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1.
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
2.
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
3.
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
4.
Ann Biol Clin (Paris) ; 77(5): 525-531, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31512576

RESUMO

In order to perform biological analysis, clinical laboratories apply the instructions of reagent suppliers. For culture media these instructions are often incomplete and poorly adapted to the variety of clinical samples and micro-organisms. The REMIC can help to overcome these shortcomings. Required time of incubation for culture media are proposed based on the nature of the sample and the type of micro-organism suspected. Nevertheless, they are most often expressed in multiple of 24 hours and they are often considered as minimal by the laboratories. As the samples are inoculated "continuously", while the readings are most often done at a single definite time of the day, we propose a strategy to optimize incubation duration of cultures medium. A time of incubation in the day so-called "limit" is defined. From this, the incubations are stopped or prolonged according to the results of the culture and the direct examination. As the instructions of suppliers of culture media are not adapted, it appears necessary that these suppliers relies on the repositories of professional societies as this is the case for agars medias used for antibiotic susceptibility testing.


Assuntos
Serviços de Laboratório Clínico/normas , Meios de Cultura/normas , Técnicas Microbiológicas , Técnicas Bacteriológicas/métodos , Técnicas Bacteriológicas/normas , Calibragem , Humanos , Incubadoras/normas , Técnicas Microbiológicas/métodos , Técnicas Microbiológicas/normas , Fatores de Tempo
5.
Arch Microbiol ; 201(10): 1427-1433, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31414157

RESUMO

We controlled and observed individual magneto-tactic bacteria (Magnetospirillum gryphiswaldense) inside a [Formula: see text]-high microfluidic channel for over 4 h. After a period of constant velocity, the duration of which varied between bacteria, all observed bacteria showed a gradual decrease in their velocity of about [Formula: see text]. After coming to a full stop, different behaviour was observed, ranging from rotation around the centre of mass synchronous with the direction of the external magnetic field, to being completely immobile. Our results suggest that the influence of the high-intensity illumination and the presence of the channel walls are important parameters to consider when performing observations of such long duration.


Assuntos
Técnicas Bacteriológicas/métodos , Magnetospirillum/fisiologia , Microfluídica , Técnicas Bacteriológicas/normas , Fatores de Tempo
6.
J Environ Public Health ; 2019: 5340263, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31360175

RESUMO

Background: Tuberculosis (TB) is a major public health problem in Liberia. Little is known about the TB laboratory performance of Liberia and the challenges after the 14 years of civil war which ended in 2003. The purpose of the study was to evaluate the TB laboratory performance of Liberia. Methods: A cross-sectional study was conducted from 2014 to 2015. The study was conducted using quantitative data of TB case findings, sputum microscopy proficiency testing, and on-site assessment of sputum microscopy laboratories in Liberia. 80 laboratories participated in the proficiency testing. Besides, four years' (2012-2015) TB case finding data obtained from the National Leprosy and Tuberculosis Control Programme (NLTCP) were used to complement the study. The data were analysed using descriptive statistics. Results: From the 80 TB sputum microscopy testing laboratories participating in proficiency testing, only 20 (25%) scored acceptable performance. 46 (58%) TB microscopy laboratories reported quantification errors for the proficiency panel slide 6 which was 3+. The national TB smear-positive cases notified were 4342 in 2012 but decreased to 3820 and 2448 in 2013 and 2014, respectively. The TB smear case detection rate showed an increase from 68% in 2010 to 78% in 2011 and a decrease to 60%, 57%, and 42% in 2012, 2013, and 2014, respectively. Conclusion: Between 2010 and 2013, the NLTCP succeeded in increasing the number of TB sputum microscopy laboratories. At most of the TB microscopy sites, the TB laboratory quality system was not implemented. The NLTCP of Liberia should develop strategies to overcome its challenges in TB laboratory testing.


Assuntos
Controle de Doenças Transmissíveis/organização & administração , Laboratórios/normas , Tuberculose/prevenção & controle , Técnicas Bacteriológicas/normas , Controle de Doenças Transmissíveis/normas , Estudos Transversais , Humanos , Ensaio de Proficiência Laboratorial , Libéria/epidemiologia , Microscopia/normas , Escarro/microbiologia , Tuberculose/diagnóstico , Tuberculose/epidemiologia
8.
Eur J Clin Microbiol Infect Dis ; 38(10): 1961-1968, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31342215

