BED Assay Research Articles

856 A Pilot Study of a Point-of-care Wound Bed Assay to Predict Graft Take

Abstract Introduction Deciding when to graft a burn wound is instrumental in acute reconstruction. A technology capable of predicting autograft success would help optimize timing of initial debridement and grafting. In prior work, members of our team developed a point-of-care proteinase assay capable of predicting delayed wound healing in chronic wounds. In the work reported here, we seek to quantify these proteolytic enzymes in acute burn wounds in an effort to ultimately determine whether a quantitative assessment of proteinase activity in a burn wound might predict successful healing of autograft. Methods Eight burn patients were taken to the operating room for autografting. Tissue samples were obtained from unique sites, as well as swabs of wounds pre- and post-excision. A point-of-care proteinase assay determined initial rates of fluorescence, which correlates with the amount of proteolytic enzyme activity. Autograft sites were assessed after two weeks, with ≥50% take considered successful and < 50% take deemed unsuccessful. Initial rates of fluorescence were compared. These groups were analyzed using two-sided student’s t tests, with p< 0.05 identified as significant. Results The pre-excision group initial rate was significantly higher than the post-excision group (p=0.0006). The tissue group initial rate was also significantly higher than the post-excision group (p = 0.0028). We also compared pre-excision initial rates of enzymatic activity between successful autografts (n=10, 18.34±20.90 RFU/min) and unsuccessful autografts (n=5, 4.32±4.62 RFU/min) with p = 0.169. Conclusions Multiple findings indicate the potential of this assay to differentiate burn wounds based on proteinase activity. Pre-excision tissue beds contained significantly higher rates of proteinase activity compared to excised and debrided tissue beds (p = 0.0006). This is consistent with burn wound morphology, as proteolytic enzymes function to debride damaged tissue and promote angiogenesis in acute wounds. Interestingly, initial rates of proteolytic enzymatic activity in pre-excised wounds of successful autografts trended towards significance compared to unsuccessful autografts (p = 0.169), suggesting that these proteolytic enzymes reflect an overall healthy wound bed, the presence of which could portend a heightened chance for autograft success. Applicability of Research to Practice This pilot study confirms the presence of proteinase activity in acute burn wounds utilizing a point-of-care test. Future work will investigate whether this test can assist physicians in predicting amenability of burn wounds to autografting.

Sequence ambiguity determined from routine pol sequencing is a reliable tool for real-time surveillance of HIV incidence trends

Identifying individuals recently infected with HIV has been of great significance for close monitoring of HIV epidemic dynamics. Low HIV sequence ambiguity (SA) has been described as a promising marker of recent infection in previous studies. This study explores the utility of SA defined as a proportion of ambiguous nucleotides detected in baseline pol sequences as a tool for routine real-time surveillance of HIV incidence trends at a national level.A total of 353 partial HIV-1 pol sequences obtained from persons diagnosed with HIV infection in Slovenia from 2000 to 2012 were studied, and SA was reported as a percentage of ambiguous base calls. Patients were characterized as recently infected by examining anti-HIV serological patterns and/or using commercial HIV-1 incidence assays (BED and/or LAg-Avidity assay). A mean SA of 0.29%, 0.14%, and 0.19% was observed for infections classified as recent by BED, LAg, or anti-HIV serological results, respectively. Welch's t-test showed a significant difference in the SA of recent versus long-standing infections (p < 0.001). CD4+ T-cell counts ≤250 cells/mm3 significantly correlated with higher SA (p < 0.001), whereas the homo/bisexual transmission route significantly correlated with lower SA (p = 0.005). When the LAg-assay was used as an indicator of recent infection, a receiver operating characteristic curve with the largest area under the curve (0.896) was observed for SA (sensitivity and specificity of 79%), indicating the best correlation of the data. A reliable estimation of the trends of HIV incident infection could be inferred from measuring SA irrespective of the cutoff used; however, in Slovenia it seems that lower cutoffs are more appropriate.Our data suggest that SA could be used as a real-time surveillance tool for close monitoring of HIV incidence trends, especially in countries where baseline HIV resistance genotyping is performed routinely, rendering this approach cost-effective.

