First Danish report of TR34/L98H A. fumigatus in three non-cystic fibrosis patients despite apparent lack of resistant isolates in recent soil samples

Astvad, K.M.T.(1)*; Jensen, R.H.(1); Hassan, T.M.(1); Mathiasen, E. G.(2); Thomsen, G.M.(3); Pedersen, U.G.(4); Christensen, M.(5); Arendrup, M.C.(2);


OBJECTIVE: Pan-azole resistant Aspergillus fumigatus (AF) harbouring the TR34/L98H or TR46/Y121F/T289A mechanism has been isolated in environmental samples and in azole naïve patients. In Denmark TR34/L98H has only been demonstrated in azole exposed cystic fibrosis patients and in soil samples (obtained June-August 2009). We present the first report of TR34/L98H in three non-cystic fibrosis patients and the data from two Danish environmental surveys. METHOD: Elleven clinical mould isolates from three patients (May 2012- June 2013) and 102 environmental AF isolates (March-April 2010 and October-September 2013) from 239 soil samples (organic (55 AF) and conventional fields (142 AF), an amusement park (17 AF), potted plants in hospitals (25 AF)) were included. Isolates were screened for resistance using azole containing agars (itraconazole 4 mg/L, voriconazole 1 mg/L and posaconazole 0.5 mg/L). Resistant isolates were susceptibility tested by EUCAST E.DEF 9.1 or Etest (table). CYP51A gene sequencing and STRAf typing were undertaken for Aspergillus isolates. RESULTS: Cases: A 73-year old woman with mammary cancer disseminated to lungs, oesophagus, skin and retroperitoneal glands was admitted to have an oesophageal stent endoscopically inserted. She received no immunosuppressants. A bronchoalveolar lavage (BAL) two days later grew a TR34/L98H AF. She died day 7 without receiving any anti-mould treatment. A 77-year old woman with Wegener's granulomatosis on high dosage prednisolone and mycophenolatmofetil was admitted following a kidney biopsy complicated by septic shock and pneumonia. Day 5 voriconazole treatment was instituted (TDM day 18: 1.09 μg/mL). Serum galactomannan antigen day 6 was 2.4 (cut-off <0.5). BALs day 7 grew a TR34/L98H and a wild type (wt) AF, followed by the TR34/L98H only day 17. Day 19 treatment was changed to L-AMB. The patient died day 30. A 60-year old woman with a grade 1-2 follicular lymphoma and paraneoplastic pemphigus was admitted following chemotherapy due to respiratory insufficiency. Day 33-40 she received caspofungin followed by voriconazole (TDM mean 2.61 (0.73-5.34 μg/mL) due to neutropenic fever. Day 44 a BAL grew a wt AF. A respiratory sample day 90 grew a pan-azole resistant Rhizomucor pusillus and a TR34/L98H AF. Despite combination with caspofungin day 91-103 followed by L-AMB from day 119 cultures remained mould positive (table). She died day 131. All 102 AF isolates from both environmental surveys were azole susceptible. CONCLUSION: These clinical cases diagnosed within a 13 months period suggest that TR34/L98H azole resistance is emerging in Denmark. Two patients were azole naïve and two had resistant and wt AF, stressing the importance of susceptibility testing of every clinical mould isolate. Interestingly, the environmental surveys demonstrated no resistance, contrasting the findings in 2009; however the European and Danish studies documenting resistance in soil isolates were all conducted during the summer months suggesting timing of sampling may be essential.

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