Induction of Artesunate Resistance in Plasmodium falciparum 3D7 Strain Using Intermittent Exposure Method and Comparing P.fk13 Sequence between Susceptible and Resistant Strains.

Background: Resistance to artemisinin has threatened major achievements in malaria control, more investigations is needed about resistant strains and related genes. We aimed to induce resistance to artesunate in the Plasmodium falciparum 3D7 strain using intermittent exposure method and comparing P.fk13 gene sequence between susceptible and resistance strains. Methods: P. falciparum 3D7 strain was cultured according to Trager & Jensen method with some modifications. Serial concentrations between 10-2 mol/l, to 10-7mol/l were prepared, then P. falciparum 3D7 was exposed to each of the dilution to determine IC50 and lethal dose. Sensitivity reduction process was started from the concentration of 10-7mol/l and ended at 10-2mol/l. Exposed parasites were collected after at least 27 days after cultivation in each drug concentration. DNA extraction, PCR and sequencing process were performed to investigate any possible mutations in the P.fk13 gene sequence. Results: Effectiveness of 10-2mol/l concentration of artemisinin was found as a lethal dose. IC50 value was equal to 5×10-4 mol/l. The resistant strain was provided in the lab, sequenced and registered in the gene bank as P.f Art -2, (accession number MH796123. 1). Alignment of this registered sample showed no mutation in P.f kelch13 gene in comparison with standard strain submitted in the GenBank. Conclusion: Resistance to artesunate in malaria parasite may occur but with no mutation in the P.f kelch13 gene. Therefore, whole genome sequencing should be applied to determine mutations in resistant strains.

Therapeutic Strategies for COVID-19 Patients: An Update.

The novel coronavirus SARS-coV-2, which emerged in Wuhan in November 2019, has increasingly spread, causing a global pandemic that infected more than 444 million people, resulting in severe social and economic ramifications, and claimed more than 6,010,000 lives by March 5, 2022. The pandemic attracted global attention with consequential multiple economic, social, and clinical studies. Among causes of poor clinical outcomes of the disease are therapeutic challenges, leading to spirals of studies in search of better therapeutic alternatives. Despite the worsening circumstances of the pandemic, no drug has yet shown remarkable efficacy in the clinical management of COVID-19 patients in large-scale trials. Many potential therapeutic strategies, including the use of nucleotide analogs, chloroquine phosphate, arbidol, protease inhibitors (lopinavir/ritonavir), plasma, monoclonal antibodies, plastic antibodies based on molecularly imprinted polymers (MIPs), traditional Chinese medicine (TCM), nanomaterials, vaccine, and mesenchymal stem cells (MSCs), have emerged with various degrees of successes. Remdesivir and dexamethasone have now been licensed based on the results of randomized controlled trials. Baricitinib, the Janus kinase (JAK) 1/2 inhibitor, is also an attractive candidate due to its properties as a potent anti-inflammatory agent and its hypothesized offtarget antiviral effects against SARS-CoV-2. Besides, human plasma from recovered COVID-19 patients is theoretically expected to be safe and effective for both therapy and post-exposure prophylaxis. In light of the literature, the correlation between the reduction of C5aR1/C5aR2 and the IL6-IL6R axis, using the available anti-IL6R mAb would be crucial. Moreover, MSCs are a potential therapeutic choice for patients with COVID-19 pneumonia. The coronavirus spike (S) protein that mediates the process of the infection via binding of host cells to the virus receptor is an essential focus for vaccine development. Importantly, with the number of patients increasing daily, there is an urgent need for effective therapeutic intervention. In this review, we expatiated on several strategies deployed for the treatment of COVID-19 infection.

Serological responses to a soluble recombinant circumsporozoite protein-VK210 of Plasmodium vivax (rPvCSP-VK210) among Iranian malaria patients.

