Multicountry Validation of SAMBA - A Novel Molecular Point-of-Care Test for HIV-1 Detection in Resource-Limited Setting.

INTRODUCTION: Early diagnosis of HIV-1 infection and the prompt initiation of antiretroviral therapy are critical to achieving a reduction in the morbidity and mortality of infected infants. The Simple AMplification-Based Assay (SAMBA) HIV-1 Qual Whole Blood Test was developed specifically for early infant diagnosis and prevention of mother-to-child transmission programs implemented at the point-of-care in resource-limited settings. METHODS: We have evaluated the performance of this test run on the SAMBA I semiautomated platform with fresh whole blood specimens collected from 202 adults and 745 infants in Kenya, Uganda, and Zimbabwe. Results were compared with those obtained with the Roche COBAS AmpliPrep/COBAS TaqMan (CAP/CTM) HIV-1 assay as performed with fresh whole blood or dried blood spots of the same subjects, and discrepancies were resolved with alternative assays. RESULTS: The performance of the SAMBA and CAP/CTM assays evaluated at 5 laboratories in the 3 countries was similar for both adult and infant samples. The clinical sensitivity, specificity, positive predictive value, and negative predictive value for the SAMBA test were 100%, 99.2%, 98.7%, and 100%, respectively, with adult samples, and 98.5%, 99.8%, 99.7%, and 98.8%, respectively, with infant samples. DISCUSSION: Our data suggest that the SAMBA HIV-1 Qual Whole Blood Test would be effective for early diagnosis of HIV-1 infection in infants at point-of-care settings in sub-Saharan Africa.

Evaluation of the performance of Abbott m2000 and Roche COBAS Ampliprep/COBAS Taqman assays for HIV-1 viral load determination using dried blood spots and dried plasma spots in Kenya.

BACKGROUND: Routine HIV viral load testing is not widely accessible in most resource-limited settings, including Kenya. To increase access to viral load testing, alternative sample types like dried blood spots (DBS), which overcome the logistic barriers associated with plasma separation and cold chain shipment need to be considered and evaluated. The current study evaluated matched dried blood spots (DBS) and dried plasma spots (DPS) against plasma using the Abbott M 2000 (Abbott) and Roche Cobas Ampliprep/Cobas TaqMan (CAP/CTM) quantitative viral load assays in western Kenya. METHODS: Matched plasma DBS and DPS were obtained from 200 HIV-1 infected antiretroviral treatment (ART)-experienced patients attending patient support centers in Western Kenya. Standard quantitative assay performance parameters with accompanying 95% confidence intervals (CI) were assessed at the assays lower detection limit (400cps/ml for CAP/CTM and 550cps/ml for Abbott) using SAS version 9.2. Receiver operating curves (ROC) were further used to assess viral-load thresholds with best assay performance (reference assay CAP/CTM plasma). RESULTS: Using the Abbott test, the sensitivity and specificity, respectively, for DPS were (97.3%, [95%CI: 93.2-99.2] and 98.1% [95%CI: 89.7-100]) and those for DBS (93.9% [95%CI: 88.8-97.2] and 88.0% [95%CI: 82.2-92.4]). The correlation and agreement using paired plasma and DPS/DBS were strong, with r2 = 90.5 and rc = 68.1. The Bland-Altman relative percent change was 95.3 for DPS, (95%CI: 90.4-97.7) and 73.6 (95%CI: 51.6-86.5) for DBS. Using the CAP/CTM assay, the sensitivity for DBS was significantly higher compared to DPS (100.0% [95% CI: 97.6-100.0] vs. 94.7% [95%CI: 89.8-97.7]), while the specificity for DBS was lower: 4%, [95% CI: 0.4-13.7] compared to DPS: 94.0%, [95% CI: 83.5-98.7]. When compared under different clinical relevant thresholds, the accuracy for the Abbott assay was 95% at the 1000cps/ml cut-off with a sensitivity and specificity of 96.6% [95% CI 91.8-98.7] and 90.4% [95% CI 78.2-96.4] respectively. The optimum threshold was at 3000 cps/ml with an accuracy of 95.5%, sensitivity and specificity of 94.6% [95%CI 89.3-97.5] and 98.1% [95%CI 88.4-99.9]) respectively. The best threshold for CAP/CTM was at 4000 copies /mL, with 92.5% accuracy (sensitivity of 96.0% [95%CI 91.0-98.3] and specificity of 82.7% [95%CI 69.2-91.3]). CONCLUSIONS: There was similar performance between matched DBS, DPS and plasma using the Abbott test, and good correlation for matched DPS and plasma using the CAPCTM test. The findings suggest that DBS and DPS may be reliably used as alternative specimens to plasma to measure HIV-1 VL using Abbott, and DPS may be reliably used with CAP/CTM in resource-limited settings.

Field Evaluation of Dried Blood Spots for HIV-1 Viral Load Monitoring in Adults and Children Receiving Antiretroviral Treatment in Kenya: Implications for Scale-up in Resource-Limited Settings.

