Diagnostic dilemma in COVID-19: Identical twin of Pangolin-CoV

Outbreak of pneumonia in the third week of December-2019, in Wuhan City of Hubei Province of China led to the isolation of a new type of coronavirus in January 2020. The same was named as novel coronavirus-2019 (2019-nCoV) by Chinese Health Authorities. Pathogen free 'Human Airway Epithelial (HAE) cells' in tissue culture bottles were apically inoculated with bronchoalveolar lavage fluids and/or throat swabs from 9 patients. Apical supernatants from these cultures were the source of viral RNA. Genomic sequencing of 2019-nCoV was done by Chinese Scientists using ‘Sanger Sequencing’ facility and made available to GenBank and is registered under GenBank accession ID: MN908947. In 5 cases out of 9 Illumina and Oxford Nanopore Sequencing was also done. All the 9 patients whose samples were used to isolate virus for genomic sequencing had exposure to Huanan Seafood Market in Wuhan City. One of these patients stayed near Huanan Seafood Market.

Genomic implications:

There exists homology in genetic sequences of six strains on 2019-nCoV. The GenBank accession IDs of these strains are: EPI_ISL_402019, EPI_ISL_402020, EPI_ISL_402021, EPI_ISL_402022, EPI_ISL_402023 and EPI_ISL_402024. These epidemic strains of COVID-19 virus are just like human siblings.

It has been documented that Genomic sequences of 2019-nCoV obtained from nine patients of severe pneumonia at Wuhan City of Hubei Province of China had 88% identity with two bat-derived 'Severe Acute Respiratory Syndrome’ (SARS) like coronaviruses with genomic IDs: bat-SL-CoVZC45 and bat-SL-CoVZXC21. Genetic sequence of 2019-nCoV has been found very distinct from SARS-CoV (2002-03). Findings were published by Chinese group of scientists in the Lancet, volume 395 in the month of February 2020.

A group of veterinary scientists working under the guidance of Yongyi Shen of the College of Veterinary Medicine, South China Agricultural University, Guangzhou-510642, China, have explored through extensive study on 25 Malayan pangolins (Manis javanica) and 4 Chinese pangolins (Manis pantadactyla) that 2019-nCoV has very close relationship with coronavirus isolated from 17 out of 25 Malayan pangolins. However, Chinese pangolins were found negative for 2019-nCoV infection by RT-PCR. Coronavirus isolated from Malayan pangolins showed 100%, 98.2%, 96.7% and 90.4% amino acid identity with 2019-nCoV in E, M, N and S genes, respectively. The study reflects that Malayan pangolins are the natural host of this zoonotic coronavirus. Yongyi Shen and his team has proved through this marvelous study that 2019-nCoV is an identical twin of Pangolin-CoV.

Analysis of plasma samples from 8 of the Malayan pangolins showed high positivity for antibodies against 2019-nCoV by double antigen sandwich ELISA. Plasma sample of one of the Malayan pangolins showed positivity for antibodies against 2019-nCoV even at 1:80 dilution of plasma.

Diagnostic dilemma:

The Real Time-Reverse Transcription-Polymerase Chain Reaction (rRT-PCR) was developed for early detection of 2019-nCoV infection using nasopharyngeal and/or oropharyngeal swabs from patients having clinical symptoms of viral infection. RT-PCR has limitations that it could not be used for detecting past infections.

Diagnostic dilemma is that rRT-PCR covers all the six strains of 2019-nCoV. There could be more unknown strains of 2019-nCoV having identical genomic expression. Molecular diagnostic technology is very new and sensitive but has its limitations. It provides indirect evidence of a causative organism. Real Time RT-PCR for 2019-nCoV does not distinguish between active infection or positive results due to unknown SARS Related Coronaviruses (SARSr-CoV).  

The only validated method for diagnosis of 2019-nCoV by World Health Organization (WHO) is rRT-PCR and nobody could question its accuracy. Collection of sample, extraction and purification of viral RNA, storage of reagents of RT-PCR Kits, setting up of rRT-PCR Assay, operation of PCR-Machine and interpretation of rRT-PCR results need extensive training and experience.

