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Table of Contents
REVIEW ARTICLE
Year : 2020  |  Volume : 11  |  Issue : 3  |  Page : 117-123

COVID diagnostics: Do we have sufficient armamentarium for the present and the unforeseen?


1 Department of Microbiology, University College of Medical Sciences and Guru Teg Bahadur Hospital, New Delhi, India
2 Department of Microbiology, G B Pant Institute of Postgraduate Medical Education and Research, New Delhi, India
3 Department of Medicine, Lady Hardinge Medical College and SSK Hospital, New Delhi, India

Date of Submission26-Jul-2020
Date of Acceptance14-Aug-2020
Date of Web Publication10-Sep-2020

Correspondence Address:
Dr. Bineeta Kashyap
Flat No. C-402, Vimal CGHS LTD., Plot-3, Sector-12, Dwarka, New Delhi - 110 078
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/INJMS.INJMS_92_20

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  Abstract 


The COVID-19 pandemic has taken the world by storm, and nations world over are battling this unprecedented health crisis. Diagnostics play the most important part in the “test, track, and treat” strategy being used in most of the nations to combat COVID-19. Although viral culture is the gold standard, it is not pursued because of the associated biohazard risks. Short of that, nucleic acid amplification tests (NAATs) are the present gold standard and are being used in several ways. Real-time reverse transcriptase-polymerase chain reaction is being widely used, although cartridge-based NAAT and TrueNat™ testing are also in vogue. Serological testing is also being used as an adjunct specially for screening (rapid antigen testing kits), while antibody (specially IgG) testing is being used as a serosurveillance strategy. Radiological investigations, especially computed tomography scan of the thorax, give peculiar peripheral ground-glass opacities which are quite characteristic in the present COVID pandemic and need to be ascertained together with other clinical features and diagnostic tools. Although the present tools have been able to support the diagnosis of COVID to quite an extent, there are limitations, and as the whole spectrum of COVID disease unfolds, the diagnostic armamentarium will also continue to expand, and we will need to use the diagnostic strategies further to be able to contain this pandemic at the earliest.

Keywords: Coronavirus, COVID-19, polymerase chain reaction, serology, viral pneumonia


How to cite this article:
Kashyap B, Goyal N, Prakash A. COVID diagnostics: Do we have sufficient armamentarium for the present and the unforeseen?. Indian J Med Spec 2020;11:117-23

How to cite this URL:
Kashyap B, Goyal N, Prakash A. COVID diagnostics: Do we have sufficient armamentarium for the present and the unforeseen?. Indian J Med Spec [serial online] 2020 [cited 2020 Sep 28];11:117-23. Available from: http://www.ijms.in/text.asp?2020/11/3/117/294805



We will not be going back to the “old normal.” The pandemic has already changed the way we live our lives. Part of adjusting to the “new normal” is finding ways to live our lives safely.

Dr. Tedros, Director-General, World Health Organization in his media briefing on July 23, 2020.[1]


  Testing is the Way Out… Top


Since its origin in the end of December 2019, SARS-CoV-2 has affected 15,296,926 individuals and is responsible for 628,903 deaths globally. India alone has reported 1,287,945 confirmed cases and 30,601 deaths due to COVID-19 till date.[1] There is a wide consensus over the perception of “test, track, and treat strategy” to be the only possible way to limit the COVID-19 spread and save precious human lives. The majority of COVID-19 cases develop mild illness and eventually recover uneventfully. However, some develop severe disease with progression to pneumonia, hypoxemia, systemic inflammatory illness, and hypercoagulability.[2] It is crucial to scale up the testing for SARS-CoV-2 and make it available even in distant outreach areas. This review intends to explore the selection of the most appropriate specimens and also discuss the availability, utility, or accuracy of the most reliable diagnostic methods for COVID-19 so as to make the clinicians better aware and also timely manage the patients.


