Overview of the different methods for RNA preparation in COVID-19 diagnosis process during the pandemic.

The COVID-19 pandemic brought to light the impact of a widespread disease on various aspects of human relationships, communities, and economies. One notable consequence was the increased demand for diagnostic kits, laboratory reagents, and personal health equipment. This surge in testing capacity worldwide led to shortages in the supply of essential items, including RNA extraction kits, which are crucial for detecting COVID-19 infections. To address this scarcity, researchers have proposed alternative and cost-effective strategies for RNA extraction, utilizing both chemical and physical solutions and extraction-free methods. These approaches aim to alleviate the challenges associated with the overwhelming number of tests being conducted in laboratories. The purpose of this review is intends to provide a comprehensive summary of the various kit-free RNA extraction methods available for COVID-19 diagnosis during the pandemic.

Correlation of vitamin D levels with serum parameters in Covid-19 patients.

BACKGROUND: It is well-established that vitamins have many beneficial roles and protect humans against inflammatory diseases. Vitamin D, a lipid-soluble vitamin, plays a crucial role in viral infections. Therefore, this study aimed to investigate if serum 25(OH)D levels affect morbidity, mortality, and levels of inflammatory parameters in COVID-19 patients. METHODS: 140 COVID-19 patients participated in this study (65 outpatients and 75 inpatients). Their blood samples were collected to determine TNFα, IL-6, D-dimer, zinc, Ca2+, and 25(OH)D levels. Patients with O2 saturation <93% were admitted and hospitalized in the infectious disease ward (inpatient group). Patients with O2 saturation >93% received routine treatment and were discharged (Outpatient group). RESULTS: The serum levels of 25(OH)D in the inpatient group were significantly lower than those in the outpatient group (p < 0.001). Serum TNF-α, IL-6, and D-dimer levels in the inpatient group were significantly higher than those in the outpatient group (p < 0.001). Serum TNF-α, IL-6, and D-dimer levels were inversely correlated with 25(OH)D levels. No significant differences were observed in the serum levels of zinc and Ca2+ between the studied groups (p = 0.96, p = 0.41 respectively). Ten out of 75 patients in the inpatient group were admitted to ICU (intubated). Nine out of them lost their lives (the mortality rate in ICU-admitted patients was 90%). CONCLUSIONS: The lower mortality and severity of COVID-19 patients with higher 25(OH)D levels represented that this vitamin alleviates the severity of COVID-19.

Significant alteration of IFN stimulated genes expression in MA104 cells infected with bovine rotavirus RF strain.

The pattern recognition receptors (PRRs) trigger signaling cascades, such as nuclear factor kappa B (NF-κB) and interferon regulatory factors (IRFs). Rotavirus (RV) countermeasures against innate responses and understanding of these processes will improve our knowledge regarding immunopathogenesis of RV infection. In this study, we investigated the effect of RV RF strain on the important ISG candidate genes engaging in virus infections for which little information is known in RV RF strain. To this end, MA104 cells were mock/infected with RF followed by incubation in the presence or absence of IFN-α and the expression of MX1, OAS1, STAT1, ISG15, and ISG56 mRNA was analyzed by real-time PCR. All of ISGs' mRNAs showed higher expression levels in IFN I treated cells compared to virus-infected cells except for ISG56. Infecting the cells with RV and treatment with IFN type I led to overexpression of ISG56 compared to cells were either infected with the virus or only treated with IFN I. In conclusion, we showed that the RV RF strain efficiently blocks type I IFN-induced gene expression particularly ISG15, MX1, STAT, and OSA1 as antiviral proteins. Furthermore, viruses may use some ISGs such as ISG 56 to regulate IFN I signaling pathway, negatively.

An update on enterovirus 71 infection and interferon type I response.

Enteroviruses are members of Pichornaviridae family consisting of human enterovirus group A, B, C, and D as well as nonhuman enteroviruses. Hand, foot, and mouth disease (HFMD) is a serious disease which is usually seen in the Asia-Pacific region in children. Enterovirus 71 and coxsackievirus A16 are two important viruses responsible for HFMD which are members of group A enterovirus. IFN α and ß are two cytokines, which have a major activity in the innate immune system against viral infections. Most of the viruses have some weapons against these cytokines. EV71 has two main proteases called 2A and 3C, which are important for polyprotein processing and virus maturation. Several studies have indicated that they have a significant effect on different cellular pathways such as interferon production and signaling pathway. The aim of this study was to investigate the latest findings about the interaction of 2A and 3C protease of EV71 and IFN production/signaling pathway and their inhibitory effects on this pathway.

