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Background: Crimean-Congo hemorrhagic fever virus (CCHFV) is a highly lethal virus that causes hemorrhagic fever in humans and is endemic in many countries, including Iran. Therefore, fast, accurate, and reliable diagnosis is crucial for patient management and outbreak control. Objectives: This study aims to optimize a TaqMan multiplex real-time RT-PCR for the rapid and specific diagnosis of CCHFV. Methods: In this study, the L (NC_005301.3) and S (NC_005302.1) fragments were used as reference sequences for blast analysis. The L and S sequence segments of CCHFV with more than 90% identity from different areas were downloaded from the Genbank database. Primers and probes were designed based on the best-conserved regions of CCHFV L and S sequence segments. To construct the plasmid, a 1751 bp fragment from the MS2 phage that was previously amplified using cloning primers was inserted into the pET-32a plasmid. The S and L segments of the CCHFV, which were 110 bp and 135 bp, respectively, were inserted downstream of the MS2 phage sequence from HindIII to NotI. The Viral-like particles (VLPs) were produced in Escherichia coli, strain BL-21(DE3), in the presence of 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG). The stability of VLP particles was confirmed in the presence of the ribonuclease enzyme. The fabrication of VLPs was approved by transmission electron microscopy (TEM) with negative staining (1% phosphotungstic acid). To validate the specificity of the primers and probes sequences, we compared them to the NCBI database and tested them experimentally using extracted DNA and RNA samples from healthy subjects and an infectious panel. Results: The VLPs showed complete resistance in the presence of the ribonuclease enzyme, and the TEM results confirmed that the VLPs were correctly produced. The TaqMan multiplex real-time RT-PCR confirmed that the primers and probes were designed correctly and were completely specific to the CCHFV. The limit of detection (LOD) of the multiplex assay for the L and S genes was one copy of the VLPs per µL. Conclusions: This TaqMan assay is reliable for amplifying CCHFV due to its design on conserved regions of the CCHFV sequences, which have minimal variability and high specificity.

Background: Abnormal position and presentation are challenges for obstetricians in about 10% of all pregnancies. Objectives: The purpose of this study was to compare ultrasound with transvaginal finger examination in detecting abnormal position and presentation of fetal head. Methods: This was a cross-sectional study performed in Ali ibn-e Abitaleb Hospital of Zahedan in 2018. The women underwent vaginal finger examination in the first phase of delivery, and then trans-abdominal ultrasound was performed for pregnant women. All examinations and ultrasounds were performed by one person with the same ultrasound device. Data were analyzed with SPSS software. Results: In this study, 100 pregnant women were studied in the first phase of pregnancy. The mean age of the subjects was 24.7 ± 5.5 years old. Fetal head position was determined in 81 and 93% of women with vaginal examination and ultrasound, respectively. There was weak agreement (kappa = 0.416). Fetal presentation was determined by finger examination in 93% of cases and by ultrasound in 100% of cases. There was moderate agreement (kappa = 0.783). Conclusions: In general, the results of this study showed that there was no significant difference between ultrasound and finger examination in the presentation and position of fetal head.

Background: Arginine metabolism is an important factor involved in tumorigenesis, progression, and survival of tumor cells. Besides, other metabolites produced in the arginine metabolism process, such as polyamines, nitric oxide, argininosuccinate, and agmatine, play key roles in different stages of tumor development. On the other hand, herbal metabolites are widely used to treat cancer. One of these herbal flavonoids is quercetin. Methods: In this study, according to MTT assay data, two concentrations of quercetin flavonoid were selected (57.5 and 115 µM) to treat human embryonic kidney 293 (HEK293) cells. Then RNA was extracted from the cells and used as a template for cDNA synthesis. Using real-time PCR, the expression of key enzymes involved in arginine metabolism was evaluated, including arginase 2 (Arg2), ornithine carbamoyl transferase (OTC), agmatinase (AGMAT), arginase 1 (Arg1), nitric oxide synthase 1 (nNOS), arginine decarboxylase (ADC), ornithine decarboxylase 1 (ODC), ornithine carbamoyl transferase (OCT), spermidine synthase (SRM), spermine synthase (SMS), argininosuccinate synthase 1 (ASS1), and argininosuccinate lyase (ASL). The Student t-test was used to analyze the data considering a P value of < 0.05 as the significance level. Results: Our results indicated significant changes in the expression of arginine metabolism enzymes after quercetin exposure, confirming a role for quercetin plant flavonoid in regulating arginine metabolism in HEK293 cells. Conclusions: Quercetin could alter the gene expression of the key enzymes involved in arginine metabolism. This was the first study investigating the effects of quercetin on arginine metabolism in HEK293 cells.

