A US-based national surveillance study for the susceptibility and epidemiology of Clostridioides difficile isolates with special reference to ridinilazole: 2020-2021.

We have previously reported on the susceptibility and epidemiology of Clostridioides difficile isolates from six geographically dispersed medical centers in the United States. This current survey was conducted with isolates collected in 2020-2021 from six geographically dispersed medical centers in the United States, with specific attention to susceptibility to ridinilazole as well as nine comparators. C. difficile isolates or stools from patients with C. difficile antibiotic-associated diarrhea were collected and referred to a central laboratory. After species confirmation of 300 isolates at the central laboratory, antibiotic susceptibilities were determined by the agar dilution method [M11-A9, Clinical and Laboratory Standards Institute (CLSI)] against the 10 agents. Ribotyping was performed by PCR capillary gel electrophoresis on all isolates. Ridinilazole had a minimum inhibitory concentration (MIC) 90 of 0.25 mcg/mL, and no isolate had an MIC greater than 0.5 mcg/mL. In comparison, fidaxomicin had an MIC 90 of 0.5 mcg/mL. The vancomycin MIC 90 was 2 mcg/mL with a 0.7% resistance rate [both CLSI and European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria]. The metronidazole MIC 90 was 1 mcg/mL, with none resistant by CLSI criteria, and a 0.3% resistance rate by EUCAST criteria. Among the 50 different ribotypes isolated in the survey, the most common ribotype was 014-020 (14.0%) followed by 106 (10.3%), 027 (10%), 002 (8%), and 078-126 (4.3%). Ridinilazole maintained activity against all ribotypes and all strains resistant to any other agent tested. Ridinilazole showed excellent in vitro activity against C. difficile isolates collected between 2020 and 2021 in the United States, independent of ribotype.

The vapor-phase multi-stage CMD test for characterizing contaminant mass discharge associated with VOC sources in the vadose zone: Application to three sites in different lifecycle stages of SVE operations.

Vapor-phase multi-stage contaminant mass discharge (CMD) tests were conducted at three field sites to measure mass discharge associated with contaminant sources located in the vadose zone. The three sites represent the three primary stages of the soil vapor extraction (SVE) operations lifecycle-pre/initial-SVE, mid-lifecycle, and near-closure. A CMD of 32g/d was obtained for a site at which soil vapor SVE has been in operation for approximately 6years, and for which mass removal is currently in the asymptotic stage. The contaminant removal behavior exhibited for the vapor extractions conducted at this site suggests that there is unlikely to be a significant mass of non-vapor-phase contaminant (e.g., DNAPL, sorbed phase) remaining in the advective domains, and that most remaining mass is likely located in poorly accessible domains. Given the conditions for this site, this remaining mass is hypothesized to be associated with the low-permeability (and higher water saturation) region in the vicinity of the saturated zone and capillary fringe. A CMD of 25g/d was obtained for a site wherein SVE has been in operation for several years but concentrations and mass-removal rates are still relatively high. A CMD of 270g/d was obtained for a site for which there were no prior SVE operations. The behavior exhibited for the vapor extractions conducted at this site suggest that non-vapor-phase contaminant mass (e.g., DNAPL) may be present in the advective domains. Hence, the asymptotic conditions observed for this site most likely derive from a combination of rate-limited mass transfer from DNAPL (and sorbed) phases present in the advective domain as well as mass residing in lower-permeability ("non-advective") regions. The CMD values obtained from the tests were used in conjunction with a recently developed vapor-discharge tool to evaluate the impact of the measured CMDs on groundwater quality.

An international, prospective, multicenter evaluation of the combination of AdvanDx Staphylococcus QuickFISH BC with mecA XpressFISH for detection of methicillin-resistant Staphylococcus aureus isolates from positive blood cultures.

