Corneal ulcers are estimated to cause 7.2% of corneal blindness ¹ and are thus a significant contributor to blindness. In Kenya, corneal injuries have been reported to account for an estimated 2.7% of corneal blindness. ² While corneal injuries are common, access to treatment and medical care is limited, and this may be due to a lack of awareness of treatment options, lack of accessibility and high cost. ³ Lack of appropriate and prompt management of ulcers leads to poor wound healing and subsequent formation of scars, resulting in partial or total vision loss.

Antibiotics act by interfering with the metabolic processes or with the organism’s structures. However, there are growing concerns about the number of drug-resistant bacterial strains ⁴ and many disease conditions are becoming more difficult to treat due to the emergence of antibiotic-resistant bacteria. ⁵ In 2010, the World Health Organization (WHO) recommended that all countries implement control procedures to mitigate the effects of multi-drug-resistant bacteria. ⁶ It emphasized the urgent need to identify alternative therapies against drug-resistant microorganisms in low- and middle-income countries, such as Kenya. In addition, Kenya has been reported to have a high utilization rate of unorthodox medical treatment due to challenges of access to orthodox medicine, cost and cultural beliefs. ³

A mushroom is a fruiting body of fungi that is non-photosynthetic and feeds on organic matter and plant organisms. ⁷ Ganoderma lucidum is a type of mushroom which has basidiocarp, mycelia and spores that contain approximately 400 different bioactive compounds. ⁸ G. lucidum as a mushroom is in the family of Ganodermataceae, a family consisting of a large group of tree fungi of the genus Ganoderma. G. lucidum has been classified into Kingdom Fungi, Phylum Basidiomycota, Class Basidiomycetes, Sub-class Homobasidiomycetes, Order Polyporales, Family Ganodermataceae, Genus Ganoderma and Species lucidum. ^{9 , 10} The mushroom has been reported to have several pharmacological effects, such as immunomodulation, analgesic, anti-atherosclerotic, anti-inflammatory and antibacterial, among other medicinal benefits to the human body. ⁸ The antibacterial effect of G. lucidum has been shown to have a healing effect on non-ocular related inflammatory injuries and infective ulcers in other parts of the human body. ¹¹ Many ulcerative conditions are becoming difficult to manage due to the increasing numbers of drug-resistant bacteria strains such as Staphylococcus aureus, Pseudomonas pyocyanea, Streptococcus pneumoniae, Pseudomonas aureginosa. ^{5 , 12} G. lucidum has been proven to be effective in managing bacterial infections that have shown resistance, ¹³ with reports indicating it to be an effective alternative to treating bacterial infections and inflammatory injuries to the human body. ¹⁴ G. lucidum is commonly available in Kenya. ⁷ This study aimed to investigate the effects of G. lucidum on chemical-induced and bacterial-infected corneal ulcers in rabbits’ eyes. Furthermore, the study aimed to compare the effects of G. lucidum with those of standard orthodox treatment approaches to chemical and bacterial corneal ulcers to establish a possible relatively cheaper alternative and/or complementary treatment to these corneal injuries.

Data were collected from 16 healthy New Zealand rabbits weighing an average of 1.4±0.42 kg. Ocular parameters for various observations following treatment for 168 hours after the introduction of chemical injuries and subsequent infection with P. aeruginosa were coded according to the grading scale of 0 to 5 and these are presented in Tables 1 to 4. Table 1 shows a comparison of the healing effects of G. lucidum extract on various ocular parameters following induction of chemical injury, using 1 Molar sodium hydroxide, for animals in group A (experiment group 1) at two-time points: at the 24 ^th and 168 ^th hours of treatment. The statistical presentation shows that although treatment with G. lucidum extract had a significant anti-inflammatory effect on just a few ocular structures: eyelid and conjunctival status changes, reduced lacrimation, reduced corneal staining and oedema, the clinical effect of treatment with the extract in all ocular structures observed were highly significant, particularly with the reduction in corneal infiltrates (effect size=74%). These pictural findings are shown in Figure 1.

Table 2 shows the manifestations of various ocular parameters for animals in group B (experiment group 2) at the first 24 ^th hour following injury with 1 Molar sodium hydroxide, and subsequent infection with P. aeruginosa 12 hours thereafter (to induce bacterial ulcer), and at the last 168 ^th hour of treatment with G. lucidum. The results show that treatment with G. lucidum extract had no significant difference (p>0.05), in the statuses of most of the ocular parameters after the 168 ^th hours, except for a significant reduction in photophobia and lacrimation (p<0.05). However, the clinical effects of treatment with the extract of G. lucidum for bacterial infected corneal ulcers in all ocular parameters observed were clinically significant (effect size>5%), with reduction of ocular discharges being most significant (effect size=93%). This finding is also shown pictorially in Figure 2.

