Drug repurposing based on a quantum-inspired method versus classical fingerprinting uncovers potential antivirals against SARS-CoV-2.

The COVID-19 pandemic has accelerated the need to identify new antiviral therapeutics at pace, including through drug repurposing. We employed a Quadratic Unbounded Binary Optimization (QUBO) model, to search for compounds similar to Remdesivir, the first antiviral against SARS-CoV-2 approved for human use, using a quantum-inspired device. We modelled Remdesivir and compounds present in the DrugBank database as graphs, established the optimal parameters in our algorithm and resolved the Maximum Weighted Independent Set problem within the conflict graph generated. We also employed a traditional Tanimoto fingerprint model. The two methods yielded different lists of lead compounds, with some overlap. While GS-6620 was the top compound predicted by both models, the QUBO model predicted BMS-986094 as second best. The Tanimoto model predicted different forms of cobalamin, also known as vitamin B12. We then determined the half maximal inhibitory concentration (IC50) values in cell culture models of SARS-CoV-2 infection and assessed cytotoxicity. We also demonstrated efficacy against several variants including SARS-CoV-2 Strain England 2 (England 02/2020/407073), B.1.1.7 (Alpha), B.1.351 (Beta) and B.1.617.2 (Delta). Lastly, we employed an in vitro polymerization assay to demonstrate that these compounds directly inhibit the RNA-dependent RNA polymerase (RdRP) of SARS-CoV-2. Together, our data reveal that our QUBO model performs accurate comparisons (BMS-986094) that differed from those predicted by Tanimoto (different forms of vitamin B12); all compounds inhibited replication of SARS-CoV-2 via direct action on RdRP, with both models being useful. While Tanimoto may be employed when performing relatively small comparisons, QUBO is also accurate and may be well suited for very complex problems where computational resources may limit the number and/or complexity of possible combinations to evaluate. Our quantum-inspired screening method can therefore be employed in future searches for novel pharmacologic inhibitors, thus providing an approach for accelerating drug deployment.

The Effect of Respiratory Viral Infections on Breakthrough Hemolysis in Patients with Paroxysmal Nocturnal Hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by hemolysis and thrombosis and is associated with significant morbidity and mortality. Although complement inhibitors have significantly changed the outcomes in PNH patients, breakthrough hemolysis (BTH) may still occur as a response to stress factors such as pregnancy, surgery, and infections. Despite the well-described association between bacterial infections and hemolysis in PNH patients, little is known about the effect of respiratory viruses on triggering hemolytic episodes. This is the first study, to our knowledge, addressing this question. We retrospectively analyzed 34 patients with PNH disease between 2016 and 2018, who were on eculizumab treatment and who presented with respiratory symptoms and were subsequently tested for 10 respiratory viruses (influenza A, influenza B, parainfluenza, respiratory syncytial virus, adenovirus, rhinovirus, and human metapneumovirus). NTS+ patients had higher inflammatory markers, with the majority requiring antibiotics. Acute hemolysis, along with a significant drop in hemoglobin, was noted in the NTS+ group, with three of them requiring a top-up transfusion and two requiring an extra dose of eculizumab. Furthermore, the time from the last eculizumab dose was longer in the NTS+ patients who had BTH, than those who did not. Our data indicate that respiratory virus infections pose a significant risk for BTH in PNH patients on complement inhibitor treatment, underlining the need for regular screening and close monitoring of patients with respiratory symptoms. Furthermore, it implies a higher risk for patients who are not established on complement inhibitors, suggesting the necessity for greater vigilance in these patients.

A Study of Universal Severe Acute Respiratory Syndrome Coronavirus 2 RNA Testing Among Residents and Staff in a Large Group of Care Homes in South London.

BACKGROUND: Care homes have experienced a high number of coronavirus disease 2019 (COVID-19)-related deaths among residents since the onset of the pandemic. However, up to May 2020, there has been a lack of information about the extent of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection among residents and staff in care homes and limited testing in this setting. METHODS: Combined nose and throat swab testing for SARS-CoV-2 RNA was carried out in 2455 residents and staff across 37 care homes in the London Borough of Bromley across a 3-week period. Results were reported within 24 hours of sample delivery, and data were collected on the presence or absence of symptoms. RESULTS: Overall, the point prevalence of SARS-CoV-2 infection was 6.5%, with a higher rate in residents (9.0%) than in staff (4.7%). A key finding was the high proportion of asymptomatic infection detected in staff (69%) and residents (51%), with evidence of underdetection of symptoms by care home staff. CONCLUSIONS: The high proportion of asymptomatic infection combined with underdetection of symptoms by care home staff indicates that offering a test to all residents and staff in care homes with rapid reporting of results would assist accurate identification of infected individuals, facilitating prompt infection prevention and control action.

