Sex differences in responses to antiretroviral treatment in South African HIV-infected children on ritonavir-boosted lopinavir- and nevirapine-based treatment

  • Stephanie Shiau1, 2,

    Affiliated with

    • Louise Kuhn1, 2,

      Affiliated with

      • Renate Strehlau3,

        Affiliated with

        • Leigh Martens3,

          Affiliated with

          • Helen McIlleron4, 5,

            Affiliated with

            • Sandra Meredith4,

              Affiliated with

              • Lubbe Wiesner4,

                Affiliated with

                • Ashraf Coovadia3,

                  Affiliated with

                  • Elaine J Abrams2, 6, 7 and

                    Affiliated with

                    • Stephen M Arpadi1, 2, 6, 7Email author

                      Affiliated with

                      BMC Pediatrics201414:39

                      DOI: 10.1186/1471-2431-14-39

                      Received: 16 October 2013

                      Accepted: 6 February 2014

                      Published: 12 February 2014

                      Abstract

                      Background

                      While studies of HIV-infected adults on antiretroviral treatment (ART) report no sex differences in immune recovery and virologic response but more ART-associated complications in women, sex differences in disease progression and response to ART among children have not been well assessed. The objective of this study was to evaluate for sex differences in response to ART in South African HIV-infected children who were randomized to continue ritonavir-boosted lopinavir (LPV/r)-based ART or switch to nevirapine-based ART.

                      Methods

                      ART outcomes in HIV-infected boys and girls in Johannesburg, South Africa from 2005–2010 were compared. Children initiated ritonavir-boosted lopinavir (LPV/r)-based ART before 24 months of age and were randomized to remain on LPV/r or switch to nevirapine-based ART after achieving viral suppression. Children were followed for 76 weeks post-randomization and then long-term follow up continued for a minimum of 99 weeks and maximum of 245 weeks after randomization. Viral load, CD4 count, lipids, anthropometrics, drug concentrations, and adherence were measured at regular intervals. Outcomes were compared between sexes within treatment strata.

                      Results

                      A total of 323 children (median age 8.8 months, IQR 5.1-13.5), including 168 boys and 155 girls, initiated LPV/r-based ART and 195 children were randomized. No sex differences in risk of virological failure (confirmed viral load >1000 copies/mL) by 156 weeks post-randomization were observed within either treatment group. Girls switched to nevirapine had more robust CD4 count improvement relative to boys in this group through 112 weeks post-randomization. In addition, girls remaining on LPV/r had higher plasma concentrations of ritonavir than boys during post-randomization visits. After a mean of 3.4 years post-randomization, girls remaining on LPV/r also had a higher total cholesterol:HDL ratio and lower mean HDL than boys on LPV/r.

                      Conclusions

                      Sex differences are noted in treated HIV-infected children even at a young age, and appear to depend on treatment regimen. Future studies are warranted to determine biological mechanisms and clinical significance of these differences.

                      Trial registration

                      ClinicalTrials.gov Identifier: NCT00117728

                      Keywords

                      HIV Children Sex differences Antiretroviral treatment outcomes Pharmacokinetics

                      Background

                      Studies of sex differences in the course of HIV infection in adults report lower HIV-1 RNA levels and higher CD4 counts in women compared to men, but similar disease progression and clinical outcomes between sexes [1, 2]. Responses to antiretroviral treatment (ART), including immune reconstitution and virologic response, are also similar [3, 4]. However, ART-associated complications, including hepatotoxicity, pancreatitis, as well as metabolic abnormalities and lipodystrophy are consistently reported more often in women than men [511]. The differential effects in ART response between sexes appear to be driven by sex-specific physiological and hormonal influences as well as by differences in drug pharmacokinetics [12, 13]. Social and behavioral factors may also play a role, as rates of adherence and treatment discontinuation vary between men and women [14].

                      Less is known about sex differences in disease progression and response to ART among children. Differences in CD4 count and HIV-1 RNA between boys and girls have been reported in treatment-naïve HIV-infected children [15]. In treated children, one study reported lower HIV-1 RNA in girls than boys and no differences in CD4 count [16] while two observational studies reported a more rapid immunologic response to ART in girls [17, 18]. Sex differences in incidence of ART-associated complications reported in children [1923] have rarely been assessed.

                      A better understanding of sex differences in the pathobiology of HIV and its complications would be aided by evaluation of these differences during early childhood when biologic, social, and behavioral differences are less pronounced than in adulthood. The objective of this study was to evaluate for sex differences in response to ART in South African HIV-infected children who were randomized to continue ritonavir-boosted lopinavir (LPV/r)-based ART or switch to nevirapine-based ART.

                      Methods

                      Study design

                      In order to assess for sex differences in immunologic, virologic, anthropometric, and other responses to antiretroviral regimens, we performed a secondary analysis of data collected as part of Neverest 2, a clinical trial (ClinicalTrials.gov: NCT00117728) assessing the reuse of nevirapine in nevirapine-exposed HIV-infected children conducted from 2005 to 2010 [24, 25]. Children previously exposed to single-dose nevirapine prophylaxis at birth, and <24 months of age at the time of initiation of LPV/r-based ART, were identified at Rahima Moosa Mother and Child Hospital in Johannesburg, South Africa. Those who achieved and sustained plasma HIV-1 RNA <400 copies/mL for ≥3 months during the first 12 months of treatment were eligible for randomization to either continue on LPV/r or switch to nevirapine-based ART in combination with stavudine and lamivudine. Specific treatment regimens have been previously reported [24, 25]. Children were followed for 76 weeks post-randomization and then re-enrolled in extended follow up. In this analysis, we evaluated sex differences pretreatment, in the pre-randomization phase, at the time of randomization, at post-randomization study visits, and at participants’ final visit. This study was approved by the Institutional Review Boards of Columbia University (New York, New York) and the University of the Witwatersrand (Johannesburg, South Africa). Informed consent was provided by each child’s parent or guardian.

