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2.
PLoS Pathog ; 16(8): e1008705, 2020 08.
Article in English | MEDLINE | ID: covidwho-732988

ABSTRACT

The recent outbreak of human infections caused by SARS-CoV-2, the third zoonotic coronavirus has raised great public health concern globally. Rapid and accurate diagnosis of this novel pathogen posts great challenges not only clinically but also technologically. Metagenomic next-generation sequencing (mNGS) and reverse-transcription PCR (RT-PCR) have been the most commonly used molecular methodologies. However, each has their own limitations. In this study, we developed an isothermal, CRISPR-based diagnostic for COVID-19 with near single-copy sensitivity. The diagnostic performances of all three technology platforms were also compared. Our study aimed to provide more insights into the molecular detection of SARS-CoV-2, and also to present a novel diagnostic option for this new emerging virus.


Subject(s)
Betacoronavirus/genetics , CRISPR-Cas Systems/genetics , Clinical Laboratory Techniques , Coronavirus Infections/diagnosis , Coronavirus Infections/genetics , Pneumonia, Viral/diagnosis , Pneumonia, Viral/genetics , Bacteria/genetics , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Genes, Viral/genetics , Genome, Viral/genetics , High-Throughput Nucleotide Sequencing/methods , Humans , Molecular Diagnostic Techniques/economics , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/economics , Nucleic Acid Amplification Techniques/methods , Pandemics , Reverse Transcriptase Polymerase Chain Reaction/methods , Sensitivity and Specificity
3.
mSphere ; 5(4)2020 08 26.
Article in English | MEDLINE | ID: covidwho-730989

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak urgently necessitates sensitive and convenient COVID-19 diagnostics for the containment and timely treatment of patients. We aimed to develop and validate a novel reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay to detect SARS-CoV-2. Patients with suspected COVID-19 and close contacts were recruited from two hospitals between 26 January and 8 April 2020. Respiratory samples were collected and tested using RT-LAMP, and the results were compared with those obtained by reverse transcription-quantitative PCR (RT-qPCR). Samples yielding inconsistent results between these two methods were subjected to next-generation sequencing for confirmation. RT-LAMP was also applied to an asymptomatic COVID-19 carrier and patients with other respiratory viral infections. Samples were collected from a cohort of 129 cases (329 nasopharyngeal swabs) and an independent cohort of 76 patients (152 nasopharyngeal swabs and sputum samples). The RT-LAMP assay was validated to be accurate (overall sensitivity and specificity of 88.89% and 99.00%, respectively) and diagnostically useful (positive and negative likelihood ratios of 88.89 and 0.11, respectively). RT-LAMP showed increased sensitivity (88.89% versus 81.48%) and high consistency (kappa, 0.92) compared to those of RT-qPCR for SARS-CoV-2 screening while requiring only constant-temperature heating and visual inspection. The time required for RT-LAMP was less than 1 h from sample preparation to the result. In addition, RT-LAMP was feasible for use with asymptomatic patients and did not cross-react with other respiratory pathogens. The developed RT-LAMP assay offers rapid, sensitive, and straightforward detection of SARS-CoV-2 infection and may aid the expansion of COVID-19 testing in the public domain and hospitals.IMPORTANCE We developed a visual and rapid reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay targeting the S gene for SARS-CoV-2 infection. The strength of our study was that we validated the RT-LAMP assay using 481 clinical respiratory samples from two prospective cohorts of suspected COVID-19 patients and on the serial samples from an asymptomatic carrier. The developed RT-LAMP approach showed an increased sensitivity (88.89%) and high consistency (kappa, 0.92) compared with those of reverse transcription-quantitative PCR (RT-qPCR) for SARS-CoV-2 screening while requiring only constant-temperature heating and visual inspection, facilitating SARS-CoV-2 screening in well-equipped labs as well as in the field. The time required for RT-LAMP was less than 1 h from sample preparation to the result (more than 2 h for RT-qPCR). This study showed that the RT-LAMP assay was a simple, rapid, and sensitive approach for SARS-CoV-2 infection and can facilitate COVID-19 diagnosis, especially in resource-poor settings.


