COVENANT JOURNAL OF HEALTH AND LIFE SCIENCES, VOL. 1, NO.

1, JUNE 2023

COMPARATIVE EVALUATION OF GENOTYPING AND CULTURE-BASED TECHNIQUES FOR FUNGAL

KERATITIS DETECTION

Oladejo M. Janet1 , Oladejo O. J2, Odetoyin B.3 , Akinduti P. A.4 Oluwaloniola V. M.1 Tangkat T1, Deji-Agboola
A. M.4

Department of Medical Microbiology University of Ilorin Teaching Hospital

1
2
Department of Ophthalmology, Ladoke Akintola University of Technology Ogbomoso, Oyo State, Nigeria.
3Department of Medical Microbiology and Parasitology, Faculty of Basic Medical Sciences, College of

Health Sciences, Obafemi Awolowo University, Ile-Ife, Osun State, Nigeria.

4
Department of Biological Sciences, Covenant University, Canaanland, Ota, Ogun State, Nigeria.

: [email protected]; +(234) 7062292642

Received: 28.03.2023; Accepted: 20.06.2023

Date of publication: June 2023

Abstract:
The study aims to compare the direct polymerase chain reaction with microbial culture for the detection and fungal pathogens in
infectious keratitis. A total of 81 corneal ulcers were culture and analyzed prospectively. PCR was performed with all corneal
scrapping with fungal and bacteria specific primers. PCR products were analysed and compared with the culture results using
standard methods. Of the 81 samples, 80 were positive by PCR, 51 for fungi and 29 for bacteria. Out of 51 PCR positive samples,
22 samples were culture positive and 29 were culture negative. The majority of PCR genotyped samples matched the positive
culture results. The positive detection rate of 80/81 (98.8%) with high suspicion of fungal keratitis and positive detection rate of
direct PCR 50/51(98.0%) were observed. The sensitivities for the diagnosis of fungal keratitis with direct PCR and culture were
98.0% (50/51) and 43.1% (22/51) (p< 0.001) whereas the specificities were 100.0% (2/2) and 100.0% (1/1) respectively. The time
required to complete the direct PCR was only 3 hours. The direct PCR assay is a rapid diagnostic technique with high sensitivity
and specificity for infectious keratitis and it is expected to have impact on the diagnosis and treatment of infectious keratitis.

Keywords: Infectious, pathogens, detection, universal, strains

microbiological evaluation [10,11] which includes cultures,

1. Introduction smear examination with microscope have limitations despite
Infectious keratitis is a significant public health problem caused the suggestive appearance and course of corneal ulcer produced
by bacteria, fungi, viruses and parasites [1]. World Health by certain pathogens.
Organization estimated that in every year, about 1.5-2.0 million
new cases of monocular blindness that occurred in developing Newer techniques like Analytical Profile Index, a commercially
countries are secondary to corneal ulceration and are common available phenotypic qualitative miniaturized systems API 20E
among outdoor workers and densely populated continents of (Bio Merieux, France) for Enterobacteriaceae and API Staph
Africa and Asia [2,3]. Rapid and aggressive treatment is needed (Bio Merieux, France) have been used for biochemical
to halt the progression and prevent loss of vision [4]. The identification of the bacterial isolates [12]. Polymerase chain
principal causes of microbial keratitis are bacteria and fungi [5], reaction (PCR) is an advanced laboratory technique that
more than 50% of infectious keratitis cases are fungal keratitis involves enzymatic amplification of specific sequences of
(FK), especially in developing countries [6]. Fungal keratitis DNA [13]. The major advantage of PCR is its ability to detect
develops rapidly, and easily induces corneal perforation which DNA from microorganisms which cannot be cultured easily or
usually requires surgical intervention [6,7]. The predisposing require a longer time [14,15,16]. Hence, PCR can detect
factors for infectious keratitis varies with geographical location, microbial DNA and is much more sensitive and specific than
corneal abrasion and ocular surface disorders such as dry eye, conventional methods [17,18,19].
trichiasis are commoner in Iraq [8] while previous trauma to In this study we evaluated the use of culture-based techniques
cornea and corneal opacity is prevalent in Nigeria [9]. Cornea for phenotypic characterization and PCR for genotyping fungal
scrapping which is the mainstay sample for diagnosis of isolates associated with corneal keratitis.
infectious keratitis is obtained in small quantity from the patient
eye and is usually insufficient for the conventional 2. Materials And Methods
microbiological diagnosis hence; there is need for a fast and
accurate diagnostic method of infectious keratitis. Definitive Clinical samples totaling 81 were collected between July 2015
diagnosis of an infectious keratitis is generally confirmed by and July 2018 from patients attending the Ophthalmology
COMPARATIVE EVALUATION OF GENOTYPING AND CULTURE-BASED TECHNIQUES FOR FUNGAL KERATITIS DETECTION