                      Measurements

                      Socio-demographic information, medical history, weight (kg), height (cm), and blood samples (for CD4 T-cell determination and HIV-1 RNA quantity) were collected at a pretreatment visit. Subsequent visits were scheduled at 2, 4, 8, 12, and every 12 weeks thereafter up to 52 weeks until randomization and at 2, 4, 8, 16, 24, 36, 52, 64, 76 and every 12 weeks thereafter. Blood samples for plasma viral load tests by quantitative HIV-1 RNA PCR (Roche, Cobas Ampliprep Taqman V2, Branchburg, NJ) were collected at 4, 16, 24, 36, 52, 64, and 76 weeks post-randomization and for absolute CD4 count and percentages (Beckman Coulter Flow Analyzer) at 16, 24, 36, 52, 64, and 76 weeks post-randomization, and for both tests at every 12 weeks thereafter until the final study visit. Lopinavir, ritonavir, and nevirapine plasma concentrations were determined using validated LC-MS/MS assays developed in the Division of Clinical Pharmacology, Cape Town, South Africa. An AB Sciex 4000 mass spectrometer was operated at unit resolution in the multiple reaction monitoring mode. The assays were validated over the concentration range of 0.16-20 μg/ml for lopinavir, 0.10-20 μg/ml for nevirapine, and 0.04-5 μg/ml for ritonavir.

                      Non-fasting total cholesterol, low-density lipoprotein (LDL), high-density lipoprotein (HDL), and triglycerides in mg/dL were measured at pretreatment, time of randomization, and 36, 88, and 136 weeks post randomization (Roche, Cobas Integra 400, Branchburg, NJ). At the final study visit following an overnight fast, total cholesterol, LDL, HDL, triglycerides, C-reactive protein in mg/L, and insulin in mg/dL were measured. Venous blood glucose in mg/dL was measured with a handheld glucometer and homeostasis model assessment of insulin resistance (HOMA-IR) was calculated [26]. Classification of biochemical results has been described previously [22]. Assessments for drug-related rashes and hepatotoxicity by alanine aminotransferase (ALT) were performed at post-randomization visits [24, 25].

                      Weight (kg) and height (cm) were measured using a digital scale and stadiometer, respectively. Weight-for-age (WAZ) and height-for-age z-scores (HAZ) were calculated using World Health Organization growth standards [27]. Underweight was defined as WAZ < −2 and stunting was defined as HAZ < −2. Circumferences at the mid-upper arm, mid-upper thigh, mid-waist, and maximum hip were measured using a flexible tape measure with a spring tension attachment. Bicep, tricep, subscapular, suprailiac, umbilical, and mid-thigh skinfolds were measured with a Harpendon caliper (Baty International, England). As previously reported, total body fat percent (%BF) was estimated by single-frequency bioimpedance analysis (BIA) (Quantum II, RJL Systems, Clinton Township, MI) [28] and children were classified as having lipodystrophy, possible lipodystrophy, or no signs of lipodystrophy by two physicians (RS, LM) [22].

                      All children with HIV-1 RNA >1000 copies/mL were recalled and retested within 4 weeks. Additional counseling was provided if adherence difficulties were detected. Children in the switch group were returned to the LPV/r-based regimen if HIV-1 RNA remained >1000 copies/mL despite enhanced adherence counseling. To evaluate adherence, caregivers were asked to return drug at all scheduled visits. Percentage of medication return was calculated and poor adherence was defined as 20% greater than expected medication return for each drug.

                      Statistical analysis

                      All outcomes were compared between sexes within treatment strata as well as between treatment groups within sex strata. Intent-to-treat comparisons were made for treatment groups. Kaplan-Meier methods and log-rank tests were used to describe time to virologic failure. For comparisons between groups at specific time points, we used the Wilcoxon rank-sum test for non-normally distributed continuous variables, t-test for normally-distributed continuous variables, and Chi-square or Fisher exact tests for categorical variables. Generalized estimating equation models were used to compare outcomes with repeated measurements over time using a first order autoregressive correlation structure, adjusting for baseline differences. Tests for interaction between sex and treatment group were performed. All p-values are 2-tailed and p-values <0.05 were considered statistically significant. Analyses were performed using SAS version 9.1.3 (Cary, North Carolina, USA).

                      Results

                      Study population

                      The flow of participants is shown in Figure 1. A total of 323 HIV-infected children, including 168 (52%) boys, were enrolled in the study and initiated LPV/r-based ART. In the period between initiating LPV/r-based treatment and randomization to either remain on LPV/r or switch to nevirapine (pre-randomization phase), 38 (11.8%) died, 40 (12.4%) did not remain in follow up, and 50 (15.5%) did not meet criteria for randomization. Of the 195 children randomized in the study, 99 remained on LPV/r, including 50 (50.5%) boys, and 96 were switched to nevirapine, including 54 (56.3%) boys. Children were followed for 76 weeks post-randomization during which time four (2.1%) children died and 15 (7.7%) children were lost to follow up. Long-term follow up continued for a minimum of 99 weeks and maximum of 245 weeks after randomization. At study completion in June 2010, six (3.1%) children died, 28 (14.4%) were lost to follow-up, and five (2.6%) transferred out. 156 (80%) completed the final study visit.
                      http://static-content.springer.com/image/art%3A10.1186%2F1471-2431-14-39/MediaObjects/12887_2013_978_Fig1_HTML.jpg
                      Figure 1

                      Flow diagram of study participants in Neverest 2. Flow diagram of study participants in Neverest 2; Abbreviations: LPV/r, lopinavir-boosted ritonavir; NVP, nevirapine; ART, antiretroviral therapy; LTFU, lost to follow up; B, boys; G, girls.

                      Pretreatment characteristics

                      The pretreatment characteristics of the 323 children initiating LPV/r-based ART are presented in Table 1. Overall, more boys initiated ART after 6 months of age (p = 0.009). Boys also had a lower mean WAZ and HAZ and a greater proportion of boys were underweight and stunted. The pretreatment anthropometric differences remained after adjusting for age at treatment initiation. No differences between sexes were seen in other pretreatment characteristics. In the pre-randomization period, similar proportions of boys and girls died, were lost to follow up, or did not meet criteria for randomization (data not shown). Results were similar if only the 195 children randomized were analyzed, but the mean WAZ of boys and the proportion of boys underweight and stunted were no longer significantly different than girls.
                      Table 1

                      Pretreatment characteristics of 323 perinatally HIV-infected South African children enrolled in Neverest 2 by sex

                      Characteristics

                      Enrolled children (n = 323)

                       
                       

                      Boys (n = 168)

                      Girls (n = 155)

                      P-value

                      Age at treatment start, N (%)

                         

                        <6 months

                      41 (24.4)

                      61 (39.4)

                       

                        6-12 months

                      70 (41.7)