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 QuickFISH BC and the new mecA XpressFISH 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 QuickFISH, a 20-min assay. If only S. aureus was detected, mecA XpressFISH testing followed. The recovered S. aureus isolates were tested by cefoxitin disk diffusion as the reference method. The QuickFISH 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 QuickFISH assay and were not tested further. The 458 compliant samples positive exclusively for S. aureus by the QuickFISH assay were tested by the mecA XpressFISH assay, which detected 209 of 211 methicillin-resistant S. aureus organisms (sensitivity, 99.1%; specificity, 99.6%). The mecA XpressFISH 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 QuickFISH BC and mecA XpressFISH 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.

Multicenter evaluation of the Sepsityper™ extraction kit and MALDI-TOF MS for direct identification of positive blood culture isolates using the BD BACTEC™ FX and VersaTREK(®) diagnostic blood culture systems.

AIMS: (i) Evaluation of delayed time to blood culture extraction by the Sepsityper kit and impact of shipping pellets off-site for MALDI-TOF MS analysis. (ii) Comparison of Sepsityper and laboratory-developed extraction methods from a literature review. METHODS AND RESULTS: Using two blood culture systems (BD BACTEC and VersaTREK), we extracted 411 positive blood cultures using the Sepsityper kit to mimic a potential protocol for institutions without a MALDI-TOF MS. Extracted pellets were shipped and analysed on the Bruker UltraflexIII. Successful extraction of 358 (87·1%) samples was determined by the presence of detectable proteins. MALDI-TOF MS correctly identified 332 (80·8%) samples. CONCLUSIONS: Delayed time to extraction did not affect Sepsityper extraction or MALDI-TOF MS accuracy. The extracted pellets remain stable and provide accurate results by MALDI-TOF MS when shipped at room temperature to off-site reference laboratories. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to show that institutions without a MALDI-TOF MS can take advantage of this innovative technology by shipping a volume of blood to an off-site laboratory for extraction and MALDI-TOF MS analysis. We also performed a literature review to compare various extraction methods.

Application of a Persistent Dissolved-phase Reactive Treatment Zone for Mitigation of Mass Discharge from Sources Located in Lower-Permeability Sediments.

The purpose of this study is to examine the development and effectiveness of a persistent dissolved-phase treatment zone, created by injecting potassium permanganate solution, for mitigating discharge of contaminant from a source zone located in a relatively deep, low-permeability formation. A localized 1,1-dichloroethene (DCE) source zone comprising dissolved- and sorbed-phase mass is present in lower permeability strata adjacent to a sand/gravel unit in a section of the Tucson International Airport Area (TIAA) Superfund Site. The results of bench-scale studies conducted using core material collected from boreholes drilled at the site indicated that natural oxidant demand was low, which would promote permanganate persistence. The reactive zone was created by injecting a permanganate solution into multiple wells screened across the interface between the lower-permeability and higher-permeability units. The site has been monitored for nine years to characterize the spatial distribution of DCE and permanganate. Permanganate continues to persist at the site, and a substantial and sustained decrease in DCE concentrations in groundwater has occurred after the permanganate injection.. These results demonstrate successful creation of a long-term, dissolved-phase reactive-treatment zone that reduced mass discharge from the source. This project illustrates the application of in-situ chemical oxidation as a persistent dissolved-phase reactive-treatment system for lower-permeability source zones, which appears to effectively mitigate persistent mass discharge into groundwater.

Persistence of a Groundwater Contaminant Plume after Hydraulic Source Containment at a Chlorinated-Solvent Contaminated Site.