Severe Acute Respiratory Syndrome Coronavirus-2 Infections in Critical Care Staff: Beware the Risks Beyond the Bedside.

OBJECTIVES: Critical care workers were considered to be at high risk of severe acute respiratory syndrome coronavirus-2 infection from patients during the first wave of the pandemic. Staff symptoms, previous swab testing, and antibody prevalence were correlated with patient admissions to investigate this assumption. DESIGN: Cross-sectional study. SETTING: A large critical care department in a tertiary-care teaching hospital in London, United Kingdom. SUBJECTS: Staff working in critical care. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Participants completed a questionnaire and provided a serum sample for severe acute respiratory syndrome coronavirus-2 antibody testing over a 3-day period in April 2020. We compared the timing of symptoms in staff to the coronavirus disease 2019 patient admissions to critical care. We also identified factors associated with antibody detection. Of 625 staff 384 (61.4%) reported previous symptoms and 124 (19.8%) had sent a swab for testing. Severe acute respiratory syndrome coronavirus-2 infection had been confirmed in 37 of those swabbed (29.8%). Overall, 21% (131/625) had detectable severe acute respiratory syndrome coronavirus-2 antibody, of whom 9.9% (13/131) had been asymptomatic. The peak onset of symptoms among staff occurred 2 weeks before the peak in coronavirus disease 2019 patient admissions. Staff who worked in multiple departments across the hospital were more likely to be seropositive. Staff with a symptomatic household contact were also more likely to be seropositive at 31.3%, compared with 16.2% in those without (p < 0.0001). CONCLUSIONS: Staff who developed coronavirus disease 2019 were less likely to have caught it from their patients in critical care. Other staff, other areas of the hospital, and the wider community are more likely sources of infection. These findings indicate that personal protective equipment was effective at preventing transmission from patients. However, staff also need to maintain protective measures away from the bedside.

A 9-month retrospective evaluation of the aetiology and management of patients presenting with encephalitis/meningoencephalitis at a South London hospital.

Encephalitis causes high morbidity and mortality. An incidence of 4.3 cases of encephalitis/100 000 population has been reported in the UK. We performed a retrospective evaluation of the diagnosis and management of adults admitted to hospital with a clinical diagnosis of encephalitis/meningoencephalitis. Clinical, laboratory and radiological data were collated from electronic records. Thirty-six patients, median age 55 years and 24 (67%) male were included. The aetiology was confirmed over nine months in 25 (69%) of whom 16 were infections (six viral, seven bacterial, two parasitic and one viral and parasitic co-infection); 7 autoimmune; 1 metabolic and 1 neoplastic. Of 24 patients with fever, 15 (63%) had an infection. The median time to computed topography, magnetic resonance imaging and electroencephalography (EEG) was 1, 8 and 3 days respectively. Neuroimaging was abnormal in 25 (69%) and 17 (89%) had abnormal EEGs. Only 19 (53%) received aciclovir treatment. Six (17%) made good recoveries, 16 (44%) had moderate disability, 8 (22%) severe disability and 6 (17%) died. Outcomes were worse for those with an infectious cause. In summary, a diagnosis was made in 69.4% of patients admitted with encephalitis/meningoencephalitis. Autoimmune causes are important to consider at an early stage due to a successful response to treatment. Only 53% of patients received aciclovir on admission. Neuroimaging and EEG studies were delayed. The results of this work resulted in further developing the clinical algorithm for managing these patients.

Epstein-Barr Virus and Monoclonal Gammopathy of Clinical Significance in Autologous Stem Cell Transplantation for Multiple Sclerosis.

