Enteric Protozoan Research Articles

Rho5 regulates lysophosphatidic acid induced macropinocytosis in Entamoeba histolytica

Entamoeba histolytica is a unicellular enteric protozoan responsible for variety of gastrointestinal pathologies. The pathogenicity of the parasite is attributed to its dynamic cytoskeleton. Rho family GTPases, a major class of molecular switches, play an indispensable role in controlling cytoskeleton remodelling. Rho GTPases are known to be activated by specific Guanine nucleotide Exchange Factors (GEF) upon stimulation by various extracellular stimuli to regulate signalling.We have established that EhRho5 is important for LPA induced micropinocytosis in the amoebic trophozoites. EhRho5 translocated from cytosol to plasma membrane and endomembranous compartments when stimulated with LPA, a bioactive phospholipid known to activate Rho subfamily members (1,2). Our findings were strengthened when FRAP (Fluorescence recovery after photobleaching) study showed that LPA treated cells show 30% faster recovery of Rho5 when compared to serum starved cells. The translocation is accompanied by activation of the GTPase as revealed by effector pull down assay(2). LPA stimulation enhanced the macropinocytic efficiency of trophozoites in a Rho5 dependent manner. We then identified the GEF for Rho5 which too, shows translocation upon stimulation with LPA. Also, depletion of the GEF leads to decreased pinocytic efficiency of the trophozoites. Using inhibitor based studies, we further demonstrated that PI3K acts as an intermediate cue for EhRho5 activation during LPA induced macropinocytosis.Moreoever, Rho5 expression showed contrary relationship with random migration of the trophozoites suggesting a pivotal role of the GTPase in mutally antagonistic cellular processes(3) in the amoeba. Reference: Fleming IN, Elliott CM, Exton JH. Differential Translocation of Rho Family GTPases by Lysophosphatidic Acid, Endothelin‐1, and Platelet‐derived Growth Factor, J. Biol. Chem. 1996, 271: 33067–33073 Van Leeuwen FN, Olivo C, Grivell S, Giepmans BNG, Collard JG, Moolenaar WH. Rac Activation by Lysophosphatidic Acid LPA1 Receptors through the Guanine Nucleotide Exchange Factor Tiam1, J. Biol. Chem. 2003, 278:400–406 Veltman DM, Lemieux MG, Knecht DA, Insall RH. PIP3‐dependent macropinocytosis is incompatible with chemotaxis. Journal of Cell Biology. 2014 Feb 17;204(4):497‐505.

Cryptosporidium and Giardia infections in humans in Tigray, Northern Ethiopia: an unexpectedly low occurrence of anthropozoonotic transmission

Enteric protozoans Cryptosporidium spp. and Giardia duodenalis are among the leading causes of diarrhoea in children. These parasites have particular impact in low- and middle-income countries. In these countries, people often live in close contact with their animals, highlighting the potential role of zoonotic routes of transmission in disease spread. The occurrence and species/genotypes of Cryptosporidium and Giardia duodenalis infecting humans in Tigray, Ethiopia were investigated, along with the risk associated with infection. Stool samples from 249 asymptomatic people (4-80 years of age) in four rural districts in Tigray and 58 from symptomatic young children (1-33 months) attending health centres in Mekelle, Tigray's main city, were analysed for Cryptosporidium oocysts and Giardia cysts. Participants in the rural areas completed questionnaires regarding potential risk factors, with emphasis on livestock contact and sources of water. The occurrence of Cryptosporidium infection was 6% and 5% in people in the rural districts and young children from Mekelle, respectively; equivalent figures for Giardia infection were 29% and 14%. Molecular characterization of Cryptosporidium isolates revealed C. ubiquitum, subtype XIIa in a sample from rural districts, and C. hominis subtype IdA17 (1 sample) and IbA9G3 (2 samples) in infants from Mekelle with diarrhoea. For Giardia, Assemblage B predominated (22/25; 88%), but we also identified three samples with Assemblage A (AII).Our major finding was that, despite the close contact between people and livestock in our rural study sites, transmission of Cryptosporidium and Giardia between humans and their animals seems to be surprisingly uncommon. Our results are discussed in relation to other relevant studies, and also draws attention to the possibility that introduction of zoonotic species and/or subtypes, such as C. parvum, could have serious consequences for both human and animal health. As our study was conducted in Tigray, further investigation in different settings in Ethiopia could provide relevant information on transmission and zoonotic potential, and the potential for spread of zoonotic transmission. In addition, given the importance of these two parasites in causing diarrhoea in children, this information is vital for developing effective appropriate interventions against transmission that can be applied not only in Tigray or Ethiopia, but throughout Africa and beyond.

