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1.
J Endocrinol Invest ; 44(12): 2675-2684, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1504521

ABSTRACT

PURPOSE: Due to relevant repercussions on reproductive medicine, we aimed to evaluate feasibility of RT-PCR as a detection method of SARS-CoV-2 RNA in seminal fluid. METHODS: A qualitative determination of the RT-PCR assays in semen was performed through different approaches: (1) efficiency of RNA extraction from sperm and seminal plasma was determined using PRM1 and PRM2 mRNA and a heterologous system as control; (2) samples obtained by diluting viral preparation from a SARS-CoV-2 panel (virus cultured in Vero E6 cell lines) were tested; (3) viral presence in different fractions of seminal fluid (whole sample, seminal plasma and post-centrifugation pellet) was evaluated. Semen samples from mild and recovered COVID-19 subjects were collected by patients referring to the Infectious Disease Department of the Policlinico Umberto I Hospital - "Sapienza" University of Rome. Control subjects were recruited at the Laboratory of Seminology-Sperm Bank "Loredana Gandini'' of the same hospital. RESULTS: The control panel using viral preparations diluted in saline and seminal fluid showed the capability to detect viral RNA presence with Ct values depending on the initial viral concentration. All tested semen samples were negative for SARS-CoV-2, regardless of the nasopharyngeal swab result or seminal fluid fraction. CONCLUSION: These preliminary data show that RT-PCR for SARS-CoV-2 RNA testing appears to be a feasible method for the molecular diagnosis of SARS-CoV-2 in seminal fluid, supported by results of the control panel. The ability to detect SARS-CoV-2 in semen is extremely important for reproductive medicine, especially in assisted reproductive technology and sperm cryopreservation.


Subject(s)
COVID-19/diagnosis , Pathology, Molecular/methods , Semen/virology , Adult , Animals , Chlorocebus aethiops , Feasibility Studies , Humans , Male , RNA, Messenger/chemistry , RNA, Viral/chemistry , Real-Time Polymerase Chain Reaction , Reproductive Techniques , Vero Cells
2.
J AOAC Int ; 104(4): 872-888, 2021 Aug 20.
Article in English | MEDLINE | ID: covidwho-1387921

ABSTRACT

BACKGROUND: The Eurofins GeneScan Technologies' VIRSeek SARS-CoV-2 Mplex kit is a RT (reverse transcription) real-time polymerase chain reaction (RT-qPCR) assay for the detection of two targets on the N-gene (nucleocapsid) of SARS-CoV-2. An extraction control, that allows monitoring of the extraction procedure and PCR inhibition, is included. OBJECTIVE: In silico analysis and wet testing showed inclusivity and exclusivity of the assay. The complete workflow starting from surface swabbing (VIRSeek PATHOSwab kit), RNA extraction (VIRSeek RNAExtractor), RT-PCR (VIRSeek SARS-CoV-2 Mplex), and evaluation with FastFinder was validated in comparison to the CDC method for detection of SARS-CoV-2 on stainless steel. METHOD: In silico analysis was performed by using the MFOLD online program. The matrix study was performed for stainless steel inoculated with SARS-CoV-2 isolated from the first documented US case of a traveler from Wuhan, China. RESULTS: For inclusivity, 15 764 sequences were analyzed and all mismatches (0.37% of the sequences had single mismatches) were considered non-critical. Cross reactivity for closely related viruses and background organisms was performed, resulting in correct exclusion of all. No significant differences were observed for the probability of detection (POD) study when comparing to the CDC method. CONCLUSIONS: Results of the inclusivity and exclusivity study show that the assay is specific for detection of SARS-CoV-2. The POD study showed no statistically significant difference compared to the CDC reference method, results were identical for the uninoculated and the high level. For the fractional recovery level, the candidate method detected 9/17 samples leading to a POD of 0.47, the reference method detected 11/20 samples leading to a POD of 0.55. HIGHLIGHT: The complete workflow starting from swabbing of the surface (VIRSeek PATHOSwab kit), RNA extraction (VIRSeek RNAExtractor), RT-PCR (VIRSeek SARS CoV-2 Mplex) and evaluation with FastFinder was validated in comparison to the US Centers for Disease Control and Prevention method for detection of SARS-CoV-2 on Stainless Steel.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction , Sensitivity and Specificity , Stainless Steel
3.
J Med Virol ; 93(9): 5538-5543, 2021 Sep.
Article in English | MEDLINE | ID: covidwho-1363694

