Microbiology 2.0-A "behind the scenes" consideration for artificial intelligence applications for interpretive culture plate reading in routine diagnostic laboratories.

Laboratory automation with Artificial Intelligence (AI) features have now emerged into routine diagnostic clinical use to interpret growth on agar plates. Applications are currently limited to urine samples and infection control screens, yet some of the details around the development of algorithms remain entrenched with AI development specialists and are not well understood by laboratorians. The generation of algorithms is not a trivial task and is a highly structured process, with several considerations needed to develop the appropriate data for specific intended uses. Understanding these considerations highlights the limitations of any algorithm created and informs better design practices so that algorithm objectives can be thoroughly tested prior to routine use.

Neurological phenomenology of the IRF2BPL mutation syndrome: Analysis of a new case and systematic review of the literature.

BACKGROUND: IRF2BPL is an intronless gene that was mapped to 14q24.3 chromosome in 2000 and codes for the interferon regulatory factor 2 binding like protein. OBJECTIVE: To analyses the clinical characteristics of the patients reported in the literature and of an additional patient we observed in order to better delineate the phenomenological spectrum of the disease and provide indications to improve clinical recognition and facilitate diagnosis. METHODS: We reported on 28 patients carrying the IRF2BPL mutation who were identified in 10 papers (n.27), using PUBMED as the search engine, and in our hospital (n. 1). RESULTS: All patients shared developmental delay/regression. Additional neurological symptoms were present in a large proportion of patients and reflected the involvement of five main neurological domains, i.e. epilepsy, dystonia, ataxia, spasticity, and ocular disturbances. Correlation analysis suggested a significant positive correlation between the number of affected neurological domains and the presence of MRI abnormalities (rho = 0.45, p = 0.02), while no significant correlation emerged between the number of affected clinical domains and age at disease onset (rho = 0.18, p = 0.35) or variant type (rho = 0.30, p = 0.12). CONCLUSIONS: Our analysis highlights that the IRF2BPL mutation syndrome is highly specific to the central nervous system. Diagnostic work-up should consider the clinical picture of the IRF2BPL mutation syndrome herein delineated and the existence of conditions that share developmental delay/regression and result from acquired/genetic or unidentifiable underlying etiology.

Effects of ocean acidification on acid-base physiology, skeleton properties, and metal contamination in two echinoderms from vent sites in Deception Island, Antarctica.

Antarctic surface waters are expected to be the first to experience severe ocean acidification (OA) with carbonate undersaturation and large decreases in pH forecasted before the end of this century. Due to the long stability in environmental conditions and the relatively low daily and seasonal variations to which they are exposed, Antarctic marine organisms, especially those with a supposedly poor machinery to eliminate CO2 and protons and with a heavily calcified skeleton like echinoderms, are hypothesized as highly vulnerable to these environmental shifts. The opportunities offered by the natural pH gradient generated by vent activities in Deception Island caldera, Western Antarctic Peninsula, were used to investigate for the first time the acid-base physiologies, the impact of OA on the skeleton and the impact of pH on metal accumulation in the Antarctic sea star Odontaster validus and sea urchin Sterechinus neumayeri. The two species were sampled in four stations within the caldera, two at pH (total scale) 8.0-8.1 and two at reduced pH 7.8. Measured variables were pH, alkalinity, and dissolved inorganic carbon of the coelomic fluid; characteristic fracture force, stress and Young's modulus using Weibull statistics and Cd, Cu, Fe, Pb and Zn concentrations in the integument, gonads and digestive system. Recorded acid-base characteristics of both studied species fit in the general picture deduced from temperate and tropical sea stars and sea urchins but conditions and possibly confounding factors, principally food availability and quality, in the studied stations prevented definitive conclusions. Reduced seawater pH 7.8 and metals had almost no impact on the skeleton mechanical properties of the two investigated species despite very high Cd concentrations in O. validus integument. Reduced pH was correlated to increased contamination by most metals but this relation was weak. Translocation and caging experiments taking into account food parameters are proposed to better understand future processes linked to ocean acidification and metal contamination in Antarctic echinoderms.

MicroRNA-551b expression profile in low and high-grade cervical intraepithelial neoplasia.

