Direct Molecular Diagnosis of Aspergillosis and CYP51A Profiling from Respiratory Samples of French Patients

Abstract

Background: Microbiological diagnosis of aspergillosis and triazole resistance is limited by poor culture yield. To better estimate this shortcoming, we compared culture and molecular detection of A. fumigatus in respiratory samples from French patients at risk for aspergillosis.Methods: A total of 97 respiratory samples including bronchoalveolar lavages (BAL), bronchial aspirates (BA), tracheal aspirates, sputa, pleural fluids, and lung biopsy were collected from 33 patients having invasive aspergillosis (n = 12), chronic pulmonary aspergillosis (n = 3), allergic bronchopulmonary aspergillosis (n = 7), or colonization (n = 11) and 28 controls. Each specimen was evaluated by culture, pan-Aspergillus qPCR, and CYP51A PCR and sequencing.Results: One A. flavus and 19 A. fumigatus with one multiazole resistant strain (5.3%) were cultured from 20 samples. Culture positivity was 62.5, 75, 42.9, and 15.8% in ABPA, CPA, IA, and colonized patients, respectively. Aspergillus detection rate was significantly higher by pan-Aspergillus qPCR than by culture in IA (90.5 vs. 42.9%; P < 0.05) and colonization group (73.7 vs. 15.8%; P < 0.05). The CYP51A PCR found one TR34/L98H along with 5 novel cyp51A mutations (4 non-synonymous and 1 promoter mutations), yet no association can be established currently between these novel mutations and azole resistance. The analysis of 11 matched pairs of BA and BAL samples found that 9/11 BA carried greater fungal load than BAL and CYP51A detection was more sensitive in BA than in BAL.Conclusion: Direct molecular detection of Aspergillus spp. and azole resistance markers are useful adjunct tools for comprehensive aspergillosis diagnosis. The observed superior diagnostic value of BAs to BAL fluids warrants more in-depth study.

Post tuberculosis treatment infectious complications

“…The sensitivity of galactomannan in bronchoalveolar lavage (BAL) is far superior to that in serum (Sehgal et al, 2019;Shin et al, 2014). PCR-based detection of Aspergillus species for CPA diagnosis has been reported to be more sensitive than culture; however false-positive results may occur (Fraczek et al, 2014;Vergidis et al, 2019;Zhao et al, 2016).…”

Development and Validation of a High-Resolution Melting Assay To Detect Azole Resistance in Aspergillus fumigatus

“…Tuohy et al developed a HRM assay to characterize the G54 codon in cyp51A, but other mutations associated with resistance were not included (42). Nested PCR assays for detecting mutations at position M220 (without discriminating among different base changes), L98H, and TR 34 have also been described (43,44). In contrast to nested PCR assays, our assay involves a closed-tube technique that reduces the possibility of contamination and the time to obtain results, since there is no need for sequencing.…”

“…Three studies detecting cyp51A mutations directly from respiratory samples have been published recently (44)(45)(46). However, those methods also have limitations; one of the studies detected only TR 34 , L98H, Y121F, and T289A mutations via melting curve analysis (45,46) and the other used nested PCR assays combined with sequencing (44).…”

Detecting Azole-Antifungal Resistance in Aspergillus fumigatus by Pyrosequencing

“…Molecular tests for the detection of azole-resistant cyp51A genotypes of A. fumigatus in clinical specimens have been developed over the past 20 years. Numerous in-house assays ranging from nested PCR, mixed-format and real-time PCR approaches have been developed and tested [103][104][105][106][107][108][109][110]. Other molecular approaches (reviewed in [105]) include the analysis of high resolution melt curves [111,112].…”