This prospective and analytical study was carried out at Microbiology Laboratory, Department of Microbiology, Mahatma Gandhi Mission (MGM) Medical College and Hospital, Navi Mumbai, India, over a period of one year from July 2014 to June 2015. Informed consent was taken from each patient and parent/care taker if age below 18 years before starting the study. Total 350 patients were enrolled for this study out of which males were 232 and females 118 and the age group from 10 years to 70 years with mean age group 40 years, the samples distribution was sputum samples (n=99), nasal and paranasal sinus (n=65), pus samples (n=63), urine (n=51), Bronchoalveolar Lavage (BAL) (n=18), ear (n=29) and eye (n=25). All 350 samples were collected from suspected patients of invasive aspergillosis attending MGM hospital with symptoms of wheezing, shortness of breath, chest pain, cough, headache, weight loss and fever. Patients already on antifungal treatment were excluded from the study. Total 400 environmental samples were taken for the study, the samples were taken from chest TB ward (n=22), medicine ward n=23), ENT ward (n=21), surgery ward (n=13), burns ward (n=17), dermatology ward (n=53), ophthalmology ward (n=34), Medical Intensive Care Unit (MICU) (n=71), Surgical Intensive Care Unit (SICU) (n=78), Intensive Care Unit (ICU) (n=39) and Pediatric Intensive Care Unit (PICU) (n=29). All samples were collected from environment by sterile swabs moisted with sterile peptone water by using template with dimensions 10 × 10 cm [13].

ATCC control strain of Aspergillus niger (ATCC^® 6275^TM), Aspergillus oryzae (ATCC^® 10124^TM) and Aspergillus brasiliensis (ATCC^® 16404^TM) was obtained from Microbiologics Inc, USA.

For detection of virulence factors in Aspergillus species in patients samples – 3-5 ml sputum, 10-20 ml urine collected in sterile container, swabs from nasal and paranasal sinuses, pus, ear and eye were collected by sterile swabs and 20-30 ml Bronchoalveolar Lavage (BAL) samples were collected with bronchoscope by trained physicians.

All patient samples (sputum, nasal and paranasal sinuses, pus, ear swab and eye swab and BAL) and swabs from environmental samples (table, light, anaesthesia trolley, floor and wall), were inoculated onto two plates of Sabouraud’s Dextrose Agar (SDA) with chloramphenicol; one inoculated plate was incubated at 28°C and the other at 37°C for two to seven days. All isolates were identified by the colony characteristics and morphological appearance. The identification was confirmed by the conidial morphology by preparing slides with one drop of lactophenol cotton blue stain and observed under 40X by light microscope. The identification of Aspergillus species was conducted according to Raper KB et al., Ellis D et al., and Afzal H et al., as colonies were detected [14-16]; they were subcultured on Czapeck’s Dox Agar (CZA) media for specific species identification according to colony characteristics and microscopical characteristics.

Various virulence factors were studied in Aspergillus isolates from patients and environment by standard phenotypic methods such as biofilm formation (by tube method) [17] adherent biofilm layer was scored visually as either negative or weakly (1+), moderately (2+ or 3+), or strongly (4+) positive, lipase production (using T80 agar plates with phenol red) [18], a change in the colour from pink to lemon yellow was observed. α-amylase production (using Starch Agar Plates) [19], observation of hydrolysis, pectinolytic activity (using pectin plates) [20], iodine solution was added to detect the clear zone, proteinolytic activity (using biuret method) [21] recorded the value “x” of unknown from graph corresponding to the optical density reading of the test sample [Table/Fig-1,2], phospholipase activity (using egg yolk agar) [11] the dense white zone of precipitation around the colonies of phospholipase – positive isolates was distinctive and well defined, haemolysis activity (using blood plate assay) was assessed [11]. Similar methods were used for ATCC control of Aspergillus niger (ATCC^® 6275^TM), Aspergillus oryzae (ATCC^® 10124^TM) and Aspergillus brasiliensis (ATCC^® 16404^TM) for detection of different virulence factors [11,17-21].

Ethical clearance was obtained from the Institutional Review Ethics Committee of MGM Institute of Health Sciences, Navi Mumbai, once reviewal of the scientific and ethical consideration of proposal for the clinical study was done. Written informed consent was obtained from each patient separately before starting the project.

Comparison of categorical variables was made by using chi-square test, p-values <0.05 were considered statistically significant. The statistical analyses were performed using SPSS, version 16.0.

Out of 750 samples only in 78 samples showed pure growth of Aspergillus species and 672 did not show pure growth, however they showed mixed growth of other fungi and bacteria which was not included in the study. Out of 350 patients samples only 39 Aspergillus isolates and from 400 environmental samples only 39 Aspergillus isolates were found suitable for the study of virulence factor.

