The potential for quality assurance systems to save costs and lives: the case of early infant diagnosis of HIV.

OBJECTIVES: Scaling up of point-of-care testing (POCT) for early infant diagnosis of HIV (EID) could reduce the large gap in infant testing. However, suboptimal POCT EID could have limited impact and potentially high avoidable costs. This study models the cost-effectiveness of a quality assurance system to address testing performance and screening interruptions, due to, for example, supply stockouts, in Kenya, Senegal, South Africa, Uganda and Zimbabwe, with varying HIV epidemics and different health systems. METHODS: We modelled a quality assurance system-raised EID quality from suboptimal levels: that is, from misdiagnosis rates of 5%, 10% and 20% and EID testing interruptions in months, to uninterrupted optimal performance (98.5% sensitivity, 99.9% specificity). For each country, we estimated the 1-year impact and cost-effectiveness (US$/DALY averted) of improved scenarios in averting missed HIV infections and unneeded HIV treatment costs for false-positive diagnoses. RESULTS: The modelled 1-year costs of a national POCT quality assurance system range from US$ 69 359 in South Africa to US$ 334 341 in Zimbabwe. At the country level, quality assurance systems could potentially avert between 36 and 711 missed infections (i.e. false negatives) per year and unneeded treatment costs between US$ 5808 and US$ 739 030. CONCLUSIONS: The model estimates adding effective quality assurance systems are cost-saving in four of the five countries within the first year. Starting EQA requires an initial investment but will provide a positive return on investment within five years by averting the costs of misdiagnoses and would be even more efficient if implemented across multiple applications of POCT.

OBJECTIFS: L'intensification du dépistage au point des soins (DPS) pour le diagnostic précoce du VIH chez le nourrisson (DPVN) pourrait réduire le grand écart dans le dépistage des nourrissons. Cependant, un DPVN DPS sous-optimal pourrait avoir un impact limité et des coûts évitables potentiellement élevés. Cette étude modélise la rentabilité d'un système d'assurance qualité pour traiter les performances des tests et les interruptions de dépistage, dues par exemple à des ruptures de stock, au Kenya, au Sénégal, en Afrique du Sud, en Ouganda et au Zimbabwe, avec des épidémies variables du VIH et des systèmes de santé différents. MÉTHODES: Nous avons modélisé une qualité de DPVN soulevée par le système d'assurance qualité à partir de niveaux sous-optimaux: c'est-à-dire des taux d'erreurs de diagnostic de 5%, 10% et 20% et des interruptions des tests de DPVN en mois, à des performances optimales ininterrompues (sensibilité de 98,5%, spécificité de 99,9%). Pour chaque pays, nous avons estimé l'impact sur un an et la rentabilité (en USD/DALY évitée) de scénarios améliorés pour éviter les infections à VIH manquées et les coûts inutiles de traitement du VIH pour les diagnostics faux positifs. RÉSULTATS: Les coûts modélisés sur un an d'un système national d'assurance qualité DPS vont de 69.359 USD en Afrique du Sud à 334.341 USD au Zimbabwe. Au niveau des pays, les systèmes d'assurance de la qualité pourraient potentiellement éviter entre 36 et 711 infections manquées (c'est-à-dire des faux négatifs) par an et des coûts de traitement inutiles entre 5.808 et 739.030 USD. CONCLUSIONS: Le modèle estime que l'ajout de systèmes d'assurance qualité efficaces permet de réaliser des économies dans quatre des cinq pays au cours de la première année. Le lancement de l'assurance qualité nécessite un investissement initial, mais fournira un retour sur investissement positif dans les cinq ans en évitant les coûts des diagnostics erronés et serait encore plus efficace s'il était mis en œuvre dans plusieurs applications de DPS.

Acceptability and feasibility of a social entrepreneurship testing model to promote HIV self-testing and linkage to care among men who have sex with men.