Background: Waterborne and foodborne diseases are considered to be major public health issues, which are associated with significant mortality each year worldwide. Viruses and most noroviruses (NoVs) are important causes of gastrointestinal infections. Objectives: The present study aimed to evaluate the gastroenteritis outbreak in Eslamabad-e Gharb in Kermanshah, Iran for the epidemiological, clinical, and laboratory assessment of the disease. Methods: This descriptive, cross-sectional study was conducted with an emphasis on the outbreak of waterborne gastroenteritis in Eslamabad-e Gharb in Kermanshah province in 2016. The sample population included 3,362 patients referring to the public and private centers, who completed the linear list. In addition, 87 water microbial samples, 205 chlorine assessment samples, and 293 human samples were examined. Data were collected using the linear list of the patients, and data analysis was performed in Excel and SPSS version 21 using chi-square. Results: Among 3,362 patients, 57.1% were female, and 42.9% were male. Most of the patients (21.1%) were aged 20 - 29 years, with the mean age of 26.8%. In total, 73.4% of the patients had diarrhea, while 3.2%, 73%, 50%, 48.8%, 29.1%, and 30.6% had bloody diarrhea, vomiting, nausea, abdominal cramps, fever, and headaches, respectively. None of the microbial water samples (n = 87) collected from the rural and urban regions of the city were infected with thermotolerant coliforms. Moreover, the chlorine assessment demonstrated that 7.8% of the samples were unfavorable and above the limited range. In total, 10 blood and 10 fecal samples were evaluated in Alebouyeh Laboratory. Furthermore, six out of 10 fecal samples had non-pathogenic E. coli, while three samples were positive for the NoV molecular test of real-time polymerase chain reaction. Conclusions: According to the results, NoV was the main cause of the disease outbreak in the studied region, and the clinical and epidemiological characteristics of the sample population, season, and environment were consistent with the diagnosis. Some of the factors that should be considered in the prevention and control of similar outbreaks include the development of a comprehensive and practical crisis management model in the epidemics, conservation of drinking water supplies and pipelines, and highlighting the importance of health education.

Background: The 3C protease is one of the most important proteases in enteroviruses which is responsible for several cleavages on viral polyproteins and maturation processes. Some reports indicated that this protease disrupts the interferon (IFN) signaling pathway; however, another report shows that 3C protease does not have such inhibitory effects on IFN signaling. Objectives: In this study, a construction was designed consisting of 3C protease to test its effects on Interferon type I signaling pathway. Methods: The designed construction containing 3C protease was transfected to rhabdomyosarcoma (RD) cells for evaluation of its effects on interferon signaling pathway. Rhabdomyosarcoma cells were treated with Interferon β for 4 hours and then the expression of two ISGs including MxA and OAS were analyzed by real-time PCR. All the proteins in interferon type I signaling pathway were also evaluated by Western blot analysis. Results: The results showed that 3C protease does not affect ISG expression after IFN induction. Western blot analysis also indicated that there is no difference in IFN pathway protein level between 3C protease treated and control groups. Conclusions: This report suggests that 3C protease alone does not have a significant effect on IFN pathway, in contrast to any of the previous reports.

