Molecular diagnosis of echinococcosis in patients based on frozen paraffin tissue samples and fixed formalin and hydatid cysts isolated from livestock in a slaughterhouse.

Background: Various genotypes of Echinococcus granulosus have been studied in high-disease-risk areas and identified as causative agents of cystic echinococcosis (CE). This study was performed to examine and identify the molecular hydatid cyst in the dissected human specimens in paraffin tissue, and the dissected animal cyst was characterized using the DNA polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of internal transcribed spacer 1 (ITS1). Materials and Methods: To determine the molecular properties of E. granulosus, 20 hydatid cyst samples (including 6 sheep samples, 9 camel samples, and 10 human paraffin samples) were collected from Zahedan and Zabol cities. After DNA extraction, molecular PCR was performed, and RFLP was evaluated. In this study, the Taq1 endonuclease cleavage enzyme was used. Results: The patterns of DNA bands found in the isolates from human CE and animal bladder cysts were the same, as indicated by the results of ribosomal DNA-ITS1 amplification from E. granulosus. Two nested primer pairs were used. The rough size of the enhanced ITS1 piece was 444 and 391 base pairs (bp), individually. After cutting the PCR product with the Taq1 enzyme, the patterns of the fragments revealed that the samples had two identical RFLP patterns. The aftereffects of this study showed that the parasite genotypes confined to sheep, camels, and people had hereditary changes. Conclusion: The transcendent type of E. granulosus sensu lato in the area is E. granulosus sensu stricto, which featured the meaning of the sheep/canine cycle in human transmission. Albeit the band profile in the camel is now and again like the sheep strain, RLFP can be recognized utilizing the PCR strategy, and two differentiating band profiles using the chemical were found in this review.

Iranian Hydatid Disease Registry: Establishment and Implementation of a Neglected Tropical Disease Registry.

BACKGROUND: Cystic echinococcosis (CE) or hydatid disease is a global public health concern which imposes considerable economic costs on the communities in endemic regions. CE surveillance data are not adequately reliable. The present study reports the development and outcomes of a CE registry in Iran. METHODS: Hydatid Registry (HydatidReg) was initially established as a single-center registry in 2014 after the ethical approval of KMU. Following a call from MoHME to promote registry of different diseases and health outcomes, a call for participation was announced and all the Iranian Universities of Medical Sciences were requested to contribute to the registry. Subsequently, a nation-wide registry of hydatid disease was established in 2016. With a global perspective, HydatidReg joined the European Register of Cystic Echinococcosis (ERCE). A data collection form based on minimum dataset was designed and standard operating procedures (SOPs) were prepared to ensure standardized patient enrolment in the registry. A biobank system with two-dimensional barcoding was established along with HydatidReg for management and organization of biological specimens. RESULTS: As of March 2021, a total of 690 patients were enrolled in the registry. HydatidReg registered 362 (17.3%) out of the total 2097 patients enrolled in ERCE. Quality control (QC) of the data demonstrated 91.2% completeness and 80% timeliness. In the biobank, 322 biological specimens from 184 CE patients have been deposited including 70 blood, 96 sera and 156 parasite materials. CONCLUSION: High-quality data in the HydatidReg registry provided opportunities for health professionals to improve quality of care and organize meaningful research.

Evaluation of G6PD deficiency in malaria patients in the south-east of Iran.

Glucose phosphate dehydrogenasis (G6PD), the most prevalent enzymatic disease in humans, exists in south-eastern Iran. The geographic correlation of its distribution with the historic malaria endemic suggests that G6PD has increased in frequency as a result of natural selection by malaria. Based on studies, there is a controversy in terms of different analytical methods in terms of resistance to malaria. Fifty malaria patients and 50 healthy individuals from several cities south-east of Iran were included in the study and after obtaining consent, blood samples were taken from them. G6PD enzyme deficiency was investigated using a fluorescent stain test. The age, gender, and nationality of malaria patients were also assessed. The results were analyzed using SPSS software and appropriate statistical tests, and the value of P<0.05 was considered significant. The results showed that in malaria patients only one person had G6PD deficiency, while this number was six in the control group, which is significantly higher than the malaria group (P<0.05). Age group 27-42 years, men and people with Iranian citizenship also showed the highest incidence of malaria. Based on the results, it can be concluded that G6PD enzyme deficiency causes resistance to malaria and the frequency of this enzyme deficiency in malaria patients is significantly lower than in other people.

Identification and Phylogenetic Classification of Fasciola species Isolated from Sheep and Cattle by PCR-RFLP in Zabol, in Sistan and Baluchistan Province, Southeast Iran.

