Comparison of PCR success rates.

<p>Partial <i>pol</i> genes were amplified for 47 group M viruses at six sites. The whole <i>pol</i> gene was amplified at the USA_FDA site.</p

Comparison of subtyping results from all sites.

<p>Comparison of subtyping results from all sites.</p

Recombination patterns in the <i>pol</i> gene and the near full length genome.

<p>The recombination patterns of near full-length sequences determined at EQAPOL are shown and indicated on the far right. The <i>pol</i> gene region amplified at each site is indicated by the colored box. Subtypes are color coded (shown at the bottom).</p

Phylogenetic analysis of partial <i>pol</i> gene sequences from all sites.

<p>All group M virus sequences (47) from six sites were analyzed together with the EQAPOL reference sequences. The phylogenetic tree was constructed using the neighbor-joining (NJ) method with Kimura two-parameter model. Sequences from the USA_FDA site were not included due to divergent sequence fragments. Ambiguous bases in DEURF07ES002 (*) and DEURF07BR002 (#) from Canada and Brazil sites resulted in independent branches. The short sequences (⌘) of DEMD07UG002 and DEURF10US008 from the Malaysia site were excluded from the analysis.</p

Phylogenetic clusters of CRF sequences.

<p>Partial <i>pol</i> gene sequences were aligned with all subtype references from the HIV sequence database. The phylogenetic tree was constructed using the neighbor-joining (NJ) method with Kimura two-parameter model. CRFs (06, 12, 14, 24 and 47) form distinct clusters and are depicted in different colors. The 50 sequences in the EQAPOL diversity panel are indicated by black dots. Asterisks indicate the EQAPOL sequences that are derived from the same strains used to define CRF24 and CRF47.</p

Comparison of subtype results across all sites.

<p>The subtype results from each site were compared to those determined by analyzing the 47 group M whole genome sequences at EQAPOL.</p

Major drug resistance mutations in seven viruses.

<p>Major drug resistance mutations in seven viruses.</p

Estimation of HIV Incidence in a Large, Community-Based, Randomized Clinical Trial: NIMH Project Accept (HIV Prevention Trials Network 043)

<div><p>Background</p><p>National Institute of Mental Health Project Accept (HIV Prevention Trials Network [HPTN] 043) is a large, Phase III, community-randomized, HIV prevention trial conducted in 48 matched communities in Africa and Thailand. The study intervention included enhanced community-based voluntary counseling and testing. The primary endpoint was HIV incidence, assessed in a single, cross-sectional, post-intervention survey of >50,000 participants.</p><p>Methods</p><p>HIV rapid tests were performed in-country. HIV status was confirmed at a central laboratory in the United States. HIV incidence was estimated using a multi-assay algorithm (MAA) that included the BED capture immunoassay, an avidity assay, CD4 cell count, and HIV viral load.</p><p>Results</p><p>Data from Thailand was not used in the endpoint analysis because HIV prevalence was low. Overall, 7,361 HIV infections were identified (4 acute, 3 early, and 7,354 established infections). Samples from established infections were analyzed using the MAA; 467 MAA positive samples were identified; 29 of those samples were excluded because they contained antiretroviral drugs. HIV prevalence was 16.5% (range at study sites: 5.93% to 30.8%). HIV incidence was 1.60% (range at study sites: 0.78% to 3.90%).</p><p>Conclusions</p><p>In this community-randomized trial, a MAA was used to estimate HIV incidence in a single, cross-sectional post-intervention survey. Results from this analysis were subsequently used to compare HIV incidence in the control and intervention communities.</p><p>Trial Registration</p><p>ClinicalTrials.gov <a href="http://www.clinicaltrials.gov/ct2/show/NCT00203749" target="_blank">NCT00203749</a></p></div

Sample collection and in-country laboratory analysis.

a<p>Excludes participants who were not contacted, declined participation, or did not meet enrollment criteria.</p>b<p>Samples were not obtained for 2,744 eligible participants (2,310 no consent, 439 blood draw failure, 19 excluded for other reasons). The HIV status of study participants was initially characterized based on the results of the two HIV rapid tests performed in-country (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068349#s2" target="_blank">Methods</a>): HIV POS: two reactive HIV rapid tests. HIV DISC: one reactive and one non-reactive HIV rapid test. HIV NEG: two non-reactive HIV rapid tests.</p>c<p>An initial estimate of HIV prevalence was based on in-country testing (calculated as # HIV POS samples/total # samples×100).</p

Investigation of sample cross-contamination at a study site.

<p>The figure shows two examples of results from two Western blot runs that were performed at the central laboratory as part of an investigation of discordant test results. Results from various laboratory tests are shown above the Western blot strips. HIV rapid tests were performed at a laboratory at the study site in Soweto, South Africa using whole blood; N indicates that both rapid tests were non-reactive, R indicates that both rapid tests were reactive. Samples were subsequently processed to produce plasma aliquots for storage which were later shipped to a central laboratory in the United States for analysis. Results from the ARCHITECT Combo HIV Ag/Ab test are shown (COMBO); N indicates that the Combo test was non-reactive, R indicates the Combo test was reactive. Samples were also tested using the Vitros EIA Human Immunodeficiency Virus Type 1 and/or 2 (HIV-1/2) Antibody Detection in Human Serum and Plasma (EIA); N indicates that the EIA test was non-reactive, R indicates the EIA test was reactive. Western blots were interpreted as negative (N) or positive (P) based on the pattern of bands observed. The banding pattern typically varies among different HIV-positive samples. The panel on the left shows that samples 11–15 were likely to have been cross-contaminated by transfer of plasma from sample 10 into those samples during aliquot preparation (sequential unintended transfer of plasma from tube to tube). Similar findings are shown in the panel on the right; samples 17–19 were likely to have been cross-contaminated by transfer of plasma from sample 16 into those samples. Further investigation at the study site confirmed that a technologist working at the study site prepared sample aliquots without changing pipette tips. All of the samples that may have been processed on the days that this technologist was working in the laboratory were excluded from the endpoint analysis.</p