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Table of Contents
Year : 2020  |  Volume : 2  |  Issue : 1  |  Page : 3-8

Unveiling the novel coronavirus in the laboratory

1 Clinical Microbiologist, Department of Laboratory Medicine, MIOT International, Chennai, Tamil Nadu, India
2 Director of Laboratory Medicine, Department of Laboratory Medicine, MIOT International, Chennai, Tamil Nadu, India

Date of Submission20-Jun-2020
Date of Decision23-Jul-2020
Date of Acceptance04-Jun-2021
Date of Web Publication7-Jul-2021

Correspondence Address:
Dr. KrishnaRao Arthi
Department of Laboratory Medicine, MIOT International, 4/112, Mount Poonamalle High Rd., Sathya Nagar, Manapakkam, Chennai - 600 089, Tamil Nadu
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/QAIJ.QAIJ_4_20

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Coronavirus Disease (COVID 19) outbreak is threatening the world as a whole. A novel bat coronavirus, whose pathogenesis is unknown to the medical fraternity is posing a challenge. The world is left with little option in the absence of a definitive treatment modality. Laboratory diagnosis of the severe acute respiratory syndrome CoV2 virus plays a pivotal role in categorizing patients for treatment, isolation, and quarantine. Real-time reverse transcriptase-polymerase chain reaction is the main modality of diagnosing the disease complimented by serological methods and biochemical parameters. Repeat testing for suspected cases remains our slogan.

Keywords: COVID, quarantine, SARS CoV2Introduction

How to cite this article:
Arthi K, Srinivas CN, Preethii N. Unveiling the novel coronavirus in the laboratory. QAI J Healthc Qual Patient Saf 2020;2:3-8

How to cite this URL:
Arthi K, Srinivas CN, Preethii N. Unveiling the novel coronavirus in the laboratory. QAI J Healthc Qual Patient Saf [serial online] 2020 [cited 2023 Mar 27];2:3-8. Available from: https://www.QAIJ.org/text.asp?2020/2/1/3/320809

Wuhan, a Hubei province in China encountered a cluster of pneumonia cases in December 2019, which was named the novel Coronavirus Disease (COVID 19) by the Centres for Disease Control (CDC) in January 2020. Severe Acute Respiratory Syndrome CoV 2 (SARS CoV 2) was the new name suggested by the International Committee on Taxonomy of Viruses due to its genetic relatedness to the SARS virus outbreak in 2003. Finally, the World Health Organization (WHO) named it COVID-19 on February 11, 2020. Coronaviruses are enveloped positive-stranded RNA viruses in the order of Nidovirales. Coronaviridae is further classified into four genera: Alpha, Beta, Delta, and Gamma coronavirus. Common human coronaviruses include Beta coronavirus OC43 and HKU1 and Alpha coronavirus 229E and NL63. Zoonotic coronaviruses that have emerged and caused outbreaks in humans are the SARS-CoV (2002) which caused the SARS and the Middle East Respiratory syndrome CoV (MERS-CoV) (2012) known as MERS.[1],[2] The primary target for the virus is the epithelial cells of the respiratory and intestinal tract and therefore, the spread of infection is through droplets or from fomites. The incubation period for the infection varies between 2 and 14 days with a median incubation period of 5.2 days.[3] The clinical presentation ranges from mild flu-like symptoms to pneumonia leading to severe morbidity and mortality in some cases. Hence, it is mandatory to “detect and treat” the affected to “protect” the vulnerable. This article is an evidence-based review on the laboratory diagnosis of SARS CoV2- the methodologies, challenges, and innovations.

  Methods Top

A thorough and detailed search was made on the internet on Google scholar, National Library of Medicine, Science direct, and Research gate using the keywords, “COVID-19 laboratory diagnosis”, “RT-PCR for COVID-19”, “Gene Xpert for SARS-CoV2”and “Serological tests for COVID-19.”

