Enterovirus D68 pneumonia in two older patients: A case report and literature review

Abstract

Enterovirus D68 mainly causes respiratory tract infections, with children being the main infected population and most adults experiencing latent infections. This report presents the cases of two older patients with enterovirus D68 pneumonia. Both of them were diagnosed using next-generation sequencing, and one of them died of mediastinal emphysema despite undergoing various treatments. This report also reviewed the biological characteristics and epidemiological features of enterovirus D68 as well as the clinical diagnosis and treatment of the viral pneumonia. Currently, there are no specific antiviral drugs or vaccines, and the treatment mainly focuses on symptomatic support and anti-inflammatory therapy. Although there exists certain understanding of enterovirus D68 pneumonia, there are challenges in aspects such as a deeper understanding of the viral mechanism, development of drugs and vaccines, and optimization of diagnosis and treatment methods. Strengthening monitoring and health education is crucial for preventing and controlling the spread of this infection.

Introduction

Enterovirus D68 (EV-D68) is a member of the enterovirus family and is classified as species D. In contrast to other enteroviruses commonly linked to systemic diseases such as hand–foot–mouth disease, EV-D68 is predominantly associated with respiratory tract infections.^1,2 The clinical manifestations of EV-D68 infections are primarily similar to those of influenza-like illness. However, there are reports of life-threatening infection cases. ³ Since the virus was first isolated from throat swab samples of children with bronchiolitis and pneumonia in California in 1962, there have been only a few reports of EV-D68 infections in the subsequent decades. ⁴ Recently, an increasing number of EV-D68 infection reports have emerged from multiple countries across Africa, America, Asia, and Europe. ⁴ Since August 2014, the United States has reported >1000 cases of EV-D68 infection, with at least 14 potentially fatal cases. ⁵ The general population is vulnerable to EV-D68; however, children are the main affected group, with most adults experiencing latent infections. ⁴ In the initial outbreak regions, outbreaks were also observed among adults.

A study conducted in Hong Kong analyzed a total of 10,695 nasopharyngeal aspirate (NPA) samples from hospitalized pediatric patients between September 2014 and December 2015. EV-D68 was detected in 15 (1.70%) samples from patients presenting with clinical symptoms ranging from wheezing to pneumonia, and these isolates belonged to subclade B3. ⁶ Another study explored the epidemic situation of EV-D68 infections in pediatric patients hospitalized due to community-acquired pneumonia in Shanghai, China, from 2013 to 2020. The results indicated that 9 (0.22%) of the 3997 nasopharyngeal swab or bronchoalveolar lavage fluid (BALF) samples tested positive for EV-D68. ⁷

Herein, we report two cases of older pneumonia patients infected with EV-D68 who were admitted to the Hangzhou First People’s Hospital in Hangzhou, Zhejiang Province, China, in November 2024. Additionally, we reviewed the biological characteristics and epidemiological features of the virus as well as the clinical diagnosis and treatment of the viral pneumonia. Written informed consent for special tests and treatments was obtained from the patients. The reporting of this study conforms to the Case Report (CARE) guidelines. ⁸ We have de-identified all patient details, and written informed consent for publication was obtained from the patients or their legally authorized person.

Case presentation

Case 1

An older man in his late 80s presented with a 1-week history of cough accompanied with chest pain. The patient developed a dry cough without obvious inducement and without expectoration or fever. He experienced pain in the front chest while coughing as well as chest tightness and dyspnea after mild physical activity. Oral administration of levofloxacin tablets was ineffective.

The patient had a history of atrial fibrillation for 18 years and had been taking warfarin tablets orally for a long time. He also had a 10-year history of primary hypertension and had been taking antihypertensive drugs for a long time. Moreover, he had a 30-year history of benign prostatic hyperplasia.

On physical examination, the patient was conscious but enervated. His body temperature was normal. The trachea was in the midline. The pharynx was congested. He had tachypnea. The breath sounds of both lungs were coarse, and a few moist rales could be heard. The heart rhythm was regular, and no obvious murmur was heard. The abdomen was soft without tenderness. There was mild edema of the lower extremities.

Blood routine on admission showed a white blood cell count of 10.0 × 10⁹/L (3.5–9.5 × 10⁹/L), neutrophil count of 7.5 × 10⁹/L (1.8–6.3 × 10⁹/L), brain natriuretic peptide (BNP) level of 375 (0–100) pg/mL, D-dimer level of 910 (0–550) μg/L, albumin level of 29.1 (40–55) g/L, and potassium level of 3.07 (3.50–5.30) mmol/L. The oxygenation index was 280. Immunoglobulin G (IgG) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was positive, while IgM was negative. The ferritin level was 715.4 (15–200) μg/L, and the interleukin-6 (IL-6) level was 27.6 (0–20) pg/mL.

