RESUMO
A patient with asplenia and multiple red blood cell transfusions acquired babesiosis infection with Babesia divergens-like/MO-1 organisms and not Babesia microti, the common United States species. He had no known tick exposure. This is believed to be the first transfusion-transmitted case and the fifth documented case of B. divergens-like/MO-1 infection.
Assuntos
Babesiose/transmissão , Transfusão de Sangue , Idoso de 80 Anos ou mais , Arkansas , Babesia/isolamento & purificação , Babesiose/tratamento farmacológico , Babesiose/parasitologia , Clindamicina/administração & dosagem , Clindamicina/uso terapêutico , Doxiciclina/administração & dosagem , Doxiciclina/uso terapêutico , Evolução Fatal , Humanos , Masculino , Transfusão de Plaquetas , Quinina/administração & dosagem , Quinina/uso terapêutico , Estados UnidosRESUMO
We have previously described a novel taxon of the genus Ehrlichia (type strain WisconsinT), closely related to Ehrlichia muris, that causes human ehrlichiosis among patients with exposures to ticks in the upper midwestern USA. DNA from this bacterium was also detected in Ixodes scapularis and Peromyscus leucopus collected in Minnesota and Wisconsin. To determine the relationship between the E. muris-like agent (EMLA) and other species of the genus Ehrlichia phenotypic, genotypic and epidemiologic comparisons were undertaken, including sequence analysis of eight gene loci (3906 nucleotides) for 39 EMLA DNA samples and the type strain of E. muris AS145T. Three loci were also sequenced from DNA of nine strains of E. muris from mouse spleens from Japan. All sequences from E. muris were distinct from homologous EMLA sequences, but differences between them were less than those observed among other species of the genus Ehrlichia. Phenotypic comparison of EMLA and E. muris revealed similar culture and electron microscopic characteristics, but important differences were noted in their geographic distribution, ecological associations and behavior in mouse models of infection. Based on these comparisons, we propose that type strain WisconsinT represents a novel subspecies, Ehrlichia murissubsp. eauclairensis,subsp. nov. This strain is available through the Centers for Disease Control and Prevention Rickettsial Isolate Reference Collection (CRIRC EMU002T) and through the Collection de Souches de l'Unité des Rickettsies (CSURP2883 T). The subspecies Ehrlichia murissubsp. muris subsp. nov. is automatically created and the type strain AS145T is also available through the same collections (CRIRC EMU001T, CSUR E2T). Included is an emended description of E. muris.
Assuntos
Ehrlichia/classificação , Ixodes/microbiologia , Filogenia , Animais , Técnicas de Tipagem Bacteriana , DNA Bacteriano/genética , Ehrlichia/genética , Ehrlichia/isolamento & purificação , Ehrlichiose/microbiologia , Feminino , Humanos , Japão , Camundongos , Minnesota , Peromyscus/microbiologia , Análise de Sequência de DNA , WisconsinRESUMO
There is an increasing need for highly sensitive and quantitative diagnostics at the point-of-care. The lateral flow immunoassay (LFA) is one of the most widely used point-of-care diagnostic tests; however, LFAs generally suffer from low sensitivity and lack of quantification. To overcome these limitations, thermal contrast amplification (TCA) is a new method that is based on the laser excitation of gold nanoparticles (GNPs), the most commonly used visual signature, to evoke a thermal signature. To facilitate the clinical translation of the TCA technology, we present the development of a TCA reader, a platform technology that significantly improves the limit of detection and provides quantification of disease antigens in LFAs. This TCA reader provides enhanced sensitivity over visual detection by the human eye or by a colorimetric reader (e.g., BD Veritor System Reader). More specifically, the TCA reader demonstrated up to an 8-fold enhanced analytical sensitivity and quantification among LFAs for influenza, malaria, and Clostridium difficile. Systematic characterization of the laser, infrared camera, and other components of the reader and their integration into a working reader instrument are described. The development of the TCA reader enables simple, highly sensitive quantification of LFAs at the point-of-care.
