scholarly journals Molecular Detection of Aspergillus: Application of a Real-Time PCR Multiplex Assay in Tissue Samples

2020 ◽  
Vol 6 (1) ◽  
pp. 11 ◽  
Author(s):  
Raquel Sabino ◽  
Helena Simões ◽  
Cristina Veríssimo
Keyword(s):  
Aspergillus Fumigatus ◽  
Multiplex Pcr ◽  
Real Time ◽  
Fungal Infections ◽  
Sensu Stricto ◽  
Azole Resistance ◽  
Tissue Samples ◽  
Pcr Multiplex ◽  
Resistance Patterns ◽  
Panfungal Pcr

Diagnosis of invasive fungal infections is complex, and the lack of standardization of molecular methods is still a challenge. Several methods are available for the diagnosis of invasive aspergillosis, but their effectiveness will depend on the studied population, the patients’ comorbidities, and the use of mold active prophylaxis, among others. The ability to determine the identity of the infecting Aspergillus species, and to detect mutations conferring specific resistance patterns directly from DNA extracted from the biological product, is an advantage of nucleic acid testing compared with antigen-based assays. In this study, we present laboratory cases where the diagnosis of aspergillosis was performed using a real-time multiplex PCR for the detection of Aspergillus DNA in tissue samples, showing its usefulness as one more tool in the diagnosis of aspergillosis in tissue samples. Aspergillus real-time multiplex PCR was also used to detect azole-resistance in some cases. In the majority of the PCR positive cases, cultures remained negative after 60 days. The PCR assay directed to Aspergillus gave positive signals for Aspergillus fumigatus sensu stricto. Results were confirmed by panfungal PCR, followed by sequencing, revealing 100% homology with Aspergillus fumigatus sensu stricto. Mutations conferring azole resistance were not detected.

Azole resistance among clinical isolates of Aspergillus fumigatus in Lima-Peru

2019 ◽  
Vol 58 (1) ◽  
pp. 54-60 ◽  
Author(s):  
Beatriz Bustamante ◽  
Luis Ricardo Illescas ◽  
Andrés Posadas ◽  
Pablo E Campos
Keyword(s):  
Aspergillus Fumigatus ◽  
Latin American ◽  
Pcr Amplification ◽  
Sensu Stricto ◽  
Clinical Isolates ◽  
Azole Resistance ◽  
Molecular Testing ◽  
Naive Patient ◽  
In Vitro Susceptibility

Abstract Azole resistance among Aspergillus fumigatus isolates, which is mainly related to mutations in the cyp51A gene, is a concern because it is rising, worldwide disseminated, and associated with treatment failure and death. Data on azole resistance of aspergillus from Latin American countries is very scarce and do not exist for Peru. Two hundred and seven Aspergillus clinical isolates collected prospectively underwent mycology and molecular testing for specie identification, and 143 isolates were confirmed as A. fumigatus sensu stricto (AFSS). All AFSS were tested for in vitro azole susceptibility, and resistant isolates underwent PCR amplification and sequencing of the whole cyp51A gene and its promoter. The in vitro susceptibility showed a minimal inhibitory concentration (MIC) range, MIC50 and MIC90 of 0.125 to >16, 0.25, and 0.5 μg/ml for itraconazole; 0.25 to 2, 0.5, and 0.5 μg/ml for voriconazole; and 0.003 to 1, 0.06, and 0.125 μg/ml for posaconazole. Three isolates (2%) showed resistance to itraconazole and exhibited different mutations of the cyp51A gene. One isolate harbored the mutation M220K, while a second one exhibited the G54 mutation plus a modification in the cyp51A gene promoter. The third isolate, from an azole naive patient, presented an integration of a 34-bp tandem repeat (TR34) in the promoter region of the gene and a substitution of leucine 98 by histidine (L98H). The three source patients had a diagnosis or suspicion of chronic pulmonary aspergillosis.

