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

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.

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

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.

scholarly journals Quantitative PCR with 16S rRNA-Gene-Targeted Species-Specific Primers for Analysis of Human Intestinal Bifidobacteria

2004 ◽  
Vol 70 (1) ◽  
pp. 167-173 ◽  
Author(s):  
Takahiro Matsuki ◽  
Koichi Watanabe ◽  
Junji Fujimoto ◽  
Yukiko Kado ◽  
Toshihiko Takada ◽  
...  
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
Real Time Pcr ◽  
Intestinal Flora ◽  
Healthy Adults ◽  
Pcr Detection ◽  
Distribution Analysis ◽  
Specific Primers ◽  
Cell Counts ◽  
Species Specific

ABSTRACT A highly sensitive quantitative PCR detection method has been developed and applied to the distribution analysis of human intestinal bifidobacteria by combining real-time PCR with Bifidobacterium genus- and species-specific primers. Real-time PCR detection of serially diluted DNA extracted from cultured bifidobacteria was linear for cell counts ranging from 106 to 10 cells per PCR assay. It was also found that the method was applicable to the detection of Bifidobacterium in feces when it was present at concentrations of >106 cells per g of feces. Concerning the distribution of Bifidobacterium species in intestinal flora, the Bifidobacterium adolescentis group, the Bifidobacterium catenulatum group, and Bifidobacterium longum were found to be the three predominant species by examination of DNA extracted from the feces of 46 healthy adults. We also examined changes in the population and composition of Bifidobacterium species in human intestinal flora of six healthy adults over an 8-month period. The results showed that the composition of bifidobacterial flora was basically stable throughout the test period.

Diagnosis of intramammary infection in samples yielding negative results or minor pathogens in conventional bacterial culturing

2010 ◽  
Vol 78 (1) ◽  
pp. 49-55 ◽  
Author(s):  
Ricardo Bexiga ◽  
Mikko T Koskinen ◽  
Jani Holopainen ◽  
Carla Carneiro ◽  
Helena Pereira ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Real Time ◽  
Real Time Pcr ◽  
Gram Positive ◽  
Milk Samples ◽  
Negative Results ◽  
Streptococcus Uberis ◽  
Cell Counts ◽  
Significant Difference ◽  
Culture Negative

Up to half of quarter milk samples submitted for mastitis diagnosis are culture-negative results or lead to identification of coagulase-negative staphylococci or Corynebacterium bovis in conventional culturing, the so-called minor pathogens. The interpretation and usefulness of these results in terms of udder and animal health management is limited, even though the amount of resources spent is relatively high. This work aimed to test two methods of analysis of milk samples with the goal of increasing detection of intramammary pathogens. In the first study, 783 milk samples were processed in duplicate: before and after freezing at −20°C for 24 h, using standard bacteriological techniques. There was a significant difference between the two methods with samples frozen for 24 h yielding significantly fewer Gram-positive catalase-positive cocci, Gram-negative bacilli, Gram-positive bacilli and significantly more samples leading to no growth, than samples before freezing. The number of samples yielding Gram-positive catalase-negative cocci was not significantly affected by freezing. In the second study, a real-time PCR-based test was performed on milk samples with an individual quarter somatic cell count above 500 000 cells/ml that were either negative (n=51 samples) or that led to the isolation of minor pathogens in culturing: Corynebacterium bovis (n=79 samples) or non-aureus staphylococci (NAS, n=32). A mastitis pathogen, beyond the result obtained with standard bacteriology, was detected on 47% of the no-growth samples, on 35% of the samples from which C. bovis had been isolated and on 25% of the samples from which NAS had been isolated. The most commonly detected major pathogen was Escherichia coli, followed by Streptococcus uberis, Arcanobacterium pyogenes/Peptoniphilus indolicus and Streptococcus dysgalactiae. These results suggest that simply freezing milk samples for 24 h does not increase the detection of intramammary bacteria in milk samples and therefore should not be recommended. However, use of the real-time PCR-based test may be useful in diagnosing intramammary infections when milk samples with high somatic cell counts are culture-negative or when culturing results in the detection of minor pathogens.

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

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.

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

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.

Enumeration of viable Escherichia coli by real-time PCR with propidium monoazide

2012 ◽  
Vol 66 (10) ◽  
pp. 2065-2073 ◽  
Author(s):  
N. Yokomachi ◽  
J. Yaguchi
Keyword(s):  
Escherichia Coli ◽  
Wastewater Treatment ◽  
Real Time ◽  
Real Time Pcr ◽  
Wastewater Treatment Plants ◽  
Propidium Monoazide ◽  
E Coli ◽  
Cell Counts ◽  
Viable Cells ◽  
Heat Treated

A photo-inducible DNA-binding dye, propidium monoazide (PMA), was used to distinguish viable and dead Escherichia coli cells. Microscopic observations using a combination of the dyes 4′,6-diamidino-2-phenylindole and PMA indicated that PMA stained only dead cells, with membrane damage, red. Mixtures of viable and heat-treated E. coli cells were subjected to real-time polymerase chain reaction (PCR) with PMA treatment. Viable cell counts were linearly related to real-time PCR threshold cycle values for PMA-treated cells in the mixtures of viable and heat-treated cells, as long as the ratio of dead cells to viable cells was no greater than 10. In the wastewater treatment plants, total, viable and culturable E. coli were enumerated by real-time PCR, real-time PCR coupled with PMA treatment and the most probable number method using EC-MUG medium, respectively. The concentrations of viable E. coli in the wastewater treatment plants were much higher than those of culturable cells. In addition, viable cells were even more chlorine resistant than culturable ones.

