Rinite em pré-escolares / Rhinitis in preschool children

A rinite no pré-escolar configura um grande desafio diagnóstico e terapêutico, tanto para pediatras, como para especialistas. Os poucos dados existentes nesta faixa etária, além da sobreposição dos sintomas também comuns às doenças respiratórias virais, tornam o diagnóstico de rinite extremamente raro e/ou frequentemente ignorado. A melhor compreensão e identificação da rinite nos pré-escolares pode ajudar a melhorar a qualidade de vida destes pacientes, através da instituição do diagnóstico e tratamento corretos. Além disso, o diagnóstico mais precoce, possivelmente possibilitará caracterizar melhor a história natural da rinite, comorbidades, fatores de risco e o acompanhamento do desenvolvimento dos diferentes fenótipos da rinite ao longo da vida.

Rhinitis in preschool children is a major diagnostic and therapeutic challenge for both pediatricians and specialists. The diagnosis of rhinitis is extremely rare and/or often ignored in this specific age group, due to the few data available and the overlapping of symptoms common to viral respiratory diseases. A better understanding and identification of rhinitis in preschool children could improve the quality of life of these patients by making diagnosis more accurate and delivering appropriate treatment. In addition, an earlier diagnosis may help better understand the natural history of rhinitis, comorbidities, risk factors and follow-up of different phenotypes throughout life.

Clinical evaluation and molecular screening of a large consecutive series of albino patients.

Oculocutaneous albinism (OCA) is characterized by hypopigmentation of the skin, hair and eye, and by ophthalmologic abnormalities caused by a deficiency in melanin biosynthesis. In this study we recruited 321 albino patients and screened them for the genes known to cause oculocutaneous albinism (OCA1-4 and OCA6) and ocular albinism (OA1). Our purpose was to detect mutations and genetic frequencies of the main causative genes, offering to albino patients an exhaustive diagnostic assessment within a multidisciplinary approach including ophthalmological, dermatological, audiological and genetic evaluations. We report 70 novel mutations and the frequencies of the major causative OCA genes that are as follows: TYR (44%), OCA2 (17%), TYRP1 (1%), SLC45A2 (7%) and SLC24A5 (<0.5%). An additional 5% of patients had GPR143 mutations. In 19% of cases, a second reliable mutation was not detected, whereas 7% of our patients remain still molecularly undiagnosed. This comprehensive study of a consecutive series of OCA/OA1 patients allowed us to perform a clinical evaluation of the different OCA forms.

Performance of real-time PCR Xpert ®MTB/RIF in diagnosing extrapulmonary tuberculosis.

The real time PCR Xpert ® MTB/RIF is fundamental for rapid diagnosis in paucibacillary respiratory samples and for the detection of multidrug-resistant TB cases. This paper aimed to determine its performance on different extrapulmonary samples. We determined sensitivity, specificity, positive and negative predictive value on respiratory and non-respiratory samples collected from January 2010 to June 2014. The protocol for the Xpert ® MTB/RIF PCR suggested by Cepheid was strictly followed for all specimens. In 12257 respiratory samples we observed a sensitivity of 87.1% and a specificity of 99.9%. There were 2818 extrapulmonary specimens, of which 250 were followed by a positive culture for Mycobacterium tuberculosis complex, whereas 72 samples were culture-negative: tuberculosis was clinically confirmed in 71 of them and was excluded for one sample. The sensitivity of the test on urine, pus and CSF samples was 88.2%, 95.6% and 100% respectively. In contrast, the sensitivity of gastric aspirates and biopsies was 81.8% and 83.6% respectively, whereas results of total cavitary fluids were significantly worse than expected (53.7% sensitivity). Our experience shows that Xpert MTB/RIF assay is an accurate, sensitive, and specific test for the rapid detection of pulmonary and extra-pulmonary TB with the only exception of cavitary fluids.

Epidemiology and outcome of Clostridium difficile infections in patients hospitalized in Internal Medicine: findings from the nationwide FADOI-PRACTICE study.

