Development and evaluation of a loop-mediated isothermal amplification (LAMP) technique for rapid, accurate, and specific detection of Blastocystis spp. in AIDS patients.

BACKGROUND: Blastocystis spp. is one of the most prevalent intestinal parasites with worldwide distribution. Various diagnostic methods with different sensitivities and specificities have been used to detect Blastocystis in clinical samples. The present study aims to develop and evaluate a LAMP assay to detect Blastocystis spp. in AIDS patients for the first time. METHODS: In this cross-sectional study, 98 AIDS patients with an average CD4 + T lymphocyte count lower than 150 cells/mm3 participated in the study. The presence of Blastocystis spp. in the stool samples of AIDS patients was examined by parasitology (direct wet mount and concentration assays) and molecular (PCR and LAMP) methods. The 18 SSU rRNA genomic target was used to design the specific primers for the PCR and LAMP assays. The specificity of designed primers for the LAMP assay was evaluated using the sequencing of a conventional PCR product by the external LAMP primers. The data were analyzed using the SPSS software and chi-square test and Fischer's exact tests were used and Cohen's Kappa calculates the agreement of the molecular tests. Associations were tested using odd ratios (OR) and 95% confidence intervals (CI) after adjustments. RESULTS: Out of 98 stool samples from patients with AIDS, 9 (9.18%), 13 (13.26%), and 15 (15.30%) samples were detected positive for Blastocystis spp. by parasitology, PCR, and LAMP techniques, respectively. PCR amplification and subsequent sequencing of the product sequences revealed that the obtained partial sequences were identical to the corresponding 18 SSU rRNA sequences reported in GenBank. The higher positivity rate for Blastocystis spp. among studied AIDS patients by LAMP technique compared to other diagnostic methods showed the higher potential and effectiveness of this relatively new described molecular assay for the detection of Blastocystis spp. in AIDS patients. CONCLUSION: The accurate and rapid detection of emerging intestinal protozoa such as Blastocystis is of clinical importance for better prevention and timely treatment of the disease, especially in immunocompromised patients. The results obtained for the first time showed that the sensitivity and accuracy of the LAMP technique in the diagnosis of Blastocystis spp. in AIDS patients is very high.

Gastrointestinal parasites in immunocompromised patients; A comparative cross-sectional study.

Gastrointestinal parasites (GIPs), including helminths and protozoa species, are a major health problem in many parts of the world. About 3.5 billion people are affected by the parasites worldwide. GIPs are one of the leading causes of death among immunocompromised individuals and can cause serious clinical complications, especially in people with Human Immunodeficiency Virus (HIV)/AIDS, hemodialysis patients, and transplant recipients. This study aimed to compare the prevalence of GIPs among immunocompromised patients and immunocompetent individuals in Lorestan province, West Iran. In the current study, with a sampling of 232 participants (114 hemodialysis, AIDS, and organ transplantation immunocompromised patients and 118 immunocompetent individuals as the control group), demographic characteristics and risk factors for GIPs were collected through a pre-designed questionnaire. Stool samples of patients and the control group were examined for GIPs using different diagnostic methods including direct smear (saline and Lugol's iodine), Ziehl-Neelsen staining, agar-plate culture, and concentration method (formalin ether sedimentation). To evaluate the relative status of the immune system, TCD4+ cells were counted in the blood samples of the subjects by flow cytometry. The results were analyzed using SPSS 21 software, Fisher exact, and chi-square statistical tests. Multivariate modeling of the data was performed using logistic regression. The prevalence of GIPs in immunocompromised patients was more than twice that of immunocompetent individuals in the control group (42.06% vs. 17.79%). The most prevalent parasites identified among immunocompromised patients were Cryptosporidium sp. (27.1%), Blastocystis sp. (16.7%), and Entamoeba coli (14.6%) respectively. Cryptosporidium sp. had the highest frequency among hemodialysis patients (6.49%), AIDS patients (26.92%), and transplant recipients (18.18%) respectively. Patients with AIDS had the highest positive results for Cryptosporidium sp. followed by Microsporidia sp. (23.7%). In immunocompetent individuals, the highest prevalence of GIPs was related to Blastocystis sp and Trichomonas hominis (28.57%). Statistical analysis of the data showed that there was a statistically significant difference between various age groups regarding infection with GIPs so the highest rate of GIPs infection was observed in the age group lower than 50 years (P = 0.035). The statistical difference between the variable of location and infection with GIPs was insignificant but remarkable (P = 0.070). According to the results, it can be concluded that GIP is more common in immunocompromised patients than in immunocompetent individuals with cryptosporidium sp. predominance. Due to the favorable conditions of immunocompromised patients for GIPs and considering them as one of the important sources of parasitic infections and parasite transmission in society, control, prevention, and monitoring of their social behaviors along with health issues are inevitable.

