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On university campuses and in similar congregate environments, surveillance testing of asymptomatic persons is a critical strategy (1,2) for preventing transmission of SARS-CoV-2, the virus that causes coronavirus disease 2019 (COVID-19). All students at Duke University, a private research university in Durham, North Carolina, signed the Duke Compact (3), agreeing to observe mandatory masking, social distancing, and participation in entry and surveillance testing. The university implemented a five-to-one pooled testing program for SARS-CoV-2 using a quantitative, in-house, laboratory-developed, real-time reverse transcription-polymerase chain reaction (RT-PCR) test (4,5). Pooling of specimens to enable large-scale testing while minimizing use of reagents was pioneered during the human immunodeficiency virus pandemic (6). A similar methodology was adapted for Duke University's asymptomatic testing program. The baseline SARS-CoV-2 testing plan was to distribute tests geospatially and temporally across on- and off-campus student populations. By September 20, 2020, asymptomatic testing was scaled up to testing targets, which include testing for residential undergraduates twice weekly, off-campus undergraduates one to two times per week, and graduate students approximately once weekly. In addition, in response to newly identified positive test results, testing was focused in locations or within cohorts where data suggested an increased risk for transmission. Scale-up over 4 weeks entailed redeploying staff members to prepare 15 campus testing sites for specimen collection, developing information management tools, and repurposing laboratory automation to establish an asymptomatic surveillance system. During August 2-October 11, 68,913 specimens from 10,265 graduate and undergraduate students were tested. Eighty-four specimens were positive for SARS-CoV-2, and 51% were among persons with no symptoms. Testing as a result of contact tracing identified 27.4% of infections. A combination of risk-reduction strategies and frequent surveillance testing likely contributed to a prolonged period of low transmission on campus. These findings highlight the importance of combined testing and contact tracing strategies beyond symptomatic testing, in association with other preventive measures. Pooled testing balances resource availability with supply-chain disruptions, high throughput with high sensitivity, and rapid turnaround with an acceptable workload.

Background Child with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection typically have mild symptoms that do not require medical attention, leaving a gap in our understanding of the spectrum of SARS-CoV-2-related illnesses that the viruses causes in children. Methods We conducted a prospective cohort study of children and adolescents (aged Results Of 382 children, 293 (77%) were SARS-CoV-2-infected. SARS-CoV-2-infected children were more likely to be Hispanic (P < .0001), less likely to have asthma (P = .005), and more likely to have an infected sibling contact (P = .001) than uninfected children. Children aged 6-13 years were frequently asymptomatic (39%) and had respiratory symptoms less often than younger children (29% vs 48%; P = .01) or adolescents (29% vs 60%; P < .001). Compared with children aged 6-13 years, adolescents more frequently reported influenza-like (61% vs 39%; P < .001) , and gastrointestinal (27% vs 9%; P = .002), and sensory symptoms (42% vs 9%; P < .0001) and had more prolonged illnesses (median [interquartile range] duration: 7 [4-12] vs 4 [3-8] days; P = 0.01). Despite the age-related variability in symptoms, wWe found no difference in nasopharyngeal viral load by age or between symptomatic and asymptomatic children. Conclusions Hispanic ethnicity and an infected sibling close contact are associated with increased SARS-CoV-2 infection risk among children, while asthma is associated with decreased risk. Age-related differences in clinical manifestations of SARS-CoV-2 infection must be considered when evaluating children for coronavirus disease 2019 and in developing screening strategies for schools and childcare settings.

Background: The United Kingdom National External Quality Assessment Service (UK NEQAS) for Leucocyte Immunophenotyping Immune Monitoring Programme, provides external quality assessment (EQA) to non-U.S. laboratories affiliated with the NIH NIAID Division of AIDS (DAIDS) clinical trials networks. Selected laboratories are required to have oversight, performance monitoring and remediation undertaken by Immunology Quality Assessment (IQA) staff under the DAIDS contract. We examined whether laboratory accuracy improves with longer EQA participation and whether IQA remediation is effective. Methods: Laboratory accuracy, defined by the measurement residuals from trial sample medians, was measured on four outcomes: both CD4+ absolute counts (cells/μL) and percentages; and CD8+ absolute counts (cells/μL) and percentages. Three laboratory categories were defined: IQA monitored (n=116), United Kingdom/non-DAIDS (n=137), and non-DAIDS/non-UK (n=1034). For absolute count outcomes, the groups were subdivided into single platform (SPT) and dual platform (DPT) users. Results: Increasing EQA duration was found to be associated with increasing accuracy for all groups in all four lymphocyte subsets (p

