Childhood screening for type 1 diabetes comparing automated multiplex Antibody Detection by Agglutination-PCR (ADAP) with single plex islet autoantibody radiobinding assays.

BACKGROUND: Two or more autoantibodies against either insulin (IAA), glutamic acid decarboxylase (GADA), islet antigen-2 (IA-2A) or zinc transporter 8 (ZnT8A) denote stage 1 (normoglycemia) or stage 2 (dysglycemia) type 1 diabetes prior to stage 3 type 1 diabetes. Automated multiplex Antibody Detection by Agglutination-PCR (ADAP) assays in two laboratories were compared to single plex radiobinding assays (RBA) to define threshold levels for diagnostic specificity and sensitivity. METHODS: IAA, GADA, IA-2A and ZnT8A were analysed in 1504 (54% females) population based controls (PBC), 456 (55% females) doctor's office controls (DOC) and 535 (41% females) blood donor controls (BDC) as well as in 2300 (48% females) patients newly diagnosed (1-10 years of age) with stage 3 type 1 diabetes. The thresholds for autoantibody positivity were computed in 100 10-fold cross-validations to separate patients from controls either by maximizing the χ2-statistics (chisq) or using the 98th percentile of specificity (Spec98). Mean and 95% CI for threshold, sensitivity and specificity are presented. FINDINGS: The ADAP ROC curves of the four autoantibodies showed comparable AUC in the two ADAP laboratories and were higher than RBA. Detection of two or more autoantibodies using chisq showed 0.97 (0.95, 0.99) sensitivity and 0.94 (0.91, 0.97) specificity in ADAP compared to 0.90 (0.88, 0.95) sensitivity and 0.97 (0.94, 0.98) specificity in RBA. Using Spec98, ADAP showed 0.92 (0.89, 0.95) sensitivity and 0.99 (0.98, 1.00) specificity compared to 0.89 (0.77, 0.86) sensitivity and 1.00 (0.99, 1.00) specificity in the RBA. The diagnostic sensitivity and specificity were higher in PBC compared to DOC and BDC. INTERPRETATION: ADAP was comparable in two laboratories, both comparable to or better than RBA, to define threshold levels for two or more autoantibodies to stage type 1 diabetes. FUNDING: Supported by The Leona M. and Harry B. Helmsley Charitable Trust (grant number 2009-04078), the Swedish Foundation for Strategic Research (Dnr IRC15-0067) and the Swedish Research Council, Strategic Research Area (Dnr 2009-1039). AL was supported by the DiaUnion collaborative study, co-financed by EU Interreg ÖKS, Capital Region of Denmark, Region Skåne and the Novo Nordisk Foundation.

Advances in risk predictive performance of pre-symptomatic type 1 diabetes via the multiplex Antibody-Detection-by-Agglutination-PCR assay.

Introduction: Achieving early diagnosis of pre-symptomatic type 1 diabetes is critical to reduce potentially life-threatening diabetic ketoacidosis (DKA) at symptom onset, link patients to FDA approved therapeutics that can delay disease progression and support novel interventional drugs development. The presence of two or more islet autoantibodies in pre-symptomatic type 1 diabetes patients indicates high-risk of progression to clinical manifestation. Method: Herein, we characterized the capability of multiplex ADAP assay to predict type 1 diabetes progression. We obtained retrospective coded sera from a cohort of 48 progressors and 44 non-progressors from the NIDDK DPT-1 study. Result: The multiplex ADAP assay and radiobinding assays had positive predictive value (PPV)/negative predictive value (NPV) of 68%/92% and 67%/66% respectively. The improved NPV stemmed from 12 progressors tested positive for multiple islet autoantibodies by multiplex ADAP assay but not by RBA. Furthermore, 6 out of these 12 patients tested positive for multiple islet autoantibodies by RBA in subsequent sampling events with a median delay of 2.8 years compared to multiplex ADAP assay. Discussion: In summary, multiplex ADAP assay could be an ideal tool for type 1 diabetes risk testing due to its sample-sparing nature (4µL), non-radioactiveness, compatibility with widely available real-time qPCR instruments and favorable risk prediction capability.

Characterization of antibodies to SARS-CoV-2 in lyophilized plasma prepared with amotosalen-UVA pathogen reduction.