Prophylactic effects of biogenic selenium nanoparticles on acute toxoplasmosis: An in vivo study.

BACKGROUND: In this investigation, the in vivo efficacy and safety of biogenic selenium nanoparticles (SeNPs) are assessed against acute toxoplasmosis caused by Toxoplasma gondii (Sarcocystidae) in the mice. METHODS: Male NMRI mice were orally treated with normal saline (control group) and SeNPs at the doses of 5 and 10 mg/kg once a day for 14 days. On the 15th day, the mice were infected with 104 tachyzoites of T. gondii RH strain by the intraperitoneal route. The mortality rate and parasite load were determined in the infected mice. The mRNA levels of IFN-γ, IL10, IL12, and inducible nitric oxide synthase were also examined in the infected mice by quantitative real-time PCR. RESULTS: The rate of mortality in the infected mice receiving SeNPs at the doses of 5 and 10 mg/kg compared with the mice in the control group was 100% on the 9 and 10 days after the administration. The mean number of tachyzoites in the infected mice receiving SeNPs was significantly lower than that in the control group. No significant difference (p > 0.05) was found in the biochemical parameters between the mice treated with SeNPs and the mice in the control group. The results revealed that mRNA levels significantly improved in the infected mice treated with SeNPs compared with those in the control group. CONCLUSION: Findings of the present investigation showed the considerable efficacy of SeNPs with no important toxicity for curing acute toxoplasmosis in the mice model. However, further studies are needed to clarify the accurate anti-Toxoplasma mechanisms of SeNPs.

Occurrence of Tetratrichomonas gallinarum (Trichomonadida: Trichomonadidae) in chicken feces from Lorestan Province, Western Iran.

Species of Tetratrichomonas gallinarum and Trichomonas gallinae, are found in the digestive tract of birds. To investigate trichomonads of a lower intestinal tract of native chickens, we examined 72 feces samples from the different life stages of fowl species in Khorramabad County, Lorestan Province, Western Iran. Using microscopical examination, out of 72 collected samples, 26 (31.6%) bird feces samples were found infected with a trichomonad similar to T. gallinarum. A 527-bp fragment of 18S rRNA gene of T. gallinarum was amplified by PCR. The 18S constructed phylogeny indicates two different clades within T. gallinarum, suggesting that commensal T. gallinarum-like of the bird hosts may be the reason for the genetic divergence in T. gallinarum species complex.

Prevalence and associated risk factors of intestinal helminthic infections in children from Lorestan province, Western Iran.

BACKGROUND: Intestinal helminthic infections are among the most important global socioeconomic and health problems. This study aimed to estimate the frequency of intestinal parasites in 366 children aged 2 to 15 years referred to the main pediatric health center of Lorestan Province, Iran. METHODS: Microscopic analysis was performed on 366 stool samples. We applied direct smear, scotch tape, and formol-ether methods. Moreover, a questionnaire was filled in by parents or guardians of the children. RESULTS: The results showed that 36 children (9.8%) were infected with at least one or more intestinal parasites. The most prevalent parasites were Enterobius vermicularis (6.8%), Hymenolepis nana (1.9%), and Ascaris lumbricoides (0.55%), in the order of their appearance. Statistical analysis showed that several risk factors were significantly associated with the prevalence intestinal helminthic parasites, including male sex (OR = 2.9; 95% CI: 1.2-6.2; p < 0.05), residing in rural regions (OR = 4.2; 95% CI: 2.1-10.6; p < 0.001), no handwashing habit before eating (OR = 5.2; 95% CI: 2.2-12.5; p < 0.001), and consuming raw or unwashed vegetables and fruits (OR = 4.8; 95% CI: 2.3-11.2; p < 0.001). CONCLUSION: The present study showed a high overall frequency of intestinal helminthic infections among the children in Lorestan province, Iran. The results of the risk factor analysis suggest that improving environmental hygiene and health education would be important for effective control of intestinal parasitic infections.