RESUMO

Mycobacterial culture remains the gold standard for detection of Mycobacterium tuberculosis (MTB) in clinical samples. However, no external quality assessment (EQA) tools exist to validate results obtained using this sophisticated method. Therefore, we developed EQA panels to assess the quality of mycobacterial culture results produced by designated TB hospitals in China. Artificial sputum containing methylcellulose was used to supplement quantified mycobacterial solutions to simulate culture-negative and culture-positive clinical sputum samples of low or high mycobacterial concentration, respectively. After storage of the quantified simulated EQA panels for 4 weeks at 4 °C, experimental bacterial quantification of the panels was again conducted, with no impact of artificial sputum on mycobacterial culture results observed. Next, 47 tuberculosis (TB) hospitals were recruited for evaluation of the EQA panels. Overall, 29 hospitals (61.7%) produced mycobacterial culture test results matching expected results for the EQA panels, while the remaining 18 (38.3%) hospitals did not. False-negative results for the low mycobacterial concentration panel sample accounted for 33 (73.3%) diagnostic errors. Compared with hospitals using solid culture methods as a control group, hospitals using the liquid culture method were less likely to produce uncertified results (aOR 0.064, 95% CI 0.005-0.770). In conclusion, we first developed then evaluated EQA panels for validation of mycobacterial culture testing in China. Our data demonstrate that approximately one-third of TB hospitals failed to produce results that met criteria for classification as certified mycobacterial culture testing providers, emphasizing the importance of quality control and quality assurance in TB diagnostics.


Assuntos
Técnicas Bacteriológicas/métodos , Testes Diagnósticos de Rotina/métodos , Ensaio de Proficiência Laboratorial/métodos , Mycobacterium tuberculosis/isolamento & purificação , Escarro/microbiologia , Tuberculose/diagnóstico , Técnicas Bacteriológicas/normas , China , Testes Diagnósticos de Rotina/normas , Hospitais , Humanos
9.
Diagn Microbiol Infect Dis ; 95(1): 20-24, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31129008

RESUMO

Diagnostic tests for Clostridioides difficile infection (CDI) lack either specificity (nucleic acid amplification tests) or sensitivity (enzyme immunoassays; EIAs). The performance of the Singulex Clarity® C. diff toxins A/B assay was compared to cell cytotoxicity neutralization assay. Testing was also performed using an EIA for glutamate dehydrogenase (GDH) and C. difficile toxins A and B (C. Diff Quik Chek Complete®), polymerase chain reaction (PCR) (BD MAX™ Cdiff Assay), and 2 multistep algorithms: algorithm 1 (discordant GDH/toxin results arbitrated by PCR) and algorithm 2 (PCR-positive samples tested with toxin EIA). The Clarity assay and PCR both had 97% sensitivity, while specificity was 100% for Clarity and 79% for PCR. Algorithm 1 yielded 41% discordant results, and both toxin EIA and algorithm 2 had 58% sensitivity. Median toxin concentrations, as measured by the Clarity C. difficile toxin assay, were 3590, 11.5, 0.4, and 0 pg/mL for GDH+/toxin+, GDH+/toxin-/PCR+, GDH+/toxin-/PCR-, and GDH-/toxin- samples, respectively (P < 0.001). The Clarity assay may offer a single-test solution for CDI.


Assuntos
Proteínas de Bactérias/análise , Toxinas Bacterianas/análise , Técnicas Bacteriológicas/normas , Clostridium difficile/isolamento & purificação , Enterocolite Pseudomembranosa/diagnóstico , Enterotoxinas/análise , Imunoensaio/normas , Algoritmos , Clostridium difficile/química , Fezes/química , Fezes/microbiologia , Glutamato Desidrogenase/análise , Humanos , Reação em Cadeia da Polimerase/normas , Sensibilidade e Especificidade
10.
Clin Microbiol Infect ; 25(12): 1553-1559, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31096045

