Real-time Reverse Transcription-polymerase Chain Reaction Test Research Articles

Recognition of COVID-19 disease from X-ray images by hybrid model consisting of 2D curvelet transform, chaotic salp swarm algorithm and deep learning technique.

The novel coronavirus disease 2019 (COVID-19), detected in Wuhan City, Hubei Province, China in late December 2019, is rapidly spreading and affecting all countries in the world. Real-time reverse transcription-polymerase chain reaction (RT-PCR) test has been described by the World Health Organization (WHO) as the standard test method for the diagnosis of the disease. However, considering that the results of this test are obtained between a few hours and two days, it is very important to apply another diagnostic method as an alternative to this test. The fact that RT-PCR test kits are limited in number, the test results are obtained in a long time, and the high probability of healthcare personnel becoming infected with the disease during the test, necessitates the use of other diagnostic methods as an alternative to these test kits. In this study, a hybrid model consisting of two-dimensional (2D) curvelet transformation, chaotic salp swarm algorithm (CSSA) and deep learning technique is developed in order to determine the patient infected with coronavirus pneumonia from X-ray images. In the proposed model, 2D Curvelet transformation is applied to the images obtained from the patient's chest X-ray radiographs and a feature matrix is formed using the obtained coefficients. The coefficients in the feature matrix are optimized with the help of the CSSA and COVID-19 disease is diagnosed by the EfficientNet-B0 model, which is one of the deep learning methods. Experimental results show that the proposed hybrid model can diagnose COVID-19 disease with high accuracy from chest X-ray images.

Current laboratory diagnosis of coronavirus disease 2019.

Coronavirus disease 2019 (COVID-19) emerged in December 2019 in Wuhan, China; it has since caused a pandemic, with more than 10,000 confirmed cases (> 800,000 tests) in Korea as of May 2020. Real-time reverse transcription polymerase chain reaction (RT-PCR) is currently the most commonly used method for the diagnosis of COVID-19 worldwide. The Korean Society for Laboratory Medicine and Korea Centers for Disease Prevention and Control regularly update the guidelines for COVID-19 diagnosis. Emergency use authorization for some laboratory diagnostic kits has been granted, enabling the timely diagnosis and treatment of COVID-19, and the isolation of infected patients. Due to the collective efforts of the government, medical professionals, local authorities, and the public, Korea’s response to the COVID-19 outbreak has been accepted widely as a model. Here, we summarize the currently available laboratory tests for COVID-19 diagnosis. Although RT-PCR tests are used widely to confirm COVID-19, antibody tests could provide information about immune responses to the virus.

Chest CT features of coronavirus disease 2019 (COVID-19) pneumonia: key points for radiologists

COVID-19 is an emerging infection caused by a novel coronavirus that is moving so rapidly that on 30 January 2020 the World Health Organization declared the outbreak a Public Health Emergency of International Concern and on 11 March 2020 as a pandemic. An early diagnosis of COVID-19 is crucial for disease treatment and control of the disease spread. Real-time reverse-transcription polymerase chain reaction (RT-PCR) demonstrated a low sensibility; therefore chest computed tomography (CT) plays a pivotal role not only in the early detection and diagnosis, especially for false negative RT-PCR tests, but also in monitoring the clinical course and in evaluating the disease severity. This paper reports the CT findings with some hints on the temporal changes over the course of the disease: the CT hallmarks of COVID-19 are bilateral distribution of ground glass opacities with or without consolidation in the posterior and peripheral lung, but the predominant findings in later phases include consolidations, linear opacities, “crazy-paving” pattern, “reversed halo” sign and vascular enlargement. The CT findings of COVID-19 overlap with the CT findings of other diseases, in particular the viral pneumonia including influenza viruses, parainfluenza virus, adenovirus, respiratory syncytial virus, rhinovirus, human metapneumovirus, etc. There are differences as well as similarities in the CT features of COVID-19 compared with those of the severe acute respiratory syndrome. The aim of this article is to review the typical and atypical CT findings in COVID-19 patients in order to help radiologists and clinicians to become more familiar with the disease.

