Showing papers by "Derrick W. Crook published in 2013"

Whole-genome sequencing to delineate Mycobacterium tuberculosis outbreaks: a retrospective observational study

Abstract: Summary Background Tuberculosis incidence in the UK has risen in the past decade. Disease control depends on epidemiological data, which can be difficult to obtain. Whole-genome sequencing can detect microevolution within Mycobacterium tuberculosis strains. We aimed to estimate the genetic diversity of related M tuberculosis strains in the UK Midlands and to investigate how this measurement might be used to investigate community outbreaks. Methods In a retrospective observational study, we used Illumina technology to sequence M tuberculosis genomes from an archive of frozen cultures. We characterised isolates into four groups: cross-sectional, longitudinal, household, and community. We measured pairwise nucleotide differences within hosts and between hosts in household outbreaks and estimated the rate of change in DNA sequences. We used the findings to interpret network diagrams constructed from 11 community clusters derived from mycobacterial interspersed repetitive-unit–variable-number tandem-repeat data. Findings We sequenced 390 separate isolates from 254 patients, including representatives from all five major lineages of M tuberculosis . The estimated rate of change in DNA sequences was 0·5 single nucleotide polymorphisms (SNPs) per genome per year (95% CI 0·3–0·7) in longitudinal isolates from 30 individuals and 25 families. Divergence is rarely higher than five SNPs in 3 years. 109 (96%) of 114 paired isolates from individuals and households differed by five or fewer SNPs. More than five SNPs separated isolates from none of 69 epidemiologically linked patients, two (15%) of 13 possibly linked patients, and 13 (17%) of 75 epidemiologically unlinked patients (three-way comparison exact p Interpretation Whole-genome sequencing can delineate outbreaks of tuberculosis and allows inference about direction of transmission between cases. The technique could identify super-spreaders and predict the existence of undiagnosed cases, potentially leading to early treatment of infectious patients and their contacts. Funding Medical Research Council, Wellcome Trust, National Institute for Health Research, and the Health Protection Agency.

Diverse Sources of C. difficile Infection Identified on Whole-Genome Sequencing

Abstract: Background It has been thought that Clostridium difficile infection is transmitted predominantly within health care settings. However, endemic spread has hampered identification of precise sources of infection and the assessment of the efficacy of interventions. Methods From September 2007 through March 2011, we performed whole-genome sequencing on isolates obtained from all symptomatic patients with C. difficile infection identified in health care settings or in the community in Oxfordshire, United Kingdom. We compared single-nucleotide variants (SNVs) between the isolates, using C. difficile evolution rates estimated on the basis of the first and last samples obtained from each of 145 patients, with 0 to 2 SNVs expected between transmitted isolates obtained less than 124 days apart, on the basis of a 95% prediction interval. We then identified plausible epidemiologic links among genetically related cases from data on hospital admissions and community location. Results Of 1250 C. difficile cases that wer...

Differences in outcome according to Clostridium difficile testing method: a prospective multicentre diagnostic validation study of C difficile infection

Abstract: Summary Background Diagnosis of Clostridium difficile infection is controversial because of many laboratory methods, compounded by two reference methods. Cytotoxigenic culture detects toxigenic C difficile and gives a positive result more frequently (eg, because of colonisation, which means that individuals can have the bacterium but no free toxin) than does the cytotoxin assay, which detects preformed toxin in faeces. We aimed to validate the reference methods according to clinical outcomes and to derive an optimum laboratory diagnostic algorithm for C difficile infection. Methods In this prospective, multicentre study, we did cytotoxigenic culture and cytotoxin assays on 12 420 faecal samples in four UK laboratories. We also performed tests that represent the three main targets for C difficile detection: bacterium (glutamate dehydrogenase), toxins, or toxin genes. We used routine blood test results, length of hospital stay, and 30-day mortality to clinically validate the reference methods. Data were categorised by reference method result: group 1, cytotoxin assay positive; group 2, cytotoxigenic culture positive and cytotoxin assay negative; and group 3, both reference methods negative. Findings Clinical and reference assay data were available for 6522 inpatient episodes. On univariate analysis, mortality was significantly higher in group 1 than in group 2 (72/435 [16·6%] vs 20/207 [9·7%], p=0·044) and in group 3 (503/5880 [8·6%], p Interpretation We noted no increase in mortality when toxigenic C difficile alone was present. Toxin (cytotoxin assay) positivity correlated with clinical outcome, and so this reference method best defines true cases of C difficile infection. A new diagnostic category of potential C difficile excretor (cytotoxigenic culture positive but cytotoxin assay negative) could be used to characterise patients with diarrhoea that is probably not due to C difficile infection, but who can cause cross-infection. Funding Department of Health and Health Protection Agency, UK.

