Correlation Of Pcr Ct Values With Symptom Onset Across Diagnostic Platforms For Sars-Cov-2

Introduction/Objective Following the definition of SARS-CoV2 outbreak as a pandemic by WHO, FDA gave EUA approval to the CDC real time polymerase chain reaction (PCR) assay and soon to other vendors to increase test availability. Most of testing platforms are PCR based, which test for multiple gene targets. We aimed to compare distribution of crossing threshold (cT) values of Diasorin (DIA), NeuMoDX (NDX) and Cepheid GenXpert (GX) for symptomatic and asymptomatic patients and assess performance of individual gene targets within the assays. We also correlated cT values with time from symptom onset. Methods/Case Report Retrospective review of medical and laboratory records of patients who tested positive for SARS-CoV2 between 08/01/2020 and 10/10/2020 on DIA, NDX, and GX platforms. Results (if a Case Study enter NA) We included 212 patients in our study. Days since symptom onset included 1 to 16 days. For DIA, mean Ct values for 46 symptomatic patients were 21.75 (S gene) and 22.74 (ORF1 gene); whereas 23.49 (S gene) and 25.49 (ORF1 gene) for 12 asymptomatic patients. Similarly, on NDX mean was 22.21 (N gene) and 23.13 (NSP2 gene) for 69 symptomatic, though 28.09 (N gene) and 28.61 (NSP2 gene) for 35 asymptomatic patients. GX manifested mean Ct value of 27.13 (E gene) and 31.22 (N2 gene) for 19 symptomatic; while 33.85 (E gene) and 36.42 (N2 gene) for 31 asymptomatic patients. Correlation coefficient for cT values versus days since symptom onset are DIA (r2 0.19), NDX (r2 0.22), and GX (r2 0.02). Conclusion The difference in cT values was statistically significant for symptomatic versus asymptomatic patients. There was positive correlation between days since symptom onset and cT values for DIA and NDX but not for GX, which may be due to difference in population tested in these platforms. These observations may be used to predict viral load and thus infectivity of patients who test positive for SARS-CoV2.

Genome-based Detection of Novel Coronavirus: An Overview Study

The Coronavirus Disease 2019, named COVID-19 is a global problem. According to the declaration of the World Health Organization (WHO), it is a novel and extreme outbreak, spreading worldwide. Firstly, numerous patients reported exposure in Wuhan City, China at a large animals and seafood market. Accordingly, the first idea is suggesting the animal-to-human transmission of this infection pathogen. Next, since then, numerous patients have decelerated no exposure to animal shops, indicating that human-to-human transmission is occurring. The new coronavirus (2019-nCoV) is a positive RNA virus with a 29.8 kb genome and S, E, M, N, and Orf1 gene fragments. The most popular method for 2019-nCoV detection is genome-based approaches, like Polymerase Chain Reaction (PCR)-based tests that require expensive experimental equipment, a controlled working environment, and high-trained technicians; they are often lacking in massive viral outbreaks. Therefore, another rapid and simple genome-based assay was evaluated for this pathogen. In this review study, the RT-LAMP technique, as the main isothermal amplification assays with less time consumption and without the need for expensive equipment, compared to conventional PCR-based methods to 2019-nCoV identification, was discussed. Accordingly, the advantage or disadvantage of these techniques was compared. The obtained data indicated that this molecular and isothermal method could be used as a successful one-step process for portable screening and the rapid identification of 2019-nCoV.

