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What is the educational challenge?Medical schools invest significant resources into the creation of multiple-choice items for assessments. This process is costly and requires faculty training. Recently ChatGPT has been used in various areas to improve content creation efficiency, and it has otherwise been used to answer USMLE-style assessment items.What are the proposed solutions?We proposed the use of ChatGPT to create initial drafts of multiple-choice items.What are the potential benefits to a wider global audience?The use of ChatGPT to generate assessment items can decrease resources required, allowing for the creation of more items, and freeing-up faculty time to perform higher level assessment activities. ChatGPT is also able to consistently produce items using a standard format while adhering to item writing guidelines, which can be very challenging for faculty teams.What are the next steps?We plan to pilot ChatGPT drafted questions and compare item statistics for those written by ChatGPT with those written by our content experts. We also plan to further identify the types of questions that ChatGPT is most appropriate for, and incorporate media into assessment items (e.g. images, videos).
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Docentes , Faculdades de Medicina , Humanos , Escolaridade , Gravação de Videoteipe , RedaçãoRESUMO
Early detection of novel pathogens can prevent or substantially mitigate biological incidents, including pandemics. Metagenomic next-generation sequencing (mNGS) of symptomatic clinical samples may enable detection early enough to contain outbreaks, limit international spread, and expedite countermeasure development. In this article, we propose a clinical mNGS architecture we call "Threat Net," which focuses on the hospital emergency department as a high-yield surveillance location. We develop a susceptible-exposed-infected-removed (SEIR) simulation model to estimate the effectiveness of Threat Net in detecting novel respiratory pathogen outbreaks. Our analysis serves to quantify the value of routine clinical mNGS for respiratory pandemic detection by estimating the cost and epidemiological effectiveness at differing degrees of hospital coverage across the United States. We estimate that a biological threat detection network such as Threat Net could be deployed across hospitals covering 30% of the population in the United States. Threat Net would cost between $400 million and $800 million annually and have a 95% chance of detecting a novel respiratory pathogen with traits of SARS-CoV-2 after 10 emergency department presentations and 79 infections across the United States. Our analyses suggest that implementing Threat Net could help prevent or substantially mitigate the spread of a respiratory pandemic pathogen in the United States.
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Derramamento de Material Biológico , Surtos de Doenças , Humanos , Simulação por Computador , Surtos de Doenças/prevenção & controle , Serviço Hospitalar de Emergência , Hospitais , Sequenciamento de Nucleotídeos em Larga Escala , Sensibilidade e EspecificidadeRESUMO
The COVID-19 pandemic caused significant disruption in medical education. With disruption comes the opportunity for innovation. Telehealth had been growing rapidly in many fields of medicine prior to the pandemic; however, the necessities of social distancing, scarcity of personal protective equipment, and mandates to prevent unnecessary exposures for healthcare workers and patients alike, brought opportunities for the exponential expansion of telehealth. With the expansion of telehealth services came the need to expand the curriculum in telehealth to prepare medical students to return to vastly transformed clinical settings as well as prepare them for a future clinical landscape likely to incorporate telehealth to a much greater degree. The University of Colorado School of Medicine (CUSOM) rapidly developed a course in telehealth to prepare students for this changing clinical environment. Simultaneously, a faculty development curriculum was created to support clinical faculty new to telehealth in basic skills and teaching in a virtual environment. Lastly, adaptations were made to the summative Clinical Practice Exam administered to students at the completion of clerkships to incorporate telehealth. Recognizing the importance of achieving competence in telehealth, the CUSOM has taken steps to invest in the development of comprehensive and integrated telehealth curricula. Many creative and innovative solutions have been adopted in the wake of this pandemic to allow medical education to continue despite many hurdles and barriers; many of these will not persist past the pandemic. However, we expect telehealth clinical skills and the curricula developed to support them to remain relevant long past the time when the COVID-19 pandemic has faded into history.