The objective of this study was to characterize the behavior of a groundwater contaminant (trichloroethene) plume after implementation of a source-containment operation at a site in Arizona. The plume resides in a quasi three-layer system comprising a sand/gravel unit bounded on the top and bottom by relatively thick silty clayey layers. The system was monitored for 60 months beginning at start-up in 2007 to measure the change in contaminant concentrations within the plume, the change in plume area, the mass of contaminant removed, and the integrated contaminant mass discharge. Concentrations of trichloroethene in groundwater pumped from the plume extraction wells have declined significantly over the course of operation, as have concentrations for groundwater sampled from 40 monitoring wells located within the plume. The total contaminant mass discharge associated with operation of the plume extraction wells peaked at 0.23 kg/d, decreased significantly within one year, and thereafter began an asymptotic decline to a current value of approximately 0.03 kg/d. Despite an 87% reduction in contaminant mass and a comparable 87% reduction in contaminant mass discharge for the plume, the spatial area encompassed by the plume has decreased by only approximately 50%. This is much less than would be anticipated based on ideal flushing and mass-removal behavior. Simulations produced with a simplified 3-D numerical model matched reasonably well to the measured data. The results of the study suggest that permeability heterogeneity, back diffusion, hydraulic factors associated with the specific well field system, and residual discharge from the source zone are all contributing to the observed persistence of the plume, as well as the asymptotic behavior currently observed for mass removal and for the reduction in contaminant mass discharge.

Characterizing long-term contaminant mass discharge and the relationship between reductions in discharge and reductions in mass for DNAPL source areas.

The objective of this study was to characterize the temporal behavior of contaminant mass discharge, and the relationship between reductions in contaminant mass discharge and reductions in contaminant mass, for a very heterogeneous, highly contaminated source-zone field site. Trichloroethene is the primary contaminant of concern, and several lines of evidence indicate the presence of organic liquid in the subsurface. The site is undergoing groundwater extraction for source control, and contaminant mass discharge has been monitored since system startup. The results show a significant reduction in contaminant mass discharge with time, decreasing from approximately 1 to 0.15 kg/d over five years. Two methods were used to estimate the mass of contaminant present in the source area at the initiation of the remediation project. One was based on a comparison of two sets of core data, collected 3.5 years apart, which suggests that a significant (~80%) reduction in aggregate sediment-phase TCE concentrations occurred between sampling events. The second method was based on fitting the temporal contaminant mass discharge data with a simple exponential source-depletion function. Relatively similar estimates, 784 and 993 kg, respectively, were obtained with the two methods. These data were used to characterize the relationship between reductions in contaminant mass discharge (CMDR) and reductions in contaminant mass (MR). The observed curvilinear relationship exhibits a reduction in contaminant mass discharge essentially immediately upon the initiation of mass reduction. This behavior is consistent with a system wherein significant quantities of mass are present in hydraulically poorly accessible domains for which mass removal is influenced by rate-limited mass transfer. The results obtained from the present study are compared to those obtained from other field studies to evaluate the impact of system properties and conditions on mass-discharge and mass-removal behavior. The results indicate that factors such as domain scale, hydraulic-gradient status (induced or natural), and flushing-solution composition had insignificant impact on the CMDR-MR profiles and thus on underlying mass-removal behavior. Conversely, source-zone age, through its impact on contaminant distribution and accessibility, was implicated as a critical factor influencing the nature of the CMDR-MR relationship.

Prospective evaluation of a matrix-assisted laser desorption ionization-time of flight mass spectrometry system in a hospital clinical microbiology laboratory for identification of bacteria and yeasts: a bench-by-bench study for assessing the impact on time to identification and cost-effectiveness.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been found to be an accurate, rapid, and inexpensive method for the identification of bacteria and yeasts. Previous evaluations have compared the accuracy, time to identification, and costs of the MALDI-TOF MS method against standard identification systems or commercial panels. In this prospective study, we compared a protocol incorporating MALDI-TOF MS (MALDI protocol) with the current standard identification protocols (standard protocol) to determine the performance in actual practice using a specimen-based, bench-by-bench approach. The potential impact on time to identification (TTI) and costs had MALDI-TOF MS been the first-line identification method was quantitated. The MALDI protocol includes supplementary tests, notably for Streptococcus pneumoniae and Shigella, and indications for repeat MALDI-TOF MS attempts, often not measured in previous studies. A total of 952 isolates (824 bacterial isolates and 128 yeast isolates) recovered from 2,214 specimens were assessed using the MALDI protocol. Compared with standard protocols, the MALDI protocol provided identifications 1.45 days earlier on average (P < 0.001). In our laboratory, we anticipate that the incorporation of the MALDI protocol can reduce reagent and labor costs of identification by $102,424 or 56.9% within 12 months. The model included the fixed annual costs of the MALDI-TOF MS, such as the cost of protein standards and instrument maintenance, and the annual prevalence of organisms encountered in our laboratory. This comprehensive cost analysis model can be generalized to other moderate- to high-volume laboratories.