Household transmission of SARS-CoV-2

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused an epidemic in China and many other countries. Many infected clusters have been found within familial households, but the data about secondary transmission among household contacts is limited.MethodsIn this retrospective case series, we enrolled 85 patients infected with SARS-CoV-2 and their household members in Wuhan. Patients were confirmed infected with SARS-CoV-2 by real-time reverse transcription polymerase chain reaction (RT-PCR) assays on throat swabs. Epidemiological, clinical and laboratory data of the household members were collected.ResultsThere were 155 close contacts in total. 104 contacts received RT-PCR assays, with 47 (30%) positive cases and 57 (37%) negative cases. 51 (33%) cases did not received RT-PCR tests for they showed no symptoms of pneumonia during the 2 weeks of quarantine. The infection rate of close contacts was 38% for households with 1 contact, 50% for households with 2 contacts, and 31% for households with 3 contacts.ConclusionsThe rate of secondary transmission among household contacts of patients with SARS-CoV-2 infection was 30%. Our data provide insight into the rate of secondary transmission of SARS-CoV-2 in home.

CT on the Diamond Princess: What Might This Tell Us About Sensitivity for COVID-19?

HomeRadiology: Cardiothoracic ImagingVol. 2, No. 2 PreviousNext Letters to the EditorFree AccessCT on the Diamond Princess: What Might This Tell Us About Sensitivity for COVID-19?Masis Isikbay* , Michael D. Hope*,†, Constantine A. Raptis‡, Amar Shah§, Andrew J. Bierhals‡, Sanjeev Bhalla‡, Mark M. Hammerǁ, Seth J. Kligerman#, Jean Jeudy**, Peter D. Filev††, Travis S. Henry*Masis Isikbay* , Michael D. Hope*,†, Constantine A. Raptis‡, Amar Shah§, Andrew J. Bierhals‡, Sanjeev Bhalla‡, Mark M. Hammerǁ, Seth J. Kligerman#, Jean Jeudy**, Peter D. Filev††, Travis S. Henry*Author AffiliationsDepartment of Radiology and Biomedical Imaging, University of California San Francisco, 505 Parnassus Ave, Room M391, Box 0628, San Francisco, CA 94143*Department of Radiology, San Francisco Veterans Affairs Medical Center, San Francisco, Calif†Mallinckrodt Institute of Radiology, Washington University School of Medicine, St Louis, Mo‡Department of Radiology, Zucker School of Medicine at Hofstra/Northwell, Manhasset, NY§Department of Radiology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MassǁDepartment of Radiology, University of California San Diego, San Diego, Calif#Department of Diagnostic Radiology and Nuclear Medicine, University of Maryland School of Medicine, Baltimore, Md**Department of Radiology and Imaging Sciences, Emory University School of Medicine, Atlanta, Ga††e-mail: [email protected]Masis Isikbay* Michael D. Hope*,†Constantine A. Raptis‡Amar Shah§Andrew J. Bierhals‡Sanjeev Bhalla‡Mark M. HammerǁSeth J. Kligerman#Jean Jeudy**Peter D. Filev††Travis S. Henry*Published Online:Apr 20 2020https://doi.org/10.1148/ryct.2020200155MoreSectionsPDF ToolsImage ViewerAdd to favoritesCiteTrack Citations ShareShare onFacebookTwitterLinked In Editor:We read with interest the recent article by Dr Inui and colleagues in the April 2020 issue of Radiology: Cardiothoracic Imaging describing chest CT findings in patients with coronavirus disease 2019 (COVID-19) infection aboard the Diamond Princess cruise ship (1). The study consisted of 104 patients with polymerase chain reaction (PCR)–confirmed COVID-19, who also underwent chest CT. The authors focused on characterizing findings in symptomatic versus asymptomatic patients, concluding that there was a high incidence of subclinical changes on CT scans performed in patients with confirmed COVID-19.However, we believe the authors overlooked an important conclusion that has implications regarding the sensitivity of CT for the diagnosis of COVID-19. In this cohort of real-time reverse-transcription polymerase chain reaction (RT-PCR)–confirmed patients, 39% had no lung opacities, including 21% of symptomatic patients. This means chest CT had only a 61% sensitivity for detecting any lung abnormalities in COVID-19, and a 20% false-negative rate in symptomatic patients.These data starkly contrast with widely cited literature reporting CT sensitivity for COVID-19 as high as 97%–98% (2,3). The differences in sensitivities between studies likely reflects the unique cohorts studied; we believe that the earlier literature from China was likely biased toward symptomatic patients imaged in later stages of disease. The current findings are more in line with Bernheim et al, who found that 56% of CT examinations were normal in the first 2 days after symptom onset (4).The article by Dr Inui and colleagues reinforces a limited diagnostic role for CT. Even when RT-PCR may be in limited supply, we endorse the consensus statements provided by the American College of Radiology and other societies (5), especially in populations where the prevalence of disease is low. This determination is based on a significant percentage of normal CT scans in RT-PCR-confirmed patients with COVID-19 (asymptomatic and symptomatic), and the lack of specific imaging findings that are definitive for COVID-19 pneumonia. The RT-PCR test remains the reference standard for diagnosis; it is a molecular test that identifies a finding not present in healthy patients or those without the disease. This is of particular importance given the logistical and safety issues of imaging patients suspected of having or known to have COVID-19 with CT, and the persistent need to isolate patients with clinical suspicion, even if imaging findings are negative or inconclusive.Disclosures of Conflicts of Interest: M.I. disclosed no relevant relationships. M.D.H. disclosed no relevant relationships. C.A.R. disclosed no relevant relationships. A.S. disclosed no relevant relationships. A.J.B. Activities related to the present article: disclosed no relevant relationships. Activities not related to the present article: author provides expert testimony for Secrecy Denney Clifford Law; author received grant from CORVEYS. Other relationships: disclosed no relevant relationships. S.B. Activities related to the present article: disclosed no relevant relationships. Activities not related to the present article: Washington University. Other relationships: disclosed no relevant relationships. M.M.H. disclosed no relevant relationships. S.J.K. disclosed no relevant relationships. J.J. disclosed no relevant relationships. P.D.F. disclosed no relevant relationships. T.S.H. disclosed no relevant relationships.

