Study Design
The multicenter, cross-sectional study was conducted from May 9, 2023, to June 15, 2023, among ophthalmic patients with suspected ocular infections of fungal etiology at three health facilities in Ghana: Anglican Eye Hospital, Jachie; St. Michaels Hospital, Pramso; and Kumasi South Hospital, Agogo. Patients' biographical and clinical data were collected using a structured questionnaire. Ocular specimens, specifically conjunctival swabs and corneal scrapings were examined for fungal pathogens. Antifungal susceptibility testing was performed using clinically used standard antifungal agents and native medicinal plants being investigated as potential antifungal agents.
Study population
The study population consisted of patients who sought ophthalmic care/services for exterior eye infections at the Anglican Eye Hospital, Jachie; St. Michaels Hospital, Pramso; and Kumasi South Hospital from May 9 to June 15, 2023. Following informed consent (and written parental consent for minors), a purposive sampling strategy was used to enroll all patients presenting with signs and symptoms of external ocular infections. Patients presenting solely for optical correction, receiving systemic/topical antibiotics, or with one week history of ocular surgery were excluded.
Study setting
The Anglican Eye Hospital, St. Michael's Hospital, and Kumasi South Hospital were selected for this study because of their high outpatient department (O.P.D.) utilization, large catchment area, and rural-urban interactions. The Anglican Eye Hospital and St. Michael's Hospital are in Bosomtwe, a rural district in Ghana, while Kumasi South Hospital is in the Asokwa Municipality, an urban settlement in the Ashanti Region of Ghana. All three facilities have either a permanent or visiting ophthalmologist, optometrists, ophthalmic nurses and opticians. They provide comprehensive eye care services, including medical history, visual acuity assessment, refraction, dispensing of refractive glasses, management of anterior and posterior segment pathologies, prescription of medications, and performance of scheduled surgeries. St. Michael's Hospital and Kumasi South Hospital serve as immediate referral hospitals for the surrounding private and polyclinics. However, all clinical emergencies are referred to the Komfo Anokye Teaching Hospital, the only tertiary health facility in the region. The study facilities do not have a microbiology laboratory, so clinicians rely on empirical approaches for routine diagnosis and management of external ocular and periocular infections.
Study variables
Operation definition
Blepharitis was characterized by symptoms such as gritty, itchy, and sore eyes with crusting and/or collarettes around the base of the eyelashes, as well as clogging of the meibomian gland and loss of eyelashes. Conjunctivitis was defined by conjunctival lesions with symptoms including hyperemia, chemosis, purulent discharge, and hemorrhage. Blepharoconjunctivitis presented with redness, dry and scaly eyelids, and symptoms of itchiness and burning. Keratoconjunctivitis was associated with complaints of dryness, itching, and mucous discharge in both the cornea and conjunctiva. Keratitis was characterized by corneal lesions with symptoms such as edema, cellular infiltration, pain, redness, and photophobia. Hordeolum was defined as swelling and tenderness of the eyelid with acute pain, photophobia, and mild epiphora. Ophthalmia neonatorum is a type of neonatal conjunctivitis that occurs within the first 28 days of life and is characterized by signs such as eyelid edema, erythema, and purulent discharge. Preseptal and orbital cellulitis showed similar features of painful swelling and/or tenderness of the eyelid with the later distinguished from the former by decreased vision and pain on eye motility14.
Data collection
Biographic and clinical data
The patients sociodemographic, socioeconomic, and health status variables were gathered by the principal investigator and a trained research assistant using a structured questionnaire. A comprehensive vision assessment including visual acuity, slit lamp bio microscopy, and ophthalmoscopy was performed by a registered optometrist on all study participants. Subsequently, patient medical history, primary and secondary diagnosis, and antimicrobial therapy prescribed were extracted using a data collection form.
