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1.
J Clin Microbiol ; 62(6): e0014424, 2024 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-38775470

RESUMO

The 4th Clinical Microbiology Open (CMO) took place in Carlsbad, California, on 10 and 11 February 2023. This event facilitated discussion between clinical and public health laboratory directors, government agencies, and industry representatives from the companies that make up ASM's Corporate Council. While many topics were discussed, much of the discussion focused on pandemic preparedness. There were four major questions addressed: (i) When is the perfect the enemy of good in pandemic testing? (ii) What other types of pathogens might cause another pandemic and how would this affect laboratory response? (iii) What research is needed to better understand the effectiveness of the pandemic response? (iv) What have we learned about the utility of self and at-home testing in future pandemics? This review serves as a summary of these discussions.


Assuntos
Pandemias , Humanos , Pandemias/prevenção & controle , COVID-19/prevenção & controle , COVID-19/epidemiologia , Preparação para Pandemia
2.
J Clin Microbiol ; 61(10): e0115422, 2023 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-37791777

RESUMO

Accurate antimicrobial susceptibility testing (AST) and reporting are essential for guiding appropriate therapy for patients and direction for public health prevention and control actions. A critical feature of AST reporting is the interpretation of AST results using clinical breakpoints for reporting as susceptible, susceptible-dose dependent, intermediate, or resistant. Breakpoints are subject to continuous adjustment and updating to best reflect current clinical data. These breakpoint changes can benefit patients and public health only if adopted in a timely manner. A recent survey identified that up to 70% of College of American Pathologists (CAP)-accredited U.S. laboratories and 45% of CAP-accredited laboratories outside the U.S. use various obsolete clinical breakpoints to interpret AST results to guide patient care. The reason for the ongoing use of obsolete breakpoints is multifactorial, including barriers encountered by laboratories, commercial AST device manufacturers, standards development organizations, and regulatory bodies alike. To begin to address this important patient safety issue, CAP implemented checklist requirements for CAP-accredited laboratories to ensure up-to-date clinical breakpoint use. Furthermore, the topic was discussed at the June 2022 American Society for Microbiology Clinical Microbiology Open (CMO) with various stakeholders to identify potential solutions. This minireview summarizes the breakpoint setting process in the U.S. and highlights solutions to close the gap between breakpoint revisions and implementation in clinical and public health laboratories. Solutions discussed include clarification of data requirements and minimum inhibitory concentration only reporting for regulatory clearance of AST devices, clinical data generation to close breakpoints gaps, advocacy, education, and greater dialogue between stakeholders.


Assuntos
Antibacterianos , Laboratórios , Humanos , Estados Unidos , Antibacterianos/farmacologia , Testes de Sensibilidade Microbiana
3.
N Engl J Med ; 380(24): 2327-2340, 2019 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-31189036

RESUMO

BACKGROUND: Metagenomic next-generation sequencing (NGS) of cerebrospinal fluid (CSF) has the potential to identify a broad range of pathogens in a single test. METHODS: In a 1-year, multicenter, prospective study, we investigated the usefulness of metagenomic NGS of CSF for the diagnosis of infectious meningitis and encephalitis in hospitalized patients. All positive tests for pathogens on metagenomic NGS were confirmed by orthogonal laboratory testing. Physician feedback was elicited by teleconferences with a clinical microbial sequencing board and by surveys. Clinical effect was evaluated by retrospective chart review. RESULTS: We enrolled 204 pediatric and adult patients at eight hospitals. Patients were severely ill: 48.5% had been admitted to the intensive care unit, and the 30-day mortality among all study patients was 11.3%. A total of 58 infections of the nervous system were diagnosed in 57 patients (27.9%). Among these 58 infections, metagenomic NGS identified 13 (22%) that were not identified by clinical testing at the source hospital. Among the remaining 45 infections (78%), metagenomic NGS made concurrent diagnoses in 19. Of the 26 infections not identified by metagenomic NGS, 11 were diagnosed by serologic testing only, 7 were diagnosed from tissue samples other than CSF, and 8 were negative on metagenomic NGS owing to low titers of pathogens in CSF. A total of 8 of 13 diagnoses made solely by metagenomic NGS had a likely clinical effect, with 7 of 13 guiding treatment. CONCLUSIONS: Routine microbiologic testing is often insufficient to detect all neuroinvasive pathogens. In this study, metagenomic NGS of CSF obtained from patients with meningitis or encephalitis improved diagnosis of neurologic infections and provided actionable information in some cases. (Funded by the National Institutes of Health and others; PDAID ClinicalTrials.gov number, NCT02910037.).


