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Time to first positive HIV-1 DNA PCR may differ with antiretroviral regimen in infants infected with non-B subtype HIV-1

Balasubramanian, Rajia; Fowler, Mary Glennb; Dominguez, Kennethc; Lockman, Shahind,e,f; Tookey, Pat A.g; Huong, Nicole Ngo Giange,h; Nesheim, Stevenc; Hughes, Michael D.i; Lallemant, Marce,h; Tosswill, Jenniferj; Shaffer, Nathank; Sherman, Gaylel; Palumbo, Paulm; Shapiro, David E.n

doi: 10.1097/QAD.0000000000001640
CLINICAL SCIENCE
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SDC

Objective: To evaluate the association of type and timing of prophylactic maternal and infant antiretroviral regimen with time to first positive HIV-1 DNA PCR test, in nonbreastfed HIV-infected infants, from populations infected predominantly with HIV-1 non-B subtype virus.

Design: Analysis of combined data on nonbreastfed HIV-infected infants from prospective cohorts in Botswana, Thailand, and the United Kingdom (N = 405).

Methods: Parametric models appropriate for interval-censored outcomes estimated the time to first positive PCR according to maternal or infant antiretroviral regimen category and timing of maternal antiretroviral initiation, with adjustment for covariates.

Results: Maternal antiretroviral regimens included: no antiretrovirals (n = 138), single-nucleoside analog reverse transcriptase inhibitor (n = 165), single-dose nevirapine with zidovudine (n = 66), and combination prophylaxis with 3 or more antiretrovirals [combination antiretroviral therapy (cART), n = 36]. Type of maternal/infant antiretroviral regimen and timing of maternal antiretroviral initiation were each significantly associated with time to first positive PCR (multivariate P < 0.0001). The probability of a positive test with no antiretrovirals compared with the other regimen/timing groups was significantly lower at 1 day after birth, but did not differ significantly after age 14 days. In a subgroup of 143 infants testing negative at birth, infant cART was significantly associated with longer time to first positive test (multivariate P = 0.04).

Conclusion: Time to first positive HIV-1 DNA PCR in HIV-1-infected nonbreastfed infants (non-B HIV subtype) may differ according to maternal/infant antiretroviral regimen and may be longer with infant cART, which may have implications for scheduling infant HIV PCR-diagnostic testing and confirming final infant HIV status.

Supplemental Digital Content is available in the text

aDepartment of Biostatistics and Epidemiology, University of Massachusetts-Amherst, Amherst, Massachusetts

bDepartment of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland

cDivision of HIV/AIDS Prevention, National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention, Centers for Disease Control and Prevention, Atlanta, Georgia

dDivision of Infectious Diseases, Department of Medicine, Brigham and Women's Hospital, Boston

eDepartment of Immunology and Infectious Diseases, Harvard University, T. H. Chan School of Public Health, Boston, Massachusetts, USA

fBotswana Harvard AIDS Institute Partnership, Gaborone, Botswana

gUniversity College Institute of Child Health, London, UK

hInstitut de recherche pour le développement (IRD) UMI 174-PHPT, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand

iDepartment of Biostatistics, Harvard University T. H. Chan School of Public Health, Boston, Massachusetts, USA

jVirus Reference Department, National Infection Service, Public Health England, London, UK

kDepartment of HIV/AIDS, World Health Organization, Geneva, Switzerland

lDepartment of Paediatrics and Child Health, Faculty of Health Sciences, University of the Witwatersrand and National Institute for Communicable Diseases, Johannesburg, South Africa

mSection of Infectious Diseases and International Health, Geisel School of Medicine at Dartmouth, 1 Medical Center Dr, Lebanon, New Hampshire

nCenter for Biostatistics in AIDS Research, Harvard University T. H. Chan School of Public Health. Boston, Massachusetts, USA.

Correspondence to David E. Shapiro, Center for Biostatistics in AIDS Research, Harvard University T. H. Chan School of Public Health, 651 Huntington Avenue, Boston, MA 02115, USA. Tel: +1 617 432 2426; fax: +1 617 432 3163; e-mail: shapiro@sdac.harvard.edu

Received 25 January, 2017

Revised 4 August, 2017

Accepted 3 September, 2017

Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Website (http://www.AIDSonline.com).

