Impact of antiretroviral therapy during acute or early HIV infection on virologic and immunologic outcomes: results from a multinational clinical trial.

OBJECTIVE: To assess how antiretroviral therapy (ART) initiation during acute or early HIV infection (AEHI) affects the viral reservoir and host immune responses. DESIGN: Single-arm trial of ART initiation during AEHI at 30 sites in the Americas, Africa, and Asia. METHODS: HIV DNA was measured at week 48 of ART in 5 million CD4+ T cells by sensitive qPCR assays targeting HIV gag and pol. Peripheral blood mononuclear cells were stimulated with potential HIV T cell epitope peptide pools consisting of env, gag, nef, and pol peptides and stained for expression of CD3, CD4, CD8, and intracellular cytokines/chemokines. RESULTS: From 2017 to 2019, 188 participants initiated ART during Fiebig stages I (n = 6), II (n = 43), III (n = 56), IV (n = 23), and V (n = 60). Median age was 27 years (interquartile range 23-38), 27 (14%) participants were female, and 180 (97%) cisgender. Among 154 virally suppressed participants at week 48, 100% had detectable HIV gag or pol DNA. Participants treated during Fiebig I had the lowest HIV DNA levels (P < 0.001). Week 48 HIV DNA mostly did not correlate with concurrent CD4+ or CD8+ T cell HIV-specific immune responses (rho range -0.11 to +0.19, all P > 0.025). At week 48, the magnitude, but not polyfunctionality, of HIV-specific T cell responses was moderately reduced among participants who initiated ART earliest. CONCLUSION: Earlier ART initiation during AEHI reduced but did not eliminate the persistence of HIV-infected cells in blood. These findings explain the rapid viral rebound observed after ART cessation in early-treated individuals with undetectable HIV DNA by less sensitive methods.

Recommendations on data sharing in HIV drug resistance research.

• Human immunodeficiency virus (HIV) drug resistance has implications for antiretroviral treatment strategies and for containing the HIV pandemic because the development of HIV drug resistance leads to the requirement for antiretroviral drugs that may be less effective, less well-tolerated, and more expensive than those used in first-line regimens. • HIV drug resistance studies are designed to determine which HIV mutations are selected by antiretroviral drugs and, in turn, how these mutations affect antiretroviral drug susceptibility and response to future antiretroviral treatment regimens. • Such studies collectively form a vital knowledge base essential for monitoring global HIV drug resistance trends, interpreting HIV genotypic tests, and updating HIV treatment guidelines. • Although HIV drug resistance data are collected in many studies, such data are often not publicly shared, prompting the need to recommend best practices to encourage and standardize HIV drug resistance data sharing. • In contrast to other viruses, sharing HIV sequences from phylogenetic studies of transmission dynamics requires additional precautions as HIV transmission is criminalized in many countries and regions. • Our recommendations are designed to ensure that the data that contribute to HIV drug resistance knowledge will be available without undue hardship to those publishing HIV drug resistance studies and without risk to people living with HIV.

The largest HIV-1-infected T cell clones in children on long-term combination antiretroviral therapy contain solo LTRs.

Combination antiretroviral therapy (cART) suppresses viral replication but does not cure HIV infection because a reservoir of infectious (intact) HIV proviruses persists in long-lived CD4+T cells. However, a large majority (>95%) of HIV-infected cells that persist on effective cART carry defective (non-infectious) proviruses. Defective proviruses consisting of only a single LTR (solo long terminal repeat) are commonly found as endogenous retroviruses in many animal species, but the frequency of solo-LTR HIV proviruses has not been well defined. Here we show that, in five pediatric donors whose viremia was suppressed on cART for at least 5 years, the proviruses in the nine largest clones of HIV-infected cells were solo LTRs. The sizes of five of these clones were assayed longitudinally by integration site-specific quantitative PCR. Minor waxing and waning of the clones was observed, suggesting that these clones are generally stable over time. Our findings show that solo LTRs comprise a large fraction of the proviruses in infected cell clones that persist in children on long-term cART. IMPORTANCE This work highlights that severely deleted HIV-1 proviruses comprise a significant proportion of the proviral landscape and are often overlooked.