Frequency and Distribution of Chest Radiographic Findings in Patients Positive for COVID-19.

BackgroundCurrent COVID-19 radiological literature is dominated by CT and a detailed description of chest x-ray (CXR) appearances in relation to the disease time course is lacking.PurposeTo describe the time course and severity of the CXR findings of COVID-19 and correlate these with real time reverse transcription polymerase chain reaction (RT-PCR) testing for SARS-Cov-2 nucleic acid.Materials and MethodsRetrospective study of COVID-19 patients with RT-PCR confirmation and CXRs admitted across 4 hospitals evaluated between January and March 2020. Baseline and serial CXRs (total 255 CXRs) were reviewed along with RT-PCRs. Correlation with concurrent CTs (total 28 CTs) was made when available. Two radiologists scored each CXR in consensus for: consolidation, ground glass opacity (GGO), location and pleural fluid. A severity index was determined for each lung. The lung scores were summed to produce the final severity score.ResultsThere were 64 patients (26 men, mean age 56±19 years). Of these, 58, 44 and 38 patients had positive initial RT-PCR (91%, [CI: 81-96%]), abnormal baseline CXR (69%, [CI: 56-80%]) and positive initial RT-PCR with abnormal baseline CXR (59 [CI:46-71%]) respectively. Six patients (9%) showed CXR abnormalities before eventually testing positive on RT-PCR. Sensitivity of initial RT-PCR (91% [95% CI: 83-97%]) was higher than baseline CXR (69% [95% CI: 56-80%]) (p = 0.009). Radiographic (mean 6 ± 5 days) and virologic recovery (mean 8 ± 6 days) were not significantly different (p= 0.33). Consolidation was the most common finding (30/64, 47%), followed by GGO (21/64, 33%). CXR abnormalities had a peripheral (26/64, 41%) and lower zone distribution (32/64, 50%) with bilateral involvement (32/64, 50%). Pleural effusion was uncommon (2/64, 3%). The severity of CXR findings peaked at 10-12 days from the date of symptom onset.ConclusionChest x-ray findings in COVID-19 patients frequently showed bilateral lower zone consolidation which peaked at 10-12 days from symptom onset.

