Application of ultrasensitive digital ELISA for p24 enables improved evaluation of HIV-1 reservoir diversity and growth kinetics in viral outgrowth assays.

The advent of combined antiretroviral therapy (cART) has been instrumental in controlling HIV-1 replication and transmission and decreasing associated morbidity and mortality. However, cART alone is not able to cure HIV-1 due to the presence of long-lived, latently infected immune cells, which re-seed plasma viremia when cART is interrupted. Assessment of HIV-cure strategies using ex vivo culture methods for further understanding of the diversity of reactivated HIV, viral outgrowth, and replication dynamics are enhanced using ultrasensitive digital ELISA based on single-molecule array (Simoa) technology to increase the sensitivity of endpoint detection. In viral outgrowth assays (VOA), exponential HIV-1 outgrowth has been shown to be dependent upon initial virus burst size surpassing a critical growth threshold of 5100 HIV-1 RNA copies. Here, we show an association between ultrasensitive HIV-1 Gag p24 concentrations and HIV-1 RNA copy number that characterize viral dynamics below the exponential replication threshold. Single-genome sequencing (SGS) revealed the presence of multiple identical HIV-1 sequences, indicative of low-level replication occurring below the threshold of exponential outgrowth early during a VOA. However, SGS further revealed diverse related HIV variants detectable by ultrasensitive methods that failed to establish exponential outgrowth. Overall, our data suggest that viral outgrowth occurring below the threshold necessary for establishing exponential growth in culture does not preclude replication competence of reactivated HIV, and ultrasensitive detection of HIV-1 p24 may provide a method to detect previously unquantifiable variants. These data strongly support the use of the Simoa platform in a multi-prong approach to measuring latent viral burden and efficacy of therapeutic interventions aimed at an HIV-1 cure.

Standardized evaluation of Zika nucleic acid tests used in clinical settings and blood screening.

Early detection of Zika virus (ZIKV) transmission within geographic regions informs implementation of community mitigation measures such as vector reduction strategies, travel advisories, enhanced surveillance among pregnant women, and possible implementation of blood and organ donor screening or deferral. Standardized, comparative assessments of ZIKV assay and testing lab performance are important to develop optimal approaches to ZIKV diagnostic testing and surveillance. We conducted an expanded blinded panel study to characterize and compare the analytical performance of fifteen diagnostic and blood screening ZIKV NAT assays, including detection among single- and multiplex assays detecting ZIKV, dengue virus (DENV) and chikungunya virus (CHIKV). A 300 member blinded panel was constructed, consisting of 11 serial half-log dilutions ranging from ~104 to 10-1 genome equivalents/mL in 25 replicates each of the Tahitian Asian ZIKV isolate in ZIKV-negative human serum. Additionally, clinical samples from individuals with DENV-like syndrome or suspected ZIKV infection in Brazil were evaluated. The majority of assays demonstrated good specificity. Analytical sensitivities varied 1-2 logs, with a substantially higher limit of detection (LOD) in one outlier. Similar analytical sensitivity for ZIKV RNA detection in singleplex and multiplex assays of the Grifols and ThermoFisher tests were observed. Coefficient of Assay Efficiency (CE), calculated to characterize assays' RNA extraction and amplification efficiency, ranged from 0.13 for the Certest VIASURE multiplex and 0.75 for the Grifols multiplex assays. In general, assays using transcription mediated amplification (TMA) technology had greater CE compared to assays using conventional PCR technology. Donor screening NAT assays were significantly more sensitive than diagnostic RT-qPCR assays, primarily attributable to higher sample input volumes. However, ideal assays to maximize sensitivity and throughput may not be a viable option in all contexts, with other factors such as cost, instrumentation, and regulatory approval status influencing assay availability and selection, particularly in resource constrained settings.

Plasma transfusion-transmission of Zika virus in mice and macaques.

