Identification and genotyping of Echinococcus granulosus from human clinical samples in Guilan province, north of Iran.

Cystic echinococcosis (CE) is a significant health problem in both human and veterinary medicine. It is caused by the tapeworm Echinococcus granulosus (E. granulosus). The objective of this study was to investigate molecular diversity of E. granulosus from the paraffin-embedded human (FFPE) tissue samples using sequencing of mitochondrial genes. Thirty-five FFPE tissue samples were collected from different regions of Guilan province, north of Iran. Demographic data were recorded using a questionnaire. Five sections (1 mm) of the tissue were prepared and deparaffined using xylene and ethanol methods. Molecular analysis was performed using the Nad1 and Cox1 genes using PCR and DNA sequencing. Totally, 25 cases (71.43%) were women and 10 cases (28.57%) were men. The most affected age group was 21-30 yr old. The most of cysts were isolated from the liver (n = 19; 54.29%) and others in the lung (n = 16; 45.71%). The Cox1 and Nad1 genes were successfully amplified in 16 (45.71%) and 12 (34.28%) DNA samples from FFPE tissue. Sequencing analysis revealed that all samples were E. granulosus sensu stricto complex (G1 and G3). In this study, E. granulosus sensu stricto complex G1 and G3 were identified in human hydatid cysts and showed the presence of sheep/dog cycle in human infection. This finding confirmed and completed previous studies on the geospatial distribution of E. granulosus sensu stricto complex G1 and G3 in the southern and coastal areas of the Caspian Sea region.

Assessment of species distribution and virulence factors of oral fungal carriage among hospitalized patients with COVID-19: a case-control study.

Background: The COVID-19 pandemic highlighted the need to study oral fungal carriage and its potential impact. In oral fungal environments, factors like changes in respiratory epithelium, increased pathogen attachment, local inflammation, and virulence factors could influence COVID-19 severity. The authors conducted a study to explore oral fungal carriage in COVID-19 patients and compare it to a healthy control group. Methods: The authors executed a case-control investigation including 144 COVID-19 patients and an equivalent number of 144 healthy controls. The matching criteria encompassed age, sex, body mass index, and the history of antibiotic and antiviral medication intake. This research was performed over a span of 12 months from May 2021 to May 2022. The mouth area was sampled with a cotton-tipped swab. Subsequently, all the samples underwent fungal culture and PCR-sequencing procedures. Results: In COVID-19 patients, oral fungal carriage was three times higher compared to healthy controls. Candida was the exclusive genus found in both groups, with Candida albicans being the most frequently isolated species (90.79%). Among COVID-19 patients, Candida species showed significantly higher esterase, proteinase, and hemolysin activity compared to healthy individuals. Both groups exhibited elevated levels of C. albicans virulence factors compared to non-albicans species. Conclusions: It is crucial to understand the way that virulence factors of oral fungal carriage act in COVID-19 patients in order to come up with novel antifungal medications, identify the contributing factors to drug resistance, and manage clinical outcomes.

Characterisation of extracellular vesicles isolated from hydatid cyst fluid and evaluation of immunomodulatory effects on human monocytes.

Hydatidosis is a disease caused by the larval stage of Echinococcus granulosus, which involves several organs of intermediate hosts. Evidence suggests a communication between hydatid cyst (HC) and hosts via extracellular vesicles. However, a little is known about the communication between EVs derived from HC fluid (HCF) and host cells. In the current study, EVs were isolated using differential centrifugation from sheep HCF and characterized by western blot, electron microscope and size distribution analysis. The uptake of EVs by human monocyte cell line (THP-1) was evaluated. The effects of EVs on the expression levels of pro- and anti-inflammatory cytokines were investigated using quantitative real-time PCR (RT-PCR), 3 and 24 h after incubation. Moreover, the cytokine level of IL-10 was evaluated in supernatant of THP-1 cell line at 3 and 24 h. EVs were successfully isolated and showed spherical shape with size distribution at 130.6 nm. After 3 h, the expression levels of pro-inflammatory cytokine genes (IL1Β, IL15 and IL8) were upregulated, while after 24 h, the expression levels of pro-inflammatory cytokines were decreased and IL13 gene expression showed upregulation. A statistically significant increase was seen in the levels of IL-10 after 24 h. The main mechanism of the communication between EVs derived from HCF and their host remains unclear; however, time-dependent anti-inflammatory effects in our study suggest that HC may modulate the immune responses via EVs.

Formulation of Neem oil-loaded solid lipid nanoparticles and evaluation of its anti-Toxoplasma activity.

