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1.
BMC Infect Dis ; 22(1): 330, 2022 Apr 04.
Article in English | MEDLINE | ID: covidwho-1775313

ABSTRACT

BACKGROUND: Respiratory infections among children, particularly community-acquired pneumonia (CAP), is a major disease with a high frequency among outpatient and inpatient visits. The causes of CAP vary depending on individual susceptibility, the epidemiological characteristics of the community, and the season. We performed this study to establish a nationwide surveillance network system and identify the causative agents for CAP and antibiotic resistance in Korean children with CAP. METHODS: The monitoring network was composed of 28 secondary and tertiary medical institutions. Upper and lower respiratory samples were assayed using a culture or polymerase chain reaction (PCR) from August 2018 to May 2020. RESULTS: A total of 1023 cases were registered in patients with CAP, and PCR of atypical pneumonia pathogens revealed 422 cases of M. pneumoniae (41.3%). Respiratory viruses showed a positivity rate of 65.7% by multiplex PCR test, and human rhinovirus was the most common virus, with 312 cases (30.5%). Two hundred sixty four cases (25.8%) were isolated by culture, including 131 cases of S. aureus (12.8%), 92 cases of S. pneumoniae (9%), and 20 cases of H. influenzae (2%). The cultured, isolated bacteria may be colonized pathogen. The proportion of co-detection was 49.2%. The rate of antibiotic resistance showed similar results as previous reports. CONCLUSIONS: This study will identify the pathogens that cause respiratory infections and analyze the current status of antibiotic resistance to provide scientific evidence for management policies of domestic respiratory infections. Additionally, in preparation for new epidemics, including COVID-19, monitoring respiratory infections in children and adolescents has become more important, and research on this topic should be continuously conducted in the future.


Subject(s)
COVID-19 , Community-Acquired Infections , Pneumonia, Mycoplasma , Adolescent , Child , Community-Acquired Infections/microbiology , Humans , Multiplex Polymerase Chain Reaction/methods , Staphylococcus aureus
2.
Sci Rep ; 12(1): 3480, 2022 03 03.
Article in English | MEDLINE | ID: covidwho-1730307

ABSTRACT

The COVID-19 pandemic has resulted in significant diversion of human and material resources to COVID-19 diagnostics, to the extent that influenza viruses and co-infection in COVID-19 patients remains undocumented and pose serious public-health consequences. We optimized and validated a highly sensitive RT-PCR based multiplex-assay for the detection of SARS-CoV-2, influenza A and B viruses in a single-test. This study evaluated clinical specimens (n = 1411), 1019 saliva and 392 nasopharyngeal swab (NPS), tested using two-assays: FDA-EUA approved SARS-CoV-2 assay that targets N and ORF1ab gene, and the PKamp-RT-PCR based assay that targets SARS-CoV-2, influenza viruses A and B. Of the 1019 saliva samples, 17.0% (174/1019) tested positive for SARS-CoV-2 using either assay. The detection rate for SARS-CoV-2 was higher with the multiplex assay compared to SARS-specific assay [91.9% (160/174) vs. 87.9% (153/174)], respectively. Of the 392 NPS samples, 10.4% (41/392) tested positive for SARS-CoV-2 using either assay. The detection rate for SARS-CoV-2 was higher with the multiplex assay compared to SARS-specific assay [97.5% (40/41) vs. 92.1% (39/41)], respectively. This study presents clinical validation of a multiplex-PCR assay for testing SARS-CoV-2, influenza A and B viruses, using NPS and saliva samples, and demonstrates the feasibility of implementing the assay without disrupting the existing laboratory workflow.


Subject(s)
Influenza A virus/isolation & purification , Influenza B virus/isolation & purification , Multiplex Polymerase Chain Reaction/methods , Nasopharynx/virology , SARS-CoV-2/isolation & purification , Saliva/virology , Humans , Limit of Detection , Reproducibility of Results
3.
PLoS One ; 17(1): e0262258, 2022.
Article in English | MEDLINE | ID: covidwho-1606499

ABSTRACT

Although patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A, influenza B and respiratory syncytial virus (RSV) show comparable or very similar manifestations, the therapeutic approaches of these respiratory viral infections are different, which requires an accurate diagnosis. Recently, the novel multiplex real-time reverse transcription-polymerase chain reaction assay AMPLIQUICK® Respiratory Triplex (BioSynex SA, Illkirch-Graffenstaden, France) allows simultaneous detection and differentiation of SARS-CoV-2, influenza A, influenza B, and RSV in respiratory tract samples. We herein evaluated the performance of the AMPLIQUICK® Respiratory Triplex for the detection of the four viruses in respiratory specimens, using Allplex™ Respiratory Panel 1 and 2019-nCoV assays (Seegene, Seoul, Korea) as reference comparator assays. A total of 359 archived predetermined respiratory samples, including 83, 145, 19 and 95 positive specimens for SARS-CoV-2, influenza A, influenza B and RSV respectively, were included. The AMPLIQUICK® Respiratory Triplex showed high concordance with the reference assays, with an overall agreement for SARS-CoV-2, influenza A, influenza B, and RSV at 97.6%, 98.8%, 98.3% and 100.0%, respectively, and high κ values ranging from 0.93 to 1.00, indicating an almost perfect agreement between assays. Furthermore, high correlations of cycle threshold (Ct) values were observed for positive samples of the four viruses between the AMPLIQUICK® Respiratory Triplex and comparator assays, with an overall high agreement between Ct values assessed by Bland-Altman analyses. In conclusion, these observations demonstrate that the multiplex AMPLIQUICK® Respiratory Triplex is a reliable assay for the qualitative detection and differentiation of SARS-CoV-2, influenza A, influenza B, and RSV in respiratory specimens, which may prove useful for streamlining diagnostics during the winter influenza-seasons.


