Impact of molecular ciprofloxacin resistance testing in management of gonorrhoea in a large urban clinic.

OBJECTIVES: Antibiotic resistance in gonorrhoea is of significant public health concern with the emergence of resistance to last-line therapies such as ceftriaxone. Despite around half of Neisseria gonorrhoeae isolates tested in the UK being susceptible to ciprofloxacin, very little ciprofloxacin is used in clinical practice. Testing for the S91F mutation associated with ciprofloxacin resistance is now available in CE-marked assays and may reduce the requirement for ceftriaxone, but many patients are treated empirically, or as sexual contacts, which may limit any benefit. We describe the real-world impact of such testing on antimicrobial use and clinical outcomes in people found to have gonorrhoea in a large urban UK sexual health clinic. METHODS: Molecular ciprofloxacin resistance testing (ResistancePlus GC assay (SpeeDx)) was undertaken as an additional test after initial diagnosis (m2000 Realtime CT/NG assay (Abbott Molecular)) in those not already known to have had antimicrobial treatment. Data from a 6-month period (from March to September 2022) were analysed to determine treatment choice and treatment outcome. RESULTS: A total of 998 clinical samples tested positive for N. gonorrhoeae in 682 episodes of infection. Of the 560 (56%) samples eligible for resistance testing, 269 (48.0%) were reported as wild-type, 180 (32.1%) were predicted to be resistant, 63 (11.3%) had an indeterminate resistance profile, and in 48 (8.6%) samples, N. gonorrhoeae was not detected. Ciprofloxacin was prescribed in 172 (75%) of 228 episodes in which the wild-type strain was detected. Four (2%) of those treated with ciprofloxacin had a positive test-of-cure sample by NAAT, with no reinfection risk. All four had ciprofloxacin-susceptible infection by phenotypic antimicrobial susceptibility testing. CONCLUSIONS: In routine practice in a large UK clinic, molecular ciprofloxacin resistance testing led to a significant shift in antibiotic use, reducing use of ceftriaxone. Testing can be targeted to reduce unnecessary additional testing. Longer term impact on antimicrobial resistance requires ongoing surveillance.

Top Tips for Designing and Managing a Public Engagement Laboratory.

Public engagement with science is a core facet of the broader science ecosystem, in particular the science research and science education sectors. In this article we demarcate the benefits of dedicated laboratories along with practitioner advice pertaining to the design and running of a public engagement learning environment. A practicing public engagement laboratory and one that is currently being developed are used as illustrative cases to provide real-world insights to public engagement practitioners.

Consideration of within-patient diversity highlights transmission pathways and antimicrobial resistance gene variability in vancomycin-resistant Enterococcus faecium.

BACKGROUND: WGS is increasingly being applied to healthcare-associated vancomycin-resistant Enterococcus faecium (VREfm) outbreaks. Within-patient diversity could complicate transmission resolution if single colonies are sequenced from identified cases. OBJECTIVES: Determine the impact of within-patient diversity on transmission resolution of VREfm. MATERIALS AND METHODS: Fourteen colonies were collected from VREfm positive rectal screens, single colonies were collected from clinical samples and Illumina WGS was performed. Two isolates were selected for Oxford Nanopore sequencing and hybrid genome assembly to generate lineage-specific reference genomes. Mapping to closely related references was used to identify genetic variations and closely related genomes. A transmission network was inferred for the entire genome set using Phyloscanner. RESULTS AND DISCUSSION: In total, 229 isolates from 11 patients were sequenced. Carriage of two or three sequence types was detected in 27% of patients. Presence of antimicrobial resistance genes and plasmids was variable within genomes from the same patient and sequence type. We identified two dominant sequence types (ST80 and ST1424), with two putative transmission clusters of two patients within ST80, and a single cluster of six patients within ST1424. We found transmission resolution was impaired using fewer than 14 colonies. CONCLUSIONS: Patients can carry multiple sequence types of VREfm, and even within related lineages the presence of mobile genetic elements and antimicrobial resistance genes can vary. VREfm within-patient diversity could be considered in future to aid accurate resolution of transmission networks.

