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Trial By Error: Did the IBS Trial Really Show that Web-Based CBT Offered Significant Clinical Effectiveness?

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By David Tuller, DrPH

I wrote some posts last year about the ACTIB trial–a major study of telephone-delivered and web-delivered cognitive behavior therapy (TCBT and WCBT) for irritable bowel syndrome (IBS). Contrary to how the results have been framed by those with reputational and financial interests in promoting them, the study demonstrated that WCBT did not provide clinically significant benefits over treatment as usual on its two primary outcomes. At least that’s how I interpret the data.

Both of these primary outcome measures were self-reported—a symptom severity scale and a measure of impact on work and social functioning. (More on these below.) As no less an authority than the Journal of Psychosomatic Research has recently noted, these sorts of outcomes are at particular risk of bias when blinding is not rigorous. In this case, with blinding not possible given the nature of the interventions, the results should already be assumed to be infused with an unknown amount of bias. In other words, marginal or modest reported benefits would be well within the range expected given the bias inherent in an unblinded study relying on subjective outcomes.

In January of 2020, Mahana Therapeutics, a San-Francisco start-up, announced that it had licensed the rights to the web-based program. In the trial, the program was called Regul8; Mahana rechristened it as Parallel. (Does anyone get the new name? Parallel to what?) I noted in blog posts and in letters to Mahana officials and advisers that the company was making inflated claims about the purported benefits of the program.

Since then, the US Food andDrug Administration has approved it for marketing–although a product like this is not assessed by anything approaching the (purportedly) rigorous standards used for pharmaceuticals. It has also received a CE mark in the UK, which apparently allows it to be sold there as well. (Given Brexit, the implications of the CE mark for European sales are not fully clear to me–or, I presume, to anyone.) This is a very minimal regulatory bar. According to the relevant UK government agency, “the CE mark is not a quality mark, nor a guarantee that the product meets all of the requirements of relevant EU product safety law.”

More recently, the same research team has published cost-effectiveness data from the IBS study. My friend and colleague Keith Geraghty, a research fellow at the University of Manchester, has tweeted about this new paper and some of its assumptions. I haven’t looked at it yet in depth, but what I don’t get is how an intervention can be cost-effective if it isn’t really clinically effective. Cost-effective at what?

The abstract of the new cost-effectiveness study starts with the following sentence: “Telephone therapist delivered CBT (TCBT) and web-based CBT (WCBT) have been shown to be significantly more clinically effective than treatment as usual (TAU) at reducing IBS symptom severity and impact at 12 months in adults with refractory IBS.”

It is important to note that the results for TCBT and WCBT in the IBS study were not the same. TCBT scored better overall on primary and secondary measures. That is not surprising. The greater personal contact could potentially lead to greater therapeutic effects; it could also lead to greater bias in participant responses. Teasing the two phenomena apart would be challenging.

But Mahana is marketing WCBT, not TCBT—and key academic investigators have financial interests in the product. The statement about the interventions being “significantly more clinically effective than treatment as usual” in the new cost-effectiveness paper prompted me to take another look at ACTIB’s 12-month results for the two co-primary outcomes. (Secondary outcomes are secondary outcomes; they should not be advanced as proving success when primary outcomes generate null results.) And when it comes to “reducing IBS symptom severity and impact at 12 months,” the above sentence from the cost-effectiveness paper does not seem to be consistent with the specific WCBT data for the primary outcomes.

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Two co-primary outcomes for the IBS study

The two co-primary outcomes were the 12-month results for the IBS-Symptom Severity Scale (IBS-SSS) and the Work and Social Adjustment Scale (WSAS). In previous posts, I focused on the results for the first–a 500-point scale in which higher scores represent more severe symptoms. A reduction of at least 50 points represents what is considered a clinically significant reduction in symptom severity for an individual. In this trial, at 12 months, the WCBT group had a mean score of 35.2 points lower than the TAU arm. That is quite a bit less than the 50-point difference considered to be clinically significant for an individual.

Despite this 50-point threshold, the investigators seem to have created something of a statistical out. Here’s what they wrote: “IBS-SSS is widely used in IBS studies and a 50-point within-participant change from baseline regarded as clinically significant. We used a 35-point between-group change as our minimal clinically important difference (MCID) assuming some improvement in TAU arm (15 points placebo response) as in the MIBS trial.”

Given that the 35.2-point finding for the WSAS in the WCBT arm squeaks past the 35-point threshold designated by the investigators, they might consider these results to represent clinical significance. But it is unclear why they should reduce the MCID target for their intervention by 15 points because of a presumed placebo response–even if a previous trial reported such a finding.

