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Free-living amoebae (FLA) thrive in diverse environmental conditions. The present study aimed to define the FLA distribution from the Caspian Sea as well as from hospital ward dust from Guilan, Iran. Seawater (20) and hospital ward dust samples (100) were collected from May to June 2018. Seawater samples were vacuum filtered through a 0.45 μm pore-size membrane. Dust was collected using sterile gauze, washed with sterile distilled water, with washings collected thereafter. Washings were similarly filtered as seawater samples. FLA from the filtered material was cultivated in non-nutrient agar. Molecular analysis was performed by PCR and sequencing using specific primers for Acanthamoeba, Naegleria, and Vermamoeba/Hartmanella. Culture and PCR returned 50 and 65% positivity, respectively, for seawater samples where sequencing revealed Acanthamoeba T2, T5 and T6 genotypes and A. palestinensis and A. lenticulata, as well as N. dobsoni and N. clarki. In addition, 30% amoebic growth and 16% PCR detection were observed from hospital ward dust samples where sequencing revealed Acanthamoeba T2, T4 and T11 genotypes and A. castellanii, A. palestinensis and A. stevensoni as well as N. clarki. For both seawater and dust samples, Acanthamoeba was the dominant isolate. The detection of potentially pathogenic FLA from seawater may pose a threat to the public, while the presence of the same in dust spells threats to both hospital staff and patients, in particular, immunocompromised individuals. Public education, awareness, improved sanitation and hygiene, and the crafting of diagnostic strategies for the early detection of FLA in humans are necessary for the mitigation and management of potential human infection cases. HIGHLIGHTS Free-living amoebae (FLA) detected in environmental and infrastructure-derived samples.; Pathogenic genotypes of FLA detected in the Caspian Sea.; Pathogenic FLA detected in hospital wards for immunocompromised patients.; Pathogenic genotypes of FLA detected in hospital ward dust.; Hygiene and policies are necessary to protect humans against FLA infections.

Due to defects and drawbacks of most conventional diagnostic methods including serology for the diagnosis of toxoplasmosis as a dangerous opportunistic infection in immunocompromised individuals, the accurate, rapid, and sensitive detection of infection in such patients is essential. In this study, the TaqMan probe-based real-time PCR and, a relatively new nucleic acid amplification method, the loop-mediated isothermal amplification (LAMP) technique was compared based on the repetitive elements (RE) sequence to detect Toxoplasma gondii (T. gondii) DNA in blood samples of immunocompromised individuals. During this study, 119 blood samples from immunocompromised cancer patients with renal failure, undergoing dialysis were studied. After DNA extraction from blood samples using the salt extraction method, the molecular techniques of TaqMan probe-based real-time PCR and LAMP were used to investigate the contamination of the samples with T. gondii, based on the 529 bp (RE) sequence of T. gondii. The analytical sensitivity of LAMP and real-time PCR was evaluated by duplicating the five-step serial dilutions of T. gondii tachyzoites from 0.25 to 5×105 spiked tachyzoites per milliliter of the Toxoplasma seronegative blood sample. The extracted DNA from other parasites and human chromosomal DNA were used to determine the specificity of the molecular methods. The obtained results were analyzed using Kappa statistical test and SPSS22 software. Out of 119 studied samples, 7 (5.8%) and 5 (4.2%) samples were positive for Toxoplasma by TaqMan probe-based real-time PCR and LAMP, respectively. The limits of detection of TaqMan probe-based real-time PCR and RE-LAMP in negative serum samples were one and five tachyzoites (CT 38), respectively. Both real-time PCR and LAMP methods were 100% specific for Toxoplasma detection. Positive results were obtained only with T. gondii DNA, while other DNA samples were negative. The TaqMan probe-based real-time PCR based on the RE sequence showed higher sensitivity to T. gondii DNA detection in blood samples of cancer patients and serial dilutions of parasitic tachyzoites. The results show that TaqMan probe-based real-time PRC is a sensitive and specific method for the detection of toxoplasmosis in immunocompromised individuals, as well as the LAMP assay, which can be used as a suitable alternative diagnostic method for the detection of toxoplasmosis in such patients, without need the for any expensive equipment.

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

The incidence of metabolic syndrome (MetS) in menopausal women is one of the main health care concerns. MetS clusters are related to an imbalance in pro- and anti-inflammatory adipokines such as secreted frizzled-related protein 5 (SFRP5) and wingless-type mammary tumor virus integration site family, member 5A (WNT5A). WNT5A induces an inflammatory state to induce insulin resistance and further pathologic consequences. Recent strategies to prevent progression of MetS to diabetes have focused on conservative treatments such as exercise and herbal medicine. The aim of this study was to investigate the mechanistic effects of cotreatment with cinnamon extract and 12-wk high-intensity endurance training on MetS components considering the non-canonical WNT5A signaling.Thirty-two female ovariectomized Wistar rats were divided into the following four groups (n = 8/group): exercise (Ova+Exe), cinnamon extract (Ova+Cin), exercise with cinnamon extract (Ova+Exe+Cin) and saline (Ova+Sal). One group of rats undergoing surgery without removal of the ovaries was considered as a sham. After 3 mo of experimental intervention, waist circumference, serum concentrations of glucose, insulin, lipid profile, tumor necrosis factor-α, WNT5A, and SFRP5 were measured.Data showed a significant reduction in serum glucose, low-density lipoprotein, homeostasis model assessment estimate of insulin resistance, and tumor necrosis factor-α, but an increase in SFRP5 level in Ova+Exe, Ova+Cin and Ova +Exe+Cin groups compared with Ova+Sal group (P0.05). Serum WNT5A significantly was reduced only in Ova+Exe+Cin group (P = 0.02).The present study indicated that high-endurance training combined with aqueous cinnamon extract supplementation for 12 wk more efficiently alleviated insulin resistance and metabolic dysfunctions via reduction in noncanonical WNT signaling in ovariectomized rats.

