A shotgun metagenomic analysis of the fecal microbiome in humans infected with Giardia duodenalis.

BACKGROUND: The mechanisms underlying the clinical outcome disparity during human infection with Giardia duodenalis are still unclear. In recent years, evidence has pointed to the roles of host factors as well as parasite's genetic heterogeneity as major contributing factors in the development of symptomatic human giardiasis. However, it remains contested as to how only a small fraction of individuals infected with G. duodenalis develop clinical gastrointestinal manifestations, whereas the majority of infected individuals remain asymptomatic. Here, we demonstrate that diversity in the fecal microbiome correlates with the clinical outcome of human giardiasis. METHODS: The genetic heterogeneity of G. duodenalis clinical isolates from human subjects with asymptomatic and symptomatic giardiasis was determined using a multilocus analysis approach. We also assessed the genetic proximity of G. duodenalis isolates by constructing phylogenetic trees using the maximum likelihood. Total genomic DNA (gDNA) from fecal specimens was utilized to construct DNA libraries, followed by performing paired-end sequencing using the HiSeq X platform. The Kraken2-generated, filtered FASTQ files were assigned to microbial metabolic pathways and functions using HUMAnN 3.04 and the UniRef90 diamond annotated full reference database (version 201901b). Results from HUMAnN for each sample were evaluated for differences among the biological groups using the Kruskal-Wallis non-parametric test with a post hoc Dunn test. RESULTS: We found that a total of 8/11 (72.73%) human subjects were infected with assemblage A (sub-assemblage AII) of G. duodenalis, whereas 3/11 (27.27%) human subjects in the current study were infected with assemblage B of the parasite. We also found that the parasite's genetic diversity was not associated with the clinical outcome of the infection. Further phylogenetic analysis based on the tpi and gdh loci indicated that those clinical isolates belonging to assemblage A of G. duodenalis subjects clustered compactly together in a monophyletic clade despite being isolated from human subjects with asymptomatic and symptomatic human giardiasis. Using a metagenomic shotgun sequencing approach, we observed that infected individuals with asymptomatic and symptomatic giardiasis represented distinctive microbial diversity profiles, and that both were distinguishable from the profiles of healthy volunteers. CONCLUSIONS: These findings identify a potential association between host microbiome disparity with the development of clinical disease during human giardiasis, and may provide insights into the mechanisms by which the parasite induces pathological changes in the gut. These observations may also lead to the development of novel selective therapeutic targets for preventing human enteric microbial infections.

Cutaneous Leishmaniasis Lesion on the Ear from Kashan, Central Iran: A Case Report.

Cutaneous leishmaniasis (CL) frequently occurs in many rural and urban areas of Iran. Leishmania major and L. tropica are principally two causative agents of CL in Iran. We report here a case of leishmaniasis of the ear in a 61-year-old man referred to the Reference laboratory, Kashan, central Iran, in Jan 2022. He suffered from a 2-month history of a 1×3 cm lesion on the left ear. In the microscopy examination, amastigotes forms of Leishmania spp. were observed. L. tropica was confirmed using a single PCR with species-specific primers. The patient was introduced to a physician to begin the treatment protocol. It is recommended that physicians, especially in an endemic area, investigate any atypical lesion for CL.

Molecular detection and genotype identification of Acanthamoeba species from bronchoalveolar lavage of patients with pulmonary symptoms suspected of cancer.

Acanthamoeba spp. are the most common free-living amoeba worldwide, inducing life-threatening diseases such as Granulomatous Amoebic Encephalitis, pulmonary infection, and amoebic keratitis. This study aimed to identify the FLA and Acanthamoeba genotypes in patients with pulmonary symptoms suspected of cancer in Kashan's hospitals, Kashan, Iran. This cross-sectional study was conducted on 97 bronchoalveolar lavage samples of patients with respiratory symptoms suspected of lung cancer, who were admitted to the Shahid Beheshti Hospital of Kashan from 2019 to 2020. The samples were cultured onto 1.5% non-nutrient agar enriched with killed Escherichia coli and examined for the presence of FLA. Following amoeba isolation and DNA extraction, Acanthamoeba spp. were determined by Polymerase Chain Reaction using JDP1 and JDP2 primers, which amplified a 490 bp fragment from the 18 S rDNA gene. Eighteen Acanthamoeba isolates were sequenced, and the genotypes were identified. The prevalence of FLA and Acanthamoeba and the relationship between symptoms and demographic variables were analyzed with SPSS Software version 16. The prevalence rates of FLA and Acanthamoeba in the BAL samples was 86.6% and 73.2%, respectively. All Acanthamoeba isolates belonged to the T4 genotype. The most symptoms among Acanthamoeba-positive patients were dyspnea and cough; however, their difference was not statistically significant. The findings indicated the high prevalence of FLA and Acanthamoeba in BAL in the population suspected of cancer in Kashan. Since the T4 genotype is a pathogenic genotype of Acanthamoeba, training health and improving sanitation levels would help to prevent infection.

