RESUMEN
BACKGROUND: Early and accurate diagnosis of acute infections can help minimize the overprescription of antibiotics and improve patient outcomes. Discrimination between bacterial and viral etiologies in acute infection based on changes in host gene expression has been described. Unfortunately, established technologies used for gene expression profiling are typically expensive and slow, confounding integration into clinical workflows. Here we report the development of an ultra-rapid test system for host gene expression profiling from blood based on quantitative reverse transcription followed by loop-mediated isothermal amplification (qRT-LAMP). METHODS: We developed 10 messenger ribonucleic acid-specific assays based on qRT-LAMP targeting 7 informative biomarkers to discriminate viral from bacterial infections and 3 housekeeping reference genes. We optimized qRT-LAMP formulations to achieve a turnaround time of 12 min without sacrificing specificity or precision. The accuracy of the test system was verified utilizing blood samples from 57 patients and comparing qRT-LAMP results to profiles obtained using an orthogonal reference technology. RESULTS: We observed a Pearson coefficient of 0.90 between bacterial/viral metascores generated by qRT-LAMP and the reference technology. CONCLUSIONS: qRT-LAMP assays can provide sufficiently accurate gene expression profiling data to enable discrimination between bacterial and viral etiologies using an established set of biomarkers and a classification algorithm.
Asunto(s)
Transcripción Reversa , Virosis , Humanos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , ARN Viral/genética , Sensibilidad y Especificidad , Virosis/diagnóstico , Virosis/genéticaRESUMEN
Although widely prevalent, Lyme disease is still under-diagnosed and misunderstood. Here we followed 73 acute Lyme disease patients and uninfected controls over a period of a year. At each visit, RNA-sequencing was applied to profile patients' peripheral blood mononuclear cells in addition to extensive clinical phenotyping. Based on the projection of the RNA-seq data into lower dimensions, we observe that the cases are separated from controls, and almost all cases never return to cluster with the controls over time. Enrichment analysis of the differentially expressed genes between clusters identifies up-regulation of immune response genes. This observation is also supported by deconvolution analysis to identify the changes in cell type composition due to Lyme disease infection. Importantly, we developed several machine learning classifiers that attempt to perform various Lyme disease classifications. We show that Lyme patients can be distinguished from the controls as well as from COVID-19 patients, but classification was not successful in distinguishing those patients with early Lyme disease cases that would advance to develop post-treatment persistent symptoms.
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Leucocitos Mononucleares/inmunología , Enfermedad de Lyme/genética , Adulto , COVID-19/genética , COVID-19/inmunología , Citocinas/genética , Citocinas/inmunología , Femenino , Estudios de Seguimiento , Humanos , Leucocitos Mononucleares/química , Enfermedad de Lyme/sangre , Enfermedad de Lyme/inmunología , Aprendizaje Automático , Masculino , Persona de Mediana Edad , Estudios Prospectivos , RNA-SeqRESUMEN
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.
Asunto(s)
Grupo Borrelia Burgdorferi , Borrelia burgdorferi , Enfermedad de Lyme , Enfermedades por Picaduras de Garrapatas , Borrelia burgdorferi/genética , Grupo Borrelia Burgdorferi/genética , Humanos , Enfermedad de Lyme/diagnóstico , Reacción en Cadena de la PolimerasaRESUMEN
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 terapéutico , Borrelia burgdorferi/efectos de los fármacos , Borrelia burgdorferi/patogenicidad , Enfermedad de Lyme/tratamiento farmacológico , Enfermedad de Lyme/microbiología , Borrelia burgdorferi/genética , Genotipo , Humanos , Reacción en Cadena de la Polimerasa , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
We introduce a new electrochemical measurement method compatible with an enzymatic biosensor that is capable of analyte sensing down to the low nanomolar concentration regime. This method is termed accumulation mode sensing and utilizes an immobilized redox polymer mediator wired to an oxidoreductase enzyme to store charge during a premeasurement charge concentration step, followed by a measurement step in which this accumulated charge is quantified. We demonstrate this new method using a model glucose sensor and show how the sensitivity of a sensor can be modified simply by adjusting the time duration of the charge concentration step. We achieve a limit of detection of 4.7 ± 1.4 nM using accumulation mode sensing, which represents a 25-fold improvement over traditional amperometry.
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Técnicas Biosensibles , Técnicas Electroquímicas , Oxidorreductasas/análisis , Electrodos , Glucosa/química , Oxidorreductasas/metabolismo , Plata/química , Compuestos de Plata/químicaRESUMEN
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.
