Department of Animal Production, Faculty of Agriculture, Baghdad University, Baghdad, Iraq

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1 World Journal of Pharmaceutical Sciences ISSN (Print): ; ISSN (Online): Published by Atom and Cell Publishers All Rights Reserved Available online at: Original Article Study of fungal infection in Iraqi patients suffering from chronic cough and antifungal sensitivity test in vitro for the fungi isolated from these patients Heba F. Hassan, 1 Nada S. Hassain, 2 Zainab A. Aldhaher, 1 Rasha M. Shaker, 1 Jamal A. Tawfeeq 3 and Ghada Ibraheem 1 1 Department of Basic Sciences. College of Dentistry. University of Baghdad, Iraq. 2 Ministry of health, Medical City, Teaching Laboratory, Baghdad, Iraq. 3 Department of Animal Production, Faculty of Agriculture, Baghdad University, Baghdad, Iraq ABSTRACT Received: / Revised: / Accepted: / Published: Aim and Objective: This study was undertaken to determine isolation of Aspergillus species and candida species from sputum samples in patients suffering from chronic cough and used antifungal sensitivity test in vitro for the fungi isolated from these patients and for the purpose of access to the remarkable progress in the field of fungal treatment. Methods: Sputum samples were collected from 62 patients and controls for isolation of Aspergillus species and yeast and in vitro antifungal sensitivity test Results: A total of forty sputum samples from the patient with chronic cough were isolated and identified, the result of fungal growth of the sputum samples, the isolates are Aspergillus fumigatus 14 (35%) and Candida. albicans 12(30%), it was revealed that Amphotericin B 24(60%) was more sensitive and effective antifungal drugs while Ketoconazole 27(67.5%) was more resistant and poor antifungal drug. Conclusion: The present study revealed that Aspergillus fumigatus was he present study revealed that Aspergillus fumigatus was more species isolates from sputum samples from the patients with chronic cough and Amphotericin B was more sensitive and effective antifungal drugs for the fungi isolated from sputum samples. Keywords: Chronic cough, sputum, antifungal sensitivity test, Aspergillus fumigatus, Candida. albicans INTRODUCTION Cough is defensive reflex mechanism that clears secretions from the upper airways of the respiratory tract by stimulation of a complex reflex arc due to respond to chemical or mechanical irritant stimulation [1]. Cough can be classified as acute, subacute, or chronic depend on duration. Acute cough lasts up to 3weeks, subacute cough lasts 3-8 weeks, and chronic cough lasts longer than 8 weeks The prevalence of chronic cough is estimate between 11 and 20% [2,3].This type of cough is observed commonly in women and in obese individuals [4].Chronic cough that cannot be controlled after basic evaluation is a common reason for referral to respiratory outpatient clinics [5].Despite heedful diagnostic evaluation and numerous treatment regulations [6], a diagnosis cannot be made in a subgroup of chronic cough patients. This condition is termed by chronic idiopathic cough (CIC) [7,8], which is defined as an uncontrolled cough that difficult treated by any current treatment [9]. Recently encountered a groups of patients with allergic fungal cough (AFC), which is difficult and is characterized by sensitization to Bjerkandera adusta [10], among a new clinical disease concept termed fungus associated chronic cough (FACC) with the following manifestations: chronic cough,the presence of environmental fungi or in the sputum and good clinical response to antifungal drugs [11].In the last three decades, fungal infection especially pulmonary aspergillosis has become important cause respiratory tract infection. The increase in frequency is mainly due to use of broad spectrum antibiotics, intensive cytotoxic therapy, corticosteroids and Immuno-suppresants [12].Diagnosis of pulmonary aspergillosis is usually missed as test of their detection cannot carry out in routine diagnostic laboratories [13]. Asergillus species are ubiquitous fungi, usually occurring in water, soil and decaying vegetation. Route of *Corresponding Author Address: Zainab A. Aldhaher, Department of Basic Sciences. College of Dentistry. University of Baghdad, Iraq. zainaba.aldhaher@gmail.com