Echovirus 30 and coxsackievirus A9 infection among young neonates with sepsis in Iran.

BACKGROUND AND OBJECTIVES: Human enteroviruses (EV) are the most common causes of neonatal sepsis-like disease. The frequencies of EV including coxsackievirus A, coxsackievirus B and Echovirus serotypes have been studied in young infants (younger than three months) with sepsis. So far, the role of enteroviruses among neonates with sepsis was not determined in Ahvaz, Iran. Therefore, this study was aimed to evaluate the frequency of EV among hospitalized young infants with clinical signs and symptoms of sepsis in Ahvaz. MATERIALS AND METHODS: Blood specimens from 128 neonates (younger than 90 days), including 56 (43.75%) girls and 72 (56.25%) boys, were collected from hospitalized neonates with clinical signs and symptoms of sepsis-like symptoms. All blood samples were negative for bacterial culture. RNA was extracted from all sera and tested for detection of 5'UTR (Untranslated Region) of the EV by RT-PCR. To determine specific strains of EV, positive 5'UTR samples were further tested for detection of the VP1 region of EV by RT-PCR. RESULTS: Overall, 50/128 (39.06%) specimens, including 24 (48%) girls and 26 (52%) boys, were positive for EV. 21/50 (42%) specimens were positive for the VP1 region. Randomly, 8 positive VP1 were selected and sequenced. Analysis of sequencing data showed 7/21 (33.33%) samples were positive for Echovirus 30 and 1/21 (4.76%) samples were positive for CVA9. CONCLUSION: The results of this survey indicate high prevalence of 39.06% of EV among young neonates with sepsis. A high prevalence of 33.3% Echoviruses 30 and a low rate of 4.76% coxsackievirus A9 infection has been observed in neonatal patients with viral sepsis. This outbreak is probably one of the first Enterovirus outbreaks to be reported in Ahvaz, Iran. The results of this survey will help to minimize unneeded use of antimicrobial drugs and reduce unnecessary hospitalization.

Human Rhinoviruses A9, A49, B14 and Echovirus 3, 9 among the patients with acute respiratory infection.

  Background: Acute respiratory infection result in high mortality and morbidity worldwide. There are several viral factors that originate respiratory diseases among them Enteroviruses(EVs) and Human Rhinoviruses(HRVs) can be mentioned. HRVs and EVs belong to Picornaviridae family and they have been recently classified under Enteroviruses. The pattern of respiratory infections generating organisms varies according to geographical locations. Therefore, it seems necessary to organize an appropriate plan to manage common viral diseases exclusively about Rhinoviruses and Enteroviruses. PATIENT AND METHODS: A total of 100 samples were collected from patients with acute respiratory infections (ARIs) who were hospitalized in Ahvaz city hospitals during December 2012 to November 2013 (one year longitude). Semi-Nested PCR was done on samples for detection of HRVs and EVs using region gene of VP4/VP2. Phylogenetic and molecular evolutionary analyses performed with MEGA version 5 software find out the sequence homology among the detected HRV and EV serotype. RESULTS: The results of this study revealed that from of 100 cases of ARIs 19 patients (19%) were HRV positive and 3 (3%) patients positive for EVs. Most positive cases of HRVs were observed in the autumn season while 3 positive cases of EVs were equally found in spring, summer and autumn. Phylogenetic analyses showed that the HRV strains were HRV-A9, HRV-A49, HRV-B14 and EV strains were Echo3 and 9. CONCLUSION: The results of this study revealed that high prevalence of 19% HRVs, HRV-A9, HRV-A49, HRV-B14 serotypes and low frequency of 3% Echo Viruses, Echo3 and Echo 9 serotypes have been detected in patients with ARI.

Three cases of mumps virus and enterovirus coinfection in children with enteroviral meningitis.

