Performances of two different panfungal PCRs to detect mould DNA in formalin-fixed paraffin-embedded tissue: what are the limiting factors?

BMC Infectious Diseases

D Goldenberger²  R Frei²  M Battegay³  L Bubendorf¹  S Savič Prince¹  M Weisser³  B Babouee Flury³ 

[1] Division of Pathology, University Hospital Basel, Schönbeinstrasse 40, Basel, 4031, Switzerland;Division of Clinical Microbiology, University Hospital Basel, Petersgraben 4, Basel, 4031, Switzerland;Division of Infectious Diseases and Hospital Epidemiology, University Hospital Basel, Petersgraben 4, Basel, 4031, Switzerland

关键词: Beta-globin gene;    Analytical sensitivity;    Formalin-fixed paraffin-embedded tissue;    Invasive fungal disease;    Internal transcribed spacer (ITS);    Panfungal PCR;   

Others  :  1090566
DOI  :  10.1186/s12879-014-0692-z

 received in 2014-04-09, accepted in 2014-12-09,  发布年份 2014

【 摘 要 】

Background

Detection of fungal DNA from formalin-fixed, paraffin-embedded (FFPE) tissue is challenging due to degradation of DNA and presence of PCR inhibitors in these samples. We analyzed FFPE samples of 26 patients by panfungal PCR and compared the results to the composite diagnosis according to the European Organization for Research and Treatment of Cancer (EORTC) criteria. Additionally we analyzed the quality of human and fungal DNA and their level of age-dependent degradation, as well as the existence of PCR inhibition in these tissue samples.

Methods

We evaluated two 45-cycle panfungal PCR tests that target the internal transcribed spacer 2 (ITS2) as well as the ITS1-5.8S-ITS2 (ITS1-2) region. The PCRs were applied to 27 FFPE specimens from 26 patients with proven invasive fungal disease (IFD), and one patient with culture and histologically negative but PCR-positive fungal infection collected at our institution from 2003 to 2010. Quality of DNA in FFPE tissue samples was evaluated using fragments of the beta-globin gene for multiplex PCR, inhibition of PCR amplification was evaluated by spiking of C. krusei DNA to each PCR premix.

Results

In 27 FFPE samples the ITS2 PCR targeting the shorter fragment showed a higher detection rate with a sensitivity of 53.8% compared to the ITS1-2 fragment (sensitivity 38%). Significant time-dependent degradation of human DNA in FFPE sample extracts was detected based on partial beta-globin gene amplification which was not in correlation to successful panfungal PCR identification of fungal organisms. The analytical sensitivity of both assays compared with culture was 60 CFU/ml of a Candida krusei reference strain. The performance of the two tests in an Aspergillus proficiency panel of an international external quality assessment programme showed considerable sensitivity.

Conclusion

Panfungal diagnostic PCR assays applied on FFPE specimens provide accurate identification of molds in highly degraded tissue samples and correct identification in samples stored up to 7 years despite sensitivity limitations, mainly caused by partial PCR inhibition and DNA degradation by formalin.

