Real-time RT-PCR Test Research Articles

Relationship of Hypocalciamia and Severity of Disease in COVID-19 Patients in Lahore Pakistan

Introduction: In Pakistan, a wave of coronavirus has started in February which was first reported in Wahun City China. Abnormal calcium blood level is considered one of the important and common electrolyte disturbances in patients with severe infection. The effect of blood calcium level on the severity of COVID-19 and the possible immune regulation mechanism is considered to be an important factor related to severity of disease. Aim of the Study: To find out the correlation of blood calcium levels and related risk factors of patients suffering from COVID-19 and provide a basis for early intervention in patients with relevant clinical characteristics and further reduce the incidence of critical illness and mortality in COVID-19 patients. Method: 180 COVID-19 patients were admitted to Gulab Devi Teaching Hospital, Lahore between February to July 2020. Subject index were tested on real-time fluorescent RT-PCR test. On the basis of the result of RT-PCR, they were divided into two groups according to clinical symptoms as mild (control/placebo group) and critical (experimental group). The patient with the mild symptom were included in the placebo group, whereas patient with the severe symptoms were included in the experimental group. Exclusion criteria: The patients who do not show the detection level of viral infection with the RT-PCR are excluded from the study. Results: The comparison has been carried out between the placebo group and the experimental group on the basis of various parameters. The patients serum calcium level was tested and the value for hypocalcemia was declared as per universal recommendation which is < 2.13mmol/L. According to the corrected serum total calcium level, COVID-19 patients can be divided into hypocalcemia group and normal blood calcium group. The result showed that hypocalcemia and immune dysfunction was found high in experimental group as compare to control group which shows a strong correlation between hypocalcemia and COVID-19 disease. Conclusion: It has been concluded from the study that patients with COVID-19 have obvious hypocalcemia and immune dysfunction, especially in experimental group patients. In this study, it is easy to be combined with multiple infections caused by pathogenic bacteria under certain conditions. Close monitoring of blood calcium levels can predict the severity of the disease more effectively.

Clinical significance of smell and taste dysfunction and other related factors in COVID-19.

PurposeThe objective of this study is to evaluate smell and taste dysfunction (STD) in coronavirus disease 2019 (COVID-19) positive and negative patients, and to assess the factors associated with STD in COVID-19 positive patients.MethodsPatients who had been tested with the real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for COVID-19 were identified, and according to the RT-PCR test results, patients were separated into Positive and Negative Groups. A telephone-based assessment was applied to both groups using the American Academy of Otolaryngology–Head and Neck Surgery Anosmia Reporting Tool. Patients in Positive Group were also asked to rate STD, nasal breathing, and anxiety in three different time periods (pre-/during-/post-COVID) using the visual analog scale (VAS).ResultsA total of 53 COVID-19 positive and 51 negative patients completed the surveys. STD was eightfold more frequent (OR 8.19; CI 95% 3.22–20.84) in the Positive Group. Of the 53 COVID-19 positive patients, 32 reported STD and 21 did not. ‘Ground-glass appearance’ on chest-computed tomography was more frequent and median lymphocyte count was significantly lower in COVID-19 positive patients with STD. During-COVID STD and nasal breathing VAS scores were significantly lower than the pre- and post-COVID scores. During-COVID STD scores were significantly correlated with anxiety scores (Spearman’s rho-0.404, p = 0.022) but not correlated with nasal breathing scores.ConclusionSTD may be related to increased inflammatory response as well as damage of olfactory neuronal pathway or non-neuronal olfactory mucosa. Understanding the exact cause of chemosensory impairment in COVID-19 can be helpful in explaining the pathophysiology of the disease.Electronic supplementary materialThe online version of this article (10.1007/s00405-020-06503-9) contains supplementary material, which is available to authorized users.

