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001-es BibID:BIBFORM074742
Első szerző:Fidler Gábor (molekuláris biológus, genetikus)
Cím:Validation of a simplex PCR assay enabling reliable identification of clinically relevant Candida species / Fidler Gabor, Leiter Eva, Kocsube Sandor, Biro Sandor, Paholcsek Melinda
Dátum:2018
ISSN:1471-2334 1471-2334
Megjegyzések:Background: Fungal bloodstream infections (BSI) may be serious and are associated with drastic rise in mortality and health care costs. Candida spp. are the predominant etiological agent of fungal sepsis. The prompt and species-level identification of Candida may influence patient outcome and survival. The aim of this study was to develop and evaluate the CanTub-simplex PCR assay coupled with Tm calling and subsequent high resolution melting (HRM) analysis to barcode seven clinically relevant Candida species. Methods: Efficiency, coefficient of correlation and the limit of reliable detection were estimated on purified Candida EDTA-whole blood (WB) reference panels seeded with Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis, Candida krusei, Candida guilliermondii, Candida dubliniensis cells in a 6-log range. Discriminatory power was measured on EDTA-WB clinical panels on three different PCR platforms; LightCycler?96, LightCycler? Nano, LightCycler? 2.0. Inter- and intra assay consistencies were also calculated. Results: The limit of reliable detection proved to be 0.2?2 genomic equivalent and the method was reliable on broad concentration ranges (106 ?10 CFU) providing distinctive melting peaks and characteristic HRM curves. The diagnostic accuracy of the discrimination proved to be the best on Roche LightCycler?2.0 platform. Repeatability was tested and proved to be % C.V.: 0.14 ? 0.06 on reference- and % C.V.: 0.14 ? 0.02 on clinical-plates accounting for a very high accuracy. Reproducibility was % C.V.: 0.11 between reference- and % C.V.: 0.12between clinicalpanels which is highly acceptable. Conclusion: Our assay demonstrates recent advances on Tm calling and HRM analysis for the molecular identification of relevant Candida species. This unique, simplex PCR assay may be capable to outperform conventional phenotypic methods by reducing time and providing accurate and reliable results directly from blood (2 h) or from whole blood culture bottles (12?24 h).
Tárgyszavak:Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
Candida
High resolution melting
Tm calling
Species level identification
Simplex PCR
Megjelenés:BMC Infectious Diseases. - 18 : 393 (2018), p. 1-13. -
További szerzők:Leiter Éva (1976-) (biológus) Kocsubé Sándor Biró Sándor (1949-) (molekuláris genetikus) Paholcsek Melinda (1984-) (molekuláris biológus, genetikus)
Pályázati támogatás:GINOP-2.3.2-15-2016-00042
GINOP
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001-es BibID:BIBFORM059207
Első szerző:Paholcsek Melinda (molekuláris biológus, genetikus)
Cím:Combining standard clinical methods with PCR showed improved diagnosis of invasive pulmonary aspergillosis in patients with hematological malignancies and prolonged neutropenia / Melinda Paholcsek, Gabor Fidler, Jozsef Konya, Laszlo Rejto, Gabor Mehes, Evelin Bukta, Juergen Loeffler, Sandor Biro
Dátum:2015
ISSN:1471-2334
Megjegyzések:Background: We assessed the diagnostic value of standard clinical methods and combined biomarker testing(galactomannan assay and polymerase chain reaction screening) in a prospective case?control study to detectinvasive pulmonary aspergillosis in patients with hematological malignancies and prolonged neutropenia.Methods: In this observational study 162 biomarker analyses were performed on samples from 27 febrile neutropenicepisodes. Sera were successively screened for galactomannan antigen and for Aspergillus fumigatus specific nucleic acidtargets. Furthermore thoracic computed tomography scanning was performed along with bronchoscopy with lavagewhen clinically indicated. Patients were retrospectively stratified to define a case-group with "proven" or "probable"invasive pulmonary aspergillosis (25.93 %) and a control-group of patients with no evidence for of invasive pulmonaryaspergillosis (74.07 %). In 44.44 % of episodes fever ceased in response to antibiotic treatment (group II). Empiricalantifungal therapy was administered for episodes with persistent or relapsing fever (group I). 48.15 % of patientsdied during the study period. Postmortem histology was pursued in 53.85 % of fatalities.Results: Concordant negative galactomannan and computed tomography supported by a polymerase chainreaction assay were shown to have the highest discriminatory power to exclude invasive pulmonary aspergillosis.Bronchoalveolar lavage was performed in 6 cases of invasive pulmonary aspergillosis and in 15 controls. Althoughbronchoalveolar lavage proved negative in 93 % of controls it did not detect IPA in 86 % of the cases. Remarkablypost mortem histology convincingly supported the presence of Aspergillus hyphae in lung tissue from a single casewhich had consecutive positive polymerase chain reaction assay results but was misdiagnosed by both computedtomography and consistently negative galactomannan assay results. For the galactomannan enzyme-immunoassay thediagnostic odds ratio was 15.33 and for the polymerase chain reaction assay it was 28.67. According to Cohen's kappaour in-house polymerase chain reaction method showed a fair agreement with the galactomannan immunoassay.Combined analysis of the results from the Aspergillus galactomannan enzyme immunoassay together with thosegenerated by our polymerase chain reaction assay led to no misdiagnoses in the control group.Conclusion: The data from this pilot-study demonstrate that the consideration of standard clinical methods combinedwith biomarker testing improves the capacity to make early and more accurate diagnostic decisions.
Tárgyszavak:Orvostudományok Klinikai orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
Invasive pulmonary aspergillosis
Biomarkers
Combination testing
Acute leukemia
Neutropenic fever
Megjelenés:BMC Infectious Diseases. - 15 : 1 (2015), p. 251. -
További szerzők:Fidler Gábor (1987-) (molekuláris biológus, genetikus) Kónya József (1964-) (szakorvos, klinikai mikrobiológus) Rejtő László (1963-) (belgyógyász, haematológus) Méhes Gábor (1966-) (patológus) Bukta Evelin Loeffler, Juergen Biró Sándor (1949-) (molekuláris genetikus)
Pályázati támogatás:SROP-4.2.2.B-15/1/KONV-2015-0001
Egyéb
TÁMOP-4.2.4.A/ 2-11/1-2012-0001
TÁMOP
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Intézményi repozitóriumban (DEA) tárolt változat
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