High-resolution computed tomography (HRCT) of the chest showed multiple patchy shadows in both lungs, which were more obvious on the right side. There was mild enlargement of mediastinal lymph nodes, and the heart was enlarged. There was a small amount of pleural effusion on both sides (Figure 1(a1) to (a3)).

Figure 1.

Chest X-ray and HRCT images of case 1. (a1–a3) images obtained upon admission to the hospital. (b1–b3) images obtained 3 days after admission, with the shadows in both lungs significantly increased compared with those observed previously (red arrows). (c1–c3) images on day 11 after admission (day 8 after anti-inflammatory and antiviral treatment), with the shadows in both lungs significantly absorbed and reduced. However, pneumothorax, mediastinal and subcutaneous emphysema occurred. HRCT: high-resolution computed tomography.

The initial diagnosis was community-acquired pneumonia and cardiac insufficiency. After admission, oxygen inhalation was given, and levofloxacin injection was administered empirically. Three days later, the patient’s dyspnea worsened. Repeat chest HRCT showed that the patchy exudative shadows in both lungs increased compared with those observed previously (Figure 1(b1) to (b3)). Then, a bronchoscopy was performed. Congestion of the mucous membranes of both bronchi was observed, and no neoplasm or obvious secretion was noted. The nucleated cell count in the BALF was 495 cells/μL, among which neutrophils accounted for 46%, lymphocytes accounted for 31%, eosinophils accounted for 15%, and alveolar macrophages accounted for 8%. Among the lymphocytes, the CD3+ T lymphocytes accounted for 97.7%, CD8+ T lymphocytes accounted for 75.78%, and CD4+ T lymphocytes accounted for 22.18%; moreover, the CD4+/CD8+ T lymphocyte ratio was 0.29. The day after the bronchoscopy, the patient’s dyspnea further worsened, and the oxygenation index dropped to 90. The next day, next-generation sequencing (NGS) detected enterovirus D68 in the BALF sample.

The diagnosis was revised as viral pneumonia, enterovirus D68 infection, type 1 respiratory failure, and cardiac insufficiency. The treatment plan was adjusted as follows: high-flow nasal cannula oxygen supplement was administered, levofloxacin injection was discontinued, methylprednisolone injection (40 mg) was administered intravenously twice a day, and the monoclonal antibody tocilizumab (antagonist of the IL-6 receptor) (400 mg) was administered intravenously once a day, with a lower dose (200 mg) administered intravenously once the next day. Oral fluoxetine (a selective serotonin reuptake inhibitor) (20 mg daily) was used for antiviral treatment. The compound sulfamethoxazole was administered orally once a day to prevent Pneumocystis carinii infection. Oral furosemide (20 mg) and spironolactone tablets (20 mg) were used for diuresis daily.

On the third day after adjusting the treatment, compared with previous findings, the dyspnea was relieved compared, the oxygenation index gradually increased, and ferritin and IL-6 levels decreased. Eight days later, chest HRCT showed a small amount of pneumothorax on the left side accompanied with mediastinal and subcutaneous emphysema on the left side of the neck. The exudation in the upper lobes of both lungs was significantly absorbed, and the exudative shadow in the lower left lobe increased slightly (Figure 1(c1) to (c3)). The methylprednisolone injection (40 mg) was changed to once a day intravenously, and the oxygen concentration of the inhaled gas was gradually decreased. One week later, it was changed to nasal catheter oxygen inhalation. Unfortunately, the patient eventually died of the deteriorating mediastinal emphysema and pneumothorax.

Case 2

A woman in her late 70s presented with a 4-day history of fever accompanied with cough without obvious inducement. The highest body temperature reached 39°C. Physical examination showed that the patient was conscious but enervated. There was no rash all over the body. The trachea was in the midline. The breath sounds of both lungs were coarse, and no obvious rales were heard. The heart rhythm was regular, and no murmur was heard. The abdomen was soft without tenderness. There was no edema of the lower extremities, and the limbs moved freely.

