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The Brazilian Journal of Infectious... 2024Chlamydia psittaci ‒ related community-acquired pneumonia associated to acute myocarditis was diagnosed in a young man with no medical history, and a professional...
Chlamydia psittaci ‒ related community-acquired pneumonia associated to acute myocarditis was diagnosed in a young man with no medical history, and a professional exposition to birds. The diagnosis was confirmed with positive specific polymerase chain reaction in bronchoalveolar lavage. The patient was treated with spiramycin for two weeks with anti-inflammatory treatment for myocarditis for three months. Clinical and biological improvement was rapidly observed followed by normalization of electrocardiogram and chest CT scan. No relapse was reported for over a two-year follow-up.
Topics: Humans; Male; Myocarditis; Psittacosis; Chlamydophila psittaci; Adult; Polymerase Chain Reaction; Community-Acquired Infections; Acute Disease; Young Adult
PubMed: 38679059
DOI: 10.1016/j.bjid.2024.103739 -
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 -
Journal of Feline Medicine and Surgery Aug 2023Feline infectious peritonitis (FIP), a common disease in cats caused by feline coronavirus (FCoV), is usually fatal once clinical signs appear. Successful treatment of...
OBJECTIVES
Feline infectious peritonitis (FIP), a common disease in cats caused by feline coronavirus (FCoV), is usually fatal once clinical signs appear. Successful treatment of FIP with oral GS-441524 for 84 days was demonstrated recently by this research group. The aim of this study was to evaluate the long-term outcome in these cats.
METHODS
A total of 18 successfully treated cats were followed for up to 1 year after treatment initiation (9 months after completion of the antiviral treatment). Follow-up examinations were performed at 12-week intervals, including physical examination, haematology, serum biochemistry, abdominal and thoracic ultrasound, FCoV ribonucleic acid (RNA) loads in blood and faeces by reverse transciptase-quantitative PCR and anti-FCoV antibody titres by indirect immunofluorescence assay.
RESULTS
Follow-up data were available from 18 cats in week 24, from 15 cats in week 36 and from 14 cats in week 48 (after the start of treatment), respectively. Laboratory parameters remained stable after the end of the treatment, with undetectable blood viral loads (in all but one cat on one occasion). Recurrence of faecal FCoV shedding was detected in five cats. In four cats, an intermediate short-term rise in anti-FCoV antibody titres was detected. In total, 12 cats showed abdominal lymphadenomegaly during the follow-up period; four of them continuously during the treatment and follow-up period. Two cats developed mild neurological signs, compatible with feline hyperaesthesia syndrome, in weeks 36 and 48, respectively; however, FCoV RNA remained undetectable in blood and faeces, and no increase in anti-FCoV antibody titres was observed in these two cats, and the signs resolved.
CONCLUSIONS AND RELEVANCE
Treatment with GS-441524 proved to be effective against FIP in both the short term as well as the long term, with no confirmed relapse during the 1-year follow-up period. Whether delayed neurological signs could be a long-term adverse effect of the treatment or associated with a 'long FIP syndrome' needs to be further evaluated.
Topics: Cats; Animals; Feline Infectious Peritonitis; Follow-Up Studies; Polymerase Chain Reaction; RNA, Viral; Coronavirus, Feline; Cat Diseases
PubMed: 37548535
DOI: 10.1177/1098612X231183250 -
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 -
Archives of Virology Aug 2023Mpox (formerly monkeypox) is a zoonotic disease caused by monkeypox virus (MPXV), which, like smallpox, is characterised by skin rashes. While the world is currently... (Review)
Review
Mpox (formerly monkeypox) is a zoonotic disease caused by monkeypox virus (MPXV), which, like smallpox, is characterised by skin rashes. While the world is currently grappling with the coronavirus disease 2019 pandemic, the appearance of MPXV has presented a global threat and raised concerns worldwide. Since May 2022, MPXV has spread rapidly in non-endemic mpox areas. As of 27 June 2023, the virus has spread to more than 112 countries and regions, with over 88,060 laboratory-confirmed cases and 147 deaths. Thus, measures to control the mpox epidemic are urgently needed. As the principal methods for identifying and monitoring mpox, laboratory detection techniques play an important role in mpox diagnosis. This review summarises the currently-used laboratory techniques for MPXV detection, discusses progress in improving these methods, and compares the benefits and limitations of various diagnostic detection methods. Currently, nucleic acid amplification tests, such as the polymerase chain reaction, are the most commonly used. Immunological methods have also been applied to diagnose the disease, which can help us discover new features of MPXV, improve diagnostic accuracy, track epidemic trends, and guide future prevention and control strategies, which are also vital for controlling mpox epidemics. This review provides a resource for the scientific community and should stimulate more research and development in alternative diagnostics to be applied to this and future public health crises.
