OPEN LETTER OF CONCERN 4 March 2025

ARE FALSE POSITIVES IN CURRENT PCR TESTING SYSTEMS SUFFICIENTLY MITIGATED AGAINST?

HART hasn’t written much about bird flu though we did recently note the similarities between the fear propaganda of covid and the first whole scale culling of chickens in the UK – One million chickens culled

Carl Heneghan and Tom Jefferson have also written about a number of FOI responses from the MHRA on the topic of the stock-piled avain flu vaccines here and several American authors have been covering the futility of testing and culling whole flocks of chickens and herds of cattle, the only predictable result of which will be to increase the price of food. It is encouraging to hear that Robert F Kennedy Junior is seriously questionning the US policy and indeed the role of vaccination in poultry.

Thus the letter below from recently retired Systematic Review Director, Katherine MacGilchrist is particularly timely and we are reproducing it in full.

From: Katherine S. MacGilchrist, BSc (Hons) Pharmacology (Bristol 1993), MSc Epidemiology (Edinburgh, 2001), Former CEO/Systematic Review Director, Epidemica Ltd.

To: Dr. Jenny Stewart, Interim Chief Executive, Animal & Plant Health Agency (APHA), UK E: APHA.CorporateCorrespondence@apha.gov.uk, enquiries@apha.gov.uk

Dr. Christine Middlemiss BVMS, MRCVS, Chief Veterinary Officer, VMD, UK E: Christine.Middlemiss@defra.gov.uk

Re. H5N1 Highly Pathogenic Avian Influenza Polymerase Chain Reaction testing

Dear Dr. Stewart and Dr. Middlemiss,

The concern

I write to express my concern that ill-defined Polymerase Chain Reaction (PCR) testing systems where false positives may be insufficiently guarded against and subsequent unethical, unevidenced-based, mandated culling of healthy poultry/livestock may needlessly jeopardise the farming sector and thus food security in the United Kingdom (UK).

In an ill-defined, non-transparent system nothing can be concluded from a ‘positive PCR test’. The greater the uncertainties the less justified are any measures. There should certainly be no mandated consequences for healthy poultry/livestock imposed by the State, following a PCR test used in isolation.

Just one, of several, recent examples of mass poultry culling in UK and USA occurred at Orchard Organic Farm, Devon, UK where over 4000 chickens with no signs of disease and in a separate barn were slaughtered and thousands of eggs destroyed following a positive H5N1 test and a financially-crippling 12-month restocking restriction imposed. A veterinarian’s response suggests these measures may be less than evidence-based ¹.

The questions

Due to the current level of uncertainty, I append a series of questions that I would be grateful if you could answer, to establish:

1. The degree to which the current PCR testing system design guards against false positives;

2. How positive PCR test results are currently interpreted. Of note, PCR tests cannot categorically distinguish between past infection/exposure (degraded nucleotides) and active infection (presence of a whole replicating virus);

3. What evidence APHA/DEFRA has for mass culling of healthy livestock vs limited culling of the symptomatic or alternative actions?

4. Whether actions taken are done so on the basis solely of a positive PCR test? Any ‘suspected case’ should ordinarily be properly confirmed, including virus isolation and culture and/or other serological tests and sequencing, plus relevant symptoms and clinical diagnosis.

The questions raised are directed at PCR tests for H5N1 in poultry, but apply equally to PCR tests used in other diseases in poultry, animals and, I might add, humans.

Importance of getting to grips with these questions

A misinterpreted or false positive PCR test result would mean that measures taken at a farm would be completely unwarranted, risking farm viability and the food supply, not to mention farmers’ stress levels, needlessly.

Such false positives in poultry could also risk an unfounded rollout of H5N1 vaccine in humans. Human immunization rollouts have been triggered by false alarms in the animal sector before, with negative results (deaths and Guillain-Barrė Syndrome), e.g. the unneeded swine flu alarm in 1976 ². Of note, it is not known how individuals who have received a COVID- 19 injection (who may be in an inflammo-thrombotic state) will react to a haemagglutinin antigen, as in the H5N1 vaccine stockpiled in USA: the Biologics Licence Application (BLA) was submitted in 2019 (before the COVID era) ³. I understand that the UK has also stockpiled an H5N1 vaccine.

