Temocillin and meropenem to discriminate resistance mechanisms leading to decreased carbapenem susceptibility with focus on OXA-48 in Enterobacteriaceae

A temocillin minimal inhibitory concentration ≥ 128 mg/L combined with the results of meropenem double disc synergy testing was used to (i) discriminate carbapenemase production from other resistance mechanisms leading to decreased carbapenem susceptibility; and (ii) differentiate Ambler classes in carbapenemase-producing enterobacteriaceae (CPE). The suggested test algorithm discriminated all extended spectrum β-lactamase/AmpC from CPE isolates, which could further be divided correctly into Ambler classes A and B enzymes as well as OXA-48 in all cases. The algorithm is simple to implement as part of the daily routine in a standard microbiology laboratory with limited access to or resources for molecular biological tools.     

KPC-2 and OXA-48 carbapenemase-harbouring Enterobacteriaceae detected in an Austrian wastewater treatment plant

Multiresistant Enterobacteriaceae, like carbapenemase-producing strains, have their primary reservoir in medical institutions. They can also be found with increasing tendency in other reservoirs. One possible way for entrance of multiresistant Enterobacteriaceae into the environment is via waste water. The aim of the study was to screen isolates from a wastewater treatment plant for the presence of carbapenemase-producing Enterobacteriaceae. Three isolates harboured carbapenemase genes, one Klebsiella pneumoniae harboured KPC-2 and one K. pneumoniae and one Escherichia coli harboured OXA-48. This is the first report of carbapenemase-harbouring Enterobacteriaceae isolated outside medical institutions in Austria and the first detection of KPC-harbouring K. pneumonia MLST ST 1245.     

NDM-1, OXA-48 and OXA-181 carbapenemase-producing Enterobacteriaceae in Sultanate of Oman

Twenty-two carbapenem-resistant enterobacterial isolates were recovered from patients hospitalized between October 2010 and March 2011 at the Royal Hospital of Muscat, Sultanate of Oman. Eleven NDM-1, five OXA-48 and one NDM-1 plus OXA-181 producers of diverse ST types were recovered from clinical samples. All carbapenemase genes were located on self-conjugative plasmids and were nearly always associated with other resistance determinants, including extended-spectrum β-lactamases and the ArmA methylase encoding resistance to aminoglycosides. This work highlights the dissemination of NDM-1 and OXA-48-type producers in the Middle East.     

Comparison of the boronic acid disk potentiation test and cefepime-clavulanic acid method for the detection of ESBL among AmpC-producing Enterobacteriaceae

Purpose: Extended spectrum β-lactamase (ESBL) and AmpC β-lactamase are important mechanisms of betalactam resistance among Enterobacteriaceae. The ESBL confirmation test described by Clinical Laboratory Standards Institute (CLSI) is in routine use. This method fails to detect ESBL in the presence of AmpC. Therefore, we compared two different ESBL detection methods against the CLSI confirmatory test. Materials and Methods: A total 200 consecutive clinical isolates of Enterobacteriaceae from various clinical samples were tested for ESBL production using (i) CLSI described phenotypic confirmatory test (PCT), (ii) boronic acid disk potentiation test and (iii) cefepime–CA disk potentiation method. AmpC confirmation was done by a modified three-dimensional test. Results: Among total 200 Enterobacteriaceae isolates, 82 were only ESBL producers, 12 were only AmpC producers, 55 were combined ESBL and AmpC producers, 14 were inducible AmpC producers and 37 isolates did not harboured any enzymes. The CLSI described PCT detected ESBL-producing organisms correctly but failed to detect 36.3% of ESBLs among combined enzyme producers. The boronic acid disk potentiation test reliably detected all ESBL, AmpC, and combined enzyme producers correctly. The cefepime–CA method detected all ESBLs correctly but another method of AmpC detection has to be adopted. Conclusion: The use of boronic acid in disk diffusion testing along with the CLSI described PCT enhances ESBL detection in the presence of AmpC betalactamases.     