Table 3 shows a comparison between manifestations of various ocular parameters for the animals in groups B and C after 168 hours of treatments with G. lucidum in group B (experimental group 2) and the standard treatment protocol in group C (positive group). The results show that although treatment in group C had better clinical effects than in group B, the difference in the treatment was, nonetheless, not statistically significant (p>0.05). This finding is also shown pictorially in Figure 3.

Table 4 shows a comparison between the manifestations of various ocular parameters for animals in group B (experiment group 2) and those in group D (negative control group) after 168 hours of treatments with G. lucidum on the former (group B) and atropine alone for the latter (group D). The results show that the difference in the treatment in both groups was not statistically significant (p>0.05). This finding is also shown pictorially in Figure 4.

In this study, we found a progressively improved healing effect of the extract of G. lucidum, from the first 24 ^th hour to the 168 ^th (seven days of treatment). The indices adopted for the study to show improvement in the healing process of a deliberately induced corneal injury and then subsequently infected using P. aureginosa, a common, normal floral, but opportunistic microorganism, were eyelid status, conjunctival status, lacrimation, epithelial eruption, corneal staining, photophobia, ocular discharge, corneal oedema, and corneal infiltrates. In group A animals ( Table 1), there were observable changes in this first experimental group exposed to chemical injury alone using 1 molar sodium hydroxide. Our findings showed that treatment with crude aqueous extract of G. lucidum had significant healing effects on just a few ocular parameters: eyelid status change, reduced lacrimation, reduced corneal staining and oedema. Nonetheless, there were improved clinical effects of treatment with the extract in observed ocular parameters as seen in Figure 1. This thus demonstrates that treatment with G. lucidum after the 168 ^th hour improved the corneal healing process following an induced chemical (alkaline) injury. However, as can be seen from Figure 1, and as is typical with most chemical injuries, specifically from sodium hydroxide that causes corneal damage through pH change, ulceration, proteolysis and collagen synthesis defects to the cornea, healing from such injuries takes time, often longer than 7 days to achieve significant physically observable signs of healing. ¹⁹ This time limitation was a key challenge in this study. Still, and as noted by Singh, ²⁰ chemical injuries, particularly those of alkaline origin, to the cornea are potentially blinding ocular injuries and constitute a true ocular emergency requiring immediate assessment and initiation of treatment. ²⁰ Our finding demonstrates that instituting treatment with an extract of G. lucidum as early as within the first 24 ^th hour of occurrence has the potential to prevent extensive and penetrating damage to the cornea and other ocular surface tissues that present a risk of irreversible vision loss.

Furthermore, among animals in group B (experiment group 2), as shown in Table 2, our finding demonstrates that treatment with crude extract of G. lucidum did not significantly change the ocular morphology of most of the animals after the 168 ^th hours, except for a significant reduction in photophobia and lacrimation. Even though the differences in the quantitative morphological parameters at the two-time points, following the initiation of treatment with the crude aqueous extract of G. lucidum, compared were not significant, the converse ( Table 2), was true for the clinical effects (effect size>5%). This, therefore, implies that the clinical effect of treatment with crude aqueous extract of G. lucidum for bacterial infected corneal ulceration is significant. Additionally, the clinical (healing) effects were found to be most significant with a reduction of ocular discharges, a good morphological indication of a reduction in microbial load. ¹³ This finding, also shown pictorially in Figure 2, confirmed findings of previous studies, ^{5 , 11 , 12} that the antibacterial effect of G. lucidum on non-ocular related inflammatory injuries and infective ulcers in other parts of the human body is clinically effective. In addition, our finding demonstrates that G. lucidum has proven to be effective in managing bacterial infections that have shown resistance, ¹³ as well as an effective alternative to treating bacterial infections and inflammatory injuries to the human body, ¹⁴ may as well be an effective alternative to the management of ulcerative conditions of the cornea resulting from bacterial infections.

