This document discusses advances and limitations in the diagnosis of tuberculosis. It outlines how microscopy has been the standard diagnostic method for over a century but has limitations in sensitivity. Molecular diagnostics like the Xpert MTB/RIF test have improved sensitivity over microscopy but still have limitations for diagnosing TB in children and extrapulmonary cases. Current molecular tests and culture-based drug susceptibility testing can be challenging to implement widely due to infrastructure and technical limitations. Overall, diagnosis of TB, particularly for difficult cases, remains imperfect even with new technologies.
1. The document discusses various advances in clinical diagnosis, radiographic assessment, microbiological analysis, and characterization of the host response that can aid in the diagnosis of periodontitis.
2. New diagnostic tools discussed include methods to analyze gingival bleeding, temperature, probing depth, digital radiography, subtraction radiography, microbiological assays, and markers in gingival crevicular fluid and saliva.
3. These diagnostic advances provide more sensitive, specific, and quantitative methods for detecting periodontal disease activity and microbial pathogens compared to traditional clinical and radiographic exams.
Traditional methods of diagnosing infectious diseases like isolation, serology, and direct detection have limitations that influence their clinical utility. Molecular methods using nucleic acid detection and characterization have advantages like faster results, increased sensitivity and specificity, and ability to adapt to new instrumentation. Introducing molecular diagnostic testing requires evaluating needs, identifying suitable changes, developing protocols, providing resources, educating staff, and ensuring continuous quality improvement.
This document discusses using a bioinformatic approach for DNA signature-based diagnostics. It describes three main molecular diagnostic methods - biochemical, antibody, and DNA-based. DNA-based methods such as PCR and DNA probes are highlighted as being highly sensitive and specific. The use of DNA barcoding to identify species via unique DNA sequences is discussed. Developing DNA signatures for pathogens like Ug99 stem rust fungus is presented as an important application of bioinformatics.
Vitreous biopsy can aid in diagnosing uveitis when clinical presentation is non-specific or atypical. A vitreous specimen can be obtained via vitreous aspiration or pars plana vitrectomy, with the latter preferred as it removes more vitreous and reduces risks. The vitreous can be analyzed through microbiological and PCR analysis, cytopathological analysis, flow cytometry, immunohistochemistry, and determining antibody and cytokine levels to diagnose infectious agents, malignancies, or inflammatory conditions causing uveitis.
Maintaining quality in molecular Diagnostics final layoutMohamed Elsawy
The document discusses key components of establishing and maintaining a quality system for molecular genetic testing laboratories. It outlines the need for quality assurance in molecular diagnostics given the increasing applications and complexity. The major components covered include planning, validation of test performance, pre-examination, examination and post-examination processes, and implementing quality system essentials with a focus on information management and assessment. The goal is to provide guidance for laboratories to deliver accurate and reliable genetic testing services.
1. The document discusses various laboratory diagnostic techniques for infectious diseases including microscopy, staining, culture-based, and molecular methods.
2. Key techniques covered include wet mount microscopy, Gram staining, acid-fast staining, immunofluorescence staining, and molecular methods like nucleic acid hybridization and PCR.
3. The document emphasizes the importance of proper specimen collection for maximizing recovery of pathogens and minimizing contamination. Adequate specimen quantity and avoidance of antimicrobial treatment prior to collection are important.
1. The document discusses various advances in clinical diagnosis, radiographic assessment, microbiological analysis, and characterization of the host response that can aid in the diagnosis of periodontitis.
2. New diagnostic tools discussed include methods to analyze gingival bleeding, temperature, probing depth, digital radiography, subtraction radiography, microbiological assays, and markers in gingival crevicular fluid and saliva.
3. These diagnostic advances provide more sensitive, specific, and quantitative methods for detecting periodontal disease activity and microbial pathogens compared to traditional clinical and radiographic exams.
Traditional methods of diagnosing infectious diseases like isolation, serology, and direct detection have limitations that influence their clinical utility. Molecular methods using nucleic acid detection and characterization have advantages like faster results, increased sensitivity and specificity, and ability to adapt to new instrumentation. Introducing molecular diagnostic testing requires evaluating needs, identifying suitable changes, developing protocols, providing resources, educating staff, and ensuring continuous quality improvement.
This document discusses using a bioinformatic approach for DNA signature-based diagnostics. It describes three main molecular diagnostic methods - biochemical, antibody, and DNA-based. DNA-based methods such as PCR and DNA probes are highlighted as being highly sensitive and specific. The use of DNA barcoding to identify species via unique DNA sequences is discussed. Developing DNA signatures for pathogens like Ug99 stem rust fungus is presented as an important application of bioinformatics.
Vitreous biopsy can aid in diagnosing uveitis when clinical presentation is non-specific or atypical. A vitreous specimen can be obtained via vitreous aspiration or pars plana vitrectomy, with the latter preferred as it removes more vitreous and reduces risks. The vitreous can be analyzed through microbiological and PCR analysis, cytopathological analysis, flow cytometry, immunohistochemistry, and determining antibody and cytokine levels to diagnose infectious agents, malignancies, or inflammatory conditions causing uveitis.
Maintaining quality in molecular Diagnostics final layoutMohamed Elsawy
The document discusses key components of establishing and maintaining a quality system for molecular genetic testing laboratories. It outlines the need for quality assurance in molecular diagnostics given the increasing applications and complexity. The major components covered include planning, validation of test performance, pre-examination, examination and post-examination processes, and implementing quality system essentials with a focus on information management and assessment. The goal is to provide guidance for laboratories to deliver accurate and reliable genetic testing services.
1. The document discusses various laboratory diagnostic techniques for infectious diseases including microscopy, staining, culture-based, and molecular methods.
2. Key techniques covered include wet mount microscopy, Gram staining, acid-fast staining, immunofluorescence staining, and molecular methods like nucleic acid hybridization and PCR.
3. The document emphasizes the importance of proper specimen collection for maximizing recovery of pathogens and minimizing contamination. Adequate specimen quantity and avoidance of antimicrobial treatment prior to collection are important.
For over 10 decades, agents of infectious diseases have been identified through their phenotype directly in specimen and after a growth in culture.
Today, we are in a molecular era, there is an opportunity to detect organisms more rapidly and accurately based on their genetic signatures.
Biomedical science research discovery offers a growing numbers of a nucleic acid amplification tests (NAATS) among which is polymerase chain reaction (PCR) for detection and identification of bacterial, parasitic, fungi and viral pathogens.
These assays improve patient care, reduce antibiotic usage, enhance test utilization and increase laboratory and hospital efficiency.
In this seminar, we will explore the clinical usefulness and potential of both conventional and real-time PCR assays in Clinical Microbiology.
This document discusses new technologies for the diagnosis of tuberculosis. It describes how microscopy using light emitting diodes has advanced diagnosis by providing a simple, robust method. Molecular tests like PCR and line probe assays can rapidly detect TB and drug resistance from samples, but are more expensive and complex. The WHO endorses tests like Xpert MTB/RIF that can simultaneously detect TB and rifampicin resistance in a few hours. While promising, molecular methods still have limitations around cost, availability, and cannot replace clinical assessment.
