Bead Array Direct rRNA Capture Assay (rCapA) for Amplification Free Speciation of Mycobacterium Cultures

Mycobacterium cultures, from patients suspected of tuberculosis or nontuberculous mycobacteria (NTM) infection, need to be identified. It is most critical to identify cultures belonging to the Mycobacterium tuberculosis complex, but also important to recognize clinically irrelevant or important NTM to allow appropriate patient management. Identification of M. tuberculosis can be achieved by a simple and cheap lateral flow assay, but identification of other Mycobacterium spp. generally requires more complex molecular methods. Here we demonstrate that a paramagnetic liquid bead array method can be used to capture mycobacterial rRNA in crude lysates of positive cultures and use a robust reader to identify the species in a direct and sensitive manner. We developed an array composed of paramagnetic beads coupled to oligonucleotides to capture 16 rRNA from eight specific Mycobacterium species and a single secondary biotinilated reporter probe to allow the captured rRNA to be detected. A ninth less specific bead and its associated reporter probe, designed to capture 23S rRNA from mycobacteria and related genera, is included as an internal control to confirm the presence of bacterial rRNA from a GC rich Gram variable genera. Using this rRNA capture assay (rCapA) with the array developed we were already able to confirm the presence of members of the M. tuberculosis complex and to discriminate a range of NTM species. This approach is not based on DNA amplification and therefore does not require precautions to avoid amplicon contamination. Moreover, the new generation of stable and cost effective liquid bead readers provides the necessary multiplexing potential to develop a robust and highly discriminatory assay

Etest® versus broth microdilution for ceftaroline MIC determination with Staphylococcus aureus: results from PREMIUM, a European multicentre study

Objectives: To compare the concordance of ceftaroline MIC values 24 by reference broth microdilution (BMD) and Etest (BioMérieux, France) for MSSA and MRSA isolates, respectively, in isolates from PREMIUM (D372SL00001), a European multi-centre study.  Methods: Ceftaroline MICs were determined by reference BMD and by Etest for 1,242 MSSA and MRSA from adult patients with community-acquired pneumonia or complicated skin and soft tissue infections collected between February and May 2012; tests were performed across six European laboratories. Selected isolates with ceftaroline resistance in broth (MIC >1 mg/L) were retested in three central laboratories to confirm their behaviour.  Results: Overall concordance between BMD and Etest was good, with >97% essential agreement and >95% categorical agreement. Nevertheless, 12 of the 26 MRSA isolates found resistant by BMD scored as susceptible by Etest, with MICs ≤1 mg/L, thus counting as very major errors, whereas only five of 380 MRSA found ceftaroline susceptible in BMD were mis-categorised as resistant by Etest. Twenty-one of the 26 isolates with MICs of 2 mg/L by BMD were then re-tested twice by each of three central laboratories: BMD MICs of 2 mg/L were consistently found for 19 of the 21 isolates. Among 147 Etest results for these 21 isolates (original plus six repeats per isolate) 112 were >1 mg/L.  Conclusions: BMD and Etest have good overall agreement for ceftaroline against Staphylococcus aureus; nevertheless, reliable Etest-based discrimination of the minority of ceftaroline-resistant (MIC 2 mg/L) MRSA is extremely challenging, requiring careful reading of strips, ideally with duplicate testing

Towards Multitarget Testing in Molecular Microbiology

Advantages of PCR assays over more conventional culture-based diagnostics include significantly higher sensitivities and shorter turnaround times. They are particularly useful when patient treatment has already been initiated or for specimens that may contain microorganisms that are slow-growing, difficult to culture, or for which culture methods do not exist. However, due to genome variability, single target testing might lead to false-negative results. This paper focuses on examples from our own experiences and the literature to provide insight into the limitations of single target testing in molecular biology. Lessons learned from these experiences include the careful design of diagnostic assays, preferably multitargeted, the importance of investigating the incidence and epidemiology of infection in detail, the frequent participation in appropriate quality assurance schemes, and the importance of continuous attentiveness by investigators when confronted with inconsistent results. In conclusion, multitargeted testing in microbiological molecular assays should be a rule

First Case of Staphylococcus pseudintermedius Infection in a Human

We present the first clinical report of a Staphylococcus pseudintermedius infection in a human. Biochemically, S. pseudintermedius can be easily misidentified as S. aureus. Therefore, the final microbiological identification requires the combination of phenotypic and genotypic tests

Are VIDAS® anti-HEV IgM and IgG assays fit for reliable diagnosis of hepatitis E virus infections? Comparison & case story telling

