A modern approach to viral diagnosis of Covid

of Covid

Prof. Carlo Federico Perno

Bambino Gesù Children’s Hospital

A modern approach to viral diagnosis

«COUGH OF PERINTHUS»

First influenza epidemic of human history

Corpus Hippocraticum: Books Epidemics II

Alexandria; late 2°-1° c. BC

Pathology of the lower respiratory track insed

Corpus Hippocraticum.

Lung anatomy, Anatomie del l’homme, Bourgery and Jacob, Guérin Editions, Paris 1862

Amodern approach to viral diagnosis of Covid

Discovery of human influenza virus (Smith, Andrewes, Iaidlaw)

Scagnolari et al., Expert review of anti-infective therapy, 2016

Amodern approach to viral diagnosis of Covid

Scagnolari et al., Expert review of anti-infective therapy, 2016

Discover of PCR (Salki, Mullis)

Amodern approach to viral diagnosis of Covid

Influenza A

R&D Systems, a Biotechne Brand

Amodern approach to viral diagnosis of Covid

 Seasonal determinants in the epidemics of respiratory viruses include seasonal changes in temperature, absolute humidity (AH), sunlight, vitamin status, and host behavior.

 Environmental factors affect host susceptibility by modulating airway defense mechanisms and affect viability and transmission of respiratory viruses.

 Human behavioral patterns affect the contact rates between infected individuals and susceptible individuals.

Amodern approach to viral diagnosis of Covid

Moriyama et al., Annu. Rev. Virol. 2020

 The extrathoracic and tracheal mucosal surface defense is directly affected by the seasonal changes in temperature and water content of the inhaled air on both infected and susceptible hosts.

 Proper mucus hydration is required for the efficient mucous transport. Dehydration caused by dry breathing air leads to increased viscoelasticity of the mucous layer and immobilizes cilia, which are pressed down by the reduced height of the dehydrated periciliary layer.

Amodern approach to viral diagnosis of Covid

Moriyama et al., Annu. Rev. Virol. 2020

Respiratory viruses are classified according to their seasonal epidemics

Replication conflicts among those respiratory viruses can contribute to the nonoverlapping peak incidence with respect to one another. Interference between respiratory viruses has been recognized by epidemiological observation that influenza viruses and RSV do not share peaks during the same period even though both are prevalent in winter

Moriyama et al., Annu. Rev. Virol. 2020

Gomez GB et al., Science 2021

During the pandemic, circulation patterns of respiratory viruses other than severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been disrupted.

This could mean a future shift in the epidemiology of respiratory diseases, potential for new epidemic threats, or larger outbreaks than previously observed.

Amodern approach to viral diagnosis of Covid

• Since WHO declared the COVID-19 pandemic on March 11th 2020, non-pharmaceutical interventions (NPI), including mask wearing and social distancing, have been implemented worldwide to counteract the spread of SARS-CoV-2

• These measures have resulted in a reduced circulation of other respiratory viruses commonly responsible for winter seasonal infections in the pediatric population

But... the scenario has changed once again

Number of different viral pathogens detected in January-December 2019 (a), in January-December 2020 (b) and January-October 2021(c).

All virus can cause overlapping clinical presentation making it difficult to distinguish the causative agents without a solid laboratory analysis

Acute respiratory tract infections are one of the leading causes of childhood morbidity and mortality worldwide, and it has been estimated that globally, respiratory infections are responsible for about 2 million deaths in children between 0 and 5 years of age.

Approximately 80% of these respiratory infection cases are caused by viral pathogens such as influenza A and B, respiratory syncytial virus (RSV) A and B, parainfluenza virus types 1–3, adenovirus, rhinovirus, human metapneumovirus (hMPV), and others.

The non-specific clinical presentation of respiratory infections poses a considerable challenge to the differential diagnosis of these pathogens.

Early and accurate diagnosis of the causative pathogens in respiratory infections is essential to administer appropriate antiviral or antibacterial therapy, initiate effective infection control measures, and reduce the length of hospital stay.

