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1.
Sheng Wu Gong Cheng Xue Bao ; 36(5): 908-919, 2020 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-32567274

RESUMO

A multiplex PCR method was developed to detect the main pathogens of Qinghai Tibetan sheep endometritis. First, the genomes of five standard bacterial strains were extracted and specific primers were selected; the multiplex PCR method was established by using the genome of the standard strain as a template. The samples were collected by sterile cotton swab from Tibetan sheep uterus, and then placed in LB medium and numbered. After 48 h, the genomes of cultured bacteria were extracted and detected by single PCR method, then the positive samples were recorded. The positive samples detected by single PCR were selected for multiplex PCR detection and recorded again. The coincidence rate between these two methods was calculated to measure the accuracy of multiplex PCR. In order to identify the species of the pathogen, 30 positive samples verified by single and multiplex PCR were randomly selected for bacterial isolation and identification. In the 600 samples, the infected ratio of Streptococcus agalactiae (GBS) was 47.33%, Escherichia coli 34.83%, Staphylococcus aureus 6.5%, Salmonella and Trueperella pyogenes were negatively detected. Among the positive samples detected by multiplex PCR, the positive ratio of GBS was 45.50%, E. coli 33.50%, S. aureus 6.5%. Comparison of two detection results, Multiplex PCR detection coincidence rate is more than 95%. The isolated pathogens were identified as E. coli, GBS and S. aureus, which was consistent with the results of two methods. The multiplex PCR method was successfully established and the main pathogens of endometritis in Qinghai Tibetan sheep were GBS, E. coli and S. aureus.


Assuntos
Bactérias , Técnicas Bacteriológicas , Endometrite , Reação em Cadeia da Polimerase Multiplex , Doenças dos Ovinos , Animais , Bactérias/genética , Bactérias/isolamento & purificação , Técnicas Bacteriológicas/métodos , Endometrite/microbiologia , Endometrite/veterinária , Feminino , Reação em Cadeia da Polimerase Multiplex/normas , Reação em Cadeia da Polimerase/veterinária , Sensibilidade e Especificidade , Ovinos , Doenças dos Ovinos/microbiologia , Tibet
2.
New Microbiol ; 43(1): 13-16, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32334488

RESUMO

The long incubation time required for Mycobacteria detection may allow cultures to become overgrown by contaminating organisms. Therefore, samples need to be decontaminated before solid and liquid culture. MYCO-TB is a ready-to-use digestion and decontamination kit with single-sample formulation developed by Copan. Sample processing time (3 minutes) is shorter than that of other commercial kits. The aim of this study was to compare the performance of MYCO-TB with MycoPrep, both based on N-acetyl-Lcysteine and sodium hydroxide solution, in terms of culture contamination and Mycobacterial detection by culture. We tested 162 respiratory samples: the overall proportions of contamination of both liquid and solid media were 1.8% for MYCO-TB and 1.8% for MycoPrep. Mycobacterial growth was detected without significant differences in times to positivity (TTP) in liquid culture: 10.5 days for MYCO-TB and 11.1 days for MycoPrep. Samples decontaminated with MYCO-TB were suitable for molecular assays such as Xpert MTB/RIF Ultra and GenoType CMdirect. Extending decontamination times (up to 10 minutes) with MYCO-TB of 20 Mycobacteria-positive specimens did not produce any difference in TTP in liquid culture or in Ultra IS1081/IS6110 probe Ct values. In conclusion, the MYCO-TB kit proved to be effective for the rapid digestion and decontamination of respiratory materials for the detection of Mycobacteria, making it possible to reduce the manual skills required and lower the risk of contamination. Longer decontamination time could be used for samples with a high level of contamination, such as those from cystic fibrosis patients.


Assuntos
Técnicas Bacteriológicas , Mycobacterium tuberculosis , Kit de Reagentes para Diagnóstico , Tuberculose , Técnicas Bacteriológicas/métodos , Técnicas Bacteriológicas/normas , Descontaminação , Humanos , Mycobacterium tuberculosis/isolamento & purificação , Kit de Reagentes para Diagnóstico/normas , Sensibilidade e Especificidade , Tuberculose/diagnóstico , Tuberculose/microbiologia
3.
Microbes Environ ; 35(1)2020.
Artigo em Inglês | MEDLINE | ID: mdl-32009018

RESUMO

We previously demonstrated that a simple modification in the preparation of agar media, i.e., autoclaving phosphate and agar separately (termed the "PS protocol"), improved the culturability of aerobic microorganisms by reducing the generation of reactive oxygen species. We herein investigated the effects of the PS protocol on the cultivation of anaerobic microorganisms using sludge from a wastewater treatment system as a microbial source. The application of the PS protocol increased colony numbers and the frequency of phylogenetically novel isolates under aerobic, nitrate reduction, and fermentation conditions. The PS protocol is useful for isolating both aerobic and anaerobic microorganisms.


