RESUMEN
This study aims to identify RNA biomarkers distinguishing neuromyelitis optica (NMO) from relapsing-remitting multiple sclerosis (RRMS) and explore potential therapeutic applications leveraging machine learning (ML). An ensemble approach was developed using differential gene expression analysis and competitive ML methods, interrogating total RNA-sequencing data sets from peripheral whole blood of treatment-naïve patients with RRMS and NMO and healthy individuals. Pathway analysis of candidate biomarkers informed the biological context of disease, transcription factor activity, and small-molecule therapeutic potential. ML models differentiated between patients with NMO and RRMS, with the performance of certain models exceeding 90% accuracy. RNA biomarkers driving model performance were associated with ribosomal dysfunction and viral infection. Regulatory networks of kinases and transcription factors identified biological associations and identified potential therapeutic targets. Small-molecule candidates capable of reversing perturbed gene expression were uncovered. Mitoxantrone and vorinostat-two identified small molecules with previously reported use in patients with NMO and experimental autoimmune encephalomyelitis-reinforced discovered expression signatures and highlighted the potential to identify new therapeutic candidates. Putative RNA biomarkers were identified that accurately distinguish NMO from RRMS and healthy individuals. The application of multivariate approaches in analysis of RNA-sequencing data further enhances the discovery of unique RNA biomarkers, accelerating the development of new methods for disease detection, monitoring, and therapeutics. Integrating biological understanding further enhances detection of disease-specific signatures and possible therapeutic targets.
Asunto(s)
Biomarcadores , Aprendizaje Automático , Neuromielitis Óptica , Análisis de Secuencia de ARN , Neuromielitis Óptica/genética , Neuromielitis Óptica/diagnóstico , Neuromielitis Óptica/tratamiento farmacológico , Humanos , Femenino , Biomarcadores/sangre , Análisis de Secuencia de ARN/métodos , Masculino , Mitoxantrona/uso terapéutico , Adulto , Diagnóstico Diferencial , Persona de Mediana Edad , Esclerosis Múltiple Recurrente-Remitente/genética , Esclerosis Múltiple Recurrente-Remitente/tratamiento farmacológico , Esclerosis Múltiple Recurrente-Remitente/sangre , Esclerosis Múltiple Recurrente-Remitente/diagnóstico , Perfilación de la Expresión Génica/métodos , Esclerosis Múltiple/genética , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/diagnóstico , Esclerosis Múltiple/sangreRESUMEN
Microbiological diagnosis is pivotal to the appropriate management and treatment of infective endocarditis. We evaluated PCR-electrospray ionization mass spectrometry (PCR/ESI-MS) for bacterial and candidal detection using 83 formalin-fixed paraffin-embedded heart valves from subjects with endocarditis who had positive valve and/or blood cultures, 63 of whom had positive valvular Gram stains. PCR/ESI-MS yielded 55% positivity with concordant microbiology at the genus/species or organism group level (e.g., viridans group streptococci), 11% positivity with discordant microbiology, and 34% with no detection. PCR/ESI-MS detected all antimicrobial resistance encoded by mecA or vanA/B and identified a case of Tropheryma whipplei endocarditis not previously recognized.
