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Objective: Immune activation is associated with morbidity and mortality during human immunodeficiency virus (HIV) infection, despite receipt of antiretroviral therapy (ART). We investigated whether microbial translocation drives immune activation in HIV-infected Ugandan children. Methods: Nineteen markers of immune activation and inflammation were measured over 96 weeks in HIV-infected Ugandan children in the CHAPAS-3 Trial and HIV-uninfected age-matched controls. Microbial translocation was assessed using molecular techniques, including next-generation sequencing. Results: Of 249 children included, 142 were infected with HIV; of these, 120 were ART naive, with a median age of 2.8 years (interquartile range [IQR], 1.7-4.0 years) and a median baseline CD4+ T-cell percentage of 20% (IQR, 14%-24%), and 22 were ART experienced, with a median age of 6.5 years (IQR, 5.9-9.2 years) and a median baseline CD4+ T-cell percentage of 35% (IQR, 31%-39%). The control group comprised 107 children without HIV infection. The median increase in the CD4+ T-cell percentage was 17 percentage points (IQR, 12-22 percentage points) at week 96 among ART-naive children, and the viral load was <100 copies/mL in 76% of ART-naive children and 91% of ART-experienced children. Immune activation decreased with ART use. Children could be divided on the basis of immune activation markers into the following 3 clusters: in cluster 1, the majority of children were HIV uninfected; cluster 2 comprised a mix of HIV-uninfected children and HIV-infected ART-naive or ART-experienced children; and in cluster 3, the majority were ART naive. Immune activation was low in cluster 1, decreased in cluster 3, and persisted in cluster 2. Blood microbial DNA levels were negative or very low across groups, with no difference between clusters except for Enterobacteriaceae organisms (the level was higher in cluster 1; P < .0001). Conclusion: Immune activation decreased with ART use, with marker clustering indicating different activation patterns according to HIV and ART status. Levels of bacterial DNA in blood were low regardless of HIV status, ART status, and immune activation status. Microbial translocation did not drive immune activation in this setting. Clinical Trials Registration: ISRCTN69078957.
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Translocação Bacteriana/imunologia , Biomarcadores/sangue , Infecções por HIV/imunologia , Translocação Bacteriana/genética , Contagem de Linfócito CD4 , Criança , Pré-Escolar , DNA Bacteriano/sangue , DNA Ribossômico , Feminino , Infecções por HIV/sangue , Infecções por HIV/tratamento farmacológico , Infecções por HIV/microbiologia , Humanos , Lactente , Inflamação , Masculino , Uganda , Carga ViralRESUMO
Background: Norovirus is a leading cause of worldwide and nosocomial gastroenteritis. The study aim was to assess the utility of molecular epidemiology using full genome sequences compared to routine infection prevention and control (IPC) investigations. Methods: Norovirus genomes were generated from new episodes of norovirus at a pediatric tertiary referral hospital over a 19-month period (n = 182). Phylogeny identified clusters of related sequences that were verified using epidemiological and clinical data. Results: Twenty-four clusters of related norovirus sequences ("sequence clusters") were observed, including 8 previously identified by IPC investigations ("IPC outbreaks"). Seventeen sequence clusters (involving 77/182 patients) were corroborated by epidemiological data ("epidemiologically supported clusters"), suggesting transmission between patients. Linked infections were identified among 44 patients who were missed by IPC investigations. Thirty-three percent of norovirus sequences were linked, suggesting nosocomial transmission; 24% of patients had nosocomial infections from an unknown source; and 43% were norovirus positive on admission. Conclusions: We show there are frequent introductions of multiple norovirus strains with extensive onward nosocomial transmission of norovirus in a pediatric hospital with a high proportion of immunosuppressed patients nursed in isolation. Phylogenetic analysis using full genome sequences is more sensitive than classic IPC investigations for identifying linked cases and should be considered when investigating norovirus nosocomial transmission. Sampling of staff, visitors, and the environment may be required for complete understanding of infection sources and transmission routes in patients with nosocomial infections not linked to other patients and among patients with phylogenetically linked cases but no evidence of direct contact.
