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Pediatric Urgent Start of HAART (PUSH)

Grant: Post-Stabilization vs Urgent Start of HAART in HIV-1 Infected Children with Severe Co-infections

Sponsored byNational Institutes of Health (2 R01 HD023412-21)

Protocol ChairsGrace John-Stewart, MD, PhD

University of Washington, Department of Global HealthSeattle, Washington, USA

Dalton Wamalwa, MBChB, MMed, MPHUniversity of Nairobi, Kenyatta National Hospital

Nairobi, Kenya

Version 619 February 2015

PUSH Protocol19 February, 2015Version 6

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Signature Page

The signatures below constitute the approval of this protocol and the attachments, and provide the necessary assurances that this trial will be conducted according to all stipulations in the protocol, including all statements regarding confidentiality, and according to legal and regulatory requirements.

Co-Principal Investigator:

02/19/15Signed: ____________________________________________ Date: ________________

Grace John Stewart, MD, PhD

Co-Principal Investigator:

Signed: ___________________________________________Date: __02/19/2015______________

Dalton Wamalwa, MBChB, MMed (Paeds), MPH


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Table of Contents

Signature Page.................................................................................................................................2List of Abbreviations and Acronyms...............................................................................................6Key Roles.........................................................................................................................................7Summary........................................................................................................................................10Figure 1. Overview of study design..............................................................................................111 Background and literature review...............................................................................................121.1 HIV-1 infected individuals are often diagnosed with HIV-1 in the setting of a severe co-infection.........................................................................................................................................121.2 Children with HIV-1 present for care late and late-presenters have high mortality following ART...............................................................................................................................................121.3 The ideal timing of ART in the context of severe co-infection is unknown and may differ by coinfecting pathogen......................................................................................................................121.4 There are limited data on pediatric IRIS following ART.......................................................131.5 The mechanisms underlying IRIS in children may differ from adults....................................131.6 Challenges to initiating ART in Kenyan children...................................................................132 Rationale.....................................................................................................................................143 Hypothesis..................................................................................................................................144 Study objectives..........................................................................................................................145 Study design and methodology...................................................................................................155.1 Study design............................................................................................................................15Table 1. Summary of proposed outcomes, eligibility, and procedures........................................155.2 Study area description.............................................................................................................155.3 Study population.....................................................................................................................155.4 Inclusion and exclusion criteria..............................................................................................155.5 Representation of women, children and minorities................................................................165.6 Co-enrollment guidelines........................................................................................................165.7 Sample size considerations......................................................................................................16Table 2. Sample size calculations*................................................................................................166 Recruitment, screening, consent, and enrollment procedures....................................................166.1 Recruitment.............................................................................................................................166.2 Screening................................................................................................................................176.3 List of screening data collection procedures.....................................................................17Figure 2. Flow chart of recruitment, screening, enrollment and randomization procedures........196.4 Enrollment..............................................................................................................................196.5 List of enrollment data collection procedures........................................................................206.6 Management of Study participants during hospitalization.....................................................21Table 3. Pre-enrollment, enrollment and post-randomization procedures administered during hospitalization by hospital and Study staff....................................................................................217 Randomization...........................................................................................................................227.1 Randomization scheme...........................................................................................................228 Post-randomization administration of ART...............................................................................228.1 ART prescription: formulation, regimen and dosage.............................................................228.2 ART initiation.........................................................................................................................228.3 Adherence and disclosure counseling and documentation.....................................................22


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8.4 Concomitant medication.........................................................................................................228.5 Monitoring response to therapy.........................................................................................238.6 Treatment interruption.......................................................................................................238.7 Changing therapy....................................................................................................................23Table 4. Potential toxicity that may require changing antiretroviral regimens............................23Table 5: Suggested ARV substitution in the event of significant toxicity....................................248.8 Drugs supply and accountability............................................................................................248.9 ART initiation for discharged subjects...................................................................................249 Post-randomization follow-up data collection procedures........................................................259.1 Data and specimen collection during post randomization follow-up.....................................25Table 6. Schedule of intensive data and specimen collection.......................................................259.2 List of follow-up procedures..................................................................................................269.7 Participant retention................................................................................................................279.8 Participant withdrawal............................................................................................................2710 Safety monitoring and adverse event reporting........................................................................2710.1 Data safety and monitoring plan...........................................................................................2710.2 Recording adverse events.....................................................................................................2810.3 Reporting serious adverse events..........................................................................................2810.4 Study discontinuation...........................................................................................................2811 Laboratory considerations........................................................................................................2811.1 Local laboratory tests.............................................................................................................29Table 7. Planned assays to identify immunologic covariates of mortality and IRIS....................3011.2 Quality assurance and quality control...................................................................................3011.3 Specimen storage...................................................................................................................3011.4 Biohazard containment.........................................................................................................3012 Human subjects considerations.................................................................................................3012.1 Institutional Review Board...................................................................................................3012.2 Risks to subjects...................................................................................................................3112.3 Adequacy of protection against risks....................................................................................3112.4 Potential benefits of the proposed research to the subjects and others.................................3212.5 Importance of the knowledge to be gained...........................................................................3312.6 Care following study completion..........................................................................................3313 Statistical analysis plans..........................................................................................................3313.1 Endpoints..............................................................................................................................3313.2 Data analysis.........................................................................................................................3313.2.1 Aim 1..................................................................................................................................3313.2.2 Aim 2.................................................................................................................................3413.2.3 Secondary aim....................................................................................................................3414 Administrative procedures.......................................................................................................3414.1 Protocol compliance.............................................................................................................3414.2 Protocol deviations/violations..............................................................................................3414.3 Study coordination.................................................................................................................3514.4 Study monitoring...................................................................................................................3514.5 Study records.........................................................................................................................3514.6 Use of Information and publications.....................................................................................35References......................................................................................................................................37


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Appendix........................................................................................................................................39


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List of Abbreviations and Acronyms

3TC Lamivudine ABC Abacavir ALT Alanine transaminaseART Antiretroviral therapyAZT ZidovudineCBC Complete blood countCDC Centers for Disease Control and PreventionCMV CytomegalovirusCRP C-reactive proteind4T Stavudine ddI Didanosine DNA Deoxyribonucleic AcidEBV Epstein Barr virusEFV Efavirenz ERC Ethical Review CommitteeEQA External quality assuranceGCLP Good Clinical and Laboratory PracticeHAART Highly Active Antiretroviral TherapyHDL High Density LipoproteinHIV Human Immunodeficiency VirusIRB Institutional Review BoardIRIS Immune reconstitution inflammatory syndromeKNH Kenyatta National HospitalLAM LipoarabinomannanLDL Low Density Lipoprotein LPV-RTV Lopinavir-ritonavir (LPV/r)MCH Maternal Child HealthNCC Nairobi City CouncilNRTI Nucleoside reverse transcriptase inhibitor NNRTI Non- nucleoside reverse transcriptase inhibitorNVP Nevirapine OPH Optimizing Pediatric HAARTPBMC Peripheral blood mononuclear cellPI Protease InhibitorPITC Provider-initiated testing and counselingPMTCT Prevention of Mother-to-Child TreatmentRNA Ribonucleic AcidSAE Serious Adverse EventTB TuberculosisTST Tuberculin skin testTDF TenofovirUN University of NairobiUW University of WashingtonUA UrinalysisVCT Voluntary counseling and testingVL Viral load (HIV-1 RNA copies/ml)WHO World Health Organization


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ZDV Zidovudine


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Key Roles

InstitutionsFunding Agency: National Institutes of Health (2 RO1 HD023412-21)Collaborating Institutions: University of Washington, Seattle, WA, USA

University of Nairobi, Nairobi, KenyaClinical laboratories: University of Nairobi, Nairobi, Kenya

Research Laboratory at KEMRI CDC, Kisumu Kenya

Protocol Development Co-ChairsGrace John-Stewart, MD, PhDProfessorDivision of Allergy & Infectious Disease, Department of MedicineInternational AIDS Research and Training Program, Department of Global HealthUniversity of Washington325 Ninth Avenue, Seattle WA 98104Tel: [email protected]

Dalton Wamalwa, MBChB, MMed (Paeds), MPHSenior LecturerPaediatrician, EpidemiologistDepartment of Paediatrics and Child HealthKenyatta National HospitalUniversity of NairobiP.O.Box 19676, Nairobi,00202Tel: +254-020-2714851, [email protected]

Co-Investigators

Sarah Benki-Nugent, MS, PhDSenior Fellow Department of MedicineUniversity of Washington325 9th AvenueSeattle, WA 98104Tel: [email protected]

Lisa Cranmer, MD, MPHSenior Pediatric Infectious Disease FellowDept. of Pediatrics University of Washington325 Ninth Avenue, Box 359300Seattle, WA 98104Tel: [email protected]


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mailto:[email protected]




Barbara Lohman Payne, PhDResearch Assistant ProfessorDepartment of MedicineUniversity of Washington325 Ninth Avenue, Box 359909Seattle WA [email protected]

Lisa A. Mills MD, MScChief, HIV Research BranchKEMRI-CDC Research and Public Health CollaborationP.O Box 1578, Kisumu [email protected]

Hellen Moraa, BSNStudy CoordinatorDepartment of Paediatrics and Child HealthUniversity of NairobiP.O. Box 19676 Nairobi, 00202Tel: [email protected]

Irene Njuguna MBChB, MScStudy PhysicianDepartment of Paediatrics and Child HealthUniversity of NairobiP.O. Box 19676 Nairobi, 00202Tel: [email protected]

Julianna Otieno, MBChB, MMed (Paeds),Pediatrician/Chief AdministratorJaramogi Oginga Odinga Teaching &Referral HospitalP.O Box 849 Kisumu 40100+254 [email protected]

Vincent Otieno, MBChBStudy PhysicianDepartment of Paediatrics and Child HealthUniversity of NairobiP.O. Box 19676 Nairobi, 00202Tel: [email protected]

Barbra Richardson, PhDResearch Associate Professor,


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Department of BiostatisticsUniversity of Washington325 Ninth Ave, Box 359909, Seattle WA, 98104Tel: 206 731-2425, 206 [email protected]

Jennifer Slyker, PhDActing InstructorDepartment of Global Health, University of Washington325 9th AvenueSeattle, WA 98104Tel: [email protected]

Clement Zeh Ph.DDirector, HIV Research LaboratoryKEMRI-CDC Research and Public Health CollaborationP.O Box 1578, Kisumu [email protected]

CollaboratorsLoice MutaiHead of Department, PaediatricsMbagathi District HospitalP.O Box 20725, 0202+254721330169

Beatrice MutaiPaediatricianMbagathi District HospitalP.O Box 20725, 0202+254733250209


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Summary

Design: Randomized clinical trial involving hospitalized HIV-1 infected children. Children will be randomized to randomized to urgent (<48 hours) versus early antiretroviral therapy (7-14 days). This trial will be unblinded.

