Purpose: The purpose of this research study is to learn about two HIV tests - a clinical "presumptive diagnosis" (PD) that a trained healthcare provider can quickly use to determine if a child is likely to be HIV-infected and in need of HIV medicines and an "expedited" gold standard RNA-PCR test (expedited PCR) that is done at the UNC Project lab located at the hospital and the result given within 48 hours. Both of these tests can obtain results quickly while the current test called dried blood spot DNA-PCR goes to a lab and the result may take up to one month. The performance of PD and expedited PCR will be compared to one another with respect to HIV-infected infants correctly initiating life-saving antiretroviral therapy.

Participants: Hospitalized children younger than 12 months of age who are HIV DNA-PCR eligible at Kamuzu Central Hospital (KCH), in Lilongwe, Malawi. Other participants will be patient caregivers and clinical officers who provide healthcare for children that could be HIV-infected. Clinical officers will be trained to conduct the presumptive diagnosis test.

Procedures (methods): Patients will be randomized to either standard of care (PD and dried blood spot DNA-PCR) or expedited PCR. A consultant pediatrician and a clinical officer will perform the PD. If the PD or expedited PCR test results are positive, hospital care could include HIV medicine.

Background A majority of HIV-infected children die by two years of age without antiretroviral therapy (ART). Unfortunately, diagnosing HIV in this age group is complex as it requires virologic testing. This has served as a barrier to younger HIV-infected (HIV+) children accessing ART in resource-constrained generalized epidemic countries. Malawi, a high HIV-prevalence country in sub-Saharan Africa, implemented a national early infant diagnosis (EID) program utilizing virologic testing with dried blood spots (DBS) DNA PCR technology in 2007 to address this gap.

In coordination with the national EID program in 2008, we implemented a routine HIV testing strategy within the pediatric inpatient ward at Kamuzu Central Hospital (KCH) that incorporated DBS. While our initial program results have been encouraging, we have observed that only 50.2% of DBS recipients have returned for their test results after hospital discharge, draining resources and reducing the potential impact of definitive virologic testing and early diagnosis. DBS technology is a contributing factor to low patient follow-up as it requires burdensome transport and lengthy processing by highly trained laboratory personnel, requiring patients to return several weeks after testing for their results. Currently in Malawi, as is the case throughout sub-Saharan Africa, definitive virologic testing that provides test results prior to hospital discharge is not routinely available.

Two alternatives that could potentially strengthen EID and pediatric ART access are the routine use of presumptive diagnosis (PD), or a clinical HIV diagnostic algorithm in young children, as an adjunct to DBS and definitive inpatient virologic testing that provides results before hospital discharge ("expedited PCR") instead of PD and DBS. This research proposal primarily aims to validate PD testing as well as assess the impact of PD and expedited PCR upon patient outcomes within the established inpatient pediatric HIV testing system at KCH.

RESEARCH QUESTION TO BE ADDRESSED BY THIS PROPOSAL Is presumptive diagnosis, as an adjunct to dried blood spot DNA PCR, a valid approach for diagnosing HIV in hospitalized Malawian children younger than 12 months of age, and how does this strategy, compared to an expedited inpatient virologic diagnosis, affect patient outcomes?

RATIONALE FOR RESEARCH

Globally, an estimated 2.2 million children younger than 15 years of age were HIV-infected in 2007, with approximately 90% living in sub-Saharan Africa. Malawi, a sub-Saharan African country of 13.2 million people, reports an adult HIV prevalence of 11.9%. While 91,000 HIV+ children were estimated to be living in Malawi in 2007, less than 8% of all Malawian children have ever been HIV tested. Additionally, HIV+ children comprised just 8% of all national ART recipients, well below the 2014 goal of 15%. To address this gap, Malawi approved the routine offering of HIV testing, or provider initiated HIV testing and counseling (PITC), in line with global recommendations in 2007. While there is a high HIV prevalence in hospitalized Malawian children and current guidelines support routine inpatient HIV testing, no clear implementation strategy for hospitals has been offered.

Substantial percentages of HIV+ infants suffer from high mortality without early infant diagnosis (EID) and ART initiation, irrespective of CD4%, leading to the recommendation that all HIV+ children younger than 1 year of age should be universally initiated on ART. Despite the simplification of ART eligibility in this age group, diagnosing HIV remains complex. Maternal HIV antibodies can circulate in the blood stream of children until 12 months of age, confounding HIV antibody test results unable to discriminate between the mother's and child's antibodies. Accordingly, DNA PCR testing that detects HIV proviral DNA is the gold standard for HIV diagnosis in this age group. To date the PCR technology utilized in EID programs throughout sub-Saharan Africa, including Malawi, is DBS. Malawi piloted an EID program starting in 2007 and approved a policy of universal ART for all HIV+ children younger than 1 year of age in 2008. In Malawi, a uniform first line fixed-dose combination ART regimen of stavudine, lamivudine, and nevirapine is provided free of charge by the Malawian government for all eligible patients.The EID program has since expanded to 14 of 28 districts. Importantly, the national EID program identified the pediatric inpatient wards as a critical access point for routine DBS testing, but recently reported that few EID sites have established routine inpatient testing programs.