BACKGROUND: Zika virus (ZIKV) epidemics with infections in pregnant women are associated with severe neurological disease in newborns. Although an arbovirus, ZIKV is also blood transfusion-transmitted (TT). Greater knowledge of the efficiency of ZIKV TT would aid decisions on testing and pathogen reduction technologies (PRT). STUDY DESIGN AND METHODS: Plasma units from ZIKV RNA-reactive blood donors were used to study infectivity in vitro, in mice, and in macaques. Furthermore, plasma units were subjected to PRT using amotosalen/ultraviolet light A (A/UVA) before transfusion. RESULTS: In vitro infectivity of ZIKV RNA-reactive plasma varied between 100 and 1000 international units (IU) of ZIKV RNA. Immunodeficient mice were more sensitive with as low as 32 IU sufficient to infect 50% of mice. 50-5500 IU of RNA led to TT in macaques using dose escalation of three different RNA-positive, seronegative plasma units. In contrast, RNA-reactive units collected postseroconversion were not infectious in macaques, even at a dose of 9 million IU RNA. After A/UVA PRT, transfusion of plasma containing up to 18 million IU was no longer infectious in vitro and did not result in ZIKV TT in macaques. CONCLUSION: Significant risks of ZIKV TT are likely confined to a relatively short viremic window before seroconversion, and that sensitive nucleic acid amplification testing likely identifies the majority of infectious plasma. PRT was demonstrated to be effective at preventing ZIKV TT. Considering that there is no approved ZIKV vaccine, these data are relevant to mitigate the risk of TT during the future ZIKV outbreaks.

Surveillance for Zika, Chikungunya, and Dengue Virus Incidence and RNAemia in Blood Donors at 4 Brazilian Blood Centers During 2016-2019.

BACKGROUND: Except for public health case reports, the incidence of Zika virus (ZIKV), chikungunya virus (CHIKV), and dengue virus (DENV) infection are not available to assess the potential blood transfusion safety threat in Brazil. METHODS: Pools of 6 donation samples (MP6) left over from human immunodeficiency virus, hepatitis B virus, and hepatitis C virus nucleic acid testing were combined to create MP18 pools (3 MP6 pools). Samples were tested using the Grifols triplex ZIKV, CHIKV, and DENV real-time transcription mediated amplification assay to estimate prevalence of RNAemia and incidence, and to compare these results to case reports in São Paulo, Belo Horizonte, Recife, and Rio de Janeiro, from April 2016 through June 2019. RESULTS: ZIKV, CHIKV, and DENV RNAemia were found from donors who donated without overt symptoms of infection that would have led to deferral. The highest RNAemic donation prevalence was 1.2% (95% CI, .8%-1.9%) for DENV in Belo Horizonte in May 2019. Arbovirus infections varied by location and time of year, and were not always aligned with annual arbovirus outbreak seasons in different regions of the country. CONCLUSIONS: Testing donations for arboviruses in Brazil can contribute to public health. Transfusion recipients were likely exposed to ZIKV, CHIKV, and DENV viremic blood components during the study period.

Blood Center Testing Allows the Detection and Rapid Treatment of Acute and Recent HIV Infection.

Blood donations in South Africa are tested for HIV RNA using individual donation NAT (ID-NAT), allowing detection and rapid antiretroviral therapy (ART) of acute HIV infections. We enrolled a cohort of acute and recent HIV-infected blood donation candidates in South Africa in 2015-2018, measured HIV antibody, ID-NAT, and recency of infection <195 days (Sedia LAg) at enrollment and initiated early ART. A small cohort of HIV elite controllers was followed without treatment. HIV reservoir measurements included ultrasensitive plasma RNA, cell-associated HIV RNA, and total DNA. Enrollment of 18 Fiebig I-III and 45 Fiebig IV-VI HIV clade C subjects occurred a median of 18 days after index blood donation. ART was administered successfully and compliance with follow-up visits was excellent. There were only minimal differences in HIV reservoir between ART initiation in Fiebig stages I-III vs. IV-VI, but ART noncompliance increased HIV reservoir. In 11 untreated HIV elite controllers, HIV reservoir levels were similar to or higher than those seen in our early treated cohort. National blood services can identify acute HIV cohorts for subsequent HIV cure research studies. Among HIV clade C-infected donors, HIV reservoir differed little by Fiebig stage at treatment initiation, but was smaller than in chronically treated HIV and those with ART noncompliance.

A high-throughput screening assay to identify inhibitory antibodies targeting alphavirus release.

