Molecular Characterization of Giardia intestinalis and Cryptosporidium spp. Detected in Humans in Agri, Türkiye.

Background: We aimed to determine the prevalence of Giardia intestinalis and Cryptosporidium spp. in patients who admitted hospital with diarrhea and to gain information about the transmission of these parasites in Agri, Türkiye. Methods: This study included 184 patients who applied to Agri-Diyadin State Hospital, Türkiye in 2022. The immunochromatographic card test was used for detection of the G. intestinalis and Cryptosporidium spp. Nested PCR-RFLP analysis of the COWP gene and sequence analysis of the gp60 gene were used to genotype and subtype Cryptosporidium spp., whereas Nested PCR and sequence analyses of ß-giardin gene were used genotype G. intestinalis. Results: Of the 184 stool specimens examined, 12 (14.29%) and 7 (3.80%) were positive for G. intestinalis and Cryptosporidium spp., respectively. The Cryptosporidium species were identified as C. parvum belonging to the IId sub-type family. The G. intestinalis were identified assemblages A. Conclusion: Assemblage A, which is associated with diarrhea, is responsible for giardiasis and C. parvum IId subtype, often found in sheep, goats and cattle, is responsible for cryptosporidiosis in Agri, Türkiye.

Selection of ssDNA aptamers and construction of aptameric electrochemical biosensor for the detection of Giardia intestinalis trophozoite protein.

Giardia intestinalis is one of the most widespread intestinal parasites and is considered a major cause of epidemic or sporadic diarrhea worldwide. In this study, we aimed to develop a rapid aptameric diagnostic technique for G. intestinalis infection. First, the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) process generated DNA aptamers specific to a recombinant protein of the parasite's trophozoite. Ten selection rounds were performed; each round, the DNA library was incubated with the target protein conjugated to Sepharose beads. Then, the unbound sequences were removed by washing and the specific sequences were eluted and amplified by Polymerase Chain Reaction (PCR). Two aptamers were selected, and the dissociation constants (Kd), were determined as 2.45 and 16.95 nM, showed their high affinity for the G. intestinalis trophozoite protein. Subsequently, the aptamer sequence T1, which exhibited better affinity, was employed to develop a label-free electrochemical biosensor. A thiolated aptamer was covalently immobilized onto a gold screen-printed electrode (SPGE), and the binding of the targeted protein was monitored using square wave voltammetry (SWV). The developed aptasensor enabled accurate detection of the G. intestinalis recombinant protein within the range of 0.1 pg/mL to 100 ng/mL, with an excellent sensitivity (LOD of 0.35 pg/mL). Moreover, selectivity studies showed a negligible cross-reactivity toward other proteins such as bovine serum albumin, globulin, and G. intestinalis cyst protein.

Distribution of Intestinal Parasites in Patients Admitted to University Hospital: Four Year Retrospective Review.

Objective: In this study, it was aimed to examine the distribution of intestinal parasites detected in people who applied to the parasitology laboratory of Firat University Faculty of Medicine between January 2018 and December 2021. Methods: Parasitological examination reports of a total of 24,432 patients who applied to the Parasitology Laboratory of Firat University Faculty of Medicine between January 2018 and December 2021 were examined retrospectively for the presence of intestinal parasites. Results: A total of 24,432 (male: 12,887, 52.75%; female: 11,545, 47.25%) patients whose stool and cellophane tape samples were analyzed during the four-year period between January 2018 and December 2021 were included in the study. Intestinal parasites were found in 335 (1.4%) of the 24,432 patients examined. The most frequently detected parasite was Giardia intestinalis (n=149, 46.6%), followed by Entamoeba coli (n=123, 38.5%) and Enterobius vermicularis (n=28, 8.6%). When the distribution of parasite detection rates by years was examined, it was seen that the highest rate was in 2021 and the lowest rate was in 2019. Conclusion: Intestinal parasitic infections (IPE) are one of the most important public health problems in the world and in our country. Various factors such as the education level of the society, socio-economic status, infrastructure and climate affect the distribution of IPE. When we look at the distribution of parasites by years, it is 1.3% in 2018; 1.13% in 2019; 1.18% in 2020; In 2021, we found it to be 2.03%. We think that this increase in intestinal parasites is caused by the infrastructure and sheltering problems caused by the earthquake in our region.

