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CONTEXT.: Laboratory testing practices for diagnosis of Clostridioides difficile infection (CDI) have evolved in response to published guidelines, availability of highly sensitive nucleic acid amplification tests (NAATs), perceived problems with the specificity of NAATs, and CDI reporting requirements. OBJECTIVE.: To assess the current state of laboratory practice for diagnostic CDI testing. DESIGN.: An optional 8-item supplemental questionnaire was distributed in December 2019 to the 1374 laboratories participating in the College of American Pathologists C difficile Detection (CDF) proficiency testing program challenge CDF-C. RESULTS.: Of 1374 CDF-C participants, 1160 (84.4%) responded, predominantly representing laboratories based in the United States (1077 of 1160; 92.8%). The majority reported using a multistep testing algorithm (684 of 1159; 59.0%). Initial testing with a glutamate dehydrogenase and toxin A/B combination test followed by NAAT for discrepant results was the most common testing method (360 of 1146; 31.4%). NAAT alone (299 of 1146; 26.1%) was next, then NAAT followed by an assay that included toxin A/B enzyme immunoassay if NAAT is positive (258 of 1146; 22.5%). Only 5.4% (62 of 1146) reported using toxin A/B immunoassay alone. Most respondents (1093 of 1131; 96.6%) reported rejecting CDI tests on formed stool, but rejection of CDI testing in pediatric patients was uncommon (211 of 1131; 18.7%). Rejection of CDI testing in patients using laxatives was reported more often by US-based respondents (379 of 1054 [36.0%] versus 9 of 77 [11.7%], P < .001). CONCLUSIONS.: Multistep algorithms for CDI diagnosis are widely used in line with published recommendations. Most respondents reported rejection of formed stool for CDI testing, but few reported rejection of testing in infants and patients taking laxatives, suggesting these may be areas of opportunity for laboratories to pursue in improving CDI testing practices.
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Objectives: This study aims to assess the presence of pathogenic microorganisms in the corneal epithelial layer of keratoconus patients. Methods: DNA was extracted from corneal epithelial samples procured from ten individual keratoconus eyes and three healthy controls. Metagenomic next-generation sequencing (mNGS) was performed to detect ocular microbiota using an agnostic approach. Results: Metagenomic sequencing revealed a low microbial read count in corneal epithelial samples derived from both keratoconus eyes (average: 530) and controls (average: 622) without a statistically significant difference (p = 0.29). Proteobacteria were the predominant phylum in both keratoconus and control samples (relative abundance: 72% versus 79%, respectively). Conclusions: The overall low microbial read count and the lack of difference in the relative abundance of different microbial species between keratoconus and control samples do not support the hypothesis that a chronic corneal infection is implicated in the pathogenesis of keratoconus. These findings do not rule out the possibility that an acute infection may be involved in the disease process as an initiating event.
ABSTRACT
There are increasing reports of carbapenem-resistant Enterobacterales (CRE) that test as cefepime-susceptible (S) or susceptible-dose dependent (SDD). However, there are no data to compare the cefepime testing performance of BD Phoenix automated susceptibility system (BD Phoenix) and disk diffusion (DD) relative to reference broth microdilution (BMD) against carbapenemase-producing (CPblaKPC-CRE) and non-producing (non-CP CRE) isolates. Cefepime susceptibility results were interpreted according to CLSI M100Ed32. Essential agreement (EA), categorical agreement (CA), minor errors (miEs), major errors (MEs), and very major errors (VMEs) were calculated for BD Phoenix (NMIC-306 Gram-negative panel) and DD relative to BMD. Correlates were also analyzed by the error rate-bounded method. EA and CA for CPblaKPC-CRE isolates (n = 64) were <90% with BD Phoenix while among non-CP CRE isolates (n = 58), EA and CA were 96.6%, and 79.3%, respectively. CA was <90% with DD for both cohorts. No ME or VME was observed for either isolate cohort; however, miEs were >10% for CPblaKPC-CRE and non-CP CRE with BD Phoenix and DD tests. For error rate-bounded method, miEs were <40% for IHigh + 1 to ILow - 1 ranges for CPblaKPC-CRE and non-CP CRE with BD Phoenix. Regarding disk diffusion, miEs were unacceptable for all MIC ranges among CPblaKPC-CRE. For non-CP CRE isolates, only IHigh + 1 to ILow - 1 range was acceptable at 37.2%. Using this challenge set of genotypic-phenotypic discordant CRE, the BD Phoenix MICs and DD susceptibility results trended higher (toward SDD and resistant phenotypes) relative to reference BMD results yielding lower CA. These results were more prominent among CPblaKPC-CRE than non-CP CRE.