Predicting antimicrobial susceptibilities for Escherichia coli and Klebsiella pneumoniae isolates using whole genomic sequence data.

Abstract: sitivityofgenome-basedresistancepredictionacrossallantibioticsforbothspecieswas0.96(95%CI:0.94‐0.98)and the specificity was 0.97 (95% CI: 0.95‐0.98). Very major and major error rates were 1.2% and 2.1%, respectively. Conclusions: Our method was as sensitive and specific as routinely deployed phenotypic methods. Validation against larger datasets and formal assessments of cost and turnaround time in a routine laboratory setting are warranted.

Relationship Between Bacterial Strain Type, Host Biomarkers, and Mortality in Clostridium difficile Infection

Abstract: (See the Editorial Commentary by Gerding and Johnson on pages 1601–3.) The widespread emergence of hypervirulent polymerase chain reaction (PCR) ribotype 027/NAP1/BI/sequence type (ST) 1 [1] strains in the early 2000s [2, 3] substantially increased Clostridium difficile infection (CDI) incidence. PCR ribotype 027 has also been associated with more severe outcomes in most [2, 4, 5] but not all [6–9] studies. Outcome variation across non-027 strains has rarely been investigated, invariably with small numbers, although these now account for most new CDIs. One study [6] (n = 395) found significantly more complicated disease outcomes with PCR ribotypes 018 (ST 17 from [10]; n = 23) and 056 (ST 34/58 [10]; n = 6), whereas another [11] (n = 168) reported similar 30-day mortality in PCR ribotype-027 (n = 46) and 017 (ST 37 [10]; n = 57). Although PCR ribotype 078 (ST 11), common in livestock [12] and rising in incidence [6, 13], is denoted hypervirulent on the basis of increased toxin production [14] and individual case severity [15], supporting clinical data are few. Attributable mortality and severe diarrhea was similar in PCR ribotype 078 (n = 54) and 027 (n = 124) in 1 study (both greater than in 501 non-027/078 cases) [13], but PCR ribotype 078 (n = 31) was not associated with complicated CDI in another [6]. Although scores to predict CDI severity, complications, or recurrence have variably included biomarkers (eg, white blood count [WBC], C-reactive protein [CRP]) [16], no studies have investigated associations between CDI strains and biomarkers. We aimed therefore to investigate whether the genotype of C. difficile clinical isolates from multilocus sequence typing (MLST) was associated with mortality and severity biomarkers using a large population-based database of CDI cases and to explore associations between strain-specific effects on host biomarkers and mortality to provide insights into infection pathogenesis.