Advancing HIV Drug Resistance Technologies and Strategies: Insights from South Africa's Experience and Future Directions for Resource-Limited Settings.

Monitoring of HIV drug resistance (HIVDR) remains critical for ensuring countries attain and sustain the global goals for ending HIV as a public health threat by 2030. On an individual patient level, drug resistance results assist in ensuring unnecessary treatment switches are avoided and subsequent regimens are tailored on a case-by-case basis, should resistance be detected. Although there is a disparity in access to HIVDR testing in high-income countries compared to low- and middle-income countries (LMICS), more LMICs have now included HIVDR testing for individual patient management in some groups of patients. In this review, we describe different strategies for surveillance as well as where HIVDR testing can be implemented for individual patient management. In addition, we briefly review available technologies for HIVDR testing in LMICs, including Sanger sequencing, next-generation sequencing, and some point-of-care options. Finally, we describe how South Africa has implemented HIVDR testing in the public sector.

HIV drug resistance in various body compartments.

PURPOSE OF REVIEW: HIV drug resistance testing using blood plasma or dried blood spots forms part of international guidelines. However, as the clinical utility of assessing drug resistance in other body compartments is less well established, we review this for blood cells and samples from other body compartments. RECENT EVIDENCE: Although clinical benefit is not clear, drug resistance testing in blood cells is often performed when patients with suppressed plasma viral loads require a treatment substitution. In patients with HIV neurocognitive disease, cerebral spinal fluid (CSF) drug resistance is rarely discordant with plasma but has nevertheless been used to guide antiretroviral drug substitutions. Cases with HIV drug resistance in genital fluids have been documented but this does not appear to indicate transmission risk when blood plasma viral loads are suppressed. SUMMARY: Drug-resistant variants, which may be selected in tissues under conditions of variable adherence and drug penetration, appear to disseminate quickly, and become detectable in blood. This may explain why drug resistance discordance between plasma and these compartments is rarely found. Partial compartmentalization of HIV populations is well established for the CSF and the genital tract but other than blood plasma, evidence is lacking to support drug resistance testing in body compartments.

NanoHIV: A Bioinformatics Pipeline for Producing Accurate, Near Full-Length HIV Proviral Genomes Sequenced Using the Oxford Nanopore Technology.

HIV-1 proviral single-genome sequencing by limiting-dilution polymerase chain reaction (PCR) amplification is important for differentiating the sequence-intact from defective proviruses that persist during antiretroviral therapy (ART). Intact proviruses may rebound if ART is interrupted and are the barrier to an HIV cure. Oxford Nanopore Technologies (ONT) sequencing offers a promising, cost-effective approach to the sequencing of long amplicons such as near full-length HIV-1 proviruses, but the high diversity of HIV-1 and the ONT sequencing error render analysis of the generated data difficult. NanoHIV is a new tool that uses an iterative consensus generation approach to construct accurate, near full-length HIV-1 proviral single-genome sequences from ONT data. To validate the approach, single-genome sequences generated using NanoHIV consensus building were compared to Illumina® consensus building of the same nine single-genome near full-length amplicons and an average agreement of 99.4% was found between the two sequencing approaches.

HIV-1 Persistence in Children during Suppressive ART.

There is a growing number of perinatally HIV-1-infected children worldwide who must maintain life-long ART. In early life, HIV-1 infection is established in an immunologically inexperienced environment in which maternal ART and immune dynamics during pregnancy play a role in reservoir establishment. Children that initiated early antiretroviral therapy (ART) and maintained long-term suppression of viremia have smaller and less diverse HIV reservoirs than adults, although their proviral landscape during ART is reported to be similar to that of adults. The ability of these early infected cells to persist long-term through clonal expansion poses a major barrier to finding a cure. Furthermore, the effects of life-long HIV persistence and ART are yet to be understood, but growing evidence suggests that these individuals are at an increased risk for developing non-AIDS-related comorbidities, which underscores the need for an HIV cure.