Amodern approach to viral diagnosis of Covid

Das et al, frontiers in microbiology, 2018

DIAGNOSIS

Amodern approach to viral diagnosis of Covid

The cornerstones of SARS-CoV-2 diagnosis

DIRECT DETECTION OF (PARTS OF) THE VIRUS

GOLD STANDARD

• Detection of nucleic acids via amplification (RT-PCR), ± sequencing

IMMUNOLOGICAL TESTS

• Detection of virusspecific antibodies

SCREENING,

PRESUMPTIVE

• Detection of one or more proteins (antigenic test)

• Definition of cellular immune response

• Culture

for SARS-CoV-2

• Rapid diagnostic test (RDT)

• Simple and rapid test, based on lateral flow immunoassay (LFIA) technology. It can be a point-of-care (POC) test or a self-test, with results available in 10-30 min.

• Chemiluminescence immunoassay (CLIA)

• High-throughput laboratory-based assay with high level of analytical agreement, and average time-to-result of 1-2 h.

• Enzyme-linked immunosorbent assay (ELISA)

• High-throughput laboratory-based assay with high level of analytical agreement, and average time-to-result of 2-5 h.

• Neutralization assay

• Lab-based test that uses live virus and cell culture methods. This test must be performed in laboratories with designated biosafety certificates to culture SARS-CoV-2-infected cells and has a time-toresult of 3–5 days.

There are four major types of serological diagnostic tests

Cell culture has a lower sensitivity than molecular techniques, the turnaround time and hands-on time required to perform cell culture compared to molecular testing are increased, and the technical expertise for performing cell culture is often not available.

Positive viral culture results would not impact the management of healthy children hospitalized for illness attributed to community acquired respiratory viral infection due to the delay in time to culture positivity

modern approach to viral diagnosis of Covid

DFA and IFA assays do not require viable viruses and the turnaround times are short (4 h)

DFA/IFA technologies are labor-intensive, require a skilled technologist to read and interpret results, require a fluorescence microscope, and are subject to reader error. Furthermore, the hands-on time required per test is not structured for high-throughput result reporting.

Compared to molecular detection methods, DFA and IFA assays have significantly reduced sensitivity and specificity

Charlton et al. Clinical Microbiology Reviews January 2019

The mainstay of laboratory diagnosis of viral respiratory infections is the rapid identification of viral antigens and/or genetic material (either DNA or RNA) in freshly collected respiratory specimens, by a number of molecular assays:

Molecular tests are more sensitive, specific and accurate than other rapid diagnostic tests such as immunochromatography, allowing the detection of one or more respiratory viruses in biological samples

Nucleic Acid Amplification Test (NAAT)

The clinical signs and symptoms of acute respiratory tract infections (RTIs) are not pathogen specific.

Highly sensitive and specific nucleic acid amplification tests have become the diagnostic reference standard for viruses, and translation of bacterial assays from basic research to routine clinical practice represents an exciting advance in respiratory medicine. How best to use newer molecular tests for RTI in combination with clinical judgment and traditional methods can be bewildering given the plethora of available assays and rapidly evolving technologies.

The detection power of PCR was better than that of traditional antigen tests and virus cultures when considering the detection of respiratory viruses

Amodern approach to viral diagnosis of Covid

Amodern approach to viral diagnosis of Covid

Amodern approach to viral diagnosis of Covid

RT-PCR cycle-threshold values <20 were one of the strongest predictors of inhospital death, both in univariate (HR: 8.38 [2.66-26.37], p=0.00015), both in multivariate-analysis (HR: 3.94 [1.758.87], p=0.001; along with comorbidities presence, creatinine, D-dimers, and Creactive protein).

Alteri C. et al., Clin Inf Dis 2020

Kaplan-Meier curves showed a progressive increase of 30day in-hospital mortality, by increase in nasopharyngeal SARS-CoV-2 load

Multiplex RT-qPCR method advantages

 Although single respiratory viruses can be detected in patients with symptoms, other respiratory viruses may also exist simultaneously.

 Children, especially those under 5 years of age, present with a higher frequency of coinfections.

 Multiplex assays that contain more than one viral gene target in a single tube have the advantage of rapid detection of several potential viral pathogens simultaneously.

 Multiplex real‐time PCR has allowed simultaneous detection of multiple respiratory viruses in a short time.

 Compared with the singleplex approach, the multiplex diagnostic approach has higher sample throughput (96 samples per run), shorter turnaround time (5 hours), and a smaller amount of sample requirement

Amodern approach to viral diagnosis of Covid

Sindromic Respiratory Panel

Amodern approach to viral diagnosis of Covid

POCT o Rapid Test:

Results in less than 30 min, relatively inexpensive and Easy to perform.

