Chitosan nanoparticles improve the effectivity of miltefosine against Acanthamoeba.

BACKGROUND: Acanthamoeba keratitis (AK) is a corneal sight-threatening infection caused by the free-living amoebae of the genus Acanthamoeba. Early and appropriate treatment significantly impacts visual outcomes. Mucoadhesive polymers such as chitosan are a potential strategy to prolong the residence time and bioavailability of the encapsulated drugs in the cornea. Regarding the recent administration of miltefosine (MF) for treating resistant AK, in the present study, we synthesized miltefosine-loaded chitosan nanoparticles (MF-CS-NPs) and evaluated them against Acanthamoeba. METHODOLOGY/PRINCIPAL FINDINGS: Chitosan nanoparticles (CNPs) were prepared using the ionic gelation method with negatively charged tripolyphosphate (TPP). The zeta-potential (ZP) and the particle size of MF-CS-NPs were 21.8±3.2 mV and 46.61±18.16 nm, respectively. The release profile of MF-CS-NPs indicated linearity with sustained drug release. The cytotoxicity of MF-CS-NPs on the Vero cell line was 2.67 and 1.64 times lower than free MF at 24 and 48 hours. This formulation exhibited no hemolytic activity in vitro and ocular irritation in rabbit eyes. The IC50 of MF-CS-NPs showed a significant reduction by 2.06 and 1.69-fold in trophozoites at 24 and 48 hours compared to free MF. Also, the MF-CS-NPs IC50 in the cysts form was slightly decreased by 1.26 and 1.21-fold at 24 and 48 hours compared to free MF. CONCLUSIONS: The MF-CS-NPs were more effective against the trophozoites and cysts than free MF. The nano-chitosan formulation was more effective on trophozoites than the cysts form. MF-CS-NPs reduced toxicity and improved the amoebicidal effect of MF. Nano-chitosan could be an ideal carrier that decreases the cytotoxicity of miltefosine. Further analysis in animal settings is needed to evaluate this nano-formulation for clinical ocular drug delivery.

Eimeria leuckarti in equid coprolites from the Sassanid Era (2nd-6th century CE) excavated in Chehrabad Salt Mine archaeological site, Iran.

OBJECTIVE: This study reports coccidian oocysts in an equid coprolite dated to the Sassanid Empire (2nd-6th century CE) recovered in Chehrabad Salt Mine archaeological site, Iran. METHODS: Between 2015 and 2017, an archaeoparasitological investigation led to the discovery of an equid coprolite in the Chehrabad Salt Mine archeological site, (Douzlakh), western Iran. Samples were rehydrated using trisodium phosphate solution and were examined by light microscopy. RESULTS: Seven oocysts of Eimeria leuckarti (Flesch, 1883) were identified; they were in various stages of sporulation. CONCLUSION: This is the first report of ancient coccidian oocysts from equids. The importance of this observation is discussed, and current knowledge of eimeriid oocysts at archaeological sites is reviewed. SIGNIFICANCE: The observations of E. leuckarti increases current knowledge of parasite biodiversity in ancient Iran when it rested along the Silk Road, a network of trade routes connecting the East and West that was central to economic, cultural, political, and religious interactions between these regions, and to livestock movement that could contribute to the transmission of the parasites from/to other regions. LIMITATIONS: The contextual information about animal species present in and around the Salt Mine during its working periods, including Achaemenid dynasty (6th to 4th century BCE) and Sassanid era (2nd to 6th century CE), is very limited and does not allow secure conclusions regarding the host origin of the coprolites. SUGGESTIONS FOR FURTHER RESEARCH: Application of molecular biology tools to identify the correct host origin of the coprolites and to detect more parasite species is advocated.

Gastrointestinal Parasites of Domestic Mammalian Hosts in Southeastern Iran.

