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1.
Microbiol Spectr ; 10(6): e0280822, 2022 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-36374034

RESUMO

The incidence of tickborne infections in the United States has risen significantly. Automation is needed for the increasing demand for testing. The Panther Fusion (Fusion) has an Open Access functionality to perform lab developed tests (LDTs) on a fully automated system. Our laboratory adapted two LDTs on Fusion; a multiplex real-time PCR for Anaplasma phagocytophilum and Ehrlichia chaffeensis (AP/EC) and a Babesia microti (BM) PCR. Limits of detection (LODs) were performed with target region plasmid panels spiked into whole blood. The LODs for AP, BM, and EC on the Fusion were 11, 17, and 10 copies/reaction, respectively. The performance of AP/EC was evaluated with 80 whole blood specimens, including 50 specimens previously positive for AP by our test of record (TOR) and 30 specimens (including 20 AP positive) spiked with EC plasmid. AP was detected in 49 out of 50 positive specimens and EC was detected in all 30 spiked specimens. BM PCR on Fusion was evaluated with 75 whole blood samples, including 16 specimens previously shown to be positive for BM and 59 negative specimens, of which 29 were spiked with BM plasmid DNA. BM was detected in 45 samples as expected. AP/EC and BM PCRs were successfully developed and optimized on the Panther Fusion with performance characteristics comparable to our TOR. These assays complement each other and allow for a modular testing approach for tickborne diseases which have differing clinical presentation. Furthermore, automation of these assays will help the lab meet the increasing demand for testing. IMPORTANCE Since the incidence of tickborne diseases has been accelerating in the United States, automation for testing has become essential in affected regions. Unfortunately, because the need is regional, commercial test manufacturers have not yet provided answers for clinical laboratories. Here, we describe the development of PCR tests on the highly automated Panther Fusion for three tickborne diseases. The Panther Fusion assays were evaluated using 155 archived whole blood (WB) specimens previously tested for Anaplasma phagocytophilum, Ehrlichia chaffeensis, and Babesia microti, while WB spiked with DNA from plasmid clones of the target regions were used for analytical sensitivity. We demonstrated that the Panther Fusion assays performed similar to the manual PCR tests used clinically in our laboratory and that automation of these tests had no adverse effect on the performance.


Assuntos
Anaplasma phagocytophilum , Doenças Transmitidas por Carrapatos , Humanos , Reação em Cadeia da Polimerase em Tempo Real , Acesso à Informação , Anaplasma phagocytophilum/genética , Técnicas de Amplificação de Ácido Nucleico , Limite de Detecção , Doenças Transmitidas por Carrapatos/diagnóstico
2.
J Clin Virol ; 141: 104879, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34153860

RESUMO

Highly sensitive nucleic acid amplification tests (NAATs) designed to detect SARS-CoV-2 RNA are the standard of care for the diagnosis of COVID-19. However, the accuracy of these methods for the quantitation of active virus rather than non-infectious RNA fragments that can persist for extended periods of time has been unclear. This issue is particularly relevant for congregate care patients who are unable to return to their home residence until fully negative by NAATs. We tested paired samples from individual patients for the presence of virus at both early and later stages of disease. Culture of nasopharyngeal swab samples for 10 days in Vero E6 cells revealed active virus in only 4 out of 14 (28.6%) patients. The ability to isolate viral plaque-forming units (PFU) correlated with viral RNA loads of >6.79 log genomic copies/ml and only occurred in samples collected from patients early after symptom onset and before development of antibody. Culture in Vero E6 cells lacking the STAT1-dependent interferon signaling pathway increased the numbers of viral PFU detected but did not affect the incidence of positive cultures. We conclude that culturable virus is correlated with SARS-CoV-2 NAATs detection only during early symptom onset and with high viral titers/low antibody titers in non-immunosuppressed patients.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Nasofaringe , Reação em Cadeia da Polimerase , RNA Viral/genética
3.
J Clin Microbiol ; 58(6)2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32229600