Molecular methods are used to detect viruses that cannot be cultured in vitro by detecting their nucleic acids. Techniques like PCR and nucleic acid-based amplification are used to detect viral genomes. Other techniques include dot blot, Southern blot, Northern blot, and in situ hybridization. Newer techniques include PCR, ligase chain reaction, branched DNA, and nucleic acid-based amplification. Nucleic acid probes are short labeled DNA strands that anneal to complementary target DNA strands through base pairing. Southern blotting separates DNA fragments by size and transfers them to a filter for detection. Northern blotting separates mRNA and transfers it to a filter. Western blotting separates proteins by size and transfers them to a filter for immunodetection.
Pcr technology and its importance in covid 19 pandemicAnupam Maity
Since the discovery of the PCR technology, its application in the various fields is increased gradually. Based on to this principle, many variations of the PCR have been established. Year by year, it is upgraded very much. It is established as a most common and accurate technique for the detection of the various diseases in the field of medicine. Now it is a ‘Gold standard’ for the detection of covid-19 also, which is much needed to contain the spread of the virus. Though various detection techniques are there for detection, but real time RT-PCR (variation of PCR) is most reliable. Viral detection is based on a simple principle of nucleic acid (viral) amplification. Various manufacturing companies are manufacturing the PCR instrument. Though the accuracy of the instruments are slightly differ to each other.
The document describes a biosensor called BREATHSAFE® for detecting H1N1 infections. It uses anti-hemagglutinin and anti-neuraminidase protein fragments immobilized on a glass slide as bioreceptors to detect the H1N1 viral proteins hemagglutinin and neuraminidase in respiratory secretions. When the viral proteins bind to the bioreceptors, secondary antibodies tagged with fluorophores bind, and fluorescence is excited and detected with a CCD camera, allowing for diagnosis. The sensor hardware will be inexpensive to manufacture and the disposable membranes will generate continuous revenue.
Addressing the Pre-PCR Analytical Variability of FFPE SamplesCandy Smellie
Despite technical advances, assessing the accuracy of pre-PCR steps, which include DNA extraction from formalin-fixed paraffin-embedded (FFPE) tissues, DNA quantitation and DNA quality control, remain a key challenge in external quality assurance.
In the webinar we will discuss the latest results from recent studies and look at ways that the accuracy of pre-PCR workflows can be improved.
Flow Cytometry Training talks - part 1
This forms the first session of the Garvan Flow , Flow Cytometry Training course. this is a 1 1/2 day training course aimed at giving new and experienced researchers a better understanding of cytometry in medical and biological research.
This document discusses molecular diagnosis of tuberculosis through nucleic acid amplification (NAA) methods like polymerase chain reaction (PCR) and transcription mediated amplification (TMA). NAA allows direct detection of mycobacterial DNA or RNA from specimens before culture results. PCR amplifies a specific DNA sequence using primers and DNA polymerase, resulting in exponential multiplication of the target sequence. TMA is an isothermal method that uses RNA transcription and DNA synthesis to amplify nucleic acids. Commercial NAA tests for tuberculosis detection include Amplicor and Enhanced Mycobacterium Tuberculosis Direct test, which have reported sensitivities of 79.4-95.2% and specificities of 98.8-100% compared to culture.
Newer methods in diagnosis of tuberculosis in childrenDr Naveen kumar
This document discusses various diagnostic tests for tuberculosis in children. It notes that bacteriological diagnosis is difficult in children due to their inability to produce sputum and the often extra-pulmonary and paucibacillary nature of the disease in children. It reviews sputum induction methods, as well as microscopy, culture, nucleic acid amplification tests, and interferon-gamma release assays as diagnostic tools and their limitations in pediatric populations. It emphasizes the need for improved diagnostics that are feasible and effective for use in children.
Flyer of our new product Cleanpid® Easy Purification Kit for detecting Legionella. Cleanpid is for purification the sample before doing the PCR. After using CLEANPID you can collect the Legionella cells and with freeze/thaw cycles you can obtain the DNA and with PCR, the customer could say if there is or not Legionella presence.
Legionellae DNA extraction is particularly problematic when different types of microorganisms may be present.
Cleanpid® Easy Purification kit is the simple method for the separation and purification of Legionella in water samples. The method is based on Legionella binding magnetic beads for capturing cells (immunomagnetic separation).
No columns or centrifugations are necessary.
A summary of the advantages of Cleanpid is:
Quick and Easy-To-Use
The kit only requires a magnetic rack (we provide it)
Improves the sensitivity and reproducibility of qPCR
Reduction of false positive rate for PCR:
o Detects only legionella viable cells (not exogenous DNA, not other microbiota...)
Reduction of false negative rate for PCR:
o Obtains a larger amount of DNA from the water samples (avoid dilution of the DNA samples for eliminating inhibitors)
The purified organism is additionally suitable for DNA extraction by simply freeze/thaw cycles (simply and cost-efficient lysis)
http://www.biotica.es/en/Products/Cleanpid%20Easy%20Purification%20kit%20for%20Legionella
Back to Basics: Fundamental Concepts and Special Considerations in gDNA Isola...QIAGEN
This document provides an overview of genomic DNA (gDNA) isolation. It discusses key considerations for gDNA isolation including sample stabilization, disruption, and storage. Common isolation technologies like silica membrane and magnetic bead kits are described. The document reviews measuring gDNA concentration and quality via UV spectroscopy and gel electrophoresis. It also provides guidance on selecting appropriate QIAGEN gDNA isolation kits based on sample type.
The document discusses nucleic acid amplification techniques, specifically polymerase chain reaction (PCR). It explains that PCR involves denaturing DNA, annealing primers, and extending DNA strands through repeated heating and cooling cycles to exponentially amplify a specific DNA target. Key aspects of PCR covered include its invention by Kary Mullis, use of Taq polymerase, and applications such as disease diagnosis using techniques like real-time PCR.
The document announces the Cell Based Assays Conference to be held on June 26-27, 2012 in London. The conference will feature keynote speakers who will provide updates on cell based assays and their importance in pharmaceutical research. Sessions over the two days will cover topics like high-content screening, 3D cell models, GPCR signaling pathways, and applications of cell based assays in drug discovery. The conference is aimed at researchers in fields related to screening, pharmacology, and assay development.
Optical forward-scattering for identification of bacteria within microcoloniesPierre R. Marcoux
3rd International Conference on Bio-Sensing Technology 2013
This work won the Award for Outstanding Oral Presentation at the 3rd International Conference on Bio-Sensing Technology 2013.
Pierre R. Marcoux, Mathieu Dupoy
Department of Technology for Biology and Healthcare, CEA-LETI MINATEC, 17 avenue des Martyrs, 38054 Grenoble, France.
Antoine Cuer, Joe-Loïc Kodja, Arthur Lefebvre, Florian Licari, Robin Louvet, Anil Narassiguin
These authors contributed equally to this work.
Ecole Centrale de Lyon, 36 avenue Guy de Collongue, 69134 Ecully, France.
Frédéric Mallard
bioMérieux SA, Innovation & Systems / Technology Research / Sample Prep & Processing Lab, 5 rue des Berges, 38000 Grenoble, France.
