Vitamin D status, nutrition and growth in HIV-infected mothers and HIV-exposed infants and children in Botswana

Alyssa M Tindall; Joan I Schall; Boitshepo Seme; Bakgaki Ratshaa; Michael Tolle; Maria S Nnyepi; Loeto Mazhani; Richard M Rutstein; Andrew P Steenhoff; Virginia A Stallings

doi:10.1371/journal.pone.0236510

. 2020 Aug 13;15(8):e0236510. doi: 10.1371/journal.pone.0236510

Vitamin D status, nutrition and growth in HIV-infected mothers and HIV-exposed infants and children in Botswana

Alyssa M Tindall ¹, Joan I Schall ¹, Boitshepo Seme ², Bakgaki Ratshaa ², Michael Tolle ³, Maria S Nnyepi ⁴, Loeto Mazhani ⁵, Richard M Rutstein ^6,⁷, Andrew P Steenhoff ^2,^5,^7,^8,^*, Virginia A Stallings ^1,⁷

Editor: Emma K Kalk⁹

¹Gastroenterology, Hepatology and Nutrition, Children’s Hospital of Philadelphia, Philadelphia, PA, United States of America

²Botswana-UPenn Partnership, Gaborone, Botswana

³Botswana-Baylor Children’s Clinical Centre of Excellence, Gaborone, Botswana

⁴Department of Nutrition, University of Botswana, Gaborone, Botswana

⁵Department of Pediatrics and Adolescent Health, School of Medicine, University of Botswana, Gaborone, Botswana

⁶General Pediatrics, Children’s Hospital of Philadelphia, Philadelphia, PA, United States of America

⁷Department of Pediatrics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, United States of America

⁸Divisions of Infectious Diseases, Children’s Hospital of Philadelphia, Philadelphia, PA, United States of America

⁹University of Cape Town, SOUTH AFRICA

Competing Interests: The authors have declared that no competing interests exist.

^✉

* E-mail: steenhoff@email.chop.edu

Roles

Alyssa M Tindall: Formal analysis, Writing – original draft, Writing – review & editing

Joan I Schall: Conceptualization, Formal analysis, Investigation, Methodology, Project administration, Writing – original draft, Writing – review & editing

Boitshepo Seme: Investigation, Writing – review & editing

Bakgaki Ratshaa: Investigation, Writing – review & editing

Michael Tolle: Investigation, Writing – review & editing

Maria S Nnyepi: Investigation, Writing – review & editing

Loeto Mazhani: Investigation, Writing – review & editing

Richard M Rutstein: Investigation, Writing – review & editing

Andrew P Steenhoff: Conceptualization, Formal analysis, Investigation, Methodology, Project administration, Supervision, Writing – original draft, Writing – review & editing

Virginia A Stallings: Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Writing – original draft, Writing – review & editing

Emma K Kalk: Editor

PMCID: PMC7425960 PMID: 32790765

Abstract

Background

Poor vitamin D status is a global health problem and common in patients with human immunodeficiency virus (HIV) in high-income countries. There is less evidence on prevalence of vitamin D deficiency and nutrition and growth in HIV-infected and -exposed children in low- and middle-income countries.

Objectives

To determine the vitamin D status in Batswana HIV-infected mothers and their children, differences among HIV-infected mothers and between HIV-exposed and -infected infants and children, and associations between vitamin D and disease-related outcomes, nutrition, and growth.

Methods

This was a cross-sectional study of HIV+ mothers and HIV-exposed infants and unrelated children (1–7.9 years). Serum 25-hydroxyvitamin D (25(OH)D) was measured, among other nutritional indicators, for mothers, infants and children. Vitamin D status for HIV-infected mothers and children, and an immune panel was assessed. History of HIV anti-retroviral medications and breastfeeding were obtained. Data were collected prior to universal combination antiretroviral therapy in pregnancy.

Results

Mothers (n = 36) had a mean serum 25(OH)D of 37.2±12.4ng/mL; 11% had insufficient (<20ng/mL), 17% moderately low (20.0–29.9ng/mL) and 72% sufficient (≥30ng/mL) concentrations. No infants (n = 36) or children (n = 48) were vitamin D insufficient; 22% of HIV- and no HIV+ infants had moderately low concentrations and 78% of HIV- and 100% of HIV+ infants had sufficient status, 8% of HIV- and no HIV+ children had moderately low concentrations and 92% of HIV- and 100% HIV+ children had sufficient concentrations. HIV+ children had significantly lower length/height Z scores compared to HIV- children. Length/height Z score was positively correlated with serum 25(OH)D in all children (r = 0.33, p = 0.023), with a stronger correlation in the HIV+ children (r = 0.47 p = 0.021). In mothers, serum 25(OH)D was positively associated with CD4% (r = 0.40, p = 0.016).

Conclusions

Results showed a low prevalence of vitamin D insufficiency in Botswana. Growth was positively correlated with vitamin D status in HIV-exposed children, and HIV+ children had poorer linear growth than HIV- children.

Introduction

Vitamin D is a fat-soluble, essential vitamin obtained primarily through ultraviolent B rays and plays a critical role in the regulation of bone metabolism, cell growth, immune and neuromuscular function. Vitamin D status correlates with numerous communicable and non-communicable diseases that are major public health problems worldwide. Poor vitamin D status is common in patients with human immunodeficiency virus (HIV) in the United States [1], but there is not substantial evidence on prevalence of vitamin D deficiency in HIV infected and exposed populations in low- and middle-income countries [2], such as Botswana [3]. Botswana has the third highest prevalence of HIV in the world and over 15% of children are HIV-exposed [4, 5]. Determining the prevalence of vitamin D insufficiency in this population is of great importance.

Vitamin D sufficiency can be effectively achieved using vitamin D supplementation [6], however, there are limited data on the prevalence of deficiency among HIV-infected mothers and no data on exposed and infected infants and young children in Botswana. Black race is a risk factor for insufficient vitamin D [7] and HIV-infected mothers are at greater risk of vitamin D deficiency due to tenofovir- and efavirenz-containing combination antiretroviral therapy (cART), which are associated with increased risk of vitamin D deficiency [8–13]. HIV-exposed children account for more than half of 24-month mortality in Botswana, but contributing factors, such as vitamin D status, have not been determined [14]. Vitamin D stores are low at birth in all infants and vitamin D is obtained through breast milk and sunlight exposure. Vitamin D deficiency is more prevalent in children with perinatally acquired HIV infections [15]. Determining the vitamin D status of HIV-infected mothers and HIV-infected or HIV-exposed infants and children in Botswana is critical. Determining if there are any confounding variables, such as feeding practices or nutritional status, that affect vitamin D concentrations will identify areas that could be targeted for intervention.

The primary objective of this urban, Botswana-based observational study was to determine the vitamin D status in HIV-infected mothers and their children–including HIV-exposed or HIV-infected. The secondary objectives were to examine differences in vitamin D status among HIV-infected mothers and between HIV-exposed and infected infants and children, and to explore associations between vitamin D status and disease-related outcomes, breastfeeding, and growth.