Analysis of Viral Diversity in Relation to the Recency of HIV-1C Infection in Botswana.

BackgroundCross-sectional, biomarker methods to determine HIV infection recency present a promising and cost-effective alternative to the repeated testing of uninfected individuals. We evaluate a viral-based assay that uses a measure of pairwise distances (PwD) to identify HIV infection recency, and compare its performance with two serologic incidence assays, BED and LAg. In addition, we assess whether combination BED plus PwD or LAg plus PwD screening can improve predictive accuracy by reducing the likelihood of a false-recent result.MethodsThe data comes from 854 time-points and 42 participants enrolled in a primary HIV-1C infection study in Botswana. Time points after treatment initiation or with evidence of multiplicity of infection were excluded from the final analysis. PwD was calculated from quasispecies generated using single genome amplification and sequencing. We evaluated the ability of PwD to correctly classify HIV infection recency within <130, <180 and <360 days post-seroconversion using Receiver Operator Characteristics (ROC) methods. Following a secondary PwD screening, we quantified the reduction in the relative false-recency rate (rFRR) of the BED and LAg assays while maintaining a sensitivity of either 75, 80, 85 or 90%.ResultsThe final analytic sample consisted of 758 time-points from 40 participants. The PwD assay was more accurate in classifying infection recency for the 130 and 180-day cut-offs when compared with the recommended LAg and BED thresholds. A higher AUC statistic confirmed the superior predictive performance of the PwD assay for the three cut-offs. When used for combination screening, the PwD assay reduced the rFRR of the LAg assay by 52% and the BED assay by 57.8% while maintaining a 90% sensitivity for the 130 and 180-day cut-offs respectively.ConclusionPwD can accurately determine HIV infection recency. A secondary PwD screening reduces misclassification and increases the accuracy of serologic-based assays.

Distance to testing sites and its association with timing of HIV diagnosis

ABSTRACTEarly HIV diagnosis enables prompt treatment initiation, thereby contributing to decreased morbidity, mortality, and transmission. We aimed to describe the association between distance from residence to testing sites and HIV disease stage at diagnosis. Using HIV surveillance data, we identified all new HIV diagnoses made at publicly funded testing sites in central North Carolina during 2005–2013. Early-stage HIV was defined as acute HIV (antibody-negative test with a positive HIV RNA) or recent HIV (normalized optical density <0.8 on the BED assay for non-AIDS cases); remaining diagnoses were considered post-early-stage HIV. Street distance between residence at diagnosis and (1) the closest testing site and (2) the diagnosis site was dichotomized at 5 miles. We fit log-binomial models using generalized estimating equations to estimate prevalence ratios (PR) and robust 95% confidence intervals (CI) for post-early-stage diagnoses by distance. Models were adjusted for race/ethnicity and testing period. Most of the 3028 new diagnoses were black (N = 2144; 70.8%), men who have sex with men (N = 1685; 55.7%), and post-early-stage HIV diagnoses (N = 2010; 66.4%). Overall, 1145 (37.8%) cases traveled <5 miles for a diagnosis. Among cases traveling ≥5 miles for a diagnosis, 1273 (67.6%) lived <5 miles from a different site. Residing ≥5 miles from a testing site was not associated with post-early-stage HIV (adjusted PR, 95% CI: 0.98, 0.92–1.04), but traveling ≥5 miles for a diagnosis was associated with higher post-early HIV prevalence (1.07, 1.02–1.13). Most of the elevated prevalence observed in cases traveling ≥5 miles for a diagnosis occurred among those living <5 miles from a different site (1.09, 1.03–1.16). Modest increases in post-early-stage HIV diagnosis were apparent among persons living near a site, but choosing to travel longer distances to test. Understanding reasons for increased travel distances could improve accessibility and acceptability of HIV services and increase early diagnosis rates.

Characteristics of Transmitted Drug-Resistant HIV-1 in Recently Infected Treatment-Naive Patients in Japan.

