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1.
Discov Med ; 29(157): 129-137, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33002409

RESUMO

Sepsis is a life-threatening clinical condition demanding accurate and rapid diagnosis of the culprit pathogen, thereby to improve prognosis. Pathogen determination through blood culture is the gold standard for diagnosis but has limitations due to low sensitivity. Recently, circulating DNAs derived from pathogenic organisms were found in the plasma of patients with sepsis and were further proved to be more sensitive biomarkers for the diagnosis of the pathogen origin in sepsis. However, the fundamental molecular characteristics of circulating DNA in patients with sepsis remain unclear. Here, we used specific PCR and Sanger sequencing to verify the microbiology culture results via the corresponding plasma circulating DNA. We analyzed the composition and molecular characteristics of circulating DNA in septic patients using next-generation sequencing technology. We showed the presence of pathogen-derived circulating DNA in the plasma of patients with sepsis. The sizes of circulating DNA fragments derived from pathogenic bacteria showed a skewed unimodal distribution, while those derived from host cells showed a normal unimodal distribution. Lengths of fragments at peak concentration for both origins ranged from 150 bp to 200 bp, and reads mapping to pathogenic bacteria genome distributed uniformly on the reference. Our findings have improved our understanding of microbial circulating DNA in patients with sepsis as a potential methodology for the accurate diagnosis of sepsis, especially in light of an urgent need for such a diagnosis associated with the COVID-19 infection.


Assuntos
Infecções Bacterianas/microbiologia , Ácidos Nucleicos Livres/sangue , DNA Bacteriano/sangue , Sepse/microbiologia , Adulto , Idoso , Infecções Bacterianas/complicações , Infecções Bacterianas/diagnóstico , Betacoronavirus , Ácidos Nucleicos Livres/análise , Técnicas de Laboratório Clínico , Infecções por Coronavirus/diagnóstico , Técnicas de Cultura , DNA Bacteriano/análise , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias/complicações , Pandemias , Pneumonia Viral , Reação em Cadeia da Polimerase , Sepse/complicações , Sepse/diagnóstico , Análise de Sequência de DNA
2.
PLoS Negl Trop Dis ; 14(9): e0008662, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32986693

RESUMO

BACKGROUND: Leptospirosis is a widespread zoonosis with global impact, particularly among vulnerable populations in resource-poor settings in tropical countries. Rodents have been considered to be the main reservoir of the disease; however, a wide variety of mammals can act as hosts as well. Here we examine the genetic diversity of Leptospira strains from biological samples of patients and animals in French Polynesia (FP) from 2011 to 2019. METHODOLOGY/PRINCIPAL FINDINGS: From 2011 to 2019, we have collected 444 blood samples from patients diagnosed as having leptospirosis. The limited volume of clinical material and low amount of leptospiral DNA in blood samples led us to develop a nested PCR targeting the secY locus that enabled us to amplify and sequence 244 samples (55%). In addition, 20 Leptospira strains recovered from the blood of patients from 2002 to 2011 were sequenced and fully characterized at the serogroup level and used as reference strains for the association of different phylogenetic branches with respective serogroups. The secY sequences were compared with publicly available sequences from patients and animal reservoirs in FP (n = 79). We identified rats as the main source of infection for L. borgpetersenii serogroup Ballum and L. interrogans serogroup Icterohaemorrhagiae, dogs as the main source of infection for L. interrogans serogroup Australis, and farm pigs as the main source of infection for L. interrogans serogroups Pomona or Canicola. L. interrogans was associated with the most severe infections with 10 and 5 fatal cases due to serogroups Icterohaemorrhagiae and Australis, respectively. Mortality was significantly associated with older age (p-value < 0.001). CONCLUSIONS/SIGNIFICANCE: We described the population dynamics of leptospires circulating among patients in FP, including two patients who were reinfected with unrelated Leptospira genotypes, and clarified the local role of the animal reservoirs in the transmission route of leptospirosis to humans. Routine Leptospira genotyping directly on biological samples should allow the epidemiological follow-up of circulating strains and assess the impact of control interventions on disease transmission.


