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Urinary tract infections (UTIs) are a worldwide health problem. Fast and accurate detection of bacterial infection is essential to provide appropriate antibiotherapy to patients and to avoid the emergence of drug-resistant pathogens. While the gold standard requires 24h to 48h of bacteria culture prior MALDI-TOF species identification, we propose a culture-free workflow, enabling a bacterial identification and quantification in less than 4 hours using 1mL of urine. After a rapid and automatable sample preparation, a signature of 82 bacterial peptides, defined by machine learning, was monitored in LC-MS, to distinguish the 15 species causing 84% of the UTIs. The combination of the sensitivity of the SRM mode on a triple quadrupole TSQ Altis instrument and the robustness of capillary flow enabled us to analyze up to 75 samples per day, with 99.2% accuracy on bacterial inoculations of healthy urines. We have also shown our method can be used to quantify the spread of the infection, from 8x104 to 3x107 CFU/mL. Finally, the workflow was validated on 45 inoculated urines and on 84 UTI-positive urine from patients, with respectively 93.3% and 87.1% of agreement with the culture-MALDI procedure at a level above 1x105 CFU/mL corresponding to an infection requiring antibiotherapy.
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OBJECTIVE: To identify tools that predict the risk of complications in patients presenting to outpatient clinics or emergency departments (ED) with acute infectious diarrhea. METHODS: Medline, Embase, Cochrane Library, Web of Science and CINAHL were searched from inception to July 2021. Articles reporting on the derivation or validation of a score to stratify the risk of intravenous rehydration or hospitalization among patients with acute infectious diarrhea in the ED or outpatient clinic were retained for analysis. RESULTS: Five articles reporting on two different tools were identified. Developed to assess the risk of hospitalization of children, the EsVida scale has not been externally validated. Developed originally to assess the level of dehydration in children, the Clinical Dehydration Scale (CDS) was evaluated as a risk stratification tool. For predicting intravenous rehydration, a CDS score ≥ 1 showed a sensitivity between 0.73 and 0.88 and specificity between 0.38 and 0.69, whereas a CDS score ≥ 5 showed a sensitivity between 0.06 and 0.32 and specificity between 0.94 and 0.99. For predicting hospitalization, a CDS score ≥ 1 showed a sensitivity between 0.74 and 1.00 and specificity between 0.34 and 0.38, whereas a CDS score ≥ 5 showed a sensitivity between 0.26 and 0.62 and specificity between 0.66 and 0.96. High heterogeneity among studies and unclear risk of bias precluded meta-analysis. CONCLUSION: As a risk-stratification tool, the CDS has been validated only for children. Further research is needed to develop and validate a tool suitable for adults in the ED.
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Desidratação , Hidratação , Criança , Adulto , Humanos , Desidratação/complicações , Desidratação/diagnóstico , Hidratação/efeitos adversos , Hospitalização , Viés , Diarreia/complicaçõesRESUMO
OBJECTIVES: To evaluate the spermicidal efficacy of non-hormonal vaginal gel in vitro and in a post-coital test, and to evaluate its contraceptive efficacy in Canadian women of childbearing age. METHODS: We conducted single-centre trial to assess spermicidal and contraceptive efficacy of vaginal gel. Participants were healthy, sexually active women aged 18-49 years and their regular male sexual partners (30 couples). Measured outcomes included effect of vaginal gel on sperm motility in vitro, its effect on sperm in a post-coital test, and its effect on pregnancy prevention over 3 months. RESULTS: For in vitro spermicidal effect, 98% and 67% of sperm were immotile in the presence of the gel with sodium lauryl sulfate (gel-SLS) and gel alone, respectively. For the post-coital test, 99% and 93% of sperm were immotile in the presence of gel-SLS and gel alone, respectively. In the second part of trial, a total of 410 instances of vaginal intercourse in 95 menstrual cycles were protected (during 3-month period of gel-SLS use before each sexual intercourse with probability of 24 conceptions prevented according to Wilcox's table). Four women became pregnant during the study period; 2 during unprotected vaginal intercourse around the time of ovulation, and 2 attributed to user failure. CONCLUSION: Based on our results, the vaginal gel demonstrated important spermicidal and contraceptive effect. A larger phase III contraceptive efficacy trial is warranted. The vaginal gel may represent a non-hormonal spermicide/contraceptive option for women.
