β LACTA test performance for detection of extended-spectrum β-lactamase-producing Gram-negative bacilli directly on bronchial aspirates samples: a validation study

ObjectivesIncidence of extended-spectrum β-lactamase-producing Gram-negative bacilli (ESBL-PE-GNB)-related infections is worryingly increasing worldwide. ESBL-PE-GNB detection directly on bronchial aspirate samples (BAS) performed for suspected pneumonia may help save empirical carbapenems. Our objectives were to optimize β-LACTA™ test (BLT) realization and evaluate BLT performance for ESBL-PE-GNB detection directly on BAS.MethodsWe studied BLT technical optimization using BAS of different matrix types spiked with increasing concentrations of CTX-M-15-producing Klebsiella pneumoniae; in vitro validation of BLT diagnostic performance on 17 ESBL enzymes, belonging to CTX-M, SHV, TEM, OXA, GES, VEB and PER groups; and clinical validation of BLT performance on 126 BAS prospectively collected from seven intensive care units.ResultsAfter optimization, BLT detected with 100% sensitivity the presence of CTX-M-15-producing K. pneumoniae spiked in sterile BAS for inoculums upon two or more GNB per field upon microscopic Gram staining examination (MGSE). The BLT accurately detected the 17 ESBLs tested at 10⁶ CFU/mL and all ESBLs except Pseudomonas aeruginosa–related OXA-14 at 10⁴ CFU/mL. Among the 126 BAS of the validation cohort, 21 (17%) gave positive BLT (ten in BAS positive and 11 in BAS negative on MGSE). All BLT-positive BAS grew with ESBL-PE-GNB, including five hyper-L2-producing Stenotrophomonas maltophilia strains. BLT detected ESBL-PE-GNB directly on clinical BAS positive for GNB on MGSE and/or growing with ≥10⁴ CFU/mL with 100% sensitivity, specificity, and positive and negative predictive values.ConclusionsBLT is an accurate tool for ESBL-PE-GNB detection directly on BAS. Further studies are needed to evaluate the impact of BLT-guided early antimicrobial de-escalation strategies.     

Clinical significance of infections caused by extended-spectrum β-lactamase-producing Enterobacteriaceae blood isolates with inducible AmpC β-lactamase

Few studies have investigated the clinical features and outcomes for extended-spectrum β-lactamase (ESBL)-producing Enterobacter spp., Citrobacter spp., Serratia spp., and Morganella morganii (ECSM) bloodstream infections. This study was performed to investigate the clinical features and outcomes for ESBL-producing ECSM bloodstream infections. Patients with ECSM bloodstream infection were enrolled from October 2006 to March 2008. Of 124 patients with ECSM bacteremia, 30 cases (24.2%) were ESBL-producing isolates. Immunosuppressive drugs use within 30 days (p=0.028), indwelling device at the time of bacteremia (p=0.042) and antibiotics use within 3 months (p=0.022) were independently associated with ESBL production in multivariate analysis. Overall 30-day mortality rate was 19.4% (24/124). When the 30-day mortality rate was evaluated, no significant difference was found between the ESBL group (16.6%; 5/30) and non-ESBL group (20.2%; 19/94). Hospitalization was longer in the ESBL group than in the non-ESBL group (65.4±92.8 vs. 32.9±37.8 days, respectively; p=0.007). The recent use of antibiotics (especially broad-spectrum cephalosporins and other β-lactam antibiotics) was an important risk factor for ESBL among ECSM bacteremia. ESBL production of ECSM isolates was not significantly associated with mortality but ESBL-producing organisms have an important impact on the duration of hospital stay and subsequent medical cost.     

