ABSTRACT
OBJECTIVE: With the current SARS-CoV2 outbreak, countless tests need to be performed on potential symptomatic individuals, contacts and travellers. The gold standard is a quantitative polymerase chain reaction (qPCR)-based system taking several hours to confirm positivity. For effective public health containment measures, this time span is too long. We therefore evaluated a rapid test in a high-prevalence community setting. STUDY DESIGN: Thirty-nine randomly selected individuals at a COVID-19 screening centre were simultaneously tested via qPCR and a rapid test. Ten previously diagnosed individuals with known SARS-CoV-2 infection were also analysed. METHODS: The evaluated rapid test is an IgG/IgM-based test for SARS-CoV-2 with a time to result of 20 min. Two drops of blood are needed for the test performance. RESULTS: Of 49 individuals, 22 tested positive by repeated qPCR. In contrast, the rapid test detected only eight of those positive correctly (sensitivity: 36.4%). Of the 27 qPCR-negative individuals, 24 were detected correctly (specificity: 88.9%). CONCLUSION: Given the low sensitivity, we recommend not to rely on an antibody-based rapid test for public health measures such as community screenings.
Subject(s)
Betacoronavirus/isolation & purification , Clinical Laboratory Techniques/standards , Community Health Services , Coronavirus Infections/diagnosis , Disease Outbreaks , Mass Screening/standards , Pneumonia, Viral/diagnosis , Point-of-Care Testing , Adult , Aged , COVID-19 , COVID-19 Testing , Coronavirus Infections/epidemiology , Female , Humans , Male , Mass Screening/methods , Middle Aged , Pandemics , Pneumonia, Viral/epidemiology , Polymerase Chain Reaction , SARS-CoV-2 , Sensitivity and Specificity , Time FactorsABSTRACT
In response to a recent outbreak in China, detection assays for a novel avian influenza A(H7N9) virus need to be implemented in a large number of public health laboratories. Here we present real-time reverse-transcription polymerase chain reaction (RT-PCR) assays for specific detection of this virus, along with clinical validation data and biologically-safe positive controls.
Subject(s)
Influenza A virus/genetics , Influenza in Birds/virology , Influenza, Human/virology , Real-Time Polymerase Chain Reaction/methods , Animals , Birds/virology , China , Humans , Influenza A virus/isolation & purification , Influenza in Birds/transmission , Influenza, Human/diagnosisABSTRACT
We present a rigorously validated and highly sensitive confirmatory real-time RT-PCR assay (1A assay) that can be used in combination with the previously reported upE assay. Two additional RT-PCR assays for sequencing are described, targeting the RdRp gene (RdRpSeq assay) and N gene (NSeq assay), where an insertion/deletion polymorphism might exist among different hCoV-EMC strains. Finally, a simplified and biologically safe protocol for detection of antibody response by immunofluorescence microscopy was developed using convalescent patient serum.
Subject(s)
Coronavirus Infections/diagnosis , Coronavirus/isolation & purification , Reverse Transcriptase Polymerase Chain Reaction/methods , Coronavirus/classification , Coronavirus/genetics , Coronavirus Infections/virology , Fluorescent Antibody Technique , Germany , Humans , Laboratories/standards , Polymorphism, Restriction Fragment Length , RNA, Viral/blood , RNA, Viral/genetics , Sensitivity and Specificity , Sequence Analysis, DNA , Virology/methodsABSTRACT
We present two real-time reverse-transcription polymerase chain reaction assays for a novel human coronavirus (CoV), targeting regions upstream of the E gene (upE) or within open reading frame (ORF)1b, respectively. Sensitivity for upE is 3.4 copies per reaction (95% confidence interval (CI): 2.56.9 copies) or 291 copies/mL of sample. No cross-reactivity was observed with coronaviruses OC43, NL63, 229E, SARS-CoV, nor with 92 clinical specimens containing common human respiratory viruses. We recommend using upE for screening and ORF1b for confirmation.
Subject(s)
Coronavirus Infections/virology , Real-Time Polymerase Chain Reaction/methods , Severe acute respiratory syndrome-related coronavirus/genetics , Severe acute respiratory syndrome-related coronavirus/isolation & purification , Coronavirus 229E, Human/genetics , Coronavirus 229E, Human/isolation & purification , Coronavirus Infections/diagnosis , Coronavirus Infections/genetics , Coronavirus NL63, Human/genetics , Coronavirus NL63, Human/isolation & purification , Coronavirus OC43, Human/genetics , Coronavirus OC43, Human/isolation & purification , Humans , Open Reading Frames , Saudi Arabia , Sensitivity and Specificity , Travel , Viral Envelope Proteins , Viroporin ProteinsABSTRACT
The lantibiotic Pep5 is produced by Staphylococcus epidermidis 5. Pep5 production and producer immunity are associated with the 20-kb plasmid pED503. A 1.3-kb KpnI fragment of pED503, containing the Pep5 structural gene pepA, was subcloned into the Escherichia coli-Staphylococcus shuttle vector pCU1, and the recombinant plasmid pMR2 was transferred to the Pep5- and immunity-negative mutant S. epidermidis 5 Pep5- (devoid of pED503). This clone did not produce active Pep5 but showed the same degree of insensitivity towards Pep5 as did the wild-type strain. Sequencing of the 1.3-kb KpnI-fragment and analysis of mutants demonstrated the involvement of two genes in Pep5 immunity, the structural gene pepA itself and pepI, a short open reading frame upstream of pepA. To identify the 69-amino-acid pepI gene product, we constructed an E. coli maltose-binding protein-PepI fusion clone. The immunity peptide PepI was detected in the soluble and membrane fractions of the wild-type strain and the immune mutants (harboring the plasmids pMR2 and pMR11) by immunoblotting with anti-maltose-binding protein-PepI antiserum. Strains harboring either pepI without pepA or pepI with incomplete pepA were not immune and did not produce PepI. Washing the membrane with salts and EDTA reduced the amount of PepI in this fraction, and treatment with Triton X-100 almost completely removed the peptide. Furthermore, PepI was hydrolyzed by proteases added to osmotically stabilized protoplasts. This suggests that PepI is loosely attached to the outside of the cytoplasmic membrane. Proline uptake and efflux experiments with immune and nonimmune strains also indicated that PepI may act at the membrane site.
