CoERG11 A395T mutation confers azole resistance in Candida orthopsilosis clinical isolates.

Background: Candida orthopsilosis is a human fungal pathogen responsible for a wide spectrum of symptomatic infections. Evidence suggests that C. orthopsilosis is mainly susceptible to azoles, the most extensively used antifungals for treatment of these infections. However, fluconazole-resistant clinical isolates are reported. Objectives: This study evaluated the contribution of a single amino acid substitution in the azole target CoErg11 to the development of azole resistance in C. orthopsilosis. Methods: C. orthopsilosis clinical isolates (n = 40) were tested for their susceptibility to azoles and their CoERG11 genes were sequenced. We used a SAT1 flipper-driven transformation to integrate a mutated CoERG11 allele in the genetic background of a fluconazole-susceptible isolate. Results: Susceptibility testing revealed that 16 of 40 C. orthopsilosis clinical isolates were resistant to fluconazole and to at least one other azole. We identified an A395T mutation in the CoERG11 coding sequence of azole-resistant isolates only that resulted in the non-synonymous amino acid substitution Y132F. The SAT1 flipper cassette strategy led to the creation of C. orthopsilosis mutants that carried the A395T mutation in one or both CoERG11 alleles (heterozygous or homozygous mutant, respectively) in an azole-susceptible genetic background. We tested mutant strains for azole susceptibility and for hot-spot locus heterozygosity. Both the heterozygous and the homozygous mutant strains exhibited an azole-resistant phenotype. Conclusions: To the best of our knowledge, these findings provide the first evidence that the CoErg11 Y132F substitution confers multi-azole resistance in C. orthopsilosis.

Susceptibility Testing of Common and Uncommon Aspergillus Species against Posaconazole and Other Mold-Active Antifungal Azoles Using the Sensititre Method.

We tested 59 common and 27 uncommon Aspergillus species isolates for susceptibility to the mold-active azole antifungal agents itraconazole, voriconazole, and posaconazole using the Sensititre method. The overall essential agreement with the CLSI reference method was 96.5% for itraconazole and posaconazole and was 100% for voriconazole. By the Sensititre method as well as the CLSI reference method, all of 10 A. fumigatus isolates with a cyp51 mutant genotype were classified as being non-wild-type isolates (MIC > epidemiological cutoff value [ECV]) with respect to triazole susceptibility.

Serum Endotoxin Activity Measured with Endotoxin Activity Assay Is Associated with Serum Interleukin-6 Levels in Patients on Chronic Hemodialysis.

BACKGROUND: This study aims to evaluate, in patients on chronic hemodialysis (PHD), the levels of endotoxin through a chemiluminescent bioassay based on the oxidative burst reaction of activated neutrophils to complement coated LPS-IgM immune complexes and define the variables possibly correlated. METHODS: In 61 PHD, we measured serum endotoxin activity (EA) with the Endotoxin Activity Assay (EAA™) and we defined the possible association with demographic, clinical and laboratory variables. RESULTS: Mean serum EA was 0.43 ± 0.26 UI. EA was low (<0.40) in 29 patients (47.5%), intermediate (0.40-0.60) in 14 (23%) and high (>0.60) in 18 (29.5%). A significant exponential relationship was detected between EA and serum interleukin-6 (IL-6) levels (r = 0.871). At the multiple regression analysis, intermediate-high EA was directly associated only with serum IL-6 levels. In a second model of multiple regression analysis without the variable serum IL-6 levels, intermediate-high EA was directly associated with constipation and serum troponin levels and inversely associated with serum albumin and the monthly number of sevelamer tablets. CONCLUSIONS: A high percentage of PHD has intermediate or high EA. Intermediate-high EA is significantly associated with serum IL-6 levels.

Advanced tools for BRCA1/2 mutational screening: comparison between two methods for large genomic rearrangements (LGRs) detection.