Population: Hospitalized HIV-1 infected children who are antiretroviral therapy (ART) naïve ≤ 12 years of age.

Sample size: 360 children will be randomized (180 per arm).

Treatment: All children will be treated with ART according to World Health Organization (WHO) and Kenyan national guidelines.

Study duration: Enrollment into the study will occur over the course of 36-48 months and each child will be routinely followed for a maximum of 6 months.

Study sites: Kenyatta National Hospital, Kisumu District Hospital, Nyanza Provincial Hospital, Mbagathi District Hospital (and at least 1 of the following hospitals: Homabay District Hospital, Kisii Level 5 Hospital)

Primary hypothesis:

HIV-1 infected children hospitalized with severe co-infection either may be unsalvageable due to too far advanced immunosuppression/co-infection or may benefit from urgent ART.

Secondary hypotheses:Urgent ART during an acute infection could potentially result in increased risk of immune reconstitution inflammatory syndrome (IRIS) or drug toxicities/interactions.

Specific aims:1. To compare the 6 month all-cause mortality rate, incidence of IRIS, and incidence of

drug toxicity in HIV-1 infected children (≤ 12 years old) presenting to the hospital with a serious infection randomized to urgent (<48 hours) versus early ART (7-14 days).

2. To determine co-factors for mortality, IRIS, and drug toxicity. Potential cofactors will include: baseline weight-for-age, height-for-age, weight-for-height (Z-scores), CD4, HIV-1 RNA, type of co-infection, age, rate of viral load and CD4 change following ART, immune activation markers, pathogen and HIV-1 specific immune responses.

Secondary aim: To determine etiologies of IRIS and to compare immune reconstitution to HIV, TB, EBV and CMV following ART overall and in each trial arm.


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Figure 1. Overview of study design


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All hospitalized children receive routine HIV-1 testing

Recruitment:Children with positive HIV-1 test result

Screening: Confirmatory HIV-1 test

Enrollment (N = 360): Children with positive confirmatory HIV-1 test result

Randomization

Early ART arm (N = 180):Initiate ART 7-14 days from enrollment

Children with negative HIV-1 test result

Children with negative HIV-1 test result

Urgent ART arm (N = 180):Initiate ART < 48 hours from enrollment

Follow-up: 6 Months of follow-up with intensive specimen and data collection at weeks 1, 2, 4, 8, 12, 16, 20, 24

Monitor for mortality, IRIS, drug toxicity

1 Background and literature review 1.1 HIV-1 infected individuals are often diagnosed with HIV-1 in the setting of a severe co-infection The majority of HIV-1 infected adults are diagnosed and present for care late in disease. In a recent analysis of the North American ACCORD study of 44,491 HIV-1 infected individuals, 54% of adults presented for care at a disease stage eligible for treatment [1]. Although opportunistic


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and co-infections in HIV-1 have decreased dramatically following highly active antiretroviral therapy (HAART), many HIV-1 infected individuals continue to be diagnosed with HIV-1 late at the time of a symptomatic co-infection [2]. While this reality motivates public health interventions to encourage earlier HIV-1 testing, late presenters will continue to be prevalent and require strategic approaches to best salvage health outcomes.

1.2 Children with HIV-1 present for care late and late-presenters have high mortality following ART Pediatric HIV-1 has a much more aggressive course than adult HIV-1, resulting in 2-year mortality for 50% in ART-untreated children [3-5]. However pediatric ART appears to have comparable benefit in children as adults, with marked survival benefit [6]. In the CHER study in South Africa, empiric early ART in asymptomatic infants resulted in 4% 1-year mortality, which was significantly lower than among infants randomized to initiate treatment when they met CD4 or clinical criteria for ART [7]. Although efforts are expanding to detect infant HIV-1, there are fewer efforts to diagnose older children who have asymptomatic HIV-1 infection. Most HIV-1 voluntary counseling and testing (VCT) centers cater solely to adults and even HIV-1 infected caregivers may perceive an asymptomatic child as being unlikely to carry HIV-1. Thus, most children with HIV-1 in Africa remain undiagnosed and when they do present for care, it is in the context of severe co-infection. In a recent systematic review of studies on pediatric ART initiation involving over 19,000 children, the majority from resource-limited settings (17,875), children from resource-limited settings had much lower CD4% at ART initiation (12% vs. 23%, p=0.01) and higher mortality (7.6% vs. 1.6%) than developed countries [8]. The management of hospitalized newly HIV-1 diagnosed children includes aggressive management of co-infection with stabilization or resolution prior to ART initiation. In the process of medical stabilization, ART preparation and adherence counseling, children die. Our proposed trial will test the concept that highly accelerated (urgent) ART in children with severe co-infection will provide survival benefit offsetting the potential risk of IRIS or drug toxicity/failure.

1.3 The ideal timing of ART in the context of severe co-infection is unknown and may differ by coinfecting pathogen Recently randomized clinical trials in adults have compared survival benefit of earlier versus later ART during acute co-infection. Zolopa and colleagues conducted a US study in HIV-1 infected adults with severe infection (mostly pneumocystis) and found that earlier ART resulted in improved survival [9]. In 2010, Karim et al found that integrated ART in South African TB-coinfected individuals resulted in survival benefit compared with sequential ART (following completion of TB therapy) [10]. Rates of IRIS in the two trial arms were comparable. In contrast to these studies, early ART in cryptococcal meningitis was associated with increased mortality [11]. In addition, an ongoing trial among adults with TB meningitis has not shown benefits of early ART and has noted increased drug adverse events with early ART [12]. These latter studies suggest that CNS infections may be more prone to inflammatory sequelae from earlier ART.

1.4 There are limited data on pediatric IRIS following ART Immune reconstitution inflammatory syndrome (IRIS) occurs as a result of an exaggerated dysregulatory immune response to either a subclinical or treated co-infection. In the absence of HIV-1, IRIS has been observed in TB-treated children and in re-feeding syndrome, but more recently has been described in ART treated HIV-infected individuals. Because children do not have as many pre-existing latent infections, it is plausible that IRIS incidence and pathogenesis would differ markedly between children and adults. In the few studies


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conducted on pediatric IRIS to date, incidence rates in children (11-21%) appear similar to those in adults (20%) and vary by pathogen. IRIS is a diagnosis of exclusion and as a result, it is difficult to precisely estimate incidence of IRIS. In both adults and children, IRIS is typically identified in the first 2 weeks to 3 months of therapy, but may appear much later (>12 months after ART initiation). In the limited studies among children, mycobacterium and varicella zoster virus (VZV) are the most common pathogens associated with IRIS [13-15]. There are only 2 prospective studies of pediatric IRIS, both of which excluded children with active opportunistic infections. Unfortunately, most late-presenting children have an active infection and are at risk for IRIS. Determining incidence of and co-factors for IRIS in this broad denominator of children is important for appropriate clinical management.

1.5 The mechanisms underlying IRIS in children may differ from adults IRIS occurs due to immune dysregulation and correlates with severity of immunosuppression (CD4 <100 cells/mm), infecting pathogen, and type of immune response [16]. T-cell responses to pathogens may be lower in children than in adults, which could result in higher pathogen loads and increased susceptibility to IRIS [17, 18]. IFN-gamma responses may be actively suppressed during fetal development [19, 20], but appear to increase rapidly during the first year of life, and it is unknown how evolving Th1-type responses contribute to IRIS during infancy [17, 21]. Dysregulation of regulatory T cell responses appears to be important in some types of IRIS; the frequency of Tregs is highest in cord blood and infancy and may potentially affect the risk of developing IRIS [22, 23]. Additionally, neonatal dendritic cells (DC) differ from adult cells in phenotype and function; an impaired ability to prime antigen-specific Th1 and CD8 T cell responses during infancy could affect propensity for further dysregulation during immune reconstitution [24]. Due to the potential severity of IRIS in children and the large number of children at risk in HIV endemic areas, a detailed evaluation of immune responses and immunoregulatory processes during the development of IRIS will be important in developing strategies to prevent and treat IRIS in children.

