Evaluation of the NG-Test CARBA 5 Kit for Rapid Detection of Carbapenemase Resistant Enterobacteriaceae.

OBJECTIVE: We evaluated NG-Test CARBA 5, a new phenotypic carbapenemase detection assay, and compared it to the routine Xpert CARBA-R polymerase chain reaction assay. Furthermore, we tested the kit's performance after bacterial growth on 4 different solid media. METHODS: Seventy carbapenem resistant Enterobacteriaceae (CRE) isolates (60 were carbapenemase producers) were collected at the Poriya Baruch Padeh Medical Center. All isolates were grown on 4 types of agar media-BD BBL CHROMagar carbapenem resistant Enterobacteriaceae, BD CHROMagar Orientation, BD MacConkey II agar, and BD Trypticase Soy Agar II with 5% sheep blood-and were then subjected to NG-Test CARBA 5 kit analysis. RESULTS: The NG-Test CARBA 5 specificity was 100% for all 4 media. However, the sensitivity was higher when bacteria were grown on TSA with 5% sheep blood (98.3%) as compared with the Orientation medium (88.3%), the CPE medium (84.7%), and the MacConkey medium (83.6%). In addition, some of the carbapenemase mechanisms such as Verona Integron-Mediated Metallo-ß-lactamase were detected with low agreement levels in specific media but higher agreement levels in the other media. CONCLUSION: NG-Test CARBA 5 may enable faster detection of carbapenemase producing CRE, which will be of value for treatment adjustment and prevention control. However, the medium type on which the bacteria are grown affects kit sensitivity.

The diagnostic performance and accuracy of 3 molecular assays for the detection of Clostridium difficile in stool samples, compared with the Xpert® C. difficile assay.

This research compares the performance and diagnostic accuracy of three molecular tests for the detection of Clostridium difficile in stool samples, with the Xpert® C. difficile assay. Fifty-nine positive and twenty-five stool negative samples were analyzed by the BDmax™ Cdiff, the Simplexa C. difficile Direct, and the GenomEra™ C. difficile, and compared with the Xpert® C. difficile routinely used in our laboratory. The highest sensitivity was 94.9% for the BDmax™ Cdiff, followed by GenomEra™ C. difficile with 93.2%, and Simplexa™ C. difficile Direct with 89.8%. The specificities of all assays were 100%. GenomEra™ C. difficile had the highest retesting rate (12%). Simplexa™ C. difficile Direct benefits from both short hands-on time and total-turnaround time and 0% retesting. The differences in performance and accuracy between these three molecular assays are insignificant and all may be used as part of the routine algorithm for detection of C. difficile.

High Rates of Fosfomycin Resistance in Gram-Negative Urinary Isolates from Israel.

BACKGROUND: Increasing antimicrobial resistance is a major problem worldwide. Many urinary tract infection (UTI) isolates are resistant to all oral antimicrobial agents, necessitating intravenous treatment even for cystitis. Fosfomycin is a broad-spectrum antibiotic that achieves high and prolonged urinary drug concentration and is considered first-line treatment for uncomplicated cystitis. Our aim was to investigate fosfomycin susceptibility among urinary isolates and search for demographic or bacterial characteristics associated with fosfomycin nonsusceptibility. MATERIALS AND METHODS: This is a retrospective study of all Gram-negative urinary isolates at Padeh-Poriya Medical Center in northern Israel. A total of 1503 isolates were tested for fosfomycin susceptibility, as well as susceptibility to other antimicrobial agents, by VITEK2 system and disk diffusion testing. Demographic and clinical data were obtained from patient electronic files. RESULTS: A total of 1,503 isolates from patients' urine were included. Mean patient age was 64.6 years, 937 (62.3%) were female, 913 (60.7%) were Jews, and in 1,058 (70.4%) cases, the infection was community acquired; 28.1% were extended-spectrum beta lactamase (ESBL)-positive. A total of 1099 (73.1%) isolates were susceptible to fosfomycin. Fosfomycin nonsusceptibility was significantly correlated to year: 124 (20.7%) in 2015 versus 280 (30.9%) in 2016; patient age: 17.6% in patients ≤50 years versus 30% in patients >50 years; hospital-acquired UTI: 34.2% versus 23.8%; and presence of ESBL positivity: 31.1% in ESBL-positive versus 20.9% in ESBL-negative isolates (p for all <0.001). CONCLUSIONS: Fosfomycin nonsusceptibility among urine culture isolates is a worrisome phenomenon that is on the rise and is more often found in elderly patients, patients with nosocomial UTI, and isolates that are ESBL positive.

