Prevalence of fungal pathogens among respiratory tract infections and associated risk factors

INTRODUCTION

Respiratory tract infections constitute a significant global health concern, contributing to one-third of infectious disease-related deaths worldwide, with an estimated annual toll of 4.3 million lives. Despite medical interventions, a majority of invasive pulmonary fungal infections exhibit alarmingly high mortality rates exceeding 50%.^[1] The prevalence of allergic bronchopulmonary aspergillosis is estimated at 2.0 million, while chronic pulmonary aspergillosis affects around 1.74 million individuals.^[2] Various factors influence the prevalence of fungal infections, including the susceptibility of certain individuals, socioeconomic conditions, and specific occupations such as farming or construction. Engaging in activities that involve exposure to soil and organic matter, residing in damp or mold-infested environments, and living in overcrowded conditions further contribute to the risk of fungal infections.^[3] Respiratory tract fungal infections, traditionally associated with invasive pulmonary diseases caused by Aspergillus spp. and Mucor spp., now encompass a broader spectrum. Uncommon filamentous fungi such as Fusarium and Penicillium have also been identified as causative agents for well-defined respiratory disorders.^[4] In chronic rhinosinusitis, fungal presence is seen in the sinuses in 63% of patients, with Aspergillus being the predominant genus.^[5] Mucormycosis is a severe infection that poses a significant risk to individuals with diabetic ketoacidosis, neutropenia, organ transplantation, and/or elevated levels of accessible iron in the bloodstream.^[6] The frequency of Mucormycosis has increased due to rising immunosuppression states, such as those in cancer and transplant patients, as well as expanded use of immunosuppressive medications for autoimmune diseases.^[7] In addition, individuals receiving prolonged antibiotic treatment, those with chronic lung diseases like chronic obstructive pulmonary 2 disease (COPD), and patients using inhaled corticosteroids may be more susceptible to fungal infections in the respiratory tract.^[8] Penicilliosis can affect both healthy and immunocompromised individuals, particularly those with human immunodeficiency virus (HIV).^[9] The term “respiratory mycosis” has expanded beyond invasive diseases to include lesser-recognized conditions such as fungal ball, Severe Asthma with Fungal Sensitization, Fungus-associated Chronic Cough, Allergic Bronchopulmonary Mycosis, and Allergic Fungal Rhinosinusitis.^[10] Determining the exact burden of fungal respiratory tract infections is essential, as the associated spectrum of risk factors becomes crucial, given the limited available classes of antifungals for systemic treatment and the growing threat of resistance to antifungals, both intrinsic and acquired, in healthcare.

MATERIAL AND METHODS

Respiratory samples, including sputum, bronchoalveolar lavage (BAL), endotracheal tube (ETT) secretions, lung tissue, and endobronchial ultrasound (EBUS), were collected from patients suspected of fungal infections. Samples were processed using standard mycological procedures.^[11]

