RESUMO
Nucleic acid tests (NATs) are considered as gold standard in molecular diagnosis. To meet the demand for onsite, point-of-care, specific and sensitive, trace and genotype detection of pathogens and pathogenic variants, various types of NATs have been developed since the discovery of PCR. As alternatives to traditional NATs (e.g., PCR), isothermal nucleic acid amplification techniques (INAATs) such as LAMP, RPA, SDA, HDR, NASBA, and HCA were invented gradually. PCR and most of these techniques highly depend on efficient and optimal primer and probe design to deliver accurate and specific results. This chapter starts with a discussion of traditional NATs and INAATs in concert with the description of computational tools available to aid the process of primer/probe design for NATs and INAATs. Besides briefly covering nanoparticles-assisted NATs, a more comprehensive presentation is given on the role CRISPR-based technologies have played in molecular diagnosis. Here we provide examples of a few groundbreaking CRISPR assays that have been developed to counter epidemics and pandemics and outline CRISPR biology, highlighting the role of CRISPR guide RNA and its design in any successful CRISPR-based application. In this respect, we tabularize computational tools that are available to aid the design of guide RNAs in CRISPR-based applications. In the second part of our chapter, we discuss machine learning (ML)- and deep learning (DL)-based computational approaches that facilitate the design of efficient primer and probe for NATs/INAATs and guide RNAs for CRISPR-based applications. Given the role of microRNA (miRNAs) as potential future biomarkers of disease diagnosis, we have also discussed ML/DL-based computational approaches for miRNA-target predictions. Our chapter presents the evolution of nucleic acid-based diagnosis techniques from PCR and INAATs to more advanced CRISPR/Cas-based methodologies in concert with the evolution of deep learning (DL)- and machine learning (ml)-based computational tools in the most relevant application domains.
Assuntos
Aprendizado Profundo , Humanos , Sistemas CRISPR-Cas , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA/genética , Aprendizado de Máquina , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genéticaRESUMO
The use of direct nucleic acid amplification of pathogens from food matrices has the potential to reduce time to results over DNA extraction-based approaches as well as traditional culture-based approaches. Here we describe protocols for assay design and experiments for direct amplification of foodborne pathogens in food sample matrices using loop-mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR). The examples provided include the detection of Escherichia coli in milk samples and Salmonella in pork meat samples. This protocol includes relevant reagents and methods including obtaining target sequences, assay design, sample processing, and amplification. These methods, though used for specific example matrices, could be applied to many other foodborne pathogens and sample types.
Assuntos
DNA Bacteriano , Microbiologia de Alimentos , Leite , Técnicas de Amplificação de Ácido Nucleico , Reação em Cadeia da Polimerase , Salmonella , Técnicas de Amplificação de Ácido Nucleico/métodos , Microbiologia de Alimentos/métodos , Animais , Leite/microbiologia , Salmonella/genética , Salmonella/isolamento & purificação , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Doenças Transmitidas por Alimentos/microbiologia , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Técnicas de Diagnóstico Molecular/métodos , SuínosRESUMO
A low-cost, lab-made polytetrafluoroethylene micro-cell, equipped with three electrodes, wasd eveloped for the impedimetric detection of SARS-CoV-2. The gold working electrode was modified with a double-ended thiolated poly-adenine probe, which was conjugated with magnetic Fe3O4@Au nanoparticles (Fe3O4@Au-(S-polyA-S)-Au). After the loop-mediated isothermal amplification (LAMP) of viral RNA, the single-guide RNA (sgRNA), specifically bound to the SARS-CoV-2 target sequence, activates Cas12a. Cas12a then cleaved the immobilized probe. As a result, the magnetic Fe3O4@Au nanoparticles were released and adsorbed onto the gold electrode surface, using an external magnet. This process increased the physical surface area of the gold electrode, facilitating redox ion ([FeIII/II(CN)6]3-/4-) electron transfer. The decrease in the charge transfer resistance was utilized for SARS-CoV-2 detection. Our LAMP-CRISPR/Cas12a-based impedimetric biosensor, powered by Fe3O4@Au-(S-polyA-S)-Au, demonstrated impressive capabilities, including a remarkable detection limit of 0.8 aM (0.48 copies/µL) and a linear range of 0.01 to 36.06 fM.