Assuntos
Líquido Cefalorraquidiano/microbiologia , Encefalite/microbiologia , Genoma Microbiano , Meningite/microbiologia , Metagenômica , Adolescente , Adulto , Líquido Cefalorraquidiano/virologia , Criança , Pré-Escolar , Encefalite/diagnóstico , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Infecções/diagnóstico , Tempo de Internação , Masculino , Meningite/diagnóstico , Meningoencefalite/diagnóstico , Meningoencefalite/microbiologia , Pessoa de Meia-Idade , Mielite/diagnóstico , Mielite/microbiologia , Estudos Prospectivos , Análise de Sequência de DNA , Análise de Sequência de RNA , Adulto Jovem
4.
Genome Res ; 29(5): 831-842, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30992304

RESUMO

Metagenomic next-generation sequencing (mNGS) for pan-pathogen detection has been successfully tested in proof-of-concept case studies in patients with acute illness of unknown etiology but to date has been largely confined to research settings. Here, we developed and validated a clinical mNGS assay for diagnosis of infectious causes of meningitis and encephalitis from cerebrospinal fluid (CSF) in a licensed microbiology laboratory. A customized bioinformatics pipeline, SURPI+, was developed to rapidly analyze mNGS data, generate an automated summary of detected pathogens, and provide a graphical user interface for evaluating and interpreting results. We established quality metrics, threshold values, and limits of detection of 0.2-313 genomic copies or colony forming units per milliliter for each representative organism type. Gross hemolysis and excess host nucleic acid reduced assay sensitivity; however, spiked phages used as internal controls were reliable indicators of sensitivity loss. Diagnostic test accuracy was evaluated by blinded mNGS testing of 95 patient samples, revealing 73% sensitivity and 99% specificity compared to original clinical test results, and 81% positive percent agreement and 99% negative percent agreement after discrepancy analysis. Subsequent mNGS challenge testing of 20 positive CSF samples prospectively collected from a cohort of pediatric patients hospitalized with meningitis, encephalitis, and/or myelitis showed 92% sensitivity and 96% specificity relative to conventional microbiological testing of CSF in identifying the causative pathogen. These results demonstrate the analytic performance of a laboratory-validated mNGS assay for pan-pathogen detection, to be used clinically for diagnosis of neurological infections from CSF.


Assuntos
Encefalite/diagnóstico , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Meningite Asséptica/diagnóstico , Metagenômica/métodos , Mielite/diagnóstico , Criança , Biologia Computacional , Encefalite/líquido cefalorraquidiano , Humanos , Meningite Asséptica/líquido cefalorraquidiano , Mielite/líquido cefalorraquidiano , Sensibilidade e Especificidade , Vírus/isolamento & purificação
5.
Clin Infect Dis ; 71(11): 2996-3001, 2020 12 31.
Artigo em Inglês | MEDLINE | ID: mdl-32511679

RESUMO

Diagnostic testing has played and will continue to play a major role in the coronavirus disease 2019 (COVID-19) pandemic. The ability to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in respiratory secretions is essential to determine when an individual is infected and potentially infectious to others. Viral detection is used for the identification, management, and isolation of individual patients. Viral detection is also used to determine when the virus has entered a community and how rapidly it is spreading. As communities attempt to reopen following periods of shutdown, the detection of both SARS-CoV-2 and specific antibodies that recognize the virus will become increasingly important as a means to assess infection and immunity in individuals and communities. Here, we discuss questions commonly asked by clinicians about COVID-19 diagnostic testing.


