Guidelines for Diagnostic Tests

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This page provides the complete Documentaion regarding Diagnostic tests used for COVID-19 caused by the recently emerged new strain of coronavirus, i.e. SARS-COV-2. Here, users can find possible Diagnostic tests, Collection and Storage of specimen, Procedure for Diagnosis and how it can be diagnosed with RT-PCR (which is currently in use).

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Diagnosis of COVID-19:

Coronavirus Diagnostic Tests:

Coronavirus infection symptoms include, fever, cough, sore throat, shortness of breath and headache. The suspected patient's nasal aspirate/tracheal aspirate sample, nose/throat swab sample, sputum and blood samples are collected for diagnostic testing [1]. Preliminary identification can be done by observing viral morphology through electron microscope [2]. Several nucleic acid amplification tests are then carried out for confirmatory diagnosis [3]. These tests are based on detection of unique viral nucleic acid sequences by techniques like RT-PCR (reverse transcription PCR), RT-LAMP (reverse transcription loop-mediated isothermal amplification), rRT-LAMP (real-time reverse transcription loop-mediated isothermal amplification) [4], RT-iiPCR (Reverse-transcription-insulated isothermal PCR), qRT-PCR (quantitative real-time reverse transcription PCR) and rtRT-PCR (real-time reverse transcription PCR) [2]. Serological tests can be done for diagnosis. These tests require paired serum samples in acute and convalescent phase from the patient [3]. Two ELISA (enzyme- linked immunosorbent assay) tests are then performed for detection of antibody against viral nucleocapsid protein and spike protein in the suspected patient and a confirmatory microneutralization assay is performed to measure the antibodies that can neutralize the virus [5]. Several immunological assays can be carried out for diagnostic purpose like immunofluorescence assay, direct fluorescent antibody assay, protein microarray, monoclonal antibody based rapid nucleocapsid protein based detection, microneutralization test (measure binding of antigen from virus and corresponding antibody), microneutralization ppNT (pseudo-particle neutralization) assay, etc. [2]. Whole genome sequencing (WGS) is yet another technique widely used for diagnostic purposes [6]. The data obtained from WGS can serves as an important tool to guide therapeutic intervention. Other tests like MCoV-MS (multiplexed CoV mass spectrometry) that uses MALDI-TOF MS system to accurately identify known human coronaviruses and provide phylogenetic evidences for newer emerging viruses can be used. Arch-shaped multiple-target sensor is a test method which can be used for detection of viruses since it helps in identification of pathogens in clinical samples [2].



Collection, transport and storage of 2019-nCoV specimen

Due to an outbreak of a coronavirus infection (by a virus named 2019-nCoV) in December 2019 in mainland China, quick collection, transport and storage of specimens from suspected patients becomes a priority. There are several guidelines available for specimen collection so that further downstream diagnostic processing could be performed. Nasopharyngeal and oropharyngeal specimens are collected using Dacron or polyester flocked swabs, transported to laboratory at 4 degree C and stored at 4 degree C for nearly 5 days or less and at -70 degree C for more than 5 days. Bronchoalveolar lavage specimen is collected in sterile container, transported to laboratory at 4 degree C and stored at 4 degree C for nearly 48 hours and at -70C degree for more than 48 hours. Endotracheal aspirate, nasopharyngeal aspirate or nasal wash specimens are collected in sterile containers, transported to laboratory at 4C degree and stored at 4C degree for approximately 48 hours and at -70C degree for more than 48 hours. Sputum specimen is collected in sterile container, transported to laboratory at 4C degree and stored at 4C degree for nearly 48 hours and at -70C degree for more than 48 hours. Tissue specimens from biopsy or autopsy including from lung are collected in sterile containers with saline, transported to laboratory at 4C degree and stored at 4C degree for nearly 24 hours and at -70C degree for more than 24 hours. Two samples of serum, one in acute phase other in convalescent phase are collected in serum separator tubes, transported to laboratory at 4C degree and stored at 4C degree for more than 5 days and at -70C degree for more than 5 days. Whole blood sample is collected in collection tube, transported to laboratory at 4C degree and stored at 4C degree for 5 days or less and at -70C degree for 5 days or more. Urine sample is collected in urine collection container, transported to laboratory at 4C degree and stored at 4C degree for 5 days or less and at - 70C degree for more than 5 days [3].



