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Camila Loredana P. A. M. Bezerra, Barbara de Almeida Lessa Castro, Anna Sara Levin, Lindissy Luara Baldi,Ana Paula Cury, Flavia Rossi, Natashia Reese, Fernanda C. Lessa, Susan Bollinger,Matias Chiarastelli Salomão
ASM Journals - Microbiology Spectrum 8 August 2025
Abstract
Carbapenemase-producing carbapenem-resistant Enterobacterales (CP-CRE) poses a public health issue. Rapid detection of CP-CRE colonization is challenging; existing methods are either expensive or time-consuming. We evaluated an immunochromatographic test (ICT) for detecting carbapenemases directly from broth-enriched rectal swabs. One hundred intensive care patients provided 178 pairs of rectal swabs. One swab was tested using the GeneXpert Carba-R PCR assay; the other was inoculated into brain-heart infusion broth. After 4 and 6 h of incubation at 37°C, the broth was tested with the RESIST-5 O.K.N.V.I. ICT for Klebsiella pneumoniae carbapenemase (KPC), New Delhi metallo-β-lactamase (NDM), oxacillinase-48 (OXA-48), Verona integron-encoded metallo-β-lactamase (VIM), and imipenemase (IMP) carbapenemases. Broths were subcultured after overnight incubation, and recovered carbapenem-resistant Enterobacterales isolates were tested using GeneXpert Carba-R PCR. Sensitivity, specificity, and accuracy were calculated in comparison to culture positive for CP-CRE, confirmed by PCR for KPC, NDM, OXA-48, VIM, and IMP. Of the 178 swabs, 60 were culture positive for CP-CRE. After 6 h, the ICT demonstrated a sensitivity of 65%, specificity of 97.5%, and accuracy of 86.8%. Among heavily soiled swabs, sensitivity reached 81.8% for ICT after 6 h, and the specificity was 100%. The mean execution time for carbapenemase detection using ICT was reduced by 60 h compared to culture. The ICT after 6 h incubation offersoffersreduced execution time for detecting CP-CREs. This method may serve as a valuable rapid screening tool, especially in resource-limited settings.
https://journals.asm.org/doi/full/10.1128/spectrum.01313-25
Bošnjak Z ., Hasman H. , Hansen F. , Hammerum AM. , Roer L. , Jurić I. , Budimir A.
J Chemother. 2025 Feb;37(1):10-14
Abstract
Two Enterobacter hormaechei isolates harbouring three carbapenemase genes each, were isolated from two patients from different ICUs at University Hospital Centre Zagreb, Croatia, which is to our knowledge, the first report of triple carbapenemase (blaVIM-2, blaNDM-1, and blaOXA-48) co-existence in E. hormachei strains and also among Enterobacterales members in Croatia. Antimicrobial susceptibility testing showed susceptibility only to colistin and amikacin. The production of carbapenemases was phenotypically tested by immunochromatographic assay and confirmed by PCR. Detailed analysis by Whole Genome Sequencing (WGS) of short reads by Illumina and long reads by Oxford Nanopore Technologies (ONT) was additionally performed and showed that both isolates belonged to ST200. They were separated by 98 Single Nucleotide Polymorphisms (SNPs) having variations in the number of blaVIM-2 genes on the chromosome, the number of blaNDM-1 genes on the plasmid, non-identical blaNDM-1 plasmids, different plasmid content in general, and only one isolate carried a 94 kb prophage.