A56 TUFT CELL RESPONSES DURING ACUTE- AND LATE-STAGE GIARDIA INFECTION

BackgroundEpithelial tuft cells can detect and respond to enteric infections and appear to help clear some intestinal parasites. Through tuft cell luminal surface receptors, tuft cells can sense ligands directly supplied by a parasite, or indirectly via excretory/secretory products. We hypothesize that microbiome alterations may also modulate tuft cell-derived gut responses. Tuft cells release the alarmin cytokine IL-25 which, upon acting on type 2 innate lymphoid cells (ILC2), ultimately lead to tuft and goblet cell hyperplasia. Upon helminth infections, tuft and goblet cell hyperplasia occurred concurrently, and coincided with the peak of infection. The role of tuft cells in infections with the enteric protozoan parasite Giardia sp. is unknown. The aim of our study is to characterize how tuft cells may be implicated in the pathophysiology of giardiasis, in an attempt to uncover novel regulatory pathways of intestinal physiology.AimsIn this study, we aim to characterise the tuft cell response to Giardia infection during acute and late stages of infection and to assess goblet cell hyperplasia.Methods5–7-week-old C57BL/6 mice and tuft cell-deficient mice ( Pou2f3-/-) were orally gavaged with 5x104Giardia muris trophozoites and scarified at days 4, 11 and 21 post-infection. Parasite burden was assessed in the duodenum. Immunofluorescence (IF) staining of doublecortin-like kinase 1 (DCLK1) – a marker of tuft cells – was performed on C57BL/6 mice jejunum tissue sections and the number of tuft cells was quantified. Goblet cells were quantified in PAS/AB-stained jejunum sections. Quantitative PCR (qPCR) was performed on Dclk1, the epithelial secretory cell transcription factor Atoh1, and the mucus gene Muc2.Results G. muris infected C57BL/6 mice displayed high parasite load at days 4 ( p<0.05) and 11 ( p<0.05), with no or low parasite burden at day 21 ( p<0.05). Pou2f3-/- mice showed less robust parasite burden at days 4 and 11, and similar low parasite burden at day 21, compared to WT mice. At day 21, tuft cell (DCLK1+) counts ( p<0.05) and Dclk1 mRNA expression levels were increased in Giardia infected mice in the jejunum. Goblet cell number and Atoh1 and Muc2 expression were increased at day 4 post-infection.ConclusionsThe data demonstrate that tuft cells expand late in Giardia infection, suggesting that upon parasite infection, tuft cells may possess roles in tissue repair or clearance of infection. Tuft cell-deficient mice ( Pou2f3-/-) had lower parasite burden early in Giardia infection, counter-intuitively suggesting that tuft cells may facilitate trophozoite colonization, further highlighting the novelty of these findings. The crosstalk between tuft cells, Giardia, and other host responses during acute and late stages of infection remain to be fully characterised.Funding AgenciesNSERC

Host-parasite interactions in infections due to Entamoeba histolytica: A tale of known and unknown.