RESUMO

Nucleic acid amplification tests, such as PCR, are the method of choice for respiratory virus testing, due to their superior diagnostic accuracy and fast turnaround time. The Panther Fusion (Fusion; Hologic) system has an array of highly sensitive in vitro diagnostic (IVD) real-time PCR assays for respiratory viruses, including an assay for influenza A (FluA) virus, influenza B (FluB) virus, and respiratory syncytial virus (RSV) (FFABR assay). The Fusion system has Open Access functionality to perform laboratory-developed tests (LDTs) alongside IVD assays. We developed two LDTs for FluA virus strain typing on the Panther Fusion instrument, enabling side-by-side testing with the FFABR assay. The LDT-FAST assay uses proprietary primers and probes designed by Hologic for the Prodesse ProFAST+ (PFAST) assay. The exWHO-FAST assay is an expanded redesign of the WHO-recommended reverse transcriptase PCRs (RT-PCRs). To evaluate the performance of these two LDTs, 110 FluA virus-positive samples were tested. Of these, 104 had been subtyped previously; 54 were H3, 46 were 09H1, and 4 were fsH1. All were appropriately subtyped by both LDTs. Of the untyped FluA virus samples, three were subtyped as H3 by both LDTs and two were subtyped as H3 by the LDT-FAST assay only. The sample not subtyped by either LDT was retested with the FFABR assay and was now negative. Limit-of-detection (LOD) analyses were performed with five FluA virus strains. The LDT-FAST LODs were similar to the FFABR assay LODs, while the exWHO-FAST LODs were higher for two H3N2 strains, findings that were explained by analysis of primer/probe homology. In conclusion, either FluA virus typing assay would be a valuable complement to the Panther Fusion respiratory menu given the performance of these LDTs, the system's full automation, and the ability to split eluates for both IVD and LDT testing.


Assuntos
Vírus da Influenza A , Influenza Humana , Acesso à Informação , Humanos , Vírus da Influenza A Subtipo H3N2 , Vírus da Influenza A/genética , Vírus da Influenza B/genética , Influenza Humana/diagnóstico , Laboratórios , Sensibilidade e Especificidade
4.
J Clin Virol ; 124: 104262, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32007841

RESUMO

BACKGROUND: In 2014, enterovirus D68 (EV-D68) was responsible for an outbreak of severe respiratory illness in children, with 1,153 EV-D68 cases reported across 49 states. Despite this, there is no commercial assay for its detection in routine clinical care. BioFire® Syndromic Trends (Trend) is an epidemiological network that collects, in near real-time, deidentified. BioFire test results worldwide, including data from the BioFire® Respiratory Panel (RP). OBJECTIVES: Using the RP version 1.7 (which was not explicitly designed to differentiate EV-D68 from other picornaviruses), we formulate a model, Pathogen Extended Resolution (PER), to distinguish EV-D68 from other human rhinoviruses/enteroviruses (RV/EV) tested for in the panel. Using PER in conjunction with Trend, we survey for historical evidence of EVD68 positivity and demonstrate a method for prospective real-time outbreak monitoring within the network. STUDY DESIGN: PER incorporates real-time polymerase chain reaction metrics from the RPRV/EV assays. Six institutions in the United States and Europe contributed to the model creation, providing data from 1,619 samples spanning two years, confirmed by EV-D68 gold-standard molecular methods. We estimate outbreak periods by applying PER to over 600,000 historical Trend RP tests since 2014. Additionally, we used PER as a prospective monitoring tool during the 2018 outbreak. RESULTS: The final PER algorithm demonstrated an overall sensitivity and specificity of 87.1% and 86.1%, respectively, among the gold-standard dataset. During the 2018 outbreak monitoring period, PER alerted the research network of EV-D68 emergence in July. One of the first sites to experience a significant increase, Nationwide Children's Hospital, confirmed the outbreak and implemented EV-D68 testing at the institution in response. Applying PER to the historical Trend dataset to determine rates among RP tests, we find three potential outbreaks with predicted regional EV-D68 rates as high as 37% in 2014, 16% in 2016, and 29% in 2018. CONCLUSIONS: Using PER within the Trend network was shown to both accurately predict outbreaks of EV-D68 and to provide timely notifications of its circulation to participating clinical laboratories.