Assuntos
Técnicas Biossensoriais , Sistemas CRISPR-Cas , Ouro , Técnicas de Amplificação de Ácido Nucleico , RNA Viral , SARS-CoV-2 , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Técnicas Biossensoriais/métodos , Ouro/química , Técnicas de Amplificação de Ácido Nucleico/métodos , Humanos , RNA Viral/análise , COVID-19/diagnóstico , COVID-19/virologia , Limite de Detecção , Eletrodos , Poli A/química , Proteínas Associadas a CRISPR , Nanopartículas de Magnetita/química , Endodesoxirribonucleases/química , Nanopartículas Metálicas/química , Proteínas de Bactérias , Técnicas de Diagnóstico MolecularRESUMO
Respiratory pathogens infecting the human respiratory system are characterized by their diversity, high infectivity, rapid transmission, and acute onset. Traditional detection methods are time-consuming, have low sensitivity, and lack specificity, failing to meet the needs of rapid clinical diagnosis. Nucleic acid aptamers, as an emerging and innovative detection technology, offer novel solutions with high specificity, affinity, and broad target applicability, making them particularly promising for respiratory pathogen detection. This review highlights the progress in the research and application of nucleic acid aptamers for detecting respiratory pathogens, discussing their selection, application, potential in clinical diagnosis, and future development. Notably, these aptamers can significantly enhance the sensitivity and specificity of detection when combined with detection techniques such as fluorescence, colorimetry and electrochemistry. This review offers new insights into how aptamers can address the limitations of traditional diagnostic methods and advance clinical diagnostics. It also highlights key challenges and future research directions for the clinical application of nucleic acid aptamers.
Assuntos
Aptâmeros de Nucleotídeos , Infecções Respiratórias , Sensibilidade e Especificidade , Humanos , Infecções Respiratórias/diagnóstico , Infecções Respiratórias/virologia , Infecções Respiratórias/microbiologia , Vírus/isolamento & purificação , Vírus/genética , Vírus/classificação , Bactérias/isolamento & purificação , Bactérias/genética , Técnica de Seleção de Aptâmeros/métodos , Viroses/diagnóstico , Viroses/virologia , Técnicas de Diagnóstico Molecular/métodosRESUMO
While HC2 and GP5+/6+ PCR-EIA were pivotal in test validation of new HPV assays, they represent the first generation of comparator tests based upon technologies that are not in widespread use anymore. In the current guideline, criteria for second-generation comparator tests are presented that include more detailed resolution of HPV genotypes. Second-generation comparator tests should preferentially target only the 12 genotypes classified as carcinogenic (IARC-group I), and show consistent non-inferior sensitivity for CIN2+ and CIN3+ and specificity for ≤CIN1 compared to one of the first-generations comparators, in at least three validation studies using benchmarks of 0.95 for relative sensitivity and 0.98 for relative specificity. Validation should take into account used storage media and other sample handling procedures. Meta-analyses were conducted to identify the assays that fulfill these stringent criteria. Four tests fulfilled the new criteria: (1) RealTime High-Risk HPV Test (Abbott), (2) Cobas-4800 HPV test (Roche Molecular System), (3) Onclarity HPV Assay (BD Diagnostics), and (4) Anyplex II HPV HR Detection (Seegene), each evaluated in three to six studies. Whereas the four assays target 14 carcinogenic genotypes, the first two identify separately HPV16 and 18, the third assay identifies five types separately and the fourth identifies all the types separately.
Assuntos
Detecção Precoce de Câncer , Papillomaviridae , Infecções por Papillomavirus , Sensibilidade e Especificidade , Neoplasias do Colo do Útero , Feminino , Humanos , DNA Viral/genética , Detecção Precoce de Câncer/métodos , Genótipo , Testes de DNA para Papilomavírus Humano/métodos , Testes de DNA para Papilomavírus Humano/normas , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/normas , Papillomaviridae/genética , Papillomaviridae/classificação , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/diagnóstico , Infecções por Papillomavirus/virologia , Neoplasias do Colo do Útero/diagnóstico , Neoplasias do Colo do Útero/virologiaRESUMO
Background: The tcdA gene codes for an important toxin produced by Clostridioides difficile (C. difficile), but there is currently no simple and cost-effective method of detecting it. This article establishes and validates a rapid and visual loop-mediated isothermal amplification (LAMP) assay for the detection of the tcdA gene. Methods: Three sets of primers were designed and optimized to amplify the tcdA gene in C. difficile using a LAMP assay. To evaluate the specificity of the LAMP assay, C. difficile VPI10463 was used as a positive control, while 26 pathogenic bacterial strains lacking the tcdA gene and distilled water were utilized as negative controls. For sensitivity analysis, the LAMP assay was compared to PCR using ten-fold serial dilutions of DNA from C. difficile VPI10463, ranging from 207 ng/µl to 0.000207 pg/µl. The tcdA gene of C.difficile was detected in 164 stool specimens using both LAMP and polymerase chain reaction (PCR). Positive and negative results were distinguished using real-time monitoring of turbidity and chromogenic reaction. Results: At a temperature of 66 °C, the target DNA was successfully amplified with a set of primers designated, and visualized within 60 min. Under the same conditions, the target DNA was not amplified with the tcdA12 primers for 26 pathogenic bacterial strains that do not carry the tcdA gene. The detection limit of LAMP was 20.700 pg/µl, which was 10 times more sensitive than that of conventional PCR. The detection rate of tcdA in 164 stool specimens using the LAMP method was 17% (28/164), significantly higher than the 10% (16/164) detection rate of the PCR method (X2 = 47, p < 0.01). Conclusion: LAMP method is an effective technique for the rapid and visual detection of the tcdA gene of C. difficile, and shows potential advantages over PCR in terms of speed, simplicity, and sensitivity. The tcdA-LAMP assay is particularly suitable for medical diagnostic environments with limited resources and is a promising diagnostic strategy for the screening and detection of C. difficile infection in populations at high risk.
