A survey on the clinical spectrum of Aspergillus infections in Isfahan, Iran, during 2010-2018

Background Aspergillosis is a fungal disease affecting millions of individuals worldwide. The genus Aspergillus comprises various complexes found in the nature, and cause a wide spectrum of diseases from superficial infections in immunocompetent hosts to life-threatening acute invasive or disseminated infections among immunocompromised patients. Methods A total of 2385 suspected cases were included in this retrospective study from January 2010 to December 2018. Direct microscopic examination with potassium hydroxide, culture on Sabouraud dextrose agar containing chloramphenicol and czapekdox agar media, and PCR-sequencing of ITS1-5.8SrDNA-ITS2 region were applied for identification of etiologic agents. Results One hundred and thirty-two out of 2385 suspected cases had Aspergillus infection (5.5%). Fifty-four patients were male and 78 patients were female. Patients in the age groups of 41–50 and 21-30 years had the highest and lowest frequencies, respectively. Leukaemia (6.8%), hemodialysis (6.8%), diabetes mellitus (6%), and neutropenia (5.3%) were the most predisposing factors among patients. Aspergillus flavus (n=54), A. fumigatus (n=24), A. niger (n=15), and A. terreus (n=12) were the most prevalent Aspergillus species, respectively.


Introduction
Aspergillosis is a wide spectrum fungal disease affecting millions of individuals worldwide, ranging from mycotoxicosis, onychomycosis, and allergic syndromes in immunocompetent hosts, to life-threatening acute invasive or systemic infections among immunosuppressed patients, such as solid organ transplant recipients, patients undergoing haematopoietic stem cell transplant (HSCT) or chemotherapy, and who are taking corticosteroids (1,2).
Aspergillus species are ubiquitous saprophytic molds contains four subgenera, which are subdivided into several Sect. (3). In clinical mycology laboratories, identification of Aspergillus species relies principally on morphological criteria, including microscopic traits such as the shape of conidiogenous cell, conidiophore, conidial germination, and mycelial structures and macroscopic features like color, surface topography, and texture of colonies (4). In recent years, molecular diagnosis in clinical laboratories has been applied for precise identification and better management of infection (5,6). In this 9-year retrospective study, we aimed to investigate the prevalence of Aspergillus infections, species identification, and clinical manifestations of patients registered in a mycology reference laboratory in Isfahan, Iran, during 2010-2018. These cases were housekeepers (n=691), farmers (n=471), freelancers (n=382), employees (n=308), students (n=193), unemployed (n=106), retirees (n=98), children (69), and unknown (n=67). Patients, who had taken antifungal drugs for the past 7 days, were excluded from the study.
The specimens obtained from superficial lesions, and deep tissues were incubated at 30°C, and 37°C, respectively, and checked for the fungal growth up to 21 days (7).
Morphological characters for identification of Aspergillus species contain the rate of colony growth, degree of sporulation, production of cleistothecia or sclerotia, texture of colonies, colors of the hypha, pigmentation, and colony reverses (8). Discriminative features of conidiophore and conidial heads among various Aspergillus species are: the dimension, shape, texture, and color of the stipe, the shape and size of vesicles, phialides (uniseriate or biseriate), the absence or presence of metulae between phialides and vesicle, conidia formation, and Hülle-cells (if present).

Molecular species identification
Between January 2016 and December 2018, all clinical isolates were identified using PCRsequencing technique.

PCR
Genomic DNA from clinical sample was extracted using a phenol / chloroform technique an initial denaturation phase at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 45 s, and extension at 72°C for 1 min, with a final extension phase at 72°C for 7 min. Six microliter of PCR products was loaded on 1.5% agarose gel, and stained with 0.5 μg/ml ethidium bromide, then visualized by gel documentation system (UVITEC, UK) and photographed.

Data analysis
The results were analyzed by Chi square and Fisher's exact test in the SPSS version 23.

Results
One hundred and thirty-two out of 2385 suspected cases had Aspergillus infection (5.5%) based on the positive DME and culture (n = 90), and molecular analysis (n = 42). Figure 1 shows the microscopic futures of some Aspergillus spp.      (14), Brazil (15) and arid regions of the Middle East (16). A. niger and A. terreus rank second in Korea, and Austria, respectively (17,18). Contrary to these findings, A. flavus was the most common Aspergillus species in the present investigation. Most of the time, we infected with this fungal disease by daily inhalation of hundreds of spores, and colonization of fungus in the respiratory tract; if our immune system is suppressed. Agree with this concept, we also obtained the most Aspergillus species from BAL samples in the present study. The fungal cell wall component galactomannans (GM) are polysaccharides including a mannose backbone with galactose as side groups that are released during the hyphal growth (19). It is a biologic marker and used as a noninvasive method for the detection of aspergillosis in blood or BAL. One of the major limitations of this study was the lack of information about this test among patients. CPA is a chronic disease which may last for years and found mostly in middle-aged individuals who are not immunocompromised. The common clinical manifestations are not specific and include fever, weight loss, malaise, fatigue, chronic cough, empyema, pleural thickening, and haemoptysis (20). In the present study, two CPA patients recognized with chronic cough, empyema, and haemoptysis, however; one of them had leukemia. The causative agents of two cases were A. flavus. Aspergillus-induced asthma (AIA) has a wide variation among different populations of 16-38% (21,22), nevertheless; only 3% of patients had AIA in the present investigation. Cystic fibrosis is the most popular congenital disease in white populations, resulting in viscous sputum which encourages the fungal conidia trapping (23). ABPA is the most important sign of allergic aspergillosis with worldwide distribution 9 that often seen in patients with cystic fibrosis and asthma (24

Conclusion
With regard to the present study, Aspergillus infections contain a wide spectrum of clinical manifestations, and often occur in immunocompromised patients. Since, people with immunodeficiency disorders are on the rise, therefore, opportunistic fungi have been noticed as one of the main concerns of morbidity and mortality in such patients and one should pay much attention to this subject. Epidemiological data from various areas seems to be essential for physicians and health professionals to find suitable clues to control and break the cycle of transmission which ultimately helps the treatment and management of