  Results Top

Full free text articles pertaining to the above keywords were included. Non-English language articles, articles downloaded in duplicate, articles not pertaining to the topic of review and those where only abstracts could be accessed were not taken for reference.

  Discussion Top

Diagnostic methods for detection of SARS CoV2 can be broadly classified as Molecular and Serological [Figure 1]. The WHO has recommended the real-time reverse transcriptase-polymerase chain reaction (rtRT-PCR) for the detection of this RNA virus.RT-PCR is a molecular test that amplifies a tiny amount of viral genetic material in a sample and is currently considered, the gold standard for the identification of the SARS-CoV-2 virus. Deep sequencing molecular methods such as next-generation sequencing and metagenomic next-generation sequencing may be needed in future to determine the mutations in SARS-CoV-2 but are currently impractical for diagnosing COVID-19. Laboratory diagnosis begins from collecting the right sample to issuing the right report.
Figure 1: Diagnostic modalities for COVID 19

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Samples collected

Upper respiratory tract samples:

  1. Nasopharyngeal (NP)/Oropharyngeal swab
  2. Nasal swab.

Lower respiratory tract samples:

  1. Sputum
  2. Bronchoalveolar lavage (BAL)
  3. Endotracheal aspirate.

NP swabs have been researched to show better recovery of the RNA (63%) compared to the oropharyngeal swabs (32%). Likewise, better yield has been demonstrated with lower respiratory tract samples. Some studies have also isolated the virus from stool samples, however, stool PCR is not done routinely.[3] A single NP swab can be preferred for testing as it is tolerated better by the patient and is safer to the operator compared to the throat swab. NP swabs have an inherent quality, to reach the correct area to be tested in the nasal cavity. CDC recommends the use of synthetic swabs with plastic or wire shafts as calcium alginate swabs may contain inhibitory substances that may inactivate the virus.[4]

How to collect a nasopharyngeal swab specimen?

  1. Label the specimen container with the patient's details
  2. Don appropriate personal protective equipment (PPE)
  3. Tilt the patient's head back slightly, so that the nasal passages become more accessible
  4. Gently insert the swab along the nasal septum, just above the floor of the nasal passage, to the nasopharynx, until resistance is felt
  5. Leave the swab in place for several seconds to absorb secretions and slowly remove the swab while rotating it.[5]

Transport of samples

After collection, the swabs are placed in a viral transport medium (VTM) to preserve the integrity of the nucleic acid and is transported to the testing facility. Lower respiratory tract samples may be sent in a sterile container. All samples should be transported to the testing laboratory at 2°C–8°C or may be frozen to −20°C if a delay is anticipated.[6]

Molecular testing methods

Current diagnostic tests to identify the SARS CoV2 RNA include real-time RT-PCR, and automated self-contained in vitro diagnostic assays such as the TRUENAT and Cartridge Based Nucleic Acid Amplification Test (CBNAAT). Molecular methods that are under evaluation are reverse transcription loop-mediated isothermal amplification ), multiplex isothermal amplification followed by microarray detection, and CRISPR (clustered regularly interspaced short palindromic repeats)-based assays. Laboratories should be equipped with a Level II or III biosafety cabinet in a negative pressure room. The technologist involved in the processing of samples should also don an N95 mask, goggles/face shield, gloves, cap, and a disposable gown. Coronaviruses have a number of molecular targets within their positive-sense, single-stranded RNA genome that can be used for PCR assays. These include genes encoding structural proteins, including envelope glycoproteins spike (S), envelope (E), transmembrane (M), helicase (Hel), and nucleocapsid (N). Furthermore, there are species-specific genes required for viral replication which include RNA-dependent RNA polymerase (RdRp), hemagglutinin-esterase, and open reading frame 1a (ORF1a) and ORF1b. In the United States, the CDC recommends two nucleocapsid protein targets (N1 and N2) while the WHO recommends first-line screening with an E gene assay followed by a confirmatory assay using the RdRp gene.[7]

