scholarly journals Development and Evaluation of Alternative Methods to Identify the Three Most Common Serotypes of Salmonella enterica Causing Clinical Infections in Kazakhstan

2021 ◽  
Vol 9 (11) ◽  
pp. 2319
Author(s):  
Sabyrkhan M. Barmak ◽  
Yuriy A. Sinyavskiy ◽  
Aidar B. Berdygaliev ◽  
Turegeldy Sh. Sharmanov ◽  
Irina S. Savitskaya ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Salmonella Enterica ◽  
Alternative Methods ◽  
Clinical Samples ◽  
Pcr Analysis ◽  
Screening Methods ◽  
Conventional Pcr ◽  
Diagnostic Efficacy ◽  
Microbial Cells

In this study, we aimed to compare the performance of conventional PCR and real-time PCR assays as screening methods for identification of three frequent, clinically significant Salmonella serovars in Kazakhstan. We determined the diagnostic efficacy of three molecular methods for detection of S. enterica subsp. enterica and typing S. Typhimurium, S. Enteritidis, and S. Virchow. A total of 137 clinical samples and 883 food samples were obtained in Almaty in 2018–2019. All tests showed high analytical specificity for detecting S. enterica and its corresponding serovariants (100%). The sensitivity of real-time PCR for each of the tested targets was 1–10 microbial cells and in conventional PCR 10–100 microbial cells. The trials with conventional PCR and real-time PCR had a diagnostic efficacy (DE) of 100% and 99.71%, respectively. The DE of real-time PCR and conventional PCR for detecting S. Enteritidis and S. Typhimurium was 99.90%, while the DE of conventional PCR and real-time PCR for detecting S. Virchow was 99.31% and 99.80%, respectively. The RAPD-PCR analysis of the genomic DNA of Salmonella enterica showed the genetic kinship of S. Enteritidis isolates, and the genetic heterogeneity of S. Typhimurium and S. Virchow isolates. Thus, the developed methods can be considered as alternatives to classical serotyping using antisera.

scholarly journals Molecular characterization of resistance mechanisms in Pseudomonas aeruginosa isolates resistant to carbapenems

2018 ◽  
Vol 11 (12) ◽  
pp. 935-943 ◽  
Author(s):  
Mona Shaaban ◽  
Ahmed Al-Qahtani ◽  
Mohammed Al-Ahdal ◽  
Rasha Barwa
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Real Time ◽  
Real Time Pcr ◽  
Treatment Options ◽  
Carbapenem Resistance ◽  
Pulse Field ◽  
Diffusion Method ◽  
Clinical Samples ◽  
Pcr Analysis ◽  
Carbapenem Resistant

Introduction: Emergence of carbapenem resistance in Pseudomonas aeruginosa increases the therapeutic dilemma. In this study, we investigated various mechanisms involved in the resistance of P. aeruginosa clinical isolates to carbapenems. Methodology: P. aeruginosa isolates were isolated from different clinical samples. The antimicrobial susceptibility was evaluated by disc diffusion method. Carbapenemases were detected among carbapenem resistant isolates. Expression level of mexB and oprD was determined by real-time PCR. Molecular relatedness among isolates was detected based on pulse-field gel electrophoresis (PFGE). Results: Ninety P. aeruginosa isolates were purified from clinical specimens. High levels of resistance to imipenem and meropenem were detected in 16 isolates. PCR analysis of carbapenemases indicated the prevalence of Verona integron-encoded metallo-beta-lactamase (VIM); four isolates produced only VIM enzymes (VIM-1 or VIM-2), while the remaining twelve co-produced both VIM-1 or VIM-2 and NDM enzymes. Additionally, real-time PCR analysis elucidated high expression levels of mexB in seven of the carbapenem resistant isolates and low expression of oprD in seven isolates. The identified carbapenem-resistant isolates were clustered into eleven PFGE profiles where clusters E1 and E2 involved isolates exhibiting multiple carbapenemase genes (blaNDM-1, blaVIM-1 and blaVIM-2). Conclusion: Various mechanisms underlying carbapenem resistance have been detected in our P. aeruginosa cohort of isolates. Emergence of P. aeruginosa as a reservoir of multiple carbapenemases is increasing over time limiting the treatment options to this serious infection. This increases the urgency for infection control practices to reduce the incidence of this infection.