1.6 Challenges to initiating ART in Kenyan children IRIS presents significant challenges to initiating ART in HIV-infected children, particularly those with substantial immunosuppression or acute opportunistic infections. In Kenya, many children are not diagnosed with HIV-1 until they become critically ill; since standard practice is to stabilize acute infections before initiating ART, many children die before ever starting ART [6, 25]. Initiation of ART during acute illness may improve prognosis by speeding immune reconstitution and restoring growth [6]. However, the potential benefits of urgent initiation of ART must be balanced against the potential risk of developing IRIS; which though uncommonly fatal, may present significant morbidity and result in poorer virologic suppression on ART [13]. To date, there have been no randomized trials of timing of ART and the development of IRIS. Urgent ART during an acute infection could potentially result in increased risk of IRIS, particularly if ART begins before total clearance of the offending pathogen. Alternately, delaying ART until the resolution of acute infection leaves the immunosuppressed child at risk for the development of other novel opportunistic infections, and may not reduce the risk of developing “unmasking-IRIS” which occurs when immune responses target a previously undetected pathogen.

2 Rationale

Over 90% of the world’s 2.2 million HIV-1 infected children reside in Africa and public health programs are expanding to both decrease the number of new pediatric HIV-1 infections and to PUSH Protocol19 February, 2015Version 6

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treat children with HIV-1 infection. Unfortunately, HIV-1 infection is under-recognized among children and pediatric HIV-1 infection is often diagnosed late in the setting of a severe co-infection. For these children, there is need to define the optimal time to initiate ART. The primary goal of this study is to determine whether urgent initiation of ART has the potential to reduce mortality in late-diagnosed HIV-1-infected children. Urgent initiation of ART, concurrent with treatment of the acute co-infection, may be associated with increased drug side-effects, difficulty in administration, and increased immune reconstitution inflammatory syndrome (IRIS). These risks may be outweighed by increased survival, prompt decrease in viral replication, faster immune recovery, and better control of both HIV-1 and the concomitant infection.

3 Hypothesis

Primary Hypothesis:HIV-1 infected children hospitalized with severe co-infection either may be unsalvageable due to too far advanced immunosuppression/co-infection or may benefit from urgent ART.

Secondary hypotheses:Urgent ART during an acute infection could potentially result in increased risk of IRIS or drug toxicities/interactions.

4 Study objectives

Specific aims:

1 To compare the 6 month all-cause mortality rate, incidence of IRIS, and incidence of drug toxicity in HIV-1 infected children (≤ 12 years old) presenting to hospital with a serious infection randomized to urgent (<48 hours) versus early ART (7-14 days).

2 To determine co-factors for mortality, IRIS, and drug toxicity. Potential cofactors will include: baseline weight-for-age, height-for-age, weight-for-height (Z-scores), CD4, HIV-1 RNA, type of co-infection, age, rate of viral load and CD4 change following ART, immune activation markers, pathogen and HIV-1 specific immune responses.

Secondary aim: To determine etiologies of IRIS and to compare immune reconstitution to HIV, TB, EBV and CMV following ART overall and in each trial arm.


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5 Study design and methodology

5.1 Study designThe proposed study will enroll 360 Kenyan children diagnosed with HIV-1 while admitted to hospital and compare rates of mortality and immune reconstitution syndrome (IRIS) in groups randomized to either urgent ART initiation (within 2 days of enrollment) or early ART (7-14 days). Study outcomes will include mortality, IRIS, and toxicity.

Table 1. Summary of proposed outcomes, eligibility, and proceduresStudy Endpoints All-cause mortality, IRIS, toxicityProjected enrollment 360 (180/randomization arm)Age range 0 - 12 yearsHealth status Acutely ill, hospitalizedSource Hospital wardsStudy follow-up 6 monthsSpecimens obtained Blood, saliva, urine, stoolSpecimen collection Baseline and weeks 2/4/12/24Use of specimens HIV viral load, CD4, diagnostics, immunologic assays, virologic

studiesART Randomized to: Urgent ART versus Early ARTProphylaxis for OI According to Kenyan National GuidelinesInclusion criteria Hospitalized, HIV-1 infected, no history of ART, < 12 years oldExclusion criteria Suspected central nervous system infection (meningitis,

encephalitis or other)Study exit referral plan

Referral to Comprehensive Care Clinic pediatric HIV-1 treatment program

5.2 Study area descriptionThe catchment area for this study includes Nairobi, Homa Bay, Kisii, Kisumu and their surrounding areas. Nairobi and Kisumu are primarily urban, although the hospital wards attract patients from more outlying, rural regions as well. Homa Bay and Kisii serve a rural population.

5.3 Study populationThis study will enroll 360 HIV-1 infected children who are aged ≤ 12 years, are ART naïve, and who have been admitted to the specified hospitals in Kenya.

5.4 Inclusion and exclusion criteriaInclusion Criteria

a) Aged ≤ 12 years old (reported)b) HIV-1 positive (for example, two rapid HIV-1 antibody tests for children >18 months and not breastfeeding, or one HIV-1 DNA/RNA test for children ≤18 months or who are breastfeeding)c) No prior ART (history of pMTCT antiretroviral treatment does not affect eligibility)d) Eligible to receive ART, according to current WHO guidelinese) Caregiver plans to reside in study catchment area for at least 6 months (reported)f) Caregiver provides sufficient locater information


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Exclusion Criteria: Suspected central nervous system infection (meningitis, encephalitis or other)

5.5 Representation of women, children and minorities Inclusion of women: The study will include male and female children. Based on previous studies we anticipate an even gender distribution, no exclusions will be made based on sex.

Inclusion of Minorities: The study will involve Kenyan children recruited from a diverse sample of ethnic groups residing in Kenya, including children from minority ethnic populations.

Inclusion of Children: All participants in this study will be children.

5.6 Co-enrollment guidelines Participants in this study may not take part in other concurrent HIV-1 treatment trials or studies involving experimental products, HIV prevention strategies such as vaccine trials.

5.7 Sample size considerationsOur power to detect a difference between trial arms is dependent upon the cumulative mortality in the cohort. The cumulative 6-month mortality in the OPH cohort, which enrolled both asymptomatic and symptomatic HIV-1 infected children was ~32%. Since the study will exclusively enroll hospitalized children older than those in OPH, we anticipate that mortality will be similar or slightly lower. Based on Table 6 below, we will aim to enroll 360 children in the study. With an estimate of 28% cumulative mortality in the early ART arm in the first 6 months of follow-up, we will have 80% power to detect a minimum 2-fold difference between study arms, assuming ~15% attrition.


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Table 2. Sample size calculations*

*Based on various estimated cumulative mortality rates, assuming a hazard ratio of 0.5, 80% power and alpha = 0.05.

Sampling method: not applicable

Definition of cases and controls: not applicable

6 Recruitment, screening, consent, and enrollment procedures

6.1 RecruitmentThis trial will be conducted at Kenyan hospitals. The first study site to be established will be Kenyatta National Hospital (KNH) in Nairobi, where site-specific clinical trials expertise has been established through the Optimizing Pediatric HAART Study. We will also expand recruitment activities to hospitals in areas with higher HIV prevalence. Potential sites for expansion include Kisii and Mbagathi District Hospitals, which have an ongoing collaboration between University of Washington and University of Nairobi as clinical pediatric training sites, and Homa Bay District Hospital, which is also a University of Nairobi Department of Pediatrics collaborative site. Kisumu Provincial General Hospital and Kisumu East District Hospital are additional potential sites.

We will recruit hospitalized children with an HIV-1 diagnosis who have not previously taken antiretroviral therapy. In Kenyan hospitals, routine HIV-1 diagnostic testing is conducted on all admitted children. Study staff will pre-screen hospital records to identify the number of children admitted to the hospital, the number of children who are tested for HIV-1, and the number of children who are HIV-1 positive. The study team will leverage established links with pediatricians, clinical officers, and nurses within the hospital pediatric wards. Hospital staff will be sensitized to the study, and will refer interested parents/caregivers (referred to as caregivers throughout) of HIV-1 infected children. Interested caregivers will be referred to Study staff for eligibility assessment and enrollment.

6.2 Screening If confirmatory HIV-1 antibody testing has not been conducted by hospital staff, or if two rapid HIV-1 antibody test results are not documented in the hospital record, caregivers will be provided with counseling regarding the possibility of the child’s HIV-1 infection. Following written informed consent, a sample will be obtained for confirmatory HIV-1 testing (for example, a second rapid HIV-1 antibody test for children >18 months and not breastfeeding, or a second HIV-1 DNA/RNA test for children ≤18 months). Performing confirmatory HIV-1 testing may include repeat HIV-1 testing with a DNA/RNA assay if an initial HIV-1 rapid antibody test result is indeterminate. Some HIV-1 rapid antibody test results are indeterminate, even afte repating the rapid antibody assay. In order to identify children who are HIV-1 positive, a DNA/RNA test is necessary for children who have an indeterminate rapid antibody test. Caregivers of children with a positive confirmatory HIV-1 test will be provided with post-test counseling.


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Cumulative incidence

Expected cases

Sample size

Final sample size assuming 15% attrition

0.30 65 293 3370.28 65 314 3610.25 65 352 405

Recruitment of HIV-1 positive children includes screening HIV-1 exposed infants ≤18 months old that have an HIV-1 negative rapid antibody test. Some children ≤18 months may test negative on a rapid HIV-1 antibody test but test positive on an HIV-1 DNA/RNA test. In order to identify children who are HIV-1 positive, a DNA/RNA test is necessary for HIV-1 exposed infants ≤18 months, even if they have a negative rapid antibody test result.