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Infecções por Caliciviridae/transmissão , Infecções por Caliciviridae/virologia , Infecção Hospitalar/transmissão , Infecção Hospitalar/virologia , Genoma Viral , Norovirus/genética , Criança , Surtos de Doenças , Gastroenterite/virologia , Genótipo , Hospitais Pediátricos , Humanos , FilogeniaRESUMO
Background: Varicella zoster virus (VZV) may cause encephalitis, both with and without rash. Here we investigate whether viruses recovered from the central nervous system (CNS; encephalitis or meningitis) differ genetically from those recovered from non-CNS samples. Methods: Enrichment-based deep sequencing of 45 VZV genomes from cerebral spinal fluid (CSF), plasma, bronchoalveolar lavage (BAL), and vesicles was carried out with samples collected from 34 patients with and without VZV infection of the CNS. Results: Viral sequences from multiple sites in the same patient were identical at the consensus level. Virus from vesicle fluid and CSF in cases of meningitis showed low-level diversity. By contrast, plasma, BAL, and encephalitis had higher numbers of variant alleles. Two CSF-encephalitis samples had high genetic diversity, with variant frequency patterns typical of mixed infections with different clades. Conclusions: Low viral genetic diversity in vesicle fluid is compatible with previous observations that VZV skin lesions arise from single or low numbers of virions. A similar result was observed in VZV from cases of VZV meningitis, a generally self-limiting infection. CSF from cases of encephalitis had higher diversity with evidence for mixed clade infections in 2 cases. We hypothesize that reactivation from multiple neurons may contribute to the pathogenesis of VZV encephalitis.
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DNA Viral/líquido cefalorraquidiano , Encefalite por Varicela Zoster/virologia , Herpesvirus Humano 3/classificação , Herpesvirus Humano 3/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Estudos de Coortes , Coinfecção/virologia , Vesículas Citoplasmáticas/virologia , Variação Genética , Genoma Viral/genética , Humanos , Pessoa de Meia-Idade , Carga Viral , Adulto JovemRESUMO
Background: Adenoviruses are significant pathogens for the immunocompromised, arising from primary infection or reinfection. Serotyping is insufficient to support nosocomial transmission investigations. We investigate whether whole-genome sequencing (WGS) provides clinically relevant information on transmission among patients in a pediatric tertiary hospital. Methods: We developed a target-enriched adenovirus WGS technique for clinical samples and retrospectively sequenced 107 adenovirus-positive residual diagnostic samples, including viremias (>5 × 104 copies/mL), from 37 patients collected January 2011-March 2016. Whole-genome sequencing was used to determine genotype and for phylogenetic analysis. Results: Adenovirus sequences were recovered from 105 of 107 samples. Full genome sequences were recovered from all 20 nonspecies C samples and from 36 of 85 species C viruses, with partial genome sequences recovered from the rest. Whole-genome phylogenetic analysis suggested linkage of 3 genotype A31 cases and uncovered an unsuspected epidemiological link to an A31 infection first detected on the same ward 4 years earlier. In 9 samples from 1 patient who died, we identified a mixed genotype adenovirus infection. Conclusions: Adenovirus WGS from clinical samples is possible and useful for genotyping and molecular epidemiology. Whole-genome sequencing identified likely nosocomial transmission with greater resolution than conventional genotyping and distinguished between adenovirus disease due to single or multiple genotypes.
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Adenoviridae/genética , Infecções por Adenovirus Humanos/virologia , Infecção Hospitalar/virologia , Genótipo , Hospedeiro Imunocomprometido , Sequenciamento Completo do Genoma , Adenoviridae/classificação , Infecções por Adenovirus Humanos/transmissão , Adolescente , Criança , Pré-Escolar , Infecção Hospitalar/transmissão , Genômica , Humanos , Lactente , Epidemiologia Molecular , FilogeniaRESUMO
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Norovirus is acknowledged to be a leading cause of acute gastroenteritis worldwide, and its importance as a cause of chronic infection in immune deficient hosts is increasingly recognised. Current evidence suggests that a coordinated response of innate immune mechanisms, CD8+ cytotoxicity and a humoral response, with CD4+ orchestration, is necessary for norovirus clearance. We explain how primary immune deficiency impairs these host defences and predisposes to chronic infection, associated with protracted diarrhoea, weight loss, and requirement for parenteral nutrition. The mucosal villous atrophy frequently seen in norovirus infection appears to be immune mediated, suggesting that some functional immune response is required in order for chronic norovirus infection to become symptomatic in primary immune deficiency. We provide a comprehensive summary of published cases of norovirus infection in patients with primary immune deficiency. Spontaneous viral clearance has been described; however, the majority of reported cases have had prolonged and severe illness. Treatment strategies are discussed in detail. Approaches that have been tried in patients with primary immune deficiency include exclusion diets, enteral and intravenous immunoglobulins, breast milk, immunosuppressants, ribavirin, and nitazoxanide. To date, only ribavirin has been used with apparent success to achieve clearance of chronic norovirus in primary immune deficiency, and randomised controlled trials are needed to evaluate a number of promising therapies that are discussed.