Within-Host Evolution of Staphylococcus aureus during Asymptomatic Carriage

Abstract: Background Staphylococcus aureus is a major cause of healthcare associated mortality, but like many important bacterial pathogens, it is a common constituent of the normal human body flora. Around a third of healthy adults are carriers. Recent evidence suggests that evolution of S. aureus during nasal carriage may be associated with progression to invasive disease. However, a more detailed understanding of within-host evolution under natural conditions is required to appreciate the evolutionary and mechanistic reasons why commensal bacteria such as S. aureus cause disease. Therefore we examined in detail the evolutionary dynamics of normal, asymptomatic carriage. Sequencing a total of 131 genomes across 13 singly colonized hosts using the Illumina platform, we investigated diversity, selection, population dynamics and transmission during the short-term evolution of S. aureus. Principal Findings We characterized the processes by which the raw material for evolution is generated: micro-mutation (point mutation and small insertions/deletions), macro-mutation (large insertions/deletions) and the loss or acquisition of mobile elements (plasmids and bacteriophages). Through an analysis of synonymous, non-synonymous and intergenic mutations we discovered a fitness landscape dominated by purifying selection, with rare examples of adaptive change in genes encoding surface-anchored proteins and an enterotoxin. We found evidence for dramatic, hundred-fold fluctuations in the size of the within-host population over time, which we related to the cycle of colonization and clearance. Using a newly-developed population genetics approach to detect recent transmission among hosts, we revealed evidence for recent transmission between some of our subjects, including a husband and wife both carrying populations of methicillin-resistant S. aureus (MRSA). Significance This investigation begins to paint a picture of the within-host evolution of an important bacterial pathogen during its prevailing natural state, asymptomatic carriage. These results also have wider significance as a benchmark for future systematic studies of evolution during invasive S. aureus disease.

Improved workflows for high throughput library preparation using the transposome-based nextera system

Abstract: Background The Nextera protocol, which utilises a transposome based approach to create libraries for Illumina sequencing, requires pure DNA template, an accurate assessment of input concentration and a column clean-up that limits its applicability for high-throughput sample preparation. We addressed the identified limitations to develop a robust workflow that supports both rapid and high-throughput projects also reducing reagent costs.

Secular Trends in Nosocomial Bloodstream Infections: Antibiotic-Resistant Bacteria Increase the Total Burden of Infection

Abstract: BACKGROUND: It is unknown whether rising incidence rates of nosocomial bloodstream infections (BSIs) caused by antibiotic-resistant bacteria (ARB) replace antibiotic-susceptible bacteria (ASB), leaving the total BSI rate unaffected. METHODS: We investigated temporal trends in annual incidence densities (events per 100 000 patient-days) of nosocomial BSIs caused by methicillin-resistant Staphylococcus aureus (MRSA), ARB other than MRSA, and ASB in 7 ARB-endemic and 7 ARB-nonendemic hospitals between 1998 and 2007. RESULTS: 33 130 nosocomial BSIs (14% caused by ARB) yielded 36 679 microorganisms. From 1998 to 2007, the MRSA incidence density increased from 0.2 to 0.7 (annual increase, 22%) in ARB-nonendemic hospitals, and from 3.1 to 11.7 (annual increase, 10%) in ARB-endemic hospitals (P = .2), increasing the incidence density difference between ARB-endemic and ARB-nonendemic hospitals from 2.9 to 11.0. The non-MRSA ARB incidence density increased from 2.8 to 4.1 (annual increase, 5%) in ARB-nonendemic hospitals, and from 1.5 to 17.4 (annual increase, 22%) in ARB-endemic hospitals (P < .001), changing the incidence density difference from -1.3 to 13.3. Trends in ASB incidence densities were similar in both groups (P = .7). With annual increases of 3.8% and 5.4% of all nosocomial BSIs in ARB-nonendemic and ARB-endemic hospitals, respectively (P < .001), the overall incidence density difference of 3.8 increased to 24.4. CONCLUSIONS: Increased nosocomial BSI rates due to ARB occur in addition to infections caused by ASB, increasing the total burden of disease. Hospitals with high ARB infection rates in 2005 had an excess burden of BSI of 20.6 per 100 000 patient-days in a 10-year period, mainly caused by infections with ARB.

Asymptomatic Clostridium difficile colonisation and onward transmission.

Abstract: 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.