ABSTRACT

In the current coronavirus disease 2019 (COVID-19) pandemic there is a mass screening of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) happening around the world due to the extensive spread of the infections. There is a high demand for rapid diagnostic tests to expedite the identification of cases and to facilitate early isolation and control spread. Hence this study evaluates six different rapid nucleic acid detection assays that are commercially available for SARS-CoV-2 virus detection. Nasopharyngeal samples were collected from 4981 participants and were tested for the SARS-CoV-2 virus by the gold standard real-time reverse-transcription polymerase chain reaction (RT-PCR) method and with one of these six rapid methods of detection. Evaluation of the rapid nucleic acid detection assays was done by comparing the results of these rapid methods with the gold standard RT-qPCR results for SARS-COV-2 detection. AQ-TOP had the highest sensitivity (98%) and a strong kappa value of 0.943 followed by Genechecker and Abbot ID NOW. The POCKIT (ii RT-PCR) assay had the highest test accuracy of 99.29% followed by Genechecker and Cobas Liat. Atila iAMP showed the highest percentage of invalid reports (35.5%) followed by AQ-TOP with 6% and POCKIT with 3.7% of invalid reports. Genechecker system, Abbott ID NOW, and Cobas Liat were found to have the best performance and agreement when compared with the standard RT-PCR for COVID-19 detection. With further research, these rapid tests have the potential to be employed in large-scale screening of COVID-19.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , SARS-CoV-2/isolation & purification , COVID-19 Nucleic Acid Testing/instrumentation , COVID-19 Nucleic Acid Testing/standards , Humans , Nasopharynx/virology , Real-Time Polymerase Chain Reaction , SARS-CoV-2/genetics , Sensitivity and Specificity , United Arab Emirates
4.
JAMA Pediatr ; 175(10): e212025, 2021 10 01.
Article in English | MEDLINE | ID: covidwho-1265361

ABSTRACT

Importance: The association between COVID-19 symptoms and SARS-CoV-2 viral levels in children living in the community is not well understood. Objective: To characterize symptoms of pediatric COVID-19 in the community and analyze the association between symptoms and SARS-CoV-2 RNA levels, as approximated by cycle threshold (Ct) values, in children and adults. Design, Setting, and Participants: This cross-sectional study used a respiratory virus surveillance platform in persons of all ages to detect community COVID-19 cases from March 23 to November 9, 2020. A population-based convenience sample of children younger than 18 years and adults in King County, Washington, who enrolled online for home self-collection of upper respiratory samples for SARS-CoV-2 testing were included. Exposures: Detection of SARS-CoV-2 RNA by reverse transcription-polymerase chain reaction (RT-PCR) from participant-collected samples. Main Outcomes and Measures: RT-PCR-confirmed SARS-CoV-2 infection, with Ct values stratified by age and symptoms. Results: Among 555 SARS-CoV-2-positive participants (mean [SD] age, 33.7 [20.1] years; 320 were female [57.7%]), 47 of 123 children (38.2%) were asymptomatic compared with 31 of 432 adults (7.2%). When symptomatic, fewer symptoms were reported in children compared with adults (mean [SD], 1.6 [2.0] vs 4.5 [3.1]). Symptomatic individuals had lower Ct values (which corresponded to higher viral RNA levels) than asymptomatic individuals (adjusted estimate for children, -3.0; 95% CI, -5.5 to -0.6; P = .02; adjusted estimate for adults, -2.9; 95% CI, -5.2 to -0.6; P = .01). The difference in mean Ct values was neither statistically significant between symptomatic children and symptomatic adults (adjusted estimate, -0.7; 95% CI, -2.2 to 0.9; P = .41) nor between asymptomatic children and asymptomatic adults (adjusted estimate, -0.6; 95% CI, -4.0 to 2.8; P = .74). Conclusions and Relevance: In this community-based cross-sectional study, SARS-CoV-2 RNA levels, as determined by Ct values, were significantly higher in symptomatic individuals than in asymptomatic individuals and no significant age-related differences were found. Further research is needed to understand the role of SARS-CoV-2 RNA levels and viral transmission.


Subject(s)
COVID-19/complications , COVID-19/diagnosis , RNA, Viral/metabolism , SARS-CoV-2/isolation & purification , Viral Load , Adolescent , Adult , Age Factors , Aged , Aged, 80 and over , COVID-19 Nucleic Acid Testing , Child , Child, Preschool , Cross-Sectional Studies , Female , Humans , Infant , Male , Middle Aged , Reverse Transcriptase Polymerase Chain Reaction , Symptom Assessment , Washington , Young Adult
5.
J Med Virol ; 93(7): 4405-4410, 2021 07.
Article in English | MEDLINE | ID: covidwho-1263105