Subject(s)
COVID-19/diagnosis , Influenza, Human/diagnosis , Multiplex Polymerase Chain Reaction/methods , Real-Time Polymerase Chain Reaction/methods , Respiratory Syncytial Virus Infections/diagnosis , COVID-19/virology , Humans , Influenza, Human/virology , Molecular Diagnostic Techniques , Nasopharynx/virology , Respiratory Syncytial Virus Infections/virology , Retrospective Studies , Sensitivity and Specificity
4.
Microbiol Spectr ; 9(3): e0069521, 2021 12 22.
Article in English | MEDLINE | ID: covidwho-1597074

ABSTRACT

Bacterial pneumonia is a challenging coronavirus disease 2019 (COVID-19) complication for intensive care unit (ICU) clinicians. Upon its implementation, the FilmArray pneumonia plus (FA-PP) panel's practicability for both the diagnosis and antimicrobial therapy management of bacterial pneumonia was assessed in ICU patients with COVID-19. Respiratory samples were collected from patients who were mechanically ventilated at the time bacterial etiology and antimicrobial resistance were determined using both standard-of-care (culture and antimicrobial susceptibility testing [AST]) and FA-PP panel testing methods. Changes to targeted and/or appropriate antimicrobial therapy were reviewed. We tested 212 samples from 150 patients suspected of bacterial pneumonia. Etiologically, 120 samples were positive by both methods, two samples were culture positive but FA-PP negative (i.e., negative for on-panel organisms), and 90 were negative by both methods. FA-PP detected no culture-growing organisms (mostly Staphylococcus aureus or Pseudomonas aeruginosa) in 19 of 120 samples or antimicrobial resistance genes in two culture-negative samples for S. aureus organisms. Fifty-nine (27.8%) of 212 samples were from empirically treated patients. Antibiotics were discontinued in 5 (33.3%) of 15 patients with FA-PP-negative samples and were escalated/deescalated in 39 (88.6%) of 44 patients with FA-PP-positive samples. Overall, antibiotics were initiated in 87 (72.5%) of 120 pneumonia episodes and were not administered in 80 (87.0%) of 92 nonpneumonia episodes. Antimicrobial-resistant organisms caused 78 (60.0%) of 120 episodes. Excluding 19 colistin-resistant Acinetobacter baumannii episodes, AST confirmed appropriate antibiotic receipt in 101 (84.2%) of 120 episodes for one or more FA-PP-detected organisms. Compared to standard-of-care testing, the FA-PP panel may be of great value in the management of COVID-19 patients at risk of developing bacterial pneumonia in the ICU. IMPORTANCE Since bacterial pneumonia is relatively frequent, suspicion of it in COVID-19 patients may prompt ICU clinicians to overuse (broad-spectrum) antibiotics, particularly when empirical antibiotics do not cover the suspected pathogen. We showed that a PCR-based, culture-independent laboratory assay allows not only accurate diagnosis but also streamlining of antimicrobial therapy for bacterial pneumonia episodes. We report on the actual implementation of rapid diagnostics and its real-life impact on patient treatment, which is a gain over previously published studies on the topic. A better understanding of the role of that or similar PCR assays in routine ICU practice may lead us to appreciate the effectiveness of their implementation during the COVID-19 pandemic.


Subject(s)
COVID-19/complications , Hospitals , Multiplex Polymerase Chain Reaction/methods , Pneumonia, Bacterial/diagnosis , Pneumonia, Bacterial/drug therapy , Aged , Anti-Bacterial Agents/therapeutic use , Bacteria/genetics , COVID-19/diagnosis , COVID-19 Testing/methods , Critical Illness , Female , Humans , Intensive Care Units , Male , Middle Aged , Pandemics , Patient Acuity , Pneumonia, Bacterial/microbiology , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification
5.
Aging (Albany NY) ; 13(23): 24931-24942, 2021 12 12.
Article in English | MEDLINE | ID: covidwho-1573020