BACKGROUND: Toxoplasmosis is caused by an intracellular zoonotic protozoan, Toxoplasma gondii, which could be lethal in immunocompromised patients. This study aimed to synthesize Neem oil-loaded solid lipid nanoparticles (NeO-SLNs) and to evaluate the anti-Toxoplasma activity of this component. METHODS: The NeO-SLNs were constructed using double emulsification method, and their shape and size distribution were evaluated using transmission electron microscope (TEM) and dynamic light scattering (DLS), respectively. An MTT assay was employed to evaluate the cell toxicity of the component. The anti-Toxoplasma activity of NeO-SLNs was investigated using vital (trypan-blue) staining. Anti-intracellular Toxoplasma activity of NeO-SLNs was evaluated in T. gondii-infected Vero cells. RESULTS: The TEM analysis represented round shape NeO-SLNs with clear and stable margins. DLS analysis showed a mean particle size 337.6 nm for SLNs, and most of nanoparticles were in range 30 to 120 nm. The cell toxicity of NeO-SLNs was directly correlated with the concentration of the component (P-value = 0.0013). The concentration of NeO-SLNs, which was toxic for at least 50% of alive T. gondii (cytotoxic concentration (CC50)), was > 10 mg/mL. The ability of NeO-SLNs to kill Toxoplasma was concentration-dependent (P-value < 0.0001), and all concentrations killed at least 70% of alive tachyzoites. Furthermore, the viability of T. gondii- infected Vero cells was inversely correlated with NeO-SLNs concentrations (P-value = 0.0317), and in the concentration 100 µg/mL at least 75% of T. gondii- infected Vero cells remained alive. CONCLUSIONS: Overall, our findings demonstrated that the NeO-SLNs was able to kill T. gondii tachyzoites in concentration 100 µg/mL with a cell toxicity lower than 20%. Such results suggest that employing SLNs as carrier for NeO can effectively kill T. gondii tachyzoites with acceptable cell toxicity. Our findings also showed that SLNs capsulation of the NeO can lead to prolonged release of the extract, suggesting that NeO-SLNs could be also employed to clear cyst stages, which should be further investigated in animal models.

Acute eosinophilic appendicitis caused by Taenia saginata: A case report.

INTRODUCTION: The role of parasites in the pathogenesis of appendicitis has been debated for a long time. To date, several gastrointestinal parasites have been reported as the causes of appendicitis in humans. Taenia infestation of the appendix is uncommon and few cases have been reported in the literature. PRESENTATION OF CASE: We reported a case of acute eosinophilic appendicitis (AEA) in a 42-year-old woman caused by T. saginata in northern Iran. The patient was admitted to the emergency department with a 2-day history of acute abdominal pain in her lower right quadrant. Abdominal ultrasonography showed intra-abdominal bleeding and endometrium cysts. Routine hematological tests showed increases in white blood cell (WBC) count of 19.8 × 103 per mcL with 3% eosinophilia. During abdominal laparotomy, peritoneal fluid was bulked with abdominal bleeding due to rupture of the uterine cyst. After investigation of inflammation in the appendix region, patient underwent appendectomy. Histopathological findings showed acute inflammation with eosinophils and a large number of round eggs with flattened segments of the genus Taenia. It is impossible to distinguish between T. saginata and T. solium based solely on egg morphology in the specimens. Therefore, based on history of the patient, which included no consumption of pork, the species was identified as T. saginata. At the three months follow-up, the patient was in good health. CONCLUSION: In the current study, a case of AEA by T. saginata was reported. However, this was not the first case of acute appendicitis by T. saginata. Further studies are necessary to show roles of parasites in pathogenesis of AEA.

Strongyloides stercoralis and other intestinal parasites in patients receiving immunosuppressive drugs in northern Iran: a closer look at risk factors.

OBJECTIVES: The objective of this study was to evaluate the prevalence of Strongyloides stercoralis and other intestinal parasites in patients receiving immunosuppressive drugs in northern Iran and to investigate related risk factors. METHODS: This cross-sectional study was conducted among 494 patients receiving immunosuppressive drugs, including cancer patients undergoing chemotherapy (n=188) and those treated with prolonged corticosteroid administration (n=306). All fresh fecal samples were examined using the direct wet-mount, formalin ethyl acetate concentration, and agar plate culture techniques. RESULTS: In total, 16.8% of patients were positive for at least 1 intestinal parasite; the helminthic and protozoan infection rates were 5.1% and 12.3%, respectively. The infection rate was significantly higher in corticosteroid-treated individuals (19.6%) than cancer patients (12.2%) (p<0.05). The prevalence rate of S. stercoralis among patients receiving chemotherapy and those treated with corticosteroids were 4.3% and 5.2%, respectively. The prevalence rate of S. stercoralis infection was significantly higher in older patients (p<0.05). CONCLUSIONS: Strongyloidiasis is one of the most common parasites among patients receiving immunosuppressive drugs in northern Iran. Early diagnosis and proper treatment of these patients are necessary to minimize the complications of severe strongyloidiasis.

