Diagnostic Considerations for Non-Acanthamoeba Amoebic Keratitis and Clinical Outcomes

Cases of amoebic keratitis involving species other than Acanthamoeba are hypothesised to be underdiagnosed and poorly understood. Amoebic keratitis is debilitating and associated with chronic visual impairment. Understanding associated symptoms of non-Acanthamoeba amoebic keratitis could facilitate new diagnostic procedures and enable prompt treatment, ultimately leading to improved patient outcomes. Thus, a review of the literature was undertaken surrounding non-Acanthamoeba amoebic keratitis. Cases were geographically widespread and mostly confined to contact lens wearers ≤ 30 years old exposed to contaminated water sources and/or demonstrating poor lens hygiene. Vermamoeba vermiformis (previously Hartmanella vermiformis) was the most common causative agent, and a moderate number of mixed keratitis cases were also reported. A crucial disease indicator was early onset stromal deterioration/ulcerations, reported in 10 of the studies, usually only occurring in advanced Acanthamoeba keratitis. Mixed infections were the most difficult to treat, often requiring keratoplasty after unsuccessful combination treatment regimens. New diagnostic measures for non-Acanthamoeba amoebic keratitis should consider early onset stromal disease as a key disease indicator. Deep corneal scrapes are also necessary for accurate amoebic identification. Moreover, a combination approach to diagnosis is advised and should involve culture, microscopy and PCR techniques. In vitro drug sensitivity tests should also be conducted to help develop patient-specific treatment regimes.


Introduction
Amoebic keratitis is of growing clinical concern given the recent rise in cases globally and the difficulties associated with accurate diagnosis and treatment [1]. A recent outbreak of amoebic keratitis has been described in the UK, with other developed countries following similar trends [1][2][3][4][5]. Acanthamoeba spp. are most commonly associated with amoebic keratitis, and most cases can be linked to contact lens wear and poor contact lens hygiene [6][7][8]. Although not the only risk pathways of infection, exposure of contact lenses to contaminated water, overwearing lenses and improper cleaning and storage of lenses and cases (e.g., using tap water for cleaning purposes) enable amoebic adhesion and growth on lens surfaces and subsequently increase the risk of corneal infection [7].
While Acanthamoeba keratitis is the most common form of amoebic keratitis and as such has been the subject of extensive research, there are other potential contributing organisms that are less considered [1]. Despite a lack of conclusive evidence demonstrating that amoebae, with the exception of Acanthamoeba, are capable of causing keratitis in isolation, there have been several reported cases of positively identified non-Acanthamoeba amoeba species during keratitis infections documented in the literature. For example, Vermamoeba vermiformis (previously Hartmannella vermiformis), Vannella and Vahlkampfia species have all been associated with amoebic keratitis, and a lack of knowledge surrounding their potential for infections may hinder effective diagnostic or treatment protocols [9]. cies have all been associated with amoebic keratitis, and a lack of knowledge surr their potential for infections may hinder effective diagnostic or treatment protoc Because of the poorly understood role of non-Acanthamoeba amoebic kera have conducted an extensive review of the literature to further detail its prevalen noses, disease symptoms and treatments. In doing this, we aim to further the acc diagnostic procedures that might aid in the rapid diagnosis and treatment of the ogens.

Results
A total of 18 articles satisfied the inclusion guidelines set for this review (F and key information was extracted from each article (Table 1). Studies were cond various locations worldwide, and study participants were diverse with respect to age and history of contact lens wear (Table 2). Of all the identified case studies, pa specifics were only detailed in the study conducted by Hajialilo et al. [10]; conse they are excluded from Table 2's participant section. Cases were described m quently in females with 61.5% (8 female to 5 male) and also more frequent in par within the 30-and-under age category (69.2% of cases, 9 ≤ 30 y/o to 4 > 30 y/o). Ca much more apparent in contact lens wearers than in non-contact lens wearers, w of cases linked to contact lens wear (61 to 5).   CR-Case Report, CS-Case Series, CL-contact lens(es), F-female, M-male, PCR-polymerase chain reaction, AK-Acanthamoeba keratitis.  Of all infections, 21% were identified in conjunction with an additional amoebic infection, with half being a co-infection with an Acanthamoeba spp. Isolated infections with Vermamoeba vermiformis were most frequent, followed by isolated Vahlkampfia infections (53% and 23%, respectively). It should also be noted that in many cases, non-amoebic co-infective agents were identified during diagnoses, and in others, evidence of extensive screening to exclude the presence of additional microbial or viral pathogens was not provided. Thus, the role of these amoebae as causative agents of keratitis and not as vectors for the transmission of other microbial pathogens, as has been described in other free-living amoebae [27], remains inconclusive. Nonetheless, the most common clinical manifestations of non-Acanthamoeba amoebic keratitis included pain (61% of studies), inflammation and irritation (61%), stromal keratitis and ulcerations (56%), epithelial abnormalities (50%), ring infiltrates (50%), impaired vision (44%) and photophobia (44%) ( Table 3). Diagnostic techniques vary between studies, with earlier studies relying primarily on culture and microscopy techniques to identify suspected pathogens. More recent diagnoses, however, have involved the use of 18s rRNA sequencing techniques to provide more accurate diagnoses with genus-specific primers ( Figure 2). Additionally, temperature tolerance tests have been performed, as they are deemed to be good indicators of pathogenic virulence [26,28].
Therapeutic approaches were broad and predominantly included drug combinations for all forms of amoebic keratitis (Table 1). This approach is similar to the approach taken with AK because no single anti-amoebic agent exists that can successfully eradicate all Acanthamoeba genotypes; hence, drug combinations are used [7,29]. Medical interventions included anti-amoebic agents (topical biguanides and aromatic diamidines), antibiotics, antifungals, antivirals and anti-inflammatory medications. Notably, several studies reported the need for keratoplasty due to poor treatment responses (Table 1) [12][13][14]17,18,22,24,26]. This emphasises the difficulty of finding an effective therapeutic agent to cure amoebic keratitis and highlights the possibility of drug resistance in other FLA besides Acanthamoeba.

