:

Question 1- I answered "partly" for the following reasons

a) I would recommend the authors define the variables collected further in the Methods section. As an example, was marital status self-report? Same for HIV status of their partner and disclosure. Specifically, what variables were from interviews and which were from medical records. This information will allow readers to better assess the results.

Thank you for the recommendation to clarify data collection methods for the different variables. We have amended the data collection section to indicate which variables were collected through structured interviews with pregnant women and variables extracted from medical charts and clinic registers.

b) On lines 245-247, the authors mention HIV-exposed infants being more likely to die antepartum. While I don't disagree with the point, follow-up of women would need to begin at conception to make this determination. Rather only some antenatal period is captured (time since first ANC visit).

Thank you for this important comment. We have added a sentence and the paragraph now reads as follows:

We found that HEI were more likely to die antepartum (1.9% versus 0.6%), consistent with medical complications, while HUI were more likely to die during the intrapartum period (2.6% versus 1.3%), consistent with obstetrical complications. However, it may be important to note that these women were followed up only from their first ANC visit and we may have missed some of the antepartum deaths which occurred before women were enrolled in the study.

Comment 2:

Question 2- I would have answered partly, but it wasn't an option. There are two points I would like to raise.

a) I would encourage the authors to reconsider their usage of p-values and statistical significance regarding interpretations. See the recent issues of The American Statistician. For the interpretation of p-values used by the authors, it requires that all error is random error. However, there is likely systematic error (confounding), since this is observational data and potential confounders are unaccounted for. The presence of the systematic error invalidates the interpretation of p-values used. It may be better to describe the general trends without an appeal to statistical significance.

Related, the systematic errors of the study should be discussed further in the limitations. While selection bias is discussed, there remains measurement error (newborn maturity is difficult to measure), missing data (women/infants lost to follow-up), and confounding (differences in maturity between HIV-exposed and HIV-unexposed may be due to some common cause).

We take note of the reviewer’s concern on the p-value interpretations and the existence of systematic error due to potential measurement error and confounding. We have adjusted the text accordingly and acknowledge potential biases from the systematic errors.

b) Have the authors considered using Kaplan-Meier or similar estimators to report a survival curve instead? This would convey more information than survival at a single time point. It would also clarify the time-scale of interest (time since birth or time since first ANC visit)

We have now included Kaplan-Meier as figure 2 to report survival.

Comment 3:

I am confused by the numbers on Line 138-141. The second and third/fourth sentence of the paragraph seem to repeat the same information. Furthermore, both n=652 and n=631 are said to be the number of HIV-exposed infants. It is not clear to me why these numbers are different.

Thank you for the observation. We have corrected the numbers and the paragraph now reads as follows:

A total of 1594 pregnant women (941 HIV-negative and 653 HIV-positive) were enrolled in the study with their infants (Figure 1). Eight HIV negative women seroconverted before delivery. Delivery information was available for 95.4% of HIV positive women (623/653 and 92.2% of HIV negative women (868/941). 623 HIV positive women gave birth to 631 HIV exposed infants (HEI) and 868 HIV negative women gave birth to 879 HIV unexposed infants (HUI). Six-week follow up information was available for 577 and 831 HEI and HUI respectively.

Comment 4:

I would encourage the authors to write out HIV-exposed and HIV-unexposed infants rather than use abbreviations.

We accept the recommendation.

Comment 5:

Line 178, the p-value does not match Table 2.

Thank you for identifying error. We have corrected the p-value to match the value in Table 2 which is the correct value.

Comment 6:

Why were both means and standard deviation, and median and interquartile range used? Based on the Results, it seems that only mean and standard deviations were used, but both are mentioned in the Methods.

We have edited the method to remove median and interquartile range. Indeed, the results reported on means and standard deviation.

Reviewer #2:

Comment 7:

Great manuscript - well written.

Thank you very much for your kind words.

Comment 8:

Minor detail: table 4 - the author uses (*) in the table and (+) in the footnote

This has been corrected.

Reviewer #3:

General Comment

Thank you for the opportunity to review this manuscript that presents a prospective cohort study of women living with (N=653) and without (N=941) HIV and their infants up to 6 weeks of age in Lesotho receiving care within routine health services. The study compared birth outcomes (stillbirth, preterm birth, low birth weight, congenital anomalies) and survival at 6 weeks of age in HIV exposed and HIV unexposed infants and also described HIV acquisition by 6 weeks of age in HIV exposed infants. The study found substantially higher rates of preterm and very preterm birth in HEI compared to HUI and a remarkably low rate of HIV-acquisition of 1% in HEI at 6 weeks of age.

