Large-volume RT-LAMP enables extraction-free amplification of HIV RNA from fingerstick plasma.

BACKGROUND: Point-of-care (POC) nucleic acid amplification tests (NAAT) can significantly expand testing coverage, which is critical for infectious disease diagnostics and monitoring. The development of various isothermal amplification techniques greatly simplifies NAATs, but the cumbersome nucleic acid extraction step remains a bottleneck for the POC. Alternatively, extraction-free amplification, where crude samples are directly added into the assay, substantially simplifies the workflow. However, sample dilution is often needed in extraction-free amplification to reduce assay inhibition from sample matrices. Since NAATs are typically run at small volumes around 20 µL, the input sample quantity is therefore limited, resulting in an inevitable sensitivity loss. RESULTS: Here we explore the potential to perform isothermal amplification in larger reaction volumes to accommodate larger sample quantities, thereby improving sensitivity in extraction-free amplification. We demonstrated the approach by developing large-volume reverse transcription loop-mediated isothermal amplification (RT-LAMP) for HIV RNA detection from fingerstick plasma. We found that LAMP at reaction volumes up to 1 mL maintained the same performance. We then identified plasma dilution conditions needed to maintain the limit of detection in RT-LAMP. Subsequently, using inactivated HIV virus, we showed the successful detection of 24 HIV RNA copies in a 500 µL RT-LAMP reaction in the presence of 20 µL plasma (fingerstick volumes), translating to a viral load of 1200 copies per mL. To reduce the increased reagent cost with expanded reaction volumes, we further identified lower-cost reagents with maintained assay performance. Moreover, we showed that large-volume LAMP, compared to 20 µL reactions, could tolerate higher concentrations of various inhibitors in the sample, such as albumin and GuSCN. SIGNIFICANCE AND NOVELTY: NAATs are conventionally conducted at small reaction volumes. Here we demonstrated that LAMP can be run at large reaction volumes (over 100 µL) with maintained assay performance, allowing sample inhibition to be mitigated while accommodating larger sample quantities. The same strategy of expanding reaction volumes could be applied to other isothermal amplification methods and various POC applications, to streamline test workflows and/or improve assay sensitivity.

Sensitive Pathogen Detection and Drug Resistance Characterization Using Pathogen-Derived Enzyme Activity Amplified by LAMP or CRISPR-Cas.

Pathogens encapsulate or encode their own suite of enzymes to facilitate replication in the host. The pathogen-derived enzymes possess specialized activities that are essential for pathogen replication and have naturally been candidates for drug targets. Phenotypic assays detecting the activities of pathogen-derived enzymes and characterizing their inhibition under drugs offer an opportunity for pathogen detection, drug resistance testing for individual patients, and as a research tool for new drug development. Here, we used HIV as an example to develop assays targeting the reverse transcriptase (RT) enzyme encapsulated in HIV for sensitive detection and phenotypic characterization, with the potential for point-of-care (POC) applications. Specifically, we targeted the complementary (cDNA) generation activity of the HIV RT enzyme by adding engineered RNA as substrates for HIV RT enzyme to generate cDNA products, followed by cDNA amplification and detection facilitated by loop-mediated isothermal amplification (LAMP) or CRISPR-Cas systems. To guide the assay design, we first used qPCR to characterize the cDNA generation activity of HIV RT enzyme. In the LAMP-mediated Product-Amplified RT activity assay (LamPART), the cDNA generation and LAMP amplification were combined into one pot with novel assay designs. When coupled with direct immunocapture of HIV RT enzyme for sample preparation and endpoint lateral flow assays for detection, LamPART detected as few as 20 copies of HIV RT enzyme spiked into 25µL plasma (fingerstick volume), equivalent to a single virion. In the Cas-mediated Product-Amplified RT activity assay (CasPART), we tailored the substrate design to achieve a LoD of 2e4 copies (1.67fM) of HIV RT enzyme. Furthermore, with its phenotypic characterization capability, CasPART was used to characterize the inhibition of HIV RT enzyme under antiretroviral drugs and differentiate between wild-type and mutant HIV RT enzyme for potential phenotypic drug resistance testing. Moreover, the CasPART assay can be readily adapted to target the activity of other pathogen-derived enzymes. As a proof-of-concept, we successfully adapted CasPART to detect HIV integrase with a sensitivity of 83nM. We anticipate the developed approach of detecting enzyme activity with product amplification has the potential for a wide range of pathogen detection and phenotypic characterization.

