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.

Current status and future prospects of Echinococcus multilocularis vaccine candidates: A systematic review.

The larval stages of Echinococcus multilocularis (E. multilocularis) are what cause the zoonotic disease known as alveolar echinococcosis (AE). Identifying the antigens that trigger immune responses during infection is extremely important for the development of vaccines against Echinococcus infections. Several studies conducted in recent decades have described the specific traits of the protective antigens found in E. multilocularis and their role in immunizing different animal hosts. The objective of the current systematic review was to summarize the findings of relevant literature on this topic and unravel the most effective vaccine candidate antigens for future research. A comprehensive search was conducted across five databases, including ProQuest, PubMed, Scopus, ScienceDirect, and Web of Science, until March 1, 2023. Two reviewers autonomously conducted the screening and evaluation of data extraction and quality assessment. In the present study, a total of 41 papers matched the criteria for inclusion. The study findings indicate that the combination of Em14-3-3 and BCG is widely considered the most often employed antigens for E. multilocularis immunization. In addition, the study describes antigen delivery, measurement of immune responses, adjuvants, animal models, as well as routes and doses of vaccination. The research indicated that recombinant vaccines containing EMY162, EM95, and EmII/3-Em14-3-3 antigens and crude or purified antigens containing ribotan-formulated excretory/secretory antigens exhibited the most favorable outcomes and elicited protective immune responses.

Comparison of the Diagnostic Performance of Antigen B Purified from Sheep Hydatid Cyst Fluid (HCF) with Commercial ELISA Kit.

INTRODUCTION: Cystic echinococcosis (CE) is a zoonotic parasitic disease caused by the metacestode of Echinococcus granulosus. CE is a health problem in Middle Eastern countries, such as Iran. The purpose of this study was to purify subunit 8 KDa antigen B from crude sheep hydatid cyst fluid (HCF) and compare its sensitivity and specificity with a commercial human ELISA kit (PT-Hydatid-96). METHODS: 28 sera samples were collected from hydatid cyst patients who had surgery for a hydatid cyst and had their disease confirmed by pathology after the surgery. Furthermore, 35 samples of healthy individuals with no history of hydatid cysts were collected, as were nine serum samples from parasite-infected non-CE patients. HCF was obtained from sheep fertile cysts at a Sari slaughterhouse and used as an antigen. In an indirect ELISA test, the B antigen was employed, and the results were compared to those from a commercial ELISA kit. RESULTS: The results of this study were analyzed using the Kappa test. The commercial ELISA kit showed 17 cases (23.6%) positive, 44 cases (61.1%) negative, and 11 cases (15.3%) borderline. B antigen showed that 18 (25%), 43 (59.7 %), and 11 (15.3%) were positive, negative, and borderline, respectively. One sample (1.4% of 72 total samples) of 35 serum samples from healthy individuals was positive using B antigen-based ELISA. In addition, all nine serum samples from parasite-infected non-CE patients were negative for both tests. The sensitivity and specificity of the commercial ELISA kit have been evaluated at 60.7% and 100%, respectively. For B antigenbased ELISA, these values are 64.3 and 97.7%, respectively. CONCLUSION: Antigen B produced from hydatid cyst fluid is a promising option for serological identification of hydatid cysts in both infected and healthy individuals. In an indirect ELISA test, hydatid fluid antigen could be used as a precise source of detection.

Global status of intestinal parasitic infections among diabetic patients: A systematic review and meta-analysis.

Intestinal parasitic infections (IPIs) have been identified as a disease agent responsible for infections in immunocompromised patients such as diabetics. We searched six electronic databases and reviewed 38 related studies using the following keywords alone or in combination: "intestinal parasites", "diabetes", "immunocompromised", "prevalence", and "human."  The pooled prevalence of IPIs in diabetic patients was 24.4% worldwide. These patients with IPIs are advised to go to health centers and perform the relevant checkups with the advent of the first symptoms of the disease, such as diarrhea and abdominal pain. Moreover, early diagnosis and treatment of IPIs in diabetic patients are highly recommended to maintain quality of life.

Investigating intestinal parasitic infections with emphasis on molecular identification of Strongyloides stercoralis and Trichostrongylus colubriformis in north of Iran.