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Introduction

To assess HIV infection in infants, serologic tests are only reliable when performed beyond 15–18 months of age because infants can carry maternal antibodies for more than a year after birth. In contrast, virologic diagnostic tests that detect the presence of HIV can be used at earlier ages. These tests include viral culture, viral antigen (p24), proviral DNA using PCR, and HIV DNA/RNA amplification and detection [1]. HIV-1 DNA or RNA amplification assays are recommended for diagnosis of HIV in infants under 15 months of age. Therefore, knowledge of the performance of these assays is essential to inform HIV diagnosis guidelines.

Previous investigations have evaluated the performance characteristics of these virologic assays for the early diagnosis of HIV infection in various individual cohorts. [2–17]. PCR assays typically achieve high specificity, and thus a positive test result is indicative of HIV infection with high probability. However, the sensitivity of PCR assays in newborns is lower in the first few weeks of life and increases thereafter. This is likely related to intrapartum transmission, which is not being detected in the newborn sample. For example, in a population of infants infected with subtype B HIV-1, Dunn et al.[18] estimate that 38% [95% confidence interval (CI) 29%, 46%] of all perinatally infected infants test positive within a day after birth. By 14 days after birth, 93% (95% CI 76%, 97%) test positive. This heterogeneity in the timing of first positive DNA PCR likely reflects the varying timing of infection (in utero vs. intrapartum) and the sensitivity of early detection of intrapartum infections (for HIV exposure at labor/delivery, actual establishment of infection with viremia, and/or viral DNA integration has not yet occurred). It is also possible that the interplay of elements such as type of antiretroviral prophylaxis regimen, maternal or infant host-mediated factors that may suppress viral replication at delivery, or the sensitivity of different diagnostic assays, may affect detection of virus in the newborn period.

No previous work provides an assessment of the association of combination antiretroviral prophylaxis regimen and/or infant prophylaxis with the time to positive signal for DNA assays. Current WHO and other guidelines for clinical management of HIV-infected pregnant women include use of combination antiretroviral regimens. [19–23] Conversely, current recommendations regarding the scheduling of diagnostic tests in HIV-exposed infants are based on studies conducted prior to the era of combination antiretroviral regimens [24,25]. These prophylactic regimens, being potent suppressors of viral replication, may delay the detection of HIV infection by virus-based assays in infants. Therefore, knowledge of the performance of these assays at different infant ages according to type of prophylaxis (particularly maternal and infant combination antiretroviral prophylaxis) is essential to inform HIV diagnosis guidelines.

Whereas several previous studies addressed the performance of HIV-1 DNA assays in HIV-infected infants infected with HIV-1 subtype B virus, relatively few studies have evaluated the performance of HIV-1 DNA assays in infants infected with non-B subtypes [1–3,5,10–15]. Non-B subtypes are prevalent in regions such as sub-Saharan Africa and Asia, which bear the major burden of mother-to-child transmission (MTCT) of HIV. Previous studies on mother–infant pairs infected with non-B subtype HIV-1 have been limited in sample size, ranging from 38 infants in a study conducted in South Africa [16], where subtype C HIV-1 infection is predominant, to 98 infected infants in a study conducted in Thailand [6], where subtype E HIV-1 or CRF01_AE is most prevalent.

Studying nonbreastfed infants provides particular insight regarding the time to positive signal associated with amplification assays, as there is no continuing HIV exposure after birth via breast milk, and timing of transmission is limited to in-utero and intrapartum infection. This study presents results from the analysis of combined data on HIV-infected, nonbreastfed infants and their HIV-infected mothers from prospective studies conducted in three countries in which non-B subtype virus is prevalent.

