Evaluation of two RT-PCR techniques for SARS-CoV-2 RNA detection in serum for microbiological diagnosis

Presence of SARS-CoV-2 RNA in serum (viraemia) in COVID-19 patients has been related to poor prognosis and death. The aim of this study was to evaluate the ability of two commercial reverse real-time-PCR (rRT-PCR) kits, cobas SARS-CoV-2 (Cobas test) and TaqPath COVID-19 CE-IVD RT-PCR Kit (Taqpath test), to detect viraemia in COVID-19 patients and their implementation as routine diagnosis in microbiology laboratory. This retrospective cohort study was conducted with 203 adult patients admitted to Hospital Universitario de La Princesa, (89 Intensive Care Unit and 114 ward) with at least one serum sample collected in the first 48 hours from admission. A total 265 serum samples were included for study. Evaluation of both rRT-PCR techniques was performed comparing with the gold standard, a Novel Coronavirus (2019-nCoV) Nucleic Acid Diagnostic Kit; considering at least one target as a positive result. Comparison of Cobas test and Taqpath test with the gold standard method, showed high values of specificity (93.75 and 92.19 respectively) and Positive Predictive Value (92.92 and 99.88 respectively). Nevertheless, sensitivity (53.72 and 73.63 respectively) and Negative Predictive Value (32.53 and 42.99 respectively) were lower; Kappa values were 0.35 for cobas test and 0.56 for Taqpath test. For both techniques, differences of viraemia detection between the ICU and non-ICU patients were significant (p<0.001). Consequently, SARS-CoV-2 viraemia positive results obtained by both rRT-PCR should be considered good tools and may help in handling COVID-19 patients. Moreover, these methods could be easily integrated in the routine laboratory COVID-19 diagnosis and may open new strategies based on an early COVID-19 treatment.

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Superiority of MALDI-TOF Mass Spectrometry over Real-Time PCR for SARS-CoV-2 RNA Detection

Viruses ◽

10.3390/v13050730 ◽

2021 ◽

Vol 13 (5) ◽

pp. 730

Author(s):

Magda Rybicka ◽

Ewa Miłosz ◽

Krzysztof Piotr Bielawski

Keyword(s):

Mass Spectrometry ◽

Gold Standard ◽

Viral Genome ◽

False Negative ◽

Rt Pcr ◽

Rna Detection ◽

Maldi Tof ◽

Negative Results ◽

False Negative Results ◽

Pcr Test

At present, the RT-PCR test remains the gold standard for early diagnosis of SARS-CoV-2. Nevertheless, there is growing evidence demonstrating that this technique may generate false-negative results. Here, we aimed to compare the new mass spectrometry-based assay MassARRAY® SARS-CoV-2 Panel with the RT-PCR diagnostic test approved for clinical use. The study group consisted of 168 suspected patients with symptoms of a respiratory infection. After simultaneous analysis by RT-PCR and mass spectrometry methods, we obtained discordant results for 17 samples (10.12%). Within fifteen samples officially reported as presumptive positive, 13 were positive according to the MS-based assay. Moreover, four samples reported by the officially approved RT-PCR as negative were positive in at least one MS assay. We have successfully demonstrated superior sensitivity of the MS-based assay in SARS-CoV-2 detection, showing that MALDI-TOF MS seems to be ideal for the detection as well as discrimination of mutations within the viral genome.

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The Progesterone Receptor in Human Term Amniochorion and Placenta Is Isoform C

Endocrinology ◽

10.1210/en.2005-0510 ◽

2006 ◽

Vol 147 (2) ◽

pp. 687-693 ◽

Cited By ~ 20

Author(s):

Anthony H. Taylor ◽

Penny C. McParland ◽

David J. Taylor ◽

Stephen C. Bell

Keyword(s):

Progesterone Receptor ◽

Western Blotting ◽

Cell Types ◽

Specific Role ◽

Fetal Membranes ◽

Rt Pcr ◽

Reproductive Tissues ◽

Pcr Techniques ◽

Human Parturition ◽

Extraembryonic Tissues

The mechanism that initiates human parturition has been proposed to be functional progesterone withdrawal whereby the 116-kDa B isoform of the progesterone receptor (PR-B) switches in favor of the 94-kDa A isoform (PR-A) in reproductive tissues. Recently other PR isoforms, PR-S, PR-C, and PR-M generated from the same gene have been identified and partially characterized. Using immunohistochemical, Western blotting, and RT-PCR techniques, evidence is provided that the major PR isoform present in human term fetal membranes (amnion and chorion) and syncytiotrophoblast of the placenta is neither of the classical nuclear PR-B or PR-A isoforms but is the N terminally truncated 60-kDa PR-C isoform. Evidence is also provided that the PR-C isoform resides in the cytoplasm of the expressing cell types. Data are also presented to show that PR-B, PR-A, and PR-S isoforms are essentially absent from the amnion and chorion, whereas PR isoforms A, B, C, and S are all present in the decidua, with PR-A being the major isoform. The syncytiotrophoblast of the placenta contains the cytoplasmic PR-C isoform but not PR-A, PR-B, or PR-S. The major PR isoform in the amnion, chorion, and placenta is PR-C, suggesting that the cytoplasmic PR-C isoform has a specific role in extraembryonic tissues and may be involved in the regulation of human parturition.

