How to tackle single target gene positive results of SARS-CoV-2 real-time PCR: A cross-sectional study.

Background and Aims: Real-time reverse-transcriptase polymerase chain reaction (real-time RT-PCR) is the gold standard test for diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, when the test result is near the detection limit of the assay the possibility of getting false positive or negative results is high. In addition, it might result in single target gene positive (STGP) results which should be interpreted with caution. Methods: This study was performed on 29,962 nasal swabs from July 1 to August 31, 2020. Ct values less than 40 for each or both of N and RdRp genes were recommended to be selected as positive. Positive samples for one gene with the Cts more than 35 were rechecked by adding more templates. Results: The results showed that 1016 (3.39%) samples were positive just for one gene with high Ct values. The results of the second reactions showed that 325 (31.99%) samples were positive for both N and RdRp which were reported positive, 301 (29.65%) were positive only for one gene which were considered as suspicious cases and resampling was suggested for them. Finally, 390 (38.385%) samples were negative for both genes. Conclusion: In conclusion, tracking weak positive results of SARS-CoV-2 real-time RT-PCR revealed that most of the individuals who were STGP clean the infection completely in less than a week which showed they were in the convalescent phase of infection. However, some of them who were in the beginning of infection showed a decrease in Ct value during a week, so they could spread the virus in the society.

Comparison of clinical outcomes, demographic, and laboratory characteristics of hospitalized COVID-19 patients during major three waves driven by Alpha, Delta, and Omicron variants in Tehran, Iran.

Introduction: This study is the first study in which demographic, laboratory data, and outcomes of coronavirus disease-2019 (COVID-19) patients due to the circulating SARS-CoV-2 infections caused by different variants (Alpha, Delta, and Omicron) are compared in Iran. Methods: We conducted a retrospective study of confirmed hospitalized COVID-19 cases from April 9, 2021, to May 22, 2022. Demographic data and laboratory findings were extracted from patients' electronic medical records on the first day of admission to the hospital. All patients were followed up for outcomes related to COVID-19 including intensive care unit (ICU) admission and mortality rate. Results: Of 760 confirmed hospitalized COVID-19 cases, 362, 298, and 100 represented patients during waves 4-6, respectively. During the Omicron wave, hospitalized patients were older than the other two waves and had a lower median level of C-reactive protein (CRP), alanine transaminase (ALT), aspartate transaminase (AST), and erythrocyte sedimentation rate (ESR). The median length of hospital stay during waves 4-6 was 5 days (interquartile range [IQR]: 4.0-8.0), 7 days (IQR: 6.0-11), and 6 days (IQR: 5.0-9.0), respectively (p < 0.001). The rate of ICU admission during waves 4-6 significantly increased. Conclusions: Although the Omicron variant caused less severe disease, in older patients who were hospitalized due to Omicron infection, longer hospital and ICU stays were reported, which could be attributed to their old age. In particular, elderly patients are more vulnerable to severe COVID-19; otherwise, as expected, other laboratory parameters and clinical outcomes were in accordance with differences in pathogenicity and infectivity of these variants.

Evaluation of SARS-CoV-2 genome detection by real-time PCR assay using pooled specimens.

Objective: To evaluate SARS-CoV-2 genome detection using pooled samples by RT-qPCR assay, compared to individual samples. Method: At first all samples were tested individually using two commercial methods targeting ORF1ab, NP and E genes. Then, four experimental groups of samples were pooled and evaluated using the same detection methods. Findings: Compared to the individual sample testing, the sample pooling conserved the sensitivity of the detection in all groups of pooled samples when the Ct value in single test was lower than 33. Conclusion: Specimen pooling may fail to detect positive samples with high Ct values. However, in scarcity of reagents or in population surveys, it could be considered as an alternative method in low prevalence settings.

Assessment of influenza A (H1N1, H3N2) oseltamivir resistance during 2017-2019 in Iran.

Background and Objectives: Neuraminidase inhibitors (NAIs) as an imperative antiviral for influenza prophylaxis and treatment are being consumed worldwide. Increasing use of these antivirals might be associated with drug resistance. Regarding the significance of these variations, this study aimed to investigate the mutations occurring in the NA gene of influenza A viruses leading to oseltamivir resistance during 2017-2019 in Iran. Materials and Methods: In this cross-sectional study, 40 influenza A (H1N1, H3N2) strains, isolated in National Influenza Center (NIC) from patients with Severe Acute Respiratory Infection (SARI) during 2017-2019 were subjected to RT-PCR and sequencing of NA complete gene. The frequency of oseltamivir resistance and variation of NA amino acids in these strains were investigated. Results: No significant mutation conferring oseltamivir resistance was detected. However, NA antigenic sites in these strains depicted minor changes compared to the vaccine strains. Among H3N2 isolates, mutations at 329, 344, 346 and 385 and among H1N1 isolates mutations at 143 and 188 residues occurred in NA antigenic regions. Conclusion: Evaluation of NA gene sequences, showed no resistant viruses to oseltamivir. Given that the viruses in the present study were the last viruses circulating in Iran before COVID-19 pandemic, the results will be beneficial to have a worthy comparison with the strains circulating after the pandemic. Constant monitoring for the emergence of drug-resistant variants and antigenic changes are crucial for all countries.

