

If you have been following the coverage on this blog about
Covid-19 false positives you will be interested to read that we have recently
discovered that UK laboratories have been routinely recording a large
proportion of Covid-19 test results as positive based on the presence of one
target gene alone, when there should have been two or more, as required to
comply with WHO rules and manufacturer instructions. For example, an average of 35% across the whole of the UK during week of 25 Jan 2021 were based on one gene only. Obviously, there is higher risk of encountering false positives
when testing for single genes alone, because of the possibility of
cross-reactivity with other HCOVs and prevalent nasopharyngeal bacteria or
reagent contamination. Unless UK laboratories have performed diagnostic
validation of their single gene call, for both the original and the B1.1.7
variant, and there is no evidence of this in the public domain, it can only be
assumed that, in the absence of confirmatory testing, many of the reported
positive results may in fact be inconclusive, negative or from people who
suffered past infection for SARS-COV-2.
An academic pre-print of article available here
The full article is reproduced below:
Positive
results from UK single gene testing for SARS-COV-2 may be inconclusive, negative,
or detecting past infections
Prof.
Martin Neil, School of Electronic Engineering and Computer Science, Queen Mary,
University of London
25
February 2021 (version 2)
Abstract
The UK Office for
National Statistics (ONS) publish a regular infection survey that reports data
on positive RT-PCR test results for SARS-COV-2 virus. This survey reports that a
large proportion of positive test results are based on the detection of a
single gene rather than on two or more genes as required in the manufacturer
instructions for use, and by the WHO in their emergency use assessment. The
proportion of positives called on single genes increased from mid-November to
mid-December 2020, suggesting a shift in testing policy coincident with the reported
significant increase in transmission of the new variant B1.1.7, and again
starting January 2021. Without diagnostic validation of the single gene call, for
both the original and the B1.1.7 variant it can only be assumed that, in the
absence of confirmatory testing, many of the reported positive results may in
fact be inconclusive, negative or from people who suffered past infection for SARS-COV-2.
Background
The ONS publish a regular
infection survey [1] that includes data from two UK lighthouse laboratories,
based in Glasgow and Milton Keynes, where both use the same RT-PCR test kit, to
detect the SARS-COV-2 virus. This survey includes data on the cycle threshold
(Ct) used to detect positive samples, the percentage of positive test results
arising from using RT-PCR, and the combinations of the SARS-COV-2 virus genes
tested that gave rise to positives between 21 September 2020 and 30 January
2021 across the whole of the UK.
ThermoFisher TaqPath kit is used by the Glasgow and
Milton Keynes lighthouse laboratories to test for the presence of three genes
from SARS-COV-2.
Despite Corman et al [2] originating the use of PCR testing for SARS-COV-2
genes there is no agreed
international standard for SARS-COV-2 testing. Instead, the World Health
Organisation (WHO) leaves it up to the manufacturer to determine what genes to
use and merely requires end users to adhere to the manufacturer instructions
for use (IFU). As a result of this we now have an opaque plethora of
commercially available testing kits, that can be applied using a variety of
test criteria. Other UK laboratories use different testing kit, and test for
different genes.
The WHO’s emergency use
assessment (EUA) for the ThermoFisher TaqPath kit [3], used by the Glasgow and
Milton Keynes lighthouse laboratories, includes the instruction manual and
contained therein is an interpretation algorithm describing an unequivocal
requirement that two-or-more genes be detected before a positive result can be
declared. The WHO have been so concerned about correct use of RT-PCR kit that
on 20 January 2021 they issued a notice for PCR users imploring them to review
manufacturer IFUs carefully and adhere to them fully [4].
Increasing proportion of
single gene “calls”
The ONS’s report [1]
lists SARS-COV-2 positive results for valid two and three gene combinations from the Glasgow and
Milton Keynes lighthouse laboratories. However, it also lists inconclusive
single gene detections as positive results. This use of single gene
“calls” therefore suggests that Glasgow and Milton Keynes lighthouse
laboratories may have breached WHO emergency use assessment (EUA) and may have
violated the manufacturer instructions for use (IFU). Indeed, Section 10 of
this ONS Covid-19 Infection survey on the 8 January 2020 [16] stated that
(emphasis mine):
“Swabs
are tested for three genes present in the coronavirus: N protein, S protein and
ORF1ab. Each swab can have any one, any two or all three genes detected.
Positives are those where one or more of these genes is detected in the
swab …..”
