Quantitating SARS-CoV-2 Neutralizing Antibodies from Human Dried Blood Spots.

Background: In the earliest days of COVID-19 pandemic, the collection of dried blood spots (DBS) enabled public health laboratories to undertake population-scale seroprevalence studies to estimate rates of SARS-CoV-2 exposure. With SARS-CoV-2 seropositivity levels now estimated to exceed 94% in the United States, attention has turned to using DBS to assess functional (neutralizing) antibodies within cohorts of interest. Methods: Contrived DBS eluates from convalescent, fully vaccinated and pre-COVID-19 serum samples were evaluated in SARS-CoV-2 plaque reduction neutralization titer (PRNT) assays, a SARS-CoV-2 specific 8-plex microsphere immunoassay, a cell-based pseudovirus assay, and two different spike-ACE2 inhibition assays, an in-house Luminex-based RBD-ACE2 inhibition assay and a commercial real-time PCR-based inhibition assay (NAB-Sure™). Results: DBS eluates from convalescent individuals were compatible with the spike-ACE2 inhibition assays, but not cell-based pseudovirus assays or PRNT. However, the insensitivity of cell-based pseudovirus assays was overcome with DBS eluates from vaccinated individuals with high SARS-CoV-2 antibody titers. Conclusion: SARS-CoV-2 neutralizing titers can be derived with confidence from DBS eluates, thereby opening the door to the use of these biospecimens for the analysis of vulnerable populations and normally hard to reach communities.

Demographic and Clinical Factors Associated With SARS-CoV-2 Spike 1 Antibody Response Among Vaccinated US Adults: the C4R Study.

This study investigates correlates of anti-S1 antibody response following COVID-19 vaccination in a U.S. population-based meta-cohort of adults participating in longstanding NIH-funded cohort studies. Anti-S1 antibodies were measured from dried blood spots collected between February 2021-August 2022 using Luminex-based microsphere immunoassays. Of 6245 participants, mean age was 73 years (range, 21-100), 58% were female, and 76% were non-Hispanic White. Nearly 52% of participants received the BNT162b2 vaccine and 48% received the mRNA-1273 vaccine. Lower anti-S1 antibody levels are associated with age of 65 years or older, male sex, higher body mass index, smoking, diabetes, COPD and receipt of BNT16b2 vaccine (vs mRNA-1273). Participants with a prior infection, particularly those with a history of hospitalized illness, have higher anti-S1 antibody levels. These results suggest that adults with certain socio-demographic and clinical characteristics may have less robust antibody responses to COVID-19 vaccination and could be prioritized for more frequent re-vaccination.

Use of Self-Collected Dried Blood Spots and a Multiplex Microsphere Immunoassay to Measure IgG Antibody Response to COVID-19 Vaccines.

Serosurveys can determine the extent and spread of a pathogen in populations. However, collection of venous blood requires trained medical staff. Dried blood spots (DBS) are a suitable alternative because they can be self-collected and stored/shipped at room temperature. As COVID-19 vaccine deployment began in early 2021, we rapidly enrolled laboratory employees in a study to evaluate IgG antibody levels following vaccination. Participants received a DBS collection kit, self-collection instructions, and a brief questionnaire. Three DBS were collected by each of 168 participants pre- and/or postvaccination and tested with a multiplex microsphere immunoassay (MIA) that separately measures IgG antibodies to SARS-CoV-2 spike-S1 and nucleocapsid antigens. Most DBS (99.6%, 507/509) were suitable for testing. Participants with prior SARS-CoV-2 infection (n = 7) generated high S antibody levels after the first vaccine dose. Naïve individuals (n = 161) attained high S antibody levels after the second dose. Similar antibody levels were seen among those vaccinated with Moderna (n = 29) and Pfizer-BioNTech (n = 137). For those receiving either mRNA vaccine, local side effects were more common after the first vaccine dose, whereas systemic side effects were more common after the second dose. Individuals with the highest antibody levels in the week prior to the second vaccine dose experienced more side effects from the second dose. Our study demonstrated that combining self-collected DBS and a multiplex MIA is a convenient and effective way to assess antibody levels to vaccination and could easily be used for population serosurveys of SARS-CoV-2 or other emerging pathogens. IMPORTANCE Serosurveys are an essential tool for assessing immunity in a population (1, 2). However, common barriers to effective serosurveys, particularly during a pandemic, include high-costs, resources required to collect venous blood samples, lack of trained laboratory technicians, and time required to perform the assay. By utilizing self-collected dried blood spots (DBS) and our previously developed high-throughput microsphere immunoassay, we were able to significantly reduce many of these common challenges. Participants were asked to self-collect three DBS before and/or after they received their COVID-19 vaccines to measure antibody levels following vaccination. Participants successfully collected 507 DBS that were tested for IgG antibodies to the spike and nucleocapsid proteins of SARS-CoV-2. When used with self-collected DBS, our relatively low-cost assay significantly reduced common barriers to collecting serological data from a population and was able to effectively assess antibody response to vaccination.

