High dietary K+ intake inhibits proximal tubule transport.

The impact of chronic dietary K+ loading on proximal tubule (PT) function was measured using free-flow micropuncture along with measurements of overall kidney function, including urine volume, glomerular filtration rate, and absolute and fractional Na+ and K+ excretion in the rat. Feeding animals a diet with 5% KCl [high K+ (HK)] for 7 days reduced glomerular filtration rate by 29%, increased urine volume by 77%, and increased absolute K+ excretion by 202% compared with rats on a 1% KCl [control K+ (CK)] diet. HK did not change absolute Na+ excretion but significantly increased fraction excretion of Na+ (1.40% vs. 0.64%), indicating that fractional Na+ absorption is reduced by HK. PT reabsorption was assessed using free-flow micropuncture in anesthetized animals. At 80% of the accessible length of the PT, measurements of inulin concentration indicated volume reabsorption of 73% and 54% in CK and HK, respectively. At the same site, fractional PT Na+ reabsorption was 66% in CK animals and 37% in HK animals. Fractional PT K+ reabsorption was 66% in CK and 37% in HK. To assess the role of Na+/H+ exchanger isoform 3 (NHE3) in mediating these changes, we measured NHE3 protein expression in total kidney microsomes as well as surface membranes using Western blots. We found no significant changes in protein in either cell fraction. Expression of the Ser552 phosphorylated form of NHE3 was also similar in CK and HK animals. Reduction in PT transport may facilitate K+ excretion and help balance Na+ excretion by shifting Na+ reabsorption from K+-reabsorbing to K+-secreting nephron segments.NEW & NOTEWORTHY In rats fed a diet rich in K+, proximal tubules reabsorbed less fluid, Na+, and K+ compared with those in animals on a control diet. Glomerular filtration rates also decreased, probably due to glomerulotubular feedback. These reductions may help to maintain balance of the two ions simultaneously by shifting Na+ reabsorption to K+-secreting nephron segments.

Explainable discovery of disease biomarkers: The case of ovarian cancer to illustrate the best practice in machine learning and Shapley analysis.

OBJECTIVE: Ovarian cancer is a significant health issue with lasting impacts on the community. Despite recent advances in surgical, chemotherapeutic and radiotherapeutic interventions, they have had only marginal impacts due to an inability to identify biomarkers at an early stage. Biomarker discovery is challenging, yet essential for improving drug discovery and clinical care. Machine learning (ML) techniques are invaluable for recognising complex patterns in biomarkers compared to conventional methods, yet they can lack physical insights into diagnosis. eXplainable Artificial Intelligence (XAI) is capable of providing deeper insights into the decision-making of complex ML algorithms increasing their applicability. We aim to introduce best practice for combining ML and XAI techniques for biomarker validation tasks. METHODS: We focused on classification tasks and a game theoretic approach based on Shapley values to build and evaluate models and visualise results. We described the workflow and apply the pipeline in a case study using the CDAS PLCO Ovarian Biomarkers dataset to demonstrate the potential for accuracy and utility. RESULTS: The case study results demonstrate the efficacy of the ML pipeline, its consistency, and advantages compared to conventional statistical approaches. CONCLUSION: The resulting guidelines provide a general framework for practical application of XAI in medical research that can inform clinicians and validate and explain cancer biomarkers.

Quantitative flow ratio versus fractional flow reserve for guiding percutaneous coronary intervention: design and rationale of the randomised FAVOR III Europe Japan trial.

