Microbial and Molecular Screening of Swimmers Associated with Conjunctivitis from Public Swimming Pools in Erbil Province

Abstract

Public swimming pools, if not treated well could work as a reservoir of many microorganisms that cause infections among swimmers. Conjunctivitis is one of those common infections that resulted from microbial and non-microbial agents, microbial conjunctivitis caused by viral (mainly Human adenovirus HAdVs) and bacterial infections. This study aims to investigate the prevalence of microbial causative agents of swimming pool conjunctivitis and evaluating the swimming pools in terms of health and the extent of contamination in Erbil province. Eighty-eight specimens were isolated and identified from the swimmers showing signs and symptoms of swimming pool conjunctivitis from different public swimming pools in Erbil city from January to the end of February 2020. Sample identified using bacteriological methods, serology test, and nested PCR for detection of HAdVs. The swimmers samples consisted of 60 males and 28 females, and they were aged between 16-56 years. The obtained results showed that, out of 88 samples, 36 (40.91%) detected as a viral infection and 29 (32.95%) as bacterial infection, while, 23 (26.13%) showed no growth (non-microbial infection). Frequency of swimming pool conjunctivitis among male and female was 60 (68.2%) and 28 (31.8%) respectively. Depending on the obtained results, it can be concluded that conjunctivitis could result from viral, bacterial, and non-microbial agents, a viral infection is the main cause followed by a bacterial infection, also public swimming pools are not a safe place and swimmers are subjected to infection by different pathogens.

Full text

Al-Mustansiriyah Journal of Science
ISSN: 1814-635X (print), ISSN:2521-3520 (online)
Volume 1, Issue 4, 2020
DOI: http://doi.org/10.23851/mjs.v31i4.903
36
Copyright © 2020 Al-Mustansiriyah Journal of Science. This work is licensed under a Creative Commons Attribution
Noncommercial 4.0 International License.
Research Article
Open Access
Microbial and Molecular Screening of Swimmers Associated with
Conjunctivitis from Public Swimming Pools in Erbil Province
Sazan Q. Maulud1, Lawin A. Omar1, Ahmed N. Hassan2*,
Rastee H. Saeed1
1Department of Biology, College of Education, Salahaddin University-Erbil, Erbil, IRAQ
2Department of Medical Laboratory Technology, Erbil Health Technical College, Erbil Polytechnic University, Erbil, IRAQ
*Contact email: ahmed.nawzad@epu.epu.iq
A r t i c l e I n f o
ABSTRACT
Received
12/09/2020
Public swimming pools, if not treated well could work as a reservoir of many microorganisms
that cause infections among swimmers. Conjunctivitis is one of those common infections that
resulted from microbial and non-microbial agents, microbial conjunctivitis caused by viral
(mainly Human adenovirus HAdVs) and bacterial infections. This study aims to investigate the
prevalence of microbial causative agents of swimming pool conjunctivitis and evaluating the
swimming pools in terms of health and the extent of contamination in Erbil province. Eighty-
eight specimens were isolated and identified from the swimmers showing signs and symptoms
of swimming pool conjunctivitis from different public swimming pools in Erbil city from
January to the end of February 2020. Sample identified using bacteriological methods, serology
test, and nested PCR for detection of HAdVs. The swimmers samples consisted of 60 males and
28 females, and they were aged between 16-56 years. The obtained results showed that, out of
88 samples, 36 (40.91%) detected as a viral infection and 29 (32.95%) as bacterial infection,
while, 23 (26.13%) showed no growth (non-microbial infection). Frequency of swimming pool
conjunctivitis among male and female was 60 (68.2%) and 28 (31.8%) respectively. Depending
on the obtained results, it can be concluded that conjunctivitis could result from viral, bacterial,
and non-microbial agents, a viral infection is the main cause followed by a bacterial infection,
also public swimming pools are not a safe place and swimmers are subjected to infection by
different pathogens.
Accepted
20/09/2020
Published
20/12/2020
KEYWORDS: Swimming Pool Conjunctivitis, Viral Conjunctivitis (HAdVs), Bacterial
Conjunctivitis, Non-microbial Conjunctivitis, Molecular Diagnosis (Nested PCR).
ةصخلا
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Sazan et al.
