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1.
Mem Inst Oswaldo Cruz ; 115: e200370, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33174903

RESUMO

BACKGROUND: Bacillus anthracis is the aetiologic agent of anthrax, a re-emerging, septicaemic, haemorrhagic and lethal disease that affects humans, domestic ruminants and wildlife. Plasmids pXO1 and pXO2 are attributes that confer pathogenicity to B. anthracis strains. This bacterium was used as biological weapon in the World Wars and in the biological attack in the United States of America at 2001. B. anthracis is classified as a Tier 1 bioterrorism agent by the Centers for Diseases Control and Prevention. Anthrax is recognised as a re-emerging disease. Several studies concerning the dynamics of B. anthracis cycle in soil revealed that nonpathogenic B. anthracis strains due to lack of pXO2 plasmid are commonly found in some types of soil. OBJECTIVES: This study aimed isolation and identification of B. anthracis spores in soil samples of the state of Rio de Janeiro, Brazil. METHODS: Phenotypic and genotypic approaches were used to identify isolates including MALDI-TOF/MS, motility test, susceptibility to gamma phage and penicillin, survey for pag and cap genes as surrogates of pXO1 and pXO2 plasmids, respectively, and sequencing of 16SrRNA-encoding gene. Physicochemical analysis of the soil samples were carried out to describe soil characteristics. FINDINGS: We observed the presence of one B. anthracis pXO1+ and pXO2- isolated from clay loam soil; one B. anthracis-like strain pXO1+ and pXO2-isolated from loamy sand; and 10 Bacillus spp. strains sensitive to phage-gamma that need better characterisation to define which their species were recovered from loamy sand. MAIN CONCLUSIONS: This work showed promising results and it was the first study to report results from an active surveillance for B. anthracis in Brazil.


Assuntos
Bacillus anthracis/isolamento & purificação , DNA Bacteriano/genética , Plasmídeos/análise , Reação em Cadeia da Polimerase/métodos , Microbiologia do Solo , Esporos Bacterianos , Fatores de Virulência/genética , Antígenos de Bactérias , Bacillus anthracis/genética , Bacillus anthracis/patogenicidade , Toxinas Bacterianas , Brasil , DNA Bacteriano/análise , Humanos , Plasmídeos/genética , Análise de Sequência de DNA , Solo , Virulência
2.
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
4.
BMC Pediatr ; 20(1): 429, 2020 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-32907595

RESUMO

BACKGROUND: Central and peripheral nervous system symptoms and complications are being increasingly recognized among individuals with pandemic SARS-CoV-2 infections, but actual detection of the virus or its RNA in the central nervous system has rarely been sought or demonstrated. Severe or fatal illnesses are attributed to SARS-CoV-2, generally without attempting to evaluate for alternative causes or co-pathogens. CASE PRESENTATION: A five-year-old girl with fever and headache was diagnosed with acute SARS-CoV-2-associated meningoencephalitis based on the detection of its RNA on a nasopharyngeal swab, cerebrospinal fluid analysis, and magnetic resonance imaging findings. Serial serologic tests for SARS-CoV-2 IgG and IgA showed seroconversion, consistent with an acute infection. Mental status and brain imaging findings gradually worsened despite antiviral therapy and intravenous dexamethasone. Decompressive suboccipital craniectomy for brain herniation with cerebellar biopsy on day 30 of illness, shortly before death, revealed SARS-CoV-2 RNA in cerebellar tissue using the Centers for Disease Control and Prevention 2019-nCoV Real-Time Reverse Transcriptase-PCR Diagnostic Panel. On histopathology, necrotizing granulomas with numerous acid-fast bacilli were visualized, and Mycobacterium tuberculosis complex DNA was detected by PCR. Ventricular cerebrospinal fluid that day was negative for mycobacterial DNA. Tracheal aspirate samples for mycobacterial DNA and culture from days 22 and 27 of illness were negative by PCR but grew Mycobacterium tuberculosis after 8 weeks, long after the child's passing. She had no known exposures to tuberculosis and no chest radiographic findings to suggest it. All 6 family members had normal chest radiographs and negative interferon-γ release assay results. The source of her tuberculous infection was not identified, and further investigations by the local health department were not possible because of the State of Michigan-mandated lockdown for control of SARS-CoV-2 spread. CONCLUSION: The detection of SARS-CoV-2 RNA in cerebellar tissue and the demonstration of seroconversion in IgG and IgA assays was consistent with acute SARS-CoV-2 infection of the central nervous infection. However, the cause of death was brain herniation from her rapidly progressive central nervous system tuberculosis. SARS-CoV-2 may mask or worsen occult tuberculous infection with severe or fatal consequences.


