Search

The Streptococcus bovis group (previously group D streptococci) consists of seven distinct species and subspecies. Definitive identification within the group is important, as certain organisms have been associated with gastrointestinal carcinoma, bacteremia, infective endocarditis, meningitis, biliary tract disease, and carcinoma, among others.

Accurate mold identification by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is dependent on robust organism representation in available databases. The Mass Spectrometry Identification (MSI) platform has proven successful for mold identification in clinical and veterinary settings but has yet to be studied with a large set of environmental isolates.

Over the last two decades, rapid technological advances have led to the wide adoption of cell and gene therapy products for the treatment of a variety of disease states. In this study, we reviewed the literature between 2003 and 2021 to provide a summary of overarching trends associated with microbial contamination in hematopoietic stem cells (HSCs) derived from peripheral blood, bone marrow, and cord blood.

Testing of cellular therapy products for Mycoplasma is a regulatory requirement by the United States Food and Drug Administration (FDA) to ensure the sterility and safety of the product prior to release for patient infusion. The risk of Mycoplasma contamination in cell culture is high. Gold standard testing follows USP which requires a 28-day agar and broth cultivation method that is impractical for short shelf-life biologics. Several commercial molecular platforms have been marketed for faster raw material and product release testing; however, little performance data are available in the literature. In this study, we performed a proof-of-principle analysis to evaluate the performance of five commercial molecular assays, including the MycoSEQ Mycoplasma detection kit (Life Technologies), the MycoTOOL Mycoplasma real-time detection kit (Roche), the VenorGEM qOneStep kit (Minerva Biolabs), the ATCC universal Mycoplasma detection kit, and the Biofire Mycoplasma assay (bioMérieux Industry) using 10 cultured Mollicutes spp., with each at four log-fold dilutions (1,000 CFU/mL to 1 CFU/mL) in biological duplicates with three replicates per condition (n = 6) to assess limit of detection (LOD) and repeatability. Additional testing was performed in the presence of tumor infiltrating lymphocytes (TILs). Based on LOD alone, the Biofire Mycoplasma assay was most sensitive followed by the MycoSEQ and MycoTOOL which were comparable. We showed that not all assays were capable of meeting the ≤10 CFU/mL LOD to replace culture-based methods according to European and Japanese pharmacopeia standards. No assay interference was observed when testing in the presence of TILs.

Over the last few years, there have been numerous discussions on the American Society for Microbiology’s clinical listservs (DivC, and ClinMicroNet) regarding sterility testing of cell and gene therapy products and environmental monitoring and gloved fingertip testing for hospital pharmacies. Clinical microbiology laboratories have often been asked to assist with testing due to the on-site proximity of laboratory equipment and microbiological expertise within the hospital environment. The role of the clinical microbiology laboratory in this setting, however, is questionable due to major differences in test requirements and regulatory oversight. Here, we provide a side-by-side comparison of clinical versus current good manufacturing practices microbiology laboratories to provide guidance to those that are currently assisting with or have been asked to assist with sterility testing of manufactured biopharmaceutical products that have become common frontline treatments in modern medicine.

Many studies have shown successful performance of MALDI-TOF MS for rapid yeast and mold identification, yet very few laboratories have chosen to apply this technology into their routine clinical mycology workflow. Some of this hesitation may be attributable to the lack of standardization regarding culture methods, extraction methods, databases, and acquisition methods. This review will provide an overview of the current status of MALDI-TOF MS for fungal identification, including key findings in the literature, processing and database considerations, updates in technology, and exciting future prospects. Significant advances towards standardization have taken place over recent years; thus, accurate species level identification of yeasts and molds, particularly within species complexes and cryptic organisms, should be highly attainable, achievable, and practical in most clinical laboratories.

The U.S. Food and Drug Administration (FDA) regulates manufacturing and testing of advanced therapeutic medicinal products (ATMPs) to ensure the safety of each product for human use. Gold-standard sterility testing (USP) and alternative blood culture systems have major limitations for the detection of fungal contaminants. In this study, we evaluated the performance of iLYM (lactic acid-fermenting organisms, yeasts, and mold) medium (designed originally for the food and beverage industry) to assess its potential for use in the biopharmaceutical field for ATMP sterility testing. We conducted a parallel evaluation of four different test systems (USP, BacT/Alert, Bactec, and Sabouraud dextrose agar [SDA] culture), three different bottle media formulations (iLYM, iFA Plus, and Myco/F Lytic), and two incubation temperatures (22.5°C and 32.5 to 35°C) using a diverse set of fungi (n = 51) isolated from NIH cleanroom environments and previous product contaminants. Additionally, we evaluated the effect of agitation versus delayed-entry static preincubation on test sensitivity and time to detection (TTD). Overall, delayed entry of bottles onto the BacT/Alert or Bactec instruments (with agitation) did not improve test performance. USP and SDA culture continued to significantly outperform each automated culture condition alone. However, we show, for the first time, that a closed-system, dual-bottle combination of iLYM 22.5°C and iFA Plus 32.5°C can provide high fungal sensitivity, statistically comparable to USP, when tested against a diverse range of environmental fungi. Our study fills a much-needed gap in biopharmaceutical testing and offers a favorable testing algorithm that maximizes bacterial and fungal test sensitivity while minimizing risk of product contamination associated with laboratory handling.

