Category: 8. Health

Co-Infection dynamics between Mpox and COVID-19

Mpox is a zoonotic disease caused by the monkeypox virus [21]. The current outbreak of Mpox in July 2022 was declared a global health emergency as it spread to over 100 non-endemic countries [22]. Co-infection of Mpox with COVID-19 has been reported, with patients showing symptoms of both infections. One of the first reported cases of COVID-19 and Mpox confection was from Florida in the United States of America (USA). The patient was immunosuppressed with a history of intravenous drugs on current HAART (highly active antiretroviral therapy and coinfected with Mpox, COVID-19 and herpes [23]. Another case report, published in Barcelona, Spain, revealed a 56-year-old man to have both Mpox and COVID-19 and syphilis simultaneously [24]. According to a review done in 2022, it was found that 3 patients who had sex with men were found co-infected with Mpox and COVID-19 [25]. A 38-year-old from the USA was found to be COVID-19 and Mpox positive by PCR [25]. One more case was reported from Italy about a 36-year-old male who was coinfected and diagnosed by PCR and tested positive for Mpox and COVID-19 at the same time [25].

Epidemiological characteristics of Mpox and COVID-19 co-infections

Between 2001 and 2021, no significant data were found about the co-infection of Mpox and COVID-19, despite the COVID-19-caused pandemic in 2020. On the contrary, significant cases were found in 2022 co-infection of MpOX and COVID-19 [26].In the post-COVID-19 era around March 2022, most countries removed their travel restrictions and returned to their pre-pandemic policies, which further provided a favourable environment for co-infections [26]. Additionally, three cases of co-infection reported were of men who had engaged in sex with men before their infections. This suggests a potential risk factor for transmission of viral infections and sexual health [25]. However, it is still too early to form any definitive epidemiological trends due to a lack of data.

Outcomes of co-infection

According to one study published, 3 patients with co-infection of Mpox and COVID-19 were diagnosed through PCR and were subsequently admitted to the hospital. Their hospital stays lasted between 4 and 9 days, indicating a need for medical intervention and monitoring during their co-infection [26]. Upon admission, two patients exhibited multiple vesicular lesions on various body sites, along with tonsillar inflammation. The third patient presented with genital ulcers and inguinal lymph node enlargement. These symptoms highlight the diverse clinical manifestations that can arise from co-infection, which may complicate diagnosis and treatment [26]. However one of the reported cases of a patient with HIV co-infected with monkeypox and COVID-19, the clinical course was relatively uncomplicated despite the presence of multiple infections [26].

Shared risk factors

The symptoms of co-infection can overlap, making diagnosis challenging. A patient exhibited symptoms common to both Mpox and COVID-19, such as fever, sore throat, and lymphadenopathy [27]. This overlap can complicate clinical assessments and necessitates thorough anamnestic collection and consideration of sexual habits for accurate diagnosis [27]. Both COVID-19 and Mpox require close contact for transmission. Mpox is primarily spread through direct contact with infected skin lesions or bodily fluids, while COVID-19 spreads through respiratory droplets during close interactions [28]. This overlap in transmission dynamics increases the risk of co-infection in settings where close contact is common, such as households or social gatherings [28].

Immune response interactions

Due to limited research studies on the co-infections of COVID-19 with Mpox, there is currently a significant gap in understanding the immune response interactions each disease has concurrently or sequentially. Comprehending these interactions is crucial, especially since both viruses are known to provoke strong immune responses that may interact in intricate ways. The innate and adaptive immune response systems both play a hand in viral clearance. In the case of orthopoxvirus infections, including Mpox, bypassing the immune system is a major factor in the progression of the disease. Through the generation of proteins, the virus impedes the host’s natural antiviral defenses, including nuclear factor kappa B (NF-kB) signalling and cytokine production [29, 30]. Mpox has also been studied to repress the cytotoxicity and migration of natural killer (NK) cells, as well as the complement system [31]. A powerful type 2 immune response is set off by Mpox infection with high levels of Th2-associated cytokines. Type 1 associated cytokines, however, remain at baseline levels. This displays the complicated immune dysfunction seen in Mpox infections [32,33,34]. Adding to the list of proteins released by the Mpox virus is a protein named orthopoxvirus MHC class I-like protein (OMCP), which helps evade the immune system by avoiding the NK response and also deflecting recognition by T cells [35]. Taking into consideration the role of adaptive immunity, especially the antibody response, the presence of Mpox-specific immunoglobulin G and immunoglobulin M antibodies is generally found in infected patients and is thus also used as a diagnostic markers [31].

On the other hand, in COVID-19, which is brought about by the novel coronavirus SARS-CoV2, the innate system can occasionally produce inadvertent effects. In particular, an inflated surge in cytokine output, which is also known as a “cytokine storm”, often leads to the unfavourable aggravation of immune-mediated tissue damage [36, 37]. Monocytes especially play a vital role in cytokine storm formation because they release pro-inflammatory cytokines such as IL-6 and TNF-⍺ [38]. Eosinophils have also been studied to be involved in the immune response by releasing cytokines associated with homeostasis and type 2 immune responses [39]. Following infection with SARS-CoV-2, there have been reports of low blood eosinophil levels, which were strongly associated with poor disease prognosis and mortality [40,41,42,43,44,45]. In a study done by Ranjbar et al., they reported an increase in levels of type 2 cytokines in their patients with COVID-19. On the other hand, no notable elevations were seen in type 1 cytokine levels [38]. This coincides with the pattern of cytokines observed in Mpox infections, however, there is reliable research yet to be done on an official link in the pattern between the two.

A common factor linking the two diseases was recently studied and involves the endoglycosidase named Heparanase (HPSE). HPSE, which cleaves heparan sulfate (HS), is produced by both SARS-CoV-2 and Mpox, and the interplay between the two molecules leads to the release of pro-inflammatory cytokines and thus the evolution of a cytokine storm, endothelial dysfunction and thrombotic events [46]. Furthermore, activated HPSE increases the polarization of macrophages, T cells and NK cells through the expression of TLR4 [47]. HPSE can initiate NK cells through natural cytotoxic receptors and simultaneously can get rid of HS, which antagonizes NK cell activation [48]. As a result of these findings, treatments targeting HPSE with specific inhibitors like low molecular weight heparin (LMWH) could lower the risk of complications in coinfection with Mpox and Covid 19 [46]. Additionally, HS mimetic compounds like pixatimod may serve as important therapeutic tools by inhibiting HPSE and reducing its induced inflammation and blood clotting issues [49] (Table 1 shows a summary of all studies).

Disease severity and complications

Mpox, previously endemic to the African region, commonly presents with a prodromal stage manifesting as fever, body ache, back pain, sore throat, chills, cough, and fatigue. Following that, is the emergence of the characteristic Mpox rash, starting typically on the face. As the infection progresses, the rash becomes generalized and typically spreads centrally. It starts with the involvement of the oral mucosa, eyes, and then prominently in the genital area. The vesiculopapular skin lesions can range from a few to thousands and are typically elevated and filled with clear/yellowish fluid [50,51,52]. The lesions undergo gradual desquamation and completely resolve 4 weeks after initial symptoms [52].

In contrast, the current global outbreak of Mpox since 2022 has been labelled as atypical as it has proved to be mostly a mild version of its previous type. The clinical presentation varies occasionally, manifesting with the absence of the classic prodromal symptoms before the rash, while the observed skin lesions are most commonly found and primarily limited to the genital and perianal region. Nonetheless, lymphadenopathy presenting as painful and enlarged lymph nodes in the maxillary, cervical, or inguinal region continues to be a distinctive sign even in the current atypical presentation [52, 53]. The interleukin-1 receptor antagonist-like protein pathway is a shared link, leading to a sustained immune response in Mpox while causing a rapid remission of COVID-19 [14, 54]. This indicates that while both these infections may co-exist, it is unlikely to find any severe instances of COVID-19 cases in monkeypox infections, and any co-infection will most likely follow the pattern of decreased clinical severity [14]. The re-emergence of Mpox is still relatively new, and the reported cases in the literature of its co-infection with COVID-19 are rare, with a small sample size to conclude from. A systematic review summarized the effects of co-infection of Mpox and COVID-19 as reported in three different case reports. It is to be noted that all patients also had multiple co-morbid illnesses (such as HIV, herpes, syphilis, type 2 diabetes mellitus, depression, and bipolar disorder) before co-infection and in some cases tri-infection with other viruses [25].

It is to be considered that all 3 patients observed in a study25 were male and most often presented with minor systemic symptoms of fever, lymphadenopathy, headache, sore throat, and fatigue, which are common overlapping symptoms of both Mpox and COVID-19. However, due to these non-specific symptoms mostly being attributed to COVID-19, the presence of vesicular and ulcerative lesions, especially in the genital area, is what confirmed the Mpox diagnosis [25]. The systematic review outlined that all 3 of the cases were hospitalized for the provision of proper care and further monitoring. The hospital stay was uncomplicated, lasting for around 4–9 days, and no severe outcomes were observed [25]. Another reported case of co-infection had an asymptomatic presentation of COVID-19, further confirming that concurrent infection does not mean more severe symptoms or complications [55]. Complications of Mpox vary in intensity and include, but are not limited to, keratitis, bronchopneumonia, altered levels of consciousness, secondary bacterial infections of the skin lesions, and eye infection with corneal scarring so severe that it leads to vision loss [56, 57]. Previously reported co-morbidities that exacerbated COVID-19 outcomes now had no such severe effect during the co-infection [58]. The current atypical Mpox outbreak has followed a mild clinical course, with estimated mortality in non-endemic regions being 0.01% [25].

Mpox cases have not had any intensive care unit admissions, and co-infection with COVID-19 did not alter the favourable outcome [23]. So far, no documented cases of co-infection have reported any complications, and all patients made a recovery and were swiftly discharged [25]. It can be inferred, from all the evidence provided in the limited research available, that Mpox does not lead to drastic patient outcomes whether it manifests alone or in concurrence witCOVID-1919. However, further research and documentation of cases are required to fully understand co-infection, as it can vary from person to person [25].

Impact on transmission dynamics

A study done to correlate cell culture infectivity with viral load in an Mpox clinical sample showed that viral load was increased in skin lesions in comparison to those in throat or nasopharyngeal samples [59]. Additionally, samples from the anal region showed a high viral load in comparison to the throat or nasopharyngeal samples [59]. Similarly, another study done to evaluate the relationship between viral load and the course of COVID-19 showed that 5 days after the symptom onset, the viral load was significantly higher in the fatal cases in comparison to those cases which were symptomatic or asymptomatic [60]. Additionally, people who had a worse prognosis were older in comparison to those in the symptomatic or asymptomatic groups [60]. A cohort study done to see pre-symptomatic viral shedding in high-risk Mpox individuals [61] showed that presymptomatic Mpox DNA was seen as early as 4 days before the symptom onset [61]. Another study done to understand temporal dynamics in viral shedding and transmissibility of COVID-19 showed that viral shedding might begin 5–6 days before the appearance of first symptoms [62].