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Methods

Prospective studies of HIV-infected mothers and their nonbreastfed infants from Botswana, Thailand, and the United Kingdom were included. All these studies previously received corresponding ethics committee approvals. Because this study represents a secondary analysis of pooled, de-identified data across the aforementioned studies, the Human Subjects Research Protection offices of the lead institutions involved in the analysis (University of Massachusetts at Amherst and Harvard T. H. Chan School of Public Health) approved this study under a nonhuman-subjects-research determination. All studies provided individual data on all HIV-infected infants satisfying the following inclusion criteria: at least one HIV-1 DNA PCR result available within 3 months of birth; mothers diagnosed with HIV no later than 2 days following delivery; and infants that were replacement-fed (did not breastfeed).

The dataset included 405 HIV-infected, nonbreastfed infants with complete data on maternal and infant antiretroviral regimen. One infant with missing data regarding maternal antiretroviral regimen was excluded from the analysis. All available diagnostic DNA PCR test results for the 405 infants were included in the analysis. A brief description of each included study is provided below, with further details in the Supplemental Digital Content (http://links.lww.com/QAD/B158).

  1. Botswana – The Mashi Study: This 2 × 2 factorial randomized clinical trial enrolled in Botswana between 2001 and 2003 [26], including 91 DNA PCR test results from 32 HIV-infected infants born to mother–infant pairs randomized to the formula-feeding arm of the trial (subtype C HIV-1 infection is prevalent).
  2. Thailand – Bangkok Collaborative Perinatal HIV Transmission Study Group (CDC): Data from two perinatal studies [7,27,28], enrolled in Thailand during 1992–1998 including 370 DNA PCR test results on 122 HIV-infected nonbreastfed infants (subtype E HIV-1 infection is prevalent among heterosexual women).
  3. Thailand – Program for HIV Prevention and Treatment (PHPT): Data on 678 DNA PCR test results from 177 HIV-infected nonbreastfed infants from two studies conducted in Thailand between 1997 and 2003 were included in this analysis [29,30].
  4. United Kingdom – National Study of HIV in Pregnancy and Childhood/Health Protection Agency Collaboration (NSHPC): Data comprising 181 DNA PCR test results from 74 HIV-infected nonbreastfed infants collected during the period 2000–2009 were analyzed [31–33] (all mothers acquired HIV in countries with predominantly non-B subtype).
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Timing of maternal antiretroviral initiation

The timing of maternal antiretroviral initiation was categorized as follows: no antiretrovirals initiated during current pregnancy or at the time of labor/delivery (n = 138); antiretrovirals initiated during labor/delivery (n = 26); antiretrovirals initiated during trimester of delivery (n = 219); antiretrovirals initiated prior to the trimester of delivery (n = 22).

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Maternal antiretroviral regimen

Antiretroviral regimen given to the mother during the trimester closest to delivery and during labor/delivery was used and categorized as: noantiretroviral regimen (n = 138); singlenucleoside reverse transcriptase inhibitor (NRTI) (n = 165); single-dose nevirapine (sdNVP) with zidovudine (ZDV) (n = 66); and three or more antiretroviral regimens [combination antiretroviral therapy (cART)] (n = 36).

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Infant antiretroviral regimen

Infant prophylactic antiretroviral regimen was categorized as follows: noantiretrovirals (n = 143); single NRTI (n = 176); sdNVP with ZDV (n = 59); and three or more antiretrovirals (cART) (n = 27).

Details regarding the operating definition of HIV infection in infants and other variables are included in the Supplemental Digital Content (http://links.lww.com/QAD/B158). The times of DNA PCR tests did not appear to differ by maternal/infant antiretroviral regimen or by time to maternal antiretroviral initiation (see Figs. S1–S3 in the Supplemental Digital Content, http://links.lww.com/QAD/B158).