Assuntos
Genótipo , Leptospira/genética , Leptospirose/epidemiologia , Epidemiologia Molecular , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Proteínas de Bactérias/genética , Criança , DNA Bacteriano/sangue , Cães , Feminino , Seguimentos , Variação Genética , Humanos , Leptospira/classificação , Leptospira/isolamento & purificação , Leptospirose/microbiologia , Leptospirose/transmissão , Masculino , Pessoa de Meia-Idade , Tipagem Molecular , Filogenia , Polinésia/epidemiologia , Ratos , Análise de Sequência de DNA , Sorogrupo , Suínos , Adulto Jovem , Zoonoses/epidemiologia
3.
BMC Infect Dis ; 20(1): 657, 2020 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-32894079

RESUMO

BACKGROUND: Tuberculosis is still a significant diagnostic and therapeutic challenge with high proportion of smear- and culture- negative incidences worldwide. The conventional diagnostic tests are time-consuming and have a low sensitivity. Digital PCR is a novel technology which can detect target sequences with relatively low abundance and obtain the absolute copy numbers of the targets. METHODS: We assessed the accuracy of dPCR in TB diagnosis using more than 250 specimens, and for the first time, we selected M.tuberculosis-specific IS1081 in addition to widely used IS6110 as the amplification targets for dPCR. The quantification of target DNA was calculated using QuantaSoft Version 1.7.4.0917 (BioRad), and SPSS version 13.0 software (SPSS Inc., Chicago, IL, USA) was used for statistical analyses. RESULTS: IS6110-dPCR was more sensitive than IS1081, with the sensitivity and specificity accounting for 40.6 and 93.4% respectively. When we classified the TB patients by personal factors for high copy number of M.tuberculosis derived DNA in plasma: bilateral TB, extrapulmonary TB and disseminated TB, the sensitivity of both IS6110- and IS1081- dPCR was the highest in patients with disseminated TB (IS6110, 100%; IS1081, 68.8%), while their sensitivity was a bit higher in patients with extrapulmonary TB (IS6110, 50.0%; IS1081, 39.3%) than that in bilateral TB (IS6110, 43.3%; IS1081, 33.3%). Compared with traditional TB diagnostic tests, joint detection IS6110 & IS1081-dPCR was not as sensitive as smear microscope or mycobacterial culture, but it was higher than IS6110 qPCR (p < 0.05) and was able to detect 47.4% of smear-negative TB patients. CONCLUSION: Our study suggested that plasma IS6110-dPCR is a rapid, moderate accurate and less-invasive method to detect M.tuberculosis DNA in plasma of TB patients and IS6110 & IS1081-dPCR has a potential to aid diagnosis of smear-negative TB.


Assuntos
Elementos de DNA Transponíveis/genética , DNA Bacteriano/genética , Mycobacterium tuberculosis/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Tuberculose Pulmonar/diagnóstico , Adolescente , Adulto , Idoso , DNA Bacteriano/sangue , Confiabilidade dos Dados , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Adulto Jovem
4.
Science ; 369(6508): 1210-1220, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32788292

RESUMO

Coronavirus disease 2019 (COVID-19) represents a global crisis, yet major knowledge gaps remain about human immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We analyzed immune responses in 76 COVID-19 patients and 69 healthy individuals from Hong Kong and Atlanta, Georgia, United States. In the peripheral blood mononuclear cells (PBMCs) of COVID-19 patients, we observed reduced expression of human leukocyte antigen class DR (HLA-DR) and proinflammatory cytokines by myeloid cells as well as impaired mammalian target of rapamycin (mTOR) signaling and interferon-α (IFN-α) production by plasmacytoid dendritic cells. By contrast, we detected enhanced plasma levels of inflammatory mediators-including EN-RAGE, TNFSF14, and oncostatin M-which correlated with disease severity and increased bacterial products in plasma. Single-cell transcriptomics revealed a lack of type I IFNs, reduced HLA-DR in the myeloid cells of patients with severe COVID-19, and transient expression of IFN-stimulated genes. This was consistent with bulk PBMC transcriptomics and transient, low IFN-α levels in plasma during infection. These results reveal mechanisms and potential therapeutic targets for COVID-19.


Assuntos
Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Pneumonia Viral/imunologia , Citocinas/sangue , DNA Bacteriano/sangue , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Feminino , Citometria de Fluxo , Antígenos HLA-DR/análise , Humanos , Imunidade , Imunidade Inata , Imunoglobulinas/sangue , Imunoglobulinas/imunologia , Mediadores da Inflamação/sangue , Interferon Tipo I/metabolismo , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Lipopolissacarídeos/sangue , Masculino , Células Mieloides/imunologia , Células Mieloides/metabolismo , Pandemias , Transdução de Sinais , Análise de Célula Única , Biologia de Sistemas , Serina-Treonina Quinases TOR/metabolismo , Transcrição Genética , Transcriptoma
5.
Nat Commun ; 11(1): 2607, 2020 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-32451375

