Analyzing the expression pattern of the noncoding RNAs (HOTAIR, PVT-1, XIST, H19, and miRNA-34a) in PBMC samples of patients with COVID-19, according to the disease severity in Iran during 2022-2023: A cross-sectional study.

Background and aims: MicroRNAs (miRNAs) and long noncoding RNAs (lncRNAs) are well-known types of noncoding RNAs (ncRNAs), which have been known as the key regulators of gene expression. They can play critical roles in viral infection by regulating the host immune response and interacting with genes in the viral genome. In this regard, ncRNAs can be employed as biomarkers for viral diseases. The current study aimed to evaluate peripheral blood mononuclear cell (PBMC) ncRNAs (lncRNAs-homeobox C antisense intergenic RNA [HOTAIR], -H19, X-inactive-specific transcript [XIST], plasmacytoma variant translocation 1 [PVT-1], and miR-34a) as diagnostic biomarkers to differentiate severe COVID-19 cases from mild ones. Methods: Candidate ncRNAs were selected according to previous studies and assessed by real-time polymerase chain reaction in the PBMC samples of patients with severe coronavirus disease 2019 (COVID-19) (n = 40), healthy subjects (n = 40), and mild COVID-19 cases (n = 40). Furthermore, the diagnostic value of the selected ncRNAs was assessed by analyzing the receiver-operating characteristic (ROC). Results: The results demonstrated that the expression pattern of the selected ncRNAs was significantly different between the studied groups. The levels of HOTAIR, XIST, and miR-34a were remarkably overexpressed in the severe COVID-19 group in comparison with the mild COVID-19 group, and in return, the PVT-1 levels were lower than in the mild COVID-19 group. Interestingly, the XIST expression level in men with severe COVID-19 was higher compared to women with mild COVID-19. ROC results suggested that HOTAIR and PVT-1 could serve as useful biomarkers for screening mild COVID-19 from severe COVID-19. Conclusions: Overall, different expression patterns of the selected ncRNAs and ROC curve results revealed that these factors can contribute to COVID-19 pathogenicity and can be considered diagnostic markers of COVID-19 severe outcomes.

Evaluation of MicroRNA Expression Pattern (miR-28, miR-181a, miR-34a, and miR-31) in Patients with COVID-19 Admitted to ICU and Diabetic COVID-19 Patients.

INTRODUCTION: MicroRNAs, or miRNAs, with regulatory performance in inflammatory responses and infection are the prevalent manifestations of severe coronavirus disease (COVID-19). This study aimed to evaluate whether PBMC miRNAs are diagnostic biomarkers to screen the ICU COVID-19 and diabetic COVID-19 subjects. METHODS: Candidate miRNAs were selected through previous studies, and then the PBMC levels of selected miRNAs (miR-28, miR-31, miR-34a, and miR-181a) were measured via quantitative reverse transcription PCR. The diagnostic value of miRNAs was determined by the receiver operating characteristic (ROC) curve. The bioinformatics analysis was utilized to predict the DEM genes and relevant bio-functions. RESULTS: The COVID-19 patients admitted to ICU had significantly greater levels of selected miRNAs compared to non-hospitalized COVID-19 and healthy people. Besides, the mean miR-28 and miR-34a expression levels in the diabetic COVID-19 group were significantly upregulated when compared with the non-diabetic COVID-19 group. ROC analyses demonstrated the role of miR-28, miR-34a, and miR-181a as new biomarkers to discriminate the non-hospitalized COVID-19 group from the COVID-19 patients admitted to ICU samples, and also miR-34a can probably act as a useful biomarker for screening diabetic COVID-19 patients. Using bioinformatics analyses, we found the performance of target transcripts in many bioprocesses and diverse metabolic routes such as the regulation of multiple inflammatory parameters. DISCUSSION: The difference in miRNA expression patterns between the studied groups suggested that miR-28, miR-34a, and miR-181a could be helpful as potent biomarkers for diagnosing and controlling COVID-19.