Merits and demerits of different molecular methods

RTPCR requires expensive equipment and the turnaround time may vary from 3 to 24 h. The extraction of nucleic acid is performed separately, after which the extracted material is amplified and detected real-time in a thermal cycler. A study by He et al. reported a sensitivity of 79% and specificity of 100%, for RT-PCR. The RT-PCR testing accuracy may be affected by a number of factors including viral load in the respiratory tract, specimens source, sampling procedures and timing, quality control of the test, and inherent performance of the testing kits.[8]

The TRUENAT is a nucleic acid detection assay in which two steps are employed. The first one is to screen for the E gene common to sarbecoviruses followed by the use of a second cartridge to detect the RdRp gene only when the screening test is positive.[9]

The GeneXpert is a CBNAAT that automates and integrates sample preparation, nucleic acid extraction and amplification, and detection of the target sequences in simple or complex samples using real-time PCR assays. The systems require the use of single-use disposable cartridges that hold the RT-PCR reagents and host the RT-PCR process. Because the cartridges are self-contained, cross-contamination between samples is minimized. The results are given out in a short time span of 45 minutes compared to RTPCR.[10]

Interpretation of molecular test results

In individuals with COVID-19 infection, viral RNA in the NP swab is measured by the cycle threshold (Ct). RNA becomes detectable as early as day 1 of symptoms and peaks within the 1st week of symptom onset. The Ct is the number of replication cycles required to produce a fluorescent signal. Lower Ct values represent higher viral RNA loads. A Ct value less than 40 for the confirmatory genes (N, RdRp, S, ORF) is clinically reported as PCR positive. The positivity usually starts to decline by week 3 and subsequently becomes undetectable. It has to be remembered that a “positive” PCR result reflects only the detection of viral RNA and does not necessarily indicate the presence of the viable virus. Some patients have tested positive for SARS CoV2, by RT-PCR even beyond week 6 following the first positive test and some have also been reported positive after 2 consecutive negative PCR tests performed 24 h apart. There is no clarity if this is a testing error, reinfection, or reactivation.[11],[12] In a study of 205 patients with confirmed COVID-19 infection, RT-PCR positivity was the highest in BAL specimens (93%), followed by sputum (72%), nasal swab (63%), and pharyngeal swab (32%).[13] An important issue we face with the real-time RT-PCR test is the risk of false-negative and false-positive results. Studies have reported that many “suspected” cases with typical clinical characteristics of COVID-19 and features of atypical pneumonia on computed tomography (CT) Chest images were not diagnosed positive. Thus, a negative result does not exclude the possibility of COVID-19 infection and should not be used as the only criterion for treatment or patient management decisions. It is always safe to consider a combination of real-time RT-PCR and clinical features for the management of the SARS-CoV-2 outbreak.[14]

False positives may occur occasionally due to:

  • Reagent contamination with previously amplified DNA
  • Cross-contamination during collection, transportation and aliquoting and
  • Technical errors.

Liberal use of negative control samples in each assay can help ensure that laboratory contamination is detected and that specimens are not inappropriately labeled as SARS-CoV2 positive.[15]

False-negative reports may be due to:

  • Improperly collected sample
  • Type of specimen - Lower respiratory tract samples yielded the highest recovery followed by NP swab
  • Stage of disease- The viral load is highest in the first 5 days after symptoms onset and wanes gradually. However, in severe disease, BAL and sputum may be positive but the virus may not be isolated from NP swabs, whereas the yield is good from an NP swab in mild-to-moderate cases[12]
  • Error in storage and transport- Prompt transport to testing laboratory and maintenance of cold chain in case of anticipated delay is mandatory[16]
  • Prolonged nucleic acid conversion - A study Xiao et al. reported that 21% of its patients turned positive by RTPCR aftertwo consecutive negatives. They have taken a longer time for conversion and may have been detected negative in the early stage of the disease. Such group of patients requires longer period of observation.[17] Clinical correlation along with radiological features suggestive of atypical pneumonia may be helpful in pointing out the diagnosis in some of the patients.[14]