ABSTRACT

One year into the coronavirus disease 2019 (COVID-19) pandemic, diagnostic strategies, although central for contact tracing and other preventive measures, are still limited. To meet the global demand, lower cost and faster antigen tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection are a convenient alternative to the gold standard reverse transcription-polymerase chain reaction (RT-PCR) assay. We tested laboratory-based RT-PCR RNA detection and two rapid antigen detection (RAD) tests, based on the immunochromatography test for nucleocapsid protein of SARS-CoV-2 (COVID-19 Ag ECO Test, ECO Diagnóstica, and Panbio COVID-19 Ag Rapid Test Abbott). Paired collection and testing were done in a small prospective open study in three clinical services in São Paulo, constituted of mostly symptomatic volunteers at collection (97%, 109/112) for a median of 4 days (interquartile range: 3-6), ranging from 1 to 30. Among the 108 paired RT-PCR/RAD tests, results were concordant in 96.4% (101/108). The test's performance was comparable, with an overall sensitivity of 87% and a specificity of 96%. These observations add to other data that suggest that antigen tests may provide reasonable sensitivity and specificity and deserve a role to improve testing strategies, especially in resource-limited settings.


Subject(s)
Antigens, Viral/analysis , COVID-19 Nucleic Acid Testing/methods , COVID-19 Serological Testing/methods , COVID-19/diagnosis , RNA, Viral/analysis , Adolescent , Adult , Aged , Aged, 80 and over , Brazil , Child , Child, Preschool , Diagnostic Tests, Routine , Female , Humans , Infant , Male , Middle Aged , Nasopharynx/virology , Prospective Studies , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Sensitivity and Specificity , Young Adult
6.
Pathologe ; 42(2): 208-215, 2021 Mar.
Article in German | MEDLINE | ID: covidwho-1235730

ABSTRACT

BACKGROUND: Analyses for the presence of SARS-CoV­2 in the tissues of COVID-19 patients is important in order to improve our understanding of the disease pathophysiology for interpretation of diagnostic histopathological findings in autopsies, biopsies, or surgical specimens and to assess the potential for occupational infectious hazard. MATERIAL AND METHODS: In this review we identified 136 published studies in PubMed's curated literature database LitCovid on SARS-CoV­2 detection methods in tissues and evaluated them regarding sources of error, specificity, and sensitivity of the methods, taking into account our own experience. RESULTS: Currently, no sufficiently specific histomorphological alterations or diagnostic features for COVID-19 are known. Therefore, three approaches for SARS-CoV­2 detection are used: RNA, proteins/antigens, or morphological detection by electron microscopy. In the preanalytical phase, the dominant source of error is tissue quality, especially the different intervals between sample collection and processing or fixation (and its duration) and specifically the interval between death and sample collection in autopsies. However, this information is found in less than half of the studies (e.g., in only 42% of autopsy studies). Our own experience and first studies prove the significantly higher sensitivity and specificity of RNA-based detection methods compared to antigen or protein detection by immunohistochemistry or immunofluorescence. Detection by electron microscopy is time consuming and difficult to interpret. CONCLUSIONS: Different methods are available for the detection of SARS-CoV­2 in tissue. Currently, RNA detection by RT-PCR is the method of choice. However, extensive validation studies and method harmonization are not available and are absolutely necessary.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , RNA, Viral , Sensitivity and Specificity
7.
J Med Virol ; 93(9): 5538-5543, 2021 Sep.
Article in English | MEDLINE | ID: covidwho-1231858

ABSTRACT

In the current coronavirus disease 2019 (COVID-19) pandemic there is a mass screening of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) happening around the world due to the extensive spread of the infections. There is a high demand for rapid diagnostic tests to expedite the identification of cases and to facilitate early isolation and control spread. Hence this study evaluates six different rapid nucleic acid detection assays that are commercially available for SARS-CoV-2 virus detection. Nasopharyngeal samples were collected from 4981 participants and were tested for the SARS-CoV-2 virus by the gold standard real-time reverse-transcription polymerase chain reaction (RT-PCR) method and with one of these six rapid methods of detection. Evaluation of the rapid nucleic acid detection assays was done by comparing the results of these rapid methods with the gold standard RT-qPCR results for SARS-COV-2 detection. AQ-TOP had the highest sensitivity (98%) and a strong kappa value of 0.943 followed by Genechecker and Abbot ID NOW. The POCKIT (ii RT-PCR) assay had the highest test accuracy of 99.29% followed by Genechecker and Cobas Liat. Atila iAMP showed the highest percentage of invalid reports (35.5%) followed by AQ-TOP with 6% and POCKIT with 3.7% of invalid reports. Genechecker system, Abbott ID NOW, and Cobas Liat were found to have the best performance and agreement when compared with the standard RT-PCR for COVID-19 detection. With further research, these rapid tests have the potential to be employed in large-scale screening of COVID-19.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , SARS-CoV-2/isolation & purification , COVID-19 Nucleic Acid Testing/instrumentation , COVID-19 Nucleic Acid Testing/standards , Humans , Nasopharynx/virology , Real-Time Polymerase Chain Reaction , SARS-CoV-2/genetics , Sensitivity and Specificity , United Arab Emirates
8.
Transbound Emerg Dis ; 2021 May 16.
Article in English | MEDLINE | ID: covidwho-1231149

