Diagnosis and monitoring of HIV in infants: investigating the first fourth generation rapid test and two viral load technologies for use in the South African setting

Thesis (M.Sc. (Med.))--University of the Witwatersrand, Faculty of Health Sciences, 2013.Human immune deficiency virus (HIV) infection contributes to child mortality rates in South Africa. Investigations of newer technologies for improving early infant diagnosis of HIV in the South African setting could reduce child mortality as life saving treatment can be accessed early in life. This study investigated three technologies: a fourth generation rapid HIV test and two viral load (VL) platforms. Determine Combo (DC) is a qualitative fourth generation rapid test that is able to detect HIV antibodies and p24 antigen simultaneously. The performance of DC was evaluated in the field on samples from pregnant and postpartum women; in the laboratory, on stored samples from children and with the addition of heat denaturation. In the maternal DC study 90 (8 .8%) of 1019 women tested HIV positive of whom 59 (17.1% prevalence) were pregnant and 31 % (4.6% prevalence) were postpartum. The sensitivity and specificity of the antibody component of DC on plasma was 100%(Confidence Interval (CI): 95.9- 100%) and 99.8%(CI: 99.2-99.9%) respectively. Three postpartum patients tested false positive for HIV antibodies (n=2) and p24 antigen (n=1). No true positive p24 antigen was detected DC was performed on stored samples from 182 (90%) HIV-exposed and 20 (10%) HIV-unexposed children aged from birth to six years. The DC HIV antibody component returned false negative results in 2 HIV-infected children; one clinically symptomatic and one asymptomatic aged 7 and 23 months respectively. The sensitivity of DC HIV antibody was 100% (CI :94.3-100%) in infants aged 6 months and younger with a specificity of 100% (CI:81.6-100%) for all ages. Of the 61 HIV infected infants tested , the DC p24 antigen was reactive in only one clinically symptomatic infant resulting in a sensitivity for detection of HIV infection of 1.7% (CI 0.3-8.9%). A heat denaturation technique designed to improve p24 antigen detection was applied to HIVinfected samples but failed to enhance p24 antigen detection on DC. HIV viral load (VL) molecular assays are used to confirm an HIV-infected diagnosis and for VL monitoring. In South Africa, plasma is the gold standard sample for VL monitoring in infants even though dried blood spots (OBS) are the preferred specimen type in resource-constrained settings and for early infant diagnosis. The use of OBS specimens for HIV VL monitoring would convenience resource limited settings. The OBS matrix therefore requires validation to determine accuracy (for establishing diagnosis) and precision (for VL monitoring) compared to plasma VL. This study investigated the accuracy and precision limits of OBS VL on the Roche Cobas AmpliPrep-Cobas TaqMan HIV-1 v2.0 assay (CAP/CTM) and the Abbott RealTime HIV-1 assay (m2000) platforms on samples from HIV-infected adults and children. The CAP/CTM was investigated on OBS containing 751J1 blood and the m2000 was investigated using one (50IJI) and two (2x501J1) OBS. Compared to plasma VL, OBS VL from adults and children were higher in the lower range «310g,510g, >185,000copies/ml) on the CAP/CTM in the study of OBS VL accuracy. Additionally, OBS VL values were >log1.0 higher in 42/100 (42%) of adult and 16/49 (33%) of measurements from children, which will have clinical significance. On the m2000 platform, the differences between plasma and OBS VL were lower in the range >5 log and higher in the range 2 log copies/ml (100 copies/ml) to 4 log copies/ml (10000 copies/ml). Compared to plasma VL, OBS VL values were >log1.0 higher in 20/82 (24%) adult and 7/43 (16%) of measurements from children. Both platforms demonstrated 100% specificity in testing stored OBS from HIV-uninfected infants who were diagnosed negative on HIV DNA PCR. Acceptable limits for plasma VL precision is a coefficient of variation (CV) <35% and standard deviation (SO) :50.19 log. Where plasma VL :5510g, OBS VL demonstrated poor precision with CV>40% in 8/10 patients and total SO>0.30 log in 4/10 patients on the CAP/CTM. The m2000 total SO was >210g between adult plasma and OBS VLs under the 4 log copies/ml cut-off, irrespective of the number of DBS used. DBS VLs were unreliable when using precision limits used on plasma VLs on both platforms. In conclusion the DC test does not offer any advantage over currently available rapid tests in diagnosing new infection in women and children. The two VL platforms can be used to establish an HIV status in treatment naive patients in view of the 100% specificity. HIV-infected patients on treatment with undetectable plasma VL will always have detectable DBS VL on CAP/CTM, but equally undetectable DBS VL on the m2000. With DBS, the CAP/CTM assay generates higher VL values in the lower VL range than on plasma likely due to amplification of proviral DNA. Both platforms display poor intra- and inter- assay precision, using plasma VL based criteria and the variances would potentially affect clinical decision making. The acceptable limits for plasma VL precision cannot be applied to DBS VL on either platform

Identifying HIV infection in South African women: How does a fourth generation HIV rapid test perform?

Background: HIV rapid tests (RT) play an important role in tackling the HIV pandemic in South Africa. Third generation RT that detect HIV antibodies are currently used to diagnose HIV infection at the point of care. Determine Combo (DC) is the first fourth generation RT that detects both p24 antigen (p24Ag) and HIV antibodies (Ab), theoretically reducing the window period and increasing detection rates. Early detection of maternal HIV infection is important to mitigate the high risk of vertical transmission associated with acute maternal infection. Objectives: We assessed the performance of the DC RT against third generation RT in antenatal and post-partum women. Methods: Third generation RT Advance Quality and Acon were used in a serial algorithm to diagnose HIV infection in antenatal and post-partum women over six months at a tertiary hospital in Johannesburg, South Africa. This data provided the reference against which the DC RT was compared on plasma and whole blood samples. Results: The 1019 participants comprised 345 (34%) antenatal and 674 (66%) post-partum women. Ninety women (8.8%) tested HIV-positive of whom 59 (66%) were tested antenatally, and 31 (34%) post-partum yielding prevalence rates of 17.1% and 4.6% respectively. The sensitivity and specificity of the Ab component of DC on plasma antenatally was 100% (93.8% – 100%) and 100% (98.6% – 100%) respectively and post-partum was 100% (88.9% – 100%) and 99.6% (98.8% – 99.9%) respectively. One false positive and not a single true positive p24Ag was detected. Of 505 post-partum women who tested HIV-negative 6–12 months prior to enrolment, 12 (2.4%) seroconverted. Conclusion: The fourth generation DC offered no advantage over current third generation RT in the diagnosis of HIV infection