OBJECTIVES: HIV self-testing (HIVST) offers an opportunity to increase HIV testing among people not reached by facility-based services. However, the promotion of HIVST is limited as a consequence of insufficient community engagement. We built a social entrepreneurship testing (SET) model to promote HIVST linkage to care among Chinese men who have sex with men (MSM) in Guangzhou. METHODS: The SET model includes a few key steps. Each participant first completed an online survey, and paid a US$23 (refundable) deposit to receive an HIVST kit and a syphilis self-testing (SST) kit. After the testing, the results were sent to the platform by the participants and interpreted by Center for Disease Control and Prevention (CDC) staff. Meanwhile, the deposit was returned to each participant. Finally, the Community based organizations (CBO) contacted the participants to provide counselling services, confirmation testing and linkage to care. RESULTS: During April-June 2015, a total of 198 MSM completed a preliminary survey and purchased self-testing kits. The majority were aged < 34 years (84.4%) and met partners online (93.1%). In addition, 68.9% of participants had ever been tested for HIV, and 19.5% had ever performed HIVST. Overall, feedback was received from 192 participants (97.0%). Of these participants, 14 people did not use the kits; among those who did use the kits, the HIV and syphilis prevalences were 4.5% (eight of 178) and 3.7% (six of 178), respectively. All of the screened HIV-positive individuals sought further confirmation testing and were linked to care. CONCLUSIONS: Using an online SET model to promote HIV and syphilis self-testing among Chinese MSM is acceptable and feasible, and this model adds a new testing platform to the current testing service system.

Pluronic-PEI copolymers enhance exon-skipping of 2'-O-methyl phosphorothioate oligonucleotide in cell culture and dystrophic mdx mice.

A series of small-size polyethylenimine (PEI)-conjugated pluronic polycarbamates (PCMs) have been investigated for the ability to modulate the delivery of 2'-O-methyl phosphorothioate RNA (2'-OMePS) in vitro and in dystrophic mdx mice. The PCMs retain strong binding capacity to negatively charged oligomer as demonstrated by agarose gel retardation assay, with the formation of condensed polymer/oligomer complexes at a wide-range weight ratio from 1:1 to 20:1. The condensed polymer/oligomer complexes form 100-300 nm nanoparticles. Exon-skipping effect of 2'-OMePS was dramatically enhanced with the use of the most effective PCMs in comparison with 2'-OMePS alone in both cell culture and in vivo, respectively. More importantly, the effective PCMs, especially those composed of moderate size (2k-5kDa) and intermediate hydrophilic-lipophilic balance (7-23) of pluronics, enhanced exon-skipping of 2'-OMePS with low toxicity as compared with Lipofectamine-2000 in vitro or PEI 25k in vivo. The variability of individual PCM for delivery of antisense oligomer and plasmid DNA indicate the complexity of interaction between polymer and their cargos. Our data demonstrate the potential of PCMs to mediate delivery of modified antisense oligonucleotides to the muscle for treating muscular dystrophy or other appropriate myodegenerative diseases.

Performance by spring and fall-calving cows grazing with full, limited, or no access to toxic Neotyphodium coenophialum-infected tall fescue.

