Diagnosis of COVID-19 by Serology in Admitted Patients with Negative RT-PCR Assay

Considering the increasing prevalence and burden of coronavirus disease 2019 (COVID-19) disease and false-negative results in routine reverse transcription-polymerase chain reaction (RT-PCR) tests, additional diagnostic methods are needed to diagnose active cases of this disease. This prospective study was conducted on patients, in whom clinical and radiological symptoms/signs were in favor of COVID-19 while their first PCR test was negative. Later on, a second RT-PCR was performed and serological evaluation was carried out and results were compared with each other. Out of 707 patients who had been referred to the hospital and were clinically and radiologically suspicious of disease, 137 patients with negative RT-PCR tests entered the study.
RT-PCR assay became positive for the second time in 45 (32.8%). Anti-COVID-19 IgM and IgG antibodies were positive in 83 (60.6%) and 86 (62.8%) patients, respectively. Finally, it was determined that serological test was diagnostic in 73% of patients and the diagnostic yield of serology was significantly higher after the first week of illness (54.8% in the first week and 88% after that). Taking advantage of both serological tests and RT-PCR helps in diagnosing 83.9% of cases. Based on the present study, the serology may be useful as a complementary test and in parallel to RT-PCR assay for diagnosis of COVID-19 among admitted symptomatic cases.

Analysis of Nucleotide Changes in RT-PCR Primer/Probe Binding Regions in SARS-CoV-2 Isolates Reported from Turkey

The SARS-CoV-2 virus, which caused the COVID-19 epidemic, caused more than 55 million cases and nearly 1.5 million deaths worldwide. For the microbiological diagnosis of the disease, the most valid method is detecting the presence of the viral genome by real-time reverse transcription polymerase chain reaction (rRT-PCR). However, due to the nature of the RNA viruses, frequent mutations may affect the sensitivity of the analyses made on the genetic material of the virus, such as PCR. In this study, we aimed to investigate the mutations in the primer-probe binding regions of the rRT-PCR panels used in COVID-19 diagnosis. SARS-CoV-2 whole genome sequence data (n= 194) isolated from COVID-19 cases in Turkey and uploaded on GISAID database from the centers in İstanbul (n= 78), Ankara (n= 58), Kars (n= 47), Bursa (n= 2), Adıyaman (n= 2), Erciyes (n= 1) and Kocaeli (n= 1) between March 17-September 14, 2020 were analyzed.
In order to determine the nucleotide changes, SARS-CoV-2 sequences from Turkey were compared to the reference genome sequence (NC_045512.1) present in “GenBank” website. The constructed data set was aligned using the MAFFT program and was checked manually if the sequences were in the same frame by using the AliView program. Primer-probe binding sites of the thirteen SARS-CoV-2 rRT-PCR panels from seven different institutes (US CDC, China CDC, Charite CDC, Pasteur, HKU, Thailand, NIID) that are being used in COVID-19 diagnosis were evaluated in terms of nucleotide changes within the corresponding regions compared to the reference genome. Sequence diversities in the viral genomes were determined via positional nucleotide numerical calculator and entropy calculator modules and nucleotide and entropy changes in primer-probe binding regions for each rRT-PCR panel were examined.
Among thirteen different primer-probe panels, nucleotide changes in the target regions of the seven primer-probe panels were determined. When viral sequences with nucleotide changes in the primer-probe binding regions were examined, the most common changes were observed in the “China CDC” N-forward primer and “US CDC” N3-forward primer binding regions. It is important that the kits to be used as diagnostic tests are designed specific to the regions with less nucleotide changes. Nucleotide changes may not be critical for DNA amplification for most PCR panels, but should be carefully monitored as they may affect the sensitivity of the assay. If the risk of alteration of the designed region is high, the primer – probe binding sites should be checked frequently and updated when necessary.

Detection of Long Noncoding RNA Expression by Real-Time PCR

Recent advances in sequencing technology and bioinformatic analysis promote the identification of long noncoding RNA (lncRNA), a novel form of untranslated RNA transcript. Long coding RNA has been extensively investigated for its fundamental biological functions in a broad spectrum of pathogenetic and therapeutic studies. The level of lncRNA expression varies among different tissue types and contexts.
Therefore, a quantitative method to accurately measure lncRNA expression is highly desired. Real-time PCR analysis enables us to conveniently determine the identity and abundance of lncRNA in a time-efficient manner. Here, we introduce how to use real-time PCR to analyze and quantify a defined lncRNA.


Comparison of Real Time PCR Cycle Threshold Values in Symptomatic and Asymptomatic COVID-19 Patients

Patients infected with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) show different clinical courses ranging from asymptomatic to severe infection requiring intensive care treatment and death. Real-time reverse transcription polymerase chain reaction (rRT-PCR), used in the diagnosis, screening and surveillance of coronavirus-2019 (COVID-19), provides the viral load as a cycle threshold (Ct) value. It has been reported that the Ct value may be related to the course of the infection and the clinical condition of the patient. In this study, it was aimed to compare the Ct and C reactive-protein (CRP) results of symptomatic and asymptomatic patients who were found to be positive with rRT-PCR. Between 14 April and 29 August 2020, a total of 355 patients aged 18 years and older with positive SARS-CoV-2 rRT-PCR test were included in the study.
The COVID-19 rRT-PCR test was performed with Bio-speedy SARS-CoV-2 rRT-PCR kit (Bioeksen, Turkey) versions, the kit targeting the RdRp gene region, and the dual gene kit versions targeting the N and ORF1ab gene regions were used. Patients were classified as symptomatic and asymptomatic according to their clinical findings. Ct and CRP results of the patients were analyzed statistically. Of the 355 patients included in the study, 237 (66.7%) were symptomatic and 118 (33.2%) were asymptomatic patients. The mean age of symptomatic patients (46.68 ± 18.03) was observed significantly higher than asymptomatic patients (38.27 ± 13.82) (p<0.001).
When the patients are evaluated according to the age groups, the rate of asymptomatic patients was significantly higher in the 21-39 age group, while the rate of symptomatic patients was significantly higher in 65 years and older group (p<0.05). The rate of comorbidity was significantly higher in symptomatic patients (n= 69, 29.1%) than in asymptomatic patients (n= 11, 9.3%) (p<0.001). Hypertension (12.2%), diabetes mellitus (9.7%), chronic respiratory disease (9.3%) and cardiovascular diseases (5.5%) were the most common diseases in symptomatic patients.
However, among these, hypertension and chronic respiratory disease were found significantly higher in symptomatic patients (p<0.05). Increased CRP rate in symptomatic patients (64.6%) was found significantly higher than asymptomatic patients (27.3%) (p<0.001). The median of Ct value was found significantly higher in asymptomatic patients (26.34, IQR= 19.78-35.48), than in symptomatic patients (21.77, IQR= 17.81-26.51) (p<0.001). Regarding the medians of Ct values obtained from target genes; RdRp gene Ct value was found significantly higher in asymptomatic patients than in symptomatic patients (p<0.001).
However, no statistical difference was found between symptomatic and asymptomatic patients in the ORF1ab and N genes Ct value medians (p> 0.05). As a result, it was observed that SARS-CoV-2 PCR positive patients were symptomatic in the presence of advanced age and comorbidity. Increased CRP value at the time of admission to the hospital was found significantly higher in symptomatic patients. Ct value has been shown to be lower in symptomatic patients, as expected. Although Ct and CRP values are thought to be useful in monitoring the clinical course and prognosis of patients with COVID-19, more detailed studies are needed to prove their clinical value.


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