Subject(s)
Anti-Bacterial Agents , Carbapenem-Resistant Enterobacteriaceae , Cefepime , Microbial Sensitivity Tests , Cefepime/pharmacology , Anti-Bacterial Agents/pharmacology , Microbial Sensitivity Tests/methods , Humans , Carbapenem-Resistant Enterobacteriaceae/drug effects , Disk Diffusion Antimicrobial Tests/methods , Enterobacteriaceae Infections/microbiology , Cephalosporins/pharmacologyABSTRACT
BACKGROUND: The treatment of carbapenem-resistant Acinetobacter baumannii/calcoaceticus complex (CRAB) presents significant treatment challenges. METHODS: We report the case of a 42-year-old woman with CRAB meningitis who experienced persistently positive cerebrospinal fluid (CSF) cultures for 13 days despite treatment with high-dose ampicillin-sulbactam and cefiderocol. On day 13, she was transitioned to sulbactam-durlobactam and meropenem; four subsequent CSF cultures remained negative. After 14 days of sulbactam-durlobactam, she was cured of infection. Whole genome sequencing investigations identified putative mechanisms that contributed to reduced cefiderocol susceptibility observed during cefiderocol therapy. Blood and CSF samples were collected pre-dose and 3-hours post initiation of a sulbactam-durlobactam infusion. RESULTS: The CRAB isolate belonged to sequence type 2. An acquired blaOXA-23 and an intrinsic blaOXA-51-like (i.e., blaOXA-66) carbapenemase gene were identified. The paradoxical effect (i.e., no growth at lower cefiderocol dilutions but growth at higher dilutions) was observed by broth microdilution after 8 days of cefiderocol exposure but not by disk diffusion. Potential markers of resistance to cefiderocol included mutations in the start codon of piuA and piuC iron transport genes and a A515V substitution in PBP3, the primary target of cefiderocol. Sulbactam and durlobactam were detected in CSF at both timepoints, indicating CSF penetration. CONCLUSIONS: This case describes successful treatment of refractory CRAB meningitis with the administration of sulbactam-durlobactam and meropenem and highlights the need to be cognizant of the paradoxical effect that can be observed with broth microdilution testing of CRAB isolates with cefiderocol.
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BACKGROUND: Patients infected with difficult-to-treat Pseudomonas aeruginosa are likely to receive meropenem (MEM) empirically before escalation to ceftolozane/tazobactam (C/T). We assessed whether pre-exposure to MEM affected C/T resistance development on C/T exposure. MATERIALS AND METHODS: Nine clinical P. aeruginosa isolates were exposed to MEM 16â mg/L for 72â h. Then, isolates were serially passaged in the presence of C/T (concentration of 10â mg/L) for 72â h as two groups: an MEM-exposed group inoculated with MEM pre-exposed isolates and a non-MEM control group. At 24â h intervals, samples were plated on drug-free and drug-containing agar (C/T concentration 16/8â mg/L) and incubated to quantify bacterial densities (log10 cfu/mL). Growth on C/T agar indicated resistance development, and resistant population was calculated by dividing the cfu/mL on C/T plates by the cfu/mL on drug-free agar. RESULTS: At 72â h, resistant populations were detected in 6/9 isolates. In five isolates, MEM exposure significantly increased the prevalence of ceftolozane/tazobactam-resistance development; the percentages of resistance population were 100%, 100%, 53.5%, 31% and 3% for the MEM-exposed versus 0%, 0%, 2%, 0.35% and ≤0.0003% in the unexposed groups. One isolate had a similar resistant population at 72â h between the two groups. The remaining isolates showed no development of resistance, regardless of previous MEM exposure. CONCLUSIONS: MEM exposure may pre-dispose to C/T resistance development and thus limit the therapeutic utility of this ß-lactam/ß-lactamase inhibitor. Resistance may be a result of stress exposure or molecular-level mutations conferring cross-resistance. Further in vivo studies are needed to assess clinical implications of these findings.
