Search

Detection of patients with intestinal colonization of carbapenem-resistant organisms (CRO), or more specifically carbapenemase-producing (CP) CRO, can prevent their transmission in healthcare facilities and aid with outbreak investigations. The objective of this work was to further develop and compare methods that combine selective culture and/or PCR to rapidly detect and recover CRO from fecal specimens. Molecularly characterized Gram-negative bacilli (n = 62) were used to spike fecal samples to establish limit of detection (LOD; n = 12), sensitivity (n = 28), and specificity (n= 21) for 3 methods to detect CP-CRO: direct MacConkey (MAC) plate and Xpert Carba-R (Cepheid) on growth, MAC broth and Carba-R testing of the broth, and direct testing by Carba-R. This was followed by a clinical study comparing methods in parallel for 286 fecal specimens. The LOD ranged from 102-105 CFU/mL depending on the carbapenemase gene and method. Combined culture/PCR methods had a sensitivity of 100%, whereas direct Carba-R testing had a sensitivity of 96% for the detection of CP-CRO. All methods had specificities of 100%. The prevalence of CP-CRO (0.7%) and non-CP-CRO (5.2 %) were low in the clinical study, where all methods demonstrated 100% agreement. The three methods performed comparably in detecting CP-CRO. Direct Carba-R testing had a higher LOD than the combined selective culture methods, but this may be offset by its rapid turnaround time for detection of CP-CRO. The selective culture methods provide the benefit of simultaneously isolating CP-CRO in culture for follow-up testing and detecting non-CP-CRO.

Transmission of livestock-associated Staphylococcus aureus clonal complex 9 (LA-SA CC9) between pigs raised on industrial hog operations (IHOs) and humans in the United States is poorly understood. We analyzed whole-genome sequences from 32 international S. aureus CC9 isolates and 49 LA-SA CC9 isolates from IHO pigs and humans who work on or live near IHOs in 10 pig-producing counties in North Carolina, USA. Bioinformatic analysis of sequence data from the 81 isolates demonstrated 3 major LA-SA CC9 clades. North Carolina isolates all fell within a single clade (C3). High-resolution phylogenetic analysis of C3 revealed 2 subclades of intermingled IHO pig and human isolates differing by 0–34 single-nucleotide polymorphisms. Our findings suggest that LA-SA CC9 from pigs and humans share a common source and provide evidence of transmission of antimicrobial-resistant LA-SA CC9 between IHO pigs and humans who work on or live near IHOs in North Carolina.

Objectives Traditional antimicrobial susceptibility testing (AST) is growth dependent and time-consuming. With rising rates of drug-resistant infections, a novel diagnostic method is critically needed that can rapidly reveal a pathogen’s antimicrobial susceptibility to guide appropriate treatment. Recently, RNA sequencing has been identified as a powerful diagnostic tool to explore transcriptional gene expression and improve AST. Methods RNA sequencing was used to investigate the potential of RNA markers for rapid molecular AST using Klebsiella pneumoniae and ciprofloxacin as a model. Downstream bioinformatic analysis was applied for optimal marker selection. Further validation on 11 more isolates of K. pneumoniae was performed using quantitative real-time PCR. Results From RNA sequencing, we identified RNA signatures that were induced or suppressed following exposure to ciprofloxacin. Significant shifts at the transcript level were observed as early as 10 min after antibiotic exposure. Lastly, we confirmed marker expression profiles with concordant MIC results from traditional culture-based AST and validated across 11 K. pneumoniae isolates. recA, coaA and metN transcripts harbour the most sensitive susceptibility information and were selected as our top markers. Conclusions Our results suggest that RNA signature is a promising approach to AST development, resulting in faster clinical diagnosis and treatment of infectious disease. This approach is potentially applicable in other models including other pathogens exposed to different classes of antibiotics.

Empiric broad‐spectrum antimicrobial treatments of urinary tract infections (UTIs) have contributed to widespread antimicrobial resistance. Clinical adoption of evidence‐based treatments necessitates rapid diagnostic methods for pathogen identification (ID) and antimicrobial susceptibility testing (AST) with minimal sample preparation. In response, a microfluidic droplet‐based platform is developed for achieving both ID and AST from urine samples within 30 min. In this platform, fluorogenic hybridization probes are utilized to detect 16S rRNA from single bacterial cells encapsulated in picoliter droplets, enabling molecular identification of uropathogenic bacteria directly from urine in as little as 16 min. Moreover, in‐droplet single‐bacterial measurements of 16S rRNA provide a surrogate for AST, shortening the exposure time to 10 min for gentamicin and ciprofloxacin. A fully integrated device and screening workflow were developed to test urine specimens for one of seven unique diagnostic outcomes including the presence/absence of Gram‐negative bacteria, molecular ID of the bacteriaas Escherichia coli, an Enterobacterales, or other organism, and assessment of bacterial susceptibility to ciprofloxacin. In a 50‐specimen clinical comparison study, the platform demonstrates excellent performance compared to clinical standard methods (areas‐under‐curves, AUCs >0.95), within a small fraction of the turnaround time, highlighting its clinical utility., Single‐cell measurements of bacterial 16S rRNA are leveraged in picoliter droplets to achieve simultaneous molecular detection of bacteria and phenotypic assessment of antimicrobial susceptibility, unlocking a 30 min sample‐to‐answer diagnostic platform for urinary tract infections.

