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1.
Cochrane Database Syst Rev ; 1: CD012768, 2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33448348

RESUMO

BACKGROUND: Xpert MTB/RIF Ultra (Xpert Ultra) and Xpert MTB/RIF are World Health Organization (WHO)-recommended rapid nucleic acid amplification tests (NAATs) widely used for simultaneous detection of Mycobacterium tuberculosis complex and rifampicin resistance in sputum. To extend our previous review on extrapulmonary tuberculosis (Kohli 2018), we performed this update to inform updated WHO policy (WHO Consolidated Guidelines (Module 3) 2020). OBJECTIVES: To estimate diagnostic accuracy of Xpert Ultra and Xpert MTB/RIF for extrapulmonary tuberculosis and rifampicin resistance in adults with presumptive extrapulmonary tuberculosis. SEARCH METHODS: Cochrane Infectious Diseases Group Specialized Register, MEDLINE, Embase, Science Citation Index, Web of Science, Latin American Caribbean Health Sciences Literature, Scopus, ClinicalTrials.gov, the WHO International Clinical Trials Registry Platform, the International Standard Randomized Controlled Trial Number Registry, and ProQuest, 2 August 2019 and 28 January 2020 (Xpert Ultra studies), without language restriction. SELECTION CRITERIA: Cross-sectional and cohort studies using non-respiratory specimens. Forms of extrapulmonary tuberculosis: tuberculous meningitis and pleural, lymph node, bone or joint, genitourinary, peritoneal, pericardial, disseminated tuberculosis. Reference standards were culture and a study-defined composite reference standard (tuberculosis detection); phenotypic drug susceptibility testing and line probe assays (rifampicin resistance detection). DATA COLLECTION AND ANALYSIS: Two review authors independently extracted data and assessed risk of bias and applicability using QUADAS-2. For tuberculosis detection, we performed separate analyses by specimen type and reference standard using the bivariate model to estimate pooled sensitivity and specificity with 95% credible intervals (CrIs). We applied a latent class meta-analysis model to three forms of extrapulmonary tuberculosis. We assessed certainty of evidence using GRADE. MAIN RESULTS: 69 studies: 67 evaluated Xpert MTB/RIF and 11 evaluated Xpert Ultra, of which nine evaluated both tests. Most studies were conducted in China, India, South Africa, and Uganda. Overall, risk of bias was low for patient selection, index test, and flow and timing domains, and low (49%) or unclear (43%) for the reference standard domain. Applicability for the patient selection domain was unclear for most studies because we were unsure of the clinical settings. Cerebrospinal fluid Xpert Ultra (6 studies) Xpert Ultra pooled sensitivity and specificity (95% CrI) against culture were 89.4% (79.1 to 95.6) (89 participants; low-certainty evidence) and 91.2% (83.2 to 95.7) (386 participants; moderate-certainty evidence). Of 1000 people where 100 have tuberculous meningitis, 168 would be Xpert Ultra-positive: of these, 79 (47%) would not have tuberculosis (false-positives) and 832 would be Xpert Ultra-negative: of these, 11 (1%) would have tuberculosis (false-negatives). Xpert MTB/RIF (30 studies) Xpert MTB/RIF pooled sensitivity and specificity against culture were 71.1% (62.8 to 79.1) (571 participants; moderate-certainty evidence) and 96.9% (95.4 to 98.0) (2824 participants; high-certainty evidence). Of 1000 people where 100 have tuberculous meningitis, 99 would be Xpert MTB/RIF-positive: of these, 28 (28%) would not have tuberculosis; and 901 would be Xpert MTB/RIF-negative: of these, 29 (3%) would have tuberculosis. Pleural fluid Xpert Ultra (4 studies) Xpert Ultra pooled sensitivity and specificity against culture were 75.0% (58.0 to 86.4) (158 participants; very low-certainty evidence) and 87.0% (63.1 to 97.9) (240 participants; very low-certainty evidence). Of 1000 people where 100 have pleural tuberculosis, 192 would be Xpert Ultra-positive: of these, 117 (61%) would not have tuberculosis; and 808 would be Xpert Ultra-negative: of these, 25 (3%) would have tuberculosis. Xpert MTB/RIF (25 studies) Xpert MTB/RIF pooled sensitivity and specificity against culture were 49.5% (39.8 to 59.9) (644 participants; low-certainty evidence) and 98.9% (97.6 to 99.7) (2421 participants; high-certainty evidence). Of 1000 people where 100 have pleural tuberculosis, 60 would be Xpert MTB/RIF-positive: of these, 10 (17%) would not have tuberculosis; and 940 would be Xpert MTB/RIF-negative: of these, 50 (5%) would have tuberculosis. Lymph node aspirate Xpert Ultra (1 study) Xpert Ultra sensitivity and specificity (95% confidence interval) against composite reference standard were 70% (51 to 85) (30 participants; very low-certainty evidence) and 100% (92 to 100) (43 participants; low-certainty evidence). Of 1000 people where 100 have lymph node tuberculosis, 70 would be Xpert Ultra-positive and 0 (0%) would not have tuberculosis; 930 would be Xpert Ultra-negative and 30 (3%) would have tuberculosis. Xpert MTB/RIF (4 studies) Xpert MTB/RIF pooled sensitivity and specificity against composite reference standard were 81.6% (61.9 to 93.3) (377 participants; low-certainty evidence) and 96.4% (91.3 to 98.6) (302 participants; low-certainty evidence). Of 1000 people where 100 have lymph node tuberculosis, 118 would be Xpert MTB/RIF-positive and 37 (31%) would not have tuberculosis; 882 would be Xpert MTB/RIF-negative and 19 (2%) would have tuberculosis. In lymph node aspirate, Xpert MTB/RIF pooled specificity against culture was 86.2% (78.0 to 92.3), lower than that against a composite reference standard. Using the latent class model, Xpert MTB/RIF pooled specificity was 99.5% (99.1 to 99.7), similar to that observed with a composite reference standard. Rifampicin resistance Xpert Ultra (4 studies) Xpert Ultra pooled sensitivity and specificity were 100.0% (95.1 to 100.0), (24 participants; low-certainty evidence) and 100.0% (99.0 to 100.0) (105 participants; moderate-certainty evidence). Of 1000 people where 100 have rifampicin resistance, 100 would be Xpert Ultra-positive (resistant): of these, zero (0%) would not have rifampicin resistance; and 900 would be Xpert Ultra-negative (susceptible): of these, zero (0%) would have rifampicin resistance. Xpert MTB/RIF (19 studies) Xpert MTB/RIF pooled sensitivity and specificity were 96.5% (91.9 to 98.8) (148 participants; high-certainty evidence) and 99.1% (98.0 to 99.7) (822 participants; high-certainty evidence). Of 1000 people where 100 have rifampicin resistance, 105 would be Xpert MTB/RIF-positive (resistant): of these, 8 (8%) would not have rifampicin resistance; and 895 would be Xpert MTB/RIF-negative (susceptible): of these, 3 (0.3%) would have rifampicin resistance. AUTHORS' CONCLUSIONS: Xpert Ultra and Xpert MTB/RIF may be helpful in diagnosing extrapulmonary tuberculosis. Sensitivity varies across different extrapulmonary specimens: while for most specimens specificity is high, the tests rarely yield a positive result for people without tuberculosis. For tuberculous meningitis, Xpert Ultra had higher sensitivity and lower specificity than Xpert MTB/RIF against culture. Xpert Ultra and Xpert MTB/RIF had similar sensitivity and specificity for rifampicin resistance. Future research should acknowledge the concern associated with culture as a reference standard in paucibacillary specimens and consider ways to address this limitation.