  Sars-Cov-2 the Virus! Top


Coronaviruses (CoVs), enveloped viruses with nonsegmented positive sense RNA, belong to the family Coronaviridae and order Nidovirales. Four genera of CoVs (α, β, γ, and δ) have been identified; the human CoVs (HCoVs) are included in the α-coronavirus (HCoV-229E and NL63) and β-coronavirus (Middle-East respiratory syndrome coronavirus [MERS-CoV], SARS-CoV, HCoV-OC43, and HCoV-HKU1) genera. SARS-CoV-2, classified under the subgenus Sarbecovirus, subfamily Orthocoronavirinae, is a group 2B coronavirus. The immune-dominant spike (S) protein, envelope (E) protein, nucleocapsid (N) protein, and membrane (M) protein constitute the four structural proteins of CoVs. The transmembrane spike glycoprotein, homotrimers projecting from the viral surface of coronavirus, mediates its entry into the host cells. S consists of two functional subunits: S1 subunit is involved in binding to the host cell receptor, whereas the S2 subunit is responsible for the fusion of viral and host cell membranes. In several CoVs, S cleaves at the border connecting the S1 and S2 subunits which have noncovalent bonding in prefusion conformation.[3] The distal S1 subunit besides containing the receptor-binding domain (s), also provides the stabilization to membrane-anchored S2 subunit in prefusion state.[4] Host proteases further cleave the S at S2 cleavage site that leads to exhaustive irreversible conformational modifications responsible for the activation of protein associated with membrane fusion.[4],[5] Thus, the entry of coronavirus into susceptible host cell is mediated through a complex process involving the binding to receptor and proteolytic processing of S protein. Coronavirus S glycoprotein, being surface exposed, is the primary target for the neutralizing antibodies and also the central point for therapeutic and vaccine research.[4] Distinct domains inside S1 subunit are utilized by various CoVs for the identification of different attachment and entry receptors. S domain A (SA) attaches to 5-N-acetyl-9-O-acetyl-sialosides of host cell surface to gain entry in endemic human CoVs OC43 and HKU1.[6] In MERS-CoV, SA is utilized for the identification of nonacetylated sialoside attachment receptors that may further promote the binding of SB to dipeptidyl peptidase 4 entry receptor.[7] SB connects directly with angiotensin-converting enzyme 2 (ACE2) for entry in target cells in SARS-CoV and several SARS-related CoVs. ACE2 is also the functional receptor for SARS-CoV-2 that mediates its entry into host cell via SARS-CoV-2 S. The analogous affinity of SARS-CoV-2 SB and SARS-CoV SB for human ACE2, partly explains the similar efficient transmission in humans of SARS-CoV-2 as that of SARS-CoV.[3]


  Diagnostic Strategies for Sars-Cov-2: A Review of Available Approaches Top


Nucleic acid amplification tests

[Table 1] depicts the various diagnostic techniques available for COVID-19 and [Table 2] shows the interpretation of various microbiological test results in COVID-19. The primary challenges for nucleic acid amplification tests (NAATs) are (i) to minimize the false negatives by efficiently detecting the small number of viral RNA copies in the sample, (ii) to reduce the false positives by effectively differentiating the positive signals emitted by other pathogens, and (iii) to have the ability to test a large number of samples accurately in limited time.
Table 1: Diagnostic techniques for COVID-19

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Table 2: Interpretation of various microbiological test results in COVID-19