Assuntos
Toxinas Bacterianas , Clostridioides difficile , Infecções por Clostridium , Enterotoxinas , Fezes , Técnicas de Amplificação de Ácido Nucleico , Sensibilidade e Especificidade , Clostridioides difficile/genética , Clostridioides difficile/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/métodos , Humanos , Toxinas Bacterianas/genética , Infecções por Clostridium/diagnóstico , Infecções por Clostridium/microbiologia , Fezes/microbiologia , Fezes/química , Enterotoxinas/genética , Primers do DNA/genética , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase/métodos , Adulto , Pessoa de Meia-IdadeRESUMO
Background: Although the emerging NGS-based assays, metagenomic next-generation sequencing (mNGS) and targeted next-generation sequencing (tNGS), have been extensively utilized for the identification of pathogens in pulmonary infections, there have been limited studies systematically evaluating differences in the efficacy of mNGS and multiplex PCR-based tNGS in bronchoalveolar lavage fluid (BALF) specimens. Methods: In this study, 85 suspected infectious BALF specimens were collected. Parallel mNGS and tNGS workflows to each sample were performed; then, we comparatively compared their consistency in detecting pathogens. The differential results for clinically key pathogens were confirmed using PCR. Results: The microbial detection rates of BALF specimens by the mNGS and tNGS workflows were 95.18% (79/83) and 92.77% (77/83), respectively, with no significant difference. mNGS identified 55 different microorganisms, whereas tNGS detected 49 pathogens. The comparative analysis of mNGS and tNGS revealed that 86.75% (72/83) of the specimens were complete or partial concordance. Particularly, mNGS and tNGS differed significantly in detection rates for some of the human herpesviruses only, including Human gammaherpesvirus 4 (P<0.001), Human betaherpesvirus 7 (P<0.001), Human betaherpesvirus 5 (P<0.05) and Human betaherpesvirus 6 (P<0.01), in which tNGS always had higher detection rates. Orthogonal testing of clinically critical pathogens showed a total coincidence rate of 50% for mNGS and PCR, as well as for tNGS and PCR. Conclusions: Overall, the performance of mNGS and multiplex PCR-based tNGS assays was similar for bacteria and fungi, and tNGS may be superior to mNGS for the detection of DNA viruses. No significant differences were seen between the two NGS assays compared to PCR.
Assuntos
Líquido da Lavagem Broncoalveolar , Sequenciamento de Nucleotídeos em Larga Escala , Metagenômica , Líquido da Lavagem Broncoalveolar/microbiologia , Líquido da Lavagem Broncoalveolar/virologia , Humanos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Metagenômica/métodos , Feminino , Masculino , Pessoa de Meia-Idade , Adulto , Reação em Cadeia da Polimerase Multiplex/métodos , Idoso , Bactérias/isolamento & purificação , Bactérias/genética , Bactérias/classificação , Infecções Respiratórias/diagnóstico , Infecções Respiratórias/virologia , Infecções Respiratórias/microbiologia , Vírus/isolamento & purificação , Vírus/genética , Vírus/classificação , Técnicas de Diagnóstico Molecular/métodos , Adulto JovemRESUMO
BACKGROUND: This study aimed to establish a method for the rapid detection of highly virulent Klebsiella pneumoniae (hvKP) by using multienzyme isothermal rapid amplification (MIRA) technology. The laboratory can quickly, accurately, and conveniently diagnose highly virulent Klebsiella pneumoniae infection. METHODS: For this study, 7 laboratory standard strains and 184 clinical isolates (including 70 strains of Klebsiella pneumoniae) were collected and screened for highly virulent Klebsiella pneumoniae based on its colony morphology, wire drawing test, and next-generation sequencing (NGS) results. Based on the nucleic acid sequence of the peg344 gene of highly virulent Klebsiella pneumoniae on GenBank (no. AP006726.1), specific conserved regions were selected to design MIRA and real-time fluorescence quantitative PCR (qPCR) specific primers and probes. The MIRA and qPCR methods were used to detect the tested strain, and the specificity, sensitivity, and clinical performance of the MIRA method for detecting hvKP were evaluated. RESULTS: In total, 21 cases of hvKP were screened from clinical isolates. The MIRA detection method utilizes specific primers and probes to transmit significant fluorescence signals at 39°C, and the detection process takes 30 minutes. The specificity test results showed that only hvKP had a specific amplification curve, while the rest of non-highly virulent Klebsiella pneumoniae (non-hvKP) had no specific amplification curve. The sensitivity test results showed that the sensitivity of MIRA for detecting hvKP is 7 × 102 CFU/mL, which is consistent with the sensitivity of the real-time fluorescence qPCR method. A simultaneous detection of 184 clinical isolates was accomplished by using MIRA and qPCR methods. Twenty-one strains of hvKP have specific amplification curves, while the remaining 163 strains of non-hvKP have no specific amplification curves. The accuracy of both methods for detecting hvKP is 100%. CONCLUSIONS: The established multienzyme isothermal rapid amplification (MIRA) has the following characteristics: a short detection time, high sensitivity, and a strong specificity, and it can be used as a powerful tool for an early diagnosis and epidemiological monitoring of hvKp.