Cheaper, practical alternatives to culling

As the migratory bird population is likely the reservoir, which is not going away, the logical solution surely is to build longer term immunity and general health condition of flocks, to lower susceptibility. Mass culling of symptom-free birds, who may have natural immunity to H5N1, will be culling precisely the birds one would most want to retain in order to promote host resistance ⁴. Building longer term immunity would also make efficiency savings in the not insubstantial taxpayer funds spent on culling compensation. Vaccine injections are not the answer: European Food Standards Agency documentation states they do not fully stop transmission in chickens ⁵ and they would be impractical from a cost/time perspective. Furthermore, the whole concept of using an injection (in deeper layers) to induce immunity at a barrier layer (skin, gut, lungs) has been questioned ⁶.

Cheaper, practical options include:

1. For birds raised in better conditions (in which H5N1 may be less pathogenic), allow H5N1 to go through flocks and select survivors ⁷;

2. Incentivise (through subsidy) raising poultry in cleaner and less tightly packed conditions (in which they will be more resistant to the spread of disease);

3. Using ultraviolet light indoors to inactivate pathogens, decrease mortality and increase productivity;

4. Use of chlorine dioxide solution (CDS) in closed drinking water drip systems ⁸.

Call for transparency, evidence-based measures and equity for farmers

Please put the testing system and consequences of it on a transparent and evidence-based footing, and make it equitable for farmers. A range of experts and stakeholders are cc’d to this letter from whom you may wish to invite contributions. Such a system would cover how tests/systems were designed, evaluated, sampled, analysed and interpreted. Any measures would differentiate between tightly packed, and free-range, poultry. Recommendations would be referenced and decision-making power retained by farmers, particularly for healthy livestock, to ensure continued viability of farm businesses.

While uncertainties remain

While uncertainties remain, farmers and veterinarians may wish to consider their participation in PCR testing if used in isolation. They may also find this Notice ⁹ regarding free-range poultry of interest or the Guard the Gate Initiative

https://guardthegate.online,

a campaign to peacefully support farmers.

Legislative changes needed

Redress in the balance of power between State and the farming sector is needed and removal of inconsistent and controversial Government positions, to prevent further damage to poultry/livestock farming.

1. Amendment of Regulation (EU) 2016/429, the ‘Animal Health Law’ ¹⁰ is required.

2. Repeal of the 2008 Climate Change Act, which put in place the self-sabotaging net zero goals for greenhouse gas (GHG) emissions. On the one hand we have the now Minister of State DEFRA, Daniel Zeichner, MP, stating, rightly, that feeding the nation is a matter of national security and a top priority ¹¹. On the other we have net zero-related stated goals of dramatically reducing dairy and meat consumption by 2030/2049 (section 6.4.1. Table 3 of ¹², Table 7.4.1 of the Seventh Climate Budget¹³) and reducing livestock numbers in the UK by 38% by 2050 versus 2023, particularly of cattle and sheep (page 187 and Table 7.4.1). These livestock reduction goals are based on an apparent need to reduce GHG emissions. Many scientists do not agree with this premise, however, nor that there is a climate emergency, as recent conference proceedings and CLINTEL Declaration in Prague attest ¹⁴. Even if there were a consensus, science is not made by consensus but rather by predictions from theories agreeing with actual observations ¹⁵. Hypotheses other than carbon dioxide (CO2) and methane emissions explain and predict the climate on this, and other, planets ¹⁶ whereas GHG-based International Panel on Climate Change (IPCC) models apparently do not. The latter also involve questionable positive feedback loops (page 21-26 of (14)), when most feedback systems in nature involve negative feedbacks (15). Of note, a grand solar minimum is predicted for the period 2020-2053 ¹⁷ before resumption to the overall trend of gradual (likely beneficial) warming (all independent of CO2). This imminent period of cooling, if correct, will require focused attention on food supplies, especially during 2031-2042 ¹⁸: we will need our poultry/livestock farmers more than ever and land retained for fodder supplies.