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SARS-CoV-2 is an enveloped virus containing positive-sense RNA as its genetic material. The genome of SARS-CoV-2 consists of approximately 30,000 nucleotides and 15 genes. Many of these genes, such as spike (S), nucleocapside (N), envelop (E), RNA-dependent RNA polymerase (RdRp), helicase (Hel), nonstructural protein 10 (nsp10), and nonstructural protein 14 (nsp14), have been utilized as probes or primer targets in the diagnostic NAATs. Initial researches demonstrated that by targeting the S gene, SARS-CoV-2 could be differentiated from SARS-CoV-1 with good specificity but low sensitivity.[2] In order to improve the sensitivity, additional viral-specific genes, such as RdRp/Hel, were integrated.[8] E and RdRp primers were considered to be most sensitive and were extensively used all over Europe, though RdRp primers were found to be cross reactive to SARS-CoV RNA.[2],[9] The WHO recommends the usage of E, N, S, and RdRp genes in various combinations for optimum diagnosis.[10] Similar to other RNA viruses, SARS-CoV-2 also has a tendency to mutate. However, the proofreading property of Nsp14 restricts the nucleotide misincorporation rate.[2] Variation in nucleotide sequence may lead to diminished recognition by distinct primer-probe set. The use of minimum two molecular targets, ideally at least one targeting the conserved/specific region, would help to mitigate the effects of a potential SARS-CoV-2 genetic drift or its cross-reaction with any other circulating coronavirus.

Real-time reverse transcription-polymerase chain reaction

Real-time reverse transcription-polymerase chain reaction (rRT-PCR) remains the validated assay for prompt diagnosis in suspected SARS-CoV-2 infection. Amplification and analysis being carried out together in a closed system, the chances of false positives in these assays are minimized. The approximate turnaround time (TAT) is 4–5 h. The high diagnostic accuracy of rRT-PCR along with the capacity to test up to ninety specimens in a single run makes it the frontline test for diagnosis of COVID-19. The need of trained workforce, specialized laboratory equipment, and specific biosafety requirements, limits its use at peripheral centers in developing countries like India.

Loop-mediated isothermal amplification is another alternative to RT-PCR which uses amplification under isothermal condition with more rapidity and specificity.

With the surge in cases of COVID-19 in India and over 150 countries across the globe, a rapid point-of-care assay is considered to be a major tool toward the containment of cases. To reduce the TAT of NAATs, many rapid platforms of microarray or sequencing solutions on multi-RT-PCR panels with automation are being developed. Though initially intended to be used for the diagnosis of tuberculosis and other infectious diseases, TrueNat™ and cartridge-based nucleic acid amplification test (CBNAAT) are now widely deployed for the detection of COVID-19 cases. The real-time micro-PCR system is achieved through a combination of the cartridge-based RNA extraction system and real-time micro-PCR analyzer. These are cartridge-based closed NAAT systems that can be performed with minimal hands-on training. CBNAAT (GeneXpert® SARS-CoV-2 test Cepheid®) is a simple, highly performing test with a short TAT. TrueNat is a chip-based RT-PCR test for the semi-quantitative detection of beta-coronavirus and SARS coronavirus RNA. The target sequence is the E gene of Sarbecovirus for beta-coronavirus and Rdrp gene for SARS coronavirus. As the viral lysis buffer used in the processing inactivates the virus, these techniques impose a minimal biosafety hazard. The TAT for these platforms is approximately 1 h. However, only 1–4 samples can be processed in a single run, limiting the samples that can be tested in a single day to 24–48. These systems have the capability of being utilized at grass-root levels.

Cycle threshold value

Real time-PCR assay measures the viral RNA in terms of cycle threshold (Ct), which is the number of cycles the fluorescent signal requires to become detectable and is inversely proportional to the viral load. The results are interpreted based on the Ct values and value <40 are generally considered positive. In RT-PCR, false-negative result may occur due to sampling error or incorrect timing of sampling.

The overall limit of detection for NAATs ranges from 100 to 1000 copies. Thus, these tests have much desired high analytical sensitivity along with very high specificity.[11]