                      45 (29.0)

                      0.009

                        12-24 months

                      57 (33.9)

                      49 (31.6)

                       

                        Median (range) in months

                      9.6 (2.2-24.2)

                      7.9 (2.0-24.9)

                      0.098

                      HIV-1 RNA quantity, N (%)

                        

                      0.475

                        <100,000 copies/ml

                      16 (10.6)

                      9 (6.8)

                        100,000-750,000 copies/ml

                      39 (25.8)

                      39 (29.6)

                        ≥750,000 copies/ml

                      96 (63.6)

                      84 (63.6)

                      CD4 count in cells/mm3, Mean (SD)

                      1001 (724)

                      1011 (786)

                      0.909

                      CD4 percent, N (%)

                         

                        <10

                      30 (19.1)

                      25 (17.0)

                       

                        10-14.9

                      35 (22.3)

                      26 (17.7)

                      0.463

                        ≥15

                      92 (58.6)

                      96 (65.3)

                       

                        Median (range)

                      16.8 (0.8-43.0)

                      19.1 (1.2-39.1)

                      0.163

                      WHO Stage, N (%)

                         

                        I/II

                      29 (21.0)

                      22 (17.5)

                      0.465

                        III/IV

                      109 (79.0)

                      104 (82.5)

                      Weight for age Z-score

                         

                        Mean (SD)

                      −2.70 (1.8)

                      −2.28 (1.6)

                      0.041

                        Z-score < −2, N (%)

                      98 (66.7)

                      71 (49.7)

                      0.003

                      Height for age Z-score

                         

                        Mean (SD)

                      −3.72 (1.8)

                      −3.11 (1.6)

                      0.003

                        Z-score < −2, N (%)

                      125 (86.2)

                      108 (76.6)

                      0.037

                      Note: Categorical variables were compared across groups using X2 tests; median age, CD4 percentage, and time on therapy were compared using Wilcoxon tests; CD4 count and height and weight for age z-scores were compared using t-tests. Denominators are as shown.

                      Abbreviations: LPV/r Lopinavir-boosted ritonavir, NVP Nevirapine, ART Antiretroviral therapy, LTFU Lost to follow up.

                      Viral load

                      There were no differences between boys and girls in either the proportion who attained viral load suppression to <400 copies/mL by 6 months into the pre-randomization phase (78.9 vs. 76.9%, p = 0.747), or the proportion with viral load <50 copies/mL at randomization (61.5 vs. 64.8%, p = 0.634) when all children were on LPV/r. The probability of virologic failure (confirmed viral load >1000 copies/mL) by 52 weeks post-randomization was similar between boys and girls who remained on LPV/r (0 vs. 0.043, p = 0.162) as well as between boys and girls who switched to nevirapine (0.224 vs. 0.172, p = 0.575). The probability of virologic failure by 156 weeks was also similar between boys and girls who remained on LPV/r (0.115 vs. 0.104, p = 0.996) as well as between boys and girls who switched to nevirapine (0.240 vs. 0.238, p = 0.954). Proportions of boys and girls in viremia categories (<50, 50–1000, >1000 copies/mL) within treatment groups were similar at all scheduled post-randomization visits.

                      CD4 percentage and count

                      While the mean CD4 percentage and cell count increased in the pre-randomization phase for boys and girls in both treatment groups, differences in CD4 response between sexes were noted among those switched to nevirapine post-randomization. Girls who switched to nevirapine had a more robust CD4 count response than boys switched to nevirapine from 24 weeks up to 112 weeks post randomization (on average CD4 count 306 cells/μL higher, p = 0.045), with significantly higher mean CD4 cell counts at 24, 64, and 100 weeks post-randomization (p = 0.027, 0.036, 0.043 respectively) and higher CD4 percentage at 64, 76, and 100 weeks post-randomization (p = 0.028, 0.035, 0.047, respectively) (Figure 2). After adjustment for age at ART initiation, pretreatment WAZ, HAZ, and CD4 count, on average CD4 count remained significantly higher for girls than boys from 24 to 112 weeks after randomization to nevirapine (p = 0.0089). The observed differences were no longer detectable at 148 weeks.
                      http://static-content.springer.com/image/art%3A10.1186%2F1471-2431-14-39/MediaObjects/12887_2013_978_Fig2_HTML.jpg
                      Figure 2

                      Immunologic and anthropometric outcomes over time. Mean CD4 percent (A) and CD4 count (B), weight-for-age z-score (WAZ) (C) and height-for-age z-score (HAZ) (D) before treatment, at randomization, and at scheduled visits with measurements through 148 weeks after randomization in Neverest 2 by sex and randomization group; Abbreviations: WAZ, weight-for-age z-score; HAZ, height-for-age z-score; LPV/r, lopinavir-boosted ritonavir; NVP, nevirapine.

                      Metabolic and laboratory assessments

                      No significant differences in mean total cholesterol, LDL, HDL, and triglycerides were found pretreatment, at randomization, or at scheduled visits 36, 88, and 136 weeks post-randomization between boys and girls (data not shown) [23]. Upon exit from the study, girls continuing on LPV/r had a higher total cholesterol:HDL ratio and lower mean HDL concentration than boys on LPV/r, as well as compared to girls on nevirapine (3.8 vs. 3.0, p = 0.002 and 48 vs. 57 mg/dL, p = 0.007, respectively), and girls on LPV/r had a higher mean HOMA-IR than boys on LPV/r (Table 2). There were no differences in proportions of boys and girls within treatment groups with drug related rashes or Grade 3 or 4 ALT at any scheduled post-randomization visits (data not shown).
                      Table 2

                      Metabolic and other laboratory assessments of 156 perinatally HIV-infected South African children at the final study visit of Neverest 2 by sex within treatment randomization group

                      Measurement

                      LPV/r (n = 85)

                       

                      NVP (n = 71)

                       
                        

                      Boys (n = 41)

                      Girls (n = 44)

                      P-value

                      Boys (n = 40)

                      Girls (n = 31)

                      P-value

                      Total Cholesterol, mg/dL

                      Mean (SD)

                      170 (39)

                      172 (39)

                      0.816

                      163 (25)

                      159 (38)

                      0.533

                       Acceptable: <170

                      N (%)

                      24 (58.5)

                      21 (47.7)

                      0.603

                      29 (72.5)

                      23 (74.2)

                      0.884

                       Borderline: 170-199

                      10 (24.4)