BACKGROUND: Product safety assurance is crucial for the clinical use of manufactured cellular therapies. A rational approach for delivering products which fail release criteria (due to potentially false-positive sterility results) is important to avoid unwarranted wastage of highly personalized and costly therapies in critically ill patients where benefits may outweigh risk. Accurate and timely interpretation of microbial sterility assays represents a major challenge in cell therapies. METHODS: We developed a systematic protocol for the assessment of positive microbial sterility test results using retrospective data from 2007 to 2016. This protocol was validated and applied prospectively between October 2016 and September 2017 to 13 products from which positive sterility results had been reported. Viable and non-viable environmental monitoring (EM) data were collected concurrently as part of facility control assessment. RESULTS: Three of 13 (23%) positive sterility results were attributable to bone marrow collections that had been contaminated with skin flora during harvest; all were infused without pertinent infectious sequelae. Of the remaining 10, one was deemed a true positive and was discarded prior to infusion, while nine were classified as false positives attributed to laboratory sampling and/or culturing processes. Three products deemed false positive were infused and six were withheld due to patient issues unrelated to microbial sterility results. No post-infusion associated infectious complications were documented. Almost half of the positive EM findings were skin flora. Paired detection of an organism in both product and associated EM was identified in one case. CONCLUSION: Application of our validated protocol to positive product sterility test results allowed for systematic data compilation for regulatory evaluation and provided comprehensive information to clinical investigators to ensure timely and strategic management for product recipients.

BACKGROUND: Plumbing systems are an infrequent but known reservoir for opportunistic microbial pathogens that can infect hospitalized patients. In 2016, a cluster of clinical sphingomonas infections prompted an investigation. METHODS: We performed whole-genome DNA sequencing on clinical isolates of multidrug-resistant Sphingomonas koreensis identified from 2006 through 2016 at the National Institutes of Health (NIH) Clinical Center. We cultured S. koreensis from the sinks in patient rooms and performed both whole-genome and shotgun metagenomic sequencing to identify a reservoir within the infrastructure of the hospital. These isolates were compared with clinical and environmental S. koreensis isolates obtained from other institutions. RESULTS: The investigation showed that two isolates of S. koreensis obtained from the six patients identified in the 2016 cluster were unrelated, but four isolates shared more than 99.92% genetic similarity and were resistant to multiple antibiotic agents. Retrospective analysis of banked clinical isolates of sphingomonas from the NIH Clinical Center revealed the intermittent recovery of a clonal strain over the past decade. Unique single-nucleotide variants identified in strains of S. koreensis elucidated the existence of a reservoir in the hospital plumbing. Clinical S. koreensis isolates from other facilities were genetically distinct from the NIH isolates. Hospital remediation strategies were guided by results of microbiologic culturing and fine-scale genomic analyses. CONCLUSIONS: This genomic and epidemiologic investigation suggests that S. koreensis is an opportunistic human pathogen that both persisted in the NIH Clinical Center infrastructure across time and space and caused health care-associated infections. (Funded by the NIH Intramural Research Programs.)

We evaluated saliva (SAL) specimens for SARS-CoV-2 reverse transcriptase PCR (RT-PCR) testing by comparison of 459 prospectively paired nasopharyngeal (NP) or midturbinate (MT) swabs from 449 individuals with the aim of using saliva for asymptomatic screening. Samples were collected in a drive-through car line for symptomatic individuals (n = 380) and in the emergency department (ED) (n = 69)., We evaluated saliva (SAL) specimens for SARS-CoV-2 reverse transcriptase PCR (RT-PCR) testing by comparison of 459 prospectively paired nasopharyngeal (NP) or midturbinate (MT) swabs from 449 individuals with the aim of using saliva for asymptomatic screening. Samples were collected in a drive-through car line for symptomatic individuals (n = 380) and in the emergency department (ED) (n = 69). The percentages of positive and negative agreement of saliva compared to nasopharyngeal swab were 81.1% (95% confidence interval [CI], 65.8% to 90.5%) and 99.8% (95% CI, 98.7% to 100%), respectively. The percent positive agreement increased to 90.0% (95% CI, 74.4% to 96.5%) when considering only samples with moderate to high viral load (cycle threshold [CT] for the NP, ≤34). Pools of five saliva specimens were also evaluated on three platforms, bioMérieux NucliSENS easyMAG with ABI 7500Fast (CDC assay), Hologic Panther Fusion, and Roche Cobas 6800. The average loss of signal upon pooling was 2 to 3 CT values across the platforms. The sensitivities of detecting a positive specimen in a pool compared with testing individually were 94%, 90%, and 94% for the CDC 2019-nCoV real-time RT-PCR, Panther Fusion SARS-CoV-2 assay, and Cobas SARS-CoV-2 test, respectively, with decreased sample detection trending with lower viral load. We conclude that although pooled saliva testing, as collected in this study, is not quite as sensitive as NP/MT testing, saliva testing is adequate to detect individuals with higher viral loads in an asymptomatic screening program, does not require swabs or viral transport medium for collection, and may help to improve voluntary screening compliance for those individuals averse to various forms of nasal collections.

Sterility testing of cellular therapy products along with the associated environmental monitoring requirements for aseptic facilities, including compounding pharmacies, continues to impact clinical microbiology laboratories, as evidenced by the numerous discussions recurring on American Society for Microbiology Division C and ClinMicroNet listservs. This minireview provides an overview of this complex field of current good manufacturing practices (cGMP) based on biopharmaceutical industry standards and summarizes the compendial and alternative rapid microbial test methods available for product sterility and Mycoplasma testing. In addition, this minireview highlights major overarching regulatory requirements governing any laboratory performing product testing as regulated by the United States Food and Drug Administration (FDA). These requirements are different from the more familiar clinical requirements of the Clinical Laboratory Improvement Act of 1988 (CLIA ’88), the College of American Pathologists (CAP), and the Joint Commission on Accreditation of Healthcare Organizations (JCAHO), all of which have no jurisdiction in this area. As the cellular therapy field continues to advance and an increasing number of medical centers participate in clinical trials of these novel therapies, it is critical that laboratories have a sound understanding of the major regulations and cGMP practices governing microbiological testing in the biopharmaceutical industry.

