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During the past thirty years, there has been a significant increase in the contamination of drinking water by enteric pathogenic protozoa, particularly Giardia and Cryptosporidium spp. Such microbial contamination has been responsible for disease outbreaks and increased background rates of disease in developed and developing countries worldwide. As such, controlling waterborne diseases is a critical aspect of public health policy and the primary objective of drinking water treatment plants (DWTPs). Limited studies applied real-time PCR (qPCR) and/or Immunofluorescence assay (IFA) for monitoring Giardia and Cryptosporidium spp., particularly in developing countries like Egypt. Samples of water from two conventional drinking water treatment plants and two compact units (CUs) were analyzed using both IFA and qPCR methods to detect Giardia and Cryptosporidium. The conventional DWTPs showed complete removal of Giardia and Cryptosporidium gene copies, whereas Mansheyat Alqanater and Niklah CUs achieved only partial removal. Specifically, Cryptosporidium gene copies removal rates were 33.33% and 60% for Mansheyat Alqanater and Niklah CUs, respectively. Niklah CU also removed 50% of Giardia gene copies, but no Giardia gene copies were removed by Mansheyat Alqanater CU. Conventional DWTPs were more effective than CUs in removing enteric protozoa. The contamination of drinking water by enteric pathogenic protozoa remains a significant issue globally, leading to increased disease rates. Infectious disease surveillance in drinking water is an important epidemiological tool to monitor the health of a population.

The first report about intestinal microsporidia from wild mammals in Egypt is herein present. A total of 134 fecal samples were separately collected from seven different mammalian species located in fifteen different localities,represented Western Desert, Nile Valley and Delta and Eastern Desert of Egypt. Microsporidian spores were obtained from the collected samples usingflotation technique and finally stained with modified trichrome (MT). Intestinal microsporidia were detected in 72.39% (97/134) of wild mammals. Moreover, rate of infection with intestinal microsporidia was 72.84% (59/81) in males and 73.08% (38/52) in females.The infection rates of intestinal microsporidia were 91.1%, 73.9%, 73.6%, 70%, 55.6% and 48.1% in red foxVulpes vulpes, the lesser Egyptian gerbilGerbillus gerbillus,the house mouseMus musculus, Sundevall's jirdMeriones crassus, Mackilligin's gerbilDipodillus mackilliginiand the Cairo spiny mouseAcomys cahirinus, respectively. The results also showed that the highest percentage of microsporidia was recorded in two mammalian species closely related to human habitats: red fox V. vulpes and G. gerbillus. In conclusion, the wild mammalian species which live in association with humans and fed on their contaminated diets were more susceptible to be infected with several microsporidian species than the other species that live in natural habitats away from humans.

EnvironmentAsia, 12, 1, 178-186

Responding to the water shortage in Egypt, the use of submerged membrane bioreactor (SMBR) for wastewater reclamation has been studied. The impact of hydraulic retention time (HRT), as the most influential operational parameter has been investigated. A bench-scale module was operated at three HRT: 6, 10 &15hs. The results demonstrated that total COD and BOD removal rates were not affected by changing the HRT or by variations in raw wastewater strength. Average residual COD values in the membrane permeate ranged from 14 to 20.3 mg O2 L-1. Corresponding residual BOD was below 3.0 mgL-1. Nitrification capacity of the SMBR was high. HRT was found to be a key parameter for fouling. Decreasing the HRT enhances membrane fouling. At HRT of 6h, fouling happened after 6 days. Corresponding values for HRTs 10 and 15 were 13 day and more than 42 days, respectively. Total coliform bacteria counts in most of the MBR permeate samples were below the detection limit of 10 CFU/100 mL. All protozoan parasitic stages detected in raw wastewater were removed by the SMBR. At HRT of 15h, both human rotaviruses and adenoviruses genome copies were reduced by 2 to 3 log10. This ratio was reduced by decreasing the HRT.