Our study also compared the treatment effect of the crude aqueous extract of G. lucidum with the existing standard treatment protocol for chemical (alkaline) induced ocular injury and bacterial-induced corneal ulcers consisting of two hourly drops of fluoroquinolone (ciprofloxacin), six hourly drops of corticosteroid (betamethasone), and eight hourly drops of atropine. ^{17 , 18} We found that after the 168 ^th hour of treatments, while animals in group C had better clinical healing (effect size>5%) , the difference between the two treatment protocols was not statistically significant (p>0.05) as can be seen in Table 3. The implication of this finding, therefore, is a demonstration that treatment with the crude aqueous extract of G. lucidum may be an effective approach to the treatment of bacterial-induced corneal ulcerative and inflammatory injuries. This further justifies a previous finding on the healing effects of extracts of G. lucidum on infective and inflammatory injuries to other non-ocular tissues of the human body. ¹⁴

Finally, we sort to further demonstrate the healing effects of crude aqueous extract of G. lucidum on ulcerative injuries to the cornea and other ocular adnexa. To do this, we compared the results, after 168 hours of treatment, of animals treated with the crude extract to those of a negative control group that only received atropine treatment ( Table 4). As shown, even though we found no significant difference (p>0.05) in the two groups, treatment with the crude extract showed less clinical healing effect ( Figure 4). This was an interesting finding of our study that the animals in group D (negative control) that were administered with atropine sulphate alone showed better clinical improvement than those treated with G. lucidum. Atropine sulphate was purposively administered to control ciliary spasms in the eyes of the animals in the negative control group. It was interesting to observe that atropine (primarily a cycloplegic agent) also known to show anti-inflammatory secondary effects, ^{21 , 22} resulted in significantly better healing effects compared with the crude aqueous extract of G. lucidum. This finding may explain the importance of introducing an anti-inflammatory treatment protocol as early as possible in the management of both chemical and bacteria-induced corneal ulcerations.

To summarize, we note that there is a dearth of empirical knowledge on the ocular-related healing effects of extracts of G. lucidum in literature to compare our findings with. Nonetheless, our study demonstrates an observable reduction in the severity of damages to the ocular adnexa from induced ulcerative chemical injury and bacterial infection. These reductions are evident from the positive changes in the ocular parameters affected following the induction of chemical injury and subsequent bacterial infection of the cornea. Specifically, we note a significant reduction in eyelid swelling and lacrimation in animals treated with G. lucidum after 168 hours of treatment. This is evident with the animals in groups A and B, where the eyelid and conjunctival severity were significantly reduced progressively ( Figures 1 and 2). Although no significant change was noticeable in a few parameters (epithelial eruption, epithelial staining, corneal fluorescein staining, and corneal oedema), we found the clinical healing effects of the crude aqueous extract of G. lucidum extracts to be highly significant, particularly for the reductions observed with ocular discharges, photophobia and corneal infiltrations. This we found, while not consistent with the healing effect observed in a study on paw oedema following intra-muscular instillation of G. lucidum, ²³ was nonetheless consistent with the healing effect observed in another study on gastric ulcers. ²⁴ Other studies that compared the clinical healing effects of extracts of G. lucidum with commonly used antibiotics, such as gentamycin sulfate ¹³ and fluoroquinolones, ²² found it to be equally effective in other body structures. We found this to not be the case with ocular healing effects, as standard treatment with a combination of fluoroquinolone, corticosteroid and atropine sulfate, and even with using atropine sulphate alone, turned out to show better clinical effect than treatment with the crude aqueous extract of G. lucidum alone.

Crude aqueous extracts of G. lucidum have shown the potential to be an alternative treatment approach for chemical (alkaline) and bacterial ( P. aeruginosa) induced corneal ulceration. The healing (anti-inflammatory and anti-infective) effects of G. lucidum have shown clinically significant healing effects after 168 hours of treatment compared with that of 24 hours. Although the cornea of animals treated with standard protocol showed better clinical effects than those treated with G. lucidum, the current investigation has shown the importance of the early institution of the anti-infective and anti-inflammatory protocol in the management of corneal ulcers. Therefore, G. lucidum, where available, may be a useful anti-infective alternative for chemical-induced and bacterial-infected corneal ulcerative conditions. In this regard, therefore, our study notes that despite the limitation of time, crude aqueous extract of G. lucidum has bioactive compounds that have the potential to be used as an alternative treatment approach for chemical-induced and bacterial-infected corneal ulcerations in resource-constrained settings. Hence, the study recommends further research to explore the anti-inflammatory potentials of crude extract G. lucidum in population-controlled settings. The study further recommends research to characterize the bioactive compounds in the crude aqueous extract as well as cytotoxicity assay of the bioactive compounds in the crude aqueous extract, as with tests to demonstrate the effectivity levels of extracts of the mushroom from other less polar and non-polar solvents.

EOV was involved in the study design, data collection, data analysis, drafting and reviewing of the manuscript. AKM was involved in the study design, data collection, data analysis, drafting and reviewing of the manuscript. POO was involved in data collection, drafting and reviewing the manuscript. AVD was involved in drafting and reviewing the manuscript to its final stage. KPM was involved in drafting and significant review of the manuscript to its final stage. All authors have read and approved the final version of the manuscript.