Entamoeba histolytica (E. histolytica) is an enteric microaerophilic protozoan parasite responsible for millions of cases worldwide. Majority of the infections due to E. histolytica remain asymptomatic; however, it can cause an array of symptoms ranging from devastating dysentery, colitis, and abscesses in different vital organs. The interactions between the E. histolytica and its host are a multifaceted chain of events rather than merely destruction and invasion. There are manifold decisive steps for the establishment of infections by E. histolytica which includes degradation of mucosal layer, adherence to the host epithelium, invasion into the host tissues, and dissemination to vital organs. It is widely hypothesized that, for establishment of infections, the interactions at the intestinal mucosa decides the fate of the disease. The delicate communications between the parasite, the host factors, and the associated bacterial microflora play a significant role in the pathogenesis of E. histolytica. In this review, we summarize the interactions between the E. histolytica and it's host at the genetic and immunological interphases emphasizing the crucial role of microbiota in these interactions.

Molecular Prevalence of Blastocystis sp. in Anatolian Water Buffaloes in Diyarbakır, Turkey

The water buffaloes raised in Turkey originate from the Mediterranean water buffalo, a subgroup of the river buffalo, and are called the Anatolian buffalo. Blastocystis spp. is an anaerobic enteric protozoan parasite that lives in the gastrointestinal tract of a wide variety of hosts, including humans. This study aims to determine the molecular prevalence of Blastocystis spp. in Anatolian buffaloes in Diyarbakır province. Fresh fecal samples from animals were placed in individual fecal sample containers. The sex and age of the animal were recorded for each sample collected. As a result of PCR analysis, specific bands of 500 bp were obtained in 34 (17%) of 200 samples. Prevalence by age groups was found 27.69% in the 0-1 age group and 11.85% in those older than one year (P&lt;0.05). The prevalence by sex was 17.46% in females and 16.22% in males (P&gt;0.05). As a result of this study, the presence of Blastocystis spp. was revealed in Anatolian Water Buffaloes in Diyarbakır. It is thought that further studies are needed to determine the zoonotic subtype potential of the agent in the region.

Gut Microbiome Profiles in Colonizations with the Enteric Protozoa Blastocystis in Korean Populations.

The influence of unicellular eukaryotic microorganisms on human gut health and disease is largely unexplored. Blastocystis species commonly colonize the gut, but their clinical significance and ecological role are unclear. We evaluated the effect of Blastocystis colonization on the fecal microbiota of Koreans. In total, 39 Blastocystis-positive and -negative fecal samples were analyzed. The fecal microbiome was assessed by targeting the V3–V4 region of the bacterial 16S ribosomal gene. Bacterial diversity was greater in the Blastocystis-positive than in the Blastocystis-negative group. The bacterial community structure and phylogenetic diversity differed according to the presence of Blastocystis. The mean proportions of Faecalibacterium species and Ruminococcaceae were larger in the Blastocystis-positive group, and that of Enterococcus species was larger in the Blastocystis-negative group. Linear discriminant analysis showed that Faecalibacterium, Prevotella 9, Ruminococcaceae UCG-002, Muribaculaceae, Rikenellaceae, Acidaminococcaceae, Phascolarctobacterium, and Ruminococcaceae UCG-005 were highly enriched in the Blastocystis-positive group, whereas Enterococcus hirae, Enterococcus faecalis, Enterococcus durans, Enterococcaceae, Lactobacillales, and Bacilli were highly abundant in the Blastocystis-negative group. Overall, our results enlighten the notion that Blastocystis colonization is associated with a healthy gut microbiota.

Persistent Cryptosporidium parvum Infection Leads to the Development of the Tumor Microenvironment in an Experimental Mouse Model: Results of a Microarray Approach.

Cryptosporidium spp. are enteric protozoa parasites that infect a variety of vertebrate hosts. These parasites are capable of inducing life-threatening gastrointestinal disease in immunocompromised individuals. With the rising epidemiological evidence of the occurrence of Cryptosporidium infections in humans with digestive cancer, the tumorigenic potential of the parasite has been speculated. In this regard, Cryptosporidium parvum has been reported to induce digestive adenocarcinoma in a rodent model of chronic cryptosporidiosis. However, the processes by which the parasite could induce this carcinogenesis are still unknown. Therefore, the transcriptomes of C. parvum infected ileo-cecal regions of mice developing tumors were analyzed in the current study. For the first time, downregulation of the expression of α-defensin, an anti-microbial target of the parasite in response to C. parvum infection was observed in the transformed tissues. This phenomenon has been speculated to be the result of resistance of C. parvum to the host defense through the upregulated expression of interferon γ-stimulated genes. The inflammatory response generated as result of attenuated expression of anti-microbial peptides highlights the role of immune evasion in the C. parvum-induced tumorigenesis. The study has also succeeded in the characterization of the tumor microenvironment (TME) which is characterized by the presence of cancer associated fibroblasts, myeloid-derived suppressor cells, tumor-associated macrophages and extracellular matrix components. Identification of immune suppressor cells and accumulation of pro-inflammatory mediators speculates that chronic inflammation induced by persistent C. parvum infection assists in development of an immunosuppressive tumor microenvironment.

