Molecular Typing of Dermatophytes Isolated from Pupils and Staff Members of Some Selected Schools in Anambra State, Nigeria

Dermatophytes are the fungal pathogens of human and animals infecting the keratinized tissues of the body namely skin, hair and nails. They include species of Trichophyton, Microsporum and Epidermophyton . This study was aimed at molecular typing of dermatophytes isolated from school pupils and staff members of some selected Schools in Anambra East and Ayamelum L.G.A s of Anambra State in Nigeria. One thousand (1000) samples (scalp/hair, nail, feet, glabrous skin and groin/perianal) were collected from pupils and staff members of both gender and age bracket of (1 to 10, 11 to 20, 21 to 30 etc ) years that showed visible signs of skin infection located in these two Local Government Areas. Standard procedures were employed in processing of test samples and inoculation on dermatophyte test medium. The plates were incubated at room temperature (25 – 27 0 C) for 7 – 10days for observation of fungal growth. Colonial, morphology, and molecular studies and sequencing were used for identification. Sensitivity was performed using sterilized discs (6mm) prepared from whatman No. 1 filter paper, impregnanted with different concentrations (25mg, 50mg, 100mg and 200mg) of terbinafine, itraconazole, ketoconazole, fluconazole and griseofulvin dissolved in 2% dimethylsuphuroxide (DMSO). Molecular studies were used as confirmatory tests on dermatophytic isolates using Sanger sequencing method. The results show that the dermatophytic isolates includes: Trichophyton tonsurans, Microsporum audounii , Trichophyton rubrum , Microsporum canis, Trichophyton violaceum, Trichophyton verrucosum, Trichophyton mentagrophytes, and Epidermorphyton flocossum. Results also revealed the nucleotide sequences of the dermatophytes and genetic relationships between isolated dermatophytes from different pupils and schools. PCR-RFLP was used as gold standard for the diagnosis and Confirmation of Source of infection of dermatophytes and can aid in initiating prompt and appropriate antifungal therapy. Phylogenetic tree was drawn to show the relationships between the isolates.