Gastrointestinal parasites (GIP) are a major cause of disease and production loss in livestock. Some have zoonotic potential, so production animals can be a source of human infections. We describe the prevalence of GIP in domestic mammals in Southeastern Iran. Fresh fecal samples (n = 200) collected from cattle (n = 88), sheep (n = 50), goats (n = 23), camels (n = 30), donkeys (n = 5), horse (n = 1), and dogs (n = 3) were subjected to conventional coprological examination for the detection of protozoan (oo)cysts and helminth ova. Overall, 83% (166/200) of the samples were positive for one or more GIP. Helminths were found in dogs, donkeys, sheep (42%), camels (37%), goats (30%), and cattle (19%), but not in the horse. Protozoa were found in cattle (82%), goats (78%), sheep (60%), and camels (13%), but not in donkeys, dogs, or the horse. Lambs were 3.5 times more likely to be infected by protozoa than sheep (OR = 3.5, 95% CI: 1.05-11.66), whereas sheep were at higher odds of being infected by helminths than lambs (OR = 4.09, 95% CI: 1.06-16.59). This is the first study assessing the prevalence of GIP in domestic mammals in Southeastern Iran.

Genomic palaeoparasitology traced the occurrence of Taenia asiatica in ancient Iran (Sassanid Empire, 2th cent. CE-6th cent. CE).

Palaeoparasitology investigates parasitological infections in animals and humans of past distance by examining biological remains. Palaeofaeces (or coprolites) are biological remains that provide valuable information on the disease, diet, and population movements in ancient times. Today, advances in detecting ancient DNA have cast light on dark corners that microscopy could never reach. The archaeological site of the Chehrabad salt mine of Achaemenid (550-330 BC) and Sassanid (third-seventh century AD) provides remains of various biotic and abiotic samples, including animal coprolites, for multidisciplinary studies. In the present work, we investigated coprolites for helminth eggs and larvae by microscopy and traced their biological agents' DNA by Next Generation Sequencing. Our results revealed various helminths, including Taenia asiatica, the species introduced in the 1990s. Implementing advanced modern molecular techniques like NGS gives a paramount view of pathogenic agents in space and time.

Prevalence and associated risk factors of human intestinal parasitic infections: a population-based study in the southeast of Kerman province, southeastern Iran.

BACKGROUND: Determination of the prevalence and distribution pattern of intestinal parasites is a fundamental step to set up an effective control program to improve the health status. This study aimed to determine the prevalence of intestinal parasitic infections and associated risk factors among inhabitants of Rudbar-e Jonub county, southeast of Kerman province, southeastern Iran. METHODS: In this cross-sectional study, 861 stool specimens were collected from inhabitants of Rudbar-e Jonub county through a multistage cluster sampling method in 2018. The collected specimens were examined by parasitological methods including, direct wet-mounting (for the fresh specimens with a watery consistency), formalin-ethyl acetate sedimentation and agar plate culture. RESULTS: The prevalence of intestinal parasites was 34.2% (95% CI 30.1 to 38.2). The prevalence of protozoan parasites 32.3% (95% CI 28.4 to 36.5) was significantly higher than helminthic parasites 3.2% (95% CI 2.1 to 4.7). Blastocystis sp. (13.3%), Entamoeba coli (11.4%) and Giardia lamblia (10.6%) as protozoan parasite and Hymenolepis nana (2.4%) as helminthic parasite were the most common detected intestinal parasites in the study. Entamoeba histolytica/dispar (1.5%), Iodamoeba bütschlii (1.0%), Chilomastix mesnili (0.5%), Entamoeba hartmanni (0.4%), Enterobius vermicularis (0.3%) and Ascaris lambercoides (0.3%) were other detected parasites. Multiple logistic regression revealed a significant association of intestinal parasitic infections with source of drinking water and residency status (rural/urban). Multiple infections with 2 or 3 parasitic agents constituted 22.7% of 295 infected cases. CONCLUSIONS: This study revealed a high prevalence of intestinal protozoan infections among inhabitants of Rudbar-e Jonub county. Intestinal parasites especially protozoans remain a challenging public health problem wherever sanitation and health measures are limited in Iran.

Protein Detection of Excretory-Secretory Products and Somatic Extracts from Fasciola hepatica and F. gigantica Using Two-Dimensional Electrophoresis.