Prevalence and Molecular Characterization of the Zoonotic Enteric Protozoans Cryptosporidium spp., Enterocytozoon bieneusi, and Blastocystis from Pallas's Squirrels (Callosciurus erythraeus) in Kanagawa Prefecture, Japan.

ABSTRACTPallas’s squirrel (Callosciurus erythraeus) was introduced in Japan in the 1930s and has since established itself in several areas across the country. Although wild Sciuridae populations have been demonstrated to be potential reservoirs for zoonotic enteric protozoa, epidemiological studies of such pathogens in Japan are scarce. Here, we examined 423 fecal samples from Pallas’s squirrels captured in Kanagawa Prefecture, Japan, using PCR and DNA sequencing to determine the occurrence of Cryptosporidium spp., Enterocytozoon bieneusi, and Blastocystis. The overall prevalence of Cryptosporidium spp., E. bieneusi, and Blastocystis was 4.3% (18/423 samples), 13.0% (55/423 samples), and 44.0% (186/423 samples), respectively. The prevalence of Blastocystis and E. bieneusi was significantly higher in spring (60.1% and 17.4%, respectively) than in winter (27.6% and 8.6%, respectively [P < 0.01]). Sequence analysis of Cryptosporidium spp., targeting the partial small subunit ribosomal RNA gene (SSU rDNA), showed 100% identity (541/541 bp) to Cryptosporidium ubiquitum, and analysis of the gp60 gene showed 99.76% (833/835 bp) identity to C. ubiquitum subtype XIIh. The sequences of the ribosomal internal transcribed spacer region of E. bieneusi and the partial SSU rDNA of Blastocystis were identified as E. bieneusi genotype SCC-2 and Blastocystis subtype 4, respectively. This study confirmed the presence of C. ubiquitum, E. bieneusi, and Blastocystis in Pallas’s squirrels in Kanagawa Prefecture. Because Pallas’s squirrels inhabit urban areas, living close to humans, the species may serve as a potential source of infection in human populations.IMPORTANCE Pallas’s squirrel is designated a “regulated organism” under the Invasive Alien Species Act in Japan, and municipal authorities are introducing control measures to reduce its populations. It has been suggested that wild mammals may play a role in contaminating the environment with zoonotic pathogens. The present study detected the enteric pathogens Cryptosporidium ubiquitum, Enterocytozoon bieneusi, and Blastocystis in the feces of Pallas’s squirrels inhabiting Kanagawa Prefecture, Japan. These pathogens persist in the environment and contaminate soils and water, which may potentially infect humans. Because Pallas’s squirrels in Kanagawa Prefecture are found in urban areas, where they are in close contact with human populations, continued monitoring of zoonotic diseases among squirrel populations will be important for evaluating the significance of wildlife in pathogen transmission.

Fecal screening of Dientamoeba fragilis: relative efficacy of permanent smear and culture

Abstract Dientamoeba fragilis (D. fragilis) is an enteric trichmonad protozoan parasite that remains obscure and neglected. The aim of this study is to detect D. fragilis as a neglected pathogen in children aged 6-12 years old complaining of gastrointestinal illness by stool culture and light microscopy with comparison between the results of both techniques. A total of 100 fresh stool samples were included in this current study. All specimens were subjected to microscopic examination using iron- hematoxylin stained stool smears and stool culture using a Loeffler’s culture medium. Culture detected 2 positive stool samples (2%) while microscopy detected (1%). Sensitivities of culture and microscopy were 100% and 50% respectively. Specificity of culture and microscopy were 100% and 95% respectively. There is a moderate agreement between culture and microscopy (K = 0.4). In conclusion, culture had a high performance compared to microscopy in the diagnosis of D. fragilis infection. Culture can be applied for routine diagnosis for the detection of D. fragilis in clinical samples.

