2022.07.03.25
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Submission and Phylogenetical of Local Isolated Trichophyton interdigitale of Iraqi Patients in NCBI.

College
of Education for Pure Science Ibn-Al-Haitham / University of Baghdad / Baghdad
-Iraq
Corresponding
author: atyafexeter@gmail.com,
Available from: http://dx.doi.org/10.21931/RB/2022.07.03.25
ABSTRACT
Dermatophytes are species with slight genetic variation, and
are yet several uncertainties about the differences among species. This study
aims to isolate and diagnose the Trichophyton interdigitale by molecular
technique and to reveal the phylogenetic distance and similarity of the Iraqi
isolates to other isolates from the globe, in addition, to submit the obtained
sequences to the NCBI database. This study included 86 with multiple
lesions on different parts of the body. The results showed different variations
within the ITS gene between the isolates. It was concluded that Trichophyton
interdigitale in Iraqi isolates had two types of substitution variations
(Transition and Transversion) different than global isolates. Moreover, it
appeared according to the phylogenetic tree, the similarity was 97% with
isolates from the Czech Republic and Japan, whereas 98% with the isolates from
France and Germany.
Keywords. Dermatophytes, genetic
variations, ITS gene, multiple lessons
INTRODUCTION
The keratinophilic group is categorized
by several genera such as Trichophyton, Microsporum, Epidermophyton, Nannizzia,
Paraphyton, Lophophyton, and Arthroderma; additionally, one of the well-known
fungi which belong to the genera are dermatophytes. Those fungi can infect both
humans and animals and can cause severe conditions 1
Dermatophytes are fungi that can
cause dermatophytosis as they can produce protease and keratinase, which
degrade keratin and thus lead to invasion and infection of different body sites.
Genetic factors have a central role in the pathogenesis of dermatophytosis 2. One of the examples
of anthropophilic is Trichophyton interdigitale, which causes
dermatophytosis. Keratomycosis, also known as fungal keratitis, is a cornea
infection. If this infection has been misdiagnosed or has been delayed to
diagnosis can lead to loss of vision 3. Several studies
have been done to know the incidence distribution in the different geographical
areas of the world, including Iraq, specifically, Baghdad governarate, by Minnat and Jinan4, Jordan 5,
Kuwait and Saudi Arabia mentioned in
previous study 6.
The most well-known technique to diagnose and differentiate
between species and sub-species is
PCR-based assays, considered a quick method 7.
Following PCR, sequencing is an effective method and informative for diagnosing
fungal infections. Classification and taxonomy can be further improved by
phylogenetic analysis of internal transcribed spacer
(ITS) gene 8. Therefore, the current research
aimed to isolate Trichophyton
interdigitale, sequence the
ITS1 gene, and compare the resulting sequence with globally NCBI database
sequences.
MATERIALS AND METHODS
Subjects: this study included 86 people admitted
to the dermatology department of (Al-Yarmook teaching hospital) with multiple
lesions on different body parts. One of the subjects has lost vision in a single
eye. Dermatological examinations of all the subjects showed extensive
erythematous plaques with clear borders and scales, scattered red papules with
ulceration, and scabs throughout the body. The patients complained of mild
itchiness over the lesions and pain in the left eye.
The ethic
consent
The consent for the collection of multiple lesions on
different parts of the body was obtained from the participants, and also the
study was consented to by ethical committees in the Iraqi Ministry of Health/
Al-Yarmook teaching hospital/Baghdad-Iraq)April 2021-August 2021).
Excluded criteria: Patients
with diabetes, eye trauma, or other significant medical disorders have been
excluded.
DNA extraction
DNA has been extracted from all the samples by using a commercial
kit (ZR Fungal/Yeast/Bacterial DNA MiniPrep, Zymo research) .
Amplification
of Gene ITS: the
amplification of the ITS gene was done by oligonucleotides that specify
a region of 600bp a sense
primer (ITS F: 5′- TCCGTAGGTGAACCTGCGG -3′) and antisense primer sequence (ITS
R:5′ TCCTCCGCTTATTGATATGC-3′) (Xie et al., 2008) Primers supplied by IDT (Canada). The final volume of PCR was 25 µl which included 3µl of eluted DNA, 10 µl PCR Pre Mix (Intron,
Korea), 0.5 µl of primer sense and antisense then nuclease-free water was used
to complete the volume to 25 µl. After the addition of all the components, the
prepared samples were placed into a thermal cycler machine and programmed as
follows;: 3 min of denaturation at 94, followed by 35 cycles of 94 °C for 45s, 52°C
for 1 min and 72 °C for 1min with final incubation at 72 °C for 7 min using a Thermal Cycler (Gene Amp, PCR system
9700; Applied Biosystems).
RESULTS
The DNA was
extracted successfully as the electrophoresis results of the eluted DNA showed
a single band by all the samples. After the PCR run, the amplified product also
resulted in a single band in electrophoresis at 600 bp for the positive
samples, as shown in figure (1; A), and the results showed that 10 out of the
86 samples (11.6%) were positive with Trichophyton interdigitale. The
results of electrophoresis are shown in figure (1).