Progress in antiretroviral treatment has led to fewer virological failure cases, but 10%-20% of treatment-naive HIV/AIDS cases are reported to harbor drug-resistant strains, suggesting transmission of drug-resistant HIV. We aimed to determine the trend in prevalence of transmitted drug-resistant (TDR) HIV in Japan, particularly in recently infected patients. Drug-resistance test was performed on 3904 HIV-1-infected cases newly diagnosed between 2007 and 2012. The number of cases infected within 6 months [recent seroconverters (RS)] was estimated by BED assay of 2700 plasma samples. Characteristics of RS cases were further analyzed. The overall prevalence of TDR was 9.1%, ranging from 7.3% in 2008% to 12.5% in 2010. Among 1403 subtype B/E/D cases with >50 CD4 T cell counts and >1000 HIV copies per milliliter, 468 (33.4%) were estimated to be RS. The prevalence of RS was significantly higher among cases who were male, Japanese, and men who have sex with men. The prevalence of TDR did not differ significantly between recent and long-term seroconverters (8.5% vs. 9.2%, respectively, P = 0.68). Common mutations in both groups were M46I/L and T215 revertants. Furthermore, sequences with these mutations, K103N and D30N/N88D formed clusters on phylogenetic trees. Our study clarified an increase in prevalence of TDR in Japan from 2007 to 2012. The phylogenetic clustering of cases with M46I/L or T215 revertants suggests that HIV with these mutations have become circulating strains. Furthermore, detailed analyses showed that Japanese men who have sex with men are more aware of their risk of HIV infection.

Transmitted drug resistance of HIV-1 strains among individuals attending voluntary counselling and testing in Taiwan.

Genotypic drug resistance testing for HIV-1 has been integrated into voluntary counselling and testing (VCT) programmes to investigate the trends of transmitted drug resistance (TDR), including integrase mutations, among individuals with recent or chronic HIV infections in Taiwan. Between 2006 and 2014, 745 of 21 886 subjects (3.4%) tested HIV positive in the VCT service. The BED assay was used to identify recent HIV infections. Genotypic resistance mutations were interpreted using the WHO 2009 list. Integrase resistance mutations were analysed using the Stanford HIV Drug Resistance Database. Three-hundred-and-sixty (48.3%) patients were recently infected with HIV-1. Of 440 patients linked to HIV care with analysable reverse transcriptase and protease genes, 49 (11.1%) were infected with HIV-1 harbouring at least one resistance-associated mutation (RAM). The prevalence of TDR to NRTIs, NNRTIs and PIs was 4.1%, 6.4% and 2.3%, respectively. TDR prevalence did not change significantly during the study period. CD4 counts ≤500 cells/mm(3) and hepatitis B surface antigen positivity were independent factors associated with acquiring drug-resistant HIV. The prevalence of integrase mutations was 3.2%. Among the seven major integrase mutations (T66I, E92Q, G140S, Y143C/H/R, S147G, Q148H/K/R and N155H), only one strain harbouring the Q148R mutation was detected. We found no statistically significant difference between patients with chronic infection and those with recent infection in the prevalence of drug-resistant mutations to any of the four classes of antiretroviral agents. The prevalence of TDR of HIV-1 strains to available antiretroviral agents is moderately high, but transmission of HIV-1 with drug-resistant mutations remains stable in Taiwan.

Seroincidence of HIV and prevalence of transmitted drug resistance of HIV-1 strains among persons seeking voluntary counselling and testing in Taiwan.