Currently, parasitic infections are one of the important health problems in the world, especially in developing countries. This study aims to investigate intestinal parasites with an emphasis on molecular identification through the analysis of mitochondrial COX1 and ITS2 gene sequences of Strongyloides stercoralis (S. stercoralis) and Trichostrongylus spp. in north of Iran. Five hundred forty stool samples were collected from medical diagnostic laboratories affiliated with Mazandaran University of Medical Sciences in Sari city, north of Iran. First, all the samples were examined using direct smear, formalin-ether sedimentation, and trichrome staining technique. Suspected samples of Strongyloides larvae were cultured in agar plate. Then, DNA was extracted from samples containing Trichostrongylus spp. eggs and Strongyloides larvae. To amplify DNA, PCR was performed and the samples with a sharp band in electrophoresis were sequenced by Sanger method. Overall, the prevalence of parasitic infections in the study population was 5.4%. The highest and the lowest level of infection was observed with Trichostrongylus spp. and S. stercoralis at 3% and 0.2%, respectively. No traces of live Strongyloides larvae were seen in the culture medium of the agar plate. The six isolates obtained from the amplification of the ITS2 gene of Trichostrongylus spp. were sequenced, all of which were Trichostrongylus colubriformis. The sequencing results of COX1 gene indicated S. stercoralis. In the present study, the prevalence of intestinal parasitic infections in north of Iran has relatively decreased that its main reason can be due to the coronavirus epidemic and compliance with health principles. However, the prevalence of Trichostrongylus parasite was relatively high that it requires special attention to apply appropriate control and treatment strategies in this field.

Global distribution of Echinococcus granulosus genotypes in domestic and wild canids: a systematic review and meta-analysis.

The current systematic review and meta-analysis demonstrate the genotypic distribution of canine echinococcosis worldwide. Studies published from the inception until 21 May 2021 were screened, relevant articles were selected and the random-effect model was used to draw forest plots with 95% confidence intervals (CIs). Totally, 44 articles were included, mostly examined dogs (37 records), followed by wolf (8 records), jackal (7 records), fox (3 records), pump fox (3 records) and coyote (1 record). Echinococcus granulosus sensu stricto (G1­G3) and G6/7 cluster of Echinococcus canadensis were the most common genotypes among canids. Most studies were conducted in Asia and Europe with 17 and 15 datasets, respectively. Exclusively, Iran possessed the highest number of studies (10 records). Meta-analysis showed that the pooled molecular prevalence of echinococcosis was 33.82% (95% CI 24.50­43.83%). Also, the highest and lowest prevalence of canine echinococcosis was calculated for South America (66.03%; 95% CI 25.67­95.85%) and Europe (19.01%; 95% CI 9.95­30.16%). Additionally, there were statistically significant differences between the global prevalence of echinococcosis in canines and publication year, continent, country, sample type, host and molecular test. These findings will elevate our knowledge on the poorly known canine echinococcosis worldwide.

Promising effects of parasite-derived compounds on tumor regression: a systematic review of in vitro and in vivo studies.

The parasites are repeatedly confronting their host to take advantage of nutrients for multiplication and survival. In this sense, a wide spectrum of molecules is released from both sides, with immune-regulatory activity, accompanying this biological battle. Such parasites and their valuable molecules can be directed toward microbial-based cancer therapy. Herein, we contrived a systematic review to gather information on the antitumor activity of parasite-derived compounds. Following systematic search in Web of Science, ScienceDirect, Scopus, PubMed, ProQuest and Embase until 31 December 2019, a total number of 51 articles (54 datasets) were finally included in this review. Thirteen parasitic agents were found to possess possible antitumor activity, comprising protozoan species Toxoplasma gondii, Trypanosoma cruzi, Trichomonas vaginalis, Acanthamoeba castellanii, Besnoitia jellisoni, Leishmania major, Plasmodium yoelii, and Plasmodium lophurae, as well as parasitic helminths Toxocara canis, Echinococcus granulosus, Taenia crassiceps, Trichinella spiralis, and Schistosoma mansoni. Most experiments were done based on antigenic preparations from T. gondii (16 studies), E. granulosus (10 studies), T. spiralis (8 studies), and T. cruzi (6 studies). Possible antitumor properties of the selected parasites were revealed in this review. However, precise molecular basis of anticancer activity for each parasite remains to be elucidated in the future.