Assuntos
Clostridioides difficile/isolamento & purificação , Imunoensaio/métodos , Influenza Humana/diagnóstico , Malária/diagnóstico , Testes Imediatos , Temperatura , Ouro/química , Humanos , Nanopartículas Metálicas/químicaRESUMO
Lyme borreliosis (LB) is a multisystem disease caused by spirochetes in the Borrelia burgdorferisensu lato (Bbsl) genospecies complex. We previously described a novel Bbsl genospecies (type strain MN14-1420T) that causes LB among patients with exposures to ticks in the upper midwestern USA. Patients infected with the novel Bbsl genospecies demonstrated higher levels of spirochetemia and somewhat differing clinical symptoms as compared with those infected with other Bbsl genospecies. The organism was detected from human specimens using PCR, microscopy, serology and culture. The taxonomic status was determined using an eight-housekeeping-gene (uvrA, rplB, recG, pyrG, pepX, clpX, clpA and nifS) multi-locus sequence analysis (MLSA) and comparison of 16S rRNA gene, flaB, rrf-rrl, ospC and oppA2 nucleotide sequences. Using a system threshold of 98.3 % similarity for delineation of Bbsl genospecies by MLSA, we demonstrated that the novel species is a member of the Bbsl genospecies complex, most closely related to B. burgdorferisensu stricto (94.7-94.9 % similarity). This same species was identified in Ixodes scapularis ticks collected in Minnesota and Wisconsin. This novel species, Borrelia mayonii sp. nov, is formally described here. The type strain, MN14-1420, is available through the Deutsche Sammlung von Mikroorganismen und Zelkulturen GmbH (DSM 102811) and the American Type Culture Collection (ATCC BAA-2743).
Assuntos
Grupo Borrelia Burgdorferi/classificação , Ixodes/microbiologia , Filogenia , Animais , Técnicas de Tipagem Bacteriana , Grupo Borrelia Burgdorferi/genética , Grupo Borrelia Burgdorferi/isolamento & purificação , DNA Bacteriano/genética , Feminino , Genes Bacterianos , Humanos , Doença de Lyme , Meio-Oeste dos Estados Unidos , Minnesota , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , WisconsinRESUMO
An Ehrlichia muris-like (EML) pathogen was detected among 4 patients in Minnesota and Wisconsin during 2009. We characterized additional cases clinically and epidemiologically. During 2004-2013, blood samples from 75,077 patients from all 50 United States were tested by PCR from the groEL gene for Ehrlichia spp. and Anaplasma phagocytophilum. During 2007-2013, samples from 69 (0.1%) patients were positive for the EML pathogen; patients were from 5 states: Indiana (1), Michigan (1), Minnesota (33), North Dakota (3), and Wisconsin (31). Most (64%) patients were male; median age was 63 (range 15-94) years; and all 69 patients reported likely tick exposure in Minnesota or Wisconsin. Fever, malaise, thrombocytopenia, and lymphopenia were the most common symptoms. Sixteen (23%) patients were hospitalized (median 4 days); all recovered, and 96% received doxycycline. Infection with the EML pathogen should be considered for persons reporting tick exposure in Minnesota or Wisconsin.
