scholarly journals Evaluation of a real-time nucleic acid sequencebased amplification (nasba) assay for diagnosis of urogenital chlamydial infection

2005 ◽  
Vol 54 (4) ◽  
pp. 17-21
Author(s):  
Е. V. Shipitsyna ◽  
А. М. Savicheva ◽  
N. E. Vorobyova ◽  
Е. V. Sokolovsky ◽  
А. Е. Guschin ◽  
...  
Keyword(s):  
Cell Culture ◽  
Nucleic Acid ◽  
Real Time ◽  
Chlamydial Infection ◽  
Culture Method ◽  
Specific Test ◽  
Predictive Values ◽  
Chlamydia Trachomatis Infection ◽  
Urogenital Infection ◽  
Urogenital Chlamydial Infection

This study was aimed to evaluate a new realtime nucleic acid sequence-based amplification (NASBA-Real- Time) assay for diagnosis of urogenital Chlamydia trachomatis infection. A total of 193 patients aged 16 to 42 (mean age, 22,8 years) were examined, with most of them having symptoms of urogenital infection. Cervical and urethral swabs from women and men, respectively, were investigated with the use of cell culture method, polymerase chain reaction (PCR) and NASBA- Real-Time. C. trachomatis infection was diagnosed in 29 patients (15 %): in 21 patients - by all the three methods, whereas 8 samples were culture negative. Sensitivity of PCR and NASBA- Real-Time methods were 100 %, cell culture method - 78,4 %. Negative predictive value of PCR and NASBA-Real-Time was found to be 100 %, and that of cell culture - 95,3 %. Specificity as well as positive predictive values of all the three methods equaled 100%. Thus, the new real-time NASBA-Real-Time assay was shown to be a very sensitive and specific test, which can be recommended as a confirmatory method for diagnosis of urogenital chlamydial infection.

scholarly journals Evaluation of StrepBSelectChromogenic Medium and the Fast-Track Diagnostics Group B Streptococcus (GBS) Real-Time PCR Assay Compared to Routine Culture for Detection of GBS during Antepartum Screening

2017 ◽  
Vol 55 (7) ◽  
pp. 2137-2142 ◽  
Author(s):  
Deirdre L. Church ◽  
Heather Baxter ◽  
Tracie Lloyd ◽  
Oscar Larios ◽  
Daniel B. Gregson
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Fast Track ◽  
Group B Streptococcus ◽  
Culture Method ◽  
Predictive Values ◽  
Content Type ◽  
Standard Culture ◽  
Group B ◽  
The Cost

ABSTRACTLife-threatening infection in neonates due to group BStreptococcus(GBS) is preventable by screening of near-term pregnant women and treatment at delivery. A total of 295 vaginal-rectal swabs were collected from women attending antepartum clinics in Calgary, Alberta, Canada. GBS colonization was detected by the standard culture method (Strep B Carrot Broth subcultured to blood agar with a neomycin disk) and compared to recovery with Strep Group B Broth (Dalynn Biologicals) subcultured to StrepBSelectchromogenic medium (CM; Bio-Rad Laboratories) and the Fast-Track Diagnostics GBS real-time PCR (quantitative PCR [qPCR]) assay (Phoenix Airmid Biomedical Corp.) performed with broth-enriched samples and the Abbottm2000sp/m2000rt system. A total of 62/295 (21%) women were colonized with GBS; 58 (19.7%) cases were detected by standard culture, while CM and qPCR each found 61 (20.7%) cases. The qPCR and CM were similar in performance, with sensitivities, specificities, and positive and negative predictive values of 98.4 and 98.4%, 99.6 and 99.6%, 98.4 and 98.4%, and 99.6 and 99.6%, respectively, compared to routine culture. Both qPCR and CM would allow more rapid reporting of routine GBS screening results than standard culture. Although the cost per test was similar for standard culture and CM, the routine use of qPCR would cost approximately four times as much as culture-based detection. Laboratories worldwide should consider implementing one of the newer methods for primary GBS testing, depending on the cost limitations of different health care jurisdictions.