Asunto(s)
Anaplasma phagocytophilum/aislamiento & purificación , Babesia/aislamiento & purificación , Borrelia burgdorferi/aislamiento & purificación , Ixodes/microbiología , Ixodes/parasitología , Rickettsia/aislamiento & purificación , Infestaciones por Garrapatas , Anaplasma phagocytophilum/genética , Animales , Artiodáctilos , Arvicolinae , Babesia/clasificación , Babesia/genética , Aves , Borrelia burgdorferi/genética , República Checa , Ciervos , Humanos , Ratones , Rickettsia/clasificación , Rickettsia/genética , Sciuridae , Encuestas y Cuestionarios , Sus scrofaRESUMEN
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/clasificación , Anaplasmataceae/aislamiento & purificación , Biodiversidad , Ixodes/microbiología , Anaplasmataceae/genética , Anaplasmataceae/fisiología , Animales , California , Filogenia , Rickettsia/aislamiento & purificación , Rickettsia/fisiología , Análisis de Secuencia de ADN , SimbiosisRESUMEN
The predominant human-biting tick throughout the southeastern United States is Amblyomma americanum. Its ability to transmit pathogens causing Lyme disease-like illnesses is a subject of ongoing controversy. Results of previous testing by the Department of Defense Human Tick Test Kit Program and other laboratories indicated that it is highly unlikely that A. americanum transmits any pathogen that causes Lyme disease. In contrast, a recent publication by Clark and colleagues (K. L. Clark, B. Leydet, and S. Hartman, Int. J. Med. Sci. 10:915-931, 2013) reported detection of Lyme group Borrelia in A. americanum using a nested-flagellin-gene PCR. We evaluated this assay by using it and other assays to test 1,097 A. americanum ticks collected from humans. Using the Clark assay, in most samples we observed nonspecific amplification and nonrepeatability of results on subsequent testing of samples. Lack of reaction specificity and repeatability is consistent with mispriming, likely due to high primer concentrations and low annealing temperatures in this protocol. In six suspect-positive samples, Borrelia lonestari was identified by sequencing of an independent gene region; this is not a Lyme group spirochete and is not considered zoonotic. B. burgdorferi was weakly amplified from one pool using some assays, but not others, and attempts to sequence the amplicon of this pool failed, as did attempts to amplify and sequence B. burgdorferi from the five individual samples comprising this pool. Therefore, B. burgdorferi was not confirmed in any sample. Our results do not support the hypothesis that A. americanum ticks are a vector for Lyme group Borrelia infections.
Asunto(s)
Borrelia burgdorferi/aislamiento & purificación , Ixodidae/microbiología , Animales , Entomología/métodos , Femenino , Humanos , Masculino , Técnicas de Diagnóstico Molecular/métodos , Reacción en Cadena de la Polimerasa/métodos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Sudeste de Estados UnidosRESUMEN
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/clasificación , Borrelia/aislamiento & purificación , Ixodes/microbiología , Animales , Borrelia/genética , Europa (Continente) , Genotipo , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/métodos , Prevalencia , Espectrometría de Masa por Ionización de Electrospray , Estados UnidosRESUMEN
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.
Asunto(s)
Babesia microti/crecimiento & desarrollo , Anaplasma phagocytophilum/genética , Anaplasma phagocytophilum/aislamiento & purificación , Animales , Vectores Artrópodos/clasificación , Vectores Artrópodos/crecimiento & desarrollo , Vectores Artrópodos/microbiología , Babesia/genética , Babesia/crecimiento & desarrollo , Babesia/aislamiento & purificación , Babesia microti/genética , Babesia microti/aislamiento & purificación , Borrelia/genética , Borrelia/aislamiento & purificación , ADN Bacteriano/genética , Alemania/epidemiología , Humanos , Ixodes/microbiología , Ixodes/parasitología , Reacción en Cadena de la Polimerasa/métodos , Prevalencia , Rickettsia/genética , Rickettsia/aislamiento & purificación , Espectrometría de Masa por Ionización de Electrospray/métodos , Enfermedades por Picaduras de GarrapatasRESUMEN