Whole Genome Sequencing and Molecular Epidemiology of Clinical Isolates of Staphylococcus aureus from Algeria.

Staphylococcus aureus is an important pathogen responsible for various healthcare- and community-acquired infections. In this study, whole genome sequencing (WGS) was used to genotype S. aureus clinical isolates from two hospitals in Algeria and to characterize their genetic determinants of antimicrobial resistance. Seventeen S. aureus isolates were included in this study. WGS, single-nucleotide polymorphism (SNP)-based phylogenetic analysis, in silico multilocus sequence typing (MLST), spa and staphylococcal cassette chromosome mec (SCCmec) typing and in silico antimicrobial resistance profiling were performed. Phenotypic antibiotic susceptibility testing was performed using the Vitek 2 system and the disk diffusion method. The isolates were separated into sequence types (STs), with ST80 being predominant; five clonal complexes (CCs); four spa types (t044, t127, t368, t386); and two SCCmec types (IVc and IVa). Whole genome analysis revealed the presence of the resistance genes mecA, blaZ, ermC, fusB, fusC, tetK, aph(3')-IIIa and aad(6) and mutations conferring resistance in the genes parC and fusA. The rate of multidrug resistance (MDR) was 64%. This work provides a high-resolution characterization of methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) isolates and emphasizes the importance of continuous surveillance to monitor the spread of S. aureus in healthcare settings in the country.

Development of an amplicon-based sequencing approach in response to the global emergence of mpox.

The 2022 multicountry mpox outbreak concurrent with the ongoing Coronavirus Disease 2019 (COVID-19) pandemic further highlighted the need for genomic surveillance and rapid pathogen whole-genome sequencing. While metagenomic sequencing approaches have been used to sequence many of the early mpox infections, these methods are resource intensive and require samples with high viral DNA concentrations. Given the atypical clinical presentation of cases associated with the outbreak and uncertainty regarding viral load across both the course of infection and anatomical body sites, there was an urgent need for a more sensitive and broadly applicable sequencing approach. Highly multiplexed amplicon-based sequencing (PrimalSeq) was initially developed for sequencing of Zika virus, and later adapted as the main sequencing approach for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Here, we used PrimalScheme to develop a primer scheme for human monkeypox virus that can be used with many sequencing and bioinformatics pipelines implemented in public health laboratories during the COVID-19 pandemic. We sequenced clinical specimens that tested presumptively positive for human monkeypox virus with amplicon-based and metagenomic sequencing approaches. We found notably higher genome coverage across the virus genome, with minimal amplicon drop-outs, in using the amplicon-based sequencing approach, particularly in higher PCR cycle threshold (Ct) (lower DNA titer) samples. Further testing demonstrated that Ct value correlated with the number of sequencing reads and influenced the percent genome coverage. To maximize genome coverage when resources are limited, we recommend selecting samples with a PCR Ct below 31 Ct and generating 1 million sequencing reads per sample. To support national and international public health genomic surveillance efforts, we sent out primer pool aliquots to 10 laboratories across the United States, United Kingdom, Brazil, and Portugal. These public health laboratories successfully implemented the human monkeypox virus primer scheme in various amplicon sequencing workflows and with different sample types across a range of Ct values. Thus, we show that amplicon-based sequencing can provide a rapidly deployable, cost-effective, and flexible approach to pathogen whole-genome sequencing in response to newly emerging pathogens. Importantly, through the implementation of our primer scheme into existing SARS-CoV-2 workflows and across a range of sample types and sequencing platforms, we further demonstrate the potential of this approach for rapid outbreak response.

Directions of change in intrinsic case severity across successive SARS-CoV-2 variant waves have been inconsistent.

