RESUMO
Treponema pallidum subsp. pallidum is a fastidious spirochete and the etiologic agent of syphilis, a sexually transmitted infection (STI). Syphilis diagnoses and disease staging are based on clinical findings and serologic testing. Moreover, according to most international guidelines, PCR analysis of swab samples from genital ulcers is included in the screening algorithm where possible. It has been suggested that PCR might be omitted from the screening algorithm due to low added value. As an alternative to PCR, IgM serology might be used. In this study, we wanted to establish the added value of PCR and IgM serology for diagnosing primary syphilis. Added value was defined as finding more cases of syphilis, preventing overtreatment, or limiting the extent of partner notification to more recent partners. We found that both PCR and IgM immunoblotting could aid the timely diagnosis of early syphilis in ~24% to 27% of patients. PCR has the greatest sensitivity and can be applied to cases with an ulcer with suspected reinfection or primary infection. In the absence of lesions, the IgM immunoblot could be used. However, the IgM immunoblot has better performance in cases with suspected primary infection than in reinfections. The target population, testing algorithm, time pressures, and costs should determine whether either test provides sufficient value to be implemented in clinical practice.
Assuntos
Testes Diagnósticos de Rotina , Imunoglobulina M , Sífilis , Humanos , Immunoblotting/normas , Imunoglobulina M/análise , Reação em Cadeia da Polimerase/normas , Sífilis/diagnóstico , Sífilis/imunologia , Sífilis/microbiologia , Treponema pallidum/genética , Testes Sorológicos/normas , Testes Diagnósticos de Rotina/economia , Testes Diagnósticos de Rotina/métodos , Testes Diagnósticos de Rotina/normas , Sensibilidade e EspecificidadeRESUMO
Toxoplasmosis can be a life-threatening infection, particularly during pregnancy and in immunocompromised patients. The biological diagnosis of toxoplasmosis is challenging and has been revolutionized by molecular detection methods. This article summarizes the data of a multicenter study involving four centers to assess the performances of a commercial PCR assay as compared with four in-house PCR assays using Toxoplasma gondii standards, 20 external quality control specimens, and 133 clinical samples. This clinical cohort includes well-characterized clinical samples corresponding to different clinical situations: confirmed congenital toxoplasmosis (44 samples), toxoplasmosis in immunocompromised patients (25 samples), and chorioretinitis (5 samples). Furthermore, 59 samples from patients without toxoplasmosis were included as negative controls. The analytical sensitivities of the five methods tested were very similar; and the limit of Toxoplasma DNA detection was around 0.01 T. gondii genome per reaction for all the methods. The overall concordance between the commercial PCR and the four in-house PCR assays was 97.7% (130/133). The clinical sensitivity and specificity were >98% and could be increased for the commercial kit when PCR was performed in multiplicate to detect low parasitic loads. In conclusion, the commercial PCR assay shows suitable performances to diagnose the different clinical forms of toxoplasmosis.
Assuntos
Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/normas , Kit de Reagentes para Diagnóstico/normas , Toxoplasma/genética , Toxoplasmose/diagnóstico , Toxoplasmose/parasitologia , Humanos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/normas , Controle de Qualidade , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Herpes simplex virus (HSV) infections of the lung are rare, but HSV is occasionally detected in bronchoalveolar lavage (BAL) specimens. We assessed whether routinely performing HSV PCR tests in BAL specimens is warranted. HSV was detected in 7% (52/722) of BALs. In 47% of HSV-positive patients a typical respiratory virus or pathologic microorganism was identified. Oral HSV reactivation was identified in 27%; however, anti-HSV therapy was initiated in just three patients following the positive HSV test. Patients undergoing BAL for transplant surveillance received anti-HSV prophylaxis more often than those with acute respiratory failure, but both groups did not differ significantly in terms of patient outcome or co-infections. No patient was diagnosed with HSV pneumonia. These findings suggest that positive HSV PCR results in BAL specimens most commonly represents contamination from oral HSV reactivation, and that HSV PCR should be ordered selectively, rather than routinely, as part of a test panel.