Discussion
This review demonstrates that cases of non-Acanthamoeba amoebic keratitis are occurring and are geographically widespread; no single location showed a significantly higher incidence rate than any other (Table 2). Notably, however, disease incidence appears to be confined to developed countries, thus following a similar pattern to AK occur-

Discussion
This review demonstrates that cases of non-Acanthamoeba amoebic keratitis are occurring and are geographically widespread; no single location showed a significantly higher incidence rate than any other (Table 2). Notably, however, disease incidence appears to be confined to developed countries, thus following a similar pattern to AK occurrences [7]. Although it is unclear, this might be the result of differences in diagnostic techniques between countries. In terms of patient demographics, Table 2 shows that the majority of individuals diagnosed with non-Acanthamoeba amoebic keratitis were ≤30 years old, and a significantly high proportion of positive cases were associated with contact lens wearers (61 cases vs. 5 non-CL wearers who had suffered ocular trauma). These findings also coincide with typical AK observations in which amoebic keratitis predominantly occurs in young, economically active adults who are most likely to wear contact lenses [2,7].
The similarities to AK infections complicate diagnoses. Generally, treatment outcomes of AK infections coincide with the speed by which accurate diagnoses can be made [7,30]; thus, a similar emphasis should be placed on rapid diagnosis in non-Acanthamoeba amoebic keratitis. Differentiation between amoebic infections is difficult; despite this, early onset stromal keratitis and ulcerations are indicative of Vahlkampfia, Vermamoeba and Vannella sp. involvement and could be useful during early observations (see Table 1) [9,11,[13][14][15]17,19,20,22,24]. While microscopic and culture techniques can be applied during diagnostics, species identification using PCR tests remains the most accurate strategy for diagnosis and should be utilised at the earliest possible convenience [1]. We provide a more comprehensive diagnostic workflow for suspected amoebic keratitis in Figure 2. The presence of co-infectious agents in amoebic keratitis is common, and several non-amoebic species were also identified that can complicate treatment, diagnosis and the overall understanding of amoebic influence during infections [11,12,17,18]. For example, Kennedy et al. (1995) [11] reported growth of Staphylococcus aureus from corneal swabs, while Aitken et al. (1996) [12] noted the presence of yeast-like fungi on the corneal surface of the patient. Advances in molecular techniques allow more comprehensive screening for co-infectious agents and should be conducted in tandem with a traditional culture-based approach to assess the most appropriate course of action for the patient.
Despite the rarity of non-Acanthamoeba amoebic keratitis, the literature compiled within this review demonstrates that care must be applied during the initial diagnoses of amoebic keratitis to ensure the most suitable treatment method is applied. It is also worth noting the recent rise in AK infections globally [2][3][4][5] and that consideration must also be given as to whether non-Acanthamoeba causative agents of amoebic keratitis follow a similar trend. Overall, we demonstrate here that non-Acanthamoeba amoebic keratitis follows a similar pattern with regard to patient demographics and treatment regimens and that infections generally coincide with contact lens wear. We also highlight differences in disease presentation and methods of diagnoses. Despite its low prevalence, a broadspectrum approach should be applied to the initial treatment of amoebic keratitis, which will necessitate further investigation into how AK therapies interact with other causative agents of amoebic keratitis. Furthermore, the role of non-Acanthamoeba amoebic species in keratitis infections has yet to be fully elucidated and, therefore, warrants additional research.

Materials and Methods
Four key databases were accessed: Scopus, Science Direct, Google Scholar and The National Center for Biotechnology Information (NCBI). The search criteria narrowly focused on Vahlkampfia, Vermamoeba (Hartmannella) and Vannella sp. and their involvement in keratitis. No date restrictions were applied due to the elusive nature of non-Acanthamoeba amoebic infections and the scarcity of relevant literature. Key words combined with Boolean operators and nested search strings were used to ensure results were confined to the topic of interest. Figure 1 details the flow path by which studies of non-Acanthamoeba amoebic keratitis infections were identified. All reported cases of keratitis associated