Assuntos
COVID-19 , Teste para COVID-19 , Técnicas de Laboratório Clínico , Humanos , Pandemias , SARS-CoV-2
6.
J Clin Microbiol ; 58(8)2020 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-32350048

RESUMO

Multiple laboratory-developed tests (LDTs) and commercially available assays have emerged to meet diagnostic needs related to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. To date, there is limited comparison data for these different testing platforms. We compared the analytical performance of a LDT developed in our clinical laboratory based on CDC primer sets and four commercially available, FDA emergency use authorized assays for SARS-CoV-2 (Cepheid, DiaSorin, Hologic Panther, and Roche Cobas) on a total of 169 nasopharyngeal swabs. The LDT and Cepheid Xpert Xpress SARS-CoV-2 assays were the most sensitive assays for SARS-CoV-2 with 100% agreement across specimens. The Hologic Panther Fusion, DiaSorin Simplexa, and Roche Cobas 6800 failed to detect positive specimens only near the limit of detection of our CDC-based LDT assay. All assays were 100% specific, using our CDC-based LDT as the gold standard. Our results provide initial test performance characteristics for SARS-CoV-2 reverse transcription-PCR (RT-PCR) and highlight the importance of having multiple viral detection testing platforms available in a public health emergency.


Assuntos
Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , Betacoronavirus/genética , COVID-19 , Teste para COVID-19 , Humanos , Nasofaringe/virologia , Pandemias , SARS-CoV-2 , Sensibilidade e Especificidade
7.
J Clin Microbiol ; 58(3)2020 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-31852767

RESUMO

Molecular testing of cerebrospinal fluid (CSF) using the BioFire FilmArray meningitis/encephalitis (FA-M/E) panel permits rapid, simultaneous pathogen detection. Due to the broad spectrum of targeted organisms, FA-M/E testing may be restricted to patients with abnormal CSF findings. We sought to determine if restriction is appropriate in our previously healthy and/or immunocompromised pediatric patients. FA-M/E was ordered on 1,025 CSF samples from 948 patients; 121 (11.8%) specimens were FA-M/E positive. Of these, 89 (73.6%) were virus positive, and 30 (24.8%) were bacterium positive. The most common targets detected were enterovirus (n = 38), human herpesvirus 6 (HHV-6) (n = 30), and Streptococcus pneumoniae (n = 14). Pleocytosis with white blood cell (WBC) levels of ≥5 cells/mm3 and ≥10 cells/mm3 were found in 33.1% and 24.3% of all specimens, respectively. Using WBC levels of ≥5 cells/mm3, 63.4% (59/93) of positive specimens exhibited pleocytosis, compared to 29.5% (233/789) of negative specimens. Among positive specimens, 54.4% (37/68) of viral and 87% (20/23) of bacterial cases had pleocytosis. The use of a pleocytosis cutoff of ≥10 cells/mm3 would have missed an additional enterovirus, one cytomegalovirus (CMV), and two HHV-6 diagnoses. CSF glucose and protein levels were normal for 83/116 (75.2%) and 51/116 (44%) positive specimens. Abnormal glucose in combination with WBC levels of ≥10 cells/mm3 showed high specificity (94.5%) and was a better predictor of FA-M/E positivity than abnormal protein. Sensitivity and positive predictive values were <90% for all biomarkers. CSF pleocytosis and abnormal glucose/protein were poor predictors of FA-M/E. Restricting FA-M/E orders based on pleocytosis or other abnormal parameters would have resulted in missed diagnostic opportunities, particularly for the detection of viruses in both previously healthy and immunocompromised patients.


