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 Table of Contents  
ORIGINAL ARTICLE
Year : 2016  |  Volume : 1  |  Issue : 2  |  Page : 1-5

Isolation of acanthamoeba species from fresh water sources in southern regions of Saudi Arabia


1 Department of Microbiology and Clinical Parasitology, College of Medicine, King Khalid University, Abha, Saudi Arabia
2 Medical student, College of Medicine, King Khalid University, Abha, Saudi Arabia

Date of Web Publication8-Aug-2020

Correspondence Address:
MBBS, MSc, PhD Ahmed Mossa Al-Hakami
Department of Microbiology and Clinical Parasitology College of Medicine, King Khalid University P.O.Box 641, Abha
Saudi Arabia
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Source of Support: None, Conflict of Interest: None


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  Abstract 


Objective: The present study aimed to investigate the occurrence of Acanthamoeba species and other FLAs in freshwater sources in southern regions of the Kingdom of Saudi Arabia and to estimate the prevalence of their existence in these areas.
Methods: Water samples from both fresh water course and hot springs (n = 15) were collected from five different zones in Aseer region, southern western parts of the kingdom. Isolation and identification of Acanthamoeba species and other FLAs were carried out from these water sources (fresh water course and hot spring). The methods included filtration, in vitro culture and incubation for up to three weeks for the selected samples according to standard methods.
Results: Examination of water samples by direct microscopic methods showed no amoebic parasites among all of the 15 samples collected from five different sites. However, when the water samples were cultured on non-nutrient agar (NNA), four (26.7%) of the 15 samples showed growth of Acanthamoeba species. Principle component analysis (PCA) indicated that the water bodies/ sources that were positive for Acanthamoeba spp. shared a common natural characteristic that the water bodies were exposed to contamination.
Conclusion: The study demonstrated that Acanthamoeba spp. were prevalent in some sites (3 out 5 sites were positives; 60%), low in two sites (1 out 3 samples) while absent in two sites (0 out of six samples). More comprehensive research is needed to find out the true prevalence of these parasites and factors affecting their existence.

Keywords: Free Living Amoeba (FLAs), Acanthamoeba spp., non-nutrient agar (NNA)


How to cite this article:
Al-Hakami AM, Al-Shehri SH, Al-Shahrani SM, Al-Qarni MA, Kadasah AS, Alghamdi SA, Hamid ME. Isolation of acanthamoeba species from fresh water sources in southern regions of Saudi Arabia. King Khalid Univ J Health Scii 2016;1:1-5

How to cite this URL:
Al-Hakami AM, Al-Shehri SH, Al-Shahrani SM, Al-Qarni MA, Kadasah AS, Alghamdi SA, Hamid ME. Isolation of acanthamoeba species from fresh water sources in southern regions of Saudi Arabia. King Khalid Univ J Health Scii [serial online] 2016 [cited 2020 Oct 21];1:1-5. Available from: https://www.kkujhs.org/text.asp?2016/1/2/1/291739




  Introduction Top


Acanthamoebae are free-living aerobic amoebae and has a world-wide distribution. Acanthamoeba species have cysts and trophozoite stages in its life-cycle without the flagellated stage. The trophozoites replicate by mitosis. The trophozoite is 20-50μm in size with rough exterior with several spines like projections (acanthopoda) while the cyst is spherical (15μm in diameter). Equally these two forms can be the source of infection.[1],[2],[3],[4] A. polyphaga, A. rhysodes, A. astronyxis, and A. divionensis.

Acanthamoeba species along with its closely related free-living amoebae Balamuthia mandrillaris are causal agents of fatal chronic disease called granulomatous amoebic encephalitis (GAE). This is particularly serious among the immunocompromised individuals. Furthermore, Acanthamoeba causes Acanthamoeba keratitis which is a vision-threatening disease. Both Acanthamoeba species and Balamuthia mandrillaris cause infections of the lungs and skin.[7],[8]

Acanthamoeba spp. are abundant in nature and have worldwide distribution. They have been isolated from soil, fresh and brackish waters, bottled mineral water and many other sources of human and animal use. Acanthamoeba species have are tolerant of a wide range of osmolarity, enabling them to survive in distilled water, tissue culture media, mammalian body fluids, and sea water.[5],[9]


  Methods Top



  Study Location, Sampling, isolation, and Identification of Amoebae Top


A total of 5 liters of water sample were collected in a sterile plastic container from five different sites at Aseer region [Figure 1]. Samples were transferred to the laboratory immediately. FLA was isolated from the samples as per recommended procedures.[6]
Figure 1: Map of Aseer region, KSA and sites of soil collection sites. Map was adapted from Google Earth (US State Department of Geography, 2016; http://earth.google.com/)

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  Examination of water for parasite Top


Water sample was mixed thoroughly and 12 mL was transferred to a clean tube and centrifuged at 1500 rpm for 10 min. The supernatant was discarded and a drop of sediment was placed on the centre of glass slide. A cover glass was placed and the slide was examined under 100X and with 400X magnifications to look for the presence of amoeba parasites. For iodine preparation, a drop of sediment was placed on centre of glass slide and a drop of iodine was added. Then a cover glass was placed and the slide before examination.