Another study on the estimation of Mpox spread in non-endemic countries with contact tracing showed delay in contact tracing led to a higher number of cases [63]. When the primary affected individual self-reports, the number of infections only rises by 11%; however, if the primary affected individual does not self-report, the average number of infections would rise by 40%. Similarly, an increase in the number of cases was seen if an unreported individual had contact with more people [63]. Another study showed the impact of delay on effective contact tracing strategies for COVID-19 [64]. With a 0-day tracing delay, prevention can reach up to 79.9%, but if a 3-day tracing delay occurs, the figure drops to 41.8 and similarly decreases to 4.9% with a 7-day treatment delay [64]. A study showed that all 20 patients who tested positive for SARS-COV2 had positive respiratory samples; similarly, among 20 stool samples, the SARS-COV-2 genome was found to be positive in 10 stool samples. In most patients, the ability to diagnose SARS-COV2 in the respiratory tract disappears after 2–3 weeks, but it can still be detected in stool samples for more than 4 weeks, thereby showing that stool can be used as an additional source of diagnosis [65].

Increased compliance with facemask usage can reduce the transmission of both COVID-19 and Mpox by limiting the spread of respiratory droplets. Vaccination provides immunity against both diseases, thereby reducing the number of susceptible individuals in the population. Similarly, practicing social distancing among infected individuals can prevent the incidence of coinfection. The use of personal protective equipment (PPE) can reduce the occurrence of healthcare-associated transmissions and the incidence of Mpox cases. Additionally, the use of rodenticides which target the vector of Mpox can help in reducing the overall reservoir population of the vectors, thereby decreasing the likelihood of spillover events [66].

There is no data available which shows whether infection with one virus affects the transmissibility of the other hence, further research should be done on this to understand the dynamics between these two viruses. (Table 2 shows the summary of all the studies).

Vaccine efficacy and cross-reactivity

The rising co-infection of COVID-19 and increased Mpox infection rate has led to the development of only the approved third-generation smallpox/monkeypox vaccine JYNNEOS, which is based on the highly attenuated modified vaccinia Ankara (MVA) vector [67]. COH04S1 is a clinically evaluated, multiantigen COVID-19 vaccine candidate built on a fully synthetic platform of the highly attenuated modified vaccinia Ankara (MVA) vector, representing the only FDA-approved smallpox/mpox vaccine, JYNNEOS [68]. The immune responses elicited by COH04S1 were compared to those from individuals vaccinated with JYNNEOS, the only FDA-approved smallpox/mpox vaccine. The results showed that the MPXV cross-reactive humoral responses from COH04S1 were comparable to those from JYNNEOS-vaccinated subjects. This indicates that COH04S1 could serve as an effective alternative or complement to existing mpox vaccines [68].

In a Phase 1 clinical trial, healthy adults who received COH04S1 exhibited substantial humoral and cellular immune responses. Notably, 45% of these subjects developed MPXV cross-neutralizing antibodies, indicating a significant level of cross-reactivity. This suggests that vaccination with COH04S1 not only protects against COVID-19 but may also confer some level of immunity against MPXV [68]. (Table 3 shows all the listed vaccines). While the above studies primarily focus on the immune response to MPXV and COVID-19 vaccines, the findings imply that vaccination against one virus may influence the immune response to the other. The presence of cross-reactive antibodies could potentially alter disease outcomes in individuals co-infected with both viruses. Dual-purpose vaccines like COH04S1 could streamline vaccination efforts by reducing the number of vaccines needed, thus conserving resources and simplifying logistics. The ability to provide immunity against multiple pathogens could be particularly beneficial in endemic regions or during concurrent outbreaks, enhancing public health responses [67].

However, further research is needed to fully understand the implications of such co-infections and the role of vaccination in modulating immune responses.

Open Access Government sits down with a researcher from the Department of Human Health and Nutritional Sciences to discuss their groundbreaking work on nonalcoholic steatohepatitis (NASH) and insulin resistance. Their research delves into the molecular underpinnings of these increasingly prevalent conditions, offering new avenues for understanding, prevention, and treatment

Given your research focuses on nonalcoholic steatohepatitis (NASH) and insulin resistance, what do you believe are the most critical public health implications of these conditions today?

NASH and insulin resistance are increasing public health problems with widespread social and economic consequences. Public health efforts must shift toward early detection, improved education, and targeted interventions that address the metabolic origins of the disease.

By addressing obesity and promoting lifestyle changes, healthcare systems can help mitigate the growing impact of these interrelated chronic conditions.

How do you envision your research findings translating into practical strategies for improving human health and nutrition at a population level? Are there any immediate applications you foresee?

Our studies expand the fundamental knowledge of liver health by integrating the regulation of lipid metabolism, epigenetics, and therapeutic interventions into a unified framework for better understanding and treating non-alcoholic steatohepatitis (NASH).

They reinforce the importance of membrane phospholipid homeostasis in preventing fat accumulation and the development of insulin resistance and introduce epigenetic modulations as a promising avenue for reversing disease progression.

Examining: Pcyt2 deficiency causes age-dependent development of nonalcoholic steatohepatitis and insulin resistance, that could be attenuated with phosphoethanolamine.

Your work identifies Pcyt2 deficiency as a causative factor in NASH and insulin resistance. Could you elaborate on the significance of this specific molecular pathway in the context of metabolic disease development?

The study elucidates the pivotal role of the Kennedy pathway for phosphatidylethanolamine (PE) synthesis in maintaining metabolic homeostasis. In this pathway, the enzyme Pcyt2 catalyzes the rate-limiting step, and in conditions of Pcyt2 deficiency, as shown in the heterozygous mouse model (Pcyt2+/-), the reduced flux through this pathway sets off a cascade of metabolic dysfunctions that affect gene expression and signal transduction, contributing to altered glucose and lipid metabolism. Importantly, even before overt liver disease is detectable, young mice with Pcyt2 deficiency exhibit altered expression of the key metabolic regulators. As they age, mice develop NASH characterized by insulin resistance, liver fibrosis, and inflammation. The supplementation with phosphonoethanolamine (PEA), an artificial substrate for Pcyt2, can reverse the metabolic derangements caused by its deficiency.

This suggests that in scenarios where the Kennedy pathway is compromised, restoring or bypassing its rate-limiting step could ameliorate liver steatosis, inflammation, and insulin resistance. Immediate applications could involve developing pharmacological agents or nutritional supplements that enhance or mimic Pcyt2 activity, which might be especially beneficial in high-risk populations predisposed to NASH and related metabolic disorders.

Your study highlights the age-dependent development of these conditions. What implications does this age dependency have for preventative or therapeutic strategies, particularly for an ageing population?

The study showed that early defence mechanisms may buffer against the full-blown development of NASH. As the body ages, the cumulative impact of altered membrane dynamics, reduced energy metabolism, and increased oxidative stress overturns the balance, leading to liver pathology and systemic metabolic dysfunction. The gradual, age-dependent disease progression indicates a critical window for early intervention before compensatory mechanisms begin to fail. Screening for the subtle metabolic changes or biomarker shifts in individuals at risk could enable preventative measures before irreversible damage occurs.

Therapeutic regimens tailored for older individuals might require a combination approach that not only incorporates nutrition modulation but also addresses inflammation and oxidative stress. Stratifying individuals based on their metabolic profile and age could help in fine-tuning intervention strategies. For instance, older patients demonstrating early biochemical signs of membrane dysfunction might be prioritized for targeted therapies, whereas younger at-risk individuals might focus primarily on preventive lifestyle changes.

Beyond the molecular findings, how might the insights from this paper influence our understanding of dietary recommendations or nutritional interventions for individuals at risk of NASH?

The impairments in the Kennedy pathway for phospholipid PE synthesis result in a dramatic imbalance in membrane composition and play a significant role in NASH development. Individuals at risk of NASH, especially those whose metabolic profiles indicate impaired phospholipid profiles, might benefit from diets that optimize not only macronutrients but also specific bioactive compounds that ensure proper phospholipid metabolism. The demonstration that supplementation with PEA can mitigate the progression of NASH in an animal model paves the way for considering similar strategies in humans. However, further research is necessary to confirm the safety and efficacy of PEA in clinical settings.

Examining: Epigenome-wide methylation analysis shows phosphonoethylamine alleviates aberrant DNA methylation in NASH caused by Pcyt2 deficiency.

This publication delves into epigenome-wide methylation changes. How does the concept of epigenetics, and specifically DNA methylation, offer a new lens through which to understand the progression and potential treatment of NASH?

In the context of NASH, the discovery of widespread shifts in methylation patterns suggests that the progression of liver pathology is not solely driven by permanent genetic mutations but also by reversible epigenetic changes. Unlike genetic alterations, these modifications can potentially be corrected or even re-programmed with appropriate interventions.

Pcyt2 deficiency is associated with widespread aberrant epigenetic reprogramming in genes crucial for energy metabolism and cellular homeostasis. As such, epigenetic changes compound the metabolic dysfunction by further altering gene expression, potentially leading to inflammation, fibrosis, and insulin resistance seen in NASH.

Treatment with PEA dramatically attenuates abnormal DNA methylation, suggesting that targeted nutritional or pharmacological interventions can not only ameliorate metabolic disturbances but also reverse detrimental epigenetic modifications. In practical terms, developing treatments that modulate DNA methylation could improve gene expression patterns associated with lipid metabolism and inflammation, thereby halting or even reversing the progression of liver disease.

The finding that phosphonoethylamine alleviates aberrant DNA methylation is significant. Could you explain the practical implications of targeting epigenetic modifications for the treatment of NASH?

The proof-of-concept that PEA can mitigate abnormal DNA methylation opens an avenue for the development of new drugs targeting epigenetic modifiers. Future agents could be designed to either mimic the action of PEA or directly inhibit aberrant methylation processes, offering another therapeutic tactic to manage or reverse NASH. This not only broadens the therapeutic arsenal but also allows for continuous innovation in the field of metabolic disease treatments.

The field of epigenetic modifications offers a promising and multifaceted strategy for treating NASH. It provides the possibility to reverse pathological gene expression, create early diagnostic tools, and implement personalized therapies, all of which could dramatically impact patient outcomes. This approach signifies a shift from merely managing symptoms towards addressing the root molecular disturbances that drive liver disease.

How might the insights from your epigenome-wide methylation analysis contribute to the development of personalized nutrition or precision medicine approaches for individuals with NASH?

Aberrant DNA methylation is an early indicator of NASH progression.

By mapping these changes, especially in genes regulating insulin signaling, inflammation, and lipid metabolism, researchers can identify which individuals are at heightened risk even before clinical symptoms become apparent. This opens the possibility of developing blood-based epigenetic biomarkers that enable clinicians to monitor disease progression and therapeutic efficacy in real-time, tailoring interventions to each patient’s molecular profile.

Dietary interventions could be designed not only to focus on nutrient balance but also to provide the right substrates to correct or prevent deleterious changes affecting liver metabolism. This precision approach could, for instance, target those with a predisposition to altered methylation in pathways critical for insulin signaling and energy metabolism, thereby mitigating the risk of full-blown NASH. Treating epigenetic modifications as dynamic biomarkers and therapeutic targets not only enriches our understanding of NASH pathophysiology but also offers a blueprint for precision medicine.

The potential to adjust dietary interventions based on an individual’s unique methylation profile represents a significant leap forward in personalized healthcare for metabolic diseases.

How do you see the research in the two publications contributing to the broader scientific dialogue surrounding liver health and metabolic disorders?