Asunto(s)
Bacterias/aislamiento & purificación , Candida/aislamiento & purificación , Endocarditis/diagnóstico , Válvulas Cardíacas/microbiología , Técnicas Microbiológicas/métodos , Reacción en Cadena de la Polimerasa/métodos , Espectrometría de Masa por Ionización de Electrospray/métodos , Bacterias/clasificación , Bacterias/efectos de los fármacos , Bacterias/genética , Candida/clasificación , Candida/efectos de los fármacos , Candida/genética , Farmacorresistencia Bacteriana , Farmacorresistencia Fúngica , Endocarditis/microbiología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Técnicas de Diagnóstico Molecular/métodosRESUMEN
The Bruker Biotyper and Vitek MS matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) instruments were evaluated for the identification of nonfermenting gram-negative bacilli (NFGNB) by a blinded comparison to conventional biochemical or molecular methods. Two hundred NFGNB that were recovered from cultures from cystic fibrosis patients in the University of Iowa Health Care (UIHC) Microbiology Laboratory between 1 January 2006 and 31 October 2010 were sent to Mayo Clinic for analysis with the Bruker Biotyper (software version 3.0) and to bioMérieux for testing with Vitek MS (SARAMIS database version 3.62). If two attempts at direct colony testing failed to provide an acceptable MALDI-TOF identification, an extraction procedure was performed. The MS identifications from both of these systems were provided to UIHC for comparison to the biochemical or molecular identification that had been reported in the patient record. Isolates with discordant results were analyzed by 16S rRNA gene sequencing at UIHC. After discrepancy testing, the Bruker Biotyper result agreed with the biochemical or molecular method, with 72.5% of isolates to the species level, 5.5% to the complex level, and 19% to the genus level (3% not identified). The level of agreement for Vitek MS was 80% species, 3.5% complex, 6% genus, and 3.5% family (7% not identified). Both MS systems provided rapid (≤3 min per isolate) and reliable identifications. The agreement of combined species/complex/genus-level identification with the reference method was higher for the Bruker Biotyper (97% versus 89.5%, P = 0.004) but required an extraction step more often. Species-level agreement with the reference method was similar for both MS systems (72.5% and 80%, P = 0.099).
Asunto(s)
Técnicas Bacteriológicas/métodos , Fibrosis Quística/complicaciones , Bacterias Gramnegativas/clasificación , Bacterias Gramnegativas/aislamiento & purificación , Infecciones por Bacterias Gramnegativas/diagnóstico , Infecciones por Bacterias Gramnegativas/microbiología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Bacterias Aerobias/química , Bacterias Aerobias/clasificación , Bacterias Aerobias/aislamiento & purificación , Bacterias Aerobias/metabolismo , Bacterias Gramnegativas/química , Bacterias Gramnegativas/metabolismo , Humanos , Iowa , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
Using data from 23,313 patients, we assessed whether two blood culture sets of three bottles per set would detect more pathogens than two sets of two bottles per set and achieve similar sensitivity to collecting three sets of two bottles per set. We also compared the yield of aerobic and anaerobic bottles. Thirty milliliters of blood was distributed to one anaerobic and two aerobic bottles. Among 26,855 collections of ≥ 60 ml within 30 min, 1,379 (5.1%) were positive for a pathogen not requiring detection in more than one set to be considered a pathogen, with 72 additional distinct pathogens detected using two 30-ml compared to two 20-ml sets of one aerobic and one anaerobic bottle (increased yield, 7.9%; 95% confidence interval [CI], 6.2 to 9.8%). For conditional pathogens requiring detection in at least two positive blood cultures for classification as pathogens (i.e., otherwise classified as contaminants), there were 162 positive detections with two 30-ml sets, of which 16 would not have been detected by two 20-ml sets (increased yield, 11.0% [95% CI, 6.4 to 17.2%]). Among 134 subjects who had three sets of 30 ml each within a 30-min interval, there was complete concordance between 60 ml of blood drawn in the first two sets of 30 ml and three 20-ml sets (P = 1.0). One aerobic bottle plus one anaerobic bottle yielded more pathogens than two aerobic bottles for organisms requiring a single (P < 0.001) and two (P = 0.04) positive sets to be defined as pathogens. In conclusion, we showed that collection of two aerobic and one anaerobic blood culture bottles per set results in improved yield compared to two bottles per set. We also confirmed that an anaerobic bottle should be included in blood culture sets.