We investigated the possible association between Parkinson's disease (PD), the second most common neurodegenerative disorder and Toxoplasma gondii infection, the most common neurotropic protozoan parasitic infection, using serological and molecular techniques. One hundred and fifteen patients with confirmed PD and 115 healthy subjects in the same age and sex distribution were enrolled in this study. Blood samples were taken from each participant and the sera was screened for anti- Toxoplasma antibodies (IgG and IgM). PCR assay was performed in duplicate using the primer pair targeting the B1 gene of Toxoplasma . Amplicons were directly sequenced to conduct the phylogenetic analysis. The prevalence of Toxoplasma infection based on IgG titer was 53% in case and 55.6% in the control groups, revealing no statistically significant association between Toxoplasma seropositivity and PD (OR = 0.90; 95% CI = 0.54–1.51; P = 0.691). According to PCR assay, the prevalence of Toxoplasma infections was 19.3% in the case and 10.4% in control groups which the difference was statistically significant (OR = 3.02; 95% CI = 1.46–6.27; P = 0.002). Multiple sequence alignment of Toxoplasma gondii isolates manifested a common haplotype by the identity: 93.6–100% and divergence: 0–6.7%. We concluded that T. gondii infection not only could not be a risk factor to PD, but even it could be concluded that patients with PD are in more risk to acquisition of infection. These results provide fresh insights into the ambiguous association between T. gondii infection and PD.

In the microevolutionary scales of Entamoeba isolates, the gene migration shows how Entamoeba spp. has epidemiologically drifted among border countries. Five hundred fecal samples were taken from patients suffering gastrointestinal disorders, abdominal pain, and diarrhea at Saggez, northwest Iran located within the border Iraq country. Following parasitological techniques, DNA samples were extracted and amplified by polymerase chain reaction (PCR) of 18S rRNA region to identify Entamoeba infections. To distinguish the Entamoeba spp., a multiplex PCR was conducted. Amplicons were sequenced to reconfirm their heterogeneity traits and phylogenetic analysis. Additionally, Entamoeba histolytica sequences of Iraq were retrieved from GenBank database. The suspected isolates were diagnosed as E. histolytica (2.2 %), Entamoeba moshkovskii (1 %), and Entamoeba dispar (0.4 %). Mixed Entamoeba infections did not detect among isolates. A parsimonious network of the sequence haplotypes displayed star-like features in the overall isolates containing E.h1, E.d2, and E.m3 as the most common haplotypes. According to analysis of molecular variance (AMOVA) test, high partial value of haplotype diversity (0.700 to 0.800) of E. histolytica was shown the total genetic variability within populations while nucleotide diversity was low among Iranian and Iraqi metapopulations. Neutrality indices of the 18S rRNA were shown negative values in E. histolytica populations which indicating significant deviations from neutrality. A pairwise fixation index (F-statistics [Fst]) as a degree of gene flow had a low value for all populations (0.001) while the number of migrants was 2.48. The statistically Fst value indicates that E. histolytica isolates are not genetically differentiated among shared isolates of Iran and Iraq. Occurrence of E.h1 between two regional populations indicates that there is dawn of Entamoeba flow due to transfer of alleles from one population to another population through host mobility and ecological alterations. To evaluate the hypothetical evolutionary scenario, further study is required to analyze Entamoeba spp. in the neighboring Middle East countries.

More recently, detection of circulating tumor cells (CTCs) has been considered as an appealing prognostic and diagnostic approach for cancer patients. CTCs as a type of tumor-derived cells are secreted by the tumor and released into the blood circulation. Since the migration of CTCs is an early event in cancer progression, patients who still have tumor-free lymph nodes have to be well examined for the CTCs presence in their blood circulation. Nowadays, there is a broad range of detection methods available to identify CTCs. As artificial RNA oligonucleotides or single-stranded DNA with receptor and catalytic characteristics, aptamers have been standing out, owing to their target-induced conformational modifications, elevated stability, and target specificity to be implemented in biosensing techniques. To date, several sensitivity-enhancement methods alongside smart nanomaterials have been used for the creation of new aptasensors to address the limit of detection (LOD), and improve the sensitivity of numerous analyte identification methods. The present review article supports a focused overview of the recent studies in the identification and quantitative determination of CTCs by aptamer-based biosensors and nanobiosensors.