ABSTRACT

Since the Coronavirus 19 (COVID-19) pandemic, several SARS-CoV-2 variants of concern (SARS-CoV-2 VOC) have been reported. The B.1.1.7 variant has been associated with increased mortality and transmission risk. Furthermore, cluster and possible co-infection cases could occur in the next influenza season or COVID-19 pandemic wave, warranting efficient diagnosis and treatment decision making. Here, we aimed to detect SARS-CoV-2 and other common respiratory viruses using multiplex RT-PCR developed on the LabTurbo AIO 48 open system. We performed a multicenter study to evaluate the performance and analytical sensitivity of the LabTurbo AIO 48 system for SARS-CoV-2, influenza A/B, and respiratory syncytial virus (RSV) using 652 nasopharyngeal swab clinical samples from patients. The LabTurbo AIO 48 system demonstrated a sensitivity of 9.4 copies/per PCR for N2 of SARS-CoV-2; 24 copies/per PCR for M of influenza A and B; and 24 copies/per PCR for N of RSV. The assay presented consistent performance in the multicenter study. The multiplex RT-PCR applied on the LabTurbo AIO 48 open platform provided highly sensitive, robust, and accurate results and enabled high-throughput detection of B.1.1.7, influenza A/B, and RSV with short turnaround times. Therefore, this automated molecular diagnostic assay could enable streamlined testing if COVID-19 becomes a seasonal disease.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , Influenza, Human/diagnosis , Multiplex Polymerase Chain Reaction/methods , Respiratory Syncytial Virus Infections/diagnosis , Adult , Aged , COVID-19/virology , Female , Humans , Influenza A virus/genetics , Influenza A virus/isolation & purification , Influenza, Human/virology , Influenzavirus B/genetics , Influenzavirus B/isolation & purification , Male , Middle Aged , Respiratory Syncytial Virus Infections/virology , Respiratory Syncytial Viruses/genetics , Respiratory Syncytial Viruses/isolation & purification , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Sensitivity and Specificity , Young Adult
6.
J Korean Med Sci ; 36(48): e328, 2021 Dec 13.
Article in English | MEDLINE | ID: covidwho-1572278

ABSTRACT

BACKGROUND: In the coronavirus disease 2019 (COVID-19) pandemic era, the simultaneous detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza virus (Flu), and respiratory syncytial virus (RSV) is important in the rapid differential diagnosis in patients with respiratory symptoms. Three multiplex real-time reverse transcription polymerase chain reaction (rRT-PCR) assays have been recently developed commercially in Korea: PowerChek™ SARS-CoV-2, Influenza A&B Multiplex Real-time PCR Kit (PowerChek; KogeneBiotech); STANDARD™ M Flu/SARS-CoV-2 Real-time Detection Kit (STANDARD M; SD BioSensor); and Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay (Allplex; Seegene). We evaluated the analytical and clinical performances of these kits. METHODS: A limit of detection tests were performed and cross-reactivity analysis was executed using clinical respiratory samples. Ninety-seven SARS-CoV-2-positive, 201 SARS-CoV-2-negative, 71 influenza A-positive, 50 influenza B-positive, 78 RSV-positive, and 207 other respiratory virus-positive nasopharyngeal swabs were tested using the three assays. The AdvanSure™ respiratory viruses rRT-PCR assay (AdvanSure; LG Life Sciences) was used as a comparator assay for RSV. RESULTS: Except in influenza B, in SARS-CoV-2 and influenza A, there were no significant differences in detecting specific genes of the viruses among the three assays. All three kits did not cross-react with common respiratory viruses. All three kits had greater than 92% positive percent agreement and negative percent agreement and ≥ 0.95 kappa value in the detection of SARS-CoV-2 and flu A/B. Allplex detected RSV more sensitively than AdvanSure. CONCLUSION: The overall performance of three multiplex rRT-PCR assays for the concurrent detection of SARS-CoV-2, influenza A/B, and RSV was comparable. These kits will promote prompt differential diagnosis of COVID-19, influenza, and RSV infection in the COVID-19 pandemic era.


Subject(s)
COVID-19/diagnosis , Influenza, Human/diagnosis , Multiplex Polymerase Chain Reaction/methods , Nasopharynx/virology , RNA, Viral/analysis , Respiratory Syncytial Virus Infections/diagnosis , COVID-19/virology , Cross Reactions , Humans , Influenza A virus/genetics , Influenza A virus/isolation & purification , Influenza B virus/genetics , Influenza B virus/isolation & purification , Influenza, Human/virology , Limit of Detection , Nucleocapsid Proteins/genetics , Polyproteins/genetics , RNA, Viral/metabolism , Reagent Kits, Diagnostic , Republic of Korea , Respiratory Syncytial Virus Infections/virology , Respiratory Syncytial Virus, Human/genetics , Respiratory Syncytial Virus, Human/isolation & purification , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Viral Matrix Proteins/genetics , Viral Proteins/genetics
7.
Microbiol Spectr ; 9(3): e0126721, 2021 12 22.
Article in English | MEDLINE | ID: covidwho-1522928