As is possible, peer counselors will be involved in post-test counseling to provide support to caregivers regarding their HIV diagnosis and to explain the voluntary nature of research studies. These children will be further assessed for enrollment on the basis of the other inclusion and exclusion criteria (described in Section 5.4). Study staff will confer with hospital clinicians and review the child’s medical records to confirm eligibility for enrollment. A community advisory board will be convened to provide guidance on key issues to address during post-test counseling.

6.3 List of screening data collection procedures

6.3.1 Children > 18 months or not breastfed in the last 6 weeks:

Listed below are all the required screening procedures for this study1. a) Study nurse will read Recruitment Script to caregiver.b) Counseling of caregiver before confirmatory testing for HIV-11.c) Written informed consent from caregiver for confirmatory HIV-1 testing1.d) Collection of blood for HIV-1 confirmatory test1.e) Provide the child’s HIV-1 test results to the caregiver1 and provide post-test

counseling.f) Verification of child and caregiver information for eligibility for enrollment.g) Obtaining locator information from caregiver.h) Written informed consent for enrollment in the study from the caregiver.

1 Will only be conducted if a confirmatory hospital rapid HIV-1 antibody test result cannot be documented in the hospital record.

6.3.2 Children ≤ 18 months or breastfed in the last 6 weeks:

Listed below are all the required screening procedures for this study. a) Study nurse will read Recruitment Script to caregiver.b) Counseling of caregiver before DNA/RNA HIV-1 test.c) Written informed consent from caregiver for first DNA/RNA HIV-1 test.d) Collection of blood for first DNA/RNA HIV-1 test.e) Provide the child’s DNA/RNA HIV-1 test results to the caregiver and provide post-test

counseling (study nurse).f) Verification of child and caregiver information for eligibility for enrollment.g) Obtaining locator information from caregiver.h) Written informed consent for enrollment in the study from the caregiver.


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Hospital staff administer routine HIV-1 testing and provide caregivers with test results and counseling

Hospital staff inform caregivers about the Study

Study staff provide information regarding the Study

Study staff provide counseling, and obtain written informed consent for testing, children provide blood specimen (Section 6.3, a-d)

Study staff provide test results and post-test counseling (Section 6.3, e)

Study staff verify enrollment eligibility and obtain written informed consent for enrollment (Section 6.3, f, g)

Study staff administer enrollment procedures (Section 6.5)

All hospitalized children

Children who test HIV-1 positive

Children whose caregivers are interested in the Study

Children whose caregivers are interested in enrolling in the Study

Caregivers meet with study Staff

Children who are HIV-1 positive

Enrolled children

Early ART arm

Children who test HIV-1 negative

Children with a negative confirmatory HIV-1 test

Urgent ART arm Study staff assign randomization allocation (Section 7.1)

Figure 2. Flow chart of recruitment, screening, enrollment and randomization procedures

6.4 Enrollment Caregivers will be given a follow-up appointment to meet with Study staff for receipt of HIV-1 test results and to discuss informed consent for study enrollment. Study staff will offer caregivers the opportunity to speak with a peer counselor again at the time of informed consent. Following written informed consent for study enrollment, a detailed medical history will be


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collected to document past infections, hospitalizations and treatments. Maternal Child Health (MCH) cards will be reviewed, if possible, to assess exposure to PMTCT. Medical records will be used to abstract relevant history, physical examination and laboratory data of enrolled children. Questionnaires will be administered to assess history of TB, zoster, herpes, meningitis, vaccination record, and skin infections. A physical examination with a developmental assessment will be administered.

Blood will be collected for HIV-1 RNA, CD4, CD8, complete blood count (CBC), C-reactive protein (CRP), blood chemistries, cryopreservation of viable cells, plasma and serum, TSpot TB test and diagnostics for other co-pathogens. Saliva will be collected for diagnosis of herpesvirus infections. A chest x-ray, a TST, and TB cultures (using a sputum or gastric aspirate specimen) will be performed for TB diagnosis, if possible. When possible, pretreatment blood, urine, and stool specimens collected during clinical procedures will be stored for future molecular pathogen testing.

6.5 List of enrollment data collection proceduresListed below are enrollment procedures for this study.

Procedures for the child Medical history, including history of TB, zoster, herpes, meningitis, vaccination

record, and skin infections Physical examination including growth and developmental progress Blood collection for the following tests as possible:

CBC Blood chemistries including glucose, electrolytes, albumin, lipids (total

cholesterol, HDL, LDL, and triglycerides), amylase, alanine transaminase (ALT), and creatinine

CRP CD4 and CD8 HIV-1 RNA quantification Serology for co-pathogens including CMV, EBV, HBV, and toxoplasma on stored

samples Cryptococcal antigen test Blood cultures if clinically indicated Malaria smear if clinically indicated T-SPOT.TB on stored samples Cryopreservation of all viable cells, plasma, and serum Molecular assays to evaluate host and pathogen genetic characteristics

(including DNA/RNA microarray) on stored samples Additional specimen collection and/or procedures will be performed as possible:

Saliva collection for herpesvirus diagnosis on stored samples Nasal swab for respiratory viruses including RSV, influenza on stored samples Chest X-ray Tuberculin skin test (TST) Sputum collection for TB culture, as clinically indicated (performed via gastric

lavage if the child is unable to produce a sputum sample) Urine samples for urinalysis and urinary lipoarabinomannan (LAM) for all

subjects, bacterial culture as clinically indicated, and a pregnancy test (hCG) for pubertal females.


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Stool samples as possible Dried blood spot (filter paper)

Procedures for caregivera) Socio-demographic informationb) PMTCT historyc) Medical history of the enrolling child’s familyd) Information on attitude regarding disclosure, and knowledge and attitudes about HIVe) Counseling regarding disclosure of child’s HIV status to child

6.6 Management of Study participants during hospitalizationTo maximize generalizability of the RCT and to optimize prompt management of co-infections, children will be managed by hospital clinicians during their hospitalization. Diagnostic tests and medication for intercurrent illnesses will be ordered per ward protocol with results transferred to study files. However, to ensure adherence to study protocol, Study staff will oversee orders for ART medication, study-related laboratory tests, and specimen collection. Table 2 summarizes hospital-based management of care by hospital and Study staff.

Table 3. Pre-enrollment, enrollment and post-randomization procedures administered during hospitalization by hospital and Study staff

Hospital Staff Study StaffManagement of intercurrent illnesses

Manage co-infections and other conditions per ward protocol

Extract data regarding management of other illnesses/conditions from medical records

Concomitant medication

Prescribe cotrimoxazole (for prophylaxis) and medication for intercurrent illnesses

Extract data regarding concomitant medication from medical records; confer with hospital staff regarding drug interactions

Enrollment, post-randomization

Standard care Oversee orders for study-related lab tests and specimen collection, and extract relevant laboratory data from medical record

ART prescription Provide ART to caregiver Prescribe ART; communicate with hospital staff regarding ART prescription

Adherence counseling and documentation

Document administration of ART in medical record

Document administration of ART; Extract data regarding administration of ART from medical records; provide adherence counseling and support to caregivers

Treatment interruption or change of regimen (toxicity monitoring)

Manage treatment and regimens per hospital protocol

Review medical records for interactions between concomitant medication and ART; review medical records for signs of drug toxicity, and IRIS; provide orders for regimen change or treatment interruption

Ward exit procedure Discharge study participants per ward protocol

Provide counseling, obtain locator information


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7 Randomization

7.1 Randomization schemeChildren will be randomized to either urgent ART initiation (within 48 hours of enrollment) or early ART (7-14 days) at enrollment. A block randomization scheme with variable block sizes will be generated using STATA 8.0 (ralloc.ado v2.2.1). Treatments will be allocated in 1:1 ratio. Study investigators and staff will be blinded to the block number, block size and sequence in the block. Arms will be assigned via pre-prepared sealed, opaque envelopes and the envelopes will be ordered in the sequence of arm assignments generated by the STATA code. Once a child’s eligibility for enrollment has been determined, the first available allocation envelope will be assigned to the child. The child will be randomized to the treatment arm indicated inside the envelope.

The randomization code will be maintained by the Study Coordinator at the study site. The treatment allocation will be un-blinded once it is assigned.

8 Post-randomization administration of ART

8.1 ART prescription: formulation, regimen and dosageART regimens consistent with the Kenyan Ministry of Health guidelines will be initiated with appropriate adaptation for concurrent TB medications, and other medications. Exposure to antiretrovirals for PMTCT will be considered in determining ART regimen. Study staff will oversee prescription of the ART regimen. In the instance of a local drug shortage, the PIs will oversee any clinical decision for potential drug substitutions, based on current medical literature.

8.2 ART initiation ART will be prescribed by Study staff and will be administered by hospital staff during hospitalization (Table 2). For children randomized to urgent ART who are unable to take oral medications, ART will be administered by nasogastric tube. If necessary, a Study physician will be available to consult with hospital staff about administration of ART through a nasogastric tube. Data on administration of ART by hospital and Study staff will be recorded in study files.

8.3 Adherence and disclosure counseling and documentation Adherence counseling for caregivers will begin in hospital at enrollment and at post-randomization follow-up visits, and will continue at each visit following discharge. Following hospital discharge, caregivers will be asked structured questions to assess ART adherence. Syrup bottles will be weighed upon dispensing the drug and when the used bottle is returned. Weighing the bottle will provide an estimate of volume of drug used. If pills are given, adherence will be measured by pill count. Medication diaries to record administration of drug will be encouraged and this data entered into the clinic chart at return visits. For caregivers in whom adherence is an issue, detailed adherence counseling will be given in an effort to optimize therapy.

