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

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

scholarly journals Pengembangan Prekultur Oxgall sebagai Sampel Klinis untuk Deteksi Salmonella typhi dengan Metode Real-time PCR

2018 ◽  
Vol 7 (2) ◽  
pp. 71-77
Author(s):  
Annisa Pratiwi Gunawan ◽  
Ai Djuminar ◽  
Ernawati Ernawati ◽  
Lidya Chaidir
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Salmonella Enterica ◽  
Salmonella Typhi ◽  
Rt Pcr

Demam tifoid merupakan beban kesehatan masyarakat yang signifikan di negara berpenghasilan rendah yang disebabkan oleh Salmonella enterica serotype typhi (S.typhi). Manifestasi klinis demam tifoid bervariasi dan tidak spesifik, sehingga membuat diagnosis menjadi sulit. Dengan menggunakan oxgall untuk pra-inkubasi sebagai media kultur selektif sebelum amplifikasi Real-time PCR (RT-PCR) pada wholeblood menghasilkan diagnostic yang cepat dan sensitif. Tujuan dari penelitian ini adalah untuk mengetahui kinerja oxgall sebelum amplifikasi  RT-PCR untuk deteksi Salmonella sp . Sebelum proses pengambilan sampel, dilakukan optimasi spike sampel untuk mengetahui bahwa reagen yang  digunakan baik untuk spesimen klinis. Dalam proses sampel, sampel wholeblood diambil dari  30 pasien dengan positif Widal tes. Sampel darah vena dari pasien demam tifoid diambil pada hari diagnosis; 5 ml untuk kultur darah, dan 5 ml untuk RT-PCR. Bakteri ditanam di oxgall 10% (standar mikrobiologi laboratorium klinik) dan diinkubasi selama 6 jam (37 ° C) sebelum DNA bakteri diisolasi untuk deteksi RT-PCR. Hasil penelitian menunjukkan bahwa reagen RT-PCR baik digunakan untuk sampel klinis dan kultur darah lebih baik daripada RT-PCR dengan menggunakan oxgall (hasil kultur darah positif lebih dari 24 jam). Hal ini menunjukkan bahwa harus ada penelitian lebih lanjut mengenai durasi inkubasi dan konsentrasi oxgall pada RT-PCR dan pemilihan sampel klinis.

scholarly journals DIAZO TEST AS A SCREENING TEST OF TYPHOID FEVER A PRACTICAL APPROACH

2018 ◽  
Vol 17 (2) ◽  
pp. 63
Author(s):  
J. Nugraha ◽  
Meiti Muljanti
Keyword(s):  
Blood Culture ◽  
Typhoid Fever ◽  
Gold Standard ◽  
Screening Test ◽  
Medical Service ◽  
Acute Infection ◽  
Salmonella Typhi ◽  
Blood Cultures ◽  
Serological Tests ◽  
Higher Sensitivity

Typhoid fever represents an endemic acute infection with a high mortality, in this case a laboratory test is needed to establish theearly diagnosis. For this study the researchers prefer urine diazo test which is a relatively easy, simple and inexpensive test. The aimof this study is to know whether the urine diazo test can be used for screening typhoid fever and whether there is a concordance withthe Widal and TUBEX® TF serological tests. Forty patients aged 2–18 years, attending to the Jeremy Medical Service Clinic, Surabaya,suffering of fever more than 3 days were studied from June up to August 2010. Urine samples were tested by diazo test, while bloodsamples as comparison were tested by Widal and TUBEX® TF tests, and blood culture was carried out as the gold standard. The sensitivityand specificity of those tests were then recorded. From the 40 samples, 12 patients showed positive Salmonella typhi blood cultures,26 diazo positive, 22 Widal positive and 14 TUBEX® TF positive. Of the 12 positive blood cultures, 10 (83%) diazo positive, 7 (58%)Widal positive and 9 (75%) TUBEX® TF positive were found. The sensitivity and specificity of diazo test was 83% and 43%, Widal test58% and 46%, TUBEX® TF test 75% and 82%. It is shown that the Diazo test had a higher sensitivity value, while the TUBEX® TF testshowed a higher level of specificity. In conclusion, so far it can be concluded that the Diazo test is quite reliable in aiding the diagnosisof typhoid fever and can be considered as a screening test for typhoid fever.

scholarly journals The Comparison of Widal Slide Examination Results between Tubex TF on Febrile Observation Patients Over 3 Days

2019 ◽  
Vol 5 (1) ◽  
pp. 54
Author(s):  
Nosa Ika Cahyariza ◽  
Rofiatu Sholihah
Keyword(s):  
Mortality Rate ◽  
Typhoid Fever ◽  
Diagnostic Test ◽  
High Mortality ◽  
Salmonella Enterica ◽  
Salmonella Typhi ◽  
Bacterial Disease ◽  
Serological Tests ◽  
Salmonella Enterica Serovar Typhi ◽  
The Difference