Sulfate reduction in groundwater: characterization and applications for remediation.

Sulfate is ubiquitous in groundwater, with both natural and anthropogenic sources. Sulfate reduction reactions play a significant role in mediating redox conditions and biogeochemical processes for subsurface systems. They also serve as the basis for innovative in situ methods for groundwater remediation. An overview of sulfate reduction in subsurface environments is provided, along with a brief discussion of characterization methods and applications for addressing acid mine drainage. We then focus on two innovative, in situ methods for remediating sulfate-contaminated groundwater, the use of zero-valent iron and the addition of electron-donor substrates. The advantages and limitations associated with the methods are discussed, with examples of prior applications.

Impact of in situ chemical oxidation on contaminant mass discharge: linking source-zone and plume-scale characterizations of remediation performance.

A large-scale permanganate-based in situ chemical oxidation (ISCO) effort has been conducted over the past ten years at a federal Superfund site in Tucson, AZ, for which trichloroethene (TCE) is the primary contaminant of concern. Remediation performance was assessed by examining the impact of treatment on contaminant mass discharge, an approach that has been used for only a very few prior ISCO projects. Contaminant mass discharge tests were conducted before and after permanganate injection to measure the impact at the source-zone scale. The results indicate that ISCO caused a significant reduction in mass discharge (approximately 75%). The standard approach of characterizing discharge at the source-zone scale was supplemented with additional characterization at the plume scale, which was evaluated by examining the change in contaminant mass discharge associated with the pump-and-treat system. The integrated contaminant mass discharge decreased by approximately 70%, consistent with the source-zone-scale measurements. The integrated mass discharge rebounded from 0.1 to 0.2 kg/d within one year after cessation of permanganate injections, after which it has been stable for several years. Collection of the integrated contaminant mass discharge data throughout the ISCO treatment period provided a high-resolution, real-time analysis of the site-wide impact of ISCO, thereby linking source-zone remediation to impacts on overall risk. The results indicate that ISCO was successful in reducing contaminant mass discharge at this site, which comprises a highly heterogeneous subsurface environment. Analysis of TCE sediment concentration data for core material collected before and after ISCO supports the hypothesis that the remaining mass discharge is associated in part with poorly accessible contaminant mass residing within lower-permeability zones.

Rapidly progressive cutaneous Rhizopus microsporus infection presenting as Fournier's gangrene in a patient with acute myelogenous leukemia.

Members of the genus Rhizopus within the class Zygomycetes can cause devastating opportunistic infections. Cutaneous disease arising from direct inoculation of fungal spores has the potential to disseminate widely. Here, we describe a dramatic case of cutaneous Rhizopus infection involving the penis in a patient with acute myelogenous leukemia. Despite aggressive surgical debridement, systemic antifungal therapy, and donor lymphocyte infusion, the infection was ultimately fatal. This case illustrates the unique diagnostic and therapeutic challenges in the clinical management of cutaneous Rhizopus infection.

Comparison of an automated repetitive sequence-based PCR microbial typing system to pulsed-field gel electrophoresis for analysis of outbreaks of methicillin-resistant Staphylococcus aureus.