ABSTRACT

Active epidemiological surveillance of infectious agents represents a fundamental tool for understanding the transmission dynamics of pathogens and establishing public policies that can reduce or limit their expansion. Epidemiological surveillance of emerging agents, such as the recently recognized severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of COVID-19, is essential to establish the risk of transmission between species. Recent studies reveal that companion animals are organisms susceptible to being infected by this pathogen due to the close contact they have with their owners. For this reason, the aim of the present work was to detect the presence of SARS-CoV-2 in dogs and cats in the state of Veracruz, Mexico, where there is active transmission of this microorganism in human populations. Oral and nasopharyngeal swab samples were collected from dogs and cats with a history of exposure to patients with COVID-19. Total RNA was extracted and detection of viral genes N1 and N2 was performed by reverse transcription polymerase chain reaction (RT-qPCR). All 130 samples of companion animals tested by RT-qPCR for SARS-CoV-2 were negative at the time they were collected. This study represents the second active surveillance of SARS-CoV-2 in populations of domestic dogs and cats in Latin America and the first approach in Mexico. Given that coronaviruses have shown a high capacity to be transmitted between species, it is imperative to establish measures to prevent this agent from entering and establishing in populations of companion animals.

9.
Int J Infect Dis ; 107: 268-270, 2021 Jun.
Article in English | MEDLINE | ID: covidwho-1228044

ABSTRACT

BACKGROUND: Healthcare workers (HCWs) are at increased risk of exposure to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the cause of coronavirus disease 2019 (COVID-19), compared with the general population. Therefore, they are given priority for the COVID-19 vaccine in the national COVID-19 vaccination campaign in Indonesia. However, while the daily number of new COVID-19 cases remains high, and data regarding the efficacy of the vaccine in healthcare settings remain unavailable, vaccinated HCWs remain at risk of COVID-19 infection and further transmission. OBJECTIVE: To identify cases of COVID-19 among vaccinated HCWs at Siloam Teaching Hospital, Indonesia via active and passive surveillance conducted by the hospital's COVID-19 infection prevention and control unit. RESULTS: Of 1040 HCWs who had received two doses of the COVID-19 vaccine, 13 (1.25%) tested positive for SARS-CoV-2 RNA on reverse transcriptase polymerase chain reaction between 2 and 11 days (median 5 days) after the second vaccination. CONCLUSION: Laboratory-confirmed COVID-19 among vaccinated HCWs soon after the second vaccination indicates that HCWs remain at risk of COVID-19. Therefore, the presence of symptoms soon after full vaccination cannot be considered as vaccine-related symptoms, and regular COVID-19 testing should be conducted among HCWs.


Subject(s)
COVID-19 Vaccines/immunology , COVID-19/epidemiology , Health Personnel , SARS-CoV-2 , Vaccination , Adult , COVID-19/prevention & control , COVID-19 Testing , Female , Hospitals, Teaching , Humans , Indonesia/epidemiology , Male
10.
MMWR Morb Mortal Wkly Rep ; 70(19): 702-706, 2021 May 14.
Article in English | MEDLINE | ID: covidwho-1227229

ABSTRACT

Prompt and accurate detection of SARS-CoV-2, the virus that causes COVID-19, has been important during public health responses for containing the spread of COVID-19, including in hospital settings (1-3). In vitro diagnostic nucleic acid amplification tests (NAAT), such as real-time reverse transcription-polymerase chain reaction (RT-PCR) can be expensive, have relatively long turnaround times, and require experienced laboratory personnel.* Antigen detection tests can be rapidly and more easily performed and are less expensive. The performance† of antigen detection tests, compared with that of NAATs, is an area of interest for the rapid diagnosis of SARS-CoV-2 infection. The Quidel Sofia 2 SARS Antigen Fluorescent Immunoassay (FIA) (Quidel Corporation) received Food and Drug Administration Emergency Use Authorization for use in symptomatic patients within 5 days of symptom onset (4). The reported test positive percentage agreement§ between this test and an RT-PCR test result is 96.7% (95% confidence interval [CI] = 83.3%-99.4%), and the negative percentage agreement is 100.0% (95% CI = 97.9%-100.0%) in symptomatic patients.¶ However, performance in asymptomatic persons in a university setting has shown lower sensitivity (5); assessment of performance in a clinical setting is ongoing. Data collected during June 30-August 31, 2020, were analyzed to compare antigen test performance with that of RT-PCR in a hospital setting. Among 1,732 paired samples from asymptomatic patients, the antigen test sensitivity was 60.5%, and specificity was 99.5% when compared with RT-PCR. Among 307 symptomatic persons, sensitivity and specificity were 72.1% and 98.7%, respectively. Health care providers must remain aware of the lower sensitivity of this test among asymptomatic and symptomatic persons and consider confirmatory NAAT testing in high-prevalence settings because a false-negative result might lead to failures in infection control and prevention practices and cause delays in diagnosis, isolation, and treatment.


