Discordant Antigenic Properties of Soluble and Virion SARS-CoV-2 Spike Proteins.

Efforts to develop vaccine and immunotherapeutic countermeasures against the COVID-19 pandemic focus on targeting the trimeric spike (S) proteins of SARS-CoV-2. Vaccines and therapeutic design strategies must impart the characteristics of virion S from historical and emerging variants onto practical constructs such as soluble, stabilized trimers. The virus spike is a heterotrimer of two subunits: S1, which includes the receptor binding domain (RBD) that binds the cell surface receptor ACE2, and S2, which mediates membrane fusion. Previous studies suggest that the antigenic, structural, and functional characteristics of virion S may differ from current soluble surrogates. For example, it was reported that certain anti-glycan, HIV-1 neutralizing monoclonal antibodies bind soluble SARS-CoV-2 S but do not neutralize SARS-CoV-2 virions. In this study, we used single-molecule fluorescence correlation spectroscopy (FCS) under physiologically relevant conditions to examine the reactivity of broadly neutralizing and non-neutralizing anti-S human monoclonal antibodies (mAbs) isolated in 2020. Binding efficiency was assessed by FCS with soluble S trimers, pseudoviruses and inactivated wild-type virions representing variants emerging from 2020 to date. Anti-glycan mAbs were tested and compared. We find that both anti-S specific and anti-glycan mAbs exhibit variable but efficient binding to a range of stabilized, soluble trimers. Across mAbs, the efficiencies of soluble S binding were positively correlated with reactivity against inactivated virions but not pseudoviruses. Binding efficiencies with pseudoviruses were generally lower than with soluble S or inactivated virions. Among neutralizing mAbs, potency did not correlate with binding efficiencies on any target. No neutralizing activity was detected with anti-glycan antibodies. Notably, the virion S released from membranes by detergent treatment gained more efficient reactivity with anti-glycan, HIV-neutralizing antibodies but lost reactivity with all anti-S mAbs. Collectively, the FCS binding data suggest that virion surfaces present appreciable amounts of both functional and nonfunctional trimers, with neutralizing anti-S favoring the former structures and non-neutralizing anti-glycan mAbs binding the latter. S released from solubilized virions represents a nonfunctional structure bound by anti-glycan mAbs, while engineered soluble trimers present a composite structure that is broadly reactive with both mAb types. The detection of disparate antigenicity and immunoreactivity profiles in engineered and virion-associated S highlight the value of single-virus analyses in designing future antiviral strategies against SARS-CoV-2.

HRP-conjugated-nanobody-based cELISA for rapid and sensitive clinical detection of ASFV antibodies.

African swine fever (ASF), which is caused by the ASF virus (ASFV), is a highly contagious hemorrhagic disease that causes high mortality to domestic porcine and wild boars and brings huge economic losses to world swine industry. Due to the lack of an effective vaccine, the control of ASF must depend on early, efficient, and cost-effective detection and strict control and elimination strategies. Traditional serological testing methods are generally associated with high testing costs, complex operations, and high technical requirements. As a promising alternative diagnostic tool to traditional antibodies, nanobodies (Nb) have the advantages of simpler and faster generation, good stability and solubility, and high affinity and specificity, although the system is dependent on the immunization of Bactrian camels to obtain the specific VHH library of the target protein. The application of Nbs in the detection of ASFV antibodies has not yet been reported yet. Using a phage display technology, one Nb against the ASFV p54 protein that exhibited high specificity and affinity, Nb8, was successfully screened. A HEK293T cell line stably expressing Nb8-horseradish peroxidase (HRP) fusion protein was established using the lentiviral expression system. Following the optimization of the reaction conditions, the Nb8-HRP fusion protein was successfully used to establish a competitive enzyme-linked immunosorbent assay (cELISA) to detect ASFV-specific antibodies in pig serum, for the first time. There was no cross-reaction with healthy pig serum, porcine pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), porcine epidemic diarrhea virus (PEDV), and classical swine fever virus (CSFV) positive sera. The optimal cut-off value for the cELISA by ROC analysis was 52.5%. A total of 209 serum samples were tested using the developed cELISA and a commercial ELISA kit. The results showed that the relative specificity of the cELISA was 98.97%, and the relative sensitivity of the cELISA was 93.3%, with the percent agreement between the two ELISA methods being 98.56%. In conclusion, a specific, sensitive, and repeatable cELISA was successfully developed based on the Nb8 as a probe, providing a promising method for the detection of anti-ASFV antibodies in clinical pig serum. KEY POINTS: • We successfully screened a specific, high affinity nanobody against ASFV p54 protein. • We establish a method for continuous and stable expression of Nb-HRP fusion protein using a lentiviral packaging system. • We establish a nanobody cELISA detection method that can monitor an ASF infection.

