Why why the negative covid19 PCR test is a misguide results?

1. Why the negative COVID19 PCR test is
a misguide result?
Presented by:
Amany Mohamed Elshamy
Chemistry Department, Faculty of science, Moeunfia University
MSc. Molecular Diagnostics of human diseases, University of Sadat City
MBA Student, the University of Suffolk, United Kingdom

2. Significance
The Americas confirmed cases
are 9,981,204
Deaths were 372,008
IFR=3.72%
The Western Pacific confirmed
cases=347,593
Deaths=8,577
IFR=2.46%
Confirmed cases =
19,936,210 cases
Death tolls= 732,499
Infection Fatality Rate (IFR)
(12th August 2020) = 3.67 %
The confirmed cases in Africa
= 848 053 cases
Deaths= 15,252
IFR= 1.79%
Until July 30 2020 and among 133
countries, 53% (n=3 129 345) were
male and 47 % (n=2 782 474) were
female
Eastern Mediterranean
confirmed cases=1,598,640
Deaths=42,052
IFR= 2.63%
6th August
2020

3. • RT-PCR test sensitivity values was about 70%- 90% at the beginning of the current
crisis
In the US, 43% of
businesses had
temporary closed
In the US, the employment
had fallen by 40%
• The Estimation of the rapid kit performance that relies on many factors ex; targeted
protein, the manufacture design and prevalence was (50.4%-100%).
Loss of Index of Industrial Production (IIP) across countries:
Loss IIP in High-income countries =30%
Loss IIP in Upper middle-income =24%
Loss IIP in Lower low-income =22%

4. Modes of transmission
Airborne
droplets
fecal-
oral
Direct
Contact
Animal-to-
Human
Mother
-to-
Child
Fomite
Symptomatic
patients
At least 23%
Asymptomatic
cases/infection
Cause
Asymptomatic transmission
23% - 41%
Infection
Pre-symptomatic transmission
No
develop
symptoms
yet
Up to 50%˜

5. The common COVID19
diagnostic methods
Clinical
symptoms
Radiology
Chest CTLaboratory
• Laboratory tests:
1) Nucleic acid Amplification Test (NAAT)
2) Antigen Based Immunoassay
3) Antibody Based Immunoassay

6. What are the most accurate COVID19
diagnostic tools?

7. ( Kubina & Dziedzic 2020)
SARS-CoV-2 genome
• SARS-Cov2 genome encodes different types of structural and non-structural
proteins.
• The central structural proteins are Spike (S) protein, Nucleocapsid (Nc)
protein, Membrane (M) protein and Envelope (E) protein (mainly responsible
for Genome maintenance, Cell membrane attachment, Immune evasion,
Antibodies neutralization, and Viral pathogenesis)
• The non-structural proteins, such as SARS polymerase (RdRp) and SARS
protease (3CL) ( translation, transcription, replication and viral assembly) .

8. SARS-CoV-2 genome
• (Wang, Hozumi, Yin, & Wei, 2020), Michigan State University,
collected 6156 genome samples across many countries, they
found that there are 5 clusters of the COVID-19 based on
4459 mutations that occurred in 8 different (structural and
non-structural) SARS-CoV-2 proteins.
Figure showed the distribution of 5 distinct COVID19 clusters in the world

9. The SARS-CoV-2 subtypes
The COVID19 is clustered into 5 subtypes
Cluster
I
cluster
II
Cluster
III
Cluster
IV
Cluster
V
Asian countries
Japan, Korean
United States, Canada and Australia
European countries
The dominant one at
the early of the crisis
Table showed the
distribution of
COVID19 subtypes
across 11 countries
(Wang, Hozumi, Yin,
& Wei, 2020)

10. Genome SARS-CoV-2 sequence mutations
• Since June 2020 (Wang, Hozumi, Yin, & Wei,
2020) study have traced the highly mutations
ratio occurred in the three dominant PCR-
based viral proteins (genes of N ,E and RNA-
dependent RNA polymerase (RdRP) in
ORF1ab region) .
• The nucleocapsid (N) protein is the worst
choice to be detected as it has 235 unique
mutations.
• The S protein is the most divergent with 385
unique mutations among the 6156 SARS-CoV-
2 genomes.
• The E protein has 13 mutations.
• RdRP gene has 223 mutations.
Table showed the
variations in the
COVID19 genome
across 11 countries
Thus may be responsible for the false
negative PCR results in some patients
samples.
(Wang, Hozumi, Yin, & Wei,
2020)

11. They could identify
the 5775 distinct
genome variants
particularly in S
and ORFab1
regions
Koyama et al.,
downloaded the
10,022 SARS CoV-2
genomes from
different databases
that infected cases
in 68 countries.