  Choosing the Right Specimens Top


Selection of an appropriate specimen is very crucial for the correct diagnosis of infected COVID-19 patients. COVID-19 patients typically shed high load of culturable virus starting from about 5–6 days of becoming symptomatic though viral RNA remains detectable in the respiratory samples for longer. Severely ill patients can continue to shed the virus for weeks to months. Fecal shedding contributing to the spread of infection remains a concern too. Nasopharyngeal (NP) swab collected by trained health-care workers (HCWs) constitutes a standard sample for RT-PCR as per the recommendation by the Centers for Disease Control and Prevention. The process of obtaining an NP swab is uncomfortable for the patient and may elicit coughing or sneezing. Hence, the use of adequate personal protective equipment and adherence to standard protocol for infection prevention and control is warranted by the HCW for this procedure. Operational difficulty in the collection of NP swabs has led to the assessment of other comparatively easily available alternative samples such as saliva, oropharyngeal swabs, nasal swabs, NP wash/aspirate, and mid-turbinate swabs. Oropharyngeal swabs are comparatively less sensitive than NP and nasal swabs.[12] Testing of simultaneously collected nasal and oropharyngeal swabs, either independently or together in a single aliquot, is an attractive alternative option to increase the chances of positivity. The collection and processing of saliva, though an appealing sample, possesses its own challenges. Nasal swabs have comparable sensitivity to NP swabs. Many studies have reported a comparable sensitivity of self-collected samples by patients with those collected by health-care personnel.[13],[14] Any upper respiratory specimen, however, may miss early infection; when a repeat testing must be performed preferably on a lower respiratory specimen, as the main site of replication by then might possibly be the lower respiratory tract. Moreover, sputum and bronchoalveolar lavage (BAL) have shown higher sensitivity in comparison to upper respiratory samples, probably due to the presence of greater viral loads in these specimens.[15] Collecting different specimen types in highly clinically suspected cases will improve detection rate by reducing false negativity. However, due to these being invasive procedures with the enhanced associated risk of aerosol generation, collection of these samples is done in selective instances.[2] Shipping of all these specimens to the reference laboratory must be done following triple packaging system ensuring appropriate labeling and sealing of the samples as per the standard protocol.

A false-negative nucleic acid amplification test: Significance?

It is of paramount importance to understand that a negative RT-PCR test result does not rule out COVID-19. There are several factors influencing the positivity of a RT-PCR test result, some of which include: low viral load in case of an incubation period or convalescent stage; or primary replication of the virus at other sites in the body (lower respiratory tract). There have been negative RT-PCR test reports with upper respiratory tract specimens in cases with suggestive pulmonary computed tomography (CT) scan findings.[16] This viral tropism for the lower respiratory tract is probably due to the inconsistent distribution of ACE2 viral receptors throughout the respiratory tract.[2],[17] A suboptimal sampling technique may also affect the RT-PCR test results. In the instances of high clinical suspicion, it is prudent to repeat testing, as the sensitivity of NP swab is below ideal. Furthermore, in a high prevalence milieu, researchers have shown a significant increase in the positivity of these tests.

A positive nucleic acid amplification test previously declared negative

The criterion most often applied for discontinuation of isolation is two negative RT-PCR test results at least 24 h apart. Nevertheless, some of such cases report positive again despite having two negative test results.[18] This may be due to the alterations in the shedding of viral RNA during convalescence. However, the prognosis of such cases seems to be good in the absence of an actual clinical or virological relapse.[2]

Does nucleic acid amplification test positivity measure infectiousness?

As the viral RNA can be demonstrated from the samples of patients during recovery, nucleic acid amplification tests are not very useful in monitoring the infectivity of COVID-19 cases.[19] Although the ability of virus present in the sample to grow in culture constitutes a better measure of infectivity, it is rarely practiced due to biosafety concerns.[20]

Quantitative nucleic acid amplification test: Any prognostic value?

Instances show the presence of high viral loads even among asymptomatic cases as determined by the real-time PCR Ct values. Therefore, the prognostic utility of viral load in isolation is limited.[21] Though some correlations have been revealed between the severity of the disease and viral load, the viral load determined by these assays in terms of Ct value should not be used for prognosis or monitoring treatment response.[22] In all probabilities, irrespective of the course of the disease, viral loads typically regress with time.[23],[24] Lower Ct values indicate high viral load and hence can be suggestive of transmissibility.


