Impact of Ascaris lumbricoides infection on the development of chronic pulmonary aspergillosis in patients with COPD

Abstract

The aetiopathogenesis of chronic obstructive pulmonary disease (COPD) remains unclear. The aim of our study was to determine the possible influence of Ascaris lumbricoides on the development of chronic pulmonary aspergillosis (CPA) in patients with COPD. The prevalence of A. lumbricoides in patients with COPD with CPA (19.05%) was significantly higher than that in those without (9.20%) and controls (4.9%) (p < 0.05). Trends in levels of Interleukin-1β and of tumour necrosis factor α suggest ascariasis increases susceptibility to Aspergillus sp. in patients with COPD and can be considered an additional risk factor for CPA.

Keywords

COPD chronic pulmonary aspergillosis cytokines

Introduction

Chronic obstructive pulmonary disease (COPD) is the most frequent non-communicable incurable lung disease with a high mortality rate, mainly in low-income countries.¹ Most patients with COPD receive broad-spectrum antimicrobials and corticosteroids, and have high susceptibility to pulmonary aspergillosis.^2,3 Chronic pulmonary aspergillosis (CPA) is often misdiagnosed, especially among patients with COPD. Early diagnosis and identification of factors predisposing to pulmonary aspergillosis are therefore critical to reduce mortality from fungal infections.⁴

Ascaris spp. induce immunological and structural changes in the lungs during larval migration owing to the switch to Th2 from Th1 response.^5–8 Fluid antigens of adult Ascaris spp. inhibit cytokine secretion and co-stimulatory molecule expression in classically activated but not alternatively activated macrophages.⁹ So Ascaris spp. may inhibit the Th1 response at various stages of the disease, providing a long-term increased susceptibility to concomitant infections. During severe infection with Ascaris spp., chronic emphysema-like lung disease may ensue, similar to COPD.⁸ Data on the long-term effects of Ascaris lumbricoides on lung function in humans are scarce and unclear.

Regulatory T cells control the balance between Th1, Th2, and Th17 responses, which are essential for the clearance of fungal infections from the host. The Th1 response plays a protective role in pulmonary aspergillosis, whereas Th2 reactions are associated with a higher fungal burden.¹⁰ Therefore, we hypothesize that co-infection with A. lumbricoides activating the Th2 response and frequent corticosteroid use in patients with COPD may be considered additional risk factors for development of pulmonary aspergillosis. Moreover, Uzbekistan is known among the regions endemic for ascariasis.¹¹

Materials and methods

Patients with COPD in the acute exacerbation phase were admitted to the Department of Respiratory Medicine of the Republican Specialized Research and Practical Medical Center of Tuberculosis and Pulmonology between January 2015 and July 2023. COPD was diagnosed on the basis of clinical examination, spirometry, chest radiography, and laboratory data according to the Global Initiative for Chronic Obstructive Lung Disease criteria.¹² Inclusion criteria of patients with COPD were age >30 years, exacerbation of the III and IV COPD stage, prolonged or frequent use of corticosteroids and antibiotics. Patients with acute and chronic infections (except CPA and ascariasis), oncological and autoimmune diseases were excluded from the study. Finally, 300 patients with COPD met the required inclusion and exclusion criteria. CPA in patients with COPD was diagnosed according to the guidelines established by the European Society for Clinical Microbiology and Infectious Diseases/European Respiratory Society and Infectious Disease Society of America.¹³ Determined by the results of a health check-up, 61 healthy subjects were included in the study as a control group.

Our study complied with the Declaration of Helsinki Ethical Principles for Medical Research Involving Human Subjects. All patients with COPD and control subjects were provided written informed consent to participate in the study. The trial was registered at the US National Institutes of Health (ClinicalTrials.gov #NCT 05783544).