Background Members of the genus Naegleria are free-living amoebae distributed in various aquatic environments. Naegleria fowleri is the only species that can cause fatal primary amoebic meningoencephalitis in humans. Materials and methods A total of 48 Nile water samples were collected from the water stream passing though Cairo. The samples were processed for the detection of Naegleria spp. using non-nutrient agar at 45°C. The isolates of Naegleria spp. were identified based on the morphologic criteria of trophozoite, flagellated and cyst stages. Molecular characterization of the isolates was performed using PCR. Results The obtained results showed that Naegleria spp. were found in 45.8% of Nile water samples by means of microscopic examination. Seasonally, the highest prevalence of Naegleria spp. was recorded in summer (66.7%). Moreover, the highest prevalence of N. fowleri was recorded in summer (25%). Conclusion The occurrence of heat-tolerant Naegleria spp., especially N. fowleri, in Nile water should be considered as a potential health threat.

Microsporidia comprises various pathogenic species can infect humans by means of water. Moreover, chlorine disinfection of drinking-water has limitations against this protozoan pathogen. A total of 48 water samples were collected from two drinking water treatment plants having two different filtration systems (slow sand filter and rapid sand filter) during one year period. Samples were collected from inlet and outlet of each plant. Samples were separately filtrated through nitrocellulose membrane (142 mm, 0.45 µm), then eluted and centrifuged. The obtained pellet from each sample was subjected to DNA extraction, then, amplification using genus-specific primer for microsporidia. Each microsporidia-PCR positive sample was performed by two species specific primers for Enterocytozoon bieneusi and Encephalitozoon intestinalis. The results of the present study showed that the percentage of removal for microsporidia through different treatment processes reached its highest rate in the station using slow sand filters (100%), while the removal by rapid sand filter system was 81.8%. Statistically, the two different drinking water treatment plants (slow and rapid) had significant effect for removal of microsporidia. Molecular identification of microsporidia-PCR positive samples using two different primers for Enterocytozoon bieneusi and Encephalitozoon intestinalis showed the presence of the two pervious species in the inlet water of the two stations, while Encephalitozoon intestinalis was detected in the outlet water only. In conclusion, the appearance of virulent microsporidia in treated drinking water may cause potential health threat., {"references":["L. Susskind, and R. Jain, Drinking Water Treatment. Springer Science 2011.pp 3-25.","E. S. 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Background: The aim of this study was to compare between slow and rapid sand filters for the removal of free-living amoebae during drinking water treatment production. Methods: Overall, 48 water samples were collected from two drinking water treatment plants having two different filtration systems (slow and rapid sand filters) and from inlet and outlet of each plant. Water samples were collected from Fayoum Drinking Water and Wastewater Holding Company, Egypt, during the year 2015. They were processed for detection of FLAs using non-nutrient agar (NNA). The isolates of FLAs were microscopically identified to the genus level based on the morphologic criteria and molecularly confirmed by the aid of PCR using genus-specific primers. Results: The percentage of removal for FLAs through different treatment processes reached its highest rate in the station using slow sand filters (83%), while the removal by rapid sand filter system was 71.4%. Statistically, there was no significant difference (P=0.55) for the removal of FLAs between the two different drinking water treatment systems. Statistically, seasons had no significant effect on the prevalence of FLAs in the two different drinking water treatment plants. Morphological identification of the isolated FLAs showed the presence of 3 genera namely Acanthamoeba, Naegleria, and Vermamoeba (Hartmannella) confirmed by PCR. Conclusion: The appearance of FLAs especially pathogenic amoebae in completely treated drinking water may cause potential health threat although there is no statistical difference between the two examined drinking water filtration systems.