Assuntos
Bactérias Anaeróbias/crescimento & desenvolvimento , Bactérias Anaeróbias/isolamento & purificação , Técnicas Bacteriológicas/métodos , Meios de Cultura/química , Ágar , Bactérias Anaeróbias/classificação , Bactérias Anaeróbias/metabolismo , Contagem de Colônia Microbiana , Fermentação , Nitratos/metabolismo , Fosfatos , Filogenia , RNA Ribossômico 16S/genética , Esgotos/microbiologia , Esterilização
4.
BMC Infect Dis ; 20(1): 90, 2020 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-32000692

RESUMO

BACKGROUND: Plague is a highly fatal disease caused by Yersinia pestis. Late diagnosis hampers disease outcome and effectiveness of control measures, induces death and disease spread. Advance on its diagnosis was the use of lateral flow rapid diagnostic test (RDT). METHODS: We assessed the performance of the plague RDT based on Y. pestis F1 antigen detection more than 15 years after its deployment in Madagascar. We compared the RDT with bacteriological culture results, using data from plague notified cases collected during the periods for which both tests were performed independently and systematically. RESULTS: Used with bubonic plague (BP) patient samples, RDTs had a sensitivity of 100% (95% CI: 99.7-100%), a specificity of 67% (95% CI: 64-70%) with a good agreement between bacteriology and RDT results (86%; κ = 0.70, 95% CI 0.67-0.73). For pneumonic plague (PP), RDT had a sensitivity of 100% (95% CI: 91-100%) and a specificity of 59% (95% CI: 49-68%) and concordance between the bacteriological and plague RDT results was moderate (70%; κ = 0.43, 95% CI 0.32-0.55). Analysis focusing on the 2017-2018 plague season including the unprecedented epidemic of PP showed that RDT used on BP samples still had a sensitivity of 100% (95% CI: 85-100%) and a specificity of 82% (95% CI: 48-98%) with a very good agreement with bacteriology 94% (κ = 0.86, 95% CI 0.67-1); for PP samples, concordance between the bacteriological and plague RDT results was poor (61%; κ = - 0.03, 95% CI -0.17 - 0.10). CONCLUSIONS: RDT performance appeared to be similar for the diagnosis of BP and PP except during the 2017 PP epidemic where RDT performance was low. This RDT, with its good sensitivity on both plague clinical forms during a normal plague season, remained a potential test for alert. Particularly for BP, it may be of great value in the decision process for the initiation of therapy. However, for PP, RDT may deliver false negative results due to inconsistent sample quality. Plague diagnosis could be improved through the development of next generation of RDTs.


Assuntos
Técnicas Bacteriológicas/métodos , Peste/microbiologia , Proteínas de Bactérias/imunologia , Testes Diagnósticos de Rotina , Epidemias , Humanos , Madagáscar/epidemiologia , Peste/epidemiologia , Estudos Retrospectivos , Yersinia pestis/imunologia
5.
BMC Infect Dis ; 20(1): 11, 2020 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-31906870

RESUMO

BACKGROUND: Brucellosis is endemic in many areas in China. The current diagnosis of Brucellosis predominantly relies on the traditional bacterial culture and serum agglutination test. In this study, we aimed to explore the value of ELISA in the diagnosis of Brucellosis in Chinese population. METHODS: We recruited 235 patients with a diagnosis of Brucellosis at different clinical stages: 117 in acute, 78 in subacute, and 40 in chronic. We also recruited 248 control patients who presented with similar clinical symptoms but with a different diagnosis other than Brucellosis. In addition, 90 healthy volunteers were also recruited. Bacterial culture, agglutination test and ELISA assay were performed to detect Brucella spp. RESULTS: Among 235 patients with Brucellosis, 51 (21.7%) was positive for bacterial culture, 150 (63.8%) were positive by agglutination test, and 232 (98.7%) were positive by ELISA (IgG and/or IgM). When we stratified the patients based on the disease stages (acute, subacute and chronic), ELISA was the most sensitive method and showed a highest positive rate in all stages. By Receiver Operating Characteristic Curve analysis of ELISA results, we found that measurement of IgG level was superior to measurement of IgM level (AUC, 0.993 versus 0.877). Since the measurement of IgG itself missed rare cases in acute phase, we recommended measuring IgG and IgM simultaneously by ELISA for the diagnosis of Brucellosis. In term of the specificity of ELISA in the diagnosis of Brucellosis, our study showed that only 1.6% (4/248) non-Brucellosis patients were positive by ELISA; all positive cases were IgM only and none showed positive IgG. Similar results were found in healthy volunteers. In summary, our study concluded that ELISA is the most sensitive and specific method to detect Brucellosis in Chinese population. CONCLUSIONS: ELISA assay is sensitive, fast, and convenient to detect Brucellosis. It shows the high sensitivity and specifity and should be used as a routine lab test when Brucellosis is suspected in clinical practice.


Assuntos
Técnicas Bacteriológicas/métodos , Técnicas Bacteriológicas/normas , Brucella/isolamento & purificação , Brucelose/diagnóstico , Ensaio de Imunoadsorção Enzimática/normas , Adulto , Idoso , Testes de Aglutinação/normas , Anticorpos Antibacterianos/sangue , Brucella/crescimento & desenvolvimento , Brucella/imunologia , Brucelose/sangue , Brucelose/microbiologia , China , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Masculino , Pessoa de Meia-Idade , Sensibilidade e Especificidade
6.
Am J Trop Med Hyg ; 102(2): 388-391, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31769397

RESUMO

Burkholderia pseudomallei infections are prevalent in Southeast Asia and northern Australia and often misdiagnosed. Diagnostics are often neither sensitive nor rapid, contributing up to 50% mortality rate. In this 2018 pilot study, we enrolled 100 patients aged 6 months-79 years from Kapit Hospital in Sarawak, Malaysia, with symptoms of B. pseudomallei infection. We used three different methods for the detection of B. pseudomallei: a real-time polymerase chain reaction (PCR) assay, a rapid lateral flow immunoassay, and the standard-of-care bacterial culture-the gold standard. Among the 100 participants, 24 (24%) were positive for B. pseudomallei by one or more of the detection methods. Comparing the two individual diagnostic methods against the gold standard-bacterial culture-of any positive test, there was low sensitivity for each test (25-44%) but high specificity (93-98%). It seems clear that more sensitive diagnostics or a sensitive screening diagnostic followed by specific confirmatory diagnostic is needed for this disease.