Subject(s)
Betacoronavirus/genetics , Clinical Laboratory Techniques/methods , Coronavirus Infections/diagnosis , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , Pneumonia, Viral/diagnosis , Adult , Asymptomatic Diseases , Female , Humans , Male , Middle Aged , Pandemics , Prospective Studies , Reverse Transcriptase Polymerase Chain Reaction/methods , Sensitivity and Specificity
4.
J Int Med Res ; 48(8): 300060520949067, 2020 Aug.
Article in English | MEDLINE | ID: covidwho-729470

ABSTRACT

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acid test is currently the gold standard for diagnosing coronavirus disease 2019 (COVID-19). This disease requires high-quality viral nucleic acid tests, and selecting the type of specimen from patients, who are at different disease stages, to use in the nucleic acid test is challenging. This article reports in detail the diagnosis and treatment process for two patients with confirmed COVID-19 and analyzes the results of the SARS-CoV-2 nucleic acid tests that were used for different types of specimens (sputum from deep cough, nasopharyngeal swab, and feces). The nucleic acid testing results of sputum from deep cough showed the best performance for positive detection. Our findings provide a reference for selecting the most suitable specimen for the clinical diagnosis of COVID-19 and improving the positive detection rate.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/diagnosis , Molecular Diagnostic Techniques/methods , Pneumonia, Viral/diagnosis , Aged , Humans , Male , Middle Aged , Pandemics , RNA, Viral/analysis
5.
Genes (Basel) ; 11(8)2020 08 17.
Article in English | MEDLINE | ID: covidwho-725327

ABSTRACT

The COVID-19 pandemic has spread very fast around the world. A few days after the first detected case in South Africa, an infection started in a large hospital outbreak in Durban, KwaZulu-Natal (KZN). Phylogenetic analysis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes can be used to trace the path of transmission within a hospital. It can also identify the source of the outbreak and provide lessons to improve infection prevention and control strategies. This manuscript outlines the obstacles encountered in order to genotype SARS-CoV-2 in near-real time during an urgent outbreak investigation. This included problems with the length of the original genotyping protocol, unavailability of reagents, and sample degradation and storage. Despite this, three different library preparation methods for Illumina sequencing were set up, and the hands-on library preparation time was decreased from twelve to three hours, which enabled the outbreak investigation to be completed in just a few weeks. Furthermore, the new protocols increased the success rate of sequencing whole viral genomes. A simple bioinformatics workflow for the assembly of high-quality genomes in near-real time was also fine-tuned. In order to allow other laboratories to learn from our experience, all of the library preparation and bioinformatics protocols are publicly available at protocols.io and distributed to other laboratories of the Network for Genomics Surveillance in South Africa (NGS-SA) consortium.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/diagnosis , Genome, Viral , High-Throughput Nucleotide Sequencing/methods , Molecular Diagnostic Techniques/methods , Pneumonia, Viral/diagnosis , Whole Genome Sequencing/methods , Betacoronavirus/pathogenicity , Coronavirus Infections/virology , High-Throughput Nucleotide Sequencing/standards , Humans , Molecular Diagnostic Techniques/standards , Pandemics , Pneumonia, Viral/virology , Reproducibility of Results , Sensitivity and Specificity , Whole Genome Sequencing/standards
6.
Epidemiol Infect ; 148: e178, 2020 08 18.
Article in English | MEDLINE | ID: covidwho-720809

ABSTRACT

Different countries have adopted strategies for the early detection of SARS-CoV-2 since the declaration of community transmission by the World Health Organization (WHO) and timely diagnosis has been considered one of the major obstacles for surveillance and healthcare. Here, we report the increase of the number of laboratories to COVID-19 diagnosis in Brazil. Our results demonstrate an increase and decentralisation of certified laboratories, which does not match the much higher increase in the number of COVID-19 cases. Also, it becomes clear that laboratories are irregularly distributed over the country, with a concentration in the most developed state, São Paulo.


