Patients with head-and-neck malignancies receive a high dose of radiation in the oral cavity during radiotherapy, which can change their oral environment and commonly cause early complications such as mucositis and xerostomia, and subsequent colonization of the oral cavity and infection with different Candida species.^[1-3] Candidiasis is the most common infection of the oral cavity in patients under radiotherapy for head-and-neck malignancies. Although the majority of Candida infections in such patients are caused by Candida albicans, infection with non-albicans species has also been reported in such patients since 1990.^[2,4-6] Candida species are present in the normal oral flora of approximately 80% of the population. However, they can cause clinical candidiasis following alteration of the oral environment and systemic conditions after radiotherapy.^[7] The main changes caused in the oral environment by radiotherapy include quantitative and qualitative changes in the saliva due to degeneration and decreased function of the salivary glands.^[1-3] Considering the high prevalence of oral candidiasis and its potential morbidity and mortality, prompt diagnosis and management of such cases is highly important.^[8]

Currently, several groups of topical and systemic antifungal medications with different formulations are available for the treatment of candidiasis. Polyenes such as nystatin and amphotericin B, and azoles such as fluconazole, ketoconazole, and itraconazole are among the most important groups of antifungal medications. Among antifungal agents, fluconazole is more extensively used for the treatment of local and systemic cases of candidiasis due to its optimal dissemination in almost all body tissues. Due to the need for long-term intake of antifungal medications, such patients often suffer from the medication side effects, and are at risk of developing resistance to routine antifungals such as fluconazole.^[9] Some recent studies evaluated the antifungal activity of statins. This hypothesis was developed after noticing a reduction in the number of reported cases of zygomycosis in diabetic patients since 1990 following the widespread use of statins.^[10]

Statins are among the serum cholesterol-lowering medications. They are successfully used by patients with hypercholesterolemia and have significantly decreased the incidence of cardiovascular diseases. Statins prevent cholesterol synthesis by inhibition of 3-hydroxy-3-methyl-glutaryl-CoA reductase and lower the serum cholesterol level as such. Yeasts use the same enzymatic pathway to produce ergosterol, instead of cholesterol, as a final product. Furthermore, statins impair the production of isoprenoid and subsequently the mitochondrial function and cell respiration in Candida. They also change the lipid structure and metabolism and dynamically alter the cell membrane of C. albicans.^[11-13]

Considering the shift in microorganisms causing Candida infection in patients with malignancy from C. albicans to non-albicans species, increased risk of Candida infection with species resistant to routine antifungal therapy in such patients, and the inhibitory effects of atorvastatin on the enzymatic pathway of yeasts, this study aimed to compare the antifungal effects of fluconazole and atorvastatin on Candida species isolated from patients undergoing head-and-neck radiotherapy.

This clinical in vitro study was conducted from February 2019 to March 2019 (ethical code: IR.MUI.RESEARCH.REC.1398.152). The methodology of this study has been described in detail in the previous study.^[14] Candida species used in this study had been isolated from patients and stored in the Parasitology and Mycology Department of Isfahan University of Medical Sciences. These species had been clinically isolated from 33 patients hospitalized in Isfahan Seyed-O-Shohada Hospital before and after 2 weeks of head-and-neck radiotherapy. The strain type had been identified by polymerase chain reaction-restriction fragment length polymerization. The study population included 18 females and 15 males between 38 and 74 years. Twenty-one out of 33 patients were positive for Candida before radiotherapy. At 2 weeks after the onset of radiotherapy, six patients were excluded (4 males and 2 females); of the remaining 27 patients, 19 were positive for Candida. To prepare the fungal suspension, Candida species were first cultured on Sabouraud dextrose agar, and incubated at 35°C for 24 h.

The antifungal efficacy of fluconazole and atorvastatin is reported in Tables 1 and 2. The frequency value could not be calculated for Candida parapsilosis, and Candida krusei since they were isolated from only one patient each. Furthermore, since these two species were not isolated from any patient before initiation of radiotherapy, the antifungal efficacy of fluconazole and atorvastatin before and during radiotherapy could not be compared for these two species, and only the following values could be calculated for them:

Antifungal efficacy of fluconazole against C. parapsilosis: MIC24 = 1 μg/mL, MIC48 >64 μg/mL, and MFC = 64 μg/mL. Antifungal efficacy of fluconazole against C. krusei: MIC24 = 32 μg/mL, MIC48 >64 μg/mL, and MFC = 64 μg/mL. Antifungal efficacy of atorvastatin against C. parapsilosis: MIC24 = 32 μg/mL, MIC48 = 256 μg/mL, and MFC = 512 μg/mL. Antifungal efficacy of atorvastatin against C. krusei: MIC24 = 256 μg/mL, MIC48 = 512 μg/mL, and MFC = 768 μg/mL.