Serological tests

Serology testing for COVID-19 is defined as the analysis of plasma, serum, or whole blood for the detection of antibodies, especially immunoglobulin G (IgG), IgM, and IgA, that are specific for SARS-CoV-2 antigens.[18] Two SARS CoV2 proteins that are notably important antigenic targets include the S and N genes.

S proteins are for receptor binding and fusion and N protein, a plays an important role in viral pathogenesis, replication, and RNA packaging. Antibodies to the N protein have been frequently detected in COVID-19 patients and hence, may be one of the immunodominant antigens in the early diagnosis of disease.[7] Seroconversion occurs approximately 7–14 days after symptom onset. Generally in the classical immune response to viruses, IgM is produced first, accompanied by IgA, and then followed by a shift toward IgG production. IgM does not play a primary role in COVID-19 antibody testing due to high false-positive rates. IgG is a longer-lasting antibody with potential viral neutralizing activity. Many manufacturers have therefore focused their efforts on developing immunoassays against IgG rather than IgM.[18]

The antibody tests:

  • Can be done on blood/serum/plasma samples
  • Test result is available within 30 min
  • Test may come positive after 7–10 days of infection
  • The test may remain positive for several weeks after infection
  • Positive test indicates exposure to SARS-CoV-2
  • Negative test does not rule out COVID-19 infection.[19]

The Indian Council for Medical Research has validated and approved an indigenous IgG ELISA for serosurveys and surveillance in high-risk population such as healthcare workers, immunocompromised persons, and frontline workers.[20]

Inflammatory biomarkers

Routine biochemical and hematological laboratory testing is essential for assessing disease severity, selecting therapeutic options, and monitoring treatment response [Table 1]. Several inflammatory biomarkers have been implicated in severe COVID-19, causing the “cytokine storm.”[18] A series of immune-mediated events occur as a response to the SARS CoV2 virus, resulting in the release of proinflammatory cytokines that correlate directly with clinical severity.[21]
Table 1: Inflammatory biomarkers in COVID

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Higher levels of procalcitonin in COVID-19, suggest the onset of bacterial infection in critically ill patients. Elevation in the D-dimer coagulation parameter has been associated with worsening disease and a higher risk of developing thromboembolic complications in COVID-19 patients. Overall, it has been studied that severe cases of COVID-19 are characterized by a massive pro-inflammatory response or cytokine storm that is estimated to progress to multiple organ damage and failure in severe cases. Biochemical monitoring of COVID-19 patients will thus involve assessing the inflammatory profile, as well as early recognition of cardiac, renal, and hepatic injury through routine laboratory testing.[22]

A study conducted in China showed that RT-PCR yielded a sensitivity of 79% and specificity of 100%. The RT-PCR testing accuracy may be affected by a number of factors such as the viral load in the respiratory tract, specimens source, sampling technique, quality control of the test, and inherent performance of the testing kits. Chest CT has proved to play an important role in early detection, evaluation, and treatment response monitoring of COVID-19 infection. However, chest CT manifestation of COVID-19 pneumonia overlaps with other types of viral pneumonia, questioning its specificity.[8] Therefore, correlating clinical symptoms of the patient along with radiological features, RTPCR, biochemical and hematological parameters will benefit the patient as a whole.

COVID-19 shows a wide range of clinical manifestations, so early diagnosis will help guide physicians to provide prompt intervention for patients who are at higher risk for developing more serious complications from COVID-19 illness. Availability of a commercial vaccine may take time and so, it is important to identify individuals who have been infected with SARS-CoV-2, with or without accompanying symptoms, and who have developed antiviral immunity.[23]

Quality control forpolymerase chain reaction

Commercial QCs are usually preferred but in the absence of commercial controls, laboratories can use:

  • Negative control: Water/universal transport media/VTM
  • Positive control: A known positive patient sample with a Ct value between 25 and 30.[24]

Newly-received lot of test kits should be tested using a panel of known positive and negative samples to confirm its performance along with the lot in use.