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Statistical methods

Time to first positive HIV-1 DNA PCR test among all nonbreastfed HIV-infected infants was estimated using parametric models appropriate for interval-censored outcomes. The models provide estimates of the probabilities of testing positive by HIV-1 DNA PCR among HIV-infected, nonbreastfed infants, by age of infant. Time to first positive HIV-1 DNA PCR was estimated according to maternal or infant antiretroviral regimen category and timing of maternal antiretroviral initiation. These variables could not be modeled jointly due to their high concordance (Table 1). Stratified Weibull models were fit to evaluate the association of each primary variable (maternal/infant antiretroviral regimen, timing of maternal antiretroviral initiation) with time to first positive HIV-1 DNA PCR [34]. Models were adjusted for other covariates including maternal CD4+ cell count and viral load closest to the time of delivery, mode of delivery, gestational age, and infant birth weight. Models could not be adjusted for country because the primary variables were each confounded with country (e.g. 94.4% of the patients in the maternal cART group were from the NSHPC study, and none were from the studies in Thailand; see Table S1 in the Supplemental Digital Content, http://links.lww.com/QAD/B158). As a sensitivity analysis, analyses were repeated for the subgroup of 299 HIV-infected infants from the studies in Thailand, to assess whether the results changed when analyses were restricted to one country. Further details on the analyses are included in the Supplemental Digital Content (http://links.lww.com/QAD/B158).

Table 1

Table 1

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Results

The dataset included 405 HIV-infected, nonbreastfed infants. Maternal antiretroviral regimen had a high degree of concordance with infant antiretroviral regimen (81%; Table 1). The timing of maternal antiretroviral initiation tended to be earlier in pregnancy for more complex regimens; 42% of women who received cART and 5% or less of women who initiated less complex antiretroviral regimens started prior to the trimester of delivery (Table 1). A similar relationship between infant antiretroviral regimen and timing of maternal antiretroviral initiation was observed (data not shown).

Maternal and infant characteristics according to maternal antiretroviral regimen category are shown in Table S1 of the Supplemental Digital Content (http://links.lww.com/QAD/B158). The maternal antiretroviral regimen groups differed significantly with respect to several characteristics: Women in the cART group were primarily from the NSHPC cohort and enrolled later, had lower CD4+ cell counts, lower viral loads, were more often diagnosed during pregnancy, and more often had subtype C or mixed subtype than women in the other antiretroviral regimen groups.

Although each of the primary variables (maternal/infant antiretrovirals, timing of antiretroviral initiation) was significantly associated with time to first positive HIV-1 DNA PCR (P < 0.0001), none of the following maternal and infant characteristics was significant in univariate models: CD4+ cell count closest to delivery (P = 0.24), viral load closest to delivery (P = 0.47), mode of delivery (P = 0.62), gestational age (P = 0.58), and birth weight (P = 0.78).

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Maternal antiretroviral regimen and time to first positive HIV-1 DNA PCR

Maternal antiretroviral regimen was significantly associated with time to first positive HIV-1 DNA PCR in a univariate model (P < 0.0001). The probability of a positive HIV-1 DNA PCR test at 1 day after birth (as opposed to later) was significantly lower in the HIV-infected babies in the maternal ‘no antiretroviral’ group (48%; 95% CI 38–59%) when compared to HIV-infected infants in the single NRTI group (85%; 95% CI 80–90%) and the sdNVP + ZDV group (76%; 95% CI 65–85%). The probability of a positive test at 1 day after birth in the maternal cART group was 66% (95% CI 49–81%). However, the probability of a positive test at or beyond 14 days of age did not differ significantly according to maternal antiretroviral regimen (Table 2, Fig. 1a). Whereas overall MTCT rates are lower in infants whose mothers received any antiretrovirals, our analyses were restricted to HIV-infected infants, so the higher probability of a positive test at 1 day after birth among HIV-infected infants whose mothers received any antiretrovirals compared with those whose mothers received no antiretrovirals may reflect the effect of antenatal and intrapartum treatment with antiretrovirals in reducing intrapartum transmissions; that is, among HIV-infected infants whose mothers received any antiretrovirals, most are in-utero infections (with positive DNA PCR at day 1 after birth) and relatively few are intrapartum infections (with negative DNA PCR at day 1 after birth), whereas among HIV-infected infants whose mothers received no antiretrovirals, both in-utero and intrapartum infections are frequent.

Table 2

Table 2

Fig. 1

Fig. 1

In a multivariable model, maternal antiretroviral regimen remained statistically significant after simultaneously adjusting for CD4+ cell count and viral load closest to delivery, mode of delivery, gestational age, and birth weight (P < 0.0001). Results were similar when restricted to data from the studies in Thailand (Supplemental Digital Content, Table S3, http://links.lww.com/QAD/B158).