The development of methods for the rapid identification of pathogenic bacteria is a major step towards accelerated clinical diagnosis of infectious diseases and efficient food and water safety control. Methods for identification of bacterial colonies on gelified nutrient broth have the potential to bring an attractive solution, combining simple optical instrumentation, no need for sample preparation or labelling, in a non-destructive process. Here, we studied the possibility of discriminating different bacterial species at a very early stage of growth (6 hours of incubation at 37°C), on thin layers of agar media (1mm of Tryptic Soy Agar), using light forward-scattering and learning algorithms (Bayes Network, Continuous Naive Bayes, Sequential Minimal Optimisation). A first database of more than 1000 scatterograms acquired on seven Gram-negative strains yielded a recognition rate of nearly 80%, after only 6 hours of incubation. We investigated also the prospect of identifying different strains from a same species through forward scattering. We discriminated thus four strains of Escherichia coli with a recognition rate reaching 82%. Finally, we show the discrimination of two species of coagulase-negative Staphylococci (S. haemolyticus and S. cohnii), on a commercial selective pre-poured medium used in clinical diagnosis (ChromID MRSA, bioMérieux), without opening lids during the scatterogram acquisition. This shows the potential of this method – non-invasive, preventing cross-contaminations and requiring minimal dish handling – to provide early clinically-relevant information in the context of fully automated microbiology labs.
Sensitivity assay of polymerase chain reaction for detection of canine adeno ...Alexander Decker
This study aimed to determine the minimum detection limit of canine adeno virus (CAV) in clinical samples using polymerase chain reaction (PCR). PCR was performed on 40 blood samples from different regions of India using reported primers for CAV detection. Positive PCR samples were serially diluted and used as templates to determine the lowest concentration detectable by PCR. The minimum detection limit was found to be a 1:1000 dilution, equivalent to 0.20 ng of DNA per microliter. The study demonstrated PCR is a sensitive method for early detection of CAV infection in clinical samples.
From Sample to Result – Workflow Solutions for Genotyping and Pathogen DetectionQIAGEN
This document discusses pathogen detection methods using high-resolution melting (HRM) analysis and multiplex real-time PCR. It begins with an overview of challenges in pathogen detection workflows, including issues with sample collection/extraction and PCR inhibition. Methods for DNA extraction and real-time PCR detection assays are presented, including QIAGEN solutions. Applications discussed include developing assays for cannabis contamination detection and bovine respiratory/GI pathogen detection. Validation strategies for new assays include testing sensitivity/specificity using synthetic templates.
2013 Merck Millipore Best Practices in Nucleic Acid Removal from Vaccine Proc...Frank Appel
The document discusses various methods for removing nucleic acids like DNA from vaccine manufacturing processes. It describes how density gradient centrifugation, depth filtration using Millistak+ filters, and nuclease treatment with Benzonase can each reduce DNA levels. It also outlines methods for removing residual Benzonase like chromatography using Fractogel resins and tangential flow filtration with Pellicon cassettes. The document provides an overview of regulatory guidelines for DNA limits and analytical techniques for detecting residual Benzonase.
DETECTION OF HELMINTHS BY USING RADIOACTIVE.SUMBUL AWAN
The document discusses various molecular techniques used to diagnose helminth parasites, including Southern blotting to detect target DNA using labeled probes, polymerase chain reaction (PCR) to amplify parasite DNA, and radioallergosorbent testing (RAST) for serology-based detection. It provides details on extracting and purifying parasite RNA and DNA, performing Southern blotting and PCR, and the basic principles and steps involved in each technique.
This document provides information about a plant pathogen detection kit from Norgen Biotek Corp. that allows for the isolation and detection of fungal pathogens from plant samples using PCR. The kit contains components for isolating DNA from plant tissues using spin column chromatography. It also contains master mixes for amplifying fungal DNA, as well as controls. The kit is a ready-to-use system for detecting pathogens like Aspergillus niger, Botrytis cinerea, Cladosporium cladosporioides, Penicillium sp., and others from plant samples in under 3 hours.
This document summarizes recent developments in tuberculosis (TB) diagnostics. It discusses the need for point-of-care tests and tests that are universally applicable to all patients to improve TB control through quick identification. While new goals aim to increase diagnosis of active TB, many cases remain undiagnosed due to problems with sample collection, transport, and record keeping. Special groups like HIV-infected individuals, women, and children present additional challenges to diagnosis. The document reviews various diagnostic methods and their effectiveness, including microscopy, culture-based tests, and new rapid tests. It highlights priorities and policies from the WHO to improve universal access to quality TB diagnosis.
Newer Diagnostic Modality in Tuberculosis.pptxBIMALESHYADAV2
The document discusses various conventional and recent advanced methods for tuberculosis diagnosis. Conventional methods discussed include microscopy using Ziehl-Neelsen staining and auramine rhodamine staining, culture on media like Lowenstein-Jensen and Middlebrook, and measurement of ADA levels. Recent advanced methods discussed include LED fluorescence microscopy, IGRA for infection detection, radiometric BACTEC system and MGIT system for faster culture, FASTPlaque TB test, Xpert MTB/RIF and Ultra assays using PCR, Truenat assays, automated NAATs, and line probe assays for molecular diagnosis and detection of drug resistance.
For over 10 decades, agents of infectious diseases have been identified through their phenotype directly in specimen and after a growth in culture.
Today, we are in a molecular era, there is an opportunity to detect organisms more rapidly and accurately based on their genetic signatures.
Biomedical science research discovery offers a growing numbers of a nucleic acid amplification tests (NAATS) among which is polymerase chain reaction (PCR) for detection and identification of bacterial, parasitic, fungi and viral pathogens.
These assays improve patient care, reduce antibiotic usage, enhance test utilization and increase laboratory and hospital efficiency.
In this seminar, we will explore the clinical usefulness and potential of both conventional and real-time PCR assays in Clinical Microbiology.
This document discusses new technologies for the diagnosis of tuberculosis. It describes how microscopy using light emitting diodes has advanced diagnosis by providing a simple, robust method. Molecular tests like PCR and line probe assays can rapidly detect TB and drug resistance from samples, but are more expensive and complex. The WHO endorses tests like Xpert MTB/RIF that can simultaneously detect TB and rifampicin resistance in a few hours. While promising, molecular methods still have limitations around cost, availability, and cannot replace clinical assessment.
Molecular methods are used to detect viruses that cannot be cultured in vitro by detecting their nucleic acids. Techniques like PCR and nucleic acid-based amplification are used to detect viral genomes. Other techniques include dot blot, Southern blot, Northern blot, and in situ hybridization. Newer techniques include PCR, ligase chain reaction, branched DNA, and nucleic acid-based amplification. Nucleic acid probes are short labeled DNA strands that anneal to complementary target DNA strands through base pairing. Southern blotting separates DNA fragments by size and transfers them to a filter for detection. Northern blotting separates mRNA and transfers it to a filter. Western blotting separates proteins by size and transfers them to a filter for immunodetection.
Pcr technology and its importance in covid 19 pandemicAnupam Maity
Since the discovery of the PCR technology, its application in the various fields is increased gradually. Based on to this principle, many variations of the PCR have been established. Year by year, it is upgraded very much. It is established as a most common and accurate technique for the detection of the various diseases in the field of medicine. Now it is a ‘Gold standard’ for the detection of covid-19 also, which is much needed to contain the spread of the virus. Though various detection techniques are there for detection, but real time RT-PCR (variation of PCR) is most reliable. Viral detection is based on a simple principle of nucleic acid (viral) amplification. Various manufacturing companies are manufacturing the PCR instrument. Though the accuracy of the instruments are slightly differ to each other.