These two publications contribute to the broader knowledge surrounding liver health and metabolic disorders. The studies integrate lipid metabolism, epigenetics, and therapeutic interventions into a unified framework for understanding and treating NASH. They reinforce the importance of phospholipid homeostasis and epigenetic modulations as a promising avenue for reversing disease progression. These insights could reshape clinical approaches, leading to more effective, personalized treatments for metabolic liver disorders. Key contributions include:

1. the establishment of Pcyt2 deficiency as a novel mechanism in age- dependent metabolic dysfunction, which links impaired membrane phospholipid metabolism to the progression of NASH, reinforcing the idea that lipid composition plays a fundamental role in liver disease beyond simple fat accumulation.
2. evidence that PEA supplementation can reverse metabolic and inflammatory damage caused by Pcyt2 deficiency and that targeting phospholipid biosynthesis could be a viable therapeutic strategy for NASH.
3. advancing the epigenetic perspective in metabolic disorders by revealing that aberrant DNA methylation plays a significant role in NASH pathogenesis.
4. demonstrating the reversibility of DNA methylation by PEA showing that epigenetic interventions, whether through diet, supplements, or pharmacological agents, could be used to restore normal gene function and prevent disease progression.

Final messages and the power of the liver

If I had to distill the key message from these research publications into something accessible to the public, it would be this:

Your Liver’s Hidden Protector: How Molecular Balance Could Be the Key to Better Health.

Did you know that liver disease isn’t just about sugar and fat? Recent research reveals a surprising connection between your liver’s health and crucial molecular processes of phospholipid metabolism and epigenetic regulation. Scientists have uncovered that when this balance is disrupted, it can lead to nonalcoholic steatohepatitis (NASH), a serious liver condition linked to insulin resistance and metabolic disorders. But here’s the most exciting finding: this damage might not be permanent. A new discovery reveals that PEA supplementation can reverse harmful changes in DNA methylation, thereby restoring normal liver function at the cellular level.

Produced on 9 July 2025 based on data submitted up to 2 July 2025

Epidemiological summary

In 2025, and as of 2 July 2025, no countries in Europe reported any locally acquired[1] human cases of WNV infection with known place of infection. In the previous five years, the first locally acquired cases of the WNV transmission season usually had symptom onset in June. However, the absence of notification of locally acquired cases of WNV in the EU/EEA and EU-neighbouring countries is not unexpected at this time of the year. This could either be due to the absence of WNV infections in humans or due to a delay in diagnosis and reporting of cases of WNV infection. Furthermore, a majority of WNV infections in humans remain asymptomatic or pauci-symptomatic. From the veterinary perspective, 2 WNV outbreaks among equids and 3 outbreaks among birds have been reported in Europe in 2025. The earliest start date of an outbreak among equids and birds was on 15 January 2025 in Germany and 16 February 2025 in Italy, while the latest onset of an outbreak among equids and birds was, respectively, on 12 June 2025 in Hungary and 11 June 2025 in Italy. The number of outbreaks in birds and equids reported during this first period of 2025 is below the mean monthly outbreak count for the same time frame (calculated from 2015–2024). During the same period in 2024, 16 outbreaks were reported. In 2025, as of 2 July, this is the lowest number of outbreaks in birds and equids reported during the same period since 2022. All three countries (and their associated regions) reported WNV outbreaks in birds and/or equids in 2024 and in prior years, indicating endemic WNV activity in these regions. In temperate regions like Europe, WNV transmission typically occurs from mid-June to mid-November, when mosquito activity is highest. Off-season reports of WNV outbreaks in birds and equids should be carefully evaluated as they raise questions about the timing of infection. The two early-season WNV outbreak reports (Germany’s equid case in January and Italy’s bird case in February) require cautious interpretation, as they may reflect residual detection (e.g. lingering antibodies or viral RNA from prior infections) rather than active transmission in 2025. The absence of reported human West Nile virus infections in Europe as of 2 July 2025, alongside a notably lower number of outbreaks in birds and equids compared to 2024, suggests a reduced level of viral circulation in the environment during the early transmission season in 2025. Natural fluctuations in virus prevalence can occur year to year, influenced by immunity levels in bird populations and ecological conditions. Human cases are expected to occur in the coming weeks.

[1] Locally acquired cases refer to cases acquired within the reporting country

Hi, it’s Michelle in New York. You may have heard of “Ozempic blindness,” when obesity drugs are linked to rare vision loss. Does this discovery change the risk-benefit analysis for the drugs? More in a moment, but first …

In June, EU regulators said that people with type 2 diabetes taking semaglutide, the active ingredient in Novo Nordisk’s Ozempic and Wegovy, are at risk of developing a rare eye condition that can cause vision loss. This could possibly affect as many as 1 in 10,000 people taking the medicine.

Research has identified key mechanisms driving aging and actionable targets for promoting longevity. A promising strategy is to preserve the cell’s ability to produce energy, repair DNA, and stress resilience, with nicotinamide adenine dinucleotide (NAD+) playing a central role. Here, Dr. Rebecca Crews presents a multi-target approach to support healthy aging

Decades of research have uncovered key mechanisms driving the aging process, identifying actionable targets to support longevity. One of the most promising strategies is preserving the cell’s capacity for energy production, DNA repair, and stress resilience. Central to all of these processes is nicotinamide adenine dinucleotide (NAD+), a coenzyme that fuels hundreds of metabolic reactions, including mitochondrial ATP production and sirtuin-mediated cellular maintenance.

It is known that NAD+ levels significantly diminish with age. This decline is tightly linked to the hallmarks of aging, contributing to mitochondrial dysfunction, impaired repair, cellular senescence, and age-related damage. Restoring NAD+ levels closer to youthful norms has, therefore, become a major focus within longevity science.

However, simply boosting NAD+ with precursors addresses only one aspect of a complex issue. A truly effective strategy tackles the root causes of NAD+ decline and maximizes outcomes. Therefore, a thoughtful strategy involves a multi-pronged approach: slowing NAD+ degradation, supporting its synthesis, and improving how NAD+-dependent pathways function (Sharma et al., 2023).

The foundation: NAD+ precursors

The most straightforward way to boost NAD+ levels is by supplying the body with its molecular precursors.

The two most widely used options are:

• Nicotinamide Riboside (NR) or Nicotinamide Mononucleotide (NMN): Both convert efficiently into NAD+ via the salvage pathway. Human trials consistently report 40–60% increases in blood NAD+ at daily doses of 250–1,000 mg (Conlon & Ford, 2022).

Choosing between NR, NMN, or using both typically depends on individual goals and cost. Consistent, daily dosing is key to maintaining elevated NAD+ levels.

Enhancing efficiency: Sirtuin activators

Many of NAD+’s health benefits stem from its role in fueling sirtuins, a family of NAD+-dependent enzymes that drive DNA repair, metabolic balance, stress resilience, and inflammation control. Sirtuin Activating Compounds (STACs) amplify these protective functions:

• Resveratrol:
  • A grape polyphenol that directly stimulates SIRT1. Its poor bioavailability is improved when taken with dietary fat or via advanced delivery systems (e.g., liposomal encapsulation, and solid-lipid nanoparticles).
• Pterostilbene:
  • A blueberry-derived analog of resveratrol that achieves higher plasma levels and may exert stronger SIRT1 activation.

Combining NAD+ precursors with STACs ensures both ample substrate and maximized sirtuin function (Sharma et al., 2023).

Protecting the pool: CD38 Inhibitors

CD38 is a major NADase whose activity increases with age and chronic inflammation, accelerating NAD+ depletion. Inhibiting CD38 conserves existing NAD+, keeping it available for beneficial pathways like sirtuin mediated repair.

Natural flavonoids have emerged as promising CD38 inhibitors:

• Apigenin:
  • Abundant in chamomile, parsley, and celery, apigenin blocks CD38 in preclinical models, elevating NAD+ and downstream sirtuin activity. It also delivers anti-inflammatory and antioxidant benefits (Kramer & Johnson, 2024).
• Quercetin:
  • A common flavonoid found in onions, apples, and berries, quercetin inhibits CD38 and offers potent antioxidant, anti-inflammatory, and senolytic benefits (Chini et al., 2018).

Flavonoid CD38 inhibitors offer multiple benefits: they simultaneously preserve NAD+, reduce oxidative stress, and curb inflammatory signaling.

Clearing the way: Senolytics

Senescent cells accumulate with age, acting as cellular “zombies” that resist death while secreting pro-inflammatory factors (SASP). This SASP, in turn, boosts CD38 in nearby cells, leading to faster NAD+ depletion.

Key senolytics (compounds that selectively eliminate these “zombie” cells) include:

• Fisetin: Found in strawberries and apples, this flavonoid has demonstrated the ability to reduce senescent cell burden, enhance health span, and extend lifespan in aged mice (Yousefzadeh et al., 2018).
• Spermidine: This polyamine, present in fermented foods and legumes, induces autophagy and may support the clearance of senescent cells. It’s associated with improved cardiovascular health and lifespan in mice (Hofer et al., 2022).

Calming the storm: Anti-inflammatory support

Chronic inflammation, a hallmark of aging, further depletes NAD+ by increasing CD38 activity. Resolving this inflammation is key to preserving the NAD+ pool and creating a healthier cellular environment:

• Curcumin: The active compound in turmeric, suppresses NF-κB and COX-2 signaling and may indirectly support sirtuins. Due to poor absorption, high-bioavailability formulations are essential (Hegde et al., 2023).
• Omega-3 Fatty Acids (EPA & DHA): These fish oil–derived fats integrate into cell membranes to reduce inflammatory signals and serve as building blocks for specialized pro resolving mediators (SPMs) that actively shut down inflammation (Kavani et al., 2022).

Synergistic anti-inflammatory effects have been observed when curcumin and omega-3 fatty acids are administered together (Saw et al., 2010).

Integrating the stack: Synergy and practical considerations

This multi-component stack represents a strategic and comprehensive approach to NAD+ metabolism. NAD+ precursors ensure ample substrate supply, STACs optimize its efficient use, CD38 inhibitors protect against its premature breakdown, senolytics reduce the detrimental burden of senescent ‘zombie’ cells, and targeted anti-inflammatory compounds re- establish cellular homeostasis. The goal is a synergistic effect that promotes overall cellular resilience.

However, implementation requires attention:

• Lifestyle first: Supplements cannot replace a foundation of a healthy diet, regular exercise, quality sleep, social connection, and stress management.
• Gradual introduction: Start supplements one by one (“start low, go slow”) to gauge individual tolerance.
• Quality is key: Opt for reputable brands that provide third-party testing for purity and potency.
• Personalization: Monitor biomarkers and subjective well being.

Conclusion: A balanced perspective on NAD+ optimization

Supporting NAD+ levels is a promising strategy in the effort to maintain cellular function and health with age. The approach outlined here – boosting NAD+ production, reducing its breakdown, improving how it’s used, and supporting the broader cellular environment – reflects the current understanding of NAD+ as a dynamic and interconnected system.

NAD+ boosting strategies should be seen as a flexible starting point, not a one-size-fits-all solution. As research advances, more personalized strategies will likely become available, guided by individual health data and deeper insights into NAD+ biology.

Importantly, NAD+support works best as part of a bigger picture. Lasting health and longevity depend just as much on diet, exercise, sleep, stress management, and social connection. Keeping these foundations strong while staying informed about new science is the most practical way to approach NAD+ optimization today.

Elinzanetant offers a new, non-hormonal treatment option for menopausal hot flushes and night sweats. Now approved by the MHRA, it provides symptom relief for women seeking alternatives to hormone therapy

The MHRA has become the first medical regulator in the world to approve elinzanetant, a novel non-hormonal oral agent, for the treatment of moderate to severe vasomotor symptoms associated with menopause. This approval introduces a new therapeutic option targeting neurokinin receptor pathways, expanding treatment possibilities beyond hormone replacement therapy.

The new marketing authorisation was granted on 8 July 2025 to Bayer plc. 