The sensitivities of these assays in pediatric and adult populations varied but were considered to be poor, and the assays could not be used to rule out infection

The lateral-flow immunoassay:

Das et al, frontiers in microbiology, 2018

The use of POCT can increase the efficiency of a hospital ward, however…….

…

.the competence of the staff performing the tests can impact the quality and accuracy of

POCT results:

 To ensure that POCT is performed safely and correctly, all hospital POCTs must be supervised by the central laboratory;

 The laboratory director must monitor performance standards in all domains of the POCT, including pre-analytical, analytical and post-analytical aspects.

Amodern approach to viral diagnosis of Covid

Point-of-care tests (POCTs) o Rapid Test:

Manufacturer Product Virus Detected

Luminex VERIGENE® Respiratory Pathogens Flex Test

GenMark Diagnostics eSensor® Respiratory Viral Panel (RVP)

Hologic Panther Fusion® Respiratory Assays

Seegene Allplex™ Respiratory Panel Assays

Adenovirus, Parainfluenza 1-4, Human Metapneumovirus, Influenza A (Flu AH1, Flu A-H1pdm09, Flu A-H3), Influenza B, Rhinovirus, Respiratory syncytial virus A-B

Adenovirus, Human Metapneumovirus, Influenza A (Flu A-H1, Flu A-H3), Influenza B, Rhinovirus, Respiratory syncytial virus A-B

Personalized Respiratory Testing: Influenza A virus, influenza B virus, respiratory syncytial virus, adenovirus, human metapneumovirus, rhinovirus, parainfluenza 1-4, SARS-CoV-2

Influenza A virus (Flu A-H1, Flu A-H1pdm09, Flu A-H3), Influenza B virus (Flu B), Respiratory syncytial virus A-B, Adenovirus, Enterovirus, Metapneumovirus, Parainfluenza 1-4, Bocavirus 1-4, Human rhinovirus, Coronavirus (229E, NL63, OC43)

Applied BioCode

BioCode® Respiratory Pathogen Panel (RPP)

Siemens Healthcare FTD Respiratory Assays

Adenovirus, Coronavirus (229E, OC43, HKU1, and NL63), Parainfluenza1-4, Respiratory Syncytial Virus A/B, Rhinovirus/Enterovirus, Human Metapneumovirus A/B, Influenza A (including subtypes H1, H1 2009 Pandemic, and H3), Influenza B

Adenovirus, Bocavirus, Coronavirus (229E; HKU1; NL63; OC43)

Enterovirus, Human metapneumovirus A/B, Parechovirus, Influenza A (H1N1), Influenza B, Mycoplasma pneumoniae, Parainfluenza virus 1-4, Respiratory syncytial virus A/B, Rhinovirus

“ a diagnostic test that is performed near the patient or treatment facility, has a fast turnaround time, and may lead to a change in patient management” .

Point-of-care tests are particularly suitable to detect COVID-19 positive

individuals in order to start self-isolation and contact tracing as soon as possible, with benefits on the spread of the infection

Forest plot of studies evaluating rapid antigenic tests

Forest plot of studies evaluating rapid molecular tests

Dinnes et al., Cochrane Database Syst Rev. 2021

Antigenic assays

There is a wide variety of commercially available FDA approved Rapid Antigenic Test (RATs) for the laboratory diagnosis of viral respiratory infection, based on either immune-chromatographic or immunofluorescence techniques.

They do not require laboratory support and the time to result is generally less than 30 minutes, so they are usually performed as POCTs, allowing their inclusion into the clinical decision-making algorithm.

Currently, commercial RATs availability is limited to the detection of influenza A virus, influenza B virus, RSV and SARS-CoV-2  These include latex agglutination tests, horizontal flow devices, lateral flow devices and optical immunoassay

Validation study of a rapid antigen test as a screening tool for SARS-CoV-2 infection.

Regrettably, their suboptimal sensitivity limits the clinical utility in the viral respiratory infection diagnosis

Is COVID-19 Testing going HOME?

By mid-September, the US Food and Drug Administration (FDA) had authorized emergency, overthe-counter use of 7 at home rapid antigen tests by both symptomatic and asymptomatic individuals.

Speed vs Sensitivity

Rapid antigen tests are increasingly used worldwide

Rapid antigen tests are cheaper and provide answers within minutes instead of a day or 2 (or 5 or 6) like PCR tests, which detect genetic material from SARS-CoV-2.