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Infant antiretroviral regimen and time to first positive HIV-1 DNA PCR

Similar results were obtained from univariate models for the association of infant antiretroviral regimen and time to first positive HIV-1 DNA PCR (Table 2, Fig. 1b). In a multivariable model, infant antiretroviral regimen remained statistically significant after simultaneously adjusting for potential confounders listed above (P < 0.0001). Results were similar when restricted to data from the studies in Thailand (Supplemental Digital Content, Table S3, http://links.lww.com/QAD/B158)

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Timing of maternal antiretroviral initiation and time to first positive HIV DNA PCR

The timing of maternal antiretroviral initiation was significantly associated with time to first positive HIV-1 DNA PCR in a univariate model (P < 0.0001). The probability of a positive test at 1 day after birth was significantly lower in the ‘no antiretroviral’ group (48%; 95% CI 38–59%) when compared to infants whose mothers initiated an antiretroviral regimen during the trimester of delivery (82%; 95% CI 77–87%) or during labor/delivery (86%; 95% CI 72–95%), and did not differ significantly when compared with infants whose mothers initiated an antiretroviral regimen prior to the trimester of delivery (57%; 95% CI 37–78%). However, the probability of a positive test at or beyond 14 days of age did not differ significantly according to timing of antiretroviral initiation (Table 2, Fig. 1c). As noted above, the higher probability of a positive test at day 1 after birth among HIV-infected infants whose mothers started an antiretroviral regimen either during the antenatal period or during labor and delivery when compared with HIV-infected infants whose mothers received no antiretrovirals may reflect the effect of antenatal and intrapartum treatment with antiretrovirals in reducing intrapartum transmissions.

In a multivariable model, timing of maternal antiretroviral initiation remained statistically significant after simultaneously adjusting for CD4+ cell count and viral load closest to delivery, mode of delivery, gestational age, and birth weight (P < 0.0001). Results were similar when restricted to data from the studies in Thailand (Supplemental Digital Content, Table S3, http://links.lww.com/QAD/B158).

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Subgroup analysis of infants testing negative at birth

The association of timing and type of maternal/infant antiretroviral regimen with time to first positive DNA PCR may be clearer if the analysis is restricted to a homogeneous subgroup of infants who likely acquired HIV infection during the intrapartum period. In a subgroup analysis of 143 infants who tested negative by DNA PCR within 1 day following birth, maternal antiretroviral regimen type was significantly associated with time to first positive HIV-1 DNA PCR in a univariate model (P = 0.02; Fig. 2a). However, this association was no longer statistically significant in a multivariable model after adjusting for maternal CD4+ cell count and viral load closest to delivery, mode of delivery, gestational age, and birth weight (P = 0.09).

Fig. 2

Fig. 2

The association of infant antiretroviral regimen was statistically significant in a univariate model (P = 0.04; Fig. 2b), and remained significant in a multivariable model after adjusting for maternal CD4+ cell count and viral load closest to delivery, mode of delivery, gestational age, and birth weight (P = 0.04). By 28 days after birth, the probability of a positive test was 73% (95% CI 60–85%) in the ‘no antiretroviral’ group (n = 57), 66% (95% CI 53–77%) in the ‘single NRTI’ group (n = 68), 68% (95% CI 44–90%) in the sdNVP + ZDV group (n = 12), and 31% (95% CI 14–61%) in the cART group (n = 6) (Fig. 2b). At 60 days after birth, similar patterns across infant antiretroviral regimen categories were observed.

The timing of maternal antiretroviral regimen was not statistically significant in univariate and multivariate models (P > 0.48).