COVID-19 pneumonia: When negative RT-PCR testing does not rule out the disease

In diagnosing COVID-19, false negative findings from the biological sample taken from a mucosal swab of the upper respiratory tract and tested with the real-time reverse-transcription polymerase chain reaction (RT-PCR) technique have been reported. This patient has had a proven contact with an infected person, clear symptoms of viral respiratory disease, yet negative test results on the fifth day of self-isolation. On repeated test after 48 hours, on the 7th day of isolation, due to persistence of some symptoms, he tested positive for SARS-CoV-2. The existence of symptoms and characteristic sings after laboratory and radiological analysis of the patient prompted the repetition of the tests, which at the end led to the confirmed diagnosis and the possibility for adequate treatment of the patient as well.

Ultra-Rapid Real-Time RT-PCR Method for Detecting Middle East Respiratory Syndrome Coronavirus Using a Mobile PCR Device, PCR1100.

Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV) is usually diagnosed through highly sensitive and specific genetic tests such as real-time reverse transcription polymerase chain reaction (RT-PCR). Currently, two real-time RT-PCR assays targeting the upE and ORF1a regions of the MERS-CoV genome are widely used, and these are the standard assays recommended by the World Health Organization (WHO). The MERS outbreaks to date suggest that rapid diagnosis and subsequent isolation of infected patients, particularly superspreaders, are critical for containment. However, conventional real-time RT-PCR assays require large laboratory instruments, and amplification takes approximately 2 h. These disadvantages limit rapid diagnosis. Here, an ultra-rapid real-time RT-PCR test was established comprising a multiplex assay for upE and ORF1a running on a mobile PCR1100 device. As few as five copies of the MERS-CoV RNA can be detected within 20 min using the standard WHO assays in the mobile PCR device, with the sensitivity and specificity being similar to those of a conventional real-time PCR instrument such as the LightCyler, thereby enabling timely intervention to control MERS-CoV infection.

Evaluating the Effect of the Within-Flock Disease Transmission Rate on Premovement Active Surveillance in Low Pathogenicity Avian Influenza-Infected Flocks.

Premovement active surveillance for low pathogenicity avian influenza (LPAI) may be a useful risk management tool for producers during high-risk periods, such as during an LPAI outbreak, or in areas where there is a recognized high risk for LPAI spread. The effectiveness of three active-surveillance protocols in mitigating LPAI spread risk related to the movement of spent broiler breeders to processing was evaluated in this study. Each protocol differed in the amount of real-time reverse transcription polymerase chain reaction (RRT-PCR) and serology testing conducted. The protocols were evaluated with the use of disease transmission and active surveillance simulation models parametrized specifically for broiler breeders to estimate the probability of detecting a current or past infection and the mean proportion of infectious birds at the time of sampling in houses where the infection remains undetected at the time of movement after exposure to the virus. The two values were estimated considering flock infection for 1-28 days prior to the day of scheduled movement. A distribution for the adequate contact rate, a parameter that controls the rate of within-house spread in the disease transmission model, was estimated for this study by a novel forward simulation approach with the use of serology data from three LPAI-infected broiler breeder flocks in the United States. The estimated distribution suggests that the lower contact-rate estimates from previously published studies were not a good fit for the serology results observed in these U.S. flocks, though considerable uncertainty remains in the parameter estimate. The results for the probability of detection and mean proportion of infectious, undetected birds suggest that RRT-PCR testing is most beneficial during the early stages of infection postexposure, and serology testing is most beneficial during the later stages of infection, results that are expected to hold for flocks outside the United States as well. Thus, protocols that combine RRT-PCR and serology testing can offer a more balanced approach with good performance over the disease course in a flock.

Performance Evaluation of Commercial Dengue Diagnostic Tests for Early Detection of Dengue in Clinical Samples.

The shattering rise in dengue virus infections globally has created a need for an accurate and validated rapid diagnostic test for this virus. Rapid diagnostic test (RDT) and reverse transcription-polymerase chain reaction (RT-PCR) diagnostic detection are useful tools for diagnosis of early dengue infection. We prospectively evaluated the diagnostic performance of nonstructural 1 (NS1) RDT and real-time RT-PCR diagnostic kits in 86 patient serum samples. Thirty-six samples were positive for dengue NS1 antigen while the remaining 50 were negative when tested with enzyme-linked immunosorbent assay (ELISA). Commercially available RDTs for NS1 detection, RTK ProDetect™, and SD Bioline showed high sensitivity of 94% and 89%, respectively, compared with ELISA. GenoAmp® Trioplex Real-Time RT-PCR and RealStar® Dengue RT-PCR tests presented a comparable kappa agreement with 0.722. The result obtained from GenoAmp® Real-Time RT-PCR Dengue test showed that 14 samples harbored dengue virus type 1 (DENV-1), 8 samples harbored DENV-2, 2 samples harbored DENV-3, and 1 sample harbored DENV-4. 1 sample had a double infection with DENV-1 and DENV-2. The NS1 RDTs and real-time RT-PCR tests were found to be a useful diagnostic for early and rapid diagnosis of acute dengue and an excellent surveillance tool in our battle against dengue.