BACKGROUND: Several studies have demonstrated neutralizing antibodies to be highly effective against alphavirus infection in animal models, both prophylactically and remedially. In most studies, neutralizing antibodies have been evaluated for their ability to block viral entry in vitro but recent evidence suggests that antibody inhibition through other mechanisms, including viral budding/release, significantly contributes to viral control in vivo for a number of alphaviruses. RESULTS: We describe a BSL-2, cell-based, high-throughput screening system that specifically screens for inhibitors of alphavirus egress using chikungunya virus (CHIKV) and Mayaro virus (MAYV) novel replication competent nano-luciferase (nLuc) reporter viruses. Screening of both polyclonal sera and memory B-cell clones from CHIKV immune individuals using the optimized assay detected several antibodies that display potent anti-budding activity. CONCLUSIONS: We describe an "anti-budding assay" to specifically screen for inhibitors of viral egress using novel CHIKV and MAYV nLuc reporter viruses. This BSL-2 safe, high-throughput system can be utilized to explore neutralizing "anti-budding" antibodies to yield potent candidates for CHIKV and MAYV therapeutics and prophylaxis.

Frequent detection but lack of infectivity of SARS-CoV-2 RNA in presymptomatic, infected blood donor plasma.

Respiratory viruses such as influenza do not typically cause viremia; however, SARS-CoV-2 has been detected in the blood of COVID-19 patients with mild and severe symptoms. Detection of SARS-CoV-2 in blood raises questions about its role in pathogenesis as well as transfusion safety concerns. Blood donor reports of symptoms or a diagnosis of COVID-19 after donation (post-donation information, PDI) preceded or coincided with increased general population COVID-19 mortality. Plasma samples from 2,250 blood donors who reported possible COVID-19-related PDI were tested for the presence of SARS-CoV-2 RNA. Detection of RNAemia peaked at 9%-15% of PDI donors in late 2020 to early 2021 and fell to approximately 4% after implementation of widespread vaccination in the population. RNAemic donors were 1.2- to 1.4-fold more likely to report cough or shortness of breath and 1.8-fold more likely to report change in taste or smell compared with infected donors without detectable RNAemia. No infectious virus was detected in plasma from RNAemic donors; inoculation of permissive cell lines produced less than 0.7-7 plaque-forming units (PFU)/mL and in susceptible mice less than 100 PFU/mL in RNA-positive plasma based on limits of detection in these models. These findings suggest that blood transfusions are highly unlikely to transmit SARS-CoV-2 infection.

The Impact of Early Antiretroviral Treatment (ART) for HIV on the Sensitivity of the Latest Generation of Blood Screening and Point of Care Assays.

INTRODUCTION: Rapid initiation of antiretroviral therapy (ART) in early HIV infection is important to limit seeding of the viral reservoir. A number of studies have shown that if ART is commenced prior to seroconversion, the seroconversion may, or may not, occur. We aimed to assess whether seroreversion or no seroconversion occurs using samples collected during an early treatment study in South Africa. METHODS: We tested 10 longitudinal samples collected over three years from 70 blood donors who initiated ART after detection of acute or early HIV infection during donation screening on fourth- and fifth-generation HIV antibody and RNA assays, and three point of care (POC) rapid tests. Donors were allocated to three treatment groups: (1) very early, (2) early, and (3) later. Longitudinal samples were grouped into time bins post-treatment initiation. RESULTS: On all three high-throughput HIV antibody assays, no clear pattern of declining signal intensity was observed over time after ART initiation in any of the treatment initiation groups and 100% detection was obtained. The Abbott Determine POC assay showed 100% detection at all time points with no seroreversion. However, the Abbott ABON HIV1 and OraSure OraQuick POC assays showed lower proportions of detection in all time bins in the very early treated group, ranging from 50.0% (95% CI: 26.8-73.2%) to 83.1% (95% CI: 64.2-93.0%), and moderate detection rates in the early and later-treated groups. CONCLUSION: While our findings are generally reassuring for HIV detection when high-throughput serological screening assays are used, POC assays may have lower sensitivity for detection of HIV infection after early treatment. Findings are relevant for blood safety and other settings where POC assays are used.

Mitigating the risk of transfusion-transmitted infections with vector-borne agents solely by means of pathogen reduction.

