INTERNAL TRANSCRIBED SPACER (ITS) OF FUNGI ISOLATES OBTAINED FROM

HVS COLLECTED FROM CHILD AGE BEARING FEMALE STUDENTS AT DELSU,

ABRAKA

BY

OTOWAN OTEKPEME JOY

FOS/ 20/21/272422

A RESEARCH PROJECT SUBMITTED TO THE DEPARTMENT OF MICROBIOLOGY

FACULTY OF SCIENCE, DELTA STATE UNIVERSITY, ABRAKA

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE AWARD OF BACH

ELOR OF SCIENCE HONORS, B.Sc. (Hons) IN MICROBIOLOGY

AUGUST 2024

1
 CERTIFICATION

This is to certify that this work was carried out by OTOWAN OTEKPEME JOY
(FOS/20/21/272422) in the Department of Microbiology, Delta State University, Abraka.

____________________. ———————————
Prof. BEN U. OWHE-UREGHE DATE
(Project Supervisor)

______________________ _______________________
DR. (MRS) ADOMI DATE
(Head of Department)

2
 DEDICATION
This project work is dedicated to God Almighty for granting me the strength, wisdom, and perse

verance to complete my research. Without His guidance and blessings, this accomplishment woul

d not have been possible. I also dedicate this work to my family, whose unwavering support and

encouragement have been a constant source of motivation throughout this journey

3
 ACKNOWLEDGEMENT
I am deeply grateful to God Almighty for granting me the strength and guidance to complet
e this research project. His grace has been my constant source of inspiration throughout this jour
ney.

I would like to express my sincere appreciation to the Head of the Department of Microbiology,
Dr. Mrs. Adomi, for her leadership and support. My profound thanks go to my project supervisor,
Prof. Ben U. Owhe-Ureghe, whose unwavering enthusiasm and insightful guidance were instrum
ental in the successful completion of this work. I am also thankful to the dedicated staff members
of the Department of Microbiology for their assistance and encouragement.

A special thank you to my father, mother, and siblings for their boundless love, care, and unwave
ring support. Their belief in me has been a cornerstone of my success, and I am forever grateful.

4
 TABLE OF CONTENT
CERTIFICATION............................................................................................................................2
DEDICATION.................................................................................................................................3
ACKNOWLEDGEMENT...............................................................................................................4
LIST OF TABLES...........................................................................................................................8
LIST OF IMAGES...........................................................................................................................8
LIST OF FIGURES.........................................................................................................................8
ABSTRACT.....................................................................................................................................9
CHAPTER ONE............................................................................................................................9
INTRODUCTION..........................................................................................................................9
1.1 Background to the Study.....................................................................................................9
1.2 Statement of the Problem..................................................................................................11
1.3 General Aim and Objectives.............................................................................................11
1.3.1 Aim:.........................................................................................................................11
1.3.2 The Specific Objectives Were To:...........................................................................11
1.4 Research Questions...........................................................................................................12
1.5 Significance of the Study..................................................................................................12
1.6 Scope of the Study............................................................................................................12
1.7 Working Definition of Terms............................................................................................13
CHAPTER TWO.........................................................................................................................13
LITERATURE REVIEW............................................................................................................13
2.1 Fungal Species Isolated from High Vaginal Swabs..........................................................13
2.2.1 Isolation and Identification Techniques..................................................................13
2.2.2 Common Fungal Species........................................................................................14
2.2 Identification of Fungal Species Using Internal Transcribed Spacer (ITS) Sequencing. .14
2.2.1 Overview of ITS Sequencing..................................................................................14
2.2.2 Importance of ITS Sequencing...............................................................................14
2.2.3 Methodology of ITS Sequencing............................................................................15

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 2.2.4 Advantages and Limitations of ITS Sequencing.....................................................15
2.3 Prevalence and Diversity of Fungi in Vaginal Microbiota................................................16
2.3.1 Prevalence of Fungal Infections..............................................................................16
2.3.2 Factors Influencing Fungal Diversity.....................................................................16
2.3.3 Implications of Fungal Diversity............................................................................16
CHAPTER THREE.....................................................................................................................17
MATERIALS AND METHODS.................................................................................................17
3.0 Study Area.........................................................................................................................17
3.1 Materials...........................................................................................................................17
3.1.1 Materials Used for Isolation....................................................................................17
3.1.2 Materials Used in the DNA Extraction Process......................................................17
3.1.3 Materials Used in the DNA Quantification Process...............................................18
3.1.4 Materials and Equipment Used in the ITS Amplification Process.........................18
3.1.5 Materials for Sequencing........................................................................................19
3.1.6 Materials for Phylogenetic Analysis.......................................................................19
3.2 Study Design and Duration...............................................................................................20
3.3 Ethical Approval...............................................................................................................20
3.4 Sample Collection.............................................................................................................20
3.5 Sterilization of Media and Glassware...............................................................................20
3.6 Laboratory Diagnostic Methods.......................................................................................20
3.6.1 Culturing of Samples..............................................................................................20
3.6.2 Purification of Isolates............................................................................................21
3.7 Molecular Identification....................................................................................................21
3.7.1 DNA Extraction......................................................................................................21
3.7.2 DNA Quantification................................................................................................21
3.7.3 Internal Transcribed Spacer (ITS) Amplification...................................................21
3.7.4 Sequencing..............................................................................................................22