Objectives: The aim of this study was to evaluate the etiologic agents of a hand, foot, and mouth disease (HFMD) outbreak in Ahvaz, Southwest Iran and their evolutionary analysis by phylogenetic construction and Simplot analysis. Methods: We collected 16 serum samples in an outbreak of HFMD in Ahvaz in October 2013. RNA was extracted from the samples and subjected to reverse transcription polymerase chain reaction for detection of Enterovirus group A and B. Positive cases were sequenced and subjected to phylogenetic and Simplot analysis for detecting the signs of recombination. Results: Of the 16 specimens, nine (56.25) were PCR-positive with the universal primers for the Enterovirus 5‘UTR region. Coxsackievirus A6 was detected as a predominant agent of HFMD with two cases of Echovirus 6 and Echovirus 30. In the case of Echovirus 6, the signs of recombination in the 5‘UTR region were observed based on phylogenetic and Simplot analysis. Conclusions: Coxsackievirus A6 is the main agent of HFMD in Ahvaz. The evidence of recombination in this isolate of Echovirus 6 in this study emphasizes common hygiene practices and sanitation to prevent the circulation of this isolate in community and the advent of new strains. © 2019, Archives of Clinical Infectious Diseases.

Background: Nowadays a number of methods are used for the extraction of genomic DNA from fecal specimens. Identification and selection of an effective DNA extraction method is considered as one of the most important steps in molecular assays of Giardia duodenalis. Objectives: We compared the effects of 3 different DNA extraction techniques in PCR amplification of a specific area of SSU rRNA gene. Methods: A total of 20 fecal samples containing purified cysts were aliquoted in 3 sub-samples. DNA extraction was performed using the Phenol-Chloroform Isoamyl alcohol (PCI), QIAamp DNA stool mini kit, and YTA Stool DNA Isolation mini Kit. The quantity and purity of extracted DNA were compared. The potency of extracted DNA was tested. Results: The results showed that the most concentrated DNA was obtained from phenol/chloroform/Isoamyl alcohol method and the best purity based on comparing the ratio of A260/230 was obtained from QIAamp DNA stool mini kit. In this study the diagnostic sensitivity of QIAamp DNA stool mini kit, phenol/chloroform/Isoamyl alcohol, and YTA Stool DNA Isolation mini Kit methods was 60%, 70%, and 60%, respectively. Conclusions: The application of a proper DNA extraction method leads to obtaining reliable and reproducible results in molecular assays and supports treatment and control strategies of G. duodenalis. In this study, PCR amplification targeting a 350-bp fragment of SSUrRNA gene demonstrated phenol/chloroform/Isoamyl alcohol as the most efficient method.

Background: Cutaneous leishmaniasis is one of the most important tropical diseases in many parts of the world caused by two main species of parasites including Leishmania major and L. tropica in the old world. Several attempts have been made to reach an effective and reliable vaccine against the disease with a spectrum of success. Selection of an effective target for vaccination needs to recognize a protein antigen with minor diversity. Leishmania activated C kinase (LACK) antigen is one of the good targets for this purpose that needs to be evaluated for its diversity in local study areas. Objectives: This study was conducted to evaluate the diversity in LACK protein among L. major species. Methods: We collected 30 L. major isolates from different parts of Khuzestan province, southwest of Iran. Leishmania activated C kinase gene was selected for amplifying by PCR and sequencing. Results: Phylogenetic tree showed that there is no diversity in LACK gene in this area. Conclusions: Leishmania activated C kinase gene is a very conservative gene, suitable for vaccination.

Background: Hepatitis E virus (HEV) is able to induce fulminant hepatitis E infection in immunosuppressed individuals. Previous studies showed that the Baculovirus expression system (BES) could be used for developing hepatitis E virus-like particles (VLPs). Objectives: In the present study, the formation of VLPs of the recombinant proteins of HEV truncated open reading frame 2 (ORF2; 112 - 607) and Rotavirus non-structural protein 4 (NSP4) (OSU-a) were investigated in BES and Escherichia. coli. Methods: To construct VLPs, the truncated ORF2-NSP4 protein was expressed in BES. The expression of protein was confirmed by western blot. This protein was expressed in E. coli. The truncated ORF2-NSP4 gene was subcloned in pET28a, and then transformed into E. coli DH5α. The confirmed colonies were transformed into E. coli BL21. The solubility of protein were checked by SDS-PAGE and western blot analysis. In the final step, to verify the VLP formation in BES and E. coli, the recombinant proteins were stained with 2% uranyl acetate and checked by transmission electron microscopy (TEM). Results: The 75 KDa truncated ORF2-NSP4 protein was successfully expressed in Sf9 cells, assembled, and formed VLP according to TEM results. In the prokaryotic expression system (E. coli), the ORF2-NSP4 gene was successfully subcloned and expressed in BL21 but VLP was not detected in TEM analysis. Conclusions: The truncated ORF2-NSP4 VLP were efficiently expressed in the SF9 cells, as a potential mucosal vaccine against HEV and Rotavirus. In the prokaryotic expression system (E. coli), the ORF2-NSP4 gene was successfully expressed.