BACKGROUND: The World Health Organization recommends viral load (VL) as the preferred method for diagnosing antiretroviral therapy failure; however, operational challenges have hampered the implementation of VL monitoring in most resource-limited settings. This study evaluated the accuracy of dried blood spot (DBS) VL testing under field conditions as a practical alternative to plasma in determining virologic failure (VF). METHODS: From May to December 2013, paired plasma and DBS specimens were collected from 416 adults and 377 children on antiretroviral therapy for ≥6 months at 12 clinics in Kenya. DBSs were prepared from venous blood (V-DBS) using disposable transfer pipettes and from finger-prick capillary blood using microcapillary tubes (M-DBS) and directly spotting (D-DBS). All samples were tested on the Abbott m2000 platform; V-DBS was also tested on the Roche COBAS Ampliprep/COBAS TaqMan (CAP/CTM) version 2.0 platform. VF results were compared at 3 DBS thresholds (≥1000, ≥3000, and ≥5000 copies/mL) and a constant plasma threshold of ≥1000 copies/mL. RESULTS: On the Abbott platform, at ≥1000-copies/mL threshold, sensitivities, specificities, and kappa values for VF determination were ≥88.1%, ≥93.1%, and ≥0.82%, respectively, for all DBS methods, and it had the lowest percentage of downward misclassification compared with higher thresholds. V-DBS performance on CAP/CTM had significantly poorer specificity at all thresholds (1000%-33.0%, 3000%-60.9%, and 5000%-77.0%). No significant differences were found between adults and children. CONCLUSIONS: VL results from V-DBS, M-DBS, and D-DBS were comparable with those from plasma for determining VF using the Abbott platform but not with CAP/CTM. A 1000-copies/mL threshold was optimal and should be considered for VF determination using DBS in adults and children.

Viral and Host Characteristics of Recent and Established HIV-1 Infections in Kisumu based on a Multiassay Approach.

Integrated approaches provide better understanding of HIV/AIDS epidemics. We optimised a multiassay algorithm (MAA) and assessed HIV incidence, correlates of recent infections, viral diversity, plus transmission clusters among participants screened for Kisumu Incidence Cohort Study (KICoS1) (2007-2009). We performed BED-CEIA, Limiting antigen (LAg) avidity, Biorad avidity, and viral load (VL) tests on HIV-positive samples. Genotypic analyses focused on HIV-1 pol gene. Correlates of testing recent by MAA were assessed using logistic regression model. Overall, 133 (12%, 95% CI: 10.2-14.1) participants were HIV-positive, of whom 11 tested recent by MAA (BED-CEIA OD-n < 0.8 + LAg avidity OD-n < 1.5 + VL > 1000 copies/mL), giving an incidence of 1.46% (95% CI: 0.58-2.35) per year. This MAA-based incidence was similar to longitudinal KICoS1 incidence. Correlates of testing recent included sexually transmitted infection (STI) treatment history (OR = 3.94, 95% CI: 1.03-15.07) and syphilis seropositivity (OR = 10.15, 95% CI: 1.51-68.22). Overall, HIV-1 subtype A (63%), D (15%), C (3%), G (1%) and recombinants (18%), two monophyletic dyads and intrinsic viral mutations (V81I, V81I/V, V108I/V and K101Q) were observed. Viral diversity mirrored known patterns in this region, while resistance mutations reflected likely non-exposure to antiretroviral drugs. Management of STIs may help address ongoing HIV transmission in this region.

Prevalence and dynamics of the K65R drug resistance mutation in HIV-1-infected infants exposed to maternal therapy with lamivudine, zidovudine and either nevirapine or nelfinavir in breast milk.

BACKGROUND: K65R is a relatively rare drug resistance mutation (DRM) selected by the NRTIs tenofovir, didanosine, abacavir and stavudine and confers cross-resistance to all NRTIs except zidovudine. Selection by other NRTIs is uncommon. OBJECTIVES: In this study we investigated the frequency of emergence of the K65R mutation and factors associated with it in HIV-1-infected infants exposed to low doses of maternal lamivudine, zidovudine and either nevirapine or nelfinavir ingested through breast milk, using specimens collected from the Kisumu Breastfeeding Study. METHODS: Plasma specimens with viral load ≥1000 copies/mL collected from HIV-infected infants at 0-1, 2, 6, 14, 24 and 36 weeks of age and maternal samples at delivery were tested for HIV drug resistance using Sanger sequencing of the polymerase gene. Factors associated with K65R emergence were assessed using Fisher's exact test and the Wilcoxon rank-sum test. RESULTS: K65R was detected in samples from 6 of the 24 infants (25%) who acquired HIV-1 infection by the age of 6 months. K65R emerged in half of the infants by 6 weeks and in the rest by 14 weeks of age. None of the mothers at delivery or the infants with a positive genotype at first time of positivity had the K65R mutation. Infants with K65R had low baseline CD4 cell counts (P = 0.014), were more likely to have DRMs earlier (≤6 weeks versus ≥14 weeks, P = 0.007) and were more likely to have multiclass drug resistance (P = 0.035). M184V was the most common mutation associated with K65R emergence. K65R had reverted by 3 months after cessation of breastfeeding. CONCLUSIONS: A high rate of K65R emergence may suggest that ingesting low doses of lamivudine via breast milk could select for this mutation. The presence of this mutation may have a negative impact on future responses to NRTI-based ART. More in vitro studies are, however, needed to establish the molecular mechanism for this selection.