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 3.7.5 Phylogenetic Analysis.............................................................................................22
3.8 Data Presentation..............................................................................................................22
CHAPTER FOUR........................................................................................................................23
RESULTS......................................................................................................................................23
4.1 Agarose Gel Electrophoresis of Fungal Isolates...............................................................23
4.2 Molecular Identification....................................................................................................23
4.2.1 ITS Sequence Analysis...........................................................................................23
4.3 Prevalence and Frequency of Fungal Isolates...................................................................25
CHAPTER FIVE.........................................................................................................................26
DISCUSSION, CONCLUSION, AND RECOMMENDATIONS............................................26
5.1 Discussion.........................................................................................................................26
5.1.1 Fungal Diversity and Prevalence............................................................................26
5.1.2 ITS Sequencing and Molecular Identification........................................................27
5.1.3 Implications for Vaginal Health..............................................................................27
5.2 Conclusion........................................................................................................................28
5.3 Recommendations.............................................................................................................28
References.....................................................................................................................................29

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 LIST OF TABLES
Table 1 : ITS Sequence Similarity and Accession Numbers.............................................................24
Table 2 : Prevalence and Frequency of Fungal Isolates....................................................................27

8
 LIST OF IMAGES
1 Plate 1: Agarose gel electrophoresis of fungal isolates. Lane 1-5 represent ITS gene bands (550
bp). Lane L represents the 100 bp Molecular ladder....................................................................24
2 Phylogenetic Tree Showing Evolutionary Distance......................................................................26

9
 LIST OF FIGURES
Figure 1 : Prevalence and Frequency of Fungal Isolates..................................28

Figure 2 : Frequency Distribution of Candida Species....................................................35

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 ABSTRACT
This study investigated the isolation and molecular identification of fungal species from high vag

inal swabs (HVS) of child-bearing female students at Delta State University, Abraka. The primar

y objective was to enhance the understanding of vaginal fungal infections by characterizing and i

dentifying fungal isolates using Internal Transcribed Spacer (ITS) sequencing. High vaginal swa

bs were collected from participants and cultured on Sabouraud agar medium media before under

going ITS sequencing analysis. The results revealed a high prevalence of Candida albicans, pres

ent in 80% of samples, and Nakaseomyces glabratus in 20%. The study underscores the efficacy

of ITS sequencing in precisely identifying fungal species, offering valuable insights into the fung

al diversity of the vaginal microbiota. These findings have significant implications for clinical pr

actice, highlighting the importance of advanced molecular techniques in improving the

diagnosis and management of vaginal fungal infections.

CHAPTER ONE

INTRODUCTION
1.1 Background to the Study
The vaginal ecosystem in healthy women is primarily dominated by various bacterial species, es

pecially those from the genus Lactobacillus. This bacterial community is essential for maintainin

g vaginal health through the production of lactic acid, which inhibits the growth of pathogenic mi

croorganisms and stabilizes the vaginal microenvironment. Fungi also contribute significantly to

vaginal health, although their role is less thoroughly investigated.

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Fungi, particularly yeasts of the genus Candida, are part of the normal vaginal flora but can beco

me pathogenic under certain conditions. These conditions include changes in vaginal pH, antibiot

ic use that disrupts bacterial flora, hormonal fluctuations, or immunocompromised states (Giovan

ni et al., 2015; Agada et al., 2017). Candida albicans, the most frequently identified fungal speci

es in the vagina, is responsible for a significant proportion of vulvovaginal candidiasis (VVC) ca

ses, estimated to account for 40% to 50% of all VVC incidents (Umeh & Emelugo, 2011; Yang e

t al., 2015). Furthermore, C. albicans is known to be sexually transmissible, adding complexity t

o its management (Lisboa et al., 2010; Nnadi et al., 2012; Nsofor et al., 2016).

