|Year : 2014 | Volume
| Issue : 1 | Page : 28-34
Candidal carriage, isolation and species variation in patients undergoing radiotherapy and chemotherapy for head and neck tumours
Shoba Rani Bakki1, Lalith Prakash Chandra Kantheti2, Kiran Kumar Kuruba2, Chandrasekhar Poosarla2, Venkat Ramana Reddy Baddam2, Raja Rajeswari Mulakaluri3
1 Department of Oral Pathology, Meghana Institute of Dental Sciences, Nizamabad, Andhra Pradesh, India
2 Department of Oral Pathology, Sibar Institute of Dental Sciences, Takkellapadu, Guntur, Andhra Pradesh, India
3 Department of Microbiology, Guntur Medical College, Guntur, Andhra Pradesh, India
|Date of Web Publication||10-Mar-2014|
Lalith Prakash Chandra Kantheti
Department of Oral Pathology, Sibar Institute of Dental Sciences, Takkellapadu, Guntur - 522 509, Andhra Pradesh
Source of Support: None, Conflict of Interest: None
Background: Opportunistic fungal infections, mainly candidiasis, is common in immunocompromised patients such as those undergoing chemo or radiatiotherapy and pose a great threat. Hence this study was done, attempting to find out the candidal carrier state and species variation, in such patients.
Aims and Objectives: To determine the prevalence and speciation of Candida colonizing in the oral cavity of individuals attending to the cancer clinics in south India.
Materials and Methods: Study groups comprised of 50 healthy, asymptomatic individuals as controls (Group I); 50 patients on : chemotherapy (Group - II) and 50 patients undergoing radiotherapy for head and neck cancers (Group - III). Mycological tests for the candidal isolation and speciation were done.
Results: A total of 49 cases showed candidal culture positivity (32.67%). Candidal carriage was highest in Group II and Group III (38%). C. albicans was the most prevalent species (30%). The culture positivity and the colony forming unit/ml from the subjects of control group and the study group were highly significant, in pair-wise comparisons.
Conclusion: Patients undergoing chemotherapy and radiotherapy for head and neck cancers are at a risk of developing oral candidiasis due to immunosuppression. C. albicans is the predominant isolate, but non-albicans species are also emerging as opportunistic pathogens. All may cause a similar spectrum of disease but differences in disease severity and antifungal susceptibility are noted. Hence, speciation can help to target the treatment of the disease in a much better way.
Keywords: Candida albicans , chemotherapy, oral cancer, oral candidiasis, radiotherapy
|How to cite this article:|
Bakki SR, Kantheti LC, Kuruba KK, Poosarla C, Baddam VR, Mulakaluri RR. Candidal carriage, isolation and species variation in patients undergoing radiotherapy and chemotherapy for head and neck tumours. J NTR Univ Health Sci 2014;3:28-34
|How to cite this URL:|
Bakki SR, Kantheti LC, Kuruba KK, Poosarla C, Baddam VR, Mulakaluri RR. Candidal carriage, isolation and species variation in patients undergoing radiotherapy and chemotherapy for head and neck tumours. J NTR Univ Health Sci [serial online] 2014 [cited 2020 Mar 30];3:28-34. Available from: http://www.jdrntruhs.org/text.asp?2014/3/1/28/128427
| Introduction|| |
Opportunistic fungal infections occur due to relatively nonpathogenic or contaminant fungi in a host, whose immunological defense mechanism is weakened by endogenous causes like malignancies and metabolic diseases like diabetes, or exogenous causes like immune-suppressive therapeutic procedures or as is in case of Acquired Immunodeficiency Syndrome (AIDS).
Among approximately 50,000 species of fungi, more than 200 are known to cause diseases in vertebrate animals and human beings.  Candida species are usually normal oral commensals and their transition to an opportunistic pathogen may be associated with the virulence attributes of the organism and also the host factors.  Candidal species like C. albicans; C. tropicalis; C. krusei; C. parakrusei; C. glabrata; C. guillermondii; C. parapsilosis; C. kefyr/pseudotropicalis; C. lusitaniae; C. dubliniensis; C. viswnathii; C. stellatoidea, and so on can be isolated.