INTRODUCTION
Dermatophytes are the fungal pathogens of humans and animals infecting the keratinized tissues involvement like skin, nails and hairs [1]. Dermatophytoses are fungal infections that specially only superficial keratinized tissues of the body, skin, hair, nails [2]. Dermatophyte infection is generally cutaneous and restricted to the non-living cornified layers due to of the unable of the fungi to penetrate the deeper tissues or organs of immunocompetent hosts [3]. These cutaneous mycoses are the most common fungal infection of man and are often called Tinea or ringworm [4].
Dermatophytes are transmitted mostly by direct contact with infected host (human or animal) or by direct or indirect contact with infected exfoliated skin or hair in clothing, combs, hair brushes, theatre seats, caps, furniture, bed linens, shoes, socks, towels, hotel rugs, bath house, and locker room floors [1]. Depending on the species, the organism may be active in the environment for up to 15 months. These fungi have worldwide distribution and at present there are 40 recognized species in the dermatophyte genera. About 25 species belonging to the genera Epidermophyton, Microsporum and Trichophyton are presently known to infect man [5]. But recently up to 40 species are found to infect man [6].
The problem of accurate definition and characterization of microorganisms (bacteria, fungi, viruses, parasites,) has always been of great important in phylogenetic analysis and taxonomic identity, clinical microbiology and epidemiology.
Initially, the study of dermatophytes was based on colony and microscopic morphology, but identification of these keratinophilic organisms was sometimes difficult because of their overlapping characteristics, variability and pleomorphism.
Phylogenetic relationships and species-specific sequences could not be entirely defined by the previous methods, more technique known to resolve genetic disjunctions between very closely related species was used. Sequencing of the hyper variable internal transcribed spacer (ITS) region, consisting in Ascomycetes of ITS 1, ITS 2 and the intermediary 5·8S rDNA, was a choice tool allowing the elucidation of the phylogeny of closely interrelated filamentous fungi [7]. A total of 54 strains belonging to 41 recognized species and varieties of the family Arthrodermataceae have been studied using this procedure [8]. The studies showed that geophilic species were highly separated from the other members of the Arthrodermataceae. In contrast, the genera Trichophyton and Microsporum were not resolved in the phylogeny obtained with this marker. The geophilic species of Stockdaleae was shown to be in need of reclassification. M. canis, M. Audouinii and M. equinum were found to be very closely related. The T. mentagrophytes complex and its Arthrodermarelated teleomorphs were classified into three groups: T. mentagrophytes, a Neotypified T. interdigitale related to A. Vanbreu seghemii and T. erinacei related to A. benhamiae. To accurately investigate the taxonomic structure of the T. mentagrophytes complex and T. tonsurans, the same authors studied 24 related species or varieties using ITS sequencing [9]. Their analysis revealed that these 24 taxa, many of them long-disused older names, could be reduced to only five species, including the three pre-defined members of the T. mentagrophytes complex, as well as T. simii, the anamorph of A. simii, and T. tonsurans. These data are in agreement with genotypic results obtained by mtDNA restriction fragment length polymorphism (RFLP) [10], CHS1 gene sequencing [11] and ITS 1 sequencing [10], in line with previous phenotypic observations. In addition, [1] found that physiological criteria are not in conflict with genetic groupings for most of the strains studied.
Laboratory diagnosis of dermatophytosis requires the association of a positive direct examination of clinical specimens showing characteristic septate hyphae and the dermatophyte cultural isolation on Sabouraud medium. Since direct examination does not allow species identification and dermatophytes generally grow in 1-3 weeks, new techniques allowing a fast and reliable diagnosis of dermatophytosis combined with species identification could be valuable. In addition, several keratinophilic organisms classically responsible for superficial and mild infections had been reported from deep-seated infections in patients with severe immunodeficiency [12].
The gene coding for the small ribosomal subunit (18SrRNA) is highly conserved and is thus a choice tool for detection of fungi, followed by hybridization with species-specific oligonucleotides. This PCR-based methodology was used by [12] in 69 skin and nail specimens allowing diagnostic distinction between dermatophyte and Candida infections. Restriction analysis of the PCR products could distinguish between dermatophytes, yeasts and moulds. The sensitivity of the technique, calculated at 92%, was higher than that of culture (73%). However the use of these techniques in clinical material is of debatable value. Skin, hair and nail are not sterile sites and, for example, arthroconidia could possibly be detected in the scalps of uninfected subjects close to a child with Tinea capitis. In these cases, PCR would not be able to distinguish contamination from infection. Therefore, the purpose of the present work was to undertake molecular typing of dermatophytes isolated from pupils and staff members of selected schools in Anambra East and Ayamelum L.G.As of Anambra State, Nigeria.

Study Design/Population
The study design was convenient sampling method. There are 21 Local Government Areas in Anambra State of Nigeria. Ayamelum and Anambra East Local Government Areas are included and are situated in lowland swampy area of the state. Seventeen schools were randomly selected in the two Local Government Areas sampled population were derived.
This study was conducted in Nursery and Primary Schools in Ayamelum and Anambra East Local Government Areas. Samples were collected from school children, adolescents and some staff that are adults. Five hundred (500) samples each were collected from males and females of ages 1-10, 11-20, 21-30, 31-40, 41-50, 51-60 in both LGA. Ayamelum and Anambra East L.G.A has moderate population with most of the inhabitants being traders, farmers, fisherman, civil servants and artisans but predominately farmers. "Omanbala River" which also is called Anambra River encloses Anam, Umueri, Aguleri and Nando and flows into the River Niger and this made it possible for about 10% of them being fishermen.