BACKGROUND: The detection of Fasciola species in various geographical regions is essential for health policymaking. Here, we aimed to identify livestock (cattle and sheep) related Fasciola genotypes by restriction fragment length polymorphism PCR. METHODS: Seventy adult Fasciola flukes were collected from 70 infected livers of 35 cattle and 35 sheep slaughtered in Zabol abattoir, south-east Iran (Jan-Jul 2017). Fasciola species were determined based on molecular features. For molecular detection, Fasciola ITS1 region was amplified and sequenced. A 700 bp fragment was amplified. These were digested with RasΙ enzyme. F. hepatica specific fragments were 47, 59, 68, 104, and 370, while those related to F. gigantica had 45, 55, 170, 370. RESULTS: The two main species of F. hepatica and F. gigantica are responsible for fasciolosis in sheep and cattle in our region. From 35 Fasciola isolated from cattle, 3 and 32 were F. hepatica and F. giagantica respectively. From 35 Fasciola isolated from sheep, 4 were F. hepatica and 31 were F. gigantica. CONCLUSION: All Seventy Fasciola samples from two different hosts (cattle and sheep) were identified as either F. hepatica or F. gigantica by PCR-RFLP. Genotypic variability of Fasciola species was high in our region. It is recommended to assess molecular variation of Fasciola isolates in other host livestock.

Seroepidemiology of Toxocara canis in Children under 14 Years Referring to Laboratories of Sistan and Baluchestan Province in Southeast of Iran.

BACKGROUND: The aim of the present survey was to assess thr seroepidemiologic and parasitological aspects of Toxocara canis infection in children under 14 yr old. METHODS: Overall, 963 sera were collected from children in the Sistan and Baluchistan Province, Southeast of Iran during the period from Sep 2015 to Jun 2016. IgG antibody against T. canis in the subjects' sera was evaluated using the commercial ELISA kit. RESULTS: Anti-Toxocara IgG were detected in the serum of 17 (1.7%) of the participants. In the examined children, the highest presence of anti-Toxocara antibodies was 2.1% (9/418) in 6-10-yr olds, which was higher than other age groups (P<0.05). Anti-Toxocara antibodies were significantly higher in males (2.4% or 12/492) than in females (1.1% or 5/471) (P<0.03). Highest serological prevalence of T. canis occurred in tribes (5.5% or 4/69), followed by rural areas (0.9% or 7/757), while in the urban area it was 0.1% (6/163) (P<0.01). A significant association was seen between the serological prevalence of T. canis and laboratory findings such as eosinophilia (P=0.001) and red blood cell count (P=0.02). CONCLUSION: Seroprevalence of Toxocara infection is high among children living in the poor regions of southeast Iran.

Prevalence and histopathology of Sarcocystosis in slaughtered carcasses in southeast Iran.

OBJECTIVE: Sarcocystis spp. are common parasites and in terms of economics and pathogenicity in domestic animals is important. The purpose of this work was to define the rate of contamination of slaughtered carcasses of cattle to Sarcocystis using digestive and histopathological methods in southeast Iran. MATERIAL AND METHODS: In this descriptive laboratory study for 1 year, 500 carcasses were examined and isolated bradyzoites of Sarcocystis with the digest method. Also, tissue samples from the esophagus and diaphragm were considered for pathologic studies and stained with hematoxylin and eosin of sections of histopathological. RESULTS: The results showed that the highest contaminations were in imported male animals aged 2-3 years old in the spring. There was a significant difference (p < 0.05) in the prevalence rate with the sex and race of cattle but no significant difference (p > 0.05) in the prevalence rate with age and season. CONCLUSION: Infection with Sarcocystis is common in oxen in this region. The imported cattle are more infected. It seems that racing and the environmental condition affect the prevalence of Sarcocystosis.

Epidemiological Study of Toxocar canis in Children under 14-Years-Old and Dogs in Zabol and Chabahar Districts, Southeast of Iran.