Typhoid fever is systematic bacterial disease usually occurs and has a high mortality rate each year, a disease transmitted from person to person due to contamination of feces, food, and water. The cause is bacterium Salmonella enterica serovar Typhi (S. Typhi) which is a natural host and reservoir for human. The limitations of the diagnostic test led to the increasing mortality rate due to typhoid fever. Besides ensuring infection in individuals, accurate serological tests are needed to ascertain the actual burden of the disease. Serological tests which are usually carried out in Puskesmas and hospital are Widal and Tubex Tf examinations. This study aims to determine whether there are differences in Widal and Tubex TF serological examinations in febrile patients over three days non-typhoid so patients can immediately find out whether they have typhoid fever or not. This study used a laboratory exploration method by examining 24 samples using Widal TYDAL and TUBEX® TF IDL Biotech. As many as 24 samples were examined by widal with antisera O, H, AH, and BH. Twenty-four of the same samples analyzed by TUBEX® TF. Results comparison of diagnostic from both methods will be compared using Mc Nemar test with significance = 0.05. Based on the examination which had done, it showed the difference in the results of Widal slide and lg M Anti Salmonella (Tubex Tf) in patients with febrile observation over three days. So, it can conclude that Tubex Tf examinations were better that widal slide examination because Tubex Tf uses Salmonella typhi anti-O9 antigen which can distinguish these organisms from >99% other Salmonella bacteria serotypes so that Tubex Tf examination is more specific.

Detection of Streptococcus pneumoniae in Sputum Samples by Real- Time PCR.

2020 ◽  
Vol 18 ◽  
Author(s):  
Pegah Shakib ◽  
Mohammad Reza Zolfaghari
Keyword(s):  
Streptococcus Pneumoniae ◽  
Real Time ◽  
Real Time Pcr ◽  
False Negative ◽  
Cross Sectional Study ◽  
Laboratory Culture ◽  
Cross Sectional ◽  
Negative Results ◽  
False Negative Results ◽  
Pcr Method

Background: Conventional laboratory culture-based methods for diagnosis of Streptococcus pneumoniae are time-consuming and yield false negative results. Molecular methods including real-time (RT)-PCR rapid methods and conventional PCR due to higher sensitivity and accuracy have been replaced instead traditional culture assay. The aim of the current study was to evaluate lytA gene for detection of Streptococcus pneumoniae in the cerebrospinal fluid of human patients with meningitis using real-time PCR assay. Material and Methods: In this cross-sectional study, a total of 30 clinical specimens were collected from patients in a period from September to December 2018. In order to evaluate the presence of lytA gene, conventional and real-time PCR methods were used without culture. Results: From 30 sputum samples five (16.66%) isolates were identified as S. pneumoniae by lytA PCR and sequencing. Discussion: In this research, an accurate and rapid real-time PCR method was used, which is based on lytA gene for diagnosis of bacteria so that it can be diagnosed. Based on the sequencing results, the sensitivity for detection of lytA gene was 100% (5/5).

scholarly journals A misleading false-negative result of Pneumocystis real-time PCR assay due to a rare punctual mutation: A French multicenter study

2016 ◽  
Vol 55 (2) ◽  
pp. 180-184 ◽  
Author(s):  
Solène Le Gal ◽  
Florence Robert-Gangneux ◽  
Yann Pépino ◽  
Sorya Belaz ◽  
Céline Damiani ◽  
...  
Keyword(s):  
Real Time ◽  
Multicenter Study ◽  
Real Time Pcr ◽  
False Negative ◽  
False Negative Result ◽  
Pcr Assay

scholarly journals Validation of a Novel Diagnostic Approach Combining the VersaTREK™ System for Recovery and Real-Time PCR for the Identification of Mycobacterium chimaera in Water Samples

2021 ◽  
Vol 9 (5) ◽  
pp. 1031
Author(s):  
Roberto Zoccola ◽  
Alessia Di Blasio ◽  
Tiziana Bossotto ◽  
Angela Pontei ◽  
Maria Angelillo ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Detection System ◽  
Bacterial Load ◽  
Microbial Control ◽  
Hospital Setting ◽  
Sample Volume ◽  
Analytical Sensitivity ◽  
Initial Water

Mycobacterium chimaera is an emerging pathogen associated with endocarditis and vasculitis following cardiac surgery. Although it can take up to 6–8 weeks to culture on selective solid media, culture-based detection remains the gold standard for diagnosis, so more rapid methods are urgently needed. For the present study, we processed environmental M. chimaera infected simulates at volumes defined in international guidelines. Each preparation underwent real-time PCR; inoculates were placed in a VersaTREK™ automated microbial detection system and onto selective Middlebrook 7H11 agar plates. The validation tests showed that real-time PCR detected DNA up to a concentration of 10 ng/µL. A comparison of the isolation tests showed that the PCR method detected DNA in a dilution of ×102 CFU/mL in the bacterial suspensions, whereas the limit of detection in the VersaTREK™ was <10 CFU/mL. Within less than 3 days, the VersaTREK™ detected an initial bacterial load of 100 CFU. The detection limit did not seem to be influenced by NaOH decontamination or the initial water sample volume; analytical sensitivity was 1.5 × 102 CFU/mL; positivity was determined in under 15 days. VersaTREK™ can expedite mycobacterial growth in a culture. When combined with PCR, it can increase the overall recovery of mycobacteria in environmental samples, making it potentially applicable for microbial control in the hospital setting and also in environments with low levels of contamination by viable mycobacteria.