Overall it is a valuable paper documenting the experience in Lesotho. I recommend however some re-consideration of the interpretation of the results and analyses presented. Please see detailed comments below.

Specific comments

Comment 9:

I would be cautious to make any conclusions about difference (present or absent) in congenital anomalies – the proportions were 0.6% in HUI and 1.0% in HEI, and although this isn’t a statistically significant difference in this sample, these are rare outcomes with low rates and the study is underpowered to detect this difference, the point estimate of which is approximately a 50% increased prevalence in HEI compared to HUI.

Thank you so much for your comment. We agree with you and have reviewed the text to read as follows.

The rate of congenital anomalies was 0.6% and 1.0% among HEI and HUI respectively.

Comment 10:

Similarly, for very low birth weight, by the point estimates there is an approximately 50% increased risk for VLBW in HEI compared to HUI, but the proportions are very low and thus this sample under-powered to compare these outcomes.

Thank you for the comment, we have edited the paragraph which now reads as follows:

The rates of very low weight (<1.5kgs) and low birth weight (<2.5 kgs) among women who initiated ART before conception was 1.7% and 11.6% respectively compared to 1.2% and 11.9% among women who initiated ART after conception.

Methods

Comment 11:

Please can you add to the methods section how gestational age was determined and what the definitions for preterm and very preterm birth were (later in the results the definition of very premature birth is given as < 32 weeks). Would also suggest using the standard terminology of ‘preterm birth’ rather than ‘premature birth’.

We have added the definitions and now use preterm birth as standard. Please see below.

Gestational age at birth was estimated by time between the date of last menstrual period given by the women at first ANC visit and the date of birth. Very preterm birth was defined as infant born at gestational age of 28-32 weeks while preterm birth was infant born after 32 weeks but before 37 weeks [15]

Comment 12:

Please also add to methods how miscarriages and stillbirths were defined.

Thank you. The definitions have been added and reads as follows:

In addition, miscarriage was defined as loss of pregnancy before the gestational age of 28 weeks and stillbirth was considered when the pregnancy was lost after 28 weeks [16].

Comment 13:

The rate of miscarriage is extremely low in both groups (up to 20% of pregnancies end in miscarriage) – this may be due to how miscarriages were defined or more likely due to under-ascertainment, with likely only late miscarriages documented in this sample of women who largely presented for antenatal care well after the first trimester. For these reasons I would be hesitant to present results on miscarriages at all.

Thank you for the comment. We understand the concern of the reviewer, however it will be a missed opportunity not to present this important data point despite its limitation in terms of collecting data only for women who lost pregnancy after enrolment in the study. This is a limitation of the study because the study is taking place at health facility level.

Comment 14:

The results presented illustrate very well that there is a pathway between HIV exposure, preterm birth and early mortality – HIV exposure is associated with a higher rate of preterm birth and preterm birth is associated with early mortality. I was therefore confused by the very final sentence of the results briefly presenting the only multivariable model described in the paper, that evaluated the association between preterm birth and early mortality adjusted for HIV exposure. From the earlier results presented preterm birth is on the causal pathway between HIV exposure (HIV exposure � preterm birth � early infant mortality) and thus adjusting for either HIV exposure or preterm birth in the reciprocal association with mortality is inappropriate. Lines 250-251 in the discussion that lead from this, discount that HIV exposure is still problematic as this is the risk factor for the increased preterm birth that is leading to the increased mortality.

Thank you for your comment. We agree with your observation and adjusting for HIV status is inappropriate as you rightly say. We have removed the HIV status adjusted result.

Comment 15:

From the descriptive results presented there are additional potential maternal confounders to take into consideration when evaluating HIV exposure and early infant mortality – particularly the differences in maternal age and gestation at first ANC. Were additional multivariable analyses conducted to take these differences into account.

While the distribution of both maternal age and gestational age at first ANC visit do differ by HIV exposure status, both maternal age and gestational age are not associated with early infant mortality therefore are unlikely to be confounders in the relationship between HIV exposure and early infant mortality. We went ahead and fitted a multivariate model of early infant mortality and HIV exposure adjusting for maternal age and gestational age. The unadjusted odds ratio for HIV exposure is 1.04 (95% CI: 0.58 – 1.87) while the adjusted odds ratio is 1.06 (95% CI: 0.56 – 1.99). We have included a statement in the results.

