Performance of the MSI-2 Database for Fungal Identification by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry from Cleanroom Environments.

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. Here, we performed a retrospective study using spectra collected by the Bruker MALDI Biotyper (MBT) v4.1 microflex LT instrument to evaluate the MSI-2 database alongside the combined use of the Bruker MBT (including the MBT Filamentous Fungi Library) and the National Institutes of Health (NIH) mold database (MBT/NIH databases). Analysis was performed for 462 environmental fungal isolates (representing 73 different fungi) cultured from the hospital pharmacy and cellular therapy suites as part of the current good manufacturing practices (cGMP) environmental monitoring program at the NIH. When used alone, MSI-2 identified 237 spectra (51.3%) at its higher score threshold (index A), while the MBT/NIH databases identified only 183 spectra (39.6%; P < 0.001) at the equivalent threshold of ≥2.0. The combination of all three databases at the respective high thresholds improved identification sensitivity to 327 spectra (70.8%). The combination of MSI-2 with the MBT/NIH databases at a lowered threshold (index B or ≥1.7, respectively) identified 400/462 environmental spectra (86.6%). Our results show that the MSI-2 database, in combination with existing databases, may be useful for environmental surveillance, particularly by clinical or industry laboratories involved in cGMP or current good tissue practices (cGTP) applications, such as cellular therapy manufacturing facilities and sterile compounding pharmacies.

Sterility Testing for Hematopoietic Stem Cells.

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. We provide a brief background on the regulatory context for human cells, tissues, and cellular and tissue-based products (HCT/Ps) as regulated by the US Food and Drug Administration (FDA), sterility testing expectations for autologous (Section 361) and allogeneic (Section 351) HSC products, and discuss clinical risks associated with the infusion of a contaminated HSC product. Finally, we discuss the expectations for current good tissue practices (cGTP) and current good manufacturing practices (cGMP) for the manufacturing and testing of HSC based on Section 361 and Section 351 categorization, respectively. We provide commentary on what is practiced in the field and discuss the critical need for updates to professional standards that keep pace with advancing technologies with an aim to clarify expectations for manufacturing and testing facilities to improve standardization across institutions.

Comparison of Five Commercial Molecular Assays for Mycoplasma Testing of Cellular Therapy Products.

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 63 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.

Comparative Evaluation of Current Biochemical-, Sequencing-, and Proteomic-Based Identification Methods for the Streptococcus bovis Group.

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. Definitive identification, however, remains elusive due to limitations and inconsistencies across commonly used identification platforms in the United States. Here, we compared the performance of standard biochemical (Trek Gram-positive identification [GPID] plate, Vitek 2 GPID), sequencing (16S rDNA, sodA) databases (NCBI, RDP, CDC MicrobeNet), and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) platforms (Vitek MS, Bruker Biotyper MS) using a set of eight type strains representing all seven strains within the S. bovis group. Despite the evaluation of contemporary methods, no single platform was able to definitively identify all type strains within the S. bovis group. Vitek MS (85.7%, 7/8) provided the most accurate definitive identifications, followed by sodA sequencing (75%, 6/8). Vitek 2 and Bruker Biotyper RUO platforms performed the next best (62.5%, 5/8). All remaining platforms failed to adequately differentiate type strains within the S. bovis group (range, 0 to 37.5%). Laboratorians and clinicians should be aware of the identification limitations of routine testing algorithms and incorporate reflex testing, when appropriate, to platforms such as Vitek MS and/or sodA sequencing that are more able to definitively identify S. bovis group organisms. Further clinical evaluation was conducted using 65 clinical isolates from three geographically distinct U.S. institutions. Future improvements in identification platforms may reveal new clinical and epidemiological trends for members of the S. bovis group.

Investigation of a Cluster of Sphingomonas koreensis Infections.

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.).

Comprehensive Study Identifies a Sensitive, Low-Risk, Closed-System Model for Detection of Fungal Contaminants in Cell and Gene Therapy Products.

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<71>) 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<71>, 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<71> 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<71>, 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.

Pooled Saliva Specimens for SARS-CoV-2 Testing.

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 for Cellular Therapies: What Is the Role of the Clinical Microbiology Laboratory?

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.

Prospective Evaluation of a Practical Guideline for Managing Positive Sterility Test Results in Cell Therapy Products.

Product safety assurance is crucial for the clinical use of manufactured cellular therapies. A rational approach for delivering products that fail release criteria (because of 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. 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 nonviable environmental monitoring (EM) data were collected concurrently as part of a facility control assessment. 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, 1 was deemed a true positive and was discarded before infusion, whereas 9 were classified as false positives attributed to laboratory sampling and/or culturing processes. Three products deemed false positive were infused and 6 were withheld because of patient issues unrelated to microbial sterility results. No postinfusion-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 1 case. 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.

Comprehensive Evaluation of Compendial USP<71>, BacT/Alert Dual-T, and Bactec FX for Detection of Product Sterility Testing Contaminants.

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<71> 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<71>, 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 <96 h and <144 h for bacterial and fungal detection, respectively, the compendial USP<71> 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<71> and BacT/Alert testing (95.7% versus 89.0%; P = 0.0860); both systems were better than the Bactec FX system (71.2%; P < 0.0001 and P = 0.0003, respectively). The Bactec FX and BacT/Alert systems performed equivalently for 30 isolates derived from clinical bloodstream infections, confirming system optimization for clinical organisms rather than environmental contaminants. Paired Sabouraud dextrose agar (SDA) plates were always positive for fungi within the acceptable time frame. This study shows that the Bactec FX system is suboptimal for product sterility testing, and it provides strong data to support the use of BacT/Alert testing at 32.5°C paired with a supplemental SDA plate as an acceptable alternative to the compendial USP<71> method for product sterility testing.

Diagnostic challenges of prolonged post-treatment clearance of Plasmodium nucleic acids in a pre-transplant autosplenectomized patient with sickle cell disease.

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.

Problematic Dichotomization of Risk for Intensive Care Unit (ICU)-Acquired Invasive Candidiasis: Results Using a Risk-Predictive Model to Categorize 3 Levels of Risk From a Multicenter Prospective Cohort of Australian ICU Patients.

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.

An Update on the Streptococcus bovis Group: Classification, Identification, and Disease Associations.

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.

Performance of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Identification of Aspergillus, Scedosporium, and Fusarium spp. in the Australian Clinical Setting.

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).

Clinical Performance of a Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry Method for Detection of Certain blaKPC-Containing Plasmids.

Rapid detection of blaKPC-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 blaKPC-containing plasmids that was previously shown to successfully track a clonal outbreak of blaKPC-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 blaKPC 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 blaKPC-containing carbapenem-resistant isolates, providing early and clinically actionable results.

Detection and Whole-Genome Sequencing of Carbapenemase-Producing Aeromonas hydrophila Isolates from Routine Perirectal Surveillance Culture.

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 blaKPC-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.

Clinical Performance of Check-Direct CPE, a Multiplex PCR for Direct Detection of bla(KPC), bla(NDM) and/or bla(VIM), and bla(OXA)-48 from Perirectal Swabs.

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.

Candida colonization as a risk marker for invasive candidiasis in mixed medical-surgical intensive care units: development and evaluation of a simple, standard protocol.

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.

A rapid matrix-assisted laser desorption ionization-time of flight mass spectrometry-based method for single-plasmid tracking in an outbreak of carbapenem-resistant Enterobacteriaceae.

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) Tn4401 transposon on the pKpQIL plasmid. Sequence analysis demonstrates the presence of this gene in other bla(KPC) Tn4401-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.