Genetic Characterization of Echinococcus granulosus Sensu Lato in Livestock and Human Isolates from North of Iran Indicates the Presence of E. ortleppi in Cattle.

PURPOSE: Identification of different genotypes of echinococcal cyst in various domestic herbivores and humans within the target area was the principal aim of the present study, performed using sequence data of cox1 and nad1 mitochondrial genes. METHODS: A total of 57 cystic echinococcosis (CE) cysts were isolated from indigenous livestock including 45 cattle, 9 sheep and 3 goats from several slaughterhouses in Guilan Province. Moreover, 12 formalin-fixed paraffin-embedded (FFPE) CE cyst tissues from humans were also included, obtained from the archives of several hospitals in Rasht, the capital of Guilan. Genetic sequencing was conducted using mitochondrial cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunit 1 (nad1) genes. RESULTS: Our results found that E. granulosus sensu stricto (s.s.) and E. ortleppi were present in 92.7% and 7.2% isolates, respectively. E. granulosus s.s. (genotypes G1 and G3) and E. ortleppi were isolated from various livestock whereas all CE cysts isolated from humans were E. granulosus s.s. G1 genotype. CONCLUSION: We found that E. granulosus s.s. G1 was the predominant genotype within the study region. This is the first study to report E. ortleppi in cattle in Iran.

Molecular Characterization of Echinococcus granulosus Sensu Stricto Isolated from the Livestock of Qazvin, Iran.

INTRODUCTION: Hydatidiosis is a serious parasitic disease in humans and livestock, worldwide. Echinococcus granulosus shows notable genetic variation among intermediate hosts. Several genotypes of the worm have been reported from different parts of Iran, but no information on the parasite genotypes status in the study region is available. The current study investigated the presence of different genotypes of E. granulosus in the livestock of Qazvin, Iran, by sequencing the mitochondrial Cox1 genes. METHODOLOGY: One hundred twenty E. granulosus isolates, including 30 from goats, 40 from cattle and 50 from sheep, were collected from the slaughterhouses in Qazvin province. Mitochondrial Cox1 gene region was amplified by PCR and 30 isolates were sequenced. Phylogenetic analysis was performed by using the MEGA 7.0 software. Morphological analysis was performed on rostellar hook length of protoscoleces. RESULTS: All isolates were identified as E. granulosus sensu stricto (G1-G3 complex) among 17% of the isolates clarified as G3 genotypes. G1 was the predominant genotype among the specimens. No significant difference between the rostellar hooks measurements of different genotypes was observed. CONCLUSION: Our findings confirmed the presence of E. granulosus sensu stricto in the region, although further studies are required to determine the haplotype diversity of E. granulosus using different mitochondrial and nuclear genes.

Molecular epidemiology of Enterocytozoon bieneusi and Encephalitozoon sp., among immunocompromised and immunocompetent subjects in Iran.

Intestinal microsporidiosis is known as an opportunistic infection in immunocompromised patients. The current study aimed to investigate intestinal microsporidia infection in human subjects with/without immunodeficiency. Totally, 600 stool samples were collected from immunocompromised (254) and immunocompetent (346) subjects. DNA extraction was performed and the SSU rRNA and the ITS genes were amplified to detect and characterize microsporidia and the relevant genotypes. Phylogenetic trees were drawn using MEGA7 software to illustrate the correlation between isolates. From 600 enrolled subjects, 283 and 317 were male and female, respectively. The average age ± SD of all tested subjects was 28.85 ± 26.92. The results of PCR demonstrated the presence of E. bieneusi and Encephalitozoon sp., among 10/600 (1.67%) and 26/600 (4.33%) of samples, respectively. Accordingly, E. bieneusi was seen among 4/346 (1.15%), 1/53 (1.88%), 3/124 (2.42%), and 2/63 (3.17%), and Encephalitozoon sp., was detected from 17/346 (4.91%), 3/53 (5.36%), 4/124 (3.22%) and 2/63 (3.17%) of healthy subjects, RA patients, cancer patients, and transplantation recipients, respectively. Statistical significant correlation was not seen between the presence of microsporidia and age, gender, stool appearance, and geographical region. Molecular analysis showed that all E. bieneusi were the genotype D. Phylogenetic tree demonstrated no classification according to the presence/absence of immunodeficiency, geographical locations and presence of diarrhea. The high prevalence of Encephalitozoon sp., in comparison to E. bieneusi in this study suggested the importance of this genus alongside with E. bieneusi in Iran. In addition, predominance of the genotype D highlighted the wide distribution of this genotype in Iran.

Echinococcus multilocularis and Echinococcus granulosus in canines in North-Khorasan Province, northeastern Iran, identified using morphology and genetic characterization of mitochondrial DNA.

