Moving towards a reliable HIV incidence test - current status, resources available, future directions and challenges ahead.

In 2011 the Incidence Assay Critical Path Working Group reviewed the current state of HIV incidence assays and helped to determine a critical path to the introduction of an HIV incidence assay. At that time the Consortium for Evaluation and Performance of HIV Incidence Assays (CEPHIA) was formed to spur progress and raise standards among assay developers, scientists and laboratories involved in HIV incidence measurement and to structure and conduct a direct independent comparative evaluation of the performance of 10 existing HIV incidence assays, to be considered singly and in combinations as recent infection test algorithms. In this paper we report on a new framework for HIV incidence assay evaluation that has emerged from this effort over the past 5 years, which includes a preliminary target product profile for an incidence assay, a consensus around key performance metrics along with analytical tools and deployment of a standardized approach for incidence assay evaluation. The specimen panels for this evaluation have been collected in large volumes, characterized using a novel approach for infection dating rules and assembled into panels designed to assess the impact of important sources of measurement error with incidence assays such as viral subtype, elite host control of viraemia and antiretroviral treatment. We present the specific rationale for several of these innovations, and discuss important resources for assay developers and researchers that have recently become available. Finally, we summarize the key remaining steps on the path to development and implementation of reliable assays for monitoring HIV incidence at a population level.

Field study of dried blood spot specimens for HIV-1 drug resistance genotyping.

Dried blood spots (DBS) are an alternative specimen type for HIV drug resistance genotyping in resource-limited settings. Data relating to the impact of DBS storage and shipment conditions on genotyping efficiency under field conditions are limited. We compared the genotyping efficiencies and resistance profiles of DBS stored and shipped at different temperatures to those of plasma specimens collected in parallel from patients receiving antiretroviral therapy in Uganda. Plasma and four DBS cards from anti-coagulated venous blood and a fifth card from finger-prick blood were prepared from 103 HIV patients with a median viral load (VL) of 57,062 copies/ml (range, 1,081 to 2,964,191). DBS were stored at ambient temperature for 2 or 4 weeks or frozen at -80 °C and shipped from Uganda to the United States at ambient temperature or frozen on dry ice for genotyping using a broadly sensitive in-house method. Plasma (97.1%) and DBS (98.1%) stored and shipped frozen had similar genotyping efficiencies. DBS stored frozen (97.1%) or at ambient temperature for 2 weeks (93.2%) and shipped at ambient temperature also had similar genotyping efficiencies. Genotyping efficiency was reduced for DBS stored at ambient temperature for 4 weeks (89.3%, P = 0.03) or prepared from finger-prick blood and stored at ambient temperature for 2 weeks (77.7%, P < 0.001) compared to DBS prepared from venous blood and handled similarly. Resistance profiles were similar between plasma and DBS specimens. This report delineates the optimal DBS collection, storage, and shipping conditions and opens a new avenue for cost-saving ambient-temperature DBS specimen shipments for HIV drug resistance (HIVDR) surveillances in resource-limited settings.

Update on World Health Organization HIV drug resistance prevention and assessment strategy: 2004-2011.

The HIV drug resistance (HIVDR) prevention and assessment strategy, developed by the World Health Organization (WHO) in partnership with HIVResNet, includes monitoring of HIVDR early warning indicators, surveys to assess acquired and transmitted HIVDR, and development of an accredited HIVDR genotyping laboratory network to support survey implementation in resource-limited settings. As of June 2011, 52 countries had implemented at least 1 element of the strategy, and 27 laboratories had been accredited. As access to antiretrovirals expands under the WHO/Joint United Nations Programme on HIV/AIDS Treatment 2.0 initiative, it is essential to strengthen HIVDR surveillance efforts in the face of increasing concern about HIVDR emergence and transmission.

Control of the interferon-induced 68-kilodalton protein kinase by the HIV-1 tat gene product.

