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Anaesthesia, Critical Care & Pain... Dec 2023Accuracy and timing of antibiotic therapy remain a challenge for lower respiratory tract infections. New molecular techniques using Multiplex Polymerase Chain Reaction,... (Meta-Analysis)
Meta-Analysis Review
Performance evaluation of a PCR panel (FilmArray® Pneumonia Plus) for detection of respiratory bacterial pathogens in respiratory specimens: A systematic review and meta-analysis.
BACKGROUND
Accuracy and timing of antibiotic therapy remain a challenge for lower respiratory tract infections. New molecular techniques using Multiplex Polymerase Chain Reaction, including the FilmArray® Pneumonia Plus Panel [FAPP], have been developed to address this. The aim of this study is to evaluate the FAPP diagnostic performance for the detection of the 15 typical bacteria of the panel from respiratory samples in a meta-analysis from a systematic review.
METHODS
We searched PubMed and EMBASE from January 1, 2010, to December 31, 2022, and selected any study on the FAPP diagnostic performance on respiratory samples compared to the reference standard, bacterial culture. The main outcome was the overall diagnostic accuracy with sensitivity and specificity. We calculated the log Diagnostic Odds Ratio and analyzed performance for separate bacteria, antimicrobial resistance genes, and according to the sample type. We also reported the FAPP turnaround time and the out-of-panel bacteria number and species. This study is registered with PROSPERO (CRD42021226280).
RESULTS
From 10 317 records, we identified 30 studies including 8 968 samples. Twenty-one were related to intensive care. The overall sensitivity and specificity were 94% [95% Confidence Interval (CI) 91-95] and 98% [95%CI 97-98], respectively. The log Diagnostic Odds Ratio was 6.35 [95%CI 6.05-6.65]. 9.3% [95%CI 9.2-9.5] of bacteria detected in culture were not included in the FAPP panel.
CONCLUSION
This systematic review reporting the FAPP evaluation revealed a high accuracy. This test may represent an adjunct tool for pulmonary bacterial infection diagnostic and antimicrobial stewardship. Further evidence is needed to assess the impact on clinical outcome.
Topics: Humans; Bacteria; Pneumonia; Respiratory Tract Infections; Bacterial Infections; Multiplex Polymerase Chain Reaction
PubMed: 37709201
DOI: 10.1016/j.accpm.2023.101300 -
Clinical Infectious Diseases : An... Nov 2023
Topics: Humans; Bronchoalveolar Lavage Fluid; Aspergillosis; Aspergillus; Mannans; Polymerase Chain Reaction; Cell-Free Nucleic Acids
PubMed: 37448327
DOI: 10.1093/cid/ciad421 -
Human Gene Therapy Aug 2023Recombinant adeno-associated virus (rAAV) has been utilized successfully for gene delivery for treatment of a variety of human diseases. To sustain the growth of...
Recombinant adeno-associated virus (rAAV) has been utilized successfully for gene delivery for treatment of a variety of human diseases. To sustain the growth of recombinant AAV gene therapy products, there is a critical need for the development of accurate and robust analytical methods. Fifty percent tissue culture infectious dose (TCID) assay is an cell-based method widely used to determine AAV infectivity, and this assay is historically viewed as a challenge due to its high variability. Currently, quantitative PCR (qPCR) serves as the endpoint method to detect the amount of replicated viral genome after infection. In this study, we optimize the TCID assay by adapting endpoint detection with droplet digital PCR (ddPCR). We performed TCID assays using ATCC AAV-2 reference standard stock material across 18 independent runs. The cell lysate from TCID assay was then analyzed using both qPCR and ddPCR endpoint to allow for direct comparison between the two methods. The long-term 1-year side-by-side comparison between qPCR and ddPCR as endpoint measurement demonstrated improved interassay precision when the ddPCR method was utilized. In particular, after the addition of a novel secondary set threshold for infectivity scoring of individual wells, the average infectious titer of 18 runs is 6.45E+08 with % coefficient of variation (CV) of 42.5 and 5.63E+08 with % CV of 34.9 by qPCR and ddPCR, respectively. In this study, we offer improvements of infectious titer assay with (1) higher interassay precision by adapting ddPCR as an endpoint method without the need of standard curve preparation; (2) identification of a second "set threshold" value in infectivity scoring that improves assay precision; and (3) application of statistical analysis to identify the acceptance range of infectious titer values. Taken together, we provide an optimized TCID method with improved interassay precision that is important for rAAV infectious titer testing during process development and manufacturing.