Biomarker-confirmed suboptimal adherence to isoniazid preventive therapy among children with HIV in western Kenya.

OBJECTIVES: The aim of this study was to assess the level and correlates of biomarker-confirmed adherence to isoniazid (INH) preventive therapy (IPT) among children with HIV (CLHIV). DESIGN: This prospective cohort study assessed adherence among CLHIV on IPT in public sector HIV clinics from 2019 through 2020. METHODS: Adherence was assessed by pill counts or caregiver or self-reports, and urine biomarkers (in-house dipstick and Isoscreen). Both urine biomarker tests detect INH metabolites within 48 h of ingestion. Consistent adherence was defined as having positive results on either biomarker at all visits. Correlates of biomarker-confirmed nonadherence at each visit were evaluated using generalized estimating equations. The in-house dipstick was validated using Isoscreen as the reference. RESULTS: Among 97 CLHIV on IPT with adherence assessments, median age was 10 years (IQR 7-13). All were on ART at IPT initiation (median duration 46 months [IQR 4-89]); 81% were virally suppressed (<1000 copies/ml). At all visits, 59% ( n  = 57) of CLHIV reported taking at least 80% of their doses, while 39% ( n  = 38) had biomarker-confirmed adherence. Viral nonsuppression (adjusted risk ratio [aRR] = 1.65; 95% confidence interval [95% CI] 1.09-2.49) and the sixth month of IPT use (aRR = 2.49; 95% CI 1.34-4.65) were independent correlates of biomarker-confirmed nonadherence at each visit. Sensitivity and specificity of the in-house dipstick were 98.1% ( 94.7 - 99.6%) and 94.7% ( 88.1 - 98.3%) , respectively, versus Isoscreen. CONCLUSION: Biomarker-confirmed adherence to IPT was sub-optimal and was associated with viral nonsuppression and duration of IPT. Urine dipstick testing may be useful in assessing adherence to IPT in clinical care.

A simple agglutination system for rapid antigen detection from large sample volumes with enhanced sensitivity.

Lateral flow assays (LFAs) provide a simple and quick option for diagnosis and are widely adopted for point-of-care or at-home tests. However, their sensitivity is often limited. Most LFAs only allow 50 µL samples while various sample types such as saliva could be collected in much larger volumes. Adapting LFAs to accommodate larger sample volumes can improve assay sensitivity by increasing the number of target analytes available for detection. Here, a simple agglutination system comprising biotinylated antibody (Ab) and streptavidin (SA) is presented. The Ab and SA agglutinate into large aggregates due to multiple biotins per Ab and multiple biotin binding sites per SA. Dynamic light scattering (DLS) measurements showed that the agglutinated aggregate could reach a diameter of over 0.5 µm and over 1.5 µm using poly-SA. Through both experiments and Monte Carlo modeling, we found that high valency and equivalent concentrations of the two aggregating components were critical for successful agglutination. The simple agglutination system enables antigen capture from large sample volumes with biotinylated Ab and a swift transition into aggregates that can be collected via filtration. Combining the agglutination system with conventional immunoassays, an agglutination assay is proposed that enables antigen detection from large sample volumes using an in-house 3D-printed device. As a proof-of-concept, we developed an agglutination assay targeting SARS-CoV-2 nucleocapsid antigen for COVID-19 diagnosis from saliva. The assay showed a 10-fold sensitivity enhancement when increasing sample volume from 50 µL to 2 mL, with a final limit of detection (LoD) of 10 pg mL-1 (∼250 fM). The assay was further validated in negative saliva spiked with gamma-irradiated SARS-CoV-2 and showed an LoD of 250 genome copies per µL. The proposed agglutination assay can be easily developed from existing LFAs to facilitate the processing of large sample volumes for improved sensitivity.

Patient-centered mobile tuberculosis treatment support tools (TB-TSTs) to improve treatment adherence: A pilot randomized controlled trial exploring feasibility, acceptability and refinement needs.

