Diagnosis of canine leptospirosis: evaluation of two PCR assays in comparison with the microagglutination test

ABSTRACT: Canine leptospirosis is definitely diagnosed by demonstrating seroconversion in paired serum samples from the acute and convalescent period by the microagglutination test (MAT). However, the application of a polymerase chain reaction (PCR) assay can provide earlier confirmation of suspected cases. The objective of this study was to evaluate two PCR assays used in diagnosis of human leptospirosis (lipL32 real-time PCR and rrs conventional PCR) in cultured microorganisms and experimentally contaminated samples (whole blood, serum, urine), and investigate their applicability in clinical samples from dogs with presumptive diagnosis of leptospirosis by using the MAT as a reference. The analytical sensitivity of the lipL32 real-time PCR was 1 genome equivalent per reaction, whereas that for the rrs conventional PCR was 10 genome equivalents per reaction. Both assays amplified the pathogenic strains but were negative when evaluating the DNA of other microorganisms that may be present in clinical samples. The lipL32 real-time PCR detected 100 bacteria/mL in whole blood samples, 1000 bacteria/mL in serum samples and 10 bacteria/mL in urine samples, whereas the rrs conventional PCR detected 1000 bacteria/mL in whole blood and serum samples and 100 bacteria/mL in urine samples. Seven out of the 51 samples from dogs with presumptive diagnosis of leptospirosis were considered as confirmed cases. ThelipL32 real-time PCR detected positive results in six of the seven confirmed cases, whereas the rrs conventional PCR detected four. The PCR assays evaluated proved to be useful diagnostic tools in the confirmation of canine leptospirosis when used together with the MAT.

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Real-time PCR for detecting circulating dengue virus in the Guangdong Province of China in 2006

Journal of Medical Microbiology ◽

10.1099/jmm.0.2008/003418-0 ◽

2008 ◽

Vol 57 (12) ◽

pp. 1547-1552 ◽

Cited By ~ 12

Author(s):

Zhijun Bai ◽

Licheng Liu ◽

Zeng Tu ◽

Lisi Yao ◽

Jianwei Liu ◽

...

Keyword(s):

Dengue Virus ◽

Real Time ◽

Real Time Pcr ◽

Cross Reactivity ◽

Clinical Samples ◽

Sequencing Analysis ◽

Serum Samples ◽

Serological Tests ◽

Wide Range ◽

Pcr Assays

Dengue virus (DENV) causes a wide range of diseases in humans, from the acute febrile illness dengue fever (DF) to life-threatening dengue haemorrhagic fever/dengue shock syndrome. We developed four real-time quantitative PCR assays for each serotype of DENV based on computational analysis. These assays had high sensitivity and specificity without cross-reactivity for the four serotypes. To evaluate the performance of these assays in detecting and typing the virus in clinical samples, we analysed 64 serum samples from Guangdong during 2006. The results showed that 71 % of those samples were positive by the DEN-1 assay. The DENV assay results, in agreement with the serological tests and sequencing analysis, showed that the pathogen resulting in the DF explosion in Guangdong in 2006 belonged to DEN-1. Compared to the serological assays, the real-time PCR assays that we developed were much more sensitive in the 1–3 days after onset of the symptoms.

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Comparison of Different PCR Methods for Detection of Brucella spp. in Human Blood Samples

Polish Journal of Microbiology ◽

10.33073/pjm-2011-004 ◽

2011 ◽

Vol 60 (1) ◽

pp. 27-33 ◽

Cited By ~ 21

Author(s):

HISHAM H. AL-AJLAN ◽

ABDELNASSER S.S. IBRAHIM ◽

ALI A. AL-SALAMAH

Keyword(s):

Blood Culture ◽

Real Time ◽

Real Time Pcr ◽

Brucella Melitensis ◽

Blood Analysis ◽

Clinical Samples ◽

Conventional Pcr ◽

Serum Samples ◽

Culture Methods ◽

Blood Specimens

For detection of Brucella species by PCR four DNA extraction methods and four targets were compared using pure culture of Brucella melitensis and the best conditions were applied in clinical samples. It was found that the MagNA Pure LC method was the most efficient and sensitive method showing a positive PCR reaction with DNA extracted from as low as 25 and 100 CFU suspended in one ml blood and one ml water, respectively. Detection of Brucella spp. by conventional PCR was investigated using four different targets. The results indicated that The B4-B5 amplification method was the most sensitive one as it could amplify DNA extracted from as a low as 25 and 100 CFU/ml suspended in one ml water and blood, respectively. Furthermore real-time PCR was able to detect Brucella using DNA extracted from as low as 50 CFU/ml blood and 15 CFU/ml water, respectively. The best and optimum detection conditions were applied to the clinical samples. Evaluation of conventional PCR assays on blood specimens confirmed 72% of the results obtained by conventional blood culture methods with a specificity of 95%, while serum samples had a sensitivity of 54% and specificity of 100%. Real-time PCR was generally found to be more sensitive and specific for detecting Brucella spp. in blood and serum samples compared to conventional PCR. The real-time PCR done on blood specimens confirmed 77.5% of the results obtained by conventional blood culture methods with specificity of 100%, while 60% of serum samples were found to be positive with specificity of 100%. These results suggest that serum and blood analysis by conventional and real time PCR is a convenient and safe method for rapid and accurate diagnosis of brucellosis.