BACKGROUND: Currently, multiplex ligation-dependent probe amplification (MLPA) is the most commonly used technique for the detection of large genomic rearrangements (LGRs) in the BRCA1/2 genes. However, a very fast assay, the BRCA1/2 multiplex amplicon quantification (MAQ), has been recently developed by Multiplicom. METHODS: As no data regarding the application of MAQ method to BRCA1/2 genes are available in literature, here we compared for the first time the performance of the MAQ assay with MLPA by using several positive BRCA1/2 LGRs DNA samples (previously tested by MLPA). RESULTS: MAQ method was able to detect all BRCA1/2 LGRs and no false-positive or -negative results were obtained in independent repetitive experiments. CONCLUSIONS: We can affirm that MAQ, as well as MLPA method, results to be valid and reproducible tools for molecular diagnostics and we are confident that this assay can be used for BRCA1/2 mutational screening as a fast and safe alternative to MLPA, particularly in the first line of analysis.

Functional effect of Saffron supplementation and risk genotypes in early age-related macular degeneration: a preliminary report.

BACKGROUND: To determine whether the functional effects of oral supplementation with Saffron, a natural compound that proved to be neuroprotective in early age-related macular degeneration, are influenced by complement factor H (CFH) and age-related maculopathy susceptibility 2 (ARMS2) risk genotypes. METHODS: Thirty-three early AMD patients, screened for CFH (rs1061170) and ARMS2 (rs10490924) polymorphisms and receiving Saffron oral supplementation (20 mg/day) over an average period of treatment of 11 months (range, 6-12), were longitudinally evaluated by clinical examination and focal electroretinogram (fERG)-derived macular (18°) flicker sensitivity estimate. fERG amplitude and macular sensitivity, the reciprocal value of the estimated fERG amplitude threshold, were the main outcome measures. RESULTS: After three months of supplementation, mean fERG amplitude and fERG sensitivity improved significantly when compared to baseline values (p < 0.01). These changes were stable throughout the follow-up period. No significant differences in clinical and fERG improvements were observed across different CFH or ARMS2 genotypes. CONCLUSIONS: The present results indicate that the functional effect of Saffron supplementation in individual AMD patients is not related to the major risk genotypes of disease.

Rapid detection of CFH (p.Y402H) and ARMS2 (p.A69S) polymorphisms in age-related macular degeneration using high-resolution melting analysis.

BACKGROUND: Age-related macular degeneration (AMD) is a complex disorder causing irreversible central vision loss. Complement Factor H (CFH) and age-related maculopathy susceptibility 2 (ARMS2) are now widely accepted as important AMD susceptibility genes. In particular, two specific variants, CFH p.Y402H and ARMS2 p.A69S, have been reported as strongly AMD associated. In order to perform the genetic screening of these single nucleotide polymorphisms (SNPs), we describe a high resolution melting analysis (HRM) as a rapid closed tube mutation scanning assay. METHODS: To validate HRM genotyping, 94 DNA samples from AMD patients (previously genotyped by sequence analysis) were analyzed. PCR amplification and melting curve analysis were performed in the LightCycler 480 Real-Time PCR System. In order to evaluate the accuracy of the HRM assay, we performed a blinded study of 20 unknown independent samples. RESULTS: We correctly genotyped all samples. In fact, all samples corresponded to the previous genotype assignments. CONCLUSIONS: Early identification of individuals with genetic risk variants CFH p.Y402H and ARMS2 p.A69S is clinically important for the definition of AMD status. High-resolution DNA melting is homogenous, accurate and rapid method for CFH and ARMS2 genotyping.

Molecular diagnosis of congenital adrenal hyperplasia due to 21-hydroxylase deficiency: an update of new CYP21A2 mutations.

Steroid 21-hydroxylase deficiency is present in more than 90% of patients with congenital adrenal hyperplasia, an inherited metabolic disorder of adrenal steroidogenesis. Impaired enzymatic activity leads to the accumulation of metabolic intermediates (progesterone and 17-hydroxyprogesterone), which results in excessive androgen production and varied signs of virilisation. CYP21A2 is an active gene and encodes for the steroid 21-hydroxylase enzyme, whereas CYP21A1P is an inactive pseudogene that contains a series of deleterious mutations. The major part of disease-causing mutations in CYP21A2 alleles are CYP21A1P-derived sequence transferred to the active gene by macro or microconversion events. Approximately 5% of all disease-causing CYP21A2 alleles harbour rare mutations that do not originate from the pseudogene. A list of all reported CYP21A2 mutations can be found in the CYP21A2 database created by the Human Cytochrome P450 (CYP) Allele Nomenclature Committee (http:www.imm.Ki.se/CYPalleles/cyp21.htm). Unfortunately, the last update of this database was in 2006. However, over the last 4 years many other novel CYP21A2 mutations have been reported in PubMed. The aim of this review is to provide a focus on the molecular and genetic aspects of the diagnosis of 21-hydroxylase deficiency. In addition, an updated list of the last new CYP21A2 mutations is included.

