Diagnostic Efficiency of Different Serological Tests and Real time PCR for Detecting Brucella Infection in Camels' Sera

Brucellosis, globally known bacterial zoonosis, is endemic to Pakistan. B. abortus in bovines, B. melitensis in small ruminants and B. canis in dogs mainly cause this disease. A total of 1821 sera (1196 from sheep and 625 from goats) from animal herds near the Pakistan–Afghanistan border were collected. In parallel testing of sera for anti-Brucella antibodies (B. abortus and B. melitensis) was carried out by RBPT and indirect ELISA. The presence of Brucella DNA in sera was tested by real-time PCR. The overall percentage of seropositive samples was 0.99 (18/1821) by both tests. All positive samples originated from Baluchistan territory which translated into 1.76% (18/1021). None of the positive sera had signals for Brucella DNA and none of sera from goats carried detectable antibodies. Both tests showed an almost perfect agreement with Kappa statistics. The flock size was found to be associated with the presence of anti-Brucella antibodies. The samples of Khyber Pakhtunkhwa (KPK) tested negative in both serological tests and hence were not processed for real-time PCR. The present study shows the presence of anti-Brucella antibodies in sheep in the Baluchistan region of Pakistan. Diagnostic services need to be improved and test and slaughter policies might be implemented for eradication of Brucella infection in these areas. Awareness about the infection is needed at the farmer’s level. Isolation and molecular biology of the isolates could help with understanding the prevailing etiology in a better way.

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The Light and Shadow of Rapid Serological Tests for SARS-CoV-2 Infection: Results from a Study in a Large Emergency Department

International Journal of Environmental Research and Public Health ◽

10.3390/ijerph17186493 ◽

2020 ◽

Vol 17 (18) ◽

pp. 6493

Author(s):

Daniela Loconsole ◽

Francesca Centrone ◽

Caterina Morcavallo ◽

Silvia Campanella ◽

Anna Sallustio ◽

...

Keyword(s):

Emergency Department ◽

Real Time ◽

Real Time Pcr ◽

Respiratory Symptoms ◽

Large Scale ◽

Serological Test ◽

Serological Tests ◽

Serological Assay ◽

Infected People ◽

Screening Programs

A critical point in the management of the SARS-CoV-2 pandemic is the need to promptly identify the greatest number of infected people and to implement strict public health measures. In this study, the performance of a rapid serological test in a clinical setting was evaluated. Samples from 819 consecutive patients (with or without respiratory symptoms) admitted to a large Emergency Department were tested between 23 March and 21 April 2020. Patient samples were tested in a real-time PCR assay and a serological assay. In total, 148/819 patients (18.1%) tested positive for SARS-CoV-2 by real-time PCR. The serological test revealed that 70/819 patients (8.5%) had anti-SARS-CoV-2 IgM and/or IgG. The prevalence of anti-SARS-CoV-2 antibodies was significantly higher in patients with respiratory symptoms lasting for >7 days than in those with respiratory symptoms lasting for 0–7 days (p < 0.001). The serological assay had an overall sensitivity of 35.1% and an overall specificity of 97.3%. A high negative predictive value (96.7%) was reported for patients without respiratory symptoms. The results confirm that rapid serological assays alone are not sufficient for diagnosis of SARS-CoV-2 infection but can be incorporated into large-scale screening programs during periods in which the virus circulation is low.

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Serologic and Molecular Detection of Mycoplasma gallisepticum in Layer Flocks in South Marmara Region of Turkey

Journal of Advances in Microbiology ◽

10.9734/jamb/2021/v21i1030390 ◽

2021 ◽

pp. 29-37

Author(s):