Comparison of Giardia duodenalis point-of-care antigen faecal tests to reference laboratory assays in non-symptomatic dogs.

Giardia duodenalis is one of the most prevalent enteric parasites of dogs. Point-of-care antigen tests (POC) are rapid and do not require additional equipment, or a specialised diagnostic laboratory. The aim of this study was to compare diagnostic tests available in veterinary practices and in a diagnostic laboratory for the detection of G. duodenalis on a cohort of group-housed dogs from New South Wales, Australia. Two different POC tests were used for the detection of G. duodenalis. Laboratory tests used were the multiplexed-tandem PCR panel (MT-PCR) that includes detection of G. duodenalis DNA, and two reference tests (an in-house TaqMan real-time PCR and a direct immunofluorescence assay, DFA). Canine faecal samples (n = 40) were tested simultaneously for the detection of G. duodenalis. Using either DFA or TaqMan real-time PCR as reference tests, 77.5% (31/40) and 82.5% (33/40) of dogs tested positive, respectively. Agreement (Kappa) between the DFA and TaqMan real-time PCR was 0.84 (95% CI 0.64 to 1.00). There was substantial G. duodenalis test outcome agreement between the two POC tests, Kappa = 0.75. Combining the two POC tests yielded 77% sensitivity and 100% specificity with DFA as reference, and for TaqMan real-time PCR it was 73% sensitivity and 100% specificity. The MT-PCR was in excellent agreement with each reference test, DFA or TaqMan real-time PCR. Due to the high specificity of both POC tests, they can be confidently used as rule-in diagnostics. Confirmatory testing that detects different biological parameters such as DNA, e.g. PCR (inc. MT-PCR), should be implemented before concluding that a dog is negative for the presence of G. duodenalis.

Prevalence and molecular characterization of Giardia intestinalis isolated from children and calves in Babylon province, Iraq.

Background and Aim: Giardia intestinalis is one of the most prevalent intestinal parasites in humans and animals, and children in close contact with livestock are particularly at risk of infection. This study aimed to detect assemblages of G. intestinalis and determine the origin of zoonotic transmission of Giardia in children and calves in different parts of Babylon province, Iraq. Materials and Methods: One hundred stool samples from children (68 boys and 32 girls) and 100 fecal samples from calves (46 males and 54 females) of different ages were randomly collected. Molecular techniques were used to estimate the prevalence of G. intestinalis in children and calves. A nested polymerase chain reaction (PCR) was performed by targeting the triose phosphate isomerase gene in the samples to detect G. intestinalis assemblages. Results: The overall rates of infection with G. intestinalis in children and calves were 21% and 34%, respectively, using the conventional microscopic method. The results illustrated that 61.90% (13/21) and 38.09% (8/21) of positive samples from children were allocated to assemblages A and B, respectively (p > 0.05). In calves, assemblages A and B were detecte in 82.35% (28/34) and 17.64% (6/34) of positive samples from calves, respectively (p ≤ 0.001). Ten PCR products were sequenced and submitted to the GenBank database. Phylogenetic analysis detected five human sequences each belonging to G. intestinalis assemblages A (OM850335-OM850339) and B (OM850340-OM850344). Similarly, five calf sequences each belonged to G. intestinalis assemblages A (ON75756-ON757660) and B (ON757661-ON757665). Conclusion: The detection of large numbers of G. intestinalis assemblage A in both humans and cattle indicated that cattle could be a main source of zoonotic G. intestinalis infection in children in Babylon province, Iraq.