Assuntos
Líquido da Lavagem Broncoalveolar/virologia , Herpes Simples/diagnóstico , Herpesvirus Humano 1/genética , Técnicas de Diagnóstico Molecular/normas , Infecções Respiratórias/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Herpes Simples/etiologia , Herpes Simples/virologia , Herpesvirus Humano 1/isolamento & purificação , Humanos , Masculino , Pessoa de Meia-Idade , Técnicas de Diagnóstico Molecular/economia , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase/economia , Reação em Cadeia da Polimerase/normas , Infecções Respiratórias/virologiaRESUMO
Control strategies that employ real time polymerase chain reaction (RT-PCR) tests for the diagnosis and surveillance of COVID-19 epidemic are inefficient in fighting the epidemic due to high cost, delays in obtaining results, and the need of specialized personnel and equipment for laboratory processing. Cheaper and faster alternatives, such as antigen and paper-strip tests, have been proposed. They return results rapidly, but have lower sensitivity thresholds for detecting virus. To quantify the effects of the tradeoffs between sensitivity, cost, testing frequency, and delay in test return on the overall course of an outbreak, we built a multi-scale immuno-epidemiological model that connects the virus profile of infected individuals with transmission and testing at the population level. We investigated various randomized testing strategies and found that, for fixed testing capacity, lower sensitivity tests with shorter return delays slightly flatten the daily incidence curve and delay the time to the peak daily incidence. However, compared with RT-PCR testing, they do not always reduce the cumulative case count at half a year into the outbreak. When testing frequency is increased to account for the lower cost of less sensitive tests, we observe a large reduction in cumulative case counts, from 55.4% to as low as 1.22% half a year into the outbreak. The improvement is preserved even when the testing budget is reduced by one half or one third. Our results predict that surveillance testing that employs low-sensitivity tests at high frequency is an effective tool for epidemic control.
Assuntos
Teste para COVID-19/estatística & dados numéricos , COVID-19/diagnóstico , SARS-CoV-2/genética , COVID-19/epidemiologia , COVID-19/virologia , Teste para COVID-19/economia , Teste para COVID-19/métodos , Epidemias , Reações Falso-Negativas , Humanos , Modelos Teóricos , Reação em Cadeia da Polimerase/economia , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/normas , Reação em Cadeia da Polimerase/estatística & dados numéricos , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/fisiologia , Sensibilidade e EspecificidadeRESUMO
We assessed carrageenan's potential to inhibit human papillomavirus (HPV) DNA extraction and amplification in vaginal swab samples collected in a trial, assessing the efficacy of a carrageenan-based gel against HPV infections. Experiment #1 consisted of adding gel (carrageenan-containing or placebo) to swabs and comparing HPV DNA detection by polymerase chain reaction (PCR) to unmanipulated samples collected from the same participants. For Experiments #2 and #3, we tested vaginal samples for inhibition by addition of an internal control and amplification by real-time PCR. Experiment #4 investigated carrageenan's interference with the extraction process by assessing HPV45 detectability in undiluted and diluted HPV45 positive samples (n = 3) with carrageenan versus no gel. In Experiment #1, there was a loss of HPV positivity with the addition of carrageenan (n = 9), but none with placebo gel (n = 5). In Experiments #2 and #3, the absence of the amplified product was observed in samples from the carrageenan arm: 3.3% (1/30) and 0.5% (1/199) of samples. In Experiment #4, HPV45 was not detected in undiluted carrageenan-containing samples, but after 1/50 dilution, the same HPV45 copy number was detected. Carrageenan does not affect the DNA extraction process, and inhibition of HPV DNA amplification by carrageenan occurs infrequently.
Assuntos
Carragenina/farmacologia , Testes de DNA para Papilomavírus Humano/normas , Papillomaviridae/efeitos dos fármacos , Papillomaviridae/genética , Infecções por Papillomavirus/diagnóstico , Reação em Cadeia da Polimerase/normas , Vagina/virologia , Adulto , DNA Viral/análise , Feminino , Testes de DNA para Papilomavírus Humano/métodos , Humanos , Infecções por Papillomavirus/virologia , Reação em Cadeia da Polimerase/métodos , Manejo de Espécimes/métodos , Vagina/efeitos dos fármacos , Adulto JovemRESUMO
The outbreak of novel coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide. To meet the urgent and massive demand for the screening and diagnosis of infected individuals, many in vitro diagnostic assays using nucleic acid tests (NATs) have been urgently authorized by regulators worldwide. A reference standard with a well-characterized concentration or titer is of the utmost importance for the study of limit of detection (LoD), which is a crucial feature for a diagnostic assay. Although several reference standards of plasmids or synthetic RNA have already been announced, a reference standard for inactivated virus particles with an accurate concentration is still needed to evaluate the complete procedure. Here, we performed a collaborative study to estimate the NAT-detectable units as a viral genomic equivalent quantity (GEQ) of an inactivated whole-virus SARS-CoV-2 reference standard candidate using digital PCR (dPCR) on multiple commercialized platforms. The median of the quantification results (4.6 × 105 ± 6.5 × 104 GEQ/mL) was treated as the consensus true value of GEQ of virus particles in the reference standard. This reference standard was then used to challenge the LoDs of six officially approved diagnostic assays. Our study demonstrates that an inactivated whole virus quantified by dPCR can serve as a reference standard and provides a unified solution for assay development, quality control, and regulatory surveillance.
