Introduction
Currently, 2.5 million children are living with HIV worldwide [1]. Over 90% live in sub-Saharan Africa [1], where advances in under-5 mortality rates have been reversed by the HIV pandemic [2], which causes 330 000 deaths annually [1].
The vast majority of children are infected through mother-to-child transmission (MTCT) but in areas of highest prevalence antenatal programmes to prevent MTCT reach only 10% of mothers [3]. Even if women have access to single-dose nevirapine (sd-NVP) perinatal prophylaxis, approximately10% of infants are infected before breastfeeding [4,5]. Furthermore, infected infants may progress more rapidly following sd-NVP exposure because a greater proportion are intrauterine-infected, and their mothers have advanced disease [5]. In Durban, South Africa, 85% of HIV-infected infants who received sd-NVP reached the 2006 World Health Organisation (WHO) treatment threshold (CD4 cell count ≤25%) by 6 months of age [5].
Large numbers of children will therefore require antiretroviral therapy (ART) during infancy (first 12 months of life), but little is known about the outcome of infants treated in resource-limited settings. Impressive results have been achieved in adult antiretroviral programmes in sub-Saharan Africa [6,7], despite initial concerns about likely adherence to ART [8]. The few studies that have reported outcome in paediatric HIV infection are encouraging but have, with one exception [9], focused on older children [10-17]. Treatment is substantially more challenging in infants than in older children and adults [18].
A central concern in management of HIV-infected infants is non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance mutations, induced by exposure to sd-NVP [19-21]. These have potentially deleterious effects on the efficacy of ART in infants [9]. Although current WHO guidelines [22] recommend first-line NNRTI-containing regimens for infants, some national treatment programmes avoid this class of drug [23], and data on ART outcome in infants following sd-NVP are urgently required [22].
Baseline NNRTI resistance, adherence to therapy and initial virological and clinical outcome, in a cohort of sd-NVP-exposed infants started on ART in the first year of life, are reported in this study.
Methods
Study design
The present study was a pilot randomized controlled trial, designed as a feasibility study to evaluate three approaches to antiretroviral treatment of HIV-infected infants. The primary endpoint was the proportion of infants progressing to AIDS by 3 years, and these data will be reported upon completion. The target sample was minimum 60 infants, which would allow detection of a 40% difference between immediate and deferred therapy arms, with 80% power and 95% confidence. Although the study was not designed primarily to assess initial virological response, this infant cohort provides an opportunity to describe early outcomes following initiation of ART as an unplanned analysis.
Maternal study participants
Antenatal mothers were recruited from October 2002, and paediatric study participants were enrolled between July 2003 and September 2005, at St Mary's Hospital, Mariannhill, and Prince Mshiyeni Hospital, Umlazi, both in KwaZulu-Natal, South Africa. Antenatal mothers at these sites underwent voluntary counselling and testing for HIV after 28 weeks' gestation. HIV-infected mothers gave written consent to participate. Mothers had viral load and CD4 cell count measured at median 35 weeks' gestation [interquartile range (IQR) 32-36]. Mothers attended two antenatal HIV education sessions. All other aspects of perinatal care followed local guidelines. HIV infection was not an indication for Caesarean section. At the time, sd-NVP was the only accessible prevention of mother-to-child transmission (pMTCT) intervention, and infant ART was not available in government hospitals until April 2004. Mothers were given sd-NVP (200 mg) to self-administer at the onset of labour, and infants were given sd-NVP (2 mg/kg) by a nurse within 72 h of birth.
Infant study participants
Venous blood was taken from infants on days 1 and 28 to identify intrauterine and intrapartum infection, respectively [24], and repeated (3-8 days later) to confirm the diagnosis. Some infants diagnosed as intrapartum-infected may have been infected early-postpartum through breast-milk transmission.
Infants were eligible if they were born at one of the study hospitals, had confirmed intrauterine/intrapartum HIV infection and caregivers gave consent for enrolment. Exclusion criteria were gestation less than 37 weeks, birth weight less than 2 kg, evidence of other congenital infections or severe congenital abnormalities [5].