Giardia VSPAS7 protein attenuates Giardia intestinalis-induced host macrophage pyroptosis.

BACKGROUND: The unicellular protozoan parasite Giardia intestinalis, which primarily infects humans and animals such as cattle and sheep, is having a major negative impact on public health. Giardia is able to evade the recognition and elimination of the host immune system because of the trophozoite surface and extracellular vesicles (EVs) covered by variant-specific surface proteins (VSPs). As key proteins for immune evasion, whether VSPs can regulate Giardia-induced pyroptosis and promote Giardia evasion of host immune responses has not been reported. METHODS: To examine the role of Giardia VSPAS7 on Giardia-induced activation of the signaling pathway, secretion of pro-inflammatory cytokines, pyroptosis and the mechanism involved, we constructed the pcDNA3.1-vspas7 expression plasmid and transfected this plasmid into mouse macrophages. Key proteins for pyroptosis, IL-1ß secretion and LDH release were detected in pcDNA3.1-vspas7-transfected wild-type (WT) cells and NLRP3-deficient cells by western blot, ELISA and LDH assays, respectively. The interactions of Giardia VSPAS7 and mouse NLRP3 were examined using immunofluorescence assays (IFA), co-immunoprecipitation (Co-IP) and bimolecular fluorescence complementation (BiFC) assays. RESULTS: VSPAS7 could decrease the levels of phosphorylated-p65 (P-p65), P-IκBα and P-ERK caused by Giardia and reduce the production levels of Giardia-induced pro-inflammatory cytokine IL-6, IL-12 p40 and TNF-α. The results showed that VSPAS7 inhibited Giardia-mediated activation of NF-κB, ERK/MAPK signaling and secretion of pro-inflammatory cytokines. Furthermore, VSPAS7 suppressed Giardia-induced macrophage pyroptosis by reducing GSDMD cleavage, caspase-1 activation, IL-1ß secretion and LDH release. We further found that VSPAS7 could interact with mouse NLRP3 directly, and in NLRP3-deficient cells the suppression of Giardia-induced macrophage pyroptosis by VSPAS7 was significantly attenuated. CONCLUSIONS: Overall, VSPAS7 could inhibit Giardia-induced activation of signaling pathways and pyroptosis in host macrophages, allowing Giardia evasion of host immune responses. Studies on Giardia VSP-mediated immune evasion provide an important theoretical basis for in-depth studies on Giardia pathogenicity.

The opportunistic protist, Giardia intestinalis, occurs in gut-healthy humans in a high-income country.

Giardia intestinalis, a cosmopolitan gastrointestinal protist, is detected mainly in patients with clinical giardiasis in high-income countries. In contrast, there is very little information on the presence of Giardia in asymptomatic individuals. Therefore, the aim of this study was to determine the presence and prevalence of Giardia in gut-healthy volunteers in the Czech Republic and to perform a comparative evaluation of different diagnostic methods, since Giardia diagnostics is complicated. Our results confirmed that the qPCR method is the most sensitive method for detecting Giardia and revealed a prevalence of 7% (22/296) in asymptomatic individuals. In most cases, the colonization intensity ranged from 10-1-101. A conventional PCR protocol targeting the TPI gene was used to identify the assemblages. However, this protocol had limited sensitivity for Giardia amplification, effectively detecting colonization above an intensity of 104. In addition, Giardia was detected in 19% of the animals, which were closely associated with the study participants. However, due to methodological limitations, zoonotic transmission could not be clearly confirmed. Notably, contact with animals proved to be the only factor that had a significant impact on the incidence of Giardia in gut-healthy humans.

Molecular screening of Entamoeba spp. (E. histolytica, E. dispar, E. coli, and E. hartmanni) and Giardia intestinalis using PCR and sequencing.