RESUMO

Quantification of pathogen and host biomarkers is essential for the diagnosis, monitoring, and treatment of infectious diseases. Here, we demonstrate sensitive and rapid quantification of bacterial load and cytokines from human biological samples to generate actionable hypotheses. Our digital assay measures IL-6 and TNF-α proteins, gram-negative (GN) and gram-positive (GP) bacterial DNA, and the antibiotic-resistance gene blaTEM with femtomolar sensitivity. We use our method to characterize bronchoalveolar lavage fluid from patients with asthma, and find elevated GN bacteria and IL-6 levels compared to healthy subjects. We then analyze plasma from patients with septic shock and find that increasing levels of IL-6 and blaTEM are associated with mortality, while decreasing IL-6 levels are associated with recovery. Surprisingly, lower GN bacteria levels are associated with higher probability of death. Applying decision-tree analysis to our measurements, we are able to predict mortality and rate of recovery from septic shock with over 90% accuracy.


Assuntos
Citocinas/sangue , DNA Bacteriano/sangue , Choque Séptico/imunologia , Choque Séptico/microbiologia , Asma/imunologia , Asma/microbiologia , Carga Bacteriana , Biomarcadores/análise , Biomarcadores/sangue , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/imunologia , Líquido da Lavagem Broncoalveolar/microbiologia , Estudos de Casos e Controles , Citocinas/análise , DNA Bacteriano/genética , Árvores de Decisões , Genes Bacterianos , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/isolamento & purificação , Bactérias Gram-Positivas/genética , Bactérias Gram-Positivas/isolamento & purificação , Interações entre Hospedeiro e Microrganismos/imunologia , Humanos , Interleucina-6/análise , Interleucina-6/sangue , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase Multiplex/estatística & dados numéricos , Prognóstico , Sensibilidade e Especificidade , Choque Séptico/mortalidade , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/sangue , Resistência beta-Lactâmica/genética
6.
J Parasitol ; 106(3): 360-368, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32227225

RESUMO

Anaplasmosis is caused by a Gram-negative obligate intracellular bacterium of the genus Anaplasma with the pathogen having a zoonotic impact. The study aimed to estimate the prevalence of anaplasmosis in Pakistan, to unravel the association of potential risk factors, and to investigate the effect on hematological parameters in affected small ruminants. A total of 150 (n = 75 sheep; n = 75 goats) blood samples were initially screened microscopically and then subjected to PCR targeting the amplification of the 16S rRNA gene fragment of Anaplasma. The PCR-based positive samples were then processed for sequencing. Statistical analysis regarding risk factors was performed using R software. The study revealed an overall 29.33% (44/150) prevalence of anaplasmosis in small ruminants. Sheep had higher (P > 0.05) prevalence (32%) as compared to goats (25.30%). The final statistical model resulting from backward elimination showed only tick infestation as a significant predictor of infection status. The phylogenetic analysis of 16S rRNA gene of Anaplasma spp. revealed 9 study isolates clustered together and showed a close resemblance (99%) with Anaplasma ovis isolate (DQ837600) from Hungary. One of the isolates showed (99%) similarity with the isolate of Anaplasma marginale (MH155594) from Iraq. Furthermore, the hematological parameters pack cell volume, red blood cells, hemoglobin, white blood cells, granulocytes, monocytes, lymphocytes, and platelet count were decreased in Anaplasma-positive animals. This is the first study at the molecular level to characterize Anaplasma spp. in small ruminants of Pakistan, and it will be useful in developing control strategies for anaplasmosis.


Assuntos
Anaplasma/genética , Anaplasmose/parasitologia , Doenças das Cabras/parasitologia , Doenças dos Ovinos/parasitologia , Zoonoses/parasitologia , Anaplasma/classificação , Anaplasma/fisiologia , Anaplasmose/sangue , Anaplasmose/epidemiologia , Animais , Sequência de Bases , DNA Bacteriano/sangue , DNA Bacteriano/isolamento & purificação , Feminino , Doenças das Cabras/sangue , Doenças das Cabras/epidemiologia , Cabras , Incidência , Masculino , Análise Multivariada , Paquistão/epidemiologia , Filogenia , Prevalência , RNA Ribossômico 16S/genética , Análise de Regressão , Fatores de Risco , Alinhamento de Sequência , Fatores Sexuais , Ovinos , Doenças dos Ovinos/sangue , Doenças dos Ovinos/epidemiologia , Zoonoses/sangue , Zoonoses/epidemiologia
7.
Nature ; 579(7800): 567-574, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32214244