Subject(s)
Antigens, Viral/analysis , COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , SARS-CoV-2/isolation & purification , Adolescent , Adult , Aged , Aged, 80 and over , COVID-19/epidemiology , COVID-19/prevention & control , Cross Infection/prevention & control , Female , Hospitals , Humans , Los Angeles/epidemiology , Male , Middle Aged , SARS-CoV-2/immunology , Sensitivity and Specificity , Young Adult
11.
Virol Sin ; 35(6): 758-767, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-1217478

ABSTRACT

Reverse transcription-polymerase chain reaction (RT-PCR) is an essential method for specific diagnosis of SARS-CoV-2 infection. Unfortunately, false negative test results are often reported. In this study, we attempted to determine the principal causes leading to false negative results of RT-PCR detection of SARS-CoV-2 RNAs in respiratory tract specimens. Multiple sputum and throat swab specimens from 161 confirmed COVID-19 patients were tested with a commercial fluorescent RT-PCR kit targeting the ORF1ab and N regions of SARS-CoV-2 genome. The RNA level of a cellular housekeeping gene ribonuclease P/MRP subunit p30 (RPP30) in these specimens was also assessed by RT-PCR. Data for a total of 1052 samples were retrospectively re-analyzed and a strong association between positive results in SARS-CoV-2 RNA tests and high level of RPP30 RNA in respiratory tract specimens was revealed. By using the ROC-AUC analysis, we identified Ct cutoff values for RPP30 RT-PCR which predicted false negative results for SARS-CoV-2 RT-PCR with high sensitivity (95.03%-95.26%) and specificity (83.72%-98.55%) for respective combination of specimen type and amplification reaction. Using these Ct cutoff values, false negative results could be reliably identified. Therefore, the presence of cellular materials, likely infected host cells, are essential for correct SARS-CoV-2 RNA detection by RT-PCR in patient specimens. RPP30 could serve as an indicator for cellular content, or a surrogate indicator for specimen quality. In addition, our results demonstrated that false negativity accounted for a vast majority of contradicting results in SARS-CoV-2 RNA test by RT-PCR.


Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , RNA, Viral/genetics , SARS-CoV-2/genetics , Autoantigens/genetics , COVID-19/epidemiology , COVID-19/virology , China/epidemiology , Humans , Negative Results , Polyproteins/genetics , RNA, Viral/isolation & purification , Reference Standards , Retrospective Studies , Reverse Transcriptase Polymerase Chain Reaction/methods , Ribonuclease P/genetics , SARS-CoV-2/isolation & purification , Sensitivity and Specificity , Viral Proteins/genetics
12.
Sci Total Environ ; 785: 147270, 2021 Sep 01.
Article in English | MEDLINE | ID: covidwho-1201954

ABSTRACT

Wastewater-based epidemiology is currently being utilized to monitor the dissemination of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), on a population scale. The detection of SARS-CoV-2 in wastewater is highly influenced by methodologies used for its isolation, concentration and RNA extraction. Although various viral concentration methods are currently employed, including polyethylene glycol (PEG) precipitation, adsorption-extraction, ultracentrifugation and ultrafiltration, to our knowledge, none of these methods have been standardized for use with a variety of wastewater matrices and/or different kits for RNA extraction and quantification. To address this, wastewater with different physical characteristics was seeded with gamma-irradiated SARS-CoV-2 and used to test the efficiency of PEG precipitation and adsorption-extraction to concentrate the virus from three physiochemically different wastewater samples, sourced from three distinct wastewater plants. Efficiency of viral concentration and RNA extraction was assessed by reverse-transcriptase polymerase chain reaction and the recovery yields calculated. As co-purification of inhibitors can be problematic for subsequent detection, two commonly used commercial master mixes were assessed for their sensitivity and efficiency to detect two SARS-CoV-2 target nucleocapsid (N) gene sequences. Recovery rates varied greatly between wastewater matrices and concentration methods, with the highest and most reproducible recovery rates (46.6-56.7%) observed when SARS-CoV-2 was precipitated with PEG and detected by the Luna® Universal master mix. The adsorption-extraction method was less effective (0-21.7%). This study demonstrates that PEG precipitation is the more robust method, which translates well to varying wastewater matrices, producing consistent and reproducible recovery rates. Furthermore, it is compatible with different kits for RNA extraction and quantitation.