The effect of carvacrol on respiratory syncytial virus infection in mice model: caution in the use of herbal medicines.

BACKGROUND AND OBJECTIVES: Respiratory syncytial virus (RSV) is one of the most common viruses associated with acute lower respiratory tract infections in infants, young children, and the elderly. Due to a lack of effective anti-viral drugs or vaccines, using an immunomodulatory strategy is probably the best option to decrease the burden of RSV disease. Here, we studied carvacrol as a known immunomodulator on RSV infection outcome in a mice model. MATERIALS AND METHODS: Balb/c mice were infected by intranasal inoculation of RSV-A2, and treatment started daily 24 h after infection. Mice were sacrificed on day five after infection and experimental analyses were performed to study airway immune cell influx, CD4 and CD8 subtypes, cytokine/chemokine secretion, lung histopathology, and viral load. RESULTS: Results showed that using carvacrol enhanced immune cell influx, cytokine/chemokine production, and virus titer, and aggravated lung pathology. Our result showed that carvacrol administration increased viral titer compared to the RSVPBS group. Also, carvacrol significantly induced IFN-γ production and did not induce IL-10 production. Besides, carvacrol non-significantly increased lymphocytes and monocytes count but did not affect the neutrophil count. CONCLUSION: Carvacrol at the concentration of 80 (mg/kg) did not show immunomodulatory activity to alleviate the RSV infection outcome. Further research is needed to uncover the effects of the carvacrol intervention on virus replication and immune responses following RSV infection. Many herbal remedies in use contain carvacrol. However, the use of herbal remedies to treat viral respiratory infections such as RSV has to be performed with caution.

Determination of genetic characterization and circulation pattern of Respiratory Syncytial Virus (RSV) in children with a respiratory infection, Tehran, Iran, during 2018-2019.

The RSV-associated disease accounts for a significant health burden particularly in infants and young children who need to be hospitalized. Since continuous surveillance of circulating RSV genotypes is crucial worldwide, this study aimed to investigate the genetic diversity of RSV circulating strains causing upper or lower acute respiratory infection. Our attention was geared towards studying the cases hospitalized or outpatient in children younger than 2 years of age in Iran during 2018/2019. In this study, nasopharyngeal swabs collected from 206 children who presented with respiratory infection symptoms, were admitted to the referral pediatric ward of Bahrami children's hospital in Tehran, Iran. RSV-positive samples were detected via Nested RT-PCR. The glycoprotein gene was sequenced, and virus genotypes were confirmed through phylogenetic analysis by the MEGA X program. A total of 74 (35.92%) samples tested positive for RSV. Among them, sequencing was done in 10 specimens from 2018 (RSV-A: RSV-B=4:6) and 19 specimens from 2019 (RSV-A: RSV-B=16:3). According to phylogenetic analysis, all RSV-A strains were assigned as ON1 genotype and RSV-B strains were assigned as BA9 genotype. A new N-glycosylation site in Iranian BA9 and positive selection in ON1 genotype was observed. Phylogenetic characterization of strains in the current study revealed co-circulation of ON1 and BA9 as the only prevalent genotypes of both RSV-A and -B groups.

Seroepidemiology of dengue and chikungunya fever in patients with rash and fever in Iran, 2017.

After the mass campaign of Measles and Rubella vaccination in 2003 in Iran, the cases of measles and rubella infection decreased but still, the cases of rash and fever were reported. It is worth noting that some other viral infections show signs similar to measles and rubella such as some arboviruses. Considering the epidemic outbreak of arbovirus infections in countries neighbouring Iran, we performed this study to estimate the possibility of chikungunya and dengue fever among measles and rubella IgM negative patients presenting with rash and fever from December 2016 to November 2017 in the National Measles Laboratory at Tehran University of Medical Sciences. Serum samples were selected at random from patients from eight provinces. The presence of DENV IgM and CHIKV IgM was examined by enzyme-linked immunosorbent assay. Of the 1306 sera tested, 210 were CHIKV seropositive and 82 were dengue seropositive. Statistical analysis demonstrated a significant increase in the CHIKV IgM antibody seropositivity rate in Kerman (OR = 2.07, 95% CI: 1.10-3.92; P = 0.024) and Fars (OR = 1.77, 95% CI: 1.06-2.93; P = 0.027). The DENV and CHIKV seropositivity rate in summer is higher than in other seasons (P < 0.01). Our seropositive samples suggest possible CHIKV and DENV infection in Iran. It is likely that these viruses are circulating in Iran and there is a need to study vector carriage of these two viruses.