Accurate laboratory diagnosis of human intestinal and extra-intestinal amoebiasis.

Accurate diagnosis of Entamoeba histolytica is important, as it is known as a causative agent for both invasive intestinal and extra-intestinal amoebiasis. Amoebiasis has a worldwide distribution, especially in developing countries, and it is responsible for up to 100,000 fatal cases annually. A number of diagnostic methods, including microscopy, culture, antigen detection, molecular based methods, and serological assays have been proposed to assist in diagnosing amoebiasis. The present study aimed to gather new data and review the available diagnostic tests of both intestinal and extra-intestinal amoebiasis and to highlight pitfalls and challenges of each of them. A broad literature of electronic databases was conducted and covered articles published up to March 2022. For laboratory diagnosis of intestinal amoebiasis, direct microscopy stool examinations and cultures should be held as the high-performance diagnostic strategies. Molecular and immunological-based assays are also recommended as complementary tests. To diagnose extra-intestinal infection, the use of serological tests is still considered the method of choice. However, serodiagnosis requires further improvement for the accurate differential diagnosis of active infection from past infections.

Acute appendicitis: a case report of hyperinfection with Enterobius vermicularis.

Acute appendicitis is one of the most common causes of abdominal emergent surgical disease worldwide. Enterobius vermicularis, a human intestinal parasite, is reported to be associated with acute appendicitis. We report a case of an 8-year-old girl who was admitted to the emergency unit with complaints of severe abdominal pain and was diagnosed with acute appendicitis. Microscopic pathological examination showed lymphoid follicles with prominent germinal centers and mantle zones within the appendix wall. Cross-sections of multiple female and male Enterobius vermicularis worms and a few longitudinal sections of E.vermicularis were seen. E. vermicularis is one of the most common human parasitic infections, so the possibility of infection of the appendix with E. vermicularis should be considered in the differential diagnosis of agents of appendicitis.

Molecular detection of Trichostrongylus species through PCR followed by high resolution melt analysis of ITS-2 rDNA sequences.

Polymerase chain reaction followed by high resolution melting (PCR-HRM) analysis is a simple, rapid and accurate method for molecular detection of various nematode species. The objective of the present study was, for the first time, to develop a PCR-HRM assay for the detection of various animal Trichostrongylus spp. A pair of primers targeting the ITS-2 rDNA region of the Trichostrongylus spp. was designed for the development of the HRM assay. DNA samples were extracted from 30 adult worms of Trichostrongylus spp., the ITS-2-rDNA region was amplified using PCR, and the resultant products were sequenced and characterized. Afterwards, the PCR-HRM analysis was conducted to detect and discriminate Trichostrongylus spp. Molecular sequence analysis revealed that 24, 4, and 1 of the samples were T. colubriformis, T. vitrinus and T. capricola, respectively. Results from PCR-HRM indicated that complete agreement was relatively found between speciation by HRM analysis and DNA sequencing for the detection of Trichostrongylus species. The PCR-HRM analysis method developed in the present study is fast and low-cost; the method can be comparable with other molecular detection techniques, representing a reliable tool for the identification of various species within the Trichostrongylus genus.

Scolicidal Effects of Chitosan-Curcumin Nanoparticles on the Hydatid Cyst Protoscolices.

