In Tanzania, previous studies have shown that tinea capitis is one of the major skin diseases reported in school children (Andrews and Burns, 2008). Other studies have documented prevalence rates between 12 and 55% of dermatomycosis among school-age children (Ferie et al., 2006; Komba and Mgonda, 2010). However, the prevalence and fungi associated with tinea capitis among children in Tanzania have not been sufficiently investigated. This study isolated and identified diverse fungi associated with tinea capitis in selected school children in Morogoro Municipality, Tanzania. The findings of this study will enhance awareness of skin fungal infections in schools and households and assist the educational and health sectors in formulating appropriate tinea capitis control strategies 

Polymerase chain reaction (PCR) and DNA sequencing

The composition of PCR master mix using DNA polymerase consisted of Dream Taq polymerase (10 µl), RNase-free water (7 µl), ITS 1F primer 10 µM (1 µl) and ITS 4 primer 10 µM (1 µl) for single reaction. Amplification was done by adding to the reaction mix solution 1 µl of extracted DNA as a template. Amplification of the 5.8 rRNA and flanking ITS1 and ITS2 was done using specific primers ITS 1F (5’-CTT GGT CAT TTA GAG GAA GTA A-3’) and ITS 4 (5’-TCC TCC GCT TAT TGA TAT GC-3’) (Balajee et al., 2007; Iwen and Hinrichs, 2002). The amplification conditions consisted of an initial denaturation at 94°C for 10 min followed by 40 cycles of denaturation (45 Sec at 94°C), annealing (45 s at 55°C) and extension (1 min at 72°C), and a final extension at 72°C for 10 min. Amplification was performed in an automated thermal cycler (Applied Biosystems, Foster City, CA). The PCR products were electrophoresed in a 2% agarose gel mixed with Gel Red nucleic acid stain (Phenix Research Products, Candler, NC). Visualization and imaging was done using a GelDoc-EZ Imager (Bio-Rad Laboratories, Hercules, CA). In order to verify the retrieval of fragments of fungi, PCR fragments were purified from the agarose gel using NucleoSpin gel and PCR clean-up kit (Macherey-Nagel, Duren, Germany) and subjected to dideoxynucleotide cycle sequencing using Big Dye Terminator Cycle Sequencing Kit Version 3.1 (Applied Biosystems, Foster City, CA). Products from dideoxynucleotide cycle sequencing reaction were purified by ethanol precipitation and separated on a 3500 Genetic Analyzer (Applied Biosystems, Foster City, CA). The obtained sequences were deposited at GenBank and assigned with accession numbers. In order to identify the isolated tinea capitis, ITS1-5.8S-ITS2 nucleotide sequences were aligned with sequences deposited at GenBank database using a BLASTn tool (www.ncbi.nlm.nih.gov/blast).

Morphological fungi identification

Fungal cultures on SDA were considered positive if there was growth after 3 days of incubation or negative if there was no growth following 7 days of incubation. Fungi with varying structures and color on primary SDA culture presented mixed colonies of varying texture and color, including white, cotton-like, and green crenated ones. Others appeared with radial furrows, grey cotton-like and creamy shinning colonies. Sub-cultivation of the isolates achieved pure colonies with different presentations as shown in Figure 1.

Following sequencing of the PCR products and conducting BLASTn at GenBank, the fungal isolates that were found to be associated with tinea capitis were identified as Trichoderma longibrachiatum, Cytobasidium minutum, Aspergillus spp, Ectophoma multirostrata, Aureobasidium pullulans, Aspergillus flavus, Cladosporium tenuissimum, Penicillium flavigenum, and Fusarium solani (Table 1).

Socio-demographic characteristics and prevalence of Tinea capitis

A total of 72 school children were recruited for the study subjects, 12 (16.67%) had tinea capitis. Out of the 10 primary schools surveyed, Chamino had 11 (15.3%) cases which was the highest overall prevalence and the difference was statistically significant (p < 0.05). The age range of the school children was between 6 and 14 years and boys, n=39 (54.2%), were more affected than girls, n=33 (45.8%). However, there was no statistical significant difference (p > 0.05) between the genders. The majority of children belonged to the age group of 9 to 14 years 62 (86.1%) and among school children of class five, 28 (38.9%), this difference was statistically significant (p<0.05).

Awareness of risk factors for Tinea capitis

Upon assessment of the habits and behaviors of school children regarding hair hygiene, of the 72 school children interviewed, 31 (43.1%) reported to wash their hair at least once a day and 61 (84.7%) regularly visited the barber for haircut. Only 18 (25%) of the infected school children did seek treatment at a health facility while 47 (65.3%) children used drugs to self-treat the infection. Thirty (41.7%) school children commonly shared combs at home and 39 (54.2%) children had an infected member in the family (Tables 2 and 3).