HIV-1 RNA and CD4% determination
HIV-1 diagnosis was undertaken by reverse-transcriptase polymerase chain reaction (PCR) on 500 μl plasma. Viral load was undertaken using the Roche Amplicor version 1.5 assay, with a lower limit of detection of 50 RNA copies/ml. CD4% was determined by flow cytometry.
Follow-up and treatment of HIV-infected infants
Infants were randomized at diagnosis to one of three study arms: deferred ART, started once clinical or immunological criteria were reached (Arm A); immediate ART given for 1 year, then stopped (Arm B); or immediate ART given with up to three structured treatment interruptions (STI) to 18 months of age, then stopped (Arm C). Participants included here were therefore randomized to deferred ART (Arm A) or immediate ART (Arms B and C). Arm B and C infants restarted ART after cessation if they fulfilled clinical or immunological criteria. Clinical criteria were paediatric stage III disease or advanced paediatric stage II disease, as defined in 2003 WHO guidelines [25]. Immunological criteria were confirmed CD4 20% or less (below 18 months of age), or 15% or less (above 18 months of age) [23]. If the CD4 cell count dropped below the age-related threshold, treatable or self-limiting illnesses were excluded, and CD4% was confirmed 4 weeks later. All infants received cotrimoxazole prophylaxis from 6 weeks to 1 year of age.
Infants were observed in a dedicated study clinic for monthly clinical review, CD4 cell count and viral load testing. Families had access to 24-h telephone advice and free treatment of illness. Clinic transport costs were provided. Support groups and maternal skills development workshops were organised. Before starting therapy, a home visit was undertaken and mothers completed two 'drug-readiness' training modules. Mothers were counselled about infant feeding based on best evidence available at the time of birth; women made an informed decision about breastfeeding or replacement feeding. Nursing staff supported mothers in their choice of feeding through ongoing education on days 1, 3 and 7 of life and at monthly clinic visits. Mothers with CD4 cell count less than 200 cells/μl received ART from the same clinic.
The study ART regimen was designed before South African Department of Health treatment guidelines were available. First-line ART was zidovudine, lamivudine, nelfinavir and nevirapine. A four-drug, three-class combination was chosen because of the uncertain implications of sd-NVP-exposure. Nelfinavir was chosen at that time as the protease inhibitor of choice because of availability, safety and proven efficacy (at 150 mg/kg/day) in infected neonates [26,27]. Nevirapine was discontinued when viral load was less than 50 copies/ml and the other three drugs continued. Infants with virological failure had genotypic resistance testing undertaken where possible, or started a second-line regimen based on likely resistance mutations. Most frequently, infants changed to didanosine, abacavir and lopinavir/ritonavir. If infants developed toxicity to a first-line drug, this was changed to a second-line drug.
Infants starting deferred ART (Arm A) were followed up for at least 6 months after reaching viral load less than 50 copies/ml, before transfer to a government clinic. Infants starting immediate ART (Arms B and C) were followed up until ART was restarted, and viral load was less than 50 copies/ml for at least 6 months, before transfer to a government clinic. Children starting abacavir-containing second-line regimens were transferred to a specialised tertiary ART clinic for follow-up. All children referred to the government system, and still traceable, were seen 6-monthly in the study clinic for clinical assessment, CD4 cell count and viral load.
Assessment of adherence
Verbal and measured adherence were assessed monthly by study nurses. 'Verbal' adherence was calculated as (number of prescribed doses - number of missed doses/number of prescribed doses) × 100%. 'Measured' adherence was calculated as (prescribed medication volume - returned medication volume/prescribed volume) × 100%. Discrepancies between medication returned and expected ART balance were addressed and adherence support provided. Mean measured and verbal adherence was calculated for the first year of ART, using data from all appointments.
Genotypic resistance testing
Pre-treatment plasma samples, from the first 3 months of life, were subjected to genotypic resistance testing at the National Institute for Communicable Diseases, Johannesburg, using an in-house assay certified by the Virology Quality Assessment Program (VQA) [28]. Genotypic resistance was defined as presence of mutations associated with impaired drug susceptibility or virological response, as specified by the Stanford University HIV Drug Resistance Database (http://hivdb.stanford.edu/).
