scholarly journals Novel Human Astroviruses: Prevalence and Association with Common Enteric Viruses in Undiagnosed Gastroenteritis Cases in Spain

Viruses ◽  
2019 ◽  
Vol 11 (7) ◽  
pp. 585 ◽  
Author(s):  
Diem-Lan Vu ◽  
Aurora Sabrià ◽  
Nuria Aregall ◽  
Kristina Michl ◽  
Virginia Rodriguez Garrido ◽  
...  
Keyword(s):  
Real Time ◽  
Acute Gastroenteritis ◽  
Enteric Viruses ◽  
Epidemiological Studies ◽  
Diagnostic Methods ◽  
Rt Pcr ◽  
Routine Diagnostics ◽  
Human Astroviruses ◽  
Stool Samples ◽  
Pcr Assays

A remarkable percentage of acute gastroenteritis cases remain etiologically undiagnosed. The aim of the study was to determine the prevalence of common and emerging enteric viruses, such as novel human astroviruses, among undiagnosed samples from children with acute gastroenteritis. Epidemiological studies for novel human astroviruses are still scarce. Stool samples collected over two consecutive winter seasons (2016–2017) from children with gastroenteritis in Spain, which were negative for bacteria, rotavirus, and adenovirus by routine diagnostics were screened by real-time RT-PCR assays for the presence of classical and novel astrovirus, rotavirus, norovirus GI and GII, sapovirus, and adenovirus. Overall, 220/384 stool samples (57.3%) were positive for at least one virus. Co-infections were identified in 21% of cases. Among a total of 315 viruses identified, adenovirus was the most prevalent (n = 103), followed by rotavirus (n = 51), sapovirus (n = 50), classical astrovirus (n = 43), novel astroviruses (n = 42), and norovirus (n = 26). Novel astroviruses were present in 13.3% of virus-positive cases. Most novel astroviruses were found in children <2-year-old (30/39 children, 77%, p = 0.01) and were found in co-infection (66%). Only classical astroviruses demonstrated significant differences in the Cq values during mono-infections compared to co-infections. In conclusion, common enteric viruses may be frequently found in children with undiagnosed gastroenteritis, indicating the need to implement more sensitive diagnostic methods. Novel astroviruses circulate in the community and could be the cause of gastroenteritis among young children.

Investigations on the Frequency of Norovirus Contamination of Ready-to-Eat Food Items in Istanbul, Turkey, by Using Real-Time Reverse Transcription PCR

2011 ◽  
Vol 74 (5) ◽  
pp. 840-843 ◽  
Author(s):  
AYSUN YILMAZ ◽  
KAMIL BOSTAN ◽  
EDA ALTAN ◽  
KARLO MURATOGLU ◽  
NURI TURAN ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Epidemiological Studies ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Food Items ◽  
Reverse Transcription Pcr ◽  
Significant Difference ◽  
Tomato Sample ◽  
Pcr Assays

Investigation of norovirus (NoV) contamination of food items is important because many outbreaks occur after consumption of contaminated shellfish, vegetables, fruits, and water. The frequency of NoV contamination in food items has not previously been investigated in Turkey. The aim of this study was to investigate the frequency of human NoV genogroups (G) I and II in ready-to-eat tomatoes, parsley, green onion, lettuce, mixed salads, and cracked wheat balls. RNA was extracted with the RNeasy Mini Kit, and a real-time reverse transcription (RT) PCR assay was performed using primers specific for NoV GI and GII. Among the 525 samples analyzed, NoV GII was detected in 1 green onion sample and 1 tomato sample by both SYBR Green and TaqMan real-time RT-PCR assays; no GI virus was detected. The Enterobactericaeae and Escherichia coli levels in the NoV-positive green onion were 6.56 and 1.28 log CFU/g, and those in the tomato were 5.55 and 1.30 log CFU/g, respectively. No significant difference in the bacterial levels was found between the NoV-positive and NoV-negative samples. This study is the first in which NoV GII was found in ready-to-eat food collected from Istanbul, Turkey; thus, these foods may be considered a risk to human health. Epidemiological studies and measures to prevent NoV infection should be considered.

scholarly journals Human Bocavirus Prevalence In Children With Acute Gastroenteritis From Rural Communities In The Northen Region Of South Africa

