PacBio next-generation sequencing uncovers Apicomplexa diversity in different habitats.

The phylum Apicomplexa comprises a large group of intracellular protozoan parasites. These microorganisms are known to infect a variety of vertebrate and invertebrate hosts, leading to significant medical and veterinary conditions such as toxoplasmosis, cryptosporidiosis, theileriosis, and eimeriosis. Despite their importance, comprehensive data on their diversity and distribution, especially in riverine environments, remain scant. To bridge this knowledge gap, we utilized next-generation high-throughput 18S rRNA amplicon sequencing powered by PacBio technology to explore the diversity and composition of the Apicomplexa taxa. Principal component analysis (PCA) and principal coordinate analysis (PCoA) indicated the habitat heterogeneity for the physicochemical parameters and the Apicomplexa community. These results were supported by PERMANOVA (P < 0.001), ANOSIM (P < 0.001), Cluster analysis, and Venn diagram. Dominant genera of Apicomplexa in inlet samples included Gregarina (38.54%), Cryptosporidium (32.29%), and Leidyana (11.90%). In contrast, outlet samples were dominated by Babesia, Cryptosporidium, and Theileria. While surface water samples revealed 16% and 8.33% relative abundance of Toxoplasma and Cryptosporidium, respectively. To our knowledge, the next-generation high throughput sequencing covered a wide range of parasites in Egypt for the first time, which could be useful for legislation of the standards for drinking water and wastewater reuse.

Fate of Cryptosporidium and Giardia through conventional and compact drinking water treatment plants.

Over the past three decades, a notable rise in the occurrence of enteric protozoan pathogens, especially Giardia and Cryptosporidium spp., in drinking water sources has been observed. This rise could be attributed not only to an actual increase in water contamination but also to improvements in detection methods. These waterborne pathogens have played a pivotal role in disease outbreaks and the overall escalation of disease rates in both developed and developing nations worldwide. Consequently, the control of waterborne diseases has become a vital component of public health policies and a 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. Water samples 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. Using qPCR and IFA, the conventional DWTPs showed complete removal of Giardia and Cryptosporidium, 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. Using IFA, both Mansheyat Alqanater and Niklah CUs showed a similar removal rate of 50% for Giardia cysts. Additionally, Niklah CU achieved a 50% removal of Cryptosporidium oocysts, whereas Mansheyat Alqanater CU did not show any removal of Cryptosporidium oocysts. 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.

Etiology of diarrheal disease among children under 5 years in Egypt: a high incidence of human bocavirus.

BACKGROUND: Human bocavirus (HBoV) is globally distributed and associated with respiratory and enteric infections. Limited data are available about the incidence of HBoV in Egyptian children. We aimed to investigate the association of HBoV genotypes in children with diarrheal disease and also to determine the possibility of HBoV co-infections with other human enteric pathogens. METHODS: A total of 102 stool samples were collected from children under five years old with diarrhea. Samples were analyzed for the presence of HBoV by real-time PCR. HBoV positive samples were tested for adenovirus (AdV), rotavirus (RoV), parasitic helminths, and enteric protozoa. RESULTS: HBoV was detected in 58% of examined cases. HBoV-3 was the most prevalent genotype observed (44%; 45 of 102), followed by HBoV-2/4 (33%; 34 of 102) and HBoV-1 (30%; 31 of 102). Although the incidence of HBoV was higher in males (66.6%; 34 of 51) than females (49%; 25 of 51), the analysis showed no significant difference for HBoV between genders. The average HBoV concentrations were 5.3 × 104 GC/g in males and 1.03 × 105 GC/g in females. Among the HBoV-positive samples, the single infection of HBoV was 52.5% (31/59), while the co-infections with multiple viruses were found in 1.7% (1/59) for HBoV and AdV, 33.9% (20/59) for HBoV and RoV, and 11.9% (7/59) for HBoV, and RoV and AdV. No co-infection with parasitic helminths or enteric protozoa was found. CONCLUSIONS: The single infection of HBoV in some children suffering from acute gastroenteritis indicated that HBoV could be the main etiologic agent of the disease. The study highlights the high incidence of HBoVs genotypes with remarkable multiple co-infections in the pre-school children in Egypt.

Assessment of Two Different Drinking Water Treatment Plants for the Removal of Free-living Amoebae, Egypt.

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.

Surveillance and Molecular Identification of Acanthamoeba and Naegleria Species in Two Swimming Pools in Alexandria University, Egypt.

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.

Microsporidial Spores in Fecal Samples of Some Domesticated Animals Living in Giza, Egypt.

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.

Isolation and molecular identification of Naegleria fowleri from Nile river, Egypt.

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.