Comment 16:

Was morbidity evaluated in any way – e.g. hospitalization before 6 weeks of age? There may be improving survival but with the higher rate of preterm birth there may still be a substantial hospitalization or other longer-term morbidity experienced by HEI (and the health care system) that is missing from this analysis. If not evaluated, this should be added as a limitation.

We indeed did collected hospitalization data on a subset of the infants. However, there was no substantial difference in the rate of hospitalization between HIV exposed (N=532) and unexposed (N=589) infants in the first 6-8 weeks of life (1.32 % versus 1.02%).

Minor comments:

Comment 17:

Would you consider using alternative terminology in place of “mother-to-child transmission”? Women with HIV have repeatedly expressed finding this term stigmatizing and prefer alternatives such as vertical HIV transmission or perinatal and postnatal HIV transmission. I understand that most country program names are still called PMTCT Programs, and this may be difficult to avoid, however in all other contexts alternatively terminology would be possible.

Thank you for your comment. Although, recently the term HIV care for pregnant and breastfeeding women (PBFW) is being introduced, in the context of this paper, we felt mother to child transmission was still appropriate since the overall project was about evaluating effectiveness of PMTCT program.

Comment 18:

Generally, it is also now appropriate to use people-first terminology and refer to women living with/without HIV rather than HIV positive/negative women.

Thank you for this comment. This is very well noted and we agree with the suggestion. However, since this terminology is emerging, we will plead to use HIV negative and HIV positive terms here for consistency.

Introduction

Comment 19:

If PMTCT is to be used, please write out in full when used the first time in line 44.

Thank you for the comment. This has been done.

Comment 20:

Line 52-53 seems incomplete at the end “Some studies found that more adverse birth outcomes such as....[where more common/occurred more often?]”

The sentence now reads:

Some studies found that adverse birth outcomes, such as increased preterm deliveries, stillbirths and low birth weight, occurred more frequently among HEI [3-8].

Results

Comment 21:

Lines 140-142 (pg 8) Please can you explain the denominators for both the HEI and HUI groups. Why are the denominators in parentheses not the same as the number stated in the narrative text i.e Of the 631 HEI we had information on 95.4% (623/653) – why is the 653 different to the 631? Same for the HUI why is the 941 different to the 879 HUI?

Thank you for the observation. We have corrected the numbers and the paragraph now reads as follows:

A total of 1594 pregnant women (941 HIV-negative and 653 HIV-positive) were enrolled in the study with their infants (Figure 1). Eight HIV negative women seroconverted before delivery. Delivery information was available for 95.4% of HIV positive women (623/653 and 92.2% of HIV negative women (868/941). 623 HIV positive women gave birth to 631 HIV exposed infants (HEI) and 868 HIV negative women gave birth to 879 HIV unexposed infants (HUI). Six-week follow up information was available for 577 and 831 HEI and HUI respectively.

Comment 22:

Thank you for the contribution of distinguishing macerated from fresh stillbirths. This additional classification of stillbirths has seldom been given in the HIV birth outcomes literature and this distinction helps to start to understand potential mechanistic pathways.

Thank you for your kind comment.

Comment 23:

Table 4 – column heading “Gestational Age” is unclear – gestational age when (at delivery, at ART initiation, at first presentation for ANC?)

Thank you for the observation. This has been clarified in Table 4.

Submitted filename: Response to Reviewers comment-Birth Outcomes in Lesotho.docx

Click here for additional data file.

22 Oct 2019

PONE-D-19-18206R1

Comparison of 6-week PMTCT outcomes for HIV-exposed and HIV-unexposed infants in the era of lifelong ART: results from an observational prospective cohort study

PLOS ONE

Dear Dr. Tiam,

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.

In addition to comments from the reviewer about overuse/interpretation of P-value, I also agree that further discussion of the systematic error will be to the benefit of this paper.

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Marcel Yotebieng, M.D., MPH, Ph.D

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. 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: I thank the authors for their revisions. I have a few minor comments that I believe will help improve the manuscript.

Line 48: MTCT is no longer abbreviated in the manuscript and therefore should be written out.