Engineering a multi-epitope vaccine candidate against Leishmania infantum using comprehensive Immunoinformatics methods.

Visceral leishmaniasis (VL) is a severe disease with particular endemicity in over 80 countries worldwide. There is no approved human vaccine against VL in the market. This study was aimed at designing and evaluation of a multimeric vaccine candidate against Leishmania infantum through utilization of helper T lymphocyte (HTL) and cytotoxic T lymphocyte (CTL) immunodominant proteins from histone H1, KMP11, LACK and LeIF antigens. Top-ranked mouse MHC-I, MHC-II binders and CTL epitopes were predicted and joined together via spacers. Also, a TLR-4 agonist (RS-09 synthetic protein) and His-tag were added to the N- and C-terminal of the vaccine sequence, respectively. The final chimeric vaccine had a length of 184 amino acids with a molecular weight of 18.99 kDa. Physico-chemical features showed a soluble, highly-antigenic and non-allergenic candidate. Secondary and tertiary structures were predicted, and subsequent analyses confirmed the construct stability that was capable to properly interact with TLR-4/MD2 receptor. Immunoinformatics simulation displayed potent stimulation of T cell immune responses, with particular rise in IFN-γ, upon vaccination with the proposed multi-epitope candidate. In conclusion, immunoinformatics data demonstrated a highly antigenic vaccine candidate in mouse, which could develop considerable levels clearance mechanisms and other components of cellular immune profile, and can be directed for VL prophylactic purposes. Supplementary Information: The online version contains supplementary material available at 10.1007/s11756-021-00934-3.

Confirmed cases of human Onchocerca lupi infection: a systematic review of an emerging threat.

Diverse Onchocerca species are present mostly parasitizing ungulates, with the exception of Onchocerca volvulus (O. volvulus) in humans and O. lupi in canids and cats. The human cases due to the O. lupi have been more highlighted during last years. So, the present review was performed to determine the detailed characteristics of confirmed human O. lupi case reports documented worldwide. Hence, a systematic search was done using English international databases (Scopus, PubMed, Web of Science, Embase, ProQuest, and Google Scholar). Totally, 14 confirmed human cases were documented during the last decade, mostly from the USA and Turkey with 7 and 3 cases, respectively. Most cases (7 individuals) were male with the age range of 22-month-old to 54-year-old. The parasite was frequently isolated from the right eye (5 cases), followed by the left eye (4 cases), cervical spinal canal (3 cases), scalp, and right forearm (one case each). Molecular identification of the isolated agent was the preferred way of diagnosis in most cases (9 records). In conclusion, human O. lupi cases have been more highlighted in recent years, whether due to the improved diagnostics and/or host-switching phenomenon, and both veterinarians and healthcare authorities should be alerted.

In silico analysis and prediction of immunogenic epitopes for pre-erythrocytic proteins of the deadly Plasmodium falciparum.

Malaria is the deadliest parasitic disease in tropical and subtropical areas around the world, with considerable morbidity and mortality, particularly due to the life-threatening Plasmodium falciparum. The present in silico investigation was performed to reveal the biophysical characteristics and immunogenic epitopes of the six pre-erythrocytic proteins of the P. falciparum using comprehensive immunoinformatics approaches. For this aim, different web servers were employed to predict subcellular localization, antigenicity, allergenicity, solubility, physico-chemical properties, post-translational modification sites (PTMs), the presence of signal peptide and transmembrane domains. Moreover, the secondary and tertiary structures of the proteins were revealed followed by refinement and validations. Finally, NetCTL server was used to predict cytotoxic T-lymphocyte (CTL) epitopes, followed by subsequent screening in terms of antigenicity and immunogenicity. Also, IEDB server was utilized to predict helper T-lymphocyte (HTL) epitopes, followed by screening regarding interferon gamma induction and population coverage. These proteins showed appropriate antigenicity, abundant PTMs as well as many CTL and HTL epitopes, which could be directed for future vaccination studies in the context of multi-epitope vaccine design.

The global seroprevalence of Toxoplasma gondii infection in bovines: a systematic review and meta-analysis.