Foreign language effect in decision-making: How foreign is it?

It has been shown that decisions and moral judgments differ when made using native languages compared to foreign languages. Cross-linguistic differences appeared in foreign languages that monolinguals typically acquired in school and used neither routinely nor extensively. We replicated these differences with two populations of proficient, native bilinguals (Italian-Venetian; Italian-Bergamasque). Venetian and Bergamasque are spoken in households and informal circles, unlike Italian, which is also used in more formal contexts. The findings reported in foreign languages for the Asian Disease Problem and the Footbridge Dilemma were reproduced in Venetian and Bergamasque. Our results show that language effects on decision-making and moral judgments are not restricted to foreign languages. The explanation proposed for foreign languages of cross-linguistic differences in emotion responses does not apply to our proficient, native bilinguals, who showed emotion responses of equal intensity in their languages. We propose that the contexts in which bilinguals use a language - either native, regional or foreign - could affect decisions.

Two novel CYP21A2 missense mutations in Italian patients with 21-hydroxylase deficiency: Identification and functional characterisation.

Steroid 21-hydroxylase deficiency is present in more than 90% of patients with congenital adrenal hyperplasia (CAH), an inherited metabolic disorder of adrenal steroidogenesis. Impaired enzymatic activity leads to the accumulation of metabolic intermediates (progesterone and 17-hydroxyprogesterone), which results in excessive androgen production and varied signs of virilisation. CYP21A2 is an active gene and encodes for the steroid 21-hydroxylase enzyme, whereas CYP21A1P is an inactive pseudogene that contains a series of deleterious mutations. The major part of disease- causing mutations in CYP21A2 alleles are CYP21A1P-derived sequence transferred to the active gene by macro or microconversion events. Only around 5% of all disease-causing CYP21A2 alleles harbour rare mutations that do not originate from the pseudogene. In this report, we report the characterisation of two novel CYP21A2 missense mutations (p.H119R and p.I194N) found in two unrelated Italian patients with nonclassic (NC) CAH clinical diagnosis. Functional in vitro assays for mutagenized CYP21 enzymes were performed in transiently transfected mammalian cells to test the residual enzyme activity and the apparent kinetic values. The residual activities obtained for p.H119R and p.I194N mutants allowed to classify them as NC-CAH associated mutations. These results correlate with the rate of severity of the patients' disease. Finally, the new p.H119R and p.I194N mutations should be included in the panel of those already listed for association with the NC form of 21-hydroxylase deficiency.

A new CYP21A1P/CYP21A2 chimeric gene identified in an Italian woman suffering from classical congenital adrenal hyperplasia form.

BACKGROUND: More than 90% of Congenital Adrenal Hyperplasia (CAH) cases are associated with mutations in the 21-hydroxylase gene (CYP21A2) in the HLA class III area on the short arm of chromosome 6p21.3. In this region, a 30 kb deletion produces a non functional chimeric gene with its 5' and 3' ends corresponding to CYP21A1P pseudogene and CYP21A2, respectively. To date, five different CYP21A1P/CYP21A2 chimeric genes have been found and characterized in recent studies. In this paper, we describe a new CYP21A1P/CYP21A2 chimera (CH-6) found in an Italian CAH patient. METHODS: Southern blot analysis and CYP21A2 sequencing were performed on the patient. In addition, in order to isolate the new CH-6 chimeric gene, two different strategies were used. RESULTS: The CYP21A2 sequencing analysis showed that the patient was homozygote for the g.655C/A>G mutation and heterozygote for the p.P30L missense mutation. In addition, the promoter sequence revealed the presence, in heterozygosis, of 13 SNPs generally produced by microconversion events between gene and pseudogene. Southern blot analysis showed that the woman was heterozygote for the classic 30-kb deletion producing a new CYP21A1P/CYP21A2 chimeric gene (CH-6). The hybrid junction site was located between the end of intron 2 pseudogene, after the g.656C/A>G mutation, and the beginning of exon 3, before the 8 bp deletion. Consequently, CH-6 carries three mutations: the weak pseudogene promoter region, the p.P30L and the g.655C/A>G splice mutation. CONCLUSION: We describe a new CYP21A1P/CYP21A2 chimera (CH-6), associated with the HLA-B15, DR13 haplotype, in a young Italian CAH patient.

