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.
- Klíčová slova
- Benchmark, Clinical viral metagenomics, Wet lab protocols,
- 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
- práce podpořená grantem MeSH
Metagenomic high-throughput sequencing (mHTS) is a hypothesis-free, universal pathogen detection technique for determination of the DNA/RNA sequences in a variety of sample types and infectious syndromes. mHTS is still in its early stages of translating into clinical application. To support the development, implementation and standardization of mHTS procedures for virus diagnostics, the European Society for Clinical Virology (ESCV) Network on Next-Generation Sequencing (ENNGS) has been established. The aim of ENNGS is to bring together professionals involved in mHTS for viral diagnostics to share methodologies and experiences, and to develop application recommendations. This manuscript aims to provide practical recommendations for the wet lab procedures necessary for implementation of mHTS for virus diagnostics and to give recommendations for development and validation of laboratory methods, including mHTS quality assurance, control and quality assessment protocols.
- Klíčová slova
- High-throughput sequencing, Next-generation sequencing, Recommendations, Viral metagenomics, Wet lab,
- MeSH
- metagenomika * MeSH
- viry * genetika MeSH
- vysoce účinné nukleotidové sekvenování MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Hepatitis E virus (HEV) infection is not notifiable at EU/EEA level, therefore surveillance relies on national policies only. Between 2005 and 2015, more than 20,000 cases were reported in EU/EEA countries. HEV testing is established in 26 countries and 19 countries sequence HEV viruses. OBJECTIVE AND STUDY DESIGN: WHO's European Action plan for viral hepatitis recommends harmonised surveillance objectives and case definitions. ECDC's HEV expert group developed minimal and optimal criteria for national hepatitis E surveillance to support EU/EEA countries in enhancing their capacity and to harmonise methods. RESULTS: The experts agreed that the primary objectives of national surveillance for HEV infections should focus on the basic epidemiology of the disease: to monitor the incidence of acute cases and chronic infections. The secondary objectives should be to describe viral phylotypes or subtypes and to identify potential clusters/outbreaks and possible routes of transmission. Seventeen of 20 countries with existing surveillance systems collect the minimal data set required to describe the epidemiology of acute cases. Eleven countries test for chronic infections. Twelve countries collect data to identify potential clusters/outbreaks and information on possible routes of transmission. DISCUSSION: Overall, the majority of EU/EEA countries collect the suggested data and meet the outlined requirements to confirm an acute case.
- Klíčová slova
- EU/EEA, Hepatitis E virus, Surveillance, Testing,
- MeSH
- epidemický výskyt choroby MeSH
- epidemiologické monitorování MeSH
- Evropská unie MeSH
- hepatitida E diagnóza epidemiologie MeSH
- incidence MeSH
- lidé MeSH
- RNA virová analýza MeSH
- surveillance populace MeSH
- Světová zdravotnická organizace MeSH
- virus hepatitidy E genetika MeSH
- vládní programy normy MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Evropa MeSH
- Názvy látek
- RNA virová MeSH
- MeSH
- ELISA MeSH
- infekce virem zika diagnóza MeSH
- lidé MeSH
- negativní výsledek MeSH
- neutralizační testy MeSH
- neutralizující protilátky imunologie MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- protilátky virové imunologie MeSH
- RNA virová genetika MeSH
- virus zika genetika imunologie izolace a purifikace MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- dopisy MeSH
- Geografické názvy
- Evropa MeSH
- Izrael MeSH
- Názvy látek
- neutralizující protilátky MeSH
- protilátky virové MeSH
- RNA virová MeSH
BACKGROUND: Human papillomaviruses (HPV) are a heterogeneous group of viruses classified into five genera. The beta-HPV type (beta-PV) infection is very common but mostly asymptomatic in immunocompetent individuals. However, beta-PVs play a role in Epidermodysplasia verruciformis and possibly in non-melanoma skin cancer. Head and neck cancer (HNC) is a common cancer type worldwide and high-risk alpha-PV involvement in HNC has been extensively studied but beta-PV types have rarely been the focus of such studies. OBJECTIVES: To evaluate the prevalence of beta-PV types in HNC, subjects with non-malignant or potentially pre-malignant oral lesions, and healthy controls. STUDY DESIGN: The frequency of different beta-PVs in samples from oral (n=35) and oropharyngeal (n=35) cancer patients, gender- and age-matched healthy controls (n=70), and subjects with various non-malignant or potentially pre-malignant oral lesions (n=102) was assessed by a highly sensitive, bead-based, multiplex genotyping assay. RESULTS: Overall, 54.8% of all tested samples contained at least one beta-PV type. Even though the correlation between types found in lavage and tissue specimens from cancer patients was low, there was a large statistically significant difference between oropharyngeal cancer patients and matched controls for HPV5 (P=0.003; OR=15.58) and between both oral (P=0.026; OR=5.7) and oropharyngeal cancer patients (P=0.002; OR=25.5) and controls for HPV122. In addition, there was no correlation between the prevalence of alpha and beta-PVs in the study patients. CONCLUSION: The study provides new data on the prevalence of beta-PVs in HNC. HPV5 was found significantly associated with HNC as already observed by other studies. Additionally, the significant association of HPV122 with HNC might warrant further study as this type has not been extensively studied so far.
