Antifungal Susceptibility Patterns of Aspergillus species isolated from patients with Pulmonary diseases in Iraq

© 2018 Alaa Hani Al-Charrakh et al. This is an open access article distributed under the terms of the Creative Commons Attribution License -NonCommercial-ShareAlikeUnported License (http://creativecommons.org/licenses/by-nc-sa/3.0/). *Corresponding Author Alaa Hani Al-Charrakh, Department of Microbiology, College of Medicine, Babylon University, Hilla, Babylon Governorate, Iraq. E-mail: ahani67 @ gmail.com Antifungal Susceptibility Patterns of Aspergillus species isolated from patients with Pulmonary diseases in Iraq


INTRODUCTION
Aspergillus infections are among the most feared opportunistic infections in humans because they are capable of causing several distinct pulmonary diseases (Krel et al., 2014). Aspergillus spp. is the main causal agent for fungal respiratory infections in the critically ill patient comes in second place after the fungi from the order Mucorales (Garnacho-Montero et al., 2013).
The Aspergillus genus contains many species and these are ubiquitous in our environment. They do not form part of the normal flora. Their spores are regularly inhaled without harmful consequences, but some species, notably A. fumigatus, are able to cause a range of diseases, including 1-Allergic bronchopulmonary aspergillosis (ABPA), which is, as its name suggests, an allergic response to the presence of Aspergillus antigen in the lungs and occurs in patients with asthma. ABPA occurs in some 10% of cystic fibrosis patients, 2-Aspergilloma in patients with pre-existing lung cavities or chronic pulmonary disorders. Aspergillus colonizes a cavity and grows to produce a fungal ball, a mass of entangled hyphae, and 3-Disseminated disease in the immunosuppressed patient when the fungus spreads from the lungs (Goering et al., 2013).
Aspergilloma occurs when the conidia that been inhaled enter a pre-existing cavity, germinate, and produce abundant hyphae in the abnormal pulmonary space. Those patients with previous cavitary disease (e.g. tuberculosis, sarcoidosis, emphysema) are at risk (Brooks et al., 2014). Nowadays, Voriconazole is considered the first choice for treatment of aspergillosis; amphotericin B and echinocandins (mainly caspofungin) used to treat this infection as well (Walsh et al., 2008;Mikulska and Viscoli, 2011). An emerging constraint associated with Aspergillosis therapeutics is the increasing resistance against triazole that observed in A. fumigatus isolates Snelders et al., 2011).
The aim of this study was to determine the prevalence of Aspergillus species in patients with pulmonary pneumonia, and antifungal susceptibility testing of these clinical fungal isolates by E-test technique.

MATERIALS AND METHODS Study design and duration
This cross-sectional study was designed to assess the occurrence of Aspergillus species in patients with pulmonary infections. Specimen collection and analysis was carried out for four months (1 st November 2015 to 30 th February 2016). Specimens collected from the specialized center of Chest and Pulmonary diseases in Hilla city/Iraq. Each specimen was cultured and examined macroscopically and microscopically.

Study subjects
Clinical specimens were obtained from patients (70 male and 69 female), presented with pulmonary symptoms, such as bronchiectasis, recurrent infections (with fever and malaise, dyspnea, anorexia, weight loss, and chest pain), chronic obstructive pulmonary disease (COPD), or a suspected case of lung infection. Mean age of the patients was 43.68 ± 10 years; with a wide-ranging age varied from 1 day to 90 years. The patients were diagnosed clinically by specialists and were tabulated according to their age, gender, and presence of Tuberculosis infection or another type of pulmonary disease.

Ethical approval
Verbal consent was taken from each patient before sampling. Investigative standards were rigidly preserved, primarily concerning confidentiality. Moreover, this study was undisclosed, the participation of patients was optional, and verbal consent was received before data uptake process was started.

Specimen collection and mycological analysis
A total of 311 specimens were collected from patients presented with the pulmonary complaint. Specimens included Bronchoalveolar lavage fluid (BAL) and sputum. The specimens were transported by screw-capped cups to the Microbiology lab and each specimen was inoculated by streaking on two general culture media (Sabouraud Dextrose agar-supplemented with chloramphenicol and Malt extract agar), and by pouring on third medium namely Potato dextrose agar, Then incubated at 25°C for 2-7 days (Forbes et al., 2014).

