Evaluation of three molecular carbapenemase tests : Eazyplex SuperBug complete B, Novodiag CarbaR plus , and Amplidiag CarbaR plus MCR

Background: Carbapenemase-producing Gram-negative bacilli, i.e., Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter, are of increased concern for the public health around the world. There is urgent need for rapid and accurate tests in order to provide correct treatment and to prevent bacterial spread in healthcare settings. Methods: The aim of this study was to evaluate three commercial multiplex carbapenemase tests with CE-IVD marking: Eazyplex SuperBug complete B (AmplexDiagnostics), Novodiag CarbaR+ (Mobidiag), and Amplidiag CarbaR+MCR (Mobidiag). All these tests recognize KPC, NDM, OXA-48/181 group, VIM, OXA-23 group, and OXA-24/40 group, and Novodiag CarbaR+ and Amplidiag CarbaR+MCR additionally recognize IMP, OXA-51 group (with promoter located within ISAbaI), OXA-58 group, and MCR, and Amplidiag CarbaR+MCR further recognizes GES (carbapenemase-type only). Results: The sensitivities and specificities of these tests with bacterial isolates were 100%. The sensitivity directly from clinical samples was 100%, but the specificity was lower, which is simply explained by the higher sensitivity of the molecular methods compared with culture method. Conclusions: Overall, these CE-IVD marked tests provide a good alternative in the detection of carbapenemase-producing organisms.Peer reviewe

Comparison of four commercial screening assays for the detection of blakpc, blandm, blaimp, blavim, and blaoxa48 in rectal secretion collected by swabs

The spread of carbapenem-resistant Enterobacteriaceae (CRE) has been enabled by the lack of control measures directed at carriers of multidrug-resistant organisms in healthcare settings. Screening patients for asymptomatic colonization on the one hand, and implementation of contact precautions on the other hand, reduces patient-to-patient transmission. Screening plates represents a relatively low-cost method for isolating CRE from rectal swabs; however, molecular assays have become widely available. This study compared the performance of four commercial molecular platforms in detecting clinically significant carbapenemase genes versus routine screening for CRE. A total of 1015 non-duplicated rectal swabs were cultured on a chromogenic carbapenem-resistant selective medium. All growing Enterobacteriaceae strains were tested for carbapenemase-related genes. The same specimens were processed using the following molecular assays: Allplex\u2122 Entero-DR, Amplidiag\uae CarbaR + MCR, AusDiagnostics MT CRE EU, and EasyScreen\u2122 ESBL/CPO. The prevalence of Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae detected by swab culture was 2.2%, while organisms producing oxacillinase (OXA)-48 and metallo-\u3b2-lactamases were infrequent. The cost of CRE-related infection control precautions, which must be kept in place while waiting for screening results, are significant, so the molecular tests could become cost-competitive, especially when the turnaround time is decreased dramatically. Molecular assays represent a powerful diagnostic tool as they allow the rapid detection of the most clinically relevant carbapenemases

Assessment of the performance of CHROMagar KPC and Xpert Carba-R assay for the detection of carbapenem-resistant bacteria in rectal swabs: First comparative study from Abu Dhabi, United Arab Emirates

© 2019 International Society for Antimicrobial Chemotherapy Objectives: The objective of this study was to evaluate the performance of CHROMagar™ KPC compared with Xpert® Carba-R assay for the detection of carbapenem-resistant bacterial isolates from rectal swabs. Methods: Rectal swabs were obtained from patients admitted to Cleveland Clinic Abu Dhabi (United Arab Emirates) over a period of 7 months and were screened for carbapenem resistance by either culture on CHROMagar KPC or carbapenemase production using the Xpert Carba-R molecular method. Further testing for carbapenem susceptibility of isolates recovered from CHROMagar KPC was performed using VITEK®2. Results: A total of 1813 rectal swabs were screened, of which 61 (3.4%) were positive for carbapenem resistance by either one or both methods. Both methods were equally efficient in detecting carbapenem resistance in 37/61 swabs (60.7%), mostly positive for Klebsiella pneumoniae (22 isolates), of which 40.9% (9/22) carried blaOXA-48-like and blaNDM. Xpert Carba-R assay detected 12 additional swabs with negative CHROMagar KPC culture and revealed additional carbapenemase-producing organisms carrying blaOXA-48-like and/or blaNDM. CHROMagar KPC recovered organisms in nine swabs not detected by the genotypic method, 44.4% of which were K. pneumoniae. Three swabs yielded false-positive results (carbapenem-susceptible organisms) by both methods. Sensitivity and specificity were, respectively, 75.4% and 99.8% for CHROMagar KPC and 80% and 99.8% for Xpert Carba-R. Conclusion: This comparative study of CHROMagar KPC versus Xpert Carba-R in rectal swabs showed a slightly higher sensitivity for the PCR-based method. Whilst CHROMagar KPC provides a less expensive screening method, Xpert Carba-R may be more accurate and faster