Assuntos
Betacoronavirus/genética , Coinfecção/diagnóstico , Infecções por Coronavirus/epidemiologia , DNA Bacteriano/análise , Mycobacterium tuberculosis/genética , Pandemias , Pneumonia Viral/epidemiologia , Tuberculose do Sistema Nervoso Central/diagnóstico , Pré-Escolar , Coinfecção/microbiologia , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Evolução Fatal , Feminino , Humanos , Mycobacterium tuberculosis/isolamento & purificação , Pneumonia Viral/diagnóstico , Pneumonia Viral/virologia , RNA Viral/análise , Tuberculose do Sistema Nervoso Central/microbiologia
5.
PLoS One ; 15(8): e0238119, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32845896

RESUMO

Abdominal tuberculosis (ATB) continues to pose a major diagnostic challenge for clinicians due to its nonspecific clinical presentation, variable anatomical location and lack of sensitive diagnostic tools. In spite of the development of several assays till date; no single test has proved to be adequate for ATB diagnosis. In this study, we for the first time report the detection of circulating cell-free Mycobacterium tuberculosis (M. tuberculosis) DNA (cfMTB-DNA) in ascitic fluid (AF) samples and its utility in ATB diagnosis. Sixty-five AF samples were included in the study and processed for liquid culture, cytological, biochemical and molecular assays. A composite reference standard (CRS) was formulated to categorize the patients into 'Definite ATB' (M. tuberculosis culture positive, n = 2), 'Probable ATB' (n = 16), 'Possible ATB' (n = 13) and 'Non-TB' category (n = 34). Two molecular assays were performed, namely, the novel cfMTB-DNA qPCR assay targeting M. tuberculosis devR gene and Xpert MTB/RIF assay (Xpert), and their diagnostic accuracy was assessed using CRS as reference standard. Clinical features such as fever, loss of weight, abdominal distension and positive Mantoux were found to be strongly associated with ATB disease (p<0.05). cfMTB-DNA qPCR had a sensitivity of 66.7% (95% CI:40.9,86.7) with 97.1% specificity (95% CI:84.7,99.9) in 'Definite ATB' and 'Probable ATB' group collectively. The sensitivity increased to 70.9% (95% CI:51.9,85.8) in the combined 'Definite', 'Probable' and 'Possible' ATB group with similar specificity. The cfMTB-DNA qPCR assay performed significantly better than the Xpert assay which demonstrated a poor sensitivity of ≤16.7% with 100% (95% CI:89.7,100) specificity (p<0.001). We conclude that cfMTB-DNA qPCR assay is an accurate molecular test that can provide direct evidence of M. tuberculosis etiology and has promise to pave the way for improving ATB diagnosis.


Assuntos
Líquido Ascítico/química , Ácidos Nucleicos Livres/análise , DNA Bacteriano/análise , Técnicas de Diagnóstico Molecular/métodos , Mycobacterium tuberculosis/genética , Tuberculose/diagnóstico , Abdome/microbiologia , Abdome/patologia , Adolescente , Adulto , Idoso , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo Real , Tuberculose/patologia , Adulto Jovem
6.
PLoS Negl Trop Dis ; 14(8): e0008573, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32841248

RESUMO

BACKGROUND: Leptospirosis has gained much attention in Sri Lanka since its large outbreak in 2008. However, most of the cases were clinically diagnosed and information on Leptospira genotypes and serotypes currently prevailing in the country is lacking. METHODOLOGY/PRINCIPAL FINDINGS: We retrospectively analyzed 24 Leptospira strains from human patients as well as isolated and characterized three Leptospira strains from black rats using the microscopic agglutination test with antisera for 19 serovars and multilocus sequence typing. The isolates were identified as Leptospira borgpetersenii sequence types (STs) 143 and 144; L. interrogans STs 30, 34, 43, 44, 74, 75, 80, 308, 313, 314, 316, and 317; and L. kirschneri ST318. Six of the 15 STs were identified for the first time in this study. Five serogroups such as Autumnalis, Grippotyphosa, Hebdomadis, Javanica, and Pyrogenes were detected among the isolates. Contrary to previous studies, various genotypes including novel STs were isolated during an outbreak in Southern Province. L. borgpetersenii serogroup Javanica ST143 was isolated both from a human and black rat. CONCLUSIONS/SIGNIFICANCE: This study revealed that genetically diverse Leptospira strains currently circulate in Sri Lanka: some genotypes have been circulating and others have emerged recently, which may explain the recent surge of leptospirosis patients with varying clinical manifestations and frequent outbreaks of leptospirosis. Black rats were identified as the source of infection for humans, but reservoir animals for other genotypes remain unknown.


Assuntos
Genótipo , Leptospira/classificação , Leptospira/genética , Leptospira/isolamento & purificação , Leptospirose/microbiologia , Tipagem de Sequências Multilocus/métodos , Adolescente , Adulto , Idoso , Testes de Aglutinação , Animais , Criança , DNA Bacteriano/análise , Reservatórios de Doenças , Feminino , Humanos , Leptospirose/epidemiologia , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Análise de Sequência de DNA , Sorogrupo , Sorotipagem , Sri Lanka/epidemiologia , Adulto Jovem
7.
PLoS One ; 15(8): e0237232, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32776951