Figure 1. A; PCR product the
band size 600 bp. The product was electrophoresis on 2% agarose at 5
volt/cm2. 1x TBE buffer for 1:30 hours. N: DNA ladder (1000 plus).
B; Gel electrophoresis of
genomic DNA extraction from fungi, 1.5% agarose gel at 5 vol /cm for 1 hour.
The amplified
region of the 10 positive samples has been sequenced by micro gen company
(Korea), and each resulted sequence aligned to a high score nucleotides
sequence within the BLAST database; the results in figure (2) showed the
sequence of the sample (2) aligned to a sequence that shown a high score (568-bit score) and 98% compatibility which accompanied with zero
gaps. For further analysis, nucleotides 347bp (corresponding Trichophyton
interdigitale isolate AJJM7 internal transcribed spacer gene positions of
the GenBank accession no. MH383047) of the ITS gene were used. The sequences
were analyzed by using NCBI/BLAST. This study's obtained sequence of ITS gene was
deposited in GenBank under accession nos. OL673018.
Summarized details for two types of substitution
variations are shown in table (1); sample sequences have been labeled by
numbers as shown in the table. The various sequence percentage was 2% for both
samples 2 and 5. And this percentage of difference includes 5 variations; two
Transition (first one is T\ C located in the 74bp and the second one is A
\G located in the 238bp) and three transversions (T\ G in 73bp, T\ G in 72bp
and A \C in 235bp). Sample 6 showed variations in two transitions (T\ C in the
50bp and A\G in 207 bp) and three transversions (T\G in 81bp, T\G 83bp and A\C
in 210bp). Both samples 7 showed three transversions (at 594 bp G\ T, at 600bp C\ G and 602bp T\ G)
while the other samples showed 100% similarity.

Figure 2.
Alignment analysis of ITS1 gene. Query symbolizes the gene sequence of the sample;
Subject represents the gene sequence by the database of NCBI.

Table 1. Variations of sequences with ITS1 aligned sequence
The phylogenetic tree analysis has been done using the Maximum
Likelihood method and is represented in figure (3). The Iraqi isolate of
this study (OL673018) is shown in the same clade as the Iranian isolate
(KT192478.1), and this clade share 98% similarity with another clade, including
France isolate and Germany isolate (MW8980291 and AJ270790.1, respectively)
both clades showed 97% similarity with both separate isolates one from Japan
and one from the Czech Republic (AB193720.1 and LT897808.1, respectively).

Figure 3.
Evolutionary analysis tree by Maximum Likelihood method for the Iraqi isolated
in this study with other global isolates
DISCUSSION
Detecting dermatophytes by the usual
microscopic methods can cause problems such as false negative results. Due to
morphological similarity and existing intermediate forms and variants,
unequivocally separating these dermatophytes is not always straightforward, and
sampling appropriate isolates for research is often troublesome 9. The
molecular study has therefore been used to compare the sequence of the ITS1
gene with the sequences of this gene within the public databases of the
globe. For those reasons, this study aimed to diagnose dermatophytes by
molecular technique and to compare the resulted sequence of ITS1 gene
with the sequences of this gene within the public databases of the globe.
In a previous study by de Hoog et
al. 1, many genes were detected and sequenced. And the results
revealed a new taxonomy of dermatophytes. T. interdigitale only included
the anthropophilic species, and zoophilic T. interdigitale isolates
belong to the species T. mentagrophytes. Another study was done by 10 that sequenced four T. mentagrophytes genomes and
exhibited that T. mentagrophytes and T. interdigitale belong to
the same phylogenetic species. In this study, according to the overall average
obtained from intraspecies and interspecies pairwise distances of combination
concerning the seven loci, T. interdigitale and T. mentagrophytes
were regarded as species complexes that may be affected by the epigenetic
change during the localization of the animal and human body. Furthermore, they
likely share a common ancestor, and T. interdigitale species are
descendants of T. mentagrophytes species. This study confirms the
results of the study undertaken by 10. The present study successfully confirmed that ITS1 region is
suitable for providing target genes for molecular identification of the Trichophyton
interdigitale. Variation in
the nucleotide composition of the ITS1 region was successfully employed for
recognition among the samples. Various targets have been employed for DNA-based
identification and discrimination of pathogenic Trichophyton
interdigitale. To the best
of our knowledge, this study was one of the first studies that isolated
Trichophyton interdigitale and was diagnosed by molecular technique in Iraq.
Another study diagnosed Yoshikawa et
al., 11 Trichophyton rubum from Al-Dewaniyah General Hospital
and found positivity of dermatophyte characters in only 5 clinical samples out
of 30. A previous study in Iraq found that most of the organisms
isolated from people suspected of dermatophytes belong to the genera Trichophyton
12.
CONCLUSIONS
It was concluded that Trichophyton
interdigitale in Iraqi isolates had two types of substitution variations
(Transition and Transversion) different than global isolates. Moreover,
according to the phylogenetic tree, the similarity was 97% with isolates from the
Czech Republic and Japan, whereas 98%
with the isolates from France and Germany.
Conflict of
interest
The authors declare no conflict of interest.
Acknowledgments
We are thankful to the participants and the Ministry of health /Iraq.
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Received: 19 March 2022 / Accepted: 7
April 2022 / Published:15 Agoust 2022
Citation: Saied
Hamied A. Submission
and Phylogenetical of Local Isolated Trichophyton interdigitale of Iraqi
Patients in NCBI. Revis Bionatura 2022;7(25) 2.
http://dx.doi.org/10.21931/RB/2022.07.03.25