A Pooling Strategy for Detecting Carbapenem Resistance Genes by the Xpert Carba-R Test in Rectal Swab Specimens

Rapid and accurate detection of carriers of carbapenemase-producing organisms (CPO) in hospitalized patients is critical for infection control and prevention. This study aimed to evaluate a pooling strategy for the detection of carbapenem resistance genes (CRG) in multiple specimens using the Xpert Carba-R test. Two rectal swabs each were collected from 415 unique patients. One swab was tested by Carba-R on the five specimen-pooled strategy. The other swab was tested individually by culture followed by DNA sequence analysis for CRG as the reference. At the first 5:1 pooling testing, 22 of 83 pools were positive, which yielded 34 positives from individual specimens when positive pools were subsequently retested. All individual specimens in the 61 negative pools were retested as negative by Carba-R. Among the 34 Carba-R-positive samples, 30 and four were positive and negative, respectively, by culture and sequencing. The remaining 381 Carba-R-negative specimens were also negative by culture and sequencing. Overall sensitivity, specificity, positive predictive value, and negative predictive value of the 5:1 pooled screening were 100.0% (95% confidence interval [CI] = 85.9% to 100%), 99.0% (95% CI = 97.2% to 99.7%), 88.2% (95% CI = 71.6% to 96.2%), and 100.0% (95% CI = 98.8% to 100%), respectively. Using the 5:1 pooling strategy, our study completed CRG screening in 414 patients with 193 reagents with significant cost savings. The 5:1 pooling strategy using the Carba-R test showed a potential method for screening CRG from rectal swabs with good sensitivity and decreased cost

Rapid identification of carbapenemase-producing enterobacteriaceae: comparison of two cultivation methods

We evaluated the performance of chromID CARBA compared with direct plating onto MacConkey agar supplemented with meropenem disk (MCM) for the screening and detection of carbapenemase-producing Enterobacteriaceae (CPE) from rectal swabs. Sensitivity and specificity values were 89.9% and 98.7% for MCM, and 92.4% and 98.8% for chromID CARBA

Multisite Evaluation of Cepheid Xpert Carba-R Assay for Detection of Carbapenemase-Producing Organisms in Rectal Swabs.

Rapid identification of patients who are colonized with carbapenemase-producing organisms (CPO) is included in multiple national guidelines for containment of these organisms. In a multisite study, we evaluated the performance of the Cepheid Xpert Carba-R assay, a qualitative diagnostic test that was designed for the rapid detection and differentiation of the blaKPC, blaNDM, blaVIM, blaOXA-48, and blaIMP-1 genes from rectal swab specimens. A double rectal swab set was collected from 383 patients admitted at four institutions (2 in the United States, 1 in the United Kingdom, 1 in Spain). One swab was used for reference culture (MacConkey broth containing 1 mg/liter of meropenem and subcultured to a MacConkey agar plate with a 10-μg meropenem disk) and for sequencing of DNA obtained from carbapenem-nonsusceptible isolates for carbapenemase identification. The other swab was used for the Xpert Carba-R assay. In addition to the clinical rectal swabs, 250 contrived specimens (108 well-characterized CPO and 142 negative controls spiked onto negative rectal swabs) were tested. Overall, 149/633 (23.5%) samples were positive by the Xpert Carba-R assay. In 6 samples, multiple targets were detected (4 VIM/OXA-48, 1 IMP-1/NDM, and 1 NDM/KPC). The Xpert Carba-R assay detected 155 targets (26 IMP-1, 30 VIM, 27 NDM, 33 KPC, 39 OXA-48) within a time range of 32 to 48 min. The sensitivity, specificity, and positive and negative predictive values of the Xpert Carba-R assay compared to those of the reference culture and sequencing results were 96.6% (95% confidence interval [CI], 92.2% to 98.9%), 98.6% (95% CI, 97.1% to 99.4%), 95.3%, and 99.0%, respectively. The Cepheid Xpert Carba-R assay is an accurate and rapid test to identify rectal colonization with CPO, which can guide infection control programs to limit the spread of these organisms