Assuntos
Antibióticos Antituberculose/uso terapêutico , Farmacorresistência Bacteriana , Mycobacterium tuberculosis/efeitos dos fármacos , Técnicas de Amplificação de Ácido Nucleico , Rifampina/uso terapêutico , Tuberculose/diagnóstico , Adulto , Viés , Reações Falso-Negativas , Reações Falso-Positivas , Humanos , Mycobacterium tuberculosis/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/métodos , Técnicas de Amplificação de Ácido Nucleico/estatística & dados numéricos , Kit de Reagentes para Diagnóstico , Sensibilidade e Especificidade , Tuberculose/líquido cefalorraquidiano , Tuberculose/tratamento farmacológico , Tuberculose dos Linfonodos/líquido cefalorraquidiano , Tuberculose dos Linfonodos/diagnóstico , Tuberculose dos Linfonodos/tratamento farmacológico , Tuberculose Meníngea/líquido cefalorraquidiano , Tuberculose Meníngea/diagnóstico , Tuberculose Meníngea/tratamento farmacológico , Tuberculose Resistente a Múltiplos Medicamentos/líquido cefalorraquidiano , Tuberculose Resistente a Múltiplos Medicamentos/diagnóstico , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Pleural/líquido cefalorraquidiano , Tuberculose Pleural/diagnóstico , Tuberculose Pleural/tratamento farmacológico
2.
Biosens Bioelectron ; 172: 112766, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33126177

RESUMO

The 2019 novel coronavirus disease (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected all aspects of human life. Rapid, accurate, sensitive and user friendly detection method is urgently needed to facilitate early intervention and control the spread of SARS-CoV-2. Here, we propose a one-pot visual SARS-CoV-2 detection system named "opvCRISPR" by integrating reverse transcription loop-mediated isothermal amplification (RT-LAMP) and Cas12a cleavage in a single reaction system. We demonstrate that the collateral activity against single-stranded DNA (ssDNA) reporters of activated Cas12a triggered by RT-LAMP amplicon increases detection sensitivity and makes detection results observable with naked eye. The opvCRISPR enables detection at nearly single molecule level in 45 min. We validate this method with 50 SARS-CoV-2 potentially infected clinical samples. The opvCRISPR diagnostic results provide 100% agreement with the Centers for Disease Control and Prevention (CDC)-approved quantitative RT-PCR assay. The opvCRISPR holds great potential for SARS-CoV-2 detection in next-generation point-of-care molecular diagnostics.


Assuntos
/métodos , Sistemas CRISPR-Cas , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , /genética , Sequência de Bases , /instrumentação , Humanos , Técnicas de Diagnóstico Molecular/instrumentação , Técnicas de Diagnóstico Molecular/estatística & dados numéricos , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Técnicas de Amplificação de Ácido Nucleico/estatística & dados numéricos , Pandemias , RNA Viral/genética , Sensibilidade e Especificidade
3.
Virology ; 549: 1-4, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32758712

RESUMO

The current outbreak of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was reported in China firstly. A rapid, highly sensitive, specific, and simple operational method was needed for the detection of SARS-CoV-2. Here, we established a real-time reverse-transcription recombinase-aided amplification assay (RT-RAA) to detect SARS-CoV-2 rapidly. The primers and probe were designed based on the nucleocapsid protein gene (N gene) sequence of SARS-CoV-2. The detection limit was 10 copies per reaction in this assay, which could be conducted within 15 min at a constant temperature (39 °C), without any cross-reactions with other respiratory tract pathogens, such as other coronaviruses. Furthermore, compared with commercial real-time RT-PCR assay, it showed a kappa value of 0.959 (p < 0.001) from 150 clinical specimens. These results indicated that this real-time RT-RAA assay may be a valuable tool for detecting SARS-CoV-2.


Assuntos
Betacoronavirus/genética , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/virologia , Genes Virais , Técnicas de Amplificação de Ácido Nucleico/métodos , Proteínas do Nucleocapsídeo/genética , Pneumonia Viral/virologia , Betacoronavirus/isolamento & purificação , China/epidemiologia , Técnicas de Laboratório Clínico/estatística & dados numéricos , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/epidemiologia , Humanos , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico/estatística & dados numéricos , Pandemias , Fosfoproteínas , Pneumonia Viral/diagnóstico , Pneumonia Viral/epidemiologia , RNA Viral/análise , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase em Tempo Real/estatística & dados numéricos , Recombinases , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/estatística & dados numéricos , Sensibilidade e Especificidade
4.
Sci Transl Med ; 12(556)2020 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-32719001