OBJECTIVE: To evaluate the expression of microRNA (miR)-551b in patients with low and high grade cervical intraepithelial neoplasia (CIN) and to find an association with high-risk Human Papillomavirus (HR-HPV) infection-related prognostic biomarkers. PATIENTS AND METHODS: The expression level of miR-551b was determined in 50 paraffin-embedded cervical specimens (10 normal squamous epithelium, 18 condylomas, 8 CIN1, and 14 CIN2-3) using quantitative Real-time polymerase chain reaction (qRT-PCR). χ2-test compared miR-551b expression in different diagnosis groups. An Ordered Logistic Regression and a Probit correlation were made to correlate miR-551b expression levels with the cervical tissue histological findings. The immunohistochemical distribution of p16 and Ki-67 according to histopathological findings was also assessed. RESULTS: The distribution of the miR-551b expression profile was significantly lower in CIN1-3 samples compared to other histological diagnosis groups (condyloma and negative). The expression levels were inversely correlated to the cervical pathological grade, from negative to CIN2-3. A 1% increase in miR-551b expression level produced an increase of 19% to the probability of a minor histological grade diagnosis in a range from negative to CIN2-3 and an increase of 13% to the probability of a negative histological grade diagnosis. Among the cases with miR-551b expression < 0.02 (considered as cut-off value) a significant statistical correlation was found between p16 and Ki-67 expression and the diagnosis of CIN2-3. CONCLUSIONS: Our data showed a significant inverse correlation between miR-551b expression and the histological grading of the lesions, suggesting a tumor suppressive function in the different stages of cervical dysplasia.

Diagnostic application of a capture based NGS test for the concurrent detection of variants in sequence and copy number as well as LOH.

Whole exome sequencing (WES) has made the identification of causative SNVs/InDels associated with rare Mendelian conditions increasingly accessible. Incorporation of softwares allowing CNVs detection into the WES bioinformatics pipelines may increase the diagnostic yield. However, no standard protocols for this analysis are so far available and CNVs in non-coding regions are totally missed by WES, in spite of their possible role in the regulation of the flanking genes expression. So, in a number of cases the diagnostic workflow contemplates an initial investigation by genomic arrays followed, in the negative cases, by WES. The opposite workflow may also be applied, according to the familial segregation of the disease. We show preliminary results for a diagnostic application of a single next generation sequencing panel permitting the concurrent detection of LOH and variations in sequences and copy number. This approach allowed us to highlight compound heterozygosity for a CNV and a sequence variant in a number of cases, the duplication of a non-coding region responsible for sex reversal, and a whole-chromosome isodisomy causing reduction to homozygosity for a WFS1 variant. Moreover, the panel enabled us to detect deletions, duplications, and amplifications with sensitivity comparable to that of the most widely used array-CGH platforms.

MYOCLONIC ASTATIC EPILEPSY IN A PATIENT WITH A DE NOVO 4q21.22q21.23 MICRODUPLICATION.

Myoclonicastatic epilepsy (MAE) is a rare form of symptomatic generalized epilepsy of uncertain etiology. To search the possible genetic basis of the disorder, here we investigate a 15 year-old patient with MAE, who is the only person presenting epilepsy in the family. High resolution array-CGH analysis was conducted on DNA extracted from peripheral blood of the patient and the parents. The copy number variant(s) (CNVs) identified were further confirmed by Fluorescent In Situ Hybridization (FISH). The array-CGH identified a de novo microduplication of about 778 Kb in the chromosome region 4q21.22-q21.23, involving 11 genes. This is the first report of a de novo CNV in MAE. The genes involved in the duplication are potential candidates that can be investigated in the future to determine their exact role in the etiopathogenesis of the disorder. However, we suggest performing microarray chromosomal analysis in patients with MAE, since rare de novo CNVs could be identified, and this is known to affect the diagnostic process and recurrence risk assessment.

Comprehensive investigation in patients affected by sperm macrocephaly and globozoospermia.