Rectal screening of carbapenemase-producing Enterobacteriaceae: a proposed workflow

Abstract Objectives Active screening is a crucial element for the prevention of carbapenemase-producing Enterobacteriaceae (CPE) transmission in healthcare settings. Here, we proposed a culture-based protocol for rectal swab CPE screening that combines the detection of CPE and the identification of carbapenamase type. Methods The workflow integrates an automatic digital analysis of selective chromogenic media (WASPLab, Copan), with subsequent rapid tests for the confirmation of carbapenemase production (i.e. detection of Klebsiella pneumoniae KPC-specific peak by MALDI-TOF mass spectrometry; a multiplex immunochromatographic assay identifying the five commonest carbapenemase types). To in-depth evaluate the performance of this protocol, data about 21 162 rectal swabs submitted for CPE screening at the Microbiology of S. Orsola-Malpighi Hospital in Bologna were analyzed. Results Considering its ability to correctly segregate plates with/without Enterobacteriaceae, WASPLab Image Analysis Software showed globally a sensitivity and a specificity of 100% and 79.4%, respectively. Out of the plates with a bacterial growth (n = 901), 76.9% were found positive for CPE by MALDI-TOF (specific KPC-peak for K. pneumoniae) or by the immunochromatographic assay. Only 2.8% of KPC-positive K. pneumoniae strains were missed by the specific MALDI-TOF MS algorithm, being detected by the immunochromatographic assay. The mean turn-around-time needed from the sample arrival to the final report ranged between 18 to 24 hours, with a significant time saving compared to a manual reading. Conclusions This workflow proved to be fast and reliable, being particularly suitable for KPC-K. pneumoniae endemic areas and for high-throughput laboratories

Exploring the antibiotic resistance profile of clinical Klebsiella pneumoniae isolates in Portugal

While antibiotic resistance is rising to dangerously high levels, resistance mechanisms are spreading globally among diverse bacterial species. The emergence of antibiotic-resistant Klebsiella pneumoniae, mainly due to the production of antibiotic-inactivating enzymes, is currently responsible for most treatment failures, threatening the effectiveness of classes of antibiotics used for decades. This study assessed the presence of genetic determinants of β-lactam resistance in 102 multi-drug resistant (MDR) K. pneumoniae isolates from patients admitted to two central hospitals in northern Portugal from 2010 to 2020. Antimicrobial susceptibility testing revealed a high rate (>90%) of resistance to most β-lactam antibiotics, except for carbapenems and cephamycins, which showed antimicrobial susceptibility rates in the range of 23.5–34.3% and 40.2–68.6%, respectively. A diverse pool of β-lactam resistance genetic determinants, including carbapenemases- (i.e., blaKPC-like and blaOXA-48-like), extended-spectrum β-lactamases (ESBL; i.e., blaTEM-like, blaCTX-M-like and blaSHV-like), and AmpC β-lactamases-coding genes (i.e., blaCMY-2-like and blaDHA-like) were found in most K. pneumoniae isolates. blaKPC-like (72.5%) and ESBL genes (37.3–74.5%) were the most detected, with approximately 80% of K. pneumoniae isolates presenting two or more resistance genes. As the optimal treatment of β-lactamase-producing K. pneumoniae infections remains problematic, the high co-occurrence of multiple β-lactam resistance genes must be seen as a serious warning of the problem of antimicrobial resistance.This work was financially supported by: LA/P/0045/2020 (ALiCE), UIDB/00511/2020 and UIDP/00511/2020 (LEPABE), funded by national funds through FCT/MCTES (PIDDAC). This study was also supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of the UIDB/04469/2020 unit, LABELS—Associate Laboratory in Biotechnology, Bioengineering and Microelectromechanical Systems—LA/P/0029/2020, Inov4Agro—Associate Laboratory for Innovation, Capacity Building and Sustainability in Agri-Food Production—LA/P/0126/2020, and projects UIDB/04033/2020 and UIDP/04033/2020 (CITAB). The authors also thank FCT for the Ph.D. Fellowship SFRH/BD/138883/2018.info:eu-repo/semantics/publishedVersio

National survey of colistin resistance among carbapenemase-producing Enterobacteriaceae and outbreak caused by colistin-resistant OXA-48-producing Klebsiella pneumoniae, France, 2014