BACKGROUND: The aim of this research was to compare excretory-secretory and somatic extract materials of Fasciola hepatica and F. gigantica to detect protein maps of two species. METHODS: Twenty infected livers were collected from sheep in industrial slaughterhouse in Tehran, 2017-2019. Worms were detached from bile ducts, then recognized according to morphologic and morphometric criteria. After three times washing, worms were incubated in RPMI culture media and excretory-secretory products were collected. Worms were crushed and homogenized for preparation of somatic extract. Two Dimensional Electrophoresis gels were accomplished for both excretory-secretory material and somatic extracts. Gels were scanned with densitometer and analyzed with Same Spots software and protein spots were identified with Expasy database. RESULTS: For both excretory-secretory products and somatic extract, protein spots were appeared with two-dimensional electrophoresis technique. Quantitative analysis showed 40 and 28 protein spots for excretory-secretory of F. hepatica and F. gigantica respectively. For somatic extract 19 and 12 protein spots were recognized for F. hepatica and F. gigantica in that order. CONCLUSION: The rate of expression of some proteins were more in F. hepatica while expression of other proteins was high in F. gigantica. The expression of protease enzyme was higher in F. gigantica than F. hepatica. These data could be considered for biochemical differentiation of Fasciola species and subsequently to design and prepare of antigens for diagnosis/vaccine development.

Intestinal Protozoa in Domestic Cats (Carnivora: Felidae, Felis catus) in Northwestern Iran: A Cross-Sectional Study with Prevalent of Microsporidian and Coccidian Parasites.

BACKGROUND: In this study, some microsporidial and coccidian parasites were isolated from 103 domestic cats in the Meshkin Shahr area, northwestern Iran during the Jun 2014 to Jun 2015, and their genera were identified using parasitological methods with emphasis on their zoonotic importance. METHODS: One hundred and three fecal samples of domestic cats were collected and preserved in formalin (10%) and conserved in phosphate buffer saline solution, finally examined by microscopy after formalin-ether concentration and specific staining. Preservation in dichromate potassium (2.5%) was performed for all coccidian positive samples and then sporulated coccidian oocysts were investigated. RESULTS: The detected parasites were Isospora spp. 6/103(5.8%). Microsporidian spores were identified in 46/103 (44.6%) of all samples post-stained by the aniline blue staining method. CONCLUSION: Microsporidial infections were more prevalent in domestic cats. Further studies are needed in the identification of microsporidial spores isolated from infected cats.

Molecular Detection and Genotyping of Intestinal Microsporidia from Stray Dogs in Iran.

BACKGROUND: Microsporidia as one of the most important pathogens in veterinary and agricultural settings, have emerged in immunocompromised patients in Iran. To date, different Enterocytozoon bieneusi genotypes have been identified in humans and animals, supporting the possibility of zoonotic zoonosis transmission potential. The aim of this study was to evaluate the distribution of E. bieneusi genotypes among overpopulated stray dogs in vicinity of Tehran, the capital city of Iran. METHODS: Totally, 75 stool and 75 urine samples were obtained from 75 stray dogs during the time period from Mar 2015 to Oct 2015. DNA extraction was performed on all the samples and specific fragment of small subunit ribosomal RNA gene of E. bieneusi and Encephalitozoon spp. was amplified. Furthermore, specific primers targeting the internal transcribed spacer region of E. bieneusi were applied to determine the genotype of the microorganism. RESULTS: Microsporidia was detected in 5.3% of stool samples, while none of the urine samples was positive for microsporidia species. Overall, 440 bp fragment of E. bieneusi was amplified in all the samples and there was no amplification for Encephalitozoon spp. The results of sequencing of 410 bp fragment of internal transcribed spacer region showed that all the E. bieneusi were genotype D. CONCLUSION: E. bieneusi was the most prevalent microsporidian species in the stray dogs and all the positive isolates were characterized as genotype D.

Molecular characterization of bacterial, viral and fungal endosymbionts of Acanthamoeba isolates in keratitis patients of Iran.

