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.

'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.

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.

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.

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.

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.

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.

Natural variation of drug susceptibility in wild-type human immunodeficiency virus type 1.

Wild-type viruses from the ViroLogic phenotype-genotype database were evaluated to determine the upper confidence limit of the drug susceptibility distributions, or "biological cutoffs," for the PhenoSense HIV phenotypic drug susceptibility assay. Definition of the natural variation in drug susceptibility in wild-type human immunodeficiency virus (HIV) type 1 isolates is necessary to determine the prevalence of innate drug resistance and to assess the capability of the PhenoSense assay to reliably measure subtle reductions in drug susceptibility. The biological cutoffs for each drug, defined by the 99th percentile of the fold change in the 50% inhibitory concentration distributions or the mean fold change plus 2 standard deviations, were lower than those previously reported for other phenotypic assays and lower than the clinically relevant cutoffs previously defined for the PhenoSense assay. The 99th percentile fold change values ranged from 1.2 (tenofovir) to 1.8 (zidovudine) for nucleoside reverse transcriptase RT inhibitors (RTIs), from 3.0 (efavirenz) to 6.2 (delavirdine) for nonnucleoside RTIs, and from 1.6 (lopinavir) to 3.6 (nelfinavir) for protease inhibitors. To evaluate the potential role of intrinsic assay variability in the observed variations in the drug susceptibilities of wild-type isolates, 10 reference viruses with different drug susceptibility patterns were tested 8 to 30 times each. The median coefficients of variation in fold change for the reference viruses ranged from 12 to 18% for all drugs except zidovudine (32%), strongly suggesting that the observed differences in wild-type virus susceptibility to the different drugs is related to intrinsic virus variability rather than assay variability. The low biological cutoffs and assay variability suggest that the PhenoSense HIV assay may assist in defining clinically relevant susceptibility cutoffs for resistance to antiretroviral drugs.

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.

Comparison of 2 modifications of the twin-block appliance in matched Class II samples.

The purpose of this study was to compare the skeletal and dental changes contributing to Class II correction with 2 modifications of the Twin-block appliance: Twin-block appliances that use a labial bow (TB1) and Twin-block appliances that incorporate high-pull headgear and torquing spurs on the maxillary central incisors (TB2). After pretreatment equivalence was established, a total of 36 consecutively treated patients with the TB1 modification were compared with 27 patients treated with the TB2 modification. Both samples were treated in the same hospital department and the same technician made all the appliances. The cephalostat, digitizing package, and statistical methods were common to both groups. The results demonstrated that the addition of headgear to the appliance resulted in effective vertical and sagittal control of the maxillary complex and thus maximized the Class II skeletal correction in the TB2 sample. Use of the torquing springs resulted in less retroclination of the maxillary incisors in the TB2 sample when compared with the TB1 sample; however, this difference did not reach the level of statistical significance.

Vertical transmission of multidrug-resistant human immunodeficiency virus type 1 (HIV-1) and continued evolution of drug resistance in an HIV-1-infected infant.

To confirm the vertical transmission of multidrug-resistant (MDR) human immunodeficiency virus type 1 (HIV-1) and to assess its impact on further evolution of drug-resistant virus in an infant, proviral DNA amplified from infected peripheral blood mononuclear cell cultures was sequenced to identify reverse transcriptase (RT) and protease (PR) mutations. The infant had proviral DNA with evidence of RT mutations (M41L, L74V, and T215Y) and 3 PR substitutions (K20R, M36I, and V82A). After delivery, the mother's proviral DNA had the same substitutions. Phylogenetic analyses of these HIV-1 RT and PR sequences indicated epidemiological linkage. Plasma drug susceptibility was determined by using a recombinant virus assay. Plasma HIV-1 obtained after the infant's birth demonstrated reduced susceptibility to zidovudine and ritonavir. Thus, vertical transmission of MDR HIV-1 was demonstrated in the setting of detectable maternal plasma viremia. Further accumulation of broad MDR in the infant's virus to 3 antiretroviral classes occurred, despite postnatal therapy.

A multiple-site-specific heteroduplex tracking assay as a tool for the study of viral population dynamics.

Rapidly evolving entities, such as viruses, can undergo complex genetic changes in the face of strong selective pressure. We have developed a modified heteroduplex tracking assay (HTA) capable of detecting the presence of single, specific mutations or sets of linked mutations. The initial application of this approach, termed multiple-site-specific (MSS) HTA, was directed toward the detection of mutations in the HIV-1 pro gene at positions 46, 48, 54, 82, 84, and 90, which are associated with resistance to multiple protease inhibitors. We demonstrate that MSS HTA is sensitive and largely specific to all targeted mutations. The assay allows the accurate and reproducible quantitation of viral subpopulations comprising 3% or more of the total population. Furthermore, we used MSS HTA in longitudinal studies of pro gene evolution in vitro and in vivo. In the examples shown here, populations turned over rapidly and more than one population was present frequently. To demonstrate the versatility of MSS HTA, we also constructed a probe sensitive to changes at positions 181 and 184 of the RT coding domain. Changes at these positions are involved in resistance to nevirapine and 2',3'-dideoxy-3'-thiacytidine (3TC), respectively. This assay easily detected the evolution of resistance to 3TC. MSS HTA provides a rapid and sensitive approach for detecting the presence of and quantifying complex mixtures of distinct genotypes, including genetically linked mutations, and, as one example, represents a useful tool for following the evolution of drug resistance during failure of HIV-1 antiviral therapy.

A novel phenotypic drug susceptibility assay for human immunodeficiency virus type 1.

Although combination antiretroviral therapy has resulted in a considerable improvement in the treatment of human immunodeficiency virus (HIV) type 1 (HIV-1) infection, the emergence of resistant virus is a significant obstacle to the effective management of HIV infection and AIDS. We have developed a novel phenotypic drug susceptibility assay that may be useful in guiding therapy and improving long-term suppression of HIV replication. Susceptibility to protease (PR) and reverse transcriptase (RT) inhibitors is measured by using resistance test vectors (RTVs) that contain a luciferase indicator gene and PR and RT sequences derived from HIV-1 in patient plasma. Cells are transfected with RTV DNA, resulting in the production of virus particles that are used to infect target cells. Since RTVs are replication defective, luciferase activity is measured following a single round of replication. The assay has been automated to increase throughput and is completed in 8 to 10 days. Test results may be useful in facilitating the selection of optimal treatment regimens for patients who have failed prior therapy or drug-naive patients infected with drug-resistant virus. In addition, the assay can be used to evaluate candidate drugs and assist in the development of new drugs that are active against resistant strains of HIV-1.

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.