Background: Digital adherence technologies hold promise to improve patient-centered tuberculosis (TB) monitoring, yet few studies have incorporated direct adherence monitoring or assessed patients' experiences with these technologies. We explored acceptability, feasibility, and refinement needs of the TB Treatment Support Tools (TB-TSTs) intervention linking a mobile app, a urine drug metabolite test, and interactive communication with a treatment supporter. Methods: This pilot study was a parallel-designed single-center randomized controlled trial with exit interviews. Newly diagnosed TB patients were randomized 1:1 using a treatment allocation button in the REDCap software preloaded with a random allocation sequence to usual care or usual care plus the TB-TSTs intervention from a respiratory medicine hospital in the province of Buenos Aires, Argentina and followed for 6-months. Due to the nature of the intervention, blinding to the group allocation could not be achieved for the recruiter or patients. The treatment outcome data extractor was blinded to the group allocation of the participants. Intervention participants used the app to report self-administering medication, potential side effects, submit photos of the urine test, and interact with a treatment supporter. Outcomes were feasibility, acceptability, and treatment outcomes. Findings: Forty-two patients were enrolled and evenly assigned to each group. Intervention participants submitted 147·2±58 (mean, SD) medication self-administration and 144·5±55 side effect reports out of 180 and 47.5±38·4 photos of the urine test out of 77. Treatment success for usual care was 81% [17/21] and 95% [20/21] for the TB-TSTs intervention. Thirty-three themes were identified within the main categories of motivation, what worked, issues experienced, and recommendations. Participants (n=12) rated it as 'easy to use' (4.57/5), 'would highly recommend to others' (4·43/5) and reported that access to the treatment support was a critical component. Recommendations included adding an alarm, appointment reminders, and off-line functionality. Interpretation: Findings suggest that the TB-TSTs intervention was feasible and acceptable and further refinement and testing is warranted. Funding: National Institute of Health K23NR017210.

Multiplex Target-Redundant RT-LAMP for Robust Detection of SARS-CoV-2 Using Fluorescent Universal Displacement Probes.

The increasing prevalence of variant lineages during the COVID-19 pandemic has the potential to disrupt molecular diagnostics due to mismatches between primers and variant templates. Point-of-care molecular diagnostics, which often lack the complete functionality of their high-throughput laboratory counterparts, are particularly susceptible to this type of disruption, which can result in false-negative results. To address this challenge, we have developed a robust Loop Mediated Isothermal Amplification assay with single tube multiplexed multitarget redundancy and an internal amplification control. A convenient and cost-effective target-specific fluorescence detection system allows amplifications to be grouped by signal using adaptable probes for pooled reporting of SARS-CoV-2 target amplifications or differentiation of the Internal Amplification Control. Over the course of the pandemic, primer coverage of viral lineages by the three redundant sub-assays has varied from assay to assay as they have diverged from the Wuhan-Hu-1 isolate sequence, but aggregate coverage has remained high for all variant sequences analyzed, with a minimum of 97.4% (Variant of Interest: Eta). In three instances (Delta, Gamma, Eta), a high-frequency mismatch with one of the three sub-assays was observed, but overall coverage remained high due to multitarget redundancy. When challenged with extracted human samples the multiplex assay showed 87% or better sensitivity (of 30 positive samples), with 100% sensitivity for samples containing greater than 30 copies of viral RNA per reaction (of 21 positive samples), and 100% specificity (of 60 negative samples). These results are further evidence that conventional laboratory methodologies can be leveraged at the point of care for robust performance and diagnostic stability over time. IMPORTANCE The COVID-19 pandemic has had tremendous impact, and the ability to perform molecular diagnostics in resource limited settings has emerged as a key resource for mitigating spread of the disease. One challenge in COVID-19 diagnosis, as well as other viruses, is ongoing mutation that can allow viruses to evade detection by diagnostic tests. We developed a test that detects multiple parts of the virus genome in a single test to reduce the chance of missing a virus due to mutation, and it is designed to be simpler and faster than typical laboratory tests while maintaining high sensitivity. This capability is enabled by a novel fluorescent probe technology that works with a simple constant temperature reaction condition.

Simultaneous monitoring of HIV viral load and screening of SARS-CoV-2 employing a low-cost RT-qPCR test workflow.