  Adjuncts to Molecular Diagnosis Top


Serological evidence of SARS-CoV-2 diagnosis

Immunological tests can either measure the antibodies produced during the host immune response to infection or the antigenic viral particles in the respiratory specimens. The techniques commonly used for the demonstration of SARS-CoV-2-specific antibodies are immunochromatographic tests, enzyme-linked immunosorbent assays (ELISA), neutralization assays, and chemilumiscent immunoassays. Serologic tests are less dependable than the NAATs for the detection of SARS-CoV-2. The prevalence of infection also plays a crucial role in determining the positive or negative predictive values of a given test. In a low-prevalence setting, a positive serologic test with limited specificity is more likely to be a false-positive test result. Concerns were raised regarding the cross-reactivity of antibodies against SARS-CoV-2 and various related or distant viral families. Other human CoVs (such as HKU1, OC43, 229E, and NL63) causing mild-to-moderate seasonal respiratory symptoms are antigenically closely related to SARS-CoV-2.[25] The chances of cross-reactions are furthermore plausible with SARS-CoV-1 or MERS-CoV. However, majority of the commercially available serologic assays demonstrate a specificity above 98%. The target antigen used in a serological assay also influences the sensitivity and specificity of that assay. S protein, produced at a much advanced stage of COVID-19, has lower sensitivity but higher specificity (particularly with S1 subunit) in comparison to N protein targets.[26]

Serological evidence: The relevance?

Serologic tests are mostly used to determine the exposure to SARS-CoV-2 in the past. These tests may also prove useful in establishing the diagnosis of COVID-19 in cases with negative NAATs with high clinical suspicion. Though IgM and IgG antibodies have been demonstrated as early as 3–6 days following the onset of symptoms, the seroconversion has been reported to occur by 3 weeks in majority of cases.[27] IgM is generally the first class of antibody to be produced in any infection followed by IgG immunity. IgM can be detected from the 2nd week, with the titers touching the peak in the 3rd week from the onset of symptoms and then slowly declining over time. IgG is reported to stabilize around 4 weeks.[28] However, in COVID-19, it is believed that IgM may be short lasting and IgG response may occur earlier than usual, and how long this IgG lasts is yet not known. IgM and IgG against SARS-CoV-2 can be determined qualitatively by immunochromatography assays and quantitatively by ELISA. Detection rates improve with the progression of illness.[29] A recent study has reported a higher accuracy of IgM and IgG ELISA in comparison to lateral flow assays.[30]

Though the antibody responses are demonstrable in majority of the COVID-19 cases, seroconversion may not be observed in immunocompromised patients or in a few with asymptomatic/very mild infections. The presence of specific antibodies against SARS-CoV-2 is most probably linked with some level of protection, though cutoff values of these antibodies are yet not established.[31] Neutralizing antibodies are generally considered more directly connected with the protective immunity. Furthermore, the production of neutralizing antibodies is complemented with T-cell responses. Low titers of antibodies are not considered protective and high titers are often encountered in severe COVID-19.[2] The recovery of mild cases even with low antibody levels and persistence of the disease in the presence of high antibody titers in severe cases raise queries about the role of neutralizing antibodies in providing immunity. Therefore probably, the therapeutic benefit of convalescent plasma has been attributed to other components by some researchers.[32]

Several lateral flow assays have been developed to detect the antigens of COVID-19 as point-of-care platform. The widely used antigen detection tests have a moderate sensitivity (ranging from 50.6% to 84%) with a high specificity (99.3% to 100%). The rapid chromatographic immunoassay may aid in the qualitative determination of SARS-CoV-2-specific antigens. The positive test results by antigen detection test can be regarded as true positives. However, the negative test results in symptomatic cases need further confirmation by real-time PCR test.[33] Although these assays have the theoretical advantage of being rapid and low cost, the viral load of the patient and the variability in specimen collection could result into lower sensitivities of these tests, early in infection.

