Evaluation of structural changes of Echinococcus granulosus protoscoleces following exposure to different protoscolicidal solutions evaluated by differential interference contrast microscopy.

The present study was aimed to assess the structural changes in protoscoleces of Echinococcus granulosus sensu stricto following exposure to different natural and chemical protoscolicidal agents using differential interference contrast (DIC)/Nomarski microscopy. Protoscoleces of sheep's liver cysts were collected aseptically. Individually, about 1000 protoscoleces were exposed to 0.5% silver nitrate, 20% hypertonic saline solution, 0.5% cetrimide solution and two different concentrations of garlic chloroformic extraction as well as phosphate-buffered saline (PBS). The protoscoleces viability was assessed using 0.1% eosin solution, and structural modifications in the protoscoleces were examined by DIC/Nomarski microscopy. The results revealed the degeneration of the tegument, disorganization of the hooks, and reduction of the size of the protoscoleces exposed to cetrimide, hypertonic sodium chloride, and silver nitrate. Furthermore, calcareous corpuscles became blurred and opaque and their numbers decreased in all the exposed samples except, those in PBS. The exposed protoscoleces to cetrimide and hypertonic sodium chloride solution showed extensive degeneration of the tegument and disorganization of the hooks. In the group exposed to 200 mg/ml chloroformic garlic extract, the protoscoleces' width decreased. The length, width, and number of calcareous corpuscles also decreased significantly in the silver nitrate-exposed protoscoleces. The study concludes that protoscoleces exposed to different solutions; cetrimide 0.5% and hypertonic sodium chloride 20% caused more pronounced structural changes in the exposed protoscoleces. These changes were well demonstrated by DIC microscopy and can be used as a supplementary tool to evaluate the effects of protoscolicidal agents. Supplementary Information: The online version contains supplementary material available at 10.1007/s12639-023-01632-4.

Comparison of Native Hydatid Cyst Fluid (HCF), Lyophilized HCF, Antigen B (AgB) and Lyophilized AgB (LAgB) Originated from Echinococcus granulosus Sensu Stricto for Sero-Diagnosis of Active, Transitional and Inactive Human Liver Cystic Echinococcosis.

Background: Cystic echinococcosis (CE) is an important zoonotic parasitic disease caused by the larval stage or metacestode of the tapeworm Echinococcus granulosus sensu lato. Due to treatment protocols for different liver cysts, diagnosis of cyst stages is very important. Different antigens have been used for CE diagnosis. However, each one is more sensitive and effective for the diagnosis of specific CE stages is not known well. We aimed to compare Native Hydatid Cyst Fluid (HCF), Lyophilized Hydatid Cyst Fluid (LHCF), antigen B (AgB) and Lyophilized antigen B (LAgB) originated from E. granulosus sensu stricto (G1-G3) genotype, for sero- diagnosis of active, transitional and inactive human liver CE using ELISA technique. Methods: The HCF was collected aseptically from liver CE cysts of sheep slaughtered from 2018 to 2019 in Shiraz slaughterhouse, Southern, Iran. The cysts were characterized by PCR and sequencing for genotype specification. Four types of antigens were used: HCF, LHCF, AgB and LAgB originated from E. granulosus sensu stricto (G1-G3) genotype. Thirty-three serum samples from active, transitional, and inactive human cysts were collected. Overall, 48 samples from other parasitic diseases and 60 samples from healthy subjects as negative controls were checked using four antigens by ELISA method. Results: The best diagnostic sensitivity with 96.97% was observed by anti-LHCF IgG ELISA test. The best specificity with 95.37% was observed in ELISA test using LAgB. Conclusion: Simultaneous test of sera with anti-LHCF IgG ELISA and anti-LAgB IgG ELISA would be the best in the diagnosis of human liver cystic echinococcosis.

Application and evaluation of native antigen B from Echinococcus granulosus sensu stricto and E. canadensis alone or mixture for serodiagnosis of human G1-G3 and G6/G7 genotypes cystic echinococcosis sera, using ELISA and Western blotting.

