[Show abstract][Hide abstract] ABSTRACT: Increased incidence of infections due to Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae (KPC-Kp) was noted among patients undergoing endoscopic retrograde cholangiopancreatography (ERCP) at a single hospital. An epidemiologic investigation identified KPC-Kp and non-KPC-producing, extended-spectrum β-lactamase (ESBL)-producing Kp in cultures from 2 endoscopes. Genotyping was performed on patient and endoscope isolates to characterize the microbial genomics of the outbreak. Genetic similarity of 51 Kp isolates from 37 patients and 3 endoscopes was assessed by pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). Five patient and 2 endoscope isolates underwent whole genome sequencing (WGS). Two KPC-encoding plasmids were characterized by single molecule, real-time sequencing. Plasmid diversity was assessed by endonuclease digestion. Genomic and epidemiologic data were used in conjunction to investigate the outbreak source. Two clusters of Kp patient isolates were genetically related to endoscope isolates by PFGE. A subset of patient isolates were collected post-ERCP, suggesting ERCP endoscopes as a possible source. A phylogeny of 7 Kp genomes from patient and endoscope isolates supported ERCP as a potential source of transmission. Differences in gene content defined 5 ST258 subclades and identified 2 of the subclades as outbreak-associated. A novel KPC-encoding plasmid, pKp28 helped define and track one endoscope-associated ST258 subclade. WGS demonstrated high genetic relatedness of patient and ERCP endoscope isolates suggesting ERCP-associated transmission of ST258 KPC-Kp. Gene and plasmid content discriminated the outbreak from endemic ST258 populations and assisted with the molecular epidemiologic investigation of an extended KPC-Kp outbreak.
[Show abstract][Hide abstract] ABSTRACT: Neisseria meningitidis is a leading bacterial cause of sepsis and meningitis globally with dynamic strain distribution over time. Beginning with an epidemic among Hajj pilgrims in 2000, serogroup W (W) sequence type (ST) 11 emerged as a leading cause of epidemic meningitis in the African ‘meningitis belt’ and endemic cases in South America, Europe, Middle East and China. Previous genotyping studies were unable to reliably discriminate sporadic W ST-11 strains in circulation since 1970 from the Hajj outbreak strain (Hajj clone). It is also unclear what proportion of more recent W ST-11 disease clusters are caused by direct descendants of the Hajj clone. Whole genome sequences of 270 meningococcal strains isolated from patients with invasive meningococcal disease globally from 1970 to 2013 were compared using whole genome phylogenetic and major antigen-encoding gene sequence analyses. We found that all W ST-11 strains were descendants of an ancestral strain that had undergone unique capsular switching events. The Hajj clone and its descendants were distinct from other W ST-11 strains in that they shared a common antigen gene profile and had undergone recombination involving virulence genes encoding factor H binding protein, nitric oxide reductase, and nitrite reductase. These data demonstrate that recent acquisition of a distinct antigen-encoding gene profile and variations in meningococcal virulence genes were associated with the emergence of the Hajj clone. Importantly, W ST-11 strains unrelated to the Hajj outbreak contribute a significant proportion of W ST-11 cases globally. This study helps illuminate genomic factors associated with meningococcal strain emergence and evolution.
[Show abstract][Hide abstract] ABSTRACT: We describe a new preservation modality combining machine perfusion (MP) at subnormothermic conditions (21°C) with a new hemoglobin-based oxygen carrier (HBOC) solution. MP (n = 6) was compared to cold static preservation (CSP; n = 6) in porcine orthotopic liver transplants after 9 h of cold ischemia and 5-day follow-up. Recipients' peripheral blood, serial liver biopsies, preservation solutions and bile specimens were collected before, during and after liver preservation. Clinical laboratorial and histological analyses were performed in addition to mitochondrial functional assays, transcriptomic, metabolomic and inflammatory mediator analyses. Compared with CSP, MP animals had: (1) significantly higher survival (100% vs. 33%; p < 0.05); (2) superior graft function (p < 0.05); (3) eight times higher hepatic O2 delivery than O2 consumption (0.78 mL O2 /g/h vs. 0.096 mL O2 /g/h) during MP; and (4) significantly greater bile production (MP = 378.5 ± 179.7; CS = 151.6 ± 116.85). MP down-regulated interferon (IFN)-α and IFN-γ in liver tissue. MP allografts cleared lactate, produced urea, sustained gluconeogenesis and produced hydrophilic bile after reperfusion. Enhanced oxygenation under subnormothermic conditions triggers regenerative and cell protective responses resulting in improved allograft function. MP at 21°C with the HBOC solution significantly improves liver preservation compared to CSP.