Background: Transmission of carbapenemase-producing organisms (CPO) threatens patient safety in healthcare facilities. As a result of a 2011 outbreak of blaKPC+ Klebsiella pneumoniae, the NIH Clinical Center (NIHCC) has prioritized early detection and isolation of CPO carriers, using point-prevalence surveys and targeted high-risk ward surveillance since 2011 and admission surveillance since 2013. We describe our experience over 6 years of admission surveillance. Methods: The NIHCC is a 200-bed research hospital that provides care for a highly immunocompromised patient population. From September 2013 to September 2019, perirectal swabs were ordered automatically for all patients on admission to nonbehavioral health wards. Swabs were ordered twice weekly for ICU patients, weekly in other high-risk wards, and monthly for hospital-wide point prevalence (excluding behavioral health). Patients hospitalized in the United States in the previous week or abroad in the previous 6 months were considered high risk for carriage and isolated pending results from 2 swabs. Most swabs (n = 37,526) were cultured onto HardyCHROM CRE. If gram-negative bacilli (GNB) were present, a molecular screen for carbapenemases was performed on a sweep of cultured material (day 1) pending organism isolation. GNB were identified by MALDI-TOF MS. Prior to June 2019, isolates were screened by blaKPC/blaNDM PCR. Starting in June 2019, Enterobacteriaceae and Pseudomonas aeruginosa were screened using the phenotypic modified carbapenem inactivation method (mCIM), reflexing to the GeneXpert CARBA-R molecular assay if positive; other GNB were tested directly with CARBA-R. Selected GNB underwent susceptibility testing (Sensititre). Whole-genome sequencing was used to assess relatedness among CPO isolates. Swabs from high-risk patients were tested directly by blaKPC PCR (n = 699) until August 2019 (most in parallel with culture) and thereafter by CARBA-R (n = 13). Results: Among 54,188 orders for perirectal swabs, 38,238 were collected from 14,497 patients (compliance 71%). Among 33 CPO-colonized patients identified from September 2013 through September 2019, 15 were identified on admission, 6 were identified in point-prevalence surveys, 8 were identified from high-risk ward surveillance, and 4 were identified from clinical cultures. Sequencing demonstrated no relatedness among CPO isolates. Although only 1.4% of patients sampled on admission were colonized with CPO, those meeting high-risk criteria were 21 times as likely to be colonized. Conclusion: Admission surveillance for CPO identified a low rate of colonization, but it detected nearly half of known CPO-colonized NIHCC patients over the past 6 years. Modest compliance with swab collection leaves room for improvement and likely results in missed instances of colonization. Although we cannot determine its effectiveness, we view our strategy as one of several key safety measures for our highly vulnerable patient population.Funding: NoneDisclosures: None

Antibiotic-resistant bacteria are a serious threat to medically fragile patient populations. The spread of antibiotic resistance through plasmid-mediated mechanisms is of grave concern as it can lead to the conversion of endogenous patient-associated strains to difficult-to-treat pathogens., Antibiotics, which are used both to prevent and to treat infections, are a mainstay therapy for lifesaving procedures such as transplantation. For this reason, and many others, increased antibiotic resistance among human-associated pathogens, such as the carbapenem-resistant Enterobacteriaceae species, is of grave concern. In this study, we report on a hematopoietic stem cell transplant recipient in whom cultures detected the emergence of carbapenem resistance and spread across five strains of bacteria that persisted for over a year. Carbapenem resistance in Citrobacter freundii, Enterobacter cloacae, Klebsiella aerogenes, and Klebsiella pneumoniae was linked to a pair of plasmids, each carrying the Klebsiella pneumoniae carbapenemase gene (blaKPC). Surveillance cultures identified a carbapenem-susceptible strain of Citrobacter freundii that may have become resistant through horizontal gene transfer of these plasmids. Selection of a multidrug-resistant Klebsiella pneumoniae strain was also detected following combination antibiotic therapy. Here we report a plasmid carrying the blaKPC gene with broad host range that poses the additional threat of spreading to endogenous members of the human gut microbiome.

The emergence of cell therapy programs in large academic centers has led to an increasing demand for clinical laboratories to assist with product sterility testing. Automated blood culture systems have shown promise as alternatives to the manual USP compendial method, but current published data are limited by small organism test sets, particularly for molds. In 2015, failure of the Bactec FX system to detect mold contamination in two products prompted us to evaluate three test systems (compendial USP, Bactec FX, and BacT/Alert Dual-T) over seven different culture combinations, using 118 challenge organisms representative of the NIH current good manufacturing practice (cGMP) environment. At method significantly outperformed the Bactec FX system (84.7% versus 64.4%; P = 0.0006) but not the BacT/Alert system at 32.5°C (78.8%; P = 0.3116). Extended incubation to 360 h with terminal visual inspection improved sensitivity, without a significant difference between compendial USP and BacT/Alert testing (95.7% versus 89.0%; P = 0.0860); both systems were better than the Bactec FX system (71.2%; P method for product sterility testing.