RESUMO

The coronavirus disease 2019 (COVID-19) pandemic caused by the SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) coronavirus is a major public health challenge. Rapid tests for detecting existing SARS-CoV-2 infections and assessing virus spread are critical. Approaches to detect viral RNA based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) have potential as simple, scalable, and broadly applicable testing methods. Compared to RT quantitative polymerase chain reaction (RT-qPCR)-based methods, RT-LAMP assays require incubation at a constant temperature, thus eliminating the need for sophisticated instrumentation. Here, we tested a two-color RT-LAMP assay protocol for detecting SARS-CoV-2 viral RNA using a primer set specific for the N gene. We tested our RT-LAMP assay on surplus RNA samples isolated from 768 pharyngeal swab specimens collected from individuals being tested for COVID-19. We determined the sensitivity and specificity of the RT-LAMP assay for detecting SARS-CoV-2 viral RNA. Compared to an RT-qPCR assay using a sensitive primer set, we found that the RT-LAMP assay reliably detected SARS-CoV-2 RNA with an RT-qPCR cycle threshold (CT) number of up to 30, with a sensitivity of 97.5% and a specificity of 99.7%. We also developed a swab-to-RT-LAMP assay that did not require a prior RNA isolation step, which retained excellent specificity (99.5%) but showed lower sensitivity (86% for CT < 30) than the RT-LAMP assay. In addition, we developed a multiplexed sequencing protocol (LAMP-sequencing) as a diagnostic validation procedure to detect and record the outcome of RT-LAMP reactions.


Assuntos
Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Pneumonia Viral/diagnóstico , Pneumonia Viral/virologia , Colorimetria/métodos , Colorimetria/estatística & dados numéricos , Infecções por Coronavirus/epidemiologia , Humanos , Técnicas de Diagnóstico Molecular/estatística & dados numéricos , Técnicas de Amplificação de Ácido Nucleico/estatística & dados numéricos , Proteínas do Nucleocapsídeo/genética , Pandemias , Fosfoproteínas , Pneumonia Viral/epidemiologia , RNA Viral/genética , RNA Viral/isolamento & purificação , RNA-Seq , Sensibilidade e Especificidade , Pesquisa Médica Translacional
5.
J Clin Microbiol ; 58(9)2020 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-32636214

RESUMO

The clinical performances of six molecular diagnostic tests and a rapid antigen test for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were clinically evaluated for the diagnosis of coronavirus disease 2019 (COVID-19) in self-collected saliva. Saliva samples from 103 patients with laboratory-confirmed COVID-19 (15 asymptomatic and 88 symptomatic) were collected on the day of hospital admission. SARS-CoV-2 RNA in saliva was detected using a quantitative reverse transcription-PCR (RT-qPCR) laboratory-developed test (LDT), a cobas SARS-CoV-2 high-throughput system, three direct RT-qPCR kits, and reverse transcription-loop-mediated isothermal amplification (RT-LAMP). The viral antigen was detected by a rapid antigen immunochromatographic assay. Of the 103 samples, viral RNA was detected in 50.5 to 81.6% of the specimens by molecular diagnostic tests, and an antigen was detected in 11.7% of the specimens by the rapid antigen test. Viral RNA was detected at significantly higher percentages (65.6 to 93.4%) in specimens collected within 9 days of symptom onset than in specimens collected after at least 10 days of symptoms (22.2 to 66.7%) and in specimens collected from asymptomatic patients (40.0 to 66.7%). Self-collected saliva is an alternative specimen option for diagnosing COVID-19. The RT-qPCR LDT, a cobas SARS-CoV-2 high-throughput system, direct RT-qPCR kits (except for one commercial kit), and RT-LAMP showed sufficient sensitivities in clinical use to be selectively used in clinical settings and facilities. The rapid antigen test alone is not recommended for an initial COVID-19 diagnosis because of its low sensitivity.


Assuntos
Técnicas de Laboratório Clínico , Infecções por Coronavirus/diagnóstico , Imunoensaio , Técnicas de Amplificação de Ácido Nucleico , Pneumonia Viral/diagnóstico , Saliva/virologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos Virais/análise , Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico/métodos , Técnicas de Laboratório Clínico/normas , Técnicas de Laboratório Clínico/estatística & dados numéricos , Feminino , Humanos , Imunoensaio/métodos , Imunoensaio/normas , Imunoensaio/estatística & dados numéricos , Masculino , Pessoa de Meia-Idade , Técnicas de Amplificação de Ácido Nucleico/métodos , Técnicas de Amplificação de Ácido Nucleico/normas , Técnicas de Amplificação de Ácido Nucleico/estatística & dados numéricos , Pandemias , RNA Viral/análise , RNA Viral/genética , Sensibilidade e Especificidade , Manejo de Espécimes , Adulto Jovem
6.
Pediatrics ; 146(2)2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32636237

RESUMO

OBJECTIVE: The Centers for Disease Control and Prevention recommend testing for Chlamydia trachomatis in sexually active female patients <25 years old using nucleic-acid amplification tests (NAAT) from a vaginal swab. Our providers were typically testing using the less sensitive urine NAATs. We aimed to increase the percentage of urogenital C trachomatis NAATs performed by using vaginal swabs in adolescent female patients ages 10 through 20 years from 1.4% to 25%. METHODS: We implemented 3 interventions at 3 pediatric practices over 12 months including education, process standardization, and cross-training. We used statistical process control to analyze the effect of interventions on our primary outcome: the percentage of urogenital C trachomatis tests performed with a vaginal swab. Our balance measure was the total number of urogenital C trachomatis tests. RESULTS: There were 818 urogenital C trachomatis tests performed: 289 before and 529 after the first intervention. Of urogenital C trachomatis tests in the preintervention time period, 1.4% were performed by using vaginal swabs. We surpassed our aim of 25% 6 weeks after the first intervention. We noted sustained improvement after the second intervention, with an average of 68.3% of tests performed by using vaginal swabs for the remaining postintervention period. There was no difference in the overall number of urogenital C trachomatis tests pre- and postintervention. CONCLUSIONS: Using quality improvement methodology and implementing easily replicable interventions, we significantly and sustainably increased use of vaginal swabs. The interventions standardizing processes were associated with a higher impact than the educational intervention.