Background: Nontypeable Haemophilus influenzae (NTHi) is responsible for diseases such as otitis media, sinusitis, bronchitis, and pneumonia. Unlike H. influenzae Serotype b (Hib), there is no vaccine against diseases induced by NTHi. High molecular weight1 (HMW1), high molecular weight 2 (HMW2), and H. influenzae type a (Hia) proteins are critical protein adhesions of NTHi with the potentiality to provide the protection. Objectives: The current study aimed at investigating the potential of recombinant HMW1555-914, HMW2553-916, and Hia585-705 proteins as subunit vaccines to induce immune responses after active immunization in Bagg albino (inbred research mouse strain) or Balb/c mice. Methods: HMW1555-914, HMW2553-916, and Hia585-705 amplified by polymerase chain reaction (PCR) were cloned into pET28a. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the Western blotting analysis were carried out to investigate the characteristics of proteins. Proteins were purified by Ni-nitrilotriacetic acid (Ni-NTA) as a gel matrix. Mice (Balb/c) were immunized subcutaneously with HMW1555-914, HMW2553-916, and Hia585-705 proteins, alone or in binary and ternary combinations. Eventually, specific antibody responses were evaluated by the enzyme-linked immunosorbent assay (ELISA) and serum bactericidal assay (SBA). Results: Antibody responses significantly increased in Balb/c immunized by ternary combination compared with the control group. IgG1/IgG2a ratio indicated that proteins directed immune responses toward T-helper 2. Antisera produced against purified proteins in ternary combination showed the highest bactericidal activity against NTHi strains with the titer of 1:32. Conclusions: The obtained results suggested that HMW1555-914, HMW2553-916, and Hia585-705 adhesins were the potential subunit vaccine candidates of NTHi and after further investigation could be considered for protection against NTHi infections.

Objectives: The study aimed to compare the efficacy and side effects of intravenous magnesium sulfate (MgSO4) and oral nifedipine for inhibition of preterm labor. Methods: This randomized controlled trial was performed on 220 women with preterm labor between 32 and 34 weeks of gestation who were randomly assigned to receive either MgSO4 or nifedipine. The primary outcome was inhibition of preterm labor, defined as prevention of delivery for 48 hours with inhibition of uterine contraction, and the secondary outcome was maternal side effects. Results: From 220 patients, 110 received nifedipine and 110 received MgSO4. There were no differences in suppression of labor pain in 24 hours and 48 hours between the two groups. Also, there were not statistically significant differences in one-minute and five-minute Apgar scores, neonatal respiratory distress syndrome, and NICU admission between the two groups. Maternal hypotension was higher in the nifedipine group, but the difference was not significant (P = 0.08). Dyspnea (P = 0.01) and minor maternal side effects (P ≤ 0.001) were significantly higher in the MgSO4 group than the nifedipine group. Serious maternal adverse effects and severe hypotension were not seen in any of the groups. Conclusions: Nifedipine is as effective as MgSO4 in arresting labor and delaying delivery for 48 hours. However, nifedipine is associated with significantly fewer maternal adverse effects.