Parasitological diagnosis included A. lumbricoides detection in patients with COPD and control subjects. Three stool samples for parasitological analysis were taken from all participants at 2-day intervals. Stool samples were collected in individual containers with 5 mL of Turdiev's preservative,¹⁴ ensuring and preserving worm eggs for staining and conservation for a year. Triple coproscopy was performed by the formalin-ethyl acetate concentration technique and iodine-stained smears.

On the first day of admission, before any drugs had been administered, 5 mL of whole blood was taken from each subject. Serum was separated for immediate testing or stored at −20 °C for later testing. Commercial ELISA kits (Vector-Best, Novosibirsk, RF) were used to detect Ascaris IgG, Aspergillus IgG, Interleukin (IL) 1β, IL-4, IL-6, tumour necrosis factor (TNF) α and IFN-γ. Enzyme-linked immunosorbent assay (ELISA) was performed according to the manufacturer's instructions.

Data were expressed as the mean ± standard deviation (SD) for continuous variables, whereas categorical variables are summarized as percentages. Categorical data were analyzed by odds ratios (OR) with 95% confidence intervals (CI) of the mean. Results were analyzed by analysis of variance and the Tukey–Kramer's multiple comparisons test for the differences among the groups. A value of p < 0.05 was considered statistically significant. Origin 8 software (OriginLab, Northampton, MA) was applied for the statistical analysis of the data.

Results

A total of 437 participants were evaluated for eligibility, of whom 76 did not meet one or more inclusion criteria, so 300 patients with COPD and 61 healthy subjects were included in the study. No significant differences in age and body mass index were observed among the groups. The patients with COPD demonstrated male predominance (79%). Most patients were ex-smokers (64%), with few being nonsmokers (28%) and the remainder current smokers (8%). Patients with COPD included used inhaled (58%), oral or intravenous (42%) corticosteroids during the acute exacerbation of the disease. Cultures were positive in 42 such patients, and Aspergillus fumigatus was the most frequently isolated species, followed by Aspergillus niger and Aspergillus flavus. Although 72 patients with COPD were Aspergillus IgG positive, only 63 met the diagnostic criteria for CPA (Table 1).

Table 1.

Baseline characteristics of participants of the study.

Characteristics	Patients with COPD (n = 300)	Control subjects (n = 61)
Age, years mean ± SD	64.9 ± 9.8	59.7 ± 11.2
Gender, n (%)
Male/female	237 (79%)/63 (21%)	40 (66%)/21 (34%)
Body mass index, kg/m²	25.6 ± 4.1	27.1 ± 3.0
Smoking status, %
Never smokers	84 (28%)	36 (59%)
Ex-smokers (at least 1 year)	192 (64%)	20 (32%)
Current smoking	24 (7%)	5 (8%)
Duration of the disease, years ± SD	14.7 ± 5.1	–
COPD GOLD stages, n (%)
Stage III	129 (43%)	–
Stage IV	171 (57%)	–
Corticosteroids use, n (%)
Inhale corticosteroids	174 (58%)	–
Oral or intravenous corticosteroids	126 (42%)	–
Positive Aspergillus culture, n (%)
Aspergillus fumigatus	28 (9.33%)	–
Aspergillus niger	12 (4%)	–
Aspergillus flavus	2 (0.67%)	–
Aspergillus IgG positive, n (%)	72 (24%)	–
Diagnostic criteria CPA, n (%)
Clinical + radiographic + serological	21 (7%)	–
Clinical + radiographic + serological + microbiology	42 (14%)	–

COPD: chronic obstructive pulmonary disease; CPA: chronic pulmonary aspergillosis; GOLD: the Global Initiative for Chronic Obstructive Lung Disease criteria; SD: standard deviation.

To assess the influence of concomitant diseases on the course of COPD, patients were divided into the following groups: patients with COPD without comorbidities (n = 214), patients with COPD with CPA (n = 51), patients with COPD with ascariasis (n = 23), and patients with COPD with CPA and ascariasis (n = 12).

