Genotypic characterization of amoeba isolated from Acanthamoeba keratitis in Poland

Amoebae belonging to the genus Acanthamoeba are free-living organisms, widespread in the environment, and excellently adjusted to various environmental conditions (Stockman et al. 2011). They occur in two developmental stages: trophozoites and cysts, resistant to environmental conditions (Marciano-Cabral and Cabral 2003). The invasive forms for the human are cysts as well as trophozoites of amoebae. Infection occurs most often in the warm seasons and is associated with contact with water. In particular cases, the infection may take place through damaged skin or accidently injured cornea, e.g., during earth works or playing with sand or water, or through contact lenses.

The most significant and most common disease caused by the free-living amoebae is Acanthamoeba keratitis (AK).

The first cases of the amoeba’s invasion of the cornea were described in 1974 (Naginton et al. 1974). In subsequent years, only a few cases of AK were described. After 1981, a rapid increase in the number of cases of keratitis was observed, which reached a peak in 1985. Most cases were described in the USA.

Within the last two decades, amoebae of the Acanthamoeba genus have become a well-known and significant risk factor for the keratitis. Contributory factors include global climate warming and the increase in the population of patients with immunological deficiencies. The number of diagnosed cases of AK in the USA increased from 22 in 1999 to 170 in 2007 (Yoder et al. 2012). A proportional increase of the number of diagnosed cases of AK also occurred in Poland.

So far, apart from the USA, AK cases have been described in Europe, Australia, Asia, and Africa. The approximate number of infections is counted in thousands. The majority of cases, as many as 85–88 %, are connected with the wearing of contact lenses (Dart et al. 2009). Among this group, 88 % of cases are connected with wearing hydrogel contact lenses and 12 % with wearing rigid lenses. Seal (2003) stated that the probability of contracting AK each year is 1:30,000 in persons wearing contact lenses.

In persons not wearing contact lenses, AK is usually diagnosed late, and in these cases, the disease is usually already at an advanced stage.

AK usually affects one eye. The first symptoms are blurred vision, photophobia, and severe pain in the eye not explained by corneal injury. Conjunctival swelling and eyelid swelling also appear. Patients have bleary and teary eyes. In the front layers of the corneal stroma, there are diffuse, ring-shaped, or crescent infiltrates and less specific satellite infiltrates. The ring-shaped infiltrates are single or numerous. The epithelium covering the corneal stroma can be unharmed or may have punctate erosions. In the late stage of the disease, pus appears in the eye. Together with the development of the disease, the corneal edema increases (Kosik-Bogacka et al. 2010).

The omnipresence of amoeba in the surrounding environment is undeniable. These cosmopolitan organisms are found in samples of soil, air, water, and animal tissues. Acanthamoeba spp. have been isolated and identified in many countries around the world, for example in Bulgaria (Tsetkova et al. 2004), Iran (Mahmoudi et al. 2012), Spain (Magnet et al. 2012), Brazil (Duarte et al. 2013), Japan (Edagawa et al. 2009), the USA (John and Howard 1995), Switzerland (Gianinazzi et al. 2009), and also in Poland (Kasprzak and Mazur 1972).

Recognition of invading amoeba of the genus Acanthamoeba in the eye is difficult. Numerous corneal infections caused by viruses, fungi, or bacteria cause symptoms similar to amoeba infection. Very often, an infection of the eye is mistakenly diagnosed as an infection caused by herpes simplex viruses.

In the case of inflammation of the cornea caused by Acanthamoeba, there are no uniform standards for diagnostic procedures. The most common diagnostic method is culture of corneal scrapings on NN agar coated with bacteria (Borin et al. 2013). Methods of visualization of the cornea using confocal microscopy and immunofluorescence are also used (Aniśko-Słomińska et al. 2011; Kokot et al. 2012). Additional methods of detection include molecular analysis of nuclear and mitochondrial DNA and methods of testing restriction fragment length polymorphism analysis after digestion of DNA (Kosik-Bogacka et al. 2010).

The taxonomy of the small amoeba species is not well established. Consequently, little is known about the genetic relationships between pathogenic and nonpathogenic strains of Acanthamoeba. During the last few years, molecular techniques (such as polymerase chain reaction—PCR) are increasingly being used in genotype identification (Booton et al. 2005; Caumo et al. 2009; Goldschmidt et al. 2012; Howe et al. 1997; Itahashi et al. 2011; Jain and Tilak 2011; Laummaunwai et al. 2012; Le Calvez et al. 2012). Our previous study confirmed the usefulness of PCR for the early, rapid, and sensitive diagnosis of pathogenic Acanthamoeba spp. strains (Derda et al. 2014). The best highly specific marker that distinguishes pathogenic Acanthamoeba strains in water samples in Poland is a pair of primers of Aca 16. The first results confirming the sensitivity and specificity of a PCR assay for the detection of AK were demonstrated by Lehmann et al. (1998), using two different pairs of primers (P1GP, P2GP). An interesting study showed that a combination of primer pairs (ACARNA, JDP, and NELSON) in the PCR-based method for diagnosing AK is more sensitive than culture and microscopy techniques (Yera et al. 2007).

Acanthamoeba as a genus of facultative human parasites is currently classified into 17 genotypes (T1–T17), each of which arguably represents a species (Stothard et al. 1998; Horn et al. 1999; Gast 2001; Hewett et al. 2003; Nuprasert et al. 2010; Corsaro and Venditti 2010).To date, isolates belonging to seven genotypes (T2, T3, T4, T5, T6, T11, and T15) have been found to be associated with AK (Stothard et al. 1998; Booton et al. 2005; Maghsood et al. 2005; Spanakos et al. 2006; Sharifi et al. 2010; Risler et al. 2013). One genotype in particular, T4, was found to be overrepresented in human disease. The main finding of our study is that in Poland, AK is almost always associated with the T4 genotype.

The correlation between Acanthamoeba genotype and clinical presentation of AK or susceptibility of the parasite to treatment has not been well explored. Khan (2003) reported that isolates belonging to AK-related genotypes exhibited significantly higher binding and cytotoxicity to corneal epithelial cells than nonclinical isolates. AK is an uncommon diagnosis in Poland. The increased number of positive cases observed in our laboratory may reflect the growing at-risk population of contact lens wearers. The present study was performed on a low number of isolates and may not reflect the entire spectrum of Acanthamoeba genotypes occurring in patients in Poland. However, our results indicate that the distribution of genotypes does not differ from that reported from other countries, with T4 as the predominant finding in AK cases.