Replacing toxic, wild-type Neotyphodium coenophialum-infected tall fescue (E+) with nontoxic, N. coenophialum-infected tall fescue (NE+) has improved cow performance, but producer acceptance of NE+ has been slow. The objective was to compare performance by spring- and fall-calving cows grazing either E+ or NE+ at different percentages of the total pasture area. Gelbvieh×Angus crossbred cows (n=178) were stratified by BW and age within calving season and allocated randomly to 1 of 14 groups representing 5 treatments for a 3-yr study: i) Fall-calving on 100% E+ (F100); ii) Spring-calving on 100% E+ (S100); iii) Fall-calving on 75% E+ and 25% NE+ (F75); iv) Spring-calving on 75% E+ and 25% NE+ (S75); and v) Spring-calving on 100% NE+ (SNE100). Groups allocated to F75 and S75 grazed E+ until approximately 28 d before breeding and weaning, then were then moved to their respective NE+ pasture area for 4 to 6 wk; those allocated to F100, S100, and SNE100 grazed their pastures throughout the entire year. Samples of tall fescue were gathered from specific cells within each pasture at the time cows were moved into that particular cell (∼1 sample/mo). Blood samples were collected from the cows at the start and end of the breeding season. Stocking rate for each treatment was 1 cow/ha. Forage IVDMD, CP, and total ergot alkaloid concentrations were affected (P<0.05) by the treatment×sampling date interaction. Hay offered, cow BW, and BCS at breeding, end of breeding, and at weaning were greater (P<0.05) from fall-calving vs. spring-calving. Cow BW at weaning was greater (P<0.05) from F75 and S75 vs. F100 and S100. The calving season×NE+ % interaction affected (P<0.05) calving rates. Preweaning calf BW gain, actual and adjusted weaning BW, ADG, sale price, and calf value at weaning were greater (P<0.05) from fall-calving vs. spring-calving and from SNE100 vs. S75 except for sale price which was greater (P<0.05) from S75 vs. SNE100. Cow concentrations of serum prolactin at breeding and serum NEFA at the end of breeding were affected (P<0.05) by the calving season×NE+ % interaction. Serum Zn and Cu concentrations from cows were affected (P<0.05) by calving season. A fall-calving season may be more desirable for cows grazing E+, resulting in greater calving rates, cow performance, and calf BW at weaning, whereas limited access to NE+ may increase calving rates, serum prolactin, and NEFA concentrations during certain times in the production cycle, particularly in spring-calving cows.

Molecular mechanisms of micronucleus, nucleoplasmic bridge and nuclear bud formation in mammalian and human cells.

Micronuclei (MN) and other nuclear anomalies such as nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) are biomarkers of genotoxic events and chromosomal instability. These genome damage events can be measured simultaneously in the cytokinesis-block micronucleus cytome (CBMNcyt) assay. The molecular mechanisms leading to these events have been investigated over the past two decades using molecular probes and genetically engineered cells. In this brief review, we summarise the wealth of knowledge currently available that best explains the formation of these important nuclear anomalies that are commonly seen in cancer and are indicative of genome damage events that could increase the risk of developmental and degenerative diseases. MN can originate during anaphase from lagging acentric chromosome or chromatid fragments caused by misrepair of DNA breaks or unrepaired DNA breaks. Malsegregation of whole chromosomes at anaphase may also lead to MN formation as a result of hypomethylation of repeat sequences in centromeric and pericentromeric DNA, defects in kinetochore proteins or assembly, dysfunctional spindle and defective anaphase checkpoint genes. NPB originate from dicentric chromosomes, which may occur due to misrepair of DNA breaks, telomere end fusions, and could also be observed when defective separation of sister chromatids at anaphase occurs due to failure of decatenation. NBUD represent the process of elimination of amplified DNA, DNA repair complexes and possibly excess chromosomes from aneuploid cells.

Long term depleted uranium exposure in Gulf War I veterans does not cause elevated numbers of micronuclei in peripheral blood lymphocytes.

Depleted uranium (DU) is a high density heavy metal that has been used in military munitions since the 1991 Gulf War. DU is weakly radioactive and chemically toxic. Long term exposure can cause adverse health effects. This study assessed genotoxic effects in DU exposed Gulf War I veterans as a function of uranium (U) body burden. Levels of urine U were used to categorize the cohort into low and high exposure groups. Exposure to DU occurred during friendly fire incidents in 1991 involving DU munitions resulting in inhalation and ingestion exposure to small particles of DU and soft tissue DU fragments from traumatic injuries. All of these Veterans are enrolled in a long term health surveillance program at the Baltimore Veterans Administration Medical Center. Blood was drawn from 35 exposed male veterans aged 36-59 years, then cultured and evaluated for micronuclei (MN) using the cytokinesis block method. The participants were divided into two exposure groups, low and high, based on their mean urine uranium (uU) concentrations. Poisson regression analyses with mean urine U concentrations, current smoking, X-rays in the past year and donor age as dependent variables revealed no significant relationships with MN frequencies. Our results indicate that on-going systemic exposure to DU occurring in Gulf War I Veterans with DU embedded fragments does not induce significant increases in MN in peripheral blood lymphocytes compared to MN frequencies in Veterans with normal U body burdens.

