Detection of mixed infection from bacterial whole genome sequence data allows assessment of its role in Clostridium difficile transmission

Bacterial whole genome sequencing offers the prospect of rapid and high precision investigation of infectious disease outbreaks. Close genetic relationships between microorganisms isolated from different infected cases suggest transmission is a strong possibility, whereas transmission between cases with genetically distinct bacterial isolates can be excluded. However, undetected mixed infections-infection with ≥2 unrelated strains of the same species where only one is sequenced-potentially impairs exclusion of transmission with certainty, and may therefore limit the utility of this technique. We investigated the problem by developing a computationally efficient method for detecting mixed infection without the need for resource-intensive independent sequencing of multiple bacterial colonies. Given the relatively low density of single nucleotide polymorphisms within bacterial sequence data, direct reconstruction of mixed infection haplotypes from current short-read sequence data is not consistently possible. We therefore use a two-step maximum likelihood-based approach, assuming each sample contains up to two infecting strains. We jointly estimate the proportion of the infection arising from the dominant and minor strains, and the sequence divergence between these strains. In cases where mixed infection is confirmed, the dominant and minor haplotypes are then matched to a database of previously sequenced local isolates. We demonstrate the performance of our algorithm with in silico and in vitro mixed infection experiments, and apply it to transmission of an important healthcare-associated pathogen, Clostridium difficile. Using hospital ward movement data in a previously described stochastic transmission model, 15 pairs of cases enriched for likely transmission events associated with mixed infection were selected. Our method identified four previously undetected mixed infections, and a previously undetected transmission event, but no direct transmission between the pairs of cases under investigation. These results demonstrate that mixed infections can be detected without additional sequencing effort, and this will be important in assessing the extent of cryptic transmission in our hospitals

Increasing burden of community-acquired pneumonia leading to hospitalisation, 1998-2014

BACKGROUND: Community-acquired pneumonia (CAP) is a major cause of mortality and morbidity in many countries but few recent large-scale studies have examined trends in its incidence. METHODS: Incidence of CAP leading to hospitalisation in one UK region (Oxfordshire) was calculated over calendar time using routinely collected diagnostic codes, and modelled using piecewise-linear Poisson regression. Further models considered other related diagnoses, typical administrative outcomes, and blood and microbiology test results at admission to determine whether CAP trends could be explained by changes in case-mix, coding practices or admission procedures. RESULTS: CAP increased by 4.2%/year (95% CI 3.6 to 4.8) from 1998 to 2008, and subsequently much faster at 8.8%/year (95% CI 7.8 to 9.7) from 2009 to 2014. Pneumonia-related conditions also increased significantly over this period. Length of stay and 30-day mortality decreased slightly in later years, but the proportions with abnormal neutrophils, urea and C reactive protein (CRP) did not change (p>0.2). The proportion with severely abnormal CRP (>100 mg/L) decreased slightly in later years. Trends were similar in all age groups. Streptococcus pneumoniae was the most common causative organism found; however other organisms, particularly Enterobacteriaceae, increased in incidence over the study period (p<0.001). CONCLUSIONS: Hospitalisations for CAP have been increasing rapidly in Oxfordshire, particularly since 2008. There is little evidence that this is due only to changes in pneumonia coding, an ageing population or patients with substantially less severe disease being admitted more frequently. Healthcare planning to address potential further increases in admissions and consequent antibiotic prescribing should be a priority

Macular Telangiectasia Type 2: A Classification System Using MultiModal Imaging MacTel Project Report Number 10