ABSTRACT

The objective of this study was to construct a novel strategy for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants using multiplex PCR-mass spectrometry minisequencing technique (mPCR-MS minisequencing). Using the nucleic acid sequence of a SARS-CoV-2 nonvariant and a synthetic SARS-CoV-2 variant-carrying plasmid, a matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) method based on the single-base mass probe extension of multiplex PCR amplification products was established to detect 9 mutation types in 7 mutated sites (HV6970del, N501Y, K417N, P681H, D614G, E484K, L452R, E484Q, and P681R) in the receptor-binding domain of the spike protein of SARS-CoV-2 variants. Twenty-one respiratory tract pathogens (9 bacteria and 12 respiratory viruses) and nucleic acid samples from non-COVID-19 patients were selected for specific validation. Twenty samples from COVID-19 patients were used to verify the accuracy of this method. The 9 mutation types could be detected simultaneously by triple PCR amplification coupled with MALDI-TOF MS. SARS-CoV-2 and six variants, B.1.1.7 (Alpha), B.1.351 (Beta), B.1.429 (Epsilon), B.1.526 (Iota), P.1 (Gamma) and B.1.617.2 (Delta), could be identified. The detection limit for all 9 sites was 1.5 × 103 copies. The specificity of this method was 100%, and the accuracy of real-time PCR cycle threshold (CT) values less than 27 among positive samples was 100%. This method is open and extensible, and can be used in a high-throughput manner, easily allowing the addition of new mutation sites as needed to identify and track new SARS-CoV-2 variants as they emerge. mPCR-MS minisequencing provides a new detection option with practical application value for SARS-CoV-2 and its variant infection. IMPORTANCE The emergence of SARS-CoV-2 variants is the key factor in the second wave of the COVID-19 pandemic. An all-in-one SARS-CoV-2 variant identification method based on a multiplex PCR-mass spectrometry minisequencing system was developed in this study. Six SARS-CoV-2 variants (Alpha, Beta, Epsilon, Iota, Gamma, and Delta) can be identified simultaneously. This method can not only achieve the multisite simultaneous detection that cannot be realized by PCR coupled with first-generation sequencing technology and quantitative PCR (qPCR) technology but also avoid the shortcomings of time-consuming, high-cost, and high technical requirements of whole-genome sequencing technology. As a simple screening assay for monitoring the emergence and spread of SARS-CoV-2 and variants, mPCR-MS minisequencing is expected to play an important role in the detection and monitoring of SARS-CoV-2 infection as a supplementary technology.


Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , Mass Spectrometry/methods , Multiplex Polymerase Chain Reaction/methods , SARS-CoV-2/isolation & purification , Base Sequence , Humans , Mutation , Polymorphism, Single Nucleotide , Protein Binding , Real-Time Polymerase Chain Reaction , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/isolation & purification , Whole Genome Sequencing
8.
Sci Rep ; 11(1): 20494, 2021 10 14.
Article in English | MEDLINE | ID: covidwho-1469989

ABSTRACT

The emergence of a novel SARS-CoV-2 B.1.1.7 variant sparked global alarm due to increased transmissibility, mortality, and uncertainty about vaccine efficacy, thus accelerating efforts to detect and track the variant. Current approaches to detect B.1.1.7 include sequencing and RT-qPCR tests containing a target assay that fails or results in reduced sensitivity towards the B.1.1.7 variant. Since many countries lack genomic surveillance programs and failed assays detect unrelated variants containing similar mutations as B.1.1.7, we used allele-specific PCR, and judicious placement of LNA-modified nucleotides to develop an RT-qPCR test that accurately and rapidly differentiates B.1.1.7 from other SARS-CoV-2 variants. We validated the test on 106 clinical samples with lineage status confirmed by sequencing and conducted a country-wide surveillance study of B.1.1.7 prevalence in Slovakia. Our multiplexed RT-qPCR test showed 97% clinical sensitivity and retesting 6,886 SARS-CoV-2 positive samples obtained during three campaigns performed within one month, revealed pervasive spread of B.1.1.7 with an average prevalence of 82%. Labs can easily implement this test to rapidly scale B.1.1.7 surveillance efforts and it is particularly useful in countries with high prevalence of variants possessing only the ΔH69/ΔV70 deletion because current strategies using target failure assays incorrectly identify these as putative B.1.1.7 variants.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , COVID-19/virology , Multiplex Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , Alleles , COVID-19/epidemiology , Humans , Mutation , Prevalence , RNA, Viral/genetics , SARS-CoV-2/isolation & purification , Slovakia/epidemiology
9.
Viruses ; 13(10)2021 10 08.
Article in English | MEDLINE | ID: covidwho-1463840

ABSTRACT

The emergence of variants of SARS-CoV-2 has created challenges for the testing infrastructure. Although large-scale genome sequencing of SARS-CoV-2 has facilitated hospital and public health responses, access to sequencing facilities globally is variable and turnaround times can be significant, so there is a requirement for rapid and cost-effective alternatives. Applying a polymerase chain reaction (PCR)-based single nucleotide polymorphism (SNP) approach enables rapid (<4 h) identification of SARS-CoV-2 lineages from nucleic acid extracts, through the presence or absence of a panel of defined of genomic polymorphisms. For example, the B.1.1.7 lineage ("UK", "Alpha", or "Kent" variant) is characterised by 23 mutations compared to the reference strain, and the most biologically significant of these are found in the S gene. We have developed a SARS-CoV-2 typing assay focused on five positions in the S gene (HV69/70, N501, K417, E484 and P681). This configuration can identify a range of variants, including all the "Variants of Concern" currently designated by national and international public health bodies. The panel has been evaluated using a range of clinical isolates and standardised control materials at four UK hospitals and shows excellent concordance with the known lineage information derived from full sequence analysis. The assay has a turnaround time of about three hours for a set of up to 24 samples and has been utilised to identify emerging variants in a clinical setting.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , Multiplex Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Genome, Viral/genetics , Humans , Nucleic Acid Amplification Techniques/methods , Polymorphism, Single Nucleotide/genetics , Sensitivity and Specificity , Spike Glycoprotein, Coronavirus/genetics , Whole Genome Sequencing/methods
10.
J Med Virol ; 94(1): 327-334, 2022 01.
Article in English | MEDLINE | ID: covidwho-1410052