12. The Commercial RT-PCR kits for testing COVID19 with
FDA approval or WHO recommendation
Target geneName of kitCompany
ORF1ab and NCOVID-19 Real-time PCR
Testing Kit
Edinburgh Genetics
RdRPCOVID-19 genesis Real-Time
PCR assay
Primerdesign Ltd.
E , ORF1ab and NLiferiver Novel Coronavirus
Multiplex RT-PCR
Shanghai ZJ Bio-Tech
ORF1ab and NPerkinElmer® SARS-CoV-2 Real time
RT-PCR
PerkinElmer Inc.
ORF1a, ECobas® SARS-CoV-2Roche Molecular Dia
N and RdRPAbbott RealTime SARS-CoV-2 testAbbott
E, ORF1a, N and RdRPXpert Xpress SARS-CoV-2Cepheid (NAT-POC)
E, ORF1ab and NDMed 2019-nCoV RT-qPCR
Detection Kit
3D Biomedicine
AutomatedLab-based

13. Technical Limitations of the COVID19
commercial PCR kits
PCR results are
positive in the
COVID19
infected and
symptomatic
patients
Pre-
symptomatic
COVID19
infected person
or has no
symptoms at all
Confirmedmethod
FalseNegativePCRresults
PCR
Advantages
PCR
Drawbacks
The most commercial kits
have a certificate of
Research Use Only (RUO).
Various kind of samples could be
examined:
1. Sputum (sensitivity 90.3%-97.2%)
2. Tracheal aspirate
3. nasal swabs and throat swabs
(sensitivity 73.3%)
4. Stool/ rectal samples (up to 23%)
5. Urine
• Sample collection ,Transport conditions , well-skilled
and labor intensive and insufficient amount of viral
genome in the specimen or in the respiratory tract
epithelium, handling (RNA virus) and the limit of
detection for each kit.
• False negative results are common within the first 7
days of infection and undetectable after 15 days.
Factors

14. What are the PCR Commercial kit limitations for
suspected cases?
• Generally, PCR is an antigen based test and considerable as
a gold diagnostic tool in the identifying of the COVID19
infected and symptomatic patients. Sensitivity = 90.3 –
99.7%The approval commercial
kits are designed to detect
ORF1ab,E, N, or RdRP
genes.
˜200 COVID19 testing kits
for Research Use Only
(RUO(, the most targeted
genes are E and RdRp.
The limit of detection for
each kit.
Test time or day, Sample
collection & Transport
conditions
High genetic diversity
and evolution
Lack of validation “quality”
So that there is an urgent
need to re-establish the
robust kits via SARS-CoV-2-
dedicated primers to
precisely detect the vast
Coronavirus genotypes to
mitigate the transmission
and rightly diagnose the
asymptomatic people.

15. CT scan images
• The typical chest CT images
is the second common tool
and more reliable
comparing to PCR results.
• CT shows the unique
bilateral pulmonary
ground-glass opacities and
consolidation shapes with
high sensitivity (Karam et
al.,2020).

17. Principle of Serology test
The titer of IgM and IgG
antibodies against a new SARS-
COV-2 infection have been raised
simultaneously or sequentially in
the circulation after 2 days and
disappeared between 2-3 months
of infection , however, till now the
longevity is unknown (Long et al.,
2020).
Figure shows the seroconversion of antibodies after symptoms onset (Long et al., 2020)
Figure shows the antibodies titer in sever and non-sever clinical symptoms (Long et al., 2020)
SensitivityAntibodies IgG/IgM
per week
<30.1%1st week
72.2%2nd week
91.4%3rd week
96.0%After 35 days
(Deeks et al., 2020)