Assuntos
Infecções por Klebsiella , Klebsiella pneumoniae , Técnicas de Amplificação de Ácido Nucleico , Sensibilidade e Especificidade , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/isolamento & purificação , Klebsiella pneumoniae/patogenicidade , Infecções por Klebsiella/diagnóstico , Infecções por Klebsiella/microbiologia , Humanos , Técnicas de Amplificação de Ácido Nucleico/métodos , Técnicas de Diagnóstico Molecular/métodos , Virulência/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reprodutibilidade dos TestesRESUMO
Background: Metagenomic next-generation sequencing (mNGS) has been widely reported to identify pathogens in infectious diseases (IDs). In this work, we intended to investigate the diagnostic value and clinical acceptance of paired-samples mNGS as compared to the culture method. Methods: A total of 361 patients with suspected infection were retrospectively included. With reference to the clinical diagnosis, we compared the diagnostic performance and clinical acceptance in pathogen detection between mNGS and culture tests. Moreover, the pathogen concordance of paired blood and respiratory tract (RT) samples in mNGS assay was investigated. Results: Among 511 samples, 62.04% were shown to be pathogen positive by mNGS, and that for clinical diagnosis was 51.86% (265/511). When compared to culture assay (n = 428), mNGS had a significantly higher positivity rate (51.87% vs. 33.18%). With reference to the clinical diagnosis, the sensitivity of mNGS outperformed that of culture (89.08% vs. 56.72%). Importantly, mNGS exhibited a clinically accepted rate significantly superior to that of culture. In addition, the mNGS result from 53 paired blood and RT samples showed that most pairs were pathogen positive by both blood and RT, with pathogens largely being partially matched. Conclusion: Through this large-scale study, we further illustrated that mNGS had a clinically accepted rate and sensitivity superior to those of the traditional culture method in diagnosing infections. Moreover, blood and paired RT samples mostly shared partial-matched positive pathogens, especially for pathogens with abundant read numbers in RT, indicating that both blood and RT mNGS can aid the identification of pathogens for respiratory system infection.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Metagenômica , Sensibilidade e Especificidade , Humanos , Estudos Retrospectivos , Metagenômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Feminino , Masculino , Pessoa de Meia-Idade , Adulto , Doenças Transmissíveis/diagnóstico , Doenças Transmissíveis/microbiologia , Idoso , Adulto Jovem , Adolescente , Criança , Técnicas de Diagnóstico Molecular/métodos , Infecções Respiratórias/diagnóstico , Infecções Respiratórias/microbiologia , Pré-EscolarRESUMO
The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered global difficulties for both individuals and economies, with new variants continuing to emerge. The Delta variant of SARS-CoV-2 remains most prevalent worldwide, and it affects the efficacy of coronavirus disease 2019 (COVID-19) vaccination. Expedited testing to detect the Delta variant of SARS-CoV-2 and monitor viral transmission is necessary. This study aimed to develop and evaluate a colorimetric reverse-transcription loop-mediated isothermal amplification (RT-LAMP) technique targeting the L452R mutation in the S gene for the specific detection of the Delta variant. In the test, positivity was indicated as a color change from purple to yellow. The assay's 95% limit of detection was 57 copies per reaction for the L452R (U1355G)-specific standard plasmid. Using 126 clinical samples, our assay displayed 100% specificity, 97.06% sensitivity, and 98.41% accuracy in identifying the Delta variant of SARS-CoV-2 compared to real-time RT-PCR. To our knowledge, this is the first colorimetric RT-LAMP assay that can differentiate the Delta variant from its generic SARS-CoV-2, enabling it as an approach for studying COVID-19 demography and facilitating proper effective control measure establishment to fight against the reemerging variants of SARS-CoV-2 in the future.
