Implementation of an In-House Platform for Rapid Screening of SARS-CoV-2 Genome Variations.

BACKGROUND: Global real-time monitoring of SARS-CoV-2 variants is crucial to controlling the COVID-19 outbreak. The purpose of this study was to set up a Sanger-based platform for massive SARS-CoV-2 variant tracking in laboratories in low-resource settings. METHODS: We used nested RT-PCR assay, Sanger sequencing and lineage assignment for 930-bp of the SARS-CoV-2 spike gene, which harbors specific variants of concern (VOCs) mutations. We set up our platform by comparing its results with whole genome sequencing (WGS) data on 137 SARS-CoV-2 positive samples. Then, we applied it on 1028 samples from March-September 2021. RESULTS: In total, 125 out of 137 samples showed 91.24% concordance in mutation detection. In lineage assignment, 123 out of 137 samples demonstrated 89.78% concordance, 65 of which were assigned as VOCs and showed 100% concordance. Of 1028 samples screened by our in-house method, 78 distinct mutations were detected. The most common mutations were: S:D614G (21.91%), S:P681R (12.19%), S:L452R (12.15%), S:T478K (12.15%), S:N501Y (8.91%), S:A570D (8.89%), S:P681H (8.89%), S:T716I (8.74%), S:L699I (3.50%) and S:S477N (0.28%). Of 1028 samples, 980 were attributed as VOCs, which include the Delta (B.1.617.2) and Alpha (B.1.1.7) variants. CONCLUSION: Our proposed in-house Sanger-based assay for SARS-CoV-2 lineage assignment is an accessible strategy in countries with poor infrastructure facilities. It can be applied in the rapid tracking of SARS-CoV-2 VOCs in the SARS-CoV-2 pandemic.

A Retrospective Study of Serum Calcium Status in Tehran, Iran (105,128 Samples, from 2009-2018).

Background: Calcium is a necessary mineral for life to keep the body and bones healthy. Various factors including hormones, diet, age, and gender affect serum calcium status. The aim of this sturdy was to assess the serum calcium level (SCL) of Tehran population, which has about 10 million multi-Ethnic populations and represents from the whole country. Methods: In this retrospective study, the measured SCL of 105,128 individuals referred to different laboratories of Tehran, Iran were evaluated and its relationship with the age, gender, seasons, and different years during 2009-2018, were analyzed. Results: After excluding outliers, 91,257samples remained, which 61162 (58.64%) and 30,095 (41.36%) were female and male, respectively. The mean SCL was 9.36 (9.35, 9.37) mg/dl (95%CI). The highest and lowest SCLs were 3.1 and 18.2mg/dl, respectively. From the total study population, 74127 (81.23%) had normal SCLs, 14110 (15.46%) had hypocalcemia, and 3020 (3.31%) had hypercalcemia. SCLs were normal in 83.6% of men and 79.66% of women. Women had a significantly higher frequency of hypocalcemia compared to men (17.2% vs. 12.83%, p<0.0001). Conclusion: Normal and abnormal SCLs were significantly different in age groups and in both genders. It means that gender and age affect SCLs. Every year of increasing age, reduces the chance of hypercalcemia by 40%, significantly. Age seems to affect hypercalcemia more than hypocalcemia. Age in men increases the risk of hypocalcemia, and reduces the risk of hypocalcemia in women. Therefore, it is recommended to encourage dietary calcium intake among premenopausal women and older men.

Disease Waves of SARS-CoV-2 in Iran Closely Mirror Global Pandemic Trends.

BACKGROUND: Complete SARS-CoV-2 genome sequencing in the early phase of the outbreak in Iran showed two independent viral entries. Subsequently, as part of a genome surveillance project, we aimed to characterize the genetic diversity of SARS-CoV-2 in Iran over one year after emerging. METHODS: We provided 319 SARS-CoV-2 whole-genome sequences used to monitor circulating lineages in March 2020-May 2021 time interval. RESULTS: The temporal dynamics of major SARS-CoV-2 clades/lineages circulating in Iran is comparable to the global perspective and represent the 19A clade (B.4) dominating the first disease wave, followed by 20A (B.1.36), 20B (B.1.1.413), 20I (B.1.1.7), leading the second, third and fourth waves, respectively. We observed a mixture of circulating B.1.36, B.1.1.413, B.1.1.7 lineages in winter 2021, paralleled in a fading manner for B.1.36/B.1.1.413 and a growing rise for B.1.1.7, prompting the fourth outbreak. Entry of the Delta variant, leading to the fifth disease wave in summer 2021, was detected in April 2021. This study highlights three lineages as hallmarks of the SARS-CoV-2 outbreak in Iran; B4, dominating early periods of the epidemic, B.1.1.413 (B.1.1 with the combination of [D138Y-S477N-D614G] spike mutations) as a characterizing lineage in Iran, and the co-occurrence of [I100T-L699I] spike mutations in half of B.1.1.7 sequences mediating the fourth peak. It also designates the renowned combination of G and GR clades' mutations as the top recurrent mutations. CONCLUSION: In brief, we provided a real-time and comprehensive picture of the SARS-CoV-2 genetic diversity in Iran and shed light on the SARS-CoV-2 transmission and circulation on the regional scale.