Assuntos
Anaplasma phagocytophilum/patogenicidade , Anaplasmataceae/patogenicidade , Testes Sorológicos/métodos , Carrapatos/parasitologia , Zoonoses/epidemiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Anaplasma phagocytophilum/genética , Anaplasmataceae/genética , Animais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Minnesota/epidemiologia , Wisconsin/epidemiologia , Zoonoses/transmissão , Zoonoses/virologiaRESUMO
BACKGROUND: Ehrlichiosis is a clinically important, emerging zoonosis. Only Ehrlichia chaffeensis and E. ewingii have been thought to cause ehrlichiosis in humans in the United States. Patients with suspected ehrlichiosis routinely undergo testing to ensure proper diagnosis and to ascertain the cause. METHODS: We used molecular methods, culturing, and serologic testing to diagnose and ascertain the cause of cases of ehrlichiosis. RESULTS: On testing, four cases of ehrlichiosis in Minnesota or Wisconsin were found not to be from E. chaffeensis or E. ewingii and instead to be caused by a newly discovered ehrlichia species. All patients had fever, malaise, headache, and lymphopenia; three had thrombocytopenia; and two had elevated liver-enzyme levels. All recovered after receiving doxycycline treatment. At least 17 of 697 Ixodes scapularis ticks collected in Minnesota or Wisconsin were positive for the same ehrlichia species on polymerase-chain-reaction testing. Genetic analyses revealed that this new ehrlichia species is closely related to E. muris. CONCLUSIONS: We report a new ehrlichia species in Minnesota and Wisconsin and provide supportive clinical, epidemiologic, culture, DNA-sequence, and vector data. Physicians need to be aware of this newly discovered close relative of E. muris to ensure appropriate testing, treatment, and regional surveillance. (Funded by the National Institutes of Health and the Centers for Disease Control and Prevention.).
Assuntos
Ehrlichia/classificação , Ehrlichiose/microbiologia , Ixodes/microbiologia , Zoonoses/microbiologia , Animais , Ehrlichia/genética , Ehrlichia/isolamento & purificação , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Minnesota , Filogenia , Reação em Cadeia da Polimerase , Wisconsin , Adulto JovemRESUMO
A 65-year-old female with a history of multiple tick bites presented with fever and pancytopenia. Intracytoplasmic rickettsial morulae were detected on peripheral smear and bone marrow biopsy specimens, and PCR amplified Ehrlichia ewingii DNA from both specimens. To our knowledge, this is the first report of E. ewingii infection of human bone marrow.
Assuntos
Ehrlichia/isolamento & purificação , Ehrlichiose/diagnóstico , Ehrlichiose/patologia , Osteomielite/diagnóstico , Osteomielite/patologia , Idoso , Sangue/microbiologia , Medula Óssea/microbiologia , Medula Óssea/patologia , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Ehrlichiose/microbiologia , Feminino , Granulócitos/microbiologia , Humanos , Microscopia , Osteomielite/microbiologia , Reação em Cadeia da Polimerase , Picadas de Carrapatos/complicaçõesRESUMO
The detection of pathogens associated with gastrointestinal disease may be important in certain patient populations, such as immunocompromised hosts, the critically ill, or individuals with prolonged disease that is refractory to treatment. In this study, we evaluated two commercially available multiplex panels (the FilmArray gastrointestinal [GI] panel [BioFire Diagnostics, Salt Lake City, UT] and the Luminex xTag gastrointestinal pathogen panel [GPP] [Luminex Corporation, Toronto, Canada]) using Cary-Blair stool samples (n = 500) submitted to our laboratory for routine GI testing (e.g., culture, antigen testing, microscopy, and individual real-time PCR). At the time of this study, the prototype (non-FDA-cleared) FilmArray GI panel targeted 23 pathogens (14 bacterial, 5 viral, and 4 parasitic), and testing of 200 µl of Cary-Blair stool was recommended. In contrast, the Luminex GPP assay was FDA cleared for the detection of 11 pathogens (7 bacterial, 2 viral, and 2 parasitic), but had the capacity to identify 4 additional pathogens using a research-use-only protocol. Importantly, the Luminex assay was FDA cleared for 100 µl raw stool; however, 100 µl Cary-Blair stool was tested by the Luminex assay in this study. Among 230 prospectively collected samples, routine testing was positive for one or more GI pathogens in 19 (8.3%) samples, compared to 76 (33.0%) by the FilmArray and 69 (30.3%) by the Luminex assay. Clostridium difficile (12.6 to 13.9% prevalence) and norovirus genogroup I (GI)/GII (5.7 to 13.9% prevalence) were two of the pathogens most commonly detected by both assays among prospective samples. Sapovirus was also commonly detected (5.7% positive rate) by the FilmArray assay. Among 270 additional previously characterized samples, both multiplex panels demonstrated high sensitivity (>90%) for the majority of targets, with the exception of several pathogens, notably Aeromonas sp. (23.8%) by FilmArray and Yersinia enterocolitica (48.1%) by the Luminex assay. Interestingly, the FilmArray and Luminex panels identified mixed infections in 21.1% and 13.0% of positive prospective samples, respectively, compared to only 8.3% by routine methods.
