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Introduction: Viral load testing is essential to manage HIV disease, especially in infants and children. Early infant diagnosis is performed using nucleic-acid testing in children under 18 months. Resource-limited health systems face severe challenges to scale-up both viral load and early infant diagnosis to unprecedented levels. Streamlining laboratory systems would be beneficial to improve access to quality testing and to increase efficiency of antiretroviral treatment programmes. We evaluated the performance of viral load testing to serve as an early infant diagnosis assay in children younger than 18 months. Methods: This study was an observational, prospective study, including children between one and 18 months of age who were born to HIV-positive mothers in 134 health facilities in Maputo City and Maputo Province, Mozambique. Dried blood spot specimens from heel or toe pricks were collected between January and April 2018, processed using SPEX buffer for both assays, and tested for routine EID and VL testing using the Roche CAP/CTM HIV-1 Qualitative v2 and Roche CAP/CTM HIV-1 Quantitative v2 assays respectively. The sensitivity, specificity and positive and negative predictive values were estimated using the EID results as the reference standard. Results: A total of 1021 infants were included in the study, of which 47% were female. Over 95% of mothers and children were on antiretroviral treatment or received antiretroviral prophylaxis respectively. The sensitivity and specificity of using the viral load assay to detect infection were 100% (95% CI: 96.2 to 100%) and 99.9% (95% CI: 99.4 to 100%). The positive and negative predictive values were 99.0% (95% CI: 94.3 to 100%) and 100% (95% CI: 99.6 to 100%). The McNemar’s test was 1.000 and Cohen’s kappa was 0.994. Conclusions: The comparable performance suggests that viral load assays can be used as an infant diagnostic assay. Infants with either low levels of viraemia or high cycle threshold values should be repeat tested to ensure the result is truly positive prior to treatment initiation, regardless of assay used. Viral load assays could replace traditional early infant diagnosis testing, substantially streamlining molecular laboratory services for children and lowering costs, with the additional advantage of providing baseline viral load results for antiretroviral treatment management.
This was an observational, prospective study that included infants between one and 18 months of age born to HIV‐1‐positive mothers in need of a routine HIV Early Infant Diagnosis (EID) test. Infants excluded from the study were those less than one month and older than 18 months of age or with low quality specimen, according to the rejection criteria used for HIV EID routine testing in Mozambique. Samples were collected from 134 health facilities that attend to infants born to HIV‐positive mothers in Maputo City and Maputo Province between January and April 2018. Dried blood spot specimens were collected and referred to the National Institute of Health Reference laboratory in Maputo for EID routine testing and viral load testing. Demographic and clinical data for study participants were collected using a routine EID form, including gender, age and exposure to maternal treatment and infant prophylaxis. Because patient identifiers were not collected, remnant spots from routine clinical samples were used, and only standard clinical test results were provided to caregivers and clinicians, individual consent was waived and approval by the Institutional Review Boards that reviewed the protocol. Dried blood spot specimens (Whatman 903, GE Healthcare Biosciences, Pittsburgh, PA, USA) were drawn from the heel or toe pricks of eligible infants and transported within three weeks to the reference laboratory for early infant diagnosis HIV‐1 PCR testing using the Roche CAP/CTM 96 HIV‐1 Qualitative Test v2 (Roche Molecular Diagnostics, Branchburg, NJ, USA). Low quality specimens were excluded, including those without full dried blood spots and when two or more cards were in the same ziplock bag without glassine paper between the cards. This test detects extracellular and intracellular HIV‐1 RNA and proviral DNA in whole blood specimens. The Roche software automatically corrects for the haematocrit value in dried blood spot specimens. Infants were determined positive when the Roche CAP/CTM qualitative EID assay reported a detectable result with a cycle threshold less than 31. National policy states that laboratories should implement an indeterminate range that includes results with a cycle threshold (Ct) value of 31 or greater using the Roche CAP/CTM EID assay; infants with an initial EID cycle threshold value in this range received a second EID test, if possible, either on the same or a new sample before a definitive test result is determined. The EID definitive result was determined based on the test result of the second (repeat) Roche CAP/CTM EID test. If the repeat EID test result was target not detected, the infant was determined to be HIV negative. If the repeat EID test result was a detectable result, the infant was determined to be HIV positive. In this study, the viral load results were not used to determine positivity nor were they returned to the healthcare facility or caregiver. After the EID test result, HIV‐1 viral load testing was performed using the Roche CAP/CTM 96 HIV‐1 Quantitative Test v2 (Roche Molecular Diagnostics, Branchburg, NJ, USA) using remnant specimens. The Sample Pre‐Extraction (SPEX) solution was used for DBS elution for both qualitative and quantitative testing. The routine EID test results were returned to the healthcare facility and caregiver per national guidelines. Viral load test results were not provided to health care facilities or caregivers and were used for study purposes only. The reference laboratory routinely participated in and passed external quality assessment programmes for both EID and viral load (provided by the Center for Disease Control and Prevention, Atlanta, USA) prior to and during the study period. The sensitivity and specificity of quantitative testing (HIV viral load) as well as the positive and negative predictive values were estimated using the qualitative assay (EID) as the reference. Cohen’s kappa and McNemar’s test were also performed. Only valid results (detectable or undetectable) were used for these calculations; invalid results were excluded. Additionally, we conducted a sub‐analysis to determine the diagnostic accuracy of the viral load and EID assays if the indeterminate range is not implemented and the EID definitive result is based solely on the initial EID test result. We compared demographic data between HIV‐positive and HIV‐negative groups using the Chi‐square test. Data were analysed using SAS/STAT software version 9.4 (SAS Institute Inc, Cary, NC, USA). This study was approved by the Mozambique National Health Bioethics Committee, Advarra Institutional Review Board in the USA, and the Ethics Review Committee from the World Health Organization, Geneva, Switzerland.