Quantitative flow ratio (QFR) is a computation of fractional flow reserve (FFR) based on invasive coronary angiographic images. Calculating QFR is less invasive than measuring FFR and may be associated with lower costs. Current evidence supports the call for an adequately powered randomised comparison of QFR and FFR for the evaluation of intermediate coronary stenosis. The aim of the FAVOR III Europe Japan trial is to investigate if a QFR-based diagnostic strategy yields a non-inferior 12-month clinical outcome compared with a standard FFR-guided strategy in the evaluation of patients with intermediary coronary stenosis. FAVOR III Europe Japan is an investigator-initiated, randomised, clinical outcome, non-inferiority trial scheduled to randomise 2,000 patients with either 1) stable angina pectoris and intermediate coronary stenosis, or 2) indications for functional assessment of at least 1 non-culprit lesion after acute myocardial infarction. Up to 40 international centres will randomise patients to either a QFR-based or a standard FFR-based diagnostic strategy. The primary endpoint of major adverse cardiovascular events is a composite of all-cause mortality, any myocardial infarction, and any unplanned coronary revascularisation at 12 months. QFR could emerge as an adenosine- and wire-free alternative to FFR, making the functional evaluation of intermediary coronary stenosis less invasive and more cost-effective.

Angiography-derived physiology guidance vs usual care in an All-comers PCI population treated with the healing-targeted supreme stent and Ticagrelor monotherapy: PIONEER IV trial design.

BACKGROUND: Current ESC guidelines recommend the use of intra-coronary pressure guidewires for functional assessment of intermediate-grade coronary stenoses. Angiography-derived quantitative flow ratio (QFR) is a novel method of assessing these stenoses, and guiding percutaneous coronary intervention (PCI). METHODS/DESIGN: The PIONEER IV trial is a prospective, all-comers, multi-center trial, which will randomize 2,540 patients in a 1:1 ratio to PCI guided by angiography-derived physiology or usual care, with unrestricted use in both arms of the Healing-Targeted Supreme sirolimus-eluting stent (HT Supreme). The stent's fast, biologically healthy, and robust endothelial coverage allows for short dual-antiplatelet therapy (DAPT); hence the antiplatelet regimen of choice is 1-month DAPT, followed by ticagrelor monotherapy. In the angiography-derived physiology guided arm, lesions will be functionally assessed using on-line QFR, with stenting indicated in lesions with a QFR ≤0.80. Post-stenting, QFR will be repeated in the stented vessel(s), with post-dilatation or additional stenting recommended if the QFR<0.91 distal to the stent, or if the delta QFR (across the stent) is >0.05. Usual care PCI is performed according to standard clinical practice. The primary endpoint is a non-inferiority comparison of the patient-oriented composite endpoint (POCE) of all-cause death, any stroke, any myocardial infarction, or any clinically, and physiologically driven revascularization with a non-inferiority risk-difference margin of 3.2%, at 1-year post-procedure. Clinical follow-up will be up to 3 years. SUMMARY: The PIONEER IV trial aims to demonstrate non-inferiority of QFR-guided PCI to usual care PCI with respect to POCE at 1-year in patients treated with HT Supreme stents and ticagrelor monotherapy. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov UNIQUE IDENTIFIER: NCT04923191 CLASSIFICATIONS: Interventional Cardiology.

Reproducibility of quantitative flow ratio: the QREP study.

BACKGROUND: Quantitative flow ratio (QFR) is a tool for physiological lesion assessment based on invasive coronary angiography. AIMS: We aimed to assess the reproducibility of QFR computed from the same angiograms as assessed by multiple observers from different, international sites. METHODS: We included 50 patients previously enrolled in dedicated QFR studies. QFR was computed twice, one month apart by five blinded observers. The main analysis was the coefficient of variation (CV) as a measure of intra- and inter-observer reproducibility. Key secondary analysis was the identification of clinical and procedural characteristics predicting reproducibility. RESULTS: The intra-observer CV ranged from 2.3% (1.5-2.8) to 10.2% (6.6-12.0) among the observers. The inter-observer CV was 9.4% (8.0-10.5). The QFR observer, low angiographic quality, and low fractional flow reserve (FFR) were independent predictors of a large absolute difference between repeated QFR measurements defined as a difference larger than the median difference (>0.03). CONCLUSIONS: The inter- and intra-observer reproducibility for QFR computed from the same angiograms ranged from high to poor among multiple observers from different sites with an average agreement of 0.01±0.08 for repeated measurements. The reproducibility was dependent on the observer, angiographic quality and the coronary artery stenosis severity as assessed by FFR.