Cefotaxime for the detection of extended-spectrum β-lactamase or plasmid-mediated AmpC β-lactamase and clinical characteristics of cefotaxime-non-susceptible Escherichia coli and Klebsiella pneumoniae bacteraemia

We investigated the performance of cefotaxime for the detection of extended-spectrum β-lactamase (ESBL) or plasmid-mediated AmpC β-lactamase (pAmpC) and the clinical characteristics of cefotaxime-non-susceptible Escherichia coli or Klebsiella pneumoniae (CTXNS-EK) bacteraemia. All of the consecutive bloodstream isolates between 2005 and 2010 in a Japanese university hospital were characterised using polymerase chain reaction (PCR). Risk factors and outcomes of CTXNS-EK were analysed by multivariate logistic regression analysis. We identified 58 CTXNS-EK (15.6%) from 249 E. coli and 122 K. pneumoniae. Cefotaxime with a minimum inhibitory concentration (MIC) of >1 μg/mL had a sensitivity of 98.3% and a specificity of 99.7% for the detection of ESBL or pAmpC. CTXNS-EK had increased from 4.5% in 2005 to 23% in 2009. Risk factors for CTXNS-EK were previous isolation of multidrug-resistant bacteria, use of oxyimino-cephalosporins or fluoroquinolones, and high Sequential Organ Failure Assessment (SOFA) score. Patients with CTXNS-EK bacteraemia less frequently received appropriate empirical therapy than patients with cefotaxime-susceptible EK bacteraemia (81% vs. 97%, p<0.001) and died within 30 days (21% vs. 5%, p=0.001). Using the current breakpoints of the Clinical and Laboratory Standards Institute (CLSI) or the European Committee on Antimicrobial Susceptibility Testing (EUCAST), cefotaxime alone can identify ESBL or pAmpC producers. CTXNS-EK is an important and increasingly prevalent bacteraemia pathogen.     

Detection of extended-spectrum β-lactamase in Enterobacter spp.--evaluation of six phenotypic tests

Extended-spectrum β-lactamases (ESBL) are plasmid-mediated enzymes that hydrolyze cephalosporins and monobactams. The lack of a standard method to detect ESBL in Enterobacter spp. has led to underestimating its frequency. The aim of this study was to evaluate ESBL detection in Enterobacter spp. By the double-disk synergy test (DDST) and combined disk test (CDT) assay using cefepime, cefotaxime, and ceftazime as substrates for ESBL, plus AmpC inhibitors in different associations. A total of 83 Enterobacter spp. ESBL and 31 non-ESBL Enterobacter spp. were tested, and a cutoff point ≥3?mm was defined using a receiver operating characteristic (ROC) curve for combined disc methods. All tests showed 100% specificity. The sensitivity was 89.2% for DDST and CDT without AmpC inibitor, 90.4% in the combined disc test in Mueller-Hinton agar containing phenylboronic acid (CDT-PBAA), and 94% in the combined disc test in Mueller-Hinton agar containing cloxacillin (CDT-CLXA). Cefepime was the best substrate, mainly when AmpC inhibitors were not used. However, superior results were achieved when all cephalosporins were evaluated together. In conclusion, to improve ESBL detection in Enterobacter spp., some modifications in phenotypic tests are needed, such as to reduce the distance between the discs to 20?mm in DDST, to use a cutoff point for ≥3?mm on the CDT, and to include a cefepime disk or an inhibitor of AmpC in all tests.     

Multi-centre evaluation of a phenotypic extended spectrum β-lactamase detection guideline in the routine setting

This study aimed to evaluate the routine setting performance of a guideline for phenotypic detection of extended spectrum β-lactamases (ESBLs) in Enterobacteriaceae, recommending ESBL confirmation with Etest or combination disc for isolates with a positive ESBL screen test (i.e. cefotaxime and/or ceftazidime MIC >1 mg/L or an automated system ESBL warning). Twenty laboratories submitted 443 Enterobacteriaceae with a positive ESBL screen test and their confirmation test result (74% Escherichia coli, 12% Enterobacter cloacae, 8% Klebsiella pneumoniae, 3% Proteus mirabilis, 2% Klebsiella oxytoca). Presence of ESBL genes was used as reference test. Accuracy of local phenotypic ESBL detection was 88%. The positive predictive value (PPV) of local screen tests was 70%, and differed per method (Vitek-2: 69%, Phoenix: 68%, disc diffusion: 92%), and species (95% K. pneumoniae-27% K. oxytoca). A low PPV (3%) was observed for isolates with automated system alarm but third-generation cephalosporin MICs <2 mg/L. Local ESBL confirmation had a PPV and negative predictive value (NPV) of 93% and 90%, respectively. Compared with centrally performed confirmation tests, 7% of local tests were misinterpreted. Combination disc was more specific than Etest (91% versus 61%). Confirmation tests were not reliable for P. mirabilis and K. oxytoca (PPV 33% and 38%, respectively, although NPVs were 100%). In conclusion, performance of Etests could be enhanced by education of technicians to improve their interpretation, by genotypic ESBL confirmation of P. mirabilis and K. oxytoca isolates with positive phenotypic ESBL confirmation, and by interpreting isolates with a positive ESBL alarm but an MIC <2 mg/L for cefotaxime and ceftazidime as ESBL-negative.     