A cross-sectional study of hepatitis C prevalence and correlates among persons who inject drugs in rural and non-rural communities.

Persons who inject drugs (PWID) have been experiencing a higher burden of new hepatitis C (HCV) due to the opioid epidemic. The greatest increases in injection have been in rural communities. However, less is known about the prevalence of HCV or its risk factors in rural compared to non-rural communities. This study compared HCV infection history, current infection, and associated behavioural and sociodemographic correlates among PWID recruited from rural and non-rural communities from Upstate New York (NY). This cross-sectional study recruited 309 PWID, using respondent-driven sampling. Blood samples were collected through finger stick for HCV antibody and RNA tests. A survey was also self-administered for HCV infection history, sociodemographics and behavioural correlates to compare by setting rurality. HCV seropositivity was significantly higher among PWID from rural than non-rural communities (71.0% vs. 46.8%), as was current infection (41.4% vs. 25.9%). High levels of past year syringe (44.4%) and equipment (62.2%) sharing were reported. Factors associated with infection history include syringe service program utilization, non-Hispanic white race, sharing needles and methamphetamine injection, which was higher in rural vs. non-rural communities (38.5% vs. 15.5%). HCV burden among PWID appears higher in rural than non-rural communities and may be increasing possibly due to greater levels of methamphetamine injection. On-going systematic surveillance of HCV prevalence and correlates is crucial to respond to the changing opioid epidemic landscape. Additionally, improving access to harm reduction services, especially with special focus on stimulants, may be important to reduce HCV prevalence among PWID in rural settings.

Utility of Newborn Dried Blood Spots to Ascertain Seroprevalence of SARS-CoV-2 Antibodies Among Individuals Giving Birth in New York State, November 2019 to November 2021.

Importance: Serosurveys can be used to monitor population-level dynamics of COVID-19 and vaccination. Dried blood spots (DBSs) collected from infants contain maternal IgG antibodies and are useful for serosurveys of individuals recently giving birth. Objectives: To examine SARS-CoV-2 antibody prevalence in pregnant individuals in New York State, identify associations between SARS-CoV-2 antibody status and maternal and infant characteristics, and detect COVID-19 vaccination among this population. Design, Setting, and Participants: A population-based, repeated cross-sectional study was conducted to detect SARS-CoV-2 nucleocapsid (N) and spike (S) IgG antibodies. Deidentified DBS samples and data submitted to the New York State Newborn Screening Program between November 1, 2019, and November 30, 2021, were analyzed. Exposures: Prenatal exposure to SARS-CoV-2 antibodies. Main Outcomes and Measures: The presence of IgG antibodies to SARS-CoV-2 N and S antigens was measured using a microsphere immunoassay. Data were analyzed by geographic region and compared with reported COVID-19 cases and vaccinations among reproductive-aged females (15-44 years of age). Data were stratified by infant birth weight, gestational age, maternal age, and multiple birth status. Results: Dried blood spot samples from 415 293 infants (median [IQR] age, 1.04 [1.00-1.20] days; 210 805 [51.1%] male) were analyzed for SARS-CoV-2 antibodies. The first known antibody-positive infant in New York State was born on March 29, 2020. SARS-CoV-2 seroprevalence reflected statewide and regional COVID-19 cases among reproductive-aged females in the prevaccine period. From February through November 2021, S seroprevalence was strongly correlated with cumulative vaccinations in each New York State region and in the state overall (rs = 0.92-1.00, P ≤ .001). S and N seroprevalences were significantly lower in newborns with very low birth weight (720 [14.8%] for S and 138 [2.8%] for N, P < .001) and low birth weight (5160 [19.3%] for S and 1233 [4.6%] for N, P = .009) compared with newborns with normal birth weight (77 116 [20.1%] for S and 19 872 [5.2%] for N). Lower N and higher S seroprevalences were observed in multiple births (odds ratio [OR], 0.84; 95% CI, 0.75-0.94; P = .002 for N and OR, 1.24; 95% CI, 1.18-1.31; P < .001 for S) vs single births and for maternal age older than 30 years (OR, 0.87; 95% CI, 0.80-0.94; P < .001 for N and OR, 1.17; 95% CI, 1.11-1.23; P < .001 for S) vs younger than 20 years. Conclusions and Relevance: In this study, seroprevalence in newborn DBS samples reflected COVID-19 case fluctuations and vaccinations among reproductive-aged women during the study period. These results demonstrate the utility of using newborn DBS testing to estimate SARS-CoV-2 seroprevalence in pregnant individuals.