Changes in Escherichia coli to enteric protozoa ratios in rivers: Implications for risk-based assessment of drinking water treatment requirements

Minimum treatment requirements are set in response to established or anticipated levels of enteric pathogens in the source water of drinking water treatment plants (DWTPs). For surface water, contamination can be determined directly by monitoring reference pathogens or indirectly by measuring fecal indicators such as Escherichia coli (E. coli). In the latter case, a quantitative interpretation of E. coli for estimating reference pathogen concentrations could be used to define treatment requirements. This study presents the statistical analysis of paired E. coli and reference protozoa (Cryptosporidium, Giardia) data collected monthly for two years in source water from 27 DWTPs supplied by rivers in Canada. E. coli/Cryptosporidium and E. coli/Giardia ratios in source water were modeled as the ratio of two correlated lognormal variables. To evaluate the potential of E. coli for defining protozoa treatment requirements, risk-based critical mean protozoa concentrations in source water were determined with a reverse quantitative microbial risk assessment (QMRA) model. Model assumptions were selected to be consistent with the World Health Organization (WHO) Guidelines for drinking-water quality. The sensitivity of mean E. coli concentration trigger levels to identify these critical concentrations in source water was then evaluated. Results showed no proportionalities between the log of mean E. coli concentrations and the log of mean protozoa concentrations. E. coli/protozoa ratios at DWTPs supplied by small rivers in agricultural and forested areas were typically 1.0 to 2.0-log lower than at DWTPs supplied by large rivers in urban areas. The seasonal variations analysis revealed that these differences were related to low mean E. coli concentrations during winter in small rivers. To achieve the WHO target of 10−6 disability-adjusted life year (DALY) per person per year, a minimum reduction of 4.0-log of Cryptosporidium would be required for 20 DWTPs, and a minimum reduction of 4.0-log of Giardia would be needed for all DWTPs. A mean E. coli trigger level of 50 CFU 100 mL−1 would be a sensitive threshold to identify critical mean concentrations for Cryptosporidium but not for Giardia. Treatment requirements higher than 3.0-log would be needed at DWTPs with mean E. coli concentrations as low as 30 CFU 100 mL−1 for Cryptosporidium and 3 CFU 100 mL−1 for Giardia. Therefore, an E. coli trigger level would have limited value for defining health-based treatment requirements for protozoa at DWTPs supplied by small rivers in rural areas.

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

BackgroundHuman 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.MethodsA 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.ResultsHBoV 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.ConclusionsThe 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.

Molecular characterization of Cryptosporidium spp. in dogs and cats in the city of Rio de Janeiro, Brazil, reveals potentially zoonotic species and genotype.

Intestinal cryptosporidiosis is a diarrheal disease caused by protists of genus Cryptosporidium that infect a wide variety of hosts, primarily vertebrates. Due to the close contact between humans and their companion animals, especially dogs and cats, there is concern about the potential for zoonotic transmission of this enteric protozoan parasite by infected animals. This study aimed to perform a microscopic and molecular diagnosis of Cryptosporidium spp. in fecal samples from domiciled dogs and cats. One hundred and nineteen fecal samples were processed using sugar centrifugal flotation followed by molecular detection of Cryptosporidium spp. DNA using nested PCR. Subtyping of isolates positive for C. parvum was performed by sequence analysis of the 60 kDa glycoprotein gene (GP60). Cryptosporidium oocysts were detected in 7.8% (5/64) and 5.4% (3/55) of the fecal samples from dogs and cats, respectively. Cryptosporidium canis (n = 3) and C. parvum (n = 2) were the main species found in dogs, whereas C. felis (n = 3) was prevalent in cats. Subtype IIaA17G2R2 (potentially zoonotic) was identified in samples positive for C. parvum. Despite the low prevalence of Cryptosporidium observed in the domiciled dogs and cats, the presence of potentially zoonotic C. parvum in dogs evidences a public health concern. Further research is needed to better understand the epidemiology, source, and potential impacts of Cryptosporidium infection in cats and dogs.