Free-living amoebae belong to the genus Acanthamoeba; can feed on microbial population by phagocytosis, and with the capability to act as a reservoir and a vehicle of microorganisms to susceptible host. Therefore, the role of endosymbiosis in the pathogenesis of Acanthamoeba is complex and not fully understood. The aim of the present study was to identify bacterial, fungal, and human adenovirus (HADV) endosymbionts as well as evaluating the endosymbionts role of such organisms in the pathogenesis of Acanthamoeba in keratitis patients living in Iran. Fifteen Acanthamoeba (T4 genotype) isolates were recovered from corneal scrapes and contact lenses of patients with keratitis. Cloning and purification was performed for all isolate. Gram staining was performed to identify bacterial endosymbionts. DNA extraction, PCR, and nested PCR was set up to identify endosymbiont of amoeba. Evaluation of pathogenicity was conducted by osmo-tolerance and thermo-tolerance assays and cell culture, and then CPE (cytopathic effect) was survey. Statistical analysis was used between Acanthamoeba associated endosymbionts and Acanthamoeba without endosymbiont at 24, 48, 72, and 96 h. A p value < 0.05 was considered as significant, statistically. A total of 9 (60%) Acanthamoeba (T4 genotypes) isolates were successfully cloned for detecting microorganism endosymbionts. The only isolate negative for the presence of endosymbiont was ICS9. ICS7 (Pseudomonas aeruginosa, Aspergillus sp., and human adenovirus endosymbionts) and ICS2 (Escherichia coli endosymbiont) isolates were considered as Acanthamoeba associated endosymbionts. ICS7 and ICS2 isolates were highly pathogen whereas ICS9 isolate showed low pathogenicity in pathogenicity evaluated. Positive CPE for ICS7 and ICS2 isolates and negative CPE for ICS9 isolate were observed in cell culture. The average number of cells, trophozoites, and cysts among ICS7, ICS2, and ICS9 isolates at 24, 48, 72, and 96 h was significant. This is the first survey on microbial endosymbionts of Acanthamoeba in keratitis patients of Iran, and also the first report of Aspergillus sp, Achromobacter sp., Microbacterium sp., Brevibacillus sp, Brevundimonas sp and Mastadenovirus sp in Acanthamoeba as endosymbionts. Our study demonstrated that microbial endosymbionts can affect the pathogenicity of Acanthamoeba; however, further research is required to clarify the exact pattern of symbiosis, in order to modify treatment protocol.

Anti-Acanthamoeba Effects of Silver and Gold Nanoparticles and Contact Lenses Disinfection Solutions.

BACKGROUND: This study aimed to investigate the anti-Acanthamoeba effects of the most used marketed disinfecting solutions in Iran. Moreover, the efficacy of some nano-compounds was tested against pathogenic Acanthamoeba. METHODS: The present study was conducted in the School of Public Health, Tehran University of Medical sciences, Tehran, Iran during 2015-2016. Cysts of Acanthamoeba T4 genotype (7 × 104 /ml) mixed at the equal volume with contact lens solutions including Opti-free, Ginza, ReNu, Maxima, Light, and Cyclean for the recommended time by the manufacturers. Nano-silver and nano-gold compounds were also treated with the amoebae. Chlorhexidine 0.02% and normal saline were used as positive and negative controls, respectively. Dead and alive amoebae were determined using vital stain and suspension was cultured in non-nutrient agar. The entire process was repeated at least three times. RESULTS: In none of the solutions in the manufacturer's brochure recommended time, full cytotoxic effect was observed on the cysts of Acanthamoeba. Opti free express solution destroyed the cysts after 6 days. Nanosilver and nano-gold compounds showed no cytotoxic effect on the cysts of Acanthamoeba. CONCLUSION: None of the Nanoparticles compounds as well as contact lenses disinfecting solutions which studied was effective on Acanthamoeba cysts in the manufacturer's brochure recommended time. However, continuing study on Nano-silver and Nano-gold compounds to find effective ingredients against Acanthamoeba are highly recommended.

Synchronous Identification of Entamoeba histolytica, Giardia intestinalis, and Cryptosporidium spp. in Stool Samples Using a Multiplex PCR Assay.

BACKGROUND: Diarrheal disease annually causes 760000 deaths in children, and 1700 million new cases are reported each year worldwide. Among the parasites, Entamoeba histolytica, Giardia intestinalis, and Cryptosporidium spp. are the most important infectious agents leading to diarrhea. Clinical presentations due to these parasites are more or less similar, and microscopy is not as much as sensitive for the detection. The aim of this study was to set up and evaluate a Multiplex PCR Assay for Synchronous Identification of Entamoeba histolytica, Giardia intestinalis, and Cryptosporidium spp. in Stool Samples. METHODS: Samples were obtained from different sources such as culture media and patient stool samples. Primer pairs were designed using primer-BLAST, and for the extraction of DNA, the QIAamp DNA stool mini kit was used. The study was conducted in Tehran, Iran and completed in 2016. RESULTS: The current multiplex PCR assay for the detection of E. histolytica achieved sensitivity and specificity of 86.36% (95% CI: 65.09% to 97.09) and 95.74 % (95% CI: 85.46% to 99.48%), respectively. Sensitivity and specificity of the test for G. intestinalis was 90.91% (95% CI: 70.84% to 98.88%) and 95.74% (95%CI: 85.46% to 99.48%), respectively, and for the detection of Cryptosporidium, multiplex PCR showed a sensitivity of 90.91% (95% CI: 70.84% to 98.88%) and specificity of 95.74% (95%CI: 85.46% to 99.48%). CONCLUSION: Multiplex PCR in this study showed admissible sensitivity and specificity for the detection of E. histolytica, G. intestinalis, and Cryptosporidium spp. in fecal samples.

