Fatma BUDAK, Keçeli Sema ÖZCAN, Birsen MUTLU, Haluk VAHABOĞLU

Febril nötropenili hastalarda kandidaya özgül taq-man pzr'ın invaziv fungal infeksiyon tanısındaki değeri

Kocaeli Üniversitesi Tıp Fakültesi Hastanesi'nde izlenen, 3 gün antibakteriyel tedaviye yanıt vermeyen, European Organisation for Research and Treatment of Cancer/Mycosis Study Group (EORTC/MSG) kriterlerine göre olası ve yüksek olasılıklı fungal infeksiyonu düşünülen febril nötropenik hastalarda, Taq-Man Polimeraz Zincir Reaksiyonu yöntemi (Taq-Man PZR) ile Candida türlerinin varlığı araştırılmıştır. 2002-2004 yılları arasında 33 hastadan, 8'i pediatrik olmak üzere 34 serum örneği alınmıştır. Tüm mantar türlerinde ortak olan 5.8S ve 28S rDNA'ya yönelik primerler ve tüm Candida türlerini saptamaya yönelik probe (All-CAN-TET) kullanılmıştır. Serum örnekleri, primer ve SYBR Green I boyası kullanılarak gerçek zamanlı PZR metodu ile tekrar edilmiştir. Bu yöntem ile sadece bir (% 2.9) rabdomiyosarkomlu çocuk hastada Candida DNA'sı saptanmıştır. Hiç bir hastanın kan kültüründe üreme olmamıştır. İnvaziv kandidozun tanısında kültür ve serolojik testler ile birlikte hastalığın erken döneminde yapılan Candida özgün PZR testi prognoz ve tedavide yol gösterici olacaktır.

Anahtar Kelimeler:

Nötropeni, Duyarlılık ve özgüllük, Polimeraz zincir reaksiyonu, Candida albicans, Nöbetler, febril, Kültür ortamı

The value of Candida specific taq-man PCR in diagnosis of invaziv fungal infections of febrile neutropenic patients

In the febrile neutropenic patients who were not responsive to antibiotic therapy for three days and suspicious of probable and high risk fungal infection were investigated for the presence of Candida spp. by Polimerase Chain Reaction (PCR) according to European Organisation for Research and Treatment of Cancer/Mycosis Study Group (EORTC/MSG). Totally 34 serum samples (8 pediatric) from 33 febrile neutropenic patients followed in the University Hospital of Kocaeli University were collected from 2002 to 2004. Candida DNA was investigated by Taq-Man PCR using common fungal primers that detect 5.8S and 28s rDNA and a probe (ALL CAN-TET) that detects all Candida spp. All serum samples were also studied by real-time PCR using primer and SYBR Green I dye. Candida DNA was positive only in one (2.9 %) pediatric patient with rabdomyosarcoma. There was no growth in blood cultures of patients. In the diagnosis of invasive candidiasis, Candida spesific PCR tests which were carried out at early stages with culture and serologic tests will be helpful in prognosis and treatment.

Keywords:

Neutropenia, Sensitivity and Specificity, Polymerase Chain Reaction, Candida albicans, Seizures, Febrile, Culture Media,

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Alexander BD: Diagnosis of fungal infection: new technologies for the mycology laboratory, Transplant Infect Dis 2002;4(Suppl 3):32-7.
Ascioglu S, Rex JH, de Pauw B et al and Invasive Fungal Infections Cooperative Group of the European Organization for Research and Treatment of Cancer; Mycoses Study Group of the National Institute of Allergy and Infectious Diseases: Defining opportunistic invasive fungal infections in immunocompromised patients with cancer and hematopoietic stem cell transplants: an international concensus, Clin Infect Dis 2002;34(1):7-14.
Bougnoux M, Dupont C, Mateo J et al: Serum is more suitable than whole blood for diagnosis of systemic candidiasis by nested PCR, J Clin Microbiol 1999;37(4):925-30.
Brandt ME, Warnock DW: Laborator aspects of medical mycology, "Dismukes WE, Pappas PG, Sobel JD (eds): Clinical Mycology" kitabında s.1-22, Oxford University Press,New York (2003).
Einsele H, Hebart H, Roller G et al: Detection and identification of fungal pathogens in blood by using molecular probes, J Clin Microbiol 1997;35(6):1353-60.
Işık N, Ağaçfidan A, Ağırbaşlı H ve ark: Bağışıklık sistemi baskılanmış hastalarda real time polimeraz zincir reaksiyonu ile mantar infeksiyonlarmm tara ve takibi, İnfeksiyon Derg 2004;18(1):79-84.
Işık N, Mills K: Using PCR and Real-Time PCR (Light Cycler) for diagnosis and follow up of invasive fungal infections in patients with hematological malignancies and transplantation, Turk J Haematol 2003;20(2):63-8.
Jones ME, Fox AJ, Barnes AJ et al: PCR-ELISA for the early diagnosis of invasive pulmonary aspergillosis infection in neutropenic patients, J Clin Pathol 1998;51(9):652-6.
Klingspor L, Jalal S: Molecular detection and identification of Candida and Aspergillosis spp. from clinical samples using real time PCR, Clin Microbiol Infect 2006;12(8):745-53.
Loeffler J, Hebart H, Henke N, Schmidt K, Einsele H: Quantification and specification of fungal DNA in clinical specimens using the light cycler instrument, "Reischl U, Witter C, Cockerill F (eds): Rapid Cycle Real-Time PZR. Methods and Applications" kitabında s. 179-86, Springer Verlag, New York (2002).
Maaroufi Y, Ahariz N, Husson M, Crokaert F: Comparison of different methods of isolation DNA of commonly encountered Candida spp. and its quantitation by using a real time PCR-based assay, J Clin Microbiol 2004;42(7):3159-63.
McLintock LA, Jones BL: Advances in the molecular and serological diagnosis of invasive fungal infection in haemato-oncology patients, Br J Haematol 2004;126(3):289-97.
Morace G, Pagano L, Sanguinetti M et al: PCR-restriction enzyme analysis for detection of Candida DNA in blood from febrile patients with hematological malignancies, J Clin Microbiol 1999;37(6):1871-5.
Sandhu GS, Kleine BC, Stockman L, Roberts GD: Molecular probes for diagnosis of fungal infections, J Clin Microbiol 1995;33(11):2913-9.
Shin JH, Nolte FS, Morrison CJ: Rapid identification of Candida species from blood culture using a clinically useful PCR method, J Clin Microbiol 1997;35(6):1454-9.
Taylor JW, Geiser DM, Burt A, Koufopanou V: The evolutionary biology and population genetics underlying fungal strain typing, Clin Microbiol Rev 1999;12(1):126-46.
White PL, Archer AE, Barnes R: Comparison of non-culture based methods for detection of systemic fungal infections, with an emphasis on invasive Candida infections, J Clin Microbiol 2005;43(5):2181-7.
White TJ, Burns TD, Lee SB, Taylor JW: Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics, "Innis MA, Helfand DH, Sninsky JJ, White TJ (eds): PCR Protocols" kitabında s.315-22, Academic Press Inc, San Diego (1990).