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Abstract Pulmonary mycosis (PM) is an infectious process in the lungs caused by one or more opportunistic fungi. Fungal colonization and infection occur following the inhalation of spores. Case mortality associated with PM can be as high as 75% in immunodeficient persons. Fungal infections in lung often pose a great diagnostic challenge due to the absence of characteristic pathognomonic and radiological features. In the last decade, mycosis became a significant healthcare concern due to the rampant use of broad- spectrum antimicrobials, prolong use of immunosuppressive drugs, increased cases of critically ill, and immunodeficient persons such as HIV/AIDS. Fungi produce mycotoxins as nonvolatile secondary metabolites and volatile organic compounds as byproducts of metabolism, The volatile organic compounds are potential asthma triggers, pose a serious health hazard for humans and animals. Presence of allergenic and toxigenic fungi has previously been reported in damp homes and offices. The aim of the present study was to: isolate and identify fungal isolates from the respiratory tract of COPD patients, investigate the predisposing factors, clinical characteristics, microbiology and suggest the most effective antifungal agents against the recovered isolates. Also the present study aimed at confirming the severity of fungal infection through a histopathological study and investigating the mycotoxigenic capacity by the isolated fungal species from the post mortem lung samples in albino mice. Summary 133 The study is a laboratory-based study conducted between October 2017 to September 2020 at Ain Shams University Hospitals (Ain-Shams Specialized Hospital, Al-Demrdash hospital) located in Cairo and Zagazaig University Hospitals located in Al Sharkia governorate. This is a prospective cross-sectional analytical study. All the procedures used in this study were approved by the research ethics committee of faculty of Science, Ain Shams University, Egypt. This study was carried out on Two hundred and fifty Egyptian (250) patients. Their ages were more than 20 years old, they were admitted to the emergency room, hospital wards, intensive care units (ICU), with chronic obstructive pulmonary disease (COPD), and have history, examination and investigations suggestive of fungal infection. Their microbiology and pathology laboratories records were reviewed daily. Clinical samples composed of sputum, Bronchoalveolar lavage (BAL) and endotracheal tube (ETT). The following data were collected from patient’s medical files: Causes of admission, duration of mechanical ventilation, length of ICU stay, underlying disease, and recovery of another respiratory pathogen. The distribution of fungal isolates on the basis of some risk factors showed that the highest percentage (36.6%) was for diabetes mellitus. The second most common risk factor identified in this study was smoking (25.9%). Prolonged antibiotic usage was found to be an important risk factor (11.8 % of the patients). Prolonged corticosteroid antibiotic usage also a risk factor for pulmonary fungal infection (11.8 % of the patients). HIV infection and chemotherapy administration were also risk factors for developing respiratory fungal disease, with percentage of 7.5% and 5.4% respectively. Summary 134 Sputum samples were collected in 150 patients by instructing them to cough and expectorate about 5–10 ml in a sterile container usually early in the morning. Bronchoalveolar lavage (BAL): BAL & endotracheal tube ETT samples were collected only from 100 patients who were mechanically ventilated, with the help of a white light flexible bronchoscope attached to a light source and digital camera. All samples were labeled with individual identity numbers and were opened aseptically at the time of analysis. For recovery of fungal pathogens, the specimens were cultured onto Sabouraud dextrose agar (Himedia-India) plates with 0.05 mg/ml of chloramphenicol disc and other media. Plates were incubated at 28±2°C for 1-4 weeks and were examined twice weekly. Macro and Micro-Morphological as well as molecular identification for the fungal species were performed for the fungal isolates. Out of the 250 patients included in this study, only 93 cases (37.20%) were identified as having respiratory fungal infections. In this study, most of the fungal agents were of the filamentous type (70.97%), whereas Candida spp. comprised 29.03% of the causative agents. A. fumigatus and A. flavus were the most prevalent mould fungal pathogens (18.28% for each). Also, molecular identification of the most potent producers of mycotoxins, fungal pathogens was conducted. Agarose gel electrophoresis was performed for the PCR amplified DNA product obtained from the fungal isolates. Phylogenetic tree based on 18S rRNA sequences depicting the relationship between the isolated strains was also performed. Summary 135 Susceptibility testing of pathogenic fungi to different antifungal drugs was conducted. The antifungal testing revealed that Ketoconazole exhibited the highest effect against Candida albicans. Itraconazole exhibited the highest effect against Candida parapsilosis. Amphotricin B and Itraconazole exhibited the most effect against Candida tropicalis. Caspofungin and Itraconazole exhibited the most effect against Candida krusei. Ketoconazole exhibited the highest effect against A. niger, A. flavus and A. fumigatus. Caspofungin and Flucytosine exhibited the highest effect against A. cristatus. Ketoconazole exhibited the highest effect against Pencillium spp. Ketoconazole had the highest antifungal action against the commonest fungal pathogens; Aspergillus spp., Pencillium spp. and Candida albicans. Four groups of isolates (Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, and