scholarly journals A35 The first laboratory confirmation of chikungunya outbreak in Ethiopia

2019 ◽  
Vol 5 (Supplement_1) ◽  
Author(s):  
Mesfin Mengesha Tsegaye ◽  
Aadamu Tayachew ◽  
Desalegn Belay ◽  
Abebe Alemu ◽  
Berhane Beyene
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Laboratory Investigation ◽  
Emergency Preparedness ◽  
Zika Virus ◽  
Chikungunya Virus ◽  
Febrile Illness ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Serum Samples

Abstract Chikungunya is a viral disease (genus Alphavirus) which is transmitted to humans by infected mosquitoes—including Aedes aegypti and A. albopictus. An outbreak of febrile illness, suspected to have been caused by chikungunya, was reported in June 2016 from Dolloado district, Suuf Kebele, in the Somalia regional state of Ethiopia that borders the Mandera county of Kenya where a confirmed chikungunya outbreak was ongoing. Laboratory investigation was carried out to confirm if the outbreak in Ethiopia was caused by Chikungunya virus. Ten serum samples were collected from suspected patients visiting a health center in Suuf Kebel, who were then sent to the Nation laboratory in Ethiopian Public Health Institute. RNA was extracted from the serum samples using QIAgene RNA Mini kit, and PCR detection of dengue, chikungunya, and Zika virus nucleic acid was done using Trioplex Real-time RT-PCR Assay following the protocol from the Center for Disease Control (CDC). The Trioplex Real-time RT-PCR assay, for detection and differentiation of RNA from dengue, Chikungunya and Zika, was provided by CDC as part of the zika emergency preparedness effort. Of the nine samples tested, eight (88.88%) were found to be positive for chikungunya virus nucleic acid but negative for dengue and Zika virus nucleic acids. The median age of the affected sampled patients was 40 years, and males appear to be more affected (66.6% of sampled patients). The laboratory investigation confirmed that the outbreak was caused by chikungunya virus. Even though further molecular characterization of the positive isolates will provide more information as to the circulating genotypes and elucidate the origin of the outbreak virus, it is also possible to assume that the outbreak was an extension of the outbreak in neighboring countries in Kenya and, therefore, warrants that cross-border integration efforts to control chikungunya should be implemented by the concerned countries.

scholarly journals Development and evaluation of one-step multiplex real-time RT-PCR assay for simultaneous detection of Zika virus and Chikungunya virus

2017 ◽  
Vol 90 (3) ◽  
pp. 389-396 ◽  
Author(s):  
Si-Qing Liu ◽  
Xiao Li ◽  
Cheng-Lin Deng ◽  
Zhi-Ming Yuan ◽  
Bo Zhang
Keyword(s):  
Real Time ◽  
Zika Virus ◽  
Chikungunya Virus ◽  
Simultaneous Detection ◽  
Rt Pcr ◽  
Pcr Assay ◽  
One Step

Detection and Quantification of Chikungunya Virus by Real-Time RT-PCR Assay

2016 ◽  
pp. 105-117 ◽  
Author(s):  
Seok Mui Wang ◽  
Ummul Haninah Ali ◽  
Shamala Devi Sekaran ◽  
Ravindran Thayan
Keyword(s):  
Real Time ◽  
Chikungunya Virus ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Detection And Quantification

scholarly journals Zika virus isolation, propagation, and quantification using multiple methods

PLoS ONE ◽  
2021 ◽  
Vol 16 (7) ◽  
pp. e0255314
Author(s):  
Worawat Dangsagul ◽  
Kriengsak Ruchusatsawat ◽  
Apiwat Tawatsin ◽  
Don Changsom ◽  
Pirom Noisumdaeng ◽  
...  
Keyword(s):  
Real Time ◽  
Zika Virus ◽  
Immunofluorescence Assay ◽  
Rt Pcr ◽  
Serum Samples ◽  
Cell Monolayers ◽  
Viral Genomes ◽  
Mosquito Cells ◽  
Autopsy Specimens ◽  
Intrathoracic Inoculation