OBJECTIVES: To determine how the intrinsic severity of successively dominant SARS-CoV-2 variants changed over the course of the pandemic. METHODS: A retrospective cohort analysis in the NHS Greater Glasgow and Clyde (NHS GGC) Health Board. All sequenced non-nosocomial adult COVID-19 cases in NHS GGC with relevant SARS-CoV-2 lineages (B.1.177/Alpha, Alpha/Delta, AY.4.2 Delta/non-AY.4.2 Delta, non-AY.4.2 Delta/Omicron, and BA.1 Omicron/BA.2 Omicron) during analysis periods were included. Outcome measures were hospital admission, ICU admission, or death within 28 days of positive COVID-19 test. We report the cumulative odds ratio; the ratio of the odds that an individual experiences a severity event of a given level vs all lower severity levels for the resident and the replacement variant after adjustment. RESULTS: After adjustment for covariates, the cumulative odds ratio was 1.51 (95% CI: 1.08-2.11) for Alpha versus B.1.177, 2.09 (95% CI: 1.42-3.08) for Delta versus Alpha, 0.99 (95% CI: 0.76-1.27) for AY.4.2 Delta versus non-AY.4.2 Delta, 0.49 (95% CI: 0.22-1.06) for Omicron versus non-AY.4.2 Delta, and 0.86 (95% CI: 0.68-1.09) for BA.2 Omicron versus BA.1 Omicron. CONCLUSIONS: The direction of change in intrinsic severity between successively emerging SARS-CoV-2 variants was inconsistent, reminding us that the intrinsic severity of future SARS-CoV-2 variants remains uncertain.

Analysis of the ARTIC V4 and V4.1 SARS-CoV-2 primers and their impact on the detection of Omicron BA.1 and BA.2 lineage-defining mutations.

The ARTIC protocol uses a multiplexed PCR approach with two primer pools tiling the entire SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) genome. Primer pool updates are necessary for accurate amplicon sequencing of evolving SARS-CoV-2 variants with novel mutations. The suitability of the ARTIC V4 and updated V4.1 primer scheme was assessed using whole genome sequencing of Omicron from clinical samples using Oxford Nanopore Technology. Analysis of Omicron BA.1 genomes revealed that 93.22 % of clinical samples generated improved genome coverage at 50× read depth with V4.1 primers when compared to V4 primers. Additionally, the V4.1 primers improved coverage of BA.1 across amplicons 76 and 88, which resulted in the detection of the variant-defining mutations G22898A, A26530G and C26577G. The Omicron BA.2 sub-variant (VUI-22JAN-01) replaced BA.1 as the dominant variant by March 2022, and analysis of 168 clinical samples showed reduced coverage across amplicons 15 and 75. Upon further interrogation of primer binding sites, a mutation at C4321T [present in 163/168 (97 %) of samples] was identified as a possible cause of complete dropout of amplicon 15. Furthermore, two mutations were identified within the primer binding regions for amplicon 75: A22786C (present in 90 % of samples) and C22792T (present in 12.5 % of samples). Together, these mutations may result in reduced coverage of amplicon 75, and further primer updates would allow the identification of the two BA.2-defining mutations present in amplicon 75: A22688G and T22679C. This work highlights the need for ongoing surveillance of primer matches as circulating variants evolve and change.

The SARS-CoV-2 Alpha variant was associated with increased clinical severity of COVID-19 in Scotland: A genomics-based retrospective cohort analysis.