Prolonged Detection of Zika Virus RNA in Pregnant Women.

Zika virus infection during pregnancy is a cause of microcephaly and other fetal brain abnormalities. Reports indicate that the duration of detectable viral RNA in serum after symptom onset is brief. In a recent case report involving a severely affected fetus, Zika virus RNA was detected in maternal serum 10 weeks after symptom onset, longer than the duration of RNA detection in serum previously reported. This report summarizes the clinical and laboratory characteristics of pregnant women with prolonged detection of Zika virus RNA in serum that were reported to the U.S. Zika Pregnancy Registry. Data were obtained from the U.S. Zika Pregnancy Registry, an enhanced surveillance system of pregnant women with laboratory evidence of confirmed or possible Zika virus infection. For this case series, we defined prolonged detection of Zika virus RNA as Zika virus RNA detection in serum by real-time reverse transcription-polymerase chain reaction (RT-PCR) 14 or more days after symptom onset or, for women not reporting signs or symptoms consistent with Zika virus disease (asymptomatic), 21 or more days after last possible exposure to Zika virus. Prolonged Zika virus RNA detection in serum was identified in four symptomatic pregnant women up to 46 days after symptom onset and in one asymptomatic pregnant woman 53 days postexposure. Among the five pregnancies, one pregnancy had evidence of fetal Zika virus infection confirmed by histopathologic examination of fetal tissue, three pregnancies resulted in live births of apparently healthy neonates with no reported abnormalities, and one pregnancy is ongoing. Zika virus RNA was detected in the serum of five pregnant women beyond the previously estimated timeframe. Additional real-time RT-PCR testing of pregnant women might provide more data about prolonged detection of Zika virus RNA and the possible diagnostic, epidemiologic, and clinical implications for pregnant women.

Development and Validation of RT-PCR Tests for the Detection and S1 Genotyping of Infectious Bronchitis Virus and Other Closely Related Gammacoronaviruses Within Clinical Samples

SummaryTwo tests were developed that allow the detection and genotyping of infectious bronchitis virus (IBV) and other closely related gammacoronaviruses. The first test employs a one‐step, reverse transcription‐polymerase chain reaction (RT‐PCR) assay in which the amplification is monitored in real time using a TaqMan® probe. This real‐time RT‐PCR test was used to examine a panel of field samples and its performance compared to virus isolation in embryonated fowls’ eggs. A total of 323 field samples were tested; 176 samples were positive using the real‐time RT‐PCR method, but only three were positive by virus isolation. Sequencing was used to confirm the positive real‐time RT‐PCR results for a subset of samples. The test is suitable for swabs and post‐mortem samples and has been shown to be highly sensitive and specific. The second test, a genotyping method, was developed for identification of the strain of IBV present in field samples based on nucleotide variations within the gene encoding the S1 subunit of the surface spike (S) glycoprotein. This method was developed to provide a tool to inform vaccination decisions and for ongoing surveillance to detect new and emerging strains of IBV within the UK. The performance of the test was evaluated using laboratory isolates of IBV and field samples. Both tests are suitable for use in a high‐throughput diagnostic laboratory.