The document describes a biosensor called BREATHSAFE® for detecting H1N1 infections. It uses anti-hemagglutinin and anti-neuraminidase protein fragments immobilized on a glass slide as bioreceptors to detect the H1N1 viral proteins hemagglutinin and neuraminidase in respiratory secretions. When the viral proteins bind to the bioreceptors, secondary antibodies tagged with fluorophores bind, and fluorescence is excited and detected with a CCD camera, allowing for diagnosis. The sensor hardware will be inexpensive to manufacture and the disposable membranes will generate continuous revenue.
Addressing the Pre-PCR Analytical Variability of FFPE SamplesCandy Smellie
Despite technical advances, assessing the accuracy of pre-PCR steps, which include DNA extraction from formalin-fixed paraffin-embedded (FFPE) tissues, DNA quantitation and DNA quality control, remain a key challenge in external quality assurance.
In the webinar we will discuss the latest results from recent studies and look at ways that the accuracy of pre-PCR workflows can be improved.
Flow Cytometry Training talks - part 1
This forms the first session of the Garvan Flow , Flow Cytometry Training course. this is a 1 1/2 day training course aimed at giving new and experienced researchers a better understanding of cytometry in medical and biological research.
This document discusses molecular diagnosis of tuberculosis through nucleic acid amplification (NAA) methods like polymerase chain reaction (PCR) and transcription mediated amplification (TMA). NAA allows direct detection of mycobacterial DNA or RNA from specimens before culture results. PCR amplifies a specific DNA sequence using primers and DNA polymerase, resulting in exponential multiplication of the target sequence. TMA is an isothermal method that uses RNA transcription and DNA synthesis to amplify nucleic acids. Commercial NAA tests for tuberculosis detection include Amplicor and Enhanced Mycobacterium Tuberculosis Direct test, which have reported sensitivities of 79.4-95.2% and specificities of 98.8-100% compared to culture.
Newer methods in diagnosis of tuberculosis in childrenDr Naveen kumar
This document discusses various diagnostic tests for tuberculosis in children. It notes that bacteriological diagnosis is difficult in children due to their inability to produce sputum and the often extra-pulmonary and paucibacillary nature of the disease in children. It reviews sputum induction methods, as well as microscopy, culture, nucleic acid amplification tests, and interferon-gamma release assays as diagnostic tools and their limitations in pediatric populations. It emphasizes the need for improved diagnostics that are feasible and effective for use in children.
Flyer of our new product Cleanpid® Easy Purification Kit for detecting Legionella. Cleanpid is for purification the sample before doing the PCR. After using CLEANPID you can collect the Legionella cells and with freeze/thaw cycles you can obtain the DNA and with PCR, the customer could say if there is or not Legionella presence.
Legionellae DNA extraction is particularly problematic when different types of microorganisms may be present.
Cleanpid® Easy Purification kit is the simple method for the separation and purification of Legionella in water samples. The method is based on Legionella binding magnetic beads for capturing cells (immunomagnetic separation).
No columns or centrifugations are necessary.
A summary of the advantages of Cleanpid is:
Quick and Easy-To-Use
The kit only requires a magnetic rack (we provide it)
Improves the sensitivity and reproducibility of qPCR
Reduction of false positive rate for PCR:
o Detects only legionella viable cells (not exogenous DNA, not other microbiota...)
Reduction of false negative rate for PCR:
o Obtains a larger amount of DNA from the water samples (avoid dilution of the DNA samples for eliminating inhibitors)
The purified organism is additionally suitable for DNA extraction by simply freeze/thaw cycles (simply and cost-efficient lysis)
http://www.biotica.es/en/Products/Cleanpid%20Easy%20Purification%20kit%20for%20Legionella
Back to Basics: Fundamental Concepts and Special Considerations in gDNA Isola...QIAGEN
This document provides an overview of genomic DNA (gDNA) isolation. It discusses key considerations for gDNA isolation including sample stabilization, disruption, and storage. Common isolation technologies like silica membrane and magnetic bead kits are described. The document reviews measuring gDNA concentration and quality via UV spectroscopy and gel electrophoresis. It also provides guidance on selecting appropriate QIAGEN gDNA isolation kits based on sample type.
The document discusses nucleic acid amplification techniques, specifically polymerase chain reaction (PCR). It explains that PCR involves denaturing DNA, annealing primers, and extending DNA strands through repeated heating and cooling cycles to exponentially amplify a specific DNA target. Key aspects of PCR covered include its invention by Kary Mullis, use of Taq polymerase, and applications such as disease diagnosis using techniques like real-time PCR.
The document announces the Cell Based Assays Conference to be held on June 26-27, 2012 in London. The conference will feature keynote speakers who will provide updates on cell based assays and their importance in pharmaceutical research. Sessions over the two days will cover topics like high-content screening, 3D cell models, GPCR signaling pathways, and applications of cell based assays in drug discovery. The conference is aimed at researchers in fields related to screening, pharmacology, and assay development.
Optical forward-scattering for identification of bacteria within microcoloniesPierre R. Marcoux
3rd International Conference on Bio-Sensing Technology 2013
This work won the Award for Outstanding Oral Presentation at the 3rd International Conference on Bio-Sensing Technology 2013.
Pierre R. Marcoux, Mathieu Dupoy
Department of Technology for Biology and Healthcare, CEA-LETI MINATEC, 17 avenue des Martyrs, 38054 Grenoble, France.
Antoine Cuer, Joe-Loïc Kodja, Arthur Lefebvre, Florian Licari, Robin Louvet, Anil Narassiguin
These authors contributed equally to this work.
Ecole Centrale de Lyon, 36 avenue Guy de Collongue, 69134 Ecully, France.
Frédéric Mallard
bioMérieux SA, Innovation & Systems / Technology Research / Sample Prep & Processing Lab, 5 rue des Berges, 38000 Grenoble, France.
The development of methods for the rapid identification of pathogenic bacteria is a major step towards accelerated clinical diagnosis of infectious diseases and efficient food and water safety control. Methods for identification of bacterial colonies on gelified nutrient broth have the potential to bring an attractive solution, combining simple optical instrumentation, no need for sample preparation or labelling, in a non-destructive process. Here, we studied the possibility of discriminating different bacterial species at a very early stage of growth (6 hours of incubation at 37°C), on thin layers of agar media (1mm of Tryptic Soy Agar), using light forward-scattering and learning algorithms (Bayes Network, Continuous Naive Bayes, Sequential Minimal Optimisation). A first database of more than 1000 scatterograms acquired on seven Gram-negative strains yielded a recognition rate of nearly 80%, after only 6 hours of incubation. We investigated also the prospect of identifying different strains from a same species through forward scattering. We discriminated thus four strains of Escherichia coli with a recognition rate reaching 82%. Finally, we show the discrimination of two species of coagulase-negative Staphylococci (S. haemolyticus and S. cohnii), on a commercial selective pre-poured medium used in clinical diagnosis (ChromID MRSA, bioMérieux), without opening lids during the scatterogram acquisition. This shows the potential of this method – non-invasive, preventing cross-contaminations and requiring minimal dish handling – to provide early clinically-relevant information in the context of fully automated microbiology labs.
