By integrating PIMA testing into AHD care protocols, healthcare providers can potentially streamline patient assessment, expedite treatment decisions, and improve overall patient outcomes. This approach aligns with global efforts to reduce HIV related morbidity and mortality by ensuring timely identification and management of individuals with advanced disease. In this study, we determined the sensitivity, specificity, positive and negative predictive values, and likelihood ratios of PIMA against FACSCalibur flow cytometry as the gold standard for monitoring advanced HIV patients with CD4 threshold of ≤ 200 cells/µL using capillary blood.
A cross-sectional study was conducted between April and July 2015 at Mildmay Uganda, which is a specialist HIV/AIDS care, prevention, and training center located in Lweza along Entebbe Road Uganda. Mildmay Uganda provides care to over 16,000 HIV infected clients with close to 8,000 currently receiving various HAART combinations. We included 110 HIV-positive adult patients seeking care from Mildmay Uganda who were required to determine a CD4 cell count of ≤ 200 cells/µL of blood during the study period and provided informed consent to participate in the study. Individuals who were unable to provide suitable samples for analysis using both methods and those whose results were invalid for at least one of the methods were excluded from the analysis. The sample size was derived from Buderer's formula based on both specificity and sensitivity. Age and sex were recorded and CD4 counts were measured and recorded by a well-trained team of laboratory technicians.
Patient demographic data were obtained from the Hospital Information Management System (HIMS) by pre-trained research assistants using pre-tested study tools, after which they were deleted from the subject by assigning a unique study ID. The results from the analysis of samples from the FACSCalibur were extracted from the hospital laboratory information system by the researcher. PIMA results were recorded in a laboratory notebook by pre-trained research assistants and verified against instrument printouts by the researcher. The results for CD4 counts were linked using a unique identifier, entered into EPI data V 3.1 and exported to Excel. Excel files were imported to the STATA software for analysis.
Two blood samples [venous and capillary] were collected from each subject using EDTA evacuated tubes and capillary tubes for venous and capillary blood, respectively, using Mildmay Uganda standard practice as detailed in the Mildmay Uganda sample collection manual.
Venous blood was analysed using the BD FACSCalibur following the Mildmay Uganda standard operating procedure for the determination of CD4 + cell counts. Briefly, 50 µl of EDTA anti-coagulated blood was stained with 20 µl of BD MultiTest CD3/CD8/CD45/CD4 fluorescence labelled antibody mixture in BD true count tubes for 15 min in the dark at room temperature, and then lysed with BD lysing solution. Analysis was performed on the BD FACSCalibur using the BD MultiTest software, and the results were directly exported into LIMS. The BD FACSCalibur was calibrated using BS calbrite beads (BD Biosciences), and the quality of reagents and the specimen processing procedures were controlled using low and normal controls (BD Biosciences) on everyday samples. Capillary blood was analysed using the PIMA following the Mildmay Uganda standard operating procedure for the use of the PIMA point-of-care CD4 testing device. Briefly, 25 µl of capillary blood was drawn into the sample collector by holding the cartridge at 45° until the cartridge was filled. The filled cartridge was capped and inserted into an Alere Pima Analyzer. The Analyzer automatically started analysing the sample once the cartridge was inserted and displayed absolute CD4 counts after 20 min. The displayed results were printed, filled, and entered into a laboratory information system (LIMS). On each day, the samples were tested, and equipment performance was checked using valid Pima Bead Standards whose results were recorded in the LIMS. Samples were tested only if the results of the Pima Bead Standards passed. All methods were conducted following the relevant guidelines and regulations.
To determine the reliability of the PIMA POC in predicting a CD4 cell count of ≤ 200 cells/µL of blood, a classification based on the FACSCalibur results as the reference was used with subjects classified as True Positive (TP), True Negative (TN), False Positive (FP), and false negative (FN). We calculated the performance characteristics as follows: sensitivity (TP÷ (TP + FN) ×100), specificity (TN÷ (TN + FP) × 100), and diagnostic efficiency (TP + TN) ÷ (TP + TN + FN + FP) ×100. Positive and negative likelihood ratios were determined using standard methods. Upward and downward misclassification probabilities were calculated for a cutoff of 200 cells/µL. The upward misclassification probability was defined as one minus the sensitivity (1-Sn), and the downward misclassification probability was defined as one minus the specificity (1-Sp) of the PIMA CD4 analyzer.