A Clinicomycological Study of Pityriasis Versicolor with a Special Referance to the Calcofluor White Stain
Correspondence Address :
Jyoti S. Kabbin
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Background: The mycological study of Pityriasis versicolor by doing Potassium hydroxide (KOH) wet mount preparations and cultures is simple, but both the techniques have their own limitations. The isolation of the organism is very difficult because it is lipophilic and it needs special media to grow, which takes time. The present study suggests that the Calcoflour white stain can be used for the preliminary identification of the fungal elements, so that clinicians can start with the treatment.
Aim: The aim of this study was to know the advantage of Calcofluor white staining versus the conventional methods which are used for the demonstration of the fungi in cases of Pityriasis versicolor.
Methods: Over a period of one year, from October 2004 to November 2005, a total of 100 clinically diagnosed cases of Pityriasis versicolor were referred from the Department of Dermatology to the Department of Microbiology, Bowring and Lady Curzon Hospital which is attached to the Bangalore Medical College and Research Institute, Bangalore. Skin scrapings from affected lesions were processed by doing KOH preparations, Calcoflour white staining and cultures.
Results: Out of the 100 cases which were studied, the maximum number of cases were in age group of 12 to 21 yrs (51%). Males 71(71%) were affected more than females 29(29%). 98(98%) samples were positive for the fungi by Calcoflour white staining, 92(92%) by the KOH preparation and 46(46%) by culture.
Conclusion: Calcoflour white staining picked up an additional four samples which were reported as negative by the KOH preparation and the culture. Two other samples were reported to be positive by Calcoflour white staining and culture and to be negative by the KOH preparation. Calcoflour white staining is a rapid, simple, sensitive and highly reliable method for identifying fungi, as it provides a good definition of the fine fungal structures and a better contrast from the background debris, cells and tissue fragments.
Calcoflour white stain (CFW) , Pityriasis versicolor, Potassium hydroxide (KOH) wet mount preparation
Pityriasis versicolor is a cutaneous, superficial, fungal infection which is characterized by skin pigmentary changes due to the colonization of the stratum corneum by dimorphic, lipophilic fungi in the normal flora of the skin, which is known as Malassezia furfur (1). The genus, Malassezia, has been recently shown to consist of seven species (2). The one lipid independent species is Malassezia pachydermatis and the six lipid dependent species are Malasezia furfur, Malaseezia sympodialis, Malassezia globosa, Malassezia obtuse, Malassezia restrica, and Malassezia sloffiae, which were identified by partial, ribosomal RNA sequence comparisons (3). The aim of this study was to know the advantage of the Calcofluor white staining versus the conventional methods for the demonstration of fungi in cases of Pityriasis versicolor.
This study was conducted over a period of one year from October 2004 to November 2005. A total of 100 clinically diagnosed cases of Pityriasis versicolor were referred from the Department of Dermatology, Bowring and Lady Curzon Hospital which is attached to the Bangalore Medical College, Bangalore. A detailed history elicitation and clinical examination were performed on each case. The affected skin area was thoroughly sponged with 70% alcohol to remove the surface contaminants. After drying, the active edges of the lesions were scraped with a flame sterilized, No. 15 scalpelblade. The skin scrapings were processed by direct microscopic examination which included a 10% KOH preparation, a 10% KOH with Parker ink preparation, a 10% KOH with Methylene blue preparation and a Calcofluor white stain preparation. Two paired sets of Sabouraudâ€™s Dextrose Agar (SDA) with cyclohexamide, one paired set with olive oil and the other paired set without olive oil were inoculated and incubated for 2 weeks at 25o C and 37oC.
The results were compiled and statistically analyzed.
The study group comprised of 100 clinically diagnosed cases of Pityriasis versicolor. The ages of the study group subjects ranged from 11 to 42 years. A maximum number of patients (51%) belonged to the age group of 12-21 years. Males (71%) were affected more than the females (29%). Most of the patients were of the hypopigmented variety (67%) as compared to the hyperpigmented variety 31(31%) and 2(2%) patients were of mixed variety. The distribution of the skin lesions showed that they were seen more commonly on the chest and back [87(87%) and 13(13%)] over other sites like the face and the abdomen.
Out of the 100 suspected cases of Pityriasis versicolor, 92 (92%) were positive for the fungi by the KOH preparation, as shown in (Table/Fig 1), 98% were positive for fungi by Calcoflour white staining as shown in (Table/Fig 2) and 46(46%) were culture positiveas shown in (Table/Fig 3). Other tests which were used, that is, the Parker ink preparation and the Methylene blue preparation were similar to the KOH preparation in the detection of the organism, as shown in (Table/Fig 3).