RESUMO

Until recently the in utero environment of pregnant women was considered sterile. Recent high-sensitivity molecular techniques and high-throughput sequencing lead to some evidence for a low-biomass microbiome associated with the healthy placenta. Other studies failed to reveal evidence for a consistent presence of bacteria using either culture or molecular based techniques. Comparing conflicting "placental microbiome" studies is complicated by the use of varied and inconsistent protocols. Given this situation, we undertook an evaluation of the in utero environment sterility using several controlled methods, in the same study, to evaluate the presence or absence of bacteria and to explain contradictions present in the literature. Healthy pregnant women (n = 38) were recruited in three maternity wards. Placenta were collected after cesarean section with or without Alexis® and vaginal delivery births. For this study we sampled fetal membranes, umbilical cord and chorionic villi. Bacterial presence was analyzed using bacterial culture and qPCR on 34 fetal membranes, umbilical cord and chorionic villi samples. Shotgun metagenomics was performed on seven chorionic villi samples. We showed that the isolation of meaningful quantities of viable bacteria or bacterial DNA was possible only outside the placenta (fetal membranes and umbilical cords) highlighting the importance of sampling methods in studying the in utero environment. Bacterial communities described by metagenomics analysis were similar in chorionic villi samples and in negative controls and were dependent on the database chosen for the analysis. We conclude that the placenta does not harbor a specific, consistent and functional microbiota.


Assuntos
Bactérias/isolamento & purificação , Vilosidades Coriônicas/microbiologia , Membranas Extraembrionárias/microbiologia , Placenta/microbiologia , Cordão Umbilical/microbiologia , Adulto , Bactérias/genética , Cesárea , Amostra da Vilosidade Coriônica , DNA Bacteriano/análise , DNA Bacteriano/genética , Parto Obstétrico , Feminino , Humanos , Microbiota , Gravidez , Manejo de Espécimes
9.
J S Afr Vet Assoc ; 91(0): e1-e7, 2020 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-32787420

RESUMO

Chickens have been implicated in most Salmonella disease outbreaks because they act as carriers of the pathogen in their gut. There are over 2500 serotypes of Salmonella that have been reported worldwide and 2000 of these serovars can be found in chickens. The main objective of this study was to determine the Salmonella serotypes found in poultry farms around Mafikeng district, South Africa. Salmonella was identified according to the guidelines of the International Organization for Standardization (ISO) (ISO 6579:2002) standard techniques. Faecal samples were collected and analysed for Salmonella using conventional cultural methods and polymerase chain reaction targeting the 16S Ribosomal Deoxyribonucleic acid (rDNA) gene for Salmonella identification. Out of 130 presumptive Salmonella isolates determined by urease and triple sugar iron tests, only 46 isolates were identified as Salmonella serotypes of which S. Typhimurium was the most frequent with 18 (39.1%), followed by S. Heidelberg with 9 (19.6%), S. bongori with 7 (15.2%), S. Enteritidis with 6 (13.0%) and both S. Paratyphi B and S. Newport with 3 (6.5%) each. Seven virulence genes including invA 100%, spy 39%, hilA 9%, misL 30%, sdfI 13%, orfL 11% and spiC 9% were detected from these Salmonella isolates in this study. The presence of these virulence genes indicates high pathogenicity potential of these isolates which is a serious public health concern because of zoonotic potential of Salmonella.


Assuntos
Galinhas , Doenças das Aves Domésticas/epidemiologia , Salmonelose Animal/epidemiologia , Salmonella/genética , Animais , DNA Bacteriano/análise , DNA Ribossômico/análise , Fezes/microbiologia , Reação em Cadeia da Polimerase/veterinária , Doenças das Aves Domésticas/microbiologia , Prevalência , Salmonella/isolamento & purificação , Salmonella/patogenicidade , Salmonelose Animal/microbiologia , Sorogrupo , África do Sul/epidemiologia , Virulência
10.
Am J Obstet Gynecol ; 223(1): 114.e1-114.e20, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32591087