Asunto(s)
Sangre/microbiología , Sepsis/diagnóstico , Manejo de Especímenes/métodos , Adolescente , Adulto , Aerobiosis , Anciano , Anciano de 80 o más Años , Anaerobiosis , Femenino , Humanos , Masculino , Técnicas Microbiológicas/métodos , Persona de Mediana Edad , Sensibilidad y Especificidad , Adulto JovenRESUMEN
Laboratory testing is an important component in the diagnosis of respiratory tract infections such as with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). However, specimen collection not only risks exposure of health care workers and other patients to infection, but also necessitates use of personal protective equipment that may be in short supply during periods of heightened disease activity. Self-collection of nasal or oropharyngeal swabs offers an alternative to address these drawbacks. Although studies in the past decade have demonstrated the utility of this approach for respiratory infections, it has not been widely adopted in routine clinical practice. The rapid spread of coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, has focused attention on the need for safe, convenient, timely, and scalable methods for collecting upper respiratory specimens for testing. The goals of this article are to highlight the literature regarding self-collected nasal or oropharyngeal specimens for respiratory pathogen testing; discuss the role of self-collection in helping prevent the spread of the COVID-19 disease from infected patients and facilitating a shift toward "virtual" medicine or telemedicine; and describe the current and future state of self-collection for infectious agents, and the impacts these approaches can have on population health management and disease diagnosis and prevention.
Asunto(s)
COVID-19 , Gestión de la Salud Poblacional , Manejo de Especímenes/métodos , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , COVID-19/diagnóstico , COVID-19/prevención & control , COVID-19/virología , Niño , Preescolar , Humanos , Lactante , Persona de Mediana Edad , Infecciones del Sistema Respiratorio/diagnóstico , Infecciones del Sistema Respiratorio/prevención & control , Infecciones del Sistema Respiratorio/virología , SARS-CoV-2 , Autocuidado , Telemedicina , Adulto JovenRESUMEN
BACKGROUND: Culturing of samples of periprosthetic tissue is the standard method used for the microbiologic diagnosis of prosthetic-joint infection, but this method is neither sensitive nor specific. In prosthetic-joint infection, microorganisms are typically present in a biofilm on the surface of the prosthesis. We hypothesized that culturing of samples obtained from the prosthesis would improve the microbiologic diagnosis of prosthetic-joint infection. METHODS: We performed a prospective trial comparing culture of samples obtained by sonication of explanted hip and knee prostheses to dislodge adherent bacteria from the prosthesis with conventional culture of periprosthetic tissue for the microbiologic diagnosis of prosthetic-joint infection among patients undergoing hip or knee revision or resection arthroplasty. RESULTS: We studied 331 patients with total knee prostheses (207 patients) or hip prostheses (124 patients); 252 patients had aseptic failure, and 79 had prosthetic-joint infection. With the use of standardized nonmicrobiologic criteria to define prosthetic-joint infection, the sensitivities of periprosthetic-tissue and sonicate-fluid cultures were 60.8% and 78.5% (P<0.001), respectively, and the specificities were 99.2% and 98.8%, respectively. Fourteen cases of prosthetic-joint infection were detected by sonicate-fluid culture but not by prosthetic-tissue culture. In patients receiving antimicrobial therapy within 14 days before surgery, the sensitivities of periprosthetic tissue and sonicate-fluid culture were 45.0% and 75.0% (P<0.001), respectively. CONCLUSIONS: In this study, culture of samples obtained by sonication of prostheses was more sensitive than conventional periprosthetic-tissue culture for the microbiologic diagnosis of prosthetic hip and knee infection, especially in patients who had received antimicrobial therapy within 14 days before surgery.