Genetic Characterization of Hydatid Cysts Isolated from Domestic Animals in Lorestan Province, Western Iran.

BACKGROUND: Regarding hydatid cyst (cystic echinococcosis, CE) as a human public health problem in the West of Iran, molecular data related to the genotypes of Echinococcus granulosus in cattle and sheep in these regions are still insufficient. Here, we evaluated the genotypes of E. granulosus infecting sheep and cattle in western Iran. METHODS: Totally, 36 hydatid cysts including 18 hydatid cysts of sheep and 18 hydatid cysts of cattle were collected from Khorramabad slaughterhouse (Lorestan Province), Western Iran between May to September 2014. Protoscoleces or germinal layers were collected from individual cysts, DNA was extracted, and genotyping was performed by sequencing and analyzing mitochondrial cytochrome c oxidase subunit 1 (cox1) gene. RESULTS: In sequencing analysis, all of sheep isolates belonged to genotype G1 (sheep strain). Among cattle hydatid cyst isolates, 16/18 (88.9%) were belonged to genotype G1 and 2/18 (11.1%) were belonged to G3 genotype. The phylogenetic analysis showed two clusters; one of the clusters includes cattle G3 genotype and the other cluster represents sheep and cattle G1 genotype that were isolated. CONCLUSION: The common sheep strain/G1 is predominant genotype in the western part of Iran, followed by G3 genotype, circulating among the animal hosts in this region. Further studies covering a larger number of isolates might be necessary to see if there are other genotypes in the hydatid cyst population in this region of Iran.

Epidemiology of pathogenic parasite Histomonas meleagridis in poultry in Lorestan province, western Iran.

Histomonas meleagridis is a flagellate protozoan parasite living in the cecum of birds digestive system and is the causative agent of histomoniasis. In this study 110 poultry fresh stool samples were assessed in order to detect H. meleagridis, and egg or adult worm of Heterakis gallinarum in Lorestan province, Iran. The results showed that the prevalence of H. meleagridis was 31%. Also, 19.5% of infected poultry had a watery stool. The eggs or adult worms of H. gallinarum were not seen in any of the samples. The results showed the high prevalence of this parasite, and the factors such as temporary hosts, susceptible hosts, and cloacal transmission with the involvement of water containing the parasite are the effective risk factors in prevalence. It is better to considered H. meleagridis as waterborne parasites, so further epidemiological studies on other birds are suggested to determine the parasite pathogenic strains using molecular methods.

Efficacy of Pistacia khinjuk Fruits on Viability of Hydatid Cyst Protoscoleces and Its Acute Toxicity in Mice Model.

BACKGROUND: This investigation aimed to evaluate the in vitro scolicidal effects of Pistacia khinjuk methanolic extract against protoscoleces of hydatid cysts and its acute toxicity in mice NMRI model. METHODS: Protoscoleces were aseptically extracted from sheep livers having hydatid cysts. Various concentrations of the essential oil (12.5-100 mg/mL) were used for 10 to 60 min. Viability of protoscoleces was confirmed using eosin exclusion test (0.1% eosin staining). Twenty-four male NMRI mice were used to assess the acute toxicity of P. khinjuk. RESULTS: P. khinjuk extract at the concentrations of 100 mg/mL after 10 min of exposure killed 100% of protoscoleces. Similarly, the mean of mortality rate of protoscoleces after 20 min of exposure to the concentration of 50 mg/mL was 100%. The LD50 of the intraperitoneal injection of the P. khinjuk methanolic extract was 2.8 g/kg and the maximum non-fatal dose was 1.7 g/kg. CONCLUSION: The findings demonstrated effective scolicidal effects of P. khinjuk extract with no considerable toxicity that might be a natural source for the producing of new scolicidal agent.

