The COVID19 PCR Test: Facts, Oddities & other Discrepancies

Written by Barbara Davie on February 19, 2021. Posted in Current News

Are common human coronaviruses causing mild respiratory and self – limiting symptoms, being presented and reported under the guise of SARS CoV-2?

This article relates to the validity of Australian PCR testing of the viral pathogen SARS CoV-2, resulting in a worldwide pandemic of symptoms or disease known as COVID-19. RT-PCR gene targets SARS CoV-2 will be discussed, SARS CoV-2 case definitions and finally addressing the validity of such testing.

“Common Sense Pathology” (published 8^th May 2020) supported by funding from the Australian Government Department of Health and was developed by the Royal College of Pathologists of Australia & supported by Australian Doctor Group. “Common Sense Pathology” describes the rudiments of the RT-PCR, Reverse Transcriptase Polymerase Chain Reaction test utilised in the “detection” of SARS CoV-2 / COVID-19. https://www.rcpa.edu.au

The rudimentary procedure of the RT-PCR test is (1) a nasopharyngeal and throat swab is collected. (2) viral RNA is extracted and converted to complimentary DNA by reverse transcriptase for testing. (3) The PCR test involves using one or more “specific” primers  that are able to bind to “virus-specific” sequences on the cDNA and then repeatedly copying (amplifying) a target sequence / gene.

It is important to note the introduction of this article clearly defines COVID-19 is caused by the virus SARS CoV-2.  SARS CoV-2 is the coronavirus that causes COVID-19. In other words, COVID-19 is the terminology used for non- specific symptoms, not peculiar to SARS CoV-2 or any other pathogen. COVID-19 being one or more symptoms is NOT transmissible nor contagious!

In Australia SARS CoV-2 testing is performed in pathology laboratories using Reverse Transcriptase Polymerase Chain Reaction, RT-PCR.  The RT-PCR tests only for the presence amino acid / protein sequences of viral RNA, NOT a whole virus. That is RT-PCR testing does not culture any whole viable virus.

The Public Health Laboratory Network (PHLN), under the auspices of the Australian Government – Department of Health have developed a standard case definition for the diagnosis of diseases which are notifiable in Australia, that is laboratory case definitions for diagnosis of communicable diseases, (page last updated on the 10^th of October 2020). www1.health.gov.au

Note: there is NO laboratory case definition for (SARS CoV-2 / COVID-19 / Human coronavirus with pandemic potential). SARS CoV is listed!  Surely during a global pandemic of SARS CoV-2, sending more than 1000² individuals to a merciless death, there must be a laboratory case definition.

The Communicable Diseases Network Australia (CDNA), summarises recommendations for surveillance, infection control, laboratory testing and contact management for coronavirus disease 2019 (COVID-19). www1.health.gov.au

www1.health.gov.au

From page 6, section 2 of the above CDNA publication

2. The disease.

Infectious agent

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the infective agent that

causes coronavirus disease 2019 (COVID-19). SARS-CoV-2 is a novel coronavirus that was

first identified in humans in Wuhan, China, in December 2019. SARS-CoV-2 shares 79.6%

sequence identity to SARS-CoV-1 ⁽¹⁾

Reference (1) from the above CDNA publication reference list is – 1. Chang D, Xu H, Rebaza A, Sharma L, Dela Cruz CS. Protecting health-care workers from subclinical coronavirus infection. The Lancet Respiratory Medicine. 2020;8(3):e13

www.ncbi.nlm.nih.gov

www.ncbi.nlm.nih.gov

Information from reference (1) is as follows – “Group A infections—a category reserved for highly infectious pathogens, such as cholera and plague. WHO confirmed 8098 cases and 774 (9·6%) deaths during the SARS outbreak in 2002, of which health-care workers accounted for 1707 (21%) cases.”

This reference is also noted on the CDNA publication, page 13 –

4. Cases Definition

Confirmed case – A confirmed case requires laboratory definitive evidence.