Purpose: To develop a severity classification for macular telangiectasia type 2 (MacTel) disease using multimodal imaging. Design: An algorithm was used on data from a prospective natural history study of MacTel for classification development. Subjects: A total of 1733 participants enrolled in an international natural history study of MacTel. Methods: The Classification and Regression Trees (CART), a predictive nonparametric algorithm used in machine learning, analyzed the features of the multimodal imaging important for the development of a classification, including reading center gradings of the following digital images: stereoscopic color and red-free fundus photographs, fluorescein angiographic images, fundus autofluorescence images, and spectral-domain (SD)-OCT images. Regression models that used least square method created a decision tree using features of the ocular images into different categories of disease severity. Main Outcome Measures: The primary target of interest for the algorithm development by CART was the change in best-corrected visual acuity (BCVA) at baseline for the right and left eyes. These analyses using the algorithm were repeated for the BCVA obtained at the last study visit of the natural history study for the right and left eyes. Results: The CART analyses demonstrated 3 important features from the multimodal imaging for the classification: OCT hyper-reflectivity, pigment, and ellipsoid zone loss. By combining these 3 features (as absent, present, noncentral involvement, and central involvement of the macula), a 7-step scale was created, ranging from excellent to poor visual acuity. At grade 0, 3 features are not present. At the most severe grade, pigment and exudative neovascularization are present. To further validate the classification, using the Generalized Estimating Equation regression models, analyses for the annual relative risk of progression over a period of 5 years for vision loss and for progression along the scale were performed. Conclusions: This analysis using the data from current imaging modalities in participants followed in the MacTel natural history study informed a classification for MacTel disease severity featuring variables from SD-OCT. This classification is designed to provide better communications to other clinicians, researchers, and patients. Financial Disclosure(s): Proprietary or commercial disclosure may be found after the references

Asymptomatic Clostridium difficile Colonisation and Onward Transmission

Introduction Combined genotyping/whole genome sequencing and epidemiological data suggest that in endemic settings only a minority of Clostridium difficile infection, CDI, is acquired from other cases. Asymptomatic patients are a potential source for many unexplained cases. Methods We prospectively screened a cohort of medical inpatients in a UK teaching hospital for asymptomatic C. difficile carriage using stool culture. Electronic and questionnaire data were used to determine risk factors for asymptomatic carriage by logistic regression. Carriage isolates were compared with all hospital/community CDI cases from the same geographic region, from 12 months before the study to 3 months after, using whole genome sequencing and hospital admission data, assessing particularly for evidence of onward transmission from asymptomatic cases. Results Of 227 participants recruited, 132 provided ≥1 stool samples for testing. 18 participants were culture-positive for C. difficile, 14/132(11%) on their first sample. Independent risk factors for asymptomatic carriage were patient reported loose/frequent stool (but not meeting CDI criteria of ≥3 unformed stools in 24 hours), previous overnight hospital stay within 6 months, and steroid/immunosuppressant medication in the last 6 months (all p≤0.02). Surprisingly antibiotic exposure in the last 6 months was independently associated with decreased risk of carriage (p = 0.005). The same risk factors were identified excluding participants reporting frequent/loose stool. 13/18(72%) asymptomatically colonised patients carried toxigenic strains from common disease-causing lineages found in cases. Several plausible transmission events to asymptomatic carriers were identified, but in this relatively small study no clear evidence of onward transmission from an asymptomatic case was seen. Conclusions Transmission events from any one asymptomatic carrier are likely to be relatively rare, but as asymptomatic carriage is common, it may still be an important source of CDI, which could be quantified in larger studies. Risk factors established for asymptomatic carriage may help identify patients for inclusion in such studies

Swab pooling enables rapid expansion of high-throughput capacity for SARS-CoV-2 community testing