High resolution melting curve analysis for rapid detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants.

Since the emergence of the original Wuhan SARS-CoV-2 strain, several new variants of the virus have emerged. Alpha, Beta, Gamma, Delta and the most recent Omicron variants have been introduced during this pandemic. Several methods including, but not restricted to, allele-specific PCR, ligation with rolling circle amplification and real-time PCR with allele-specific probes are able to detect mutations as low as a single nucleotide polymorphism. High-resolution melting curve analysis is ano-ther technique to assess any mutations in a nucleic acid chain. Confirmed samples with SARS-CoV-2 infection were subjected to variant identification using a de novo-designed HRM assay. In order to select for mutations with the highest effect on Tm of the amplicon, deletion mutations of NSP6 (Del 3675-3677), and S1 (Del 144) were chosen for HRM analysis. HRM analysis for the amplicon of the primer set-1 (NSP6) resulted in Tm differences of -0.39°C, +0.4°C, and -0.6°C between Alpha, Delta, and Omicron variants, respectively, in comparison to the original Wuhan strain. Moreover, HRM analysis of the amplification performed by primer set-2 (S1) led to Tm differences of +0.32°C, -0.26°C, and +0.24°C between Alpha, Delta, and Omicron variants, respectively, in comparison to original Wuhan strain. The test was able to specify each sample to its variant group with more than 90 percent of confidence. The results obtained in this study demonstrate that using a single closed-tube strategy with a HRM-equipped machine, screening new variants of the virus is possible in a fast and reliable way. Keywords: high resolution melting; SARS coronavirus 2; mutation; variant; genotyping.

The Relationship Between Population-Level SARS-CoV-2 Cycle Threshold Values and Trend of COVID-19 Infection: Longitudinal Study.

BACKGROUND: The distribution of population-level real-time reverse transcription-polymerase chain reaction (RT-PCR) cycle threshold (Ct) values as a proxy of viral load may be a useful indicator for predicting COVID-19 dynamics. OBJECTIVE: The aim of this study was to determine the relationship between the daily trend of average Ct values and COVID-19 dynamics, calculated as the daily number of hospitalized patients with COVID-19, daily number of new positive tests, daily number of COVID-19 deaths, and number of hospitalized patients with COVID-19 by age. We further sought to determine the lag between these data series. METHODS: The samples included in this study were collected from March 21, 2021, to December 1, 2021. Daily Ct values of all patients who were referred to the Molecular Diagnostic Laboratory of Iran University of Medical Sciences in Tehran, Iran, for RT-PCR tests were recorded. The daily number of positive tests and the number of hospitalized patients by age group were extracted from the COVID-19 patient information registration system in Tehran province, Iran. An autoregressive integrated moving average (ARIMA) model was constructed for the time series of variables. Cross-correlation analysis was then performed to determine the best lag and correlations between the average daily Ct value and other COVID-19 dynamics-related variables. Finally, the best-selected lag of Ct identified through cross-correlation was incorporated as a covariate into the autoregressive integrated moving average with exogenous variables (ARIMAX) model to calculate the coefficients. RESULTS: Daily average Ct values showed a significant negative correlation (23-day time delay) with the daily number of newly hospitalized patients (P=.02), 30-day time delay with the daily number of new positive tests (P=.02), and daily number of COVID-19 deaths (P=.02). The daily average Ct value with a 30-day delay could impact the daily number of positive tests for COVID-19 (ß=-16.87, P<.001) and the daily number of deaths from COVID-19 (ß=-1.52, P=.03). There was a significant association between Ct lag (23 days) and the number of COVID-19 hospitalizations (ß=-24.12, P=.005). Cross-correlation analysis showed significant time delays in the average Ct values and daily hospitalized patients between 18-59 years (23-day time delay, P=.02) and in patients over 60 years old (23-day time delay, P<.001). No statistically significant relation was detected in the number of daily hospitalized patients under 5 years old (9-day time delay, P=.27) and aged 5-17 years (13-day time delay, P=.39). CONCLUSIONS: It is important for surveillance of COVID-19 to find a good indicator that can predict epidemic surges in the community. Our results suggest that the average daily Ct value with a 30-day delay can predict increases in the number of positive confirmed COVID-19 cases, which may be a useful indicator for the health system.