Assuntos
Encefalite , Meningite , Vírus , Bactérias , Líquido Cefalorraquidiano , Criança , Encefalite/microbiologia , Encefalite/virologia , Feminino , Humanos , Masculino , Meningite/microbiologia , Meningite/virologia , Técnicas de Diagnóstico Molecular
8.
J Clin Microbiol ; 57(12)2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31462553

RESUMO

Methicillin (ß-lactam) resistance in Staphylococcus epidermidis is mediated by the mecA gene, with resistance reported to be as high as 90%. The goal of this study was to evaluate oxacillin and cefoxitin disk diffusion (DD) and broth microdilution (BMD) methods for the detection of mecA-mediated ß-lactam resistance in 100 human isolates of S. epidermidis (48 mecA-positive isolates and 52 mecA negative isolates). Oxacillin DD tests using the Clinical and Laboratory Standards Institute (CLSI) M100-S28 breakpoints for S. pseudintermedius/S. schleiferi accurately differentiated mecA-positive and -negative S. epidermidis isolates, with categorical agreement (CA) of 100% and no very major errors (VMEs) or major errors (MEs) identified. Likewise, oxacillin BMD and cefoxitin DD tests using the coagulase-negative Staphylococcus species (CoNS) breakpoints were highly reliable for detecting mecA-mediated ß-lactam resistance in S. epidermidis isolates. For cefoxitin DD and BMD results interpreted using S. aureus/S. lugdunensis breakpoints, the CA was 97.6% and 96.2%, respectively. There were 4.9% VMEs for cefoxitin DD with 0% MEs, and 3.6% VMEs and 3.9% MEs for cefoxitin BMD. Oxacillin BMD using S. aureus/S. lugdunensis breakpoints yielded the highest VMEs at 17.4% and 90% CA. Our findings demonstrate that oxacillin DD tests using the CLSI M100-S28 breakpoints for S. pseudintermedius/S. schleiferi and oxacillin BMD and cefoxitin DD tests using the CoNS breakpoints reliably identified mecA-mediated ß-lactam resistance in S. epidermidis Using mecA PCR as the gold standard, the PBP2a SA culture colony test (Abbott Diagnostics) exhibited 100% sensitivity and specificity whereas 2 false negatives were identified using the PBP2' latex agglutination test kit (Thermo Fisher Scientific) with sensitivity and specificity of 95.8% and 100%, respectively.


Assuntos
Antibacterianos/farmacologia , Cefoxitina/farmacologia , Testes de Sensibilidade Microbiana/métodos , Oxacilina/farmacologia , Staphylococcus epidermidis/efeitos dos fármacos , Resistência beta-Lactâmica , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Humanos , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade , Infecções Estafilocócicas/microbiologia , Staphylococcus epidermidis/enzimologia , Staphylococcus epidermidis/isolamento & purificação
9.
J Clin Microbiol ; 57(9)2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31315955

RESUMO

We applied metagenomic next-generation sequencing (mNGS) to detect Zaire Ebola virus (EBOV) and other potential pathogens from whole-blood samples from 70 patients with suspected Ebola hemorrhagic fever during a 2014 outbreak in Boende, Democratic Republic of the Congo (DRC) and correlated these findings with clinical symptoms. Twenty of 31 patients (64.5%) tested in Kinshasa, DRC, were EBOV positive by quantitative reverse transcriptase PCR (qRT-PCR). Despite partial degradation of sample RNA during shipping and handling, mNGS followed by EBOV-specific capture probe enrichment in a U.S. genomics laboratory identified EBOV reads in 22 of 70 samples (31.4%) versus in 21 of 70 (30.0%) EBOV-positive samples by repeat qRT-PCR (overall concordance = 87.1%). Reads from Plasmodium falciparum (malaria) were detected in 21 patients, of which at least 9 (42.9%) were coinfected with EBOV. Other positive viral detections included hepatitis B virus (n = 2), human pegivirus 1 (n = 2), Epstein-Barr virus (n = 9), and Orungo virus (n = 1), a virus in the Reoviridae family. The patient with Orungo virus infection presented with an acute febrile illness and died rapidly from massive hemorrhage and dehydration. Although the patient's blood sample was negative by EBOV qRT-PCR testing, identification of viral reads by mNGS confirmed the presence of EBOV coinfection. In total, 9 new EBOV genomes (3 complete genomes, and an additional 6 ≥50% complete) were assembled. Relaxed molecular clock phylogenetic analysis demonstrated a molecular evolutionary rate for the Boende strain 4 to 10× slower than that of other Ebola lineages. These results demonstrate the utility of mNGS in broad-based pathogen detection and outbreak surveillance.