Elinzanetant significantly reduced the number and intensity of hot flushes

Approximately 13 million women in the UK are going through perimenopause or menopause, with up to 80% expected to experience hot flushes during the menopause transition, and many remaining untreated.

When oestrogen levels drop during menopause, specific brain cells become overactive and interrupt the body’s ability to control temperature, which leads to hot flushes and night sweats. 

Elinzanetant works by calming these signals in the brain, helping to bring the body’s temperature back to a normal level. The medication can also help alleviate sleep problems and is administered in tablet form.

Julian Beach, MHRA Interim Executive Director of Healthcare Quality and Access, said:  “Hot flushes and night sweats associated with menopause can have a significant negative impact on quality of life.  

“We are therefore pleased to announce our approval of elinzanetant, which has met the MHRA’s standards for safety, quality, and effectiveness. 

Elinzanetant offers a non-hormonal alternative for those who may not be able to, or prefer not to, take hormone-based therapies. As with all licensed medicines, we will continue to monitor its safety closely as it becomes more widely used.” 

Successful in-human clinical trial

Elinzanetant’s approval is based on results from the OASIS clinical trials, which involved over 1,400 women aged 40 to 65 across several countries.

The Phase III OASIS 1 and 2 clinical trials were randomised, double-blind, placebo-controlled studies evaluating the efficacy and safety of elinzanetant in menopausal women experiencing moderate to severe vasomotor symptoms, such as hot flushes and night sweats. Participants received a daily oral dose of 120 mg elinzanetant or placebo for 12 weeks. Results showed that elinzanetant significantly reduced both the frequency and severity of hot flushes compared to placebo, with improvements observed early in the treatment course. Secondary outcomes also demonstrated enhancements in sleep quality and overall menopause-related quality of life.

“Menopausal symptoms are frequent side effects of endocrine therapy for breast cancer, often leading to treatment discontinuation, which is why management of these symptoms can play an important role in breast cancer treatment,” said Dr. Fatima Cardoso, Principal Investigator of OASIS-4, from Lisbon, Portugal. “With no currently approved treatments for this indication, there is an unmet medical need for therapeutic options.”

“The robust efficacy and favourable safety profile of elinzanetant reinforces its potential as a non-hormonal treatment for women experiencing menopause,” said Dr. Christian Rommel, member of the Executive Committee of Bayer AG’s Pharmaceutical Division and Global Head of Research and Development. “We look forward to submitting applications to health authorities for marketing authorisations of elinzanetant to treat moderate to severe VMS associated with menopause in women, building upon our extensive legacy and commitment to women’s healthcare.” 

Introduction

Acquired immunodeficiency syndrome (AIDS), resulting from HIV infection, poses a significant threat to human health and remains a critical public health concern. HIV mainly infected through blood transfusion, sexual contact, and mother-to-child transmission. In China, the HIV/AIDS epidemic has similarly become a major public health concern. According to an assessment by the Chinese Center for Disease Control and Prevention, as of 2018, approximately 1.25 million individuals had been infected with HIV across the country.¹ Notably, the Joint United Nations Program on HIV/AIDS (UNAIDS) reports suggested that global AIDS-related mortality and new infection rates have declined year by year.² However, the AIDS epidemic among young people is not optimistic. The AIDS-related mortality of male adolescents has risen against the trend,³ particularly among adolescent college students.⁴ Hence, there is a crucial need to raise awareness and enhance knowledge regarding HIV infection in this population.

Awareness of HIV infection refers to an individual’s general understanding or consciousness of the existence of HIV, its modes of transmission, and its impact on society.⁵ While HIV-related knowledge encompasses a deeper understanding of HIV/AIDS, including its biology, epidemiology, prevention strategies, treatment options, and societal implications.⁶ It goes beyond mere awareness and delves into specifics such as the importance of viral load and CD4 count monitoring, strategies for reducing HIV transmission rates (like PrEP and PEP), and the social and cultural factors influencing HIV prevalence and stigma.⁷ Nowadays, the characteristics of adolescents in terms of health awareness and related behaviors may be significantly different from those of other groups, due to the change of traditional and modern media.⁸ However, most previous researches on adolescents’ health perception and related behaviors investigation focused on men who have sex with men (MSM).^9–11To enhance awareness and knowledge of HIV infection, it’s imperative to ascertain the current level of awareness and knowledge about HIV within this demographic. Although studies on HIV-related knowledge among university students exist,^12,13 research specifically targeting medical students remains limited, particularly in China and similar sociocultural settings.

To address this gap, we conducted a study to assess awareness, knowledge, practices, and sources of information on HIV infection among college students. Our objective is to assess the HIV awareness and knowledge among young college students, categorized into medical and non-medical students. Thus, this study aims to provide valuable data on this specific population and to support improvements in medical education, especially for adolescent students who are increasingly at risk of HIV infection.

Subjects and Methods

Subjects

This multicenter study was conducted via an online survey at two universities in China, namely Xiamen University and Southern Medical University. A total of 490 college students were recruited using a convenience sampling method through online platforms, with all participation being entirely voluntary. Among the participants, 261 were medical students and 229 were non-medical students (Figure 1). The medical students were mainly enrolled in clinical medicine program and the program were required to complete coursework in infectious diseases, including content on HIV prevention and management. In contrast, the non-medical students, who majored in science, technology, engineering, and mathematics (STEM) fields, did not receive formal instruction on HIV-related topics as part of their curriculum. This educational difference was considered when interpreting their knowledge levels. Additionally, socio-demographic data of all participants enrolled were also recorded, including gender, age, education.

This questionnaire-based study was approved by the Ethics Committee of Nanfang Hospital, Southern Medical University (NFEC-2021-334). Informed consent was waived for all questionnaire participants, as all responses were collected anonymously. All procedures in our study were in accordance with the ethical standards of the responsible committee on human experimentation and with the Helsinki Declaration of 1975, as revised in 2008. Informed consent was obtained from all participants for inclusion in the study.

Questionnaire

The National AIDS Sentinel Surveillance Implementation Plan¹⁴ released by the National Center for STD and AIDS Prevention and Control of China identified eight key target populations, including youth and university students, and designed tailored questionnaires specifically for them. We adapted this questionnaire with specific modifications to better align with the objectives and target population of our study. We collected their socio-demographic data and investigate the awareness of and knowledge about HIV infection, the practice of preventive measures, and sources of HIV-related information. The questionnaire was divided into five sections and listed as followed:

1. HIV awareness and knowledge: 1). Will HIV be transmitted by blood transfusion? 2). Will HIV be transmitted by sharing dishes? 3). Will HIV be transmitted through insect bites? 4). Will HIV be transmitted by sexual contact? 5). What is the window period for HIV antibody testing? 6). Does HIV mostly damage the immune system? 7). Can most AIDS patients be cured? 8) Does correct use of condoms can reduce the risk of HIV transmission? 9) Do you have awareness on risk of HIV infection? HIV awareness and knowledge were treated as a unified construct representing participants’ overall understanding of HIV. This included concepts such as transmission routes, prevention methods, and treatment possibilities. A total of 9 questions were used to assess this knowledge level, with each question offering three response options: “Yes”, “No”, or “Don’t know”. Correct answers were coded as 1, and incorrect or “Don’t know” responses as 0. Participants who answered all 9 questions correctly were classified into the group not lacking HIV-related knowledge.
2. Sources of HIV-related information: 1). Friends; 2). Internet; 3). Schools; 4). TV medias; 5). Doctors; 6). Parents; 7). Others. (School Sources: Education through lectures, educational materials, and school-based programs; Internet Sources: Access to websites, online forums, and educational videos; Friends: Personal Experiences and Conversations; TV Media: Public service announcements, documentaries, and news coverage addressing HIV; Doctors: Medical advice, counseling, and participate in referrals for HIV testing, treatment options, and specialized care providers; Parents: Family discussions and parental guidance; Other Sources: Community resources and printed materials providing information about HIV, or sources other than those mentioned above.
3. Attitude towards HIV: 1). Will you test HIV antibody if necessary 2). Have you ever been tested for HIV? 3). Are you afraid of HIV-infected patients? 4). Do you feel lack knowledge of HIV?
4. Type of HIV knowledge most interested: 1). How to prevent HIV infection? 2). How to use condoms correctly? 3). More knowledge of reproductive health? 4). Types of high-risk behaviors? 5). Knowledge of HIV testing methods?
5. Important part of a good HIV education should be: 1). Novelty of HIV related knowledge. 2). Types of HIV related knowledge. 3). Depth of HIV related knowledge. 4). Wider audience. 5). Numbers of HIV related education. 6) Tailor content to audience.

Statistical Analysis

Missing data accounted for less than 5% of the dataset and were handled using multiple imputation. Data were expressed as mean ± standard deviation for continuous variables or numbers (percentages) for categorical variables. Prior to statistical testing, the normality of continuous variables was assessed using the Shapiro–Wilk test. Independent samples t-tests were used when normality assumptions were met; otherwise, nonparametric tests (Mann–Whitney U) were applied. Chi-square tests were used to compare categorical variables. We also used univariate and multivariate logistic regression analysis to determine factors related to the lack of HIV-related knowledge. The significance level was set as P <0.05 (two-tailed). Data analysis and quality control procedures were performed using SPSS 23.0 (Chicago, USA).

Results

Demographic Data

We enrolled 490 students. Of these, a total of 261 student were medical students and 229 were non-medical students. As shown in Table 1, the average age of medical students was 21.41 ± 1.71 years, while that of non-medical students was 20.15 ± 2.61 years. Among the medical students, 39.8% were male and 60.2% were female; in contrast, 54.6% of non-medical students were male and 45.4% were female. Regarding academic grade, most medical students were in their third (33.7%), fourth (27.6%), or postgraduate (33.0%) years. Non-medical students were primarily in their second year (46.7%) and third year (24.9%), with 10.0% being postgraduates.

Table 1 Demographics of the Two Groups

HIV Awareness and Related Knowledge

Differences in HIV transmission knowledge between medical and non-medical students are shown in Figure 2A. There is no significantly different of self-report awareness on risk of HIV infection between medical students and non-medical students (P = 0.919). In terms of HIV transmission routes, compared with medical students, a higher proportion of non-medical students believed that HIV would be transmitted by shared dishes (P=0.016) and insects bites (P=0.037).

Figure 2 (A) Proportion of correct answers about knowledge of HIV transmission routes. (B) Proportion of correct answers about HIV knowledges. (C) Proportion of student with sources of HIV knowledge. (D) Attitudes towards HIV in students. (E) Types of HIV knowledges most interested in students. (F) The comments from students on important parts as a good HIV education.

As shown in Figure 2B, while no significant difference was found in the knowledge of condom use for HIV prevention (P = 0.197), medical students demonstrated significantly better understanding in several other areas. These included awareness of the HIV antibody window period (P < 0.001), knowledge that HIV primarily damages the immune system (P = 0.002), and recognition that most HIV infections cannot be cured (P = 0.015).

Sources of HIV-Related Knowledge and Attitudes Towards HIV

As shown in Figure 2C, there were significant differences in the sources of HIV-related information between medical students and non-medical students. Non-medical students were more likely to obtain HIV-related knowledge from the Internet (P=0.029), TV medias (P=0.027) and others (P=0.032). The proportion of medical students acquiring HIV-related knowledge from doctors was higher than that of non-medical students (P<0.001).

Most of college students in both groups expressed no fear of HIV patients, with a relatively higher proportion among medical students (P<0.001). The testing rate for HIV among college students in both groups is relatively low, although both groups expressed their willingness to undergo HIV testing if necessary. Interestingly, a higher percentage of non-medical students indicated a lack of HIV knowledge and expressed a need for more education (P=0.002, Figure 2D).

