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Here we describe a method for the detection of Clostridium difficile from stool using a novel low-complexity and rapid extraction process called Heat Elution (HE). The HE method is two-step and takes just 10 minutes, no specialist instruments are required and there is minimal hands-on time. A test method using HE was developed in conjunction with Loop-mediated Isothermal Amplification (LAMP) combined with the real-time bioluminescent reporter system known as BART targeting the toxin B gene (tcdB). The HE-LAMP-BART method was evaluated in a pilot study on clinical fecal samples (tcdB(+), n = 111; tcdB(-), n= 107). The HE-LAMP-BART method showed 95.5% sensitivity and 100% specificity against a gold standard reference method using cytotoxigenic culture and also a silica-based robotic extraction followed by tcdB PCR to control for storage. From sample to result, the HE-LAMP-BART method typically took 50 minutes, whereas the PCR method took >2.5 hours. In a further study (tcdB(+), n = 47; tcdB(-), n= 28) HE-LAMP-BART was compared to an alternative commercially available LAMP-based method, Illumigene (Meridian Bioscience, OH), and yielded 87.2% sensitivity and 100% specificity for the HE-LAMP-BART method compared to 76.6% and 100%, respectively, for Illumigene against the reference method. A subset of 27 samples (tcdB(+), n = 25; tcdB(-), n= 2) were further compared between HE-LAMP-BART, Illumigene, GeneXpert (Cepheid, Sunnyvale, CA) and RIDA®QUICK C. difficile Toxin A/B lateral flow rapid test (R-Biopharm, Darmstadt, Germany) resulting in sensitivities of HE-LAMP-BART 92%, Illumigene 72% GeneXpert 96% and RIDAQuick 76% against the reference method. The HE-LAMP-BART method offers the advantages of molecular based approaches without the cost and complexity usually associated with molecular tests. Further, the rapid time-to-result and simple protocol means the method can be applied away from the centralized laboratory settings.

Here we describe a method for the detection of Clostridium difficile from stool using a novel low-complexity and rapid extraction process called Heat Elution (HE). The HE method is two-step and takes just 10 minutes, no specialist instruments are required and there is minimal hands-on time. A test method using HE was developed in conjunction with Loop-mediated Isothermal Amplification (LAMP) combined with the real-time bioluminescent reporter system known as BART targeting the toxin B gene (tcdB). The HE-LAMP-BART method was evaluated in a pilot study on clinical fecal samples (tcdB+, n = 111; tcdB-, n = 107). The HE-LAMP-BART method showed 95.5% sensitivity and 100% specificity against a gold standard reference method using cytotoxigenic culture and also a silica-based robotic extraction followed by tcdB PCR to control for storage. From sample to result, the HE-LAMP-BART method typically took 50 minutes, whereas the PCR method took >2.5 hours. In a further study (tcdB+, n = 47; tcdB-, n = 28) HE-LAMP-BART was compared to an alternative commercially available LAMP-based method, Illumigene (Meridian Bioscience, OH), and yielded 87.2% sensitivity and 100% specificity for the HE-LAMP-BART method compared to 76.6% and 100%, respectively, for Illumigene against the reference method. A subset of 27 samples (tcdB+, n = 25; tcdB-, n = 2) were further compared between HE-LAMP-BART, Illumigene, GeneXpert (Cepheid, Sunnyvale, CA) and RIDA registered QUICK C. difficile Toxin A/B lateral flow rapid test (R-Biopharm, Darmstadt, Germany) resulting in sensitivities of HE-LAMP-BART 92%, Illumigene 72% GeneXpert 96% and RIDAQuick 76% against the reference method. The HE-LAMP-BART method offers the advantages of molecular based approaches without the cost and complexity usually associated with molecular tests. Further, the rapid time-to-result and simple protocol means the method can be applied away from the centralized laboratory settings.

The burden and influence of health-care associated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections is unknown. We aimed to examine the use of rapid SARS-CoV-2 sequencing combined with detailed epidemiological analysis to investigate health-care associated SARS-CoV-2 infections and inform infection control measures. In this prospective surveillance study, we set up rapid SARS-CoV-2 nanopore sequencing from PCR-positive diagnostic samples collected from our hospital (Cambridge, UK) and a random selection from hospitals in the East of England, enabling sample-to-sequence in less than 24 h. We established a weekly review and reporting system with integration of genomic and epidemiological data to investigate suspected health-care associated COVID-19 cases. Between March 13 and April 24, 2020, we collected clinical data and samples from 5613 patients with COVID-19 from across the East of England. We sequenced 1000 samples producing 747 high-quality genomes. We combined epidemiological and genomic analysis of the 299 patients from our hospital and identified 35 clusters of identical viruses involving 159 patients. 92 (58%) of 159 patients had strong epidemiological links and 32 (20%) patients had plausible epidemiological links. These results were fed back to clinical, infection control, and hospital management teams, leading to infection-control interventions and informing patient safety reporting. We established real-time genomic surveillance of SARS-CoV-2 in a UK hospital and showed the benefit of combined genomic and epidemiological analysis for the investigation of health-care associated COVID-19. This approach enabled us to detect cryptic transmission events and identify opportunities to target infection-control interventions to further reduce health-care associated infections. Our findings have important implications for national public health policy as they enable rapid tracking and investigation of infections in hospital and community settings. COVID-19 Genomics UK (supported by UK Research and Innovation, the National Institute of Health Research, the Wellcome Sanger Institute), the Wellcome Trust, the Academy of Medical Sciences and the Health Foundation, and the National Institute for Health Research Cambridge Biomedical Research Centre.