SARS-CoV-2 has presented a rapidly accelerating global public health crisis. The ability to detect and analyze viral RNA from minimally invasive patient specimens is critical to the public health response. Metagenomic next-generation sequencing (mNGS) offers an opportunity to detect SARS-CoV-2 from nasopharyngeal (NP) swabs. This approach also provides information on the composition of the respiratory microbiome and its relationship to coinfections or the presence of other organisms that may impact SARS-CoV-2 disease progression and prognosis. Here, using direct Oxford Nanopore long-read third-generation metatranscriptomic and metagenomic sequencing of NP swab specimens from 50 patients under investigation for COVID-19, we detected SARS-CoV-2 sequences by applying the CosmosID bioinformatics platform. Further, we characterized coinfections and detected a decrease in the diversity of the microbiomes in these patients. Statistically significant shifts in the microbiome were identified among COVID-19-positive and -negative patients, in the latter of whom a higher abundance of Propionibacteriaceae and a reduction in the abundance of Corynebacterium accolens were observed. Our study also corroborates the growing evidence that increased SARS-CoV-2 RNA detection from NP swabs is associated with the early stages of disease rather than with severity of disease. This work illustrates the utility of mNGS for the detection and analysis of SARS-CoV-2 from NP swabs without viral target enrichment or amplification and for the analysis of the respiratory microbiome., Metagenomic next-generation sequencing (mNGS) offers an agnostic approach for emerging pathogen detection directly from clinical specimens. In contrast to targeted methods, mNGS also provides valuable information on the composition of the microbiome and might uncover coinfections that may associate with disease progression and impact prognosis. To evaluate the use of mNGS for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and/or other infecting pathogens, we applied direct Oxford Nanopore long-read third-generation metatranscriptomic and metagenomic sequencing. Nasopharyngeal (NP) swab specimens from 50 patients under investigation for CoV disease 2019 (COVID-19) were sequenced, and the data were analyzed by the CosmosID bioinformatics platform. Further, we characterized coinfections and the microbiome associated with a four-point severity index. SARS-CoV-2 was identified in 77.5% (31/40) of samples positive by RT-PCR, correlating with lower cycle threshold (Ct) values and fewer days from symptom onset. At the time of sampling, possible bacterial or viral coinfections were detected in 12.5% of SARS-CoV-2-positive specimens. A decrease in microbial diversity was observed among COVID-19-confirmed patients (Shannon diversity index, P = 0.0082; Chao richness estimate, P = 0.0097; Simpson diversity index, P = 0.018), and differences in microbial communities were linked to disease severity (P = 0.022). Furthermore, statistically significant shifts in the microbiome were identified among SARS-CoV-2-positive and -negative patients, in the latter of whom a higher abundance of Propionibacteriaceae (P = 0.028) and a reduction in the abundance of Corynebacterium accolens (P = 0.025) were observed. Our study corroborates the growing evidence that increased SARS-CoV-2 RNA detection from NP swabs is associated with the early stages rather than the severity of COVID-19. Further, we demonstrate that SARS-CoV-2 causes a significant change in the respiratory microbiome. This work illustrates the utility of mNGS for the detection of SARS-CoV-2, for diagnosing coinfections without viral target enrichment or amplification, and for the analysis of the respiratory microbiome.

Blood culture (BC) often fails to detect bloodstream microorganisms in sepsis. However, molecular diagnostics hold great potential. The molecular method PCR/electrospray ionization-mass spectrometry (PCR/ESI-MS) can detect DNA from hundreds of different microorganisms in whole blood. The aim of the present study was to evaluate the performance of this method in a multicenter study including 16 teaching hospitals in the United States (n = 13) and Europe (n = 3)., Blood culture (BC) often fails to detect bloodstream microorganisms in sepsis. However, molecular diagnostics hold great potential. The molecular method PCR/electrospray ionization-mass spectrometry (PCR/ESI-MS) can detect DNA from hundreds of different microorganisms in whole blood. The aim of the present study was to evaluate the performance of this method in a multicenter study including 16 teaching hospitals in the United States (n = 13) and Europe (n = 3). First, on testing of 2,754 contrived whole blood samples, with or without spiked microorganisms, PCR/ESI-MS produced 99.1% true-positive and 97.2% true-negative results. Second, among 1,460 patients with suspected sepsis (sepsis-2 definition), BC and PCR/ESI-MS on whole blood were positive in 14.6% and 25.6% of cases, respectively, with the following result combinations: BC positive and PCR/ESI-MS negative, 4.3%; BC positive and PCR/ESI-MS positive, 10.3%; BC negative and PCR/ESI-MS positive, 15.3%; and BC negative and PCR/ESI-MS negative, 70.1%. Compared with BC, PCR/ESI-MS showed the following sensitivities (coagulase-negative staphylococci not included): Gram-positive bacteria, 58%; Gram-negative bacteria, 78%; and Candida species, 83%. The specificities were >94% for all individual species. Patients who had received prior antimicrobial medications (n = 603) had significantly higher PCR/ESI-MS positivity rates than patients without prior antimicrobial treatment—31% versus 22% (P

The rise of antimicrobial resistant pathogens can be attributed to the lack of a rapid pathogen identification (ID) or antimicrobial susceptibility testing (AST), resulting in delayed therapeutic decision at the point of care. Gonorrhea is usually empirically treated with no AST results available before treatment, thus contributing to rapid rise in drug resistance. Herein we present a rapid AST platform using RNA signatures for Neisseria gonorrhoeae (NG). RNA-seq followed by bioinformatic tools were applied to explore potential markers in transcriptome profile of NG upon minutes of azithromycin exposure. Validation of candidate markers using PCR showed that two markers ( arsR (NGO1562) and rpsO ) can deliver accurate AST results across 14 tested isolates. Further validation of our cutoff in comparison to MIC across 64 more isolates confirmed reliability of our platform. Our RNA markers combined with emerging molecular point-of-care systems has the potential to greatly accelerate both ID and AST to inform treatment. Article Summary Line In this study, we present a rapid antimicrobial susceptibility testing platform using RNA signatures for Neisseria gonorrhoeae upon minutes of azithromycin exposure.

Methicillin-resistantWe searched relevant papers in PubMed for the last 10 years. In major papers, we scanned the bibliographies to ensure that important articles were included. This review describes screening and diagnostic test methods for MRSA and their analytical performances with a focus on rapid molecular-based assays including those that are on the horizon. Future novel technologies will allow more rapid detection of phenotypic resistance. In the case of whole-genome sequencing, detection of mutations may predict resistance, transmission, and virulence.Currently there are many diagnostic options for the detection of MRSA in surveillance and clinical samples. In general, these are highly accurate and have resulted in improvements in targeted management and reduction in hospital or intensive care unit length of stay for both MSSA and MRSA. Impact on mortality has been variable. Promising novel technologies will not only accurately identify pathogens and detect their resistance markers but will allow discovery of virulence determinants that might further affect patient management.