IntroductionThe total case number of persons who are newly diagnosed with HIV continues to increase in Taiwan and men who have sex with men (MSM) have re-emerged as the leading risk group for HIV transmission. In this study, we aimed to estimate the incidence rate of HIV infection among those individuals who sought voluntary counselling and testing (VCT) service at a university hospital.MethodsBetween 1 April, 2006 and 31 December, 2013, 18,246 tests for HIV antibody were performed among 12143 individuals at the VCT service. A total of 2157 individuals who tested negative for anti-HIV antibody had subsequent follow-up tests at the same VCT service, which composed the study population for estimation of incidence rate of recent HIV infection. The BED assays were used to identify recent HIV infections that occurred within the previous six months before seeking VCT service.ResultsDuring the 6.5-year study period, 647 individuals were diagnosed as being HIV-positive, with an overall HIV seroprevalence of 3.55% (95% CI 3.27–3.82). The overall incidence rate of HIV infection was estimated 4.13 per 100 person-years of follow-up (95% CI 3.67–4.69 per 100 person-years of follow-up). MSM had an estimated 10-fold higher seroprevalence and seroincidence of HIV than heterosexuals. Of 647 clients testing positive for HIV, 603 clients were MSM (93.2%) and 477 patients (70.8%) subsequently sought HIV care at the hospital; 226 (47.4%) were diagnosed as having recent HIV infections by the BED assay, while 244 (51.2%) long-term infection and 7 without data by the BED assay. Of those patients, 173 (75.6%) and 178 patients (73.0%) with recent HIV infection and long-term infection had data of transmitted drug resistance mutations, respectively. The prevalence of transmitted drug resistance mutations to any class of antiretroviral therapy was 9.0% and 10.6% (p=0.68), respectively, of the HIV-1 strains from the patients with recent HIV infection and long-term infection, respectively.ConclusionsThe seroincidence rate of HIV among persons seeking VCT was estimated 4.13 per 100 person-years of follow-up. The prevalence of transmitted drug resistance to any class of antiretroviral agents was similar between those who were recently infected with HIV and those who had long-term infection in Taiwan.

Development of a Proficiency Testing Program for the HIV-1 BED Incidence Assay in China

The HIV-1 BED incidence assay was adopted in China in 2005 for HIV-1 incidence surveillance. A proficiency testing (PT) program was established in 2006 to provide quality assurance services. The BED PT program consisted of two components, an international program provided by the U.S. Centers for Disease Control and Prevention from 2006 and a domestic program started by the National HIV/HCV Reference Laboratory in 2011. Each PT panel consisted of eight coded specimens distributed to participating laboratories semi-annually, and testing results were collected and analyzed. The number of participating laboratories increased progressively from 2006 to 2012. The Chinese HIV-1 incidence laboratory network performed satisfactorily both in international and domestic PT programs. We also demonstrated that the BED assay was highly reproducible among participating laboratories. Our success and lessons learned can be readily replicated in other countries or regions contemplating the establishment of a PT program for assay-based HIV incidence estimation.

The Impact of Wild-Type Reversion on Transmitted Resistance Surveillance

The Impact of Wild-Type Reversion on Transmitted Resistance Surveillance

Social Network–Based Recruitment Successfully Reveals HIV-1 Transmission Networks Among High-Risk Individuals in El Salvador

HIV in Central America is concentrated among certain groups such as men who have sex with men (MSM) and female sex workers (FSWs). We compared social recruitment chains and HIV transmission clusters from 699 MSM and 787 FSWs to better understand factors contributing to ongoing HIV transmission in El Salvador. Phylogenies were reconstructed using pol sequences from 119 HIV-positive individuals recruited by respondent-driven sampling (RDS) and compared with RDS chains in 3 cities in El Salvador. Transmission clusters with a mean pairwise genetic distance ≤ 0.015 and Bayesian posterior probabilities =1 were identified. Factors associated with cluster membership were evaluated among MSM. Sequences from 34 (43%) MSM and 4 (10%) FSW grouped in 14 transmission clusters. Clusters were defined by risk group (12 MSM clusters) and geographic residence (only 1 spanned separate cities). In 4 MSM clusters (all n = 2), individuals were also members of the same RDS chain, but only 2 had members directly linked through recruitment. All large clusters (n ≥ 3) spanned >1 RDS chain. Among MSM, factors independently associated with cluster membership included recent infection by BED assay (P = 0.02), sex with stable male partners (P = 0.02), and sex with ≥ 3 male partners in the past year (P = 0.04). We found few HIV transmissions corresponding directly with the social recruitment. However, we identified clustering in nearly one-half of MSM suggesting that RDS recruitment was indirectly but successfully uncovering transmission networks, particularly among recent infections. Interrogating RDS chains with phylogenetic analyses may help refine methods for identifying transmission clusters.