Sex difference in kidney electrolyte transport III: Impact of low K intake on thiazide-sensitive cation excretion in male and female mice.

We compared the regulation of the NaCl cotransporter (NCC) in adaptation to a low-K (LK) diet in male and female mice. We measured hydrochlorothiazide (HCTZ)-induced changes in urine volume (UV), glomerular filtration rate (GFR), absolute (ENa, EK), and fractional (FENa, FEK) excretion in male and female mice on control-K (CK, 1% KCl) and LK (0.1% KCl) diets for 7 days. With CK, NCC-dependent ENa and FENa were larger in females than males as observed previously. However, with LK, HCTZ-induced ENa and FENa increased in males but not in females, abolishing the sex differences in NCC function as observed in CK group. Despite large diuretic and natriuretic responses to HCTZ, EK was only slightly increased in response to the drug when animals were on LK. This suggests that the K-secretory apparatus in the distal nephron is strongly suppressed under these conditions. We also examined LK-induced changes in Na transport protein expression by Western blotting. Under CK conditions females expressed more NCC protein, as previously reported. LK doubled both total (tNCC) and phosphorylated NCC (pNCC) abundance in males but had more modest effects in females. The larger effect in males abolished the sex-dependence of NCC expression, consistent with the measurements of function by renal clearance. LK intake did not change NHE3, NHE2, or NKCC2 expression, but reduced the amount of the cleaved (presumably active) form of γENaC. LK reduced plasma K to lower levels in females than males. These results indicated that males had a stronger NCC-mediated adaptation to LK intake than females.

Resting distal to aortic pressure ratio and fractional flow reserve discordance affects the diagnostic performance of quantitative flow ratio: Results from an individual patient data meta-analysis.

OBJECTIVE: To evaluate the diagnostic performance of quantitative flow ratio (QFR) related to fractional flow reserve (FFR) and resting distal-to-aortic pressure ratio (resting Pd/Pa) concordance. BACKGROUND: QFR is a method for computation of FFR based on standard coronary angiography. It is unclear how QFR is performed in patients with discordance between FFR and resting pressure ratios (distal-to-aortic pressure ratio [Pd/Pa]). MATERIALS AND METHODS: The main comparison was the diagnostic performance of QFR with FFR as reference stratified by correspondence between FFR and resting Pd/Pa. Secondary outcome measures included distribution of clinical or procedural characteristics stratified by FFR and resting Pd/Pa correspondence. RESULTS: Four prospective studies matched the inclusion criteria. Analysis was performed on patient level data reaching a total of 759 patients and 887 vessels with paired FFR, QFR, and resting Pd/Pa. Median FFR was 0.85 (IQR: 0.77-0.90). Diagnostic accuracy of QFR with FFR as reference was higher if FFR corresponded to resting Pd/Pa: accuracy 90% (95% CI: 88-92) versus 72% (95% CI: 64-80), p < .001, and sAUC 0.95 (95% CI: 0.92-0.96) versus 0.73 (95% CI: 0.69-0.77), p < .001. Resting Pd/Pa and FFR discordance were related to age, sex, hypertension, and lesion severity. CONCLUSION: Diagnostic performance of QFR with FFR as reference is reduced for lesions with discordant FFR (≤0.80) and resting Pd/Pa (≤0.92) measurements.

Diagnostic performance of quantitative flow ratio in prospectively enrolled patients: An individual patient-data meta-analysis.