Sensitivity assay of polymerase chain reaction for detection of canine adeno ...Alexander Decker
This study aimed to determine the minimum detection limit of canine adeno virus (CAV) in clinical samples using polymerase chain reaction (PCR). PCR was performed on 40 blood samples from different regions of India using reported primers for CAV detection. Positive PCR samples were serially diluted and used as templates to determine the lowest concentration detectable by PCR. The minimum detection limit was found to be a 1:1000 dilution, equivalent to 0.20 ng of DNA per microliter. The study demonstrated PCR is a sensitive method for early detection of CAV infection in clinical samples.
From Sample to Result – Workflow Solutions for Genotyping and Pathogen DetectionQIAGEN
This document discusses pathogen detection methods using high-resolution melting (HRM) analysis and multiplex real-time PCR. It begins with an overview of challenges in pathogen detection workflows, including issues with sample collection/extraction and PCR inhibition. Methods for DNA extraction and real-time PCR detection assays are presented, including QIAGEN solutions. Applications discussed include developing assays for cannabis contamination detection and bovine respiratory/GI pathogen detection. Validation strategies for new assays include testing sensitivity/specificity using synthetic templates.
2013 Merck Millipore Best Practices in Nucleic Acid Removal from Vaccine Proc...Frank Appel
The document discusses various methods for removing nucleic acids like DNA from vaccine manufacturing processes. It describes how density gradient centrifugation, depth filtration using Millistak+ filters, and nuclease treatment with Benzonase can each reduce DNA levels. It also outlines methods for removing residual Benzonase like chromatography using Fractogel resins and tangential flow filtration with Pellicon cassettes. The document provides an overview of regulatory guidelines for DNA limits and analytical techniques for detecting residual Benzonase.
DETECTION OF HELMINTHS BY USING RADIOACTIVE.SUMBUL AWAN
The document discusses various molecular techniques used to diagnose helminth parasites, including Southern blotting to detect target DNA using labeled probes, polymerase chain reaction (PCR) to amplify parasite DNA, and radioallergosorbent testing (RAST) for serology-based detection. It provides details on extracting and purifying parasite RNA and DNA, performing Southern blotting and PCR, and the basic principles and steps involved in each technique.
This document provides information about a plant pathogen detection kit from Norgen Biotek Corp. that allows for the isolation and detection of fungal pathogens from plant samples using PCR. The kit contains components for isolating DNA from plant tissues using spin column chromatography. It also contains master mixes for amplifying fungal DNA, as well as controls. The kit is a ready-to-use system for detecting pathogens like Aspergillus niger, Botrytis cinerea, Cladosporium cladosporioides, Penicillium sp., and others from plant samples in under 3 hours.
This document summarizes recent developments in tuberculosis (TB) diagnostics. It discusses the need for point-of-care tests and tests that are universally applicable to all patients to improve TB control through quick identification. While new goals aim to increase diagnosis of active TB, many cases remain undiagnosed due to problems with sample collection, transport, and record keeping. Special groups like HIV-infected individuals, women, and children present additional challenges to diagnosis. The document reviews various diagnostic methods and their effectiveness, including microscopy, culture-based tests, and new rapid tests. It highlights priorities and policies from the WHO to improve universal access to quality TB diagnosis.
Newer Diagnostic Modality in Tuberculosis.pptxBIMALESHYADAV2
The document discusses various conventional and recent advanced methods for tuberculosis diagnosis. Conventional methods discussed include microscopy using Ziehl-Neelsen staining and auramine rhodamine staining, culture on media like Lowenstein-Jensen and Middlebrook, and measurement of ADA levels. Recent advanced methods discussed include LED fluorescence microscopy, IGRA for infection detection, radiometric BACTEC system and MGIT system for faster culture, FASTPlaque TB test, Xpert MTB/RIF and Ultra assays using PCR, Truenat assays, automated NAATs, and line probe assays for molecular diagnosis and detection of drug resistance.
1) Tuberculosis (TB) is commonly diagnosed through direct microscopy, culture, immunodiagnostic tests, molecular tests, and histopathology using samples from sputum, BAL, CSF, tissues, and other body fluids.
2) Direct microscopy has low sensitivity but is quick, while culture has higher sensitivity and allows drug susceptibility testing but takes 1-2 weeks for results. Newer liquid culture systems can provide results in only a few days.
3) Molecular tests like PCR and interferon-gamma release assays provide rapid results within hours and are also used for diagnosis, but many have high costs.
Recent advances in Tuberculosis diagnosisNishantTawari
This document discusses recent advances in tuberculosis diagnosis. It notes that in 2017 there were over 10 million new TB cases globally, including 2.8 million in India. New diagnostic techniques have been developed to improve detection of both drug-sensitive and drug-resistant TB. These include nucleic acid amplification tests like Xpert MTB/RIF, which can detect TB and rifampin resistance in under 3 hours. Other techniques discussed are line probe assays, automated liquid culture systems, and urine lipoarabinomannan tests. The document examines the advantages and limitations of various methods for directly and indirectly detecting active TB.
Molecular biology test for Tuberculosis SomaMajumdar6
This document discusses molecular biology tests for tuberculosis (TB), specifically the Xpert MTB/RIF assay. The assay uses polymerase chain reaction (PCR) to amplify TB bacterial DNA and detect resistance to the drug rifampicin. It has advantages of being rapid, requiring minimal training, and having a closed cartridge system that minimizes contamination. The assay directly detects TB and rifampicin resistance from sputum samples in under two hours.
This document discusses molecular assays for tuberculosis diagnosis and drug resistance testing. It begins with an introduction to the global burden of TB and importance of accurate diagnosis. It then describes various microbiological diagnostic methods including smear microscopy, culture techniques, nucleic acid amplification tests (NAATs), and molecular methods like whole genome sequencing (WGS). Specific NAATs discussed in detail are Xpert MTB/RIF and targeted sequencing approaches. Centralized high-throughput diagnostic tests are also mentioned. Overall the document provides an overview of established and emerging molecular methods that can improve TB diagnosis and detection of drug resistance.
Salivary antibodies as markers of recent acute cryptosporidiosis in children ...Michelo Simuyandi
This document outlines a study to evaluate salivary antibodies as markers for recent Cryptosporidium infection in children under 5 years old in Zambia. It proposes a case-control study comparing saliva samples to standard diagnostic methods using serum and stool samples. The study aims to assess the sensitivity and specificity of salivary antibodies for detecting recent Cryptosporidium infection up to 6 months prior. It also proposes a retrospective study using existing saliva samples from a cohort study to examine antibody profiles over time and associations with other factors.
This document provides information on the laboratory diagnosis of tuberculosis. It discusses the classification of mycobacteria, specimen collection, and the various diagnostic methods used which include smear microscopy, culture, and molecular tests. Smear microscopy has limitations but is used in low-resource settings due to its low cost. Culture is the gold standard but is complex and requires biosafety. Liquid culture systems allow for faster results than solid media. Drug sensitivity testing determines resistance and is important for treatment. Molecular tests like line probe assays and GeneXpert can rapidly detect M. tuberculosis and resistance, with GeneXpert suitable for both pulmonary and extrapulmonary samples. Microcare Laboratory in Surat, India provides various tuberculosis diagnostic services and
This document provides information on the laboratory diagnosis of tuberculosis. It discusses the classification of mycobacteria, specimen collection, and the various diagnostic methods used which include smear microscopy, culture, and molecular tests. Smear microscopy has limitations but is widely used due to its low cost. Culture is the gold standard but is more complex and requires biosafety. Liquid culture systems allow for faster results than solid media. Drug sensitivity testing determines resistance and is important for treatment. Molecular tests like line probe assays and GeneXpert can rapidly detect M. tuberculosis and resistance, with GeneXpert suitable to test pulmonary and some extrapulmonary samples directly. The document concludes with details about Microcare Laboratory which provides accredited tuberculosis diagnostic services
Laser scanning cytometry and liquid based cytologyanaonline
Liquid based cytology (LBC) has been introduced to improve cervical screening by limiting some of the limitations of conventional pap smears. With LBC, a sample is collected in a preservative solution rather than directly smeared onto a slide. This liquid sample is then processed using an automated system that disperses cells, collects them on a filter, and transfers them onto a slide. LBC results in slides with cleaner backgrounds and better cell dispersion, making screening easier and more accurate. However, LBC equipment and solutions can be more expensive compared to conventional pap smears. Laser scanning cytometry is a technique that can be used to analyze cells collected with LBC.