Despite the wide availability of antiretroviral drugs, more than 250,000 infants are vertically infected with HIV-1 annually, emphasizing the need for additional interventions to eliminate pediatric HIV-1 infections. Here, we aimed to define humoral immune correlates of risk of mother-to-child transmission (MTCT) of HIV-1, including responses associated with protection in the RV144 vaccine trial. Eighty-three untreated, HIV-1–transmitting mothers and 165 propensity score–matched nontransmitting mothers were selected from the Women and Infants Transmission Study (WITS) of US nonbreastfeeding, HIV-1–infected mothers. In a multivariable logistic regression model, the magnitude of the maternal IgG responses specific for the third variable loop (V3) of the HIV-1 envelope was predictive of a reduced risk of MTCT. Neutralizing Ab responses against easy-to-neutralize (tier 1) HIV-1 strains also predicted a reduced risk of peripartum transmission in secondary analyses. Moreover, recombinant maternal V3–specific IgG mAbs mediated neutralization of autologous HIV-1 isolates. Thus, common V3-specific Ab responses in maternal plasma predicted a reduced risk of MTCT and mediated autologous virus neutralization, suggesting that boosting these maternal Ab responses may further reduce HIV-1 MTCT.

The Center for HIV/AIDS Vaccine Immunology (CHAVI) consortium was established to determine the host and virus factors associated with HIV transmission, infection and containment of virus replication, with the goal of advancing the development of an HIV protective vaccine. Studies to meet this goal required the use of cryopreserved Peripheral Blood Mononuclear Cell (PBMC) specimens, and therefore it was imperative that a quality assurance (QA) oversight program be developed to monitor PBMC samples obtained from study participants at multiple international sites. Nine site-affiliated laboratories in Africa and the USA collected and processed PBMCs, and cryopreserved PBMC were shipped to CHAVI repositories in Africa and the USA for long-term storage. A three-stage program was designed, based on Good Clinical Laboratory Practices (GCLP), to monitor PBMC integrity at each step of this process. The first stage evaluated the integrity of fresh PBMCs for initial viability, overall yield, and processing time at the site-affiliated laboratories (Stage 1); for the second stage, the repositories determined post-thaw viability and cell recovery of cryopreserved PBMC, received from the site-affiliated laboratories (Stage 2); the third stage assessed the long-term specimen storage at each repository (Stage 3). Overall, the CHAVI PBMC QA oversight program results highlight the relative importance of each of these stages to the ultimate goal of preserving specimen integrity from peripheral blood collection to long-term repository storage.

Background: The aim of clinical laboratories is to produce accurate and reproducible results to enable effective and reliable clinical practice and patient management. The standard approach is to use both internal quality control (IQC) and external quality assessment (EQA). IQC serves, in many instances, as a “go, no go” tool to provide real time assurance that instruments and reagent or test systems are performing within defined specifications. EQA however, takes a snapshot at a specific point in time of the full testing process, results are compared to other laboratories performing similar testing but inevitably has some built in delay from sample issue to performance data review. In addition, if IQC or EQA identify areas of concern it can be difficult to determine the exact nature of the problem. In an attempt to address this problem, we have developed an instant QA panel that we have termed VERIQAS™, specifically for CD4+ T lymphocyte counting, and have undertaken a “proof of principle” pilot study to examine how the use of VERIQAS™ could result in improvement of laboratory performance. In addition, we have examined how this approach could be used as a training and education tool (in a domestic/international setting) and potentially be of value in instrument validation/switch studies (a switch study being defined as a laboratory changing from one method/instrument to a new method/instrument with the VERIQAS™ panel being used as an adjunct to their standard switch study protocol). Methods: The basic panel consists of 20 stabilized samples, with predefined CD4+ T lymphocyte counts, that span low clinically relevant to normal counts, including some blinded replicates (singlet up to quadruplicate combinations). The CD4+ T lymphocyte target values for each specimen is defined as the trimmed mean ± 2 trimmed standard deviations, where the trimmed values are derived from the CD4+ T lymphocyte counts reported by the participating centers (∼780 laboratories) that receive each UK NEQAS for Leucocyte Immunophenotyping send out. Results for the VERIQAS™ panel were returned online, via a specially designed website, and the participant was provided with an immediate assessment (pass or fail). Results: To date, the panel has been preliminary trialed by eight laboratories to (i) assess pre-EQA qualification (two laboratories); (ii) address performance issues (two laboratories); or (iii) validate new instruments or techniques (four laboratories). Interestingly, even in this pilot study, the panel has been instrumental in identifying specific technical problems in laboratories with EQA performance issues as well as confirming that implementation of new techniques or instruments have been successful. Conclusion: We report here a new and novel “proof of principle” pilot study to quality assessment, that we have termed VERIQAS™, designed to provide instant feedback on performance. Participating laboratories receive 20 “blinded” samples that are in singlet up to quadruplicate combinations. Once a centre reports its results via a website, immediate feedback is provided to both the participant and the EQA organizers, enabling, if required, the initiation of targeted remedial action. We have also shown that this approach has the potential to be used as a tool for prequalification, troubleshooting, training and instrument verification. Pilot phase field trials with VERIQAS™ have shown that the panel can highlight laboratory performance problems, such as suboptimal instrument set up, pipetting and gating strategies, in a rapid and efficient manner. VERIQAS™ will now be introduced, where appropriate, as a second phase study within UK NEQAS for Leucocyte Immunophenotyping to assist those laboratories that have performance issues and also made available to laboratories for training and education of staff and instrument validation studies. © 2011 International Clinical Cytometry Society.

