Elsevier

Environmental Research

Volume 133, August 2014, Pages 20-26
Environmental Research

Occurrence of triazole-resistant Aspergillus fumigatus with TR34/L98H mutations in outdoor and hospital environment in Kuwait

https://doi.org/10.1016/j.envres.2014.05.009Get rights and content

Highlights

  • Triazole-resistance in A. fumigatus is a major public health concern.

  • This study reports triazole-resistant A. fumigatus in the environment in Kuwait.

  • Eight of 115 (7%) A. fumigatus isolates were resistant to triazoles.

  • All resistant isolates contained TR34/L98H mutations in cyp51A.

  • A multiplex allele-specific-PCR assay detected L98H mutation in resistant isolates.

Abstract

Background: Invasive aspergillosis due to triazole-resistant Aspergillus fumigatus, a common airborne fungal pathogen, has emerged in some European and Asian countries, likely due to exposure to azole fungicides in the environment. This study determined the occurrence of triazole-resistant A. fumigatus in outdoor and hospital environment in Kuwait, a semi-arid desert country in Arabian Peninsula.

Methods

Outdoor/indoor air, water and cotton swab samples were cultured on malt extract agar and A. fumigatus colonies were identified by phenotypic and molecular methods. Drug susceptibility of A. fumigatus isolates to itraconazole, posaconazole and voriconazole was carried out by an Etest and a broth microdilution method. Resistance mechanisms involving cyp51A mutations were probed by mixed-format real-time (MF-rt)-PCR assays. Triazole-resistant isolates were typed by nine-locus microsatellite analysis. A multiplex allele-specific (MAS)-PCR assay was developed for detection of L98H mutation in cyp51A.

Results

Of 115 A. fumigatus isolates obtained from 362 environmental samples from across Kuwait, 8 isolates were resistant to itraconazole, posaconazole and voriconazole. All itraconazole-resistant isolates contained a 34-bp tandem repeat (TR34) in the promoter region and a L98H mutation at codon 98 (TR34/L98H) in cyp51A. These mutations were absent in all itraconazole-susceptible isolates. MAS-PCR accurately detected L98H mutation in all triazole-resistant isolates. Three microsatellite patterns were observed among resistant isolates with one pattern clustering with Indian clinical and environmental isolates.

Conclusions

Triazole-resistant A. fumigatus with TR34/L98H mutations in cyp51A is prevalent in Kuwait. Although triazole-resistant A. fumigatus has not yet been isolated from clinical specimens, its presence in the environment suggests that the possibility of susceptible individuals getting infected with such strains exists and may pose therapeutic challenges in its management.

Introduction

Invasive aspergillosis (IA) is a life-threatening infection in patients with prolonged neutropenia, transplant recipients and other severely immunocompromised patients (Kontoyiannis et al., 2010). Aspergillus fumigatus, one of the most prevalent and ubiquitous airborne fungal pathogens, is the principal etiological agent implicated in IA. Therapeutic options are limited due to variable efficacy of different classes of antifungal drugs against clinical A. fumigatus isolates (Walsh et al., 2008, Pfaller et al., 2011). Oral triazole (itraconazole, posaconazole and voriconazole) antifungal drugs are highly effective against A. fumigatus isolates in vitro and represent first-line therapy in the management and prophylaxis of IA (Mikolajewska et al., 2012). Acquired triazole resistance during therapy of IA was initially perceived as a manageable problem due to very low frequency of isolation of triazole-resistant clinical A. fumigatus isolates (Verweij et al., 2002). However, clinical failures have been increasingly reported in recent years and the frequency of isolation of triazole-resistant clinical A. fumigatus isolates has increased dramatically in some European and Asian countries (Pfaller et al., 2011, van der Linden et al., 2011, Howard et al., 2009, Camps et al., 2012, Chowdhary et al., 2012a, Lockhart et al., 2011, Rath et al., 2012, Hamprecht et al., 2012, Rocchi et al., 2014). Since culture positivity even in confirmed cases of IA is typically less than 50% (Arendrup et al., 2012), the actual resistance rates among clinical A. fumigatus isolates are likely to be much higher. This dramatic shift has been attributed to the exposure of environmental fungi to 14α-demethylase inhibitors which are structurally related to clinically licensed triazoles and are widely used in crop plant protection and ornamental flower preservation to control fungal growth in agriculturally important countries (Snelders et al., 2009, Verweij et al., 2009).