Sample processing

Three hundred and eighty respiratory samples were collected from symptomatic patients at the Adesh Institute of Medical Sciences and Research, Punjab. All the samples were processed by direct microscopy, by potassium hydroxide (KOH) wet mount, and by Gram staining. The specimen was placed on a clean glass slide, and a drop of 20% KOH was added. Biopsy material was kept in a tube with 40% KOH in an incubator overnight at 37°C) and then a wet mount was prepared and examined under the microscope for fungal structures such as hyphae, spores, or yeast cells for confirmation. Gram staining was also done to look for Gram-positive oval budding yeast cells. Culture was done on Sabouraud Dextrose Agar (SDA), which contains digests of animal tissues (peptones), which provide a nutritious source of amino acids and nitrogenous compounds for the growth of fungi and yeasts. Dextrose is added as the energy and carbon source. Agar is the solidifying agent. Chloramphenicol and/or tetracycline may be added as broad-spectrum antimicrobials to inhibit the growth of a wide range of Gram-positive and Gram-negative bacteria. Gentamicin is added to further inhibit the growth of Gram-negative bacteria. The pH is adjusted to approximately 5.6 to enhance the growth of fungi. Three tubes, two plain sabouraud dextrose agar (SDA) at 25°C and 37°C, and one SDA with antibiotic at 25°C (blue top) were used for every sample. Tubes were inoculated using a bent wire for processed tissue and a loop for liquid samples. All routine fungal cultures were incubated at 37°C and 25°C for 4 weeks before they were discarded. Culture was not discarded when a fungus was first isolated but held for the entire period of 4 weeks so that slow-growing fungi are not overlooked. All culture tubes kept at 37°C and 25°C were examined for growth every day for the 1^st week and thrice weekly thereafter for 3 weeks. Findings were recorded as no growth/growth/yeast/ mold. Isolates were further identified depending on the type of growth. Gram staining was done, if Gram-positive oval budding yeast cells approximately 4–8 um in diameter with pseudo hyphae were seen, further germ tube test was performed for distinguishing Candida albicans from other Candida species: 12 × 75 mm test tubes were labeled and with a Pasteur pipette, three drops of freshly pooled human serum were dispensed into the tubes. Using a sterile wooden applicator stick or straight nichrome wire, the yeast colony was emulsified into the serum and incubated the test at 35°C for 2.5–3 h, and then examined under the microscope to confirm the presence or absence of germ tubes. In case of mold, Lactophenol Cotton Blue Mount was prepared on an oil-free or grease-free slide. Placed a drop of Lactophenol blue on the slide and added the fungal specimen over this drop using sterile inoculating needles. Using two sterile dissecting needles, the fungus was gently teased apart and evenly spread in the Lactophenol. It was then examined under the microscope for fungal morphological features. This was followed by microculture technique, slide culture was directly inoculated on the deficient media plate. Small pieces of agar blocks from the corner of the plate were cut and placed on the same plate, inoculation was done in the same way as in slide culture on four corners of the block, incubated for 5–7 days at 25°C, and observed for the morphological forms [Figure 1, Supplementary Material Color plate 1].

Supplementary Material

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Figure 1: Flowchart showing processing of respiratory samples. KOH: Potassium hydroxide, SDA: Sabouraud dextrose agar, LCB: Lactophenol cotton blue, EBU: Endobronchial ultrasound.

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RESULTS

The present study was conducted in the Mycology Section of the Central laboratory, Department of Microbiology, Adesh Institute of Medical Sciences and Research, Bathinda, Punjab. A total of 380 respiratory samples that included 201 BAL samples, 91 sputum samples, 54 endotracheal secretions, 20 EBUS samples, and 14 lung tissue samples, and these samples were obtained from symptomatic patients hospitalized in various pulmonary units. Out of the total 380 samples, 147 (38.6%) showed growth, and no growth was seen in 233 (61.3 %) samples. Among the total 147 growth-positive samples, 72.10% were male while 27.9% were female. The highest incidence of pulmonary fungal infection was found to be among patients in the age group of 50–79 years, while the lowest incidence was found in patients ≤30 years of age [Figures 2 and 3, Tables 1 and 2].

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Figure 2: Culture positivity rate (n = 380).

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Figure 3: Sex-wise distribution of patients (n = 147).

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Cough (94.16%), dyspnea (81.02%), breathlessness (69.18%), fever (60%), weight loss (56.2%), and chest pain (43%) were the predominant symptoms in the patients, while fatigue and joint pain accounted for the minor symptoms. About 73% of positive patients had history of diabetes mellitus, 51% of patients were on prolonged antibiotic use, 47% of patients had history of allergy, 35% of patients had asthma while 15% of patients had history of usage of steroids, 11% of patients had prior history of tuberculosis, while 3% of them were on chemotherapeutic agents. Around 3% of patients had coinfection of tuberculosis (TB) with human immunodeficiency virus (HIV) [Table 3 and Figure 4].