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We report acute retinal necrosis caused by the vaccine Oka strain following immunization of a 78-year-old woman with live zoster vaccine. Whole genome sequencing confirmed the ocular vOka strain to be derived from the vaccine and excluded the presence of new mutations or recombination with wild-type Varicella zoster virus.
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Vacina contra Herpes Zoster/efeitos adversos , Herpes Zoster/complicações , Herpesvirus Humano 3/isolamento & purificação , Síndrome de Necrose Retiniana Aguda/virologia , Vacinação/efeitos adversos , Aciclovir/administração & dosagem , Aciclovir/análogos & derivados , Aciclovir/uso terapêutico , Idoso , Antivirais/administração & dosagem , Antivirais/uso terapêutico , Vírus de DNA/genética , Vírus de DNA/isolamento & purificação , DNA Viral , Olho/citologia , Olho/patologia , Olho/virologia , Feminino , Herpes Zoster/prevenção & controle , Vacina contra Herpes Zoster/administração & dosagem , Herpesvirus Humano 3/genética , Humanos , Síndrome de Necrose Retiniana Aguda/diagnóstico , Síndrome de Necrose Retiniana Aguda/tratamento farmacológico , Síndrome de Necrose Retiniana Aguda/etiologia , Valaciclovir , Valina/administração & dosagem , Valina/análogos & derivados , Valina/uso terapêutico , Corpo Vítreo/citologia , Corpo Vítreo/imunologia , Corpo Vítreo/virologia , Sequenciamento Completo do GenomaRESUMO
Routine childhood vaccination against measles, mumps and rubella has virtually abolished virus-related morbidity and mortality. Notwithstanding this, we describe here devastating neurological complications associated with the detection of live-attenuated mumps virus Jeryl Lynn (MuVJL5) in the brain of a child who had undergone successful allogeneic transplantation for severe combined immunodeficiency (SCID). This is the first confirmed report of MuVJL5 associated with chronic encephalitis and highlights the need to exclude immunodeficient individuals from immunisation with live-attenuated vaccines. The diagnosis was only possible by deep sequencing of the brain biopsy. Sequence comparison of the vaccine batch to the MuVJL5 isolated from brain identified biased hypermutation, particularly in the matrix gene, similar to those found in measles from cases of SSPE. The findings provide unique insights into the pathogenesis of paramyxovirus brain infections.
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Encéfalo/virologia , Encefalite Viral/virologia , Vacina contra Caxumba/efeitos adversos , Vírus da Caxumba/isolamento & purificação , Biópsia , Encéfalo/diagnóstico por imagem , Encéfalo/patologia , Doença Crônica , Encefalite Viral/complicações , Encefalite Viral/diagnóstico por imagem , Encefalite Viral/terapia , Evolução Fatal , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Masculino , Vírus da Caxumba/genética , Imunodeficiência Combinada Severa/complicações , Imunodeficiência Combinada Severa/diagnóstico por imagem , Imunodeficiência Combinada Severa/terapiaRESUMO
Norovirus incidence was compared between severe combined immunodeficiency children with (n = 10) and without (n = 8) B cells. 60% of B+ and 63% of B- patients developed norovirus infections therefore norovirus replication in B lymphocytes is not essential for infection.