The TAU condition is meant to replicate, more or less, the usual care that patients receive. To be considered effective, the intervention should demonstrate a clinically significant benefit when compared with TAU—not when compared with baseline. In fact, the first sentence of the abstract of the new cost-effectiveness paper specifically references the comparison with TAU—not with baseline numbers. After all, clinical trials are designed to assess the effects of interventions over and above changes in those who don’t receive them.

To be fair, the investigators also reported that a greater percentage of those who answered the IBS-SSS at 12 months in the WCBT arm reported an improvement of at least 50 points, compared to the percentage in the TAU arm. But 30% of the participants did not provide 12-month responses for the IBS-SSS, so their responses are unknown. That means interpreting these data can be challenging. Perhaps those who received the intervention were more inspired to report positive results than those who received TAU alone, even if the latter also felt they’d improved to the same extent. We simply don’t know.

In other words, these reported findings do not take into account the large amount of missing data. In contrast, the designated primary outcomes—the differences in the mean scores of the arms at 12 months–were specifically adjusted to address that issue.

The other co-primary outcome measure, the WSAS, is a 40-point scale to assess how an illness or medical condition impacts work and social domains. As with the IBS-SSS, lower scores represent better results. In the IBS study, the differences in mean scores at 12 months between the intervention arms and the TAU arms at 12 months were modest—a 3.5-point difference for TCBT and a 3-point difference for WCBT. The paper did not include an MCID for that measure, unlike for the IBS-SSS.

However, members of the same research team have recently published a study of CBT as a treatment for so-called “persistent physical symptoms” (PPS), a category that includes IBS; in fact, the trial recruited a substantial number of participants from gastroenterology. In this instance, the investigators designated the WSAS as the sole primary outcome and further identified a difference of 3.6-points as the MCID for that measure. As reported, the benefits for the intervention fell well below the MCID for the measure.

If the MCID of a 3.6-point difference on the WSAS is applied to the IBS study findings—and I see no reason why it shouldn’t be, especially given the overlap in research teams and patient populations–the conclusion is clear. Neither the TCBT nor the WCBT provided clinically significant benefits over TAU on that measure.

Yet the investigators still claim in the first sentence of the abstract of their new cost-effectiveness paper that both TCBT and WCBT were shown to have a clinically significant effect on reducing symptom severity and impact. And the WCBT program is being marketed with the same argument. Given that the co-primary outcomes of the major study of WCBT found no clinically significant reduction of symptom severity and no clinically significant impact on improving work and social adjustment at 12 months, these claims are certainly open to question.

Trial By Error: Null Outcomes Presented as Success in Yet Another CBT Trial from Prof Trudie Chalder

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By David Tuller, DrPH

Trudie Chalder, a professor of cognitive behavior therapy (CBT) at King’s College London, has recently published yet another high-profile paper: the main results for “efficacy” from a trial of CBT for patients with so-called “persistent physical symptoms” (PPS) in secondary care. As usual with this group of investigators, things haven’t turned out well. But despite null results for the primary outcome, Professor Chalder and her like-minded colleagues have cast the findings in a positive light in their article, published in Psychological Medicine. 

(Psychological Medicine also published the bogus 2013 “recovery” paper from the PACE team; Professor Chalder was one of the three lead investigators for this classic of likely research misconduct, in which participants could get worse on the primary outcomes and still be deemed. to be “recovered.” When I complained to the editors about it a few years ago, I was advised to replicate the PACE trial; instead, I wrote a letter to the journal that demanded an immediate retraction and garnered more than 100 signatories.)

The new study, called the PRINCE Secondary Trial, is separate from another trial of people with persistent physical symptoms in primary care—the PRINCE Primary Trial. Both are part of the ongoing campaign by Professor Chalder and her colleagues to provide an evidentiary base to justify the expansion of psychological services to anyone suffering from PPS, a category also frequently referred to as “medically unexplained symptoms” (MUS). For Professor Chalder and her colleagues, PPS and MUS include chronic fatigue syndrome, irritable bowel syndrome, fibromyalgia, and pretty much anything else that resists easy clinical assessment and diagnosis and could be inferred to be psychologically driven and/or perpetuated by experts predisposed toward such an interpretation.

First, let’s note that PRINCE Secondary is an unblinded trial relying on self-reported outcomes—a study design fraught with potential and actual bias. This is not just the opinion of people who dislike the PACE trial and believe that Psychological Medicine publishes a lot of crap research. As I’ve noted, the current editor-in-chief of the Journal of Psychosomatic Research, along with his two predecessors, published an editorial earlier this year in which they clearly indicated that subjective outcomes were subject to enormous bias in studies that were not rigorously blinded. (That hasn’t stopped the journal from continuing to publish such problematic research, such as recent output from Professor Chalder’s PACE colleague, Professor Peter White.)  

On the basis of this laudable stance from the Journal of Psychosomatic Research, any positive findings from PRINCE Secondary would be suspect from the start. But that’s not even at issue here, given the null results for the primary outcome—the Work and Social Adjustment Scale (WSAS) at 52-weeks. (The invaluable blog CBT Watch has published a critique of the trial.)

