Investigating the Effect of Satureja khuzestanica Essential oil on MDR1 Gene Expression in Leishmania major.

BACKGROUND: Cutaneous leishmaniasis is among the neglected diseases in the world. Pentavalent antimonial compounds are considered the first-line treatment for this disease. However, using alternative natural products has received great attention due to the side effects of chemical drugs and drug resistance of the Leishmania parasite. The present study aims to investigate the effect of Satureja khuzestanica essential oil (SKEO) on MDR1 gene expression. METHODS: In this study, standard strains of Leishmania major promastigotes were exposed to 5, 10, 15, and 20 µg/ml of SKEO. MDR1 gene expression of parasites exposed to essential oil was evaluated using real-time PCR. GAPDH was employed as the housekeeping gene for internal control. RESULTS: Despite the increase, no statistically significant difference was observed in the relative expression of the MDR1 gene between the control group and the groups containing 5, 10, and 20 µg/ml of SKEO (P > 0.05). The relative expression of the MDR1 gene significantly increased in the group containing 15 µg/ml of essential oil compared to the control one (P < 0.05). CONCLUSION: This study showed that the use of essential oil of Satureja khuzestanica plant can have an increasing effect on the expression of MDR1 gene of Leishmania promastigotes, which is the best case if Satureja khuzestanica essential oil reduces the expression of MDR1 gene. So it seems that the use of essential oil of Satoria plant is effective in controlling Leishmania parasite, but its concentrations induce drug resistance. As a result, concentrations of essential oil should be used that have a controlling effect on the growth and proliferation of Leishmania parasite and also have the least effect on the induction of MDR1 gene expression.

Identification and genotyping of Acanthamoeba spp. in the water resources of western Iran.

Background: Acanthamoeba spp. is opportunistic amoeba that resides in water, soil, and air. Some pathogenic genotypes of the genus of Acanthamoeba can cause granulomatous amoebic encephalitis (GAE) in people with a defective immune system. The parasite can also cause Acanthamoeba keratitis (AK) among contact lens users. This study was conducted to isolate and identify the Acanthamoeba genotypes in water resources in Lorestan province, western Iran. Methods: Collected 72 water samples from surface and groundwater (springs and aqueducts) in Lorestan province. Samples were filtered and cultured in non-nutrient 1.5% agar medium covered with Escherichia coli (E. coli) at 25 °C. DNA extraction was done and the PCR reaction was performed to detect the Acanthamoeba spp. The positive PCR products were sequenced to determine the genotypes of Acanthamoeba. Results: Out of 72 examined water samples, 23.61% were positive for Acanthamoeba sp. by PCR. From PCR-positive samples, 8 (47.05%) samples were T4 genotypes and others were other Acanthamoeba genotypes (T1-T23). Therefore, approximately half of the genotypes belong to the pathogenic T4 genotype. Conclusions: The water examined samples in western provinces of Iran have the potential risk factor for public health. Therefore, the efforts of healthcare providers are needed to identify, train, and prevention from human infections.

Rapid, sensitive, and specific detection of SARS-CoV-2 in nasopharyngeal swab samples of suspected patients using a novel one-step loop-mediated isothermal amplification (one-step LAMP) technique.