Specimen collection and transport
A total of 237 ocular samples were collected from patients with external eye infections utilizing aseptic procedures. To obtain the conjunctival samples, the patients' eyes were positioned upward, and the lower tarsal plate of the eyelids and the fornix of the conjunctiva were gently rolled with a moistened saline cotton bud in repeated strokes from nasal to temporal and vice versa. For corneal ulcer and keratitis infections, a modified version of an earlier protocol was employed, which involved utilizing slit-lamp biomicroscopy and topical local anesthesia (1-2 drops of 0.5% fresh proparacaine) to firmly scrape the edges of the ulcer. However, no patients in the study had dacryocystitis and/or blepharitis, thus puncture and/or aspiration of the lacrimal sac and swabbing of the eyelids were not performed. The samples were then placed in a sterile, freshly prepared nutrient broth and transported within 1-3 hours in a standard triple packaging system (an absorbent cotton wrapping primary container enclosed in a sealed bag and kept in an insulated icebox with icepacks) from the study sites to the Faculty of Pharmacy and Pharmaceutical Sciences Laboratory at the Kwame Nkrumah University of Science and Technology for fungal microbial investigation.
Laboratory methods
Characterization of fungal ocular isolates
Ocular specimen was investigated for fungal pathogens using standard microbiology techniques. Briefly, ocular specimens were inoculated on a potato dextrose agar (Oxoid Ltd. Basingstoke, Hants United Kingdom) and incubated at 37°C for 96 hours. Fungal positive culture was primarily confirmed by sub-culturing on Potato Dextrose Agar (PDA) supplemented with 100 µg/ml streptomycin, and 100µg/ml penicillin (Fisher Scientific, Schwerte, Germany). Morphologic characterisation, and microscopic identification were performed as seen in Table 9.
Table 9: Identification and characterisation of clinically recovered ocular fungal isolates among ophthalmic patients from Ghana.
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Isolate code
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Colony characteristics
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Microscopic characteristics
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Isolate name
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KS1
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Deep cream color, round shape, pasty and smooth texture with a smell
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Pseudo-hyphae with terminal chlamydospores; clusters of blastoconidia at septa
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Candida albicans
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4
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Cream color, round, and smooth surface
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Clusters of small, budding, ovoid, and elongated cells. No pseudo-hyphae.
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Candida auris
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SM14
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Bluish-green color with a white border, powdery surface, and reverse color is yellow
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Septate hyphae with conidiophores and metulae forming flask-shaped phialides bearing round conidia (“brush” appearance)
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Penicillium citrinum
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SM22
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Cream color, round shape, pasty, and smooth surface
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Round and oval cells parallel to pseudo-hyphae (appearance of logs)
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Candida albicans
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AE5
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Green color with a white border, and smooth surface, and the reverse color is white
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Septate hyphae, with smooth and short conidiophores and an equal length of biseriate phialides and metulae forming the upper half of the vesicle.
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Aspergillus nomius
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K1
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Cream color with rough, mycelial fringe (wrinkle) surface
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Oval and elongated cells
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Candida krusei
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KS3
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Bluish-green color with a powdery or velvety surface and the reverse color is white.
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Septate hyphae, smooth and short conidiophores. Compact uniseriate phialides (occupies upper two-thirds of the vesicle)
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Aspergillus fumigatus
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DNA extraction and quantification
Ocular fungal genomic DNA was extracted using the Quick-DNATM Fungal/Bacterial MiniPrep Kit (Zymo Research Group, California, USA) according to the manufacturer's instructions. The quality of the extracted DNA was evaluated by agarose gel electrophoresis. The concentration of isolated DNA was determined using a Nanodrop ND 2000 at 260 nm (Thermo Scientific, USA).
Polymerase chain reaction
The fungal internal transcribed spacer region (ITS1-5.8S rDNA-ITS2) and the D1/D2 domain of the 26S rRNA gene were amplified from genomic DNA isolated from ocular fungal samples using PCR ITS1 and ITS4 (Forward[F]d: ITS1'-5' TCCGTAGGTGAACCTGCG G3' and reverse[R]: ITS4-5'- TCCTCC GCTTATTGATATGC3') and NL-1 and NL-4 (F: NL1- 5'-GCA TATCAATAAGCG GAGGAAAAG and R: NL4- 5'- GGTCCGTGTTTCAAGACGG) primer pairs (New England BioLabs Inc). PCR reactions were performed in a 20 µL reaction volume. Briefly, 1 µL of template DNA was added to a 19 µL master mix containing 12.5 µL of 10x Dream TaqTM Green Buffer (KCl, [NH4]2SO4 and 20 mM MgCl2), 0.5 µL each of forward and reverse primers, and 5.5 µL nuclease-free water. The denaturation step was performed at 95˚C for 5 minutes, followed by 25 amplification cycles of 95°C for 1 minute, 49°C (ITS1-F and ITS4-R) or 52°C (NL1-F and NL4-R) for 30 seconds, and 72°C for 2 minutes, with a final extension step of 10 minutes at 72°C. PCR products were examined on 1.5% agarose gels run in 1 x Tris-borate-EDTA (TBE) buffer and visualized by ethidium bromide staining under UV light.