                      14 (31.8)

                      8 (20.0)

                      5 (16.1)

                       Elevated: ≥200

                      7 (17.1)

                      9 (20.5)

                      3 (7.5)

                      3 (9.7)

                      HDL, mg/dL

                      Mean (SD)

                      54 (14)

                      48 (12)

                      0.044

                      60 (15)

                      57 (16)

                      0.352

                       Abnormal: <35

                      N (%)

                      2 (4.9)

                      5 (11.4)

                      0.277

                      0 (0.0)

                      2 (6.4)

                      0.103

                       Normal: ≥35

                      39 (95.1)

                      39 (88.6)

                      40 (100.0)

                      29 (93.6)

                      LDL, mg/dL

                      Mean (SD)

                      98 (31)

                      101 (38)

                      0.664

                      89 (24)

                      87 (29)

                      0.738

                       Acceptable: <110

                      N (%)

                      28 (68.3)

                      28 (63.6)

                      0.895

                      31 (77.5)

                      25 (80.7)

                      0.516

                       Borderline: 110-129

                      7 (17.1)

                      9 (20.5)

                      7 (17.5)

                      3 (9.7)

                       Elevated: ≥130

                      6 (14.6)

                      7 (15.9)

                      2 (0.5)

                      3 (9.7)

                      Total Cholesterol: HDL Ratio

                      Mean (SD)

                      3.3 (1.0)

                      3.8 (1.1)

                      0.049

                      2.8 (0.7)

                      3.0 (1.0)

                      0.547

                      Triglycerides, mg/dL

                      Mean (SD)

                      88 (34)

                      99 (42)

                      0.207

                      69 (27)

                      74 (32)

                      0.457

                       Normal: ≤150

                      N (%)

                      37 (90.2)

                      37 (84.1)

                      0.398

                      39 (97.5)

                      30 (96.8)

                      0.855

                       Abnormal: >150

                      4 (9.8)

                      7 (15.9)

                      1 (2.5)

                      1 (3.2)

                      C-Reactive Protein, mg/L

                      Mean (SD)

                      3.2 (5.6)

                      3.8 (6.5)

                      0.453

                      9.5 (23.8)

                      9.8 (18.1)

                      0.423

                       Normal: ≤3

                      N (%)

                      33 (80.5)

                      36 (81.8)

                      0.875

                      27 (67.5)

                      19 (61.3)

                      0.587

                       Elevated: >3

                      8 (19.5)

                      8 (18.2)

                      13 (32.5)

                      12 (38.7)

                      Glucose, mg/dL

                      Mean (SD)

                      96.9 (7.1)

                      96.4 (7.8)

                      0.736

                      97.6 (9.2)

                      93.7 (8.5)

                      0.070

                       Normal: <110

                      N (%)

                      80 (98.8)

                      41 (100.0)

                      0.332

                      74 (98.7)

                      39 (97.5)

                      0.375

                       Impaired: ≥110

                      1 (1.2)

                      0 (0.0)

                      1 (1.3)

                      1 (2.5)

                      HOMA-IR

                      Mean (SD)

                      0.90 (0.5)

                      1.16 (0.6)

                      0.036

                      1.17 (1.0)

                      0.96 (0.6)

                      0.289

                       Normal: ≤3.16

                      N (%)

                      41 (100.0)

                      44 (100.0)

                      1.0

                      36 (92.3)

                      31 (100.0)

                      0.115

                       Resistance: >3.16

                       

                      0 (0.0)

                      0 (0.0)

                       

                      3 (7.7)

                      0 (0.0)

                       

                      Abbreviations: LPV/r Lopinavir-boosted ritonavir, NVP Nevirapine, HDL High-density lipoprotein, LDL Low-density lipoprotein, HOMA-IR Homeostatic model assessment of insulin resistance.

                      Anthropometrics

                      WAZ for boys and girls increased rapidly in the pre-randomization phase (Figure 2); the mean change in WAZ for boys was greater than girls, though not significant (1.69 vs. 1.50, p = 0.386). After randomization, boys on LPV/r consistently had a lower WAZ than boys on nevirapine through 148 weeks, but this difference was not statistically significant. There were no sex differences in likelihood of dropping more than one Z score in WAZ before 52 weeks post-randomization.

                      Mean change in HAZ was greater for girls than boys in the pre-randomization phase when all children were on LPV/r, though this difference was not significant (0.14 vs. -0.06, p = 0.473). After randomization, height for boys and girls on both treatment regimens improved through 148 weeks (Figure 2). Mean HAZ was significantly lower for boys than girls on nevirapine at randomization (−3.38 vs. -2.99, p = 0.021). HAZ stayed lower for boys on nevirapine than girls on nevirapine through 52 weeks post-randomization (b = −0.57, p = 0.031). The association remained when adjusted for age at initiation of ART (b = −0.54, p = 0.026), but not when adjusted for pretreatment HAZ (b = −0.38, p = 0.145).

                      At the final study visit, girls had a larger %BF by BIA than boys (18.7 vs. 12.8%, p < 0.0001). A similar pattern was observed by cumulative skinfold sum, though this difference was not significant (42.9 vs. 39.7 mm, p = 0.076). Patterns of fat were similar between boys and girls in both treatment randomization groups (Table 3). There were no differences in proportions of children with lipodystrophy between boys and girls.
                      Table 3

                      Body composition measures of 156 perinatally HIV-infected South African children at the final study visit of Neverest 2 by sex within treatment randomization group, Mean (SD)

                      Measurement

                      LPV/r (n = 85)

                       

                      NVP (n = 71)

                       
                       

                      Boys (n = 41)

                      Girls (n = 44)

                      P-value

                      Boys (n = 40)

                      Girls (n = 31)

                      P-value

                      MUAC, cm

                      16.1 (15.2)

                      15.5 (2.0)

                      0.288

                      15.2 (1.8)

                      15.1 (2.1)

                      0.844

                      MWC: MHC Ratio

                      1.25 (1.9)

                      0.93 (0.1)

                      0.287

                      0.95 (0.1)

                      0.95 (0.1)

                      0.773

                      SFS, mm

                      41.9 (12.1)

                      44.0 (10.1)

                      0.408

                      37.2 (9.7)

                      41.3 (10.3)

                      0.120

                      %BF by BIA

                      14.5 (6.2)

                      19.2 (7.0)