https://pubmed.ncbi.nlm.nih.gov/38741515/
Shahinda Rezk, Nourhan Ashraf, Hassan Heshmat, Gamal Elsawaf, Amel Elsheredy
Acta Microbiol Immunol Hung . 2024 Apr 4;71(2):140-147
Abstract
Bloodstream infections (BSIs) caused by multidrug-resistant bacteria are a critical life-threatening challenge which necessitates the urgency to trigger life-saving treatment in a timely manner. This study aimed to evaluate the time required for rapid detection of carbapenemase-producing Enterobacterales (CPE) directly from blood culture bottles to optimize empirical treatment of BSI, especially in pediatric and infant patients, using a cost-effective method. This study included 419 Gram-negative bacteria, of which Klebsiella pneumoniae and Escherichia coli were the most common CPE causing BSI in pediatric and neonatal patients. Phenotypic and genotypic resistance of the selected isolates (45 K. pneumoniae and 9 E. coli) were determined by VITEK-2 Compact system and PCR, respectively. BACT/ALERT bottles were spiked with isolates. Finally, colorimetric RESIST-BC assay and Vitek-2 compact system were evaluated for the rapid detection of carbapenem-resistant bacteria directly from positive blood culture bottles. All selected isolates were phenotypically resistant to carbapenems. PCR showed that blaNDM and blaOXA-48 were present in all isolates, blaVIM was present in 44.4%, while blaKPC and blaIMP were entirely absent. The RESIST-BC kit showed good agreement with PCR for blaNDM and blaOXA-48, demonstrating high sensitivity and specificity, but not with blaVIM. These findings point out that RESIST-BC assay demonstrated an exceptionally short detection time for CPE, completing all cases within the first hour after the blood culture bottles flagged positive. It is also superior in providing a clue for clinicians on antibiotic combinations that can be administered, depending on the type of β-lactamases detected, promptly and efficiently, with low expenses.
https://pubmed.ncbi.nlm.nih.gov/38573768/
Kalaivani R, Kali A, Surendran R, Sujaritha T, Ganesh Babu CP.
Indian J Med Microbiol. 2024 Jan-Feb;47:100530
Abstract
Purpose: The choice of antibiotics for treatment of Carbapenem-Resistant Enterobacterales (CRE) is increasing becoming limited due to co-expression of Metallo-beta-lactamases (MBL) along with other carbapenemases in these isolates. The study aimed to investigate the occurrence of CRE and to determine the in-vitro synergy and clinical outcomes of Ceftazidime-Avibactam and Aztreonam combination in CRE infections in adult Intensive Care Units (ICUs).
Methods: 79 CRE isolates recovered from adult ICUs during January to March 2023 were tested by O.K.N.V.I. RESIST-5, a lateral flow multiplex assay for rapid detection of OXA-48-like, NDM, IMP, VIM, and KPC carbapenemases. Ceftazidime-Avibactam MIC was determined by microbroth dilution method and in vitro synergy between Ceftazidime-Avibactam and Aztreonam was assessed by Modified E-test/disc diffusion method for these isolates.
Results: The study revealed 7.5 % occurrence of CRE in our hospital, with high occurrence of NDM (n = 42, 53.1 %) and OXA-48-like (n = 63, 79.7 %) carbapenemase. Production of more than one type of carbapenemases was found in 44 isolates. A total of 57 isolates (72 %) had Ceftazidime-Avibactam resistance and 44 of them displayed Ceftazidime-Avibactam and Aztreonam in-vitro synergy. Successful clinical outcome was observed in two patients who received Ceftazidime-Avibactam and Aztreonam combination therapy for 7 days or more.
Conclusions: Despite the preponderance of Ceftazidime-Avibactam resistant CRE expressing NDM and OXA-48-like carbapenemase in our hospital, 77.2 % of them displayed in-vitro synergy of Ceftazidime-Avibactam with Aztreonam. It emphasizes the potential therapeutic utility of this combination in CRE strains showing coproduction of MBL and serine carbapenemases. Greater therapeutic potential of Ceftazidime-Avibactam and Aztreonam combination was observed with extended duration of therapy. However, further clinical evidence is needed to establish the efficacy of this combination and consider other factors that influence treatment outcomes.
https://pubmed.ncbi.nlm.nih.gov/38246242/
Diac I, Neculai-Cândea L, Horumbă M, Dogăroiu C, Costescu M, Keresztesi AA.
Exp Ther Med. 2023 Nov 15;27(1):14.