Antifungal susceptibility testing
Antifungal susceptibility testing was performed using the agar well diffusion method, as previously documented67,68, for common clinically used antifungal agents and medicinal plant extracts. To commence the procedure, a microbial inoculum of isolated ocular fungi with a McFarland standard of 0.5 (1x107 CFU/mL) was employed to create an even lawn of cells on a 20mm sterile plate with potato dextrose agar. Subsequently, equal diameter wells (6mm) were created using a sterile cork-borer, and each well was respectively seeded with 0.1ml of crude medicinal plant extracts of four different concentrations (100, 50, 25, 12.5 mg/ml). Clinical antifungal agents used as reference drugs were prepared by dissolving in DMSO (amphotericin B [AMB], terbinafine [TER], itraconazole [ITZ]) and distilled water (fluconazole [FLZ])31. Following incubation, the clear zones around the wells containing extracts and anti-fungal drugs (zone of inhibition) were measured in millimeters using a caliper and meter rule. The entire procedure was performed in triplicates to obtain the mean zone of growth inhibition. DMSO was and distilled water were tested as negative controls67,68.
Determination of Minimum Inhibitory Concentration and Minimum Fungicidal Concentration
The minimum inhibitory concentration (MIC) of crude medicinal plant extracts were evaluated using the broth dilution assay as previously described67,69,70. Two-fold serial dilutions of the extracts or standard antifungal agents (AMB, FLZ, ITZ, and TER) were prepared in sterile 96-well microtiter plates. One hundred microliters of each two-fold serial dilution of crude extract were transferred into the wells, followed by the addition of 100 μl of double strength nutrient broth containing an inoculum size of approximately 2.0 x 105 CFU/ml. The plates were incubated for 24 hours at 37°C. Afterward, 20 µL of a 1.25 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (M.T.T) solution were added to each well, and the plates were further incubated for 30 minutes at 37°C. The MIC was determined as the lowest concentration of extract or antibiotic that completely inhibited the growth of the organism in the microdilution wells, as detected by the absence of purple coloration after MTT addition during a 24-hour incubation period at 37°C. All assays were performed in triplicate. Furthermore, the minimum fungicidal concentration (M.F.C) was performed as previously described69. For each well that displayed no obvious purple coloration indicative of growth from the MIC assay, 20 µL of suspension was transferred into new plates containing 100 µL freshly prepared nutrient broth. The microtiter plates were then incubated at 37°C for 24 hours. Following the incubation period, 25 µL of 125 mg/mL MTT was introduced into each well and incubated at 37°C for 30 minutes to facilitate the proliferation of microorganisms. MFC was defined as concentration at which no visible growth was observed after the introduction of the MTT dye67,69,70.
Collection, authentication, and preparation of medicinal plants
The fresh leaves of S. alata, C. odorata, and Aspilia africana were collected from Kwahu-Asakraka (6°38′05.2″N 0°41′20.6″W) in the Eastern Region of Ghana between June and July 2023. Similarly, dried flower buds of S. aromaticum and powdered stem bark of C. verum were purchased from a spice retail shop from a local market in the Ashanti Region of Ghana (latitude 6.6886o N, longitude 1.6156o W), in the Ashanti region of Ghana in July 2023. The authentication of the medicinal plants was performed by an expert botanist and the herbarium accession number of the S. alata (KNUST/HMI/2023 L016), A. africana (KNUST/HMI/2023 L017), C. odorata (KNUST/HMI/2023 L018) C. verum (KNUST/HMI/2023 SB027) and S. aromaticum (KNUST/HMI/2023 FB001) respectively stored at the reference repository of the Department of Herbal Medicine at the Kwame Nkrumah University of Science and Technology in Kumasi, Ghana. After collection, the fresh leaves of S. alata, C. odorata, and A. africana were washed under running tap water and left to air-dry at room temperature for six days to remove moisture. The dried leaves and dried flower buds of S. aromaticum and C. verum were homogenized into a fine powder and stored in an airtight plastic container at room temperature until use.