Sincerely,

Katherine S. MacGilchrist, BSc (Hons) Pharmacology, MSc Epidemiology

Former CEO/Systematic Review Director, Epidemica Ltd., Founder/Director Herborium Ltd.

Author Note: No grant or funding has been received by the author in relation to this letter.

ADDENDUM: QUESTIONS RELATING TO GUARDING AGAINST FALSE POSITIVES, INTERPRETATION OF A POSITIVE RESULT AND MEASURES FOLLOWING A POSITIVE H5N1 HPAI PCR TEST

Conceptual questions

1. What evidence is there that a pathogen identified/suggested by an oropharyngeal swab sample is the same as the causative agent of an infection in the lung of symptomatic poultry?

2. What is the methodological body of work that supports the supposition that the presence of a genetic fragment or fragments (which is what PCR detects) can be inferred to mean that an active infection is present and that the genetic fragment(s) is/are the cause of said infection?

3. What evidence do you have to discount that the PCR tests are simply picking up a background signal of DNA/RNA?

4. What evidence do you have to support the inference that a novel signal/influenza A subtype genetic sequence equates with having a novel RNA virus?

5. What evidence do you have that an RNA virus can be pandemic?

Transparency

1. Does APHA publish a list of diseases for which the only tests available are PCR tests?

Confirmatory testing following a positive PCR test result

1. Does APHA use virus isolation and culture to verify any/all PCR positive test results, or any other serological tests? A Freedom of Information Request for records held by CVO Scotland suggests the Scottish Government may not use virus isolation for H5NI HPAI ¹⁹.

Intrinsic false positive rate of the H5N1 HPAI PCR test

Please note I am not questioning staff expertise in running tests to ISO17025 lab standards.

1. What is the intrinsic false positive rate of the test in your laboratories?

2. How many H5N1 PCR tests in poultry has APHA/UK labs run in 2024 and 2025?

3. How many positives were obtained?

4. From the answers to the above three questions are the positives obtained simply due to the intrinsic false positive rate of the test (i.e. likely to just be false positives)? Do you report such results publicly? If so please reference where.

5. For the positive PCR tests obtained was confirmatory testing (virus isolation and culture or other serological tests) performed prior to action being taken on farms?

6. Please provide the PCR test manufacturer’s instructions for use (IFU).

• Please provide the peer-reviewed publication documenting the evaluation of the H5N1 PCR test in the UK, i.e. evaluating its false positive rate and other performance parameters. Please specify the sample sizes used in evaluation studies and confidence intervals around the false positive rate estimates.

• Please state in which setting/population of chickens the published evaluation of the test was performed, e.g. country (UK?); barn, free-range or organic hens; large-scale commercial or smaller-scale set-up. Of note, the intrinsic false positive rate will only be valid within the tested setting, so any recommendations following a positive PCR test can only be deemed evidence- based if the test has been evaluated in the specific setting. Generalisation between very different farm settings is insufficient to warrant measures.

• Are the PCR tests used in the UK for H5N1 HPAI made by different manufacturers? If so, how many such tests are there? Please answer Questions 1, 6-8 for each manufacturer’s test.

Design of the H5N1 HPAI PCR test(s)

1. Exactly which nucleotide sequence(s) does the test identify? I’m asking here for the exact viral genome nucleotide sequence(s) that the complementary primers/probes are targeted to find.