scholarly journals Comparative Evaluation of Six Molecular Assays based on RT-PCR and Cross Primer Isothermal Amplification for SARS-CoV-2 RNA Detection

2021 ◽  
Author(s):  
Shuang Wu ◽  
Xiaolu Shi ◽  
Qiongcheng Chen ◽  
Yixiang Jiang ◽  
Le Zuo ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Comparative Study ◽  
Reverse Transcriptase ◽  
Real Time ◽  
Isothermal Amplification ◽  
Nucleic Acid Amplification ◽  
Clinical Samples ◽  
Predictive Values ◽  
Health Crisis ◽  
Sensitivity Specificity

Abstract Background: SARS-CoV-2 is a newly emerged coronavirus, causing the coronavirus disease 2019 (COVID-19) outbreak in December, 2019. As drugs and vaccines of COVID-19 remain in development, accurate virus detection plays a crucial role in the current public health crisis. Quantitative real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) kits have been reliably used for detection of SARS-CoV-2 RNA since the beginning of the COVID-19 outbreak, whereas isothermal nucleic acid amplification-based point-of-care automated kits have also been considered as a simpler and rapid alternative. However, as these kits have only been developed and applied clinically within a short timeframe, their clinical performance has not been adequately evaluated to date. We describe a comparative study between a newly developed cross-priming isothermal amplification (CPA) kit (Kit A) and five RT-qPCR kits (Kits B–F) to evaluate their sensitivity, specificity, predictive values and accuracy. Methods: Fifty-two clinical samples were used including throat swabs (n=30), nasal swabs (n=7), nasopharyngeal swabs (n=7) and sputum specimens (n=8), comprising confirmed (n=26) and negative cases (n=26). SARS-CoV-2 detection was simultaneously performed on each sample using six nucleic acid amplification kits. The sensitivity, specificity, positive/negative predictive values (PPV/NPV) and the accuracy for each kit were assessed using clinical manifestation and molecular diagnoses as the reference standard. Reproducibility for RT-qPCR kits was evaluated in triplicate by three different operators using a SARS-CoV-2 RNA-positive sample. On the basis of the six kits’ evaluation results, CPA kit (Kit A) and two RT-qPCR Kits (Kit B and F) were applied to the SARS-CoV-2 detection in close-contacts of COVID-19 patients. Results: For Kit A, the sensitivity, specificity, PPV/NPV and accuracy were 100%. Among the five RT-qPCR kits, Kits B, C and F had good agreement with the clinical diagnostic reports (Kappa≥0.75); Kits D and E were less congruent (0.4≤Kappa<0.75). Differences between all kits were statistically significant (P<0.001). The reproducibility of RT-qPCR kits was determined using a coefficients of variation (CV) between 0.95% and 2.57%, indicating good reproducibility. Conclusions: This is the first comparative study to evaluate CPA and RT-qPCR kits’ specificity and sensitivity for SARS-CoV-2 detection, and could serve as a reference for clinical laboratories, thus informing testing protocols amid the rapidly progressing COVID-19 pandemic. Keywords: SARS-CoV-2; COVID-19; nucleic acid detection; real-time reverse transcriptase PCR (RT-qPCR); cross-priming isothermal amplification (CPA)

Chlamydia trachomatis infection positivity rates determined by nucleic acid amplification test in patients of hospitals in the northeastern region of Ukraine

2017 ◽  
Vol 28 (14) ◽  
pp. 1405-1409
Author(s):  
Alexei Belozorov ◽  
Olga Fedets ◽  
Tatjana Chastii ◽  
Elena Milutina ◽  
Oksana Sokol ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Chlamydia Trachomatis ◽  
Chlamydial Infection ◽  
Age Groups ◽  
Nucleic Acid Amplification Test ◽  
Nucleic Acid Amplification ◽  
Test Results ◽  
Chlamydia Trachomatis Infection ◽  
Northeastern Region ◽  
Medical Institutions