The goals of this study were to optimize processing methods of cryopreserved peripheral blood mononuclear cells (PBMC) for immunological assays, identify acceptance parameters for the use of cryopreserved PBMC for functional and phenotypic assays, and to define limitations of the information obtainable with cryopreserved PBMC. Blood samples from 104 volunteers (49 human immunodeficiency virus-infected and 55 uninfected) were used to assess lymphocyte proliferation in response to tetanus, candida, and pokeweed-mitogen stimulation and to enumerate CD4 + and CD8 + T cells and T-cell subpopulations by flow cytometry. We determined that slowly diluting the thawed PBMC significantly improved viable cell recovery, whereas the use of benzonase improved cell recovery only sometimes. Cell storage in liquid nitrogen for up to 15 months did not affect cell viability, recovery, or the results of lymphocyte proliferation assays (LPA) and flow cytometry assays. Storage at −70°C for ≤3 weeks versus storage in liquid nitrogen before shipment on dry ice did not affect cell viability, recovery, or flow cytometric results. Storage at −70°C was associated with slightly higher LPA results with pokeweed-mitogen but not with microbial antigens. Cell viability of 75% was the acceptance parameter for LPA. No other acceptance parameters were found for LPA or flow cytometry assay results for cryopreserved PBMC. Under optimized conditions, LPA and flow cytometry assay results for cryopreserved and fresh PBMC were highly correlated, with the exception of phenotypic assays that used CD45RO or CD62L markers, which seemed labile to freezing and thawing.

Background The global HIV/AIDS pandemic and guidelines for initiating anti-retroviral therapy (ART) and opportunistic infection prophylaxis demand affordable, reliable, and accurate CD4 testing. A simple innovative approach applicable to existing technology that has been successfully applied in resource-challenged settings, PanLeukogated CD4 (PLG), could offer solutions for cost saving and improved precision. Methods Day-old whole blood from 99 HIV+ donors was simultaneously studied in five North-American laboratories to compare the performance of their predicate methods with the dual-platform PLG method. The predicate technology included varying 4-color CD45/CD3/CD4/CD8 protocols on different flow cytometers. Each laboratory also assayed eight replicate specimens of day-old blood from 10 to 14 local donors. Bias and precision of predicate and PLG methods was studied between- and within-participating laboratories. Results Significantly (P < 0.0001) improved between-laboratory precision/coefficient of variation (CV%) was noted using the PLG method (overall median 9.3% vs. predicate median CV 13.1%). Within-laboratory precision was also significantly (P < 0.0001) better overall using PLG (median 4.6% vs. predicate median CV 6.2%) and in 3 of the 5 laboratories. PLG counts tended to be 11% smaller than predicate methods (P < 0.0001) for shipped (median of predicate—PLG = 31) and local specimens (median of predicate—PLG = 23), both overall and in 4 of 5 laboratories (median decreases of 4, 16, 20, and 21% in shipped specimens); the other laboratory had a median increase of 5%. Conclusion Laboratories using predicate CD4 methods similar to those in this study could improve their between-laboratory and their within-laboratory precision, and reduce costs, by switching to the PLG method after adequate training, if a change (usually, a decrease) in CD4 counts is acceptable to their health systems. © 2008 Clinical Cytometry Society