Microbial and Molecular Screening of Swimmers Associated with Conjunctivitis from Public Swimming Pools in Erbil Province
2020
37
INTRODUCTION
People visit swimming pools for different reasons;
the majority of people swim as recreational
activities, others swim as sports, while some swim
for rehabilitative treatment and physiotherapy. In
the last few years, swimming pools became
increasingly popular [1]. However, it should be
regarded that public swimming pools are
subjected to different types of contaminations, and
not all swimming pools follows the health
standards instructions. Public health problems
may occur as a result of possible maintenance
failure [2]. The contamination of the pools by the
microorganisms resulted in different infections
and complications that appear especially in
swimmers with immune system deficiencies,
diseases, such as diarrhea, skin, ear and upper
respiratory infections, particularly if the
swimmer's head is submerged [1].
Conjunctivitis is an inflammation or infection of
the conjunctiva, which is the thin clear layer that
lines the inner surface of the eyelid and covers the
white part of the eye [3, 4]. There are many types
of swimming pool conjunctivitis, but based on the
etiology we can divide them into bacterial
conjunctivitis, viral conjunctivitis, fungal
conjunctivitis, and parasitic conjunctivitis [5].
Generally, an increased amount of tears, thick
yellow discharge, itchy eyes, burning eyes, and
blurred vision are among symptoms of
conjunctivitis [3, 6]. Viral conjunctivitis (pink or
red-eye), the main cause of microbial
conjunctivitis, refers to those types of swimming
pool conjunctivitis caused by adenovirus, namely
human adenovirus (HAdVs), which cause
different clinical manifestations [7, 8]. HAdVs
can survive for prolonged periods in water and
show high stability in the environment; its
presence indicates the fecal contamination of
water. Ingestion or direct contact with
contaminated water is among routes of HAdVs
transmission in swimming pools [2, 9].
Bacterial conjunctivitis, the second cause of
microbial conjunctivitis, main causative agents of
bacterial conjunctivitis includes; Staphylococcus,
in first place, followed by Streptococcus,
Corynebacterium, and finally Moraxella species.
The correct diagnosis of causative agents of
microbial conjunctivitis is important for correct
treatment [6, 10, 11].
Non-bacterial conjunctivitis, is the third common
cause of swimming pool conjunctivitis, which
includes the chemical substance that added to the
water for disinfection purpose [11]. The most
important and widely used disinfectant is chlorine
compounds. Improper uses of chlorine cause
allergic, irritation, and damage of the eye and tear
film, leads to conjunctivitis (redness) of the eyes
[12].
Recently, public swimming pools became very
popular in Erbil city, but unfortunately some of
them do not follow standard heath instructions and
not all of them monitored or supervised by
competent authorities and health committees.
Continues monitoring and investigations are
required to characterize the contaminants of these
pools. The present study aimed to determine the
main causative agents (viral, bacterial, and non-
microbial) of conjunctivitis among swimmers of
public swimming pools and to evaluate swimming
pools from a healthy point of view.
MATERIALS AND METHODS
Specimen collection
Conjunctiva swabs were collected from eighty-
eight swimmers who were suspected of having
conjunctivitis infection depending on the clinical
signs and complications, like; sudden eye
pinkness or redness, discomfort, pain, tearing,
burning, conjunctiva haemorrhages, eyelid
swelling for two months. Samples included 60
male, and 28 female aged from sixteen to fifty-six
in swimming pools selected randomly in the
center of Erbil province from January to the end
of February 2020. Specimens were taken from
each participant using two sterile Dacron swabs,
one dry collected for bacteriological study and the
other collected in 1 ml Phosphate-buffered saline
(PBS) and transported immediately to the
laboratory on ice and stored at -20 ◦C [13].
Bacteriological Examination of Samples
Preparation of Bacterial Media
MacConkey, blood, and Chocolate agar (Oxoid,
UK), were used to cultivate the isolates.
Preparation of the media were prepared according
to manufacturer’s instructions and sterilized with
an autoclave at 121˚C for 15 minute [14].
Isolation and identification of bacteria
The conjunctiva swabs were inoculated on Blood,
MacConkey, and Chocolate agar and incubated at
37 ºC for 24hrs. After incubation, slides prepared

Al-Mustansiriyah Journal of Science
ISSN: 1814-635X (print), ISSN:2521-3520 (online)
Volume 1, Issue 4, 2020
DOI: http://doi.org/10.23851/mjs.v31i4.903
38
Copyright © 2020 Al-Mustansiriyah Journal of Science. This work is licensed under a Creative Commons Attribution
Noncommercial 4.0 International License.
for positive culture to identify the morphology
using Gram stain. Biochemical tests for
identification was used, IMVIC test, urease,
oxidase, motility, and catalase test was used to
distinguish between Staphylococcus (catalase +ve)
and Streptococcus (catalase -ve). Coagulase test
used for identification of different species of
Staphylococcus, coagulase positive
Staphylococcus aureus (the pathogenic strains)
and the coagulase negative Staphylococcus
species (the nonpathogenic strains) [15].