Efficient direct extended-spectrum β-lactamase detection by multiplex real-time PCR: accurate assignment of phenotype by use of a limited set of genetic markers

The number and diversity of genes potentially complicate genetic approaches to the rapid detection of transmissible extended-spectrum β-lactamase genes. We developed a robust multiplexed real-time PCR assay based on targets identified in a prior survey and used this to detect relevant genes in 617 consecutive clinical isolates of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae.     

Comparison of isoelectric focusing and polymerase chain reaction for the detection of β-lactamases

Extended spectrum β-lactamases (ESBLs) have been observed in virtually all the species of family Enterobacteriaceae. Threat posed by antibiotic resistance because of ESBLs is more serious as a number of technical problems are associated with the detection of these enzymes. Although a number of detection methods have been designed for ESBLs, every method has its own benefits and shortcomings as well. In earlier days, isoelectric focusing (IEF) was used as the gold standard for ESBL detection. This study was undertaken to compare IEF with polymerase chain reaction, a method which has been extensively used for ESBL detection these days.     

A novel framework to compare the effectiveness of β-lactamase inhibitors against extended-spectrum β-lactamase-producing Enterobacteriaceae

ObjectivesExtended-spectrum β-lactamases (ESBLs) present a serious challenge in the treatment of Gram-negative bacterial infections. ESBLs mediate resistance to most β-lactams, which may be reversed with the addition of an active β-lactamase inhibitor (such as tazobactam, relebactam and avibactam). However, various ESBLs may exhibit different susceptibilities to these inhibitors, which could impact efficacy. We proposed a framework for comparing the efficacy of these inhibitors when combined with the same β-lactam.MethodsThree clinical isolates of Klebsiella pneumoniae harbouring CTX-M-15 and one Escherichia coli isolate with SHV-12 were examined. Piperacillin MICs were determined by broth dilution using escalating concentrations of tazobactam, relebactam and avibactam. The resulting MICs were characterized as response to inhibitor concentrations using an inhibitory sigmoid E_max model. Using the best-fit parameter values, the model was conditioned with fluctuating inhibitor concentrations to simulate instantaneous MIC_i profiles for each isolate–inhibitor pair. Using a simulated exposure of 4 g piperacillin every 8 h, %fT > MIC_i was estimated for each piperacillin/inhibitor combination. A hollowfibre infection model was subsequently used to validate the predicted effectiveness of selected combinations.ResultsIn all scenarios, piperacillin MIC reductions were well characterized with increasing inhibitor concentrations. As predicted by %fT > MIC_i, combining piperacillin with avibactam (61.4%–73.6%) was found to be superior to tazobactam (13.5%–44.5%) for suppressing bacterial growth over time.ConclusionWe illustrated a practical approach to compare the performance of different inhibitors. This platform may be used clinically to identify the optimal pairing of various β-lactams and β-lactamase inhibitors for individual isolates producing ESBLs.     

Multi-center evaluation of the VITEK® MS system for mass spectrometric identification of non-Enterobacteriaceae Gram-negative bacilli

Studies have demonstrated that matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a rapid, accurate method for the identification of clinically relevant bacteria. The purpose of this study was to evaluate the performance of the VITEK MS v2.0 system (bioMérieux) for the identification of the non-Enterobacteriaceae Gram-negative bacilli (NEGNB). This multi-center study tested 558 unique NEGNB clinical isolates, representing 18 genera and 33 species. Results obtained with the VITEK MS v2.0 were compared with reference 16S rRNA gene sequencing and when indicated recA sequencing and phenotypic analysis. VITEK MS v2.0 provided an identification for 92.5 % of the NEGNB isolates (516 out of 558). VITEK MS v2.0 correctly identified 90.9 % of NEGNB (507 out of 558), 77.8 % to species level and 13.1 % to genus level with multiple species. There were four isolates (0.7 %) incorrectly identified to genus level and five isolates (0.9 %), with one incorrect identification to species level. The remaining 42 isolates (7.5 %) were either reported as no identification (5.0 %) or called “mixed genera” (2.5 %) since two or more different genera were identified as possible identifications for the test organism. These findings demonstrate that the VITEK MS v2.0 system provides accurate results for the identification of a challenging and diverse group of Gram-negative bacteria.     

