This presentation constitutes the classification of corona viruses and various diagnostic modalities of COVID

1. Diagnostics & classification
of corona viruses
Dr. Shinjan Patra

2. Outline
• Basic structure & Genome
• Classifications & Phylogenetics
• Basic Diagnostic approach
• Nucleic Acid based assays
• Serology based assays
• Home based assays
• Clinical & Radiographic tools
• Mass pool screening
• Metagenomic profile

3. Corona viruses
• Group of related RNA viruses that cause diseases in mammals and birds
• In humans and birds, cause respiratory tract infections that can range from
mild to lethal
• Mild illnesses in humans like common cold (like rhinoviruses), while more
lethal varieties can cause SARS, MERS, and COVID-19
• Cause diarrhea in cows and pigs
• In mice cause hepatitis and encephalomyelitis
• First reported human coronavirus the HCoV‐229E: identified through the
isolation of the causative agent of a common cold in some patients in the
United States in 60’s

5. Basic structure
• Enveloped viruses with a positive-sense single-stranded RNA genome
and a nucleocapsid of helical symmetry
• Genome size ranges from approximately 26 to 32 kb
• One of the largest among RNA viruses
• Characteristic club-shaped spikes that project from their surface,
which in electron micrographs create an image reminiscent of the
solar corona

6. Genome & proteins
• Genomes organized in a fixed pattern in which the 5' two‐thirds part
of the genome occupied by the nonstructural proteins
• 3' one‐third of the genome is occupied with the major structural
proteins (S, E, M, and N)
• Spike glycoprotein (S) key protein
• Important role in virus replication, pathogenesis, and immune
response
• S protein composed of two subunits, S1 and S2
• S1 much more variable while the S2 conserved
• Usually inert until it infects its host

7. Others
• Lipid bilayer envelope, membrane proteins, and nucleocapsid protect
the virus when it is outside the host cell
• Lipid bilayer in which the membrane (M), envelope (E) and spike (S)
structural proteins are anchored
• M protein crucial during the assembly, budding, envelope formation,
and pathogenesis stages of the virus lifecycle
Almeida JD, Berry DM, Cunningham CH, Hamre D, Hofstad MS, Mallucci L, McIntosh K, Tyrrell DA (November 1968). "Virology: Coronaviruses"
(https://doi.org/10.1038%2F220650b0). Nature. 220 (5168): 650. Bibcode:1968Natur.220..650

9. Classification
• Genus was split into four genera in 2009 namely
Alphacoronavirus
Betacoronavirus
Deltacoronavirus
Gammacoronavirus
• As of 2021, 45 species officially recognised

10. Alphacoronavirus: Species
• Alphacoronavirus 1 (TGEV, Feline coronavirus, Canine coronavirus)
• Human coronavirus 229E
• Human coronavirus NL63
• Miniopterus bat coronavirus 1
• Miniopterus bat coronavirus HKU8
• Porcine epidemic diarrhea virus
• Rhinolophus bat coronavirus HKU2
• Scotophilus bat coronavirus 512

11. Betacoronavirus : Species
• Severe acute respiratory syndrome–related coronavirus (SARS-CoV, SARS-
CoV-2)
• Middle East respiratory syndrome-related coronavirus (MERS)
• Betacoronavirus 1 (Bovine Coronavirus, Human coronavirus OC43)
• Hedgehog coronavirus 1
• Human coronavirus HKU1
• Murine coronavirus
• Pipistrellus bat coronavirus HKU5
• Rousettus bat coronavirus HKU9
• Tylonycteris bat coronavirus HKU4

12. Gammacoronavirus
• Avian coronavirus
• Beluga whale coronavirus SW1

13. Deltacoronavirus
• Bulbul coronavirus HKU11
• Porcine coronavirus HKU15

14. Phylogenetic tree

15. Reservoir
• Bats for alphacoronaviruses and betacoronavirus
• Birds for gammacoronaviruses and deltacoronaviruses

16. Human coronaviruses
• Human coronavirus OC43 (HCoV-OC43)
• Human coronavirus HKU1 (HCoV-HKU1)
• Human coronavirus 229E (HCoV-229E)
• Human coronavirus NL63 (HCoV-NL63)
• Severe acute respiratory syndrome coronavirus (SARSCoV) (identified in
2003)
• Middle East respiratory syndrome-related coronavirus (MERS-CoV)
(identified in 2012)
• Severe acute respiratory syndrome coronavirus 2 (SARSCoV-2) (identified in
2019)
Wertheim JO, Chu DK, Peiris JS, Kosakovsky Pond SL, Poon LL (June 2013). "A case for the ancient origin of coronaviruses“ (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3676139).
Journal of Virology. 87 (12): 7039–45. doi:10.1128/JVI.03273-12

