Stringent HIV Viral Load Threshold for Virological Failure Using Dried Blood Spots: Is the Perfect the Enemy of the Good?

To the Editors: Since 2013, World Health Organization (WHO) recommends plasma HIV viral load (VL) as the preferred monitoring strategy of antiretroviral therapy (ART), complementing clinical and immunological criteria. VL monitoring enables accurate detection of virological failure prompting timely adherence counseling and regimen switches before drug resistance accumulates, and also prevention of inappropriate switching to more costly second-line regimens. Despite significant progress, there are major obstacles to scaling up VL testing in low-income and middleincome countries (LMICs), including high costs, complex sample logistics, limited laboratory infrastructure, and shortage of skilled staff. With point-of-care applications still largely unavailable, dried blood spots (DBS), rather than plasma, are propagated, because of the advantages of easy sample collection through finger prick (eliminating phlebotomy and centrifugation) and easy shipment to a reference laboratory at ambient temperature (eliminating cold-chain transportation). However, potential limitations of DBS include lower amplification sensitivity (due to small sample volumes) and lower specificity (contribution by cell-associated viruses may overestimate VL). To account for these limitations, WHO previously recommended a VL threshold for virological failure of 3000– 5000 copies per milliliter with DBS, compared to 1000 copies per milliliter with plasma. However, a 2014 WHO technical update revised the threshold with DBS to 1000 copies per milliliter, citing provisional data from a review by Clinton Health Access Initiative on 5 commercial VL assays, which suggested sufficient sensitivities for this threshold. We here assessed the public health implications of the revised, more stringent virological failure threshold with DBS, in terms of patient misclassification and clinical decision-making, using routine data from western Kenya. To this end, we projected the degree of patient misclassification, in terms of unnecessary test repeats and treatment switches, at the 1000 copies per milliliter, compared to the 5000 copies per milliliter threshold. For this study, we used routinely collected standard plasma VL data from western Kenya collected real-time in the national web-based database (http://www. nascop.org/eid/viraloverall.php), obtained either using the Abbott real-time HIV-1 or Roche Cobas-Ampliprep/TaqMan (CAP/CTM) v2.0. We used reported sensitivity and specificity of 5 common VL test at the 1000 copies per milliliter threshold from the Clinton Health Access Initiative review and at the 5000 copies per milliliter threshold from individual studies from sub-Saharan Africa, with the exception of Siemens kPCR (Table 1). We also included data for finger-prick–based DBS, which was available for the Abbott and BioMérieux NucliSENSE assays. All comparisons were performed against plasma according to the assay standard procedures. Under the current guidelines, the initial VL test (routine or targeted), if above threshold, is followed by intensive adherence counseling and repeat test after 3 months. Thus, a false-positive initial test will lead to an unnecessary repeat test and a false-positive postadherence test to an unnecessary regimen switch. A false-negative test will lead to undetected virological failure with potential for developing drug resistance. Projected misclassifications were calculated as follows: overall misclassification [(1-sensitivity) · (total positive) + (1-specificity) · total negatives/total tests], unnecessary repeats [(1-specificity) · (total negatives initial test)/total initial tests], unnecessary switches [(1-specificity) · (total negatives post-adherence test)/total post-adherence tests], and undetected failures [(1sensitivity) · (total positives)/total tests]. The analysis included 79,566 VL test results from 78,604 patients receiving ART (97% on first-line) at 514 facilities in western Kenya, collected between January 2013 and November 2014. The reasons for testing were routine initial test (n = 56,006; 70.4%), targeted initial test (n = 8517; 10.7%), postadherence test (n = 4833; 6.1%), and unrecorded (n = 10,210; 12.8%). VL test results were categorized as,1000 copies per milliliter for 79.5%, 1000–5000 copies per milliliter for 4.6%, and . 5000 copies per milliliter for 15.9%. Thus, at the 1000 copies per milliliter threshold, the proportion of patients with virological failure was 20.5%; among those, 22.4% had 1000–5000 copies per milliliter. Table 1 summarizes the projected proportions of misclassifications for each VL assay at the 1000 and 5000 copies per milliliter thresholds. Overall, misclassifications were substantially more frequent at the 1000 copies per milliliter (range, 4.1%–45.0%) threshold compared with 5000 copies per milliliter (range, 1.2%–38.0%), and this varied between assays used. The projected proportions of unnecessary repeat VL tests ranged from 1.8% (BioMérieux NucliSENSE, Craponne, France) to 45.0% (Roche CAP/CTMSPEX) at 1000 copies per milliliter and 0.0% (BioMérieux NucliSENSE) to 36.1% (Roche CAP/CTM-SPEX, Basel, Switzerland) at 5000 copies per milliliter (Table 1). The 1000 copies per milliliter threshold led to a relative increase of 82.3% for Abbott real-time (venous blood); 56.9% for Abbott real-time (Abbott, Abbott Park, IL) (finger prick); 96.4% for BioMérieux NucliSENSE (venous blood); 100% for BioMérieux NucliSENSE (finger SI is supported by a grant from the European Union through the Erasmus Mundus program. RLH is supported by a grant from the Netherlands Organization for Scientific Research through the Innovational Research Incentives Scheme. The authors have no funding or conflicts of interest to disclose. T.F. R.d. W. , S.C. I. , R.L. H. , and C.E. Z. conceived and designed the study. S.C. I. performed the data analysis and drafted the manuscript. S.C. I. , R.L. H. , and T.F. R.d. W. provided valuable input to interpretation of the data and critically reviewed the paper for important intellectual content. All authors reviewed and approved the final version of the manuscript.