Severe acute respiratory syndrome Corona virus and intraocular fluid sampling

Dear Editor, We congratulate the authors for the excellent documentation of their cases.[1] We would like to share our experiences dealing with similar cases and would also like to seek clarifications from the authors. We have also been routinely doing reverse transcriptase polymerase chain reaction (RT-PCR) for severe acute respiratory syndrome Corona virus (SARS-CoV-2) in our patients with retinal vascular occlusion, endogenous endophthalmitis, panophthalmitis (unpublished data), and with posterior uveitis.[23] The authors statement “To the best of our knowledge, no study to date evaluated the intraocular fluids for the presence of SARS-CoV-2 in live humans is not correct.” Bilgic et al.[4] have isolated the SARS-CoV-2 in patients with endophthalmitis from the vitreous sample. In an another study by Koo et al.[5], six (19.4%) patients demonstrated detectable SARS-CoV-2 RNA in the aqueous sample, and fortunately, all these individuals did not have any systemic symptoms. We were not able to isolate the SARS-CoV-2 virus in either aqueous or vitreous sample. We have been adopting the following procedure for processing the samples. Our samples are collected in a 1 mL tubeculin syringe and capped, transported in an viral transport medium available from Bhat Bio-Scan™ to the laboratory maintaining a temperature between 2 and 8°C. TaqPath™ Corona Virus Disease-19 (COVID-19) Multiplex Diagnostic Solution from Thermo Fisher Scientific™ is used for multiplex real-time RT-PCR test. This helps in qualitative detection of nucleic acid from SARS-CoV-2. Three regions of the SARS-CoV-2 single-stranded RNA genome – The Orf1ab, N gene, and S gene – are amplified and detected with the help of primer and the probe. It is of importance as SARS-CoV-2 is either not detected or detected in a very low percentage, of ocular samples in patients who have COVID-19.[6] We would also like to suggest that a positive control may be useful by adding a known quantity of viral RNA to ocular fluid to rule out the presence of polymerase chain reaction inhibitors. It would also be useful if the authors could share if SARS-CoV-2 antibodies and other inflammatory markers were raised in their seven patients. Our patients who were not vaccinated had raised levels of antibodies in addition to raised levels of D-dimer, serum ferritin, and lactate dehydrogenase. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest.

Evaluation of a SARS-CoV-2 rapid antigen test: Potential to help reduce community spread?

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can spread from symptomatic patients with COVID-19, but also from asymptomatic individuals. Therefore, robust surveillance and timely interventions are essential for the control of virus spread within the community. In this regard the frequency of testing and speed of reporting, but not the test sensitivity alone, play a crucial role. In order to reduce the costs and meet the expanding demands in real-time RT-PCR (rRT-PCR) testing for SARS-CoV-2, complementary assays, such as rapid antigen tests, have been developed. Rigorous analysis under varying conditions is required to assess the clinical performance of these tests and to ensure reproducible results. We evaluated the sensitivity and specificity of a recently licensed rapid antigen test using 137 clinical samples in two institutions. Test sensitivity was between 88.2-89.6 % when applied to samples with viral loads typically seen in infectious patients. Of 32 rRT-PCR positive samples, 19 demonstrated infectivity in cell culture, and 84 % of these samples were reactive with the antigen test. Seven full-genome sequenced SARS-CoV-2 isolates and SARS-CoV-1 were detected with this antigen test, with no cross-reactivity against other common respiratory viruses. Numerous antigen tests are available for SARS-CoV-2 testing and their performance to detect infectious individuals may vary. Head-to-head comparison along with cell culture testing for infectivity may prove useful to identify better performing antigen tests. The antigen test analyzed in this study is easy-to-use, inexpensive, and scalable. It can be helpful in monitoring infection trends and thus has potential to reduce transmission.

Rapid Implementation of Severe Acute Respiratory Syndrome Coronavirus 2 Emergency Use Authorization RT-PCR Testing and Experience at an Academic Medical Institution

An epidemic caused by an outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in China in December 2019 has since rapidly spread internationally, requiring urgent response from the clinical diagnostics community. We present a detailed overview of the clinical validation and implementation of the first laboratory-developed real-time RT-PCR test offered in the NewYork-Presbyterian Hospital system following the Emergency Use Authorization issued by the US Food and Drug Administration. Nasopharyngeal and sputum specimens (n = 174) were validated using newly designed dual-target real-time RT-PCR (altona RealStar SARS-CoV-2 Reagent) for detecting SARS-CoV-2 in upper respiratory tract and lower respiratory tract specimens. Accuracy testing demonstrated excellent assay agreement between expected and observed values and comparable diagnostic performance to reference tests. The limit of detection was 2.7 and 23.0 gene copies per reaction for nasopharyngeal and sputum specimens, respectively. Retrospective analysis of 1694 upper respiratory tract specimens from 1571 patients revealed increased positivity in older patients and males compared with females, and an increasing positivity rate from approximately 20% at the start of testing to 50% at the end of testing 3 weeks later. Herein, we demonstrate that the assay accurately and sensitively identifies SARS-CoV-2 in multiple specimen types in the clinical setting and summarize clinical data from early in the epidemic in New York City.