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Faculty Opinions recommendation of Evaluation of conventional and real-time PCR assays using two targets for confirmation of results of the COBAS AMPLICOR Chlamydia trachomatis/Neisseria gonorrhoeae test for detection of Neisseria gonorrhoeae in clinical samples.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1025709.374387 ◽

2006 ◽

Author(s):

David Persing

Keyword(s):

Chlamydia Trachomatis ◽

Neisseria Gonorrhoeae ◽

Real Time ◽

Real Time Pcr ◽

Clinical Samples ◽

Pcr Assays

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Detection of Human Polyomaviruses in Urine from Bone Marrow Transplant Patients: Comparison of Electron Microscopy with PCR

Clinical Chemistry ◽

10.1373/clinchem.2003.024539 ◽

2004 ◽

Vol 50 (2) ◽

pp. 306-312 ◽

Cited By ~ 24

Author(s):

Stefan S Biel ◽

Andreas Nitsche ◽

Andreas Kurth ◽

Wolfgang Siegert ◽

Muhsin Özel ◽

...

Keyword(s):

Electron Microscopy ◽

Bone Marrow ◽

Real Time ◽

Bone Marrow Transplant ◽

Real Time Pcr ◽

Marrow Transplant ◽

Test System ◽

Urine Samples ◽

Clinical Samples ◽

Transplant Patients

Abstract Background: We studied electron microscopy (EM) as an appropriate test system for the detection of polyomavirus in urine samples from bone marrow transplant patients. Methods: We evaluated direct EM, ultracentrifugation (UC) before EM, and solid-phase immuno-EM (SPIEM). The diagnostic accuracy of EM was studied by comparison with a real-time PCR assay on 531 clinical samples. Results: The detection rate of EM was increased by UC and SPIEM. On 531 clinical urine samples, the diagnostic sensitivity of EM was 47% (70 of 149) with a specificity of 100%. We observed a linear relationship between viral genome concentration and the proportion of urine samples positive by EM, with a 50% probability for a positive EM result for urine samples with a polyomavirus concentration of 106 genome-equivalents (GE)/mL; the probability of a positive EM result was 0% for urine samples with <103 GE/mL and 100% for urine samples containing 109 GE/mL. Conclusions: UC/EM is rapid and highly specific for polyomavirus in urine. Unlike real-time PCR, EM has low sensitivity and cannot quantify the viral load.

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Novel multiplex real-time PCR assays reveal a high prevalence of diarrhoeagenic Escherichia coli pathotypes in healthy and diarrhoeal children in the South of Vietnam

10.21203/rs.3.rs-18430/v1 ◽

2020 ◽

Author(s):

Vu Thuy Duong ◽

Le Thi Phuong Tu ◽

Ha Thanh Tuyen ◽

Le Thi Quynh Nhi ◽

James I Campbell ◽

...

Keyword(s):

Escherichia Coli ◽

Real Time ◽

Real Time Pcr ◽

Clinical Samples ◽

Middle Income ◽

Middle Income Countries ◽

Virulence Markers ◽

Low Middle Income Countries ◽

High Prevalence ◽

Pcr Assays

Abstract BackgroundDiarrhoeagenic Escherichia coli (DEC) infections are common in children in low-middle income countries (LMICs). However, detecting the various DEC pathotypes is complex as they cannot be differentiated by classical microbiology. We developed four multiplex real-time PCR assays were to detect virulence markers of six DEC pathotypes; specificity was tested using DEC controls and other enteric pathogens. PCR amplicons from the six E. coli pathotypes were purified and amplified to be used to optimize PCR reactions and to calculate reproducibility. After validation, these assays were applied to clinical samples from healthy and diarrhoeal Vietnamese children and associated with clinical data. ResultsThe multiplex real-time PCRs were found to be reproducible, and specific. At least one DEC variant was detected in 34.7% (978/2,815) of the faecal samples from diarrhoeal children; EAEC, EIEC and atypical EPEC were most frequent Notably, 41.2% (205/498) of samples from non-diarrhoeal children was positive with a DEC pathotype. In this population, only EIEC, which was detected in 34.3% (99/289) of diarrhoeal samples vs. 0.8% (4/498) non-diarrhoeal samples (p<0.001), was significantly associated with diarrhoea. Multiplex real-time PCR when applied to clinical samples is an efficient and high-throughput approach to DEC pathotypes. ConclusionsThis approach revealed high carriage rates of DEC pathotypes among Vietnamese children. We describe a novel diagnostic approach for DEC, which provides baseline data for future surveillance studies assessing DEC burden in LMICs.

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