Extraction and phytochemical analysis of plants
Ethanolic extracts of medicinal plants were obtained using cold maceration method as previously documented. Phytochemical screening performed on plant extracts to identify bioactive compounds as described previously66,67,71,. Briefly, to elucidate the presence of tannins, a 100 mg sample of each plant extract was mixed with 3 ml of distilled water and 2 drops of 10% alcoholic FeCl3 solution. The formation of a deep blue or green coloration signified the presence of tannins. For saponins, a 100 mg of plant extract was mixed with 3 ml of distilled water, resulting in persistent foam formation as a marker of saponins. To test for alkaloids, a 100 mg sample extract was mixed with 3 ml of distilled water and vigorously shaken. A 1 ml sample of each solvent extract was then treated with 4 drops of 1% HCl, heated at 60°C for 60 seconds, and cooled. Next, 5 drops of Wagner's reagent were added to the solution, and the formation of a reddish-brown precipitate indicated the presence of alkaloids. To test for terpenoids, A mass of 100 mg of each of the plant extract was added to three (3) mL of distilled water and mixed vigorously. A volume of 0.5 mL of chloroform was added to one (1) mL of the solvent extracts in different test tubes and treated with two (2) drops of concentrated H2SO4 slowly down the test tube wall. A ring formation of reddish-brown precipitate showed the occurrence of terpenoids. To test for flavonoids, a mass of 100 mg of each of the plant extract was added to three (3) mL of distilled water and mixed vigorously. One (1) mL of each solvent extract was treated in their separate test tubes with a few drops of 20% NaOH. The presence of flavonoids was demonstrated by the production of a bright yellow color that became colorless with the addition of dilute HCl. Liebermann’s test was performed to ascertain the presence of glycosides. Briefly, crude extract was combined with equal volumes of chloroform and acetic acid (2ml), and the resulting mixture was cooled in an ice bath and cautiously added with concentrated sulfuric acid. The presence of a color change from violet to blue to green indicated the presence of glycoside66,68,70.
Quality assurance and control
The study used a modified structured questionnaire to collect demographic and clinical data. Questionnaires were written in English and administered by the principal investigator and a trained research assistant. The questionnaire was explained in the local dialect for study participants who could not understand instructions in English. Study data were double-checked for accuracy and completeness. At the facility, all test tubes containing eye samples were well labeled to prevent mix-ups. Laboratory reagents and culture media for the experiments were checked for expiration dates, and sterility control was performed to determine the integrity of the media, such as freedom from contamination. Evaluation of media performance and/or functionality was performed using the American Type Culture Collection (A.T.C.C.) standard reference strains of C. albicans ATCC 10231. Medicinal plant species used for studies were accurately identified and verified by an experienced botanist.
Data protection and management
The hardcopy of the completed questionnaires was securely stored in a locked location, accessible only to the principal investigator and research consultant. Similarly, the non-aggregated data set in softcopy was protected by a strong alphanumeric password. The research report presented to institutions and for publication was aggregated, making it impossible to trace individual study participants.
Ethical considerations
A hierarchical ethical review protocol was followed to conduct this study. The study protocol was modified from a previous investigation and approved by the Committee on Human Research, Publication and Ethics (C.H.R.P.E.) of the Kwame Nkrumah University of Science and Technology and the Komfo Anokye Teaching Hospital (reference number: CHRPE/AP/116/24). Written informed consent was obtained from all adult participants. For minors, written informed consent was obtained from caregivers after the study protocol was explained to the best of their understanding. The study adhered to the tenets of the Declaration of Helsinki, and all laboratory procedures were performed in accordance with the guidelines of the Clinical Laboratory Standard Institute (C.L.S.I.).
Statistical analysis
Data were entered and processed in Microsoft Excel before being exported to a Windows-compatible version of a statistical package and service solution (IBM Corporation IBM® SPSS® Statistics for Windows, version 25.0 Armonk, NY). The Kolmogorov-Smirnov statistic was used to determine normality. Biographical, medical, and ophthalmic characteristics were presented, and the sex difference was examined using chi-squared analysis. Cross-tabulations with frequencies and percentages were used to illustrate clinical diagnosis, cultural status, and antifungal therapy. All antifungal susceptibility assays were performed in triplicate with identical colonies, and inhibition zones are presented as mean ± standard deviation. The association between sample characteristics and the prevalence of ocular fungal infections was examined using bivariate logistic regression with a significance level of p <0.05.