Small-scale risk assessment of transmission of parasites from wastewater treatment plant to downstream vegetable farms

Aim: The aim of the present study was to simultaneously investigate parasitic contamination of treated wastewater and downstream vegetable farms that are irrigated with treated sewage, during a year

Background: [Oo] Cysts and eggs of parasites are resistant to most of routine wastewater treatment process. Irrigation of vegetables farms with either treated wastewater or illegally use of raw wastewaters enhances the risk of contamination with enteric pathogens

Methods: The treated wastewater samples were taken after chlorination from a wastewater treatment plant located at the south of Tehran. In addition, 60 vegetable samples [5 samples from each farm] were collected from the selected downstream farms that routinely used treated wastewater for irrigation of crops. Parasitological tests were performed using Ziehl-Neelsen, conventional lugol's iodine staining and direct microscopical examination

Results: Parasites including free living larvae, eggs of Toxoascaris leonina, egg of Toxocara sp. Trichuris sp, Trichostrongylus sp and amoeboid trophozoite were seen in 5/12 [41.7%] of vegetable samples gathered during a year. There was no statistically significant correlation between the season and parasitic contamination of the vegetables [P= 1]. Furthermore, parasitic contamination was observed in 7/12 [53.8%] of treated wastewater samples. The correlation between season and parasitic contamination of treated wastewater was evaluated that the results showed a higher contamination of treated wastewater in spring and autumn [P<0.05].Fisher's exact test also showed that there was no significant correlation between parasitic contaminations of vegetable samples and treated wastewater according to seasonal change

Conclusion: The results showed parasites in both treated wastewater plant and downstream crops farms that suggests the public health importance of the quality of water resources that routinely used for irrigation of vegetable farms

Epidemiologiccal study of intestinal parasites in referred individuals to the medical centers' laboratories of Haji-Abad city, Hormozgan Province, Iran, 2015

Background: Intestinal parasitic infection is one of the most prevalent health problems in developing countries. This study was conducted to determine the prevalence of intestinal parasitic infection and its correlation with socio-demographic parameters in Haji-abad, 2015

Materials and Methods: This cross-sectional descriptive study was conducted on 635 samples. After completing questionnaires, stool samples were assessed macroscopically, and microscopically using direct slide smear with saline and lugol, formalin-ether concentration, Ziehl-Neelsen staining to track Cryptosporidium species and Trichrome staining for the samples suspected to amoeba and other indeterminate cases. PCR using specific primers was conducted for Entamoebahistolytica/E. dispar suspected samples. The results were analyzed using SPSS[ver.16] software

Results: Of total 635 samples, 198 cases [31.2%] were infected by at least one intestinal parasite. The most common parasites in this area were: Blastocystis sp. [105, 16.5%], Endolimax nana [43, 6.8%], Entamoeba coli [32, 5.0%], Giardia lamblia [31, 4.9%], and Iodamoeba butschlii [11, 1.7%]. Enterobius vermicularis [1, 0.2%] was the only detected helminthic infection. Regarding socio-demographic variables, age, residence, sampling month, and job showed a significant correlation with IPIs [p-value=0.031, 0.019, 0.014, 0.012; respectively]. None of nine microscopically suspected E. histolytica/E. dispar cases were confirmed by molecular investigations [PCR method] and were considered as E. coli

Conclusion: In agreement with previous studies, helminthes infections show a dramatic decline compare to protozoa in this study. The relatively high incidence of intestinal protozoan infections in studies performed in Iran supports strategies for preventing the transmission and expansion of these parasites as a priority

Prevalence and subtype identification of Blastocystis isolated from humans in Ahvaz, Southwestern Iran

Aim: The aim of the present study was to determine the prevalence and subtype distribution of Blastocystis and its relation with demographic data and symptoms in humans referred to medical centers in Ahvaz 2014-2015

Background: Infections with intestinal parasites are one of the most important threats to human health worldwide, especially in tropical and subtropical areas. Blastocystis sp. is a common parasite of humans with a vast variety of non-human hosts. We aimed to study the prevalence and subtypes of Blastocystis sp. in individuals referred to medical laboratories in Ahvaz city, southwest Iran

