Molecular Detection and Species-Specific Identification of Medically Important Aspergillus Species by Real-Time PCR in Experimental Invasive Pulmonary Aspergillosis

Abstract

Diagnosis of invasive pulmonary aspergillosis (IPA) remains a major challenge to clinical microbiology laboratories. We developed rapid and sensitive quantitative PCR (qPCR) assays for genus-and species-specific identification of Aspergillus infections by use of TaqMan technology. In order to validate these assays and understand their potential diagnostic utility, we then performed a blinded study of bronchoalveolar lavage (BAL) fluid specimens from well-characterized models of IPA with the four medically important species. A set of real-time qPCR primers and probes was developed by utilizing unique ITS1 regions for genus-and species-specific detection of the four most common medically important Aspergillus species (Aspergillus fumigatus, A. flavus, A. niger, and A. terreus). Pan-Aspergillus and species-specific qPCRs with BAL fluid were more sensitive than culture for detection of IPA caused by A. fumigatus in untreated (P < 0.0007) and treated (P < 0.008) animals, respectively. For infections caused by A. terreus and A. niger, culture and PCR amplification from BAL fluid yielded similar sensitivities for untreated and treated animals. Pan-Aspergillus PCR was more sensitive than culture for detection of A. flavus in treated animals (P ‫؍‬ 0.002). BAL fluid pan-Aspergillus and species-specific PCRs were comparable in sensitivity to BAL fluid galactomannan (GM) assay. The copy numbers from the qPCR assays correlated with quantitative cultures to determine the pulmonary residual fungal burdens in lung tissue. Pan-Aspergillus and species-specific qPCR assays may improve the rapid and accurate identification of IPA in immunocompromised patients.

Aerial Prevalence of Aspergillus calidoustus Isolates in and around a Tertiary Care Hospital in Kuwait and Assessment of Their Pathogenicity

“…Consistent with published reports (2,13,14), our clinical and environmental isolates showed reduced susceptibility to triazoles. In fact, itraconazole, posaconazole, and voriconazole exhibited relatively higher MIC values against our solitary clinical isolate (Table 1).…”

Early diagnosis and treatment of invasive aspergillosis as a main determinant of outcome – review of literature according to the presented case report

“…However, this traditional approach enables the testing of susceptibility, and expands the number of species possible to reveal, as far as possible, a basis for others tests [21]. Furthermore, the results of positive cultures may distinguish the difference between an active disease, while viable fungi can be found, and the period after the initiation of treatment, when the GM assay may still be positive [22].…”

The Effect Analysis of Different Experimental Methods for the Diagnosis of Invasive Pulmonary Aspergillosis in a Rat Model

“…The sensitivity of the GM assay ranges from 60% to 100% for infected Aspergillus samples, and the specificity ranges from 80% to 100% [10][11][12][13][14]. The cut-off value has a significant impact on diagnosis because of cross-reactivity [8,12,[15][16][17]. Furthermore, treatment with anti-fungal therapies decreases the fungal load and reduces the GM concentration, which can fall below the detectable limit of the GM assay [18].…”

“…Assays that detect fungal antigens such as GM by enzyme-linked immunosorbent assay (GM assay) or Aspergillus DNA by polymerase chain reaction (PCR) are emerging diagnostic methods; however, their specificity and sensitivity require additional characterization and refinement. The sensitivity of the GM assay ranges from 60% to 100% for infected Aspergillus samples, and the specificity ranges from 80% to 100% [10][11][12][13][14]. The cut-off value has a significant impact on diagnosis because of cross-reactivity [8,12,[15][16][17].…”