Early in the COVID-19 pandemic, doctors recognized that patients who developed a “cytokine storm” – a surge of pro-inflammatory immune proteins – were often the sickest and at highest risk of dying.
But a cytokine storm can also occur in other illnesses, such as influenza. Today, scientists report preliminary results on a sweat sensor that acts as an early warning system for an impending cytokine storm, which could help doctors more effectively treat patients.
The researchers will present their results today at the spring meeting of the American Chemical Society (ACS).
“Especially now in the context of COVID-19, if you could monitor pro-inflammatory cytokines and see them trending upwards, you could treat patients early, even before they develop symptoms,” says Shalini Prasad, Ph.D., the project’s principal investigator, who is presenting the work at the meeting.
Early detection is important because once a cytokine storm has been unleashed, the excessive inflammation can damage organs, causing severe illness and death. In contrast, if doctors could administer steroidal or other therapies as soon as cytokine levels begin to rise, hospitalizations and deaths could be reduced.
Although blood tests can measure cytokines, they are difficult to perform at home, and they can’t continuously monitor the proteins’ levels. Cytokines are excreted in sweat at lower levels than in blood. To collect enough sweat for testing, scientists have asked patients to exercise, or they have applied a small electrical current to patients’ skin.
However, these procedures can themselves alter cytokine levels, Prasad notes. “When it comes to cytokines, we found that you have to measure them in passive sweat. But the big challenge is that we don’t sweat much, especially in air-conditioned environments,” she says. Prasad, who is at the University of Texas at Dallas, estimates that most people produce only about 5 microliters, or one-tenth of a drop, of passive sweat in a 0.5-inch square of skin in 10 minutes.
So the researchers wanted to develop an extremely sensitive method to measure cytokine levels in tiny amounts of passive sweat. They drew on their previous work on a wearable sweat sensor to monitor markers of inflammatory bowel disease (IBD). The wristwatch-like device, which is being commercialized by EnLiSense LLC (a company co-founded by Prasad), measures the levels of two proteins that spike during IBD flare-ups.
When the device is worn on the arm, passive sweat diffuses onto a disposable sensor strip that is attached to an electronic reader. The sensor strip, which contains two electrodes, is coated with antibodies that bind to the two proteins. Binding of the proteins to their antibodies changes the electrical current flowing through the e-reader.
The reader then wirelessly transfers these data to a smartphone app that converts electrical measurements to protein concentrations. After a few minutes, the old sweat diffuses out, and newly excreted sweat enters the strip for analysis.
For their new cytokine sensor (called the SWEATSENSER Dx), the researchers made sensor strips with antibodies against seven pro-inflammatory proteins: interleukin-6 (IL-6), IL-8, tumor necrosis factor-α (TNF-α), TNF-related apoptosis-inducing ligand, IL-10, interferon-γ-induced protein-10 and C-reactive protein. They inserted the strips into their device and, in a small observational study, they tested them on six healthy people and five people with influenza. Two of the sick people showed elevated cytokine levels, and in all participants, cytokines in passive sweat correlated with levels of the same proteins in serum.
The SWEATSENSER Dx was even sensitive enough to measure cytokines in patients taking anti-inflammatory drugs, who excrete cytokines in the low-picogram-per-milliliter concentration range. The device tracked cytokine levels for up to 168 hours before the sensor strip needed to be replaced.
EnLiSense, in partnership with the researchers, is now planning clinical trials of the cytokine sensor in people with respiratory infections.
“Access to COVID-19 patients has been a challenge because healthcare workers are overwhelmed and don’t have time to test investigational devices,” Prasad says. “But we’re going to continue to test it for all respiratory infections because the disease trigger itself doesn’t matter—it’s what’s happening with the cytokines that we’re interested in monitoring.”
Monitoring the Cytokine Storm
SARS-CoV-2 infects epithelial lung cells via specific interactions with the angiotensin converting enzyme 2 (ACE2).10 While efforts are being made worldwide to better understand the cytokine storm that characterizes the progression to severe pneumonia or ARDS, previous information from SARS-CoV and MERS-CoV infections, shown in Figure1, indicates the main factors.11−13 It is known that the virus replicates very quickly in the early stages of infection. This means that high levels of viral proteins known to antagonize interferon (IFN) responses are generated, which results in a strong yet delayed proinflammatory response at the site of infection.