OBJECTIVES: We aimed to provide robust performance estimates for quantitative flow ratio (QFR) in assessment of intermediary coronary lesions. BACKGROUND: Angiography-based functional lesion assessment by QFR may appear as a cost saving and safe approach to expand the use of physiology-guided percutaneous coronary interventions. QFR was proven feasible and showed good diagnostic performance in mid-sized off-line and on-line studies with fractional flow reserve (FFR) as reference standard. METHODS: We performed a collaborative individual patient-data meta-analysis of all available prospective studies with paired assessment of QFR and FFR using the CE-marked QFR application. The main outcome was agreement of QFR and FFR using a two-step analysis strategy with a multilevel mixed model accounting for study and center level variation. RESULTS: Of 16 studies identified, four studies had prospective enrollment and provided patient level data reaching a total of 819 patients and 969 vessels with paired FFR and QFR: FAVOR Pilot (n = 73); WIFI II (n = 170); FAVOR II China (n = 304) and FAVOR II Europe-Japan (n = 272). We found an overall agreement (mean difference 0.009 ± 0.068, I2 = 39.6) of QFR with FFR. The diagnostic performance was sensitivity 84% (95%CI: 77-90, I2 = 70.1), specificity 88% (95%CI: 84-91, I2 = 60.1); positive predictive value 80% (95%CI: 76-85, I2 = 33.4), and negative predictive value 95% (95%CI: 93-96, I2 = 75.9). CONCLUSIONS: Diagnostic performance of QFR was good with FFR as reference in this meta-analysis of high quality studies. QFR could provide an easy, safe, and cost-effective solution for functional evaluation of coronary artery stenosis.

The CAMH Neuroinformatics Platform: A Hospital-Focused Brain-CODE Implementation.

Investigations of mental illness have been enriched by the advent and maturation of neuroimaging technologies and the rapid pace and increased affordability of molecular sequencing techniques, however, the increased volume, variety and velocity of research data, presents a considerable technical and analytic challenge to curate, federate and interpret. Aggregation of high-dimensional datasets across brain disorders can increase sample sizes and may help identify underlying causes of brain dysfunction, however, additional barriers exist for effective data harmonization and integration for their combined use in research. To help realize the potential of multi-modal data integration for the study of mental illness, the Centre for Addiction and Mental Health (CAMH) constructed a centralized data capture, visualization and analytics environment-the CAMH Neuroinformatics Platform-based on the Ontario Brain Institute (OBI) Brain-CODE architecture, towards the curation of a standardized, consolidated psychiatric hospital-wide research dataset, directly coupled to high performance computing resources.

Diagnostic Performance of In-Procedure Angiography-Derived Quantitative Flow Reserve Compared to Pressure-Derived Fractional Flow Reserve: The FAVOR II Europe-Japan Study.

BACKGROUND: Quantitative flow ratio (QFR) is a novel modality for physiological lesion assessment based on 3-dimensional vessel reconstructions and contrast flow velocity estimates. We evaluated the value of online QFR during routine invasive coronary angiography for procedural feasibility, diagnostic performance, and agreement with pressure-wire-derived fractional flow reserve (FFR) as a gold standard in an international multicenter study. METHODS AND RESULTS: FAVOR II E-J (Functional Assessment by Various Flow Reconstructions II Europe-Japan) was a prospective, observational, investigator-initiated study. Patients with stable angina pectoris were enrolled in 11 international centers. FFR and online QFR computation were performed in all eligible lesions. An independent core lab performed 2-dimensional quantitative coronary angiography (2D-QCA) analysis of all lesions assessed with QFR and FFR. The primary comparison was sensitivity and specificity of QFR compared with 2D-QCA using FFR as a reference standard. A total of 329 patients were enrolled. Paired assessment of FFR, QFR, and 2D-QCA was available for 317 lesions. Mean FFR, QFR, and percent diameter stenosis were 0.83±0.09, 0.82±10, and 45±10%, respectively. FFR was ≤0.80 in 104 (33%) lesions. Sensitivity and specificity by QFR was significantly higher than by 2D-QCA (sensitivity, 86.5% (78.4-92.4) versus 44.2% (34.5-54.3); P<0.001; specificity, 86.9% (81.6-91.1) versus 76.5% (70.3-82.0); P=0.002). Area under the receiver curve was significantly higher for QFR compared with 2D-QCA (area under the receiver curve, 0.92 [0.89-0.96] versus 0.64 [0.57-0.70]; P<0.001). Median time to QFR was significantly lower than median time to FFR (time to QFR, 5.0 minutes [interquartile range, -6.1] versus time to FFR, 7.0 minutes [interquartile range, 5.0-10.0]; P<0.001). CONCLUSIONS: Online computation of QFR in the catheterization laboratory is clinically feasible and is superior to angiographic assessment for evaluation of intermediary coronary artery stenosis using FFR as a reference standard. CLINICAL TRIAL REGISTRATION: URL: https://www.clinicaltrials.gov. Unique identifier: NCT02959814.