Assuntos
COVID-19 , Colorimetria , Mutação , Técnicas de Amplificação de Ácido Nucleico , SARS-CoV-2 , SARS-CoV-2/genética , Humanos , Técnicas de Amplificação de Ácido Nucleico/métodos , Colorimetria/métodos , COVID-19/virologia , COVID-19/diagnóstico , COVID-19/genética , Sensibilidade e Especificidade , Técnicas de Diagnóstico Molecular/métodos , Glicoproteína da Espícula de Coronavírus/genética , RNA Viral/genética , Teste de Ácido Nucleico para COVID-19/métodosRESUMO
BACKGROUND: Endometrial Tuberculosis is one of the most common gynecological problems known to have serious implications for the quality of life like infertility. The commonly practiced histopathology solely relies on the suggestive feature of Tuberculosis (TB) with low specificity. Regarding the alternative bacteriological and molecular detection tools, little evidence was generated on their utility in the diagnosis of endometrial tuberculosis in Ethiopia. Therefore, we aim to investigate the detection rate of molecular and bacteriological detection methods on formalin-fixed paraffin-embedded biopsy samples for the diagnosis of endometrial and lymph node TB. METHODS: A retrospective cross-sectional study was conducted on 90 formalin fixed paraffin embedded biopsy samples from patients with gynecologic and lymph problems collected between 2018 and 2022 at St. Paul's Hospital Millennium Medical College, Addis Ababa, Ethiopia. SPSS version 26 was used for statistical analysis. The diagnostic performance was calculated using the histopathology method as the reference standard. Cohen's Kappa value was used to measure the level of agreement. A test with a P-value of < 0.05 was considered statistically significant. RESULTS: A total of 90 samples were analyzed in the current study. Auramine O, GeneXpert MTB/RIF assay, and Real-Time PCR tests have shown a detection rate of 32/90 (36%), 43/90 (47.8%), and 54/90 (60%) respectively (P ≤ 0.01). The sensitivity and specificity of AO were 38.1% and 95% respectively. RT PCR showed superior sensitivity followed by GeneXpert MTB/RIF assay, 70% and 58.6%. AO and molecular methods have shown a similarly low level of agreement with histopathology (Kappa value = 0.2). CONCLUSIONS: In a resource-limited setting, the selection of diagnostic tools needs careful attention. Putting the patients on anti-TB treatments based solely on histopathological findings may lead to undesired and adverse complications. Therefore, applying molecular and bacteriological detection methods along with histopathology, could help minimize inappropriate antimicrobial use.
Assuntos
Endométrio , Mycobacterium tuberculosis , Inclusão em Parafina , Sensibilidade e Especificidade , Tuberculose dos Linfonodos , Humanos , Feminino , Estudos Transversais , Estudos Retrospectivos , Adulto , Endométrio/microbiologia , Endométrio/patologia , Biópsia , Pessoa de Meia-Idade , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Tuberculose dos Linfonodos/diagnóstico , Tuberculose dos Linfonodos/microbiologia , Tuberculose dos Linfonodos/patologia , Adulto Jovem , Etiópia , Linfonodos/microbiologia , Linfonodos/patologia , Formaldeído , Técnicas de Diagnóstico Molecular/métodos , Tuberculose dos Genitais Femininos/diagnóstico , Tuberculose dos Genitais Femininos/patologia , Tuberculose dos Genitais Femininos/microbiologia , AdolescenteRESUMO
BACKGROUND: The diagnosis of peripheral isolated nodular lesions that are suspected as pulmonary tuberculosis (PTB) is challenging, which are not easily accessible via conventional bronchoscopy. This study evaluated the combined use of Xpert MTB/RIF assay and endobronchial ultrasonography with a guide sheath (EBUS-GS) for detecting MTB infection in peripheral lung bands, for early detection of PTB. METHODS: The clinical data of 232 patients with suspected peripheral nodular PTB who underwent EBUS-GS between June 2020 and October 2023 were retrospectively reviewed. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and area under the curve (AUC) of acid-fast bacilli smear, culture, Xpert MTB/RIF assay, and pathological examination were calculated. To assess diagnostic accuracy, the results of the four methods were directly compared with the final clinical diagnosis. RESULTS: In total, 146 and 86 patients were clinically diagnosed with peripheral nodular PTB and non-PTB, respectively. The sensitivity, specificity, PPV, NPV, and AUC values of combined Xpert MTB/RIF assay and EBUS-GS were 47.26%, 100.0%, 100.0%, 52.76%, and 0.74; those of acid-fast bacilli smear were 8.22%, 97.67%, 85.71%, 38.53%, and 0.53; those of culture were 31.51%, 100.0%, 100.0%, 46.24%, and 0.66; and those of pathological examination were 23.97%, 97.67%, 94.59%, 43.08%, and 0.61, respectively. CONCLUSION: The diagnostic accuracy of the combined Xpert MTB/RIF assay and EBUS-GS was significantly better than that of other conventional tests. Hence, this novel technique can be routinely applied for diagnosing and managing peripheral nodular PTB.