RESUMO

OBJECTIVES: TB-EASM (Howsome, Shanghai, China), an automated system combining smear preparation, staining and microscopy in a single platform, was evaluated for tuberculosis (TB) diagnosis in a high disease-burden setting. METHODS: Sputum samples from individuals with pulmonary TB were processed in parallel using conventional manual smear microscopy (MS), TB-EASM, liquid culture and GeneXpert. Method sensitivity and specificity were compared with Mycobacterium tuberculosis detection by mycobacteria growth indicator tube (MGIT) and/or GeneXpert MTB/RIF. RESULTS: Of 524 samples, 496 met evaluation criteria for study inclusion. The proportion of M. tuberculosis detected by TB-EASM was 28.2% (150/496), significantly higher than for MS (111/496, 21.2%, p 0.01) and comparable to the rate for MGIT (163/496, 32.9%, p > 0.05). For 190 M. tuberculosis-positive cases identified using MGIT and/or GeneXpert MTB/RIF, the reference standard detection methods, TB-EASM detected 140 positive cases, for an overall sensitivity rate of 73.7% (140/190, 95% CI 67.4-79.9), which was significantly higher than for MS (105/190, 55.3%, 95% CI 48.2-62.3, p < 0.01). Specificities were 96.7% (296/306, 95% CI 94.7-98.7) for TB-EASM and 98.0% (300/306, 95% CI 96.5-99.6) for MS. CONCLUSION: TB-EASM outperformed conventional MS for M. tuberculosis detection in sputum specimens.


Assuntos
Técnicas Bacteriológicas/métodos , Testes Diagnósticos de Rotina/métodos , Mycobacterium tuberculosis/isolamento & purificação , Tuberculose Pulmonar/diagnóstico , Adulto , Idoso , Automação Laboratorial , Técnicas Bacteriológicas/instrumentação , Técnicas Bacteriológicas/normas , China/epidemiologia , Testes Diagnósticos de Rotina/instrumentação , Testes Diagnósticos de Rotina/normas , Feminino , Humanos , Masculino , Microscopia , Pessoa de Meia-Idade , Mycobacterium tuberculosis/citologia , Sensibilidade e Especificidade , Escarro/microbiologia , Tuberculose Pulmonar/epidemiologia , Adulto Jovem
11.
Enferm. infecc. microbiol. clín. (Ed. impr.) ; 37(supl.1): 1-18, mayo 2019. tab
Artigo em Espanhol | IBECS | ID: ibc-189723

RESUMO

Se presenta el análisis anual de los resultados remitidos durante el año 2016 por los participantes inscritos en el Programa de Control de Calidad de la Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica (SEIMC), que incluye las áreas de bacteriología, serología, micología, parasitología, micobacterias, virología, microbiología molecular y genotipos de resistencia bacteriana. Los resultados obtenidos por los centros participantes destacan, de nuevo, la adecuada capacitación de la inmensa mayoría de los laboratorios españoles de microbiología clínica, como ya iba sucediendo en los últimos años. Sin embargo, el programa muestra que es posible obtener un resultado erróneo, incluso en determinaciones de la mayor trascendencia y en cualquier laboratorio. Una vez más, se resalta la importancia de complementar el control interno que lleva a cabo cada laboratorio con estudios de intercomparación externos, como los que ofrece el Programa de Control de Calidad SEIMC. Información sobre el suplemento: este artículo forma parte del suplemento titulado "Programa de Control de Calidad Externo SEIMC. Año 2016", que ha sido patrocinado por Roche, Vircell Microbiologists, Abbott Molecular y Francisco Soria Melguizo, S.A


The External Quality Control Programme of the Spanish Society of Infectious Diseases and Clinical Microbiology (SEIMC) includes controls for bacteriology, serology, mycology, parasitology, mycobacteria, virology, molecular microbiology, and genotypic bacterial resistance. As in previous years, the results obtained in 2016 confirm the excellent skill and good technical standards in the vast majority of clinical microbiology laboratories in Spain. However, erroneous results can be obtained in any laboratory and in clinically relevant determinations. Once again, the results of this programme highlight the need to implement both internal and external controls. Supplement information: This article is part of a supplement entitled "SEIMC External Quality Control Programme. Year 2016", which is sponsored by Roche, Vircell Microbiologists, Abbott Molecular and Francisco Soria Melguizo, S.A


Assuntos
Humanos , Avaliação de Resultados em Cuidados de Saúde/normas , Sociedades Médicas/normas , Controle de Qualidade , Análise de Dados , Sorologia/normas , Biologia Molecular , Bacteriologia/normas , Técnicas Bacteriológicas/normas , Virologia/normas
12.
Sci Data ; 6(1): 43, 2019 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-31028276

RESUMO

Cation adjusted-Mueller Hinton Broth (CA-MHB) is the standard bacteriological medium utilized in the clinic for the determination of antibiotic susceptibility. However, a growing number of literature has demonstrated that media conditions can cause a substantial difference in the efficacy of antibiotics and antimicrobials. Recent studies have also shown that minimum inhibitory concentration (MIC) tests performed in standard cell culture media (e.g. RPMI and DMEM) are more indicative of in vivo antibiotic efficacy, presumably because they are a better proxy for the human host's physiological conditions. The basis for the bacterial media dependent susceptibility to antibiotics remains undefined. To address this question, we characterized the physiological response of methicillin-resistant Staphylococcus aureus (MRSA) during exposure to sub-inhibitory concentrations of the beta-lactam antibiotic nafcillin in either CA-MHB or RPMI + 10% LB (R10LB). Here, we present high quality transcriptomic, exo-metabolomic and morphological data paired with growth and susceptibility results for MRSA cultured in either standard bacteriologic or more physiologic relevant medium.