Subject(s)
Clinical Laboratory Techniques/statistics & numerical data , Coronavirus Infections/diagnosis , Laboratories/supply & distribution , Pneumonia, Viral/diagnosis , Betacoronavirus , Brazil/epidemiology , Coronavirus Infections/epidemiology , Humans , Incidence , Molecular Diagnostic Techniques , Pandemics , Pneumonia, Viral/epidemiology
8.
Int J Mol Sci ; 21(16)2020 Aug 07.
Article in English | MEDLINE | ID: covidwho-714483

ABSTRACT

Sensitive molecular assays are critical for coronavirus disease 2019 (COVID-19) diagnosis. Here, we designed and evaluated two single-tube nested (STN) real-time RT-PCR assays, targeting SARS-CoV-2 RdRp/Hel and N genes. Both STN assays had a low limit of detection and did not cross react with other human coronaviruses and respiratory viruses. Using 213 initial respiratory specimens from suspected COVID-19 patients, the sensitivity of both the STN COVID-19-RdRp/Hel and the STN COVID-19-N assays was 100% (99/99), while that of the comparator non-nested N assay was 95% (94/99). Among 108 follow-up specimens from confirmed COVID-19 patients who tested negative by the non-nested COVID-19-RdRp/Hel assay, 28 (25.9%) were positive for SARS-CoV-2 by the STN COVID-19-RdRp/Hel or the STN COVID-19-N assay. To evaluate the performance of our novel STN assays in pooled specimens, we created four sample pools, with each pool consisting of one low positive specimen and 49 negative specimens. While the non-nested COVID-19-RdRp/Hel assay was positive in only one of four sample pools (25%), both of the STN assays were positive in two of four samples pools (50%). In conclusion, the STN assays are highly sensitive and specific for SARS-CoV-2 detection. Their boosted sensitivity offers advantages in non-traditional COVID-19 testing algorithms such as saliva screening and pooled sample screening during massive screening.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/virology , Molecular Diagnostic Techniques/methods , Pneumonia, Viral/virology , Real-Time Polymerase Chain Reaction/methods , Betacoronavirus/pathogenicity , Coronavirus Infections/diagnosis , Humans , Molecular Diagnostic Techniques/standards , Nucleocapsid Proteins/genetics , Pandemics , Pneumonia, Viral/diagnosis , RNA Replicase/genetics , Real-Time Polymerase Chain Reaction/standards , Respiratory Mucosa/virology , Sensitivity and Specificity , Viral Nonstructural Proteins/genetics
9.
Viruses ; 12(8)2020 08 07.
Article in English | MEDLINE | ID: covidwho-713633

ABSTRACT

Rapid large-scale testing is essential for controlling the ongoing pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The standard diagnostic pipeline for testing SARS-CoV-2 presence in patients with an ongoing infection is predominantly based on pharyngeal swabs, from which the viral RNA is extracted using commercial kits, followed by reverse transcription and quantitative PCR detection. As a result of the large demand for testing, commercial RNA extraction kits may be limited and, alternatively, non-commercial protocols are needed. Here, we provide a magnetic bead RNA extraction protocol that is predominantly based on in-house made reagents and is performed in 96-well plates supporting large-scale testing. Magnetic bead RNA extraction was benchmarked against the commercial QIAcube extraction platform. Comparable viral RNA detection sensitivity and specificity were obtained by fluorescent and colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) using a primer set targeting the N gene, as well as RT-qPCR using a primer set targeting the E gene, showing that the RNA extraction protocol presented here can be combined with a variety of detection methods at high throughput. Importantly, the presented diagnostic workflow can be quickly set up in a laboratory without access to an automated pipetting robot.