Assuntos
Mycobacterium tuberculosis , Sensibilidade e Especificidade , Tuberculose Pulmonar , Humanos , Tuberculose Pulmonar/diagnóstico , Tuberculose Pulmonar/microbiologia , Masculino , Feminino , Pessoa de Meia-Idade , Estudos Retrospectivos , Mycobacterium tuberculosis/isolamento & purificação , Mycobacterium tuberculosis/genética , Adulto , Idoso , Broncoscopia/métodos , Endossonografia/métodos , Técnicas de Diagnóstico Molecular/métodos , Valor Preditivo dos Testes , Pulmão/microbiologia , Pulmão/diagnóstico por imagem , Pulmão/patologiaRESUMO
Candida auris is a pathogenic yeast frequently exhibiting multidrug resistance and thus warrants special attention. The prompt detection and proper identification of this organism are needed to prevent its spread in healthcare facilities. The authors of this paper had previously developed LAMPAuris, a loop-mediated isothermal amplification assay, for the specific detection of C. auris. LAMPAuris is evaluated in this report for its ability to identify C. auris from five clades and to detect it from clinical specimens. A total of 103 skin swab samples were tested in comparison with a culture-based method and C. auris-specific SYBR green qPCR. The results show that the LAMPAuris assay had specificities ranging from 97 to 100% and sensitivities ranging from 66 to 86%. The lower sensitivity could be attributed to DNA degradation caused by the prolonged storage of the samples. In conclusion, LAMPAuris proved to be a rapid and reliable method for identifying C. auris and for detecting it in clinical specimens. Fresh specimens should ensure better yield and higher sensitivities.
Assuntos
Candida auris , Candidíase , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , Sensibilidade e Especificidade , Humanos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Candidíase/diagnóstico , Candidíase/microbiologia , Candida auris/genética , Candida auris/isolamento & purificação , Pele/microbiologia , Fatores de Tempo , Candida/isolamento & purificação , Candida/genética , Candida/classificaçãoRESUMO
Detection and identification of microorganisms are the first steps to guide susceptibility testing and enable clinicians to confirm diseases and guide therapy. The faster the pathogen identification is determined, the quicker the appropriate treatment can be started. In the clinical microbiology laboratory, multiple methodologies can be used to identify organisms, such as traditional biochemical testing or more recent methods like MALDI TOF MS and nucleic acid detection/identification assays. Each of these techniques has advantages and limitations, and clinical laboratories need to determine which methodology is best suited to their particular setting in terms of clinical needs, availability of technical expertise and cost. This article presents a concise review of the history, utilization, advantages and limitations of the main methods used for identifying microorganisms in microbiology laboratories.