The prevalence of A. lumbricoides in patients with COPD without CPA (9.70%) was higher than in controls (4.9%) (OR: 2.07; 95% CI: 0.60 to 7.10; p > 0.1), but a significantly high percentage was in patients with COPD with CPA (19.05%) (OR: 4.54; 95% CI: 1.21 to 17.02; p < 0.05). The prevalence of A. lumbricoides in patients with COPD and control subjects is shown in Figure 1.

Figure 1.

The prevalence of Ascaris lumbricoides in patients with COPD and control subjects. The data show the percentage of A. lumbricoides in patients with COPD without CPA (n = 237), patients with COPD with CPA (n = 63), and control subjects (n = 61). *Compared with the control subjects (p < 0.05); ‡ compared with the patients with COPD. COPD: chronic obstructive pulmonary disease; CPA: chronic pulmonary aspergillosis.

As shown in Figure 2, the level of IL-1β in patients with COPD with ascariasis (24.3 ± 11.6 pg/mL) was significantly higher than that in patients with COPD with (11.5 ± 3.1 pg/mL; p < 001) and without CPA (5.6 ± 8.7 pg/mL; p < 001) and control subjects (1.9 ± 2.0 pg/mL; p < 001). The highest level of IL-1β was found in patients with COPD with concomitant CPA and ascariasis (29.6 ± 26.6 pg/mL).

Figure 2.

The level of serum IL-1β in patients with COPD and controls. The figure presents the level of IL-1β in patients with COPD without comorbidities (n = 214), patients with COPD with CPA (n = 51), patients with COPD with ascariasis (n = 23), patients with COPD with CPA and ascariasis (n = 12), and control subjects (n = 61). *Compared with the control subjects (p < 0.005); # compared with the patients with COPD (p < 0.0005); ‡ compared with the patients with COPD with ascariasis (p < 0.005). COPD: chronic obstructive pulmonary disease; CPA: chronic pulmonary aspergillosis; IL: interleukin.

Figure 3 demonstrates that the level of IL-4 was significantly higher in all COPD groups than that in controls (0.46 ± 0.80 pg/mL; p < 0.01). The highest level of IL-4 was found in patients with COPD with ascariasis (3.3 ± 0.9 pg/mL) and patients with COPD with concomitant CPA and ascariasis (2.9 ± 0.9 pg/mL). There was a 1.8-fold lower level of IL-4 in patients with COPD with CPA (1.8 ± 1.4 pg/mL) than in patients with COPD with ascariasis (3.4 ± 0.9 pg/mL).

Figure 3.

The level of serum IL-4 in patients with COPD and controls. The figure presents the level of IL-4 in patients with COPD without comorbidities (n = 214), patients with COPD with CPA (n = 51), patients with COPD with ascariasis (n = 23), patients with COPD with CPA and ascariasis (n = 12), and control subjects (n = 61). *Compared with the control subjects (p < 0.005); # compared with the patients with COPD (p < 0.0005). COPD: chronic obstructive pulmonary disease; CPA: chronic pulmonary aspergillosis; IL: interleukin.

According to Figure 4, the IL-6 concentration was not significantly different in patients with COPD with CPA (10.0 ± 5.2 pg/mL) and without CPA (10.5 ± 13.3 pg/mL). The highest level of IL-6 was in patients with COPD with ascariasis (39.8 ± 8.5 pg/mL), as well as in patients with COPD with CPA and ascariasis (21.1 ± 3.6 pg/mL). In all cases, IL-6 was significantly higher than in controls (2.6 ± 2.78 pg/mL) (p < 001).

Figure 4.

The level of serum IL-6 in patients with COPD and controls. The figure presents the level of IL-6 in patients with COPD without comorbidities (n = 214), patients with COPD with CPA (n = 51), patients with COPD with ascariasis (n = 23), patients with COPD with CPA and ascariasis (n = 12), and control subjects (n = 61). *Compared with the control subjects (p < 0.005); # compared with the patients with COPD (p < 0.0005); † compared with the patients with COPD with ascariasis and CPA (p < 0.005); ‡ compared with the patients with COPD with ascariasis (p < 0.005). COPD: chronic obstructive pulmonary disease; CPA: chronic pulmonary aspergillosis; IL: interleukin.

