Sains Malaysiana 39(2)(2010): 321–327

 

Penggunaan Kaedah Tindak Balas Rantai Polimerase dalam Penentuan Prognosis Infeksi Sistemik Kandidiasis pada Tikus

(Determining Prognosis of Systemic Candidiasis Infection in Rats with the Polymerase Chain Reaction Method)

 

Jacinta Santhanam*, Siti Azura Zainon, Chin Chook Fung & Faezah Shekh Abdullah

Jabatan Sains Bioperubatan, Fakulti Sains Kesihatan Bersekutu

Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Malaysia

 

Diserahkan: 28 April 2009 / Diterima: 4 Ogos 2009

 

ABSTRAK

 

Infeksi kulat sistemik yang paling kerap berlaku pada pesakit hospital adalah infeksi kandidiasis yang disebabkan oleh Candida spp. Pendiagnosan infeksi ini melalui pengkulturan dan ujian serologi mengambil masa ataupun kurang sensitif dan spesifik. Oleh itu, tindak balas rantai polimerase (PCR) yang mengesan DNA kulat telah diperkembangkan untuk mendapatkan diagnosis yang cepat dan tepat. Dalam kajian ini, keupayaan asai PCR ‘seminested’ untuk mengesan infeksi sistemik Candida albicans pada haiwan makmal telah ditentukan. Tikus dewasa Sprague-Dawley diinfeksi dengan C. albicans secara suntikan intravena sel yis tersebut. Darah tikus diperolehi setiap 3 atau 4 hari untuk ekstraksi DNA. Setiap minggu, 3 ekor tikus dikorbankan dan organ-organ dalamannya dikultur untuk memastikan kehadiran infeksi sistemik C. albicans. Tempoh kajian ini adalah selama 4 minggu (28 hari). PCR ‘seminested’ dijalankan ke atas sampel DNA dengan menggunakan primer fungus universal, iaitu ITS1 dan ITS3 serta primer spesifik C. albicans (CALB1). Produk PCR yang terhasil dikesan dengan elektroforesis gel agaros. Asai PCR ‘seminested’ berjaya mengesan DNA C. albicans di dalam sampel darah haiwan terinfeksi dari hari ke-2 sehingga hari ke-25 pos-infeksi secara tekal. Pengkulturan organ ginjal, hati dan limpa menunjukkan haiwan tersebut diinfeksi secara sistemik sehingga hari ke-21 pos-infeksi dan telah pulih (keputusan kultur negatif) pada hari ke-28 pos-infeksi. Kesimpulannya, kajian ini menunjukkan PCR ‘seminested’ adalah satu kaedah yang berupaya mengesan infeksi sistemik Candida albicans sepanjang tempoh infeksi. Oleh itu, asai PCR ini mempunyai nilai sebagai kaedah diagnosis yang berkesan dan juga mampu menentukan prognosis apabila memantau status infeksi.

 

Kata kunci: Candida; infeksi sistemik; prognosis; seminested

 

ABSTRACT

 

Candidiasis infections caused by the fungi Candida spp. are currently the most common systemic fungal infection in hospitalised patients. Diagnostic procedures involving culture or serological tests are either slow or lacking sensitivity and specificity. Thus polymerase chain reaction (PCR) assays which detect fungal DNA have been developed to provide a rapid, accurate diagnosis. In this study we evaluated a seminested PCR assay for the detection of systemic Candida albicans infection in laboratory animals. Adult Sprague-Dawley rats were infected with C. albicans by intravenous injection of yeast cells. Blood was collected from the animals every 3 to 4 days for extraction of DNA. Each week, 3 animals were sacrificed and their organs were cultured to confirm systemic C. albicans infection. The study period was 4 weeks (28 days). A seminested PCR was performed on DNA samples using universal fungal primers (ITS1 and ITS3) and a C. albicans specific primer (CALB1). The PCR product was detected by agarose gel electrophoresis. The PCR assay was able to detect C. albicans DNA in the blood samples of infected animals consistently from day 2 until day 25 post-infection. Organ culture of kidneys, liver and spleen revealed that the rats were systemically infected until day 21 post-infection and had recovered from infection (negative culture results) by day 28 post-infection. This study shows that the seminested PCR was an effective method to detect systemic C. albicans infection throughout the infection period. Therefore this assay is not only a diagnostic tool but has prognostic value as well for the monitoring of infection status.