Assuntos
Coinfecção/epidemiologia , Surtos de Doenças , Ebolavirus/classificação , Doença pelo Vírus Ebola/epidemiologia , Doença pelo Vírus Ebola/virologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Metagenômica/métodos , Adulto , Coinfecção/parasitologia , Coinfecção/patologia , Coinfecção/virologia , República Democrática do Congo/epidemiologia , Ebolavirus/genética , Ebolavirus/isolamento & purificação , Feminino , Doença pelo Vírus Ebola/parasitologia , Doença pelo Vírus Ebola/patologia , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Adulto Jovem
10.
J Clin Microbiol ; 56(5)2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29540454

RESUMO

Early establishment of infectious processes allows for expedited clinical management of meningitis and encephalitis. The FilmArray meningitis/encephalitis (FA-M/E) panel provides rapid detection of potential pathogens associated with encephalitis/meningitis in both immunocompetent and compromised patients. Here, we conducted a 1-year review of the performance of the FA-M/E panel at a tertiary care children's hospital. Two hundred sixty-five samples from 251 patients were tested. We found 87.25% (219/251) were negative, 9.96% (25/251) were positive for viral analytes, and 3.19% (8/251) were positive for bacterial analytes. When possible, positive results were confirmed by alternate testing; 4/6 available bacterial positives and 17/20 available viral positives were confirmed by retrospective culture or molecular testing.


Assuntos
Encefalite/diagnóstico , Meningite/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Bactérias/isolamento & purificação , Líquido Cefalorraquidiano/microbiologia , Líquido Cefalorraquidiano/virologia , Criança , Encefalite/líquido cefalorraquidiano , Humanos , Los Angeles , Meningite/líquido cefalorraquidiano , Avaliação de Programas e Projetos de Saúde , Kit de Reagentes para Diagnóstico , Reprodutibilidade dos Testes , Estudos Retrospectivos , Atenção Terciária à Saúde , Vírus/isolamento & purificação
11.
Clin Infect Dis ; 65(9): 1477-1485, 2017 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-29020199

RESUMO

BACKGROUND: Twelve percent of all acute liver failure (ALF) cases are of unknown origin, often termed indeterminate. A previously unrecognized hepatotropic virus has been suspected as a potential etiologic agent. METHODS: We compared the performance of metagenomic next-generation sequencing (mNGS) with confirmatory nucleic acid testing (NAT) to routine clinical diagnostic testing in detection of known or novel viruses associated with ALF. Serum samples from 204 adult ALF patients collected from 1998 to 2010 as part of a nationwide registry were analyzed. One hundred eighty-seven patients (92%) were classified as indeterminate, while the remaining 17 patients (8%) served as controls, with infections by either hepatitis A virus or hepatitis B virus (HBV), or a noninfectious cause for their ALF. RESULTS: Eight cases of infection from previously unrecognized viral pathogens were detected by mNGS (4 cases of herpes simplex virus type 1, including 1 case of coinfection with HBV, and 1 case each of HBV, parvovirus B19, cytomegalovirus, and human herpesvirus 7). Several missed dual or triple infections were also identified, and assembled viral genomes provided additional information on genotyping and drug resistance mutations. Importantly, no sequences corresponding to novel viruses were detected. CONCLUSIONS: These results suggest that ALF patients should be screened for the presence of uncommon viruses and coinfections, and that most cases of indeterminate ALF in the United States do not appear to be caused by novel viral pathogens. In the future, mNGS testing may be useful for comprehensive diagnosis of viruses associated with ALF, or to exclude infectious etiologies.