BACKGROUND: Circumsporozoite protein (CSP) has a central immune domain that includes short regions of repeating amino acid sequences. This immunodynamic region is an epitope of B cells that can elicit an immune response in human and laboratory animals. The aim of the present study was to express the recombinant PvCSP-VK210 antigen and evaluate it for assaying antibodies obtained during human P. vivax infection by Western blotting and indirect ELISA (enzyme-linked immunosorbent assay). METHOD: Genomic DNA of P. vivax was isolated from a blood sample of an Iranian person with vivax malaria, and by PCR, the fragment of the PvCSP-VK210 gene was amplified. The gene fragment was cut after gel purification by BamHI and HindIII enzymes and then cloned into pET28a expression vector. Finally, the recombinant pET28a was transformed into the E. coli BL21 (DE3) as the expression host. In order to produce His-tagged protein, the expression host was cultured in LB medium. The protein was purified by Ni-NTA columns and immobilized metal affinity chromatography, and after confirmation by Western blotting technique, was used as the antigen in the indirect ELISA test. RESULTS: The recombinant protein was expressed and purified as a 32-kDa protein. The sensitivity and specificity of the indirect ELISA test with the recombinant PvCSP-VK210 antigen were 61.42% and 97.14%, respectively, based on OD = 0.313. Between the results of the microscopic test and the indirect ELISA test with the recombinant PvCSP-VK210 antigen there was a Kappa coefficient of 0.586. The positive and negative predictive value and validity of the ELISA test with the recombinant PvCSP-VK210 antigen were 95.55%, 71.57%, 79.28%, respectively. CONCLUSION: The sensitivity of the indirect ELISA method with the recombinant PvCSP-VK210 antigen was 61.42%, which is the first report from Iran.

Human Toxocariasis in individuals with blood disorders and cancer patients: the first seroepidemiological study in Iran.

Toxocara is one of the most prevalent nematodes in Iran, which infect humans as an intermediate host. Infection complications result from the larva migration. Human toxocariasis prevalence was various in Iran according to the area of study and population. This study was designed to evaluate the seropositivity of Toxocara IgG in patients with blood disorders and cancer patients in southwest Iran. Moreover, the study of the associated risk factors for this infection. A total of 1122 serum samples, from February 8, 2019 to August 21, 2019, including 600 healthy individuals and 522 individuals with cancer and blood disorders patients were collected. Serum samples were collected for detection of Toxocara IgG by using ELISA (Enzyme-Linked Immunosorbent Assay) kit. Sociodemographic data of all participants were collected and examined to determine their association with the infection. Out of 101 individuals with white blood cell disorders (5.94%), red blood cell disorders (7.48%) and cancer patients (11.06%) were seropositive for Toxocara IgG antibodies. The infection rate among all study population revealed that (10.76%) were positive for Toxocara IgG. This study showed the fundamental role of contact with pets and infection in groups with blood cell disorders (P-value ≤ 0.05%); while in cancer patients the association wasn't significant. Other factors such as age, location of residence, and sex showed that the association with this infection wasn't significant.

Interaction between Chitosan and Chloroquine against Plasmodium berghei and P. falciparum Using In-Vivo and In-Vitro Tests.

BACKGROUND: The use of antimalarial drugs with number of compounds in combination form may potentiate each other's activity. METHODS: This study was conducted in the School of Public Health, Tehran University of Medical Sciences, Tehran, Iran in 2018. It was based on two methods including in vivo and in vitro tests with aim of considering interaction between chitosan and chloroquine against Plasmodium berghei and P. falciparum parasites using different ratios of the agents with ED50s and IC50s baselines. RESULTS: Administrating 10 and 20 mg/kg (mouse body weight) of chitosan alone to the P. berghei -infected mice up to 4 successive days resulted in 37% and 45% inhibition of P. berghei respectively, while employing the compound with chloroquine in combination form with ratios of 90/10 and 70/30 (chloroquine/chitosan) had a considerable potentiation including 71.58% and 83.85% inhibition effectiveness against P. berghei. Moreover, 20 mg/L (CCM) concentration of chitosan alone could eliminate 69.55% of P. falciparum in culture medium while in combination with chloroquine in ratios of 90/10 (chloroquine/chitosan) had considerable potentiation including 79.14% inhibition effectiveness. Mean survival time of those mice received combination therapy in ratios of 90/10 and 70/30 (chloroquine/chitosan) was longer than those took up mono therapy of either chloroquine or chitosan based on their ED50s doses. CONCLUSION: Interaction between chloroquine and chitosan showed considerable potentiation in combination form against either P. berghei or P. falciparum using in vivo and in vitro tests respectively. Meanwhile, interaction between the above mentioned agents resulted in a notable survival time for those P. berghei-infected mice treated with the combination.