BACKGROUND: In the absence of effective antiviral drugs or vaccines, early and accurate detection of SARS-CoV-2 infection is essential to the COVID-19 pandemic. This study developed and evaluated a novel rapid One-Step LAMP assay to directly detect the SARS-CoV-2 RNA from nasopharyngeal (NP) swab samples of patients with suspected SARS-CoV-2 infection living in deprived areas in comparison to One-Step Real-time PCR. METHODS: Two hundred fifty-four NP swab samples from patients suspected of COVID-19 infection living in deprived western areas of Iran were tested by TaqMan One-Step RT-qPCR and fast One-Step LAMP assays. Tenfold serial dilutions of SARS-CoV-2 RNA standard strain where the viral copy number in each dilution was previously determined using the qPCR and various templates were used to investigate the analytical sensitivity and specificity of the One-Step LAMP assay in triplicate. Also, the efficacy and reliability of the method compared to TaqMan One-Step RT-qPCR were evaluated using SARS-CoV-2 positive and negative clinical samples. RESULTS: The results of the One-Step RT-qPCR and One-Step LAMP tests were positive in 131 (51.6%) and 127 (50%) participants, respectively. Based on Cohen's kappa coefficient (κ), the agreement between the two tests was 97%, which was statistically significant (P < 0.001). The detection limit for the One-Step LAMP assay was 1 × 101 copies of standard SARS-CoV-2 RNA per reaction in less than an hour in triplicates. Negative results in all samples with non-SARS-CoV-2 templates represent 100% specificity. CONCLUSIONS: The results showed that the One-Step LAMP assay is an efficient consistent technique for detecting SARS-CoV-2 among suspected individuals due to its simplicity, speed, low cost, sensitivity, and specificity. Therefore, it has great potential as a useful diagnostic tool for disease epidemic control, timely treatment, and public health protection, especially in poor and underdeveloped countries.

New Strategies in the Treatment of Plasmodium berghei Based on Nanoparticles: A Systematic Review.

BACKGROUND: Drug resistance is a current issue affecting parasites caused by Plasmodium. Therefore, researchers have expanded their studies on nanoparticles to find new and effective drugs that can treat drug-resistant strains. The present study systematically investigates the effect of different nanoparticles, including metal, polymer, and lipid nanoparticles, on Plasmodium berghei. METHODS: In this study, English-language online literature was obtained from the databases Science Direct, PubMed, Scopus, Ovid, and Cochrane to conduct a systematic review. In the search, we used the keywords: (Plasmodium Berghei) AND (Malaria) AND (Parasitemia) AND (antimalarial activity) AND (nanoparticles) AND (Solid lipid NPS) AND (Nano lipid carriers) AND (Artemether) AND (Chloroquine) AND (intraperitoneal) AND (in vivo). Initially, a total of 160 studies were retrieved from the search. After removing duplicates, 80 studies remained. After reviewing the title and abstract of each study, 45 unrelated studies were eliminated. RESULTS: The remaining 35 studies were thoroughly reviewed using the full texts. The final result was 21 studies that met the inclusion/exclusion criteria. CONCLUSION: Using these findings, we can conclude that various nanoparticles possess antiparasitic effects that may be applied to emerging and drug-resistant parasites. Together, these findings suggest that nanostructures may be used to design antiparasitic drugs that are effective against Plasmodium berghei.

Molecular evaluation of Cryptosporidium spp. among breeding calves of Lorestan province Western Iran.

BACKGROUND: Cryptosporidium spp. are opportunistic intestinal protozoans with global distribution and are of great importance as zoonotic protozoans are common to humans and domestic animals, including cattle and calves. Identification and detection of parasite species using precise methods including molecular methods can be an effective step in treating and controlling parasites. OBJECTIVES: This study aimed to investigate the prevalence of Cryptosporidium among breeding calves of Khorramabad city, Lorestan province, Western Iran, using PCR. METHODS: The faecal samples were taken from 181 healthy and diarrhoeal calves and after the Ziehl Neelsen Acid-fast staining and microscopic evaluation, the genomic DNA was extracted for molecular evaluations. To detect Cryptosporidium species, specific primers targeting the SAM-1 gene of Cryptosporidium and a commercial master mix were used for PCR. RESULTS: Out of 181 faecal samples of breeding calves in Khorramabad city, 9 samples (5%) were positive for Cryptosporidium spp. using the PCR method. Statistical analysis of the data showed that there was no significant statistical relationship between Cryptosporidium infection of the calves and variables of age, breed, type of water consumption, clinical signs of diarrhoea, and sampling location, while parasite infection had a significant relationship with calf gender so that all Cryptosporidium positive samples were from male calves (p ≤ 0.05). CONCLUSIONS: Considering the presence of Cryptosporidium infection, the region's traditional grazing system, and the close relationship between livestock and humans, there is a possibility of human infection in the region. So preventive measures such as periodic animal testing with sensitive and accurate diagnostic techniques including PCR, pharmacological treatment of livestock, water hygiene and the use of industrial grazing instead of traditional grazing to improve the hygiene of food consumed by livestock are recommended.

