Mutation on lysX from Mycobacterium avium hominissuis impacts the host–pathogen interaction and virulence phenotype

The lysX gene from Mycobacterium avium hominissuis (MAH) is not only involved in cationic antimicrobial resistance but also regulates metabolic activity. An MAH lysX deficient mutant was shown to exhibit a metabolic shift at the extracellular state preadapting the bacteria to the conditions inside host-cells. It further showed stronger growth in human monocytes. In the present study, the LysX activity on host–pathogen interactions were analyzed. The lysX mutant from MAH proved to be more sensitive toward host-mediated stresses such as reactive oxygen species. Further, the lysX mutant exhibited increased inflammatory response in PBMC and multinucleated giant cell (MGC) formation in human macrophages during infection studies. Coincidentally, the lysX mutant strain revealed to be more reproductive in the Galleria mellonella infection model. Together, these data demonstrate that LysX plays a role in regulating the bacillary load in host organisms and the lack of lysX gene facilitates MAH adaptation to intracellular host-habitat, thereby suggesting an essential role of LysX in the modulation of host–pathogen interaction.Peer Reviewe

A multicentre external quality assessment: A first step to standardise PCR protocols for the diagnosis of histoplasmosis and coccidioidomycosis

Background: In-house real-time PCR (qPCR) is increasingly used to diagnose the so-called endemic mycoses as commercial assays are not widely available. Objectives: To compare the performance of different molecular diagnostic assays for detecting Histoplasma capsulatum and Coccidioides spp. in five European reference laboratories. Methods: Two blinded external quality assessment (EQA) panels were sent to each laboratory that performed the analysis with their in-house assays. Both panels included a range of concentrations of H. capsulatum (n = 7) and Coccidioides spp. (n = 6), negative control and DNA from other fungi. Four laboratories used specific qPCRs, and one laboratory a broad-range fungal conventional PCR (cPCR) and a specific cPCR for H. capsulatum with subsequent sequencing. Results: qPCR assays were the most sensitive for the detection of H. capsulatum DNA. The lowest amount of H. capsulatum DNA detected was 1-4 fg, 0.1 pg and 10 pg for qPCRs, specific cPCR and broad-range cPCR, respectively. False positive results occurred with high concentrations of Blastomyces dermatitidis DNA in two laboratories and with Emergomyces spp. in one laboratory. For the Coccidioides panel, the lowest amount of DNA detected was 1-16 fg by qPCRs and 10 pg with the broad-range cPCR. One laboratory reported a false positive result by qPCR with high load of Uncinocarpus DNA. Conclusion: All five laboratories were able to correctly detect H. capsulatum and Coccidioides spp. DNA and qPCRs had a better performance than specific cPCR and broad-range cPCR. EQAs may help standardise in-house molecular tests for the so-called endemic mycoses improving patient management.This work was supported by research project PI21CIII/00007 from Spanish Fondo de Investigaciones Sanitarias of the Instituto de Salud Carlos IIIS

Phenotypic Diversity of Cryptococcus neoformans var. neoformans Clinical Isolates from Localized and Disseminated Infections

Cryptococcus neoformans var. neoformans is the second most prevalent agent of cryptococcosis in central Europe. Infections mostly present with localized skin and disseminated infections. Previous studies did not find these presentations to be determined by the fungal genotype as detected by multilocus sequence typing (MLST). However, phenotypic fungal traits may impact clinical presentation. Here, we studied the growth and virulence factors of C. neoformans var. neoformans isolates from disseminated and localized infections and an environmental isolate. We used coincubation with Acanthamoeba castellanii and the Galleria mellonella infection model to identify phenotypic characteristics potentially associated with clinical presentation. Clinical isolates of C. neoformans var. neoformans present a substantial phenotypic variability. Median survival of G. mellonella varied between 6 and 14 days. C. neoformans var. neoformans isolates from disseminated infections showed stronger melanization and larger capsules. They demonstrated superior uptake into an amoeba and increased cytotoxicity for the amoeba. Differences of strains from localized and disseminated infections in coincubation with amoeba are in line with the importance of phagocytes in the pathogenesis of disseminated cryptococcosis. Phenotypic traits and non-vertebrate infection models may help understand the virulence potential of C. neoformans var. neoformans isolates.Peer Reviewe

Disseminated coccidioidomycosis: Monitoring of serologic markers for treatment response

We describe a patient with a disseminated coccidioidomycosis. Biomarkers in serum during itraconazole therapy showed a rapid clearing of Coccidioides DNA as detected by PCR. Coccidioides antibody detection by lateral flow assay became negative after one year and decreased from 1:64 to 1:8 in the complement fixation test after two years. The (1 → 3)-ß-D-glucan levels normalised after two years without increase after cessation of antifungal therapy. Biomarkers in serum may guide treatment decisions in disseminated coccidioidomycosis.Peer Reviewe