Assuntos
Técnicas Bacteriológicas/métodos , Burkholderia pseudomallei/isolamento & purificação , Melioidose/diagnóstico , Melioidose/microbiologia , Humanos , Malásia , Melioidose/epidemiologia , Sensibilidade e Especificidade
7.
Am J Ophthalmol ; 212: 34-42, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31770517

RESUMO

PURPOSE: Rapid identification of virulent pathogens is essential to strengthen the therapeutic strategy of acute endophthalmitis. OBJECTIVES: This study sought to compare the contribution of a combination of polymerase chain reaction (PCR)-based tests to culture methods, in patients with postoperative endophthalmitis. DESIGN: Prospective multicenter study diagnostic evaluation. METHODS: Setting: university referral centers. PARTICIPANTS: 153 consecutive patients presenting with acute or delayed-onset postoperative endophthalmitis, between 2008 and 2015. There were a total of 284 aqueous humor (AH) and/or vitreous fluid (VF) samples. Outcomes and measurements: microbiological tests of intraocular samples included bacterial culturing of pediatric blood culture bottles; 16SrDNA amplification and sequencing (panbacterial PCR) for detection and identification of all bacterial species; real-time PCR (qPCR) assays targeting the femA or lytA gene for detection of Staphylococcus aureus (S. aureus) or Streptococcus pneumoniae (S. pneumoniae), respectively; and a qPCR assay targeting the tuf gene for detection and quantification of Staphylococcus epidermidis (S. epidermidis). RESULTS: At the time of admission, the rate of detection of microorganisms by PCR-based tests was not significantly different than that by culturing (38% versus 30% in AH samples [n = 69]; 66% versus 63% in VF samples [n = 82], respectively). In contrast, after 1 intravitreal injection (IVI) of antibiotics, the identification rate by PCR-based tests was higher than that in VF by culturing (62% vs 48%, respectively; n = 94; P = 0.05). Bacteria were identified in 70% of patients, with a predominance of Gram-positive bacteria (93%). Specific qPCR tests targeting S. aureus and S. pneumoniae did not provide additional diagnoses but provided earlier results. The S. epidermidis load in vitreous at the time of patients' admission was higher in cases of final visual acuity (VA) of <20/40 (127,118 ± 125,848 DNA copies/mL) in patients with a VA of ≥20/40 (40350,000 ± 46,912 DNA copies/mL; P = 0.09). No significant changes in S. epidermidis load was found after one IVI. CONCLUSIONS: Patients with acute or delayed-onset endophthalmitis should benefit from microbiological identification in vitreous samples by combined analysis using bacterial cultures in pediatric blood culture bottles and panbacterial PCR. The last test was more effective than cultures in vitreous samples collected after an IVI of antibiotics. The qPCR tests targeting S. aureus and S. pneumoniae gave earlier results than culture and panbacterial PCR but did not provide additional diagnoses. As for S. epidermidis infections, determination of bacterial load using the qPCR test targeting the tuf gene could help evaluation of the visual prognosis of patients. Its role in the follow-up of patients after antibiotic treatment needs further investigation.


Assuntos
Endoftalmite/diagnóstico , Infecções Oculares Bacterianas/diagnóstico , Complicações Pós-Operatórias/diagnóstico , Infecções Estafilocócicas/diagnóstico , Doença Aguda , Idoso , Antibacterianos/uso terapêutico , Humor Aquoso/microbiologia , Técnicas Bacteriológicas/métodos , Extração de Catarata/efeitos adversos , DNA Bacteriano , DNA Ribossômico , Endoftalmite/tratamento farmacológico , Feminino , Humanos , Masculino , Técnicas de Amplificação de Ácido Nucleico/normas , Reação em Cadeia da Polimerase/normas , Estudos Prospectivos , Reação em Cadeia da Polimerase em Tempo Real/normas , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/isolamento & purificação , Staphylococcus epidermidis/isolamento & purificação , Streptococcus pneumoniae/isolamento & purificação , Trabeculectomia/efeitos adversos , Acuidade Visual/fisiologia , Vitrectomia/efeitos adversos , Corpo Vítreo/microbiologia
8.
J Med Microbiol ; 69(1): 49-51, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31750812

RESUMO

Introduction. Burkholderia pseudomallei (melioidosis) is an important cause of community-acquired pneumonia (CAP) in the tropics. Selective medium is recommended for laboratory diagnosis with non-sterile respiratory samples, while PCR is not routinely used due to variable reported performance. The effectiveness of these diagnostic modalities varies by site.Aim. To compare selective media and real-time PCR (qPCR) with routine media in detecting B. pseudomallei in CAP respiratory samples in a low-incidence setting in Kuala Lumpur, Malaysia.Methodology. Respiratory samples were routinely cultured on blood, chocolate and MacConkey agar (RESP-ROUTINE), and compared to culture on selective Ashdown medium (RESP-SELECTIVE) and qPCR. The gold standard was routine culture of B. pseudomallei from any site (ALL-ROUTINE).Results. B. pseudomallei was detected in 8/204 (3.9 %) samples. Overall sensitivity rates differed (P=0.03) for qPCR (100%), RESP-SELECTIVE (87.5%) and RESP-ROUTINE (50%). There was a trend towards lower median days to positive culture for RESP-SELECTIVE (1 day) compared to RESP-ROUTINE (2 days, P=0.08) and ALL-ROUTINE (2 days, P=0.06). Reagent costs for each additional detection were USD59 for RESP-SELECTIVE and USD354 for PCR.Conclusions. In a low-incidence setting, selective culture of respiratory samples on Ashdown was more sensitive and allowed quicker identification than routine media, at reasonable cost. Blood cultures are critical, confirming four cases missed by routine respiratory culture. Selective medium is useful in early pneumonia (pre-sepsis) and resource-limited settings where blood cultures are infrequently done. Real-time PCR is costly, but highly sensitive and useful for high-risk patients with diabetes, cancer or immunosuppressants, or requiring ventilation or intensive care.