Laboratory definitive evidence:

1. Detection of SARS-CoV-2 by nucleic acid testing¹ ; OR ………….

Note: the reference, Chang D, Xu H, Rebaza A, Sharma L, Dela Cruz CS. Protecting health-care workers from subclinical coronavirus infection. The Lancet Respiratory Medicine. 2020;8(3):e13, has no information regarding the nucleic acid testing of SARS CoV-2. This reference labels COVID-19 as the novel coronavirus.

This opposes the CDNA publication, page 6 – “Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the infective agent that causes coronavirus disease 2019 (COVID-19).”

COVID-19 a term making reference to non-specific symptoms.

The Public Health Laboratory Network Statement on Asymptomatic Testing for SARS-CoV-2 – 6 August 2020.  “Diagnostic testing for SARS-CoV-2 (the virus that causes COVID-19) is vital to containing the COVID-19 pandemic in Australia.”

www.health.gov.au

www.health.gov.au

Page 2 – “RT-PCR tests are the gold standard for diagnostic SARS-CoV-2 testing in Australia. Available evidence for the reliability of RT-PCR tests mainly comes from symptomatic patients. The test’s clinical role in detecting asymptomatic carriers is still unclear given these in vitro diagnostic devices were not designed for screening.”

“As community prevalence of COVID-19 falls and the rate of asymptomatic testing increases, the proportion of false positive results may increase.”

“PHLN is of the strong view that testing is targeted toward population cohorts where the maximum value is derived, and emphatically discourage non-clinically indicated asymptomatic testing.”

PHLN guidance on laboratory testing for SARS-CoV-2 (the virus that causes COVID-19), documents, “commercial NAT assays have been available for SARS-CoV-2 testing in Australia since March 2020. The majority of diagnostic laboratories now employ either commercial or in-house developed assays for testing. The turnaround times are less than 24 hours after the laboratory receives a specimen. Faster turnaround times can be achieved when using rapid, but low throughput, RT-assays. The commonly circulating coronaviruses can be usually detected by commercial assays (e.g. NL63, 229E strains).”  www.health.gov.au

To minimise the risks of false positive results in low prevalence settings, confirming positive results is done with either:

• RT-PCR assays detecting a different target gene (particularly for assays with a single target); • Sequencing (Whole Genome Sequencing WGS)

False positive results can be confirmed by RT-PCR assays detecting a different target gene (particularly for assays with a single target). For example, the pan-sarbecovirus E gene target utilised in some assays is NOT specific for SARS CoV-2!  That is other coronaviruses and common human circulating viruses share the same target E gene. www.health.gov.au

No single gene target is specific for SARS CoV-2!   If a single gene target is non-specific for SARS CoV-2, how can multiple gene targets be specific for SARS CoV-2? SARS CoV-2 RT-PCR assays based on multiple gene targets are merely a series of single gene targets.

The Public Health Laboratory Network, 17^th November 2014, provide a summary laboratory definition for Severe acute respiratory syndrome coronavirus (SARS-CoV) infection. https://www1.health.gov.au/internet/main/publishing.nsf/Content/cda-phlncd-sars.htm.

The above PHLN publication describes some common and routinely circulating human coronaviruses (eg. HKU1, OC43, NL63, 229E), causing mainly mild respiratory tract infections. These common human coronaviruses have a lipoprotein envelope that surrounds the nucleocapsid (N) protein and genomic RNA.  The envelope contains membrane (M) proteins, envelope (E) proteins and spike (S) proteins. S proteins form projections giving the virus the characteristic corona appearance.

The Public Health Laboratory Network Guidance on the Nucleic Acid Test Result Interpretation for SARS-CoV-2, list a range of NAT used in Australian laboratories, including: – https://www.health.gov.au/sites/default/files/documents/2020/07/phln-guidance-on-nucleic-acid-test-result-interpretation-for-sars-cov-2.pdf

• In-house and commercial assays,
• Assays targeting different parts of the SARS-CoV-2 genome, including the N gene, E gene, S gene, RdRp gene and Orf-1ab gene;
• Single gene target assays, or assays that detect multiple SARS-CoV genes in one test.