Assuntos
Infecções Bacterianas/diagnóstico , Fezes/microbiologia , Fezes/parasitologia , Gastroenteropatias/diagnóstico , Enteropatias Parasitárias/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Viroses/diagnóstico , Animais , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Infecções Bacterianas/microbiologia , Fezes/virologia , Gastroenteropatias/microbiologia , Gastroenteropatias/parasitologia , Gastroenteropatias/virologia , Humanos , Enteropatias Parasitárias/parasitologia , Parasitos/classificação , Parasitos/genética , Parasitos/isolamento & purificação , Estudos Prospectivos , Sensibilidade e Especificidade , Viroses/virologia , Vírus/classificação , Vírus/genética , Vírus/isolamento & purificaçãoRESUMO
Ehrlichiosis is a tick-borne disease that ranges in severity from asymptomatic infection to fatal sepsis. Ehrlichiosis acquired from transfusion of blood products has not been documented in the literature to date. A case of Ehrlichia ewingii infection likely transmitted by transfusion of leukoreduced platelets is described, and public health implications are discussed.
Assuntos
Ehrlichia/isolamento & purificação , Ehrlichiose/transmissão , Transfusão de Plaquetas/efeitos adversos , Doadores de Sangue , Criança , Ehrlichia/imunologia , Ehrlichiose/diagnóstico , Ehrlichiose/tratamento farmacológico , Humanos , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras/microbiologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapiaRESUMO
BACKGROUND: The gold standard for malaria diagnosis is the examination of thick and thin blood films. Thick films contain 10 to 20 times more blood than thin films, correspondingly providing increased sensitivity for malaria screening. A potential complication of thick film preparations is sloughing of the blood droplet from the slide during staining or rinsing, resulting in the loss of sample. In this work, two methods for improving thick film slide adherence ('scratch' (SCM) and 'acetone dip' (ADM) methods) were compared to the 'standard method' (SM) of thick film preparation. METHODS: Standardized blood droplets from 26 previously examined EDTA whole blood specimens (22 positive and four negative) were concurrently spread on glass slides using the SM, ADM, and SCM. For the SM and ADM prepared slides, the droplet was gently spread to an approximate 22 millimeters in diameter spot on the slide using the edge of a second glass slide. For the SCM, the droplet was spread by carefully grinding (or scratching) it into the slide with the point of a second glass slide. Slides were dried for one hour in a laminar flow hood. For the ADM, slides were dipped once in an acetone filled Coplin jar and allowed to air dry. All slides were then Giemsa-stained and examined in a blinded manner. Adherence was assessed by blinded reviewers. RESULTS: No significant or severe defects were observed for slides prepared with the SCM. In contrast, 8 slides prepared by the ADM and 3 prepared using the SM displayed significant or severe defects. Thick films prepared by the three methods were microscopically indistinguishable and concordant results (positive or negative) were obtained for the three methods. Estimated parasitaemia of the blood samples ranged from 25 to 429,169 parasites/µL of blood. CONCLUSIONS: The SCM is an inexpensive, rapid, and simple method that improves the adherence of thick blood films to standard glass slides without altering general slide preparation, microscopic appearance or interpretability. Using the SCM, thick films can be reliably examined less than two hours after sample receipt. This represents a significant diagnostic improvement over protocols requiring extended drying periods.