Viral suppression is associated with HIV-antibody level and HIV-1 DNA detectability in early treated children at 2 years of age.

OBJECTIVES: Early infant HIV diagnosis and antiretroviral therapy (ART) initiation are now implemented shortly after birth. Maintaining and monitoring ART adherence is difficult and requires frequent visits. We, therefore, investigated whether HIV antibodies and HIV-1 DNA levels are markers of cumulative viremia. DESIGN: We conducted a cross sectional investigation at 2 years of age of HIV antibodies and HIV-1 DNA levels in a well characterized cohort of 31 children who started ART shortly after birth. METHODS: HIV antibodies were measured by a combination of the Abbott ARCHITECT HIV Ag/Ab Combo and Geenius HIV 1/2 supplemental assays; and total HIV-1 DNA quantified using a sensitive quantitative PCR (qPCR) assay targeting the HIV-1 integrase gene. RESULTS: Infant post-exposure prophylaxis consisted of zidovudine (AZT) and nevirapine (NPV) (or NVP only, in one child) within 1 day of birth, transitioning, after positive diagnosis, to three-drug ART, at a median [interquartile range (IQR)] of 7 (4-9.5) days. Twelve of 31 children had well suppressed HIV plasma viral loads (HIVVL) and the remainder periods of viremia (HIVVL > 100 copies/ml after 3 months of ART), classified as non-suppressed. At 24 months of age: 11 of 12 (92%) of well suppressed children had undetectable HIV-1 antibodies versus 3 of 19 (16%) non-suppressed children (P < 0.001) and 7 of 12 (58%) well suppressed children had undetectable HIV-1 DNA versus 3 of 19 (16%) non-suppressed children (P = 0.02). CONCLUSION: Considering low assay costs and the high proportion of well suppressed children with undetected antibody levels at 2 years, HIV antibody levels may be a valuable marker of cumulative adherence in children who start treatment shortly after birth and could prompt adherence and viral load investigation.

HIVIntact: a python-based tool for HIV-1 genome intactness inference.

The characterisation of the HIV-1 reservoir, which consists of replication-competent integrated proviruses that persist on antiretroviral therapy (ART), is made difficult by the rarity of intact proviruses relative to those that are defective. While the only conclusive test for the replication-competence of HIV-1 proviruses is carried out in cell culture, genetic characterization of genomes by near full-length (NFL) PCR and sequencing can be used to determine whether particular proviruses have insertions, deletions, or substitutions that render them defective. Proviruses that are not excluded by having such defects can be classified as genetically intact and, possibly, replication competent. Identifying and quantifying proviruses that are potentially replication-competent is important for the development of strategies towards a functional cure. However, to date, there are no programs that can be incorporated into deep-sequencing pipelines for the automated characterization and annotation of HIV genomes. Existing programs that perform this work require manual intervention, cannot be widely installed, and do not have easily adjustable settings. Here, we present HIVIntact, a python-based software tool that characterises genomic defects in NFL HIV-1 sequences, allowing putative intact genomes to be identified in-silico. Unlike other applications that assess the genetic intactness of HIV genomes, this tool can be incorporated into existing sequence-analysis pipelines and applied to large next-generation sequencing datasets.

Early Emergence and Long-Term Persistence of HIV-Infected T-Cell Clones in Children.