Virological Examination of Samples
HAdV Immunochromatographic test (IC)The
Immunochromatographic test was performed
according to the manufacturer's instruction (Aden
test, SA Scientific TM, USA).
Directly after sample collection using sterile swab
this test performed in which 4-four drops
(approximately 150μL) of the specimen were put
into the kit specimen well. The proper time
allowed for the specimen to be filtered through the
kit to the specimen position and the control
position. The results were read within 15 minutes.
Within 30 minute from the application of
specimen to the kit, the readings were finalized
[8].
Molecular Test PCR
Primer design
Two sets of primers were used ADHEX1,
ADHEX2, to approve that the positive results of
Immunochromatographic test are HAdVs [16, 17],
first primer ADHEX1 used for nested PCR, while
the second one ADHEX2 used as a specific
primer to detect HAdV DNA. Sequence and base
pairs of the primers (see Table 1).
Table 1. Primers name, sequences, location, and product size used for detection of HAdV DNA Hexon gene.
Primer
Sequence (5'–3')
Location*
Product size (base pairs)
ADHEX1F
AAC ACC TAY GAC TAC ATG AAC
20380–20400
930 bp.
ADHEX1R
AAT GGG GTA AAG CAT GTT TGC
20836–20854
ADHEX2F
CCC ATT GAA CCA CCA CCG ATC
20485–20503
473 bp.
ADHEX2R
ACA TCC TTA CCG AAG TTC CAC
20632–20652
*Positions as referred to adenovirus 2 Hexon protein-coding region sequence [16].
HAdV DNA extraction
The genomic matter of HAdV and RNAs were
extracted only in thirty-six isolates with positive
IC Adenovirus test result using QIAGEN DNA
extraction kit (Hilden, Germany). The reagents of
the kit were reconstituted before use according to
the protocol of the manufacturers’ instructions [8].
Adenovirus Hexon gene amplification
A partial Hexon sequence of HAdV was amplified
from all 36 isolates that showed positive results by
Immunochromatographic test using nested PCR,
described by Avellón, Pérez, Aguilar, Lejarazu
and Echevarría [16] with some optimization [8,
18]. The primers used for first-round PCR and
nested PCR were specific for the detection of
HAdV DNA (see Table 1). A fifty microliter
reaction volume for the first-round PCR was
prepared. Cycling was carried out by (Gradient
thermal cycler Alpha Cycler PCRmax series).
PCR conditions (steps, time, and temperature) for
the first primer performed as described (see Table
2). One microliter of the first-round PCR was
transferred to a nested PCR mixture containing:
31 µl nuclease-free water, 5 µl 10X PCR buffer, 8
µl of MgCl2, 1.5 µl of 10 mmol dNTP mixes, 0.5
µl primer ADHEX2F (100 pmol), 0.5 µl primer
ADHEX2R 100 pmol and 2.5 U Taq polymerase
(Promega). PCR condition (steps, time, and
temperature) for the second primer was carried out
as described (see Table 2).
Detection of PCR products
After amplification, 5µl of PCR product was
analyzed using electrophoresis (BioTech-USA) in
1.2% agarose at 75 V for 45 minutes. Safe stain
(EvaGreen Fluorescent Gel Stain-Jena
Bioscience) was used to visualize the band, DNA
marker (1000 bp.) was used as a ruler and bands
were visualized under UV trans-illuminator
(Benchtop UV-Transilluminator-BioTech-USA).

Sazan et al.
Microbial and Molecular Screening of Swimmers Associated with Conjunctivitis from Public Swimming Pools in Erbil Province
2020
39
Results documented using a 16-megapixel camera
for photography.
Statistical analysis
Chi-square test was performed for variables and
P<0.05 values were regarded as significant
differences.
Table 2. PCR protocol and conditions, including time and temperature of all steps for ADHEX1 and ADHEX2 primers.
