Ultrasensitive Clostridioides difficile Toxin Testing for Higher Diagnostic Accuracy.

Currently available diagnostic tests for Clostridioides difficile infection (CDI) lack specificity or sensitivity, which has led to guideline recommendations for multistep testing algorithms. Ultrasensitive assays for detection of C. difficile toxins provide measurements of disease-specific markers at very low concentrations. These assays may show improved accuracy compared to that of current testing methods and offer a potential standalone solution for CDI diagnosis, although large studies of clinical performance and accuracy are lacking.

Evaluation of Cycle Threshold, Toxin Concentration, and Clinical Characteristics of Clostridioides difficile Infection in Patients with Discordant Diagnostic Test Results.

Clostridioides difficile infection (CDI) is one of the most common health care-associated infections that can cause significant morbidity and mortality. CDI diagnosis involves laboratory testing in conjunction with clinical assessment. The objective of this study was to assess the performance of various C. difficile tests and to compare clinical characteristics, Xpert C. difficile/Epi (PCR) cycle threshold (CT ), and Singulex Clarity C. diff toxins A/B (Clarity) concentrations between groups with discordant test results. Unformed stool specimens from 200 hospitalized adults (100 PCR positive and 100 negative) were tested by cell cytotoxicity neutralization assay (CCNA), C. diff Quik Chek Complete (Quik Chek), Premier Toxins A and B, and Clarity. Clinical data, including CDI severity and CDI risk factors, were compared between discordant test results. Compared to CCNA, PCR had the highest sensitivity at 100% and Quik Chek had the highest specificity at 100%. Among clinical and laboratory data studied, prevalences of leukocytosis, prior antibiotic use, and hospitalizations were consistently higher across all subgroups in comparisons of toxin-positive to toxin-negative patients. Among PCR-positive samples, the median CT was lower in toxin-positive samples than in toxin-negative samples; however, CT ranges overlapped. Among Clarity-positive samples, the quantitative toxin concentration was significantly higher in toxin-positive samples than in toxin-negative samples as determined by CCNA and Quik Chek Toxin A and B. Laboratory tests for CDI vary in sensitivity and specificity. The quantitative toxin concentration may offer value in guiding CDI diagnosis and treatment. The presence of leukocytosis, prior antibiotic use, and previous hospitalizations may assist with CDI diagnosis, while other clinical parameters may not be consistently reliable.

High Agreement Between an Ultrasensitive Clostridioides difficile Toxin Assay and a C. difficile Laboratory Algorithm Utilizing GDH-and-Toxin Enzyme Immunoassays and Cytotoxin Testing.

The Singulex Clarity C. diff toxins A/B (Clarity) assay is an automated, ultrasensitive immunoassay for the detection of Clostridioides difficile toxins in stool. In this study, the performance of the Clarity assay was compared to that of a multistep algorithm using an enzyme immunoassay (EIA) for detection of glutamate dehydrogenase (GDH) and toxins A and B arbitrated by a semiquantitative cell cytotoxicity neutralization assay (CCNA). The performance of the assay was evaluated using 211 residual deidentified stool samples tested with a GDH-and-toxin EIA (C. Diff Quik Chek Complete; Techlab), with GDH-and-toxin discordant samples tested with CCNA. The stool samples were stored at -80°C before being tested with the Clarity assay. For samples discordant between Clarity and the standard-of-care algorithm, the samples were tested with PCR (Xpert C. difficile; Cepheid), and chart review was performed. The testing algorithm resulted in 34 GDH+/toxin+, 53 GDH-/toxin-, and 124 GDH+/toxin- samples, of which 39 were CCNA+ and 85 were CCNA- Clarity had 96.2% negative agreement with GDH-/toxin- samples, 100% positive agreement with GDH+/toxin+ samples, and 95.3% agreement with GDH+/toxin-/CCNA- samples. The Clarity result was invalid for one sample. Clarity agreed with 61.5% of GDH+/toxin-/CCNA+ samples, 90.0% of GDH+/toxin-/CCNA+ (high-positive) samples, and 31.6% of GDH+/toxin-/CCNA+ (low-positive) samples. The Singulex Clarity C. diff toxins A/B assay demonstrated high agreement with a testing algorithm utilizing a GDH-and-toxin EIA and CCNA. This novel automated assay may offer an accurate, stand-alone solution for C. difficile infection (CDI) diagnostics, and further prospective clinical studies are merited.

Ultrasensitive Detection of Clostridium difficile Toxins Reveals Suboptimal Accuracy of Toxin Gene Cycle Thresholds for Toxin Predictions.