Statistical methods
Comparisons between groups were made using Student's t test or the Mann-Whitney U test for quantitative variables, Fisher's exact test for qualitative variables, and log rank test for survival data. Other statistical tests used are indicated in the text. All statistical analyses were undertaken using GraphPad Prism Version 4.0a.
Ethical approval
The study was approved by the Biomedical Research Ethics Committee of the University of KwaZulu-Natal, Durban and the Institutional Review Board of the Massachusetts General Hospital, Boston, USA.
Results
MTCT rate and infant randomization
As previously reported [5], 719 HIV-infected mothers gave birth to 740 babies. Seventy-five were identified as HIV-infected by day 28 (transmission rate 10.3%; 69% intrauterine, 31% intrapartum/early postpartum). Twelve infants were ineligible, as previously described [4].
The remaining 63 infected infants were randomized to immediate (n = 43) or deferred ART (n = 20). In the immediate group, one infant withdrew, one was lost to follow-up, and one died before receiving ART. Of the 40 remaining infants, four died in infancy whilst on ART and 36 were followed up for 1 year or more after starting ART (Fig. 1). Of the 20 infants in the deferred group, three did not meet the criteria to start ART in infancy; 13 of the 17 meeting criteria in year 1 started ART in infancy; of the remaining four infants, three started ART in the second year of life, and one died in infancy (Fig. 1). The data presented here relate to the 49 infants who commenced ART in infancy and were followed up for 1 year or more. This report focuses on the initial virological response to therapy and does not include details of subsequent response to therapy in infants restarting treatment post-STI or after the period of early ART; this will form the basis of a separate report.
Demographics of HIV-infected mothers and infants
Data were available for the 60 mothers whose infants were randomized and started follow-up. Median age was 26 years (range 16-39); women mostly lived without their partner (44/60; 73%), had median two dependents (range 0-16) and no formal source of income (52/60; 87%). Housing conditions were frequently crowded and basic (Table 1). Twenty-eight out of 46 (61%) households were living on less than $1 per person per day, the World Bank definition of extreme poverty [29]. Median maternal CD4 cell count was 312 cells/μl (IQR 154-449). Thirteen mothers (22%) started ART postnatally.
Resistance mutations following single-dose nevirapine prophylaxis
All infants were exposed to sd-NVP: in 60 out of 63 (95%) cases maternal sd-NVP was taken; in 62 out of 63 (98%) cases administration of infant sd-NVP was documented. Pre-treatment plasma samples (median day 31; range 1-90), were available for 53 out of 63 (84%) infants. In two cases, a PCR product could not be amplified; resistance results were therefore available for 51 out of 63 (81%) infants. All infants were infected with HIV-1 subtype C. Genotypic NNRTI resistance mutations were found in 20 out of 51 (39%) infants. The most common mutation was Y181C in 10 out of 51 (20%) infants (Fig. 2a). Two participants had multiple NNRTI resistance mutations.
As described previously [21], viral load was significantly higher in mothers of infants with NNRTI resistance than in mothers of infants without resistance mutations (median 131 500 copies/ml as compared with 33 800 copies/ml; P = 0.024; Mann-Whitney test; Fig. 2b). There was no significant difference in CD4 cell count between mothers of infants with and without NNRTI resistance mutations (median 293 cells/μl as compared with 321 cells/μl respectively; P = 0.77; Mann-Whitney test; Fig. 2c). There was no difference in the frequency of genotypic NNRTI resistance mutations between intrauterine-infected and intrapartum-infected infants [13/33 (39%) intrauterine-infected as compared with seven out of 18 (39%) intrapartum-infected; P = 1.0; Fisher's Exact test].
Adherence to antiretroviral therapy
Data were available on 438 verbal adherence checks and 481 measured adherence checks undertaken on 49 infants during the first year of ART. Overall, mean verbal adherence was 99.3% (95% CI 99.0-99.7); mean measured adherence was 95.4% (95% CI 94.1-96.7). Although verbal adherence therefore marginally overestimated true adherence, there was a strong correlation between the two measures (r = 0.54 Pearson; P < 0.0001).
