Role of Comorbidities in Establishment of Pulmonary Mycosis in Chronic Obstructive Pulmonary Disease Patients in and Around Rewa

Background: Viruses and bacterias have been already considered as a major cause of chronic obstructive pulmonary disease (COPD) exacerbations; whereas, the major role of fungal colonization and infection is poorly understood. Objective: Keeping this fact in mind the present study was designed to find out the microbes responsible for acute exacerbation of COPD, which is one the common disorder of chronic lung disease in and around Rewa along with the profile of pulmonary fungal infection among COPD patients with and without comorbidities to determine their prevalence, risk factors, and outcome among those patients. Patients and methods: In this prospective cross-sectional analytic study, different samples (sputum , bronchoalveolar lavage, blood, and others) from 180 COPD patients at risk for pulmonary fungal infection were examined using mycological analysis (direct microscopy with lactophenol cotton blue and culture in SDA media). Broncho alveolar lavage and blood samples were examined using the human 1,3-β-d-glucan and galactomannan ELISA tests. Results: The prevalence of pulmonary fungal infection was significantly higher in COPD patients with comorbidities (77.5%) whereas COPD patients without comorbidities (53%) (P < 0.001), with a predominance of Candida and Aspergillus spp. in both groups. Mechanical ventilation, corticosteroid therapy, ICU admission and age were major risk factors for pulmonary fungal infection in COPD patients with comorbidities [P = 0.013, odds ratio (ODR) = 2.24; P = 0.029, ODR = 2.02; P = 0.024, ODR = 1.98; and P = 0.036, ODR = 2.64; respectively]. COPD patients with comorbidities had significantly higher mortality rate (13.8%) compared with COPD patients without comorbidities (3.6%; P < 0.05). Blood galactomannan antigen was positive in 19 (10.5%) COPD patients with comorbidities versus 8 (4.4%) in COPD patients without comorbidities (P < 0.05). Conclusion: COPD patients with comorbidities had a higher prevalence of pulmonary fungal infection rate compared with COPD patients without comorbidities. Age, mechanical ventilation, corticosteroid therapy, and ICU admission were independent risk factors for pulmonary fungal infection in COPD patients with comorbidities.


INTRODUCTION
Comorbidities frequently impact chronic obstructive pulmonary disease (COPD) patients and significantly affect the patients' survival, quality of life, and exacerbation frequency (Putcha et al., 2013). It is believed that chronic inflammatory state in COPD may accelerate the natural history of some comorbidities, and hence COPD is considered as a systemic disorder (Divo et al., 2012). Airways of COPD patients are often colonized with potential pathogenic microorganisms (Sethi & Murphy, 2008) and may lead to increased airway inflammation (O'Donnell et al., 2006). The potential role of fungal colonization and infection in the pathogenesis of COPD is poorly understood as bacteria and viruses were usually considered as the major cause of COPD exacerbations. Aspergillus spp. is the most common fungal genus to cause pulmonary-associated fungal infections in COPD patients (Bulpa et al., 2007).
The primary objective of this study was to screen COPD patients with comorbidities and COPD without comorbidities for microbiological and serological pieces of evidence for pulmonary fungal infection to determine the prevalence of pulmonary fungal infection among those patients. The second objective was to identify the risk factor for pulmonary function infection among those patients. The third objective was to investigate the frequency of positive fungal culture and the clinical outcomes among those patients.

MATERIALS AND METHODS
This prospective cross-sectional analytic study included 180 COPD patients at risk for pulmonary fungal infection who were admitted in the Medicine Department Intensive Care Unit. After meeting the inclusion criteria, informed consent was obtained from all study participants and relatives according the clinical condition of the patients. Criteria for Patients Selection COPD patients who were eligible for enrolment displayed a combination of the following host factors: 1. A history of pre-existing COPD and immunosuppression from corticosteroids or other underlying conditions (e.g. diabetes, malnutrition, and liver cirrhosis). 2. Clinical signs and/or symptoms suggestive of invasive mycosis. 3. Any of the following symptoms of lower respiratory tract infection: expectoration, dyspnoea, or haemoptysis; pleuritic chest pain etc. 4. Fever persists even after minimum 3 days of appropriate antibiotics, or fever relapsing after 5 days or along with continuation of antibiotics 5. Steroid use: at least 4 mg of methylprednisolone (or equivalent) a day for at least 7 days in the past 3 weeks before admission or during the course of the ICU stay for at least 5 days, or a cumulative dose of at least 250 mg of methylprednisolone (or equivalent) in the past 3 months before enrollment (Dutkiewicz & Hage, 2010); Garnacho-Montero et al., 2013).
Exclusion criteria included patients who received systemic antifungal therapy within 3 days before sample collection and patients who refused to participate in the study.
All patients were subjected to clinical and routine laboratory investigations. Radiology, including plain chest radiograph, and high resolution CT of the chest were performed for patients.
Source of the specimen and method of examination according to the site of the lesion involved the following: direct microscopic examination of sputum samples, bronchoalveolar lavage (BAL) samples, and sterile catheter samples,culture examination of pleural fluid and blood samples (Xavier et al., 2009). Sabouraud's Dextrose agar was routinely used. HiChrome agar was used for the identification of some species of Candida. Serologic diagnosis was carried out using the human 1,3-β-d-glycosidase (Wright et al., 2011) and human galactomannan (GM) ELISA tests (Pfeiffer et al., 2006) of blood and BAL samples.

Procedures
Collection of sputum samples from the patients according to universal precautions (Xavier et al., 2009) were transported and stored in sealed tubes (Stynen et al., 1995), unexposed to air.

