Comparison of BED and avidity test results obtained at enrollment and at study follow-up.

<p>The figure shows results obtained for the BED assay (Panels A and B) and the avidity assay (Panels C and D) at enrollment and at follow-up (12–24 months after enrollment) for two groups of women: women identified as recently infected at enrollment using the MAA (MAA recent, Panels A and C), and women who were known to have non-recent HIV infection at enrollment (known non-recent, Panels B and D). Data are shown only for women who had test results obtained at both study visits (paired data). Dotted lines indicate the cutoffs used for the BED and avidity assay in the MAA (1.0 OD-n and 80% avidity index, respectively).</p

Comparison of HIV diversity in women with MAA recent versus known non-recent HIV infection at enrollment.^*

*<p>HIV diversity was measured using a high resolution melting (HRM) diversity assay, which expresses the genetic diversity in each region analyzed as a single numeric HRM score. The median and IQR values for HRM scores are shown.</p>a<p>IQR: Interquartile range.</p>b<p>P values were calculated using Wilcoxon rank sum test.</p

Samples used for analysis (see text).

<p>Samples collected at study enrollment were available for 2,561 (76.8%) of the 3,335 women enrolled in the PEPI-Malawi trial. The figure shows the number of samples available for analysis using the BED assay (BED), the avidity assay (avidity index, AI) and the HRM diversity assay (HRM) at enrollment and follow-up (12–24 months after enrollment). Different subsets of women were evaluated, including women identified as recently infected at enrollment using the multi-assay algorithm (MAA recent), women identified as not recently infected at enrollment using the MAA (MAA non-recent), and women who were known to have non-recent HIV infection at enrollment (known non-recent).</p

Comparison of the change in HIV diversity between enrollment and follow-up in women in the MAA recent and known non-recent groups (paired analysis).

**<p>HIV diversity was measured using a high resolution melting (HRM) diversity assay, which expresses the genetic diversity in each region analyzed as a single numeric HRM score.</p>a<p>Median paired difference of HRM scores at enrollment and follow-up; interquartile ranges are shown in parentheses.</p>b<p>P values were calculated using Wilcoxon sign rank test for within group change being zero.</p>c<p>P values were calculated using Wilcoxon rank sum test for changes being the same for the MAA recent and known non-recent groups.</p

Comparison of HRM diversity in women in the PEPI-Malawi trial and adults in the United States.

<p>The figure compares HRM test results obtained for two groups: women in the PEPI-Malawi trial (PEPI, analyzed in this report) and adults in the United States (US, analyzed in a previous study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057350#pone.0057350-Cousins1" target="_blank">[15]</a>). Results from the PEPI-Malawi trial are shown for women identified as recently infected at enrollment using the MAA (MAA recent, N = 73), and women who were known to have non-recent HIV infection at enrollment (known non-recent, N = 54); these data were obtained by testing samples collected at the time of study enrollment. Results from adults in the US are shown for adults with known recent HIV infection (N = 102, samples collected at the time of HIV seroconversion, median time since last negative HIV test: 189 days, range 14–540 days) and adults with known non-recent HIV infection (N = 67, known to be infected for 2 years or more, including 32 with CD4 cell counts <50 cells/mm³ and 30 on antiretroviral therapy) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057350#pone.0057350-Cousins1" target="_blank">[15]</a>. P values were calculated using the Wilcoxon rank sum test.</p

Samples used for analysis.

a<p>Samples were obtained from the following clinical cohorts (see Methods): CAPRISA: the CAPRISA 004 Study/TRAPS <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078818#pone.0078818-AbdoolKarim1" target="_blank">[32]</a>; FHI/Uganda and FHI/Zimbabwe: the FHI360 Hormonal Contraception and HIV (HC-HIV) Trial <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078818#pone.0078818-Morrison1" target="_blank">[24]</a>; HPTN 039: the HIV Prevention Trials Network 039 Trial <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078818#pone.0078818-Celum1" target="_blank">[33]</a>; Partners: the Partners in Prevention HSV/HIV Transmission Study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078818#pone.0078818-Celum2" target="_blank">[34]</a>; PEPI: the Pre-Exposure Prophylaxis in Infants – Malawi Trial <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078818#pone.0078818-Kumwenda1" target="_blank">[35]</a>; Rakai: the Rakai Health Sciences Program <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078818#pone.0078818-Wawer1" target="_blank">[36]</a>.</p

BED-CEIA and avidity assay results for HIV subtypes A, C, and D.