Rapid Detection of Listeria monocytogenes by Real-Time PCR in Processed Meat and Dairy Products

2014 ◽  
Vol 77 (3) ◽  
pp. 453-458 ◽  
Author(s):  
EUN JEONG HEO ◽  
BO RA SONG ◽  
HYUN JUNG PARK ◽  
YOUNG JO KIM ◽  
JIN SAN MOON ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Real Time ◽  
Infant Formula ◽  
Real Time Pcr ◽  
Processed Meat ◽  
Ground Meat ◽  
Rt Pcr ◽  
Hybridization Probe ◽  
Cell Counts ◽  
Two Samples

The objectives of this study were to evaluate the detection of Listeria monocytogenes in different ready-to-eat foods using real-time PCR (RT-PCR). Various concentrations (100 to 105 CFU/ml) of L. monocytogenes ATCC 19115 were inoculated into ham, sausage, ground meat, processed milk, cheese, and infant formula. L. monocytogenes ATCC 19115 in the samples was then enumerated on Oxford agar, and DNA was extracted from the samples before and after incubation at 36°C for 4 h. A set of primers and hybridization probe designed in this study was then used to detect the pathogen. The standard curve was then prepared by plotting cycle threshold values for each dilution versus L. monocytogenes cell counts (log CFU). The specificity of the set of primers and hybridization probe was appropriate. A 4-h incubation at 36°C before DNA extraction produced optimum standard curves in comparison to the results for a 0-h incubation. Thus, a 4-h incubation at 36°C was applied for monitoring L. monocytogenes in collected food samples. To monitor L. monocytogenes in foods, 533 samples (ham, 129; sausage, 226; ground meat, 72; processed cheese, 54; processed milk, 42; and infant formula, 10) were collected from retail markets and from the step before pasteurization in plants. Of all 533 samples, 4 samples (0.8%) showed positive signals in RT-PCR. Two samples from hams (1.6%) and two samples from sausages (0.9%) were determined to be positive for L. monocytogenes at &lt;100 CFU/g. The results indicate that the RT-PCR detection method with the set of primers and hybridization probe designed in this study should be useful in monitoring for L. monocytogenes in processed meat and milk products.

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

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.

scholarly journals Revisiting Brucellosis in Small Ruminants of Western Border Areas in Pakistan

Pathogens ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 929 ◽  
Author(s):  
Tariq Jamil ◽  
Khushal Khan Kasi ◽  
Falk Melzer ◽  
Muhammad Saqib ◽  
Qudrat Ullah ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Small Ruminants ◽  
Kappa Statistics ◽  
Flock Size ◽  
Serological Tests ◽  
Perfect Agreement ◽  
Parallel Testing ◽  
Brucella Infection ◽  
Western Border

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.

scholarly journals Standardization of a multiplex real-time PCR test for the identification of Angiostrongylus cantonensis, A. costaricensis and A. vasorum

Biomédica ◽  
2018 ◽  
Vol 38 (1) ◽  
pp. 111 ◽  
Author(s):  
Rubén E. Varela-M ◽  
Jinney Stefany Arias ◽  
Luz Elena Velásquez
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Angiostrongylus Cantonensis ◽  
Pcr Test

Introducción. En el mundo, las angiostrongilosis de mayor impacto en salud humana y animal son ocasionadas por Angiostrongylus cantonensis, A. costaricensis y A. vasorum. En las personas, las formas clínicas son la meningitis eosinofílica y la angiostrongilosis abdominal, y, en los mamíferos cánidos, el daño cardiopulmonar. Se las consideran enfermedades emergentes debido a la propagación mundial del caracol africano Lissachatina fulica, un huésped intermediario de los parásitos. Los escasos métodos de identificación de Angiostrongylus spp. no son muy específicos ni sensibles y son costosos. Se necesita urgentemente una herramienta diagnóstica asequible, sensible y específicapara el manejo de las angiostrongilosis humana y la animal.Objetivo. Desarrollar una prueba de PCR múltiple en tiempo real (qPCR) para identificar las tres especies patógenas de Angiostrongylus.Materiales y métodos. Mediante un análisis bioinformático se seleccionó una secuencia del genoma ITS-2 de Angiostrongylus para garantizar la especificidad del cebador y las sondas. El ADN de los parásitos adultos (control positivo) y de las larvas se extrajo con el estuche DNeasyBlood & Tissue®.Las reacciones de la PCR cuantitativa se ejecutaron en un termociclador Smartcycler Cepheid®, usando el estuche de mezcla maestra QuantiTect®. Como control negativo, se utilizó ADN humano, de otros parásitos y del caracol africano.Resultados. Los valores del ciclo umbral para los controles positivos de ADN fueron: 21 para Angiostrongylus cantonensis, 22 para A. costaricensis y 31 para A. vasorum. En los controles negativos, el ciclo umbral fue cero. La qPCR mostró una eficiencia de amplificación de 2 (100 %).Conclusiones. En el laboratorio se estandarizó una qPCR múltiple para tres especies clínicamente significativas de Angiostrongylus.

Sign in / Sign up

Export Citation Format

Share Document