High Sensitivity and Specificity (With 95% Confidence Interval) of RealDetect™ COVID-19 RT-PCR Kit Clinical Trial
Official title:
Performance Evaluation of RealDetect™ COVID-19 RT-PCR Kit for the Detection of SARS-CoV-2 Virus
The novel Severe acute respiratory syndrome coronavirus 2 (SARS-C0V 2) originated in Wuhan,
China in December 2019. As of April 15 2020, the virus has spread across 213
countries/territories with 1,914,916 cases and 123,010 deaths and a crude case fatality ratio
(CFR) of 6.4%. In Bangladesh, the situation is also grave. As of May 14, 2020, there were
18,863 cases and 283 deaths.
In order to suppress COVID-19 transmission, it is important to diagnose COVID-19 patients,
which would help in the process of quarantine and isolation of the patients and also in
contact tracing. COVID-19 testing can identify the SARS-CoV-2 virus and includes methods that
detect the presence of virus itself such as real time reverse-transcription-polymerase chain
reaction (RT-PCR), isothermal nucleic acid amplification, antigen) and those that detect
antibodies produced in response to infection. Until now, RT-PCR has been known as the best
approach for - detection. It would be very useful if Bangladesh had its own locally produced
RT-PCR kits, provided that the kits are no less in quality than imported kits in terms of
sensitivity, specificity, price etc. The present study aims to carry out the performance
evaluation of RealDetect RT-PCR Kit for COVID-19 detection.
The RealDetect™ COVID-19 RT-PCR diagnostic panel is a locally produced real-time RT-PCR test
intended for the qualitative detection of nucleic acid from the SARS-CoV-2 in nasopharyngeal
swab specimens collected from individuals who meet SARS-CoV-2 clinical criteria. The approach
is based on the RT-PCR method which uses two (Nucleocapsid 1, Nucleocapsid 2) sets of
gene-specific primers and corresponding fluorescent probes to detect two specific regions
within the novel coronavirus (SARS-CoV-2) nucleocapsid protein Nucleocapsid gene. This RT-PCR
panel detects SARS-CoV-2 Ribonucleic acid (RNA) specifically. The approach does not generate
any false positives to other coronaviruses or human microflora. The kit also contains a
primer-probe set which detects human housekeeping gene, ribonuclease Protein (RNase P). That
is, the Ribonuclease Protein (RNase P) serves as an internal reference control to monitor
sample collection, ribonucleic acid (RNA) extraction, and amplification.
This is a case control study. The study will analyze 120 samples (60 COVID-19 positive and 60
COVID-19 negative both fresh and frozen) from Institute of Epidemiology, Disease Control and
Research (IEDCR). These specimens will be blinded before handing over to Institute for
Developing Science & Health Initiatives (ideSHi) for RealDetect Kit. All samples will be
analyzed by Real Time PCR System. Necessary validation will also be carried out at the
COVID-19 laboratory of the Dhaka Medical College Hospital and an external validation expert
will be involved. The Principal Investigator (PI) will also receive the sample information
regarding positive/negative status from Institute for Epidemiology, Disease Control and
Research (IEDCR) and compare ideSHi and IEDCR data. Unpaired t-test, Wilcox's test, Rank
test, Compare test, Mean test, Sensitivity/Specificity test, Regression analysis and
Geometric mean with 95% Confidence Interval (CI) will be used to analyze the data. It needs
to conduct a univariate analysis.
Considering the current global pandemic, just like any other countries it is becoming very
difficult for Bangladesh to fight the COVID-19 spread. An increased number of testing is
needed in Bangladesh and to cope with this demand an increase in assay kits are needed
urgently in the country.
Currently we are totally dependent on imported test kits. At the time when the whole world is
facing a shortage of RT-PCR test kits, import from other countries seems to be very
difficult. Primarily, countries manufacturing these kits are focused on meeting the demand in
their own countries first and thus those countries impose a ban on exporting these kits. So,
availability of the kits will become a big problem for Bangladesh to increase testing in the
country.
Secondly, due to lockdown situation worldwide, transporting these kits from another country
seems second big issue we are facing right now. The kits are stranded in the airports for
weeks before reaching in our country. Not to mention, the quality of the test kits is being
jeopardized in the whole uncertain transportation process. The RT-PCR test kits are very
temperature sensitive and maintaining the cold chain is mandatory in order to perform the
test properly.