KCH is a referral hospital with a 215 bed pediatric ward located in the capital of Lilongwe with more than 10,000 pediatric admissions annually. In 2007, less than 10% of pediatric inpatients at KCH were estimated to have accessed voluntary HIV counseling and testing (VCT) services offered by lay counselors (Pediatric Department, KCH, unpublished data). In January 2008, we implemented a PITC program utilizing DBS testing in the KCH inpatient pediatric department in collaboration with the Baylor International Pediatric AIDS Initiative (BIPAI), KCH, and Lighthouse. The overall program objectives were to improve inpatient pediatric HIV diagnosis and care, establish EID as a routine inpatient service, and provide national guidance for inpatient pediatric PITC program expansion.

Over the first 12 months of implementation, the PITC program offered HIV testing to 45.2% of admissions, or 10,245 mothers and their hospitalized children, representing a four-fold increase in comparison to pre-PITC estimates1. 98.7% of mother-child pairs accepted testing, and we observed the HIV prevalence of hospitalized children to be 8.5%, nearly two-fold higher than the estimated community pediatric prevalence of 4.8% in Malawi. Furthermore, 73.9% of HIV+ hospitalized children had not yet accessed ART , despite over 70% of these patients clinically or immunologically eligible. The program also completed 476 DBS tests during the same time period, representing the highest volume EID site in Malawi, with 40.1% testing positive.

Unfortunately, only 239 DBS recipients, or 50.2% of children tested, enrolled into outpatient care at the Baylor College of Medicine-Abbott Fund Children's Clinical Centre of Excellence clinic at KCH. Of these, 48.3% were initiated on ART a median of 2.4 months after DBS collection. Low follow-up has been experienced at other EID sites throughout Malawi. One contributing factor is the several weeks of processing time required for DBS samples. In Malawi, DBS tests are transported to and from central laboratories to be processed in higher volumes by sophisticated equipment and highly specialized laboratory personnel. Therefore, counselors instruct caregivers to return in at least one month for their child's DBS test results. If caregivers return for their child's results, they are then commonly re-referred for HIV care at another site, often at an even later date, further delaying evaluation for ART eligibility. Currently, a similar system exists at KCH. Despite substantial investment into EID, the processing and transport delays inherent to DBS have lessened the impact of EID and have resulted in a continued barrier to pediatric ART access in this age group.

Two diagnostic alternatives that could reduce the delays complicating the current DBS systems are PD and point of care virologic testing. While PD is a less sensitive and specific diagnostic alternative to PCR, it requires limited resources and provides a strategy to immediately offer ART to hospitalized patients that fulfill PD criteria, avoiding the one-month wait time and resultant losses to follow-up. Malawi National guidelines recommend the use of PD to identify and initiate symptomatic HIV+ infants younger than 12 months of age on ART when PCR is unavailable. While the sensitivity and specificity of PD has been previously reported from Rwanda and Kenya, these studies are only partly applicable to Malawi due to the lower estimated malaria burdens in Rwandese children, and the mixture of inpatients and outpatients within the Kenyan study cohort. Both scenarios are likely to alter the reported sensitivities and specificities of PD as compared to Malawi, justifying the need to generate locally applicable data. To date, PD has not been widely practiced in Malawi, and data generated from this research could provide guidance to increase the usefulness of PD. The primary limitation to PD is that HIV-uninfected patients could be incorrectly started on ART. However, it is possible that such patients might still experience lower mortality and HIV transmission rates, with little adverse effects, as suggested by the recent outcomes of extended post-natal antiretroviral prophylaxis studies.

Point of care virologic testing, on the other hand, has the potential to revolutionize pediatric HIV care as HIV+ hospitalized infants could receive same day, accurate diagnostic results, allowing immediate evaluation for ART eligibility and initiation. While no point of care virologic test has yet been approved for routine use, the enhanced laboratory services available at University of North Carolina Project (UNC Project) can offer HIV RNA PCR test results in less than 48 hours. "Expedited" virologic testing is likely to provide definitive HIV test results to nearly all hospitalized HIV-exposed infants prior to discharge, therefore replicating the affect that point of care virologic testing will produce in the hospitalized setting. Understanding how expedited PCR testing affects inpatient ART uptake, outpatient follow-up, and longer term outcomes, compared to the current standard of care, will be critical for further system development and strengthening in anticipation of future point of care test availability.

This research proposal seeks to utilize the current inpatient pediatric PITC system established at KCH to validate PD within the hospital setting, as well as investigate patient outcomes utilizing current EID standard of care (PD and DBS) compared to expedited PCR.

Inclusion Criteria:

• Male and female children
• Less than 12 months of age
• Inpatient at KCH
• HIV DNA-PCR test eligible
• No prior definitive PCR test result

Exclusion Criteria:

• Caregiver does not want to be contacted by study team
• Mother initiated on ART prior to pregnancy