As stated above, the coronavirus gene targets, N, E and S  genes are not specific to SARS CoV-2.

Gulholm. T (Department of Infectious Diseases, Prince of Wales Hospital, Randwick, NSW, Australia), Basile K (Centre for Infectious Diseases and Microbiology Laboratory Services, NSW Health Pathology – Institute of Clinical Pathology and Medical Research, Westmead Hospital, Westmead, NSW, Australia), Kok. J (Centre for Infectious Diseases and Microbiology – Public Health, Westmead Hospital, Westmead, NSW, Australia), Chen. SCA (Marie Bashir Institute for Infectious Diseases and Biosecurity, The University of Sydney Westmead Hospital, Westmead, NSW, Australia ) and Rawlinson.W (NSW Health Pathology, Serology and Virology Division, Prince of Wales Hospital, Randwick, NSW, Australia) describe the Coronaviridae family – (Coronavirus  genome)  is approximately 30 kb in size, , containing seven known genes – ORF1a, ORF1b, ORF3, E, M, N and S. Published in Laboratory diagnosis of severe acute respiratory syndrome coronavirus – 2. Pathology, (December 2020): 52(7):745–753.

ORF1a and ORF1b, represents two-thirds of the SARS CoV-2 genome, with the remaining third encoding structural proteins. The E, M, N and S genes encode E (envelope protein), M (membrane protein), N (nucleocapsid of nucleoprotein) and S (spike protein). These proteins are common to coronaviruses of the Coronaviridae family.

https://www.pathologyjournal.rcpa.edu.au/article/S0031-3025(20)30938-7/fulltext, https://www.pathologyjournal.rcpa.edu.au/action/showPdf?pii=S0031-3025%2820%2930938-7.

Note: SARS CoV-2 and common human coronaviruses belong to the Coronaviridae family!

All members of the coronavirus family possess single-stranded RNA genomes ranging in size from 27 to 33 kb. The coronavirus genomes encode polyproteins from Open Reading Frames (ORF1a/ORF1ab) and four structural proteins, from the spike (S), envelope (E), membrane (M) and nucleocapsid (N) genes, which are common to all coronaviruses. (Michel. J, Mayar C et al)  https://virologyj.biomedcentral.com/track/pdf/10.1186/s12985-020-01402-1.pdf

The coronavirus Open Reading Frames, ORF1a, ORF1b and overlap of ORF1a and ORF1b (ORF1ab) gives rise in the host cell polyproteins (polypeptides) pp1a and pp1ab. (Llanes A, Restrepo CM et al) https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7352669/pdf/ijms-21-04546.pdf

Parts of the coronavirus genome, ORF1a, ORF1b and ORF1ab will produce polyproteins pp1a and pp1ab inside a host cell.  The ORF1ab gene is not specific to SARS CoV-2!

Coronavirus polypeptides pp1a and pp1ab undergo further processing leading to the production of a non-structural polypeptide, RNA dependent RNA polymerase (RdRp). (Artika et al)  https://www.cell.com/action/showPdf?pii=S2405-8440%2820%2931586-3

RdRp gene is not specific to SARS CoV-2!

As described above by the Public Health Laboratory Network (PHLN),  RT-PCR assays  for the “detection” and “diagnosis” of SARs CoV-2, can target single or multiple gene targets; the N, E, S, RdRp and ORF-1ab. https://www.health.gov.au/sites/default/files/documents/2020/07/phln-guidance-on-nucleic-acid-test-result-interpretation-for-sars-cov-2.pdf   However these genes are NOT specific to SARS CoV-2, they are common to all coronaviruses of the Coronaviridae family.