Assuntos
Infecções por Chlamydia/diagnóstico , Chlamydia trachomatis/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/métodos , Pediatras/educação , Padrões de Prática Médica/tendências , Vagina/microbiologia , Esfregaço Vaginal/tendências , Adolescente , Criança , Infecções por Chlamydia/epidemiologia , Feminino , Humanos , Massachusetts/epidemiologia , Técnicas de Amplificação de Ácido Nucleico/estatística & dados numéricos , Folhetos , Guias de Prática Clínica como Assunto , Padrões de Prática Médica/estatística & dados numéricos , Utilização de Procedimentos e Técnicas/tendências , Avaliação de Programas e Projetos de Saúde , Melhoria de Qualidade , Comportamento Sexual , Adulto Jovem
7.
Biosens Bioelectron ; 164: 112316, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32553350

RESUMO

Recent research suggests that SARS-CoV-2-infected individuals can be highly infectious while asymptomatic or pre-symptomatic, and that an infected person may infect 5.6 other individuals on average. This situation highlights the need for rapid, sensitive SARS-CoV-2 diagnostic assays capable of high-throughput operation that can preferably utilize existing equipment to facilitate broad, large-scale screening efforts. We have developed a CRISPR-based assay that can meet all these criteria. This assay utilizes a custom CRISPR Cas12a/gRNA complex and a fluorescent probe to detect target amplicons produced by standard RT-PCR or isothermal recombinase polymerase amplification (RPA), to allow sensitive detection at sites not equipped with real-time PCR systems required for qPCR diagnostics. We found this approach allowed sensitive and robust detection of SARS-CoV-2 positive samples, with a sample-to-answer time of ~50 min, and a limit of detection of 2 copies per sample. CRISPR assay diagnostic results obtained nasal swab samples of individuals with suspected COVID-19 cases were comparable to paired results from a CDC-approved quantitative RT-PCR (RT-qPCR) assay performed in a state testing lab, and superior to those produced by same assay in a clinical lab, where the RT-qPCR assay exhibited multiple invalid or inconclusive results. Our assay also demonstrated greater analytical sensitivity and more robust diagnostic performance than other recently reported CRISPR-based assays. Based on these findings, we believe that a CRISPR-based fluorescent application has potential to improve current COVID-19 screening efforts.


Assuntos
Betacoronavirus/isolamento & purificação , Sistemas CRISPR-Cas , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , Sequência de Bases , Betacoronavirus/genética , Técnicas Biossensoriais/métodos , Técnicas Biossensoriais/estatística & dados numéricos , Infecções por Coronavirus/virologia , Corantes Fluorescentes , Genes Virais , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sequenciamento de Nucleotídeos em Larga Escala/estatística & dados numéricos , Humanos , Técnicas de Amplificação de Ácido Nucleico/métodos , Técnicas de Amplificação de Ácido Nucleico/estatística & dados numéricos , Pandemias , Pneumonia Viral/virologia , Valor Preditivo dos Testes , RNA Viral/análise , RNA Viral/genética , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/estatística & dados numéricos , Sensibilidade e Especificidade
8.
Small ; 16(32): e2002169, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32578378

RESUMO

The ongoing global novel coronavirus pneumonia COVID-19 outbreak has engendered numerous cases of infection and death. COVID-19 diagnosis relies upon nucleic acid detection; however, currently recommended methods exhibit high false-negative rates and are unable to identify other respiratory virus infections, thereby resulting in patient misdiagnosis and impeding epidemic containment. Combining the advantages of targeted amplification and long-read, real-time nanopore sequencing, herein, nanopore targeted sequencing (NTS) is developed to detect SARS-CoV-2 and other respiratory viruses simultaneously within 6-10 h, with a limit of detection of ten standard plasmid copies per reaction. Compared with its specificity for five common respiratory viruses, the specificity of NTS for SARS-CoV-2 reaches 100%. Parallel testing with approved real-time reverse transcription-polymerase chain reaction kits for SARS-CoV-2 and NTS using 61 nucleic acid samples from suspected COVID-19 cases show that NTS identifies more infected patients (22/61) as positive, while also effectively monitoring for mutated nucleic acid sequences, categorizing types of SARS-CoV-2, and detecting other respiratory viruses in the test sample. NTS is thus suitable for COVID-19 diagnosis; moreover, this platform can be further extended for diagnosing other viruses and pathogens.


Assuntos
Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Nanoporos , Técnicas de Amplificação de Ácido Nucleico/métodos , Pneumonia Viral/diagnóstico , Pneumonia Viral/virologia , Betacoronavirus/classificação , Infecções por Coronavirus/epidemiologia , DNA Viral/genética , DNA Viral/isolamento & purificação , Genes Virais , Humanos , Limite de Detecção , Mutação , Nanotecnologia , Técnicas de Amplificação de Ácido Nucleico/estatística & dados numéricos , Pandemias , Pneumonia Viral/epidemiologia , RNA Viral/genética , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Infecções Respiratórias/diagnóstico , Infecções Respiratórias/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade
9.
Malar J ; 19(1): 129, 2020 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-32228615

RESUMO

BACKGROUND: The World Health Organization (WHO) recommends parasite-based diagnosis of malaria. In recent years, there has been surge in the use of various kinds of nucleic-acid amplification based tests (NAATs) for detection and identification of Plasmodium spp. to support clinical care in high-resource settings and clinical and epidemiological research worldwide. However, these tests are not without challenges, including lack (or limited use) of standards and lack of reproducibility, due in part to variation in protocols amongst laboratories. Therefore, there is a need for rigorous quality control, including a robust external quality assessment (EQA) scheme targeted towards malaria NAATs. To this effect, the WHO Global Malaria Programme worked with the UK National External Quality Assessment Scheme (UK NEQAS) Parasitology and with technical experts to launch a global NAAT EQA scheme in January 2017. METHODS: Panels of NAAT EQA specimens containing five major species of human-infecting Plasmodium at various parasite concentrations and negative samples were created in lyophilized blood (LB) and dried blood spot (DBS) formats. Two distributions per year were sent, containing five LB and five DBS specimens. Samples were tested and validated by six expert referee laboratories prior to distribution. Between 37 and 45 laboratories participated in each distribution and submitted results using the online submission portal of UK NEQAS. Participants were scored based on their laboratory's stated capacity to identify Plasmodium species, and individual laboratory reports were sent which included performance comparison with anonymized peers. RESULTS: Analysis of the first three distributions revealed that the factors that most significantly affected performance were sample format (DBS vs LB), species and parasite density, while laboratory location and the reported methodology used (type of nucleic acid extraction, amplification, or DNA vs RNA target) did not significantly affect performance. Referee laboratories performed better than non-referee laboratories. CONCLUSIONS: Globally, malaria NAAT assays now inform a range of clinical, epidemiological and research investigations. EQA schemes offer a way for laboratories to assess and improve their performance, which is critical to safeguarding the reliability of data and diagnoses especially in situations where various NAAT methodologies and protocols are in use.