Assuntos
COVID-19/diagnóstico , Reação em Cadeia da Polimerase/métodos , RNA Viral/análise , SARS-CoV-2/genética , COVID-19/virologia , Teste de Ácido Nucleico para COVID-19/métodos , Teste de Ácido Nucleico para COVID-19/normas , Proteínas do Nucleocapsídeo de Coronavírus/genética , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , Proteínas do Nucleocapsídeo de Coronavírus/normas , Humanos , Limite de Detecção , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosfoproteínas/normas , Reação em Cadeia da Polimerase/normas , Poliproteínas/genética , Poliproteínas/metabolismo , Poliproteínas/normas , Controle de Qualidade , RNA Viral/metabolismo , RNA Viral/normas , Kit de Reagentes para Diagnóstico , Padrões de Referência , SARS-CoV-2/isolamento & purificação , Proteínas Virais/genética , Proteínas Virais/metabolismo , Proteínas Virais/normas , Vírion/genética , Vírion/isolamento & purificaçãoRESUMO
Phlebotomine sand flies (Diptera: Psychodidae: Phlebotominae) are a group of small insects of great concern for Public Health. These dipterous are intensely studied worldwide due to their involvement in the transmission of several pathogens, mainly Leishmania spp. parasites. Nowadays, the molecular tools have been included in Phlebotomine sand flies studies and has shown to be powerful tools in bioecology studies of these dipterous. Thereby, when molecular approaches are employed, there is a great concern regarding the amount and quality of the DNA obtained for analysis. Here, seven methods of DNA extraction, between commercial kits and in house extraction protocols were evaluated. We considered measure of DNA concentration and purity ratios using a spectrophotometer to check the performance of each protocol. In addition, the quality evaluation of the DNA extracted was performed by endogenous gene PCR on samples. The results of the seven evaluated DNA extraction protocols and their implications are discussed.
Assuntos
DNA/isolamento & purificação , Psychodidae/genética , Análise de Variância , Animais , Custos e Análise de Custo , DNA/análise , DNA/normas , Eletroforese em Gel de Ágar , Feminino , Masculino , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/normas , Cloreto de Sódio , Espectrofotometria , Fatores de TempoRESUMO
Trichuriasis is a serious threat to the economic development of animal husbandry. This research aimed to establish a droplet digital PCR (ddPCR) method to detect Trichuris spp. for the early diagnosis and prevention of trichuriasis in sheep. The real-time quantitative PCR (qPCR) and ddPCR methods were used for the detection of nematodes by targeted amplification of the ITS gene. Each means was evaluated to optimize the limit of detection and reproducibility. For a recombinant plasmid, the qPCR results showed that the detection limit was 31.7 copies per reaction. In contrast to qPCR, ddPCR was able to detect concentrations below 3.17 copies per reaction. Both assays exhibited good reproducibility. However, the ddPCR method was more stable for low-copy-number detection. This new assay was specific for Trichuris spp. and did not cross-react with other relevant gastrointestinal nematodes. A total of 98 clinical samples were tested with both assays. The results showed that the positive rate of ddPCR (80.6%) was higher than that of qPCR (72.4%). This method could be used as an efficient molecular biology tool to test for Trichuris spp. and could be a new valuable tool for the clinical diagnosis and prevention of trichuriasis.