Postpartum Mother-To-Child Transmission of HIV in a Breastfeeding Infant.

This article is an examination of MTCT of HIV through breastfeeding in a mother who seroconverted postnatally.

High-Throughput Multiplex SARS-CoV-2 IgG Microsphere Immunoassay for Dried Blood Spots: A Public Health Strategy for Enhanced Serosurvey Capacity.

Early in the pandemic when diagnostic testing was not widely available, serosurveys played an important role in estimating the prevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in different populations. Dried blood spots (DBS), which can be collected in nonclinical settings, provide a minimally invasive alternative to serum for serosurveys. We developed a Luminex-based SARS-CoV-2 microsphere immunoassay (MIA) for DBS that detects IgG antibodies to nucleocapsid (N) and spike subunit 1 (S1) antigens. The assay uses a 384-well plate format and automated liquid handlers for high-throughput capacity. Specificity was assessed using a large collection of prepandemic DBS and well-characterized sera. Sensitivity was analyzed using serology data from New York State SARS-CoV-2 serosurvey testing and matched diagnostic test results. For DBS, the specificity was 99.5% for the individual N and S1 antigens. Median fluorescence intensity (MFI) values for DBS and paired sera showed a strong positive correlation for N (R2 = 0.91) and S1 (R2 = 0.93). Sensitivity, assessed from 1,134 DBS with prior laboratory-confirmed SARS-CoV-2 infection, ranged from 83% at 0 to 20 days to 95% at 61 to 90 days after a positive test. When stratified using coronavirus disease 2019 (COVID-19) symptom data, sensitivity ranged from 90 to 96% for symptomatic and 77 to 91% for asymptomatic individuals. For 8,367 health care workers reporting detailed symptom data, MFI values were significantly higher for all symptom categories. Our results indicate that the SARS-CoV-2 IgG DBS MIA is sensitive, specific, and well-suited for large population-based serosurveys. The ability to readily modify and multiplex antigens is important for ongoing assessment of SARS-CoV-2 antibody responses to emerging variants and vaccines. IMPORTANCE Testing for antibodies to SARS-CoV-2 has been used to estimate the prevalence of COVID-19 in different populations. Seroprevalence studies, or serosurveys, were especially useful during the early phase of the pandemic when diagnostic testing was not widely available, and the resulting seroprevalence estimates played an important role in public health decision making. To achieve meaningful results, antibody tests used for serosurveys should be accurate and accessible to diverse populations. We developed a test that detects antibodies to two different SARS-CoV-2 proteins in dried blood spots (DBS). DBS require only a simple fingerstick and can be collected in nonclinical settings. We conducted a robust validation study and have demonstrated that our test is both sensitive and specific. Furthermore, we demonstrated that our test is suitable for large-scale serosurveys by testing over 56,000 DBS collected in a variety of community-based venues in New York State during the spring of 2020.

Temporal Analysis of Serial Donations Reveals Decrease in Neutralizing Capacity and Justifies Revised Qualifying Criteria for Coronavirus Disease 2019 Convalescent Plasma.