BACKGROUND: Clostridium difficile (CD) is a leading cause of diarrhoea among hospitalized patients. The objective of this study was to evaluate the rate, the optimal diagnostic work-up, and outcome of CD infections (CDI) in Internal Medicine (IM) wards in Italy. METHODS: PRACTICE is an observational prospective study, involving 40 IM Units and evaluating all consecutive patients hospitalized during a 4-month period. CDI were defined in case of diarrhoea when both enzyme immunoassay for GDH, and test for A/B toxin were positive. Patients with CDI were followed-up for recurrences for 4 weeks after the end of therapy. RESULTS: Among the 10,780 patients observed, 103 (0.96 %) showed CDI, at admission or during hospitalization. A positive history for CD, antibiotics in the previous 4 weeks, recent hospitalization, female gender and age were significantly associated with CDI (multivariable analysis). In-hospital mortality was 16.5 % in CD group vs 6.7 % in No-CD group (p < 0.001), whereas median length of hospital stay was 16 (IQR = 13) vs 8 (IQR = 8) days (p < 0.001) among patients with or without CDI, respectively. Rate of CD recurrences was 14.6 %. As a post-hoc evaluation, 23 out of 34 GDH+/Tox- samples were toxin positive, when analysed by molecular method (a real-time PCR assay). The overall CD incidence rate was 5.3/10,000 patient-days. CONCLUSIONS: Our results confirm the severity of CDI in medical wards, showing high in-hospital mortality, prolonged hospitalization and frequent short-term recurrences. Further, our survey supports a 2-3 step algorithm for CD diagnosis: EIA for detecting GDH, A and B toxin, followed by a molecular method in case of toxin-negative samples.

Multi-center and multi-method evaluation of in vitro activities of ceftaroline against S. aureus.

This five-site study was performed to assess the reproducibility of ceftaroline MIC and disk results for Staphylococcus aureus. Three commercial broth microdilution, three gradient diffusion and ceftaroline 5µg disk diffusion methods were compared to a reference broth microdilution method against challenge isolates (n = 41) and isolates collected at four European sites (n = 30/site). For four MIC methods (Sensititre and three gradient diffusion methods), 99.0% of consolidated MIC results were within +/- 1 dilution of the reference MIC. Categorical agreement rates based on EUCAST breakpoints for the challenge isolates were 75.6-100% and for disk testing were 78.0-92.7%. There was no clear distinction between isolates with MIC results of 1 and 2mg/L with regard to variation in MIC or molecular genotyping results. The addition of an intermediate category for isolates with MIC results of 2mg/L would help to identify these isolates as borderline susceptible/non-susceptible isolates.

Screening of PAX6 gene in Italian congenital aniridia patients revealed four novel mutations.

PURPOSE: To uncover underlying mutations in a cohort of Italian patients with aniridia, a rare congenital panocular condition with an incidence ranging from 1:64,000 to 1:100,000. The disease may be found isolated or in association with other syndromes characterized by partial or complete absence of the iris and iris hypoplasia. METHODS: We analyzed the PAX6 gene in 11 patients with aniridia fulfilling the following inclusion criteria: partial or complete absence of the iris and age < 18 years at the time of diagnosis. DNA sequence analysis was integrated with Multiple Ligation Probe Assay (MLPA) analysis. RESULTS: We identified seven PAX6 mutations, including four novel ones. The majority of mutations lie in the DNA-binding domain and all produce a truncated protein. All tested patients did not have WT1 gene deletions thus excluding the WAGR syndrome. We present the clinical findings in the four cases harboring novel mutations. We were unable to identify mutations in four cases with complete aniridia thus indicating that other gene/s could be involved in the disease. CONCLUSIONS: It is important to establish the molecular diagnosis early to avoid repeated and long-term screening for Wilms tumor. Our work further emphasizes that a wide range of ocular phenotypes are associated with loss of function PAX6 mutations. In addition to the possibility of stochastic variations, other genetic variations could play a role as modifier genes, thus giving rise to the observed different ocular phenotypes.

Automated Scoring of Chromogenic Media for Detection of Methicillin-Resistant Staphylococcus aureus by Use of WASPLab Image Analysis Software.

Recently, systems have been developed to create total laboratory automation for clinical microbiology. These systems allow for the automation of specimen processing, specimen incubation, and imaging of bacterial growth. In this study, we used the WASPLab to validate software that discriminates and segregates positive and negative chromogenic methicillin-resistant Staphylococcus aureus (MRSA) plates by recognition of pigmented colonies. A total of 57,690 swabs submitted for MRSA screening were enrolled in the study. Four sites enrolled specimens following their standard of care. Chromogenic agar used at these sites included MRSASelect (Bio-Rad Laboratories, Redmond, WA), chromID MRSA (bioMérieux, Marcy l'Etoile, France), and CHROMagar MRSA (BD Diagnostics, Sparks, MD). Specimens were plated and incubated using the WASPLab. The digital camera took images at 0 and 16 to 24 h and the WASPLab software determined the presence of positive colonies based on a hue, saturation, and value (HSV) score. If the HSV score fell within a defined threshold, the plate was called positive. The performance of the digital analysis was compared to manual reading. Overall, the digital software had a sensitivity of 100% and a specificity of 90.7% with the specificity ranging between 90.0 and 96.0 across all sites. The results were similar using the three different agars with a sensitivity of 100% and specificity ranging between 90.7 and 92.4%. These data demonstrate that automated digital analysis can be used to accurately sort positive from negative chromogenic agar cultures regardless of the pigmentation produced.

Community-acquired Clostridium difficile infection in children: A retrospective study.