8.4 Concomitant medication All children will receive cotrimoxazole (Septrin) for prophylaxis of infections, as per Kenyan MOH guidelines. Hospital staff will prescribe treatments for intercurrent illnesses as needed.


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8.5 Monitoring response to therapyResponse to therapy will be assessed using the following clinical and immunologic and virologic measures, during the 6 months of treatment with ART for all subjects. Growth: At each monthly visit, children will have height, weight, mid-arm and head

circumference assessed. Growth will be plotted on standard growth charts and analyzed as z-scores for age.

Morbidity: Intercurrent illnesses will be documented at each Study visit, with particular attention to infectious diseases.

Immunologic response: CD4% and count will be assessed at each visit. Development: Age appropriate gross motor and cognitive milestones will be assessed.

8.6 Treatment interruptionART may be interrupted for a participant by the site investigator due to any adverse reactions to the drug (see Section 8.7 for additional detail) or poor compliance to treatment.

8.7 Changing therapy Children may need to have regimens changed because of toxicity, poor clinical response or poor immunologic response. The first line regimens listed are notable for toxicities listed in Table 3. For children with toxicity from a single-drug without clinical or immunologic poor response, the offending drug will be switched to an appropriate substitute. If toxicity is related to an identifiable drug in a regimen, a drug from the same class that does not have the same adverse effect will replace the offending drug. Table 4 provides detail regarding potential drug substitution options.

Poor response to treatment will be considered in children with poor clinical and/or immunological response to ART. Caregivers will be interviewed regarding compliance to ascertain if poor drug response stemmed from poor adherence rather than lack of efficacy. For mothers in whom adherence is an issue, detailed adherence counseling will be given in an effort to optimize therapy. If compliance is persistently poor, antiretrovirals will be withdrawn.

Table 4. Potential toxicity that may require changing antiretroviral regimensDrug Potential toxicity Parameters

ZidovudineNausea, myelosuppression Hb <7.5 gm/dl,

platelets < 49,000, neutrophils <250

Lamivudine Pancreatitis, neuropathy, hepatitis, neutropenia

Amylase 2X normal, ALT 10X normal, neutrophils <250

Stavudine Neuropathy, pancreatitis Amylase, lipase 2X normal

Nevirapine Severe rash, hepatitis ALT 10X normalAbacavir Allergic reactions such as skin

rashes, hypersensitivity reactions

ALT 10X normal

Lopinavir/Ritonavir GI toxicity, glucose intolerance Lipids 3x normal


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Table 5: Suggested ARV substitution in the event of significant toxicityARV Most frequent significant

toxicitySuggested ARV substitution

Abacavir Hypersensitivity reaction AZTZidovudine Severe anaemiaa or neutropeniab D4T or ABC

Lactic acidosis ABCd

Severe gastrointestinal intolerancec D4T or ABC

Stavudine Lactic acidosis ABCd

Peripheral neuropathy AZT or ABCf

Pancreatitis substituteLipoatrophy/metabolic syndromee AZT

Lopinavir/ritonavir N/A N/A

Nevirapine Acute symptomatic hepatitisg

Lopinavir/ritonavirHypersensitivity reactionSevere or life-threatening rash (Stevens-Johnson syndrome)

a. Exclude malaria in places of stable malaria, severe anaemia is defined as Hb 7.5g/dlb. Defined as neutrophil count of <500/mm3c. Defined as severe refractory intestinal intolerance that prevents ingestion of ARV drug regimen (i.e., persistent nausea and vomiting).d. Reinitiation of ART should not include AZT or d4T so ABC is preferrede. Substitution of d4T typically may not reverse lipoatrophyf. In children ABC or AZT can be considered as an alternative.g. Symptomatic NVP-associated hepatitis is rare in HIV infected children before adolescence

8.8 Drugs supply and accountability The hospital and pediatric treatment clinic pharmacies will provide antiretroviral medicines for all children. Medicines will be obtained through the CDC/PEPFAR program. These may be branded antiretroviral drugs or WHO pre-qualified, US Food and Drug Administration (US FDA)-approved generic drugs depending on the prevailing PEPFAR, US government policy at the time. After hospital discharge, the caregiver will be asked to return the bottles of used or remaining antiretroviral drugs at every visit. The details of the drugs including brand name, volume, pre-weight of the medicine bottles, batch number, and expiry date will be recorded against each participant ID. The bottles will be disposed of by the hospital in accordance with the pharmacy bio-safety regulations.

8.9 ART initiation for discharged subjectsIn the event that subjects are discharged from hospital prior to initiation of ART, a visit will be scheduled at the Study Clinic for ART initiation. Study staff may conduct a home-based ART initiation visit, with the caregiver’s permission, if the child is too ill to return to the Study Clinic within the appropriate window.


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9 Post-randomization follow-up data collection procedures

9.1 Data and specimen collection during post randomization follow-upFollow-up visits for data and specimen collection will occur either in the hospital or at the Study Clinic (for discharged subjects). Following discharge, children will be referred to the local pediatric HIV-1 clinic for follow-up, in which a study clinician will be based. Caregivers will be escorted to this Study Clinic prior to discharge from the hospital.

Children will be followed-up for a total of 6 months after enrollment. Children will be seen at 8 intensive Study visits, including at enrollment while in the hospital and at weeks 1, 2, 4, 8, 12, 16, 20 and 24.

At the first Study Clinic visit, detailed home addresses will be obtained from caregivers. In addition, ‘up-country’ address information will be collected. These home addresses will be tracked for families who are lost to follow-up. For caregivers who agree to a home visit, GPS coordinates will be obtained, if possible.

At each study visit, a study nurse will administer a standardized questionnaire and abstract relevant clinical and laboratory information from the medical record. Counseling regarding adherence and disclosure of the child’s own HIV-1 status will be provided. Blood specimens will be collected at weeks 2, 4, 12, and 24. Saliva, nasal swab, urine and stool specimens will be stored as is possible.

Peripheral blood mononuclear cells (PBMCs) will only be cyropreserved for a subset of study participants. The study will process and store PBMCs at all available blood collection time-points for at least 50 participants. Other subjects may only have plasma and serum stored from their blood collection visits. Assays for cellular activation and immune suppression will be performed on the PMBCs collected from the subset of study participants.

Table 6. Schedule of intensive data and specimen collectionStudy visit

Enroll ARTstart

Week 1

Week 2

Week 4

Week 8

Week 12

Week 16

Week 20

Week 24**

Clinical ProceduresMedical history* XPhysical exam, development, growth X X X X X X X X X

Extract clinical data from hospital records or clinic as needed

X X X X X X X X X X

Real-time Lab AssaysCBC with differential X X X XALT X X X XBUN/creatinine X X X XAlbumin X XCRP X X XCD4 and CD8 X X XHIV-1 DNA(≤ 18 months old)*** X

HIV-1 RNA X XCryptococcal antigen XUrine LAM X


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TB AFB and Culture (sputum/gastric lavage)Stool GeneXpertTSTChest X-ray, as possibleBlood/Cultures, if clinically indicated X

Malaria smear, if clinically indicated X

Specimen StoragePBMC X X X XPlasma X X X X XSerum X X X XSaliva XNasal swab XPAX gene tube(as is possible) Dried blood spot X X X X

Notes. *Medical history will focus on past illnesses, hospitalizations, and treatments; CBC, complete blood count; LFT, liver function tests; CRP, C-reactive protein; TST, tuberculin skin test; **Week 52 post-ART status of children exited from the Study clinic will be abstracted from the local pediatric HIV-1 clinic, with focus on late IRIS or mortality. *** Also occurs at screening visit, before enrollment.

9.2 List of follow-up procedures Assess adherence to ART Counseling regarding adherence and disclosure of the child’s own HIV-1 status Physical examination including growth and developmental progress Clinical diagnoses Morbidities since last study visit Blood collection for the following tests as possible (Note: blood will not be collected

at every visit. See Table 6 for the visits at which different tests will be performed) CBC Blood chemistries including glucose, electrolytes, albumin, lipids (total

cholesterol, HDL, LDL, and triglycerides), amylase, alanine transaminase (ALT), and creatinine

CRP CD4 and CD8

HIV-1 RNA quantification T-SPOT.TB Cryopreservation of all viable cells, plasma, and serum Molecular assays to evaluate host and pathogen genetic characteristics

(including DNA/RNA microarray) Dried blood spot (filter paper)

Blood samples and other specimens will be obtained according to the decision of the Study Clinician based on the clinical examination of the child.

9.3 Interim visits Caregivers can bring their child to the Study Clinic anytime in between two scheduled visits. A visit will be considered interim if it falls between two schedules visits.

Interim visit procedures include:


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Child illness history Adherence to ART Physical examination and developmental progress Blood samples and other specimens will be obtained according to the decision of the Study Clinician based on the clinical examination of the child.

9.4 Missed and delayed/late visits Visits may be completed within a three-day window around the target date for the week 1 and week 2 visits. Visits may be completed within a 1-week window around the target date for subsequent visits.

For participants who do not complete scheduled visits within the visit window, the visit will be considered “missed”. The missed visit CRF will be completed to document the missed visit. For participants who miss specimen collection visits, procedures specified to take place at these visits will be conducted at the participants’ next visit.

9.5 Reimbursement for travelTravel reimbursement will be provided to caregivers to compensate them for the cost of transport to Study clinic for Study visits. Reimbursement will be at least KSh 300 (~US $5.00) for each visit.