Recent advancements in molecular techniques, such as internal transcribed spacer (ITS) sequenci

ng, have provided deeper insights into the fungal diversity in the vaginal environment (Feng et a

l., 2014; Sharma et al., 2014). These techniques allow the identification of fungal species that are

not easily detected through traditional culture methods. For example, studies have shown that IT

S sequencing can uncover a broader range of fungal species, including less common or previousl

y unidentified species, which may be associated with various vaginal conditions (Criseo et al., 20

15).

The focus on fungal pathogens is particularly significant in the context of female reproductive he

alth. Alterations in the vaginal flora, including the overgrowth of fungal species, can lead to cons

iderable health issues such as recurrent infections and altered treatment responses (Borman et al.,

2008; Romeo and Criseo, 2011). Understanding the composition of the fungal microbiome throu

gh ITS sequencing can aid in identifying potential pathogens and developing targeted treatment s

trategies.

All in all, while research has traditionally been centered on bacterial communities in the vagina, f

ungi play an equally crucial role. Internal Transcribed Spacer (ITS) sequencing provides a valuab

12
le tool for exploring fungal diversity and understanding its impact on vaginal health, which is vit

al for improving the diagnosis and treatment of fungal infections in women (Shruti, 2014; Giova

nni et al., 2015; Yang et al., 2015).

1.2 Statement of the Problem

The vaginal microbiome, while primarily bacterial, also includes fungal species like Candida, w

hich can turn pathogenic under specific conditions. Despite its importance, the diversity of fungi

and its impact on vaginal health are not as thoroughly studied as bacterial communities. Tradition

al culture methods often fail to detect less common fungal species, making molecular techniques

such as ITS sequencing vital for a comprehensive understanding. Molecular identification has re

vealed emerging strains of Candida albicans in cases of candidiasis, which is essential for accura

te diagnosis and treatment (Li et al., 2008; Criseo et al., 2015). This knowledge gap hampers effe

ctive diagnosis and treatment of fungal infections, highlighting the necessity for advanced micro

bial analysis. This study intends to investigate the vaginal diversity within the vaginal environme

nt of child-bearing female students at Delta State University, Abraka, using ITS sequencing.

1.3 General Aim and Objectives

1.3.1 Aim:
To investigate the yeast flora of vigina (HVS) of child-bearing female students at Delta State Uni

versity, Abraka, Nigeria, using Internal Transcribed Spacer (ITS) sequencing.

1.3.2 The Specific Objectives Were To:

i. Isolate fungi from HVS samples of child-bearing female students at Delta State Universit

y, Abraka.

ii. Determine fungal species using ITS sequencing.

iii. Analyze the prevalence and diversity of the identified yeast in the study population

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1.4 Research Questions
i. What fungal species can be isolated from high vaginal swabs (HVS) of child-bearing fem

ale students at Delta State University, Abraka?

ii. What is the prevalence and diversity of the identified fungi among the study population?

1.5 Significance of the Study

This study is significantly important for clinical practice and public health. Understanding the fu

ngal community in vaginal flora can lead to improved diagnostic methods and treatment options

for fungal infections, which are frequently underdiagnosed and undertreated. Insights from this st

udy can enhance the accuracy of fungal identification, resulting in more effective management of

infections and potentially lowering recurrence rates. Additionally, this research will contribute to

the broader fields of mycology and reproductive health by emphasizing the importance of fungal

microbiota in vaginal health, which is essential for developing targeted interventions and health p

olicies. The findings may also inform educational and preventive measures for young women, en

hancing their overall reproductive health and well-being. Lastly, this study provides a foundation

for future research and clinical practices aimed at addressing fungal infections and improving he

alth outcomes in similar populations.

1.6 Scope of the Study

This study focuses on analyzing the Internal Transcribed Spacer (ITS) of fungal isolates obtained

from high vaginal swabs of child-bearing female students at Delta State University, Abraka. It ex

amines fungal diversity and prevalence within this specific population. However, the study does

not cover bacterial infections or non-fungal pathogens, nor does it address broader reproductive h

ealth issues beyond fungal infections. Furtheremore, the number of samples ollected are too few t

o justify a significant inference of thhe Study.

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1.7 Working Definition of Terms

Abraka: Refers to a town within Delta State, Nigeria, where Delta State University is situate

d.

Delta State University (DELSU): Refers to an institution of higher learning located in Abra

ka, Delta State, Nigeria.

Female Students: These are females who are currently enrolled in academic programs at DE

LSU, Abraka.