Assuntos
Testes Diagnósticos de Rotina/estatística & dados numéricos , Malária/diagnóstico , Técnicas de Amplificação de Ácido Nucleico/estatística & dados numéricos , Plasmodium/isolamento & purificação , Garantia da Qualidade dos Cuidados de Saúde/estatística & dados numéricos , Humanos , Controle de Qualidade , Reprodutibilidade dos Testes , Organização Mundial da Saúde
10.
PLoS One ; 14(12): e0226413, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31830129

RESUMO

OBJECTIVE: Given rising incidence of Neisseria gonorrhoeae and Chlamydia trachomatis (GC/CT), development of efficacious screening strategies is critical to interruption of the infection cycle. However, a small proportion of nucleic acid amplification testing (NAAT) results are inconclusive-resulting in delays in diagnosis and treatment. As such, this study seeks to evaluate factors associated with inconclusive rectal GC/CT NAAT. METHODS: This is a retrospective chart review of individuals who received an inconclusive rectal GC/CT NAAT result at a single institution from 3/2016-6/2018. Inconclusive GC/CT NAAT was defined as presence of PCR amplification inhibitors using Roche Cobas v2.0 CT/NG assay. Clinical charts were abstracted for age, gender, HIV status, GC/CT (urogenital, rectal, pharyngeal) and syphilis screening results during the study period, clinic type (HIV clinic, university student health center, other), and whether repeat testing occurred within 6 months following an inconclusive result. Logistic regression analysis was used to calculate adjusted and unadjusted odds ratios of factors associated with receipt of repeat testing following an inconclusive rectal GC/CT NAAT result. RESULTS: During the study period, 6.1% (852/14,015) of rectal GC/CT NAAT were inconclusive for one or both of GC and CT. Among the 413 patients whose inconclusive rectal GC/CT NAAT results that were included in our analysis, 66.6% (275/413) received repeat testing within 6 months, of which 8.7% (24/275) were positive (compared to 5.4% positivity rate of all rectal samples). In multivariable analysis, individuals living with HIV had lower odds of receiving repeat testing following inconclusive rectal GC/CT NAAT compared to HIV uninfected individuals (adj OR 0.25; p = 0.001). CONCLUSIONS: Despite being disproportionately affected by the STI epidemic, individuals living with HIV had 75% lower odds of receiving repeat testing following inconclusive rectal GC/CT NAAT compared to HIV-uninfected individuals, representing potentially missed opportunities for treatment and prevention of ongoing STI transmission.


Assuntos
Infecções por Chlamydia/diagnóstico , Chlamydia trachomatis/isolamento & purificação , Gonorreia/diagnóstico , Infecções por HIV/complicações , Programas de Rastreamento/normas , Neisseria gonorrhoeae/isolamento & purificação , Reto/microbiologia , Adolescente , Adulto , Idoso , Infecções por Chlamydia/epidemiologia , Infecções por Chlamydia/etiologia , Feminino , Gonorreia/epidemiologia , Gonorreia/etiologia , HIV/isolamento & purificação , Humanos , Masculino , Pessoa de Meia-Idade , Técnicas de Amplificação de Ácido Nucleico/estatística & dados numéricos , Reto/virologia , Estudos Retrospectivos , Estados Unidos/epidemiologia , Adulto Jovem
11.
Infect Control Hosp Epidemiol ; 40(3): 269-275, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30786942

RESUMO

OBJECTIVE: We evaluated whether a diagnostic stewardship initiative consisting of ASP preauthorization paired with education could reduce false-positive hospital-onset (HO) Clostridioides difficile infection (CDI). DESIGN: Single center, quasi-experimental study. SETTING: Tertiary academic medical center in Chicago, Illinois. PATIENTS: Adult inpatients were included in the intervention if they were admitted between October 1, 2016, and April 30, 2018, and were eligible for C. difficile preauthorization review. Patients admitted to the stem cell transplant (SCT) unit were not included in the intervention and were therefore considered a contemporaneous noninterventional control group. INTERVENTION: The intervention consisted of requiring prescriber attestation that diarrhea has met CDI clinical criteria, ASP preauthorization, and verbal clinician feedback. Data were compared 33 months before and 19 months after implementation. Facility-wide HO-CDI incidence rates (IR) per 10,000 patient days (PD) and standardized infection ratios (SIR) were extracted from hospital infection prevention reports. RESULTS: During the entire 52 month period, the mean facility-wide HO-CDI-IR was 7.8 per 10,000 PD and the SIR was 0.9 overall. The mean ± SD HO-CDI-IR (8.5 ± 2.0 vs 6.5 ± 2.3; P < .001) and SIR (0.97 ± 0.23 vs 0.78 ± 0.26; P = .015) decreased from baseline during the intervention. Segmented regression models identified significant decreases in HO-CDI-IR (Pstep = .06; Ptrend = .008) and SIR (Pstep = .1; Ptrend = .017) trends concurrent with decreases in oral vancomycin (Pstep < .001; Ptrend < .001). HO-CDI-IR within a noninterventional control unit did not change (Pstep = .125; Ptrend = .115). CONCLUSIONS: A multidisciplinary, multifaceted intervention leveraging clinician education and feedback reduced the HO-CDI-IR and the SIR in select populations. Institutions may consider interventions like ours to reduce false-positive C. difficile NAAT tests.