Increased frequency of chromosome translocations in airline pilots with long-term flying experience.

BACKGROUND: Chromosome translocations are an established biomarker of cumulative exposure to external ionising radiation. Airline pilots are exposed to cosmic ionising radiation, but few flight crew studies have examined translocations in relation to flight experience. METHODS: We determined the frequency of translocations in the peripheral blood lymphocytes of 83 airline pilots and 50 comparison subjects (mean age 47 and 46 years, respectively). Translocations were scored in an average of 1039 cell equivalents (CE) per subject using fluorescence in situ hybridisation (FISH) whole chromosome painting and expressed per 100 CE. Negative binomial regression models were used to assess the relationship between translocation frequency and exposure status and flight years, adjusting for age, diagnostic x ray procedures, and military flying. RESULTS: There was no significant difference in the adjusted mean translocation frequency of pilots and comparison subjects (0.37 (SE 0.04) vs 0.38 (SE 0.06) translocations/100 CE, respectively). However, among pilots, the adjusted translocation frequency was significantly associated with flight years (p = 0.01) with rate ratios of 1.06 (95% CI 1.01 to 1.11) and 1.81 (95% CI 1.16 to 2.82) for a 1- and 10-year incremental increase in flight years, respectively. The adjusted rate ratio for pilots in the highest compared to the lowest quartile of flight years was 2.59 (95% CI 1.26 to 5.33). CONCLUSIONS: Our data suggests that pilots with long-term flying experience may be exposed to biologically significant doses of ionising radiation. Epidemiological studies with longer follow-up of larger cohorts of pilots with a wide range of radiation exposure levels are needed to clarify the relationship between cosmic radiation exposure and cancer risk.

Technique for culturing Macaca mulatta peripheral blood lymphocytes for fluorescence in situ hybridization of whole chromosome paints.

The rhesus monkey (Macaca mulatta) has long been an important model in biomedical and behavioral research. The biomedical importance of M. mulatta is due to its 93% genetic similarity with humans and its complex social behavior. The recent sequencing of the M. mulatta genome has enhanced its role in biological research. However, the use of the macaque as an experimental model in cytogenetic assays has been problematic due to difficulties in obtaining large numbers of well-spread cells in metaphase without the use of extremely toxic mitogens such as staphylococcal enterotoxin A (SEA). Here we describe a technique for culturing and producing sufficient numbers of cells in metaphase using the common mitogens phytohemagglutinin (PHA), concanavalin A (ConA), and T-cell growth factor (TCGF) which act synergistically to induce M. mulatta T-lymphocyte division. Using this method we have obtained a mitotic index in 48 h cultures of 12.0+/-2.2 metaphase cells/100 cells (n=5 animals). Fluorescence in situ hybridization with whole chromosome painting of M. mulatta cells was performed with human whole-chromosome probes that labeled the following chromosomes for human (M. mulatta): 1(1), 2q(12), 2p(13), 4(5) pairs in red, and 3(2), 5(6) and 6(4) pairs in green. In humans this probe combination simultaneously paints 3 chromosome pairs in red and 3 in green, whereas in M. mulatta 4 chromosome pairs are labeled in red and 3 pairs are labeled in green. Using this method we show a baseline frequency of 0.026 translocations per 100 whole-genome cell equivalents in peripheral blood lymphocytes obtained from unexposed adolescent non-human primates. This method will add to the usefulness of M. mulatta as an animal model in biomedical research.

Detection of acute HIV infections among sexually transmitted disease clinic patients: a practice in Guangxi Zhuang Autonomous Region, China.