Zika virus (ZIKV) was isolated from the archival urine, serum, and autopsy specimens by intrathoracic inoculation of Toxorhynchitis splendens and followed by three blind sub-passaging in C6/36 mosquito cells. The virus isolates were identified using an immunofluorescence assay and real-time reverse transcription-polymerase chain reaction (real-time RT-PCR). This study analyzed 11 ZIKV isolates. One isolate (0.6%) was obtained from 171 urine samples, eight (8.7%) from 92 serum samples and two from tissues of an abortive fetus. After propagation in C6/36 cells, ZIKV was titrated by plaque and focus forming unit (FFU) assays in Vero cell monolayers, and viral genomes were determined via real-time and digital RT-PCR. Plaque and FFU assay quantitations were comparable, with the amount of infectious viruses averaging 106−107 PFU or FFU/ml. Real-time RT-PCR semi-quantified the viral genome numbers, with Ct values varying from 12 to 14. Digital RT-PCR, which precisely determines the numbers of the viral genomes, consistently averaged 10–100 times higher than the number of infectious units. There was good correlation between the results of these titration methods. Therefore, the selection of a method should be based on the objectives of each research studies.

scholarly journals Evaluation of real-time nucleic acid sequence-based amplification for detection of Chikungunya virus in clinical samples

2009 ◽  
Vol 58 (9) ◽  
pp. 1168-1172 ◽  
Author(s):  
J.-N. Telles ◽  
K. Le Roux ◽  
P. Grivard ◽  
G. Vernet ◽  
A. Michault
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Chikungunya Virus ◽  
False Negative ◽  
Nucleic Acid Sequence ◽  
Clinical Samples ◽  
Acid Extraction ◽  
Plasma Samples ◽  
Rt Pcr ◽  
Nucleic Acid Extraction

The Chikungunya virus (CHIKV) is a member of the genus Alphavirus that is transmitted to humans by Aedes mosquitoes. In 2005 and 2006, the Indian Ocean island of La Réunion was hit with an unprecedented CHIKV fever outbreak that infected 300 000 people. In the present study, we describe the evaluation of real-time nucleic acid sequence-based amplification (RT-NASBA) for the detection of CHIKV in clinical samples. A co-extracted and co-amplified chimerical CHIKV RNA sequence was used as an internal control to eliminate false-negative results. The detection threshold of the assay was determined from quantified CHIKV-positive plasma, and estimated to be 200 copies per NASBA reaction. The specificity of the assay was determined using blast analyses and non-cross-reactivity using an O'nyong-nyong virus culture and 250 CHIKV RT-PCR-negative plasma samples. A 100 % specificity was found and no invalid result was obtained, showing the good quality of the nucleic acid extraction. The assay was then evaluated using 252 CHIKV-positive RT-PCR plasma samples. The samples were all tested positive, including those with low viral load. This evaluation showed that the RT-NASBA is a rapid (5 h from sample nucleic acid extraction to detection), sensitive, specific and reliable method for the routine diagnosis of CHIKV in clinical samples.

scholarly journals Improved detection of Zika virus RNA in human and animal specimens by a novel, highly sensitive and specific real-time RT-PCR assay targeting the 5′-untranslated region of Zika virus

2017 ◽  
Vol 22 (5) ◽  
pp. 594-603 ◽  
Author(s):  
Jasper Fuk-Woo Chan ◽  
Cyril Chik-Yan Yip ◽  
Kah-Meng Tee ◽  
Zheng Zhu ◽  
Jessica Oi-Ling Tsang ◽  
...  
Keyword(s):  
Real Time ◽  
Zika Virus ◽  
Untranslated Region ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Virus Rna ◽  
Highly Sensitive

scholarly journals Using the LN34 Pan-Lyssavirus Real-Time RT-PCR Assay for Rabies Diagnosis and Rapid Genetic Typing from Formalin-Fixed Human Brain Tissue

Viruses ◽  
2020 ◽  
Vol 12 (1) ◽  
pp. 120 ◽  
Author(s):  
Rene Edgar Condori ◽  
Michael Niezgoda ◽  
Griselda Lopez ◽  
Carmen Acosta Matos ◽  
Elinna Diaz Mateo ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Dominican Republic ◽  
Real Time ◽  
Brain Tissue ◽  
Border Region ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Fixed Tissue ◽  
Genetic Typing ◽  
Formalin Fixed

Human rabies post mortem diagnostic samples are often preserved in formalin. While immunohistochemistry (IHC) has been routinely used for rabies antigen detection in formalin-fixed tissue, the formalin fixation process causes nucleic acid fragmentation that may affect PCR amplification. This study reports the diagnosis of rabies in an individual from the Dominican Republic using both IHC and the LN34 pan-lyssavirus real-time RT-PCR assay on formalin-fixed brain tissue. The LN34 assay generates a 165 bp amplicon and demonstrated higher sensitivity than traditional PCR. Multiple efforts to amplify nucleic acid fragments larger than 300 bp using conventional PCR were unsuccessful, probably due to RNA fragmentation. Sequences generated from the LN34 amplicon linked the case to the rabies virus (RABV) strain circulating in the Ouest Department of Haiti to the border region between Haiti and the Dominican Republic. Direct sequencing of the LN34 amplicon allowed rapid and low-cost rabies genetic typing.

