Identification of Candida species isolated from cancer patients by Polymerase Chain Reaction – Restriction Fragment Length Polymorphism in Apeksha Hospital, Maharagama, Sri Lanka

Candida infections pose a significant challenge in managing cancer patients. A rapid and effective method for Candida species identification is crucial to address this issue. This study at Apeksha Hospital, Maharagama, aimed to identify Candida species using Polymerase Chain Reaction – Restriction Fragment Length Polymorphism. ITS1-5.8S-ITS2 rDNA regions of 52 clinical Candida isolates and standard strains were amplified using ITS1 and ITS4 primers and digested with Msp I enzyme. Candida tropicalis (38%), Candida parapsilosis (31%), Candida albicans (13%), Candida glabrata (8%), Candida krusei (4%), Candida haemulonii (4%), and Candida intermedia (2%) were identified. PCR-RFLP proves to be a rapid, and reliable method for identifying medically important Candida species in cancer patients.


Introduction
Candida infections, collectively known as candidiasis, are opportunistic fungal infections caused by various Candida species.These infections can range from superficial skin infections, such as oral thrush and vaginal yeast infections, to more serious systemic infections in immunocompromised individuals [1].Candida infections are particularly concerning in immunocompromised cancer patients, as they can lead to bloodstream infections (candidemia) and other severe complications, resulting in significant morbidity and mortality [2,3] (Key words: Candida, PCR-RFLP, cancer patients) Rapid molecular identification of Candida species is important for timely therapy decisions, outbreak control, managing antifungal resistance, reducing mortality, and conducting epidemiological investigations [4].
Various molecular identification methods are available for the diagnosis of Candida infections.However, the Polymerase Chain Reaction -Restriction Fragment Length Polymorphism (PCR-RFLP) is favored for its costeffectiveness, sensitivity, specificity, and rapid identification of Candida species [5,6,7] In the PCR-RFLP technique, ITS amplification targets the highly conserved ribosomal RNA genes present in all Candida species, allowing for the amplification of DNA.PCR products of ITS amplification can then be subjected to Restriction fragment length polymorphism analysis using restriction enzymes such as MspI, BushII, and SfilI [5].
This study aimed to identify 52 Candida isolates from cancer patients with candidemia (blood infection) using PCR-RFLP at Apeksha Hospital Maharagama, Sri Lanka.

Methods
In this study, 52 clinical isolates of Candida were investigated from the blood of cancer patients with candidemia at Apeksha Hospital Maharagama.These patients, spanning various ages and cancer types, were included in the analysis.In the microbiology laboratory at the Apeksha Hospital, Candida were originally isolated and cultured on blood agar from these blood samples.Then Candida isolates were sub-cultured in Sabouraud Dextrose Agar and Sabouraud Dextrose Broth media.DNA was extracted using the Qiagen DN easy Blood and Tissue Extraction Kit (Quiagen India 2019).
Finally, all PCR products and restriction fragments were visualized using 0.8% agarose gels stained with ethidium bromide, under UV light.The BioRad GelDoc system was used to obtain photographs of the gels.
The expected sizes of DNA bands after ITS amplification and PCR-RFLP are given in Table 1.

Discussion
In this study, ITS1 and ITS4 universal primers amplified ITS1-5.8S-ITS2rDNA regions at all 52 Candida clinical isolates, with some bands overlapping in size.However, MspI enzyme digestion of these products enabled the differentiation of Candida species that were difficult to distinguish before.This study, along with previous research by Amin et al. (2012), Gharaghani et al. (2018), and Mirhendi et al. (2006), confirm that PCR-RFLP is a highly sensitive, specific, and direct method for detecting Candida species.Combining PCR's specificity with RFLP's discriminatory power makes this molecular method efficient for Candida identification.
PCR-RFLP is a cost-effective and feasible method for routine clinical laboratory testing of Candida identification.It is more affordable than other advanced molecular methods, despite requiring specific reagents and enzymes.PCR-RFLP offers a moderate turnaround time, and it is relatively simple, needing only a thermal cycler and gel electrophoresis setup [5,6,7,8].In contrast, methods like PCR followed by sequencing, MALDI-TOF MS, and multiplex PCR are more expensive and require specialized equipment and expertise.Therefore, limiting  their routine use in clinical laboratories.Importantly, the use of this technique can help guide treatment decisions by identifying the specific species present, laying the groundwork for future antifungal susceptibility testing [5,10].
This study offers valuable insights into the prevalence of Candida species among cancer patients at Apeksha Hospital, providing novel and clinically relevant information.This information can significantly contribute to the management and treatment of candidemia in this vulnerable population.Overall, identifying Candida species in cancer patients is essential for tailoring treatment strategies, enhancing patient outcomes, and deepening our understanding of fungal infections in this susceptible group.

Figure 2 .
Figure 2. Patterns of ITS1/ITS4 amplification products of Candida isolates after digestion with MspI enzyme.

Table 1 . Expected sizes of DNA bands after ITS amplification and PCR-RFLP
[6,7]