CT-scan findings of COVID-19 pneumonia based on the time elapsed from the beginning of symptoms to the CT imaging evaluation: a descriptive study in Iran.

Background. Coronavirus disease 2019 (COVID-19) was initially detected in Wuhan city, China. Chest CT features of COVID-19 pneumonia have been investigated mostly in China, and there is very little information available on the radiological findings occurring in other populations. In this study, we aimed to describe the characteristics of chest CT findings in confirmed cases of COVID-19 pneumonia in an Iranian population, based on a time classification.Methods. Eighty-nine patients with COVID-19 pneumonia, confirmed by a real-time RT-PCR test, who were admitted to non-ICU wards and underwent a chest CT scan were retrospectively enrolled. Descriptive evaluation of radiologic findings was performed using a classification based on the time interval between the initiation of the symptoms and chest CT-scan.Results. The median age of patients was 58.0 years, and the median time interval from the onset of symptoms to CT scan evaluation was 7 days. Most patients had bilateral (94.4%) and multifocal (91.0%) lung involvement with peripheral distribution (60.7%). Also, most patients showed involvement of all five lobes (77.5%). Ground-glass opacities (GGO) (84.3%) and mixed GGO with consolidation (80.9%) were the most common identified patterns. We also found that as the time interval between symptoms and CT scan evaluation increased, the predominant pattern changed from GGO to mixed pattern and then to elongated-containing and band-like-opacities-containing pattern; on the other hand, the percentage of lung involvement increased.Conclusions. Bilateral multifocal GGO, and mixed GGO with consolidation were the most common patterns of COVID-19 pneumonia in our study. However, these patterns might change according to the time interval from symptoms.

SARS-CoV-2 Among Military and Civilian Patients, Metro Manila, Philippines.

ABSTRACTIntroductionSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) belonging to the family Coronaviridae and genus Betacoronavirus is the causative agent of COVID-19 disease and was first identified in Wuhan, China. SARS-CoV-2 spread globally with >28 million cases and 911,000 deaths recorded worldwide as of September 12, 2020. The Philippines reported the first case of community transmission on March 5, 2020, and despite the government imposing one of the longest and strictest lockdowns in Southeast Asia, the number of confirmed COVID-19 cases still surged with >250,000 cases and 4,000 deaths reported as of September 12, 2020. It is important to estimate the burden and impact of SARS-CoV-2 on the military population since this can affect the military readiness.Materials and MethodsNasopharyngeal and oropharyngeal swabs were collected and SARS-CoV-2 real-time RT-PCR testing was performed on the samples. Phylogenetic analysis was performed using sequences from 23 SARS-CoV-2-positive specimens from this study and sequences retrieved from GenBank and GISAID databases.ResultsFrom April 14 to August 15, 2020, a total of 12,432 specimens were tested with 763 (6%) unique individuals testing positive for SARS-CoV-2 by rRT-PCR. In the military population, majority of the patients who were tested (80%) and those who tested positive for SARS-CoV-2 (86%) were male. Military and civilian status was available for 7,672 patients with 515/5,042 (10%) positive among military patients and 248/2,630 (9%) positive among civilian patients. Both military and civilian populations had the highest case counts of SARS-CoV-2-positive cases in the 21- to 30- and 31- to 40-year-old age groups, while the 71- to 80-year-old age group had the highest proportion (18%) of SARS-CoV-2-positive cases. Sequencing analysis showed 19 different variants in the 23 genomes. Twenty of the 23 genomes were classified under clade GR/B1.1, 2 genomes were classified under clade GR/B1.1.28, and 1 genome was classified under Clade O/B.6. Twenty-two of the 23 sequences collected after June 25, 2020, contained the D614G mutation.ConclusionWe describe here the results of SARS-CoV-2 testing for military and civilian patients and personnel. The 21- to 30- and 31- to 40-year-old age groups had the highest case counts of SARS-CoV-2-positive cases. Sequencing results showed the presence of the D614G mutation in the spike protein in a majority of specimens collected from the end of June to July 2020.