scholarly journals A multiplex real-time PCR assay targeting virulence and resistance genes in Salmonella enterica serotype Typhimurium

2011 ◽  
Vol 11 (1) ◽  
pp. 151 ◽  
Author(s):  
Marie Bugarel ◽  
Sophie A Granier ◽  
François-Xavier Weill ◽  
Patrick Fach ◽  
Anne Brisabois
Keyword(s):  
Real Time ◽  
Resistance Genes ◽  
Real Time Pcr ◽  
Salmonella Enterica ◽  
Pcr Assay

Rapid detection and quantitation of hepatitis B virus DNA by real-time PCR using a new fluorescent (FRET) detection system

2004 ◽  
Vol 30 (2) ◽  
pp. 191-195 ◽  
Author(s):  
Sani Hussein Aliyu ◽  
Muktar Hassan Aliyu ◽  
Hamisu M Salihu ◽  
Surendra Parmar ◽  
Hamid Jalal ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Real Time ◽  
Real Time Pcr ◽  
Rapid Detection ◽  
Detection System ◽  
Hepatitis B Virus Dna ◽  
B Virus

scholarly journals False Negative Results in High Viremia Parvovirus B19-Samples Tested with Real-Time PCR

2010 ◽  
Vol 59 (2) ◽  
pp. 129-132 ◽  
Author(s):  
PIOTR GRABARCZYK ◽  
ALEKSANDRA KALIŃSKA ◽  
EWA SULKOWSKA ◽  
EWA BROJER
Keyword(s):  
Positive Result ◽  
Real Time ◽  
Real Time Pcr ◽  
Parvovirus B19 ◽  
False Negative ◽  
Immunocompromised Patients ◽  
Negative Results ◽  
False Negative Results ◽  
Parvovirus B 19 ◽  
The Way

Extremely high viremia is observed during some viruses infection, especialy in immunocompromised patients. False negative results of Parvovirus B 19 DNA tests performed with real-time PCR in high viremic samples are reported. The way of fluorescence diagrams analysis and algorithm of positive result confirmation to exclude such phenomenon are proposed.

scholarly journals Rapid Detection of Campylobacter coli, C. jejuni, and Salmonella enterica on Poultry Carcasses by Using PCR-Enzyme-Linked Immunosorbent Assay

2003 ◽  
Vol 69 (6) ◽  
pp. 3492-3499 ◽  
Author(s):  
Yang Hong ◽  
Mark E. Berrang ◽  
Tongrui Liu ◽  
Charles L. Hofacre ◽  
Susan Sanchez ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Salmonella Enterica ◽  
Rapid Detection ◽  
Diarrheal Disease ◽  
False Negative ◽  
Enzyme Linked Immunosorbent Assay ◽  
Campylobacter Coli ◽  
Culture Methods ◽  
Isolation And Identification ◽  
Serological Tests

ABSTRACT Contamination of retail poultry by Campylobacter spp. and Salmonella enterica is a significant source of human diarrheal disease. Isolation and identification of these microorganisms require a series of biochemical and serological tests. In this study, Campylobacter ceuE and Salmonella invA genes were used to design probes in PCR-enzyme-linked immunosorbent assay (ELISA), as an alternative to conventional bacteriological methodology, for the rapid detection of Campylobacter jejuni, Campylobacter coli, and S. enterica from poultry samples. With PCR-ELISA (40 cycles), the detection limits for Salmonella and Campylobacter were 2 � 102 and 4 � 101 CFU/ml, respectively. ELISA increased the sensitivity of the conventional PCR method by 100- to 1,000-fold. DNA was extracted from carcass rinses and tetrathionate enrichments and used in PCR-ELISA for the detection of Campylobacter and S. enterica, respectively. With PCR-ELISA, Salmonella was detected in 20 of 120 (17%) chicken carcass rinses examined, without the inclusion of an enrichment step. Significant correlation was observed between PCR-ELISA and cultural methods (kappa = 0.83; chi-square test, P < 0.001) with only one false negative (1.67%) and four false positives (6.67%) when PCR-ELISA was used to screen 60 tetrathionate enrichment cultures for Salmonella. With PCR-ELISA, we observed a positive correlation between the ELISA absorbance (optical density at 405 nm) and the campylobacter cell number in carcass rinse, as determined by standard culture methods. Overall, PCR-ELISA is a rapid and cost-effective approach for the detection and enumeration of Salmonella and Campylobacter bacteria on poultry.

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