A Solve-RD ClinVar-based reanalysis of 1522 index cases from ERN-ITHACA reveals common pitfalls and misinterpretations in exome sequencing.

PURPOSE: Within the Solve-RD project (https://solve-rd.eu/), the European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies aimed to investigate whether a reanalysis of exomes from unsolved cases based on ClinVar annotations could establish additional diagnoses. We present the results of the "ClinVar low-hanging fruit" reanalysis, reasons for the failure of previous analyses, and lessons learned. METHODS: Data from the first 3576 exomes (1522 probands and 2054 relatives) collected from European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies was reanalyzed by the Solve-RD consortium by evaluating for the presence of single-nucleotide variant, and small insertions and deletions already reported as (likely) pathogenic in ClinVar. Variants were filtered according to frequency, genotype, and mode of inheritance and reinterpreted. RESULTS: We identified causal variants in 59 cases (3.9%), 50 of them also raised by other approaches and 9 leading to new diagnoses, highlighting interpretation challenges: variants in genes not known to be involved in human disease at the time of the first analysis, misleading genotypes, or variants undetected by local pipelines (variants in off-target regions, low quality filters, low allelic balance, or high frequency). CONCLUSION: The "ClinVar low-hanging fruit" analysis represents an effective, fast, and easy approach to recover causal variants from exome sequencing data, herewith contributing to the reduction of the diagnostic deadlock.

The RD-Connect Genome-Phenome Analysis Platform: Accelerating diagnosis, research, and gene discovery for rare diseases.

Rare disease patients are more likely to receive a rapid molecular diagnosis nowadays thanks to the wide adoption of next-generation sequencing. However, many cases remain undiagnosed even after exome or genome analysis, because the methods used missed the molecular cause in a known gene, or a novel causative gene could not be identified and/or confirmed. To address these challenges, the RD-Connect Genome-Phenome Analysis Platform (GPAP) facilitates the collation, discovery, sharing, and analysis of standardized genome-phenome data within a collaborative environment. Authorized clinicians and researchers submit pseudonymised phenotypic profiles encoded using the Human Phenotype Ontology, and raw genomic data which is processed through a standardized pipeline. After an optional embargo period, the data are shared with other platform users, with the objective that similar cases in the system and queries from peers may help diagnose the case. Additionally, the platform enables bidirectional discovery of similar cases in other databases from the Matchmaker Exchange network. To facilitate genome-phenome analysis and interpretation by clinical researchers, the RD-Connect GPAP provides a powerful user-friendly interface and leverages tens of information sources. As a result, the resource has already helped diagnose hundreds of rare disease patients and discover new disease causing genes.

Differences in Expression of IQSEC2 Transcript Isoforms in Male and Female Cases with Loss of Function Variants and Neurodevelopmental Disorder.

Pathogenic hemizygous or heterozygous mutations in the IQSEC2 gene cause X-linked intellectual developmental disorder-1 (XLID1), characterized by a variable phenotype including developmental delay, intellectual disability, epilepsy, hypotonia, autism, microcephaly and stereotypies. It affects both males and females typically through loss of function in males and haploinsufficiency in heterozygous females. Females are generally less affected than males. Two novel unrelated cases, one male and one female, with de novo IQSEC2 variants were detected by trio-based whole exome sequencing. The female case had a previously undescribed frameshift mutation (NM_001111125:c.3300dup; p.Met1101Tyrfs*5), and the male showed an intronic variant in intron 6, with a previously unknown effect (NM_001111125:c.2459+21C>T). IQSEC2 gene expression study revealed that this intronic variant created an alternative donor splicing site and an aberrant product, with the inclusion of 19bp, confirming the pathogenic effect of the intron variant. Moreover, a strong reduction in the expression of the long, but also the short IQSEC2 isoforms, was detected in the male correlating with a more severe phenotype, while the female case showed no decreased expression of the short isoform, and milder effects of the disease. This suggests that the abnormal expression levels of the different IQSEC2 transcripts could be implicated in the severity of disease manifestations.

De novo small deletion affecting transcription start site of short isoform of AUTS2 gene in a patient with syndromic neurodevelopmental defects.