The aim of this study was to provide a comprehensive genetic/phenotypic characterization of subjects suffering infertility owing to sperm macrocephaly (n = 3) or globozoospermia (n = 9) and to investigate whether the patients' genetic status was correlated with the alteration of various sperm parameters. AURKC was sequenced in case of sperm macrocephaly while the DPY19L2 status has been analyzed by multiple approaches including a novel qPCR-based copy number assay in case of globozoospermia. Globozoospermic patients were also analyzed for SPACA1, a novel candidate gene herein tested for the first time in humans. The effect of the patients' genetic status was interrogated by implementing the molecular screening with the characterization of several sperm parameters: (i) routine sperm analysis, integrated with transmission electron microscopy; (ii) sperm fluorescent in situ hybridization (FISH) analysis; (iii) sperm DNA fragmentation (DF) analysis. Moreover, for the first time, we performed microsatellite instability analysis as a marker of genome instability in men with sperm macrocephaly and globozoospermia. Finally, artificial reproductive technology (ART) history has been reported for those patients who underwent the treatment. Macrocephalic patients had an AURKC mutation and >89% tetraploid, highly fragmented spermatozoa. DPY19L2 was mutated in all patients with >80% globozoospermia: the two homozygous deleted men and the compound heterozygous showed the severest phenotype (90-100%). The newly developed qPCR method was fully validated and has the potential of detecting also yet undiscovered deletions. DPY19L2 status is unlikely related to FISH anomalies and DF, although globozoospermic men showed a higher disomy rate and DF compared with internal reference values. No patient was mutated for SPACA1. Our data support the general agreement on the negative correlation between macro/globozoospermia and conventional intracytoplasmic sperm injection outcomes. Microsatellites were stable in all patients analyzed. The comprehensive picture provided on these severe phenotypes causing infertility is of relevance in the management of patients undergoing ART.

Proton nuclear magnetic resonance spectroscopy of urine samples in preterm asphyctic newborn: a metabolomic approach.

In order to highlight differences in the metabolic profile of healthy (control) compared with asphyxiated newborns, by using untargeted metabolomic approach coupled with (1)H NMR spectroscopy, we evaluated the effects of asphyxia on newborn urine metabolites. Our results showed that lactate, glucose and TMAO, together with threonine plus 3-hydroxyisovalerate are the metabolites more characterizing the asphyxiated group; lower contribute to discrimination is related to other metabolites such as dimethylglycine, dimethylamine, creatine, succinate, formate, urea and aconitate. After 24-48h from resuscitation preterm asphyctic neonates showed their recovery pattern that still can be differentiated by the controls.

Community and hospital acquired Clostridium difficile in South Australia - ribotyping of isolates and a comparison of laboratory detection methods.

UNLABELLED: A total of 274 samples were screened for toxigenic Clostridium difficile using a combination of several commercially available assays, and positive isolates ribotyped. A two-step algorithm assisted in demonstrating an increased prevalence of C. difficile infection in South Australia of 9·8%, most of which were ribotypes 014 and 052. A glutamate dehydrogenase assay followed by the detection of genes associated with toxin production was the most sensitive and specific algorithm for screening for toxigenic C. difficile. SIGNIFICANCE AND IMPACT OF THE STUDY: Rapid and accurate detection of toxigenic Clostridium difficile is important for the diagnosis of C. difficile colitis and the management of patients in healthcare institutions to minimize the spread of disease. A critical review of currently available commercial methods supports a recommendation for a 2-step algorithm that is relatively inexpensive and amenable to the routine pathology laboratory. It is anticipated that the detection of C. difficile will increase with improved detection methods, and the ribotype prevalence presented in this manuscript will be useful for any current and future source tracking purposes.

Focal dysplasia of the cerebral cortex and infantile spasms associated with somatic 1q21.1-q44 duplication including the AKT3 gene.

Somatic and germline duplications or activating mutations of AKT3 have been reported in patients with hemimegalencephaly and megalencephaly. We performed array comparative genomic hybridization on brain tissue and blood in 16 consecutive patients with symptomatic epilepsy due to focal or multilobar malformations of cortical development who underwent surgical treatment of epilepsy. One patient with infantile spasms and a dysplastic left frontal lobe harboured a somatic trisomy of the 1q21.1-q44 chromosomal region, encompassing the AKT3 gene, in the dysplastic brain tissue but not in blood and saliva. Histopathology revealed severe cortical dyslamination, a thin cortex in the premotor area with microgyri and microsulci, immature neurons with disoriented dendrites and areas of cortical heterotopia in the sub-cortical white matter. These cytoarchitectural changes are close to those defining type Ib focal cortical dysplasia. Immunohistochemistry in brain specimens showed hyperactivation of the PI3K/AKT/mTOR pathway. These findings indicate that AKT3 upregulation may cause focal malformations of cortical development. There appears to be an etiologic continuum between hemimegalencephaly and focal cortical dysplastic lesions. The extent of brain malformations due to AKT3 upregulation may be related to the embryonic stage when the post-zygotic gene alteration occurs.