Comparison of disk diffusion,MIC test strip and broth microdilution methods for cefiderocol susceptibility testing on carbapenem-resistant enterobacterales

ObjectivesCefiderocol is a catechol-substituted cephalosporin dedicated to the treatment of infections caused by multidrug resistant gram-negative rods. Cefiderocol susceptibility testing might be complex. We compared cefiderocol susceptibility testing methods on a relevant collection of carbapenem-resistant Enterobacterales.MethodsCE-IVD (European CE marking required for all in vitro diagnostic (IVD)) broth microdilution (BMD) plate (ThermoFisher, Waltham, MA, USA) using regular Mueller-Hinton broth, MIC test strip (Liofilchem, Teramo, Italy), and disk diffusion (Liofilchem) were compared to a frozen BMD plate prepared with iron depleted Mueller-Hinton broth. First, a collection of 100 entirely sequenced carbapenem-resistant Enterobacterales was used to compare these methods. Then, a prospective comparison of disk diffusion and CE-IVD BMD was performed on 827 consecutive carbapenem non-susceptible Enterobacterales including 634 carbapenemase-producers.ResultsCompared to reference method, CE-IVD BMD plate gave 95.0% (95% CI, 88.8–97.9) categorisation agreement (CA), 2.8% (95% CI, 0.4–14.2) very major errors (VME), and 1.6% (95% CI, 0.3–8.7) major errors (ME) with high reproducibility. MIC strip gave only 63% (95% CI, 53.2–71.8) of CA and 94.9% (95% CI, 83.1–98.6) of VME due to critical underestimation of the MICs. Disk diffusion gave 77% (95% CI, 67.9–84.2) CA with additional 8% of the isolates within the area of technical uncertainty of 18–22 mm.Prospectively, disk diffusion gave 81.7% (95% CI, 79.0–84.2) CA, 23.3% (95% CI, 15.1–34.2%)VME, and 4.9% (95% CI, 3.6–6.7) ME. Additionally, 21.3% (95% CI, 18.6–24.2) of CRE were within the area of technical uncertainty.DiscussionCommercial CE-IVD BMD (ThermoFisher) is accurate for cefiderocol MIC determination in difficult-to-treat Enterobacterales whereas MIC test strip (Liofilchem), that was formulated for Pseudomonas aeruginosa only, should be avoided. Disk diffusion might be useful for screening, but many of these CRE have to be re-tested using BMD to assess definitive categorization.     

Comparison of five methods for detection of carbapenemases in Enterobacterales with proposal of a new algorithm

ObjectivesThe aim of this study was to evaluate the performance of five different carbapenemase tests and to develop an algorithm which will permit the detection of most common and rare carbapenemases in routine microbiology laboratories.MethodsThe immunochromatographic tests CARBA-5 (NG), RESIST-4 O.K.N.V. (Coris), the colorimetric β-CARBA (BioRad), a newly developed carbapenem-inactivation method (CIM) supplemented with zinc (zCIM), and the Xpert Carba-R (Cepheid) were challenged with a collection of 189 molecularly characterized Enterobacterales isolates, including 146 carbapenemase producers (CPE): VIM (n = 48), OXA-48-like (n = 40), NDM (n = 29), KPC (n = 13), IMI (n = 9), IMP (n = 9), OXA-58 (n = 2), and GES (n = 2).ResultsThe overall sensitivity/specificity values for the five carbapenemase detection tests were 84.2% (CI 77.6–89.2%)/100% (CI 91.8–100%) for RESIST-4, 88.2% (CI 82.1–92.4%)/100% (CI 91.8–100%) for CARBA-5, 88.2% (CI 82.1–92.4%)/100% (CI 91.8–100%) for Xpert Carba-R, 73.7% (CI 66.2–80.0%)/100% (CI 93.4–99.0%) for β-CARBA, and 97.4% (CI 87.9–99.6%)/97.7% (CI 87.9–99.6%) for zCIM. The four common carbapenemases (KPC, OXA-48-like, NDM, and VIM) were detected with ≥97.6% sensitivity by all tests except for β-CARBA (76.6% (CI 68.4–83.2%)). IMI and GES were only detected by zCIM (sensitivity 90.9% (CI 62.3–98.4%)). Based on these results a new algorithm was developed, consisting of an immunochromatographic assay as the first test followed by zCIM, which allows detection of 99.3% of all carbapenemases assessed.ConclusionsExcept for β-CARBA, all methods showed excellent sensitivity/specificity for the detection of the four most frequent carbapenemases. With the new algorithm, rare variants can also be detected. It is rapid, simple, and inexpensive and can be performed in any microbiology laboratory, as no PCR equipment is required.     