Child-Bearing: Refers to the physiological capability of a female who is of reproductive age

to conceive and bear children.

High Vaginal Swabs (HVS): Refers to samples collected from the vaginal area for the purpo

se of detecting and analyzing microbial or fungal infections.

Fungal Isolates: Refers to fungi obtained from clinical samples, such as high vaginal swabs,

and cultured for further analysis and identification.

Internal Transcribed Spacer (ITS): Refers to a region of DNA located between the ribosom

al RNA (rRNA) genes, used for identifying and classifying fungi due to its variability among

different species.

CHAPTER TWO

LITERATURE REVIEW
2.1 Fungal Species Isolated from High Vaginal Swabs
2.2.1 Isolation and Identification Techniques
The isolation of C. Albicans involves inoculating HVS samples on Sabouraud Dextrose Agar (S

DA) and incubating at 30°C for three days (Agada et al., 2017). Morphological and biochemical

15
characteristics, including colony morphology, microscopy, Gram staining, and germ tube tests, ar

e used for preliminary identification (Ochei and Kolhatkar, 2000). Molecular techniques like PC

R and ITS sequencing are employed for accurate identification (Li et al., 2008; Criseo et al., 201

5).

2.2.2 Common Fungal Species

High vaginal swabs (HVS) are commonly used to diagnose infections in the urogenital tract of w

omen. Studies have shown that Candida albicans is the most frequently isolated fungal species fr

om HVS samples (Lisboa et al., 2010; Nnadi et al., 2012; Nsofor et al., 2016). Other Candida sp

ecies, such as Candida glabrata, Candida tropicalis, and Candida parapsilosis, are also prevalen

t but less common (Yang et al., 2015).

2.2 Identification of Fungal Species Using Internal Transcribed Spacer (ITS) Sequencing
2.2.1 Overview of ITS Sequencing
The Internal Transcribed Spacer (ITS) region of fungal rRNA genes is a widely used molecular

marker for fungal identification and phylogenetic studies. The ITS region, which includes ITS1,

5.8S rRNA, and ITS2, is highly variable among fungal species, making it an effective target for s

pecies-level identification. ITS sequencing involves amplifying the Internal Transcribed Spacer

(ITS) region using polymerase chain reaction (PCR) and sequencing the PCR products. The resul

ting sequences are compared to reference databases to identify the fungal species.

2.2.2 Importance of ITS Sequencing

The ITS region of fungal rDNA is a highly variable sequence that provides species-specific signa

tures, making it an essential tool for fungal identification (Feng et al., 2014). ITS sequencing hel

ps distinguish between closely related species and provides insights into genetic diversity and ev

olutionary relationships (Sharma et al., 2014).

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2.2.3 Methodology of ITS Sequencing
The process of ITS sequencing typically involves several steps:

1. Sample Collection and DNA Extraction: High vaginal swabs are collected from the study pop

ulation, and fungal DNA is extracted using commercial DNA extraction kits, such as the ZR f

ungal/bacterial DNA mini prep extraction kit (Inqaba, South Africa).

2. PCR Amplification: The ITS region is amplified using specific primers, such as ITS1 and IT

S4. PCR conditions, including annealing temperature and cycle number, are optimized to ens

ure efficient and specific amplification of the target region (Wahyuningsih et al., 2000).

3. Sequencing: The amplified ITS products are sequenced using Sanger sequencing or next-gen

eration sequencing (NGS) platforms. Sanger sequencing is often used for smaller studies, whi

le NGS is preferred for larger-scale analyses due to its higher throughput.

4. Sequence Analysis: The obtained sequences are aligned and compared to reference sequences

in databases such as GenBank or UNITE. Bioinformatics tools, such as BLAST, are used to i

dentify the fungal species based on sequence similarity (Mirhendi et al., 2010).

2.2.4 Advantages and Limitations of ITS Sequencing

Sequencing Internal Transcribed Spacer (ITS) offers several advantages, including high resolutio

n for species identification (Criseo et al., 2015), broad applicability across fungal taxa, and the a

vailability of extensive reference databases. It is a rapid and reliable method that can be used in c

linical and research settings to improve diagnostic accuracy and guide appropriate treatment strat

egies (Sharma et al., 2014). However, there are also limitations. PCR amplification can be affect

ed by primer biases and contamination, and the accuracy of species identification depends on the

quality and comprehensiveness of reference databases. Additionally, ITS sequencing may not dis

tinguish closely related species or detect cryptic species complexes.