Subject(s)
Anti-Bacterial Agents , Cephalosporins , Meropenem , Pseudomonas Infections , Pseudomonas aeruginosa , Tazobactam , Pseudomonas aeruginosa/drug effects , Cephalosporins/pharmacology , Meropenem/pharmacology , Tazobactam/pharmacology , Anti-Bacterial Agents/pharmacology , Humans , Pseudomonas Infections/microbiology , Pseudomonas Infections/drug therapy , Microbial Sensitivity Tests , Serial PassageSubject(s)
Blood Culture , Multiplex Polymerase Chain Reaction , Rhodotorula , Humans , Multiplex Polymerase Chain Reaction/methods , Blood Culture/methods , Rhodotorula/genetics , Rhodotorula/isolation & purification , False Positive Reactions , Molecular Diagnostic Techniques/methods , Fungemia/diagnosis , Fungemia/microbiologyABSTRACT
Next-generation sequencing (NGS) applications for the diagnostics of infectious diseases has demonstrated great potential with three distinct approaches: whole-genome sequencing (WGS), targeted NGS (tNGS), and metagenomic NGS (mNGS, also known as clinical metagenomics). These approaches provide several advantages over traditional microbiologic methods, though challenges still exist.
Subject(s)
High-Throughput Nucleotide Sequencing , Metagenomics , Whole Genome Sequencing , Sensitivity and SpecificityABSTRACT
BACKGROUND: Investigations into antibiotics for extended-spectrum ß-lactamase-producing Enterobacterales (ESBL-E) bloodstream infections (BSIs) have focused on blaCTX-M genes. Outcomes of patients with non-CTX-M-producing ESBL-E BSIs and optimal treatment are unknown. METHODS: A multicenter observational study investigating 500 consecutive patients with ceftriaxone-resistant Enterobacterales BSIs during 2018-2022 was conducted. Broth microdilution and whole genome sequencing confirmed antibiotic susceptibilities and ESBL gene presence, respectively. Inverse probability weighting (IPW) using propensity scores was employed to ensure patients infected with non-CTX-M and CTX-M ESBL-E BSIs were similar prior to evaluation of outcomes. RESULTS: 396 patients (79.2%) were confirmed to have an ESBL-E BSI. ESBL gene family prevalence was as follows: blaCTX-M (n=370), blaSHV (n=16), blaOXY (n=12), and blaVEB (n=5). ESBL gene identification was not limited to Escherichia coli and Klebsiella species. In the IPW cohort, there was no difference in 30-day mortality or ESBL-E infection recurrence between the non-CTX-M and CTX-M groups (OR=.99, 95% CI 0.87-1.11; p=0.83) and (OR=1.10, 95% CI 0.85--1.42; p=0.47), respectively. In an exploratory analysis limited to the non-CTX-M group, 86% of the 21 patients receiving meropenem were alive on day 30; none of the 5 patients receiving piperacillin-tazobactam were alive on day 30. CONCLUSIONS: Our findings suggest that non-CTX-M and CTX-M ESBL-producing Enterobacterales BSIs are equally concerning and associated with similar clinical outcomes. Meropenem may be associated with improved survival in patients with non-CTX-M ESBL-E BSIs, underscoring the potential benefit of comprehensive molecular diagnostics to enable early antibiotic optimization for patients with ESBL-E BSI, beyond just blaCTX-M genes.