Assuntos
Antibacterianos/farmacologia , Técnicas Bacteriológicas , Meios de Cultura , Staphylococcus aureus Resistente à Meticilina , Testes de Sensibilidade Microbiana/normas , Nafcilina/farmacologia , Técnicas Bacteriológicas/métodos , Técnicas Bacteriológicas/normas , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/crescimento & desenvolvimento , Transcriptoma
14.
Eur J Clin Microbiol Infect Dis ; 38(6): 1171-1178, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30859358

RESUMO

Faster respiratory pathogen detection and antibiotic resistance identification are important in critical care due to the severity of illness, significant prior antibiotic exposure and infection control implications. Our objective was to compare the performance of the commercial Unyvero P55 Pneumonia Cartridge (Curetis AG) with routine bacterial culture methods and in-house bacterial multiplex real-time PCR assays. Seventy-four bronchoalveolar lavage specimens from patients admitted to a Scottish intensive care unit (ICU) over a 33-month period were tested prospectively by routine culture and viral PCR and retrospectively by Unyvero P55 and in-house bacterial PCR. Sensitivity/specificity was 56.9%/58.5% and 63.2%/54.8% for the Unyvero P55 and in-house bacterial PCR panels respectively; sensitivity for in-panel targets was 63.5 and 83.7% respectively. Additional organisms were detected by Unyvero P55 and in-house bacterial PCR panels in 16.2% specimens. Antibiotics were changed on the basis of routine test results in 48.3% cases; of these, true-positive or true-negative results would have been obtained earlier by Unyvero P55 or in-house bacterial PCR panel in 15 (53.6%) and 17 (60.7%) cases respectively. However, a false-negative molecular test result may have been acted upon in six (21.4%) cases with either assay. Sensitivity/specificity of Unyvero P55 antibiotic resistance detection was 18.8%/94.9% respectively. Molecular testing identified a number of respiratory pathogens in this patient cohort that were not grown in culture, but resistance detection was not a reliable tool for faster antibiotic modification. In their current set-up, molecular tests may only have benefit as additional tests in the ICU pneumonia setting.


Assuntos
Bactérias/isolamento & purificação , Técnicas Bacteriológicas/normas , Líquido da Lavagem Broncoalveolar/microbiologia , Resistência Microbiana a Medicamentos/genética , Reação em Cadeia da Polimerase Multiplex/normas , Pneumonia/diagnóstico , Adulto , Idoso , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Bactérias/efeitos dos fármacos , Bactérias/genética , Testes Diagnósticos de Rotina , Feminino , Humanos , Unidades de Terapia Intensiva , Masculino , Pessoa de Meia-Idade , Pneumonia/tratamento farmacológico , Pneumonia/microbiologia , Estudos Retrospectivos , Sensibilidade e Especificidade
15.
Eur J Clin Microbiol Infect Dis ; 38(5): 859-864, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30715666

RESUMO

The filtration method (FM) is the most effective isolation technique for Epsilobacteriaceae from stool samples. FM's different adaptations make it difficult to compare data between studies. This study was performed in three phases to optimize FM from a routine laboratory perspective. In July-September 2014 (part I), FM was performed on Mueller-Hinton agar containing 5% sheep blood and Columbia agar containing 5% sheep blood. In July 2016 (part II), FM was performed using 0.60-µm pore size polycarbonate filters (0.6-PC filter) and 0.45-µm pore size cellulose acetate filters (0.45-AC filter); in January 2018 (part III), the addition of hydrogen to incubators was studied. On 1146 stools analyzed in part I, the positive samples that showed no growth on the Butzler medium (n = 22/72, 30.6%) had improved growth of Epsilobacteriaceae when using the Columbia instead of the Mueller-Hinton medium (21/22 strains vs. 11/22, p < 0.05). In part II, on 718 stools, 91 strains grew with FM (12.7%), more with 0.6-PC filter (90/91) than with 0.45-AC filter (44/91) (p < 0.05). In part III, 578 stools were cultured, 98 Epsilobacteriaceae strains grew with FM, and 7% hydrogen finding significantly more Epsilobacteriaceae than without hydrogen (90/98, 91.8%, vs. 72/98, 73.5%; p < 0.05). The use of a Columbia medium containing 5% sheep blood with 0.6-PC filters incubated at 37 °C in a 7% hydrogen-enriched atmosphere led to an almost fourfold increase in the isolation rate of Epsilobacteriaceae among the studied combinations. Reference centers for Campylobacter should use standardized protocols to enable the comparison of prevalence in space and time.