Abstract
In recent years, the emergence of carbapenem-resistant strains has been increasing worldwide, including in Romania. Rapid tests for post-mortem examinations have been researched and currently have several applications. In the present study, we aimed to test the performance of O.K.N.V.I. RESIST-5 tests on impure post-mortem biological samples compared with a standard of pure cultures. When a death occurs during hospitalization and the issue of malpractice arises, the medico-legal practice would benefit from rapid tests applicable to post-mortem samples. Thus, detection and differentiation of the five targeted carbapenemases, namely oxacilinase-48, Klebsiella pneumoniae carbapenemase, New Delhi metallo-β-lactamase, Verona integron-encoded metallo-β-lactamase and imipenemase, could be useful in guiding sampling for third-party microbiological assessment and could also be an asset from an epidemiological standpoint. The present prospective and observational pilot study included medico-legal autopsy cases performed at Mina Minovici National Institute of Legal Medicine (Romania) between June and July 2022. A total of two sets of O.K.N.V.I. RESIST-5 tests were performed: Test I, which was performed on-site from biological samples obtained during autopsy; and Test II, which was performed on pure cultures after sample inoculation and incubation. Total of 39 O.K.N.V.I. RESIST-5 rapid tests were performed on 19 biological samples, at least one sample per case. The O.K.N.V.I. RESIST-5 tests performed on-site showed an overall sensitivity of 92.3% with a 100% specificity. The results obtained through rapid tests using post-mortem impure samples were comparable to the results obtained from sample cultures with good sensitivity and specificity. Through post-mortem screening for carbapenem resistance, it would be possible to narrow down the number of cases that require further bacteriological assessment.
https://pubmed.ncbi.nlm.nih.gov/38125340/
BERNABEU S., BONNIN RA., DORTET L.
42e Réunion interdisciplinaire de chimiothérapie anti-infectieuse 12-13 décembre, 2022 Paris.
Comparison of three lateral flow immunochromatographic assays
Yehouenou CL, Soleimani R, Kpangon AA, Simon A, Dossou FM, Dalleur O.
Trop Med Infect Dis. 2022 Aug 21;7(8):200.
Abstract
An alarming worldwide increase in antimicrobial resistance is complicating the management of surgical site infections (SSIs), especially in low-middle income countries. The main objective of this study was to describe the pattern of carbapenem-resistant bacteria in hospitalized patients and to highlight the challenge of their detection in Benin. We collected pus samples from patients suspected to have SSIs in hospitals. After bacterial identification by MALDI-TOF mass spectrometry, antimicrobial susceptibility was performed according to the Kirby-Bauer method. Carbapenem-resistant strains were characterized using, successively, the Modified Hodge Test (MHT), the RESIST-5 O.K.N.V.I: a multiplex lateral flow and finally the polymerase chain reaction. Six isolates were resistant to three tested carbapenems and almost all antibiotics we tested but remained susceptible to amikacin. Four (66.7%) of them harbored some ESBL genes (blaCTX-M-1 and blaTEM-1). The MHT was positive for Carbapenems but not for Pseudomonas aeruginosa and Acinetobacter baumannii. As surgical antimicrobial prophylaxis, five of the six patients received ceftriaxone. The following carbapenems genes were identified: bla OXA-48(33.3%, n = 2), blaNDM (33.3%, n = 2) and blaVIM (33.3%, n = 2). These findings indicate a need for local and national antimicrobial resistance surveillance and the strengthening of antimicrobial stewardship programs in the country.
https://pubmed.ncbi.nlm.nih.gov/36006292/
Hong J, Kang D, Kim D.
Antibiotics (Basel). 2021 Apr 19;10(4):460.
Abstract
The objective of this study was to evaluate the performance of the RESIST-5 O.K.N.V.I. assay for identifying these five common domestic carbapenemases among a large number of clinical isolates in South Korea. A total of 268 non-duplicated clinical isolates of gram-negative bacilli were included in this study as follows: 258 carbapenemase-producing (CP) strains (OXA-48-like, KPC, NDM, VIM, IMP, GES, OXA-23 and two or more carbapenemase producers) and 10 non-CP carbapenem-resistant Enterobacterales (non-CP CREs). Overall sensitivity and specificity were 98.4% and 100%, respectively. In addition, all non-targeted carbapenemase producers including GES and OXA-23 producers and non-CP CREs were correctly identified as negative results. There were only four discrepant cases in which three VIM carbapenemase producers and one NDM carbapenemase producer were not detected. The RESIST-5 O.K.N.V.I. assay as an in vitro diagnostic test for detecting five common carbapenemases provided rapid and accurate results in a short time, indicating that this method could provide an innovative solution for early detection, resulting in appropriate antimicrobial treatment in the clinical field.
https://pubmed.ncbi.nlm.nih.gov/33921669/
Bernabeu S, Bonnin RA, Dortet L.