Detection of people with acute HIV infection (AHI) affords an important opportunity for early HIV treatment and prevention. HIV RNA reverse transcriptase-polymerase chain reaction (RT-PCR) testing with two-stage pooling scheme was used to detect the AHI in specimens collected from sexually transmitted disease (STD) clinic patients in Guangxi, China. A total of 246 HIV RNA tests were required to screen 11 395 samples negative for conventional enzyme immunoassay (EIA) and Western blot assays, and five AHI cases (0.04%, 95%CI 0.02% to 0.10%) with a high viral load (median of 265,677 copies per ml) were detected. The total expenditure for RT-PCR testing reflected an added cost of $2.9 per specimen screened and $6575 per additional case of AHI identified among the study population. This study supports the feasibility of pooled RNA testing in addition to detection of HIV infections among patients at STD clinics in China, but the cost effectiveness should be carefully considered.

Sex worker incarceration in the People's Republic of China.

Tens of thousands of commercial sex workers in China are administratively detained each year in female re-education through labor (RTL) centres for moral education and vocational training. Recent increases in syphilis and heterosexual HIV make tailored HIV prevention efforts for sex workers increasingly important in many regions of China. However, RTL centres focused on detaining commercial sex workers have not traditionally been linked to sexually transmitted infections (STI)/HIV programmes. The stigma of being incarcerated and selling sex complicates STI/HIV prevention for these women. Incarcerated sex workers represent a particularly marginalized HIV risk group that has been excluded from domestic and international HIV programmes to date. Although several laws and administrative decrees provide a legal mandate for sex worker STI/HIV testing, treatment and rights, there is still substantial variation in how laws are implemented. Creating devoted medical services and legal aid for incarcerated sex workers is important in curbing the spread of heterosexual HIV and other STIs in China. Recent legal and social developments suggest that China's RTL system will be transformed in the near future, gaining momentum for reform that could improve the sexual and human rights of incarcerated sex workers.

Retrospective assessment of radiation exposure using biological dosimetry: chromosome painting, electron paramagnetic resonance and the glycophorin a mutation assay.

Biological monitoring of dose can contribute important, independent estimates of cumulative radiation exposure in epidemiological studies, especially in studies in which the physical dosimetry is lacking. Three biodosimeters that have been used in epidemiological studies to estimate past radiation exposure from external sources will be highlighted: chromosome painting or FISH (fluorescence in situ hybridization), the glycophorin A somatic mutation assay (GPA), and electron paramagnetic resonance (EPR) with teeth. All three biodosimeters have been applied to A-bomb survivors, Chernobyl clean-up workers, and radiation workers. Each biodosimeter has unique advantages and limitations depending upon the level and type of radiation exposure. Chromosome painting has been the most widely applied biodosimeter in epidemiological studies of past radiation exposure, and results of these studies provide evidence that dose-related translocations persist for decades. EPR tooth dosimetry has been used to validate dose models of acute and chronic radiation exposure, although the present requirement of extracted teeth has been a disadvantage. GPA has been correlated with physically based radiation dose after high-dose, acute exposures but not after low-dose, chronic exposures. Interindividual variability appears to be a limitation for both chromosome painting and GPA. Both of these techniques can be used to estimate the level of past radiation exposure to a population, whereas EPR can provide individual dose estimates of past exposure. This paper will review each of these three biodosimeters and compare their application in selected epidemiological studies.

Harmonisation of European tests for serological diagnosis of Brucella infection in bovines.

The principal methods for the serological diagnosis of bovine brucellosis are the complement fixation test (CFT), serum agglutination test (SAT), Rose-Bengal test (RBT), indirect enzyme-linked immunosorbent assay (iELISA) and more recently the competitive ELISA (cELISA) and the fluorescent polarisation assay (FPA). Guidelines set by the World Organisation for Animal Health (OIE) describe methods and diagnostic thresholds for each of these tests. Many countries have adopted these methods for the purposes of eradication of brucellosis and have legislated for the use of these tests (the CFT and SAT in particular) for the prevention of the spread of the disease through international trade. Within the European Union (EU) each member state has a National Reference Laboratory which regulates the quality of brucellosis diagnosis and works to the recommendations set by the OIE. This article describes the results from the first three EU ring trials assessing the harmonisation of diagnostic tests between each member state. The general level of harmony for SAT, CFT, and iELISA was found to be good, but issues of standardisation of the RBT, cELISA and FPA remain. The cELISA and FPA in particular need further work to create European harmony. The ring trials also proved successful at providing specific evidence of poor performance in some areas. The decision on whether or not to take action on the basis of these results rested with the individual laboratories concerned. The increase in the number of participants in these trials over time reflected the enlargement of the EU and increased the need for quality assurance.