ATCC control of A. niger, A. oryzae and A. brasiliensis showed presence of all virulence factors (biofilm formation, α-amylase production, pectinolytic activity, proteinolytic activity, lipase activity, phospholipase activity and haemolysis activity) which were tested for patients and environmental samples.

Total 78 Aspergillus isolates were included for study out of which 39 from patient samples and 39 from environment samples. Maximum number of Aspergillus niger (n=38), followed by Aspergillus fumigatus (n=18), Aspergillus flavus (n=12), Aspergillus brasiliensis (n=6) and Aspergillus terrus (n=4) were isolated.

In patient samples maximum number of Aspergillus species isolated from sputum (n=15) followed by nasal and paranasal sinuses (n=7), pus (n=7), bronchoalveolar lavage (n=3), ear (n=3), eye (n=2), urine (n=2).

In environment samples maximum number of Aspergillus species isolated from chest TB ward (n=7), medicine ward (n=7) followed by ENT ward (n=6), surgery ward (n=3), dermatology ward (n=3), opthalmology ward (n=3), MICU (n=3), burns ward (n=2), SICU (n=2), ICU (n=2), PICU (n=1).

Comparison of the production of virulence factors in patient as well as environment samples has been depicted in [Table/Fig-3].

Biofilm production was found in 32 out of 39 patient samples i.e., 82.05% [Table/Fig-4] as compared to environmental samples 15 out of 39 i.e., 38.46%. Lipase activity was seen in 26 out of 39 [Table/Fig-5]. Samples of patients i.e., 66.66% as compared to environmental samples 11 out of 39 i.e., 28.20%. In patients samples α amylase activity was seen in 35 out of 39 [Table/Fig-6] i.e., 89.74% as compared to environmental samples 14 out of 39 i.e., 35.89%. Pectinolytic activity was seen in 24 out of 39 [Table/Fig-7] patients samples i.e., 61.53% as compared to environmental samples 9 out of 39 i.e., 23.07%. Proteinolytic activity was seen in 34 out of 39 patients [Table/Fig-8] samples i.e., 87.17% as compared to environmental samples 16 out of 39 i.e., 41.02%. Phospholipase activity was seen in 24 out of 39 patients samples [Table/Fig-9] i.e., 61.53% as compared to environmental samples 10 out of 39 i.e., 25.64%. Haemolysin activity was seen in 31 out of 39 patients samples [Table/Fig-10] i.e., 79.48% as compared to environmental samples 13 out of 39 i.e., 33.33%. Comparison of virulence factors in Aspergillus species from patent and environmental samples showed, statistically significant (p-value <0.05) [Table/Fig-3].

[Table/Fig-4]:

Showing biofilm production by Aspergillus species, tube 1- negative, Tube 2 to tube 3 weak (+), tube 4 – moderate (2 or 3+) and tube 5 – stong (4+) biofilm production.

[Table/Fig-5]:

Showing lipase activity on Tween 80 agar medium: a) Showed negative for lipase activity; b) Showed positive for lipase activity.

[Table/Fig-6]:

Aspergillus spp. showing alpha amylase production: a) Plate: control; b) Plate: positive for alpha amylase.

[Table/Fig-7]:

Aspergillus spp. showing pectinolytic esterase activity: a) Plate: negative for pectinolytic esterase activity; b) Plate: positive for pectinolytic esterase activity.

[Table/Fig-8]:

Aspergillus spp. showing proteinase activity. Tube 4: control, Tube 5: negative for proteinase activity, Tube 6: positive for proteinase activity;

[Table/Fig-9]:

Aspergillus niger showing phospholipase activity. Colony 1: negative control, colony 2: negative for phospholipase activity, colony 3 and 4: positive for phospholipase activity, colony 5: positive control. (Images left to right)

[Table/Fig-10]:

Aspergillus species showing beta type of haemolytic activity on blood agar. a) Front view of blood agar, b) Back view of blood agar.

Biofim production, lipase activity, pectinolytic activity, proteinolytic activity, phospholipase activity and haemolysin activity for all isolates of aspergillus in patient samples and environment samples are shown in [Table/Fig-11,12] respectively.

Virulence factors i.e., biofilm, lipase, α-amylase, pectinase, proteinase, phospholipase and haemolysin in patients and environmental samples showed great variation. In patients samples, the virulence factors showed in maximum number, whereas in environmental samples it presents in less number [Table/Fig-11,12].

Various virulence factors – biofilm, proteinase, haemolysin, α amylase, phospholipase, lipase, pectinase were studied. The principle mode of action of these virulence factors is as follows – Various virulence factors cause degradation of tissue carbohydrate (α–amylase), protein (proteinase), phospholipids (phospholipase), pectins (pectinase), and lipids (lipase). Biofilm formation helps prevention of phagocytosis of aspergilli and allows exporential growth [22]. Haemolysin causes lysis of red blood cells. Similarly, cytotoxic and cytolytic lesions are produced by aspergilli.