Exclusion of Mucorales Co-Infection in a Patient with Aspergillus flavus Sinusitis by Fluorescence In Situ Hybridization (FISH)

Invasive fungal infections are associated with increased mortality in hematological patients. Despite considerable advances in antifungal therapy, the evaluation of suspected treatment failure is a common clinical challenge requiring extensive diagnostic testing to rule out potential causes, such as mixed infections. We present a 64-year-old patient with secondary AML, diabetes mellitus, febrile neutropenia, and sinusitis. While cultures from nasal tissue grew Aspergillus flavus, a microscopic examination of the tissue was suggestive of concomitant mucormycosis. However, fluorescence in situ hybridization (FISH) using specific probes targeting Aspergillus and Mucorales species ruled out mixed infection. This was confirmed by specific qPCR assays amplifying the DNA of Aspergillus, but not of Mucorales. These results provided a rational basis for step-down targeted therapy, i.e., the patient received posaconazole after seven days of calculated dual therapy with liposomal amphotericin B and posaconazole. Despite clinical response to the antifungal therapy, he died due to the progression of the underlying disease within two weeks after diagnosis of fungal infection. Molecular diagnostics applied to tissue blocks may reveal useful information on the etiology of invasive fungal infections, including challenging situations, such as with mixed infections. A thorough understanding of fungal etiology facilitates targeted therapy that may improve therapeutic success while limiting side effects.Peer Reviewe

Identification of Mucormycosis by Fluorescence In Situ Hybridization Targeting Ribosomal RNA in Tissue Samples

Mucormycosis is an invasive fungal infection associated with high mortality, partly due to delayed diagnosis and inadequate empiric therapy. As fungal cultures often fail to grow Mucorales, identification of respective hyphae in tissue is frequently needed for diagnosis but may be challenging. We studied fluorescence in situ hybridization (FISH) targeting specific regions of the fungal ribosomal RNA (rRNA) of Mucorales to improve diagnosis of mucormycosis from tissue samples. We generated a probe combination specifically targeting Mucorales. Probe specificity was verified in silico and using cultivated fungi. Mucorales hyphae in tissue of a mouse model demonstrated a bright cytoplasmatic hybridization signal. In tissue samples of patients with mucormycosis, a positive signal was seen in 7 of 12 (58.3%) samples. However, autofluorescence in 3 of 7 (42.9%) samples impaired the diagnostic yield. Subsequent experiments suggested that availability of nutrients and antifungal therapy may impact on the FISH signal obtained with Mucorales hyphae. Diagnosis of mucormycosis from tissue might be improved by rRNA FISH in a limited number of cases only. FISH signals may reflect different physiological states of fungi in tissue. Further studies are needed to define the value of FISH to diagnose mucormycosis from other clinical samples.Peer Reviewe

Cryptococcus bacillisporus (VGIII) Meningoencephalitis Acquired in Santa Cruz, Bolivia

We describe a case of chronic meningoencephalitis with hydrocephalus caused by Crypto- coccus bacillisporus (VGIII) in an immunocompetent patient from Santa Cruz, Bolivia. This first report of a member of the Cryptococcus gattii species complex from Bolivia suggests that C. bacillisporus (VGIII) is present in this tropical region of the country and complements our epidemiological and clinical knowledge of this group of emerging fungal pathogens in South America.Peer Reviewe

Comparison of quantitative real time PCR with Sequencing and ribosomal RNA-FISH for the identification of fungi in Formalin fixed, paraffin-embedded tissue specimens

Background: Identification of the causative agents of invasive fungal infections (IFI) is critical for guiding antifungal therapy. Cultures remain negative in a substantial number of IFI cases. Accordingly, species identification from formalin fixed, paraffin embedded (FFPE) tissue specimens by molecular methods such as fluorescence in situ hybridisation (FISH) and PCR provides an appealing approach to improve management of patients. Methods: We designed FISH probes targeting the 28S rRNA of Aspergillus and Candida and evaluated them with type strains. Fluorescence microscopy (FM), using FISH probes and quantitative broadrange fungal PCR targeting the rRNA gene were applied to FFPE tissue specimens from patients with proven IFI in order to explore benefits and limitations of each approach. Results: PCR followed by sequencing identified a broad spectrum of pathogenic fungi in 28 of 40 evaluable samples (70%). Hybridisation of FISH probes to fungal rRNA was documented in 19 of 40 tissue samples (47.5%), including 3 PCR negative samples with low fungal burden. The use of FISH was highly sensitive in invasive yeast infections, but less sensitive for moulds. In samples with hyphal elements, the evaluation of hybridisation was impaired due to autofluorescence of hyphae and necrotic tissue background. Conclusions: While PCR appears to be more sensitive in identifying the causative agents of IFI, some PCR negative and FISH positive samples suggest that FISH has some potential in the rapid identification of fungi from FFPE tissue samples