2 infection by inhalation of fungal spores. From lungs, spread takes place leading to systemic spergillosis. Appearance of aspergillus infection in association with chronic lung disease like pulmonary TB, asthma, lung abscess, bronchopneumonia, and lung malignancy has been documented. There are 185 species of genus aspergillus, only 20 can cause human infection. Aspergillus fumigatus is the most common species causes human infection all over the world [14]. Antifungal therapy have provided a new treatment strategy for chronic difficult cough, included in unexplained chronic cough the efficacy of antifungal treatment for chronic idiopathic cough has not been evaluated yet [15].Now in vitro antifungal susceptibility tests are mainly used for epidemiological study, determination of the degree of antifungal activity, and the prediction of clinical outcome depend on an optimization of antifungal treatment [16]. METHODS AND METHODOLOGY Patients and controls: Forty patients attending the respiratory outpatient clinic at medical city in Baghdad suffering from chronic cough from October 2013 till April 2014 were eligible in this study; they were all diagnosed by specialist physician. In addition thirty apparently healthy volunteers were considered as control group their age and gender matched with patients group were enrolled in current study. Collection of the samples: Sputum sample was collected from each patient and control in sterile cup then this sputum samples was separated from saliva by used centrifuge then streaked in duplicate plates on Sabouraud Dextrose Agar (SDA) media with chloramphenicol, one plate of inoculated incubated at 25 C and the other at 37 C for 2-5 days. The isolates were transferred in vial contain distilled water (D.W) and stored at room temperature. Fungal growth: Fungal growth was identified by colony morphology and lacto phenol cotton blue stain while yeast growth was differentiated by use (CHROM candida agar), germ tube test and use of API for candida species diagnosis. Antifungal susceptibility testing: Disc diffusion method by using different antifungal disks at the following potencies: Clotrimazole (10mcg/disk), Fluconazole (25mcg/disk), Ketoconazole (10mcg/disk) and Miconazole (10mcg/disk), Amphotericin B (20mcg/disk), Nystatin (100 IU/disk), Econazole(10mcg/disk). The isolates were transferred from D.W stocks to SDA agar to enhance sporulation. Then 0.1ml was taken of 14 growth suspension (10^6 cells / ml) was put on surface of Sabouraud Dextrose Agar (SDA) media and spread by using glass spreader L-shaped, left to dry for (10-20) minutes, then antifungal disks were applied to the plates, after which the plates were incubated at (25-37)C for (24-48) hr. after the colonies grew, the activity (susceptibility& resistance) of each antifungal disks were measured of diameter of the inhibition zone around each discs using the ruler [17]. Statistical analysis: The statistical analysis of this prospective study performed with the statistical package for social sciences (SPSS) 21.0 and Microsoft Excel Categorical data formulated as count and percentage. Chi-square test used to describe the association of these data. RESULTS AND DISCUSSION Distribution of Patients and Controls According To Age and Gender: The mean age of patients with chronic cough was 39.8 ± years, there was statistically significant difference (p>0.05) in gender between both studied groups (figure 1), but there was male predominance among patients, there were 24 (60%) of chronic cough patients males, while only 16 (40%) of patients were females (table 1, figure 2). The present result showed that chronic cough is more prevalent in patients with with mean age (39.8) and this result is consistent with other study reported by Shrimali et al. [12]. Also the current results denoted a predominance of chronic cough among males than females which is comparison with other study conducted by Rai who indicated that chronic cough occurs more commonly in females [3]. The isolation: A total of forty sputum samples from the patient with chronic cough were isolated and identified, the result of fungal growth of the sputum samples are shown in table (3), the isolates are Aspergillus fumigatus 14 (35%), Candida. albicans 12(30%), mixed infection with Aspergillus fumigatus & Candida. albicans 3(7.5%) and no growth 11(27.5%) in patients samples and no growth in healthy controls as clearly shown in Table 2. these results agree with Shrimali et al. showed that among 61 positive culture of sputum samples, 18 culture shows Aspergillus fumigates, 7 are Aspergillus niger, 3 culture shows Aspergillus flavus and 1 case show Aspergillus nidulans (12). Also Daleine et al. was observed that prevalence of Aspergillus species was 15.1% in sputum specimens [18].Pursell and Paredes were found that 4.8% had been colonised with Aspergillus species depend on positive culture of the sputum samples [19]. Johnson concluded that fungal growth and Candida in respiratory

