Multisite Evaluation of Point of Care CD4 Testing in Papua New Guinea

<div><p>Laboratory-based CD4 monitoring of HIV patients presents challenges in resource limited settings (RLS) including frequent machine breakdown, poor engineering support and limited cold chain and specimen transport logistics. This study assessed the performance of two CD4 tests designed for use in RLS; the Dynal assay and the Alere PIMA test (PIMA). Accuracy of Dynal and PIMA using venous blood was assessed in a centralised laboratory by comparison to BD FACSCount (BD FACS). Dynal had a mean bias of −50.35 cells/µl (r² = 0.973, p<0.0001, n = 101) and PIMA −22.43 cells/µl (r² = 0.964, p<0.0001, n = 139) compared to BD FACS. Similar results were observed for PIMA operated by clinicians in one urban (n = 117) and two rural clinics (n = 98). Using internal control beads, PIMA precision was 10.34% CV (low bead mean 214.24 cells/µl) and 8.29% (high bead mean 920.73 cells/µl) and similar %CV results were observed external quality assurance (EQA) and replicate patient samples. Dynal did not perform using EQA and no internal controls are supplied by the manufacturer, however duplicate testing of samples resulted in r² = 0.961, p<0.0001, mean bias = −1.44 cells/µl. Using the cut-off of 350 cells/µl compared to BD FACS, PIMA had a sensitivity of 88.85% and specificity of 98.71% and Dynal 88.61% and 100%. A total of 0.44% (2/452) of patient samples were misclassified as “no treat” and 7.30% (33/452) “treat” using PIMA whereas with Dynal 8.91% (9/101) as “treat” and 0% as “no treat”. In our setting PIMA was found to be accurate, precise and user-friendly in both laboratory and clinic settings. Dynal performed well in initial centralized laboratory evaluation, however lacks requisite quality control measures, and was technically more difficult to use, making it less suitable for use at lower tiered laboratories.</p></div

Accuracy of PIMA compared to BD FACS.

<p>Linear regression analysis plots (right column) and Bland Altman bias plots (left column) with upper and lower 95% limits of agreement (LOA) shown as broken lines and mean bias shown as an unbroken line. A. and B. PIMA versus BD FACS both performed at an urban (reference) laboratory, by a single laboratory technician, C. and D. BD FACS versus PIMA both performed at the rural laboratory by a single laboratory technician, E. and F. BD FACS performed by laboratory technician at an urban laboratory PIMA versus PIMA performed by clinical staff at an urban clinic, G. and PIMA. BD FACS performed at by laboratory technician at a rural laboratory versus PIMA performed at two rural clinics by clinical staff.</p

Accuracy and Precision of Dynal assay.

<p>Linear regression analysis plots (right column) and Bland Altman bias plots (left column) with upper and lower 95% limits of agreement (LOA) shown as broken lines and mean bias shown as an unbroken line. A. and B. Dynal accuracy versus BD FACS in urban (reference) laboratory C. and D. Precision of measurements using the Dynal assay performed in duplicate by the same laboratory technician in urban (reference) laboratory.</p

Quality Assurance and precision of PIMA.

<p>A. Internal Quality control using manufacturer provided bead standard controls Normal (mean 957, range 670–1244 cells/µl) and Low (mean 192, range 132–252 cells/µl). (Site 1: Urban Laboratory, Site 2: Urban Clinic, Site 3: Rural Laboratory, Site 4: Rural Clinic, Site 5: Rural Clinic). B. QASI External Quality Assurance Panel each sample tested in replicate by a single laboratory technician at the reference laboratory, Central Public Health Laboratory (CPHL). C. Assessment of assay precision using replicates from the same blood sample run in duplicate by a single laboratory technician. n = sample size,</p><p>B. SD = standard deviation, CV = coefficient of variation, QASI = Quality Assurance Scheme for Immunology (Public Health Agency of Canada).</p><p>Quality Assurance and precision of PIMA.</p

Operator Precision of PIMA testing.

<p>Linear regression analysis plots (right column) and Bland Altman bias plots (left column) with upper and lower 95% limits of agreement (LOA) shown as broken lines and mean bias shown as an unbroken line. A and B PIMA 1 operated by laboratory technicians at an urban (reference) laboratory, versus PIMA 2 operated by clinicians at an urban clinic, C and D PIMA 1 operated by laboratory technicians a rural laboratory compared to PIMA 2 operated by clinicians at 2 rural clinics.</p

Clinical Misclassification Analysis using PIMA and Dynal compared to BD FACS.

<p>A. 2×2 Tables CD4 count compared to BD FACS clinical misclassification using 350 cells/µl cut off for determination of ART Eligibility at each site and total for PIMA 1,2,3,4 and Dynal 5. B. BD FACS and PIMA CD4 count results for misclassified samples from each site 1,2,3,4 and 5. Dynal. Values in bold font indicate samples misclassified by PIMA as non-treat (<350 cells/µl) and values in normal font indicate those samples misclassified by PIMA or Dynal as treat (<350 cells/µl).</p><p>Clinical Misclassification Analysis using PIMA and Dynal compared to BD FACS.</p

Summary of observed transmitted HIV drug resistance mutations (Port Moresby, National Capital District).

<p>Summary of observed transmitted HIV drug resistance mutations (Port Moresby, National Capital District).</p

Demographic characteristics of survey participants.

<p>Demographic characteristics of survey participants.</p

Summary of observed transmitted HIV drug resistance mutations (Mount Hagen, Western Highlands Province).

<p>Summary of observed transmitted HIV drug resistance mutations (Mount Hagen, Western Highlands Province).</p

Neighbor-joining tree with WHO Reference Sequences (Mount Hagen, Western Highlands Province Sequences with one of more NNRTI SDRM denoted by squares and sequences with one or more NNRTI and NRTI SDRM are denoted by circles.

<p>Subtype reference sequences are included.</p