Functional analysis of two rare CYP21A2 mutations detected in Italian patients with a mildest form of congenital adrenal hyperplasia.

BACKGROUND: More than 90% of all cases of congenital adrenal hyperplasia (CAH) result from steroid 21-hydroxylase gene (CYP21A2) mutations. Most of these mutations result from intergenic recombinations between CYP21A2 and closely linked CYP21A1P pseudogene. Rare mutations not generated by gene conversion account for 5-10% of 21-hydroxylase deficiency alleles. OBJECTIVE: Functional analysis of two novel CYP21A2 missense mutations (p.R224W and p.D407N) was performed. DESIGN: Our study was composed of two Italian patients suffering from a very mild form of nonclassic CAH (NC-CAH). To assay the enzymatic activity of mutants, the in vitro analysis was performed in transiently transfected COS-1 cells. RESULTS: The residual activities obtained for p.R224W and p.D407N mutants allow their classification as NC-CAH mutations. These results correlate with the rate of severity of the patients' disease. CONCLUSIONS: In this paper, we report two novel CYP21A2 mutations in two Italian individuals affected by 21-hydroxylase deficiency. Based on the functional in vitro analysis we can classify these mutations as NC-CAH variants.

A new CYP21A2 nonsense mutation causing severe 21-hydroxylase deficiency.

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder caused primarily by defects in the steroid 21-hydroxylase gene (CYP21A2). The CYP21A2 gene is located in the HLA class III region on the short arm of chromosome 6p21.3, along with an inactive pseudogene, CYP21A1P, that is 98% homologous with CYP21A2 in its coding sequence. Most mutations found in the CYP21A2 gene are normally present in the pseudogene, implying that recombination events (microconversion) between these two genes may lead to the transfer of mutations from the pseudogene to the functional gene. Approximately only 5% of all CYP21A2 alleles causing disease harbour rare mutations not originating from the pseudogene. However, detection of these rare and spontaneous mutations has continued to expand worldwide. In this report, we describe the clinical and genetic findings in a Romanian newborn suffering from classic salt wasting form of CAH due to severe 21-hydroxylase deficiency.

Potential Use of MALDI-ToF Mass Spectrometry for Rapid Detection of Antifungal Resistance in the Human Pathogen Candida glabrata.

The echinocandins are relatively new antifungal drugs that represent, together with the older azoles, the recommended and/or preferred agents to treat candidaemia and other forms of invasive candidiasis in human patients. If "time is of the essence" to reduce the mortality for these infections, the administration of appropriate antifungal therapy could be accelerated by the timely reporting of laboratory test results. In this study, we attempted to validate a MALDI-ToF mass spectrometry-based assay for the antifungal susceptibility testing (AFST) of the potentially multidrug-resistant pathogen Candida glabrata against anidulafungin and fluconazole. The practical applicability of the assay, reported here as MS-AFST, was assessed with a panel of clinical isolates that were selected to represent phenotypically and genotypically/molecularly susceptible or resistant strains. The data show the potential of our assay for rapid detection of antifungal resistance, although the MS-AFST assay performed at 3 h of the in vitro antifungal exposure failed to detect C. glabrata isolates with echinocandin resistance-associated FKS2 mutations. However, cell growth kinetics in the presence of anidulafungin revealed important cues about the in vitro fitness of C. glabrata isolates, which may lead to genotypic or phenotypic antifungal testing in clinical practice.

A rapid diagnostic workflow for cefotaxime-resistant Escherichia coli and Klebsiella pneumoniae detection from blood cultures by MALDI-TOF mass spectrometry.

