Rapid detection and identification of mucormycetes in bronchoalveolar lavage samples from immunocompromised patients with pulmonary infiltrates by use of high-resolution melt analysis.

Rapid differential diagnostics of pulmonary infiltrates suspected of invasive fungal disease in an immunocompromised host and early initiation of effective antifungal therapy are crucial for patient outcomes. There are no serological tests available to detect mucormycetes; therefore, PCR-based methods are highly suitable. We validated our previously published PCR followed by high-resolution melt analysis (PCR/HRMA) to detect Rhizopus spp., Rhizomucor pusillus, Lichtheimia corymbifera, and Mucor spp. in bronchoalveolar lavage (BAL) samples from immunocompromised patients who were at risk of invasive fungal disease. All PCR/HRMA-positive samples were retested using novel real-time quantitative PCR (RQ PCR) assays specific to the species identified. In total, between January 2009 and December 2012 we analyzed 99 BAL samples from 86 patients with pulmonary abnormalities using PCR/HRMA. Ninety (91%) BAL samples were negative, and 9 (9%) samples were positive. The sensitivity and specificity of PCR/HRMA were 100% and 93%, respectively. By combining the positive results of PCR/HRMA with positive RQ PCR results, the specificity was raised to 98%. PCR/HRMA, due to its high negative predictive value (99%), represents a fast and reliable tool for routine BAL sample screening for the differential diagnosis of pulmonary infiltrates in immunocompromised patients for the four most clinically important mucormycetes.

[Invasive fungal infections in immunocompromised patients with focus on aspergillosis and its causative agents]. / Invazivní mykatické infekce u imunokompromitovaných nemocných se zamerením na aspergilózu a její puvodce.

Invasive fungal diseases (IFD) are a life-threatening infectious complications in immunocompromised patients and are associated with high rate of morbidity and mortality. The most common invasive mycosis in patients who underwent an allogeneic hematopoietic stem cell transplantation is invasive aspergilosis (IA), most frequently caused by the clinically dominant species Aspergillus fumigatus and, rarely, also by Aspergillus flavus, Aspergillus terreus and Aspergillus niger. In recent years, other related Aspergillus species were also reported to cause IFD, phenotypically similar to A. fumigatus and moreover, frequently exhibiting resistance towards various antifungals. For example, it is Aspergillus lentulus, Aspergillus viridinutans, Neosartoya fischeri, etc. Classical microbiological methods such as direct microscopy or culture are usually used for the identification of Aspergillus species. The application of PCR-based molecular techniques and monitoring of secondary metabolites production enable detection and identification of species, which are not distinguishable solely by their morphology. PCR methods are also useful for molecular strain typing of aspergilli and can reveal the genetic diversity of isolates.

[Detection and identification of filamentous fungi causing mycoses using molecular genetic methods]. / Detekce a identifikace puvodcu mykotických infekcí zpusobených vlaknitými houbami pomocí metod molekulární genetiky.

Methods of molecular genetics offer rapid and sensitive detection and identification of fungal pathogens. The currently used methods are based mainly on PCR. With regard to the ubiquitous presence of fungi, it is important to prevent contamination during the whole process, from sampling to laboratory analyses. Molecular genetic methods are not included among the EORTC/MSG criteria used for the diagnosis of invasive fungal diseases since interlaboratory standardization is still missing. Another reason is the use of different target genes for PCR. ITS sequences from rDNA clusters are recommended for DNA barcoding of fungi. The use of DNA sequencing for identification of fungi in clinical samples has certain limitations and interpretation of results could be problematic in some cases. DNA sequences are searched and compared in public databases on the Internet, the best known of them being the GenBank. However, more reliable data for identification of fungi are offered by specialized mycological databases.

Common variable immunodeficiency patients display elevated plasma levels of granulocyte activation markers elastase and myeloperoxidase.

Common variable immunodeficiency disorders (CVIDs) represent a group of primary immunodeficiency diseases characterized by hypogammaglobulinemia and dysfunctional immune response to invading pathogens. Previous studies have indicated that CVID is associated with microbial translocation and systemic myeloid cell activation. The goal of this study was to determine whether patients with CVID display elevated systemic levels of markers of granulocyte activation and whether the levels are further influenced by intravenous immunoglobulin (IVIg) infusions. The plasma levels of granulocyte activation markers elastase and myeloperoxidase were determined using enzyme-linked immunosorbent assay (ELISA) in 46 CVID patients and 44 healthy controls. All CVID patients were in a stable state with no apparent acute infection. In addition, granulocyte activation markers' plasma levels in 24 CVID patients were determined prior to and 1 h following IVIg administration. Neutrophil elastase and myeloperoxidase plasma levels were significantly higher in CVID patients than in healthy controls. Systemic elastase levels were further increased following IVIg administration. In vitro stimulation of 13 CVID patients' whole blood using IVIg in a therapeutically relevant dose for 2 h resulted in a significant increase in plasma elastase levels compared to unstimulated blood. The data presented here indicate that CVID is associated with chronic granulocytic activation which is further exacerbated by administering IVIg. Increased myeloperoxidase and elastase levels may contribute to associated comorbidities in CVID patients.