Detection of virulence factors and antifungal susceptibility of human and avian Aspergillus flavus isolates

Aspergillus flavus is the second leading cause of invasive and non-invasive aspergillosis. Secretion of hydrolytic enzymes is considered as a virulence factor in this species. Our work aimed to study in vitro production of some virulence factors, to evaluate the biofilm production against human and avian A. flavus isolates and to investigate the antifungal susceptibility agents. Hydrolytic enzymes, biofilm production and molecular typing were studied for 62 human and 36 avian A. flavus isolates by specific solid media and six microsatellite markers. The susceptibility to antifungal agents was evaluated for 37 human isolates. All human and avian A. flavus isolates showed positive activities of extracellular hydrolase: phospholipase, protease and hemolysin. A positive elastase activity was seen in 64.51% of human A. flavus isolates and 86.1% of avian A. flavus isolates. All A. flavus in these two populations formed biofilms. Statistical significant difference was observed for the mean phospholipase activities (P = 0.025) and biofilm quantification (P = 0.0001) between human and avian A. flavus isolates. The in vitro susceptibility results showed a resistance in 83.7%, 81.08% and 16.21% of A. flavus isolates respectively to amphotericin B, itraconazole and posaconazole. No association was noted between all virulence factors and the genotypes of human and avian isolates. Our study allowed us to show that human strains have a higher production of extracellular hydrolases and biofilm then avian strains. These virulence factors appear to act synergistically to contribute to the virulence of A. flavus strains. Moreover, significant correlation between virulence patterns and antifungal susceptibility profiles was observed.     

Pathogenic free-living amoebae: Epidemiology and clinical review

Free-living amoebae are widely distributed in soil and water. Small number of them was implicated in human disease: Acanthamoeba spp., Naegleria fowleri, Balamuthia mandrillaris and Sappinia diploidea. Some of the infections were opportunistic, occurring mainly in immunocompromised hosts (Acanthamoeba and Balamuthia encephalitis) while others are non opportunistic (Acanthamoeba keratitis, Naegleria meningoencephalitis and some cases of Balamuthia encephalitis). Although, the number of infections caused by these amoebae is low, their diagnosis was still difficult to confirm and so there was a higher mortality, particularly, associated with encephalitis. In this review, we present some information about epidemiology, ecology and the types of diseases caused by these pathogens amoebae.     

Osteoma of the calvaria in L-2-hydroxyglutaric aciduria

L-2-Hydroxyglutaric aciduria (L-2-OHGA) is a rare autosomal recessive neurometabolic disease linked to chromosome 14q21.1 and is caused by mutations in the gene that most likely encodes L: -2-hydroxyglutarate dehydrogenase, which normally catalyses L: -2-hydroxyglutarate to alpha-ketoglutarate. It is characterized by progressive mental deterioration, pyramidal and cerebellar syndromes, macrocephaly and marked polycystic white-matter degeneration mainly involving frontal lobes. Brain tumours of variable nature have frequently been observed in L-2-OHGA. We report a patient affected by this disease who at the age of 20 years developed a bone tumour involving the right frontal region of the calvaria. He had first presented at the age of 10 years with psychomotor delay, clumsy gait and moderate mental impairment. Examination showed macrocephaly, cerebellar ataxia and quadripyramidal syndrome. Brain MRI showed low signal intensities on T1-weighted images and high signal intensities on T2-weighted images in cerebral subcortical white matter. Serum and urinary amino acid assay was normal. Urinary 2-hydroxyglutaric acid was 1418 mmol/mol creatinine (controls <25). Analysis of the L-2-hydroxyglutarate dehydrogenase gene revealed a homozygous mutation in exon 2 (A320G). At the age of 20 years, an osteoma of the right frontal bone was diagnosed. This finding reinforces the opinion concerning the association of L-2-OHGA and tumorigenesis and prompted us to verify the possible responsibility of some overproduced substances in this disease for the development of tumours and to look for any correlation between the type of mutation in the L-2-OHGA gene and the tumorigenic potential observed in some patients affected by this disease.     