The use of nucleic acid amplification tests (NAATs) for the diagnosis of Clostridium (Clostridioides) difficile infection (CDI) leads to overdiagnosis. To improve the clinical specificity of NAATs, there has been a recent interest in using toxin gene cycle thresholds (CT s) to predict the presence and absence of toxins. Although there is an association between CT values and fecal toxin concentrations, the predictive accuracy of the former is suboptimal for use in clinical practice. Ultrasensitive toxin immunoassays to quantify free toxins in stool offer a novel option for high-sensitivity fecal toxin detection rather than using surrogate markers for prediction.

Increased Clinical Specificity with Ultrasensitive Detection of Clostridioides difficile Toxins: Reduction of Overdiagnosis Compared to Nucleic Acid Amplification Tests.

Clostridioides difficile infection (CDI) is one of the most common health care-associated infections, resulting in significant morbidity, mortality, and economic burden. Diagnosis of CDI relies on the assessment of clinical presentation and laboratory tests. We evaluated the clinical performance of ultrasensitive single-molecule counting technology for detection of C. difficile toxins A and B. Stool specimens from 298 patients with suspected CDI were tested with the nucleic acid amplification test (NAAT; BD MAX Cdiff assay or Xpert C. difficile assay) and Singulex Clarity C. diff toxins A/B assay. Specimens with discordant results were tested with the cell cytotoxicity neutralization assay (CCNA), and the results were correlated with disease severity and outcome. There were 64 NAAT-positive and 234 NAAT-negative samples. Of the 32 NAAT+/Clarity- and 4 NAAT-/Clarity+ samples, there were 26 CCNA- and 4 CCNA- samples, respectively. CDI relapse was more common in NAAT+/toxin+ patients than in NAAT+/toxin- and NAAT-/toxin- patients. The clinical specificity of Clarity and NAAT was 97.4% and 89.0%, respectively, and overdiagnosis was more than three times more common in NAAT+/toxin- than in NAAT+/toxin+ patients. The Clarity assay was superior to NAATs for the diagnosis of CDI, by reducing overdiagnosis and thereby increasing clinical specificity, and the presence of toxins was associated with negative patient outcomes.

Ultrasensitive Detection of Clostridioides difficile Toxins in Stool by Use of Single-Molecule Counting Technology: Comparison with Detection of Free Toxin by Cell Culture Cytotoxicity Neutralization Assay.

Laboratory tests for Clostridioides difficile infection (CDI) rely on the detection of free toxin or molecular detection of toxin genes. The Singulex Clarity C. diff toxins A/B assay is a rapid, automated, and ultrasensitive assay that detects C. difficile toxins A and B in stool. We compared CDI assays across two prospective multicenter studies to set a cutoff for the Clarity assay and to independently validate the performance compared with that of a cell culture cytotoxicity neutralization assay (CCCNA). The cutoff was set by two sites testing fresh samples from 897 subjects with suspected CDI and then validated at four sites testing fresh samples from 1,005 subjects with suspected CDI. CCCNA testing was performed at a centralized laboratory. Samples with discrepant results between the Clarity assay and CCCNA were retested with CCCNA when the Clarity result agreed with that of at least one comparator method; toxin enzyme immunoassays (EIA), glutamate dehydrogenase (GDH) detection, and PCR were performed on all samples. The cutoff for the Clarity assay was set at 12.0 pg/ml. Compared to results with CCCNA, the Clarity assay initially had 85.2% positive agreement and 92.4% negative agreement. However, when samples with discrepant results between the Clarity assay and CCCNA in the validation study were retested by CCCNA, 13/17 (76.5%) Clarity-negative but CCCNA-positive samples (Clarity+/CCCNA-) became CCCNA-, and 5/26 (19.2%) Clarity+/CCCNA- samples became CCCNA+, resulting in a 96.3% positive agreement and 93.0% negative agreement between Clarity and CCCNA results. The toxin EIA had 59.8% positive agreement with CCCNA. The Clarity assay was the most sensitive free-toxin immunoassay, capable of providing CDI diagnosis in a single-step solution. A different CCCNA result was reported for 42% of retested samples, increasing the positive agreement between Clarity and CCCNA from 85.2% to 96.3% and indicating the challenges of comparing free-toxin results to CCCNA results as a reference standard.

Ultrasensitive Detection of Clostridioides difficile Toxins A and B by Use of Automated Single-Molecule Counting Technology.