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

2019 ◽  
Vol 39 (4) ◽  
pp. 255-262
Author(s):  
Paula L. Martin ◽  
Nestor O. Stanchi ◽  
Bibiana F. Brihuega ◽  
Estela Bonzo ◽  
Lucía Galli ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Whole Blood ◽  
Urine Samples ◽  
Clinical Samples ◽  
Conventional Pcr ◽  
Serum Samples ◽  
Presumptive Diagnosis ◽  
Microagglutination Test ◽  
Pcr Assays

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.

scholarly journals Detection of food hazards in foods: comparison of real time polymerase chain reaction and cultural methods

2016 ◽  
Vol 5 (1) ◽  
Author(s):  
Paolo Bonilauri ◽  
Lia Bardasi ◽  
Roberto Leonelli ◽  
Mattia Ramini ◽  
Andrea Luppi ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Reference Method ◽  
Food Samples ◽  
Alternative Methods ◽  
Animal Origin ◽  
Screening Methods ◽  
Molecular Screening ◽  
Cultural Methods ◽  
Pcr Method

Foodstuffs should not contain microorganisms or their toxins or metabolites in quantities suggesting an unacceptable risk for human health. The detection of food hazards in foods is performed by several tests that produce results dependent on the analytical method used: an analytical reference method, defined as standard, is associated with each microbiological criterion laid down in Regulation 2073/2005, but, analytical methods other than the reference ones, in particular more rapid methods, could be used. Combined screening methods performed by real time PCR are currently validated as alternative methods according to the ISO 16140:2003 and certified by the Association Française de Normalisation. However, the positive results obtained with these alternative methods, the investigated molecular relations that resulted positive have to be confirmed with cultural methods using the same enrichment media in which the molecular screening was performed. Since it is necessary to assess if these testing schemes provide equivalent guarantees of food safety, the aim of this retrospective study is to analyze the data collected, from 2012 to 2014, by Emilia Romagna Region in the field of <em>Piano Regionale Alimenti</em> (Food Regional Plan), during official controls monitoring food samples, of animal and other than animal origin. Records performed by combined methods of molecular screening of <em>Salmonella</em> spp., <em>Listeria monocytogenes</em> and thermophilic <em>Campylobacter</em> and cultural confirmation results were gathered together and the results were compared in order to assess the sensitivity of the methods. A total of 10.604 food samples were considered in this study: the comparison of the data revealed that the RT-PCR method detected <em>Salmonella</em>, <em>L. monocytogenes</em>, and thermophilic <em>Campylobacter</em> in 2.18, 3.85 and 3.73% of the samples, respectively, whereas by using cultural method these pathogens were isolated in 0.43, 1.57 and 1.57 % of samples, respectively. In spite of the use of the same enrichment broth, the real time PCR method disclosed a percentage of positive samples that were negative to cultural examination ranging between 20 and 43%, with a PCR/culture ratio between 2.37 to 5.00. In conclusion, the results of this study pose a doubt about the sensitivity of the official cultural methods regarding the isolation of the three investigated foodborne pathogens. Moreover this study may be a useful tool for Veterinary Authorities to assess appropriate sampling plans to control the risk relating to the consumption of contaminated foods.

scholarly journals A Multi-Country, Single-Blinded, Phase 2 Study to Evaluate a Point-of-Need System for Rapid Detection of Leishmaniasis and Its Implementation in Endemic Settings

2021 ◽  
Vol 9 (3) ◽  
pp. 588
Author(s):  
Prakash Ghosh ◽  
Abhijit Sharma ◽  
Narayan Bhattarai ◽  
Kumar Abhishek ◽  
Thilini Nisansala ◽  
...  
Keyword(s):  
Sri Lanka ◽  
Real Time ◽  
Real Time Pcr ◽  
Evaluation Study ◽  
Nucleic Acid Amplification ◽  
Clinical Samples ◽  
Lower Sensitivity ◽  
Skin Sample ◽  
Diagnostic Efficacy ◽  
Kala Azar