The COVID-19 pandemic interrupted routine care for individuals living with HIV, putting them at risk of virologic failure and HIV-associated illness. Often this population is at high risk for exposure to SARS-CoV-2 infection, and once infected, for severe disease. Therefore, close monitoring of HIV plasma viral load (VL) and screening for SARS-CoV-2 infection are needed. We developed a non-proprietary method to isolate RNA from plasma, nasal secretions (NS), or both. The extracted RNA is then submitted to RT-qPCR to estimate the VL and classify HIV/SARS-CoV-2 status (i.e., HIV virologic failure or suppressed; SARS-CoV-2 as positive, presumptive positive, negative, or indeterminate). In contrived samples, the in-house RNA extraction workflow achieved a detection limit of 200-copies per mL for HIV RNA in plasma and 100-copies per mL for SARS-CoV-2 RNA in NS. Similar detection limits were observed for HIV and SARS-CoV-2 in pooled plasma/NS contrived samples. When comparing in-house with standard extraction methods, we found high agreement (>0.91) between input and measured RNA copies for HIV LTR in contrived plasma; SARS-CoV-2 N1/N2 in contrived NS; and LTR, N1, and N2 in pooled plasma/NS samples. We further evaluated this workflow on 133 clinical specimens: 40 plasma specimens (30 HIV-positive), 67 NS specimens (31 SARS-CoV-2-positive), and 26 combined plasma/NS specimens (26 HIV-positive with 10 SARS-CoV-2-positive), and compared the results obtained using the in-house RNA extraction to those using a commercial kit (standard extraction method). The in-house extraction and standard extraction of clinical specimens were positively correlated: plasma HIV VL (R2 of 0.81) and NS SARS-CoV-2 VL (R2 of 0.95 and 0.99 for N1 and N2 genes, respectively); and pooled plasma/NS HIV VL (R2 of 0.71) and SARS-CoV-2 VL (R2 of 1 both for N1 and N2 genes). Our low-cost molecular test workflow ($1.85 per pooled sample extraction) for HIV RNA and SARS-CoV-2 RNA could serve as an alternative to current standard assays ($12 per pooled sample extraction) for laboratories in low-resource settings.

Aptamer Sandwich Lateral Flow Assay (AptaFlow) for Antibody-Free SARS-CoV-2 Detection.

The COVID-19 pandemic is among the greatest health and socioeconomic crises in recent history. Although COVID-19 vaccines are being distributed, there remains a need for rapid testing to limit viral spread from infected individuals. We previously identified the SARS-CoV-2 spike protein N-terminal domain (NTD) binding DNA aptamer 1 (SNAP1) for detection of SARS-CoV-2 virus by aptamer-antibody sandwich enzyme-linked immunoassay (ELISA) and lateral flow assay (LFA). In this work, we identify a new aptamer that also binds at the NTD, named SARS-CoV-2 spike protein NTD-binding DNA aptamer 4 (SNAP4). SNAP4 binds with high affinity (<30 nM) for the SARS-CoV-2 spike protein, a 2-fold improvement over SNAP1. Furthermore, we utilized both SNAP1 and SNAP4 in an aptamer sandwich LFA (AptaFlow), which detected SARS-CoV-2 UV-inactivated virus at concentrations as low as 106 copies/mL. AptaFlow costs <$1 per test to produce, provides results in <1 h, and detects SARS-CoV-2 at concentrations that indicate higher viral loads and a high probability of contagious transmission. AptaFlow is a potential approach for a low-cost, convenient antigen test to aid the control of the COVID-19 pandemic.

The impact of a rapid home test on telehealth decision-making for influenza: a clinical vignette study.

BACKGROUND: Home testing for influenza has the potential to aid triage and management decisions for patients with influenza-like illness. As yet, little is known about the effect of the home influenza testing on clinical decision-making via telehealth. The goal of this study was to determine the clinicians' decision thresholds for influenza and whether the availability of a home influenza test affects clinical decisions. METHODS: We identified primary care physicians at 4 different sites in the US, largely via in-person continuing education meetings. Clinicians were asked for each vignette whether to treat empirically ("rule in"), ask the patient come to the clinic for further evaluation ("test"), or neither test nor treat ("rule out"). They were then given the results of a home influenza test, and were again asked to select from these three options. We measured the agreement of physician estimates of the likelihood of influenza with the probability based on a clinical prediction model. The test and treatment thresholds of influenza were determined based on mixed-effect logistic regressions. RESULTS: In total, 202 clinicians made 570 sets of clinical decisions. Agreement between estimated and actual probability of influenza was fair. The test and treatment thresholds were 24% (95% CI: 22% to 25%) and 63% (95% CI: 58% to 65%) before revealing the actual likelihood of influenza. After providing the results of a home flu test the thresholds were similar, 26% (95% CI: 24% to 29%) and 59% (95% CI: 56% to 62%). However, approximately half of clinicians changed their cliical management decision after being given the home influenza test result, largely by categorizing more patients in the "rule out" and "rule in" groups, and reducing the need for in-person evaluation from 41% of patients to only 20%. CONCLUSION: In the context of a telehealth visit for a patient with influenza-like illness, we identified a test threshold of approximately 25% and a treatment threshold of approximately 60%. Adding the home influenza test results reduced uncertainty and significantly decreased the need for in-person visits.