First report of birds infection by intestinal parasites in Khorramabad, west Iran.

Parasitic infections in birds are omnipresent, even when they occur in low amounts, may result in subclinical diseases. There aren't any studies, based on Iranian data, investigating the prevalence of intestinal parasitic infections in some birds' species. We conducted a cross-sectional study between December 2011 and December 2012. The fecal samples were taken from 451 birds including hen, turkey, sparrow, pigeon and decorative birds. The samples screened for intestinal parasitic infections using direct smear, formalin-ether concentration technique, modified Ziehl-Neelsen staining, Culture in RPMI 1640 medium, sporulation with potassium dichromate and Trichrome and Giemsa staining. Out of 451 birds' species, 157 (34.8 %), were infected with one or more type of intestinal parasites. We identified two nematode, two cestoda species and five protozoan parasites species. No trematodes were found in the samples studied. The parasites identified among birds involved Raillietina spp. (4.2 %) and Eimeria spp. (7.1 %) were the most common helminthes and protozoa respectively. From total of birds study, 12 (2.7 %) and 6 (1.3 %) have two and three mixed infections respectively. Intestinal parasitic infections are common in birds in west Iran. The future studies are needed in order to determine to which extent the infections influence mortality and performance of the birds.

Genetic diversity of blastocystis isolated from cattle in khorramabad, iran.

BACKGROUND: Blastocystis is a zoonotic protozoan parasite living in the digestive system of some vertebrates. This parasite has some subtypes, pathogenicity status of which has still remained controversial. OBJECTIVES: The aim of this study was to determine the subtype of Blastocystis in infected cattle. MATERIALS AND METHODS: This descriptive cross-sectional study was performed on 196 isolates from cattle stool samples collected from slaughterhouse in Khorramabad city, Iran, in 2012. Genomic DNA was extracted and to determine the Blastocystis subtype, seven pairs of sequence-tagged sites (STS) primers were used. RESULTS: Of 196 specimens, 19 (9.6%) were infected with Blastocystis. Among the 19 positive samples, the most common subtype was ST5 (47.36 %), followed by ST3 (10.53%) and ST6 (10.53%). Two (10.53%) samples had mixed infections by ST3 and ST5. The four isolates not amplified by any STS primers were probably unknown genotypes. CONCLUSIONS: In the present study, the highest prevalence was for ST5, which is so important for epidemiology and risk of human infection. The report related to ST3 in cattle as a subtype of human showed mutual infection between human and cattle. Another important point in this study was the ST6 report. Finally, it seems that gathering epidemiological data is needed for a better understanding of the potential animal reservoirs for human infection.

Genetic diversity of human blastocystis isolates in khorramabad, central iran.

BACKGROUND: There are some genetic differences in Blastocystis that show the existence of species or genotypes. One of these genes that help in identifying Blastocystis is SSUrRNA. The aim of this study was assessment of genetic diversity of Blastocystis by PCR with seven pairs of STS primers. METHODS: This study was done on 511 stool samples collected from patients referred to the health care centers of Khorramabad, Central Iran, in 2012. Genomic DNA was extracted and in order to determine the Blastocystis subtype in contaminated samples, seven pairs of primers STS (subtype specific sequence-tagged site) were used. RESULTS: Out of 511 samples, 33 (6.5%) samples were infected with Blastocystis. Subtype (ST) of 30 samples was identified and three subtypes 2, 3 and 4 were determined. Mix infection was reported 10% which 3.33% of the infection was for the mixture of ST 3 and ST5 and 6.67% was for the mixture of ST 2 and ST 3. CONCLUSION: The predominant subtype was ST3 that is the main human subtype. The dominance of ST2 and 5 are important in this study. This superiority has been reported in some of the studies in ST 2 which is different from the studies in other countries, because they have announced priorities of the ST1 and ST6 after ST3.