Heteroresistance to Itraconazole Alters the Morphology and Increases the Virulence of Cryptococcus gattii

Cryptococcus gattii is the main etiological agent of cryptococcosis in immunocompetent individuals. The triazole drug itraconazole is one of the antifungals used to treat patients with cryptococcosis. Heteroresistance is an adaptive mechanism to counteract the stress of increasing drug concentrations, and it can enhance the ability of a microorganism to survive under antifungal pressure. In this study, we evaluated the ability of 11 C. gattii strains to develop itraconazole heteroresistance. Heteroresistant clones were analyzed for drug susceptibility, alterations in cell diameter, capsule properties, and virulence in a murine model. Heteroresistance to itraconazole was intrinsic in all of the strains analyzed, reduced both the capsule size and the cell diameter, induced molecular heterogeneity at the chromosomal level, changed the negatively charged cells, reduced ergosterol content, and improved the antioxidant system. A positive correlation between surface/volume ratio of original cells and the level of heteroresistance to itraconazole (LHI) was observed in addition to a negative correlation between capsule size of heteroresistant clones and LHI. Moreover, heteroresistance to itraconazole increased the engulfment of C. gattii by macrophages and augmented fungal proliferation inside these cells, which probably accounted for the reduced survival of the mice infected with the heteroresistant clones and the higher fungal burden in lungs and brain. Our results indicate that heteroresistance to itraconazole is intrinsic and increases the virulence of C. gattii. This phenomenon may represent an additional mechanism that contributes to relapses of cryptococcosis in patients during itraconazole therapy.     

Molecular and Serological Diversity of Neisseria meningitidis Carrier Strains Isolated from Italian Students Aged 14 to 22 Years

Neisseria meningitidis is an obligate human commensal that commonly colonizes the oropharyngeal mucosa. Carriage is age dependent and very common in young adults. The relationships between carriage and invasive disease are not completely understood. In this work, we performed a longitudinal carrier study in adolescents and young adults (173 subjects). Overall, 32 subjects (18.5%) had results that were positive for meningococcal carriage in at least one visit (average monthly carriage rate, 12.1%). Only five subjects tested positive at all four visits. All meningococcal isolates were characterized by molecular and serological techniques. Multilocus sequence typing, PorA typing, and sequencing of the 4CMenB vaccine antigens were used to assess strain diversity. The majority of positive subjects were colonized by capsule null (34.4%) and capsular group B strains (28.1%), accounting for 23.5% and 29.4% of the total number of isolates, respectively. The fHbp and nhba genes were present in all isolates, while the nadA gene was present in 5% of the isolates. The genetic variability of the 4CMenB vaccine antigens in this collection was relatively high compared with that of other disease-causing strain panels. Indications about the persistence of the carriage state were limited to the time span of the study. All strains isolated from the same subject were identical or cumulated minor changes over time. The expression levels and antigenicities of the 4CMenB vaccine antigens in each strain were analyzed by the meningococcal antigen typing system (MATS), which revealed that expression can change over time in the same individual. Future analysis of antigen variability and expression in carrier strains after the introduction of the MenB vaccine will allow for a definition of its impact on nasopharyngeal/oropharyngeal carriage.     

Panencephalopathic Creutzfeldt‐Jakob Disease with Distinct Pattern of Prion Protein Deposition in a Patient with D178N Mutation and Homozygosity for Valine at Codon 129 of the Prion Protein Gene

Prion diseases include sporadic, acquired and genetic forms linked to mutations of the prion protein (PrP) gene (PRNP). In subjects carrying the D178N PRNP mutation, distinct phenotypes can be observed, depending on the methionine/valine codon 129 polymorphism. We present here a 53‐year‐old woman with D178N mutation in the PRNP gene and homozygosity for valine at codon 129. The disease started at age 47 with memory deficits, progressive cognitive impairment and ataxia. The clinical picture slowly worsened to a state of akinetic mutism in about 2 years and the disease course was 6 years. The neuropathologic examination demonstrated severe diffuse cerebral atrophy with neuronal loss, spongiosis and marked myelin loss and tissue rarefaction in the hemispheric white matter, configuring panencephalopathic Creutzfeldt‐Jakob disease. PrP deposition was present in the cerebral cortex, basal ganglia and cerebellum with diffuse synaptic‐type pattern of immunoreactivity and clusters of countless, small PrP deposits, particularly evident in the lower cortical layers, in the striatum and in the molecular layer of the cerebellum. Western blot analysis showed the presence of type 1 PrP^Sc (Parchi classification). These findings underline the clear‐cut distinction between the neuropathological features of Creutzfeldt‐Jakob disease associated with D178N PRNP mutation and those of fatal familial insomnia.