BACKGROUND: Next-generation sequencing sample preparation requires nanogram to microgram quantities of DNA; however, many relevant samples are comprised of only a few cells. Genomic analysis of these samples requires a whole genome amplification method that is unbiased and free of exogenous DNA contamination. To address these challenges we have developed protocols for the production of DNA-free consumables including reagents and have improved upon multiple displacement amplification (iMDA). RESULTS: A specialized ethylene oxide treatment was developed that renders free DNA and DNA present within Gram positive bacterial cells undetectable by qPCR. To reduce DNA contamination in amplification reagents, a combination of ion exchange chromatography, filtration, and lot testing protocols were developed. Our multiple displacement amplification protocol employs a second strand-displacing DNA polymerase, improved buffers, improved reaction conditions and DNA free reagents. The iMDA protocol, when used in combination with DNA-free laboratory consumables and reagents, significantly improved efficiency and accuracy of amplification and sequencing of specimens with moderate to low levels of DNA. The sensitivity and specificity of sequencing of amplified DNA prepared using iMDA was compared to that of DNA obtained with two commercial whole genome amplification kits using 10 fg (~1-2 bacterial cells worth) of bacterial genomic DNA as a template. Analysis showed >99% of the iMDA reads mapped to the template organism whereas only 0.02% of the reads from the commercial kits mapped to the template. To assess the ability of iMDA to achieve balanced genomic coverage, a non-stochastic amount of bacterial genomic DNA (1 pg) was amplified and sequenced, and data obtained were compared to sequencing data obtained directly from genomic DNA. The iMDA DNA and genomic DNA sequencing had comparable coverage 99.98% of the reference genome at ≥1X coverage and 99.9% at ≥5X coverage while maintaining both balance and representation of the genome. CONCLUSIONS: The iMDA protocol in combination with DNA-free laboratory consumables, significantly improved the ability to sequence specimens with low levels of DNA. iMDA has broad utility in metagenomics, diagnostics, ancient DNA analysis, pre-implantation embryo screening, single-cell genomics, whole genome sequencing of unculturable organisms, and forensic applications for both human and microbial targets.
Asunto(s)
Contaminación de ADN , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Análisis de Secuencia de ADN/métodos , ADN/análisis , Óxido de Etileno/farmacología , Genoma Bacteriano , Genoma Humano , Humanos , Indicadores y Reactivos , Reacción en Cadena de la Polimerasa , Reproducibilidad de los ResultadosRESUMEN
BACKGROUND: Animal studies suggest that Borrelia burgdorferi, the agent of Lyme disease, may persist after antibiotic therapy and can be detected by various means including xenodiagnosis using the natural tick vector (Ixodes scapularis). No convincing evidence exists for the persistence of viable spirochetes after recommended courses of antibiotic therapy in humans. We determined the safety of using I. scapularis larvae for the xenodiagnosis of B. burgdorferi infection in humans. METHODS: Laboratory-reared larval I. scapularis ticks were placed on 36 subjects and allowed to feed to repletion. Ticks were tested for B. burgdorferi by polymerase chain reaction (PCR), culture, and/or isothermal amplification followed by PCR and electrospray ionization mass spectroscopy. In addition, attempts were made to infect immunodeficient mice by tick bite or inoculation of tick contents. Xenodiagnosis was repeated in 7 individuals. RESULTS: Xenodiagnosis was well tolerated with no severe adverse events. The most common adverse event was mild itching at the tick attachment site. Xenodiagnosis was negative in 16 patients with posttreatment Lyme disease syndrome (PTLDS) and/or high C6 antibody levels and in 5 patients after completing antibiotic therapy for erythema migrans. Xenodiagnosis was positive for B. burgdorferi DNA in a patient with erythema migrans early during therapy and in a patient with PTLDS. There is insufficient evidence, however, to conclude that viable spirochetes were present in either patient. CONCLUSIONS: Xenodiagnosis using Ixodes scapularis larvae was safe and well tolerated. Further studies are needed to determine the sensitivity of xenodiagnosis in patients with Lyme disease and the significance of a positive result. Clinical Trials Registration NCT01143558.
Asunto(s)
Vectores Arácnidos/microbiología , Ixodes/microbiología , Enfermedad de Lyme/diagnóstico , Xenodiagnóstico/métodos , Animales , Borrelia burgdorferi/genética , Borrelia burgdorferi/aislamiento & purificación , Femenino , Glositis Migratoria Benigna/microbiología , Humanos , Enfermedad de Lyme/transmisión , Masculino , Ratones , Ratones SCID , Persona de Mediana EdadRESUMEN
Early Lyme disease is often difficult to diagnose. Left untreated, symptoms can last for many years leading to chronic health problems. Serological tests for the presence of antibodies that react to Borrelia burgdorferi antigens are generally used to support a clinical diagnosis. Due to the biologically delayed antibody response, serology is negative in many patients in the initial 3 weeks after infection and a single test cannot be used to demonstrate active disease, although certain specialized tests provide strong correlation. Because of these limitations there exists a need for better diagnostics for Lyme disease that can detect Borrelia genomic material at the onset of symptoms.