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Figure 4: Underlying risk factors and co-morbidities among patients (n = 147). TB: Tuberculosis, HIV: Human Immunodeficiency virus, H/O: History of

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Aspergillus flavus (38.51%) and Aspergillus fumigatus (29.20%) represented the two most commonly isolated fungi in the current study followed by Aspergillus niger (10.20%), C. albicans (8.39%), Mucor (6.90%), and Penicillium (4.70%) while Aspergillus versicolor (2.0%) was the least commonly isolated fungus [Figure 5, Supplementary Material Color plate 2].

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Figure 5: Percentage of fungi isolated (n = 147).

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Among 147 patients with respiratory symptoms, chest radiography was available for 110. Typical radiological patterns compatible with pulmonary fungal infections were observed in a large number of cases – multiple nodular opacities in 42 (38%), halo sign in 21 (19%), and cavitary lesions or air crescent sign in 14 (13%). Consolidation was the most common feature, seen in 51 (46%), while bronchiectatic changes and fibrosis consistent with prior tuberculosis were present in 33 (30%). Mediastinal lymphadenopathy was detected in 8 (7%) patients. Serum β-D glucan levels and galactomannan testing were performed in 98 and 82 patients, respectively. The positivity rate was 63% for β-D glucan and 56% for galactomannan. BAL galactomannan testing was conducted in 46 patients, with a positivity rate of 85%. Concordant positivity of BAL galactomannan, serum galactomannan, and β-D glucan was observed in 51.7% of patients, providing strong evidence of invasive infection. For treatment, voriconazole was used as the first-line antifungal in 91.2% of patients, while liposomal amphotericin B was administered to 8.8% who had hepatic intolerance or azole contraindications resistance.

Out of 147 positive isolates, 55.78% of positive isolates were obtained from the BAL sample following 20.40% from the ETT sample, 14.96% from the sputum sample, 5.45% from the EBUS sample, and 3.41% from the lung tissue sample [Figure 6].

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Figure 6: Sample-wise distribution of positive isolates (n = 147). BAL: Bronchoalveolar lavage, ETT: Endotracheal tube

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Eighty-two isolates were obtained from the BAL sample out of which 34 isolates were A. flavus, 20 were A. fumigatus, seven were A. niger, seven were C. albicans, five were Mucor, six were Penicillium, and three were A. versicolor. Twenty-two isolates were obtained from sputum samples out of which seven were A. flavus, eight were A. fumigatus, three were A. niger, and four were C. albicans. Forty-nine positive isolates were obtained from the ETT out of which ten isolates were A. flavus, 11 were A. fumigatus, five were niger, and one isolate was C. albicans and two isolates were Mucor. Eight isolates were obtained from the EBUS sample, out of which three were A. flavus, three were A. fumigatus, and two were Mucor. Finally, five positive isolates were obtained from lung tissues, out of which two were A. flavus, two were A. fumigatus, and one was Mucor [Figure 7].

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Figure 7: Sample-wise distribution of isolates (n = 147).