Little is known about the emergence and persistence of human immunodeficiency virus (HIV)-infected T-cell clones in perinatally infected children. We analyzed peripheral blood mononuclear cells (PBMCs) for clonal expansion in 11 children who initiated antiretroviral therapy (ART) between 1.8 and 17.4 months of age and with viremia suppressed for 6 to 9 years. We obtained 8,662 HIV type 1 (HIV-1) integration sites from pre-ART samples and 1,861 sites from on-ART samples. Expanded clones of infected cells were detected pre-ART in 10/11 children. In 8 children, infected cell clones detected pre-ART persisted for 6 to 9 years on ART. A comparison of integration sites in the samples obtained on ART with healthy donor PBMCs infected ex vivo showed selection for cells with proviruses integrated in BACH2 and STAT5B Our analyses indicate that, despite marked differences in T-cell composition and dynamics between children and adults, HIV-infected cell clones are established early in children, persist for up to 9 years on ART, and can be driven by proviral integration in proto-oncogenes.IMPORTANCE HIV-1 integrates its genome into the DNA of host cells. Consequently, HIV-1 genomes are copied with the host cell DNA during cellular division. Pediatric immune systems differ significantly from adults, consisting primarily of naive T cells, which have low expression of the HIV-1 coreceptor CCR5. This difference may result in variances in the number or size of infected cell clones that persist in children on ART. Here, we provide the most extensive analysis of the integration landscape of HIV-1 in children. We found that, despite the largely naive cell populations in neonatal immune systems, patterns of HIV-1 integration and the size of infected cell clones are as large and widespread as those in adults. Furthermore, selection for integration events in proto-oncogenes were observed in children despite early ART. If such cell clones persist for the life span of these individuals, there may be long-term consequences that have yet to be realized.

HIV false positive screening serology due to sample contamination reduced by a dedicated sample and platform in a high prevalence environment.

Automated testing of HIV serology on clinical chemistry analysers has become common. High sample throughput, high HIV prevalence and instrument design could all contribute to sample cross-contamination by microscopic droplet carry-over from seropositive samples to seronegative samples resulting in false positive low-reactive results. Following installation of an automated shared platform at our public health laboratory, we noted an increase in low reactive and false positive results. Subsequently, we investigated HIV serology screening test results for a period of 21 months. Of 485 initially low positive or equivocal samples 411 (85%) tested negative when retested using an independently collected sample. As creatinine is commonly requested with HIV screening, we used it as a proxy for concomitant clinical chemistry testing, indicating that a sample had likely been tested on a shared high-throughput instrument. The contamination risk was stratified between samples passing the clinical chemistry module first versus samples bypassing it. The odds ratio for a false positive HIV serology result was 4.1 (95% CI: 1.69-9.97) when creatinine level was determined first, versus not, on the same sample, suggesting contamination on the chemistry analyser. We subsequently issued a notice to obtain dedicated samples for HIV serology and added a suffix to the specimen identifier which restricted testing to a dedicated instrument. Low positive and false positive rates were determined before and after these interventions. Based on measured rates in low positive samples we estimate that before the intervention, of 44 117 HIV screening serology samples, 753 (1.71%) were false positive, declining to 48 of 7 072 samples (0.68%) post-intervention (p<0.01). Our findings showed that automated high throughput shared diagnostic platforms are at risk of generating false-positive HIV test results, due to sample contamination and that measures are required to address this. Restricting HIV serology samples to a dedicated platform resolved this problem.

Novel Criteria for Diagnosing Acute and Early Human Immunodeficiency Virus Infection in a Multinational Study of Early Antiretroviral Therapy Initiation.