Assuntos
Técnicas Bacteriológicas/métodos , Burkholderia pseudomallei/isolamento & purificação , Meios de Cultura/química , Melioidose/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Infecções Comunitárias Adquiridas/diagnóstico , Infecções Comunitárias Adquiridas/epidemiologia , Humanos , Incidência , Malásia/epidemiologia , Melioidose/epidemiologia , Pneumonia Bacteriana/diagnóstico , Pneumonia Bacteriana/epidemiologia , Sensibilidade e Especificidade
9.
J Hosp Infect ; 104(3): 381-389, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31790743

RESUMO

Recently, molecular assays have been demonstrated to be reliable for rapid detection of carbapenemase-producing Enterobacteriaceae (CPE) directly from positive blood cultures, reducing significantly the time for identification. Few studies have tested their performance on rectal swabs and no comprehensive conclusions have been reached regarding their utility for infection control management. Our aim was to review and assess the overall diagnostic test accuracy of polymerase chain reaction for the detection of CPE in rectal swabs. The electronic database PubMed was searched, up to October 1st, 2019, without language restriction or publication date restrictions. First, the concepts of the research questions were defined: 'carbapenemase-producing Enterobacteriaceae', 'molecular testing', 'test detection', and 'rectal screening'. Two reviewers independently screened studies, extracted data, and assessed quality using the QUADAS-2 instrument. Statistical analyses were carried out in Stata software using the bivariate model. In all, 143 articles were screened and 16 studies were included. Five (31%) of the studies were conducted in the context of a CPE outbreak; one study (6%) included patients pre-identified with CPE in clinical samples (blood or tracheal secretions), whereas the rest (63%) collected rectal swabs from patients considered at high risk of colonization. The molecular assays evaluated had a relatively good sensitivity of 0.95 (95% confidence interval (CI): 0.902-0.989), and an excellent specificity of 0.994 (95% CI: 0.965-1). Molecular techniques seem to be a useful, accurate diagnostic tool in screening for carriage of CPE in contact patients around a fortuitous discovery of a non-isolated hospitalized carrier patient.


Assuntos
Infecções por Enterobacteriaceae/diagnóstico , Enterobacteriaceae/isolamento & purificação , Reto/microbiologia , beta-Lactamases/metabolismo , Técnicas Bacteriológicas/métodos , Enterobacteriaceae/enzimologia , Humanos , Programas de Rastreamento/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Fatores de Tempo
10.
J Med Microbiol ; 69(1): 46-48, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31789587

RESUMO

Introduction. The question of whether a single day of incubation is sufficient for urine cultures has been a matter of debate.Aim. The aim of this study was to investigate the potential benefit of prolonged incubation for initially culture-negative urines.Methodology. Eight hundred and twelve urine specimens with no growth after incubation for 20 h were incubated for an additional 20 h to detect slower growing uropathogenic organisms.Results. This study included a considerable number of urine cultures from immunocompromised and/or kidney-transplanted patients. For 99.9 % of the specimens, there was no difference in the interpretation of results.Conclusion. Twenty hours of incubation did not have any negative effect on the detection of uropathogens.


Assuntos
Bactérias/isolamento & purificação , Técnicas Bacteriológicas/métodos , Testes Diagnósticos de Rotina/métodos , Infecções Urinárias/diagnóstico , Urina/microbiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Fatores de Tempo , Adulto Jovem
11.
Ann Lab Med ; 40(3): 259-263, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31858767

RESUMO

There is an urgent need for accurate and rapid diagnostic assays capable of identifying carbapenemase-producing Enterobacteriaceae (CPE). We assessed the performance of the RESIST-4 O.K.N.V. (OKNV) assay (Coris BioConcept, Gembloux, Belgium) for the identification of oxacillinase (OXA)-48-like-, Klebsiella pneumoniae carbapenemase (KPC)-, New Delhi metallo-ß-lactamase (NDM)-, and Verona integron-encoded metallo-ß-lactamase (VIM)-producing Enterobacteriaceae grown on sheep blood agar (SBA) and the CHROMagar KPC medium. Sixty-five carbapenem-resistant Enterobacteriaceae (CRE) isolates with characterized carbapenemase content were used to evaluate the OKNV assay. The assay correctly identified all 30 isolates that produced one of the four targeted carbapenemase families. Additionally, it correctly identified 15 isolates that co-produced KPC and NDM, VIM and NDM or OXA-48-like and NDM, but failed to identify an NDM-1 and OXA-232 co-producing Klebsiella pneumoniae isolate. All 16 non-carbapenemase-producing CRE and four CPE isolates exhibited negative results, and no cross-reaction was observed. Overall, the sensitivity and specificity of the assay were 97.8% and 100%, respectively. The OKNV assay is an accurate and rapid assay for identifying OXA-48-like, KPC, NDM, and VIM carbapenemases produced by Enterobacteriaceae isolates cultured on both SBA and the CHROMagar KPC media in the clinical microbiology laboratory.