BACKGROUND: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-infected patients exhibit disease ranging from asymptomatic to severe pneumonia, multi-organ failure, and death. convalescent COVID plasma (CCP) from recovered patients with high levels of neutralizing antibodies has demonstrated therapeutic efficacy to reduce the morbidity of coronavirus disease 2019 (COVID-19) in some studies. The development of assays to characterize the activity of CCP to neutralize SARS-CoV-2 infectivity offers the possibility to improve potential therapeutic efficacy. Lyophilization of CCP may increase the availability of this therapy. We hypothesized that SARS-CoV-2 antibody profiles of pooled lyophilized pathogen-reduced CCP from COVID-19-recovered blood donors retains virus-neutralizing efficacy as reported for frozen pathogen-reduced CCP. METHODS: Pooled lyophilized pathogen-reduced plasma was prepared from recovered COVID plasma donors. Antibodies to SARS-CoV-2 were characterized in each donor plasma prior to pathogen reduction and lyophilization and after lyophilization of individual CCP, and in the lyophilized CCP pool. Several complimentary assays were used to characterize antibody levels, neutralizing capacity, and the spectrum of antigen reactivity. The mean values for individual plasma samples and the value in the pool were compared. RESULTS: The mean ratio for antibody binding to SARS-CoV-2 antigens before and after treatment was 0.95 ± 0.22 mean fluorescent intensity (MFI) units. Antibody activity to an array of influenza virus antigens demonstrated a mean activity ratio of 0.92 ± 0.12 MFI before and after treatment. CONCLUSIONS: The antibody activity in pooled pathogen-reduced lyophilized CCPs demonstrated minimal impact due to pathogen reduction treatment and lyophilization.

Shadow of a Pandemic: Persistence of Prenatal SARS-CoV-2 Antibodies in Newborn Blood Spots.

To investigate COVID-19 surveillance among pregnant women, the California Genetic Disease Screening Program conducted a screening performance and seroprevalence evaluation of maternal SARS-CoV-2 antibodies detected in banked newborn dried blood spots (DBS). We obtained seropositive results for 2890 newborn DBS from cohorts in 2020 and 2021 using Enable Bioscience's Antibody Detection by Agglutination-PCR (ADAP) assay for SARS-CoV-2 antibodies. To infer maternal infection, we linked 312 women with a known laboratory-confirmed COVID-19 episode with their newborn's DBS SARS-CoV02 antibody result. Among 2890 newborns, we detected 453 (15.7%) with SARS-CoV-2 antibodies in their DBS. Monthly snapshot statewide seroprevalence among neonates was 12.2% (95% CI 10.3-14.1%, n =1156) in December 2020 and 33.3% (95% CI 29.1-37.4%, n = 26) in March 2021. The longest time recorded from COVID-19 infection to a seropositive neonatal result was 11.7 months among the 312 mothers who had an available SARS-CoV-2 PCR test result. Approximately 94% (153/163) of DBS were seropositive when a known maternal infection occurred earlier than 19 days before birth. The estimated relative sensitivity of DBS to identify prevalent maternal infection was 85.1%, specificity 98.5% and PPV 99.2% (n = 312); the sensitivity was lowest during the December 2021 surge when many infections occurred within 19 days of birth. Fifty pre-pandemic specimens (100% seronegative) and 23 twin-pair results (100% concordant) support an intrinsic specificity and PPV of ADAP approaching 100%. Maternal infection surveillance is limited by a time lag prior to delivery, especially during pandemic surges.

Characterization of pathogen-inactivated COVID-19 convalescent plasma and responses in transfused patients.