The tat-responsive region (TAR) of the human immunodeficiency virus-1 (HIV-1) exhibits a trans-inhibitory effect on translation in vitro by activating the interferon-induced 68-kilodalton protein kinase (p68 kinase). Productive infection by HIV-1 was shown to result in a significant decrease in the amount of cellular p68 kinase. The steady-state amount of p68 kinase was also reduced in interferon-treated HeLa cell lines stably expressing tat, as compared to the amount of the kinase in interferon-treated control HeLa cells. Thus, the potential translational inhibitory effects of the TAR RNA region mediated by activation of p68 kinase may be downregulated by tat during productive HIV-1 infection.

cis-acting translational effects of the 5' noncoding region of c-myc mRNA.

We have previously shown that the 5' noncoding region of mouse c-myc mRNA has a negative effect on translational efficiency in a rabbit reticulocyte lysate (A. Darveau, J. Pelletier, and N. Sonenberg, Proc. Natl. Acad. Sci. USA 82:2315-2319, 1985). We wanted to localize and characterize the inhibitory translational element(s) in the mRNA and to study its effect in other in vitro and in vivo systems. Here we report that the restrictive element is confined to a 240-nucleotide sequence of the 5' noncoding region of mouse c-myc mRNA and that this sequence acts in cis to inhibit the translation of a heterologous mRNA. In addition, we report that the cis-inhibitory effect is also exhibited in microinjected Xenopus oocytes and wheat-germ extracts but not in HeLa cell extracts. Transfection of corresponding plasmid DNA constructs into several established cell lines did not produce the cis-inhibitory effect. A model to explain these results is presented.

The feminisation of the orthodontic workforce.

OBJECTIVE: To describe the status and activity of women in the UK orthodontic workforce. DESIGN AND SETTING: Postal questionnaire based on the UK orthodontic workforce. SUBJECTS: All orthodontic providers in the UK. MATERIALS AND METHODS: A questionnaire was circulated to the total study population. The variables studied relating to sex were numbers, age, number of sessions worked, productivity, professional status and retirement intentions. RESULTS: The response rate was 72.7%. 31.4% of the participants were female. The average age of female providers was 42.7 (SE 0.48) years, who were on average 4 years younger than males. Sixty-six percent of specialist trainees are women and 34% men. 41.5% of male providers and 31.6% of female providers plan to retire in the next 15 years. The mean number of sessions worked by women was 7.2 (SE 0.1) and men 8.2 (SE 0.1). Women completed 24.2 (SE 1.9) cases per session and men 25.6 (SE 1.3). CONCLUSIONS: The orthodontic workforce is becoming increasingly feminised. The cumulative effect of more women completing fewer cases will mean that workforce planners will need to consider increasing numbers to allow for this feminisation.

Identification of a protein that binds specifically to RNA from the first exon of c-myc.

A 55 kilodalton protein present in HeLa and MEL cells, which binds specifically to RNA derived from the first exon of the human c-myc gene, has been identified using a UV-induced crosslinking assay. This protein, called p55, is found in both cytoplasmic and nuclear fractions. The binding site on the RNA, defined using deletion analysis and synthetic oligoribonucleotides, is a purine rich region located between the two major transcriptional initiation sites P1 and P2. Possible involvement of p55 in a number of regulatory events is discussed.

Developmental regulation of translation by the 5' noncoding region of murine c-myc mRNA in Xenopus laevis.

The translational efficiency of chloramphenicol acetyltransferase (CAT) mRNA containing a 5' noncoding sequence derived from exon 1 of the murine c-myc gene (360CAT) has been examined at different stages of Xenopus egg development. In contrast to its reduced translation in the Xenopus oocyte, 360CAT mRNA is translated as efficiently as CAT mRNA when injected into either mature Xenopus eggs or Xenopus embryos. No significant alteration of 360CAT mRNA stability was observed up to 10 h post-fertilization in Xenopus embryos as compared to that of CAT mRNA. The increase in 360CAT mRNA translational efficiency in Xenopus embryos was not observed with CAT mRNAs possessing other inhibitory 5' noncoding sequences. The increase in 360CAT mRNA translational efficiency is attributed to a trans-acting factor synthesized or activated following Xenopus oocyte maturation. The possible significance of the 5' noncoding region of c-myc mRNA in developmental expression of c-myc is discussed.