Current tests for the detection of Clostridioides (formerly Clostridium) difficile free toxins in feces lack sensitivity, while nucleic acid amplification tests lack clinical specificity. We have evaluated the Singulex Clarity C. diff toxins A/B assay (currently in development), an automated and rapid ultrasensitive immunoassay powered by single-molecule counting technology, for detection of C. difficile toxin A (TcdA) and toxin B (TcdB) in stool. The analytical sensitivity, analytical specificity, repeatability, and stability of the assay were determined. In a clinical evaluation, frozen stool samples from 311 patients with suspected C. difficile infection were tested with the Clarity C. diff toxins A/B assay, using an established cutoff value. Samples were tested with the Xpert C. difficile/Epi assay, and PCR-positive samples were tested with an enzyme immunoassay (EIA) (C. Diff Quik Chek Complete). EIA-negative samples were further tested with a cell cytotoxicity neutralization assay. The limits of detection for TcdA and TcdB were 0.8 and 0.3 pg/ml in buffer and 2.0 and 0.7 pg/ml in stool, respectively. The assay demonstrated reactivity to common C. difficile strains, did not show cross-reactivity to common gastrointestinal pathogens, was robust against common interferents, allowed detection in fresh and frozen stool samples and in samples after three freeze-thaw cycles, and provided results with high reproducibility. Compared to multistep PCR and toxin-testing procedures, the Singulex Clarity C. diff toxins A/B assay yielded 97.7% sensitivity and 100% specificity. The Singulex Clarity C. diff toxins A/B assay is ultrasensitive and highly specific and may offer a standalone solution for rapid detection and quantitation of free toxins in stool.

Simple Real-Time PCR and Amplicon Sequencing Method for Identification of Plasmodium Species in Human Whole Blood.

Malaria is the leading identifiable cause of fever in returning travelers. Accurate Plasmodium species identification has therapy implications for P. vivax and P. ovale, which have dormant liver stages requiring primaquine. Compared to microscopy, nucleic acid tests have improved specificity for species identification and higher sensitivity for mixed infections. Here, we describe a SYBR green-based real-time PCR assay for Plasmodium species identification from whole blood, which uses a panel of reactions to detect species-specific non-18S rRNA gene targets. A pan-Plasmodium 18S rRNA target is also amplified to allow species identification or confirmation by sequencing if necessary. An evaluation of assay accuracy, performed on 76 clinical samples (56 positives using thin smear microscopy as the reference method and 20 negatives), demonstrated clinical sensitivities of 95.2% for P. falciparum (20/21 positives detected) and 100% for the Plasmodium genus (52/52), P. vivax (20/20), P. ovale (9/9), and P. malariae (6/6). The sensitivity of the P. knowlesi-specific PCR was evaluated using spiked whole blood samples (100% [10/10 detected]). The specificities of the real-time PCR primers were 94.2% for P. vivax (49/52) and 100% for P. falciparum (51/51), P. ovale (62/62), P. malariae (69/69), and P. knowlesi (52/52). Thirty-three specimens were used to test species identification by sequencing the pan-Plasmodium 18S rRNA PCR product, with correct identification in all cases. The real-time PCR assay also identified two samples with mixed P. falciparum and P. ovale infection, which was confirmed by sequencing. The assay described here can be integrated into a malaria testing algorithm in low-prevalence areas, allowing definitive Plasmodium species identification shortly after malaria diagnosis by microscopy.

Netrin-1 promotes glioblastoma cell invasiveness and angiogenesis by multiple pathways including activation of RhoA, cathepsin B, and cAMP-response element-binding protein.

Glioblastomas are very difficult tumors to treat because they are highly invasive and disseminate within the normal brain, resulting in newly growing tumors. We have identified netrin-1 as a molecule that promotes glioblastoma invasiveness. As evidence, netrin-1 stimulates glioblastoma cell invasion directly through Matrigel-coated transwells, promotes tumor cell sprouting and enhances metastasis to lymph nodes in vivo. Furthermore, netrin-1 regulates angiogenesis as shown in specific angiogenesis assays such as enhanced capillary endothelial cells (EC) sprouting and by increased EC infiltration into Matrigel plugs in vivo, as does VEGF-A. This netrin-1 signaling pathway in glioblastoma cells includes activation of RhoA and cyclic AMP response element-binding protein (CREB). A novel finding is that netrin-1-induced glioblastoma invasiveness and angiogenesis are mediated by activated cathepsin B (CatB), a cysteine protease that translocates to the cell surface as an active enzyme and co-localizes with cell surface annexin A2 (ANXA2). The specific CatB inhibitor CA-074Me inhibits netrin-1-induced cell invasion, sprouting, and Matrigel plug angiogenesis. Silencing of CREB suppresses netrin-1-induced glioblastoma cell invasion, sprouting, and CatB expression. It is concluded that netrin-1 plays an important dual role in glioblastoma progression by promoting both glioblastoma cell invasiveness and angiogenesis in a RhoA-, CREB-, and CatB-dependent manner. Targeting netrin-1 pathways may be a promising strategy for brain cancer therapy.