Diagnosis of Toxoplasma Infection in Allogenic Pre HCTSP Patients Using Molecular Methods.

Background: We aimed to estimate the incidence of Toxoplasma infection in T. gondiiseropositive patients under allogeneic hematopoietic stem cell transplantation (HSCT). Methods: The present research was a prospective study on 54 whole blood samples of allogeneic HSCT recipients, who were referring to bone narrow transplantation centers affiliated to Shahid Beheshti University of Medical Sciences, Tehran, Iran in 2018. All patients were IgG positive against T. gondii. Results: Overall, 54 Toxoplasma positive pre-HCTSP patients were enrolled. 53.7% (n= 29) were male, also 1.9% (n=1) had germ-line type of the disease. The Multiple myeloma patients had higher age in comparison with other disease, but pairwise comparison showed the difference of age between Multiple myeloma patients were statistically significant with Acute lymphoblastic leukemia, Acute myeloblastic leukemia and Huntington's disease (P< 0.05). The results of PCR assay showed 5.6% (n= 3) of the patients were infected with Toxoplasma. Conclusion: PCR method has detected considerable incidence of Toxoplasma infection for monitoring HSCT recipients at risk for toxoplasmosis, and many patients who showed the incidence of toxoplasmosis had previous infections with the Toxoplasma parasite.

Diagnostic power of one-step and two-step RT-qPCR methods to SARS­CoV­2 detection.

BACKGROUND: Coronavirus-2019 (COVID-2019) is a novel coronavirus known as Acute Respiratory Syndrome (SARS-CoV-2). The premier standard test for SARS-CoV-2 diagnosis is a one-step RT-qPCR method, which requires specific probes and reagents. Therefore, detection on a large scale is expensive and cannot be very accurate. METHODS: A cost-effective technique based on SYBR green was evaluated in the current study. The specific primers for S and N genes were designed, then performed the cross-reactivity test with other coronavirus and respiratory viruses positive samples. Moreover, the analytical sensitivity test was carried out with 8 dilutions (1:10). Lastly, the SARS-CoV-2 clinical samples (n = 210) were tested by these two methods, and receiver operating characteristic (ROC) analysis was performed to investigate the incremental diagnostic value of each gene in the study methods. RESULTS: The two-step method detected up to 6th dilutions of the SARS-CoV-2 samples and did not show any amplification of the positive samples of other respiratory viruses. ROC analysis revealed a diagnostic ability of the two-step method for SARS-CoV-2 with an area under the ROC curve of ≥ 0.7 (P Ë‚ 0.05) and relatively high sensitivity and specificity. The combination of N and S genes increased the sensitivity up to 88%, specificity up to 86%, and area under the ROC curve up to 0.85 (95% confidence interval (95% CI) 0.72 to 0.93, P = 0.0461). CONCLUSION: Our findings indicated that the two-step method has comparable sensitivity and specificity to the one-step method. Therefore, this method can be considered a potential diagnostic method for diagnosing and monitoring COVID-19 patients. It suggests that when the one-step RT-qPCR method is not available, the two-step RT-qPCR can be used to identify SARS-CoV-2.

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.

Utility of blood as the clinical specimen for the diagnosis of ocular toxoplasmosis using uracil DNA glycosylase-supplemented loop-mediated isothermal amplification and real-time polymerase chain reaction assays based on REP-529 sequence and B1 gene.