A comparison of the antifungal efficacy of fluconazole and atorvastatin is presented in Tables 3 and 4. The Mann–Whitney U-test revealed that both before and during radiotherapy, the MIC24, MIC48, and MFC of fluconazole were significantly lower than the MIC24 of atorvastatin for all three Candida species (P < 0.001 to P = 0.003).

Since 7 out of 33 patients were positive for C. albicans both before and during radiotherapy, the difference in all three parameters of MIC24, MIC48, and MFC of fluconazole and atorvastatin against C. albicans was calculated before and during radiotherapy and analyzed by the Mann–Whitney U-test [Table 5]. The Mann–Whitney U-test revealed that the MIC24 of fluconazole and atorvastatin increased significantly (P = 0.007) following treatment, while MIC48 and MFC had no significant alteration (P > 0.05).

The present results showed that fluconazole had higher antifungal effects on all Candida species isolated from patients under head-and-neck radiotherapy than atorvastatin both before and during radiotherapy, such that the MIC24, MIC48, and MFC of fluconazole were lower than those of atorvastatin.

In the present study, in addition to the assessment of MIC24, MIC48, and MFC, susceptibility to fluconazole was also evaluated such that the breakpoint of 8 μg/mL was defined for fluconazole MIC.^[16] In other words, species with MIC values equal or higher than 8 μg/mL were considered resistant, while those below this threshold were considered sensitive to fluconazole. Since atorvastatin is not an antifungal medication, a breakpoint is not defined for it.

To date, no previous study has compared the antifungal effects of fluconazole and atorvastatin on patients under radiotherapy. The results of the present study are different from Esfahani et al.^[17] study. For C. albicans both MIC and MFC of fluconazole were lower than Esfahani et al. study.^[17] On the other hand, both MIC and MFC of atorvastatin were higher than in that study. Of note, in the present study, MIC and MFC of fluconazole were lower than that of atorvastatin, while in Esfahani et al.’s study, it was vice versa. For C. glabrata comparing the results of these two studies comes into the same trend.^[17]

The possible reasons for the difference in the results of the present study and those of Esfahani et al.^[17] can be the difference in treatment protocols of the study populations which included radiotherapy and chemotherapy in their study, and the role of radiotherapy in xerostomia and its impact on oral microorganisms. The effects of radiotherapy on the oral environment include xerostomia, changes in the quality of the saliva, changes in oral pH, mucositis, changes in the physiological behavior and morphology of Candida species, and changes in pathogenesis and virulence of yeasts.^[7,18-20]

Szenzenstein et al.^[21] evaluated the effect of atorvastatin alone (without comparing it to fluconazole) on Candida species isolated from the blood of hospitalized patients. They evaluated C. parapsilosis, Candida orthopsilosis, and Candida metapsilosis. Since in the present study, C. parapsilosis was isolated from only one patient 2 weeks after the initiation of radiotherapy, the results of their study and the present investigation can only be compared regarding C. parapsilosis. In the present study, the MIC of atorvastatin for C. parapsilosis was found to be 32 μg/mL which was close to the value of the study by Szenzenstein et al. which was 25 μg/mL.^[21]

Brilhante et al.^[22] showed that atorvastatin had widely variable effects on different Candida species such that its mean MIC was 52.6 μg/mL for C. albicans, 165.34 μg/mL for C. tropicalis, 755.06 μg/mL for C. krusei, and 1491.37 μg/mL for C. parapsilosis. In contrast to their findings, the present results revealed that the MIC of atorvastatin for C. parapsilosis and C. krusei was lower than that for other species, and much lower than the value reported by Brilhante et al.^[22] However, the present results were close to the findings of Brilhante et al.^[22] regarding C. albicans and C. tropicalis.