Real-time polymerase chain reaction for rapid detection of genes encoding SHV extended-spectrum β-lactamases

Purpose: This study aimed to develop an improved method for the detection of bacterial SHV-type extended-spectrum β-lactamases (ESBLs). Materials and Methods: Our method was based on real-time polymerase chain reaction (PCR) in which the amplification of the product was monitored with a fluorescent probe. This method enabled the detection of bla_SHV genes with high degrees of sensitivity and specificity. Results: Based on ESBL phenotyping methods and bla gene DNA sequencing, we identified 240 bla genes from 662 Enterobacteriaceae isolated from clinical culture specimens. Of these 240 isolates, 26 had the blaSHV-28 genotype and three had the blaSHV-1 genotype. With our new real-time PCR assay, we detected 29 out of 29 bla_SHV genes in ESBL-producing isolates. Conclusion: This method represents a powerful tool for epidemiological studies of SHV ESBLs. Furthermore, it has potential for use in diagnostic microbiology.     

Frequency of extended spectrum β-lactamase producing urinary isolates of Gram-negative bacilli among patients seen in a multispecialty hospital in Vellore district,India

Extended-spectrum beta-lactamase (ESBL) producing strains of Coliform bacilli are on the rise and present a major threat especially in India. We assessed the frequency of ESBL producers among urinary isolates from patients presenting urinary tract infections. ESBL screening was done using Double Disk Synergy Test (DDST) and confirmed using E-test and Polymerase Chain Reaction (PCR). With E-test, 92.2% were positive for ESBL. In PCR, 100% strains were positive for any of the three gene targets tested. CTX-M was positive in majority of the strains followed by TEM and SHV. Two (3.22%) strains were positive for all the three genes; 21% strains were positive for both TEM and CTX-M genes. There was no statistically significant difference in the findings of E-test and PCR testing in the determination of ESBL producers (Fisher exact test P = 0.15). The strength of agreement between them was ‘fair’ (k = 0.252). Continuous monitoring of ESBL producers among Indian strains is important to rationalize the antibiotic policy to be followed.     

Comparison of BACTEC™ blood culture media for the detection of fungemia

The aim of the present study was to investigate whether addition of the BACTEC? Mycosis bottle to the standard BACTEC? aerobic and anaerobic bottles contributed to a higher detection rate and a faster time to detection (TTD) of fungi. This was a retrospective cohort study of all patients with a positive blood culture with Candida species delivered to the Department of Clinical Microbiology, Herlev and Gentofte Hospital, Denmark in the 8-year period 2006 through 2014. The patients had at least one BACTEC? aerobic and one Mycosis bottle sampled at the same time and at least one of the bottles yielded growth of fungi. Among 184 patients included, 173 were examined using BACTEC? aerobic, anaerobic and Mycosis bottles. The anaerobic vial generally had the lowest detection rate and the longest TTD. The detection rate of BACTEC? aerobic plus anaerobic with the BACTEC? Mycosis bottle was significantly higher than the detection rate of BACTEC? aerobic plus anaerobic without BACTEC? Mycosis bottle for all species after 1–5 days, and specially for Candida glabrata at 2, 3, 4 and 5 days. TTD for C. glabrata was significantly shorter for BACTEC? Mycosis than TTD for BACTEC? aerobic or anaerobic bottles after ½ to 4 days. When combining “first or only” detection, the BACTEC? Mycosis bottle had a significantly higher detection as compared to the aerobic bottle. Addition of the BACTEC? Mycosis bottle to the standard BACTEC? aerobic and anaerobic bottles significantly contributed to a higher detection rate and a faster TTD of fungemia.     