Assuntos
Surtos de Doenças , Enterovirus Humano D , Infecções por Enterovirus/diagnóstico , Infecções por Enterovirus/epidemiologia , Infecções Respiratórias/diagnóstico , Infecções Respiratórias/epidemiologia , Algoritmos , Criança , Infecções por Enterovirus/virologia , Monitoramento Epidemiológico , Europa (Continente)/epidemiologia , Humanos , Infecções Respiratórias/virologia , Sensibilidade e Especificidade , Estados Unidos/epidemiologia
5.
J Clin Virol ; 121: 104204, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31743836

RESUMO

BACKGROUND: Nucleic acid amplification tests (NAATs), such as PCR, are preferred for respiratory virus testing, due to superior diagnostic accuracy and faster turnaround time. Panther Fusion® Respiratory Assays (Fusion), which includes FluA/B/RSV (FFABR), Paraflu and AdV/hMPV/RV, offers a modular approach to syndromic testing on a fully automated platform while improving gene targets and expanding the test menu. OBJECTIVES AND STUDY DESIGN: We evaluated Fusion using 275 consecutive nasopharyngeal specimens previously used in an analysis of five PCRs, as well as 225 archived specimens. RESULTS: Of the combined 500 specimens, 134 were positive for influenza A (FluA), 54 for FluB, 65 for RSV, 64 for parainfluenza (PIV), 24 for adenovirus (AdV), 21 for humanmetapneumovirus (hMPV), and 40 for rhinovirus (RV) with Fusion. Of the positive samples Fusion correlated with historical results for all but one, despite multiple freeze-thaws cycles of this collection. Fusion was positive for an additional 33 samples, including 11 FluAs, 7 RSVs, 3 PIV3s, 3 AdV, 6 hMPV and 3 RVs. These samples were retested with corresponding Prodesse (Pro) assays using quadruple sample volume. This resolver test confirmed Fusion results for an additional 4 FluAs, 4 RSVs, 1 PIV3 and 3 AdVs. The sensitivity and specificity ranges of Fusion were 99-100% and 98-100%. Limit of detection (LOD) analyses were performed on a variety of Flu isolates. The LODs ranged from 2.69 to 2.99 log copies/ml and demonstrated superior LOD as compared to previously published data for some assays or to concurrent analyses with two new commercial tests.


Assuntos
Técnicas de Amplificação de Ácido Nucleico/métodos , Infecções Respiratórias/diagnóstico , Viroses/diagnóstico , Vírus/isolamento & purificação , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Automação Laboratorial , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Limite de Detecção , Masculino , Pessoa de Meia-Idade , Nasofaringe/virologia , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Infecções Respiratórias/virologia , Sensibilidade e Especificidade , Viroses/virologia , Adulto Jovem
6.
J Clin Virol ; 107: 11-18, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30103162

RESUMO

BACKGROUND: Emerging influenza A/H3N2 clades have been associated with M1 gene mutations which affect the performance of commercial PCR assays. OBJECTIVES AND STUDY DESIGN: The evolution and prevalence of problematic M1 mutations, and their associated viral clades, were investigated. All European and USA isolates from the GISAID database with both HA and M1 sequences available, collected during the respiratory seasons from the Fall of 2007 through January of 2018, were analyzed. RESULTS: Five M1 target region patterns, designated A-E, were observed in more than 10% of the isolates during a season, with patterns that appeared sequentially, each having one additional mutation. The C153T mutation was universal. Pattern A, which only had the single mutation, predominated between 2007/08 and 2009/10. Dual- and triple-mutation patterns (B and C) emerged in 2010/11 and 2011/12 respectively, and pattern C predominated for one season (2012/13). In 2012/13, the problematic quadruple-mutation containing C163T first appeared in 3C.2 viruses. Seasons 2013/14 and 14/15 were associated with significant viral diversity with five clades and four M1 patterns co-circulating, with different rates in Europe and the USA. Since 2014, clade 3C.2a with M1 pattern D has emerged as the predominant type. During 2016/17 season, a new quintuplet mutation pattern (E) emerged in cluster 3C.2a1 isolates. CONCLUSIONS: M1 target region mutations have been prevalent for more than ten years, with the number of mutations continually increasing. Often population inferences of M1 mutations can be made based on viral clade. However, gene segment reassortment can affect predictive abilities.