The level of TNF-α was significantly higher in all groups of patients with COPD than in the controls (p ≤ 0.01). The highest TNF-α level was observed in patients with COPD with CPA and ascariasis (12.05 ± 2.89 pg/mL) and patients with COPD with ascariasis (6.74 ± 3.30 pg/mL) (Fig. 5).

Figure 5.

The level of serum TNF-α in patients with COPD and controls. The figure presents the level of TNF-α in patients with COPD without comorbidities (n = 214), patients with COPD with CPA (n = 51), patients with COPD with ascariasis (n = 23), patients with COPD with CPA and ascariasis (n = 12), and control subjects (n = 61). *Compared with the control subjects (p < 0.005); # compared with the patients with COPD (p < 0.0005); † compared with the patients with COPD with ascariasis and CPA (p < 0.005). COPD: chronic obstructive pulmonary disease; CPA: chronic pulmonary aspergillosis; TNF: tumour necrosis factor.

The level of IFN-γ was lower in patients with COPD with ascariasis (6 ± 3.42 pg/mL) and patients with COPD with CPA and ascariasis (4.5 ± 3.6 pg/mL) than in the controls (10.0 ± 4.8 pg/mL). The highest level of IFN-γ was observed in patients with COPD without comorbidity (15.4 ± 3.6 pg/mL) and patients with COPD with CPA (13.4 ± 5.9 pg/mL) (Fig. 6).

Figure 6.

The level of serum IFN-γ in patients with COPD and controls. The figure presents the level of IFN-γ in patients with COPD without comorbidities (n = 214), patients with COPD with CPA (n = 51), patients with COPD with ascariasis (n = 23), patients with COPD with CPA and ascariasis (n = 12), and control subjects (n = 61). *Compared with the control subjects (p < 0.005); # compared with the patients with COPD (p < 0.0005); † compared with the patients with COPD with ascariasis and CPA (p < 0.005); ‡ compared with the patients with COPD with ascariasis (p < 0.005). COPD: chronic obstructive pulmonary disease; CPA: chronic pulmonary aspergillosis.

Discussion

Detection of A. lumbricoides in patients with COPD with CPA was of special interest because it could indicate a possible association of ascariasis with CPA development in patients with COPD. The latter is an incurable disease, but it is important to identify concomitant infections that may aggravate its course and can be treated.⁴ Uzbekistan is endemic for intestinal parasites, including A. lumbricoides; thus, it is likely that patients with COPD are infected with intestinal helminths.¹¹ We found that the prevalence of A. lumbricoides in patients with COPD complicated with CPA was 3.8 times as high as that in the control group and 2.0 times higher than that in patients with COPD without CPA. Our data indicate that A. lumbricoides significantly increases susceptibility to Aspergillus sp. and may be considered as additional risk factor for CPA in patients with COPD.