C. albicans is the most common species detected in the oral cavity of both healthy and medically compromised individuals. , But recent studies have shown other species of Candida also to be emerging pathogens. Apart from this, the antifungal susceptibility patterns of albicans and also the newer species of Candida has been changing. Hence, the isolation and speciation of the causative species of Candida is gaining importance. ,
Since the recent past, saliva is gaining importance as an effective and a noninvasive diagnostic aid. It is a well-established fact, based on many previous studies that saliva can be a good means of identifying the oral candidal carriage. Studies with unstimulated salivary samples taken from chronic and acute oral candidiasis cases showed increased colony forming unit count per ml CFU/mL compared with the normal healthy individuals. 
Oral yeast colonization studies conducted by Leung et al.,  using salivary samples in irradiated, dentate, and xerostomic patients showed a greater candidal carriage in the irradiated individuals. According to Ueta et al.,  the virulence of Candida strain in chemotherapy and irradiated individuals is largely determined by activities of adherence, multiplication and release of a cytoplasmic antigen, enolase & Candida aspartic proteinase. A study on the candidal speciation by Redding et al.,  in patients with head and neck cancers undergoing radiation therapy revealed C. albicans to be the predominant species.
Dahiya et al., examined nonalbicans Candida oral infections in patients with head-and-neck cancer receiving external beam radiotherapy with or without concurrent chemotherapy and concluded that nonalbicans Candida is emerging as a relatively common cause of oropharyngeal candidiasis in head and neck cancer patients and that chromogenic media are helpful to screen these infections.  Bruno et al.,  assessed the colonization of the candidal species in head and neck irradiated patients and concluded that candida colonization continuously increased during radiation therapy. Apart from the C. albicans, an increased incidence of the nonalbicans species like C. tropicalis, C. krusei and C. lusitanae were also observed. 
With this as the back ground, a study was done attempting to find out the candidal carrier state and the species variation in patients undergoing chemotherapy and radiation therapy for head and neck cancers attending to the chemotherapy and radiation therapy units at Government General Hospital (GGH), Guntur, Andhra Pradesh, India.
Aims and objectives
To find out the candidal carrier state in individuals undergoing chemotherapy and radiation therapy.
- To isolate the most common candidal species prevalent in those individuals and to compare the results with the control group.
- To calculate the CFUs count/mL, which relates to the immune status of the individuals in study population.
| Materials and Methods|| |
The present study was carried out in the Department of Microbiology, Guntur Medical College, Guntur, Andhra Pradesh, India. A total of 100 patients attending to the radiotherapy and chemotherapy units and 50 patients attending the outpatient department at the GGH, Guntur, were the study sample. Patients who were on antifungals for a period of 15 days before the sample collection, patients who had any history of recent usage of corticosteroids or any immunosuppressant drugs, patients with removable or fixed dentures and those with any previous history of mucosal diseases were excluded from the study. All findings were recorded in a standardized format. The data were coded and entered into the database program for statistical analysis by SPSS package. The samples were collected from three groups of the study population, divided as follows:
- Group I: 50 patients who were age- and sex-matched with study groups and apparently healthy with no systemic diseases after routine investigations.
- Group II: 50 patients, 20-80 years of age who were on chemotherapy for head and neck cancers.
- Group III: 50 patients, 20-80 years of age who were undergoing radiotherapy for head and neck cancers.
After an informed consent, a detailed history was taken, including the name, age, gender, IP/OP number, occupation, address, predisposing factors, onset & duration of complaint, treatment, and so on. Ethical considerations about the underlying disease were accounted.
Unstimulated saliva samples were collected from the subjects of all three groups by way of oral rinse, with the following protocol: 10 mL of sterile phosphate buffer saline solution (phosphate buffered saline 0.1M, pH 7.2) was supplied to each individual as oral rinse, for 60 secs. A sterile plastic container (Speci-can 30 mL) was used to collect the expelled oral contents, which was transported to the laboratory immediately, for the various mycological tests. Standard mycological procedures given by Milne et al.,  were followed for processing all the samples. Budding yeast cells were identified using direct microscopic examination of KOH wet mount and a gram-stained smear [Figure 1].
|Figure 1: Gram's stained smear showing budding yeast cells and pseudohyphae|
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After inoculation of the specimen onto Sabouraud's dextrose agar (SDA), the plates were incubated at 37 0 C for up to 48-72 hours. Within 1-3 days, creamy white, smooth pasty colonies and opaque colonies with yeasty odor were seen to be growing. The formula: CFU/mL = 1000 × Number of colonies/4, was used to calculate the CFU count.