Routine identification of fungal pathogens from positive blood cultures by culture-based methods can be time-consuming, delaying treatment with appropriate antifungal agents. The GenMark Dx ePlex investigational use only blood culture identification fungal pathogen panel (BCID-FP) rapidly detects 15 fungal targets simultaneously in blood culture samples positive for fungi by Gram staining. We aimed to determine the performance of the BCID-FP in a multicenter clinical study. Blood culture samples collected at 10 United States sites and tested with BCID-FP at 4 sites were compared to the standard-of-care microbiological and biochemical techniques, fluorescence in situ hybridization using peptide nucleic acid probes (PNA-FISH) and matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS)., Routine identification of fungal pathogens from positive blood cultures by culture-based methods can be time-consuming, delaying treatment with appropriate antifungal agents. The GenMark Dx ePlex investigational use only blood culture identification fungal pathogen panel (BCID-FP) rapidly detects 15 fungal targets simultaneously in blood culture samples positive for fungi by Gram staining. We aimed to determine the performance of the BCID-FP in a multicenter clinical study. Blood culture samples collected at 10 United States sites and tested with BCID-FP at 4 sites were compared to the standard-of-care microbiological and biochemical techniques, fluorescence in situ hybridization using peptide nucleic acid probes (PNA-FISH) and matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). Discrepant results were analyzed by bi-directional PCR/sequencing of residual blood culture samples. A total of 866 clinical samples, 120 retrospectively and 21 prospectively collected, along with 725 contrived samples were evaluated. Sensitivity and specificity of detection of Candida species (C. albicans, C. auris, C. dubliniensis, C. famata, C. glabrata, C. guilliermondii, C. kefyr, C. krusei, C. lusitaniae, C. parapsilosis, and C. tropicalis) ranged from 97.1 to 100% and 99.8 to 100%, respectively. For the other organism targets, sensitivity and specificity were as follows: 100% each for Cryptococcus neoformans and C. gattii, 98.6% and 100% for Fusarium spp., and 96.2% and 99.9% for Rhodotorula spp., respectively. In 4 of the 141 clinical samples, the BCID-FP panel correctly identified an additional Candida species, undetected by standard-of-care methods. The BCID-FP panel offers a faster turnaround time for identification of fungal pathogens in positive blood cultures that may allow for earlier antifungal interventions and includes C. auris, a highly multidrug-resistant fungus.

Rapid identification from positive blood cultures is standard of care (SOC) in many clinical microbiology laboratories. The GenMark Dx ePlex Blood Culture Identification Gram-Positive (BCID-GP) Panel is a multiplex nucleic acid amplification assay based on competitive DNA hybridization and electrochemical detection using eSensor technology. This multicenter study compared the investigational-use-only (IUO) BCID-GP Panel to other methods of identification of 20 Gram-positive bacteria, four antimicrobial resistance genes, and both Pan Candida and Pan Gram-Negative targets that are unique to the BCID-GP Panel., Rapid identification from positive blood cultures is standard of care (SOC) in many clinical microbiology laboratories. The GenMark Dx ePlex Blood Culture Identification Gram-Positive (BCID-GP) Panel is a multiplex nucleic acid amplification assay based on competitive DNA hybridization and electrochemical detection using eSensor technology. This multicenter study compared the investigational-use-only (IUO) BCID-GP Panel to other methods of identification of 20 Gram-positive bacteria, four antimicrobial resistance genes, and both Pan Candida and Pan Gram-Negative targets that are unique to the BCID-GP Panel. Ten microbiology laboratories throughout the United States collected residual, deidentified positive blood culture samples for analysis. Five laboratories tested both clinical and contrived samples with the BCID-GP Panel. Comparator identification methods included each laboratory’s SOC, which included matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) and automated identification systems as well as targeted PCR/analytically validated real-time PCR (qPCR) with bidirectional sequencing. A total of 2,342 evaluable samples (1,777 clinical and 565 contrived) were tested with the BCID-GP Panel. The overall sample accuracy for on-panel organisms was 89% before resolution of discordant results. For pathogenic Gram-positive targets (Bacillus cereus group, Enterococcus spp., Enterococcus faecalis, Enterococcus faecium, Staphylococcus spp., Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus lugdunensis, Listeria spp., Listeria monocytogenes, Streptococcus spp., Streptococcus agalactiae, Streptococcus anginosus group, Streptococcus pneumoniae, and Streptococcus pyogenes), positive percent agreement (PPA) and negative percent agreement (NPA) ranged from 93.1% to 100% and 98.8% to 100%, respectively. For contamination rule-out targets (Bacillus subtilis group, Corynebacterium, Cutibacterium acnes, Lactobacillus, and Micrococcus), PPA and NPA ranged from 84.5% to 100% and 99.9% to 100%, respectively. Positive percent agreement and NPA for the Pan Candida and Pan Gram-Negative targets were 92.4% and 95.7% for the former and 99.9% and 99.6% for the latter. The PPAs for resistance markers were as follows: mecA, 97.2%; mecC, 100%; vanA, 96.8%; and vanB, 100%. Negative percent agreement ranged from 96.6% to 100%. In conclusion, the ePlex BCID-GP Panel compares favorably to SOC and targeted molecular methods for the identification of 20 Gram-positive pathogens and four antimicrobial resistance genes in positive blood culture bottles. This panel detects a broad range of pathogens and mixed infections with yeast and Gram-negative organisms from the same positive blood culture bottle.

The ideal sampling method and benefit of qualitative versus quantitative culture for carbapenem-resistant Enterobacteriaceae (CRE) recovery in hospitalized patient rooms and bathrooms is unknown. Although the use of nylon-flocked swabs improved overall gram-negative organism recovery compared with cellulose sponges, they were similar for CRE recovery. Quantitative culture was inferior and unrevealing beyond the qualitative results.

