Purpose: Aspergillus fumigatus is an opportunistic fungal pathogen responsible for high mortality rates. After inhaling conidia dispersed in the air patients become colonized or infected, depending on their immunological status. Infections, like aspergilloma or invasive aspergillosis, may result from colonization that can occur due a mix of different A. fumigatus genotypes. Azole therapy may have the ability to eradicate these microorganisms. However, prolonged exposure to these drugs may result in emergence of resistance to them. Increasingly, A. fumigatus multi-azole-resistant isolates are making surveillance mandatory and several methods have been developed to detect azole resistance in A. fumigatus: culture and susceptibility testing and molecular based methods, usually cyp51A gene sequencing. The University Hospital of Campinas (HC-UNICAMP) is a tertiary care teaching institution where many high-risk patients are susceptible to opportunistic infectious agents such as A. fumigatus.
Methods: This study evaluated 109 A. fumigatus strains isolated from 39 patients that had at least two sequential isolates, cared at HC-UNICAMP during a period of 15 years. These isolates were obtained from sputum, pleural liquid, bronchoalveolar lavage, biopsies and other clinical specimens. Morphological identification was confirmed trough β-tubulin DNA sequencing. Susceptibility profiles were determined according to CLSI document (M38-A2) for itraconazole, voriconazole, amphotericin B, micafungin and caspofungin. Cyp51A sequencing was performed for isolates with high minimum inhibitory concentrations (MICs) to azoles. All isolates were submitted to microsatelites typing (STRAfs 2AB, 3AB, 4AB, BDA, BDB, BDD) to assess if the serial isolates were genetically related.
Results: Nine isolates from 9 different patients showed resistance to at least one azole, but amino acid changes in cyp51A gene were found in six of them. Following e-Burst V.3 software analysis, 97 different sequence types (STs) were found. A snapshot plotted with the 109 isolates, showed a clonal complex, involving 6 isolates from 3 different patients (P11, P2 and P4) (Figure 1). These 6 isolates carry the already known point mutations F46Y, M127V, N248T, D225E and E427K. The 3 patients had immunosuppressive diseases, proven A. fumigatus infection and other A. fumigatus isolates that showed azole resistance without point mutations, and fell outside the clonal complex (Figure 1). Patient 11 and 4, were at the hospital concomitantly, but never shared the same room. Interestingly, the P2 patient was hospitalized 2 years before the other two.
Figure 1: Clonal Complex involving 11 isolates from 3 patients (P2, P4 and P11); red squares highlight isolates that showed point mutations and black lines indicate 1 ST difference.
Conclusion: Being included in a clonal complex means that the isolates may have the same clonal origin but, in this case, with 1 to 3 STs of difference. More studies are needed to clarify these facts: perhaps an environmental source outside or inside the hospital has served as a reservoir for the isolates of these patients.
Full conference title:
- AAA 8th (2018)