Subject(s)
Betacoronavirus/chemistry , Betacoronavirus/genetics , Clinical Laboratory Techniques/methods , Coronavirus Infections/virology , Pneumonia, Viral/virology , RNA, Viral/isolation & purification , Betacoronavirus/isolation & purification , Coronavirus Infections/diagnosis , Humans , Magnetic Phenomena , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , Pandemics , Pneumonia, Viral/diagnosis , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction/methods , Reverse Transcription , Sensitivity and Specificity
12.
J Clin Microbiol ; 58(8)2020 Jul 23.
Article in English | MEDLINE | ID: covidwho-709926

ABSTRACT

The recent emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has posed formidable challenges for clinical laboratories seeking reliable laboratory diagnostic confirmation. The swift advance of the crisis in the United States has led to Emergency Use Authorization (EUA) facilitating the availability of molecular diagnostic assays without the more rigorous examination to which tests are normally subjected prior to FDA approval. Our laboratory currently uses two real-time reverse transcription-PCR (RT-PCR) platforms, the Roche Cobas SARS-CoV2 and the Cepheid Xpert Xpress SARS-CoV-2. The two platforms demonstrate comparable performances; however, the run times for each assay are 3.5 h and 45 min, respectively. In search for a platform with a shorter turnaround time, we sought to evaluate the recently released Abbott ID Now COVID-19 assay, which is capable of producing positive results in as little as 5 min. We present here the results of comparisons between Abbott ID Now COVID-19 and Cepheid Xpert Xpress SARS-CoV-2 using nasopharyngeal swabs transported in viral transport media and comparisons between Abbott ID Now COVID-19 and Cepheid Xpert Xpress SARS-CoV-2 using nasopharyngeal swabs transported in viral transport media for Cepheid and dry nasal swabs for Abbott ID Now. Regardless of method of collection and sample type, Abbott ID Now COVID-19 had negative results in a third of the samples that tested positive by Cepheid Xpert Xpress when using nasopharyngeal swabs in viral transport media and 45% when using dry nasal swabs.


Subject(s)
Betacoronavirus/isolation & purification , Clinical Laboratory Techniques/methods , Coronavirus Infections/diagnosis , Molecular Diagnostic Techniques/methods , Nasal Mucosa/virology , Nasopharynx/virology , Nucleic Acid Amplification Techniques/methods , Pneumonia, Viral/diagnosis , Adult , Aged , Aged, 80 and over , Animals , Betacoronavirus/genetics , Coronavirus Infections/virology , Female , Humans , Male , Middle Aged , New York City , Pandemics , Pneumonia, Viral/virology , Sensitivity and Specificity , Specimen Handling/methods , Time Factors
14.
Curr Med Sci ; 40(4): 614-617, 2020 Aug.
Article in English | MEDLINE | ID: covidwho-696956

ABSTRACT

The novel Coronavirus SARS-CoV-2 caused an outbreak of pneumonia in Wuhan, Hubei province of China in January 2020. This study aims to investigate the effects of different temperature and time durations of virus inactivation on the results of PCR testing for SARS-CoV-2. Twelve patients at the Renmin Hospital of Wuhan University suspected of being infected with SARS-CoV-2 were selected on February 13, 2020 and throat swabs were taken. The swabs were stored at room temperature (20-25°C), then divided into aliquots and subjected to different temperature for different periods in order to inactivate the viruses (56°C for 30, 45, 60 min; 65, 70, 80°C for 10, 15, 20 min). Control aliquots were stored at room temperature for 60 min. Then all aliquots were tested in a real-time fluorescence PCR using primers against SARS-CoV-2. Regardless of inactivation temperature and time, 7 of 12 cases (58.3%) tested were positive for SARS-CoV-2 by PCR, and cycle threshold values were similar. These results suggest that virus inactivation parameters exert minimal influence on PCR test results. Inactivation at 65°C for 10 min may be sufficient to ensure safe, reliable testing.