9.6 End of study/final contact Since the children's last Study follow-up visit will include a number of laboratory tests, a final contact will be made with the caregiver of each child to provide the child's test results, and end of study counseling. Children will be referred to the PEPFAR-funded HIV Treatment Program to continue their HIV-1 care. At 1 year post-randomization we will trace children through the HIV treatment Program to enable abstraction of data regarding late IRIS and survival up to a year after ART initiation. Children who are not in active follow-up at the HIV Treatment Program will be traced to obtain endpoint information among survivors and verbal autopsy data regarding children who die. Our current studies have integrated clinical referral for children between care and research programs to facilitate transfer of clinical information between the two programs.

9.7 Participant retention Once a child/caregiver pair is enrolled in the study, the Study staff will make every reasonable effort to retain the pair in follow-up for 6 months of treatment with HAART.

9.8 Participant withdrawal Participants may voluntarily withdraw from the study for any reason at any time. The site investigator may also withdraw participants from the study in order to protect their safety and/or if the caregiver is unable to ensure that the child is showing good adherence to HAART.


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10 Safety monitoring and adverse event reporting

10.1 Data safety and monitoring plan A Data Safety and Monitoring Board (DSMB) will be convened prior to initiation of the study. The DSMB will involve pediatricians, HIV-1 experts with pediatric HIV-1 experience, biostatisticians, and epidemiologists.

Serious adverse event reports will be compiled and reported as described in Section 10.3.

The Study team will review monthly pooled summaries of serious adverse events (SAEs). The DSMB will review interim comparisons of the trial arms at approximately 6-monthly intervals after enrolment has begun. The DSMB will determine threshold values for the pooled monthly data to determine criteria for convening an emergency DSMB meeting. If pooled SAEs on monthly summaries are higher than anticipated (as pre-defined by the DSMB at first meeting), the DSMB will be urgently convened to evaluate study progress. Dr. Barbra Richardson will oversee preparation of interim study analyses and presentation to the DSMB. O’Brien-Fleming stopping rules will be used to determine whether early trial cessation is indicated.

10.2 Recording adverse eventsAll adverse events, including serious adverse events will be recorded on case report forms. Adverse events will be graded using the Division of AIDS (DAIDS) Table for Grading the Severity of Adult and Pediatric Adverse Events (Version 1.0, 2004, Clarification, 2009).

10.3 Reporting serious adverse eventsSerious adverse event (SAE) reports will be compiled within 72 hours of occurrence and disseminated to the Study team, and to the KNH ERC. SAEs that are possibly related to study intervention will be reported to the UW IRB. An SAE is an event associated with death, admission to hospital, prolongation of hospitalization, a persistent or significant disability or incapacity or an otherwise life threatening condition. In addition, urticaria or hepatoxicity grade>=2, any sign or symptom or biological result with grade >=3 will be considered an SAE.

10.4 Study discontinuationThis study may be discontinued at any time by the Protocol Chairs, the NIH, Kenyan Government or regulatory authorities or the IRBs.

11 Laboratory considerations

All specimen transport, processing, testing and results reporting will be conducted in compliance with standards of good clinical and laboratory practice. The study site will establish standard operating procedures, including specimen chain of custody and quality assurance and quality control procedures, for all protocol-specified laboratory tests prior to study initiation. All specimens will be transported in accordance with International Air Transport Association.

Blood will be used for diagnostic bacterial cultures, HIV-1 testing, to monitor immunologic and virologic response to ART, to monitor toxicity before and during ART, to measure inflammation, and to measure pathogen-specific changes in immune responses during immune reconstitution. Blood will also be used for the diagnosis of co-infections using serology (for EBV, CMV, toxoplasmosis, and Cryptococcus). Saliva will be used for herpesvirus diagnostics, nasal swab


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will be used for other respiratory virus diagnostics, sputum will be used for TB cultures, and urine and stool specimens will be used for clinical diagnostics. The following sections suggest potential laboratories that will conduct specific tests; however these may change subject to current local capacity to conduct the assays.

Variables: See Section 13.1 on study endpoints.

11.1 Local laboratory tests HIV-1 DNA PCR assays Rapid HIV-1 antibody testing CD4 and CD8 percent and count determination CBC CRP

Blood chemistries as possible including blood glucose, electrolytes, lipid profile, amylase, ALT, and creatinine

T-SPOT.TB Assays for inflammation, immune activation, and immune responses Blood cultures HIV-1 viral load assays

Materials and assays: Details of the above-mentioned tests are given below:

Rapid HIV-1 antibody test: Children (no longer considered to be exposed to maternal antibody) who are recruited to the study on the basis of a single rapid HIV-1 antibody test will have a confirmatory test at screening, using commercially available kits.

HIV-1 viral load assays: Viral load assays for the purpose of HIV-1 diagnosis will be conducted in local laboratories that have external quality assessment (EQA) in place as part of ongoing good clinical and laboratory practice (GCLP) accreditation. HIV-1 RNA for the purpose of assessing virological response to ART will be quantified using the Gen-Probe TMA assay in Dr. Overbaugh’s laboratory in Seattle, WA. This assay is sensitive for subtypes A, C, and D, which are prevalent in Nairobi [26] and can detect low copy numbers of HIV-1 in plasma, as low as ~10-25 copies per reaction [27].

CD4 and CD8: CD4 and CD8 percent and count will be determined in local laboratories using standard flow cytometry instrumentation in local laboratories with EQA as part of GCLP.

Assays for CBC and blood chemistries: glucose, electrolytes, lipid profile (total cholesterol, triglycerides, HDL, LDL), amylase, ALT, and creatinine will be conducted in a Kenyan nationally certified commercial diagnostic laboratories with internal and external quality assurance documentation.

Assays for inflammation, immune activation, and immune responses, and inflammation: Plasma and PBMC will be stored for the ascertainment of inflammation and pathogen-specific immune activation and responses. Table 7 summarizes samples, assays and rationale for these tests.


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Inflammation: C-reactive Protein (CRP) levels have been shown to increase during the development of IRIS [16]. CRP will be used as a general marker of inflammation, and will be measured using a commercial ELISA kit.

Immune activation: Frequencies of activated CD4 and CD8 T cells may predict mortality and are hypothesized to play a role in the immunopathogenesis of IRIS. We will use flow cytometry to quantify the frequency of activated CD4 and CD8 T cells, using HLA-DR/CD38 co-expression to identify activated cells.

Cellular immune responses: Baseline HIVgag-specific CD8 T cell responses and TB-specific (tuberculin and ESAT-6) CD4 T cell responses will be measured using IFN-gamma ELISpot assays.

Table 7. Planned assays to identify immunologic covariates of mortality and IRISSample & test

Factor Rationale Predicted association

Plasma ELISA

CRP level Marker of inflammation

Positive association with IRIS

PBMC flow cytometry

% HLA-DR+ CD38+(on CD4 &CD8)

Cellular activation Positive association with IRIS and/or mortality

PBMC ELISpot

HIV-specific IFN-gamma T cell response (CD4 & CD8)

Immune suppression

Positive association with IRIS, negative association with mortality

PBMCELISpot

TB-specific IFN-gamma T cell response (CD4)

Immunesuppression

Positive association with IRIS, negative association with mortality

Training procedures: All laboratory staff will be trained to follow all relevant guidelines and procedures.

Data collection instruments: See Section 14.3 regarding case report forms.

11.2 Quality assurance and quality controlFor HIV-1 diagnostic filter paper testing, quality assessment will include a repeat assay on-site as well as on-site control samples spiked with HIV-1 DNA that are run in parallel. Laboratory tests for HIV-1 RNA, CD4 and CD8, and blood chemistries will be conducted by laboratories with EQA systems in place.

11.3 Specimen storageStudy site staff will collect blood at enrollment and at visits specified in Section 9 from which all viable cells will be separated and stored at University of Nairobi

11.4 Biohazard containmentAs the transmission of HIV can occur through contact with contaminated needles, blood and blood products, appropriate precautions will be employed by all personnel in the drawing of


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blood, handling and shipping of blood samples during this study, as currently recommended by the United States Centers for Disease Control and Prevention.

12 Human subjects considerations

12.1 Institutional Review Board This is a collaborative research proposal that will involve field and laboratory procedures in Nairobi, Kenya and laboratory and data analyses in Seattle, Washington. The study will be reviewed by the Institutional Review Board (IRB) at the University of Washington and the Kenyatta National Hospital (KNH) Ethical Review Committee (ERC). The study will not recruit subjects prior to approval from both the University of Washington IRB and the KNH ERC.

12.2 Risks to subjectsRandomization armsThis is a randomized trial that will involve the initiation of ART during acute co-infection, or following stabilization of acute co-infection. The optimal time for ART initiation in children with advanced disease is unknown. Initiation of ART during treatment for an acute co-infection (Urgent ART arm) could result in an increased risk of side effects, hepatotoxicity, drug interactions (particularly for TB), and immune reconstitution inflammatory syndrome (IRIS). In order to avoid potential life-threatening cryptococcal-IRIS, we will exclude all children with suspected cryptococcal meningitis. Initiation of oral ART may require the placement of a nasogastric (NG) tube, which can cause vomiting, discomfort, nose bleeds, sinusitis, and sore throat; less commonly severe complications of NG tubes include ulcerations, esophageal perforation and aspiration. Initiation of early ART (within 7-14 days) could result in slower immune reconstitution and an increased risk for developing new opportunistic infections (including nosocomial). Additionally, if patients are referred to the Study Clinic to initiate ART following hospital discharge, there may be an increased risk of default (Eg. never seeking ART and HIV-1 care) in children who require ART.