Due to the kinetics of antibody formation or variabilities in the sensitivities of these assays, clinical decision-making should not be solely relied upon these tests unless strong evidence exists. Though impractical in early stage, antibody detection tests may be used for retrospective evaluation and epidemiological surveillance in terms of the burden of infection, significance of asymptomatic infections, basic reproduction number of the virus, or the overall mortality.

Computed tomography scan

CT scan is often regarded as an important auxiliary investigation for COVID-19. The researchers from Wuhan have reported a considerably higher sensitivity of CT scan in comparison to PCR tests in COVID-19 cases.[34] CT scan plays a pivotal role in the early diagnosis and timely management of COVID-19 cases. The characteristic features of COVID-19 infection comprise bilateral multi-lobar ground-glass opacities with differential distribution, subpleural ascendance, thickened lobular septa with inconsistent alveolar filling, and amalgation.[34],[35] However, these findings of CT scan are suggestive and not confirmatory for COVID-19 diagnosis.

Viral culture

Though viral culture is the gold standard for the isolation and characterization of the virus, it is not used for the diagnosis of COVID-19 due to the process being labor intensive and also due to the requirement of biosafety level 3 facility with skilled workforce. Vero, Huh, and human airway epithelial cells lines have been used by several researchers to observe the cytopathic effects, which are confirmed by RT-PCR.[36],[37],[38],[39]

Biomarkers

Several biomarkers are routinely used in clinical practice for their possible predictive role in the assessment of disease progression. They are crucial in identifying the cases at higher risk of developing complications. In addition, these also help in deciding the treatment protocols in COVID-19 cases. [Figure 1] shows the commonly used biomarkers in COVID-19 patients.
Figure 1: Biomarkers with predictive role in COVID-19 progression

Click here to view



  Diagnosis of Sars-Cov-2: Challenges and Limitations! Top


Despite an upstanding accomplishment of validated NAATs, there are some inherent challenges. NAATs carry the risk of false-negative results due to several pre-analytical factors that can influence the end results such as the inappropriate timing of collection of specimen (too early or too late in the course of illness); poor-quality specimen; type of sample (lower respiratory tract specimens, such as BAL and induced sputum, have better sensitivity than upper respiratory tract specimens such as NP and oropharyngeal swabs); and lapse in sample transportation (unsuitable container, inappropriate viral transport medium, or inadequate maintenance of cold chain, etc.).[11] Serological assays have a low sensitivity in the early course of COVID-19. The clinical utility of serodiagnosis is confined to the convalescent patients with negative molecular test results. In addition, researchers have documented the ongoing evolution of SARS-CoV-2 genome through active mutations and genetic recombination.[40],[41] Being RNA virus, SARS-CoV-2 is also deficient in effective proofreading machinery needed to secure the RNA replication fidelity.[41] Mutations may change the sequence of primers hybridizing regions, thus yielding false-negative results.[41],[42] However, this issue can be addressed by targeting more than one (two or three) sequence in viral genome.


  What Do We Need to Remember? Top


  • The complex scenario of the ongoing and rapidly evolving COVID-19 pandemic warrants the concerted use of various available modalities and their interpretation in relation to the clinical milieu of individual case
  • The diagnosis not only needs to be timely and accurate, but should also contribute toward providing relevant epidemiological information so as to assess the actual burden and spread of the disease. In this context, the serological assays can complement molecular diagnosis, especially among those who are still asymptomatic and not hospitalized or may be used as screening assays as adjunct to diagnosis, though NAAT continues to remain the reference standard for COVID-19 diagnosis
  • Monitoring the viral load along with being observant of the technique and timing of sample collection will help better interpret different stages of the disease. The type of sample to be collected, whether upper or lower respiratory, will further reduce the false negativity
  • Clinical corroboration completed by serological evidence will help to reach a prompt management decision.
  • The patients' microbiota and the immune system will finally contribute to the varied clinical manifestations present and the prognosis. Hence, continuous addition to the existing knowledge needs to be made as further studies get published.


Financial support and sponsorship

None.

Conflicts of interest

There are no conflicts of interest.



 
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