A wide range of intestinal protozoan parasites inhabit the human gut. To establish a more comprehensive molecular screening, we designed PCR-sequencing screening methods for Entamoeba spp., including commensal species, and Giardia intestinalis, and performed such methods using 174 stool samples collected from Kenyan children. The prevalences of the target species were as follows: E. histolytica (2/174, 1.1%), E. dispar (20/174, 11.5%), E. coli (107/174, 61.5%), E. hartmanni (77/174, 44.3%), and G. intestinalis (54/174, 31.0%). PCR amplicons specific to G. intestinalis was differentiated to assemblages A (8/174, 4.6%) and B (46/174, 26.4%). PCR specificity for Entamoeba spp. was quite high, except for some cross-reactions between E. hartmanni detection primers and G. intestinalis, although the false-positive amplicons were discernible by the band size. The 18S rRNA PCR primers that was designed by Monis et al. in 1999 for G. intestinalis, have specificity issue, therefore amplicon sequencing was essential not only to determine assemblage classifications but also to confirm the positive results by eliminating potential non-specific reactions. The detection sensitivity of both the Entamoeba universal PCR and the G. intestinalis PCR was more than 100 copies of the target loci, which is sufficient for detecting a single trophozoite or cyst of both species.

An Improved Method to Enrich Large Extracellular Vesicles Derived from Giardia intestinalis through Differential Centrifugation.

Giardia intestinalis is a flagellated unicellular protozoan that colonizes the small intestine, causing the diarrheal disease called giardiasis. The production of extracellular vesicles (EVs) by G. intestinalis and the role of these EVs in the parasite's interaction with the host have been described. According to biogenesis, EVs are grouped mainly into large (microvesicles-derived from the plasma membrane) and small (exosomes-derived from multivesicular bodies). Populations of EVs are heterogeneous, and improved methods to separate and study them are needed to understand their roles in cell physiology and pathologies. This work aimed to enrich the large extracellular vesicles (LEVs) of G. intestinalis in order to better understand the roles of these vesicles in the interaction of the parasite with the host. To achieve the enrichment of the LEVs, we have modified our previously described method and compared it by protein dosage and using Nano tracking analysis. Giardia intestinalis vesiculation was induced by incubation in a TYI-S-33 medium without serum, to which 1 mM of CaCl2 was added at 37 °C for 1 h. Then, the supernatant was centrifuged at 15,000× g for 1 h (15 K 1 h pellet), 15,000× g for 4 h (15 K 4 h pellet) and 100,000× g for 1.5 h (100 K 1h30 pellet). The pellet (containing EVs) was resuspended in 1× PBS and stored at 4 °C for later analysis. The EVs were quantified based on their protein concentrations using the Pierce BCA assay, and by nanoparticle tracking analysis (NTA), which reports the concentration and size distribution of the particles. The NTA showed that direct ultracentrifugation at 100,000× g for 1.5 h and centrifugation at 15,000× g for 4 h concentrated more EVs compared to centrifugation at 15,000× g for 1 h. Additionally, it revealed that centrifugation at 15,000× g 4 h was able to concentrate at the same particle concentration levels as a direct ultracentrifugation at 100,000× g for 1.5 h. As for the enrichment of LEVs, the NTA has shown a higher concentration of LEVs in direct ultracentrifugation at 100,000× g for 1.5 h, and in centrifugation at 15,000× g for 4 h, compared to centrifugation at 15,000× g for 1 h. Our results have shown that the most used method at 15,000× g for 1 h is not enough to obtain a representative population of large EVs, and we suggest that LEVs released by G. intestinalis can be better enriched by direct ultracentrifugation at 100,000× g for 1.5 h, or by centrifugation at 15,000× g for 4 h.

The Influence of Selected Factors on the Detection of Giardia intestinalis by Microscopic and Immunoenzymatic Methods.