Correlation between interleukin-28 gene polymorphism with interleukin-28 cytokine levels and viral genotypes among HCV patients in Yazd, Iran.

OBJECTIVES: Recent studies using genome-wide association studies (GWAS) have shown the strong association between polymorphisms near the interleukin-28B (IL-28B) gene and spontaneous clearance of hepatitis C virus (HCV). The present study was designed to evaluate the association of interleukin-28 gene polymorphism with interleukin-28 cytokine levels in different viral genotypes among HCV patients in Yazd, Iran. RESULT: The most prevalent genotype in chronic cases was genotype 3a, and the lowest one was 2/3a. There were statistically significant differences in genotype frequency between the two studied groups for IL-28B rs12979860C/T. The frequency of CC genotype of IL-28B at rs12979860 SNP was higher in spontaneously cleared patients in comparison with chronic HCV patients. Significant association was found when serum levels of IL28B were compared to various IL-28 genotypes. There was a significant difference between IL-28 polymorphism and HCV genotypes (p = 0.003).

Prevalence of Influenza A(H1N1)pdm09 Virus Resistant to Oseltamivir in Shiraz, Iran, During 2012 - 2013.

BACKGROUND: Oseltamivir has been used as a drug of choice for the prophylaxis and treatment of human influenza A(H1N1)pdm09 infection across the world. However, the most frequently identified oseltamivir resistant virus, influenza A(H1N1)pdm09, exhibit the H275Y substitution in NA gene. OBJECTIVES: This study aimed to determine the prevalence and phylogenetic relationships of oseltamivir resistance in influenza A(H1N1)pdm09 viruses isolated in Shiraz, Iran. PATIENTS AND METHODS: Throat swab samples were collected from 200 patients with influenza-like disease from December 2012 until February 2013. A total of 77 influenza A(H1N1)pdm09 positive strains were identified by real-time polymerase chain reaction (PCR). Oseltamivir resistance was detected using quantal assay and nested-PCR method. The NA gene sequencing was conducted to detect oseltamivir-resistant mutants and establish the phylogeny of the prevalent influenza variants. RESULTS: Our results revealed that A(H1N1)pdm09 viruses present in these samples were susceptible to oseltamivir, and contained 5 site specific mutations (V13G, V106I, V241I, N248D, and N369K) in NA gene. These mutations correlated with increasing expression and enzymatic activity of NA protein in the influenza A(H1N1)pdm09 viruses, which were closely related to a main influenza A(H1N1)pdm09 cluster isolated around the world. CONCLUSIONS: A(H1N1)pdm09 viruses, identified in this study in Shiraz, Iran, contained 5 site specific mutations and were susceptible to oseltamivir.

Antigenic Variation of the Haemagglutinin Gene of the Influenza A (H1N1) pdm09 Virus Circulating in Shiraz, February-April 2013.

BACKGROUND: A new pandemic influenza A (H1N1) emerged in April 2009, causing considerable morbidity and mortality. Since mutations in the haemagglutinin (HA) may influence the antigenicity and pathogenicity of the virus, continued epidemiological and molecular characterization for the effective control of pandemic flu and developing of more appropriate vaccine is crucial. OBJECTIVE: To monitor the molecular evolution of A (H1N1) pdm09 viruses in a specific time period in Shiraz, Southern Iran. METHODS: A total of 200 samples were collected from February-April 2013. HA gene of the isolates was amplified and sequenced. Phylogenetic analysis of the HA gene was performed. RESULTS: Out of 200 samples, a total of 77 (38.5%) samples were confirmed as A (H1N1) pdm09 virus using Real-time PCR method. Nucleotide similarity of our study strains with respect to reference strain A/California/07/2009 (H1N1) was 97.5%-98.5%. Phylogenetic analysis of our study strains indicated that the dominant A (H1N1) pdm09 clade was clade 7 and the dominant genetic group in circulating strains in Shiraz was genetic group 6. Some of our study strains showed substitutions at or in the vicinity of the antigenic sites of the HA1 region which may affect the efficacy of the vaccine. CONCLUSION: Our study strains showed a high homology to the vaccine strain. Our findings confirm the genetic variability of influenza A (H1N1) pdm09 and highlight the necessity of continuous molecular study of the virus for effective management of influenza.