Asunto(s)
Técnicas Bacteriológicas/métodos , Prótesis de Cadera/microbiología , Prótesis de la Rodilla/microbiología , Infecciones Relacionadas con Prótesis/diagnóstico , Sonicación , Adulto , Anciano , Anciano de 80 o más Años , Antiinfecciosos/uso terapéutico , Bacterias/aislamiento & purificación , Diagnóstico Diferencial , Femenino , Prótesis de Cadera/efectos adversos , Humanos , Prótesis de la Rodilla/efectos adversos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Falla de Prótesis , Infecciones Relacionadas con Prótesis/microbiología , Sensibilidad y EspecificidadRESUMEN
Since some strains of Brucella species may require carbon dioxide for growth, a multilaboratory study was conducted to compare broth microdilution susceptibility results using ambient air (AA) and 5% CO2 incubation conditions. Six antimicrobial agents were tested against 39 Brucella isolates. Aminoglycoside MICs tended to be 1 log2 dilution higher in CO2 than in AA; tetracycline-class MICs to be 1 log2 dilution lower in CO2.
Asunto(s)
Antibacterianos/farmacología , Brucella/efectos de los fármacos , Dióxido de Carbono/metabolismo , Brucella/crecimiento & desarrollo , Humanos , Pruebas de Sensibilidad Microbiana/métodosAsunto(s)
Química Clínica/métodos , Genómica/métodos , Técnicas de Diagnóstico Molecular/métodos , Análisis de Secuencia de ADN , Enfermedad/genética , Humanos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Análisis de Secuencia de ADN/métodos , Análisis de Secuencia de ADN/normas , Análisis de Secuencia de ADN/tendenciasRESUMEN
The acquisition of beta-lactamases in members of the Enterobacteriaceae family poses a challenge to antimicrobial susceptibility testing in the clinical laboratory. We correlated the distribution of the MICs for Klebsiella spp. and Escherichia coli with the presence of extended-spectrum beta-lactamase (ESBL) and plasmid-mediated AmpC beta-lactamase (pAmpC) genes. A total of 264 isolates were subjected to cefazolin, ceftriaxone, cefotaxime, ceftazidime, cefepime, and aztreonam agar dilution MIC determination; ESBL screening and confirmatory testing by the methods of the Clinical and Laboratory Standards Institute (CLSI); and for isolates for which the MICs of extended-spectrum cephalosporins were > or =1 microg/ml or the MICs of cefpodoxime were >4 microg/ml, PCR amplification and sequencing of the ESBL and pAmpC genes. PCR was positive for 73/81 isolates (45 isolates with an ESBL gene alone, 24 isolates with a pAmpC gene alone, with 4 isolates with both genes). Compared to PCR, confirmatory testing by the CLSI method yielded a sensitivity and a specificity of 98.0 and 96.3%, respectively; there were six false-positive results and one false-negative result. No distinction in the MIC distribution was apparent between isolates with the ESBL gene and isolates with the pAmpC gene. A substantial percentage of the isolates with PCR-confirmed ESBL and/or pAmpC genes fell within the current CLSI susceptible category. For a ceftazidime, ceftriaxone, or cefotaxime MIC of > or =2 microg/ml, a dichotomy existed between isolates with and without ESBL and pAmpC genes in most cases. This suggests that the presence of the ESBL and the pAmpC enzymes may yield similar MICs of extended-spectrum cephalosporins, many of which fall within the current nonresistant categories. Lowering of the current CLSI breakpoints for cephalosporins appears to be warranted.