Background: Giardia intestinalis is one of the most common parasites in humans. Contaminated food and water can be a source of infection. Substances added to food are intended to increase its safety. We aimed to determination of the influence of various microorganisms and compounds that stimulate digestive functions, as well as preservatives and antioxidants on the detection of G. intestinalis by microscopic and immunoenzymatic methods. Methods: Twenty stool samples, archived in 1998-2018 in the Provincial Sanitary and Epidemiological Station in Bydgoszcz (Poland), collected both from patients referred for parasitic examinations by a doctor of a medical facility and from private individuals, were used to assess the impact of selected factors (such as bacterial strains, viruses and substances added to food) on the detection of G. intestinalis by microscopic and immunoenzymatic methods. Results: G. intestinalis was detected by both microscopic and immunoenzymatic methods with the same sensitivity (100%). The result of the G. intestinalis determination was positive in 90% of the samples after the addition of potassium sorbate, and in 25% of the samples after the addition of citric acid. Conclusion: The presence of other microorganism such as bacteria and viruses does not influence on the detection of G. intestinalis by microscopic and immunoenzymatic methods in stool samples. Citric acid as an antioxidant added to foods affects the detection of G. intestinalis. Due to the small number of samples used, it is necessary to continue research on the impact of various factors on the detection of protozoa.

Refractory giardiasis in a child with steroid-resistant nephrotic syndrome.

Giardiasis is an infection of the small intestine caused by the protozoan parasite Giardia intestinalis and one of the most common parasitic intestinal diseases in humans worldwide. It mainly manifests as a self-limited illness in the case of immunocompetent patients and usually does not require treatment. However, immunodeficiency is a risk factor for the onset of severe Giardia infection. In this report, a case of recurrent giardiasis refractory to nitroimidazole therapy is presented. A 7-year-old male patient with steroid-resistant nephrotic syndrome came to our hospital because of chronic diarrhoea. The patient was on long-term immunosuppressive therapy. Microscopic examination of stool showed a significant number of trophozoites and cysts of G. intestinalis. Treatment with metronidazole for longer duration than recommended has failed to clear the parasite in the present case.

Novel therapeutic opportunities for Toxoplasma gondii, Trichomonas vaginalis, and Giardia intestinalis infections.

INTRODUCTION: Toxoplasma gondii, Trichomonas vaginalis, and Giardia intestinalis are the causative agents of toxoplasmosis, trichomoniasis, and giardiasis, three important infections threatening human health and affecting millions of people worldwide. Although drugs and treatment are available to fight these protozoan parasites, side effects and increasing drug resistance require continuous efforts for the development of novel effective drugs. AREAS COVERED: The patents search was carried out in September/October 2022 with four official scientific databases (Espacenet, Scifinder, Reaxys, Google Patents). Treatments for toxoplasmosis, trichomoniasis, and giardiasis (2015-2022) have been grouped according to their chemotypes. In particular, novel chemical entities have been reported and investigated for their structure-activity relationship, when accessible. On the other hand, drug repurposing, extensively exploited to obtain novel antiprotozoal treatment, has been in-depth described. Finally, natural metabolites and extracts have also been reported. EXPERT OPINION: T. gondii, T. vaginalis, and G. intestinalis are protozoan infections usually controlled by immune system in immunocompetent patients; however, they could represent a threatening health for immunocompromised people. The needs of novel effective drugs, endowed with new mechanisms of actions, arises from the increasing drug resistance affecting antibiotic as well as antiprotozoal therapies. In this review different therapeutic approaches to treat protozoan infections have been reported.

Inactivation of Giardia intestinalis cysts in water using a sonochemical method based on mid-high-frequency waves.