'Who does what' in the orthodontic workforce.

The contraction of the economy in the United Kingdom and constraints on the National Health Service (NHS) together with new opportunities for the delivery of orthodontic treatment has resulted in an increasing number of dental personnel across the different registrant groups. This article focuses on the changes that have taken place in the orthodontic workforce over the past decade. Although others help deliver orthodontic services such as material suppliers, treatment coordinators and those involved in marketing, this article will restrict itself to informing the reader specifically about which dental registrants are doing what at the clinical interface. How health professionals have developed their skills to undertake the role they play within the team and possible threats arising because of these changes are also discussed.

Loss of antiretroviral drug susceptibility at low viral load during early virological failure in treatment-experienced patients.

BACKGROUND: Clinical studies have demonstrated a correlation between the response to second-line antiretroviral therapy and the number of drugs in the regimen to which the virus is susceptible. These studies have largely been performed in patients with viral loads over 1000 copies/ml. OBJECTIVES: To examine the evolution of resistance during early virological failure, and the potential role of susceptibility testing in patients with low viral loads (below 1000 copies/ml), in treatment-experienced patients. METHODS: Drug susceptibility and genotypes of HIV-1 from indinavir-experienced patients undergoing therapy with nelfinavir, saquinavir, abacavir and either a second nucleoside reverse transcriptase inhibitor (NRTI) or nevirapine were determined. RESULTS: Sixteen subjects were studied. Five of the ten subjects treated with nevirapine, and one of six treated with a second NRTI, achieved and maintained plasma HIV RNA < 500 copies/ml. Virus from the treatment failures lost susceptibility to one or more treatment drugs, including nelfinavir and/or saquinavir, after 4 to 36 weeks of treatment. In six of the ten failures, virus with new reductions in drug susceptibility was detected prior to failure. In five of the six failures who had at least one plasma sample with a viral load between 50 and 1000 copies/ml, reductions in susceptibility to one or more treatment drugs were detected (viral load range: 260 to 630 copies/ml). CONCLUSIONS: Drug resistance can be detected at viral loads below 1000 copies/ml which may be predictive of treatment failure. Failure of a second line regimen was typically associated with early evolution of resistance in HIV protease.

Temperature sensitive mutants of influenza A virus generated by reverse genetics and clustered charged to alanine mutagenesis.

Temperature sensitive (ts) mutants of influenza A virus have the potential to serve as live attenuated (att) virus vaccines. Previously, ts mutants were isolated by chemical mutagenesis or arose spontaneously, and most likely contained point mutations in one or more genes. While sufficiently attenuated, even the most genetically stable of these viruses was found to revert to a more virulent form in a seronegative vaccinee. Recently developed technology, however, allows the introduction of engineered mutations into the genome of influenza A and B viruses, permitting the rational design of attenuated mutants with the potential for increased genetic stability. To accomplish this goal, we have introduced ts mutations into the PB2 gene of A/Los Angeles/2/87 (H3N2) and rescued the mutated genes into infectious viruses. We have used clustered charged to alanine mutagenesis (substitution of alanine for charged amino acid residues which are present in clusters) of the PB2 gene to generate novel ts mutants. Viruses containing such ts PB2 genes were attenuated in mice and ferrets. This approach has thus yielded several vaccine candidates with ts and attenuated characteristics in animal models. Combination of these mutations with each other or with other ts mutations may lead to a high level of genetic stability.

Polymorphisms in p1-p6/p6* of HIV type 1 can delay protease autoprocessing and increase drug susceptibility.