INTRODUCTION: Autologous hematopoietic stem cell transplantation (AHSCT) with anti-thymocyte globulin (ATG) conditioning as treatment of active multiple sclerosis (MS) is rapidly increasing across Europe (EBMT registry data 2017). Clinically significant Epstein-Barr virus reactivation (EBV-R) following AHSCT with ATG for severe autoimmune conditions is an underrecognized complication relative to T-cell deplete transplants performed for hematological diseases. This retrospective study reports EBV-R associated significant clinical sequelae in MS patients undergoing AHSCT with rabbit ATG. METHODS: Retrospective data were analyzed for 36 consecutive MS-AHSCT patients at Kings College Hospital, London. All patients routinely underwent weekly EBV DNA polymerase chain reaction monitoring and serum electrophoresis for monoclonal gammopathy (MG or M-protein). EBV-R with rising Epstein-Barr viral load, M-protein, and associated clinical sequelae were captured from clinical records. RESULTS: All patients had evidence of rising EBV DNA-emia, including 7 who were lost to long-term follow-up, with a number of them developing high EBV viral load and associated lymphoproliferative disorder (LPD). Nearly 72% (n = 18/29) developed de novo MG, some with significant neurological consequences with high M-protein and EBV-R. Six patients required anti-CD20 therapy (rituximab) with complete resolution of EBV related symptoms. Receiver operating characteristics estimated a peak EBV viremia of >500 000 DNA copies/mL correlated with high sensitivity (85.5%) and specificity (82.5%) (area under the curve: 0.87; P = .004) in predicting EBV-R related significant clinical events. CONCLUSION: Symptomatic EBV reactivation increases risk of neurological sequelae and LPD in MS-AHSCT. We recommend regular monitoring for EBV and serum electrophoresis for MG in MS patients in the first 3 months post-AHSCT.

Resolution by deep sequencing of a dual hepatitis E virus infection transmitted via blood components.

Hepatitis E virus (HEV) is a zoonotic infection, with consumption of processed pork products thought to be the major route of transmission in England. The clinical features of HEV infection range from asymptomatic infection to mild hepatitis to fulminant liver failure. Persistent, chronic hepatitis is increasingly recognized in immunocompromised patients. Infection via HEV-containing blood components and organs has been reported and measures to reduce this transmission risk were introduced into the blood service in England in 2016. We report here the sequence and phylogenetic findings from investigations into a transmission event from an HEV-infected donor to two recipients. Phylogenetic analysis of HEV genome sequence fragments obtained by Sanger sequencing showed that, whilst most of the sequences from both recipients' samples grouped with the sequence from the blood donor sample, the relationship of five sequences from recipient 2 were unresolved. Analysis of Illumina short-read deep sequence data demonstrated the presence of two divergent viral populations in the donor's sample that were also present in samples from both recipients. A clear phylogenetic relationship was established, indicating a probable transmission of both populations from the donor to each of the immunocompromised recipients. This study demonstrates the value of the application of new sequencing technologies combined with bioinformatic data analysis when Sanger sequencing is not able to clarify a proper phylogenetic relationship in the investigation of transmission events.

Functional and genetic predisposition to rhinovirus lower respiratory tract infections in prematurely born infants.

Term born infants are predisposed to human rhinovirus (HRV) lower respiratory tract infections (LRTI) by reduced neonatal lung function and genetic susceptibility. Our aim was to investigate whether prematurely born infants were similarly predisposed to HRV LRTIs or any other viral LRTIs. Infants born less than 36 weeks of gestational age were recruited. Prior to neonatal/maternity unit discharge, lung function (functional residual capacity by helium gas dilution and multiple breath washout, lung clearance index and compliance (Crs), and resistance (Rrs) of the respiratory system) was assessed and DNA samples assessed for eight single nucleotide polymorphisms (SNPs) in seven genes: ADAM33, IL10, MMP16 NFκB1A,SFTPC, VDR, and NOS2A. Infants were prospectively followed until 1 year corrected age. Nasopharyngeal aspirates (NPAs) were sent whenever an infant developed a LRTI and tested for 13 viruses. One hundred and thirty-nine infants were included in the analysis. Infants who developed HRV LRTIs had reduced Crs (1.6 versus 1.2 mL/cmH2O/kg, p = 0.044) at 36 weeks postmenstrual age. A SNP in the gene coding for the vitamin D receptor was associated with the development of HRV LRTIs and any viral LRTIs (p = 0.02). CONCLUSION: Prematurely born infants may have both a functional and genetic predisposition to HRV LRTIs. What is Known: • Term born infants are predisposed to rhinovirus lower respiratory tract (HRV LRTIs) infection by reduced neonatal lung function. • Term born infants requiring hospitalisation due to HRV bronchiolitis were more likely to have single nucleotide polymorphism (SNP) in the IL-10 gene. What is New: • Prematurely born infants who developed a HRV LRTI had lower C rs before maternity unit discharge. • A SNP in the gene coding for the vitamin D receptor was associated with the development of HRV LRTIs and overall respiratory viral LRTIs in prematurely born infants.