2015 The Authors. American Journal of Transplantation Published by The American Society of Transplantation and the American Society of Transplant Surgeons.
Full-text · Article · Jan 2015 · American Journal of Transplantation
[Show abstract][Hide abstract] ABSTRACT: Background: Despite strategies to reduce transmission, Clostridium difficile (CD) remains a leading cause of morbidity in health care settings. The aims of our study were (1) to determine if asymptomatic inpatient CD carriers can be liked by molecular epidemiology to other inpatients with CD infection (CDI) and (2) to determine if active surveillance testing (AST) criteria to identify inpatients at risk for vancomycin-resistant Enterococcus (VRE) colonization are sufficiently sensitive to capture asymptomatic CD carriage.
Methods: Over a 5 day period, all inpatients at University of Pittsburgh Medical Center – Presbyterian Hospital (UPMC) were targeted for one-time AST for CD via perirectal sampling. Archived stool specimens were obtained from (1) clinical CDI cases 7 weeks prior to AST for CD, (2) clinical CDI cases during AST for CD, and (3) clinical hospital-associated CDI (HA-CDI) cases 12 weeks after AST for CD. CD carriage was defined as AST positivity in the absence of clinical CDI during or +/- 3 months from AST; clinical CDI was defined as clinician-requested, nucleic stool test positivity for toxigenic CD. Specimens were cultured for CD using broth enrichment. CD isolates were typed using tcdC and multilocus variable number tandem repeat (MLVA) genotyping. Isolates whose MLVA genotypes had a summed tandem-repeat difference of ≤2 were considered highly related. “Probable” transmission events were inferred when patients exhibited simultaneous occupancy of the same inpatient ward and their isolates were highly related. “Possible” transmission events were inferred when patients exhibited simultaneous occupancy of the hospital, but not the same inpatient ward, and their isolates were highly related. AST for VRE was performed according to UPMC policy throughout.
Results: 53 (10%) inpatients were identified as CD carriers. 123 clinical CDI cases were identified; isolates from 101 (83%) of these episodes were obtained. 6 (5.9%) clinical CDI isolates were highly related to CD carrier isolates, 4 of which were HA and could be linked epidemiologically to CD carriers – 2 by probable and 2 by possible transmission events. None of the CD carriers implicated in transmission events underwent VRE AST.
Conclusion: Hospital-wide AST for CD carriage may identify a reservoir of CD involved in CDI.
[Show abstract][Hide abstract] ABSTRACT: Previous studies suggested that 7 to 15% of healthy adults are colonized with toxigenic Clostridium difficile. To investigate the epidemiology, genetic diversity, and duration of C. difficile colonization in asymptomatic persons, we recruited healthy adults from the general population in Allegheny County, Pennsylvania.
Participants provided epidemiological and dietary intake data and submitted stool specimens. The presence of C. difficile in stool specimens was determined by anaerobic culture. Stool specimens yielding C. difficile underwent nucleic acid testing of the tcdA gene segment with a commercial assay; tcdC genotyping was performed on C. difficile isolates. Subjects positive for C. difficile by toxigenic anaerobic culture were asked to submit additional specimens. One hundred six (81%) of 130 subjects submitted
specimens, and 7 (6.6%) of those subjects were colonized with C. difficile. Seven distinct tcdC genotypes were observed among the 7 C. difficile-colonized individuals, including tcdC genotype 20, which has been found in uncooked ground pork in this region. Two (33%) out of 6 C. difficile-colonized subjects who submitted additional specimens tested positive for identical C. difficile strains on successive occasions, 1 month apart. The prevalence of C. difficile carriage in this healthy cohort is concordant with prior estimates. C. difficile-colonized individuals may be important reservoirs for C. difficile and may falsely test positive for infections due to C. difficile when evaluated for community-acquired diarrhea caused by other enteric pathogens.
[Show abstract][Hide abstract] ABSTRACT: Although the rate of ceftriaxone resistance in methicillin-sensitive Staphylococcus aureus (MSSA) is reported at approximately 3% in the literature, findings at our institution suggest it is grossly underestimated. Eighty-seven percent of MSSA isolates tested were non-suceptible to ceftriaxone and activity of this antibiotic could not be predicted using other beta-lactams.