Exploring College Students’ Interest in HIV Knowledge and Attitudes Towards Related Education

We investigated which aspects of HIV-related knowledge students were most interested in, as shown in Figure 2E. We noted that medical students were more interested in HIV testing methods than non-medical students (P=0.002).

We also evaluate among two groups of college students to identify the characteristics of effective HIV education. Interestingly, non-medical students believe that good HIV education should encompass a wider range of HIV knowledge in different fields (P=0.046), whereas medical students think it should involve more frequent sessions with longer durations, rather than just covering the basics (P=0.038, Figure 2F).

The Understanding of HIV Transmission Routes and Knowledge Sources Among College Students

The source of HIV-related information was also a key factor affecting the lack of HIV knowledge. As a result, we assessed the percentage of students who accurately answered questions across different sources of knowledge. In medical students, a higher percentage of those who answered all HIV transmission route questions correctly reported that their knowledge originated from school (P=0.013, Figure 3A).

Figure 3 (A) Proportion of medical students lacking HIV transmission knowledge among different sources. (B) Proportion of non-medical students lacking HIV transmission knowledge among different sources.

Similarly, among non-medical students, we observed a parallel trend. A greater number of non-medical students who answered correctly indicated that their knowledge was acquired from school (P=0.001). Concurrently, non-medical students who did not fully comprehend HIV transmission mentioned that their knowledge came from TV and other medias (p=0.022) and other sources (P<0.001, Figure 3B).

Risk Factors for Lack of HIV-Related Knowledge

We conducted a multivariate logistic regression analysis, as shown in Table 2. After adjusting for gender, age, grade, and various sources of HIV knowledge (including doctors, TV/media, school, internet, friends, parents, and other sources), we found that being a non-medical student was an independent risk factor for insufficient HIV-related knowledge (OR = 0.421, 95% CI: 0.195–0.912, P = 0.028). This indicates that medical students were approximately 58% less likely than non-medical students to lack adequate HIV knowledge.

Table 2 Multivariable Analysis for HIV Related Knowledges and Awareness

Impact of Clinical Practice in HIV Wards on HIV Knowledge

A total of 192 medical students (192/229) participated in clinical practice in HIV wards. We compared the HIV related knowledge and behavior before and after the clinical practice. We observe an enhancement in HIV knowledge and awareness, as shown in Figure 4A and B. There is also a notable increase in the proportion of student familiar with PEP and PrEP (P<0.001), and a greater willingness to utilize PEP and PrEP when necessary (P<0.001, Figure 4C). Elevated self-rating scores indicate that more student expressed their enthusiasm for helping HIV-infected people (P=0.015, Figure 4D).

Figure 4 (A) Difference of HIV awareness after education and clinical practice in HIV wards. (B) Difference of HIV knowledges after education and clinical practice in HIV wards. (C) Difference of attitudes on PEP/PrEP after education and clinical practice in HIV wards. (D) Difference of attitudes on HIV infected patients after education and clinical practice in HIV wards.

Discussion

In 2014, The Joint United Nations Programme on HIV/AIDS (UNAIDS) launched the 95–95-95 targets. The aim was to diagnose 95% of all HIV-positive individuals, provide antiretroviral therapy (ART) for 95% of those diagnosed and achieve viral suppression for 95% of those treated by 2030. To accomplish this objective, enhancing awareness and knowledge concerning HIV infection among college students is paramount, especially considering the gradual rise in HIV infection rates in this population. Currently, the extent of awareness and knowledge regarding HIV infection among college students remains uncertain. Therefore, to achieve this goal of the WHO, it is important to analyze the level of awareness and knowledge about HIV among this population.

This study suggested that there were indeed more students who wanted to learn more HIV related knowledge. For the source of HIV-related knowledge, more non-medical students obtained HIV-related knowledge from the Internet and TV media, while the proportion of medical students acquiring knowledge from doctors was higher than that of non-medical students. Particularly, medical students were more interested in HIV testing methods than non-medical students. Our research can provide data support for further improving the methods and contents of health education for young people.

Previous studies have suggested that HIV-infected adolescents in Africa generally have sexual and reproductive health needs.^15,16 Nevertheless, due to insufficient resources of the health system and limited skills among health workers, there is still a huge gap between the services adolescents received and their actual health needs.^15,17 Among non-HIV adolescents, it is necessary to raise their awareness of HIV infection to avoid HIV infection due to high-risk sexual contact. Although in our data, self-report HIV infection awareness of medical students and non-medical students were similar. It is still important to the popularization of adolescent health and increase HIV infection awareness among young people. At the stage of adolescence, the demand for sexual and reproductive health will gradually arise. Popularizing health knowledge helps prevent HIV infection. In this study, we found that non-medical students’ knowledge of HIV transmission is still not satisfactory. Some non-medical students believe that share dishes and insects bite will cause the spread of HIV. Popularizing HIV-related knowledge among non-medical students still needs to continue.

In our study, we noticed a higher percentage of non-medical students lacking HIV knowledge. The discrepancy in HIV knowledge between medical and non-medical students could indeed be attributed to the absence of specialized HIV education among non-medical students. Perhaps several methods can be used, including but not limited to increasing HIV-related lectures, interdisciplinary collaboration, or opening some online education resources of medical students to non-medical students, etc. By these strategies, educational institutions can thereby promote better understanding and awareness of this important public health issue among non-medical students.

The study showed that the essential way for non-medical students to acquire HIV-related knowledge were the Internet and TV medias. Medical students have also increased the access to HIV knowledge at school and doctors. Other studies have also shown that the Internet is playing an increasingly important role in obtaining health information.^18–20 The Internet is the important way for college students to obtain relevant knowledge.²¹ Malaysian research suggests that up to 57.1% of young Malaysians have searched for information about sexually transmitted diseases on the Internet.²² The most important sources for Iranian teenagers to obtain health information related to high-risk behaviors are the Internet and virtual social media.^23,24 According to the results of this study, medical students who obtained the knowledge from school have a more adequate understanding of HIV-related knowledge. HIV-related knowledge education requires high accuracy and professionalism. However, the reality is that the quality of information on the Internet is currently uneven.^25–27 In addition, the results of this study also suggest that for non-medical students, schools should enhance the publicity of HIV-related knowledge in order to raise students’ awareness and knowledge of HIV.

In this study, we also found medical students and non-medical students are also different in the content of interest of HIV-related knowledge. For medical students, the professional knowledge of HIV testing can be increased to better suit their interests. We also observed that non-medical students expressed a greater need for additional HIV knowledge and education. However, regarding the characteristics of good HIV education, the two groups of college students held different perspectives. Non-medical students expressed the need for more comprehensive HIV knowledge, while medical students emphasized the necessity for more in-depth and extensive hours of HIV education.

The discrepancy in HIV knowledge acquisition between medical and non-medical students, with medical students receiving more information from doctors, underscores the importance of targeted educational interventions. Medical students, due to their exposure to clinical settings, have access to healthcare professionals who can provide firsthand knowledge and guidance on HIV-related topics. In contrast, non-medical students may lack similar access to healthcare environments, resulting in a gap in their HIV knowledge. To address this issue, educational institutions could consider implementing initiatives to bridge this gap, such as providing non-medical students with opportunities to interact with healthcare professionals or offering specialized HIV education programs tailored to their needs. Such efforts could contribute to promoting a more comprehensive understanding of HIV across diverse student populations.

To the best of our knowledge, this study is the first to investigate the acquisition of HIV-related knowledge among medical students and non-medical students. At the same time, we investigated the accuracy and preferences of their HIV-related knowledge. The current situation provides basic reference materials for the popularization of youth health. However, our study has some limitations. First, the small sample size of our study may induce bias. Second, due to resource constraints, we refrained from conducting a before-and-after comparison among students with HIV knowledge popularization. Third, whether young people in other regions have similar conditions requires further verification. Extent to which health education has improved students’ attitudes towards HIV-related knowledge and behaviors remains to be further studied. In addition, medical students are expected to perform better in HIV-related knowledge, future studies should consider including students from more closely related fields or stratifying by academic year to reduce baseline knowledge gaps and enhance comparability.

Conclusions

In our study, we found that the medical students were more aware of HIV related knowledge than the non-medical students. Our research can provide relevant data support for further improving the methods and content of health education for young people.

Abbreviations

AIDS, Acquired immunodeficiency syndrome; HIV, human immunodeficiency virus; MSM, men who have sex with men.

Data Sharing Statement

The data used in the current study are available from the corresponding author upon reasonable request.

Ethical Approval and Consent to Participate

This retrospective study was approved by the Ethics Committee of Nanfang Hospital, Southern Medical University (NFEC-2021-334). Informed consent was waived for all questionnaire participants, as all responses were collected anonymously.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Funding

This work received no funding.

Disclosure

The authors declare no conflicts of interest in this work.

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Introduction

The primary prevention strategy for acute coronary syndromes (ACS) after percutaneous coronary intervention (PCI) is mainly based on dual antiplatelet therapy (DAPT), whose standard regimen consists of aspirin in combination with a P2Y12 receptor inhibitor (eg, clopidogrel, prasugrel, or ticagrelor). As a widely used antiplatelet drug, clopidogrel can be used to treat coronary heart disease and stroke by inhibiting adenosine diphosphate (ADP) receptors to reduce platelet activation and aggregation. However, poor control of platelet activity, known as clopidogrel resistance (CR), may occur in some patients after clopidogrel therapy,¹ which may lead to in-stent thrombosis and myocardial infarction. In addition, the most common adverse effect caused by clopidogrel is the risk of bleeding, especially gastrointestinal bleeding. One study found that among patients who underwent PCI and drug-eluting stents (DES) and completed 12 months of DAPT, complications such as gastrointestinal bleeding and cerebral hemorrhage could be induced in both the aspirin and clopidogrel groups (incidence: 1.5% in the aspirin group vs 1.7% in the clopidogrel group, P=0.160), but the clopidogrel group was superior to the aspirin group in terms of secondary prevention.² Other studies have shown that P2Y12 receptor inhibitors, specifically clopidogrel, were superior in reducing the incidence of myocardial infarction (RR=0.77, 95% CI: 0.67–0.89) and hemorrhagic stroke (RR=0.53, 95% CI: 0.30–0.92) and did not significantly increase the risk of major bleeding (RR=0.96), as compared with aspirin alone (RR=0.96, 95% CI: 0.71–1.30).³ Notably, in ACS patients treated with PCI, ticagrelor and prasugrel were associated with a high risk of bleeding compared to clopidogrel, with prasugrel exhibiting a higher risk of short-term bleeding at 90 days (HR=1.66, 95% CI: 1.11–2.48).⁴ In recent years, several studies have found that with clopidogrel, patients may also suffer from rare adverse effects such as severe fatigue and bleeding from small bowel ulcers. In clinical individualised treatment, drug efficacy can be assessed by means of genetic polymorphism testing, platelet function testing and plasma concentration monitoring, so that dosage can be adjusted to achieve better therapeutic effects and reduce the occurrence of adverse reactions.^5–7

Although new antiplatelet agents are emerging, clopidogrel remains important in the treatment of cardiovascular disease (CVD). Current clinical practice guidelines (2023 ACC/AHA and 2023 ESC) point to clopidogrel as an alternative choice for patients at high bleeding risk and actively recommend genetic testing to guide clopidogrel use. As the precision medicine paradigm advances, dynamic dose adjustment based on pharmacogenomics may further reshape its clinical status, which provides an important direction for future research.