Middle-aged and elderly patients with COVID-19 pneumonia arising from asymptomatic carriers: A report of six cases.

Middle-aged and elderly patients with COVID-19 pneumonia arising from asymptomatic carriers: A report of six cases.

Impact of Pool Testing in Detection of Asymptomatic Patients with COVID-19.

ObjectiveDuring the current pandemic, COVID-19 has been detected in patients using real-time reverse transcriptase-polymerase chain reaction (RT-PCR) that confirms the presence of SARS-CoV-2 RNA. The demand for increased testing, particularly for asymptomatic individuals required alternative approaches to single-patient RT-PCR testing, such as pooling.MethodsThis study explored the impact of dilution on the detectability of SARS-CoV-2 in asymptomatic patients using RT-PCR and demonstrated that pooling can be effective in low prevalence populations.ResultsThe RT-PCR results for the 3:1, 5:1, and 7:1 aliquot samples showed little differences in CT values, confirming detection capability at these dilutions.ConclusionBased on the results of the present study, a pooled approach with up to 5:1 sample aliquots and using the current RT-PCR methodology likely will detect SARS CoV2 RNA among asymptomatic patients.

Performance of Severe Acute Respiratory Syndrome Coronavirus 2 Real-Time RT-PCR Tests on Oral Rinses and Saliva Samples

Access to rapid and accurate detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA is essential for controlling the current global pandemic of coronavirus disease 2019. In this study, the use of oral rinses (ORs) and posterior oropharyngeal saliva as an alternative to swab collection methods from symptomatic and asymptomatic health care workers for the detection of SARS-CoV-2 RNA by RT-PCR was evaluated. For saliva samples, the overall agreement with oropharyngeal swabs was 93% (Ƙ = 0.84), with a sensitivity of 96.7% (95% CI, 83.3%–99.8%). The agreement between saliva and nasopharyngeal swabs was 97.7% (Ƙ = 0.93), with a sensitivity of 94.1% (95% CI, 73.0%–99.7%). ORs were compared with nasopharyngeal swabs only, with an overall agreement of 85.7% (Ƙ = 0.65), and a sensitivity of 63% (95% CI, 46.6%–77.8%). The agreement between a laboratory-developed test based on the CDC RT-PCR and two commercial assays, the Xpert Xpress SARS-CoV-2 and the Cobas SARS-CoV-2, was also evaluated. The overall agreement was >90%. Finally, SARS-CoV-2 RNA in saliva samples was shown to be stable, with no changes in viral loads over 24 hours at both room temperature and 4°C. Although the dilution of SARS-CoV-2 in ORs precluded its acceptability as a sample type, posterior oropharyngeal saliva was an acceptable alternative sample type for SARS-CoV-2 RNA detection.

Genetic analysis of the 2019 coronavirus pandemic with from real-time reverse transcriptase polymerase chain reaction.

Corona viruses (CoV) are known to cause extreme pandemics in the globe. The year 2020 will be a pandemic with the spread of the novel coronavirus (SARS-CoV-2) across the globe. Coronavirus 2019 (COVID-19) has been a part of our scary life for more than a quarter of a year in 2020. The Wuhan market and China have been the most commonly used terms in the world for at least a quarter of 2020. A zoonotic coronavirus has entered organisms to affect organisms for the third season in several centuries. CoV is a global pandemic prompted a drastic and rapid reconfiguration of society. CoV have extraordinary broad genomes of about 30 kilobases of RNA. There is no genetic relationship between the SARS-CoV, MERS and SARS-CoV-2. For health care strategies and for anticipating and preventing potential outbreaks, adequate description of the international spread of COVID-19 virus is imperative. The WHO has declared COVID-19 as endemic to pandemic in the first trimester of 2020. The biggest issues for diagnosis COVID-19 is not established apart from Real-time reverse transcriptase polymerase chain reaction (RT-PCR). In order to monitor the COVID-19 pandemic, testing of active SARS-CoV-2 infections is a fundamental public health method. The vast use of SARS-CoV-2 RT-PCR tests around the world has led to increased availability of test kits, which is also a major bottleneck. The technique RT-PCR was generally agreed in the present scenario to detect SARS-CoV-2 in the human body. This review discusses about the importance of molecular technique for diagnosing the pandemic disease of 2019. In conclusion, RT-PCR was found to be an apt technique for identification of SARS-CoV-2.