J Antimicrob Chemother. 2022 Nov 15
https://pubmed.ncbi.nlm.nih.gov/36376016/
Gallah S, Villageois-Tran K, Godmer A, Arlet G, Rottman M, Benzerara Y, Garnier M.
J Clin Microbiol. 2021 May 19;59(6):e02973-20.
Abstract
The increasing incidence of carbapenemase-producing Gram-negative bacilli (C-PGNB) represents a major public health challenge. Rapid detection of digestive colonization with C-PGNB is fundamental to control their spread. We performed the validation of a rapid protocol for C-PGNB detection directly on rectal swabs. We developed a protocol combining enrichment by a rapid selective subculture of the rectal swab medium and realization of a Resist-4 O.K.N.V. K-SeT test on the bacterial pellet obtained. The limit of detection and performances of this protocol were validated in vitro on 52 C-PGNB strains spiked on a calibrated sample suspension and confirmed in clinical settings on 144 rectal swabs sampled from patients with C-PGNB digestive colonization (n = 48) and controls (patients with extended-spectrum beta-lactamase [ESBL] colonization [n = 48] and without carbapenemase/ESBL [n = 48]). The protocol detected, with 100% sensitivity, the presence of the 15 OXA-48-, 14 KPC-, 13 NDM-, and 10 VIM-producing GNB from 103 CFU/ml. The limit of detection was 2 × 102 CFU/ml. Among the 48 C-PGNB-containing rectal swabs of the validation cohort, 46 were accurately detected. False negative were observed for 1 NDM-producing Acinetobacter baumannii strain and 1 OXA-48-producing Escherichia coli strain. The 96 control swabs were negative. Sensitivity and specificity for C-PGNB detection were 97.7% (95% confidence interval [CI], 87.7 to 100) and 100% (95% CI, 96.2 to 100). The negative likelihood ratio was 0.04 (95% CI, 0.01 to 0.16). Considering a C-PGNB digestive colonization prevalence between 0.01% and 0.1%, positive and negative predictive values were 100%. Our protocol is a rapid and low-cost method detecting accurately the digestive colonization with carbapenemase-producing Enterobacteriaceae in 4 h without any requirement for specific equipment.
https://pubmed.ncbi.nlm.nih.gov/33789958/
Pitout JDD, Peirano G, Kock MM, Strydom KA, Matsumura Y
Clin Microbiol Rev. 2019 Nov 13;33(1):e00102-19
Abstract
Surveillance studies have shown that OXA-48-like carbapenemases are the most common carbapenemases in Enterobacterales in certain regions of the world and are being introduced on a regular basis into regions of nonendemicity, where they are responsible for nosocomial outbreaks. OXA-48, OXA-181, OXA-232, OXA-204, OXA-162, and OXA-244, in that order, are the most common enzymes identified among the OXA-48-like carbapenemase group. OXA-48 is associated with different Tn1999 variants on IncL plasmids and is endemic in North Africa and the Middle East. OXA-162 and OXA-244 are derivatives of OXA-48 and are present in Europe. OXA-181 and OXA-232 are associated with ISEcp1, Tn2013 on ColE2, and IncX3 types of plasmids and are endemic in the Indian subcontinent (e.g., India, Bangladesh, Pakistan, and Sri Lanka) and certain sub-Saharan African countries. Overall, clonal dissemination plays a minor role in the spread of OXA-48-like carbapenemases, but certain high-risk clones (e.g., Klebsiella pneumoniae sequence type 147 [ST147], ST307, ST15, and ST14 and Escherichia coli ST38 and ST410) have been associated with the global dispersion of OXA-48, OXA-181, OXA-232, and OXA-204. Chromosomal integration of bla OXA-48 within Tn6237 occurred among E. coli ST38 isolates, especially in the United Kingdom. The detection of Enterobacterales with OXA-48-like enzymes using phenotypic methods has improved recently but remains challenging for clinical laboratories in regions of nonendemicity. Identification of the specific type of OXA-48-like enzyme requires sequencing of the corresponding genes. Bacteria (especially K. pneumoniae and E. coli) with bla OXA-48, bla OXA-181, and bla OXA-232 are emerging in different parts of the world and are most likely underreported due to problems with the laboratory detection of these enzymes. The medical community should be aware of the looming threat that is posed by bacteria with OXA-48-like carbapenemases.
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