Pharmacokinetics and Pharmacological Effects of Clopidogrel

Clopidogrel, with the molecular formula C₁₆H₁₆CINO₂S, acts as an ADP receptor inhibitor and is an inhibitor of platelet aggregation, platelet inhibition results from irreversible binding of the P2Y12 receptor. Clopidogrel inhibits platelet aggregation by selectively inhibiting ADP binding to platelet receptors and inhibiting ADP-mediated activation of the glycoprotein IIb/IIIa (GPIIb/IIIa) complex.

Clopidogrel, a thienopyridine prodrug, is inactive in vitro and must be biotransformed in vivo to be effective, with a number of different genetically encoded metabolising enzymes involved in the multi-step biotransformation of clopidogrel.⁸ Clopidogrel is mainly metabolised by the liver after ingestion by two metabolic pathways, one is hydrolysed to inactive carboxylic acid derivatives via an esterase-dependent pathway (CES 1). The other is through the hepatic cytochrome P450-dependent pathway (CYP2C19: 44.9%, CYP2B6: 19.4%, CYP1A2: 35.8%), which first converts clopidogrel to the 2-oxo-clopidogrel intermediate, and then to clopidogrel-AM through CYP2C9, CYP2B6, CYP2C19 and CYP 3A 4/5 which ultimately acts systemically⁹ (Figure 1 and Table 1).

Table 1 Summary of Pharmacokinetics of Clopidogrel

Figure 1 Mechanism of action of clopidogrel in humans. Clopidogrel, as a precursor drug, needs to be metabolized by the liver before it can exert its antiplatelet effect. There are two metabolic pathways, one of which is hydrolyzed to inactive carboxylic acid derivatives by an esterase-dependent pathway (CES 1). The other is bioactivation via the hepatic cytochrome P450-dependent pathway (mainly catalyzed by CYP2C19, CYP2C9, and CYP3A4 enzymes) to produce the active metabolite clopidogrel-AM. This figure summarizes the key enzymatic steps and metabolic transformations that underlie the pharmacological activity of clopidogrel.

Clinical Use of Clopidogrel in Related Diseases

Coronary Artery Disease

Coronary artery disease (CAD) is defined as a condition with one or more of the following: a history of myocardial infarction, percutaneous coronary intervention (PCI) or coronary artery bypass grafting (CABG), coronary artery stenosis of ≥50%, and chest pain accompanied by myocardial ischaemia.¹⁰ CAD includes acute coronary syndrome (ACS) and stable coronary heart disease. According to the 2023 ESC Guidelines for the management of acute coronary syndromes, DAPT needs to be initiated as early as possible after ACS, and clopidogrel in combination with aspirin reduces the risk of early ischemic events. The guidelines emphasize that in the acute phase of ACS, ticagrelor or prasugrel are preferentially recommended for their more potent antiplatelet effects, but clopidogrel remains the logical choice for patients at high risk of bleeding or with contraindications, such as advanced age, prior history of bleeding, and inability to tolerate ticagrelor (eg, dyspnea) or prasugrel (eg, prior history of stroke). In patients requiring long-term anticoagulation after atrial fibrillation or mechanical valve replacement, clopidogrel in combination with an anticoagulant (eg, rivaroxaban) is associated with a lower risk of bleeding than ticagrelor/ prasugrel (Class I recommendation, Level of Evidence A).¹¹ In 2020, the safety and efficacy of clopidogrel, ticagrelor, and prasugrel were compared in patients with st-segment elevation myocardial infarction. All-cause mortality and ischaemic event rates were reduced with ticagrelor and prasugrel compared to clopidogrel. However, since clopidogrel is cheaper, it will still dominate in the clinic.^12,13 In 2023, Kim, MC et al¹⁴ found that in the treatment of patients with acute myocardial infarction with a high risk of bleeding, clopidogrel, as compared to ticagrelor, would reduce adverse clinical outcomes, such as bleeding, and was safer to use. In 2024, Kim, SH et al¹⁵ found that patients with acute myocardial infarction often experienced dyspnoea with the use of ticagrelor, and that switching to clopidogrel in patients with such symptoms resulted in an improvement in dyspnoea without increasing the risk of ischaemic events.In 2024, Li et al¹⁶ found that in dual-risk ACS patients who completed 9 to 12 months of DAPT after drug-eluting stent implantation (DAPT) and were free of adverse events for at least 6 months prior to randomization to a subgroup, a 9-month extension of the clopidogrel monotherapy regimen was superior to continuation of clopidogrel DAPT in reducing clinically relevant bleeding without increasing ischemic events.

In 2021, clopidogrel was found to reduce morbidity and mortality in stable coronary heart disease and has been widely used in such patients.¹⁷ According to the 2024 ESC Guidelines and the 2023 AHA/ACC/ACCP/ASPC/NLA/PCNA guidelines for the management of chronic coronary syndromes, clopidogrel monotherapy (75 mg/d) is the standard alternative (Class I recommendation) to aspirin in chronic coronary disease (CCS) for those who are not aspirin tolerant patients (Class I recommendation), and there is insufficient evidence to support the superiority of novel P2Y12 inhibitors over clopidogrel in long-term monotherapy, which is more suitable for long-term management because of its lower cost and lower bleeding risk. In patients at high bleeding risk for PCI, especially those with normal CYP2C19 genes, clopidogrel is the first choice for switching to monotherapy after a short period of dual resistance (1–3 months) (Class I recommendation).^18,19 Kang et al²⁰ found that clopidogrel monotherapy was superior to aspirin in reducing both thrombotic and bleeding risks in patients who were event-free 6–18 months after PCI and required long-term antithrombotic therapy, and that the advantages were independent of the patient’s risk of bleeding or the complexity of PCI, with a wide range of applicability and without the need to adjust treatment strategies.

Stroke

Stroke is defined as the sudden onset of a focal neurological deficit at a site consistent with the extent of the large cerebral arteries.¹² Currently, clopidogrel is increasingly used for secondary prevention of ischaemic stroke, and can be used as a reasonable monotherapy option in patients with non-embolic ischaemic stroke or as a 21-day dual antiplatelet therapy in combination with aspirin after minor ischaemic stroke or transient ischaemic attack (TIA) in patients at high risk of stroke recurrence.⁸ Carotid endarterectomy (CEA) improves carotid blood flow and reduces the risk of stroke. In 2023, a study found that patients with a history of symptomatic carotid artery disease were more likely to use clopidogrel at the time of surgery. From 2010 to 2017, a total of 1066 patients were treated with CEA. During the study period, clopidogrel use increased by 24.9% over these seven years, which equates to an annual increase of 11%, and clopidogrel was not associated with an increased risk of postoperative complications, including bleeding. These data suggest that clopidogrel should not be discontinued before CEA and should be considered part of the “optimal pharmacological treatment” for patients undergoing CEA.²¹

Central Retinal Vein Occlusion

Retinal vascular occlusions are the second most common vascular disorder in the retina after diabetic retinopathy.²² There are two major forms of retinal vein occlusion: branch retinal vein occlusion (BRVO) and central retinal vein occlusion (CRVO). Based on fundus fluorescein angiography (FFA), CRVO can also be classified into ischemic or nonischemic.²³ Al Ghaithi et al reported a case of a 54-year-old male with nonischemic CRVO and multiple systemic comorbidities, including diabetes, hypertension, and dyslipidemia. After initial treatment with aspirin failed, switching to clopidogrel demonstrated significant efficacy in improving visual acuity, resolving macular edema, and alleviating retinal vascular pathology, suggesting that clopidogrel may be superior to aspirin in select CRVO patients and providing strong evidence for its potential new indications in CRVO management.²⁴

Adverse Effects of Clopidogrel in Clinical Practice

Gastrointestinal Bleeding

Clopidogrel inhibits platelet aggregation and increases the risk of bleeding, the most common is gastrointestinal bleeding. One study found 2,252 reports of clopidogrel adverse events through the US Food and Drug Administration Adverse Event Reporting System (FAERS) database, and clopidogrel leads to the highest rate of hospitalised gastrointestinal bleeding events.²⁵ Clopidogrel does not directly damage the gastric mucosa, but on the one hand inhibits platelet production of vascular endothelial growth factor, which in turn inhibits the proliferative capacity of gastric mucosal epithelial cells. On the other hand, it induces apoptosis of gastric mucosal cells, which reduces the expression of intercellular tight junction proteins, thus affecting the repair ability of gastric mucosa. Therefore, clopidogrel also has a damaging effect on the gastric mucosa to some extent, which increases with increasing dose.²⁶

Liver Injury

Clopidogrel-induced liver injury has been seen in the form of hepatocellular or mixed cholestatic/hepatocellular patterns. The most common pattern of liver injury is mixed, followed by hepatocellular damage and cholestatic injury. It has been shown that hepatotoxicity occurs 35 days after initiation of clopidogrel (range 3–180 days) and the clinical presentation is more inclined to a dose-dependent response.²⁷

Neutropenia

A PubMed search for “clopidogrel” and “neutropenia” revealed 17 cases since 2000. Clopidogrel administration resulted in a significant decrease in haemoglobin and white blood cells, leading to an increased risk of bleeding. An 84-year-old female patient with a previous asymptomatic acute myocardial infarction, who had coronary stenting, experienced a significant decrease in haemoglobin and white blood cells approximately 40 days after clopidogrel administration. Subsequent replacement administration of ticagrelor resulted in a rise in markers after the switch.^28,29

Thrombotic Microangiopathy

Immune thrombocytopenic purpura, and thrombotic thrombocytopenic purpura are all thrombotic microangiopathies, which may be induced by the use of clopidogrel. Lizondo López, T et al³⁰ reported an example of thrombotic microangiopathy in a patient who developed microangiopathic haemolytic anaemia and thrombocytopenia after one month of treatment with clopidogrel and aspirin. After extensive clinical and laboratory investigations, it was shown that his thrombotic microangiopathy was induced by clopidogrel. Both Grossman, K and Távora, C et al^31,32 reported cases of clopidogrel-induced immune thrombocytopenic purpura, which can be considered to be caused by clopidogrel in patients presenting with isolated thrombocytopenia. Ndulue, CN et al³³ reported a case of thrombotic thrombocytopenic purpura triggered by clopidogrel in a Nigerian patient with chronic kidney disease (CKD), which resolved quickly after discontinuation of clopidogrel.

Rare Adverse Reactions

In addition to the common adverse reactions described above, many rare adverse reactions to clopidogrel have been reported. Severe fatigue syndrome is a rare but clinically significant side effect of overreaction to clopidogrel in patients undergoing neurovascular endovascular intervention. In 2021, Bass et al³⁴ assessed patient response to clopidogrel using the VerifyNow assay, which is expressed in terms of P2Y12 reaction units (PRUs), with lower PRU values equating to a greater degree of inhibition of the P2Y12 receptor and a lower presumed probability of platelet aggregation, with overresponse to clopidogrel defined as PRUs ≤60. The diagnosis of clopidogrel-induced severe fatigue was made when symptoms appeared after clopidogrel treatment and resolved with dose reduction.

To date, seven cases of insulin autoimmune syndrome (IAS) induced by clopidogrel have been reported, and the sulfhydryl group of clopidogrel metabolites can induce IAS with hypoglycemia as the main symptom. Shi Chen et al³⁵ conducted a meta-analysis of six trials involving a total of 61,399 participants and found that clopidogrel-associated hypoglycemia may occur in Asian participants. Du, W et al³⁶ reported recurrent hypoglycemic episodes in a patient after 23 days of antiplatelet therapy with clopidogrel, suggesting that physicians should be vigilant for hypoglycemia-related symptoms in clopidogrel users.