scholarly journals Molecular Identification, Antifungal Susceptibility Testing, and Mechanisms of Azole Resistance in Aspergillus Species Received within a Surveillance Program on Antifungal Resistance in Spain

2019 ◽  
Vol 63 (9) ◽  
Author(s):  
Olga Rivero-Menendez ◽  
Juan Carlos Soto-Debran ◽  
Narda Medina ◽  
Jose Lucio ◽  
Emilia Mellado ◽  
...  
Keyword(s):  
Aspergillus Terreus ◽  
Fungal Infections ◽  
Novel Mutation ◽  
Antifungal Susceptibility ◽  
Resistance Mechanism ◽  
Sensu Stricto ◽  
Antifungal Resistance ◽  
Surveillance Program ◽  
Azole Resistance ◽  
Content Type

ABSTRACT Antifungal resistance is one of the major causes of the increasing mortality rates for fungal infections, especially for those caused by Aspergillus spp. A surveillance program was established in 2014 in the Spanish National Center for Microbiology for tracking resistance in the most prevalent Aspergillus species. A total of 273 samples were included in the study and were initially classified as susceptible or resistant according to EUCAST breakpoints. Several Aspergillus cryptic species were found within the molecularly identified isolates. Cyp51 mutations were characterized for Aspergillus fumigatus, Aspergillus terreus, and Aspergillus flavus sensu stricto strains that were classified as resistant. Three A. fumigatus sensu stricto strains carried the TR34/L98H resistance mechanism, while two harbored G54R substitution and one harbored the TR46/Y121F/T289A mechanism. Seventeen strains had no mutations in cyp51A, with ten of them resistant only to isavuconazole. Three A. terreus sensu stricto strains harbored D344N substitution in cyp51A, one of them combined with M217I, and another carried an A249G novel mutation. Itraconazole-resistant A. flavus sensu stricto strains harbored P220L and H349R alterations in cyp51A and cyp51C, respectively, that need further investigation on their implication in azole resistance.

scholarly journals Nationwide surveillance of azole-resistant Aspergillus fumigatus environmental isolates in Greece: detection of pan-azole resistance associated with the TR46/Y121F/T289A cyp51A mutation

2020 ◽  
Vol 75 (11) ◽  
pp. 3181-3188
Author(s):  
Maria Siopi ◽  
Olga Rivero-Menendez ◽  
Georgios Gkotsis ◽  
Anthi Panara ◽  
Nikolaos S Thomaidis ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Sensu Stricto ◽  
Resistant Isolate ◽  
Azole Resistance ◽  
Medical Settings ◽  
Nationwide Surveillance ◽  
The Mean ◽  
The One ◽  
Organically Grown ◽  
Of Greece

Abstract Background Acquired azole resistance (AR) in Aspergillus fumigatus emphasizes the importance of the One Health multisectorial approach. The prevalence of azole-resistant A. fumigatus in the environment of Greece is unknown. Methods Between October 2016 and September 2017, a total of 716 soil samples were collected from 23 provinces and screened for AR using azole-containing agar plates. Recovered isolates were macro-/microscopically identified and colonies were counted. Azole susceptibility testing of A. fumigatus species complex (SC) isolates was performed (EUCAST E.DEF9.3.1). Azole-resistant A. fumigatus isolates were subjected to confirmatory molecular identification and sequencing of the cyp51A gene. Results No yeasts were recovered, while multiple moulds grew on 695 (97%) samples. Overall, zygomycetes (most non-Mucor genera) grew on 432 (60%) samples, while Aspergillus spp. grew on 500 (70%) [410 (57%) Aspergillus niger SC; 120 (17%) Aspergillus terreus SC; 101 (14%) A. fumigatus SC; 34 (5%) Aspergillus flavus SC]. The mean ± SD soil load of Aspergillus spp. was 2.23 ± 0.41 log10 cfu/g (no differences among species). No azole-resistant non-A. fumigatus spp. isolate was detected. Itraconazole, voriconazole, isavuconazole and posaconazole MIC50/MIC90 (MIC range) of A. fumigatus SC strains were 0.25/0.5 (0.25 to >8), 0.5/1 (0.25 to >8), 1/1 (0.125 to >8) and 0.06/0.125 (0.06–1) mg/L, respectively. Overall, 1/500 (0.2%) of Aspergillus isolates, and 1/101 (1%) of A. fumigatus SC isolates, was pan-azole-resistant (itraconazole, voriconazole, isavuconazole and posaconazole MIC >8, >8, >8 and 1 mg/L, respectively). The resistant isolate was recovered from organically grown raisin grapes treated with homemade compost and it was an A. fumigatus sensu stricto isolate harbouring the TR46/Y121F/T289A mutation. The soil’s load was higher compared with azole-susceptible strains (3.74 versus 2.09 log10 cfu/g). Conclusions This is the first known report of environmental pan-azole-resistant A. fumigatus in Greece. Since data on Greek clinical isolates are lacking, this finding must alarm the systematic local surveillance of AR in medical settings.