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2.3 Prevalence and Diversity of Fungi in Vaginal Microbiota
2.3.1 Prevalence of Fungal Infections
The prevalence of fungal infections in the vaginal microbiota varies based on geographical locati

on, age, and health status of the population. Studies have reported varying prevalence rates of C.

Albicans and non-albicans species among different populations (Giovanni et al., 2015; Agada et

al., 2017).

2.3.2 Factors Influencing Fungal Diversity

Several factors, including antibiotic use, hormonal changes, and sexual activity, influence the div

ersity and composition of vaginal fungi (Yang et al., 2015). Antibiotic use disrupts the normal va

ginal flora, leading to overgrowth of opportunistic fungi like C. Albicans (Umeh and Emelugo, 2

011). Hormonal changes during pregnancy and menstrual cycles also affect fungal prevalence an

d diversity (Lisboa et al., 2010).

2.3.3 Implications of Fungal Diversity

Understanding the diversity of vaginal fungi is crucial for developing targeted treatments and pre

vention strategies. The identification of non-albicans species is particularly important, as they m

ay exhibit different antifungal resistance profiles and pathogenicity compared to C. Albicans (Bo

rman et al., 2008; Romeo and Criseo, 2011).

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 CHAPTER THREE

MATERIALS AND METHODS

3.0 Materials
3.0.1 Materials Used for Isolation

 A) Materials: The materials used for isolation included sterile swabs, Sabouraud Dextros

e Agar (SDA) sourced from Oxoid Ltd, UK, Chloramphenicol antibiotics from Sigma-Al

drich, USA, and Petri dishes.

 B) Equipment: The incubation process was carried out using an incubator manufactured

by Memmert, Germany.

3.0.2 Materials Used in the DNA Extraction Process

 A) Materials: The DNA extraction process utilized the ZR Fungal/Bacterial DNA Mini P

rep Extraction Kit from Inqaba Biotechnological, South Africa, which included ZR Bashi

ng Bead Lysis Tubes, isotonic buffer, lysis solution, collection tubes, bacterial DNA bindi

ng buffer, DNA pre-wash buffer, bacterial DNA wash buffer, DNA elution buffer, the Zy

mo-Spin IV Spin Filter (orange top), and the Zymo-Spin IIC Column (all supplied by Zy

mo Research, USA). Additionally, 1.5 microliter centrifuge tubes were used.

 B) Equipment: The equipment used for DNA extraction included an Eppendorf minispin

plus centrifuge (by Eppendorf, Model: 5452), Vortex-Genie 2 mixer (by Scientific Industr

ies Inc., Model: SL-D258), and Four E’s Scientific laboratory Vortex mixer shaker (by Fo

ur E’s Scientific, Model: M10101002).

3.0.3 Materials Used in the DNA Quantification Process

 A) Materials: The materials employed in the DNA quantification process included sterile

distilled water, normal saline, and the extracted genomic DNA.

19
  B) Equipment: The quantification process was conducted using a Thermo Scientific Nan

odrop 1000 UV spectrophotometer (by Thermo Fisher Scientific, USA, Model: ND100),

along with a computer equipped with Nanodrop software and pedestals specifically desig

ned for holding the DNA samples.

3.0.4 Materials and Equipment Used in the ITS Amplification Process

 A) Materials: The ITS amplification process required ITS1F primer (5'-CTTGGTCATTT

AGAGGAAGTAA-3') and ITS4 primer (5'-TCCTCCGCTTATTGATATGC-3'), both fro

m Integrated DNA Technologies, USA. The process also utilized X2 Dream Taq Master

Mix from Inqaba Biotechnological, South Africa, Taq polymerase from Thermo Fisher Sc

ientific, USA, and agarose gel from Sigma-Aldrich, USA.

 B) Equipment: The equipment used for ITS amplification included the ABI 9700 Applie

d Biosystems thermal cycler from Applied Biosystems, USA, and an electrophoresis appa

ratus from Bio-Rad, USA.

3.0.5 Materials for Sequencing

 A) Materials: The materials for sequencing comprised the BigDye Terminator kit, BigDy

e® Terminator v1.1/v3.1, and 5x BigDye sequencing buffer, all from Applied Biosystems,

USA, along with PCR primer (10 µM) from Integrated DNA Technologies, USA, and P

CR template (2-10 ng per 100 bp) from Thermo Fisher Scientific, USA.

 B) Equipment: Sequencing was performed using the 3510 ABI sequencer provided by In

qaba Biotechnological, Pretoria, South Africa.