Objectives: Hepatitis E virus (HEV) genotype 3 is an emerging pathogen in developed countries. We evaluated the performance of two new serological assays for the detection of HEV, VIDAS (R) anti-HEV IgM and IgG. Methods: VIDAS (R) assays were performed on 77 clinical samples: 68 samples from patients suspected for HEV infection and 9 samples which previously tested positive for HEV IgM, IgG or HEV PCR. All samples were also tested using Wantai HEV assays. Cross-reactivity was assessed. To get a better view on the natural course of HEV infections, three clinical cases are described. Results: The concordance rate between VIDAS (R) and Wantai assays was good for HEV IgM (0.75,CI 0.52-0.98) and very good for HEV IgG (0.85,CI 0.72-0.98). Four samples tested borderline/positive with Wantai IgM but negative with VIDAS (R) IgM. All of these samples were HEV RNA negative, HEV IgG was positive in 2/4 samples. Five samples produced conflicting HEV IgG results. These tested positive with VIDAS (R) but negative with Wantai IgG. All five samples were HEV IgM and RNA negative. We detected no cross-reactivity. The clinical cases illustrate that HEV serology can still be negative in the very beginning of an acute infection. Conclusions: There is a good agreement between VIDAS (R) and Wantai anti-HEV IgM and IgG assays. Discrepant HEV IgM results probably reflect false positive Wantai IgM results (RNA-/IgG- samples) and longer-lasting positive Wantai IgM (RNA-/IgG+ samples). Discrepant HEV IgG results, could either represent resolved HEV infections (false negative Wantai IgG results) or false positive VIDAS (R) HEV IgG results

A model-based analysis of the predictive performance of different renal function markers for cefepime clearance in the ICU

Several population pharmacokinetic models for cefepime in critically ill patients have been described, which all indicate that variability in renal clearance is the main determinant of the observed variability in exposure. The main objective of this study was to determine which renal marker best predicts cefepime clearance. A pharmacokinetic model was developed using NONMEM based on 208 plasma and 51 urine samples from 20 ICU patients during a median follow-up of 3 days. Four serum-based kidney markers (creatinine, cystatin C, urea and uromodulin) and two urinary markers [measured creatinine clearance (CLCR) and kidney injury molecule-1] were evaluated as covariates in the model. A two-compartment model incorporating a renal and non-renal clearance component along with an additional term describing haemodialysis clearance provided an adequate description of the data. The Cockcroft-Gault formula was the best predictor for renal cefepime clearance. Compared with the base model without covariates, the objective function value decreased from 1971.7 to 1948.1, the median absolute prediction error from 42.4% to 29.9% and the between-subject variability in renal cefepime clearance from 135% to 50%. Other creatinine- and cystatin C-based formulae and measured CLCR performed similarly. Monte Carlo simulations using the Sanford guide dose recommendations indicated an insufficient dose reduction in patients with a decreased kidney function, leading to potentially toxic levels. The Cockcroft-Gault formula was the best predictor for cefepime clearance in critically ill patients, although other creatinine- and cystatin C-based formulae and measured CLCR performed similarly

An interlaboratory comparison of ITS2-PCR for the identification of yeasts, using the ABI Prism 310 and CEQ8000 capillary electrophoresis systems

<p>Abstract</p> <p>Background</p> <p>Currently, most laboratories identify yeasts routinely on the basis of morphology and biochemical reactivity. This approach has quite often limited discriminatory power and may require long incubation periods. Due to the increase of fungal infections and due to specific antifungal resistence patterns for different species, accurate and rapid identification has become more important. Several molecular techniques have been described for fast and reliable identification of yeast isolates, but interlaboratory exchangeability of identification schemes of molecular techniques has hardly been studied. Here, we compared amplified ITS2 fragment length determination by an ABI Prism 310 (Applied Biosystems, Foster City, Ca.) capillary electrophoresis system with that obtained by a CEQ8000 (Beckman Coulter, Fullerton, Ca.) capillary electrophoresis system.</p> <p>Results</p> <p>Although ITS2 size estimations on both systems differed and separate libraries had to be constructed for each system, both approaches had the same discriminatory power with regard to the 44 reference strains, identical identifications were obtained for 39/ 40 clinical isolates in both laboratories and strains from 51 samples were correctly identified using CEQ8000, when compared to phenotypic identification.</p> <p>Conclusion</p> <p>Identification of yeasts with ITS2-PCR followed by fragment analysis can be carried out on different capillary electrophoresis systems with comparable discriminatory power.</p

Detection of fastidious mycobacteria in human intestines by the polymerase chain reaction

SCOPUS: ar.jinfo:eu-repo/semantics/publishe