scholarly journals Azole resistant Aspergillus fumigatus clinical isolate screening in azole-containing agar plates (EUCAST E.Def 10.1): low impact of plastic trays used and poor performance in cryptic species

2021 ◽  
Author(s):  
Julia Serrano-Lobo ◽  
Ana Gómez ◽  
Belén Rodríguez-Sánchez ◽  
Patricia Muñoz ◽  
Pilar Escribano ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Cryptic Species ◽  
Screening Method ◽  
Fungal Growth ◽  
Poor Performance ◽  
Sensu Stricto ◽  
Azole Resistance ◽  
Major Error ◽  
Agar Method ◽  
The Impact

Azole-containing agar is used in routine Aspergillus fumigatus azole resistance screening. We evaluated the impact of the type of plastic used to prepare in-house agar plates on the procedurés performance against A. fumigatus sensu stricto and cryptic species. A. fumigatus sensu stricto (n=91) and cryptic species (n=52) were classified as susceptible or resistant (EUCAST E.Def 9.3.2; clinical breakpoints v10). In-house azole-containing agar plates were prepared following EUCAST E.Def 10.1 on three types of multi-dish plates. We assessed the sensitivity, specificity, and agreement values of the agar plates to screen for azole resistance. Overall, sensitivity and specificity values of the agar screening method were 100% and 93.3%, respectively. The type of tray used did not affect these values. All isolates harbouring TR 34 -L98H substitutions were classified as resistant to itraconazole and voriconazole by the agar method; however, false susceptibility (very major error) to posaconazole was not uncommon and happened in isolates with posaconazole MICs of 0.25 mg/L. Isolates harbouring G54R and TR 46 -Y121F-T289A substitutions were correctly classified by the agar method as itraconazole/posaconazole resistant and voriconazole-resistant, respectively. False resistance (major error) occurred in isolates showing tiny fungal growth. Finally, agreements between both procedures against cryptic species were much lower. Azole-containing agar plates are a convenient and reliable tool to screen for resistance in A. fumigatus sensu stricto ; the type of plastic tray used minimally affects the method. On the contrary, the performance against cryptic species is rather poor.

Diagnosis of Invasive Fungal Infections by a Real-Time Panfungal PCR Assay in Pediatric Patients Undergoing Intensive Chemotherapy or Allogeneic Stem Cell Transplantion.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 343-343
Author(s):  
Christine Landlinger ◽  
Lenka Baskova ◽  
Sandra Preuner ◽  
Martine Van Grotel ◽  
Nico G. Hartwig ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Predictive Value ◽  
Pediatric Patients ◽  
Fungal Infections ◽  
Invasive Fungal Infections ◽  
Immunocompromised Patients ◽  
Pcr Assay ◽  
Highly Sensitive ◽  
Panfungal Pcr