COVID-19 Molecular Testing in Korea: Practical Essentials and Answers From Experts Based on Experiences of Emergency Use Authorization Assays.

Coronavirus disease 2019 (COVID-19) is a respiratory disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Early detection of COVID-19 and immediate isolation of infected patients from the naive population are important to prevent further pandemic spread of the infection. Real-time reverse transcription (RT)-PCR to detect SARS-CoV-2 RNA is currently the most reliable diagnostic method for confirming COVID-19 worldwide. Guidelines for clinical laboratories on the COVID-19 diagnosis have been recently published by Korean Society for Laboratory Medicine and the Korea Centers for Disease Control and Prevention. However, these formal guidelines do not address common practical laboratory issues related to COVID-19 real-time RT-PCR testing and their solutions. Therefore, this guideline is intended as a practical and technical supplement to the “Guidelines for Laboratory Diagnosis of COVID-19 in Korea”.

Incidence of influenza during pregnancy and association with pregnancy and perinatal outcomes in three middle-income countries: a multisite prospective longitudinal cohort study

Influenza vaccination during pregnancy prevents influenza among women and their infants but remains underused among pregnant women. We aimed to quantify the risk of antenatal influenza and examine its association with perinatal outcomes. We did a prospective cohort study in pregnant women in India, Peru, and Thailand. Before the 2017 and 2018 influenza seasons, we enrolled pregnant women aged 18 years or older with expected delivery dates 8 weeks or more after the season started. We contacted women twice weekly until the end of pregnancy to identify illnesses with symptoms of myalgia, cough, runny nose or nasal congestion, sore throat, or difficulty breathing and collected mid-turbinate nasal swabs from symptomatic women for influenza real-time RT-PCR testing. We assessed the association of antenatal influenza with preterm birth, late pregnancy loss (≥13 weeks gestation), small for gestational age (SGA), and birthweight of term singleton infants using Cox proportional hazards models or generalised linear models to adjust for potential confounders. Between March 13, 2017, and Aug 3, 2018, we enrolled 11 277 women with a median age of 26 years (IQR 23-31) and gestational age of 19 weeks (14-24). 1474 (13%) received influenza vaccines. 310 participants (3%) had influenza (270 [87%] influenza A and 40 [13%] influenza B). Influenza incidences weighted by the population of women of childbearing age in each study country were 88·7 per 10 000 pregnant woman-months (95% CI 68·6 to 114·8) during the 2017 season and 69·6 per 10 000 pregnant woman-months (53·8 to 90·2) during the 2018 season. Antenatal influenza was not associated with preterm birth (adjusted hazard ratio [aHR] 1·4, 95% CI 0·9 to 2·0; p=0·096) or having an SGA infant (adjusted relative risk 1·0, 95% CI 0·8 to 1·3, p=0·97), but was associated with late pregnancy loss (aHR 10·7, 95% CI 4·3 to 27·0; p<0·0001) and reduction in mean birthweight of term, singleton infants (-55·3 g, 95% CI -109·3 to -1·4; p=0·0445). Women had a 0·7-0·9% risk of influenza per month of pregnancy during the influenza season, and antenatal influenza was associated with increased risk for some adverse pregnancy outcomes. These findings support the added value of antenatal influenza vaccination to improve perinatal outcomes. US Centers for Disease Control and Prevention. For the Thai, Hindi, Marathi and Spanish translations of the abstract see Supplementary Materials section.

Risk factors associated with adverse fetal outcomes in pregnancies affected by Coronavirus disease 2019 (COVID-19): a secondary analysis of the WAPM study on COVID-19.