BACKGROUND: Coronavirus disease 2019 (COVID-19) convalescent plasma (CCP) received an Emergency Use Authorization by the US Food and Drug Administration (FDA). CCP with a signal-to-cutoff ratio of ≥12 using the Ortho VITROS severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunoglobulin G (IgG) test (OVSARS2IgG) is permitted to be labeled "high titer." Little is known about the relationship between OVSARS2IgG ratio and neutralizing capacity of plasma/sera against genuine SARS-CoV-2. METHODS: Nine hundred eighty-one samples from 196 repeat CCP donors 0-119 days post-initial donation (DPID) were analyzed. Neutralizing capacity was assessed for 50% (PRNT50) and 90% (PRNT90) reduction of infectious virus using the gold standard plaque reduction neutralization test (PRNT). A subset of 91 donations was evaluated by OVSARS2IgG and compared to PRNT titers for diagnostic accuracy. RESULTS: Of donations, 32.7%/79.5% (PRNT90/PRNT50) met a 1:80 titer initially but only 14.0%/48.8% (PRNT90/PRNT50) met this cutoff ≥85 DPID. Correlation of OVSARS2IgG results to neutralizing capacity allowed extrapolation to CCP therapy results. CCP with OVSARS2IgG ratios equivalent to a therapeutically beneficial group had neutralizing titers of ≥1:640 (PRNT50) and/or ≥1:80 (PRNT90). Specificity and positive predictive value of the OVSARS2IgG for qualifying highly neutralizing CCP was optimal using ratios significantly greater than the FDA cutoff. CONCLUSIONS: This information provides a basis for refining the recommended properties of CCP used to treat COVID-19.

Cumulative incidence and diagnosis of SARS-CoV-2 infection in New York.

PURPOSE: New York State (NYS) is an epicenter of the SARS-CoV-2 pandemic in the United States. Reliable estimates of cumulative incidence in the population are critical to tracking the extent of transmission and informing policies. METHODS: We conducted a statewide seroprevalence study in a 15,101 patron convenience sample at 99 grocery stores in 26 counties throughout NYS. SARS-CoV-2 cumulative incidence was estimated from antibody reactivity by first poststratification weighting and then adjusting by antibody test characteristics. The percent diagnosed was estimated by dividing the number of diagnoses by the number of estimated infection-experienced adults. RESULTS: Based on 1887 of 15,101 (12.5%) reactive results, estimated cumulative incidence through March 29 was 14.0% (95% confidence interval [CI]: 13.3%-14.7%), corresponding to 2,139,300 (95% CI: 2,035,800-2,242,800) infection-experienced adults. Cumulative incidence was highest in New York City 22.7% (95% CI: 21.5%-24.0%) and higher among Hispanic/Latino (29.2%), non-Hispanic black/African American (20.2%), and non-Hispanic Asian (12.4%) than non-Hispanic white adults (8.1%, P < .0001). An estimated 8.9% (95% CI: 8.4%-9.3%) of infections in NYS were diagnosed, with diagnosis highest among adults aged 55 years or older (11.3%, 95% CI: 10.4%-12.2%). CONCLUSIONS: From the largest U.S. serosurvey to date, we estimated >2 million adult New York residents were infected through late March, with substantial disparities, although cumulative incidence remained less than herd immunity thresholds. Monitoring, testing, and contact tracing remain essential public health strategies.

Three Years of Shared Service HIV Nucleic Acid Testing for Public Health Laboratories: Worthwhile for HIV-1 but Not for HIV-2.

BACKGROUND: In 2016, HIV-2 nucleic acid testing (NAT) was added to a shared service program that conducts HIV-1 NAT for public health laboratories performing the recommended algorithm for diagnosing HIV. Here, we evaluate the usefulness of HIV-2 NAT in this program as compared with HIV-1 NAT. METHODS: Specimens eligible for HIV-1 NAT were reactive on an HIV-1/2 antibody or antigen/antibody initial test and nonreactive or indeterminate on a supplemental antibody test or were reactive for HIV-1 antigen-only on an HIV-1/2 antigen/antibody initial test. Specimens eligible for HIV-2 NAT were reactive on an initial test, HIV-2 indeterminate or HIV indeterminate on a supplemental antibody test and had no detectable HIV-1 RNA or were reactive for HIV-2 antibody on an HIV-1/2 antigen/antibody test, and this reactivity was not confirmed with a supplemental antibody assay. All specimens were tested in a reference laboratory using APTIMA HIV-1 qualitative RNA and/or a validated qualitative HIV-2 RNA real-time PCR assay. RESULTS: During 2016 to 2019, HIV-1 RNA was detected in 234 (14%) of 1731 specimens tested. HIV-2 RNA was not detected in 52 specimens tested. Median time from specimen collection to reporting of HIV-1 and HIV-2 NAT results by year ranged from 9 to 10 days and from 22 to 27 days, respectively. Two specimens with HIV-2 indeterminate results on a supplemental antibody test had detectable HIV-1 RNA. CONCLUSIONS: A shared service model for HIV-1 NAT is both feasible and beneficial for public health laboratories. However, because no HIV-2 infections were detected, our data suggest that this program should reconsider the usefulness of HIV-2 NAT testing.