PURPOSE: In the current era, cystic echinococcosis (CE), as larval stage of Echinococcus granulosus, is considered as a threat to human health. Scolicidal agents used in the surgery of cysts have different side effects. Therefore, the present study aimed to assess the effects of chitosan nanoparticles containing curcumin (Ch-Cu NPs) on the protoscolices of the hydatid cyst in vitro. METHODS: Ch-Cu NPs were synthesized using a simple co-precipitation method and their structural and morphological properties were characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), zeta analyzer, and Fourier transform infrared (FT-IR) spectroscopy. Then, the effects of different concentrations of Ch-Cu NPs (0.25, 0.05, 1, 2, and 4 mg/mL) on the fatality rate, and the length and width of protoscolices in different times (5, 10, 20, 30, and 60 min) were investigated. In addition, the SEM technique was used to evaluate the structure of the protoscolices after treatment. RESULTS: Based on the results, the presence of curcumin on the chitosan nanoparticles was confirmed by FT-IR analysis. Further, XRD analysis approved the crystal structure of chitosan NPs. Furthermore, the highest fatality rate was 68% in 4 mg/mL concentration of Ch-Cu NPs. The length and width of protoscolices decreased based on the high concentrations of Ch-Cu NPs, compared to the control group. CONCLUSION: Finally, Ch-Cu NPs expressed good scolicidal activities, which made them suitable to be considered as an anti-protoscolex agent.

Giardia lamblia infection: review of current diagnostic strategies.

Giardiasis has a global distribution and it is a common cause of diarrhea in both children and adults and is transmitted via the fecal-oral route through direct or indirect ingestion of cysts. The laboratory diagnosis of Giardia spp. is mainly based on demonstration of microscopic cyst or trophozoite in stool samples but several immunological-based assays and molecular methods are also available for giardiasis diagnosis. The aim of this study was to conduct a review of the applied methods in medical laboratory and to highlight pitfalls and challenges of them for diagnosis of giardiasis. In this article we have evaluated the Giardia diagnostic methods with a broad review of literature, electronic databases and books. The search has covered the articles and some textbooks that have published up to 2018. It has been concluded that traditional microscopy combination with stool concentration method should still be held in the routine medical laboratory due to economical and high sensitivity and immunological-based assay and molecular methods which are recommended to use as a complementary test to the traditional technique.

Prevalence of intestinal parasites among food handlers in Kashan, central Iran, 2017-2018.

The most important transmission path of intestinal parasites is the consumption of contaminated water and food. This survey was conducted to determine the prevalence of intestinal parasites among food handlers in Kashan, central Iran. This cross-sectional study was carried on 1018 food handlers who referred to the health centers in order to receive health certificate in 2017-2018. Stool samples of food handlers were collected and examined using direct and formalin-ethyl acetate methods. Results were recorded in information form along with some demographic data such as sex, age. The data were analyzed based on the tests of descriptive statistics by SPSS 16. Of the 1018 individuals examined 851 (83.6%) were men and 167 (16.4%) were female. Infection rate was 10.2% among the population and 9 types of parasites were diagnosed totally. Rate of infection to protozoan intestinal parasites were: Blastocystis sp. 7.17%, Giardia duodenalis 1.2%, Entamoeba coli 1.27%, Endolimax nana 1.08%, Dientamoeba fragilis 0.3%, Iodamoeba butschlii 0.3%, Entamoeba histolytica/E. dispar 0.2%, Chilomastix mesnili 0.1%. Hymenolepis nana (0.1%) was the only intestinal worm that observed. In this investigation, 8.9% of the individual were infected by one parasite, 1.08% by two and 0.2% by 3 or more parasites. This study showed that infection to intestinal helminthic is rare among food handlers in Kashan but the prevalence of protozoan intestinal parasites is high that similar to other regions of Iran. To reduce the rate of parasitic infections and inhibition of their transmission, continuing the process of promoting the health level is recommended.

Induction of Apoptosis by Alcoholic Extract of Combination Verbascum thapsus and Ginger officinale on Iranian Isolate of Trichomonas vaginalis.

BACKGROUND: The protozoan Trichomonas vaginalis is a sexually transmitted disease (STD). Metronidazole is a chosen drug for the treatment. This study evaluated the anti trichomonal activity of alcoholic extracts of combination Verbascum thapsus and Ginger officinale. METHODS: This experimental study was conducted in the Parasitology Laboratory, Kashan University of Medical Sciences, Kashan, Iran in 2015, on 23 women with suspected trichomoniasis referring to Kashan clinical centers. Medium TYI-S-33 was used for culture of three T. vaginalis isolates. Different concentrations (25, 50, 100, 200, 400, 800 µg/ml) of V. thapsus and G. officinale ethanol extract added to Trichomonas trophozoites in 48-well plates and metronidazole considered as positive control and the negative control was TYI-S33 containing Trichomonas trophozoites without any drug. In all of mentioned groups, trophozoites number counted 12, 24, 48 h after culture. Results were analyzed using ANOVA statistical test, to evaluate the toxicity of extract, measured by MTT assay. Induced apoptosis of T. vaginalis after treatment with different concentrations of extract was determined by Flow Cytometry. RESULTS: IC50 of alcoholic extract of combination V. thapsus and G. officinale and metronidazole after 24h was 73.80 µg/ml and 0.0326 µg/ml, respectively. The toxicity percentage of 25-800 µg/ml concentrations of this combination were between 0.2-1.98. In different concentrations of extract (25,50,100,200 and 400 µg/ml) apoptosis percent after 48h was 18.97 to 77.19 and necrosis percent was calculated 1.35, 3.18, 3.10, 1.16 and 4.09, respectively. CONCLUSION: Alcoholic extract of combination V. thapsus and G. officinale induces programmed death in T. vaginalis. Due to no toxicity on macrophages, it can be examined in vivo studies.