The Prevalence of Trichomoniasis in Women Referred to Clinical Centers in South of Tehran, Iran during 2015-2016.

BACKGROUND: This study aimed to estimate the prevalence of trichomoniasis infection among females in Tehran, Iran. METHODS: This study was conducted on 482 women referred to the 6 obstetrics and gynecology centers of Tehran during 2015-2016. Some information including education, occupation, and number of sexual partners was obtained and clinical signs and symptoms of the genital tract were diagnosed by clinical examination. Two swabs were collected from the posterior fornix of patients. Two laboratory techniques, wet mount, and culture were carried out. Finally, statistical analysis test was performed using SPSS software version 16.0. RESULTS: Age distribution of patients was 15-60 yr. Trichomonas vaginalis was detected in 2 out of 482 participants (0.41%). All of the infected individuals were married (0.43%) and they had unique sexual partner and all of them had clinical symptoms. Significant association was observed between incidence of T. vaginalis infection and educational levels (P= 0.03), occupation (P=0.006), clinical symptoms (P=0.001), marriage (P=0.006) and bacterial infection (P=0.018). CONCLUSION: The prevalence of trichomoniasis was low and its incidence was associated with several risk factors.

Detection of Pseudocyst Forms of Trichomonas muris in Rodents from Iran.

BACKGROUND: Trichomonas muris is one of the most common protozoa diagnosed in rodents. The trichomonads are generally described as presenting only trophozoite form while pseudocyst is another morphological form of trichomonads identified among gastrointestinal and genitourinary trichomonads. We identified and described different shapes of T. muris pseudocysts and trophozoite in stool samples were collected from rodents including Merinos persicus, Mus musculus and Cricetulus migratorius. METHODS: In this cross-sectional study, stool samples from 204 trapped rodents were collected from Meshkin Shahr during Mar to Dec 2014. Samples were preserved in formalin 10% and PVA solution and transferred to Department of Medical Protozoology and Mycology, School of Public Health, Tehran University of Medical Sciences. Formalin-ether concentration method was used for the samples. The slides were stained with tri-chrome staining method and observed under light microscope. RESULTS: The trophozoites were classified as T. muris based on size (18 to 24 µm), presence of three anterior flagella, recurrent flagellum, undulating membrane, and axostyle in direct examination and stained slides with trichrome staining method. Fifty-five out of 204 (27%) rodents were infected with T. muris in which 51(25%) samples pseudocysts form were observed. The spherical bodies of pseudocyst with almost 8 µm size, contained internalized flagella, an undulating membrane with recurrent flagellum, axostyle, and costa were seen. The pseudocysts were more prevalent than trophozoite form and pseudocysts were found with different shapes in this study. CONCLUSION: T. muris pseudocysts were found in stool samples of caught rodents for the first time in northwestern Iran.

Emerging Intestinal Microsporidia Infection in General Population in Jiroft District, Southeastern Iran: A Cross-sectional Study in 2013-2014.

BACKGROUND: Microsporidia have been reported as the cause of opportunistic infections in immunocompromised patients in Iran and other countries. There is no data on prevalence of intestinal microsporidia in healthy population of Iran. This study aimed to provide preliminary data on the present status of microsporidia infection in the local healthy population in Jiroft, Kerman Province from southeastern Iran in 2013-2014. METHODS: Fresh stool samples were randomly collected from 418 residents in rural 209 (50%) and urban 209 (50%) areas of Jiroft. All of the collected samples were concentrated with conventional formalin-ether, stained with Ryan blue. Microscopic examination was performed with high magnification on each sample separately for the demonstration of microsporidia spores. RESULTS: Microsporidial spores were identified in 41 out of 418 (9.8%) samples including 16.41(39%) from rural areas and 25.41(61%) from urban areas. In general, there was no significant difference between sex, age, job, education, and contact with soil and livestock, water supply, gastrointestinal disorders and microsporidia infection among general population in Jiroft. CONCLUSION: Intestinal microsporidia infection without clinical manifestations is prevalent in general population resident in southeastern Iran. Appropriate molecular methods are needed for microsporidia species identification.

