POLYMERASE CHAIN REACTION IN SERUM FOR DIAGNOSIS OF ASPERGILLOSIS
Keywords:
Aspergillus, aspergillosis, PCR, serum, diagnosisAbstract
Introduction: Aspergillosis is a major threat for immunocompromised and critically ill patients. Early diagnosis and initiation of appropriate antifungal therapy are essential for favorable clinical outcome.
Aim: The aim of this study was to evaluate the performance of a PCR assay in serum, compared to conventional method, for the diagnosis of aspergillosis.
Material and methods: Specimens of 125 patients divided into 4 groups, classified according to clinical diagnosis and EORTC/MSG criteria, were analysed at the Institute of Microbiology and Parasitology, Faculty of Medicine, Skopje, with conventional and molecular methods, during a period of two years (2014-2016).
Results: Seventy-one isolates of Aspergillus were confirmed in BAL culture. Aspergillus was detected in 63.33% of patients with chronic aspergillosis, followed by 56.67% of cystic fibrosis patients, 51.43% of patients with primary immunodeficiency, and 43.33% of patients with prolonged stay in ICU. Sensitivity and specificity of BAL culture were: 64.29% and 100%, 59.09% and 100%, 54.55% and 12.5%, 100% and 54.17%, in the I, II, III and IV group, respectively. PCR performed in serum yielded the following results for sensitivity and specificity: 53.57% and 100% in group I, 36.36% and 100% in group II, 9.09% and 100% in group III, respectively. Serum PCR was not performed in the group with cystic fibrosis.
Conclusion: Our results indicate that PCR in serum could be a useful adjunct test for diagnosis of aspergillosis, along with results from conventional mycological method, so a timely antifungal treatment with a favorable clinical outcome, is achieved.
References
Geiser DM. Sexual structures in Aspergillus: morphology, importance and genomics. Med Mycol 2009; 47(Suppl 1): S21-S26. doi: 10.1080/13693780802139859.
Bongomin F, Gago S, Oladele RO, Denning DW. Global and Multi-National Prevalence of Fungal Diseases-Estimate Precision. J Fungi (Basel) 2017; 3(4): 57. doi: 10.3390/jof3040057.
Jenks JD, Hoenigl M. Treatment of Aspergillosis. J Fungi (Basel) 2018; 4(3): 98. doi: 10.3390/jof4030098.
Janssens I, Lambrecht BN, Van Braeckel E. Aspergillus and the Lung. Semin Respir Crit Care Med 2024; 45(1): 3-20. doi: 10.1055/s-0043-1777259.
Barton RC. Laboratory diagnosis of invasive aspergillosis: from diagnosis to prediction of outcome. Scientifica (Cairo) 2013; 2013: 459405. doi: 10.1155/2013/459405.
De Pauw B, Walsh TJ, Donnelly JP, Stevens DA, Edwards JE, Calandra T, 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. doi: 10.1086/588660.
White PL, Bretagne S, Klingspor L, Melchers WJ, McCulloch E, Schulz B, et al. Aspergillus PCR: one step closer to standardization. J Clin Microbiol 2010; 48(4): 1231-1240. doi: 10.1128/JCM.01767-09.
Imbert S, Gauthier L, Joly I, Brossas JY, Uzunov M, Touafek F, et al. Aspergillus PCR in serum for the diagnosis, follow-up and prognosis of invasive aspergillosis in neutropenic and nonneutropenic patients. Clin Microbiol Infect 2016; 22(6): 562.e1-8. doi: 10.1016/j.cmi.2016.01.027.
Ullmann AJ, Aguado JM, Arikan-Akdagli S, Denning DW, Groll AH, Lagrou K, et al. Diagnosis and management of Aspergillus diseases: executive summary of the 2017 ESCMID-ECMM-ERS guideline. Clin Microbiol Infect 2018; 24(Suppl 1): e1-e38. doi: 10.1016/j.cmi.2018.01.002.