No preview · Article · Mar 2014 · Clinical Infectious Diseases
[Show abstract][Hide abstract] ABSTRACT: The use of molecular methods to diagnose Clostridium difficile infection (CDI) has improved diagnostic yield compared to conventional methods. However, PCR testing can detect colonization and has introduced several practical challenges pertaining to need for treatment and isolation of cases.
For all new cases detected by real-time PCR, concurrent cytotoxin assay was performed and genetic characterization with MLVA (multi-locus variable number tandem repeat analysis) was done to determine relatedness. We used PCR cycle threshold (Ct) of detection as surrogate marker for bacterial burden in stool.
Overall, 54 cases of CDI were detected during the study period. 42 were concurrently tested by CYT and characterized by MLVA .MLVA analysis revealed marked genetic diversity with no ongoing outbreaks; four cases were due to NAP1 strain. CYT -/PCR + cases had a higher median Ct value of detection compared to CYT+/PCR + cases (28.2 vs 22.5; p = 0.01). Among 25 strains that were genetically related, 9/11 isolates in this dominant cluster were positive by CYT compared to 4/14 in non-dominant clusters (p = 0.02).
CYT-/PCR+ cases contribute to hospital based transmission. However, the risk of transmission of C. difficile from CYT +/PCR+ cases may be higher than those that are CYT-/PCR+.
[Show abstract][Hide abstract] ABSTRACT: The detection of meningococcal outbreaks relies on serogrouping and epidemiologic definitions. Advances in molecular epidemiology have improved the ability to distinguish unique Neisseria meningitidis strains, enabling the classification of isolates into clones. Around 98% of meningococcal cases in the United States are believed to be sporadic.
Meningococcal isolates from 9 Active Bacterial Core surveillance sites throughout the United States from 2000 through 2005 were classified according to serogroup, multilocus sequence typing, and outer membrane protein (porA, porB, and fetA) genotyping. Clones were defined as isolates that were indistinguishable according to this characterization. Case data were aggregated to the census tract level and all non-singleton clones were assessed for non-random spatial and temporal clustering using retrospective space-time analyses with a discrete Poisson probability model.
Among 1,062 geocoded cases with available isolates, 438 unique clones were identified, 78 of which had ≥2 isolates. 702 cases were attributable to non-singleton clones, accounting for 66.0% of all geocoded cases. 32 statistically significant clusters comprised of 107 cases (10.1% of all geocoded cases) were identified. Clusters had the following attributes: included 2 to 11 cases; 1 day to 33 months duration; radius of 0 to 61.7 km; and attack rate of 0.7 to 57.8 cases per 100,000 population. Serogroups represented among the clusters were: B (n = 12 clusters, 45 cases), C (n = 11 clusters, 27 cases), and Y (n = 9 clusters, 35 cases); 20 clusters (62.5%) were caused by serogroups represented in meningococcal vaccines that are commercially available in the United States.
Around 10% of meningococcal disease cases in the U.S. could be assigned to a geotemporal cluster. Molecular characterization of isolates, combined with geotemporal analysis, is a useful tool for understanding the spread of virulent meningococcal clones and patterns of transmission in populations.
[Show abstract][Hide abstract] ABSTRACT: During 2003-2012, 8 clusters of meningococcal disease were identified in Rio de Janeiro State, Brazil, all caused by serogroup C Neisseria meningitidis. The isolates were assigned to 3 clonal complexes (cc): cc11, cc32, and cc103. These hyperinvasive disease lineages were associated with endemic disease, outbreaks, and high case-fatality rates.
Full-text · Article · Nov 2013 · Emerging Infectious Diseases
[Show abstract][Hide abstract] ABSTRACT: Background. Previous studies have suggested that asymptomatic carriers of toxigenic C. difficile are a source of hospital-associated (HA) infections. MLVA (multi-locus variable number of tandem repeats analysis) is a highly discriminatory molecular subtyping tool that helps to determine possible transmission sources. Methods. C. difficile isolates were recovered from peri-rectal swabs collected for vancomycin resistant enterococcus (VRE) surveillance as well as from clinical C. difficile toxin-positive stool samples from July - November 2009 at UPMC Presbyterian. MLVA was performed to determine the genetic relationships between isolates from asymptomatic carriers and patients with HA-C. difficile infections (HA-CDI). Asymptomatic carriage and HA-CDI isolates were considered to be associated if the carriage isolate was collected before the HA-CDI isolate and if the MLVA genotypes had a summed tandem repeat difference <2. Results. Of 3006 patients screened, 314 (10.4%) were positive for toxigenic C. difficile, of whom 226 (7.5%) were detected only by VRE surveillance cultures. Of 56 incident cases of CDI classified as HA at UPMC during the study with available isolates, 17 (30%) cases were associated with CDI patients while 16 (29%) cases were associated with carriers. Transmission events from prior bed occupants with CDI (n=2) or carriers (n=2) were identified in 4/56 cases. Conclusions. In our hospital with an established infection control program designed to contain transmission from symptomatic CDI patients, asymptomatic carriers appear to have played an important role in transmission. Identification and isolation of carriers may be necessary to further reduce transmission of C. difficile in such settings.