Disruption of the autism susceptibility candidate 2 (AUTS2) gene through genomic rearrangements, copy number variations (CNVs), and intragenic deletions and mutations, has been recurrently involved in syndromic forms of developmental delay and intellectual disability, known as AUTS2 syndrome. The AUTS2 gene plays an important role in regulation of neuronal migration, and when altered, associates with a variable phenotype from severely to mildly affected patients. The more severe phenotypes significantly correlate with the presence of defects affecting the C-terminus part of the gene. This article reports a new patient with a syndromic neurodevelopmental disorder, who presents a deletion of 30 nucleotides in the exon 9 of the AUTS2 gene. Importantly, this deletion includes the transcription start site for the AUTS2 short transcript isoform, which has an important role in brain development. Gene expression analysis of AUTS2 full-length and short isoforms revealed that the deletion found in this patient causes a remarkable reduction in the expression level, not only of the short isoform, but also of the full AUTS2 transcripts. This report adds more evidence for the role of mutated AUTS2 short transcripts in the development of a severe phenotype in the AUTS2 syndrome.

Preparing Data at the Source to Foster Interoperability across Rare Disease Resources.

The ability to combine heterogeneous data distributed across the globe is critically important to boost research on rare diseases, but it presents a number of methodological, representational and automation challenges. In this scenario, biomedical ontologies are of critical importance for enabling computers to aid in information retrieval and analysis across data collections.This chapter presents an approach to preparing rare disease data for integration through the application of a global standard for computer-readable data and knowledge. This includes the use of common data elements, ontological codes and computer-readable data. This approach was developed under a number of domain-relevant requirements, such as controlled access to data, independence of the original sources, and the desire to combining the data sources with other computational workflows and data platforms.

Hyperkinetic Movement Disorder Caused by the Recurrent c.892C>T NACC1 Variant.

BACKGROUND: Genetic syndromes of hyperkinetic movement disorders associated with epileptic encephalopathy and intellectual disability are becoming increasingly recognized. Recently, a de novo heterozygous NACC1 (nucleus accumbens-associated 1) missense variant was described in a patient cohort including one patient with a combined mitochondrial oxidative phosphorylation (OXPHOS) deficiency. OBJECTIVES: The objective is to characterize the movement disorder in affected patients with the recurrent c.892C>T NACC1 variant and study the NACC1 protein and mitochondrial function at the cellular level. METHODS: The movement disorder was analyzed on four patients with the NACC1 c.892C>T (p.Arg298Trp) variant. Studies on NACC1 protein and mitochondrial function were performed on patient-derived fibroblasts. RESULTS: All patients had a generalized hyperkinetic movement disorder with chorea and dystonia, which occurred cyclically and during sleep. Complex I was found altered, whereas the other OXPHOS enzymes and the mitochondria network seemed intact in one patient. CONCLUSIONS: The movement disorder is a prominent feature of NACC1-related disease.

CERT1 mutations perturb human development by disrupting sphingolipid homeostasis.

Neural differentiation, synaptic transmission, and action potential propagation depend on membrane sphingolipids, whose metabolism is tightly regulated. Mutations in the ceramide transporter CERT (CERT1), which is involved in sphingolipid biosynthesis, are associated with intellectual disability, but the pathogenic mechanism remains obscure. Here, we characterize 31 individuals with de novo missense variants in CERT1. Several variants fall into a previously uncharacterized dimeric helical domain that enables CERT homeostatic inactivation, without which sphingolipid production goes unchecked. The clinical severity reflects the degree to which CERT autoregulation is disrupted, and inhibiting CERT pharmacologically corrects morphological and motor abnormalities in a Drosophila model of the disease, which we call ceramide transporter (CerTra) syndrome. These findings uncover a central role for CERT autoregulation in the control of sphingolipid biosynthetic flux, provide unexpected insight into the structural organization of CERT, and suggest a possible therapeutic approach for patients with CerTra syndrome.

New insights into antibody levels against SARS-CoV-2 for healthcare personnel vaccinated with tozinameran (Comirnaty).