RESUMO

BACKGROUND: Preterm prelabor rupture of the membranes (PPROM) is frequently complicated by intraamniotic inflammatory processes such as intraamniotic infection and sterile intraamniotic inflammation. Antibiotic therapy is recommended to patients with PPROM to prolong the interval between this complication and delivery (latency period), reduce the risk of clinical chorioamnionitis, and improve neonatal outcome. However, there is a lack of information regarding whether the administration of antibiotics can reduce the intensity of the intraamniotic inflammatory response or eradicate microorganisms in patients with PPROM. OBJECTIVE: The first aim of the study was to determine whether antimicrobial agents can reduce the magnitude of the intraamniotic inflammatory response in patients with PPROM by assessing the concentrations of interleukin-6 in amniotic fluid before and after antibiotic treatment. The second aim was to determine whether treatment with intravenous clarithromycin changes the microbial load of Ureaplasma spp DNA in amniotic fluid. STUDY DESIGN: A retrospective cohort study included patients who had (1) a singleton gestation, (2) PPROM between 24+0 and 33+6 weeks, (3) a transabdominal amniocentesis at the time of admission, and (4) intravenous antibiotic treatment (clarithromycin for patients with intraamniotic inflammation and benzylpenicillin/clindamycin in the cases of allergy in patients without intraamniotic inflammation) for 7 days. Follow-up amniocenteses (7th day after admission) were performed in the subset of patients with a latency period lasting longer than 7 days. Concentrations of interleukin-6 were measured in the samples of amniotic fluid with a bedside test, and the presence of microbial invasion of the amniotic cavity was assessed with culture and molecular microbiological methods. Intraamniotic inflammation was defined as a bedside interleukin-6 concentration ≥745 pg/mL in the samples of amniotic fluid. Intraamniotic infection was defined as the presence of both microbial invasion of the amniotic cavity and intraamniotic inflammation; sterile intraamniotic inflammation was defined as the presence of intraamniotic inflammation without microbial invasion of the amniotic cavity. RESULTS: A total of 270 patients with PPROM were included in this study: 207 patients delivered within 7 days and 63 patients delivered after 7 days of admission. Of the 63 patients who delivered after 7 days following the initial amniocentesis, 40 underwent a follow-up amniocentesis. Patients with intraamniotic infection (n = 7) and sterile intraamniotic inflammation (n = 7) were treated with intravenous clarithromycin. Patients without either microbial invasion of the amniotic cavity or intraamniotic inflammation (n = 26) were treated with benzylpenicillin or clindamycin. Treatment with clarithromycin decreased the interleukin-6 concentration in amniotic fluid at the follow-up amniocentesis compared to the initial amniocentesis in patients with intraamniotic infection (follow-up: median, 295 pg/mL, interquartile range [IQR], 72-673 vs initial: median, 2973 pg/mL, IQR, 1750-6296; P = .02) and in those with sterile intraamniotic inflammation (follow-up: median, 221 pg/mL, IQR 118-366 pg/mL vs initial: median, 1446 pg/mL, IQR, 1300-2941; P = .02). Samples of amniotic fluid with Ureaplasma spp DNA had a lower microbial load at the time of follow-up amniocentesis compared to the initial amniocentesis (follow-up: median, 1.8 × 104 copies DNA/mL, 2.9 × 104 to 6.7 × 108 vs initial: median, 4.7 × 107 copies DNA/mL, interquartile range, 2.9 × 103 to 3.6 × 107; P = .03). CONCLUSION: Intravenous therapy with clarithromycin was associated with a reduction in the intensity of the intraamniotic inflammatory response in patients with PPROM with either intraamniotic infection or sterile intraamniotic inflammation. Moreover, treatment with clarithromycin was related to a reduction in the load of Ureaplasma spp DNA in the amniotic fluid of patients with PPROM <34 weeks of gestation.


Assuntos
Antibacterianos/uso terapêutico , Infecções Bacterianas/prevenção & controle , Corioamnionite/prevenção & controle , Claritromicina/uso terapêutico , Clindamicina/uso terapêutico , Ruptura Prematura de Membranas Fetais , Penicilina G/uso terapêutico , Adulto , Líquido Amniótico/química , Infecções Bacterianas/etiologia , Corioamnionite/etiologia , Estudos de Coortes , DNA Bacteriano/análise , Feminino , Humanos , Interleucina-6/análise , Gravidez , Estudos Retrospectivos , Ureaplasma/genética
11.
Artigo em Inglês | MEDLINE | ID: mdl-32520211

RESUMO

Simple, low-cost and effective diagnostic tests for tuberculosis (TB) are needed especially in TB-high burden settings. The present study evaluated the performance of an in-house loop-mediated isothermal amplification (LAMP) for diagnosing TB by comparing it to Xpert MTB/RIF, microscopy and culture. In Thailand, a total of 204 excess sputum samples volume after the processing of cultures were used for Mycobacterium tuberculosis (MTB) detection by Xpert MTB/RIF and LAMP. Based on culture results as the gold standard, the overall sensitivity of LAMP and Xpert MTB/RIF were 82.1% (126/153; 95% confidential interval [CI]: 75.4-88.98%) and 86.9 % (133/153; 95% CI: 80.5-90.8%) respectively, and the specificity of both tests was 100% (51/51; 95% CI: 93.0-100.0%). In comparison with Xpert MTB/RIF, the sensitivity and specificity of LAMP were 94.7% (126/133; 95% CI: 89.5-97.9%), and 100.0% (73/73; 95% CI: 94.9-100.0%), respectively. The average threshold cycle (Ct) of Xpert MTB/RIF detection for positive and negative LAMP results was statistically different, of 18.4 and 27.0, respectively (p < 0.05). In comparison with the acid-fast staining technique, and analyzing LAMP and Xpert MTB/RIF in smear-negative/culture-positive specimens, there was an increase of the detection rate by 47.7% (21/44) and 54.6% (24/44). The diagnostic sensitivity and specificity of LAMP appeared to be comparable to those of Xpert MTB/RIF. We claim that this LAMP has potential to provide a sensitive diagnostic test for the rapid TB diagnosis. It allowed a fast detection of MTB before the cultures and it could be used in resource-limited laboratory settings.