Information Brochures/Manuals of all the Real Time RT-PCR Kits used for detecting 2019-nCoV immaculately exhibit that the kits are meant for Research use only (RUO) . Information is available online regarding recommended use of kits. I would like to share an adapted image from the Brochure of RealStar SARS CoV-2 RT-PCR Kit 1.0 of Altona Diagnostics GmbH, Hamburg (Figure-1) and another image from the Manual of Liferiver Novel Coronavirus (2019-nCoV) Real Time RT-PCR Kit of Shanghai ZJ Bio-Tech Co. Ltd., China (Figure-2), for attesting my point that diagnostic use of RT-PCR kits for 2019-nCoV (SARS CoV-2) is not valid or ethical.

Figure-1 For Research use only (RUO)

Figure-2: For Research use only (RUO)

What happens to your sample?

Extraction of RNA is the start point of all the kits for RT-PCR analysis of nasopharyngeal or oropharyngeal swabs as well as bronchoalveolar lavage samples. The purity of RNA extracted could only ensure the accuracy of results. It is highly important to use the RNA Extraction Kit compatible with the kit being used for rRT-PCR. Some most extensively used RNA Extraction Kits and Systems are:

§  AltoStar Automation System AM 16 (Altona Diagnostics)

§  QIAamp Viral RNA Mini kit (QIAGEN)

§  QIAsymphony (QIAGEN)

§  MagNA Pure 96 System (Roche)

§  Maxwell 16 IVD Instrument (Promega)

§  VERSANT kPRC Molecular System SP (Siemens Healthcare)

If you use spin column based, RNA extraction procedure, extra precaution should be taken to get rid of any traces of ethanol in the RNA purified; since ethanol is an inhibitor of rRT-PCR.

Real Time RT-PCR:

The rRT-PCR is a complex assay for detection of target genes: Target-E Gene (for B βCoV specific RNA), Target-S Gene (for SARS CoV-2 specific RNA) and Target-IC (for Internal Control). Different companies have different reporters (fluorescent dye labels) with or without quencher. The RealStar SARS CoV-2 RT-PCR Kit has used FAM^TM, Cy5 and JOE^TM without any quencher respectively for above mentioned target genes.

The RT-PCR comprises three steps, which are completed in Thermal Cycler at different temperatures ranging from 55^oC to 95^oC. The dye acquisition takes place during amplification cycle. In brief:

Step-I is Reverse Transcription; it is done at 55^oC at hold for single cycle of 20 minutes.

Step-II is Denaturation; it is done at 95^oC at hold for single cycle of 2 minutes.

Step-III is Amplification of DNA; it is a multicycle process (at least 30-45 cycles). One cycle of Amplification is completed in holds at three different temperatures: 95^oC for 15 seconds, 55^oC for 45 seconds and 72^OC for 15 seconds. So, one amplification cycle is completed in 1 minute and 15 seconds. 45 cycles for amplification will be completed in 56 minutes and 25 seconds (75x45=3375 seconds). So, the total turnaround time for rRT-PCR is 3 hours including sample preparation. However, it would take longer as the kit is meant for 14 samples and 6 controls. Preparation of multiple specimens and setting up machines needs extra time and precautions to avoid contaminations.

Some most commonly used RT-PCR machines are:

• ABI Prism 7500 SDS (Applied Biosystems)
• CFX96^TM Deep Well Real-Time PCR Detection System (Bio-Rad)
• CFX96^TM Deep Well Dx System (Bio-Rad)
• CFX96^TM Real-Time PCR Detection System (Bio-Rad)
• CFX96^TM Dx System (Bio-Rad)
• LightCycler 480 Instrument II (Roche)
• Rotor-Gene 6000 (Corbett Research)
• Rotor-Gene Q5/6 Plex Platform (QIAGEN)
• Mx 3005^TM QPCR System (Stratagene)
• VERSANT kPCR Molecular System AD (Siemems Healthcare)

The data analysis on Real Time-PCR machines needs expertise as per instrument manufacturer’s instructions. Qualitative Analysis of fluorescence signals captured for genes under detection is done to conclude the outcome of rRT-PCR assay.

Each amplification cycle doubles the copies of DNA molecule. If we start with single copy of cDNA after transcription from viral RNA; after completion of 31 amplification cycles; there will be more than one billion (1x10⁹) copies of DNA. After completion of 41 cycles of amplification; more than one trillion (1X10¹²) copies of DNA will be available for detection and analysis.

Past infection with 2019-nCoV could be detected trough tests for detecting IgM and IgG type antibodies against the virus. Double antigen sandwich ELISA could be very sensitive and specific assay for detecting both the IgM and IgG type antibodies.