From January 2014 to December 2014, 972 consecutive non-replicate carbapenemase-producing Enterobacteriaceae isolates from colonised or infected patients were collected at the Associated French National Reference Centre as part of the French national survey on antimicrobial resistance. It included 577 Klebsiella spp. (59%), 236 Escherichia coli (24%), 108 Enterobacter spp. (11%), 50 Citrobacter spp. (5%), and a single Salmonella spp. isolate (0.1%). Of 561 K. pneumoniae isolates, 35 were found to be resistant to colistin (6.2%). PFGE analysis revealed a clonal outbreak involving 15 K. pneumoniae isolates belonging to sequence type ST11, recovered in a single hospital in the Picardie region in northern France. Those clonally related isolates showed variable levels of resistance to colistin, ranging from 4 to 64 mg/L. They harboured the blaOXA-48 carbapenemase gene and the blaCTX-M-15 extended-spectrum beta-lactamase gene. Among the 91 Enterobacter cloacae isolates, seven were resistant to colistin and produced different types of carbapenemases. Surprisingly, none of the E. coli and Citrobacter spp. isolates showed resistance to colistin. This national survey including carbapenemase-producing isolates recovered in 2014 reported a high rate of colistin resistance in K. pneumoniae and E. cloacae (6.2% and 7.7%, respectively) in France

Molecular characterization of antibiotic resistant bacteria in newly HIV diagnosed adults in a community setting in Tanzania. Implications for infection prevention and control in HIV : Antibiotic resistant bacteria, HIV, Community, Sub-Saharan Africa