VEGF: a potential target for hydrocephalus.

Growth factors are primarily responsible for the genesis, differentiation and proliferation of cells and maintenance of tissues. Given the central role of growth factors in signaling between cells in health and in disease, it is understandable that disruption of growth factor-mediated molecular signaling can cause diverse phenotypic consequences including cancer and neurological conditions. This review will focus on the specific questions of enlarged cerebral ventricles and hydrocephalus. It is also well known that angiogenic factors, such as vascular endothelial growth factor (VEGF), affect tissue permeability through activation of receptors and adhesion molecules; hence, recent studies showing elevations of this factor in pediatric hydrocephalus led to the demonstration that VEGF can induce ventriculomegaly and altered ependyma when infused in animals. In this review, we discuss recent findings implicating the involvement of biochemical and biophysical factors that can induce a VEGF-mimicking effect in communicating hydrocephalus and pay particular attention to the role of the VEGF system as a potential pharmacological target in the treatment of some cases of hydrocephalus. The source of VEGF secretion in the cerebral ventricles, in periventricular regions and during pathologic events including hydrocephalus following hypoxia and hemorrhage is sought. The review is concluded with a summary of potential non-surgical treatments in preclinical studies suggesting several molecular targets including VEGF for hydrocephalus and related neurological disorders.

Epidemiology of malaria in a village in the Rufiji River Delta, Tanzania: declining transmission over 25 years revealed by different parasitological metrics.

BACKGROUND: Assessments of the epidemiology of malaria over time are needed to understand changes in transmission and guide control and elimination strategies. METHODS: A longitudinal population study was established in 1985 in Nyamisati village in the Rufiji River Delta, Tanzania. A physician and research team lived in the village 1984-2000. Parasite prevalence by microscopy and two PCR methods, spleen rates and haemoglobin levels were measured in repeated cross-sectional surveys between 1985 and 2010. Passive surveillance of malaria cases was maintained until end 1999. Bed nets were distributed after the surveys 1993, 1999 and 2010. RESULTS: In 1985, overall parasite prevalence by microscopy was 70% (90% in children ages two to nine years). The prevalence decreased gradually by microscopy (38.9% 1994, 26.7% 1999) and msp2-PCR (58.7% 1994, 44.8% 1999), whereas real-time PCR prevalence remained higher throughout the 1990s (69.4% 1994, 64.8% 1999). In 2010, parasite prevalence was 17.8% by real-time PCR and 16.3% by msp2-PCR, and estimated to 4.8% by microscopy. Spleen rates in children ages two to nine years decreased earlier than parasite prevalence, from >75 to 42% in the 1980s, to nil during the 1990s. The prevalence of severe and moderate anaemia decreased from 41.1 to 13.1%. No deaths at the time of acute malaria were recorded when the research team lived in the village. CONCLUSIONS: A marked decline in malaria transmission was observed over 25 years. The decrease was detected after the arrival of the research team and continued gradually both before and after distribution of bed nets. Spleen rates and microscopy identified early changes when transmission was still intense, whereas real-time PCR was a more sensitive metric when transmission was reduced. The study provides historical data on malaria within a closely monitored rural village and contributes to the understanding of changing epidemiology in sub-Saharan Africa.

Bacteraemia caused by Actinobaculum schaalii: An overlooked pathogen?

Actinobaculum schaalii is a uropathogen resistant to ciprofloxacin and trimethoprim-sulfamethoxazole. It requires a long culture time and specific conditions, and is therefore easily overgrown by other bacteria and regarded as part of the normal bacterial flora. We review 17 cases of A. schaalii bacteraemia, demonstrating its invasive potential. A. schaalii should always be ruled out as causative agent in patients with urinary tract infection or urosepticaemia with treatment failure.

Child mask mandates for COVID-19: a systematic review.