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Linfócitos B/virologia , Infecções por Caliciviridae/patologia , Norovirus/imunologia , Imunodeficiência Combinada Severa/virologia , Adolescente , Linfócitos B/patologia , Infecções por Caliciviridae/imunologia , Criança , Pré-Escolar , Humanos , Lactente , Estudos RetrospectivosAssuntos
Agamaglobulinemia/imunologia , Agamaglobulinemia/virologia , Doenças Genéticas Ligadas ao Cromossomo X/imunologia , Doenças Genéticas Ligadas ao Cromossomo X/virologia , Kobuvirus/imunologia , Infecções por Picornaviridae/imunologia , Infecções por Picornaviridae/virologia , Viroses/imunologia , Adolescente , Doença Crônica , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/virologia , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Masculino , Viroses/virologiaRESUMO
Norovirus full-genome sequencing is challenging due to sequence heterogeneity among genomes. Previous methods have relied on PCR amplification, which is problematic due to primer design, and transcriptome sequencing (RNA-Seq), which nonspecifically sequences all RNA, including host and bacterial RNA, in stool specimens. Target enrichment uses a panel of custom-designed 120-mer RNA baits that are complementary to all publicly available norovirus sequences, with multiple baits targeting each position of the genome, which overcomes the challenge of primer design. Norovirus genomes are enriched from stool RNA extracts to minimize the sequencing of nontarget RNA. SureSelect target enrichment and Illumina sequencing were used to sequence full genomes from 507 norovirus-positive stool samples with reverse transcription-real-time PCR cycle threshold (CT) values of 10 to 43. Sequencing on an Illumina MiSeq system in batches of 48 generated, on average, 81% on-target reads per sample and 100% genome coverage with >12,000-fold read depth. Samples included genotypes GI.1, GI.2, GI.3, GI.6, GI.7, GII.1, GII.2, GII.3, GII.4, GII.5, GII.6, GII.7, GII.13, GII.14, and GII.17. When outliers were accounted for, we generated >80% genome coverage for all positive samples, regardless of CT values. A total of 164 samples were tested in parallel with conventional PCR genotyping of the capsid shell domain; 164/164 samples were successfully sequenced, compared to 158/164 samples that were amplified by PCR. Four of the samples that failed capsid PCR analysis had low titers, which suggests that target enrichment is more sensitive than gel-based PCR. Two samples failed PCR due to primer mismatches; target enrichment uses multiple baits targeting each position, thus accommodating sequence heterogeneity among norovirus genomes.
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Fezes/virologia , Genoma Viral , Norovirus/isolamento & purificação , Hibridização de Ácido Nucleico/métodos , RNA Viral/genética , Análise de Sequência de DNA/métodos , Manejo de Espécimes/métodos , Infecções por Caliciviridae/virologia , Humanos , Masculino , Norovirus/genéticaRESUMO
BACKGROUND: An 18-month-old boy developed encephalopathy, for which extensive investigation failed to identify an etiology, 6 weeks after stem cell transplant. To exclude a potential infectious cause, we performed high-throughput RNA sequencing on brain biopsy. METHODS: RNA-Seq was performed on an Illumina Miseq, generating 20 million paired-end reads. Nonhost data were checked for similarity to known organisms using BLASTx. The full viral genome was sequenced by primer walking. RESULTS: We identified an astrovirus, HAstV-VA1/HMO-C-UK1(a), which was highly divergent from human astrovirus (HAstV 1-8) genotypes, but closely related to VA1/HMO-C astroviruses, including one recovered from a case of fatal encephalitis in an immunosuppressed child. The virus was detected in stool and serum, with highest levels in brain and cerebrospinal fluid (CSF). Immunohistochemistry of the brain biopsy showed positive neuronal staining. A survey of 680 stool and 349 CSF samples identified a related virus in the stool of another immunosuppressed child. CONCLUSIONS: The discovery of HAstV-VA1/HMO-C-UK1(a) as the cause of encephalitis in this case provides further evidence that VA1/HMO-C viruses, unlike HAstV 1-8, are neuropathic, particularly in immunocompromised patients, and should be considered in the differential diagnosis of encephalopathy. With a turnaround from sample receipt to result of <1 week, we confirm that RNA-Seq presents a valuable diagnostic tool in unexplained encephalitis.