2. Who and which institutions selected those nucleotide sequences for the test? The reason for asking this is because in SARS-CoV-2 testing two of the three genes that the PCR test contained were from Charité in Berlin (E gene) and from US CDC (N2 gene) and these apparently tested positive for all samples tested, even for control samples (e.g. water) ²⁰,²¹, so if sequences have been selected by these institutions for the H5N1 PCR test we should ensure that evaluation of the test for those gene sequences has occurred across different samples including controls/blanks. Careful scrutiny of such validation studies is also required as serious methodological flaws have been observed previously, which result in cross-reactivity being presented as less of a problem than it actually is (21).

3. How long are each of the gene sequences (how many nucleotides)? Shorter sequences are more likely to cross-react and increase false positives.

4. Where in the Influenza A/H5N1 genome do the gene sequences sought reside? At the 5’-end or at the 3’-end and/or in the middle? Which location of the RNA is more stable? Greater stability would mean that the nucleotide fragment would not degrade as quickly, resulting in a longer period of time over which a test would result in a positive, even long after a chicken had recovered from any infection. So, selection of the gene sequences can impact the positivity rate.

5. Are the H5-specific gene targets in the PCR test used in UK from North American H5 isolates? As per the WOAH Terrestrial Manual 2021 page 10, some primer/probe sequences have been designed to detect North American H5 isolates and may not be suitable for all isolates.

6. Do any of the gene sequences sought cross-react with other pathogens? Which ones? Low Pathogenic Avian Influenza (LPAI) H9N2, H11N1, H4N6? Please provide the cross-reactivity rates for each, the cycle thresholds used to detect the competing pathogen(s) and the peer- reviewed publication(s) or reports documenting the validation work. Please specify the sample sizes used and confidence intervals around the cross-reactivity rate estimates. By way of example, one of the SARS-CoV-2/2019-nCoV PCR tests was demonstrated to cross react with HCoV-229E and HCoV-OC43 ²² and the problem of cross-reactivity of PCR tests as applied to SARS-CoV-2 has been discussed further here (21).

7. How many gene sequences does the test look for? Normally at least 3 sequences should be used, from regions across the viral genome.

8. Is there evidence of contamination of primer and probe batches?

9. What independent quality control has the PCR kit been subject to?

10. What assurances are there that the PCR test in use in UK is detecting contemporary viruses and has been validated for use specifically in the UK, as recommended in the World Organisation of Animal Health (WOAH) Terrestrial Manual 2021?

Question about timing of use of the H5N1 PCR test within a test system

1. If seasonal testing were performed only, when H5N1 in poultry is known to have a higher true prevalence in the UK, this would help lower the likelihood of false positives. January- March (colder ambient temperatures) is documented as the main season for H5N1 in poultry in several countries, coinciding also with circulation of seasonal influenza virus ²³. Out-of- season testing, when the true prevalence of disease is low and more likely to be lower than the intrinsic false positive rate of the test, will increase the likelihood of positives being false. Has APHA considered limiting farm poultry testing to high prevalence season only?

Questions about how the sample material is obtained

1. Does APHA advise veterinarians/farmers to only take samples from birds with (relevant) symptoms? This is important because if the intrinsic false positive rate of the test is higher than the true prevalence of the disease in the population tested then a majority of positives will be false positive. Testing should only be performed in poultry WITH symptoms, therefore. This is a well-known concept in epidemiology. Mass testing in asymptomatic populations can result in false positive pseudo-epidemics. Incidentally, this likely contributed to what was observed in 2020 in human populations where mass testing of people without symptoms was encouraged in summer.

2. Do you advise veterinarians/farmers on sample taking methods to reduce the likelihood of cross-contamination? E.g. to reduce the possibility of contamination of the sample with migratory bird faeces (that may contain influenza viruses)?

Questions about how the sample is analysed

1. Does APHA declare a positive based on detecting the presence of a single gene target? This would lead to more false positives, due to cross-reactivity with other pathogens or reagent contamination, as was observed when single gene testing was employed in UK Lighthouse Laboratories for SARS-CoV-2 ²⁴.

2. Do you declare a positive based on detecting at least two or more gene sequences, as was recommended by the World Health Organization (WHO) regarding PCR testing for COVID- 19 (please see WHO guidelines page 60 of ²⁵). Requiring all three gene sequences to be present to declare a positive would further lower the risk of false positives.