Assuntos
Técnicas de Diagnóstico Molecular , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Humanos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/normas , Bactérias/genética , Bactérias/classificação , Bactérias/isolamento & purificação , Técnicas Microbiológicas/métodosRESUMO
Assuntos
Filtração , Mycobacterium tuberculosis , Técnicas de Amplificação de Ácido Nucleico , Tuberculose Pulmonar , Humanos , Tuberculose Pulmonar/diagnóstico , Tuberculose Pulmonar/microbiologia , Mycobacterium tuberculosis/isolamento & purificação , Feminino , Masculino , Adulto , Pessoa de Meia-Idade , Filtração/instrumentação , Escarro/microbiologia , Sensibilidade e Especificidade , Aerossóis , Máscaras , Técnicas de Diagnóstico Molecular , Idoso , Adulto Jovem , Polipropilenos , Gelatina , Manejo de Espécimes/instrumentação , Manejo de Espécimes/métodosRESUMO
Loop-mediated isothermal amplification (LAMP) is a cost-effective, rapid, and highly specific method of replicating nucleic acids. Adding multiple targets into a single LAMP assay to create a multiplex format is highly desirable for clinical applications but has been challenging due to a need to develop specific detection techniques and strict primer design criteria. This study describes the evaluation of a rapid triplex LAMP assay, MAST ISOPLEX®VTEC, for the simultaneous detection of Shiga toxin/verotoxin 1 and 2 (stx1/vt1 and stx2/vt2) genes in verotoxigenic Escherichia coli (E. coli) (VTEC) isolates with inhibition control (IC) synthetic DNA using a single fluorophore-oligonucleotide probe, MAST ISOPLEX®Probes, integrated into the primer set of each target. MAST ISOPLEX®Probes used in the MAST ISOPLEX®VTEC kit produce fluorescent signals as they integrate with reaction products specific to each target, allowing tracking of multiple amplifications in real time using a real-time analyzer. Initial validation on DNA extracts from fecal cultures and synthetic DNA sequences (gBlocks) showed that the MAST ISOPLEX®VTEC kit provides a method for sensitive simultaneous triplex detection in a single assay with a limit of detection (LOD) of less than 100 target copies/assay and 96% and 100% sensitivity and specificity, respectively.
Assuntos
Técnicas de Amplificação de Ácido Nucleico , Técnicas de Amplificação de Ácido Nucleico/métodos , Humanos , Sensibilidade e Especificidade , Toxina Shiga I/genética , Técnicas de Diagnóstico Molecular/métodos , Toxina Shiga II/genética , Limite de Detecção , Escherichia coli Shiga Toxigênica/genética , Escherichia coli Shiga Toxigênica/isolamento & purificação , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/diagnóstico , Kit de Reagentes para DiagnósticoRESUMO
Monitoring yellow fever in non-human primates (NHPs) is an early warning system for sylvatic yellow fever outbreaks, aiding in preventing human cases. However, current diagnostic tests for this disease, primarily relying on RT-qPCR, are complex and costly. Therefore, there is a critical need for simpler and more cost-effective methods to detect yellow fever virus (YFV) infection in NHPs, enabling early identification of viral circulation. In this study, an RT-LAMP assay for detecting YFV in NHP samples was developed and validated. Two sets of RT-LAMP primers targeting the YFV NS5 and E genes were designed and tested together with a third primer set to the NS1 locus using NHP tissue samples from Southern Brazil. The results were visualized by colorimetry and compared to the RT-qPCR test. Standardization and validation of the RT-LAMP assay demonstrated 100% sensitivity and specificity compared to RT-qPCR, with a detection limit of 12 PFU/mL. Additionally, the cross-reactivity test with other flaviviruses confirmed a specificity of 100%. Our newly developed RT-LAMP diagnostic test for YFV in NHP samples will significantly contribute to yellow fever monitoring efforts, providing a simpler and more accessible method for viral early detection. This advancement holds promise for enhancing surveillance and ultimately preventing the spread of yellow fever.
Assuntos
Técnicas de Amplificação de Ácido Nucleico , Sensibilidade e Especificidade , Febre Amarela , Vírus da Febre Amarela , Animais , Vírus da Febre Amarela/genética , Vírus da Febre Amarela/isolamento & purificação , Brasil/epidemiologia , Febre Amarela/diagnóstico , Febre Amarela/virologia , Febre Amarela/epidemiologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Técnicas de Diagnóstico Molecular/métodos , Primatas/virologiaRESUMO
A simple method has been developed for semi-quantitative analysis of the colorimetric output of loop-mediated isothermal amplification (LAMP) using a 3D-printed tube holder with a smartphone and notebook for the detection of Raillietina, which is the cause of Raillietiniasis affecting free-range chicken farming. In this method, a light is directed from a notebook screen to the LAMP products in the tube holder and the color absorption of the LAMP products is measured by using the appropriate smartphone application. It was found that the malachite green dye-coupled LAMP (MaG-LAMP) assay showed the highest sensitivity and specificity for detecting Raillietina without any cross-reaction with other related parasites and hosts. The limit of detection was 10 fg/µL of DNA. A total of 60 fecal samples were infectively confirmed by microscopic examination and the results of microscopy compared with those of MaG-LAMP and triplex PCR assays. Microscopy and MaG-LAMP based on the color absorption demonstrated high agreement in Raillietina detection with kappa = 1. Rapid, simple, cost-effective, and easy interpretation of colorimetric LAMP assays and their high sensitivity make them superior to PCR and morphological investigation, demonstrating the feasibility of this assay in point-of-care screening to support farm management and solve chicken health problems. Our study presents is an alternative diagnostic method using semi-quantitative analysis of colorimetric LAMP based on the differing solution color absorptions between positive and negative reactions for infectious disease diagnosis.