A Multiplex Real-Time Reverse Transcription Polymerase Chain Reaction Assay for Detection and Differentiation ofBluetongue VirusandEpizootic Hemorrhagic Disease VirusSerogroups

Bluetongue virus (BTV) causes disease in domestic and wild ruminants and results in significant economic loss. The closely related Epizootic hemorrhagic disease virus (EHDV) has been associated with bluetongue-like disease in cattle. Although U.S. EHDV strains have not been experimentally proven to cause disease in cattle, there is serologic evidence of infection in cattle. Therefore, rapid diagnosis and differentiation of BTV and EHDV is required. The genetic sequence information and bioinformatic analysis necessary to design a real-time reverse transcription polymerase chain reaction (RT-PCR) assay for the early detection of indigenous and exotic BTV and EHDV is described. This sequence data foundation focused on 2 conserved target genes: one that is highly expressed in infected mammalian cells, and the other is highly expressed in infected insect cells. The analysis of all BTV and EHDV prototype strains indicated that a complex primer design was necessary for both a virus group-comprehensive and virus group-specific gene amplification diagnostic test. This information has been used as the basis for the development of a rapid multiplex BTV-EHDV real-time RT-PCR that detects all known serotypes of both viruses and distinguishes between BTV and EHDV serogroups. The sensitivity of this rapid, single-tube, real-time RT-PCR assay is sufficient for diagnostic application, without the contamination problems associated with standard gel-based RT-PCR, especially nested RT-PCR tests.

Detection of H5N1 High-Pathogenicity Avian Influenza Virus in Meat and Tracheal Samples from Experimentally Infected Chickens

The Asian H5N1 highly pathogenic avian influenza (HPAI) virus causes a systemic disease with high mortality of poultry and is potentially zoonotic. In both chickens and ducks, the virus has been demonstrated to replicate in both cardiac and skeletal muscle cells. Experimentally, H5N1 HPAI virus has been transmitted to chickens through the consumption of raw infected meat. In this study, we investigated virus replication in cardiac and skeletal muscle and in the trachea of chickens after experimental intranasal inoculation with the H5N1 HPAI virus. The virus was detected in tissues by real-time reverse transcription-polymerase chain reaction (RRT-PCR) and virus isolation, and in the trachea by RRT-PCR and a commercial avian influenza (AI) viral antigen detection test. A modified RNA extraction protocol was developed for rapid detection of the virus in tissues by RRT-PCR. The H5N1 HPAI virus was sporadically detected in meat and the tracheas of infected birds without any clinical sign of disease as early as 6 hr postinfection (PI), and was detected in all samples tested at 24 hr PI and later. No differences in sensitivity were seen between virus isolation and RRT-PCR in meat samples. The AI viral antigen detection test on tracheal swabs was a useful method for identifying infected chickens when they were sick or dead, but was less sensitive in detecting infected birds when they were preclinical. This study provides data indicating that preslaughter tracheal swab testing can identify birds infected with HPAI among the daily mortality and prevent infected flocks from being sent to processing plants. In addition, the modified RNA extraction and RRT-PCR test on meat samples provide a rapid and sensitive method of identifying HPAI virus in illegal contraband or domestic meat samples.

Development and Bench Validation of Real-Time Reverse Transcription Polymerase Chain Reaction Protocols for Rapid Detection of the Subtypes H6, H9, and H11 of Avian Influenza Viruses in Experimental Samples

Real-time reverse transcription polymerase chain reaction (RRT-PCR) is commonly used for the rapid detection, as well as to determine the subtype, of avian influenza viruses (AIVs). There are 16 known serologically distinct hemagglutinin (HA) subtypes of AIV described. Currently, determination of the subtypes of AIVs by RRT-PCR tests has been limited to the H5 and H7 subtypes. In this study, RRT-PCR assays were developed in simplex formats for rapid detection of AIV subtypes H6, H9, and H11. The primers and probes for RRT-PCR were designed from nucleotide sequences of the HA genes, which were either downloaded from GenBank (for H6 and H9) or sequenced for this study. The specificity and sensitivity of the RRT-PCR assays were determined based on the detection of the virus from a proficiency panel consisting of 15 different HA subtypes of AIVs and from serial dilutions of target viral RNA. The subtype-specific RRT-PCR assays were used to detect the virus in cloacal and oropharyngeal swabs of experimental chickens inoculated with H6, H9, and H11 AIVs, and the test results were compared with validated RRT-PCR assays based on the amplification of AI matrix (MA) gene. A high correlation of the matrix test and the specific H6, H9, and H11 by the RRT-PCR assays was observed; kappa coefficients for the agreement of test results in cloacal and oropharyngeal swabs combined were 0.927, 0.962, and 0.981, respectively.