The present study investigated the prevalence, socio-demographic characteristics and identification of fungi associated with Tinea capitis in school children in primary schools of Morogoro. This study has shown an overall prevalence of tinea capitis in 10 primary school children in Morogoro region to be 16.67%. This prevalence is slightly lower than in previous reports, 48.86% in the Tanzania (Chikoi et al., 2018; Komba and Mgonda, 2010). The lower prevalence observed might be due to differences in the study populations of the studies with respect to, their socio-demographic and behavioral characteristics.

Identification of fungi isolates by morphological and molecular techniques has indicated that the following fungi are associated with tinea capitis: Trichoderma longibrachiatum, Cytobasidium minutum. Aspergillus spp, Ectophoma multirostrata, Aureobasidium pullulans, Pichia terricola, Aspergillus flavus, Cladosporium tenuissimum, Penicillium flavigenum and Fusarium solani. The two most common fungal agents of tinea capitis infection have been reported to be Trichophyton tonsurans  and  Microsporum canis (Komba and Mgonda, 2010). Some of the dermatophytes identified in the study such as Aspergillus spp have been reported elsewhere to be causative agents of the tinea capitis in children (Jia et al., 2019; Wiegand et al., 2017). The study findings are in agreement with studies conducted in Uganda in which reported moulds A. niger, F. oxysporum, and S. brevicaulis in 12 cases by culture methods. The Uganda studies raised the question whether these moulds were potential agents of tinea capitis in the children or if were mere  contaminants.  So   far   there   is   no  confirmatory evidence that moulds are capable of causing tinea capitis (Wiegand et al., 2017). Recently, (Chokeva et al., 2016) discussed the possible role of A. niger in tinea capitis infection and concluded that pathogenic moulds could be ‘new’ etiologic agents of Tinea capitis in children, especially in areas of social deprivation and poor living environments.

This study has revealed that tinea capitis is particularly prevalent in children between the ages of 9 and 14 years. This  is  in  agreement  with  other  studies,  that  children below the age of 10 years were highly infected (George and Altraide, 2008), suggesting that dermatophytosis, especially tinea capitis, is predominantly a pre-pubertal disease (Moto et al., 2015). The infection was more prevalent  in   male   school   children   (54.2%),  possibly because parents/guardians are more insistent on the hygiene of female than their male children (Falahati et al., 2003). Furthermore, frequency of hair wash among the children could reduce tinea capitis because children who washed more frequently presented lower infestation rates (Afolabi et al., 2018; Chikoi et al., 2018). It was also reported that 84.7% of the barber’s shops did not clean the shaving devices regularly after a haircut.

Hospital attendance was reported by only 25% of the infected children with tinea capitis, possibly due to lack of resources to pay for treatment by parents/guardians, who resorted  to  applying  herbs  or  other  cheaper remedies, with questionable efficacy (Chikoi et al., 2018; Ferie et al., 2006). This study further, showed a significant association between tinea capitis infection and contact with domestic animals such as cats and dogs, (p<0.05). It is suspected that the infection may be shared between children and domestic animals (Ameh and Olkolo, 2004). Studies elsewhere have  reported  that sharing of clothes, towels, and combs facilitate transmission of tinea capitis (Chikoi et al., 2018). In this study, there was no significant association between infection and sharing of hair combs, beds or possible contact with an infected family member (p> 0.05). This is in line with previous findings in the southern part of Tanzania (Chikoi et al., 2018).

The findings of this study indicate a high level of tinea capitis infection among children attending government-owned public primary schools in the Morogoro Municipality, Tanzania. Apart from T. tonsurans and M. canis, diverse dermatophytes are potentially opportunistic etiologic agents of tinea capitis in children, especially those living in environment with poor hygienic practice. The severity of tinea capitis among school going children is moderately high and that socio-demographic and behavioral factors profoundly influence the development and occurrence of the infection in children. There was a significant association between tinea capitis and certain unhygienic practices, and contact with domestic animals. This suggests an animal–human transmission of tinea capitis is likely in the study area; however, this study did not carry out a detailed investigation to confirm this transmission route. Majority of the affected children did not seek for medical assistance; hence, school health programs must emphasize on the importance of skin and hair hygiene to enable early detection, prevention and eventual treatment of dermatophytosis among school children. Since the study population was small and this being a point prevalence cross-section study, the tinea capitis infection is most likely underestimated.

The authors have not declared any conflict of interests.

This work was supported by the Government of the United Republic of Tanzania through the Higher Education Student Loan Board (HESLB). The authors appreciate the College of Veterinary Medicine and Biomedical Sciences of Sokoine University of Agriculture for granting permission to conduct this research in the Department of Veterinary Microbiology, Parasitology and Biotechnology. The authors are also grateful to individuals who assisted with data collection and critical review of the final drafts of the manuscript.