PRINCE Secondary, according to the trial protocol, was “an RCT designed to evaluate the efficacy and cost-effectiveness of a transdiagnostic cognitive behavioural intervention for adults with PPS in secondary care.” The intervention—”therapist-delivered transdiagnostic” CBT, or TDT-CBT—was offered in addition to standard medical care (SMC). It was specifically developed to address the following concerns, as outlined in the published paper: “Patients with PPS can develop unhelpful cognitions and behaviour which can consequently lead to a reduction in daily functioning, reduced quality of life, and an increased susceptibility towards developing depression and anxiety.” The comparison arm received SMC alone.

The protocol touted the trial as a major initiative: “The PRINCE Secondary study will be the first trial worldwide to address the efficacy and cost-effectiveness of a manual-based, transdiagnostic, approach…If it proves to be efficacious, this treatment approach could significantly improve overall functioning in patients with PPS and may lead to substantial long-term economic benefits to the NHS.”

That last point is important. Professor Chalder and many of her colleagues have promoted the expansion of the National Health Service’s Improving Access to Psychological Therapies program to people with MUS. After this publication, it will be hard to cite PRINCE Secondary as proof that CBT for MUS is an “efficacious” treatment–on the contrary, the results undermine any such claim. But that probably won’t make Professor Chalder or any other members of the CBT ideological brigades question their own assertions about their favored interventions. [In research, “efficacy” and “efficacious” refer to how interventions perform in controlled studies like clinical trials; “effectiveness” and “effective” refer to how interventions perform in the real world.]

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Let’s forget about “efficacy” and cite “helpfulness” instead

The protocol for the PRINCE Secondary Trial, published in BMC Pyschiatry, clearly stated: “Efficacy will be assessed by examining the difference between arms in the primary outcome Work and Social Adjustment Scale (WSAS) at 52 weeks after randomisation.” The trial itself notes that the WSAS “was chosen as the primary outcome because the focus of therapy was on targeting processes which might result in a reduction of the impact of symptoms.” In other words, the TDT-CBT was aimed specifically at influencing the cognitive and behavioral factors that were presumed to be preventing PPS patients from full engagement in their work and social lives.

Ok, then. After conducting their due diligence and assessing all the earlier studies and possible outcome measures in the process of developing an authoritative protocol, the investigators determined how they wanted everyone to definitively measure the impact of their intervention. The WSAS is a 40-point scale. The investigators calculated that a difference of 3.6 points or more on the scale would be considered clinically significant. That is, any change of less than 3.6 points would be of insignificant clinical benefit to an individual—it would be an essentially meaningless blip that would not translate into a noticeable improvement.

At 52 weeks, the mean WSAS score of those who received the intervention was only 1.48 points lower than those did not. (Lower WSAS scores represent improvement.) The p-value was 0.139—far from the 0.05 threshold needed to be considered statistically significant. So the WSAS 52-week findings were both clinically and statistically insignificant. Moreover, the entire confidence interval range (-3.44 to 0.48) fell below the designated 3.6-point threshold for clinical significance. These are pretty unequivocal results. They are definitely not useful to those seeking to promote the use of CBT as a treatment for PPS and MUS.

That’s why the conclusion of the paper’s abstract is so striking, and so bizarre. An abstract’s conclusion is what many people who scan a paper will likely remember the most. Here is the entire conclusion of this abstract: “We have preliminary evidence that TDT-CBT + SMC may be helpful for people with a range of PPS. However, further study is required to maximise or maintain effects seen at end of treatment.”

Anyone who takes the time to review the paper should be mystified by this conclusion. This full-scale trial was approved because a lot of earlier research, as outlined in the protocol, had produced ample “preliminary evidence” of the kind mentioned in the conclusion. The protocol, unless I misread it, did not propose to produce more “preliminary evidence” that the TDT-CBT intervention “may be helpful.” PRINCE Secondary was presented in the protocol and received funding based on the notion that it would produce hard data about “the efficacy and cost-effectiveness” of the intervention. (The Psychological Medicine paper did not include the “cost-effectiveness” data.)

It should be noted that “helpfulness” is not the same as “efficacy” and is not defined in the protocol or the trial itself. An intervention might be “helpful” in some way as a supportive strategy while having no “efficacy” as an actual treatment. In this trial, the method of assessing the “efficacy” of the treatment was clearly designated; the results did not achieve that metric, so the treatment cannot be described as “efficacious.” As a vague stand-in, “helpfulness” sounds positive but can mean more or less anything—as it seems to here.