Background. Delayed antifungal therapy for invasive candidiasis (IC) contributes to poor outcomes. Predictive risk models may allow targeted antifungal prophylaxis to those at greatest risk. Methods. A prospective cohort study of 6685 consecutive nonneutropenic patients admitted to 7 Australian intensive care units (ICUs) for ≥72 hours was performed. Clinical risk factors for IC occurring prior to and following ICU admission, colonization with Candida species on surveillance cultures from 3 sites assessed twice weekly, and the occurrence of IC ≥72 hours following ICU admission or ≤72 hours following ICU discharge were measured. From these parameters, a risk-predictive model for the development of ICU-acquired IC was then derived. Results. Ninety-six patients (1.43%) developed ICU-acquired IC. A simple summation risk-predictive model using the 10 independently significant variables associated with IC demonstrated overall moderate accuracy (area under the receiver operating characteristic curve = 0.82). No single threshold score could categorize patients into clinically useful high- and low-risk groups. However, using 2 threshold scores, 3 patient cohorts could be identified: those at high risk (score ≥6, 4.8% of total cohort, positive predictive value [PPV] 11.7%), those at low risk (score ≤2, 43.1% of total cohort, PPV 0.24%), and those at intermediate risk (score 3-5, 52.1% of total cohort, PPV 1.46%). Conclusions. Dichotomization of ICU patients into high- and low-risk groups for IC risk is problematic. Categorizing patients into high-, intermediate-, and low-risk groups may more efficiently target early antifungal strategies and utilization of newer diagnostic tests.

The Streptococcus bovis group has undergone significant taxonomic changes over the past 2 decades with the advent of new identification methods with higher discriminatory power. Although the current classification system is not yet embraced by all researchers in the field and debate remains over the performance of molecular techniques for identification to the species level within the group, important disease associations for several members of the group have been clarified. Here, we provide a brief overview of the history of the S. bovis group, an outline of the currently accepted classification scheme, a review of associated clinical syndromes, and a summary of the performance and diagnostic accuracy of currently available identification methods.

Perirectal surveillance cultures and a stool culture grew Aeromonas species from three patients over a 6-week period and were without epidemiological links. Detection of the bla KPC-2 gene in one isolate prompted inclusion of non- Enterobacteriaceae in our surveillance culture workup. Whole-genome sequencing confirmed that the isolates were unrelated and provided data for Aeromonas reference genomes.

We evaluated the clinical performance of Check-Direct CPE for carbapenemase detection directly from 301 perirectal swabs (258 patients) in a nonoutbreak setting. Culture of a PCR-confirmed, carbapenemase-containing organism, or history of colonization with such organism within the previous 2 weeks, was used as the reference standard. Check-Direct CPE demonstrated a sensitivity value, specificity value, positive predictive value (PPV), and negative predictive value (NPV) of 100% (all bla KPC ), 88%, 21%, and 100%, respectively. False positives accounted for 79% ( n = 34) of samples for which a cycle threshold ( C T ) value was reached. Simulated studies to evaluate specimen pooling as an approach to minimize costs showed no difference in C T values for pooled groups of three or five that each contained a single specimen spiked with ∼1,500 CFU bla KPC Klebsiella pneumoniae ; however, the detection rate dropped to 60% at a seeded concentration of ∼150 CFU. When data were pooled, C T values for bla KPC were higher for heavy-feces-containing than for light-feces-containing liquid-suspended specimens. Furthermore, C T values for liquid-suspended specimens were 4 to 5 C T values lower (i.e., represented greater sensitivity) than those seen in direct swab analysis. Culture was equivalent to or better than Check-Direct CPE for 13/15 (87%) isolates tested in a limit-of-detection analysis. Detection of a carbapenemase gene at a C T cutoff value of ≤35 was culture confirmed in 23/24 (96%) of cases; however, C T values of >35 overlapped broadly between culture-positive ( n = 21) and culture-negative ( n = 36) specimens. Check-Direct CPE will likely prove most useful in high-prevalence areas or in outbreak settings where rapid carbapenemase detection is critical for infection control management.

Enterobacter cloacae is an important nosocomial bacterium that is prevalent in hospital intensive care units (ICUs) ([1][1][–][2][4][3]). The presence of carbapenem-hydrolyzing beta-lactamases, such as Klebsiella pneumoniae carbapenemase (KPC) encoded by the bla KPC gene, underlies multidrug

Background Autosplenectomy, as a result of sickle cell disease, is an important risk factor for severe malaria. While molecular methods are helpful in providing rapid and accurate infection detection and species identification, the effect of hyposplenism on result interpretation during the course of infection should be carefully considered. Case presentation A 32-year old autosplenectomized Nigerian male with severe sickle cell disease was referred to the National Institutes of Health for allogenic hematopoietic stem cell transplant. Despite testing negative for malaria by both smear and PCR 2 weeks after arrival in the USA, the patient developed fever and diffuse bilateral lower rib cage and upper abdominal pain 2 weeks later and subsequently tested positive for Plasmodium falciparum. Parasitaemia was tracked over time by microscopy and nucleic acid tests to evaluate the therapeutic response in the setting of hyposplenism. The patient showed prompt resolution of patent infection by microscopy but remained positive by molecular methods for > 30 days after treatment initiation. Conclusion While molecular testing can provide sensitive Plasmodium nucleic acid detection, the persistence of Plasmodium nucleic acids following adequate treatment in functionally asplenic patients can lead to a diagnostic dilemma. In such patients, clinical response and peripheral blood smears should guide patient management following treatment. Nonetheless, in pre-transplant patients at high-risk for pre-existing Plasmodium infections, highly sensitive molecular assays can be useful to rule out infection prior to transplantation.