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Anticoncepcionais , Cremes, Espumas e Géis Vaginais , Adolescente , Adulto , Canadá , Preservativos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Gravidez , Motilidade dos Espermatozoides , Adulto JovemRESUMO
DNA hybridization phenomena occurring on solid supports are not understood as clearly as aqueous phase hybridizations and mathematical models cannot predict some empirically obtained results. Ongoing research has identified important parameters but remains incomplete to accurately account for all interactions. It has previously been shown that the length of the overhanging (dangling) end of the target DNA strand following hybridization to the capture probe is correlated to interactions with the complementary strand in solution which can result in unbinding of the target and its release from the surface. We have developed an instrument for real-time monitoring of DNA hybridization on spherical particles functionalized with oligonucleotide capture probes and arranged in the form of a tightly packed monolayer bead bed inside a microfluidic cartridge. The instrument is equipped with a pneumatic module to mediate displacement of fluid on the cartridge. We compared this system to both conventional (passive) and centrifugally-driven (active) microfluidic microarray hybridization on glass slides to establish performance levels for the detection of single nucleotide polymorphisms. The system was also used to study the effect of the dangling end's length in real-time when the immobilized target DNA is exposed to the complementary strand in solution. Our findings indicate that increasing the length of the dangling end leads to desorption of target amplicons from bead-bound capture probes at a rate approaching that of the initial hybridization process. Finally, bead bed hybridization was performed with Streptococcus agalactiae cfb gene amplicons obtained from randomized clinical samples, which allowed for identification of group B streptococci within 5-15 min. The methodology presented here is useful for investigating competitive hybridization mechanisms on solid supports and to rapidly validate the suitability of microarray capture probes.
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DNA , Microfluídica , DNA/genética , Sondas de DNA/genética , Hibridização de Ácido Nucleico , Sondas de Oligonucleotídeos/genéticaRESUMO
Fast identification of microbial species in clinical samples is essential to provide an appropriate antibiotherapy to the patient and reduce the prescription of broad-spectrum antimicrobials leading to antibioresistances. MALDI-TOF-MS technology has become a tool of choice for microbial identification but has several drawbacks: it requires a long step of bacterial culture before analysis (≥24 h), has a low specificity and is not quantitative. We developed a new strategy for identifying bacterial species in urine using specific LC-MS/MS peptidic signatures. In the first training step, libraries of peptides are obtained on pure bacterial colonies in DDA mode, their detection in urine is then verified in DIA mode, followed by the use of machine learning classifiers (NaiveBayes, BayesNet and Hoeffding tree) to define a peptidic signature to distinguish each bacterial species from the others. Then, in the second step, this signature is monitored in unknown urine samples using targeted proteomics. This method, allowing bacterial identification in less than 4 h, has been applied to fifteen species representing 84% of all Urinary Tract Infections. More than 31,000 peptides in 190 samples were quantified by DIA and classified by machine learning to determine an 82 peptides signature and build a prediction model. This signature was validated for its use in routine using Parallel Reaction Monitoring on two different instruments. Linearity and reproducibility of the method were demonstrated as well as its accuracy on donor specimens. Within 4h and without bacterial culture, our method was able to predict the predominant bacteria infecting a sample in 97% of cases and 100% above the standard threshold. This work demonstrates the efficiency of our method for the rapid and specific identification of the bacterial species causing UTI and could be extended in the future to other biological specimens and to bacteria having specific virulence or resistance factors.
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Bactérias/classificação , Proteínas de Bactérias/urina , Bacteriúria/urina , Cromatografia Líquida/métodos , Aprendizado de Máquina , Espectrometria de Massas em Tandem/métodos , Bactérias/isolamento & purificação , Humanos , Peptídeos/urina , Proteômica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The MI, Chromocult® coliform, and Compass CC chromogenic culture-based methods used to assess water quality by the detection of Escherichia coli and total coliforms were compared in terms of their specificity and sensitivity, using 16S rRNA sequencing for colony identification. A sewage water sample was divided in 2-µL subsamples for testing by all three culture-based methods. All growing colonies were harvested and subjected to 16S rRNA sequencing. Test results showed that all E. coli colonies were correctly identified by all three methods, for a specificity and a sensitivity of 100%. However, for the total coliform detection, the MI agar, Chromocult® coliform agar, and Compass CC agar were specific for only 69.2% (9/13), 47.2% (25/53), and 40.5% (17/42), whereas sensitive for 97.8% (45/46), 97.5% (39/40), and 85.7% (24/28), respectively. Thus, given the low level of specificity of these methods for the detection of total coliforms, confirming the identity of total coliform colonies could help to take public health decisions, in particular for cities connected to a public drinking water distribution system since the growth of few putative total coliform colonies on chromogenic agar is problematic and can lead to unnecessary and costly boiling notices from public health authorities.
