An Infinitely Expandable Cloning Strategy plus Repeat-Proof PCR for Working with Multiple shRNA

Vector construction with restriction enzymes (REs) typically involves the ligation of a digested donor fragment (insert) to a reciprocally digested recipient fragment (vector backbone). Creating a suitable cloning plan becomes increasingly difficult for complex strategies requiring repeated insertions such as constructing multiple short hairpin RNA (shRNA) expression vectors for RNA interference (RNAi) studies. The problem lies in the reduced availability of suitable RE recognition sites with an increasing number of cloning events and or vector size. This report details a technically simple, directional cloning solution using REs with compatible cohesive ends that are repeatedly destroyed and simultaneously re-introduced with each round of cloning. Donor fragments can be made by PCR or sub-cloned from pre-existing vectors and inserted ad infinitum in any combination. The design incorporates several cloning cores in order to be compatible with as many donor sequences as possible. We show that joining sub-combinations made in parallel is more time-efficient than sequential construction (of one cassette at a time) for any combination of 4 or more insertions. Screening for the successful construction of combinations using Taq polymerase based PCR became increasingly difficult with increasing number of repeated sequence elements. A Pfu polymerase based PCR was developed and successfully used to amplify combinations of up to eleven consecutive hairpin expression cassettes. The identified PCR conditions can be beneficial to others working with multiple shRNA or other repeated sequences, and the infinitely expandable cloning strategy serves as a general solution applicable to many cloning scenarios

Multiple shRNA combinations for near-complete coverage of all HIV-1 strains

<p>Abstract</p> <p>Background</p> <p>Combinatorial RNA interference (co-RNAi) approaches are needed to account for viral variability in treating HIV-1 with RNAi, as single short hairpin RNAs (shRNA) are rapidly rendered ineffective by resistant strains. Current work suggests that 4 simultaneously expressed shRNAs may prevent the emergence of resistant strains.</p> <p>Results</p> <p>In this study we assembled combinations of highly-conserved shRNAs to target as many HIV-1 strains as possible. We analyzed intersecting conservations of 10 shRNAs to find combinations with 4+ matching the maximum number of strains using 1220+ HIV-1 sequences from the Los Alamos National Laboratory (LANL). We built 26 combinations of 2 to 7 shRNAs with up to 87% coverage for all known strains and 100% coverage of clade B subtypes, and characterized their intrinsic suppressive activities in transient expression assays. We found that all combinations had high combined suppressive activities, though there were also large changes in the individual activities of the component shRNAs in our multiple expression cassette configurations.</p> <p>Conclusion</p> <p>By considering the intersecting conservations of shRNA combinations we have shown that it is possible to assemble combinations of 6 and 7 highly active, highly conserved shRNAs such that there is always at least 4 shRNAs within each combination covering all currently known variants of entire HIV-1 subtypes. By extension, it may be possible to combine several combinations for complete global coverage of HIV-1 variants.</p

Barriers and facilitators to HIV and syphilis rapid diagnostic testing in antenatal care settings in low-income and middle-income countries: a systematic review.

BACKGROUND: Testing and treatment during pregnancy is a well-established and cost-effective prevention strategy, which relies largely on use of rapid diagnostic tests (RDTs). Yet, in many low-income and-middle-income countries, the uptake of RDTs is suboptimal. A qualitative meta-synthesis was conducted to identify the barriers and enablers to use of HIV and syphilis RDTs among pregnant women in low-income and middle-income countries. METHODS: This review was conducted using PRISMA guidelines. Eligible studies included peer-reviewed publications, which used qualitative methods to explore HIV and syphilis RDT in antenatal care clinics in low-income and middle-income countries. Studies focusing on perspectives of pregnant women, healthcare workers and/or stakeholders were included. We used an inductive approach informed by a modified socioecological model to synthesise the data. RESULTS: 62 manuscripts met the eligibility criteria. For pregnant women, initial acceptance of the RDT and continuation in antenatal care depends on the perception that engaging in testing will be a beneficial experience for their baby and themselves, often influenced by the provision of services that are gender-sensitive, confidential, respectful, flexible and considers their well-being into the future. Local sociocultural beliefs about pregnancy and diseases, awareness of diseases and gender roles in society also influenced RDT acceptability among pregnant women. For healthcare workers, the ability to provide high-quality RDT care required ongoing training, accurate and easy to use tests, support from supervisors and communities, sufficient resources and staffing to provide services, and reliable salary. At the stakeholder level, well-developed guidelines and health system infrastructures were imperative to the delivery of RDT in antenatal clinics. CONCLUSION: Our findings highlight clear gaps to the provision of sustainable and culturally acceptable maternal HIV and/or syphilis screening using RDTs. In addition, greater attention needs to be paid to community stakeholders in promoting the uptake of RDT in antenatal clinics. PROSPERO REGISTRATION NUMBER: CRD42018112190

The costs of scaling up HIV and syphilis testing in low- and middle-income countries: a systematic review.