Viral lower respiratory tract infections and preterm infants' healthcare utilisation.

UNLABELLED: The aim of this study was to determine whether respiratory syncytial virus (RSV) and other viral lower respiratory tract infections (LRTI) in prematurely born infants were associated with similar effects on healthcare utilisation and related cost of care in the second compared to the first year after birth. Thirteen infants who had RSV LRTIs (RSV), 21 who had other viral LRTIs (other viral) and 25 had no viral LRTIs (no LRTI) were prospectively followed. Nasopharyngeal aspirates were collected whenever an infant had an LRTI regardless of whether it was in the hospital or in the community. Healthcare utilisation and the health-related cost of care were determined. Only the RSV group compared to the no LRTI group had higher overall respiratory costs in both year 1 (mean, £3,917 versus £24; p < 0.041) and year 2 (mean, £1,164 versus £61; p = 0.012). Only the RSV group required respiratory admissions; the RSV admission rate in year 2 was 3.4 % (number needed to treat 59). CONCLUSION: RSV LRTIs are associated with increased healthcare utilisation and cost of care in the first and second year; nevertheless, if prophylaxis is to be cost-effective in the second year, a high risk group needs to be identified.

Respiratory outcome of prematurely born infants following human rhinovirus A and C infections.

UNLABELLED: Human rhinoviruses (HRVs) are a common cause of lower respiratory tract infections (LRTIs) and are associated with chronic respiratory morbidity. Our aim was to determine whether HRV species A or C were associated with chronic respiratory morbidity and increased health care utilisation in prematurely born infants. A number of 153 infants with a median gestational age of 34 (range 23-35) weeks were prospectively followed. Nasopharyngeal aspirates were collected whenever the infants had LRTIs regardless of hospitalisation status. Parents completed a respiratory diary card and health questionnaire about their infant when they were 11 and 12 months corrected age, respectively. The health-related cost of care during infancy was calculated from the medical records using the National Health Service (NHS) reference costing scheme and the British National Formulary for children. There were 32 infants that developed 40 HRV LRTIs; samples were available from 23 of the 32 infants for subtyping. Nine infants had HRV-A LRTIs, 13 HRV-C LRTIs, and one infant had a HRV-B LRTI. Exclusion of infants who also had RSV LRTIs revealed that the infants who had a HRV-C LRTI were more likely to wheeze (p < 0.0005) and use respiratory medications (p < 0.0005) and had more days of wheeze (p = 0.01) and used an inhaler (p = 0.02) than the no LRTI group. In addition, the respiratory cost of care was greater for the HRV-C LRTI than the no LRTI group (p < 0.0005). CONCLUSION: Our results suggest HRV-C is associated with chronic respiratory morbidity during infancy in prematurely born infants.

Lung function of preterm infants before and after viral infections.

UNLABELLED: Our aim was to determine whether viral lower respiratory tract infections (LRTIs) adversely affect prematurely born infants' lung function at follow up. Seventy infants, median gestational age 34 (range, 24-35) weeks were prospectively followed; 32 had an RSV (n = 14) or another respiratory viral (n = 18) LRTI (viral LRTI group) and 38 had no LRTI (no LRTI group). Six of the viral LRTI and five of the no LRTI group had been hospitalised. Nasopharyngeal aspirates (NPAs) obtained whenever the infants had an LRTI. Lung function (functional residual capacity [FRCHe], compliance [Crs] and resistance [Rrs] of the respiratory system) was measured at 36 weeks postmenstrual age (PMA) and 1 year corrected. At 1 year, lung volume (FRCpleth) and airways resistance (Raw) were also assessed. There were no significant differences in the lung function of the two groups at 36 weeks PMA but at 1 year, the viral LRTI compared to the no LRTI group had a higher mean Raw (23 versus 17 cm H2O/l/s, p = 0.0068), the differences remained significant after adjustment. CONCLUSION: These results suggest viral LRTIs, regardless of whether hospitalisation is required, adversely affect prematurely born infants' airway resistance at follow up.