Background Small-colony variants (SCVs) are a distinct phenotype of Staphylococcus aureus, known for their role in chronic, difficult to treat infections, including cystic fibrosis (CF) lung disease. The goal of this study was to characterize SCV MRSA infection in an adult and pediatric CF population and to identify antibiotic susceptibility patterns unique to SCV MRSA. Methods We recovered methicillin-resistant S. aureus (MRSA) from respiratory culture samples from CF patients at the Johns Hopkins Hospital during a 6 month study period. Results Of 1161 samples, 200 isolates (17%) were identified as MRSA, and 37 isolates from 28 patients were identified as SCV MRSA. A higher proportion of MRSA was found among SCV isolates (37/66, 56%) compared to normal colony variant (NCV) isolates (163/417, 39%), p = 0.02. All SCV MRSA isolates from individual patients were susceptible to vancomycin and ceftaroline, but they demonstrated higher rates of antibiotic resistance to trimethoprim/sulfamethoxazole, moxifloxacin, and erythromycin, compared to NCV MRSA isolates. Additionally, individuals with SCV MRSA had lower lung function, higher rates of persistent MRSA infection, and higher rates of previous antibiotic use, compared to individuals with NCV MRSA. Conclusions A significant proportion of MRSA isolates recovered from patients with CF have the SCV morphology. Compared to individuals with NCV MRSA, those with SCV MRSA have higher rates of persistent MRSA infection and lower lung function. SCV MRSA isolates were more resistant than NCV, but they are highly susceptible to vancomycin, linezolid and ceftaroline.

The Staphylococcus intermedius group is a collection of coagulase-positive staphylococci composed of 5 members, including Staphylococcus pseudintermedius, a zoonotic pathogen often associated with exposure to dogs, and Staphylococcus delphini, which has not previously been recovered from humans. Here, we describe the first human case of S. delphini infection.

Bloodstream infection with Bacillus cereus/thuringiensis can be life threatening, particularly in patients who are severely immunocompromised. In this report we describe a case that progressed from asymptomatic to fatal over approximately 5 hours despite extensive resuscitation efforts. We identify the pathogen and assemble its genome, in which we find genes for toxins that may have contributed to the precipitous demise. In the context of this and other cases we discuss the possible indication for rapid appropriate antibiotic administration and potentially antitoxin treatment or toxin removal in fulminant illness in immunocompromised patients.

Accurate detection of carbapenemase-producing glucose-nonfermenting Gram-negative bacilli (CPNFs), including Pseudomonas aeruginosa and Acinetobacter baumannii , is necessary to prevent their dissemination within health care settings. We performed a method comparison study of 11 phenotypic carbapenemase detection assays to evaluate their accuracy for the detection of CPNFs. A total of 96 carbapenem-resistant glucose-nonfermenting isolates were included, of which 29% produced carbapenemases. All CPNFs were molecularly characterized to identify β-lactamase genes. A total of 86% of the carbapenemase-producing P. aeruginosa isolates produced class B carbapenemases. Several assays performed with a sensitivity of >90% for the detection of carbapenemase-producing P. aeruginosa , including all rapid chromogenic assays and the modified carbapenem inactivation method. Most included assays, with the exception of the Manual Blue Carba assay, the Modified Carba NP assay, the boronic acid synergy test, and the metallo-β-lactamase Etest, had specificities of >90% for detecting carbapenemase-producing P. aeruginosa . Class D carbapenemases were the most prevalent carbapenemases among the carbapenemase-producing A. baumannii strains, with 60% of the carbapenemase-producing A. baumannii isolates producing acquired OXA-type carbapenemases. Although several assays achieved >90% specificity in identifying carbapenemase-producing A. baumannii , no assays achieved a sensitivity of greater than 90%. Our findings suggest that the available phenotypic tests generally appear to have excellent sensitivity and specificity for detecting carbapenemase-producing P. aeruginosa isolates. However, further modifications to existing assays or novel assays may be necessary to accurately detect carbapenemase-producing A. baumannii .

The epidemiology of Staphylococcus aureus infection in children is dynamic. We conducted a retrospective observational study on pediatric clinical cultures, performed between 2005 and 2017, that grew S aureus to determine temporal trends in antibiotic resistance. Although methicillin resistance declined, clindamycin and trimethoprim-sulfamethoxazole resistance increased significantly, especially among community-onset isolates.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Arcobacter spp. are commonly associated with shellfish and have been increasingly implicated in human gastrointestinal disease. We report the first case of human bacteremia with Malacobacter (previously Arcobacter) mytili acquired after exposure to Maryland crab. Arcobacter spp. should be considered in febrile illnesses when the history indicates exposure to seafood.

Industrial hog operation (IHO) workers are at increased risk of carrying Staphylococcus aureus in their nares, particularly strains that are livestock-associated (LA) and multidrug-resistant. The pathogenicity of LA-S. aureus strains remains unclear, with some prior studies suggesting reduced transmission and virulence in humans compared to community-associated methicillin-resistant (CA-MRSA) S. aureus. The objective of this study was to determine the degree to which LA-S. aureus strains contracted by IHO workers cause disease relative to a representative CA-MRSA strain in a mouse model of skin and soft tissue infection (SSTI). Mice infected with CC398 LA-S. aureus strains (IHW398-1 and IHW398-2) developed larger lesion sizes with higher bacterial burden than mice infected with CA-MRSA (SF8300) (p S. aureus infected mice had decreased IL-1β protein levels compared with CA-MRSA-infected mice (p S. aureus SSTIs. WGSA revealed heterogeneity in virulence factor and antimicrobial resistance genes carried by LA-S. aureus and CA-MRSA strains. The observed pathogenicity suggest that more attention should be placed on preventing the spread of LA-S. aureus into human populations.

The GenMark Dx ePlex Respiratory Pathogen Panel (RP) is a multiplexed nucleic acid test for the qualitative detection of common viral and a few bacterial causes of respiratory tract infections. The ePlex RP has received FDA clearance for nasopharyngeal swab (NPS) specimens collected in viral transport media. In this study, we evaluated the performance of the ePlex RP panel in comparison to the NxTAG Respiratory Pathogen Panel (NxTAG-RPP) from Luminex in use in our laboratory, not only for NPS but also for bronchoalveolar lavage specimens (BAL). We also evaluated the impact of implementing the ePlex RP on the test turn-around time (TAT). The newest panel from GenMark Dx, the ePlex Respiratory Pathogen Panel 2 (RP2), which added the SARS-CoV-2 target to the RP was also evaluated for NPS. Verification of the performance of the ePlex RP for both NPS and BAL showed 93.3 % and 84.9 % total agreement with the NxTAG-RPP respectively. An overall comparison of the TAT after implementing the ePlex RP as compared to the NxTAG-RPP assay showed an average decrease of almost seven-fold.