Vaccine with bacterium-like particles displaying HIV-1 gp120 trimer elicits specific mucosal responses and neutralizing antibodies in rhesus macaques.

Preclinical studies have shown that the induction of secretory IgA (sIgA) in mucosa and neutralizing antibodies (NAbs) in sera is essential for designing vaccines that can effectively block the transmission of HIV-1. We previously showed that a vaccine consisting of bacterium-like particles (BLPs) displaying Protan-gp120AE-MTQ (PAM) could induce mucosal immune responses through intranasal (IN) immunization in mice and NAbs through intramuscular (IM) immunization in guinea pigs. Here, we evaluated the ability of this vaccine BLP-PAM to elicit HIV-1-specific mucosal and systemic immune responses through IN and IM immunization combination strategies in rhesus macaques. First, the morphology, antigenicity and epitope accessibility of the vaccine were analysed by transmission electron microscopy, bio-layer interferometry and ELISA. In BLP-PAM-immunized macaques, HIV-1-specific sIgA were rapidly induced through IN immunization in situ and distant mucosal sites, although the immune responses are relatively weak. Furthermore, the HIV-1-specific IgG and IgA antibody levels in mucosal secretions were enhanced and maintained, while production of serum NAbs against heterologous HIV-1 tier 1 and 2 pseudoviruses was elicited after IM boost. Additionally, situ mucosal responses and systemic T cell immune responses were improved by rAd2-gp120AE boost immunization via the IN and IM routes. These results suggested that BLP-based delivery in combination with the IN and IM immunization approach represents a potential vaccine strategy against HIV-1.

Diastolic dysfunction in people with HIV without known cardiovascular risk factors in Western Kenya.

OBJECTIVES: Diastolic dysfunction (DD) has been reported to be highly prevalent in people living with HIV (PLWH) on antiretroviral therapy (ART) leading to the hypothesis that it may be an early marker of myocardial disease. Our objective was to evaluate the prevalence of DD in people living with human immunodeficiency virus without known history of diabetes or hypertension in Western Kenya. METHODS: In this cross-sectional study in western Kenya, 110 PLWH on ART and without known diabetes or hypertension were matched for age ±5 years and sex to HIV-uninfected controls. Study participants underwent a comprehensive two-dimensional echocardiogram and laboratory testing. RESULTS: The mean (SD) age in the HIV-positive group was 42.9 (8.6) years compared with 42.1 (12.9) years in the HIV-uninfected group. Mean (SD) CD4 +T cell count for the HIV-positive group was 557 (220) cells/ml. Mean systolic and diastolic blood pressures were within the normal range and comparable between the two groups. Mean body mass index was 25.2 (5.4) kg/m2 and 26.3 (5.4) kg/m2 in HIV-positive and uninfected participants, respectively. There was only 1 (0.9 %) case of DD in each group. Despite low prevalence of DD, PLWH had 5.76 g/m2 higher left ventricular mass index (p=0.01) and 2.77 mL/m2 larger left atrial volume (p=0.02) compared with the HIV-negative group after adjusting for risk factors associated with DD. CONCLUSION: Contrary to prior reports, DD in PLWH was low. Environmental and cardiovascular disease risk factors such as diabetes and hypertension may be significant modifiers for development and progression of DD in PLWH.

Comparison of SARS-CoV-2 Variants of Concern 202012/01 (U.K. Variant) and D614G Variant Transmission by Different Routes in Syrian Hamsters.

Introduction: Many SARS-CoV-2 variants of concern (VOC) have been reported recently that were linked to increased transmission. In our earlier study using VOC 202012/01 (U.K. variant) and D614G variant in the hamster model, we observed higher viral RNA shedding through nasal wash in the case of U.K. variant with lower pathogenicity in lung. In this study, we have studied transmission of these two variants by direct contact, aerosol, and fomite routes in Syrian hamsters and compared the viral load and body weight changes in hamsters exposed by both variants to understand the transmission efficiency. Methods: Nasal, throat, and rectal swabs were collected sequentially to assess viral load till 14 days. Results: Transmission could be established by direct, aerosol, and fomite contact in Syrian hamsters. Body weight loss or viral load in the contact animals exposed did not show any statistical significance. Conclusion: The study demonstrated comparable transmission of both U.K. and D614G variants of SARS-CoV-2 in Syrian hamsters in the given conditions. Provided these data, it seems that all the routes of exposure are effective leading to higher transmission.

Cesium Lead Bromide (CsPbBr3) Perovskite Quantum Dot-Based Photosensor for Chemiluminescence Immunoassays.