Flu@home: the Comparative Accuracy of an At-Home Influenza Rapid Diagnostic Test Using a Prepositioned Test Kit, Mobile App, Mail-in Reference Sample, and Symptom-Based Testing Trigger.

At-home testing with rapid diagnostic tests (RDTs) for respiratory viruses could facilitate early diagnosis, guide patient care, and prevent transmission. Such RDTs are best used near the onset of illness when viral load is highest and clinical action will be most impactful, which may be achieved by at-home testing. We evaluated the diagnostic accuracy of the QuickVue Influenza A+B RDT in an at-home setting. A convenience sample of 5,229 individuals who were engaged with an on-line health research platform were prospectively recruited throughout the United States. "Flu@home" test kits containing a QuickVue RDT and reference sample collection and shipping materials were prepositioned with participants at the beginning of the study. Participants responded to daily symptom surveys. If they reported experiencing cough along with aches, fever, chills, and/or sweats, they used their flu@home kit following instructions on a mobile app and indicated what lines they saw on the RDT. Of the 976 participants who met criteria to use their self-collection kit and completed study procedures, 202 (20.7%) were positive for influenza by qPCR. The RDT had a sensitivity of 28% (95% CI = 21 to 36) and specificity of 99% (98 to 99) for influenza A, and 32% (95% CI = 20 to 46) and 99% (95% CI = 98 to 99), for influenza B. Our results support the concept of app-supported, prepositioned at-home RDT kits using symptom-based triggers, although it cannot be recommended with the RDT used in this study. Further research is needed to determine ways to improve the accuracy and utility of home-based testing for influenza.

Diagnostic Accuracy of an At-Home, Rapid Self-test for Influenza: Prospective Comparative Accuracy Study.

BACKGROUND: Rapid diagnostic tests (RDTs) for influenza used by individuals at home could potentially expand access to testing and reduce the impact of influenza on health systems. Improving access to testing could lead to earlier diagnosis following symptom onset, allowing more rapid interventions for those who test positive, including behavioral changes to minimize spread. However, the accuracy of RDTs for influenza has not been determined in self-testing populations. OBJECTIVE: This study aims to assess the accuracy of an influenza RDT conducted at home by lay users with acute respiratory illness compared with that of a self-collected sample by the same individual mailed to a laboratory for reference testing. METHODS: We conducted a comparative accuracy study of an at-home influenza RDT (Ellume) in a convenience sample of individuals experiencing acute respiratory illness symptoms. Participants were enrolled in February and March 2020 from the Greater Seattle region in Washington, United States. Participants were mailed the influenza RDT and reference sample collection materials, which they completed and returned for quantitative reverse-transcription polymerase chain reaction influenza testing in a central laboratory. We explored the impact of age, influenza type, duration, and severity of symptoms on RDT accuracy and on cycle threshold for influenza virus and ribonuclease P, a marker of human DNA. RESULTS: A total of 605 participants completed all study steps and were included in our analysis, of whom 87 (14.4%) tested positive for influenza by quantitative reverse-transcription polymerase chain reaction (70/87, 80% for influenza A and 17/87, 20% for influenza B). The overall sensitivity and specificity of the RDT compared with the reference test were 61% (95% CI 50%-71%) and 95% (95% CI 93%-97%), respectively. Among individuals with symptom onset ≤72 hours, sensitivity was 63% (95% CI 48%-76%) and specificity was 94% (95% CI 91%-97%), whereas, for those with duration >72 hours, sensitivity and specificity were 58% (95% CI 41%-74%) and 96% (95% CI 93%-98%), respectively. Viral load on reference swabs was negatively correlated with symptom onset, and quantities of the endogenous marker gene ribonuclease P did not differ among reference standard positive and negative groups, age groups, or influenza subtypes. The RDT did not have higher sensitivity or specificity among those who reported more severe illnesses. CONCLUSIONS: The sensitivity and specificity of the self-test were comparable with those of influenza RDTs used in clinical settings. False-negative self-test results were more common when the test was used after 72 hours of symptom onset but were not related to inadequate swab collection or severity of illness. Therefore, the deployment of home tests may provide a valuable tool to support the management of influenza and other respiratory infections.