Materials and methods

Participants

This was an observational, age- and HIV-status stratified survey of 36 HIV-infected (HIV+) mothers and their infants (0–11.9 months), and 48 unrelated children (1–7.9 years). Mothers, infants and children were recruited from Princess Marina Hospital, Gaborone, and Bamalete Lutheran Hospital, Ramotswa, in Botswana where they were followed for medical care. Mothers and children were recruited over a 10-month period from November, 2012 through August, 2013. Mothers with HIV infection and infants and children with HIV exposure in usual state of good health were eligible and consent was obtained. Mothers, infants or children with a health condition unrelated to HIV infection likely to affect nutritional status, hospitalization, accident or emergency visit, or unscheduled sick visit to a clinic in the two weeks prior to recruitment were not eligible. The protocol was approved by the Institutional Review Boards of the Children’s Hospital of Philadelphia (CHOP), University of Pennsylvania, Botswana Ministry of Health, Princess Marina Hospital, Bamalete Lutheran Hospital, and Baylor College of Medicine.

Prevention of mother to child transmission guidelines

All HIV infected pregnant women were assessed for eligibility for triple prophylaxis/cART according to Botswana’s national HIV guidelines at the time. HIV infected women were evaluated for cART eligibility based on laboratory testing (CD4 cell count) or clinical presentation (WHO clinical stage). Women with CD4 cell count less than 350 or WHO clinical stage 3 or 4 were started on cART through the national ARV program as soon as possible, regardless of the stage of pregnancy. Women attending healthcare facilities that provided triple ARV prophylaxis, who were not eligible for cART were provided with triple ARV prophylaxis starting from 14 weeks of gestation. Women who chose to breastfeed continued triple ARV prophylaxis until their infants were at least six months of age and completed weaning. Both women on cART and those that were on ARV prophylaxis were given supplementary AZT 300 mg every 3 hours during labor and delivery [16].

Similarly by 2009, >95% of infants born to HIV infected mothers were given ARV prophylaxis at birth according to national guidelines; they were given nevirapine as a single dose as soon as possible after birth (within 72 hours) plus four weeks of AZT [16].

Sampling

A convenience sample was used. For balanced representation across infant and child ages and HIV status groups, 18 mother-infant pairs with infants aged 0–5.9 months and 18 pairs with infants aged 6–11.9 months attending routine medical visits were enrolled. Given the rate of perinatal transmission of HIV in Botswana was less than 3% at the time of data collection [16], the younger infants (aged 0–5.9 months) were categorized as HIV-. Removing the younger infants did not change the results, therefore, we included them in the dataset. The older infants, ages 6–11.9 months, were balanced by HIV status as determined by the HIV PCR result in their medical record. Forty-eight unrelated children with HIV-infected mothers were also enrolled, 24 at ages 1–3.9 years and 24 at ages 4–7.9 years, balanced by HIV status. An attempt was made to balance for equal sex representation of infants and children.

Clinical data collection

Following consent, participant study visits consisted of a structured questionnaire, physical measurements, and a blood draw. Clinical data, including HIV status and current medications, were extracted from medical records. HIV status was classified using the Centers for Disease Control and Prevention (CDC) clinical classifications [17, 18] and CD4 count. cART regimens were characterized as tenofovir-containing, and either protease inhibitor-based (PI) or non-nucleoside reverse transcriptase inhibitor-based (NNRTI), i.e., efavirenz or nevirapine. Information on whether infants and children were breast or bottle fed was also obtained from the medical records.

Trained research nurses completed anthropometric measurements [19] and equipment was calibrated weekly. Height and length in centimeters were measured using a stadiometer (Seca, UK) and weight in kilograms by digital standing scale (Adam Medical) for adults and wheel-chair digital scale (Seca, UK) for children. Anthropometric measurements were completed in triplicate and the mean was used for analysis. Body mass index (BMI unit) was calculated as kg/m². For infants and young children up to age two years where length was measured, length and weight were converted into Z scores (standard deviation units) using the WHO growth charts (http://www.cdc.gov/growthcharts/), which is recommended by the CDC [20]. For older children the CDC recommends using the CDC algorithms to convert height and weight into Z scores [21].

Laboratory methods

Vitamin D status biomarkers included serum 25-hydroxyvitamin D (25(OH)D), 1,25-dihydroxyvitamin D (1,25(OH)D), intact parathyroid hormone (PTH), and other nutritional biomarkers associated with vitamin D (serum magnesium, phosphorous, albumin, calcium). Immune status was determined in all women and those children living with HIV. Serum 25(OH)D was measured using liquid chromatography tandem mass spectrometry (Clinical Laboratory, CHOP, Philadelphia, PA, USA) with intra- and inter-assay coefficients of variation (CV) below 8%; serum 1,25(OH)D and PTH were assessed by radioimmunoassay using a radio-iodinated tracer (Heartland Assays, Ames, IA, USA) with intra- and inter-assay CV of 9.8% and 12.6% for 1,25(OH)D and 2.7% and 4.3% for PTH. Serum magnesium, phosphorus, albumin, and calcium (corrected for albumin) were measured using standard techniques (Diagnofirm Diagnostic Laboratory, Gaborone, Botswana). The immunological indicators and HIV-1 RNA VL were measured by Diagnofirm Diagnostic Laboratories (Gaborone, Botswana).

Statistical methods

Serum 25(OH)D concentration was categorized as: insufficient, < 20 ng/mL; moderately low, 20–29.9 ng/mL; and sufficient, ≥ 30 ng/mL. CD4% was categorized as: low, < 15%; moderate, 15–24.9%; and high, ≥ 25%, where a high CD4% indicates a favorable immunological profile. Characteristics of the mothers, infants, and children were analyzed using conventional parametric statistics (means, SD, percentages) and comparisons made between HIV status groups using unpaired (two-sample) student’s t-tests for continuous variables and chi-square or Fisher’s exact tests for categorical data. Z scores for length, height and weight were compared for infants and children by analysis of covariance (ANCOVA), adjusting for chronological age, to account for growth trends with age. Breastfeeding (ever breast fed) and sex were also examined as covariates. Season of examination was grouped into two categories: Spring/Summer (August-January) and Autumn/Winter (February-July). Vitamin D-related laboratory values were compared using analysis of covariance (ANCOVA), adjusting for season of examination (Spring/Summer, Autumn/Winter) to account for possible differences in vitamin D related to sunlight exposure. Given the effect of efavirenz on vitamin D status [8, 9, 11–13], a unique category was created for efavirenz-containing regimens to control for confounding treatment effects. An undetectable RNA VL was set to < 25 copies/mL (RNA log = 1.4). Associations between serum 25(OH)D and outcomes were explored using Pearson correlation coefficients or Spearman rank correlations depending upon skewness. Levels of statistical significance were set at α = 0.05.

Results

Thirty-six HIV+ mothers of infants aged 0–11.9 months were enrolled. HIV+ mothers gave birth to 27 (75%) HIV negative (HIV-) and 9 (25%) HIV+ infants. Forty-eight children, aged 1–7.9 years with HIV exposure (HIV+ mothers) were recruited. Half of the children recruited were HIV- and half HIV+ by study design.