Once approved, this qualitative RT-PCR based kit can be a principal tool for Bangladesh in
effective diagnosis and clinical management of COVID-19 along with collective actions made by
the govt. of Bangladesh. Being developed and manufactured in Bangladesh means that there will
be no shortage of testing kits during this pandemic and it will also be possible to export
the kit after fulfilling domestic needs. The kit will definitely be cost-effective compared
to other commercially available kits & the price will be around 30% less than the
commercially available kits in the country. Current purchase price of Bangladesh govt. per
test of the imported COVID-19 RT-PCR test is approx. Tk. 2780/= & RealDetect™ COVID-19 RT-PCR
per test cost will be approx. Tk. 1940/=
However, this type of performance evaluation study on RT-PCR method for the detection of
SARS-CoV-2 virus has not yet been done in our country. This is
the rationale why we have designed this protocol for performance evaluation of RealDetect™
COVID-19 RT-PCR kit in Bangladesh for the SARS-CoV-2 virus.
Hypothesis:
RealDetect™ COVID-19 RT-PCR will be an effective diagnostic tool for the detection of SARS
CoV-2 virus using nasopharyngeal swab specimens.
Methodology:
Study type & purpose:
This is a case control study. This study is expected to validate whether the locally
developed RealDetect COVID-19 RT-PCR kit is suitable for the use in Bangladesh for the
detection of SARS-CoV-2 in specimens collected from suspected COVID-19 patients.
Study Population and samples Testing and analysis of a total of 120 specimens will be carried
out. Of the 120 samples, 60 will be stored samples (30 COVID-19 positive samples and 30
COVID-19 negative samples) from IEDCR. The positive/negative status of the samples will be
blinded before sending to ideSHi. The samples have already been stored at -80 degree Celsius
in the IEDCR freezer.
In addition, 60 fresh specimens which will be tested at IEDCR will also be tested at ideSHi
using RealDetect on a real time basis. These samples will also be blinded by IEDCR and sent
to the testing laboratory (ideSHi) as they become available. Of these 60 samples, 30 will be
COVID-19 positive and 30 COVID- 19 negative samples. These fresh samples will be provided to
ideSHi for testing and analysis for performance evaluation of RealDetect COVID-19 RT-PCR kit.
The PI will coordinate these activities.
IEDCR will provide ideSHi with the samples needed for the performance evaluation study of
RealDetect RT-PCR Kit for COVID-19 detection. It should be mentioned here that IEDCR is the
government reference laboratory for COVID-19 detection in Bangladesh. There is a MOU between
ideSHi and IEDCR and these two organizations have been doing collaborative work from 2014.
Thus IEDCR has been involved in this study.
To avoid biasness, ideSHi (CRO) investigators will be blinded to the samples provided by
IEDCR. That is, ideSHi investigators will not know which of the 60 samples are positive and
which of the 60 samples are negative. After RT- PCR run using RealDetect RT-PCR Kit, ideSHi
will send the results to the Principal investigator. The Principal investigator will also
receive IEDCR results for the same samples. Finally, the PI will compare the results from
ideSHi and IEDCR and determine the sensitivity, specificity, positive predictive value and
negative predictive value.
Study Design: Case control study.
Statistical Basis of Sample Size:
Formulae:
Variable Notations:
Α (the probability of type I error) (significance level) is the probability of rejecting the
true null hypothesis (0.05) Β (the probability of type II error) (1 - power of the test) is
the probability of failing to reject the false null hypothesis (0.80).
PA (proportion of discordant pairs of type A), proportion of discordant pairs of type A among
discordant pairs (1/4) PD (proportion of discordant pairs) among all pairs (0.11)
Npairs, required sample size pairs 2x60=120 A matched-pair design is used, with one patient
in a matched pair assigned to RT-PCR positive for COVID-19 and the other one to RT-PCR
negative for COVID-19. How many matched pairs need to be enrolled in the study to have a 95%
chance of finding a significant difference using a two-sided test with type I error = 0.05?
So, we will use a total of 120 samples; 60 COVID-19 positive samples and 60 COVID-19 negative
samples.
Diagnostic Criteria for selection of COVID-19 positive and negative specimens:
IEDCR uses many different kits for COVID-19 detection. These kits include, (1) A*Star
Fortitute Kit 2 for COVID-19 detection (Singapure), (2) Sansure Biotec Limited (China), (3)
ModularDx Kit (TIB Molecular Biology, Germany, and (4) DaAn Kit (China). Among these 2 kits,
A*Star Fortitude Kit 2 has been known as the best kit in IEDCR depending on its performance
because it rarely generates false positive or false negative results.
A*Star Fortitude Kit 2 is used for confirmation of any discordant results for other kits in
IEDCR. That is, IEDCR uses A*Star Fortitude Kit for COVID detection as the Gold Standard. In
our case, this is the main reason for the selection of this kit in order to compare the
performances of RealDetect™ COVID-19 RT-PCR kit.