BACKGROUND: This study evaluated whether pathogen reduction technology (PRT) in plasma and platelets using amotosalen/ultraviolet A light (A/UVA) or in red blood cells using amustaline/glutathione (S-303/GSH) may be used as the sole mitigation strategy preventing transfusion-transmitted West Nile (WNV), dengue (DENV), Zika (ZIKV), and chikungunya (CHIKV) viral, and Babesia microti, Trypanosoma cruzi, and Plasmodium parasitic infections. METHODS: Antibody (Ab) status and pathogen loads (copies/mL) were obtained for donations from US blood donors testing nucleic acid (NAT)-positive for WNV, DENV, ZIKV, CHIKV, and B. microti. Infectivity titers derived from pathogen loads were compared to published PRT log10 reduction factors (LRF); LRFs were also reviewed for Plasmodium and T. cruzi. The potential positive impact on donor retention following removal of deferrals from required questioning and testing for WNV, Babesia, Plasmodium, and T. cruzi was estimated for American Red Cross (ARC) donors. RESULTS: A/UVA and S-303/GSH reduced infectivity to levels in accordance with those recognized by FDA as suitable to replace testing for all agents evaluated. If PRT replaced deferrals resulting from health history questions and/or NAT for WNV, Babesia, Plasmodium, and T. cruzi, 27,758 ARC donors could be retained allowing approximately 50,000 additional donations/year based on 1.79 donations/donor for calendar year 2019 (extrapolated to an estimated 125,000 additional donations nationally). CONCLUSION: Pathogen loads in donations from US blood donors demonstrated that robust PRT may provide an opportunity to replace deferrals associated with donor questioning and NAT for vector-borne agents allowing for significant donor retention and likely increased blood availability.

The Recipient Epidemiology and Donor Evaluation Study-IV-Pediatric (REDS-IV-P): A research program striving to improve blood donor safety and optimize transfusion outcomes across the lifespan.

BACKGROUND: The Recipient Epidemiology and Donor Evaluation Study-IV-Pediatric (REDS-IV-P) is a new iteration of prior National Heart, Lung, and Blood Institute (NHLBI) REDS programs that focus on improving transfusion recipient outcomes across the lifespan as well as the safety and availability of the blood supply. STUDY DESIGN AND METHODS: The US program includes blood centers and hospitals (22 including 6 free-standing Children's hospitals) in four geographic regions. The Brazilian program has 5 participating hemocenters. A Center for Transfusion Laboratory Studies (CTLS) and a Data Coordinating Center (DCC) support synergistic studies and activities over the 7-year REDS-IV-P program. RESULTS: The US is building a centralized, vein-to-vein (V2V) database, linking information collected from blood donors, their donations, the resulting manufactured components, and data extracts from hospital electronic medical records of transfused and non-transfused patients. Simultaneously, the Brazilian program is building a donor, donation, and component database. The databases will serve as the backbone for retrospective and prospective observational studies in transfusion epidemiology, transfusion recipient outcomes, blood component quality, and emerging blood safety issues. Special focus will be on preterm infants, patients with sickle cell disease, thalassemia or cancer, and the effect of donor biologic variability and component manufacturing on recipient outcomes. A rapid response capability to emerging safety threats has resulted in timely studies related to Severe Acute Respiratory Syndrome Corona Virus-2 (SARS-CoV-2). CONCLUSIONS: The REDS-IV-P program endeavors to improve donor-recipient-linked research with a focus on children and special populations while also maintaining the flexibility to address emerging blood safety issues.

Minipool testing for SARS-CoV-2 RNA in United States blood donors.

BACKGROUND: SARS-CoV-2 RNA prevalence in blood donors from large geographic areas of high community transmission is limited. We tested residual donor plasma minipools (MPs) to determine SARS-CoV-2 RNAemia prevalence in six United States areas. STUDY DESIGN/METHODS: Blood donations collected from 7 March 2020 to 25 September 2020 were tested for SARS-CoV-2 RNA (vRNA) in MP of 6 or 16 donations using the Grifols Procleix SARS-CoV-2 research-use only (RUO) transcription-mediated amplification (TMA) assay. Reactive results were confirmed using an alternate target region TMA assay. Reactive MPs were tested by TMA after serial dilution to estimate viral load. Testing for anti-SARS-CoV-2 antibodies and infectivity was performed. RESULTS: A total of 17,995 MPs corresponding to approximately 258,000 donations were tested for vRNA. Three confirmed reactive MP16 were identified. The estimated prevalence of vRNA reactive donations was 1.16/100,000 (95% CI 0.40, 3.42). The vRNA-reactive samples were non-reactive for antibody, and the estimated viral loads of the (presumed single) positive donations within each MP ranged from <1000 to <4000 copies/ml. When tested, no infectivity was observed in inoculated permissive cell cultures. DISCUSSION: Blood donation MP-nucleic acid testing (NAT) indicated that SARS-CoV-2 RNAemia is infrequent and, when detected, the vRNA was at low concentrations. Only one RNA-reactive MP could be tested for infectivity for operational reasons and was not infectious in cell culture. These findings support current recommendations from international and national regulatory agencies to not screen donors by NAT.