Over the period reported
the maximum percentage of positives on a single gene is 35% for the
whole of the UK for the week of 25 January. The overall UK average is 21%. The
maximum percentage reported is 65%, in East England in the week beginning 5
October. In Wales it is 48%, in Northern Ireland it is 47% and in Scotland it
is 49%. The full data including averages and maxima/minima are given in Table
1.
Figures 1 and 2 show the percentage
of weekly single gene positives across the UK nations and English regions. There
was a significant peak across the whole of the UK (except NI) from mid-November
to early December, coincident with the reported significant increase in
transmission of the new variant B1.1.7 [5]. However, given this new variant was
concentrated in Kent and NE London, with limited spread into the rest of
London, Anglia and Essex its presence cannot explain why single gene calls
increased in other regions such as the North East and Yorks. & Humber. There
has been another significant increase in the percentage of single gene
positives since the end of 2020, rising throughout January, and here the rise
is steady across all English regions and UK nations.
In startling
contradiction to the ONS reports Professor Alan McNally, Director of the
University of Birmingham Turnkey laboratory, who helped set up the Milton
Keynes lighthouse laboratory, reported in the Guardian newspaper, in an article
about the new variant, that all lighthouse laboratories operated a policy that
adhered to the manufacturer instructions for use: requiring two-or-more genes
for positive detection [6] (this policy is also documented in the supplementary
material provided in [7]).
Table
1: Percentage of weekly single gene positives from 21 September 2020 to 25
January 2021, including averages and maxima/minima
Figure
1: Percentage of weekly single gene positives from 21 September 2020 to 25
January 2021 (UK nations)
Figure
2: Percentage of weekly single gene positives from 21 September 2020 to 25
January 2021 (English regions)
In April 2020, the UK
lighthouse laboratories were testing for single genes and discounted the S gene
as early as mid-May, months before the discovery of the new variant B1.1.7
(emphasis mine):
“Swabs
were analysed at the UK’s national Lighthouse Laboratories at Milton Keynes
(National Biocentre) (from 26 April) and Glasgow (from 16 August) …., with
swabs from specific regions sent consistently to one laboratory. RT-PCR for
three SARS-CoV-2 genes (N protein, S protein and ORF1ab) ..... Samples are
called positive in the presence of at least single N gene and/or ORF1ab
but may be accompanied with S gene (1, 2 or 3 gene positives). S gene is not
considered a reliable single gene positive (as of mid-May 2020).”
Indeed, in Table 1 of
[17] 18% of tests were positive on one gene only and it was concluded, in Table
2 of [17], that for people with single gene positives, when Ct > 34, none
had symptoms and for people with Ct < 34 only 33% had symptoms.
Furthermore in [16],
published January 8th 2021, it states the goal of using one gene was
explicitly to approximate the growth of the new variant (emphasis mine):
“There
has recently been an increase in the percentage of positive cases where only
the ORF1ab- and N-genes were found and a decrease in the percentage of cases
with all three genes. We can use this information to approximate the
growth of the new variant.”
Quality control and cross
reactivity
Quality control problems
have already been reported in UK laboratories [8, 9, 10] and there have been
concerns expressed about the potential for false positives arising
consequently. Recent suspicion focused on problems potentially caused by
breaches in acceptable Ct thresholds (Ct > 37), suggesting no, or past,
infection. However, this new ONS data shows there may be an additional
potentially dominant source of false positives, at least within the period
covered by the ONS report, if not from April 2020; specifically, positives
caused by potential breach of WHO end user assessment and manufacturer
instructions for use. These sources of false positives appear not to be caused
by statistical error but might instead be categorised as systematic
non-compliance.
Concerns about testing in
commercial laboratories were documented by the ONS as early as May 2020 [11],
when the REACT study discovered that circa 40% of positive tests from
commercial laboratories were in fact false positives. A similar false positive
rate (44%) was reported in Australia [12] in April 2020. More recently Nicholas Lewis claims that,
despite very low false positive rates (0.033%) from testing done by
non-commercial and academic laboratories, there may be good reason to suspect
the operational false positive rates from lighthouse laboratories are worse
than these by some orders of magnitude [13].
Obviously, there is
higher risk of encountering false positives when testing for single genes alone,
because of the possibility of cross-reactivity with other HCOVs and prevalent
nasopharyngeal bacteria or reagent contamination. The potential for cross
reactivity when testing for SARS-COV-2 has already been confirmed by the German
Instand laboratory report from April 2020 [14]. This report describes the
systematic blind testing of positive and negative samples anonymously sent to
many laboratories throughout Germany and evaluated for the presence of a
variety of genes associated with SARS-COV-2. They reported significant
cross reactivity and resultant false positives for OC43, and HCoV 229E (a
common cold virus) as well as for SARS-COV-2 negative samples, not containing any
competing pathogen.