Similar prevalence of low-abundance drug-resistant variants in treatment-naive patients with genotype 1a and 1b hepatitis C virus infections as determined by ultradeep pyrosequencing.

BACKGROUND AND OBJECTIVES: Hepatitis C virus (HCV) variants that confer resistance to direct-acting-antiviral agents (DAA) have been detected by standard sequencing technology in genotype (G) 1 viruses from DAA-naive patients. It has recently been shown that virological response rates are higher and breakthrough rates are lower in G1b infected patients than in G1a infected patients treated with certain classes of HCV DAAs. It is not known whether this corresponds to a difference in the composition of G1a and G1b HCV quasispecies in regards to the proportion of naturally occurring DAA-resistant variants before treatment. METHODS: We used ultradeep pyrosequencing to determine the prevalence of low-abundance (<25% of the sequence reads) DAA-resistant variants in 191 NS3 and 116 NS5B isolates from 208 DAA-naive G1-infected patients. RESULTS: A total of 3.5 million high-quality reads of ≥ 200 nucleotides were generated. The median coverage depth was 4150x and 4470x per NS3 and NS5B amplicon, respectively. Both G1a and G1b populations showed Shannon entropy distributions, with no difference between G1a and G1b in NS3 or NS5B region at the nucleotide level. A higher number of substitutions that confer resistance to protease inhibitors were observed in G1a isolates (mainly at amino acid 80 of the NS3 region). The prevalence of amino acid substitutions that confer resistance to NS5B non-nucleoside inhibitors was similar in G1a and G1b isolates. The NS5B S282T variant, which confers resistance to the polymerase inhibitors mericitabine and sofosbuvir, was not detected in any sample. CONCLUSION: The quasispecies genetic diversity and prevalence of DAA-resistant variants was similar in G1a and G1b isolates and in both NS3 and NS5B regions, suggesting that this is not a determinant for the higher level of DAA resistance observed across G1a HCV infected patients upon treatment.

Non-nucleoside reverse transcriptase inhibitor (NNRTI) cross-resistance: implications for preclinical evaluation of novel NNRTIs and clinical genotypic resistance testing.

OBJECTIVES: The introduction of two new non-nucleoside reverse transcriptase inhibitors (NNRTIs) in the past 5 years and the identification of novel NNRTI-associated mutations have made it necessary to reassess the extent of phenotypic NNRTI cross-resistance. METHODS: We analysed a dataset containing 1975, 1967, 519 and 187 genotype-phenotype correlations for nevirapine, efavirenz, etravirine and rilpivirine, respectively. We used linear regression to estimate the effects of RT mutations on susceptibility to each of these NNRTIs. RESULTS: Sixteen mutations at 10 positions were significantly associated with the greatest contribution to reduced phenotypic susceptibility (≥10-fold) to one or more NNRTIs, including: 14 mutations at six positions for nevirapine (K101P, K103N/S, V106A/M, Y181C/I/V, Y188C/L and G190A/E/Q/S); 10 mutations at six positions for efavirenz (L100I, K101P, K103N, V106M, Y188C/L and G190A/E/Q/S); 5 mutations at four positions for etravirine (K101P, Y181I/V, G190E and F227C); and 6 mutations at five positions for rilpivirine (L100I, K101P, Y181I/V, G190E and F227C). G190E, a mutation that causes high-level nevirapine and efavirenz resistance, also markedly reduced susceptibility to etravirine and rilpivirine. K101H, E138G, V179F and M230L mutations, associated with reduced susceptibility to etravirine and rilpivirine, were also associated with reduced susceptibility to nevirapine and/or efavirenz. CONCLUSIONS: The identification of novel cross-resistance patterns among approved NNRTIs illustrates the need for a systematic approach for testing novel NNRTIs against clinical virus isolates with major NNRTI-resistance mutations and for testing older NNRTIs against virus isolates with mutations identified during the evaluation of a novel NNRTI.