20
3.0.6 Materials for Phylogenetic Analysis

 A) Software: The software utilized for phylogenetic analysis included Trace Edit (a bioin

formatics algorithm for editing sequences developed by CEMB, Pakistan), BLASTN fro

m the National Center for Biotechnology Information (NCBI), USA, ClustalX from the E

uropean Bioinformatics Institute, UK, and MEGA 6.0 (Molecular Evolutionary Genetics

Analysis) from the USA.

 B) Methods: The methods used included the Neighbor-Joining method for inferring evol

utionary history as described by Saitou and Nei (1987), the bootstrap consensus tree for r

epresenting evolutionary history as proposed by Felsenstein (1985), and the Jukes-Cantor

method for computing evolutionary distances, based on the work of Jukes and Cantor (19

69).

21
3.1 Study Area
The research was conducted at Delta State University (DELSU), located in Abraka, Nigeria.

Founded in 1992, DELSU is a well-regarded institution offering a large range of undergraduate

and postgraduate programs across various faculties. The university is renowned for its dedication

to academic excellence and research, especially in the sciences, technology, and humanities.

Abraka is situated within Delta State, which has an estimated population of over 5 million

people. The state spans a landmass of approximately 18,050 square kilometers, characterized by

a mix of urban and rural areas. Delta State is also known for its significant oil and gas reserves,

contributing to both its economic activities and environmental challenges, including pollution.

The choice of this location is pivotal due to its access to a diverse population of female students,

essential for the objectives of this research.

3.2 Study Design and Duration

A cross-sectional study design was employed to evaluate the internal transcribed spacer (ITS) reg

ion of fungal isolates from High Vaginal Swabs (HVS). The study was conducted over a period o

f six months (from January to June 2024), encompassing sample collection, laboratory analysis, a

nd data interpretation.

3.3 Ethical Approval

Ethical approval was secured from the Institutional Review Board of Delta State University, Abr

aka. Informed consent was obtained from all participants before sample collection, ensuring conf

identiality and allowing participants the option to withdraw from the study at any point.

3.4 Sample Collection

High Vaginal Swabs (HVS) were collected from female students at Delta State University, Abrak

a. Samples were collected from participants by a trained Medical Laboratory Scientist. The swab

22
s were transported to Nucleomatrix laboratory Bayelsa in sterile containers and processed immed

iately to maintain sample integrity.

3.5 Sterilization of Media and Glassware

All media and glassware used in the study were sterilized by autoclaving at 121°C for 15 minutes.

This procedure ensured the elimination of any contaminants that could potentially impact the ac

curacy of the results.

3.6 Laboratory Diagnostic Methods

3.6.1 Culturing of Samples

Upon arrival at the laboratory, the samples were inoculated onto selective and differential media.

Media included Nutrient Agar, Sabouraud Dextrose Agar, and Blood Agar, which support the gro

wth of a wide range of bacteria and fungi. Plates were incubated at 37°C for 24 to 48 hours, and

observations were recorded based on colony morphology (Prescott, Harley, and Klein, 2002).

3.6.2 Purification of Isolates

Colonies of interest were isolated by streaking onto fresh agar plates to obtain pure cultures. Eac

h isolate was then subcultured to ensure it was free of contaminants. Pure cultures were stored at

4°C for further analysis.

3.7 Molecular Identification

3.7.1 DNA Extraction
DNA extraction was performed using the ZR fungal/bacterial DNA mini prep extraction kit from

Inqaba, South Africa. A substantial growth of the pure culture of suspected isolates was suspende

d in 200 µL of isotonic buffer and placed into ZR Bashing Bead Lysis tubes. After adding 750 µL

of lysis solution, the tubes were processed in a bead beater at maximum speed for 5 minutes and

centrifuged at 10,000 x g for 1 minute. Four hundred (400) µL of supernatant was transferred to

23
a Zymo-Spin IV spin filter and centrifuged at 7,000 x g for 1 minute. The filtrate was combined

with 1,200 µL of fungal/bacterial DNA binding buffer and applied to a Zymo-Spin IIC column. F

ollowing several centrifugation steps with washing buffers, DNA was eluted with 100 µL of eluti

on buffer and stored at -20°C (Sambrook & Russell, 2001; Ausubel et al., 2002).

3.7.2 DNA Quantification

The extracted genomic DNA was quantified using the Nanodrop 1000 spectrophotometer. The de

vice was initialized with 2 µL of sterile distilled water and blanked with normal saline. Two micr

oliters of DNA were then loaded onto the pedestal, and the concentration was measured using the

Nanodrop software (Wilfinger, Mackey, & Krueger, 1997; Glasel, 1995).