Asunto(s)
Antibacterianos/farmacología , Cefalosporinas/farmacología , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Klebsiella/efectos de los fármacos , Klebsiella/enzimología , Pruebas de Sensibilidad Microbiana/métodos , beta-Lactamasas/biosíntesis , ADN Bacteriano/química , ADN Bacteriano/genética , Errores Diagnósticos , Infecciones por Enterobacteriaceae/microbiología , Escherichia coli/aislamiento & purificación , Genes Bacterianos , Humanos , Klebsiella/aislamiento & purificación , Reacción en Cadena de la Polimerasa , Sensibilidad y Especificidad , Análisis de Secuencia de ADN , beta-Lactamasas/genéticaRESUMEN
BACKGROUND: Despite increasing concerns about antimicrobial resistance and emerging pathogens among blood culture isolates, contemporary population-based data on the age- and sex-specific incidence of bloodstream infections (BSIs) are limited. METHODS: Retrospective, population-based, cohort study of all residents of Olmsted County, Minnesota, with a BSI between January 1, 2003, and December 31, 2005. The medical record linkage system of the Rochester Epidemiology Project and microbiology records were used to identify incident cases. RESULTS: A total of 1051 unique patients with positive blood culture results were identified; 401 (38.2%) were classified as contaminated. Of 650 patients with cultures deemed clinically relevant, the mean +/- SD age was 63.1 +/- 23.1 years, and 52.5% were male. The most common organisms identified were Escherichia coli (in 163 patients with BSIs [25.1%]) and Staphylococcus aureus (in 108 patients with BSIs [16.6%]). Nosocomial BSIs were more common in males than females (23.8% vs 13.9%; P = .002). The age-adjusted incidence rate of BSI was 156 per 100 000 person-years for females and 237 per 100 000 person-years for males (P<.001), with an age- and sex-adjusted rate of 189 per 100 000 person-years. Rates of BSI due to gram-positive cocci were 64 per 100 000 person-years for females and 133 per 100 000 person-years for males (P<.001); gram-negative bacillus BSI rates (85/100 000 person-years for females and 79/100 000 person-years for males) were not significantly different between sexes (P = .79). The rate of S aureus BSI was 23 per 100 000 person-years for females and 46 per 100 000 person-years for males (P = .005). CONCLUSIONS: There are significant differences in the age and sex distribution of organisms among patients with BSIs. The incidence of BSI increases sharply with increasing age and is significantly higher in males, mainly because of nosocomial organisms, including S aureus.
Asunto(s)
Bacteriemia/epidemiología , Factores de Edad , Estudios de Cohortes , Infección Hospitalaria/epidemiología , Infecciones por Escherichia coli/epidemiología , Femenino , Infecciones por Bacterias Gramnegativas/epidemiología , Humanos , Masculino , Persona de Mediana Edad , Minnesota/epidemiología , Estudios Retrospectivos , Factores Sexuales , Infecciones Estafilocócicas/epidemiologíaRESUMEN
Testing for cytomegalovirus-, Epstein-Barr virus-, and BK virus-specific gene targets in specimens from solid-organ transplant recipients for DNA by quantitative real-time polymerase chain reaction has been implemented in many diagnostic facilities. This technology provides rapid, accurate, and reproducible results for early detection, monitoring, and medical management of patients with these infections. Because these assays are becoming commonly used in clinical practice, the technical variables associated with specimen processing (e.g., nucleic acid extraction, gene target, and result reporting), amplification, and unique patient characteristics (e.g., age, sex, underlying diseases, immune status, and immunosuppressive regimens received) are factors that may influence the understanding and interpretation of test results. We emphasize the need for standardization of existing variables through parallel comparative and proficiency testing, uniform units for expressing results, to provide for clinical correlation with the results of these molecular assays.
Asunto(s)
Infecciones por Citomegalovirus/diagnóstico , ADN Viral/sangre , Infecciones por Virus de Epstein-Barr/diagnóstico , Trasplante de Órganos , Reacción en Cadena de la Polimerasa/métodos , Infecciones por Polyomavirus/diagnóstico , Infecciones Tumorales por Virus/diagnóstico , Virus BK/genética , Virus BK/aislamiento & purificación , Citomegalovirus/genética , Citomegalovirus/aislamiento & purificación , Infecciones por Citomegalovirus/virología , Infecciones por Virus de Epstein-Barr/virología , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/aislamiento & purificación , Humanos , Trasplante de Órganos/normas , Infecciones por Polyomavirus/virología , Infecciones Tumorales por Virus/virología , Viremia/diagnósticoRESUMEN
THE OBJECTIVE OF THE STUDY IS TO DETERMINE THE PREVALENCE OF HIGH-RISK HUMAN PAPILLOMAVIRUS (HRHPV) INFECTION IN TONSILLAR SWABS AND TISSUE: Patients undergoing tonsillectomy for nonmalignant causes were enrolled. A flocked swab and fresh tissue were collected from the left and right tonsil of each patient. Specimens were tested for hrHPV DNA using the Roche cobas test and for the presence of E6/E7 messenger RNA using the Hologic Aptima hrHPV test. Of the 193 patients enrolled, 129 were in the pediatric group (ages 1-12years; median, 5years), and 64 were in the adult group (ages 13-55; median, 22years). All swab and tissue specimens were negative for hrHPV by both methods. Positive, negative, and internal controls performed as expected. We found a 0% rate of infection indicating that detectable hrHPV infection in tonsillar tissue appears to be uncommon in the children and adults in the population sampled.