BACKGROUND: Nowadays, the global spread of resistance to oxyimino-cephalosporins in Enterobacteriaceae implies the need for novel diagnostics that can rapidly target resistant organisms from these bacterial species. METHODS: In this study, we developed and evaluated a Direct Mass Spectrometry assay for Beta-Lactamase (D-MSBL) that allows direct identification of (oxyimino)cephalosporin-resistant Escherichia coli or Klebsiella pneumoniae from positive blood cultures (BCs), by using the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) technology. RESULTS: The D-MSBL assay was performed on 93 E. coli or K. pneumoniae growing BC samples that were shortly co-incubated with cefotaxime (CTX) as the indicator cephalosporin. Susceptibility and resistance defining peaks from the samples' mass spectra were analyzed by a novel algorithm for bacterial organism classification. The D-MSBL assay allowed discrimination between E. coli and K. pneumoniae that were resistant or susceptible to CTX with a sensitivity of 86.8% and a specificity of 98.2%. CONCLUSION: The proposed algorithm-based D-MSBL assay, if integrated in the routine laboratory diagnostic workflow, may be useful to enhance the establishment of appropriate antibiotic therapy and to control the threat of oxyimino-cephalosporin resistance in hospital.

Diagnosis of Nocardia paucivorans central nervous system infection by DNA sequencing from paraffin-embedded tissue.

Infections by Nocardia spp. are generally regarded as opportunistic diseases in immunocompromised patients, but can also affect immunocompetent subjects. Such infections represent an important diagnostic challenge for clinicians and microbiologists, and diagnosis is frequently delayed or even conducted post mortem. A 54-year-old man was admitted to our hospital because of ventriculitis and relapsing brain abscess. Five months prior, this patient had undergone external ventricular drain and surgery for a cerebellar abscess. Histopathology demonstrated pyogenic inflammatory reaction, microbiologic investigations proved negative and empiric antimicrobial therapy was administered for a total of eight weeks. Six weeks later, the patient developed relapsing neurologic manifestations. On reviewing the patient's clinical history it emerged that the patient had suffered pneumonia two months prior to neurosurgery, treated with amoxicillin/clavulanate 3g a day and levofloxacin 500mg a day for three weeks. On the CNS relapsing manifestations, nocardiosis was suspected and DNA sequencing from the formalin-fixed paraffin-embedded cerebellar tissue collected during neurosurgery allowed diagnosis of Nocardia paucivorans infection. The patient received medical therapy for 11 months. At follow-up, eight months after treatment was discontinued, the patient was aymptomatic. Nocardia spp. infections need to be suspected not only in immunocompromised, but also in immunocompetent patients. Proper samples need to be collected for proper microbiologic investigations. Paraffin-embedded tissue genomic sequencing can be a useful tool for diagnosis of nocardiosis.

CYP21A2 p.E238 deletion as result of multiple microconversion events: a genetic study on an Italian congenital adrenal hyperplasia (CAH) family.

More than 90% of congenital adrenal hyperplasia (CAH) cases are associated with mutations in the 21-hydroxylase gene (CYP21A2) in the HLA class III area on the short arm of chromosome 6p21.3. The major part of disease-causing mutations in CYP21A2 alleles are CYP21A1P-derived sequence transferred to the active gene by macroconversion or microconversion events. Only around 5% of all disease-causing CYP21A2 alleles harbor rare mutations that do not originate from the pseudogene. A complete list of all reported CYP21A2 mutations can be found in the CYP21A2 database created by the Human Cytochrome P450 (CYP) Allele Nomenclature Committee (http://www.imm.Ki.se/CYPalleles/cyp21.htm). In this report, we describe clinical and genetic findings regarding an Italian woman suffering from a classic salt-wasting form of CAH due to a severe 21-hydroxylase deficiency. A complex genetic family study was performed including a prenatal diagnosis. The patient was found to be heterozygous for p.I172N (exon 4), p.E238del (exon 6), p.M239K (exon 6), and p.F306insT (exon 7) mutations and homozygous for p.I236N (exon 6) and p.V237E (exon 6) mutations. The deletion of glutamic acid 238 is a new mutation not reported before in the literature. CYP21A2 genotyping has become a valuable complement to biochemical CAH investigation. We highlight the contribution of molecular genetic advancements to the clinical management of patients with 21-hydroxylase deficiency.

Genes, pseudogenes and like genes: the case of 21-hydroxylase in Italian population.