Tashiro M, Takazono T, Izumikawa K. Chronic pulmonary aspergillosis: comprehensive insights into epidemiology, treatment, and unresolved challenges. Ther Adv Infect Dis 2024; 11: 20499361241253751. doi: 10.1177/20499361241253751.
Agarwal R, Sehgal IS, Muthu V, Denning DW, Chakrabarti A, Soundappan K, et al. Revised ISHAM-ABPA working group clinical practice guidelines for diagnosing, classifying and treating allergic bronchopulmonary aspergillosis/mycoses. Eur Respir J 2024; 63(4): 2400061. doi: 10.1183/13993003.00061-2024.
Skladny H, Buchheidt D, Baust C, Krieg-Schneider F, Seifarth W, Leib-Mösch C, et al. Specific Detection of Aspergillus Species in Blood and Bronchoalveolar Lavage Samples of Immunocompromised Patients by Two-Step PCR. J Clin Microbiol 1999; 37(12): 3865-3871. doi: 10.1128/jcm.37.12.3865-3871.1999.
Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. 2nd ed. Cold Spring Harbor Laboratory Press; 1989.
Walsh TJ, Anaissie EJ, Denning DW, Herbrecht R, Kontoyiannis DP, Marr KA, et al. Treatment of Aspergillosis: Clinical Practice Guidelines of the Infectious Diseases Society of America. Clin Infect Dis 2008; 46(3): 327-360. doi: 10.1086/525258.
Montagna MT, Lovero G, Coretti C, Martinelli D, Delia M, De Giglio O, et al. SIMIFF study: Italian fungal registry of mold infections in hematological and non-hematological patients. Infection 2014; 42: 141-151. doi: 10.1007/s15010-013-0539-3.
Eigl S, Hoenigl M, Spiess B, Heldt S, Prattes J, Neumeister P, et al. Galactomannan testing and Aspergillus PCR in same-day bronchoalveolar lavage and blood samples for diagnosis of invasive aspergillosis. Med Mycol 2017; 55(5): 528-534. doi: 10.1093/mmy/myw102 PMID:27744310.
Tashiro T, Izumikawa K, Tashiro M, Takazono T, Morinaga Y, Yamamoto K, et al. Diagnostic significance of Aspergillus species isolated from respiratory samples in an adult pneumology ward. Med Mycol 2011; 49(6): 581-587. doi: 10.3109/13693786. 2010.548084.
Mennink-Kersten MA, Ruegebrink D, Wasei N, Melchers WJ, Verweij PE. In vitro release by Aspergillus fumigatus of galactofuranose antigens, 1,3-beta-D-glucan, and DNA, surrogate markers used for diagnosis of invasive aspergillosis. J Clin Microbiol 2006; 44(5): 1711-1718. doi: 10.1128/JCM.44.5.1711-1718.2006.
Zarrinfar H, Mirhendi H, Makimura K, Satoh K, Khodadadi H, Paknejad O. Use of mycological, nested PCR, and real-time PCR methods on BAL fluids for detection of A. fumigatus and A. flavus in solid organ transplant recipients. Mycopathologia 2013; 176(5-6): 377-385. doi: 10.1007/s11046-013-9657-9.
Ader F. Invasive pulmonary aspergillosis in patients with chronic obstructive pulmonary disease: an emerging fungal disease. Curr Infect Dis Rep 2010; 12(6): 409-416. doi: 10.1007/s11908-010-0132-1.
Perfect JR, Cox GM, Lee JY, Kauffman CA, de Repentigny L, Chapman SW, et al. The impact of culture isolation of Aspergillus species: a hospital-based survey of aspergillosis. Clin Infect Dis 2001; 33(11): 1824-1833. doi: 10.1086/323900.
Imbert S, Meyer I, Palous M, Brossas JY, Uzunov M, Touafek F, et al. Aspergillus PCR in Bronchoalveolar Lavage Fluid for the Diagnosis and Prognosis of Aspergillosis in Patients With Hematological and Non-hematological Conditions. Front Microbiol 2018; 9: 1877. doi: 10.3389/fmicb.2018.01877.