In the paper, the investigators designate eight secondary outcomes. They tout marginal improvements in three of them as indicating possible “helpfulness.” But the results suggest at best the following: Giving people eight sessions of encouragement and attention could prompt them to upgrade their answers by one step or two on some—but not most—questionnaires, compared to those who do not receive eight weeks of such encouragement and attention. That’s it. Expansive interpretations of “helpfulness” are not justified.

Let’s examine these secondary results in a bit more detail. The first is the WSAS at 20 weeks, which reported a 2.41-point difference between the groups. This is still below the 3.6-point threshold for being a clinically significant difference. And as expected because of the bias inherent with self-reported outcomes in unblinded studies, even this minimal apparent effect was not maintained by the 52-week point. (The WSAS at 20 weeks was not in fact listed as a secondary outcome in the protocol.)

Other results are also unimpressive. Five of the eight listed secondary outcomes did not produce statistically significant findings. Two others did. The intervention group achieved a 1.51-point difference from the comparison group on the 30-point Patient Health Questionnnaire 15 and a 0.55-point difference on the 9-point Global Clinical Impression scale. These minimal reported improvements do not provide convincing evidence in favor of the intervention, since they are well within the range of responses that one might expect from the kind of bias noted by the editors of the Journal of Psychosomatic Research in an unblinded study with subjective outcomes.

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The problem of multiple comparisons

And there’s another issue here. When authors engage in multiple comparisons, they increase the likelihood of obtaining some results that reach statistical significance by chance. To make up for that, it is common to correct or adjust the results with standard statistical steps—and the Bonferroni correction is the most well-known. Yet the PRINCE investigators don’t like that approach. It is too stringent for their needs, so they decided not to do it. Here’s what they say:

“Throughout this paper, we present unadjusted p values. Methods for adjusting the family-wise error by methods such as the Bonferroni correction are known to be conservative. However, if one were to use a method that controlled the false-discovery rate such as the Benjamini–Hochberg procedure then the differences on PHQ-15, WSAS at 20 weeks and CGI remained statistically significant and would therefore be considered as discoveries after correction for all nine outcomes (eight secondary plus primary outcome).“

My creative interpretation of this statement: Our findings are weak but they’re even weaker than they appear to be as reported. That’s why we didn’t bother to calculate and present p-values that took into account and corrected for how many tests we ran to try to find statistically significant results. Also, the standard method to correct for multiple tests in a study like this is really, really tough to get through, so we’re not going to use it. But we can assure you that a correction method we like better still lets us call these results “discoveries!” (We’re not presenting those corrected results, but trust us on this one–the study is a success.)

An abstract’s conclusion should at least make an effort to incorporate the findings for the primary outcome—the main results of interest. The conclusion from Chalder and colleagues should have forthrightly noted that the intervention was not efficacious. In this context, to prioritize exceedingly modest unadjusted results from a minority of secondary outcomes over the null results of the primary outcome is an insult to readers. This tactic for hiding bad news reeks of desperation—like a bald man’s comb-over.

Furthermore, it is disingenuous for investigators to assert that these meager data represent “preliminary evidence” for the intervention when the primary outcome was a bust. The call for further research to study how to maintain or extend these effects is unwarranted. The intervention failed to produce the predicted and desired effects. That’s the only credible interpretation of these disastrous results.

Peer reviewers and journal editors are supposed to act as safeguards against misrepresentations. In this case, the system failed. A conclusion that does not mention the null results for the primary outcome is unacceptable—and it should have been unpublishable.

The remarkable diversity of bat coronaviruses

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All human viruses, including SARS-CoV-2, arose from spillovers from other animals. Results of a recent study of bat samples collected in a small region of Yunnan Province, China revealed additional close relatives of SARS-CoV-2 and SARS-CoV.

After the identification of SARS-CoV-2 in early 2020, wildlife sampling and retrospective genome sequencing revealed highly related viruses in animals. The virus RaTG13, from Rhinolophus affinis, shares the highest overall genome identity with SARS-CoV-2, 96.10%. The virus RmYN02 from the bat R. malayanus is the closest relative of SARS-CoV-2 in the ORF1ab open reading frame. Pangolin viruses from Guangdong have identical amino acids at six key residues in the receptor binding domain (RBD) needed for binding to human ACE2. A more distantly related coronavirus was isolated from a bat in Japan, and two viruses from Cambodia have 92.6% genome identity with SARS-CoV-2 and share five of six critical amino acids in the RBD. Finally a bat coronavirus from Thailand is closely related to RmYN02. These observations demonstrate that bats from a broad part of Asia harbor close relatives of SARS-CoV-2.