Maturation of infectious human immunodeficiency virus type 1 (HIV-1) particles requires proteolytic cleavage of structural polyproteins by viral protease. Inhibition of protease is a powerful tool for the treatment of HIV infection. Using a well-established phenotypic drug susceptibility assay, we found that sequences outside of the protease gene can modulate the susceptibility to protease inhibitors (PIs). Chimeric viruses carrying p1-p6/p6* sequences from patient isolates in the context of an NL4-3 molecular clone exhibited increased PI susceptibility. Furthermore, this phenotype was associated with a delay in protease autoprocessing in virions and a reduction in replication capacity. We propose that the interplay between protease and the C terminus of Gag is critical for proper protease activity and mismatches between these regions can reduce viral replication and increase drug susceptibility.

Fluorides for the prevention of white spots on teeth during fixed brace treatment.

BACKGROUND: White spots can appear on teeth during fixed brace treatment because of early decay around the brace attachments. Fluoride is effective at reducing decay in susceptible individuals and is routinely prescribed in various different forms to patients during orthodontic treatment. OBJECTIVES: To evaluate the effectiveness of fluoride in preventing white spots during orthodontic treatment and to compare the different modes of delivery of fluoride. SEARCH STRATEGY: We searched the Cochrane Oral Health Group's Trials Register (to 22 August 2002); CENTRAL (The Cochrane Library Issue 3, 2002); MEDLINE (January 1966 to July 2003); EMBASE (January 1980 to week July 2003). Authors of trials were contacted for further data. SELECTION CRITERIA: Trials were selected if they met the following criteria: a randomised or quasi-randomised clinical trial, involving the use of a fluoride-containing product compared with no use or use of a non-fluoride control and enamel demineralisation was assessed during or after orthodontic treatment. DATA COLLECTION AND ANALYSIS: Six reviewers independently, in duplicate, extracted data. The primary outcome was the difference in the presence or absence of white spots between experimental and control patients for parallel design studies, and between experimental and control quadrants, for split-mouth design studies. Potential sources of heterogeneity were examined. Sensitivity analyses were undertaken for the items assessed for quality and publication bias. MAIN RESULTS: The primary outcome of the review was the presence or absence of white spots by patient at the end of treatment. Secondary outcomes included any quantitative assessment of enamel mineral loss or lesion depth. Other outcomes such as differences in size and severity of white spots, any patient based outcomes, such as perception of white spots could not be included because there were insufficient data. Fifteen trials, with 723 participants, provided data for this review. None of the studies fulfilled all of the methodological quality assessment criteria. There is some evidence that a daily sodium fluoride mouthrinse reduces the severity of enamel decay surrounding a fixed brace (weighted mean difference for lesion depth -70.0; 95% CI -118.2 to -21.8) and that use of a glass ionomer cement for bracket bonding reduces the prevalence (Peto OR 0.35; 95% CI 0.15 to 0.84) and severity of white spots (weighted mean difference for mineral loss -645 vol%.microm; 95% CI -915 to -375) compared with composite resins. REVIEWERS' CONCLUSIONS: There is some evidence that the use of topical fluoride or fluoride-containing bonding materials during orthodontic treatment reduces the occurrence and severity of white spot lesions, however there is little evidence as to which method or combination of methods to deliver the fluoride is the most effective. Based on current best practice in other areas of dentistry, for which there is evidence, we recommend that patients with fixed braces rinse daily with a 0.05% sodium fluoride mouthrinse. More high quality, clinical research is required into the different modes of delivering fluoride to the orthodontic patient.

The aetiology of hypodontia: the prevalence, severity and location of hypodontia within families.

AIMS: Previous studies have indicated that hypodontia has a significantly higher prevalence in the relatives of affected individuals than in the general population. This study aims to examine further the roles of genetic and environmental factors in the aetiology of hypodontia by investigating the relationship between the severity and distribution of hypodontia between family members, and any discernable effect of maternal health during pregnancy and birth weight. METHODS AND RESULTS: 117 first degree relatives of 41 index patients were examined clinically and radiographically to identify the presence, severity and location of hypodontia. Both siblings and parents of index patients had a higher prevalence of hypodontia than the general population. The number and location of missing teeth was not related to the number and location of missing teeth in parents or siblings. The expression of hypodontia within a family was not affected by maternal health during pregnancy. CONCLUSIONS: The variation found in the expression of hypodontia within families suggests that its occurrence is not solely determined by genetic factors, but epigenetic and environmental factors probably also are important. This finding is consistent with a multifactorial aetiology for this condition.

