MultiSero: An Open-Source Multiplex-ELISA Platform for Measuring Antibody Responses to Infection.

A multiplexed enzyme-linked immunosorbent assay (ELISA) that simultaneously measures antibody binding to multiple antigens can extend the impact of serosurveillance studies, particularly if the assay approaches the simplicity, robustness, and accuracy of a conventional single-antigen ELISA. Here, we report on the development of multiSero, an open-source multiplex ELISA platform for measuring antibody responses to viral infection. Our assay consists of three parts: (1) an ELISA against an array of proteins in a 96-well format; (2) automated imaging of each well of the ELISA array using an open-source plate reader; and (3) automated measurement of optical densities for each protein within the array using an open-source analysis pipeline. We validated the platform by comparing antibody binding to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) antigens in 217 human sera samples, showing high sensitivity (0.978), specificity (0.977), positive predictive value (0.978), and negative predictive value (0.977) for classifying seropositivity, a high correlation of multiSero determined antibody titers with commercially available SARS-CoV-2 antibody tests, and antigen-specific changes in antibody titer dynamics upon vaccination. The open-source format and accessibility of our multiSero platform can contribute to the adoption of multiplexed ELISA arrays for serosurveillance studies, for SARS-CoV-2 and other pathogens of significance.

Workshop-based learning and networking: a scalable model for research capacity strengthening in low- and middle-income countries.

Science education and research have the potential to drive profound change in low- and middle-income countries (LMICs) through encouraging innovation, attracting industry, and creating job opportunities. However, in LMICs, research capacity is often limited, and acquisition of funding and access to state-of-the-art technologies is challenging. The Alliance for Global Health and Science (the Alliance) was founded as a partnership between the University of California, Berkeley (USA) and Makerere University (Uganda), with the goal of strengthening Makerere University's capacity for bioscience research. The flagship program of the Alliance partnership is the MU/UCB Biosciences Training Program, an in-country, hands-on workshop model that trains a large number of students from Makerere University in infectious disease and molecular biology research. This approach nucleates training of larger and more diverse groups of students, development of mentoring and bi-directional research partnerships, and support of the local economy. Here, we describe the project, its conception, implementation, challenges, and outcomes of bioscience research workshops. We aim to provide a blueprint for workshop implementation, and create a valuable resource for bioscience research capacity strengthening in LMICs.

SARS-CoV-2 PCR and antibody testing for an entire rural community: methods and feasibility of high-throughput testing procedures.

BACKGROUND: Early in the pandemic, inadequate SARS-CoV-2 testing limited understanding of transmission. Chief among barriers to large-scale testing was unknown feasibility, particularly in non-urban areas. Our objective was to report methods of high-volume, comprehensive SARS-CoV-2 testing, offering one model to augment disease surveillance in a rural community. METHODS: A community-university partnership created an operational site used to test most residents of Bolinas, California regardless of symptoms in 4 days (April 20th - April 23rd, 2020). Prior to testing, key preparatory elements included community mobilization, pre-registration, volunteer recruitment, and data management. On day of testing, participants were directed to a testing lane after site entry. An administrator viewed the lane-specific queue and pre-prepared test kits, linked to participants' records. Medical personnel performed sample collection, which included finger prick with blood collection to run laboratory-based antibody testing and respiratory specimen collection for polymerase chain reaction (PCR). RESULTS: Using this 4-lane model, 1,840 participants were tested in 4 days. A median of 57 participants (IQR 47-67) were tested hourly. The fewest participants were tested on day 1 (n = 338 participants), an intentionally lower volume day, increasing to n = 571 participants on day 4. The number of testing teams was also increased to two per lane to allow simultaneous testing of multiple participants on days 2-4. Consistent staffing on all days helped optimize proficiency, and strong community partnership was essential from planning through execution. CONCLUSIONS: High-volume ascertainment of SARS-CoV-2 prevalence by PCR and antibody testing was feasible when conducted in a community-led, drive-through model in a non-urban area.

Universal Polymerase Chain Reaction and Antibody Testing Demonstrate Little to No Transmission of Severe Acute Respiratory Syndrome Coronavirus 2 in a Rural Community.

BACKGROUND: Limited systematic surveillance for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the early months of the US epidemic curtailed accurate appraisal of transmission intensity. Our objective was to perform case detection of an entire rural community to quantify SARS-CoV-2 transmission using polymerase chain reaction (PCR) and antibody testing. METHODS: We conducted a cross-sectional survey of SARS-CoV-2 infection in the rural town of Bolinas, California (population 1620), 4 weeks after shelter-in-place orders. Participants were tested between April 20 and 24, 2020. Prevalence by PCR and seroprevalence from 2 forms of antibody testing were performed in parallel (Abbott ARCHITECT immunoglobulin [Ig]G and in-house IgG enzyme-linked immunosorbent assay). RESULTS: Of 1891 participants, 1312 were confirmed Bolinas residents (>80% community ascertainment). Zero participants were PCR positive. Assuming 80% sensitivity, it would have been unlikely to observe these results (P < .05) if there were >3 active infections in the community. Based on antibody results, estimated prevalence of prior infection was 0.16% (95% credible interval [CrI], 0.02%-0.46%). The positive predictive value (PPV) of a positive result on both tests was 99.11% (95% CrI, 95.75%-99.94%), compared with PPV 44.19%-63.32% (95% CrI, 3.25%-98.64%) if 1 test was utilized. CONCLUSIONS: Four weeks after shelter-in-place, SARS-CoV-2 infection in a rural Northern California community was extremely rare. In this low-prevalence setting, use of 2 antibody tests increased seroprevalence estimate precision. This was one of the first community-wide studies to successfully implement synchronous PCR and antibody testing, particularly in a rural setting. Widespread testing remains an underpinning of effective disease control in conjunction with consistent uptake of public health measures.