Context: An immunosuppressive drug, Cyclosporine (CsA), has been commonly used in kidney transplants. A safe dosing of CsA often causes nephrotoxity, bone marrow toxicity, and infection. Pharmacokinetic characteristics of CsA and CsA marker relation to the immune suppression have not been completely described in clinical practices yet. Objectives: This review summarizes our achievements on pharmacokinetic and CsA level Marker in kidney transplant patients. Search methods and Selection criteria: A literature review was done using the National Center for Biotechnology Information’s Pubmed Medline, Ovid Medline and Embase, and Iranian registries (Iranmedex, SID, MagIran, and IranDoc). Titles and abstracts were then reviewed to select studies based upon the predefined inclusion criteria. Our study defined a population of adult solid organ transplant recipients receiving the cyclosporine that Cyclosporine monitoring was done. Data Collection and Analysis: Two reviewers independently appraised the quality of each trial and extracted the data from the included trials. Results: CsA pharmacokinetics is very different between kidney transplant recipients, in order to CsA profiling, no pharmacodynamic tools has been confirmed yet in clinical practices and the best way to individualize calcineurin inhibitor therapy is still a controversial issue. C0 levels do not exactly predict the CsA level or rejection risk, patient monitored by C2 levels have upper doses of CsA and have a lower frequency of early acute allograft rejection than patients profiled with C0 and although CA is highly heterogeneous closely post-transplant and seems to be unhelpful early after post-transplant it is more favorable after first months after. Conclusions: Establishing a biological CsA marker may be helpful in clinical decisions on the dose. It seemed to be logical that we should re-inspect the possibility of using them as a supplementary tool towards better therapeutic drug monitoring of cyclosporine or it needs to be reevaluated and needs to find a new target for a therapeutic plan in kidney transplant patients.

Background: Patients with schizophrenia have abnormal skin flush response to niacin. We aimed to evaluate the accuracy of niacin skin test in these patients. Objectives: We aimed to evaluate the accuracy of niacin skin test in these patients. Materials and Methods: This diagnostic trial with parallel-group design was conducted at the Noor university hospital in Isfahan (Iran) from January to September 2014. Participated Subjects were hospitalized schizophrenic adult and their first degree relatives, bipolar disorder patients and healthy controls (n = 25 in each group). Niacin skin test was performed using 0.5 mL of 0.1 M and 0.01 M diluted methyl nicotinate solutions applied every 5 minutes for a total of 20 minutes and graded from 0 (no redness) to 3 (extreme redness). Sensitivity, specificity, and positive and negative predictive values were calculated. Results: The time point at which there was no further significant change in the skin response was 10 minutes after the test. At this set point, schizophrenic patients had lower response to each solutions compared to others (P 0.05). A grade of ≤ 1 skin response to the 0.01 M solution of methyl nicotinate would provide sensitivity, specificity, positive and negative predictive values of 80%, 93.3%, 80%, and 93.3%, respectively, in differentiating schizophrenic from other groups. Using 0.1 M solution provide lower sensitivity (32%) and negative predictive value (81.5%), but higher specificity (100%) and positive predictive value (100%). Conclusions: Niacin skin flush response is impaired in schizophrenic patients. This phenomenon may be used as a complementary diagnostic test in psychiatric workups.

Background: The hepatitis E virus (HEV) accounts for hepatitis E infection with relatively high mortality rate in pregnant women that can lead to fulminant hepatitis. The baculovirus expression system (BES) has the capability to produce high-level recombinant proteins and could be useful for vaccine designing. Objectives: The aim of this study was designing a recombinant hepatitis E virus ORF2 and Rotavirus NSP4 (ORF2-NSP4) and to evaluating construction these recombinant proteins in the BES. Methods: The truncated ORF2 gene (112-607) and truncated ORF2-NSP4 were subcloned in pFastBac1 plasmid, separately, followed by digestion and confirmed by digestion and sequencing. Then the products were transformed into Escherichia coli DH5 alpha and retransformed in DH10Bac competent cells. Finally the white colonies containing Bacmid DNA subjected to PCR for confirming transformation. Bacmid DNA containing HEV truncated ORF2 and HEV truncated ORF2-NSP4 genes were transfected into SF9 cells using BES. The expressed proteins in the cell lysate were evaluated by SDS-PAGE and determined by the western blot assay. Results: The lengths of the subcloned genes, truncated ORF2 and truncated ORF2-NSP4 were 1500 and 2000bp, respectively. After retransforming in DH10Bac, the size of PCR products were 300 bp in Bacmid DNA without recombination while it was 4300 and 3800 bp in Bacmid truncated ORF2-NSP4 and Bacmid truncated ORF2 PCR products. The analysis of protein expression by SDS-PAGE and immunoblotting revealed the presence of 56 KDa for truncated ORF2 and 74.5 KDa for truncated ORF2-NSP4 proteins. Conclusions: The results of the present study showed that the baculovirus expression system (SF9 cells) was able to express truncated ORF2 and truncated ORF2-NSP4 proteins as a potential candidate vaccine.