Parasite-derived microRNAs as a diagnostic biomarker: potential roles, characteristics, and limitations.

MicroRNAs (miRNAs), a subclass of small regulatory RNAs that present from ancient unicellular protozoans to parasitic helminths and parasitic arthropods. MiRNAs' mode of action has attracted wide attention as a result of their unique functional importance. MiRNAs play a role in diverse physiological and pathological processes ranging from organ development, immune function to apoptosis and cancer at the post-transcription gene expression. Thus, miRNAs are known to be targets for clinical treatment and therapy. The discovery of the high stability of circulating miRNA in various types of host body fluids, such as whole blood, serum, plasma, saliva, and urine has increased great interest among researchers in the potential of circulating miRNA as a prognosis/diagnosis of infectious. Some circulating miRNAs biomarkers advanced to clinical applications related to human diseases. However, this idea starts to come only in the fields of infectious disease. The goal of this review is to enhance the current understanding of these molecules and their applicability in the field of medicine. A detailed review of the available literature consulting tools performed in online repositories such as NCBI, PubMed, Medline, ScienceDirect, and UpToDate. This review summarizes an overview of preclinical studies using circulating miRNAs biomarkers against infectious diseases affecting humans. The use of miRNA as a safe and potential tool is encouraging news, considering that until now, guidelines for the use of miRNA in clinical practice are still lacking.

Molecular Characterization and Phylogeny of Taenia hydatigena and Echinococcus granulosus from Iranian Sheep and Cattle Based on COX1 Gene.

Hydatid cyst, the larval stage of Echinococcus granulosus, and Cysticercus tenuicollis, the larval stage of Taenia hydatigena, are prevalent in domestic, livestock, and wild ruminants. The main goal of this research was to identify the isolates of E. granulosus and C. tenuicollis by partial sequencing with PCR amplification of the cytochrome C oxidase 1 (COX1) gene. During a routine veterinary inspection at a Chabahar city slaughterhouse, two samples of hydatid cysts from sheep's liver and cattle's lung and two samples of C. tenuicollis from sheep's liver were collected. After DNA extraction, the fragment of the COX1 gene was amplified by the PCR method. Sample sequences were modified and synchronized by Chromas and CLC genomic workbench 11 software. Sequence analysis was carried out by BLAST algorithms and GenBank databases. Phylogenetic trees were performed using MEGA 7 software and the neighbor-joining and maximum likelihood method for T. hydatigena and E. granulousus. The result indicated that the main genotype of parasites and the amplified fragment size were G1 and approximately 455 bp, respectively. The analysis of phylogenetic trees based on nucleic acid for four samples showed that there was a common ancestor. However, the shift in nucleotides in the two isolates in E. granulosus and the two isolates of T. hydatigena were non-synonymous type and synonymous type, respectively. The present study showed that the dominant genotype in all isolates was G1 and this report was similar to other studies in Iran and the world. Also, the partial COX1 gene sequence was matched with T. hydatigena.

Spatial analysis of Toxocara spp. eggs in soil as a potential for serious human infection.

Toxocara spp. cause one of the most widespread soil-transmitted helminthic infections worldwide. In both developed and developing countries, soil contamination with Toxocara eggs is considered as a major threat to public health. A total of 515 soil samples from 89 sampling sites were collected from different locations of public health such as Wastelands and Streets, public parks, and marginal areas. The soil samples were examined for Toxocara eggs using a centrifugal-floatation technique utilizing a saturated sodium nitrate solution. centralization of positive soil samples in the province was studied by Spatial Statistics Techniques such as Average Nearest Neighbors and Spatial Autocorrelation and Kernel Density Function Toxocara spp. eggs were found in 94 (18.25 %) out of 515 samples collected from the studied areas. According to the results obtained, marginal areas are often contaminated with eggs of Toxocara. Consequently, preventive measures including health education should be implemented to reduce the potential risk of this parasitic infection.