Assuntos
Vírus da Influenza A Subtipo H3N2/genética , Influenza Humana/virologia , Mutação , Proteínas da Matriz Viral/genética , Europa (Continente) , Evolução Molecular , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Influenza Humana/diagnóstico , Filogenia , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Fatores de Tempo , Estados Unidos
7.
JMIR Public Health Surveill ; 4(3): e59, 2018 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-29980501

RESUMO

BACKGROUND: Health care and public health professionals rely on accurate, real-time monitoring of infectious diseases for outbreak preparedness and response. Early detection of outbreaks is improved by systems that are comprehensive and specific with respect to the pathogen but are rapid in reporting the data. It has proven difficult to implement these requirements on a large scale while maintaining patient privacy. OBJECTIVE: The aim of this study was to demonstrate the automated export, aggregation, and analysis of infectious disease diagnostic test results from clinical laboratories across the United States in a manner that protects patient confidentiality. We hypothesized that such a system could aid in monitoring the seasonal occurrence of respiratory pathogens and may have advantages with regard to scope and ease of reporting compared with existing surveillance systems. METHODS: We describe a system, BioFire Syndromic Trends, for rapid disease reporting that is syndrome-based but pathogen-specific. Deidentified patient test results from the BioFire FilmArray multiplex molecular diagnostic system are sent directly to a cloud database. Summaries of these data are displayed in near real time on the Syndromic Trends public website. We studied this dataset for the prevalence, seasonality, and coinfections of the 20 respiratory pathogens detected in over 362,000 patient samples acquired as a standard-of-care testing over the last 4 years from 20 clinical laboratories in the United States. RESULTS: The majority of pathogens show influenza-like seasonality, rhinovirus has fall and spring peaks, and adenovirus and the bacterial pathogens show constant detection over the year. The dataset can also be considered in an ecological framework; the viruses and bacteria detected by this test are parasites of a host (the human patient). Interestingly, the rate of pathogen codetections, on average 7.94% (28,741/362,101), matches predictions based on the relative abundance of organisms present. CONCLUSIONS: Syndromic Trends preserves patient privacy by removing or obfuscating patient identifiers while still collecting much useful information about the bacterial and viral pathogens that they harbor. Test results are uploaded to the database within a few hours of completion compared with delays of up to 10 days for other diagnostic-based reporting systems. This work shows that the barriers to establishing epidemiology systems are no longer scientific and technical but rather administrative, involving questions of patient privacy and data ownership. We have demonstrated here that these barriers can be overcome. This first look at the resulting data stream suggests that Syndromic Trends will be able to provide high-resolution analysis of circulating respiratory pathogens and may aid in the detection of new outbreaks.

8.
Diagn Microbiol Infect Dis ; 91(3): 239-244, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29550058

RESUMO

Influenza circulating during the 2014/15 season was associated with significant drift and the emergence of new H3N2 subgroups. It was also known that mutations were accumulating in the WHO-recommended amplification region of the M1 gene, affecting the performance of many commercial polymerase chain reaction assays. However, the prevalence of M1 target mutations was unknown. To investigate this, isolates from the GISAID database from Australia (n=100), Europe (n=473), and the USA (n=1175) were analyzed. The predominate clade was 3C.2a (75%), with a higher representation among USA isolates (81%). The M1 target demonstrated four primary sequence patterns, designated A-D. Pattern D was most often observed (84%). Some clades correlated with M1 target patterns, as seen between subclades 3C.2a and 3C.3a and patterns D and C, respectively. 3C.3 isolates were more diverse, with three patterns represented. Among the 3C.3b isolates, pattern B predominated in non-USA viruses, while pattern D predominated among USA isolates.


