Cytokines IL1β, IL6, TNFα & serum cortisol may not constitute reliable biomarkers to identify [PASC], 2024, Fleischer, Kleinschnitz et al.

[SNT Gatchaman]

Cytokines IL1β, IL6, TNFα and serum cortisol levels may not constitute reliable biomarkers to identify individuals with post-acute sequelae of COVID-19
Michael Fleischer; Fabian Szepanowski; Anne K Mausberg; Livia Asan; Ellen Uslar; Denise Zwanziger; Lothar Volbracht; Mark Stettner; Christoph Kleinschnitz

BACKGROUND
Post-acute sequelae of COVID-19 (PASC) comprise a broad spectrum of symptoms such as fatigue, general weakness, compromised attention and sleep or anxiety disorders. PASC represents a medical and socio-economic challenge.

OBJECTIVES
Our study evaluated cytokines (IL-1β, IL-6 and TNFα) and cortisol levels in a cohort of typical patients with PASC, suffering concentration problems, fatigue and difficulties finding words.

DESIGN
This was a prospective cohort study. Four groups were analysed and compared: those who had never contracted SARS-CoV-2 (n = 13), infected but had no PASC (n = 34), infected with former PASC that resolved (n = 40) and patients with ongoing PASC after infection (n = 91).

METHODS
Cytokine and cortisol serum levels were determined in patients’ blood samples.

RESULTS
Cytokine levels of IL-1β, IL-6, TNFα and cortisol levels did not differ between groups analysed.

CONCLUSIONS
This may indicate a non-organic/psychosomatic genesis of PASC; further studies are needed to elucidate the underlying causes of PACS, and non-organic causes should not be overlooked.

PLAIN LANGUAGE SUMMARY
Without clear biological markers for people who will continue to present with postacute sequelae of COVID-19 (PASC) should we now focus on psychological factors?

Many people across the globe are still suffering from post-acute sequelae of COVID-19 (PASC), commonly called post-COVID. Typical symptoms of PASC include severe tiredness (fatigue), concentration deficits (brain fog) or difficulty finding words. We need a better understanding of how these symptoms arise to find ways to help patients. Our team of researchers set out to explore this. We posed the question: could measurements of immune system activity provide an identifier for people who are susceptible to postCOVID?

The participants in our study were divided into four groups: 1. A group of 13 people who had never contracted SARS-CoV-2. 2. A group of 34 people who had been infected with SARS-CoV-2 but had no PASC. 3. A group of 40 people who had been infected with SARS-CoV-2 and had already suffered from PASC that had now resolved. 4. A group of 91 people who were no longer sick with COVID-19 but were still suffering from PASC.

Serum samples from all participants were taken to measure cytokine and cortisol levels. People with PASC could not be identified by testing their blood samples for cytokines (IL-1β, IL-6, TNFα) or cortisol. No difference between the four groups was found on any marker. Measuring these cytokines or cortisol is, therefore, unlikely to be useful in predicting which patients will suffer from PASC. Continuation of symptoms long after COVID-19 has passed is distressing for many people worldwide. Psychological factors may play a role and need to be studied further in order to help this patient population.

Link | PDF (Therapeutic Advances in Neurological Disorders)

 

[Jonathan Edwards]

Serum samples from all participants were taken to measure cytokine and cortisol levels. People with PASC could not be identified by testing their blood samples for cytokines (IL-1β, IL-6, TNFα) or cortisol. No difference between the four groups was found on any marker. Measuring these cytokines or cortisol is, therefore, unlikely to be useful in predicting which patients will suffer from PASC.

Funny thing that these markers are not used for diagnosing anything else much either!

Not very good for diabetes, brain tumour, respiratory failure, kidney failure, Crohn's disease and so on. So those might also be psychological?

 

[SNT Gatchaman]

Sex ratios were comparable: 63-66% female.

Age was unbalanced: assumed uninfected 33 vs PASC 46-47.

No prior COVID-19: 33.4 ± 11.8

Never PASC: 38.7 ± 16.3

Resolved PASC: 46.6 ± 12.9

Ongoing PASC: 47.3 ± 12.6

---

Cortisol (mmol/l)

No prior COVID-19: 298.4 ± 76.1

Never PASC: 298.7 ± 97.7

Resolved PASC: 331.8 ± 105.2

Ongoing PASC: 340.5 ± 112.6

So although not significant, cortisol levels were increased in LC. Low cortisol was said to be a distinguishing feature of LC in Distinguishing features of Long COVID identified through immune profiling (2023, Nature) —

Participants with LC from two sites had significantly decreased systemic cortisol levels; this remained significant after accounting for variations in demographics and sample-collection times. Notably, the decreased cortisol did not associate with a compensatory increase in ACTH levels, suggesting that the hypothalamic–pituitary axis response to regulate cortisol may be inappropriately blunted.

 

[Hutan]

Without clear biological markers for people who will continue to present with postacute sequelae of COVID-19 (PASC) should we now focus on psychological factors?