Seroprevalence of human hydatid cyst: a cross sectional study in a rural areas of Zahedan, southeastern Iran.

Hydatidosis or cystic echinococcosis (CE) is one of the most common zoonosis diseases. Iran is one of the endemic regions in terms of this disease. For the first time, the present study was conducted to determine the seroprevalence of hydatid cyst in Zahedan rural areas due to the importance of human CE and lack of information in this region. The present study was performed on 551 people referred to seven rural health centers in Zahedan during 2019-2020. Serum samples were collected and analyzed by indirect ELISA method using recombinant antigen B subunit B8/1. Results were analyzed by SPSS (version 22) software and Chi-square test. The CE seroprevalence was 4%. The most positive cases were in the age group of 10-30 years. The highest infection was reported in homemakers. A significant relationship (P-value<0.05) was only reported between the seropositivity to hydatid cyst and the presence of dogs in the environment. The present study's findings indicated human hydatid cyst in rural areas of Zahedan is a health problem; moreover, the control and prevention principles and analysis of various epidemiological aspects of this disease should be considered.

Genotyping and Phylogenetic Analysis of Plasmodium vivax Circumsporozoite Protein (PvCSP) Gene of Clinical Isolates in South-Eastern Iran.

BACKGROUND: Circumsporozoite protein (CSP) is one of the most important surface sporozoite antigens in malaria, recently considered as a candidate for vaccination. Considering the importance of CSP, this study was conducted to investigate the polymorphism and genetic diversity of Plasmodium vivax Circumsporozoite Protein (Pvcsp) in the southeastern region of Iran during 2015-2016. METHODS: To investigate polymorphism and genetic diversity, 20 blood samples were collected from patients with P. vivax, then DNA was extracted and amplified using partial sequence of CSP gene. Polymerase chain reaction (PCR) products were sequenced and compared to sequences from genomic databases using BLAST. Genetic evaluation and phylogenic analysis were performed using MEGA7 and DnaSP5 software's on 38 sequences include 20 sequences of our study and 18 sequences of Gene Bank. RESULTS: Eleven isolates were VK210 genotype and 9 isolates contained VK247. The result of variable segregation nucleotide site indicated that the differentiation of sequences in CSP were 25.67% in our 20 samples which are less than the 38 samples with a value of 26.67%. Comparing the ratio of dN/dS regions in the CSP gene indicates that the CSP varies more synonymously and amino acid has lower variation. Out of 38 samples, 35 unique haplotypes were identified based on 1042 nucleotide sequences in CSP, showing a variation percentage of 99.4%. CONCLUSION: The Tajima D analyses showed that CSP gene in P. vivax had a positive number in the total analyzed sequences, which means that the P. vivax mutations are in order to select positive evolution.

Environmental soil contamination by Toxocara species eggs in public places of Ilam, Iran.

INTRODUCTION: The purpose of the study is to assess environmental contamination by Toxocara species eggs in public places in the city of Ilam, Ilam Province, southwest Iran. MATERIAL AND METHODS: Between September 2018 and March 2019, 130 soil samples were collected from public places of 5 district municipalities of Ilam, southwest Iran. Soil samples were examined by microscopy following flotation method by sodium nitrate. RESULTS: Soil analysis showed that 5.88% of the soils stored, 52.54% from gardens, 29.42% from rubbish, and 11.72% from green spaces were contaminated with Toxocara spp. eggs. In total, 13.08 % of soil samples (17/130) were positive for Toxocara eggs (P > 0.05). CONCLUSIONS: The findings revealed that care should be taken when using soil from gardens, green spaces and rubbish, and also should be seriously considered because of the potential issues of toxocariasis and also the risk to the public.

Seroprevalence of Toxoplasma gondii and Toxocara spp. infections among pregnant women with and without previous abortions in the west of Iran.

