PREVALENCE, IDENTIFICATION AND ANTIFUNGAL SUSCEPTIBILITY OF DERMATOPHYTES CAUSING TINEA CAPITIS IN A LOCALITY OF NORTH CENTRAL NIGERIA

Background: Tinea capitis impacts negatively on the health of children, consequently affecting their education. Its prevalence is unknown in many African communities. Tinea capitis is faced with therapeutic challenges as resistance to all classes of antifungal agents continues to emerge. This study determined the prevalence, identified dermatophytes of Tinea capitis in Okelele community in North Central Nigeria; and evaluated the susceptibility of isolates to selected antifungal drugs. Materials and Methods: Three hundred and one pupils from seven primary schools in the locality who gave assent and those with parental consent were recruited into the study. Scalp scrapings and hairs were collected from participants and subjected to microscopy and culture. Isolates identified by colonial morphology and micromorphology were subjected to disk diffusion antifungal susceptibility testing. Results: Two hundred and twenty-eight of the participants had mycologically proven Tinea capitis giving a prevalence of 75.7%. The dermatophytes identified were T. rubrum (68.0%), M. ferrugineum (22.0%), T. mentagrophytes (8.0%) and T. verrucosum (2.0%). Resistance observed with these isolates was as low as 21.2% to as high as 100% while sensitivity ranged from 78.8% to 100%. Only large family size significantly influenced the occurrence of T. capitis among the risk factors. Conclusion: Prevalence of Tinea capitis from this study is high. T. rubrum being anthropophilic and the predominant dermatophyte identified corroborates large family size as an important risk factor. Antifungal resistance as a cause of therapeutic failure was demonstrated by some isolates in this study.


Introduction
Tinea capitis, commonly called ring worm infection of the scalp, is predominantly seen among children, especially during the prepubescent age (Elewski, 2000). It affects the scalp hair follicles and surrounding skin causing significant hair loss (Nnoruka et al., 2007). Scalp ring worm is highly contagious and frequently spread among family members and classmates (Ameneh, 2010). Tinea capitis has a global distribution but it is more prevalent in hot humid climates than temperate regions (Ameneh, 2010). It may occur sporadically or in epidemics. It is endemic in many developing countries making it a significant infectious dermatological disease (Nnoruka et al., 2007). Males are more commonly affected than females, often with a frequency of about two to five times higher (Anosike et al., 2005).
The causative agents of T. capitis vary from one geographical region to another, though largely due to Microsporum and Trichophyton species. The predominant dermatophyte causing Tinea capitis in a given geographical region may also change over time (Elewski, 2000), hence there is a need for constant surveillance to determine the epidemiological trends of the disease.
Despite advances in preventing and treating Tinea capitis, it remains a worldwide public health problem, especially among school children in economically underdeveloped countries (Ayanbimpe et al., 2008). Acquired hair loss a common complication of the disease causes disfiguring and social stigma. In Nigeria, T. capitis is a major problem being endemic and also one of the most prevalent dermato-mycoses (Nweze, 2001). With the current global increase in antimicrobial resistance, therapy of Tinea capitis has also become a clinical challenge. The objective of this study therefore was to determine the prevalence and identify dermatophytes of T. capitis in Okelele community, a locality in North Central Nigeria and evaluate the susceptibility of isolates to selected antifungal drugs.