                      0.002

                      10.9 (7.6)

                      17.9 (6.9)

                      0.0002

                      Extremity Fat Area

                            

                      Arm Fat Area, cm2

                      5.52 (2.0)

                      6.05 (2.2)

                      0.259

                      4.91 (1.3)

                      5.65 (2.0)

                      0.063

                      % of Fat in Arm

                      26.6 (5.5)

                      31.5 (9.6)

                      0.006

                      27.2 (8.2)

                      30.9 (8.2)

                      0.063

                      Leg Fat Area, cm2

                      14.9 (5.5)

                      16.5 (6.5)

                      0.236

                      12.3 (4.4)

                      15.2 (6.1)

                      0.025

                      % of Fat in Leg

                      20.1 (5.6)

                      23.4 (7.4)

                      0.028

                      17.7 (4.5)

                      21.6 (6.4)

                      0.006

                      Regional Fat Proportion of Total Body Fat

                            

                      Arm: [(BSF + TSF)/SFS]

                      0.30 (0.05)

                      0.30 (0.04)

                      0.985

                      0.31 (0.05)

                      0.29 (0.03)

                      0.197

                      Trunk: [(SSF + SISF + USF)/SFS]

                      0.46 (0.07)

                      0.45 (0.06)

                      0.484

                      0.47 (0.05)

                      0.47 (0.05)

                      0.658

                      Leg: [MTSF/SFS]

                      0.24 (0.04)

                      0.25 (0.04)

                      0.297

                      0.22 (0.03)

                      0.24 (0.04)

                      0.026

                      Trunk-Extremity Skinfold Ratios

                            

                      Trunk-Arm: [(SSF + SISF)/(BSF + TSF + SSF + SISF)]

                      0.49 (0.06)

                      0.50 (0.06)

                      0.652

                      0.50 (0.04)

                      0.51 (0.06)

                      0.306

                      Trunk-Leg: [(SSF + SISF)/(MTSF + SSF + SISF)]

                      0.55 (0.06)

                      0.55 (0.07)

                      0.978

                      0.58 (0.04)

                      0.57 (0.06)

                      0.278

                      Abbreviations: LPV/r Lopinavir-boosted ritonavir, NVP Nevirapine, MUAC Mid-upper arm circumference, MWC Mid-waist circumference, MHC Maximum hip circumference, BSF Bicep skinfold, TSF Tricep skinfold, SSF Subscapular skinfold, SISF Suprailiac skinfold, USF Umbilical skinfold, MTSF Mid-thigh skinfold, SFS Skinfold sum, %BF Percent body fat, BIA Bioimpedance analysis.

                      Note: Cumulative skinfold sum (SFS) was calculated by adding BSF, TSF, SSF, SISF, USF, and MTSF. Extremity fat areas, regional fat proportions of total body fat, and trunk-extremity skinfold ratios were calculated as previously reported [22].

                      Drug concentrations and adherence

                      Of those randomized to remain on LPV/r, the plasma concentrations of ritonavir adjusted for WAZ and time since dose was on average 0.115 μg/ml higher in girls than boys (p = 0.048) in scheduled post-randomization visits. No significant sex differences in lopinavir concentrations were observed. Of those switched to nevirapine, there were no sex differences in nevirapine concentrations. There were no differences between boys and girls in the proportion of children non-adherent to their drugs at any post-randomization scheduled visits as assessed by medication return percentages.

                      Discussion

                      In this randomized clinical trial, we assessed sex differences in treatment outcomes of South African HIV-infected children initiated on LPV/r before age two and later randomized to remain on LPV/r or switch to nevirapine after attaining suppression of viremia. Though no sex differences were observed in virologic response, girls switched to nevirapine had a more robust immunologic response than boys switched to nevirapine. In addition, after a minimum of 99 and maximum of 245 weeks on ART, girls on LPV/r had a higher total cholesterol:HDL ratio and lower mean HDL concentration than boys on LPV/r. As children remain on life-long treatment, these sex disparities noted early in life may have implications for long term clinical outcomes.

                      Few pediatric studies have assessed sex differences in virologic response to ART. Similar to a recent meta-analysis of virologic outcomes in treatment-experienced adults [3] and a randomized trial comparing nevirapine and LPV/r-based treatment initiation in young children with no prior nevirapine exposure [29], our study observed no sex differences in suppression of virus in response to ART. This finding is in contrast to an earlier study in children on mono- or dual-nucleoside analogue therapy that reported lower viral load levels in girls than boys [16].

                      Our finding that girls switched to nevirapine have a greater immunologic response than boys is similar to results from two observational studies conducted in children receiving non-nucleoside reverse transcriptase inhibitors (NNRTI)-based regimens [17, 18]. A stronger CD4 cell count response in women, compared to men after treatment initiation, has also been reported in a large cohort study of HIV-infected adults, the majority of whom were initiated on NNRTI-based therapy [30], but these results appear to be explained by higher baseline CD4 count in women. In contrast, our association remained when adjusted for pretreatment CD4 count as well as age at ART initiation. The reason for the difference in CD4 response by sex among children randomly assigned to switch to nevirapine is unclear, but it does not appear to be due to better drug adherence or viral suppression or differences in drug concentrations; the difference may reflect underlying sex differences in CD4 parameters that have been reported in healthy uninfected children [31]. The consequences of this time-limited difference are also unclear. High baseline CD4 cell count has been identified as a risk factor for nevirapine-related hepatotoxicity during ART initiation in women and pregnant women [32], but we did not see increased abnormal ALT levels or drug-related rashes in girls on nevirapine. Although it is unclear if the incremental increase in CD4 response we observed among girls switched to nevirapine will have long term benefits, the simultaneous rapid improvement in growth suggests that there is a clinical benefit for these girls during this time period.

                      Although studies in adults observe more lipid abnormalities in ART-treated women [11, 3336], to our knowledge, no prior studies have reported sex differences in metabolic outcomes in children. The differences we observe do not appear to be explained by underlying sex differences in healthy children [37, 38] and may be a result of greater exposure to ritonavir in girls. Ritonavir is known to adversely affect lipid metabolism through suppression of the degradation of the nuclear form of sterol regulatory element binding proteins in the liver and other sites, resulting in increased fatty acid and cholesterol production [39]. Higher ritonavir drug concentration have been previously reported in adult women [40], and may be driven by sex differences in body size and composition as well as drug metabolism, transport, and excretion [13].