Blood routine on admission showed that the white blood cell count was 8.8 × 10⁹/L, with the proportion and count of neutrophils being 79.6% and 7.0 × 10⁹/L, respectively. The proportion and count of lymphocytes were 10% and 0.9 × 10⁹/L, respectively. The proportion and count of eosinophils were 1.5% and 0.13 × 10⁹/L, respectively. The C-reactive protein level was 134.9 (0–10) mg/L. The BNP level was 375 pg/mL, D-dimer level was 190 μg/L, alanine aminotransferase level was 15 U/L, lactate dehydrogenase (LDH) level was 196 (0–40) U/L, creatinine level was 52 (20–80) μmol/L, albumin level was 35.7 g/L, and potassium level was 3.8 mmol/L. The oxygenation index (P/F) was 280. IgG against SARS-CoV-2 was positive, whereas IgM was negative. The ferritin level was 412.4 (15–200) μg/L. The procalcitonin level was 0.08 ng/mL, and the IL-6 level was 25.9 pg/mL. Nucleic acid testing of the novel SARS-CoV-2 was negative. The antigens of both influenza A and influenza B viruses were negative. Chest HRCT showed multiple scattered patchy shadows in both lungs (Figure 2(a1) to (a3)).

Figure 2.

Chest X-ray and HRCT images of case 2. (a1–a3) images obtained upon admission to the hospital. (b1–b3) images obtained 8 days after admission, with the shadows in both lungs significantly increased (red arrows). (c1–c3) images obtained 2 weeks after the use of methylprednisolone, with the shadows in both lungs significantly absorbed and reduced. HRCT: high-resolution computed tomography.

The initial diagnosis was community-acquired pneumonia and type 1 respiratory failure. Levofloxacin tablets were administered orally empirically, and antipyretic treatment was provided. The patient gradually developed symptoms such as perioral numbness, dizziness, diarrhea, nausea, and vomiting. Therefore, levofloxacin tablets were discontinued, and omadacycline injection was administered intravenously for anti-infection treatment.

Subsequent laboratory reports showed that IgM against influenza B virus in the blood was weakly positive. IgM against influenza A virus, Mycoplasma pneumoniae, Chlamydia pneumoniae, adenovirus, respiratory syncytial virus, Legionella pneumophila serogroup 1, and parainfluenza virus types 1, 2, and 3 were all negative. Repeat chest HRCT showed that the multiple patchy shadows in both lungs were significantly aggravated compared with that at admission, and it was more obvious on the right side (Figure 2(b1) to (b3)).

The diagnosis was revised as viral pneumonia, possibly influenza B virus infection, and respiratory failure type 1. Oseltamivir was administered for antiviral treatment, and methylprednisolone injection (40 mg) was administered intravenously once a day.

Bronchoscopy showed no obvious abnormalities in both bronchi. Bronchoalveolar lavage was performed on the upper lobe of the right lung. The BALF was colorless and slightly turbid, and the nucleated cell count was 612 cells/μL. Of these, neutrophils accounted for 20%, lymphocytes for 10%, and eosinophils for 63%. he NGS showed enterovirus D68 positivity.

The diagnosis was revised as viral pneumonia and enterovirus D68 infection. The antibiotic was discontinued, and methylprednisolone injection (20 mg daily) was continued for anti-inflammation therapy. Two weeks later, follow-up chest HRCT showed that the inflammatory exudative shadows in both lungs were significantly absorbed (Figure 2(c1) to (c3)). The second arterial blood gas analysis showed that the respiratory failure had been corrected. The patient was discharged.

Discussion and review

In this research, the diagnosis of two patients with EV-D68-associated viral pneumonia was conclusively established through the detection of EV-D68 in their BALF samples through NGS.

EV-D68 virus has been recategorized under the Enterovirus genus within the Picornaviridae family since 1976 after initially being misclassified as Rhinovirus type 87 due to its resemblance in causing respiratory tract infections.^9,10 The virus exhibits a distinct morphological structure. Its minuscule viral particles, featuring a typical icosahedral symmetry with a diameter ranging from 24 to 30 nm, are readily transmitted via airborne droplets and intensive contact.^3,4 EV-D68, which lacks an envelope, demonstrates robust resistance to lipid solvents such as ether and chloroform, ensuring its stability in diverse environmental conditions.

Genomically, EV-D68 harbors a positive-sense single-stranded RNA genome with a length of approximately 7.4–7.5 kb. This genome encodes a polyprotein that is cleaved into multiple mature proteins, including structural proteins (VP1, VP2, VP3, and VP4) and nonstructural proteins (2A, 2B, 2C, 3A, 3B, 3C, and 3D) in cells infected with EV-68. ¹ These nonstructural proteins are involved in processes such as viral replication, transcription, translation, and interaction with host cells.