These virulence factors along with toxins initially cause derangement of cellular function, gradually progressing to necrosis/lysis of cells and tissues involving greater portion of organ and finally leading to organ failure.

Study of virulence factors in our work revealed that these virulence factors are present in Aspergillus isolates from patients as well as environment.

Virulence factors in decreasing order in patient isolates is as follows – α–amylase, proteinase, biofilm, haemolysin, lipase, phospholipase, pectinase.

However, the same decreasing order is not seen in environment samples and number of positivity is also less as compared to patient samples.

It appears that virulence factors are inherently present in environment samples and active in small percentage for survival in environment. But on entry in human tissues, their activity is enhanced to oppose the adverse condition, protective mechanism of human bodies which tries to eliminate the microorganisms. Hence, for survival in human tissues, the virulence factors activity is geared up to oppose human protective mechanisms.

Mezher MA et al., studied on 62 patient’s sputum samples, out of which 19 (30.6%) were showed growth of Aspergillus species [11]. Maximum isolates were A. fumigatus (12.9%) followed by A. terreus (6.5%), A. niger and A. flavus (1.6%) each. They reported that A. fumigatus showed production of haemolysin and phospholipase 62.5% each and protease production 87.5%, while A. terreus showed production of protease and phospholipase 50% each and haemolysin 25%, while A. flavus, showed no production of haemolysin, phospholipase protease. All isolates of A. niger showed phospolipase production (100%). Gharamah AA et al., sudied on 115 patients samples out of which 25 were Aspergillus species isolated, protease activity (80%), lipase (84%) and haemolysis (28%) [23]. Mythili A et al., studied on 108 clinical samples out of which 60 fungal isolates were obtained [24]. Highest lipase activity, protease activity, α-amylase activity and pectinase activity was also prominent for corneal isolate of Aspergillus species. Birinci A et al., studied on 30 A. fumigatus, nine A. flavus and four A. niger strains isolated from clinical specimens [25]. A. fumigatus showed proteinase 76.7% (23/30) and phospholipase activity 93.3% (28/30). A. flavus isolates did not exhibit proteinase or phospholipase activity. A. niger showed phospholipase activity (25%) but did not exhibit proteinase activity.

Aspergillus species also play an important role in recyclation of global carbon and nitrogen, and are mainly found in soil or decaying vegetation, the fungi can produce a small, hydrophobic conidia that can be easily circulated and remain present in the air and may survive even an unfavorable environmental condition for a long time, at this stage it can be transferred into human through inhalation. Aspergillus contains 200 identified species, out of which 20 or less than that are known to cause human infection [26].

Pathogenesis of aspergillosis is dependent on various factors of the host (immune status) and virulence factors of the pathogen. Some putative virulence factors have been identified for different Aspergillus species. These include adhesions e.g., biofilm production and haemolysin, pigments hydrolytic enzymes such as proteases, proteinase, lipase, phospholipases, α-amylase, low-molecular-weight, non-protein metabolites [4-6]. These virulence factors could play a significant role in the pathogenesis of invasive aspergillosis [7-10]. Virulence factors of Aspergillus species may confirm the pathogenicity and invasiveness nature of fungi. Detection of virulence factor can help to differentiate pathogenic from non-pathogenic aspergilli also help the physican to start antifungal drug whenever needed.

Various immunosuppressive mycotoxins such as gliotoxin are mainly produced by aspergilli. Gliotoxin may produce better under certain culture conditions, such as temperature at 37°C and presence of high oxygen content, which is closely related to the host environment. Gliotoxin is produced in the organs of patients in case of aspergillosis. The present study did not take any account on mycotoxin production. A study reported that the significance of mycotoxins as virulence factors will hopefully be clarified in the near future [27].

Further studies on large sample size are needed to highlight the roles of various virulence factors to confirm the pathogenecity and invasiveness nature of Aspergillus species. Detection of Alpha toxin (aflR gene) in Aspergillus isolates by Polymerase Chain Reaction (PCR) may useful for early detection of virulence factors. Studies on gliotoxin are required that may help in the diagnosis of Aspergillus associated colonization in context of cystic fibrosis.

Virulence factors are very important determinants of any microorganism that help to differentiate pathogenic from non pathogenic species. The presence of virulence factors in aspergilli isolates may give the signal of invasiveness of the Aspergillus species. As regards the differences in virulence factors produced by various Aspergillus species, it can be seen that protienase, α–amylase, haemolysin, biofilm and phospholipase are most important virulence factors (activity ranges from 42.85% to 100%). These findings are common in Aspergillus species isolated from patient and environment samples. This study revealed that aspergilli isolated from environmental samples showed production of virulence factors in less number, but when aspergilli enters into humans, it produces much more virulence factors which can add to the pathogenicity of aspergilli in producing tissue damage.