3. What is the cycle threshold (Ct) you use in the H5N1 PCR test gene sequences above which you would not declare a positive?

4. Please provide the test development and validation process work for the above Ct value.

5. In the setting of this/these Ct value(s) is the mean Ct value taken for each gene sequence or another basis used? In the setting of Ct value beyond which a positive would not be declared, has consideration been given to the consequences of declaring a positive test result, given the current apparent policy of ‘slaughter on suspicion’ of healthy poultry? The more adverse the consequence the more stringent the Ct value setting should be in terms of avoiding false positive results, i.e. the Ct value above which a positive would not be declared should be set at a lower value. The higher the Ct value of a test the less adverse any consequences should be, e.g. isolate/treat/cull a symptomatic hen vs culling all poultry at a site whether symptomatic or not.

6. Do you use nested primers?

7. Following amplification, do you conduct sequencing to check that what has been amplified is indeed the gene fragment targeted?

Reporting the test result and interpretation

1. When reporting results to veterinarians/farmers, do you report the Ct value(s)? Without this, veterinarians and farmers cannot interpret results.

2. When reporting results to veterinarians/farmers, do you report which of the specific nucleotide sequences/gene targets sought by the test were identified in the sample?

3. Do you provide any interpretation guidance? For example, the intrinsic false positive rate of the test, and cross-reactivity rates in the relevant setting/population might add context to a positive test result.

4. Do you check what setting the sample being reported on was from and whether the validation work you have for the PCR test is valid in that setting? Do you report this to veterinarians/farmers?

5. The PCR test detects specific nucleotide sequences. It does not distinguish clearly between an active infection/exposure and a past infection/exposure. Do you state this qualification when reporting results to veterinarians/farmers? If the test is designed to detect three gene targets from H5N1 HPAI, from different regions across the viral genome, if all three are required to be detected to declare a positive and if the Ct number was low for the test, then an active infection might be more likely, but still could not be confirmed definitively. If only a single gene target is required in order to declare a positive and a high Ct number (e.g. above 30) is used the ‘positive’ result will have little practical clinical meaning (due to higher likelihood of cross- reactivity or contamination) and will only serve to decimate poultry farming.

Assessing false positive rate across testing system from contamination

1. Have known blanks/negatives been put through the whole system from a farm, through vet offices, distribution and through the laboratory to test the operative false positive rate? This addresses the issue of cross-contamination (as opposed to cross-reactivity). If so, what was the result and when/where was it performed?

Definition of a case/suspected case of H5N1

1. Please provide how you define a ‘case’ of H5N1, which would result in measures/culling being undertaken at a farm currently.

2. Is the current definition of a ‘case’ based on a positive PCR test only, without the need for relevant symptoms in the chicken and a veterinarian’s clinical diagnosis and confirmatory testing (viral isolation and culture or other serological tests)?

3. What is the definition of a ‘suspected case’ vs a ‘confirmed case’?

Slaughter on suspicion and farm restriction actions

1. Please provide the evidence on which slaughter of healthy poultry and of healthy poultry within a radius of a ‘positive case’ is demonstrated to be a better policy – in terms of long-term effectiveness (in preventing spread of infection and reducing flock genetic susceptibility) and cost-effectiveness (from a societal perspective) – compared to a policy of isolating symptomatic ‘positive’ poultry and treating them or culling only those affected symptomatic birds.

2. Please provide the evidence on which farms are restricted for 12 months after a ‘positive case’ is identified.

3. Please provide the evidence for the destruction of eggs and the destruction of feed after a ‘positive case’ is identified.

4. Please provide the evidence for the disinfection requirements after a ‘positive case’ is identified. Has chlorine dioxide been considered? ²⁶

5. Please provide the evidence for housing orders and bird flu prevention zones.

6. Has APHA/DEFRA systematically reviewed alternatives to culling? If so, when?

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