Topics: Humans; Dependovirus; Polymerase Chain Reaction; Genome, Viral; Real-Time Polymerase Chain Reaction
PubMed: 37276150
DOI: 10.1089/hum.2023.014 -
Biosensors Jan 2024Single-cell analysis provides an overwhelming strategy for revealing cellular heterogeneity and new perspectives for understanding the biological function and disease... (Review)
Review
Single-cell analysis provides an overwhelming strategy for revealing cellular heterogeneity and new perspectives for understanding the biological function and disease mechanism. Moreover, it promotes the basic and clinical research in many fields at a single-cell resolution. A digital polymerase chain reaction (dPCR) is an absolute quantitative analysis technology with high sensitivity and precision for DNA/RNA or protein. With the development of microfluidic technology, digital PCR has been used to achieve absolute quantification of single-cell gene expression and single-cell proteins. For single-cell specific-gene or -protein detection, digital PCR has shown great advantages. So, this review will introduce the significance and process of single-cell analysis, including single-cell isolation, single-cell lysis, and single-cell detection methods, mainly focusing on the microfluidic single-cell digital PCR technology and its biological application at a single-cell level. The challenges and opportunities for the development of single-cell digital PCR are also discussed.
Topics: Polymerase Chain Reaction; Microfluidics; DNA; RNA; Single-Cell Analysis
PubMed: 38391982
DOI: 10.3390/bios14020064 -
Clinical Chemistry Sep 2023Partition classification is a critical step in the digital PCR data analysis pipeline. A range of partition classification methods have been developed, many motivated by... (Review)
Review
BACKGROUND
Partition classification is a critical step in the digital PCR data analysis pipeline. A range of partition classification methods have been developed, many motivated by specific experimental setups. An overview of these partition classification methods is lacking and their comparative properties are often unclear, likely impacting the proper application of these methods.
CONTENT
This review provides a summary of all available digital PCR partition classification approaches and the challenges they aim to overcome, serving as a guide for the digital PCR practitioner wishing to apply them. We additionally discuss strengths and weaknesses of these methods, which can further guide practitioners in vigilant application of these existing methods. This review provides method developers with ideas for improving methods or designing new ones. The latter is further stimulated by our identification and discussion of application gaps in the literature, for which there are currently no or few methods available.
SUMMARY
This review provides an overview of digital PCR partition classification methods, their properties, and potential applications. Ideas for further advances are presented and may bolster method development.
Topics: Polymerase Chain Reaction
PubMed: 37401391
DOI: 10.1093/clinchem/hvad063 -
Frontiers in Immunology 2023Immunosequencing has emerged as a newer clinical test for assessment of T-cell clonality in the blood and skin of cutaneous T-cell lymphoma (CTCL) patients. Utilization... (Review)
Review
Immunosequencing has emerged as a newer clinical test for assessment of T-cell clonality in the blood and skin of cutaneous T-cell lymphoma (CTCL) patients. Utilization of immunosequencing, also known as high-throughput sequencing of the T-cell receptor (HTS-TCR), enables identification and quantification of the precise genetic signature of dominant T-cell clones. Although immunosequencing is more sensitive than commonly used methods such as polymerase chain reaction (PCR) paired with capillary electrophoresis or flow cytometry, it remains underutilized for CTCL management. Nonetheless, incorporation of HTS-TCR in clinical practice offers distinct advantages compared to other molecular analyses that may improve diagnostic evaluation, prognostication, and disease monitoring in CTCL. The objective of this comprehensive review is to provide a thorough explanation of the application of immunosequencing in the context of CTCL. We describe the significance of T-cell clonality and the methods used to detect it, including a detailed comparison between PCR paired with capillary electrophoresis and HTS-TCR. The utilization of immunosequencing in the blood and skin of CTCL patients is discussed in depth, specifically outlining how HTS-TCR can assist in diagnosing CTCL, predicting outcomes, and tracking disease progression. Finally, we address the potential applications of immunosequencing in clinical management and research as well as the novel challenges it presents.