Assuntos
Sangue/parasitologia , Técnicas de Laboratório Clínico/métodos , Malária/diagnóstico , Microscopia/métodos , Parasitologia/métodos , Manejo de Espécimes/métodos , Humanos , Parasitemia/diagnóstico , Coloração e Rotulagem/métodosRESUMO
Human adenoviruses (HAdVs) are ubiquitous double-stranded DNA viruses that cause a wide array of diseases in humans including pharyngitis, pneumonia, gastroenteritis, hemorrhagic cystitis, and keratoconjunctivitis. They also cause life-threatening opportunistic infections in immunocompromised individuals and are responsible for outbreaks in certain populations. Diagnosis is traditionally by cell culture or antigen detection methods. However, some HAdVs can take up to 4 weeks to isolate, and diarrheagenic types 40 and 41 will not grow in routine cell culture. Therefore, the goal of this study was to design a rapid, real-time PCR assay to detect all known 57 HAdV types from multiple different specimen sources. Primers and fluorescence resonance energy transfer hybridization probes were designed to target a 185-bp region of the penton base gene of HAdV. The analytical sensitivity was determined to be 10 copies/µl for HAdV types showing exact primer/probe homology in specimen matrix. Using whole-virus strains, the analytical sensitivity for representative HAdV types ranged from 10(-1) to 10(3) 50% tissue culture infective dose (TCID(50))/ml. The assay demonstrated 100% sensitivity and 99% specificity. This real-time PCR assay provides a rapid method for the detection of all 57 known HAdV types from respiratory specimens, blood, stool, urine, and ocular swabs.
Assuntos
Infecções por Adenovirus Humanos/diagnóstico , Infecções por Adenovirus Humanos/virologia , Adenovírus Humanos/classificação , Adenovírus Humanos/isolamento & purificação , Técnicas de Laboratório Clínico/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Virologia/métodos , Adenovírus Humanos/genética , Primers do DNA/genética , Transferência Ressonante de Energia de Fluorescência , Humanos , Sondas de Oligonucleotídeos/genética , Sensibilidade e EspecificidadeRESUMO
Campylobacter jejuni and Salmonella, Shigella, and Yersinia species (along with Shiga toxin-producing Escherichia coli) are the most common causes of acute bacterial diarrheal disease in the United States. Current detection techniques are time-consuming, limiting usefulness for patient care. We developed and validated a panel of rapid PCR assays for the detection and identification of C. jejuni, C. coli, Salmonella, and Yersinia species and Shigella and enteroinvasive E. coli in stool samples. A total of 392 archived stool specimens, previously cultured for enteric pathogens, were evaluated by PCR. Overall, 104 stool specimens had been culture positive (C. jejuni/coli [n = 51], Salmonella species [n = 42], Shigella species [n = 6], and Yersinia species [n = 5]). Compared to culture, the overall sensitivity and specificity of PCR detection of these organisms were 92 and 98% (96/104 and 283/288), respectively, from fresh or Cary Blair stool (P = 0.41); 87 and 98% (41/47 and 242/246), respectively, from fresh stool (P = 0.53); and 96 and 98% (55/57 and 41/42), respectively, from Cary Blair stool (P = 0.56). For individual genera, PCR was as sensitive as the culture method, with the exception of Salmonella culture using selenite enrichment for which PCR was less sensitive than culture from fresh, but not Cary Blair (P = 0.03 and 1.00, respectively) stools. This PCR assay panel for the rapid diagnosis of acute infectious bacterial diarrheal pathogens has a sensitivity and specificity equivalent to that of culture for stools in Cary Blair transport medium. Paired with reflexive culture of stools testing positive, this should provide an improvement in care for patients with acute infectious diarrheal disease.