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Contagem de Colônia Microbiana/métodos , Escherichia coli/isolamento & purificação , Esgotos/microbiologia , DNA Bacteriano/análise , DNA Bacteriano/genética , Enterobacteriaceae/genética , Enterobacteriaceae/isolamento & purificação , Escherichia coli/genética , RNA Ribossômico 16S/análise , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
The mEI, Chromocult® enterococci, and m-Enterococcus culture-based methods used to assess water quality by the detection of Enterococcus spp. were first compared in terms of sensitivity using (1) 41 different type strains of Enterococcus spp. and (2) environmental colonies identified by 16S rRNA sequencing. Then, two specific-rtPCR assays targeting Enterococcus spp. and Enterococcus faecalis/faecium were tested for their ability to confirm the identity of putative enterococcal colonies. The mEI, Chromocult® enterococci, and m-Enterococcus methods detected ß-glucosidase activity for 28 (68.3%), 32 (78.0%), and 12 (29.3%) of the 41 reference enterococcal strains tested, respectively. Analysis with environmental colonies showed that mEI and Chromocult® enterococci media had false positive rates of 4.3% and 5.0%, respectively. Finally, the two rtPCR assays showed a specificity of 100%. Only two (2/19) colonies of E. faecium isolated from mEI agar were not detected by the Enterococcus faecium rtPCR assay, for a sensitivity of 89.5%. Our results showed that Chromocult® enterococci medium recovered more E. faecalis/faecium cells than the two other methods. Thus, the use of Chromocult® enterococci combined with the Enterococcus faecalis/faecium rtPCR assay showed the best combination to decrease the high false-positive rate obtained when the entire Enterococcus genus is targeted.
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Carga Bacteriana/métodos , Enterococcus/isolamento & purificação , Monitoramento Ambiental/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Microbiologia da Água , Enterococcus faecalis/genética , Enterococcus faecalis/isolamento & purificação , Enterococcus faecium/genética , Enterococcus faecium/isolamento & purificação , Monitoramento Ambiental/instrumentação , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Sensibilidade e Especificidade , Qualidade da ÁguaRESUMO
BACKGROUND: First introduced in 2006, recombinase polymerase amplification (RPA) has stirred great interest, as evidenced by 75 publications as of October 2015, with 56 of them just in the last 2 years. The widespread adoption of this isothermal molecular tool in many diagnostic fields represents an affordable (approximately 4.3 USD per test), simple (few and easy hands-on steps), fast (results within 5-20 min), and sensitive (single target copy number detected) method for the identification of pathogens and the detection of single nucleotide polymorphisms in human cancers and genetically modified organisms. CONTENT: This review summarizes the current knowledge on RPA. The molecular diagnostics of various RNA/DNA pathogens is discussed while highlighting recent applications in clinical settings with focus on point-of-care (POC) bioassays and on automated fluidic platforms. The strengths and limitations of this isothermal method are also addressed. SUMMARY: RPA is becoming a molecular tool of choice for the rapid, specific, and cost-effective identification of pathogens. Owing to minimal sample-preparation requirements, low operation temperature (25-42 °C), and commercial availability of freeze-dried reagents, this method has been applied outside laboratory settings, in remote areas, and interestingly, onboard automated sample-to-answer microfluidic devices. RPA is undoubtedly a promising isothermal molecular technique for clinical microbiology laboratories and emergence response in clinical settings.