With the advancement of isothermal nucleic acid amplification techniques, detection of the pathogenic DNA in clinical samples at point-of-need is no longer a dream. The newly developed recombinase polymerase amplification (RPA) assay incorporated in a suitcase laboratory has shown promising diagnostic efficacy over real-time PCR in detection of leishmania DNA from clinical samples. For broader application of this point-of-need system, we undertook a current multi-country diagnostic evaluation study towards establishing this technique in different endemic settings which would be beneficial for the ongoing elimination programs for leishmaniasis. For this study purpose, clinical samples from confirmed visceral leishmaniasis (VL) and post-kala-azar dermal leishmaniasis (PKDL) patients were subjected to both real-time PCR and RPA assay in Bangladesh, India, and Nepal. Further skin samples from confirmed cutaneous leishmaniasis (CL) patients were also included from Sri Lanka. A total of 450 clinical samples from VL patients, 429 from PKDL patients, 47 from CL patients, and 322 from endemic healthy/healthy controls were under investigation to determine the diagnostic efficacy of RPA assay in comparison to real-time PCR. A comparative sensitivity of both methods was found where real-time PCR and RPA assay showed 96.86% (95% CI: 94.45–98.42) and 88.85% (95% CI: 85.08–91.96) sensitivity respectively in the diagnosis of VL cases. This new isothermal method also exhibited promising diagnostic sensitivity (93.50%) for PKDL cases, when a skin sample was used. Due to variation in the sequence of target amplicons, RPA assay showed comparatively lower sensitivity (55.32%) than that of real-time PCR in Sri Lanka for the diagnosis of CL cases. Except for India, the assay presented absolute specificity in the rest of the sites. Excellent concordance between the two molecular methods towards detection of leishmania DNA in clinical samples substantiates the application of RPA assay incorporated in a suitcase laboratory for point-of-need diagnosis of VL and PKDL in low resource endemic settings. However, further improvisation of the method is necessary for diagnosis of CL.

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

2011 ◽  
Vol 60 (1) ◽  
pp. 27-33 ◽  
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.

scholarly journals Development of a Cell Culture Method To Isolate and Enrich Salmonella enterica Serotype Enteritidis from Shell Eggs for Subsequent Detection by Real-Time PCR

2009 ◽  
Vol 75 (16) ◽  
pp. 5321-5327 ◽  
Author(s):  
J. B. Day ◽  
U. Basavanna ◽  
S. K. Sharma
Keyword(s):  
Cell Culture ◽  
Real Time ◽  
Real Time Pcr ◽  
Salmonella Enterica ◽  
International Organization ◽  
Pcr Analysis ◽  
Intracellular Replication ◽  
Microbiological Method ◽  
Shell Eggs ◽  
Animal Feeding

ABSTRACT Salmonella enterica serotype Enteritidis is a major cause of nontyphoidal salmonellosis from ingestion of contaminated raw or undercooked shell eggs. Current techniques used to identify Salmonella serotype Enteritidis in eggs are extremely laborious and time-consuming. In this study, a novel eukaryotic cell culture system was combined with real-time PCR analysis to rapidly identify Salmonella serotype Enteritidis in raw shell eggs. The system was compared to the standard microbiological method of the International Organization for Standardization (Anonymous, Microbiology of food and animal feeding stuffs—horizontal method for the detection of Salmonella, 2002). The novel technique utilizes a mouse macrophage cell line (RAW 264.7) as the host for the isolation and intracellular replication of Salmonella serotype Enteritidis. Exposure of macrophages to Salmonella serotype Enteritidis-contaminated eggs results in uptake and intracellular replication of the bacterium, which can subsequently be detected by real-time PCR analysis of the DNA released after disruption of infected macrophages. Macrophage monolayers were exposed to eggs contaminated with various quantities of Salmonella serotype Enteritidis. As few as 10 CFU/ml was detected in cell lysates from infected macrophages after 10 h by real-time PCR using primer and probe sets specific for DNA segments located on the Salmonella serotype Enteritidis genes sefA and orgC. Salmonella serotype Enteritidis could also be distinguished from other non-serogroup D Salmonella serotypes by using the sefA- and orgC-specific primer and probe sets. Confirmatory identification of Salmonella serotype Enteritidis in eggs was also achieved by isolation of intracellular bacteria from lysates of infected macrophages on xylose lysine deoxycholate medium. This method identifies Salmonella serotype Enteritidis from eggs in less than 10 h compared to the more than 5 days required for the standard reference microbiological method of the International Organization for Standardization (Microbiology of food and animal feeding stuffs—horizontal method for the detection of Salmonella, 2002).

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