Methods: From September 2014 to September 2015, 618 stool samples were collected from 16 medical laboratories in Ahvaz, and examined using direct wet mount, formalin-ether concentration, a modified version of the Ziehl-Neelsen staining technique, and cultivation in xenic HSr + S medium. Subtypes of positive Blastocysts sp. were obtained using the "barcoding" method. The results were analyzed using SPSS software, version 16, with Chi-square and Fisher's exact test

Results: Totally, 325 [52.6%] of the referred individuals were men and 293 [47.4%] were women. Blastocystis sp. was observed in 146 [23.6%] samples. Co-infections with other intestinal parasites were found in 32 [5.17%] cases. Out of the 146 positive isolates, 20.83%, 20.83% and 58.34% belonged to ST1, ST2, ST3 respectively

Conclusion: Blastocystis sp. was quite common in the study population, with a carrier rate corresponding to nearly one in every four individuals. The subtype distribution identified in the present study was largely identical to that reported from other studies in Iran, with ST3 being the most common

Contamination of swimming pools and park ponds with free living amoebae in Tehran

Background and Objectives: Free-living amoebae [FLA] include various genera habitat in water sources; some FLA can lead to severe complications in high risk people. The present study aimed to isolate free living amoebae using morphological methods in recreational water sources of Tehran

Materials and Methods: This cross sectional study was performed during 8 months in 2014. Seventy five samples from Tehran were collected and filtered. Samples were cultured in 1.5% non-nutrient bacto-agar. Plates were then monitored for the presence of amoebae daily and positive plates were cloned. In the present study, identification was based on morphological criteria and page key. The page key is based on morphological character of free living amoebae such as trophozoites shape, pseudopodia shape, amoebae nucleus, and endo and ecto-cysts in the cystic form, These criteria could lead to identification of amoeba at the family and genus level

Results: Out of 75 water samples, 18 [24%] were positive for free living amoebae. Of 40 pond waters, 13 [32,5%] were positive including Acanthamoeba, Hartmcmnella and Vahlkampfiids [Naegleria] and out of 35 samples 5 [14.2%] strain belonging io Acanthamoeba were identified based on morphological criteria

Conclusion: According to the presence of free living amoebae in recreational water sources, it is necessary to alert swimming pools authorities and high risk people. Additionally, posting alarming signs and educating the high risk people is of utmost importance to prevent free living amoebae-related infections

[Sero-prevalence study of visceral leishmaniasis using Direct Agglutination Test (DAT) in children up to 12 years old in Delphan City, Lorestan Province: introduce a new focus of VL In Iran]

Background and Objectives: Two clinical forms of leishmaniasis exist in Iran: cutaneous and visceral. According to the sporadic reports of new cases of Visceral Leishmaniasis [VL] in Lorestan province, real status of VL is not clear, so this study aimed to describe the seroprevalence of VL in Delphan city

Materials and Methods: In this descriptive analytic study, blood samples were collected from children /= 1/3200 accompanied with clinical symptoms was considered as VL diseas

Results: 800 collected serum samples, 21[2.62%] showed anti-Leishmania antibodies at titers of 1/800 and 1/1600, whereas 5[0.62%] showed anti-Leishmania antibodies at titers of ?1/3200. But just one of them showed clinical symptoms [anemia and large abdominal] which is under treatment with miltefosine

Conclusion: A new focus of VL with low endemicity is going to be formed in our region, which showed that further studies on vector and reservoirs is necessary in the region and other parts of Lorestan province

Different morphologies of Leishmania major amastigotes with no molecular diversity in a neglected endemic area of zoonotic cutaneous leishmaniasis in Iran