SARS-CoV-2 outbreak in Iran: The dynamics of the epidemic and evidence on two independent introductions.

The SARS-CoV-2 virus has been rapidly spreading globally since December 2019, triggering a pandemic, soon after its emergence. While Iran was among the first countries confronted with rapid spread of virus in February 2020, no real-time SARS-CoV-2 whole-genome tracking in early phase of outbreak was performed in the country. To address this issue, we provided 50 whole-genome sequences of viral isolates ascertained from different geographical locations in Iran during March-July 2020. The corresponding analysis on origins, transmission dynamics and genetic diversity of SARS-CoV-2 virus, represented at least two introductions of the virus into the country, constructing two major clusters defined as B.4 and B.1*. The first entry of the virus might have occurred around very late 2019/early 2020, as suggested by the time to the most recent common ancestor, followed by a rapid community transmission that led to dominancy of B.4 lineage in early epidemic till the end of June. Gradually, reduction in dominancy of B.4 occurred possibly as a result of other entries of the virus, followed by surge of B.1* lineages, as of mid-May. Remarkably, variation tracking of the virus indicated the increase in frequency of D614G mutation, along with B.1* lineages, which showed continuity till October 2020. The increase in frequency of D614G mutation and B.1* lineages from mid-May onwards predicts a rapid viral transmission that may push the country into a critical health situation followed by a considerable change in composition of viral lineages circulating in the country.

Assessment of Key Elements in the Innate Immunity System Among Patients with HIV, HCV, and Coinfections of HIV/HCV.

BACKGROUND: Coinfection of Hepatitis C virus (HCV) with human immunodeficiency virus (HIV) has a higher risk of mortality than HCV or HIV monoinfection. HCV and HIV infections are specified by systemic inflammation, but the inflammation process in HCV/HIV coinfection is much complicated and is not well characterized. OBJECTIVE: The aim of this study was to analyze the expression of TLR-3, TLR-7, IL-10, IFN-1 (IFN-α, IFN-ß), and TNF-α in HIV, HCV and HIV/HCV co-infected patients. METHODS: Forty-five patients including HIV group (n=15), HCV group (n=15), HIV/HCV coinfection group (n=15) and healthy control group (n=15) participated. Peripheral blood mononuclear cells (PBMCs) were obtained. PBMC-RNA, HCV and HIV RNA were extracted from all subjects and cDNA was synthesized. The viral load analyzed by reverse transcription-quantitative PCR (RT-qPCR), and the expression levels of IFN-α, IFN-ß, TLR-3, TLR-7, TNF, and IL-10 mRNA were quantified in PBMCs. RESULTS: The levels of IFN-I, IL-10, and TNF-α were overexpressed in all patients' groups (p<0.05), TLR-7 was upregulated in all groups, but this upregulation was not statistically significant (p>0.05). TLR-3 showed a decrease in all patient groups (p<0.05). The statistical analysis demonstrated that TLR-3 has a negative correlation with HIV load, whereas other genes positively correlated with HIV load. In addition, TLR-3, TNF-α, and IFN-I were negatively correlated with HCV load, whereas TLR-7 and IL-10 s were positively correlated with HCV load. CONCLUSION: Our results showed a significant relationship between the expression level of innate immunity genes and inflammation in HCV, HIV, and HIV/HCV coinfected patients.

A Comprehensive, Epidemiological and Ecological Descriptive Study on Vitamin D Status in Iran (308005 People, from 2009-2018).

BACKGROUND: Vitamin D is an essential substance for absorption of calcium and phosphorus from intestine so it is vital for muscles and skeletal development. Deficiency of this vitamin is pandemic. The vitamin D status depends on the different factors such as UV exposure, diet, and ecological features of living location, age and gender. The aim of this study was to describe the vitamin D level in different provinces of Iran and to investigate the association between vitamin D status and multiple variables. METHODS: We collected the serum 25(OH)D (Vitamin D) level data of 308,005 people referred to different laboratories from 30 provinces of Iran and organized them by each province, year, age, gender, precipitation, latitude and longitude, and humidity over 10 yr (2009-2018). Data were analyzed to find out the correlation between age, gender, longitude and latitude, humidity and sum of precipitation. RESULTS: West Azerbaijan had the highest level of vitamin D with a mean level of 33.24 and a standard deviation of 32.001, and North Khorasan had the lowest level with a mean level of 14.46 and a standard deviation of 8.980 among 30 provinces of Iran. The correlation between all studied variables (age, and gender, latitude and longitude, humidity, the sum of precipitation) was significant (P<0.001). CONCLUSION: The average total vitamin D level in Iran is 25.41 ng/ml, which is within the area of deficiency. Vitamin D is associated with age, and gender, latitude and longitude, humidity, the sum of precipitation. So changes in any of these variables can lead to vitamin D alteration.