Phenotypic heterogeneity and mutational spectrum in a cohort of 45 Italian males subjects with X-linked ectodermal dysplasia.

Ectodermal dysplasias (EDs) are a group of genetic disorders characterized by the abnormal development of the ectodermal-derived structures. X-linked hypohidrotic ectodermal dysplasia, resulting from mutations in ED1 gene, is the most common form. The main purpose of this study was to characterize the phenotype spectrum in 45 males harboring ED1 mutations. The study showed that in addition to the involvement of the major ectodermal tissues, the majority of patients also have alterations of several minor ectodermal-derived structures. Characterizing the clinical spectrum resulting from ED1 gene mutations improves diagnosis and can direct clinical care.

Therapeutic implications of novel mutations of the RFX6 gene associated with early-onset diabetes.

Identification of the genetic defect underlying early-onset diabetes is important for determining the specific diabetes subtype, which would then permit appropriate treatment and accurate assessment of recurrence risk in offspring. Given the extensive genetic and clinical heterogeneity of the disease, high-throughput sequencing might provide additional diagnostic potential when Sanger sequencing is ineffective. Our aim was to develop a targeted next-generation assay able to detect mutations in several genes involved in glucose metabolism. All 13 known MODY genes, genes identified from a genome-wide linkage study or genome-wide association studies as increasing the risk of type 2 diabetes and genes causing diabetes in animal models, were included in the custom panel. We selected a total of 102 genes by performing a targeting re-sequencing in 30 patients negative for mutations in the GCK, HNF1α, HNF4α, HNF1ß and IPF1 genes at the Sanger sequencing analysis. Previously unidentified variants in the RFX6 gene were found in three patients and in two of them we also detected rare variants in WFS1 and ABCC8 genes. All patients showed a good therapeutic response to dipeptidyl peptidase-4 (DPP4) inhibitors. Our study reveals that next-generation sequencing provides a highly sensitive method for identification of variants in new causative genes of diabetes. This approach may help in understanding the molecular etiology of diabetes and in providing more personalized treatment for each genetic subtype.

Y-chromosome microdeletions are not associated with SHOX haploinsufficiency.

STUDY QUESTION: Are Y-chromosome microdeletions associated with SHOX haploinsufficiency, thus representing a risk of skeletal anomalies for the carriers and their male descendents? SUMMARY ANSWER: The present study shows that SHOX haploinsufficiency is unlikely to be associated with Y-chromosome microdeletions. WHAT IS KNOWN ALREADY: Y-chromosome microdeletions are not commonly known as a major molecular genetic cause of any pathological condition except spermatogenic failure. However, it has been recently proposed that they are associated not only with infertility but also with anomalies in the pseudoautosomal regions (PAR), among which SHOX haploinsufficiency stands out with a frequency of 5.4% in microdeletion carriers bearing a normal karyotype. This finding implies that sons fathered by men with Y-chromosome defects will not only exhibit fertility problems, but might also suffer from SHOX-related conditions. STUDY DESIGN: Five European laboratories (Florence, Münster, Barcelona, Padova and Ancona), routinely performing Y-chromosome microdeletion screening, were enrolled in a multicenter study. PARTICIPANTS/MATERIALS, SETTING, METHODS: PAR-linked and SHOX copy number variations (CNVs) were analyzed in 224 patients carrying Y-chromosome microdeletions and 112 controls with an intact Y chromosome, using customized X-chromosome-specific array-CGH platforms and/or qPCR assays for SHOX and SRY genes. MAIN RESULTS AND THE ROLE OF CHANCE: Our data show that 220 out of 224 (98.2%) microdeletion carriers had a normal SHOX copy number, as did all the controls. No SHOX deletions were found in any of the examined subjects (patients as well as controls), thus excluding an association with SHOX haploinsufficiency. SHOX duplications were detected in 1.78% of patients (n = 4), of whom two had an abnormal and two a normal karyotype. This might suggest that Y-chromosome microdeletions have a higher incidence for SHOX duplications, irrespective of the patient's karyotype. However, the only clinical condition observed in our four SHOX-duplicated patients was infertility. LIMITATIONS, REASONS FOR CAUTION: The number of controls analyzed is rather low to assess whether the SHOX duplications found in the two men with Y-chromosome microdeletions and a normal karyotype represent a neutral polymorphism or are actually associated with the presence of the microdeletion. WIDER IMPLICATIONS OF THE FINDINGS: Men suffering from infertility due to the presence of Y-chromosome microdeletions can resort to artificial reproductive technology (ART) to father their biological children. However, infertile couples must be aware of the risks implied and this makes genetic counseling a crucial step in the patient's management. This study does not confirm previous alarming data that showed an association between Y-chromosome microdeletions and SHOX haploinsufficiency. Our results imply that deletion carriers have no augmented risk of SHOX-related pathologies (short stature and skeletal anomalies) and indicate that there is no need for radical changes in genetic counseling of Yq microdeletion carriers attempting ART, since the only risk established so far for their male offspring remains impaired spermatogenesis. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Italian Ministry of University (grant PRIN 2010-2012 to C.K.), Tuscan Regional Health Research Program ('Progetto Salute 2009') to G.F., the Spanish Ministry of Health (grant FIS-11/02254) and the European Union 'Reprotrain' Marie Curie Network (project number: 289880 to C.K.). The authors declare that no conflicting interests exist.