Early, accurate and effective diagnosis of toxoplasmosis can make an important contribution to the prevention and control of disease, especially in people who are at risk. In this study, two commonly used genomic repeats of Toxoplasma gondii, RE (GenBank accession number AF146527) and B1, were compared to each other in nested-PCR assay. Five hundred and thirty-five blood samples from children with leukemia were tested for the presence of T. gondii antibodies using enzyme immunoassays. One hundred and ten DNA samples of these patients (50 IgM+, IgG+, 10 IgM-, IgG+, and 50 IgM-, IgG-) were analyzed by nested-PCR. The specificity of two nested PCR assays was determined using the DNA samples of other parasites and human chromosomal DNA. As a result, 82% (41/50) and 68% (34/50) of the IgM+, IgG+ samples were positive on duplicate RE and B1-nested PCR analyses, respectively. None of the 10 IgM-, IgG+ seropositive samples was detected positive after testing RE and B1-nested PCR assays in duplicate. One (2%) of the 50 seronegative samples was positive by duplicate RE-nested PCR but none of them were positive by duplicate B1-nested PCR. The detection limit of the RE-nested PCR assay was 640 fg of T. gondii DNA whereas this rate for B1-nested PCR was 5.12 pg of the DNA template. No cross-reactivity with the DNA of other parasites and human chromosomal DNA was found. The results indicate that an RE-based nested PCR assay is more sensitive than B1 genomic target, of those tested, for detection of T. gondii. It is noteworthy that in comparison with B1-nested PCR, RE-nested PCR could detect the T. gondii DNA in seronegative samples too.

Entamoeba histolytica, Giardia lamblia and Cryptosporidium spp. are common causes of diarrheal and intestinal diseases all over the world. Microscopic methods are useful in the diagnosis of intestinal parasites (IPs), but their sensitivity was assessed approximately 60 percent. Recently, molecular techniques have been used increasingly for the identification and characterization of the parasites. Among those, in this study we have used multiplex PCR and Real-time PCR with melting curve analysis (qPCR-MCA) for simultaneous detection and differentiation of E. histolytica, E. dispar, E. moshkovskii, G. lamblia and Cryptosporidium spp. in human fecal samples. Twenty DNA samples from 12 E. histolytica and 8 E. dispar samples and twenty stool samples confirmed positive for G. lamblia and Cryptosporidium spp. were analyzed. After DNA extraction from the samples, multiplex PCR was done for detection and differentiation of above mentioned parasites. QPCR-MCA was also performed for the detection and differentiation of 11 isolates of above mentioned parasite in a cycle with a time and temperature. Multiplex PCR was able to simultaneous detect and differentiate of above mentioned parasite in a single reaction. QPCR-MCA was able to differentiate genus and species those five protozoa using melting temperature simultaneously at the same time and temperature programs. In total, qPCR-MCA diagnosed 7/11 isolation of E. histolytica, 6/8 isolation of E. dispar, 1/1 E. moshkovskii Laredo, 10/11 G. Lamblia and 6/11 Cryptosporidium spp. Application of multiplex PCR for detection of more than one species in a test in developing countries, at least in reference laboratories has accurate diagnosis and plays a critical role in differentiation of protozoan species. Multiplex PCR assay with a template and multi template had different results and it seems that using a set of primers with one template has higher diagnostic capability in compare with multi template. The results of this study showed that the use of the qPCR-MCA can be an effective method to simultaneous distinguish of the above mentioned parasites.

We studied the prevalence of intestinal parasites (IPs), their risk factors and associated symptoms among patients with gastrointestinal disorders. A total of 1,301 participants aged 22 days-90 years were enrolled in this study. We used a structured questionnaire to obtain socio-demographic and stool examination to investigate intestinal parasite infections. Data analysis was performed using SPSS16. The overall prevalence of intestinal parasites (IPs) was 32.2% (419/1,301). Three hundred and fifty nine cases/1,301 (27.6%) were infected with a single parasite and 60/1,301 cases (4.6%) presented polyparasitism. The most common IP was Blastocystis sp. 350/1,301 (26.9%), followed by Entamoeba coli 38/1,301 (2.92%), Giardia lamblia 30/1,301 (2.3%) and Cryptosporidium spp. 17/1,301 (1.3%). Regarding the socio-demographic variables, educational status (p = 0.001), contact with domestic animals and soil (p = 0.02), age above 15 years (p = 0.001) and seasons (p = 0.001) were significantly associated to intestinal parasitic infections. Concerning clinical characteristics, the presence of IPs was significantly associated to diarrhea (OR = 1.57; CI 95% = 1.24-1.98; p < 0.001) and dysentery (OR = 1.94; CI 95% = 1.03-3.66; p < 0.04). Our findings suggest that IPs are one of the main causal agents of gastrointestinal disorders. Improving the knowledge on local risk factors such as poverty, low level of education, poor sanitation, contact with soil and contact with domestic animal is warranted.