- Klíčová slova
- Cancer, Lesion, Oral, Oropharyngeal, alpha-HPV, beta-HPV,
- MeSH
- Betapapillomavirus klasifikace genetika izolace a purifikace MeSH
- dospělí MeSH
- genotyp * MeSH
- genotypizační techniky MeSH
- infekce papilomavirem epidemiologie virologie MeSH
- lidé středního věku MeSH
- lidé MeSH
- nemoci úst epidemiologie virologie MeSH
- orofarynx virologie MeSH
- prevalence MeSH
- senioři MeSH
- ústa virologie MeSH
- zdraví dobrovolníci pro lékařské studie MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Hepatitis E virus (HEV) is endemic in EU/EEA countries, but the understanding of the burden of the infection in humans is inconsistent as the disease is not under EU surveillance but subject to national policies. STUDY: Countries were asked to nominate experts and to complete a standardised questionnaire about the epidemiological situation and surveillance of HEV in their respective EU/EEA country. This study reviewed surveillance systems for human cases of HEV in EU/EEA countries and nominated experts assessed the epidemiology in particular examining the recent increase in the number of autochthonous cases. RESULTS: Surveillance systems and case definitions across EU/EEA countries were shown to be highly variable and testing algorithms were unreliable. Large increases of autochthonous cases were reported from Western EU/EEA countries with lower case numbers seen in Northern and Southern European countries. Lack of clinical awareness and variability in testing strategies might account for the observed differences in hepatitis E incidence across EU/EEA countries. Infections were predominantly caused by HEV genotype 3, the most prevalent virus type in the animal reservoirs. CONCLUSION: Discussions from the expert group supported joint working across countries to better monitor the epidemiology and possible changes in risk of virus acquisition at a European level. There was agreement to share surveillance strategies and algorithms but also importantly the collation of HEV data from human and animal populations. These data collected at a European level would serve the 'One Health' approach to better informing on human exposure to HEV.
- Klíčová slova
- Epidemiology, Europe, Hepatitis E virus, Surveillance, Zoonotic infections,
- MeSH
- endemické nemoci * MeSH
- hepatitida epidemiologie MeSH
- lidé MeSH
- osobní újma zaviněná nemocí MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Evropa epidemiologie MeSH
- Klíčová slova
- Fatal case, Haemophagocytic lymphohistiocytosis, Immunosuppressed patient, Tick-borne encephalitis,
- MeSH
- imunokompromitovaný pacient * MeSH
- klíšťová encefalitida virologie MeSH
- lidé MeSH
- moč virologie MeSH
- viremie virologie MeSH
- viry klíšťové encefalitidy genetika fyziologie MeSH
- vylučování virů * MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- dopisy MeSH
- komentáře MeSH
- práce podpořená grantem MeSH
BACKGROUND: Fourth-generation HIV assays detect both antigen and antibody, facilitating detection of acute/early HIV infection. The Bio-Rad GS HIV Combo Ag/Ab assay (Bio-Rad Combo) is an enzyme immunoassay that simultaneously detects HIV p24 antigen and antibodies to HIV-1 and HIV-2 in serum or plasma. OBJECTIVE: To evaluate the performance of the Bio-Rad Combo assay for detection of HIV infection in adults from Southern Africa. STUDY DESIGN: Samples were obtained from adults in Soweto and Vulindlela, South Africa and Dar es Salaam, Tanzania (300 HIV-positive samples; 300 HIV-negative samples; 12 samples from individuals previously classified as having acute/early HIV infection). The samples were tested with the Bio-Rad Combo assay. Additional testing was performed to characterize the 12 acute/early samples. RESULTS: All 300 HIV-positive samples were reactive using the Bio-Rad Combo assay; false positive test results were obtained for 10 (3.3%) of the HIV-negative samples (sensitivity: 100%, 95% confidence interval [CI]: 98.8-100%); specificity: 96.7%, 95% CI: 94.0-98.4%). The assay detected 10 of the 12 infections classified as acute/early. The two infections that were not detected had viral loads<400 copies/mL; one of those samples contained antiretroviral drugs consistent with antiretroviral therapy. CONCLUSIONS: The Bio-Rad Combo assay correctly classified the majority of study specimens. The specificity reported here may be higher than that seen in other settings, since HIV-negative samples were pre-screened using a different fourth-generation test. The assay also had high sensitivity for detection of acute/early infection. False-negative test results may be obtained in individuals who are virally suppressed.