Objectives To evaluate the strength of association between maternal and pregnancy characteristics and the risk of adverse perinatal outcomes in pregnancies with laboratory confirmed COVID-19. Methods Secondary analysis of a multinational, cohort study on all consecutive pregnant women with laboratory-confirmed COVID-19 from February 1, 2020 to April 30, 2020 from 73 centers from 22 different countries. A confirmed case of COVID-19 was defined as a positive resulton real-time reverse-transcriptase-polymerase-chain-reaction(RT-PCR) assay of nasal and pharyngeal swab specimens.The primary outcome was a composite adverse fetal outcome, defined as the presence of either abortion (pregnancy loss before 22weeks of gestations), stillbirth (intrauterine fetal death after 22weeks of gestation), neonatal death (death of a live-born infant within the first 28days of life), and perinatal death (either stillbirth or neonatal death). Logistic regression analysis was performed to evaluate parameters independently associated with the primary outcome. Logistic regression was reported as odds ratio (OR) with 95% confidence interval (CI). Results Mean gestational age at diagnosis was 30.6±9.5weeks, with 8.0% of women being diagnosed in the first, 22.2% in the second and 69.8% in the third trimester of pregnancy. There were six miscarriage (2.3%), six intrauterine device (IUD) (2.3) and 5 (2.0%) neonatal deaths, with an overall rate of perinatal death of 4.2% (11/265), thus resulting into 17 cases experiencing and 226 not experiencing composite adverse fetal outcome. Neither stillbirths nor neonatal deaths had congenital anomalies found at antenatal or postnatal evaluation. Furthermore, none of the cases experiencing IUD had signs of impending demise at arterial or venous Doppler. Neonatal deaths were all considered as prematurity-related adverse events. Of the 250 live-born neonates, one (0.4%) was found positive at RT-PCR pharyngeal swabs performed after delivery. The mother was tested positive during the third trimester of pregnancy. The newborn was asymptomatic and had negative RT-PCR test after 14days of life. At logistic regression analysis, gestational age at diagnosis (OR: 0.85, 95% CI 0.8-0.9 per week increase; p<0.001), birthweight (OR: 1.17, 95% CI 1.09-1.12.7 per 100g decrease; p=0.012) and maternal ventilatory support, including either need for oxygen or CPAP (OR: 4.12, 95% CI 2.3-7.9; p=0.001) were independently associated with composite adverse fetal outcome. Conclusions Early gestational age at infection, maternal ventilatory supports and low birthweight are the main determinants of adverse perinatal outcomes in fetuses with maternal COVID-19 infection. Conversely, the risk of vertical transmission seems negligible.

Development and validation of a real-time RT-PCR test for screening pepper and tomato seed lots for the presence of pospiviroids

Potato spindle tuber viroid and other pospiviroids can cause serious diseases in potato and tomato crops. Consequently, pospiviroids are regulated in several countries. Since seed transmission is considered as a pathway for the introduction and spread of pospiviroids, some countries demand for the testing of seed lots of solanaceous crops for the presence of pospiviroids. A real-time RT-PCR test, named PospiSense, was developed for testing pepper (Capsicum annuum) and tomato (Solanum lycopersicum) seeds for seven pospiviroid species known to occur naturally in these crops. The test consists of two multiplex reactions running in parallel, PospiSense 1 and PospiSense 2, that target Citrus exocortis viroid (CEVd), Columnea latent viroid (CLVd), pepper chat fruit viroid (PCFVd), potato spindle tuber viroid (PSTVd), tomato apical stunt viroid (TASVd), tomato chlorotic dwarf viroid (TCDVd) and tomato planta macho viroid (TPMVd, including the former Mexican papita viroid). Dahlia latent viroid (DLVd) is used as an internal isolation control. Validation of the test showed that for both pepper and tomato seeds the current requirements of a routine screening test are fulfilled, i.e. the ability to detect one infested seed in a sample of c.1000 seeds for each of these seven pospiviroids. Additionally, the PospiSense test performed well in an inter-laboratory comparison, which included two routine seed-testing laboratories, and as such provides a relatively easy alternative to the currently used tests.