Assuntos
Técnicas Bacteriológicas/métodos , Infecções por Campylobacter/diagnóstico , Campylobacter/isolamento & purificação , Fezes/microbiologia , Filtração/métodos , Técnicas Bacteriológicas/normas , Campylobacter/crescimento & desenvolvimento , Infecções por Campylobacter/microbiologia , Celulose/análogos & derivados , Meios de Cultura , Testes Diagnósticos de Rotina/normas , Filtração/normas , Humanos , Hidrogênio , Filtros Microporos , Cimento de Policarboxilato
16.
Eur J Clin Microbiol Infect Dis ; 38(6): 1087-1093, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30783889

RESUMO

Campylobacter diagnosis is hampered because many laboratories continue to use traditional stool culture, which is slow and suffers false-negative results. This large multi-site study used a composite reference method consisting of a new FDA-cleared immunoassay and four molecular techniques to compare to culture. Prospectively collected patient fecal specimens (1552) were first preliminarily categorized as positive or negative by traditional culture. All specimens were also tested by EIA, and any EIA-positive or culture-discrepant results were further characterized by 16S rRNA qPCR, eight species-specific PCR assays, bidirectional sequencing, and an FDA-cleared multiplex PCR panel. The five non-culture methods showed complete agreement on all positive and discrepant specimens which were then assigned as true-positive or true-negative specimens. Among 47 true-positive specimens, culture incorrectly identified 13 (28%) as negative, and 1 true-negative specimen as positive, for a sensitivity of 72.3%. Unexpectedly, among the true-positive specimens, 4 (8%) were the pathogenic species C. upsaliensis. Culture had a 30% false result rate compared to immunoassay and molecular methods. More accurate results lead to better diagnosis and treatment of suspected campylobacteriosis.


Assuntos
Técnicas Bacteriológicas/normas , Infecções por Campylobacter/diagnóstico , Campylobacter/isolamento & purificação , Técnicas de Diagnóstico Molecular/normas , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Campylobacter upsaliensis/isolamento & purificação , Criança , Pré-Escolar , Fezes/microbiologia , Feminino , Humanos , Imunoensaio/normas , Lactente , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Sensibilidade e Especificidade , Adulto Jovem
17.
J Immunol Methods ; 469: 1-10, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30710562

RESUMO

A major challenge to tuberculosis (TB) vaccine development is the lack of a validated immune correlate of protection. Mycobacterial growth inhibition assays (MGIAs) represent an unbiased measure of the ability to control mycobacterial growth in vitro. A successful MGIA could be applied to preclinical and clinical post-vaccination samples to aid in the selection of novel vaccine candidates at an early stage and provide a relevant measure of immunogenicity and protection. However, assay harmonisation is critical to ensure that comparable information can be extracted from different vaccine studies. As part of the FP7 European Research Infrastructures for Poverty Related Diseases (EURIPRED) consortium, we aimed to optimise the direct MGIA, assess repeatability and reproducibility, and harmonise the assay across different laboratories. We observed an improvement in repeatability with increased cell number and increased mycobacterial input. Furthermore, we determined that co-culturing in static 48-well plates compared with rotating 2 ml tubes resulted in a 23% increase in cell viability and a 500-fold increase in interferon-gamma (IFN-γ) production on average, as well as improved reproducibility between replicates, assay runs and sites. Applying the optimised conditions, we report repeatability to be <5% coefficient of variation (CV), intermediate precision to be <20% CV, and inter-site reproducibility to be <30% CV; levels within acceptable limits for a functional cell-based assay. Using relevant clinical samples, we demonstrated comparable results across two shared sample sets at three sites. Based on these findings, we have established a standardised operating procedure (SOP) for the use of the direct PBMC MGIA in TB vaccine development.