CoercionParticipants will receive laboratory tests that are above the standard of care free of charge. Caregivers may feel coerced to enroll in the study in order to receive care within a research setting during the hospitalization of their child and during the initial months of follow-up on ART.

Specimen collectionThe study involves serial blood specimen sampling at enrollment, and weeks 1, 2, 4, 12 and 24. Phlebotomy can cause pain and bruising. At enrollment, saliva, nasal swabs, urine and stool specimens will be collected. Collection of these types of specimens causes little discomfort or is painless and is not associated with known risks to patients. Collection of a sputum sample can cause pain and discomfort. If sputum collection is performed through a gastric lavage, there is a risk of vomiting and, in rare cases, of aspiration. If a child is severely ill at the time of ART initiation, a nasogastric tube may be needed to administer ART. Using this tube may cause vomiting and discomfort.

Medical managementParticipation in the study has the potential to compromise care for hospitalized children, if it is prioritized above urgent clinical care for the acute infection.

Confidentiality of HIV status and data


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There is a non-negligible risk of loss of confidentiality of HIV-1 status or other medical information.

12.3 Adequacy of protection against risksRandomization armsSevere adverse events will be reported to the Study team and to the KNH ERC within 48 hrs of occurrence. A Data Safety Monitoring Board (DSMB) will review cumulative adverse events at 4 monthly intervals and make recommendations on continuation of the study or necessary adaptations based on interim analyses. Urgent ART may result in increased risk of IRIS, drug toxicity, and side effects. Early ART may result in increased mortality, development of OIs or slower immune reconstitution. These outcomes will be monitored closely to determine whether either treatment allocation is associated with adverse outcomes.

CoercionIn order to minimize the risk of coercion, Study staff will not be recruiting participants directly from the hospital. Patients will be managed by hospital attending physicians (with no involvement in the clinical trial) who will inform caregivers about the study, and offer to liaise interested caregivers with Study staff to discuss enrolment.

Medical managementHospital: In order to minimize the possibility that participation in the study will interfere with medical management of acute infections during hospitalization, Study personnel will have no role in the clinical management of hospitalized children other than randomization to treatment arm and ensuring administration of ART within the timeframes detailed in Section 7.1. Children will be treated and managed by hospital staff (who are not involved in the study) in accordance with standard procedures. Study clinic: Participants receiving HIV-1 care in the Study Clinic will be managed according to Kenyan National Guidelines and WHO recommendations for ART and the prevention of opportunistic infections. Participants experiencing adverse events while in the trial will be treated by the Study Clinicians or referred for care as appropriate. Adverse events will be reported to the Principal Investigators IRB, and DSMB as described in Section 10.3.

Confidentiality of HIV status and dataHIV status: All Key Personnel have been trained in the Protection of Human Subjects and HIPAA. The Nairobi Clinical Trials field team has more than 5 years experience managing HIV-1 infected children in the setting of clinical trials, and will take every precaution to protect participants’ and their caregivers’ confidentiality. Information regarding HIV-1 status will only be provided to the participants and their caregivers. For home-based visits, study staff will visit in plain clothes, and will keep the reason for their visit confidential. Data: Study identifiers will be linked to coded data; clinical staff will have access to patient identifiers, but the laboratory and analysts will receive only coded data. Links between patient identifiers and study codes will be kept for a period of 5 years after the end of the study, at which time the link between patient IDs and codes will be destroyed.

Disclosure:A CAB will be convened to advise on issues related to disclosure of the child’s HIV status to themselves, caregivers, and other family members.


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12.4 Potential benefits of the proposed research to the subjects and othersDirect benefits to participation in the study will include individualized HIV-1 care and intensive management during initiation of ART. Although many HIV-1 related treatments and services are provided for free by PEPFAR and/or the Kenyan government (including ART, tuberculosis treatment, and prophylaxis for opportunistic infections), the population accessing public Kenyan hospitals is very poor, and the cost of diagnostic tests and treatments is sometimes an obstacle to good medical care. Although the study cannot assume the entire cost of medical care for each participant, the study will provide study-specific tests and treatments. This is expected to result in better than standard level of care in Kenyan hospitals. Since we do not know the optimal time to initiate ART in very sick children, this study has great potential to inform national and international guidelines for ART initiation in children.

12.5 Importance of the knowledge to be gainedThere is no way of determining the optimal timing of ART initiation without conducting a randomized trial. Participation in a randomized clinical trial is not without risk. However, we do not know whether urgent or post-stabilization ART initiation will be better for severely ill HIV-infected children. Urgent ART may be associated with an increased risk of side effects, hepatotoxicity, drug interactions, and immune reconstitution inflammatory syndrome (IRIS). However, delaying ART until stabilization of acute co-infection could result in slower immune reconstitution, an increased risk for developing new opportunistic infections, and increased likelihood of defaulting therapy. It is also important to note that post-stabilization ART more closely approximates normal practice in starting very ill children on ART; and mortality in this group is very high. The results of this trial will inform treatment guidelines, and will have the potential to benefit the millions of children with undiagnosed HIV-1.

12.6 Care following study completionAll children who exit the study will be referred for continued treatment in the local Pediatric HIV-1 Treatment Clinic or a health facility of their choice. Dr. Dalton Wamalwa, co-investigator, will facilitate links to ensure that children are maintained on appropriate protocols for treatment and monitoring. Local Pediatric HIV-1 treatment clinics receive PEPFAR Funding to provide free antiretroviral therapy. Currently available regimens include NVP-containing and PI-containing HAART.

13 Statistical analysis plans

13.1 EndpointsThe study endpoints are mortality, IRIS, and drug toxicity. A comprehensive review of current literature will guide definition of IRIS. For example, the following published pediatric IRIS case definition may be used [28]: 1. Response to ART by receiving HIV ART and virologic response with >1 log decrease in HIV-1 RNA; 2. Clinical deterioration of an infectious or inflammatory condition temporally related to ART initiation; 3. Symptoms cannot be explained by expected clinical course of a previously recognized and successfully treated infection, medication side effect or toxicity, treatment failure, or complete non-adherence. Drug toxicity definitions will be measured using DAIDS toxicity tables.


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Mortality in the urgent vs early ART arms will be compared at 1, 3, and 6 months. Similarly, IRIS and drug toxicity in the urgent vs early ART arms will be compared at 1, 3, and 6 months.

13.2 Data analysis Primary clinical trial analyses will be intent-to-treat based on randomized allocation. Secondary “as-treated” analyses will also be conducted.

13.2.1 Aim 1To compare the 6 month all-cause mortality rate, incidence of IRIS, and incidence of drug toxicity in HIV-1 infected children with a serious infection randomized to urgent (within 48 hours) versus early (within 7-14 days) ARTPrimary clinical trial analyses will be intent-to-treat based on randomized allocation. Secondary “as-treated” analyses will also be conducted. To determine if the randomization to urgent versus early ART resulted in balanced groups, baseline measurements of the children will be compared between the randomization groups using Chi-square tests for categorical variables and the Mann-Whitney U test for continuous variables. Loss to follow-up in the two arms will be compared using Kaplan Meier survival analysis. The primary outcome of the study, mortality will be compared at 1, 3, and 6 months post-randomization using Kaplan-Meier survival analysis, and cumulative incidence of mortality compared using Cox proportional hazards regression analysis. Similar analyses will be conducted using either IRIS or drug-toxicity as an endpoint. Potential cofactors of interest include age, immunosuppression (low CD4%), high viral load, low WHZ, immune activation, and pathogen-specific immune responses. In addition, a combined adverse events analysis will be conducted evaluating time to mortality, IRIS, or toxicity and cofactors for combined adverse outcome. Cox proportional hazards regression analyses will be conducted to determine cofactors for mortality, IRIS, or toxicity. If there are sufficient numbers of a specific pathogen-related IRIS, subset analyses to explore cofactors for IRIS within the pathogen of interest will also be conducted.

13.2.2 Aim 2To determine co-factors for mortality, IRIS, and drug toxicityBaseline measurements of immune parameters will be used in multivariate Cox regression models (including randomization arm, CD4, age, and Z-scores) to test for associations with the risk of mortality or IRIS. Baseline immune activation and pathogen-specific immune responses will be compared for children who did and did not develop IRIS using t-tests and non-parametric statistical tests. 1- 2- and 4- week T-cell activation and pathogen-specific immune responses will be compared between the trial arms. A subset of specimens will be used for longitudinal studies of HIV-1 and pathogen specific immune responses in the two study arms and among children with suspected IRIS. In addition, the two trial arms will allow comparison of baseline T-cell phenotype and activation markers and impact in the context of ART administered either concurrent with co-pathogen vs. following clearance of co-pathogen, lending unique insights into the development of IRIS.

13.2.3 Secondary aimTo determine etiologies of IRIS and to compare immune reconstitution to HIV, TB, EBV and CMV following ART overall and in each trial armAmong children meeting the case-definition of IRIS, medical histories, clinical records, and laboratory diagnostics will be used to identify the etiologies of pediatric IRIS. Pediatric IRIS


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reflects diverse etiologies and the study will provide important prospective data for clinicians. Pathogen-specific immune responses using ELISpot and flow cytometry and pathogen burden (if applicable) for HIV-1, TB, EBV, and CMV will be longitudinally compared between children who did and did not develop IRIS.

14 Administrative procedures

14.1 Protocol complianceThis study will be conducted in compliance with the protocol. Any protocol amendments will be reviewed and approved by the relevant IRBs/ECs prior to implementing the amendments except when necessary to protect the safety, the rights or welfare of the children and their caregivers, or to eliminate apparent immediate hazards to participants.