Multiplex PCR amplification assay for the detection of blaSHV, blaTEM and blaCTX-M genes in Enterobacteriaceae

Extended-spectrum beta-lactamases (ESBLs) are often mediated by (bla-)SHV, (bla)TEM and (bla)CTX-M genes in Enterobacteriaceae and other Gram-negative bacteria. Numerous molecular typing methods, including PCR-based assays, have been developed for their identification. To reduce the number of PCR amplifications needed we have developed a multiplex PCR assay which detects and discriminates between (bla-)SHV, (bla)TEM and (bla)CTX-M PCR amplicons of 747, 445 and 593 bp, respectively. This multiplex PCR assay allowed the identification of (bla-)SHV, (bla)TEM and (bla)CTX-M genes in a series of clinical isolates of Enterobacteriaceae with previously characterised ESBL phenotype. The presence of (bla)SHV, (bla)TEM and (bla)CTX-M genes was confirmed by partial DNA sequence analysis. Apparently, the universal well-established CTX-M primer pair used here to reveal plasmid-encoded (bla)CTX-M genes would also amplify the chromosomally located K-1 enzyme gene in all Klebsiella oxytoca strains included in the study.     

Rapid detection of tenuazonic acid in cereal and fruit juice using a lateral-flow immunochromatographic assay strip

A lateral-flow immunochromatographic assay (ICA) strip was developed for determination of tenuazonic acid (TeA) using both semi-quantitative and quantitative methods. The ICA strip was based on produced anti-TeA monoclonal antibody, and could be used for TeA analysis in cereal and fruit juice, with visual limits of detection of 1600?ng/g and 400?ng/mL, and cut-off values of 6400?ng/g and 3200?ng/mL, respectively. The calculated limits of detection values were 126.4?ng/g and 65.3?ng/mL for wheat and apple juice samples, respectively. The recovery rates ranged from 90% to 129%. In summary, the developed strip was effective for TeA detection and would be suitable for on-site detection and rapid screening of samples.     

Sensitive enzyme-linked immunosorbent assay for detection of Clostridium botulinum neurotoxins A, B, and E using signal amplification via enzyme-linked coagulation assay.

A new immunoassay amplification method has been applied to the measurement of toxins A, B, and E from Clostridium botulinum. The technique is a modified enzyme-linked immunosorbent assay (ELISA) which relies on the detection of sandwich complexes on microtiter plates by a solid-phase coagulation assay known as ELCA, or enzyme-linked coagulation assay. In the method, a coagulation activating enzyme (RVV-XA) isolated from the venom of Russell's viper is conjugated to affinity-purified horse antibodies specific for toxin type A, B, or E. Plates are coated with affinity-purified antibodies, and standard captag (capture-tag) protocols using labeled antibody are employed to bind the toxin from solution. Complexes are detected by adding a modified plasma substrate which contains all the coagulation factors mixed with alkaline phosphatase-labeled fibrinogen and solid-phase fibrinogen; deposition of solid-phase, enzyme-labeled fibrin on the solid phase is then a reflection of formation of toxin-RVV-XA-antibody complexes on the solid phase. Because of the ability to detect RVV-XA by this coagulation assay at concentrations < 0.1 pg/ml, it was possible to measure C. botulinum toxins A, B, and E at mouse bioassay levels (< 10 pg/ml, or < 0.07 pM) for both purified neurotoxin and crude culture filtrates obtained from strains known to produce appropriate single toxins. ELISA-ELCA should be applicable to measurement of toxins in most of the materials (contaminated food, blood, and excreta) for which the comparably sensitive mouse bioassay is currently employed. This method has the potential of broad application to the measurement of low concentrations of any antigen for which appropriate immunochemical reagents are available, in a color test format.     