The HIV-infected mothers ranged in age from 21 to 41 (mean±SD, 31.8±5.1 yrs), and had been diagnosed with HIV 3.7±3.2 yrs (range 0.5 to 10 years) prior: 69% were taking cART and 42% were on efavirenz-containing regimens (Table 1). Serum 25(OH)D was 37.2±12.4 ng/mL; 11% had insufficient (<20 ng/mL), 17% moderately low (20.0–29.9 ng/mL) and 72% sufficient (≥ 30 ng/mL) serum 25(OH)D concentrations. The mothers of HIV- vs. HIV+ infants did not differ in terms of current age, height, weight, BMI, or current use of cART, including the use of efavirenz-containing regimens (Table 1). There was a significant difference in the season of examination between mothers with HIV- or HIV+ infants. In terms of vitamin D status, even adjusting for season of examination, the mothers of infants who were HIV+ had significantly lower concentrations of serum 25(OH)D and serum albumin and had a smaller proportion of serum 25(OH)D concentrations in the sufficient range (≥ 30 ng/mL) compared to the mothers of HIV- infants (Table 1). Further adjusting for current use of efavirenz-containing regimens did not alter the significance of these results.

Table 1. Maternal characteristics of HIV positive mothers (n = 36) stratified by HIV status of their infants.

	HIV+ Mothers		p-value
	HIV- Infants	HIV+ Infants
Maternal Characteristic	n = 27	n = 9
Age, y	32.6±5.0	30.9±5.0	0.38
Height, cm	159.6±6.3	158.9±7.7	0.78
Body weight, kg	60.6±9.8	57.2±9.3	0.36
BMI, kg/m²	23.8±3.8	22.6±4.0	0.43
< 18.5, %	4	11	0.71
18.5–24.9, %	63	67
25.0–29.9, %	26	22
≥ 30.0, %	7	0
Season of examination
Spring/Summer, %	11	78	<0.001
Fall/Winter, %	89	22	<0.001
cART, %	70	67	0.84
Efavirenz-containing regimens, %	37	56	0.33
Serum vitamin D and nutrition indicators^*
25(OH)D, ng/mL	40.5±2.5	27.2±4.9	0.03
< 20.0, %	11	11	0.002
20–29.9, %	4	56
≥ 30.0, %	85	33
1,25(OH)D, pg/mL^†	41.8±4.9	44.8±9.4	0.79
PTH, pg/mL^†	35.7±2.7	24.6±5.2	0.09
Magnesium, mmol/L	0.96±0.02	0.87±0.04	0.06
Phosphorus, mmol/L	1.10±0.03	1.14±0.05	0.53
Calcium, mmol/L	2.26±0.02	2.25±0.04	0.49
Albumin, g/L	39.1±0.7	35.6±1.3	0.04

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Data are presented as mean ± standard deviation (SD) or a least square means (LSM) ± standard error of the mean (SEM) for continuous variables, and as percent for distribution of categorical variables. HIV = human immunodeficiency virus; BMI = body mass index; cART = combined antiretroviral therapy; 25(OH)D = 25-hydroxyvitamin D; 1,25(OH)D = 1,25-dihydroxyvitamin D; PTH = intact parathyroid hormone.

*Results are reported as LSM ± SEM from analyses of covariance, adjusting for season of examination.

†Sample sizes are n = 26 for mothers of HIV- infants and n = 9 for mothers of HIV+ infants.

Comparison of the infants who were HIV- or HIV+ indicated a tendency for HIV+ infants to be smaller in both length and weight by approximately -0.9 Z score, but the differences were not statistically significant (Table 2). There were no differences in sex, and few of either group were ever breastfed. Adjusting for season of examination, there was no difference in concentration of serum 25(OH)D, and no infant, whether HIV- or HIV+ had insufficient concentration of serum 25(OH)D. Serum 1,25(OH)D was significantly higher in infants who were HIV+.

Table 2. Infant (n = 36) and child (n = 48) characteristics stratified by HIV status.

	Infants			Children
	HIV-	HIV+	p-value	HIV-	HIV+	p-value
Characteristic	n = 27	n = 9	p-value	n = 24	n = 24	p-value
Age, mo	5.1±3.6	8.5±2.4	0.02	42.6±15.0	54.3±26.0	0.06
Length/height Z Score^*	-0.89±0.37	-1.73±0.67	0.31	-0.83±0.20	-1.43±0.20	0.04
Weight Z Score^*	-0.19±0.24	-1.08±0.42	0.10	-1.15±0.24	-0.90±0.24	0.49
Sex, male, %	37	33	0.84	50	38	0.38
Breastfed, %	7	11	0.73	0	54	<0.001
Season of examination
Spring/Summer, %	11	78 ^‡	<0.001	50	54	0.77
Fall/Winter, %	89	22	<0.001	50	46	0.77
Serum vitamin D and nutrition indicators^*
25(OH)D, ng/mL	45.4±2.8	50.2±5.6	0.49	47.9±2.8	51.4±2.8	0.37
< 20.0 ng/mL, %	0	0	0.12	0	0	0.15
20–29.9 ng/mL, %	22	0		8	0
≥ 30.0 ng/mL, %	78	100		92	100
1,25(OH)D, pg/mL^†	43.6±5.4	82.6±12.6	0.02	51.1±5.9	57.2±6.1	0.51
PTH, pg/mL^†	24.4±2.6	24.2±5.4	0.97	22.0±1.7	26.3±1.8	0.09
Magnesium, mmol/L	1.04±0.03	0.94±0.05	0.16	1.14±0.04	1.07±0.04	0.14
Phosphorus, mmol/L	1.97±0.06	1.94±0.05	0.85	1.59±0.04	1.52±0.04	0.24
Calcium, mmol/L	2.52±0.02	2.51±0.04	0.80	2.29±0.02	2.33±0.02	0.04
Albumin, g/L	37.4±0.7	38.1±1.3	0.67	38.0±0.5	38.5±0.5	0.49

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Data are presented as mean ± standard deviation (SD) or a least square means (LSM) ± standard error of the mean (SEM) for continuous variables, and as percent for distribution of categorical variables. HIV = human immunodeficiency virus; 25(OH)D = 25-hydroxyvitamin D; 1,25(OH)D = 1,25-dihydroxyvitamin D; PTH = intact parathyroid hormone.

*Results are reported as least square means (LSM) ± standard error of the means (SEM) from analyses of covariance, adjusting for age (in months) for length/height z-scores and weight and for season of examination for the serum vitamin D and nutrition indicators.

†Sample sizes are n = 27 for HIV- infants and n = 8 for HIV+ infants; sample sizes are n = 24 for HIV- children and n = 23 for HIV+ children.

Children aged 1–7.9 years had poor growth status overall (approximately -1.0 Z scores for length/height and weight). HIV+ children were significantly shorter, after controlling for age. When examined separately, males and females had similar growth outcomes, with HIV+ children shorter than their HIV- counterparts. There were no HIV- children who were ever breastfed, but a little over half of the HIV+ were breastfed as infants (Table 2). Both sex and whether ever breastfed were tested as covariates, and neither significantly contributed to model or explained the difference in length/height z scores between HIV+ and HIV- children. Furthermore, in the HIV+ group, there were no significant differences in growth status in length or weight between children who were breastfed as infants and those that were not. Vitamin D status in HIV+ was comparable to children who were HIV-, and no child had an insufficient (< 20 ng/mL) 25(OH)D concentration. Serum calcium was significantly higher in children who were HIV+. Length/height Z score was positively correlated with serum 25(OH)D in all 1–7.9 year old children (r = 0.33, p = 0.023), with a stronger correlation in the HIV+ children (r = 0.47 p = 0.021).