                      Sex differences in growth parameters during ART were also detected. Though height remained significantly lower for boys on nevirapine than girls on nevirapine up to 52 weeks post-randomization after adjusting for age at initiation of ART, it was no longer significant when adjusted for pretreatment height. We have previously reported that earlier age of ART initiation can affect growth [41]. Few studies have compared growth responses between sexes; one study of older children reported higher rates of stunting in boys than girls [42]. In our study, we noted higher levels of fat in girls than boys which may reflect underlying sex differences in total body fat observed in healthy children [4345]. Greater fat accumulation in girls, as reported in studies of older HIV-infected children during puberty, may be related to estrogen effects on fat accumulation [46, 47]. In the present study in which all children were pre-pubertal, however, we did not observe sex differences in prevalence of lipohypertrophy or other forms of lipodystrophy [46, 48].

                      Although our study is one of the first to evaluate sex differences in childhood response to potent combination ART, it has limitations. Children in this trial met eligibility criteria for treatment based on South African guidelines in place at the time and only those who attained viral suppression in the first year of therapy were randomized [49]. Thus, our study has limited generalizability to populations with suboptimal adherence or virologic response. In addition, we were not able to assess the potential relationship between drug pharmacokinetics and body composition, or detect rare adverse events.

                      Conclusions

                      In this study, we observed no sex differences in viral suppression; however, compared to boys switched to nevirapine, girls switched to nevirapine had a more robust immune recovery. In addition, girls on LPV/r had a more unfavorable lipid profile compared to boys on LPV/r at the final study visit. This study provides a glimpse into early childhood sex differences related to ART. The course and long term consequences of these differences with respect to immunologic and metabolic outcomes is unknown. Future studies of sex differences among HIV-infected children should focus on pharmacokinetics as well as pathophysiologic pathways including chronic inflammation, immune senescence and mitochondrial dysfunction which may influence the therapeutic response and risk of complications. The potential effects of factors such as malnutrition, infections, co-morbidity, viral sub-type, and underlying genetic profiles also warrant further investigation during childhood and adolescence.

                      Abbreviations

                      ALT: 

                      Alanine aminotransferase

                      ART: 

                      Antiretroviral treatment

                      BIA: 

                      Bioimpedance analysis

                      HAZ: 

                      Height-for-age z-score

                      HDL: 

                      High-density lipoprotein

                      HOMA-IR: 

                      Homeostasis model assessment of insulin resistance

                      LPV/r: 

                      Ritonavir-boosted lopinavir

                      LDL: 

                      Low-density lipoprotein

                      NNRTI: 

                      Non-nucleoside reverse transcriptase inhibitor

                      WAZ: 

                      Weight-for-age z-score

                      %BF: 

                      Total body fat percent.

                      Declarations

                      Acknowledgements

                      This study was supported in part by grants from the National Institutes of Child Health and Human Development (HD 47177, HD 61255, HD 073952, HD 073977) and Secure the Future Foundation (grant number RES 219). None of the authors have any conflict of interest to declare.

                      Authors’ Affiliations

                      (1)
                      Gertrude H. Sergievsky Center, College of Physicians and Surgeons, Columbia University
                      (2)
                      Department of Epidemiology, Mailman School of Public Health, Columbia University
                      (3)
                      Empilweni Services and Research Unit, Rahima Moosa Mother and Child Hospital, University of the Witwatersrand
                      (4)
                      Department of Medicine, Division of Clinical Pharmacology, University of Cape Town
                      (5)
                      Institute of Infectious Disease and Molecular Medicine, University of Cape Town
                      (6)
                      ICAP, Mailman School of Public Health, Columbia University
                      (7)
                      Department of Pediatrics, College of Physicians and Surgeons, Columbia University

