HIV drug resistance mutation Profiles of DBS specimens collected on W-903, A-226, and M-TFN filter papers.

<p>HIV-1 drug resistance genotyping analyses of the <i>pol</i> region were performed for all the DBS specimens with a viral load ≥1,000 copies/mL and with all three types of the filter papers using a broadly sensitive genotyping assay (N = 26). Drug resistance mutations against nucleoside reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs) were identified using the HIVdb program, and HIV-1 drug resistance profiles were determined by the HIValg program at the Stanford HIV Drug Resistance Database website. Discordant mutations that were identified in only one type of filter paper are shown in boldface type. Specimens that had a difference in drug susceptibility ratings with one of the filter paper types are indicated by asterisk (*). Eight specimens with no mutations detected in any of the filter paper types were excluded from the table.</p><p>HIV drug resistance mutation Profiles of DBS specimens collected on W-903, A-226, and M-TFN filter papers.</p

Genotyping efficiency and nucleotide sequence identities of 26 DBS specimens with a VL ≥1000 copies/mL and collected on W-903, A-226, and M-TFN filter papers.

<p>*:Fisher's exact test; ^#SD: Standard deviation.</p><p>Genotyping efficiency and nucleotide sequence identities of 26 DBS specimens with a VL ≥1000 copies/mL and collected on W-903, A-226, and M-TFN filter papers.</p

EV71 A3 or A8-specific CD4+ T cells respond to the poliovirus 3 Sabin (PV3) homolog, A3v peptide.

<p>PBMCs from two adult subjects were cultured in the presence of EV71 A3, EV71 A8, PV3 A3v, or SP4 peptides for 4 days and then with recombinant human IL-2 for additional 3 days. The cells were washed and re-stimulated with SP4, EV71 A3 or PV A3′, then surface-stained with fluorescent antibodies against CD3 and CD4 molecules and intracellularly for the production of IFNγ. Gated CD3+ cells are shown. (A) SP4 or PV3 A3v-stimulated cells were re-stimulated with the respective peptides. (B) EV71 A3-stimulated cells were re-stimulated with SP4, EV71 A3 or PV3 A3v, respectively. (C) EV71 A8-stimulated cells were re-stimulated with SP4, EV71 A8 or PV3 A3v, respectively. The above results were representative data from three independent experiments.</p

Proportional distribution of mutated and ambiguous mutated amino acids at HIVDR sites.

<p>The mutation score at each of the drug resistance sites [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077649#B9" target="_blank">9</a>] was proportionally calculated with the mutated and ambiguous mutated amino acids for all the sequences in the datasets. A mutated or ambiguous mutated amino acid was defined as an amino acid had mutated from a wild type to a pure non-synonymous mutation or an ambiguous mutation in the mixture allele. The scores were summed by 1 for a pure amino acid mutation and 0.5 for an ambiguous amino acid mutation, and then converted to percentages against the total number of wild-type amino acids at the site. The distribution of drug resistance mutation scores was plot by the dataset of Threshold (bottom panel), Vietnam (VN, central panel) and Baseline (top panel). The <i>x</i>-axis is the wild-type amino acids at drug resistance sites; the <i>y</i>-axis is the drug resistance mutation score (%). The sites with obvious score changes across the 3 datasets from bottom to top panel were labeled by up-triangle (increased), rhombus (remained), and down- triangle (decreased). Amino acids of protease gene (Prt) were top-dash lined, and of reverse transcriptase gene (RT) were top-solid lined.</p

Descriptive statistics of ambiguous mutation in various sequence datasets.

<p>Plot of ambiguous mutations with descriptive statistics was performed using online statistical tool (<u><a href="http://www.physics.csbsju.edu/stats/" target="_blank">http://www.physics.csbsju.edu/stats/</a></u>). Individual country dataset was described for minimal and maximal ranges (short horizontal line at the bottom and top of the box), interquartile range (IQR, at 1^st to 3^rd quartile, box), median (line inside box), suspected outlier (open dot), and outlier (solid dot). Number in the bracket is the number of sequences from the country, Angola (AO), Botswana (BW), China (CN), Kenya (KE), Malawi (MW), Tanzania (TZ), Vietnam (VN), Nigeria (NG), and Canada (CA) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077649#B24" target="_blank">24</a>]. Numbers with asterisk were calculated without the outlier in dash square box. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077649#pone-0077649-g001" target="_blank">Figure 1</a>-insert shows the descriptive statistics of ambiguous mutation index in the dataset based on subtype (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077649#pone-0077649-t003" target="_blank">Table 3</a>).</p

Both A3- and A8-specific T cells are HLA-DR-restricted CD4+ T cells.

<p>(A) and (B) PBMCs from adult donors were stimulated with A3 or A8 peptides and then expanded with recombinant IL-2. (A) The stimulated PBMCs were stained with fluorescently labeled antibodies against CD3, CD4, CD8 and IFNγ before flow cytometric analysis. Antigen-specific IFNγ production was primarily associated with CD4+ T cells. (B) CD8+ T cells produced negligible level of IFNγ in response to A3 or A8. (C) A3 and A8 can induce strong CD4+ cell responses in HFMD patients infected by EV71. (D) and (E) ELISpot assays were performed to examine IFNγ production by PBMCs which were stimulated with A3 (D) or A8 (E) peptides alone or in presence of blocking antibodies against HLA-DR, HLA-DP, or HLA-A, B and –C (HLA-ABC). The results were shown as average spot forming units ± standard deviation (n = 3). Only anti-HLADR antibodies abrogated the ELISpot responses, demonstrating that the responses to EV71 are Class II-restricted.</p

Factors associated with viral suppression using univariable and multivariable logistic regression models.

<p>Factors associated with viral suppression using univariable and multivariable logistic regression models.</p

Flowchart describing the number of patients with first and second VL test and percent of virologic suppression among them.

<p>Flowchart describing the number of patients with first and second VL test and percent of virologic suppression among them.</p

Demographic, clinical, virologic, and immunological characteristics of study participants at the time of the first viral load testing (n = 2,313).

<p>Demographic, clinical, virologic, and immunological characteristics of study participants at the time of the first viral load testing (n = 2,313).</p

Difference of mixture chromatographs generated independently by 3 different operators using the optimized in-house assay from one PT sample.

<p>Panel A shows 2 codons (37 and 41 of RT) with nucleotide base calling of AYR; Panel B shows the AWR at codon 41 (the second peaks at codon 37 were not detected in this replicate); Panel C shows ACR at codon 37 (minor T was not called by the ReCall at the cutoff of 15%) and AHR at codon 41 (almost equal height of second and third peak at the 2^nd position).</p