Background: The challenges of rapid upscaling of testing capacity were a major lesson from the COVID-19 pandemic response. The need for process adjustments in high-throughput testing laboratories made sample pooling a challenging option to implement. / Objective: This study aimed to evaluate whether pooling samples at source (swab pooling) was as effective as qRT-PCR testing of individuals in identifying cases of SARS-CoV-2 in real-world community testing conditions using the same high-throughput pipeline. / Methods: Two cohorts of 10 (Pool10: 1,030 participants and 103 pools) and 6 (Pool6: 1,284 participants and 214 pools) samples per pool were tested for concordance, sensitivity, specificity, and Ct value differences with individual testing as reference. Results: Swab pooling allowed unmodified application of an existing high-throughput SARS-Cov-2 testing pipeline with only marginal loss of accuracy. For Pool10, concordance was 98.1% (95% Confidence interval: 93.3–99.8%), sensitivity was 95.7% (85.5–99.5%), and specificity was 100.0% (93.6–100.0%). For Pool6, concordance was 97.2% (94.0–99.0%), sensitivity was 97.5% (93.7–99.3%), and specificity was 96.4% (87.7–99.6%). Differences of outcomes measure between pool size were not significant. Most positive individual samples, which were not detected in pools, had very low viral concentration. If only individual samples with a viral concentration > 400 copies/ml (i.e. Ct value < 30) were considered positive, the overall sensitivity of pooling increased to 99.5%. / Conclusion: The study demonstrated high sensitivity and specificity by swab pooling and the immediate capability of high-throughput laboratories to implement this method making it an option in planning of rapid upscaling of laboratory capacity for future pandemics

Short-term genome stability of serial Clostridium difficile ribotype 027 isolates in an experimental gut model and recurrent human disease

Background Clostridium difficile whole genome sequencing has the potential to identify related isolates, even among otherwise indistinguishable strains, but interpretation depends on understanding genomic variation within isolates and individuals. Methods Serial isolates from two scenarios were whole genome sequenced. Firstly, 62 isolates from 29 timepoints from three in vitro gut models, inoculated with a NAP1/027 strain. Secondly, 122 isolates from 44 patients (2–8 samples/patient) with mostly recurrent/on-going symptomatic NAP-1/027 C. difficile infection. Reference-based mapping was used to identify single nucleotide variants (SNVs). Results Across three gut model inductions, two with antibiotic treatment, total 137 days, only two new SNVs became established. Pre-existing minority SNVs became dominant in two models. Several SNVs were detected, only present in the minority of colonies at one/two timepoints. The median (inter-quartile range) [range] time between patients’ first and last samples was 60 (29.5–118.5) [0–561] days. Within-patient C. difficile evolution was 0.45 SNVs/called genome/year (95%CI 0.00–1.28) and within-host diversity was 0.28 SNVs/called genome (0.05–0.53). 26/28 gut model and patient SNVs were non-synonymous, affecting a range of gene targets. Conclusions The consistency of whole genome sequencing data from gut model C. difficile isolates, and the high stability of genomic sequences in isolates from patients, supports the use of whole genome sequencing in detailed transmission investigations

Review of cigarette smoking and tuberculosis in China: intervention is needed for smoking cessation among tuberculosis patients

<p>Abstract</p> <p>Background</p> <p>As a risk factor of tuberculosis (TB), tobacco smoking has increased substantially over the past three decades, especially in developing countries. However, the association between smoking and TB, which has been shown to exist in different studies with different ethnic background, has not yet received sufficient attention in terms of TB care standards and research in China.</p> <p>Methods</p> <p>An observational study was conducted in two rural areas of China. A total of 613 TB patients frequency matched with 1226 controls were interviewed by using a structured questionnaire. The associations between cigarette smoking and risk of TB were estimated by computing odds ratios (ORs) and 95% confidence intervals (95% CIs) from logistic regression model. Patients' smoking behavior and patterns of smoking cessation were followed after TB diagnosis. Multivariate Cox proportional hazards model was applied to calculate hazard ratios (HRs) and 95% confidence intervals (95% CIs) in analyzing the risk factors for smoking relapse. The Kaplan-Meier estimate was computed to plot the ability of smoking-free after cessation among different groups, with the Log-rank test being used to compare the difference.</p> <p>Results</p> <p>The proportion of cigarette smoking was 54.6% in TB cases, which was significantly higher than that in controls (45.1%) with adjusted OR of 1.93(95% CI: 1.51–2.48). Though 54.9% smokers stopped smoking after being diagnosed with TB, more than 18% relapsed during the follow-up period. The proportion of relapse was higher within 6–9 months (6%) and 12–15 months (11%) after cessation. In the Cox regression estimates adjusted for age and gender, compared with those highly educated and previously treated patients, the hazard ratios of smoking relapse were 3.48(95% CI: 1.28–9.47) for less educated (< 6 years) and 4.30(95% CI: 1.01–18.30) for newly treated patients, respectively.</p> <p>Conclusion</p> <p>Cigarette smoking is associated with TB in the Chinese. Interventions of smoking cessation are recommended to be included in the current TB control practice.</p