Around two-thirds of all new HIV infections and 90% of syphilis cases occur in low- and middle-income countries (LMICs). Testing is a key strategy for the prevention and treatment of HIV and syphilis. Decision-makers in LMICs face considerable uncertainties about the costs of scaling up HIV and syphilis testing. This paper synthesizes economic evidence on the costs of scaling up HIV and syphilis testing interventions in LMICs and evidence on how costs change with the scale of delivery. We systematically searched multiple databases (Medline, Econlit, Embase, EMCARE, CINAHL, Global Health and the NHS Economic Evaluation Database) for peer-reviewed studies examining the costs of scaling up HIV and syphilis testing in LMICs. Thirty-five eligible studies were identified from 4869 unique citations. Most studies were conducted in Sub-Saharan Africa (N = 17) and most explored the costs of rapid HIV in facilities targeted the general population (N = 19). Only two studies focused on syphilis testing. Seventeen studies were cost analyses, 17 were cost-effectiveness analyses and 1 was cost-benefit analysis of HIV or syphilis testing. Most studies took a modelling approach (N = 25) and assumed costs increased linearly with scale. Ten studies examined cost efficiencies associated with scale, most reporting short-run economies of scale. Important drivers of the costs of scaling up included testing uptake and the price of test kits. The 'true' cost of scaling up testing is likely to be masked by the use of short-term decision frameworks, linear unit-cost projections (i.e. multiplying an average cost by a factor reflecting activity at a larger scale) and availability of health system capacity and infrastructure to supervise and support scale up. Cost data need to be routinely collected alongside other monitoring indicators as HIV and syphilis testing continues to be scaled up in LMICs

Sofosbuvir/velpatasvir for 12 vs. 6 weeks for the treatment of recently acquired hepatitis C infection.

BACKGROUND AND AIMS Shortened duration therapy for acute and recent hepatitis C virus (HCV) infection has been shown to be highly effective in several small non-randomised studies with direct-acting antiviral regimens, however large randomised studies are lacking. METHODS REACT was an NIH-funded multicentre international, open-label, randomised, phase 4 non-inferiority trial examining the efficacy of short course (6 weeks) versus standard course (12 weeks) therapy with sofosbuvir-velpatasvir for recent HCV infection (estimated duration of infection <= 12 months). Randomisation occurred at week 6. The primary endpoint was SVR12 in the intention-to treat (ITT) population. A total of 250 participants were planned for enrolment. On advice of the data safety and monitoring board the study was halted early. RESULTS Primary analysis population consisted of 188 randomised participants at termination of study enrolment; short arm (n=93), standard arm (n=95). Ninety seven percent were male and 69% HIV positive. ITT SVR12 was 76/93, 81.7% (95% CI 72.4-89.0) in the short arm and 86/95, 90.5% (95% CI 82.7-95.6) in the standard arm. The difference between the arms was -8.8 (95% CI: -18.6, 1.0). By modified ITT analysis in which non-virological reasons for failure were excluded (death, reinfection, lost to follow-up) SVR12 was 76/85, 89.4% (95% CI 80.8-95.0) in the short arm and 86/88, 97.7% in the standard arm (95% CI 92.0-99.7; difference -8.3%, p=0.025). CONCLUSIONS In this randomised study in recent HCV infection, 6 weeks sofosbuvir-velpatasvir did not meet the criteria for non-inferiority to standard 12 weeks duration. LAY SUMMARY In this randomised trial one hundred and eighty people with recently acquired hepatitis C infection were randomly assigned to treatment using either a short 6-week course (93 people) or standard 12-week course (95 people) of the hepatitis C treatment sofosbuvir/velpatasvir. There were nine cases of relapse after treatment in the short course and two using the standard course. A shortened course of 6 weeks therapy for hepatitis C infection was considered not as effective as a standard twelve week course in people with recently acquired hepatitis C infection. TRIAL REGISTRATION Clinicaltrials.gov Identifier: NCT02625909

Hepatitis C virus transmission between eight high-income countries among men who have sex with men: a whole-genome analysis.