Subject(s)
COVID-19 , Viruses , Humans , SARS-CoV-2 , Waste Water
13.
J AOAC Int ; 104(4): 914-923, 2021 Aug 20.
Article in English | MEDLINE | ID: covidwho-1192012

ABSTRACT

BACKGROUND: The VERIPRO® SARS-CoV-2 Env assay uses reverse transcriptase (RT) PCR to detect SARS-CoV-2, the causative agent of COVID-19, from stainless-steel environmental sample swabs. OBJECTIVE: To validate the VERIPRO SARS-CoV-2 Env assay as part of the AOAC Research Institute's Emergency Response Validation Performance Tested Method(s)SM program. METHOD: The VERIPRO SARS-CoV-2 Env assay was evaluated for specificity using in silico analysis of 15 764 SARS-CoV-2 sequences and 65 exclusivity organisms (both near neighbors and background organisms). The candidate method was evaluated in an unpaired study design for one environmental surface (stainless steel) and compared to the U.S. Centers for Disease Control and Prevention 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel, Instructions for Use (Revision 4, Effective 6/12/2020). RESULTS: Results of the in silico analysis demonstrate the specificity of the method in being able to detect target sequences and discriminate them from near neighbors. In the matrix study, the candidate method demonstrated statistically significant better recovery of the target analyte than the reference method. CONCLUSIONS: The VERIPRO SARS-CoV-2 Env assay is a rapid and accurate method that can be utilized by food producers to detect the causative agent of COVID-19 on food contact surfaces. HIGHLIGHTS: The VERIPRO SARS-CoV-2 Env assay can be performed without the need for an optional RNA purification step to detect SARS-CoV-2 from environmental surfaces.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19 Testing , Humans , RNA, Viral , Sensitivity and Specificity , Stainless Steel
14.
J King Saud Univ Sci ; 33(4): 101439, 2021 Jun.
Article in English | MEDLINE | ID: covidwho-1185114

ABSTRACT

By the end of year 2019, the new virus SARS-CoV-2 appeared, causing the Coronavirus Disease 2019 (COVID-19), and spread very fast globally. A continuing need for diagnostic tools is a must to contain its spread. Till now, the gold standard method, the reverse transcription polymerase chain reaction (RT-PCR), is the precise procedure to detect the virus. However, SARS-CoV-2 may escape RT-PCR detection for several reasons. The development of well-designed, specific and sensitive serological test like enzyme immunoassay (EIA) is needed. This EIA can stand alone or work side by side with RT-PCR. In this study, we developed several EIAs including plates that are coated with either specially designed SARS-CoV-2 nucleocapsid or surface recombinant proteins. Each protein type can separately detect anti-SARS-CoV-2 IgM or IgG antibodies. For each EIAs, the cut-off value, specificity and sensitivity were determined utilizing RT-PCR confirmed Covid-19 and pre-pandemic healthy and other viruses-infected sera. Also, the receiver operator characteristic (ROC) analysis was performed to define the specificities and sensitivities of the optimized assay. The in-house EIAs were validated by comparing against commercial EIA kits. All in-house EIAs showed high specificity (98-99%) and sensitivity (97.8-98.9%) for the detection of IgG/IgM against RBD and N proteins of SARS-CoV-2. From these results, the developed Anti-RBD and anti-N IgG and IgM antibodies EIAs can be used as a specific and sensitive tool to detect SARS-CoV-2 infection, calculate the burden of disease and case fatality rates.

15.
Ann Clin Biochem ; 58(4): 368-376, 2021 07.
Article in English | MEDLINE | ID: covidwho-1175245

ABSTRACT

BACKGROUND: Quantitative antibody tests are expected to be useful in diagnostics of COVID-19 and investigation of herd immunity against SARS-CoV-2. To make it proper to perform them, understanding of the immunological aspects is critically important. The present study aimed to assess humoral responses in COVID-19 using various quantitative antibody tests. METHODS: Four quantitative antibody tests that are different in targeted antigens, detectable immunoglobulin classes and avidity were used. Diagnosis was confirmed by RT-PCR for SARS-CoV-2 detection. Antibody titres of 117 samples collected from 24 COVID-19 patients and 23 non-COVID-19 patients were measured to evaluate correlations between different tests. For 24 COVID-19 patients, antibody titres measured at various time points after the onset or the RT-PCR diagnosis were subjected to assessment of humoral responses. RESULTS: Correlations between tests were observed to some degree, although there were discrepancies putatively due to differences in measurement principle. Seronegative COVID-19 was diagnosed for some patients, in whom antibody titres were less than the cut-off value in each test throughout the time courses. IgG seroconversion without prior IgM seroconversion most frequently occurred, while predominance of IgM responses over IgG responses was observed in some severe cases. Viral burdens estimated according to threshold cycle values at the RT-PCR seemed to impact antibody responses. CONCLUSIONS: The results provide insights into the nature of humoral responses to SARS-CoV-2 and diagnostic performance of antibody tests.