OBJECTIVES: The SARS-CoV-2 Alpha variant was associated with increased transmission relative to other variants present at the time of its emergence and several studies have shown an association between Alpha variant infection and increased hospitalisation and 28-day mortality. However, none have addressed the impact on maximum severity of illness in the general population classified by the level of respiratory support required, or death. We aimed to do this. METHODS: In this retrospective multi-centre clinical cohort sub-study of the COG-UK consortium, 1475 samples from Scottish hospitalised and community cases collected between 1st November 2020 and 30th January 2021 were sequenced. We matched sequence data to clinical outcomes as the Alpha variant became dominant in Scotland and modelled the association between Alpha variant infection and severe disease using a 4-point scale of maximum severity by 28 days: 1. no respiratory support, 2. supplemental oxygen, 3. ventilation and 4. death. RESULTS: Our cumulative generalised linear mixed model analyses found evidence (cumulative odds ratio: 1.40, 95% CI: 1.02, 1.93) of a positive association between increased clinical severity and lineage (Alpha variant versus pre-Alpha variants). CONCLUSIONS: The Alpha variant was associated with more severe clinical disease in the Scottish population than co-circulating lineages.

Development of an amplicon-based sequencing approach in response to the global emergence of human monkeypox virus.

The 2022 multi-country monkeypox (mpox) outbreak concurrent with the ongoing COVID-19 pandemic has further highlighted the need for genomic surveillance and rapid pathogen whole genome sequencing. While metagenomic sequencing approaches have been used to sequence many of the early mpox infections, these methods are resource intensive and require samples with high viral DNA concentrations. Given the atypical clinical presentation of cases associated with the outbreak and uncertainty regarding viral load across both the course of infection and anatomical body sites, there was an urgent need for a more sensitive and broadly applicable sequencing approach. Highly multiplexed amplicon-based sequencing (PrimalSeq) was initially developed for sequencing of Zika virus, and later adapted as the main sequencing approach for SARS-CoV-2. Here, we used PrimalScheme to develop a primer scheme for human monkeypox virus that can be used with many sequencing and bioinformatics pipelines implemented in public health laboratories during the COVID-19 pandemic. We sequenced clinical samples that tested presumptive positive for human monkeypox virus with amplicon-based and metagenomic sequencing approaches. We found notably higher genome coverage across the virus genome, with minimal amplicon drop-outs, in using the amplicon-based sequencing approach, particularly in higher PCR cycle threshold (lower DNA titer) samples. Further testing demonstrated that Ct value correlated with the number of sequencing reads and influenced the percent genome coverage. To maximize genome coverage when resources are limited, we recommend selecting samples with a PCR cycle threshold below 31 Ct and generating 1 million sequencing reads per sample. To support national and international public health genomic surveillance efforts, we sent out primer pool aliquots to 10 laboratories across the United States, United Kingdom, Brazil, and Portugal. These public health laboratories successfully implemented the human monkeypox virus primer scheme in various amplicon sequencing workflows and with different sample types across a range of Ct values. Thus, we show that amplicon based sequencing can provide a rapidly deployable, cost-effective, and flexible approach to pathogen whole genome sequencing in response to newly emerging pathogens. Importantly, through the implementation of our primer scheme into existing SARS-CoV-2 workflows and across a range of sample types and sequencing platforms, we further demonstrate the potential of this approach for rapid outbreak response.

Bacterial and fungal communities in tracheal aspirates of intubated COVID-19 patients: a pilot study.

Co-infections with bacterial or fungal pathogens could be associated with severity and outcome of disease in COVID-19 patients. We, therefore, used a 16S and ITS-based sequencing approach to assess the biomass and composition of the bacterial and fungal communities in endotracheal aspirates of intubated COVID-19 patients. Our method combines information on bacterial and fungal biomass with community profiling, anticipating the likelihood of a co-infection is higher with (1) a high bacterial and/or fungal biomass combined with (2) predominance of potentially pathogenic microorganisms. We tested our methods on 42 samples from 30 patients. We observed a clear association between microbial outgrowth (high biomass) and predominance of individual microbial species. Outgrowth of pathogens was in line with the selective pressure of antibiotics received by the patient. We conclude that our approach may help to monitor the presence and predominance of pathogens and therefore the likelihood of co-infections in ventilated patients, which ultimately, may help to guide treatment.

Medicine Maker: An Outreach Activity for Pharmaceutical Manufacturing and Health Literacy.