Molecular Detection of Soil-Transmitted Helminths and Enteric Protozoa Infection in Children and Its Association with Household Water and Sanitation in Manhiça District, Southern Mozambique.

Intestinal parasite infections can have detrimental health consequences in children. In Mozambique, soil-transmitted helminth (STH) infections are controlled through mass drug administration since 2011, but no specific control program exists for enteric protozoa. This study evaluates STH and protozoan infections in children attending healthcare in Manhiça district, Southern Mozambique, and its association with water and sanitation conditions. We conducted a cross-sectional study in children between 2 and 10 years old in two health centers (n = 405). A stool sample and metadata were collected from each child. Samples were analyzed by multi-parallel real-time quantitative PCR (qPCR). We fitted logistic regression-adjusted models to assess the association between STH or protozoan infection with household water and sanitation use. Nineteen percent were infected with at least one STH and 77.5% with at least one enteric protozoon. qPCR detected 18.8% of participants with intestinal polyparasitism. Protected or unprotected water well use showed a higher risk for at least one protozoan infection in children (OR: 2.59, CI: 1.01–6.65, p-value = 0.010; OR: 5.21, CI: 1.56–17.46, p-value = 0.010, respectively) compared to household piped water. A high proportion of children had enteric protozoan infections. Well consumable water displayed high risk for that.

Epidemiology and clinical features of intestinal protozoan infections detected by Real-time PCR in non-native children within an Italian tertiary care children's hospital: A cross-sectional study

BackgroundEnteric parasite infections are underestimated due to the limited sensitivity and specificity of microscopy, which remains the diagnostic gold standard in routine clinical practice. This could be a major problem in high-income countries, where the burden of parasitic diseases is low. In recent years, Multiplex Real-Time polymerase chain reaction (RT-PCR) based methods have been implemented. Therefore, the aim of this study was to evaluate the prevalence of four enteric protozoan species detected by RT-PCR in non-native children in Italy, and to describe their clinical characteristics. MethodsAdopted and immigrant children, evaluated for migration health assessment between 2017 and 2020 in a tertiary care children's hospital in Italy, were enrolled. Molecular analysis for Giardia lamblia, Dientamoeba fragilis, Blastocystis hominis, and Entamoeba histolytica, was conducted by in-house RT-PCR. ResultsOverall, 209 children were enrolled and 70% of them resulted positive by RT-PCR for at least one enteric parasite. B. hominis (47.8%) was the most commonly identified protozoa, followed by D. fragilis (44.5%). Co-infections with multiple pathogens were detected in 35.4% of the samples. Almost 80% of parasite-positive children were asymptomatic and the most common symptom was flatulence (60.7% of symptomatic children). Eosinophils were significantly increased in RT-PCR positive children compared to the negative ones and children with D. fragilis presented the highest eosinophils count. ConclusionsThe In-house Multiplex RT-PCR assay provides a valid molecular detection system for selected enteric parasites. This novel and accurate diagnostic method can help in increasing the detection rate of parasite infection, especially in high-risk population.

CRISPR/Cas12a-based on-site diagnostics of Cryptosporidium parvum IId-subtype-family from human and cattle fecal samples

