Systematic Approach Revealed SERPING1 Splicing-Affecting Variants to be Highly Represented in the Czech National HAE Cohort.

Hereditary angioedema due to C1 inhibitor deficiency (C1-INH-HAE) is a rare and life-threatening condition characterized by recurrent localized edema. We conducted a systematic screening of SERPING1 defects in a cohort of 207 Czech patients from 85 families with C1-INH-HAE. Our workflow involved a combined strategy of sequencing extended to UTR and deep intronic regions, advanced in silico prediction tools, and mRNA-based functional assays. This approach allowed us to detect a causal variant in all families except one and to identify a total of 56 different variants, including 5 novel variants that are likely to be causal. We further investigated the functional impact of two splicing variants, namely c.550 + 3A > C and c.686-7C > G using minigene assays and RT-PCR mRNA analysis. Notably, our cohort showed a considerably higher proportion of detected splicing variants compared to other central European populations and the LOVD database. Moreover, our findings revealed a significant association between HAE type 1 missense variants and a delayed HAE onset when compared to null variants. We also observed a significant correlation between the presence of the SERPING1 variant c.-21 T > C in the trans position to causal variants and the frequency of attacks per year, disease onset, as well as Clinical severity score. Overall, our study provides new insights into the genetic landscape of C1-INH-HAE in the Czech population, including the identification of novel variants and a better understanding of genotype-phenotype correlations. Our findings also highlight the importance of comprehensive screening strategies and functional analyses in improving the C1-INH-HAE diagnosis and management.

Manifestations of cutaneous mycobacterial infections in patients with inborn errors of IL-12/IL-23-IFNγ immunity

Background: Inborn errors of IL-12/IL-23-IFNγ immunity underlie Mendelian susceptibility to mycobacterial diseases (MSMD), a group of immunodeficiencies characterized by a highly selective susceptibility to weakly virulent strains of mycobacteria, such as non-tuberculous mycobacteria (NTM) and bacillus Calmette-Guérin (BCG). Cutaneous mycobacterial infections are common in MSMD and may represent a red flag for this immunodeficiency. Objectives: We present a case series of four paediatric patients with MSMD, specifically with IFNγR1 and STAT1 deficiencies, and cutaneous NTM/BCG infections to increase awareness of this immunodeficiency, which may, in some cases, be intercepted by the dermatologist and thus timely referred to the immunologist. Materials & Methods: Clinical, laboratory and genetic investigations of the four paediatric patients with MSMD are presented. Results: All four presented patients experienced early complications after BCG vaccination. Two patients suffered recurrent mycobacteriosis, one patient experienced delayed BCG reactivation, and one patient died of disseminated avian mycobacteriosis. The dermatological manifestation in these patients included destructive nasal ulcerations, scrofuloderma of various sites and lupus vulgaris. All patients had a normal basic immune phenotype. Conclusion: The presented cases demonstrate that NTM/BCG infections in otherwise seemingly immunocompetent patients should raise suspicion of MSMD. This is of utmost importance as specific therapeutic approaches, such as IFNγ treatment or haematopoietic stem cell transplantation, may be employed to improve the disease outcome.

Detection and identification of fungi in bronchoalveolar lavage fluid from immunocompromised patients using panfungal PCR.

Rapid diagnostics of fungal pneumonia and initiation of appropriate therapy are still challenging. In this study, we used two panfungal assays to test bronchoalveolar lavage fluid (BALF) samples to prove their ability to confirm invasive fungal disease diagnosis and identify causative agents. Two methods targeting different fungal rDNA regions were used, and the obtained PCR products were sequenced directly or after cloning. In total, 106 BALF samples from 104 patients were tested. After sequencing, we obtained 578 sequences. Four hundred thirty-seven sequences were excluded from further analysis due to duplication (n = 335) or similarity with sequences detected in the extraction control sample (n = 102); 141 unique sequences were analyzed. Altogether, 23/141 (16%) of the fungi detected belonged to pathogenic species, and 63/141 (45%) were identified as various yeasts; a variety of environmental or very rare fungal human pathogens represented 29/141 (21%) of the total and 26/141 (18%) were described as uncultured fungus. Panfungal PCR detected fungal species that would be missed by specific methods in only one case (probable cryptococcosis). Panfungal PCR followed by sequencing has limited use for testing BALF samples due to frequent commensal or environmental fungal species pickup.

Rapid detection of fungal pathogens in bronchoalveolar lavage samples using panfungal PCR combined with high resolution melting analysis.

Despite advances in the treatment of invasive fungal diseases (IFD), mortality rates remain high. Moreover, due to the expanding spectrum of causative agents, fast and accurate pathogen identification is necessary. We designed a panfungal polymerase chain reaction (PCR), which targets the highly variable ITS2 region of rDNA genes and uses high resolution melting analysis (HRM) for subsequent species identification. The sensitivity and specificity of this method was tested on a broad spectrum of the most clinically important fungal pathogens including Aspergillus spp., Candida spp. and mucormycetes. Despite the fact that fluid from bronchoalveolar lavage (BAL) is one of the most frequently tested materials there is a lack of literature sources aimed at panfungal PCR as an IFD diagnostic tool from BAL samples. The applicability of this method in routine practice was evaluated on 104 BAL samples from immunocompromised patients. Due to high ITS region variability, we obtained divergent melting peaks for different fungal species. Thirteen out of 18 patients with proven or probable IFD were positive. Therefore, the sensitivity, specificity, positive predictive value and negative predictive value of our method were 67%, 100%, 100%, and 94%, respectively. In our assay, fungal pathogens identification is based on HRM, therefore omitting the expensive and time consuming sequencing step. With the high specificity, positive and negative predictive values, short time needed to obtain a result, and low price, the presented assay is intended to be used as a quick screening method for patients at risk of IFD.

Disseminated fusariosis by Fusarium proliferatum in a patient with aplastic anaemia receiving primary posaconazole prophylaxis - case report and review of the literature.

Disseminated fusariosis is a life-threatening, invasive, opportunistic infection in immunocompromised patients, especially those with haematological malignancies. The prognosis is poor because these fungi are resistant to many of the available antifungal agents. We present a case of disseminated fusariosis caused by Fusarium proliferatum in a patient with severe aplastic anaemia complicated by a secondary infection of Aspergillus flavus, with a fatal outcome. We also review the documented Fusarium infections in immunocompromised hosts.

Detecting human cytomegalovirus drug resistant mutations and monitoring the emergence of resistant strains using real-time PCR.

BACKGROUND: Antiviral resistance development is a serious complication of human cytomegalovirus virostatic therapy caused by mutations in UL 97 and/or UL54 genes. OBJECTIVES: To determinate the presence of sensitive and resistant strains in patients developing antiviral resistance. STUDY DESIGN: We used three different molecular biological methods for mutation analysis-restriction fragment length polymorphism, sequencing and real-time PCR approach. RESULTS: We describe three allogeneic hematopoietic stem cell transplant patients developing the GCV resistant HCMV strains manifested by virostatic treatment failure. In these patients we identified UL97 mutations L595S, A594V and A594T and monitored the dynamics of coexisted sensitive/resistant strains. We confirmed the presence of mixed HCMV populations and in two patients a phenomenon of sensitive strain repopulation which occurred after 6.5 months and 1 month after removing GCV pressure. CONCLUSIONS: Our results show changes in proportions of sensitive/resistant subpopulations over time but other studies would be required to demonstrate the beneficial impact of their monitoring on clinical outcome.

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.