Further studies of neuroangiostrongyliasis (rat lungworm disease) in Australian dogs: 92 new cases (2010–2020) and results for a novel, highly sensitive qPCR assay

Parasitology ◽  
2020 ◽  
pp. 1-9
Author(s):  
Rogan Lee ◽  
Tsung-Yu Pai ◽  
Richard Churcher ◽  
Sarah Davies ◽  
Jody Braddock ◽  
...  
Keyword(s):  
Infectious Disease ◽  
Quantitative Polymerase Chain Reaction ◽  
Enzyme Linked Immunosorbent Assay ◽  
Chain Reaction ◽  
Cell Counts ◽  
Highly Sensitive ◽  
Elisa Testing ◽  
Rat Lungworm ◽  
Polymerase Chain ◽  
Sydney Australia

Abstract The principal aim of this study was to optimize the diagnosis of canine neuroangiostrongyliasis (NA). In total, 92 cases were seen between 2010 and 2020. Dogs were aged from 7 weeks to 14 years (median 5 months), with 73/90 (81%) less than 6 months and 1.7 times as many males as females. The disease became more common over the study period. Most cases (86%) were seen between March and July. Cerebrospinal fluid (CSF) was obtained from the cisterna magna in 77 dogs, the lumbar cistern in f5, and both sites in 3. Nucleated cell counts for 84 specimens ranged from 1 to 146 150 cells μL−1 (median 4500). Percentage eosinophils varied from 0 to 98% (median 83%). When both cisternal and lumbar CSF were collected, inflammation was more severe caudally. Seventy-three CSF specimens were subjected to enzyme-linked immunosorbent assay (ELISA) testing for antibodies against A. cantonensis; 61 (84%) tested positive, titres ranging from <100 to ⩾12 800 (median 1600). Sixty-one CSF specimens were subjected to real-time quantitative polymerase chain reaction (qPCR) testing using a new protocol targeting a bioinformatically-informed repetitive genetic target; 53/61 samples (87%) tested positive, C T values ranging from 23.4 to 39.5 (median 30.0). For 57 dogs, it was possible to compare CSF ELISA serology and qPCR. ELISA and qPCR were both positive in 40 dogs, in 5 dogs the ELISA was positive while the qPCR was negative, in 9 dogs the qPCR was positive but the ELISA was negative, while in 3 dogs both the ELISA and qPCR were negative. NA is an emerging infectious disease of dogs in Sydney, Australia.

scholarly journals Analysis and validation of a highly sensitive one-step nested quantitative real-time polymerase chain reaction assay for specific detection of severe acute respiratory syndrome coronavirus 2

2020 ◽  
Vol 17 (1) ◽  
Author(s):  
Yang Zhang ◽  
Chunyang Dai ◽  
Huiyan Wang ◽  
Yong Gao ◽  
Tuantuan Li ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Quantitative Polymerase Chain Reaction ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Qrt Pcr ◽  
Highly Sensitive ◽  
One Step ◽  
Polymerase Chain

Abstract Background Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is posing a serious threat to global public health. Reverse transcriptase real-time quantitative polymerase chain reaction (qRT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. Due to technical limitations, the reported positive rates of qRT-PCR assay of throat swab samples vary from 30 to 60%. Therefore, the evaluation of alternative strategies to overcome the limitations of qRT-PCR is required. A previous study reported that one-step nested (OSN)-qRT-PCR revealed better suitability for detecting SARS-CoV-2. However, information on the analytical performance of OSN-qRT-PCR is insufficient. Method In this study, we aimed to analyze OSN-qRT-PCR by comparing it with droplet digital PCR (ddPCR) and qRT-PCR by using a dilution series of SARS-CoV-2 pseudoviral RNA and a quality assessment panel. The clinical performance of OSN-qRT-PCR was also validated and compared with ddPCR and qRT-PCR using specimens from COVID-19 patients. Result The limit of detection (copies/ml) of qRT-PCR, ddPCR, and OSN-qRT-PCR were 520.1 (95% CI: 363.23–1145.69) for ORF1ab and 528.1 (95% CI: 347.7–1248.7) for N, 401.8 (95% CI: 284.8–938.3) for ORF1ab and 336.8 (95% CI: 244.6–792.5) for N, and 194.74 (95% CI: 139.7–430.9) for ORF1ab and 189.1 (95% CI: 130.9–433.9) for N, respectively. Of the 34 clinical samples from COVID-19 patients, the positive rates of OSN-qRT-PCR, ddPCR, and qRT-PCR were 82.35% (28/34), 67.65% (23/34), and 58.82% (20/34), respectively. Conclusion In conclusion, the highly sensitive and specific OSN-qRT-PCR assay is superior to ddPCR and qRT-PCR assays, showing great potential as a technique for detection of SARS-CoV-2 in patients with low viral loads.

scholarly journals Analysis and validation of a highly sensitive one-step nested quantitative real-time polymerase chain reaction assay for specific detection of severe acute respiratory syndrome coronavirus 2