Gene
Initial
Denaturation
Denaturation
Annealing
Extension
Final extension
No. of
cycles
Temp
Time
Temp
Time
Temp
Time
Temp
Time
Temp
Time
ADHEX1
94◦C
2min
93◦C
1min
50 ◦C
1min
72◦C
1min
72◦C
6min
30
ADHEX2
94◦C
2min
93◦C
1min
55 ◦C
1min
72◦C
1min
72◦C
6min
30
RESULTS
Eighty-eight conjunctiva samples were isolated
and identified from the swimmers suffering from
pink eyes conjunctivitis at the different public
swimming pools in Erbil city. These patients
consisted of 60 (68.2%) males and 28 (31.8%)
females, and they were aged between 16-56 years
(see Table 3).
Results of bacterial cultivation on Blood (BA),
MacConkey, and Chocolate agar, morphological
identification, and biochemical tests detected 29
positive isolates, in which 22 (25%) of the
samples confirmed as Staphylococcus aureus (β-
hemolysis on BA, gram-positive cocci in clusters,
positive urease, catalase and coagulase, negative
for oxidase and motility), and 7 (7.95%) of the
samples were Streptococcus pneumonia (ɑ-
hemolysis on BA, gram-positive diplococci,
negative for urease, oxidase, motility, catalase,
and coagulase), results of detecting HAdV using
Immunochromatographic test (IC) detected 36
(40.91%) as positive samples, (see Table 3).
Table 3. The age, gender, and infectious agents of swimmers according to the present study.
Age/Years
Gender Bacterial Viral Non-microbial
No. % No. % No. % No. %
16-56
Male 60*** 68.2
Staphylococcus aureus 22*** 25
HAdVs 36 40.91
23 26.14
Female 28*** 31.8
Streptococcus pneumonia 7*** 7.95
Total
88 100
29* 32.95
36* 40.91
23* 26.14
*** p < .0001; ***p < .0002;* p < .0376
The results of PCR amplification for HAdVs
detection, in which 36 isolates amplified using
specific primers, all 36 samples showed positive
results when the amplified product were analyzed
by agarose gel electrophoresis, as shown in Figure
1.
The overall result showed both viral and bacterial
infections, out of 88 samples, 36 (40.91%) viral
infected, 29 (32.95%) bacterial infection, and 23
(26.13%) no growth (non-microbial infections), as
shown in Figure 2.
Results of statistical analysis using Chi-square test
showed high significance differences (𝒑 <
. 𝟎𝟎𝟎𝟏) between males and females. Also, there
were significant differences (𝒑 < . 𝟎𝟑𝟕𝟔) among
conjunctivitis types (viral, bacterial, and non-
microbial infections), and (𝒑 < . 𝟎𝟎𝟎𝟐) between
bacterial conjunctivitis (Staphylococcus aureus
and Streptococcus pneumoniae).
Figure 1. Agarose gel electrophoresis analysis 1.2% (stained
with safe stain) of PCR products presenting gene amplified
with ADHEX2 primer that showed the expected size 473 bp.
Lane 1: is 1000 bp. DNA Ladder; Lane 2 negative control:
Lanes 3-18: gene product amplified from HAdV genome run
on 75V for 45min including samples from 1 to 16. All
samples showed positive results for HAdV Hexon gene.

Al-Mustansiriyah Journal of Science
ISSN: 1814-635X (print), ISSN:2521-3520 (online)
Volume 1, Issue 4, 2020
DOI: http://doi.org/10.23851/mjs.v31i4.903
40
Copyright © 2020 Al-Mustansiriyah Journal of Science. This work is licensed under a Creative Commons Attribution
Noncommercial 4.0 International License.
Figure 2. The percentage of infections in swimmer
infected conjunctivitis.
DISCUSSIONS
The present study detected microbial (viral and
bacterial), and non-microbial eye conjunctivitis
among swimmers (males and females) in public
swimming pools in different frequencies, in which
viral conjunctivitis detected as main causative
agent followed by bacterial and non-microbial
agents, respectively.
Regarding the results of sex group of the present
study, there were significant differences between
males and females (𝒑 < . 𝟎𝟎𝟎𝟏); males showed
higher frequency than the females with (68.2%)
and (31.8), respectively. The explanation of male
prevalence over female could be understood
regarding the cultural differences and rules, as
Erbil population are highly restricted to cultural
rules and female not allowed to visit public
swimming pools like the male. Detecting a higher
rate in male rather than the female is approved by
other studies as well [19, 20].
