COVID-19 PCR test performance on samples stored at ambient temperature.

The WHO-named Coronavirus Disease 2019 (COVID-19) infection had become a pandemic within a short time period since it was detected in Wuhan. The outbreak required the screening of millions of samples daily and overwhelmed diagnostic laboratories worldwide. During this pandemic, the handling of patient specimens according to the universal guidelines was extremely difficult as the WHO, CDC and ECDC required cold chain compliance during transport and storage of the swab samples. The aim of this study was to compare the effects of two different storage conditions on the COVID-19 real-time PCR assay on 30 positive nasopharyngeal and/or oropharyngeal samples stored at both ambient temperature (22 ± 2 °C) and +4 °C. The results revealed that all the samples stored at ambient temperature remain PCR positive for at least six days without any false-negative result. In conclusion, transporting and storing these types of swab samples at ambient temperature for six days under resource-limited conditions during the COVID-19 pandemics are acceptable.

Does sampling saliva increase detection of SARS-CoV-2 by RT-PCR? Comparing saliva with oro-nasopharyngeal swabs.

The gold standard method in the diagnosis of SARS-CoV-2 infection is the detection of viral RNA in the nasopharyngeal sample by RT-PCR. Recently, saliva samples have been suggested as an alternative sample. In the present study, we aimed to compare RT-PCR results in nasopharyngeal, oro-nasopharyngeal and saliva samples of COVID-19 patients. 98 of 200 patients were positive in RT-PCR analysis performed before the hospitalization. On day 0, at least one sample was positive in 67 % of 98 patients. The positivity rate was 83 % for both oro-nasopharyngeal and nasopharyngeal samples, while it was 63 % for saliva samples (p < 0.001). On day 5, RT-PCR was performed in 59 patients, 34 % had at least one positive result. The positivity rate was 55 % for both saliva and nasopharyngeal samples, while it was 60 % for oro-nasopharyngeal samples. Our study shows that the sampling saliva does not increase the sensitivity of RT-PCR tests at the early stages of infection. However, on the 5th day, viral RNA detection rates in saliva were similar to nasopharyngeal and oro-nasopharyngeal samples. In conclusion, we suggest that, in patients receiving treatment, RT-PCR in saliva, in addition to the standard samples, is important to determine the isolation period and control transmission.

The origin of SARS-CoV-2 in Istanbul: Sequencing findings from the epicenter of the pandemic in Turkey.

OBJECTIVE: Turkey is one of the latest countries that COVID-19 disease was reported, with the first case on March 11, 2020, and since then, Istanbul became the epicenter of the pandemic in Turkey. Here, we reveal sequences of the virus isolated from three different patients with various clinical presentations. METHODS: Nasopharyngeal swab specimens of the patients were tested positive for the COVID-19 by qRT-PCR. Viral RNA extraction was performed from the same swab samples. Amplicon based libraries were prepared and sequenced using the Illumina NextSeq platform. Raw sequencing data were processed for variant calling and generating near-complete genome sequences. All three genomes were evaluated and compared with other worldwide isolates. RESULTS: The patients showed various clinics (an asymptomatic patient, patient with mild disease, and with severe pulmonary infiltration). Amplicon-based next-generation sequencing approach successfully applied to generate near-complete genomes with an average depth of 2.616. All three viral genomes carried the D614G variant (G clade according to GISAID classification) with implications for the origin of a spread first through China to Europe then to Istanbul. CONCLUSION: Here, we report the viral genomes circulating in Istanbul for the first time. Further sequencing of the virus isolates may enable us to understand variations in disease presentation and association with viral factors if there is any. In addition, the sequencing of more viral genomes will delineate the spread of disease and will guide and ease the necessary measures taken to stem the spread of the novel coronavirus.

Roscovitine-treated HeLa cells finalize autophagy later than apoptosis by downregulating Bcl­2.

The cell cycle is tightly regulated by the family of cyclin-dependent kinases (CDKs). CDKs act as regulatory factors on serine and threonine residues by phosphorylating their substrates and cyclins. CDK­targeting drugs have previously demonstrated promising effects as cancer therapeutics both in vitro and in vivo. Roscovitine, a purine­derivative and specific CDK inhibitor, has been demonstrated to arrest the cell cycle and induce apoptosis in a number of different cancer cell lines, including HeLa cervical cancer cells. In the present study, roscovitine was able to decrease both the cell viability and cell survival as well as induce apoptosis in a dose­dependent manner in HeLa cells by modulating the mitochondrial membrane potential. The decrease of anti­apoptotic B-cell lymphoma 2 (Bcl­2) and Bcl-2 extra large protein expression was accompanied by the increase in pro­apoptotic Bcl-2-associated X protein and P53-upregulated modulator of apoptosis expression. The marked decrease in Bcl­2 following exposure to roscovitine (20 µM) for 48 h prompted us to determine the autophagic regulation. The outcome revealed that roscovitine triggered Beclin­1 downregulation and microtubule-associated light chain 3 cleavage starting from 12 h of incubation. Another biomarker of autophagy, p62, a crucial protein for autophagic vacuole formation, was diminished following 48 h. In addition, monodansyl cadaverin staining of autophagosomes also confirmed the autophagic regulation by roscovitine treatment. The expression levels of different Bcl­2 family members determined whether apoptosis or autophagy were induced following incubation with roscovitine for different time periods. Downregulation of pro­apoptotic Bcl­2 family members indicated induction of apoptosis, while the downregulation of anti­apoptotic Bcl­2 family members rapidly induced autophagosome formation in HeLa cells.