BACKGROUND: Efficacy of donated COVID-19 convalescent plasma (dCCP) is uncertain and may depend on antibody titers, neutralizing capacity, timing of administration, and patient characteristics. STUDY DESIGN AND METHODS: In a single-center hypothesis-generating prospective case-control study with 1:2 matched dCCP recipients to controls according to disease severity at day 1, hospitalized adults with COVID-19 pneumonia received 2 × 200 ml pathogen-reduced treated dCCP from 2 different donors. We evaluated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies in COVID-19 convalescent plasma donors and recipients using multiple antibody assays including a Coronavirus antigen microarray (COVAM), and binding and neutralizing antibody assays. Outcomes were dCCP characteristics, antibody responses, 28-day mortality, and dCCP -related adverse events in recipients. RESULTS: Eleven of 13 dCCPs (85%) contained neutralizing antibodies (nAb). PRT did not affect dCCP antibody activity. Fifteen CCP recipients and 30 controls (median age 64 and 65 years, respectively) were enrolled. dCCP recipients received 2 dCCPs from 2 different donors after a median of one hospital day and 11 days after symptom onset. One dCCP recipient (6.7%) and 6 controls (20%) died (p = 0.233). We observed no dCCP-related adverse events. Transfusion of unselected dCCP led to heterogeneous SARS CoV-2 antibody responses. COVAM clustered dCCPs in 4 distinct groups and showed endogenous immune responses to SARS-CoV-2 antigens over 14-21 days post dCCP in all except 4 immunosuppressed recipients. DISCUSSION: PRT did not impact dCCP anti-virus neutralizing activity. Transfusion of unselected dCCP did not impact survival and had no adverse effects. Variable dCCP antibodies and post-transfusion antibody responses indicate the need for controlled trials using well-characterized dCCP with informative assays.

CalScope: Monitoring Severe Acute Respiratory Syndrome Coronavirus 2 Seroprevalence From Vaccination and Prior Infection in Adults and Children in California May 2021-July 2021.

Background: Understanding the distribution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies from vaccination and/or prior infection is critical to the public health response to the pandemic. CalScope is a population-based serosurvey in 7 counties in California. Methods: We invited 200 000 randomly sampled households to enroll up to 1 adult and 1 child between April 20, 2021 and June 16, 2021. We tested all specimens for antibodies against SARS-CoV-2 nucleocapsid and spike proteins, and each participant completed an online survey. We classified participants into categories: seronegative, antibodies from infection only, antibodies from infection and vaccination, and antibodies from vaccination only. Results: A total of 11 161 households enrolled (5.6%), with 7483 adults and 1375 children completing antibody testing. As of June 2021, 33% (95% confidence interval [CI], 28%-37%) of adults and 57% (95% CI, 48%-66%) of children were seronegative; 18% (95% CI, 14%-22%) of adults and 26% (95% CI, 19%-32%) of children had antibodies from infection alone; 9% (95% CI, 6%-11%) of adults and 5% (95% CI, 1%-8%) of children had antibodies from infection and vaccination; and 41% (95% CI, 37%-45%) of adults and 13% (95% CI, 7%-18%) of children had antibodies from vaccination alone. Conclusions: As of June 2021, one third of adults and most children in California were seronegative. Serostatus varied regionally and by demographic group.

Detection of neutralizing antibodies against multiple SARS-CoV-2 strains in dried blood spots using cell-free PCR.

An easily implementable serological assay to accurately detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralizing antibodies is urgently needed to better track herd immunity, vaccine efficacy and vaccination rates. Herein, we report the Split-Oligonucleotide Neighboring Inhibition Assay (SONIA) which uses real-time qPCR to measure the ability of neutralizing antibodies to block binding between DNA-barcoded viral spike protein subunit 1 and the human angiotensin-converting enzyme 2 receptor protein. The SONIA neutralizing antibody assay using finger-prick dried blood spots displays 91-97% sensitivity and 100% specificity in comparison to the live-virus neutralization assays using matched serum specimens for multiple SARS-CoV-2 variants-of-concern. The multiplex version of this neutralizing antibody assay, using easily collectable finger-prick dried blood spots, can be a valuable tool to help reveal the impact of age, pre-existing health conditions, waning immunity, different vaccination schemes and the emergence of new variants-of-concern.

Multiplex agglutination-PCR (ADAP) autoantibody assays compared to radiobinding autoantibodies in type 1 diabetes and celiac disease.