Department of the University of Ilorin Teaching Hospital cornea using 23G sterile needle under Slit Lamp
(UITH), Sobi Specialist Hospital and Civil Service Clinic Biomicroscope after instillation of non-preservative topical
which serve as referral hospitals in Kwara State, North Central, anaesthesia into the infected eye. Immediately after collection,
Nigeria. Ethical permission for the study was obtained. the samples were introduced into brain heart infusion broth
(BHB), yeast extract broth (YEB), and Tris EDTA buffer which
Eyes with clinically suspected viral and parasitic corneal ulcers was stored at temperature of -800C. Also smears of the samples
were excluded. History of pain, photophobia, watering, and were made on the slide for Gram staining and the remaining
redness were taken as inclusion criteria. Duration of symptoms sample was streaked directly on Blood agar, Chocolate and
and history of predisposing factors like trauma, contact lens MacConkey agar.
wear, dry eye, surgery, etc. were noted. Ocular examination
included visual acuity (VA) of both eyes, and slit lamp
examination of the cornea for size, site, and depth of the ulcer,
presence or absence of perforation. Fluorescein staining of the
corneal ulcer for epithelial defect and the presence or absence
of hypopyon were determined. After taking informed consent
and explaining the procedure to the patient, the corneal
scrapings were collected by the Ophthalmologist from infected

81 total samples

80 culture positive 1 culture negative

51 fungal PCR positive 29 fungal culture

positive

Fig. 1. PCR and Culture results of samples collected from infectious keratitis patients

Gram stained for cellular morphology, biochemical test carried positive to phenotypic confirmatory test were grown on
out according to Sagar Aryal [20]. nutrient agar for 24 hours.
The yeast extract broths (YEB) were subcultured onto A single colony growth was picked, transferred to 0.1 mls of
Sabouraud dextrose agar (SDA) and were incubated at 28 sterile water, boiled for 10 minutes to lyse the cells. The lysate
degree Celsius for five days. Lactophenol cotton blue wet was centrifuge and 3 ml of the supernatant was used as the
mount preparation was carried out on the growth from DNA sample for the PCR. All primers were prepared by Iqaba
Sabouraud dextrose agar (SDA) plates while Mycology Atlas Biotechnology, West African Limited. They were used in 4 sets
and biochemical tests were used for the identification of the of PCR as follows:
fungi. Suspected yeast isolates from Sabouraud dextrose agar Extracted DNA was genotyped for bacterial and fungal isolates
(oxoid, UK) were Gram’s stained and confirmed yeast isolates using the universal bacterial primer (27f, 1525r)
were further characterized as reported by Matare [21]. (5’-AGAGTTTGATCCTGGCTCAG-3,
5’-AAGGAGGTGATCCARCC-3); Universal fungi (F, R)
Phenotypical bacteria and fungi isolates (GTG AAA TTG TTG AAA GGGAA, GAC TCC TTG GTC
Bacterial DNA was isolated by boiling method according to the CGT GTT); Species Specific primer (Pseudomonas aeruginosa
steps previously described by Grupta [18]. Isolates that were ( PA-SS-F, PA-SS-R), Staphylococcus aureus (S4F, S4R) and
Staphylococcus epidermidis (91E-F, 3B-R); Primers specific
for Candida albicans (CABF59F, CADBR125R) to identify 25 cycles (GeneAmp PCR System 9700; Applied Biosystems,
samples that gave positive results in the second set of PCR. For Foster City, CA, USA). After an initial denaturation at 95
all PCR protocols, a reaction mixture without sample DNA was degree Celsius for 5 minutes, each cycle consisted of
used as a negative control, in addition to using DNA from the denaturation at 94 degree Celsius for 1 minute, annealing at 50
most common cause of microbial keratitis as a positive control. degree. Amplification was performed in a thermal cycler for a
Amplification was performed in a thermal cycler for a total of total of 25 cycles (GeneAmp PCR System 9700; Applied
2
COMPARATIVE EVALUATION OF GENOTYPING AND CULTURE-BASED TECHNIQUES FOR FUNGAL KERATITIS DETECTION

Biosystems, Foster City, CA, USA). After an initial then electrophorised in 1% of agarose gel then stained with
denaturation at 95 degree Celsius for 1 minute, and extension at E2-vision blue light DNA dye and visualized in an ultraviolet
72 degree Celsius for 5 minutes. The amplified product was transilluminator [15].