BACKGROUND: Ocular infection with Toxoplasma gondii is a major preventable cause of blindness, especially in young people. The aim of the present study was to assess detection rate of T. gondii DNA in blood samples of clinically diagnosed of ocular toxoplasmosis using uracil DNA glycosylase-supplemented loop-mediated isothermal amplification (UDG-LAMP) and real-time quantitative PCR (qPCR) based on REP-529 and B1. METHODS: One hundred and seventeen patients with clinically diagnosed ocular toxoplasmosis (OT) were participated in the study as well as 200 control patients. Peripheral blood samples were assessed using UDG-LAMP and qPCR techniques targeting REP-529 and B1. RESULTS: Detection limits of qPCR using REP-529 and B1 were estimated as 0.1 and 1 fg of T. gondii genomic DNA, respectively. The limits of detection for UDG-LAMP using REP-529 and B1 were 1 and 100 fg, respectively. In this study, 18 and 16 patients were positive in qPCR using REP-529 and B1, respectively. Based on the results of UDG-LAMP, 15 and 14 patients were positive using REP-529 and B1, respectively. Results of the study on patients with active ocular lesion showed that sensitivity of REP-529 and BI targets included 64 and 63%, respectively using qPCR. Sensitivity of 62 and 61%, were concluded from UDG-LAMP using REP-529 and B1 in the blood cases of active ocular lesion. qPCR was more sensitive than UDG-LAMP for the detection of Toxoplasma gondii DNA in peripheral blood samples of patients with clinically diagnosed toxoplasmic chorioretinitis. Furthermore, the REP-529 included a better detection rate for the diagnosis of ocular toxoplasmosis in blood samples, compared to that the B1 gene did. Moreover, the qPCR and UDG-LAMP specificity assessments have demonstrated no amplifications of DNAs extracted from other microorganisms based on REP-529 and B1. CONCLUSIONS: Data from the current study suggest that qPCR and UDG-LAMP based on the REP-529 are promising diagnostic methods for the diagnosis of ocular toxoplasmosis in blood samples of patients with active chorioretinal lesions.

Immunodiagnosis and molecular validation of Toxoplasma gondii-recombinant dense granular (GRA) 5 protein for the detection of toxoplasmosis in hemodialysis patients.

Toxoplasmosis causes serious complications in immunocompromised and pregnant women. Serological tests for the detection of toxoplasmosis are often designed from parasitic tachyzoites antigens. The process of producing these antigens is very difficult. The purpose of this study was evaluation of T. gondii-rGRA5 for the immunodiagnosis and molecular detection of Toxoplasma infection using enzyme-linked immunosorbent assay (ELISA) and LAMP methods in hemodialysis patients. The GRA5 gene was successfully expressed and purified by affinity chromatography assay and evaluated by western blot. Then it was used to design an ELISA assay. A total of 260 samples were tested for anti-Toxoplasma IgG and IgM antibodies using a commercial ELISA kit and designed ELISA kit. Finally, the LAMP method was used to evaluate the precision and reliability of the results obtained by commercial and designed ELISA kits. The consistency of the results of two methods was analyzed using the Kappa coefficient of agreement. The rGRA5 revealed higher immunoreactivity with 1:100 dilution of sera from toxoplasmosis patients. The specificity and sensitivity of the assay were 93% and 96%, respectively. According to the Kappa coefficient, there was a substantial correlation between the results of ELISA and LAMP based on rGRA5 (≈98%, p < 0.001). Also it showed that rGRA5 protein can be used as an antigenic protein for designing sero-diagnostic tests to identify Toxoplasma infection especially in hemodialysis patients.

Accurate and rapid detection of Fasciola hepatica copro-DNA in sheep using loop-mediated isothermal amplification (LAMP) technique.

Fascioliasis is a parasitic infection caused by Fasciola spp. in humans and animals. Despite significant advances in vaccination and new therapeutic agents, little attention has been paid to validate methods for the diagnosis of fascioliasis in animals. This study aimed to compare the loop-mediated isothermal amplification (LAMP) technique with PCR assay for the diagnosis of F. hepatica in sheep. In this cross-sectional study, 195 stool samples were collected from sheep for 3 months in Lorestan province, West of Iran. Specimens' parasitological examination was performed by using the direct wet mount and formalin-ether concentration method. After DNA extraction from the samples, molecular analysis was done using PCR and LAMP techniques based on the Fasciola ribosomal intergenic spacer (IGS) sequence. Of 195 specimens of sheep, 11 specimens were identified as F. hepatica-positive infection by using microscopic, PCR and LAMP assays. Kappa agreement test results showed that there was a significant agreement between the results of microscopic examination diagnostic tests, PCR and LAMP (Kappa = 0.51-0.72 and p < .001). According to the results of chi-square comparisons between parasite prevalence applying different techniques and variables of age, sex breed, and type of drinking water, there was no significant relationship (p ≥ .05). However, most of the infected sheep with Fasciola were 3- to 4-year-old females, of the Lori breed and consumed tap water. In many endemic areas, successful prevention and treatment of fascioliasis in animals depend on rapid and accurate diagnosis. Based on the results of the Kappa agreement, the significant agreement among the results of the microscopic examination, PCR and LAMP indicates the accuracy and reliability of these tests in the diagnosis of F. hepatica in sheep. However, molecular methods, especially the LAMP technique, are suggested because of their higher sensitivity and reliability for the diagnosis of F. hepatica even under field conditions.