Multiplex Antibody-Detection by Agglutination-PCR (ADAP) assay was compared to singleplex standard radiobinding assays (RBA) to detect autoantibodies against insulin (IAA), GAD65 (GADA), islet antigen-2 (IA-2A), ZnT8 (ZnT8A) and tissue transglutaminase (TGA). Serum samples from 273 (114F/158M), 15-73 years of age healthy controls and 227 (109F/118M) newly diagnosed type 1 diabetes children, 1-11 years of age, were analyzed in both assay systems.The original WHO standard 97/550 and in-house reference standards for RBA were compared to ADAP. The ADAP and RBA generated parallel reference standards in all assays except TGA. Lower detection limits were observed in the ADAP assay for GADA,IAA and ZnT8A, markedly for TGA, but not for IA-2A. The Receiver Operating Characteristics (ROC) curve AUC analyses for pairwise comparison of ADAP with RBA showed no difference for GADA (n.s.), ADAP greater AUC for IAA (p = 0.005), RBA greater AUC for IA-2A (p = 0.0004) and ZnT8A (p < 0.0001) while ADAP TGA had a greater AUC compared to both RBA TGA-IgG (p < 0.0001) and TGA-IgA (p < 0.0001). These data suggest that the ADAP and RBA assays are comparable with equal performance for GADA, better ADAP performance for IAA while the RBA showed better performance in both IA-2A and ZnT8A associated with greater heterogeneity in autoantibody levels. The simultaneous analysis of 5 different autoantibodies by ADAP in sample volume reduced to only 4 µL and at an increased lower detection limit in all assays except IA-2A makes the ADAP automated autoantibody assay a distinct advantage for high throughput screening.

Antibody profiles in COVID-19 convalescent plasma prepared with amotosalen/UVA pathogen reduction treatment.

BACKGROUND: COVID-19 convalescent plasma (CCP), from donors recovered from severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection, is one of the limited therapeutic options currently available for the treatment of critically ill patients with COVID-19. There is growing evidence that CCP may reduce viral loads and disease severity; and reduce mortality. However, concerns about the risk of transfusion-transmitted infections (TTI) and other complications associated with transfusion of plasma, remain. Amotosalen/UVA pathogen reduction treatment (A/UVA-PRT) of plasma offers a mitigation of TTI risk, and when combined with pooling has the potential to increase the diversity of the polyclonal SARS-CoV-2 neutralizing antibodies. STUDY DESIGN AND METHODS: This study assessed the impact of A/UVA-PRT on SARS-CoV-2 antibodies in 42 CCP using multiple complimentary assays including antigen binding, neutralizing, and epitope microarrays. Other mediators of CCP efficacy were also assessed. RESULTS: A/UVA-PRT did not negatively impact antibodies to SARS-CoV-2 and other viral epitopes, had no impact on neutralizing activity or other potential mediators of CCP efficacy. Finally, immune cross-reactivity with other coronavirus antigens was observed raising the potential for neutralizing activity against other emergent coronaviruses. CONCLUSION: The findings of this study support the selection of effective CCP combined with the use of A/UVA-PRT in the production of CCP for patients with COVID-19.

Automation of a multiplex agglutination-PCR (ADAP) type 1 diabetes (T1D) assay for the rapid analysis of islet autoantibodies.

Screening for islet autoantibody markers to identify individuals who are at high risk for developing type 1 diabetes (T1D), often years in advance of clinical symptoms, is both a challenge and a necessity. Identifying high-risk individuals not only reduces hospitalization and rates of life-threatening diabetes ketoacidosis (DKA), but also directs enrollment into prevention trials that require patients who are in the early stages of disease. Here we describe an automated high-throughput multiplex islet autoantibody assay that integrates antibody detection by agglutination-PCR (ADAP) chemistry on the Hamilton Microlab STAR liquid handling platform. The automated system features on-deck thermal cycling and plate sealing to minimize the level of human intervention. The automated multiplex ADAP T1D assay performed similarly to that of manual methods using two distinct cohorts of clinical specimens obtained from the Lucile Packard Children's Hospital at Stanford University and the 2018 Islet Autoantibody Standardization Program (IASP). Notably, the automated assay requires only 4 µL of serum sample for the simultaneous analysis of GAD, IA-2 and insulin autoantibodies. Up to 96 samples may be processed in as little as 3 hours, and the only user intervention required is to transfer a final sealed 96-well plate containing PCR amplicons onto a quantitative PCR (RT-qPCR) instrument for quantification. The automated system is particularly well suited for large-scale analysis of islet autoantibodies in a reproducible, timely, and cost-effective manner.

Effectiveness of Abbott BinaxNOW Rapid Antigen Test for Detection of SARS-CoV-2 Infections in Outbreak among Horse Racetrack Workers, California, USA.

