Comparison of cross-sectional HIV incidence assay results from dried blood spots and plasma

<div><p>Background</p><p>Assays have been developed for cross-sectional HIV incidence estimation using plasma samples. Large scale surveillance programs are planned using dried blood spot (DBS) specimens for incidence assessment. However, limited information exists on the performance of HIV cross-sectional incidence assays using DBS.</p><p>Methods</p><p>The assays evaluated were: Maxim HIV-1 Limiting Antigen Avidity EIA (LAg-Avidity), Sedia HIV-1 BED-Capture EIA (BED-CEIA), and CDC modified BioRad HIV-1/2 Plus O Avidity-based Assay (CDC-BioRad Avidity) using pre-determined cutoff values. 100 matched HIV-1 positive plasma and DBS samples, with known duration of infection, from the Consortium for the Evaluation and Performance of HIV Incidence Assays repository were tested. All assays were run in duplicate. To examine the degree of variability within and between results for each sample type, both categorical and continuous results were analyzed. Associations were assessed with Bland Altman, R² values and Cohen’s kappa coefficient (ĸ).</p><p>Results</p><p>Intra-assay variability using the same sample type was similar for all assays (R² 0.96 to 1.00). The R² values comparing DBS and plasma results for LAg-Avidity, BED-CEIA, and CDC-BioRad Avidity were 0.96, 0.94, and 0.84, respectively. The concordance and ĸ values between DBS and plasma for all three assays were >87% and >0.64, respectively. The Bland-Altman analysis showed significant differences between plasma and DBS samples. For all three assays, a higher number of samples were classified as recent infections using DBS samples.</p><p>Conclusions</p><p>DBS and plasma sample results were highly correlated. However, when compared to plasma, each assay performed somewhat differently in DBS at the lower and higher ends of the dynamic range. DBS samples were more likely to be classified as recently infected by all three assays, which may lead to overestimation of incidence in surveys using performance criteria derived for plasma samples.</p></div

R² values for replicate plasma and DBS samples tested with LAg-Avidity, BED-CEIA, and CDC-BioRad Avidity.

<p>R² values for replicate plasma and DBS samples tested with LAg-Avidity, BED-CEIA, and CDC-BioRad Avidity.</p

Bland-Altman plots of results obtained from testing matched plasma and DBS samples with LAg-Avidity, BED-CEIA, and CDC-BioRad Avidity.

<p>Bland-Altman plots of results obtained from testing matched plasma and DBS samples with LAg-Avidity, BED-CEIA, and CDC-BioRad Avidity.</p

Average differences (standard deviations) of replicate plasma samples and matched plasma and DBS samples tested with LAg-Avidity, BED-CEIA, and CDC-BioRad Avidity.

<p>Average differences (standard deviations) of replicate plasma samples and matched plasma and DBS samples tested with LAg-Avidity, BED-CEIA, and CDC-BioRad Avidity.</p

Characteristics of matched plasma and DBS samples.

<p>Characteristics of matched plasma and DBS samples.</p

Correlation of results from matched plasma and DBS samples tested with LAg-Avidity, BED-CEIA, and CDC-BioRad Avidity.

<p>Correlation of results from matched plasma and DBS samples tested with LAg-Avidity, BED-CEIA, and CDC-BioRad Avidity.</p

Comparison of categorical results obtained from epidemiologic data and results obtained by testing plasma and DBS samples.

<p>Results from LAg-Avidity (highlighted in blue), BED-CEIA (highlighted in green), and CDC-BioRad Avidity (highlighted in pink).</p

Fluorescence-activated cell sorting (FACS) strategies for PBMC samples (A-G) and GI tract samples (H-M; ileum shown as example).

<p><b> </b>For PBMC, all cells were included (panel A), doublets excluded (panel B), and residual non-viable cells excluded by LIVE/DEAD violet cell staining ("Viab dump," panel C). Low-frequency CCR5+ events were then collected in one sorting tube (inside box gate, panel D). Of the remaining, CCR5- events (outside box gate, panel D), CD3+ events negative for CD14 and CD11c were included for further gating (inside polygon gate, panel E), with remaining events collected in a second sorting tube (outside polygon gate, panel E). CCR5-CD3+ events negative for CD14 and CD11c that were also CD8- (panel F) and T cell receptor-γδ-, CD20-, and CD56- ("Lin dump," panel G) were collected in a third sorting tube as presumptive CD4+ T cells. Remaining CD3+ events that were either CD8+ or Lin dump+ were combined in a fourth sorting tube. For ileum and rectum, all cells were included (panel H) and then doublets excluded (panel I). Viable CD45+ events were included for further gating (inside polygon gate, panel J), with all events outside this gate collected in one sorting tube as non-hematopoietic cells. CD3+ events negative for CD14 and CD11c were included for further gating (inside polygon gate, panel K), with remaining events collected in a second sorting tube (outside polygon gate, panel K). CD3+ events negative for CD14 and CD11c that were also CD8- (panel L) and T cell receptor-γδ-, CD20-, and CD56- ("Lin dump," panel M) were collected in a third sorting tube as presumptive CD4+ T cells. Remaining CD3+ events that were either CD8+ or Lin dump+ were combined in a fourth sorting tube. Numbers in upper-right corners of flow plots indicate the percentages of events on plots falling inside gates shown. </p

HIV-specific antibodies.

<p>Blood from four time points was tested for HIV specific antibody levels using the HIV-1/2 VITROS assay (3A), a detuned version of the HIV-1 VITROS assay (3B), and the Limiting Antigen avidity assay (3C). The y-axis shows the relative level of total HIV-specific antibody, as expressed as the signal to cutoff ratio (3A–B) or normalized optical density, ODn (3C). The x axis represents months since transplant. In <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003347#ppat-1003347-g003" target="_blank">Figure 3A</a>, the dotted line represents the diagnostic HIV antibody assay cut-off level used to classify individuals as HIV-positive or HIV-negative. For purposes of comparison, HIV antibody responses were also measured in HIV-uninfected adults, untreated HIV-infected adults, and ART-treated chronically-infected adults using the detuned HIV-1 VITROS assay (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003347#ppat-1003347-g003" target="_blank">Figure 3D</a>). The bars in the scatterplots represent the median and interquartile ranges of distributions of seroreactivity for each group. Finally, samples from the Berlin Patient were tested for antibodies to other infectious diseases (3E). Tests included antibodies to CMV (strong positive, above the limit of detection), EBV, measles, and hepatitis B (all within the range of detection) as well as VZV, mumps, rubella, and toxoplasmosis (all negative, below the limit of detection). Only the results within detectable range of the assay are shown. S/CO = signal/cutoff ratio; ODn = normalized optical density; AI = antibody index.</p

Timeline for clinical treatments and study samples.

<p>Timeline for clinical treatments and study samples.</p