These pro-inflammatory cytokines and chemokines attract macrophages and neutrophils that also release pro-inflammatory agents. This amplifies the inflammation, giving rise to ARDS, sepsis, or multiorgan dysfunction syndrome (MODS), all of which are associated with poor outcomes.14
A comprehensive review of the cytokines and chemokines involved in the COVID-19 cytokine storm is already available, although studies performed in large cohorts of patients have not been performed.12 These are required in order to determine cutoff values for diagnosis and prognosis purposes.
Pro-inflammatory cytokines such as IL-1, IL-6, IL-8, and tumor necrosis factor alpha (TNF-α) have been found elevated in the sera of severely and critically ill COVID-19 patients.15−24 This indicates that these cytokines could be good prognosis biomarkers; that is, they could indicate the progression to severe or critical COVID-19.
Many recent cytokine profiling studies, however, have only been performed in small populations, and therefore cannot be broadly generalized.17,22 Larger studies are required to establish cutoff values as well as to determine the intricate relationships between pro- and anti-inflammatory factors in COVID-19 progression.
Based on other hyperinflammatory syndromes such as sepsis, the levels of these biomarkers will likely fluctuate rapidly. This means that patients with inflammation biomarkers below a certain threshold value may evolve quickly and require anti-inflammatory treatments even though their initial biomarker measurements indicated otherwise.
A solution to this problem could be to perform periodic measurements of biomarkers in order to evaluate their kinetics.23 This would require biosensors that are inexpensive and easy to use at the point of care in order to not overburden healthcare workers. It should also be noted that, although cytokines are also present in saliva, urine, and sputum,24 no information is currently available about their function and usefulness as biomarkers for cytokine storm monitoring in COVID-19. Measuring cytokines in sputum could be particularly valuable because it would reveal information about local inflammation. However, only 30–40% of COVID-19 patients produce sputum.16
Anti-inflammatory cytokines such as IL-10 have also been found in the sera of COVID-19 patients.22−25 It is known from other hyperinflammatory syndromes that dysregulated inflammation is often followed by immune suppression,26 putting these patients at high risk of opportunistic infections from bacteria, fungi, or even latent viruses such as human cytomegalovirus (CMV).27−29
This is particularly problematic in the context of the ICU, since nosocomial infections by multiresistant pathogens can be lethal if not promptly detected. Because of this, we anticipate that biosensors aimed at detecting coinfections will also be required in order to manage critical COVID-19 patients. For example, biosensors that detect procalcitonin (PCT) could be useful to detect opportunistic bacterial infections.30−32
Procalcitonin is produced in parenchymal cells in response to bacterial toxins. In healthy individuals, PCT is considered undetectable (below 0.05 ng mL–1). PCT levels above 1–2 ng mL–1 are usually considered as a warning of potential bacterial sepsis.33 Since PCT levels do not rise to such a large extent in viral infections, biosensors for this biomarker could indicate the onset of bacterial infections in COVID-19 patients.
Potential Biosensor-Guided Therapies
Table 1 summarizes some anti-inflammatory treatments proposed so far for ameliorating the cytokine storm in COVID-19 patients. It should be noted that the evidence supporting these treatments is weak. In other words, randomized clinical trials for these drugs in the context of COVID-19 care have not yet been completed. Their use is not recommended in official protocols by WHO, only for clinical trials or compassionate motives. Many are repurposed drugs already in use for other diseases and syndromes, but not for infections.34 It is important to highlight this as many of these pharmaceuticals are immunosuppressive. Since COVID-19 patients are already immunosuppressed due to IFN attenuation and anti-inflammatory responses, the administration of these immunomodulators may exacerbate the risk of an opportunistic infection or reduce the effect of antiviral drugs.