Assuntos
Proteínas de Bactérias/análise , Enterobacteriaceae/enzimologia , beta-Lactamases/análise , Técnicas Bacteriológicas/métodos , Enterobacteriáceas Resistentes a Carbapenêmicos/enzimologia , Enterobacteriáceas Resistentes a Carbapenêmicos/isolamento & purificação , Farmacorresistência Bacteriana/genética , Enterobacteriaceae/isolamento & purificação , Infecções por Enterobacteriaceae/diagnóstico , Infecções por Enterobacteriaceae/microbiologia , Humanos , Klebsiella pneumoniae/enzimologia , Klebsiella pneumoniae/isolamento & purificação , Kit de Reagentes para Diagnóstico , Sensibilidade e Especificidade
12.
PLoS One ; 14(12): e0225475, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31790434

RESUMO

Rapid sample preparation is one of the leading bottlenecks to low-cost and efficient sample component detection. To overcome this setback, a technology known as Lyse-It has been developed to rapidly (less than 60 seconds) lyse Gram-positive and-negative bacteria alike, while simultaneously fragmenting DNA/RNA and proteins into tunable sizes. This technology has been used with a variety of organisms, but the underlying mechanism behind how the technology actually works to fragment DNA/RNA and proteins has hitherto been studied. It is generally understood how temperature affects cellular lysing, but for DNA/RNA and protein degradation, the temperature and amount of energy introduced by microwave irradiation of the sample, cannot explain the degradation of the biomolecules to the extent that was being observed. Thus, an investigation into the microwave generation of reactive oxygen species, in particular singlet oxygen, hydroxyl radicals, and superoxide anion radicals, was undertaken. Herein, we probe one aspect, the generation of reactive oxygen species (ROS), which is thought to contribute to a non-thermal mechanism behind biomolecule fragmentation with the Lyse-It technology. By utilizing off/on (Photoinduced electron transfer) PET fluorescent-based probes highly specific for reactive oxygen species, it was found that as oxygen concentration in the sample and/or microwave irradiation power increases, more reactive oxygen species are generated and ultimately, more oxidation and biomolecule fragmentation occurs within the microwave cavity.


Assuntos
Métodos Analíticos de Preparação de Amostras/métodos , Técnicas Bacteriológicas/métodos , Fragmentação do DNA/efeitos dos fármacos , Detergentes/farmacologia , Estabilidade de RNA/efeitos dos fármacos , Fragmentação do DNA/efeitos da radiação , DNA Bacteriano/química , DNA Bacteriano/efeitos dos fármacos , DNA Bacteriano/efeitos da radiação , Hidrólise/efeitos da radiação , Listeria monocytogenes/efeitos dos fármacos , Listeria monocytogenes/genética , Listeria monocytogenes/efeitos da radiação , Micro-Ondas , Oxirredução/efeitos dos fármacos , Oxirredução/efeitos da radiação , Oxigênio/análise , Oxigênio/metabolismo , Proteólise/efeitos dos fármacos , Proteólise/efeitos da radiação , Estabilidade de RNA/efeitos da radiação , RNA Bacteriano/química , RNA Bacteriano/efeitos dos fármacos , RNA Bacteriano/efeitos da radiação , Espécies Reativas de Oxigênio/análise , Espécies Reativas de Oxigênio/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Staphylococcus aureus/efeitos da radiação , Temperatura , Fatores de Tempo , Vibrio cholerae/efeitos dos fármacos , Vibrio cholerae/genética , Vibrio cholerae/efeitos da radiação
13.
BMC Infect Dis ; 19(1): 1037, 2019 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-31818261

RESUMO

BACKGROUND: Shigella spp. and entero-invasive E. coli (EIEC) use the same invasive mechanism to cause diarrheal diseases. Public health regulations apply only to Shigella spp. infections, but are hampered by the lack of simple methods to distinguish them from EIEC. In the last decades, molecular methods for detecting Shigella spp. and EIEC were implemented in medical microbiological laboratories (MMLs). However, shigellosis cases identified with molecular techniques alone are not notifiable in most countries. Our study investigates the impact of EIEC versus Shigella spp. infections and molecular diagnosed shigellosis versus culture confirmed shigellosis for re-examination of the rationale for the current public health regulations. METHODS: In this multicenter cross-sectional study, fecal samples of patients suspected for gastro-enteritis, referred to 15 MMLs in the Netherlands, were screened by PCR for Shigella spp. or EIEC. Samples were cultured to discriminate between the two pathogens. We compared risk factors, symptoms, severity of disease, secondary infections and socio-economic consequences for (i) culture-confirmed Shigella spp. versus culture-confirmed EIEC cases (ii) culture positive versus PCR positive only shigellosis cases. RESULTS: In 2016-2017, 777 PCR positive fecal samples with patient data were included, 254 of these were culture-confirmed shigellosis cases and 32 were culture-confirmed EIEC cases. EIEC cases were more likely to report ingestion of contaminated food and were less likely to be men who have sex with men (MSM). Both pathogens were shown to cause serious disease although differences in specific symptoms were observed. Culture-negative but PCR positive cases were more likely report travel or ingestion of contaminated food and were less likely to be MSM than culture-positive cases. Culture-negative cases were more likely to suffer from multiple symptoms. No differences in degree of secondary infections were observed between Shigella spp. and EIEC, and culture-negative and culture-positive cases. CONCLUSIONS: No convincing evidence was found to support the current guidelines that employs different measures based on species or detection method. Therefore, culture and molecular detection methods for Shigella spp. and EIEC should be considered equivalent for case definition and public health regulations regarding shigellosis. Differences were found regarding risks factors, indicating that different prevention strategies may be required.