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Discussion

Maternal and infant antiretroviral regimen were both significantly associated with time to first positive HIV-1 DNA PCR, in multivariable models, after adjusting for potential confounders (P < 0.0001). The probability of a positive test at 1 day of age in the ‘no antiretroviral’ group was significantly lower when compared to each of the other antiretroviral groups. The timing of maternal antiretroviral initiation was also significantly associated with time to first positive HIV-1 DNA PCR even after adjustment for potential confounders (P < 0.0001). The probability of a positive test at 1 day of age was significantly lower among infants whose mothers received no antiretroviral, when compared to infants whose mothers initiated an antiretroviral regimen during the trimester of delivery or infants whose mothers initiated an antiretroviral regimen during the time of labor/delivery. However, these differences were not statistically significant when infants were tested beyond 2 weeks of age (Table 2). These findings may reflect the prophylactic effects of antiretrovirals in reducing the risk of intrapartum transmissions, resulting in a larger proportion of in-utero transmissions in antiretroviral-exposed infants when compared to infants not exposed to antiretrovirals [27]. An additional potential explanation for this finding could be a true increase in the sensitivity of the birth PCR for detecting in-utero infection, due to a longer period between transmission and testing (if transmission occurred prior to antiretroviral initiation). Whether and to what extent either of the above hypotheses is true cannot be determined by this analysis.

Our results for infants exposed to maternal single NRTI prophylaxis benefit from a larger sample size than in most other studies, for which small sample size might explain the variability in sensitivities found. In our study, the probability of a positive test at 1 day after birth was 85% (95% CI 80–88%) among infants whose mothers received single NRTI during pregnancy. Due to small sample sizes ranging from 11 to 24 infants, previous reports of DNA PCR test positivity rates at birth among infants exposed to single maternal NRTI in populations with subtype B infections have been variable, and ranged from 11 to 27% [3,15]. Our findings in the subgroup of infants unexposed to antiretrovirals are similar to those reported in the literature in similarly antiretroviral-unexposed infants, infected with subtype B HIV-1. In our study, the probabilities of a positive test by HIV-1 DNA PCR among infants unexposed to antiretrovirals were 48% (95% CI 38–59%) and 80% (95% CI 74–86%) within 1 and 14 days after birth, respectively. Among antiretroviral-unexposed infants infected with subtype B HIV-1, Dunn et al.[18]. reported 38% (95% CI 29–46%) and 93% (95% CI 76–97%) testing positive by DNA PCR at 1 and 14 days after birth, respectively. Other reports among antiretroviral-unexposed infants infected with subtype B HIV-1 concur with these estimates [5,11–14,17].

Smaller studies of the sensitivity of DNA PCR for early infant diagnosis of HIV infection have also been conducted in populations infected with non-B subtype virus [6,7,16,17,35]. Based on 65 infected infants born to HIV-positive mothers enrolled in the French multicenter prospective cohort, Burgard et al.[17] reported that the sensitivity of DNA PCR at birth, and 1 and 3 months were 55%, 89%, and 100%, respectively. The results reported in the study by Burgard et al.[17] are consistent with the results in the ‘no antiretroviral’ group in our study. However, contradictory to our findings, Burgard et al.[17] reported that neither the presence nor type of maternal/infant ART was significantly associated with time to first positive HIV-1 DNA PCR at birth and at 1 month. Previous reports from two small, prospective studies conducted in South Africa were consistent with the findings of our study. In a prospective cohort of 38 infected infants exposed to at least maternal ZDV prophylaxis, sdNVP and infant ZDV, the proportions testing positive by HIV-1 DNA PCR at birth and at 4 weeks were 68% (95% CI 53–81%) and 88% (95% CI 69–96%), respectively [16]. In another South African study of 58 infected, nonbreastfed infants exposed to sdNVP, Sherman et al.[35] reported that the proportion of HIV-infected infants testing positive by HIV-1 DNA PCR at 6 weeks of age was 99%. Data from two previously reported studies conducted in Thailand have been included in our analysis [6,7].