Smears were also prepared from the sediment and Giemsa staining was performed on methanol fixed, dried slides. 10% Giemsa stain was added and slide was left for 25 minutes. Slide was then washed with distilled water, dried and examined under 1000X magnification

Cultivation of Acanthamoeba spp

Water samples (approximately 5 liters were filtered through 0.45μm pore size cellulose nitrate membrane filter (47mm in diameter) under a vacuum. The membrane filter for each water sample was scraped and the collected materials were placed in 15mL of sterile cover tubes containing 10mL phosphate buffered saline (PBS). Tubes were incubated at room temperature overnight and then centrifuged for ten minutes at 1500rpm to collect particles on filters. After centrifugation, the supernatant solution was discarded and the pellet was inoculated onto 1.5% non-nutrient agar (NNA) plates. A dense suspension of heat inactivated Escherichia coli, prepared in saline was seeded onto NNA plates to grow amoebae. After the inoculation of the samples, all plates were incubated at 30°C and examined daily for the presence of FLAs for up to 15 days using a light microscope (100x).

Morphological characterization of Acanthamoeba isolates

Amoeba cysts that were isolated from culture were stained with haematoxylin/eosin[10] and were directly examined under the microscope and identified. Acanthamoeba cysts (Acanthamoeba sp. I to III) were established as per as known scheme.[11] Principle component analysis (PCA) was done to see whether water sources cluster with the presence of Acanthamoeba spp. PCA was done using PAST software (PAST Version 3.14; Øyvind Hammer, Natural History Museum, University of Oslo, 1999-2016).


  Results Top


In this study out of five sites in Aseer, three sites demonstrated the presence of Acanthamoeba spp., whereas 2 sites were negative with all of the three testes used. The overall prevalence of Acanthamoeba spp. in the five sites was estimated to be 26.7% (4/15*100) [Table 1].
Table 1: Detection of Acanthamoeba spp. in five sites in Aseer region, Saudi Arabia using microscopic and culture methods

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Examination of water samples by direct microscopic methods showed no amoebic parasites among all of the 15 samples collected from five different sites (Table 1). When the samples cultured on non-nutrient agar (NNA), four (26.7%) of the 15 samples showed growth of Acanthamoeba species and were demonstrated microscopically [Figure 2].
Figure 2: Different Acanthamoeba spp. shown on NNA incubated at room temperature for 2 weeks

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PCA indicated that water sources that were positive for Acanthamoeba spp. shared a common natural characteristic. This included water sources exposed to contamination [Figure 3].
Figure 3: Plot of water bodies/ sources against results of Acanthamoeba spp. Principle component analysis (PCA) was done using PAST software (version 3.14). Note the clustering of sites with positive Acanthamoeba spp. (A1, E1).

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  Discussion Top


In the present study 26.7% prevalence of Acanthamoeba spp. was demonstrated microscopically from all 15 samples representing 5 geographical sites in Aseer region. NNA culture method has proven more sensitive than direct examination of the water deposits [Table 1]. FLAs are distributed worldwide and the composition of these species at certain locations depends on the surroundings. FLAs are getting increasing attention as reservoirs and potential vectors for the transmission of pathogenic bacteria.[12],[13] Studies have shown that the food-borne and opportunistic pathogens are able to interact with species of Acanthamoeba and more so in A. polyphaga. These pathogens multiply intracellularly within the FLAs and in theory able to spread in the environment and to contribute to food contamination.[14]