HIV+ participants were evaluated for immunological markers, with comparisons made between HIV+ mothers with HIV- and HIV+ infants (Table 3). HIV+ mothers of HIV+ infants had significantly lower CD4%, greater percentage with CD4% < 15%, and higher RNA VL. In the HIV-infected mothers as a group, serum 25(OH)D was positively correlated with CD4% (r = 0.40, p = 0.016). Table 3 also presents the HIV disease status for the 9 infants and the 24 children in this survey who were HIV+. Nearly all of the HIV+ infants (89%) and all of the HIV+ children were on cART, and half of the infants and 79% of the children had CD4% at or greater than 25%.

Table 3. Immunological and clinical characteristics of HIV positive mothers, infants, and children.

	HIV+ Mothers			HIV+
	HIV- Infants	HIV+ Infants	p-value	Infants	Children
Characteristic	n = 27	n = 9	p-value	n = 9	n = 24
Age at diagnosis, y	28.4±5.4	28.7±4.6	0.86	0.2±0.1	1.3±1.3
Years since diagnosis, y	4.2±3.4	2.2±2.1	0.09	0.5±0.3	3.2±2.0
cART, %	70	67	0.84	89	100
CD4 count^*	489±223	345±239	0.11	1771±1408	1341±771
CD4%	28.1±7.8	15.9±5.2	<0.001	25.2±11.0	35.5±10.9
< 15.0, %	7^†	56		25	4
15.0–24.9, %	30	33		25	17
≥ 25.0, %	63	11	0.003	50	79
CD8 count^*	807±408	1003±618	0.28	2173±1364	1171±457
RNA, log copies/mL^*	2.07±1.40	3.63±1.81	0.01	3.10±1.53	1.64±0.54

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Data are presented as mean ± standard deviation (SD), and as percent for distribution of categorical variables. HIV = human immunodeficiency virus; SD = standard deviation; cART = combined antiretroviral therapy.

*For CD4 count, sample sizes are n = 27 for mothers of HIV- infants, n = 9 for mothers of HIV+ infants, n = 8 for HIV+ infants, and n = 24 for HIV+ children. For CD8 count, sample sizes are n = 27 for mothers of HIV- infants, n = 9 for mothers of HIV+ infants, n = 8 for HIV+ infants, and n = 22 for HIV+ children. For RNA log copies, sample sizes are n = 27 for mothers of HIV- infants, n = 9 for mothers of HIV+ infants, n = 4 for HIV+ infants, and n = 15 for HIV+ children.

Discussion

The aims of this cross-sectional, observational study were to determine the vitamin D status of HIV-infected Batswana mothers and their HIV-exposed infants and young children. Overall, 11% of HIV+ mothers had an insufficient vitamin D status and mothers with HIV+ infants had lower 25(OH)D concentrations compared to mothers with HIV- infants. The mothers of HIV+ infants also had poorer HIV disease status with lower CD4% and higher RNA viral load than their counterparts with HIV- infants. This was the first study of vitamin D status in Batswana infants and young children exposed to HIV and we found no infants or children were vitamin D insufficient based upon a serum 25(OH)D concentration <20.0 ng/mL, whether they were HIV+ or HIV-. Vitamin D status positively correlated with length/height Z score in both HIV+ and HIV- children and also with CD4% in HIV+ mothers.

This study contributes to understanding the prevalence of vitamin D deficiency among HIV-infected individuals in Botswana. Previous reports on vitamin D status in Batswana adults and children reported a large range of insufficiency; 17–78% of participants had insufficient vitamin D status [6, 22, 23]. Findings from the present study indicate a lower prevalence of vitamin D insufficiency in Batswana HIV+ mothers (11%) compared to previous reports in adults and children with and without HIV infections. Our results show better vitamin D status in Batswana mothers, infants and children compared to other African countries, such as Ethiopia and Algeria [24]. These differences may be due to demographics and dietary habits. One study reported consumption of traditional and indigenous Batswana foods was associated with better dietary diversity and food security [25]. Traditional Batswana foods are more nutrient dense compared to Western-style foods, which are energy-dense and nutrient-poor [26]. Western foods are becoming available in Botswana and could contribute to vitamin D status [26]. Future studies exploring the relation between dietary intake and vitamin D status in HIV+ individuals may provide insight to the range of vitamin D insufficiency reported in Botswana and other African countries.

The proportion of vitamin D insufficiency we observed in HIV+ mothers was not reflected in HIV-exposed and infected infants in our study. No infants were deficient, regardless of whether infants were breastfed or not, and although mothers with HIV- infants had significantly higher 25(OH)D concentrations, there were no significant differences in 25(OH)D concentrations between HIV+ and HIV- infants. This was an unexpected finding given the group of mothers with insufficient vitamin D concentrations and breastfed infants, which supplies minimal vitamin D. Another study in Botswana reported 19% of infants and children under 2 years (n = 80) with and without tuberculosis were vitamin D insufficient [27] and a study in otherwise healthy Tanzanian infants reported 76% had insufficient vitamin D [28]. HIV+ infants in the present study had significantly greater 1,25(OH)D compared to HIV-, however, 1,25(OH)D is not considered a good surrogate for vitamin D status [29] and so the implications of this difference are unclear. Similarly, there were no differences in vitamin D status between HIV+ or HIV- children. There was a statistically significant difference in calcium concentrations between HIV+ and HIV- children, but the difference is not clinically significant and both groups had concentrations in a safe range. Findings from this study show HIV-exposed infants and children in Botswana are not vitamin D insufficient according to the < 20 ng/mL cut-point.

Serum 25(OH)D was also correlated with length/height Z-score in HIV+ and HIV- children 1–7.9 years; children with better Z-scores had higher vitamin D concentrations. There is existing evidence that low serum 25(OH)D is more prevalent in stunted populations [30, 31], which may be related to the integral role of vitamin D in muscle and bone metabolism [32]. However, maternal vitamin D status versus child vitamin D status may differentially affect the risk of growth faltering. Sudfeld and colleagues [33] reported low vitamin D concentrations (<10 ng/mL) in Tanzanian infants exposed to HIV were associated with wasting. However, Powis et al. [22] reported no association between maternal insufficient vitamin D concentrations (<32 ng/mL) and infant growth in Botswana. Identifying variables that affect vitamin D status in infancy and early childhood, such as dietary vitamin D, would help understand the relation between vitamin D status and stunting in HIV-exposed Batswana offspring. For example, HIV-exposed and–infected children may have generally poorer health and appetite, which could affect their intake. Findings from our survey demonstrate the potential impact of vitamin D concentrations on childhood growth. Even when vitamin D concentrations were sufficient, the correlation between vitamin D and growth status was still significant.