ABSTRACT

Genomic surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays an important role in COVID-19 pandemic control and elimination efforts, especially by elucidating its global transmission network and illustrating its viral evolution. The deployment of multiplex PCR assays that target SARS-CoV-2 followed by either massively parallel or nanopore sequencing is a widely-used strategy to obtain genome sequences from primary samples. However, multiplex PCR-based sequencing carries an inherent bias of sequencing depth among different amplicons, which may cause uneven coverage. Here we developed a two-pool, long-amplicon 36-plex PCR primer panel with ~1000-bp amplicon lengths for full-genome sequencing of SARS-CoV-2. We validated the panel by assessing nasopharyngeal swab samples with a <30 quantitative reverse transcription PCR cycle threshold value and found that ≥90% of viral genomes could be covered with high sequencing depths (≥20% mean depth). In comparison, the widely-used ARTIC panel yielded 79%-88% high-depth genome regions. We estimated that ~5 Mbp nanopore sequencing data may ensure a >95% viral genome coverage with a ≥10-fold depth and may generate reliable genomes at consensus sequence levels. Nanopore sequencing yielded false-positive variations with frequencies of supporting reads <0.8, and the sequencing errors mostly occurred on the 5' or 3' ends of reads. Thus, nanopore sequencing could not elucidate intra-host viral diversity.


Subject(s)
Genome, Viral/genetics , Multiplex Polymerase Chain Reaction/methods , Nanopore Sequencing/methods , SARS-CoV-2/genetics , Whole Genome Sequencing/methods , COVID-19 , High-Throughput Nucleotide Sequencing/methods , Humans , Nasopharynx/virology , RNA, Viral/genetics , Sequence Analysis, RNA/methods
11.
Sci Rep ; 11(1): 18126, 2021 09 13.
Article in English | MEDLINE | ID: covidwho-1406407

ABSTRACT

COVID-19 has emerged as global pandemic with largest damage to the public health, economy and human psyche.The genome sequence data obtained during the ongoing pandemic are valuable to understand the virus evolutionary patterns and spread across the globe. Increased availability of genome information of circulating SARS-CoV-2 strains in India will enable the scientific community to understand the emergence of new variants and their impact on human health. The first case of COVID-19 was detected in Chambal region of Madhya Pradesh state in mid of March 2020 followed by multiple introduction events and expansion of cases within next three months. More than 5000 COVID-19 suspected samples referred to Defence Research and Development Establishment, Gwalior, Madhya Pradesh were analyzed during the nation -wide lockdown and unlock period. A total of 136 cases were found positive over a span of three months that included virus introduction to the region and its further spread. Whole genome sequences employing Oxford nanopore technology were generated for 26 SARS-CoV-2 circulating in 10 different districts in Madhya Pradesh state of India. This period witnessed index cases with multiple travel histories responsible for introduction of COVID-19 followed by remarkable expansion of virus. The genome wide substitutions including in important viral proteins were identified. The detailed phylogenetic analysis revealed the circulating SARS-CoV-2 clustered in multiple clades including A2a, A4 and B. The cluster-wise segregation was observed, suggesting multiple introduction links and subsequent evolution of virus in the region. This is the first comprehensive whole genome sequence analysis from central India, which revealed the emergence and evolution of SARS-CoV-2 during thenation-wide lockdown and unlock.


Subject(s)
COVID-19/diagnosis , Mutation, Missense , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , COVID-19/epidemiology , COVID-19/virology , Evolution, Molecular , Genome, Viral/genetics , India , Multiplex Polymerase Chain Reaction/methods , Pandemics/prevention & control , Phylogeny , SARS-CoV-2/classification , SARS-CoV-2/physiology , Whole Genome Sequencing/methods
12.
J Mol Diagn ; 23(9): 1078-1084, 2021 09.
Article in English | MEDLINE | ID: covidwho-1386076

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is highly contagious and has caused significant medical/socioeconomic impacts. Other than vaccination, effective public health measures, including contact tracing, isolation, and quarantine, is critical for deterring viral transmission, preventing infection progression and resuming normal activities. Viral transmission is affected by many factors, but the viral load and vitality could be among the most important ones. Although in vitro studies have indicated that the amount of virus isolated from infected individuals affects the successful rate of virus isolation, whether the viral load carried at the individual level would determine the transmissibility was unknown. We examined whether the cycle threshold (Ct) value, a measurement of viral load by RT-PCR assay, could differentiate the spreaders from the non-spreaders in a population of college students. Our results indicate that while at the population level the Ct value is lower, suggesting a higher viral load, in the symptomatic spreaders than that in the asymptomatic non-spreaders, there is a significant overlap in the Ct values between the two groups. Thus, Ct value, or the viral load, at the individual level could not predict the transmissibility. Instead, a sensitive method to detect the presence of virus is needed to identify asymptomatic individuals who may carry a low viral load but can still be infectious.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/transmission , COVID-19/virology , Multiplex Polymerase Chain Reaction/methods , Universities/statistics & numerical data , COVID-19/epidemiology , Carrier State/virology , Contact Tracing , Female , Humans , Louisiana/epidemiology , Male , Nasopharynx/virology , Public Health , Quarantine , Retrospective Studies , Students/statistics & numerical data , Viral Load , Young Adult
13.
Sci Rep ; 11(1): 16355, 2021 08 11.
Article in English | MEDLINE | ID: covidwho-1354115