Development of a New Multiplex Real-Time RT-PCR Assay for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Detection

This research describes the development of a new multiplex real-time RT-PCR test for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with primers designed to amplify a 108 bp target on the spike surface glycoprotein (S gene) and a hydrolysis TaqMan probe designed to specifically detect SARS-CoV-2. The limit of detection (LOD) and clinical performance of this new assay were evaluated. A LOD study with inactivated virus exhibited performance equal to the modified CDC assay, with a final LOD of 1301 ± 13 genome equivalents/mL for the Northwell Health Laboratories laboratory-developed test (NWHL LDT) versus 1249 ± 14 genome equivalents/mL for the modified CDC assay. In addition, a clinical evaluation with 270 nasopharyngeal swab specimens exhibited 98.5% positive percent agreement and 99.3% negative percent agreement compared with the modified CDC assay. The NWHL LDT multiplex design allows testing of 91 patients per plate, versus a maximum of 29 patients per plate on the modified CDC assay, providing the benefit of testing significantly more patients per run and saving reagents, during a time when both of these parameters are critical. The results show that the NWHL LDT multiplex assay performs as well as the modified CDC assay but is more efficient and cost-effective and can be used as a diagnostic assay and for epidemiologic surveillance and clinical management of SARS-CoV-2.

Evaluation of performance of two SARS-CoV-2 Rapid IgM-IgG combined antibody tests on capillary whole blood samples from the fingertip.

The SARS-CoV-2 (Severe Acute Respiratory Syndrome CoronaVirus 2) is responsible for the infectious respiratory disease called COVID-19 (COronaVIrus Disease 2019). In response to the growing COVID-19 pandemic, point-of-care (POC) tests have been developed to detect specific antibodies, IgG and IgM, to SARS-CoV-2 virus in human whole blood. We conducted a prospective observational study to evaluate the performance of two POC tests, COVID-PRESTO® and COVID-DUO®, compared to the gold standard, RT-PCR (real-time reverse transcriptase polymerase chain reaction). RT-PCR testing of SARS-Cov-2 was performed from nasopharyngeal swab specimens collected in adult patients visiting the infectious disease department at the hospital (Orléans, France). Capillary whole blood (CWB) samples from the fingertip taken at different time points after onset of the disease were tested with POC tests. The specificity and sensitivity of the rapid test kits compared to test of reference (RT-PCR) were calculated. Among 381 patients with symptoms of COVID-19 who went to the hospital for a diagnostic, 143 patients were RT-PCR negative. Results of test with POC tests were all negative for these patients, indicating a specificity of 100% for both POC tests. In the RT-PCR positive subgroup (n = 238), 133 patients were tested with COVID-PRESTO® and 129 patients were tested with COVID-DUO® (24 patients tested with both). The further the onset of symptoms was from the date of collection, the greater the sensitivity. The sensitivity of COVID-PRESTO® test ranged from 10.00% for patients having experienced their 1st symptoms from 0 to 5 days ago to 100% in patients where symptoms had occurred more than 15 days before the date of tests. For COVID-DUO® test, the sensitivity ranged from 35.71% [0-5 days] to 100% (> 15 days). COVID-PRESTO® and DUO® POC tests turned out to be very specific (none false positive) and to be sensitive enough after 15 days from onset of symptom. These easy to use IgG/IgM combined test kits are the first ones allowing a screening with CWB sample, by typing from a finger prick. These rapid tests are particularly interesting for screening in low resource settings.

Prolonged SARS-CoV-2 detection and reversed RT-PCR results in mild or asymptomatic patients

Background The clinical course and viral detection period in mild or asymptomatic coronavirus disease 2019 (COVID-19) patients are not yet known. The presumed low diagnostic sensitivity of upper respiratory specimens for Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) makes it difficult to confirm infection and recommend de-isolation. Methods We retrospectively reviewed real-time reverse transcriptase-polymerase chain reaction (RT-PCR) test results of mild or asymptomatic COVID-19 patients who were admitted at the Daegu-Gyeongbuk 7th community treatment centre in Korea between 9 March 2020 and 10 April 2020. Patients underwent an upper respiratory RT-PCR test every week until discharge. From the RT-PCR results, we evaluated the rate of prolonged (>3 weeks) SARS-CoV-2 RNA positivity. We analysed the proportion of reversed results, defined as a positive or indeterminate result one day after a negative RT-PCR result, according to time (<14, 15–21, 22–28, >28 days) from the initial positive RT-PCR result. Results In 23% (69/300) of patients, SARS-CoV-2 was detected more than 3 weeks after the initial positive RT-PCR. In 14% (42/300) of patients, the RT-PCR results were positive for more than 4 weeks. For 37.5% (152/405) of negative RT-PCR results, the results were reversed in the next day’s test. And 43.5% (123/283) of negative RT-PCR results were reversed within 3 weeks of diagnosis. Conclusions The detection of SARS-CoV-2 lasting more than 3 weeks was common in mild or asymptomatic patients. Upper respiratory RT-PCR results were frequently reversed from negative to positive.