Background “Green” construction approaches may place hospitals at risk for long-term contamination of potable water, presenting hazards for immunosuppressed patients. Remediation may be needed to manage these unanticipated consequences. An indolent, 11-year cluster of Sphingomonas koreensis infections at the NIH Clinical Center was traced to potable water. During the investigation, 4 sinks in 2 ICU rooms were found to have intractable contamination. Despite replacement of sink fixtures, faucet swabs and water continued to grow S. koreensis, and mean free chlorine residual (FCR) from the sinks was 0.27 mg/L (goal > 0.5). We undertook a major project to replace and reroute sections of pipes leading to the sinks (Figures 1 and 2). Methods Hot and cold water pipes were removed from the sinks to the supply pipes and replaced. Pipes were rerouted so that hot and cold water supplying the sinks returned in a loop to the domestic hot water heater to eliminate 10-meter drop-down sections of dead-leg pipe. Automatic faucets were replaced with manual faucets. Faucet biofilm was swabbed and cultured monthly for S. koreensis. Environmental samples were cultured on sheep blood agar for 5 days. Yellow colonies were subcultured and identified by MALDI-TOF MS (Bruker). Results Demolition of the wall behind the sinks revealed hot and cold water piping containing stagnant water. Water pooled in cold water pipe had heavy growth of S. koreensis; water from the hot water pipe could not be collected. Hot and cold water FCR for the sinks after piping changes averaged 0.74 and 1.07 mg/L, respectively, compared with 0.27 mg/L simultaneously from automated faucets in unmodified ICU rooms. Faucet cultures were negative for S. koreensis after replumbing, and have remained negative for >6 months. Conclusion New hospital construction strategies appear to increase potable water contamination risks; novel remediation approaches are needed. Replacing contaminated water pipes and rerouting pipes to minimize stagnation eradicated longstanding contamination of 2 ICU sinks. Although the experiment was conducted on a small scale, it demonstrates that plumbing flaws that jeopardize patient safety can be corrected through multidisciplinary collaboration and creative plumbing strategies. Disclosures All authors: No reported disclosures.

Here, we report the genome sequence of a bla NDM-1 -positive Klebsiella pneumoniae AATZP isolate cultured from a perirectal surveillance swab collected upon admission of a patient to the NIH Clinical Center in 2014. Genome sequencing of this isolate revealed three plasmids, including one carrying the bla NDM-1 gene encoding resistance to carbapenems.

Carbapenem-resistant Klebsiella pneumoniae strains are formidable hospital pathogens that pose a serious threat to patients around the globe due to a rising incidence in health care facilities, high mortality rates associated with infection, and potential to spread antibiotic resistance to other bacterial species, such as Escherichia coli. Over 6 months in 2011, 17 patients at the National Institutes of Health (NIH) Clinical Center became colonized with a highly virulent, transmissible carbapenem-resistant strain of K. pneumoniae. Our real-time genomic sequencing tracked patient-to-patient routes of transmission and informed epidemiologists’ actions to monitor and control this outbreak. Two of these patients remained colonized with carbapenemase-producing organisms for at least 2 to 4 years, providing the opportunity to undertake a focused genomic study of long-term colonization with antibiotic-resistant bacteria. Whole-genome sequencing studies shed light on the underlying complex microbial colonization, including mixed or evolving bacterial populations and gain or loss of plasmids. Isolates from NIH patient 15 showed complex plasmid rearrangements, leaving the chromosome and the blaKPC-carrying plasmid intact but rearranging the two other plasmids of this outbreak strain. NIH patient 16 has shown continuous colonization with blaKPC-positive organisms across multiple time points spanning 2011 to 2015. Genomic studies defined a complex pattern of succession and plasmid transmission across two different K. pneumoniae sequence types and an E. coli isolate. These findings demonstrate the utility of genomic methods for understanding strain succession, genome plasticity, and long-term carriage of antibiotic-resistant organisms., IMPORTANCE In 2011, the NIH Clinical Center had a nosocomial outbreak involving 19 patients who became colonized or infected with blaKPC-positive Klebsiella pneumoniae. Patients who have intestinal colonization with blaKPC-positive K. pneumoniae are at risk for developing infections that are difficult or nearly impossible to treat with existing antibiotic options. Two of those patients remained colonized with blaKPC-positive Klebsiella pneumoniae for over a year, leading to the initiation of a detailed genomic analysis exploring mixed colonization, plasmid recombination, and plasmid diversification. Whole-genome sequence analysis identified a variety of changes, both subtle and large, in the blaKPC-positive organisms. Long-term colonization of patients with blaKPC-positive Klebsiella pneumoniae creates new opportunities for horizontal gene transfer of plasmids encoding antibiotic resistance genes and poses complications for the delivery of health care.

We developed an Australian database for the identification of Aspergillus , Scedosporium , and Fusarium species ( n = 28) by matrix-assisted laser desorption ionization−time of flight mass spectrometry (MALDI-TOF MS). In a challenge against 117 isolates, species identification significantly improved when the in-house-built database was combined with the Bruker Filamentous Fungi Library compared with that for the Bruker library alone ( Aspergillus , 93% versus 69%; Fusarium , 84% versus 42%; and Scedosporium , 94% versus 18%, respectively).

We applied multiplex-tandem PCR (MT-PCR) to 255 EDTA whole-blood specimens, 29 serum specimens, and 24 plasma specimens from 109 patients with Candida bloodstream infection (candidemia) to determine whether a diagnosis could be expedited in comparison with the time to diagnosis by the use of standard blood culture. Overall, the MT-PCR performed better than blood culture with DNA extracted from whole blood from 52/74 (70%) patients, accelerating the time to detection (blood culture flagging) and determination of the pathogenic species (by use of the API 32C system [bioMérieux, Marcy l'Etoile, France]) by up to 4 days (mean, 2.2 days; range, 0.5 to 8 days). Candida DNA was detected more often in serum (71%) and plasma (75%) than in whole blood (54%), although relatively small numbers of serum and plasma specimens were tested. The sensitivity, specificity, positive predictive value, and negative predictive value of the assay with whole blood were 75%, 97%, 95%, and 85%, respectively. Fungal DNA was not detected by MT-PCR in 6/24 (25%) whole-blood samples drawn simultaneously with the positive blood culture sample. MT-PCR performed better with whole-blood specimens stored at −20°C (75%) and when DNA was extracted within 1 week of sampling (66%). The molecular and culture identification results correlated for 61 of 66 patients (92%); one discrepant result was due to misidentification by culture. All but one sample from 53 patients who were at high risk of candidemia but did not have proven disease were negative by MT-PCR. The results demonstrate the good potential of MT-PCR to detect candidemia, to provide Candida species identification prior to blood culture positivity, and to provide improved sensitivity when applied to with serum and plasma specimens.