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DNA/genética , Técnicas e Procedimentos Diagnósticos , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA/genética , Humanos , Sistemas Automatizados de Assistência Junto ao LeitoRESUMO
Oxidants were shown to contribute to the lethality of bactericidal antibiotics in different bacterial species, including the laboratory strain Streptococcus pneumoniae R6. Resistance to penicillin among S. pneumoniae R6 mutants was further shown to protect against the induction of oxidants upon exposure to unrelated bactericidal compounds. In the work described here, we expanded on these results by studying the accumulation of reactive oxygen species in the context of antibiotic sensitivity and resistance by including S. pneumoniae clinical isolates. In S. pneumoniae R6, penicillin, ciprofloxacin, and kanamycin but not the bacteriostatic linezolid, erythromycin, or tetracycline induced the accumulation of reactive oxygen species. For the three bactericidal compounds, resistance to a single molecule prevented the accumulation of oxidants upon exposure to unrelated bactericidal antibiotics, and this was accompanied by a reduced lethality. This phenomenon does not involve target site mutations but most likely implicates additional mutations occurring early during the selection of resistance to increase survival while more efficient resistance mechanisms are being selected or acquired. Bactericidal antibiotics also induced oxidants in sensitive S. pneumoniae clinical isolates. The importance of oxidants in the lethality of bactericidal antibiotics was less clear than for S. pneumoniae R6, however, since ciprofloxacin induced oxidants even in ciprofloxacin-resistant S. pneumoniae clinical isolates. Our results provide a clear example of the complex nature of the mode of action of antibiotics. The adaptive approach to oxidative stress of S. pneumoniae is peculiar, and a better understanding of the mechanism implicated in response to oxidative injury should also help clarify the role of oxidants induced by antibiotics.
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Antibacterianos/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/metabolismo , Ciprofloxacina/farmacologia , Eritromicina/farmacologia , Linezolida/farmacologia , Testes de Sensibilidade Microbiana , Mutação/genética , Penicilinas/farmacologia , Streptococcus pneumoniae/genética , Tetraciclina/farmacologiaRESUMO
OBJECTIVES: The objective of this study was to characterize chromosomal mutations associated with resistance to tetracycline in Streptococcus pneumoniae. METHODS: Chronological appearance of mutations in two S. pneumoniae R6 mutants (R6M1TC-5 and R6M2TC-4) selected for resistance to tetracycline was determined by next-generation sequencing. A role for the mutations identified was confirmed by reconstructing resistance to tetracycline in a S. pneumoniae R6 WT background. RNA sequencing was performed on R6M1TC-5 and R6M2TC-4 and the relative expression of genes was reported according to R6. Differentially expressed genes were classified according to their ontology. RESULTS: WGS of R6M1TC-5 and R6M2TC-4 revealed mutations in the gene rpsJ coding for the ribosomal protein S10 and in the promoter region and coding sequences of the ABC genes patA and patB. These cells were cross-resistant to ciprofloxacin. Resistance reconstruction confirmed a role in resistance for the mutations in rpsJ and patA. Overexpression of the ABC transporter PatA/PatB or mutations in the coding sequence of patA contributed to resistance to tetracycline, ciprofloxacin and ethidium bromide, and was associated with a decreased accumulation of [(3)H]tetracycline. Comparative transcriptome profiling of the resistant mutants further revealed that, in addition to the overexpression of patA and patB, several genes of the thiamine biosynthesis and salvage pathway were increased in the two mutants, but also in clinical isolates resistant to tetracycline. This overexpression most likely contributes to the tetracycline resistance phenotype. CONCLUSIONS: The combination of genomic and transcriptomic analysis coupled to functional studies has allowed the discovery of novel tetracycline resistance mutations in S. pneumoniae.
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Mutação , RNA Bacteriano/biossíntese , RNA Mensageiro/biossíntese , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/genética , Resistência a Tetraciclina , Tetraciclina/farmacologia , DNA Bacteriano/química , DNA Bacteriano/genética , Genoma Bacteriano , Dados de Sequência Molecular , RNA Bacteriano/genética , RNA Mensageiro/genética , Seleção Genética , Análise de Sequência de DNARESUMO
The combination of molecular diagnostic technologies is increasingly used to overcome limitations on sensitivity, specificity or multiplexing capabilities, and provide efficient lab-on-chip devices. Two such techniques, PCR amplification and microarray hybridization are used serially to take advantage of the high sensitivity and specificity of the former combined with high multiplexing capacities of the latter. These methods are usually performed in different buffers and reaction chambers. However, these elaborate methods have high complexity and cost related to reagent requirements, liquid storage and the number of reaction chambers to integrate into automated devices. Furthermore, microarray hybridizations have a sequence dependent efficiency not always predictable. In this work, we have developed the concept of a structured oligonucleotide probe which is activated by cleavage from polymerase exonuclease activity. This technology is called SCISSOHR for Structured Cleavage Induced Single-Stranded Oligonucleotide Hybridization Reaction. The SCISSOHR probes enable indexing the target sequence to a tag sequence. The SCISSOHR technology also allows the combination of nucleic acid amplification and microarray hybridization in a single vessel in presence of the PCR buffer only. The SCISSOHR technology uses an amplification probe that is irreversibly modified in presence of the target, releasing a single-stranded DNA tag for microarray hybridization. Each tag is composed of a 3-nucleotide sequence-dependent segment and a unique "target sequence-independent" 14-nucleotide segment allowing for optimal hybridization with minimal cross-hybridization. We evaluated the performance of five (5) PCR buffers to support microarray hybridization, compared to a conventional hybridization buffer. Finally, as a proof of concept, we developed a multiplexed assay for the amplification, detection, and identification of three (3) DNA targets. This new technology will facilitate the design of lab-on-chip microfluidic devices, while also reducing consumable costs. At term, it will allow the cost-effective automation of highly multiplexed assays for detection and identification of genetic targets.
