The performance of using dried blood spot samples for HIV-1 viral load testing: a systematic review and meta-analysis.

Systematic review

A total of 60 studies were identified for inclusion (Figure 1 and Table 1) [19–69]. Thirteen studies were excluded due to use of a qualitative assay, 8 studies were drug resistance testing studies, 2 studies used panels as primary sample types, 2 studies were review manuscripts lacking primary data, 2 studies used random samples. spiked blood samples and 1 study used an incorrect sample. comparator There was low to moderate heterogeneity in clinical and analytical performance comparisons within technologies, as well as in viral load medians and distributions (Table 2).

Study quality

There was some risk of bias in patient selection, however low risk of bias with the reference standard and index test (S2 Fig). Participants in most studies were not recruited consecutively or did not report the patient recruitment process, and only 5% of studies reported the patient recruitment process. There was high applicability in patient selection, index test, and reference standard; however, there were some concerns as the majority of the studies (58%) were conducted in Africa; most studies (>90%) used laboratory-prepared venous blood from a pipette; and most studies (>90%) used only 1 dried blood spot filter paper (Whatman 903).

Systematic review analysis

Mean bias was the most frequently reported analytical measure in all studies included in the systematic review (82%); therefore, the forest plots of each study were developed by technology (Fig. S3). Half of the studies included patients on antiretroviral therapy [21–23,29,33,35–37,39,40,42,43,45–47,51–54,56–59,62,64,65,68,69], while the remaining studies included patients who were not receiving antiretroviral treatment or did not indicate such information. Study characteristics, such as sample size, median viral loads, and patient viral load distributions, are summarized in Table 2.

Meta-analysis

A total of 40 studies provided 45 datasets across the 6 technologies, resulting in a total of 10,871 plasma and dried blood spot viral load results. [22,24,26,28,30,31,33,34,36–45,48–53,56,58,59,62–66,68,69]. The studies not included in the systematic review were due to the inability of the main authors to share the data. Of these, 58% of the pairs were analyzed with Roche COBAS TaqMan technology [22,26,40,43,48,49,51,56,58,65,66,69]25% with Abbott RealTimetere HIV-1 technology [26,28,31,37–39,42,45,58,59,63,69]10% with bioMérieux NucliSENS EasyQ technology [24,30,31,33,34,36,40,42,53,62]5% with Biocentric Generic HIV Charge Virale technology [41,52,64]1% with Hologic Aptima [68]and 1% with Siemens VERSANT HIV-1 RNA technology [50]. Approximately 70% of the paired data points were from studies conducted in Africa [22,24,26,28,33,34,37,39,40,42,43,48,53,56,58,59,64,65,74]of which 36% were from the Southern African Development Community region [22,26,28,42,43,56,59,64] and 24% from the East African Community region [24,33,37,48,65,69].

The viral load distribution for the 10,871 plasma samples tested was relatively evenly distributed across all viral load ranges (Fig. 2A). While approximately 41% of all plasma samples were undetectable (below the detection limit of the technology), 30% of all study plasma samples were between detectable (at the detection limit of the technology or more) and 10,000 copies/mL. Furthermore, by including only plasma samples from patients known to be on ART, we found that just over 40% of patients had undetectable levels of viral load (Fig. 2B). Approximately 31% of plasma samples from patients on ART were between detectable and 10,000 copies/mL.

Median dried blood spot viral loads were higher than median plasma viral loads for all but 2 technologies. Overall, the mean difference was 1.03 log copies/mL (Table 3) . The Abbott RealTimeterand Colon HIV-1, Abbott RealTimeterand HIV-1 one-spot, Biocentric Generic HIV Charge Virale, bioMérieux NucliSENS EasyQ HIV-1, Hologic Aptima, Roche COBAS TaqMan FVE, Roche COBAS TaqMan SPEX, and Siemens VERSANT HIV-1 RNA had a difference between median dry blood viral loads from spot and plasma samples of 0.09, 0.04, 0.17, −0.30, 0.12, 0.33, 1.99, and −0.13 log copies/mL, respectively. The mean bias for each technology was calculated by pooling all the primary data for each technology as if it were a study (Table 3). Mean biases between dried blood spot values ​​and plasma viral load varied significantly by technology. The overall mean bias was 0.30 log copies/mL. The Abbott RealTimeterand Colon HIV-1 (−0.12 log copies/mL), Abbott RealTimeterBiases for the one-point HIV-1 (0.02 log copies/mL) and Roche COBAS TaqMan FVE (0.06 log copies/mL) assays were closer to zero, while bioMerieux NucliSENS EasyQ HIV-1 ( −0.41 log copies/mL) and the Roche COBAS TaqMan SPEX Assay biases (1.03 log copies/mL) were furthest from zero. The Abbott RealTimeterThe HIV-1 two-spot, bioMerieux NucliSENS EasyQ HIV-1, and Siemens VERSANT HIV-1 RNA technologies had negative mean biases indicating underquantification compared to the plasma viral load result, which was expected due to the smaller volume. input sample. The positive mean biases of Biocentric Generic HIV Charge Virale and Roche COBAS TaqMan SPEX reflect excessive quantitation compared to the plasma viral load result, likely due to chemical extraction and processing processes that result in amplification of acids. Total intracellular and extracellular nucleic.

