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Invasive Pulmonary Aspergillosis Diagnosed by Molecular Test

By Labmedica International staff writers
Posted on 12 Jun 2019
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Image: The PLATELIA ASPERGILLUS Ag Test (Photo courtesy of Bio-Rad Laboratories).
Image: The PLATELIA ASPERGILLUS Ag Test (Photo courtesy of Bio-Rad Laboratories).
Invasive aspergillosis (IA) is a common opportunistic fungal infection, mainly affecting patients with severe and prolonged neutropenia. Early diagnosis of invasive pulmonary aspergillosis (IPA) is notoriously difficult, but crucial for prompt treatment initiation required to improve patient outcome.

The mycological evidence used for the diagnosis of probable IPA includes traditional microbiological microscopy and culture of a respiratory specimen, along with non-culture-based serological tests, like that of GM antigen in serum and bronchoalveolar lavage (BAL) fluid, as well as β-d-Glucan in serum.

Medical scientists from Rambam Health Care Campus (Haifa, Israel) and their associates performed bronchoscopies using a fiber-optic bronchoscope with cardiopulmonary monitoring. The procedure was conducted under conscious sedation and local anesthesia. Laboratory analysis of the broncho-alveolar lavage (BAL) fluid included the following: cytological staining for the detection of fungal elements, Pneumocystis jirovecii (PJ) bodies and viral inclusion bodies in alveolar cells; bacterial stains and cultures including specific growth media for Mycobacterial spp., Legionella spp.; fungal cultures; viral cultures for herpes simplex virus (HSV), and cytomegalovirus (CMV); polymerase chain reaction (PCR) for the detection of Aspergillus spp., Legionella spp., Mycobacterial spp., PJ, HSV, CMV and respiratory viruses (influenza, parainfluenza, respiratory syncytial virus, adenovirus, human metapneumovirus) nucleic acid.

The GM antigen in serum and BAL fluid was measured using ELISA. Total DNA was prepared from 5 mL of a BAL fluid sample using QIAamp DNA mini kit and was PCR-amplified using a two-step (nested) PCR assay that specifically amplifies a highly conserved Aspergillus species-specific region of the 18S ribosomal RNA gene. A 232bp PCR fragment encoded by the human β-globin gene was amplified in parallel as a control for the presence of host DNA. Total DNA products were amplified in a T3 Thermocycler.

During the 12-year study period, January 2005 to December 2016, 1,072 patients underwent 1,248 bronchoscopies with BAL for a suspected opportunistic lung infection. Of the study population, 630/1072 (59%) were males; median age was 55 (1–90) years. Hematological malignancy was found in 77%, of them 40% had AML and 35.6% underwent hematopoietic stem cell transplantation (HSCT). IPA was diagnosed in 531 patients (42.5%), seven-proven, 280-probable and 244-possible. PCR was positive in 266 cases, of them 213 had IPA, indicating a true positive rate of 80% (213/266) and a false positive rate of 20% (53/266). These results establish the diagnostic performance of PCR to have sensitivity of 40%, specificity of 93%, PPV- 80% and NPV-68%. Of 244 patients with possible IPA, 80 had positive PCR. Including PCR in the diagnostic criteria would move 80 cases from the possible group to the probable one. A combination of positive PCR and/or BAL-GM increases sensitivity to 74%, while positivity of both tests elevates PPV to 99.4%.

The authors concluded that including PCR test for the detection of Aspergillus DNA in BAL in the mycological criteria of the EORTC/MSG definitions increases the rate and the certainty of IPA diagnosis. The study was published in the June 2019 issue of the International Journal of Infectious Diseases.

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