Isothermal Amplification with a Target-Mimicking Internal Control and Quantitative Lateral Flow Readout for Rapid HIV Viral Load Testing in Low-Resource Settings.

Point-of-care diagnostics often use isothermal nucleic acid amplification for qualitative detection of pathogens in low-resource healthcare settings but lack sufficient precision for quantitative applications such as HIV viral load monitoring. Although viral load (VL) monitoring is an essential component of HIV treatment, commercially available tests rely on relatively high-resource chemistries like real-time polymerase chain reaction and are thus used on an infrequent basis for millions of people living with HIV in low-income countries. To address the constraints of low-resource settings on nucleic acid quantification, we describe a recombinase polymerase amplification and lateral flow detection approach that quantifies HIV-1 DNA or RNA by comparison to a competitive internal amplification control (IAC) of a known copy number, which may be set to any useful threshold (in our case, a clinically relevant threshold for HIV treatment failure). The IAC is designed to amplify alongside the HIV target with a similar efficiency, allowing for normalization of the assay to variation or inhibition and enabling an endpoint readout that is compatible with commercially available kits for nucleic acid lateral flow detection and interpretable with minimal instrumentation or by the naked eye. We find that this approach can reliably differentiate ≤600 or ≥1400 copies of HIV DNA from a 1000-copy threshold when lateral flow strips are imaged with a conventional office scanner and analyzed with free densitometry software. We further demonstrate a user-friendly adaptation of this analysis to process cell phone photographs with an automated script. Alternatively, we show via a survey that 21 minimally trained volunteers could reliably resolve ≥10-fold (log10) differences of HIV DNA or RNA by naked eye interpretation of lateral flow results. This amplification and detection workflow requires minimal instrumentation, takes just 30 min to complete, and when combined with a suitable sample preparation method, may enable HIV VL testing while the patient waits or a self-test, which has the potential to improve care. This approach may be adapted for other applications that require quantitative analysis of a nucleic acid target in low-resource settings.

Requirements and Study Designs for U.S. Regulatory Approval of Influenza Home Tests.

Home testing for infectious disease has come to the forefront during the COVID-19 pandemic. There is now considerable commercial interest in developing complete home tests for a variety of viral and bacterial pathogens. However, the regulatory science around home infectious disease test approval and procedures that test manufacturers and laboratory professionals will need to follow have not yet been formalized by the U.S. Food and Drug Administration (FDA), with the exception of Emergency Use Authorization (EUA) guidance for COVID-19 tests. We describe the state of home-based testing for influenza with a focus on sample-to-result home tests, discuss the various regulatory pathways by which these products can reach populations, and provide recommendations for study designs, patient samples, and other important features necessary to gain market access. These recommendations have potential application for home use tests being developed for other viral respiratory infections, such as COVID-19, as guidance moves from EUA designation into 510(k) requirements.

Implementation of an interactive mobile application to pilot a rapid assay to detect HIV drug resistance mutations in Kenya.