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IMPORTANCE: Testing for enteric bacterial pathogens in patients hospitalized for more than 3 days is almost always inappropriate. Our study validates the utility of the 3-day rule and the use of clinical decision support tools to decrease unnecessary testing of enteropathogenic bacteria other than C. difficile. Overriding the restriction was very low yield. Our study highlights the importance of diagnostic stewardship and further refines the criteria for allowing providers to override the restriction while monitoring the impact of the interventions.
Subject(s)
Clostridioides difficile , Humans , Diarrhea/microbiology , EnterobacteriaceaeABSTRACT
Accurate antimicrobial susceptibility testing (AST) and reporting are essential for guiding appropriate therapy for patients and direction for public health prevention and control actions. A critical feature of AST reporting is the interpretation of AST results using clinical breakpoints for reporting as susceptible, susceptible-dose dependent, intermediate, or resistant. Breakpoints are subject to continuous adjustment and updating to best reflect current clinical data. These breakpoint changes can benefit patients and public health only if adopted in a timely manner. A recent survey identified that up to 70% of College of American Pathologists (CAP)-accredited U.S. laboratories and 45% of CAP-accredited laboratories outside the U.S. use various obsolete clinical breakpoints to interpret AST results to guide patient care. The reason for the ongoing use of obsolete breakpoints is multifactorial, including barriers encountered by laboratories, commercial AST device manufacturers, standards development organizations, and regulatory bodies alike. To begin to address this important patient safety issue, CAP implemented checklist requirements for CAP-accredited laboratories to ensure up-to-date clinical breakpoint use. Furthermore, the topic was discussed at the June 2022 American Society for Microbiology Clinical Microbiology Open (CMO) with various stakeholders to identify potential solutions. This minireview summarizes the breakpoint setting process in the U.S. and highlights solutions to close the gap between breakpoint revisions and implementation in clinical and public health laboratories. Solutions discussed include clarification of data requirements and minimum inhibitory concentration only reporting for regulatory clearance of AST devices, clinical data generation to close breakpoints gaps, advocacy, education, and greater dialogue between stakeholders.
Subject(s)
Anti-Bacterial Agents , Laboratories , Humans , United States , Anti-Bacterial Agents/pharmacology , Microbial Sensitivity TestsABSTRACT
The advancement of infectious disease diagnostics, along with studies devoted to infections caused by gram-negative and gram-positive bacteria, is a top scientific priority of the Antibacterial Resistance Leadership Group (ARLG). Diagnostic tests for infectious diseases are rapidly evolving and improving. However, the availability of rapid tests designed to determine antibacterial resistance or susceptibility directly in clinical specimens remains limited, especially for gram-negative organisms. Additionally, the clinical impact of many new tests, including an understanding of how best to use them to inform optimal antibiotic prescribing, remains to be defined. This review summarizes the recent work of the ARLG toward addressing these unmet needs in the diagnostics field and describes future directions for clinical research aimed at curbing the threat of antibiotic-resistant bacterial infections.
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Gram-Negative Bacterial Infections , Leadership , Humans , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Gram-Positive Bacteria , Drug Resistance, Bacterial , Gram-Negative Bacteria , Microbial Sensitivity Tests , Gram-Negative Bacterial Infections/drug therapyABSTRACT
IMPORTANCE: Nosocomiicoccus species are recently described as members of the Staphylococcaceae family. With their inclusion in commercial matrix-assisted laser desorption/ionization-time of flight mass spectrometry databases, Nosocomiicoccus species can now be identified when Gram-positive cocci in clusters are detected in positive blood cultures. However, their clinical significance is not known, making it difficult for the clinical microbiology laboratory to decide the extent of work-up. Based on our study, Nosocomiicoccus species demonstrate low pathogenicity and opportunistic potential. If isolated from a single blood culture set, limited work-up should be performed to an extent similar to other possible blood culture contaminants.