Management of Tuberculosis: Diagnostic Approach

Accurate and timely diagnosis of Tuberculosis (TB) is a prerequisite for the treatment and control of spread of infection to other family members of the patient and health professionals dealing with patients. In the recent past several advances in the diagnosis and management of Tuberculosis (TB) have come out. Tools and technology are of great help in understanding of pathogenesis, demonstration of Mycobacterium, drug sensitivity testing and evaluation of prophylaxis.

Tuberculosis is a major health problem requiring early management at diagnostic and treatment level to bring down the mortality rate. In the developing countries the current mortality rate is 1 death per 100,000 population. The global incidence rate of Tuberculosis (TB) reported in the year 2010 was 128/100,000/year. To sustain the control and elimination of Tuberculosis (TB) there is a need for efficient testing and treatment regimens.

Diagnosis of Tuberculosis (TB) has conventionally been relied upon sputum microscopy of Micobacterium tuberculosis (an Acid Fast Bacilli) by Ziehl-Neelsen Staining Technique. The technique is very specific but has a poor sensitivity (around 50%). More sensitive technique used for demonstrating Acid Fast Bacilli (AFB) is by culture on Lowenstein Jensen Medium. Isolation of mycobacteria by culture method is considered to be gold standard in-spite of being time consuming.

The scenario of multi drug resistant TB (MDR-TB) has given a challenge to biomedical scientists to develop new drugs and diagnostic methods. Advances in molecular techniques for the diagnosis of TB have revolutionized diagnostic approach to this public health problem. Various new diagnostic modalities are based on the DNA extraction from the mycobacterial isolates. The nucleotide sequences of DNA are amplified and multiplied millions of times by polymerase chain reaction (PCR) for comparative diagnosis through detection of amplified DNA.

The tests that detect Mycobacterium tuberculosis antigens in clinical specimens could provide rapid and direct evidence of infection. The major targeted antigen of Mycobacterium tuberculosis is Lipo-arabino-mannan (LAM). LAM is detected in the urine of patients suspected of having pulmonary TB (Tuberculosis of lungs) as well as extra-pulmonary TB (Tuberculosis of organs other than lungs).

Molecular Tests are very sensitive and specific for diagnosis of infection and monitoring of treatment as well as for evaluating drug resistance. Uniplex-PCR (single insertion sequence IS6110 of 38 kDa)) or Multiplex-PCR (for multiplex targeting like IS6110 and MPB 64) are of great help for detecting drug resistance. Multiplex-PCR is more sensitive than Uniplex-PCR. Multiplex-PCR is useful in early detection, species differentiation (Mycobacterium tuberculosis or Mycobacterium avium) and epidemiology.

Two Molecular Assays used for rapid diagnosis of a case of TB and drug-resistance testing are:  i) X-pert MTB/RIF, and ii) Line Probe Assay (LPA)

i)                    X-pert MTB/RIF: X-pert MTB/RIF detects Mycobacterium tuberculosis (MTB) and resistance to Rifampicin (RIF) using Real-Time PCR (RT-PCR) Assay by amplifying MTB-specific sequence of the rpoB gene (inherent of MTB genome) that is probed with molecular beacons for mutations within the RIF-resistance determining region. Diagnosis of TB can be determined within 2 hours from the sputum samples with minimal health hazard. X-pert MTB/RIF test has 99% sensitivity and 100% specificity.

ii)                  Line Probe Assay (LPA):  Rapid detection of anti-TB drug resistance by Mycobacterium tuberculosis is the need of the hour for effective treatment and management of patient care. Line Probe Assays have been developed for rapid detection of rifampicin resistance and/or MTB-DR (especially rifampicin in combination with isoniazid). The Line Probe Assay (LPA) employs the hybridization of labeled PCR products with oligonucleotide probe on a strip and reading by colorimeter. The genotype MTB-DRplus Assay also simultaneously detects specific mutations in the katG gene conforming high level isoniazid resistance as well as in the inhA gene conforming low level isoniazid resistance.

The Molecular Assays are labeled for use on isolates from solid and liquid culture as well as directly on sputum smear positive pulmonary specimens. Mycobacterium tuberculosis ‘strain typing’ is very important for the analysis of the spread of tuberculosis as well as for monitoring the evolution of antibiotic resistance. These assays are also used to assess the bacterial load for monitoring of anti-TB treatment (ATT).

Just click the following link to update your knowledge about Management of Tuberculosis through Therapeutic Approach: http://drugsense.blogspot.in/2014/06/management-of-tuberculosis-therapeutic.html