Elçin Günaydın ◽

Özlem Kardoğan ◽

Gülşen Goncagül ◽

Yavuz Çokal

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Molecular Detection ◽

Preventive Measures ◽

Mycoplasma Gallisepticum ◽

Marmara Region ◽

Time Loss ◽

Serological Tests ◽

Primary Screening ◽

Pooled Samples

Background: Due to the economic impacts of Mycoplasma gallisepticum (MG) infection in poultry, it is essential to have a fast, reliable and accurate diagnostic test to diagnose the infection. Aims: It was aimed to examine the presence of MG in the South Marmara Region of Turkey where extensive commercial layer flocks exist by RPA, ELISA and real-time PCR. Materials and Methods: In the study, 981 sera and 160 tracheal swab samples (20 swabs per each flock) obtained from eight layer flocks were examined for the presence of MG-antibody by RPA, ELISA, and the presence of MG by real-time PCR, respectively. Results: MG-seropositive flock rate was determined to be 100% by RPA. Twenty-three of the RPA positive sera in each flock LA, LB, LC, LD, LF, LG, and 17 RPA positive sera in flock LE (due to 17 positive RPA sera obtained) were examined for the presence of MG antibody by ELISA, and MG-seropositive flock rate was determined to be 87.5%. As a result of the examination of a total of 32 tracheal swab samples (20 swabs perflock/5 swabs=4 pooled samples, 8 flocksX4 pooled samples= 32 samples) for the presence of MG, real-time PCR positive flock rate was found to be 75%. Conclusion: To decide the flock whether it is infected or not and the initiate effective preventive measures against MG infection as soon as possible; serology should be applied simultaneously with bacteriology and/or PCR to prevent time loss due to shortcomings of serological tests used as primary screening test such as cross reactions, sensitivity and specificity problems.

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A Novel Real-Time PCR-Based Screening Test with Pooled Fecal Samples for Bovine Johne’s Disease

Journal of Clinical Microbiology ◽

10.1128/jcm.01761-20 ◽

2020 ◽

Vol 58 (12) ◽

Author(s):

Satoko Kawaji ◽

Reiko Nagata ◽

Yasutaka Minegishi ◽

Yumi Saruyama ◽

Akiko Mita ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Johne's Disease ◽

Johne’S Disease ◽

Enzyme Linked Immunosorbent Assay ◽

Pcr Assay ◽

Serological Tests ◽

Fecal Samples ◽

Content Type ◽

Cattle Herds

ABSTRACT Johne’s disease (JD) is an economically important infectious disease in livestock farming caused by Mycobacterium avium subsp. paratuberculosis. As an alternative to serological tests, which are used mainly for the screening of whole herds, we developed a novel ResoLight-based real-time PCR (RL-PCR) assay with pooled fecal samples for the detection of fecal shedders in cattle herds. The RL-PCR assay included an internal amplification control (IC) which was amplified using the same primer pair as the target molecule M. avium subsp. paratuberculosis IS900 and differentiated based on melting temperatures. Individual fecal suspensions were pooled and concentrated by centrifugation to avoid a loss of sensitivity by the dilution effect. Combined with a DNA extraction kit (Johne-PureSpin; FASMAC), no inhibition of PCR amplification was observed with up to 15 fecal samples in a pool. The detection limit of RL-PCR at a pool size of 10 was 10 M. avium subsp. paratuberculosis organisms per gram of feces, which was comparable to that of individual testing. A total of 2,654 animals in 12 infected herds were screened by individual antibody-enzyme-linked immunosorbent assay (ELISA) and the RL-PCR assay using pooled feces. Fifty animals were diagnosed with JD through the screening by RL-PCR, compared with only 5 by ELISA (which were also positive in RL-PCR). In 7 JD-free herds, the results of 4 out of 327 pools (1.2%) were invalid due to the lack of IC amplification, and then animals were confirmed negative individually. Our results suggest that implementation of herd screening by pooled RL-PCR would advance the monitoring and control of JD in cattle herds.

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Real time PCR assay for differentiation of Brucella abortus and Brucella melitensis

Indian Journal of Animal Research ◽

10.18805/ijar.v0iof.8464 ◽

2017 ◽

Author(s):

Vinay Kumar ◽

Sushila Maan ◽

Aman Kumar ◽

Kanisht Batra ◽

Deepika Deepika ◽

...

Keyword(s):