BackgroundCryptosporidium parvum is an enteric protozoan parasite with zoonotic importance and can cause cryptosporidiosis in humans as well as domestic and wild animals worldwide. The IId subtype family (SF) is one of the most prevalent subtypes of C. parvum. Some clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) protein systems have been developed to detect nucleic acid with high flexibility, sensitivity and specificity.MethodsBy integrating recombinase polymerase amplification and the Cas12a/crRNA trans-cleavage system (termed ReCTC), we established end-point diagnostics by observing fluorescence readouts with the naked eye under blue light and on-site diagnostics using a lateral flow strip (LFS) biosensor.ResultsOur ReCTC-based diagnoses can detect as little as a single copy of a cloned C. parvum 60-kDa glycoprotein (GP60) gene, 10 oocysts per gram (OPG), clinical fecal sample without tedious extraction of genomic DNA and have no cross-reactivity with other SFs of C. parvum or other common enteric parasitic protozoa.ConclusionsThis study provided a new strategy for direct identification of the IId SF of C. parvum free of highly trained operators and expensive special equipment.Graphical

Methods, Protocols, Guidance and Standards for Performance Evaluation for Point-of-Use Water Treatment Technologies: History, Current Status, Future Needs and Directions

It is estimated that 780 million people do not have access to improved drinking water sources and approximately 2 billion people use fecally contaminated drinking water. Effective point-of-use water treatment systems (POU) can provide water with sufficiently reduced concentrations of pathogenic enteric microorganisms to not pose significant health risks to consumers. Household water treatment (HWT) systems utilize various technologies that physically remove and/or inactivate pathogens. A limited number of governmental and other institutional entities have developed testing protocols to evaluate the performance of POU water treatment systems. Such testing protocols are essential to documenting effective performance because inferior and ineffective POU treatment technologies are thought to be in widespread use. This critical review examines specific practices, procedures and specification of widely available POU system evaluation protocols. Testing protocols should provide standardized and detailed instructions yet be sufficiently flexible to deal with different treatment technologies, test microbe priorities and choices, testing facility capabilities and public health needs. Appropriate infectivity or culture assays should be used to quantify test enteric bacteria, viruses and protozoan parasites, or other appropriate surrogates or substitutes for them, although processes based on physical removal can be tested by methods that detect microbes as particles. Recommendations include further research of stock microbe production and handling methods to consistently yield test microbes in a realistic state of aggregation and, in the case of bacteria, appropriately physiologically stressed. Bacterial quantification methods should address the phenomenon of bacterial injury and repair in order to maximally recover those that are culturable and potentially infectious. It is only with harmonized national and international testing protocols and performance targets that independent and unbiased testing can be done to assure consumers that POU treatment technologies are able to produce water of high microbial quality and low health risk.

DISTRIBUTION OF ENTERIC PROTOZOA IN THE RIVER NILE DRAINS: EGYPT USING FIELD AND REMOTE SENSING STUDIES

Polluted water is a massive international issue that needs to be monitored and evaluated at allconditions for water management policy. This study identified enteric protozoa in three drains;as well as the physicochemical characteristics, heavy metals of such freshwater bodies in seasonalbiotic status in Abu Youssef, El-Rahaway, and El-Qalyubia in the River Nile, Egypt. Also,the spatial distribution of the pathogenic protozoa along the three drains was investigatedusing remote sensing (RS) and the geographic information system (GIS). Applying these approachesgave a useful epidemiological of protozoa in drains. The results showed Entamoebahistolytica and Giardia intestinalis in the three drains. Their survival depended on oxygen demand(COD) concentration, organic matters and coliform bacteria existence despite the heavymetal pollution. This study gave valuable information within the GIS framework to accuratelyquantify the spatial distribution of aquatic biota and water chemistry and thus determined therisks to water bodies and proposing the proper mitigation measure to save such vital resource.

THERAPEUTIC AND IMMUNO-MODULATORY EFFECT OF SILVER NANOPARTICLES-LOADED WITH METRONIDAZOLE ON EXPERIMENTAL BLASTOCYSTOSIS