AIM: The aim of this study is to determine the levels of spike protein IgG and total antibodies in subjects vaccinated against SARS-CoV-2 (both infected and non-infected) and the titer evolution over time. In addition, we also addressed the performance of each of the included platforms in the study, as they are intended to measure antibody levels in naturally infected patients. MATERIALS AND METHODS: An observational study including 288 volunteer healthcare professionals vaccinated against SARS-CoV-2 (Comirnaty™) at the Andújar Alto Guadalquivir Hospital. Serum samples were obtained in September 2020 and 14 and 90 days after administration of the second dose. The following in vitro methods were used: Elecsys Anti-SARS-CoV-2 N and Elecsys Anti-SARS-CoV-2 S (Roche, Germany) and EliA SARS-CoV-2-Sp1 IgG (Thermo Fisher Scientific, Germany). RESULTS: For the Elecsys S method at 1/10 dilution and for the EliA Sp1 IgG method at 1/5 dilution, 54% and 19% of samples were out of range, respectively. The vaccine activated a high humoral response- 0 to 3000 BAU/mL being the "normal titer range" in all volunteers. Patients vaccinated after COVID-19 exhibited higher total S antibody load values than non-vaccinated volunteers while showing the same response for S IgG isotype. Titers decreased up to 86% in the case of S IgG neutralizing antibodies. CONCLUSIONS: The characterization of human response to SARS-CoV-2 vaccines is still far from being completely elucidated. It is important to increase the methods dynamic range to study humoral response evolution in depth and decide whether booster doses or seasonal vaccination plans will be necessary to definitively control the pandemic.

Five new cases of syndromic intellectual disability due to KAT6A mutations: widening the molecular and clinical spectrum.

BACKGROUND: Pathogenic variants of the lysine acetyltransferase 6A or KAT6A gene are associated with a newly identified neurodevelopmental disorder characterized mainly by intellectual disability of variable severity and speech delay, hypotonia, and heart and eye malformations. Although loss of function (LoF) mutations were initially reported as causing this disorder, missense mutations, to date always involving serine residues, have recently been associated with a form of the disorder without cardiac involvement. RESULTS: In this study we present five new patients, four with truncating mutations and one with a missense change and the only one not presenting with cardiac anomalies. The missense change [p.(Gly359Ser)], also predicted to affect splicing by in silico tools, was functionally tested in the patient's lymphocyte RNA revealing a splicing effect for this allele that would lead to a frameshift and premature truncation. CONCLUSIONS: An extensive revision of the clinical features of these five patients revealed high concordance with the 80 cases previously reported, including developmental delay with speech delay, feeding difficulties, hypotonia, a high bulbous nose, and recurrent infections. Other features present in some of these five patients, such as cryptorchidism in males, syndactyly, and trigonocephaly, expand the clinical spectrum of this syndrome.

Usefulness of the "Candida score" for discriminating between Candida colonization and invasive candidiasis in non-neutropenic critically ill patients: a prospective multicenter study.

OBJECTIVE: To assess the usefulness of the "Candida score" (CS) for discriminating between Candida species colonization and invasive candidiasis (IC) in non-neutropenic critically ill patients. A rate of IC <5% in patients with CS <3 was the primary end point. DESIGN: Prospective, cohort, observational study. SETTING: Thirty-six medical-surgical intensive care units of Spain, Argentina, and France. PATIENTS: A total of 1,107 non-neutropenic adult intensive care unit patients admitted for at least 7 days between April 2006 and June 2007. MEASUREMENTS AND MAIN RESULTS: Clinical data, surveillance cultures for fungal growth, and serum levels of (1-3)-beta-d-glucan and anti-Candida antibodies (in a subset of patients) were recorded. The CS was calculated as follows (variables coded as absent = 0, present = 1): total parenteral nutrition x1, plus surgery x1, plus multifocal Candida colonization x1, plus severe sepsis x2. A CS >or=3 accurately selected patients at high risk for IC. The colonization index was registered if >or=0.5. The rate of IC was 2.3% (95% confidence interval [CI] 1.06-3.54) among patients with CS <3, with a linear association between increasing values of CS and IC rate (p 7 days, with a CS <3 and not receiving antifungal treatment, the rate of IC was <5%. Therefore, IC is highly improbable if a Candida-colonized non-neutropenic critically ill patient has a CS <3.

SpainUDP: The Spanish Undiagnosed Rare Diseases Program.