The sources of EV-D68 infection encompass patients, asymptomatic carriers, and healthy virus shedders. Unlike the majority of enteroviruses, EV-D68 is predominantly transmitted through the respiratory tract and intensive contact.^3,4 This mode of transmission renders it more likely to spread in densely populated and poorly ventilated areas such as schools, kindergartens, and hospitals. ⁴ In this report, neither of the two patients has a definite and known source of infection. It is speculated that their source of infection may be asymptomatic infectors.

EV-D68 infections are more common in late summer, early autumn, and winter, although sporadic cases also occur in other seasons. ³ However, differences are observed across various regions due to factors such as climate and population activity patterns. ⁴ This indicates that the virus’s transmission season is affected by a combination of multiple local environmental, virus mutation, immune status of the population, and social factors.

The two cases reported herein occurred during the transition period between autumn and winter, which is exactly the season when respiratory virus infections are prevalent. In terms of population susceptibility, children are more inclined to develop symptomatic infections, while adults frequently experience asymptomatic infections. Infants, children, and adolescents with pre-existing respiratory conditions, such as asthma, are at particularly elevated risk of severe illness. ⁴ The two patients reported herein were both older individuals, which may be related to the decline in immunity of the older population.

EV-D68 primarily targets the respiratory system, causing bronchiolitis, pneumonia, and even respiratory failure in severe cases. EV-D68 infections are associated with a higher morbidity rate and a greater likelihood of necessitating intensive care than non-EV-D68 and human rhinovirus infections. In recent years, an association between EV-D68 and severe neurological complications, such as acute flaccid myelitis, has been identified, further accentuating its uniqueness within the Enterovirus genus. ¹¹

The clinical manifestations of adult patients with EV-D68 viral pneumonia vary substantially. In mild cases, patients typically present with low- or moderate-grade fever lasting 2–3 days, accompanied with chills, dry cough with scant white mucoid sputum, sore throat, muscle ache, fatigue, and upper respiratory tract symptoms resembling those of the common cold. Physical examination may reveal pharyngeal congestion, lymphoid follicle hyperplasia on the posterior pharyngeal wall, tonsillar enlargement, and a few scattered dry or wet rales in the lungs.

In severe cases, high fever persists for 5–7 days or longer. Dyspnea is a key symptom, with respiratory rates reaching 30–40 breaths per minute. The cough is more intense, with increased sputum production. Lung auscultation reveals extensive dry and wet rales. Systemic symptoms and complications may also manifest, including gastrointestinal symptoms such as nausea, vomiting, abdominal pain, and diarrhea as well as neurological manifestations such as altered mental status, somnolence, coma, and delirium due to cerebral hypoxia and hypercapnia.

Imaging studies such as chest X-ray or CT are indispensable. Chest X-ray may reveal increased and thickened lung markings in the early stage; as the disease progresses, patchy infiltrates or large-scale consolidation may be observed. Chest CT can show ground-glass opacities, interlobular septal thickening, and areas of consolidation, providing more precise information for early diagnosis and disease assessment.

Diagnosis mainly relies on laboratory testing. For example, real-time reverse transcription polymerase chain reaction can be used to detect the virus in nasopharyngeal aspirates. A novel technology, droplet digital PCR assay (ddPCR), has been developed. ¹² ddPCR directly quantifies the absolute number of copies within a given sample, which in turn accurately quantifies viral loads in tissues and body fluids such as plasma and lung or nasal aspirates. In addition, NGS technology can be used for accurate virus detection and mutation analysis. In the two older patients reported in this study, enterovirus D68 infection was confirmed via NGS of the BALF samples. Meanwhile, by combining the patients’ clinical symptoms, physical signs, and chest imaging findings, they were finally diagnosed with EV-D68-related viral pneumonia. In case 2 reported in this study, the patient’s serum anti-influenza B virus IgM was weakly positive, but the nucleic acid tests of pharyngeal swabs and BALF samples were both negative, which does not support the diagnosis of influenza B virus infection. In the clinical diagnosis of adult EV-D68 viral pneumonia, a multimodal diagnostic system, which amalgamates clinical symptoms (fever, cough, and dyspnea), physical signs (lung rales), imaging findings, and laboratory results (virus nucleic acid and serological antibody tests) should be comprehensively applied to improve the accuracy and timeliness of diagnosis.