Molecular diversity of Leishmania major and its morphological changes have become a controversial issue among researchers. Some aspects of polymorphic shapes of amastigotes in clinical manifestations along with molecular variation were evaluated among suspected patients of some exceptional zoonotic cutaneous leishmaniasis locations in Northern Khuzestan, Southwestern Iran. Suspected patients [n = 165] were sampled in zoonotic cutaneous leishmaniasis foci over two consecutive years during 2012- 2014. Prepared smears were stained, scaled and measured by ocular micrometer. DNA was extracted from smears; ITS-rDNA and Cytochrome b [Cyt b] markers were amplified, and PCR products were digested by BsuR1 restriction enzyme. Then the RFLP and sequencing were employed. Only L. major was identified in patients containing regular amastigotes' shapes [oval or round] with a size of 2-4 microm in each of classical wet, dry, mixed lesions. Meanwhile, irregular shapes [spindle, pear, or cigarette] were observed separately in non-classical wet lesions with more than 4 microm. Interestingly, a few amastigotes with an external flagellum were observed in some lesions. All sequenced ITS-rDNA and Cyt b genes of L. major did not show any molecular variation [chi [2] P > 0.05], including only one common haplotype [GenBank access no. EF413075]. Findings proved that unlike other endemic foci, there is not a meaningful correlation between phenotypic and genotypic features of L. major isolates. This study is considered as the first comprehensive report to incriminate morphometric shapes of L. major amastigotes, which enhances our knowledge concerning their relevance with various clinical appearances and genotypic traits

Frequency of intestinal parasitic infections among individuals referred to the medical center laboratories in Nahavand City, Hamadan Province, Western Iran

Intestinal parasitic infections [IPIs] cause serious public health problem in the world, especially those located in tropical and subtropical areas. This study was conducted with the aim of obtaining frequency of intestinal parasites in referred people to the Nahavand city laboratories, Hamadan province, western Iran. A comparative cross-sectional study was conducted among checkup individuals and patients referred to laboratories of Nahavand County. A total of 371 stool samples [150 from checkup individuals and 221 from patients] were selected by using systematic random sampling during summer 2014. The stool specimens were examined macroscopically, and microscopically by using direct slide smear [saline wet mount and lugol staining], formaldehyde - diethyl ether concentration, trichrome staining and modified Ziehl-Neelsen staining techniques. The results were analyzed using SPSS version 16 and Chi-square test. Ninety two patients [24.8%] were infected with single or multiple intestinal parasites. The overall prevalence of IPIs in checkup individuals and patients was 21.3% and 27.1%, respectively. The frequency of the observed intestinal parasites was: Blastocystis spp. 72 [19.4%], Entamoeba coli 7 [1/9%], Endolimax nana 7 [1/9%], Giardia lamblia 5 [1/3%], Cryptosporidium spp. 3 [0.8%], Entamoeba hartmanni 3 [0.8%], Entamoeba histolitica/E. dispar 1 [0.3%], Trichomonas hominies 1 [0.3%], Chilomastix mesnili 1 [0.3%], Iodamoeba butschlii 1 [0.3%] and Enterobius vermicularis egg l [0.3%]. The proportion of observed protozoan parasites 91 [24.5%] is higher than helminthes infection 1 [0.3%]. The worm infections in Nahavand city was dramatically decreased over the past decades, induced increases in public health at the community level. Blastocystis spp. was the predominant intestinal parasite in people referred to the Nahavand city laboratories. Proportion of pathogenic IPIs among patients 4.07% [9 of 221] was higher in compare to the checkup individuals in which only one out of 150 [0.66%] Giardia lamblia was observed

[Cutaneous leishmaniasis in suspected refereed patients to health centers of varamin and determination of sand flies species during 2012-2013]

Leis mania is one of the most important diseases in Iran, with high prevalence in some part of country including, Tehran province this study was aimed to investigate the cutaneous lesions of patients studies was also referred to different 1aporatory of Health center of Vermin [a city In Tehran province], sand flies species m selected area. In this cross sectional study demographic data was collected by special questionnaire. Smears of suspected patients to stained and examined by microscope Sand flies were collected by sticky traps CDC light haps and aspirators, female specimens mere mounted and identified by diagnostic keys at species level Leis mania parasites were observed m 56 820 of cases. Disease was more common among male [64%] the majority of patients [3094] aged between 25-39 year old. Most lesions [50%] observed in hands and legs of patients, 76% of affected people had lust one lesion Half of them had a history of travel to endemic areas. More than 2500 sand flies captured during this study; among them 1100 female specimens were mounted and identified The prevalent spicies was Sergentomyia spp [65/9%], the second was papatasi [33/20%]. It should be mentioned that Ph sergenti, Ph alexandri and Ph caucasicus group also were identified in lower percentage According to current results and the status of the studied region including it seems that [Agricultural Activities animal husbandry and migration] cutaneous leishmaniasis could be a health threatening problem