Topics: Animals; COVID-19; Mpox (monkeypox); Pandemics; Polymerase Chain Reaction; Zoonoses
PubMed: 37543543
DOI: 10.1007/s00705-023-05848-w -
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 -
Ugeskrift For Laeger Apr 2024Leishmaniasis is transmitted by sandflies and involves cutaneous, mucocutaneous, or visceral disease. Sporadic, imported cases in Denmark emphasize the need for greater... (Review)
Review
Leishmaniasis is transmitted by sandflies and involves cutaneous, mucocutaneous, or visceral disease. Sporadic, imported cases in Denmark emphasize the need for greater awareness. The incidence is stable with at least ten verified cases per year. Diagnostic methods include PCR- and antibody tests with a high positivity rate for PCR (17%) and a low positivity rate for antibody (1.4%). The latter should be used only when visceral disease is suspected. Immunosuppressed patients are at particular risk. Treatment strategies are chosen according to the severity of the condition, as argued in this review.
Topics: Humans; Denmark; Leishmaniasis; Communicable Diseases, Imported; Antiprotozoal Agents; Polymerase Chain Reaction; Leishmaniasis, Cutaneous
PubMed: 38704708
DOI: 10.61409/V09230568 -
Medicine Jul 2023The present study aimed to evaluate the radiological findings of coronavirus patients who had positive computed tomography of the lung following real-time reverse...
The present study aimed to evaluate the radiological findings of coronavirus patients who had positive computed tomography of the lung following real-time reverse transcriptase-polymerase chain reaction testing. The data of 1727 patients who had reverse-transcriptase-polymerase chain reaction (RT-PCR) testing between May 2020 and August 2021 and had thoracic computed tomography (CT) on Days 7th to 8th were analyzed retrospectively. The Radiological Society of North America's recommended reporting system was used to categorize CT findings. Of the 1727 patients who underwent RT-PCR testing, there were 1417 patients with positive CT results. Of these 1417 patients, 679 (47.9%) were female. When patients with high blood values were evaluated, the number of CT-positive patients was significantly higher than CT-negative patients (P < .05). The number of patients with low lymphocyte and albumin values was significantly higher (P < .05). In 75.7% of those who had positive CT results, the PCR result was positive. Thoracic CT is a critical diagnostic tool in Coronavirus Disease 2019 patients with RT-PCR negative. It also depicts the progression of lung involvement in RT-PCR-positive patients. Performing it late in the disease's progression may increase the risk of contracting the disease.
Topics: Humans; Female; Male; COVID-19; SARS-CoV-2; Retrospective Studies; Sensitivity and Specificity; Lung; Reverse Transcriptase Polymerase Chain Reaction
PubMed: 37443487
DOI: 10.1097/MD.0000000000034267 -
Pathologica Oct 2023COVID-19 identification is routinely performed on fresh samples, such as nasopharyngeal and oropharyngeal swabs, even if, the detection of the virus in formalin-fixed... (Review)
Review
COVID-19 identification is routinely performed on fresh samples, such as nasopharyngeal and oropharyngeal swabs, even if, the detection of the virus in formalin-fixed paraffin-embedded (FFPE) autopsy tissues could help to underlie mechanisms of the pathogenesis that are not well understood. The gold standard for COVID-19 detection in FFPE samples remains the qRT-PCR as in swab samples, contextually other methods have been developed, including immunohistochemistry (IHC), and in situ hybridization (ISH). In this manuscript, we summarize the main data regarding the methods of COVID-19 detection in pulmonary and extra-pulmonary post-mortem samples, and especially the sensitivity and specificity of these assays will be discussed.
Topics: Humans; COVID-19; Polymerase Chain Reaction
PubMed: 38054901
DOI: 10.32074/1591-951X-933 -
Cell Reports. Medicine Aug 2023It is often challenging to distinguish cancerous from non-cancerous lesions in the brain using conventional diagnostic approaches. We introduce an analytic technique...
It is often challenging to distinguish cancerous from non-cancerous lesions in the brain using conventional diagnostic approaches. We introduce an analytic technique called Real-CSF (repetitive element aneuploidy sequencing in CSF) to detect cancers of the central nervous system from evaluation of DNA in the cerebrospinal fluid (CSF). Short interspersed nuclear elements (SINEs) are PCR amplified with a single primer pair, and the PCR products are evaluated by next-generation sequencing. Real-CSF assesses genome-wide copy-number alterations as well as focal amplifications of selected oncogenes. Real-CSF was applied to 280 CSF samples and correctly identified 67% of 184 cancerous and 96% of 96 non-cancerous brain lesions. CSF analysis was considerably more sensitive than standard-of-care cytology and plasma cell-free DNA analysis in the same patients. Real-CSF therefore has the capacity to be used in combination with other clinical, radiologic, and laboratory-based data to inform the diagnosis and management of patients with suspected cancers of the brain.
Topics: Humans; Polymerase Chain Reaction; Central Nervous System Neoplasms; Nucleic Acid Amplification Techniques; Short Interspersed Nucleotide Elements; Central Nervous System
PubMed: 37552989
DOI: 10.1016/j.xcrm.2023.101148