It should be noted that
these issues are not unique to the UK. Likewise, 70 Dutch laboratories were
surveyed in November 2020 [15], by the National Institute for Public Health and
the Environment, with 76 diagnostic workflows reported as using only one target
gene to diagnose the presence of SARS-COV-2 (46% of all workflows).
Conclusions
Unless the UK lighthouse
laboratories have performed diagnostic validation of their single gene call, for
both the original and the B1.1.7 variant, and there is no evidence of this in
the public domain, it can only be assumed that, in the absence of confirmatory
testing, many of the reported positive results may be inconclusive, negative or
from people who suffered past infection for SARS-COV-2. Even with diagnostic
validation of the single gene call, the UK lighthouse laboratories appear to be
in breach of both the WHO emergency use assessment and, also, to have potentially
violated the ThermoFisher TaqPath kit instructions for use.
References
[1] Steel
K. and Fordham E. Office for National Statistics. Coronavirus (Covid-19)
Infection Survey. 5 December 2020.
https://www.ons.gov.uk/peoplepopulationandcommunity/healthandsocialcare/conditionsanddiseases/datasets/coronaviruscovid19infectionsurveydata
[2] Corman
V., Landt O. et al “Detection of 2019 novel coronavirus (2019-nCoV) by
real-time RT-PCR” Euro Surveillance. 2020 Jan;25(3):2000045. doi: 10.2807/1560-7917.ES.2020.25.3.2000045.
[3] WHO
Emergency Use Assessment Coronavirus disease (COVID-19) IVDs. PUBLIC REPORT.
Product: TaqPath COVID‑19 CE‑IVD RT‑PCR Kit. EUL Number: EUL-0525-156-00. Page
60
https://www.who.int/diagnostics_laboratory/eual/200921_final_pqpr_eul_0525_156_00_taqpath_covid19_ce_ivd_rt_pcr_kit.pdf?ua=1
[4] WHO
Information Notice for IVD Users 2020/05. Nucleic acid testing (NAT)
technologies that use polymerase chain reaction (PCR) for detection of
SARS-CoV-2. 20 January 2021
https://www.who.int/news/item/20-01-2021-who-information-notice-for-ivd-users-2020-05
[5] Public
Health England “Investigation of novel SARS-COV-2 variant”, 202012/01.
https://assets.publishing.service.gov.uk/government/uploads/system/uploads/attachment_data/file/959438/Technical_Briefing_VOC_SH_NJL2_SH2.pdf
[6] Alan
McNally. “It's vital we act now to suppress the new coronavirus variant”
Opinion section the Guardian Newspaper, 22 Dec 2020. https://amp.theguardian.com/commentisfree/2020/dec/22/new-coronavirus-variant-b117-transmitting?CMP=Share_AndroidApp_Other&__twitter_impression=true
[7] Richter,
A., Plant, T., Kidd, M. et al. How to establish an academic
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[8] Daily
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https://www.dailymail.co.uk/news/article-8746663/Chaos-Britains-Covid-labs-Scientist-lifts-lid-government-facilities.html
[9]
Channel 4 Dispatches: Lockdown Chaos: How the Government Lost Control. 15th
November 2020
https://origin-corporate.channel4.com/press/news/dispatches-uncovers-serious-failings-one-uks-largest-covid-testing-labs
[10] BBC
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https://www.bbc.co.uk/news/health-54552620
[11] Riley
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[12]
Rahman, H., Carter, I., Basile, K., Donovan, L., Kumar, S., Tran, T., ... &
Rockett, R. (2020). Interpret with caution: An evaluation of the commercial
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virus. Journal of Clinical Virology, 104374
[13]
Lewis. N. “Rebuttal of claims by Christopher Snowden about False Positive
Covid-19 test results”. February 2020. https://www.nicholaslewis.org/a-rebuttal-of-claims-by-christopher-snowdon-about-false-positive-covid-19-test-results/
[14]
Zeichhardt H., and Kammel M. “Comment on the Extra ring test Group 340
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Qualitätssicherung in medizinischen Laboratorien e.V. (INSTAND Society for the
Promotion of Quality Assurance in Medical Laboratories e.V.) 3rd June 2020.
https://www.instand-ev.de/System/rv-files/340%20DE%20SARS-CoV-2%20Genom%20April%202020%2020200502j.pdf
[15] External
Quality Assessment of laboratories Performing SARS-CoV-2 Diagnostics for the
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[16] ONS Coronavirus
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[17]
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