Development of ic-ELISA and lateral-flow immunochromatographic assay strip for the detection of folic acid in energy drinks and milk samples

Folic acid (FA) is an important vitamin for human growth and development, especially for pregnant women. A sensitive, rapid, and accurate FA detection method is required to assess the nutritional quality and safety of foods. A monoclonal antibody against FA was prepared for the development of indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and lateral-flow immunochromatographic assay (ICA) strip. The 50% inhibitory concentration and limit of detection of ic-ELISA were 0.12 and 0.018?ng/ml, respectively. The visual limit of detection and cut-off values of the lateral-flow ICA strip were 0.5 and 2.5?ng/ml, respectively. Using the ICA strip, FA recovery rates were 89–98% from energy drinks and 73–87% for milk samples and were in good agreement with those obtained from the conventional microbiological assay method. Our developed methods are sensitive, convenient, effective, and suitable for on-site detection and rapid mass screening of food samples.     

Comparison of a new neuraminidase detection assay with an enzyme immunoassay, immunofluorescence, and culture for rapid detection of influenza A and B viruses in nasal wash specimens

The performance of a new, rapid, easy-to-perform assay based on neuraminidase enzyme activity for detection of influenza virus types A and B was compared to detection by culture, indirect immunofluorescence, and enzyme immunoassay in 479 nasal wash specimens from children with respiratory infections. Compared to isolation of influenza virus by culture, the neuraminidase assay had a sensitivity of 70.1%, specificity of 92.4%, positive predictive value of 76.3%, and negative predictive value of 89.9%. There was a higher sensitivity for the detection of influenza A virus (76.4%) than for influenza B virus (40.9%). Indirect immunofluorescence showed a sensitivity of 59.8% and specificity of 97% compared to culture isolation for detection of influenza A and B viruses. Enzyme immunoassay showed a sensitivity of 89.7% and specificity of 98.1% for the detection of influenza A alone. The quality of the nasal wash specimen had a significant effect on the detection of influenza virus by all of the assays. A strong response of the neuraminidase assay was more likely to represent a culture-confirmed influenza infection. This new rapid neuraminidase assay was useful for the detection of influenza A and B viruses in nasal wash specimens.     

Performance evaluation of a rapid molecular diagnostic,MultiCode based,sample-to-answer assay for the simultaneous detection of Influenza A,B and respiratory syncytial viruses

BackgroundClinical signs and symptoms of different airway pathogens are generally indistinguishable, making laboratory tests essential for clinical decisions regarding isolation and antiviral therapy. Immunochromatographic tests (ICT) and direct immunofluorescence assays (DFA) have lower sensitivities and specificities than molecular assays, but have the advantage of quick turnaround times and ease-of-use.ObjectiveTo evaluate the performance of a rapid molecular assay, ARIES FluA/B & RSV, using laboratory developed RT-PCR assays (LDA), ICT (BinaxNOW) and DFA.MethodsAnalytical and clinical performance were evaluated in a retrospective study arm (stored respiratory samples obtained between 2006–2015) and a prospective study arm (unselected fresh clinical samples obtained between December 2015 and March 2016 tested in parallel with LDAs).ResultsGenotype inclusivity and analytical specificity was 100%. However, ARIES was 0.5 log, 1–2logs and 2.5logs less sensitive for fluA, RSV and fluB respectively, compared to LDA. In total, 447 clinical samples were included, of which 15.4% tested positive for fluA, 9.2% for fluB and 26.0% for RSV, in both LDA and ARIES. ARIES clinical sensitivity compared to LDA was 98.6% (fluA), 93.3% (fluB) and 95.1% (RSV). Clinical specificity was 100% for all targets. ARIES detected 10.6% (4 fluA, 8 fluB, 11 RSV) and 26.9% (7 fluA, 3 fluB, 22 RSV) more samples compared to DFA and ICT, all confirmed by LDA.ConclusionAlthough analytically ARIES is less sensitive than LDA, the clinical performance of the assay in our tertiary care setting was comparable, and significantly better than that of the established rapid assays.     

Comparison of the direct fluorescence assay and real-time polymerase chain reaction for the detection of influenza virus A and B in immunocompromised patients

OBJECTIVE:

This study evaluated the diagnostic performance of two methods for the detection of influenza virus in immunocompromised transplant patients.