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Infecções por Astroviridae/virologia , Encéfalo/patologia , Encefalite Viral/diagnóstico , Encefalite Viral/patologia , Hospedeiro Imunocomprometido , Mamastrovirus/patogenicidade , Infecções por Astroviridae/diagnóstico , Infecções por Astroviridae/patologia , Sequência de Bases , Biópsia , Encéfalo/ultraestrutura , Encefalite Viral/virologia , Fezes/virologia , Genoma Viral , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Masculino , Mamastrovirus/genética , Mamastrovirus/isolamento & purificação , Filogenia , Prevalência , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Análise de Sequência de RNA , Transplante de Células-TroncoAssuntos
Linfócitos B/imunologia , Infecções por Caliciviridae/imunologia , Imunodeficiência de Variável Comum/imunologia , Hospedeiro Imunocomprometido , Norovirus/fisiologia , Linfócitos T/imunologia , Adulto , Antígenos CD19/metabolismo , Antivirais/uso terapêutico , Infecções por Caliciviridae/diagnóstico , Infecções por Caliciviridae/tratamento farmacológico , Células Cultivadas , Imunodeficiência de Variável Comum/diagnóstico , Imunodeficiência de Variável Comum/tratamento farmacológico , Enterocolite , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Nitrocompostos , Receptor de Morte Celular Programada 1/metabolismo , Recidiva , Ribavirina/uso terapêutico , Especificidade da Espécie , Tiazóis/uso terapêutico , Resultado do Tratamento , Regulação para CimaRESUMO
BACKGROUND: Chlamydia trachomatis is a pathogen of worldwide importance, causing more than 100 million cases of sexually transmitted infections annually. Whole-genome sequencing is a powerful high resolution tool that can be used to generate accurate data on bacterial population structure, phylogeography and mutations associated with antimicrobial resistance. The objective of this study was to perform whole-genome enrichment and sequencing of C. trachomatis directly from clinical samples. METHODS: C. trachomatis positive samples comprising seven vaginal swabs and three urine samples were sequenced without prior in vitro culture in addition to nine cultured C. trachomatis samples, representing different serovars. A custom capture RNA bait set, that captures all known diversity amongst C. trachomatis genomes, was used in a whole-genome enrichment step during library preparation to enrich for C. trachomatis DNA. All samples were sequenced on the MiSeq platform. RESULTS: Full length C. trachomatis genomes (>95-100% coverage of a reference genome) were successfully generated for eight of ten clinical samples and for all cultured samples. The proportion of reads mapping to C. trachomatis and the mean read depth across each genome were strongly linked to the number of bacterial copies within the original sample. Phylogenetic analysis confirmed the known population structure and the data showed potential for identification of minority variants and mutations associated with antimicrobial resistance. The sensitivity of the method was >10-fold higher than other reported methodologies. CONCLUSIONS: The combination of whole-genome enrichment and deep sequencing has proven to be a non-mutagenic approach, capturing all known variation found within C. trachomatis genomes. The method is a consistent and sensitive tool that enables rapid whole-genome sequencing of C. trachomatis directly from clinical samples and has the potential to be adapted to other pathogens with a similar clonal nature.
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Infecções por Chlamydia/microbiologia , Chlamydia trachomatis/genética , Sequência de Bases , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Filogenia , Análise de Sequência de DNARESUMO
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.
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Benchmarking , Metagenômica , Sensibilidade e Especificidade , Vírus , Metagenômica/métodos , Metagenômica/normas , Humanos , Vírus/genética , Vírus/classificação , Vírus/isolamento & purificação , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sequenciamento de Nucleotídeos em Larga Escala/normas , Viroses/diagnóstico , Viroses/virologia , Biologia Computacional/métodosAssuntos
Agamaglobulinemia/complicações , Doenças Genéticas Ligadas ao Cromossomo X/complicações , Infecções por Picornaviridae/diagnóstico , Infecções por Picornaviridae/etiologia , Tirosina Quinase da Agamaglobulinemia/genética , Agamaglobulinemia/diagnóstico , Agamaglobulinemia/genética , Agamaglobulinemia/imunologia , Biomarcadores , Pré-Escolar , Doença Crônica , Doenças Genéticas Ligadas ao Cromossomo X/diagnóstico , Doenças Genéticas Ligadas ao Cromossomo X/genética , Doenças Genéticas Ligadas ao Cromossomo X/imunologia , Humanos , Imunofenotipagem , Kobuvirus/imunologia , Masculino , Mutação , Infecções por Picornaviridae/tratamento farmacológico , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
Metagenomic next-generation sequencing (mNGS) is an untargeted technique capable of detecting all microbial nucleic acid within a sample. This protocol outlines our wet laboratory method for mNGS of cerebrospinal fluid (CSF) specimens and tissues from sterile sites. We use this method routinely in our clinical service, processing 178 specimens over the past 2.5 years in a laboratory that adheres to ISO:15189 standards. We have successfully used this protocol to diagnose multiple cases of encephalitis and hepatitis.
