HIV viral load assays when used with whole blood perform well as a diagnostic assay for infants

Objective Over the past several years, only approximately 50% of HIV-exposed infants received an early infant diagnosis test within the first two months of life. While high attrition and mortality account for some of the shortcomings in identifying HIV-infected infants early and putting them on life-saving treatment, fragmented and challenging laboratory systems are an added barrier. We sought to determine the accuracy of using HIV viral load assays for infant diagnosis of HIV. Methods We enrolled 866 Ugandan infants between March–April 2018 for this study after initial laboratory diagnosis. The median age was seven months, while 33% of infants were less than three months of age. Study testing was done using either the Roche or Abbott molecular technologies at the Central Public Health Laboratory. Dried blood spot samples were prepared according to manufacturer-recommended protocols for both the qualitative and quantitative assays. Viral load test samples for the Roche assay were processed using two different buffers: phosphate-buffered saline (PBS: free virus elution viral load protocol [FVE]) and Sample Pre-Extraction Reagent (SPEX: qualitative buffer). Dried blood spot samples were processed for both assays on the Abbott using the manufacturer’s standard infant diagnosis protocol. All infants received a qualitative test for clinical management and additional paired quantitative tests. Results 858 infants were included in the analysis, of which 50% were female. Over 75% of mothers received antiretroviral therapy, while approximately 65% of infants received infant prophylaxis. The Roche SPEX and Abbott technologies had high sensitivity (>95%) and specificity (>98%). The Roche FVE had lower sensitivity (85%) and viral load values. Conclusions To simplify and streamline laboratory practices, HIV viral load may be used to diagnose HIV infection in infants, particularly using the Roche SPEX and Abbott technologies. This was a blinded, cross-sectional, prospective study to investigate the diagnostic accuracy of laboratory-based viral load quantitative assays to determine HIV infection compared to laboratory-based, qualitative infant diagnosis assays. All testing occurred at the Central Public Health Laboratory in Kampala, Uganda using remnant samples from routinely collected dried blood spot samples. Samples were received in the laboratory through the national infant diagnosis system from any health care facility in the country submitting a clinical sample from an HIV-exposed infant less than 18 months of age for routine diagnosis. Sample receipt, processing, and testing occurred between March and August 2018. All clinical samples were tested using the Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 Qualitative Test, v2.0 (total nucleic acid detected)–these results were provided to the health care facility, health care workers, and caregivers to manage the infant’s care. Samples were purposefully selected in that all consecutively collected positive samples and an equal number of randomly selected negative samples were included and blindly tested each week until the target sample size was met. Most (179) of the negative samples were used for both the Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 Test, v2.0 and Abbott RealTime HIV-1 viral load assays (RNA only detected); however, 70 additional consecutive negative samples were collected for testing using the Abbott viral load assay, as the original samples were insufficient for testing with both assays. Separate sets of consecutively collected positive samples were used for the two technologies (Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 Test, v2.0 and Abbott RealTime HIV-1 viral load), because the majority of positive samples did not have sufficient remaining spots available as all positive samples in routine clinical care are repeat tested in the laboratory prior to result dispatch. Demographic and clinical data were collected from each patient using routine national requisition forms, including age, sex, maternal treatment, infant prophylaxis, and breastfeeding status. The cycle threshold of both qualitative and quantitative assays were captured as well as the qualitative result (detected or not detected) and viral load result from the quantitative assay. Dried blood spot preparation and testing for the qualitative assays were conducted as previously described for the Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 Qualitative Test, v2.0 [18]. Dried blood spots were prepared in two ways for the Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 Test v2.0, using SPEX and PBS (free virus elution: FVE protocol) buffers [18, 19]. In brief, one spot was cut out using a pair of scissors or 12mm circular punch, transferred with forceps to an S-tube and 1100 ul of Sample Pre-Extraction Reagent (SPEX) was added; the tubes were incubated in a thermomixer at 56°C and shaken at 1000 rpm for ten minutes before being loaded on to the sample rack for testing. For the COBAS AmpliPrep/COBAS TaqMan HIV-1 Qualitative Test, v2.0 using the FVE protocol, one spot was cut out using a pair of scissors or 12 mm circular punch, transferred with forceps to an S-tube and 1000 ul of calcium- and magnesium-free Phosphate buffered saline (PBS) buffer added; the tubes were incubated at room temperature for at least 30 minutes or overnight. The tubes were gently tapped at the bottom to homogenize the solution before being loaded on to the sample rack for testing. Dried blood spots for the Abbott RealTime HIV-1 Viral Load assay were prepared similarly to those prepared for the Abbott RealTime HIV-1 Qualitative assay [20]. In brief, one spot was punched from the card using a sterile pipet tip, placed in a tube, and 1300 ul of mSample Preparation System buffer added; the tubes were manually swirled to ensure the spot was fully submerged, and incubated in a thermomixer at 55°C for 30 minutes. Tubes were then manually swirled again before being transferred directly to the sample rack for testing. Alternatively, as a sub-analysis to determine if a different sample preparation might improve performance, we also processed a separate set of samples using a modified dried blood spot sample preparation protocol, in which two spots were submerged in 1500 ul of mDBS buffer (all other steps remaining consistent). The sensitivity and specificity of using the viral load assays to accurately diagnose HIV infection were calculated using the Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 Qualitative Test, v2.0 assay as this assay is currently the standard test used for clinical management in Uganda. The score-based Wilson method [21] was used to construct confidence intervals for sensitivity and specificity. Confidence intervals for Cohen’s Kappa were estimated [22]. McNemar’s chi-squared test for symmetry of rows and columns in a two-dimensional contingency table was estimated [23]. Further, a sub-analysis was conducted comparing the performance of the quantitative assay with the qualitative assay in infants exposed to antiretroviral drugs–either through infant prophylaxis or maternal treatment. All statistical analyses were performed in the R statistical computing environment. This study was approved by the Uganda National Council for Science and Technology; the Higher Degrees, Research and Ethics Committee from Makerere University, Uganda; Chesapeake International Review Board in the United States; and the Ethics Review Committee from the World Health Organization, Geneva, Switzerland. Informed consent was waived by each ethical review committee because of the use of routine, leftover clinical samples. The data were fully anonymized prior to access and analysis. Viral load test results were not provided to patients. The routine clinical qualitative infant diagnosis test results were returned to the health care facility and caregiver per national guidelines.