Clopidogrel-induced small bowel ulcers and bleeding are uncommon, and in 2021 Lee, SH. et al³⁷ reported a case of bleeding small bowel ulcers from clopidogrel use in an 86-year-old male clopidogrel-resistant patient, which was found to be expected to increase in older patients with risk factors. Rowell’s syndrome, a combination of polymorphic lupus erythematosus and lupus erythematosus, was first reported in the literature in 2024 in a 52-year-old woman with sjogren’s syndrome who was taking paquinimod for two months but developed this symptom when she mistakenly took clopidogrel for one week.³⁸ Clopidogrel induced arthritis is a rare instance, Faiza Javed et al³⁹ reported the development of inflammatory arthritis in a male patient after 5 days of clopidogrel, and after diagnosis by exclusion it was determined that clopidogrel was responsible for the development of the induced arthritis.

Impact of Genetic Polymorphisms on Clinical Use of Clopidogrel

Several genes are involved in the biotransformation of clopidogrel to the active drug, and their polymorphisms may interfere with the biotransformation, leading to a decrease or increase in the amount of active metabolite, thereby affecting the drug’s efficacy.

CYP2C19 Gene Polymorphisms

Some patients develop residual high platelet reactivity (HPR) during antiplatelet therapy. HPR has been shown to be significantly associated with thrombotic events. One of the major causes of residual HPR after antiplatelet therapy is the CYP2C19 polymorphism, the most common genetic variant associated with clopidogrel resistance. Recent studies have shown that patients with one or more CYP2C19 loss-of-function alleles (LoF, *2 or *3) with low levels of active metabolites have an increased incidence of HPR, clopidogrel resistance and major adverse cardiovascular events (MACE).⁴⁰ Clopidogrel efficacy decreases with increasing number of CYP2C19 LoF alleles. Individuals carrying one CYP2C19 LoF allele are referred to as intermediate metabolizers (IM) and those carrying two CYP2C19 LoF alleles are referred to as poor metabolizers (PM).^41,42 Several studies have shown that increasing the dose of clopidogrel improves platelet inhibition and overcomes resistance to clopidogrel in patients with IM (*2) but not in patients with PM (*2).⁴³ According to the Clinical Pharmacogenetics Implementation Consortium (CPIC) guidelines for clopidogrel treatment based on the CYP2C19 genotype, replacement therapy is recommended for patients who are PM.⁴⁴ There are significant racial and regional differences in the distribution of CYP2C19 polymorphisms, and it has been estimated that approximately 15% of white and black populations carry at least one LoF allele (*2 or *3) compared to 29–35% in Asian populations.⁸

Mohitosh Biswas et al^45,46 compared the clinical efficacy of clopidogrel versus other P2Y12 receptor antagonist treatments after PCI in CAD patients who inherited the CYP2C19 LoF allele. The results showed that CAD patients who inherited the CYP2C19 LoF allele and were treated with clopidogrel had a significant 62% increased risk of MACE compared with patients treated with other P2Y12 receptor antagonists such as prasugrel or ticagrelor. In another article, he assessed the overall risk of recurrent stroke in stroke or TIA patients who carried the CYP2C19 LoF allele and were taking clopidogrel, and the risk of recurrent ischemic stroke was significantly increased in Asian patients with stroke or TIA compared with non-carriers.⁴⁷ It has also been shown that in patients with coronary artery disease or stroke, clopidogrel users who carry the CYP2C19 LoF allele have a significantly higher risk of MACE than clopidogrel-treated individuals alone who do not carry a defect in this gene.⁴⁸

Fu et al⁴⁹ found that the proportion of clopidogrel-resistant individuals among carriers of one or more CYP2C19 loss-of-function alleles was estimated to be 71.7%, which was significantly different from 32.1% among non-carriers. Similarly, Jia et al⁵⁰ demonstrated a higher incidence of platelet hyperreactivity during clopidogrel treatment of ischemic stroke in CYP2C19 *2 or *3 carriers than in non-carriers.

Liu et al⁵¹ demonstrated that the risk of clopidogrel resistance was higher in carriers of the CYP2C19*2 allele than in non-carriers, but did not increase the risk of vascular events or recurrence rates.

A gain-of-function (GoF) allele, CYP2C19*17, was first described in 2006.⁵² This variant allele increases gene transcription, thereby increasing enzyme activity and clopidogrel responsiveness. Individuals carrying one or both *17 alleles have been described as ultra-rapid metabolizers (UM) based on the genotype and phenotype of CYP2C19.^41,42 A meta-analysis by Li, Y et al⁵³ showed that carriers of the CYP2C19*17 allele had better clinical outcomes with clopidogrel and a lower risk of cardiovascular events, but carriers of CYP2C19*17 had a higher risk of bleeding. In 2010, the US Food and Drug Administration (FDA) recommended that individuals should be considered for alternative antiplatelet agents or higher doses of clopidogrel depending on their CYP2C19 genotype or poor metabolism genotype.⁵⁴

CYP3A4 Gene Polymorphisms

The CYP3A4 enzymes plays a role in the metabolism of the clopidogrel prodrug molecule and is primarily responsible for the bioconversion of approximately 40% of 2-oxoclopidogrel (the inactive metabolite) to the active metabolite cis-thiol-clopidogrel, which is responsible for P2Y12 receptor inhibition.^55,56 The expression of the CYP3A4 enzyme is derived from the CYP3A4 gene, and the CYP3A4*1G mutation is thought to be a protective factor against clopidogrel resistance in ischemic stroke patients, as this mutation increases the concentration of the active clopidogrel-related substance, thereby increasing platelet inhibition. Liu⁵¹ showed that the estimated risk of clopidogrel resistance was significantly lower in CYP3A4*1G carriers than in non-carriers.

ABCB1 Gene Polymorphisms

The p-glycoprotein (P-gp) encoded by the ABCB1 gene regulates the absorption of clopidogrel in the small intestine.⁵⁷ P-gp is a transmembrane protein whose main function is to pump drugs out of cells and into the circulation, and this pumping mechanism may affect the bioavailability of the drug. Mega et al⁵⁸ found that ABCB1 gene polymorphisms affect the degree of platelet inhibition, which is strongly correlated with the risk of MACE. Simon et al⁵⁹ reported an association between adverse events in patients taking clopidogrel and the ABCB1 (C3435T, rs1045642) polymorphism, with carriers of the GG and AA genotypes showing a significantly increased incidence of adverse events. ABCB1 polymorphisms have been found to influence ADP-induced platelet aggregation, and carriers of the G allele are more prone to exhibit hyporesponsiveness to antiplatelet therapy. The ABCB1 C3435T polymorphism could be considered as a potential genetic biomarker for the risk of MACE in CAD patients on clopidogrel after receiving PCI. One study confirmed that patients carrying the ABCB1 C3435T double mutation (TT) had a significantly increased risk of MACE compared with patients carrying the CC genotype or the CC+CT genotype.⁶⁰

However, there are studies that suggest otherwise, Mugosa, S et al⁶¹ conducted a study related to ABCB1 gene polymorphisms in a population in Montenegro, Europe, but found no significant correlation between ABCB1 gene polymorphisms and clopidogrel efficacy and safety.

CYP1A2 Gene Polymorphisms

A Korean study found an enhanced response to clopidogrel in smokers, which has been referred to as the “smoker’s paradox”. However, this phenomenon is not universal and was only observed in carriers of the cytochrome P450 CYP1A2 allele, suggesting a genotype-dependent effect of smoking on clopidogrel response.⁶² Cresci et al⁶³ recruited 2732 patients diagnosed with myocardial infarction and taking clopidogrel and evaluated the correlation between long-term clinical efficacy and safety of clopidogrel in patients carrying the CYP1A2*1c allele. The results showed that patients carrying the CYP1A2*1c allele were significantly more responsive to clopidogrel, but they had a worse prognosis and a significantly higher mortality rate due to major bleeding events.

PON1 Gene Polymorphisms

The paraoxonase 1 (PON1) gene may play an important role in clopidogrel resistance. The PON1 gene is involved in high-density lipoprotein (HDL) antioxidant processes such as platelet-activating factor acetylhydrolase and lecithin-cholesterol acyltransferase, which has the ability to hydrolyse oxidised low-density lipoprotein (LDL) cholesterol and cleave phospholipid peroxidation adducts, leading to potential prevention of atherosclerosis. Several studies have shown that reduced PON1 activity affects serum glucose, increases the risk of diabetes and reduces platelet inhibition.^64,65 PON1 is also involved in the esterification and subsequent inactivation of clopidogrel, which is more likely to lead to clopidogrel resistance.⁶⁶

It has been shown that single nucleotide polymorphisms in the PON1 gene are associated with lower clopidogrel responsiveness in patients with atherosclerosis, and that the PON1 Q192R polymorphism is associated with clopidogrel biotransformation. However, the above conclusions have been challenged by a large number of studies that have failed to replicate these results, possibly due to epigenetic changes.^67,68 Mohitosh Biswas et al⁶⁹ in assessing the overall risk of MACE associated with harboring the PON1 Q192R gene variant in patients taking clopidogrel, found that the PON1 Q192R gene polymorphism did not have a significant effect on the risk of MACE or bleeding events in patients treated with clopidogrel.

KDR Gene Polymorphism

The KDR gene is responsible for the transcription of vascular endothelial growth factor receptor 2 (VEGFR2), which plays an important role in cardiovascular disease and platelet aggregation. Al Awaida, W. et al⁷⁰ found that the KDR (rs1870377) gene correlates with CR in cardiovascular disease (CVD) patients admitted for percutaneous coronary intervention as a potential genetic biomarker.

Table 2 Clopidogrel-Related Gene Polymorphisms and Their Clinical Significance

Clopidogrel-related gene polymorphisms and their clinical significance are shown in Table 2. A panel of experts assembled by the American College of Cardiology Foundation (ACCF) and the American Heart Association (AHA) has suggested that the current methodologies for genotyping are still a matter of considerable debate.⁷¹ The main advantage of genotypic testing is that it does not require measurement after drug administration.⁷² Proponents argue that there are common genetic polymorphisms that have been shown to affect platelet response to clopidogrel and its clinical efficacy in randomized clinical trials and registration experience, and that genotypic assessment is justified. In contrast, opponents argue that the available evidence does not demonstrate that CYP2C19 carriers can improve efficacy by adjusting clopidogrel dosage or using alternative antiplatelet agents. Furthermore, in clinical practice, genotype-guided antiplatelet therapy selection strategies do not yield immediate results, which severely limits the usefulness of these data in the acute care setting.⁵⁰ In addition, further studies have recommended treating patients based on their clinical indications rather than genetic testing.⁷³ In conclusion, there are limited examples of pharmacogenomic testing requirements or recommendations in detailed clinical practice guidelines (CPGs), and the presence of a standardized method for assessing the evidence for clinical application of pharmacogenomic testing could increase the recommendation of pharmacogenomic testing in CPGs to some extent. Consistent recommendations for pharmacogenomic testing in CPGs may enhance the clinical utilization of testing, provide more effective treatments, and benefit society.⁷⁴

Monitoring Methods

Platelet Function Test

The Platelet Function Test (PFT) provides a rapid assessment of platelet function and an estimate of the degree of platelet inhibition. Thus, the test is applicable for assessing clopidogrel-treated patients at elevated thrombotic risk to gauge antiplatelet therapy efficacy and residual platelet activity, guiding optimal medication and dosing selection.¹³ Wadhwa et al⁷⁵ evaluated the economic viability of platelet function testing (PFT) in dual antiplatelet therapy (DAPT). They recommended PFT, followed by a switch from clopidogrel to prasugrel in combination with aspirin if resistance is identified, citing the reasonable cost of PFT for tailoring DAPT regimens to individual patients. The primary PFTs for clopidogrel include the VerifyNow assay, light transmission aggregometry (LTA), thromboelastography (TEG) platelet labelling system, rotational thromboelastography (ROTEM) and vasodilator-associated stimulated phosphoprotein (VASP) assay.