Cystic echinococcosis (CE) is one of the most important helminthic diseases in the world with different genotypes distribution. The application of specific genotype antigens together with sera from patients with specific cyst genotypes have not been reported, so far. The present study aimed to apply and evaluate native AgB from Echinococcus granulosus sensu stricto (Eg) and Echinococcus canadensis (Ec) alone or mixture for serodiagnosis of human G1-G3 and G6/G7 genotypes cystic echinococcosis sera, using ELISA and Western blotting. A total of 47 human sera along with 47 human CE cysts were collected. CE genotypes were determined. Native AgB were prepared from E. granulosus s.s and E. canadensis genotypes. ELISA and Western blot were performed on human specific G1-G3 and G6/G7 genotypes sera. Species specific native AgB were used alone or mixed. The sensitivity of ELISA using alone and mixed 1Eg-1Ec, 1Eg-2Ec, and 2Eg-1Ec of native AgB from E. granulosus s.s and E. canadensis genotypes for human G1-G3 sera were 92.10, 89.47, 97.37, 100, and 100%, respectively; while using AgBs, alone and mixed for human G6/G7 sera were 100%. The sensitivity of Western blotting using native AgB of E. granulosus s.s and E. canadensis genotypes alone and mixed 2Eg-1Ec were 78.95% and 100% for human G1-G3 and G6/G7 genotypes sera, respectively. The mixture of AgB from Echinoccus granulosus sensu stricto and Echinococcus canadensis genotypes increased ELISA sensitivity for the diagnosis of human CE. Preparation and application of native AgB from specific and prevalent genotypes of CE in endemic regions is recommended.

Comparison of Isoenzyme Pattern of Echinococcus granulosus sensu stricto (G1-G3) and E. canadensis (G6/G7) Protoscoleces

Background: Different genotypes of Echinococcus granulosus sensu lato (s.l.) infect humans and ungulate animals, causing cystic echinococcosis. Simultaneous isoenzyme, as well as molecular characterizations of this parasite, has not yet been investigated in Iran. The present study aimed to evaluate the isoenzyme pattern of the E. granulosus sensu stricto (s.s.) and E. canadensis genotypes in Iran. Methods: A total of 32 (8 humans and 24 animals) cystic echinococcosis cysts were isolated from Shiraz, Tehran, Ilam, and Birjand from May 2018 to December 2020. The DNAs were extracted and their genotypes were determined by molecular methods. Enzymes were extracted from the cysts and subjected to polyacrylamide gel electrophoresis. The activities of glucose-6-phosphate sehydrogenase (G6PD), malate dehydrogenase (MDH), malic enzyme (ME), nucleoside hydrolyse 1 (NH1), and isocitrate dehydrogenase (ICD) were examined in the cyst samples using isoenzyme method and compared it with the genotyping findings. Results: DNA sequence analysis of the samples showed that the specimens contained 75% E. granulosus s.s. (G1) and 25% E. canadensis (G6) genotypes. The isoenzyme pattern of ICD in both genotypes produced a six-band pattern with different relative factors. The G6PD also produced two bands with different relative migrations in both genotypes. The MDH and NH1 systems revealed a two-band pattern, while only one band was generated in the ME enzyme in the E. granulosus s.s. genotype. In the E. canadensis, the MDH and NH1 enzymes showed one band, and the ME enzyme represented a two-band pattern. Conclusion: Our findings suggest that E. granulosus s.s. and E. canadensis genotypes have entirely different isoenzyme patterns for NH1, G6PD, MDH, and ME.

Cystic Echinococcosis/Hydatid Cyst Coinfection with HIV: A Report from Shiraz, Iran.