Review of Trichomonas vaginalis in Iran, Based on Epidemiological Situation.

Trichomoniasis, which is caused by Trichomonas vaginalis, is the most common non-viral sexually transmitted infection (STI) in the world including Iran. There were roughly 250 million new cases all over the world in a year. T. vaginalis as an important disease has been associated with HIV (in terms of exposure to sexually transmitted infection, STI) which increases the number of high-risk members, and thus it is an important public health problem. Additionally, this pathogen has been associated with serious health consequences. For instance, it may cause a woman to deliver a low-birth-weight or premature infant, and increase chances of cervical cancer. Because little information is available about the prevalence of T. vaginalis infection in Iranian population, this review was carried out to determine the prevalence of T. vaginalis among Iranian population. For this systematic review, data about epidemiology of T. vaginalis in different parts of Iran with different populations were systematically collected from 1992 to 2017 through the international databases such as PubMed, Scirus, ISI Web of Science, Scopus, EMBASE, Science Direct and Google Scholar and Islamic World Science Citation Center (ISC). National database searching included Iran Medex, Iran Doc, Magiran and Scientific Information Database (SID). A total of 39 clinical and laboratory investigations about the prevalence of Trichomoniasis from different regions of Iran were analyzed. The overall prevalence rate of T. vaginalis infection in Iranian population was estimated to be minimally 0.4% and maximally 42%. The present review showed that T. vaginalis infection rate is relatively high among the Iranian population. The control strategies, including personal hygienic education, simultaneous couple treatment, the sensitivity of diagnostic methods, appropriate preventive tool (condom) in sexual contacts could lead to the disruption of transmission.

Epidemiology and economic loss of fasciolosis and dicrocoeliosis in Arak, Iran.

AIM: Fasciolosis and dicrocoeliosis are important parasitic diseases worldwide, causing significant financial losses due to decrease in production and viscera condemnation in animals. We performed the current research to assess the epidemiology of these infections and determine their significance from an economic perspective in Arak, Iran. MATERIALS AND METHODS: In total, we evaluated 118,463 sheep, 207,652 goats, and 43,675 cattle through necropsic analysis at the slaughterhouses. The average weight of sheep, goat, and cattle liver was 1000, 900, and 5000 g, respectively. The average price of liver in the market was 8 USD/kg. Moreover, the elimination of fundamental nutrients and vitamins was evaluated in infected livers. The prevalence of fasciolosis and dicrocoeliosis was determined. Analysis of variance test was applied for the statistical analysis, and the significance level was <0.05. RESULTS: In total, Fasciola hepatica and Dicrocoelium dendriticum were detected in 0.56% (confidence interval CI, 0.54-0.59) and 0.77% (CI, 0.75-0.81) of the animals, respectively (p=0.1). The annual economic loss attributed to fasciolosis and dicrocoeliosis was 26698.4 and 30479.2 USD, respectively. The total economic loss was 10,880, 9079.2, and 10,520 dollars in sheep, cattle, and goats, respectively. On the other hand, financial loss resulting from fasciolosis was 7160, 6098.4, and 13,440 dollars in sheep, goats, and cattle, respectively. In addition, economic loss due to dicroceliasis was 10,880, 9079.2, and 10,520 dollars, respectively. CONCLUSION: Overall, fasciolosis and dicrocoeliosis in Iran always remain common in sheep, goats, and cattle that afford major economic loss of all the country also exist in Arak province. The present study could provide basic information for further examination of liver fluke infections in Iran.