Investigations in Tuberculosis and advancesNirish Vaidya
This document discusses various techniques for investigating Mycobacterium tuberculosis and advances in the field. It summarizes key characteristics of M. tuberculosis and the global burden of tuberculosis. It then describes several laboratory techniques for detecting and diagnosing tuberculosis, including sputum smear microscopy, mycobacterial culture methods, tuberculin skin testing, and newer molecular techniques such as nucleic acid amplification tests and interferon-gamma release assays. Advances in rapid molecular diagnostics and their applications for tuberculosis detection and drug resistance testing are also discussed.
This document discusses molecular diagnostic methods for detecting drug resistance in gram-positive bacteria. It covers several key points:
1) Traditional phenotypic methods for antimicrobial susceptibility testing require pure culture and take several days for results, while genotypic methods can provide results within hours directly from patient specimens without culturing.
2) Important resistance mechanisms in clinically relevant gram-positive pathogens include beta-lactamases in Staphylococcus and penicillin-binding protein mutations conferring methicillin resistance.
3) Newer molecular techniques for antimicrobial susceptibility testing include PCR, real-time PCR, and next-generation sequencing, which can more rapidly detect resistance genes compared to traditional phenotypic methods.
Laser scanning cytometry and liquid based cytologyanaonline
Liquid based cytology (LBC) has been introduced to improve cervical screening. It uses a liquid preservative instead of smearing cells directly on a slide. This allows automated processing to disperse cells and transfer them evenly to a slide. LBC reduces sampling errors, improves cell preservation and slide quality compared to conventional smears. However, it requires specialized equipment and is more expensive. Laser scanning cytometry is a technique that can analyze individual cells from LBC samples. It provides quantitative measurements and complements flow cytometry by allowing analysis of adherent cells and solid tissues.
- Tuberculosis remains a major global health problem, with an estimated 9 million new cases in 2013. Early diagnosis is important to curb transmission and initiate optimal treatment.
- The World Health Organization endorses several tests for TB diagnosis including microscopy, culture-based tests, and molecular tests. Specific techniques recommended include LED fluorescence microscopy, liquid culture systems, line probe assays, and the Xpert MTB/RIF test.
- New diagnostic technologies aim to provide rapid, accurate, and accessible diagnosis to help reach more of the estimated 3 million TB cases that go undiagnosed or unreported each year.
This presentation is about lab diagnosis of tuberculosis. It highlights use of currently available diagnostic methods in identifying pulmonary and extrapulmonary tuberculosis.
This document discusses various methods for identifying bacteria, including traditional phenotypic methods, immunochemical methods, and genotypic molecular methods. Phenotypic methods involve examining bacterial morphology, staining characteristics, growth requirements, and biochemical reactions. Immunochemical methods like immunofluorescence and ELISA use antigen-antibody reactions for identification. Molecular identification methods analyze bacterial DNA sequences. Correct specimen collection, handling, and transport are essential for accurate identification. Identification determines clinical significance and appropriate treatment.
This document provides an overview of tuberculosis diagnosis and treatment in India. It discusses:
1) India's goal to end TB by 2025 and new programs to support TB patients and educate communities.
2) Recommendations that all medical colleges have facilities for multidrug-resistant TB management and that 5 whole genome sequencing facilities be established for surveillance and research.
3) Diagnostic tests for TB including smear microscopy, molecular tests like CBNAAT and line probe assay, and culture. It provides details on each test's methodology, turnaround time, and sensitivity.
4) Classification of TB cases as new, previously treated, clinically diagnosed or microbiologically confirmed. Treatment regimens are outlined according
The document discusses the GeneXpert MTB/RIF instrument, which is an automated diagnostic test that can identify Mycobacterium tuberculosis (MTB) DNA and resistance to rifampicin (RIF) using nucleic acid amplification. It describes how the instrument works, the types of samples it can process, how samples are loaded and tested, and how results are interpreted. The summary also mentions that the GeneXpert MTB/RIF assay has been implemented in Nepal through a project to improve tuberculosis case detection using this technology.
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1. TUBERCOLOSI:UNA
MALATTIA
SOCIALE
21-‐22
SETTEMBRE
2012
Ia
tappa:
da
Kock
a
Xpert
D
Cirillo
San
Raffaele
Scien/fic
Ins/tute
Milan
Italy
2. Outline
• Urgent
need
for
an
improved
diagnosis
• Microscopy
• Culture
and
DST
needs
for
standards
and
EQAs
• Molecular
diagnosis
is
a
reality
• What
can
be
achieved
with
current
molecular
tools
• What
cannot
be
achieved
with
the
current
molecular
tools
• Global
prospec/ve
3. Urgent
need
for
new
diagnosGcs
• TB
case
detec/on
gaps:
– cases
undiagnosed
• Inaccessible
facili/es
• Not
self
repor/ng,
not
returning
• Wrong
diagnosis
– Cases
diagnosed
in
private
care
and
not
reported
• Infec/on
control
(Stop
TB/MDR-‐TB
transmission)
• Guide
treatment
• Monitoring
treatment
4. Microscopy
:
a
century
old
procedure
Rapid test
Inexpensive
n Does not allow species identification
n Not applicable to all samples
n Specificity for Mycobacterium spp:
>95%
Sensitivity: 25-65% (90 % of higly
n
infectious cases) Fluorescence Ziehl-Neelsen staining
Positive Predictive Value for TB
n 1st AFB smear 80-82 %
depends on epidemiological situation
2nd AFB smear 10-14 %
LED
microscopy
recommended
over
light
and
fluorescent
3rd AFB smear 5-8 %
microscopy
5. TB
Culture
Advantages Disadvantages
• Definitive diagnosis of TB • Complex and expensive
compared to microscopy
• Increases case finding of • Requires complex handling of
30-50% specimens
• Early detection of cases • Skilled technicians
• Provide strains for DST • Appropriate infrastructure and
biosafety levels
and epidemiological
studies
LIMITATIONS: need for decontamination and identification
*coverage 500.000/1000000
6. Culture:
solid/
liquid
solid liquid
• Low cost for reagents, not • Complex and expensive can be
automated automated (MGIT)
• Highest infrastructure and
• Culture level infrastructure biosafety levels
• Low contamination rate • Case finding increased 10% over
• Long time to positivity solid
• Colony morphology • Diagnostic delay reduced to days
• ID required • ID required
• DST only for selected drugs
• DST only for selected drugs
Strip speciation tests for fast ID of Tbcomplex
Molecular test for speciation of most common mycobacteria
7. M. tuberculosis identification
Morphology/ Molecular
Immuno-‐
Biochemical
tests
cromatographic
tests
LPAs
test
Probes
on
liquid
phase
Sequencing
EQA
and
standardiza/on
Spoligotyping
Enzyme
7
restric/on
8. DST
• Definitive diagnosis of DRTB
3
main
methods
Absolute
Propor/on
concentra/on
Resistant
Ra/o
methods
method
method
9. DST:
Liquid/solid
mtedia
comparison
Liquid
media
compared
o
solid
media
Advantages
compared
to
solid
media:
•
more
rapid
•
high
quality
of
media
•
fully
automated
system
•
tes/ng
of
1st,
2nd
,
and
new
drugs
(Linezolid)
•
safety:
plas/c
tubes
•
pyrazinamide
sensi/vity
test
Break
points
for
2nd
line
drugs
Disadvantages:
recently
revised
•
expensive
S/ll
poor
correla/on
with
clinical
•
higher
contamina/on
rate
outcome,
some
tes/ng
not
fully
•
dependency
on
a
company
reliable
•
no
DST
for
Cycloserine
12. Is
new
real-‐Gme
technology
improving
the
sensiGvity
of
molecular
tests?