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DNA Viral/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Hibridização de Ácido Nucleico/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Oligonucleotídeos/química , Reação em Cadeia da Polimerase/métodos , Fluorescência , Humanos , Vírus da Influenza A/genética , Vírus da Influenza B/genética , Sondas de Oligonucleotídeos/química , Vírus Sinciciais Respiratórios/genéticaRESUMO
Recombinase polymerase amplification (RPA) technology relies on three major proteins, recombinase proteins, single-strand binding proteins, and polymerases, to specifically amplify nucleic acid sequences in an isothermal format. The performance of RPA with respect to sequence mismatches of closely-related non-target molecules is not well documented and the influence of the number and distribution of mismatches in DNA sequences on RPA amplification reaction is not well understood. We investigated the specificity of RPA by testing closely-related species bearing naturally occurring mismatches for the tuf gene sequence of Pseudomonas aeruginosa and/or Mycobacterium tuberculosis and for the cfb gene sequence of Streptococcus agalactiae. In addition, the impact of the number and distribution of mismatches on RPA efficiency was assessed by synthetically generating 14 types of mismatched forward primers for detecting five bacterial species of high diagnostic relevance such as Clostridium difficile, Staphylococcus aureus, S. agalactiae, P. aeruginosa, and M. tuberculosis as well as Bacillus atropheus subsp. globigii for which we use the spores as internal control in diagnostic assays. A total of 87 mismatched primers were tested in this study. We observed that target specific RPA primers with mismatches (n > 1) at their 3'extrimity hampered RPA reaction. In addition, 3 mismatches covering both extremities and the center of the primer sequence negatively affected RPA yield. We demonstrated that the specificity of RPA was multifactorial. Therefore its application in clinical settings must be selected and validated a priori. We recommend that the selection of a target gene must consider the presence of closely-related non-target genes. It is advisable to choose target regions with a high number of mismatches (≥36%, relative to the size of amplicon) with respect to closely-related species and the best case scenario would be by choosing a unique target gene.
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Pareamento Incorreto de Bases , Recombinases/química , Bactérias , Humanos , Reação em Cadeia da Polimerase/métodos , Sensibilidade e EspecificidadeRESUMO
The MI agar, Colilert(®), Chromocult coliform(®) agar, and DC with BCIG agar chromogenic culture-based methods used to assess microbiological quality of drinking water were compared in terms of their ubiquity, sensitivity, ease of use, growth of atypical colonies and affordability. For ubiquity, 129 total coliform (representing 76 species) and 19 Escherichia coli strains were tested. Then, 635 1-L well water samples were divided into 100 mL subsamples for testing by all four methods. Test results showed that 70.5, 52.7, 36.4, and 23.3% of the non-E. coli total coliform strains and 94.7, 94.7, 89.5, and 89.5% of the 19 E. coli strains yielded a positive signal with the four methods, respectively. They also yielded a total coliform positive signal for 66.5, 51.7, 64.9, and 55.0% and an E. coli positive signal for 16.1, 14.8, 17.3, and 13.4% of the 635 well water samples tested, respectively. Results showed that Colilert(®) is the most expensive method tested in terms of reactants, yet it is the easiest to use. Large numbers of atypical colonies were also often observed on Chromocult coliform(®) and DC with BCIG, thereby challenging the target microorganism count. Thus, the MI agar method seems to be the best option for the assessment of drinking water quality.