Gulholm et al, have tabled (table 2) a list of SARS CoV-2 nucleic acid tests (NAT) in routine use in Australia. https://www.pathologyjournal.rcpa.edu.au/action/showPdf?pii=S0031-3025%2820%2930938-7 / https://www.pathologyjournal.rcpa.edu.au/article/S0031-3025(20)30938-7/fulltext

The above NAT (RT-PCR) assays for SARS CoV-2 use target genes – RdRp, N, ORF1a, ORF8, N1, N2, ORF1ab, and E.

Pyrc K et al, (in their minireview – the Novel Human Coronaviruses NL63 and HKU1) describe the human coronaviruses HCoV-229E and HCoV-OC43 as responsible for inducing a “common cold” in healthy volunteers. 1466-06.pdf (nih.gov)  The Centre for Disease Control and Prevention state, “the common human coronaviruses, including types 229E, NL63, OC43, and HKU1, usually cause mild to moderate upper-respiratory tract illnesses, like the common cold. Most people get infected with one or more of these viruses at some point in their lives.” Common Human Coronaviruses (cdc.gov)

Coronaviruses are positive-stranded RNA viruses, with the largest viral genome of the RNA viruses (27 to 33 kb). The virus particle is enveloped and carries extended spike proteins on the membrane surface, providing the typical crown-like structure (crown corona) seen by electron microscopy. The coronaviruses share a conserved organization of their RNA genomes. Within the coronavirus genomes there are the (Open Reading Frames) ORF1a, ORF 1b, ORF1ab genes, spike protein (S), envelope protein (E), membrane protein (M) and nucleocapsid protein (N) genes. (Pyrc K et al)

A diagram representation of coronavirus genomes shows HCoV-HKU1 as having an open reading frame 8 (ORF8). (Pyrc et al)   Others have listed the genome order of HCoV-HKU1 as ORF1a, ORF1b, HE, S, ORF4, E, M, N and ORF8. (Zhao X et al)

The genome of SARS-CoV-2 is nearly 30,000 nucleotides. The characteristic gene order is ORF1a, ORF1b, S, ORF3a/3 b, E, M, ORF6, ORF7a/7 b, ORF8, N, ORF9a, ORF9b and ORF10.  (Zhao X et al)

NOTE: From a process of deduction, why are the open reading frame, ORF9a, ORF9b and ORF10 genes not being utilised as targets in the RT-PCR testing of SARS CoV-2 ????

The authors (Zhao X et al), of “2020 update on human coronaviruses: one health, one world” main.pdf (nih.gov), state precisely, “a high conservation was observed for structural proteins E, M and A across the Betacoronavirus genus, while accessory proteins ORF8 seem to have much stricter evolutionary constraints.”  This means across the Betacoronavirus genus including  SARS CoV-2, SARS CoV, and the “common head cold” human coronaviruses HCoV-HKU1 & HCoV-OC43 there is a high degree of protein sequence commonality within the test envelope (E), membrane (M) and especially more with the open reading frame gene (ORF8) gene targets.

The coronavirus nucleocapsid (N) protein has been shown to have 3 distinct and highly conserved domains (parts). Sequence comparison indicates that a particular domain of the N protein is conserved at least among the alpha, beta and gamma groups of coronaviruses, suggesting a common structural and functional role for this domain. (Artika et al)

A genomic diagram of HCoV-HKU1 and HCoV-OC43 shows ORF9 and ORF7 respectively. Note: If SARS CoV-2 has ORF10 gene, why not use ORF10 as a target when using RT-PCR? (Artika et al)

Note: SARS CoV-2 is a betacoronavirus with HCoV-HKU1 and HCoV-OC43.  The human coronavirus HCoV-229E and HCoV-NL63 are alphacoronaviruses.  HCoV-HKU1, HCoV-OC43, HCoV-229E and HCoV-NL63 are human coronaviruses causing common cold symptoms, more severe symptoms are seen in the immunocompromised.