A highly multiplexed droplet digital PCR assay to measure the intact HIV-1 proviral reservoir.

Quantifying the replication-competent HIV reservoir is essential for evaluating curative strategies. Viral outgrowth assays (VOAs) underestimate the reservoir because they fail to induce all replication-competent proviruses. Single- or double-region HIV DNA assays overestimate it because they fail to exclude many defective proviruses. We designed two triplex droplet digital PCR assays, each with 2 unique targets and 1 in common, and normalize the results to PCR-based T cell counts. Both HIV assays are specific, sensitive, and reproducible. Together, they estimate the number of proviruses containing all five primer-probe regions. Our 5-target results are on average 12.1-fold higher than and correlate with paired quantitative VOA (Spearman's ρ = 0.48) but estimate a markedly smaller reservoir than previous DNA assays. In patients on antiretroviral therapy, decay rates in blood CD4+ T cells are faster for intact than for defective proviruses, and intact provirus frequencies are similar in mucosal and circulating T cells.

Evaluating Differences in Whole Blood, Serum, and Urine Screening Tests for Zika Virus, Puerto Rico, USA, 2016.

We evaluated nucleic acid amplification testing (NAAT) for Zika virus on whole-blood specimens compared with NAAT on serum and urine specimens among asymptomatic pregnant women during the 2015-2016 Puerto Rico Zika outbreak. Using NAAT, more infections were detected in serum and urine than in whole blood specimens.

Impact of Anti-PD-1 and Anti-CTLA-4 on the Human Immunodeficiency Virus (HIV) Reservoir in People Living With HIV With Cancer on Antiretroviral Therapy: The AIDS Malignancy Consortium 095 Study.

BACKGROUND: Antibodies to programmed cell death 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) may perturb human immunodeficiency virus (HIV) persistence during antiretroviral therapy (ART) by reversing HIV latency and/or boosting HIV-specific immunity, leading to clearance of infected cells. We tested this hypothesis in a clinical trial of anti-PD-1 alone or in combination with anti-CTLA-4 in people living with HIV (PLWH) and cancer. METHODS: This was a substudy of the AIDS Malignancy Consortium 095 Study. ART-suppressed PLWH with advanced malignancies were assigned to nivolumab (anti-PD-1) with or without ipilimumab (anti-CTLA-4). In samples obtained preinfusion and 1 and 7 days after the first and fourth doses of immune checkpoint blockade (ICB), we quantified cell-associated unspliced (CA-US) HIV RNA and HIV DNA. Plasma HIV RNA was quantified during the first treatment cycle. Quantitative viral outgrowth assay (QVOA) to estimate the frequency of replication-competent HIV was performed before and after ICB for participants with samples available. RESULTS: Of 40 participants, 33 received nivolumab and 7 nivolumab plus ipilimumab. Whereas CA-US HIV RNA did not change with nivolumab monotherapy, we detected a median 1.44-fold increase (interquartile range, 1.16-1.89) after the first dose of nivolumab and ipilimumab combination therapy (P = .031). There was no decrease in the frequency of cells containing replication-competent HIV, but in the 2 individuals on combination ICB for whom we had longitudinal QVOA, we detected decreases of 97% and 64% compared to baseline. CONCLUSIONS: Anti-PD-1 alone showed no effect on HIV latency or the latent HIV reservoir, but the combination of anti-PD-1 and anti-CTL-4 induced a modest increase in CA-US HIV RNA and may potentially eliminate cells containing replication-competent HIV. CLINICAL TRIALS REGISTRATION: NCT02408861.

Assessing the Suitability of Next-Generation Viral Outgrowth Assays to Measure Human Immunodeficiency Virus 1 Latent Reservoir Size.