Blastocystis is one of the commonest enteric protozoa in many hosts. This study evaluatedthe effect of silver nanoparticles (AgNps) loaded with metronidazole (MTZ) on mice infectedwith Blastocystis. They were subjected to parasitological, histopathological, immunological,and immunohistochemical studies. The parasitological results revealed a powerful effect of AgNPs loaded with MTZ over either MTZ or Ag Nps; where the reduction of cysts count in stoolwere (90. 7%, 87.33% & 59.33%) respectively as compared to infected non-treated group. Histopathologicalresults showed that AgNPs loaded with MTZ were the best in reducing inflammatorychanges followed by MTZ then AgNPs. Regarding IgA, IgM & IgG, Ag NPs loadedwith MTZ treated mice showed the best result followed by MTZ treated mice then Ag NPstreated mice; where the reduction of mean concentration of IgA were (30.5%, 26.24% &2.13%) respectively, while that of IgM were (15.2%, 12.8% & 2.8%) respectively and IgG were(23.1%, 12.3% & 5.2%) respectively as compared to infected non-treated group. The synergisticeffect of both Ag NPS & MTZ provoked the best reduction of CD3 & CD20 expression andaugmented counter modulation of the immune response to restore normal immune homeostasis.

Intestinal protozoan infections among Egyptian neutropenic patients with acute leukemia.

Several enteric protozoan species are linked to diarrhea in humans, with some causing debilitating illnesses, essentially in immunocompromised and neutropenic patients as in acute leukemias. The aim of this study was to detect intestinal protozoa in Egyptian neutropenic patients with acute leukemia. The study comprised two groups; 40 newly diagnosed neutropenic acute leukemia patients and 30 controls. Stool samples were collected from all participants and subjected to routine microscopic examination, special staining and detection of copro-antigen using rapid diagnostic test (RDT) RIDA®QUICK Entamoeba/ Giardia/ Cryptosporidium Combi. Cases were tested post-chemotherapy at the nadir of neutropenia (absolute neutrophil count ANC<= 0.5x109/L) and 19 cases were also tested initially prior to chemotherapy. Of examined patients, 15/40 (37%) were positive for Blastocystis hominis by wet mount, 10/40 (25%) had microsporidia using modified trichrome stain and only 2 cases (5%) of Cryptosporidium spp. by Ziehl-Neelsen stain. By RDT, 8/40 cases (20%) were positive compared to entirely negative controls. The positive cases included 4 patients with G. intestinalis 2 with Entamoeba and 2 with Cryptosporidium.19/40 cases were tested both pre- and post-chemotherapy. microsporidian spp. was diagnosed in 6/19 cases at the nadir of neutropenia compared to none of the cases pre-chemotherapy and the difference was statistically significant (p= 0.031*). Intestinal protozoa in acute leukemia patients post-chemotherapy are common especially B. hominis. Furthermore, RDT might be helpful for diagnosing intestinal protozoa in acute leukemia. Attention is highly required as intestinal protozoa infection can emerge after chemotherapy such as microsporidia.

Molecular detection of Giardia duodenalis and Cryptosporidium spp. from stray dogs residing in monasteries in Bangkok, Thailand.

Both Cryptosporidium spp. and Giardia duodenalis are enteric protozoan parasites that infect a wide variety of domestic animals as well as humans worldwide, causing diarrheal diseases. Giardia duodenalis assemblages C and D are specific to canine hosts and zoonotic assemblages A and B are also found in dogs as a reservoir host. In dogs, Cryptosporidium canis is the host-specific species while humans are infected by C. hominis and C. parvum and at least another 16 zoonotic Cryptosporidium species have been reported causing human infections, with C. meleagridis, C. viatorum, and C. ubiquitum being the most frequent. The objective of this study was to determine the prevalence of Cryptosporidium spp. and G. duodenalis from stray dogs in areas of Bangkok and to identify the species and assemblages. Fecal samples (540) were collected from dogs residing in 95 monasteries in 48 districts in the Bangkok metropolitan area. Nested Polymerase Chain Reaction (PCR) was performed using the ssu-rRNA gene for both parasites. In total, 3.0% (16/540) samples were positive for G. duodenalis, with most being G. duodenalis assemblage D (7/16) followed by assemblage C (7/16) and zoonotic assemblage A (2/16). The prevalence of Cryptosporidium spp. was 0.7% (4/540) based on the PCR results and all were the dog genotype C. canis. These results indicated that dogs residing in Bangkok monasteries poses a limited role as source of human giardiosis and cryptosporidiosis.