Assuntos
Reações Falso-Negativas , Vírus da Influenza A Subtipo H3N2/genética , Influenza Humana/diagnóstico , Proteínas Mutantes/genética , Mutação , Reação em Cadeia da Polimerase/métodos , Proteínas da Matriz Viral/genética , Austrália , Europa (Continente) , Genótipo , Humanos , Incidência , Vírus da Influenza A Subtipo H3N2/isolamento & purificação , Estados Unidos
9.
J Clin Microbiol ; 56(3)2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29305549

RESUMO

Influenza is associated with rapid evolution due to lack of RNA polymerase proofreading, immunogenic selection, and frequent rearrangement of gene segments. Evolutionary changes affecting the performance of diagnostic testing have long been recognized. Hence, it is not surprising that such challenges apply to nucleic acid amplification tests, even though they are designed to target highly conserved regions. Initially, case reports involved single isolates of A(H1N1)pdm09. Over the past 4 years, subtype H3N2 viruses evolved to viral clades with mutations in the WHO-recommended target region, such that almost all isolates worldwide have significantly reduced sensitivities with many commercial reverse transcription-PCR tests.


Assuntos
Deriva Genética , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H3N2/genética , Influenza Humana/diagnóstico , Técnicas de Diagnóstico Molecular/estatística & dados numéricos , Humanos , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Vírus da Influenza A Subtipo H3N2/isolamento & purificação , Técnicas de Diagnóstico Molecular/normas , Mutação , Kit de Reagentes para Diagnóstico/normas , Kit de Reagentes para Diagnóstico/estatística & dados numéricos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Reação em Cadeia da Polimerase Via Transcriptase Reversa/estatística & dados numéricos , Proteínas Virais/genética
10.
J Clin Microbiol ; 55(11): 3258-3266, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28878009

RESUMO

The purpose of this study was to perform a multisite evaluation to establish the performance characteristics of the BD Max extended enteric bacterial panel (xEBP) assay directly from unpreserved or Cary-Blair-preserved stool specimens for the detection of Yersinia enterocolitica, enterotoxigenic Escherichia coli (ETEC), Vibrio, and Plesiomonas shigelloides The study included prospective, retrospective, and prepared contrived specimens from 6 clinical sites. BD Max xEBP results were compared to the reference method, which included standard culture techniques coupled with alternate PCR and sequencing, except for ETEC, for which the reference method was two alternate PCRs and sequencing. Alternate PCR was also used to confirm the historical results for the retrospective specimens and for discrepant result analysis. A total of 2,410 unformed, deidentified stool specimens were collected. The prevalence in the prospective samples as defined by the reference method was 1.2% ETEC, 0.1% Vibrio, 0% Y. enterocolitica, and 0% P. shigelloides Compared to the reference method, the positive percent agreement (PPA) (95% confidence interval [CI]), negative percent agreement (NPA) (95% CI), and kappa coefficient (95% CI) for the BD Max xEBP assay for all specimens combined were as follows: ETEC, 97.6% (87.4 to 99.6), 99.8% (99.5 to 99.9), and 0.93 (0.87 to 0.99); Vibrio, 100% (96.4 to 100), 99.7% (99.4 to 99.8), and 0.96 (0.93 to 0.99); Y. enterocolitica, 99.0% (94.8 to 99.8), 99.9% (99.8 to 99.9), and 0.99 (0.98 to 1); P. shigelloides, 100% (96.4 to 100), 99.8% (99.5 to 99.9), and 0.98 (0.95 to 1), respectively. In this multicenter study, the BD Max xEBP showed a high correlation (kappa, 0.97; 95% CI, 0.95 to 0.98) with the conventional methods for the detection of ETEC, Vibrio, Y. enterocolitica, and P. shigelloides in stool specimens from patients suspected of acute gastroenteritis, enteritis, or colitis.