Rapid and sensitive methods for accurate strain delineation are essential for monitoring and preventing transmission of methicillin-resistant Staphylococcus aureus (MRSA). Pulsed-field gel electrophoresis (PFGE) has been the standard technique for strain typing most bacterial species including MRSA. The goal of this study was to compare the performance of the DiversiLab microbial typing system (Bacterial BarCodes, Inc., Houston, TX) (rep-PCR) to that of PFGE for typing MRSA isolates from five well-defined outbreaks. The DiversiLab rep-PCR assay is a rapid, semiautomated method based on PCR amplification of specific regions between noncoding repetitive sequences in the bacterial genome. rep-PCR was performed according to the manufacturer's recommendations, and the results were analyzed and dendrograms were generated using the DiversiLab analysis software (version 2.1.66 a). PFGE was performed and interpreted according to published procedures. rep-PCR results using similarity indices (SI) of 80%, 85%, and 90% were compared to PFGE analysis. In addition, intra- and interrun reproducibility was determined for rep-PCR. Overall, correct assignment to outbreak versus nonoutbreak clusters occurred for 91 of 109 isolates (85% agreement) when using a SI of 85%. For each specific outbreak, concordance between rep-PCR and PFGE ranged from 73% to 100%. There were 18 discrepant results (17%). Fourteen isolates were unique by PFGE, but they were placed in clusters by rep-PCR; the other 4 were placed in clusters different from those assigned by PFGE. Intra- and interrun reproducibility was excellent. Times to results were 12 to 24 h for rep-PCR compared to 2 to 4 days for PFGE. Rapid, standardized results and excellent reproducibility make rep-PCR a valuable tool for use in MRSA investigations. However, since rep-PCR was less discriminatory than PFGE, we recommend that it be used to screen isolates, followed by testing isolates which share the same rep-PCR pattern with a more sensitive method, such as PFGE or multilocus sequence typing.

Staphylococcus aureus with reduced susceptibility to vancomycin.

Infections with Staphylococcus aureus with reduced susceptibility to vancomycin continue to be reported, including 2 cases caused by S. aureus isolates with full resistance to vancomycin. This review first outlines the definitions of vancomycin-intermediate S. aureus (VISA) and vancomycin-resistant S. aureus (VRSA) and risk factors for infection. Next, we describe the mechanisms of resistance and methods of laboratory detection of the organisms. Finally, we address infection control and management issues associated with isolation of VISA and VRSA.

Comparison of a commercial reversed passive latex agglutination assay to an enzyme immunoassay for the detection of Shiga toxin-producing Escherichia coli.

A multicenter study was performed to compare the performance of a prototypic reversed passive latex agglutination assay (VTEC Screen "Seiken"; Denka-Seiken, Japan) with the Premier EHEC Enzyme Immunoassay (Meridian Diagnostics, USA) for the detection of Shiga toxin in 554 diarrheal stool samples. Standard culture on sorbitol MacConkey agar and the use of latex agglutination reagents were included to identify the Escherichia coli O157, O26 and O111 serotypes. There was 99% agreement between the VTEC screen and enzyme immunoassay (kappa=0.823). Seventeen samples were positive for toxin by one or both assays. One toxin-positive sample using the enzyme immunoassay and four positive samples using the VTEC Screen could not be confirmed. Serotypes identified included: O157:H7 (n=8), O26 (n=2), O111 (n=1) and O45:H2 (n=1). The VTEC screen is easy to perform and comparable to the Meridian EHEC test for detection of Shiga toxin in clinical samples.

Identification of Mycobacterium spp. by using a commercial 16S ribosomal DNA sequencing kit and additional sequencing libraries.

Current methods for identification of Mycobacterium spp. rely upon time-consuming phenotypic tests, mycolic acid analysis, and narrow-spectrum nucleic acid probes. Newer approaches include PCR and sequencing technologies. We evaluated the MicroSeq 500 16S ribosomal DNA (rDNA) bacterial sequencing kit (Applied Biosystems, Foster City, Calif.) for its ability to identify Mycobacterium isolates. The kit is based on PCR and sequencing of the first 500 bp of the bacterial rRNA gene. One hundred nineteen mycobacterial isolates (94 clinical isolates and 25 reference strains) were identified using traditional phenotypic methods and the MicroSeq system in conjunction with separate databases. The sequencing system gave 87% (104 of 119) concordant results when compared with traditional phenotypic methods. An independent laboratory using a separate database analyzed the sequences of the 15 discordant samples and confirmed the results. The use of 16S rDNA sequencing technology for identification of Mycobacterium spp. provides more rapid and more accurate characterization than do phenotypic methods. The MicroSeq 500 system simplifies the sequencing process but, in its present form, requires use of additional databases such as the Ribosomal Differentiation of Medical Microorganisms (RIDOM) to precisely identify subtypes of type strains and species not currently in the MicroSeq library.