Our results also show a correlation between vitamin D status and HIV disease status. This finding agrees with existing literature that showed vitamin D supplementation improved HIV disease status [6, 34–36]. We found a significant, positive correlation between serum 25(OH)D and CD4% in the HIV infected mothers. This correlation suggests adequate vitamin D status may be associated with improved HIV disease status. Although there was a small percentage of mothers with insufficient vitamin D status, the immunological benefits of vitamin D may only be observed at sufficient or greater plasma 25(OH)D concentrations. Data were collected prior to universal cART in pregnancy, which may have affected the mean CD4%. The present study echoes the favorable relation between vitamin D status and immune outcomes reported elsewhere [34, 35].

There were significant differences in length/height Z-scores between HIV+ and HIV- children; HIV+ children had poorer linear growth than HIV-uninfected children aged 1–7.9. The HIV+ infants had lower length Z scores by almost a full Z score, although this did not reach significance due to small sample size. It is important to recognize there may be confounding variables, such as diet, that were not captured as part of this study. However, these results corroborate with other reports of stunting among HIV+ children [33, 37, 38]. Overall, our results show HIV-exposed infants and children had poorer overall growth regardless of whether they are HIV- or HIV+. Sudfeld et al. [33] reported HIV-exposed Batswana children had an increased risk of stunting compared to HIV-unexposed peers in children less than 5 years old. Linear growth faltering serves as a marker of malnutrition and is associated with increased morbidity and mortality [39]. Our survey is in agreement with the current literature and showed HIV+ children had poorer linear growth compared to HIV- children.

Strengths of this study include the under-studied population of infants and children exposed to HIV in Botswana and nutrition and growth-related outcomes explored in relation to both HIV and vitamin D status. The study limitations were the difference in the season of examination between the two cohorts of mothers with HIV- or HIV+ infants, though this was adjusted for during statistical analysis using ANCOVA, and there may be confounding variables that were not captured in this cross-sectional study. Additionally, we used a convenience sample recruited in 2012–2013 that may not be representative of the population, the sample size was modest and future larger studies are needed to confirm these data.

Conclusion

In this urban Botswana-based observational study, only 11% of HIV-infected mothers exhibited insufficient serum vitamin D status, yet none of their infants were vitamin D insufficient. Further, no HIV-exposed children 1–7.9 years were vitamin D insufficient. Children living with HIV had poorer linear growth than HIV-uninfected children, with significantly lower length/height status in 1–7.9 year old children. However, additional data such as diet may provide a better understanding of HIV status on growth. There was also a significant association between vitamin D status and HIV disease status among mothers living with HIV. Results from this study show a low prevalence of vitamin D insufficiency in a smaller cohort and emphasize the need for further research to identify confounding variables that affect vitamin D status in HIV infected and exposed individuals.

Supporting information

S1 Data. Botswana-vitamin D study data.

(XLSX)

Click here for additional data file.^{(107.6KB, xlsx)}

Acknowledgments

Authors are grateful to the subjects and their families for study participation. In addition we wish to thank our colleagues in the Ministry of Health who provide day-to-day care for these patients, as well as to the leadership of all organizations involved whose work makes cross-institutional partnerships and collaborations possible, including Dr. Marape Marape who played a role in enabling some of the work with the Botswana Baylor Center of Excellence. The authors would also like to recognize Julia Samuel and Dr. Mary Hediger for their extensive help on this project.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This research was supported by a grant from the Penn Center for AIDS Research (CFAR), an NIH-funded program (P30 AI 045008) to VAS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0236510.r001

Decision Letter 0

Emma K Kalk

30 Mar 2020

PONE-D-20-05581

Vitamin D status, nutrition and growth in HIV-infected mothers and HIV-exposed infants and children in Botswana

PLOS ONE

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Reviewer #1: The manuscript titled: “Vitamin D status, nutrition and growth in HIV-infected mothers and HIV-exposed infants and children in Botswana.” presented by Tindall et al is reviewed below.

The authors present a paper about vitamin D status and its relation to growth and HIV disease state in women living with HIV and exposed infants. It is a cross-sectional quantitative study. The authors demonstrate that vitamin D insufficiency was relatively rare but that the level of vitamin D did correlate with growth and HIV indicators in mothers.

The information is useful and the work seems to be scientifically sound. The paper is very well written and clear but there are a few issues to address

Methods:

Line 95: Please consider adding details about where mothers were recruited from, was it from their ART follow-up clinics?

Line 111-112: The sentence “the younger infants” seems to conflict with the results in that the sentence says that PCR results were not yet available yet the results clearly distinguish positive and negative. I would say that you cannot present the results as “neg” vs “pos” infants if the negative group actually is largely unknown in terms of HIV status. Please also explain why HIV testing was not offered as part of the study if results were not available on enrolment.

Line 114: The 48 children, were they at all related to the infants and mothers? Were they older siblings or totally unrelated. Please clarify also how and where they were recruited.

Line 132-133: In terms of using both the CDC as well as WHO charts, please explain why and also whether this may cause an effect on the results.

Discussion:

Line 248-249 and also line 264-265: Please consider explaining the relationship between cause and effect of Vit D and various clinical outcomes. Is there evidence that clearly links vitamin D as the cause and height as the effect?

Line 273-274: What comparison are you using the make this statement “Our results also..”

Minor:

Line 45: consider removing one “and” and replace with comma,

Line 86: add the word “vitamin”

Line 262: the word “disease” seems to be missing after HIV.

Line 302: remove “the”

Reviewer #2: The authors are presenting a research study which was designed to determine the vitamin D status in HIV-infected mothers and their children, and to compare vitamin serum levels between HIV positive and negative children.

Major comments

1. The number of participants enrolled is small, only 36 mothers and 48 children. In addition to these small numbers the children of the 36 HIV positive numbers are further divided to HIV-infected to HIV-exposed, with only 9 being HIV infected. This makes it difficult to interpret the significance of findings in this study. How was the sample size calculated, and what were the assumptions made to calculate the sample size?

2. In children, they have stratified them to HIV-positive and HIV-negative, why was the same stratification not done for the mothers?

3. In presenting the Tables, authors have pointed on variables that showed statistical significance using different symbols. It will be much easier for readers to understand the Tables better if they can add a column with p-values.

4. In Table 2, for the differences notes in length and height Z scores, did they adjust for potential confounders e.g. sex, and breast feeding.

5. Table 3 does not reflect on what was being studied, that is vitamin D. Secondly the numbers presented for HIV positive infants and children are confusing. For example for CD4 count there are 2 values, what does this mean. On the row of CD4% the numbers in brackets (8) and (24) are referring to what?

6. In making their conclusion, they need to adjust for potential confounders before they can conclude that children with HIV had poorer linear growth than HIV-uninfected children.

7. The conclusion that there was a significant association between vitamin D status and HIV disease is supported by which results?

8. The authors need to reconsider their statement that the findings from this study show a 'REASSURINGLY LOW PREVALENCE" of vitamin D insufficiency, when they had such a low sample size.

Minor comment

They must try and paraphase some of the sentences in the report as they are often long and one has to read them many times before one can understand their meaning. Below are some of the sentences to be considered for paraphrasing.