BACKGROUND: Mask mandates for children during the COVID-19 pandemic varied in different locations. A risk-benefit analysis of this intervention has not yet been performed. In this study, we performed a systematic review to assess research on the effectiveness of mask wearing in children. METHODS: We performed database searches up to February 2023. The studies were screened by title and abstract, and included studies were further screened as full-text references. A risk-of-bias analysis was performed by two independent reviewers and adjudicated by a third reviewer. RESULTS: We screened 597 studies and included 22 in the final analysis. There were no randomised controlled trials in children assessing the benefits of mask wearing to reduce SARS-CoV-2 infection or transmission. The six observational studies reporting an association between child masking and lower infection rate or antibody seropositivity had critical (n=5) or serious (n=1) risk of bias; all six were potentially confounded by important differences between masked and unmasked groups and two were shown to have non-significant results when reanalysed. Sixteen other observational studies found no association between mask wearing and infection or transmission. CONCLUSIONS: Real-world effectiveness of child mask mandates against SARS-CoV-2 transmission or infection has not been demonstrated with high-quality evidence. The current body of scientific data does not support masking children for protection against COVID-19.

Bacterial coinfections in travelers with malaria: rationale for antibiotic therapy.

Malaria predisposes children in areas where malaria is endemic to concurrent bacteremia, often with severe outcomes. The importance of bacterial coinfections in patients diagnosed with malaria in nonendemic settings has, however, not been reported. A retrospective analysis of microbiology data was performed in 755 travelers diagnosed with malaria in Sweden. Bacterial cultures from blood and other locations were correlated to clinical outcome and antibiotic treatment. Blood cultures were drawn from 417 (55%) patients (88% of whom were >15 years old), and bacterial isolates of clinical relevance (Salmonella enterica serovar Enteritidis and Escherichia coli) were detected in 2 patients (0.3%). Cultures from other locations (mainly urine, nasopharyngeal, and fecal samples) were obtained from 44% of the patients with 4.9% positivity. Of the 38 patients given antibiotics, 47% had neither severe malaria nor positive cultures and/or radiology signs indicative of treatment. C-reactive protein levels were associated with bacterial infections but had only a fair predictive value. Bacterial coinfections are uncommon among travelers with malaria. These data suggest a weaker association between malaria and bacteremia than previously described in endemic settings and might indicate different patient populations with different pathophysiological mechanisms and microbial environments. The study supports a restrictive antibiotic policy in returning travelers with malaria.

Osteopontin but not parathyroid hormone-related protein predicts prognosis in human renal cell carcinoma.

OBJECTIVE: To evaluate the relationship between osteopontin (OPN) in serum and plasma and parathyroid hormone-related protein (PTHrP) in serum, plasma and tumour tissue, and to assess the prognostic impact of OPN and PTHrP in human renal cell carcinoma (RCC). MATERIAL AND METHODS: The study included 269 patients with RCC. In 189 patients, immunohistochemical (IHC) PTHrP tumour tissue expression was evaluated, and OPN and PTHrP in serum were assessed. In 80 patients, plasma OPN and PTHrP were analysed. Tumour type, TNM stage, nuclear grade and RCC-specific survival were also registered. In a sub-group, IHC expression of CD 31 was assessed. The prognostic information of the factors was analysed using uni- and multivariate analyses. RESULTS: The median OPN level was 2.3 times higher in plasma than in serum. Serum OPN was significantly higher in patients with papillary RCC compared to clear cell RCC and chromophobe RCC. Both serum and plasma OPN levels were positively correlated to TNM stage and nuclear grade. Multivariate analysis showed that serum and plasma OPN levels were independent prognostic factors for RCC-specific survival, along with TNM stage. Immunohistochemical expression of PTHrP associated to TNM stage but not to nuclear grade or serum OPN. Furthermore, IHC expression of PTHrP was positively correlated to serum PTHrP but inversely to tumour CD31 expression. Plasma PTHrP was increased in 20% of the patients and related to TNM stage but not to nuclear grade. Plasma OPN was significantly higher in patients with increased PTHrP levels, compared to those with normal levels. CONCLUSION: Plasma OPN levels differed between RCC types, and in clear cell RCC, both serum and plasma OPN levels were independent predictors of survival. We found no evidence for prognostic value related to circulating levels or the IHC expression of PTHrP.

Hypoxia-inducible factor-2alpha mRNA expression in human renal cell carcinoma.