Assuntos
Genoma Viral/genética , Vírus de Hepatite/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Falência Hepática Aguda/virologia , Metagenômica/métodos , Adolescente , Adulto , DNA Viral/genética , Feminino , Hepatite Viral Humana/diagnóstico , Hepatite Viral Humana/genética , Humanos , Masculino , Pessoa de Meia-Idade , RNA Viral/genética , Estudos Retrospectivos , Adulto Jovem
13.
N Engl J Med ; 370(25): 2408-17, 2014 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-24896819

RESUMO

A 14-year-old boy with severe combined immunodeficiency presented three times to a medical facility over a period of 4 months with fever and headache that progressed to hydrocephalus and status epilepticus necessitating a medically induced coma. Diagnostic workup including brain biopsy was unrevealing. Unbiased next-generation sequencing of the cerebrospinal fluid identified 475 of 3,063,784 sequence reads (0.016%) corresponding to leptospira infection. Clinical assays for leptospirosis were negative. Targeted antimicrobial agents were administered, and the patient was discharged home 32 days later with a status close to his premorbid condition. Polymerase-chain-reaction (PCR) and serologic testing at the Centers for Disease Control and Prevention (CDC) subsequently confirmed evidence of Leptospira santarosai infection.


Assuntos
Encéfalo/patologia , Líquido Cefalorraquidiano/microbiologia , DNA Bacteriano/análise , Leptospira/genética , Leptospirose/diagnóstico , Meningoencefalite/diagnóstico , Análise de Sequência de DNA/métodos , Adenosina Desaminase/deficiência , Adolescente , Agamaglobulinemia/complicações , Biópsia , Febre/etiologia , Cefaleia/etiologia , Humanos , Leptospira/isolamento & purificação , Leptospirose/complicações , Leptospirose/microbiologia , Masculino , Meningoencefalite/complicações , Meningoencefalite/microbiologia , Imunodeficiência Combinada Severa/complicações
14.
Genome Res ; 24(7): 1180-92, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24899342

RESUMO

Unbiased next-generation sequencing (NGS) approaches enable comprehensive pathogen detection in the clinical microbiology laboratory and have numerous applications for public health surveillance, outbreak investigation, and the diagnosis of infectious diseases. However, practical deployment of the technology is hindered by the bioinformatics challenge of analyzing results accurately and in a clinically relevant timeframe. Here we describe SURPI ("sequence-based ultrarapid pathogen identification"), a computational pipeline for pathogen identification from complex metagenomic NGS data generated from clinical samples, and demonstrate use of the pipeline in the analysis of 237 clinical samples comprising more than 1.1 billion sequences. Deployable on both cloud-based and standalone servers, SURPI leverages two state-of-the-art aligners for accelerated analyses, SNAP and RAPSearch, which are as accurate as existing bioinformatics tools but orders of magnitude faster in performance. In fast mode, SURPI detects viruses and bacteria by scanning data sets of 7-500 million reads in 11 min to 5 h, while in comprehensive mode, all known microorganisms are identified, followed by de novo assembly and protein homology searches for divergent viruses in 50 min to 16 h. SURPI has also directly contributed to real-time microbial diagnosis in acutely ill patients, underscoring its potential key role in the development of unbiased NGS-based clinical assays in infectious diseases that demand rapid turnaround times.


Assuntos
Biologia Computacional/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Metagenômica/métodos , Bases de Dados de Ácidos Nucleicos , Humanos , Curva ROC , Reprodutibilidade dos Testes , Software
15.
PLoS Pathog ; 11(12): e1005325, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26658760