3.7.3 Internal Transcribed Spacer (ITS) Amplification

The ITS region of the isolates DNA was amplified using ITS1F (5’-CTTGGTCATTTAGAGGA

AGTAA-3’) and ITS4 (5’-TCCTCCGCTTATTGATATGC-3’) primers on an ABI 9700 thermal c

ycler at a final volume of 30 µL for 35 cycles. The PCR mix contained X2 Dream Taq Master Mi

x, primers at 0.4 µM, and the DNA template. PCR conditions included initial denaturation at 95°

C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 53°C for 30 seconds, extensio

n at 72°C for 30 seconds for 35 cycles, and final extension at 72°C for 5 minutes. The product w

as resolved on a 1% agarose gel at 120V for 15 minutes and visualized on a blue light transillumi

nator (White et al., 1990; Gardes & Bruns, 1993).

3.7.4 Sequencing
Sequencing was carried out using the BigDye Terminator kit on a 3510 ABI sequencer by Inq

aba Biotechnological, Pretoria, South Africa. The final volume was 10 µL, including 0.25 µL Big

Dye® terminator v1.1/v3.1, 2.25 µL of 5x BigDye sequencing buffer, 10 µM Primer PCR primer,

and 2-10 ng PCR template per 100 bp. Sequencing conditions included 32 cycles of 96°C for 10

24
seconds, 55°C for 5 seconds, and 60°C for 4 minutes (Sanger, Nicklen, & Coulson, 1977; McCo

mbie et al., 1992).

3.7.5 Phylogenetic Analysis

The obtained sequences were edited using the bioinformatics tool Trace Edit. Similar sequences

were downloaded from the National Center for Biotechnology Information (NCBI) database usin

g BLASTN. These sequences were aligned using ClustalX. The evolutionary history was inferre

d using the Neighbor-Joining method in MEGA 6.0 (Saitou and Nei, 1987). The bootstrap consen

sus tree inferred from 500 replicates (Felsenstein, 1985) represents the evolutionary history of th

e taxa analyzed. The evolutionary distances were computed using the Jukes-Cantor method (Juke

s and Cantor, 1969).

3.8 Data Presentation

The data obtained from the study were presented using tables and charts to effectively communic

ate the findings.

CHAPTER FOUR

RESULTS
4.1 Agarose Gel Electrophoresis of Fungal Isolates

The agarose gel electrophoresis of the ITS region from fungal isolates, as depicted in Plate 4.1,

shows that lanes 1 through 5 exhibit ITS gene bands at 550 bp, while lane K contains the 100 bp

molecular ladder.

25
1 Plate 1: Agarose gel electrophoresis of fungal isolates. Lane 1-5 represent ITS gene bands

(550 bp). Lane L represents the 100 bp Molecular ladder.

4.2 Molecular Identification

4.2.1 ITS Sequence Analysis
Table 1: ITS Sequence Similarity and Accession Numbers

Isolate Code Identified Species Closest Known Accession Number

Relative

T1 Candida albicans Candida albicans OR394128

T2 Nakaseomyces Nakaseomyces OR647617

glabratus glabratus

26
 T3 Candida albicans Candida albicans OR394128

T4 Candida albicans Candida albicans OR394128

T5 Candida albicans Candida albicans OR394128

The ITS sequences from the isolates matched exactly with sequences in the NCBI nr/nt database.

The ITS sequences demonstrated 100% similarity to known Candida species. The evolutionary

distances computed using the Jukes-Cantor method supported the phylogenetic placement of the

ITS sequences within the Candida species, with close relatedness to Candida albicans and

Candida tropicalis.

Table 1: ITS Sequence Similarity and Accession Numbers

A phylogenetic tree was constructed to illustrate the evolutionary relationships between the

fungal isolates and closely related species. The tree shows the evolutionary distance among the

isolates and their related Candida species.

27
 Figure 1: Prevalence and Frequency of Fungal Isolates

Phylogenetic Tree Showing Evolutionary Distance

The study identified various Candida species with the following prevalence and frequency:

Table 2: Prevalence and Frequency of Fungal Isolates

Pathogen Number of Samples Prevalence (%)

Candida albicans 4 80%

Nakaseomyces glabratus 1 20%

28
 CHAPTER FIVE

DISCUSSION, CONCLUSION, AND RECOMMENDATIONS

5.1 Discussion
The study aimed to explore the fungal diversity in the vaginal environment of child-bearing fema

le students at Delta State University, Abraka, using Internal Transcribed Spacer (ITS) sequencing.

