Sensitive HIV-1 DNA Pol Next-Generation Sequencing for the Characterisation of Archived Antiretroviral Drug Resistance.

Modern HIV-1 treatment effectively suppresses viral amplification in people living with HIV. However, the persistence of HIV-1 DNA as proviruses integrated into the human genome remains the main barrier to achieving a cure. Next-generation sequencing (NGS) offers increased sensitivity for characterising archived drug resistance mutations (DRMs) in HIV-1 DNA for improved treatment options. In this study, we present an ultra-sensitive targeted PCR assay coupled with NGS and a robust pipeline to characterise HIV-1 DNA DRMs from buffy coat samples. Our evaluation supports the use of this assay for Pan-HIV-1 analyses with reliable detection of DRMs across the HIV-1 Pol region. We propose this assay as a new valuable tool for monitoring archived HIV-1 drug resistance in virologically suppressed individuals, especially in clinical trials investigating novel therapeutic approaches.

Effectiveness of rapid SARS-CoV-2 genome sequencing in supporting infection control for hospital-onset COVID-19 infection: Multicentre, prospective study.

Background: Viral sequencing of SARS-CoV-2 has been used for outbreak investigation, but there is limited evidence supporting routine use for infection prevention and control (IPC) within hospital settings. Methods: We conducted a prospective non-randomised trial of sequencing at 14 acute UK hospital trusts. Sites each had a 4-week baseline data collection period, followed by intervention periods comprising 8 weeks of 'rapid' (<48 hr) and 4 weeks of 'longer-turnaround' (5-10 days) sequencing using a sequence reporting tool (SRT). Data were collected on all hospital-onset COVID-19 infections (HOCIs; detected ≥48 hr from admission). The impact of the sequencing intervention on IPC knowledge and actions, and on the incidence of probable/definite hospital-acquired infections (HAIs), was evaluated. Results: A total of 2170 HOCI cases were recorded from October 2020 to April 2021, corresponding to a period of extreme strain on the health service, with sequence reports returned for 650/1320 (49.2%) during intervention phases. We did not detect a statistically significant change in weekly incidence of HAIs in longer-turnaround (incidence rate ratio 1.60, 95% CI 0.85-3.01; p=0.14) or rapid (0.85, 0.48-1.50; p=0.54) intervention phases compared to baseline phase. However, IPC practice was changed in 7.8 and 7.4% of all HOCI cases in rapid and longer-turnaround phases, respectively, and 17.2 and 11.6% of cases where the report was returned. In a 'per-protocol' sensitivity analysis, there was an impact on IPC actions in 20.7% of HOCI cases when the SRT report was returned within 5 days. Capacity to respond effectively to insights from sequencing was breached in most sites by the volume of cases and limited resources. Conclusions: While we did not demonstrate a direct impact of sequencing on the incidence of nosocomial transmission, our results suggest that sequencing can inform IPC response to HOCIs, particularly when returned within 5 days. Funding: COG-UK is supported by funding from the Medical Research Council (MRC) part of UK Research & Innovation (UKRI), the National Institute of Health Research (NIHR) (grant code: MC_PC_19027), and Genome Research Limited, operating as the Wellcome Sanger Institute. Clinical trial number: NCT04405934.

Clinical Validation of a Rapid Variant-Proof RT-RPA Assay for the Detection of SARS-CoV-2.

The COVID-19 pandemic has unveiled a pressing need to expand the diagnostic landscape to permit high-volume testing in peak demand. Rapid nucleic acid testing based on isothermal amplification is a viable alternative to real-time reverse transcription polymerase chain reaction (RT-PCR) and can help close this gap. With the emergence of SARS-CoV-2 variants of concern, clinical validation of rapid molecular tests needs to demonstrate their ability to detect known variants, an essential requirement for a robust pan-SARS-CoV-2 assay. To date, there has been no clinical validation of reverse transcription recombinase polymerase amplification (RT-RPA) assays for SARS-CoV-2 variants. We performed a clinical validation of a one-pot multi-gene RT-RPA assay with the E and RdRP genes of SARS-CoV-2 as targets. The assay was validated with 91 nasopharyngeal samples, with a full range of viral loads, collected at University College London Hospitals. Moreover, the assay was tested with previously sequenced clinical samples, including eleven lineages of SARS-CoV-2. The rapid (20 min) RT-RPA assay showed high sensitivity and specificity, equal to 96% and 97%, respectively, compared to gold standard real-time RT-PCR. The assay did not show cross-reactivity with the panel of respiratory pathogens tested. We also report on a semi-quantitative analysis of the RT-RPA results with correlation to viral load equivalents. Furthermore, the assay could detect all eleven SARS-CoV-2 lineages tested, including four variants of concern (Alpha, Beta, Delta, and Omicron). This variant-proof SARS-CoV-2 assay offers a significantly faster and simpler alternative to RT-PCR, delivering sensitive and specific results with clinical samples.

