Browsing by Author "Hsiao, Marvin"

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    Classification of HIV virological failure using whole blood versus plasma viral load
    (2016) Khan, Aabida; Hsiao, Marvin
    Introduction: HIV viral load testing is the preferred monitoring approach for HIV infected patients on combination antiretroviral therapy (cART) as it is more sensitive than CD4 count and clinical monitoring. In resource limited settings, timely plasma separation and transportation to testing laboratories is a major barrier to the access of HIV viral load testing. The 2015 World Health Organisation guidelines recommend that cART should be initiated in all adults and children living with HIV regardless of disease stage or CD4 count, thereby escalating the demand for HIV viral load testing. Potential solutions to expand implementation and scale up of viral load testing in low and middle income countries are whole blood testing through point of care (POC) viral load assays or dried blood spots (DBS) collected at the health facility. Utilization of whole blood instead of plasma would simplify sample collection, storage and transportation requirements and be cost effective. However, the paucity of studies comparing whole blood HIV viral load across different test platforms, especially in the correct classification of virological failure, has resulted in the lack of a standardised programmatic approach to whole blood viral load testing. Methods: We evaluated four HIV whole blood viral load test methods namely Alere q HIV-1/2 POC, Abbott RealTime HIV-1 DBS original and updated protocols, and Roche CAP/CTM DBS free virus elution (FVE) protocol, against the standard of care, plasma viral load, on 299 samples across the viral load spectrum from South African patients on cART. Virological failure was defined at >1000 copies/ml. Proportions of correct classification of virological failure and overall correlation with plasma were used for evaluating each method's performance. Results: Alere q, Abbott original and updated, and Roche FVE correctly classified virological failure in 61%, 89%, 87% and 76% of all samples tested respectively. The performance varied across plasma viral load categories. Alere q showed good correlation above plasma viral load of 1000 copies/ml, with correct classification of virological failure in 100% of samples. However, below the plasma threshold of 1000 copies/ml, Alere q demonstrated significant over-quantification, resulting in reduced specificity and upward misclassification of virological failure in 39% of all samples tested. Abbott original and updated also had good sensitivity of 98% and 91% respectively and the best overall correlation with plasma (r² = 0.76 and 0.72 respectively), but there was upward misclassification in 10% and 8% of samples tested respectively. Roche FVE had the best specificity of 99% but with significantly reduced sensitivity of 53%, especially between 1000–10,000 copies/ml of plasma, resulting in downward misclassification in 24% of all samples tested. Greatest variability between the different testing methods was seen when plasma viral load was 40-1000 copies/ml. Correlation was best for all whole blood viral load assays at >10,000 copies/ml. Conclusion: The key finding highlighted by this study is the great variability between the different whole blood test methods. Various factors influence the ability to quantify whole blood HIV viral load such as input volume used in each assay vary, sample treatment/processing (DBS versus fresh blood samples versus FVE), extraction (RNA selective, total nucleic acid extraction), amplification target and detection methods are different for each of the platforms tested. Based on our study, Alere q and Abbott DBS need to raise their whole blood threshold for virological failure in order to reduce upward misclassification and Roche FVE needs to achieve better sensitivity around its limit of detection. Receiver operating characteristic curve analysis can be used to determine the optimum threshold of virological failure for each assay.
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    Droplet digital PCR detection and next-generation sequencing of HIV-1 in breastmilk of breastfeeding women on antiretroviral treatment
    (2025) Hurree, Jennah Nivashni ; Abrahams, Melissa-Rose; Hsiao, Marvin
    Persistence of HIV-1 in a stable latent reservoir in breastmilk has been associated with low- level mother to child transmission despite maternal antiretroviral therapy (ART). We aimed to evaluate and optimize the sensitivity of HIV-1 DNA quantitation in breastmilk using droplet digital PCR (ddPCR) and sequencing of viral variants in breastmilk using Illumina MiSeq next generation sequencing to inform transmission risk and efficacy of maternal ART clinical trials. HIV-negative breastmilk samples were spiked with 8E5 cells (a cell line where each cell contains one replication-defective HIV genome) at concentrations ranging from 10 000 - 1 8E5 cell(s)/million. Genomic DNA was extracted with the QIAGEN AllPrep DNA/RNA Mini Kit (QIAGEN, Hilden, Denmark) and ddPCR was performed using the ddPCR Supermix for Probes (No dUTP) (BIO-RAD, California, USA), HIV-1 gag and/or pol and housekeeping gene rpp30 primers and probes, BanII restriction enzyme, CutSmart buffer (New England Biolabs, Massachusetts, USA) and the QX200TM ddPCR System (BIO-RAD, California, USA). The envelope gene V1-V2 region was amplified using the KAPA2G Fast Multiplex Mix (Roche, Basel, Switzerland) and Expand High Fidelity PCR enzyme (Roche diagnostics, Basel Switzerland) with Nextera DNA CD indexes and sequenced via the Illumina Miseq v3 platform. Genomic DNA recovery was low (ranging from 25.12 - 106.85 ng/µl with an average of 67.18 ng/µl) when spiking in a background of 1 million cells, resulting in HIV DNA being detectable by ddPCR only in samples spiked with the highest 8E5 cell concentration. When repeating this experiment with 8E5 cell spike-in concentrations ranging from 50 000 - 5 8E5 