Fecal samples, oral swabs, and urine samples collected from mainly horseshoe bats in a small area of Yunnan Province between May 2019 – November 2020 were analyzed by RNA sequencing. The results revealed 9 novel betacoronaviruses and 17 alphacoronaviruses. Four of the betacoronaviruses were related to SARS-CoV-2 and three were related to SARS-CoV. Rhinolophus pusillus virus RpYN06 was the closest relative of SARS-CoV-2 in most of the genome with the exception of the spike gene. The other three SARS-CoV-2 related coronaviruses encode a spike gene that could weakly bind to the hACE2 receptor in vitro. Patches of high sequence identity in different regions of the genome illustrate the work of extensive recombination events.

While all the viruses identified in this study, as well as the previously discovered RmYN01 and RmYN02, were identified in a 1100 hectare part of Yunnan province, the results of ecological modeling show that many species of rhinolophid bats are present in much of Southeast Asia and southern China. Numerous other bat species and other wildlife inhabit this area, many of which can be infected by SARS-CoV-2. Hence much more wildlife sampling is needed to track potential spillovers of viruses into humans.

These findings also demonstrate that a very large part of Asia might harbor the direct ancestor of SARS-CoV-2. Remember that HIV/AIDS was first detected in Los Angeles in 1981 but the virus spilled over from a chimpanzee to a human in Africa in ~1920.

Trial By Error: #MEAction’s Chronic Illness Survey; Patients’ Research Informs CDC’s Long-COVID Advice

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By David Tuller, DrPH

New survey to assess links between chronic conditions

Jaime Seltzer, #MEAction’s Director of Scientific & Medical Outreach, has been pretty busy lately. This week, she appeared on a panel at the World Health Organization’s Long COVID webinar—alongside no less than the distinguished and always affable Professor Sir Simon Wessely. Seltzer has also just been named to the Independent Advisory Group for Cochrane’s new exercise therapy review. And she recently launched, in cooperation with Dr Mady Hornig, an epidemiologist at Columbia University, the Chronic Illness Survey Adventure (Symptom Cluster Characterization in Complex Chronic Disease), an effort to examine possible links and overlaps between ME/CFS and long COVID, POTS, hEDS, and MCAS.

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Holiday travel explains spread of a SARS-CoV-2 variant

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coronavirus Spike

The emergence and spread throughout Europe of a SARS-CoV-2 variant, 20E (EU1) in the summer of 2020 illustrates how a virus may become dominant not by increased transmissibility but through travel and lack of effective containment and screening.

The SARS-CoV-2 variant 20E (EU1) emerged in Spain in the summer of 2020 and spread to multiple European countries. By the fall of 2020 most of the sequences in Europe were from 20E (EU1). The variant bears a number of amino acid changes including A222V in the N-terminal domain of the spike protein (pictured).

Results of binding and neutralization assays revealed that the A222V change did not affect interaction of the spike protein with polyclonal or monoclonal antibodies. Lentivirus particles bearing the spike with A222V did not reproduce with higher efficiency in cells in culture. Authors conclude that these observations are not consistent with increased transmissibility of the 20E (EU1) variant. However, I would argue that studying the infectivity of a pseudotyped virus – in this case a lentivirus bearing the SARS-CoV-2 spike – in 293 cells (possibly human embryonic kidney) has virtually no relevance to what occurs in humans. At the very least, authentic SARS-CoV-2 infection of human respiratory tract cells must be examined. Even then, the results may have no direct bearing on what occurs in humans.

In contrast, epidemiological evidence explains the spread of 20E (EU1). This variant arose first in Spain in early summer 2020. It then spread extensively in Spain and also spread to other European countries. Results of modeling indicate that the spread of the variant can be explained by holiday travel-associated transmission (many EU countries opened their borders to travel on 15 June), and human behaviors such as failure to distance, mask, restrict gatherings, and adequately test for infections.

It seems likely that human behavior might also account for the global spread of other SARS-CoV-2 variants (e.g. alpha and beta). Authors believe that such spread is due to increased inherent transmission of the viruses, but the data in support of this conclusion are not convincing. Increased reproduction of pseudotyped viruses in cells in culture, as pointed out above, is likely irrelevant. Increased shedding of viral RNA from the nasopharynx, detected by PCR, is also irrelevant as it does not represent infectious virus. In no case has shedding of infectious virus from the nasopharyngeal tract been studied to address potential mechanisms of increased transmission. It is claimed that the reproductive index of variants is increased, but these calculations are flawed. The reproductive index is determined by a formula that includes both viral and host factors. However, host factors are never included when this index is determined for individual variants. As the study above indicates, human activities can substantially affect predominance of a virus in a population.

Why do variants out-compete and displace other viruses? It is because the variants have increased fitness, the ability of a virus to reproduce in the host. Fitness can be altered in many ways, including evasion of antibody responses, increased particle stability, and even person to person transmission. No experiments have been done to explain the increased fitness of variants. Fitness is not the same as transmission.