2020 ◽  
Author(s):  
Yang Zhang ◽  
Chunyang Dai ◽  
Huiyan Wang ◽  
Yong Gao ◽  
Tuantuan Li ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Quantitative Polymerase Chain Reaction ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Qrt Pcr ◽  
Highly Sensitive ◽  
One Step ◽  
Polymerase Chain

Abstract Background Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is posing a serious threat to global public health. Reverse transcriptase real-time quantitative polymerase chain reaction (qRT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. Due to technical limitations, the reported positive rates of qRT-PCR assay of throat swab samples vary from 30–60%. Therefore, the evaluation of alternative strategies to overcome the limitations of qRT-PCR is required. A previous study reported that one-step nested (OSN)-qRT-PCR revealed better suitability for detecting SARS-CoV-2. However, information on the analytical performance of OSN-qRT-PCR is insufficient. Method: In this study, we aimed to analyze OSN-qRT-PCR by comparing it with droplet digital PCR (ddPCR) and qRT-PCR by using a dilution series of SARS-CoV-2 pseudoviral RNA and a quality assessment panel. The clinical performance of OSN-qRT-PCR was also validated and compared with ddPCR and qRT-PCR using specimens from COVID-19 patients. Result The LoD (copies/ml) of qRT-PCR, ddPCR, and OSN-qRT-PCR were 520.1 (95% CI): 363.23–1145.69) for ORF1ab and 528.1 (95% CI: 347.7–1248.7) for N, 401.8 (95% CI: 284.8–938.3) for ORF1ab and 336.8 (95% CI: 244.6–792.5) for N, and 194.74 (95% CI: 139.7–430.9) for ORF1ab and 189.1 (95% CI: 130.9–433.9) for N, respectively. Of the 34 clinical samples from COVID-19 patients, the positive rates of OSN-qRT-PCR, ddPCR, and qRT-PCR were 82.35% (28/34), 67.65% (23/34), and 58.82% (20/34), respectively. Conclusion In conclusion, the highly sensitive and specific OSN-qRT-PCR assay is superior to ddPCR and qRT-PCR assays, showing great potential as a technique for detection of SARS-CoV-2 in patients with low viral loads.

scholarly journals Airway Bacteria Quantification Using Polymerase Chain Reaction Combined with Neutrophil and Eosinophil Counts Identifies Distinct COPD Endotypes

Biomedicines ◽  
2021 ◽  
Vol 9 (10) ◽  
pp. 1337
Author(s):  
Augusta Beech ◽  
Simon Lea ◽  
Jian Li ◽  
Natalie Jackson ◽  
Alex Mulvanny ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Quantitative Polymerase Chain Reaction ◽  
Bacterial Species ◽  
Bacterial Load ◽  
Bacterial Colonisation ◽  
Chain Reaction ◽  
Cell Counts ◽  
Copd Patients ◽  
Polymerase Chain ◽  
Eosinophil Counts

Background: Chronic obstructive pulmonary disease (COPD) inflammatory endotypes are associated with different airway microbiomes. We used quantitative polymerase chain reaction (qPCR) analysis of sputum samples to establish the bacterial load upper limit in healthy controls; these values determined the bacterial colonisation prevalence in a longitudinal COPD cohort. Bacteriology combined with sputum inflammatory cells counts were used to investigate COPD endotypes. Methods: Sixty COPD patients and 15 healthy non-smoking controls were recruited. Sputum was analysed by qPCR (for Haemophilus influenzae, Moraxella catarrhalis, Streptococcus pneumoniae and Psuedomonas aeruginosa) and sputum differential cell counts at baseline and 6 months. Results: At baseline and 6 months, 23.1% and 25.6% of COPD patients were colonised with H. influenzae, while colonisation with other bacterial species was less common, e.g., S. pneumoniae—1.9% and 5.1%, respectively. H. influenzae + ve patients had higher neutrophil counts at baseline (90.1% vs. 67.3%, p < 0.01), with similar results at 6 months. COPD patients with sputum eosinophil counts ≥3% at ≥1 visit rarely showed bacterial colonisation. Conclusions: The prevalence of H. influenzae colonisation was approximately 25%, with low colonisation for other bacterial species. H. influenzae colonisation was associated with sputum neutrophilia, while eosinophilic inflammation and H. influenzae colonisation rarely coexisted.

Screening of toxoplasmosis in cancer patients: a concern

2018 ◽  
Vol 49 (1) ◽  
pp. 31-34 ◽  
Author(s):  
Amir Abdoli ◽  
Mohammad Barati ◽  
Majid Pirestani ◽  
Abdolhossein Dalimi
Keyword(s):  
Infectious Disease ◽  
Polymerase Chain Reaction ◽  
Cancer Patients ◽  
Enzyme Linked Immunosorbent Assay ◽  
Immunocompromised Patients ◽  
Chain Reaction ◽  
Nested Polymerase Chain Reaction ◽  
Blood Samples ◽  
Different Types ◽  
Polymerase Chain

Toxoplasmosis is an opportunistic infectious disease in immunocompromised patients, including cancer patients, whose detection is by molecular and serological methods. A total of 106 blood samples from patients with different types of cancer were evaluated for anti- Toxoplasma gondii IgG and IgM antibodies by the enzyme-linked immunosorbent assay (ELISA) and the parasite DNA by nested polymerase chain reaction (PCR). These were detected in 41.51% (44/106) and 0.94% (1/106), respectively, but T. gondii IgM antibody was not detected at all. These results suggest that the screening of toxoplasmosis should be considered more routinely in cancer patients.