cells/million in a total of 5 million cells, genomic DNA recovery was higher (ranging from 498.99 - 757.28 ng/µl with an average of 631.32 ng/µl) resulting in a detection sensitivity of 44.3 gag copies/million cells with a lowest per reaction HIV copy number detection of 7 gag copies. We noted that 8E5 cell line HIV genome copies/cell varied across different passages, ranging from 0.4 - 0.8 copies/cell instead of the expected 1 copy/cell. HIV-negative breastmilk samples spiked with 8E5 cells ranging from 50 000 - 50 8E5 cells/million were likewise successfully PCR amplified and sequenced (gDNA input at 1000 ng), with sequences matching the 8E5 HIV-1 /LAV genome, as expected. Additionally, this method was successfully applied to genomic DNA from PBMCs of women on ART from the CAPRISA 002 cohort, Kwa-Zulu Natal, confirming the sensitivity of this method to amplify low-copy number HIV templates. ddPCR and Illumina MiSeq methods were applied to cells from 2 ml breastmilk samples from three women with blood viral loads of 20 - 331 copies/ml from the Dolutegravir in pregnant HIV infected mothers and their neonates (DolPHIN-2) trial, yet no HIV genome copies above background level were detected and no HIV targets could be PCR amplified. Viral quantification and sequencing from breastmilk is difficult particularly when viral load is low in the presence of ART and no guidelines have been established for processing breastmilk. Both ddPCR and Illumina Miseq were successful at quantifying and sequencing low levels (1.3log10) of HIV from spiked-in breastmilk samples. HIV copies below background levels obtained from breastmilk samples of the DolPHIN-2 study potentially demonstrate effective viral suppression in breastmilk by integrase inhibitor Dolutegravir-based ART. However, the number of samples analysed was low and cell pellets available for analysis possibly contained too few cells for ddPCR detection and Illumina Miseq of HIV, based on our observations from the spike-in experiments.
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    Laboratory investigation of low positive and discrepant HIV serology results
    (2014) Hans, Lucia; Korsman, Stephen; Hsiao, Marvin
    In our diagnostic virology laboratory, we test on average 1500 samples for HIV antibody/antigen each month, of which 0.6% produces problematic results. These problematic samples produce either weakly reactive screening and confirmatory results or, discrepant screening and confirmatory results. Both scenarios require additional tests to confirm HIV status thus increasing cost and turnaround time. There is a need to devise an optimal strategy within the laboratory to rapidly and easily manage these samples with minimal additional cost. The WHO recommends three HIV testing strategies. Strategy I ensures blood transfusion safety while strategies II and III are used for both surveillance and diagnostics in high prevalence and low prevalence areas respectively.1The 2010 National antenatal sentinel HIV & syphilis prevalence study reported the South African HIV prevalence as 30.2%.2 There were 1.8 million new cases of HIV infection in Sub-Saharan Africa in 2011.3South Africa (SA) is a high prevalence country and therefore the national HIV testing guideline is based on strategy II. The HIV screening and confirmatory strategy at Groote Schuur is based on these recommendations.
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    Phylogenetic exploration of nosocomial transmission chains of 2009 influenza A/H1N1 among children admitted at Red Cross War Memorial Children's Hospital, Cape Town, South Africa in 2011
    (Public Library of Science, 2015) Valley-Omar, Ziyaad; Nindo, Fredrick; Mudau, Maanda; Hsiao, Marvin; Martin, Darren Patrick
    Traditional modes of investigating influenza nosocomial transmission have entailed a combination of confirmatory molecular diagnostic testing and epidemiological investigation. Common hospital-acquired infections like influenza require a discerning ability to distinguish between viral isolates to accurately identify patient transmission chains. We assessed whether influenza hemagglutinin sequence phylogenies can be used to enrich epidemiological data when investigating the extent of nosocomial transmission over a four-month period within a paediatric Hospital in Cape Town South Africa. Possible transmission chains/channels were initially determined through basic patient admission data combined with Maximum likelihood and time-scaled Bayesian phylogenetic analyses. These analyses suggested that most instances of potential hospital-acquired infections resulted from multiple introductions of Influenza A into the hospital, which included instances where virus hemagglutinin sequences were identical between different patients. Furthermore, a general inability to establish epidemiological transmission linkage of patients/viral isolates implied that identified isolates could have originated from asymptomatic hospital patients, visitors or hospital staff. In contrast, a traditional epidemiological investigation that used no viral phylogenetic analyses, based on patient co-admission into specific wards during a particular time-frame, suggested that multiple hospital acquired infection instances may have stemmed from a limited number of identifiable index viral isolates/patients. This traditional epidemiological analysis by itself could incorrectly suggest linkage between unrelated cases, underestimate the number of unique infections and may overlook the possible diffuse nature of hospital transmission, which was suggested by sequencing data to be caused by multiple unique introductions of influenza A isolates into individual hospital wards. We have demonstrated a functional role for viral sequence data in nosocomial transmission investigation through its ability to enrich traditional, non-molecular observational epidemiological investigation by teasing out possible transmission pathways and working toward more accurately enumerating the number of possible transmission events.