At what point did the BPS brigade actually not focus on psychological factors?

Cytokine levels of IL-1β, IL-6, TNFα and cortisol levels did not differ between groups analysed.

Oh, please. We've been fending off poorly based claims that cortisol is somehow different in people with ME/CFS and LC, compared to normal people, for ages now. And this difference (sometimes higher, sometimes lower, always of highly questionable validity) was supposed to prove that our HPA axis was deranged, due to, well, stress, and not being very good at coping with stress i.e. that the illnesses are psychosomatic.

And now, they are suggesting that the fact that cortisol is not different is somehow proof that there is no biological cause, and the illnesses are psychosomatic?

And, don't get me started on cytokines... Researchers have been measuring them for ages, almost certainly poorly, with all sorts of results but mostly null ones in terms of finding a difference. Anyone who has been paying attention to the literature must surely know that nothing useful will come from measuring these cytokines in blood, at least not without some careful management of a whole range of factors including activity levels and measuring momentary and localised effects.

Oh, I suppose possibly these researchers were paying attention... i.e. 'What can we measure that won't show a difference between normal people and those people bleating on about their vague fatigue symptoms, so we can parade the results around as evidence of no real pathology?'

 

[poetinsf]

The weird thing about these papers is that their conclusions/interpretations have nothing to do with the results or their hypotheses. In fact, there is no hypothesis to begin with. It seems they are randomly poking around and then come to random conclusions.

 

[Sean]

At what point did the BPS brigade actually not focus on psychological factors?

Indeed.

It is clear that one of their current strategies is to try rewriting history to make out that the BPS view is the underdog and a 'new BPS approach' is needed after the 'long dominant biomedical approach' has failed.

There was another one of these pieces of nonsense late last year, IIRC, saying the same thing. It is so brazenly dishonest. These people are just shameless.

 

[rvallee]

Kleinschnitz has been a prominent and especially insulting Long Covid denier from the start. He is basically doing much of the same in Germany as Wessely did in the UK for ME, I guess including doing tiny studies checking at a few things to push for the conclusion he started with. He has written many articles completely dismissive of LC, always pushing the psychosocial angle and has shown profound disrespect for long haulers. It's actually an indictment of medical research that he was involved in any research like this.

What's especially weird is that this should not even get a passing grade in any school beyond the first year. To argue that checking a few things that aren't biomarkers of any disease means that not finding something there means this completely unrelated and irrational ideological construct is nonsense. Even in first year essays this could somehow pass if the writing is good and there is a logical structure to the argument, but barely. It's very similar to arguing that the Earth must be flat since if you put something round on the floor it doesn't roll away to the curvature.

 

[Sean]

It's very similar to arguing that the Earth must be flat since if you put something round on the floor it doesn't roll away to the curvature.

I like that comparison.

 

[Sid]

It’s time for these long covid researchers to read the goddamn literature on ME/CFS and acknowledge the decades of work that came before them. Stop wasting time on useless avenues like cytokines which are not useful biomarkers. This has been known for decades.

 

[SNT Gatchaman]

Open peer review by Prof. Matthias Kohl (having published previously with Mark Vink, criticising this group's work). Abridged —

The authors state that this is a prospective cohort study, which is obviously observational in nature. Based on the reference numbers given for the local ethics committee, the study appears to be closely related to the study by Fleischer et al. (2022), where the same reference numbers are given and which was also described by the authors as a prospective observational cohort study. Unfortunately, neither of the two studies was deposited in a registry for clinical studies, even though this should be the standard for observational studies today.

Furthermore, it is not explained in more detail in which sense the study was planned prospectively. For the four biomarkers investigated and their significance in the context of PASC, reference is made to studies whose results were published in 2021 and 2022. In particular, reference is also made to the WHO Delphi consensus criteria of October 2021 for the definition of PASC, although the connection between the consensus criteria and the publication cited in the article, which deals with “a clinically based classification of disease severity for paediatric COVID-19”, is unclear. Since the inclusion of patients began in January 2021, at a time when neither the definition of the PASC group was possible nor the significance of the selected biomarkers in this context was known, it must be assumed that the study objectives were originally different. Again it is not possible to verify this as the study was not registered.

It is not clear, for example, how an attempt was made to avoid selection bias, whether and how potential confounders were examined and how exactly the 178 participants examined in the study were came about (e.g. numbers examined for eligibility, confirmed, included in the study, and analyzed).

Since a non-parametric test was used to compare the groups, it is also unclear why the ShapiroWilk test (Shapiro and Wilk (1965)) was also applied, which is a test of normality (not parametric distribution!). Based on the data distributions shown in Figure 1 of Fleischer et al. (2024), it can be assumed that there was clearly no normal distribution, at least for the cytokines. Accordingly, it is also highly doubtful that Pearson’s correlation is applicable in this case, as the necessary assumption of a linear relationship between the parameters investigated is extremely questionable.