Giardia intestinalis is a pollutant of food and water, resistant to conventional disinfection treatments and its elimination requires effective methods action. Herein, mid-high-frequency ultrasound (375 kHz), which produces HO• and H2O2, was used as an alternative method of treatment to inactivate Giardia intestinalis cysts in water. The effect of ultrasound power (4.0, 11.2, 24.4 W) on the sonogeneration of radicals was tested, showing that 24.4 W was the condition most favorable to treat the parasite. The viability of the protozoan cysts was evaluated using the immunofluorescence technique and vital stains, showing this protocol was useful to quantify the parasite. The sonochemical method (at 375 kHz and 24.4 W) was applied at different treatment times (10, 20, and 40 min). A significant decrease in the protozoan concentration (reduction of 52.4% of viable cysts) was observed after 20 min of treatment. However, the extension of treatment time up to 40 min did not increase the inactivation. Disinfecting action was associated with attacks on the Giardia intestinalis cyst by sonogenerated HO• and H2O2 (which may induce structural damage, even the cell lysis). For future work is recommended to test combinations with UVC or Fenton process to enhance the inactivating action of this method.•Mid-high-frequency ultrasound produces HO• and H2O2 profitable to inactivate Giardia intestinalis.•Immunofluorescence technique and vital stains allowed us to quantify the parasite viability.•Giardia intestinalis cysts concentration decreased by 52.4% after only 20 min of sonication.

Wastewater treatment plants discharges disseminated more Giardia than Cryptosporidium.

Parasitic protozoa Giardia intestinalis and Cryptosporidium parvum are causative agents for giardiasis and cryptosporidiosis, respectively. These infections are mostly associated with waterborne diseases. The discharges from wastewater treatment plants (WWTPs) that reach surface waters cause waterborne transmission because there are no regulations for monitoring these protozoa. This emphasizes how crucial the removal capacities of WWTPs to prevent the spread of infectious parasitic pathogens. For this reason, in this study, five different types of WWTPs including conventional activated sludge (CAS), biological nutrient removal (BNR), sequencing batch reactor (SBR), membrane bioreactor (MBR), and WWTP with coagulation-flocculation and UV disinfection (CoFlUV) units were investigated over a year, seasonally in terms of their G. intestinalis and C. parvum removal capacities. The seasonal abundances of these protozoa-specific genes in both the influents and effluents of each WWTP were determined by qPCR. The reduction of protozoan rDNA copies in the effluent wastewater samples compared with the influent wastewater samples was assessed as log10 reduction values (LRVs). LRVs >3 were reachable for C. parvum in all types of WWTPs tested. However, only LRVs 1-2 were reachable for G. intestinalis in CAS, SBR, CoFlUV, and MBR. Significant seasonal variations were just observed in SBR and CAS for G. intestinalis and C. parvum (p < 0.05), respectively. The findings depicted that WWTPs tested disseminated more giardiasis causative agents than cryptosporidiosis. Therefore, G. intestinalis needs to be monitored in WWTPs' discharges to reduce any potential damage of this parasite to public health. PRACTITIONER POINTS: Removal of G. intestinalis and C. parvum in WWTPs was affected by the process. LRV 2.92 was the highest LRV achieved for G. intestinalis. LRV >3 was reachable for C. parvum. WWTPs discharges disseminated more G. intestinalis than C. parvum. WWTPs effluents should be monitored in terms of G. intestinalis.

Influence of Selected Factors on the Survival Assessment and Detection of Giardia intestinalis DNA in Axenic Culture.

Giardia intestinalis is one of the most common food-borne protozoa. The sensitivity of pathogens to physical and chemical factors is the basis for developing measures to reduce the incidence of the population. Several methods are available to detect the presence of G. intestinalis. The study determines the influence of 22 selected factors on the survival assessment and detection of G. intestinalis DNA in trophozoites axenically cultured. The influence of a given factor on the test result was observed in the case of 17 factors (77.3%) in the microscopic method, while only in the case of 3 (13.6%) substances in the real-time PCR method. Prevention of G. intestinalis infections, e.g., by ensuring food and water safety, is a crucial issue affecting public health. The experiment was conducted on trophozoites as the first approach. It is necessary to continue research and observe the epidemiological situation. In future studies, the impact of the studied factors on the survival assessment and detection of Giardia intestinalis DNA in axenically cultured cysts should be determined.