【 授权许可】

   
2014 Babouee Flury et al.; licensee BioMed Central Ltd.

【 参考文献 】

• [1]Barnes RA: Early diagnosis of fungal infection in immunocompromised patients. J Antimicrob Chemother 2008, 61(Suppl 1):i3-i6.
• [2]Auberger J, Lass-Florl C, Ulmer H, Nogler-Semenitz E, Clausen J, Gunsilius E, Einsele H, Gastl G, Nachbaur D: Significant alterations in the epidemiology and treatment outcome of invasive fungal infections in patients with hematological malignancies. Int J Hematol 2008, 88(5):508-515.
• [3]Tarrand JJ, Lichterfeld M, Warraich I, Luna M, Han XY, May GS, Kontoyiannis DP: Diagnosis of invasive septate mold infections. A correlation of microbiological culture and histologic or cytologic examination. Am J Clin Pathol 2003, 119(6):854-858.
• [4]Greer CE, Wheeler CM, Manos MM: Sample preparation and PCR amplification from paraffin-embedded tissues. Genome Res 1994, 3:S113-S122.
• [5]Inadome Y, Noguchi M: Selection of higher molecular weight genomic DNA for molecular diagnosis from formalin-fixed material. Diagn Mol Pathol 2003, 12(4):231-236.
• [6]White TBT, Lee S, Taylor JW: Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. Academic Press, San Diego; 1990.
• [7]Cabaret O, Toussain G, Abermil N, Alsamad IA, Botterel F, Costa JM, Papon JF, Bretagne S: Degradation of fungal DNA in formalin-fixed paraffin-embedded sinus fungal balls hampers reliable sequence-based identification of fungi. Med Mycol 2011, 49(3):329-332.
• [8]Lau A, Chen S, Sorrell T, Carter D, Malik R, Martin P, Halliday C: Development and clinical application of a panfungal PCR assay to detect and identify fungal DNA in tissue specimens. J Clin Microbiol 2007, 45(2):380-385.
• [9]Munoz-Cadavid C, Rudd S, Zaki SR, Patel M, Moser SA, Brandt ME, Gomez BL: Improving molecular detection of fungal DNA in formalin-fixed paraffin-embedded tissues: comparison of five tissue DNA extraction methods using panfungal PCR. J Clin Microbiol 2010, 48(6):2147-2153.
• [10]Paterson PJ, Seaton S, McHugh TD, McLaughlin J, Potter M, Prentice HG, Kibbler CC: Validation and clinical application of molecular methods for the identification of molds in tissue. Clin Infect Dis 2006, 42(1):51-56.
• [11]Rickerts V, Khot PD, Myerson D, Ko DL, Lambrecht E, Fredricks DN: Comparison of quantitative real time PCR with Sequencing and ribosomal RNA-FISH for the identification of fungi in formalin fixed, paraffin-embedded tissue specimens. BMC Infect Dis 2011, 11:202. BioMed Central Full Text
• [12]Willinger B, Obradovic A, Selitsch B, Beck-Mannagetta J, Buzina W, Braun H, Apfalter P, Hirschl AM, Makristathis A, Rotter M: Detection and identification of fungi from fungus balls of the maxillary sinus by molecular techniques. J Clin Microbiol 2003, 41(2):581-585.
• [13]De Pauw B, Walsh TJ, Donnelly JP, Stevens DA, Edwards JE, Calandra T, Pappas PG, Maertens J, Lortholary O, Kauffman CA, Denning DW, Patterson TF, Maschmeyer G, Bille J, Dismukes WE, Herbrecht R, Hope WW, Kibbler CC, Kullberg BJ, Marr KA, Muñoz P, Odds FC, Perfect JR, Restrepo A, Ruhnke M, Segal BH, Sorrell JD, Sobel JD, Sorrell TC, Viscoli C, et al.: Revised definitions of invasive fungal disease from the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) Consensus Group. Clin Infect Dis 2008, 46(12):1813-1821.
• [14]Clinical microbiology procedures handbook, 3rd edn. USA: ASM Press Washington; 2010.
• [15]De Hoog GS, Guarro J, Gené J, Figueras MJ: Atlas of clinical fungi, 2nd and 3rd edn.