RESUMO

Systematic characterization of the cancer microbiome provides the opportunity to develop techniques that exploit non-human, microorganism-derived molecules in the diagnosis of a major human disease. Following recent demonstrations that some types of cancer show substantial microbial contributions1-10, we re-examined whole-genome and whole-transcriptome sequencing studies in The Cancer Genome Atlas11 (TCGA) of 33 types of cancer from treatment-naive patients (a total of 18,116 samples) for microbial reads, and found unique microbial signatures in tissue and blood within and between most major types of cancer. These TCGA blood signatures remained predictive when applied to patients with stage Ia-IIc cancer and cancers lacking any genomic alterations currently measured on two commercial-grade cell-free tumour DNA platforms, despite the use of very stringent decontamination analyses that discarded up to 92.3% of total sequence data. In addition, we could discriminate among samples from healthy, cancer-free individuals (n = 69) and those from patients with multiple types of cancer (prostate, lung, and melanoma; 100 samples in total) solely using plasma-derived, cell-free microbial nucleic acids. This potential microbiome-based oncology diagnostic tool warrants further exploration.


Assuntos
Microbiota/genética , Neoplasias/diagnóstico , Neoplasias/microbiologia , Plasma/microbiologia , Estudos de Casos e Controles , Estudos de Coortes , DNA Bacteriano/sangue , DNA Viral/sangue , Conjuntos de Dados como Assunto , Feminino , Humanos , Biópsia Líquida , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/microbiologia , Masculino , Melanoma/sangue , Melanoma/diagnóstico , Melanoma/microbiologia , Neoplasias/sangue , Neoplasias da Próstata/sangue , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/microbiologia , Reprodutibilidade dos Testes
8.
Nat Microbiol ; 5(3): 465-472, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32066959

RESUMO

In this single-arm non-comparative trial, 13 patients in an Australian hospital with severe Staphylococcus aureus infections were intravenously administered a good manufacturing practice-quality preparation of three Myoviridae bacteriophages (AB-SA01) as adjunctive therapy. AB-SA01 was intravenously administered twice daily for 14 d and the clinical, haematological and blood biochemical parameters of the recipients were monitored for 90 d. The primary outcome was the assessment of safety and tolerability (that is, pain and redness at the infusion site and systemic adverse reactions, such as fever, tachycardia, hypotension, diarrhoea or abdominal pain and the development of renal or hepatic dysfunction). No adverse reactions were reported, and our data indicate that AB-SA01 administered in this way is safe in severe S. aureus infections, including infective endocarditis and septic shock. Future controlled trials will be needed to determine the efficacy of AB-SA01 but no phage resistance evolved in vivo and the measurements of bacterial and phage kinetics in blood samples suggest that 12 h dosing of 109 plaque-forming units may be a rational basis for further studies.


Assuntos
Terapia por Fagos/efeitos adversos , Terapia por Fagos/métodos , Infecções Estafilocócicas/terapia , Staphylococcus aureus/virologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Austrália , Bacteriófagos , DNA Bacteriano/sangue , DNA Viral/sangue , Endocardite/microbiologia , Feminino , Humanos , Cinética , Masculino , Pessoa de Meia-Idade , Myoviridae , Choque Séptico/microbiologia , Resultado do Tratamento , Adulto Jovem
9.
J Biotechnol ; 310: 80-88, 2020 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-32017954

RESUMO

We have identified 24 molecular markers, based on circulating nucleic acids (CNA) originating from the human genome, which in combination can be used in a quantitative real-time PCR (qPCR) assay to identify the presence of human sepsis, starting two to three days before the first clinical signs develop and including patients who meet the SEPSIS-3 criteria. The accuracy was more than 87 % inside of the same patient cohort for which the markers were developed and up to 81 % in blind studies of patient cohorts which were not included in the marker development. As our markers are host-based, they can be used to capture bacterial as well as fungal sepsis, unlike the current PCR-based tests, which require species-specific primer sets for each organism causing human sepsis. Our assay directly uses an aliquot of cell-free blood as the substrate for the PCR reaction, thus allowing to obtain the diagnostic results in three to four hours after the collection of the blood samples.


Assuntos
DNA Bacteriano , DNA Fúngico , Reação em Cadeia da Polimerase em Tempo Real , Sepse , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Estudos de Coortes , DNA Bacteriano/sangue , DNA Bacteriano/genética , DNA Fúngico/sangue , DNA Fúngico/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Sepse/sangue , Sepse/genética , Sepse/microbiologia
10.
Artigo em Inglês | MEDLINE | ID: mdl-31936370

RESUMO

This study was carried out to determine the risk factors of leptospirosis infection among local urban service workers in Sabah. This is a cross-sectional study involving 394 workers in Kota Kinabalu City, Sabah, conducted from February to March 2017. Information on demography, occupational exposures and environmental factors was obtained by a modified validated questionnaire. Polymerase Chain Reaction (PCR) was used to determine the prevalence of positive leptospirae. The overall figure for positive leptospirae was 9.4% (95% CI: 6.8-12.8). Urban sweepers and lorry drivers made up the highest proportion of positive leptospirae respondents, contributing 15.5% and 9.4%, respectively. The significant risk factors for positive leptospirae were older age (p-value = 0.001), higher monthly salary (p-value = 0.039), longer duration of employment (p-value = 0.011) and working as an urban sweeper (p-value = 0.021). Leptospirae was prevalent among healthy urban service workers and relates to their working activities.