Our study evaluated the association between timing and type of maternal/infant antiretroviral regimen and time to first positive HIV-1 DNA PCR, in the subgroup of 143 HIV-infected nonbreastfed infants who tested DNA PCR-negative within 1 day of birth. In this subgroup of infants, all HIV transmissions are likely to have occurred during the intrapartum period. Both maternal and infant antiretroviral regimen had statistically significant associations with time to first positive HIV-1 DNA PCR in univariate models (P = 0.02 and 0.04); the association remained significant in multivariable models for infant antiretroviral regimens, but not maternal antiretroviral regimens (P = 0.09 and 0.04). In this subgroup, a longer time to detection of infection by HIV-1 DNA PCR was observed among infants who received cART when compared to infants who received either no antiretroviral or single NRTI. Although the sample size in the infant cART group is limited (n = 6), these results are consistent with the hypothesis that the lag time between infection and DNA PCR test positivity may be prolonged among infants exposed to highly potent combination antiretroviral regimens when compared to infants unexposed to antiretrovirals or those exposed to only monotherapy regimens. These observations may lend support to testing paradigms with more repeat and/or delayed repeat testing among HIV-exposed infants who test negative at birth and are exposed to potent antiretroviral regimens. Although our analyses were restricted to data from nonbreastfed infants so that all infants had a known time of cessation of exposure to maternal HIV-1 infection, these results equally apply to breastfed infants, suggesting that HIV-diagnostic testing scheduled immediately after the end of breastfeeding may be subject to an increased rate of false-negatives among infants exposed to cART when compared to infants exposed to less potent antiretroviral regimens. Larger studies of the effects of potent antiretroviral regimens on timing of DNA PCR test positivity are needed.

When infection with nonsubtype B or group O HIV is considered a possibility, it is recommended that both HIV DNA and RNA assays should be performed on infant samples for diagnostic purposes – preferably using DNA and RNA assays that can pick up non-clade B or 0 subtypes [21]. Whereas HIV DNA PCR assays are not as sensitive as HIV RNA assays at detecting nonsubtype B or group O HIV and may have underestimated such infections, HIV RNA assays may be less sensitive than HIV DNA PCR in detecting HIV in the presence of combination antiretroviral drugs [21]. HIV DNA PCR testing was the preferred HIV testing method technology among infants in this study. The most recent WHO infant HIV-diagnostic recommendations acknowledge that DNA PCR is currently the standard method for diagnosis of HIV infection in infants [36]. Therefore, although the universe of HIV-infected infants in this study may have been underestimated based on our testing methodology, among those who were found to be HIV-infected, HIV DNA PCR testing may have increased the likelihood of detection of HIV infection among infants on cART and the sensitivity of our overall analysis in comparing various perinatal prophylaxis regimens. Therefore, despite a lack of use of RNA testing in this study, we feel that this study provides pertinent results from the point of view of common practice and in more accurately estimating the effect of combination perinatal antiretroviral prophylaxis compared to that of noncombination antiretroviral prophylaxis or no antiretroviral prophylaxis.

Limitations of this analysis include the small number of cART-exposed infants, varying sample collection and handling procedures across studies, and different DNA PCR tests used. Although the analyses were adjusted for potential confounders, there might be residual confounding by study (and by HIV subtype). To address this issue, we repeated the analyses for the subgroup of infants from the studies in Thailand and observed similar trends as in the main analysis (see Tables S2 and S3 in the Supplemental Digital Content, http://links.lww.com/QAD/B158). Lastly, breastfeeding status was assessed by self-report; however, the possibility of unreported breastfeeding cannot be ruled out definitively.

Earlier testing for all HIV-exposed infants is clearly desirable, as it has been shown that early treatment of infected infants has a significant effect in reducing morbidity and mortality [37,38]. However, the (very) low rate of HIV transmission with potent antiretroviral prophylaxis might suggest that early testing has limited yield and so repeat testing of almost all infants would be needed [39]. A delayed or additional later repeat testing schedule might be especially important among infants exposed to cART regimens, due to the potent effects of cART regimens on suppressing viral replication. Alternate approaches involving the use of sensitive assays and larger amounts of DNA should increase diagnosis of HIV infection at birth [40]. As the sample size in the cART group was limited in this study, future work evaluating the effects of cART regimens on DNA PCR test positivity in a larger dataset is warranted. Studies among breastfed infants are also crucial in determining the optimal scheduling of testing to facilitate early infant diagnosis of HIV infection [41].