In the environment, there exist many genera of pathogenic protozoa of both human and animal origin but only limited information is available regarding these organisms in developing countries.[15],[16] The spread of FLAs species depends on its tolerance to survive under adverse conditions. Therefore, the ecological importance of FLAs must be adequately studied to prevent occurrence of fatal human diseases.[17],[18] The presence of FLAs in tap water may represent a health risk to both immunocompromised and immunocompetent individuals.[19] One of the earlier studies from this part of the kingdom has emphasized upon the hazards of water sources to human regarding the prevalence of Acanthamoeba spp. In their study, authors could isolate 33 strains of Acanthamoeba species from water sources at Wadi Hanifah, Saudi Arabia using non-nutrient agar with 1%NaCl added. The isolates were identified as A. astronyxis, A. comandoni, A. culbertsoni and A. quina. The optimum and maximum tolerated temperatures for the growth as well as the pathogenicity of each strain for mice were also recorded.[20] Acanthamoeba species are now becoming opportunists particularly in patients infected with (Human Immunodeficiency Virus (HIV) and a lack of effective therapeutic treatment represents a major problem and future emphasis must be placed on clinical trials dealing with the management of Acanthamoeba infections in patients with AIDS.[21]

The study concluded that Acanthamoeba spp. were common in some sites (66.7%) while absent or not detected in others or recorded low occurrence (33.3%). Further investigation covering larger area and sites is required to uncover the true prevalence and factors affecting the presence Acanthamoeba spp. in this part of the country.

Acknowledgments

The authors thank Mr Salah Abdullah and Mr Ihab AbdelRahim for their kind help in laboratory work and technical assistance.

Conflict of interest

The authors declare that they have no conflicts of interest.



 
  References Top

1.
Band RN Mohrlok S. The cell cycle and induced amitosis in Acanthamoeba. J Protozool; 1973; 20: 654-657.  Back to cited text no. 1
    
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Alves Dde S, Moraes AS, Alves LM, Gurgel- Goncalves R, Lino Junior Rde S, Cuba-Cuba CA, et al. Experimental infection of T4 Acanthamoeba genotype determines the pathogenic potential. Parasitol Res; 2016; 115: 3435-3440.  Back to cited text no. 2
    
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Cabello-Vilchez AM, Reyes-Batlle M, Montalban- Sandoval E, Martin-Navarro CM, Lopez-Arencibia A, Elias-Letts R, et al. The isolation of Balamuthia mandrillaris from environmental sources from Peru. Parasitol Res; 2014; 113: 2509-2513.  Back to cited text no. 3
    
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Visvesvara GS, Moura H Schuster FL. Pathogenic and opportunistic free-living amoebae: Acanthamoeba spp., Balamuthia mandrillaris, Naegleria fowleri, and Sappinia diploidea. FEMS Immunol Med Microbiol; 2007; 50: 1-26.  Back to cited text no. 5
    
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Grossniklaus HE, Waring GOt, Akor C, Castellano- Sanchez AA Bennett K. Evaluation of hematoxylin and eosin and special stains for the detection of acanthamoeba keratitis in penetrating keratoplasties. Am J Ophthalmol; 2003; 136: 520-526.  Back to cited text no. 10
    
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Kong HH Chung DI. A riboprinting scheme for identification of unknown Acanthamoeba isolates at species level. Korean J Parasitol; 2002; 40: 25-31.  Back to cited text no. 11
    
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Anacarso I, de Niederhausern S, Messi P, Guerrieri E, Iseppi R, Sabia C, et al. Acanthamoeba polyphaga, a potential environmental vector for the transmission of food-borne and opportunistic pathogens. J Basic Microbiol; 2012; 52: 261-268.  Back to cited text no. 14
    
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Bonilla-Lemus P, Caballero Villegas AS, Carmona Jimenez J Lugo Vazquez A. Occurrence of free-living amoebae in streams of the Mexico Basin. Exp Parasitol; 2014; 145 Suppl: S28-33.  Back to cited text no. 15
    
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Shanan S, Abd H, Bayoumi M, Saeed A Sandstrom G. Prevalence of protozoa species in drinking and environmental water sources in Sudan. Biomed Res Int; 2015; 2015: 345619.  Back to cited text no. 16
    
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Adekambi T, Ben Salah S, Khlif M, Raoult D Drancourt M. Survival of environmental mycobacteria in Acanthamoeba polyphaga. Appl Environ Microbiol; 2006; 72: 5974-5981.  Back to cited text no. 17
    
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Thomas V, McDonnell G, Denyer SP Maillard JY. Free-living amoebae and their intracellular pathogenic microorganisms: risks for water quality. FEMS Microbiol Rev; 2010; 34: 231-259.  Back to cited text no. 19
    
20.
Al-Herrawy AZ Al-Rasheid KA. Identification of Acanthamoeba strains isolated from a freshwater course in Saudi Arabia. J Egypt Public Health Assoc; 1998; 73: 621-633.  Back to cited text no. 20
    
21.
Bottone EJ. Free-living amebas of the genera Acanthamoeba and Naegleria: an overview and basic microbiologic correlates. Mt Sinai J Med; 1993; 60.  Back to cited text no. 21
    


    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
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