BACKGROUND: Antiretroviral therapy (ART) initiation during acute and early human immunodeficiency virus infection (AEHI) limits HIV reservoir formation and may facilitate post-ART control but is logistically challenging. We evaluated the performance of AEHI diagnostic criteria from a prospective study of early ART initiation. METHODS: AIDS Clinical Trials Group A 5354 enrolled adults at 30 sites in the Americas, Africa, and Asia who met any 1 of 6 criteria based on combinations of results of HIV RNA, HIV antibody, Western blot or Geenius assay, and/or the signal-to-cutoff (S/CO) ratio of the ARCHITECT HIV Ag/Ab Combo or GS HIV Combo Ag/Ab EIA. HIV status and Fiebig stage were confirmed by centralized testing. RESULTS: From 2017 through 2019, 195 participants were enrolled with median age of 27 years (interquartile range, 23-39). Thirty (15.4%) were female. ART was started by 171 (87.7%) on the day of enrollment and 24 (12.3%) the next day. AEHI was confirmed in 188 (96.4%) participants after centralized testing, 4 (2.0%) participants were found to have chronic infection, and 3 (1.5%) found not to have HIV discontinued ART and were withdrawn. Retrospectively, a nonreactive or indeterminate HIV antibody on the Geenius assay combined with ARCHITECT S/CO ≥10 correctly identified 99 of 122 (81.2%) Fiebig II-IV AEHI cases with no false-positive results. CONCLUSIONS: Novel AEHI criteria that incorporate ARCHITECT S/CO facilitated rapid and efficient ART initiation without waiting for an HIV RNA result. These criteria may facilitate AEHI diagnosis, staging, and immediate ART initiation in future research studies and clinical practice. CLINICAL TRIALS REGISTRATION: NCT02859558.

Pooled testing: A tool to increase efficiency of infant HIV diagnosis and virological monitoring.

BACKGROUND: Pooled testing, or pooling, has been used for decades to efficiently diagnose relatively rare conditions, such as infection in blood donors. Programmes for the prevention of mother-to-child transmission of HIV and for antiretroviral therapy (ART) are being rolled out in much of Africa and are largely successful. This increases the need for early infant diagnosis (EID) of HIV using qualitative nucleic acid testing and for virological monitoring of patients on ART using viral load testing. While numbers of patients needing testing are increasing, infant HIV infections and ART failures are becoming rarer, opening an opportunity for pooled testing approaches. AIM: This review highlights the need for universal EID and viral load coverage as well as the challenges faced. We introduce the concept of pooled testing and highlight some important considerations before giving an overview of studies exploring pooled testing for EID and virological monitoring. RESULTS: For ART monitoring, pooling has been shown to be accurate and efficient; for EID it has not been tried although modelling shows it to be promising. The final part attempts to place pooling into the context of current mother-to-child transmission of HIV and ART programmes and their expected trajectories over the next years. CONCLUSION: Several points warrant consideration: pre-selection to exclude samples with an elevated pre-test probability of positivity from pooled testing, the use of dried blood or plasma spots, and choosing a pooling strategy that is both practically feasible and economical. Finally, novel ideas are suggested to make pooling even more attractive.

Seroprevalence of Toxoplasma gondii infection among pregnant women in Windhoek, Namibia, in 2016.

BACKGROUND: When a pregnant woman contracts Toxoplasma gondii (T. gondii) infection during pregnancy, it may be vertically transmitted to the foetus. Information on the incidence of congenital toxoplasmosis (CT) in developing countries is scarce. Most studies focus on the seroprevalence of T. gondii infection among pregnant women. This study aimed to determine the seroprevalence of T. gondii infection among pregnant women attending public antenatal care in Windhoek, Namibia, in 2016. METHODS: In this descriptive study, 344 urban pregnant women attending public antenatal care were voluntarily enrolled in the study. Seroprevalence of anti-T. gondii Immunoglobulin G (IgG) was determined by automated immunoassay. Samples with a positive T. gondii IgG result were tested for T. gondii Immunoglobulin M (IgM) and specific IgG avidity by using an enzyme-linked immunosorbent assay (ELISA) test. A questionnaire captured demographic data and exposure to risk factors. Data were analysed using Statistical Package for the Social Sciences (SPSS) and R. RESULTS: Anti-T. gondii IgG was found in nine (2.61%) pregnant women. There was no association of anti-T. gondii IgG with demographic characteristics or exposure to risk factors.Anti-T. gondii IgM was positive in one (0.3%) woman, while three (0.9%) women had borderline anti-T. gondii IgM results. Specific IgG avidity was low, equivocal and high in 0%, 33% and 67% of seropositive pregnant women, respectively. CONCLUSION: Seroprevalence of anti-T. gondii IgG is much lower in Namibia than is reported in other developing countries. Investigation into specific IgM seropositivity and IgG avidity showed that pregnant women in the central region of Namibia are at low risk of vertical transmission and development of CT.