Saliva as a potential clinical specimen for diagnosis of SARS-CoV-2

Background It is almost nine months, still there is no sign to stop the spreading of the COVID-19 pandemic. Rapid and early detection of the virus is the master key to cease the rapid spread and break the human transmission chain. There are very few studies in search of an alternate and convenient diagnostic tool which can substitute nasopharyngeal swab (NPS) specimen for detection of SARS-CoV-2. We aimed to analyse the comparison and agreement between the feasibility of using the saliva in comparison to NPS for diagnosis of SARS-CoV-2. Methods A total number of 74 patients were enrolled for this study. We analysed and compared the NPS and saliva specimen collected within 48 h after the symptom onset. We used real time quantitative polymerase chain reaction (RT-qPCR), gene sequencing for the detection and determination SARS-CoV-2 specific genes. Phylogenetic tree was constructed to establish the isolation of viral RNA from saliva. We use Bland-Altman model to identify the agreement between two sampling methods. Findings This study shows a lower CT mean value for the detection of SARS-CoV-2 ORF1 gene (27.07; 95% CI, 25.62 to 28.52) in saliva methods than that of NPS (28.24; 95% CI, 26.62 to 29.85) sampling method. Bland-Altman analysis produces relatively smaller bias and high agreement between these specimen tools. Phylogenetic analysis with the RdRp and Spike gene confirmed the presence of SARS-CoV-2 in the saliva samples. Interpretation: In conclusion, our study highlights that saliva represents a promising tool in COVID-19 diagnosis and would reduce the exposure risk of frontline health workers which is one of biggest concern in primary healthcare settings.

Comparative analysis of primer-probe sets for the laboratory confirmation of SARS-CoV-2

Coronavirus disease 2019 (COVID-19) is newly emerging human infectious diseases, which is caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2, also previously known as 2019-nCoV). Within two months of the outbreak, more than 80,000 cases of COVID-19 have been confirmed worldwide. Since the human to human transmission occurred easily and the human infection is rapidly increasing, the sensitive and early diagnosis is essential to prevent the global outbreak. Recently, World Health Organization (WHO) announced various primer and probe sets for SARS-CoV-2 previously developed in China, Germany, Hong Kong, Japan, Thailand, and USA. In this study, we compared the ability to detect SARS-CoV-2 RNA among the seven primer-probe sets for N gene and the three primer-probe sets for Orf1 gene. The result of the comparative analysis represented that the ‘2019-nCoV_N2, N3’ of USA and the ‘ORF1ab’ of China are the most sensitive primer-probe sets for N and Orf1 genes, respectively. Therefore, the appropriate combination from ORF1ab (China), 2019-nCoV_N2, N3 (USA), and NIID_2019-nCOV_N (Japan) sets should be selected for the sensitive and reliable laboratory confirmation of SARS-CoV-2.

An Asymptomatic Patient with Fatal Infertility Carried a Swedish Strain of Chlamydia trachomatis with Additional Deletion in The Plasmid orf1 that Belonged to A Different MLST Sequence Type

Here, we present the first case of asymptomatic genital Chlamydial infection caused by the new emerging Chlamydia trachomatis (C.t.) ST13 strain genovar E, which has a double deletion of 377 bp and 17 bp in orf1 gene of the cryptic plasmid (ddCT). This case occurred in an infertile patient (case-patient) with a detectable level of Chlamydial antibodies and a spermatozoa deficiency known as azoospermia. Additionally, the ddCT strain showed the presence of a duplication of 44 bp in the plasmid orf3 and SNP in orf4, which were known as the typical characteristics of the Swedish variant of C.t. (nvCT) genovar E. Multilocus sequence typing (MLST) determined a significant difference between ddCT and nvCT in four alleles (oppA, hfiX, gitA and enoA). Both ddCT and nvCT were assigned to different genetic lineages and could be allocated to two different non-overlapping clonal complexes. Furthermore, ddCT demonstrated a considerable difference among 4-5 alleles in comparison with other C.t. strains of genovar E of ST4, ST8, ST12, and ST94, including the founder of a single relevant cluster, wtCT E/SW3 (Swedish genetic lineage). In contrast to other genovar E strains, ddCT had identical alleles with seven out of seven loci found in ST13 strains of genovars D and G, including the founder for this clonal group, D/UW-3/CX, and six out of seven loci found in its derivatives, such as ST6, ST10, and ST95 of genovars G and H. Nevertheless, MSTree V2 showed that ddCT and nvCT could have a common early ancestor, which is a parental C.t. G/9301 strain of ST9. A significant difference between ddCT and nvCT of genovar D (nvCT-D) that was recently found in Mexico was also determined as: (i) ddCT belonged to genovar E but not to genovar D; (ii) ddCT had a 44 bp duplication within the orf3 of the plasmid typical for nvCT; (iii) ddCT possessed an additional 17 bp deletion in the orf1. In conclusion, improved case management should include the clinical physician’s awareness of the need to enhance molecular screening of asymptomatic Chlamydia patients. Such molecular diagnostics might be essential to significantly reducing the global burden of Chlamydial infection on international public health.