14.2 Protocol deviations/violationsAll protocol deviations/violations will be documented by the Study coordinator with an explanation of the deviation. The staff responsible for the protocol deviation/violation will write a memorandum listing the deviation to the Study coordinator as soon as possible. This memorandum will include a description of the deviation, background information and the solution to prevent such a deviation in the future. A summary of protocol violations will be reported to the Sponsor (NIH), University of Washington IRB and KNH ERC and included in the interim data reports.

14.3 Study coordinationStudy implementation will be directed by this protocol and the study standard operating procedure manual.

Standard operating procedures (SOPs): SOPs will be developed for all clinical, laboratory, pharmacy, and data operations and will be reviewed by an independent monitor prior to their use. Clinical and laboratory procedures will be conducted using GCLP guidelines.

Case report forms will be developed by the protocol team. Data will be entered and cleaned on site. Data quality control reports and queries will be routinely generated and reviewed by the lead investigators. The on-site Study team will resolve any queries. Case report forms will form part of the study source documents.

14.4 Study monitoringAn independent NIH contracted Study Monitor will review the protocol and SOPs prior to trial initiation. It is anticipated that the Study Monitor will review the trial at 6-month intervals throughout the duration of the study.

Study Monitors will conduct periodic visits to the study site to: Verify compliance with the human subjects and other research regulations and guidelines Assess adherence to the study protocol and study procedures manual Confirm the quality and accuracy of information collected at the study site and entered into the study database

The Site Investigators will allow Study Monitors to inspect study facilities and documentation (e.g., informed consent forms, clinic and laboratory records, case report forms), as well as PUSH Protocol19 February, 2015Version 6

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observe the performance of study procedures (if participants consent). A site visit log will be maintained at the study site to document all visits.

14.5 Study recordsSite Investigators will maintain, and store in a secure manner, complete, accurate, and current study records throughout the study. The investigator will retain all study records for at least five years after completion of the study. Study records include administrative documentation and regulatory documentation as well as documentation related to each participant screened and enrolled in the study, including informed consent forms, locator forms and case report forms from all visit during the course of the study.

14.6 Use of Information and publicationsThe results of this study will be published collaboratively by the University of Nairobi and University of Washington in compliance with the National Institute of Child Health and Human Development (NICHD) guidelines.

15 Study limitations

Recruitment for this study may be difficult as potential subjects are likely to be severely ill in the hospital. As a result, participants might be a self-selected group prone towards higher ART adherence and care seeking behaviors. The relatively small difference in timing of ART initiation between the two randomization groups could limit the effect observed.

16 Time frame

Year 1: Preparation of protocol; obtain necessary ethical and regulatory approvalsYears 2-3: 24 month recruitment periodYear 4: 6-12 month follow-up of participantsYear 5: Study close-out; data analysis; preparation of results for publication


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References1. Althoff KN, Gange SJ, Klein MB, et al. Late presentation for human immunodeficiency virus care in the United States and Canada. Clin Infect Dis;50:1512-20.2. Brooks JT, Kaplan JE, Holmes KK, Benson C, Pau A, Masur H. HIV-associated opportunistic infections--going, going, but not gone: the continued need for prevention and treatment guidelines. Clin Infect Dis 2009;48:609-11.3. Newell ML, Coovadia H, Cortina-Borja M, Rollins N, Gaillard P, Dabis F. Mortality of infected and uninfected infants born to HIV-infected mothers in Africa: a pooled analysis. Lancet 2004;364:1236-43.4. Obimbo EM, Mbori-Ngacha DA, Ochieng JO, et al. Predictors of early mortality in a cohort of human immunodeficiency virus type 1-infected african children. Pediatr Infect Dis J 2004;23:536-43.5. Obimbo EM, Wamalwa D, Richardson B, et al. Pediatric HIV-1 in Kenya: pattern and correlates of viral load and association with mortality. J Acquir Immune Defic Syndr 2009;51:209-15.6. Wamalwa DC, Farquhar C, Obimbo EM, et al. Early response to highly active antiretroviral therapy in HIV-1-infected Kenyan children. J Acquir Immune Defic Syndr 2007;45:311-7.7. Violari A, Cotton MF, Gibb DM, et al. Early antiretroviral therapy and mortality among HIV-infected infants. N Engl J Med 2008;359:2233-44.8. Peacock-Villeda E RB, John-Stewart G. Clinical Outcomes of HAART in Pediatric Populations: A Systematic Comparison of Developing and Developed Countries. . Conference on Retrovirology and Opportunistic Infections 2009.9. Zolopa A, Andersen J, Powderly W, et al. Early antiretroviral therapy reduces AIDS progression/death in individuals with acute opportunistic infections: a multicenter randomized strategy trial. PLoS One 2009;4:e5575.10. Abdool Karim S NK, Grobler A, Padayatchi N, Baxter C, Gray A, Gengiah T, Nair G, Bamber S, Singh A, Khan M, Pienaar J, El-Sadr W, Friedland G, Abdool Karim Q. . Timing of Initiation of Antiretroviral Drugs during Tuberculosis Th. N Engl J Med 2010;362:697-706.11. Makadzange AT, Ndhlovu CE, Takarinda K, et al. Early versus delayed initiation of antiretroviral therapy for concurrent HIV infection and cryptococcal meningitis in sub-saharan Africa. Clin Infect Dis;50:1532-8.12. Torok ME YN, Chau T, et al. Randomized controlled trial of immediate versus deferred antiretroviral therapy in HIV-associated tuberculous meningitis. 49th Interscience Conference on Antimicrobial Agents and Chemotherapy 2009:Abstract H 1224.13. Smith K, Kuhn L, Coovadia A, et al. Immune reconstitution inflammatory syndrome among HIV-infected South African infants initiating antiretroviral therapy. Aids 2009;23:1097-107.14. Puthanakit T, Oberdorfer P, Akarathum N, Wannarit P, Sirisanthana T, Sirisanthana V. Immune reconstitution syndrome after highly active antiretroviral therapy in human immunodeficiency virus-infected thai children. Pediatr Infect Dis J 2006;25:53-8.15. Puthanakit T, Oberdorfer P, Punjaisee S, Wannarit P, Sirisanthana T, Sirisanthana V. Immune reconstitution syndrome due to bacillus Calmette-Guerin after initiation of antiretroviral therapy in children with HIV infection. Clin Infect Dis 2005;41:1049-52.16. Meintjes G, Rabie H, Wilkinson RJ, Cotton MF. Tuberculosis-associated immune reconstitution inflammatory syndrome and unmasking of tuberculosis by antiretroviral therapy. Clin Chest Med 2009;30:797-810, x.


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17. Lohman BL, Slyker JA, Richardson BA, et al. Longitudinal assessment of human immunodeficiency virus type 1 (HIV-1)-specific gamma interferon responses during the first year of life in HIV-1-infected infants. J Virol 2005;79:8121-30.18. Shalekoff S, Meddows-Taylor S, Gray GE, et al. Identification of human immunodeficiency virus-1 specific CD8+ and CD4+ T cell responses in perinatally-infected infants and their mothers. Aids 2009;23:789-98.19. Wilson CB, Westall J, Johnston L, Lewis DB, Dower SK, Alpert AR. Decreased production of interferon-gamma by human neonatal cells. Intrinsic and regulatory deficiencies. J Clin Invest 1986;77:860-7.20. White GP, Watt PM, Holt BJ, Holt PG. Differential patterns of methylation of the IFN-gamma promoter at CpG and non-CpG sites underlie differences in IFN-gamma gene expression between human neonatal and adult CD45RO- T cells. J Immunol 2002;168:2820-7.21. Lohman-Payne B, Slyker JA, Richardson BA, et al. Infants with late breast milk acquisition of HIV-1 generate interferon-gamma responses more rapidly than infants with early peripartum acquisition. Clin Exp Immunol 2009;156:511-7.22. Michaelsson J, Mold JE, McCune JM, Nixon DF. Regulation of T cell responses in the developing human fetus. J Immunol 2006;176:5741-8.23. Legrand FA, Nixon DF, Loo CP, et al. Strong HIV-1-specific T cell responses in HIV-1-exposed uninfected infants and neonates revealed after regulatory T cell removal. PLoS One 2006;1:e102.24. Willems F, Vollstedt S, Suter M. Phenotype and function of neonatal DC. Eur J Immunol 2009;39:26-35.25. Wamalwa DC, Obimbo EM, Farquhar C, et al. Predictors of mortality in HIV-1 infected children on antiretroviral therapy in Kenya: a prospective cohort. BMC Pediatr;10:33.26. Emery S, Bodrug S, Richardson BA, et al. Evaluation of performance of the Gen-Probe human immunodeficiency virus type 1 viral load assay using primary subtype A, C, and D isolates from Kenya. Journal of clinical microbiology 2000;38:2688-95.27. DeVange Panteleeff D, Emery S, Richardson BA, et al. Validation of performance of the gen-probe human immunodeficiency virus type 1 viral load assay with genital swabs and breast milk samples. Journal of clinical microbiology 2002;40:3929-37.28. Boulware DR, Callens S, Pahwa S. Pediatric HIV immune reconstitution inflammatory syndrome. Curr Opin HIV AIDS 2008;3:461-7.


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Appendix1. Consent for HIV-1 Testing2. Assent for HIV-1 Testing3. Consent for Enrollment4. Assent for Enrollment5. Study Budget


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