Assuntos
Automação Laboratorial/métodos , Técnicas Bacteriológicas/métodos , Diarreia/diagnóstico , Fezes/microbiologia , Bactérias Gram-Negativas/isolamento & purificação , Infecções por Bactérias Gram-Negativas/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Diarreia/microbiologia , Feminino , Bactérias Gram-Negativas/classificação , Infecções por Bactérias Gram-Negativas/microbiologia , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase/métodos , Estudos Prospectivos , Estudos Retrospectivos , Adulto Jovem
12.
J Clin Virol ; 93: 25-29, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28600949

RESUMO

BACKGROUND: Nucleic acid amplification assays have become the method of choice for influenza (Flu) testing due to superior accuracy and faster turnaround time. Although assays are designed to detect highly conserved genomic targets, mutations can influence test sensitivity. Most of the circulating viruses in the United States during the 2014-2015 season were associated with significant genetic drift; however, the effect on testing was unknown. OBJECTIVES AND STUDY DESIGN: We compared the performance of Prodesse ProFlu+/ProFAST+ (PFlu/PFAST), FilmArray Respiratory Panel (RP), cobas® Influenza A/B test (cIAB), and Xpert® Flu (Xpt) in a retrospective analysis of consecutive nasopharyngeal specimens received for a two-week period during the winter of 2015. Furthermore, limits of detection (LOD) were determined with six isolates of Flu. RESULTS: Of the 275 specimens, 63 were positive for FluA by PFAST, 60 were positive by RP, 58 were positive by cIAB and 52 were positive by Xpt. Only a subset of 135 specimens was tested by PFlu, of which 32 were positive. The sensitivity/specificity for PFAST, RP, cIAB, Xpt and PFlu was 100/99.1%, 96.7/99.5%, 91.8/99.1%, 85.2%/100%, and 75.6%/98.9%, respectively. LOD analyses demonstrated assay performance variations were strain associated. Specifically, PFlu's and cIAB's LODs were higher with A/Texas/50/2012-like and A/Switzerland/9715293/2013-like strains, while Xpt's highest LOD was with the Swiss strain. CONCLUSIONS: Strain-associated assay performance variation is known to occur with other Flu test methods; hence, it is not surprising that such variation would be observed with molecular tests. Careful monitoring and reporting for strain-associated variances are warranted for all test methods.


Assuntos
Vírus da Influenza A/genética , Vírus da Influenza B/genética , Influenza Humana/diagnóstico , Sequência de Bases , Evolução Molecular , Deriva Genética , Genoma Viral , Humanos , Influenza Humana/virologia , Limite de Detecção , Técnicas de Diagnóstico Molecular , Reação em Cadeia da Polimerase em Tempo Real , Estudos Retrospectivos , Análise de Sequência de DNA , Estados Unidos , Carga Viral
13.
Pediatrics ; 130(6): e1695-9, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23147965

RESUMO

Palivizumab is a humanized monoclonal antibody used to decrease the threat of respiratory syncytial virus (RSV) infection among children at high risk. There are no standard guidelines due to conflicting data on palivizumab's use in the treatment of RSV lower respiratory tract infections. Intravenous (IV) palivizumab was shown to be well tolerated and associated with decreased mortality in high-risk children who have RSV disease. However, it did not prevent lower respiratory tract infections and did not affect the survival rate of allogeneic stem cell transplant recipients who had RSV infection. We present 2 children with acute lymphocytic leukemia (ALL) and persistent RSV infection while receiving chemotherapy. Patient A is a 4-year-old male with Down syndrome, ALL, and persistent RSV infection for at least 3 months. Patient B is a 3-year-old female with pre-B cell ALL whose chemotherapy intensification phase was delayed due to a month-long RSV infection. RSV infections were determined by using real-time polymerase chain reaction assays from nasopharyngeal swabs before IV palivizumab therapy; patient A was positive for RSV at 36 cycles and patient B was positive for RSV at 29 cycles. RSV infection was cleared in both patients within 72 hours after receiving IV palivizumab (patient A: 16 mg/kg; patient B: 15 mg/kg). IV palivizumab may be a treatment option for persistent RSV infection among immunocompromised patients.