Assuntos
Galinhas , Colorimetria , Técnicas de Amplificação de Ácido Nucleico , Impressão Tridimensional , Smartphone , Colorimetria/métodos , Colorimetria/instrumentação , Técnicas de Amplificação de Ácido Nucleico/métodos , Animais , Limite de Detecção , Corantes de Rosanilina/química , Técnicas de Diagnóstico Molecular/instrumentação , Técnicas de Diagnóstico Molecular/métodos , Fezes/química , Fezes/microbiologiaRESUMO
Patients with neurogenic rosacea (NR) frequently demonstrate pronounced neurological manifestations, often unresponsive to conventional therapeutic approaches. A molecular-level understanding and diagnosis of this patient cohort could significantly guide clinical interventions. In this study, we amalgamated our sequencing data (n = 46) with a publicly accessible database (n = 38) to perform an unsupervised cluster analysis of the integrated dataset. The eighty-four rosacea patients were partitioned into two distinct clusters. Neurovascular biomarkers were found to be elevated in cluster 1 compared to cluster 2. Pathways in cluster 1 were predominantly involved in neurotransmitter synthesis, transmission, and functionality, whereas cluster 2 pathways were centered on inflammation-related processes. Differential gene expression analysis and WGCNA were employed to delineate the characteristic gene sets of the two clusters. Subsequently, a diagnostic model was constructed from the identified gene sets using linear regression methodologies. The model's C index, comprising genes PNPLA3, CUX2, PLIN2, and HMGCR, achieved a remarkable value of 0.9683, with an area under the curve (AUC) for the training cohort's nomogram of 0.9376. Clinical characteristics from our dataset (n = 46) were assessed by three seasoned dermatologists, forming the NR validation cohort (NR, n = 18; non-neurogenic rosacea, n = 28). Upon application of our model to NR diagnosis, the model's AUC value reached 0.9023. Finally, potential therapeutic candidates for both patient groups were predicted via the Connectivity Map. In summation, this study unveiled two clusters with unique molecular phenotypes within rosacea, leading to the development of a precise diagnostic model instrumental in NR diagnosis.
Assuntos
Aprendizado de Máquina , Rosácea , Transcriptoma , Humanos , Rosácea/genética , Rosácea/diagnóstico , Feminino , Masculino , Adulto , Pessoa de Meia-Idade , Perfilação da Expressão Gênica , Técnicas de Diagnóstico Molecular , Análise por Conglomerados , BiomarcadoresRESUMO
The indiscriminate use of antibiotics has resulted in a growing resistance to drugs in Pseudomonas aeruginosa. The identification of antibiotic resistance genes holds considerable clinical significance for prompt diagnosis. In this study, we established and optimized a Recombinase-Aided Amplification (RAA) assay to detect two genes associated with drug resistance, oprD and arr, in 101 clinically collected P. aeruginosa isolates. Through screening for the detection or absence of oprD and arr, the results showed that there were 52 Imipenem-resistant P. aeruginosa (IRPA) strains and 23 Rifampin-resistant P. aeruginosa (RRPA) strains. This method demonstrated excellent detection performance even when the sample concentration is 10 copies/µL at isothermal conditions and the results could be obtained within 20 minutes. The detection results were in accordance with the results of conventional PCR and Real-time PCR. The detection outcomes of the arr gene were consistently with the resistance spectrum. However, the antimicrobial susceptibility results revealed that 65 strains were resistant to imipenem, while 49 strains sensitive to imipenem with oprD were identified. This discrepancy could be attributed to genetic mutations. In summary, the RAA has higher sensitivity, shorter time, and lower-cost instrument requirements than traditional detection methods. In addition, to analyze the epidemiological characteristics of the aforementioned drug-resistant strains, we conducted Multilocus Sequence Typing (MLST), virulence gene, and antimicrobial susceptibility testing. MLST analysis showed a strong correlation between the sequence types ST-1639, ST-639, ST-184 and IRPA, while ST-261 was the main subtype of RRPA. It was observed that these drug-resistant strains all possess five or more virulence genes, among which exoS and exoU do not coexist, and they are all multidrug-resistant strains. The non-coexistence of exoU and exoS in P.aeruginosa is related to various factors including bacterial regulatory mechanisms and pathogenic mechanisms. This indicates that the relationship between the presence of virulence genes and the severity of patient infection is worthy of attention. In conclusion, we have developed a rapid and efficient RAA (Recombinase-Aided Amplification) detection method that offers significant advantages in terms of speed, simplicity, and cost-effectiveness (especially in time and equipment aspect). This novel approach is designed to meet the demands of clinical diagnostics.