BACKGROUND: Community acquired-Clostridium difficile infection (CDI) has increased also in children in the last years. AIMS: To determine the incidence of community-acquired CDI and to understand whether Clostridium difficile could be considered a symptom-triggering pathogen in infants. METHODS: A five-year retrospective analysis (January 2007-December 2011) of faecal specimens from 124 children hospitalized in the Niguarda Ca' Granda Hospital for prolonged or muco-haemorrhagic diarrhoea was carried out. Stool samples were evaluated for common infective causes of diarrhoea and for Clostridium difficile toxins. Patients with and without CDI were compared for clinical characteristics and known risk factors for infection. RESULTS: Twenty-two children with CDI were identified in 5 years. An increased incidence of community-acquired CDI was observed, ranging from 0.75 per 1000 hospitalizations in 2007 to 9.8 per 1000 hospitalizations in 2011. Antimicrobial treatment was successful in all 19 children in whom it was administered; 8/22 CDI-positive children were younger than 2 years. No statistically significant differences in clinical presentation were observed between patients with and without CDI, nor in patients with and without risk factors for CDI. CONCLUSIONS: Our study shows that Clostridium difficile infection is increasing and suggests a possible pathogenic role in the first 2 years of life.

Two novel splicing mutations in the SLC45A2 gene cause Oculocutaneous Albinism Type IV by unmasking cryptic splice sites.

Oculocutaneous albinism (OCA) is characterized by hypopigmentation of the skin, hair and eye, and by ophthalmologic abnormalities caused by a deficiency in melanin biosynthesis. OCA type IV (OCA4) is one of the four commonly recognized forms of albinism, and is determined by mutation in the SLC45A2 gene. Here, we investigated the genetic basis of OCA4 in an Italian child. The mutational screening of the SLC45A2 gene identified two novel potentially pathogenic splicing mutations: a synonymous transition (c.888G>A) involving the last nucleotide of exon 3 and a single-nucleotide insertion (c.1156+2dupT) within the consensus sequence of the donor splice site of intron 5. As computer-assisted analysis for mutant splice-site prediction was not conclusive, we investigated the effects on pre-mRNA splicing of these two variants by using an in vitro minigene approach. Production of mutant transcripts in HeLa cells demonstrated that both mutations cause the almost complete abolishment of the physiologic donor splice site, with the concomitant unmasking of cryptic donor splice sites. To our knowledge, this work represents the first in-depth molecular characterization of splicing defects in a OCA4 patient.

SOX2, OTX2 and PAX6 analysis in subjects with anophthalmia and microphthalmia.

Anophthalmia (A) and microphthalmia (M) are rare developmental anomalies that have significant effects on visual activity. In fraction of A/M subjects, single genetic defects have been identified as causative. In this study we analysed 65 Italian A/M patients, 21 of whom are syndromic, for mutations in SOX2, OTX2 and PAX6 genes. In syndromic patients the presence of genome imbalances through array CGH was also investigated. No mutations were found for OTX2 and PAX6 genes. Three causative SOX2 mutations were found in subjects with syndromic A. In a subject with syndromic signs and monolateral M, two de novo 6.26 Mb and 1.37 Mb deletions in 4q13.2q13.3 have been identified. A SOX2 missense (p.Ala161Ser) mutation was found in 1 out of 39 a subject with non-syndromic monolateral M. Alanine at position 161 is conserved along phylogeny and the p.Ala161Ser mutation is estimated pathogenic by in silico analysis. However, this mutation was also present in the unaffected patient's daughter.

PDCD10 gene mutations in multiple cerebral cavernous malformations.

Cerebral cavernous malformations (CCMs) are vascular abnormalities that may cause seizures, intracerebral haemorrhages, and focal neurological deficits. Familial form shows an autosomal dominant pattern of inheritance with incomplete penetrance and variable clinical expression. Three genes have been identified causing familial CCM: KRIT1/CCM1, MGC4607/CCM2, and PDCD10/CCM3. Aim of this study is to report additional PDCD10/CCM3 families poorly described so far which account for 10-15% of hereditary cerebral cavernous malformations. Our group investigated 87 consecutive Italian affected individuals (i.e. positive Magnetic Resonance Imaging) with multiple/familial CCM through direct sequencing and Multiplex Ligation-Dependent Probe Amplification (MLPA) analysis. We identified mutations in over 97.7% of cases, and PDCD10/CCM3 accounts for 13.1%. PDCD10/CCM3 molecular screening revealed four already known mutations and four novel ones. The mutated patients show an earlier onset of clinical manifestations as compared to CCM1/CCM2 mutated patients. The study of further families carrying mutations in PDCD10/CCM3 may help define a possible correlation between genotype and phenotype; an accurate clinical follow up of the subjects would help define more precisely whether mutations in PDCD10/CCM3 lead to a characteristic phenotype.