The Abbott BinaxNOW rapid antigen test is cheaper and faster than real-time reverse transcription PCR (rRT-PCR) for detecting severe acute respiratory syndrome coronavirus 2. We compared BinaxNOW with rRT-PCR in 769 paired specimens from 342 persons during a coronavirus disease outbreak among horse racetrack workers in California, USA. We found positive percent agreement was 43.3% (95% CI 34.6%-52.4%), negative percent agreement 100% (95% CI 99.4%-100%), positive predictive value 100% (95% CI 93.5%-100%), and negative predictive value 89.9% (95% CI 87.5%-92.0%). Among 127 rRT-PCR-positive specimens, the 55 with paired BinaxNOW-positive results had a lower mean cycle threshold than the 72 with paired BinaxNOW-negative results (17.8 vs. 28.5; p<0.001). Of 100 specimens with cycle threshold <30, a total of 51 resulted in positive virus isolation; 45 (88.2%) of those were BinaxNOW-positive. Our comparison supports immediate isolation for BinaxNOW-positive persons and confirmatory testing for negative persons.

Prospective Cohort of Fluvoxamine for Early Treatment of Coronavirus Disease 19.

We report a real-world experience using fluvoxamine for coronavirus disease 19 (COVID-19) in a prospective cohort in the setting of a mass outbreak. Overall, 65 persons opted to receive fluvoxamine (50 mg twice daily) and 48 declined. Incidence of hospitalization was 0% (0 of 65) with fluvoxamine and 12.5% (6 of 48) with observation alone. At 14 days, residual symptoms persisted in 0% (0 of 65) with fluvoxamine and 60% (29 of 48) with observation.

Sensitive detection of multiple islet autoantibodies in type 1 diabetes using small sample volumes by agglutination-PCR.

Islet autoantibodies are predominantly measured by radioassay to facilitate risk assessment and diagnosis of type 1 diabetes. However, the reliance on radioactive components, large sample volumes and limited throughput renders radioassay testing costly and challenging. We developed a multiplex analysis platform based on antibody detection by agglutination-PCR (ADAP) for the sample-sparing measurement of GAD, IA-2 and insulin autoantibodies/antibodies in 1 µL serum. The assay was developed and validated in 7 distinct cohorts (n = 858) with the majority of the cohorts blinded prior to analysis. Measurements from the ADAP assay were compared to radioassay to determine correlation, concordance, agreement, clinical sensitivity and specificity. The average overall agreement between ADAP and radioassay was above 91%. The average clinical sensitivity and specificity were 96% and 97%. In the IASP 2018 workshop, ADAP achieved the highest sensitivity of all assays tested at 95% specificity (AS95) rating for GAD and IA-2 autoantibodies and top-tier performance for insulin autoantibodies. Furthermore, ADAP correctly identified 95% high-risk individuals with two or more autoantibodies by radioassay amongst 39 relatives of T1D patients tested. In conclusion, the new ADAP assay can reliably detect the three cardinal islet autoantibodies/antibodies in 1µL serum with high sensitivity. This novel assay may improve pediatric testing compliance and facilitate easier community-wide screening for islet autoantibodies.

A serological assay to detect SARS-CoV-2 antibodies in at-home collected finger-prick dried blood spots.

Accurate surveillance of coronavirus disease 2019 (COVID-19) incidence requires large-scale testing of the population. Current testing methods require in-person collection of biospecimens by a healthcare worker, limiting access of individuals who do not have access to testing facilities while placing both patients and healthcare workers at risk of exposure to infection. We report the development and validation of a at-home finger-prick dried blood spot collection kit and an analysis method. We demonstrated 100% sensitivity and specificity using at-home collected specimens across the US. Such methods may facilitate the conduct of unbiased serosurveys within hard to reach populations and help reduce the sample collection burden of serological testing on both health care systems and individuals alike.

Sensitive and Specific Detection of SARS-CoV-2 Antibodies Using a High-Throughput, Fully Automated Liquid-Handling Robotic System.