Natural Intestinal Protozoa in Rodents (Rodentia: Gerbillinae, Murinae, Cricetinae) in Northwestern Iran.

BACKGROUND: Majority of parasitic infections in rodents have zoonotic importance. This study aimed to determine the frequency and intensity of intestinal protozoa infections of rodents including Meriones persicus, Mus musculus and, Cricetulus migratorius. METHODS: This survey was conducted in Meshkin Shahr district in northwestern Iran from Mar. to Dec. of 2014. Intestinal samples of 204 rodents including M. persicus (n=117), M. musculus (n=63) and C. migratorius (n=24) were parasitologically examined. Formalin-ether concentration method was done for all of rodents stool samples and observed with light microscope. All of suspected cases were stained with trichorome staining Method. Cultivation in dichromate potassium 2.5% was carried out for all of coccidian positive samples. Acid fast and aniline blue staining methods were used for detecting of coccidian oocysts and intestinal microsporidial spores, respectively. RESULTS: About 121(59.3%) of the caught rodents were generally infected with intestinal protozoa. Entamoeba muris 14(6.9%), Trichomonas muris 55(27.0%), Chilomastix betencourtti 17 (8.3%), Giardia muris 19(9.3%), Eimeria spp. 46(22.5%), Isospora spp. 4(2%) and Cryptosporidium spp. 1(0.5%) were found from the collected rodents. Microsporidian spores were identified in 63 (31%) out of the 204 collected rodents using aniline blue staining method. CONCLUSION: Since some of the infections are zoonotic importance thus, control of rodents can be decreased new cases of the parasitic zoonoses in humans.

Dicrocoelium Egg Identified in an Ancient Cemetery in Kiasar Archeological Site, Northern Iran, Dated Back 247 BC-224 AD.

BACKGROUND: Along with the newly emergence of paleoparasitology research in Iran, findings of parasites from Northern part of the county have not been reported so far. In this study tracking for the lancet liver fluke dates back 250 BC is addressed. METHODS: Samples were taken from grave crypts of the soil layers attached to the pelvic bones from above-mentioned site in 2015. The laboratory examinations were conducted in the Dept. of Medial Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran. Current rehydration technique using TSP 0.5% was utilized for examining the samples. RESULTS: Out of 10 burial soil samples examined, one individual was seen parasitized with a Dicrocoelium egg. The burial belonged to an adolescent male 20-22 yr old. The egg was in brown color and the length/width parameters of 36×22/5 µm. Parthian coins found in nearby the burials in Kiasar Cemetery, declared the time of the skeleton about 247 BC - 224 AD confidently. CONCLUSION: Although the possibility of transit infection with D. dendriticum is high, yet the environmental and geographical conditions in that time are in favor of a normal human transmission in northern Iran.

Subtypes Distribution and Frequency of Blastocystis sp. Isolated from Diarrheic and Non-diarrheic Patients.

BACKGROUND: Blastocystis is one of the most common parasites, reported from both human and animals. This parasite is more prevalent in regions with low levels of hygiene, close contact with animal and unsuitable disposal systems. The aim of the study was to subtype Blastocystis sp., isolated from diarrheic and non-diarrheic patients using sequencing of 18S ribosomal DNA. METHODS: Totally, 300 stool samples were collected from diarrheic and nondiarrheic patients referred to Imam Reza Hospital, Tehran from Apr to Aug 2015. All samples were concentrated using conventional Formalin - ether technique and recognized under light microscope. The fresh stool samples were also cultivated in clotted fetal bovine medium and examined for growing of Blastocystis every 48 h with direct smear slides for 10 d. DNA extraction was performed on all positive samples. Amplified DNA fragment of 18S rDNA was sequenced and compared with reference genes, previously deposited in Genbank database. RESULTS: The number of diarrheic and non-diarrheic patients participated in the study was 134 (44.66%) and 166 (55.34%), respectively. Three subtypes 1, 2, 3 were identified from positive samples. Subtype 2 was the most prevalent (36.5%) followed by subtype 1 (33.3%) and subtype 3 (30.2%). There were no mixed subtypes. Furthermore, the most prevalent subtypes in diarrheic and non-diarrheic patients were subtype 2 (39.28%) and subtype 1 (37.14%), respectively. CONCLUSION: Blastocystis sp., is one of the most prevalent unicellular parasites among diarrheic and non-diarrheic patients. Indeed, ST2 was the most prevalent subtype particularly in those samples collected from diarrheic patients.