Sample Collection
Clinical samples of hair, nail, skin and groin were collected from the participants. The infected areas in the body were swabbed with 70% ethanol to remove surface contaminants. The skin scraping was taken from the border areas of lesions with the help of a sterile scalpel and placed in sterile Petri dishes or between two clean microscopic slides in clean envelopes and transported to the laboratory. While collecting skin specimens moist exudates present on the lesions were also collected and examined.
The nail was collected by shaving nails that have been cleaned with 70% ethanol. The scrapings were taken from the proximal to the distal end of the nail. The samples collected were labeled with the patient's Name, Age, Sex, Date of collection, Code of the patient, and location of the infection and was taken to the laboratory for processing.

Direct microscopy
A potassium hydroxide mount was prepared by placing a few drops of 10% potassium hydroxide on a clean glass slide. The specimen was placed in the solution and allowed to stand for 30 minutes. A gentle heat was applied through a Bunsen flame to facilitate softening and clearing of the keratin found in the specimen.

Culture and isolation
Sterile Petri plates containing dermatophyte test medium were inoculated with scalp scraping and hair samples that were collected from infected subjects. This medium is a selective and chromogenic medium that permits the growth of dermatophytes and these organisms give the medium a reddish coloration. These plates were incubated at room temperature of about 25 -27 0 C for up to 10 days during which the plates were observed for growth. Each fungal growth was subcultured on SDA to obtain a pure culture which was then stored in agar slants for molecular studies.

Identification
Colonial morphology on dermatophyte test medium was used for preliminary identification of the dermatophytes. Pure fungal colonies were also subjected to lactophenol blue staining for microscopic observation of specialized hyphae and the morphology of their macronidia, micro conidia and chlamydospores. IIC Column and centrifuged at 10,000 x g for 1 minute. The Zymo-Spin TM IIC Column was transferred to a clean 1.5 ml microcentrifuge tube and 100 (35 minimum) DNA Elution Buffer added directly to the column matrix, and centrifuged at 10,000 x g for 30 seconds to elute the DNA.

PCR protocol
Ten(10) μl of One Taq Quick-Load 2X Master Mix with Standard Buffer (New England Biolabs Inc.); 1 μl each of forward and reverse primers; 7 μl of Nuclease free water and 1μl of DNA template was used to prepare 20 μl reaction volume of the PCR cocktail.
The reaction was gently mixed and transferred to a preheated thermal cycler.
Amplification conditions for the PCR were as follows: 5 min at 94 0 C to denature the DNA, followed by 35 cycles of denaturation at 94 0 C for 30 secs, primer annealing (internal transcribed spacer 1 and 2) at 50 0 C for 30 secs and strand extension at 68 0 C for 10 minutes on an Eppendorf nexus gradient Mastercycler (Germany). PCR products were separated on a 2% agarose gel and DNA bands were visualized with syber gold.

Sequencing protocol
PCR products were cleaned using ExoSAP Protocol as follows: The Exo/SAP master mix was prepared by adding the following to a 0.6ml micro-centrifuge tube:-50μl Exonuclease I ( The cleaned products were injected on the Applied Biosystems ABI 3500XL Genetic Analyser with a 50cm array, using POP7 Sequence chromatogram analysis was performed using Finch TV analysis software.

RESULTS
Out of the total of 1000 cultured samples collected, 320(32%) yielded growth for dermatophytes. Out of this 120 were The prevalence of dermatophytosis is shown on Table 1 and T. tonsurans is the most prevalent in the two Local Government Areas followed by T. rubrum , M.audounii , T. violaceum , T. verrucosum , T. mentagrophytes, M. canis and E. floccosum . Total number of samples collected and cultured were 1000 and out of this 320 were positive for dermatophytes. Table 2

Phylogenetic Tree
The phylogenetic tree portrays the genetic relatedness of the dermatophytic isolates. This is achieved by using paralogues and homologues. The paralogues showed that the organisms are not related while homologues showed that the isolates have common ancestors: Dermatophyte isolate 13 (Epidermophyton flocossum) is paralogue to dermatophytic isolates 5 (Microsporum audounii); Dermatophyte isolate 2 (Microsporum audounii) and Dermatophyte isolate 10 (Microsporum canis).