Variants of influenza virus arise frequently, and these variants displace existing viruses because they have a fitness advantage. For influenza virus, a fitness advantage is often conferred by HA amino acid changes that allow escape from antibody neutralization. Antigenic variants can infect a slightly larger number of hosts and that is enough natural selection advantage for the new variants to outcompete the older ones. No one ever says that these influenza virus variants have increased transmission.

Unfortunately the narrative that the variants have increased transmission is dominating the media. This situation has arisen because virologists, epidemiologists, and evolutionary biologists are not talking to one another. In addition, what we know about other viruses, as illustrated here for influenza virus, is also ignored.

Trial By Error: More Disinformation from Professor White in Journal of Psychosomatic Research

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By David Tuller, DrPH

The Journal of Psychosomatic Research seems to be suffering from some sort of identity crisis. Earlier this year, the editor and his two immediate predecessors published an admirable editorial in which they noted the serious risk of bias in subjective outcomes in studies that are not rigorously blinded. Yet the journal’s editorial advisory board is studded with investigators who specialize in unblinded studies that rely solely on subjective outcomes—in particular, Professors Peter White and Michael Sharpe, two of the lead PACE investigators, and Professor Per Fink, their Danish colleague.

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Trial By Error: Norway Rejects New Clinical Trial of Woo-Woo Lightning Process

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By David Tuller, DrPH

In a welcome display of scientific acumen, Norwegian research ethics authorities have rejected a proposed study of the woo-woo called the Lightning Process as a treatment for ME/CFS. Since Norway generally appears to be a hotbed of biopsychosocial thinking, this excellent decision is a bit of a surprise. It follows a heated public debate about the issue over the last year, both before and after regional ethics authorities granted initial approval to the study in November.

(Dagbladet, a popular daily publication, ran an opinion piece of mine last June about the issue. In April, Codastory.com, an international news site, published my lengthy investigation of the Lightning Process, which highlighted the proposed Norwegian study.)

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When Viruses Collide with Parasitic Worms

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by Gertrud U. Rey

The absence of infection with some parasitic worms (also known as helminths) often coincides with the development of asthma, allergy, and autoimmune disorders, suggesting that these worms may have co-evolved a commensal relationship with their human hosts. However, infection with these worms may also lead to an impaired immune response against pathogenic infections by other parasites, bacteria, and some viruses. 

Based on such observations, the authors of a recent study explored the effect of infection with Heligmosomoides polygyrus bakeri (Hpb) – a naturally occurring intestinal roundworm of rodents – on West Nile virus (WNV) infection outcome in mice. WNV is a member of the Flavivirus genus, along with Dengue virus, Zika virus, yellow fever virus, tick-borne encephalitis virus, and Powassan virus. These viruses all infect their human hosts through the bite of an arthropod and ultimately target cells of the central nervous system.

To assess the effect of intestinal Hpb infection on the pathogenesis of WNV, the authors used three groups of mice: the first group was only fed Hpb larvae to initiate an Hpb infection, the second group was only infected with WNV by inoculation in the foot pad, and the third group was infected with both Hpb and WNV. Infection with Hpb alone had no adverse effect on body weight or survival of the mice, and infection with WNV alone caused moderate weight loss and 86% survival. In contrast, infection with both Hpb and WNV led to greater weight loss and only 28% survival. To determine the reason for this enhanced pathogenicity, the mice were euthanized, and different organs were assessed for viral titers. Mice infected only with WNV, or with both WNV and Hpb, had similar levels of virus in the spleen, an organ that filters the blood and produces immune cells in response to the presence of infectious pathogens. However, mice infected with both Hpb and WNV had higher levels of virus in different parts of the colon and the mesenteric lymph nodes, which serve as a firewall to prevent systemic spread of microorganisms. The presence of such high viral titers in these regions suggested that co-infection with Hpb and WNV leads to the infection of cells in gut-proximal places.

To determine which gut cells are targeted by WNV during a Hpb/WNV co-infection, the authors stained pieces of gut tissue from Hpb/WNV co-infected mice with a combination of fluorescent antibodies that bind WNV antigens or neuronal cell markers. Analysis of these tissues by fluorescence microscopy revealed that co-infected animals have higher levels of viral antigens in intestinal neurons compared to control animals infected only with Hpb or WNV, suggesting that infection with both Hpb and WNV increases the frequency of infection of gut neuronal cells. Microscopic analysis also revealed certain gut abnormalities in co-infected animals, including changes in the composition of intestinal villi, which are small, finger-like projections that extend into the lumen of the small intestine. Co-infected animals had shortened villi, a phenomenon typically associated with pathology and inflammation. Co-infected animals also had commensal bacteria inside their spleens, so it is possible that the shortened villi enabled translocation of the bacteria across the epithelial gut barrier, which normally restricts the entry of harmful substances by separating the intestinal organs from the external environment.