Materials and Methods
This cross-sectional descriptive study was carried out in Okelele community, a locality in North Central Nigeria. Three hundred and one pupils aged 5-14 years attending the seven primary schools in the community were recruited via a multistage sampling technique. Ethical clearance was obtained from the Ethics and Research committee, University of Ilorin Teaching Hospital (U.I.T.H) vide a certificate UITH/CAT/189/14/239, and written informed consent from parent/guardian of children or assent of children older than seven years were obtained before recruitment.
Pupils with clinical evidence of Tinea capitis excluding those who have been on antifungal use within fourteen days prior to recruitment were selected. Tinea capitis was identified clinically by the presence of scaly scalp, alopecia or hair loss and pus filled sores (Ali et al., 2007). A proforma designed for the study was used to obtain information on socio-demographic characteristics, clinical profile and risk factors.
Hairs and scalp scrapings were collected from each participant, into clean brown envelopes (Forbes et al., 2007) while swab stick was used to obtain sample from pus filled sores (Winin et al., 2006). All specimens were properly labeled to indicate the code for each subject, the type of specimen and date of collection. Specimens were then transported to the Mycology laboratory, Department of Microbiology, U.I.T.H., Ilorin and processed immediately.
A wet mount of each specimen was prepared in 20% potassium hydroxide (KOH). Each treated slide was examined under low(10x) and high (40x) objectives of a light microscope for the presence of spores and their distribution pattern in hairs (ectothrix, endothrix and favic type) and hyphae in scalp scrapings, crusts or pus. All specimens irrespective of microscopy results (whether positive or negative) were cultured on a pair of Mycosel agar plates (Monica, 2006). For each specimen one plate was incubated at room temperature and the other at between 35-37 o C. The culture plates were examined every other day for evidence of growth for up to a maximum of four weeks thereafter cultures were considered negative for growth (Baron et al., 2003). Isolates on culture were identified using colonial morphology and micromorphology using lactophenol cotton blue preparation. Macroscopic and microscopic features were compared to those contained in a practical mycology text (Ellis et al., 2007). Further characterization of the isolates was done using the hair perforation and urease tests.
Isolates obtained were subjected to Agar Disk-diffusion antifungal susceptibility test (Rex et al., 2006). The standardized inocula of the test organisms were prepared from fresh cultures made on chloramphenicol supplemented Sabouraud Dextrose Agar (SDA) slants (Barry et al., 2000). On to each young culture of fungal isolate, 3mls of 0.9% normal saline was dispensed. With the aid of a sterile glass rod the dermatophyte isolates were gently scrapped to make a suspension. The organisms' mixtures were filtered using whatmann filter paper No 1 to remove mycelia and hyphae.
The filtrate which contained the spores was then standardized using 0.5 Mac Farland turbidity standard that corresponds to a cell density of 1.5x10 8 cfu/ml.Using sterile swab sticks SDA plates were then streaked evenly with the standardized inocula suspensions (Rex et al., 2006). The antifungal discs namely Griseofulvin 25µg, Terbinafine 30µg, Itraconazole 8µg, Fluconazole 25µg by Oxoid, Ketoconazole 10µg, Miconazole 10µg and Clotrimazole 10µg from Mast Diagnostica were then applied to the surface of the inoculated agar plates. The inoculum of T. mentagrophytes ATCCMYA 4439 was similarly prepared and inoculated as control. All plates were incubated at room temperature for two to five days. Plates were examined daily for growth. After growth, the zones of inhibition for the test and control organisms were measured using a meter rule calibrated in millimeters. These measurements were compared with the CLSI standards.
Statistical analysis was performed using the Statistical Package for Social Sciences (SPSS) version 16 software. Descriptive analyses of variables were used to summarize data. The frequencies and mean (±sd) were generated for categorical and continuous variables respectively. Quantitative and qualitative demographic characteristics were summarized. Results were presented in tabular and pictorial forms. Categorical data were compared using Pearson's chi square test. To determine the predictor(s) of the presence of Tinea capitis, binary logistic regression analysis was done. Odds ratio were determined with respective confidence intervals. A p-value ˂ 0.05 was taken as statistically significant. Conclusions and recommendations were based on scientific evidence from the results.

Results
A total of 301 pupils participated in this study and 228 had mycologically proven Tinea capitis giving a prevalence of 75.7% (Confidence Interval = 73.0-78.5). Sixty-six (21.9%) out of the 301 participants were from Okelele A school, with the highest population of pupils; and the largest number 67 (22.3%) of the participants were from primary three class. Two hundred and fifty-nine (86.0%) of these children were males while 42 (14.0%) were females, giving a male to female ratio of 6.2: 1.0. The mean age of the pupils was 9.81±2.41. Nineteen (6.3%) of the pupils belonged to high socio-economic class (SEC) while majority 219 (72.8%) were from low SEC (Table 1).  Table 2 presents the prevalence of Tinea capitis according to age and sex. There was no significant difference in the infection rate between the two age groups (p=0.270) and across gender (p=0.275).  5 The antifungal susceptibility assay shows that 78.8% of the T. rubrum isolates were sensitive to clotrimazole and miconazole, 21.1% were resistant to both drugs, while all isolates (100.0%) of T. rubrum were resistant to ketoconazole, fluconazole, itraconazole ,griseofulvin, and terbinafine. M. ferrugineum and T. mentagrophytes were 100.0% sensitive to clotrimazole, miconazole and terbinafine. In addition, T. mentagrophytes showed 100.0% intermediate susceptibility to fluconazole. T. verrucosum was 100.0% resistant to all the antifungal drugs (Table 3).   Table 4 shows that large family size had significant association with the occurrence of T. capitis (p< 0.05). Other risk factors were not significantly associated with its occurrence (p> 0.05). The odds of Tinea capitis occurring in children with large family size was two times higher compared to those with small family size (OR=2.025, 95% CI=1.185-3.461, p=0.010).  (Adegbola, 2008), *** ≥ 3 people living per room is taken as overcrowding (Park, 2005), OR = Odds ratio, C I= confidence interval.