Short Communication: Effect of Short-Course Antenatal Zidovudine and Single-Dose Nevirapine on the BED Capture Enzyme Immunoassay Levels in HIV Type 1 Subtype C Infection

Cross-sectional prevalence studies based on immunoassays that discriminate between recent and long-term infections, such as the BED assay, have been widely used to estimate HIV incidence. However, individuals receiving highly active antiretroviral therapy tend to have lower BED levels and are associated with a higher risk for being mistakenly classified as recent infections. To assess the effect of short-term antenatal zidovudine (ZDV) and single-dose nevirapine (sdNVP) on the BED levels in HIV-1C infection, we measured longitudinal BED normalized optical density (OD-n) levels using stored plasma samples collected prenatally and postnatally from 159 pregnant HIV-infected women in Botswana who participated in the randomized clinical Mother-to-Child-Prevention study, the Mashi study. All women received ZDV from 34 weeks gestation through delivery and were randomized to receive either sdNVP or placebo during labor. Among 159 subjects, the OD-n levels decreased from baseline to delivery in 93 subjects (p=0.039), suggesting that short-course ZDV may decrease OD-n levels. sdNVP at delivery did not affect longitudinal BED OD-n levels postdelivery. However, sdNVP appeared to modify the association between CD4 count at delivery and OD-n levels postdelivery. When estimating HIV incidence with the BED assay, special care may be required regarding women who received short-term ZDV for prevention of mother-to-child transmission.

Towards Estimation of HIV-1 Date of Infection: A Time-Continuous IgG-Model Shows That Seroconversion Does Not Occur at the Midpoint between Negative and Positive Tests

Estimating date of infection for HIV-1-infected patients is vital for disease tracking and informed public health decisions, but is difficult to obtain because most patients have an established infection of unknown duration at diagnosis. Previous studies have used HIV-1-specific immunoglobulin G (IgG) levels as measured by the IgG capture BED enzyme immunoassay (BED assay) to indicate if a patient was infected recently, but a time-continuous model has not been available. Therefore, we developed a logistic model of IgG production over time. We used previously published metadata from 792 patients for whom the HIV-1-specific IgG levels had been longitudinally measured using the BED assay. To account for patient variability, we used mixed effects modeling to estimate general population parameters. The typical patient IgG production rate was estimated at r = 6.72[approximate 95% CI 6.17,7.33]×10−3 OD-n units day−1, and the carrying capacity at K = 1.84[1.75,1.95] OD-n units, predicting how recently patients seroconverted in the interval ∧ t = (31,711) days. Final model selection and validation was performed on new BED data from a population of 819 Swedish HIV-1 patients diagnosed in 2002–2010. On an appropriate subset of 350 patients, the best model parameterization had an accuracy of 94% finding a realistic seroconversion date. We found that seroconversion on average is at the midpoint between last negative and first positive HIV-1 test for patients diagnosed in prospective/cohort studies such as those included in the training dataset. In contrast, seroconversion is strongly skewed towards the first positive sample for patients identified by regular public health diagnostic testing as illustrated in the validation dataset. Our model opens the door to more accurate estimates of date of infection for HIV-1 patients, which may facilitate a better understanding of HIV-1 epidemiology on a population level and individualized prevention, such as guidance during contact tracing.

Development of a Novel Rapid HIV Test for Simultaneous Detection of Recent or Long-Term HIV Type 1 Infection Using a Single Testing Device