RESUMO

Hepatitis C virus (HCV) and human pegivirus (HPgV), formerly GBV-C, are the only known human viruses in the Hepacivirus and Pegivirus genera, respectively, of the family Flaviviridae. We present the discovery of a second pegivirus, provisionally designated human pegivirus 2 (HPgV-2), by next-generation sequencing of plasma from an HCV-infected patient with multiple bloodborne exposures who died from sepsis of unknown etiology. HPgV-2 is highly divergent, situated on a deep phylogenetic branch in a clade that includes rodent and bat pegiviruses, with which it shares <32% amino acid identity. Molecular and serological tools were developed and validated for high-throughput screening of plasma samples, and a panel of 3 independent serological markers strongly correlated antibody responses with viral RNA positivity (99.9% negative predictive value). Discovery of 11 additional RNA-positive samples from a total of 2440 screened (0.45%) revealed 93-94% nucleotide identity between HPgV-2 strains. All 12 HPgV-2 RNA-positive cases were identified in individuals also testing positive for HCV RNA (12 of 983; 1.22%), including 2 samples co-infected with HIV, but HPgV-2 RNA was not detected in non-HCV-infected individuals (p<0.0001), including those singly infected by HIV (p = 0.0075) or HBV (p = 0.0077), nor in volunteer blood donors (p = 0.0082). Nine of the 12 (75%) HPgV-2 RNA positive samples were reactive for antibodies to viral serologic markers, whereas only 28 of 2,429 (1.15%) HPgV-2 RNA negative samples were seropositive. Longitudinal sampling in two individuals revealed that active HPgV-2 infection can persist in blood for at least 7 weeks, despite the presence of virus-specific antibodies. One individual harboring both HPgV-2 and HCV RNA was found to be seronegative for both viruses, suggesting a high likelihood of simultaneous acquisition of HCV and HPgV-2 infection from an acute co-transmission event. Taken together, our results indicate that HPgV-2 is a novel bloodborne infectious virus of humans and likely transmitted via the parenteral route.


Assuntos
Infecções por Flaviviridae/virologia , Vírus GB C/genética , Hepacivirus/genética , Hepatite C/virologia , Hepatite Viral Humana/virologia , Sequência de Bases , Coinfecção/genética , Coinfecção/virologia , Feminino , Infecções por Flaviviridae/genética , Hepatite C/genética , Hepatite Viral Humana/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
16.
Nucleic Acids Res ; 43(7): e46, 2015 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-25586223

RESUMO

Next-generation sequencing (NGS) approaches rapidly produce millions to billions of short reads, which allow pathogen detection and discovery in human clinical, animal and environmental samples. A major limitation of sequence homology-based identification for highly divergent microorganisms is the short length of reads generated by most highly parallel sequencing technologies. Short reads require a high level of sequence similarities to annotated genes to confidently predict gene function or homology. Such recognition of highly divergent homologues can be improved by reference-free (de novo) assembly of short overlapping sequence reads into larger contigs. We describe an ensemble strategy that integrates the sequential use of various de Bruijn graph and overlap-layout-consensus assemblers with a novel partitioned sub-assembly approach. We also proposed new quality metrics that are suitable for evaluating metagenome de novo assembly. We demonstrate that this new ensemble strategy tested using in silico spike-in, clinical and environmental NGS datasets achieved significantly better contigs than current approaches.


Assuntos
Bactérias/genética , Metagenômica , Análise de Sequência/métodos , Vírus/genética , Genoma Bacteriano , Genoma Viral , Humanos
17.
Emerg Infect Dis ; 22(10): 1788-92, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27448188

RESUMO

Sequencing of isolates from patients in Bahia, Brazil, where most Zika virus cases in Brazil have been reported, resulted in 11 whole and partial Zika virus genomes. Phylogenetic analyses revealed a well-supported Bahia-specific Zika virus lineage, which indicates sustained Zika virus circulation in Salvador, Bahia's capital city, since mid-2014.


Assuntos
Infecção por Zika virus/virologia , Zika virus/classificação , Adulto , Idoso , Brasil/epidemiologia , DNA Viral , Feminino , Genoma Viral , Humanos , Masculino , Tipagem Molecular , Filogenia , Análise de Sequência de DNA , Zika virus/genética , Infecção por Zika virus/epidemiologia
18.
J Clin Microbiol ; 54(9): 2348-53, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27413190

RESUMO

Metagenomic next-generation sequencing (mNGS) of samples from 15 patients with documented Zika virus (ZIKV) infection in Bahia, Brazil, from April 2015 to January 2016 identified coinfections with chikungunya virus (CHIKV) in 2 of 15 ZIKV-positive cases by PCR (13.3%). While generally nonspecific, the clinical presentation corresponding to these two CHIKV/ZIKV coinfections reflected infection by the virus present at a higher titer. Aside from CHIKV and ZIKV, coinfections of other viral pathogens were not detected. The mNGS approach is promising for differential diagnosis of acute febrile illness and identification of coinfections, although targeted arbovirus screening may be sufficient in the current ZIKV outbreak setting.