Detection of severe acute respiratory syndrome coronavirus 2 in nasopharynx according to clinical phenotype of affected patients

ObjectivesDuration of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the upper respiratory tract is extremely variable, but its relation to disease severity is unknown. We investigated this relation in the 530 000 inhabitants of the northeastern Italian province of Udine. MethodsWe analysed real-time RT-PCR tests for SARS-CoV-2 in upper respiratory specimens conducted at the Virology Laboratory of the University Hospital of Udine, Italy (which serves the whole province) from 1 March to 30 April 2020 Specimens were from positive individuals in four groups characterized by different disease severity (critically ill patients admitted to intensive care units, patients admitted to infectious disease units, symptomatic patients visiting the emergency department and not hospitalized, and asymptomatic individuals tested during contact tracing or screening activities). Duration of viral positivity was assessed from the first positive test to the day of the first of two consecutive negative tests. Univariate and multivariate analyses were conducted to investigate differences in the four groups. ResultsFrom 1 March to 30 April, 39 483 RT-PCR tests for SARS-CoV-2 were conducted on 23 778 individuals, and 974 individuals had a positive test result. Among those with multiple tests (n = 878), mean time to negativity was 23.7 days (standard error 0.3639; median 23, interquartile range 16–30 days). Mean time to negativity was longer in the group admitted to the intensive care unit than in the others, whereas no difference was observed between asymptomatic patients and those with mild disease. ConclusionsDisease control measures should not be adjusted to account for differences in viral shedding according to symptomatic status.

Comparison of SARS-CoV-2 detection from combined nasopharyngeal/oropharyngeal swab samples by a laboratory-developed real-time RT-PCR test and the Roche SARS-CoV-2 assay on a cobas 8800 instrument

ObjectiveAlthough several assays have been developed to detect SARS-CoV-2 RNA in clinical specimens, their relative performance is unknown.MethodsThe concordance between the cobas 8800 SARS-CoV-2 and a laboratory developed (LD) reverse transcriptase-polymerase chain reaction (RT-PCR) assay was assessed on 377 combined nasopharyngeal/oropharyngeal swabs in Hanks medium.ResultsThe positive and negative agreement between these assays were 99.3 % (95 % CI, 97.3–99.9) and 77.1 % (95 % CI, 67.7–84.4), respectively, for an overall agreement of 93.6 % (95 % CI, 90.7–95.7) beyond random chance (kappa of 0.82, 95 % CI, 0.75−0.85). Of the 22 samples positive by cobas SARS-CoV-2 only, 9 were positive only for ORF-1 gene and had Cycle thresholds (Ct) > 35.1, 8 were positive only for the E gene with Ct > 35.5 and 5 were positive for both targets with Ct > 33.9. Samples positive only with the cobas assay were more often positive with only one gene target (77.3 %) than samples positive in both assays (16.9 %, p < 0.0001). Ct values in the cobas SARS-CoV-2 assay were significantly higher in the 279 samples testing positive in both assays (32.9 %, 95 % CI 32.3–33.6) compared to the 22 samples with discordant results (36.6 %, 95 % CI 36.2–37.1; p = 0.0009). An excellent correlation (r2 = 0.98) was obtained between Ct values of the ORF-1 and E targets in the cobas assays and a good correlation was obtained between LD RT-PCR test and cobas SARS CoV-2 ORF-1 target (r2 = 0.82).ConclusionOur study demonstrated an excellent concordance between a LD RT-PCR and the cobas SARS-CoV-2 tests on the 8800 platform.