Laboratory assays for the detection of recent HIV infection for HIV incidence surveillance are essential to HIV prevention efforts worldwide because they can identify populations with a high incidence and allow targeting of resources and monitoring of incidence trends over time. This study describes the development of a novel rapid HIV-1 incidence-prevalence (I-P) test that can be used for the simultaneous detection and discrimination of prevalent (long-term) or incident (recent) HIV infections using a single device. A lateral flow assay was developed that uses a multisubtype recombinant gp41 protein applied at two concentrations of antigen (high and low). Prevalent and incident HIV-1 infections can be distinguished based on differential antibody binding at the two antigen concentrations. High level/high avidity antibodies present in prevalent infections bind to and are detected at both antigen concentrations while low level/low avidity antibodies present in recent HIV infections are detected only at the higher antigen concentration line. A total of 205 HIV-positive specimens with known status (recent=105, long-term=100), including 57 specimens from seroconversion panels, were tested by the rapid I-P assay and the results were compared to the HIV-1 BED capture enzyme immunoassay (CEIA). There was a 95.1% agreement of final classification (recent or long-term) with the BED assay (kappa=0.910) (mean recency period=162 days) and a high correlation between the intensity score of the low antigen line with the BED OD-n (Pearson correlation=0.89). The new rapid I-P test has great potential to simplify HIV surveillance efforts by simultaneously providing information on both HIV prevalence and incidence using a single, rapid test device.

HIV Prevalence and Incidence Among Women at Higher Risk of Infection in Addis Ababa, Ethiopia

This study aimed to estimate HIV incidence among women at higher HIV risk in Addis Ababa, Ethiopia using cross-sectional data. To refine the estimate, we sought to determine a local correction factor by estimating a false recent rate for the BED assay. The overall study had two parallel phases: cross-sectional incidence and BED false recent (BED FR). A total of 1856 women at higher HIV risk were enrolled into the cross-sectional phase. For the BED FR, 500 women and 70 men with known HIV infection of greater than 12 months were enrolled. Rapid HIV tests were used to assess participant HIV status. Samples from women with positive results on HIV rapid testing were then analyzed using the BED assay to determine incident HIV infections. The overall HIV prevalence was 11.3%, with women greater than 25 years old having a higher prevalence (26.5% vs. 7.9% for younger women). The overall unadjusted HIV incidence was 3.3%. The estimated HIV incidence was 2.6% when adjusted using the locally derived false recent rate of 3.5%. Similar incidence rates were found between the younger and older age groups. This is one of the first field-based studies of HIV incidence in Ethiopia; it demonstrates that there is sufficient incidence to warrant further HIV prevention efforts among women at higher risk of HIV infection.

Primary resistance of HIV to antiretrovirals among individuals recently diagnosed at voluntary counselling and testing centres in the metropolitan region of Recife, Pernambuco

Determining the prevalence and type of antiretroviral (ARV) resistance among ARV-naïve individuals is important to assess the potential responses of these individuals to first-line regimens. The prevalence of primary resistance and the occurrence of recent infections among individuals with human immunodeficiency virus (HIV)/acquired immune deficiency syndrome (AIDS) were identified among recently diagnosed patients at five sexually transmitted disease/AIDS testing and counselling centres in the metropolitan region of Recife (RMR), Pernambuco, Brazil, between 2007-2009. One-hundred and eight samples were analysed using the Calypte® BED assay. Males predominated (56%), as did patients aged 31-50 years. Twenty-three percent presented evidence of a recent HIV infection. The median CD4+ T lymphocyte count was 408 cells/mm³ and the median viral load was 3.683 copies/mL. The prevalence of primary resistance was 4.6% (confidence interval 95% = 1-8.2%) based on criteria that excluded common polymorphisms in accordance with the surveillance drug resistance mutation criteria. The prevalence of resistance to non-nucleoside reverse transcriptase, nucleoside/nucleotide reverse transcriptase and protease inhibitors were 3.8%, 1.5% and 0.8%, respectively. Fifty-seven percent of strains were from clade B, 37.7% were clade F and 3.1% were clade C; there were no statistically significant differences with respect to resistance between clades. Recent infection tended to be more common in men (p = 0.06) and in municipalities in the south of the RMR (Jaboatão dos Guararapes and Cabo de Santo Agostinho) (p = 0.046). The high prevalence of recent infection and the high prevalence of non-B strains in this poor Brazilian region merit further attention.