Asunto(s)
Genotipo , Tonsila Palatina/virología , Papillomaviridae/clasificación , Papillomaviridae/aislamiento & purificación , Infecciones por Papillomavirus/epidemiología , Infecciones por Papillomavirus/virología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Preescolar , ADN Viral/análisis , ADN Viral/genética , ADN Viral/aislamiento & purificación , Femenino , Humanos , Lactante , Masculino , Persona de Mediana Edad , Papillomaviridae/genética , Prevalencia , ARN Mensajero/análisis , ARN Mensajero/genética , ARN Mensajero/aislamiento & purificación , ARN Viral/análisis , ARN Viral/genética , ARN Viral/aislamiento & purificación , Adulto JovenRESUMEN
The events surrounding September 11, 2001, and its aftermath have compelled the public health and medical community to face the hitherto unfamiliar reality of bioterrorism. Physicians and public health personnel are frontline soldiers in this new form of warfare. This article provides a general overview of the pathophysiology, clinical presentation, diagnosis, and management of patients infected with the 6 highest priority agents that could potentially be used in bioterrorism. The diseases discussed include anthrax, smallpox, tularemia, plague, botulism, and viral hemorrhagic fevers. Despite the unpredictable nature of bioterrorism, disaster preparedness and knowledge of essential diagnostic and epidemiological principles can contribute substantially toward combating this new threat.
Asunto(s)
Bioterrorismo/prevención & control , Enfermedades Transmisibles/diagnóstico , Enfermedades Transmisibles/tratamiento farmacológico , Notificación de Enfermedades , Brotes de Enfermedades/prevención & control , Rol del Médico , Carbunco/diagnóstico , Carbunco/tratamiento farmacológico , Botulismo/diagnóstico , Botulismo/tratamiento farmacológico , Enfermedades Transmisibles/microbiología , Enfermedades Transmisibles/virología , Personal de Salud , Fiebres Hemorrágicas Virales/diagnóstico , Fiebres Hemorrágicas Virales/tratamiento farmacológico , Humanos , Control de Infecciones , Peste/diagnóstico , Peste/tratamiento farmacológico , Salud Pública , Viruela/diagnóstico , Viruela/tratamiento farmacológico , Tularemia/diagnóstico , Tularemia/tratamiento farmacológico , Estados UnidosRESUMEN
OBJECTIVE: To determine whether autoclaving suspensions of vaccinia virus, herpes simplex virus (HSV), varicella-zoster virus (VZV), and Bacillus anthracis inactivate infectivity of these agents but allow detection of target DNA by LightCycler polymerase chain reaction (PCR). MATERIAL AND METHODS: Swabs were inserted into tubes containing serial 10-fold dilutions (10(-1) to 10(-5); 500 microL; 6 samples per dilution) of vaccinia virus, HSV, VZV, or a single suspension of 10(8) colony-forming units of B anthracis (2 samples). One half of the samples were autoclaved, and the remainder were not. An aliquot of each not autoclaved sample served as a control for infectivity. RESULTS: Autoclaving swabs saturated with suspensions of vaccinia virus, HSV, or VZV eliminated the infectivity of these agents; however, DNA was detectable in most autoclaved samples in dilutions of 10(-1) to 10(-4) by LightCycler PCR. All not autoclaved specimens were detected by culture (infectivity) except for VZV and, in most dilutions of 10(-1) to 10(-3), by assay of target DNA by LightCycler PCR. Similarly positive results were obtained for PCR assessment of sporulated B anthracis. CONCLUSIONS: Standard autoclaving procedures eliminated the infectivity of viruses (and B anthracis), but target DNA was often retained for detection by LightCycler PCR. Current recommendations indicate that the laboratory diagnosis of smallpox virus infection be performed only within Biosafety Level 4 facilities. We suggest that, in addition to the requirement for immediate coordination with public health officials, the federal government consider expanding the existing guidelines for processing these specimens to encourage immediate collection, autoclaving, and testing by LightCycler PCR to differentiate smallpox virus from other dermal pathogens such as HSV and VZV by specific qualified laboratories.