ABSTRACT

Rapid diagnostic tests are tools of paramount impact both for improving patient care and in antimicrobial management programs. Particularly in the case of respiratory infections, it is of great importance to quickly confirm/exclude the involvement of pathogens, be they bacteria or viruses, while obtaining information about the presence/absence of a genetic target of resistance to modulate antibiotic therapy. In this paper, we present our experiences with the use of the Biofire® FilmArray® Pneumonia Panel Plus (FAPP; bioMérieux; Marcy l'Etoile, France) to assess coinfection in COVID-19 patients. A total of 152 respiratory samples from consecutive patients were examined, and 93 (61%) were found to be FAPP positive, with the detection of bacteria and/or viruses. The patients were 93 males and 59 females with an average age of 65 years who were admitted to our hospital due to moderate/severe acute respiratory symptoms. Among the positive samples were 52 from sputum (SPU) and 41 from bronchoalveolar lavage (BAL). The most representative species was S. aureus (most isolates were mecA positive; 30/44, 62%), followed by gram-negative pathogens such as P. aeruginosa, K. pneumoniae, and A. baumannii. Evidence of a virus was rare. Cultures performed from BAL and SPU samples gave poor results. Most of the discrepant negative cultures were those in which FAPP detected pathogens with a microbial count ≤ 105 CFU/mL. H. influenzae was one of the most common pathogens lost by the conventional method. Despite the potential limitations of FAPP, which detects a defined number of pathogens, its advantages of rapid detection combined with predictive information regarding the antimicrobial resistance of pathogens through the detection of some relevant markers of resistance could be very useful for establishing empirical targeted therapy for the treatment of patients with respiratory failure. In the COVID era, we understand the importance of using antibiotics wisely to curb the phenomenon of antibiotic resistance.


Subject(s)
COVID-19/complications , Coinfection , Diagnostic Tests, Routine , Respiratory Tract Infections/complications , Respiratory Tract Infections/diagnosis , Aged , Female , Humans , Male , Multiplex Polymerase Chain Reaction/methods , Respiratory Tract Infections/microbiology , Respiratory Tract Infections/virology
14.
J Mol Diagn ; 23(9): 1078-1084, 2021 09.
Article in English | MEDLINE | ID: covidwho-1349527

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is highly contagious and has caused significant medical/socioeconomic impacts. Other than vaccination, effective public health measures, including contact tracing, isolation, and quarantine, is critical for deterring viral transmission, preventing infection progression and resuming normal activities. Viral transmission is affected by many factors, but the viral load and vitality could be among the most important ones. Although in vitro studies have indicated that the amount of virus isolated from infected individuals affects the successful rate of virus isolation, whether the viral load carried at the individual level would determine the transmissibility was unknown. We examined whether the cycle threshold (Ct) value, a measurement of viral load by RT-PCR assay, could differentiate the spreaders from the non-spreaders in a population of college students. Our results indicate that while at the population level the Ct value is lower, suggesting a higher viral load, in the symptomatic spreaders than that in the asymptomatic non-spreaders, there is a significant overlap in the Ct values between the two groups. Thus, Ct value, or the viral load, at the individual level could not predict the transmissibility. Instead, a sensitive method to detect the presence of virus is needed to identify asymptomatic individuals who may carry a low viral load but can still be infectious.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/transmission , COVID-19/virology , Multiplex Polymerase Chain Reaction/methods , Universities/statistics & numerical data , COVID-19/epidemiology , Carrier State/virology , Contact Tracing , Female , Humans , Louisiana/epidemiology , Male , Nasopharynx/virology , Public Health , Quarantine , Retrospective Studies , Students/statistics & numerical data , Viral Load , Young Adult
15.
Trop Biomed ; 38(3): 283-288, 2021 Sep 01.
Article in English | MEDLINE | ID: covidwho-1346923

ABSTRACT

Various methods have been developed for rapid and high throughput full genome sequencing of SARS-CoV-2. Here, we described a protocol for targeted multiplex full genome sequencing of SARS-CoV-2 genomic RNA directly extracted from human nasopharyngeal swabs using the Ion Personal Genome Machine (PGM). This protocol involves concomitant amplification of 237 gene fragments encompassing the SARS-CoV-2 genome to increase the abundance and yield of viral specific sequencing reads. Five complete and one near-complete genome sequences of SARS-CoV-2 were generated with a single Ion PGM sequencing run. The sequence coverage analysis revealed two amplicons (positions 13 751-13 965 and 23 941-24 106), which consistently gave low sequencing read coverage in all isolates except 4Apr20-64- Hu. We analyzed the potential primer binding sites within these low covered regions and noted that the 4Apr20-64-Hu possess C at positions 13 730 and 23 929, whereas the other isolates possess T at these positions. The genome nucleotide variations observed suggest that the naturally occurring variations present in the actively circulating SARS-CoV-2 strains affected the performance of the target enrichment panel of the Ion AmpliSeq™ SARS CoV 2 Research Panel. The possible impact of other genome nucleotide variations warrants further investigation, and an improved version of the Ion AmpliSeq™ SARS CoV 2 Research Panel, hence, should be considered.