Performance of the Alere Determine™ HIV-1/2 Ag/Ab Combo Rapid Test with algorithm-defined acute HIV-1 infection specimens.

BACKGROUND: The capacity of HIV Antigen/Antibody (Ag/Ab) immunoassays (IA) to detect HIV-1 p24 antigen has resulted in improved detection of HIV-1 infections in comparison to Ab-only screening assays. Since its introduction in the US, studies have shown that the Determine HIV-1/2 Ag/Ab Combo assay (Determine Ag/Ab) detects HIV infection earlier than laboratory-based IgM/IgG-sensitive IAs, but its sensitivity for HIV-1 p24 Ag detection is reduced compared to laboratory-based Ag/Ab assays. However, further evaluation is needed to assess its capacity to detect acute HIV-1 infection. OBJECTIVE: To assess the performance of Determine Ag/Ab in serum from acute HIV-1 infections. STUDY DESIGN: Select serum specimens that screened reactive on a laboratory-based Ag/Ab IA or IgM/IgG Ab-only IA, with a negative or indeterminate supplemental antibody test and detectable HIV-1 RNA were retrospectively tested with Determine Ag/Ab. Results were compared with those of the primary screening immunoassay to evaluate concordance within this set of algorithm-defined acute infections. RESULTS: Of 159 algorithm-defined acute HIV-1 specimens, Determine Ag/Ab was reactive for 105 resulting in 66.0% concordance. Of 125 that were initially detected by a laboratory-based Ag/Ab IA, 81 (64.8%) were reactive by Determine Ag/Ab. A total of 34 acute specimens were initially detected by a laboratory-based IgM/IgG Ab-only IA and 24 (70.6%) of those were reactive by Determine Ag/Ab. CONCLUSIONS: Due to their enhanced sensitivity, laboratory-based Ag/Ab IAs continue to be preferred over the Determine Ag/Ab as the screening method used by laboratories conducting HIV diagnostic testing on serum and plasma specimens.

Evaluation of dried blood spot protocols with the Bio-Rad GS HIV Combo Ag/Ab EIA and Geenius™ HIV 1/2 Supplemental Assay.

OBJECTIVE: FDA-approved antigen/antibody combo and HIV-1/2 differentiation supplemental tests do not have claims for dried blood spot (DBS) use. We compared two DBS-modified protocols, the Bio-Rad GS HIV Combo Ag/Ab (BRC) EIA and Geenius™ HIV-1/2 (Geenius) Supplemental Assay, to plasma protocols and evaluated them in the CDC/APHL HIV diagnostic algorithm. METHODS: BRC-DBS p24 analytical sensitivity was calculated from serial dilutions of p24. DBS specimens included 11 HIV-1 seroconverters, 151 HIV-1-positive individuals, including 20 on antiretroviral therapy, 31 HIV-2-positive and one HIV-1/HIV-2-positive individuals. BRC-reactive specimens were tested with Geenius using the same DBS eluate. Matched plasma specimens were tested with BRC, an IgG/IgM immunoassay and Geenius. DBS and plasma results were compared using the McNemar's test. A DBS-algorithm applied to 348 DBS from high-risk individuals who participated in surveillance was compared to HIV status based on local testing algorithms. RESULTS: BRC-DBS detects p24 at a concentration 18 times higher than in plasma. In seroconverters, BRC-DBS detected more infections than the IgG/IgM immunoassay in plasma (p=0.0133), but fewer infections than BRC-plasma (p=0.0133). In addition, the BRC/Geenius-plasma algorithm identified more HIV-1 infections than the BRC/Geenius-DBS algorithm (p=0.0455). The DBS protocols correctly identified HIV status for established HIV-1 infections, including those on therapy, HIV-2 infections, and surveillance specimens. CONCLUSIONS: The DBS protocols exhibited promising performance and allowed rapid supplemental testing. Although the DBS algorithm missed some early infections, it showed similar results when applied to specimens from a high-risk population. Implementation of a DBS algorithm would benefit testing programs without capacity for venipuncture.