17. Diagnostics

18. Introduction
• Growing list of over 400 diagnostic tests either in development or
approved for clinical use
• Oropharyngeal and nasopharyngeal swabs recommended for the detection
of early infection
• Other lower respiratory tract specimens like the sputum and
bronchoalveolar lavage used for late detection and monitoring of patients
with severe pneumonia
• Mean incubation period of the disease 5.2 days
• Real-time RT-PCR based molecular assay remains the test of choice
• Serological tests being introduced as supplementary tools

21. Nucleic acid based assays: RT-PCR & it’s
newer congeners

22. URT specimen collection
• URT specimens collected within 5-6 days of the onset of the symptoms
• Timeline demonstrated high viral loads
• Swabs should have flocked non-toxic synthetic fibers, such as polyester, as well as
synthetic nylon handles
• Detection rate of oropharyngeal swabs significantly lower than nasal swabs
• Order to increase the sensitivity, the WHO recommends the collection of a
combined nasopharyngeal and oropharyngeal swab
• Detection rate infected people approached 100%
Wang W, Xu Y, Gao R, et al. Detection of SARS-CoV-2 in different types of clinical specimens. JAMA. 2020;11.

23. Transport & storage
• Once collected, swabs should be placed in vials containing Viral
Transport Medium (VTM) and transported to the laboratory within
24-72 hours
• Ideally under refrigerated conditions (4 C)
• If delays in transport are expected, the samples should be stored at
-70 C

24. Other samples
• BAL yield the highest SARS-CoV-2 RNA
• Patients requiring emergent intubation, a LRT specimen can be collected
during the intubation procedure (specially important in newly discovered
mutant strains)
• In advanced COVID-19 cases, rectal swabs may be preferred sample for
diagnosis
Leung WK, TO KF, Chan PK, et al. Enteric involvement of severe acute respiratory syndrome-associated coronavirus infection. Gastroenterology. 2003;125:1011e1017

25. Viral load & detection
• Viral load in respiratory samples highest during the initial stages of the
disease, reaching a peak in the second week, followed by lowered viral
loads
• Throat swabs from disease-recovered individuals show positive results for
up to 50 days
• RNA shown to be present in fecal and anal swabs weeks after respiratory
samples were found negative
• Ranges from 104 to 107 copies/ml
• Positivity rates for blood, saliva and tears 88, 78 and 16% respectively
Yongchen, Z. et al. Different longitudinal patterns of nucleic acid and serology testing results based on disease severity of COVID-19 patients. Emerg. Microbes Infect. 9, 833–836 (2020)

26. Target selection
• Molecular targets within their positive-sense, single-stranded RNA genome
• Include structural proteins: (S), (E), (M), helicase (Hel) and nucleocapsid (N)
• RNA dependent RNA polymerase (RdRp), hemagglutinin-esterase (HE) and
open reading frames 136 ORF1a and ORF1b
• CDC recommends two nucleocapsid targets (N1 and N2)
• WHO recommends initial screening with E gene followed by confirmation
using the RdRp
• Ideal design would include at least one conserved region and one specific
region to mitigate against the effects of genetic drift

27. Result interpretation
• If all the targets in the RT-PCR assay test positive, a case considered to
be laboratory confirmed
• Cycle threshold value (Ct-value) less than 40 defined as a positive
test, while a Ct value of 40 or more is defined as a negative test
• Ct-value <40 only one of the two targets is defined as indeterminant
and requires confirmation by retesting
• The assays with three targets, positives for two or more targets are
considered positive
• CT values have nothing to do with clinical manifestations
Bain W, Lee JS, Watson AM, Stitt‐Fischer MS. Practical guidelines for collection. Manipulation and inactivation of SARS‐CoV‐2 and COVID‐19 clinical specimens. Curr Protoc Cytom.
2020;93:e77