Reporting of T4 genotype of acanthamoeba isolates in recreational water sources of Gilan province, northern Iran

Acanthamoeba spp. is the causative agent of blindness keratitis and fatal encephalaitis. Presence of Acanthamoeba spp. in a wide variety of niches such as different water types can lead to exposure of high risk people such as contact lens wearers. The main aim of the present study was to explore the occurrence of Acanthamoeba genotypes in the recreational water sources using both morphological and molecular approaches in Gilan province, Iran. Overall, 50 samples were collected from recreational water sources including man- made and natural waters in Gilan province. Filtration and cultivation of samples was performed using non-nutrient agar. Cloning of Acanthamoeba spp. was done to eliminate bacterial and fungi contamination. PCR amplification and sequencing were performed using genus-specific primer pair. Genotype identification was based on homology analysis of 18S rRNA gene [DF3] of the obtained sequences with the available genes in the gene bank data base. Out of 50 water samples, 15 [30%] were positive for Acanthamoeba trophozoites and cysts according to morphological criteria. Cloning of 13 isolates [26%] was done successfully. Molecular analysis of 13 Acanthamoeba strain revealed that all isolates were belonged to potentially pathogenic T4 genotype. T4 genotype is the main cause of Acanthamoeba-related infections. Presence of Acanthamoeba belonged to T4 genotype in recreational water sources is of concern for high risk people. Alarming sign and education to high risk people is of utmost importance to prevent such infections

Challenging loop-mediated isothermal amplification (LAMP) technique for molecular detection of Toxoplasma gondii

OBJECTIVE@#To compare analytical sensitivity and specificity of a newly described DNA amplification technique, LAMP and nested PCR assay targeting the RE and B1 genes for the detection of Toxoplasma gondii (T. gondii) DNA.@*METHODS@#The analytical sensitivity of LAMP and nested-PCR was obtained against10-fold serial dilutions of T. gondii DNA ranging from 1 ng to 0.01 fg. DNA samples of other parasites and human chromosomal DNA were used to determine the specificity of molecular assays.@*RESULTS@#After testing LAMP and nested-PCR in duplicate, the detection limit of RE-LAMP, B1-LAMP, RE-nested PCR and B1-nested PCR assays was one fg, 100 fg, 1 pg and 10 pg of T. gondii DNA respectively. All the LAMP assays and nested PCRs were 100% specific. The RE-LAMP assay revealed the most sensitivity for the detection of T. gondii DNA.@*CONCLUSIONS@#The obtained results demonstrate that the LAMP technique has a greater sensitivity for detection of T. gondii. Furthermore, these findings indicate that primers based on the RE are more suitable than those based on the B1 gene. However, the B1-LAMP assay has potential as a diagnostic tool for detection of T. gondii.

Challenging loop-mediated isothermal amplification (LAMP) technique for molecular detection of Toxoplasma gondii

Objective: To compare analytical sensitivity and specificity of a newly described DNA amplification technique, LAMP and nested PCR assay targeting the RE and B1 genes for the detection of Toxoplasma gondii (T. gondii) DNA. Methods: The analytical sensitivity of LAMP and nested-PCR was obtained against10-fold serial dilutions of T. gondii DNA ranging from 1 ng to 0.01 fg. DNA samples of other parasites and human chromosomal DNA were used to determine the specificity of molecular assays. Results: After testing LAMP and nested-PCR in duplicate, the detection limit of RE-LAMP, B1-LAMP, RE-nested PCR and B1-nested PCR assays was one fg, 100 fg, 1 pg and 10 pg of T. gondii DNA respectively. All the LAMP assays and nested PCRs were 100% specific. The RE-LAMP assay revealed the most sensitivity for the detection of T. gondii DNA. Conclusions: The obtained results demonstrate that the LAMP technique has a greater sensitivity for detection of T. gondii. Furthermore, these findings indicate that primers based on the RE are more suitable than those based on the B1 gene. However, the B1-LAMP assay has potential as a diagnostic tool for detection of T. gondii.