Assuntos
Gestão de Antimicrobianos/estatística & dados numéricos , Infecções por Clostridium/diagnóstico , Educação em Saúde/estatística & dados numéricos , Pacientes Internados/estatística & dados numéricos , Ensaios Clínicos Controlados não Aleatórios como Assunto/estatística & dados numéricos , Técnicas de Amplificação de Ácido Nucleico/estatística & dados numéricos , Adulto , Infecções por Clostridium/tratamento farmacológico , Infecção Hospitalar/diagnóstico , Infecção Hospitalar/tratamento farmacológico , Reações Falso-Positivas , Feminino , Humanos , Masculino
12.
Diagn Microbiol Infect Dis ; 94(2): 157-159, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30642719

RESUMO

Perinatal group B Streptococcus (GBS) disease prevention guidelines in 2010 allowed for processing of screening specimens by nucleic acid amplification tests (NAATs); however, the extent of NAAT use is unknown. A 2016 laboratory survey sent to 10 surveillance sites found that 18.7% of responding laboratories offered NAAT for GBS screening (antenatal only: 7.3%; intrapartum only: 4.1%; both: 3.4%).


Assuntos
Técnicas de Diagnóstico Molecular/estatística & dados numéricos , Técnicas de Amplificação de Ácido Nucleico/estatística & dados numéricos , Complicações Infecciosas na Gravidez/diagnóstico , Diagnóstico Pré-Natal/estatística & dados numéricos , Utilização de Procedimentos e Técnicas , Infecções Estreptocócicas/diagnóstico , Streptococcus agalactiae/isolamento & purificação , Feminino , Humanos , Gravidez , Streptococcus agalactiae/genética , Estados Unidos
13.
Mil Med ; 184(7-8): e275-e280, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-30690497

RESUMO

INTRODUCTION: US Military and civilian personnel regularly deploy to regions that are endemic for the Hepatitis B virus (HBV), including the Western Pacific, Africa, Eastern Mediterranean, Southeast Asia, and Europe. When patients have life-threatening injuries that require any blood component that is not immediately available, they are typically transfused with locally collected fresh whole blood from a walking blood bank. Currently, there is no simple and easy method for sensitively screening fresh blood in deployed theaters of conflict. MATERIALS AND METHODS: In order to fill the gap, we have developed a loop-mediated isothermal amplification (LAMP) assay to detect the presence of HBV in blood products. The primers were designed to target the gene of the pre-Surface/Surface antigen region of HBV. The amplification reaction mixture was incubated at 60°C for 60 min. The amplicon can be detected by a handheld fluorescence tube scanner or an immune-chromatography test strip. RESULTS: We were able to detect down to 10 copies of viral DNA by LAMP reaction for HBV DNA extracted from HBV-positive plasma. We also identified the optimal heat treatment condition (125°C for 10 min) for plasma specimens without requiring DNA extraction for the LAMP assay. The sensitivity of the assay was evaluated with polymerase chain reaction (PCR) confirmed HBV-positive samples. Using LAMP, we detected HBV in 107 out of 127 (84%) samples. CONCLUSION: This LAMP assay has the potential to be used in resource-limited settings to improve the safety of locally collected blood in endemic regions.


Assuntos
Hepatite B/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Vírus da Hepatite B , Humanos , Técnicas de Diagnóstico Molecular/normas , Técnicas de Diagnóstico Molecular/estatística & dados numéricos , Técnicas de Amplificação de Ácido Nucleico/normas , Técnicas de Amplificação de Ácido Nucleico/estatística & dados numéricos , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/estatística & dados numéricos , Sensibilidade e Especificidade
14.
J Infect Dev Ctries ; 13(12): 1135-1141, 2019 12 31.
Artigo em Inglês | MEDLINE | ID: mdl-32088701

RESUMO

INTRODUCTION: Human respiratory syncytial virus (hRSV) is a common respiratory virus closely related to respiratory tract infection (RTI). Rapid and accurate detection of hRSV is urgently needed to reduce the high morbidity and mortality due to hRSV infection. METHODOLOGY: Here, we established a highly sensitive and specific reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for the rapid detection of A and B group hRSV simultaneously. The specific primer sets for hRSV A and B groups were designed in the M and M2-2 gene, respectively. SYTO 9 was used as the fluorescent dye for real-time monitoring of the amplification of hRSV RNA without cross reaction between hRSV A and B. RESULTS: The limit of detection (LOD) of our new method was 281.17 50% tissue culture infective doses (TCID50)/mL for hRSV A and 1.58 TCID50/mL for hRSV B. Using 90 clinical samples, a comparison to traditional RT-PCR was performed to validate this assay. The positivity rate of RT-LAMP and RT-PCR were 67.8% and 55.6%, respectively, and the positivity rate of RT-LAMP was significantly higher than RT-PCR (χ2 test, P < 0.01). CONCLUSIONS: Compared with traditional RT-PCR method, the newly developed fluorescent RT-LAMP combined with well-designed primers and SYTO 9 is quite sensitive, specific, rapid and well applicable to hRSV clinical diagnosis.


Assuntos
Técnicas de Amplificação de Ácido Nucleico/métodos , Infecções por Vírus Respiratório Sincicial/diagnóstico , Vírus Sincicial Respiratório Humano/isolamento & purificação , Infecções Respiratórias/diagnóstico , Pré-Escolar , China , Humanos , Lactente , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/estatística & dados numéricos , Técnicas de Amplificação de Ácido Nucleico/estatística & dados numéricos , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/classificação , Vírus Sincicial Respiratório Humano/genética , Infecções Respiratórias/virologia , Transcrição Reversa , Sensibilidade e Especificidade
15.
Trans R Soc Trop Med Hyg ; 112(6): 285-293, 2018 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-29992299

RESUMO

Background: In Zimbabwe, while the Xpert MTB/RIF assay is being used for diagnosing tuberculosis and rifampicin-resistance, re-treatment tuberculosis (TB) patients are still expected to have culture and drug sensitivity testing (CDST) performed at national reference laboratories for confirmation. The study aim was to document the Xpert MTB/RIF assay scale-up and assess how the CDST system functioned for re-treatment TB patients. Methods: We performed an ecologic study using national aggregate data. Results: Use of the Xpert MTB/RIF assay increased from 11 829 to 68 153 between 2012 and 2016. Xpert assays worked well, with successful tests in more than 90% of cases, TB detection rates at 15-17% and rifampicin resistance in <10%. During Xpert scale-up, the number of sputum specimens from re-treatment TB patients reaching national reference laboratories for CDST increased from 12% to 51%. In terms of laboratory performance, culture contamination increased from 3% to 17%, positive cultures from 13% to 17% and successful CDST from 6% to 14%: the proportion of CDST showing any resistance to rifampicin averaged 44%. From 2009 to 2016, the proportion of notified re-treatment TB patients with successful CDST increased from <1% to 7%. Conclusions: While components of Zimbabwe's CDST system for re-treatment TB patients showed some changes during the scale-up of the Xpert MTB/RIF assay, overall performance was poor. The country must either invest in improving CDST performance or in advanced molecular diagnostic technology.