Recombinational switching of the Clostridium difficile S-Layer and a novel glycosylation gene cluster revealed by large-scale whole-genome sequencing

Abstract: Background Clostridium difficile is a major cause of nosocomial diarrhea, with 30-day mortality reaching 30% The cell surface comprises a paracrystalline proteinaceous S-layer encoded by the slpA gene within the cell wall protein (cwp) gene cluster Our purpose was to understand the diversity and evolution of slpA and nearby genes also encoding immunodominant cell surface antigens Methods Whole-genome sequences were determined for 57 C difficile isolates representative of the population structure and different clinical phenotypes Phylogenetic analyses were performed on their genomic region (>63 kb) spanning the cwp cluster Results Genetic diversity across the cwp cluster peaked within slpA, cwp66 (adhesin), and secA2 (secretory translocase) These genes formed a 10-kb cassette, of which 12 divergent variants were found Homologous recombination involving this cassette caused it to associate randomly with genotype One cassette contained a novel insertion (length, approximately 24 kb) that resembled S-layer glycosylation gene clusters Conclusions Genetic exchange of S-layer cassettes parallels polysaccharide capsular switching in other species Both cause major antigenic shifts, while the remainder of the genome is unchanged C difficile genotype is therefore not predictive of antigenic type S-layer switching and immune escape could help explain temporal and geographic variation in C difficile epidemiology and may inform genotyping and vaccination strategies

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

Abstract: 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.

Effect of Age on Treatment Outcomes in Clostridium difficile Infection

Abstract: OBJECTIVES: To determine the effect of advancing age on the clinical outcomes of Clostridium difficile (CDI) treatment. DESIGN: Regression modeling of results from two double-blind randomized multicenter studies on the treatment of primary and first recurrent cases of CDI to examine for effects of age and study drug on outcomes of cure (resolution of diarrhea), recurrence within 4 weeks of completing successful therapy, and cure without recurrence. SETTING: Participants were randomized into studies in the United States, Canada, and Europe. PARTICIPANTS: Nine hundred ninety-nine individuals with toxin-positive CDI were randomized to receive vancomycin (125 mg 4 times daily) or fidaxomicin (200 mg twice daily) for 10 days. MEASUREMENTS: The effect of advancing age in those aged 18 to 40 years and in 10-year increments thereafter was examined. RESULTS: The model predicts a 17% lower clinical cure, 17% greater recurrence, and 13% lower sustained clinical response by advancing decade than in those younger than 40 (P < .01 each). Clinical cure was similar in the fidaxomicin and vancomycin treatment groups, although fidaxomicin was associated with a more than 50% lower relative risk for recurrence than vancomycin (P < .001). Multivariate regression modeling showed that risk factors accounting for poorer outcomes with advancing age include infection with the BI strain type, inpatient status, renal insufficiency, leukocytosis, hypoalbuminemia, and concomitant medication exposure. CONCLUSION: Measurable and progressive deterioration in CDI treatment outcomes occurred with advancing age in those aged 40 and older, highlighting the need for prevention and treatment strategies. Fidaxomicin treatment was associated with a 60% lower risk of recurrence than vancomycin after adjusting for age, concomitant antibiotics, and C. difficile strain.

Comparison of Multilocus Variable-Number Tandem-Repeat Analysis and Whole-Genome Sequencing for Investigation of Clostridium difficile Transmission

Abstract: No study to date has compared multilocus variable-number tandem-repeat analysis (MLVA) and whole-genome sequencing (WGS) in an investigation of the transmission of Clostridium difficile infection. Isolates from 61 adults with ongoing and/or recurrent C. difficile infections and 17 asymptomatic carriage episodes in children (201 samples), as well as from 61 suspected outbreaks affecting 2 to 41 patients in 31 hospitals in the United Kingdom (300 samples), underwent 7-locus MLVA and WGS in parallel. When the first and last samples from the same individual taken for a median (interquartile range [IQR]) of 63 days (43 to 105 days) apart were compared, the estimated rates of the evolution of single nucleotide variants (SNVs), summed tandem-repeat differences (STRDs), and locus variants (LVs) were 0.79 (95% confidence interval [CI], 0.00 to 1.75), 1.63 (95% CI, 0.00 to 3.59), and 1.21 (95% CI, 0.00 to 2.67)/called genome/year, respectively. Differences of >2 SNVs and >10 STRDs have been used to exclude direct case-to-case transmission. With the first serial sample per individual being used to assess discriminatory power, across all pairs of samples sharing a PCR ribotype, 192/283 (68%) differed by >10 STRDs and 217/283 (77%) by >2 SNVs. Among all pairs of cases from the same suspected outbreak, 1,190/1,488 (80%) pairs had concordant results using >2 SNVs and >10 STRDs to exclude transmission. For the discordant pairs, 229 (15%) had ≥2 SNVs but ≤10 STRDs, and 69 (5%) had ≤2 SNVs but ≥10 STRDs. Discordant pairs had higher numbers of LVs than concordant pairs, supporting the more diverse measure in each type of discordant pair. Conclusions on whether the potential outbreaks were confirmed were concordant in 58/61 (95%) investigations. Overall findings using MLVA and WGS were very similar despite the fact that they analyzed different parts of the bacterial genome. With improvements in WGS technology, it is likely that MLVA locus data will be available from WGS in the near future.