1. On page 4, Lines 80-83

2. On page 5, Lines 95-98

3. On page 9, Lines 180-184

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Reviewer #1: No

Reviewer #2: No

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Attachment

Submitted filename: PONE-D-20-05581.pdf

Click here for additional data file.^{(87.3KB, pdf)}

PLoS One. 2020 Aug 13;15(8):e0236510. doi: 10.1371/journal.pone.0236510.r002

Author response to Decision Letter 0

2 Jun 2020

The information is useful and the work seems to be scientifically sound. The paper is very well written and clear but there are a few issues to address

Methods:

Line 95: Please consider adding details about where mothers were recruited from, was it from their ART follow-up clinics?

Thank you for this suggestion. We have changed the previous sentence to read: Mothers, infants and children were recruited from Princess Marina Hospital, Gaborone, and Bamalete Lutheran Hospital, Ramotswa, in Botswana where they were followed for medical care.

The HIV PCR testing was provided by the well-established, Botswana Pediatric HIV Program and the study was designed and budgeted for utilizing the results provided through this program. The Botswana 2013 Global AIDS Response Report (when study data were collected) reported that 47% of infants born to HIV positive mothers received a virological HIV test within two months after birth, this is likely why at the time of data collection, the younger infants did not have available results. However, the report also documents that the rate of transmission from mother to child at this time was less than 3%. Given the low transmission rates in this population, these infants were categorized as HIV- at the time of data collection.

Further, when we removed the infants who did not have a confirmatory test result from the analysis, the significance of the results remained the same for both the analysis between mothers with HIV+ and HIV- infants and between HIV+ and HIV- infants. Specifically, we performed analysis of the data for a subsample of the older infants (6-11.9 months old, n=18) for whom HIV status was known (10 were HIV- and 8 were HIV+). The analyses for this subgroup confirmed our findings presented in the paper: 1) mothers of HIV+ infants had significantly lower 25(OH)D levels and significantly lower CD4% than mothers of HIV- infants; 2) HIV+ infants had poorer growth status than their HIV- counterparts by almost 1 Z score for both length and weight Z scores, although likely due to small sample size this did not reach statistical significance.

Line 114: The 48 children, were they at all related to the infants and mothers? Were they older siblings or totally unrelated. Please clarify also how and where they were recruited.

The children enrolled in this study were unrelated to the mothers and infants. We have clarified this in the methods section: This was an observational, age- and HIV-status stratified survey of 36 HIV-infected (HIV+) mothers and their infants (0-11.9 months), and 48 unrelated children (1-7.9 years).

Line 132-133: In terms of using both the CDC as well as WHO charts, please explain why and also whether this may cause an effect on the results.

This is the recommendation of the CDC – the CDC recommends using WHO growth charts for children 0-2 years and the CDC growth charts for children 2-18 years. We have amended the sentence in our methods to read: For infants and young children up to age two years where length was measured, length and weight were converted into Z scores (standard deviation units) using the WHO growth chart data (http://www.cdc.gov/growthcharts/), as recommended by the CDC(18). For older children the CDC recommends using the CDC algorithms to convert height and weight into Z scores. Given this is the recommendation for these age groups, we suggest would be inappropriate to use one of the reference charts for all ages.

Discussion:

Thank you for this question. The relation between vitamin D and height is being explored in other clinical research settings. A recently published prospective study examining the effect of micronutrient status and growth in exclusively breastfed children reported:

“Children who were exclusively breastfed for longer than 4 months without proper supplement were more likely to have persistent vitamin D insufficiency and have relatively slower growth after infancy compared to mix-fed children.”

Although these children were not exposed to HIV, there was still a relation between vitamin D status and height. The children in this study were Taiwanese and in the discussion of our study, we present studies that report on populations closer to the population we studied to compare vitamin D status and growth, particularly in HIV exposed infants and children.

Line 273-274: What comparison are you using the make this statement “Our results also..”

Thank you for this comment, we have changed the sentence to read: Overall, our results show HIV-exposed infants and children had poorer overall growth regardless of whether they were HIV- or HIV+.

Minor:

Line 45: consider removing one “and” and replace with comma,

We have changed the sentence to read: Length/height and weight Z scores determined growth status.

Line 86: add the word “vitamin”

We have added this.

Line 262: the word “disease” seems to be missing after HIV.

We have added this.

Line 302: remove “the”

We have removed this.

Major comments

The primary objective of this study was to determine the vitamin D status of HIV-infected mothers and their infants and children. This was an observational study or survey of vitamin D in Botswana mothers, infants and children. Sample sizes were not calculated based upon effect sizes for the outcomes. Secondary objectives were to examine potential differences in vitamin D status and other outcomes among each of the groups and associations were exploratory.

2. In children, they have stratified them to HIV-positive and HIV-negative, why was the same stratification not done for the mothers?

Thank you for this question. All of the mothers were HIV+ by study design, so the same stratification is not possible.

We have added the p-values to the table and removed the symbols that described the significance level.

4. In Table 2, for the differences notes in length and height Z scores, did they adjust for potential confounders e.g. sex, and breast feeding.

Thank you for this question. We did examine males and females separately and they had similar growth outcomes. We did adjust for age in the analysis of covariance as this was significant when added to the model. When added to the models, neither sex nor breastfeeding (ever breastfed) were significant covariates, that is, they did not significantly contribute to the growth outcomes and therefore we did not include them in the final model. It is worth noting that very few infants were breastfed (2 in the HIV- group and 1 in the HIV+ group). For the older children, there were no HIV- children who were ever breastfed. A little over half of the HIV+ children had been breastfed. Therefore, we examined using student’s unpaired t tests whether there were differences in growth (length and weight Z scores) among HIV+ children only who were breastfed vs not breastfed and found no significant differences by breastfeeding. Since breastfeeding only occurred in the HIV+ children, we cannot rule out that it may have contributed to the poorer growth status in these children along with other factors, and further studies are be needed to tease this out.

Thank you for this comment. Table 3 was included to provide the information on HIV disease status (both immunological and clinical characteristics) for the whole sample. Mothers of HIV exposed (but HIV-) and HIV+ infants could be compared, and mothers of HIV+ infants had poor immunological status (lower CD4%). Therefore, we examined the association in these HIV-infected mothers of CD4% with vitamin D concentration, our primary outcome, and found a significant correlation.

6. In making their conclusion, they need to adjust for potential confounders before they can conclude that children with HIV had poorer linear growth than HIV-uninfected children.

We appreciate this comment and understand the reviewer’s concern. We have amended the language in the conclusion to address the fact that there may be confounding variables. Breastfeeding may be a confounding variable and a larger sample size and more even distribution amongst children who were breastfed vs formula fed in each group would lend to a better understanding. Additionally, we do not have dietary data to adjust for dietary intake and have listed this as a limitation of this study. We have also suggested future studies should examine confounding factors that could affect this relation as currently, we do not know what other variables may be confounders and this study is not designed for subgroup analyses.

7. The conclusion that there was a significant association between vitamin D status and HIV disease is supported by which results?