Unmanned aerial vehicles (UAVs) could potentially be used to transport microbiological specimens. To examine the impact of UAVs on microbiological specimens, blood and sputum culture specimens were seeded with usual pathogens and flown in a UAV for 30 ± 2 min. Times to recovery, colony counts, morphologies, and matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS)-based identifications of the flown and stationary specimens were similar for all microbes studied.

Like a bottle of fine wine that has been allowed to age, the 40-year career in clinical microbiology of Patrick R. Murray, Ph.D., has yielded a unique and sublime blend of contributions to our discipline. From clinical laboratory director to editor extraordinaire to innovator and discoverer of

Two screening methods to detect staphylococcal colonization in humans were compared. Direct plating to CHROMagar (BD Diagnostics) was compared to a broth preenrichment followed by plating to Baird-Parker agar. The broth-enrichment method was comparable to CHROMagar for methicillin-resistant Staphylococcus aureas (MRSA) detection, but the enrichment method was optimum for recovery of coagulase-positive Staphylococcus spp.

Rectal swabs from high-risk patients were screened for carbapenem-resistant organisms (CROs) using several methods. The direct MacConkey plate method was the most sensitive for CROs (95%), while chromID CARBA and the Check-Direct CPE screen assay were the most sensitive for the detection of carbapenemase-producing organisms (CPOs) (100%; all bla KPC ). All methods had a specificity of >90% for CROs, and for CPOs, the specificity ranged from 85 to 98%. Broth enrichment methods performed poorly compared to direct inoculation methods, negating the need for the broth enrichment step.

Background: Corynebacterium species and gram-positive coryneform-like bacteria (coryneforms) are increasingly reported causes of opportunistic infections in immunocompromised patients. Biochemical identification methods for these bacteria are often inaccurate. We evaluated matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) for routine Corynebacterium and coryneform identification. Methods: A total of 286 Corynebacterium species and coryneforms recovered from patients were identified by MALDI-TOF MS analysis using the Bruker Microflex instrument, Biotyper software version 3.0, and database version 3.1.66 (Bruker Daltonics, Billerica, MA) following formic acid–based, direct on-plate extraction. The spectral cutoff scores used for interpretation were 2.0 or more for species-level identification and 1.7 or more for genus level. Scores lower than 1.7 were considered as no reliable identification. The results were compared with API Coryne (bioMerieux, Durham, NC) and sequencing of 16S ribosomal RNA genes. Results: Of the 231 Corynebacterium (19 species), 99.6% were correctly identified to the genus level and 88.7% to the species level. Of the 55 coryneforms (14 genera), 90.9% were correctly identified to the genus level and 67.3% to the species level. API Coryne was able to identify 89.2% of Corynebacterium species (species level) and 63.6% of coryneforms (genus level). Conclusions: Rapid on-plate testing yielded identification of more Corynebacterium species and related bacteria than biochemical methods.

During a 14-month period of using matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) for group B streptococcus (GBS) identification, we recovered 32 (1%) Streptococcus pseudoporcinus isolates from 3,276 GBS screening cultures from female genital sources (25 isolates from pregnant women and 7 from nonpregnant women). An additional two S. pseudoporcinus isolates were identified from a urine culture and a posthysterectomy wound culture. These isolates were found to cross-react with three different GBS antigen agglutination kits, PathoDx (Remel) (93%), Prolex (Pro-Lab Diagnostics) (38%), and Streptex (Remel) (53%). New approaches to bacterial identification in routine clinical microbiology laboratories may affect the prevalence of S. pseudoporcinus .

We report a case of bypass graft infection and bacteremia caused by Anaerostipes caccae. A review of the literature shows no reported human infection caused by this microorganism to date. The patient was initially treated with vancomycin and piperacillin-tazobactam on admission and with amoxicillin-clavulanate upon discharge. The slow-growing organism was subsequently found to be susceptible to metronidazole and ertapenem.

The critical nature of the microbiology laboratory in infectious disease diagnosis calls for a close, positive working relationship between the physician/advanced practice provider and the microbiologists who provide enormous value to the healthcare team. This document, developed by experts in laboratory and adult and pediatric clinical medicine, provides information on which tests are valuable and in which contexts, and on tests that add little or no value for diagnostic decisions. This document presents a system-based approach rather than specimen-based approach, and includes bloodstream and cardiovascular system infections, central nervous system infections, ocular infections, soft tissue infections of the head and neck, upper and lower respiratory infections, infections of the gastrointestinal tract, intra-abdominal infections, bone and joint infections, urinary tract infections, genital infections, and other skin and soft tissue infections; or into etiologic agent groups, including arthropod-borne infections, viral syndromes, and blood and tissue parasite infections. Each section contains introductory concepts, a summary of key points, and detailed tables that list suspected agents; the most reliable tests to order; the samples (and volumes) to collect in order of preference; specimen transport devices, procedures, times, and temperatures; and detailed notes on specific issues regarding the test methods, such as when tests are likely to require a specialized laboratory or have prolonged turnaround times. In addition, the pediatric needs of specimen management are also emphasized. There is intentional redundancy among the tables and sections, as many agents and assay choices overlap. The document is intended to serve as a guidance for physicians in choosing tests that will aid them to quickly and accurately diagnose infectious diseases in their patients.

OBJECTIVES: In this study, we characterize a concurrent disseminated infection with a virulent hypermucoviscous (HMV) Klebsiella pneumoniae and an OXA-181-producing XDR K. pneumoniae from a patient with recent hospitalization in India. During exposure to meropenem therapy, the highly susceptible HMV K. pneumoniae became resistant to carbapenems, consistent with the acquisition of bla(OXA-181). METHODS: Twelve K. pneumoniae isolates were recovered from the patient and the hospital room environment over a 3 month hospitalization. Phenotypic and molecular studies were completed to characterize the isolates. Oxford Nanopore and Illumina MiSeq WGS were performed to study phylogeny (MLST and SNPs), plasmids and virulence genes and demonstrate changes in the organism’s resistome that occurred over time. RESULTS: WGS revealed that the HMV K. pneumoniae belonged to ST23 and harboured an IncH1B virulence plasmid, while the XDR K. pneumoniae belonged to ST147 and possessed two MDR plasmids (IncR and IncFII), the bla(OXA-181)-bearing ColKP3 plasmid and chromosomal mutations conferring the XDR phenotype. Sequential isolates demonstrated plasmid diversification (fusion of the IncR and IncFII plasmids), mobilization of resistance elements (ompK35 inactivation by ISEcp1-bla(CTX-M-15) mobilization, varying numbers of resistance genes on plasmid scaffolds) and chromosomal mutations (mutations in mgrB) leading to further antibiotic resistance that coincided with antibiotic pressure. Importantly, the HMV strain in this study was unable to preserve the carbapenem-resistant phenotype without the selective pressure of meropenem. CONCLUSIONS: To the best of our knowledge, we are the first to report a carbapenem-resistant HMV K. pneumoniae strain in the USA. Ultimately, this case demonstrates the role of antibiotic pressure in the acquisition and loss of important genetic elements.