Assuntos
Leptospirose/epidemiologia , Doenças Profissionais/epidemiologia , Adulto , Monitoramento Biológico , Cidades , Estudos Transversais , DNA Bacteriano/sangue , Emprego , Feminino , Humanos , Leptospira/genética , Leptospirose/sangue , Leptospirose/microbiologia , Malásia , Masculino , Pessoa de Meia-Idade , Doenças Profissionais/sangue , Doenças Profissionais/microbiologia , Exposição Ocupacional/análise , Reação em Cadeia da Polimerase , Prevalência , Fatores de Risco , Saúde da População Urbana
12.
Parasitol Res ; 119(1): 299-315, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31734862

RESUMO

The capability of imidacloprid 10% + flumethrin 4.5% (Seresto®) collars to prevent transmission of Borrelia burgdorferi sensu lato (Bbsl) and Anaplasma phagocytophilum (Ap) by naturally infected ticks was evaluated in two studies with 44 dogs. In each study, one group served as non-treated control, whereas the other groups were treated with the Seresto® collar. All dogs were exposed to naturally Bbsl- and Ap-infected hard ticks (Ixodes ricinus, Ixodes scapularis). In study 1, tick infestation was performed on study day (SD) 63 (2 months post-treatment [p.t.]); in study 2, it was performed on SD 32 (one month p.t.) respectively SD 219 (seven months p.t.). In situ tick counts were performed 2 days after infestation. Tick counts and removals followed 6 (study 1) or 5 days (study 2) later. Blood sampling was performed for the detection of specific Bbsl and Ap antibodies and, in study 1, for the documentation of Ap DNA by PCR. Skin biopsies were examined for Bbsl by PCR and culture (only study 1). The efficacy against Ixodes spp. was 100% at all time points. In study 1, two of six non-treated dogs became infected with Bbsl, and four of six tested positive for Ap; none of the treated dogs tested positive for Bbsl or Ap. In study 2, ten of ten non-treated dogs became infected with Bbsl and Ap; none of the treated dogs tested positive for Bbsl or Ap; 100% acaricidal efficacy was shown in both studies. Transmission of Bbsl and Ap was successfully blocked for up to 7 months.


Assuntos
Acaricidas/uso terapêutico , Transmissão de Doença Infecciosa/veterinária , Doenças do Cão/tratamento farmacológico , Ehrlichiose/veterinária , Doença de Lyme/veterinária , Infestações por Carrapato/veterinária , Acaricidas/administração & dosagem , Anaplasma phagocytophilum/genética , Anaplasma phagocytophilum/imunologia , Anaplasma phagocytophilum/fisiologia , Animais , Anticorpos Antibacterianos/sangue , Vetores Aracnídeos/microbiologia , Borrelia burgdorferi/genética , Borrelia burgdorferi/imunologia , Borrelia burgdorferi/fisiologia , DNA Bacteriano/sangue , Transmissão de Doença Infecciosa/prevenção & controle , Doenças do Cão/prevenção & controle , Doenças do Cão/transmissão , Cães , Ehrlichiose/prevenção & controle , Ehrlichiose/transmissão , Ixodes/microbiologia , Doença de Lyme/prevenção & controle , Doença de Lyme/transmissão , Neonicotinoides/administração & dosagem , Nitrocompostos/administração & dosagem , Piretrinas/administração & dosagem , Infestações por Carrapato/tratamento farmacológico , Infestações por Carrapato/microbiologia , Infestações por Carrapato/parasitologia , Resultado do Tratamento
13.
J Matern Fetal Neonatal Med ; 33(3): 359-367, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29909752