Subject(s)
Genome, Viral , High-Throughput Nucleotide Sequencing , Multiplex Polymerase Chain Reaction , SARS-CoV-2/genetics , Whole Genome Sequencing , Base Sequence , COVID-19 , High-Throughput Nucleotide Sequencing/methods , Humans , Multiplex Polymerase Chain Reaction/methods , Whole Genome Sequencing/methods
16.
ACS Appl Mater Interfaces ; 13(26): 30295-30305, 2021 Jul 07.
Article in English | MEDLINE | ID: covidwho-1337092

ABSTRACT

As viruses have been threatening global public health, fast diagnosis has been critical to effective disease management and control. Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) is now widely used as the gold standard for detecting viruses. Although a multiplex assay is essential for identifying virus types and subtypes, the poor multiplicity of RT-qPCR makes it laborious and time-consuming. In this paper, we describe the development of a multiplex RT-qPCR platform with hydrogel microparticles acting as independent reactors in a single reaction. To build target-specific particles, target-specific primers and probes are integrated into the particles in the form of noncovalent composites with boron nitride nanotubes (BNNTs) and carbon nanotubes (CNTs). The thermal release characteristics of DNA, primer, and probe from the composites of primer-BNNT and probe-CNT allow primer and probe to be stored in particles during particle production and to be delivered into the reaction. In addition, BNNT did not absorb but preserved the fluorescent signal, while CNT protected the fluorophore of the probe from the free radicals present during particle production. Bicompartmental primer-incorporated network (bcPIN) particles were designed to harness the distinctive properties of two nanomaterials. The bcPIN particles showed a high RT-qPCR efficiency of over 90% and effective suppression of non-specific reactions. 16-plex RT-qPCR has been achieved simply by recruiting differently coded bcPIN particles for each target. As a proof of concept, multiplex one-step RT-qPCR was successfully demonstrated with a simple reaction protocol.


Subject(s)
Hydrogels/chemistry , Multiplex Polymerase Chain Reaction/methods , Nanotubes, Carbon/chemistry , RNA, Viral/analysis , Reverse Transcriptase Polymerase Chain Reaction/methods , Boron Compounds/chemistry , Coronavirus/chemistry , DNA Primers/chemistry , DNA, Single-Stranded/chemistry , Fluorescent Dyes/chemistry , Graphite/chemistry , Influenza A virus/chemistry , Newcastle disease virus/chemistry , Proof of Concept Study , RNA, Viral/chemistry , Virus Diseases/diagnosis
17.
Curr Issues Mol Biol ; 43(2): 728-748, 2021 Jul 20.
Article in English | MEDLINE | ID: covidwho-1319597

ABSTRACT

The ongoing coronavirus disease (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a severe threat to human health and the global economy and has resulted in overwhelming stress on health care systems worldwide. Despite the global health catastrophe, especially in the number of infections and fatalities, the COVID-19 pandemic has also revolutionized research and discovery with remarkable success in diagnostics, treatments, and vaccine development. The use of many diagnostic methods has helped establish public health guidelines to mitigate the spread of COVID-19. However, limited information has been shared about these methods, and there is a need for the scientific community to learn about these technologies, in addition to their sensitivity, specificity, and limitations. This review article is focused on providing insights into the major methods used for SARS-CoV-2 detection. We describe in detail the core principle of each method, including molecular and serological approaches, along with reported claims about the rates of false negatives and false positives, the types of specimens needed, and the level of technology and the time required to perform each test. Although this study will not rank or prioritize these methods, the information will help in the development of guidelines and diagnostic protocols in clinical settings and reference laboratories.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19 Serological Testing/methods , Clustered Regularly Interspaced Short Palindromic Repeats , Enzyme-Linked Immunosorbent Assay/methods , Gold Colloid , High-Throughput Nucleotide Sequencing/methods , Humans , Immunoassay/methods , Molecular Diagnostic Techniques/methods , Multiplex Polymerase Chain Reaction/methods , Nucleic Acid Amplification Techniques/methods , Real-Time Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification
18.
Arch Virol ; 166(9): 2551-2561, 2021 Sep.
Article in English | MEDLINE | ID: covidwho-1309044