BACKGROUND: Evaluations of human immunodeficiency virus (HIV) curative interventions require reliable and efficient quantification of replication-competent latent reservoirs. The "classic" quantitative viral outgrowth assay (QVOA) has been regarded as the reference standard, although prohibitively resource and labor intensive. We compared 6 "next-generation" viral outgrowth assays, using polymerase chain reaction or ultrasensitive p24 to assess their suitability as scalable proxies for QVOA. METHODS: Next-generation QVOAs were compared with classic QVOA using single leukapheresis-derived samples from 5 antiretroviral therapy-suppressed HIV-infected participants and 1 HIV-uninfected control; each laboratory tested blinded batches of 3 frozen and 1 fresh sample. Markov chain Monte Carlo methods estimated extra-Poisson variation at aliquot, batch, and laboratory levels. Models also estimated the effect of testing frozen versus fresh samples. RESULTS: Next-generation QVOAs had similar estimates of variation to QVOA. Assays with ultrasensitive readout reported higher infectious units per million values than classic QVOA. Within-batch testing had 2.5-fold extra-Poisson variation (95% credible interval [CI], 2.1-3.5-fold) for next-generation assays. Between-laboratory variation increased extra-Poisson variation to 3.4-fold (95% CI, 2.6-5.4-fold). Frozen storage did not substantially alter infectious units per million values (-18%; 95% CI, -52% to 39%). CONCLUSIONS: The data offer cautious support for use of next-generation QVOAs as proxies for more laborious QVOA, while providing greater sensitivities and dynamic ranges. Measurement of latent reservoirs in eradication strategies would benefit from high throughput and scalable assays.

Everolimus, an mTORC1/2 inhibitor, in ART-suppressed individuals who received solid organ transplantation: A prospective study.

Pharmacologic inhibition of the mammalian target of rapamycin (mTOR) in the setting of renal transplantation has previously been associated with lower human immunodeficiency virus 1 (HIV-1) DNA burden, and in vitro studies suggest that mTOR inhibition may lead to HIV transcriptional silencing. Because prospective clinical trials are lacking, we conducted an open-label, single-arm study to determine the impact of the broad mTOR inhibitor, everolimus, on residual HIV burden, transcriptional gene expression profiles, and immune responses in HIV-infected adult solid organ transplant (SOT) recipients on antiretroviral therapy. Whereas everolimus therapy did not have an overall effect on cell-associated HIV-1 DNA and RNA levels in the entire cohort, participants who maintained everolimus time-averaged trough levels >5 ng/mL during the first 2 months of therapy had significantly lower RNA levels up to 6 months after the cessation of study drug. Time-averaged everolimus trough levels significantly correlated with greater inhibition of mTOR gene pathway transcriptional activity. Everolimus treatment also led to decreased PD-1 expression on certain T cell subsets. These data support the rationale for further study of the effects of mTOR inhibition on HIV transcriptional silencing in non-SOT populations, either alone or in combination with other strategies. Trial Registration: ClinicalTrials.gov NCT02429869.

Declining antibody levels to Trypanosoma cruzi correlate with polymerase chain reaction positivity and electrocardiographic changes in a retrospective cohort of untreated Brazilian blood donors.

BACKGROUND: Although infection with Trypanosoma cruzi is thought to be lifelong, less than half of those infected develop cardiomyopathy, suggesting greater parasite control or even clearance. Antibody levels appear to correlate with T. cruzi (antigen) load. We test the association between a downwards antibody trajectory, PCR positivity and ECG alterations in untreated individuals with Chagas disease. METHODOLOGY/PRINCIPAL FINDINGS: This is a retrospective cohort of T. cruzi seropositive blood donors. Paired blood samples (index donation and follow-up) were tested using the VITROS Immunodiagnostic Products Anti-T.cruzi (Chagas) assay (Ortho Clinical Diagnostics, Raritan NJ) and PCR performed on the follow-up sample. A 12-lead resting ECG was performed. Significant antibody decline was defined as a reduction of > 1 signal-to-cutoff (S/CO) unit on the VITROS assay. Follow-up S/CO of < 4 was defined as borderline/low. 276 untreated seropositive blood donors were included. The median (IQR) follow-up was 12.7 years (8.5-16.9). 56 (22.1%) subjects had a significant antibody decline and 35 (12.7%) had a low/borderline follow-up result. PCR positivity was lower in the falling (26.8% vs 52.8%, p = 0.001) and low/borderline (17.1% vs 51.9%, p < 0.001) antibody groups, as was the rate of ECG abnormalities. Falling and low/borderline antibody groups were predominantly composed of individuals with negative PCR and normal ECG findings: 64% and 71%, respectively. CONCLUSIONS/SIGNIFICANCE: Low and falling antibody levels define a phenotype of possible spontaneous parasite clearance.