Background Pandemic COVID-19 caused by the coronavirus SARS-CoV-2 has a high incidence of patients with severe acute respiratory syndrome (SARS). Many of these patients require admission to an intensive care unit (ICU) for invasive ventilation and are at significant risk of developing a secondary, ventilator-associated pneumonia (VAP). Objectives To study the incidence of VAP and bacterial lung microbiome composition of ventilated COVID-19 and non-COVID-19 patients. Methods In this retrospective observational study, we compared the incidence of VAP and secondary infections using a combination of microbial culture and a TaqMan multi-pathogen array. In addition, we determined the lung microbiome composition using 16S RNA analysis in a subset of samples. The study involved 81 COVID-19 and 144 non-COVID-19 patients receiving invasive ventilation in a single University teaching hospital between March 15th 2020 and August 30th 2020. Results COVID-19 patients were significantly more likely to develop VAP than patients without COVID (Cox proportional hazard ratio 2.01 95% CI 1.14–3.54, p  = 0.0015) with an incidence density of 28/1000 ventilator days versus 13/1000 for patients without COVID ( p  = 0.009). Although the distribution of organisms causing VAP was similar between the two groups, and the pulmonary microbiome was similar, we identified 3 cases of invasive aspergillosis amongst the patients with COVID-19 but none in the non-COVID-19 cohort. Herpesvirade activation was also numerically more frequent amongst patients with COVID-19. Conclusion COVID-19 is associated with an increased risk of VAP, which is not fully explained by the prolonged duration of ventilation. The pulmonary dysbiosis caused by COVID-19, and the causative organisms of secondary pneumonia observed are similar to that seen in critically ill patients ventilated for other reasons.

Background Although Mycoplasma genitalium is a common sexually transmitted pathogen causing clinically distinct diseases both in male and females, few genomes have been sequenced up to now, due mainly to its fastidious nature and slow growth. Hence, we lack a robust phylogenetic framework to provide insights into the population structure of the species. Currently our understanding of the nature and diversity of M. genitalium relies on molecular tests targeting specific genes or regions of the genome and knowledge is limited by a general under-testing internationally. This is set against a background of drug resistance whereby M. genitalium has developed resistance to mainly all therapeutic antimicrobials. Results We sequenced 28 genomes of Mycoplasma genitalium from temporally (1980–2010) and geographically (Europe, Japan, Australia) diverse sources. All the strain showed essentially the same genomic content without any accessory regions found. However, we identified extensive recombination across their genomes with a total of 25 regions showing heightened levels of SNP density. These regions include the MgPar loci, associated with host interactions, as well as other genes that could also be involved in this role. Using these data, we generated a robust phylogeny which shows that there are two main clades with differing degrees of genomic variability. SNPs found in region V of 23S rRNA and parC were consistent with azithromycin/erythromycin and fluoroquinolone resistances, respectively, and with their phenotypic MIC data. Conclusions The sequence data here generated is essential for designing rational approaches to type and track Mycoplasma genitalium as antibiotic resistance increases. It represents a first approach to its population genetics to better appreciate the role of this organism as a sexually transmitted pathogen.

The aim of this systematic review is to provide an overview of the literature on the effects of bariatric surgery on obesity-associated electrocardiogram (ECG) abnormalities and cardiac arrhythmias. Fourteen studies were included with a methodological quality ranging from poor to good. Majority of the studies showed a significant decrease of QT interval and related measures after bariatric surgery. Seven studies were included in the meta-analysis on effects of bariatric surgery on QTc interval and a significant decrease in QTc interval of − 33.6 ms, 95%CI [− 49.8 to − 17.4] was seen. Bariatric surgery results in significant decrease in QTc interval and P-wave dispersion, i.e., a normalization of initial pathology. The effects on atrial fibrillation are conflicting and not yet fully understood.