Assuntos
Reação em Cadeia da Polimerase/veterinária , Doenças dos Ovinos/diagnóstico , Tricuríase/veterinária , Animais , DNA de Helmintos/isolamento & purificação , Fezes/parasitologia , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/normas , Reação em Cadeia da Polimerase em Tempo Real/normas , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Ovinos , Doenças dos Ovinos/parasitologia , Doenças dos Ovinos/prevenção & controle , Tricuríase/diagnóstico , Tricuríase/prevenção & controleRESUMO
The World Health Organization has declared coronavirus disease 2019 (COVID-19) a pandemic. Polymerase chain reaction testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the diagnostic gold standard of COVID-19. We have developed a simulation-based training program for mobile prehospital diagnostic teams in the province of Styria, Austria, and performed a prospective observational study on its applicability and effectivity.The 1-day curriculum uses theoretical instruction, technical skills training, and simulator-based algorithm training to teach and train prehospital patient identification and communication, donning the personal protective equipment, collection of naso-/oropharyngeal swabs for SARS-CoV-2 polymerase chain reaction testing, doffing the personal protective equipment, and sample logistics. Trainings were conducted at the SIM CAMPUS simulation hospital, Eisenerz, using high-fidelity patient simulation. To ensure achievement of predefined learning outcomes, participants had to undergo a final simulator-based objective structured clinical examination.In March 2020, 45 emergency medical assistants and 1 physician of the Austrian Red Cross participated on a voluntary basis. Forty-five of the 46 participants (97.8%) completed the curriculum successfully, with mean objective structured clinical examination ratings of 98.6%.Using several proven educational concepts, we have successfully drafted and implemented a training program for mobile prehospital SARS-CoV-2 diagnostic teams. Based on simulation-based objective structured examinations, it has prepared participants effectively for preclinical duties.
Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , Treinamento por Simulação/métodos , Ambulâncias/estatística & dados numéricos , Áustria/epidemiologia , Betacoronavirus/isolamento & purificação , Líquidos Corporais/virologia , COVID-19 , Infecções por Coronavirus/epidemiologia , Currículo , Feminino , Pessoal de Saúde/educação , Humanos , Masculino , Pandemias , Equipamento de Proteção Individual , Pneumonia Viral/epidemiologia , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/normas , Estudos Prospectivos , SARS-CoV-2 , Manejo de Espécimes/métodosRESUMO
While the FilmArray Respiratory Panel EZ has been proven to reduce inappropriate antibiotic use in the outpatient pediatric setting, it is unclear whether its implementation will also reduce downstream health costs such as provider visits and telephone calls. This analysis will help pediatricians make more informed decisions on the implementation and judicious use of the Respiratory Panel EZ in their clinical practice.
Assuntos
Instituições de Assistência Ambulatorial/estatística & dados numéricos , Sistemas Automatizados de Assistência Junto ao Leito/normas , Reação em Cadeia da Polimerase/normas , Infecções Respiratórias/diagnóstico , Infecções Respiratórias/virologia , Vírus/genética , Vírus/isolamento & purificação , Criança , Pré-Escolar , Registros Eletrônicos de Saúde , Florida , Seguimentos , Implementação de Plano de Saúde , Humanos , Reação em Cadeia da Polimerase/instrumentação , Reação em Cadeia da Polimerase/métodos , Infecções Respiratórias/economiaRESUMO
Droplet digital PCR (ddPCR) has attracted much attention in the detection of genetic signatures of cancer present at low levels in circulating tumor DNA (ctDNA) in blood. A growing number of laboratory-developed liquid biopsy tests based on such technology have become commercially available for clinical settings. To obtain consistent and comparable results, an international standard is necessary for validation of the analytical performance. In this study, a novel and SI-traceable "ctDNA" reference material (RM) carrying BRAF V600E was prepared by gravimetrically mixing a 152 bp PCR amplicon and sonicated wild-type genomic DNA. The ddPCR performance was evaluated by analyzing serial "ctDNA" dilutions using a competitive MGB assay. The mutant frequency concordance (k) between ddPCR and the gravimetrical value was 1.03 in the range from 53.9% to 0.1%. The limit of blank (LoB), detection (LoD) and quantification (LoQ) of ddPCR assay were determined to be 0.01%, 0.02% and 0.1%, respectively. Results from the interlaboratory study, using challenging low levels of BRAF V600E ctDNA RMs, demonstrated that the participating laboratories had the appropriate technical competency to perform accurate ddPCR-based low level of ratio measurements. However, a systematic error caused by uncorrected droplet volume in Naica Crystal ddPCR platform was found by using the ctDNA RM. Between-laboratory consistency in copy number measurement was greatly improved when a correct droplet volume was applied for the ddPCR measurement by using the ctDNA RM. This confirms that the "ctDNA" RM is fit for the validation of ddPCR systems for ctDNA quantification. This would also support translation of tests for circulating tumor DNA by ddPCR into routine use.