Lines 108-112: While the information is available in the tables, it may be useful to include the categories of responses in the text. Something along the lines of "HIV status of spouse (yes; no)".

Line 133: Only means are mentioned in the edited version but standard deviations are still calculated and presented in tables.

Lines 136-137: I see that the authors removed most of the p-values as previously suggested. Therefore, I am not sure why statistical tests (Chi-square and t-test/rank-sum) are discussed in the analysis section. Additionally, the authors may want to mention the use of the Kaplan-Meier estimator to generate the survival curves.

Lines 166, 168, 202: It appears that p-values are still used for interpreting some descriptive results.

Lines 239, 240, 242, 283: The word "significant" is used to describe the results. While maybe not what the authors intend, this may easily be misread by readers as statistically significant, so I would suggest replacing or removing the word "significant".

Lines 302-312: While the authors did add to their limitations, I believe that further discussion of systematic errors such as measurement error would be beneficial.

Reviewer #2: Minor comment: Line 26 in the abstract, the authors conclude that “Implementation of universal maternal ART lowers MTCT BY 6 weeks of age ...”. This conclusion is not supported by the findings. Did you mean “at 6 weeks of age? as is consistent with teh conclusion in the main paper (line 288)

Reviewer #3: Authors have addressed my initial comments adequately and I have no additional comments. Recommend manuscript for publication.

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Amy L. Slogrove

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

15 Nov 2019

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:

I thank the authors for their revisions. I have a few minor comments that I believe will help improve the manuscript.

Line 48: MTCT is no longer abbreviated in the manuscript and therefore should be written out.

Thank you, MTCT has been written in full in the manuscript.

Lines 108-112: While the information is available in the tables, it may be useful to include the categories of responses in the text. Something along the lines of "HIV status of spouse (yes; no)".

The following sentence has been inserted: “Concerning HIV status of spouses, 4.2% of HIV negative women had HIV positive partners while 29.6% of HIV positive women had an HIV negative partner.”

Line 133: Only means are mentioned in the edited version but standard deviations are still calculated and presented in tables.

This has been corrected.

Lines 136-137: I see that the authors removed most of the p-values as previously suggested. Therefore, I am not sure why statistical tests (Chi-square and t-test/rank-sum) are discussed in the analysis section. Additionally, the authors may want to mention the use of the Kaplan-Meier estimator to generate the survival curves.

This has been corrected and the paragraph now reads as follows: “HIV-free survival was estimated as the proportion of children alive and HIV-negative among all exposed children. The precision around survival estimates was assessed by 95% confidence intervals. We used the Kaplan Meier curves to graphically display infant mortality, infection, and HIV free survival. We performed complete case analysis, and missing data were not imputed.”

Lines 166, 168, 202: It appears that p-values are still used for interpreting some descriptive results.

This has been corrected.

Lines 239, 240, 242, 283: The word "significant" is used to describe the results. While maybe not what the authors intend, this may easily be misread by readers as statistically significant, so I would suggest replacing or removing the word "significant".

The language has been corrected.

Lines 302-312: While the authors did add to their limitations, I believe that further discussion of systematic errors such as measurement error would be beneficial.

Thank you. We have added additional explanation.

Reviewer #2: Minor comment: Line 26 in the abstract, the authors conclude that “Implementation of universal maternal ART lowers MTCT BY 6 weeks of age ...”. This conclusion is not supported by the findings. Did you mean “at 6 weeks of age? as is consistent with the conclusion in the main paper (line 288)

Thank you. The sentence in the abstract has been corrected to align with the conclusion of the manuscript.

Reviewer #3: Authors have addressed my initial comments adequately and I have no additional comments. Recommend manuscript for publication.

Thank you very much.

Submitted filename: Response to Reviewers comment-Birth Outcomes in Lesotho_2.docx

Click here for additional data file.

26 Nov 2019

Comparison of 6-week PMTCT outcomes for HIV-exposed and HIV-unexposed infants in the era of lifelong ART: results from an observational prospective cohort study

PONE-D-19-18206R2

Dear Dr. Tiam,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

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With kind regards,

Marcel Yotebieng, M.D., MPH, Ph.D

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

13 Dec 2019

PONE-D-19-18206R2

Comparison of 6-week PMTCT outcomes for HIV-exposed and HIV-unexposed infants in the era of lifelong ART: results from an observational prospective cohort study

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