This conclusion is from the correlation between the HIV-infected mothers and CD4%: In the HIV-infected mothers as a group, serum 25(OH)D was positively associated with CD4% (r=0.40, p=0.016).

8. The authors need to reconsider their statement that the findings from this study show a 'REASSURINGLY LOW PREVALENCE" of vitamin D insufficiency, when they had such a low sample size.

We appreciate this suggestion. We have amended the sentence to: Results from this study show a low prevalence of vitamin D insufficiency in this small cohort and emphasize the need for further research to identify confounding variables that affect vitamin D status in HIV infected and exposed individuals.

Minor comment

1. On page 4, Lines 80-83

2. On page 5, Lines 95-98

3. On page 9, Lines 180-184

We have restructured these sentences to be shorter and read easier.

Attachment

Submitted filename: Bots-D Response to Reviewers 050620.docx

Click here for additional data file.^{(27.3KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0236510.r003

Decision Letter 1

Emma K Kalk

4 Jun 2020

PONE-D-20-05581R1

Vitamin D status, nutrition and growth in HIV-infected mothers and HIV-exposed infants and children in Botswana

PLOS ONE

Dear Dr. Tindall,

==============================

Thank you for submitting the revised manuscript. The methodology is clearer and discussion more measured.

I did not see a response to the notes in the pdf attached to the initial review.

Below please find further remarks which I hope with strengthen the submission.

==============================

Please submit your revised manuscript by Jul 19 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Emma K. Kalk

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

PONE-D-20-05581R1

Line 46: “Immune panel” implies more than CD4 count

Line 43: The groups of infant v child participants have been clarified in the text. Please do so in the abstract.

Line 80: Climate and sunlight are referenced; this could be applied to Botswana. There is already some data on vit D in pregnant women and children and HIV published by your group. you could mention this here.

Line 89: The authors mention nutritional status and feeding in the introduction but seem to have little data on this. Only length/height is discussed and only breast feeding as a binary variable. Duration of breast feeding may also be relevant; age of introduction of other foods, variety of foods etc. It is better not to introduce these terms if you have no related data.

Line 110: Sampling. Please provide more detail on how were these women recruited? Were they attending routine adult HIV services and the asked about off-spring? Were the infants/children recruited first? Was the sample selected randomly? Children >12m living with and without HIV were balanced. How was this done practically, and could it not introduce bias? If the women/children were recruited in a different manner, this could bias the results (we know already they were recruited in different seasons).

Is the hospital site a reflection of the population as a whole? You note that it is an urban population. This limitation should be discussed. As should the omission of all acutely or chronically unwell children who may have a different vitamin D status. It should be clearly stated that these findings are limited to quite a specific group.

Line 113: The amended sentence is not grammatically correct. You could preface this by saying that infants of unconfirmed HIV status were categorized as negative ‘given the rate of perinatal transmission etc.’ Sensitivity analyses are described in the response to the reviewer comment. IT might be useful to mention that sensitivity analyses were performed which did not change the observed associations.

With all the stratification the numbers become very small and once must be careful not to overstate the findings. Caution is required in interpreting these data.

Line 118: adding “unrelated” children would make the groups explicit.

Line 125: HIV status – this is usually positive, negative or indeterminate; could this refer to the clinical and/or immunological stage of women living with HIV?

Line 145: ‘Immunological outcomes’, like ‘an immune panel’ in abstract and ‘immunological markers’ in line is a bit misleading if you mean CD4 and VL only. The terms imply additional assays (cytokines? T cell phenotype?) For example, “Immune status was determined in all women and those children living with HIV…” may be more suitable here.

Line 160: were all data normally distributed?

Line 167: Why did you choose ANCOVA over regression analysis?

Did you check the model?

Please add a description of relevant PMTCT and ART guidelines at the time as these speak to additional exposures.

Line 182: This is a low proportion – is ART not indicated for all pregnant women in Botswana?

Line 187: As presented the sentence states that the HIV infected infants were examined in on season and the HEU in another. Is this correct?

Line 197: breastfeeding. Does this refer to current BF or ever BF?

Line 283: The association was weak; a weak but significant positive correlation. Reviewer 2 had also noted that the original statement is very emphatic.

Are CD4 values presented for the mothers or the infants/children? Conventionally, CD4 is presented as an absolute number in adults (and children >5years) and only as % in the under-fives.

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

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 PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2020 Aug 13;15(8):e0236510. doi: 10.1371/journal.pone.0236510.r004

Author response to Decision Letter 1

9 Jun 2020

Additional Editor Comments:

PONE-D-20-05581R1

Line 46: “Immune panel” implies more than CD4 count

CD4, CD8 and viral load were also measured (HIV-1 RNA VL). We describe the methods in lines 137-145.

Line 43: The groups of infant v child participants have been clarified in the text. Please do so in the abstract.

We have clarified this: This was a cross-sectional study of HIV+ mothers and HIV-exposed infants and unrelated children (1-7.9 years).

We have included an additional citation and sentence here: Vitamin D deficiency is more prevalent in children with perinatally acquired HIV infections(15).

We agree, there is a lot of nutritional information that would be helpful in assessing this population. We have amended the sentence to include “breastfeeding” instead of “nutrition”.

We have added information to better describe our sample: A convenience sample was used. For balanced representation across infant and child ages and HIV status groups, 18 mother-infant pairs with infants aged 0-5.9 months and 18 pairs with infants aged 6-11.9 months attending routine medical visits were enrolled. (lines 108-110).

We also added the sample as a limitation in the discussion: Additionally, we used a convenience sample that may not be representative of the population the sample size was modest and future larger studies are needed to confirm these data. (line 299)

With all the stratification the numbers become very small and once must be careful not to overstate the findings. Caution is required in interpreting these data.

Thank you for this comment. We have altered the sentence and added detail about the additional analysis: Given the rate of perinatal transmission of HIV in Botswana was less than 3% at the time of data collection(16), the younger infants (aged 0-5.9 months) were categorized as HIV-. Removing the younger infants did not change the results, therefore, we included them in the dataset.

Line 118: adding “unrelated” children would make the groups explicit.

We have added this.

Line 125: HIV status – this is usually positive, negative or indeterminate; could this refer to the clinical and/or immunological stage of women living with HIV?

We are also classifying how well-controlled/compliant with medication participants are (using CD4 count, viral load).

We have changed the sentence to your recommended language. (line 147)

Line 160: were all data normally distributed?

Variables were assessed for normality using a Skewness and Kurtosis test (Stata) and all primary outcome variables were normally distributed. Some variables were slightly skewed according to the SK test, but quantile plots appeared normal, so variables were not transformed.

Line 167: Why did you choose ANCOVA over regression analysis?

Did you check the model?

We used ANCOVA primarily because we were interested in generating the adjusted means (LS means) for the variables. In the case of the nutritional status variables, such as serum vitamin D, we were interested in the difference between groups (mothers with HIV+ vs. HIV- infants or HIV+ vs. HIV- infants and children) adjusted for the seasonal effect which is known to occur. In the case of the growth variables in the infants and children, we were interested in what the mean differences were in the Z scores between HIV+ and HIV- groups after adjusting for the effect of age. Regression models give much the same results in terms of the significance of the groups differences in these variables, however they do not generate the adjusted means.