VerifyNow Assay

The VerifyNow assay is a whole blood point-of-care test that measures P2Y12 receptor activity by turbidimetric optical detection of platelet aggregation. The test is rapid bedside assay, completed in less than 5 minutes, which is an advantage over LTA and VASP phosphorylation assays. The VerifyNow assay also allows direct monitoring the effect from clopidogrel on P2Y12 receptors, helping determine the dosage of clopidogrel in patients due to undergo coronary artery stenting. In addition, the analysis is technically simple and the results are easy to interpret.⁷⁶ The results are expressed in PRUs and the effective therapeutic window for clopidogrel is 85 < PRU < 208.⁷⁷

Light Transmission Aggregometry (LTA)

LTA is considered gold standard in all PFTs. In LTA, the optical density of platelet aggregates in a sample is detected in the optical channel, whereas in the impedance aggregometry method, the change in resistance between two electrodes is measured. Although LTA has been included in many studies, it has been criticized for being time-consuming and lacking standardization and reproducibility.^78,79 The results of LTA are expressed as the maximum platelet aggregation rate. A study has shown that a maximum platelet aggregation rate greater than 40% is the optimal LTA platelet function threshold for clopidogrel when it is used to treat intracranial aneurysm shunts and prevent thromboembolic events.⁸⁰

Thromboelastography (TEG) Platelet Labelling System and Rotational Thromboelastometry (ROTEM)

Thromboelastography combined with platelet mapping (TEG-PM) produces more complex data than PRU. The output of this test is four real-time curves of clot development as a function of time, showing clot initiation, expansion, and lysis. The values extracted from these curves represent multiple aspects of the coagulation cascade, with enzyme-promoted clot formation, intensity of clot formation, and rate of clot breakdown all being part of the underlying TEG curve. Under these conditions, platelet aggregation and the resulting clot intensity are determined exclusively by ADP-dependent platelet aggregation, mediated by the P2Y12 ADP receptor.⁸¹ The rate of ADP-induced platelet inhibition was calculated from TEG to determine the incidence of CR and the factors influencing it.⁸² Using the TEG-PM assay, the following reference values were formulated under the effective therapeutic window for clopidogrel, with amplitude (MA) values of 31 mm-47 mm; arachidonic acid (AA) greater than 50% and ADP greater than 30%. Individualized antiplatelet therapy based on TEG-PM results reduces the risk of ischaemic events in patients with non-cardioembolic ischaemic stroke without increasing the risk of bleeding events and mortality.⁸³

Conventional TEG techniques can overproduce thrombin and therefore their lack of ability to determine ADP receptor inhibition is considered a major problem associated with conventional TEG. However, ROTEM is able to monitor clopidogrel without thrombin production and Schultz-Lebahn, A et al⁸⁴ used ROTEM to analyse platelet function.

Vasodilator-Associated Stimulated Phosphoprotein (VASP) Assay

The most pharmacologically specific assay for assessing clopidogrel therapy is VASP, which targets only the P2Y12 receptor.^79,85 The VASP assay is based on a physical principle: quantitative flow cytometry measures levels of phosphorylated and dephosphorylated VASP, which correlate with inhibition or activation of P2Y12. However, we must consider that VASP phosphorylation is regulated by a number of factors, which are impaired in certain comorbidities. In addition, VASP assays are time-consuming.^86,87 Aleil et al⁸⁸ implemented the first VASP assay in 2005 for the detection of clopidogrel-resistant patients with ischaemic cardiovascular events. The effective therapeutic window for clopidogrel is formulated with a reference platelet reactivity index (PRI) of 16–50 and requires the study of scatter plots to interpret the conditions.⁷⁶

Therapeutic Drug Monitoring

High-performance liquid chromatography (HPLC) is usually used for the identification and quantification of compounds, and the determination of drug levels in patients’ plasma using HPLC becomes difficult due to the low levels of prodrugs in plasma after clopidogrel ingestion and the instability of sulfhydryl derivatives. Therefore, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been used to assay the blood of three patients with coronary artery disease who underwent stent implantation, which allowed the simultaneous determination of clopidogrel, 2-oxo-clopidogrel and clopidogrel thiol metabolites in human plasma.⁸⁹ In 2024, Li et al⁹⁰ studied 100 patients with ischemic cerebrovascular disease diagnosed by a neurologist as needing clopidogrel therapy, and all the patients were genotyped for CYP2C19, the plasma concentration and plasma clopidogrel clearance of different groups of patients before and after clopidogrel treatment were detected by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), the platelet aggregation rate of patients with different genotypes was determined by turbidimetric assay, and CR and stent thrombosis were analyzed in all the groups after 3 months of treatment.

Clinically, the genotypes of the patients were tested before administration, and after the end of the administration, the results of the PFT test were used to reflect the clinical results together with the plasma concentration determined by HPLC-MS/MS to provide a basis for judgement of the clinical administration of clopidogrel.⁹¹

Discussion

The clinical application of clopidogrel, as a major drug in antiplatelet therapy, requires a balance between optimization of efficacy and risk control. In this paper, we synthesized the available evidence that genetic polymorphisms and individual differences in drug metabolism are key factors contributing to CR and adverse events.The CYP2C19 LOF allele has been identified as a major genetic marker affecting the production of active metabolites of clopidogrel, and gene-directed therapy significantly reduces the risk of in-stent thrombosis and myocardial infarction, especially in patients with CAD.⁸⁸ However, the association of PON1 Q192R polymorphism with CR remains controversial, and a recent Meta-analysis showed that it had no significant effect on the risk of MACE and bleeding,⁶⁵ suggesting that future studies need to focus on epigenetic regulation and combined multigene effects.

In terms of clinical monitoring strategies, the combination of PFT and blood drug concentration provides a double guarantee for individualized treatment.VerifyNow and VASP assays can dynamically assess the level of P2Y12 receptor inhibition, whereas HPLC-MS/MS technology provides a direct basis for dosage adjustment by quantifying the concentration of active metabolites of clopidogrel.^71,72,86,87 Notably, the lack of harmonized standards for PFT thresholds and the limitations of VASP assays to be interfered by comorbidities need to be carefully considered in clinical interpretation. In addition, the applicability of genotype-guided immediate testing (eg, bedside CYP2C19 rapid typing) for patients with acute PCI still needs to break through technical bottlenecks.⁵⁰

The gene-efficacy relationship shows heterogeneity across the disease spectrum: switching to ticagrelor in CYP2C19 LOF carriers in stroke patients reduces the risk of recurrence, whereas the genetic evidence for peripheral arterial disease (PAD) is not yet sufficient, suggesting that clinical decision-making needs to be combined with disease-specific evidence.⁸⁸ Notably, while novel P2Y12 inhibitors circumvent some of the metabolic defects, their bleeding risk (eg, prasugrel short-term bleeding HR=1.66)⁴ limits the advantages of their use in older or high-risk bleeding populations, highlighting the unique position of clopidogrel in balancing thrombotic/bleeding risk.

Conclusion

Recent studies have shown that platelet aggregation plays a key role in both the initiation and progression of thrombosis. Despite the emergence of new antiplatelet agents, clopidogrel remains the basic drug in current clinical practice due to its favorable safety profile, including a low risk of bleeding and high tolerability. However, the individual variability of this drug is of concern-gene polymorphisms leading to clopidogrel resistance can increase the risk of thrombotic events, suggesting the importance of precise dosing. In CAD, secondary prevention of ischemic stroke, gene-directed therapeutic strategies have shown significant benefits, with carriers of the CYP2C19 LOF allele switching to ticagrelor or prasugrel experiencing a lower incidence of major ischemic events (myocardial infarction, in-stent thrombosis, etc.) compared with conventional regimens, while non-carriers continue to use clopidogrel to not only achieve comparable thrombotic efficacy to that achieved with the newer P2Y12 inhibitors but also significantly reduce the risk of major hemorrhage and significantly reduced the risk of major bleeding. Of particular note, in areas with limited medical resources, targeted genetic testing may be more cost-effective than “empirically reinforced antithrombotic” regimens. Clinical assessment of clopidogrel efficacy is currently based on the determination of optimal thresholds for platelet inhibition by light turbidimetric assay (LTA), platelet function assays such as VerifyNow, and in vivo measurement of active metabolite concentrations by HPLC-MS/MS. However, the standardization of these methods still faces challenges, with a lack of uniform consensus on critical values for functional assays and effective concentration thresholds for pharmacokinetic monitoring still requiring large-scale cohort validation.

In summary, this review aims to help achieve individualized and precise drug administration, reduce or avoid the occurrence of clinical adverse events, reduce the severity of the corresponding symptoms, and ensure that patients’ treatment progresses smoothly, as well as to improve the quality of life and confidence of patients during the treatment period. Therefore, it has significant social and economic benefits.

Abbreviations

ADP, adenosine diphosphate; CR, clopidogrel resistance; GPIIb/IIIa, glycoprotein IIb/IIIa; CAD, Coronary artery disease; PCI, percutaneous coronary intervention; CABG, coronary artery bypass grafting; ACS, acute coronary syndrome; TIA, transient ischaemic attack; CEA, carotid endarterectomy; BRVO, branch retinal vein occlusion; CRVO, central retinal vein occlusion; FFA, fundus fluorescein angiography; FAERS, the US Food and Drug Administration Adverse Event Reporting System; CKD, chronic kidney disease; PRUs, P2Y12 reaction units; IAS, insulin autoimmune syndrome; HPR, high platelet reactivity; LOF, loss-of-function; GOF, gain-of-function; UM, ultra-rapid metabolizers; FDA, the US Food and Drug Administration; P-gp, p-glycoprotein; MACE, major adverse cardiovascular event; HDL, high-density lipoprotein; LDL, low-density lipoprotein; CVD, cardiovascular disease; CPGs, clinical practice guidelines; PFT, platelet function test; DAPT, dual antiplatelet therapy; LTA, light transmission aggregometry; TEG, thromboelastography; ROTEM, rotational thromboelastography; VASP, vasodilator-associated stimulated phosphoprotein; AA, arachidonic acid; HPLC, High-performance liquid chromatography; LC-MS/MS, liquid chromatography-tandem mass spectrometry; HPLC-MS/MS, high-performance liquid chromatography-tandem mass spectrometry; CYP2C19, cytochrome P450 Family 2 Subfamily C Member 19 gene; CYP3A4, cytochrome P450 Family 3 Subfamily A Member 4 gene; ABCB1, ATP Binding CassetteSubfamily B Member 1 gene; CYP1A2, cytochrome P450 Family 1 Subfamily A Member 2 gene; PON1, paraoxonase 1; KDR, Kinase Insert Domain Receptor.

Acknowledgments

This work was supported by Wu Jieping Medical Foundation (320.6750.2023-26-3).

Disclosure

The authors report no conflicts of interest in this work.

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