Metagenomics is gradually being implemented for diagnosing infectious diseases. However, in-depth protocol comparisons for viral detection have been limited to individual sets of experimental workflows and laboratories. In this study, we present a benchmark of metagenomics protocols used in clinical diagnostic laboratories initiated by the European Society for Clinical Virology (ESCV) Network on NGS (ENNGS). A mock viral reference panel was designed to mimic low biomass clinical specimens. The panel was used to assess the performance of twelve metagenomic wet lab protocols currently in use in the diagnostic laboratories of participating ENNGS member institutions. Both Illumina and Nanopore, shotgun and targeted capture probe protocols were included. Performance metrics sensitivity, specificity, and quantitative potential were assessed using a central bioinformatics pipeline. Overall, viral pathogens with loads down to 104 copies/ml (corresponding to CT values of 31 in our PCR assays) were detected by all the evaluated metagenomic wet lab protocols. In contrast, lower abundant mixed viruses of CT values of 35 and higher were detected only by a minority of the protocols. Considering the reference panel as the gold standard, optimal thresholds to define a positive result were determined per protocol, based on the horizontal genome coverage. Implementing these thresholds, sensitivity and specificity of the protocols ranged from 67 to 100 % and 87 to 100 %, respectively. A variety of metagenomic protocols are currently in use in clinical diagnostic laboratories. Detection of low abundant viral pathogens and mixed infections remains a challenge, implying the need for standardization of metagenomic analysis for use in clinical settings.

• MeSH
• benchmarking * MeSH
• lidé MeSH
• metagenomika * metody   normy   MeSH
• senzitivita a specificita * MeSH
• virové nemoci diagnóza   virologie   MeSH
• viry * genetika   klasifikace   izolace a purifikace   MeSH
• výpočetní biologie metody   MeSH
• vysoce účinné nukleotidové sekvenování metody   normy   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• multicentrická studie MeSH

BACKGROUND: Epilepsy is a heterogeneous disease with a broad phenotypic spectrum and diverse genotypes. A significant proportion of epilepsies has a genetic aetiology. In our study, a custom designed gene panel with 112 genes known to be associated with epilepsies was used. In total, one hundred and fifty-one patients were tested (86 males / 65 females). RESULTS: In our cohort, the highest probability for the identification of the cause of the disease was for patients with a seizure onset within the first four weeks of life (61.9% clarification rate) - about two times more than other groups. The level of statistical significance was determined using a chi-square analysis. From 112 genes included in the panel, suspicious and rare variants were found in 53 genes (47.3%). Among the 151 probands included in the study we identified pathogenic variants in 39 patients (25.8%), likely pathogenic variants in three patients (2%), variants of uncertain significance in 40 patients (26.5%) and likely benign variants in 69 patients (45.7%). CONCLUSION: Our report shows the utility of diagnostic genetic testing of severe childhood epilepsies in a large cohort of patients with a diagnostic rate of 25.8%. A gene panel can be considered as a method of choice for the detection of pathogenic variants within patients with unknown origin of early onset severe epilepsy.

• MeSH
• dítě MeSH
• epilepsie genetika   MeSH
• genetická predispozice k nemoci genetika   MeSH
• kojenec MeSH
• křeče u dětí genetika   MeSH
• lidé MeSH
• mutace MeSH
• předškolní dítě MeSH
• rozdělení chí kvadrát MeSH
• záchvaty genetika   MeSH
• Check Tag
• dítě MeSH
• kojenec MeSH
• lidé MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: Once the outbreak with Burkholderia cenocepacia ST32 was identified in the Prague cystic fibrosis (CF) centre, molecular tools were implemented into diagnostic routine in order to complement infection control measures with as accurate as possible microbiological service. In addition, genotyping techniques were applied as part of an infection surveillance program to assign species and strain status in samples positive for Burkholderia cepacia complex (Bcc). We sought to evaluate a usefulness of Bcc-specific PCR in infection control and to map evolution of the outbreak. METHODS: Since 2001, 6109 respiratory samples from 299 CF patients were examined not only by conventional culture, but also by PCR, detecting Bcc directly in sputum. RESULTS: Diagnosis of Bcc infection was established by culture in 54 patients already prior to 2001. As 39 more patients were diagnosed by culture and/or PCR during 2001-2010, this represented annual prevalence of 18.5%-28.9%. Twelve of 39 patients were culture negative at the time of their first PCR positivity. Although 2/3 of them became subsequently culture positive, the time delay in diagnostics of the infection by culture ranged from 1 to 22 months. New cases of Bcc infection were detected every year, however a dramatic drop was observed for the epidemic strain ST32. CONCLUSION: A likely factor contributing to the end of ST32 epidemic was segregation of Bcc infected patients that included also patients with no culture, but PCR positivity. The diagnostic PCR led to timely identification of cases with 'culture-invisible' infection.

• MeSH
• Burkholderia cepacia komplex * genetika   izolace a purifikace   MeSH
• cystická fibróza * epidemiologie   mikrobiologie   MeSH
• dospělí MeSH
• epidemický výskyt choroby prevence a kontrola   statistika a číselné údaje   MeSH
• infekce bakteriemi rodu Burkholderia * diagnóza   epidemiologie   prevence a kontrola   MeSH
• infekce dýchací soustavy epidemiologie   mikrobiologie   prevence a kontrola   MeSH
• kontrola infekce metody   MeSH
• lidé MeSH
• polymerázová řetězová reakce * metody   statistika a číselné údaje   MeSH
• sputum mikrobiologie   MeSH
• techniky typizace bakterií MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika MeSH