Human spiruridiasis due to Physaloptera spp. (Nematoda: Physalopteridae) in a grave of the Shahr-e Sukhteh archeological site of the Bronze Age (2800-2500 BC) in Iran.

Evidence of rare human helminthiasis in paleoparasitological records is scarce. we report here the finding of Physaloptera spp. eggs in a soil sample collected in the pelvic and sacrum bones area of a skeleton excavated from a grave of Shahr-e Sukhteh archeological site dating back to the Bronze Age. The site is located in southeastern Iran and has attracted the attention of numerous archeological teams owing to its vast expanse and diverse archeological findings since 1997. The spirurid nematodes Physaloptera spp. are rarely the cause of human helminthiasis nowadays, but this infection might not have been so rare in ancient populations such as those in the Shahr-e Sukhteh. Out of 320 skeletons analyzed in this study, only one parasitized individual was detected. This surprising result led us to suspect the role of nematophagous fungi and other taphonomic processes in possible false-negative results. This is the first paleoparasitological study on human remains in this archeological site and the first record of ancient human physalopterosis in the Middle East.

First Paleoparasitological Report on the Animal Feces of Bronze Age Excavated from Shahr-e Sukhteh, Iran.

Shahr-e Sukhteh (meaning burnt city in Persian) in Iran is an archeological site dated back to around 3,200-1,800 BC. It is located in Sistan and Baluchistan Province of Iran and known as the junction of Bronze Age trade routes crossing the Iranian plateau. It was appointed as current study area for paleoparasitological investigations. Excavations at this site have revealed various archeological materials since 1967. In the present study, sheep and carnivore coprolites excavated from this site were analyzed by means of rehydration technique using TSP solution for finding helminth eggs. Dicrocoelium dendriticum, Capillaria sp., and Taenia sp. eggs were identified, while some other objects similar to Anoplocephalidae and Toxocara spp. eggs were also retrieved from the samples but their measured parameters did not match those of these species. The present paper illustrates the first paleoparasitological findings of Bronze Age in eastern Iran supporting the economic activities, peopling, and communication as well as the appropriate condition for zoonotic helminthiasis life cycle in Shahr-e Sukhteh archeological site.

Inter- and intra-subtype variation of Blastocystis subtypes isolated from diarrheic and non-diarrheic patients in Iran.

Blastocystis is a common intestinal parasitic protist infecting birds and mammals. Blastocystis comprises at least 17 subtypes (ST), of which ST1-ST9 have been detected in humans. Significant correlation between certain subtypes and pathogenicity remains to be established. Nevertheless, some studies suggest a potential linkage between subtypes (inter- and intra-subtype variation) and clinical manifestations. The aim of this study was to identify intra-subtype genetic variation of subtypes of Blastocystis in stools samples submitted by diarrheic and non-diarrheic patients. A 550-bp fragment of the nuclear small subunit ribosomal rRNA gene was amplified from 58 culture-positive samples isolated from diarrheic and non-diarrheic Iranian patients. PCR products were sequenced and sequences subjected to phylogenetic analysis. Intra-and inter-subtype variation was calculated. Based on comparison with reference sequences in GenBank, ST1, ST2 and ST3 were found in 18 (31.03%), 21 (36.22%), and 19 (32.75%) of the samples, respectively. Diarrheic stools were observed in eight (44.44%), 10 (47.61%), and nine (47.36%) patients with ST1, ST2, and ST3, respectively. No statistically significant correlation was found between subtypes and diarrhea (P=1.000). Multiple sequence alignment exhibited a within-subtype similarity of 98.76%, 97.17%, and 99.78% in ST1, ST2, and ST3, respectively. Highest similarity was seen among ST3 isolates, while lowest similarity was seen among ST2 isolates. Phylogenetic analysis did not suggest any correlation between diarrhea and intra-subtype variation. Inter- and intra-subtype variation in SSU rRNA gene appears not to reflect differences in the clinical outcome of Blastocystis carriage.