Prevalence and mechanism of triazole resistance in Aspergillus fumigatus in a referral chest hospital in Delhi, India and an update of the situation in Asia.

Anuradha Chowdhary,Cheshta Sharma,Shallu Kathuria,Jacques F. Meis,Ferry Hagen

doi:10.3389/fmicb.2015.00428

Abstract

Aspergillus fumigatus causes varied clinical syndromes ranging from colonization to deep infections. The mainstay of therapy of Aspergillus diseases is triazoles but several studies globally highlighted variable prevalence of triazole resistance, which hampers the management of aspergillosis. We studied the prevalence of resistance in clinical A. fumigatus isolates during 4 years in a referral Chest Hospital in Delhi, India and reviewed the scenario in Asia and the Middle East. Aspergillus species (n = 2117) were screened with selective plates for azole resistance. The isolates included 45.4% A. flavus, followed by 32.4% A. fumigatus, 15.6% Aspergillus species and 6.6% A. terreus. Azole resistance was found in only 12 (1.7%) A. fumigatus isolates. These triazole resistant A. fumigatus (TRAF) isolates were subjected to (a) calmodulin and β tubulin gene sequencing (b) in vitro antifungal susceptibility testing against triazoles using CLSI M38-A2 (c) sequencing of cyp51A gene and real-time PCR assay for detection of mutations and (d) microsatellite typing of the resistant isolates. TRAF harbored TR34/L98H mutation in 10 (83.3%) isolates with a pan-azole resistant phenotype. Among the remaining two TRAF isolates, one had G54E and the other had three non-synonymous point mutations. The majority of patients were diagnosed as invasive aspergillosis followed by allergic bronchopulmonary aspergillosis and chronic pulmonary aspergillosis. The Indian TR34/L98H isolates had a unique genotype and were distinct from the Chinese, Middle East, and European TR34/L98H strains. This resistance mechanism has been linked to the use of fungicide azoles in agricultural practices in Europe as it has been mainly reported from azole naïve patients. Reports published from Asia demonstrate the same environmental resistance mechanism in A. fumigatus isolates from two highly populated countries in Asia, i.e., China and India and also from the neighboring Middle East.

Highlights

Among the Aspergillus species, Aspergillus fumigatus is the leading etiologic agent of all forms of aspergillosis, which could be attributed to the ubiquitous presence of its thermo-tolerant spores that are refractory to adverse environmental conditions (Kwon-Chung and Sugui, 2013)
Barring 12 isolates of A. fumigatus none of the other Aspergillus species grew on Sabouraud dextrose agar (SDA) plates supplemented with ITC and/or VRC
We examined azole susceptibility and resistant mechanisms among A. fumigatus isolates from patients with bronchopulmonary aspergillosis in a referral Chest Institute, which caters to a vast population of Delhi and adjoining states of Uttar Pradesh, Haryana and to far remote regions of India

Summary

Introduction

Among the Aspergillus species, Aspergillus fumigatus is the leading etiologic agent of all forms of aspergillosis, which could be attributed to the ubiquitous presence of its thermo-tolerant spores that are refractory to adverse environmental conditions (Kwon-Chung and Sugui, 2013). The most common mechanism of triazole resistance has been linked to theTR34/L98H mutation with tandem repeat in the cyp51A promoter region combined with a single amino acid exchange of leucine 98 to histidine (Chowdhary et al, 2014c) This mutated allele has spread throughout the A. fumigatus population and has been reported worldwide from patients as well as the environment (Mellado et al, 2007; Verweij et al, 2007; Rodriguez-Tudela et al, 2008; Snelders et al, 2008, 2009; Baddley et al, 2009; Howard et al, 2009; Mortensen et al, 2010, 2011; Lockhart et al, 2011; van der Linden et al, 2011; Burgel et al, 2012; Chowdhary et al, 2012a,b; Hamprecht et al, 2012; Jeurissen et al, 2012; Morio et al, 2012; Rath et al, 2012; Alastruey-Izquierdo et al, 2013; Bader et al, 2013; Escribano et al, 2013; Rocchi et al, 2014; Kidd et al, 2015; Steinmann et al, 2015). We conducted a prospective study for the assessment of prevalence of TRAF and the underlying cyp51A mutations in clinical isolates of Aspergillus species collected during a 4-year (2011– 2014) period in a referral Chest Hospital in Delhi, India and reviewed the reports on TRAF isolates from environmental and clinical sources from Asia and the neighboring Middle East

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