Optimization of a Low Cost and Broadly Sensitive Genotyping Assay for HIV-1 Drug Resistance Surveillance and Monitoring in Resource-Limited Settings

Commercially available HIV-1 drug resistance (HIVDR) genotyping assays are expensive and have limitations in detecting non-B subtypes and circulating recombinant forms that are co-circulating in resource-limited settings (RLS). This study aimed to optimize a low cost and broadly sensitive in-house assay in detecting HIVDR mutations in the protease (PR) and reverse transcriptase (RT) regions of pol gene. The overall plasma genotyping sensitivity was 95.8% (N = 96). Compared to the original in-house assay and two commercially available genotyping systems, TRUGENE® and ViroSeq®, the optimized in-house assay showed a nucleotide sequence concordance of 99.3%, 99.6% and 99.1%, respectively. The optimized in-house assay was more sensitive in detecting mixture bases than the original in-house (N = 87, P<0.001) and TRUGENE® and ViroSeq® assays. When the optimized in-house assay was applied to genotype samples collected for HIVDR surveys (N = 230), all 72 (100%) plasma and 69 (95.8%) of the matched dried blood spots (DBS) in the Vietnam transmitted HIVDR survey were genotyped and nucleotide sequence concordance was 98.8%; Testing of treatment-experienced patient plasmas with viral load (VL) ≥ and <3 log10 copies/ml from the Nigeria and Malawi surveys yielded 100% (N = 46) and 78.6% (N = 14) genotyping rates, respectively. Furthermore, all 18 matched DBS stored at room temperature from the Nigeria survey were genotyped. Phylogenetic analysis of the 236 sequences revealed that 43.6% were CRF01_AE, 25.9% subtype C, 13.1% CRF02_AG, 5.1% subtype G, 4.2% subtype B, 2.5% subtype A, 2.1% each subtype F and unclassifiable, 0.4% each CRF06_CPX, CRF07_BC and CRF09_CPX

Long-lasting effect of perinatal exposure to L-tryptophan on circadian clock of primary cell lines established from male offspring born from mothers fed on dietary protein restriction.

Background aimsMaternal undernutrition programs metabolic adaptations which are ultimately detrimental to adult. L-tryptophan supplementation was given to manipulate the long-term sequelae of early-life programming by undernutrition and explore whether cultured cells retain circadian clock dysregulation.MethodsMale rat pups from mothers fed on low protein (8%, LP) or control (18%, CP) diet were given, one hour before light off, an oral bolus of L-tryptophan (125 mg/kg) between Day-12 and Day-21 of age. Body weight, food intake, blood glucose along with the capacity of colonization of primary cells from biopsies were measured during the young (45-55 days) and adult (110-130 days) phases. Circadian clock oscillations were re-induced by a serum shock over 30 hours on near-confluent cell monolayers to follow PERIOD1 and CLOCK proteins by Fluorescent Linked ImmunoSorbent Assay (FLISA) and period1 and bmal1 mRNA by RT-PCR. Cell survival in amino acid-free conditions were used to measure circadian expression of MAP-LC3B, MAP-LC3B-FP and Survivin.ResultsTryptophan supplementation did not alter body weight gain nor feeding pattern. By three-way ANOVA of blood glucose, sampling time was found significant during all phases. A significant interaction between daily bolus (Tryptophan, saline) and diets (LP, CP) were found during young (p = 0.0291) and adult (p = 0.0285) phases. In adult phase, the capacity of colonization at seeding of primary cells was twice lower for LP rats. By three-way ANOVA of PERIOD1 perinuclear/nuclear immunoreactivity during young phase, we found a significant effect of diets (p = 0.049), daily bolus (pConclusionsSequelae of early-life undernutrition and the effects of L-tryptophan supplementation can be monitored non-invasively by circadian sampling of blood D-glucose and on the expression of PERIOD1 protein in established primary cell lines

Development and Evaluation of a Decision Aid for BRCA Carriers with Breast Cancer

An explorative study of an image retrieval interface with respect to the support it offers the user to organise their search results is presented. The evaluation, involving design professionals performing practical and relevant tasks, shows that the proposed approach succeeds in encouraging the user to conceptualise their tasks better

Evolution of total blood glucose over 24 hours of rats sampled during young (red) and adult (blue) phases.