Since 1999, the National Institute of Allergy and Infectious Diseases Division of AIDS (NIAID DAIDS) has funded the Immunology Quality Assessment (IQA) Program with the goal of assessing proficiency in basic lymphocyte subset immunophenotyping for each North American laboratory supporting the NIAID DAIDS HIV clinical trial networks. Further, the purpose of this program is to facilitate an increase in the consistency of interlaboratory T-cell subset measurement (CD3(+)4(+)/CD3(+)8(+) percentages and absolute counts) and likewise, a decrease in intralaboratory variability. IQA T-cell subset measurement proficiency testing was performed over a ten-year period (January 2003-July 2012), and the results were analyzed via longitudinal analysis using mixed effects models. The goal of this analysis was to describe how a typical laboratory (a statistical modeling construct) participating in the IQA Program performed over time. Specifically, these models were utilized to examine trends in interlaboratory agreement, as well as successful passing of proficiency testing. Intralaboratory variability (i.e., precision) was determined by the repeated measures variance, while fixed and random effects were taken into account for changes in interlaboratory agreement (i.e., accuracy) over time. A flow cytometer (single-platform technology, SPT) or a flow cytometer/hematology analyzer (dual-platform technology, DPT) was also examined as a factor for accuracy and precision. The principal finding of this analysis was a significant (p0.001) increase in accuracy of T-cell subset measurements over time, regardless of technology type (SPT or DPT). Greater precision was found in SPT measurements of all T-cell subset measurements (p0.001), as well as greater accuracy of SPT on CD3(+)4(+)% and CD3(+)8(+)% assessments (p0.05 and p0.001, respectively). However, the interlaboratory random effects variance in DPT results indicates that for some cases DPT can have increased accuracy compared to SPT. Overall, these findings demonstrate that proficiency in and among IQA laboratories have, in general, improved over time and that platform type differences in performance do exist.

Functional immunologic assays using cryopreserved peripheral blood mononuclear cells (PBMC) are influenced by blood processing, storage and shipment. The objective of this study was to compare the viability, recovery and ELISPOT results of PBMC stored and shipped in liquid nitrogen (LN/LN) or stored in LN and shipped on dry ice (LN/DI) or stored at -70°C for 3 to 12 weeks and shipped on DI (70/DI 3 to 12); and to assess the effect of donor HIV infection status on the interaction between storage/shipment and the outcome measures. PBMC from 12 HIV-infected and 12 uninfected donors showed that LN/LN conferred higher viability and recovery than LN/DI or 70/DI 3, 6, 9 or 12. LN/DI PBMC had higher viability than any 70/DI PBMC. The PBMC viability and recovery linearly decreased with the duration of storage at -70°C from 3 to 12 weeks. This effect was more pronounced in samples from HIV-infected than uninfected donors. Results of ELISPOT assays using CMV pp65, CEF and Candida albicans antigens were qualitatively and quantitatively similar across LN/LN, LN/DI and 70/DI 3. However, ELISPOT values significantly decreased with the duration of storage at -70°C both in HIV-infected and uninfected donors. ELISPOT results also decreased with PBMC viability

TransFix(TM) and Cyto-Chex((R)) BCT (blood collection tube) reagents have been shown to maintain whole blood integrity for delayed immunophenotyping by flow cytometry. We evaluated the ability of these blood-stabilizing reagents to preserve HIV-seropositive blood for delayed CD4(+) T-cell quantification utilizing the Dynal((R)) Biotech T4 Quant Kit. TransFix was added to EDTA-anticoagulated whole blood and tested at a 1:10 dilution over 7 d using the Dynal (n = 21) manual method. Compared to baseline analysis, a significant decrease in mean CD4(+) counts was observed over time. Cyto-Chex BCT-preserved samples (n = 20) were tested for CD4(+) counts by Dynal over 7 d, with storage at varying temperatures: room temperature (21 degrees C), 37 degrees C, and 37 degrees C with intermittent storage at 42 degrees C. A significant decline in mean CD4(+) counts was observed in samples at all temperatures compared to baseline (p < 0.05). Increases in temperature to and above 37 degrees C resulted in a greater decline in mean CD4(+) counts over time. Our findings indicated that neither TransFix or Cyto-Chex BCT was a suitable blood stabilizer when used for delayed CD4(+) quantification with a low-cost manual CD4(+) bead-based method.

Seven Brazilian sites participating in the Pediatric AIDS Clinical Trials Group international cryopreservation quality assurance pilot program cryopreserved and shipped peripheral blood mononuclear cells (PBMC) to a central U.S. laboratory for analysis. Cell viability and recovery significantly increased over time. A wet-laboratory training session conducted at the central laboratory significantly improved the quality of the cryopreserved PBMC.