Public engagement in medicine has become more important in promoting population health management and literacy. Medicine is a topic of great societal importance, and many public engagement activities have been developed to promote this area. However, they often narrowly focus on patient groups, diseases, a singular pharmaceutical drug or analytical technique. Despite the importance of these activities, general audiences are still heavily reliant on doctors and pharmacists for information about their medicine and lack basic knowledge around medication use and personal safety. Given this, a broader engagement approach is warranted to target health literacy among the wider public. "Medicine Maker" is a hands-on public engagement workshop that provides audiences with the opportunity to "manufacture" and inspect the quality of proxy or "dummy" medicine through guided inquiry. Here, we detail the development of the Medicine Maker workshop from its origins in the teaching of Irish third-level pharmacy students, to its initial application with a variety of lay audiences. Formal and informal feedback from participants indicates that the workshop can help foster a more critical understanding of medicine manufacturing, quality control, and personal health.

Presence of optrA-mediated linezolid resistance in multiple lineages and plasmids of Enterococcus faecalis revealed by long read sequencing.

Transferable linezolid resistance due to optrA, poxtA, cfr and cfr-like genes is increasingly detected in enterococci associated with animals and humans globally. We aimed to characterize the genetic environment of optrA in linezolid-resistant Enterococcus faecalis isolates from Scotland. Six linezolid-resistant E. faecalis isolated from urogenital samples were confirmed to carry the optrA gene by PCR. Short read (Illumina) sequencing showed the isolates were genetically distinct (>13900 core SNPs) and belonged to different MLST sequence types. Plasmid contents were examined using hybrid assembly of short and long read (Oxford Nanopore MinION) sequencing technologies. The optrA gene was located on distinct plasmids in each isolate, suggesting that transfer of a single plasmid did not contribute to optrA dissemination in this collection. pTM6294-2, BX5936-1 and pWE0438-1 were similar to optrA-positive plasmids from China and Japan, while the remaining three plasmids had limited similarity to other published examples. We identified the novel Tn6993 transposon in pWE0254-1 carrying linezolid (optrA), macrolide (ermB) and spectinomycin [ANT(9)-Ia] resistance genes. OptrA amino acid sequences differed by 0-20 residues. We report multiple variants of optrA on distinct plasmids in diverse strains of E. faecalis. It is important to identify the selection pressures driving the emergence and maintenance of resistance against linezolid to retain the clinical utility of this antibiotic.

Rapid detection of SARS-CoV-2 variants using allele-specific PCR.

Tracking severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants through whole genome sequencing (WGS) is vital for effective infection control and prevention (IPC) measures, but can be time-consuming and resource-heavy. We describe an in-house validation of an allele-specific polymerase chain reaction (ASP) variant assay to detect variants of concern (VOC). ASP sensitivity for detecting Delta, Alpha and Beta was 99.45 %, 100 %, and 66.67 %, respectively, compared with WGS. Specificity was 100 % in detecting all three VOC. ASP generated results 1.3 days faster compared with WGS. These findings suggest using variant assays such as ASP may enhance epidemiological surveillance and IPC measures.

Genomics-informed outbreak investigations of SARS-CoV-2 using civet.

The scale of data produced during the SARS-CoV-2 pandemic has been unprecedented, with more than 13 million sequences shared publicly at the time of writing. This wealth of sequence data provides important context for interpreting local outbreaks. However, placing sequences of interest into national and international context is difficult given the size of the global dataset. Often outbreak investigations and genomic surveillance efforts require running similar analyses again and again on the latest dataset and producing reports. We developed civet (cluster investigation and virus epidemiology tool) to aid these routine analyses and facilitate virus outbreak investigation and surveillance. Civet can place sequences of interest in the local context of background diversity, resolving the query into different 'catchments' and presenting the phylogenetic results alongside metadata in an interactive, distributable report. Civet can be used on a fine scale for clinical outbreak investigation, for local surveillance and cluster discovery, and to routinely summarise the virus diversity circulating on a national level. Civet reports have helped researchers and public health bodies feedback genomic information in the appropriate context within a timeframe that is useful for public health.