Citywide serosurveillance of the initial SARS-CoV-2 outbreak in San Francisco using electronic health records.

Serosurveillance provides a unique opportunity to quantify the proportion of the population that has been exposed to pathogens. Here, we developed and piloted Serosurveillance for Continuous, ActionabLe Epidemiologic Intelligence of Transmission (SCALE-IT), a platform through which we systematically tested remnant samples from routine blood draws in two major hospital networks in San Francisco for SARS-CoV-2 antibodies during the early months of the pandemic. Importantly, SCALE-IT allows for algorithmic sample selection and rich data on covariates by leveraging electronic health record data. We estimated overall seroprevalence at 4.2%, corresponding to a case ascertainment rate of only 4.9%, and identified important heterogeneities by neighborhood, homelessness status, and race/ethnicity. Neighborhood seroprevalence estimates from SCALE-IT were comparable to local community-based surveys, while providing results encompassing the entire city that have been previously unavailable. Leveraging this hybrid serosurveillance approach has strong potential for application beyond this local context and for diseases other than SARS-CoV-2.

multiSero: open multiplex-ELISA platform for analyzing antibody responses to SARS-CoV-2 infection.

Serology has provided valuable diagnostic and epidemiological data on antibody responses to SARS-CoV-2 in diverse patient cohorts. Deployment of high content, multiplex serology platforms across the world, including in low and medium income countries, can accelerate longitudinal epidemiological surveys. Here we report multiSero, an open platform to enable multiplex serology with up to 48 antigens in a 96-well format. The platform consists of three components: ELISA-array of printed proteins, a commercial or home-built plate reader, and modular python software for automated analysis (pysero). We validate the platform by comparing antibody titers against the SARS-CoV-2 Spike, receptor binding domain (RBD), and nucleocapsid (N) in 114 sera from COVID-19 positive individuals and 87 pre-pandemic COVID-19 negative sera. We report data with both a commercial plate reader and an inexpensive, open plate reader (nautilus). Receiver operating characteristic (ROC) analysis of classification with single antigens shows that Spike and RBD classify positive and negative sera with the highest sensitivity at a given specificity. The platform distinguished positive sera from negative sera when the reactivity of the sera was equivalent to the binding of 1 ng mL âˆ'1 RBD-specific monoclonal antibody. We developed normalization and classification methods to pool antibody responses from multiple antigens and multiple experiments. Our results demonstrate a performant and accessible pipeline for multiplexed ELISA ready for multiple applications, including serosurveillance, identification of viral proteins that elicit antibody responses, differential diagnosis of circulating pathogens, and immune responses to vaccines.

Citywide serosurveillance of the initial SARS-CoV-2 outbreak in San Francisco.

Serosurveillance provides a unique opportunity to quantify the proportion of the population that has been exposed to pathogens. Here, we developed and piloted Serosurveillance for Continuous, ActionabLe Epidemiologic Intelligence of Transmission (SCALE-IT), a platform through which we systematically tested remnant samples from routine blood draws in two major hospital networks in San Francisco for SARS-CoV-2 antibodies during the early months of the pandemic. Importantly, SCALE-IT allows for algorithmic sample selection and rich data on covariates by leveraging electronic medical record data. We estimated overall seroprevalence at 4.2%, corresponding to a case ascertainment rate of only 4.9%, and identified important heterogeneities by neighborhood, homelessness status, and race/ethnicity. Neighborhood seroprevalence estimates from SCALE-IT were comparable to local community-based surveys, while providing results encompassing the entire city that have been previously unavailable. Leveraging this hybrid serosurveillance approach has strong potential for application beyond this local context and for diseases other than SARS-CoV-2.

Universal PCR and antibody testing demonstrate little to no transmission of SARS-CoV-2 in a rural community.

