scholarly journals Norovirus Epidemiology and Genetic Diversity in Leipzig, Germany during 2013–2017

Viruses ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 1961
Author(s):  
Nora Ennuschat ◽  
Sabine Härtel ◽  
Corinna Pietsch ◽  
Uwe G. Liebert
Keyword(s):  
Genetic Diversity ◽  
Nucleic Acid ◽  
Real Time ◽  
Older Patients ◽  
Vaccine Development ◽  
Age Groups ◽  
Rt Pcr ◽  
Viral Capsids ◽  
Stool Samples ◽  
Gastrointestinal Complaints

Globally and in all age groups, noroviruses are a main cause of gastroenteritis. To assess their local epidemiology and genetic diversity, stool samples of 7509 inpatients with gastrointestinal complaints from all age groups were analyzed. After detection of norovirus genogroup I and II RNA by real-time RT-PCR, viral capsids were genotyped by partial nucleic acid sequencing. In the case of GII.2 strains, polymerase genotypes were also assessed. Between October 2013 and September 2017, presence of norovirus RNA was shown in 611 samples (8.1%), of which 610 (99.8%) were typed successfully. Norovirus positivity rate was higher in patients aged below five years (14.8%) than in older patients (5.7%). Among the 611 norovirus positive samples, GII.4 (56.6%) strains prevailed, followed by GII.6 (11.3%), GII.3 (11.0%) and GII.2 (9.5%). The most common genogroup I (GGI) genotype was GI.3 (3.6%). In addition, rare genotypes such as GII.13, GII.14 and GII.26 were detected. Interestingly, GII.3 infections were most common in children under the age of five years. Assessment of polymerase genotypes in GII.2 viruses showed a shift from P2 to P16, with higher diversity in P2 sequences. The varying distribution of norovirus genotypes depending on season, age and setting of infection highlights the importance of frequent genotyping as a basis for vaccine development and needful adjustments.

scholarly journals Genetic Diversity of Sapoviruses among Inpatients in Germany, 2008−2018

Viruses ◽  
2019 ◽  
Vol 11 (8) ◽  
pp. 726 ◽  
Author(s):  
Mann ◽  
Pietsch ◽  
Liebert
Keyword(s):  
Genetic Diversity ◽  
Age Groups ◽  
Structural Protein ◽  
Rt Pcr ◽  
Sporadic Cases ◽  
Stool Samples ◽  
Gastrointestinal Complaints ◽  
Polymerase Chain ◽  
Infants And Young Children ◽  
Selection Of

Sapovirus enteric disease affects people of all ages across the globe, in both sporadic cases and outbreak settings. Sapovirus is seldom assessed in Germany and its epidemiology in the country is essentially unknown. Thus, sapovirus occurrence and genetic diversity were studied by real-time reverse transcription polymerase chain reaction (RT-PCR) and partial sequencing of major viral structural protein (VP1) gene in two different sets of stool samples: 1) a selection of 342 diarrheal stools collected from inpatient children during 2008−2009, and 2) 5555 stool samples collected during 2010–2018 from inpatients of all age groups with gastrointestinal complaints. Results showed year-round circulation of sapoviruses, with peaks during cooler months. In total, 30 samples (8.8%) of the first and 112 samples of the second set of samples (2.0%) were sapovirus positive. Capsid gene sequencing was successful in 134/142 samples (94.4%) and showed circulation of all known human pathogenic genogroups. Genotype GI.1 predominated (31.8%), followed by GII.1 (16.7%), GII.3 (14.5%), GI.2 (13.8%) and GV.1 (12.3%). Additionally, minor circulation of GI.3, GI.6, GII.2, GII.4, GII.6 and GIV.1 was shown. Consequently, sapovirus diagnostics need broadly reactive RT-PCR protocols and should particularly be considered in infants and young children. Further studies from other sampling sites are essential to extend our knowledge on sapovirus epidemiology in Germany.

scholarly journals Molecular Epidemiology and Genetic Diversity of Norovirus in Young Children in Phnom Penh, Cambodia

2016 ◽  
Vol 2016 ◽  
pp. 1-8 ◽  
Author(s):  
Kaewkanya Nakjarung ◽  
Ladaporn Bodhidatta ◽  
Pimmnapar Neesanant ◽  
Paphavee Lertsethtakarn ◽  
Orntipa Sethabutr ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Young Children ◽  
Real Time ◽  
Pediatric Hospital ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Phnom Penh ◽  
Stool Samples ◽  
Polymerase Chain ◽  
Predominant Strain

This study investigated the genetic diversity of noroviruses identified from a previous surveillance study conducted at the National Pediatric Hospital in Phnom Penh, Cambodia, from 2004 to 2006. In the previous study, 926 stool samples were collected from children aged 3–60 months with acute diarrhea (cases) and without diarrhea (controls) with reported 6.7% of cases and 3.2% of controls being positive for norovirus. The initial norovirus diagnostic assay was performed with real-time reverse transcription-polymerase chain reaction (real-time RT PCR) which also distinguished between genogroups I and II (GI and GII). Norovirus infection was most commonly detected in children aged 12–23 months in both cases and controls. Norovirus Genotyping Tool and phylogenetic analysis of partial sequences of the 3′ end of the RNA-dependent RNA Polymerase (RdRp) and the capsid domain region were employed to assign genotypes of the norovirus strains. GII.4 was the most predominant capsid genotype detected at 39.5% followed by GII.6 at 14.9%. The GII.4 Hunter 2004 variant was the predominant strain detected. Six RdRP/capsid recombinants including GII.P7/GII.6, GII.P7/GII.14, GII.P7/GII.20, GII.P12/GII.13, GII.P17/GII.16, and GII.P21/GII.3 were also identified. This study of norovirus infection in young children in Cambodia suggests genetic diversity of norovirus as reported worldwide.