Assuntos
Anticorpos Monoclonais Humanizados/administração & dosagem , Antivirais/administração & dosagem , Infecções Oportunistas/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/complicações , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Pré-Escolar , Feminino , Humanos , Infusões Intravenosas , Masculino , Infecções Oportunistas/diagnóstico , Palivizumab , Reação em Cadeia da Polimerase , Leucemia-Linfoma Linfoblástico de Células Precursoras B/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Infecções por Vírus Respiratório Sincicial/diagnóstico , Prevenção Secundária , Resultado do Tratamento
14.
J Mol Diagn ; 11(2): 87-92, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19225134

RESUMO

Viralload (VL) assessment of cytomegalovirus (CMV) by real-time PCR is an important tool for diagnosing and monitoring CMV viremia in patients with compromised immune systems. We report results from a sample exchange organized by members of the Association for Molecular Pathology that compared PCR results from 23 laboratories; 22 such laboratories used a laboratory-developed real-time PCR assay and one laboratory used a competitive PCR assay. The samples sent to each laboratory were comprised of a dilution panel of CMV virion-derived reference materials that ranged from 0 to 500,000 copies/ml. Accuracy, linearity, and intralaboratory precision were established for the different laboratory-developed assays. Overall, PCR results were linear for each laboratory (R(2) > 0.97 in all but two). While 13 laboratories showed no significant quantitative assay bias, 10 laboratories reported VLs that were significantly different compared with expected values (bias range, -0.82 to 1.4 logs). The intralaboratory precision [mean coefficient of variance of 2% to 5% (log-scale)] suggested that changes in VLs of less than 3- to fivefold may not be significantly different. There was no significant association between laboratory-specific technical variables (PCR platform, calibrator, extraction method) and assay linearity or accuracy. These data suggested that, within each laboratory, relative VL values were linear, but additional method standardization and a CMV DNA reference standard are needed to allow laboratories to achieve comparable numeric results.


Assuntos
Infecções por Citomegalovirus/diagnóstico , Citomegalovirus/genética , Reação em Cadeia da Polimerase/métodos , Carga Viral/métodos , DNA/genética , DNA/isolamento & purificação , Humanos , Modelos Lineares , Reação em Cadeia da Polimerase/normas , Sensibilidade e Especificidade , Carga Viral/normas
15.
Expert Rev Mol Diagn ; 4(4): 485-93, 2004 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15225096

RESUMO

Nucleic acid amplification technologies are well-characterized methods for the rapid and sensitive diagnosis of a wide variety of infectious diseases. Herpes simplex virus amplification is no exception, demonstrating as much as a ninefold enhancement in sensitivity over viral culture in some settings. Currently, there are no US Food and Drug Administration-approved systems for herpes simplex virus amplification; hence, most laboratories utilize in-house-developed PCR-based systems. Unfortunately, the utilization of herpes simplex virus amplification has been confined to the investigations of suspected herpes simplex virus encephalitis, largely due to cost and the need for appropriately trained technical staff. Recent methodological advances will help to render herpes simplex virus nucleic acid amplification technologies more applicable to routine practice. However, the use of nucleic acid amplification technologies for the diagnosis of genital herpes infection remains highly controversial.


Assuntos
Herpes Genital/diagnóstico , Herpesvirus Humano 1/genética , Herpesvirus Humano 2/genética , Técnicas de Amplificação de Ácido Nucleico , DNA Viral/análise , Corantes Fluorescentes/metabolismo , Humanos , Controle de Qualidade , Sensibilidade e Especificidade , Testes Sorológicos , Estados Unidos , United States Food and Drug Administration
16.
J Clin Microbiol ; 42(4): 1528-33, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15070999