Assuntos
Técnicas Bacteriológicas/métodos , Infecções por Campylobacter/diagnóstico , Campylobacter/isolamento & purificação , Infecções por Enterobacteriaceae/diagnóstico , Enterobacteriaceae/isolamento & purificação , Fezes/microbiologia , Reação em Cadeia da Polimerase/métodos , Infecções por Campylobacter/microbiologia , Diarreia/microbiologia , Infecções por Enterobacteriaceae/microbiologia , Humanos , Sensibilidade e Especificidade , Fatores de Tempo , Estados UnidosRESUMO
Shiga toxin (Stx)-producing Escherichia coli (STEC) bacteria are a frequent cause of food-borne gastroenteritis, hemorrhagic colitis, and hemolytic uremic syndrome. Because antimicrobial agents are generally contraindicated in patients infected with STEC, a sensitive and specific diagnostic test with rapid turnaround is essential. Current culture methods may fail to detect non-O157 STEC. We evaluated a Stx gene real-time PCR assay using hybridization probes and the LightCycler instrument with 204 prospectively collected stool specimens, which were also tested for Stx by enzyme immunoassay (EIA) (ProSpecT STEC; Remel, Lenexa, KS) and by culturing on chromogenic agar (Chromagar O157; BD BBL, Sparks, MD). In addition, 85 archived stool specimens previously tested for Stx (by EIA) and/or E. coli O157:H7 (by culture) were tested by PCR. Sample preparation for PCR included mixing the stool in sterile water and extraction of nucleic acid using the MagNA Pure LC instrument (Roche Diagnostics). The PCR assay had 100% sensitivity and specificity compared to EIA and culture for specimens collected prospectively (4 of 204 specimens were positive) and compared to culture and/or EIA for archival specimens (42 of 85 specimens were positive). Both the EIA and PCR produced positive results from a specimen containing an O103 serotype STEC in the prospective specimens, and the PCR test detected three positive specimens that contained nonviable STEC in the archived specimens. The PCR assay demonstrated 100% sensitivity and specificity compared to EIA and/or culture and more rapid turnaround than either EIA or culture.
Assuntos
Infecções por Escherichia coli/microbiologia , Fezes/microbiologia , Reação em Cadeia da Polimerase/métodos , Escherichia coli Shiga Toxigênica/isolamento & purificação , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Escherichia coli O157/genética , Escherichia coli O157/crescimento & desenvolvimento , Escherichia coli O157/isolamento & purificação , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/imunologia , Humanos , Técnicas Imunoenzimáticas/métodos , Sondas de Oligonucleotídeos/genética , Sensibilidade e Especificidade , Toxina Shiga/genética , Toxina Shiga/imunologia , Escherichia coli Shiga Toxigênica/genética , Escherichia coli Shiga Toxigênica/crescimento & desenvolvimentoRESUMO
OBJECTIVES: Pathogenic free-living amebae (FLAs) cause skin, ocular, and central nervous system (CNS) infections with significant morbidity and mortality. Diagnosis of FLA infections by pathologic examination of tissue sections can be aided using molecular assays. This study investigated the performance characteristics of a multiplex real-time polymerase chain reaction (PCR) assay (FLA-PCR) for detection and differentiation of FLAs in clinical specimens. METHODS: FLA-PCR was performed on 39 human specimens comprising one cutaneous, 14 corneal, and 24 CNS formalin-fixed, paraffin-embedded (FFPE) tissues with a histopathologic diagnosis of FLA infection and four CNS FFPE tissues with inflammation but no evidence of FLAs. In addition, clinical specificity and assay limit of detection were determined. RESULTS: FLA detection sensitivities ranged from 79% to 84% in FFPE tissues. No cross-reactivity was observed. CONCLUSIONS: While sensitivity is limited, FLA-PCR assay may serve as a useful adjunct for detection or confirmation of FLA infections in FFPE tissues.