Intrapartum human immunodeficiency virus transmission rate in a central hospital in the Western Cape province after universal antiretroviral therapy roll-out.

The national human immunodeficiency virus (HIV) mother-to-child transmission rate at 6-10 weeks post-partum was 0.9% in 2016. There is a paucity of data about the intrapartum transmission rate after lifelong antiretroviral therapy was implemented in 2015. We assessed all pregnant women living with HIV who delivered at Tygerberg Hospital in 2017. Positive polymerase chain reactions (PCRs) at birth indicated an in utero transmission rate of 0.8%. One infant with a negative PCR at birth tested positive at 6-10 weeks. The intrapartum transmission rate was low (0.08%). About 25% of infants were lost to follow-up after birth.

Intact HIV Proviruses Persist in Children Seven to Nine Years after Initiation of Antiretroviral Therapy in the First Year of Life.

In adults starting antiretroviral therapy (ART) during acute infection, 2% of proviruses that persist on ART are genetically intact by sequence analysis. In contrast, a recent report in children treated early failed to detect sequence-intact proviruses. In another cohort of children treated early, we sought to detect and characterize proviral sequences after 6 to 9 years on suppressive ART. Peripheral blood mononuclear cells (PBMC) from perinatally infected children from the Children with HIV Early antiRetroviral (CHER) study were analyzed. Nearly full-length proviral amplification and sequencing (NFL-PAS) were performed at one time point after 6 to 9 years on ART. Amplicons with large internal deletions were excluded (<9 kb). All amplicons of ≥9 kb were sequenced and analyzed through a bioinformatic pipeline to detect indels, frameshifts, or hypermutations that would render them defective. In eight children who started ART at a median age of 5.4 months (range, 2.0 to 11.1 months), 733 single NFL-PAS amplicons were generated. Of these, 534 (72.9%) had large internal deletions, 174 (23.7%) had hypermutations, 15 (1.4%) had small internal deletions, 3 (1.0%) had deletions in the packaging signal/major splice donor site, and 7 (1.0%) were sequence intact. These 7 intact sequences were from three children who initiated ART after 2.3 months of age, one of whom had two identical intact sequences, suggestive of a cell clone harboring a replication-competent provirus. No intact proviruses were detected in four children who initiated ART before 2.3 months of age. Rare, intact proviruses can be detected in children who initiate ART after 2.3 months of age and are probably, as in adults, maintained by clonal expansion of cells infected before ART initiation.IMPORTANCE There are limited data about the proviral landscape in children exhibiting long-term suppression after early treatment, particularly in Sub-Saharan Africa where HIV-1 subtype C predominates. Investigating the sequence-intact reservoir could provide insight on the mechanisms by which intact proviruses persist and inform ongoing cure efforts. Through nearly full-length proviral amplification and sequencing (NFL-PAS), we generated 733 NFL-PAS amplicons from eight children. We showed that rare, genetically intact proviruses could be detected in children who initiated ART after 2.3 months of age. The frequency of intact proviruses was lower (P < 0.05) than that reported for HIV subtype B-infected adults treated during early HIV infection. We show that cells harboring genetically intact HIV proviruses are rare in children exhibiting long-term suppression after early treatment and may require the processing of a large number of cells to assess reservoir size. This points to the need for efficient methods to accurately quantify latent reservoirs, particularly in pediatric studies where sample availability is limited.

Neurodevelopment at 11 months after starting antiretroviral therapy within 3 weeks of life.