Several viruses are responsible for aseptic meningitis; however, in the region of Southwest Iran, the role played by each virus is still not very well known. The aim of this study is to determine the relative frequencies of mumps virus, herpes viruses, and enteroviruses, as well as coinfections among them, in patients with aseptic meningitis.In this cross-sectional study, samples of cerebrospinal fluid were collected between December 2012 and December 2013 from patients under 14 years, who were hospitalized in Abuzar Children's Hospital in Ahvaz, Southwest Iran (the only children's hospital in Khuzestan province and Southwest Iran).All 66 cerebrospinal fluid samples and corresponding clinical data were collected from patients with aseptic meningitis by specialists, and with the patients' consent. The DNA and RNA were extracted from these samples and subjected to polymerase chain reaction as well as reverse transcription polymerase chain reaction (RT-PCR) for detection of mumps virus, herpes viruses, and enteroviruses. Nine of the samples (3 mumps-positive and 6 enterovirus-positive) were sequenced. The mumps virus sequences were investigated for possible mutations in the SH and partial HN regions.Up to 39 patients (59.09%) were found to be positive for enteroviruses, 3 (4.5%) for mumps virus, and 1 (1.5%) for herpes viruses (specifically, the varicella-zoster virus). Two patients (3.03%) had a mumps virus and enterovirus coinfection. Among the 3 detected mumps virus samples, 1 belonged to genotype B, while the others belonged to genotype N. Six sequenced enteroviruses indicated the highest similarity with Echovirus 30. An amino acid substitution at position 51 (N→T) was detected in the HN region of genotype N mumps virus samples, in comparison to the reference strain.

Study on Rotavirus Infection and Its Genotyping in Children Below 5 Years in South West Iran.

BACKGROUND: Human rotaviruses are the most important agents for severe dehydrating diarrhea in children below 5 years old. Rotaviruses (RV) is a serious public health problem in developing and developed countries. OBJECTIVES: The aim of this study was to determine the prevalence of rotavirus infection and their genotypes in children younger than 5 years of age with acute diarrhea in Ahvaz, Iran. MATERIALS AND METHODS: For this study, 200 stool samples from children below 5 years of age with acute diarrhea were collected between October 2011 and March 2012. Initially all stool samples were tested for rotavirus antigen by ELISA, and positive samples were confirmed by RT-PCR targeting the VP6 rotavirus gene. Determination of rotavirus genotypes was carried out by performing RT-PCR for G and P types. Altogether, 15 samples were sequenced. RESULTS: Out of 200 stool samples, 100 (50%) had rotavirus antigen detected by ELISA and 73 (36.5%) were found positive by RT-PCR. Of the rotavirus strains identified, only 63 (86.3%) were positive for both VP7 and VP4 while 10 (13.7%) strains were found nontypeable. Rotavirus infection accounts for 36.5% of gastroenteritis cases in samples from symptomatic children. The most prevalent rotavirus genotypes were G1P [8] (80%) followed by G2P [4] (20%). CONCLUSIONS: Our results suggest that group A rotavirus is a major pathogene of acute diarrhea in Ahvaz city. The genotypes circulating are similar with those of other countries.

Prevalence of Hepatitis C virus Genotype 3a in patients with Hodgkin and Non-Hodgkin Lymphoma.

BACKGROUND AND OBJECTIVES: Hepatitis C virus (HCV) is a major public health problem worldwide. Replication and persistence of HCV genome have been described in the liver tissue as well as B cells lymphocyte. Several investigations have reported that long-term persistence of HCV in B cells may result in Hodgkin and Non-Hodgkin lymphoma. This study was aimed to determine frequency of HCV RNA in histological tissues obtained from patients suffered from Hodgkin and Non-Hodgkin lymphoma. MATERIALS AND METHODS: 52 formalin-fixed paraffin-embedded tissue blocks including 23 (44.3%) Hodgkin and 29 (55.7%) Non-Hodgkin samples were collected and five micrometer sections were prepared. RNA was extracted and cDNA was synthesized. Two consecutive Nested RT-PCR assays were carried out for detection of HCV 5' UTR and core gene. RT-PCR products were sequenced and aligned to construct HCV phylogenic tree to evaluate the homology of sequences in comparison to the reference sequences retrieved from Genbank. RESULTS: Overall, 6 Non-Hodgkin (20.6%) and 3 Hodgkin lymphoma (13.04%) samples showed positive PCR results for both 5' UTR and HCV core RNA via nested PCR (P<0.469). Sequencing results revealed that all detected HCV RNA samples belonged to the genotype 3a. CONCLUSION: Despite low prevalence of HCV infection in Iran, high frequency of HCV RNA genotypes 3a (17.3%) has been found in patients with Hodgkin and Non-Hodgkin lymphoma. To improve treatment regimens, screening of HCV RNA in patients suffered from Hodgkin or Non-Hodgkin lymphoma is recommended which can be done through highly sensitive molecular means before and after immunosuppression status.

Molecular Characterization of Pre-Core/Core and S Region of Hepatitis B Virus in Hemodialysis Patients With Occult Hepatitis B Infection.