Therapeutic effects of Iranian herbal extracts against Trichomonas vaginalis

Trichomonas vaginalis is a flagellated parasite affecting about 276 million people annually worldwide. Tricomoniasis is associated with different complications in pregnant women and infants. 5'-nitroimidazole derivatives (metronidazole, ornidazole, and tinidazole) are FDA approved drugs recommended for trichomoniasis treatment. Treatment with metronidazole 5'-nitroimidazole derivatives is associated with many side effects, and drug resistance to metronidazole has been reported in some cases. Recently, many attempts have been made to evaluate the effects of plants on causative agents of vaginal infections. In our research, the national and international databases were searched and the effects of various herbal extracts on T. vaginalis in Iran were reviewed from 2006 to 2016. In articles investigated, some plants had favorable antitrichomonal effects and needed to be further investigated. All the plant extracts have only been evaluated in vitro. Surveys of different articles in this review show that the active ingredients present in different parts of plants, including aerial parts, leaves, flowers, stems, and root can be suitable sources for introducing and developing new antitrichomonal compounds.

Genetic Variation of Giardia lamblia Isolates from Food-handlers in Kashan, Central Iran.

BACKGROUND: Based on genotyping study of human isolates of Giardia lamblia; humans are mainly infected by two assemblages A and B. The present study was carried out to determine the sub-assemblages of G. lamblia isolated from food handlers referred to Kashan health centers, central Iran, 2015. METHODS: In this cross-sectional study, 3653 stool samples collected from food-handlers that annually refer to health center for getting a health certification and examined microscopically for G. lamblia cyst. Totally, 44 isolates were selected from 47 Giardia positive samples. Cysts were partially purified by the sucrose density gradient method. After freeze-thaw cycles, genomic DNA was extracted using QIAamp Stool Mini kit. A single step PCR-RFLP method was used to amplify a 458bp fragment at the glutamate dehydrogenase (gdh) locus, restriction enzymes BspLI and RsaI were used for distinguish between genotypes A and B and their subgroups. RESULTS: Of 44 isolates, 24(54.5%) were sub-assemblage AII, 9(20.5%) group B including 7(15.9%) BIII and 2(4.6%) BIV sub-assemblage and 11(25%) isolates showed a mixed pattern of AII and B. Sub-assemblage AI was not detected in this study. CONCLUSION: The higher rate of sub-assemblage AII demonstrated an anthroponotic origin of the infection so infected food-handlers could directly transmit this protozoan to consumers via contaminated food and water. For finding of pattern of transmission and distribution of Giardia assemblages and sub-assemblage, more studies in human and animal population in different regions are necessary.

Molecular and Morphological Characterizations of Echinococcus granulosus from Human and Animal Isolates in Kashan, Markazi Province, Iran.

BACKGROUND: One of the most important zoonotic helminths in the world is known as Echinococcus granulosus. Different strains of the E. granulosus have been described based on morphological and molecular characterizations, however, there is limited information regarding the characteristics of the phenotypes and genotypes of E. granulosus in Iran. METHODS: The present study was prepared to evaluate the phenotypic and genotypic diversity of E. granulosus isolates collected from human, goat, sheep, and cattle based on 19 standard morphometric parameters and mitochondrial and nuclear genes (CO1, ND1, and ITS1) in Kashan, Markazi Province, Iran during 2013-2014. RESULTS: The biometric analysis for the 19 characters revealed that the 19 morphometric values of cattle isolates were exceptionally higher than human, goat, and sheep isolates (P<0.05). Molecular analysis confirms the morphological findings. Phylogenic analysis of the CO1, NAD1 and ITS1 genes for all isolates, independent of the host, revealed that the common sheep strain (G1) is traveling among livestock in Kashan and the strains are highly adapted to goats, cattle, sheep, and humans. CONCLUSION: Both morphological and molecular results of this study indicated that the only genotype G1 of E. granulosus travels between humans and other intermediate hosts of this parasite in the area study.

Intestinal parasitic infections in different groups of immunocompromised patients in Kashan and Qom cities, central Iran.