Assuntos
DNA Tumoral Circulante/genética , Análise Mutacional de DNA/normas , Laboratórios , Reação em Cadeia da Polimerase/normas , Proteínas Proto-Oncogênicas B-raf/genética , Padrões de Referência , IncertezaRESUMO
In the prenatal period, the copy number aberrations of chromosomes 13, 18, 21, X and Y account for over 80% of the clinically significant chromosome abnormalities. Classical cytogenetic analysis is the gold standard in invasive prenatal diagnostics but the long test waiting time affects its clinical utility. Several molecular rapid tests have been developed and employed in clinical practice, however all have substantial drawbacks. The aim of the study was to design and evaluate an optimized tool for rapid molecular detection of fetal aneuploidies. We established a novel single-day method using a chip-based platform, the QuantStudio 3D Digital PCR system. In order to assess the clinical usefulness of our screening test, we analyzed 133 prenatal samples. The difference in distributions of euploid and aneuploid samples identified the ploidy of each of the target chromosomes with high precision. The distribution of the chromosome ratio for euploid and aneuploid samples showed a statistically significant result (p = 0.003 for trisomy 13, p = 0.001 for trisomies 18 and 21, Mann-Whitney U test). Our results suggest that this novel chip-based approach provides a tool for rapid, technically simple, cost-effective screening for common fetal aneuploidies.
Assuntos
Aneuploidia , Transtornos Cromossômicos/diagnóstico , Testes Genéticos/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Diagnóstico Pré-Natal/métodos , Adulto , Transtornos Cromossômicos/genética , Custos e Análise de Custo , Feminino , Testes Genéticos/economia , Testes Genéticos/normas , Humanos , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos/economia , Análise de Sequência com Séries de Oligonucleotídeos/normas , Reação em Cadeia da Polimerase/economia , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/normas , Gravidez , Diagnóstico Pré-Natal/economia , Diagnóstico Pré-Natal/normas , Sensibilidade e EspecificidadeRESUMO
The ability for safe and rapid pathogenic sample transportation and subsequent detection is an increasing challenge throughout the world. Herein, we describe and use bead-beating, vortex, sonication, 903 protein saver cards, and Lyse-It methods, aiming to inactivate Gram-positive and -negative bacteria with subsequent genome DNA (quantitative Polymerase Chain Reaction) qPCR detection. The basic concepts behind the four chosen technologies is their versatility, cost, and ease of use in developed and underdeveloped countries. The four methods target the testing of bacterial resilience, cellular extraction from general and complex media and subsequent DNA extraction for qPCR detection and amplification. These results demonstrate that conventional high temperature heating, 903 protein saver cards, and Lyse-It are all viable options for inactivating bacterial growth for safe shipping. Additionally, Lyse-It was found to be particularly useful as this technology can inactivate bacteria, extract cells from 903 protein saver cards, lyse bacterial cells, and additionally keep genomic DNA viable for qPCR detection.
Assuntos
Fracionamento Celular/métodos , DNA Bacteriano/normas , Técnicas de Diagnóstico Molecular/métodos , Fracionamento Celular/economia , Fracionamento Celular/normas , DNA Bacteriano/química , Bactérias Gram-Negativas/química , Bactérias Gram-Positivas/química , Técnicas de Diagnóstico Molecular/economia , Técnicas de Diagnóstico Molecular/normas , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/normas , Kit de Reagentes para Diagnóstico/normasRESUMO
We conducted a multilaboratory assessment to determine the suitability of a new commercially available reference material with 40 cancer variants in a background of wild-type DNA at four different variant allele frequencies (VAFs): 2%, 0.50%, 0.125%, and 0%. The variants include single nucleotides, insertions, deletions, and two structural variations selected for their clinical importance and to challenge the performance of next-generation sequencing (NGS) methods. Fragmented DNA was formulated to simulate the size distribution of circulating wild-type and tumor DNA in a synthetic plasma matrix. DNA was extracted from these samples and characterized with different methods and multiple laboratories. The various extraction methods had differences in yield, perhaps because of differences in chemistry. Digital PCR assays were used to measure VAFs to compare results from different NGS methods. Comparable VAFs were observed across the different NGS methods. This multilaboratory assessment demonstrates that the new reference material is an appropriate tool to determine the analytical parameters of different measurement methods and to ensure their quality assurance.