Assuntos
Mycobacterium tuberculosis/isolamento & purificação , Escarro/microbiologia , Tuberculose/diagnóstico , DNA Bacteriano/análise , DNA Bacteriano/genética , Testes Diagnósticos de Rotina , Humanos , Técnicas de Diagnóstico Molecular/métodos , Mycobacterium tuberculosis/genética , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade , Tailândia
12.
PLoS Negl Trop Dis ; 14(6): e0008051, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32569298

RESUMO

BACKGROUND: In Japan, Buruli ulcer cases are often advanced, requiring surgical treatment. However, extensive debridement is often difficult because of cosmetic and functional sequelae. Moreover, the lesions are complicated and composed of edematous erythema, necrotic ulcer, and erythematous skin lesions caused by a paradoxical reaction, which also make it difficult to perform adequate debridement. METHODOLOGY/PRINCIPAL FINDINGS: We performed quantitative polymerase chain reaction (PCR) analysis for IS2404 using 29 samples taken from mapping biopsy. We evaluated the relationship among mycobacterial burden, histopathological findings, and clinical outcomes using 83 tissue samples taken from mapping biopsy and debrided Buruli ulcer. On quantitative PCR, the Cp values of IS2404 amplification were substantially different in each site. The major histological findings could be divided into massive subcutaneous necrosis with scant inflammatory cell infiltration and dense inflammatory cell infiltration. Of the 84 sites, 34 were subjected to repeated histological evaluations. In these sites, histological necrosis did not disappear over time despite standard antibiotic treatment. In contrast, the ulcers were cured and no recurrences were observed without resecting the 11 biopsied sites that lacked histological necrosis. Although quantitative PCR revealed that a lower Cp value of IS2404 was associated with histological massive necrosis, sites that showed lower Cp values clinically did not always need debridement. CONCLUSION/SIGNIFICANCE: Our descriptive study revealed that the histological findings and amounts of mycobacterial DNA differed according to the sites despite being found in one lesion. Our results showed that the need for surgical debridement in each site was correlated with histological necrosis without inflammatory cell infiltration, as the inflammation is supposed to represent an active host immune response rather than mycobacterial burden. We suggest that the debridement of lesions with histological necrosis in mapping biopsy may be useful for Japanese cases with unsuccessful standard antibiotic treatment to achieve sufficient clinical improvement.


Assuntos
Carga Bacteriana , Úlcera de Buruli/microbiologia , Úlcera de Buruli/patologia , Histocitoquímica , Mycobacterium ulcerans/isolamento & purificação , Adulto , Biópsia , Elementos de DNA Transponíveis , DNA Bacteriano/análise , DNA Bacteriano/genética , Humanos , Japão , Masculino , Mycobacterium ulcerans/genética , Reação em Cadeia da Polimerase em Tempo Real , Adulto Jovem
13.
PLoS One ; 15(6): e0232610, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32497137

RESUMO

Pneumonia is one of the most important causes of morbidity and mortality in children. Identification and characterization of pathogens that cause infections are crucial for accurate treatment and accelerated recovery. However, in most cases, the causative agent cannot be identified, which is partly due to the limited spectrum of pathogens covered by current diagnostics based on nucleic acid amplification. Therefore, in this study, we explored the application of metagenomic next-generation sequencing (mNGS) for the diagnosis of children with severe pneumonia. From April to July 2017, 32 hospitalized children with severe nonresponding pneumonia in Shenzhen Children's Hospital were included in this study. Blood tests were conducted immediately after hospitalization to assess cell counts and inflammatory markers, oropharyngeal swabs were collected to identify common pathogens by qPCR and culture. After bronchoscopy, bronchoalveolar lavage fluid (BALF) samples were collected for further pathogen identification using standardized diagnostic tests and mNGS. Blood tests were normal in 3 of the 32 children. In 9 oropharyngeal swabs, bacterial pathogens were detected, in 5 of these Mycoplasma pneumoniae was detected. Adenovirus was detected in 5 BALF samples, using the Direct Immunofluorescence Assay (DFA). In 15 cases, no common pathogens were found in BALF samples, using the current standard diagnostic tests, while in all 32 BALFs, pathogens were identified using mNGS, including adenovirus, Mycoplasma pneumoniae, Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, cytomegalovirus and bocavirus. This study shows that, with mNGS, the sensitivity of detection of the causative pathogens in children with severe nonresponding pneumonia is significantly improved. In addition, mNGS gives more strain specific information, helps to identify new pathogens and could potentially help to trace and control outbreaks. In this study, we have shown that it is possible to have the results within 24 hours, making the application of mNGS feasible for clinical diagnostics.


Assuntos
Coinfecção , Metagenômica/métodos , Pneumonia Bacteriana/diagnóstico , Pneumonia Viral/diagnóstico , Contagem de Células Sanguíneas , Líquido da Lavagem Broncoalveolar/microbiologia , Líquido da Lavagem Broncoalveolar/virologia , Criança , Pré-Escolar , China/epidemiologia , Coinfecção/microbiologia , Coinfecção/virologia , DNA Bacteriano/análise , DNA Viral/análise , Feminino , Técnica Direta de Fluorescência para Anticorpo , Humanos , Lactente , Pacientes Internados , Masculino , Metagenoma , Orofaringe/microbiologia , Orofaringe/virologia , Pneumonia Bacteriana/epidemiologia , Pneumonia Bacteriana/microbiologia , Pneumonia Viral/epidemiologia , Pneumonia Viral/virologia , Estudo de Prova de Conceito , RNA Viral/análise , Sensibilidade e Especificidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
14.
PLoS One ; 15(5): e0233356, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32469988