Novel prokaryotic diversity in sediments of Tunisian multipond solar saltern

The phylogenetic diversity of a sediment microbial community from two ponds having different salinities, 150–200 g/l (M2) and 250–300 g/l (TS38), of an Sfax (Tunisia) solar saltern, was investigated using 16S rRNA clone libraries. The 16S rRNA genes from 135 bacterial clones and 105 archaeal clones were sequenced and phylogenetically analyzed. 32 operational taxonomic units (OTUs) were generated for Bacteria and 64 for Archaea. The bacterial community in M2 sediment was affiliated only with Bacteroidetes, while that in TS38 sediment was dominated by clones affiliated with Bacteroidetes, (gamma, alpha, delta) Proteobacteria and unclassified bacteria; these represented 56.52, 26.08, 4.34, 4.34 and 8.7% of the OTUs, respectively. In the M2 and TS38 sediments, 44.44 and 43.47% of the bacterial OTUs, respectively, were novel. All archaeal sequences fell into the Euryarchaeota phylum. In both sediments, 38.46 and 72.55% of the OTUs had less than 97% 16S rRNA sequence identity, representing novel OTUs. Two sequences, retrieved from TS38 sediment, were found to be affiliated with the candidate division MSBL-1 defining two OTUs. The sediment phylogenetic study revealed the presence of a highly diverse microbial population in highly salty media.     

Highly Discriminatory Variable-Number Tandem-Repeat Markers for Genotyping of Trichophyton interdigitale Strains

Trichophyton interdigitale is the second most frequent cause of superficial fungal infections of various parts of the human body. Studying the population structure and genotype differentiation of T. interdigitale strains may lead to significant improvements in clinical practice. The present study aimed to develop and select suitable variable-number tandem-repeat (VNTR) markers for 92 clinical strains of T. interdigitale. On the basis of an analysis of four VNTR markers, four to eight distinct alleles were detected for each marker. The marker with the highest discriminatory power had eight alleles and a D value of 0.802. The combination of all four markers yielded a D value of 0.969 with 29 distinct multilocus genotypes. VNTR typing revealed the genetic diversity of the strains, identifying three populations according to their colonization sites. A correlation between phenotypic characteristics and multilocus genotypes was observed. Seven patients harbored T. interdigitale strains with different genotypes. Typing of clinical T. interdigitale samples by VNTR markers displayed excellent discriminatory power and 100% reproducibility.     

Genetic heterogeneity of megaloblastic anaemia type 1 in Tunisian patients

Megaloblastic anaemia 1 (MGA1) is a rare autosomal recessive condition characterized by selective intestinal vitamin B12 malabsorption and proteinuria. More than 200 MGA1 patients have been identified worldwide, but the disease is relatively prevalent in Finland, Norway and several Eastern Mediterranean regions. MGA1 is genetically heterogeneous and can be caused by mutations in either the cubilin (CUBN) or the amnionless (AMN) gene. In the present study we investigated the molecular defect underlying MGA1 in nine Tunisian patients belonging to six unrelated consanguineous families. Haplotype and linkage analyses, using microsatellite markers surrounding both CUBN and AMN genes, indicated that four out of the six families were likely to be linked to the CUBN gene. Patients from these families were screened for the Finnish, Mediterranean and Arabian mutations already published. None of the screened mutations could be detected in our population. One family showed a linkage to AMN gene. Direct screening of the AMN gene allowed the identification of the c.208-2A>G mutation, previously described in a Jewish Israeli patient of Tunisian origin and in Turkish patients. This suggests that the c.208-2A>G mutation may derive from a single Mediterranean founder ancestor. For the last family, haplotype analysis excluded both CUBN and AMN genes, suggesting the existence of a third locus that may cause MGA1.