<p>Samples from the validation sample set were analyzed using the BED-CEIA (Panels A–C) and the avidity assay (Panels D–F). Results are shown for each assay for subtypes A, C, and D as a function of duration of HIV infection (years after HIV seroconversion). Data are shown for 50 randomly-selected samples for each 6-month interval after seroconversion. The HIV incidence testing algorithms evaluated in this report only included algorithms with BED-CEIA results ≤1.5 OD-n or avidity results ≤90% AI (dashed lines).</p

Accuracy of incidence estimates obtained using the BED-CEIA alone, the avidity assay alone, a two-assay multi assay algorithm (MAA), and two four-assay MAAs in three epidemic scenarios^*.

*<p>MAA: multi-assay algorithm; BED-CEIA: BED capture immunoassay (results expressed as normalized optical density units); AI: avidity index (results expressed as a percentage); CD4: CD4 cell count (results expressed as cells/mm³); VL: viral load (results expressed as HIV RNA copies/mL); yrs: years; Rel. bias: relative bias; RMSE: root mean square error. The lower two rows show results for MAAs (see text); for these MAAs, individuals are classified as MAA positive if they have results for all for assays that are below/above the cutoffs indicated.</p><p>The relative bias (in % of true incidence over 12 months) and precision of incidence estimates (expressed as the root mean square error for log incidence, RMSE) are shown for a 6-month cohort follow-up estimator and four cross-sectional testing algorithms in three different epidemic scenarios. The ranks show the relative ranking of each algorithm among the 403 evaluated algorithms according to precision of incidence estimation (RMSE).</p

Capacity to estimate and detect a 35% reduction in HIV incidence in the Southern African communities of Project Accept using the BED-CEIA alone, the avidity assay alone, and two multi-assay algorithms (MAAs)^*.

*<p>BED-CEIA: BED capture immunoassay (results expressed as normalized optical density units); AI: avidity assay (results expressed as a percentage, avidity index); CD4: CD4 cell count (results expressed as cells/mm³); VL: viral load (results expressed as HIV RNA copies/mL); Std: standard; RR: relative risk.</p><p>The table shows the mean estimated intervention effect, empirical standard error of log estimated intervention effect, the power to detect the 35% difference in incidence, and the coverage of the 95% confidence intervals obtained by a simulation study under the stable epidemic scenario. The lower two rows show results for MAAs (see text); for these MAAs, individuals are classified as MAA positive if they have results for all for assays that are below/above the cutoffs indicated.</p

Window periods and classification of individuals with long-standing infection as positive, for the BED-CEIA alone, the avidity assay alone, a two-assay multi-assay algorithm (MAA) and two 4-assay MAAs^*.

*<p>Window periods are shown in years for four testing algorithms for subtype A, subtype C, subtypes A and C combined, and subtype D. BED: the BED capture immunoassay (results are expressed as normalized optical density units); Avidity: the avidity assay (results are expressed as a percentage, avidity index); CD4: CD4 cell count (results are expressed as cells/mm³); VL: HIV viral load (results are expressed as HIV RNA copies/mL). The lower two rows show results for MAAs (see text); for these MAAs, individuals are classified as MAA positive if they have results for all for assays that are below/above the cutoffs indicated.</p>a<p>ND: not determined; MAAs that include viral load could not be evaluated for subtype D due to missing viral load data.</p