METHODS:

A total of 475 respiratory samples, 236 from patients in a hematopoietic stem cell transplantation program and 239 from kidney transplant patients, were analyzed by a direct fluorescence assay and the Centers for Disease Control real-time polymerase chain reaction protocol for influenza A and B detection.

RESULTS:

Influenza detection using either method was 7.6% in the hematopoietic stem cell transplant group and 30.5% in the kidney transplant patient group. Influenza detection by real-time polymerase chain reaction yielded a higher positive rate compared with fluorescence than that reported by other studies, and this difference was more pronounced for influenza A. The fluorescence assay sensitivity, specificity, positive and negative predictive values, and kappa coefficient were 17.6%, 100%, 1, 0.83, and 0.256, respectively, and lower detection rates occurred in the kidney transplant patients.

CONCLUSIONS:

The real-time polymerase chain reaction performance and the associated turnaround time for a large number of samples support the choice of this method for use in different routine diagnostic settings and influenza surveillance in high-risk patients.     

PCR 检测产 A、B、E、F 和 G 型肉毒神经毒素梭菌的神经毒素基因

肉毒神经毒素分为Ａ、Ｂ、Ｃ、Ｄ、Ｅ、Ｆ和Ｇ七个血清型，其中Ａ、Ｂ、Ｅ和Ｆ型引起人类肉毒中毒。为此，选取肉毒神经毒素的保守序列设计了一对简并引物，对１８株肉毒梭菌和８株相关梭菌进行了检测，表明该引物可特异扩增Ａ、Ｂ、Ｅ、Ｆ和Ｇ型肉毒梭菌及产Ｅ型肉毒神经毒素的丁酸梭菌，产物为２６４ｂｐ，敏感性达１０ｐｇ细菌ＤＮＡ。采有酚－氯仿抽提法、固相载体捕获法和热裂解法处理产毒菌株，结果表明热裂解法安全、简便、快速，所制模板扩增效果明显。因此，此ＰＣＲ方法可用于快速敏感地检测引起人类肉毒中毒的梭菌。     

Agreement between CBNAAT,liquid culture and line probe assay for detection of Mycobacterium tuberculosis and anti-tubercular drug resistance in extrapulmonary samples

PurposeCartridge based nucleic acid amplification test (CBNAAT) has been endorsed by the WHO as the screening test for diagnosing extrapulmonary tuberculosis (EPTB). In the present study we report the agreement between CBNAAT (Xpert MTB/RIF), liquid culture (LC) and line probe assay (LPA) for diagnosis of Mycobacterium tuberculosis and detection of drug resistance among EPTB cases.MethodsThe EP samples were subjected to CBNAAT (Xpert MTB/RIF, Cepheid, USA) and wherever possible, to LC (MGIT 960, Becton Dickinson, USA) followed sequentially by first line and second line-LPA (FL-LPA, SL-LPA, Hain Lifescience, Germany) on the isolates.ResultsTotal 566/4080 (13.9%) EP samples were detected positive for M. tuberculosis on CBNAAT. Aspirates from lymph nodes were most often positive (11/30; 36.6%), followed by pus (240/873; 27.5%) and CSF samples (166/104; 15.8%). The detection of M. tuberculosis was more in adults than children except in tissue biopsy samples. Rifampicin resistance was also higher among adults except CSF in which resistance was more in children. Total 185 of 566 (32.7%) CBNAAT positive and 770 of 3510 (21.9%) CBNAAT negative samples could be cultured of which 110/185 (59.4%) and 33/770 (4.3%) respectively turned positive. FL-LPA and SL-LPA of 143 culture isolates showed that 27 isolates had drug resistance, of which 3 (2.1%) were XDR, 11 (7.7%) were Pre-XDR (FQ) and 13 (9.1%) were MDR. Of these 27 resistant isolates, 12 were negative by CBNAAT and two were mislabeled as Rifampicin sensitive or indeterminate based on the unique RpoB gene mutation patterns on LPA. The positive and negative agreements between LC and CBNAAT for detection of M. tuberculosis were 67.1% and 92.7% respectively and between LPA and CBNAAT for rifampicin resistance detection were 98.9% and 92.9% respectively.ConclusionsFor EPTB, CBNAAT should be accompanied with LC wherever possible irrespective of the CBNAAT result.