Abstract Abstract 343 Invasive fungal infections are life threatening events in severely immunocompromised patients, and there is urgent need for reliable screening methods facilitating rapid and broad detection of pathogenic fungi. We have established a two-reaction real-time PCR assay permitting highly sensitive detection and quantitative monitoring of more than 80 fungal pathogens, covering a large spectrum of moulds, yeasts and Zygomycetes (European patent No. 06817468.9). To assess the clinical potential of the panfungal real-time PCR assay, more than 600 consecutive specimens from 126 pediatric patients carrying a high risk of invasive fungal infections were analyzed. The results revealed an excellent correlation between PCR positivity and the presence of proven, probable or possible fungal infection according to the criteria of the European Organization for Research and Treatment of Cancer (EORTC), indicating a sensitivity of the assay of 96% (95%CI: 81-99.3%). Hence, the negative predictive value of the panfungal PCR assay presented is very high, and our current data indicate that molecular screening of patients during febrile neutropenic episodes by the assay can help prevent unnecessary toxicity resulting from empirical antifungal treatment in individuals who may not be at risk of imminent fungal disease. The specificity of the assay in the test cohort of patients was in the range of 76% (95%CI:62-87%), and the observations indicate that rapid species identification may be required to assess the positive predictive value for impending fungus-related disease. The availabel data provide a basis for appropriately designed clinical studies addressing the full diagnostic potential of fungal screening by highly sensitive, broad- spectrum molecular assays in severely immunocompromised patients. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The Aspergillus fumigatus Mismatch Repair MSH2 Homolog Is Important for Virulence and Azole Resistance

mSphere ◽  
2019 ◽  
Vol 4 (4) ◽  
Author(s):  
Thaila Fernanda dos Reis ◽  
Lilian Pereira Silva ◽  
Patrícia Alves de Castro ◽  
Rafaela Andrade do Carmo ◽  
Marjorie Mendes Marini ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Mismatch Repair ◽  
Genetic Instability ◽  
Galleria Mellonella ◽  
Fungal Infections ◽  
Invasive Pulmonary Aspergillosis ◽  
Azole Resistance ◽  
Repair System ◽  
Wild Type ◽  
Content Type

ABSTRACT The genetic stability of every living organism depends on accurate DNA replication and repair systems. Here, we investigated the Aspergillus fumigatus MSH2 mismatch repair (MMR) gene MshA and how it impacts virulence and the evolution of azole resistance. We examined mshA gene variation in 62 environmental and clinical A. fumigatus strains. We have observed 12 strains with variants (18.2%), and 8 strains among them showed missense variants. We demonstrated that A. fumigatus mshA null mutants are haploid and have conserved karyotypes with discrete gross chromosomal rearrangements. The ΔmshA strains are not sensitive to several DNA-damaging agents. The lack of mshA caused a significant reduction of virulence of A. fumigatus in a neutropenic murine model of invasive pulmonary aspergillosis and in the invertebrate alternative model Galleria mellonella. Wild-type and ΔmshA populations did not show any significant changes in drug resistance acquisition after they were transferred 10 times in minimal medium in the absence of any stress. However, these populations rapidly acquired virulence in the ΔmshA background and high levels of resistance to posaconazole in the presence of this drug (at least 200-fold-higher levels of resistance than those derived from the wild-type strain). Taken together, these results suggest that genetic instability caused by ΔmshA mutations can confer an adaptive advantage, mainly increasing posaconazole resistance and virulence acquisition. IMPORTANCE Invasive aspergillosis (IA) has emerged as one of the most common life-threatening fungal diseases in immunocompromised patients, with mortality rates as high as 90%. Systemic fungal infections such as IA are usually treated with triazoles; however, epidemiological research has shown that the prevalence of azole-resistant Aspergillus fumigatus isolates has increased significantly over the last decade. There is very little information about the importance of genomic stability for A. fumigatus population structure, azole resistance, and virulence. Here, we decided to investigate whether the mismatch repair system could influence A. fumigatus azole resistance and virulence, focusing on one of the components of this system, MSH2. Although the mutation frequency of mshA (the A. fumigatus MSH2 homologue) is low in environmental and clinical isolates, our results indicate that loss of mshA function can provide increased azole resistance and virulence when selected for. These results demonstrate the importance of genetic instability in A. fumigatus as a possible mechanism of evolving azole resistance and establishing fitness in the host.