Application of multiplex PCR for detection and differentiation of entamoeba histolytica, entamoeba dispar and entamoeba moshkovskii

Entamoeba moshkovskii and E. dispar are impossible to differentiate microscopically from the pathogenic species E. histolytica. Multiplex polymerase chain reaction [Multiplex PCR] is a widespread molecular biology technique for amplification of multiple targets in a single PCR experiment. For detection and differentiation of the three-microscopy indistinguishable Entamoeba species in human, multiplex PCR assay using different DNA extraction methods was studied. A conserved forward primer was derived from the middle of the small-subunit rRNA gene, and reverse primers were designed from signature sequences specific to each of these three Entamoeba species. A 166-bp PCR product with E. histolytica DNA, a 580-bp product with E. moshkovskii DNA and a 752-bp product with E. dispar DNA were generated in a single-round and multiplex PCR reaction. We recommend this PCR assay as an accurate, rapid, and effective diagnostic method for the detection and discrimination of these three Entamoeba species in both routine diagnosis of amoebiasis and epidemiological surveys

Molecular characterization of bovine cryptosporidium using cryptosporidium oocyst wall protein [COWP] gene

Cryptosporidium is a globally distributed protozoan parasite and one of the most common causes of infection and diarrhea in humans and cattle. The aim of the present study was to determine the species of Cryptosporidium among cattle with diarrhea by a nested PCR-RFLP technique at Cryptosporidium oocyst wall protein [COWP]. Fecal samples from 158 calves aged 1-20 weeks were collected from 10 dairy farms in Qazvin province, Iran. Initial identification of Cryptosporidium was carried out by Zeihl-Neelsen acid-fast staining method of stool samples. DNA was extracted from 26 [16.45 %] positive microscopically samples and Cryptosporidium genotypes were determined. Cryptosporidium parvum were identified in 80.8% of the positive samples and, Cryptosporidium andersoni in 19.2%. In conclusion the use of COWP primers could be sensitive enough to conduct a routine detection study. The nested PCR method using the COWP gene sequence can be an alternative diagnostic method to identify infected with Cryptosporidium and its genetic diversity

Potential treatment of inflammatory bowel disease: a review of helminths therapy

An inflammatory bowel disease [IBD] is most common in highly industrialized Western countries but uncommon in less developed areas of the world where helminths are frequent. The hygiene hypothesis proposes that the recent increase in allergic and autoimmune diseases is due to modern highly hygienic life styles and medical conditions. Loss of routine exposure to parasitic helminths, as a result of increasing lifestyle-associated factors, may be one factor leading to the increased disease prevalence. In animal models and clinical trials of IBD, gastrointestinal nematodes colonization suppresses intestinal inflammation through multiple mechanisms including induction of innate and adaptive regulatory circuits. Studies using helminths like Trichuris suis or Necator americanus showed that these helminths are safe and may be effective therapeutic approaches for the control of IBD and other immune diseases. The aim of present review was to exploring the therapeutic use of helminths for the control of IBD

Ferredoxin gene mutation in Iranian Trichomonas vaginalis isolates

Trichomonas vaginalis causes trichomoniasis and metronidazole is its chosen drug for treatment. Ferredoxin has role in electron transport and carbohydrate metabolism and the conversion of an inactive form of metronidazole [CO] to its active form [CPR]. Ferredoxin gene mutations reduce gene expression and increase its resistance to metronidazole. In this study, the frequency of ferredoxin gene mutations in clinical isolates of T.vaginalis in Tehran has been studied. Forty six clinical T. vaginalis isolates of vaginal secretions and urine sediment were collected from Tehran Province since 2011 till 2012. DNA was extracted and ferredoxin gene was amplified by PCR technique. The ferredoxin gene PCR products were sequenced to determine gene mutations. In four isolates [8.69%] point mutation at nucleotide position -239 [the translation start codon] of the ferredoxin gene were detected in which adenosine were converted to thymine. Mutation at nucleotide -239 ferredoxin gene reduces translational regulatory protein's binding affinity which concludes reduction of ferredoxin expression. For this reduction, decrease in activity and decrease in metronidazole drug delivery into the cells occur. Mutations in these four isolates may lead to resistance of them to metronidazole

[Expression of a recombinant serine-rich Entamoeba Histolytica protein [SREHP]]