There are no accurate data regarding the prevalence of Chlamydia trachomatis infection in Ukraine. This study aims to estimate the prevalence in the northeastern region of the country through reviewing nucleic acid amplification test results in patients of medical institutions in the Kharkov region during 2014–2016. Samples from 6920 patients (5028 women and 1892 men) aged 12–76 years were tested. The overall positivity rate was 4.5% (95% CI 4.0–5.0): 3.9% (95% CI 3.4–4.5) in women and 6.1% (95% CI 5.1–7.3) in men. The highest prevalence was found in the 16–20 (8.5%, CI 6.3–11.4) and 21–25 (8.0%, CI 6.7–9.4) year age groups. The prevalence in men was higher than in women in all investigated groups. The results show the need for more attention to the prevention, diagnosis, and treatment of chlamydial infection in these age groups of women and men in this region.

scholarly journals Optimizing a Suspension Culture Method with a Decreased Cost to Detect Enteroviruses in Water to Increase Surveillance Access

2020 ◽  
Vol 11 (2) ◽  
pp. 35-44
Author(s):  
Stephanie Tornberg-Belanger ◽  
Jonathan Sreter ◽  
Aaron Margolin
Keyword(s):  
Cell Culture ◽  
Nucleic Acid ◽  
Water Samples ◽  
Financial Burden ◽  
Virus Detection ◽  
Culture Method ◽  
Virus Concentration ◽  
Viral Nucleic Acid ◽  
A Cell ◽  
Materials Used

Enteroviruses are a public health threat due to the high incidence of infections and potential for serious illness or death. Some laboratories in high-income countries detect enteroviruses in water by integrating cell culture and PCR (ICC/PCR). This combined method carries a high financial burden, due in part to specialized cell culture equipment. Therefore, we expanded upon a pilot study to reduce the cost by using common laboratory polypropylene tubes to create a cell culture in suspension. We optimized the protocol by determining minimal incubation periods post-infection as a function of the initial virus concentration. Cells in suspension and traditional monolayers were inoculated with poliovirus and incubated in 8-hour intervals up to 48 hours prior to extraction. Quantitative PCR (qPCR) was used to detect viral nucleic acid targets. Treated and raw water samples were seeded with virus and the suspension ICC/qPCR protocol used to ascertain whether the protocol performed similar to directly seeding cells. No variation in virus detection occurred using the suspension ICC/qPCR or monolayer ICC/qPCR (p = 0.95). In surface water samples, viral nucleic acid was successfully detected, with no significant increase after 32 h (p > 0.05). Suspension ICC/qPCR is as effective as monolayer ICC/qPCR in detecting enteroviruses in surface waters. Materials used in the suspension ICC/qPCR have a lower monetary cost than traditional cell culture materials without loss of sensitivity. More accessible testing of waters for enterovirus contamination through cost reduction has the potential to reduce human exposure and disease.

Automated cell counting of astrocytes on patterned substrates containing aliphatic and charged properties

1995 ◽  
Vol 53 ◽  
pp. 974-975
Author(s):  
W. Shain ◽  
H. Ancin ◽  
H.C. Craighead ◽  
M. Isaacson ◽  
L. Kam ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cell Attachment ◽  
Culture Method ◽  
Cell Counting ◽  
Data Sets ◽  
Nuclear Staining ◽  
Double Positive ◽  
A Cell ◽  
Wafer Test ◽  
Cell Densities

Neural protheses have potential to restore nervous system functions lost by trauma or disease. Nanofabrication extends this approach to implants for stimulating and recording from single or small groups of neurons in the spinal cord and brain; however, tissue compatibility is a major limitation to their practical application. We are using a cell culture method for quantitatively measuring cell attachment to surfaces designed for nanofabricated neural prostheses.Silicon wafer test surfaces composed of 50-μm bars separated by aliphatic regions were fabricated using methods similar to a procedure described by Kleinfeld et al. Test surfaces contained either a single or double positive charge/residue. Cyanine dyes (diIC18(3)) stained the background and cell membranes (Fig 1); however, identification of individual cells at higher densities was difficult (Fig 2). Nuclear staining with acriflavine allowed discrimination of individual cells and permitted automated counting of nuclei using 3-D data sets from the confocal microscope (Fig 3). For cell attachment assays, LRM5 5 astroglial cells and astrocytes in primary cell culture were plated at increasing cell densities on test substrates, incubated for 24 hr, fixed, stained, mounted on coverslips, and imaged with a 10x objective.