Evaluation criteria for the diagnosis of fungal infection
We used the EORTC/MSG diagnostic criteria as the reference standard for case definition of invasive aspergillosis (IA), which were classified as definite, probable, or possible (Pfeiffer et al., 2006). predicted between the two groups (P < 0.05).
COPD patients with comorbidities were associated with lower lung function (FEV1%: 46.5 ± 18.6 vs. 54.8 ± 19.9% predicted in COPD patients without comorbidities).  The prevalence of pulmonary fungal infection was significantly higher in COPD patients with comorbidities (77.5%) compared with COPD patients without comorbidities (53%) (P < 0.001) [ Table 3]. In the present study we did not get statistically significant difference as regards fungal species (P > 0.05) with predominance of Candida and Aspergillus spp., in both groups. There was a statistically significant difference as regards blood GM, which was positive in 18 (20.4%) COPD patients with comorbidities versus seven (9.2%) COPD patients without comorbidities (P < 0.05). However, 1,3-β-dglucan and BAL GM showed no statistically significant difference between the groups (P > 0.05) [ Table 4].

DISCUSSION
This study was designed to assess the profile of fungal infection among COPD patients with and without comorbidities to determine the prevalence, risk factors, and outcome of pulmonary fungal infection among those patients.
As regards comorbid diseases in this study, the common associations included DM, liver cirrhosis, cardiovascular diseases, malignancy, chronic renal failure, and anaemia. Diabetes was the most predominant comorbid disease and was recorded in 28.0% of patients, followed by liver cirrhosis and cardiovascular diseases in 21.3 and 12.3% patients, respectively. However, bronchogenic carcinoma was recorded in 8.8% of cases. The current study revealed that COPD patients with comorbidities were associated with significantly lower mean age and lower FEV1% predicted compared with COPD patients without comorbidities; however, there was no statistically significant difference as regards sex.
Multiple comorbidities may coexist in individuals with COPD and play an important role in determining clinical outcomes in COPD, even after control of the confounding factors (Putcha et al., 2014;Dal Negro et al., 2015). Although, COPD patients have normal pulmonary defense mechanisms against Aspergillus spp., such as the ingestion of conidia (the ends of some hyphae are rounded and could be confused with yeast, but that are instead called conidia or spores) by pulmonary macrophages and killing of hyphae by neutrophils. However, there many factors that predispose to colonization and infection with Aspergillus spp., including structural changes in lung architecture with the formation of bullae, and the common use of long-term steroid treatment (even inhaled steroids) increases host susceptibility by reducing oxidative killing of the organism by pulmonary macrophages and increases its linear growth by 30-40%. Moreover, comorbidity factors such as diabetes, alcoholism, and malnutrition may further enhance the risk for pulmonary fungal infection in COPD patients (Meersseman et al., 2007).
Recent studies reported that COPD may affect the function of other organs, including the heart, vasculature, muscles, liver, gastroenteric apparatus, kidney, and brain; it is frequently associated with various disorders  and accelerates lung aging (MacNee et al., 2014). Several comorbidities frequently predominate, particularly in elderly patients, but the relationship linking their prevalence to patients' sex and COPD severity is still unclear (Jo et al., 2015;Miravitlles et al., 2015).
In the present study, the prevalence of pulmonary fungal infection was significantly higher in COPD patients with comorbidities (77.5%) compared with COPD patients without comorbidities (53.0%). However, there was a predominance of Candida and Aspergillus spp., in both groups, with no statistically significant difference. There was statistically significant difference as regards blood GM, which was positive in 18 (20.4%) COPD patients with comorbidities versus seven (9.2%) COPD patients without comorbidities. However, 1,3-β-d-glucan and BAL GM showed no statistically significant difference between the two groups. In contrast, Tutar et al. (2013) found that GM with median value 0.54 ng/ml was positive in nine patients (81.8%) and 1,3-β-d-glucan was examined in five patients and was positive in three (60%) of them.
As regards the outcome, this study demonstrated that COPD patients with comorbidities at risk factors for pulmonary fungal infection in COPD patients with comorbidities versus COPD patients without comorbidities, age, mechanical ventilation therapy, corticosteroid therapy, and ICU admission were major risk factors for pulmonary fungal infection in COPD patients with comorbidities, with a statistically significant difference. However, antibiotic therapy was prominent in both groups, with no statistically significant difference. Similarly, in recent years, it has been shown that corticosteroid use plays a significant role in terms of increasing the rate of invasive pulmonary aspergillosis (IPA) incidence in COPD cases (Guinea et al. 2010). In a retrospective study, it was shown that steroid use of over 700 mg in total within the last three months in COPD patients increased the risk for IPA (Stynen et al. 1995). Moreover, it has been stated by other authors that inhaled steroids are a risk factor for IPA (Shukla & Shukla, 2017). Moreover, using three or more antibiotics for 10 days was a risk factor for IPA development in COPD (Muquim et al. 2005;He et al. 2011). Another study found that four cases had used antibiotics in the last 3 months and a history of antibiotic use was the only risk factor for pulmonary fungal infection in one case that did not receive systemic steroid treatment (Agustí et al. 2003). In another study on 1209 patients with a positive respiratory culture for Aspergillus spp., Perfect et al. (2001) showed that COPD, corticosteroid use, and DM were the three main risk factors for fungal colonization.

CONCLUSION
The prevalence of pulmonary fungal infection was significantly higher in COPD patients with comorbidities (77.8%) compared with COPD patients without comorbidities. There was predominance of Candida and Aspergillus spp., in both groups. Age, mechanical ventilation, corticosteroids therapy, and ICU admission were independent risk factors