Subject(s)
Betacoronavirus/genetics , Betacoronavirus/isolation & purification , Clinical Laboratory Techniques/methods , Coronavirus Infections/diagnosis , Coronavirus Infections/virology , Pneumonia, Viral/diagnosis , Pneumonia, Viral/virology , Real-Time Polymerase Chain Reaction/methods , Virus Inactivation , Adult , Aged , China/epidemiology , Coronavirus Infections/epidemiology , Humans , Infection Control/methods , Medical Laboratory Personnel , Middle Aged , Molecular Diagnostic Techniques/methods , Occupational Exposure/prevention & control , Pandemics , Pneumonia, Viral/epidemiology , RNA, Viral/analysis , RNA, Viral/genetics , Temperature , Time Factors
15.
Sci Transl Med ; 12(556)2020 08 12.
Article in English | MEDLINE | ID: covidwho-688785

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic caused by the SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) coronavirus is a major public health challenge. Rapid tests for detecting existing SARS-CoV-2 infections and assessing virus spread are critical. Approaches to detect viral RNA based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) have potential as simple, scalable, and broadly applicable testing methods. Compared to RT quantitative polymerase chain reaction (RT-qPCR)-based methods, RT-LAMP assays require incubation at a constant temperature, thus eliminating the need for sophisticated instrumentation. Here, we tested a two-color RT-LAMP assay protocol for detecting SARS-CoV-2 viral RNA using a primer set specific for the N gene. We tested our RT-LAMP assay on surplus RNA samples isolated from 768 pharyngeal swab specimens collected from individuals being tested for COVID-19. We determined the sensitivity and specificity of the RT-LAMP assay for detecting SARS-CoV-2 viral RNA. Compared to an RT-qPCR assay using a sensitive primer set, we found that the RT-LAMP assay reliably detected SARS-CoV-2 RNA with an RT-qPCR cycle threshold (CT) number of up to 30, with a sensitivity of 97.5% and a specificity of 99.7%. We also developed a swab-to-RT-LAMP assay that did not require a prior RNA isolation step, which retained excellent specificity (99.5%) but showed lower sensitivity (86% for CT < 30) than the RT-LAMP assay. In addition, we developed a multiplexed sequencing protocol (LAMP-sequencing) as a diagnostic validation procedure to detect and record the outcome of RT-LAMP reactions.


Subject(s)
Betacoronavirus/genetics , Betacoronavirus/isolation & purification , Coronavirus Infections/diagnosis , Coronavirus Infections/virology , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , Pneumonia, Viral/diagnosis , Pneumonia, Viral/virology , Colorimetry/methods , Colorimetry/statistics & numerical data , Coronavirus Infections/epidemiology , Humans , Molecular Diagnostic Techniques/statistics & numerical data , Nucleic Acid Amplification Techniques/statistics & numerical data , Nucleocapsid Proteins/genetics , Pandemics , Pneumonia, Viral/epidemiology , RNA, Viral/genetics , RNA, Viral/isolation & purification , RNA-Seq , Sensitivity and Specificity , Translational Medical Research
16.
JCI Insight ; 5(10)2020 05 21.
Article in English | MEDLINE | ID: covidwho-687860

ABSTRACT

BACKGROUNDThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a novel viral pneumonia (COVID-19), which is rapidly spreading throughout the world. The positive result of nucleic acid test is a golden criterion to confirm SARS-CoV-2 infection, but the detection features remain unclear.METHODSWe performed a retrospective analysis in 5630 high-risk individuals receiving SARS-CoV-2 nucleic acid tests in Wuhan, China, and investigated their characteristics and diagnosis rates.RESULTSThe overall diagnosis rate was 34.7% (1952/5630). Male (P = 0.025) and older populations (P = 2.525 × 10-39) were at significantly higher risk of SARS-CoV-2 infection. People were generally susceptible, and most cases concentrated in people of 30-79 years. Furthermore, we investigated the association between diagnosis rate and the amount of testing in 501 subjects. Results revealed a 1.27-fold improvement (from 27.9% to 35.5%) of diagnosis rate from testing once to twice (P = 5.847 × 10-9) and a 1.43-fold improvement (from 27.9% to 39.9%) from testing once to 3 times (P = 7.797 × 10-14). More than 3 testing administrations was not helpful for further improvement. However, this improvement was not observed in subjects with pneumonia (P = 0.097).CONCLUSIONAll populations are susceptible to SARS-CoV-2 infection, and male and older-aged populations are at significantly higher risk. Increasing the amount of testing could significantly improve diagnosis rates, except for subjects with pneumonia. It is recommended to test twice in those high-risk individuals whose results are negative the first time, and performing 3 tests is better, if possible.FUNDINGThis work was supported by National Mega Project on Major Infectious Disease Prevention (no. 2017ZX10103005-007) and National Key Research and Development Program of China (no. 2018YFE0204500).