Evaluation of the molecular detection of ciprofloxacin resistance in Neisseria gonorrhoeae by the ResistancePlus GC assay (SpeeDx).

There is growing concern due to the emergence of multidrug resistance in Neisseria gonorrhoeae. A rapid molecular test which guides and provides antimicrobial susceptibility knowledge prior to start of treatment is needed. This study evaluated the clinical performance of the ResistancePlus GC assay compared to in-house PCR and antimicrobial susceptibility results for ciprofloxacin resistance. Samples were selected from a range of sites with corresponding cultures isolated from the same patient episode. The ResistancePlus GC assay displayed high sensitivity for N. gonorrhoeae detection (98.5%) and gyrA detection (97.1%). There was high agreement (98.9%) between the ResistancePlus GC assay and culture phenotype. Mixed population testing showed that the assay was able to detect resistance in a sample containing a minority variant of 27% resistant. The ResistancePlus GC assay performed well and could be used to provide a clinically relevant indication of ciprofloxacin susceptibility for the treatment of gonorrhoea.

Genomic epidemiology reveals multiple introductions of SARS-CoV-2 from mainland Europe into Scotland.

Coronavirus disease 2019 (COVID-19) was first diagnosed in Scotland on 1 March 2020. During the first month of the outbreak, 2,641 cases of COVID-19 led to 1,832 hospital admissions, 207 intensive care admissions and 126 deaths. We aimed to identify the source and number of introductions of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into Scotland using a combined phylogenetic and epidemiological approach. Sequencing of 1,314 SARS-CoV-2 viral genomes from available patient samples enabled us to estimate that SARS-CoV-2 was introduced to Scotland on at least 283 occasions during February and March 2020. Epidemiological analysis confirmed that early introductions of SARS-CoV-2 originated from mainland Europe (the majority from Italy and Spain). We identified subsequent early outbreaks in the community, within healthcare facilities and at an international conference. Community transmission occurred after 2 March, 3 weeks before control measures were introduced. Earlier travel restrictions or quarantine measures, both locally and internationally, would have reduced the number of COVID-19 cases in Scotland. The risk of multiple reintroduction events in future waves of infection remains high in the absence of population immunity.

Multicenter Evaluation of QIAstat-Dx Respiratory Panel V2 for Detection of Viral and Bacterial Respiratory Pathogens.

QIAstat-Dx Respiratory Panel V2 (RP) is a novel molecular-method-based syndromic test for the simultaneous and rapid (∼70-min) detection of 18 viral and 3 bacterial pathogens causing respiratory infections. This report describes the first multicenter retrospective comparison of the performance of the QIAstat-Dx RP assay to the established ePlex Respiratory Pathogen Panel (RPP) assay, for which we used 287 respiratory samples from patients suspected with respiratory infections. The QIAstat-Dx RP assay detected 312 (92%) of the 338 respiratory targets that were detected by the ePlex RPP assay. Most of the discrepant results have been observed in the low-pathogen-load samples. In addition, the QIAstat-Dx RP assay detected 19 additional targets in 19 respiratory samples that were not detected by the ePlex RPP assay. Nine of these discordant targets were considered to represent true positives after discrepancy testing by a third method. The main advantage of the QIAstat-Dx system compared to other syndromic testing systems, including the ePlex RPP assay, is the ability to generate cycle threshold (CT ) values, which could help with the interpretation of results. Taking the data together, this study showed good performance of the QIAstat-Dx RP assay in comparison to the ePlex RPP assay for the detection of respiratory pathogens. The QIAstat-Dx RP assay offers a new, rapid, and accurate sample-to-answer multiplex panel for the detection of the most common viral and bacterial respiratory pathogens and therefore has the potential to direct appropriate therapy and infection control precautions.