The molecular basis of resistance to triazoles in A. fumigatus strains involves point mutations in the 14α-sterol demethylase encoded by cyp51A gene and/or increased cyp51A expression due to a tandem repeat alteration in the promoter region (Mellado et al., 2007, Snelders et al., 2008, Denning et al., 2011). While amino acid substitutions in cyp51A gene, particularly at codons G54, G138, P216, M220 and/or G448, have been found in clinical triazole-resistant A. fumigatus isolates, a single dominant mechanism involving a 34 bp tandem repeat (TR34) in the cyp51A promoter region together with an L98H substitution has been observed in triazole-resistant isolates recovered from environmental sources and treatment naïve patients (Howard et al., 2009, Chowdhary et al., 2012a, Lockhart et al., 2011, Rath et al., 2012, Hamprecht et al., 2012, Rocchi et al., 2014, Mellado et al., 2007, Snelders et al., 2008, Denning et al., 2011, Camps et al., 2012, van der Linden et al., 2013). The occurrence of these mutations has been detected in both, culture isolates of A. fumigatus and clinical specimens by highly sophisticated molecular techniques typically involving two rounds of PCR or real-time PCR together with specific probes/molecular beacons or DNA sequencing (Mellado et al., 2007, Snelders et al., 2008, Denning et al., 2011, Camps et al., 2012, van der Linden et al., 2013, Spiess et al., 2012, Zhao et al., 2013). In this report, we describe the isolation of triazole-resistant A. fumigatus strains from outdoor air and hospital environment in Kuwait, a semi-arid desert country in the Arabian peninsula. We also show that all 8 triazole-resistant A. fumigatus isolates contain TR34 in the cyp51A promoter region and L98H mutation in cyp51A. We also describe a novel multiplex allele-specific (MAS)-PCR assay for rapid detection of the L98H mutation in the cyp51A gene.

Section snippets

Environmental sampling, culture, identification and typing

Outdoor and indoor air sampling was performed with an Andersen 6-Stage air impactor (Graseby-Andersen, Atlanta, GA, USA) at ground level and at ~50 feet above the ground level outside a major tertiary care (Mubarak Al-Kabeer) hospital by exposing malt extract agar supplemented with chloramphenicol (50 mg/L) medium (MEA)-containing Petri plates for 15 min. Water samples collected from different wards and intensive care units of the hospital treated with sodium thiosulfate (120 mg/L) to neutralize

Results

A total of 115 A. fumigatus isolates were grown from 362 different environmental samples collected from 7 separate localities (Jabriya, Salmiya, Shuwaikh, Jaleeb Al-Shuyoukh, Fahaheel, Al-Jahra and Al-Wafra) spread across Kuwait during the course of this study (Fig. 1). It is important to point out here that Jabriya and Salmiya are mainly residential areas with road-side trees and recreational parks; Fahaheel is mainly arid residential area with adjoining petrochemical complex; Shuwaikh and

Discussion

Selective drug pressure during widespread use of antimicrobial agents in clinical settings eventually leads to emergence of drug-resistant strains of pathogenic organisms. Triazole resistance was first detected in clinical A. fumigatus strains isolated in USA more than two decades ago which has now been described from several European and Asian countries (Verweij et al., 2002, van der Linden et al., 2011, Howard et al., 2009, Camps et al., 2012, Chowdhary et al., 2012a, Lockhart et al., 2011,

Acknowledgments

The study was supported by Kuwait University Research Sector under Grant no. MI01/09. We thank Ajmal Theyyathel and Leena Joseph for providing excellent technical assistance. We are grateful to Anuradha Chowdhary, Delhi, India for providing resistant Aspergillus isolates for comparison purposes. The study was approved by the Ethical Committee, Faculty of Medicine, Kuwait University.

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