The WHO and many national clinical guidelines in resource-limited settings recommend using viral load testing as a binary result, above or below a specified threshold to identify virologic failure. Therefore, we compared various virologic failure thresholds for dried blood spot samples (1,000, 3,000, 5,000, 7,500, and 10,000 copies/mL) with the currently suggested threshold of 1,000 copies/mL for plasma samples to correctly classify patients (Table 3 and Fig. 3). Using a dried blood spot sample threshold of 1000 copies/mL, all 6 technologies had greater than 80% sensitivity of detecting viral load greater than 1000 copies/mL. At the same threshold, the specificity of detecting a viral load below 1,000 copies/mL was greater than 80% for all technologies except Biocentric Generic HIV Charge Virale (55.16%), Hologic Aptima (73.44%) and Roche COBAS TaqMan SPEX (43.86%). Using a higher virologic failure threshold, such as 5000 copies/mL, for dried blood spot samples lowered the sensitivity and increased the specificity of all technologies. Finally, HSROC curves were created for those technologies where more than 4 studies were included in the meta-analysis (Fig. S4).

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Fig3. Sensitivity and specificity forest plots of all studies included in the meta-analysis for each viral load technology using a treatment failure threshold of 1000 copies/mL.

Abbott Real Timeterand HIV-1 colon (a), Abbott RealTimeterand HIV-1 one-spot (b), Biocentric Generic HIV Charge Virale (c), bioMerieux NucliSENS EasyQ HIV-1 (d), Hologic Aptima (e), Roche COBAS TaqMan FVE (f), Roche COBAS TaqMan SPEX (g ), Siemens VERSANT HIV-1 RNA (h). The red bars and lines indicate the overall metrics for each viral load technology.

https://doi.org/10.1371/journal.pmed.1004076.g003

Additionally, to better understand the performance of dried blood spot samples at lower treatment failure thresholds (less than 1000 copies/mL), we compared the 6 predefined virologic treatment failure thresholds: detectable, 200, 400, 500 , 600, and 800 copies/mL—between plasma and dried blood spot samples for each technology and protocol (Table 4). Biocentric Generic HIV Charge Virale and Roche COBAS TaqMan SPEX technologies had poor specificity (<40%) at all lower thresholds below 1000 copies/mL. The Siemens Versant had a sensitivity and specificity greater than 85% using a threshold of 800 copies/mL; however, the specificity decreased below 80% at a threshold of 600 copies/mL and below 70% with all thresholds below 500 copies/mL. The Abbott RealTimeterThe HIV-1 two-spot and Roche COBAS TaqMan FVE protocols had high sensitivities and specificities at all lower thresholds except detectable. The new Abbott RealTimeterHowever, the HIV-1 single point protocol had high specificities at all lower thresholds, but sensitivity performance was below 85% at the 200 copies/mL and detectable thresholds. The Hologic Aptima had high sensitivities at all thresholds except detectable; however, the specificity was less than 85% at the 800 copies/mL and 200 copies/mL thresholds. Finally, the bioMérieux NucliSENS EasyQ HIV-1 had sensitivities and specificities greater than 85% at all thresholds.

Quantitative polymerase chain reaction inherently introduces a level of variability in test results, typically +/−0.3 log copies/mL [68,69]. Therefore, we sought to understand whether the observed performance with each technology was within the inherent limits of variability of the assay. For the Abbott RealTimeterand Colon HIV-1, Abbott RealTimeterand HIV-1 one-spot, Biocentric Generic HIV Charge Virale, bioMérieux NucliSENS EasyQ HIV-1, Hologic Aptima, Roche COBAS TaqMan FVE, Roche COBAS TaqMan SPEX, and Siemens VERSANT HIV-1 RNA, 59.28%, 68.71% , 38.04%, 52.54%, 50.40%, 62.03%, 33.45%, 47.22% of the dried blood sample test results were within the standard deviation of +/ −0.3 log copies/mL of the paired plasma test result, respectively (Fig. 4).

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Figure 4. A substantial proportion of dried blood spot results fall outside the plasma result +/−0.3 log copies/mL for each technology.

Abbott Real Timeterand HIV-1 colon (a), Abbott RealTimeterand HIV-1 one-spot (b), Biocentric Generic HIV Charge Virale (c), bioMerieux NucliSENS EasyQ HIV-1 (d), Hologic Aptima (e), Roche COBAS TaqMan FVE (f), Roche COBAS TaqMan SPEX (g ), Siemens VERSANT HIV-1 RNA (h). The blue bars represent +/−0.3 log copies/mL of the plasma result, while the orange triangles represent the viral load result of the paired dried blood spots.

https://doi.org/10.1371/journal.pmed.1004076.g004

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