Results from the current study regarding the
causative agents of swimming pools conjunctivitis
revealed that the most prevalence swimming pool
conjunctivitis was caused by viral infection with
(40.91%) frequency as shown in Figure 2. The
obtained results showed that viral conjunctivitis is
prevalence over bacterial and other conjunctivitis,
such a result approved by a research carried out by
Azari and Barney in 2013, that collected results
of researches that investigated causative agents
of conjunctivitis over 10 years, they showed that
viral conjunctivitis is the most common overall
cause of infectious conjunctivitis [21]. Another
explanation for the prevalence of adenovirus
infection in the current study may be resulted
from time of sample collection (January and
February), as adenovirus tend to peak in the
winter and early spring [22]. Conjunctiva
waterborne outbreaks resulted from the prevalence
of HAdVs in the swimmers such as pharyngo-
conjunctivitis and possibly gastroenteritis at these
swimming pools [23]. Several other researches
worldwide approved the result of the current study
for detecting HAdV as the main causative agent
for swimming pool conjunctivitis [2, 20, 24, 25].
Being highly stable in the environment, surviving
for prolonged periods in water, and having
different routes of transmission made HAdVs to
be the main causative agent of swimming pool
conjunctivitis [2].
The second conjunctivitis infection was a bacterial
infection in which Staphylococcus aureus showed
the highest prevalence (25%) compared to
Streptococcus pneumonia (7.95%), with
(𝒑 < . 𝟎𝟎𝟎𝟐) significant differences. Bacterial
conjunctivitis became a major issue as it increases
year after year; Cavuoto, Zutshi, Karp, Miller and
Feuer [26] reported an increase from 4.4% in 1994
to 42.9% in 2003 with bacterial conjunctivitis.
According to a study carried out in 2017 that
covers researches of 14 countries worldwide,
revealed that Staphylococcus aureus is the major
cause of bacterial conjunctivitis in significantly
higher frequencies than Streptococcus pneumonia,
which agree with the results of the current study.
In an approach in Iraq to detect the most common
pathogens for conjunctivitis a study carried out in
2017 and their results showed that Staphylococcus
aureus was the main pathogen rather than
Streptococcus spp [27]. Furthermore, Taiwo and
his colleagues in 2004, reported a prevalence rate
of 34.7% of S. aureus in the North Central part of
Nigeria [28]. Another study reported an
approximately the same result of the current study
carried by Saberianpour and Momtaz in 2016
[29].
The third causative agent for swimming pool
conjunctivitis was a non-microbial infection, with
(26.14%). Detecting of such a high level of
chemical conjunctivitis is related to using
chlorine, which is considered as the main cause of
eye irritation after swimming and consequently
causes conjunctivitis [9]. Chlorine dehydrates
eyes and consequently removes the tear film, the
redness occurs when the blood vessels near the
29
36
23
32.95
40.91
26.14
0
10
20
30
40
50
Bacterial
Infections
Viral Infection Non-microbial
Infection
No. of Positive resultes %

Sazan et al.
Microbial and Molecular Screening of Swimmers Associated with Conjunctivitis from Public Swimming Pools in Erbil Province
2020
41
surface of the eye become larger and then dilate
[30]. Most public swimming pools in Erbil city
use chlorine as a disinfectant and they do not
restrict to the standards for the amount of chlorine
that should be added regarding the volume of
water in the pool, A study carried out in 2009
approved that the amount of chlorine differed
among the public swimming pools in Erbil city
[31].
Results of the current study revealed that public
swimming pools are not a safe place, especially if
they are not sufficiently treated and disinfected,
they could play a significant role in spreading
several diseases like conjunctiva, such results are
approved by other researches [2, 32]. The
microbiological quality of all the water sources
was poor and contaminated with different
microbial organisms; they are pathogenic.
Swimmers using public swimming pools request
from pool responsible and health authorities for
more strict surveillance measures for the
protection of swimmer’s health [33].
CONCLUSIONS
Microorganisms, rather than other contaminations,
cause most conjunctivitis among swimmers.
Microbial conjunctivitis resulted from viral and
bacterial infections. It can be concluded that
public swimming pools are not a safe place, and
swimmers in public swimming pools are subjected
to viral and bacterial infections. Many swimming
pools are not obeying health instruction, and most
swimmers are not aware of the risks of
contaminated water of these pools. Further studies
are needed to determine the prevalence rate of
different microorganisms related to conjunctivitis.
RECOMMENDATIONS
1- Swimming pools must strictly apply healthy
instructions.
2- Periodic microbial tests must be done for the
water of swimming pools.
3- Swimmers must not neglect any signs and
symptoms of conjunctivitis; they must contact
their health providers as soon as possible to
treat any infections.
4- More research and investigations are
recommended in this field.
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DOI: http://doi.org/10.23851/mjs.v31i4.903
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