In this case the envelope (E), membrane (M) and open reading frame (ORF8) gene targets cannot to be utilised to detect the presence of SARS CoV-2 RNA from a RT-PCR assay. The envelope (E), membrane (M) and open reading frame (ORF8) gene targets are NOT specific for SARS CoV-2.

The genome of human coronavirus HCoV- HKU1 has been well depicted figure 1 and table 2, in the article, “Characterization and Complete Genome Sequence of a Novel Coronavirus, Coronavirus HKU1, from Patients with Pneumonia”, including ORF1a, ORF1b, Spike (S), envelope (E), membrane (M), nucleocapsid (N) and ORF8. (Woo P.C.Y, Lau. S.K.P et al)

Open Reading Frame (ORF10) is proposed as unique to SARS-CoV-2. ORF10 codes for a peptide only 38 amino acids long. There is no data yet providing evidence that the protein is expressed during SARS-CoV-2 infection. (Michel et al)

Note: This is odd and highly peculiar! Literature describes ORF10 being part of the SARS CoV-2 genome. The ONLY way to get an “isolate” of SARS CoV-2 is from an individual who is infected. Does the SARS CoV-2 open reading frame, ORF10 only exist in medical and scientific publications, without a natural wildtype occurrence in infected human populations?

As discussed, as the gene targets of the coronavirus family are highly conserved between members, this renders the RT-PCR unable to be utilised for the diagnosis or detection of SARS CoV-2.

Australian Testing RT-PCR for SARS CoV-2 / COVID-19

Rahman H, Carter I et al, from the (Centre for Infectious Diseases and Microbiology Laboratory Services, NSW Health Pathology-Institute of Clinical Pathology and Medical Research, Westmead Hospital, Westmead, NSW, 2145, Australia, Centre for Infectious Diseases and Microbiology – Public Health, Westmead Hospital, Westmead, NSW, 2145, Australia, Marie Bashir Institute for Infectious Diseases and Biosecurity, The University of Sydney Westmead Hospital, Westmead, NSW, 2145, Australia and d Centre for Virus Research, Westmead Institute for Medical Research, Westmead, NSW 2145, Australia), evaluated the commercial assay for the specific detection of SARS-CoV-2 (AusDiagnostics Pty Ltd, Mascot, NSW, Australia). The targets employed were the envelope (E), RdRp, nucleocapsid (N), open reading frames (ORF1ab, ORF1b) and membrane (M) genes. It is to be noted the AusDiagnostics  RT-PCR assay for SARS CoV-2 detected other “common cold” human coronaviruses – HCoV-NL63 and HCoV-229E.  (Rahman H, Carter I et al)

The authors concluded with “the AusDiagnostics assay IS NOT specific for the detection SARS-CoV-2.”

A new version of RT-PCR test called the Cepheid Xpert® Xpress SARS-CoV-2, has been approved and is being rolled out in sites in Australia. This is both simpler and faster than most other RT-PCR tests and can be used outside main laboratory settings. https://www.labtestsonline.org.au/learning/index-of-conditions/covid-19/what-s-ahead-for-covid-19-testing

On the 16^th April 2020, The Hon Greg Hunt MP
Minister for Health and Aged Care, released to the general public, “World first rapid COVID-19 testing to protect Aboriginal and Torres Strait Islander communities.” The media release explains, the Australian Government is investing $3.3 million to establish a rapid coronavirus (COVID-19) Remote Point of Care Testing Program for remote and rural Aboriginal and Torres Strait Islander communities.