Evolution and Functional Analysis of orf1 Within nif Gene Cluster from Paenibacillus graminis RSA19

Paenibacillus is a genus of Gram-positive, facultative anaerobic and endospore-forming bacteria. Genomic sequence analysis has revealed that a compact nif (nitrogen fixation) gene cluster comprising 9–10 genes nifBHDKENX(orf1)hesAnifV is conserved in diazotrophic Paenibacillus species. The evolution and function of the orf1 gene within the nif gene cluster of Paenibacillus species is unknown. In this study, a careful comparison analysis of the compositions of the nif gene clusters from various diazotrophs revealed that orf1 located downstream of nifENX was identified in anaerobic Clostridium ultunense, the facultative anaerobic Paenibacillus species and aerobic diazotrophs (e.g., Azotobacter vinelandii and Azospirillum brasilense). The predicted amino acid sequences encoded by the orf1 gene, part of the nif gene cluster nifBHDKENXorf1hesAnifV in Paenibacillus graminis RSA19, showed 60–90% identity with those of the orf1 genes located downstream of nifENX from different diazotrophic Paenibacillus species, but shared no significant identity with those of the orf1 genes from different taxa of diazotrophic organisms. Transcriptional analysis showed that the orf1 gene was expressed under nitrogen fixation conditions from the promoter located upstream from nifB. Mutational analysis suggested that the orf1 gene functions in nitrogen fixation in the presence of a high concentration of O2.

Molecular detection of hepatitis E virus in feces and slurry from swine farms, Rio Grande do Sul, Southern Brazil

Hepatitis E virus (HEV) is highly disseminated among swine herds worldwide. HEV is also a threat to public health, since particularly genotypes 3 and 4 may cause acute hepatitis in human beings. No previous studies were done on the occurrence of HEV in environmental samples in Rio Grande do Sul, Brazil. In the present study, reverse transcriptase-polymerase chain reaction (RT-PCR) was employed to detect the presence of HEV in swine feces and in effluents from slurry lagoons in farms located in the municipality of Teutônia, inside the area of swine husbandry in the state. Pooled fecal samples from the floor of pig barns from 9 wean-to-finish farms and liquid manure samples were collected from the slurry lagoons from 8 of these farms. From the pooled fecal samples, 8/9 were positive for the HEV ORF1 gene by RT-PCR; all the slurry lagoon samples were positive for HEV RNA (100%). The identity of the HEV ORF1 amplicons was confirmed by sequencing belonging to HEV genotype 3, which was previously shown to be circulating in South America.

The prevalence of hepatitis E virus in piglets on Czech pig production farms and phylogenetic analysis of recovered isolates

The aim of our study was to determine the prevalence of hepatitis E virus (HEV) in domestic pigs and to investigate the genetic divergence of swine HEV in the Czech Republic. To this end, a one-step real time RT-PCR assay was introduced as a screening method while nested RT-PCR was used as an additional method to obtain specific sequences from the HEV genome and thus to perform sequence analysis. A total of 63 piglets originating from 14 farms were examined. Bile and intestinal contents were collected from each animal. At least one HEV RNA positive piglet was found in ten (71.4%) of the monitored farms. HEV RNA was most frequently detected in bile samples (34.9%) compared to intestinal content samples (22.2%). In nine piglets (14.3%), both biological samples were HEV RNA-positive. Based on these results sequence and phylogenetic analysis of one randomly selected HEV isolate originating from each HEV RNA-positive farm was performed. Analysis of 287 bp PCR products of the ORF1 gene showed that all the studied HEV isolates could be classified into genotype 3 and subgenotypes 3f and 3g. The failure to find any 100% homology between our isolates and HEV isolates deposited in the GenBank confirms the significant variability within the HEV genome.