BACKGROUND: Occult hepatitis B infection (OBI) is a major public health problem worldwide, which harbors potential risk of hepatitis B virus (HBV) transmission through blood transfusion and transplantation. OBI is characterized by the presence of HBV-DNA in the blood or liver tissue without detectable hepatitis B surface antigen (HBsAg) in the serum. An important cause of OBI is the occurrence of mutations in the HBV genome, especially in the S region. OBJECTIVES: The study aims to analyze mutations in S and pre-core/core regions of HBV-DNA in hemodialysis patients. PATIENTS AND METHODS: Sera of 216 hemodialysis patients were tested for HBsAg and hepatitis B core antibody (HBcAb) by ELISA. Sera of patients that tested negative for HBsAg were evaluated by PCR for the detection of HBV-DNA in the S and pre-core/core regions. In total, six PCR products were sequenced, aligned, and compared with the HBV reference sequence. Amino acid deletion and nucleotide substitution were considered mutations in S and pre-core/core regions of HBV-DNA. RESULTS: Among 216 patients, 203 (93.98%) and 175 (81.01%) sera samples tested negative for HBsAg and HBcAb, respectively. Among all HBsAg-negative samples, six (2.9%) tested positive for HBV-DNA, including four (1.97%) for S and two (0.98%) for pre-core regions. All four (1.97%) samples that tested positive for the S region belonged to HBV-subtype awy. The amino acid sequence of all four samples showed the YMDD motif in position 204 (rtM204). There were three amino acid substitutions in the S region (T127P, P153L, and F170S) and one substitution in the RT region (Y135S). Moreover, two (0.98%) pre-core/core positive patients had an unexpected stop codon in position 1896. CONCLUSIONS: This study indicates that 2.9% of hemodialysis patients had OBI, which is considered as a major public health problem worldwide. Moreover, we observed three mutations in S region, including T127P, P153L, and F170S, which caused OBI. This study is first to report a mutation analysis of HBV in hemodialysis patients in southwestern Iran. These results indicate that current screening tests based on HBsAg detection are not reliable for detection of HBV infection in dialysis patients.

Antimicrobial Activity of Calcium Hydroxide and Betamethasone on Enterococcus faecalis; An in vitro Assessment.

INTRODUCTION: Calcium hydroxide (CH) is one of the most common intracanal medications. Corticosteroids (CS) are used in endodontics because of their anti-inflammatory activity. This study aimed to evaluate the antimicrobial effect of CH+betamethasone and CH+saline against Enterococcus faecalis (E. faecalis) using agar diffusion test and measuring the microbial zone of inhibition (ZOI). METHODS AND MATERIALS: Four plates containing Mueller-Hinton broth and E. faecalis culture media, were prepared. In each plate, 5 holes (5×3 mm) were created and a creamy mixture of CH+betamethasone was inserted into the holes (10 holes for each material). Two holes with ampicillin disks and two empty holes were used as negative and positive controls, respectively. Plates were incubated for 24 h and then the diameter of microbial ZOI was measured. The pH of each mixture was measured by pH meter. Data were analyzed using the Mann-Whitney U test. RESULTS: The mean diameter of ZOI for CH+betamethasone and CH+saline was 3.4 and 3 mm, respectively. The difference was not significant (P=0.143). The pH was 12.5 for CH+saline and 12.3 CH+betamethasone, respectively. CONCLUSION: The mixture of CH+betamethasone had good antimicrobial effects against E. faecalis. Further studies are needed to confirm the value of this mixture in clinical settings.

Evaluation of hepatitis B surface antibody and specific gamma interferon response in health care workers after vaccination.

BACKGROUND: Health care workers are at high risk of acquiring hepatitis B virus (HBV) infection through occupational exposure to blood or body fluids. Thus, the assessment of anti-HBs status after immunization is very important. OBJECTIVES: This study aimed to evaluate the measurement of HBsAb titer and specific gamma interferon response among the vaccinated health care workers in Golestan Hospital, Ahvaz, Iran. PATIENTS AND METHODS: The blood samples of 39 health care workers, including 13 general surgeons, 10 anesthesiologists, 5 neurosurgeons, 3 general physicians, 1 orthopedist, 2 urologist and 5 nurses were collected during June 2013. All the participants had received HBV vaccine. They had received last vaccine dose from 2 months to 14 years ago. Their sera were tested for anti-hepatitis B antibody and HBc-IgG by the ELISA. Also, the evaluation of specific interferon γ response against HBsAg was carried out using ELISA test. The age of health care workers were between 24 and 58 years with the mean age of 34.3 ± 7.4 y. RESULTS: Out of 39 sera, 22 (56.41%) had HBsAb titer above 100 IU/mL, 17 (43.6%) had titer below 100 IU/mL, 27 (69.2%) had positive specific HBsAg interferon γ, 8 (20.5%) cases had positive antibody response above 100IU, but negative for specific interferon γ and 3 (7.6%) cases were positive for HBc-IgG. CONCLUSIONS: Overall, 87.2% of the health care workers had immunity against HBV infection, which showed remarkable immunity response following HBV vaccination. Booster dose of HBV vaccine is recommended for those whose immunity are below 100 IU/mL.