RESUMO

Ureaplasma, spp. Mycoplasma genitalium, and Mycoplasma hominis are associated with infection of the genitourinary tract, reproductive failure, and neonatal morbidity and mortality. We have developed a multiplex PCR for the detection of these Mycoplasmatales in a single amplification reaction. The analytical sensitivities of this assay were 10.8, 10.8, and 8.8 CFU for each organism, respectively. This multiplex PCR was compared to culture on 26 cervical swabs, 2 vaginal swabs, 4 female urine specimens, 49 semen samples, 2 male urine specimens, and 1 nonspecified sample. A total of 21 specimens were culture positive (25%); 17 of these were PCR positive. An additional 11 specimens were PCR positive but culture negative. Of the 21 culture-positive specimens, 17 (81%) grew Ureaplasma spp. and 4 (19%) grew Mycoplasma spp. Of the 28 PCR-positive specimens, Ureaplasma spp. was detected in 23 (82%), M. hominis was detected in 3 (11%), and both were detected in 2 (7%). In a confirmatory analysis, all samples were tested by amplification of a second target of the ureaplasma genome. True-positive cases were defined as a positive result by culture or by both amplification assays. The multiplex PCR detected organisms in 26 of the 30 true-positive specimens, as well as in 2 other specimens. Based on a 36% prevalence of infection, the sensitivity, specificity, and positive and negative predictive values of multiplex PCR analyses were 87, 96, 94, and 93%, respectively. Multiplex PCR offers a rapid, sensitive, and easy method to detect genital mycoplasmas.


Assuntos
Doenças dos Genitais Femininos/microbiologia , Doenças dos Genitais Masculinos/microbiologia , Mycoplasmataceae/isolamento & purificação , Infecções por Mycoplasmatales/microbiologia , Reação em Cadeia da Polimerase/métodos , Meios de Cultura , DNA Bacteriano/análise , Feminino , Humanos , Masculino , Mycoplasmataceae/classificação , Mycoplasmataceae/genética , Mycoplasmataceae/crescimento & desenvolvimento , Valor Preditivo dos Testes , Sensibilidade e Especificidade
17.
Plasmid ; 51(1): 48-53, 2004 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-14711529

RESUMO

The SHV-5 extended-spectrum beta-lactamase gene of pACM1 was previously shown to reside on a segment of DNA ( approximately 7.9 kb) homologous to part of the Klebsiella pneumoniae chromosome. Regions of pACM1 overlapping the ends of the homology were sequenced. A defective copy of IS26 was found on each side of, and immediately adjacent to, the homology. The copies were oriented as direct repeats reminiscent of the compound transposon Tn2680. Other mobile elements and a putative mutagenesis gene, several of which were also defective, were also located in the vicinity of the homology. An intact precursor to the transposon remnant might have contributed to the dissemination of the SHV-5 gene.


Assuntos
Plasmídeos/genética , beta-Lactamases/genética , Elementos de DNA Transponíveis , Klebsiella/genética , Análise de Sequência de DNA
18.
J Clin Microbiol ; 41(1): 164-73, 2003 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-12517843

RESUMO

The analytical performance of the NucliSens HIV-1 QT assay, a highly sensitive test based on nucleic acid sequence-based amplification technology, was evaluated in a multicenter trial. Assay specificity was evaluated with 502 plasma (EDTA) specimens from human immunodeficiency virus type 1 (HIV-1)-seronegative volunteer donors. No HIV-1 RNA was reported in any of the donor specimens. Analytical sensitivity and reproducibility were estimated with panels prepared from a high-titer well-characterized HIV-1 RNA stock (5.84 x 10(8) RNA copies/ml). The assay's dynamic range was linear from 10(6) to 10(1) HIV-1 RNA copies, with a lower detectable limit of 25 copies/ml and a 95% detection rate of 176 copies/ml. Sensitivity of the assay to detect HIV-1 RNA in clinical specimens from patients (n = 101) and in commercially available or prepared panels (n = 24) was compared with NASBA HIV-1 RNA QT (an earlier version of NucliSens HIV-1 QT) and with the Food and Drug Administration-approved standard and ultrasensitive AMPLICOR HIV-1 MONITOR, version 1.0, assays. Detection of HIV-1 RNA was reproducible over a 5-log range (mean standard deviation = 0.15 log). The NucliSens and the standard AMPLICOR assays were equivalent in detection of HIV-1 RNA (concentration, 10(3) to 10(5) copies/ml) in 57 clinical specimens. The NucliSens assay was more sensitive in detecting HIV-1 RNA at lower concentrations (

Assuntos
HIV-1/isolamento & purificação , RNA Viral/análise , Kit de Reagentes para Diagnóstico , HIV-1/genética , Humanos , Técnicas Microbiológicas , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
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