Possible reasons for the difference between their results and the present findings include different sources of isolates, methodological differences in the measurement of MIC, and the atorvastatin solvent. A systematic review by Ting et al.^[23] showed that the MIC of statins may change depending on the type of solvent, form of statin used, methodology, culture media, and exposure time to statin. Atorvastatin has been extensively used as a blood cholesterol-lowering medication since 1990.^[10] Recently, atorvastatin was evaluated as a possible new antifungal agent that can inhibit the biosynthesis in the fungal cell wall,^[23] with the assumption that it might be a good alternative antifungal medication to control oral candidiasis in cases with drug resistance potential.

Brilhante et al.^[22] and Kurnatowski et al.^[24] demonstrated that C. albicans and C. tropicalis isolated from the oral cavity of patients with head-and-neck cancer before and during radiotherapy were resistant to fluconazole. In contrast to their studies, Bulacio et al.^[25] revealed that C. albicans and C. tropicalis species isolated during radiotherapy were sensitive to fluconazole. Consistent with the results of Bulacio et al.^[25] and in contrast to the findings of Brilhante et al.^[22] and Kurnatowski et al.,^[24] the present study indicated that C. albicans, C. tropicalis, and C. glabrata were sensitive to fluconazole both before and during radiotherapy.

Differences between studies may be due to the different origins of Candida species (e.g., isolated from animals), different sampling methods (swabbing vs. mouthwash), and different inclusion criteria.

Radiotherapy changes the oral environment and causes xerostomia, changes the saliva quality, alters the oral pH, causes mucositis, changes the physiological and morphological behavior of Candida species, and eventually changes the pathogenesis and virulence of yeasts.^[7,18,20] Karbach et al.^[26] in 2012 demonstrated that reduction in saliva following head-and-neck radiotherapy is correlated with the occurrence of treatment-resistant Candida species. Evidence shows that environmental changes cause fast alterations in gene expression in C. albicans and its adaptation to environmental conditions.^[27] Moreover, the majority of infection-dependent changes in gene expression of C. albicans are a reflection of its environmental adaptation.^[28] Paula et al.^[29] and Zida et al.^[30] reported that Candida species showed resistance to fluconazole in 13% of HIV-positive patients with no clinical evidence of candidiasis and 66.5% of symptomatic hospitalized patients. In contrast to the abovementioned theory, the present results regarding the MIC24 of fluconazole showed that all three species of C. albicans, C. tropicalis, and C. glabrata isolated from patients before and during radiotherapy were sensitive to fluconazole (MIC <8 μg/mL).

No resistance development to fluconazole following radiotherapy in the present study may be due to the fact that a minimum radiation dose of 2000 cGy is required for the parotid gland to cause a significant reduction in saliva flow.^[31] No previous study has precisely calculated the radiation dose of the parotid gland, and only the radiation dose based on the field and direction of irradiation has been estimated.^[32] The present study was the first to precisely calculate the radiation dose of the parotid gland using professional software (TiGRT, LinaTech LLC, USA) separately for each patient. The results showed that in only 39% of patients, the radiation dose of the parotid gland was over 2000 cGy in the 2 weeks. Thus, only 39% of patients received the minimum required dose to decrease the saliva flow and quality, which may also lead to the emergence of drug-resistant species.

Atorvastatin has gastrointestinal absorption and a systemic effect and may be used in the abovementioned cases at risk of candidemia. The serum level of atorvastatin in the Iranian population with a daily dose of 40 mg is reported to be 50 ± 30 ng/mL.^[33] The present results showed that the minimum concentration of atorvastatin that can inhibit Candida was 32 μg/mL. Since this concentration is almost 1000 times the serum level of atorvastatin, despite the oral bioavailability of atorvastatin, it cannot be recommended to control possible candidemia following orofacial Candida infection in patients under radiotherapy.

Another finding of the present study was that radiotherapy had a significant effect on the MIC24 of both medications and increased their efficacy since the values before radiotherapy were larger than the values during radiotherapy. Ramla et al.^[7] demonstrated an increase in the production of hydrolytic enzymes including phospholipase and proteinase by the fungi following radiotherapy, which may change the efficacy of antifungal medications following radiotherapy. However, since these enzymes are correlated with colonization of the host tissue by the fungi and tissue invasion, they do not affect the in vitro susceptibility of fungi. Future studies with a higher sample size might elucidate the explanation for such facts.

The present results indicated that considering no resistance development by the Candida species to fluconazole following radiotherapy and also high MIC of atorvastatin (1000 times its serum level), fluconazole is still recommended for treatment of oral candidiasis in patients under radiotherapy, and atorvastatin is not suitable for this purpose.