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Lyme disease is a tick-borne infection caused by the bacteria Borrelia burgdorferi Current diagnosis of early Lyme disease relies heavily on clinical criteria, including the presence of an erythema migrans rash. The sensitivity of current gold-standard diagnostic tests relies upon antibody formation, which is typically delayed and thus of limited utility in early infection. We conducted a study of blood and skin biopsy specimens from 57 patients with a clinical diagnosis of erythema migrans. Samples collected at the time of diagnosis were analyzed using an ultrasensitive, PCR-based assay employing an isothermal amplification step and multiple primers. In 75.4% of patients, we directly detected one or more B. burgdorferi genotypes in the skin. Two-tier testing showed that 20 (46.5%) of those found to be PCR positive remained serologically negative at both acute and convalescent time points. Multiple genotypes were found in three (8%) of those where a specific genotype could be identified. The 13 participants who lacked PCR and serologic evidence for exposure to B. burgdorferi could be differentiated as a group from PCR-positive participants by their levels of several immune markers as well as by clinical descriptors such as the number of acute symptoms and the pattern of their erythema migrans rash. These results suggest that within a Mid-Atlantic cohort, patient subgroups can be identified using PCR-based direct detection approaches. This may be particularly useful in future research such as vaccine trials and public health surveillance of tick-borne disease patterns.
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Grupo Borrelia Burgdorferi , Borrelia burgdorferi , Doença de Lyme , Doenças Transmitidas por Carrapatos , Borrelia burgdorferi/genética , Grupo Borrelia Burgdorferi/genética , Humanos , Doença de Lyme/diagnóstico , Reação em Cadeia da PolimeraseRESUMO
Knockout of the memory suppressor gene histone deacetylase 2 (Hdac2) in mice elicits cognitive enhancement, and drugs that block HDAC2 have potential as therapeutics for disorders affecting memory. Currently available HDAC2 catalytic activity inhibitors are not fully isoform specific and have short half-lives. Antisense oligonucleotides (ASOs) are drugs that elicit extremely long-lasting, specific inhibition through base pairing with RNA targets. We utilized an ASO to reduce Hdac2 messenger RNA (mRNA) in mice and determined its longevity, specificity, and mechanism of repression. A single injection of the Hdac2-targeted ASO in the central nervous system produced persistent reduction in HDAC2 protein and Hdac2 mRNA levels for 16 weeks. It enhanced object location memory for 8 weeks. RNA sequencing (RNA-seq) analysis of brain tissues revealed that the repression was specific to Hdac2 relative to related Hdac isoforms, and Hdac2 reduction caused alterations in the expression of genes involved in extracellular signal-regulated kinase (ERK) and memory-associated immune signaling pathways. Hdac2-targeted ASOs also suppress a nonpolyadenylated Hdac2 regulatory RNA and elicit direct transcriptional suppression of the Hdac2 gene through stalling RNA polymerase II. These findings identify transcriptional suppression of the target gene as a novel mechanism of action of ASOs.
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Borrelia burgdorferi is the etiological agent of Lyme disease. In the current study, we used direct-detection PCR and electrospray ionization mass spectrometry to monitor and genotype B. burgdorferi isolates from serially collected whole-blood specimens from patients clinically diagnosed with early Lyme disease before and during 21 days of antibiotic therapy. B. burgdorferi isolates were detected up to 3 weeks after the initiation of antibiotic treatment, with ratios of coinfecting B. burgdorferi genotypes changing over time.
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Antibacterianos/uso terapêutico , Borrelia burgdorferi/efeitos dos fármacos , Borrelia burgdorferi/patogenicidade , Doença de Lyme/tratamento farmacológico , Doença de Lyme/microbiologia , Borrelia burgdorferi/genética , Genótipo , Humanos , Reação em Cadeia da Polimerase , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Recently, a student-initiated movement to end the United States Medical Licensing Examination Step 2 Clinical Skills and the Comprehensive Osteopathic Medical Licensing Examination Level 2-Performance Evaluation has gained momentum. These are the only national licensing examinations designed to assess clinical skills competence in the stepwise process through which physicians gain licensure and certification. Therefore, the movement to end these examinations and the ensuing debate merit careful consideration. The authors, elected representatives of the Directors of Clinical Skills Courses, an organization comprising clinical skills educators in the United States and beyond, believe abolishing the national clinical skills examinations would have a major negative impact on the clinical skills training of medical students, and that forfeiting a national clinical skills competency standard has the potential to diminish the quality of care provided to patients. In this Perspective, the authors offer important additional background information, outline key concerns regarding the consequences of ending these national clinical skills examinations, and provide recommendations for moving forward: reducing the costs for students, exploring alternatives, increasing the value and transparency of the current examinations, recognizing and enhancing the strengths of the current examinations, and engaging in a national dialogue about the issue.