Assuntos
Biomarcadores Tumorais , DNA Tumoral Circulante , DNA de Neoplasias , Biópsia Líquida , Neoplasias/diagnóstico , Neoplasias/genética , Alelos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sequenciamento de Nucleotídeos em Larga Escala/normas , Humanos , Biópsia Líquida/métodos , Biópsia Líquida/normas , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/normas , Garantia da Qualidade dos Cuidados de Saúde , Padrões de ReferênciaRESUMO
The quality of PCR to detect vancomycin-resistant enterococci (VRE) was evaluated by analysing their performance in six consecutive external quality assessment (EQA) schemes, organized annually since 2013 by Quality Control for Molecular Diagnostics. VRE EQA panels consisted of 12-14 heat-inactivated samples. Sensitivity was tested with vanA-positive Enterococcus faecium (E. faecium), vanB-positive E. faecium, E. faecalis or E. gallinarum or vanC-positive E. gallinarum in different concentrations. Vancomycin-susceptible enterococci, Staphylococcus aureus or sample matrix was used to study the specificity. Participants were asked to report the VRE resistance status of each sample. The detection rate of vanA-positive samples was already 95% in the 2013 EQA panel (range 94-97%) and remained stable over the years. The 2013 detection rate of vanB-positive samples was 82% but increased significantly by more than 10% in subsequent years (96% in 2014, 95% in 2015, 92% in 2016 and 93% in 2017/2018, p < 0.05). The vanC detection rate by the limited number of assays specifically targeting this gene was lower compared to vanA/B (range 55-89%). The number of false positives in the true-negative sample (8% in 2013 to 1.4% in 2018) as well as the van-gene-negative bacterial samples (4% in 2013 to 0% in 2018) declined over the years. In the six years of VRE proficiency testing to date, the detection of vanA-positive strains was excellent and an increased sensitivity in vanB detection as well as an increase in specificity was observed. Commercial and in-house assays performed equally well.
Assuntos
Patologia Molecular/estatística & dados numéricos , Patologia Molecular/normas , Reação em Cadeia da Polimerase/normas , Controle de Qualidade , Resistência a Vancomicina/genética , Enterococos Resistentes à Vancomicina/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Carbono-Oxigênio Ligases/genética , Enterococcus faecium/genética , Infecções por Bactérias Gram-Positivas/microbiologia , Testes de Sensibilidade Microbiana , Sensibilidade e Especificidade , Enterococos Resistentes à Vancomicina/efeitos dos fármacos , Enterococos Resistentes à Vancomicina/isolamento & purificaçãoRESUMO
Anaplasma phagocytophilum, the bacterial pathogen responsible for tick-borne fever and human granulocytic anaplasmosis, can seriously affect the health of humans and a wide range of other mammals. In this study, we developed a recombinase polymerase amplification (RPA) assay to detect A. phagocytophilum in clinical samples. Following alignment of the relevant DNA sequences, a pair of specific primers based on the 16S rRNA gene was designed to specifically detect A. phagocytophilum. The assay was performed at a constant temperature of 38⯰C for 30â¯min, with a final primer concentration of 0.4⯵M. The specificity of the primers was confirmed when DNA from A. phagocytophilum was used as the positive control, and DNA from other related pathogens were used as the negative controls, with ddH2O acting as the blank control. The results showed that the primers did not cross-react with DNA from the other related pathogens. The assay's detection limit was 1.77â¯×â¯10-5â¯ng/µl, a 10â¯×â¯higher sensitivity level than that determined for nested PCR. The RPA assay's performance was evaluated using 44 clinical samples, and the prevalence results for A. phagocytophilum were found to not differ significantly between the RPA assay and the nested PCR. Thus, we have developed a specific, sensitive, rapid and cost-effective RPA method, requiring only a water bath, for the detection of A. phagocytophilum. The assay should be especially useful in resource-limited areas where access to laboratory equipment is limited.
Assuntos
Anaplasma phagocytophilum/isolamento & purificação , Ehrlichiose/veterinária , Reação em Cadeia da Polimerase/veterinária , Doenças dos Ovinos/microbiologia , Anaplasma phagocytophilum/genética , Animais , Análise Custo-Benefício , DNA Bacteriano/sangue , DNA Bacteriano/química , DNA Bacteriano/isolamento & purificação , DNA Polimerase Dirigida por DNA , Ehrlichiose/diagnóstico , Ehrlichiose/microbiologia , Sistemas Automatizados de Assistência Junto ao Leito/economia , Sistemas Automatizados de Assistência Junto ao Leito/normas , Reação em Cadeia da Polimerase/economia , Reação em Cadeia da Polimerase/normas , RNA Ribossômico 16S/genética , Recombinases , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Ovinos , Doenças dos Ovinos/diagnóstico , Fatores de TempoRESUMO
BACKGROUND: Arcobacter faecis and A. lanthieri are two newly classified species of genus Arcobacter. The prevalence and distribution of virulence, antibiotic resistance and toxin (VAT) genes in these species are required to assess their potential pathogenic health impacts to humans and animals. This study (i) developed species- and gene-specific primer pairs for the detection of six virulence, two antibiotic resistance, and three toxin genes in two target species; (ii) optimized eight single-tube multiplex and three monoplex PCR protocols using the newly developed species- and gene-specific primers; and (iii) conducted specificity and sensitivity evaluations as well as validation of eleven mono- and multiplex PCR assays by testing A. faecis (n= 29) and A. lanthieri (n= 10) strains isolated from various fecal and agricultural water sources to determine the prevalence and distribution of VAT genes and assess the degree of pathogenicity within the two species. RESULTS: Detection of all ten and eleven target VAT genes, and expression of cytolethal distending toxin (cdtA, cdtB and cdtC) genes in A. faecis and A. lanthieri reference strains with high frequency in field isolates suggest that they are potentially pathogenic strains. These findings indicate that these two species can pose a health risk to humans and animals. CONCLUSIONS: The study results show that the developed mono- and multiplex PCR (mPCR) assays are simple, rapid, reliable and sensitive for the simultaneous assessment of the potential pathogenicity and antibiotic resistance profiling of tet(O) and tet(W) genes in these two newly discovered species. Also, these assays can be useful in diagnostic and analytical laboratories to determine the pathotypes and assessment of the virulence and toxin factors associated to human and animal infections.