2019 ◽  
Author(s):  
Mpumelelo Casper Rikhotso ◽  
Ronewa Khumela ◽  
Jean Pierre Kabue ◽  
Afsatou Ndama Traoré ◽  
Natasha Potgieter
Keyword(s):  
South Africa ◽  
Rural Communities ◽  
Real Time ◽  
Real Time Pcr ◽  
Acute Gastroenteritis ◽  
Enteric Viruses ◽  
Health Care Facilities ◽  
Human Bocavirus ◽  
Stool Samples ◽  
Pcr Targeting

AbstractBACKGROUNDAcute gastroenteritis (AGE) is a leading cause of morbidity and mortality in young children worldwide. Human Bocavirus (HBoV) is an emerging virus globally associated with diarrhea. The aim of this study was to demonstrate the prevalence of HBoV genotypes in children (≤5 years) from rural communities in South Africa (SA) suffering from AGE.MATERIAL AND METHODA total of 141 fecal samples of children ≤5 years with acute gastroenteritis (AGE) were collected from rural Primary Health Care facilities in the Vhembe district of SA between June 2017 and July 2018. Clinical symptoms and demographic data were also recorded. A total of 102 (72%) were outpatients and 39 (28%) were hospitalized patients. Human Bocavirus (HBoV) genotypes were determined using Real-Time Multiplex PCR. DNA extracts of positive samples were confirmed by conventional PCR targeting the NS1 gene. Co-infection with other enteric viruses were determined in HBoV positive samples using Real-Time PCR.RESULTSHBoV was detected in 8 (5.7%) children with AGE. Children were in the age group between 1-24 months. HBoV1 and HBoV3 genotypes were each detected in 3 (37.5%) stool samples and HBoV2 in 2 (25%) stool samples. Co-infection with other enteric viruses included Rotavirus (37.5%); Adenovirus (37.5%); Norovirus (25%) and Astrovirus (12.5%).CONCLUSIONHBoV infections could be seen as a potential emerging diarrheal pathogen in South Africa. Further studies are required to understand the role of HBoV infections in children and adults with acute gastroenteritis.Author summaryAcute gastroenteritis (AGE) is recognized as a major cause for mortality in children ≤5 years of age in Africa and other developing countries. Viruses known to be involved in AGE includes Rotavirus, Norovirus, Astrovirus and Adenovirus and have been reported globally. Recently the Human Bocavirus (HBoV) have been reported in numerous studies globally as a potential cause of diarrhea. In this study, the prevalence and genetic diversity of human Bocavirus in children with AGE from rural communities in Limpopo, South Africa were investigated. In total, 141 stool samples from children ≤ 5 years with AGE were assessed for the presence of HBoV using Real-Time PCR. HBoV were detected in 8 (5.7%) patients and included 3 positive samples for HBoV1 and HBoV3 respectively and 2 positive for HBoV2. No HBoV4 were detected. Among the 8 positive HBoV samples, co-infection with other enteric viruses were found in 7 (87.5%) samples, while mono infection with HBoV alone was detected in 1 (12.5%) patient. HBoV mixed infection with Rotavirus (3/8; 37.5%); Adenovirus (3/8; 37.5%); Norovirus (2/8; 25%) and Astrovirus (1/8; 12.5%) were observed in this study. This study reported for the first time on the prevalence of human Bocavirus in children with AGE from rural communities in South Africa.

scholarly journals Detection of SARS Coronavirus in Patients with Severe Acute Respiratory Syndrome by Conventional and Real-Time Quantitative Reverse Transcription-PCR Assays

2004 ◽  
Vol 50 (1) ◽  
pp. 67-72 ◽  
Author(s):  
Leo L M Poon ◽  
Kwok Hung Chan ◽  
On Kei Wong ◽  
Timothy K W Cheung ◽  
Iris Ng ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Real Time ◽  
N Gene ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Reverse Transcription Pcr ◽  
Copy Numbers ◽  
Stool Samples ◽  
Pcr Assays