Background: Swimming in contaminated water was reported to be associated with Acanthamoeba and N. fowleri human infections. The present study was carried out with the aim of isolation and identification of the different species of Acanthamoeba and Naegleria from two swimming pools in Alexandria University. Methods: Samples were collected from the swimming pools of Alexandria University Stadium and Faculty of Agriculture-Alexandria University during the period from May 2012 to April 2013. Results: Free-living amoebae were prevalent in the collected samples. Molecular characterization confirmed the identity of ten Acanthamoeba isolates and seven Naegleria isolates. Acanthamoeba T3, T4, T5, T11 and T15 genotypes were identified. Acanthamoeba T4 was the most prevalent genotype. Conclusion: The relatively high prevalence of Acanthamoeba, especially genotype T4, indicates the presence of a health hazard to swimmers particularly those wearing contact lenses. Naegleria fowleri was not found during the present study.

Genus Acanthamoeba causes 3 clinical syndromes amebic keratitis, granulomatous amebic encephalitis and disseminated granulomatous amebic disease (eg, sinus, skin and pulmonary infections). A total of 144 tap water samples were collected from Giza governorate, Egypt. Samples were processed for detection of Acanthamoeba species using non-nutrient agar (NNA) and were incubated at 30oC. The isolates of Acanthamoeba were identified to species level based on the morphologic criteria. Molecular characterization of the Acanthamoeba isolates to genus level was performed by using PCR. The obtained results showed that the highest occurrence percentage of Acanthamoeba species in water samples was observed in summer season (38.9%), then it decreased to be 30.6% in spring and 25% in each of autumn and winter. PCR analysis showed that 100% of 43 Acanthamoeba morphologically positive samples were positive by genus specific primer. In the present study eight species of Acanthamoeba can be morphologically recognized namely Acanthamoeba triangularis, Acanthamoeba echinulata, Acanthamoeba astronyxis, Acanthamoeba comandoni, Acanthamoeba griffini, Acanthamoeba culbertsoni, Acanthamoeba quina and Acanthamoeba lenticulata. In conclusion, the most common Acanthamoeba species in tap water was Acanthamoeba comandoni

Background: The aim of the present work was to investigate the prevalence and species of intestinal microsporidiosis among animals in Giza, Egypt. Methods: A total of 869 animal fecal samples were collected from domesticated animals (dogs, cats, rabbits, cattle, buffaloes, sheep, goats, donkeys and pigs) living in Giza, Egypt. Spores of microsporidia were concentrated from collected samples by centrifugation and finally stained with modified trichrome (MT) stain to detect microsporidial spores. Microsporidial spores in microscopically-positive samples were molecularly confirmed and identified using species-specific primers. Results: Spores of microsporidia were microscopically detected in 17.0% of the examined animal fecal samples. The highest and lowest rates of infection with intestinal microsporidia were recorded in dogs (33.3%) and buffaloes (6.9%), respectively. Molecularly, the obtained microsporidial spores were classified as Enterocytozoon bieneusi and E. intestinalis. Dual infection with both identified species was observed in fecal samples from buffalo, rabbit, goat, cat, pig and dog. Conclusion: Domestic animals may play a role in dissemination of intestinal microsporidiosis in the environment. Examined animals were infected with E. bieneusi in a higher percentage than E. intestinalis.

Background: The aim of the present work was to investigate the prevalence and species of intestinal microsporidiosis among animals in Giza, Egypt. Methods: A total of 869 animal fecal samples were collected from domesticated animals (dogs, cats, rabbits, cattle, buffaloes, sheep, goats, donkeys and pigs) living in Giza, Egypt. Spores of microsporidia were concentrated from collected samples by centrifugation and finally stained with modified trichrome (MT) stain to detect microsporidial spores. Microsporidial spores in microscopically-positive samples were molecularly confirmed and identified using species-specific primers. Results: Spores of microsporidia were microscopically detected in 17.0% of the examined animal fecal samples. The highest and lowest rates of infection with intestinal microsporidia were recorded in dogs (33.3%) and buffaloes (6.9%), respectively. Molecularly, the obtained microsporidial spores were classified as Enterocytozoon bieneusi and E. intestinalis. Dual infection with both identified species was observed in fecal samples from buffalo, rabbit, goat, cat, pig and dog. Conclusion: Domestic animals may play a role in dissemination of intestinal microsporidiosis in the environment. Examined animals were infected with E. bieneusi in a higher percentage than E. intestinalis.