Real Time ◽

Brucella Abortus ◽

Real Time Pcr ◽

Brucella Melitensis ◽

Economic Losses ◽

Conventional Pcr ◽

Serological Tests ◽

Detectable Difference ◽

Pcr Assays ◽

Respective Species

Brucellosis is one of the zoonotic diseases of major concern and can cause huge economic losses to livestock industry. Serological tests and bacterial isolation are considered as the gold standard assay for diagnosis of Brucella spp. but they are time-consuming, hazardous and lack specificity. To control and eradicate a disease, a confirmatory diagnostic method which is sensitive, quick and specific is the foremost requirement. Therefore in this study, we evaluated the performances of two newly designed TaqMan real-time PCR assays targeting the BruAB_0168 gene and BMEII0466 gene for Brucella abortus and Brucella melitensis (respectively). Both the assays were found to be highly specific in differentiation of respective species. Both the assays can detect as low as 0.02 fg of DNA and there was no detectable difference found in sensitivity of these two tests. R2 value and efficiency of these tests ranged from 0.992 - 0.998 and 100- 106%, respectively showing that these assays are highly efficient. Compared to conventional PCR assays these qPCR assays were 100 times higher sensitive. In conclusion, the present study showed that the developed real-time PCR assays are more sensitive, specific, have high reproducibility and repeatability and are faster than serological and conventional PCR methods for differentiation of Brucella abortus and Brucella melitensis.

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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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Epidemiological situation of Q fever in sheep, goats and cattle in the Wielkopolska Voivodeship between the years 2011 and 2015, based on monitoring studies and cases from clinical practice

Medycyna Weterynaryjna ◽

10.21521/mw.5621 ◽

2017 ◽

Vol 73 (1) ◽

pp. 56-61

Author(s):

Piotr Kneblewski ◽

Jolanta Budzyk ◽

Leslaw Szabłoński ◽

Jan Olechnowicz ◽

Michał Majewski ◽

...

Keyword(s):

Clinical Practice ◽

Positive Result ◽

Real Time ◽

Dna Sequence ◽

Real Time Pcr ◽

Q Fever ◽

Serological Tests ◽

Veterinary Research Institute ◽

Veterinary Research ◽

National Veterinary Research Institute

The publication presents the results of monitoring Q fever in the Wielkopolska Voivodeship and three outbreaks disclosed as part of a clinical field practice. In five years (2011 – 2015) of examination in the Wielkopolska Voivodeship, 2,431 serological tests were carried out (1,851 in sheep, 343 in goats and 237 in cattle). Antibodies against Coxiella burneti were found three times. The first positive result in 2011 affected herds of goats and cattle and was confirmed in the reference laboratory of the National Veterinary Research Institute in Pulawy. A specific DNA sequence for Coxiella burneti by real-time PCR method was found. The farm consisted of 1,494 goats and 397 cattle. Serological tests were carried out to give positive results in 15.3% of the cattle and 5.77% of the goats from the whole herd. Breeding selection and the elimination of seropositive animals and double oxytetracycline treatment reduced the proportion of animals with a positive result to 5.53% in cattle and 0.96% in goats. After more than a year the elimination of seropositive animals and probable natural decline in antibody levels has led to the recognition of an outbreak of Q fever to be eliminated. The second positive result of the monitoring of Q fever was found in 2014 in one cow out of seven respondents, but the serological test was not confirmed in the reference, as a specific DNA sequence for Coxiella burneti was not found. The research conducted in sheep in 2015 showed the presence of antibodies against Coxiella burneti in two samples. The results were confirmed by the detection of genetic material of the pathogen by real-time PCR examination in the National Veterinary Research Institute in Pulawy. Three outbreaks of Q fever revealed in clinical practice related to bovine herds where clinical disturbances were observed in: reproduction, milk production decrease or increase in internal body temperature and symptoms of the respiratory system. The positive ELISA test results were the reason for the elimination of seropositive animals. Moreover, after the disclosure of infection two herds were vaccinated using an inactivated vaccine Coxevac (CEVA), which caused the improvement of production results and relief of clinical symptoms. It is worth mentioning that in two farms along with cattle there were fallow deer supported by staff cowman. Official monitoring tests of Q fever revealed an outbreak of the disease in a herd of goats and cattle, which lead to taking effective action to protect public health because of the zoonotic nature of this infection and epidemiological risk. In the disclosure of these clinical signs in cattle it is advisable to carry out laboratory tests for Q fever.