Chemiluminescence immunoassays have been widely employed for diagnosing various diseases. However, because of the extremely low intensity chemiluminescence signals, highly sensitive transducers, such as photomultiplier tubes and image sensors with cooling devices, are required to overcome this drawback. In this study, a hypersensitive photosensor was developed based on cesium lead bromide (CsPbBr3) perovskite quantum dots (QDs) with sufficient high sensitivity for chemiluminescence immunoassays. First, CsPbBr3 QDs with a highly uniform size, that is, 5 nm, were synthesized under thermodynamic control to achieve a high size confinement effect. For the fabrication of the photosensor, MoS2 nanoflakes were used as an electron transfer layer and heat-treated at an optimum temperature. Additionally, a parylene-C film was used as a passivation layer to improve the physical stability and sensitivity of the photosensor. In particular, the trap states on the CsPbBr3 QDs were reduced by the passivation layer, and the sensitivity was increased. Finally, a photosensor based on CsPbBr3 QDs was employed in chemiluminescence immunoassays for the detection of human hepatitis B surface antigen, human immunodeficiency virus antibody, and alpha-fetoprotein (AFP, a cancer biomarker). When compared with the conventionally used equipment, the photosensor was determined to be feasible for application in chemiluminescence immunoassays.

Improved HIV-positive infant survival is correlated with high levels of HIV-specific ADCC activity in multiple cohorts.

Defining immune responses that protect humans against diverse HIV strains has been elusive. Studying correlates of protection from mother-to-child transmission provides a benchmark for HIV vaccine protection because passively transferred HIV antibodies are present during infant exposure to HIV through breast milk. A previous study by our group illustrated that passively acquired antibody-dependent cellular cytotoxicity (ADCC) activity is associated with improved infant survival whereas neutralization is not. Here, we show, in another cohort and with two effector measures, that passively acquired ADCC antibodies correlate with infant survival. In combined analyses of data from both cohorts, there are highly statistically significant associations between higher infant survival and passively acquired ADCC levels (p = 0.029) as well as dimeric FcγRIIa (p = 0.002) or dimeric FcγRIIIa binding (p < 0.001). These results suggest that natural killer (NK) cell- and monocyte antibody-mediated effector functions may contribute to the observed survival benefit and support a role of pre-existing ADCC-mediating antibodies in clinical outcome.

Mucosal Priming with a Recombinant Influenza A Virus-Vectored Vaccine Elicits T-Cell and Antibody Responses to HIV-1 in Mice.

Recombinant influenza A viral (IAV) vectors are potential to stimulate systemic and mucosal immunity, but the packaging capacity is limited and only one or a few epitopes can be carried. Here, we report the generation of a replication-competent IAV vector that carries a full-length HIV-1 p24 gene linked to the 5'-terminal coding region of the neuraminidase segment via a protease cleavage sequence (IAV-p24). IAV-p24 was successfully rescued and stably propagated, and P24 protein was efficiently expressed in infected mammalian cells. In BALB/c mice, IAV-p24 showed attenuated pathogenicity compared to that of the parental A/PR/8/34 (H1N1) virus. An intranasal inoculation with IAV-p24 elicited moderate HIV-specific cell-mediated immune (CMI) responses in the airway and vaginal tracts and in the spleen, and an intranasal boost with a replication-incompetent adenovirus type 2 vector expressing the HIV-1 gag gene (Ad2-gag) greatly improved these responses. Importantly, compared to an Ad2-gag prime plus IAV-p24 boost regimen, the IAV-p24 prime plus Ad2-gag boost regimen had a greater efficacy in eliciting HIV-specific CMI responses. P24-specific CD8+ T cells and antibodies were robustly provoked both systemically and in mucosal sites and showed long-term durability, revealing that IAV-p24 may be used as a mucosa-targeted priming vaccine. Our results illustrate that IAV-p24 is able to prime systemic and mucosal immunity against HIV-1 and warrants further evaluation in nonhuman primates.IMPORTANCE An effective HIV-1 vaccine remains elusive despite nearly 40 years of research. CD8+ T cells and protective antibodies may both be desirable for preventing HIV-1 infection in susceptible mucosal sites. Recombinant influenza A virus (IAV) vector has the potential to stimulate these immune responses, but the packaging capacity is extremely limited. Here, we describe a replication-competent IAV vector expressing the HIV-1 p24 gene (IAV-p24). Unlike most other IAV vectors that carried one or several antigenic epitopes, IAV-p24 stably expressed the full-length P24 protein, which contains multiple epitopes and is highly conserved among all known HIV-1 sequences. Compared to the parental A/PR/8/34 (H1N1) virus, IAV-p24 showed an attenuated pathogenicity in BALB/c mice. When combined with an adenovirus vector expressing the HIV-1 gag gene, IAV-p24 was able to prime P24-specific systemic and mucosal immune responses. IAV-p24 as an alternative priming vaccine against HIV-1 warrants further evaluation in nonhuman primates.