Assuntos
Amebíase/diagnóstico , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Acanthamoeba , Balamuthia mandrillaris , Formaldeído , Humanos , Naegleria fowleri , Inclusão em Parafina , Sensibilidade e Especificidade , Fixação de TecidosRESUMO
We have developed a rapid real-time PCR method using fluorescence resonance energy transfer probes and the LightCycler (Roche Diagnostics), which will detect the presence of the tcdC gene of Clostridium difficile in stool samples. Our PCR method also will identify the presence of base pair deletions, one of which (18 bp) has been associated with the "epidemic" toxin-hyperproducing strains. We compared the results of this PCR with those of three C. difficile toxin-detecting enzyme immunoassays (EIAs), an EIA for the detection of glutamate dehydrogenase (GDH), and culture of C. difficile. A total of 200 stool specimens were studied by the methods under comparison. C. difficile was isolated from 49 specimens by culture, and 44 of these were confirmed as containing one of the genes associated with toxin production ("toxigenic culture"). Using toxigenic culture as the "gold standard", the sensitivities, specificities, and positive and negative predictive values, respectively, of the assays were 48%, 98%, 88%, and 87% for the Premier toxin A and B test; 48%, 99%, 91%, and 87% for the ImmunoCard toxin A & B test; 48%, 84%, 46%, and 85% for the Xpect C. difficile toxin A/B test; 32%, 100%, 100%, and 84% for the Triage C. difficile panel (for toxin A); and 86%, 97%, 90%, and 96% for the LightCycler PCR. Thus, in comparison to the sensitivity of toxigenic culture, the sensitivities of the toxin immunoassays were unacceptably low, while the LightCycler real-time PCR assay for the detection of the tcdC gene of C. difficile is sensitive and specific.
Assuntos
Proteínas de Bactérias/análise , Proteínas de Bactérias/genética , Toxinas Bacterianas/análise , Clostridioides difficile/isolamento & purificação , Meios de Cultura , Enterotoxinas/análise , Imunoensaio/métodos , Reação em Cadeia da Polimerase/métodos , Proteínas Repressoras/genética , Técnicas Bacteriológicas , Clostridioides difficile/genética , Clostridioides difficile/imunologia , Sondas de DNA , Enterocolite Pseudomembranosa/diagnóstico , Enterocolite Pseudomembranosa/microbiologia , Transferência Ressonante de Energia de Fluorescência , Humanos , Valor Preditivo dos Testes , Sensibilidade e EspecificidadeRESUMO
We performed a retrospective review of 23,777 specimens tested by our Lyme real-time polymerase chain reaction assay to determine the percent positive rates by specimen source. The percent positive rates were highest in synovial fluid (6.4%) and tissue (6.5%), and lowest in blood (0.1%) and cerebrospinal fluid (0.09%).
Assuntos
Borrelia burgdorferi/isolamento & purificação , Doença de Lyme , Reação em Cadeia da Polimerase/métodos , Proteínas de Bactérias/análise , Líquido Cefalorraquidiano/microbiologia , Humanos , Reprodutibilidade dos Testes , Estudos Retrospectivos , Líquido Sinovial/microbiologiaRESUMO
The Ehrlichia muris-like agent (EMLA) is an emerging, tick-transmitted human pathogen that occurs in the upper Midwestern United States. Here, we describe the development and validation of a p13-based quantitative real-time PCR TaqMan assay to detect EMLA in blood or tissues of ticks, humans, and rodents. The primer and probe specificities of the assay were ascertained using a large panel of various Ehrlichia species and other members of Rickettsiales. In addition to control DNA, both non-infected and EMLA-infected human blood, Mus musculus blood, and M. musculus tissue extracts were evaluated, as were non-infected and EMLA-infected Ixodes scapularis and uninfected Dermacentor variabilis DNA lysates. The specificity of the probe was determined via real-time PCR. An EMLA p13 control plasmid was constructed, and serial dilutions were used to determine the analytical sensitivity, which was found to be 1 copy per 4µl of template DNA. The sensitivity and specificity of this assay provides a powerful tool for ecological studies involving arthropod vectors and their mammalian hosts.