• research is beginning to suggest that they aren’t as sensitive in the real world as they appeared to be in clinical trials …

The third-generation LumiraDx test has a sensitivity of 91% for samples with Ct ≤ 29 (high viral-load), and of 34% for samples with Ct > 29 (low viral-load)

Not Positive About Negatives

«you could possibly still have COVID-19 even though the test is negative»

The antigen tests’ lower sensitivity translates into a higher rate of false negatives than with PCR-testing (is even lower in people who don’t have symptoms)

 If not CONFIRMED by RT-PCR you can miss diagnosis …

The mainstay of laboratory diagnosis of viral respiratory infections is the rapid identification of viral antigens and/or genetic material (either DNA or RNA) in freshly collected respiratory specimens, by a number of molecular assays:

Molecular tests are more sensitive, specific and accurate than other rapid diagnostic tests such as immunochromatography, allowing the detection of one or more respiratory viruses in biological samples

Serologic diagnosis is of considerable help epidemiologically, but it cannot detect the presence of the virus in the early stage of infection and is therefore of limited use in all those conditions that require a prompt response to infection (including the isolation of patient and/or the initiation of antiviral therapy)

Amodern approach to viral diagnosis of Covid

Diagnostic testing for respiratory viruses has been revolutionized by recent advances that have made rapid and highly accurate tests accessible to clinical laboratories

CRISPR-based DETECTR technology

A diagnostic method that can be readily adapted to detect infection from emergent viruses is urgently needed.

The future development of portable microfluidic-based cartridges and lyophilized reagents to run the assay could enable point-of-care testing outside of the clinical diagnostic laboratory, such as airports, local emergency departments and clinics and other locations.

A severe COVID-19 manifestation characterized 14 of the 19 patients with a ddPCR confirmed SARS-CoV-2 infection (73.7%)

modern approach to viral diagnosis of Covid

Evolution of «use-cases» during COVID-19 pandemics

INITIAL PHASE

• Triage of at-risk presymptomatic and symptomatic individuals

• Diagnosis of symptomatic individuals

• Differential diagnosis

PRE-VACCINE

• Triage of at-risk presymptomatic and symptomatic individuals

• Diagnosis of symptomatic individuals

• Differential diagnosis

• Confirmatory testing

• Testing of patients with previous COVID-19

• Surveillance and environmental monitoring

POST-VACCINE

• Triage of at-risk presymptomatic and symptomatic individuals

• Diagnosis of symptomatic individuals

• Differential diagnosis

• Confirmatory testing

• Testing of patients with previous COVID-19

• Surveillance and environmental monitoring

• Definition of vaccinebreakthrough infections

Amodern approach to viral diagnosis of Covid

cases had been declining, and

shifted resources to vaccinating people Weekly COVID-19 testing. Data provided by the European Centre for Disease Prevention and Control –

Many countries have now reduced testing because

they

FDA advisory panel recommends Pfizer vaccine for kids ages 5 to 11

A panel of independent advisers to the Food and Drug Administration is recommending that the agency issue an emergency use authorization for the Pfizer-BioNTech vaccine in children ages 5 to 11 years old.

The vote was 17 in favor and one abstention.

The FDA panel accepted Pfizer’s data indicating the vaccine is safe and 90.7% effective in preventing COVID-19 infections in this age group.

OPBG SARS-CoV-2 surveillance of variants

Amodern approach to viral diagnosis of Covid

First SARS-CoV-2 diagnoses with Ct<29 in the overall and in patients aged <18years from March 2020 to September 2021

The SARS-CoV-2 genome encodes approximately 25 proteins that are required for infection and replication, including four major structural proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N).

The S protein plays a critical role in fusion and entry into the host cell, and it comprises an N-terminal S1 receptor-binding domain (RBD), N-terminal domain (NTD), and a C-terminal S2 subunits. The primary function of the SARS-CoV-2 N protein (NP) is binding and packing of the viral RNA genome into a helical nucleocapsid structure during viral replication.

Studies on the serum of recovered COVID-19 patients suggest that host-neutralizing antibodies primarily work against S and N proteins. Consequently, the likelihood of predicting immunity status could increase in serological tests that target various regions of S or N proteins.