Automated Multireplicate Quantification of Persistent HIV-1 Viremia in Individuals on Antiretroviral Therapy.

Clearance of low-level viremia that persists in most HIV-1-positive individuals on antiretroviral therapy (ART) is an important milestone for efforts to cure HIV-1 infection. The level of persistent viremia on ART is generally below the lower limit of quantification (LOQ) of current FDA-cleared plasma HIV-1 RNA assays (20 to 40 copies/ml) but can be quantified by reverse transcriptase PCR (RT-PCR) assays with single-copy sensitivity. Such assays require multistep manual methods, and their low throughput limits the capacity to monitor the effects of interventions on persistent viremia. Recently, S. Bakkour, X. Deng, P. Bacchetti, E. Grebe, et al. (J Clin Microbiol 58:e01400-20, 2020, https://doi.org/10.1128/JCM.01400-20), reported the use of multiple replicates and Poisson statistics to infer HIV-1 RNA concentrations below the commercial LOQ of an automated platform (Hologic Panther Aptima). Here, we evaluate the detection and quantitation of low-level viremia using the following two adaptions of the automated platform: a multireplicate strategy (9×) and a concentrated single-replicate strategy in which 5 ml of plasma is concentrated by centrifugation (1×, concentrated). We compare these new methods to a recently reported manual integrase-targeting single-copy assay version 2 (iSCA v2). Using laboratory-generated HIV-1 RNA plasma samples at known concentrations, all three methods had similar sensitivity for HIV-1 RNA detection, although iSCA v2 was most sensitive (95% LOD, 2.3 copies/ml), 9× was marginally less sensitive (95% LOD, 3.0 copies/ml), and 1×, concentrated was least sensitive (95% LOD, 3.9 copies/ml). In contrast, for clinical plasma samples, 9× had greater sensitivity than iSCA v2 (82% of samples were quantifiable compared with 62% of samples by iSCA v2). These results support 9× as an acceptable high-throughput alternative to iSCA v2 for quantifying low-level viremia in individuals on ART.

Replicate Aptima Assay for Quantifying Residual Plasma Viremia in Individuals on Antiretroviral Therapy.

Detection of residual plasma viremia in antiretroviral therapy (ART)-suppressed HIV-infected individuals is critical for characterizing the latent reservoir and evaluating the impact of cure interventions. Ultracentrifugation-based single-copy assays are sensitive but labor intensive. Fully automated replicate testing using a standard clinical viral load assay was evaluated as a high-throughput alternative for the quantification of low-level viremia. Four plasma samples from blood donors with acute HIV-1 infection and one viral culture supernatant were serially diluted into 25-ml samples to nominal viral loads ranging from 39 to <0.5 copies (cp)/ml. Each dilution was tested with 45 replicates (reps) using 0.5 ml/rep with the Aptima HIV-1 Quant assay. The nominal and estimated viral loads based on the single-hit Poisson model were compared, and a hybrid Poisson digital model for calibrated viral load estimation was derived. Testing performed using 45 reps on longitudinal plasma samples from 50 ART-suppressed individuals in the Reservoir Assay Validation and Evaluation Network (RAVEN) study cohort (range of 1 to 19 years of continuous ART suppression) showed a median viral load of 0.54 cp/ml (interquartile range [IQR], 0.22 to 1.46 cp/ml) and a 14% (95% confidence interval [CI], 9% to 19%) decline in viral load for each additional year in duration suppressed. Within the RAVEN cohort, the expected false-negative rate for detection at lower rep numbers using 9 and 18 reps was 26% and 14%, respectively. Residual plasma viremia levels positively correlated with cell-associated HIV RNA and DNA. The performance characteristics of the replicate Aptima assay support its use for quantifying residual plasma viremia to study the latent HIV reservoir and cure interventions.