Highlights from the 2016 HIV diagnostics conference: The new landscape of HIV testing in laboratories, public health programs and clinical practice.

The 2016 HIV Diagnostics Conference, held in Atlanta, Georgia, was attended by public health officials, laboratorians, HIV testing program managers, surveillance coordinators and industry representatives. The conference addressed test performance data, the implementation of new testing algorithms, quality assurance, and the application of new tests in a variety of settings. With regard to the recommended Centers for Disease Control and Prevention/Association of Public Health Laboratories HIV laboratory testing algorithm, the conference featured performance data, implementation challenges such as a lack of test options for the second and third steps, as well as data needs for new tests that may be used as part of the algorithm. There are delays when nucleic acid testing is needed with the algorithm. Novel tests such as point of care nucleic acid tests are needed on the U.S. market to readily identify acute infection. Multiplex tests are being developed which allow for the simultaneous detection of multiple pathogens. CDC staff highlighted new guidance for testing in non-clinical settings. Innovative approaches to linking testing and care in some settings have led to identification of early infections, improved receipt of test results and expedited initiation of therapy. Work continues to optimize testing so that infections are accurately identified as early as possible and time to treatment is minimized to improve health outcomes and prevent transmission.

Two human immunodeficiency virus Type 2 cases in US blood donors including serologic, molecular, and genomic characterization of an epidemiologically unusual case.

BACKGROUND: Blood donation screening for human immunodeficiency virus Type 2 (HIV-2) has been in place in the United States since 1992. However, only three HIV-2 antibody-positive donors have been reported to date, all detected via HIV-1 cross-reactivity. STUDY DESIGN AND METHODS: Here we identify two additional HIV-2-positive donors by routine anti-HIV-1 and anti-HIV-2 screening, including a first-time male donor living in Georgia having recently immigrated to the United States from West Africa (from a 1998 donation) and a Taiwanese female repeat donor (nurse) living in California with no travel outside of Taiwan or apparent connections to West Africa (from a 2015 donation). Neither donor acknowledged any risk factors, and both remained asymptomatic through follow-up. The second donor was further investigated by serologic, molecular, and genomic assays because of her unusual demographics. She was documented to harbor HIV-2 RNA, albeit sporadically by HIV-2-specific nucleic acid tests (35%-100% of replicates) and at very low levels (<9.6 IU/mL). Metagenomic next-generation sequencing (mNGS) confirmed the identification of a Group B HIV-2 strain, with recovered reads covering 46.9% of the predicted genome. CONCLUSIONS: The estimated frequency of an HIV-2-positive blood donor in the United States is one in 57 million donations. Due to the low frequency and low pathogenicity of HIV-2, public health and blood donation screening efforts must focus on HIV-1 detection and prevention. However, detection of HIV-2 infection in a donor with no apparent link to West Africa suggests that the United States must remain vigilant for HIV-2 virus infections. Ultradeep mNGS may be useful in the future for comprehensive identification of rare transfusion-transmissible agents.

Nucleic acid testing by public health referral laboratories for public health laboratories using the U.S. HIV diagnostic testing algorithm.