The Alpha variant was not associated with excess nosocomial SARS-CoV-2 infection in a multi-centre UK hospital study.

OBJECTIVES: Recently emerging SARS-CoV-2 variants have been associated with an increased rate of transmission within the community. We sought to determine whether this also resulted in increased transmission within hospitals. METHODS: We collected viral sequences and epidemiological data of patients with community and healthcare associated SARS-CoV-2 infections, sampled from 16th November 2020 to 10th January 2021, from nine hospitals participating in the COG-UK HOCI study. Outbreaks were identified using ward information, lineage and pairwise genetic differences between viral sequences. RESULTS: Mixed effects logistic regression analysis of 4184 sequences showed healthcare-acquired infections were no more likely to be identified as the Alpha variant than community acquired infections. Nosocomial outbreaks were investigated based on overlapping ward stay and SARS-CoV-2 genome sequence similarity. There was no significant difference in the number of patients involved in outbreaks caused by the Alpha variant compared to outbreaks caused by other lineages. CONCLUSIONS: We find no evidence to support it causing more nosocomial transmission than previous lineages. This suggests that the stringent infection prevention measures already in place in UK hospitals contained the spread of the Alpha variant as effectively as other less transmissible lineages, providing reassurance of their efficacy against emerging variants of concern.

Reduced antibody cross-reactivity following infection with B.1.1.7 than with parental SARS-CoV-2 strains.

Background: The degree of heterotypic immunity induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains is a major determinant of the spread of emerging variants and the success of vaccination campaigns, but remains incompletely understood. Methods: We examined the immunogenicity of SARS-CoV-2 variant B.1.1.7 (Alpha) that arose in the United Kingdom and spread globally. We determined titres of spike glycoprotein-binding antibodies and authentic virus neutralising antibodies induced by B.1.1.7 infection to infer homotypic and heterotypic immunity. Results: Antibodies elicited by B.1.1.7 infection exhibited significantly reduced recognition and neutralisation of parental strains or of the South Africa variant B.1.351 (Beta) than of the infecting variant. The drop in cross-reactivity was significantly more pronounced following B.1.1.7 than parental strain infection. Conclusions: The results indicate that heterotypic immunity induced by SARS-CoV-2 variants is asymmetric. Funding: This work was supported by the Francis Crick Institute and the Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg.

Genomic characteristics and clinical effect of the emergent SARS-CoV-2 B.1.1.7 lineage in London, UK: a whole-genome sequencing and hospital-based cohort study.

BACKGROUND: Emergence of variants with specific mutations in key epitopes in the spike protein of SARS-CoV-2 raises concerns pertinent to mass vaccination campaigns and use of monoclonal antibodies. We aimed to describe the emergence of the B.1.1.7 variant of concern (VOC), including virological characteristics and clinical severity in contemporaneous patients with and without the variant. METHODS: In this cohort study, samples positive for SARS-CoV-2 on PCR that were collected from Nov 9, 2020, for patients acutely admitted to one of two hospitals on or before Dec 20, 2020, in London, UK, were sequenced and analysed for the presence of VOC-defining mutations. We fitted Poisson regression models to investigate the association between B.1.1.7 infection and severe disease (defined as point 6 or higher on the WHO ordinal scale within 14 days of symptoms or positive test) and death within 28 days of a positive test and did supplementary genomic analyses in a cohort of chronically shedding patients and in a cohort of remdesivir-treated patients. Viral load was compared by proxy, using PCR cycle threshold values and sequencing read depths. FINDINGS: Of 496 patients with samples positive for SARS-CoV-2 on PCR and who met inclusion criteria, 341 had samples that could be sequenced. 198 (58%) of 341 had B.1.1.7 infection and 143 (42%) had non-B.1.1.7 infection. We found no evidence of an association between severe disease and death and lineage (B.1.1.7 vs non-B.1.1.7) in unadjusted analyses (prevalence ratio [PR] 0·97 [95% CI 0·72-1·31]), or in analyses adjusted for hospital, sex, age, comorbidities, and ethnicity (adjusted PR 1·02 [0·76-1·38]). We detected no B.1.1.7 VOC-defining mutations in 123 chronically shedding immunocompromised patients or in 32 remdesivir-treated patients. Viral load by proxy was higher in B.1.1.7 samples than in non-B.1.1.7 samples, as measured by cycle threshold value (mean 28·8 [SD 4·7] vs 32·0 [4·8]; p=0·0085) and genomic read depth (1280 [1004] vs 831 [682]; p=0·0011). INTERPRETATION: Emerging evidence exists of increased transmissibility of B.1.1.7, and we found increased virus load by proxy for B.1.1.7 in our data. We did not identify an association of the variant with severe disease in this hospitalised cohort. FUNDING: University College London Hospitals NHS Trust, University College London/University College London Hospitals NIHR Biomedical Research Centre, Engineering and Physical Sciences Research Council.