Effect of a confirmatory testing algorithm on early acute HIV diagnosis in Korea.

ABSTRACT: The 17 Provincial Institutes of Health and Environment (PIHEs) in Korea use HIV antibody, antigen, and Western blot assays for confirmatory testing of HIV infection. The Korea Disease Control and Prevention Agency (KDCA) has further included p24 antigen neutralization and nucleic acid tests (NATs) since 2015. Our study aimed to investigate the effect of this new testing algorithm on the confirmation rate of HIV infection.Annual changes, from 2012 through 2017, in positive or indeterminate HIV confirmatory results were compared for the two algorithms between the PIHEs and the KDCA. Fiebig stages and Western blot p31 band were used to identify the diagnostic proportions of acute or early chronic HIV for the two algorithms.The number of positive cases in the samples requested from PIHEs for reconfirmation by the KDCA has steadily increased from 10.3% in 2014 to 33.3% in 2017. However, the number of indeterminate cases dropped sharply, from 71.9% in 2014 to 14.0% in 2017. The results for the p31 reactive band were 27.4% and 88.4% for the KDCA and PIHEs, respectively. Of positive cases reported by the KDCA, 22.9% were in the early acute stage and Fiebig stages I to II.The new testing algorithm has improved the diagnosis of HIV infections in the early acute stage. Early confirmatory diagnosis can prevent secondary transmission of HIV and provide early treatment opportunities for people living with HIV infection.

A sensitive method to quantify HIV-1 antibodies in mucosal samples.

Human immunodeficiency virus (HIV) remains a significant public health issue. In recent years, passive immunization with broadly neutralizing antibodies (bNabs) is being considered as a potentially efficacious approach for fighting HIV. One candidate that holds great promise is represented by the CD4-binding site targeted bNab capable of neutralizing over 90% of circulating HIV strains, VRC01. VRC01 along with its variants and clonal relatives - VRC01-LS and VRC07-523LS are currently being evaluated as vaccines in a number of clinical trials for HIV treatment and prevention. While mucosal areas of the body serve as major ports of HIV entry, reliable quantification of bNabs for pharmacokinetic and bioavailability analyses has been challenging due to low antibody concentrations in these samples. We developed an immunoassay on the Singulex platform which enables ultra-sensitive quantification of VRC01, VRC07, VRC01-LS and VRC07-523LS with a greater than 4-log linear dynamic range (LDR) and less than 120 pg/mL lower limit of quantitation (LLOQ). We implemented this assay to quantify VRC01 levels in rectal, cervical and oral mucosal samples in two passive immunization studies conducted with VRC01 - VRC 601 and VRC 602. Our assay was able to successfully quantify VRC01 levels in mucosal samples from all dosage groups (5 - -40 mg/kg) in these trials. VRC01 levels in a significant proportion of these samples (37% in oral and 25% in rectal mucosa) were below the lower limits of quantitation of other traditional immunoassays used for VRC01 quantification. We also measured VRC01 levels in sera from these trials and found that VRC01 measurements made using our assay exhibited excellent correlation (r2 = 0.9509) with measurements made previously using Enzyme-linked immunosorbent assay (ELISA). Our assay provides a reliable, sensitive and accurate method for quantification of clinically relevant bNabs and will help delineate antibody infiltration and bioavailability characteristics in complex biological matrices (CBM) such as mucosal tissues. This will in turn help determine clinical antibody threshold concentrations required to mediate protection against HIV acquisition and serve to inform dosing regimens and clinical trial design for future efficacy trials with these bNabs.

Prognostic Factors of Cytomegalovirus Infection Associated Retinitis in HIV-Negative Patients: A Retrospective Cohort Study.

Purpose: To summarize the prognostic factors of cytomegalovirus (CMV) retinitis (CMVR) in HIV-negative patients treated with multiple intravitreal injections (IVs) of ganciclovir.Methods: A retrospective cohort study (70 eyes) was conducted. Clinical signs, initial and final best corrected visual acuity (BCVA), initial aqueous load of CMV DNA, course of treatment, and occurrence of complications were recorded and analyzed.Results: A positive correlation was found between the baseline and the final best corrected visual acuity (P < .001) and between the initial aqueous CMV DNA load and the number of IVs (P = .01). A lesion close to the posterior pole (P < .001) and a larger retinal lesion (P = .002) remarkably led to worse visual prognosis.Conclusions: Poor visual prognosis was significantly associated with poor initial visual acuity, proximity of lesion to the posterior pole, and an extensive CMV lesion. The treatment duration was positively correlated with the initial aqueous CMV DNA load.