Assuntos
Febre de Chikungunya/epidemiologia , Vírus Chikungunya/isolamento & purificação , Coinfecção/epidemiologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Metagenômica/métodos , Infecção por Zika virus/epidemiologia , Zika virus/isolamento & purificação , Adulto , Brasil/epidemiologia , Febre de Chikungunya/diagnóstico , Febre de Chikungunya/virologia , Vírus Chikungunya/genética , Coinfecção/diagnóstico , Coinfecção/virologia , Feminino , Humanos , Pessoa de Meia-Idade , Técnicas de Diagnóstico Molecular/métodos , Zika virus/genética , Infecção por Zika virus/diagnóstico , Infecção por Zika virus/virologia
19.
Transfusion ; 56(6 Pt 2): 1560-8, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27079968

RESUMO

BACKGROUND: Blood donation screening for human immunodeficiency virus Type 2 (HIV-2) has been in place in the United States since 1992. However, only three HIV-2 antibody-positive donors have been reported to date, all detected via HIV-1 cross-reactivity. STUDY DESIGN AND METHODS: Here we identify two additional HIV-2-positive donors by routine anti-HIV-1 and anti-HIV-2 screening, including a first-time male donor living in Georgia having recently immigrated to the United States from West Africa (from a 1998 donation) and a Taiwanese female repeat donor (nurse) living in California with no travel outside of Taiwan or apparent connections to West Africa (from a 2015 donation). Neither donor acknowledged any risk factors, and both remained asymptomatic through follow-up. The second donor was further investigated by serologic, molecular, and genomic assays because of her unusual demographics. She was documented to harbor HIV-2 RNA, albeit sporadically by HIV-2-specific nucleic acid tests (35%-100% of replicates) and at very low levels (<9.6 IU/mL). Metagenomic next-generation sequencing (mNGS) confirmed the identification of a Group B HIV-2 strain, with recovered reads covering 46.9% of the predicted genome. CONCLUSIONS: The estimated frequency of an HIV-2-positive blood donor in the United States is one in 57 million donations. Due to the low frequency and low pathogenicity of HIV-2, public health and blood donation screening efforts must focus on HIV-1 detection and prevention. However, detection of HIV-2 infection in a donor with no apparent link to West Africa suggests that the United States must remain vigilant for HIV-2 virus infections. Ultradeep mNGS may be useful in the future for comprehensive identification of rare transfusion-transmissible agents.


Assuntos
Doadores de Sangue , HIV-2/imunologia , Reação Transfusional , Adulto , África Ocidental/etnologia , Feminino , Anticorpos Anti-HIV/sangue , Infecções por HIV/diagnóstico , Infecções por HIV/transmissão , HIV-1/patogenicidade , HIV-2/genética , HIV-2/patogenicidade , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Risco , Taiwan/etnologia , Estados Unidos/epidemiologia
20.
Clin Infect Dis ; 60(6): 919-23, 2015 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-25572898

RESUMO

Metagenomic next-generation sequencing (NGS) was used to diagnose an unusual and fatal case of progressive encephalitis in an immunocompromised adult presenting at disease onset as bilateral hearing loss. The sequencing and confirmatory studies revealed neuroinvasive infection of the brain by an astrovirus belonging to a recently discovered VA/HMO clade.


Assuntos
Infecções por Astroviridae/diagnóstico , Encefalite Viral/diagnóstico , Mamastrovirus/genética , Mamastrovirus/isolamento & purificação , Adulto , Encéfalo/patologia , Proteínas do Capsídeo/genética , Encefalite Viral/complicações , Evolução Fatal , Perda Auditiva Bilateral/etiologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Leucemia Linfoide/complicações , Filogenia
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