Propensity-matched analysis of the impact of extended-spectrum β-lactamase production on adults with community-onset Escherichia coli,Klebsiella species,and Proteus mirabilis bacteremia

Background

The presence of extended-spectrum β-lactamase (ESBL) in Escherichia coli, Klebsiella species, and Proteus mirabilis (EKP) is of great microbiological and clinical importance. The study dealing with the direct impact of ESBL producers on the outcome of patients with community-onset bacteremia is lacking.

Comparison of the f-HPV typing™ and Hybrid Capture II® assays for detection of high-risk HPV genotypes in cervical samples

Human papillomavirus genotyping is being considered in cervical screening programs and for monitoring the effectiveness of HPV vaccination. Both approaches require access to fast, easy and high-throughput technology. The aim of this study was to compare a new commercial assay (f-HPV typing?) with the Hybrid Capture II? (HC2) to detect HPV infection. The F-HPV typing is a multiplex fluorescent PCR method recognizing E6 and E7 regions of 13 high-risk (HR) HPV types, the same set of HR-types targeted HC2 test. A subset of 157 cervical samples was tested with both assays. The percentage of positive HR-HPV DNA samples was 24% (37/155) by HC2 and 33% (49/155) by f-HPV typing. Concordant results were found in 133/155 (overall agreement, 85.8%; Cohen's kappa=0.65). The analytical sensitivity and specificity of f-HPV were 97.6 and 93, respectively. In conclusion, this study shows that the f-HPV assay provides a good alternative to HC2 to detect HPV infection, allowing simple and rapid HPV genotyping and detecting multiple infections.     

Comparison of disc and MIC reduction methods with polymerase chain reaction for the detection of metallo-β-lactamase in Pseudomonas aeruginosa

Purpose: The present study was undertaken to evaluate the screening antibiotic, confirmatory phenotypic test and agent against PCR as gold standard and to detect the prevalent MBL gene. Materials and Methods: Three hundred and twenty-six Pseudomonas aeruginosa isolates were screened for resistance to Imipenem (IPM), Meropemem (MEM) and Ceftazidime (CAZ) by disc diffusion. Isolates resistant to any of these were considered screen test-positive for MBL and were subjected to Double disc synergy test (DDST) and Disc potentiation test (DPT: Using IPM, MEM and CAZ alone and with EDTA), Minimum inhibitory concentration (MIC) reduction [four-fold or more reduction in MIC of IPM and MEM in presence of chelators: EDTA and 1,10-phenanthroline (EPI/EPM: EDTA-phenanthroline- Imipenem/Meropenem Broth Microdilution method)] and polymerase chain reaction (PCR) for bla_IMP and bla_VIM. Results: Screen test-positives by MEM and CAZ were 19.3% as against 17.8% by IPM. MEMDDST, DPT and EPM confirmed 100% screen-test positives as against 93.7% by CAZ DDST and DPT-2, 76.2% by CAZ DPT-1, 88.9% by IPM DDST, 85.7% by IPM DPT-1 and 92.1% by EPI. IPMand CAZ DDST together confirmed 100% while IPM and CAZ DPT-2 confirmed 96.8%. All 63 screen-test positives showed the presence of bla_VIM. Conclusions: MEM was found to be the best screening and confirmatory agent for MBL detection and bla_VIM was found to be the prevalent MBL gene in this part of the country.     

Total internal reflection photoacoustic spectroscopy for the detection of β-hematin

Evanescent field sensing methods are currently used to detect many different types of disease markers and biologically important chemicals such as the HER2 breast cancer receptor. Hinoue et al. used Total Internal Reflection Photoacoustic Spectroscopy (TIRPAS) as a method of using the evanescent field to detect an optically opaque dye at a sample interface. Although their methods were successful at detecting dyes, the results at that time did not show a very practical spectroscopic technique, which was due to the less than typical sensitivity of TIRPAS as a spectroscopy modality given the low power (≈ 1 to 2 W) lasers being used. Contrarily, we have used an Nd:YAG laser with a five nanosecond pulse that gives peak power of 1 MW coupled with the TIRPAS system to increase the sensitivity of this technique for biological material sensing. All efforts were focused on the eventual detection of the optically absorbing material, hemozoin, which is created as a byproduct of a malarial infection in blood. We used an optically analogous material, β-hematin, to determine the potential for detection in the TIRPAS system. In addition, four properties which control the sensitivity were investigated to increase understanding about the sensor's function as a biosensing method.     

