Oropharyngeal candidiasis (OPC) is the most common fungal infection in cancer patients. Patients receiving head-and-neck radiotherapy are more susceptible to Candida colonization and infection. The prevalence of OPC is highly variable among patients, ranging from 11% to 55%.¹,²,³,⁴ Therapeutic radiation to the head and neck can be followed by both acute and chronic oral complications, such as radiation-induced mucositis and xerostomia. These side effects usually occur 1–2 weeks after the initiation of treatment and trigger Candida overgrowth.⁵,⁶

The most frequent Candida species causing OPC in head-and-neck cancer patients undergoing radiotherapy is Candida albicans.⁷,⁸ However, other species of the genus Candida such as Candida parapsilosis, Candida krusei, Candida lusitaniae, Candida stellatoidea, Candida guilliermondii, and Candida dubliniensis have been shown to increase the incidence of fungal infections.⁹ It is known that geographic location influences the species distribution. For instance, while Candida glabrata is the predominant non-C. albicans species in North America, Candida tropicalis is the predominant species in Brazil.¹⁰ Therefore, it seems reasonable to identify species in different areas.

The present study aimed to identify the oral Candida species type (ST), colony count (CC), and OPC in head-and-neck cancer patients undergoing radiotherapy before and 2 weeks after radiation.

This was a quasi-experimental study.

Sampling

Head-and-neck cancer patients undergoing radiotherapy at Seyed-Al-Shohada Hospital, Isfahan University of Medical Sciences, Isfahan, Iran, participated in this study. The inclusion criteria were as follows:

A minimum total radiation dose of 6000 cGy and a minimum daily radiation dose of 180 cGy

The exclusion criteria were smoking, diabetes mellitus, immunodeficiency, mental retardation, and wearing an oral prosthesis. Two samples were taken from each patient (before RT and 2 weeks after RT). Two saline moisturized swabs were contaminated with the mucosa of the oral cavity.

Laboratory procedure

The first swab was drawn on a slab and checked for the presence of Candida species through Giemsa staining. The second swab was cultured on Sabouraud dextrose agar, containing chloramphenicol 0.5% for CC, and morphological studies were performed to confirm OPC. A second culture of colonies from the former culture was prepared for identifying Candida species. For identification, polymerase chain reaction–restriction fragment length polymorphism was performed using internal transcribed spacers (ITS1 and ITS4) and Mspl restriction enzyme.

Statistical analysis

Head-and-neck cancers were divided into intraoral (oral cavity and pharynx) and extraoral (larynx, neck, thyroid, ear, and carotid body) malignancy groups. Candida species were categorized as C. albicans and non-C. albicans. The CC was also categorized into three groups: 0 CFU; 1–1000 CFU; and >1000 CFU. The parotid radiation dose was divided into under 2000cGy and above 2000cGy. Chi-square test was used for analyzing the relationship between the malignancy type and OPC, CC, and ST before and after RT and for assessing the relationship between radiation dose and OPC, CC, and ST. Finally, the kappa coefficient was measured to compare OPC, CC, and ST before and after RT. P < 0.05 was considered statistically significant.

Overall, 57% of the patients had extraoral malignancies, while 43% were diagnosed with intraoral malignancies. The total parotid radiation dose ranged from 4.66 cGy to 4104.79 cGy, with a mean of 1857.39 cGy. Table 1 shows Candida detection and ST among patients. Figure 1 shows the frequency of species in candida positive patients before and two weeks after radiation therapy.{Table 1}Figure 1

The frequency of species in Candida-positive patients before and 2 weeks after radiation therapy.

Twenty-one of 33 patients were Candida-positive before treatment. Fungal species, CC, and OPC included C. albicans (60%), C. tropicalis (22%), C. glabrata (9%), and other species (9%). Suryawanshi et al. and Kurnatowski et al. reported the same results (C. albicans 60%). In Suryawanshi et al.'s study, three and five different Candida species were detected before and after RT, respectively.¹¹ Furthermore, Kurnatowski et al. isolated six and eight different species before and after RT, respectively.¹² C. krusei and C. parapsilosis were detected as new species in our study, while C. lusitaniae and C. guilliermondii were their newly detected species. The PCR method was applied for species identification in the present study rather than only morphological and biomedical analyses.

In the present study, 21.4% of the patients with intraoral malignancies were OPC positive. Wisniewski et al. reported that 17 of 30 patients with oral cancer were Candida positive.¹³ Čanković and Bokor-Bratić found that 30% of oral cancer patients were positive for Candida, 55% of whom had species other than C. albicans.¹⁴ In our study, 57% of the patients with extraoral malignancies were OPC positive. Similarly, Krajewska-Kułak et al. revealed fungi in the oral cavity of 55.9% of patients with cancer versus 24.4% of healthy individuals.¹⁵ In the present study, the difference in terms of CC, ST, and OPC between the two malignancy types (extra/intraoral) was not significant (P = 0.49, P = 0.26, and P = 0.97, respectively).