Main Immunomodulators Proposed So Far for Managing Inflammation in COVID-19
|Corticosteroids||Nonspecific||Binding to receptor enhances or represses the transcription of inflammation genes|
|(Hydroxy)chloroquine||Nonspecific||Suppressing the activation of T cells|
|Immunoglobulins||Nonspecific||Binding to Fc receptors|
|Azithromycin||Nonspecific||Decreasing the pro-inflammatory response|
|Baricitinib||JAK||Inhibitor of Janus kinase (JAK)|
|Anankinra||IL-1||Antagonist of the IL-1 receptor|
|Tocilizumab/Sarilumab||IL-6||Binding to IL-6 receptor thus blocking the interaction with gp130|
A good example of the polemic surrounding these anti-inflammatory treatments is the use of corticosteroids. Corticosteroids such as methylprednisolone are inexpensive and globally available. They are not routinely recommended for sepsis care except in cases of septic shock. Previous evidence from SARS-CoV-1 and MERS patients treated with corticosteroids yielded widely disparate outcomes,11 and the WHO and IDSA have discouraged the use of corticosteroids for COVID-19 care. Nevertheless, several authors report good outcomes when administering these drugs at cautious doses.8,35,36
Of those reporting good outcomes, dosing and timing have been highlighted as the key factors for successful corticosteroid therapy in the context of SARS-CoV infections. Kinetic profiles of pro-inflammatory cytokines such as IL-6 could provide crucial data to guide the onset of corticosteroid treatment and adjust doses in order to reduce inflammation while minimizing side effects.37 Other nonspecific anti-inflammatory treatments that could benefit from biosensor-guided administration are (hydroxy)chloroquine,7 immunoglobulins, azithromycin,38 and convalescent plasma therapies.39
Drugs based on monoclonal antibodies and recombinant proteins such as Tocilizumab40 or Anakinra41 have also been proposed for reducing hyperinflammation caused by COVID-19. These treatments block specific pro-inflammatory signal pathways. These drugs could benefit from a “companion diagnostics” approach similar to that which is used in cancer care. That is, the administration of these drugs would be guided by measurements of the specific pro-inflammatory factors they modulate. For example, Tocilizumab binds receptors of IL-6 (both soluble and membrane-bound).
This blocks the interaction with membrane-bound gp130, which in turn prevents the activation of a downstream Janus kinase responsible for signal cascading.42 It has been proposed that blockers of the IL6-mediated inflammatory response such as Tocilizumab and Sarilumab should be guided by IL-6 measurements with a threshold value around 20 pg mL–1.43,44 These antibodies have proven to be useful to treat unwanted cytokine release syndromes in immune anticancer therapies.
Some early reports on the benefits of using Tocilizumab are encouraging, although a clinical trial for Sarilumab has recently been discontinued due to a lack of clear positive outcomes. Interestingly, serial IL-6 measurements have shown that after the administration of Tocilizumab there is a slight increase in IL-6 followed by sharp decrease over time.43 These early reports highlight the relevance of performing kinetic measurements of biomarkers for monitoring the progress of inflammatory diseases.
Current Biosensor Candidates for Managing the Cytokine Storm
Table S1 summarizes the main features of recent biosensor prototypes for cytokine detection. As highlighted above, time-dependent pro- and anti-inflammatory responses are involved in COVID-19 progression. A multisensor system capable of detecting several of these biomarkers simultaneously would provide evidence to determine disease stage and guide personalized therapies. For example, it has been reported that plasmonic nanosensor arrays can detect 6 cytokines simultaneously (IL-2, IL-6, IL-4, IL-10, IFN-γ, and TNF-α, FigureFigure22a).45
These multisensors require a minute sample size (1 μL) and show an impressive dynamic range for quantification (between 10 and 10 000 pg mL–1). The signal transduction mechanism consists of measuring changes in the localized surface plasmon resonance (LSPR) of gold nanorods with dark-field microscopy.
This provides information about cytokine binding to antibodies in real time, which is more informative than traditional end-point ELISA. The total time required to run the whole chip is only 40 min. The rapid analysis time makes this detection platform suitable for aiding clinical decision-making in emergency situations. Another interesting multisensor platform using electrochemical transducers is shown in FigureFigure22B.
It contains 32 individually addressable electrodes, each one multiplexed with an 8-port manifold to provide 256 measurements in less than 1 h.46 However, it also requires an off-line protein capture step on magnetic nanobeads. Electrochemical immunoassays for IL-6 with these devices reported a wide dynamic range between 0.1 and 104 pg mL–1. Multiplexed cytokine measurements with electrochemical biosensors have also been reported using graphene oxide to fabricate nanoprobes.47 This platform detects IL-6, IL-1β, and TNF-α spiked into the same mouse serum sample. The multiplexed detection was achieved by using antibodies bound to three different signal reporters (nile blue (NB), methyl blue (MB), and ferrocene (Fc)).