29. Limitation
• Sample storage prone to contamination
• High temperature during the processing of swabs collected from COVID‐19
patients responsible for at least 10% false‐negative results
• Problems during the collection of swabs, such as bleeding or coughing and
sneezing
• Low-quality nucleic acid purification
• Cost
• Wait time

30. RT loop-mediated isothermal amplification
(RT–LAMP)
• Based on nanotechnology
• Detected by levels of turbidity or by colorimetric or fluorescence measures
• Can be recorded by mobiles also using special software
• High specificity and sensitivity
• Whole procedure can be done in a portable machine and executed in a short time
• Ability to perform the test with RNA extraction approach or without RNA
extraction
• Less affected by the presence of inhibitors
• Limit of this assay as low as 1–10 copies of the RNA molecules
• Many advantages over other NAT assays
Zhu, X. et al. Multiplex reverse transcription loop-mediated isothermal amplification combined with nanoparticle-based lateral flow biosensor for the diagnosis of
COVID-19. Biosens Bioelectron. 166, 112437 (2020)

32. The recombinase polymerase
amplification (RPA)
• Sensitive, accurate and fast
• Isothermal amplification of the target DNA
• Nest‐RPA: two subsequent reactions, including amplification of a
flanking region of a target sequence, then the nest reaction is usually
conducted using internal primers inside the flanking region
Huang W, Lin D, Wang C, et al. The determination of release from isolation of COVID‐19 patients requires ultra‐high sensitivity nucleic acid test technology. J Infect. 2021;82:159‐198

33. CRISPR‐based assays
• Conducted in two consecutive steps
• Preamplification RNA step
• Second, applying the CRISPR/Cas13a enzyme activity to target a
specific sequence within the viral RNAs
• Conducted in about 50 min
• Detection limit of as little as two copies of RNA
Huang Z, Tian D, Liu Y, et al. Ultra‐sensitive and high‐throughput CRISPR‐p owered COVID‐19 diagnosis. Biosens Bioelectron. 2020; 164:112316

34. The specific high sensitivity enzymatic
reporter UnLOCKing (SHERLOCK) assay
• Conducted in three subsequent steps in less than an hour
• First step: isothermal incubation of the extracted RNAs
• Second step another incubation for the action of the RPA for 30 min
• Final step another incubation for the colorimetric visualization of the
results for at least 2 min enhanced by the action of the Cas‐12 or
Cas‐13
Patchsung M, Jantarug K, Pattama A, et al. Clinical validation of a Cas13‐based assay for the detection of SARS‐CoV‐2 RNA. Nat Biomed Eng. 2020;4:1140‐1149

35. Diagnostics using nanomaterials
• Requires minimal sample preparation and provides fast and direct virus
detection
• Facilitate viral RNA extraction through coprecipitation followed by
polyamine ester functionalization via (3-aminopropyl) triethoxysilane
• Lower limit of detection is 30 virus particles
• Completed in 15 min
• Can quantify virus below standard nasopharyngeal swab and saliva viral
concentrations
Hildebrandt, N. et al. Energy transfer with semiconductor quantum dot bioconjugates: A versatile platform for biosensing, energy harvesting, and other developing applications. Chem. Rev. 117, 536–
711 (2017

36. Serology based Diagnostics: Antibody &
Antigen assays

37. Basic Pathology
• Neutralizing antibodies found in up to 50% of infected individuals by day 7
and in all infected individuals by day 14
• IgM levels increase during the first week after SARS‐CoV‐2 infection, peak
after 2 weeks and then fall back to near-background levels in most
individuals
• IgG detectable after 1 week and is maintained at a high level for a long
period
• Up to 84 days and serve to protect against reinfection
• IgA responses appear between 4 and 10 d after infection
• Low levels of IgA with IgG without known exposure suggest herd immunity
Long, Q. X. et al. Antibody responses to SARS-CoV-2 in patients with COVID-19. Nat. Med. 26, 845–848 (2020)