Mennesker som lever med HIV-infeksjon i utviklingsland har økt risiko for å bli smittet med antibiotikaresistente bakterier. Høyere forbruk av antibiotika på grunn av HIV-relatert sykdom gir et seleksjonspress som kan føre til fremvekst av antibiotikaresistente bakterier. Bærerskap av antibiotikaresistente bakterier med virulensgener øker risikoen for alvorlige infeksjoner i denne populasjonen. Målet med avhandlingen var å anslå forekomsten av og karakterisere antibiotikaresistente bakterier som koloniserer voksne mennesker med nypåvist HIV-infeksjon i bydeler i Dar es Salaam, Tanzania. Resultatene av avhandlingen kan hjelpe å utvikle strategier for å forebygge og kontrollere spredning av, og infeksjoner med, antibiotikaresistente bakterier blant mennesker som lever med HIV. Penselprøver ble tatt fra rektum og fra nese og nasofarynks på voksne personer med nypåvist HIV-infeksjon og undersøkt for antibiotikaresistente bakterier, extended spectrum beta-lactamase produserende Enterobacterales (ESBLPE), karbapenemase-produserende Enterobacterales (CPE), meticillin-resistente Staphylococcus aureus (MRSA), og antibiotikaresistente pneumokokker (Streptococcus pneumoniae). Studien fant at bare 4% (22/537) av HIV-positive deltakere hadde MRSA i nese / nasofarynx. Disse MRSA-isolatene var ofte resistente mot gentamicin (95%), ciprofloksacin (91%) og erytromycin (82%), men sjeldnere resistente mot kotrimoksasol (9%). Vi fant at MRSA-isolatenes fenotypiske følsomhetsmønster stemte godt overens med funn av resistensgener og mutasjoner i bakteriene. Alle MRSA isolatene tilhørte CC8 og ST8-SCCmecIV MRSA klonene og var negative for panton-valentine leukocidin (PVL) og arginine catabolic mobile element (ACME) type 1. Ingen av ST8-SCCmecIV spa-t1476 MRSA klonene fra Tanzania var beslektet med den globalt spredde USA300 klonen. Studien viste at personer med nypåvist HIV-infeksjon hadde høy forekomst av rektalt bærerskap av ESBLPE (32.6%, 194/595), med overvekt av genotype CTX-M-15. Antibiotikabruk siste 4 uker og CD4 tall under 350 celler/μL var uavhengige risikofaktorer for fekalt bærerskap av ESBLPE. Vi fant karbapenemasen blaNDM-5 lokalisert på et IncX3 type plasmid i et E. coli ST2083 isolat fra rektalpensel fra en person med HIV-infeksjon. Isolatet hadde også 3 andre plasmider, IncFIA, IncFIB og Col(BS512). IncFA plasmidet bar gener som uttrykker resistens mot fluorokvinoloner, aminoglykosider, trimetoprim, sulfametoksazol, makrolider og tetrasykliner. Studien viste at pneumokokker fra nasofarynks på personer med HIV-infeksjon hadde høy forekomst av resistens mot penicillin (74%) og kotrimoksasol (71%), antibiotika som ofte brukes som behandling mot lungebetennelse i Tanzania. Videre var 26.3% av pneumokokkene multiresistente og kotrimoksasol-resistente pneumokokker hadde multiple mutasjoner i dihydrofolatreduktase genene. For å oppsummere fant vi at personer med nypåvist HIV-infeksjon i bydeler i Dar es Salaam ofte var bærere av antibiotikaresistente bakterier. Funnene setter søkelys på den store spredningen av antibiotikaresistente bakterier i Tanzania, ikke bare i sykehus, men også ute i samfunnet. Funn av E. coli med plasmidbårne karbapenemaser av type blaNDM-5 og flere andre resistensdeterminanter setter søkelys på behovet for å implementere intervensjoner for å hindre spredning av antibiotikaresistente bakterier.In developing countries human immunodeficiency virus infected individuals are at increased risk of acquiring antibiotic resistant bacteria. High frequency of antibiotic use associated with HIV related illness drives emergence of bacteria with antimicrobial resistance (AMR) due to selection pressure. The carriage of antibiotic resistant bacteria coupled with virulence genes increases the risk of severe infections in this population. The aim of this thesis was detection and characterization of antibiotic-resistant bacteria colonizing newly HIV diagnosed adults in a community setting in Tanzania. The findings could help in developing strategies for preventing and controlling the spread and infection of antibiotic resistant bacteria in HIV-positive individuals. Rectal and nasal/nasopharyngeal swabs collected from newly HIV diagnosed adults from the community were used for detection of antibiotic resistant bacteria including extended spectrum b-lactamase producing Enterobacterales (ESBLPE), carbapenemase producing Enterobacterales (CPE), methicillin-resistant Staphylococcus aureus (MRSA), and antibiotic-resistant Streptococcus pneumoniae. The study found that only 4% (22/537) of the HIV-positive participants carried MRSA in the nose/nasopharynx. These MRSA isolates were frequently resistant to gentamicin (95%), ciprofloxacin (91%), and erythromycin (82%), but less often to trimethoprim-sulfamethoxazole (9%). We found that the phenotypic susceptibility patterns of all MRSA isolates were highly concordant with genotypic findings (resistance genes and mutations in genome). All MRSA isolates belonged to the CC8 and ST8-SCCmecIV MRSA clone and negative for panton-valentine leukocidin (PVL) and arginine catabolic mobile element (ACME) type 1. All ST8-SCCmecIVspa-t1476 MRSA clones from Tanzania were unrelated to the globally successful USA300 clone. The study showed a high prevalence (32.6%, 194/595) of fecal carriage of ESBL-PE in newly HIV diagnosed adults in the community setting, and confirmed the predominance of the blaCTX-M-15 genotype. Antibiotic use in the last 4 weeks and CD4 count <350 cells/μL were independent risk factors for fecal carriage of ESBL-PE in this HIV-infected population. In this study, we detected blaNDM-5 carried on an IncX3 type plasmid in one E. coli ST2083 isolate obtained from an HIV-infected adult from the community setting in Tanzania. In addition, E. coli from the HIV-infected adult carried three more plasmid types; IncFIA, IncFIB and Col(BS512). The IncFA type plasmid was found to carry several genes conferring resistance against fluoroquinolone, aminoglycosides, sulfamethoxazole, trimethoprim, macrolides and tetracycline. The study found that Streptococcus pneumoniae colonizing the nasopharynx of HIV-infected adults displayed a high rate of resistance to penicillin (74%) and cotrimoxazole (71%), antibiotics commonly used as first line treatment in suspected bacterial pneumonia in Tanzania. Furthermore, 26.3% were multidrug-resistance (MDR) and cotrimoxazole-resistant Streptococcus pneumoniae had multiple mutations in the dihydrofolate reductase gene. In conclusion, we found that carriage of antibiotic resistant bacteria was common among newly diagnosed HIV-infected adults from community settings. This highlights the large-scale spread of these resistant bacteria in Tanzania, not only in hospitals, but also in the community as well. The detection of a blaNDM-5 producing E. coli carried on a plasmid with clusters of other resistance determinant genes calls for urgent implementation of intervention strategies to curb the spread of antimicrobial resistant bacteria.Doktorgradsavhandlin