Validation of FPA and cELISA for the detection of antibodies to Brucella abortus in cattle sera and comparison to SAT, CFT, and iELISA.

The fluorescence polarisation assay (FPA) is a recently described test for the serological diagnosis of Brucella infection. It has many methodological advantages over older, more established tests and can be performed in a fraction of the time. To validate the FPA, serum samples from 146 confirmed (by culture) Brucella-infected cattle were tested in conjunction with serum samples from 1947 noninfected cattle. The competitive ELISA (cELISA) was validated using these positive reference samples and 1440 negative samples, while data for the indirect ELISA (iELISA) was generated from 6957 negative samples plus the positive sera. Published diagnostic specificity (DSp) data for the complement fixation test (CFT) and serum agglutination test (SAT) was used in conjunction with the test results on the positive sera to obtain diagnostic specificity plus diagnostic sensitivity (DSn). After selection of a cutoff for the FPA and cELISA, the diagnostic specificity and sensitivity total for each test were compared. The results, with 95% confidence intervals, were: FPA (195.7+/-2.79), iELISA (195.0+/-2.70), cELISA (194.9+/-3.48), CFT (191.7+/-4.45), and SAT (180.4+/-6.33). The data presented supports the use of the FPA in diagnosis of brucellosis and questions the use of the SAT and CFT for either screening or confirmatory testing.

Detection of DNA point mutations and mRNA expression levels by rolling circle amplification in individual cells.

Rolling circle amplification has been useful for detecting point mutations in isolated nucleic acids, but its application in cytological preparations has been problematic. By pretreating cells with a combination of restriction enzymes and exonucleases, we demonstrate that rolling circle amplification in situ can detect gene copy number and single base mutations in fixed cells with efficiencies up to 90%. It can also detect and quantify transcribed RNA in individual cells, making it a versatile tool for cell-based assays.

Fish cytogenetics and the future of radiation biodosimetry.

Fluorescence in situ hybridisation (FISH) with whole chromosome paints has greatly facilitated the analysis of structural chromosome aberrations and has led to translocations replacing dicentrics as the aberration of choice for many applications. Major challenges remain if we are to go from translocations to an understanding of the health consequences of radiation exposure. Yet to be surmounted are the roles of individual susceptibility, time since exposure, and the effects of subjects age. Accomplishing these objectives will require automation, reduced costs, improved calibration, and extensive use of baseline samples.

Evaluation of three somatic genetic biomarkers as indicators of low dose radiation effects in clean-up workers of the Chernobyl nuclear reactor accident.

The goals of this study were to assess three biomarkers of genetic effect for their individual and collective ability to detect and estimate radiation exposure in Russian Chernobyl clean-up workers. Work assignments were planned to limit dose to 0.25 Gy. The three biomarkers employed were chromosome translocations detectcd in lynmphocytes by florescence in situ hybridisation (FISH), and mutation at two genes, glycophorin A (GPA) in red blood cells detected by flow cytometry and hypoxanthine phosphoribosyltransferase (HPRT) in lymphocytes detected by selective cell culture. Samples were Obtained from 1992 to 2000. The time between exposure at Chernobyl and sample acquisition was > or =5 years. The lymphocyte assays detected an elevation over controls in average outcomes it clean-up workers: translocation rates were 46% higher when adjusted for age and smoking and HPRT mutant frequencies were were 16% higher when adjusted for age. The G PA assay did not detect an exposure effect. The results indicate that measuring frequency of translocations by FISH is preferred for low dose radiation, retrospective biochemistry.