Bovines, especially cattle, are considered as one of the main sources of Toxoplasma gondii infection for humans. A more comprehensive understanding of the occurrence of T. gondii is needed to provide a global perspective on the prevalence of T. gondii in bovines. Here, we present the results of the first systematic review and meta-analysis on the global T. gondii seroprevalence in bovines. Six databases (PubMed, ScienceDirect, Web of Science, Scopus, ProQuest and Google Scholar) were comprehensively searched for relevant studies published between 1 January 1967 and 30 May 2019. Among 7691 publications searched, 178 studies (from 50 countries) with 193 datasets were included in the meta-analysis. The global pooled and weighted seroprevalence of T. gondii among bovines was 17.91% [95% confidence interval (CI): 15.32­20.6]. Weighted prevalence based on the host was as follows: cattle 16.94% (95% CI: 14.25­19.81), buffalo 22.26% (95% CI: 16.8­29), yak 23% (95% CI: 14­33) and bison 8.1% (95% CI: 3.9­13.7). Continued monitoring on the status of T. gondii seroprevalence in bovines is essential. Study on the prevalence of T. gondii in the products of bovines such as milk, meat, etc., which are considered as the source of transmission of infection to humans, is recommended.

Current situation and future prospects of Echinococcus granulosus vaccine candidates: A systematic review.

Cystic echinococcosis is a worldwide zoonotic disease, represents a threat for livestock and humans, manifests as a quiescent, subclinical and chronic hydatid cyst infection. The disease imposes high expenditures and economic losses in medical and veterinary. Prophylactic vaccination would be one of the effective preventive health care against echinococcosis. During the last decades, many studies have characterized the protective antigens of Echinococcus granulosus and their role in immunization of various animal host species. Herein, we aimed to systematically evaluate and represent the best antigens as possible vaccine candidates for cystic echinococcosis. Data were systematically searched from five databases including ProQuest, PubMed, Scopus, ScienceDirect and Web of Science, up to 1 February 2020. Two reviewers independently screened and assessed data extraction and quality assessment. A total of 47 articles were eligible for inclusion criteria in the current study. The most common antigens used for vaccination against E. granulosus were EG95 and antigen B. Freund's adjuvant and Quil A have been predominantly utilized. In addition, regarding the antigen delivery, animal models, measurement of immune responses and reduction in hydatid cyst have been discussed in the text. The data demonstrated that DNA vaccines with antigen B and recombinant protein vaccines based on EG95 antigen have the best results and elicited protective immune responses.

Global seroprevalence of Neospora spp. in horses and donkeys: A systematic review and meta-analysis.

Neospora infections due to Neospora caninum and N. hughesi are prevalent among equids of the world. The disease may affect reproductive and neurological consequences. The present systematic review and meta-analysis was performed to evaluate the global Neospora seroprevalence among horses and donkeys. A number of 57 out of 1575 total retrieved studies were included by searching the PubMed, Scopus, Science Direct, Web of Science and ProQuest. A total of 25,783 horses and 4377 donkeys were examined serologically, yielding a weighted seroprevalence of 13.46 % (95 % CI: 10.26 %-17.42 %) globally. Subgroup analysis revealed that there were statistically significant differences in the overall prevalence of Neospora spp. in the examined animals according to year, continent, WHO region, country, host, and diagnostic method. Age and sex in horses as well as sex in donkeys were significantly associated with the Neospora seroprevalence (P < 0.001). Our findings showed a relatively high exposure to Neospora spp. in horses and donkeys worldwide. Considering the possible economic impact, as well as the importance in animal health, more studies are needed to clarify the species causing equine neosporosis, correlate serological findings with clinical outcomes and assess the risk factors, in order to define adequate prevention and control strategies.

Global Status of Toxoplasma gondii Seroprevalence in Rodents: A Systematic Review and Meta-Analysis.