Assuntos
Antibióticos Antituberculose/farmacologia , Mycobacterium tuberculosis/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico , Saúde Pública , Rifampina/farmacologia , Escarro/microbiologia , Tuberculose Resistente a Múltiplos Medicamentos/diagnóstico , Antibióticos Antituberculose/uso terapêutico , Inquéritos Epidemiológicos , Humanos , Mycobacterium tuberculosis/efeitos dos fármacos , Técnicas de Amplificação de Ácido Nucleico/economia , Técnicas de Amplificação de Ácido Nucleico/estatística & dados numéricos , Saúde Pública/economia , Rifampina/uso terapêutico , Sensibilidade e Especificidade , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Resistente a Múltiplos Medicamentos/epidemiologia , Zimbábue/epidemiologia
16.
Anal Biochem ; 551: 4-6, 2018 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-29680224

RESUMO

Rapid molecular diagnostic testing for respiratory infections can improve patient care and minimize unnecessary prescriptions of antibiotics. We present the preliminary clinical evaluation of Orion GenRead® RSV, a novel, rapid, and easy-to-use molecular test for the diagnosis of respiratory syncytial virus (RSV) infection. The sensitivity and specificity of Orion GenRead RSV were 99% and 100%, respectively. Orion GenRead RSV detected RSV-positive specimens within 15 min. The performance of Orion GenRead RSV was similar to that of the reference method and this test could rapidly detect RSV within minutes. Orion GenRead RSV is applicable for near-patient testing.


Assuntos
Técnicas de Diagnóstico Molecular/métodos , Infecções por Vírus Respiratório Sincicial/diagnóstico , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/isolamento & purificação , Infecções Respiratórias/diagnóstico , Humanos , Técnicas de Diagnóstico Molecular/estatística & dados numéricos , Nasofaringe/virologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Técnicas de Amplificação de Ácido Nucleico/estatística & dados numéricos , Infecções por Vírus Respiratório Sincicial/virologia , Infecções Respiratórias/virologia , Fatores de Tempo
17.
Breast ; 39: 39-45, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29574393

RESUMO

BACKGROUND: One-step nucleic acid amplification (OSNA) assay is a molecular diagnostic method for intraoperative detection of lymph node metastasis by amplifying cytokeratin 19 mRNA. We aim to further evaluate the performance of OSNA assay for the intraoperative diagnosis of sentinel lymph node metastasis in breast cancer. METHODS: PubMed, Cochrane Library, Web of Science databases were searched to retrieve related literature published up until December 2017. This meta-analysis was performed using a random-effects model. Risks of bias and quality assessments of included studies were evaluated and subgroup analysis was performed. RESULTS: Nineteen studies were included in this meta-analysis. For overall metastasis, the pooled sensitivity, specificity and area under the summary receiver-operating characteristic curve (AUC) were 0.90, 0.96 and 0.98, respectively. For macrometastasis, the pooled sensitivity, specificity and AUC were 0.85, 0.98 and 0.94, respectively. CONCLUSION: OSNA assay is an accurate molecular diagnosis for intraoperative detection of sentinel lymph node macrometastasis in breast cancer.


Assuntos
Neoplasias da Mama/cirurgia , Cuidados Intraoperatórios/métodos , Metástase Linfática/diagnóstico , Técnicas de Diagnóstico Molecular/estatística & dados numéricos , Técnicas de Amplificação de Ácido Nucleico/estatística & dados numéricos , Área Sob a Curva , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Feminino , Humanos , Queratina-19/análise , Metástase Linfática/patologia , Pessoa de Meia-Idade , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA Mensageiro/análise , Curva ROC , Sensibilidade e Especificidade , Linfonodo Sentinela/patologia , Linfonodo Sentinela/cirurgia
18.
PLoS One ; 13(2): e0192979, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29444148

RESUMO

Francisella noatunensis subsp. orientalis (Fno) is the causative agent of piscine francisellosis in warm water fish including tilapia. The disease induces chronic granulomatous inflammation with high morbidity and can result in high mortality. Early and accurate detection of Fno is crucial to set appropriate outbreak control measures in tilapia farms. Laboratory detection of Fno mainly depends on bacterial culture and molecular techniques. Recombinase polymerase amplification (RPA) is a novel isothermal technology that has been widely used for the molecular diagnosis of various infectious diseases. In this study, a recombinase polymerase amplification (RPA) assay for rapid detection of Fno was developed and validated. The RPA reaction was performed at a constant temperature of 42°C for 20 min. The RPA assay was performed using a quantitative plasmid standard containing a unique Fno gene sequence. Validation of the assay was performed not only by using DNA from Fno, closely related Francisella species and other common bacterial pathogens in tilapia farms, but also by screening 78 Nile tilapia and 5 water samples. All results were compared with those obtained by previously established real-time qPCR. The developed RPA showed high specificity in detection of Fno with no cross-detection of either the closely related Francisella spp. or the other tested bacteria. The Fno-RPA performance was highly comparable to the published qPCR with detection limits at 15 and 11 DNA molecules detected, respectively. The RPA gave quicker results in approximately 6 min in contrast to the qPCR that needed about 90 min to reach the same detection limit, taking only 2.7-3 min to determine Fno in clinical samples. Moreover, RPA was more tolerant to reaction inhibitors than qPCR when tested with field samples. The fast reaction, simplicity, cost-effectiveness, sensitivity and specificity make the RPA an attractive diagnostic tool that will contribute to controlling the infection through prompt on-site detection of Fno.