Discussion
The prevalence (75.7%) of Tinea capitis obtained, demonstrated a high infection rate among the school children. Several factors such as low standard of living, poor sanitary and hygienic conditions that are characteristics of rural communities especially in developing countries might have accounted for this. Lack of surveillance and control measures could also account for increased incidence of T. capitis over time. This prevalence was higher than the 31.2% reported for Tinea capitis by Ayanbimpe et al. (2005). The markedly lower prevalence obtained in previous studies (Menan et al., 2002;Sidat et al., 2007 ) represented prevalence of clinically suggestive T. capitis as opposed to prevalence of mycologically proven Tinea capitis among children with clinically suggestive disease reported in the present study. The variations in prevalence could also be due to the sampling methods used and could be a reflection of people's habits, climatic conditions, standards of hygiene and levels of education all of which can influence predisposition to Tinea capitis (Fathi and Samarai, 2000). Adefemi et al. (2011) in a prevalence study among school children in Oke-oyi reported T. capitis as the predominant dermatophytosis where it was found to account for 76.1% of cases. This percentage, though comparable to the one obtained in the present study, represented the proportion of children with T. capitis. The prevalence of Tinea capitis was not influenced by age or sex. This probably indicates similar exposure to infection irrespective of age or sex.
The predominant causative agent of Tinea capitis among the children was T. rubrum. Others were M. ferrugineum, T. mentagrophytesand T. verrucosum in decreasing order. Out of these dermatophyte species T. rubrum and M. ferrugineum are anthropophilic, T. verrucosum is a zoophilic dermatophyte while T. mentagrophytes has both anthropophilic and zoophilic varieties. This finding shows a predominance of anthropophilic dermatophytes. The ecology of these dermatophytes indicates more commonly the transfer of infection from person to person and less commonly, from animals to humans. The affected children might be sharing personal care items such as combs, towels, hats and beddings which could serve as vehicles for the transfer of infective agents among them. Also, close interaction at home with infected family members and at school with infected playmates might have exposed them to infection with anthropophilic agents. Ayanbimpe et al. (2005) found T. soudanese as the predominant causative agent of Tinea capitis among other isolates. T. mentagrophtes was reported as the major isolate by Adefemi et al. (2011). In Turkey, Altindis et al. (2003) reported T. violaceum as the predominant isolate while in Ivory Coast, Menan et al. (2002) reported T. soudanese as the commonest etiological agent of Tinea capitis.
In Mozambique, M. audouinii was reported as the major isolate (Sidat et al., 2007). These findings indicate variations over time, in the aetiological agents of Tinea capitis within the same geographical area and from one geographical area to the other as earlier reported (Ameh and Okolo, 2004;Sidat et al., 2007 ).
Griseofulvin is the drug of choice for Tinea capitis and has been in use for several decades. All four dermatophyte species reported in this study were found to be resistant to griseofulvin. Repeated exposure of these fungal agents to griseofulvin and the long duration required for griseofulvin therapy with possible failure of compliance could account for the resistance. This observation was previously documented by Al-Refai (2007), thus suggesting possible reasons for treatment failure that may be experienced with the use of the drug. Similarly, all isolates were resistant to itraconazole and ketoconazole but T. mentagrophytes showed intermediate susceptibility to fluconazole. The relative safety and ease of delivery of the azoles has led to widespread use of these drugs both for prophylactic and therapeutic purposes. Repeated exposure to these drugs could result in secondary resistance (Rubio et al., 2003). The intermediate susceptibility observed with fluconazole could mean that higher drug concentration would be needed to achieve therapeutic effect. Some studies (Pakshir et al., 2007;Singh et al., 2007) however, have documented fluconazole to have less activity against dermatophytes. With the exception of T. verrucosum, the isolates were sensitive to miconazole and clotimazole. Mukherje et al. (2003) documented primary resistance of T. rubrum to terbinafine and this might suggest why all the T. rubrum isolates in this study could have intrinsic resistance to terbinafine. Although T. mentagrophytes and M. ferrugineum were sensitive to terbinafine, terbinafine is not commonly used for the treatment of Tinea capitis.
Tinea capitis, like any contagious disease, has associated risk factors. This study found a significant association between family size and Tinea capitis. Children from large families were twice as likely to have Tinea capitis compared to those with small families. Large family size with limited income for family upkeep encourages the sharing of personal care items. These items could act as vehicles facilitating the transfer of fungal spores from one infected child to the other which could lead to spread of the disease. With inadequate living space, large family size could result in overcrowding. This may encourage close interaction between infected and non-infected family members leading to transmission of infection, although this study did not find statistically significant association between overcrowding and Tinea capitis. Other risk factors such as socio-economic class, malnutrition, presence of scalp lesions in family members and exposure to domestic animals and pets were not significantly associated with Tinea capitis. These findings are in contrast with reports from previous studies (Nweze, 2001;Ameh and Okolo, 2004;Adefemi et al., 2011) where these risk factors were found to be associated with Tinea capitis.