The test, called the Xpert SARS-CoV-2 test, uses rapid technology to detect COVID-19 infections at the point-of-care by using a nasal swab polymerase chain reaction (PCR) test in the early phases of the illness. https://www.health.gov.au/ministers/the-hon-greg-hunt-mp/media/world-first-rapid-covid-19-testing-to-protect-aboriginal-and-torres-strait-islander-communities

The Cephid GeneXpert (XpertXpress) cartridge system is one example of this type of rapid PCR test that is used in Victoria. Coronavirus (COVID-19) Case and contact management guidelines for health services and general practitioners DATE 28 January 2021 Version 26.1 https://www.dhhs.vic.gov.au/sites/default/files/documents/202101/Case%20and%20contact%20management%20guidelines%20for%20health%20services%20and%20gps.pdf

12^th August 2020 – Hon. SJ MILES (Murrumba—ALP) (Deputy Premier and Minister for Health and Minister for Ambulance Services), Queensland. “We bought 35 more GeneXpert machines to rapidly test for COVID and more Panther machines so we could test right around the state.” https://www.parliament.qld.gov.au/documents/hansard/2020/2020_08_12_WEEKLY.pdf

Situational report on the Indigenous Health Response to the COVID-19 Pandemic. Prepared by ASHM, members of the Taskforce’s Indigenous Health Cluster Group, the Testing Cluster Group and the Taskforce Chair. Document last updated: May 14th 2020.  “GeneXpert®Point of Care Test machines – leading up to the COVID-19 pandemic there were 33 GeneXpert® machines available for use in healthcare settings that provide care to First Nations people; On March 20th 2020 the Federal Drug Administration in the United States authorized the use of Xpert® Xpress SARS-CoV-2 cartridge (Cepheid, Sunnyvale, United States of America)15 for use by GeneXpert® machines. The test is fully automated and can provide results within 45 minutes16 . The Therapeutic Goods Administration provided rapid approval for the test on March 22nd 202017. In response to the COVID-19 pandemic there are now 87 GeneXpert® machines available, or en route to remote primary health care services that provide care to First Nations people.” https://ashm.org.au/covid-19/clinical-care/indigenous-health/

Exciting to see @AusAirForce plane arrive with supplies to help PNG prepare for #COVID19. 12,000 new testing kits, the 3rd batch of GeneXpert kits and new advisers to join @ADFinPNG to work with PNG Defence Force. Major effort by #TeamAustralia! https://news.defence.gov.au/national/live-updates-operation-covid-19-assist-tuesday-12-may-2020-tue-05122020-0900

Government of Western Australia – GeneXpert COVID rapid test approval process. The TGA-approved GeneXpert Xpress SARS-CoV-2 test for use on the Xpert platform (Cepheid) enables the rapid molecular detection of SARS-CoV-2 infection at the point-of-care.https://ww2.health.wa.gov.au/-/media/Corp/Documents/Health-for/Infectious-disease/COVID19/COVID19-GeneXpert-COVID-rapid-test-approval-process.pdf

NSW Health Pathology has Cepheid GeneXperts in 37 laboratories throughout NSW, including all larger regional laboratories. These instruments provide rapid PCR diagnosis (approximately 60 minutes) and have been used to provide rapid flu testing. A limited number of cartridges to allow rapid on-site SARS- CoV-2 testing is now available at metropolitan, regional and rural sites with GeneXperts. These sites are: Westmead, Blacktown, Blue Mountains, Lithgow, Bathurst, Mount Druitt, Nepean, Bega, Broken Hill, Wagga Wagga, Griffith, Goulburn, Orange, Dubbo, John Hunter Hospital, Armidale, Taree, Gosford, Tweed, Tamworth, Coffs Harbour, Lismore, Calvary Mater Newcastle, Royal North Shore, Campbelltown, Liverpool, Bowral, Fairfield, Bankstown, Randwick, Wollongong, St George, Royal Princess Alfred, Concord Hospital, Kempsey, Grafton and Cooma. https://www.health.nsw.gov.au/Infectious/covid-19/communities-of-practice/Pages/guide-rapid-prc-testing.aspx

There is no doubt the GeneXpert RT-PCR testing for SARS CoV-2 / COVID-19 is being employed throughout Australia and territories.