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Enfermedad de Lyme/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Borrelia/química , Borrelia/inmunología , Borrelia/aislamiento & purificación , Humanos , Enfermedad de Lyme/microbiología , Reacción en Cadena de la Polimerasa/métodos , Pruebas Serológicas/métodos , Espectrometría de Masa por Ionización de Electrospray/métodosRESUMEN
A prospective study was performed to determine the value of direct molecular testing of whole blood for detecting the presence of culturable and unculturable bacteria and yeasts in patients with suspected bloodstream infections. A total of 464 adult and pediatric patients with positive blood cultures matched with 442 patients with negative blood cultures collected during the same period were recruited during a 10-month study. PCR amplification coupled with electrospray ionization mass spectrometry (PCR-ESI-MS) plus blood culture reached an overall agreement of 78.6% in the detection and species-level identification of bacterial and candidal pathogens. Of 33 culture-negative/PCR-ESI-MS-positive specimens, 31 (93.9%) were judged to be truly bacteremic and/or candidemic based on a medical chart review and analytical metrics. Among the 15 culture-positive specimens in which PCR-ESI-MS detected additional bacterial or yeast species, 66.7% and 20.0% of the additional positive specimens by PCR-ESI-MS were judged to be truly or possibly bacteremic and/or candidemic, respectively. Direct analysis of blood samples by PCR-ESI-MS rapidly detects bacterial and yeast pathogens in patients with bloodstream infections. When used in conjunction with blood culture, PCR-ESI-MS enhances the diagnostics of septicemia by shortening test turnaround time and improving yields.
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Bacteriemia/diagnóstico , Candidemia/diagnóstico , Técnicas Microbiológicas/métodos , Técnicas de Diagnóstico Molecular/métodos , Reacción en Cadena de la Polimerasa/métodos , Espectrometría de Masa por Ionización de Electrospray/métodos , Adulto , Anciano , Bacterias/clasificación , Bacterias/aislamiento & purificación , Sangre/microbiología , Candida/clasificación , Candida/aislamiento & purificación , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Factores de TiempoRESUMEN
Acinetobacter spp. are a diverse group of Gram-negative bacteria frequently implicated in nosocomial infections. Genotypic methods have been instrumental in studying Acinetobacter, but few offer high resolution, rapid turnaround time, technical ease and high inter-laboratory reproducibility, which has hampered understanding of disease incidence, transmission patterns and diversity within this genus. Here, we further evaluated multilocus PCR electrospray ionization/mass spectrometry (PCR/ESI-MS), a method that is simple and robust, and provides both species characterization and strain-level resolution of Acinetobacter spp. on a single platform. We examined 125 Acinetobacter isolates from 21 hospitals, laboratories and medical centres spanning four counties in Arizona, USA, using PCR/ESI-MS. We compared PCR/ESI-MS with an in-house amplified fragment length polymorphism (AFLP) genotyping scheme. PCR/ESI-MS demonstrated that Acinetobacter spp. from Arizonan hospitals had similar species and strain distributions to other US civilian hospitals. Furthermore, we showed that the PCR/ESI-MS and AFLP genotypes were highly congruent, with the former having the advantages of robust inter-laboratory reproducibility, rapid turnaround time and simple experimental set-up and data analysis. PCR/ESI-MS is an effective and high-throughput platform for strain typing of Acinetobacter baumannii and for identification of other Acinetobacter spp., including the emerging nosocomial pathogens Acinetobacter pittii and Acinetobacter nosocomialis.