Cheap and rapid in-house method for direct identification of positive blood cultures by MALDI-TOF MS technology.

BACKGROUND: Rapid and accurate pathogen identification in blood cultures is very important for septic patients and has major consequences on morbidity and mortality rates. In recent years, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS)-based technology has become useful for highly specific and sensitive identification of bacteria and yeasts from clinical samples including sterile body fluids. Additional in-house methods enabled direct identification from blood cultures following various preparation protocols. METHODS: Blood culture (5 ml) was harvested from each positive bottle following growth identification by BACTEC™ FX system and transferred into a VACUETTE® Z Serum Sep Clot Activator tube containing an inert gel, which following centrifugation separates microorganisms from the blood cells. We used MALDI-TOF MS analysis for identification of microorganisms collected from the gel surface. RESULTS: Positive blood culture bottles (186) were collected. In comparison with the routine method, 99% (184/186) and 90% (168/186) of the isolates were correctly identified by the SepsiTyper kit and the in-house method, respectively. We found high concordance (Pearson coefficient = 0.7, p <  0.0001) between our in-house method and the SepsiTyper kit. Additionally, high correlation was found in sub-groups of identified bacteria, with Pearson coefficients of 0.77 (p <  0.0001), 0.67 (p <  0.0001), and 0.73 (p <  0.007) for Gram negative, Gram positive, and anaerobic bacteria, respectively. CONCLUSIONS: Our in-house method was found to be in good agreement with the SepsiTyper kit. Considering the low costs and the rapid and easy implementation of this procedure, we propose our in-house method for the direct identification of bacteria from blood cultures.

Elevated serum immunoglobulin A levels in patients with Clostridium difficile infection are associated with mortality.

The current research investigated immunoglobulin A levels in serum of patients with Clostridium difficile infection. Our main aim was to test whether immunoglobulin A (IgA) levels at the early stage of infection can predict disease severity of C. difficile infection. Fifty-four patients were enrolled in the study. Stool samples were collected and analyzed by PCR for detection of C. difficile. IgA levels were measured in serum samples that were collected from each patient immediately after receiving a positive PCR result. Additionally, data concerning epidemiologic and clinical characteristics of the patients was retrospectively collected from the medical records. Serum IgA levels were not associated with the severity of C. difficile infection. However, a positive correlation between IgA levels and mortality during hospitalization was found. Additionally, IgA levels correlated with C-reactive protein and white blood cell levels. Although we did not find a correlation between IgA levels and C. difficile infection severity, we could associate IgA levels with the severity of general immune response and to complications of the infection, i.e. mortality. We think that this could represent a compensatory mechanism to the damage caused by C. difficile. Considering serum IgA anti-inflammatory functions, it is possible the increase in IgA levels aimed to suppress the more intense inflammatory response in patients who died during hospitalization.

Antimicrobial susceptibility of Campylobacter jejuni and Campylobacter coli: comparison between Etest and a broth dilution method.

BACKGROUND: Campylobacter is a leading cause of foodborne gasteroenteritis worldwide. Antimicrobial susceptibility testing for Campylobacter spp. is not routinely performed by most clinical laboratories. However, the emergence of resistant isolates strengthens the importance of antimicrobial susceptibility testing and the critical need for epidemiologic surveillance. The aim of this study was to compare the efficacy of Etest and Sensititre kit (a broth microdilution method) as methods for susceptibility tests and the critical need for epidemiologic surveillance. The aim of this study was to compare the efficacy of Etest and Sensititre kit (a broth microdilution method) as methods for susceptibility testing of Campylobacter spp. to tetracycline, erythromycin, and ciprofloxacin. METHODS: Sixty-six Campylobacter isolates were collected from feces samples and subjected to susceptibility testing by Etest and Sensititre, a broth microdilution kit for tetracycline, erythromycin, and ciprofloxacin. Minimal inhibitory concentration (MIC) results of each method were determined and compared. RESULTS: Similar MIC interpretations for tetracycline, erythromycin, and ciprofloxacin were found in 97%, 98.5%, and 100% of the isolates, respectively, indicating a good level of agreement between Etest and Sensititre (p < 0.0001); additionally, the correlation between the two methods was highly significant for the three tested antibiotics (p < 0.0001). CONCLUSIONS: Both the broth microdilution and the Etest are reliable and convenient methods for testing antimicrobial susceptibility of Campylobacter spp. The Sensititre kit has the advantages of high availability and the automation.

Antimicrobial susceptibility of Clostridium difficile isolates in Israel.