HIV coinfected with other parasitic diseases may cause a serious problem for the patients. A few case reports describing echinococcosis with human immunodeficiency virus (HIV) infection have been reported in the world; however, it has not been reported in Iran, so far. Here, the first case of liver hydatid cyst coinfected with HIV in Iran is reported. The patient is a 46-year-old female HIV-positive based on the laboratory report. Her clinical symptoms included abdominal pain, abdominal enlargement, and anorexia. Ultrasound showed three large hepatic hydatid cysts with hundreds of daughter cysts. Ultrasonography of the cyst revealed it as a CE2 stage according to the WHO classification. The patient went under complete anesthesia followed by complete cyst removal by surgery. Observation of the hydatid cyst fluid using eosin 0.1% revealed more than 70% viable protoscoleces. Histopathology examination, polymerase chain reaction (PCR), and viable protoscoleces confirmed the diagnosis of echinococcosis. The IgG ELISA test with native AgB for E. granulosus infection was also positive. mtDNA amplification using PCR and sequencing showed the cyst as E. granulosus sensu stricto genotype. Our observations show that huge, large, and high-pressure cysts with hundreds of daughter cysts are difficult to be completely removed, and drug treatment has not been able to reduce their size. Therefore, in HIV coinfection with hydatid cyst, surgery is preferable to other treatments.

Comparative genotyping of Blastocystis infecting cattle and human in the south of Iran.

BACKGROUND: Blastocystis is a unicellular protozoan and one of the most common parasites found in humans and many animals' intestinal tract. The present study aimed to compare the genotypes of Blastocystis infecting cattle and humans in the south of Iran. METHODS: A total of 100 human stool samples and 75 cattle stool samples were microscopically examined for Blastocystis infection. DNA was extracted from thirty-eight microscopically positive samples (13 humans and 25 cattle). PCR was performed on positive samples targeting the Blastocystis-specific SSU rDNA gene. PCR products of eight humans and eleven cattle samples were sequenced and compared with available reference sequences in GenBank by BLAST queries. Genetic diversity was measured for Blastocystis subtypes in human and cattle, based on haplotype and nucleotide diversities. RESULTS: The PCR detected Blastocystis in ten humans and twenty-four cattle samples. Blastocystis subtypes 1, 2, and 6 were found in humans whereas subtypes 5 and 10 were found in cattle. Subtype (ST) 2 was the most predominant subtypes in humans whereas, in cattle specimens, the ST5 was the most dominant subtype. Based on the Blastocystis sequences of SSU rDNA, 68 sites were polymorphic and 49 sites were parsimony informative, resulting in the identification of 15 haplotypes, 10 haplotypes in the cattle and 5 in humans. No haplotype was shared between cattle and human parasites. CONCLUSION: Human-derived Blastocystis subtypes were different from cattle subtypes in southern Iran. Nevertheless, subtype 5 in cattle can be a risk factor for human infection.

Triclabendazole (Anthelmintic Drug) Effects on the Excretory- Secretory Proteome of Fasciola hepatica in Two Dimension Electrophoresis Gel.

BACKGROUND: The aim of this study was to evaluate the protein spots of excretory - secretory products of Fasciola hepatica using two dimension electrophoresis method in the presence and absence of triclabendazole drug which can be considered to detect the target protein of the drug. METHODS: F. hepatica parasites were collected from infected cattle livers, divided in two groups and cultivated in RPMI 1640 medium. First group was treated with triclabendazole (TCBZ) and second group considered as control. The excretory-secretory (ES) products of each group were separated and total protein determined by Bradford method. To provide proteome spots, the ES proteins were precipitated and two dimension electrophoresis (2-DE) gel prepared. Protein amounts of two groups were compared using the statistical t-test and protein spots from 2-DE in test and control groups were also statistically analyzed. The protein spots of gels were identified by using protein database. RESULTS: The t-test showed a significant increase of total proteins in treated group (P<0.5). The protein spots count in the control group was less than test group however statistically not significant (p>0.05). Cathepsin L- protein (MW 36.7 pH 5.34), 14-3-3 epsilon 2 isoform (MW 28.2 pH 5.36), Cathepsin L1D (MW 36.5 pH 5.8) and Cathepsin L1D (MW 36.6 pH 6.26) were identified in test group. CONCLUSION: It seems that, these results can be considered to determine the proteins which the drug acts as a target on them.