We describe results from a multicenter study evaluating the Accelerate Pheno system, a first of its kind diagnostic system that rapidly identifies common bloodstream pathogens from positive blood cultures within 90 min and determines bacterial phenotypic antimicrobial susceptibility testing (AST) results within ∼7 h. A combination of fresh clinical and seeded blood cultures were tested, and results from the Accelerate Pheno system were compared to Vitek 2 results for identification (ID) and broth microdilution or disk diffusion for AST. The Accelerate Pheno system accurately identified 14 common bacterial pathogens and two Candida spp. with sensitivities ranging from 94.6 to 100%. Of fresh positive blood cultures, 89% received a monomicrobial call with a positive predictive value of 97.3%. Six common Gram-positive cocci were evaluated for ID. Five were tested against eight antibiotics, two resistance phenotypes (methicillin-resistant Staphylococcus aureus and Staphylococcus spp. [MRSA/MRS]), and inducible clindamycin resistance (MLSb). From the 4,142 AST results, the overall essential agreement (EA) and categorical agreement (CA) were 97.6% and 97.9%, respectively. Overall very major error (VME), major error (ME), and minor error (mE) rates were 1.0%, 0.7%, and 1.3%, respectively. Eight species of Gram-negative rods were evaluated against 15 antibiotics. From the 6,331 AST results, overall EA and CA were 95.4% and 94.3%, respectively. Overall VME, ME, and mE rates were 0.5%, 0.9%, and 4.8%, respectively. The Accelerate Pheno system has the unique ability to identify and provide phenotypic MIC and categorical AST results in a few hours directly from positive blood culture bottles and support accurate antimicrobial adjustment.

Bacteroides fragilis is a common colonic symbiote of which one subtype, enterotoxigenic Bacteroides fragilis (ETBF), causes inflammatory diarrhea. However, asymptomatic ETBF colonization is common. Through its primary virulence factor, B. fragilis toxin (BFT), ETBF causes asymptomatic, chronic colitis in C57BL/6 mice and increased colon tumorigenesis in multiple intestinal neoplasia mice. Human studies suggest an association between ETBF infection, inflammatory bowel disease, and colon cancer. Additional studies on ETBF epidemiology are, therefore, crucial. The goal of this study is to develop a reliable fecal diagnostic for ETBF. To develop a sensitive assay for ETBF, we tested multiple protocols on mouse stools spiked with serially diluted ETBF. Each assay was based on either touchdown or quantitative polymerase chain reaction (qPCR) and used primers targeted to bft to detect ETBF. Using touchdown PCR or qPCR, the mean ETBF detection limit was 1.55 × 10(6) colony-forming units (CFU)/g stool and 1.33 × 10(4) CFU/g stool, respectively. Augmentation of Bacteroides spp. growth in fecal samples using PYGB (Peptone Yeast Glucose with Bile) broth enhanced ETBF detection to 2.93 × 10(2) CFU/g stool using the touchdown PCR method and 2.63 × 10(2) CFU/g stool using the qPCR method. Fecal testing using combined culture-based amplification and bft touchdown PCR is a sensitive assay for the detection of ETBF colonization and should be useful in studying the role of ETBF colonization in intestinal diseases, such as inflammatory bowel disease and colon cancer. We conclude that touchdown PCR with culture-based amplification may be the optimal ETBF detection strategy, as it performs as well as qPCR with culture-based amplification, but is a less expensive technique.

In an effort to track and control the transmission of Staphylococcus aureus, patient isolates are saved for epidemiological studies ([1][1][–][2][3][3]). Study investigators often assume that colonies with the same morphology on the original culture plate represent the same clone. However, there

Sepsis caused by Staphylococcus aureus is a major health problem worldwide. Better outcomes are achieved when rapid diagnosis and determination of methicillin susceptibility enable early optimization of antimicrobial therapy. Eight large clinical laboratories, seven from the United States and one from Scotland, evaluated the combination of the Staphylococcus Quick FISH BC and the new mecA Xpress FISH assay (both AdvanDx, Woburn, MA, USA) for the detection of methicillin-resistant S. aureus in positive blood cultures. Blood cultures flagged as positive by automated blood culture instruments and demonstrating only Gram-positive cocci in clusters on Gram stain were tested by Quick FISH, a 20-min assay. If only S. aureus was detected, mecA Xpress FISH testing followed. The recovered S. aureus isolates were tested by cefoxitin disk diffusion as the reference method. The Quick FISH assay results were concordant with the routine phenotypic testing methods of the testing laboratories in 1,211/1,221 (99.1%) samples and detected 488/491 S. aureus organisms (sensitivity, 99.4%; specificity, 99.6%). Approximately 60% of the samples (730) contained coagulase-negative staphylococci or nonstaphylococci as assessed by the Quick FISH assay and were not tested further. The 458 compliant samples positive exclusively for S. aureus by the Quick FISH assay were tested by the mecA Xpress FISH assay, which detected 209 of 211 methicillin-resistant S. aureus organisms (sensitivity, 99.1%; specificity, 99.6%). The mecA Xpress FISH assay also showed high reproducibility, with 534/540 tests performed by 6 operators over 5 days achieving reproducible results (98.9% agreement). The combination of the Staphylococcus Quick FISH BC and mecA Xpress FISH assays is sensitive, specific, and reproducible for the detection of methicillin-resistant S. aureus and yields complete results in 2 h after the blood culture turns positive.