We evaluated a combined panfungal PCR-reverse line blot (RLB) hybridization assay based on internal transcribed spacer 1 (ITS1) and ITS2 region polymorphisms to identify 159 Candida , Cryptococcus neoformans , and Aspergillus isolates (22 species). Its utility to identify fungal pathogens directly from 27 clinical specimens was also determined. ITS sequence analysis was performed to resolve discrepant identifications or where no RLB result was obtained. Species-specific ITS2- and ITS1-based probes correctly identified 155 of 159 isolates (98%) and 149 (93.7%) isolates, respectively. All strains were unambiguously differentiated with the exception of cross-reactivity between the Candida norvegensis probe and Candida haemulonii DNA product. Species identification of the pathogen was made for all 21 specimens (sensitivity of 100%) where species-specific probes were included in the RLB; however, there was no ITS2 probe-based hybridization signal for two specimens. Results were concordant with the culture results for 18 (85.7%) specimens. The assay was able to provide species identification in the absence of a culture result (two specimens) and to detect mixed infection (one specimen). The results indicate that the RLB assay is capable of reliably detecting yeasts and Aspergillus spp. in clinical specimens and that the incorporation of both ITS1- and ITS2-targeted probes is required for optimal sensitivity. The test has potential utility in the early diagnosis of invasive fungal infection, since “fungal” DNA was detected in all 27 specimens. Prior to incorporation of probes to detect other fungal species, ITS sequencing may be performed to achieve species identification.

Given the rise in the incidence of invasive fungal infections (IFIs) and the expanding spectrum of fungal pathogens, early and accurate identification of the causative pathogen is essential. We developed a panfungal PCR assay that targets the internal transcribed spacer 1 (ITS1) region of the ribosomal DNA gene cluster to detect fungal DNA in fresh and formalin-fixed, paraffin-embedded (PE) tissue specimens from patients with culture-proven ( n = 38) or solely histologically proven ( n = 24) IFIs. PCR products were sequenced and compared with sequences in the GenBank database to identify the causal pathogen. The molecular identification was correlated with results from histological examination and culture. The assay successfully detected and identified the fungal pathogen in 93.6% and 64.3% of culture-proven and solely histologically proven cases of IFI, respectively. A diverse range of fungal genera were identified, including species of Candida , Cryptococcus , Trichosporon , Aspergillus , Fusarium , Scedosporium , Exophiala , Exserohilum , Apophysomyces , Actinomucor , and Rhizopus . For five specimens, molecular analysis identified a pathogen closely related to that identified by culture. All PCR-negative specimens ( n = 10) were PE tissues in which fungal hyphae were visualized. The results support the use of the panfungal PCR assay in combination with conventional laboratory tests for accurate identification of fungi in tissue specimens.

Rapid detection of bla KPC -containing organisms can significantly impact infection control and clinical practices, as well as therapeutic choices. Current molecular and phenotypic methods to detect these organisms, however, require additional testing beyond routine organism identification. In this study, we evaluated the clinical performance of matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) to detect pKpQIL_p019 (p019)—an ∼11,109-Da protein associated with certain bla KPC -containing plasmids that was previously shown to successfully track a clonal outbreak of bla KPC -pKpQIL- Klebsiella pneumoniae in a proof-of-principle study (A. F. Lau, H. Wang, R. A. Weingarten, S. K. Drake, A. F. Suffredini, M. K. Garfield, Y. Chen, M. Gucek, J. H. Youn, F. Stock, H. Tso, J. DeLeo, J. J. Cimino, K. M. Frank, and J. P. Dekker, J Clin Microbiol 52:2804–2812, 2014, http://dx.doi.org/10.1128/JCM.00694-14 ). PCR for the p019 gene was used as the reference method. Here, blind analysis of 140 characterized Enterobacteriaceae isolates using two protein extraction methods (plate extraction and tube extraction) and two peak detection methods (manual and automated) showed sensitivities and specificities ranging from 96% to 100% and from 95% to 100%, respectively (2,520 spectra analyzed). Feasible laboratory implementation methods (plate extraction and automated analysis) demonstrated 96% sensitivity and 99% specificity. All p019-positive isolates ( n = 26) contained bla KPC and were carbapenem resistant. Retrospective analysis of an additional 720 clinical Enterobacteriaceae spectra found an ∼11,109-Da signal in nine spectra (1.3%), including seven from p019-containing, carbapenem-resistant isolates (positive predictive value [PPV], 78%). Instrument tuning had a significant effect on assay sensitivity, highlighting important factors that must be considered as MALDI-TOF MS moves into applications beyond microbial identification. Using a large blind clinical data set, we have shown that spectra acquired for routine organism identification can also be analyzed automatically in real time at high throughput, at no additional expense to the laboratory, to enable rapid detection of potentially bla KPC -containing carbapenem-resistant isolates, providing early and clinically actionable results.