Correlation of CD81 and SCARB1 polymorphisms on virological responses in Iranian patients with chronic hepatitis C virus genotype 1.

The cluster of differentiation 81 (CD81) and scavenger receptor class B member 1 (SCARB1) plays an important role in the entry of hepatitis C virus (HCV). We assessed the correlation of five single nucleotide polymorphisms (SNPs) of CD81 (rs800136, rs2651842, rs2522012, rs800146, and rs708564) and SCARB1 rs10846744 polymorphisms with treatment responses in 395 treatment-naïve patients with chronic HCV (CHC) genotype 1 treated with pegylated interferon-α and ribavirin (pegIFN-α/RBV). The frequency of rapid virologic response (RVR), complete early virologic response (cEVR) and sustained virologic response (SVR) were 57.2%, 55.2%, and 58.2%, respectively. RVR, cEVR, and SVR were significantly associated with CD81 rs800136 (CC), CD81 rs2651842 (AA), CD81 rs708564 (TT), and SCARB1 rs10846744 (CC). High rates of RVR, cEVR, and SVR were reported for the CD81 rs800136 (CC), CD81 rs2651842 (AA), and CD81 rs708564 (TT) genotypes when correlated with higher levels of low-density lipoprotein (LDL) and lower levels of high-density lipoprotein (HDL) as well as lower levels of HDL and LDL in the SCARB1 rs10846744 (CC) genotype. In addition, patients with GG genotype had higher fasting blood glucose (FBS) level than those with CC genotype. In conclusion, CD81 and SCARB1 SNPs may serve as powerful predictor factors for treatment responses in CHC patients, and this effect is correlated with serum lipoprotein and FBS levels.

Nonselective mevalonate kinase inhibitor as a novel class of antibacterial agents.

Introduction. There are a few evidences about targeting isoprenoids biosynthesis pathway in bacteria for finding new antibiotics. This study was conducted to assess antibacterial effects of vanadyl sulfate (VS), one of the mevalonate kinase inhibitors to find a new target for killing bacteria. Materials and Methods. Antibacterial effect of VS alone and in combination with glycine or EDTA was assessed on Escherichia coli and Pseudomonas aeruginosa as Gram-negative and Staphylococcus aureus and Enterococcus faecalis as Gram-positive bacteria using serial dilution method and minimum inhibitory concentrations (MICs) identified. Result. MICs for S. aureus and E. coli were 4 and 8 mg/mL, respectively. VS could not affect the growth of two other bacteria. However, VS in combination with glycine not only inhibited the growth of E. faecalis and P. aeruginosa, but also reduced MICs for VS-sensitive bacteria (S. aureus and E. coli). EDTA could reduce MIC for E. coli and P. aeruginosa. Conclusion. VS could inhibit the growth of S. aurous and E. coli, and adding glycine or EDTA improved VS antibacterial activity presumably via instability of the cell wall and enhanced transport of VS through bacterial cell wall. Inhibition of the isoprenoid pathway might provide new tools to overcome bacterial resistance.

The prevalence of Human Papilloma Virus (HPV) infection in the oligospermic and azoospermic men.

BACKGROUND: Human papilloma virus (HPV) infection is one of the most common sexually transmitted diseases that affects men like women and infected cutaneous and mucosal squamous epithelium. The aim of the present study was to determine the prevalence of HPV in the semen of oligospermic, azoospermic and normal patients. METHODS: From June 2012 to June 2013, a total of 90 individuals were enrolled in this cross sectional comparative study. The participants were classified into three groups (oligospermia, azoosprmia and normal). This classification was based on a new WHO reference values for human semen characteristics published on 2010. After extraction of DNA from specimens L1 gene of HPV was amplified by nested polymerase chain reaction (Nested-PCR) and the PCR products of positive specimens were genotyped using INNO-LiPA HPV Genotyping Extra assay. RESULTS: Among 50 confirmed oligospermic male, 15 were HPV DNA positive (30%). In azoospemic group we had 8 HPV DNA positive (40%) and in normal group just 3 of 20(15%) samples were positive. Statistical assessment was done with SPSS v.15. Chi-square test showed no significant relationship between 3 groups results. Based on independent samples t-test, we found statistical significant relationship for sperm count (p<0.05) and sperm motility (slow) (p<0.05) in oligospermic group positive samples compared with negative. In the present study, 13 HPV genotypes were detected among positive samples. HPV genotypes 16, 45 in the high risk group and 6,11,42 in the low risk group were more frequent than the others. CONCLUSION: The current study shows that HPV infection can affect on sperm count and motility and decrease count of sperm cell and decrease motility capability of these cells.