Genetic predisposition of RSV infection-related respiratory morbidity in preterm infants.

UNLABELLED: The aim of this study was to assess whether prematurely born infants have a genetic predisposition to respiratory syncytial virus (RSV) infection-related respiratory morbidity. One hundred and forty-six infants born at less than 36 weeks of gestation were prospectively followed. Nasopharygeal aspirates were obtained on every occasion the infants had a lower respiratory tract infection (LRTI) regardless of need for admission. DNA was tested for 11 single-nucleotide polymorphisms (SNPs). Chronic respiratory morbidity was assessed using respiratory health-related questionnaires, parent-completed diary cards at a corrected age of 1 year and review of hospital notes. Lung function was measured at a post menstrual age (PMA) of 36 weeks and corrected age of 1 year. A SNP in ADAM33 was associated with an increased risk of developing RSV LRTIs, but not with significant differences in 36-week PMA lung function results. SNPs in several genes were associated with increased chronic respiratory morbidity (interleukin 10 (IL10), nitric oxide synthase 2A (NOS2A), surfactant protein C (SFTPC), matrix metalloproteinase 16 (MMP16) and vitamin D receptor (VDR)) and reduced lung function at 1 year (MMP16, NOS2A, SFTPC and VDR) in infants who had had RSV LRTIs. CONCLUSIONS: Our results suggest that prematurely born infants may have a genetic predisposition to RSV LRTIs and subsequent respiratory morbidity which is independent of premorbid lung function.

Rhinovirus infection and healthcare utilisation in prematurely born infants.

Our aim was to determine whether rhinovirus (RV) lower respiratory tract infections (LRTIs) in prematurely born infants increase health-related cost of care during infancy. 153 infants born at <36 weeks of gestation were prospectively followed to 1 year. Cost of care was calculated from the National Health Service reference costing scheme and healthcare utilisation determined by examining hospital/general practitioner records. 20 infants developed RV LRTIs (RV group), 17 respiratory syncytial virus (RSV) LRTIs (RSV group), 12 both RV and RSV LRTIs (RV/RSV group) and 74 had no LRTI (no LRTI group). Compared with the no LRTI group, the RV/RSV LRTI group had the greatest increase in adjusted mean cost (difference GBP 5769), followed by the RV LRTI group (difference GBP 278) and, finally, the RSV LRTI group (difference GBP 172) (p=0.045). The RV group had more outpatient (p<0.05) and respiratory-related general practitioner (p<0.05) attendances, more wheezed at follow-up (p<0.001) than the no LRTI group and more had respiratory-related outpatient attendances than the RSV LRTI group (p<0.05). We conclude that RV LRTIs were associated with increased health-related cost of care during infancy; our results suggest that the RV group compared with the RSV group suffered greater chronic respiratory morbidity.

Progressive multifocal leukoencephalopathy secondary to idiopathic CD4 lymphocytopenia treated with pembrolizumab.

BACKGROUND: Progressive multifocal leukoencephalopathy (PML) is a rare demyelinating disease due to a lytic infection of oligodendrocytes caused by John Cunningham polyoma virus (JCV) infection. Idiopathic CD4+ T-cell lymphocytopenia (ICL) is a very rare cause of PML. METHODS: We present an individual with PML secondary to ICL treated with 3 doses of pembrolizumab, a Programmed-Death-1 Immune Checkpoint Inhibitor following with complete resolution of symptoms and conduct a review of the literature. CONCLUSION: This report illustrates the objective clinical and radiological improvement in a patient with PML due to ICL and suggests further study of immune checkpoint inhibitors as potential treatment for patients with PML.

Effect of early EBV and/or CMV viremia on graft function and acute cellular rejection in pediatric liver transplantation.