In the BALF of the two patients reported in this study, the number of neutrophils was slightly higher than that of lymphocytes, and the number of eosinophils increased significantly. However, the number of eosinophils in the peripheral blood did not increase, and the vasculitis-related antibodies and IgE were all within the normal range. It is speculated that the increase in eosinophils in BALF may be a reactive change due to virus infection. The levels of IL-6 and ferritin in the serum were both elevated, consistent with the degree of lung inflammation, and decreased after anti-inflammatory treatment. This is consistent with the findings reported in a previous study. ¹³

Currently, there are no effective antiviral drugs or preventive vaccines against EV-D68 in humans. Treatment mainly focuses on symptomatic and supportive treatment, such as oxygen supplementation and the use of antibiotics, to prevent secondary infections, although antibiotics are ineffective against the virus itself. The cornerstone of the treatment for severe viral pneumonia is anti-inflammatory therapy. Referring to the treatment experience of coronavirus disease 2019, anti-inflammatory treatment may require the use of glucocorticoids, IL-6 receptor antagonists, and JAK inhibitors. In case 1, we used a combination of glucocorticoids, IL-6 receptor antagonists, and JAK inhibitors for anti-inflammatory treatment. For case 2, which showed a relatively mild condition, the pneumonia rapidly and significantly subsided with the use of only 40 mg of glucocorticoids per day.

In vitro studies have reported that fluoxetine can inhibit the proliferation of enterovirus D68. ¹⁴ However, its antiviral effect was not reported in the clinical study of acute myelitis caused by this virus. In case 1 of this study, we attempted to administer fluoxetine for antiviral treatment. Although the patient’s viral pneumonia improved significantly, its efficacy must be confirmed by a randomized controlled trial because the current study involved a single noncontrolled case.

Recent studies have identified MFSD6 as a key receptor for EV-D68 infection and developed an MFSD6-Fc (CH3) recombinant protein in 2025.^15,16 In vitro and in vivo neonatal mouse experiments demonstrated its inhibitory effect on virus infection. Additionally, a monoclonal antibody, EV-68-228-N, developed from recovered patients’ blood, is being evaluated in a phase 1 NIH-sponsored clinical trial at Vanderbilt University Medical Center. In laboratory models, the recombinant protein neutralizes multiple EV-D68 strains, and the trial aims to assess its safety, persistence, and optimal dose. ¹⁷ Vesatolimod (GS-9620), a Toll-like receptor 7 agonist, significantly inhibited EV-D68 replication without inducing significant cytotoxicity at virucidal concentrations. ¹⁸ In an animal model, treatment with guanidine led to a significant reduction in lung virus titers, reduced monocyte chemotactic protein-1 (MCP-1) and IL-6 levels, and prevented histological lesions in the lungs. ¹⁹ In 2024, a self-replicating RNA vaccine encoding the EV-D68 antigen was developed. This vaccine can induce a robust neutralizing antibody response in mice and nonhuman primates and prevent upper and lower respiratory tract infections of EV-D68 and neurological diseases in mice.^20,21

Researchers from Shanghai Pasteur Institute prepared two monoclonal antibodies, 2H12 and 8F12. The combination of these two monoclonal antibodies shows a balanced and highly effective neutralizing effect. It can confer broader protection in mice compared with a single monoclonal antibody and is still effective when administered 3 days after infection, which has important clinical transformation value. ²²

Supportive therapy for EV-D68 pneumonia has been refined. For dyspneic patients, appropriate respiratory support (nasal cannula, mask, and noninvasive or invasive ventilation) is selected based on severity, with continuous monitoring and parameter adjustment. Personalized nutritional plans, guided by biomarker monitoring (pre-albumin and transferrin), aim to enhance the body’s resistance. A recent clinical study proposed a comprehensive supportive care protocol including respiratory rehabilitation for patients with muscle weakness.

The prognosis of viral pneumonia caused by enterovirus D68 is usually good, and the mortality rate is low. Unfortunately, the patient in case 1 eventually died of mediastinal emphysema.

In conclusion, we reported two cases of EV-D68 pneumonia in adults, which are relatively rare. Detailed descriptions of the conditions can help improve clinicians’ understanding of this disease. As this case report is a retrospective study, some inflammatory indicators were not tested or rechecked in a timely manner. Additionally, due to the small number of cases, it is difficult to discover the patterns.

Although significant progress has been achieved in understanding EV-D68-associated pneumonia, numerous challenges persist. The development of effective antiviral drugs and vaccines remains a top priority. Further research is warranted to optimize diagnostic methods and treatment strategies and gain a deeper understanding of the virus’s pathogenesis and transmission mechanisms. Strengthening surveillance and public health education are also pivotal for preventing the spread of EV-D68.