Discrepant test findings in early infant diagnosis of HIV in a national reference laboratory in Kenya: challenges and opportunities for programs.

BACKGROUND: In Kenya, the availability of a cheap diagnostic service for HIV-exposed infants has helped scale-up access to treatment, and provided a means by which programs that support Prevention of Mother to Child Transmission of HIV can be evaluated. As expected for any large testing program, discrepant and indeterminate results present a significant challenge. METHODS: Dried Blood Spots were collected from health centers countrywide and couriered to four laboratories for tests. Results were dispatched either by email, telephone, GSM SMS printer or courier. Between 2006 and 2009, tests were conducted with the Manual Roche v. 1.5 Assay. In 2010 the labs switched fully to the Cobas® AmpliPrep/ Cobas® TaqMan® HIV-1 Qual automated Roche Test. RESULTS: Between 2006 and 2010, the KEMRI CVR EID Lab conducted 64 591 HIV tests in on children <18 months of age. HIV tests (38 834) used the manual assay, while 17 133 tests used the automated assay. Overall, 10.7% (6915) of the samples tested positive, while 86.6% (55 967) tested negative. A total of 1.6% (1041) tested indeterminate and required a re-bleed of the infant. Two hundred positive tests by the manual assay were retrieved randomly and retested using the automated assay. Among them, 192 (96%) remained positive, 5 (2.5%) were negative while 3 (1.5%) failed. A total of 160 negative samples by the manual assay were retrieved and retested with the automated assay. Among them, 154 (96.24%) remained negative, 3 (1.88%) tested positive while 3 (1.88%) failed. A total of 215 samples that gave indeterminate results by the manual assay were retested using the automated system. Among them, 62 (28.8%) gave positive results, 144 (66.97%) negative and 6 (2.8%) samples still gave discrepant results. Three (1.4%) did not amplify successfully. A few infants who were apparently positive appeared to test HIV negative with age. CONCLUSIONS: Indeterminate results are a significant challenge for HIV diagnostic services, as seen in the Kenyan EID Program. In our experience, they are more often negative than they are positive. False positive and false negative results can arise from clerical error, contamination and limitations of the technologies available. To forestall the consequences of such outcomes, the sensitivity and specificity of available assays must be further improved. All HIV positive samples should be retested for confirmation, and if confirmed, a new sample must be drawn and tested for DNA at the time the infant receives their initial results or starts antiretroviral therapy. Viral clearance is a phenomenon that requires further studies.

Technical performance evaluation of the MyT4 point of care technology for CD4+ T cell enumeration.

OBJECTIVE: Though absolute CD4+ T cell enumeration is the primary gateway to antiretroviral therapy initiation for HIV-positive patients in all developing countries, patient access to this critical diagnostic test is relatively poor. We technically evaluated the performance of a newly developed point-of-care CD4+ T cell technology, the MyT4, compared with conventional CD4+ T cell testing technologies. DESIGN: Over 250 HIV-positive patients were consecutively enrolled and their blood tested on the MyT4, BD FACSCalibur, and BD FACSCount. RESULTS: Compared with the BD FACSCount, the MyT4 had an r2 of 0.7269 and a mean bias of -23.37 cells/µl. Compared with the BD FACSCalibur, the MyT4 had an r2 of 0.5825 and a mean bias of -46.58 cells/µl. Kenya currently uses a CD4+ T cell test threshold of 350 cells/µl to determine patient eligibility for antiretroviral therapy. At this threshold, the MyT4 had a sensitivity of 95.3% (95% CI: 88.4-98.7%) and a specificity of 87.9% (95% CI: 82.3-92.3%) compared with the BD FACSCount and sensitivity and specificity of 88.2% (95% CI: 79.4-94.2%) and 84.2% (95% CI: 78.2-89.2%), respectively, compared with the BD FACSCalibur. Finally, the MyT4 had a coefficient of variation of 12.80% compared with 14.03% for the BD FACSCalibur. CONCLUSIONS: We conclude that the MyT4 performed well at the current 350 cells/µl ART initiation eligibility threshold when used by lower cadres of health care facility staff in rural clinics compared to conventional CD4+ T cell technologies.

Evaluation of PIMA™® point of care technology for CD4 T cell enumeration in Kenya.

CD4+ T cell enumeration is used to determine eligibility for antiretroviral therapy (ART) and to monitor the immune status of HIV-positive patients; however, many patients do not have access to this essential diagnostic test. Introducing point of care (POC) testing may improve access. We have evaluated Alere's PIMA™, one such POC device, against conventional CD4+ testing platforms to determine its performance and validity for use in Kenya. In our hands, Alere PIMA™ had a coefficient of variability of 10.3% and of repeatability of 175.6 cells/µl. It differed from both the BD FACSCalibur™ (r(2) = 0.762, mean bias -64.8 cells/µl), and the BD FACSCount™ (r(2) = 0.874, mean bias 7.8 cells/µl). When compared to the FACSCalibur™ at a cutoff of 350 cells/µl, it had a sensitivity of 89.6% and a specificity of 86.7% in those aged 5 years and over (Kw = 0.7566). With the BD FACSCount™, it had a sensitivity of 79.4% and a specificity of 83.4% in those aged 5 years and over (Kw = 0.7790). The device also differed from PARTEC Cyflow™ (r(2) = 0.781, mean bias -24.2 cells/µl) and GUAVA™ (r(2) = 0.658, mean bias -0.3 cells/µl) platforms, which are used in some facilities in Kenya. We conclude that with refinement, Alere PIMA™ technology has potential benefits for HIV-positive patients. This study highlights the difficulty in selecting the most appropriate reference technology for technical evaluations.