BACKGROUND: Canids are definitive hosts of Echinococcus multilocularis and Echinococcus granulosus. This study aimed to survey these two Echinococcus species in canids of North-Khorasan Province, northeastern Iran, using morphological criteria and genetic characterization of mitochondrial DNA. METHODS: The carcasses of 106 canids, namely 61 jackals (Canis aureus), 23 foxes (Vulpes vulpes), 19 dogs (Canis familiaris) and three wolves (Canis lupus) were collected from the study area in 2013-2014 and examined for Echinococcus species. Morphological features were assessed by microscopy of adult worms. For molecular characterization, DNA was extracted, mostly from the adult worms but also from eggs. DNA fragments of the cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunit 1 (nad1) mitochondrial genes were amplified and sequenced. Sequences were aligned and compared with reference sequences. Intraspecific and interspecific diversity were calculated and phylogenetic analysis was performed. RESULTS: Overall, 9.4% of the canids (eight jackals and two foxes) were found infected with E. multilocularis by molecular methods, of which seven cases were also confirmed using morphological description of the adult worms. Echinococcus granulosus was found in 6.6% of the canines (four dogs, two jackals and one wolf) as determined by both molecular methods and adult cestode morphology. All E. granulosus isolates were identified as the G1 genotype. Comparative sequence analysis indicated 0-0.7% and 0% intraspecific divergence within E. granulosus isolates and 0% and 0-0.2% within E. multilocularis isolates for cox1 and nad1, respectively. CONCLUSIONS: This study revealed the presence of E. multilocularis and E. granulosus in canids of North-Khorasan Province of Iran. Jackals were found infected with both E. multilocularis and E. granulosus, but infection with the former species was higher.

Small-scale risk assessment of transmission of parasites from wastewater treatment plant to downstream vegetable farms.

AIM: The aim of the present study was to simultaneously investigate parasitic contamination of treated wastewater and downstream vegetable farms that are irrigated with treated sewage, during a year. BACKGROUND: (Oo) Cysts and eggs of parasites are resistant to most of routine wastewater treatment process. Irrigation of vegetables farms with either treated wastewater or illegally use of raw wastewaters enhances the risk of contamination with enteric pathogens. METHODS: The treated wastewater samples were taken after chlorination from a wastewater treatment plant located at the south of Tehran. In addition, 60 vegetable samples (5 samples from each farm) were collected from the selected downstream farms that routinely used treated wastewater for irrigation of crops. Parasitological tests were performed using Ziehl-Neelsen, conventional lugol's iodine staining and direct microscopical examination. RESULTS: Parasites including free living larvae, eggs of Toxoascaris leonina, egg of Toxocara sp. Trichuris sp, Trichostrongylus sp and amoeboid trophozoite were seen in 5/12 (41.7%) of vegetable samples gathered during a year. There was no statistically significant correlation between the season and parasitic contamination of the vegetables (P= 1). Furthermore, parasitic contamination was observed in 7/12 (53.8%) of treated wastewater samples. The correlation between season and parasitic contamination of treated wastewater was evaluated that the results showed a higher contamination of treated wastewater in spring and autumn (P<0.05). Fisher's exact test also showed that there was no significant correlation between parasitic contaminations of vegetable samples and treated wastewater according to seasonal change. CONCLUSION: The results showed parasites in both treated wastewater plant and downstream crops farms that suggests the public health importance of the quality of water resources that routinely used for irrigation of vegetable farms.

Pathogenic assays of acanthamoeba belonging to the t4 genotype.

BACKGROUND: Acanthamoeba genus is introduced as opportunistic and cosmopolitan parasite. Monkey and wistar rat are appropriate models for experimental study on Acanthamoeba infection. In this study Acanthamoeba spp. were isolated from hot spring (HS), windows dust (WD) and a corneal sample of keratitis patient (KP) and their pathogenicity surveyed by in vitro and in vivo tests. METHODS: Isolates of Acanthamoeba were cultivated axenically for 12 months in PYG medium. Overall, 30 wistar rats, in 6 equal groups were used for developing experimental Acanthamoeba keratitis (AK) and Granulomatous Amoebic Encephalitis (GAE). The Keratitis and Granulomatous Encephalitis experiments were performed by intrastromal and intranasal inoculation of Acanthamoeba cysts, respectively. Pathogenicity of the three isolates was also evaluated by in vitro test using osmotolerance and temperature tolerance assays. Identification of genotypes were performed by PCR technique and sequencing. RESULT: None of the isolates could perform AK and GAE in wistar rats, although all isolates were described as T4 genotype. Isolates obtained from KP and WD could grow only in 30 °C, but not in 37 °C and 40 °C. On the other hand, HS isolate grew in 30 °C and 37 °C but not in 40 °C. Moreover, all of isolate grew in 0.5 M mannitol but not in 1 M and 1.5 M. CONCLUSION: T4 isolates with a long-term axenic culture and different factors related to host and parasite may play role in pathogenicity of these free-living amoebae.