• [16]Hall L, Wohlfiel S, Roberts GD: Experience with the MicroSeq D2 large-subunit ribosomal DNA sequencing kit for identification of filamentous fungi encountered in the clinical laboratory. J Clin Microbiol 2004, 42(2):622-626.
• [17]Babouee B, Goldenberger D, Elzi L, Lardinois D, Sadowski-Cron C, Bubendorf L, Savic Prince S, Battegay M, Frei R, Weisser M: Prospective study of a panfungal PCR assay followed by sequencing, for the detection of fungal DNA in normally sterile specimens in a clinical setting: a complementary tool in the diagnosis of invasive fungal disease? Clin Microbiol Infect 2013, 19(8):E354-E357.
• [18]Goldenberger D, Perschil I, Ritzler M, Altwegg M: A simple “universal” DNA extraction procedure using SDS and proteinase K is compatible with direct PCR amplification. PCR Methods Appl 1995, 4(6):368-370.
• [19]Conen A, Weisser M, Hohler D, Frei R, Stern M: Hormographiella aspergillata: an emerging mould in acute leukaemia patients?Clin Microbiol Infect 2010.
• [20]Balajee SA, Gribskov JL, Hanley E, Nickle D, Marr KA: Aspergillus lentulus sp. nov., a new sibling species of A. fumigatus. Eukaryot Cell 2005, 4(3):625-632.
• [21]Provan AB, Hodges E, Smith AG, Smith JL: Use of paraffin wax embedded bone marrow trephine biopsy specimens as a source of archival DNA. J Clin Pathol 1992, 45(9):763-765.
• [22]Bretagne S, Costa JM, Marmorat-Khuong A, Poron F, Cordonnier C, Vidaud M, Fleury-Feith J: Detection of Aspergillus species DNA in bronchoalveolar lavage samples by competitive PCR. J Clin Microbiol 1995, 33(5):1164-1168.
• [23]Kuykendall JR, Bogdanffy MS: Efficiency of DNA-histone crosslinking induced by saturated and unsaturated aldehydes in vitro. Mutat Res 1992, 283(2):131-136.
• [24]Abuali MM, Posada R, Del Toro G, Roman E, Ramani R, Chaturvedi S, Chaturvedi V, LaBombardi VJ: Rhizomucor variabilis var. regularior and Hormographiella aspergillata infections in a leukemic bone marrow transplant recipient with refractory neutropenia. J Clin Microbiol 2009, 47(12):4176-4179.
• [25]Lagrou K, Massonet C, Theunissen K, Meersseman W, Lontie M, Verbeken E, Van Eldere J, Maertens J: Fatal pulmonary infection in a leukaemic patient caused by Hormographiella aspergillata. J Med Microbiol 2005, 54(Pt 7):685-688.
• [26]Verweij PE, van Kasteren M, van de Nes J, de Hoog GS, de Pauw BE, Meis JF: Fatal pulmonary infection caused by the basidiomycete Hormographiella aspergillata. J Clin Microbiol 1997, 35(10):2675-2678.
• [27]Conen A, Weisser M, Hohler D, Frei R, Stern M: Hormographiella aspergillata: an emerging mould in acute leukaemia patients? Clin Microbiol Infect 2011, 17(2):273-277.
• [28]Sipos R, Szekely AJ, Palatinszky M, Revesz S, Marialigeti K, Nikolausz M: Effect of primer mismatch, annealing temperature and PCR cycle number on 16S rRNA gene-targetting bacterial community analysis. FEMS Microbiol Ecol 2007, 60(2):341-350.
• [29]Balajee SA, Nickle D, Varga J, Marr KA: Molecular studies reveal frequent misidentification of Aspergillus fumigatus by morphotyping. Eukaryot Cell 2006, 5(10):1705-1712.
• [30]Alcazar-Fuoli L, Mellado E, Alastruey-Izquierdo A, Cuenca-Estrella M, Rodriguez-Tudela JL: Aspergillus section Fumigati: antifungal susceptibility patterns and sequence-based identification. Antimicrob Agents Chemother 2008, 52(4):1244-1251.
• [31]Alhambra A, Catalan M, Moragues MD, Brena S, Ponton J, Montejo JC, del Palacio A: Isolation of Aspergillus lentulus in Spain from a critically ill patient with chronic obstructive pulmonary disease. Rev Iberoam Micol 2008, 25(4):246-249.