RESUMO

Objectives: To evaluate if midtrimester maternal serum contains microbial DNA and whether it differs between women with spontaneous preterm birth (SPTB) and those delivering at term.Study design: In this retrospective case-control study, we identified 20 healthy nulliparas with SPTB at 24-33 weeks of a nonanomalous singleton in 2014. Each case was matched by race/ethnicity to a control delivering at 39-40 weeks. Serum samples, collected at 15-20 weeks and stored at -80 C, were thawed and DNA extracted. PCR with primers targeting the 16S rDNA V4 region were used to prepare an amplicon library, sequenced using Illumina MiSeq, and analyzed using quantitative insight into microbial ecology (QIIME). Taxonomy was assigned using Ribosomal Database program (RDP) Classifier (threshold 0.8) against a modified Greengenes database. Differences in number of observed species, microbial alpha-diversity and beta-diversity, and taxa level analyses were undertaken.Results: All 40 samples were included. Women with SPTB had more unique observed species (p = .046) and higher mean alpha-diversity by Shannon index (but not Chao1 or Simpson) (p = .024). Microbial composition was different between groups by Bray-Curtis clustering (p = .03) but not by weighted (p = .13) or unweighted Unifrac (p = .11). Numerous taxa in the Firmicutes, Proteobacteria, and Actinobacteria phyla differed between groups (p < .05).Conclusions: SPTB is associated with distinct microbial DNA changes detected in midtrimester maternal serum.


Assuntos
DNA Bacteriano/sangue , Microbiota , Nascimento Prematuro/microbiologia , Adulto , Feminino , Humanos , Gravidez , Segundo Trimestre da Gravidez , Nascimento Prematuro/sangue , Estudos Retrospectivos , Adulto Jovem
14.
PLoS One ; 14(11): e0224656, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31721817

RESUMO

Repeated quantitative measurement of bacterial DNA on whole blood has been shown to be a promising method for monitoring bloodstream infection (BSI) with selected bacterial species. To enable broad use of this method, we developed a quantitative droplet digital PCR (ddPCR) method for 16S rDNA. It was validated with species-specific ddPCRs for Staphylococcus aureus (nuc), Streptococcus pneumoniae (lytA), and Escherichia coli (uidA) on spiked whole blood samples and on repeated whole blood samples (days 0, 1-2, 3-4, 6-8, and 13-15) from 83 patients with BSI with these pathogens. In these patients, 16S rDNA and species-specific DNA were detected in 60% and 61%, respectively, at least at one time-point. The highest positivity rates were seen in S. aureus BSI, where 92% of the patients were 16S rDNA-positive and 85% nuc-positive. Quantitative 16S rDNA and species-specific DNA showed strong correlations in spiked samples (r = 0.98; p < 0.0001) and clinical samples (r = 0.84; p < 0.0001). Positivity for 16S rDNA was rapidly cleared in patients with S. pneumoniae and E. coli BSI, but more slowly and sometimes persisted, in those with S. aureus BSI. The initial 16S rDNA load was higher in BSI patients with sepsis (Sepsis-3 definition) than without sepsis (median 2.38 vs. 0 lg10 copies/mL; p = 0.031) and in non-survivors than in survivors (median 2.83 vs. 0 lg10 copies/mL; p = 0.006). 16S rDNA ddPCR appears to be a promising method for bacterial DNA monitoring during BSI. The clinical value of such monitoring should be further studied.


Assuntos
DNA Bacteriano/sangue , DNA Ribossômico/análise , Sepse/diagnóstico , Infecções Estafilocócicas/diagnóstico , Escherichia coli/isolamento & purificação , Humanos , Sepse/microbiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/isolamento & purificação
15.
Am J Trop Med Hyg ; 101(6): 1263-1264, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31674302

RESUMO

In July 2018, acute Q fever (AQF) was diagnosed in two Japanese individuals from the same family. They returned to Japan from Malawi, where the epidemiology of AQF is unknown. A child presented to the hospital with high-grade fever without any symptoms, and a mother presented with fever and dry cough. Paired serum antiphase Ⅱ IgM and IgG significantly elevated in the convalescent phase in both cases. Coxiella burnetii gene (IS1111) was detected from the mother's blood sample. They had no reported direct animal contact, but the onset of symptoms coincided with the dry season in Malawi, which may have facilitated environmental dispersal. These cases may serve as an alert for high-risk people to possible AQF spread and underdiagnosis in Malawi.


Assuntos
Anticorpos Antibacterianos/sangue , Família , Febre Q/diagnóstico , Doença Relacionada a Viagens , Doença Aguda , Adulto , Pré-Escolar , Coxiella burnetii , DNA Bacteriano/sangue , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Japão , Malaui , Masculino
16.
BMC Vet Res ; 15(1): 368, 2019 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-31653234