Field evaluation of capillary blood and oral-fluid HIV self-tests in the Democratic Republic of the Congo.

BACKGROUND: HIV self-testing (HIVST) is an additional approach to increasing uptake of HIV testing services. The practicability and accuracy of and the preference for the capillary blood self-test (Exacto Test HIV) versus the oral fluid self-test (OraQuick HIV self-test) were compared among untrained individuals in the Democratic Republic of the Congo (DRC). METHODS: This multicenter cross-sectional study (2019) used face-to-face, tablet-based, structured questionnaires in a facility-based HIVST approach. Volunteers from the general public who were at high risk of HIV infection, who were between 18 and 49 years of age, and who had signed an informed consent form were eligible for the study. The successful performance and correct interpretation of the self-test results were the main outcomes of the practicability evaluation. The successful performance of the HIV self-test was conditioned by the presence of the control band. The sensitivity and specificity of the participant-interpreted results compared to the laboratory results were estimated for accuracy. Preference for either type of self-test was assessed. Logistic regression models were used to examine factors associated with participants' preference. RESULTS: A total of 528 participants were included in this survey. The rate of successful performance of the HIV self-tests was high, with the blood test (99.6%) and the oral-fluid test (99.4%) yielding an absolute difference of 0.2% (95% CI: -1.8 to 1.1; P = 0.568). The rate of correct interpretation of the HIV self-test results was 84.4% with the blood test versus 83.8% with the oral-fluid test (difference = 0.6; 95% CI: -0.2 to 1.7; P = 0.425). Misinterpretation (25.4% for the blood test and 25.6% for the oral-fluid test) and inability to interpret (20.4% for the blood test and 21.1% for the oral-fluid test) test results were significantly more prevalent with invalid tests. The Exacto Test HIV self-test and the OraQuick HIV self-test showed 100% and 99.2% sensitivity, and 98.9% and 98.1% specificity, respectively. Preference for oral-fluid-based HIVST was greater than that for blood-based HIVST (85.6% versus 78.6%; P = 0.008). Preference for the blood test was greater among participants with a university education (86.1%; aOR = 2.4 [95% CI: 1.1 to 4.9]; P = 0.016), a higher risk of HIV infection (88.1%; aOR = 2.3 [95% CI: 1.0 to 5.3]; P = 0.047), and knowledge about the existence of HIVST (89.3%; aOR = 2.2 [95% CI: 1.0 to 5.0]; P = 0.05). CONCLUSION: Our field observations demonstrate that blood-based and oral-fluid-based HIVST are both practicable approaches with a high and comparable rate of accuracy in the study setting. Although preference for the oral-fluid test was generally greater, preference for the blood test was greater among participants with a university education, a high risk of HIV infection, and knowledge about the existence of HIVST. Both approaches seem complementary in the sense that users can choose the type of self-test that best suits them for a similar result. Taken together, our observations support the use of the two HIV self-test kits in the DRC.

Use of the Bio-Rad Geenius HIV-1/2 supplemental assay for the testing of oral fluids for the presence of HIV antibody.

There is currently an absence of products which are cleared by the FDA to provide supplemental testing for oral fluid for HIV antibody. We created a procedure for the use of the BioRad Geenius HIV-1/2 as a supplemental antibody test for oral fluid specimens. The modified procedure was evaluated for its ability to detect HIV-1 antibody in oral fluid in specimens that were found to be repeatedly reactive for HIV-1 antibody by way of the Avioq HIV-1 enzyme immunoassay (EIA). Evaluated were oral fluid specimens analyzed at a local public health laboratory which were stored frozen and oral fluid specimens collected prospectively. Prospectively collected specimens were from patients whose HIV status was subsequently assessed through blood-based testing. For retrospective specimens found repeatedly EIA reactive, and positive by Western blot, the modified Geenius was found positive in 37/38 instances (97.4 %). Those specimens with a mean EIA signal-to-cutoff (S/CO) greater than 3.00 were found to be positive by Geenius in 34/34 (100 %) of instances. For specimens found repeated reactive by EIA and positive by Western blot with mean S/CO less than or equal to 3.00, the Geenius was positive in 4/5 instances (80 %) of instances. For prospectively collected specimens, the Geenius accurately confirmed infection in 22/24 cases (92 %) while prospective specimens found repeatedly reactive by EIA without supplemental Geenius testing were confirmed positive in 29/37 instances (78 %). A modified usage of the Geenius HIV-1/2 Supplemental Assay antibody test may provide utility in the supplementation of testing of oral fluid for the presence of HIV-1 antibody.