scholarly journals Validation of a New Aspergillus Real-Time PCR Assay for Direct Detection of Aspergillus and Azole Resistance of Aspergillus fumigatus on Bronchoalveolar Lavage Fluid

2015 ◽  
Vol 53 (3) ◽  
pp. 868-874 ◽  
Author(s):  
Ga-Lai M. Chong ◽  
Wendy W. J. van de Sande ◽  
Gijs J. H. Dingemans ◽  
Giel R. Gaajetaan ◽  
Alieke G. Vonk ◽  
...  
Keyword(s):  
Bronchoalveolar Lavage ◽  
Aspergillus Fumigatus ◽  
Real Time ◽  
Real Time Pcr ◽  
Gold Standard ◽  
Azole Resistance ◽  
Wild Type ◽  
Pcr Assay ◽  
Content Type ◽  
Resistant Strains

Azole resistance inAspergillus fumigatusis increasingly reported. Here, we describe the validation of the AsperGenius, a new multiplex real-time PCR assay consisting of two multiplex real-time PCRs, one that identifies the clinically relevantAspergillusspecies, and one that detects the TR34, L98H, T289A, and Y121F mutations in CYP51A and differentiates susceptible from resistantA. fumigatusstrains. The diagnostic performance of the AsperGenius assay was tested on 37 bronchoalveolar lavage (BAL) fluid samples from hematology patients and 40 BAL fluid samples from intensive care unit (ICU) patients using a BAL fluid galactomannan level of ≥1.0 or positive culture as the gold standard for detecting the presence ofAspergillus. In the hematology and ICU groups combined, there were 22 BAL fluid samples from patients with invasive aspergillosis (IA) (2 proven, 9 probable, and 11 nonclassifiable). Nineteen of the 22 BAL fluid samples were positive, according to the gold standard. The optimal cycle threshold value for the presence ofAspergilluswas <36. Sixteen of the 19 BAL fluid samples had a positive PCR (2Aspergillusspecies and 14A. fumigatussamples). This resulted in a sensitivity, specificity, and positive and negative predictive values of 88.9%, 89.3%, 72.7%, and 96.2%, respectively, for the hematology group and 80.0%, 93.3%, 80.0%, and 93.3%, respectively, in the ICU group. The CYP51A real-time PCR confirmed 12 wild-type and 2 resistant strains (1 TR34-L98H and 1 TR46-Y121F-T289A mutant). Voriconazole therapy failed for both patients. The AsperGenius multiplex real-time PCR assay allows for sensitive and fast detection ofAspergillusspecies directly from BAL fluid samples. More importantly, this assay detects and differentiates wild-type from resistant strains, even if BAL fluid cultures remain negative.

scholarly journals Diagnosis of invasive fungal infections by a real-time panfungal PCR assay in immunocompromised pediatric patients

Leukemia ◽  
2010 ◽  
Vol 24 (12) ◽  
pp. 2032-2038 ◽  
Author(s):  
C Landlinger ◽  
S Preuner ◽  
L Bašková ◽  
M van Grotel ◽  
N G Hartwig ◽  
...  
Keyword(s):  
Real Time ◽  
Pediatric Patients ◽  
Fungal Infections ◽  
Invasive Fungal Infections ◽  
Pcr Assay ◽  
Panfungal Pcr
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