PCR in Comparison with Culture Methods for The Diagnosis of Candida albicans Responsible for Candidemia in Leukemic Patients

Abstract

Background: Currently, candidemia infections represent an increasing cause of morbidity and mortality in seriously ill hospitalized patients. Because the accurate diagnosis of candidiasis remains difficult, fast and reliable assay for characterization of fungal pathogens is critical for the early initiation of adequate antifungal therapy and/or for introduction of preventive measures.

Objective: To detect candidemia in leukemic patients by molecular methods in comparing with golden standard method (culture method).

Materials and methods: A total of 60 leukemic patients were included in this study. Clinical type and other demographic data were recorded. Blood samples were taken from each patient, culture; germ tube formation and carbohydrate fermentation were done for each sample. DNA extraction and polymerase chain reaction (PCR) were used for detection of Candida albicansin cultured bottles. This study was conducted on leukemic patients admitted to four different hospitals in Baghdad city from September 2010 to March 2011.  Sixty patients suffering from acute lymphoid (ALL) and myeloid (AML) leukemia were included in this study. The age of patients were ranging between 3-46 years old. Twenty five apparently healthy individuals were enrolled in this study as control group. Three milliliters of blood were collected from each patient; 1.5 ml was inoculated in 20 ml Brain heart infusion broth (Cruikshank. 1975). The rest of blood (1.5ml) was stored in -20ᴼC for further analysis. Blood cultures were incubated at 37°C for 10 days, and examined daily for growth. DNA purification kit was purchased from QIAGENE^®Company. This method was used for the purification of genomic DNA from fresh or frozen samples of 1 ml overnight yeast cultures by using the GentraPuregene Yeast/Bact. Kit. PCR was performed to detect Candida albicans species through the amplification of specific gene (αINT1)

Results: Only one positive culture result out of 60 samples was obtained for Candida sp., (1.7%).  PCR results showed that there were only three out of sixty were positive for C. albicans (5%). In this study we obtained only one positive sample according to culture, while three samples only gave positive results according to PCR method. These results suggest that molecular analysis of candidemia is more sensitive and less time consuming than culture and other conventional methods.

Conclusions: we concluded the following: The rate of candidemia was 1.7% among leukemic patients, according to culture results, only 5% of blood cultures was positive according to PCR. Results showed 100% sensitivity and 96.6% specificity and it is rapid, easy, reliable and also applicable in clinical laboratory for identification of medically important Candida spp