The spleens and brains of Hpb/WNV co-infected mice also contained markedly reduced numbers of WNV-specific CD8+ T cells, which usually contribute to the clearance of WNV infection. The authors reasoned that the lack of CD8+ T cells was likely due to bacterial invasion, which may impact the activation of key immune cell types involved in triggering the differentiation of virus-specific CD8+ T cells. Remarkably, treatment of Hpb/WNV co-infected mice with antibiotics reversed these kinetics, leading to increased levels of WNV-specific CD8+ T cells, a result that correlated with diminished bacterial levels in the blood and spleen. This observation suggested that bacterial invasion of lymphoid tissues is largely responsible for impaired WNV-specific CD8+ T cell responses during co-infection with Hpb and WNV.   

The clearance of helminth infections is typically mediated by the activation of “type 2” immune responses, which are characterized by the production of various cytokines, including IL-4. Binding of IL-4 to its cognate cell surface receptor activates STAT6, a cytoplasmic transcription factor that ultimately signals the activation of macrophages and differentiation of T cells. To determine if these type 2 immune responses contribute to the lethality observed after co-infection with Hpb and WNV, the authors performed the next set of experiments on “STAT6 null” mice, which lack the STAT6 gene. Interestingly, STAT6 null mice co-infected with Hpb and WNV did not have any of the increased lethality observed in wild type mice. Hpb/WNV-co-infected STAT6 null mice also had reduced overall viral levels and no gut abnormalities characteristic of enhanced pathogenesis, suggesting that STAT6 signaling is needed for development of enhanced disease.

To determine the relevance of IL-4 in the STAT6 signaling cascade, wild type mice were infected with WNV alone, followed by administration of IL-4. This regimen led to increased pathogenicity like that observed in Hpb/WNV co-infected mice, including decreased survival, weight loss, and higher WNV burden in the brain and gut. In contrast, the same WNV/IL-4 regimen given to STAT6 null mice did not produce the same pathogenicity, suggesting that the effects of IL-4 are dependent on the presence of STAT6.  

Type 2 immune responses in the gut are initiated by tuft cells, which are specialized cells of the intestinal epithelium that are capable of multiplying in response to the presence of certain parasites. To see whether tuft cells are needed for Hpb-mediated enhanced WNV disease, the authors infected “tuft null” mice (mice lacking tuft cells) with both Hpb and WNV. These mice did not develop any of the effects characteristic of enhanced pathogenesis, suggesting that tuft cells are needed for development of enhanced disease.

Tuft cells selectively express a receptor for succinate, a metabolite needed for energy production. To see whether succinate sensing is involved in enhanced WNV disease, wild type mice were administered succinate and were then infected with WNV. All these mice developed enhanced disease, even in the absence of Hpb infection. However, the same succinate/WNV regimen in tuft null mice, or in mice expressing tuft cells but lacking succinate receptors, did not lead to enhanced disease. These experiments further confirm that succinate receptor-expressing tuft cells are needed to initiate the signaling pathway leading to enhanced disease. Collectively, these results suggest that Hpb-mediated activation of type 2 immune responses in intestinal epithelial cells depends on tuft cells and might be initiated by succinate sensing.

Similar enhanced disease characteristics were also observed after co-infection of mice with Hpb and Zika virus or Powassan virus, suggesting that co-infection with helminths may enhance the disease severity of multiple human flaviviruses. Although these parasitic worm/flavivirus co-infections are currently more common in tropical regions, their prevalence may spread with increasing global climate changes/warming. This study outlines a fascinating aspect of the interface between parasitic and viral infections that warrants further exploration.

[For a more detailed discussion of this paper, I recommend This Week in Parasitism episode 194]

T cells will save us from COVID-19, part two

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T cells are the other arm of the adaptive immune response (in addition to B cells) that are essential for clearing virus infections. Most studies of immunity to SARS-CoV-2 infection have focused on antibodies and their ability to neutralize virus infection. The observation that variants of concern are less effectively neutralized by antibodies against ancestral strains have led to predictions of vaccine failure and doom. This outlook is incorrect: amino acid changes in the spike protein of SARS-CoV-2 variants of concern do not impact T cell reactivity and are not likely to affect the ability of T cells to clear infection. This conclusion has been emphasized by the results of another study which show that SARS-CoV-2 variants of concern partially escape humoral but not T-cell responses in COVID-19 convalescent donors and vaccinees.

Cytotoxic T cells can sense that a cell is infected and kill it (illustrated). T cells sense infected cells by virtue of viral peptides that are presented by major histocompatibility molecules on the plasma membrane. Such T cell peptides may be produced from nearly any viral protein. In contrast, only certain viral proteins, like the spike of SARS-CoV-2, can give rise to antibodies that block infection. T cell epitopes are presented to T cells on the infected cell surface by MHC molecules, which are encoded by highly polymorphic genes. That means that your MHC is likely different from mine, and so will be the viral peptides displayed in them. So if a T cell epitope varies during your infection, it won’t matter to other people – their infected cells will be displaying different T cell peptides.