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Competência Clínica/normas , Avaliação Educacional/normas , Licenciamento em Medicina/normas , Diretores Médicos/psicologia , Avaliação Educacional/métodos , Humanos , Estados UnidosRESUMO
Ixodes ricinus ticks are vectors of numerous human and animal pathogens. They are host generalists able to feed on more than 300 vertebrate species. The prevalence of tick-borne pathogens is influenced by host-vector-pathogen interactions that results in spatial distribution of infection risk. Broad-range polymerase chain reaction electrospray ionization mass spectrometry (PCR/ESI-MS) was used to analyze 435 I. ricinus nymphs from four localities in the south of the Czech Republic for the species identification of tick-borne pathogens. Borrelia burgdorferi sensu lato spirochetes were the most common pathogen detected in the ticks; 21% of ticks were positive for a single genospecies and 2% were co-infected with two genospecies. Other tick-borne pathogens detected included Rickettsia helvetica (3.9%), R. monacensis (0.2%), Anaplasma phagocytophilum (2.8%), Babesia venatorum (0.9%), and Ba. microti (0.5%). The vertebrate host of the ticks was determined using PCR followed by reverse line blot hybridization from the tick's blood-meal remnants. The host was identified for 61% of ticks. DNA of two hosts was detected in 16% of samples with successful host identification. The majority of ticks had fed on artiodactyls (50.7%) followed by rodents (28.6%) and birds (7.8%). Other host species were wild boar, deer, squirrels, field mice and voles.
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Anaplasma phagocytophilum/isolamento & purificação , Babesia/isolamento & purificação , Borrelia burgdorferi/isolamento & purificação , Ixodes/microbiologia , Ixodes/parasitologia , Rickettsia/isolamento & purificação , Infestações por Carrapato , Anaplasma phagocytophilum/genética , Animais , Artiodáctilos , Arvicolinae , Babesia/classificação , Babesia/genética , Aves , Borrelia burgdorferi/genética , República Tcheca , Cervos , Humanos , Camundongos , Rickettsia/classificação , Rickettsia/genética , Sciuridae , Inquéritos e Questionários , Sus scrofaRESUMO
Human haploinsufficiency of the transcription factor Tcf4 leads to a rare autism spectrum disorder called Pitt-Hopkins syndrome (PTHS), which is associated with severe language impairment and development delay. Here, we demonstrate that Tcf4 haploinsufficient mice have deficits in social interaction, ultrasonic vocalization, prepulse inhibition, and spatial and associative learning and memory. Despite learning deficits, Tcf4(+/-) mice have enhanced long-term potentiation in the CA1 area of the hippocampus. In translationally oriented studies, we found that small-molecule HDAC inhibitors normalized hippocampal LTP and memory recall. A comprehensive set of next-generation sequencing experiments of hippocampal mRNA and methylated DNA isolated from Tcf4-deficient and WT mice before or shortly after experiential learning, with or without administration of vorinostat, identified "memory-associated" genes modulated by HDAC inhibition and dysregulated by Tcf4 haploinsufficiency. Finally, we observed that Hdac2 isoform-selective knockdown was sufficient to rescue memory deficits in Tcf4(+/-) mice.
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Metilação de DNA/genética , Memória , Plasticidade Neuronal/genética , Proteína 2 Semelhante ao Fator 7 de Transcrição/metabolismo , Animais , Transtorno Autístico/complicações , Transtorno Autístico/patologia , Transtorno Autístico/fisiopatologia , Ilhas de CpG/genética , Metilação de DNA/efeitos dos fármacos , Modelos Animais de Doenças , Fácies , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Hipocampo/metabolismo , Histona Desacetilase 2/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Hiperventilação/complicações , Hiperventilação/genética , Hiperventilação/patologia , Hiperventilação/fisiopatologia , Deficiência Intelectual/complicações , Deficiência Intelectual/genética , Deficiência Intelectual/patologia , Deficiência Intelectual/fisiopatologia , Potenciação de Longa Duração/efeitos dos fármacos , Masculino , Memória/efeitos dos fármacos , Camundongos , Atividade Motora/efeitos dos fármacos , Plasticidade Neuronal/efeitos dos fármacos , Inibição Pré-Pulso/efeitos dos fármacos , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , Transcrição Gênica/efeitos dos fármacos , VorinostatRESUMO