38. Antibody detection
• Commonly immune response against the viral spike (S) protein
• N-based immunoassays, SARS-CoV-2 IgG (Abbott) shows a sensitivity
of up to 100%
• S-based immunoassays, Liaison SARS-CoV-2 S1/S2 IgG and the
combination S- and N-based platform COVID-19 VIRCLIA IgG
MONOTEST demonstrated equivalent sensitivities
• Time to results can vary from 13 min (Abbott ID NOW) to 45 min
(Cepheid Xpert Xpress)
• Detected colorimetrically

39. Pros & Cons
• Provide the advantage of fast time to result and low cost of detection
• Suffer from poor sensitivities
• Nonspecific responses of IgM and weeks required to develop specific
IgG responses limit their use in active case management
• Can play a role in diagnosing late infection
• Greatest potential to understand the true scale of human-to-human
transmission
Huang, C., Wen, T., Shi, F., Zeng, X. & Jiao, Y. Rapid detection of IgM antibodies against the SARS-CoV-2 virus via colloidal gold

40. Other newer methods
• Immunofluorescent assay (IFA)
• Lateral flow immunoassays (LFIA)
• Luciferase immunoprecipitation system (LIPS) assay
• The gold‐immunochromatography assay (GICA)
• The microneutralization and the pseudoviral particle neutralization
(PPNT) assays

41. Pitfalls
• Practical way is to test two serum samples collected during the acute
febrile stage of the diseases and another sample collected about four
weeks later
• Inconsistency in the level of the seroconversion
• Patients may be asymptomatic and seroconvert while they are still
shedding the virus in high amounts
• Cross‐reactivity of most coronaviruses

42. SARS-CoV-2 antigens
• Rapid diagnostic assay
• In samples from the respiratory tract of infected individuals
• Detected antigen(s) expressed only if the virus is actively replicating
• Tests can be used to identify acute or early infection
• Useful initial screening tests
• Best of assays showed 50% sensitivity when compared to the RT‐PCR

43. Ultra-sensitive antigen assay
• Double antibody sandwich ELISA assay
• For the detection of spike antigen
• Thio‐NAD cycling allowed a high sensitivity starting 10 min of the
application of the assay
• Characterized by high specificity and ultrasensitivity
Kyosei Y, Namba M, Yamura S, et al. Proposal of de novo antigen test for COVID‐19: ultrasensitive detection of spike proteins of SARS‐CoV‐2. Diagnostics. 2020;10:594

44. Pitfalls
• Cross‐reactivity among human coronaviruses
• Used antibodies polyclonal; thus may pick up several antigens of
closely related viruses
• Hamper both specificity and sensitivity

45. Home based assay’s

46. Lucira COVID-19 all in one test kit
• Self-collected nasal swab samples in individuals aged 14 and older
• Utilizes RT-LAMP technology to detect RNA of the N gene for SARS-
CoV-2
• Can create a signal from a few copies of RNA in less than 30
minutes
• Successful amplification reaction creates a pH change and
subsequently a color change of the halochromic agent

47. Other FDA approved home based tests
• Elverlywell: First authorized by FDA ; Nasal swab needed to sent to lab
• Let’sGetChecked
• Phosphorus: Saliva samples RT-PCR
• Ellume home test

48. Coviself: Home based rapid antigen assay
• Mylab Discovery Solutions invented
• ICMR’s approval for India’s first self-use Rapid test
• Not need sample collection by a healthcare professional
• Very specific ; less sensitive
• Need to insert the nasal swab up to 2-4 cm until resistance is met
• Immunochromatographic nitrocellulose membrane assay
• Uses highly sensitive antibodies to detect N-protein
• Read results within 10-15 minutes
www.coviself.com

51. Other Clinical & Radiographic diagnostic tools

52. Saliva testing
• Presence of SARS-CoV-2 RNA in saliva samples not always associated with disease
severity, compared to nasopharyngeal swabs
• Easy directed and non-invasive method of sampling
• Can be stored in stabilizing solutions and posted several days later in the testing
centre
• May facilitate the detection of both the virus itself and the antibodies
• Major potential for COVID-19 screening
• 72% of individuals with COVID-19 unable to produce sufficient sample volume
• Mean SARS-CoV-2 titres were five times higher (P < 0.05) in saliva (n = 37)
compared with nasopharyngeal swabs (n = 46)
Azzi, L. et al. Saliva is a reliable tool to detect SARS-CoV-2. J. Infect. 81, e45–e50 (2020)

53. Fecal tests
• High incidence and viral persistence in faeces have been observed
when nasopharyngeal swab samples were virus negative
• Viral load can be detected up to four weeks
• Documented gastrointestinal symptoms support faecal–oral
transmission routes
• Live virus can be isolated
• May be tracked through wastewater, which enables community
surveillance and could be a powerful tool in tracking COVID-19 spread
Nobel, Y. R. et al. Gastrointestinal symptoms and COVID-19: case–control study from the United States. Gastroenterology 159, 373–375.e2 (2020).