Subject(s)
Antibodies, Viral , COVID-19 Nucleic Acid Testing , COVID-19 Serological Testing , COVID-19 , Immunity, Humoral , Immunoglobulin G , Immunoglobulin M , SARS-CoV-2 , Antibodies, Viral/blood , Antibodies, Viral/immunology , COVID-19/blood , COVID-19/diagnosis , COVID-19/immunology , Female , Humans , Immunoglobulin G/blood , Immunoglobulin G/immunology , Immunoglobulin M/blood , Immunoglobulin M/immunology , Male , SARS-CoV-2/immunology , SARS-CoV-2/metabolism
16.
Ocul Immunol Inflamm ; 29(4): 681-683, 2021 May 19.
Article in English | MEDLINE | ID: covidwho-1171738

ABSTRACT

Purpose: To present a a case study that aims to investigate the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the ocular tissue samples of a patient previously infected with COVID-19 and determine its transmissibility.Study Design: Case ReportResults: In this case study, SARS-CoV-2 was not detected in the vitreous and uveal tissue samples by RT-PCR for detection of three gene targets in a patient with a past COVID-19 infection 15 days prior to presention with a globe rupture.Conclusions: Our findings suggest that patients with long-term existence of SARS-CoV-2 at low detectable levels may not have active intraocular viral shedding. This is of particular importance as ophthalmic surgical procedures may potentiate virus spread from patients infected with SARS-CoV-2.


Subject(s)
COVID-19/virology , Eye Infections, Viral/diagnosis , RNA, Viral/analysis , SARS-CoV-2/genetics , Uvea/virology , Vitreous Body/virology , Adult , COVID-19/complications , COVID-19/diagnosis , Eye Infections, Viral/etiology , Eye Infections, Viral/virology , Female , Humans , Specimen Handling , Virus Shedding
17.
J Med Virol ; 93(7): 4405-4410, 2021 07.
Article in English | MEDLINE | ID: covidwho-1160182

ABSTRACT

One year into the coronavirus disease 2019 (COVID-19) pandemic, diagnostic strategies, although central for contact tracing and other preventive measures, are still limited. To meet the global demand, lower cost and faster antigen tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection are a convenient alternative to the gold standard reverse transcription-polymerase chain reaction (RT-PCR) assay. We tested laboratory-based RT-PCR RNA detection and two rapid antigen detection (RAD) tests, based on the immunochromatography test for nucleocapsid protein of SARS-CoV-2 (COVID-19 Ag ECO Test, ECO Diagnóstica, and Panbio COVID-19 Ag Rapid Test Abbott). Paired collection and testing were done in a small prospective open study in three clinical services in São Paulo, constituted of mostly symptomatic volunteers at collection (97%, 109/112) for a median of 4 days (interquartile range: 3-6), ranging from 1 to 30. Among the 108 paired RT-PCR/RAD tests, results were concordant in 96.4% (101/108). The test's performance was comparable, with an overall sensitivity of 87% and a specificity of 96%. These observations add to other data that suggest that antigen tests may provide reasonable sensitivity and specificity and deserve a role to improve testing strategies, especially in resource-limited settings.


Subject(s)
Antigens, Viral/analysis , COVID-19 Nucleic Acid Testing/methods , COVID-19 Serological Testing/methods , COVID-19/diagnosis , RNA, Viral/analysis , Adolescent , Adult , Aged , Aged, 80 and over , Brazil , Child , Child, Preschool , Diagnostic Tests, Routine , Female , Humans , Infant , Male , Middle Aged , Nasopharynx/virology , Prospective Studies , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Sensitivity and Specificity , Young Adult
18.
J Clin Microbiol ; 59(6)2021 05 19.
Article in English | MEDLINE | ID: covidwho-1158099

ABSTRACT

LamPORE is a novel diagnostic platform for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA combining loop-mediated isothermal amplification with nanopore sequencing, which could potentially be used to analyze thousands of samples per day on a single instrument. We evaluated the performance of LamPORE against reverse transcriptase PCR (RT-PCR) using RNA extracted from spiked respiratory samples and stored nose and throat swabs collected at two UK hospitals. The limit of detection of LamPORE was 10 genome copies/µl of extracted RNA, which is above the limit achievable by RT-PCR, but was not associated with a significant reduction of sensitivity in clinical samples. Positive clinical specimens came mostly from patients with acute symptomatic infection, and among them, LamPORE had a diagnostic sensitivity of 99.1% (226/228; 95% confidence interval [CI], 96.9% to 99.9%). Among negative clinical specimens, including 153 with other respiratory pathogens detected, LamPORE had a diagnostic specificity of 99.6% (278/279; 98.0% to 100.0%). Overall, 1.4% (7/514; 0.5% to 2.9%) of samples produced an indeterminate result on first testing, and repeat LamPORE testing on the same RNA extract had a reproducibility of 96.8% (478/494; 94.8% to 98.1%). LamPORE has a similar performance as RT-PCR for the diagnosis of SARS-CoV-2 infection in symptomatic patients and offers a promising approach to high-throughput testing.