3. RESULTS
The age group ranged from 10-65 years with a male to female ratio 3:1 as shown in Table 1 below.

Table 1: Age distribution for Infectious Cornea Keratitis among the patients

Age Group Number of Overall Male Percentage Female Percentage χ2 P-value

(Year) patients percentage (n) (%) (n) (%)
(n) (%)
10- 20 9 11.1 5 6.2 4 4.9

21-49 48 59.3 38 46.9 10 12.4 2.793 0.259

≥ 50 24 29.6 16 19.7 8 9.9

Total 81 100.0 59 72.8 22 27.2

The majority of patients were outdoor workers more importantly the artisans, farmers and traders which accounted for 79.1%
(Table 2). The single largest category among outdoor workers was farmers, constituting 38.3% of total patients.

Table 2: Occupation as risk factor for Infectious Cornea Keratitis among the patients

Occupation Overall Percentage Male Female χ2 P

number value
(n) (%) (n) (%) (n) (%)

Artisan 19 23.5 16 19.7 3 3.7

Farmer 31 38.3 26 32.2 5 6.2 22.035 0.001

Civil servant 7 8.6 7 8.6 0 0.0

Trader 14 17.3 6 7.4 8 9.9

Student 7 8.6 4 4.9 3 3.7

Others 3 3.7 0 0.0 3 3.7

Total 81 100.0 59 72.8 22 27.2

P <0.001 is significant

The mean duration of patients presenting to hospital was 7 days. Most patients had symptoms for at least 7-10 days. Centrally
located corneal ulcers were evident in many patients (75.0%). Majority of the ulcers (87.3%) measured 1-3mm in size while 12.7%
of patients had ulcer size > 4mm.

Table 3: PCR detection results for suspected infectious keratitis in the clinic

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COMPARATIVE EVALUATION OF GENOTYPING AND CULTURE-BASED TECHNIQUES FOR FUNGAL KERATITIS DETECTION

Clinical No of samples examined Positive samples Positive detection Total detection

diagnosis rate % rate %
Fungi Bacteria

Fungal keratitis 52 51 0 62.9% 98.8%

Bacterial 29 29 0 35.8%
keratitis

Out of 52 samples that were fungal suspected infectious keratitis one was PCR negative which means cornea was co-infected with
other microbe aside from bacteria. The total positive detection rate was 98.8% as shown in table 3 above.

Table 4: Performance of Direct PCR and Culture for diagnosis of Fungal keratitis

Direct Culture Outcome of Sensitivity % Specificity %

PCR discrepant analysis,
no

Positive Negative Positive Negative Culture PCR Culture PCR

Positive 22 29 50 1 22/51(43.1%) 50/51(98.0%) 2/2(100%) 1/1(100%)

Negative 1 1 1 1

P<0.001

The performance of culture and direct PCR with corneal scrapings from all patients were analysed. The sensitivity and specificity
of direct PCR were 95.6% (22/23) and 96.6% (29/30) respectively (Table 4). After discrepant analysis, a total of 50 true positives
were obtained. The sensitivity and specificity of direct PCR was 98.0% and 100% respectively. However, of the 50 true positives,
only 22 showed positive results by culture. Therefore, the sensitivity and specificity of culture was 43.3% and 100% respectively.

Figure1. DNA amplicons of Fungal Isolates expressed on agarose gel showed presence
of band in lane 3, 4, 5, 6, 7, 8, 9, 10 and lane 1 is the marker while no band was found in
lane 2.

4. DISCUSSION to 49 years and it indicates an active working year for most men
who strive to have sustainable socioeconomic status and also
In this study, bacterial and fungal strains associated with provide for the family. Many people were involved in active
infectious keratitis were studied among patients of various service such as farming, trading in local materials, retail and
demographic characteristics. The demographic studies showed petty business that could predisposed them to infectious
high prevalence (59.3%) of infectious keratitis among ages 21 keratitis from soil or other contaminated materials while trying
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 COVENANT JOURNAL OF HEALTH AND LIFE SCIENCES, VOL. 01, NO. 1, JUNE 2023

to remove sweat, tears or watery mucous from their eyes. 5. CONCLUSION

Studies done by Saka [9] in Ilorin recorded high participants in The direct PCR assay incorporates species-specific primers aid
the age range 20-80 years (70.3%) as well as Ameen [22] in diagnosis of bacterial and fungal agents causing corneal
Egypt that reveals the higher rate in ages between 40 and 50 keratitis. Hence, it provides useful information on the
years which disagrees with our study. The gender distribution pathogens associated with corneal ulcers. We have found that
in our study reveals higher preponderance of males (72.84%) PCR reliably discriminates bacterial and fungal pathogens. We
than female subjects which is in agreement with Ibanga [23] also find that PCR assay provided precise detection of
who reported 60.87% of the male patients with suppurative pathogens associated with keratitis compared to culture-based
corneal ulcers in Calabar, Nigeria. Joshi [24] and Seal [25] method.
reported similar findings but female preponderances were
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