Please add a description of relevant PMTCT and ART guidelines at the time as these speak to additional exposures.

We have added the following language under methods: Between 2006-2009, more than 95% of HIV+ pregnant women in Botswana received the national standard antiretrovirals (ARVs) for prevention of mother to child transmission (PMTCT). This was all HIV infected pregnant women were eligible for triple prophylaxis/ART. HIV infected women were evaluated for ART eligibility based on laboratory testing (CD4 cell count) or clinical presentation (WHO clinical stage). Women with CD4 cell count less than 350 or WHO clinical stage 3 or 4 were started on ART through the national ARV Program as soon as possible, regardless of the stage of pregnancy. Women attending healthcare facilities that provided triple ARV prophylaxis, who were not eligible for ART were provided with triple ARV prophylaxis starting from 14 weeks of gestation. Women who chose to breastfeed continued triple ARV prophylaxis until their infants were at least six months of age and completely after weaning. Both women on ART and those that were on ARV prophylaxis were given supplementary AZT 300 mg every 3 hours during labor and delivery.

Similarly by 2009, >95% of infants born to HIV infected mothers were given ARV prophylaxis at birth according to national guidelines; they were given Nevirapine as a single dose as soon as possible after birth (within 72 hours) plus four weeks of AZT.

Line 182: This is a low proportion – is ART not indicated for all pregnant women in Botswana?

The paragraphs above explain this – this study was done at the time that ART rollout for all HIV infected pregnant women was being rolled out across Botswana.

Line 187: As presented the sentence states that the HIV infected infants were examined in on season and the HEU in another. Is this correct?

This is correct.

Line 197: breastfeeding. Does this refer to current BF or ever BF?

This refers to “ever” breast fed. We have added “ever” to this sentence.

Line 283: The association was weak; a weak but significant positive correlation.

Reviewer 2 had also noted that the original statement is very emphatic.

Are CD4 values presented for the mothers or the infants/children? Conventionally, CD4 is presented as an absolute number in adults (and children >5years) and only as % in the under-fives.

We understand the editor and reviewer’s concerns. We have amended the sentence to read: This correlation suggests adequate vitamin D status may be associated with improved HIV disease status.

Additionally, we are only referring to the infected mothers for this correlation: We found a significant, positive correlation between serum 25(OH)D and CD4% in the HIV infected mothers.

Attachment

Submitted filename: Response to Editor 061120.docx

Click here for additional data file.^{(22.8KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0236510.r005

Decision Letter 2

Emma K Kalk

18 Jun 2020

PONE-D-20-05581R2

Vitamin D status, nutrition and growth in HIV-infected mothers and HIV-exposed infants and children in Botswana

PLOS ONE

Dear Dr. Tindall,

I apologize for going backwards and forwards. It is now clear that the women-infant pairs were recruited several years ago (2012 - 2013) and the samples banked. Was this part of an earlier study, perhaps with different objectives? If so, please describe the enrollment procedures of the original study and how the current sample reflects the participant profile of that study. Were the unrelated older children enrolled over the same period in 2012 - 2013? If not, please state this and justify their inclusion. From where were they recruited? As acknowledged by the authors, the sample sizes are small and it is important that there was some system supporting participant inclusion if these results are to be useful. The integrity of the comparator groups needs to be sound and the methodology explicit.

I am still uncertain with respect to the PMTCT guidelines at the time of the study: were these the same as the 2009 guidelines referenced?

Please include in your abstract and discussion that these findings reflect Vitamin D status in a population prior to the introduction of universal ART in pregnancy.

Please submit your revised manuscript by Aug 02 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Emma K. Kalk

Academic Editor

PLOS ONE

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

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 PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2020 Aug 13;15(8):e0236510. doi: 10.1371/journal.pone.0236510.r006

Author response to Decision Letter 2

29 Jun 2020

Thank you for allowing us to clarify these details. The women-infant pairs and the unrelated children were all specifically recruited for this study which was conducted in 2012-2013. We have also amended the following sentence in the discussion to better characterize the limitations of the study: “we used a convenience sample recruited in 2012-2013 that may not be representative of the population, the sample size was modest and future larger studies are needed to confirm these data.” Although data was collected during this time period, this is the only data that describes vitamin D status and growth in Batswana infants and young children. Hence these data remain important and we hope that the publication of this study will stimulate further work in this area.

I am still uncertain with respect to the PMTCT guidelines at the time of the study: were these the same as the 2009 guidelines referenced?

Thank you for the opportunity to clarify. The PMTCT guidelines section has been revised to better describe the care guidelines at the time of data collection, which does reflect that ART was available to pregnant women, but implementation was not yet universal.

Please include in your abstract and discussion that these findings reflect Vitamin D status in a population prior to the introduction of universal ART in pregnancy.

We have included this (lines 47-48 & 300-301).

Attachment

Submitted filename: Response to editor 062920.docx

Click here for additional data file.^{(13.5KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0236510.r007

Decision Letter 3

Emma K Kalk

9 Jul 2020

Vitamin D status, nutrition and growth in HIV-infected mothers and HIV-exposed infants and children in Botswana

PONE-D-20-05581R3

Dear Dr. Tindall,

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

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Emma K. Kalk

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0236510.r008

Acceptance letter

Emma K Kalk

3 Aug 2020

PONE-D-20-05581R3

Vitamin D status, nutrition and growth in HIV-infected mothers and HIV-exposed infants and children in Botswana

Dear Dr. Tindall:

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.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Emma K. Kalk

Academic Editor

PLOS ONE

Associated Data

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

Supplementary Materials

S1 Data. Botswana-vitamin D study data.

(XLSX)

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Submitted filename: PONE-D-20-05581.pdf

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Submitted filename: Bots-D Response to Reviewers 050620.docx

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Submitted filename: Response to Editor 061120.docx

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Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

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PERMALINK

Vitamin D status, nutrition and growth in HIV-infected mothers and HIV-exposed infants and children in Botswana

Alyssa M Tindall

Joan I Schall

Boitshepo Seme

Bakgaki Ratshaa

Michael Tolle

Maria S Nnyepi

Loeto Mazhani

Richard M Rutstein

Andrew P Steenhoff

Virginia A Stallings

Roles

Abstract

Background

Objectives

Methods

Results

Conclusions

Introduction

Materials and methods

Participants

Prevention of mother to child transmission guidelines

Sampling

Clinical data collection

Laboratory methods

Statistical methods

Results

Table 1. Maternal characteristics of HIV positive mothers (n = 36) stratified by HIV status of their infants.

Table 2. Infant (n = 36) and child (n = 48) characteristics stratified by HIV status.

Table 3. Immunological and clinical characteristics of HIV positive mothers, infants, and children.

Discussion

Conclusion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Emma K Kalk

Roles

Author response to Decision Letter 0

Decision Letter 1

Emma K Kalk

Roles

Author response to Decision Letter 1

Decision Letter 2

Emma K Kalk

Roles

Author response to Decision Letter 2

Decision Letter 3

Emma K Kalk

Roles

Acceptance letter

Emma K Kalk

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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