Efficacy of a Multiplex BangasureTM RT-PCR Kit for the Detection of COVID-19
Condition: COVID-19
MeSH terms: COVID-19
Study type: Observational [Patient Registry]

Summary: 
      The outbreak of the severe acute respiratory syndrome (SARS) corona virus (CoV)-2 continues
      to increase globally. To control the coronavirus disease 2019 (COVID-19) transmission,
      diagnose is very important step which would help in the process of quarantine, isolation of
      the infected person and also in contact tracing. Among the various techniques real-time
      reverse transcription polymerase chain reaction (rRT-PCR) is mostly used and considers as
      gold standard method for viral nucleic acid detection as per World Health Organization (WHO)
      and Centre for Diseases Control (CDC), USA. However, possible false-negative and
      false-positive results produce misleading consequences, making it necessary to improve
      existing methods. So, investigators need a robust and reliable PCR kit to detect different
      kinds of variant of SARS-CoV-2 virus to reduce its transmissibility and take preventive
      management plan. Beside that to manage the increasing number of testing, investigators are
      mostly dependent on imported test kit which seems to be very difficult at this pandemic
      situation and costly. Hence, virulence of SARS-CoV-2 virus may change rapidly due to mutation
      and country become hot spot so, gene detection RT-PCR kit is time demand. In this point of
      view, investigator's aim to develop an validate multiplex rRT-PCR qualitative diagnostic
      method, which targets two viral genes E (envelope protein), N (nucleocapsid gene, according
      to the CDC, USA guidelines for the development of RT-PCR kit and one human gene RNase P as
      internal control simultaneously.

      This is a case control study where 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). This specimen will be blinded before supplying over to NILMRC/Contact ROL/
      Bangladesh Clinical Trail Limited (BCTL), Dhaka, Bangladesh for Bangladesh Reference
      Institute for Chemical Measurements (BRiCM) RT-PCR Kit. Required in house validation will
      also be conducted as per International Council for Harmonization (ICH) and FDA guideline and
      also external validation will be carried in different Director General of Health Service
      (DGHS), Bangladesh approved COVID-19 diagnostic laboratory. Data will be analyzed by computer
      using Statistical Package for the Social Sciences (SPSS) software and will be presented into
      the frequency table, graph and chart according to the requirements.
    

Detailed description: 
      Introduction:

      The corona virus COVID-19 pandemic is the defining global health crisis of our time and the
      greatest challenge that have faced since World War Two. The pandemic is much more than a
      health crisis; it's also an unprecedented socio-economic crisis. Since its identification in
      the region of Wuhan, China, not only the virus has spread across 213 countries/territories
      with 209 million cases and 4.39 million globally but also could put 400million people out of
      their jobs as The International Labor Organization estimates.

      To contain the spread of disease, multidisciplinary strategies have been launched in
      different regions of the world, including implementing social distancing, maintaining
      personal hygiene, contact tracing, and implementing quarantine, travel restrictions, and lock
      downs. A widely accepted method is testing for SARS-CoV-2, typically utilizing nasopharyngeal
      swab specimens. Patients tested positive require appropriate clinical management by either
      effective isolation or quarantine at home for mild symptoms or within health care facilities
      for moderate to severe symptoms. In addition, wide-scale testing provides more informative
      epidemiologic data for drafting policies on disease monitoring and control. Many approaches
      have been proposed to detect SARS-CoV-2 virus in nasopharyngeal fluids such as multiplex
      RT-PCR, CRISPR/Cas12, and CRISPR-Cas3, lateral flow immunoassay, paper-based bimolecular
      sensors, SHERLOCK testing in one pot, DNA aptamer, loop-mediated isothermal amplification
      (LAMP), etc. Among these methods, nucleic acid amplification-based tests are the most common
      for the diagnosis of SARS-CoV-2.

      In addition, US CDC suggest a protocol for the detection of SARS-CoV-2, based on the
      amplification of two regions of the nucleocapsid gene, namely N1and N2, and a human internal
      control gene RNase P (RP). In addition to these strategies, efforts to develop SARS-CoV-2
      detection methods with high efficiency and accuracy, with less reaction time and effort, are
      still ongoing.