Comparison of Cell Culture and a Poliovirus Gene Probe Assay for the Detection of Enteroviruses in Environmental Water Samples

1993 ◽  
Vol 27 (3-4) ◽  
pp. 311-314 ◽  
Author(s):  
Aaron B. Margolin ◽  
Charles P. Gerba ◽  
Kenneth J. Richardson ◽  
Jaime E. Naranjo
Keyword(s):  
Cell Culture ◽  
Activated Sludge ◽  
Water Samples ◽  
Cdna Probe ◽  
Culture Method ◽  
Tidal River ◽  
Nucleic Acid Hybridization ◽  
Treated Wastewater ◽  
Gene Probe ◽  
Naturally Occurring

Nucleic acid hybridization provides a rapid non-cell culture method for the detection of enteric viruses in water. The purpose of this work was to compare the detection of naturally occurring enteroviruses by cell culture with their detection by a poliovirus gene probe in various types of water samples. Samples of activated sludge effluent, tertiary treated wastewater (activated sludge, filtration and passage through reverse osmosis), ground water, surface water and tidal river water were processed through 1 MDS Virozorb filters to concentrate any naturally occurring virus. Viruses were eluted from the filters with pH 9.5 beef extract and reduced in volume by flocculation to 20-30 ml. These concentrates were then assayed in the BGM cell line by the cytopathogenic effects (CPE) method and by a poliovirus cDNA probe (base pairs 115-7440) labeled with 32P. A total of 233 samples were assayed in this manner. In slightly more than 93% of the samples gene probe and cell culture yielded the same results. Of these samples 36 were positive by gene probe and 28 by cell culture assay. Positive samples for gene probe were confirmed by treatment with NaOH or RNAse and then reprobed. Samples demonstrating CPE upon primary passage were confirmed positive by subsequent passage of cell lysate on a new monolayer of BGM cells. Ten samples were positive by gene probe and negative by cell culture, and 4 samples were negative by gene probe and positive by cell culture.

scholarly journals Real-time monitoring of liver fibrosis through embedded sensors in a microphysiological system

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Hafiz Muhammad Umer Farooqi ◽  
Bohye Kang ◽  
Muhammad Asad Ullah Khalid ◽  
Abdul Rahim Chethikkattuveli Salih ◽  
Kinam Hyun ◽  
...  
Keyword(s):  
Cell Culture ◽  
Liver Fibrosis ◽  
Real Time ◽  
Hepatic Fibrosis ◽  
Transforming Growth Factor ◽  
Cell Monolayer ◽  
Embedded Sensors ◽  
Matrix Deposition ◽  
Tgf Β1

AbstractHepatic fibrosis is a foreshadowing of future adverse events like liver cirrhosis, liver failure, and cancer. Hepatic stellate cell activation is the main event of liver fibrosis, which results in excessive extracellular matrix deposition and hepatic parenchyma's disintegration. Several biochemical and molecular assays have been introduced for in vitro study of the hepatic fibrosis progression. However, they do not forecast real-time events happening to the in vitro models. Trans-epithelial electrical resistance (TEER) is used in cell culture science to measure cell monolayer barrier integrity. Herein, we explored TEER measurement's utility for monitoring fibrosis development in a dynamic cell culture microphysiological system. Immortal HepG2 cells and fibroblasts were co-cultured, and transforming growth factor β1 (TGF-β1) was used as a fibrosis stimulus to create a liver fibrosis-on-chip model. A glass chip-based embedded TEER and reactive oxygen species (ROS) sensors were employed to gauge the effect of TGF-β1 within the microphysiological system, which promotes a positive feedback response in fibrosis development. Furthermore, albumin, Urea, CYP450 measurements, and immunofluorescent microscopy were performed to correlate the following data with embedded sensors responses. We found that chip embedded electrochemical sensors could be used as a potential substitute for conventional end-point assays for studying fibrosis in microphysiological systems.