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Meningitis patients with Angiostrongylus cantonensis may present without eosinophilia in the cerebrospinal fluid in northern Vietnam

PLoS Neglected Tropical Diseases ◽

10.1371/journal.pntd.0008937 ◽

2020 ◽

Vol 14 (12) ◽

pp. e0008937

Author(s):

Tomoko Hiraoka ◽

Ngo Chi Cuong ◽

Sugihiro Hamaguchi ◽

Mihoko Kikuchi ◽

Shungo Katoh ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Clinical Syndrome ◽

Angiostrongylus Cantonensis ◽

Tertiary Referral Hospital ◽

Serological Tests ◽

Blood Eosinophilia ◽

Northern Vietnam ◽

Cell Counts ◽

Eosinophil Cell

Background Eosinophilic meningitis (EM) is a rare clinical syndrome caused by both infectious and noninfectious diseases. In tropical pacific countries, Angiostrongylus cantonensis is the most common cause. However, the EM definition varies in the literature, and its relation to parasitic meningitis (PM) remains unclear. Methodology/Principal findings Adult and adolescent patients of 13 years old or above with suspected central nervous system (CNS) infections with abnormal CSF findings were prospectively enrolled at a tertiary referral hospital in Hanoi, Vietnam from June 2012 to May 2014. Patients with EM or suspected PM (EM/PM) were defined by the presence of either ≥10% eosinophils or an absolute eosinophil cell counts of ≥10/mm3 in the CSF or blood eosinophilia (>16% of WBCs) without CSF eosinophils. In total 679 patients were enrolled: 7 (1.03%) had ≥10% CSF eosinophilia, 20 (2.95%) had ≥10/mm3 CSF eosinophilia, and 7 (1.03%) had >16% blood eosinophilia. The patients with ≥10% CSF eosinophilia were significantly younger (p = 0.017), had a lower body temperature (p = 0.036) than patients with ≥10/mm3 CSF eosinophilia among whom bacterial pathogens were detected in 72.2% (13/18) of those who were tested by culture and/or PCR. In contrast, the characteristics of the patients with >16% blood eosinophilia resembled those of patients with ≥10% CSF eosinophilia. We further conducted serological tests and real-time PCR to identify A. cantonensis. Serology or real-time PCR was positive in 3 (42.8%) patients with ≥10% CSF eosinophilia and 6 (85.7%) patients with >16% blood eosinophilia without CSF eosinophils but none of patients with ≥10/mm3 CSF eosinophilia. Conclusions The etiology of PM in northern Vietnam is A. cantonensis. The eosinophil percentage is a more reliable predictor of parasitic EM than absolute eosinophil count in the CSF. Patients with PM may present with a high percentage of eosinophils in the peripheral blood but not in the CSF.

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Optimizing Real-Time PCR methods for detection of ssaN gene Salmonella enterica subsp.enterica in the blood specimen

Journal of Biomedicine and Translational Research ◽

10.14710/jbtr.v6i2.7120 ◽

2020 ◽

Vol 6 (2) ◽

pp. 41-47

Author(s):

Oktania Sandra Puspita ◽

Andi Yasmon ◽

Beti Ernawati Dewi

Keyword(s):

Typhoid Fever ◽

Real Time ◽

Real Time Pcr ◽

Salmonella Enterica ◽

Detection System ◽

Acute Infection ◽

False Negative ◽

Salmonella Typhi ◽

Serological Tests ◽

Blood Specimen

Background Typhoid fever caused by Salmonella typhi is a common acute infection of the reticuloendothelial system, intestinal lymphoid tissue, and gall bladder. Detection of Salmonella spp. is still based on cultures and serological methods.Widal test is one of the serological tests that is still widely used, especially in developing countries including Indonesia.Widal tests have low sensitivity and specificity. They often produce false positive or false negative results.ObjectiveThe aim of this study were i) real time PCR optimization to develop a Salmonella enterica detection system. ii) molecular detection of new target gene (ssaN gene) from blood specimens in typhoid fever patients.Methods An experimental laboratory study was performed from March to October 2016. Extraction of Salmonella typhi DNA is used as templates for the optimization of real time PCR reaction.The blood sample was from patients suspected with typhoid fever obtained from the Menteng Sub-district Health Center according to the inclusion criteria.ResultsSpecificity test of real time PCR showed that the primers and probes used are not cross-react against other microorganisms. Sensitivity test obtained minimal detection is at least 10 cfu/ml of blood specimen. In blood clinical specimens, real time PCR could detect 19 (38%) positive samples of 50 blood specimen from suspected typhoid fever patients. Eleven samples with negative Widal serology gives positive results in real time PCR.ConclusionReal time PCR used in this study can increase the level of rate of positive testing by 22% of the total specimens.Keywords : Salmonella enterica subsp.enterica, typhoid fever, ssaN gene, real time PCR

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