RESUMO

BACKGROUND: The obligate intracellular bacterium Coxiella burnetii causes globally distributed zoonotic Q fever. Ruminant livestock are common reservoirs of C. burnetii. Coxiella burnetii are shed in large numbers in the waste of infected animals and are transmitted by inhalation of contaminated aerosols. This study was conducted to evaluate the prevalence of C. burnetii infection in domestic animals and ticks in areas of Slovenia associated with a history of Q fever outbreaks. RESULTS: A total of 701 ticks were collected and identified from vegetation, domestic animals and wild animals. C. burnetii DNA was detected in 17 out of 701 (2.4%) ticks. No C. burnetii DNA was found in male ticks. Ticks that tested positive in the PCR-based assay were most commonly sampled from wild deer (5.09%), followed by ticks collected from domestic animals (1.16%) and ticks collected by flagging vegetation (0.79%). Additionally, 150 animal blood samples were investigated for the presence of C. burnetii-specific antibodies and pathogen DNA. The presence of pathogen DNA was confirmed in 14 out of 150 (9.3%) blood samples, while specific antibodies were detected in sera from 60 out of 150 (40.4%) animals. CONCLUSIONS: Our results indicate that ticks, although not the primary source of the bacteria, are infected with C. burnetii and may represent a potential source of infection for humans and animals. Ticks collected from animals were most likely found to harbor C. burnetii DNA, and the infection was not lost during molting. The persistence and distribution of pathogens in cattle and sheep indicates that C. burnetii is constantly present in Slovenia.


Assuntos
Coxiella burnetii/isolamento & purificação , Febre Q/veterinária , Carrapatos/microbiologia , Animais , Anticorpos Antibacterianos/sangue , Bovinos , Doenças dos Bovinos/microbiologia , Coxiella burnetii/genética , Coxiella burnetii/imunologia , DNA Bacteriano/sangue , Cervos/microbiologia , Feminino , Masculino , Muda , Reação em Cadeia da Polimerase/veterinária , Prevalência , Febre Q/epidemiologia , Ovinos , Doenças dos Ovinos/microbiologia , Eslovênia/epidemiologia , Zoonoses
17.
Top Companion Anim Med ; 36: 12-15, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31472723

RESUMO

Vector-borne rickettsioses represent emerging threats to public health worldwide. The aim of this work was the screening for the presence of Rickettsia spp. in the blood of dogs and cats from southern Portugal. A PCR product of the expected size was amplified from DNA extracts obtained from blood samples of 29 out of 225 (12.9%) cats and in 2 out of 375 (.5%) dogs using genus-specific primers targeting Rickettsia gltA. Rickettsia conorii israelensis was identified by phylogenetical analysis of partial ompB sequences, amplified from blood samples taken from both a cat and a dog. The obtained results reinforce the idea that domestic animals may act as sentinels for the presence of vector-borne Rickettsia spp. in a given geographical area. In addition, rickettsioses should be included in the differential diagnosis of canine and feline vector-borne diseases.


Assuntos
Doenças do Gato/microbiologia , Doenças do Cão/microbiologia , Infecções por Rickettsia/veterinária , Rickettsia conorii/isolamento & purificação , Animais , Proteínas da Membrana Bacteriana Externa/genética , Gatos , Citrato (si)-Sintase/genética , DNA Bacteriano/sangue , Cães , Reação em Cadeia da Polimerase/veterinária , Portugal/epidemiologia , Infecções por Rickettsia/epidemiologia , Rickettsia conorii/genética , Análise de Sequência de DNA
18.
Int J Infect Dis ; 89: 66-71, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31521852

RESUMO

OBJECTIVES: To determine blood Brucella DNA loads between brucellosis patients and those without brucellosis. METHODS: The patient group included 350 brucellosis patients. The control was composed of 200 subjects without brucellosis. The extracted DNA from blood was tested by quantitative polymerase chain reaction (qPCR). The cutoff value was determined by receiver operating characteristic curve analysis. A portion of the brucellosis patients were monitored by qPCR during therapy. RESULTS: The detection limit of qPCR was between 1E+01cfu/µL and 1E+08cfu/µL. The standard curve R2 reached 0.998. The cutoff value was 4E+01cfu/µL, which was determined by comparison of the patient group and the control. The qPCR assay had a specificity of 100% and a sensitivity of 93.14%. The monitoring results showed that the Brucella DNA load decreased in most patients during the first 4 weeks of treatment. One patient with bad treatment compliance showed a rebound. CONCLUSIONS: The qPCR results were in accordance with the course of brucellosis in the clinic. The DNA load often reflects the situation of the Brucella-infected patient. The cutoff value provides an important reference of infection. This qPCR-based method can be used to assist in the diagnosis of brucellosis and to adjust the therapy.