The risk factors for Candida carriage and OPC include poor oral hygiene, smoking, wearing dentures, and xerostomia.¹⁶ Besides, OPC is common among patients with prolonged severe diseases, such as diabetes, organ transplantation, and a history of chemotherapy.¹⁷,¹⁸,¹⁹ The carriage of Candida in the oral cavity of cancer patients is much higher than of healthy individuals, and non-albicans species play an important role in their infection. These species are more prevalent in cancer and critically ill patients.²⁰,²¹ Although the main cause remains controversial, alterations in the oral mucosal tissue, saliva quality/quantity changes, and underlying immunodeficiencies may be involved.²²

Importantly, the current study excluded patients with local risk factors for candidiasis, including smoking, use of dentures, history of topical drug use, diabetes, and history of antibiotic therapy/chemotherapy to determine the effect of underlying cancer on OPC, CC, and ST. The site of tumor affected OPC, CC, or ST neither before treatment nor following RT. Poor oral hygiene related to lifestyle modification and underlying immunodeficiencies might explain this finding regardless of the cancer site.

Similar to our study, Lou et al. calculated the exact planning dose of parotid gland radiation. Besides, they focused on the percentage of the parotid gland volume, which received different radiation doses. They concluded that neither a low radiation dose (<20 Gy) to a high volume of the parotid gland (>60%) nor a high radiation dose (40 Gy) to a low volume of the parotid gland (<20%) was significantly related to xerostomia. Xerostomia occurred when at least 50% of the parotid tissue received more than 20 Gy radiation doses simultaneously.²³ Overall, xerostomia is a known predisposing factor for OPC, Candida species change, and CC increase.²⁴

The present study did not show any significant relationship between OPC, CC, and ST alterations and radiation dose. It seems that besides beam quantity, the volume of radiated gland also plays an important role in xerostomia and candidiasis. In this regard, Nishii et al. showed that among several factors, severe mucositis, low levels of serum leukocytes, and tumor size were significantly related to candidiasis. In line with our results, radiotherapy and total radiation dose had no effect on Candida occurrence.²⁵

The present study showed that after 2 weeks of RT, OPC and CC changed significantly (P = 0.003 and P = 0.001, respectively), whereas ST did not significantly change (P = 0.081). Similarly, Lalla et al. found that following treatment, OPC increased from 7.5% to 39.1%. It should be noted that their patients underwent combined chemotherapy/RT in their study, whereas our patients received RT alone.¹

Moreover, Kurnatowski et al., Dwornika et al., and Ptyko et al. reported an increase in Candida-positive patients following RT. In contrast to our study, they only reported positive/negative detection or growth of Candida, whereas our study focused on the detailed properties of candidiasis, including CC, ST, and OPC.¹²,²⁶,²⁷ In this regard, Kurnatowski et al. showed an increase in Candida-positive patients from 66.2% to 80% following 3 weeks of RT.¹² Ptyko et al. also reported an increase of 22% in Candida detection after 2 weeks of RT from 46.3% to 68.3%.²⁶ Moreover, Dwornika et al. reported the growth of Candida after 2 to 3 weeks of RT at two sites of the oral mucosa (cheeks and tongue). The growth of Candida was observed in 5.6% of the cases in both sites before RT, while it changed to 38.9% and 19.7% following RT, respectively.²⁷

Consistent with our findings, Kurnatowski et al. found C. albicans as the most common Candida species both before and after RT.¹² In contrast, Nucci et al. found C. glabrata to be the predominant species following RT.²⁸ Unlike other studies, ST was statistically analyzed in the present study; according to the results, although raw data showed an increased diversity following RT, it was not statistically significant. The combination of oral and biological conditions in a patient leads to the evolution or determination of OPC. The responsible factors include the quantity and quality of saliva, xerostomia, reduced oral rinse, mucositis, and inability to practice oral hygiene correctly.²²,²⁹

In general, alterations in the saliva and mucositis begin in the 1^st week of RT, resulting in oral biological changes and, therefore, the increased risk of OPC.³⁰ On the other hand, Ramla et al. found that radiotherapy could reduce fungal adherence to epithelial cells. In other words, radiotherapy might cause raw and tender mucosa in the oral cavity and eliminate the suitable conditions for many fungal cells to penetrate into the host tissue by their germ tubes in vivo;²² this controversial finding may explain variations in OPC and ST.

This study had some limitations. First, since the exclusion criteria were strictly observed, the sample size was limited in the present study. Second, an estimated dose rather than an accurate radiation dose to the gland was measured in this study, regardless of the tissue volume. It is suggested that the chronic side effects of radiation be studied on more participants using adjuvant beam calculation techniques in the future.

The present study showed that OPC, CC, and ST were not related to the malignancy site. Following RT, OPC and CC changed significantly, while ST showed no significant changes. The radiation dose and malignancy site had no effects on OPC, CC, or ST alterations following RT.

Financial support and sponsorship

Nil.

Conflicts of interest

The authors of this manuscript declare that they have no conflicts of interest, real or perceived, financial or non-financial in this article.