Our study is the first investigation that carried out in Bandar Abbas with culture and molecular methods. The results of our study showed that frequency of free-living amoebae in different water resources of Bandar Abbas city is considerable (37.3%). The study revealed that in culture method, 6% of 83 samples were positive for Acanthamoeba, while PCR detected 9.6%, which indicates the higher sensitivity of the molecular method in the diagnosis of Acanthamoeba. Studies indicate that PCR can be more sensitive and effective method in diagnosis of Acanthamoeba and can eliminate the need for skilled microscopist, but no single method is suggested [15-17].
In our study, some negative cases in culture method showed positive band in PCR test. Microscopic diagnosis of Acanthamoeba mostly rely on detecting polygonal or star shape cyst of amoeba. It may exist few cysts in some cultures that we couldn’t realize or have been misdiagnosed as artifacts.
Based on the phylogenic tree of the 18 SrRNA gene, Acanthamoeba T4 was the primary genotype detected in water samples from all the studied area, and phylogenic analysis showed valuable sensitivity and specificity for differentiation between each genotype. In previous studies, the T4 genotype was reported to be isolated from samples such as surface waters, drinking water, natural thermal water, swimming pools, hospital water, recreational water areas, and also soil and dust sources [18-20]. Also, our findings showed that positive cases of pools were mixes of two genotypes (T4, T15), but other positive cases were T4. Therefore, T4 considered as worldwide and isolated from clinical and environmental samples [21, 22].
Wegdan M. Abd El Wahab in Egypt collected 80 samples of water supplies and proved that the dominant genotype of Acanthamoeba is T4; this is in accordance with our results[23].
Magnet et al. reported that 94.6% of samples were positive for Acanthamoeba sp. by PCR, and most of them belonged to the T4 genotype, which was in line with our study [24].
Another study conducted in Italy in samples from different water matrices, Acanthamoeba T4 was the most common genotype detected in 13/18 isolates (72.2%), while T15 genotype was observed only in samples from Apulia and Basilicata [18].
Golestani in Kashan, Pezeshki in Zanjan, Fraji in Lorestan showed that frequency of Acanthamoebais between 30-80% in water samples, pools tap water, and hospital pipes [23, 25, 26]. These studies are in agreement with our results, except in amount of chlorine in Lorestan study that has a significant relation with organism growth and multiplication, but in our chlorine did not affect Acanthamoeba viability.
In most studies, sensitivity of molecular methods in the detection of free-living amoebae were higher than culture method. We used culture and molecular methods to diagnose Acanthamoeba simultaneously, and high sensitivity of molecular method was confirmed.
There was a statistically significant difference between cultivation and molecular methods in identifying positive cases of Acanthamoeba (P > 0.001).
Polymerase chain reaction (PCR) method has been used since 1996 in identifying Acanthamoeba, and a recent study on its accuracy by Boggild et al. showed that it compared favorably with smear microscopy in terms of sensitivity. Still, DNA extraction should be done in very proper ad precise manner, although specificity is slightly poorer [27].
The physicochemical properties of water samples recorded in this study included pH, residual or remnant chlorine, turbidity, or external particles (if it is present should remove by gauze) and temperature that considered for each sample before filtration and cultivation.
There was no significant relationship between chlorine amount and frequency of Acanthamoebaby culture and molecular method (P >0.05). Michael Storey showed that Acanthamoeba cysts could remain in 100 mg/l chlorine (free and combined) for 10 min[28]. In our study, Acanthamoeba isolated from places with 2 mg/l free chlorine.
The most positive detected cases of Acanthamoeba have been related to swimming pools at the temperature range of 26-30 °C. Nielsen and Naveed Ahmed Khan showed that the highest growth rate for six amoebic strains tested was close to 30-32 °C [29, 30]. Result didn't show any positive samples from geothermal hot spring water of Bandar Abbas. Some studies showed that based on the morphological characteristics of amoebae, 42% of warm spring waters of southwest of Iran are positive for Acanthamoeba, and others revealed that genotype T2, T4, T15could exist in such environments [31, 32].
No significant statistical differences were observed between pH variable and frequency of Acanthamoeba using culture and molecular methods (p-value = 0.014) and p-value (p = 0.116) respectively. In the range of pH 7-8.3, more positive cases of Acanthamoeba were identified.