Mycobacterium avium complex--the role of potable water in disease transmission.

Mycobacterium avium complex (MAC) is a group of opportunistic pathogens of major public health concern. It is responsible for a wide spectrum of disease dependent on subspecies, route of infection and patients pre-existing conditions. Presently, there is limited research on the incidence of MAC infection that considers both pulmonary and other clinical manifestations. MAC has been isolated from various terrestrial and aquatic environments including natural waters, engineered water systems and soils. Identifying the specific environmental sources responsible for human infection is essential in minimizing disease prevalence. This paper reviews current literature and case studies regarding the wide spectrum of disease caused by MAC and the role of potable water in disease transmission. Potable water was recognized as a putative pathway for MAC infection. Contaminated potable water sources associated with human infection included warm water distribution systems, showers, faucets, household drinking water, swimming pools and hot tub spas. MAC can maintain long-term contamination of potable water sources through its high resistance to disinfectants, association with biofilms and intracellular parasitism of free-living protozoa. Further research is required to investigate the efficiency of water treatment processes against MAC and into construction and maintenance of warm water distribution systems and the role they play in MAC proliferation.

Fabry disease: polymorphic haplotypes and a novel missense mutation in the GLA gene.

Fabry disease: polymorphic haplotypes and a novel missense mutation in the GLA gene. Fabry disease (FD) is an X-linked lysosomal storage disorder with a heterogeneous spectrum of clinical manifestations that are caused by the deficiency of α-galactosidase A (α-Gal-A) activity. Although useful for diagnosis in males, enzyme activity is not a reliable biochemical marker in heterozygous females due to random X-chromosome inactivation, thus rendering DNA sequencing of the α-Gal-A gene, alpha-galactosidase gene (GLA), the most reliable test for the confirmation of diagnosis in females. The spectrum of GLA mutations is highly heterogeneous. Many polymorphic GLA variants have been described, but it is unclear if haplotypes formed by combinations of such variants correlate with FD, thus complicating molecular diagnosis in females with normal α-Gal-A activity. We tested 67 female probands with clinical manifestations that may be associated with FD and 110 control males with normal α-Gal-A activity. Five different combinations of GLA polymorphic variants were identified in 14 of the 67 females, whereas clearcut pathogenetic alterations, p.Met51Ile and p.Met290Leu, were identified in two cases. The latter has not been reported so far, and both mutant forms were found to be responsive to the pharmacological chaperone deoxygalactonojirimycin (DGJ; migalastat hydrochloride). Analysis of the male control population, as well as male relatives of a suspected FD female proband, permitted the identification of seven different GLA gene haplotypes in strong linkage disequilibrium. The identification of haplotypes in control males provides evidence against their involvement in the development of FD phenotypic manifestations.