Assuntos
Doenças dos Peixes/microbiologia , Francisella/genética , Francisella/isolamento & purificação , Infecções por Bactérias Gram-Negativas/veterinária , Técnicas de Amplificação de Ácido Nucleico/métodos , Tilápia/microbiologia , Animais , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , DNA Polimerase Dirigida por DNA , Egito , Pesqueiros , Francisella/patogenicidade , Infecções por Bactérias Gram-Negativas/microbiologia , Técnicas de Amplificação de Ácido Nucleico/estatística & dados numéricos , Reação em Cadeia da Polimerase em Tempo Real , Recombinases , Especificidade da Espécie
19.
Sex Transm Dis ; 45(3): 177-182, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29420445

RESUMO

BACKGROUND: The Centers for Disease Control and Prevention 2015 Sexually Transmitted Disease Treatment Guidelines recommend that clinicians consider cephalosporin treatment failure in patients who deny interval sexual exposure and are nucleic acid amplification test (NAAT) positive for Neisseria gonorrhoeae (NG) at least 7 days after adequate treatment. We evaluate the real-world implications of the interval the Centers for Disease Control and Prevention recommends for a NAAT test-of-cure (TOC), by ascertaining the frequency of NG NAAT positivity at different anatomic sites among men who have sex with men (MSM) at TOC 7 to 30 days after treatment. METHODS: We analyzed data from the medical records of MSM with laboratory-confirmed NG who were presumptively treated for NG during the period from June 2013 to April 2016 and returned for a TOC visit within 30 days. Data examined included symptoms, site of NG specimen collection, treatment regimen, follow-up testing, and intervening sexual activity. RESULTS: There were 1027 NG-positive specimens obtained from 763 MSM patients at 889 presumptive treatment visits. Of these, 44% (337/763) MSM returned for 1 or more TOC visits, and 413 specimens were collected a median of 10 days after presumptive treatment. Three percent (14/413) of specimens collected were NG NAAT positive at TOC a median of 13 days after treatment: 5% (12/256) of urethral specimens, 1% (1/147) of anorectal specimens (P = 0.037, urethral vs. anorectal), and 10% (1/10) of oropharyngeal specimens (P = 0.40, urethral vs. oropharyngeal). CONCLUSIONS: A small percent of patients were NG NAAT positive at TOC. Compared with anorectal specimens, urethral specimens were more frequently still positive at TOC. A large proportion of MSM will return for a TOC visit as part of standard clinical care.


Assuntos
Azitromicina/uso terapêutico , Ceftriaxona/uso terapêutico , Gonorreia/diagnóstico , Neisseria gonorrhoeae/isolamento & purificação , Minorias Sexuais e de Gênero/estatística & dados numéricos , Adulto , Assistência ao Convalescente , Centers for Disease Control and Prevention, U.S. , Gonorreia/tratamento farmacológico , Gonorreia/microbiologia , Homossexualidade Masculina , Humanos , Masculino , Registros Médicos , Neisseria gonorrhoeae/genética , Cidade de Nova Iorque/epidemiologia , Técnicas de Amplificação de Ácido Nucleico/estatística & dados numéricos , Comportamento Sexual , Saúde Sexual , Manejo de Espécimes , Estados Unidos
20.
J Clin Microbiol ; 56(3)2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29305538

RESUMO

Globally, Xpert MTB/RIF (Xpert) is the most widely used PCR test for the diagnosis of tuberculosis (TB). Positive results in previously treated patients, which are due to old DNA or active disease, are a diagnostic dilemma. We prospectively retested sputum from 238 patients, irrespective of current symptoms, who were previously diagnosed to be Xpert positive and treated successfully. Patients who retested as Xpert positive and culture negative were exhaustively investigated (repeat culture, chest radiography, bronchoscopy with bronchoalveolar lavage, long-term clinical follow-up). We evaluated whether the duration since previous treatment completion, mycobacterial burden (the Xpert cycle threshold [CT ] value), and reclassification of Xpert-positive results with a very low semiquantitation level to Xpert-negative results reduced the rate of false positivity. A total of 229/238 (96%) of patients were culture negative. Sixteen of 229 (7%) were Xpert positive a median of 11 months (interquartile range, 5 to 19 months) after treatment completion. The specificity was 93% (95% confidence interval [CI], 89 to 96%). Nine of 15 (40%) Xpert-positive, culture-negative patients reverted to Xpert negative after 2 to 3 months (1 patient declined further participation). Patients with false-positive Xpert results had a lower mycobacterial burden than patients with true-positive Xpert results (CT , 28.7 [95% CI, 27.2 to 30.4] versus 17.6 [95% CI, 16.9 to 18.2]; P < 0.001), an increased likelihood of a chest radiograph not compatible with active TB (5/15 patients versus 0/5 patients; P = 0.026), and less-viscous sputum (15/16 patients versus 2/5 patients whose sputum was graded as mucoid or less; P = 0.038). All patients who initially retested as Xpert positive and culture negative ("Xpert false positive") were clinically well without treatment after follow-up. The duration since the previous treatment poorly predicted false-positive results (a duration of ≤2 years identified only 66% of patients with false-positive results). Reclassifying Xpert-positive results with a very low semiquantitation level to Xpert negative improved the specificity (+3% [95% CI, +2 to +5%]) but reduced the sensitivity (-10% [95% CI, -4 to -15%]). Patients with previous TB retested with Xpert can have false-positive results and thus not require treatment. These data inform clinical practice by highlighting the challenges in interpreting Xpert-positive results, underscore the need for culture, and have implications for next-generation ultrasensitive tests.


Assuntos
Testes Diagnósticos de Rotina/estatística & dados numéricos , Testes Diagnósticos de Rotina/normas , Mycobacterium tuberculosis/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/estatística & dados numéricos , Tuberculose/diagnóstico , Adolescente , Adulto , Idoso , Antibióticos Antituberculose/uso terapêutico , Conjuntos de Dados como Assunto , Reações Falso-Positivas , Seguimentos , Humanos , Pessoa de Meia-Idade , Mycobacterium tuberculosis/genética , Técnicas de Amplificação de Ácido Nucleico/normas , Estudos Prospectivos , Sensibilidade e Especificidade , África do Sul , Escarro/microbiologia , Tuberculose/tratamento farmacológico , Tuberculose/microbiologia , Adulto Jovem
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