One of the IRDiRC goals for 2017⁻2027 is to achieve definitive diagnosis for rare undiagnosed diseases within one year, as delay in diagnosis remains one of the pending issues in the rare diseases field. The Spanish Undiagnosed Rare Diseases Program (SpainUDP) was created in response to this challenging scenario to cover patients' needs and after seeing the success of the Undiagnosed Diseases Program (UDP) in the USA. SpainUDP offers a multidisciplinary approach to those patients who have long sought a diagnosis without any success. During the first phase of the protocol, undiagnosed cases are sent to SpainUDP by individual patients or families, patient organizations or hospitals. After careful analysis of phenotype, data from sequencing experiments (WES) is processed with a standard pipeline and detailed standardized phenotypic information (mapped to the Human Phenotype Ontology, HPO) is connected to genetic data. In addition, the participation of SpainUDP in international initiatives such as the European projects RD-Connect and Solve RD, the Undiagnosed Diseases Network International (UDNI), and the MatchMaker Exchange (MME) platform, allows the establishment of a global data sharing strategy across multiple projects submitting data to these international initiatives. From the official beginning of the program (at the end of 2015) until early 2018, 147 cases were accepted in SpainUDP. During this time, 37 cases (25%) dropped out the program due to several reasons. The remaining 110 cases are distributed as follows: phenotypic and genotypic (WES) characterization was finished in 30 cases, of which 20 (67%) were diagnosed; 21 cases are pending on variants' validation by Sanger sequencing; in 25 cases, WES is ongoing and 34 cases are being studied for deep phenotypic characterization. In conclusion, SpainUDP aims to achieve a diagnosis following two recommendations of the IRDiRC: the patients' diagnosis in as short a time as possible and the promotion of data sharing (especially genomic) at the international level.

The RD-Connect Registry & Biobank Finder: a tool for sharing aggregated data and metadata among rare disease researchers.

In rare disease (RD) research, there is a huge need to systematically collect biomaterials, phenotypic, and genomic data in a standardized way and to make them findable, accessible, interoperable and reusable (FAIR). RD-Connect is a 6 years global infrastructure project initiated in November 2012 that links genomic data with patient registries, biobanks, and clinical bioinformatics tools to create a central research resource for RDs. Here, we present RD-Connect Registry & Biobank Finder, a tool that helps RD researchers to find RD biobanks and registries and provide information on the availability and accessibility of content in each database. The finder concentrates information that is currently sparse on different repositories (inventories, websites, scientific journals, technical reports, etc.), including aggregated data and metadata from participating databases. Aggregated data provided by the finder, if appropriately checked, can be used by researchers who are trying to estimate the prevalence of a RD, to organize a clinical trial on a RD, or to estimate the volume of patients seen by different clinical centers. The finder is also a portal to other RD-Connect tools, providing a link to the RD-Connect Sample Catalogue, a large inventory of RD biological samples available in participating biobanks for RD research. There are several kinds of users and potential uses for the RD-Connect Registry & Biobank Finder, including researchers collaborating with academia and the industry, dealing with the questions of basic, translational, and/or clinical research. As of November 2017, the finder is populated with aggregated data for 222 registries and 21 biobanks.

[DEMOCOPHES SPAIN AND ITS CONTRIBUTION TO THE HARMONIZATION OF EUROPEAN HUMAN BIOMONITORING]. / DEMOCOPHES ESPAÑA Y SU CONTRIBUCIÓN A LA ARMONIZACIÓN DE LA BIOVIGILANCIA HUMANA EN EUROPA.

UNLABELLED: Objetive: contributing to demonstrate of the feasibility of a coordinated action on human biomonitoring in Europe (DEMOCOPHES project) and demonstrate the utility of HBM studies to assess the influence of diet and lifestyle in environmental exposures. METHODS: the EU protocol was adapted to the national requirements. The quality controls defined herein were followed and special care was taken to ensure the comparability of the results among participating countries. RESULTS: the protocol adaptation did not shown significant difficulties. Only minor changes were applied, so the original design of the study was respected. 134 mother- child pairs were selected in one school in Añover de Tajo (Toledo) and three schools in Madrid. All volunteers donated a urine and hair sample and complete the epidemiological questionnaire. Significant differences were found in the participation rates between the sampling locations. DISCUSSION: standardization of all steps in a human biomonitoring study is essential for its harmonized development in Europe. The results has contributed to obtain for the first time comparable data about environmental exposure in the general population within 17 EU countries showing the differences associated with diet and lifestyles. The experiences and materials developed in the fieldwork could be applied to the design and implementation of HBM studies in the future.