Timing and efficacy of ART in HIV-infected infants
In the deferred ART group, 17 out of 20 (85%) infants reached CD4 cell count below 20% during the first year of life, by median day 99 (range 44-291). Thirteen infants started ART, at median day 142 (range 81-227). Median time from reaching treatment criteria to starting ART was 41 days (range 1-106 days). Delay was commonly due to tuberculosis (TB) treatment, social problems or caregiver training. One year after ART, viral load was less than 400 copies/ml in 13/13 (100%) infants, and less than 50 copies/ml in 12 out of 13 (92%) infants.
In the immediate ART group, therapy was started on median day 28 (range 8-164) in intrauterine-infected infants, and median day 55 (range 36-90) in intrapartum-infected infants. One year post-ART, viral load was less than 400 copies/ml in 36/36 (100%) infants, and less than 50 copies/ml in 34 out of 36 (94%) infants.
First-line therapy in all but three (94%) infants was the four-drug ART combination (zidovudine, lamivudine, nelfinavir, nevirapine); in three infants nevirapine was omitted because of safety concerns (e.g. abnormal liver function) or discontinued because of toxicity. Ten infants (20%) started second-line ART within 1 year because of concurrent TB treatment (five out of 49; 10%) virological failure (four out of 49; 8%) or both (one out of 49; 2%). Three infants (6%) had single drug substitutions for zidovudine toxicity. Of the five infants with virological failure, only one had known baseline NNRTI resistance (K103N mutation); one infant had no resistance testing undertaken and three had no genotypic NNRTI mutations.
Taken together, 49 out of 49 (100%) infants suppressed viral load to less than 400 copies/ml and 46 out of 49 (94%) to less than 50 copies/ml within 1 year of starting ART (Fig. 3), despite high baseline viral load (median 952 000 copies/ml). Ten out of 49 (20%) infants needed a second-line regimen to achieve suppression, due to virological failure and/or TB treatment. On an intention-to-treat basis, therefore, 39 out of 49 (80%) infants achieved viral load less than 400 copies/ml on first-line ART. Median CD4 cell count was not significantly different between groups 12 months after ART (immediate group 33%; deferred group 32%; P = 0.70; Mann-Whitney test).
Factors impacting virological suppression
Median time to viral load less than 50 copies/ml was 121 days (range 15-308) in the immediate ART group and 115 days (range 76-204) in the deferred group. Time to viral load less than 50 copies/ml was not significantly different between groups (Fig. 4a; P = 0.39; log rank test).
Time to undetectability was inversely related to maternal CD4 cell count (r = -0.42; P = 0.005; Spearman; Fig. 4b) and directly related to maternal viral load (r = 0.32; P = 0.03; Spearman; data not shown). Time to undetectability was directly related to viral load at ART initiation (r = 0.64; P < 0.001; Spearman; Fig. 4c) and to peak viral load in infants (r = 0.52; P < 0.001; Spearman; data not shown). There was no difference in time to undetectability between infants with and without NNRTI mutations (P = 0.99, log rank test; Fig. 4d). There was no evidence for a strong effect of adherence on viral load suppression (hazard ratio 1.05 [CI 0.95-1.17] from a proportional hazards model).
Morbidity and mortality in HIV-infected infants
Mortality during infancy did not differ significantly between immediate and deferred ART groups. In the immediate group, five out of 43 (12%) children died in infancy; in the deferred treatment group one out of 20 (5%) died in infancy (P = 0.65). There was no significant difference in survival time between immediate and deferred groups by Kaplan-Meier analysis (data not shown; log rank test P = 0.15).
Infants in the immediate group who started ART by 2 months of age (n = 25) had fewer outpatient illness episodes compared with infants in the deferred treatment group (median seven as compared with 12 illness episodes per infant; P = 0.003, Mann-Whitney test) and were less likely to be admitted to hospital [four out of 25 (16%) immediate as compared with eight out of 19 (42%) deferred; P = 0.09; Fisher's Exact Test]. There was no significant difference in morbidity or mortality between breastfed and formula-fed infants (data not shown).