Antifungal susceptibility testing
Antifungal susceptibility by E-test method was done on non-supplemented Muller Hinton Agar (MHA) according to the method described in M51-A and M38-A2 CLSI documents. E-test gradient strips of Posaconazole (POS), itraconazole (ITC), voriconazole (VO), and caspofungin (CAS) were obtained from Liofilchem Lab, Italy. The concentration gradient for each drug ranged from 0.002 to 32 μg/ml. The strips were stored frozen at −20°C until they were used in the study. The E-test was performed by following the manufacturer's instructions. Each solidified medium was inoculated by dipping a nontoxic (latex-free) sterile swab into the respective undiluted stock inoculum suspension and evenly streaking it in three directions over the entire surface of a 150-mm Petri plate containing 60 ml of medium. The plates were incubated at 35°C, and the MICs were read at 24h or 48h as the concentration of drug that elicited 100% inhibition of growth (posaconazole, itraconazole, voriconazole) or as the minimum effective concentration (MEC, caspofungin).
There was a tendency of an agar-based method to produce false susceptible results after 24 hours which can be explained, e.g., as being due to the shift of the interpretive categories in some Aspergillus strains when comparing reading after 24h and 48h (Buchta et al., 2008). Therefore, in case of A. terreus, the first reading at 24h was considered while in the case of the remaining Aspergillus spp. the second reading was considered.
The 139 Aspergillus isolates were gathered from 70 male (50.3%) and 69 female (49.6%). Six patients with ongoing tuberculosis infection were all given positive Aspergillus isolates.
Ten species of Aspergillus were identified (Table 1). A. flavus, A. niger, and A. terreus were the most frequently isolated species followed by A. fumigatus in the fourth place (51%, 48.9%, 28.7%, and 11.5%), respectively. Some specimens had grown mixed Aspergillus culture (63 isolates (45.32) out of 139) therefore the total number of positive Aspergillus isolates (N = 139) does not equal the total number of the individual isolates obtained (N = 218). The creditability of direct microscopy in the diagnosis of fungal infection in sputum smears has been proven.
In a study, all of the 27 (100%) samples that were positive for Aspergillus spp. by direct sputum microscopy and culture. In contrary, examination by direct microscopy failed to detect three samples, which were later found to be positive with culture. Direct microscopy is therefore 90% sensitive in detecting Aspergillus spp. in sputum smears (Njunda et al., 2012).
Aspergillus spp. may be responsible for important clinical events from saprophytic colonization of the airways to rapidly invasive and life-threatening disseminated diseases, depending on the host immune status and the presence of underlying lung disease (Latgé et al., 2001).
Several authors found that the most frequent species of Aspergillus associated with clinical disease cases were A. flavus, A. terreus, and A. niger followed by A. fumigatus (Malik et al., 2009;Shujat et al., 2014).
Another study showed that 28.08% of total 381 isolates were belonging to the genus Aspergillus. A. fumigatus was the one having highest percentage of frequency (29.9%), followed by A. niger (28.9%), as well as the isolated species A. flavus and A. terreus and A. nidulans (18.7%, 12.14%, 2.8%), respectively (Aziz, 2014). It was found that the four most frequently isolated Aspergillus species as a causative agent in the pulmonary infections are A. fumigatus, A. flavus, A. niger, and A. terreus (Njunda et al., 2012;Barberan et al., 2012;Diba et al., 2007).
Various studies reported that A. fumigatus is more prevalent in the Western countries, while A. flavus is more prevalent in the Middle East and Asia as the leading cause of invasive aspergillosis of the lungs (Patterson et al., 2000;Groll and Kolve, 2004;Chamilos and Kontoyiannis, 2005;Singh and Paterson, 2005;Pfaller et al., 2006). However, it has been found that A. niger, A. nidulans, and A. ustus are only rarely isolated (Pfaller et al., 2006;Shao et al., 2007). A. niger was considered as a non-probable cause of pulmonary Aspergillosis.
A. niger isolates were mostly accounted as contamination (Patterson et al., 2000). This leaves us to consider A. flavus, and A. fumigatus as two most probable cause of pulmonary Aspergillosis.
In Aspergillus infections, the lung continues to represent the most frequently involved site. Pulmonary infection is a phenotypical representation of the interaction between lowered defense mechanisms in the host and the virulence of the fungus (Pagano et al., 2008).
This study result showed that patients with pulmonary diseases and patients with lower immune status are mainly at risk of infection by pathogenic Aspergillus spp. and the fact that pulmonary abnormalities are a predisposing factor for the fungal infection.
Results also found that six patients were presented with tuberculosis. These patients with TB were having a concomitant Aspergillosis infection (6/139, 4.31%). Moreover, along with those with proven tuberculosis, almost two-thirds of the patients (211/311, 67.84%) have had a history of chronic bronchitis, pneumonia, cancer, undergone COPD attack or suffering from COPD during the time when the sample has been taken or have another form of the pulmonary disease. Others have a suspected case of pneumonia with or without immunocompromisation.
In a single study, nine 9 (4.5%) patients infected with both infections (Tuberculosis and Aspergillosis). Among the nine 9 patients who were infected with Aspergillus sp. and TB, six 6 were infected with A. fumigatus, two 2 with A. niger and one 1 with A. flavus. None of the patients was infected with A. terreus (Njunda et al., 2012). Previous tuberculosis (either classical or atypical) was the most commonly identified primary underlying condition (38/126, 30.2%). The second common, primary underlying condition was ABPA (15/126, 11.9%). However, all of the 232 underlying conditions identified for 126 Chronic Pulmonary Aspergillosis (CPA) cases, COPD/emphysema was the most common (42/126, 33.3%).