Key performance indicators (KPIs) refer to the collection and analysis of data at each step of testing to serve as an indicator for the performance of the whole testing cascade. KPIs should be analyzed and reported at least once a month and should include the number of specimens tested, specimen type, number (%) of positive, negative, and invalid test results, specimen rejection rate, percentage of failed IQC results, external quality assurance or proficiency testing performance and turnaround time.[24]

During the time of this COVID-19 health crisis, College of American Pathologists (CAP) has recommended the following,

  • For emergency use authorization tests in a patient care setting, it is sufficient if QC is performed as per the manufacturer's instructions. The US Food and Drug Administration deems these tests to be Clinical Laboratory Improvement Amendments waived tests as they do not require stringent conditions and the risk of an erroneous result is insignificant.[25] The Tamil Nadu government has announced a quality assurance program for Government and private laboratories across the state whereby laboratories have to send 5 negative and 5 positive samples with the Ct value for the E gene between 25-35, fortnightly to the identified mentor laboratories across the state.[26]

  Biosafety and Infection Control Measures Top

The WHO has recommended adherence to certain biosafety laboratory practices in order to reduce exposure to the staff and prevent laboratory-acquired infection.

  • Handling and processing of specimens from suspected cases as per standard precautions described earlier in this study. Samples to be transported in double-layered packing with biohazard label. Leaking containers and those with visible contamination on the outside should be discarded and a repeat specimen requested
  • Nonpropagative laboratory work such as NAAT to be performed in BSL-2 laboratories
  • Testing to be carried out only by properly trained and competent personnel
  • Risk assessment and risk management to be conducted for laboratory personnel
  • Biosafety cabinet II or III to be used for processing of samples
  • Appropriate disinfectants with proven activity against enveloped viruses, proper dilution with recommended time of contact to be used (5% sodium hypochlorite or 70% ethanol)
  • Proper donning and doffing of appropriate PPE by laboratory personnel handling specimens
  • Technical procedures to be performed with minimal generation of aerosols.[27],[28]

  Impact of COVID-19 Top

It has been hardly 6 months since the outbreak in China, within which the novel Coronavirus has engulfed the whole world. Combatting the corona is the highest on the global agenda currently. The economy has taken a severe hit on account of lockdown and suspension of transportation. Health-care facilities are overwhelmed with COVID positive patients, that other infectious diseases like tuberculosis and HIV/AIDS have taken the backseat. Furthermore, patients with non-communicable diseases and those on continuous treatment like chemotherapy have suffered due to break in the supply chain of materials such as drugs and logistics, which have had a serious impact on the mental and physical health of these patients.[29] The pandemic on a large has created destruction worldwide and the only positive impact is on the environment.[30]

  Conclusion Top

No test gives a 100% accurate result; tests need to be evaluated to determine their sensitivity and specificity, ideally by comparison with a “gold standard.” The lack of such a clear-cut “gold-standard” for COVID-19 testing makes the evaluation of test accuracy challenging. Therefore, repeat testing in negative cases, especially in those with strong clinical suspicion, detailed history and clinical examination, correlation of clinical findings with radiological features and laboratory parameters will guide us in the management of the patient.

As there are currently no vaccines and proved antiviral agents for definitive treatment, it is necessary to strengthen the laboratory network for early diagnosis of COVID-19 not only for the management of cases but also for the isolation of asymptomatic and mildly symptomatic cases and quarantine of close contacts.

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Conflicts of interest

There are no conflicts of interest.

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[PUBMED]  [Full text]  
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