RESUMO

Plant rhizosphere-associated bacterial communities play key roles in affecting host health in response to diverse biotic stresses. Currently, the effect of continuous cropping of potato on soil bacterial communities and physiochemical parameters has not been well documented. Herein, we compared bacterial composition and diversity in rotationally and continuously (5, 10, and 30 years) cropped soils, and clarified the correlations between soil properties and the bacterial communities revealed by Illumina MiSeq sequencing. Our results demonstrated that Proteobacteria, Actinobacteria and Firmicutes were the predominant phyla in all the tested soil samples. While the abundance of Proteobacteria showed an increase, the abundance of Actinobacteria and Firmicutes displayed a reduction with the increase of continuous cropping years. At the genus level, as continuous cropping years increasing, the abundance of Pseudarthrobacter, Bacillus and Pseudomonas decreased, but the abundance of Rhodanobacte, Sphingobium, Mizugakiibacter and Devosia increased. Our results also demonstrated that the abundance of plant growth-promoting rhizobacteria in the rotationally cropped soil was significantly higher than that of continuously cropped soil. Furthermore, our results showed that soil organic matter, available nitrogen, available phosphorus and available potassium were significantly correlated with bacterial community distribution. Overall, our work provides a comprehensive view of altered structure and composition of bacterial communities between the continuously cropped soil and rotationally cropped soil.


Assuntos
Bactérias/classificação , Biodiversidade , Produtos Agrícolas/crescimento & desenvolvimento , DNA Bacteriano/análise , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Microbiologia do Solo , Solanum tuberosum/crescimento & desenvolvimento , Bactérias/genética , Filogenia , Rizosfera
15.
Zhonghua Er Ke Za Zhi ; 58(5): 403-407, 2020 May 02.
Artigo em Chinês | MEDLINE | ID: mdl-32392957

RESUMO

Objective: To explore the value of nucleic acid detection methods in pharyngeal swabs in the etiological diagnosis of Mycoplasma pneumoniae (MP) infection in children. Methods: Four hundred and fifty-four (male 210, female 244) children with pneumonia in Department of Pulmonology, Children's Hospital of Zhejiang University School of Medicine were enrolled from July, 2018 to October, 2018. Pharyngeal swabs and venous blood were obtained on the first or the second day after hospitalization. Fluorescence detection quantitative amplification of DNA, thermostatic amplification of RNA, MP culture and MP-IgM were used to detect MP simultaneously. MP infection is diagnosed if MP culture is positive or the two of the other three methods are positive. Pharyngeal swabs were acquired and detected using fluorescence quantitative amplification of DNA, thermostatic amplification of RNA and MP culture again for children with confirmed MP infection before discharge. The detection rates and quantitative changes of the three methods were compared, and χ(2) test was used for comparison among groups. Results: A total of 454 hospitalized children with pneumonia were included in this study. The detection rates of fluorescence quantitative amplification of DNA, thermostatic amplification of RNA, MP culture and MP-IgM IgM were 43.6% (198/454), 43.2% (196/454), 40.0% (180/454) and 30.6% (139/454) respectively. The difference of detection rates of the four methods was statistically significant (χ(2)=20.8, P<0.05),but no significant difference between the detection rates of fluorescence quantitative amplification of DNA and thermostatic amplification of RNA was found (χ(2)=0.018, P=0.900). They both had higher detection rates than MP-IgM or MP culture. MP infection is diagnosed if MP culture is positive or the two of the other three methods are positive, and two hundred and nine children were diagnosed as MP infection. In the second test of MP infection in 209 children before discharge, the positive rate of MP culture was 67.5% (141/209), with 39.4% (13/33) changed from negative to positive, and 27.3% (48/176) changed from positive to negative. The positive rate of thermostatic amplification of RNA was 82.3% (172/209), with 16.2% (31/191) turned from positive to negative, and 66.7% (12/18) turned from negative to positive. The positive rate of fluorescence quantitative amplification of DNA was 67.0% (140/209), with 52.9% (18/34) cases changed from negative to positive, and 30.3% (53/175) cases changed from positive to negative. MP-DNA load decreased in 141 cases (67.5%) and increased in 68 cases (32.5%) in the second test among the positive samples tested by fluorescence quantitative amplification of DNA. The detection rates of the four methods in the non-severe group and the severe group were similar, and the differences among the groups were not statistically significant (all P>0.05). In the second test, the proportion of changing from negative to positive in the severe group was higher than that in the non-severe group, but only the difference in the thermostatic amplification of RNA was statistically significant (P=0.038) and the cases of changing from negative to positive of thermostatic amplification of RNA in the severe group and non-severe group are 7 and 5 respectively. Conclusions: The methods of pharyngeal swab nucleic acid detection have high sensitivity and application value in the etiological diagnosis of acute MP infection in children. The results of fluorescence quantitative amplification of DNA and thermostatic amplification of RNA are highly consistent, and they are both more advantageous than MP-IgM. Repeated testing in the acute phase is helpful to find MP infection children whose first test is negative. The load of MP-DNA did not decrease in some children in the acute stage after antibiotic treatment.