Acute and post-acute phase of COVID-19: Analyzing expression patterns of miRNA-29a-3p, 146a-3p, 155-5p, and let-7b-3p in PBMC.

BACKGROUND: When a new pathogen, such as severe acute respiratory syndrome coronavirus 2, appears all novel information can aid in the process of monitoring and in the diagnosis of the coronavirus disease (COVID-19). The aim of the current study is to elucidate the specific miRNA profile which can act as new biomarkers for distinguishing acute COVID-19 disease from the healthy group and those in the post-acute phase of the COVID-19 disease. METHODS: The expression level of selected miRNAs including let-7b-3p, miR-29a-3p, miR-146a-3p and miR-155-5p were evaluated in peripheral blood mononuclear cells (PBMCs) of COVID-19 patients, in both the acute and post-acute COVID-19 phase of the disease and healthy groups, by real-time PCR assays. Specificity and sensitivity of miRNAs was tested by receiver operating characteristic (ROC) analysis in COVID-19 patients. RESULTS: The expression level of all miRNAs in COVID-19 patients was significantly higher than in the healthy group. Therefore, the expression pattern of miR-29a-3p, miR-146a-3p and let-7b-3p in the post-acute COVID-19 phase was significantly different from the acute COVID-19 phase. ROC analyses demonstrated that miR-29a-3p, -155-5p and -146a-3p may serve as the novel biomarker for COVID-19 diagnosis with high specificity and sensitivity. In addition, miR-29a-3p, and -146a-3p can maybe act as novel biomarkers for distinguishing acute from post-acute phase of COVID-19 disease. DISCUSSION: The difference in miRNA expression pattern between COVID-19 patients and those in the healthy group, and between acute COVID-19 with post-acute COVID-19, suggested that cellular miRNAs could be used as promising biomarkers for diagnosis and monitoring of COVID-19.

The First Detection of Co-Infection of Double-Stranded RNA Virus 1, 2 and 3 in Iranian Isolates of Trichomonas vaginalis.

BACKGROUND: The Totiviridae family includes a number of double-stranded RNA viruses that can infect Trichomonas vaginalis. Some T. vaginalis isolates are infected with one or more double-stranded RNA (dsRNA) viruses. In this study, different strains of double-stranded RNA virus in Iranian isolates of T. vaginalis were evaluated for the first time in Iran. METHODS: Vaginal swabs were collected from 1550 participants who were referred to hospitals associated with Iran University of Medical Sciences, Tehran, Iran from June to November 2018. T. vaginalis isolates were cultured in Diamond's modified medium. After the extraction of nucleic acids using a DNA/RNA extraction kit, RT-PCR was performed and PCR products were purified and sequenced. RESULTS: In general 9 (0.6%) isolates were confirmed as T. vaginalis among 1550 collected vaginal samples. Among 9 isolates of T. vaginalis, three of them were infected with TVV1. One isolate has multiple infections with T. vaginalis virus (TVV1, TVV2 and TVV3) as coinfection. The nucleotide BLAST indicated that the T. vaginalis virus 1(TVV1) isolates were most closely related to TVV1-OC5, TVV1-UR1-1.The T. vaginalis virus 2 (TVV2) sequence had also a similarity with TVV2-UR1-1, TVV2-UR1 and TVV2-OC3. The sequence of T. vaginalis virus 3(TVV3) had similarity with TVV3-OC5, TVV3-UR1-1 and TVV3-UR1. CONCLUSION: Three dsRNA viruses T. vaginalis virus (TVV1, TVV2 and TVV3) were detected using RT-PCR in T. vaginalis Iranian isolates. The coinfection of TVV1, TVV2 and TVV3 in one isolate of T.vaginalis was observed for the first time in Iran.