Trends in Genotypic HIV-1 Antiretroviral Resistance between 2006 and 2012 in South African Patients Receiving First- and Second-Line Antiretroviral Treatment Regimens.

OBJECTIVES: South Africa's national antiretroviral (ARV) treatment program expanded in 2010 to include the nucleoside reverse transcriptase (RT) inhibitors (NRTI) tenofovir (TDF) for adults and abacavir (ABC) for children. We investigated the associated changes in genotypic drug resistance patterns in patients with first-line ARV treatment failure since the introduction of these drugs, and protease inhibitor (PI) resistance patterns in patients who received ritonavir-boosted lopinavir (LPV/r)-containing therapy. METHODS: We analysed ARV treatment histories and HIV-1 RT and protease mutations in plasma samples submitted to the Tygerberg Academic Hospital National Health Service Laboratory. RESULTS: Between 2006 and 2012, 1,667 plasma samples from 1,416 ARV-treated patients, including 588 children and infants, were submitted for genotypic resistance testing. Compared with 720 recipients of a d4T or AZT-containing first-line regimen, the 153 recipients of a TDF-containing first-line regimen were more likely to have the RT mutations K65R (46% vs 4.0%; p<0.001), Y115F (10% vs. 0.6%; p<0.001), L74VI (8.5% vs. 1.8%; p<0.001), and K70EGQ (7.8% vs. 0.4%) and recipients of an ABC-containing first-line regimen were more likely to have K65R (17% vs 4.0%; p<0.001), Y115F (30% vs 0.6%; p<0.001), and L74VI (56% vs 1.8%; p<0.001). Among the 490 LPV/r recipients, 55 (11%) had ≥1 LPV-resistance mutations including 45 (9.6%) with intermediate or high-level LPV resistance. Low (20 patients) and intermediate (3 patients) darunavir (DRV) cross resistance was present in 23 (4.6%) patients. CONCLUSIONS: Among patients experiencing virological failure on a first-line regimen containing two NRTI plus one NNRTI, the use of TDF in adults and ABC in children was associated with an increase in four major non- thymidine analogue mutations. In a minority of patients, LPV/r-use was associated with intermediate or high-level LPV resistance with predominantly low-level DRV cross-resistance.

Prototypical Recombinant Multi-Protease-Inhibitor-Resistant Infectious Molecular Clones of Human Immunodeficiency Virus Type 1.

The many genetic manifestations of HIV-1 protease inhibitor (PI) resistance present challenges to research into the mechanisms of PI resistance and the assessment of new PIs. To address these challenges, we created a panel of recombinant multi-PI-resistant infectious molecular clones designed to represent the spectrum of clinically relevant multi-PI-resistant viruses. To assess the representativeness of this panel, we examined the sequences of the panel's viruses in the context of a correlation network of PI resistance amino acid substitutions in sequences from more than 10,000 patients. The panel of recombinant infectious molecular clones comprised 29 of 41 study-defined PI resistance amino acid substitutions and 23 of the 27 tightest amino acid substitution clusters. Based on their phenotypic properties, the clones were classified into four groups with increasing cross-resistance to the PIs most commonly used for salvage therapy: lopinavir (LPV), tipranavir (TPV), and darunavir (DRV). The panel of recombinant infectious molecular clones has been made available without restriction through the NIH AIDS Research and Reference Reagent Program. The public availability of the panel makes it possible to compare the inhibitory activities of different PIs with one another. The diversity of the panel and the high-level PI resistance of its clones suggest that investigational PIs active against the clones in this panel will retain antiviral activity against most if not all clinically relevant PI-resistant viruses.

Collinearity of protease mutations in HIV-1 samples with high-level protease inhibitor class resistance.