COVID-19 is a global challenge and requires technologies that are easy to implement in many different scenarios. The oversaturation of hospitals during the peak of the infection has forced some governments to decentralize COVID-19 care and manage patients in temporary field hospitals and in-home quarantine. In these scenarios, detection systems that are not bound by centralized infrastructure play a key role. For instance, a lateral flow immunoassay has been proposed for detecting IL-6 in unprocessed blood with high sensitivity.48
The ability to detect cytokines directly from blood is extremely useful in decentralized healthcare scenarios, since purifying serum or plasma requires a centrifugation step that is difficult to perform at the bedside. However, blood contains cells like erythrocytes, which can interfere with colorimetric detection schemes. The lateral flow test used antibody-decorated nanoparticles as probes and surface-enhanced Raman spectroscopy (SERS) as the signal transduction mechanism.
It showed a limit of detection of 5 pg mL–1 in whole blood. This limit of detection, however, was achieved with a DXR Raman microscope, which is not suitable for point-of-care use. Over the past decade, a growing trend in biosensors has been to interface them with mobile devices such as smartphones.49−51 This is an appealing option because smartphones already have a high global market penetration and therefore do not require the purchase of additional readers. Mobile biosensors for IL-6 have been recently proposed.52
The devices consist of a paper immunosensor for colorimetric detection using gold nanoprobes (Figure2C). The gold nanoprobes generate colored spots that are detected in real time with a smartphone app. Instead of external attachments, the app uses an augmented reality system in order to control photographic conditions. This system also compensates for variable light conditions, allowing the user to quantify colorimetric signals using only an unmodified smartphone. The biosensor was able to detect variations in IL-6 levels as small as 12.5 pg mL–1 in whole blood and with a rapid assay time of only 18 min. Furthermore, the paper substrate makes it particularly useful for monitoring infectious diseases because it can be easily disposed of by incineration.
Biosensors capable of continuously monitoring cytokine levels would be ideal for detecting COVID-19 patients progressing to severe or critical stages as well as to check the success of anti-inflammatory therapies. While to the best of our knowledge immunosensors for continuous detection of biomarkers in blood have not yet been proposed, some prototypes with the potential to achieve this feature can be found in the recent literature. For example, needle-shaped microelectrodes have been proposed for detecting alterations in IL-6 levels in real time (Figure3A).53
In this design, the interaction between the cytokine and antibodies bound to the electrode changes the impedance of the system without the need for detection antibodies or labels. The authors suggest that integrating the sensors with a cannula within the bloodstream could enable the real-time monitoring of IL-6 levels, although the initial prototype was only tested in surrogate serum samples.
Recently, an electrochemical biosensor capable of continuous monitoring in blood samples has been proposed. It consists of a wire electrode modified with aptamers that change their configuration upon binding their target (Figure3B).54 This changes the position of a redox active molecule (methylene blue) with respect to the electrode, which can be followed with square-wave voltammetry (Figure3c).
The authors demonstrated that the sensors implanted in a rat could detect vancomycin as a target molecule in real time (Figure3D). Adapting this technology for detecting cytokines could enable a precise profiling of the COVID-19 inflammation.55 Finally, a wearable detection platform has also been proposed that could be a game-changer in inflammation monitoring.56 It is based on the intradermal delivery of biocompatible near-infrared (NIR) quantum dots. An array of these nanosensors is delivered using dissolvable microneedles.
The fluorescence emission of the quantum dots was fine-tuned so that they would be invisible to the naked eye, but detectable upon illumination with NIR light. The pattern of emitted light generated by the array can be detected with a modified smartphone and evaluated with a machine learning algorithm. While this technology has initially been demonstrated as a way to keep vaccination records, adaptation for continuous monitoring of biomarkers could revolutionize the field of personalized medicine.
reference link: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7299396/
More information: Abstract Title: SWEATSENSER DX an enabling technology for on demand profiling of cytokines on passively expressed eccrine sweat