                      References

                      1. Sterling TR, Vlahov D, Astemborski J, Hoover DR, Margolick JB, Quinn TC: Initial plasma HIV-1 RNA levels and progression to AIDS in women and men. N Engl J Med 2001,344(10):720–725.PubMedView Article
                      2. Farzadegan H, Hoover DR, Astemborski J, Lyles CM, Margolick JB, Markham RB, Quinn TC, Vlahov D: Sex differences in HIV-1 viral load and progression to AIDS. Lancet 1998,352(9139):1510–1514.PubMedView Article
                      3. Soon GG, Min M, Struble KA, Chan-Tack KM, Hammerstrom T, Qi K, Zhou S, Bhore R, Murray JS, Birnkrant DB: Meta-analysis of gender differences in efficacy outcomes for HIV-positive subjects in randomized controlled clinical trials of antiretroviral therapy (2000–2008). AIDS Patient Care STDS 2012,26(8):444–453.PubMedView Article
                      4. Moore AL, Kirk O, Johnson AM, Katlama C, Blaxhult A, Dietrich M, Colebunders R, Chiesi A, Lungren JD, Phillips AN: Virologic, immunologic, and clinical response to highly active antiretroviral therapy: the gender issue revisited. J Acquir Immune Defic Syndr 2003,32(4):452–461.PubMedView Article
                      5. Ofotokun I, Pomeroy C: Sex differences in adverse reactions to antiretroviral drugs. Top HIV Med 2003,11(2):55–59.PubMed
                      6. Bersoff-Matcha SJ, Miller WC, Aberg JA, van der Horst C, Hamrick HJ Jr, Powderly WG, Mundy LM: Sex differences in nevirapine rash. Clin Infect Dis 2001,32(1):124–129.PubMedView Article
                      7. Sanne I, Mommeja-Marin H, Hinkle J, Bartlett JA, Lederman MM, Maartens G, Wakeford C, Shaw A, Quinn J, Gish RG, et al.: Severe hepatotoxicity associated with nevirapine use in HIV-infected subjects. J Infect Dis 2005,191(6):825–829.PubMedView Article
                      8. Moore RD, Keruly JC, Chaisson RE: Incidence of pancreatitis in HIV-infected patients receiving nucleoside reverse transcriptase inhibitor drugs. AIDS 2001,15(5):617–620.PubMedView Article
                      9. Currier J, Averitt Bridge D, Hagins D, Zorrilla CD, Feinberg J, Ryan R, Falcon R, Tennenberg A, Mrus J, Squires K: Sex-based outcomes of darunavir-ritonavir therapy: a single-group trial. Ann Intern Med 2010,153(6):349–357.PubMed CentralPubMedView Article
                      10. Martinez E, Mocroft A, Garcia-Viejo MA, Perez-Cuevas JB, Blanco JL, Mallolas J, Bianchi L, Conget I, Blanch J, Phillips A, et al.: Risk of lipodystrophy in HIV-1-infected patients treated with protease inhibitors: a prospective cohort study. Lancet 2001,357(9256):592–598.PubMedView Article
                      11. Pernerstorfer-Schoen H, Jilma B, Perschler A, Wichlas S, Schindler K, Schindl A, Rieger A, Wagner OF, Quehenberger P: Sex differences in HAART-associated dyslipidaemia. AIDS 2001,15(6):725–734.PubMedView Article
                      12. Umeh OC, Currier JS: Sex differences in pharmacokinetics and toxicity of antiretroviral therapy. Expert Opin Drug Metab Toxicol 2006,2(2):273–283.PubMedView Article
                      13. Floridia M, Giuliano M, Palmisano L, Vella S: Gender differences in the treatment of HIV infection. Pharmacol Res 2008,58(3–4):173–182.PubMedView Article
                      14. d’arminio Monforte A, Cozzi-Lepri A, Phillips A, de Luca A, Murri R, Mussini C, Grossi P, Galli A, Zauli T, Montroni M, et al.: Interruption of highly active antiretroviral therapy in HIV clinical practice: results from the Italian cohort of antiretroviral-naive patients. J Acquir Immune Defic Syndr 2005,38(4):407–416.PubMedView Article
                      15. Ruel TD, Zanoni BC, Ssewanyana I, Cao H, Havlir DV, Kamya M, Achan J, Charlebois ED, Feeney ME: Sex differences in HIV RNA level and CD4 cell percentage during childhood. Clin Infect Dis 2011,53(6):592–599.PubMedView Article
                      16. Foca M, Moye J, Chu C, Matthews Y, Rich K, Handelsman E, Luzuriaga K, Paul M, Diaz C: Gender differences in lymphocyte populations, plasma HIV RNA levels, and disease progression in a cohort of children born to women infected with HIV. Pediatrics 2006,118(1):146–155.PubMedView Article
                      17. Renner L, Prin M, Li FY, Goka B, Northrup V, Paintsil E: Time to and predictors of CD4+ T-lymphocytes recovery in HIV-infected children initiating highly active antiretroviral therapy in Ghana. AIDS Res Treat 2011, 2011:896040.PubMed CentralPubMed
                      18. Puthanakit T, Kerr S, Ananworanich J, Bunupuradah T, Boonrak P, Sirisanthana V: Pattern and predictors of immunologic recovery in human immunodeficiency virus-infected children receiving non-nucleoside reverse transcriptase inhibitor-based highly active antiretroviral therapy. Pediatr Infect Dis J 2009,28(6):488–492.PubMedView Article
                      19. Carter RJ, Wiener J, Abrams EJ, Farley J, Nesheim S, Palumbo P, Bulterys M: Dyslipidemia among perinatally HIV-infected children enrolled in the PACTS-HOPE cohort, 1999–2004: a longitudinal analysis. J Acquir Immune Defic Syndr 2006,41(4):453–460.PubMedView Article
                      20. Sztam KA, Jiang H, Jurgrau A, Deckelbaum RJ, Foca MD: Early increases in concentrations of total, LDL, and HDL cholesterol in HIV-infected children following new exposure to antiretroviral therapy. J Pediatr Gastroenterol Nutr 2011,52(4):495–498.PubMedView Article
                      21. Jaquet D, Levine M, Ortega-Rodriguez E, Faye A, Polak M, Vilmer E, Levy-Marchal C: Clinical and metabolic presentation of the lipodystrophic syndrome in HIV-infected children. AIDS 2000,14(14):2123–2128.PubMedView Article
                      22. Arpadi S, Shiau S, Strehlau R, Martens L, Patel F, Coovadia A, Abrams EJ, Kuhn L: Metabolic abnormalities and body composition of HIV-infected children on Lopinavir or Nevirapine-based antiretroviral therapy. Arch Dis Child 2013,98(4):258–264.PubMedView Article
                      23. Strehlau R, Coovadia A, Abrams EJ, Martens L, Arpadi S, Meyers T, Kuhn L: Lipid profiles in young HIV-infected children initiating and changing antiretroviral therapy. J Acquir Immune Defic Syndr 2012,60(4):369–376.PubMed CentralPubMedView Article
                      24. Reitz C, Coovadia A, Ko S, Meyers T, Strehlau R, Sherman G, Kuhn L, Abrams EJ: Initial response to protease-inhibitor-based antiretroviral therapy among children less than 2 years of age in South Africa: effect of cotreatment for tuberculosis. J Infect Dis 2010,201(8):1121–1131.PubMed CentralPubMedView Article
                      25. Kuhn L, Coovadia A, Strehlau R, Martens L, Hu CC, Meyers T, Sherman G, Hunt G, Persaud D, Morris L, et al.: Switching children previously exposed to nevirapine to nevirapine-based treatment after initial suppression with a protease-inhibitor-based regimen: long-term follow-up of a randomised, open-label trial. Lancet Infect Dis 2012,12(7):521–530.PubMedView Article