Assuntos
DNA Bacteriano/análise , Técnicas de Amplificação de Ácido Nucleico/métodos , Pneumonia por Mycoplasma/diagnóstico , Anticorpos Antibacterianos/sangue , Criança , Feminino , Hospitalização , Humanos , Imunoglobulina M/sangue , Masculino , Mycoplasma pneumoniae , Faringe/microbiologia , Sensibilidade e Especificidade
16.
Acta Trop ; 207: 105513, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32371220

RESUMO

Worldwide, Bartonella species are known to infect a wide range of mammalian and arthropod hosts, including humans. The current study aimed to investigate the prevalence of Bartonella spp. in synanthropic mammals captured in peri-urban areas from Central-Western and Southern Brazil and their ectoparasites. For this aim, 160 mammals belonging to four species, and 218 associated arthropods were sampled. DNA was extracted and subjected to different Bartonella screening assays. Additionally, blood samples from 48 small rodents were submitted to liquid BAPGM culture followed by qPCR assay and solid culture. Two out of 55 Rattus captured in Santa Catarina state were PCR-positive for Bartonella when targeting the nuoG, 16S, and ITS loci. Sequences showed high homology with Bartonella coopersplainsensis. Conversely, all 48 small rodents, 14 capybaras and 43 opossum DNA samples from animals trapped in Mato Grosso do Sul were Bartonella negative in the HRM real time PCR assays targeting the ITS locus and gltA gene. Additionally, all mammal-associated ectoparasites showed negativity results based on HRM real time PCR assays. The present study showed, for the first time, the occurrence of B. coopersplainsensis in Brazil, shedding some light on the distribution of rats-related Bartonella in South America. In addition, the majority of rodents and marsupials were negative for Bartonella spp. Since B. coopersplainsensis reservoirs - Rattus spp. - are widely dispersed around the globe, their zoonotic potential should be further investigated.


Assuntos
Bartonella/isolamento & purificação , Mamíferos/microbiologia , Ftirápteros/microbiologia , Carrapatos/microbiologia , Animais , Bartonella/genética , DNA Bacteriano/análise , Humanos , Mamíferos/parasitologia , Marsupiais/microbiologia , Gambás/microbiologia , Gambás/parasitologia , Ratos , Roedores/microbiologia , Roedores/parasitologia
17.
Arch Anim Nutr ; 74(4): 296-308, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32308036

RESUMO

Grape pomace (GP) is an abundant by-product from wine production and is rich in phenolic compounds, unsaturated fatty acids, dietary fibre and beneficial bacteria. In this study, weaned piglets were fed a basic diet supplemented with 5% GP for 4 weeks. Compared with those in the control (CON) group, it was found that the proportion of Lactobacillus delbrueckii, Olsenella umbonata and Selenomonas bovis in the caecum and the villus height and villus height/crypt depth ratio (VCR) of the jejunum were both significantly increased in the GP group (p < 0.05). Meanwhile, at the mRNA expression level, several proinflammatory cytokines (IL-1ß, IL-8, IL-6 and TNF-α) were significantly downregulated (p < 0.05) in piglet caecal tissue, and the short-chain fatty acid receptors (GPR41 and GPR43) were not significantly upregulated. In contrast, the levels of IgG was significantly increased (p < 0.05) in the sera of weaned piglets in the GP group. However, no difference in growth performance between the two groups of piglets was detected. These results show that GP had no adverse effects on the growth performance of piglets, but GP can promote the content of some beneficial bacteria in the caecum; this effect is conducive to improving the disease resistance potential of piglets.


Assuntos
Bactérias/metabolismo , Microbioma Gastrointestinal/efeitos dos fármacos , Sus scrofa/crescimento & desenvolvimento , Sus scrofa/microbiologia , Vitis/química , Actinobacteria/metabolismo , Ração Animal/análise , Animais , Ceco/efeitos dos fármacos , Ceco/microbiologia , DNA Bacteriano/análise , DNA Ribossômico/análise , Dieta/veterinária , Suplementos Nutricionais/análise , Feminino , Frutas/química , Jejuno/efeitos dos fármacos , Jejuno/fisiologia , Lactobacillus delbrueckii/metabolismo , Masculino , Probióticos , Distribuição Aleatória , Selenomonas/metabolismo
18.
An Acad Bras Cienc ; 92 Suppl 1: e20180557, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32348408

RESUMO

In Brazil and in other countries of the world, studies have been conducted to identify Listeria monocytogenes in cattle meat that is preferably consumed undercooked and, when marketed without meeting strict phytosanitary requirements, may cause outbreaks of listeriosis. In the such, foodborne outbreaks, the methods used for the detection of the pathogen and the efficiency associated with them are crucial for the proper assessment. In this study, we used the techniques biochemical and molecular for identification of the L. monocytogenes isolated from 30 samples of the fresh beef, marketed in ten butchers' shop of the free-fair from a municipality from the Bahia, Brazil. The results obtained from biochemical tests (catalase, motility, ß-hemolysis and carbohydrate fermentation), as well as PCR analysis for the hly gene (hemolysin production is an important factor in the pathogenesis of listeriosis) revealed that 50% of butchers shops presented bovine meat contaminated with bacteria of the Listeria sp. and confirmed that 54.16% of the analyzed meat samples were positive for L. monocytogenes. This study highlights the importance of microbiological surveillance in free-fair to minimize the exposure of consumers to this foodborne pathogen.