Study of the Association of Mutant HBsAg Gene and Hodgkin and Non-Hodgkin Lymphoma.

BACKGROUND: Hepatitis B Virus (HBV) is responsible for chronic, acute, and fulminant hepatitis, which are prevalent worldwide. Chronic HBV may lead to cirrhosis and hepatocellular carcinoma. Several epidemiological studies have indicated that hepatitis B virus is involved in B-cell Hodgkin and Non-Hodgkin Lymphoma (NHL). OBJECTIVES: The aim of this study was to evaluate the association between hepatitis B infection and Hodgkin and non-Hodgkin Lymphoma. MATERIALS AND METHODS: Paraffin embedded of 41 block samples including 12 (29.26%) Hodgkin and 29 (70.73%) non-Hodgkin patients were collected. Next, DNA extraction was carried out for all the samples followed by HBV DNA detection by the nested polymerase chain reaction (PCR). The positive HBV DNA samples were sequenced, and HBV genotypes and HBV subtypes were determined. RESULTS: Three out of 12 (25%) Hodgkin samples and seven out of 29 (24.13%) non-Hodgkin showed positive HBV DNA results. The results of sequencing revealed that the D genotype was predominant among the positive HBV patients. Interestingly an unpredictable amino acid proline was detected in position 88 of the HBs gene, which indicates a new mutation in the "S" region of HBV DNA in patients with Hodgkin and non-Hodgkin lymphoma. CONCLUSIONS: A high rate of 25% and 24.13% of HBV DNA was detected among patients with Hodgkin and non-Hodgkin lymphoma, respectively.

Occult hepatitis B virus among the patients with abnormal alanine transaminase.

BACKGROUND: The occult hepatitis B infection (OBI) is defined as the presence of hepatitis B virus (HBV) DNA in the sera or in the liver biopsy and the absence of hepatitis B surface antigen (HBsAg) by serological test. OBJECTIVES: The current study aimed to evaluate the occult HBV infection by polymerase chain reaction (PCR) and determine HBV genotyping among the patients with abnormal alanine transaminase (ALT) in Ahvaz city, Iran. PATIENTS AND METHODS: The sera of 120 patients, 54 (45%) females and 66 (55%) males, with abnormal ALT 40-152 IU were collected. All the patients were negative for HBsAg for more than one year. The patients` sera were tested by PCR using primers specified for the S region of HBV. Then the positive PCR products were sequenced to determine HBV genotyping and phylogenic tree. RESULTS: Of these 120 subjects, 12 (10%) patients including 6 (5%) males and 6 (5%) females were found positive for HBV DNA by PCR, which indicated the presence of occult HBV infection among these patients. The sequencing results revealed that genotype D was predominant with sub-genotyping D1 among OBI patients. CONCLUSIONS: Occult hepatitis B infection is remarkably prevalent in Ahvaz, Iran, and should be considered as a potential risk factor for the transmission of Hepatitis B Virus throughout the community by the carriers.

Serotype determination of adenoviruses in children with respiratory infection.

OBJECTIVE: To determine adenovirus serotypes among children with acute respiratory infection (ARI) in Khoozestan province, Iran during years 2010-2011. METHODS: One hundred sixty three nasopharyngeal swabs were collected from children between 1 and 15 y who were hospitalized for the acute respiratory infection. The viral DNA was extracted from the nasopharyngeal swabs and adenoviruses were detected by Nested PCR. Positive PCR samples were sequenced in order to confirm the adenovirus serotypes. RESULTS: Out of 163 samples, 30 (18.4 %) were positive for adenoviruses by nested PCR. Twenty two PCR products were sequenced and recognized as Ad5 and Ad2 serotypes including 19 (86.3 %) Ad5 and 3 (13.7 %) Ad2. CONCLUSIONS: This study reveals that adenoviruses with Ad5 predominance are important cause of respiratory tract infection in children.