The Panther Fusion RT-PCR for SARS CoV-2 uses gene targets – open reading frames, ORF1a region 1 and ORF1b region 2, as listed on page 3 of product instructions and manual. The manufacturer states clearly, the Panther Fusion SARS-CoV-2 assay amplifies and detects two conserved regions of the ORF1ab gene. The ORF1ab gene is highly conserved within the Coronavirus family. This means the ORF1ab gene of SARS Cov-2 is strikingly similar to any member of the Coronavirus family, that includes “common head cold” coronaviruses HCoV-HKU1, HCoV-OL43, HCoV-229E and CoV-NL63. https://www.hologic.com/sites/default/files/2020-03/AW-21159-001_002_01.pdf

The NSW GeneXpert testing targets the nucleocapsid (N) and (E) genes. Clinical reporting is solely based on the two targets genes (N) and (E). Reporting is as follows –

• detected – N gene without E gene identified

• detected presumptive – E gene only identified                 (E gene ONLY!)
• invalid – failed result (testing will be repeated locally)
• not detected – N gene and E gene negative

(https://www.health.nsw.gov.au/Infectious/covid-19/communities-of-practice/Pages/guide-rapid-prc-testing.aspx, https://www.pathology.health.nsw.gov.au/covid-19-info/rapid-testing)

The N and E gene targets are highly conserved with members of the coronavirus family. Highly conserved gene targets cannot be diagnostic for SARS CoV-2.

The Coronaviridae family includes human-infecting coronaviruses SARS-CoV, MERS-CoV, HCoV-OC43, HCoV-229E, HCoV-NL63 and 6 HCoV-HKU1. SARS-CoV-2 assays can yield false positives if they are 22 not targeted specifically to SARS-CoV-2, as the virus is closely related to other 23 Coronavirus organisms. In addition, SARS-CoV-2 may present with other respiratory 24 infections, which make it even more difficult to identify. Lopez-Rincon et al, Bulletin World Health Organisation) https://www.who.int/bulletin/online_first/20-261842.pdf

Question: Relating to the RT-PCR testing, does SARS CoV-2 superimpose on common human coronaviruses and vice versa?

Lopez-Rincon A et al document, SARS-CoV-2 assays can yield false positives through non-specific detection of other Coronaviruses, as the virus is closely related to other Coronavirus organisms. https://www.nature.com/articles/s41598-020-80363-5 Published 13th June 2021.

SARS-CoV-2 is a novel coronavirus that was first identified in humans in Wuhan, China, in December 2019. SARS-CoV-2 shares 79.6% sequence identity to SARS-CoV-1. (The Communicable Diseases Network of Australia, Coronavirus Disease 2019 (COVID-19) CDNA National Guidelines for Public Health Units, https://www1.health.gov.au/internet/main/publishing.nsf/Content/7A8654A8CB144F5FCA2584F8001F91E2/$File/COVID-19-SoNG-v4.2.pdf.

Note: SARS-CoV-2 shares 79.6% (80%) sequence identity to SARS-CoV-1! RT-PCR methods for SARS CoV-1 gives rise to “non-specific” amplification of DNA from circulating coronaviruses.  https://www1.health.gov.au/internet/main/publishing.nsf/Content/cda-phlncd-sars.htm. One can conclude from the RT-PCR methods for SARS CoV-2 will also induce unintentional “non-specific” amplification of circulating coronaviruses: HKU1, OC43, NL63, 229E. It has been documented SARS CoV-2 shares 80% homology with SARS CoV-1 and vice versa!

NSW Health Pathology, COVID-19 information, https://www.pathology.health.nsw.gov.au/covid-19-info/rapid-testing, provides a link to the NSWHP major verification report of the XpertXpress SARS CoV-2 RT-PCR testing.