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Técnicas Bacteriológicas/métodos , Enterobacteriaceae/enzimologia , Monitoramento Ambiental/métodos , Glucuronidase/metabolismo , Microbiologia da Água/normas , beta-Galactosidase/metabolismo , Enterobacteriaceae/isolamento & purificação , Especificidade da EspécieRESUMO
MI agar and Colilert(®), as well as mFC agar combined with an Escherichia coli-specific molecular assay (mFC + E. coli rtPCR), were compared in terms of their sensitivity, ease of use, time to result and affordability. The three methods yielded a positive E. coli signal for 11.5, 10.8, and 11.5% of the 968 well water samples tested, respectively. One hundred and thirty-six (136) samples gave blue colonies on mFC agar and required confirmation. E. coli-specific rtPCR showed false-positive results in 23.5% (32/136) of cases. In terms of ease of use, Colilert was the simplest method to use while the MI method provided ease of use comparable to all membrane filtration methods. However, the mFC + E. coli rtPCR assay required highly trained employees for confirmation purposes. In terms of affordability, and considering contamination rate of well water samples tested, the Colilert method and the mFC + E. coli rtPCR assay were at least five times more costly than the MI agar method. Overall, compared with the other two methods tested, the MI agar method offers the most advantages to assess drinking water quality.
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Água Potável/microbiologia , Microbiologia da Água , Ágar , Técnicas Bacteriológicas/economia , Custos e Análise de Custo , Meios de Cultura , Escherichia coli/crescimento & desenvolvimentoRESUMO
Alterations in penicillin-binding proteins, the target enzymes for ß-lactam antibiotics, are recognized as primary penicillin resistance mechanisms in Streptococcus pneumoniae. Few studies have analyzed penicillin resistance at the genome scale, however, and we report the sequencing of S. pneumoniae R6 transformants generated while reconstructing the penicillin resistance phenotypes from three penicillin-resistant clinical isolates by serial genome transformation. The genome sequences of the three last-level transformants T2-18209, T5-1983, and T3-55938 revealed that 16.2 kb, 82.7 kb, and 137.2 kb of their genomes had been replaced with 5, 20, and 37 recombinant sequence segments derived from their respective parental clinical isolates, documenting the extent of DNA transformation between strains. A role in penicillin resistance was confirmed for some of the mutations identified in the transformants. Several multiple recombination events were also found to have happened at single loci coding for penicillin-binding proteins (PBPs) that increase resistance. Sequencing of the transformants with MICs for penicillin similar to those of the parent clinical strains confirmed the importance of mosaic PBP2x, -2b, and -1a as a driving force in penicillin resistance. A role in resistance for mosaic PBP2a was also observed for two of the resistant clinical isolates.
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Genoma Bacteriano/genética , Resistência às Penicilinas/genética , Streptococcus pneumoniae/efeitos dos fármacos , Antibacterianos/uso terapêutico , DNA Bacteriano/genética , Humanos , Testes de Sensibilidade Microbiana , Penicilinas/uso terapêutico , Infecções Pneumocócicas/tratamento farmacológico , Infecções Pneumocócicas/microbiologia , Streptococcus pneumoniae/genética , Transformação Bacteriana/genéticaRESUMO
BACKGROUND: Group B streptococcal infections are the leading cause of sepsis and meningitis in newborns. A rapid and reliable method for the detection of this pathogen at the time of delivery is needed for the early treatment of neonates. Isothermal amplification techniques such as recombinase polymerase amplification have advantages relative to PCR in terms of the speed of reaction and simplicity. METHODS: We studied the clinical performance of recombinase polymerase amplification for the screening of group B streptococci in vaginal/anal samples from 50 pregnant women. We also compared the limit of detection and the analytical specificity of this isothermal assay to real-time PCR (RT-PCR). RESULTS: Compared to RT-PCR, the recombinase polymerase amplification assay showed a clinical sensitivity of 96% and a clinical specificity of 100%. The limit of detection was 98 genome copies and the analytical specificity was 100% for a panel of 15 bacterial and/or fungal strains naturally found in the vaginal/anal flora. Time-to-result for the recombinase polymerase amplification assay was <20 min compared to 45 min for the RT-PCR assay; a positive sample could be detected as early as 8 min. CONCLUSIONS: We demonstrate the potential of isothermal recombinase polymerase amplification assay as a clinically useful molecular diagnostic tool that is simple and faster than PCR/RT-PCR. Recombinase polymerase amplification offers great potential for nucleic acid-based diagnostics at the point of care.