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Expression of β-catenin in human trophoblast and its role in placenta accreta and placenta previa

Journal of International Medical Research ◽

10.1177/0300060518799265 ◽

2018 ◽

Vol 47 (1) ◽

pp. 206-214

Author(s):

Qing Han ◽

Lianghui Zheng ◽

Zhaodong Liu ◽

Jinying Luo ◽

Rongxin Chen ◽

...

Keyword(s):

Western Blotting ◽

Cell Invasion ◽

Placenta Accreta ◽

Placenta Previa ◽

Control Group ◽

Rt Pcr ◽

Lesion Area ◽

Human Trophoblast ◽

Chorionic Villi ◽

Pcr Techniques

Objectives To investigate the expression of β-catenin in chorionic villi, and to explore its roles in placenta accreta and placenta previa. Methods We compared β-catenin expression in the control group, placenta accreta group (lesion area and normal zones), and placenta previa group (placental central and placental edge zones) by immunohistochemistry, Western blotting, and RT-PCR techniques. Results Compared with the normal group, the placenta accreta group had a longer length of stay, greater bleeding volume, and lower newborn birth weight. Further, the expression of β-catenin was lower in both placenta previa and placenta accreta groups than in the control group, as measured by immunohistochemistry. Compared with the control group, expression of β-catenin was significantly lower in the placenta previa and placenta accreta groups by Western blotting and RT-PCR. Importantly, the level of placental β-catenin was significantly different when compared between the lesion and normal zones of placenta. Conclusion The expression of β-catenin in placenta accreta might play an important role in the regulation of placental cell invasion; low expression of β-catenin in placenta accreta might be responsible for excessive trophoblastic invasion.

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Use of immunodot blot and multiplex reverse transcriptase-polymerase chain reaction in dengue virus detection in macerates of Aedes aegypti larvae

Microbiology Research ◽

10.4081/mr.2012.e13 ◽

2012 ◽

Vol 3 (1) ◽

pp. 13

Author(s):

Aline T.A. Chagas ◽

Michelle D. Oliveira ◽

Jose M.S. Mezencio ◽

Eduardo A.M. Silva ◽

Leandro L. Oliveira ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Aedes Aegypti ◽

Dengue Virus ◽

Reverse Transcriptase ◽

Epidemiological Surveillance ◽

Rt Pcr ◽

Chain Reaction ◽

Denv Serotypes ◽

Polymerase Chain ◽

Pcr Techniques

The <em>Dengue virus</em> is the main arbovirus that affects man in terms of morbidity and mortality. The detection of the virus is very important for epidemiological surveillance, so here we propose to standardize and compare the immunodot blot (IDB) and multiplex reverse transcriptase-polymerase chain reaction (M-RT-PCR) techniques to detect and characterize the dengue virus (DENV) serotypes in samples of <em>Aedes aegypti</em> larvae. Thus, the IDB and M-RT-PCR techniques were standardized using macerated samples of larvae collected in nature. The use of monoclonal antibodies in IDB has not shown great results, but DENV detection through this method was possible using polyclonal antibodies. The distinction of serotypes 1, 2 and 3 was carried out by M-RT-PCR.

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A new dual-targeting real-time RT-PCR assay for hepatitis D virus RNA detection

Diagnostic Microbiology and Infectious Disease ◽

10.1016/j.diagmicrobio.2018.05.016 ◽

2018 ◽

Vol 92 (2) ◽

pp. 112-117 ◽

Cited By ~ 1

Author(s):

Yan Wang ◽

Jeffrey S. Glenn ◽

Mark A. Winters ◽

Li-ping Shen ◽

Ingrid Choong ◽

...

Keyword(s):

Real Time ◽

Rt Pcr ◽

Pcr Assay ◽

Rna Detection ◽

Hepatitis D ◽

Hepatitis D Virus ◽

Dual Targeting ◽

Virus Rna

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Digital Droplet PCR (ddPCR) for Detection and Quantitation of Hepatitis Delt Virus

10.21203/rs.3.rs-791508/v1 ◽

2021 ◽

Author(s):

Ling Xu ◽

Xiangying Zhang ◽

Yaling Cao ◽

Zihao Fan ◽

Yuan Tian ◽

...