BACKGROUNDS: Recently, we have considered two new findings in genetics of 21-hydroxylase deficiency with great interested: the existence of rare RCCX trimodular haplotypes, where the CYP21A2 like-gene downstream of the TNXA gene carries from one to six pseudogene mutations, and population specific allelic frequencies of wild-type CYP21A2 loci in the CYP21A1P pseudogene. Both these events represent a further complication in CYP21A2 genetics. Therefore, the choice of the molecular protocol becomes a crucial point when genetic analysis is required. In this regard, we must consider that the literature is still lacking consistent data on the Italian population. For this reason, we report genetic results obtained on 375 healthy individuals of Italian origin. METHODS: Different genetic protocols were compared and novel molecular strategies were performed. RESULTS: In our group, only two known haplotypes were identified. In addition, specific allelic frequencies of CYP21A2 wild-type loci in the pseudogene have been established. CONCLUSIONS: Based on our results, we can affirm that the employment of different molecular methods is necessary to ensure a correct CYP21A2 genotyping. In order to avoid mistakes both in patient diagnosis and/or in risk evaluation of the relatives, each case should be investigated in function of a careful clinical evaluation and the molecular test should be performed in specialized centres.

The first case of association between postpartum thyroiditis and thyroid hormone resistance in an Italian patient showing a novel p.V283A THRB mutation.

BACKGROUND: Postpartum thyroiditis (PPT) is characterized by the development of postpartum thyroid dysfunction, which may occur up to 12 months after delivery. The syndrome usually presents with transient thyrotoxicosis, followed by transient hypothyroidism. The association of this condition with resistance to thyroid hormones (RTH) has never been described. PATIENT FINDINGS: In this report, we describe a 30-year-old patient affected by RTH due to a novel p.V283A thyroid hormone receptor-ß (THRB) heterozygous mutation in exon 8, which affects the ligand-binding domain, never before described in literature. A simple polymorphism was excluded through screening of 100 healthy controls. SUMMARY: The patient became pregnant twice (in 2008 and in 2009) and developed PPT after both deliveries. Two months after her first pregnancy and one month after her second pregnancy, she presented with severe endogenous thyrotoxicosis and concomitant suppressed thyrotropin (TSH) levels, which represents an unusual finding in patients affected by RTH. Other causes of hyperthyroidism were excluded. After the hyperthyroid phase, she became hypothyroid (TSH >75 mU/L and low free-thyroxine and free-tri-iodothyronine levels), and eventually returned to her usual euthyroid status. During the course of PPT, no specific treatment was required, except for ß-blockers used to treat tachycardia during the hyperthyroid phase. CONCLUSIONS: We report a unique case of a woman affected by RTH, due to a novel mutation V283A in THRB, who experienced PPT with a severe thyrotoxic phase after both her pregnancies. The association between RTH and PPT has never been reported in the literature. In particular, the marked suppression of TSH occurring when levels of TH are particularly elevated is not a frequent condition during RTH.

p.H282N and p.Y191H: 2 novel CYP21A2 mutations in Italian congenital adrenal hyperplasia patients.

More than 90% of all cases of congenital adrenal hyperplasia (CAH) result from steroid 21-hydroxylase gene (CYP21A2) mutations. The CYP21A2 gene is located in the human leukocyte antigen (HLA) class III region on the short arm of chromosome 6p21.3, along with an inactive pseudogene, CYP21A1P, that is 98% homologous in its coding sequence with CYP21A2. Most CYP21A2 mutations result from intergenic recombinations between CYP21A2 and the closely linked CYP21A1P pseudogene. Rare mutations not generated by gene conversion account for only 5% to 10% of 21-hydroxylase deficiency alleles. However, detection of these rare and spontaneous mutations has continued to expand worldwide. We identified 2 novel CYP21A2 missense mutations (p.H282N and p.Y191H) in 2 Italian patients with simple-virilizing and nonclassic CAH forms. Functional analysis of these CYP21A2 mutations was performed. Functional in vitro assay for mutagenized CYP21A2 enzymes was performed in transiently transfected mammalian cells to test the residual enzyme activity and the apparent kinetic values. The residual activities obtained allowed us to classify the p.H282N and p.Y191H variants as simple-virilizing and nonclassic CAH associated mutations, respectively. These results correlate with the rate of severity of the patients' disease. This finding provides a further contribution for assisting in the diagnosis of CAH patients.