BACKGROUND: The purpose of this study was seroepidemiological and parasitological assessment of Toxocara canis infection in children and dogs in Zabol and Chabahar, Iran. METHODS: This study was a descriptive-analytic study with a simple random sampling of children under 14 yr old, referring to urban, rural, and tribal laboratories of Zabol and Chabahar, Sistan and Baluchestan Province, Iran in 2016. Demographic data, clinical, and laboratory conditions of patients were collected through interviews, questionnaires, and blood count measuring. The prevalence of IgG antibodies against T. canis was assessed by ELISA. T. canis eggs in dogs (as the original host) were also assessed by examining animal feces. Then the data were analyzed using SPSS 19 software and descriptive statistics, chi-square and ANOVA statistical tests. RESULTS: Totally, 364 patients were enrolled, of which 51.6% were female and mean±SD age of participants was 7.2 (±3.7) yr. IgG antibodies against T. canis was observed in 3.8% of cases. A significant association was found between the seroprevalence of T. canis and eosinophil (P=0.003) and red blood cell count (P=0.04). We also found a significant association between serological prevalence of T. canis and demographic parameters, such as city of residence (P=0.003), gender (P=0.04), consumption of vegetables (P=0.01), and the living place (P=0.04). Mean antibody titration was 2.2 ±1.1, with statistically significant difference among age groups (P=0.001). In addition, T. canis infection was positive in 27.5% of dogs living in the study areas. CONCLUSION: High risk of infection represented in patients referring to laboratories of Zabol and Chabahar. In addition, given the fact that dogs are the final hosts to transfer Toxocara infection to humans, this study emphasizes the need to control the population of stray dogs in the region to prevent the development of disease in the human society.

Identification of Acanthamoeba Genotypes in Pools and Stagnant Water in Ponds in Sistan Region in Southeast Iran.

OBJECTIVE: Acanthamoeba is one of the most abundant free-living amoebas that is widely distributed in natural and artificial environment resources. Acanthamoeba pathogenic genotypes cause chronic human diseases including amoebic keratitis and granulomatous amoebic encephalitis. The aim of this study was to determine and identify Acanthamoeba genotypes residing in pools and stagnant water in ponds in Sistan region in southeast Iran. This descriptive study was conducted at the Parasitology Laboratory, School of Medicine, Zabol University of Medical Sciences. METHODS: In this descriptive study, 93 water samples were collected from pools and ponds in Zabol, Zahak, Hirmand, Hamoon, and Nimrooz in Sistan region. Samples after filtering through 0.45-µm nitrocellulose paper filters were cultured in a 1.5% non-nutrient agar medium enriched with heat-killed Escherichia coli. Polymerase chain reaction (PCR) was conducted using specialized primers for detecting the genus Acanthamoeba. The sequencing of positive samples was used for determining Acanthamoeba genotypes. RESULTS: From 82 free-living amoeba positive culture samples, 38 isolates were confirmed to belong to the genus Acanthamoeba by PCR. On sequencing, 34 samples (89.47%) belonged to the T4 genotype, three (7.9%) to the T5 genotype, and one (2.63%) to the T3 genotype. CONCLUSION: All genotypes found in this study are potentially pathogenic. The T4 genotype is the main genotype of Acanthamoeba responsible for amoebic keratitis. Resource water is a potential risk factor for the distribution of free-living amoeba. Therefore, more attention of health authorities to determine, training and prevention from infection are recommended.

Study of Genetic Variation of Leishmania major Based on Internal Transcribed Spacer 1 (ITS1) in Chabahar, Iran.

BACKGROUND: Zoonotic cutaneous leishmaniasis (ZCL) is polymorphic disease that may show various clinical manifestations. OBJECTIVES: This study investigates the determination of genetic variation within the species of Leishmania major isolates from new cases in Chabahar, a port city in Southeast Iran (situated at the Iran-Pakistan border). Migration in this region indicates that leishmaniasis is spreading gradually, and a new micro-habitat focus appears each year. MATERIALS AND METHODS: A variety of nucleic acid detection methods that target both DNA and RNA have been developed. The restriction fragment length polymorphism analysis of amplified internal transcribed spacer 1 with polymerase chain reaction (ITS1-RFLP PCR) assay is a multipurpose tool for the diagnosis of Leishmania from clinical samples and for enabling the determination of the infecting Leishmania species. The goal of this study was the identification of species based on ITS1-RFLP in the ribosomal operon of L. major from clinically different forms of ZCL amplified by PCR, followed by the digestion of the PCR product with restriction enzymes. The profiles were observed and visualized in agarose gel under UV light. We used direct smears to identify the parasites. While taking the smear, samples were collected for culture or direct PCR. We used the PCR-RFLP assay of the ITS1 genes for direct identification of Leishmania species in 24 out of 33 suspected patients. PCR-ITS1 amplification was done on the 24 samples confirmed by culture via growth and parasitological methods. RESULTS: Of the 24 isolates, 21 had 350 bp bands (87.5%) and three had 450 bp bands (12.5%). After using the restriction enzyme, banding patterns including fragments of 210 and 140 bp for L. major were detected in 19 cases. CONCLUSIONS: The L. major species causing ZCL in Chabahar have limited genetic variation. There seems to be little manifestation of diversity between these lesions as a new focus of disease, and new micro-habitats for the disease are appearing in parts of this region.

Leishmania major: Genetic Profiles of the Parasites Isolated from Chabahar, Southeastern Iran by PPIP-PCR.