A modified RNA-Seq approach for whole genome sequencing of RNA viruses from faecal and blood samples.

Abstract: To date, very large scale sequencing of many clinically important RNA viruses has been complicated by their high population molecular variation, which creates challenges for polymerase chain reaction and sequencing primer design. Many RNA viruses are also difficult or currently not possible to culture, severely limiting the amount and purity of available starting material. Here, we describe a simple, novel, high-throughput approach to Norovirus and Hepatitis C virus whole genome sequence determination based on RNA shotgun sequencing (also known as RNA-Seq). We demonstrate the effectiveness of this method by sequencing three Norovirus samples from faeces and two Hepatitis C virus samples from blood, on an Illumina MiSeq benchtop sequencer. More than 97% of reference genomes were recovered. Compared with Sanger sequencing, our method had no nucleotide differences in 14,019 nucleotides (nt) for Noroviruses (from a total of 2 Norovirus genomes obtained with Sanger sequencing), and 8 variants in 9,542 nt for Hepatitis C virus (1 variant per 1,193 nt). The three Norovirus samples had 2, 3, and 2 distinct positions called as heterozygous, while the two Hepatitis C virus samples had 117 and 131 positions called as heterozygous. To confirm that our sample and library preparation could be scaled to true high-throughput, we prepared and sequenced an additional 77 Norovirus samples in a single batch on an Illumina HiSeq 2000 sequencer, recovering >90% of the reference genome in all but one sample. No discrepancies were observed across 118,757 nt compared between Sanger and our custom RNA-Seq method in 16 samples. By generating viral genomic sequences that are not biased by primer-specific amplification or enrichment, this method offers the prospect of large-scale, affordable studies of RNA viruses which could be adapted to routine diagnostic laboratory workflows in the near future, with the potential to directly characterize within-host viral diversity.

Renal impairment and clinical outcomes of Clostridium difficile infection in two randomized trials.

Abstract: Background/Aims: Patients with chronic kidney disease (CKD) have increased risk for Clostridium difficile infection (CDI) and for subsequent mo

Whole genome sequencing and de novo assembly identifies Sydney-like variant noroviruses and recombinants during the winter 2012/2013 outbreak in England