BACKGROUND: Many public health laboratories adopting the U.S. HIV laboratory testing algorithm do not have a nucleic acid test (NAT), which is needed when the third- or fourth-generation HIV screening immunoassay is reactive and the antibody-based supplemental test is non-reactive or indeterminate. OBJECTIVES: Among public health laboratories utilizing public health referral laboratories for NAT conducted as part of the algorithm, we evaluated the percentage of screening immunoassays needing NAT, the number of specimens not meeting APTIMA (NAT) specifications, time to APTIMA result, the proportion of acute infections (i.e., reactive APTIMA) among total infections, and screening immunoassay specificity. STUDY DESIGN: From August 2012 to April 2013, 22 laboratories enrolled to receive free APTIMA (NAT) at New York or Florida public health referral laboratories. Data were analyzed for testing conducted until June 2013. RESULTS: Submitting laboratories conducted a median of 4778 screening immunoassays; 0-1.3% (median 0.2%) needed NAT. Of 140 specimens received, 9 (6.4%) did not meet NAT specifications. The median time from specimen collection to reporting the 11 reactive NAT results was ten days, including six days from receipt in the submitting laboratory to shipment to the referral laboratory. Acute infections ranged from 0 to 12.5% (median 0%) of total infections. Third- and fourth-generation immunoassays met package insert specificity values. CONCLUSIONS: Public health referral laboratories provide a feasible option for conducting NAT. Reducing the time from specimen collection to submission of specimens for NAT is an important step toward maximizing the public health impact of identifying acute infections.

New York State 2010 HIV testing law: an evaluation of testing rates using laboratory data.

As of September 2010, New York State (NYS) Public Health Law mandates the offer of HIV testing to all persons aged 13-64 years receiving hospital or primary care services. Changes in the number of HIV tests 13 months before and after law enactment were assessed using HIV test volume data from 166 laboratories holding NYS permits to conduct HIV testing on specimens originating in NYS. Compared with the pre-enactment baseline, overall HIV testing volume increased by 13% following enactment, with the volume of conventional and rapid HIV screening tests increasing by 12.0% and 13.7%, respectively. These data suggest that testing law is having an impact consistent with the legislative intent to increase HIV testing in NYS. Monitoring should be continued to assess testing trends across a variety of health care venues to identify and address additional barriers to HIV testing access.

Evaluation of supplemental testing with the Multispot HIV-1/HIV-2 Rapid Test and APTIMA HIV-1 RNA Qualitative Assay to resolve specimens with indeterminate or negative HIV-1 Western blots.

BACKGROUND: The use of Western blot (WB) as a supplemental test after reactive sensitive initial assays can lead to inconclusive or misclassified HIV test results, delaying diagnosis. OBJECTIVE: To determine the proportion of specimens reactive by immunoassay (IA) but indeterminate or negative by WB that could be resolved by alternative supplemental tests recommended under a new HIV diagnostic testing algorithm. STUDY DESIGN: Remnant HIV diagnostic specimens that were reactive on 3rd generation HIV-1/2 IA and either negative or indeterminate by HIV-1 WB from 11 health departments were tested with the Bio-Rad Multispot HIV-1/HIV-2 Rapid Test (Multispot) and the Gen-Probe APTIMA HIV-1 RNA Qualitative Assay (APTIMA). RESULTS: According to the new testing algorithm, 512 (89.8%) specimens were HIV-negative, 55 (9.6%) were HIV-1 positive (including 19 [3.3%] that were acute HIV-1 and 9 [1.6%] that were positive for HIV-1 by Multispot but APTIMA-negative), 2 (0.4%) were HIV-2 positive, and 1 (0.2%) was HIV-positive, type undifferentiated. 47 (21.4%) of the 220 WB-indeterminate and 8 (2.3%) of the 350 WB-negative specimens were HIV-1 positive. CONCLUSION: Applying the new HIV diagnostic algorithm retrospectively to WB-negative and indeterminate specimens, the HIV infection status could be established for nearly all of the specimens. IA-reactive HIV-infected persons with WB-negative results had been previously misclassified as uninfected, and HIV diagnosis was delayed for those with WB-indeterminate specimens. These findings underscore the limitations of the WB to confirm HIV infection after reactive results from contemporary 3rd or 4th generation IAs that can detect HIV antibodies several weeks sooner than the WB.

Validation and clinical use of a sensitive HIV-2 viral load assay that uses a whole virus internal control.