BACKGROUND: The clinical impact of Epstein-Barr virus (EBV) and cytomegalovirus (CMV) infections in the early post-transplantation period are poorly documented. We investigated the prevalence and timing of EBV and CMV infections during the first 21 d post-transplantation in relation to graft function and acute cellular rejection in a large cohort of pediatric liver transplantation recipients. PATIENTS AND METHODS: Clinical, biochemical, virological, and histopathological data of 62 consecutive children who received a liver transplant were reviewed retrospectively. RESULTS: Seventeen patients (27%) developed EBV and 11 (18%) CMV viremia (mean interval from surgery: 7.6 d, SD 3.6 and 8.7 d, SD 6.4, respectively). EBV and CMV viremia were more common as a consequence of reactivation than of primary infection. EBV viremic recipients had more often abnormal bilirubin levels [p = 0.01; OR 5.8: 95% CI 1.3-25.5]. Acute rejection was diagnosed in 20 recipients (32.3%). No correlation was found between rejection and EBV and CMV serology before transplantation and viremia after transplantation (mean interval between the diagnosis of rejection and the detection of EBV DNA and CMV DNA: one d, SD 4.4 and five d, SD 9.2, respectively). CONCLUSION: EBV and CMV viremia occur at a very early-stage post-transplantation and do not appear to affect the short-term outcome of the transplant.

Potential pitfalls in analysing a SARS-CoV-2 RT-PCR assay and how to standardise data interpretation.

The emergence of SARS-CoV-2 in December 2019 lead to the rapid implementation of assays for virus detection, with real-time RT-PCR arguably considered the gold-standard. In our laboratory Altona RealStar SARS-Cov-2 RT-PCR kits are used with Applied Biosystems QuantStudio 7 Flex thermocyclers. Real-time PCR data interpretation is potentially complex and time-consuming, particularly for SARS-CoV-2, where the laboratory handles up to 2000 samples each day. To simplify this, an automated system that rapidly interprets the curves, developed by diagnostics.ai was introduced. QuantStudio software provides two methods for interpretation, relative threshold and baseline threshold. Many of our assays are analysed using relative threshold and directly exported into pcr.ai software, however, in some rare cases the QuantStudio software assigns positive results to 'ambiguous' curves, flagged by pcr.ai, requiring manual intervention. Due to the sample numbers processed and the proportionate increase in curves flagged by pcr.ai, the two methods were investigated. An audit was carried out to determine the frequency of these curves, involving 138 samples tested during November 2020, including 97 serial samples from 38 patients and it was determined that the relative threshold method produced unreliable results in many of these cases. In addition, we present a solution to simplify the interpretation and automate the process.

Nosocomial transmission of hepatitis E virus and development of chronic infection: The wider impact of COVID-19.

BACKGROUND: Transmission of hepatitis E virus (HEV) within the healthcare setting is extremely rare. Additionally, the development of chronic HEV infection in association with severe acute respiratory syndrome coronavirus disease 2 (SARS-CoV-2) infection and/or its immunomodulatory therapy has not been reported previously. AIMS: To describe the investigation and management of a nosocomial HEV transmission incident during the coronavirus disease 2019 (COVID-19) pandemic. METHODS: Epidemiological and molecular investigation of two individuals hospitalised with COVID-19 who were both diagnosed with HEV infection. RESULTS: Findings from our investigation were consistent with transmission of HEV from one patient with a community-acquired HEV infection to another individual (identical HEV sequences were identified in the two patients), most likely due to a breach in infection control practices whilst both patients shared a bed space on the intensive care unit (ICU). Chronic HEV infection requiring treatment with ribavirin developed in one patient with prolonged lymphopaenia attributable to COVID-19 and/or the immunomodulators received for its treatment. Further investigation did not identify transmission of HEV to any other patients or to healthcare workers. CONCLUSIONS: The extraordinary demands that the COVID-19 pandemic has placed on all aspects of healthcare, particularly within ICU settings, has greatly challenged the ability to consistently maintain optimal infection prevention and control practices. Under the significant pressures of the COVID-19 pandemic a highly unusual nosocomial HEV transmission incident occurred complicated further by progression to a chronic HEV infection in one patient.

Homebrew: Protocol for glassmilk-based nucleic-acid extraction for SARS-CoV-2 diagnostics.

The gold standard protocol for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection detection remains reverse transcription quantitative polymerase chain reaction (qRT-PCR), which detects viral RNA more sensitively than any other approach. Here, we present Homebrew, a low-cost protocol to extract RNA using widely available reagents. Homebrew is as sensitive as commercially available RNA extraction kits. Homebrew allows for sample pooling and can be adapted for automation in high-throughput settings. For complete details on the use and execution of this protocol, please refer to Page et al. (2022).