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Acknowledgements

We gratefully acknowledge the contributions of Professor Stephen Lagakos (Harvard School of Public Health, deceased), whose mentorship and support was invaluable in the conception and development of this study. We would also like to acknowledge the invaluable contribution of Janet Masters, NSHPC data manager and co-ordinator for many years, who was a member of the Collaborative Working Group for this analysis; Janet died in December 2012 (tribute at www.nshpc.ucl.ac.uk/nshpc/jmasters).

Author contributions: All authors contributed substantially to the conception, design, and data preparation for this collaborative analysis of pre-existing data. R.B., M.D.H., and D.E.S. developed the statistical analysis plan and collaborated on the statistical analyses. R.B. and D.E.S. wrote the first draft of the manuscript and all authors participated in the review and editing of the final manuscript.

NSHPC: National surveillance of obstetric and paediatric HIV in the UK is undertaken through the National Study of HIV in Pregnancy and Childhood (NSHPC) in collaboration with Public Health England, and Health Protection Scotland. The contribution of the midwives, obstetricians, genitourinary physicians, pediatricians, clinical nurse specialists, and all other colleagues who report to the NSHPC through the British Paediatric Surveillance Unit of the Royal College of Paediatrics and Child Health, and the obstetric reporting scheme originally established under the auspices of the Royal College of Obstetricians and Gynaecologists, is essential and gratefully acknowledged. The National Study of HIV in Pregnancy and Childhood has Research Ethics Committee approval (Ref: MREC/04/2/009).

The Mashi Study: We thank the following individuals who were instrumental in the design and conduct of the Mashi study: Ibou Thior, MD, MSc; Laura M Smeaton, MSc; Roger L Shapiro, MD, MPH; Carolyn Wester, MD; Lisa Stevens, MD; Trevor Peter, PhD, MPH; Erik van Widenfelt, BSc; Joseph Makhema, MBChB; Kenneth McIntosh, MD; Vladimir Novitsky, MD, PhD; Stephen Lagakos, PhD; Max Essex, PhD

Bangkok Collaborative Perinatal HIV Transmission Study Group (CDC) studies: We thank the following individuals for their contributions to the original studies: Dr Tawee Chotpitayasunondh, Dr Anuvat Roongpisuthipong, Dr R.J. Simonds, and Mary Culnane; and Dr Michelle S. McConnell for her contributions to data extraction and interpretation for this multicohort analysis.

Disclaimer: The opinions expressed by authors contributing to this article do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated.

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Conflicts of interest

R.B., M.G.F., K.D., and Dr Shapiro were supported by a grant (R21 HD072792) from the National Institutes of Health, USA. S.L. was supported by funding by the National Institutes of Health R01 HD37793. P.A.T. received a grant (GHP/003/013/003) from the Health Protection Agency, UK and from the UK National Screening Programme; the NSHPC is managed within the Population, Policy & Practice Programme at the Institute of Child Health (ICH), University College London (UCL), which previously benefited from funding support from the Medical Research Council (MRC) in its capacity as the MRC Centre of Epidemiology for Child Health (grant number G0400546); the UCL ICH receives a proportion of funding from the UK Department of Health's National Institute for Health Research Biomedical Research Centres funding scheme. P.A.T. has also received grants from Public Health England, the UK National Screening Programme, the PENTA Foundation, AbbVie Inc, and IATEC/Kendle; she is a member of the Writing Group for the British HIV Association's Guidelines for the Management of HIV Infection in Pregnant Women. N.G.G.H. and M.L. were supported by grants from The National Institutes of Health, USA (R01 HD 33326; R01 HD 39615). J.T. is a member of the Writing Group for the British HIV Association's Guidelines for the Management of HIV Infection in Pregnant Women. P.P. has served as a consultant to Merck Pharmaceuticals and serves on DSMBs for Gilead and ApoPharma. All other authors report no conflicts of interest or sources of funding.

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* Present address: Nathan Shaffer, MD, Consultant, Decatur, GA 30030, USA.

Keywords:

DNA PCR assays; early infant diagnosis of HIV; mother-to-child transmission of HIV

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