Maternal Humoral Immune Responses Do Not Predict Postnatal HIV-1 Transmission Risk in Antiretroviral-Treated Mothers from the IMPAACT PROMISE Study.

To design immune interventions that can synergize with antiretroviral therapy (ART) to reduce the rate of HIV mother-to-child transmission (MTCT), it is essential to characterize maternal immune responses in the setting of ART during pregnancy and breastfeeding and define their effect on MTCT. Prior studies reported an association between breast milk envelope (Env)-specific antibodies and antibody-dependent cell cytotoxicity (ADCC) activity with reduced postnatal transmission. In this study, we investigated whether these immune correlates were similarly associated with protection in a matched case-control study of mother-infant pairs receiving maternal ART or infant nevirapine prophylaxis during breastfeeding in the International Maternal-Pediatric-Adolescent AIDS Clinical Trials Network Promoting Maternal-Infant Survival Everywhere (PROMISE) trial, assessing postnatal transmission risk in 19 transmitting and 57 nontransmitting mothers using conditional logistic regression models adjusted for maternal plasma viral load. The odds ratios of postnatal MTCT for a 1-unit increase in an immune correlate were 3.61 (95% confidence interval [CI], 0.56, 23.14) for breast milk Env-specific secretory IgA (sIgA), 2.32 (95% CI, 0.43, 12.56) for breast milk and 2.16 (95% CI, 0.51, 9.14) for plasma Env-specific IgA, and 4.57 (95% CI, 0.68, 30.48) for breast milk and 0.96 (95% CI, 0.25, 3.67) for plasma ADCC activity, with all CIs spanning 1.0. Interestingly, although mucosal IgA responses are poor in untreated HIV-infected women, there was a strong correlation between the magnitudes of breast milk and plasma Env-specific IgA in this cohort. In this analysis of the small number of postnatal virus transmissions in the landmark PROMISE study, no single antibody response was associated with breast milk transmission risk.IMPORTANCE Each year, >150,000 infants become newly infected with HIV-1 through MTCT despite ART, with up to 42% of infections occurring during breastfeeding. Several factors contribute to continued pediatric infections, including ART nonadherence, the emergence of drug-resistant HIV strains, acute infection during breastfeeding, and poor access to ART in resource-limited areas. A better understanding of the maternal humoral immune responses that provide protection against postnatal transmission in the setting of ART is critical to guide the design of maternal vaccine strategies to further eliminate postnatal HIV transmission. In this study, we found that in women treated with antiretrovirals during pregnancy, there was a positive correlation between plasma viral load and breast milk and plasma IgA responses; however, conclusions regarding odds of MTCT risk were limited by the small sample size. These findings will inform future studies to investigate maternal immune interventions that can synergize with ART to eliminate MTCT during breastfeeding.

Evolution of an Electronic Health Record Based-Human Immunodeficiency Virus (HIV) Screening Program in an Urban Emergency Department for Diagnosing Acute and Chronic HIV Infection.

BACKGROUND: Since 2006, Centers for Disease Control and Prevention guidelines recommend routine opt-out human immunodeficiency virus (HIV) testing among sexually active 13- to 64-year-olds. Earlier diagnosis and treatment of HIV infection reduces morbidity and mortality and can limit transmission to others. OBJECTIVE: Our aim was to increase HIV testing, diagnosis, and linkage to care in the emergency department (ED). METHODS: Beginning May 4, 2015, we utilized our electronic health record (EHR) to enhance HIV testing in patients seen in the Rush University Medical Center emergency department in Chicago, IL, who were 13-64 years of age, did not have HIV listed on their problem list, and did not have an HIV antigen/antibody (Ag/Ab) test in the EHR within the past rolling 12-month period. Strategies included use of a "Best Practice Advisory" and later auto-order screening linked to a complete blood count order. RESULTS: Our baseline HIV test rate was 2.5% of the target population by age (average of 93 tests per month). From May 4, 2015 to January 31, 2019, 137,749 patients of 240,091 ED visits met our test criteria and 23,588 (17.1% of the target population) HIV Ag/Ab tests were performed, resulting in 164 positive tests. We identified 18 acute seroconverters, 51 new chronically infected persons, and 95 known infected, many of who had not disclosed their status. Our positive test rate was 0.70%, which dropped to 0.29% if only newly diagnosed individuals were counted. CONCLUSIONS: EHR enhancements in a large urban ED identifies both newly diagnosed acute and chronically HIV-infected persons. Identification of previously diagnosed patients offers an opportunity to relink them to care.