Topics: Humans; Skin Neoplasms; Polymerase Chain Reaction; Lymphoma, T-Cell, Cutaneous; Skin; Receptors, Antigen, T-Cell
PubMed: 38213330
DOI: 10.3389/fimmu.2023.1300061 -
Tierarztliche Praxis. Ausgabe K,... Dec 2023is a facultative pathogenic intestinal parasite. Giardiosis in dogs and cats may appear with or without clinical signs. Typical signs include diarrhea with or without...
is a facultative pathogenic intestinal parasite. Giardiosis in dogs and cats may appear with or without clinical signs. Typical signs include diarrhea with or without vomiting. The prevalence in young animals is high and may amount to up to 50%. There are 8 different genotypes (A - H), which are called assemblages. Assemblages C and D are most common in dogs and assemblage F most frequent in cats. However, animals may also be infected with the zoonotically effective assemblages A and B or exhibit mixed infections. The immunofluorescence test (IFA), the enzyme-linked immunosorbent assay (ELISA) and fecal centrifugation using zinc sulphate solution are currently recommended as diagnostic methods. Polymerase chain reaction (PCR) may be used to determine the corresponding assemblage. Approved treatments for giardiosis include fenbendazole and metronidazole. In addition, undertaking specific hygiene measures is warranted. Only animals showing clinical signs or those living in the same household with high-risk patients (e. g. immunosuppressed humans) are recommended to receive medication. The aim of treatment is clinical improvement of the diseased dogs and cats. Frequently, complete elimination of Giardia is not attained.
Topics: Animals; Cats; Dogs; Humans; Cat Diseases; Dog Diseases; Giardiasis; Giardia lamblia; Polymerase Chain Reaction; Feces
PubMed: 38056479
DOI: 10.1055/a-2191-1723 -
Mycopathologia Dec 2023The diagnosis of chronic pulmonary aspergillosis (CPA) is established by combined clinic-radio-microbiological criteria. Out of the different microbiological criteria, a...
The diagnosis of chronic pulmonary aspergillosis (CPA) is established by combined clinic-radio-microbiological criteria. Out of the different microbiological criteria, a positive serology for Aspergillus-specific IgG levels is the cornerstone of diagnosis. Alternatively, other microbiological evidence are sometimes sought viz., positive Aspergillus antigen (broncho-alveolar lavage fluid, i.e., BALF galactomannan ≥ 1.0), histopathological demonstration of the fungi following lung biopsy or resection, demonstration of hyaline septate hyphae in direct microscopy resembling Aspergillus spp. or its growth on a respiratory specimen. However, the exact roles of BALF- GM and the newer BALF-PCR have not been confirmed by studies till date. This study enrolled 210 patients with suspected CPA. Of the participants, 88 patients met the criteria for CPA, whereas 122 patients had an alternative diagnosis. The sensitivity-specificity of AsperGenius® PCR and "in-house" PCR were 52.27(36.69-67.54) %-33.78 (23.19-45.72) % and 36.36 (22.41-52.23) %-39.19 (28.04-51.23) % respectively. The sensitivity/specificity of BALF (> 1.0) and serum galactomannan (> 1.0) were 46.55% (33.34-60.13)/64.08% (54.03-73.3) and 29.82% (22.05-37.6)/86.84% (81.1-92.59) respectively. The optimal cut-off values for BALF-Galactomannan and serum galactomannan in diagnosing CPA were found to be 0.69 (sensitivity: 64%; specificity: 53%) and 0.458 (sensitivity: 67%; specificity: 64%) respectively. This results of this study suggests that Aspergillus PCR from BAL may not be a good "rule-in" test for diagnosing CPA. While the performances of GM in BAL and serum may be better than PCR, it should be best used in conjunction with other clinical, radiological, and other microbiological characteristics.