Abstract: Norovirus is the commonest cause of epidemic gastroenteritis among people of all ages. Outbreaks frequently occur in hospitals and the community, costing the UK an estimated £110 m per annum. An evolutionary explanation for periodic increases in norovirus cases, despite some host-specific post immunity is currently limited to the identification of obvious recombinants. Our understanding could be significantly enhanced by full length genome sequences for large numbers of intensively sampled viruses, which would also assist control and vaccine design. Our objective is to develop rapid, high-throughput, end-to-end methods yielding complete norovirus genome sequences. We apply these methods to recent English outbreaks, placing them in the wider context of the international norovirus epidemic of winter 2012. Norovirus sequences were generated from 28 unique clinical samples by Illumina RNA sequencing (RNA-Seq) of total faecal RNA. A range of de novo sequence assemblers were attempted. The best assembler was identified by validation against three replicate samples and two norovirus qPCR negative samples, together with an additional 20 sequences determined by PCR and fractional capillary sequencing. Phylogenetic methods were used to reconstruct evolutionary relationships from the whole genome sequences. Full length norovirus genomes were generated from 23/28 samples. 5/28 partial norovirus genomes were associated with low viral copy numbers. The de novo assembled sequences differed from sequences determined by capillary sequencing by <0.003%. Intra-host nucleotide sequence diversity was rare, but detectable by mapping short sequence reads onto its de novo assembled consensus. Genomes similar to the Sydney 2012 strain caused 78% (18/23) of cases, consistent with its previously documented association with the winter 2012 global outbreak. Interestingly, phylogenetic analysis and recombination detection analysis of the consensus sequences identified two related viruses as recombinants, containing sequences in prior circulation to Sydney 2012 in open reading frame (ORF) 2. Our approach facilitates the rapid determination of complete norovirus genomes. This method provides high resolution of full norovirus genomes which, when coupled with detailed epidemiology, may improve the understanding of evolution and control of this important healthcare-associated pathogen.

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

Abstract: 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 nonsynonymous, 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.

Regarding “Clostridium Difficile Ribotype Does Not Predict Severe Infection”

Abstract: To the Editor—We read with interest the recent article from Walk et al, “Clostridium difficile Ribotype Does Not Predict Severe Infection” [1]. However, their interpretation makes 2 epidemiologic errors, rendering their conclusions unreliable. First, they treat “no evidence of difference” as “evidence of no difference”: These are equivalent if, and only if, a study is adequately powered to exclude the possibility of a modest effect, for example, based on a 95% confidence interval (CI) (as in noninferiority trials). The comparison of 027/078 vs other ribotypes for severe disease had an unadjusted odds ratio (OR) of 2.33 (95% CI, 1.03–5.02; P = .035). Adjusting for age, Charlson comorbidity index, hematocrit, or platelets reduced the OR only slightly (still remaining >2.1) with P values of .06–.07 and 95% CIs of .92–4.78 (ie, essentially unchanged). The .05 P value cutoff is an arbitrary threshold from days when significance tables had to be calculated laboriously by hand [2]. Statistically, there is little difference in the strength of evidence provided from findings with P values of .035 and .07, as evidenced by the CIs in Table 3 [1], which all extend above 4, well above any plausible margin that might be considered noninferior. The lowest OR is for tcdC deletion (1.26 [95% CI, .38–4.94]); however, the meaning of this estimate is unclear as this deletion is ubiquitous among ribotype 027 isolates, and hence is completely confounded with the majority of the “hypervirulent” strains included in the dataset. Unfortunately, replicating “no evidence of difference” in a separate validation cohort [1] does not transform it into “evidence of no difference”—it simply means that 2 underpowered studies have been conducted. Another problem is adjusting for factors (biomarkers at diagnosis) on the causal pathway between an exposure (C. difficile ribotype) and an outcome (disease severity), sometimes denoted mediation or overadjustment bias [3]. Consider a pathogen in which the entire mechanism for causing disease is through raising white blood cell (WBC) count. Suppose there are only 2 strains, 1 of which causes twice the WBC count rise. By definition, all the differential mortality between these 2 strains will be due to their differential WBC count rise—adjusting for this will reduce the WBC count–adjusted effect of a strain to zero. Essentially, if there is any strain effect on a biomarker (not analyzed by Walk et al [1]), then the biomarker-adjusted OR for a strain represents an effect that is not mediated through biomarkers. It cannot be interpreted as the causal effect of strain, because it overadjusts for strain-related biomarker differences. This study shows that WBC count and albumin are strong predictors of C. difficile severity and that there is an unadjusted association between strain and severity. However, unfortunately it does not address the key question as to the causal impact of strain including biomarker-mediated and biomarker-independent effects. Our larger study of 1893 enzyme immunoassay–positive, culture-positive, multilocus sequence–typed strains shows definitively that strains vary in their overall impact on mortality (adjusted for multiple potential confounders) and in their overall impact on biomarkers (predominantly those associated with inflammatory pathways), and that residual variation in mortality risk remains even after adjusting for biomarker-mediated effects [4].