Toxoplasmosis is one of the most prevalent infections in humans and animals caused by the intracellular protozoan parasite Toxoplasma gondii (T. gondii). Rodents, as intermediate and reservoir hosts, play a key role in the maintenance and transmission of T. gondii. They can be contaminated and maintain the parasite in the form of cysts in their bodies, demonstrating an infection source for their offsprings, predators (particularly felids), and other animals. Therefore, the present systematic review and meta-analysis study was carried out to evaluate the global seroprevalence of T. gondii in these mammals. For achieving the purpose of the current study, six English databases (PubMed, Science Direct, Web of Science, Scopus, ProQuest, and Google Scholar) were systematically searched for related studies from 1970 to 2018. Finally, a total of 52,372 records were screened, 105 records including 26,221 rodents were incorporated in the present study. By random effect models, the overall seroprevalence was calculated at 6% (95% CI = 6-7%), with the highest amount was observed in Africa (24%) and South America (18%), and the lowest amount in Europe (1%). The subgroup data analysis by gender manifested that the prevalence of Immunoglobulin G antibodies did not differ between genders (P > 0.05). Due to the significant heterogeneity, meta-regression models were applied based on serological techniques and continental regions; however, the obtained values were not statistically significant (P = 0.480 and P = 0.295, respectively). The present study revealed a relatively low level of T. gondii seroprevalence in rodents; however, if they were the main food source for their predators, they would cause high transmission of T. gondii.

Sarcocystosis in Ruminants of Iran, as Neglected Food-Borne Disease: A Systematic Review and Meta-analysis.

PURPOSE: Sarcocystis is a zoonotic parasitic pathogen which endangers the safety of meat and meat products. This systematic review and meta-analysis aimed to evaluate the prevalence rate and status of Sarcocystis spp. in ruminants as important food sources in Iran. METHODS: Data were collected from papers indexed in five English language electronic databases (PubMed, Scopus, Web of Science, Science Direct, and Google Scholar) and four Persian electronic databases (IranMedex, SID, IranDoc, and Magiran) from January to April 2019. Papers were selected based on inclusion criteria. Data analysis was performed in StatsDirect statistical software, version 2.7.2. RESULTS: The searching process resulted in the identification of 73 studies. Data analyses revealed that the total prevalence (95% confidence intervals) of Sarcocystis spp. in Iranian ruminants was 74.40% (64.01-83.56). In addition, a significant association was also observed between sarcocystosis infection in Iranian ruminants and year, host, location, and diagnostic technique (P < 0.001). CONCLUSIONS: According to our data, the prevalence of Sarcocystis infection in ruminants is relatively high. High pathogenicity of some Sarcocystis spp. and the negative impact that the spread of some parasites among ruminants can have on human and animal health necessitate the direction of more attention toward monitoring, controlling, and preventing sarcocystosis.

Filarial worms: a systematic review and meta-analysis of diversity in animals from Iran with emphasis on human cases.

Current systematic review and meta-analysis demonstrate the prevalence reports of filariasis in animals in Iran along with human cases. Studies were screened, relevant papers were selected and the random-effect model was used by forest plot with 95% confidence interval (CI). Of 17 records of human case-reports, particularly from Khuzestan province (5 cases), Dirofilaria repens was the most detected parasite (10 cases) with higher involvement of the right eye (7 cases) than other organs. Eleven animal species were reported to be parasitised by filarioids in Iran. The prevalence of Dirofilaria immitis in canids was 14.69% (95% CI: 10.33-19.67), with highest rates (20.92%; 95% CI: 13.84-29.03) in free-ranging dogs. Male (10.07%; 95% CI: 5.10-16.47) and more than 1-year old (20.77%; 95% CI: 8.66-36.42) dogs were more likely to be found infected. The frequency of other filarioids of zoonotic interest was: Acanthocheilonema reconditum in dogs 2.15% (95% CI: 0.71-4.33), Dipetalonema evansi in camels 10.16% (95% CI: 4.73-17.34), Onchocerca cervicalis in horses 3.63% (95% CI: 1.44-6.75%) and Onchocerca fasciata 16.57% (95% CI: 10.12-24.24%) in camels. Still, our knowledge on parasitic filariae in Iran is limited and more investigation is needed in both human and animal populations.

Molecular Characterization of Trichomonas vaginalis Strains Based on Identifying Their Probable Variations in Asymptomatic Patients.