BACKGROUND: Human immunodeficiency virus type 2 (HIV-2) is distantly related to the more widespread HIV-1. Although HIV-2 infection is rare in the U.S., cases are concentrated in the Northeast. No FDA-approved HIV-2 viral load assays exist. A clinically validated laboratory-developed assay is currently available in the U.S., however it is not currently approved for use on New York State patients. OBJECTIVE: To develop a sensitive viral load assay to quantify HIV-2 RNA in plasma and to validate it for clinical use. METHODS: The real-time RT-PCR assay simultaneously amplifies HIV-2 and a whole virus internal control, added during the lysis step. Two extraction volumes can be used. Results are reported in HIV-2 RNA International Units (IU). RESULTS: The assay has a limit of detection of 7 IU/mL and a lower limit of quantification of 29 IU/mL. The assay detects multiple strains of HIV-2 group A and B and generates reproducible results. Samples exchanged with a comparator laboratory produced similar viral load results, with 74% of positives differing by <0.5 log10 IU/mL. To date, we have tested 52 clinical specimens from 25 individuals. Twenty-eight (54%) specimens had measurable HIV-2 viral loads (range: 1.63-5.14 log10 IU/mL), 10 (19%) were positive but not quantifiable, and 14 were negative. HIV-2 RNA was detected in at least one specimen from 19 of 25 (76%) individuals tested. CONCLUSIONS: We developed a sensitive and accurate HIV-2 viral load assay. Validation data indicate the assay is suitable for clinical use and its availability in New York State will improve clinical monitoring of HIV-2 infected patients.

Expansion of HIV screening to non-clinical venues is aided by the use of dried blood spots for Western blot confirmation.

BACKGROUND: HIV rapid testing programs in New York State (NYS) are required to collect a specimen for confirmation of a preliminary positive result; however, some venues have limited capacity to collect venous blood, and confirmation using oral fluid is restricted by cost and availability. OBJECTIVE: To evaluate the feasibility of using dried blood spots (DBS) at non-clinical HIV rapid testing sites for Western blot testing. STUDY DESIGN: The New York State Department of Health facilitated registration of 48 non-clinical HIV test sites and provided training on DBS procedures. Following a reactive rapid test, DBS were collected by fingerstick onto filter paper cards, dried and mailed to the NYS public health laboratory for Western blot testing. RESULTS: From October 2010 to December 2012, 280 DBS specimens were submitted for confirmation. Four (1.4%) were unsatisfactory for testing and 276 (98.6%) DBS were tested. Of these, 235 (85.1%) were positive, 37 (13.4%) were negative and 4 (1.4%) were indeterminate. During this period, the laboratory also received 1033 venous blood specimens for rapid test confirmation, and 35 (3.4%) were unsatisfactory. Of the 998 tested by Western blot, 784 (78.6%) were positive, 197 (19.7%) were negative and 17 (1.7%) were indeterminate. DISCUSSION: Compared to venous blood, the percentage of rapid test referral specimens with a positive Western blot was significantly greater for DBS specimens and the frequency of unsatisfactory specimens did not differ significantly. These results indicate that DBS are a suitable alternative to venous blood for confirmation of HIV rapid tests conducted at non-clinical sites.

Characterization of HIV-1 subtypes and drug resistance mutations among individuals infected with HIV in Georgia.

In order to describe HIV-1 subtypes and drug resistance mutations in Georgia, blood samples from 153 patients infected with HIV-1 collected from 2006 to 2008 were genotyped. Of these, 126 samples were from newly diagnosed, antiretroviral (ARV)-naïve patients and 27 from ARV-treated patients. Partial pol region sequences were used to identify drug resistance mutations and to conduct phylogenetic analysis for subtype determination. The results indicated that 138 (90.2%) patients harbored subtype A viruses, 11 (7.2%) carried subtype B virus, two subtype G (1.3%), one (0.6%) subtype F and one (0.6%) 03_AB recombinant. All subtype A strains clustered with the Former Soviet Union A (A FSU) subtype. Among patients with no prior exposure to ARVs, mutations associated with resistance were detected in five patients: three (2.4%) patients had reverse transcriptase (RT) inhibitor mutations and two other patients had the protease (PI) inhibitor associated mutation M46I. PI mutation V77I was found in 42 of subtype A isolates. Of 27 ARV-treated patients, 22 (81.5%) harbored at least one nucleoside reverse transcriptase inhibitors (NRTI), a non-NRTI (NNRTI) and/or a PI mutation. The most common NRTI resistance mutation was M184V/I (74.1%). Frequency of thymidine analog mutations was relatively low (25.9%). With regard to NNRTI mutations, G190S/A was the most frequent mutation, which might be a preferred mutations for subtype A. Georgia's HIV epidemic continues to be dominated by Subtype A FSU. The prevalence of transmitted drug resistance is low, but has the potential to increase with increasing use of ARVs.