UNLABELLED: Bloodstream infection (BSI) and sepsis are rising in incidence throughout the developed world. The spread of multi-drug resistant organisms presents increasing challenges to treatment. Surviving BSI is dependent on rapid and accurate identification of causal organisms, and timely application of appropriate antibiotics. Current culture-based methods used to detect and identify agents of BSI are often too slow to impact early therapy and may fail to detect relevant organisms in many positive cases. Existing methods for direct molecular detection of microbial DNA in blood are limited in either sensitivity (likely the result of small sample volumes) or in breadth of coverage, often because the PCR primers and probes used target only a few specific pathogens. There is a clear unmet need for a sensitive molecular assay capable of identifying the diverse bacteria and yeast associated with BSI directly from uncultured whole blood samples. We have developed a method of extracting DNA from larger volumes of whole blood (5 ml per sample), amplifying multiple widely conserved bacterial and fungal genes using a mismatch- and background-tolerant PCR chemistry, and identifying hundreds of diverse organisms from the amplified fragments on the basis of species-specific genetic signatures using electrospray ionization mass spectrometry (PCR/ESI-MS). We describe the analytical characteristics of the IRIDICA BAC BSI Assay and compare its pre-clinical performance to current standard-of-care methods in a collection of prospectively collected blood specimens from patients with symptoms of sepsis. The assay generated matching results in 80% of culture-positive cases (86% when common contaminants were excluded from the analysis), and twice the total number of positive detections. The described method is capable of providing organism identifications directly from uncultured blood in less than 8 hours. DISCLAIMER: The IRIDICA BAC BSI Assay is not available in the United States.
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Bactérias/isolamento & purificação , Infecções Bacterianas/sangue , Bioensaio/métodos , Candida/isolamento & purificação , Candidíase/sangue , Sepse/sangue , Algoritmos , Antibacterianos/uso terapêutico , Primers do DNA , Farmacorresistência Bacteriana , Farmacorresistência Fúngica , Humanos , Limite de Detecção , Reação em Cadeia da Polimerase , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Sepse/microbiologia , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
BACKGROUND: Rapid molecular diagnostic (RMD) platforms may lead to better antibiotic use. Our objective was to develop analytical strategies to enhance the interpretation of RMDs for clinicians. METHODS: We compared the performance characteristics of 4 RMD platforms for detecting resistance against ß-lactams in 72 highly resistant isolates of Escherichia coli and Klebsiella pneumoniae (PRIMERS I). Subsequently, 2 platforms were used in a blinded study in which a heterogeneous collection of 196 isolates of E. coli and K. pneumoniae (PRIMERS II) were examined. We evaluated the genotypic results as predictors of resistance or susceptibility against ß-lactam antibiotics. We designed analytical strategies and graphical representations of platform performance, including discrimination summary plots and susceptibility and resistance predictive values, that are readily interpretable by practitioners to inform decision-making. RESULTS: In PRIMERS I, the 4 RMD platforms detected ß-lactamase (bla) genes and identified susceptibility or resistance in >95% of cases. In PRIMERS II, the 2 platforms identified susceptibility against extended-spectrum cephalosporins and carbapenems in >90% of cases; however, against piperacillin/tazobactam, susceptibility was identified in <80% of cases. Applying the analytical strategies to a population with 15% prevalence of ceftazidime-resistance and 5% imipenem-resistance, RMD platforms predicted susceptibility in >95% of cases, while prediction of resistance was 69%-73% for ceftazidime and 41%-50% for imipenem. CONCLUSIONS: RMD platforms can help inform empiric ß-lactam therapy in cases where bla genes are not detected and the prevalence of resistance is known. Our analysis is a first step in bridging the gap between RMDs and empiric treatment decisions.