Discussion
ART has transformed the outcome of paediatric HIV in resource-rich countries [30], but in resource-limited countries treatment of children has lagged behind that of adults [31]. Where ART is available, programmes have tended to focus on older children [12-17] because of rapid disease progression in infants [32,33], difficulty of early HIV diagnosis [31], lack of appropriate drug formulations [22,31], and reluctance to start infants on long-term medication [31]. However, it is critical that infants are given access to ART, as MTCT rates remain high in many countries and untreated mortality exceeds 50% by 2 years of age [32,33]. This report focuses in detail on a small group of infants at the epicentre of the HIV pandemic and demonstrates that, despite high viral loads and baseline NNRTI resistance, excellent virological, immunological and clinical outcomes are achievable.
Despite challenging social and environmental conditions, good ART adherence is achievable in adults and children in sub-Saharan Africa [7,13,15]. However, no studies have specifically reported adherence in infants. Although there is no accepted gold standard, medication returns are frequently used as a measure of adherence [34]. Accepting the limitations of this methodology, infants' caregivers showed excellent adherence to ART, and there was a strong correlation between verbal and measured adherence. This suggests that verbal adherence checks, which are simple and quick to undertake, provide a useful estimate of true adherence. Although no strong effect of adherence on virological suppression was detected, the overall high levels of adherence limit the power of this study to determine the effect precisely.
A major concern where sd-NVP is used to reduce MTCT is NNRTI resistance, detected in 23-87% of infants, depending on the assay used [19-21]. In this study, 39% infants had baseline genotypic mutations. Previous studies report persistence of mutations beyond 12 months [20,21], and this is of concern when NNRTI-containing regimens are recommended as first-line therapy for infants [22]. In Botswana, virological failure occurred in 10 out of 15 sd-NVP-exposed infants treated with a three-drug, nevirapine-containing regimen, at median 8.5 months of age [9]. In this Durban study, infants received a protease inhibitor in addition to nevirapine, and therefore a direct comparison cannot be made to the Botswanan study. However, it is encouraging that successful virological suppression is achievable in infants despite high viral loads and high-frequency NNRTI resistance. A four-drug regimen may be more potent in infancy, as virological suppression can be difficult to achieve [35]. However, this may not be feasible in resource-limited settings, where simplified regimens are required. Further studies are needed to investigate optimal regimens following exposure to sd-NVP.
Treatment of HIV-infected infants has other challenges compared to older children: discordant responses to ART; lack of appropriate drug formulations; unpalatable liquid medication; uncertain pharmacokinetics; and reliance on caregivers [18]. It is, therefore, striking that 100% infants achieved virological suppression to less than 400 copies/ml, and 94% to less than 50 copies/ml, within 1 year of starting ART. This bears comparison with European and US studies, in which 20-70% infants achieved undetectable viral loads [35-38], although some infants in these studies presented with advanced disease. This outcome may not be so readily achieved outside a relatively well resourced study setting.
The fact that five infants (10%) switched to second-line regimens for virological failure during the first year of treatment, despite generally high levels of adherence is, however, notable. The difficulty in reducing high viral loads, and consequent risk of drug resistance, is well recognised in infants [36] and is of particular concern in resource-limited settings, where options for subsequent regimens are limited. Six infants had to undergo regimen changes to accommodate treatment for TB, which is a frequent coinfection in sub-Saharan Africa. Taking this into account, 80% infants achieved undetectable viral load (<400 copies/ml) on a first-line regimen. This result is still encouraging, although ART roll-out programmes will need to accommodate substantial numbers of children switching from first-line therapy in resource-limited settings.
The fact that six of the 63 (10%) enrolled infants died within a year, despite access to ART and free 24-h medical care, is also notable. One infant randomized to deferred ART died of gastroenteritis at 6 months of age. Although treatment criteria had been reached, ART had not yet been started at the time of death. Five infants randomized to immediate treatment died, but two patients were not on ART at the time of death. One infant died of septicaemia at 5 weeks of age, before ART had been started. One infant, who started ART on day 30, had a good initial virological response but defaulted therapy after 2 months following her mother's death; she was taken abroad by her family and died at 5 months, of probable pneumonia. Of the three infants on ART at the time of death, one died of acute gastroenteritis at 6 weeks of age, 10 days after starting ART; one infant died of septicaemia, complicated by disseminated intravascular coagulation, and hypernatraemia secondary to gastroenteritis at 3 months of age, 3 weeks after starting ART; and one died of probable sudden infant death syndrome at 5 months of age, although the family refused postmortem examination. Taken together, three out of six children were not on ART at the time of death; in the remaining three children, there was no evidence that mortality was related to ART. This high level of infant mortality is not unexpected, given high general infant mortality rates in resource-limited countries [5.9% in South Africa [(www.geographyiq.com/ranking/ranking_Infant_Mortality_Rate_aall.htm)], substantially higher mortality rates even in uninfected infants of HIV-infected mothers [39], and similar mortality rates reported from other ART roll-out programmes [10,11,13,15,16]. It is noteworthy that two-thirds of mothers reported undertaking predominant replacement feeding for their infants, following counselling based on best available evidence at the time of birth. More recent data have led to current WHO recommendations [40] that mothers of infants who are known to be infected should be strongly encouraged to continue breastfeeding to optimize infant health.