54. Use of electron microscope
• Sample of choice stool samples from humans
• Unique crown or solar morphology of coronaviruses
• Through the negative staining technique

55. Chest Xray
• Abnormalities include bilateral lower zone and peripherally
predominant consolidation and hazy opacities
• Affords 69% sensitivity
• Negative CXR alone cannot rule out lung involvement
• Features often non-specific
• Does not rule out alternative infections, especially in the context of
typical signs and symptoms
Wong, H. Y. F. et al. Frequency and distribution of chest radiographic findings in patients positive for COVID-19. Radiology 296, E72–E78 (2020)

56. HRCT Thorax
• Illustrate bilateral pulmonary parenchymal ground-glass and consolidated pulmonary
opacities with occasionally rounded morphology and marginal lung dispersal
• ‘Reversed halo’ pattern and signs of septal thickening
• With negative RT–PCR can present with abnormal chest CT and later be diagnosed with
COVID-19 specially for recent mutant strains
• In a study sensitivity of chest CT scans for COVID-19 97%, on the basis of positive RT–PCR
results, 75% (308 of 413 patients) had positive chest CT scans with negative RT–PCR
results
• CT abnormalities may precede symptom onset in 44% of the patients
• Abnormalities on chest CT may be seen even in asymptomatic individuals with positive
RT–PCR
Ai, T. et al. Correlation of chest CT and RT–PCR testing in coronavirus disease 2019 (COVID-19) in China: a report of 1014 cases. Radiology 296, E32–E40 (2020)

57. Recommendations
• CT should not be used for first-line screening of COVID-19, but should
be used sparingly for hospitalized, symptomatic patients with specific
clinical indications
Czawlytko, C., Hossain, R. & White, C. S. Covid-19 diagnostic imaging recommendations. Appl. Radiol. 49, 10–15 (2020)

58. MRI Thorax
• Provide much more detail of the pathologies in the soft tissue than CT
• American College of Radiology advises medical facilities to avoid
performing MRI
• Sanitizing MRI machines takes a long time and poses significant
challenges
• High-efficiency particulate air (HEPA) filter systems, typically used to
increase air exchange, are not compatible with MRI
ACR Guidance on COVID-19 and MR Use (Americal College of Radiology, 2020)

60. Mass pool screening
• Pooling can provide surveillance for infected individuals
• High-throughput, highly automated PCR testing and matrices are
pooling strategies
• Pool size depends upon the prevalence of the population
• Pooling eliminates up to 80% of reagent cost when tested in a
population having a prevalence of positive samples of ≤1% and hence
decreases the global costs
• Can be adopted by schools, universities, workplaces and religious
organizations that seeking to reopen
Lim, K. L. et al. A novel strategy for community screening of SARS-CoV-2 (COVID-19): sample pooling method. PLoS ONE 15, e0238417 (2020

61. Metagenomic profile
• Metagenomic next-generation sequencing (mNGS) can detect whole
viral genomes and any coinfection
• Revealed positive SARS-CoV-2 samples that matched the database of
SARS-CoV-associated viruses
• Detects all the sequences lined up to already known bacterial and
viral databases
• Enabled the study of viral origins from different sources
• Helpful in predicting future outbreaks before pandemics emerge

62. Summary
• NAB assays Gold Standard
• Newer NAB assays less time consuming & highly sensitive
• Clinical diagnosis relies on a combination of chest CT and RT–PCR
• Positive sampling rates vary widely between various samples
• Immunodiagnostic approaches most viable 7–11 d after exposure and
are therefore less useful in diagnosis of acute infection
• Home based assays can be the future cornerstone for diagnosing in
upcoming epidemic waves

63. Thank you………….