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

Abstract

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...

Measurement of viral load by the automated Abbott real-time HIV-1 assay using dried blood spots collected and processed in Malawi and Mozambique

“…In previous studies performed with the Abbott system, sensitivity for the detection of virological failure at 1 000 copies/mL was between 75% and 100% and specificity was between 82% and 97%. [10,11] In our study, very high positive and negative predictive values were observed when using a threshold of 1 000 copies/mL, suggesting that even with the use of DBS the threshold of 1 000 copies/mL can provide reliable results, given the specificity of 98.6% at this threshold.…”

“…[5] The modest overquantification that we observed with DBS can be ascribed to the presence of HIVDNA and intracellular RNA and seems to have a greater impact on samples with a low viral load (in our study <3 000 copies/mL), as previously reported. [4,11,14] The 2013 HIV treatment guidelines of the World Health Organi zation [15] indicate a threshold of 1 000 copies/mL of HIVRNA to define virological failure using plasma, and suggest use of a higher, although undefined, threshold when using DBS. In previous studies performed with the Abbott system, sensitivity for the detection of virological failure at 1 000 copies/mL was between 75% and 100% and specificity was between 82% and 97%.…”

Development of a Sensitive and Specific Serological Assay Based on Luminex Technology for Detection of Antibodies to Zaire Ebola Virus

“…For 92 of them, the date between symptom onset and blood sampling was known and the median interval between these two dates was 350 days (interquartile range [IQR]: 293 to 421 days). For each patient, five spots of 50 l of whole blood were prepared as described previously (31). Plasma was reconstituted from one DBS spot in 1 ml of incubation buffer, consisting of phosphate-buffered saline (PBS) containing 0.75 mol/liter of NaCl, 1% (wt/vol) bovine serum albumin (Sigma-Aldrich, St. Quentin Fallavier, France), 5% (vol/vol) fetal bovine serum (Gibco-Invitrogen, Cergy Pontoise, France), and 0.2% (vol/vol) Tween 20 (Sigma-Aldrich).…”

Estimation of HIV-1 DNA Level Interfering with Reliability of HIV-1 RNA Quantification Performed on Dried Blood Spots Collected from Successfully Treated Patients

“…Studies have shown that DBS specimens may be useful for early diagnosis of pediatric HIV-1 infection, treatment monitoring, external quality assessment programs, and resistance testing (2)(3)(4). HIV-1 RNA testing on whole blood collected on DBS is highly effective in determining viral failure at the clinical threshold of 5,000 copies/ml and may be used effectively at a threshold of 1,000 copies/ml, although a suboptimal sensitivity was reported for this level (5).…”

“…Fifty microliters of blood collected on EDTA tubes was spotted onto five 12-mm circles on a Whatman 903 filter paper card (Whatman GmbH, Dassel, Germany) and air dried for 1 day prior to storage at Ϫ20°C. For elution, two entire 12-mm spots were placed in 2.0 ml of lysis buffer under constant shaking for 1 h as previously described (2). DBS HIV-1 RNA was then extracted by the NucliSENS miniMag extraction system (bioMérieux, Marcy l'Etoile, France) or by the large-volume nucleic acid extraction kit using the Arrow extraction system (Biocentric, Bandol, France).…”