Candidal speciation tests were done-the Germ-tube test for rapid morphological identification of C. albicans and C. dublinensis [Figure 2]. Chlamydospore and blastoconidia production was tested on cornmeal agar by Dalmau culture plate technique. The isolates from the SDA medium were then inoculated on Chrom Agar medium and incubated at 37°C in dark for 48 hours. Only the pigmented colonies were considered for species identification [Figure 3]. Apart from this, various biochemical tests like the carbohydrate assimilation and fermentation tests were also utilized for speciation of Candida.
| Results|| |
Out of the 150 cases subjected to mycological investigations for confirming the presence of Candida species, 49 were culture positive. A total of 11 cases in the Group I (n = 50) were positive for Candida (22%). In both Group II and Group III, 19 cases were positive for candidal growth (38%). But, there was no statistical significance when the study groups and culture positivity were correlated (chi-square = 3.8801; df = 2; P = 0.1437) [Table 1].
When the study groups were correlated with respect to gender and culture positivity, it was statistically highly significant (unpaired 't' test, at df = 6, t = 9.28, P < 0.001**) [Table 2].
A statistical comparison was done between the three groups, with respect to CFU/ml by Kruskal-Wallis analysis of variance (ANOVA), and a high significance was observed (P = 0.0236*; at 5 level of significance P < 0.05). Pair wise comparisons were done for all three groups by Mann-Whitney U-Test. Group I and Group II (P = 0.0479*), and also Group I and Group III (P = 0.0471*; at 5 level of significance P < 0.05) showed statistical significance, accordingly. A similar comparison of Group II and Group III was statistically insignificant [Table 3] and [Table 4].
|Table 3: Comparision of three study groups with respect to colony forming unit/ml by kruskal-wallis analysis of variance by ranks|
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|Table 4: Comparision of three study groups with respect to colony forming unit/ml by mann-whitney u-test|
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Totally, 49% of the cases in this study were culture positive of which, C. albicans was the predominant isolate (30%), followed by C. tropicalis (8.00%), C. glabrata (5%), C. parapsilosis (2%), C. krusei (2%), C. stellatoidea (1%), and C. kefyr (1%) [Table 5].
Though there was no statistical significance, a definite predominance of C. albicans over other species was observed in the study.
| Discussion|| |
Oral candidiasis is an opportunistic infection of the oral cavity. It can also be a mark of systemic disease such as diabetes mellitus and is a common problem among the immunocompromised. Oral candidiasis is caused by an overgrowth or infection of the oral cavity by a yeast-like fungus, Candida.
Different species of Candida can cause candidiasis, which may manifest as acute or chronic and it can be either a superficial or a disseminated mycosis. It may arise due to either endogenous or exogenous causes and as a primary or secondary infection. Candida, which is normally a saprophytic organism, can become distinctly pathogenic, in patients with depressed defense mechanism. 
There has been a dramatic increase in the number of immunocompromised individuals in the past few decades, which can be attributed to treatments like chemotherapy and radiation therapy targeted against neoplasias, increased number of patients undergoing solid organ and bone marrow transplants, growing number of patients requiring intensive care and an increased number of people presenting with human immunodeficiency virus/AIDS. 
Oral candidiasis is common in patients with head and neck cancers, especially during chemotherapy and radiotherapy. The acute form usually presents as erythema, but the diagnosis may often be missed, as it may be mistaken for radiation mucositis.
According to Ueta et al.,  suppression of antifungal activity of leukocytes and the increase in candida cell virulence are the predisposing factors for systemic candidiasis in patients undergoing chemo-and radiation therapy. The compromised salivary function secondary to destruction of glandular tissue by radiation is thought to be a major factor leading to Candida infection. Changes induced by exposure to radiation may occur during and after completion of therapy, including mucositis, candidiasis, osteoradionecrosis, and radiation caries. 
A total of 150 cases were screened in the present study for oral candidal isolation and speciation, out of which, 49 were culture positive, giving an overall prevalence of 32.67%. The oral candidal carriage in normal healthy individuals (Group I) was observed to be 22%. In patients undergoing chemotherapy (Group II) and radiation therapy (Group III), oral candida carriage was found to be 38%. This indicates a definitive risk of these patients to develop oral candidiasis.
Oral candidal carriage was reported by various authors in healthy as well as in immunocompromised individuals, the findings of which were consistent with the present study. The prevalence of yeast in the oropharynx of healthy volunteers was reported to be 35%, by Cohen et al.  In his study, Bunetel and Bonnaure - Mallet  found an oral candidal prevalence of 38% in patients undergoing chemotherapy. Amador et al.,  reported 38% candidal positivity in patients undergoing oral and pharyngeal radiation therapy. The positivity could be due to the fact that the patients are often unable to maintain adequate nutritional status and oral hygiene during radiotherapy and chemotherapy, in spite of receiving instructions and care. Based on the type of sample, the size of sample and the sampling method adopted, a considerable variation may be observed in humans, with respect to the candidal prevalence. 