Vancomycin MIC as a predictor of outcome in MRSA bacteraemia in the UK context

Abstract: Objectives Raised vancomycin MICs have been associated with poor outcomes for methicillin-resistant Staphylococcus aureus (MRSA) bacteraemia in the USA and mainland Europe. We investigated if this also applies in the UK, where EMRSA-15 (clonal complex 22) dominates. Methods Isolates from UK patients receiving vancomycin therapy for MRSA bacteraemia in 2008–10 were collected, along with clinical details. Outcomes (i.e. patient survival or bacteraemia resolution) were reported 28 days after vancomycin therapy ended. The relationship between clinical outcome and MIC—as determined by CLSI and BSAC agar dilution methods—was assessed. Results Among 228 MRSA bacteraemias, 82% were caused by EMRSA-15; 65% of the patients were male and the median age was 70.5 years. MICs correlated between methods, but CLSI agar dilution testing gave a mode at 1 mg/L with only 12% of results either side, whereas the BSAC method gave a mode straddling 0.7–1 mg/L with <4% outliers. Twenty-three percent of patients died, with MRSA contributory in half; another 17% had unresolved bacteraemia at 28 days. Neither death nor unresolved bacteraemia was significantly associated with higher vancomycin MICs by either method. Rifampicin co-therapy had no quantifiable effect on outcome. The patient's age was the only significant correlate of patient outcome (P < 0.01); the underlying medical condition of the patient was important for the resolution of bacteraemia (P < 0.01), though not for overall mortality. Conclusions Subtle vancomycin MIC differences did not correlate with worse outcomes for vancomycin monotherapy or for vancomycin/rifampicin co-therapy in MRSA bacteraemia. Regardless of the exact MIC–outcome relationship, detecting such small MIC differences seems unlikely to be reliable in routine laboratories.

Annual Report of the Chief Medical Officer: Infections and the rise of antibiotic resistance

Abstract: Chapter 11: Future Challenges. Edited by Tom Fowlerwith substantial contributions from Kevin Dean, Martin Stewart-Weeks, David Cooksey,Stephen J Fowler, Paul Dark, Ashley Woodcock, SueHill, Tom Fowler, David Walker, David M Salisbury,Sharon Peacock, Danny Altman, Stephen Wyllie, MikeCatchpole, Andrew Hall, Derrick Crook

Arthroplasty Infection at Revision Microbiological Diagnosis of Prospective Evaluation of Criteria for

Abstract: A prospective study was performed to establish criteria for the microbiological diagnosis of prosthetic jointinfection at elective revision arthroplasty. Patients were treated in a multidisciplinary unit dedicated to themanagement and study of musculoskeletal infection. Standard multiple samples of periprosthetic tissue wereobtained at surgery, Gram stained, and cultured by direct and enrichment methods. With reference to histologyas the criterion standard, sensitivities, specificities, and likelihood ratios (LRs) were calculated by using differ-ent cutoffs for the diagnosis of infection. We performed revisions on 334 patients over a 17-month period, of whom297 were evaluable. The remaining 37 were excluded because histology results were unavailable or could notbe interpreted due to underlying inflammatory joint disease. There were 41 infections, with only 65% of all sam-ples sent from infected patients being culture positive, suggesting low numbers of bacteria in the samples tak-en. The isolation of an indistinguishable microorganism from three or more independent specimens was highlypredictive of infection (sensitivity, 65%; specificity, 99.6%; LR, 168.6), while Gram staining was less useful (sen-sitivity, 12%; specificity, 98%; LR, 10). A simple mathematical model was developed to predict the performanceof the diagnostic test. We recommend that five or six specimens be sent, that the cutoff for a definite diagnosisof infection be three or more operative specimens that yield an indistinguishable organism, and that because of itslow level of sensitivity, Gram staining should be abandoned as a diagnostic tool at elective revision arthroplasty.