Therefore, the characterization of specific SARS-CoV-2 antigen domains targeted by the humoral immune response becomes an integral part of the serological test development

Serological, or antibody, tests detect immunoglobulins produced by the host’s plasma B cells following exposure to foreign antigens.

• The S protein forms a transmembrane homotrimer protruding from the viral surface to attach to the host cellular receptor ACE2.

The SARS-CoV-2 spike (S) trimeric glycoprotein is a focus of vaccine development because it is the primary target of host immune defenses.

• S comprises two functional subunits: subunit S1 responsible for binding to the cell surface receptor ACE2 and subunit S2 responsible viral fusion to the cell membrane. Wrapp

The Spike (S) protein (of about 150kDa) is the major antigen presented on the surface of SARSCoV-2

965 HCWs vaccinated at the Bambino-Gesu Children Hospital, Rome, Italy (292 [30.2%] males, median age: 46 years [IQR:3656]).

 Serum samples were available before the first dose and at 7, 21, and 28 days afterwards.

 All HCWs had a negative SARS-CoV-2 status by molecular (Allplex2019-nCov, Seegene) and antibody assays

 Presence and titers of anti-spike (S) antibodies in serum samples were evaluated by Elecsys®Anti-SARS-CoV-2- S (Roche, cut-off:0.8U/mL) detecting antibody against the S1-receptor- bindingdomain (RBD) and the LIAISON® SARS-CoV-2 TrimericS IgG assay (DiaSorin, cut-off: 13 AU/mL), detecting IgG against the trimeric S protein.

Recipients were stratified according to age (2355 or ≥56 years) and sex.

The highest anti-S titers were detected in samples at day 28, when 99.8% of subjects showed a median anti-RBD titer of 2003(IQR:1089- 2,500, Similar results were observed for the anti-trimeric S titers (Fig. 1B).

At day 7, while only few individuals had low positive titers (2 to antiRBD:3.31 and 6.95 U/mL, and 5 to the S protein: median 26 [IQR:21–31]) (Fig .1A–B), a primary cellular response was already present.

At day 21 (before the second administration), 97.5% of individuals showed positive anti-RBD antibodies, though with modest titers (median:54[IQR:26–109], Fig. 1A). Similar results were observed for antitrimeric S antibodies (Fig. 1B).

Zaffina et al., 2021

Hypothetical illustration of protective immunity against COVID-19.

Study summary and key findings.(A) Cohort design and objectives. Longitudinal samples were collected from SARS-CoV-2 naïve and recovered individuals and measured for both antibodies and memory B cells. (B) Distinct patterns of antibody and memory B cell responses to mRNA vaccination in SARS-CoV-2 naïve and recovered individuals. (C) Age-associated differences in antibody and memory B cell responses to mRNA vaccination. (D) Baseline memory B cells in SARS-CoV-2 recovered individuals contribute to recall responses after mRNA vaccination. Goel

Gating strategy for the detection of memory B cells specific for Trimeric Spike.

Amodern approach to viral diagnosis of Covid

Serum specific

2. PERSISTENCE OF SPECIFIC MEMORY B CELLS

antibodies

to RBD and Trimeric Spike

decline further six months after a complete vaccine cycle

Amodern approach to viral diagnosis of Covid

The decline of serum specific antibodies months after vaccination is a physiological event.

It occurs after every vaccine and in every individual

Nature Reviews Immunology volume 21, pages83–100(2021)

In contrast, Spike-specific memory B cells continue to increase six months after vaccination

2. PERFORMANCE OF SPECIFIC MEMORY B CELLS

In 17 HCWs with breakthrough infections, specific antibodies against Spike and RBD rapidly increase in the serum and reach the highest levels 4-6 days after the positive NPS.

In contrast, Anti N antibodies start to increase after 10 days.

Upon encounter with the pathogen for which they are specific MBCs have the function of rapidly producing antibodies in the serum and at the site of invasion, the oropharinx for SARSCoV-2

Accurate and fast diagnosis of SARS CoV2 is mandatory in order to:

 Save people’s lives

Stop the epidemics

But is also part of proper management to

 Reduce unnecessary use of antibiotics

 Select appropriate treatment for fragile patients

 Accurate detection of respiratory viruses is important in patient care, as it guides both therapy and infection control measures.

 In any infectious disease outbreak, accurate and accessible diagnostic testing must be one of the pillars of control-measure policies to understand and minimize the spread of disease