Molecular evaluation of Toxocara species in stray cats using loop-mediated isothermal amplification (lamp) technique as a rapid, sensitive and simple screening assay.

Toxocara species are parasitic nematodes of dogs and cats with a worldwide distribution. The adult worm lives in the intestine, and horizontal transmission of the infection occurs through eating paratenic host or embryonated eggs. This study aimed to estimate the molecular prevalence of Toxocara species in stray cats using the loop-mediated isothermal amplification (LAMP) technique. A total of 95 stool samples were randomly collected from stray cats in Khorramabad city in western Iran. Microscopic examination was performed after the separation and extraction of supernatants. The LAMP reaction was performed using the internal transcribed spacer 2 (ITS2) gene primers of Toxocara species and the appropriate master mix. The overall prevalence of Toxocara spp. in stray cats was 20% (19/95, CI 95%: 0.2 ± 0.08) by parasitological and molecular assessments. The microscopic examination of stool samples revealed that 19 samples were positive for Toxocara. The same 19 positive samples were also positive by the LAMP technique. Interestingly, based on the results of the LAMP assay, out of 95 studied samples, 18 (18.94%; CI 95%: 0.19 ± 0.08) specimens were Toxocara canis, while only 1 (1.05%; CI 95%: 0.005 ± 0.01) sample was diagnosed as Toxocara cati. The relatively high prevalence of Toxocara species in the studied cats shows the role of this species in spreading the parasite and the role of the cats in transmitting this zoonotic parasite. Preventive measures including the control of stray cat's population by castration and protection of public gardens where children play are recommended. The easy, highly sensitive and specific LAMP method is proposed for the differential detection of Toxocara species in animals and humans.

A loop-mediated isothermal amplification (LAMP) assay for detection of Toxoplasma gondii infection in women with spontaneous abortion.

The present study aimed to use the loop-mediated isothermal amplification (LAMP) technique in comparison with serological tests to determine the rate of T. gondii infection in women suffering from spontaneous abortion (SA). A total of 140 women suffering from their first SA were included in this study. The collected aborted fetal remains and blood samples from each patient were examined in sterilized conditions using the LAMP technique and ELISA. Of the 140 women, 80 (57.1%) tested seropositive for anti-Toxoplasma antibodies by ELISA, 72 (51.4%) women tested seropositive for the IgG antibody, 8 (5.7%) tested seropositive for the IgM antibody. Among the eight women who'd had their first SA who tested seropositive for IgM antibody by ELISA, only five cases (62.5%) reported positively to the LAMP test. The difference in the frequency distribution of the LAMP results for measuring the Toxoplasma infection in pregnant women under study was statistically significant (P < 0.001) from the results of the serological test (ELISA). Although there was a significant difference between age and positivity in the LAMP test (P = 0.017), no significant difference was observed between positivity in the LAMP test and other variables. The findings of the present investigation suggest that LAMP is a preferred method for determining Toxoplasma infection in pregnant women suffering from SA compared with other routine serological tests. Even in a field with limited facilities and equipment, this technique can be effective and efficient in accurately and specifically diagnosing Toxoplasma infections in women at high risk of SA.

Loop-Mediated Isothermal Amplification (LAMP) Assay to Detect Toxoplasmosis in Schizophrenia Patients.