COVID-19 poses a major challenge to care homes, as SARS-CoV-2 is readily transmitted and causes disproportionately severe disease in older people. Here, 1167 residents from 337 care homes were identified from a dataset of 6600 COVID-19 cases from the East of England. Older age and being a care home resident were associated with increased mortality. SARS-CoV-2 genomes were available for 700 residents from 292 care homes. By integrating genomic and temporal data, 409 viral clusters within the 292 homes were identified, indicating two different patterns – outbreaks among care home residents and independent introductions with limited onward transmission. Approximately 70% of residents in the genomic analysis were admitted to hospital during the study, providing extensive opportunities for transmission between care homes and hospitals. Limiting viral transmission within care homes should be a key target for infection control to reduce COVID-19 mortality in this population.

Spanish bunch groundnut varieties occupy most of the cultivated area in Asia and Africa, and these varieties lack required 2-3 weeks of fresh seed dormancy (FSD) hampering kernel quality. Genomic breeding can help to improve commercial groundnut cultivars for FSD in a shorter time with greater precision. In this regard, a recombinant inbred line (RIL) population from the cross ICGV 02266 (non-dormant) × ICGV 97045 (dormant) was developed and genotyped with a 5 K mid-density genotyping assay. A linkage map was constructed with 325 SNP loci spanning a total map length of 2335.3 cM and five major QTLs were identified on chromosomes Ah01, Ah11, Ah06, Ah16 and Ah17. Based on differential gene expression using transcriptomic information from dormant (Tifrunner) and non-dormant (ICGV 91114) genotypes, histone deacetylases, histone-lysine N-methyltransferase, cytochrome P450, protein kinases, and ethylene-responsive transcription factor were identified as key regulators involved in the hormonal regulation of dormancy. Six Kompetitive Allele Specific PCR (KASP) markers were successfully validated in the diverse panel including selected RILs of the same population and germplasm lines. These validated KASP markers could facilitate faster breeding of new varieties with desired dormancy using marker-assisted early generation selection.

The groundnut breeding program at International Crops Research Institute for the Semi- Arid Tropics routinely performs marker-based early generation selection (MEGS) in thousands of segregating populations. The existing MEGS includes planting of segregating populations in fields or glasshouses, label tagging, and sample collection using leaf-punch from 20–25 day old plants followed by genotyping with 10 single nucleotide polymorphisms based early generation selection marker panels in a high throughput genotyping (HTPG) platform. The entire process is laborious, time consuming, and costly. Therefore, in order to save the time of the breeder and to reduce the cost during MEGS, we optimized a single seed chipping (SSC) process based MEGS protocol and deployed on large scale by genotyping >3000 samples from ongoing groundnut breeding program. In SSC-based MEGS, we used a small portion of cotyledon by slicing-off the posterior end of the single seed and transferred to the 96-deep well plate for DNA isolation and genotyping at HTPG platform. The chipped seeds were placed in 96-well seed-box in the same order of 96-well DNA sampling plate to enable tracking back to the selected individual seed. A high germination rate of 95–99% from the chipped seeds indicated that slicing of seeds from posterior end does not significantly affect germination percentage. In addition, we could successfully advance 3.5 generations in a year using a low-cost rapid generation turnover glass-house facility as compared to routine practice of two generations in field conditions. The integration of SSC based genotyping and rapid generation advancement (RGA) could significantly reduce the operational requirement of person-hours and expenses, and save a period of 6–8 months in groundnut genetics and breeding research.

Malnutrition is a major challenge globally, and groundnut is a highly nutritious self‐pollinated legume crop blessed with ample genomic resources, including the routine deployment of genomic‐assisted breeding. This study aimed to identify genomic regions and candidate genes for high iron (Fe) and zinc (Zn) content, utilizing a biparental mapping population (ICGV 00440 × ICGV 06040;). Genetic mapping and quantitative trait locus (QTL) analysis (474 mapped single‐nucleotide polymorphism loci; 1536.33 cM) using 2 seasons of phenotypic data together with genotypic data identified 5 major main‐effect QTLs for Fe content. These QTLs exhibited log‐of‐odds (LOD) scores ranging from 6.5 to 7.4, explaining phenotypic variation (PVE) ranging from 22% (qFe‐Ah01) to 30.0% (qFe‐Ah14). Likewise, four major main effect QTLs were identified for Zn content, with LOD score ranging from 4.4 to 6.8 and PVE ranging from 21.8% (qZn‐Ah01) to 32.8% (qZn‐Ah08). Interestingly, three co‐localized major and main effect QTLs (qFe‐Ah01, qZn‐Ah03, and qFe‐Ah11) were identified for both Fe and Zn contents. These genomic regions harbored key candidate genes, including zinc/iron permease transporter, bZIP transcription factor, and vacuolar iron transporter which likely play pivotal roles in the accumulation of Fe and Zn contents in seeds. The findings of this study hold potential for fine mapping and diagnostic marker development for high Fe and Zn contents in groundnut. Core Ideas The authors identified nine major main effect and two main effect QTLs for Fe and Zn content. Three co‐localized QTLs for Fe and Zn contents were detected. Genomic regions harbored key candidate genes.