The results reveal a predominance of Candida species, with a notable presence of Candida albic

ans and a lesser occurrence of Nakaseomyces glabratus.

5.1.1 Fungal Diversity and Prevalence

The agarose gel electrophoresis and ITS sequence analysis indicated a high prevalence of Candi

da albicans (80%) among the isolates, consistent with findings from previous studies that have id

entified C. albicans as the most common pathogen in vaginal infections (Lisboa et al., 2010; Nna

di et al., 2012). The high prevalence of C. albicans is supported by its well-documented role as a

frequent cause of vulvovaginal candidiasis (VVC), accounting for 40% to 50% of cases (Umeh a

nd Emelugo, 2011; Yang et al., 2015). This aligns with the study’s results showing that C. albica

ns was the dominant fungal species.

The identification of Nakaseomyces glabratus in 20% of the samples is noteworthy. N. glabratus ,

previously less frequently reported in vaginal infections compared to C. albicans, is gaining atte

ntion due to its increasing recognition in clinical settings (Giovanni et al., 2015). This finding su

ggests that non-albicans Candida species are also significant in the vaginal microbiota, which is c

orroborated by other studies emphasizing the emerging relevance of non-albicans Candida speci

es (Agada et al., 2017).

29
5.1.2 ITS Sequencing and Molecular Identification
The ITS sequencing proved effective in identifying fungal species with high resolution. The ITS

region’s variability among fungal species provided clear differentiation between C. albicans and

N. glabratus, aligning with previous research that underscores ITS sequencing as a robust tool fo

r fungal identification (Feng et al., 2014; Sharma et al., 2014). The sequences demonstrated 100

% similarity with known Candida species, confirming the accuracy of the molecular identificatio

n. This supports the findings of Criseo et al. (2015), that highlighted the reliability of ITS sequen

cing in accurately identifying fungal species and detecting previously unidentified species.

The phylogenetic tree constructed from the ITS sequences showed close evolutionary relationshi

ps among the Candida species, validating the genetic similarity and supporting the identification

results. This aligns with previous research that has used phylogenetic analysis to understand fung

al relationships and evolutionary history (Mirhendi et al., 2010; Saitou and Nei, 1987).

5.1.3 Implications for Vaginal Health

The dominance of C. albicans in the study population underscores its critical role in vaginal infe

ctions. Its prevalence reflects the findings of previous studies that have linked C. albicans to recu

rrent infections and altered treatment responses (Romeo and Criseo, 2011; Borman et al., 2008).

The presence of N. glabratus highlights the need for comprehensive diagnostic methods to identi

fy non-albicans species, which may have different antifungal resistance profiles compared to C. a

lbicans (Yang et al., 2015). This emphasizes the importance of using molecular techniques like I

TS sequencing to ensure accurate diagnosis and therefore employ effective treatment strategies.

5.2 Conclusion
This study successfully isolated, characterized and identified fungal species from high vaginal s

wabs of child-bearing female students at Delta State University, Abraka. The results revealed a hi

30
gh prevalence of Candida albicans and a notable presence of Nakaseomyces glabratus. The use o

f ITS sequencing provided precise identification and highlighted the evolutionary relationships a

mong the fungal isolates.

Understanding the fungal diversity in the vaginal environment is essential for improving diagnost

ic accuracy and therefore treatment strategies. The predominance of C. albicans confirms its sign

ificant role in vaginal infections, while the presence of non-albicans species like N. glabratus ind

icates a broader spectrum of potential pathogens.

5.3 Recommendations
1. Improved Diagnostic Techniques: It is recommended that healthcare providers incorpor

ate ITS sequencing into diagnostic protocols for fungal infections to ensure accurate ident

ification of both common and less common fungal species. This approach will improve di

agnostic accuracy and treatment outcomes.

2. Monitoring Fungal Diversity: Regular monitoring of fungal diversity in the vaginal mic

robiota should be conducted to track changes in prevalence and resistance patterns. This

will help in understanding the evolving fungal landscape and adapting treatment strategie

s accordingly.

3. Educational Programs: Educational programs should be developed to raise awareness a

bout fungal infections and the importance of early diagnosis and treatment. Emphasis sho

uld be placed on understanding the role of non-albicans Candida species and their potenti

al impact on vaginal health.

4. Further Research: Additional research is needed to explore the clinical implications of n

on-albicans Candida species and their resistance profiles. Longitudinal studies could prov

31
 ide insights into the dynamics of fungal infections and their impact on women's health ov

er time.

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APPEDIX

Figure 2: Frequency Distribution of Candida Species

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36