Assuntos
Disenteria Bacilar/epidemiologia , Disenteria Bacilar/microbiologia , Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/microbiologia , Adolescente , Adulto , Técnicas Bacteriológicas/métodos , Estudos Transversais , Diarreia/microbiologia , Disenteria Bacilar/etiologia , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Escherichia coli/patogenicidade , Infecções por Escherichia coli/etiologia , Fezes/microbiologia , Feminino , Gastroenterite/microbiologia , Homossexualidade Masculina/estatística & dados numéricos , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Países Baixos/epidemiologia , Reação em Cadeia da Polimerase , Saúde Pública , Shigella/genética , Shigella/isolamento & purificação , Shigella/patogenicidade , Adulto Jovem
14.
Ann Clin Lab Sci ; 49(6): 748-755, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31882425

RESUMO

Bacterial sepsis after platelet transfusion is a major cause of transfusion-transmitted infections in the US. The Food and Drug Administration (FDA) recommends performing quality control for platelet bacterial detection on days 4 and 5 before platelet transfusion. We assessed the feasibility of implementing the Pan Genera Detection (PGD) test, an FDA-approved immunoassay for platelet bacterial detection, for the primary and secondary bacterial screening of platelet units in a high-volume setting. Records were reviewed from January 2010 through December 2015. All apheresis platelets underwent primary screening by using culture methods. Additional screening with the PGD test was performed daily until February 2013, when PGD testing of apheresis platelets was performed at the start of storage day 5. In April 2015, PGD testing of apheresis platelet products was performed at the start of storage day 4. Post-storage pooled whole blood-derived platelets were screened by using the PGD test on the day of use. During the 6-year study period, 16,839 PGD tests were performed. If the PGD test was reactive, repeat PGD testing was performed. In cases of repeat reactivity, units were quarantined and cultured. Initially, 42 (0.25%) tests were reactive; 26/42 (61.91%) were repeatedly reactive and resulted in an overall PGD repeat reactivity rate of 0.15%. Only one sample grew coagulase-negative Staphylococcus No transfusion-transmitted infections were reported. The PGD bacterial detection assay was feasible and efficient in our high-volume transfusion service.


Assuntos
Técnicas Bacteriológicas/métodos , Plaquetas/microbiologia , Transfusão de Sangue , Imunoensaio/métodos , Bactérias/isolamento & purificação , Técnicas Bacteriológicas/estatística & dados numéricos , Humanos , Imunoensaio/estatística & dados numéricos , Fluxo de Trabalho
16.
Indian Pediatr ; 56(11): 913-916, 2019 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-31729320

RESUMO

OBJECTIVE: To find the effects of inhaled corticosteroids and the impact of different doses of inhaled corticosteroids on the isolation of nasopharyngeal flora in asthmatic children aged 1-15 years. METHODS: The study included 75 children with asthma and 25 age-matched controls. Nasopharyngeal swabs were obtained. Bacteria were identified by standard techniques. RESULTS: Pathogenic organisms were isolated from 36% of asthmatic children and 20% of controls, the difference was not significant statistically (OR=2.25, 95% CI=0.75-6.67, P=0.13). There was no statistically significant association of using a high dose of inhaled corticosteroids with the isolation of pathogenic organisms. Usage of biomass fuel for cooking in the household of asthmatic children increases the risk of colonization (OR=3.4, 95% CI= 1.26-9.10, P=0.03). CONCLUSIONS: Inhaled corticosteroids are safe in the treatment of asthma and there is no association between different doses of Inhaled corticosteroids and isolation of the pathogenic organism.


Assuntos
Asma , Bactérias , Glucocorticoides/administração & dosagem , Nasofaringe/microbiologia , Administração por Inalação , Asma/tratamento farmacológico , Asma/microbiologia , Bactérias/classificação , Bactérias/efeitos dos fármacos , Bactérias/isolamento & purificação , Técnicas Bacteriológicas/métodos , Técnicas Bacteriológicas/estatística & dados numéricos , Criança , Pré-Escolar , Correlação de Dados , Relação Dose-Resposta a Droga , Feminino , Humanos , Lactente , Masculino , Fatores de Risco
17.
Braz. j. biol ; 79(4): 555-565, Nov. 2019. tab
Artigo em Inglês | LILACS | ID: biblio-1001469

RESUMO

Abstract Different methodologies have been developed throughout the years to identify environmental microorganisms to improve bioremediation techniques, determine susceptibility profiles of bacteria in contaminated environments, and reduce the impact of microorganisms in ecosystems. Two methods of bacterial biochemical identification are compared and the susceptibility profile of bacteria, isolated from residential and industrial wastewater, is determined. Twenty-four bacteria were retrieved from the bacteria bank of the Environmental Microbiology Laboratory at the Institute of Biology (IB) of the Universidade Federal de Pelotas, Pelotas, Brazil. Bacteria were identified by conventional biochemical tests and by the VITEK ®2 automated system. Further, the susceptibility profile to antibiotics was also determined by the automated system. Six species of bacteria (Raoutella planticola, K. pneumoniae ssp. pneumoniae , Serratia marcescens, Raoutella sp., E. cloacae and Klebsiella oxytoca) were identified by conventional biochemical tests, while three species of bacteria (K. pneumoniae ssp. pneumoniae, S. marcescens and K. oxytoca ) were identified by VITEK®2 automated system. VITEK ®2 indicated agreement in 19 (79.17%) isolates and difference in five (20.83%) isolates when compared to results from conventional biochemical tests. Further, antibiotic susceptibility profile results showed that all isolates (100%) were resistant to at least one out of the 18 antibiotics tested by VITEK®2. Thus, no multi-resistant bacteria that may be used in effluent treatment systems or in bioremediation processes have been reported. Results indicate VITEK ® 2 automated system as a potential methodology in the determination of susceptibility profile and identification of environmental bacteria.