Topics: Humans; Pulmonary Aspergillosis; Aspergillus; Mannans; Bronchoalveolar Lavage Fluid; Sensitivity and Specificity; Polymerase Chain Reaction; Invasive Pulmonary Aspergillosis
PubMed: 37857979
DOI: 10.1007/s11046-023-00797-z -
Molecular Biology Reports Nov 2023Plant pathogens cause severe losses to agricultural yield worldwide. Tracking plant health and early disease detection is important to reduce the disease spread and thus... (Review)
Review
Plant pathogens cause severe losses to agricultural yield worldwide. Tracking plant health and early disease detection is important to reduce the disease spread and thus economic loss. Though visual scouting has been practiced from former times, detection of asymptomatic disease conditions is difficult. So, DNA-based and serological methods gained importance in plant disease detection. The progress in advanced technologies challenges the development of rapid, non-invasive, and on-field detection techniques such as spectroscopy. This review highlights various direct and indirect ways of detecting plant diseases like Enzyme-linked immunosorbent assay, Lateral flow assays, Polymerase chain reaction, spectroscopic techniques and biosensors. Although these techniques are sensitive and pathogen-specific, they are more laborious and time-intensive. As a consequence, a lot of interest is gained in in-field adaptable point-of-care devices with artificial intelligence-assisted pathogen detection at an early stage. More recently computer-aided techniques like neural networks are gaining significance in plant disease detection by image processing. In addition, a concise report on the latest progress achieved in plant disease detection techniques is provided.
Topics: Artificial Intelligence; Plants; Polymerase Chain Reaction; Plant Diseases
PubMed: 37823933
DOI: 10.1007/s11033-023-08838-y -
ACS Synthetic Biology Nov 2023The introduction of thermostable polymerases revolutionized the polymerase chain reaction (PCR) and biotechnology. However, many GC-rich genes cannot be PCR-amplified...
The introduction of thermostable polymerases revolutionized the polymerase chain reaction (PCR) and biotechnology. However, many GC-rich genes cannot be PCR-amplified with high efficiency in water, irrespective of temperature. Although polar organic cosolvents can enhance nucleic acid polymerization and amplification by destabilizing duplex DNA and secondary structures, nature has not selected for the evolution of solvent-tolerant polymerase enzymes. Here, we used ultrahigh-throughput droplet-based selection and deep sequencing along with computational free-energy and binding affinity calculations to evolve Taq polymerase to generate enzymes that are both stable and highly active in the presence of organic cosolvents, resulting in up to 10% solvent resistance and over 100-fold increase in stability at 97.5 °C in the presence of 1,4-butanediol, as well as tolerance to up to 10 times higher concentrations of the potent cosolvents sulfolane and 2-pyrrolidone. Using these polymerases, we successfully amplified a broad spectrum of GC-rich templates containing regions with over 90% GC content, including templates recalcitrant to amplification with existing polymerases, even in the presence of cosolvents. We also demonstrated dramatically reduced GC bias in the amplification of genes with widely varying GC content in quantitative polymerase chain reaction (qPCR). By expanding the scope of solvent systems compatible with nucleic acid polymerization, these organic solvent-resistant polymerases enable a dramatic reduction of sequence bias not achievable through thermal resistance alone, with significant implications for a wide range of applications including sequencing and synthetic biology in mixed aqueous-organic media.
Topics: DNA-Directed DNA Polymerase; DNA; Polymerase Chain Reaction; Base Composition; Solvents
PubMed: 37611245
DOI: 10.1021/acssynbio.2c00515