BACKGROUND: Leishmaniasis is important vector-borne parasitic disease worldwide, caused by the genus Leishmania. The objective of the current study was to identify genetic polymorphism in L. major, one of the species causing cutaneous leishmaniasis (CL), isolated from southeastern Iran, using Permissively Primed Intergenic Polymorphic-Polymerase Chain Reaction (PPIP-PCR) method. METHODS: Overall, 340 patients with suspected CL were examined. They referred to the Central Laboratory in Chabahar, Iran during Apr 2013 to Feb 2014. Microscopic examination of Giemsa-stained slides from lesions as well as aspirates cultured in Novy- Mac Neal-Nicolle (NNN) Media was employed in order to diagnose CL in these patients. Our analyses detected 86 suspected subjects as having CL from which 35 isolates were cultured successfully. PPIP-PCR method was performed on extracted genomic DNA from selected isolates in order to determine the genetic polymorphism among L. major isolates. RESULTS: The electrophoresis patterns demonstrated two genetic profiles including A or A1 patterns between all samples tested. Frequency of A and A1 sub-types were 33 (94.3%) and two (5.7%), respectively. CONCLUSION: Both host and parasite factors may contribute to the clinical profile of human leishmaniasis in the endemic foci of the disease. Here we showed that genetic variations pertaining to the Leishmania parasites might determine, in part, the clinical outcomes of human leishmaniasis.

Species Diversity and Distribution of Ticks (Acari: Ixodidae) in Zabol County, Eastern Iran.

BACKGROUND: Ticks are important vectors of human and animal pathogens. They are considered as main vectors for transmission of many viral, bacterial, rickettsial and parasitical pathogens. The aim of the present study was to find out species diversity of ticks, which infested the domestic ruminants in Zabol County, Eastern Iran in 2012. METHODS: Ticks were selected randomly from sheep, goats, cattle and camels. The ticks were collected from the body of infested animals and stored in 70% ethanol, then transported to the laboratory of Zabol University of Medical Sciences. Following examinations under stereomicroscope, ticks were identified using available taxonomic keys. RESULTS: In this study, a total number of 469 adult ticks (381 males and 88 females) were collected. Ticks were classified into 2 genera and 9 species including: Hyalomma dromedarii (17.3%), Hy. schulzei (1.8%), Hy. marginatum (0.5%), Hy. anatolicum excavatum (12.60%), Hy. anatolicum anatolicum (11.2%), Hy. asiaticum asiaticum (11.0%), Rhipicephalus sanguineus (21.2%), Rh. bursa (10.2%) and Rh. turacunis (13.911%). The frequency of genus Hyalomma (54.6%) was higher than Rhipicephalus. Rh. sanguineus was the predominant tick species and accounted for 21.26% of the ticks. The ratio of males was more than the female ticks. CONCLUSION: Hyalomma and Rhipicephalus species are commonly distributed in the study area. Further investigations are needed to identify the role of above tick species as vectors of pathogenic organisms.

Genetic polymorphism of Leishmania major in two hyper endemic regions of Iran revealed by PPIP-PCR and ITS- RFLP.

BACKGROUND: Zoonotic cutaneous leishmaniasis (ZCL) is a polymorphic disease that may show various clinical manifestations. Although it is suggested that the genetic variability of the parasite is one of the factors influencing clinical manifestations of leishmaniasis, no data exists regarding genetic polymorphism of Leishmania major (L.major). This study investigates the determination of genetic variations within the species of L.major isolates from different cases of ZCL in two hyper-endemic areas of Iran. METHODS: A variety of nucleic acid detection methods that target both DNA and RNA have been developed. Among these, the polymerase chain reaction (PCR) method proved to be a highly sensitive and specific technique. Species identification was based on permissively primed intergenic polymorphic-polymerase chain reaction (PPIP-PCR) and restriction fragment length polymorphism analysis of amplified internal transcribed spacer (ITS-RFLP) in the ribosomal operon of L.major from clinically different forms of ZCL. The DNA products were amplified by PCR, followed by digestion of the PCR product with restriction enzymes. The profiles were visualized in agarose gel under ultraviolet (UV) light. RESULTS: The PCR product obtained for all isolates was about 1060 bp in size. Different patterns of PPIP-PCR and ITS-RFLP in the ribosomal operon were classified as I, II, III, IV, and V. This classification was according to the number and localization of bands. Results of this research detected the genetic and clinical polymorphism of L. major, and showed that strain A was more frequent than other strains. CONCLUSION: The L.major causing ZCL in Isfahan, Iran is genetically a highly polymorphic species and PPIP-PCR exposed more genetic polymorphism among clinical samples in Isfahan, Iran.