Evaluation of a PCR assay for diagnosis of toxoplasmosis in serum and peripheral blood mononuclear cell among HIV/AIDS patients.

Cerebral toxoplasmosis is one of the neurological infections with high morbidity and mortality in patients with AIDS, so the accurate method for diagnosis of cerebral toxoplasmosis seems necessary. In this study, nested PCR assay using B1 gene was evaluated in diagnosis of toxoplasmosis in serum and peripheral blood mononuclear cell (PBMC) among HIV/AIDS patients. One hundred eight blood samples from HIV/AIDS patients, including four patients with cerebral toxoplasmosis and 104 HIV/AIDS patients without cerebral toxoplasmosis were evaluated for the Toxoplasma gondii antibodies using Enzyme Linked immunosorbent Assay. DNA of serum and PBMC of these patients were extracted and nested-PCR was carried out. Of 108 participants, 95 cases (88%) were positive for Toxoplasma IgG antibodies and one patient was found positive for Toxoplasma IgM antibody. In general, four patients, including three patients with cerebral toxoplasmosis, who were positive for Toxoplasma IgG antibodies and one patient without cerebral toxoplasmosis who was positive for Toxoplasma IgM antibody were found to be PCR positive. DNA of T. gondii was detected in both serum and PBMC in two cerebral toxoplasmosis patients; however DNA was detected in only PBMC in other cerebral toxoplasmosis patient. All cases with cerebral toxoplasmosis were also diagnosed by clinical and radiological manifestations. The results of this study showed that the numbers of positive samples by PCR in PBMC were higher than serum specimens for diagnosis of toxoplasmosis. If molecular method and immunological assay are complemented with magnetic resonance imaging, the results can be useful for diagnosis of cerebral toxoplasmosis.

Evaluation of CCR5-Δ32 mutation among individuals with high risk behaviors, neonates born to HIV-1 infected mothers, HIV-1 infected individuals, and healthy people in an Iranian population.

One of the important genetic factors related to resistance to HIV-1 infection is the presence of the C-C chemokine receptor type 5 delta 32 (CCR5-Δ32) homozygous genotype (Δ32/Δ32). The aim of this study was to evaluate the CCR5-Δ32 mutation among individuals with high-risk behaviors, neonates born to HIV-1-infected mothers in the prevention of mother-to-child transmission (PMTCT) project, HIV-1-infected individuals, and healthy people. The frequency of the CCR5-Δ32 genotype was assessed in a cross-sectional survey carried out from March 2014 to March 2019 among four different groups of the Iranian population. Genomic DNA was extracted from peripheral blood mononuclear cells of 140 Iranian healthy people, 84 neonates born to HIV-1-infected mothers in the PMTCT project, 71 people with high-risk behaviors, and 76 HIV-1-infected individuals. The polymerase chain reaction method was used for the amplification of the CCR5 gene. The CCR5-Δ32 heterozygous deletion was detected in five (6.6%) HIV-1-infected individuals, four (4.7%) neonates born to HIV-1 positive mothers, two (1.4%) healthy people, and also three (4.2%) people with high-risk behaviors whereas the CCR5-Δ32 homozygous deletion was absent in all the groups (Fisher's exact test, P = .0242). The allele of CCR5-Δ32 homozygous was not detected in the four study groups, and no significant difference was seen in the frequency of the CCR5Δ32 heterozygous allele between HIV seropositive and seronegative individuals. Therefore, it seems that this allele alone cannot explain the natural resistance to HIV-1 infection and probably several mechanisms are responsible for these processes and it should be further investigated.

High prevalence of occult hepatitis C virus infection in injection drug users with HIV infection.