Colonization with Candida species is an independent risk factor for invasive candidiasis (IC), but the minimum and most practicable parameters for prediction of IC have not been optimized. We evaluated Candida colonization in a prospective cohort of 6,015 nonneutropenic, critically ill patients. Throat, perineum, and urine were sampled 72 h post-intensive care unit (ICU) admission and twice weekly until discharge or death. Specimens were cultured onto chromogenic agar, and a subset underwent molecular characterization. Sixty-three (86%) patients who developed IC were colonized prior to infection; 61 (97%) tested positive within the first two time points. The median time from colonization to IC was 7 days (range, 0 to 35). Colonization at any site was predictive of IC, with the risk of infection highest for urine colonization (relative risk [RR] = 2.25) but with the sensitivity highest (98%) for throat and/or perineum colonization. Colonization of ≥2 sites and heavy colonization of ≥1 site were significant independent risk factors for IC (RR = 2.25 and RR = 3.7, respectively), increasing specificity to 71% to 74% but decreasing sensitivity to 48% to 58%. Molecular testing would have prompted a resistance-driven decision to switch from fluconazole treatment in only 11% of patients infected with C. glabrata , based upon species-level identification alone. Positive predictive values (PPVs) were low (2% to 4%) and negative predictive values (NPVs) high (99% to 100%) regardless of which parameters were applied. In the Australian ICU setting, culture of throat and perineum within the first two time points after ICU admission captures 84% (61/73 patients) of subsequent IC cases. These optimized parameters, in combination with clinical risk factors, should strengthen development of a setting-specific risk-predictive model for IC.

Klebsiella pneumoniae is an important nosocomial pathogen, and multidrug-resistant strains have become a worldwide concern. Here, we report the complete genome of a K. pneumoniae isolate with chromosomally integrated bla KPC genes and a colibactin synthesis locus.

Background Following a rise in nosocomial infections due to Sphingomonas, a waterborne Gram-negative organism, we undertook an epidemiological investigation to identify possible sources and develop a remediation strategy. Methods We analyzed Sphingomonas isolates from 30 inpatients in the past 11 years, and we reviewed each patient’s chart. We collected swabs of faucets, water samples, and free and total chlorine levels from rooms of Sphingomonas patients from 2016, using unrelated rooms as controls. Water samples and chlorine levels were collected from hospital pipes. Swabs were placed into 1 mL TSB and cultured to sheep blood agar. Isolates were identified by MALDI-TOF MS. Water samples were tested via membrane filtration (500 mL) and spread plate method (1 mL). Patient and environmental Sphingomonas isolates underwent whole genome sequencing, and were analyzed with Mash and Snippy for overall genomic sequence and single-nucleotide polymorphisms comparisons, respectively, to assess relatedness. Results Of 27 faucets examined, 59% grew Sphingomonas spp., and 33% grew highly-resistant S. koreensis. Of 21 water samples, 76% grew Sphingomonas spp., and 48% grew S. koreensis. Sequence analysis demonstrated strong genetic similarity among S. koreensis clinical isolates from the past 11 years and recent faucet and water isolates. One patient’s S. koreensis isolate was genetically related to isolates from faucets in his room. Sphingomonas did not grow from samples collected from municipal water or some of the far upstream water pipes within the hospital. Free chlorine levels were extremely low in hot water, leading to a program of flushing in order to restore and maintain adequate levels. Among 7 contaminated faucets that were replaced, 3 became recolonized within 4 weeks, and continued to grow Sphingomonas from water. Conclusion Investigation and genome sequencing suggest long-standing S. koreensis colonization within the hospital plumbing system that has served as a reservoir for sporadic infections among immunosuppressed patients. Remediation of Sphingomonas plumbing contamination is an ongoing challenge guided by few published data. Hospital water must be rendered safe for even the most immunosuppressed patients. Disclosures All authors: No reported disclosures.

Public health officials have raised concerns that plasmid transfer between Enterobacteriaceae species may spread resistance to carbapenems, an antibiotic class of last resort, thereby rendering common health care–associated infections nearly impossible to treat. To determine the diversity of carbapenemase-encoding plasmids and assess their mobility among bacterial species, we performed comprehensive surveillance and genomic sequencing of carbapenem-resistant Enterobacteriaceae in the National Institutes of Health (NIH) Clinical Center patient population and hospital environment. We isolated a repertoire of carbapenemase-encoding Enterobacteriaceae, including multiple strains of Klebsiella pneumoniae , Klebsiella oxytoca , Escherichia coli , Enterobacter cloacae , Citrobacter freundii , and Pantoea species. Long-read genome sequencing with full end-to-end assembly revealed that these organisms carry the carbapenem resistance genes on a wide array of plasmids. K. pneumoniae and E. cloacae isolated simultaneously from a single patient harbored two different carbapenemase-encoding plasmids, indicating that plasmid transfer between organisms was unlikely within this patient. We did, however, find evidence of horizontal transfer of carbapenemase-encoding plasmids between K. pneumoniae , E. cloacae , and C. freundii in the hospital environment. Our data, including full plasmid identification, challenge assumptions about horizontal gene transfer events within patients and identify possible connections between patients and the hospital environment. In addition, we identified a new carbapenemase-encoding plasmid of potentially high clinical impact carried by K. pneumoniae , E. coli , E. cloacae , and Pantoea species, in unrelated patients and in the hospital environment.

Carbapenem-resistant Enterobacteriaceae (CRE) have spread globally and represent a serious and growing threat to public health. Rapid methods for tracking plasmids carrying carbapenemase genes could greatly benefit infection control efforts. Here, we demonstrate that real-time, direct tracking of a single plasmid in a bacterial strain responsible for an outbreak is possible using a commercial matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) system. In this case, we retrospectively tracked the bla KPC carbapenemase gene-bearing pKpQIL plasmid responsible for a CRE outbreak that occurred at the NIH Clinical Center in 2011. An ∼11,109-Da MS peak corresponding to a gene product of the bla KPC pKpQIL plasmid was identified and characterized using a combination of proteomics and molecular techniques. This plasmid peak was present in spectra from retrospectively analyzed K. pneumoniae outbreak isolates, concordant with results from whole-genome sequencing, and absent from a diverse control set of bla KPC -negative clinical Enterobacteriaceae isolates. Notably, the gene characterized here is located adjacent to the bla KPC Tn 4401 transposon on the pKpQIL plasmid. Sequence analysis demonstrates the presence of this gene in other bla KPC Tn 4401 -containing plasmids and suggests that this signature MS peak may be useful in tracking other plasmids conferring carbapenem resistance. Plasmid identification using this MALDI-TOF MS method was accomplished in as little as 10 min from isolated colonies and 30 min from positive (spiked) blood cultures, demonstrating the potential clinical utility for real-time plasmid tracking in an outbreak.