Subject(s)
Bacteremia , Blood Culture , Humans , Clinical Relevance , Staphylococcaceae , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Bacteremia/microbiologyABSTRACT
Antimicrobial susceptibility test and report guidelines are an important tool for antimicrobial stewardship programs. Since 1972, Tables 1 within the Clinical and Laboratory Standards Institute (CLSI) M100 document have provided a general framework upon which clinical microbiologists and antimicrobial stewardship teams can build algorithms for susceptibility testing and reporting that meet the specific needs of their institution. Many changes were made to Tables 1 in M100-Ed33 to modernize the content to reflect the landscape of current clinical practice, including the growing armamentarium of antimicrobial agents, the emergence of new mechanisms of antimicrobial resistance, the increasing prevalence of infections caused by multidrug-resistant organisms, and updated consensus recommendations for first-choice and alternative agents for treatment. With these items in mind, the CLSI Table 1 ad hoc working group revised Tables 1 with the ultimate goal of supporting institutions in the creation of individualized test and report strategies that support local antimicrobial stewardship program initiatives. These strategies are built on the concepts of selective and cascade reporting. This minireview introduces the concept of CLSI M100-Ed33 Tables 1, describes the changes to Tables 1 introduced in 2023, and provides clinical vignettes that demonstrate how Tables 1 can be used in various scenarios to devise antimicrobial susceptibility test and report strategies.
Subject(s)
Anti-Infective Agents , Antimicrobial Stewardship , Humans , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Gram-Negative Bacteria , Anti-Infective Agents/pharmacology , Anti-Infective Agents/therapeutic use , Microbial Sensitivity TestsABSTRACT
Background: Cefiderocol and ceftazidime-avibactam plus aztreonam (CZA-ATM) are preferred treatment regimens for New Delhi metallo-ß-lactamase (NDM)-producing infections. Methods: We report the case of a US patient who traveled to India to receive a renal transplant. He subsequently experienced pyelonephritis by an NDM-producing Escherichia coli. Broth microdilution and the broth disk elution method indicated resistance to all ß-lactams, including cefiderocol and CZA-ATM. Whole-genome sequencing investigations were undertaken to identify resistance mechanisms. Results: An E. coli isolate belonging to sequence type (ST) 167 containing a blaNDM-5 gene was identified on a plasmid of the IncFIA/IncFIB/IncFIC replicon groups. When compared with the genome of another ST167 E. coli clinical isolate containing blaNDM-5 and exhibiting susceptibility to cefiderocol and CZA-ATM, a 12-base pair insertion in ftsI, translating to a 4-amino acid duplication in PBP3, was identified. Moreover, a blaCMY-59 gene was harbored on an IncI-γ replicon type, and frameshift mutations were identified in the cirA iron transport gene. Conclusions: This is the first clinical case of a US patient harboring an NDM-producing isolate exhibiting resistance to all available ß-lactam agents. The isolate's unexpected resistance to cefiderocol and CZA-ATM was likely due to a combination of (1) a modified PBP3 (increased MICs to both regimens), (2) truncated iron-binding protein (increased cefiderocol MIC), and (3) a blaCMY gene (reduced CZA-ATM activity). E. coli ST167 clinical isolates harboring blaNDM-5 genes are a recognized international high-risk clone. When coupled with the additional mechanisms identified in our patient's isolate, which is not uncommon for this high-risk clone, pan-ß-lactam resistance may occur.