One of the pathological forms of chronic hepatitis C is occult HCV infection (OCI), in which there is no detectable HCV RNA in plasma specimens but HCV RNA is present in PBMCs and liver biopsy specimens. The aim of this study is to estimate the prevalence of OCI in HIV-positive people who are injection drug users (IDUs). From April 2015 to August 2018, 161 Iranian IDUs with HIV infection enrolled in the study. Viral RNA was extracted from plasma and PBMC samples of participants, and the presence of HCV RNA was examined using RT nested PCR with primers from two conserved regions (5´-UTR and NS5B). HCV genotyping was performed using RFLP and sequencing methods. Of the 161 patients, 134 (83.2%) were positive for anti-HCV antibodies. All 27 patients who were negative for anti-HCV were also negative for HCV RNA in plasma, but five of them (18.5%) were positive for HCV RNA in PBMCs. Importantly, 9 out of 50 patients (18.0%) who apparently had recovered from HCV infection (i.e., were anti-HCV positive and HCV RNA negative) were positive for HCV RNA in PBMCs. Overall, 18.1% of the patients who had no signs of previous HCV infection or had apparently recovered from the disease had OCI. The HCV genotypes of the cases with OCI were as follows: five patients (35.7%) were infected with subtype 1a, eight patients (57.1%) were infected with subtype 3a, and one patient (7.1%) was infected with genotype 4. Thus, it seems that the prevalence of OCI in HIV-positive IDUs is extremely significant in Iran and is likely to delay the global eradication of HCV infection until 2030.

Detection of HCV genome in peripheral blood mononuclear cells of Iranian seropositive and HCV RNA negative in plasma of patients with beta-thalassemia major: Occult HCV infection.

Beta (ß) thalassemia major is a genetic blood disorder with a deficiency in the hemoglobin beta chain, requiring blood transfusion therapy. Multiple blood transfusions increase the risk of transmitting blood-borne infections. The aim of this study is to determine the frequency of hepatitis C virus (HCV) infection in Iranian individuals with ß-thalassemia major. A total of 164 patients with ß-thalassemia major were recruited for this study. HCV RNA testing was done on plasma and peripheral blood mononuclear cells (PBMCs) from the HCV seropositive samples (with reverse transcriptase-nested polymerase chain reaction [PCR] method using primers from the 5'-untranslated region [UTR]), and all HCV RNA positive samples were genotyped by the restriction fragment length polymorphism assay. For confirmation of the HCV genotyping in PBMCs of occult HCV infection [OCI]-positive patients, the PCR products of two different regions of HCV (5'-UTR and nonstructural protein 5B [NS5B]) were sequenced. Of 164 patients, 29.3% were positive for anti-HCV antibodies, and HCV RNA was detected in the plasma specimens of 13.4% patients and in the PBMC samples of 15.2% participants. The genomic HCV-RNA was detected in PBMC samples in 3 (6.3%) of the total 48 individuals who were HCV seropositive, and plasma HCV-RNA negative (occult HCV infection). The subtypes of HCV in the plasma and PBMC samples of three participants were not identical. This study shows that among this group of Iranian patients with ß-thalassemia major, 13.4% had active HCV infection and 6.3% had occult HCV infection as evidenced by HCV RNA detected in PBMC specimens. Therefore, the design of a prospective study that focuses on the diagnosis of OCI can be very valuable and provide more information.

Detection of HBV genome in the plasma and peripheral blood mononuclear cells of Iranian HBsAg negative patients with HIV infection: occult HBV infection.