Abnormal Retinal Reflectivity To Short-Wavelength Light In Type 2 Idiopathic Macular Telangiectasia

PURPOSE: Macular telangiectasia Type 2 (MacTel) is a bilateral, progressive, potentially blinding retinal disease characterized by vascular and neurodegenerative signs, including an increased parafoveal reflectivity to blue light. Our aim was to investigate the relationship of this sign with other signs of macular telangiectasia Type 2 in multiple imaging modalities. METHODS: Participants were selected from the MacTel Type 2 study, based on a confirmed diagnosis and the availability of images. The extent of signs in blue-light reflectance, fluorescein angiographic, optical coherence tomographic, and single- and dual-wavelength autofluorescence images were analyzed. RESULTS: A well-defined abnormality of the perifovea is demonstrated by dual-wavelength autofluorescence and blue-light reflectance in early disease. The agreement in area size of the abnormalities in dual-wavelength autofluorescence and in blue-light reflectance images was excellent: for right eyes: ρ = 0.917 (P < 0.0001, 95% confidence interval 0.855-0.954, n = 46) and for left eyes: ρ = 0.952 (P < 0.0001, 95% confidence interval 0.916-0.973, n = 49). Other changes are less extensive initially and expand later to occupy that area and do not extend beyond it. CONCLUSION: Our findings indicate that abnormal metabolic handling of luteal pigment and physical changes giving rise to increased reflectance are widespread in the macula throughout the natural history of the disease, precede other changes, and are relevant to early diagnosis

Chromosome 15q25 (CHRNA3-CHRNA5) Variation Impacts Indirectly on Lung Cancer Risk

Genetic variants at the 15q25 CHRNA5-CHRNA3 locus have been shown to influence lung cancer risk however there is controversy as to whether variants have a direct carcinogenic effect on lung cancer risk or impact indirectly through smoking behavior. We have performed a detailed analysis of the 15q25 risk variants rs12914385 and rs8042374 with smoking behavior and lung cancer risk in 4,343 lung cancer cases and 1,479 controls from the Genetic Lung Cancer Predisposition Study (GELCAPS). A strong association between rs12914385 and rs8042374, and lung cancer risk was shown, odds ratios (OR) were 1.44, (95% confidence interval (CI): 1.29–1.62, P = 3.69×10−10) and 1.35 (95% CI: 1.18–1.55, P = 9.99×10−6) respectively. Each copy of risk alleles at rs12914385 and rs8042374 was associated with increased cigarette consumption of 1.0 and 0.9 cigarettes per day (CPD) (P = 5.18×10−5 and P = 5.65×10−3). These genetically determined modest differences in smoking behavior can be shown to be sufficient to account for the 15q25 association with lung cancer risk. To further verify the indirect effect of 15q25 on the risk, we restricted our analysis of lung cancer risk to never-smokers and conducted a meta-analysis of previously published studies of lung cancer risk in never-smokers. Never-smoker studies published in English were ascertained from PubMed stipulating - lung cancer, risk, genome-wide association, candidate genes. Our study and five previously published studies provided data on 2,405 never-smoker lung cancer cases and 7,622 controls. In the pooled analysis no association has been found between the 15q25 variation and lung cancer risk (OR = 1.09, 95% CI: 0.94–1.28). This study affirms the 15q25 association with smoking and is consistent with an indirect link between genotype and lung cancer risk