scholarly journals Differing Effects of Standard and Harsh Nucleic Acid Extraction Procedures on Diagnostic Helminth Real-Time PCRs Applied to Human Stool Samples

Pathogens ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 188
Author(s):  
Tanja Hoffmann ◽  
Andreas Hahn ◽  
Jaco J. Verweij ◽  
Gérard Leboulle ◽  
Olfert Landt ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Real Time Pcr ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
Pcr Assay ◽  
Bead Beating ◽  
Stool Samples ◽  
Pcr Assays ◽  
Human Stool

This study aimed to assess standard and harsher nucleic acid extraction schemes for diagnostic helminth real-time PCR approaches from stool samples. A standard procedure for nucleic acid extraction from stool and a procedure including bead-beating as well as proteinase K digestion were compared with group-, genus-, and species-specific real-time PCR assays targeting helminths and nonhelminth pathogens in human stool samples. From 25 different in-house and commercial helminth real-time PCR assays applied to 77 stool samples comprising 67 historic samples and 10 external quality assessment scheme samples positively tested for helminths, higher numbers of positive test results were observed after bead-beating-based nucleic acid extraction for 5/25 (20%) real-time PCR assays irrespective of specificity issues. Lower cycle threshold values were observed for one real-time PCR assay after the standard extraction scheme, and for four assays after the bead-beating-based scheme. Agreement between real-time PCR results after both nucleic acid extraction strategies according to Cohen’s kappa ranged from poor to almost perfect for the different assays. Varying agreement was observed in eight nonhelminth real-time PCR assays applied to 67 historic stool samples. The study indicates highly variable effects of harsh nucleic acid extraction approaches depending on the real-time PCR assay used.

Identifying the etiologic role of Parvovirus B19 in non-immune hydrops fetalis by histopathology, immunohistochemistry and nucleic acid testing: a retrospective study

Open Medicine ◽  
2007 ◽  
Vol 2 (3) ◽  
pp. 271-279 ◽  
Author(s):  
Koray Ergunay ◽  
Gulcin Altinok ◽  
Bora Gurel ◽  
Ahmet Pinar ◽  
Arzu Sungur ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
San Francisco ◽  
Real Time Pcr ◽  
Nested Pcr ◽  
Parvovirus B19 ◽  
Etiologic Agent ◽  
Hydrops Fetalis ◽  
Nucleic Acid Detection

AbstractIntrauterine Parvovirus B19 infections may cause fetal anemia, non-immune hydrops fetalis or abortion. This study focuses on the pathogenic role of Parvovirus B19 in non-immune hydrops fetalis at Hacettepe University, a major reference hospital in Turkey. Twenty-two cases of non-immune hydrops fetalis were retrospectively selected out of a total of 431 hydrops fetalis specimens from the Department of Pathology archieves. Paraffine embedded tissue sections from placental and liver tissues from each case were evaluated by histopathology, immunohistochemistry, nested PCR and commercial quantitative Real-time PCR. Viral DNA was detected in placental tissues by Real-time PCR in 2 cases (2/22, 9.1%) where histopathology also revealed changes suggestive of Parvovirus B19 infection. No significant histopathologic changes were observed for the remaining sections. Nested PCR that targets the VP1 region of the viral genome and immunohistochemistry for viral capsid antigens were negative for all cases. As a result, Parvovirus B19 is identified as the etiologic agent for the development of non-immune hydrops fetalis for 9.1% of the cases in Hacettepe University, Turkey. Real-time PCR is observed to be an effective diagnostic tool for nucleic acid detection from paraffine embedded tissues. Part of this study was presented as a poster at XIIIth International Congress of Virology, San Francisco, USA (Abstract V-572).

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