The presence of hepatitis B virus (HBV) DNA in the absence of traceable hepatitis B surface antigen (HBsAg) in the plasma specimen of patients is defined as occult HBV infection (OBI). This study aimed to detect HBV-DNA in the plasma and peripheral blood mononuclear cells (PBMCs) of Iranian HBsAg negative patients with human immunodeficiency virus (HIV) infection. This cross-sectional study was conducted on 172 patients with HIV infection from September 2015 to August 2017. The patients were tested for serological parameters (HBsAg, HBcAb, HBeAg and HBeAb) against HBV infection. Moreover, they were tested for HBV viral load (using COBAS TaqMan 48 Kit, Roche, USA) in plasma and the presence of the HBV genome in PBMC specimens using real-time PCR. The mean age of the patients was 35.4 ± 13.4 years. Of the 172 studied patients, 109 (63.4%) were male. In this study, 151 (87.8%) patients were negative for HBsAg, 111 (64.5%) patients were negative for all HBV infection serological markers, 9 (5.2%) patients were only positive for HBsAg and 29 (16.9%) patients were only positive for HBcAb. Moreover, five (3.3%) patients with HBsAg negative had OBI (in the plasma sample of four patients and PBMC specimens of all five patients, HBV-DNA was detected). The present study revealed that 3.3% of the patients with HIV infection had occult HBV infection. Presumably, designing prospective studies to identify this infection in patients with HIV infection is informative and valuable.

Preparation of transgenic Iranian lizard Leishmania coding HIL-12.

BACKGROUND AND OBJECTIVES: Leishmania are intracellular flagellate protozoan parasites cause a wide spectrum of clinical manifestations in human. The immunological basis for resistance against leishmaniasis depends on Thl responses in the course of performance of cytokines like IL-12. In this study, a transgenic Leishmania coding human IL-12 was produced that can be used in Leishmanization. MATERIALS AND METHODS: A fragment of Iranian lizard Leishmania (I.L.L) gene, named Cysteine Peptidase C (CPC), was amplified separately as two parts with PCR reaction. Then, they were attached using SOEing PCR such that the restriction site of SalI was placed in the middle of it. SOEing PCR product was purified and cloned in HindIII restriction site of pGEM-7z-f and named pKDB-CPC. After clone optimization, the hIL-12 construct was cloned in SalI restriction site of pKDB-CPC and named pKDB-IL12. Prokaryotic section of the above construct was removed and transferred into I.L.L by electroporation. RESULTS: Production of recombinant hIL-12 in transgene parasites was proved by ELISA. rhIL-12 secreted into supernatant culture medium accumulated at concentrations up to 246.53 ± 15.92 pg.mL-1. CONCLUSION: Targeted gene replacement into the I.L.L genome using plasmid pKDB-cpc identical replacement process was successfully completed for the first time. Stabilized recombinant DNA consist of target gene didn't have any toxicity for the parasite. Transgenic I.L.L produced and secreted active human interleukin 12 and can be an appropriate candidate for Leishmanization.

Socioeconomic status and mortality after acute myocardial infarction: a study from Iran.

BACKGROUND: Studies have shown an inverse relationship between socioeconomic status (SES) and mortality due to coronary heart disease (CHD). Little is known about this association in Iran. This study aimed to investigate whether mortality after myocardial infarction (MI) varies by SES. METHODS: In a retrospective study, 1283 MI patients who hospitalized in Tehran Heart Center from March 2005 to March 2006 were followed up in March 2008. Demographic, clinical and SES data were collected from case records and by telephone interviews. Multiple logistic regression analysis was performed to estimate the predictive effect of socioeconomic factors on outcome. RESULTS: In all 664 patients were studied. Of these, 500 patients were alive and 164 were dead due to MI (64 died at hospital and 100 died at home). The results of regression analysis showed that in addition to treatment (OR = 9.52, 95%CI 4.84-18.7), having diabetes (OR = 1.78, 95% CI 1.12-2.81) or hyperlipidemia (OR = 1.82, 95% CI 1.14-2.90), socioeconomic variables including living area in square per person (lowest level vs. upper level OR = 4.92, 95% CI 2.11-11.4), unemployment (OR = 3.50, 95% CI 1.50-8.13) and education (OR for illiterate patients = 2.51, 95% CI 1.00-6.31) were the most significant contributing factors to increased mortality after MI. CONCLUSION: Although the findings should be interpreted with caution, the study results indicated that socioeconomic variables were significant contributing factors to increased mortality after myocardial infarction. The underlying role of socioeconomic status on increased mortality after MI deserves further investigation.