Acute HIV Infection in a Patient with Repeat HIV Antibody/Antigen Negative Results Presenting at an Urban Emergency Department: A Case Report.

BACKGROUND: It is not uncommon for patients with human immunodeficiency virus (HIV) infections to visit the emergency department (ED) during seroconversion. However, patients with newly acquired HIV may not have a reactive screening result. We report a case of a patient who initially screened reactive on a fourth generation HIV test and subsequently nonreactive twice, but ultimately had positive viral load tests. CASE REPORT: A 41-year-old woman experiencing symptoms of a sore throat, odynophagia, and back and flank pain for 5 days presented to the ED. The patient had a reactive HIV screen but negative confirmatory antibody test. The ED provider ordered a HIV viral load, informed the patient, and discharged with oral antibacterial agent. The patient returned the next day and after review of Visit 1 results, the ED provider ordered a second HIV screen, which had a nonreactive result. Another HIV viral load order was placed. The patient was discharged and returned a third time, 4 days after initial presentation. On this visit she was admitted, and the initial HIV viral load result returned positive. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: We report a case of a patient who initially screened reactive on a fourth generation HIV screening and then twice nonreactive on the same screening test, ultimately having positive viral loads. The most probable explanation for her series of atypical HIV results is that the patient presented during the p24 seroconversion window, which is graphically conveyed in Figure 1. If her first screening had been performed during the window, no further test would have been performed to rule out HIV, contributing to misdiagnosis. ED providers need to be aware that, at some time points during seroconversion from "negative" to "positive", patients recently infected with HIV and manifesting prodromal symptoms may nonetheless have a negative screening result.

Brief Report: Diagnostic Accuracy of Oral Mucosal Transudate Tests Compared with Blood-Based Rapid Tests for HIV Among Children Aged 18 Months to 18 Years in Kenya and Zimbabwe.

BACKGROUND: Gaps persist in HIV testing for children who were not tested in prevention of mother-to-child HIV transmission programs. Oral mucosal transudate (OMT) rapid HIV tests have been shown to be highly sensitive in adults, but their performance has not been established in children. METHODS: Antiretroviral therapy-naive children aged 18 months to 18 years in Kenya and Zimbabwe were tested for HIV using rapid OraQuick ADVANCE Rapid HIV-1/2 Antibody test on oral fluids (OMT) and blood-based rapid diagnostic testing (BBT). BBT followed Kenyan and Zimbabwean national algorithms. Sensitivity and specificity were calculated using the national algorithms as the reference standard. RESULTS: A total of 1776 children were enrolled; median age was 7.3 years (interquartile range: 4.7-11.6). Among 71 children positive by BBT, all 71 were positive by OMT (sensitivity: 100% [97.5% confidence interval (CI): 94.9% to 100%]). Among the 1705 children negative by BBT, 1703 were negative by OMT (specificity: 99.9% [95% CI: 99.6% to 100.0%]). Due to discrepant BBT and OMT results, 2 children who initially tested BBT-negative and OMT-positive were subsequently confirmed positive within 1 week by further tests. Excluding these 2 children, the sensitivity and specificity of OMT compared with those of BBT were each 100% (97.5% CI: 94.9% to 100% and 99.8% to 100%, respectively). CONCLUSIONS: Compared to national algorithms, OMT did not miss any HIV-positive children. These data suggest that OMTs are valid in this age range. Future research should explore the acceptability and uptake of OMT by caregivers and health workers to increase pediatric HIV testing coverage.

Investigating the addition of oral HIV self-tests among populations with high testing coverage - Do they add value? Lessons from a study in Khayelitsha, South Africa.