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Antibacterianos/uso terapêutico , Infecções por Enterobacteriaceae/tratamento farmacológico , Escherichia coli/efeitos dos fármacos , Klebsiella pneumoniae/efeitos dos fármacos , Testes de Sensibilidade Microbiana/métodos , Técnicas de Diagnóstico Molecular/métodos , Resistência beta-Lactâmica , Infecções por Enterobacteriaceae/microbiologia , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Técnicas de Genotipagem/métodos , Humanos , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/isolamento & purificação , Fatores de TempoRESUMO
OBJECTIVE: Early identification of causative microorganism(s) in patients with severe infection is crucial to optimize antimicrobial use and patient survival. However, current culture-based pathogen identification is slow and unreliable such that broad-spectrum antibiotics are often used to insure coverage of all potential organisms, carrying risks of overtreatment, toxicity, and selection of multidrug-resistant bacteria. We compared the results obtained using a novel, culture-independent polymerase chain reaction/electrospray ionization-mass spectrometry technology with those obtained by standard microbiological testing and evaluated the potential clinical implications of this technique. DESIGN: Observational study. SETTING: Nine ICUs in six European countries. PATIENTS: Patients admitted between October 2013 and June 2014 with suspected or proven bloodstream infection, pneumonia, or sterile fluid and tissue infection were considered for inclusion. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: We tested 616 bloodstream infection, 185 pneumonia, and 110 sterile fluid and tissue specimens from 529 patients. From the 616 bloodstream infection samples, polymerase chain reaction/electrospray ionization-mass spectrometry identified a pathogen in 228 cases (37%) and culture in just 68 (11%). Culture was positive and polymerase chain reaction/electrospray ionization-mass spectrometry negative in 13 cases, and both were negative in 384 cases, giving polymerase chain reaction/electrospray ionization-mass spectrometry a sensitivity of 81%, specificity of 69%, and negative predictive value of 97% at 6 hours from sample acquisition. The distribution of organisms was similar with both techniques. Similar observations were made for pneumonia and sterile fluid and tissue specimens. Independent clinical analysis of results suggested that polymerase chain reaction/electrospray ionization-mass spectrometry technology could potentially have resulted in altered treatment in up to 57% of patients. CONCLUSIONS: Polymerase chain reaction/electrospray ionization-mass spectrometry provides rapid pathogen identification in critically ill patients. The ability to rule out infection within 6 hours has potential clinical and economic benefits.
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Bacteriemia/microbiologia , Patógenos Transmitidos pelo Sangue/isolamento & purificação , Pneumonia Bacteriana/microbiologia , Reação em Cadeia da Polimerase/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Adulto , Idoso , Bacteriemia/diagnóstico , Líquidos Corporais/microbiologia , Cuidados Críticos/métodos , Estado Terminal , Diagnóstico Precoce , Europa (Continente) , Feminino , Bactérias Gram-Negativas/isolamento & purificação , Bactérias Gram-Positivas/isolamento & purificação , Humanos , Unidades de Terapia Intensiva , Masculino , Pessoa de Meia-Idade , Técnicas de Diagnóstico Molecular , Pneumonia Bacteriana/diagnóstico , Medição de Risco , Sensibilidade e Especificidade , Manejo de EspécimesRESUMO
Ixodes pacificus ticks can harbor a wide range of human and animal pathogens. To survey the prevalence of tick-borne known and putative pathogens, we tested 982 individual adult and nymphal I. pacificus ticks collected throughout California between 2007 and 2009 using a broad-range PCR and electrospray ionization mass spectrometry (PCR/ESI-MS) assay designed to detect a wide range of tick-borne microorganisms. Overall, 1.4% of the ticks were found to be infected with Borrelia burgdorferi, 2.0% were infected with Borrelia miyamotoi and 0.3% were infected with Anaplasma phagocytophilum. In addition, 3.0% were infected with Babesia odocoilei. About 1.2% of the ticks were co-infected with more than one pathogen or putative pathogen. In addition, we identified a novel Anaplasmataceae species that we characterized by sequencing of its 16S rRNA, groEL, gltA, and rpoB genes. Sequence analysis indicated that this organism is phylogenetically distinct from known Anaplasma species with its closest genetic near neighbors coming from Asia. The prevalence of this novel Anaplasmataceae species was as high as 21% at one site, and it was detected in 4.9% of ticks tested statewide. Based upon this genetic characterization we propose that this organism be called 'Candidatus Cryptoplasma californiense'. Knowledge of this novel microbe will provide awareness for the community about the breadth of the I. pacificus microbiome, the concept that this bacterium could be more widely spread; and an opportunity to explore whether this bacterium also contributes to human or animal disease burden.