Objetivo: contribuir a la armonización europea de la biovigilancia en humanos (proyecto DEMOCOPHES) demostrando la utilidad de los estudios de biovigilancia para valorar la influencia de la dieta y los estilos de vida como vía de exposición a contaminantes ambientales. Métodos: se adaptó el protocolo europeo a las necesidades nacionales, siguiendo los controles de calidad definidos en él y sin comprometer la obtención de datos comparables entre los países participantes. Resultados: la adaptación nacional del protocolo europeo no presentó grandes dificultades y, salvo mínimas modificaciones, se respetó el diseño original del estudio. Participaron 134 parejas madre-hijo, seleccionados en un colegio de Añover de Tajo (Toledo) y tres colegios de Madrid. Los voluntarios donaron una muestra de pelo y de orina y contestaron a las preguntas del cuestionario epidemiológico. Se observaron diferencias significativas en la participación de los voluntarios en las dos localizaciones de muestreo. Discusión: la estandarización de todas las etapas de un estudio de biovigilancia en humanos es esencial para su desarrollo armonizado a escala internacional. Los resultados obtenidos han contribuido a la obtención de datos sobre exposición ambiental, por primera vez comparables en 17 países europeos, y han permitido observar diferencias relacionadas con la dieta y los hábitos de vida. Las experiencias y el material de trabajo desarrollado para el estudio piloto serán aplicables al diseño e implementación de futuros estudios de HBM.

The influence of causal connections between symptoms on the diagnosis of mental disorders: evidence from online and offline measures.

An experiment conducted with students and experienced clinicians demonstrated very fast and online causal reasoning in the diagnosis of Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV) mental disorders. The experiment also demonstrated that clinicians' causal reasoning is triggered by information that is directly related to the causal structure that explains the symptoms, such as their temporal sequence. The use of causal theories was measured through explicit, verbal diagnostic judgments and through the online registration of participants' reading times of clinical reports. To detect both online and offline causal reasoning, the consistency of clinical reports was manipulated. This manipulation was made by varying the temporal order in which different symptoms developed in hypothetical clients, and by providing explicit information about causal connections between symptoms. The temporal order of symptoms affected the clinicians' but not the students' reading times. However, offline diagnostic judgments in both groups were influenced by the consistency manipulation. Overall, our results suggest that clinicians engage in fast and online causal reasoning processes when dealing with diagnostic information concerning mental disorders, and that both clinicians and students engage in causal reasoning in diagnostic judgment tasks.

Renewal effects in interference between outcomes as measured by a cued response reaction time task: further evidence for associative retrieval models.

Two experiments demonstrated renewal effects in interference between outcomes in human participants. Experiment 1 revealed a XYX renewal effect, whereas Experiment 2 showed a XYZ renewal effect. The results from both experiments conformed to Bouton's (1993) theory of interference and recovery from interference, and contradicted the predictions derived from alternative accounts. Unlike previous demonstration of renewal effects, a cued response reaction time (RT) task was used, able to detect the effects of fast retrieval processes based on associative activation and that allowed little impact of inferential reasoning.

Interference between outcomes, spontaneous recovery, and context effects as measured by a cued response reaction time task: evidence for associative retrieval models.

The most common associative explanation of interference is based on a retrieval failure. Retrieval, in turn, is considered as the result of an associative activation mechanism that is thought to be fast and automatic. However, up-to-date, there is no evidence of interference based on dependent measures specifically related to this kind of low level processes. The objective of the present study was to test whether interference phenomena can be observed by using a cued response task designed to detect low level retrieval processes. Experiment 1 evaluated whether the cued response task served to show a priming effect. Such effect allowed us to interpret the results found in the remaining experiments of the series. Experiment 2 aimed to find the interference effect by using the cued response task. Experiments 3 and 4 were conducted to assess whether spontaneous recovery and context-change effects could also be observed. The results showed that interference and recovery from interference phenomena can be attributable to fast retrieval processes, which is consistent with associative accounts of interference.

Molecular diagnosis of Aspergillus fumigatus endocarditis.

A 66-year-old male with ischaemic cardiomyopathy and chronic lymphocytic leukemia developed signs of severe systemic inflammatory response syndrome. Serial blood cultures were negative and a SeptiFast test detected the presence of Aspergillus fumigatus DNA. Afterwards, detection of galactomannan and 1,3-ß-D-glucan showed a positive result. Autopsy revealed the presence of branched fungal structures suggestive of Aspergillus.

Serum procalcitonin levels in critically ill patients colonized with Candida spp: new clues for the early recognition of invasive candidiasis?