Synergistic Effect of Polysaccharides from Chamomile Tea with Nitazoxanide Increases Treatment Efficacy against Giardia intestinalis.

Giardia intestinalis (syn. G. lamblia, G. duodenalis) is a protozoa parasite that produces one of the most frequent waterborne causes of diarrhea worldwide. This protozoan infects most mammals, including humans, and colonizes the small intestine, adhering to intestinal cells. The mechanism by which G. intestinalis causes diarrhea is multifactorial, causing intestinal malabsorption. The treatment of giardiasis uses chemotherapeutic drugs such as nitroimidazoles, furazolidone, paromomycin, and benzimidazole compounds. However, they are toxic, refractory, and may generate resistance. To increase efficacy, a current treatment strategy is to combine these drugs with other compounds, such as polysaccharides. Several studies have shown that polysaccharides have gastroprotective effects. Polysaccharides are high-molecular weight polymers, and they differ in structure and functions, being widely extracted from vegetables and fruits. In the present study, we show that polysaccharides found in chamomile tea (called MRW), in contact with antiparasitic agents, potentially inhibit the adhesion of parasites to intestinal cells. Moreover, at 500 µg/mL, they act synergistically with nitazoxanide (NTZ), increasing its effectiveness and decreasing the drug dose needed for giardiasis treatment.

Parasitic infections among patients hospitalized in the Tropical and Parasitic Clinic of Poznan University of Medical Sciences, Poland between 2015 and 2018. Is there a relationship between protozoa infection and gastrointestinal symptoms?

Introduction: Diarrhoea is a common reason for hospitalization among travellers returning from the tropics. The aetiology is predominantly bacterial, but it can also be caused by parasites such as Giardia intestinalis, Cryptosporidium spp., and Blastocystis spp. Aim: We analysed patients from the Poznan Tropical and Parasitic Clinic to evaluate the presence of parasitic infections and to find correlations between infections, journeys, and gastrointestinal symptoms. Material and methods: In our study we examined 2561 stool samples obtained from patients hospitalized in the Tropical and Parasitic Department of Poznan Medical University, Poland. Microscopic examinations of samples were performed based on a direct thin smear in 0.9% NaCl, which allowed the assessment of the presence of protozoa life stages. Results: In 106 (4.14%) of the 2561 examined samples we detected parasites, mainly from people coming back from tropical areas (61.32%). Mostly we detected Blastocystis sp. and Giardia intestinalis. Fifty percent of patients suffered from gastrointestinal symptoms, so careful microscopic stool examination should be performed in every case in which intestinal pathology occurs, and certainly in travelling individuals. Conclusions: Traveling is a real risk factor for protozoa infection. The most common parasites detected in the stool are Blastocystis sp. and Giardia intestinalis. Parasitic coinfection should be taken into consideration as a pathologic agent in patients suffering from abdominal signs and persistent diarrhoea. Prolonged protozoa infection and its role in microbiota alterations requires further investigation.

Do Intestinal Unicellular Parasites Have a Role in the Inflammatory and Redox Status among the Severely Obese?

The diagnosis of obesity comprises subjects with totally different phenotypes and metabolic profiles. Systemic inflammation and oxidative stress derived from the white adipose tissue are suggested as the link between this disease and the development of insulin resistance and metabolic comorbidities. The presence of unicellular eukaryotic parasites colonizing the human gut ecosystem is a common circumstance, and yet their influence on the inflammatory and redox status of the obese host has not been assessed. Herein, a set of inflammatory and redox biomarkers were assessed together with a parasitological analysis of 97 severely obese subjects. Information was also collected on insulin resistance and on the antioxidant composition of the diet. The global prevalence of intestinal unicellular parasites was 49.5%, with Blastocystis sp. the most prevalent protozoan found (42.3%). Colonized subjects displayed a higher total antioxidant capacity and a trend towards higher extracellular superoxide dismutase activity, regardless of their insulin resistance status, along with lower reduced glutathione/oxidized glutathione (GSH/GSSG) ratios in plasma in the insulin-resistant subgroup. No changes in malondialdehyde levels, or in inflammatory cytokines in plasma, were found in regard to the colonization status. In conclusion, enteric eukaryotic unicellular parasites may play an important role in modulating the antioxidant defenses of an obese host, thus could have beneficial effects with respect to the development of systemic metabolic disorders.