Background Although the immunosuppressant cyclosporine (CsA) is widely used after kidney transplantation over the long term, there is still no firm consensus on the best way to monitor of CsA blood levels. Objectives Cyclosporine (CsA) assay is critical for the management of renal transplant recipients due to inter– and intra–patient variation in CsA absorption and metabolism. Patients and Methods: In a retrospective cross sectional study, blood levels of CsA (through and 2 hours post dose) measured at least 5 times during 3 years post transplantation, in 7702 kidney transplant recipients from different transplant center of Tehran, IR Iran between 2008 and 2012. Cyclosporine absorption (CA) calculated C2/C0 ratio. Results CA had a significant correlation with allograft function (P = 0.000, r =.0.285), this correlation was stronger than its relationship with C0 and C2 blood levels (P = 0.000 and P = 0.000 as well as r = 0.033 and r = 0.090, respectively). In univariate analysis during different times after transplantation, C0 and C2 blood levels significantly decreased over three years follow up (P = 0.000), (P = 0.000); While, CA reversely increases over the time (P = 0.000). In linear regression model overall CA levels had correlation with lower age of recipient (P = 0.02), hypokalemia (P = 0.001), higher level of creatinine (P = 0.02) and triglyceride (P = 0.001). Conclusions The present study shows that CsA absorption changes trough the post-transplant time and appears to increases over time in long–term period after kidney transplantation.

INTRODUCTION: Foreign body in the vagina is a common cause of vaginal discharge, which may be either purulent or hemorrhagic. CASE PRESENTATION: This problem may produce symptoms or be asymptomatic for long periods of time and may result from ignorance, accident, malice, psychotic tendencies, attempts at sexual stimulation or sexual abuse. The current report presents the case of a girl that had inserted a foreign body in her vagina probably due to childish prank. CONCLUSIONS: The clinicians should always think of foreign bodies in the vagina in cases of chronic, antibiotic resistant vaginal discharge and lower abdominal pain especially in young girls. Language: en

Background: Rotavirus (RV) is a major cause of gastroenteritis in infants and children and is one of the most severe public health problems. Rotaviruses outer layer contains two proteins including VP4 and VP7. These proteins are necessary for host-cell binding and penetration. TLP (triple layer virus particle) of RV is a complete infectious virion that binds to the target cells and internalized at the cytoplasm. The DLP (double layer virus particle) is a non-infectious particle that is formed through exclusion of the outer layer proteins including VP4 and VP7. These DLPs are the transcriptionally active forms of rotavirus. Objectives: The aim of this study was to transfer DLP of RV into cytoplasm of MA104 cells by Lipofectamine and to analyze their replication. Materials and Methods: Initially, rotavirus was purified by CsCl discontinuous gradient and DLP was separated from TLP based on density differences. For confirmation, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the proteins were conducted Then the purified DLP of RV was transferred into MA104 cells using Lipofectamine. Results: We attempt to avoid the attachment and entry of the rotavirus by using Lipofectamine to mediate the delivery of viral particles directly into the cytoplasm. DLP was endocytosed into the cytoplasm following treatment by Lipofectamine and then replicated in cytoplasm. Conclusions: Therefore the non-infectious DLPs were became infectious if introduced into the cytoplasm of permissive and cancerous cells, without passing attachment and entry process.