Three commercial antimicrobial susceptibility testing (AST) methods were compared to broth microdilution for testing of Staphylococcus aureus and enterococci against vancomycin, daptomycin, and linezolid. Despite high levels of categorical agreement and essential agreement, vancomycin MICs determined by MicroScan were often 1 log 2 concentration higher and MICs determined by Phoenix 1 log 2 concentration lower. Daptomycin MICs were 1 to 2 log 2 concentrations higher by all AST methods, except Etest, potentially impacting definitive antimicrobial therapy for bloodstream infections due to these organisms.

Background: Use of antimicrobials in industrial food-animal production is associated with the presence of antimicrobial-resistant Staphylococcus aureus (S. aureus) among animals and humans. Hog slaughter/processing plants process large numbers of animals from industrial animal operations and are environments conducive to the exchange of bacteria between animals and workers. Objectives: We compared the prevalence of multidrug-resistant S. aureus (MDRSA) and methicillin-resistant S. aureus (MRSA) carriage among processing plant workers, their household members, and community residents. Methods: We conducted a cross-sectional study of hog slaughter/processing plant workers, their household members, and community residents in North Carolina. Participants responded to a questionnaire and provided a nasal swab. Swabs were tested for S. aureus, and isolates were tested for antimicrobial susceptibility and subjected to multilocus sequence typing. Results: The prevalence of S. aureus was 21.6%, 30.2%, and 22.5% among 162 workers, 63 household members, and 111 community residents, respectively. The overall prevalence of MDRSA and MRSA tested by disk diffusion was 6.9% and 4.8%, respectively. The adjusted prevalence of MDRSA among workers was 1.96 times (95% CI: 0.71, 5.45) the prevalence in community residents. The adjusted average number of antimicrobial classes to which S. aureus isolates from workers were resistant was 2.54 times (95% CI: 1.16, 5.56) the number among isolates from community residents. We identified two MDRSA isolates and one MRSA isolate from workers as sequence type 398, a type associated with exposure to livestock. Conclusions: Although the prevalence of S. aureus and MRSA was similar in hog slaughter/processing plant workers and their household and community members, S. aureus isolates from workers were resistant to a greater number of antimicrobial classes. These findings may be related to the nontherapeutic use of antimicrobials in food-animal production. Citation: Castillo Neyra R, Frisancho JA, Rinsky JL, Resnick C, Carroll KC, Rule AM, Ross T, You Y, Price LB, Silbergeld EK. 2014. Multidrug-resistant and methicillin-resistant Staphylococcus aureus (MRSA) in hog slaughter and processing plant workers and their community in North Carolina (USA). Environ Health Perspect 122:471–477; http://dx.doi.org/10.1289/ehp.1306741

Early identification of carbapenemase-producing Enterobacteriaceae (CPE) is essential to prevent their dissemination within health care settings. Our objective was to evaluate the accuracy of 11 phenotypic assays for the detection of CPE. Two collections of carbapenem-resistant Enterobacteriaceae (CRE) isolates were evaluated, including 191 retrospective isolates (122 CP-CRE and 69 non-CP isolates) as well as 45 prospective clinical isolates (15 CP-CRE and 30 non-CP-CRE) obtained over a 3-month period. The sensitivity and specificity of each test was determined, with molecular genotype serving as the gold standard. Among the retrospective cohort, sensitivities ranged from 72% for the boronic acid synergy test for the detection of KPC producers to ≥98% for the modified Carba NP, the Rapidec Carba NP, the manual Blue Carba, and the modified carbapenem inactivation method for the detection of any CPE. Sensitivity differed among tests across enzyme classes. All assays had excellent specificity exceeding 95%, with the exception of the boronic acid synergy test (88%) and modified Hodge test (91%). Prospectively, 45 CRE isolates were encountered over a 3-month period, including 15 CPE (33%) and 30 non-CP-CRE (67%). Results from the prospective cohort were similar. However, a decrease in specificity was observed across most tests, likely due to restricted inclusion of non-CP-CRE to assess the specificity of the assays. Overall, accuracy of CPE detection varied across phenotypic tests. Local epidemiology of CP genotypes, turnaround time, and ease of incorporation into the laboratory workflow should be considered when selecting a phenotypic assay for clinical use.

Background Co-infections of Candida auris and carbapenemase-producing carbapenem-resistant Gram-negative organisms (CP-CRO) are an increasing global concern and rarely seen in the United States. We report the case of a 59-year-old male, with recent hospitalization in India, admitted to our facility with C. auris isolated from urine and axilla/groin specimens and CP-CRO from five body sites. Methods Travel screening in the emergency department identified a patient at high risk for colonization/infection with multidrug-resistant organisms (MDRO). Contact precautions were initiated. Eight CP-CRO isolates were subsequently identified from clinical and routine surveillance cultures from five separate sites. Of the isolates, seven contained one or more carbapenemase-producing genes detected by Xpert Carba-R assay (Cepheid, Sunnyvale, CA) (Table 1). The microbiology laboratory alerted the infection control department of a presumptive positive C. auris from a clinical urine culture from the same patient. Enhanced mitigation strategies were initiated in regards to cleaning and disinfection. An exposure investigation was also conducted using a point prevalence approach. Surveillance cultures were obtained from inpatients currently admitted to the same unit as the index patient. Axilla/groin specimens were collected for C. auris testing, and rectal specimens were collected for CP-CRO gene testing (CRE Real-Time PCR). Results Eighteen patients in addition to the index patient were hospitalized on the acute medicine unit. One patient refused testing for CP-CRO; therefore, 17 patients were tested for CP-CRO, and 18 patients were tested for C. auris. Neither CP-CRO nor C. auris were recovered from any patient. Conclusion A patient co-infected with C. auris and multiple CP-CRO was identified by clinical and routine surveillance cultures at Johns Hopkins Hospital. Travel screening allowed proactive isolation upon presentation. Enhanced infection control measures were implemented and a point prevalence surveillance study was conducted on the general acute care medicine inpatient unit. No transmission of either C. auris or CP-CRO was detected, likely due in part to rapid identification and strict infection control measures. Disclosures All authors: No reported disclosures.

