Dried Blood Spot HIV-1 RNA Quantification Using Open Real-Time Systems in South Africa and Burkina Faso

Viljoen, Johannes; Gampini, Sandrine; Danaviah, Sivapragashini; Valéa, Diane; Pillay, Sureshnee; Kania, Dramane; Méda, Nicolas; Newell, Marie‐Louise; Perre, Philippe Van de; Raffi, François

doi:10.1097/qai.0b013e3181edaaf5

Cited by 52 publications

(46 citation statements)

References 27 publications

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“…The use of DBS with commercial and in-house VL assays has already been shown to have some success (20,21). Preliminary data using spiked whole-blood samples have shown that the current VFA performs well with DBS but with reduced sensitivity compared to plasma samples, which has been previously described (20,22).…”

Section: Discussionmentioning

confidence: 98%

Development and Evaluation of an Affordable Real-Time Qualitative Assay for Determining HIV-1 Virological Failure in Plasma and Dried Blood Spots

et al. 2013

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h Virological failure (VF) has been identified as the earliest, most predictive determinant of HIV-1 antiretroviral treatment (ART) failure. Due to the high cost and complexity of virological monitoring, VF assays are rarely performed in resource-limited settings (RLS). Rather, ART failure is determined by clinical monitoring and to a large extent immunological monitoring. This paper describes the development and evaluation of a low-cost, dried blood spot (DBS)-compatible qualitative assay to determine VF, in accordance with current WHO guideline recommendations for therapy switching in RLS. The assay described here is an internally controlled qualitative real-time PCR targeting the conserved long terminal repeat domain of HIV-1. This assay was applied to HIV-1 subtypes A to H and further evaluated on HIV-1 clinical plasma samples from South Africa (n ‫؍‬ 191) and Tanzania (n ‫؍‬ 42). Field evaluation was performed in Uganda using local clinical plasma samples (n ‫؍‬ 176). Furthermore, assay performance was evaluated for DBS. This assay is able to identify VF for all major HIV-1 group M subtypes with equal specificity and has a lower detection limit of 1.00E؉03 copies/ml for plasma samples and 5.00E؉03 copies/ml for DBS. Comparative testing yielded accurate VF determination for therapy switching in 89% to 96% of samples compared to gold standards. The assay is robust and flexible, allowing for "open platform" applications and producing results comparable to those of commercial assays. Assay design enables application in laboratories that can accommodate real-time PCR equipment, allowing decentralization of testing to some extent. Compatibility with DBS extends access of sampling and thus access to this test to remote settings.

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Section: Discussionmentioning

confidence: 98%

Development and Evaluation of an Affordable Real-Time Qualitative Assay for Determining HIV-1 Virological Failure in Plasma and Dried Blood Spots

et al. 2013

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“…Recently, several RLC programs have started to use DBS to expand access to HIV VL and genotypic drug resistance testing (12)(13)(14). It is thus important to evaluate to what extent this approach is reliable compared to using plasma, also considering the existing infrastructure and personnel.…”

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confidence: 99%

Field Evaluation of Dried Blood Spots for Routine HIV-1 Viral Load and Drug Resistance Monitoring in Patients Receiving Antiretroviral Therapy in Africa and Asia

et al. 2014

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h Dried blood spots (DBS) can be used in developing countries to alleviate the logistic constraints of using blood plasma specimens for viral load (VL) and HIV drug resistance (HIVDR) testing, but they should be assessed under field conditions. Between 2009 and 2011, we collected paired plasma-DBS samples from treatment-experienced HIV-1-infected adults in Burkina Faso, Cameroon, Senegal, Togo, Thailand, and Vietnam. The DBS were stored at an ambient temperature for 2 to 4 weeks and subsequently at ؊20°C before testing. VL testing was performed on the plasma samples and DBS using locally available methods: the Abbott m2000rt HIV-1 test, generic G2 real-time PCR, or the NucliSENS EasyQ version 1.2 test. In the case of virological failure (VF), i.e., a plasma VL of >1,000 copies/ml, HIVDR genotyping was performed on paired plasma-DBS samples. Overall, we compared 382 plasma-DBS sample pairs for DBS VL testing accuracy. The sensitivities of the different assays in different laboratories for detecting VF using DBS varied from 75% to 100% for the m2000rt test in labs B, C, and D, 91% to 93% for generic G2 realtime PCR in labs A and F, and 85% for the NucliSENS test in lab E. The specificities varied from 82% to 97% for the m2000rt and NucliSENS tests and reached only 60% for the generic G2 test. The NucliSENS test showed good agreement between plasma and DBS VL but underestimated the DBS VL. The lowest agreement was observed for the generic G2 test. Genotyping was successful for 96/124 (77%) DBS tested, and 75/96 (78%) plasma-DBS pairs had identical HIVDR mutations. Significant discrepancies in resistance interpretations were observed in 9 cases, 6 of which were from the same laboratory. DBS can be successfully used as an alternative to blood plasma samples for routine VL and HIVDR monitoring in African and Asian settings. However, the selection of an adequate VL measurement method and the definition of the VF threshold should be considered, and laboratory performance should be monitored.T he increasing availability of antiretroviral treatment (ART) has dramatically contributed to a reduction in mortality and morbidity related to HIV/AIDS in resource-limited countries (RLC). In order to limit the emergence of resistance to antiretroviral drugs, HIV treatment should ideally be accompanied by regular virological monitoring, including viral load (VL) and genotypic drug resistance testing (1). With the recent scaling up of ART in RLC, people from semiurban and rural areas are now also receiving treatment, and recent data have shown variable levels of virological failure (VF) and drug resistance in these areas. In certain settings, VF can be Ͼ20% after 12 months of ART (2-4), stressing the urgent need for improved virological monitoring.In the majority of developing countries with limited resources, monitoring blood plasma VL poses logistical challenges since plasma preparation, storage, and/or shipment requires personnel and laboratory infrastructure that is often lacking. Today, viral load assays still require sophistic...