Diagnostic Test for 2019-nCoV:

For rapid detection of 2019-nCoV in suspected patients, several diagnostic tests are performed nowadays like RT-PCR (nucleic acid amplification), whole genome sequencing, serological, immunological, mass spectrometric and arch-shaped multiple-target sensor. RT- PCR is by far the most commonly and widely used method. The tests are described as follows:

1. RT-PCR

Using lower respiratory tract and upper respiratory tract samples from 2019-nCoV suspected human cases. Two monoplex real time RT-PCR assays are performed targeting ORF1b gene and N gene of 2019-nCoV genome (Accession no.: MN908947), or E gene and RdRp (RNA dependent RNA polymerase) gene. N gene RT-PCR is recommended as screening assay and Orf1b is recommended as confirmatory one. However further validation suggested that N gene assay performed well but was slightly less sensitive [7]. Hence, E gene assay was later recommended as first-line screening tool followed by confirmatory testing with RdRp gene [2].

Procedure for performing RT-PCR assays:

a) Viral RNA is extracted from clinical specimens. b) Master-mix is prepared for carrying out RT-PCR by taking 8.5 µl of RNase free H 2 O, 5 µl of 4x reaction mix (TaqMan Fast Virus Master mix), 1 µl of forward primer (10 µM), 1 µl of reverse primer (10 µM), 0.5 µl of probe (10 µM) and 4 µl of RNA sample. c) Thermal cycler is set at 50°C for minutes for reverse transcription and at 95°C for 20 seconds for inactivation of reverse transcriptase, 40 cycles of PCR amplification can be set at 95°C for seconds for denaturation and at 60°C for 3 seconds for annealing. Each PCR cycle is set to run for 1 hour and 15 minutes [8]. In case of ORf1b gene and N gene RT-PCR assays, SARS-CoV is used as a positive control to perform the assay. As SARS was eliminated from human population, suspected cases that are showing positive results are considered infected with 2019-nCoV [9]. Sequencing is further performed to help in confirming and distinguishing SARS-CoV and 2019-nCoV.

2. Whole Genome sequencing

Is carried out using lower respiratory tract and upper respiratory tract samples.

3. Serological testing

Is performed using paired serum samples. The first sample is collected during first week of illness. The second sample is collected during third to fourth week of illness. However, if only one sample can be collected, the sample is tried to be collected at least three weeks after the onset of symptoms [3]. In case of 2019-nCoV, serology can be used for diagnostic purpose when 2019-nCoV antigens and their respective monoclonal antibodies will be developed [2].

4. Immunological Testing

Microneutralization test can be performed for diagnosis. It is rapid and easy to operate but have lower sensitivity and specificity [2].

5. MCoV-MS

It can be carried out to provide phylogenetic evidences for newer emerging viruses like human coronaviruses (hCoVs) [2].

6. Arch-shaped multiple-target sensor

It can be used for detection of hCoVs [2].

References:

  1. Coronavirus testing. Medline Plus, 2020.
  2. Yu F, Du L, Ojcius DM, et al., Measures for diagnosis and treating infections by a novel coronavirus responsible for a pneumonia outbreak originating in Wuhan, China. Microbes Infect, 2020.
  3. Laboratory testing for 2019 novel coronavirus (2019-CoV) in suspected human cases. World Health Organization, 2020.
  4. Zhang N, Wang L, Deng X, et al., Recent advances in the detection of respiratory virus infection in humans. Journal of Medical Virology, 2020.
  5. CDC laboratory testing for Middle East Respiratory Syndrome Coronavirus (MERS-CoV). Centre for disease control and prevention, 2019.
  6. Palacios G, Druce J, Du L, et al., A new Arenavirus in a cluster of fatal transplant-associated diseases. The New England Journal of Medicine, 2008.
  7. Corman VM, Landt O, Kaiser M, et al., Detection of novel coronavirus (2019-nCoV) by Real time RT-PCR. Euro Surveill, 2020.
  8. Chu DKW, Pan Y, Cheng SMS, et al., Molecular Diagnosis of a Novel Coronavirus (2019-nCoV) Causing an Outbreak of Pneumonia. Clinical Chemistry, 2020.
  9. Detection of 2019 novel coronavirus (2019-nCoV) in suspected human cases by RT-PCR. HKU Med, 2020.

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