BACKGROUND: Toxoplasma gondii (T. gondii) causes an important parasitic infection known as toxoplasmosis, which is a globally distributed important zoonosis. One of the major serious characteristics of T. gondii is its ability to manipulate the behavior of intermediate hosts. We performed a cross-sectional study to determine toxoplasmosis in schizophrenic patients, as one of the major neuropsychiatric disorders, using loop-mediated isothermal amplification (LAMP) technic by targeting parasite B1 gene. METHODS: Blood samples were taken from 118 schizophrenic patients hospitalized in tow hospitals including Baharan, Clinic of Psychiatric Ali-ibn-Abi-Talib Hospital (in Zahedan City), and Amir-al Momenin Psychiatric Hospital (in Zabol City), Sistan and Baluchestan Province, southeast Iran in 2016. They were analyzed using LAMP, and compared with the previous data of nested-PCR and serology. RESULTS: Out of the 118 schizophrenic individuals, 56 patients (47.4%) were found to be infected with T. gondii. The diagnosis of toxoplasmosis was confirmed in 41 patients (34.7%) via the nested-PCR. The seroprevalence of toxoplasmosis in schizophrenic patients was 55.9% (66/118). CONCLUSION: We found a high efficiency of LAMP method in identifying toxoplasmosis and its high prevalence among schizophrenic patients. Our findings could provide viable offer implications for the prevention of schizophrenia.

Depression and Toxoplasma gondii infection: assess the possible relationship through a seromolecular case-control study.

Depression disorder is one of the most common psychological recognitions that characterized by sadness, low self-confidence, and disinterest in every activity. Considering evidence showing the effects of toxoplasmosis on the psychological disease, this study conducted to investigate the serological and molecular aspects of Toxoplasma gondii infection among patients with depression. In this study, after selecting the patients with depression and control groups under the supervision of a psychologist, the blood samples were collected and the serum samples and buffy coat were separated. The specific anti-Toxoplasma IgG antibodies in serum samples were evaluated using the commercial ELISA kit. Then the desired region of the Toxoplasma B1 gene was amplified using the specific primers. To confirm the specificity of primers to amplify the B1 gene of Toxoplasma, the extracted PCR product was sequenced. The overall prevalence of toxoplasmosis in patients with depression was 59.8 and 60.19% by ELISA and PCR, respectively. In the control group, the prevalence of Toxoplasma was 56.3 and 40.2% by serology and PCR. There was a significant correlation between the prevalence of toxoplasmosis and depression. Moreover, a significant difference was found between the variables of age, sex, kind of nutrition, level of education and toxoplasmosis among the two cases and control groups. The higher prevalence of Toxoplasma infection among patients with depression compared with the control group indicates the probable impact of this parasite on depression and exacerbates its symptoms, which requires special attention of specialist physicians and patient's relatives.

Challenging TaqMan probe-based real-time PCR and loop-mediated isothermal amplification (LAMP): the two sensitive molecular techniques for the detection of toxoplasmosis, a potentially dangerous opportunistic infection in immunocompromised patients.

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.

Comparison of the RE-529 sequence and B1 gene for Toxoplasma gondii detection in blood samples of the at-risk seropositive cases using uracil DNA glycosylase supplemented loop-mediated isothermal amplification (UDG-LAMP) assay.

Diagnosis of toxoplasmosis is an important issue, especially in at-risk patients. The molecular methods showed a promising future for such diagnosis; however, the method itself and the target sequence to be detected is an important part of accurate detection of the infection. The aim of the present study was to evaluate the RE-529 sequence and B1 gene for Toxoplasma gondii detection in blood samples of the at-risk seropositive cases using uracil DNA glycosylase supplemented loop-mediated isothermal amplification (UDG-LAMP) assay. In this study, 110 T. gondii seropositive at-risk individuals (pregnant women and immunocompromised patients) and 110 seronegative controls were enrolled. The two most studied sequences (RE-529 and B1) were used and compared for accurate and reliable detection of T. gondii in blood samples using UDG-LAMP assay and compared with real-time PCR method. The detection limit, accuracy, and reliability of UDG-LAMP for the parasite's DNA were also studied. Among 110 studied cases, 39 (35.45%) and 36 (32.7%) were positive for T. gondii DNA with the RE-LAMP and B1-LAMP, respectively. The seronegative cases remained negative for T. gondii DNA with the studied genes, however, there were few false negatives compared with real-time PCR method. The detection limit of the UDG-LAMP for both DNA targets was 0.16 tachyzoite's DNA per reaction tube. Based on the results of this study, the RE-529 sequence has a better detection rate compared to the B1 gene for toxoplasmosis among at-risk people. UDG-LAMP is a highly sensitive, accurate, and reliable method with no false-positive results for the diagnosis of T. gondii infection in blood specimens, however few cases may be missed.