scholarly journals RT-PCR Method for Detecting Cowpea Mottle Carmovirus in Vigna Germ Plasm

Plant Disease ◽  
1999 ◽  
Vol 83 (7) ◽  
pp. 639-643 ◽  
Author(s):  
A. G. Gillaspie ◽  
S. E. Mitchell ◽  
G. W. Stuart ◽  
R. F. Bozarth
Keyword(s):  
Germ Plasm ◽  
Enzyme Linked Immunosorbent Assay ◽  
Mild Mosaic ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Viral Pathogens ◽  
Highly Sensitive ◽  
Pcr Method ◽  
The Common ◽  
Polymerase Chain

A highly sensitive reverse transcription-polymerase chain reaction (RT-PCR) method was developed to detect cowpea mottle carmovirus (CPMoV) in newly acquired germ plasm of Vigna spp. It detected virus in tissues diluted up to 10-9. The preferred primers were designed from the RNA replicase cDNA sequence of CPMoV. These primers were able to detect CPMoV in plants infected with 10 different isolates of the virus. There were no cross-reactions with either bean mild mosaic or melon necrotic spot carmoviruses or any of the common cowpea viral pathogens tested. The RT-PCR method was up to 105 times more sensitive than direct antigen coating enzyme-linked immunosorbent assay (DAC-ELISA) in detecting CPMoV. The RT-PCR method gave no false positive reaction as is sometimes seen with ELISA.

Salivary Diagnostics

2012 ◽  
Vol 37 (6) ◽  
pp. 562-570 ◽  
Author(s):  
DTW Wong
Keyword(s):  
Quantitative Polymerase Chain Reaction ◽  
Clinical Applications ◽  
Cardiovascular Disorders ◽  
Systemic Diseases ◽  
Chain Reaction ◽  
Salivary Biomarkers ◽  
Highly Sensitive ◽  
Polymerase Chain ◽  
Clinical Conditions ◽  
Salivary Diagnostics

SUMMARY Saliva is a noninvasive and accessible biofluid that permits early detection of oral and systemic diseases. Recent scientific and technologic advances have uncovered specific salivary biomarkers for a number of clinical conditions, including cancers, autoimmune diseases, and cardiovascular disorders. The availability of highly sensitive and high-throughput assays such as microarray, mass spectrometry, reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) and nano-scale sensors that can measure proteins and nucleic acids are poising saliva as an emerging biofluid for translational and clinical applications. This paper will discuss development of salivary biomarkers for the detection of oral and systemic diseases and the translational application of these markers for clinical applications.

scholarly journals Analysis and validation of a highly sensitive one-step nested quantitative real-time polymerase chain reaction assay for specific detection of severe acute respiratory syndrome coronavirus 2

2020 ◽  
Author(s):  
Yang Zhang ◽  
Chunyang Dai ◽  
Huiyan Wang ◽  
Yong Gao ◽  
Tuantuan Li ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Quantitative Polymerase Chain Reaction ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Qrt Pcr ◽  
Highly Sensitive ◽  
One Step ◽  
Polymerase Chain

Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is posing a serious threat to global public health. Reverse transcriptase real-time quantitative polymerase chain reaction (qRT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. Due to technical limitations, the reported positive rates of qRT-PCR assay of throat swab samples vary from 30%-60%. Therefore, the evaluation of alternative strategies to overcome the limitations of qRT-PCR is required. A previous study reported that one-step nested (OSN)-qRT-PCR revealed better suitability for detecting SARS-CoV-2. However, information on the analytical performance of OSN-qRT-PCR is insufficient. In this study, we aimed to analyze OSN-qRT-PCR by comparing it with droplet digital PCR (ddPCR) and qRT-PCR by using a dilution series of SARS-CoV-2 pseudoviral RNA and a quality assessment panel. The clinical performance of OSN-qRT-PCR was also validated and compared with ddPCR and qRT-PCR using specimens from COVID-19 patients. The LoD (copies/ml) of qRT-PCR, ddPCR, and OSN-qRT-PCR were 520.1 (95% CI): 363.23-1145.69) for ORF1ab and 528.1 (95% CI: 347.7-1248.7) for N, 401.8 (95% CI: 284.8-938.3) for ORF1ab and 336.8 (95% CI: 244.6-792.5) for N, and 194.74 (95% CI: 139.7-430.9) for ORF1ab and 189.1 (95% CI: 130.9-433.9) for N, respectively. Of the 34 clinical samples from COVID-19 patients, the positive rates of OSN-qRT-PCR, ddPCR, and qRT-PCR were 82.35% (28/34), 67.65% (23/34), and 58.82% (20/34), respectively. In conclusion, the highly sensitive and specific OSN-qRT-PCR assay is superior to ddPCR and qRT-PCR assays, showing great potential as a technique for detection of SARS-CoV-2 in patients with low viral loads.

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