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“…While standard laboratory platforms typically retain high accuracy when utilising the recommended threshold of 1 000 copies/ mL, both DBS and POC devices may need to utilise a higher limit (3 000 -5 000 copies/mL has been suggested) until better sensitivity is established at the lower limit of detection. [2] A study by Viljoen et al [3] in Durban using DBS HIV-1 RNA testing appeared accurate and feasible down to approximately 3 000 copies/mL. In a study conducted in southern India by Neogi et al, [4] DBS HIV-1 RNA testing revealed 100% sensitivity and specificity at 5 000 copies/mL, but only 50% sensitivity with 100% specificity at 1 000 copies/mL.…”

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confidence: 99%

“…[2] A study by Viljoen et al [3] in Durban using DBS HIV-1 RNA testing appeared accurate and feasible down to approximately 3 000 copies/mL. In a study conducted in southern India by Neogi et al, [4] DBS HIV-1 RNA testing revealed 100% sensitivity and specificity at 5 000 copies/mL, but only 50% sensitivity with 100% specificity at 1 000 copies/mL. Most recently, when Kleshik et al [5] quantified HIV-1 RNA in single 50 µL DBSs and limited incubation time prior to sample preparation to 30 minutes, a limit of detection of 866 copies/mL was reported.…”

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confidence: 99%

An investigation of fingerstick blood collection for point-of-care HIV-1 viral load monitoring in South Africa

et al. 2015

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Background. Viral load (VL) quantification is an important tool in determining newly developed drug resistance or problems with adherence to antiretroviral therapy (ART) in HIV-positive patients. VL monitoring is becoming the standard of care in many resource-limited settings. Testing in resource-limited settings may require sampling by fingerstick because of general shortages of skilled phlebotomists and the expense of venepuncture supplies and problems with their distribution. Objective. To assess the feasibility and ease of collecting 150 µL capillary blood needed for the use of a novel collection device following a classic fingerstick puncture. Methods. Patients were recruited by the study nurse upon arrival for routine ART monitoring at the Themba Lethu Clinic in Johannesburg, South Africa. Each step of the fingerstick and blood collection protocol was observed, and their completion or omission was recorded. Results. One hundred and three patients consented to the study, of whom three were excluded owing to the presence of callouses. From a total of 100 patients who consented and were enrolled, 98% of collection attempts were successful and 86% of participants required only one fingerstick to successfully collect 150 µL capillary blood. Study nurse adherence to the fingerstick protocol revealed omissions in several steps that may lower the success rate of capillary blood collection and reduce the performance of a subsequent VL assay. Conclusion. The findings of this study support the feasibility of collecting 150 µL of capillary blood via fingerstick for point-of-care HIV-1 VL testing in a resource-limited setting.

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Dried Blood Spot HIV-1 RNA Quantification Using Open Real-Time Systems in South Africa and Burkina Faso

Cited by 52 publications

References 27 publications

Development and Evaluation of an Affordable Real-Time Qualitative Assay for Determining HIV-1 Virological Failure in Plasma and Dried Blood Spots

Development and Evaluation of an Affordable Real-Time Qualitative Assay for Determining HIV-1 Virological Failure in Plasma and Dried Blood Spots

Field Evaluation of Dried Blood Spots for Routine HIV-1 Viral Load and Drug Resistance Monitoring in Patients Receiving Antiretroviral Therapy in Africa and Asia

An investigation of fingerstick blood collection for point-of-care HIV-1 viral load monitoring in South Africa

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