Consents from the patients are not required. Specimens will be obtained from IEDCR and so
there is no requirement of patient's enrollment and consents from the patients.
All specimens will be diagnosed as COVID-19 positive on the basis RT-PCR positive results
with Ct value ≤ 30 for the viral RNA. If the respiratory
specimens give positive results upon RT-PCR analysis at the lab targeting the conserved viral
gene(s) for SARS-CoV-2, it will be considered as confirmed positive case for COVID-19.
On the other hand, if the respiratory specimens give negative results with no amplification
upon RT-PCR analysis at the lab targeting the conserved viral gene(s) for SARS-CoV-2, it will
be considered as confirmed negative case for COVID-19.
Place of study: Institute for Developing Science & Health Initiatives (ideSHi) ; Mohakhali,
Dhaka.
Sample Collection Site(s): Virology Laboratory, Institute of Epidemiology, Disease Control
and Research (IEDCR), Mohakhali, Dhaka
Note: IEDCR will provide ideSHi with the samples needed for the performance evaluation study
of RealDetect RT-PCR Kit for COVID-19 detection. It should be mentioned here that IEDCR is
the government reference laboratory for COVID-19 detection in Bangladesh. IEDCR has its own
stored samples in - 80°C. There is a MOU between ideSHi and IEDCR and these two organizations
have been doing collaborative work from 2014.
Data Collection Procedure: COVID-19 positive samples will be confirmed by laboratory findings
of positive RT-PCR results for the viral RNA. Selection of the specimens will be carried out
following the inclusion and exclusion criteria. After data collection, data will be stored in
an Excel sheet for later analysis.
Data Analysis:
The sensitivity, specificity, positive predictive value and negative predictive value of
RealDetect™ RT-PCR kit will be determined. Alongside sensitivity, specificity, positive
predictive value and negative predictive value, Kappa coefficient, positive likelihood ratio,
and negative likelihood ratio will also be calculated
Unpaired t-test, Wilcox's test, Rank test, compare test, Mean test, Sensitivity/Specificity
test, Regression analysis and Geometric mean with 95% CI will be used to analyze the data. It
will need to conduct a univariate analysis. A p value of ≤0.05 will be considered as the
level of significant association. The sensitivity, specificity, kappa coefficient, positive
predictive value (PPV), negative predictive value (NPV), positive likelihood ratio (LR+),
negative likelihood ratio (LR-) and efficiency will be calculated using the following
formulae in which TP is the number of true positives, FP the number of false positives, False
negative (FN) the number of false negatives and True Negative (TN) the number of true
negatives: Sensitivity = True positive (TP)/[TP + FN]; Specificity = TN/ [TN + False positive
(FP)]; Kappa coefficient K=(Observed Agreement (OA)- Agreement by Chance (AC)/ (1-AC);
Accuracy = [TP + TN]/ [TP + TN +FP + FN]; Positive predictive value (PPV) = TP/[TP + FP];
Negative predictive value (NPV) = TN/[TN + FN]; LR+= sensitivity/ [100 - specificity] and LR-
= [100 - sensitivity]/specificity
Utilization of Results:
1. Bangladesh Medical Research Council (BMRC) ethical clearance is needed for performance
evaluation study of the RealDetect™ COVID-19 RT-PCR kit.
2. This will help for further processing of RealDetect™ COVID-19 RT-PCR kit for Director
General of Drug Administration (DGDA) regulations to legally market the products in
Bangladesh.
Facilities:
The Principal Investigator and other investigators of the Bangladesh University of Health
Sciences (BUHS) will be coordinating the evaluation and data analysis and publication.
However, the testing will be carried out in a separate organization using blinded specimens
from IEDCR at the ideSHi. It is one of the Government selected organizations which has been
assigned for COVID-19 testing to provide services and is also a registered Contract Research
Organization (CRO). The institute has international standard Biological Safety Levels (BSL) 2
plus and BSL facilities with sophisticated equipment like real time PCR machine which is
required for real time RT-PCR for COVID-19 detection.
Approval of the Head of the Department/Institute:
This collaborative study will be carried out in the following institutes:
Institute of Epidemiology, Disease Control and Research (IEDCR), Mohakhali, Dhaka (For sample
collection & sample storage) And Institute for Developing Science & Health Initiatives
(ideSHi); Mohakhali, Dhaka (For sample analysis, data collection, data analysis)
Ethical Implications:
To avoid the biasness, the study will be blinded to ideSHi researchers who will be involved
in the study.
Permission will be taken from Bangladesh Medical Research Council.
The privacy and confidentiality will be strictly maintained during and after data collection
and data storage.
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