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Anaplasmataceae/classificação , Anaplasmataceae/isolamento & purificação , Biodiversidade , Ixodes/microbiologia , Anaplasmataceae/genética , Anaplasmataceae/fisiologia , Animais , California , Filogenia , Rickettsia/isolamento & purificação , Rickettsia/fisiologia , Análise de Sequência de DNA , SimbioseRESUMO
Borrelia miyamotoi, a relapsing fever-related spirochete transmitted by Ixodes ticks, has been recently shown to be a human pathogen. To characterize the prevalence of this organism in questing Ixodes ticks, we tested 2,754 ticks for a variety of tickborne pathogens by PCR and electrospray-ionization mass spectrometry. Ticks were collected from California, New York, Connecticut, Pennsylvania, and Indiana in the United States and from Germany and the Czech Republic in Europe from 2008 through 2012. In addition, an isolate from Japan was characterized. We found 3 distinct genotypes, 1 for North America, 1 for Europe, and 1 for Japan. We found B. miyamotoi infection in ticks in 16 of the 26 sites surveyed, with infection prevalence as high as 15.4%. These results show the widespread distribution of the pathogen, indicating an exposure risk to humans in areas where Ixodes ticks reside.
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Borrelia/classificação , Borrelia/isolamento & purificação , Ixodes/microbiologia , Animais , Borrelia/genética , Europa (Continente) , Genótipo , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , Prevalência , Espectrometria de Massas por Ionização por Electrospray , Estados UnidosRESUMO
Abstract Ticks harbor numerous pathogens of significance to human and animal health. A better understanding of the pathogens carried by ticks in a given geographic area can alert health care providers of specific health risks leading to better diagnosis and treatments. In this study, we tested 226 Ixodes ricinis ticks from Southern Germany using a broad-range PCR and electrospray ionization mass spectrometry assay (PCR/ESI-MS) designed to identify tick-borne bacterial and protozoan pathogens in a single test. We found 21.2% of the ticks tested carried Borrelia burgdorferi sensu lato consisting of diverse genospecies; a surprisingly high percentage of ticks were infected with Babesia microti (3.5%). Other organisms found included Borrelia miyamotoi, Rickettsia helvetica, Rickettsia monacensis, and Anaplasma phagocytophilum. Of further significance was our finding that more than 7% of ticks were infected with more than one pathogen or putative pathogen.
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Babesia microti/crescimento & desenvolvimento , Anaplasma phagocytophilum/genética , Anaplasma phagocytophilum/isolamento & purificação , Animais , Vetores Artrópodes/classificação , Vetores Artrópodes/crescimento & desenvolvimento , Vetores Artrópodes/microbiologia , Babesia/genética , Babesia/crescimento & desenvolvimento , Babesia/isolamento & purificação , Babesia microti/genética , Babesia microti/isolamento & purificação , Borrelia/genética , Borrelia/isolamento & purificação , DNA Bacteriano/genética , Alemanha/epidemiologia , Humanos , Ixodes/microbiologia , Ixodes/parasitologia , Reação em Cadeia da Polimerase/métodos , Prevalência , Rickettsia/genética , Rickettsia/isolamento & purificação , Espectrometria de Massas por Ionização por Electrospray/métodos , Doenças Transmitidas por CarrapatosAssuntos
Técnicas Biossensoriais , Hospitais , Infecções/diagnóstico , Infecções Bacterianas/diagnóstico , DNA Bacteriano/análise , DNA Fúngico/análise , DNA Viral/análise , Humanos , Controle de Infecções/métodos , Espectrometria de Massas , Micoses/diagnóstico , Reação em Cadeia da Polimerase , Viroses/diagnósticoRESUMO
The rapid identification of bacteria and fungi directly from the blood of patients with suspected bloodstream infections aids in diagnosis and guides treatment decisions. The development of an automated, rapid, and sensitive molecular technology capable of detecting the diverse agents of such infections at low titers has been challenging, due in part to the high background of genomic DNA in blood. PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) allows for the rapid and accurate identification of microorganisms but with a sensitivity of about 50% compared to that of culture when using 1-ml whole-blood specimens. Here, we describe a new integrated specimen preparation technology that substantially improves the sensitivity of PCR/ESI-MS analysis. An efficient lysis method and automated DNA purification system were designed for processing 5 ml of whole blood. In addition, PCR amplification formulations were optimized to tolerate high levels of human DNA. An analysis of 331 specimens collected from patients with suspected bloodstream infections resulted in 35 PCR/ESI-MS-positive specimens (10.6%) compared to 18 positive by culture (5.4%). PCR/ESI-MS was 83% sensitive and 94% specific compared to culture. Replicate PCR/ESI-MS testing from a second aliquot of the PCR/ESI-MS-positive/culture-negative specimens corroborated the initial findings in most cases, resulting in increased sensitivity (91%) and specificity (99%) when confirmed detections were considered true positives. The integrated solution described here has the potential to provide rapid detection and identification of organisms responsible for bloodstream infections.