      However, despite being considered as the gold standard for SARS CoV-2 detection, testing has
      lagged behind in many countries because of various factors, most important being supply chain
      issues with lack of reagents and adequate test kits. Therefore, many patients (both
      symptomatic and asymptomatic) remain untested and hence are potentially contributing to
      community spread of the virus.

      If Bangladesh can produce its own RT-PCR kits which is no less in quality in terms of
      sensitivity, specificity etc. than that would mitigate the shortage of RT-PCR kit as
      investigators are totally dependent on imported test kits and also save foreign currency.
      Moreover, RT-PCR test kits are very temperature sensitive and need to maintain the cold chain
      for its quality which can be mitigated by developing local RT PCR kit. Additionally, many
      variant of SARS-CoV-2 has been developed due to mutation, which has shown more virulence and
      high transmission rate. So, development of new RT PCR kit which can detect newer strain can
      be extremely helpful.

      So, here investigator's aim to develop the three gene detectable robust RT-PCR kit that will
      be country first and give reliable result of different kinds of variant of SARS-CoV-2 virus
      for reducing its transmissibility and taking preventive management plan 2. Objectives of the
      Study:

        1. General Objectives: Primary objectives are aimed to develop multiplex real-time reverse
           transcription polymerase chain reaction (rRT-PCR) to detect of SARS-CoV-2.

        2. Secondary Objectives: For the best RT-PCR performance, the combination of E and N gene
           targets should be optimized.

           3. Methodology: 3.1. Study type & purpose: This is a case control and observational
           study in which two existing groups frozen and fresh samples are identified and compared.

           3.2. Place of study:

             -  Institute Epidemiology Disease Control and Research (IEDCR)

             -  M/S. Clinical Research Organization Ltd 3.3. Sample Collection Site(s):

             -  Institute Epidemiology Disease Control and Research (IEDCR) or

             -  National Institute of Laboratory Medicine & Referral Centre (NILMRC), Dhaka,
                Bangladesh.

           3.4. Duration of study: • 3 months (November, 2021 to January, 2022) after getting
           Ethical Clearance from Bangladesh Medical Research Council.

           3.5. Study Population/sample selection 3.5.1 CRO will receive testing samples from
           IEDCR. IEDCR and BCTL/CROL as CRO have memorandum of understanding (MoU) for different
           collaborative research work. So, NILMRC/ BCTL/CROL will receive a total 120 sample from
           IEDCR for the evaluation of RT-PCR kit efficiency. Of the 120 samples, 60 will be stored
           samples (30 COVID-19 known positive and 30 COVID-19 negative samples) which have been
           kept at -80°C lab freezers at IEDCR facilities and another 60 fresh specimens (30
           COVID-19 positive and 30 COVID-19 negative samples) which are being tested at IEDCR.
           Samples with discordant results will be rejected before testing by BRiCM RT PCR kit.
           That is, more than 120 identified samples from IEDCR might be needed to analyze by
           imported RT-PCR kits and extraction kit at BCTL/CROL. This is how 120 samples will be
           selected for performance evaluation of BRiCM RT-PCR kit.

           3.5.2 To avoid biasedness and conflicts of interest, sponsor and PI/co-investigators
           will be blinded to the sample ID and positive/negative status of the samples and
           clinical records of the patients. The positive/negative status of both stored and fresh
           samples will be identified at IEDCR before sending to BCTL/CROL. That is, BCTL/CROL
           personnel will also be blinded to sample identity document (ID) and positive
           negative/negative status before analysis. Finally, the CRO head will send the
           performance evaluation certificate to the PI or Sponsor.

           3.5.3. Statistical Basis of Sample Size:

           Formulae:

           The study is conducted on 120 participants (60 stored known positive & negative and 60
           fresh known positive & negative). To achieve desire absolute accuracy (n) in estimating
           efficiency of RT-PCR the minimum sample size estimated following following the Discrete
           Bernoulli Probability Function as follows:

           Alfa= 5% D= absolute Accuracy (%) with 95% confidence. Desired Absolute accuracy n D
           Alfa=5% Z=1.96 10 30.99% n D= (1.96)/(2* Square Root (SQRT) (n)) 20 21.91% 100 9.80% 30
           17.89% 120 8.94% 100 9.80% 200 6.93% 300 5.66% 400 4.90% 1000 3.10% 10000 0.98% 20000
           0.69% 30000 0.57% 100000 0.3% 3.6 Sample Selection Criteria 3.6.1 Inclusion criteria:
           Samples with Positives RT-PCR results with Ct value≤35 for the COVID-19 genes at IEDCR
           will be selected as COVID-19 positive. Samples with negative RT-PCR results with no
           amplification for the COVID-19 genes at IEDCR will be selected as COVID-19.