3 secretions can be associated with chronic sputum production, bronchiectasis and mucous plugging. Fungal bronchitis is typically ignored, and lead to significant morbidity and sometimes mortality. Antifungal treatment for Candidal bronchitis led to improvement in patient symptoms and clinical status, while early stopping of treatment lead to recurrence. Controlled clinical experiment are needed to determine whether therapy guidelines should be modified to support effective identification and antifungal treatment for such patients [20], and used the corticosteroids cause impaired clearance and killing of Aspergillus fumigatus conidia, and has also been associated with enhanced growth of Aspergillus species [21]. The spectrum of pulmonary fungal infection is wide. While invasive fungal infection and aspergilloma are known, chronic bronchitis and mucous plugging association to fungal infections are not. Most studies on pulmonary fungal infection refused fungi isolated from sputum, requiring fungus from tissue or pleural fluid culture [22]. There is some recognition that fungi in sputum has adverse health effects. Aspergillus tracheobronchitis characterize aspergillus in sputum with thick mucous plugs [23]. C. albicans cultured from sputum of cystic fibrosis patients is related with increased hospital-treated exacerbations [24]. Many patients had prior bacterial bronchitis or pneumonia. Fungal bronchitis may be a risk factor for bacterial infection. C. albicans is a risk factor for pseudomonas pneumonia in the intensive care unit, prolonged glucocorticoid, longterm antibiotics and diabetes use have increased colonization with C. albicans and other fungi [25]. The susceptibility to antifungal drugs : Regarding the data,it was revealed that Amphotericin B 24(60%), Econazole 19(47.5%), Clotrimazole17(42.5%) respectively were more sensitive and effective antifungal drugs while Ketoconazole 27(67.5%), Fluconazole 26(65%), Nystatin 26(65%) respectively were more resistant and poor antifungal drug activity table 3. Amphotericin B has a wide spectrum of activity that included the majority of yeast and mould isolates, there have been reports submit that some strains of Candida lusitaniae and Trichosporon spp. are less susceptible to amphotericin B than other yeast species [26]. This results agree with study in India conducted carried out antifungal susceptibility testing showed 16.7% were resistant against Fluconazole and 9.5% resistant strains of Candida against Nystatin [27]. Antifungal susceptibility testing methods are useful to detect antifungal resistance and to define the best therapy for a specific fungus. Clinical microbiology depend on these methods to select the therapy for fungal infection, and to know the local and the international epidemiology of antifungal resistance [28].Fungal infections are a major cause of morbidity and mortality in spite of the latest developments of diagnostic tools and treatment options. Early initiation of the correct antifungal treatment has been demonstrated to have a direct effect on the patient s [29]. New chronic lung infections have been described with a wide effect on the patient s quality of life, and a high cost of therapy and care [30]. Candida, Aspergillus, cryptococcus and Pneumocystis are the main etiologic factor of fungal infections [31]. The burden and mortality related with these diseases based on the area and the affected population [32]. for the time being, three main families of antifungals drugs are used clinically to treat fungal infections: polyenes represented by amphotericin B, azoles with differents derivatives such as itraconazole, fluconazole, voriconazole, posaconazole, isavuconazole; and the echinocandins caspofungin, micafungin and anidulafungin. The increased use of antifungals drugs has encouraged a higher selective pressure on fungal strains and resistance has arised in two main ways: several species have developed secondary resistance and susceptible species have been replaced by resistant ones, changing the epidemiology of fungal infections [33]. Antifungal resistance is becoming an emerging problem. On the one hand, there is the intrinsic resistance, and on the other hand the development of secondary resistance that should be diagnosed because resistant strains are related with poorer outcomes, intrinsic resistance of C. glabrata and C. krusei to fluconazole is well known [28]. During antifungal treatment acquired resistance in Candida spp. infections has also been reported and C. albicans, C. tropicalis and C. krusei, have also confirmed for developing secondary resistance [34]. Change on genes encoding the target enzymes of these therapy (14 alpha sterol demethylase (ERG11) or up regulation of multidrug efflux transporters for azoles groups [35]. CONCLUSION The present study revealed that Aspergillus fumigatus was he present study revealed that Aspergillus fumigatus was more species isolates from sputum samples from the patients with chronic cough and in vitro antifungal sensitivity test used to determine antifungal resistance and define the best therapy for a specific fungus, Amphotericin B was more sensitive and effective antifungal drugs while Fluconazole was more resistant and poor antifungal drug activity for the fungi isolated from sputum from the patients with chronic cough. 15

4 Figure -1: Distribution of chronic cough Patient and Healthy Control according the age Table-1: Distribution of chronic cough Patient and Healthy Control According the Gender Study Control Gender No. % No. % Male Female Total Figure-2: Distribution of chronic cough Patient and Healthy Control According the Gender Table-2-Isolation and identification of fungal culture from sputum samples and controls Species No. sample % Healthy control % Aspergillus.fumigatus Aspergillus + Candida albicans Candida. albicans NO growth Total Antifungal drugs Table-3- the sensitive and resistant of antifungal drugs Resistant Sensitive No. % No. % Chi-square 16 P-value AMP P<0.01 NS P<0.01 KT P<0.01 FLC P<0.01 ECO P<0.01 MIC P<0.01 CLO P<0.01 *High significant

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