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Canal Anal/microbiologia , DNA Polimerase Dirigida por DNA/química , Sistemas Automatizados de Assistência Junto ao Leito , Recombinases/química , Streptococcus agalactiae/genética , Vagina/microbiologia , DNA Bacteriano/química , DNA Bacteriano/genética , Feminino , Humanos , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico/métodos , Gravidez , Sensibilidade e Especificidade , Streptococcus agalactiae/isolamento & purificaçãoRESUMO
This work demonstrates the ability of a bacterial concentration and recovery procedure combined with three different PCR assays targeting the lacZ, wecG, and 16S rRNA genes, respectively, to detect the presence of total coliforms in 100-ml samples of potable water (presence/absence test). PCR assays were first compared to the culture-based Colilert and MI agar methods to determine their ability to detect 147 coliform strains representing 76 species of Enterobacteriaceae encountered in fecal and environmental settings. Results showed that 86 (58.5%) and 109 (74.1%) strains yielded a positive signal with Colilert and MI agar methods, respectively, whereas the lacZ, wecG, and 16S rRNA PCR assays detected 133 (90.5%), 111 (75.5%), and 146 (99.3%) of the 147 total coliform strains tested. These assays were then assessed by testing 122 well water samples collected in the Québec City region of Canada. Results showed that 97 (79.5%) of the samples tested by culture-based methods and 95 (77.9%), 82 (67.2%), and 98 (80.3%) of samples tested using PCR-based methods contained total coliforms, respectively. Consequently, despite the high genetic variability of the total coliform group, this study demonstrated that it is possible to use molecular assays to detect total coliforms in potable water: the 16S rRNA molecular assay was shown to be as efficient as recommended culture-based methods. This assay might be used in combination with an Escherichia coli molecular assay to assess drinking water quality.
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Contagem de Colônia Microbiana/métodos , Água Potável/microbiologia , Enterobacteriaceae/isolamento & purificação , Escherichia coli/isolamento & purificação , Microbiologia da Água/normas , Canadá , DNA Bacteriano/isolamento & purificação , Enterobacteriaceae/classificação , Fezes/microbiologia , Reação em Cadeia da Polimerase , RNA Ribossômico 16S/isolamento & purificaçãoRESUMO
OBJECTIVE: Conventional vaginal applicators with a single apical hole do not distribute vaginal formulations homogenously and do not cover the entire vaginal and cervical mucosa. To overcome this problem and offer women further protection against vaginal infections, we designed a unique vaginal applicator with multiple apical and lateral holes. We have previously shown that the new applicator distributes an investigational vaginal gel homogenously over the entire vaginal and cervical mucosa. In this study, we investigated (using MRI) whether the new applicator works as well with marketed vaginal gels and creams. METHODS: Eighteen women participated in the study and six vaginal gels and creams were tested. Each woman used a marketed vaginal product with its own commercial applicator (CA) once and with our universal vaginal applicator (UVA) once to deliver the same product. The applications were separated by a one-week period. Pelvic MRI was performed immediately after vaginal application to evaluate the product's distribution and mucosal coverage. RESULTS: Immediately after application of the vaginal product, the UVA homogenously distributed the six products (3 gels and 3 creams) over the entire vaginal and cervical mucosa. On the other hand, the tested CA delivered four products (3 gels and 1 cream) mainly to the cervix and the upper vagina, but not to the mid and lower vagina; for the other two creams, the distribution was similar to that of UVA. Furthermore, the UVA received the highest acceptability score. CONCLUSION: The UVA can be used to deliver different vaginal gel and cream products homogenously throughout the vagina. This was the first time the UVA had been tested with marketed vaginal gels and creams. This applicator, giving uniform mucosal coverage and being highly acceptable, may help women to better protect themselves against sexually transmitted infections.