AIM: In spite of the importance of toxoplasmosis and toxocariasis among the high-risk groups, such as pregnant women, the infections are categorized as a neglected tropical disease by the World Health Organization. Toxoplasma gondii and Toxocara spp. infections can cause systemic and ocular diseases in infants during pregnancy. In this study, we investigated seroprevalence and risk factors of toxoplasmosis, toxocariasis and their co-infection in pregnant women and non-pregnant women referred to the healthcare facilities of Ilam province, west of Iran. METHODS: A total of 378 sera samples (189 pregnant women and 189 non-pregnant women) was investigated for the presence of IgG antibodies against T. gondii and Toxocara spp. by Enzyme-linked immunosorbent assay (ELISA). The samples of all pregnant women with abortion (56 cases) were also evaluated for IgM anti-toxoplasmosis antibody by ELISA method. Moreover, associated factors were obtained from the participant's questionnaires. Data analysis for this study was performed using the spss software version 20. RESULTS: Seroprevalence of T. gondii, Toxocara spp., and their co-infection in pregnant women was 39.7%, 21.2% and 9.5%, respectively. Regarding the risk factors, the contact with a cat (P = 0.04) and dog (P = 0.00) were significantly associated with T. gondii and Toxocara spp., respectively. CONCLUSION: This study highlighted the importance of serological diagnosis before pregnancy. Moreover, we believe that more epidemiological studies are needed for a better understanding of overlaps between T. gondii and Toxocara spp. in pregnant women.

Identification of Alleles in the MSP1 Gene Related to Complicated Malaria in Patients Infected with Plasmodium falciparum in Southeast of Iran.

BACKGROUND: To overcome human malaria problem several solutions have been employed including extensive studies in the field of Plasmodia relevant antigens. The aim of this study was to determine allelic variation in the MSP1 gene of Plasmodium falciparum among some falciparum malaria-infected patients in Southeastern Iran. METHODS: Twenty P. falciparum positive cases were enrolled from Sistan and Baluchistan Province, southeastern Iran in 2013-15. From each case, 1.5ml of peripheral blood was collected into EDTA contained tubes. Thick and thin blood smears were stained with standard Giemsa stain and were checked with conventional microscopical method. DNA was extracted from blood samples and amplification of block 2 MSP1 was performed using specific primers. Gel electrophoresis was done and results showed some amplification fragments corresponding to block 2 regions of Pf MSP1 gene. Finally, four samples from different allelic types were sent for sequencing process. RESULTS: Fragments were different in size, so classified into six allelic types as kinds of 1-6 based on happening frequencies. Digestion of PCR products revealed two sub allelic types (A and B) within allelic types 2 and 3, but not in allelic types 1, 4, 5 and 6. Twenty percent of samples were sent for sequencing. Sequence alignment showed 78.95% to 91.83% identity between samples. CONCLUSION: Identity between samples and phylogenetic tree revealed that there is an extensive diversity range among isolates. Fifty percent of the isolates were under the risk of complicated malaria. Two of these patients (10%) needed special care and recovery was obtained after getting hospital services.

Risk factors and prevalence of toxocariasis in pregnant women and diabetic patients compared to healthy adults in Ilam province, western Iran.

Toxocara is one of the common intestinal nematodes in dogs and cats and is the agent of tissue migratory larvae in humans. Customarily, the prevalence of human toxocariasis hovers around 15.8 % in Iran. Furthermore, other research outcomes demonstrated a tendency for an outbreak of toxocariasis in Iran. Therefore, we carried out a cross-sectional study and assessed the seroprevalence of toxocariasis humans in Ilam Province, western of Iran. A total of 539 serum samples were collected between September 2017 and March 2018 from patients referred to the Health Centers of Ilam province, Iran. Serum samples were investigated for the presence of Toxocara using IgG antibodies, ELISA (Enzyme-Linked Immunosorbent Assay) kit. Risk factors such as contact with cats and dogs, living in rural areas were investigated among the study population. Out of 539 total samples collected, 97 cases (17.99 %) were positive for anti-toxocara IgG antibodies. These antibodies were recovered from serum samples of otherwise healthy adults (15.54 %, 49/296), pregnant women (21.16 %, 40/189) and diabetic patients (14.81 %, 8/54). This study showed significant relationship between toxocariasis and contact with animal pets in all studied groups (P value ≤ 0.05) and a significant relationship between toxocariasis and living in rural areas among pregnant women (P value ≤ 0.05).