INTRODUCTION: HIV self-testing (HIVST) offers a useful addition to HIV testing services and enables individuals to test privately. Despite recommendations to the contrary, repeat HIV testing is frequent among people already on anti-retroviral treatment (ART) and there are concerns that oral self-testing might lead to false negative results. A study was conducted in Khayelitsha, South Africa, to assess feasibility and uptake of HIVST and linkage-to-care following HIVST. METHODS: Participants were recruited at two health facilities from 1 March 2016 to 31 March 2017. People under 18 years, or with self-reported previously-diagnosed HIV infection, were excluded. Participants received an OraQuick Rapid HIV-1/2 Antibody kit, and reported their HIVST results by pre-paid text message (SMS) or by returning to the facility. Those not reporting within 7 days were contacted by phone. Electronic and paper-based clinical and laboratory records were retrospectively examined for all participants to identify known HIV outcomes, after matching for name, date of birth, and sex. These findings were compared with self-reported HIVST results where available. RESULTS: Of 639 participants, 401 (62.8%) self-reported a negative HIVST result, 27 (4.2%) a positive result, and 211 (33.0%) did not report. The record search identified that of the 401 participants self-reporting a negative HIVST result, 19 (4.7%) were already known to be HIV positive; of the 27 self-reporting positive, 12 (44%) were known HIV positive. Overall, records showed 57/639 (8.9%) were HIV positive of whom 39/57 (68.4%) had previously-diagnosed infection and 18/57 (31.6%) newly-diagnosed infection. Of the 428 participants who self-reported a result, 366 (85.5%) reported by SMS. CONCLUSIONS: HIVST can improve HIV testing uptake and linkage to care. SMS is acceptable for reporting HIVST results but negative self-reports by participants may be unreliable. Use of HIVST by individuals on ART is frequent despite recommendations to the contrary and its implications need further consideration.

HIV-1/2 screening in blood centers: implementing a two-step serological screening assay approach to reduce donor deferral.

BACKGROUND: Screening blood donations for human immunodeficiency virus 1/2 (HIV-1/2) infection in blood centers is often done with a highly sensitive 3rd-generation immunoassay which may cause false-positive results. Donations found repeatedly reactive (RR) are discarded regardless of negative HIV-1/2 nucleic acid testing (NAT) and confirmatory assays results. These donors are notified and deferred if RR in a subsequent donation. We evaluated the introduction of a secondary 4th-generation serological assay to the overall algorithm performance. METHODS: All donations collected between January 2016 and May 2018 (574,338) were screened using 3rd-generation immunoassay (PRISM HIV O Plus) and NAT (Procleix Ultrio/Ultrio Elite). Serology RR donations were tested with 4th-generation Architect HIV Ag/Ab Combo and Vidas HIV Duo Ultra and a confirmatory assay (Geenius HIV-1/2). RESULTS: The two 4th-generation assays found that 86% (179/209 on Architect) and 94% (182/193 on Vidas) of the 3rd-generation immunoassay RR were negative for HIV-1/2, which were also negative by confirmatory assay. Only 14% (30/209 on Architect) and 6% (11/193 on Vidas) that were 3rd-generation HIV-1/2 RR required confirmation, of which eight donors were confirmed as HIV positive. The probability of missing an HIV-1 infected donor by this algorithm is one in a million RR cases. CONCLUSION: The introduction of a two-step serological screening algorithm in blood centers whereby 4th-generation assay will be performed for all 3rd-generation RR blood donors will reduce the number of donations requiring confirmation, save time and money, and most importantly, reduce the number of discarded blood donations and allow re-entry processes.

Human T-cell lymphotropic virus 1/2 and human immunodeficiency virus antibodies identification among transactional sex workers and drug users in the Dominican Republic.

BACKGROUND: Human immunodeficiency virus (HIV) increases the risk of acquiring human T-cell lymphotropic virus (HTLV) and subsequently HTLV's progression to tropical spastic paraparesis/HTLV-1-associated myelopathy (TSP/HAM). Existing data have exclusively reported generalized rates of HIV and HTLV-1 chronic viral infections in the Dominican Republic. To our knowledge, no published studies have focused on the rates of HTLV-1/2 in transactional sex workers and drug users, both higher risk groups, in the Dominican Republic. METHODS: From December 2012 to April 2013 we conducted a study to estimate the seroprevalence of HTLV-1/2 immunoglobulin G (IgG) and HIV antibodies among transactional sex workers and intravenous drug users in Santo Domingo, Dominican Republic. Serological status was analysed with behaviour and demographic data. RESULTS: We collected and analysed plasma from 200 participants with a mean age of 27.4 y in men and 25.2 y in women. The overall weighted seroprevalence of HTLV-1/2 IgG antibodies was 13.91% (95% CI 7.59 to 20.23) in men and 10.59% (95% CI 4.05 to 17.13) in women. The overall weighted seroprevalence of HIV-1 was 13.91% (95% CI 7.59 to 20.23%) in men and 17.65% (95% CI 9.55 to 25.75) in women. Male intravenous drug users had an exceptionally high rate of HTLV-positive HIV co-infection, at 75% (95% CI 44.99 to 105.01). Although there an association has been found between HTLV/HIV co-infections and sex work, the adjusted odds revealed a confounding role of HIV infection. CONCLUSIONS: The results highlight the urgent need for enhanced public health preventive strategies among high-risk populations in the Dominican Republic and other resource-constrained Caribbean settings, as well as global adoption of routine screening for HTLV-associated infections, particularly in these high-risk, underserved populations.