The difference in mortality between immediate and deferred treatment groups in this small study was not statistically significant. This is in contrast to the CHER (Children with HIV Early Antiretroviral Therapy) Study [41], in which South African infants, identified as HIV-infected before 12 weeks of age, and with CD4 cell counts of 25% or more, were randomized to immediate (n = 252) or deferred (n = 125) ART. Following interim review, enrolment to the deferred treatment arm was stopped because mortality was significantly higher than in infants starting immediate ART (16% as compared with 4%, respectively). Differences between groups in this current study, may therefore, have become apparent with a larger sample size; however, factors contributing to the wellbeing of infants in the deferred treatment group included frequent follow-up, 24-h access to clinic staff and free treatment of illness. There was a modest clinical difference between groups; infants starting therapy within 2 months of life had fewer inpatient and outpatient illness episodes. No significant difference was found in virological or immunological outcome after 1 year of immediate or deferred treatment. However, differences between immediate and deferred ART may not become apparent until later time points [27,42,43].
Currently, only 5-7% of people on ART worldwide are children, and paediatric HIV represents a public health emergency in its own right. Infants are relatively neglected in current treatment programmes, although they form a substantial proportion of those in need of ART. Although the obvious solution to the paediatric HIV crisis is prevention of MTCT, multiple barriers, including uncertain maternal HIV status [31], lack of access to perinatal interventions [3] and ongoing viral transmission through the balanced advantages of prolonged breastfeeding [40], mean that many infants will continue to become infected with HIV. This study provides the much-needed evidence that early treatment is successful in resource-limited settings. Although the question of optimal regimens and timing of treatment needs to be clarified, this should not delay expansion of infant ART provision so that the success achieved in resource-rich countries can be replicated in resource-limited countries globally.
Acknowledgements
Author contributions: Study concept and design (G.T.-W., B.D.W., P.G.); data acquisition and analysis (A.P., W.M., G.T.-W., M.R., V.P., C.T., N.M., L.M., P.G.); statistical analysis (A.P., N.M.); writing and revision of article (A.P., G.T.-W., N.M., L.M., B.D.W., P.G.); study supervision (A.P., W.M., G.T.-W., B.D.W., P.G.); obtained funding (B.D.W., P.G.).
Contributions from the HPP Study Group are gratefully acknowledged. Members of the HPP Study Group: Natasha Blanckenberg, Ayanda Cengimbo, Prakash Jeena, Shabashini Reddy, Zenele Mncube, Mary Vanderstok, Krista Dong and Hoosen Coovadia.
We thank the children in this study, their caregivers, and the clinic staff: Kesia Mgwenya, Thandi Cele, Thandi Sikhakhane, Maud Mbambo, Thandekile Phahla, Deli Sindane, Thobekile Sibaya, Nicky Linda and Pretty Siphengane.
The present study was supported by the Wellcome Trust (P.G.); Medical Research Council UK (A.P.); National Institutes of Health (P.G.; Grant code 2RO1-AI-46995), Secure the Future Initiative, Bristol Myers Squibb, Doris Duke Charitable Foundation, and the Mark and Lisa Schwartz Foundation. The resistance testing was funded through a CDC Co-operative Agreement (Grant no. U62/CCU022901-1). P.G. is an Elizabeth Glaser Pediatric AIDS Foundation Scientist. B.D.W. is a Doris Duke Distinguished Clinical Science Professor.
Conflicts of interest: None.
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