Ahmad Z Al-Herrawy,1 Mohamed G Heshmat,2 Shaban H Abu Kabsha,3 Mahmoud A Gad,1 Wael M Lotfy2 1Water Pollution Research Department, National Research Centre, Cairo, Egypt; 2Parasitology Department, Medical Research Institute, Alexandria University, Alexandria, Egypt; 3Behera Water and Drainage Company, Damanhour, Egypt Abstract: Members of the genus Acanthamoeba are common free-living amoebae that may be pathogenic to humans. The aim of the present work was to evaluate the presence of Acanthamoeba species in the Damanhour Drinking Water Treatment Plant (DWTP) before and after conventional drinking water treatment. Water samples were filtered through nitrocellulose membranes of 0.45 µm pore size, cultured on nonnutrient agar plates seeded with Escherichia coli, and incubated at 37°C. The obtained amoebae, which proved to be morphologically consistent with Acanthamoeba spp., were confirmed by polymerase chain reaction (PCR). The confirmed isolates of Acanthamoeba were morphologically identified to the species level, based on assessment of the size and shape of the endo- and ectocysts and the mean number of pores per cyst. The obtained results showed that members of the genus Acanthamoeba were isolated from 33.3% and 16.7% of inlet and outlet samples, respectively. Six different species of Acanthamoeba were morphologically identified. Species morphologically identified as Acanthamoeba castellanii, Acanthamoeba culbertsoni, and Acanthamoeba polyphaga were isolated from the “finished” water of DWTP. Such species may be pathogenic to humans. In conclusion, the conventional drinking water treatment steps did not remove all the detected species of Acanthamoeba in the inlet water, and about half of them could escape to completely treated and finished water. These species may cause human infections. Keywords: free-living, amoebae, identification, morphology, PCR

Constructed wetlands have been recognized as offering a removal treatment option for high concentrations of chemical and biological contaminants in domestic wastewater. The enteric protozoan parasite Cryptosporidium is considered to be one of the most highly resistant and most infectious organisms for humans, animals and birds. Moreover, some species of Cryptosporidium are known to have a zoonotic nature. In this investigation a pilot scale for a domestic wastewater treatment system is used, consisting of the following steps: (1) up‐flow anaerobic sludge blanket (UASB) reactor; (2) free water surface (FWS) unit; and (3) sub‐surface flow (SSF) unit. This treatment system was fed with domestic wastewater to assess its efficiency in removing Cryptosporidium oocysts. The obtained Cryptosporidium oocysts were detected by two different staining techniques (acid fast trichrome ‘AFT’ and modified Ziehl Neelsen ‘MZN’ stains). Results revealed that the AFT stain was more sensitive and reliable for the detection of ...

Background: The free-living amoebae Acanthamoeba spp., have been recognized as etiologic agents of amoebic encephalitis, keratitis, otitis, lung lesions and other skin infections mainly in immuno-compromised individuals. In this study, morpho-physiological and biochemical characterization of Acanthamoeba strains isolated from the Egyptian aquatic environment were surveyed. Methods: some Acanthamoeba species were cultivated on non-nutrient agar. Isolated strains of Acanthamoeba were identification based on the morphology of trophic and cyst forms in addition to temperature and osmo-tolerance assays. Biochemical characterization of the isolated amoeba strains was performed using quantitative assay as well as qualitative determination of proteolytic activity in zymograph analysis. Results: Potentially pathogenic Acanthamoeba species were isolated from all of the examined water sources. Colorimetric assays showed protease activity in the heat-tolerant isolates of Acanthamoeba. All pathogenic isolates of Acanthamoeba exhibited higher protease activity than did the non-patho-genic ones. The zymographic protease assays showed various banding patterns for different strains of Acanthamoeba. Conclusion: The incidence and prevalence of the pathogenic Acanthamoeba species in the aquatic environment using parasitological and biochemical diagnostic tools will provide baseline data against which the risk factors associated with waterborne transmission can be identified.