BACKGROUND: Hypoxia-inducible factor (HIF)-2alpha is upregulated in hypoxia or by inactivation of the von Hippel-Lindau (VHL) tumour suppressor gene. In a number of malignancies, increased HIF-2alpha expression may indicate worse prognosis. The aim of this study was to evaluate the prognostic information of HIF-2alpha mRNA expression in renal cell carcinoma (RCC). MATERIAL AND METHODS: HIF-2alpha mRNA was quantified by real time polymerase chain reaction (rt-PCR) in tumour tissue samples from 202 patients. Samples from 50 corresponding kidney cortex tissue were analysed as controls. mRNA levels were evaluated in relation to tumour cell type, TNM stage, nuclear grade and disease specific survival. RESULTS: The levels of HIF-2alpha mRNA were significantly higher in 168 clear cell (c)RCC than in 23 papillary (p)RCC (p < 0.001) or 11 chromophobe (ch)RCC (p < 0.006). Among cRCC there was an inverse correlation between HIF-2alpha mRNA levels and TNM stage I and II-IV tumours (p=0.01), and nuclear grade (p = 0.006). After a median follow-up time of 99 months (range 34-247), 106 patients had died of RCC. No correlation of HIF-2alpha mRNA to survival was observed. A multivariate analysis of prognostic factors in cRCC showed that TNM stage alone was an independent predictor of prognosis; HIF-2alpha mRNA levels did not add further prognostic information. DISCUSSION: The results demonstrated that HIF-2alpha mRNA levels were higher in cRCC compared to pRCC and chRCC. Furthermore, HIF-2alpha mRNA levels were inversely related to TNM stage and nuclear grade in cRCC.

Laboratory comparison between cell cytotoxicity neutralization assay and ultrasensitive single molecule counting technology for detection of Clostridioides difficile toxins A and B, PCR, enzyme immunoassays, and multistep algorithms.

Diagnostic tests for Clostridioides difficile infection (CDI) lack either specificity (nucleic acid amplification tests) or sensitivity (enzyme immunoassays; EIAs). The performance of the Singulex Clarity® C. diff toxins A/B assay was compared to cell cytotoxicity neutralization assay. Testing was also performed using an EIA for glutamate dehydrogenase (GDH) and C. difficile toxins A and B (C. Diff Quik Chek Complete®), polymerase chain reaction (PCR) (BD MAX™ Cdiff Assay), and 2 multistep algorithms: algorithm 1 (discordant GDH/toxin results arbitrated by PCR) and algorithm 2 (PCR-positive samples tested with toxin EIA). The Clarity assay and PCR both had 97% sensitivity, while specificity was 100% for Clarity and 79% for PCR. Algorithm 1 yielded 41% discordant results, and both toxin EIA and algorithm 2 had 58% sensitivity. Median toxin concentrations, as measured by the Clarity C. difficile toxin assay, were 3590, 11.5, 0.4, and 0 pg/mL for GDH+/toxin+, GDH+/toxin-/PCR+, GDH+/toxin-/PCR-, and GDH-/toxin- samples, respectively (P < 0.001). The Clarity assay may offer a single-test solution for CDI.

Diversity of resistance mechanisms in carbapenem-resistant Enterobacteriaceae at a health care system in Northern California, from 2013 to 2016.

The mechanism of resistance in carbapenem-resistant Enterobacteriaceae (CRE) has therapeutic implications. We comprehensively characterized emerging mechanisms of resistance in CRE between 2013 and 2016 at a health system in Northern California. A total of 38.7% (24/62) of CRE isolates were carbapenemase gene-positive, comprising 25.0% (6/24) blaOXA-48 like, 20.8% (5/24) blaKPC, 20.8% (5/24) blaNDM, 20.8% (5/24) blaSME, 8.3% (2/24) blaIMP, and 4.2% (1/24) blaVIM. Between carbapenemases and porin loss, the resistance mechanism was identified in 95.2% (59/62) of CRE isolates. Isolates expressing blaKPC were 100% susceptible to ceftazidime-avibactam, meropenem-vaborbactam, and imipenem-relebactam; blaOXA-48 like-positive isolates were 100% susceptible to ceftazidime-avibactam; and metallo ß-lactamase-positive isolates were nearly all nonsusceptible to above antibiotics. Carbapenemase gene-negative CRE were 100% (38/38), 92.1% (35/38), 89.5% (34/38), and 31.6% (12/38) susceptible to ceftazidime-avibactam, meropenem-vaborbactam, imipenem-relebactam, and ceftolozane-tazobactam, respectively. None of the CRE strains were identical by whole genome sequencing. At this health system, CRE were mediated by diverse mechanisms with predictable susceptibility to newer ß-lactamase inhibitors.