Resumo Diferentes metodologias foram desenvolvidas ao longo dos anos para identificar microrganismos ambientais para melhorar as técnicas de biorremediação, determinar perfis de suscetibilidade de bactérias em ambientes contaminados e reduzir o impacto de microrganismos nos ecossistemas. Dois métodos de identificação bioquímica bacteriana são comparados e o perfil de susceptibilidade de bactérias, isoladas de efluentes residenciais e industriais, é determinado. Vinte e quatro bactérias foram coletadas do banco de bactérias do Laboratório de Microbiologia Ambiental do Instituto de Biologia (IB) da Universidade Federal de Pelotas, Pelotas, Brasil. As bactérias foram identificadas por testes bioquímicos convencionais e pelo sistema automatizado VITEK®2. Além disso, o perfil de suscetibilidade aos antibióticos também foi determinado pelo sistema automatizado. Seis espécies de bactérias (Raoutella planticola , K. pneumoniae ssp. pneumoniae, Serratia marcescens, Raoutella sp., E. cloacae e Klebsiella oxytoca) foram identificadas por testes bioquímicos convencionais, enquanto três espécies de bactérias (K. pneumoniae ssp. pneumoniae, S. marcescens e K. oxytoca) foram identificados pelo sistema automatizado VITEK®2. VITEK®2 indicou concordância em 19 (79,17%) isolados e diferença em cinco (20,83%) isolados quando comparados aos resultados de testes bioquímicos convencionais. Além disso, os resultados do perfil de suscetibilidade aos antibióticos mostraram que todos os isolados (100%) foram resistentes a pelo menos um dos 18 antibióticos testados pelo VITEK®2. Assim, não foram relatadas bactérias multirresistentes que possam ser usadas em sistemas de tratamento de efluentes ou em processos de biorremediação. Os resultados indicam que o sistema automatizado VITEK ® 2 é uma metodologia potencial na determinação do perfil de suscetibilidade e identificação de bactérias ambientais.


Assuntos
Bactérias/isolamento & purificação , Bactérias/efeitos dos fármacos , Técnicas Bacteriológicas/métodos , Farmacorresistência Bacteriana , Brasil , Técnicas Bacteriológicas/instrumentação , Antibacterianos/farmacologia
18.
Dis Colon Rectum ; 62(11): 1390-1400, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31596764

RESUMO

BACKGROUND: Few data are published on perianal tuberculosis. OBJECTIVE: This study aimed to determine the best method to diagnose tuberculosis in patients with fistula-in-ano and to conduct a systematic review to determine the incidence and characteristics of tuberculosis fistula-in-ano. DATA SOURCES: The prospective study data and existing literature were derived from PubMed, Google scholar, and Scopus STUDY SELECTION:: Prospective analysis of patients with tuberculous fistula-in-ano treated between 2014 and 2018 was conducted, and a systematic review of studies describing ≥3 patients with tuberculosis fistula-in-ano was completed. INTERVENTION: Testing of tuberculosis was performed by histopathology or polymerase chain reaction of tissue or pus from the fistula tract. MAIN OUTCOME MEASURES: The primary outcomes measured were the detection rate of various tests to detect tuberculosis in fistula-in-ano and the prevalence rate of tuberculosis in simple versus complex fistulas. RESULTS: In 637 samples (410 patients) tested, tuberculosis was detected in 49 samples (43 patients). Additional samples (n = 106) sent in patients with a high index of suspicion tested positive in 14 more patients. Thus, overall, 63 samples tested positive in 57 patients (total: 743 samples in 410 patients were tested). Tuberculosis was detected in 2 of 181 patients (1.1%) in tissue (histopathology), in 28 of 341 patients (8.2%) in tissue (polymerase chain reaction), and in 19 of 115 patients (16.5%) in pus (polymerase chain reaction) samples. To detect tuberculosis, tissue (polymerase chain reaction) was significantly better than tissue (histopathology) (28/341 vs 2/181, p < 0.00001) and pus (polymerase chain reaction) was significantly better than tissue (polymerase chain reaction) (19/115 vs 28/341, p < 0.0009). Tuberculosis was significantly more common in complex fistulas than in simple fistulas (20.3% vs 7.2%, p = 0.0002). The systematic review (n = 199) highlighted that tubercular fistulas are more common in recurrent and complex fistulas and in tuberculosis endemic regions. LIMITATIONS: The true sensitivity and specificity of each testing modality could not be determined because not all patients with tuberculosis fistula-in-ano were tested by every diagnostic modality studied. CONCLUSIONS: The tuberculosis detection rate of polymerase chain reaction was significantly higher than histopathology. Among polymerase chain reaction, pus had higher detection rate than tissue. Tuberculosis was associated with more complex and recurrent fistulas.