Phenotypic detection of plasmid-acquired AmpC in Escherichia coli—evaluation of screening criteria and performance of two commercial methods for the phenotypic confirmation of AmpC production

The phenotypic detection of plasmid-acquired AmpC (pAmpC) in Escherichia coli is challenging, and molecular methods are required for confirmation. In addition to cefoxitin resistance, multiresistance and high-level resistance to cephalosporins have both been suggested as criteria for targeting isolates with pAmpC, but data to support these proposed criteria are lacking. A Swedish collection of 378 isolates with either putative chromosomal hyperproduction of AmpC (cAmpC) or pAmpC were subjected to disk diffusion and minimum inhibitory concentration (MIC) determination with the Etest. The frequency of resistance to gentamicin, ciprofloxacin, and trimethoprim among cAmpC and pAmpC was compared to elucidate the issue of multidrug resistance. Lastly, methods for the phenotypic confirmation of pAmpC were compared. One in-house disk diffusion method, one method employing NeoSensitabs (Rosco), and one Etest method (bioMérieux) were compared. The analysis of histograms based on both disk diffusion and the Etest showed that resistance [according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST)] to cefotaxime and/or ceftazidime occurred in almost all isolates. By coining resistance instead of non-susceptibility, the number of isolates required to subject to phenotypic testing/genotypic confirmation dropped by more than 40 %, without compromising the sensitivity substantially. Further, almost 70 % of isolates with pAmpC were non-multidrug resistant, clearly indicating that this is an inappropriate criterion for further investigation. The phenotypic tests all had more than 90 % sensitivity, and the best sensitivities were obtained with the in-house method and with the ceftazidime ± cloxacillin NeoSensitab. In conclusion, clinical resistance to cefotaxime and/or ceftazidime seems to be an appropriate criterion for pAmpC screening, and several phenotypic methods perform well for the phenotypic confirmation of AmpC production prior to genotypic confirmation.     

Clinical characteristics of bacteraemia caused by extended-spectrum β-lactamase-producing Enterobacteriaceae in the era of CTX-M-type and KPC-type β-lactamases

Clin Microbiol Infect 2012; 18: 887-893 ABSTRACT: A multicentre, case-control study was conducted to assess risk factors and patient outcomes of bacteraemia caused by Enterobacteriaceae producing extended-spectrum β-lactamases (ESBLs) and Klebsiella pneumoniae carbapenemases (KPCs). One hundred and five and 20 patients with bacteraemia caused by ESBL-producing and KPC-producing organisms were matched to controls who had bacteraemia caused by non-ESBL/KPC-producing organisms, respectively. Independent risk factors for ESBL production included admission from a nursing home (OR 4.64; 95% CI 2.64-8.16), chronic renal failure (OR 2.09; 95% CI 1.11-3.92), the presence of a gastrostomy tube (OR 3.36; 95% CI 1.38-8.18), length of hospital stay before infection (OR 1.02; 95% CI 1.01-1.03), transplant receipt (OR 2.48; 95% CI 1.24-4.95), and receipt of antibiotics with Gram-negative activity in the preceding 30 days (OR 1.76; 95% CI 1.00-3.08). Twenty-eight-day crude mortality rates for patients infected with ESBL-producing or KPC-producing organisms and controls were 29.1% (34/117) and 19.5% (53/272), respectively (OR 1.70; 95% CI 1.04-2.80). On multivariate analysis, inadequate empirical therapy (OR 2.26; 95% CI 1.18-4.34), onset of bacteraemia while in the intensive-care unit (OR 2.74; 95% CI 1.47-5.11), Apache II score (OR 1.17; 95% CI 1.12-1.23) and malignancy (OR 2.66; 95% CI 1.31-5.41) were independent risk factors for mortality. CTX-M was the most common ESBL type in Escherichia coli, whereas SHV predominated in Klebsiella spp. and Enterobacter spp.