Determining the Genotype of Acanthamoeba Strains Isolated From the Oraland Nasal Cavities of Individuals With Immunodeficiency

Acanthamoeba is a free-living amoeba that can be found in various environments. It is found in ear, nose and pharyngeal mucosa of patients with respiratory problems .This is the most common protozoan found in the environment. The antibodies against Acanthamoeba antigens have been found in the serum of more than 80% of people with complete immunity, which indicates high human contact with this amoeba. Acanthamoeba is the cause of human diseases such as amoebic keratitis and granulomatous amebic encephalitis, which is mostly seen in immunocompromised people.Skin wounds and nasopharyngeal infections caused by this amoeba have also been observed, mostly in people with AIDS. Based on rRNA gene sequence determination, Acanthamoeba genus is divided into 12 different genotypes (T1 to T12). Most human Acanthamoeba infections are related to the T4 genotype.Studies have shown that the number of amoebic keratitis is increasing worldwide due to use of contact lenses. Acanthamoeba genotyping is a useful tool for taxonomic and epidemiological studies. It explains the relationships between infectious isolates and the phenotype of the disease. Since different strains show different pathogenic power, determining the strains with high pathogenic power can have more therapeutic importance. More studies to identify pathogenic features and genetic markers are needed to clarify this matter. The present study aims to determine the genotype of Acanthamoeba strains isolated from the oral cavity of people with immunodeficiency.

This cross-sectional descriptive study was conducted for 18 months.
The samples were collected with a sterile swab from pharyngeal or nasal secretions of 179 eligible patients in Golestan and Shafa hospitals in Ahvaz, Iran in 2019. The patients who had immunodeficiency with various underlying diseases including diabetes, AIDS, those who were under treatment with chemotherapy drugs and steroids, as well as dialysis patients were included in the study. In the university laboratory, the swab soaked in secretions was cultured on basic agar medium. Before closing the environment, it was autoclaved for 15 minutes at 121°C and divided into plates under the hood. The plates were fixed with parafilm and kept in refrigerator at 4°C. To detect Acanthamoeba, all samples were cultured separately in a non-nutrient medium (1.5% Bacto agar) along with an old medium of Escherichia coli. Then, bacteria were added to the medium to provide a good source of food for amoeba. The media were incubated at a room temperature and microscopic observation was done on days 2-14 to identify the samples positive for Acanthamoeba. All positive amoebae isolates can be cloned by subculturing method to produce pure culture for extracting pure DNA. Therefore, the existing parasites were transferred to new plates. To determine the genotype of the target sample, after cultivation using the primers presented in Table 1, polymerase chain reaction (PCR) was performed under the conditions shown in Table 2.The primers were diluted. For this purpose, 180 µL of distilled water and 20 µL of primer were added to two micro-tubes named as diluted G₁ and diluted G₂.To determine the genotype of Acanthamoeba isolated from patients, the PCR product was finally determined and the information related to sequencing of the fragment for each sample was compared with the information available in the gene bank, and the genotype of the amoeba was finally determined. Statistical analysis was done using SPSS software, version 22.

In this study, 110 men and 69 women participated. Most of them (n=48, 26.8%) were at the age group of 60-69 years, and the mean age of participants was 48±1 years. As shown in Table 4, 99 samples were taken from the throat and 80 samples were taken from the nose of patients.Among the samples examined by PCR, 6(3.4%) were positive to Acanthamoeba, collected from the oral (n=4) and nasal (n=2) cavities of patients. Acanthamoeba was identified in the medium by observing star-shaped cysts. The PCR analysis was successfully performed on these samples and the nucleotide sequence was determined for them. All positive samples of T4 genotype were obtained. The PCR analysis was confirmed by observing the 500-bp band on agarose gel (Figure 1).

The prevalence of infection with Acanthamoeba parasite in patients with immunodeficiency is higher. The Acanthamoeba positive samples identified in these people belong to the T4 genotype.