BACKGROUND: The absence of systematic surveillance for SARS-CoV-2 has curtailed accurate appraisal of transmission intensity. Our objective was to perform case detection of an entire rural community to quantify SARS-CoV-2 transmission using PCR and antibody testing. METHODS: We conducted a cross-sectional survey of the prevalence and cumulative incidence of SARS-CoV-2 infection in the rural town of Bolinas, California (population 1,620), four weeks following shelter-in-place orders. Residents and county essential workers were tested between April 20th-24th, 2020. Prevalence by PCR and seroprevalence combining data from two forms of antibody testing were performed in parallel (Abbott ARCHITECT IgG to nucleocapsid protein and in-house IgG ELISA to the receptor binding domain). RESULTS: Of 1,891 participants, 1,312 were confirmed Bolinas residents (>80% community ascertainment). Zero participants were PCR positive. Assuming 80% sensitivity, it would have been unlikely to observe these results (p<0.05) if there were >3 active infections in the community. Based on antibody results, estimated prevalence of prior infection was 0.16% (95% CrI: 0.02%, 0.46%). Seroprevalence estimates using only one of the two tests would have been higher, with greater uncertainty. The positive predictive value (PPV) of a positive result on both tests was 99.11% (95% CrI: 95.75%, 99.94%), compared to PPV 44.19%-63.32% (95% CrI range 3.25%-98.64%) if only one test was utilized. CONCLUSIONS: Four weeks following shelter-in-place, active and prior SARS-CoV-2 infection in a rural Northern California community was extremely rare. In this low prevalence setting, use of two antibody tests increased the PPV and precision of seroprevalence estimates.

SARS-CoV-2 PCR and antibody testing for an entire rural community: methods and feasibility of high-throughput testing procedures.

High-volume, community-wide ascertainment of SARS-CoV-2 prevalence by PCR and antibody testing was successfully performed using a community-led, drive-through model with strong operational support, well-trained testing units, and an effective technical platform.

Comparative Efficacy of Artemether-Lumefantrine and Dihydroartemisinin-Piperaquine for the Treatment of Uncomplicated Malaria in Ugandan Children.

BACKGROUND: In Uganda, artemether-lumefantrine (AL) and dihydroartemisinin-piperaquine (DHA-PQ) showed excellent treatment efficacy for uncomplicated malaria in prior trials. Because the frequency of resistance to artemisinins and piperaquine is increasing in Southeast Asia and the prevalence of Plasmodium falciparum polymorphisms associated with resistance has changed, we reassessed treatment efficacies at 3 sites in Uganda. METHODS: For this randomized, single-blinded clinical trial, children aged 6-59 months with uncomplicated falciparum malaria were assigned treatment with AL or DHA-PQ and followed for 42 days. Primary end points were risks of recurrent parasitemia, either unadjusted or adjusted to distinguish recrudescence from new infection. We assessed selection by study regimens of relevant P. falciparum genetic polymorphisms associated with drug resistance. RESULTS: Of 599 patients enrolled, 578 completed follow-up. There were no early treatment failures. The risk of recurrent parasitemia was lower with DHA-PQ as compared to AL at all 3 sites at 42 days (26.0% vs 47.0%; P < .001). Recrudescent infections were uncommon in both the DHA-PQ and AL arms (1.1% and 2.2%, respectively; P = .25). Neither regimen selected for pfcrt or pfmdr1 polymorphisms associated with drug resistance. CONCLUSIONS: AL and DHA-PQ remain effective for the treatment of malaria in Uganda. Neither regimen selected for genetic polymorphisms associated with drug resistance. CLINICAL TRIALS REGISTRATION: ISRCTN15793046.

Changing Molecular Markers of Antimalarial Drug Sensitivity across Uganda.

The potential spread of antimalarial drug resistance to Africa, in particular for artemisinins and key partner drugs, is a major concern. We surveyed Plasmodium falciparum genetic markers associated with drug sensitivity on 3 occasions at ∼6-month intervals in 2016 and 2017 at 10 sites representing a range of epidemiological settings in Uganda. For putative drug transporters, we found continued evolution toward wild-type sequences associated with increased sensitivity to chloroquine. For pfcrt K76T, by 2017 the prevalence of the wild type was >60% at all sites and >90% at 6 sites. For the pfmdr1 N86Y and D1246Y alleles, wild type prevalence ranged from 80 to 100%. We found low prevalence of K13 propeller domain mutations, which are associated with artemisinin resistance in Asia, but one mutation previously identified in northern Uganda, 675V, was seen in 2.0% of samples, including 5.5% of those from the 3 northernmost sites. Amplification of the pfmdr1 and plasmepsin2 genes, associated elsewhere with decreased sensitivity to lumefantrine and piperaquine, respectively, was seen in <1% of samples. For the antifolate targets pfdhfr and pfdhps, 5 mutations previously associated with resistance were very common, and the pfdhfr 164L and pfdhps 581G mutations associated with higher-level resistance were seen at multiple sites, although prevalence did not clearly increase over time. Overall, changes were consistent with the selective pressure of the national treatment regimen, artemether-lumefantrine, with increased sensitivity to chloroquine, and with poor efficacy of antifolates. Strong evidence for resistance to artemisinins was not seen. Continued surveillance of markers that predict antimalarial drug sensitivity is warranted.

Azo···phenyl stacking: a persistent self-assembly motif guides the assembly of fluorinated cis-azobenzenes into photo-mechanical needle crystals.

We describe a novel, persistent motif of molecular assembly in photo-mechanical crystals and cocrystals of fluorinated cis-azobenzenes. The azo···phenyl stacking, preserved upon either chemical substitution or halogen-bonded cocrystallization, guides the assembly of fluorinated cis-azobenzenes into columnar stacks and drives the formation of crystals with needle-like morphologies optimal for photo-mechanical motion.