Flavonoids from Piper Species as Promising Antiprotozoal Agents against Giardia intestinalis. Structure-Activity Relationship and Drug-Likeness Studies.

Diarrhea diseases caused by the intestinal protozoan parasite Giardia intestinalis are a major global health burden. Moreover, there is an ongoing need for novel anti-Giardia drugs due to drawbacks with currently available treatments. This paper reports on the isolation and structural elucidation of six new flavonoids (1-6), along with twenty-three known ones (7-29) from the Piper species. Their structures were established by spectroscopic and spectrometric techniques. Flavonoids were tested for in vitro antiprotozoal activity against Giardia intestinalis trophozoites. In addition, structure-activity relationship (SAR) and in silico ADME studies were performed to understand the pharmacophore and pharmacokinetic properties of these natural compounds. Eight flavonoids from this series exhibited remarkable activity in the micromolar range. Moreover, compound 4 was identified as having a 40-fold greater antiparasitic effect (IC50 61.0 nM) than the clinical reference drug, metronidazole (IC50 2.5 µM). This antiprotozoal potency was coupled with an excellent selectivity index (SI 233) on murine macrophages and in silico drug-likeness. SAR studies revealed that the substitution patterns, type of functional group, and flavonoid skeleton played an essential role in the activity. These findings highlight flavonoid 4 as a promising candidate to develop new drugs for the treatment of Giardia infections.

Investigation of the Precence of Entamoeba histolytica, Giardia intestinalis and Cryptosporidium spp. in Patients Who Undergone Endoscopy and Colonoscopy

Objective: In this study, it was aimed to investigate the presence of Entamoeba histolytica, Giardia intestinalis and Cryptosporidium spp. in the samples taken during the procedure from patients who underwent endoscopy and/or colonoscopy with different prediagnoses, and in the stools of the same patients, by ELISA and direct microscopy methods. Methods: A total of 88 patients' endoscopic and colonoscopic pre-washed materials, which consisted of 49 individuals who belong former group and 39 individuals to the next group, were, respectively, obtained, and the stool samples were also included to study from the same group. All the specimens were immediately transferred to the parasitology research laboratory within the same day and stored C until for the next step of ELISA applications. Results: All the samples were examined by direct microscopy and ELISA method. In the examinations performed using the ELISA method; E. histolytica was detected in 2 (2.3%) stool samples, and G. intestinalis was found in 4 (4.5%) stool samples. In the colonoscopic wash/swab samples of the patients who underwent colonoscopy, 6 (6.8%) G. intestinalis, 1 (1.1%) Cryptosporidium spp. detected. No parasites were detected by ELISA in any of the stool samples or endoscopic washing/swab samples of the patients who underwent colonoscopy. No parasites were detected in stool and wash/swab samples by the direct examination method. When the incidence of G. intestinalis in washing/swab samples taken from patients who underwent endoscopy and colonoscopy was statistically compared, the difference was found to be significant (p<0.05). When the incidence of G. intestinalis in the stools of patients who underwent endoscopy was compared, the difference between genders was found to be significant (p<0.05). Conclusion: In patients with gastrointestinal complaints and undergoing endoscopy and colonoscopy, investigation of the presence of parasites by stool examination with direct microscopy may be insufficient. In addition to the direct examination of the stool sample, it is thought that the investigation of parasite antigens in the wash/swab materials that can be easily taken during the endoscopy and colonoscopy procedure is necessary and critical in the diagnosis.