Fungal contamination of Azadi Teaching Hospital and Hevi Paediatric Hospital Environments, Duhok, Iraq

This survey study was undertaken to find out the prevalence and distribution of fungi occupying the residential surfaces in Azadi Teaching Hospital and Hevi Paediatric Hospital in Duhok province, Iraq. Three hundred samples were collected from randomly selected areas in laboratories, wards, consultation rooms, operating theatres and hospital kitchen by swabs. These samples were cultured on two selective fungal media (PDA and SDA) and also samples from air were cultured on PDA in order to determine the genus or species of the agents in the samples. The results found microorganisms that were identified included forty eight isolates in addition to non identified yeasts and sterile mycelium also detected. From 300 samples collected from period January to May 2016, molds comprised 60% and yeasts comprised 40%. All fungal isolates were identified depending on the morphological and microscopic examinations as well as biochemical tests. The environmental contaminations in Azadi Teaching and Hevi Paediatric Hospitals with fungi ( Aspergillus ) were the highest fungal isolates whereas Candida sp. was the highest yeast isolate.


Introduction
Fungi are ubiquitous in the environment but they rarely cause symptoms in human beings due to the effective defence mechanisms, such as the cellmediated response.Different fungi causing infections, some are commonly occurring infections while the others are rare 1 .Fungal contamination in healthcare facilities has been the subject of numerous studies.These have shown that high percentages of hospital infections are caused by fungi, such as Candida sp. and various species of Aspergillus, Penicillium, and Cladosporium 2 .Hospitals are considered the favourite places for the emergence of a wide variety of opportunistic and pathogenic fungi; these fungi are ubiquitous and can be acquired from host surroundings or are components of normal endogenous flora.However, any decreasing in the host organism's defensive ability due to cancer, HIV infection 3-4 , medical therapy and organ transplantation may lead to uncontrolled multiplication of fungi and consequent onset of infection.Among kidney transplant recipients, 11% of infections were of fungal origin.In 47% of such cases, infection occurred during the first three months after transplantation 5 .After solid organ transplantation, 80% of fungal infections involved Candida and Aspergillus species.Among immunocompromised patients in general, the incidence of invasive Aspergillosis and other fungal diseases have increased during the last few decades which remains a serious complication and could be fatal 6 .Airborne fungal conidia are inhaled by everyone, because their concentration is high in the air 7 , this may be the reason why nosocomial acquired infections and community acquired infections quite often develop in immunocompromised and in immunocompetent people 8 .
The aim of this study was to determine the level of fungal contamination in Azadi Teaching Hospital and Hevi Paediatric Hospital in Duhok province.

Samples collection
From first January to end of May 2016, 300 samples were taken using sterile transport media swabs from various environments including operating theatres, intensive care, laboratories, outpatient departments, kitchens, and other working environments like nurses' offices, and refectories in both hospitals.

Culturing laboratory:
Swabs cultivated directly on the media culture appropriate (Sabouroad dextrose agar, Malt extract agar and Potato dextrose agar) and exposure of PDA plates to air.The dishes were incubated at 25 and 37 ºC for 7-35 days.By the end of this period, the cultures examined for any growth and then subculture of the dishes that have shown positive results 9 .

Identification of Fungi :
During incubation period, different fungal colonies were subjected to macroscopic and microscopic to observe their growth, mycelium nature and structure of hyphae.Filamentous fungal growth -as mold and yeast-that grow on SDA, MEA and PDA were subcultured on separate SDA culture plates.One plate was incubated at 37° C, and the remaining was incubated at 25° C 10 .Pure culture growth of each mold and yeast colony was examined under magnification for their microscopic structures and cross identified by using mycological keys manuals 11- 17 .Biochemical tests used for identification of candida species 38 .

Frequency Percentage:
The percentage frequency of species isolated was calculated by applying the following formula.

Occurrence Percentage:
The percentage for the Occurrence of each species isolated was calculated according to the following equation.