BACKGROUND: The aim of this study was to identify the Trichomonas vaginalis strains/haplotypes based on identifying their probable variations in asymptomatic patients referred to Tabriz health centers, northwestern Iran. METHODS: Sampling was taken from 50-suspected women to T. vaginalis in northwestern Iran. The obtained samples were smeared and cultured. Fifty DNA samples were extracted, amplified and identified by nested polymerase chain reaction and PCR-RFLP of actin gene using two endonuclease enzymes: MseI and RsaI. To reconfirm, the amplicons of actin gene were directly sequenced in order to identify the strains/haplotypes. RESULTS: PCR-RFLP patterns, sequencing and phylogenetic analyses revealed definitely the presence of the G (n=22; 73.4%) and E (n=8; 26.6%) strains. Multiple alignments findings of genotype G showed five haplotypes and two amino acid substitutions in codons 192 and 211 although, no remarkable unique haplotype was found in genotype E. CONCLUSION: The accurate identification of T. vaginalis strains based on discrimination of their unknown haplotypes particularly those which are impacted on protein translation should be considered in parasite status, drug resistance, mixed infection with HIV and monitoring of asymptomatic trichomoniasis in the region.

The first morphometric and phylogenetic perspective on molecular epidemiology of Echinococcus granulosus sensu lato in stray dogs in a hyperendemic Middle East focus, northwestern Iran.

BACKGROUND: Hydatidosis is considered to be a neglected cyclo-zoonotic disease in Middle East countries particularly northwestern Iran which is caused by metacestode of tapeworm Echinococcus granulosus sensu lato. Human hydatidosis is a high public health priority in the area, however there is little known from a morphometric and phylogenetic perspective on molecular epidemiology of adult Echinococcus spp. in Iranian stray dogs. METHODS: 80 dogs (38 males and 42 females) were collected during June 2013 to April 2014 in northwestern Iran. The isolated parasites from each dog were distinguished by morphometric keys including small, large hook length and blade length. Subsequently, isolates were confirmed by sequencing of mitochondrial cytochrome oxidase subunit 1 gene. RESULTS: 16 (8 males and 8 females) (Prevalence 20%) out of 80 dogs were infected to genus Echinococcus. With regard to demographic factors, the frequency of parasitism in both male, female adults and their age groups showed no difference (P > 0.05). The phylogenetic analyses of cox1 sequences firmly revealed the 13 sheep strains (G1), one buffalo strain (G3), one camel strain (G6) and one mixed infection. The findings of rostellar hook morphology show an intraspecies variation range among G1 isolates. However, hook measurements in Echinococcus derived from G1 (sheep strain) were not a significant difference from those G6 and G3 strains. Six unique haplotypes were identified containing a high range of diversity (Haplotype diversity 0.873 vs. Nucleotide diversity 0.02). CONCLUSIONS: First presence of camel strain (G6) in this region seems to indicate that potential intermediate hosts play a secondary role in the maintenance of camel-dog biology. Current findings have heightened our knowledge about determination of Echinococcus prevalence, strains of taxonomy and genotypic trait of parasite in Iranian stray dogs which will also help in the development of strategies for monitoring and control of infected stray dogs in the area.

Designing and conducting in silico analysis for identifying of Echinococcus spp. with discrimination of novel haplotypes: an approach to better understanding of parasite taxonomic.

The definitive identification of Echinococcus species is currently carried out by sequencing and phylogenetic strategies. However, the application of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) patterns is not broadly used as a result of heterogeneity traits of Echinococcus genome in different regions of the world. Therefore, designing and conducting a standardized pattern should indigenously be considered in under-studied areas. In this investigation, an in silico mapping was designed and developed for eight Echinococcus spp. on the basis of regional sequences in Iran and the world. The numbers of 60 Echinococcus isolates were collected from the liver and lungs of 15 human, 15 sheep, 15 cattle, and 15 camel cases in Semnan province, Central Iran. DNA samples were extracted and examined by polymerase chain reaction of ribosomal DNA (rDNA) internal transcribed spacer 1 (ITS1) and PCR-RFLP via Rsa1 endonuclease enzyme. Moreover, 15 amplicons of cytochrome oxidase 1 (Cox1) were directly sequenced in order to identify the strains/haplotypes. PCR-RFLP and phylogenetic analyses revealed firmly the presence of the G1 and G6 genotypes with heterogeneity (three novel haplotypes) of Cox1 gene although no other expected genotypes were found in the region. Finding shows that the identification of novel haplotypes along with discrimination of Echinococcus spp. through regional patterns can unambiguously illustrate the real taxonomic status of parasite in Central Iran.