Our study has shown a male predominance in all the study groups, with an overall positivity of 34.8% and it was 29.5% in females, which was statistically highly significant (P < 0.001). A definite male predominance was observed in the candidal culture positivity, particularly in the immunodeficiency states. This could be attributed to the adverse habits like smoking, alcohol, and so on, more prevalent in men, which are also definitive risk factors for developing oropharyngeal cancers. Gooris et al.,  reported a male predominance in patients with oral cancers, receiving radiotherapy.
An attempt was made in this study to find out the significance of the CFU/ml from the oral rinse samples collected in different groups. A comparison between all the three groups with respect to CFU/mL by Kruskal-Wallis ANOVA by ranks has shown a definite statistical significance (P < 0.05). A pair-wise comparison of three study groups with respect to CFU/mL by Mann-Whitney U-test has also shown a definite statistical significance between the control group and the study groups (P < 0.05).
CFU/mL was <600 in the control group, whereas majority of chemotherapy and radiotherapy patients showed a CFU/mL of >2000. This suggests that the patients on chemotherapy and radiotherapy for head and neck cancers have an increased candidal carriage which may predispose them to clinical oral candidiasis. Similar findings were also reported in the studies by Dahiya et al., Bruno et al., Belzai et al., and Hanan et al.
The most commonly isolated species is C. albicans, but many studies have shown various nonalbicans species also to be evolving, with varying pathogenecity and antifungal susceptibility. 
In the present study, C. albicans was the most commonly isolated species in all the study groups with an overall positivity of 30% and nonalbicans being 19%. Our studies were consistent with the findings of Shaheen and Taha,  who reported 56% of C. albicans and 44% of nonalbicans in Quranian patients, and Amador et al.,  who reported 68% of C. albicans and 32% of nonalbicans species. Nicolatou-Galitis et al.,  demonstrated C. albicans to be the predominant species.
In the present study, the nonalbicans species reported were C. tropicalis, C. glabrata, C. parapsilosis, C. kefyr, C. stellatoidea, and C. krusei. Oral candidiasis caused by nonalbicans candida was well-established and reported by Dahiya et al.,  and Redding et al. The species shift toward nonalbicans is not only related to antifungal medications, as it is also observed in patients who were not treated for candidiasis.
According to Fridkin and Jarwis,  C. albicans and nonalbicans species are closely related but differ from each other with respect to epidemiology, virulence, and antifungal susceptibility. All may cause a similar spectrum of disease, but differences in disease severity and susceptibility to antifungal agents were reported.
The increased number of nonalbicans species in the present study may be attributed to the changing ecology of the pathogens and evolution of nonalbicans species as pathogens, in immune compromised individuals. Among the nonalbicans species, C. tropicalis was isolated in large numbers both in control group as well as study groups.
The oral rinse technique used in the study appears to be relatively sensitive, detecting Candida in 22% of control subjects. This is comparable to the 20%-60% detected in a similar study using various culture techniques, done by Cohen et al. 
The present study showed a higher prevalence of Candida in the oral cavity of the immune compromised individuals like patients on anticancer therapy compared with the normal individuals. The male subjects in study group had significantly increased prevalence of Candida when compared with female subjects. C. albicans was the predominant species isolated in the study group. The nonalbicans species were also isolated, indicating that other species can also be pathogens for the disease.
| Conclusion|| |
The patients undergoing head and neck radiation therapy and chemotherapy are at definitive risk of developing oral candidiasis. Immune status has a definite impact on candidal carriage. In the immune compromised individuals, the members of the normal flora may probably acquire invasiveness and become pathogenic. Though C. albicans is the predominant isolate, nonalbicans species also have been proven to be emerging pathogenic opportunistic infection. They differ from each other with respect to epidemiology, virulence, and antifungal susceptibility. All may cause a similar spectrum of disease but differences in disease severity and susceptibility to antifungal agents are seen. Due to the changing pattern of candidal species, such studies involving isolation and identification of various species of Candida in immune compromised individuals is important for much better treatment strategies and thus gain a good control over the disease. Further studies on a larger cohort prospectively would be of value in confirming the findings of this study.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]
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