As of July 22, 2020, more than 14.7 million infections of SARS-CoV-2, the virus responsible for Coronavirus Disease 2019 (COVID-19), have been confirmed globally. Serological assays are essential for community screening, assessing infection prevalence, aiding identification of infected patients, and enacting appropriate treatment and quarantine protocols in the battle against this rapidly expanding pandemic. Antibody detection by agglutination-PCR (ADAP) is a pure solution phase immunoassay that generates a PCR amplifiable signal when patient antibodies agglutinate DNA-barcoded antigen probes into a dense immune complex. Here, we present an ultrasensitive and high-throughput automated liquid biopsy assay based on the Hamilton Microlab ADAP STAR automated liquid-handling platform, which was developed and validated for the qualitative detection of total antibodies against spike protein 1 (S1) of SARS-CoV-2 that uses as little as 4 µL of serum. To assess the clinical performance of the ADAP assay, 57 PCR-confirmed COVID-19 patients and 223 control patients were tested. The assay showed a sensitivity of 98% (56/57) and a specificity of 99.55% (222/223). Notably, the SARS-CoV-2-negative control patients included individuals with other common coronaviral infections, such as CoV-NL63 and CoV-HKU, which did not cross-react. In addition to high performance, the hands-free automated workstation enabled high-throughput sample processing to reduce screening workload while helping to minimize analyst contact with biohazardous samples. Therefore, the ADAP STAR liquid-handling workstation can be used as a valuable tool to address the COVID-19 global pandemic.

Detection of SARS-CoV-2 neutralizing antibodies with a cell-free PCR assay.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to more than 4 million confirmed infections worldwide and over 300,000 deaths. While Remdesivir has recently received FDA emergency use authorization for treatment of SARS-CoV-2 infection, convalescent plasma (CP) with high titers of SARS-CoV-2 neutralizing antibodies (NAbs) from recovered donors remains a promising and widely accessible method to mitigate severe disease symptoms. Here, we describe the development and validation of a cell-free neutralization PCR assay using SARS-CoV-2 spike protein S1 and human ACE2 receptor-DNA conjugates. By comparing with samples collected prior to the outbreak, we confirmed that NAbs were specifically detected in COVID-19 cases. Using our unique assay, the NAb signals are detectable as early as 10 days after onset of symptoms and continue to rise, plateauing after 18 days. Notably, we showed that the use of licensed pathogen reduction technology to inactivate potentially contaminating infectious pathogens in CP did not alter NAb signals, paving a path to safely administer effective CP therapies. The described neutralization PCR assay can serve as a qualification tool to easily identify suitable CP donors of a potentially lifesaving therapy. In addition, this assay tool is readily deployable in standard laboratories with biosafety level 2 capability, and can yield results within 2-3 hr. This advancement can facilitate research on factors driving diverse COVID-19 disease manifestations, and to evaluate the impact of various CP processing protocols on CP therapeutic efficacy.

A serological assay to detect SARS-CoV-2 antibodies in at-home collected finger-prick dried blood spots.

Accurate surveillance of coronavirus disease 2019 (COVID-19) incidence requires large-scale testing of the population. Current testing methods require in-person collection of biospecimens by a healthcare worker, limiting access of individuals who do not have access to testing facilities while placing both the patient and healthcare worker at risk of exposure to infection. We report the development and validation of a at-home finger-prick dried blood spot collection kit and an analysis method. We demonstrated 100% sensitivity and specificity using at-home collected specimens across the US. Such methods may facilitate the conduct of unbiased serosurveys within hard to reach populations and help reduce the sample collection burden of serological testing on both health care systems and individuals alike.

Antibody detection by agglutination-PCR (ADAP) enables early diagnosis of HIV infection by oral fluid analysis.

Oral fluid (OF) is a highly effective substrate for population-based HIV screening efforts, as it is noninfectious and significantly easier to collect than blood. However, anti-HIV antibodies are found at far lower concentrations in OF compared with blood, leading to poor sensitivity and a longer period of time from infection to detection threshold. Thus, despite its inherent advantages in sample collection, OF is not widely used for population screening. Here we report the development of an HIV OF assay based on Antibody Detection by Agglutination-PCR (ADAP) technology. This assay is 1,000-10,000 times more analytically sensitive than clinical enzyme-linked immunoassays (EIAs), displaying both 100% clinical sensitivity and 100% specificity for detecting HIV antibodies within OF samples. We show that the enhanced analytical sensitivity enables this assay to correctly identify HIV-infected individuals otherwise missed by current OF assays. We envision that the attributes of this improved HIV OF assay can increase testing rates of at-risk individuals while enabling diagnosis and treatment at an earlier time point.