Assuntos
Fissura Anal , Mycobacterium tuberculosis , Fístula Retal , Estreptomicina/administração & dosagem , Tuberculose Gastrointestinal , Assistência ao Convalescente/métodos , Antituberculosos/administração & dosagem , Técnicas Bacteriológicas/métodos , Técnicas Bacteriológicas/estatística & dados numéricos , Feminino , Fissura Anal/diagnóstico , Fissura Anal/epidemiologia , Fissura Anal/microbiologia , Fissura Anal/terapia , Humanos , Incidência , Índia/epidemiologia , Imagem por Ressonância Magnética/métodos , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Avaliação de Resultados em Cuidados de Saúde , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/estatística & dados numéricos , Fístula Retal/diagnóstico , Fístula Retal/epidemiologia , Fístula Retal/microbiologia , Fístula Retal/terapia , Recidiva , Reprodutibilidade dos Testes , Procedimentos Cirúrgicos Operatórios/métodos , Procedimentos Cirúrgicos Operatórios/estatística & dados numéricos , Tuberculose Gastrointestinal/diagnóstico , Tuberculose Gastrointestinal/epidemiologia , Tuberculose Gastrointestinal/fisiopatologia , Tuberculose Gastrointestinal/terapia
19.
Anaerobe ; 60: 102107, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31647977

RESUMO

BACKGROUND: The optimal and practical laboratory diagnostic approach for detection of Clostridioides difficile to aid in the diagnosis of C. difficile infection (CDI) is controversial. A two-step algorithm with initial detection of glutamate dehydrogenase (GDH) or nucleic acid amplification test (NAAT) alone are recommended as a predominant method for C. difficile detection in developed countries. The aim of this study was to compare the performance of enzyme immunoassays (EIA) detecting toxins A and B, NAAT detecting the toxin B gene, and GDH compared to toxigenic culture (TC) for C. difficile as the gold standard, in patients prospectively and actively assessed with clinically significant diarrhea in 12 medical facilities in Japan. METHODS: A total of 650 stool specimens were collected from 566 patients with at least three diarrheal bowel movements (Bristol stool grade 6-7) in the preceding 24 h. EIA and GDH were performed at each hospital, and NAAT and toxigenic C. difficile culture with enriched media were performed at the National Institute of Infectious Diseases. All C. difficile isolates recovered were analyzed by PCR-ribotyping. RESULTS: Compared to TC, the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of EIA were 41%, 96%, 75% and 84%, respectively, and for NAAT were 74%, 98%, 91%, and 92%, respectively. In 439 specimens tested with GDH, the sensitivity, specificity, PPV, and NPV were 73%, 87%, 65%, and 91%, and for an algorithm (GDH plus toxin EIA, arbitrated by NAAT) were 71%, 96%, 85%, and 91%, respectively. Among 157 isolates recovered, 75% of isolates corresponded to one of PCR-ribotypes (RTs) 002, 014, 018/018", and 369; RT027 was not isolated. No clear differences in the sensitivities of any of EIA, NAAT and GDH for four predominant RTs were found. CONCLUSION: The analytical sensitivities of NAAT and GDH-algorithm to detect toxigenic C. difficile in this study were lower than most previous reports. This study also found low PPV of EIAs. The optimal method to detect C. difficile or its toxins to assist in the diagnosis of CDI needs further investigation.


Assuntos
Técnicas Bacteriológicas , Infecções por Clostridium/diagnóstico , Infecções por Clostridium/microbiologia , Clostridium difficile/genética , Toxinas Bacterianas/genética , Técnicas Bacteriológicas/métodos , Técnicas Bacteriológicas/normas , Infecções por Clostridium/epidemiologia , Clostridium difficile/classificação , Clostridium difficile/isolamento & purificação , Feminino , Humanos , Japão/epidemiologia , Masculino , Reação em Cadeia da Polimerase , Estudos Prospectivos , Ribotipagem , Sensibilidade e Especificidade
20.
Ann Biol Clin (Paris) ; 77(5): 537-542, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31638584

RESUMO

The cytobacteriological examination of urine (CBEU) is the most prescribed test in a microbiology laboratory. The objectives of this work were to present our experience in carrying out the CBEU and to share one of the means that we consider very useful in the daily practice of this crucial analysis. This is a 28-month prospective study (March 2016 to June 2018). The CBEU was carried out in accordance with the recommendations of the medical microbiology referential (REMIC). Antibiotic susceptibility was studied in accordance with the recommendations of the l'European committee on antimicrobial susceptibility testing (EUCAST). Cultures were positive for urinary tract infection in 5.09% (n=769) of cases and for colonization in 4.88% (n=737) of cases. E. coli alone accounted for 57.8% (n=850) of all isolates. In our experience, the display of the interpretation flowchart at the bench level, as well as the availability of clinical information and cytology results when examining urocultures, allows our technicians to decide, independently, what action to take for each CBEU according to the particular context of the patient for whom it has been prescribed. Similarly, this flowchart allows the unique microbiologist in the laboratory to contextually interpret each CBEU.


Assuntos
Técnicas Bacteriológicas , Interpretação Estatística de Dados , Design de Software , Urinálise , Infecções Urinárias/diagnóstico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Técnicas Bacteriológicas/métodos , Técnicas Bacteriológicas/estatística & dados numéricos , Criança , Pré-Escolar , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/diagnóstico , Infecções por Escherichia coli/microbiologia , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Estudos Prospectivos , Reprodutibilidade dos Testes , Urinálise/métodos , Urinálise/estatística & dados numéricos , Infecções Urinárias/microbiologia , Adulto Jovem
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