Asunto(s)
Bacillus anthracis/aislamiento & purificación , Bioterrorismo , ADN Bacteriano/aislamiento & purificación , ADN Viral/aislamiento & purificación , Herpesvirus Humano 3/aislamiento & purificación , Reacción en Cadena de la Polimerasa/instrumentación , Simplexvirus/aislamiento & purificación , Esterilización , Virus Vaccinia/aislamiento & purificación , Bacillus anthracis/genética , Guías como Asunto , Herpesvirus Humano 3/genética , Humanos , Plásmidos , Simplexvirus/genética , Estados Unidos , Virus Vaccinia/genéticaRESUMEN
Rapid-cycle real-time polymerase chain reaction has immediate and important implications for diagnostic testing in the clinical microbiology laboratory. In our experience this novel testing method has outstanding performance characteristics. The sensitivities for detecting microorganisms frequently exceed standard culture-based assays, and the time required to complete the assays is considerably shorter than that required for culture-based assays. We describe the principle of real-time polymerase chain reaction and present clinical applications, including the detection of Bacillus anthracis, the causative agent of anthrax. This latter test is commercially available as the result of a collaborative venture between Mayo Clinic and Roche Applied Science, hence the designation The Mayo-Roche Rapid Anthrax Test.
Asunto(s)
Bacillus anthracis/aislamiento & purificación , ADN Bacteriano/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Carbunco/diagnóstico , Bacillus anthracis/genética , Bacterias/aislamiento & purificación , ADN Bacteriano/análisis , Humanos , Sensibilidad y EspecificidadRESUMEN
We compared a commercial line probe assay (INNO-LiPA HCV II, Innogenetics, N.V., Ghent, Belgium, distributed by Bayer Diagnostics) to an in-house 5' untranslated region direct DNA sequencing method for genotyping hepatitis C virus (HCV). Initial evaluation demonstrated that the INNO-LiPA HCV II assay and sequencing assay assigned the same genotype for 110/132 (83.3%) patient specimens (98 subtype and 12 genotype only identifications). Following the initial evaluation, the INNO-LiPA HCV II assay was used routinely to genotype HCV from patient specimens submitted to our laboratory for genotyping (n = 1,739). During this second part of the study, novel line probe patterns have been noted and interpreted using the in-house direct sequencing assay. Reactivity at bands 1, 2, 3, 4, 5 and 8 (n = 4) or 1, 2, 3, 4, 6 and 7 (n = 2) represented HCV genotype 1. Reactivity at bands 1, 2, 5 and 9 (n = 1) represented HCV genotype 2. Reactivity at bands 1, 2, 5, 9 and 16 (n = 1) represented HCV genotype 4. Reactivity at bands 1, 2, 5, 9, 10, 11 (weak band) and 12 (n = 118) most likely represented HCV genotype 2b. This information should be of use to INNO-LiPA HCV II assay users.