Objectif : Les applicateurs vaginaux conventionnels dotés d'un seul orifice apical ne permettent pas de distribuer les formulations vaginales de façon homogène et ne couvrent pas l'intégralité de la muqueuse vaginale et cervicale. Pour surmonter ce problème et offrir aux femmes davantage de protection contre les infections vaginales, nous avons conçu un applicateur vaginal unique en son genre doté de multiples orifices apicaux et latéraux. Nous avons déjà démontré que ce nouvel applicateur permettait de distribuer un gel vaginal expérimental de façon homogène sur l'intégralité de la muqueuse vaginale et cervicale. Dans le cadre de cette étude, nous nous sommes penchés (en ayant recours à l'IRM) sur la question de savoir si ce nouvel applicateur fonctionnait tout aussi bien dans le cas des crèmes et des gels vaginaux offerts sur le marché. Méthodes : Dix-huit femmes ont participé à l'étude et six crèmes et gels vaginaux ont été mis à l'essai. Chacune de ces femmes a utilisé à deux reprises un même produit vaginal offert sur le marché : une fois au moyen de l'applicateur commercial fourni par le fabricant (AC) et une autre fois au moyen de notre applicateur vaginal universel (AVU). Une période d'une semaine séparait ces deux applications. Une IRM pelvienne a été menée immédiatement à la suite de chacune de ces applications vaginales afin d'évaluer la distribution du produit et l'aire couverte en ce qui concerne la muqueuse. Résultats : Immédiatement à la suite de l'application du produit vaginal, nous avons constaté que l'utilisation de l'AVU permettait la distribution homogène des six produits (trois gels et trois crèmes) sur l'intégralité de la muqueuse vaginale et cervicale. En revanche, dans le cas de quatre des produits en question (trois gels et une crème), l'AC mis à l'essai a donné lieu à une distribution ayant principalement atteint le col utérin et la partie supérieure du vagin (excluant ainsi les parties intermédiaire et inférieure du vagin); pour ce qui est des deux autres crèmes, la distribution obtenue était semblable à celle qu'a permise l'AVU. De surcroît, l'AVU a obtenu le score d'acceptabilité le plus élevé. Conclusion : L'AVU peut être utilisé pour assurer l'administration de divers produits vaginaux en gel et en crème de façon homogène dans tout le vagin. Il s'agissait de la première mise à l'essai de l'AVU au moyen de crèmes et de gels vaginaux offerts sur le marché. Cet applicateur, qui permet de couvrir l'aire muqueuse de façon uniforme et qui compte une acceptabilité élevée, pourrait aider les femmes à mieux se protéger contre les infections transmissibles sexuellement.
Assuntos
Sistemas de Liberação de Medicamentos/instrumentação , Imageamento por Ressonância Magnética , Cremes, Espumas e Géis Vaginais/administração & dosagem , Administração Intravaginal , Anti-Infecciosos Locais/administração & dosagem , Colo do Útero/efeitos dos fármacos , Feminino , Humanos , Vagina/efeitos dos fármacosRESUMO
Infectious diseases (IDs) are a leading cause of death. The diversity and adaptability of microbes represent a continuing risk to health. Combining vision with passion, our transdisciplinary medical research team has been focussing its work on the better management of infectious diseases for saving human lives over the past five decades through medical discoveries and innovations that helped change the practice of medicine. The team used a multiple-faceted and integrated approach to control infectious diseases through fundamental discoveries and by developing innovative prevention tools and rapid molecular diagnostic tests to fulfill the various unmet needs of patients and health professionals in the field of ID. In this article, as objectives, we put in context two main research areas of ID management: innovative infection prevention that is woman-controlled, and the rapid molecular diagnosis of infection and resistance. We also explain how our transdisciplinary approach encompassing specialists from diverse fields ranging from biology to engineering was instrumental in achieving success. Furthermore, we discuss our vision of the future for translational research to better tackle IDs.
RESUMO
Objective: To identify tools that predict the risk of complications for patients presenting to an outpatient clinic or an emergency department (ED) with influenza-like illness. Methods: We searched Medline, Embase, Cochrane Library and CINAHL from inception to July 2023. We included articles reporting on the derivation or validation of a score or algorithm used to stratify the risk of hospitalization or mortality among patients with influenza-like illness in the ED or outpatient clinic. Results: Twelve articles reporting on eight scores and six predictive models were identified. For predicting the need for hospitalization, the area under the curve (AUC) of the PMEWS and the CURB-65 ranged respectively from 0.76 to 0.94, and 0.65 to 0.88. The Community Assessment Tool had an AUC of 0.62. For predicting inpatient mortality, AUC was 0.66 for PMEWS and 0.79 for CURB-65, 0.79 for the SIRS criteria and 0.86 for the qSOFA score. Two scores were developed without external validation during the Covid-19 pandemic. The CovHos score and the Canadian Covid discharge score had an AUC ranged from 0.70 to 0.91. The predictive models performed adequately (AUC from 0.76 to 0.92) but will require external validation for clinical use. Tool diversity and study population heterogeneity precluded meta-analysis. Conclusion: Although the CURB, PMEWS and qSOFA scores appear to predict accurately the risk of complications of influenza-like illness, none were reliable enough to justify their widespread ED use. Refinement of an existing tool or development of a new tool to optimize the management of these patients is needed.