The determination of cytokine levels in patients with COPD showed that the most pronounced shifts were observed for IL-1β, and its level was significantly higher in all COPD groups than in controls. The high prevalence of ascariasis in patients with COPD with CPA is in accordance with the cytokine profile: the highest level of IL-1β was detected in patients with COPD with ascariasis and CPA, significantly exceeding the corresponding values in patients with COPD without comorbidity and in controls. The increase in IL-1β levels in patients with COPD is explained by the inclusion of NLRP3 inflammasome activation in COPD pathogenesis, which is involved in the release of the pro-inflammatory cytokines IL-1β and IL-18. Active caspase-1 or caspase-4/5/11 process pro-IL-1β and pro-IL-18 into their mature forms, inducing inflammation and pyroptotic cell death or inflammation without pyroptosis.¹⁵ It has been established that elevated levels of IL-1β correlated with common inflammatory biomarkers (white blood cell count and fibrinogen) in 89% of patients with COPD.¹⁶ Significantly increased levels of IL-1β and CPA in patients with COPD with ascariasis indicate that susceptibility to Aspergillus sp. and exacerbation of pulmonary inflammation can be mediated by A. lumbricoides. Our results are in agreement with other data¹⁷: Ascaris suum infection during pulmonary fibrosis in mice exacerbated chronic pulmonary inflammation and dysfunction but not fibrosis. These data suggest possible activation of the NLRP3 inflammasome by Ascaris spp. as well as exacerbation of the inflammatory process. The NLRP3 inflammasome mediates caspase-1 activation and the secretion of the proinflammatory cytokines IL-1β/IL-18 in response to helminth infections.¹⁸ Infection with A. lumbricoides induces a more severe course of malaria caused by Plasmodium falciparum, manifested by more severe parasitemia and significantly higher serum IL-1β levels.¹⁹ Our findings of a significant increase in serum IL-1β levels in patients with COPD with ascariasis and CPA correspond to a previous study.²⁰ Alternative activation of alveolar macrophages in aspergillosis and ascariasis (Th1 and Th2 responses, respectively) in patients with COPD may lead to fungal burden and inflammation enhancement.

In our study, IL-4 levels in all patients with COPD were higher than those in controls. The highest IL-4 level was found in patients with COPD with ascariasis, exceeding this index in patients with COPD with ascariasis and CPA. Ascariasis could suppress the Th1 response to Aspergillus sp. and worsen the CPA course in patients with COPD.

The inflammatory process in patients with COPD is accompanied by elevated levels of TNF-α, IL-6, and IL-8 in the serum.²¹ The high levels of TNF-α and IL-6 in patients with COPD with ascariasis and CPA in our study are similar to other data.^22,23

Rise in both IL-1 and IL-6 have been associated with negative health outcomes, mortality and a pro-inflammatory phenotype in COPD. IL-6 in COPD was shown to correlate negatively with lung function, and IL-1β induces airway inflammation.²⁴ Thus, a significant increase in IL-1β, IL-6, and TNF-α levels in patients with COPD with ascariasis and CPA in comparison with the control group and patients with COPD without such comorbidity indicates enhancement of inflammation induced by A. lumbricoides.

Our study has the limitation of being conducted at a single centre, with a limited sample size, and included only patients with COPD at stages III and IV during exacerbation, which may have influenced the stability of the results. Furthermore, the impossibility of studying the impact of the A. lumbricoides migration phase on CPA development in patients with COPD is acknowledged.

Conclusions

The high prevalence of ascariasis and changes in the cytokine profile in patients with COPD with CPA in comparison with healthy controls allows us to consider A. lumbricoides as an additional risk factor. The study needs to be continued to clarify the duration of immunological shifts after parasite elimination and their possible correction. Diagnosis of A. lumbricoides in patients with COPD is important not only for helminth endemic regions but also where tourism and intensive migration occur.

Footnotes

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Abdurakhim Toychiev

References

Rossaki

Hurst

van Gemert

, et al. Strategies for the prevention, diagnosis and treatment of COPD in low- and middle- income countries: the importance of primary care. Expert Rev Respir Med 2021; 15: 1563–1577.

Tudesq

Peyrony

Lemiale

, et al. Invasive pulmonary aspergillosis in nonimmunocompromised hosts. Semin Respir Crit Care Med 2019; 40: 540–547.

Martínez-García

MÁ

Oscullo

García-Ortega

, et al. Inhaled corticosteroids in adults with non-cystic fibrosis bronchiectasis: from bench to bedside. A narrative review. Drugs 2022; 82: 1453–1468.

Otu

Kosmidis

Mathioudakis

, et al. The clinical spectrum of aspergillosis in chronic obstructive pulmonary disease. Infection 2023; 20: 1–17.

Nutman

. Looking beyond the induction of Th2 responses to explain immunomodulation by helminths. Parasite Immunol 2015; 37: 304–313.