INTRODUCTION: Intestinal parasitic infections (IPIs) are important causes of morbidity and mortality in patients with immunocompromising conditions. OBJECTIVE: The aim of this study was to determine the prevalence of IPIs in different groups of immunocompromised patients, including hemodialysis patients (HD), renal transplant recipients (RTR), cancer and HIV/AIDS patients in comparison with healthy individuals in two central cities of Iran (Kashan and Qom). METHODS: In this case-control study, the stool samples of 135 HD, 50 RTR, 60 cancer patients, 20 HIV/AIDS patients and 120 healthy subjects were tested using direct-smear, formol-ether concentration, Ziehl-Neelsen staining and Agar plate method. RESULTS: The overall infection rate was 11.7% (31/265) in patient groups and 0% (0/120) in the control group. The frequency of parasites was 25% in HIV/AIDS patients, 11.9% (16/135) in HD, 12.0% (6/50) in RTR and 6.7% (4/60) in cancer patients. Blastocystis hominis (4.2%) and Giardia lamblia (3.0%) were the most prevalent parasites in patient groups. The infection rate was significantly higher in male (17.6%) than female (5.4%) patients (p = .002), but no statistically significant association was observed according to the age and educational levels. CONCLUSIONS: This study showed a high prevalence of IPIs in immunocompromised patients. The results of this study suggest that periodic stool examinations for screening of IPIs should be included as a part of routine medical care in these patients.

Ginger (Zingiber officinale) induces apoptosis in Trichomonas vaginalis in vitro.

BACKGROUND: Trichomoniasis is the most common sexually transmitted protozoan diseases in the worldwide. Metronidazole is the choice drug for trichomoniasis treatment, however, metronidazole resistant Trichomonas vaginalis (T.vaginalis) has been reported. Natural products are the source of most new drugs, and Zingiber officinale (Ginger) is widely used ingredient in the traditional medicine. OBJECTIVE: The aim of the present study was to determine the effect of different concentrations of the ginger ethanol extract on the growth of T.vaginalis trophozoites in vitro. MATERIALS AND METHODS: In this experimental study, 970 women who were attend in Kashan health centers were examined for T. vaginalis. Of them, 23 samples were infected with T.vaginalis. Three T. vaginalis isolates were cultured in a TYI-S-33 medium. The effect of ginger ethanol extracts and its toxicity in different concentrations (25, 50, 100, 200, 400, 800 µg/ml) on mouse macrophages were measured in triplicate exam by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. The effect of ginger on apoptosis induction was determined by Flow cytometry. RESULTS: The IC50 of ginger and metronidazole were 93.8 and 0.0326 µg/ml, respectively. 12, 24 and 48 hr after adding different concentrations of extract on mouse macrophages, fatality rates in maximum dose (800 µg/ml) were 0.19, 0.26 and 0.31 respectively. Flow cytometry results showed the apoptosis rate following treatment with different concentrations of the extract after 48 hr were 17, 28.5, 42.1, 58.8, 76.3 and 100% respectively, while in the control group was 2.9%. CONCLUSION: Ginger ethanol extract induces programmed death in T. vaginalis. It is recommended that due to the known teratogenic effect of metronidazole, ginger can be considered as an alternative drug for metronidazole.

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.

Detection of Bendiocarb and Carbaryl Resistance Mechanisms among German Cockroach Blattella germanica (Blattaria: Blattellidae) Collected from Tabriz Hospitals, East Azerbaijan Province, Iran in 2013.

BACKGROUND: Insecticide resistance is one of the serious problems for German cockroach control program. This study was conducted to determine the bendiocarb and Carbaryl resistance mechanisms in German cockroaches using the piperonyl butoxide (PBO). METHODS: Bioassay tests were conducted with 4 to 6 different concentrations of both insecticides with four replicates of 10 susceptible strain cockroaches per concentration to determine of discriminative concentration. After determining discriminative concentration, the result was compared to wild strain. The levels of susceptibility and resistance ratio (RR) and synergism ratio (SR) were calculated for each five wild strains. Moreover resistance mechanisms in wild strains were determined using PBO synergist in vivo. RESULTS: Hospital strains showed different levels of resistance to bendiocarb and carbaryl compared to susceptible strain. The bendiocarb and carbaryl resistance ratios ranged from 2.11 to 7.97 and 1.67 to 2 at LD50 levels, respectively. The synergist PBO significantly enhanced the toxicity of bendiocarb and carbaryl to all strains with different degrees of synergist ratio, 1.31, 1.39, 3.61, 1.78, 1.62 and 2.1 fold for bendiocarb, 1.19, 1.18, 1.12. 1.29, 1.45 and 1.11-fold for carbaryl, suggesting monooxygenase involvement in bendiocarb and carbaryl resistance. CONCLUSION: The synergetic effect of PBO had the highest effect on bendiocarb and resistance level was significantly reduced, which indicates the important role of monoxidase enzyme in creating resistance to Bendiocarb. Piperonyl butoxide did not have a significant synergistic effect on carbaryl and did not significantly break the resistance.