Short communication: the number of HIV major NRTI mutations correlates directly with other antiretroviral-associated mutations and indirectly with replicative capacity and reduced drug susceptibility.

While it is known that selection for specific HIV-1 drug resistance-associated mutations (DRM) occurs following ART failure, the patterns of resistance mutations, reduced susceptibility (RS), and replicative capacity (RC) that appear as the number of major NRTI mutations increases have been less well-studied. These changes were examined as a function of the number of major NRTI mutations using patient-derived HIV samples submitted for resistance testing between 2003-2005 (n = 35,222) that were grouped by number of NRTI-DRMs present. In the absence of NRTI-DRMs, few (3.4%) samples had RS to one or more NRTI, 33.6% to one or more NNRTI, and 12.6% to one or more PI. With one NRTI-DRM, 94% had RS to one or more NRTI, 50% to one or more NNRTI, and 33% to one or more PI. Increases in NRTI-DRMs were accompanied by increased prevalence of NNRTI and PI DRMs and RS. With one NRTI-DRM, the mean number of NRTIs with RS was 1.7, while when five NRTI-DRMs were present, RS to > or =5 NRTIs was observed. PI-DRM and RS increased at a slower rate than NNRTI-DRM and RS. RC declined from a mean of 97.8% for samples without NRTI-DRMs to 68.9% with one NRTI-DRM, possibly due to reduced fitness conferred by K65R or M184I/V, to an RC of 43.9% for samples with seven to eight NRTI-DRMs. The relatively high percent of samples with NNRTI-DRM but without NRTI-DRMs may result from selection following virologic failure, and/or transmission of virus uniquely resistant to NNRTI.

Clinical pearl: clinical tips with System-R.

The article describes the versatility and ease of use of a relatively new bracket system manufactured by GAC called System-R. This system consists of two bracket types; standard width and reduced width, both of which have an active self-ligating clip. The reduced friction offered by this system allows different mechanics to be employed. Security of ligation and absence of decaying force values allows longer treatment intervals. Fast and reliable opening and closing of the clips means reduced chairside time. Difficulties experienced personally by these brackets are highlighted and some troubleshooting tips are included.

Fluorides, orthodontics and demineralization: a systematic review.

OBJECTIVES: To evaluate the effectiveness of fluoride in preventing white spot lesion (WSL) demineralization during orthodontic treatment and compare all modes of fluoride delivery. DATA SOURCES: The search strategy for the review was carried out according to the standard Cochrane systematic review methodology. The following databases were searched for RCTs or CCTs: Cochrane Clinical Trials Register, Cochrane Oral Health Group Specialized Trials Register, MEDLINE and EMBASE. Inclusion and exclusion criteria were applied when considering studies to be included. Authors of trials were contacted for further data. DATA SELECTION: The primary outcome of the review was the presence or absence of WSL by patient at the end of treatment. Secondary outcomes included any quantitative assessment of enamel mineral loss or lesion depth. DATA EXTRACTION: Six reviewers independently, in duplicate, extracted data, including an assessment of the methodological quality of each trial. DATA SYNTHESIS: Fifteen trials provided data for this review, although none fulfilled all the methodological quality assessment criteria. One study found that a daily NaF mouthrinse reduced the severity of demineralization surrounding an orthodontic appliance (lesion depth difference -70.0 microm; 95% CI -118.2 to -21.8 microm). One study found that use of a glass ionomer cement (GIC) for bracket bonding reduced the prevalence of WSL (Peto OR 0.35; 95% CI 0.15-0.84) compared with a composite resin. None of the studies fulfilled all of the methodological quality assessment criteria. CONCLUSIONS: There is some evidence that the use of a daily NaF mouthrinse or a GIC for bonding brackets might reduce the occurrence and severity of WSL during orthodontic treatment. More high quality, clinical research is required into the different modes of delivering fluoride to the orthodontic patient.