Evaluation of the Prodesse Hexaplex multiplex PCR assay for direct detection of seven respiratory viruses in clinical specimens.

We evaluated the Hexaplex assay (Prodesse, Waukesha, WI) for the detection of 7 respiratory viruses (influenza A and B, parainfluenza 1-3, and respiratory syncytial virus [RSV] A and B). The Hexaplex assay was performed on 300 respiratory samples during the 1999-2000 respiratory virus season. Results of this assay were compared with shell vial cell culture and/or direct fluorescent antibody stain. The overall sensitivity and specificity of the assay were 96.6% and 94.1%, respectively. The respective sensitivity and specificity of the Hexaplex assay for detection of specific virus groups were as follows: influenza A, 98.6% and 97.8%; influenza B, 100% and 100%; and for parainfluenza viruses (1-3), 100% and 99.1%. The assay did not perform as well with patients infected with RSV: sensitivity and specificity were 91.0% and 98.6%, respectively. There are 2 major drawbacks to this assay: it is technically demanding (3-4 hours hands-on time), and it is expensive ($80-$90 direct cost). Nevertheless, because of the excellent sensitivity and specificity, the Hexaplex assay may be valuable in the diagnosis of respiratory viral infections in immunocompromised patients.

Comparison of three transport systems (Starplex StarSwab II, the new Copan Vi-Pak Amies Agar Gel collection and transport swabs, and BBL Port-A-Cul) for maintenance of anaerobic and fastidious aerobic organisms.

Of utmost importance in evaluations of clinical samples for infectious agents is proper specimen transport to the clinical laboratory. In the present study we compared three transport systems (the new Starplex StarSwab II, the new Copan Vi-Pak Amies Agar Gel collection and transport swabs, and the BBL Port-A-Cul) for survival of anaerobic and fastidious aerobic bacteria. The new Copan Vi-Pak system has been modified by nitrogen gas flushing to keep an ideal low E(h) condition and to prevent oxidation of the transport medium. The Copan Vi-Pak system outperformed the other two swabs evaluated by maintaining the viabilities of both anaerobic and fastidious aerobic bacteria for 24 h for the majority of the organisms evaluated. This time period should be sufficient for transport of specimens to the clinical microbiology laboratory without compromising organism recovery.

Evaluation of the roche AMPLICOR enterovirus PCR assay in the diagnosis of enteroviral central nervous system infections.

BACKGROUND: Enteroviruses cause a substantial number of cases of aseptic meningitis annually in the USA. While culture has been useful in the detection of patients with viral meningitis it is time-consuming and lacks sensitivity. Detection of viral nucleic acid in patient specimens has been demonstrated to improve enteroviral detection. OBJECTIVES: A research use only commercial amplification assay, the Roche AMPLICOR EV test, was compared to culture for the diagnosis of enteroviral meningoencephalitis. STUDY DESIGN: Four-hundred and sixty-five consecutive CSF samples sent prospectively for suspicion of enteroviral infection were evaluated by PCR and shell-vial culture. Clinical information and CSF analysis were used to resolve PCR positive, culture negative samples. Sensitivity and specificity were calculated using resolved data. RESULTS: There were 138 samples which met the definition of a true positive. Of these culture detected 77 (sensitivity 55.8%) and PCR detected 136 (sensitivity 98.6%). PCR missed two culture positive samples. Upon repeat testing, these CSF samples were found to contain inhibitors. CONCLUSIONS: The Roche AMPLICOR EV-PCR test was statistically more sensitive than culture (P<0.001) in the detection of enteroviruses in CSF in patients suspected of having enteroviral meningitis. This assay also has the advantage of a rapid turnaround time of 5-6 h compared to 3-5 days for culture.