Entamoeba histolytica antigenic markers such as Serine-Rich E. histolytica protein [SREHP] have recently been used for vaccine preparation, genetic diversity studies of Entamoeba histolytica isolates and for differentiation between E. histolytica and E. dispar species. This study was carried out with the aim of expression of a recombinant Serine Rich E. histolytica protein in the laboratory to use it in the ELISA kit. In this study which is an exploration method, an Iranian isolate of Serine-Rich E. histolytica gene which had previously been cloned in bluescript plasmid [pBSc], was cut using BamHI restriction enzyme. After extracting and purification from gel, the SREHP gene was sub cloned into pET32a expression vector. The inserted gene was confirmed with Rosconis solution, PCR and sequencing methods. PCR was performed with the SREHP specific primers as well as pET T7 promoter primer. The cloned gene was also digested with HindIII and BamHI restriction enzymes. Recombinant plasmid was conveyed to competent cell BL21 [DE3]. A colony of the plasmid including SREHP gene was cultivated and induced with IPTG. The result of expressed protein was observed on the SDS-PAGE gel. The SREHP gene was sub cloned into pET32a expression vector. A recombinant plasmid including an inserted SREHP gene was screened and confirmed with quick check method using Ruscoins solution, as well as PCR by special primers [SREHP and universal pET primer], digested with BamHI and HindIII restriction enzymes. Finally an open reading frame of 666 nucleotides from inserted SREHP gene was obtained with the sequencing method. The recombinant protein of Serine-Rich E. histolytica in presence of IPTG was expressed in five hours and the result of expressed protein in the length of 44 KDa was observed on SDS-PAGE gel. SREHP protein was successfully cloned and expressed in this study. However additional studies are recommended for preparation and purification of the SREHP in a large quantity and the using it for the ELISA test

Cloning and expression of Brucella cyclic beta-1, 2 glucan transporter gene [cgt]

Brucellosis is an important cosmopolitan infection disease caused by organisms belonging to the genus Brucella. The cgt gene [cyclic beta-1, 2 glucan transporter gene] is a virulent factor in Brucella genus. The present study was conducted with the aim of cloning and expression of Brucella cgt gene. Brucella melitensis cgt gene was amplified from extracted chromosomal DNA by PCR, then PCR product was cloned into pTZ57R and subcloned into pGEMEX-1 expression vector, then expressed in JM109 E. coli strain. Recombinant protein was confirmed by western blot analysis using patient's serum. The PCR product was cloned in pTZ57R plasmid via T/A cloning method. Recombinant plasmid was digested by Band HI and SacI restriction enzymes, the released band was purified and subcloned into pGEMEX-1 expression vector. Then, sample cells were lysed using lyses buffer and sonicated, then electrophoresed on SDS-PAGE. Protein bands were transferred on nitrocellulose membrane and reacted by patient's serum and detected by FIRP conjugated-anti human antibody. We cloned and expressed Brucella abortus cyclic beta-1, 2-glucan transporter gene [cgt] which is an important agent in brucellosis. Using cgt gene mutant may be an effective way for inhibiting or decreasing the pathogenicity of bacteria

Inhibitory Effects of Iranian Thymus vulgaris Extracts on in Vitro Growth of Entamoeba histolytica

One of the most common drugs used against a wide variety of anaerobic protozoan parasites is metronidazole. However, this drug is mutagenic for bacteria and is a potent carcinogen for rodents. Thymus vulgaris is used for cough suppression and relief of dyspepsia. Also it has antibacterial and antifungal properties. The aim of this study was to investigate antiamebic effect of Thymus vulgaris against Entamoeba histolytica in comparison with metronidazole. One hundred gram air-dried T. vulgaris plant was obtained and macerated at 25 degrees C for 14 days using n-hexane and a mixture of ethanol and water. For essential oil isolation T. vulgaris was subjected to hydrodistillation using a clevenger-type apparatus for 3 hr. E. histolytica, HM-1: IMSS strain was used in all experiments. It was found that the minimal inhibitory concentration (MIC) for T. vulgaris hydroalcoholic, hexanic extracts, and the essential oil after 24 hr was 4 mg/mL, 4 mg/mL, and 0.7 mg/mL, respectively. After 48 hr the MIC for T. vulgaris hydroalcoholic and hexanic extracts was 3 and 3 mg/mL, respectively. Therefore, it can be concluded that the Iranian T. vulgaris is effective against the trophozoites of E. histolytica.