18. Figures show the concentration titer of different Abs after the symptoms onset (Liu et al., 2020; Guo et al 2020)

19. Point-of-Care and serological assay
Tables from (Kubina &
Dziedzic 2020) display the
evaluation of serological
tests validated by the FDA
and clinical significance
results of IgM/IgG
serological test

20. Disadvantages Advantages
Friendly used and
short TAT
Low recourses
regions
costs (around £6 per
test or $ 8-10)
False +ve result
Depend on viral
activity status
Low sensitivity and
specificity at the first
week of infection
Stratify people
into groups (at-
risk or not).
Easy to be implemented in
the high burden areas with
low incomes.
Use various kinds
of samples: blood,
plasma, serum, CSF
and stool.
Serological
detection tests
It may be a non-invasive,
useful and affordable
tool for identifying of
asymptomatic or carrier
cases.
For screening
purposes in the
vital places .

22. Promising platform
CRISPR-POC platform
technology
Cost-effective and minimal training platform
Nucleic acid sensing providing a high sensitive and
accurate results
30-60 min
Loop-mediated isothermal
amplification (LAMP)
Inexpensive
ultrasensitive with high specificity as it relies on 6
primers to detect 8 targets in the same reaction
60 min to get results
Image from Feng Zhang laboratory showing SARS-COV-2 lateral
flow strip readout

23. Conclusion
To improve the approach, more accurate results and enhancing the
early reporting
PCR
Clinical
symptoms
Serology test
CT images

24. References
• Böger, B., Fachi, M. M., Vilhena, R. O., Cobre, A. F., Tonin, F. S., & Pontarolo, R. (2020). Systematic review with meta-analysis of the accuracy of
diagnostic tests for COVID-19. American journal of infection control, S0196-6553(20)30693-3. Advance online publication.
https://doi.org/10.1016/j.ajic.2020.07.011
• Deeks, J. J., Dinnes, J., Takwoingi, Y., Davenport, C., Spijker, R., Taylor-Phillips, S., Adriano, A., Beese, S., Dretzke, J., Ferrante di Ruffano, L., Harris, I.
M., Price, M. J., Dittrich, S., Emperador, D., Hooft, L., Leeflang, M. M., Van den Bruel, A., & Cochrane COVID-19 Diagnostic Test Accuracy Group
(2020). Antibody tests for identification of current and past infection with SARS-CoV-2. The Cochrane database of systematic reviews, 6(6),
CD013652. https://doi.org/10.1002/14651858.CD013652
• Karam, M., Althuwaikh, S., Alazemi, M., Abul, A., Hayre, A., & Gavin, B. (2020). Chest CT versus RT-PCR for the Detection of COVID-19: Systematic
Review and Meta-Analysis. MedRxiv, 2020.06.22.20136846. https://doi.org/10.1101/2020.06.22.20136846
• Kashir, J., & Yaqinuddin, A. (2020). Loop mediated isothermal amplification (LAMP) assays as a rapid diagnostic for COVID-19. Medical
hypotheses, 141, 109786. https://doi.org/10.1016/j.mehy.2020.109786
• Kubina, R., & Dziedzic, A. (2020). Molecular and Serological Tests for COVID-19 a Comparative Review of SARS-CoV-2 Coronavirus Laboratory and
Point-of-Care Diagnostics. Diagnostics (Basel, Switzerland), 10(6), 434. https://doi.org/10.3390/diagnostics10060434
• L. Guo et al., “Profiling Early Humoral Response to Diagnose Novel Coronavirus Disease (COVID-19).,” Clin. Infect. Dis. an Off. Publ. Infect. Dis. Soc.
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• Long, Q.-X., Liu, B.-Z., Deng, H.-J., Wu, G.-C., Deng, K., Chen, Y.-K., … Huang, A.-L. (2020). Antibody responses to SARS-CoV-2 in patients with COVID-
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• W. Liu et al., “Evaluation of Nucleocapsid and Spike Protein-based ELISAs for detecting antibodies against SARS-CoV-2.,” J. Clin. Microbiol., Mar.
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• (https://www.synthego.com/blog/crispr-coronavirus-detection)
• https://www.unido.org/stories/coronavirus-economic-impact-10-july-2020
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25. Thank you
002 01000886828
@ amanyelshamy@icloud.com