Oncogenesis and Tumor Inhibition by MicroRNAs and its Potential Therapeutic Applications: A Systematic Review.

MicroRNAs appear as small molecule modifiers, which improve many new findings and mechanical illustrations for critically important biological phenomena and pathologic events. The best-characterized non-coding RNA family consists of about 2600 human microRNAs. Rich evidence has revealed their crucial importance in maintaining normal development, differentiation, growth control, aging, modulation of cell survival or apoptosis, as well as migration and metastasis as microRNAs dysregulation leads to cancer incidence and progression. By far, microRNAs have recently emerged as attractive targets for therapeutic intervention. The rationale for developing microRNA therapeutics is based on the premise that aberrantly expressed microRNAs play a significant role in the emergence of a variety of human diseases ranging from cardiovascular defects to cancer, and that repairing these microRNA deficiencies by either antagonizing or restoring microRNA function may yield a therapeutic benefit. Although microRNA antagonists are conceptually similar to other inhibitory therapies, improving the performance of microRNAs by microRNA replacement or inhibition that is a less well- described attitude. In this assay, we have condensed the last global knowledge and concepts regarding the involvement of microRNAs in cancer emergence, which has been achieved from the previous studies, consisting of the regulation of key cancer-related pathways, such as cell cycle control and the DNA damage response and the disruption of profile expression in human cancer. Here, we have reviewed the special characteristics of microRNA replacement and inhibition therapies and discussed explorations linked with the delivery of microRNA mimics in turmeric cells. Besides, the achievement of biomarkers based on microRNAs in clinics is considered as novel non-invasive biomarkers in diagnostic and prognostic assessments.

Diagnosis of Candida albicans: conventional diagnostic methods compared to the loop-mediated isothermal amplification (LAMP) assay.

Candida species cause a wide range of opportunistic infections in humans and animals. The detection of Candida species by conventional diagnosis methods is costly and time consuming. This study was conducted for the first time to evaluate and compare a relatively new molecular assay and the loop-mediated isothermal amplification (LAMP) technique with conventional methods for detection of Candida albicans. In this study, 70 different species of Candida identified by conventional methods were cultured on Sabouraud chloramphenicol agar medium and then the genomic DNA was extracted. The LAMP technique was performed using specific primers targeting the ITS2 gene of C. albicans. The analytical sensitivity and specificity of LAMP were measured using a tenfold serial dilution prepared from extracted DNA from standard C. albicans strain from 1 ng to 1 fg and the DNA samples of other clinical Candida species and three non-Candida yeast. Out of 70 yeast samples analyzed by LAMP technique, 24 samples (34.3%) were positive for C. albicans. Comparison of the results showed that the CHROMagar Candida and germ tube production methods are quite consistent with the LAMP technique, while the agreement amount between the results of carbohydrate assimilation and chlamydoconidia generation assays and LAMP technique was 98.5% and 72.8%, respectively. The detection limits of the LAMP assay were 10 fg of the DNA from the standard C. albicans strain. No amplification was observed in the DNA samples of other yeast species and only the DNA sample of standard C. albicans strain was amplified. Based on the results, it can be concluded that the LAMP method is as specific and precise as common diagnostic methods, but is faster, easier deployable or more sensitive. Therefore, this method can be used as a suitable complementary assay for Candida diagnosis in medical diagnostic laboratories and field conditions.