Detection of Colorado Tick Fever viral RNA in acute human serum samples by a quantitative real-time RT-PCR assay

2007 ◽  
Vol 140 (1-2) ◽  
pp. 43-48 ◽  
Author(s):  
Amy J. Lambert ◽  
Olga Kosoy ◽  
Jason O. Velez ◽  
Brandy J. Russell ◽  
Robert S. Lanciotti
Keyword(s):  
Human Serum ◽  
Real Time ◽  
Viral Rna ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Serum Samples ◽  
Human Serum Samples

scholarly journals High-Volume Extraction of Nucleic Acids by Magnetic Bead Technology for Ultrasensitive Detection of Bacteria in Blood Components

2007 ◽  
Vol 53 (1) ◽  
pp. 104-110 ◽  
Author(s):  
Melanie Störmer ◽  
Knut Kleesiek ◽  
Jens Dreier
Keyword(s):  
Detection Limit ◽  
Nucleic Acid ◽  
Nucleic Acids ◽  
Real Time ◽  
High Volume ◽  
Magnetic Bead ◽  
Locked Nucleic Acid ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Probe System

Abstract Background: Nucleic acid isolation, the most technically demanding and laborious procedure performed in molecular diagnostics, harbors the potential for improvements in automation. A recent development is the use of magnetic beads covered with nucleic acid–binding matrices. We adapted this technology with a broad-range 23S rRNA real-time reverse transcription (RT)-PCR assay for fast and sensitive detection of bacterial contamination of blood products. Methods: We investigated different protocols for an automated high-volume extraction method based on magnetic-separation technology for the extraction of bacterial nucleic acids from platelet concentrates (PCs). We added 2 model bacteria, Staphylococcus epidermidis and Escherichia coli, to a single pool of apheresis-derived, single-donor platelets and assayed the PCs by real-time RT-PCR analysis with an improved primer–probe system and locked nucleic acid technology. Coamplification of human β2-microglobulin mRNA served as an internal control (IC). We used probit analysis to calculate the minimum concentration of bacteria that would be detected with 95% confidence. Results: For automated magnetic bead–based extraction technology with the real-time RT-PCR, the 95% detection limit was 29 × 103 colony-forming units (CFU)/L for S. epidermidis and 22 × 103 CFU/L for E. coli. No false-positive results occurred, either due to nucleic acid contamination of reagents or externally during testing of 1030 PCs. Conclusions: High-volume nucleic acid extraction improved the detection limit of the assay. The improvement of the primer–probe system and the integration of an IC make the RT-PCR assay appropriate for bacteria screening of platelets.

scholarly journals Development and Evaluation of a Duo Chikungunya Virus Real-Time RT-PCR Assay Targeting Two Regions within the Genome

Viruses ◽  
2019 ◽  
Vol 11 (8) ◽  
pp. 755 ◽  
Author(s):  
Laurence Thirion ◽  
Laura Pezzi ◽  
Iban Corcostegui ◽  
Audrey Dubot-Pérès ◽  
Alessandra Falchi ◽  
...  
Keyword(s):  
Real Time ◽  
Chikungunya Virus ◽  
Point Mutations ◽  
In Silico Analysis ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Genetic Lineages ◽  
Sensitivity Specificity ◽  
Dual Target ◽  
Major Sequence

Chikungunya virus (CHIKV) re-emerged as a globalized health threat fifteen years ago. There are dozens of RT-PCR assays published. An inventory of the latter was made, and after in silico analysis, two assays were selected for their ability to detect strains belonging to the five CHIKV genetic lineages. They were combined in order to provide a robust assay not affected by genetic point mutations and the resulting Duo CHIKV real-time RT-PCR assay was compared to the two parental single-plex tests against five strains belonging to the five genetic lineages. The Duo CHIKV assay performed equally, or better, in terms of sensitivity, specificity, linearity and signal intensity. Dual-target assays are better suited for viruses having the propensity to evolve into new variants via point mutations or major sequence deletions/insertions. Here, we demonstrated that combining two single systems into a dual-target assay did not impair sensitivity and specificity, and proved a potent diagnostic tool to face a potential emergence of CHIKV variants by newly evolving mutations.

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