We developed a multiplex tandem PCR (MT-PCR) assay for the rapid identification of 16 fungi directly from culture. MT-PCR results were concordant with phenotypic identification for all cultures studied ( n = 183). The colony MT-PCR assay was rapid (

Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is a powerful tool for the rapid and highly accurate identification of clinical pathogens but has not been utilized extensively in clinical mycology due to challenges in developing an effective protein extraction method and the limited databases available. Here, we developed an alternate extraction procedure and constructed a highly stringent database comprising 294 individual isolates representing 76 genera and 152 species. To our knowledge, this is the most comprehensive clinically relevant mold database developed to date. When challenged with 421 blinded clinical isolates from our institution, by use of the BioTyper software, accurate species-level (score of ≥2.0) and genus-level (score of ≥1.7) identifications were obtained for 370 (88.9%) and 18 (4.3%) isolates, respectively. No isolates were misidentified. Of the 33 isolates (7.8%) for which there was no identification (score of Penicillium species that were not represented in the database. Our library clearly outperformed the manufacturer's database that was obtained with the instrument, which identified only 3 (0.7%) and 26 (6.2%) isolates at species and genus levels, respectively. Identification was not affected by different culture conditions. Implementation into our routine workflow has revolutionized our mycology laboratory efficiency, with improved accuracy and decreased time for mold identification, eliminating reliance on traditional phenotypic features.

Multiplex, real-time PCR has become an invaluable tool for the rapid identification of pathogens in clinical specimens enabling earlier and more targeted management of antimicrobial therapy. In this chapter, we describe the methodology behind a novel multiplex-tandem PCR (MT-PCR) platform designed for the rapid identification of up to 18 fungal pathogens in blood cultures, primary isolation plates, and whole blood, serum, and plasma.

Many species of fungi can cause disease in plants, animals and humans. Accurate and robust detection and quantification of fungi is essential for diagnosis, modeling and surveillance. Also direct detection of fungi enables a deeper understanding of natural microbial communities, particularly as a great many fungi are difficult or impossible to cultivate. In the last decade, effective amplification platforms, probe development and various quantitative PCR technologies have revolutionized research on fungal detection and identification. Examples of the latest technology in fungal detection and differentiation are discussed here.

Invasive fungal infections are an important cause of infectious morbidity. Nonculture-based methods are increasingly used for rapid, accurate diagnosis to improve patient outcomes. New and existing DNA amplification platforms have high sensitivity and specificity for direct detection and identification of fungi in clinical specimens. Since laboratories are increasingly reliant on DNA sequencing for fungal identification, measures to improve sequence interpretation should support validation of reference isolates and quality control in public gene repositories. Novel technologies (e.g., isothermal and PNA FISH methods), platforms enabling high-throughput analyses (e.g., DNA microarrays and Luminex®xMAP™) and/or commercial PCR assays warrant further evaluation for routine diagnostic use. Notwithstanding the advantages of molecular tests, serological assays remain clinically useful for patient management. The serum Aspergillus galactomannan test has been incorporated into diagnostic algorithms of invasive aspergillosis. Both the galactomannan and the serum β-D-glucan test have value for diagnosing infection and monitoring therapeutic response.

To assess the generalisability of published clinical risk predictive models for invasive candidiasis in ICU patients. The performance characteristics of published clinical risk factor-only and Candida colonisation-only predictive models for invasive candidiasis were assessed in a multicentre cohort of Australian ICU patients. Clinical risk factors and Candida colonisation parameters were collected prospectively from patients. The two clinical risk factor-only predictive models applied to an Australian patient cohort (n = 615) performed less well than in published studies involving derivation populations. Model performance characteristics improved when Candida colonisation parameters were added post-hoc. Risk predictive models should factor in both clinical risk factors and Candida colonisation parameters. Integrating these models into therapeutic algorithms first requires external validation in different patient populations and settings.

We describe the first development and evaluation of a rapid multiplex tandem PCR (MT-PCR) assay for the detection and identification of fungi directly from blood culture specimens that have been flagged as positive. The assay uses a short-cycle multiplex amplification, followed by 12 simultaneous PCRs which target the fungal internal transcribed spacer 1 (ITS1) and ITS2 region, elongation factor 1-α (EF1-α), and β-tubulin genes to identify 11 fungal pathogens: Candida albicans, Candida dubliniensis, Candida glabrata, Candida guilliermondii, Candida krusei, Candida parapsilosis complex, Candida tropicalis, Cryptococcus neoformans complex, Fusarium solani, Fusarium species, and Scedosporium prolificans . The presence or absence of a fungal target was confirmed by melting curve analysis. Identification by MT-PCR correlated with culture-based identification for 44 (100%) patients. No cross-reactivity was detected in 200 blood culture specimens that contained bacteria or in 30 blood cultures without microorganisms. Fungi were correctly identified in five specimens with bacterial coinfection and in blood culture samples that were seeded with a mixture of yeast cells. The MT-PCR assay was able to provide rapid (

We thank Sally Partridge for contributing her [very insightful analysis and thoughts][1] ([1][2]). In our study ([2][3]), we found that it was possible to track a pKpQIL plasmid using a MALDI-TOF MS peak corresponding to a cleavage product of p019, a pKpQIL-encoded protein. Dr. Partridge points out