Keyword(s):

Liver Failure ◽

Lower Limit ◽

Limit Of Detection ◽

Quantitative Detection ◽

Rt Pcr ◽

Rna Detection ◽

Detection Rates ◽

Digital Droplet Pcr ◽

Droplet Pcr ◽

Hdv Infection

Abstract Background & Aims The prevalence of hepatitis delt virus (HDV) far exceeds our expected level, there remains a lack of reliable quantitative assays for HDV RNA detection. We sought to develop a new method based on digital droplet PCR (ddPCR) for HDV RNA quantitative detection. Methods With plasmid (pMD19T) containing HDV full-genome, we determined the method for ddPCR-based HDV RNA quantification. To compare various assays for HDV detection, 30 cases diagnosed hepatitis D and 14 controls were examined by ELISA, RT-PCR and ddPCR. 728 HBV-related patients including 182 chronic hepatitis B (CHB), 182 liver cirrhosis (LC), 182 hepatocellular carcinoma (HCC) and 182 liver failure (LF) were screened for HDV infection. Results The limit of detection of ddPCR for HDV is significantly low, which lower limit of detection (LLoD) and lower limit of quantitation (LLoQ) to be 5.51 copies/reaction (95% CI: 1.15–6.4*105) and 0.18 copies/reaction (95% CI: 0.0012151- 0.76436), respectively. Among the 44 samples, ELISA detected 30 cases positive for anti-HDV, ddPCR reported 24 samples and RT-PCR reported 10 samples positive for HDV RNA. Moreover, the positive rates of anti-HDV IgG were 1.1%, 3.3%, 2.7% and 7.1% in patients with CHB, LC, HCC, and LF; the detection rates of RT-PCR in HDV RNA were 0%, 16.67%, 15.4% and 20%, however, the detection rates of ddPCR were 0%, 33.33%, 30.77% and 60%. Conclusion We establish a high sensitivity and high specificity quantitative HDV RNA detection method based on ddPCR compared to RT-PCR. HBV-related end-stage liver disease, especially liver failure, are associated with a remarkably high rate of HDV infection.

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Confirmed or unconfirmed cases of 2019 novel coronavirus pneumonia in Italian patients: a retrospective analysis of clinical features

BMC Infectious Diseases ◽

10.1186/s12879-020-05504-7 ◽

2020 ◽

Vol 20 (1) ◽

Author(s):

Giulia De Angelis ◽

Brunella Posteraro ◽

Federico Biscetti ◽

Gianluca Ianiro ◽

Lorenzo Zileri Dal Verme ◽

...

Keyword(s):

Clinical Features ◽

Multivariable Analysis ◽

Etiologic Agent ◽

Nasopharyngeal Swab ◽

Imaging Features ◽

Rt Pcr ◽

Rna Detection ◽

Incubation Periods ◽

Novel Coronavirus ◽

To Receive

Abstract Background Since December 2019, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged as a novel etiologic agent of viral pneumonia. We aimed to compare clinical features of 165 Italian patients with laboratory confirmed or unconfirmed 2019-nCoV pneumonia. Methods On March 31, 2020, hospitalized patients who presented with fever and/or respiratory symptoms, exposures, and presence of lung imaging features consistent with 2019-nCoV pneumonia were included. Before admission to a hospital ward, patients underwent RT-PCR based SARS-CoV-2 RNA detection in their nasopharyngeal swab samples. Results Of 165 patients studied, 119 had positive RT-PCR results and 46 were RT-PCR negative for 2 days or longer (i.e., when the last swab sample was obtained). The median age was 70 years (IQR, 58–78), and 123 (74.6%) of 165 patients had at least one comorbidity. The majority of patients (101/165, 61.2%) had a mild pneumonia, and the remaining patients (64/165, 38.8%) a severe/critical pneumonia. We did not find any substantial difference in symptoms, incubation periods, and radiographic/CT abnormalities as well as in many of the biological abnormalities recorded. However, at multivariable analysis, higher concentrations of hemoglobin (OR, 1.34; 95% CI, 1.11–1.65; P = 0.003) and lower counts of leukocytes (OR, 0.81; 95% CI, 0.72–0.90; P < 0.001) were statistically associated with confirmed COVID-19 diagnosis. While mortality rates were similar, patients with confirmed diagnosis were more likely to receive antivirals (95% vs 19.6%, P < 0.001) and to develop ARDS (63% vs 37%, P = 0.003) than those with unconfirmed COVID-19 diagnosis. Conclusions Our findings suggest that unconfirmed 2019-nCoV pneumonia cases may be actually COVID-19 cases and that clinicians should be cautious when managing patients with presentations compatible with COVID-19.

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