In observational studies, as in the present study, confounders must always be taken into account, which can have a decisive influence on the result. This point was apparently given very little to no attention in the present study. Nothing is mentioned in the Statistics section and only a few demographic parameters are listed in Table 1, the relevance of these parameters for the results does not appear to have been examined in detail.

Due to the serious methodological and statistical weaknesses described above, it must be concluded that the reported results are highly likely to be biased and that the existence of false negative results does not seem unlikely. The final conclusion, that “the above-mentioned cytokines and cortisol are not appropriate biomarkers. The results of this study are consistent with our previous findings and those of others who did not find any laboratory changes and have suggested a non-organic/psychosomatic genesis of PASC.”, which is already implied in the title of the paper, therefore appears completely unjustified.

In particular, it seems completely illogical why one can conclude a "non-organic/ psychosomatic genesis of PASC" on the basis of only four negative biomarkers when there is contradictory evidence in the literature, as also stated in the introduction by Fleischer et al. (2024), and when there are tens of thousands of possible biomarkers that could prove an organic genesis of PASC.

 

[Sid]

Psychological factors may play a role and need to be studied further in order to help this patient population.

So, to sum up this study, we measured 4 metabolites in your body. They were normal. Therefore, you are mentally ill.

 

[NelliePledge]

I hope Prof Matthias Kohl will be able to provide such reviews of many more such papers

also then possibly publish himself on persistent flaws in psychosomatic papers

 

[SNT Gatchaman]

I hope Prof Matthias Kohl will be able to provide such reviews of many more such papers

Published criticism in https://journals.sagepub.com/doi/10.1177/17562864241254268

Furthermore, it is not explained in more detail in which sense the study was planned prospectively. For the four biomarkers investigated and their significance in the context of PASC, reference is made to studies whose results were published in 2021 and 2022. In particular, reference is also made to the WHO Delphi consensus criteria of October 2021 for the definition of PASC. Since the inclusion of patients began in January 2021, at a time when neither the definition of the PASC group was possible nor the significance of the selected biomarkers in this context was known, it must be assumed that the study objectives were originally different.

...

Due to the methodological and statistical flaws described above, it must be concluded that the reported results are highly likely to be biased and that the existence of false negative results does not seem unlikely. The final conclusion made is that ‘the above-mentioned cytokines and cortisol are not appropriate biomarkers. The results of this study are consistent with our previous findings and those of others who did not find any laboratory changes and have suggested a nonorganic/psychosomatic genesis of PASC.’ The first part of this conclusion is already implied in the title of the paper and must, therefore, be clearly questioned.

It also ignores the long-established fact that the absence of evidence is not the evidence of absence. Further studies are certainly needed to be able to exclude IL1β, IL6, TNFα, and serum cortisol as biomarkers associated with PASC. Finally, it is not sufficiently argued why one can conclude a ‘nonorganic/psychosomatic genesis of PASC’ on the basis of only four negative biomarkers when there is contradictory evidence in the literature, as also stated in the introduction of Fleischer et al.,1 and when there are tens of thousands of possible biomarkers that could prove an organic genesis of PASC.

Finally, it is not sufficiently argued why one can conclude a ‘nonorganic/psychosomatic genesis of PASC’ on the basis of only four negative biomarkers when there is contradictory evidence in the literature, as also stated in the introduction of Fleischer et al., and when there are tens of thousands of possible biomarkers that could prove an organic genesis of PASC.

(Author response at https://journals.sagepub.com/doi/10.1177/17562864241255819)

 

[Hutan]

From the authors' response, which is fairly long, the final paragraph:

Taken together, our study demonstrated that neither serum levels of Interleukin-1 beta (IL-1β), Interleukin-6 (IL-6), and Tumor necrosis factor alpha (TNFα), nor cortisol are suitable biomarkers to identify patients with PASC. Interestingly, these ‘negative’ findings have recently been confirmed by other groups5–7 underlining the urgent need of independent replication of results in post-COVID research. Given the lack of valid biomarkers and relevant organ damage,2,5,8,9 the conclusion that PASC might represent a primarily psychosomatic or functional disorder is, therefore, reasonable and appropriate.

I'm not arguing with them about their actual results - it's quite likely that none of those biomarkers in blood are suitable biomarkers to identify patients with PASC. But they completely sidestep the main problem - the likelihood that four molecules aren't biomarkers does not mean that there are no biomarkers.

Yes, the fact that the four molecules probably aren't biomarkers 'might' mean that the disease is all sorts of things. It doesn't rule out the possibility that people with Long Covid have been cursed by the spaghetti monster as retribution for the widespread popularity of pineapple on pizza... But the paper only talked up the possibility of a psychosomatic etiology, which shows incredible bias.

Absolutely bizarre.

It might be a reminder when providing formal criticism to just focus in on the strongest point. Trying to argue that the molecules might in fact be biomarkers allowed the authors to focus on that in their reply.