Assuntos
Brucella/isolamento & purificação , Brucelose/diagnóstico , DNA Bacteriano/sangue , Adulto , Testes de Aglutinação , Medula Óssea/microbiologia , Brucella/efeitos dos fármacos , Brucella/genética , Brucelose/tratamento farmacológico , Brucelose/microbiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade
19.
BMC Infect Dis ; 19(1): 796, 2019 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-31510926

RESUMO

BACKGROUND: The number of new rickettsial species are rapidly increasing, and increasing numbers of Rickettsia raoultii (R. raoultii) infection cases have been detected in humans. However, neurological abnormalities caused by R. raoultii are rarely reported, especially in northwestern China. CASE PRESENTATION: A 36-year-old Kazakh shepherd with an attached tick on part temporalis, presented with right eyelid droop, lethargy, fever, headache, fever (38.0-41.0 °C) and erythematous rash. The examination of cerebrospinal fluid (CSF) showed cerebrospinal pressure of 200 mm H2O, leukocyte count of 300.0 × 106/L, adenosine deaminase of 2.15 U/L, and total protein concentration of 0.93 g/L. The diagnosis of R. raoultii infection was confirmed by six genetic markers, and semi-quantified by enzyme-linked immunosorbent assay for rickettsial antigen. The patient gradually recovered after treatment with doxycycline and ceftriaxone. R. raoultii DNA was found both in a tick detached from this patient and in 0.18% (2/1107) of blood samples collected from local shepherds. CONCLUSIONS: This is the first reported case with neurological abnormalities caused by R. raoultii in northwestern China. It is vital to detect rickettsial agents both in blood and CSF for tick bite patients with neurological abnormalities. Public health workers and physicians should pay attention to neurological abnormalities caused by Rickettsia.


Assuntos
Doenças do Sistema Nervoso/diagnóstico , Infecções por Rickettsia/diagnóstico , Rickettsia/metabolismo , Picadas de Carrapatos/diagnóstico , Adenosina Desaminase/líquido cefalorraquidiano , Adulto , Animais , Antígenos de Bactérias/análise , Antígenos de Bactérias/imunologia , Ceftriaxona/uso terapêutico , China , DNA Bacteriano/sangue , Doxiciclina/uso terapêutico , Humanos , Contagem de Leucócitos , Masculino , Doenças do Sistema Nervoso/etiologia , Filogenia , RNA Ribossômico 16S/metabolismo , Rickettsia/classificação , Rickettsia/genética , Infecções por Rickettsia/complicações , Infecções por Rickettsia/tratamento farmacológico , Picadas de Carrapatos/complicações , Carrapatos/genética
20.
Medicine (Baltimore) ; 98(23): e15724, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31169672

RESUMO

Diagnosis of Q fever is difficult due to the lack of distinct clinical features that distinguish it from other febrile diseases. Serologic testing is the gold standard method for diagnosing Q fever, but antibody formation may not be detectable for 2 to 3 weeks from symptom onset, limiting early diagnosis. We thus evaluated the diagnostic utility of polymerase chain reaction (PCR) to detect Coxellia burnetii DNA in serum from patients with suspected acute Q fever.All adult patients with suspected acute Q fever were prospectively enrolled at a tertiary-care hospital from January 2016 through July 2018. Acute Q fever was diagnosed using clinical and laboratory criteria: fever with at least one other symptoms (myalgia, headache, pneumonia, or hepatitis) and single phase II immunoglobulin G (IgG) antibody titers ≥1:200 or immunoglobulin M (IgM) antibody titer ≥1:50 (probable), or a fourfold increase or seroconversion in phase II IgG antibody titers as measured by indirect immunofluorescence assays between paired samples (confirmed). We performed PCR targeting the transposase gene insertion element IS1111a of C. burnetii.Of the 35 patients with suspected acute Q fever, 16 (46%) were diagnosed with acute Q fever including 8 probable and 8 confirmed cases; the remaining 19 (54%) were diagnosed with other febrile diseases. The proportion of males diagnosed with Q fever was higher than those diagnosed with other febrile diseases (88% vs 44%, P = .03), but there were no other significant differences in clinical characteristics between the 2 groups. The Q fever PCR sensitivity was 81% (95% confidence interval [CI], 54-96), specificity was 90% (95% CI, 67-99), positive predictive value was 87% (95% CI, 63-96), and negative predictive value was 85% (95% CI, 67-94).Q fever PCR testing using blood from patients with suspected acute Q fever seems to be a rapid and useful test for early diagnosis of Q fever.


Assuntos
Coxiella burnetii/genética , DNA Bacteriano/sangue , Reação em Cadeia da Polimerase/estatística & dados numéricos , Febre Q/diagnóstico , Doença Aguda , Adulto , Anticorpos Antibacterianos/sangue , Coxiella burnetii/imunologia , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Estudos Prospectivos , República da Coreia , Sensibilidade e Especificidade
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