DISCUSSION
Dermatophytes are fungi infection of the scalp, skin, and hair shaft that are caused by filamentous fungi. Development in the application of nucleic acid amplification technology has enhanced the quality of dermatophyte detection. Several nucleic acid-based molecular methods have been developed to detect fungi from clinical specimen targeting 18SrDNA, ITS1 and ITS 2 regions, 5.8SrDNA and 28SrDNA. Therefore, in the present study, two targets of the fungal genomethe ITS region and 18SrDNAwere chosen as they have cleavage sites that could be of value for application of RFLP (restricted fragment length polymorphism) on the amplified product to detect dermatophytes. Out of 1000 samples collected, consisting of 500 samples each from Anambra East and Ayamelum L.G.A of Anambra State; 320 were both culture and KOH positive out of this 12 of the positive samples were subjected to molecular typing and all were positive. This agrees with the work of Mochizuki et al, [13]. PCR and RFLP is rapid and results can be obtained within 8 -9 hours from samples directly from DNA extraction to reporting by electrophoresis. This technique is more accurate, sensitive and from source detection and also for epidemiology identification of the disease.
Phylogenic tree indicates that ancestral image gave rise to all organism on the tree. A branch point indicates where the two lineage diverged. A linage that evolved early and remains unbranched is a basal taxon. When two linage stem from the same branch point, they are sister texa. It also helps in knowing the evolutionary history of organisms or groups of organisms. It shows ''How and when other branches of phylogenic tree have evolved from the main stock. It discloses the time of origin and subsequent evolution from simple to complex.
From the molecular DNA and protein extractions of samples from pupils and staff of Anambra east and Ayamelum L.G.A, there was relationship between the Tricophyton.tonsurrans isolated from the scalp lesion of 26 years old male staff working in school H, and scalp region of a 8 year old female pupil attending school E, and groin lesion of a 53 years old female staff teaching in school L in the same area. There was also a very close relationship between Microsporum audounni isolated from scalp region of a 14 year old male pupil in school K and the one isolated from skin lesion of a 39 year old male staff at school N, is of the same ancestors with the one isolated from the scalp lesion of a 12 years old female pupil at school A. Microsporum canis isolated from a skin lesion of a 7 year old female pupil in school H has no ancestral relationship with other Microsporum species isolated. Tricophyton mentagrophytes isolated from the scalp lesion of a 13 year old female pupil attending school K also has no ancestral relationship with other Tricophyton mentagrophytes in ine with [14]. From all the comparisons made from the isolates the phylogenic trees was used to estimate the relationships among the species represented by these sequences.

CONCLUSION
Dermatophytic infections are common contagious disease. Primitive diagnosis of dermatophytosis can be done by KOH mount and culture, which takes longer time to report and cannot differentiate at the level of genus and species level. Results from this study indicate that PCR -RFLP may be considered as gold standard for the diagnosis and confirmation of source of infection of dermatophytosis and can aid the clinician in initiating prompt and appropriate antifungal therapy.
In conclusion, From the 12 isolates that went through molecular typing using the DNA and protein sequences, the phylogenic trees showed a very close genetic relatedness between Tricophyton tonsurance isolates from different pupils and schools. This also include Microsporum audounii and Tricophyton rubrum, Microsporum canis and Tricophyton metagrophytes. There was no ancestral relatedness between Microsporum canis and Tricophyton metagrophytes.

ETHICAL APPROVAL AND CONSENT
Ethical clearance was sort for and was given by iyi enu mission hospital with reference number IEH/ REC/01/VOL 1/ 1. A consent letter was signed by all the Guardian and Parents of all the participant .