OBJECTIVES: To determine whether pan-protease inhibitor (PI)-resistant virus populations are composed predominantly of viruses with resistance to all PIs or of diverse virus populations with resistance to different subsets of PIs. METHODS: We performed deep sequencing of plasma virus samples from nine patients with high-level genotypic and/or phenotypic resistance to all licensed PIs. The nine virus samples had a median of 12 PI resistance mutations by direct PCR Sanger sequencing. RESULTS: For each of the nine virus samples, deep sequencing showed that each of the individual viruses within a sample contained nearly all of the mutations detected by Sanger sequencing. Indeed, a median of 94.9% of deep sequence reads had each of the PI resistance mutations present as a single chromatographic peak in the Sanger sequence. A median of 5.0% of reads had all but one of the Sanger mutations that were not part of an electrophoretic mixture. CONCLUSIONS: The collinearity of PI resistance mutations in the nine virus samples demonstrated that pan-PI-resistant viruses are able to replicate in vivo despite their highly mutated protease enzymes. We hypothesize that the marked collinearity of PI resistance mutations in pan-PI-resistant virus populations results from the unique requirements for multi-PI resistance and the extensive cross-resistance conferred by many of the accessory PI resistance mutations.

Low-level persistence of drug resistance mutations in hepatitis B virus-infected subjects with a past history of Lamivudine treatment.

We sought to determine the prevalence of hepatitis B virus (HBV) lamivudine (LAM)-resistant minority variants in subjects who once received LAM but had discontinued it prior to virus sampling. We performed direct PCR Sanger sequencing and ultradeep pyrosequencing (UDPS) of HBV reverse transcriptase (RT) of plasma viruses from 45 LAM-naive subjects and 46 LAM-experienced subjects who had discontinued LAM a median of 24 months earlier. UDPS was performed to a depth of ∼3,000 reads per nucleotide. Minority variants were defined as differences from the Sanger sequence present in ≥0.5% of UDPS reads in a sample. Sanger sequencing identified ≥1 LAM resistance mutations (rtL80I/V, rtM204I, and rtA181T) in samples from 5 (11%) of 46 LAM-experienced and none of 45 LAM-naive subjects (0%; P = 0.06). UDPS detected ≥1 LAM resistance mutations (rtL80I/V, rtV173L, rtL180M, rtA181T, and rtM204I/V) in 10 (22%) of the 46 LAM-experienced subjects, including 5 in whom LAM resistance mutations were not identified by Sanger sequencing. Overall, LAM resistance mutations were more likely to be present in LAM-experienced (10/46, 22%) than LAM-naive subjects (0/45, 0%; P = 0.001). The median time since LAM discontinuation was 12.8 months in the 10 subjects with a LAM resistance mutation compared to 30.5 months in the 36 LAM-experienced subjects without a LAM resistance mutation (P < 0.001). The likelihood of detecting a LAM resistance mutation was significantly increased using UDPS compared to Sanger sequencing and was inversely associated with the time since LAM discontinuation.

Standardized representation, visualization and searchable repository of antiretroviral treatment-change episodes.

BACKGROUND: To identify the determinants of successful antiretroviral (ARV) therapy, researchers study the virological responses to treatment-change episodes (TCEs) accompanied by baseline plasma HIV-1 RNA levels, CD4+ T lymphocyte counts, and genotypic resistance data. Such studies, however, often differ in their inclusion and virological response criteria making direct comparisons of study results problematic. Moreover, the absence of a standard method for representing the data comprising a TCE makes it difficult to apply uniform criteria in the analysis of published studies of TCEs. RESULTS: To facilitate data sharing for TCE analyses, we developed an XML (Extensible Markup Language) Schema that represents the temporal relationship between plasma HIV-1 RNA levels, CD4 counts and genotypic drug resistance data surrounding an ARV treatment change. To demonstrate the adaptability of the TCE XML Schema to different clinical environments, we collaborate with four clinics to create a public repository of about 1,500 TCEs. Despite the nascent state of this TCE XML Repository, we were able to perform an analysis that generated a novel hypothesis pertaining to the optimal use of second-line therapies in resource-limited settings. We also developed an online program (TCE Finder) for searching the TCE XML Repository and another program (TCE Viewer) for generating a graphical depiction of a TCE from a TCE XML Schema document. CONCLUSIONS: The TCE Suite of applications - the XML Schema, Viewer, Finder, and Repository - addresses several major needs in the analysis of the predictors of virological response to ARV therapy. The TCE XML Schema and Viewer facilitate sharing data comprising a TCE. The TCE Repository, the only publicly available collection of TCEs, and the TCE Finder can be used for testing the predictive value of genotypic resistance interpretation systems and potentially for generating and testing novel hypotheses pertaining to the optimal use of salvage ARV therapy.