Results and Discussion
Table-1 shows number of swabs, percentages of contamination in both Azadi Teaching and Hevi Paediatric Hospitals.As shown in the table the percentage of positive contamination in Azadi Hospital is more than the percentage of contamination in Hevi Paediatric Hospital, this could due to the high number of patients occupied in this hospital.13), and Rhizopus sp.(11).
The difference between the numbers of fungi isolated on both media was expected because PDA is a selective medium for luxuriant growth of yeasts and molds.Potato infusion encourages mold sporulation and pigment production even in some dermatophytes 18 .PDA enhances the production of reproductive structures (conidia) and colony colour when other media fail to do so; it is often recommended for slide culture or for inducing an isolate to exhibit a characteristic pigment 19 .Because of low pH SDA is useful for the cultivation of fungi particularly those associated with skin infections 20 .(1995) found that 70 -80% of the fungi in hospitals air were Aspergillus sp.Our study showed similar distribution of the mold and yeast species 23 .Penicillium was second in the number of species isolated and was represented by ten species.These species are usually found in indoor environment and cause infections to humans and animals.This study has shown Candida fungi were isolated from indoor environment in all selected units.As a rule, candidiasis is an endogenous infection; however, exogenous infections are also possible.Numerous studies have shown evidence that Candida fungi are detected in the indoor air at e.g.surgical, haematological and obstetric wards; they are potential and major source of infections, especially in risk group patients [24][25][26] .Another yeast-like organism like Geotrichum candidum and Aurobasidium are opportunistic organism that causes pulmonary and systemic infections in human and animals.Alternaria species are emerging as opportunistic pathogens.A case of Phaeohyphomycosis caused by A. infectoria in a renal transplant recipient has been reported 27 .Chrysosporium species have been isolated in this study and some were reported to cause superficial skin infection.Microascus species are considered as potentially pathogenic to human beings and are frequently isolated from floor dusts

Conclusion
In conclusion, the present study revealed the presence of potentially pathogenic and toxigenic fungi in both hospitals in Duhok province.Strict hygienic measures should therefore be undertaken to control the spread of these fungi in the environment and to avoid mycotic infection among the patients and staff alike.Check air conditioning systems for standing moisture and clean or replace as necessary.

Table 1 -The number of swabs and percentages of contamination. Hospitals Azadi Hevi Total
Table-2 shows the genera of fungi isolated from both hospitals using two media PDA and SDA by swab spreading method and exposure of plates of PDA to air for 15 minutes.The results shown thirty two species of fungi in addition to sterile mycelium and non identified yeast have been demonstrated.Thirty species of fungi were isolated on PDA using swab method; the highest number obtained was Aspergillus sp.(264), Penicillium sp.(125), Ulocladium sp.(86), Cladosporium sp.(75), followed by Candida sp.(69) and Rhizopus (35).Twenty seven species of fungi were isolated on PDA plates exposure to air, the highest number obtained was Aspergillus sp.(71), Ulocladium sp.(39), Penicillium sp.(30), followed by Cladosporium sp.(25) and Candida sp.(17).Twenty two species of fungi were isolated on SDA using swab method; the highest number obtained was Aspergillus sp.(55), Penicillium sp.(39), Candida sp.(19), Dermatophytes (16) followed by Ulocladium sp. (

Table ( 2): Isolated Fungi by different culture media.
Mucor, yeast and Fusarium species Aspergillus was represented by 12 species and showed the widest diversity among all recovered genera.A. niger, A. flavus and A. fumigatus showed the high frequency and occurrence percentages because these species are ubiquitous and the major airborne contaminant fungi.Some species can cause infection in humans and animals.The most common pathogenic species are A. fumigatus and A. flavus.A. fumigatus infections are primary pulmonary infections includes Aspergillosis, Pneumonia, Asthma and Allergy 21 .A. flavus cause infection of the external ear and Aspergillosis 22 .Overberger et al.

28-30 .
31gomycetes fungi isolated in this study include Absidia, Mucor and Rhizopus that cause brain infections via nose31.Many fungi isolated from both hospitals like A. flavus, A. ochraceus, A. parasiticus, A. fumigatus, Emericella nidulans, Fusarium and Stachybotrys secrete secondary metabolites which harm human and animals during inhalation and are known as mycotoxins 32-33 Dermatophytes also were isolated in this study including Microsporum, Epidermphyto and Trichophyton; however, some people are at greater risk than others.The fungus takes advantage of skin of those patients with reduced immune capacity.Our results in this study are in line with many studies