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.

scholarly journals Optimization of Real-time Reverse Transcriptase Polymerase Chain Reaction for Detection of Dengue Virus

2020 ◽  
pp. 1-7
Author(s):  
Kaunara A. Azizi ◽  
Arnold J. Ndaro ◽  
Athanasia Maro ◽  
Adonira Saro ◽  
Reginald A. Kavishe
Keyword(s):  
Polymerase Chain Reaction ◽  
Nucleic Acid ◽  
Dengue Virus ◽  
Reverse Transcriptase ◽  
Real Time ◽  
Acid Extraction ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Reliable Technique ◽  
Polymerase Chain

Aims: This study was set to optimize conditions for real time reverse transcriptase polymerase chain reaction (RT-PCR) for detection of dengue virus by using rapid and simple nucleic acid extraction method. Methodology: One step and two step real time RT-PCR were evaluated in different PCR thermocyclers. Extraction of viral RNA was done by using a simple boom method. Results: The real time RT-PCR technique was successfully optimized using simple and rapid method for purification of nucleic acid, ‘boom method’. The technique works better when performed in a two-step procedure and can works well with all range of real time PCR machines. The optimized real time RT-PCR used in the present study is a valuable and reliable technique for routine diagnosis of dengue. Further investigation on the cost effectiveness in adopting this technique for routine screening and monitoring of the dengue infection should be done.

scholarly journals Real-Time Detection of Noroviruses in Surface Water by Use of a Broadly Reactive Nucleic Acid Sequence-Based Amplification Assay

2006 ◽  
Vol 72 (8) ◽  
pp. 5349-5358 ◽  
Author(s):  
Saskia A. Rutjes ◽  
Harold H. J. L. van den Berg ◽  
Willemijn J. Lodder ◽  
Ana Maria de Roda Husman
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Water Samples ◽  
Rna Extraction ◽  
Sewage Treatment ◽  
Rdrp Gene ◽  
Nucleic Acid Sequence ◽  
Viral Gastroenteritis ◽  
Rt Pcr ◽  
Inhibitory Factors

ABSTRACT Noroviruses are the most common agents causing outbreaks of viral gastroenteritis. Outbreaks originating from contaminated drinking water and from recreational waters have been described. Due to a lack of cell culture systems, noroviruses are detected mostly by molecular methods. Molecular detection assays for viruses in water are often repressed by inhibitory factors present in the environment, like humic acids and heavy metals. To study the effect of environmental inhibitors on the performance of nucleic acid sequence-based amplification (NASBA), we developed a real-time norovirus NASBA targeting part of the RNA-dependent RNA polymerase (RdRp) gene. Specificity of the assay was studied with 33 divergent clones that contained part of the targeted RdRp gene of noroviruses from 15 different genogroups. Viral RNA originated from commercial oysters, surface waters, and sewage treatment plants in The Netherlands. Ninety-seven percent of the clones derived from human noroviruses were detected by real-time NASBA. Two clones containing animal noroviruses were not detected by NASBA. We compared the norovirus detection by real-time NASBA with that by conventional reverse transcriptase PCR (RT-PCR) with large-volume river water samples and found that inhibitory factors of RT-PCR had little or no effect on the performance of the norovirus NASBA. This consequently resulted in a higher sensitivity of the NASBA assay than of the RT-PCR. We show that by combining an efficient RNA extraction method with real-time NASBA the sensitivity of norovirus detection in water samples increased at least 100 times, which consequently has implications for the outcome of the infectious risk assessment.

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 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.

A clinical specimen collection and transport medium for molecular diagnostic and genomic applications

2010 ◽  
Vol 139 (11) ◽  
pp. 1764-1773 ◽  
Author(s):  
L. T. DAUM ◽  
S. A. WORTHY ◽  
K. C. YIM ◽  
M. NOGUERAS ◽  
R. F. SCHUMAN ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Genetic Characterization ◽  
Clinical Specimen ◽  
Molecular Transport ◽  
Cold Chain ◽  
Molecular Diagnostic ◽  
Rt Pcr ◽  
Transport Medium ◽  
Specimen Collection

SUMMARYPathogen detection and genetic characterization has dramatically changed in recent years. Clinical laboratories are transitioning from traditional culture and primer-specific sequencing to more robust and rapid nucleic acid testing such as real-time PCR and meta-genomic characterization, respectively. Specimen collection is the first step in any downstream molecular diagnostic procedure. PrimeStore Molecular Transport Medium (MTM) is an optimized blend of nucleic acid stabilizing reagents that includes a non-specific internal positive control that can be amplified using real-time RT–PCR for tracking the integrity of a specimen from the point of collection to detection. PrimeStore MTM is shown here to effectively kill pathogens, including highly pathogenic H5 influenza virus, inactivate nucleases and to protect and preserve released RNA at ambient temperature for up to 30 days for downstream real-time and traditional RT–PCR detection and genetic characterization. PrimeStore MTM is also compatible with a variety of commercial extraction kits. PrimeStore is suited for routine clinical specimens and has added utility for field collection in remote areas, triage centres, border crossings and during pandemics where cold-chain, transport, and dissemination of potentially infectious pathogens are a concern.

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