Background Pneumocystis jirovecii is a medically important fungal pathogen responsible for opportunistic infections in immunocompromised hosts with high morbidity and mortality. Compared with standard microscopy based assays, home-brew nucleic acid amplification tests (NAAT) have emerged as sensitive tools for the diagnosis of P. jirovecii pneumonia, but their sensitivities vary depending upon selected genetic targets. Recent studies suggest that the mitochondrial small subunit (mtSSU) is a better NAAT target given its higher copy number and stable expression in the disease process. We aimed to develop and evaluate a mtSSU-targeted MultiCode real-time PCR assay that incorporates a sample processing control (SPC) and enables detection of P. jirovecii in bronchoalveolar lavage fluid (BALF) and induced sputum. Methods Firstly, we compared manual DNA extraction using Zymo Quick DNA kit with automated extraction using the NucliSENS easyMAG system after sample pretreatment with either FastPrep mechanical grinding or vortex-based bead beating. We then determined the mouse hepatitis virus SPC (Luminex) spike-in amount, and optimized the PCR conditions on the ABI 7500 PCR system. A new Pneumocystis mtSSU run control was generated by cloning and transforming mtSSU gene into a genetically engineered E. coli strain, and quantified with a home-brew quantitative TaqMan PCR. Lastly, the performance characteristics of the MultiCode PCR assay were determined. Results Mechanical grinding of BALF or sputum before the easyMAG based extraction was better than the other extraction protocols as evidenced by lower CT of mtSSU or SPC. Diluted SPC added to samples before DNA extraction made its CT within 31–34. With the mtSSU run control, the limit of detection of the new assay was 80 copies/mL. No cross-reactivity was found with 9 respiratory viruses, 8 bacteria or 11 fungi. The assay has high reproducibility for three-day detection of the same sample aliquots for mtSSU (CT: 30.0–30.3; CV%: 0.5–1.6) and SPC (CT: 32.1–32.2; CV%: 0.8–2.4). Conclusion We developed a novel MultiCode real-time PCR assay for detection of P. jirovecii in BALF and sputum, which demonstrated high analytical sensitivity, specificity and reproducibility and warrants further clinical validation. Disclosures All authors: No reported disclosures.

The Verigene Clostridium difficile Nucleic Acid test (Verigene CDF test) (Nanosphere, Northbrook, IL) is a multiplex qualitative PCR assay that utilizes a nanoparticle-based array hybridization method to detect C. difficile tcdA and tcdB in fecal specimens. In addition, the assay detects binary toxin gene sequences and the single base pair deletion at nucleotide 117 (Δ 117) in tcdC to provide a presumptive identification of the epidemic strain 027/NAP1/BI (referred to here as ribotype 027). This study compared the Verigene CDF test with anaerobic direct and enriched toxigenic culture on stool specimens from symptomatic patients among five geographically diverse laboratories within the United States. The Verigene CDF test was performed according to the manufacturer's instructions, and the reference methods performed by a central laboratory included direct culture onto cycloserine cefoxitin fructose agar (CCFA) and enriched culture using cycloserine cefoxitin mannitol broth with taurocholate and lysozyme. Recovered isolates were identified as C. difficile using gas liquid chromatography and were tested for toxin using a cell culture cytotoxicity neutralization assay. Strains belonging to ribotype 027 were determined by PCR ribotyping and bidirectional sequencing for Δ 117 in tcdC . A total of 1,875 specimens were evaluable. Of these, 275 specimens (14.7%) were culture positive by either direct or enriched culture methods. Compared to direct culture alone, the overall sensitivity, specificity, positive predictive value, and negative predictive value for the Verigene CDF test were 98.7%, 87.5%, 42%, and 99.9%, respectively. Compared to combined direct and enriched culture results, the sensitivity, specificity, positive predictive value, and negative predictive values of the Verigene CDF test were 90.9%, 92.5%, 67.6%, and 98.3%, respectively. Of the 250 concordantly culture-positive specimens, 59 (23.6%) were flagged as “hypervirulent”; 53 were confirmed as ribotype 027, and all 59 possessed Δ 117 in tcdC . Time to results was approximately 2.5 h per specimen. The Verigene CDF test is a novel nucleic acid microarray that reliably detects both C. difficile toxins A and B in unformed stool specimens and appears to adequately identify ribotype 027 isolates.

Electrospray ionization mass spectrometry (ESI-MS) analysis of reverse transcription (RT)-PCR amplicons from human respiratory samples allows for broad pathogen identification approximately 8 h after collection. We investigated the performance characteristics of a high-throughput RT-PCR-coupled ESI-MS assay for distinguishing biothreat (BT) agents from common bacterial, fungal, and viral respiratory pathogens in bronchoalveolar lavage (BAL) fluid specimens from subjects with suspected respiratory infections. In a retrospective case series, 202 BAL fluid specimens were collected at the Johns Hopkins Hospital between August 2010 and February 2011 from patients with suspected acute respiratory infections. Samples were processed using standard bacterial, viral, and fungal testing in the clinical microbiology laboratory as part of routine care and then were blindly spiked with either water or nucleic acids from BT organisms ( Bacillus anthracis , Yersinia pestis , Francisella tularensis , Brucella spp., Burkholderia spp., and Rickettsia prowazekii ) and tested by RT-PCR–ESI-MS. The sensitivities and specificities of RT-PCR–ESI-MS versus standard clinical methods were as follows: for mock BT DNA, 98.5% sensitivity (95% confidence interval [CI], 94.2 to 99.7%) and 100% specificity (95% CI, 93.1 to 100.0%); for bacterial pathogens, 81.8% sensitivity (95% CI, 74.3 to 87.6%) and 73.6% specificity (95% CI, 64.2 to 81.4%); for viral pathogens, 93.3% sensitivity (95% CI, 66.0 to 99.7%) and 97.3% specificity (95% CI, 89.7 to 99.5%); for fungal pathogens, 42.6% sensitivity (95% CI, 29.5 to 56.7%) and 97.8% specificity (95% CI, 91.8 to 99.6%). Our data suggest that RT-PCR–ESI-MS is a useful adjunct to standard culture protocols for rapid detection of both BT and common respiratory pathogens; further study is required for assay validation, especially for fungal detection, and potential implementation.

Candida auris, an emerging multidrug-resistant yeast associated with a high mortality rate, has been increasingly reported outside the United States to cause outbreaks in hospital settings ([1][1]). Although this organism is rare in the United States, its prevalence may be underestimated because of