Reconsidering Yarbus: a failure to predict observers' task from eye movement patterns.

In 1967, Yarbus presented qualitative data from one observer showing that the patterns of eye movements were dramatically affected by an observer's task, suggesting that complex mental states could be inferred from scan paths. The strong claim of this very influential finding has never been rigorously tested. Our observers viewed photographs for 10s each. They performed one of four image-based tasks while eye movements were recorded. A pattern classifier, given features from the static scan paths, could identify the image and the observer at above-chance levels. However, it could not predict a viewer's task. Shorter and longer (60s) viewing epochs produced similar results. Critically, human judges also failed to identify the tasks performed by the observers based on the static scan paths. The Yarbus finding is evocative, and while it is possible an observer's mental state might be decoded from some aspect of eye movements, static scan paths alone do not appear to be adequate to infer complex mental states of an observer.

Automating HIV drug resistance genotyping with RECall, a freely accessible sequence analysis tool.

Genotypic HIV drug resistance testing is routinely used to guide clinical decisions. While genotyping methods can be standardized, a slow, labor-intensive, and subjective manual sequence interpretation step is required. We therefore performed external validation of our custom software RECall, a fully automated sequence analysis pipeline. HIV-1 drug resistance genotyping was performed on 981 clinical samples at the Stanford Diagnostic Virology Laboratory. Sequencing trace files were first interpreted manually by a laboratory technician and subsequently reanalyzed by RECall, without intervention. The relative performances of the two methods were assessed by determination of the concordance of nucleotide base calls, identification of key resistance-associated substitutions, and HIV drug resistance susceptibility scoring by the Stanford Sierra algorithm. RECall is freely available at http://pssm.cfenet.ubc.ca. In total, 875 of 981 sequences were analyzed by both human and RECall interpretation. RECall analysis required minimal hands-on time and resulted in a 25-fold improvement in processing speed (∼150 technician-hours versus ∼6 computation-hours). Excellent concordance was obtained between human and automated RECall interpretation (99.7% agreement for >1,000,000 bases compared). Nearly all discordances (99.4%) were due to nucleotide mixtures being called by one method but not the other. Similarly, 98.6% of key antiretroviral resistance-associated mutations observed were identified by both methods, resulting in 98.5% concordance of resistance susceptibility interpretations. This automated sequence analysis tool provides both standardization of analysis and a significant improvement in data workflow. The time-consuming, error-prone, and dreadfully boring manual sequence analysis step is replaced with a fully automated system without compromising the accuracy of reported HIV drug resistance data.

The HIVdb system for HIV-1 genotypic resistance interpretation.

The Stanford HIV Drug Resistance Database hosts a freely available online genotypic resistance interpretation system called HIVdb to help clinicians and laboratories interpret HIV-1 genotypic resistance tests. These tests are designed to assess susceptibility to nucleoside and nonnucleoside reverse transcriptase inhibitors (NRTI and NNRTI), protease inhibitors and integrase inhibitors. The HIVdb genotypic resistance interpretation system output consists of (1) a list of penalty scores for each antiretroviral (ARV) resistance mutation in a submitted sequence, (2) estimates of decreased NRTI, NNRTI, protease and integrase inhibitor susceptibility, and (3) comments about each ARV resistance mutation in the submitted sequence. The application's strengths are its convenience for submitting sequences, its quality control analysis, its transparency and its extensive comments. The Sierra Web service is an extension that enables laboratories analyzing many sequences to individualize the format of their results. The algorithm specification interface compiler makes it possible for HIVdb to provide results using a variety of different HIV-1 genotypic resistance interpretation algorithms.