Virological failure, HIV-1 drug resistance, and early mortality in adults admitted to hospital in Malawi: an observational cohort study.

BACKGROUND: Antiretroviral therapy (ART) scale-up in sub-Saharan Africa combined with weak routine virological monitoring has driven increasing HIV drug resistance. We investigated ART failure, drug resistance, and early mortality among patients with HIV admitted to hospital in Malawi. METHODS: This observational cohort study was nested within the rapid urine-based screening for tuberculosis to reduce AIDS-related mortality in hospitalised patients in Africa (STAMP) trial, which recruited unselected (ie, irrespective of clinical presentation) adult (aged ≥18 years) patients with HIV-1 at admission to medical wards. Patients were included in our observational cohort study if they were enrolled at the Malawi site (Zomba Central Hospital) and were taking ART for at least 6 months at admission. Patients who met inclusion criteria had frozen plasma samples tested for HIV-1 viral load. Those with HIV-1 RNA of at least 1000 copies per mL had drug resistance testing by ultra-deep sequencing, with drug resistance defined as intermediate or high-level resistance using the Stanford HIVDR program. Mortality risk was calculated 56 days from enrolment. Patients were censored at death, at 56 days, or at last contact if lost to follow-up. The modelling strategy addressed the causal association between HIV multidrug resistance and mortality, excluding factors on the causal pathway (most notably, CD4 cell count, clinical signs of advanced HIV, and poor functional and nutritional status). FINDINGS: Of 1316 patients with HIV enrolled in the STAMP trial at the Malawi site between Oct 26, 2015, and Sept 19, 2017, 786 had taken ART for at least 6 months. 252 (32%) of 786 patients had virological failure (viral load ≥1000 copies per mL). Mean age was 41·5 years (SD 11·4) and 528 (67%) of 786 were women. Of 237 patients with HIV drug resistance results available, 195 (82%) had resistance to lamivudine, 128 (54%) to tenofovir, and 219 (92%) to efavirenz. Resistance to at least two drugs was common (196, 83%), and this was associated with increased mortality (adjusted hazard ratio 1·7, 95% CI 1·2-2·4; p=0·0042). INTERPRETATION: Interventions are urgently needed and should target ART clinic, hospital, and post-hospital care, including differentiated care focusing on patients with advanced HIV, rapid viral load testing, and routine access to drug resistance testing. Prompt diagnosis and switching to alternative ART could reduce early mortality among inpatients with HIV. FUNDING: Joint Global Health Trials Scheme of the Medical Research Council, UK Department for International Development, and Wellcome Trust.

Bigger and Better? Representativeness of the Influenza A Surveillance Using One Consolidated Clinical Microbiology Laboratory Data Set as Compared to the Belgian Sentinel Network of Laboratories.

Infectious diseases remain a serious public health concern globally, while the need for reliable and representative surveillance systems remains as acute as ever. The public health surveillance of infectious diseases uses reported positive results from sentinel clinical laboratories or laboratory networks, to survey the presence of specific microbial agents known to constitute a threat to public health in a given population. This monitoring activity is commonly based on a representative fraction of the microbiology laboratories nationally reporting to a single central reference point. However, in recent years a number of clinical microbiology laboratories (CML) have undergone a process of consolidation involving a shift toward laboratory amalgamation and closer real-time informational linkage. This report aims to investigate whether such merging activities might have a potential impact on infectious diseases surveillance. Influenza data was used from Belgian public health surveillance 2014-2017, to evaluate whether national infection trends could be estimated equally as effectively from only just one centralized CML serving the wider Brussels area (LHUB-ULB). The overall comparison reveals that there is a close correlation and representativeness of the LHUB-ULB data to the national and international data for the same time periods, both on epidemiological and molecular grounds. Notably, the effectiveness of the LHUB-ULB surveillance remains partially subject to local regional variations. A subset of the Influenza samples had their whole genome sequenced so that the observed epidemiological trends could be correlated to molecular observations from the same period, as an added-value proposition. These results illustrate that the real-time integration of high-throughput whole genome sequencing platforms available in consolidated CMLs into the public health surveillance system is not only credible but also advantageous to use for future surveillance and prediction purposes. This can be most effective when implemented for automatic detection systems that might include multiple layers of information and timely implementation of control strategies.