Pityriasis versicolor is one of the most common disorders of pigmentation in the world (4). In the tropical areas, it has been reported in 30 to 40% of the population and the incidence is lower in temperate climates (1to 4%) (5).The disease is usually brought to the physicianâ€™s attention because of its cosmetic effects on the patient (6).The commonest clinical species is thought to be Malassezia furfur (7),(8). The Malaseezia species are fastidious fungi which require the addition of lipids to the culture media and which grow best at a temperature range of 32_37Â°C in an anaerobic environment (9). Pityriasis versicolor occurs when yeast forms convert to their mycelial forms due to certain predisposing factors. The exogenous factors include heat, moisture and occlusion of the skin due to either clothing or cosmetics (2). Occlusion results in an increased carbon dioxide concentration (10). The endogenous factors include diseased states like seborrheic dermatitis, Cushingâ€™s syndrome, malnutrition and hyperhidrosis (11).
In our study, the lesions were most commonly seen in the age group of 12-21 (51%) years, as has been reported by other workers. Marple et al (12) found the incidence to be 49(49%) in adults, Belec et al (13) during their study, found that the maximium prevalence (23.5%) was in the age group of 15-25 years, Vijaya et al (14).found that the lesions were common in the age group of 21- 30 years, (56%) and Rao et al (15) found in their study that 30% of the patients belonged to the age group of 21-30 years.
The disease may occur at any age, but it is more common during adolescence and in young adults due to an increase in the sebaceous activity (16) and hormonal changes.
The present study showed a pre-dominant involvement of males as compared to females, as in the following studies; Roberts et al (11)found the ratio of males to females to be about 3:2 among the patients in the United Kingdom, Kim and Suh et al (17) found it to be 2:1 among Korean patients, Belec et al (13) found that males were affected more than females, Vijaya et al (14) found more males (78%) than females (22%) among 100 patients and Rao et al (15) reported that more males (73.33%) were affected in comparison to the females (26.60%). The higher incidence of Pityriasis versicolor in males may be due to their outdoor activities (14). The type of lesions were more hypopigmented (67%) than hyper pigmented (31%), which was similar to that which was reported in the study of Rao et al (15). The distribution of the skin lesions commonly occurred over the chest and the upper back (87%) in our study among 100 patients. These observations were in conformity with the findings of Roberts et al, who found that 92% of the patients carried them on their trunk, Belec at el (13) who found that the lesions commonly occurred over the upper arm, and the trunk (48.6%), Vijaya et al (14), who found that the lesions were more common over the chest and the back (85%) and Rao et al (15) who found that the lesions were seen commonly over the back (70%) and the chest (58.30%). The distribution of the lesions generally parallels the density of the sebaceous gland distribution, with a greater occurrence on the chest and the back (18).
In this study, Calcoflour white staining was positive in 98(98%) patients as compared to the KOH preparation [92(92%) patients].
The causative agent was isolated in 46(46%) patients. Our results were in conformity with the findings of Maheswari et al (19), who found that 96% of the patients were positive for the KOH preparation out of 100 cases and that 70% patients were positive for culture, Shanker et al (20) who found that all their 100% cases were positive for KOH and that the culture yielded a positive growth only in 60% patients, Vijaya et al (14) in whose study, 42% out of 100 cases were positive for growth and Rao et al (15) who found 91% patients were positive for KOH and that 40% showed growth on SDA with oil. Kindo et al (21), during their study, found that 70% patients were positive KOH and that 68.75% patients showed growth on Dixonâ€™s agar.
Calcoflour white staining picked up an additional four samples which were reported as negative by KOH and culture. Two other samples were found to be positive by Calcoflour white staining and culture, which were reported as negative by the KOH preparation.
Calcoflour white staining is a rapid, simple, sensitive and a highly reliable method for identifying fungi, as it provides a good definition of the fine fungal structures and better contrast from the background debris (22), cells and tissue fragments. The disadvantage of fluorescent staining is that it requires a fluorescent microscope, expertise in reporting and is expensive. Laboratories will find that investing in a fluorescent microscope will yield better patient care (23). To determine the clinical usefulness and characteristics of each test, Calcoflour white staining was chosen as the gold standard for statistical analysis (24).
To conclude, Calcoflour staining was found to be far superior to the conventional KOH preparation for the detection of fungi in clinical specimens.
The authors are thankful to the staff of the Microbiology Department, Bowring and L.C.Hospital which is attached to Bangalore Medical College, Bangalore, for their co-operation in this study.
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