13. New
generaGon
of
tests
does
not
improve
sensiGvity
Tortoli
et
al
2012
JCM
Methods
Amplicor
n
on
NAs
amples
Taqman
vs
based
oon
15000
s detec/on
have
intrinsic
limita/ons:
• Absence
on
specificity
ubop/mal
specimen
selec/on,
quan/ty,
Improvement
i f
target:
s
quality,..
Decrease
in
invalid
results
Improvement
in
post
test
probability
• Subop/mal
sample
prepara/on
14. Are
molecular
tests
tools
for
difficult
to
diagnose
cases?
• TB
in
Children
• Extrapulmonary
Tuberculosis
17. Diagnosis
in
difficult
cases
• Children
diagnosis
is
not
microbiologically
confirmed
in
40-‐60%
of
cases,
current
molecular
tools
s/ll
subop/mal.
A
host/
pathogens
biomarkers
approach
is
probably
needed
• Extrapulmonary
TB:
the
performance
of
molecular
tools
varies
and
should
be
considered
separately
for
each
specific
specimen
type.
18. Unsolved
Problems
of
Molecular
Tests
for
Direct
Diagnosis
of
Tuberculosis
• Subop/mal
sensi/vity/specificity
versus
a
gold
standard:
– Compared
to
culture
in
liquid
media
– Compared
to
a
combined
standard
based
on
the
“inten/on
to
treat”/
response
to
treatment
Decreased
confidence
in
the
test
19. Conventional DST: technical challenges
• Adequate
infrastructures
and
biosefety
levels
• MGIT
DST:
the
gold
standard
• MDRTB
:
3-‐6
weeks;
XDRTB
:
6-‐9
weeks
• Reproducibility
and
accuracy
of
results
are
drugs
dependent:
– Rifampicin,
isoniazid
:
good
results
– Second-‐line:
fluoroquinolones
and
injectables
CorrelaGon
of
sensiGvity
test
results
and
clinical
outcome
is
difficult
to
evaluate
and
we
have
very
limited
or
no
evidence
for
Pyr,E,
and
2nd
line
drugs
other
than
INJ
and
FQs
on
MDR
cases
In
addi/on:
Capital
cost
of
facili/es
Cost
of
maintenance
Cost
of
staff
Van
Deun
A.
et
al
2011.
IJTLD
15(1):116-‐124
20. MECHANISMS
OF
DRUG
RESISTANCE
IN
M.
tuberculosis
Zhang
Y
and
Yew
W,
Int
J
Tuberc
Lung
Dis
2009
21. Commercial
Molecular
tests
for
e
Tdopted
in
WHO
Global
plan
(2006-‐2015):development
and
roll
out
of
new
technologies
to
b a
B/
MDR
TB
detecGon
endorsed
by
WHO
resources-‐limited
senngs
GenoType
MTBDRplus,
InnoLiPA
Rif.TB
• Reverse
hybridiza/on,
colorimetric
reac/on
• Results
in
6-‐7
h
•
some
flexibility
(n
probes/strip:
30-‐40)
•
Technical
exper/se:
some
• Biosafety
lev
2
Xpert
MTB/RIF
• Integrated/automated
qPCR
• Results
in
2h
• Closed
system
(limited
number
of
probes:
<10)
•
Technical
exper/se:
none
22. LPA
performance
i(n
isolates
and
clinical
LPAs:
performances
*based
on
2°
generaGon)
Rifampicin
samples
Isoniazid
Inno-‐LiPA
Rif.TB
GenoType
MTBDRplus*
GenoType
MTBDRplus
Hot-‐spot
rpoB
gene
cod.
315
katG
gene
nt
-‐8,-‐15,-‐16
inhA
gene
Morgan
M
et
al
2005.
BMC
Infect
Dis
5:62
Ling
DI
et
al
2008.
Eur
Respir
J
32:1165-‐1174
Ling
DI
et
al
2008.
Eur
Respir
J
32:1165-‐1174
Sensi.vity
95-‐98%
Sensi.vity
95-‐99%
Sensi.vity
82-‐93%
Specificity
98-‐100%
Specificity
97-‐100%
Specificity
95-‐100%
Decontaminated
clinical
specimens
(AFB-‐posi?ve)
Dec.
clin.
spec.
(AFB-‐pos)
Sensi.vity
95-‐99%
Sensi.vity
72-‐92%
Specificity
97-‐99%
Specificity
96-‐99%
23. TAT
to
Rif
–R
detecGon
and
reporGng
RIF-‐R
detecGon
Time
to
report
to
treatment
center
Boehme
CC
et
al
2011.
Lancet
377(9776):1495-‐505
Xpert
MTB/RIF:
0-‐1
d
Xpert
MTB/RIF:
0-‐1
d
(Microscopy:
1-‐2
d)
LPA:
10-‐26
d*
LPA:
27-‐53
d*
Culture
DST:
30-‐124
d**
Culture
DST:
38-‐102
d**
(culture:
42-‐62
d)
Some
results
not
reported/lost
*
test
on
AFB-‐pos
clinical
specimen
+
test
on
clinical
isolate
for
AFB-‐neg
cases
**
DST
performed
by
MGIT
+
DST
performed
on
LJ
24. What
can
be
achieved
or
parGally
achieve
with
the
current
molecular
tools
• Diagnosis
of
TB,
rifampicin
resistant
TB,
MDR-‐TB
in
smear
posi/ve/nega/ve
samples
• Iden/fica/on
of
up
to
80%
fluoroquinolones
and
up
to
40-‐80%
of
injectable
resistant
cases
among
MDR-‐
TB
cases
• Improvement
of
diagnosis
of
TB
in
extrapulmonary
samples
• Support
to
diagnosis
of
TB
in
children
• Diagnosis
of
NTMs
infec/on
26. DR
tesGng:
Rifampicin
Bactericidal
an/bio/c
that
inhibits
the
bacterial
DNA-‐
dependent
RNA
polymerase.