Detection of Recent HIV-1 Infection Using a New Limiting-Antigen Avidity Assay: Potential for HIV-1 Incidence Estimates and Avidity Maturation Studies

BackgroundAccurate and reliable laboratory methods are needed for estimation of HIV-1 incidence to identify the high-risk populations and target and monitor prevention efforts. We previously described a single-well limiting-antigen avidity enzyme immunoassay (LAg-Avidity EIA) to detect recent HIV-1 infection.MethodsWe describe here further optimization and characterization of LAg-Avidity EIA, comparing it to the BED assay and a two-well avidity-index (AI) EIA. Specimen sets included longitudinal sera (n = 393), collected from 89 seroconverting individuals from 4 cohorts representing 4 HIV-1 subtypes, and sera from AIDS patients (n = 488) with or without TB co-infections from 3 different cohorts. Ninety seven HIV-1 positive specimens were purchased commercially. The BED assay, LAg-Avidity EIA, AI-EIA and HIV serology were performed, as needed.ResultsMonitoring quality control specimens indicated high reproducibility of the LAg-Avidity EIA with coefficient of variation of <10% in the dynamic range. The LAg-Avidity EIA has an overall mean duration of recency (ω) of 141 days (95% CI 119–160) at normalized optical density (ODn) cutoff of 1.0, with similar ω in different HIV-1 subtypes and populations (132 to 143 days). Antibody avidity kinetics were similar among individuals and subtypes by both the LAg-Avidity EIA and AI-EIA compared to the HIV-IgG levels measured by the BED assay. The false recent rate among individuals with AIDS was 0.2% with the LAg-Avidity EIA, compared to 2.9% with the BED assay. Western blot profiles of specimens with increasing avidity confirm accurate detection of recent HIV-1 infections.ConclusionsThese data demonstrate that the LAg-Avidity EIA is a promising assay with consistent ω in different populations and subtypes. The assay should be very useful for 1) estimating HIV-1 incidence in cross-sectional specimens as part of HIV surveillance, 2) identifying risk factors for recent infections, 3) measuring impact of prevention programs, and 4) studying avidity maturation during vaccine trials.

Trends in early and late diagnosis of HIV-1 infections in Tokyoites from 2002 to 2010

The objective of this study was to delineate the trends in early and late diagnosis of HIV-1 infection in newly diagnosed Tokyoites. The BED assay was used to identify cases diagnosed at an early stage of infection. BED-positive non-AIDS cases with a CD4 cell count ≥ 200/μl were defined as cases with recent infection. The rates of AIDS and recent infection in 809 newly diagnosed Tokyoites during 2002-2010 were analyzed. The AIDS rate was 22.5%. AIDS patients were older (40.4 years) than non-AIDS patients (35.0 years), and a smaller proportion were men who have sex with men (MSM) in AIDS patients (81.7%) than in non-AIDS patients (89.9%). The AIDS rate was persistently lower (≤ 14.3%) in ≤ 29-year-old than in ≥ 30-year-old MSM. The rate of recent infection was 24.4%. Individuals with recent infection (33.0 years old) were younger than the others (37.2 years). The rate of recent infection was lower (≤ 18.5%) in MSM aged ≥ 40 years than in those aged ≤ 39 years during the study period, except for 2007 and 2008. Younger MSM Tokyoites appear to be aware of the risk of their sexual behavior, sufficient to take voluntary HIV testing repeatedly, resulting in early diagnosis. Older MSM did not take HIV testing frequently enough and may be a good target for campaigns promoting testing.

Correlation of Prospective and Cross-Sectional Measures of HIV Type 1 Incidence in a Higher-Risk Cohort in Ho Chi Minh City, Vietnam