OBJECTIVE: Invasive candidiasis (IC) outcomes in intensive care units (ICUs) could be improved by the early administration of antifungals. The Candida Score (CS) prediction rule has been proposed for the selection of patients who could develop IC. Procalcitonin (PCT) levels allow prompt identification of sepsis, but their behavior in the setting of IC is unclear. We hypothesize that PCT could be helpful in the early diagnosis of IC in patients with Candida sp. colonization. DESIGN: Prospective observational study. SETTING: Thirty-six ICUs in Spain, Portugal and France. PATIENTS: Every non-neutropenic critically ill patient hospitalized for more than 7 days without concurrent bacterial infection. The CS was calculated weekly. Serums were collected concomitantly. MEASUREMENTS AND RESULTS: Two hundred twenty PCT levels were measured in 136 patients [neither colonized nor infected (NCNI): n = 73; multifocal colonization (MF): n = 43; MF + IC: n = 20]. Baseline PCT levels were significantly higher in the MF + IC group than in other groups (p = 0.001). In patients with MF, the highest CS value calculated during the patient's stay was the sole independent predictor of IC. The receiver-operating curve analysis showed that the diagnosis values of PCT and CS were comparable (AUROCC = 0.713, and 0.727, respectively). Moreover, PCT increased the positive predictive value of CS from 44.7 to 59.3%. CONCLUSIONS: After 7 days of hospitalization, PCT levels in patients with MF who go on to develop IC are higher than in others. Serum PCT could also improve the predictive value of CS. PCT together with CS could therefore be considered for the assessment of IC risk.

[Diagnosis of Chlamydia trachomatis infection in a clinic for sexually transmitted disease: evaluation of cervical, urethral and rectal swab samples by polymerase chain reaction]. / Diagnóstico de la infección por Chlamydia trachomatis en un centro de diagnóstico y prevención de infecciones de transmisión sexual: evaluación de los exudados cervicales, uretrales y rectales mediante técnica de PCR.

INTRODUCTION: The aim of this study is to analyze the clinical and epidemiological characteristics of Chlamydia trachomatis infection in patients attended in a clinic for sexually transmitted disease in Seville (Spain). Microbiological diagnosis was performed in various types of samples. MATERIAL AND METHODS: The study included 3854 patients (50.8% women and 49.2% men, mean age 30.1 years) seen from 2002 to 2004. Among the total, 50% belonged to groups engaging in high risk sexual practices: female commercial sex workers (CSWs) (47%), men who maintain sexual relationships with other men (MSM) (45%), users of prostitution (4%), promiscuous heterosexual men (4%), those with a risk partner (2.7%) and injection drug users (IDU) (2.2%). We analyzed a total of 5978 samples (2384 cervical exudates, 2645 urethral exudates and 949 rectal exudates), for the detection of C. trachomatis by PCR technique with the COBAS Amplicor CT System. RESULTS: Prevalence of C. trachomatis infection was 6% (4.3% in women and 7.8% in men). Among the total in women, 51.2% of positive samples were from women with high-risk sex factors and 73.8% of the women were asymptomatic. In men, the proportions were 70.5% and 36.9%, respectively. Cervical, urethral and rectal exudates yielded positive results in 4%, 4.9% and 4.3%, respectively. CONCLUSIONS: Systematic sampling for C. trachomatis detection is necessary in symptomatic and asymptomatic patients practicing high-risk sex; periodic follow-up studies are also needed for early detection of sexually transmitted infection. Rectal sample collection is important for detecting this infection in MSM and in patients whose sexual habits make it advisable.

[Microbiological procedures for diagnosing mycoses and for antifungal susceptibility testing]. / Procedimientos de diagnóstico microbiológico de las micosis y estudios de sensibilidad a los antifúngicos.

Fungal infections are a diagnostic and therapeutic problem of increasing concern due to the frequency and severity of disseminated infection in immunocompromised patients. Culture-based methods are characteristically slow and have poor sensitivity; hence, other methods, based on the detection of fungus-specific genetic, antigenic and metabolic components are being developed to enable early diagnosis and specific treatment. Moreover, reproducible antifungal susceptibility methods that can be adapted for use in clinical laboratories have been standardized to allow in vitro detection of resistance, which correlates with a less favorable clinical outcome. In this paper we review the main microbiological procedures available for the diagnosis of fungal infections and for antifungal susceptibility testing.