Target:
β-‐subunit
of
the
RNA
polymerase
(encoded
by
rpoB),
blocking
elonga/on
of
the
RNA
chain.
codon
526/
531
high
level
resistance
to
rifampicin,
MutaGons
in
a
“hot-‐spot”
region
of
81
bp
of
rifabu/n
e
rifapen/n
rpoB
gene
(Rifampin
resistance-‐determining
516
e
522
high
PPV
for
region)
→
RIF
resistance
(>
95%)
rifabu/n
res
27. ISONIAZID
ISONIAZID
INH:
targeGng
mycolic
acid
biosysthesis
MutaGons
in
KatG
gene
prevent
INH
acGvaGon
(cod.
315,
60-‐90%)
MutaGons
in
the
direct
target
inhA
(inhA
belongs
to
the
family
of
short-‐chain
dehydrogenases/reductases.
It
is
essenGal
in
MTB)
MutaGons
in
the
promoter
of
inhA
gene
leading
to
drug
tritraGon
(direct
target
over-‐producGon)
Only
Kat
G
315
and
inhA
are
included
in
LPA
Ratan
A
et
al.
EID
1998
28. PPV
and
NPV
for
Rif
resistance
at
different
prevalence
of
Rif
resistance
WHO/HTM/TB/2011.2
29. Discrepancies
with
MGIT
DST
• In vitro growth of Rif sensitive strains from
samples identified as Rif res or MDR by LPA
• Few cases from strains tested by LPA
• rpoB absence of WT8 (codo530-533) and absence of
rpoB MUT3
• Strains bearing the mutations grow slowly in liquid
culture and are identified as Rif sensitive
• inhA mut strains sensitive to INH by MGIT DST
Discordant
reports
are
confusing
for
pa/ents
Management
if
not
appropriately
explained
30. Heteroresistance
Real
heteroresistance
False
heteroresistance
• Co-‐presence
of
mutated
• Laboratory
cross
and
wild
type
popula/on
contamina/on
due
to
• Two
different
strains
insufficient
control
of
amplicons
LPAs
can
detect
heteroresistance
from
clinical
strains:
clinical
role?
31. Role
of
point
mutaGons
in
predicGng
clinical
outcome:
embB
Codon
306
• Low
sensi/vity
of
embB306
locus
for
predic/ng
Eth
Res
and
MDR-‐TB
(35.5%),
High
specificity
for
iden/fying
MDR-‐TB
(92.6%;
87/94).
The
posi/ve
predic/ve
value
(77.4%;
24/31)
and
the
nega/ve
predic/ve
value
(66.4%;
87/131)
of
this
locus
are
moderate.
Xin
Shen,
AAC2007
• Plinke
et
al
AAC
2006,
Mioxo
et
al
ERJ
2012:
higher
PPV
for
ETH
resistance
32. Commercial
tests
for
Fluoroquinolones
and
injectables
• Commercial
LPA
tests
for
detec/on
of
resistance
to
second
line
drugs
show
a
high
PPV
and
a
low
NPV
due
to
the
limited
number
of
muta/ons
included
in
the
tests
• PPV
and
NPV
may
vary
with
the
genotypic
background
of
the
strains
and/or
with
the
prevalence
of
specific
genotypes
in
the
target
popula/on
Mioxo
et
al.
ERJ
2012
33. Molecular
test
performances
should
be
evaluated
in
the
epidemiological
contests
• PPV
and
NPV
may
vary
in
different
genotypic
backgrounds
• High
prevalence
of
specific
clones
bearing
selected
muta/ons
not
included
in
current
diagnos/c
assays
may
modify
PPV
and
NPV
34. Role
of
Molecular
typing
• To
iden/fy
epidemiological
links
between
TB
pa/ents
to
detect
and
control
outbreaks
early
and
rapidly
• Rule
out
suspected
outbreaks
and
confirm
transmission
has
NOT
occurred
• To
iden/fy
incorrect
TB
diagnosis
based
on
false-‐posi/ve
cultures
and
thus
avoid
unnecessary
inves/ga/on
and
treatment
• To
dis/nguish
exogenous
re-‐infec/on
from
endogenous
reac/va/on
in
pa/ents
with
a
past
history
of
TB
• Discover
unusual
transmission
senngs
and
transmission
between
different
regions
• Monitor
the
size
of
clusters
and
thus
monitor
progress
towards
TB
elimina/on
• Vaccine
and
DR
detec/on
implica/ons
35. Needs
for
berer
tools
Among
3037
pa/ents
with
new
cases
of
tuberculosis
and
892
with
previously
treated
cases,
5.7%
(95%
confidence
interval
[CI],
4.5
to
7.0)
and
25.6%
(95%
CI,
21.5
to
29.8),
had
mul/drug-‐resistant
(MDR)
tuberculosis.
Among
all
pa/ents
with
tuberculosis,
approximately
1
of
4
had
disease
that
was
resistant
to
isoniazid,
rifampin,
or
both,
and
1
of
10
had
MDR
tuberculosis.
Approximately
8%
of
the
pa/ents
with
MDR
tuberculosis
had
extensively
drug-‐resistant
(XDR)
tuberculosis
36. Lab-‐on
Chip
for
molecular
diagnosGcs
PCR:
• Ultra-Fast PCR
• Asymmetric Cy-5 PCR strategy
Microarray:
• Orientation probes
• Hybridization Control probes
• Hybridization Negative Controls probes
Current Lay out:
ID of MTBC, relevant NTMs
MDR phenotype
Lab-on-chip architecture
2 PCR reactors of 12.5 uL volume each (Total 25 ul)
1 Hybridization chamber of 30 uL All the reaction modules
A 126 spots DNA microarray are fluidically integrated
2 in-let ports compatible with standard micro-pipettor tips
Integrated Heaters and Sensors
37. Open
Issues
• How
to
monitor
the
response
to
therapy?
– Sputum
smear
is
s/ll
guiding
decisions
on
admission
and
discharge
– Sputum
culture
is
s/ll
the
only
reliable
monitoring
tool
for
MDR
pa/ents
• Pa/ents
with
H
monoresistance
may
go
undetected,
in
R
res
H
should
be
le{
un/l
proven
R?
• Contact
treatment
in
absence
of
H
sensi/vity
data
• Discrepancy
between
phenotypic
and
genotypic
results
• Are
all
the
muta/ons
in
rpoB
equally
contribu/ng
to
resistance?
• Muta/ons
to
key
second
line
drugs
and
cross
resistance
• How
to
report
molecular
data
in
order
to
provide
clinical
guidance?
39. Acknowledgments
Emanuele
Borroni
Andrea
M.
Cabibbe
Irene
Festoso
Paola
Mantegani
Paolo
Mioro
Luca
Norbis
Enrico
Tortoli
Ilaria
Valente
Diego
Zallocco
Emerging
Bacterial
Pathogens
Unit
WHO
Suprana/onal
Reference
Laboratory
for
TB
Control
San
Raffaele
Scien/fic
Ins/tute
Milano,
ITALY
Fulvio
Salvo
and
Delek
Hospital
Staff
AISPO
Alberto
Mareelli
Alberto
Roggi
and
SRL/
NTP
Burkina
faso
Ins/tute
of
Infec/ous
and
Tropical
Diseases
University
of
Brescia
Brescia,
ITALY
Thanks for your attention!
Lanfranco
Farorini
GB
Migliori
Is/tuto
Superiore
di
Sanità
FSM,Tradate
Roma,
ITALY