Subject(s)
COVID-19 , Nanopore Sequencing , Humans , Molecular Diagnostic Techniques , Nucleic Acid Amplification Techniques , RNA, Viral/genetics , Reproducibility of Results , SARS-CoV-2 , Sensitivity and Specificity
19.
Pathologe ; 42(Suppl 1): 81-88, 2021 Nov.
Article in English | MEDLINE | ID: covidwho-1152000

ABSTRACT

BACKGROUND: Analyses for the presence of SARS-CoV­2 in the tissues of COVID-19 patients is important in order to improve our understanding of the disease pathophysiology for interpretation of diagnostic histopathological findings in autopsies, biopsies, or surgical specimens and to assess the potential for occupational infectious hazard. MATERIAL AND METHODS: In this review we identified 136 published studies in PubMed's curated literature database LitCovid on SARS-CoV­2 detection methods in tissues and evaluated them regarding sources of error, specificity, and sensitivity of the methods, taking into account our own experience. RESULTS: Currently, no sufficiently specific histomorphological alterations or diagnostic features for COVID-19 are known. Therefore, three approaches for SARS-CoV­2 detection are used: RNA, proteins/antigens, or morphological detection by electron microscopy. In the preanalytical phase, the dominant source of error is tissue quality, especially the different intervals between sample collection and processing or fixation (and its duration) and specifically the interval between death and sample collection in autopsies. However, this information is found in less than half of the studies (e.g., in only 42% of autopsy studies). Our own experience and first studies prove the significantly higher sensitivity and specificity of RNA-based detection methods compared to antigen or protein detection by immunohistochemistry or immunofluorescence. Detection by electron microscopy is time consuming and difficult to interpret. CONCLUSIONS: Different methods are available for the detection of SARS-CoV­2 in tissue. Currently, RNA detection by RT-PCR is the method of choice. However, extensive validation studies and method harmonization are not available and are absolutely necessary.


Subject(s)
COVID-19 , SARS-CoV-2 , Autopsy , Humans , RNA, Viral , Sensitivity and Specificity
20.
Int J Gen Med ; 14: 435-440, 2021.
Article in English | MEDLINE | ID: covidwho-1133770

ABSTRACT

BACKGROUND: Early diagnosis of the novel coronavirus disease of 2019 (COVID-19) in asymptomatic and symptomatic patients is crucial to identify infectious individuals and to help prevent the spread of the virus in the community. Several assays have been developed and are in use in today's clinical practice. These assays vary in their analytical and clinical performance. For an accurate diagnosis, medical professionals must become more familiar with the test's utility to select the most appropriate test. This study aims to evaluate the analytical performance of rapid antigen tests used for the detection of SARS-CoV-2 viral antigen compared to RT-PCR SARS-CoV-2 molecular assay. METHODS: Oropharyngeal swab specimens from five COVID-19 patients were tested by seven rapid antigen tests developed by different IVD companies. RT-PCR to detect specific RNA fragments of SARS-CoV-2 was used as a confirmatory test. The cycle threshold (Ct) value, which often reflects viral load, in these specimens ranged from 15 to 35. For the analytical evaluation, extraction fluid of each antigen kit was spiked with attenuated ATCC virus at different concentrations ranging from 4.6x104/mL to 7.5x105/mL and tested with antigen testing kits. RESULTS: Out of five confirmed positive SARS-CoV-2 specimens by RT-PCR, only one sample showed a positive result by one of the seven evaluated antigen testing kits. The positive result was observed in the specimen with a Ct value of 15. All other evaluated rapid tests were negative for all five positive specimens. This was further confirmed with the spiking study using ATCC attenuated virus, where extraction fluid of each rapid test was spiked with concentrations ranging from 4.6x104/mL to 7.5x105/mL. None of these spiked specimens showed positive results, indicating very low sensitivity of these antigen kits. CONCLUSION: This comparison study shows that rapid antigen tests are less sensitive than RT-PCR tests and are not reliable tests for testing asymptomatic patients, who often carry low viral load. Analytical performance of rapid antigen tests should be thoroughly evaluated before implementing it at clinical decision level.

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