Assuntos
Arcobacter , Toxinas Bacterianas/genética , Técnicas de Tipagem Bacteriana/métodos , Resistência Microbiana a Medicamentos/genética , Infecções por Bactérias Gram-Negativas/microbiologia , Reação em Cadeia da Polimerase , Virulência/genética , Animais , Arcobacter/efeitos dos fármacos , Arcobacter/genética , Arcobacter/patogenicidade , Técnicas de Tipagem Bacteriana/normas , Genes Bacterianos/genética , Infecções por Bactérias Gram-Negativas/diagnóstico , Humanos , Reação em Cadeia da Polimerase Multiplex/normas , Reação em Cadeia da Polimerase/normas , Sensibilidade e EspecificidadeRESUMO
Quantitative Polymerase Chain Reaction (qPCR) is a molecular method commonly used to detect and quantify bacterial DNA on food but is limited by its inability to distinguish between live and dead cell DNA. To overcome this obstacle, propidium monoazide (PMA) alone or with deoxycholate (DC) was used to prevent dead cell detection in qPCR. qPCR methods were used to detect strains of Escherichia coli O157, which can cause infection in humans with an infectious dose of less than 10â¯cells. A 5 strain E. coli O157:H7 cocktail was inoculated onto beef steaks and treated with interventions used in meat facilities (lactic acid (5%), peroxyacetic acid (200 ppm) or hot water (80 °C for 10 s)). Treatment of PMA or PMA + DC was applied to samples followed by DNA extraction and quantification in qPCR. RNA was also quantified in addition to conventional plating. For lactic acid intervention, qPCR DNA quantification of E. coli O157:H7 yielded 6.59⯱â¯0.21 and 6.30⯱â¯0.11 log gene copy #/cm2 for control and lactic acid samples, respectively and after treatment with PMA or PMA + DC this was further reduced to 6.31 ± 0.21 and 5.58 ± 0.38, respectively. This trend was also observed for peroxyacetic acid and hot water interventions. In comparison, RNA quantification yielded 7.65 ± 0.13 and 7.02 ± 0.38 log reverse transcript/cm2 for rRNA control and lactic acid samples, respectively, and for plating (LB), 7.51⯱â¯0.06 and 6.86⯱â¯0.32 log CFU/cm2, respectively. Our research determined that treatment of PMA + DC in conjunction with qPCR prevented dead cell DNA detection. However, it also killed cells injured from intervention that may have otherwise recovered. RNA quantification was more laborious and results had higher variability. Overall, quantification with conventional plating proved to be the most robust and reliable method for live EHEC detection on beef.