- Klíčová slova
- Africa, Diagnosis, Enzyme immunoassay, Fourth-generation, HIV,
- MeSH
- HIV antigeny * imunologie MeSH
- HIV infekce diagnóza imunologie virologie MeSH
- HIV korový protein p24 imunologie MeSH
- HIV protilátky * imunologie MeSH
- HIV-1 imunologie MeSH
- HIV-2 imunologie MeSH
- HIV imunologie MeSH
- imunoenzymatické techniky * metody normy MeSH
- lidé MeSH
- reagenční diagnostické soupravy * normy MeSH
- reprodukovatelnost výsledků MeSH
- senzitivita a specificita MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Research Support, N.I.H., Extramural MeSH
- Geografické názvy
- jižní Afrika MeSH
- Názvy látek
- HIV antigeny * MeSH
- HIV korový protein p24 MeSH
- HIV protilátky * MeSH
- reagenční diagnostické soupravy * MeSH
BACKGROUND: Antiviral resistance development is a serious complication of human cytomegalovirus virostatic therapy caused by mutations in UL 97 and/or UL54 genes. OBJECTIVES: To determinate the presence of sensitive and resistant strains in patients developing antiviral resistance. STUDY DESIGN: We used three different molecular biological methods for mutation analysis-restriction fragment length polymorphism, sequencing and real-time PCR approach. RESULTS: We describe three allogeneic hematopoietic stem cell transplant patients developing the GCV resistant HCMV strains manifested by virostatic treatment failure. In these patients we identified UL97 mutations L595S, A594V and A594T and monitored the dynamics of coexisted sensitive/resistant strains. We confirmed the presence of mixed HCMV populations and in two patients a phenomenon of sensitive strain repopulation which occurred after 6.5 months and 1 month after removing GCV pressure. CONCLUSIONS: Our results show changes in proportions of sensitive/resistant subpopulations over time but other studies would be required to demonstrate the beneficial impact of their monitoring on clinical outcome.
- Klíčová slova
- HCMV resistance, Real-time PCR, Repopulation, Sensitive/resistant strain,
- MeSH
- antivirové látky terapeutické užití MeSH
- cytomegalovirové infekce farmakoterapie virologie MeSH
- Cytomegalovirus účinky léků genetika izolace a purifikace MeSH
- diagnostické techniky molekulární metody MeSH
- dospělí MeSH
- homologní transplantace škodlivé účinky MeSH
- kvantitativní polymerázová řetězová reakce metody MeSH
- lidé středního věku MeSH
- lidé MeSH
- missense mutace * MeSH
- neúspěšná terapie MeSH
- polymorfismus délky restrikčních fragmentů MeSH
- sekvenční analýza DNA MeSH
- transplantace hematopoetických kmenových buněk škodlivé účinky MeSH
- virová léková rezistence * MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- antivirové látky MeSH
BACKGROUND: More than 90% of cases of anal cancers are caused by high-risk human papillomavirus (HR HPV) infection and a history of cervical intraepithelial neoplasia (CIN) is established as possible risk factor. OBJECTIVES: To demonstrate relationship between anal and cervical HPV infection in women with different grades of CIN and microinvasive cervical cancer. STUDY DESIGN: A total of 272 women were enrolled in the study. The study group included 172 women who underwent conization for high-grade CIN or microinvasive cervical cancer. The control group consisted of 100 women with non-neoplastic gynecologic diseases or biopsy-confirmed CIN 1. All participants completed a questionnaire detailing their medical history and sexual risk factors and were subjected to anal and cervical HPV genotyping using Cobas and Lynear array HPV test. RESULTS: Cervical, anal, and concurrent cervical and anal HPV infections were detected in 82.6%, 48.3% and 42.4% of women in the study group, and in 28.0%, 26.0% and 8.0% of women in the control group, respectively. The prevalence of the HR HPV genotypes was higher in the study group and significantly increased with the severity of cervical lesion. Concurrent infections of the cervix and anus occurred 5.3-fold more often in the study group than in the control group. Any contact with the anus was the only significant risk factor for development of concurrent HPV infection. CONCLUSIONS: Concurrent anal and cervical HR HPV infection was found in nearly half of women with CIN 2+. The dominant genotype found in both anatomical locations was HPV 16. Any frequency and any type of contact with the anus were shown as the most important risk factor for concurrent HPV infection.
- Klíčová slova
- AIN, AIS, ASC-H, Anal cancer, Anal infection, CIN, CIN 2+, COC, Cervical infection, DNA, HIV, HPV, HR, HRT, HSIL, IUD, LR, OR, PCR, adenocarcinoma in situ, anal intraepithelial neoplasia, atypical squamous cells, cannot exclude high-grade squamous intraepithelial lesion, cervical intraepithelial neoplasia, combined oral contraception, deoxyribonucleic acid, high-grade squamous intraepithelial lesion, high-gradeCIN or microinvasive cervical cancer, high-risk, hormone replacement therapy, human immunodeficiency virus, human papillomavirus, intrauterine device, low-risk, odd ratio, polymerase chain reaction,
- MeSH
- anální kanál virologie MeSH
- cervix uteri virologie MeSH
- dospělí MeSH
- dysplazie děložního hrdla virologie MeSH
- genotyp MeSH
- infekce papilomavirem komplikace epidemiologie přenos virologie MeSH
- kohortové studie MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladý dospělý MeSH
- molekulární epidemiologie MeSH
- Papillomaviridae klasifikace genetika izolace a purifikace MeSH
- průzkumy a dotazníky MeSH
- sexuální chování MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladý dospělý MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