OBJECTIVES: An increase of Clostridium difficile isolates with reduced susceptibility to various antimicrobial agents has been observed, including isolates that are non-susceptible to antibiotics that are routinely used for treatment of C. difficile, such as vancomycin and metronidazole. We determined the susceptibility rates of C. difficile isolates from hospitals in northern Israel to various antibiotics including tigecycline, which was not previously reported from Israel. METHODS: A total of 81 stool samples were collected from three hospitals in northern Israel from patients with C. difficile infection. Specimens were screened for BI/NAP1/027 ribotype, cultured, and sensitivity tests were performed for vancomycin, metronidazole, moxifloxacin, and tigecycline. Statistical tests were applied for analysing the differences in distribution of resistance between the different antibiotics and between BI/NAP1/027 and resistance of antibiotics. RESULTS: Reduced susceptibility was found among 6/81 isolates for vancomycin, 4/81 for metronidazole, and 17/81 for moxifloxacin. Only 1 isolate had reduced susceptibility to tigecycline, with a mean MIC of 0.05µg/mL. Reduced susceptibility to moxifloxacin was significantly associated with reduced susceptibility to vancomycin (p=0.016) and to metronidazole (p=0.0276), and reduced susceptibility to metronidazole was associated with reduced susceptibility to vancomycin (p=0.0259). Eight of 81 isolates (9.9%) were positive for BI/NAP1/027 ribotype and had significantly higher non-susceptibility rates to moxifloxacin and vancomycin compared with BI/NAP1/027 negative isolates (p<0.0001 and p=0.0113, respectively). CONCLUSIONS: We found higher non-susceptibility rates to vancomycin and metronidazole than most previous studies, while tigecycline resistance rates are very low in northern Israel, rendering it a potential agent for treating CDI.

Role of Single Procalcitonin Test on Admission as a Biomarker for Predicting the Severity of Clostridium difficile Infection.

Objective: To evaluate whether serum Procalcitonin (PCT) at the early stage of infection can serve as a potential biomarker for determining Clostridium difficile infection (CDI) severity. Methods: Fifty-four patients diagnosed with CDI were enrolled in the study. Serum samples were obtained within a median time of 24-48 h of the lab result for presence of C. difficile. PCT levels were measured by chemiluminescence immunoassay. Demographic, clinical, and prognostic data concerning the patients were retrospectively collected from medical records. The illness severity score was determined according to "Score indices for C. difficile infection severity." Results: We found that serum PCT levels were significantly higher in patients with moderate disease, compared to patients with mild disease (p = 0.0032). Additionally, PCT was correlated with mortality (p = 0.0002), white blood cell count (p = 0.019), and community-acquired disease (p = 0.0345). Conclusion: Early measurement of PCT may serve as a biomarker for early prediction of CDI severity, which is of great importance due to the high risk of complications and death.

Correlation between fecal calprotectin levels, disease severity and the hypervirulent ribotype 027 strain in patients with Clostridium difficile infection.

BACKGROUND: Clostridium difficile is the most common infectious etiology of nosocomial diarrhea. Fecal calprotectin (fc) is a sensitive marker of intestinal inflammation, found to be associated with enteric bacterial infections and inflammatory bowel disease. METHODS: We evaluated fc levels using a Chemiluminescent immunoassay method, in hospitalized patients with C. difficile infection (CDI) diagnosed by molecular stool examination and assessed correlation with virulent ribotype 027 strain infection, antibiotic susceptibility by gradient Etest strip performed on C. difficile colonies and clinical and laboratory measures of disease severity. Statistical analysis was performed for correlation of fc levels with clinical and laboratory parameters, disease severity and patient outcomes. RESULTS: Overall 29 patients with CDI were admitted at the Poria medical center in northern Israel, during June 2014-May 2015. Resistance to metronidazole was found in 3 (10.3 %) isolates and to vancomycin in 5 (17.2 %) isolates. Regarding patient outcomes, within 30 days of CDI diagnosis, recurrence of disease occurred in 10 (34.5 %) patients and 2 patients (6.9 %) died. Seven (24.1 %) isolates were C. difficile ribotype 027. Mean fc level was 331.4 µg/g (21-932). Higher fc levels were found in patients with C. difficile ribotype 027 (p < 0.0005). Fc levels were also correlated with elevated peripheral blood white cell count (p = 0.0007). A trend for higher fc levels was found in patients with a higher clostridium severity score index (p = 0.0633). No correlation was found between fecal calprotectin levels and age, sex, functional status, community versus hospital acquired CDI, antibiotic susceptibility, fever, and creatinine levels. CONCLUSIONS: Our study highlights the fact that fc has a potential role as a biomarker of disease severity and binary toxin producing ribotype associated disease.