Genomic Investigations of Acute Hepatitis of Unknown Aetiology in Children (preprint)

Since the first reports of hepatitis of unknown aetiology occurring in UK children, over 1000 cases have been reported worldwide, including 268 cases in the UK, with the majority younger than 6 years old. Using genomic, proteomic and immunohistochemical methods, we undertook extensive investigation of 28 cases and 136 control subjects. In five cases who underwent liver transplantation, we detected high levels of adeno-associated virus 2 (AAV2) in the explanted livers. AAV2 was also detected at high levels in blood from 10/11 non-transplanted cases. Low levels of Adenovirus (HAdV) and Human Herpesvirus 6B (HHV-6B), both of which enable AAV2 lytic replication, were also found in the five explanted livers and blood from 15/17 and 6/9 respectively, of the 23 non-transplant cases tested. In contrast, AAV2 was detected at low titre in 6/100 whole bloods from child controls from cohorts with presence or absence of hepatitis and/or adenovirus infection. Our data show an association of AAV2 at high titre in blood or liver tissue, with unexplained hepatitis in children infected in the recent HAdV-F41 outbreak. We were unable to find evidence by electron microscopy, immunohistochemistry or proteomics of HAdV or AAV2 viral particles or proteins in explanted livers, suggesting that hepatic pathology is not due to direct lytic infection by either virus. The potential that AAV2, although not previously associated with disease, may, together with HAdV-F41 and/or HHV-6, be causally implicated in the outbreak of unexplained hepatitis, requires further investigation.

Antibiotic dispensing practices during COVID-19 and implications for Antimicrobial Resistance (AMR): Parallel mystery client studies in Uganda and Tanzania (preprint)

Background: Over-the-counter antibiotic access is common in low-and-middle-income countries, including Tanzania and Uganda, and this may accelerate antimicrobial resistance. Our study explores critical aspects of the drug seller-client interaction and antibiotic dispensing patterns for simulated COVID-19 symptoms during the pandemic in two study sites in countries with different government responses to the pandemic. Methods: : Research assistants posing as clients approached different types of drug sellers such as pharmacies (Pharms), drug shops (DSs), and accredited drug dispensing outlets (ADDOs) in Mwanza, Tanzania (nPharms= 415, nADDOs= 116) and Mbarara, Uganda (nPharms= 440, nDSs= 67) , from June 10-July 30, 2021. The mystery clients (who held no prescription) sought advice for simulated COVID-19 symptoms from the drug sellers, documented the quality of their interaction with sellers and the type of drugs dispensed. Results: : Adherence to COVID-19 preventive measures and vigilance to COVID-19 symptoms was low in both sites but significantly higher in Uganda than in Tanzania. A higher percentage of drug sellers in Mbarara (Pharms= 36%, DSs= 35%, p-value= 0.947 ) compared to Mwanza (Pharms= 9%, ADDOs= 4%, p-value= 0.112 ) identified the client’s symptoms as possibly COVID-19. More than three-quarters of drug sellers in both Mbarara (Pharms= 86%, DSs= 89%) and Mwanza (Pharms= 93%, ADDOs= 97%) sold prescription-only medicines without asking for a prescription. A relatively high percentage of drug sellers that sold prescription-only medicines in Mwanza (Pharms= 51%, ADDOs= 67%) compared to Mbarara (Pharms= 31%, DSs= 42%) sold a partial course without any objection. Among the drug sellers who sold antibiotics, more drug sellers in Mbarara (Pharms= 73%, DSs= 78%, p-value= 0.580 ) compared to Mwanza (Pharms= 40% ADDOs= 46%, p-value= 0.537 ) sold antibiotics relevant for treating secondary bacterial infections in COVID-19 patients. Conclusion: Our study highlights low levels of awareness of and vigilance towards COVID-19 symptoms and widespread propensity to dispense prescription-only antibiotics without a prescription and to dispense partial doses of antibiotics. The implications of these for antimicrobial resistance are further discussed. Our study also highlights the need for more efforts to improve antibiotic stewardship among drug sellers in response to the current pandemic and to prepare them for future health emergencies.

A SARS-CoV-2 Antigen Rapid Diagnostic Test for Resource Limited Settings (preprint)

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19 disease. RT-qPCR has been the primary method of diagnosis; however, the required infrastructure is lacking in many developing countries and the virus has remained a global challenge. More inexpensive and immediate test methods are required to facilitate local, regional, and national management strategies to re-open world economies. Here we have developed a SARS-CoV-2 antigen test in an inexpensive lateral flow format to generate a chromatographic result identifying the presence of the SARS-CoV-2 antigen, and thus an active infection, within a patient anterior nares swab sample. Our 15-minute test requires no equipment or laboratory infrastructure to administer with a limit of detection of 2.0 x 102 TCID50/mL and 87.5% sensitivity, 100% specificity when tested against 40 known positive and 40 known negative patient samples established by a validated RT-qPCR test. 

Use of the Lateral Flow Immunoassay to Characterize SARS-CoV-2 RBD-Specific Antibodies and Their Ability to React with the UK, SA and BR P.1 Variant RBDs (preprint)

Identifying anti-spike antibodies that exhibit strong neutralizing activity against current dominant circulating variants and antibodies that are escaped by these variants have important implications in the development of therapeutic and diagnostic solutions as well as in improving understanding of the humoral response to SARS-CoV-2 infection. We characterized seven anti-RBD monoclonal antibodies for their binding activity, pairing capability and neutralization activity to SARS-CoV-2 and three variant RBDs (UK, SA and BR P.1) via lateral flow immunoassays. The results allowed us to group these antibodies into three distinct epitope bins. Our studies showed that two antibodies had broadly potent neutralizing activity against SARS-CoV-2 and these variant RBDs and that one antibody did not neutralize the SA and BR P.1 RBDs. The antibody escaped by the SA and BR P.1 RBDs retained binding activity to SA and BR P.1 RBDs but was unable to induce neutralization. Further, we demonstrated that the lateral flow immunoassay can be a rapid and effective tool for antibody characterization, including epitope classification and antibody neutralization kinetics. From these studies, the potential contributions of the mutations (N501Y, E484K and K417N/T) contained in these variants’ RBDs on antibody pairing capability, neutralization activity and therapeutic antibody targeting strategy are discussed.