Arvanitis M, Ziakas PD, Zacharioudakis IM, Zervou FN, Caliendo AM, Mylonakis E. PCR in diagnosis of invasive aspergillosis: a meta-analysis of diagnostic performance. J Clin Microbiol 2014; 52(10): 3731-3742. doi: 10.1128/JCM.01365-14.
Egger M, Jenks JD, Hoenigl M, Prattes J. Blood Aspergillus PCR: The Good, the Bad, and the Ugly. J Fungi 2020;6(1):18. doi:10.3390/jof6010018.
Hope WW, Walsh TJ, Denning DW. Laboratory diagnosis of invasive aspergillosis. Lancet Infect Dis 2005; 5(10): 609-622. doi: 10.1016/S1473-3099(05)70238-3.
Zhang S, Wang S, Wan Z, Que C, Li R, Yu J. Quantitative Real-Time PCR and Platelia Galactomannan Assay for the Diagnosis of Invasive Pulmonary Aspergillosis: Bronchoalveolar Lavage Fluid Performs Better Than Serum in Non-neutropaenic Patients. Mycopathologia 2016; 181(9-10): 625-629. doi: 10.1007/s11046-016-0024-5.
Simoneau E, Kelly M, Labbe AC, Roy J, Laverdière M. What is the clinical significance of positive blood cultures with Aspergillus sp in hematopoietic stem cell transplant recipients? A 23 year experience. Bone Marrow Transplant 2005; 35(3): 303-306. doi: 10.1038/sj.bmt.1704793.
Löffler J, Hebart H, Brauchle U, Schumacher U, Einsele H. Comparison between plasma and whole blood specimens for detection of Aspergillus DNA by PCR. J Clin Microbiol 2000; 38(10): 3830-3833. doi: 10.1128/JCM.38.10.3830-3833.2000.
Costa C, Costa JM, Desterke C, Botterel F, Cordonnier C, Bretagne S. Real-time PCR coupled with automated DNA extraction and detection of galactomannan antigen in serum by enzyme-linked immunosorbent assay for diagnosis of invasive aspergillosis. J Clin Microbiol 2002; 40(6): 2224-2227. doi: 10.1128/JCM.40.6.2224-2227.2002.
Garcia ME, Blanco JL, Caballero J, Gargallo-Viola D. Anticoagulants interfere with PCR used to diagnose invasive aspergillosis. J Clin Microbiol 2002; 40: 1567-1568. doi: 10.1128/JCM.40.4.1567-1568.2002.
Bernal-Martínez L, Gago S, Buitrago MJ, Gomez-Lopez A, Rodriguez Tudela JL, Cuenca-Estrella M. Analysis of performance of a PCR-based assay to detect DNA of Aspergillus fumigatus in whole blood and serum: a comparative study with clinical samples. J Clin Microbiol 2011; 49(10): 3596-3599. doi: 10.1128/JCM.00647-11
Halliday C, Hoile R, Sorrell T, James G, Yadav S, Shaw P, et al. Role of prospective screening of blood for invasive aspergillosis by polymerase chain reaction in febrile neutropenic recipients of haematopoietic stem cell transplants and patients with acute leukaemia. Br J Haematol 2006; 132(4): 478-486. doi: 10.1111/j.1365-2141.2005.05887.x.
Raad I, Hanna H, Sumoza D, Albitar M. Polymerase chain reaction on blood for the diagnosis of invasive pulmonary aspergillosis in cancer patients. Cancer 2002; 94(4): 1032-1036. PMID: 11920473.
Mengoli C, Cruciani M, Barnes RA, Loeffler J, Donnelly JP. Use of PCR for diagnosis of invasive aspergillosis: systematic review and meta-analysis. Lancet Infect Dis 2009; 9(2): 89-96. doi:10.1016/S1473-3099(09)70019-2.