Assuntos
DNA Bacteriano/análise , Proteínas Hemolisinas/genética , Listeria monocytogenes/isolamento & purificação , Produtos da Carne/microbiologia , Animais , Brasil , Bovinos , Microbiologia de Alimentos , Proteínas Hemolisinas/análise , Listeria monocytogenes/genética
19.
Rev Bras Parasitol Vet ; 29(1): e022419, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32236336

RESUMO

This study aimed to evaluate the occurrence of diseases transmitted by Amblyomma ovale in 61 dogs monitored for three years through collections of ticks and blood, interviews, telemetry and camera traps in three areas of Serra do Mar State Park, Brazil. Blood samples were used to investigate infection by Rangelia vitalii by real-time TaqMan PCR and Rickettsia parkeri by IIFA. The collected ticks were submitted to conventional PCR to investigate the presence of R. parkeri . These data were compared with the monitoring results and interviews with the owners. Dogs considered as companion presented a risk of infection by R. parkeri strain Mata Atlantica 5.4 times higher than those not considered as companion (p = 0.009). Dogs that had at least one A. ovale collected during the campaigns had a 10 times higher risk of infection by R. parkeri strain Mata Atlantica than those who did not (p = 0.009). One dog positive for R. vitalii by real-time TaqMan PCR was parasitized by A. ovale frequently during monitoring. Sequenced ompaA - positive DNA samples had 100% identity of R. parkeri strain Mata Atlantica clone As106. From the findings, it is urgent to control domestic dogs around rainforests to reduce zoonoses transmission.


Assuntos
Doenças do Cão/epidemiologia , Ixodidae/microbiologia , Infecções por Rickettsia/veterinária , Rickettsia/isolamento & purificação , Animais , Brasil/epidemiologia , DNA Bacteriano/análise , Doenças do Cão/diagnóstico , Doenças do Cão/microbiologia , Cães , Técnica Indireta de Fluorescência para Anticorpo , Reação em Cadeia da Polimerase , Floresta Úmida , Rickettsia/classificação , Rickettsia/genética , Infecções por Rickettsia/diagnóstico , Infecções por Rickettsia/epidemiologia , Análise de Sequência de DNA , Telemetria
20.
PLoS One ; 15(4): e0230579, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32271774

RESUMO

Birds are important hosts for the first life stages of the Ixodes ricinus tick and they can transport their parasites over long distances. The aim of this study was to investigate the prevalence of Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, Neoehrlichia mikurensis and Rickettsia helvetica in ticks collected from migratory birds in Norway. A total of 815 Ixodes ricinus ticks from 216 birds trapped at Lista Bird Observatory in southern Norway during spring and autumn migration in 2008 were analysed by real-time PCR. B. burgdorferi s. l. was the most prevalent pathogen, detected in 6.1% of the ticks. The prevalence of N. mikurensis, A. phagocytophilum and R. helvetica was 1.2%, 0.9% and 0.4% respectively. In addition, one sample (0.1%) was positive for B. miyamotoi. In total, 8.2% of the ticks were infected with at least one pathogen. Co-infection with B. burgdorferi s. l. and N. mikurensis or A. phagocytophilum was found in 6.0% of the infected ticks. Our results show that all the known major tick-borne bacterial pathogens in Norway are subject to transport by migratory birds, potentially allowing spread to new areas. Our study showed a surprisingly high number of samples with PCR inhibition (57%). These samples had been extracted using standard methodology (phenol-chloroform extraction). This illustrates the need for inhibition controls to determine true prevalence rates.


Assuntos
Doenças das Aves , Aves/parasitologia , Ixodes/microbiologia , Infestações por Carrapato/microbiologia , Doenças Transmitidas por Carrapatos/microbiologia , Anaplasma phagocytophilum/genética , Anaplasma phagocytophilum/isolamento & purificação , Anaplasmataceae/classificação , Anaplasmataceae/genética , Anaplasmataceae/isolamento & purificação , Migração Animal/fisiologia , Animais , Doenças das Aves/epidemiologia , Doenças das Aves/microbiologia , Doenças das Aves/parasitologia , Aves/fisiologia , Borrelia/classificação , Borrelia/genética , Borrelia/isolamento & purificação , Borrelia burgdorferi/genética , Borrelia burgdorferi/isolamento & purificação , Coinfecção/epidemiologia , Coinfecção/microbiologia , DNA Bacteriano/análise , Noruega/epidemiologia , Reação em Cadeia da Polimerase , Prevalência , Rickettsia/classificação , Rickettsia/genética , Rickettsia/isolamento & purificação , Infestações por Carrapato/epidemiologia , Infestações por Carrapato/veterinária , Doenças Transmitidas por Carrapatos/epidemiologia , Doenças Transmitidas por Carrapatos/veterinária
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