BACKGROUND: Antiretroviral therapy (ART) started between 7 and 12 weeks of age improves neurodevelopmental outcomes in HIV-infected (HIV+) infants, but the impact of even earlier initiation is not yet described. OBJECTIVES: We assessed the early neurodevelopment of HIV+ infants who started ART within 21 days of life. METHOD: Participants were enrolled from the public sector birth HIV-diagnosis programme. Inclusion criteria included the following: birth weight > 2000 g, infant commencing ART < 6 weeks and no infant cytomegalovirus disease. Antiretroviral therapy included Zidovudine/Lamivudine/Nevirapine for the first 2 weeks, the latter then replaced by Lopinavir/Ritonavir. Once body weight > 3 kg and gestational age > 44 weeks, Abacavir replaced Zidovudine. The Griffiths mental development scales (GMDS) were administered at 10-12 months. RESULTS: Of 29 infants assessed, 23 (79%) were girls. Mean birth weight was 3002 ± 501 g. Twenty-four mothers (83%) received ART during pregnancy. Seven (24%) infants were diagnosed HIV+ within 48 h of birth. Median [interquartile range] viral load (VL) at diagnosis was 3904 [259-16 922] copies/mL, age starting ART was 6.0 [3-10] days and age at VL suppression was 19.1 [15-36] weeks. At the GMDS assessment, nine (31%) participants had detectable VL and 26 (90%) had World Health Organization (WHO) clinical stage I disease. The GMDS was performed at a mean age of 11.5 ± 0.8 months. Mean quotients were within the average range: Global Griffiths score was 103.6 ± 10.9 and mean quotients on the subscales ranged from lowest 95.9 ± 13.4 for locomotor to highest 112.8 ± 11.3 for hearing-and-language. CONCLUSION: Preliminary findings in this small group suggest that early neurodevelopmental scores are within the normal range in infants with perinatal HIV infection who started ART at a median of 6 days.

HIV-1 DNA decay is faster in children who initiate ART shortly after birth than later.

INTRODUCTION: There is limited data in children on whether persistence of HIV-1 infected cells is affected by age at initiating antiretroviral therapy (ART), its duration or any subsequent ART interruption. We therefore investigated the effects of both age of ART initiation and duration of ART interruption on HIV-1 DNA decay in children. METHODS: We investigated HIV-1 DNA decay in three groups of children on ART: Group-1 (n = 7) started uninterrupted ART within eight days of life; Group-2 (n = 8) started uninterrupted ART at a median of five months of age; and Group-3 (n = 23) started ART at a median age of 1.8 months for either 40 or 96 weeks, then interrupted ART (median of seven months), and restarted ART based on CD4 count and clinical criteria. Total HIV-1 DNA was assayed using a sensitive HIV-1 subtype C-adapted quantitative PCR for integrase. The duration of ART was square root transformed to fit the observed slowing of HIV-1 DNA decay rate. For each group, point estimates for decay rates were determined after six months of continuous suppressive ART in groups 1 and 2 or six months after restarting ART in Group-3. Groups-2 and 3 were combined using a mixed effect regression model to investigate covariates of HIV-1 DNA decay rate. RESULTS AND DISCUSSION: At six months of continuous suppressive ART, the HIV-1 DNA t½ (95% CI) was shorter in Group-1 (n = 7): 2.7 months (2.1 to 3.8), than 9.2 months (7.4 to 12.1) in Group-2 (n = 8); and 9.6 months (7.6 to 12.6) in Group-3 (n = 23) (p < 0.01). In multivariable analyses, HIV-1 DNA before treatment (p < 0.001) and the change in HIV-1 DNA during interruption (p < 0.01) were independent predictors of slower HIV-1 DNA decay. CONCLUSIONS: These data suggest that ART initiation within the first week of life can reduce the persistence of long-lived infected cells. Delaying ART is associated with slower decay of infected cells.