Antifungal susceptibility testing
Since fungal infections in hospitals are much less frequent than other microbial infections, susceptibility tests for fungi are not routinely performed in some diagnostic laboratories (Badiee et al., 2012). In the present study, ten 10 Aspergillus spp. isolates were tested by using E-test against four antifungal agents ITC, POS, O, and CAS).
The E-test was carried on non-supplemented MHA (Figure 1). Antifungal agents showed good activity against Aspergillus spp. except for some forms of resistance which have detected in some isolates even though the antifungals tested are not in use nationally and it was the first time the fungus exposing to them (Table 2). A group of authors mentioned that there was no difference in MIC or MEC values at 24 hr and 48 hr, therefore we suggest E-test be done on non-supplemented MHA in place of RPMI 1640 agar with 2% glucose and MOPS, which is also the common media available in most of the laboratories (Gupta et al., 2015).
In the present study, resistance to ITC was seen in (20%) of isolates. Badiee et al. (2012) reported that 30.6% of Aspergillus species isolates were resistant to ITC when tested by E-test. It was found that echinocandins exhibited a good activity against A. fumigatus isolates. However, azole agents had different activity in vitro. The first isolate was susceptible in vitro to ITC, VO (MIC ≤ 1 mg/L), and POS (MIC value ≤ 0.25 mg/L). The second isolate, obtained after VO therapy, was resistant in vitro to ITC, VO, and POS. Mellado et al. (2013) reported that pulmonary A. fumigatus isolates recovered from Spanish patient were multiple triazoles resistant.
Resistance to ITC is usually associated with a reduction in POS susceptibility, predictably because the 2 drugs are structurally similar (Rodriguez-Tudela et al., 2008). This study showed resistance to ITC and POS by Aspergillus section flavi. Isolates with reduced susceptibility to ITC are frequently crossresistant to other triazoles, and specific testing is recommended (Arendrup et al., 2012). The MIC results obtained by E-test methods demonstrated that POS was very active against all Aspergillus spp. (all susceptible at MIC <0.25 μg/ml). All isolates of Aspergillus spp. were inhibited with <0.5 μg/ml of VO (Rudramurthy et al., 2011).
These results demonstrate the excellent efficacy of VO against Aspergillus species and suggest that VO may be the treatment of choice in invasive aspergillosis caused by A. fumigatus and A. flavus. VO was found to have high-MIC in 9.1% Aspergillus isolates (Guinea et al., 2010). VO of a low-MIC was reported in 4.9% of A. flavus and considered as sensitive (Rudramurthy et al., 2011).
However, Kaufman et al. (2004) found that resistance of A. fumigatus to the azoles was reported to vary from a high of 52% and 38% with ITC and ravuconazole, respectively, to a low of 11% with VO. They also reported that high resistance of A. fumigatus to the azole agent POS was detected.