Assuntos
Escherichia coli O157/isolamento & purificação , Microbiologia de Alimentos/métodos , Viabilidade Microbiana , Reação em Cadeia da Polimerase/normas , Carne Vermelha/microbiologia , Animais , Azidas/química , Azidas/farmacologia , Bovinos , Contagem de Colônia Microbiana/normas , DNA Bacteriano/química , DNA Bacteriano/genética , Ácido Desoxicólico/química , Ácido Desoxicólico/farmacologia , Escherichia coli O157/efeitos dos fármacos , Escherichia coli O157/genética , Manipulação de Alimentos , Temperatura Alta , Ácido Láctico/farmacologia , Viabilidade Microbiana/efeitos dos fármacos , Ácido Peracético/farmacocinética , Propídio/análogos & derivados , Propídio/química , Propídio/farmacologia , RNA Bacteriano/genéticaRESUMO
BACKGROUND: Single nucleotide polymorphisms (SNPs) in codons 167, 198 and 200 of the beta-tubulin isotype 1 gene are associated with benzimidazoles resistance in many helminths. Codon 167 mutation has never been described in hookworms; however, polymorphisms in codons 198 and 200 have been described for Ancylostoma caninum and Necator americanus. These mutations have never been investigated in Ancylostoma braziliense; therefore, it is not known if they are present in this species and whether they are correlated with treatment resistance. The RFLP-PCR technique has been used to analyze these polymorphisms in some nematodes, but depending on the species, these alterations do not create or eliminate any restriction enzyme cleavage site, making it impossible to use this technique. Here, we describe the standardization and application of a modified RFLP-PCR technique for detecting polymorphisms in individual A. braziliense worms recovered from naturally infected dogs in two Brazilian states. RESULTS: The molecular techniques used were sensitive, specific, and easy to apply. To our knowledge, we report for the first time the presence of a polymorphism at codon 198 of the beta-tubulin gene of A. braziliense (1/81; 95% CI: 0-3.69%). CONCLUSIONS: It is not known whether the presence of the mutation in codon 198 of the beta-tubulin gene of A. braziliense has importance for this parasite. However, based on studies of other helminths, it is possible that this polymorphism is directly related to the resistance to benzimidazoles. This may be a major concern, since this nematode has considerable relevance as a parasite of canids and felids and as one of the agents of cutaneous larva migrans in humans. Standardized methodologies will be useful for screening for polymorphisms in the beta-tubulin gene of canine hookworms in a broader population. The method could also be adapted for the analysis of other SNPs in other nematode species.
Assuntos
Ancylostoma/genética , Resistência a Múltiplos Medicamentos/genética , Mutação , Reação em Cadeia da Polimerase/normas , Polimorfismo de Fragmento de Restrição/genética , Polimorfismo de Nucleotídeo Único/genética , Ancylostoma/efeitos dos fármacos , Animais , Benzimidazóis/farmacologia , Códon , Doenças do Cão/parasitologia , Cães/parasitologia , Feminino , Técnicas de Genotipagem/métodos , Masculino , Mutagênese Sítio-Dirigida/métodos , Mutagênese Sítio-Dirigida/normas , Reação em Cadeia da Polimerase/economia , Reação em Cadeia da Polimerase/métodos , Sensibilidade e Especificidade , Tubulina (Proteína)/genéticaRESUMO
BACKGROUND: Enterobacteriaceae are a common cause of hospital infections. Carbapenems are a clinically effective treatment of such infections. However, resistance is on the rise. In particular, carbapenemase-producing carbapenem-resistant Enterobacteriaceae (CP-CRE) are increasingly common. In order to limit spread in clinical settings, screening and isolation is being recommended, but many different screening methods are available. We aimed to compare the impact and costs of three algorithms for detecting CP-CRE carriage. METHODS: We developed an individual-based simulation model to compare three screening algorithms using data from a UK National Health Service (NHS) trust. The first algorithm, "Direct PCR", was highly sensitive/specific and quick (half a day), but expensive. The second, "Culture + PCR", was relatively sensitive/specific but slower, requiring 2.5 days. A third algorithm, "PHE", repeated the "Culture + PCR" three times with an additional PCR. Scenario analysis was used to compare several levels of CP-CRE prevalence and coverage of screening, different specialities as well as isolation strategies. Our outcomes were (1) days that a patient with CP-CRE was not detected and hence not isolated ("days at risk"), (2) isolation bed days, (3) total costs and (4) mean cost per CP-CRE risk day averted per year. We also explored limited isolation bed day capacity. RESULTS: We found that although a Direct PCR algorithm would reduce the number of CP-CRE days at risk, the mean cost per CP-CRE risk day averted per year was substantially higher than for a Culture + PCR algorithm. For example, in our model of an intensive care unit, during a year with a 1.6% CP-CRE prevalence and 63% screening coverage, there were 508 (standard deviation 15), 642 (14) and 655 (14) days at risk under screening algorithms Direct PCR, Culture + PCR and PHE respectively, with mean costs per risk day averted of £192, £61 and £79. These results were robust to sensitivity analyses. CONCLUSIONS: Our results indicate that a Culture + PCR algorithm provides the optimal balance of cost and risk days averted, at varying isolation, prevalence and screening coverage scenarios. Findings from this study will help clinical organisations determine the optimal screening approach for CP-CRE, balancing risk and resources.