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DISCUSSION

The respiratory tract serves as a common portal for the entry and establishment of fungal pathogens. Colonization, often asymptomatic at first, can progress to invasive fungal disease in vulnerable individuals, particularly those with underlying comorbidities or compromised immunity. In the present study, the prevalence of fungal infections among respiratory samples was found to be 38.68%, reflecting a substantial burden. Comparable findings were reported in previous studies,^[12,13] which observed fungal prevalence rates of 35.67% and 31.62%, respectively. The similarity suggests a consistent rise in fungal respiratory infections globally, possibly attributed to increased use of broad-spectrum antibiotics, steroid therapy, and the growing population of immunocompromised individuals.^[12,13] Among the different types of respiratory samples, bronchoalveolar lavage (BAL) yielded the highest number of positive fungal isolates (56%), followed by endotracheal tube (ETT) secretions (33%), endobronchial ultrasound (EBUS) (40%), sputum (14.9%), and lung tissue (3.4%). This higher recovery from bronchoalveolar lavage (BAL) could be due to its ability to sample deeper lung segments, making it a more sensitive method for detecting lower respiratory tract infections.^[14] A male predominance was noted in this study, with 72% of infected individuals being male. This gender bias might be explained by occupational exposure, especially in labor-intensive sectors like farming, construction, and mining, where dust, soil, and mold exposure are more prevalent.^[15] Furthermore, higher smoking rates and greater environmental exposure in males may contribute to increased susceptibility. Although Bitew and Bati (2021). ^[15] observed a more balanced gender distribution, local cultural or occupational differences may account for the divergence. The mean age of affected patients was 64.5 years, closely aligning with the findings of Li et al. (2016).^[16] who reported a mean age of 67 years. The elderly population is particularly vulnerable due to immunosenescence, reduced mucociliary clearance, and a higher prevalence of chronic respiratory diseases such as COPD and asthma, all of which can predispose to fungal colonization and infection. [16] Common symptoms included productive cough (87.07%), dyspnea (71.02%), breathlessness (69.18%), fever (60%), weight loss (55.1%), and chest pain (43%). These manifestations are consistent with those observed by Rafat et al. (2020)^[12] and reflect the non-specific clinical presentation of respiratory fungal infections, which often overlap with bacterial or mycobacterial pneumonia. The lack of distinctive clinical markers contributes to diagnostic delays and inappropriate treatment. The most prevalent risk factor identified was diabetes mellitus (73%), which is known to impair neutrophil function and enhance fungal proliferation, particularly in mucormycosis and candidiasis. Prolonged antibiotic use (51%) can disrupt normal respiratory flora, favoring fungal overgrowth. Allergic history (47%) and asthma (35%) may indicate underlying allergic fungal airway disease.

Tuberculosis (11%), steroid use (15%), and chemotherapy (3%) also compromise immune defenses, facilitating fungal infection.^[12] Compared to Rafat et al.’s^[12] study, which highlighted tuberculosis (24.81%) and chemotherapy (21.89%), this study emphasizes the growing role of metabolic and iatrogenic factors, especially in regions with high diabetes prevalence. Regarding fungal species, the dominant isolates were A. flavus (38.09%) and A. fumigatus (29.9%), followed by A. niger (10.51%), C. albicans (8.1%), Mucor spp. (6.8%), and Penicillium spp. (4.6%). A. versicolor (2%) was the least commonly isolated species. These findings are in line with those by Shaukat et al. (2022).^[14] and Roohani et al. (2018),^[17] who noted variability in species prevalence depending on geographic location, climatic conditions, hospital settings, and diagnostic capacities. For instance, A. flavus is more frequently isolated in warmer climates like India and parts of the Middle East, whereas A. fumigatus dominates in cooler, temperate zones.^[17] High prevalence of Aspergillosis can be due to an enriched high-risk inpatient population with chronic pulmonary disease, prior tuberculosis, prolonged corticosteroid exposure, environmental exposure, and impaired airway clearance markedly increases the yield of Aspergillus from respiratory samples. Second, the diagnosis was not based solely on culture positivity but was supported by radiological and serological evidence. Candida was considered a genuine pathogen and not a contaminant, as the culture positivity of Candida was confirmed by positive paired blood cultures and the absence of other bacterial pathogens. The colony count of more than 10⁶ colony forming unit (CFU)/ mL was considered significant. The study underscores the increasing significance of fungal infections in respiratory illnesses, driven by host factors, environmental exposure, and healthcare-associated risks. The findings highlight the urgent need for early detection strategies, risk stratification, and awareness among clinicians, especially considering the limited antifungal treatment options and growing antifungal resistance.

CONCLUSION

This study highlights the significant burden of respiratory fungal infections in hospitalized patients, particularly among older individuals with comorbidities such as diabetes and prolonged antibiotic use. The predominance of Aspergillus species underscores the need for timely diagnostic methods and targeted antifungal therapy. Incorporating fungal diagnostics into the routine evaluation of respiratory infections could enhance patient outcomes and prevent complications.