VERIFICATION OF THE XPERT^R XPRESS SARS-CoV-2 (Cepheid, Sunnyvale, CA), Jane Drury , Ian Carter, Jen Kok , Susan Maddocks , Dominic Dwyer , Sharon Chen , Hemalatha Varadhan:  Department of Microbiology, John Hunter Hospital (JHH) and the  Centre for Infectious Diseases and Microbiology Laboratory Services, ICPMR, NSW Health Pathology.

Rationale for verification – The Xpert^R Xpress SARS-CoV-2 is a rapid, real-time RT-PCR test intended for the qualitative detection of nucleic acid from the SARS-CoV-2 in either nasopharyngeal swab and/or nasal wash/aspirate specimens collected from individuals suspected of COVID-19. The gene targets comprise the (i) N2 and (ii) E genes of SARSCoV-2. There is an urgent need to perform verification of this commercial assay to augment and complement present testing capacity within NSWHP.

The NSWHP provide reporting comments with all testing for SARS CoV-2. The comments are as follows – Comment to be on all results:

1. This is not a NATA-accredited assay.
2. The assay is validated for testing of nasopharyngeal swabs and nasal aspirates/wash collected in UTM.
3. The Cepheid recommendations are that specimens returning a positive result for the “E” gene target are called a “presumptive positive”. NSW Health Pathology calls such specimens “positive” i.e. SARS-CoV-2 RNA detected. We have included this comment as a disclaimer. Sharon Chen and Hema Varadhan April 6 2020.

This equates to a single gene target (E) testing positive for SARS COV-2 will be reported as

presumptive positive, based on the fact a single gene target is not reliable. However, NSWHP

now calls this presumptive positive a definitive POSITIVE!

The question is how valid are the positive SARS CoV-2 test results in the Australian and abroad populations?  This highlighted in the Xpert® Xpress SARS-CoV-2, Instructions for Use For Use Under an Emergency Use Authorization (EUA) Only.  https://www.cepheid.com/Package%20Insert%20Files/Xpress-SARS-CoV-2/Xpert%20Xpress%20SARS-CoV-2%20Assay%20ENGLISH%20Package%20Insert%20302-3562%20Rev.%20F.pdf

The Analytical Specificity (Exclusivity), page 15  – an in silico (computer) analysis for possible cross-reactions with all the organisms listed in Table 5 was conducted by mapping primers and probes in the Xpert Xpress SARS-CoV-2 test individually to the sequences downloaded from the GISAID database (as of March 13, 2020).  It was noted E primers and probes are not specific for SARS-CoV-2 and will detect Human and Bat SARS-coronavirus.

Table 5 lists – Human coronavirus 229E, Human coronavirus OC43, Human coronavirus HKU1, Human coronavirus NL63, SARS-coronavirus, MERS-coronavirus and Bat coronavirus.

Summary

The gene targets employed in the RT-PCR testing of SARS CoV-2 (the viral pathogen causing a pandemic and the disease COVID-19) are not unique to SARS CoV-2. All gene targets are highly conserved (a high degree of commonality) across the Coronavirus family members including the “common head cold” coronaviruses HCoV-HKU1, HCoV-OL43, HCoV-229E and CoV-NL63. These gene targets are not unique to SARS CoV-2!

The question is how valid are the positive SARS CoV-2 test results in the Australian and abroad populations, when gene targets, primer and probes are not specific for SARS CoV-2?

Are common human coronaviruses causing mild respiratory and self – limiting symptoms, being presented and reported under the guise of SARS CoV-2?

References:

Artika. I.M, Dewantari. A.K & Wiyatno A. Molecular biology of coronaviruses: current knowledge. Heliyon. 2020,8(6); E04743  https://doi.org/10.1016/j.heliyon.2020.e04743, https://www.cell.com/action/showPdf?pii=S2405-8440%2820%2931586-3

Chang D, Xu H, Rebaza A, Sharma L, Dela Cruz CS. Protecting health-care workers from subclinical coronavirus infection. The Lancet Respiratory Medicine. 2020;8(3):e13 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7128440/, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7128440/pdf/main.pdf

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