The primary aim of this study was to estimate HIV incidence within a high-risk population in Ho Chi Minh City (HCMC), Vietnam using both cross-sectional and prospective methodologies. A secondary aim was to develop a local correction factor for the BED and avidity index incidence assays. The research study design consisted of three phases: (1) cross-sectional, (2) prospective, and (3) BED false recent (BED FR). A total of 1619 high-risk, sexually active individuals were enrolled in the cross-sectional phase and 355 of the opiate-negative, HIV-negative women were subsequently enrolled in the prospective phase. Four-hundred and three men and women with known HIV infection duration of greater than 12 months were enrolled in the BED FR phase. The HIV prevalence for all participants in the cross-sectional phase was 15.8%. HIV incidence in the cross-sectional group was estimated using the BED IgG capture assay and AxSYM avidity index assay for recent HIV infection and incidence within the prospective cohort was determined by observations of HIV seroconversion. HIV incidence in opiate-negative women was estimated using the BED assay to be 0.8% unadjusted and 0.5% after applying the locally derived BED false recent rate of 1.7%; no seroconversions were observed in the prospective cohort. We also screened the cross-sectional samples for evidence of acute infection using nucleic acid testing, 4th generation HIV EIA, and SMARTube coupled with Genscreen and Determine diagnostic tests; no confirmed acute infections were identified by any method. HIV incidence within this opiate-negative study population was low and incidence estimates from the two methods compared favorably with each other. Incidence estimates and false recent rates using the AxSYM assay were higher: AI FRR of 2.7% and adjusted incidence of 1.7% per year (95% CI, 0.6, 2.8). By comparison, both HIV prevalence and incidence estimates for the opiate-positive group were higher.

Statistical Power and Estimation of Incidence Rate Ratios Obtained from BED Incidence Testing for Evaluating HIV Interventions among Young People

BackgroundThe objectives of this study were to determine the capacity of BED incidence testing to a) estimate the effect of a HIV prevention intervention and b) provide adequate statistical power, when used among young people from sub-Saharan African settings with high HIV incidence rates.MethodsFirstly, after having elaborated plausible scenarios based on empirical data and the characteristics of the BED HIV-1 Capture EIA (BED) assay, we conducted statistical calculations to determine the BED theoretical power and HIV incidence rate ratio (IRR) associated with an intervention when using BED incidence testing. Secondly, we simulated a cross-sectional study conducted in a population among whom an HIV intervention was rolled out. Simulated data were analyzed using a log-linear Poisson model to recalculate the IRR and its confidence interval, and estimate the BED practical power. Calculations were conducted with and without corrections for misclassifications.ResultsCalculations showed that BED incidence testing can yield a BED theoretical power of 75% or more of the power that can be obtained in a classical cohort study conducted over a duration equal to the BED window period. Statistical analyses using simulated populations showed that the effect of a prevention intervention can be estimated with precision using classical statistical analysis of BED incidence testing data, even with an imprecise knowledge of the characteristics of the BED assay. The BED practical power was lower but of the same magnitude as the BED theoretical power.ConclusionsBED incidence testing can be applied to reasonably small samples to achieve good statistical power when used among young people to estimate IRR.

A Comprehensive Evaluation of the Proficiency Testing Program for the HIV-1 BED Incidence Assay

The HIV-1 BED incidence assay was developed at the Centers for Disease Control and Prevention and since 2005 has been available as a commercial kit for use in HIV-1 incidence surveillance. A BED-specific proficiency testing (PT) program was initiated in 2006 that included a panel of eight coded specimens (six unique and two duplicates) to participating laboratories. The number of participating laboratories increased from 12 to 38 from 2006 to 2009. Overall, 96.1% of the laboratories reported results, and 95.4% of those reporting achieved a 100% score. The observed mean normalized optical density (OD-n) values of all participants correlated well with the expected OD-n values for all specimens (R(2) = 0.98) used for seven PT rounds. BED testing demonstrated high reproducibility among all laboratories, with an agreement of 99.3% (574/578) between initial and confirmatory classification and regression statistics of R(2) = 0.96, slope = 1.022, and intercept = 0.0066. Reproducibility among duplicate specimens was very high during each PT round, with mean deviation of 1.8%. Analysis of controls and calibrator specimen for all 343 runs showed a coefficient of variation of ca. 20% for raw ODs in the dynamic range, which was reduced to <10% when the OD was normalized (OD-n). Most laboratories that failed the PT assessment had transcriptional errors, kit reagent problems, or specimen handling errors. Thus, the BED-specific PT program enabled us to track performance of different laboratories conducting the BED assay while identifying areas for improvements. This program will also serve as a template for future PT programs for new incidence assays as they become available.