BACKGROUND Microelimination of the hepatitis C virus (HCV) among men who have sex with men (MSM) could be complicated by continuous external introductions and the emergence of phylogenetic clusters harbouring clinically significant resistance-associated substitutions (RAS). To investigate international clustering and the prevalence and transmission of RAS, we aimed to analyse whole-genome HCV sequences from MSM with a recently acquired infection who participated in a large, international HCV treatment trial. METHODS For this whole-genome analysis, we obtained HCV sequences from 128 MSM who had acquired HCV within the past 12 months and were participating in the REACT trial. The participants from whom sequences were obtained were recruited at 24 sites in eight countries. We inferred maximum-likelihood phylogenies and identified transmission clusters for HCV genotypes separately. We constructed time-scaled phylogenies to estimate cluster introduction dates and used a Bayesian Skygrid approach to estimate the effective population size over the past 50 years. We calculated the prevalence of RAS and the extent of RAS transmission in the study population. FINDINGS The majority of recent HCV infections were part of international networks that arose in the late 1990s and early 2000s. Sequences obtained in the same country clustered frequently, and in 36% of subclusters since 2015 we found evidence of international transmission. European MSM were more likely than non-European MSM to be in a cluster (odds ratio 11·9 [95% CI 3·6-43·4], p<0·0001). The effective population size decreased rapidly since around 2015 in Europe. RAS associated with substantially diminished cure rates were infrequently detected and transmission of highly resistant viruses was not observed. INTERPRETATION Despite antiviral treatment becoming widely available, international transmission of HCV among MSM has still occurred over the past 8 years, which could complicate microelimination of the virus in this population. RAS-enriched clusters and widespread RAS transmission are currently not a threat to elimination goals. These findings support an international approach for HCV microelimination among MSM. FUNDING National Institutes of Health and Dr. C.J. Vaillant Fonds

AR-quiver approach to affine canonical basis elements

AbstractThis is the continuation of [Y. Li, Affine quivers of type A˜n and canonical bases, math.QA/0501175]. We describe the affine canonical basis elements in the case when the affine quiver has arbitrary orientation. This generalizes the description in [G. Lusztig, Affine quivers and canonical bases, Publ. Math. Inst. Hautes Études Sci. 76 (1992) 111–163]

Spirometry use in patientswith sickle cell disease with and without asthma and acute chest syndrome: Amulticenter study

A de‐identified data repository of electronic medical record data, i2b2 (Informatics for Integrating Biology and the Bedside), including four geographically diverse academic medical centers, was queried to determine the use of diagnostic spirometry testing in African American children and young adults 5‐34 years of age with sickle cell disease (SCD) with or without a documented history of asthma and/or acute chest syndrome (ACS). A total of 2749 patients were identified with SCD, of these 577 had asthma and 409 had ACS. Cross‐referencing the CPT code for diagnostic spirometry showed that for patients identified as having SCD, a history of ACS, and a diagnosis of asthma, only 31% across all four centers had spirometry. Having an asthma diagnosis was associated with ACS. Among SCD patients with asthma, the proportion with ACS for the four centers was 47%, 75%, 38%, and 36% respectively. The bivariate association between asthma and ACS for each Center was significant for each (P < .001). To summarize, only one third of patients with co‐morbid SCD, ACS, and asthma received the spirometry procedure as recommended in evidence‐based guidelines, suggesting limited testing for changes in pulmonary function. Future studies to determine barriers and facilitators to implementation of pulmonary testing in SCD are warranted

Sequencing of the Hepatitis C Virus: A Systematic Review

Since the identification of hepatitis C virus (HCV), viral sequencing has been important in understanding HCV classification, epidemiology, evolution, transmission clustering, treatment response and natural history. The length and diversity of the HCV genome has resulted in analysis of certain regions of the virus, however there has been little standardisation of protocols. This systematic review was undertaken to map the location and frequency of sequencing on the HCV genome in peer reviewed publications, with the aim to produce a database of sequencing primers and amplicons to inform future research. Medline and Scopus databases were searched for English language publications based on keyword/MeSH terms related to sequence analysis (9 terms) or HCV (3 terms), plus "primer" as a general search term. Exclusion criteria included non-HCV research, review articles, duplicate records, and incomplete description of HCV sequencing methods. The PCR primer locations of accepted publications were noted, and purpose of sequencing was determined. A total of 450 studies were accepted from the 2099 identified, with 629 HCV sequencing amplicons identified and mapped on the HCV genome. The most commonly sequenced region was the HVR-1 region, often utilised for studies of natural history, clustering/transmission, evolution and treatment response. Studies related to genotyping/classification or epidemiology of HCV genotype generally targeted the 5'UTR, Core and NS5B regions, while treatment response/resistance was assessed mainly in the NS3-NS5B region with emphasis on the Interferon sensitivity determining region (ISDR) region of NS5A. While the sequencing of HCV is generally constricted to certain regions of the HCV genome there is little consistency in the positioning of sequencing primers, with the exception of a few highly referenced manuscripts. This study demonstrates the heterogeneity of HCV sequencing, providing a comprehensive database of previously published primer sets to be utilised in future sequencing studies