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Acinetobacter/clasificación , Tipificación de Secuencias Multilocus/métodos , Reacción en Cadena de la Polimerasa/métodos , Espectrometría de Masa por Ionización de Electrospray/métodos , Acinetobacter/genética , Acinetobacter/aislamiento & purificación , Análisis del Polimorfismo de Longitud de Fragmentos Amplificados , Arizona , Técnicas de Tipificación Bacteriana/métodos , Genes Bacterianos , Genotipo , Humanos , Filogenia , ARN Bacteriano/genética , ARN Ribosómico 16S/genética , Reproducibilidad de los ResultadosAsunto(s)
Borrelia burgdorferi/aislamiento & purificación , Glositis Migratoria Benigna/diagnóstico , Enfermedad de Lyme/diagnóstico , Adolescente , Adulto , Anticuerpos Antibacterianos/sangre , Borrelia burgdorferi/genética , ADN Bacteriano/sangre , Diagnóstico Diferencial , Glositis Migratoria Benigna/sangre , Glositis Migratoria Benigna/microbiología , Glositis Migratoria Benigna/patología , Humanos , Enfermedad de Lyme/sangre , Enfermedad de Lyme/microbiología , Enfermedad de Lyme/patología , Reacción en Cadena de la PolimerasaRESUMEN
Molecular bioforensic research is dependent on rapid and sensitive methods such as real-time PCR (qPCR) for the identification of microorganisms. The use of synthetic positive control templates containing small modifications outside the primer and probe regions is essential to ensure all aspects of the assay are functioning properly, including the primers and probes. However, a typical qPCR or reverse transcriptase qPCR (qRT-PCR) assay is limited in differentiating products generated from positive controls and biological samples because the fluorescent probe signals generated from each type of amplicon are indistinguishable. Additional methods used to differentiate amplicons, including melt curves, secondary probes, and amplicon sequencing, require significant time to implement and validate and present technical challenges that limit their use for microbial forensic applications. To solve this problem, we have developed a novel application of electrospray ionization mass spectrometry (ESI-MS) to rapidly differentiate qPCR amplicons generated with positive biological samples from those generated with synthetic positive controls. The method has sensitivity equivalent to qPCR and supports the confident and timely determination of the presence of a biothreat agent that is crucial for policymakers and law enforcement. Additionally, it eliminates the need for time-consuming methods to confirm qPCR results, including development and validation of secondary probes or sequencing of small amplicons. In this study, we demonstrate the effectiveness of this approach with microbial forensic qPCR assays targeting multiple biodefense agents (bacterial, viral, and toxin) for the ability to rapidly discriminate between a positive control and a positive sample.
Asunto(s)
Bioterrorismo/prevención & control , ADN Bacteriano/análisis , Ciencias Forenses/métodos , ARN Viral/análisis , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Espectrometría de Masa por Ionización de Electrospray , Clostridium botulinum tipo F/genética , Bacterias Gramnegativas/genética , Virus Hendra/genética , Virus Nipah/genética , Polimorfismo de Nucleótido Simple , Sensibilidad y EspecificidadRESUMEN
Many organisms, such as insects, filarial nematodes, and ticks, contain heritable bacterial endosymbionts that are often closely related to transmissible tickborne pathogens. These intracellular bacteria are sometimes unique to the host species, presumably due to isolation and genetic drift. We used a polymerase chain reaction/electrospray ionization-mass spectrometry assay designed to detect a wide range of vectorborne microorganisms to characterize endosymbiont genetic signatures from Amblyomma americanum (L.), Amblyomma maculatum Koch, Dermacentor andersoni Stiles, Dermacentor occidentalis Marx, Dermacentor variabilis (Say), Ixodes scapularis Say, Ixodes pacificus Cooley & Kohls, Ixodes ricinus (L.), and Rhipicephalus sanguineus (Latreille) ticks collected at various sites and of different stages and both sexes. The assay combines the abilities to simultaneously detect pathogens and closely related endosymbionts and to identify tick species via characterization of their respective unique endosymbionts in a single test.
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Ixodidae/microbiología , Simbiosis , Animales , Larva/microbiología , Ninfa/microbiología , Óvulo/microbiología , Reacción en Cadena de la Polimerasa , Rickettsia/aislamiento & purificación , Especificidad de la Especie , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Technology for comprehensive identification of biothreats in environmental and clinical specimens is needed to protect citizens in the case of a biological attack. This is a challenge because there are dozens of bacterial and viral species that might be used in a biological attack and many have closely related near-neighbor organisms that are harmless. The biothreat agent, along with its near neighbors, can be thought of as a biothreat cluster or a biocluster for short. The ability to comprehensively detect the important biothreat clusters with resolution sufficient to distinguish the near neighbors with an extremely low false positive rate is required. A technological solution to this problem can be achieved by coupling biothreat group-specific PCR with electrospray ionization mass spectrometry (PCR/ESI-MS). The biothreat assay described here detects ten bacterial and four viral biothreat clusters on the NIAID priority pathogen and HHS/USDA select agent lists. Detection of each of the biothreat clusters was validated by analysis of a broad collection of biothreat organisms and near neighbors prepared by spiking biothreat nucleic acids into nucleic acids extracted from filtered environmental air. Analytical experiments were carried out to determine breadth of coverage, limits of detection, linearity, sensitivity, and specificity. Further, the assay breadth was demonstrated by testing a diverse collection of organisms from each biothreat cluster. The biothreat assay as configured was able to detect all the target organism clusters and did not misidentify any of the near-neighbor organisms as threats. Coupling biothreat cluster-specific PCR to electrospray ionization mass spectrometry simultaneously provides the breadth of coverage, discrimination of near neighbors, and an extremely low false positive rate due to the requirement that an amplicon with a precise base composition of a biothreat agent be detected by mass spectrometry.