Evaluation of the Significance of Molecular Methods in the Diagnosis of Invasive Fungal Infections: Comparison with Conventional Methods

Abstract

Direct microscopy and culture methods are still valuable standard conventional methods for the diagnosis of infections caused by true or opportunistic fungal pathogens, especially in high risk patients. However, some of the problems concerning the application and interpretation of those methods, indicate a need for more rapid, practical and reliable tests with high sensitivity and specificity. This study was conducted to compare the results obtained by molecular methods with the results of conventional methods performed simultaneously for the detection and identification of causative fungi in clinical samples. Clinical samples [24 bronchoalveolar lavage (BAL); 14 blood; 5 peritoneal, 4 pleural and 1 pericardial fluids; 1 cerebrospinal fluid (CSF), 1 urine] from 50 immunosuppressed patients were included in the study. All of the samples were cultivated on Sabouraud dextrose and brain-heart infusion agar media and incubated at 30 C and 37 C for 30 days. Samples other than blood were stained with 10-15% KOH + calcofluor white and examined by direct microscopy. Conventional identification of the isolates were performed by using basic morphological and biochemical characteristics. The isolation of fungal DNAs for polymerase chain reaction (PCR) was achieved by classical phenol-chloroform-isoamylalcohol procedure (9-10 hours) and commercial DNA extraction kit (6-7 hours) and general and species-specific primers (multiplex) from ITS1, ITS2, ITS3, ITS4, 5.85 rDNA and 28S rDNA regions were choosen for amplification. In PCR results, 550 base-paired (bp) bands obtained with universal primers were evaluated as fungal DNA positivity, and 273 bp, 320 bp, 423 bp, 357 bp, 136 bp and 385 bp bands with species-specific primers were evaluated as Candida albicans, Candida parapsilosis, Candida glabrata, Candida tropicalis, Cryptococcus neoformans and Aspergillus fumigatus positivities, respectively. Seventeen (34%) of the 50 samples yielded fungal growth on culture (C.albicans in 12 SAL, 3 blood, 1 urine sample, and C.parapsilosis in 1 urine), while seven SAL out of 36(19.4%) non-blood samples gave positive result by direct microscopy. Of the samples 27 (54%) were found positive by PCR. All of the 17 culture positive samples were also found PCR positive, and all of the 23 culture negative samples were also found PCR negative. However, fungal DNAs were detected by PCR in 10 of the samples (5 BAL, 4 peritoneal fluids, 1 CSF) which were negative by direct microscopy and culture methods. These fungi were identified as C.albicans (n = 8), C.parapsilosis (n = 1, from peritonal fluid) and C.neoformans (n = 1, from CSF) by multiplex PCR. No samples yielded PCR negative, culture positive result. All of those 10 PCR positive, culture negative samples belonged to patients who were under antifungal treatment. The detection of C.neoformans DNA from CSF sample of a patient with suspected cryptococcosis only with PCR provided the chance for rapid therapy. In statistical evaluation, the concordance between culture and PCR methods were found significantly high (k = 0.61; p < 0.001), whereas it was minimal (k = 0.24; p < 0.001) between direct microscopy and PCR. When considering culture as the reference method, the sensitivity and specificity of PCR were estimated as 100% and 69.7%, respectively. In addition, multiplex PCR was as successful as culture and conventional identification methods in the identification of all fungal species.