Mutational screening of NOTCH3 gene reveals two novel mutations: complexity of CADASIL diagnosis.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an adult onset hereditary vascular disease with neurological manifestations. The classical clinical course is relentlessly progressive with early transient ischaemic attacks (TIA) or strokes, dementia and finally death in the mid-1960s. The disorder is inherited in an autosomal dominant fashion, with high penetrance and broad variable clinical course even within family. It is caused by mutations in the NOTCH3 gene; all causative mutations result in gain or loss of a cysteine residue within the extracellular domain, with exons 3 and 4 reported as hot spot mutational sites. Mutation analysis of the NOTCH3 gene was performed through direct sequencing of the 2-23 exons containing all EGF-like domains. Patients underwent genetic counselling pre and post testing. Here, we report two novel mutations located in exons 6 and 15 of the NOTCH3 gene; clinical description for the probands and for available relatives is enclosed. No reliable data on incidence or prevalence rates of this disease are available: it is therefore essential that the diagnosis is obtained in all suspected cases through the extensive analysis of the NOTCH3 gene and that all cases are brought to the attention of the scientific community.

Functional characterization of two novel splicing mutations in the OCA2 gene associated with oculocutaneous albinism type II.

Oculocutaneous albinism (OCA) is characterized by hypopigmentation of the skin, hair and eye, and by ophthalmologic abnormalities caused by a deficiency in melanin biosynthesis. OCA type II (OCA2) is one of the four commonly-recognized forms of albinism, and is determined by mutation in the OCA2 gene. In the present study, we investigated the molecular basis of OCA2 in two siblings and one unrelated patient. The mutational screening of the OCA2 gene identified two hitherto-unknown putative splicing mutations. The first one (c.1503+5G>A), identified in an Italian proband and her affected sibling, lies in the consensus sequence of the donor splice site of OCA2 intron 14 (IVS14+5G>A), in compound heterozygosity with a frameshift mutation, c.1450_1451insCTGCCCTGACA, which is predicted to determine the premature termination of the polypeptide chain (p.I484Tfs*19). In-silico prediction of the effect of the IVS14+5G>A mutation on splicing showed a score reduction for the mutant splice site and indicated the possible activation of a newly-created deep-intronic acceptor splice site. The second mutation is a synonymous transition (c.2139G>A, p.K713K) involving the last nucleotide of exon 20. This mutation was found in a young African albino patient in compound heterozygosity with a previously-reported OCA2 missense mutation (p.T404M). In-silico analysis predicted that the mutant c.2139G>A allele would result in the abolition of the splice donor site. The effects on splicing of these two novel mutations were investigated using an in-vitro hybrid-minigene approach that led to the demonstration of the causal role of the two mutations and to the identification of aberrant transcript variants.

Tuberculosis outbreak in a primary school, Milan, Italy.

Investigation of an outbreak of tuberculosis (TB) in a primary school in Milan, Italy, found 15 schoolchildren had active TB disease and 173 had latent TB infection. TB was also identified in 2 homeless men near the school. Diagnostic delay, particularly in the index case-patient, contributed to the transmission of infection.

Rhinoscleroma in an immigrant from Egypt: a case report.

Rhinoscleroma is a chronic indolent granulomatous infection of the nose and the upper respiratory tract caused by Klebsiella rhinoscleromatis; this condition is endemic to many regions of the world including North Africa. We present a case of rhinoscleroma in a 51-year-old Egyptian immigrant with 1-month history of epistaxis. We would postulate that with increased travel from areas where rhinoscleroma is endemic to other non-endemic areas, diagnosis of this condition will become more common.

Prevalence and epidemiology of microbial pathogens causing bloodstream infections: results of the OASIS multicenter study.

Beginning on April 2007, a prospective multicenter study was performed to investigate prevalence and epidemiology of microbial pathogens causing bloodstream infections (BSIs). Twenty microbiology laboratories participated to the survey over a 1-year period. A total of 11,638 episodes of BSI occurred in 11 202 patients, with 8.5% (n=985) of episodes being polymicrobial. Of 12 781 causative organisms, aerobic Gram-negative bacteria were 47.4% (n=6058), whereas Gram-positives accounted for 43.9% (n=5608). The remaining organisms included fungal species (n=924, 7.2%) and anaerobes (n=191, 1.5%). The most prevalent agents were Escherichia coli (21.7%), Staphylococcus aureus (14.9%), Staphylococcus epidermidis (8.2%), Pseudomonas aeruginosa (7.0%), and Enterococcus faecalis (6.3%). Isolates recovered from patients admitted to medical, surgical, and intensive care units accounted for 62.9%, 17.7%, and 19.4% of cases, respectively. BSIs were classified as hospital-acquired in 67.2% of cases. Compared with previous studies, our data show an increasing role of Gram-negative bacteria among both hospital- and community-acquired blood isolates.