Penicillium sp.) were examined for the presence of 6 mycotoxins (AFB1, ochratoxin A, citrinin, patulin, cyclopiazonic acid, kojic acid). This study proved that human respiratory system is susceptible to AFB1 and accumulation from A. flavus strains, and the highest mean yield of AFB1 (93.7+0.51 μg/L) was detected in A. flavus isolate number 59 (A. flavus Ali 1), at mean dry weight of 1.03+ 0.01 g/L. Summary 136 Ochratoxin A was extracted from A. fumigatus, A. niger, and Penicillium sp. at 214 nm. The mean highest concentration of Ochratoxin A (28.7 + 0.35 μg/L) was extracted from isolate number 33 (A. fumigatus Ali 2), with dry weight of 0.83 + 0.01 g/L. While, the isolate number 90 of A. niger was produced the highest concentration of Ochratoxin A (67.9 + 0.3 μg/L), with dry weight of 1.33 + 0.46 g/L. The isolate number 23 of Penicillium sp. (Penicillium chrysogenum Ali8) was produced the highest concentration of Ochratoxin A (54+1.0 μg/L) with dry weight of 0.82+0.03 g/L. Citrinin was extracted from Penicillium sp. at 366 nm. The mean highest concentration (45+1.0 μg/L) was extracted from isolate number 73 (Penicillium crustosum Ali 3) with dry weight of 1.0+ 0.03 g/L. Patulin was extracted from Penicillium sp. at 540 nm. The mean highest concentration extracted from Penicillium sp was 63.3 + 1.5 μg/L, it was produced at mean dry weight of 1 + 0.03 g/L. Cyclopiazonic acid was extracted from A. flavus, A. fumigatus, and Penicillium sp. at 550 nm. The mean highest concentration extracted from A. flavus was 48.3 +0.58 μg/L, it was produced from the isolate number 59 (A. flavus Ali 1) at mean dry weight of 1.03+ 0.01 g/L. The mean highest concentration extracted from A. fumigatus was 38+1.0 μg/L, it was produced from the isolate number 33 (A. fumigatus Ali 2) at mean dry weight of 0.83 + 0.01g/L. The mean highest concentration extracted from Penicillium sp was 45.7+2.5 μg/L, it was produced from the isolate number 23 (Penicillium chrysogenumAli8) with fungal dry weight of 0.82+ 0.03g/L. Kojic acid was extracted from A. flavus, A. niger, and Penicillium sp. at 540 nm. The mean highest concentration of Kojic acid extracted from A. flavus was 496.3 + 2.5 μg/L, it was produced from the isolate number 59 (A. flavus Ali 1) with mycelial dry weight of 1.03+ 0.01 g/L. Summary 137 The mean highest concentration extracted from A. niger was 95.3+3.2 μg/L, it was produced from the isolate number 90 (A. nigerAli 4) with fungal dry weight of 1.33 + 0.46 g/L. The mean highest concentration extracted from Penicillium sp was 164.3 + 4.04 μg/L, it was produced from the isolate number 23 (Penicillium chrysogenumAli8) with fungal dry weight of 0.82+ 0.03g/L. In histopathological examination, 35 male albino mice Balb/c weighing about 18-25 g were obtained from the animal house unit of Suez Canal University. The rooms and cages used for housing the animals were thoroughly cleaned time to time. Mice were reared under strict hygienic conditions during and before infecting them. All mice were adjudged to be healthy. Mice were provided with food and water ad libitum. Mice were observed daily for any morbidity or mortality. Another mice model was examined for the effect of the extracted mycotoxins (Ochratoxin A, AFB1, citrinin, patulin, CPA& Kojic acid) after being immunosuppressed. Histopathological examination was completed as with fungal spores. Lung tissue changes in abino mice were evident in the form of high density fungal conidia and proliferating hyphae, surrounded by necrotic lung tissue and damaged alveoli. As regards the mycotoxins effects, there were inflammatory cells infiltration in the interstitial space, interalveolar septae, alveolar space, intrabronchial and intravascular as well as dilated congested blood vessels, thickened alveolar wall and thickened bronchial wall. Data were analyzed using the SPSS statistical software (IBM Corp., Armonk, NY, USA). Numerical data were expressed as mean+SD, while qualitative data were presented as count (%), the appropriate tests were used and statistical significance was considered at p<0.05. Conclusion 138 Conclusion A high prevalence of pulmonary fungal pathogens was found in patients with COPD in this study. Of this, A. flavus and A. fumigatus were the predominant isolates. Pulmonary fungal infections were more prevalent at the older age group and males showed slightly higher contribution. Some risk factors were found to be related to the prevalence of pulmonary fungal infections, they were mainly contributing in creation of an immunosuppression state. In order of the frequency, they were diabetes mellitus, smoking, prolonged antibiotic use, prolonged corticosteroid use, HIV and chemotherapy. Ketoconazole had the highest antifungal action against the commonest fungal pathogens; Aspergillus spp., Pencillium spp. and Candida albicans. Human respiratory system is susceptible to AFB1, Ochratoxin A, Citrinin, Patulin, CPA and Kojic acid accumulation from colonized fungal strains, which predispose to several health hazards. The inflammatory and destructive effects of the fungal infecting pathogens as well as their mycotoxins were proved in the histopathological examination. Recommendations 139 Recommendations We recommend the followings: Further studies on larger number of patients are recommended. Being life threatening, every effort should be prompted for the early detection and elimination of fungal infections. Attention should be paid to pick up cases with fungal infections superimposed on the underlying chronic lung diseases, not to add extramorbidity. Special care should be provided to COPD patients, with more focused consideration to the older age groups and patients with risk factors. Efforts should be subjected to introduce more save derivatives of the drug ketoconazole, as it proved the highest efficacy. |