Gazzinelli-Guimaraes

Nutman

. Helminth parasites and immune regulation. F1000Res 2018; 7: F1000 Faculty Rev-1685.

Long

Lanter

Pazos

, et al. Intestinal helminth infection enhances bacteria-induced recruitment of neutrophils to the airspace. Sci Rep 2019; 9: 15703.

Knight

, et al. Transient Ascaris suum larval migration induces intractable chronic pulmonary disease and anemia in mice. PLoS Negl Trop Dis 2021; 15: e0010050.

Almeida

Nejsum

Williams

. Modulation of human macrophage activity by Ascaris antigens is dependent on macrophage polarization state. Immunobiology 2018; 223: 405–412.

10.

Dewi

IMW

van de Veerdonk

Gresnigt

. The multifaceted role of T-helper responses in host defense against Aspergillus fumigatus. J Fungi (Basel) 2017; 3: 55.

11.

Toychiev

Navruzov

Pazylova

, et al. Intestinal protozoa and helminths in ulcerative colitis and the influence of anti-parasitic therapy on the course of the disease. Acta Trop 2021; 213: 105755.

12.

Mirza

Clay

Koslow

, et al. COPD guidelines: a review of the 2018 GOLD report. Mayo Clin Proc 2018; 93: 1488–1502.

13.

Salzer

Heyckendorf

Kalsdorf

, et al. Characterization of patients with chronic pulmonary aspergillosis according to the new ESCMID/ERS/ECMM and IDSA guidelines. Mycoses 2017; 60: 136–142.

14.

Toychiev

Abdujapparov

Imamov

, et al. Intestinal helminths and protozoan infections in patients with colorectal cancer: prevalence and possible association with cancer pathogenesis. Parasitol Res 2018; 117: 3715–3723.

15.

Swanson

Deng

Ting

. The NLRP3 inflammasome: molecular activation and regulation to therapeutics. Nat Rev Immunol 2019; 19: 477–489.

16.

Markelić

Hlapčić

Čeri

, et al. Activation of NLRP3 inflammasome in stable chronic obstructive pulmonary disease. Sci Rep 2022; 12: 7544.

17.

Oliveira

FMS

da Paixão Matias

Kraemer

, et al. Comorbidity associated to Ascaris suum infection during pulmonary fibrosis exacerbates chronic lung and liver inflammation and dysfunction but not affect the parasite cycle in mice. PLoS Negl Trop Dis 2019; 13: e0007896.

18.

Zakeri

Hansen

Andersen

, et al. Immunomodulation by helminths: intracellular pathways and extracellular vesicles. Front Immunol 2018; 9: 2349.

19.

Ntonifor

Chewa

Oumar

, et al. Intestinal helminths as predictors of some malaria clinical outcomes and IL-1β levels in outpatients attending two public hospitals in Bamenda, North West Cameroon. PLoS Negl Trop Dis 2021; 15: e0009174.

20.

Zhan

Zhao

, et al. The serum level of IL-1B correlates with the activity of chronic pulmonary aspergillosis. Can Respir J 2018; 2018: 8740491.

21.

Austin

Crack

Bozinovski

, et al.

COPD and stroke: are systemic inflammation and oxidative stress the missing links?

Clin Sci (Lond) 2016; 130: 1039–1050.

22.

Chi

Song

, et al. Clinical efficacy of sequential therapy with voriconazole on COPD patients in acute phase with pulmonary aspergillosis and effects on cytokines and pulmonary functions. Eur Rev Med Pharmacol Sci 2018; 22: 1837–1842.

23.

Ito

Takazono

Obase

, et al. Serum cytokines usefulness for understanding the pathology in allergic bronchopulmonary aspergillosis and chronic pulmonary aspergillosis. J Fungi (Basel) 2022; 8: 436.

24.

Garth

Barnes

Krick

. Targeting cytokines as evolving treatment strategies in chronic inflammatory airway diseases. Int J Mol Sci 2018; 19: 3402.