A study of antibody and T cell responses to SARS-CoV-2 and variants of concern B.1.1.7 and B.1.351 was carried out in a cohort of health care workers vaccinated with BNT162B2 mRNA vaccine. Some of these individuals had recovered from COVID-19 and received a single vaccine dose, while others had not been infected and received two vaccine doses.

Plaque assays were used to show that vaccine-induced antibodies neutralized the infectivity of B.1.1.7 virus as well as ancestral SARS-CoV-2. However, neutralization titers against the B.1.135 variant were about 4-fold lower.

To examine T cell responses to vaccination, blood mononuclear cells (PBMC) were stimulated with peptides from the spike protein. PBMC from immunized patients were activated equally by peptides spanning the entire spike protein from the ancestral virus, or from peptide pools covering the regions of spike that are changed in the variant viruses. As shown previously, T cell epitopes in the spike protein of variants of concern do not functionally change.

We can deduce the meaning of these findings for infection with variants of concern. In theory, should a variant fail to be efficiently neutralized by antibody in an individual previously infected with ancestral virus, or vaccinated (with ancestral spike protein), the T cells should still be able to prevent spread of infection and severe disease or death. This hypothesis is supported by the outcome of trials with the Johnson & Johnson Ad26.COV2-S vaccine, an adenovirus vectored spike vaccine. Sera from volunteers in South Africa, where B.1.351 circulates, had a tenfold reduced neutralization titer against that variant of concern. However, the vaccine was 100% effective in preventing hospitalization and death due to COVID-19 – likely a consequence of virus-specific cytotoxic T cells.

The situation with other vaccines is less clear. The AZD1222 vaccine, which showed 70% efficacy in the UK, showed only 22% efficacy in South Africa against mild to moderate COVID-19. Whether or not this vaccine would prevent severe COVID-19 could not be assessed as there was no serious disease observed during this trial.

The B.1.1.7 variant has the N501Y change in the spike protein, while B.1.351 and P.1 (Brazil) have in addition the amino acid changes K417N/T and E484K. Variant B.1.617 (India) has E484Q and L452R; the latter is also present in CAL variants. Given that none of these changes alter T cell reactivity it is likely that spike vaccines will protect against serious disease and death caused by these variants. Reports of reinfection with such variants in previously vaccinated individuals that leads to serious disease should be taken with a large grain of salt.

Novel human coronaviruses from pigs and dogs

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The seven human coronaviruses have all originated from spillover events from a variety of nonhuman animals, including bats, rodents, and camels. Recently isolated coronaviruses from humans appear to have originated in pigs and dogs.

Three of 369 plasma samples collected between May 2014 and December 2015 in two schools in Haiti were found to be positive for coronavirus. All three children had fever, and one reported cough and abdominal pain. Whole genome sequence analysis of all three isolates revealed them to be porcine deltacoronaviruses (PDCoV). The human coronaviruses known to date are alpha- and betacoronaviruses.

PDCoV was first identified in 2012 in Hong Kong and then in 2014 in the Americas. The virus infects epithelial cells in the jejunum and ileum and the large intestine, leading to gastrointestinal symptoms. No PDCoV infections have been reported in Haitian domestic pigs.

The PDCoVs isolated four months apart from two children attending the same school are closely related to a virus isolated from pigs a year later in China. The other Haitian child was infected with PDCoV that is more closely related to an isolate from pigs in Arkansas from 2015. The implication of sequence analysis is that two separate zoonotic transmissions took place. How the viruses traveled from the US and China to Haiti is not clear.

All three PDCoV human isolates share a signature of amino acid changes in the viral ORF1a/b and spike proteins that is not seen in other PDCoV isolates. These changes might have been selected during reproduction in human hosts.

In a separate study, 8 of 301 nasopharyngeal swabs from patients hospitalized between 2017-2018 with pneumonia in Malaysia were shown to harbor canine coronavirus (CCoV), an alpha coronavirus. Close analysis of the genome sequence revealed that the virus is a recombinant, with most of the genome originating from CCoV and part of the spike from feline coronavirus.

Canine and feline coronaviruses cause moderate to severe intestinal disease in dogs and cats. The viruses have a worldwide distribution and given the prevalence of these animals as pets, it is not surprising that human infections may occur, albeit rarely. The eight patients who harbored this novel CoV were children living in areas where exposure to domestic animal and jungle wildlife would be expected, possibly explaining how the infections were acquired.

These human infections with porcine and canine coronaviruses appear to be isolated incidents which did not lead to extensive human transmission. Nevertheless the findings emphasize the public health threat of animal coronaviruses and the need for improving our surveillance for these viruses.