Assuntos
Ehrlichia/isolamento & purificação , Ehrlichiose/epidemiologia , Ehrlichiose/microbiologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Ehrlichia/classificação , Ehrlichia/genética , Humanos , Meio-Oeste dos Estados Unidos , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Lyme borreliosis is the most common tick-borne disease in the northern hemisphere. It is a multisystem disease caused by Borrelia burgdorferi sensu lato genospecies and characterised by tissue localisation and low spirochaetaemia. In this study we aimed to describe a novel Borrelia species causing Lyme borreliosis in the USA. METHODS: At the Mayo clinic, from 2003 to 2014, we tested routine clinical diagnostic specimens from patients in the USA with PCR targeting the oppA1 gene of B burgdorferi sensu lato. We identified positive specimens with an atypical PCR result (melting temperature outside of the expected range) by sequencing, microscopy, or culture. We collected Ixodes scapularis ticks from regions of suspected patient tick exposure and tested them by oppA1 PCR. FINDINGS: 100â545 specimens were submitted by physicians for routine PCR from Jan 1, 2003 to Sept 30, 2014. From these samples, six clinical specimens (five blood, one synovial fluid) yielded an atypical oppA1 PCR product, but no atypical results were detected before 2012. Five of the six patients with atypical PCR results had presented with fever, four had diffuse or focal rash, three had symptoms suggestive of neurological inclusion, and two were admitted to hospital. The sixth patient presented with knee pain and swelling. Motile spirochaetes were seen in blood samples from one patient and cultured from blood samples from two patients. Among the five blood specimens, the median oppA1 copy number was 180 times higher than that in 13 specimens that tested positive for B burgdorferi sensu stricto during the same time period. Multigene sequencing identified the spirochaete as a novel B burgdorferi sensu lato genospecies. This same genospecies was detected in ticks collected at a probable patient exposure site. INTERPRETATION: We describe a new pathogenic Borrelia burgdorferi sensu lato genospecies (candidatus Borrelia mayonii) in the upper midwestern USA, which causes Lyme borreliosis with unusually high spirochaetaemia. Clinicians should be aware of this new B burgdorferi sensu lato genospecies, its distinct clinical features, and the usefulness of oppA1 PCR for diagnosis. FUNDING: US Centers for Disease Control and Prevention Epidemiology and Laboratory Capacity for Infectious Diseases (ELC) Cooperative Agreement and Mayo Clinic Small Grant programme.
Assuntos
Borrelia burgdorferi/classificação , Borrelia burgdorferi/isolamento & purificação , Doença de Lyme/diagnóstico , Infecções por Spirochaetales/sangue , Animais , Borrelia burgdorferi/genética , DNA Bacteriano/genética , Humanos , Doença de Lyme/microbiologia , Reação em Cadeia da Polimerase/métodos , Estados UnidosAssuntos
Ixodes/microbiologia , Ixodes/parasitologia , Anaplasma phagocytophilum/patogenicidade , Animais , Babesia microti/patogenicidade , Borrelia burgdorferi/patogenicidade , Vírus da Encefalite Transmitidos por Carrapatos/patogenicidade , Humanos , Infestações por Carrapato/microbiologia , Infestações por Carrapato/virologia , WisconsinRESUMO
An Ehrlichia muris-like (EML) bacterium was recently detected in humans and Ixodes scapularis ticks in Minnesota and Wisconsin. The reservoir for this agent is unknown. To investigate the occurrence of the EML agent, groEL PCR testing and sequencing was performed on blood from small mammals and white-tailed deer that were collected in areas where human and tick infections were previously demonstrated. DNA of the EML agent was detected in two Peromyscus leucopus of 146 small mammals (1.4%); while 181 O. virginianus tested negative. This report provides the first evidence that DNA from the EML agent is found in P. leucopus, the same animal that is a reservoir for Anaplasma phagocytophilum in this region. The role of white-tailed deer remains inconclusive. Further sampling is warranted to understand the spatial and temporal distribution, transmission and maintenance of this pathogen.