 

Keywords: Candida; prognosis; seminested; systemic infection

 

RUJUKAN

 

Bougnoux, M.E., Dupont, L., Mateo, J., Saulnier, P., Faivre, V., Payen, D. & Nicholas-Chonoine, M.H. 1999. serum is more suitable than whole blood for diagnosis of systemic candidiasis by nested PCR. Journal of Clinical Microbiology 37: 925-930.

Chou, Q., Russell, M., Birch, D.E., Raymond, J. & Bloch, W. 1992. Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications. Nucleic Acids Research 20: 1717-1723.

Conant, Norman, F., Smith, D.T., Baker, R.D., Callaway, J.L. 1971. Manual of clinical mycology. Edisi ketiga. W.B. Saunders company.

Dupont, B. 1990. Clinical manifestations and management of candidosis in the compromised patients. 55-84. In Fungal infection in the compromised patient, edited by D.W. Warnode & M.D. Richardson. Edisi kedua. New York: John Wiley & sons, Inc.

Einsele, H., Hebart, H., Roller, G., Loffler, J., Rothenhofer, I., Muller C.A., Bowden, R.A., van Burik, J., Engelhard, S., Kanz, L. & Shumacher U. 1997. Detection and identification of fungal pathogens in blood using molecular probes. Journal of Clinical Microbiology. 35: 1353-1360.

Emmons, C.W., Binford, C.H. & Utz, J.P. 1971. Medical Mycology. Ed. ke 2. Lea & Frebiger.

Ferrer, C., Colom, F., Frases, S., Mulet, E., Abad, J.L. & Alio, J.L. 2001. Detection and identification of fungal pathogens by PCR and by ITS2 and 5.8S ribosomal DNA typing in ocular infections. Journal of Clinical Microbiology 39: 2873-2879.

Holmes, A.R., Cannon, R.D., Shepherd, M.G. & Jenkinson, H.F. 1994. Detection of Candida albicans & other yeast in blood by PCR. Journal of Clinical Microbiology 32: 223-228

Liu, D., Coloe, S., Jones, Lloyd., Braird. R. & Pedersen, J. 1996. Genetic speciation of Candida isolates by arbitrarily primed polymerase chain reaction. FEMS Microbiology Letters 145: 23-26.

Loeffler, J., Hebart, H., Brauchele, U., Shumacher, U. & Einsele, H. 2000. Comparison between plasma and whole blood specimens for detection of Aspergillus DNA by PCR. Journal of Clinical Microbiology 38: 3830-3833.

Morace, G., Pageno. L., Sangiuretti, M., posteraro, B., Mele, L., Equitoni, F., D’Amore, G., Leona, G. & Falda, G. 1999 : PCR restriction enzyme analysis to detection of Candida DNA in blood from febrile patients with hematologic malignancies. Journal of Clinical Microbiology 37: 1871-1877.

Mousavi, S.A., Khalesi, E., Shahidi Bonjar, G.H., Aghighi, S., Sharifi, F. & Aram, F. 2007. Rapid molecular diagnosis for Candida species using PCR-RFLP. Biotechnology 6(4): 583-587.

Niesters, H.G., guessens, W.H., Meis, J.F. & Quint. W.G. 1993. Rapid PCR- based identification assays for species Candida. J. Clin. Microbiol. 3: 904-910.

Pryce, T.M., Kay, I.D., Palladino, S. & Heath, C.H. 2003. Real-time automated polymerase chain reaction (PCR) to detect Candida albicans and Aspergillus fumigatus DNA in whole blood from high-risk patients. Diag. Microbiol. Infect. Dis. 47: 487-496.

Wahyuningsih, R., Freisleber, H.J., Sonntag, H.G. & Schnitzler, P. 2000. Simple and rapid identification of Candida albicans DNA in serum by PCR for diagnosis of invasive candidiasis. J. Clin. Microbiol. 38: 3016-3021.

Walsh, T.J., Francesconi, A, Kasai, M. & Chanock, S.J. 1995. PCR and single-strand conformational polymorphism for recognition of medically important oppornistic fungi. J. Clin. Microbiol. 33: 3216-3220.

 

*Pengarang untuk surat-menyurat; email: jacinta@medic.ukm.my