Usability is an overlooked aspect of implementing lab-based assays, particularly novel assays in low-resource-settings. Esoteric instructions can lead to irreproducible test results and patient harm. To address these issues, we developed a software application based on "Aquarium", a laboratory-operating system run on a computer tablet that provides step-by-step digital interactive instructions, protocol management, and sample tracking. Aquarium was paired with a near point-of-care HIV drug resistance test, "OLA-Simple", that detects mutations associated with virologic failure. In this observational study we evaluated the performance of Aquarium in guiding untrained users through the multi-step laboratory protocol with little supervision. To evaluate the training by Aquarium software we conducted a feasibility study in a laboratory at Coptic Hope Center in Nairobi, Kenya. Twelve volunteers who were unfamiliar with the kit performed the test on blinded samples (2 blood specimens; 5 codons/sample). Steps guided by Aquarium included: CD4+ T-Cell separation, PCR, ligation, detection, and interpretation of test results. Participants filled out a short survey regarding their demographics and experience with the software and kit. None of the laboratory technicians had prior experience performing CD4+ separation and 7/12 had no experience performing laboratory-based molecular assays. 12/12 isolated CD4+ T cells from whole blood with yields comparable to isolations performed by trained personnel. The OLA-Simple workflow was completed by all, with genotyping results interpreted correctly by unaided-eye in 108/120 (90%) and by software in 116/120 (97%) of codons analyzed. In the surveys, participants favorably assessed the use of software guidance. The Aquarium digital instructions enabled first-time users in Kenya to complete the OLA-simple kit workflow with minimal training. Aquarium could increase the accessibility of laboratory assays in low-resource-settings and potentially standardize implementation of clinical laboratory tests.

Harmony COVID-19: A ready-to-use kit, low-cost detector, and smartphone app for point-of-care SARS-CoV-2 RNA detection.

RNA amplification tests sensitively detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, but their complexity and cost are prohibitive for expanding coronavirus disease 2019 (COVID-19) testing. We developed "Harmony COVID-19," a point-of-care test using inexpensive consumables, ready-to-use reagents, and a simple device. Our ready-to-use, multiplexed reverse transcription, loop-mediated isothermal amplification (RT-LAMP) can detect down to 0.38 SARS-CoV-2 RNA copies/µl and can report in 17 min for high­viral load samples (5000 copies/µl). Harmony detected 97 or 83% of contrived samples with ≥0.5 viral particles/µl in nasal matrix or saliva, respectively. Evaluation in clinical nasal specimens (n = 101) showed 100% detection of RNA extracted from specimens with ≥0.5 SARS-CoV-2 RNA copies/µl, with 100% specificity in specimens positive for other respiratory pathogens. Extraction-free analysis (n = 29) had 95% success in specimens with ≥1 RNA copies/µl. Usability testing performed first time by health care workers showed 95% accuracy.

A Systematic Review of Clinical Prediction Rules for the Diagnosis of Influenza.

BACKGROUND: Clinical prediction rules (CPRs) can assist clinicians by focusing their clinical evaluation on the most important signs and symptoms, and if used properly can reduce the need for diagnostic testing. This study aims to perform an updated systematic review of clinical prediction rules and classification and regression tree (CART) models for the diagnosis of influenza. METHODS: We searched PubMed, CINAHL, and EMBASE databases. We identified prospective studies of patients presenting with suspected influenza or respiratory infection and that reported a CPR in the form of a risk score or CART-based algorithm. Studies had to report at a minimum the percentage of patients in each risk group with influenza. Studies were evaluated for inclusion and data were extracted by reviewers working in parallel. Accuracy was summarized descriptively; where not reported by the authors the area under the receiver operating characteristic curve (AUROCC), predictive values, and likelihood ratios were calculated. RESULTS: We identified 10 studies that presented 14 CPRs. The most commonly included predictor variables were cough, fever, chills and/or sweats, myalgias, and acute onset, all which can be ascertained by phone or telehealth visit. Most CPRs had an AUROCC between 0.7 and 0.8, indicating good discrimination. However, only 1 rule has undergone prospective external validation, with limited success. Data reporting by the original studies was in some cases inadequate to determine measures of accuracy. CONCLUSIONS: Well-designed validation studies, studies of interrater reliability between telehealth an in-person assessment, and studies using novel data mining and artificial intelligence strategies are needed to improve diagnosis of this common and important infection.

Rapid Near Point-of-Care Assay for HLA-B*57:01 Genotype Associated with Severe Hypersensitivity Reaction to Abacavir.