             -  Gender: All

             -  Age: 5 to 70 years (Child, Adult, Older Adult) 7.8.2. Exclusion criteria

             -  Samples with equivocal/ambiguous RT-PCR results in terms of sigmoidal curve and Ct
                value will be excluded.

           Consents from the patients are not required because the CRO will receive the specimens
           from IEDCR as per MOU with IEDCR. So there is no requirement of patient's enrollment and
           consents and CRF from the CRO side.

           3.7 Data Collection Procedure: COVID-19 positive samples will be confirmed by laboratory
           findings of positive RT-PCR results for the viral RNA.

           3.8 Data Analysis: The sensitivity, specificity, positive predictive value and negative
           predictive value of BRiCM COVID-19 real time RT-PCR kit will be determined. The
           chi-square test (two sided) will be performed for comparing categorical variables. A p
           value of ≤0.05 will be considered as the level of significant association. Alongside
           sensitivity, specificity, positive predictive value and negative predictive value, Kappa
           coefficient, positive likelihood ratio, and negative likelihood ratio will also be
           calculated.

           3.9 The database is safeguarded against unauthorized access by established security
           procedures; appropriate backup copies of the database and related software files shall
           be maintained. Databases are backed up by the database administrator in conjunction with
           any updates or changes to the database.

           3.10 The Investigator must make study data accessible to the monitor and authorized
           representatives. The Investigator must provide interim study report with data at every
           three months interval to the principle investigator. As per requirement study report and
           data may be provided to Bangladesh Medical Research Council (BMRC) and Directorate
           General of Drug Administration (DGDA). The Investigator must ensure the reliability and
           availability of source documents.

           3.11 Monitoring visits will be conducted by principle investigator, co- investigator as
           well as the representatives according to Guidelines for Good Clinical Practice (GCP).

           3.12 In order to maintain subject confidentiality, only a site number, subject number
           and subject initials will identify all study subjects on Case Records Forms (CRF) and
           other documentation submitted to the investigator. Additional subject confidentiality
           issues (if applicable) are covered in the Clinical Study Agreement.

           3.13 All records will be kept secret to the PI, co-investigators, sponsor except IEDCR
           responsible persons who are in charge. Samples will be identified as code/ID number and
           date at IEDCR before sending to BCTL/Contact Research Organization Limited (CROL) 4. For
           this study the mentioned institute IEDCR/Bangladesh Clinical Trail Limited (BTCL)/CRO
           Ltd. has the capability to conduct the clinical trial to find out the efficacy of kit
           through their existing resource. Beside that CRO can manage the chemical, reagent and
           technology if any needed for the study as per MoU between funding agencies.

           5. Conflict of the interest BRiCM is the funding agency of proposed COVID-19 RT-PCR kit,
           is a statutory body functioning under the administrative control of the Ministry of
           Science and Technology. It is a research organization and a reference institute in the
           area of chemical measurements. It is neither a commercial enterprise nor a profit making
           organization. According to constitutional sanction the principal responsibility of BRiCM
           is to carry on research leading to such innovation that will add value to the government
           effort for ensuring welfare of the people.
    

Observational model: Case-Control
Time perspective: Prospective
Sampling method: Non-Probability Sample
Study population: 
        IEDCR and BCTL/CROL as CRO have MoU for different collaborative research work. So, NILMRC/
        BCTL/CROL will receive a total 120 sample from IEDCR for the evaluation of RT-PCR kit
        efficiency. Of the 120 samples, 60 will be stored samples (30 COVID-19 known positive and
        30 COVID19 negative samples) which have been kept at -80°C lab freezers at IEDCR facilities
        and another 60 fresh specimens (30 COVID-19 positive and 30 COVID19 negative samples) which
        are being tested at IEDCR. Samples with discordant results will be rejected before testing
        by BRiCM RT PCR kit. That is, more than 120 identified samples from IEDCR might be needed
        to analyze by imported RT-PCR kits and extraction kit at BCTL/CROL. This is how 120 samples
        will be selected for performance evaluation of BRiCM RT-PCR kit.