Subject(s)
Clinical Laboratory Techniques , Coronavirus Infections/diagnosis , Pneumonia, Viral/diagnosis , Adolescent , Adult , Age Factors , Aged , Aged, 80 and over , Child , Child, Preschool , China/epidemiology , Clinical Laboratory Techniques/methods , Coronavirus Infections/epidemiology , Female , Humans , Incidence , Infant , Infant, Newborn , Male , Middle Aged , Molecular Diagnostic Techniques , Pandemics , Pneumonia, Viral/epidemiology , RNA, Viral/analysis , Real-Time Polymerase Chain Reaction , Retrospective Studies , Sex Factors , Young Adult
18.
J Clin Microbiol ; 58(8)2020 Jul 23.
Article in English | MEDLINE | ID: covidwho-684555

ABSTRACT

An outbreak of coronavirus disease 2019 (COVID-19) caused by a novel coronavirus (severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2]) began in Wuhan, Hubei, China, in December 2019 and spread rapidly worldwide. The response by the Alberta Precision Laboratories, Public Health Laboratory (ProvLab), AB, Canada, included the development and implementation of nucleic acid detection-based assays and dynamic changes in testing protocols for the identification of cases as the epidemic curve increased exponentially. This rapid response was essential to slow down and contain transmission and provide valuable time to the local health authorities to prepare appropriate response strategies. As of May 24, 2020, 236,077 specimens were tested, with 6,475 (2.74%) positives detected in the province of Alberta, Canada. Several commercial assays are now available; however, the response from commercial vendors to develop and market validated tests is a time-consuming process. In addition, the massive global demand made it difficult to secure a reliable commercial supply of testing kits and reagents. A public health laboratory serves a unique and important role in the delivery of health care. One of its functions is to anticipate and prepare for novel emerging pathogens with a plan for pandemic preparedness. Here, we outline the response that involved the development and deployment of testing methodologies that evolved as SARS-CoV-2 spread worldwide, the challenges encountered, and mitigation strategies. We also provide insight into the organizational structure of how a public health response is coordinated in Alberta, Canada, and its benefits.


Subject(s)
Betacoronavirus/isolation & purification , Clinical Laboratory Techniques/methods , Coronavirus Infections/diagnosis , Diagnostic Services/organization & administration , Molecular Diagnostic Techniques/methods , Pneumonia, Viral/diagnosis , Public Health Administration/methods , Alberta , Humans , Pandemics
19.
J Mol Diagn ; 22(8): 968-974, 2020 08.
Article in English | MEDLINE | ID: covidwho-683077

ABSTRACT

Clinical molecular laboratory professionals are at the frontline of the response to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, providing accurate, high-quality laboratory results to aid in diagnosis, treatment, and epidemiology. In this role, we have encountered numerous regulatory, reimbursement, supply-chain, logistical, and systems challenges that we have struggled to overcome to fulfill our calling to provide patient care. In this Perspective from the Association for Molecular Pathology Infectious Disease Subdivision Leadership team, we review how our members have risen to these challenges, provide recommendations for managing the current pandemic, and outline the steps we can take as a community to better prepare for future pandemics.


Subject(s)
Betacoronavirus , Coronavirus Infections/epidemiology , Pandemics , Pathology, Molecular , Pneumonia, Viral/epidemiology , Coronavirus Infections/diagnosis , Humans , Leadership , Molecular Diagnostic Techniques , Pathology, Molecular/organization & administration , Pneumonia, Viral/diagnosis , Societies, Medical , United States/epidemiology
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