<p>Rats were from mothers fed on Low protein diet (square) with bolus of L-tryptophan (filled, LPT) or without (white, LPS) and Control diet (circle) with bolus of L-tryptophan (filled, CT) or without (white, CS). Data are expressed as means ±SEM. By three-way ANOVA we found a significant effect of the Zeitgeber (Hours) on both time series (p<0.001; A & B) and a significant interaction between Diet (Low protein, Control) and daily bolus (L-tryptophan or saline) for young phase (p = 0.0291; C) and for the adult phase (p = 0.0285; D). Note that interactions between factors shown on C and D are reversed. By applying Cosinor analysis, we found that the maximum at 16 h was representative of a rhythm for the group of rats fed as control and receiving daily bolus of L-tryptophan (Fourier analysis, autospectral plot and spectral density analysis gave a maximum at 16.7 h for CT series on A).</p

Gestational performance of dams fed on low-protein or control diet.

*<p>Unpaired Student’s t-test. Overall results are means ±SD, *<0.05.</p

Adhesion and colonization of primary cells on conventional plastics according to age.

<p>Cells were isolated by trypsinization from tail biopsies within 7 days of culture. Identical capacity to rise primary cultures were found for rats whatever their mother’s diet and perinatal treatment during the young phase. A significant loss in the capacity of colonization (p<0.05) was found for cellular preparations obtained from undernourished adult rats whatever the perinatal treatment (with or without a daily bolus of L-tryptophan).</p

Growth of offspring and amount of visceral fat at sacrifice.

<p>Evolution of body weight (A) and daily growth rate (B) of offspring receiving a daily bolus of tryptophan or of saline solution from Day-12 to 21 of age. Evolution of body weight of offspring after weaning (C) and visceral fat at day 140 (D). Four groups of rat pups are shown referred as LPS = Low-protein saline (n = 9 ); LPT = low-protein tryptophan (n = 9). Body weight s’ gain of low protein and control groups were divergent at day-7 and remained so independently of tryptophan supplementation. Data are expressed as means and ±SEM. *P<0.05; **P<0.01; ***P<0.001 by two-way ANOVA followed by Bonferroni test. (*LPS vs CS and #LPT vs CT). The body weight of offspring (n = 42) after weaning until 140-old age remained lower until the end of experiment (C) with similar visceral fat (g/100 g) at sacrifice (D). Data are expressed as means ±SEM. CS = control saline (n = 12 ); CT = control tryptophan (n = 12 ); LPS = Low-protein saline (n = 9 ); LPT = low-protein tryptophan (n = 9). *P<0.05; **P<0.01; ***P<0.001 by RM two-way ANOVA followed by Bonferroni test (*LPS vs CS and #LPT vs CT.).</p

Immunodection of PERIOD1 on primary cell monolayers from young rats over 30 hours after a 2 h serum shock according to diets and tryptophan supplementation.

<p>A. Expression of PERIOD1 was found localized in the nucleus (yellow arrows) at 6 and 30 h after serum shock. Consistent observations of the nuclear localization of PERIOD1 at 6 h were indicative of correct cell synchronization by the serum shock. Re-occurence of nuclear staining at 30 h showed PERIOD1 cycling. The yellow bar at bottom of 6H plane stands for 10 µm. B. Quantification of nuclear PERIOD1 staining on confocal images of cellular monolayers by using the Hoechst staining to delineate nuclei area and to integrate PERIOD1 staining. By three-way ANOVA, we found a significant effect of diets (p = 0.0490), of daily bolus of L-tryptophan (p<0.0001) and of Hours (p = 0.0002). All factors were significantly interacting (p = 0.0148). Data are expressed as means ±SEM. A range of 31 to 74 nuclei were measured for the intensity of PERIOD1 from at least 3 cell lines per group.</p