                      26. Keskin M, Kurtoglu S, Kendirci M, Atabek ME, Yazici C: Homeostasis model assessment is more reliable than the fasting glucose/insulin ratio and quantitative insulin sensitivity check index for assessing insulin resistance among obese children and adolescents. Pediatrics 2005,115(4):e500.PubMedView Article
                      27. Child growth standards and WHO Anthro and Macros [http://​www.​who.​int/​childgrowth/​en/​]
                      28. Horlick M, Arpadi SM, Bethel J, Wang J, Moye J, Cuff P, Pierson RN, Kotler D: Bioelectrical impedance analysis models for prediction of total body water and fat-free mass in healthy and HIV-infected children and adolescents. Am J Clin Nutr 2002,76(5):991.PubMed
                      29. Violari A, Lindsey JC, Hughes MD, Mujuru HA, Barlow-Mosha L, Kamthunzi P, Chi BH, Cotton MF, Moultrie H, Khadse S, et al.: Nevirapine versus ritonavir-boosted lopinavir for HIV-infected children. N Engl J Med 2012,366(25):2380–2389.PubMed CentralPubMedView Article
                      30. Nash D, Katyal M, Brinkhof MW, Keiser O, May M, Hughes R, Dabis F, Wood R, Sprinz E, Schechter M, et al.: Long-term immunologic response to antiretroviral therapy in low-income countries: a collaborative analysis of prospective studies. AIDS 2008,22(17):2291–2302.PubMed CentralPubMedView Article
                      31. European Collaborative Study: Are there gender and race differences in cellular immunity patterns over age in infected and uninfected children born to HIV-infected women? J Acquir Immune Defic Syndr 2003,33(5):635–641.View Article
                      32. Jamisse L, Balkus J, Hitti J, Gloyd S, Manuel R, Osman N, Djedje M, Farquhar C: Antiretroviral-associated toxicity among HIV-1-seropositive pregnant women in Mozambique receiving nevirapine-based regimens. J Acquir Immune Defic Syndr 2007,44(4):371–376.PubMedView Article
                      33. Hoffman RM, Umeh OC, Garris C, Givens N, Currier JS: Evaluation of sex differences of fosamprenavir (with and without ritonavir) in HIV-infected men and women. HIV Clin Trials 2007,8(6):371–380.PubMedView Article
                      34. Thiebaut R, Dequae-Merchadou L, Ekouevi DK, Mercie P, Malvy D, Neau D, Dabis F: Incidence and risk factors of severe hypertriglyceridaemia in the era of highly active antiretroviral therapy: the Aquitaine Cohort, France, 1996–99. HIV Med 2001,2(2):84–88.PubMedView Article
                      35. Currier JS, Martorell C, Osiyemi O, Yin MT, Ryan R, de la Rosa G, Mrus J: Effects of darunavir/ritonavir-based therapy on metabolic and anthropometric parameters in women and men over 48 weeks. AIDS Patient Care STDS 2011,25(6):333–340.PubMedView Article
                      36. Kumar PN, Rodriguez-French A, Thompson MA, Tashima KT, Averitt D, Wannamaker PG, Williams VC, Shaefer MS, Pakes GE, Pappa KA: A prospective, 96-week study of the impact of Trizivir, Combivir/nelfinavir, and lamivudine/stavudine/nelfinavir on lipids, metabolic parameters and efficacy in antiretroviral-naive patients: effect of sex and ethnicity. HIV Med 2006,7(2):85–98.PubMedView Article
                      37. Srinivasan SR, Wattigney W, Webber LS, Berenson GS: Race and gender differences in serum lipoproteins of children, adolescents, and young adults–emergence of an adverse lipoprotein pattern in white males: the Bogalusa heart study. Prev Med 1991,20(6):671–684.PubMedView Article
                      38. The Lipid Research Clinics Population Studies Data Book. Vol 1 In National Institutes of Health; Lipid Metabolism Branch; Division of Heart and Vascular Diseases. Bethesda, MD: National Heart, Lung, and Blood Institute; 1980.
                      39. Hui DY: Effects of HIV protease inhibitor therapy on lipid metabolism. Prog Lipid Res 2003,42(2):81–92.PubMedView Article
                      40. Umeh OC, Currier JS, Park JG, Cramer Y, Hermes AE, Fletcher CV: Sex differences in lopinavir and ritonavir pharmacokinetics among HIV-infected women and men. J Clin Pharmacol 2011,51(12):1665–1673.PubMed CentralPubMedView Article
                      41. Shiau S, Arpadi S, Strehlau R, Martens L, Patel F, Coovadia A, Abrams EJ, Kuhn L: Initiation of antiretroviral therapy before 6 months of age is associated with faster growth recovery in South African children perinatally infected with human immunodeficiency virus. J Pediatr 2013. [Epub ahead of print]
                      42. Rosso R, Vignolo M, Parodi A, di Biagio A, Sormani MP, Bassetti M, Aicardi G, Bassetti D: Bone quality in perinatally HIV-infected children: role of age, sex, growth, HIV infection, and antiretroviral therapy. AIDS Res Hum Retroviruses 2005,21(11):927–932.PubMedView Article
                      43. Taylor RW, Gold E, Manning P, Goulding A: Gender differences in body fat content are present well before puberty. Int J Obes Relat Metab Disord 1997,21(11):1082–1084.PubMedView Article
                      44. He Q, Horlick M, Thornton J, Wang J, Pierson RN Jr, Heshka S, Gallagher D: Sex and race differences in fat distribution among Asian, African-American, and Caucasian prepubertal children. J Clin Endocrinol Metab 2002,87(5):2164–2170.PubMedView Article
                      45. Staiano AE, Katzmarzyk PT: Ethnic and sex differences in body fat and visceral and subcutaneous adiposity in children and adolescents. Int J Obes (Lond) 2012,36(10):1261–1269.View Article
                      46. Spoulou V, Kanaka-Gantenbein C, Bathrellou I, Mora S, Mostrou G, Sidossis L, Chrousos G, Theodoridou M: Monitoring of lipodystrophic and metabolic abnormalities in HIV-1 infected children on antiretroviral therapy. Hormones (Athens) 2011,10(2):149–155.View Article
                      47. Beregszaszi M, Dollfus C, Levine M, Faye A, Deghmoun S, Bellal N, Houang M, Chevenne D, Hankard R, Bresson JL, et al.: Longitudinal evaluation and risk factors of lipodystrophy and associated metabolic changes in HIV-infected children. J Acquir Immune Defic Syndr 2005,40(2):161–168.PubMedView Article
                      48. Kinabo GD, Sprengers M, Msuya LJ, Shayo AM, van Asten H, Dolmans WM, van der Ven AJ, Warris A: Prevalence of lipodystrophy in HIV-infected children in Tanzania on highly active antiretroviral therapy. Pediatr Infect Dis J 2013,32(1):39–44.PubMedView Article
                      49. South Africa Department of Health. National antiretroviral treatment guidelines (first edition, 2004): section 2, antiretroviral treatment in children. [http://​southafrica.​usembassy.​gov/​media/​2004-doh-art-guidelines.​pdf]
                      50. Pre-publication history

                        1. The pre-publication history for this paper can be accessed here: http://​www.​biomedcentral.​com/​1471-2431/​14/​39/​prepub

                      Copyright

                      © Shiau et al.; licensee BioMed Central Ltd. 2014

                      This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://​creativecommons.​org/​publicdomain/​zero/​1.​0/​) applies to the data made available in this article, unless otherwise stated.