Assuntos
Técnicas Bacteriológicas/normas , Criopreservação/normas , Desenvolvimento de Medicamentos/normas , Testes de Liberação de Interferon-gama/estatística & dados numéricos , Leucócitos Mononucleares/microbiologia , Mycobacterium bovis/efeitos dos fármacos , Vacinas contra a Tuberculose/farmacologia , Técnicas de Cultura de Células/normas , Células Cultivadas , Interações Hospedeiro-Patógeno , Humanos , Interferon gama/metabolismo , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Mycobacterium bovis/crescimento & desenvolvimento , Mycobacterium bovis/imunologia , Reprodutibilidade dos Testes
18.
J Clin Microbiol ; 57(3)2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30626663

RESUMO

Borrelia miyamotoi disease (BMD) is a newly recognized borreliosis that is cotransmitted by ticks wherever Lyme disease is zoonotic. Unlike Borrelia burgdorferi sensu lato, the agent of Lyme disease, B. miyamotoi is closely related to relapsing fever spirochetes, such as Borrelia hermsi i Some authors have suggested that the disease caused by B. miyamotoi should be considered a hard-tick-transmitted relapsing fever, and thus, the main mode of confirming a diagnosis for that infection, microscopy to analyze a blood smear, may have clinical utility. To determine whether blood smears may detect B. miyamotoi in the blood of acute BMD patients, we made standard malariological thick smears from anticoagulated blood samples that were previously determined to contain this agent (by PCR) and analyzed them for morphological evidence of spirochetes. Spirochetes were not detected in the blood smears from 20 PCR positive patient blood samples after examination of 100 thick smear fields and only 2 of 20 demonstrated spirochetes when the examination was extended to 300 thick smear fields. Inoculation of severe combined immunodeficient (SCID) mice yielded isolates from 5 of 5 samples, but 0 of 3 BALB/c mice became infected. We conclude that in strong contrast to the diagnosis of typical relapsing fever, microscopy of blood smears is not sensitive enough for confirming a diagnosis of BMD but that SCID mouse inoculation could be a useful complement to PCR.


Assuntos
Técnicas Bacteriológicas/métodos , Técnicas Bacteriológicas/normas , Sangue/microbiologia , Borrelia/isolamento & purificação , Microscopia/normas , Febre Recorrente/diagnóstico , Animais , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Reação em Cadeia da Polimerase , Febre Recorrente/sangue , Febre Recorrente/microbiologia , Sensibilidade e Especificidade
19.
Diagn Microbiol Infect Dis ; 93(3): 191-195, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30477953

RESUMO

BACKGROUND: Targeted antimicrobial therapy can reduce morbidity in patients with sepsis. Molecular methodologies used in the clinical laboratory can provide information about infectious agents faster than traditional culture methods. Using molecular information to make clinical decisions more quickly has been shown to improve patient outcomes, and reduce length of stay and healthcare cost in adults. Its effect on pediatric care is less well described. METHODS: Blood cultures growing Gram-positive cocci or Gram-positive bacilli on Gram stain were evaluated by molecular and traditional methodologies. Results from the molecular platform, Luminex Verigene® Blood Culture - Gram-positive Panel (BC-GP) were compared to results from standard culture and susceptibility testing (Vitek™ MS, Vitek™, E-test®). Overall statistical agreement is evaluated. RESULTS: 1231 positive pediatric blood cultures grew single isolates detectable by the BC-GP panel. 899 were correctly identified to species, 282 to genus, 50 isolates were not detected. All organisms detected by BC-GP that grew in single isolate cultures were identified as the same organism by Vitek™ MS with the exception of 7 organisms.112 cultures were found to have polymicrobial growth of Gram-positive organisms. Excellent overall agreement was noted for antimicrobial resistance markers with only 5 samples displaying discordant results. DISCUSSION: In general, clinicians can use the identification and antimicrobial resistance marker data gained from Luminex Verigene® BC-GP with confidence to alter empiric coverage. Rare instances of disagreement with traditional culture data led to maintaining the empiric clinical approach and did not result in patient harm.


Assuntos
Bacteriemia/diagnóstico , Técnicas Bacteriológicas/normas , Farmacorresistência Bacteriana/genética , Bactérias Gram-Positivas/isolamento & purificação , Técnicas de Diagnóstico Molecular/normas , Adolescente , Adulto , Antibacterianos/farmacologia , Bacteriemia/microbiologia , Criança , Coinfecção/diagnóstico , Farmacorresistência Bacteriana/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Bactérias Gram-Positivas/genética , Humanos , Testes de Sensibilidade Microbiana , Reprodutibilidade dos Testes , Adulto Jovem
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