The nucleoside reverse transcriptase inhibitor abacavir (ABC) is used commonly to treat young children with HIV infection and is a component of the fixed-dose-combination Triumeq®. ABC can trigger a severe hypersensitivity reaction in people who are homozygous or heterozygous for HLA-B*57:01. Testing for HLA-B*57:01 before ABC initiation is standard-of-care in high-resource settings, but current tests are costly or difficult to access in resource-limited settings. To address these gaps, we developed an inexpensive simple-to-use rapid assay to detect HLA-B*57:01. We designed and optimized a multiplexed polymerase chain reaction (PCR) to amplify HLA-B*57 subtypes and the human beta-globin gene; employed probes and ligation to specifically tag the HLA-B*57:01 allele with biotin. Tagged-ligated products were detected by immunocapture in an enzyme-linked immunosorbent assay plate or lateral flow strip. Cell lines with known HLA genotypes were used to optimize the assay. The optimized assay was then compared with genotypes of clinical specimens (n = 60) determined by sequencing, with specimens enriched for individuals with HLA-B*57:01. The optimized assay utilizes 40-min 35-cycle multiplex PCR for B*57 and beta-globin; 20-min ligation reaction; and 15-min detection. Evaluation of the HLA-B*57:01 oligonucleotide ligation assay using clinical specimens had a sensitivity of 100% (n = 27/27 typed as B*57:01) and specificity of 100% (n = 33/33 typed as non-B*57:01) by visual interpretation of lateral flow strips. The cost is US$5.96/specimen. This rapid and economical assay accurately detects HLA-B*57:01 in clinical specimens. Use of this assay could expand access to HLA-B*57:01 genotyping and facilitate safe same-day initiation of ABC-based treatment.

Characterizing the molecular composition and diagnostic potential of Mycobacterium tuberculosis urinary cell-free DNA using next-generation sequencing.

BACKGROUND: Urine cell-free DNA (cfDNA) is an attractive target for diagnosing pulmonary Mycobacterium tuberculosis (MTB) infection, but has not been thoroughly characterized as a biomarker. METHODS: This study was performed to investigate the size and composition of urine cfDNA from tuberculosis (TB) patients with minimal bias using next-generation sequencing (NGS). A combination of DNA extraction and single-stranded sequence library preparation methods demonstrated to recover short, highly degraded cfDNA fragments was employed. Urine cfDNA from 10 HIV-positive patients with pulmonary TB and two MTB-negative controls was examined. RESULTS: MTB-derived cfDNA was identifiable by NGS from all MTB-positive patients and was absent from negative controls. MTB cfDNA was significantly shorter than human cfDNA, with median fragment lengths of ≤19-52 bp and 42-92 bp, respectively. MTB cfDNA abundance increased exponentially with decreased fragment length, having a peak fragment length of ≤19 bp in most samples. In addition, we identified a larger fraction of short human genomic cfDNA, ranging from 29 to 53 bp, than previously reported. Urine cfDNA fragments spanned the MTB genome with relative uniformity, but nucleic acids derived from multicopy elements were proportionately over-represented. CONCLUSIONS: TB urine cfDNA is a potentially powerful biomarker but is highly fragmented, necessitating special procedures to maximize its recovery and detection.

Multiplex Target-Redundant RT-LAMP for Robust Detection of SARS-CoV-2 Using Fluorescent Universal Displacement Probes.

The increasing prevalence of variant lineages during the COVID-19 pandemic has the potential to disrupt molecular diagnostics due to mismatches between primers and variant templates. Point-of-care molecular diagnostics, which often lack the complete functionality of their high throughput laboratory counterparts, are particularly susceptible to this type of disruption, which can result in false negative results. To address this challenge, we have developed a robust Loop Mediated Isothermal Amplification assay with single tube multiplexed multi-target redundancy and an internal amplification control. A convenient and cost-effective target specific fluorescence detection system allows amplifications to be grouped by signal using adaptable probes for pooled reporting of SARS-COV-2 target amplifications or differentiation of the Internal Amplification Control. Over the course of the pandemic, primer coverage of viral lineages by the three redundant sub-assays has varied from assay to assay as they have diverged from the Wuhan-Hu-1 isolate sequence, but aggregate coverage has remained high for all variant sequences analyzed, with a minimum of 97.4% (Variant of Interest: Eta). In three instances (Delta, Gamma, Eta), a high frequency mismatch with one of the three sub-assays was observed, but overall coverage remained high due to multi-target redundancy. When challenged with extracted human samples the multiplexed assay showed 100% sensitivity for samples containing greater than 30 copies of viral RNA per reaction, and 100% specificity. These results are further evidence that conventional laboratory methodologies can be leveraged at the point-of-care for robust performance and diagnostic stability over time.