[Show abstract][Hide abstract] ABSTRACT: The increase in community associated Clostridium difficile disease paired with recent data on C. difficile in retail foods has led to speculation that C. difficile is a food-borne pathogen. However, there is no current epidemiologic evidence (i.e. restaurant or food-associated outbreaks) to support this hypothesis. Rates of C. difficile recovery from food vary widely across laboratories and may be due to a number of confounding factors. This commentary discusses the results of two published investigations and suggests that higher prevalence rates observed in some food studies may be due to laboratory contamination. The conclusions are that prevalence of C. difficile in retail foods is relatively low and further investigations are required to determine if C. difficile is food-borne.
[Show abstract][Hide abstract] ABSTRACT: Recurrent Clostridium difficile infection (CDI) occurs in up to 35% of patients. Recurrences can be due to either relapse with the same strain or reinfection
with another strain. In this study, multilocus variable-number tandem-repeat analysis (MLVA) was performed on C. difficile isolates from patients with recurrent CDI to distinguish relapse from reinfection. In addition, univariate and multivariate
analyses were performed to identify risk factors associated with relapse. Among patients with a single recurrence, relapse
due to the original infecting strain was more prevalent than reinfection and the interval between episodes was shorter than
among patients who had reinfections. Among patients with >1 recurrence, equal distributions of relapse and reinfection or
a combination of the two episode types were observed. Initial infection with the BI/NAP1/027 epidemic clone was found to be
a significant risk factor for relapse. This finding may have important implications for patient therapy. Classification of
recurrent CDI episodes by MLVA can be utilized to make informed patient care decisions and to accurately define new CDI cases
for infection control and reimbursement purposes.
Preview · Article · Oct 2012 · Journal of clinical microbiology
[Show abstract][Hide abstract] ABSTRACT: Laparoscopic liver resections are being performed with increasing frequency, with several groups having reported minimally invasive approaches for major anatomic hepatic resections. Some surgeons favor a pure laparoscopic approach, while others prefer a hand-assisted approach for major laparoscopic liver resections. There are clear advantages and disadvantages to a hand-assisted technique. The purpose of this study is to summarize the literature comparing pure laparoscopic and hand-assisted approaches for minimally invasive hepatic resection, and to describe our approach in 432 laparoscopic liver resections.
Preview · Article · Oct 2012 · Journal of Hepato-Biliary-Pancreatic Sciences
[Show abstract][Hide abstract] ABSTRACT: During the 1990s, an epidemic of B:4 Neisseria meningitidis infections affected Brazil. Subsequent increase in C:4 disease suggested B → C capsular switching. This study identified B → C switches within the sequence type 32 complex. Substantial disease related to capsular switching emphasizes the need for surveillance of circulating meningococcal strains to optimize disease control.
Preview · Article · Aug 2012 · Emerging Infectious Diseases
[Show abstract][Hide abstract] ABSTRACT: In the United States, serogroup Y, ST-23 clonal complex Neisseria meningitidis was responsible for an increase in meningococcal disease incidence during the 1990s. This increase was accompanied by antigenic shift of three outer membrane proteins, with a decrease in the population that predominated in the early 1990s as a different population emerged later in that decade. To understand factors that may have been responsible for the emergence of serogroup Y disease, we used whole genome pyrosequencing to investigate genetic differences between isolates from early and late N. meningitidis populations, obtained from meningococcal disease cases in Maryland in the 1990s. The genomes of isolates from the early and late populations were highly similar, with 1231 of 1776 shared genes exhibiting 100% amino acid identity and an average π(N) = 0.0033 and average π(S) = 0.0216. However, differences were found in predicted proteins that affect pilin structure and antigen profile and in predicted proteins involved in iron acquisition and uptake. The observed changes are consistent with acquisition of new alleles through horizontal gene transfer. Changes in antigen profile due to the genetic differences found in this study likely allowed the late population to emerge due to escape from population immunity. These findings may predict which antigenic factors are important in the cyclic epidemiology of meningococcal disease.