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Introduction. Molecular techniques are used in the clinical microbiology laboratory to support culture-based diagnosis of infection and are particularly useful for detecting difficult to culture bacteria or following empirical antimicrobial treatment.Hypothesis/Gap Statement. Broad-range 16S rRNA PCR is a valuable tool that detects a wide range of bacterial species. Diagnostic yield is low for some sample types but can be improved with the addition of qPCR panels targeting common bacterial pathogens.Aim. To evaluate the performance of a broad-range 16S rRNA gene PCR and the additional diagnostic yield of targeted qPCR applied to specimens according to a local testing algorithm.Methodology. In total, 6130 primary clinical samples were collected as part of standard clinical practice from patients with suspected infection during a 17 month period. Overall, 5497 samples were tested by broad-range 16S rRNA gene PCR and a panel of targeted real-time qPCR assays were performed on selected samples according to a local testing algorithm. An additional 633 samples were tested by real-time qPCR only. The 16S rRNA gene PCR was performed using two assays targeting different regions of the 16S rRNA gene. Laboratory developed qPCR assays for seven common bacterial pathogens were also performed. Data was extracted retrospectively from Epic Beaker Laboratory Information Management System (LIMS).Results. Broad-range 16S rRNA gene PCR improves diagnostic yield in culture-negative samples and detects a large range of bacterial species. Streptococcus spp., Staphylococcus spp. and the Enterobacteriaceae family are detected the most frequently in samples with a single causative organism, but mixed samples frequently contained anaerobic species. The highest diagnostic yield was obtained from abscess, pus and empyema samples; 44.9â% were positive by 16S and 61â% were positive by the combined 16S and targeted qPCR testing algorithm. Samples with a particularly low diagnostic yield were blood, with 3.3â% of samples positive by 16S and CSF with 4.8â% of samples positive by 16S. The increased diagnostic yield of adding targeted qPCR is largest (~threefold) in these two sample types.Conclusion. Broad-range PCR is a powerful technique that can detect a very large range of bacterial pathogens but has limited diagnostic sensitivity. The data in this report supports a testing strategy that combines broad-range and targeted bacterial PCR assays for maximizing diagnosis of infection in culture-negative specimens. This is particularly justified for blood and CSF samples. Alternative approaches, such as metagenomic sequencing, are needed to provide the breadth of broad-range PCR and the sensitivity of targeted qPCR panels.
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Bactérias , Infecções Bacterianas , Humanos , Bactérias/genética , DNA Bacteriano/genética , DNA Bacteriano/análise , Genes de RNAr , Reação em Cadeia da Polimerase/métodos , Estudos Retrospectivos , RNA Ribossômico 16S/genética , Infecções Bacterianas/diagnósticoRESUMO
Background: Infections by several DNA viruses can severely impact outcomes in paediatric immunocompromised patients. Current testing, which is generally limited to singleplex qPCR assays, can miss both common and rarer viruses if they are not targeted. Objectives: To evaluate the performance of the Galileo Viral Panel (Galileo), a sample-to-result shotgun metagenomics platform for the detection and quantification of 12 DNA viruses, compared to standard of care qPCR assays. Study design: A clinical performance evaluation was carried out using 43 prospectively collected EDTA plasma samples positive for one or more DNA viruses. Agreement between assays was assessed by overall, positive, and negative percent agreement, as well as quantitative agreement by linear regression and Bland-Altman analysis. Results: Overall positive percent agreement was 84% (95% CI: 76%-90%), and negative percent agreement was 95% (95% CI: 92%-97%). There was a high correlation between Galileo and qPCR for ADV, CMV, EBV, and VZV (R2 = 0.91) and a mean difference by Bland Altman of -0.43 log10 IU or cp/ml (95% limits of agreement, -1.37 to 0.51). In addition, there was a high correlation between Galileo Signal Score and qPCR for TTV (R2 = 0.85). Conclusion: We observed high qualitative and quantitative agreement between qPCR and Galileo. Galileo identified additional viruses that were not tested with routine qPCR and could impact clinical outcomes.