Asunto(s)
Regiones no Traducidas 5'/genética , Hepacivirus/genética , Técnicas de Sonda Molecular , Análisis de Secuencia de ADN/métodos , Genoma Viral , Genotipo , Hepacivirus/aislamiento & purificación , Hepatitis C/virología , HumanosRESUMEN
OBJECTIVE: To compare the effectiveness of self-collected and health care worker (HCW)-collected nasal swabs for detection of influenza viruses and determine the patients' preference for type of collection. PATIENTS AND METHODS: We enrolled adult patients presenting with influenzalike illness to the Emergency Department at Mayo Clinic, Rochester, Minnesota, from January 28, 2011, through April 30, 2011. Patients self-collected a midturbinate nasal flocked swab from their right nostril following written instructions. A second swab was then collected by an HCW from the left nostril. Swabs were tested for influenza A and B viruses by real-time reverse transcription-polymerase chain reaction, and percent concordance between collection methods was determined. RESULTS: Of the 72 paired specimens analyzed, 25 were positive for influenza A or B RNA by at least one of the collection methods (34.7% positivity rate). When the 14 patients who had prior health care training were excluded, the qualitative agreement between collection methods was 94.8% (55 of 58). Two of the 58 specimens (3.4%) from patients without health care training were positive only by HCW collection, and 1 of 58 (1.7%) was positive only by patient self-collection. A total of 53.4% of patients (31 of 58) preferred the self-collection method over the HCW collection, and 25.9% (15 of 58) had no preference. CONCLUSION: Self-collected midturbinate nasal swabs provide a reliable alternative to HCW collection for influenza A and B virus real-time reverse transcription-polymerase chain reaction.
Asunto(s)
Líquidos Corporales/virología , Mucosa Nasal/virología , Manejo de Especímenes/métodos , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Satisfacción del Paciente , Reacción en Cadena en Tiempo Real de la Polimerasa , Adulto JovenAsunto(s)
Sistemas de Computación , Reacción en Cadena de la Polimerasa , Benzotiazoles , Diaminas , Enterococcus/aislamiento & purificación , Enterococcus/fisiología , Transferencia Resonante de Energía de Fluorescencia , Perfilación de la Expresión Génica , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Neoplasia Residual/metabolismo , Compuestos Orgánicos , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo de Nucleótido Simple , Quinolinas , Resistencia a la VancomicinaRESUMEN
OBJECTIVE: To compare rates of group A beta-hemolytic streptococci (GABHS) detection by real-time polymerase chain reaction (rtPCR) and standard culture (SCx) at different anatomic sites to determine whether a more patient-friendly site (eg, retromolar trigone or gingivobuccal sulcus) would yield results similar to the tonsillar surface. Real-time polymerase chain reaction can detect GABHS at rates equal to SCx, and results require only a few hours. DESIGN: Prospective study. SETTING: Tertiary care setting. PATIENTS: The study population comprised 130 patients undergoing tonsillectomy or adenotonsillectomy. INTERVENTION: At tonsillectomy, swabs were taken of pharyngeal tonsil surface, pharyngeal tonsillar core, inferior gingivobuccal sulcus, and retromolar trigone. Tissue samples were taken from tonsil core and adenoid. All comparisons between methods and sites were made using the McNemar test for comparing correlated proportions. All calculated P values were 2-sided. MAIN OUTCOME MEASURE: Detection of GABHS by rtPCR and SCx. RESULTS: In 41 cases (32%), GABHS was detected at 1 or more sampled sites, and 29 of those positive were detected on the tonsil surface-SCx and rtPCR results were both positive in 28 (97%). Of these 29 cases, results from the gingivobuccal site were positive by both rtPCR and SCx in 4 (14%), rtPCR only in 3 (10%), and SCx only in 3 (10%). Of the 7 tonsil surface-positive cases with retromolar trigone swabs, results were positive by rtPCR only in 1 (14%) and SCx only in 2 (29%). CONCLUSION: Whether rtPCR or SCx is used, swabs of gingivobuccal sulcus and retromolar trigone do not accurately reflect GABHS populations on the tonsil surface.