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BACKGROUND: Carbapenem-resistant Enterobacterales (CRE) are a public health concern. Among these isolates, there are reports of isolates that test as cefepime susceptible or susceptible-dose dependent (SDD) in vitro despite presence of a carbapenemase. This study aimed to evaluate the pharmacokinetic/pharmacodynamic profile of cefepime against carbapenemase-producing (CP-CRE) and non-producing (non-CP-CRE) isolates with a range of cefepime MICs. METHODS: Reference broth microdilution and modified carbapenem inactivation method (mCIM) were performed on genotypically characterized clinical CRE isolates. Ultimately, CP-CRE (nâ=â21; blaKPC) and non-CP-CRE (nâ=â19) isolates with a distribution of cefepime MICs (≤0.5 to >256 mg/L) were utilized in the murine thigh infection model. Mice were treated with cefepime human-simulated regimens (HSRs) representative of a standard dose (1 g q12h 0.5 h infusion) or the SDD dose (2 g q8h 0.5 h infusion). Efficacy was assessed as the change in bacterial growth at 24 h compared with 0 h control, where ≥1 log bacterial reduction is considered translational value for clinical efficacy. RESULTS: Among both cohorts of CRE isolates, i.e. CP-CRE and non-CP-CRE, that tested as SDD to cefepime in vitro, 1 log bacterial reduction was not attainable with cefepime. Further blunting of cefepime efficacy was observed among CP-CRE isolates compared with non-CP-CRE across both susceptible and SDD categories. CONCLUSIONS: Data indicate to avoid cefepime for the treatment of serious infections caused by CRE isolates that test as cefepime susceptible or SDD. Data also provide evidence that isolates with the same antibiotic MIC may have different pharmacokinetic/pharmacodynamic profiles due to their antimicrobial resistance mechanism.
Subject(s)
Carbapenems , Gammaproteobacteria , Humans , Animals , Mice , Cefepime , Carbapenems/pharmacology , Carbapenems/therapeutic use , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , beta-Lactamases , Enterobacteriaceae , Microbial Sensitivity TestsABSTRACT
Cefiderocol is an option for infections caused by multidrug-resistant Pseudomonas aeruginosa, but its in vitro activity against these isolates and its clinical effectiveness for isolates with MICs of >1 µg/mL is unclear. We investigated the in vitro activity of cefiderocol against P. aeruginosa isolates collected from patients treated with cefiderocol through the compassionate use program and assessed physician-reported clinical response and 28-day all-cause mortality by cefiderocol MIC values. P. aeruginosa isolates underwent susceptibility testing to cefiderocol and comparator agents by using reference broth microdilution. U.S. Food and Drug Administration (FDA; susceptible, ≤1 µg/mL) and Clinical and Laboratory Standards Institute (CLSI; susceptible, ≤4 µg/mL) cefiderocol breakpoints were applied. Additionally, molecular characterization of ß-lactamase genes was performed. Clinical response and vital status were reported by treating physicians. Forty-six patients with P. aeruginosa infections were evaluated. Twenty-nine (63%) and 42 (91%) isolates were susceptible to cefiderocol using FDA and CLSI breakpoints, respectively. Thirty-seven (80%) and 32 (70%) isolates were not susceptible to ceftolozane-tazobactam and ceftazidime-avibactam, respectively. The clinical response rate was 69% (20/29) with a cefiderocol MIC of ≤1 µg/mL, 69% (9/13) with a cefiderocol MIC of 2 to 4 µg/mL, and 100% (4/4) with an MIC of ≥8 µg/mL, while day 28 all-cause mortality rates were 23% (6/26; MIC ≤ 1 µg/mL), 33% (4/12; MIC, 2 to 4 µg/mL), and 0% (0/4; MIC ≥8 µg/mL), respectively. Cefiderocol was active in vitro against most P. aeruginosa isolated from patients with limited or no alternative therapies. Patients with cefiderocol MICs of 2 to 4 µg/mL did not have significantly worse outcomes than those with MICs of ≤1 µg/mL.