ABSTRACT

The aim of this study was to identify and validate a sensitive, high-throughput, and cost-effective SARS-CoV-2 real-time RT-PCR assay to be used as a surveillance and diagnostic tool for SARS-CoV-2 in a university surveillance program. We conducted a side-by-side clinical evaluation of a newly developed SARS-CoV-2 multiplex assay (EZ-SARS-CoV-2 Real-Time RT-PCR) with the commercial TaqPath COVID-19 Combo Kit, which has an Emergency Use Authorization from the FDA. The EZ-SARS-CoV-2 RT-PCR incorporates two assays targeting the SARS-CoV-2 N gene, an internal control targeting the human RNase P gene, and a PCR inhibition control in a single reaction. Nasopharyngeal (NP) and anterior nares (AN) swabs were tested as individuals and pools with both assays and in the ABI 7500 Fast and the QuantStudio 5 detection platforms. The analytical sensitivity of the EZ-SARS-CoV-2 RT-PCR assay was 250 copies/ml or approximately 1.75 genome copy equivalents per reaction. The clinical performance of the EZ-SARS-CoV-2 assay was evaluated using NP and AN samples tested in other laboratories. The diagnostic sensitivity of the assay ranged between 94 and 96% across the detection platforms, and the diagnostic specificity was 94.06%. The positive predictive value was 94%, and the negative predictive value ranged from 94 to 96%. Pooling five NP or AN specimens yielded 93% diagnostic sensitivity. The overall agreement between these SARS-CoV-2 RT-PCR assays was high, supported by a Cohen's kappa value of 0.93. The EZ-SARS-CoV-2 RT-PCR assay performance attributes of high sensitivity and specificity with AN sample matrix and pooled upper respiratory samples support its use in a high-throughput surveillance testing program.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , Coronavirus Nucleocapsid Proteins/genetics , Multiplex Polymerase Chain Reaction/methods , RNA, Viral/genetics , SARS-CoV-2/genetics , COVID-19/epidemiology , COVID-19/virology , COVID-19 Nucleic Acid Testing/economics , COVID-19 Nucleic Acid Testing/instrumentation , Epidemiological Monitoring , Gene Expression , Humans , Multiplex Polymerase Chain Reaction/economics , Multiplex Polymerase Chain Reaction/instrumentation , Nasal Cavity/virology , Nasopharynx/virology , Phosphoproteins/genetics , Reproducibility of Results , Sensitivity and Specificity , Specimen Handling/methods , Viral Load
19.
Int J Infect Dis ; 107: 179-181, 2021 Jun.
Article in English | MEDLINE | ID: covidwho-1300799

ABSTRACT

OBJECTIVE: The aim of this study was to evaluate the QIAstat-Dx® Respiratory SARS-CoV-2 Panel (QIAstat-SARS-CoV-2), which is a closed, fully automated, multiplex polymerase chain reaction (PCR) assay that detects severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and 21 other pathogens that cause respiratory disease. METHODS: Nasopharyngeal swabs from patients with or suspected of having coronavirus disease 2019 were collected and tested at Bichat-Claude Bernard Hospital, Paris, France. Using the World Health Organisation-approved real-time-PCR assay developed by the Charité Institute of Virology as the reference, positive percent agreement (PPA) and negative percent agreement (NPA) were calculated. RESULTS: In total, 189 negative and 88 positive samples were analyzed. QIAstat-SARS-CoV-2 had an NPA of 90.48% (95% confidence interval (CI), 85.37%, 94.26%) and a PPA of 94.32% (95% CI, 87.24%, 98.13%). Co-infections were detected by QIAstat-SARS-CoV-2 in 4/277 specimens. The methods exhibited comparable failure rates (23/307 [7.5%] vs. 6/298 [2.0%] for QIAstat-SARS-CoV-2 and reference methods, respectively). The turnaround time was shorter for QIAstat-SARS-CoV-2 compared with the reference method (difference in mean -14:30 h [standard error, 0:03:23; 95% CI, -14:37, -14:24]; P < 0.001). CONCLUSIONS: QIAstat-SARS-CoV-2 shows good agreement with the reference assay, providing faster and accurate results for detecting SARS-CoV-2.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , Multiplex Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , Adolescent , Adult , Aged , Aged, 80 and over , Child , Child, Preschool , Female , Humans , Infant , Male , Middle Aged , Nasopharynx/virology , Retrospective Studies , Young Adult
20.
Mol Cell Probes ; 58: 101744, 2021 08.
Article in English | MEDLINE | ID: covidwho-1253367

ABSTRACT

To increase the repertoire of PCR based laboratory developed tests (LDTs) for the detection of SARS-CoV-2, we describe a new multiplex assay (SORP), targeting the SARS-CoV-2's, Spike and ORF8 genes. The widely used human RNaseP internal control was modified to specifically co-amplify the RNaseP mRNA. The SORP triplex assay was tested on a cohort (n = 372; POS = 144/NEG = 228) of nasopharyngeal flocked swab (NPFS) specimens, previously tested for the presence of SARS-CoV-2 using a PCR assay targeting E and RdRp genes. The overall sensitivity and specificity of the SORP assay was: 99.31% (95% CI: 96.22-99.98%), 100.0% (95% CI: 98.4-100%) respectively. The SORP assay could also detect a panel of variants of concern (VOC) from the B1.1.7 (UK) and B1.351 (SA) lineage. In summary, access to a repertoire of new SARS-CoV-2 LDT's would assist diagnostic laboratories in developing strategies to overcome some of the testing issues encountered during high-throughput SARS-CoV-2 testing.


Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , Clinical Laboratory Techniques/methods , Multiplex Polymerase Chain Reaction/methods , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , COVID-19/virology , DNA Primers/genetics , DNA Probes/genetics , Humans , Molecular Diagnostic Techniques/methods , Reproducibility of Results , Ribonuclease P/genetics , SARS-CoV-2/physiology , Sensitivity and Specificity , Spike Glycoprotein, Coronavirus/genetics , Viral Proteins/genetics
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