Abstract Background: A novel coronavirus (CoV) was recently identified as the agent for severe acute respiratory syndrome (SARS). We compared the abilities of conventional and real-time reverse transcription-PCR (RT-PCR) assays to detect SARS CoV in clinical specimens. Methods: RNA samples isolated from nasopharyngeal aspirate (NPA; n = 170) and stool (n = 44) were reverse-transcribed and tested by our in-house conventional RT-PCR assay. We selected 98 NPA and 37 stool samples collected at different times after the onset of disease and tested them in a real-time quantitative RT-PCR specific for the open reading frame (ORF) 1b region of SARS CoV. Detection rates for the conventional and real-time quantitative RT-PCR assays were compared. To investigate the nature of viral RNA molecules in these clinical samples, we determined copy numbers of ORF 1b and nucleocapsid (N) gene sequences of SARS CoV. Results: The quantitative real-time RT-PCR assay was more sensitive than the conventional RT-PCR assay for detecting SARS CoV in samples collected early in the course of the disease. Real-time assays targeted at the ORF 1b region and the N gene revealed that copy numbers of ORF 1b and N gene sequences in clinical samples were similar. Conclusions: NPA and stool samples can be used for early diagnosis of SARS. The real-time quantitative RT-PCR assay for SARS CoV is potentially useful for early detection of SARS CoV. Our results suggest that genomic RNA is the predominant viral RNA species in clinical samples.

scholarly journals Monitoring Shedding of Five Genotypes of RotaTeq Vaccine Viruses by Genotype-Specific Real-Time Reverse Transcription-PCR Assays

2018 ◽  
Vol 56 (6) ◽  
Author(s):  
Yuki Higashimoto ◽  
Masaru Ihira ◽  
Yu Miyazaki ◽  
Ayumi Kuboshiki ◽  
Sayaka Yoshinaga ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Cross Reactivity ◽  
Rt Pcr ◽  
Fecal Shedding ◽  
Reverse Transcription Pcr ◽  
Coefficients Of Variation ◽  
Long Time ◽  
Stool Samples ◽  
Pcr Assays

ABSTRACTRotaTeq (RV5) is a widely used live attenuated pentavalent rotavirus (RV) vaccine. Although fecal shedding of RV vaccine strains persists for long time periods, it is unclear how each vaccine strain replicates in intestinal tissue and is excreted in stool. To examine this issue, we established RV5 genotype-specific real-time reverse transcription-PCR (RT-PCR) assays. Five real-time RT-PCR assays were designed for the VP7 gene in genotypes G1, G2, G3, G4, and G6. All assays exhibited excellent linearity, and the detection limit was 1 infectious unit (IU)/reaction for G2, G4, and G6 and 10 IUs/reaction for G1 and G3. No cross-reactivity was observed among G genotypes. The inter- and intra-assay coefficients of variation were less than 3%. The assays were used to examine 129 stool samples collected from eight infants who received RV5. In cases 1 and 2, who received three rounds of vaccination, RV shedding decreased gradually with the number of vaccinations. G1 and G6 shedding appeared to be predominant in comparison to shedding of the other genotypes. Patterns of fecal shedding of the five genotypes of vaccine viruses differed between the eight vaccine recipients. RV5 genotype-specific real-time RT-PCR assays will be useful to study the molecular biology of RV5 replication in infants and experimental animals.

scholarly journals Real-time RT-PCR assays to differentiate wild-type group A rotavirus strains from Rotarix®and RotaTeq®vaccine strains in stool samples

2013 ◽  
Vol 10 (3) ◽  
pp. 767-777 ◽  
Author(s):  
Rashi Gautam ◽  
Mathew D Esona ◽  
Slavica Mijatovic-Rustempasic ◽  
Ka Ian Tam ◽  
Jon R Gentsch ◽  
...  
Keyword(s):  
Real Time ◽  
Wild Type ◽  
Rt Pcr ◽  
Type Group ◽  
Group A Rotavirus ◽  
Group A ◽  
Stool Samples ◽  
Pcr Assays

scholarly journals Comprehensive Real-Time RT-PCR Assays for the Detection of Fifteen Viruses Infecting Prunus spp.

Plants ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 273
Author(s):  
Alfredo Diaz-Lara ◽  
Kristian Stevens ◽  
Vicki Klaassen ◽  
Deborah Golino ◽  
Maher Al Rwahnih
Keyword(s):  
Genetic Diversity ◽  
Real Time ◽  
Germplasm Collection ◽  
Fruit Trees ◽  
Diagnostic Methods ◽  
Detection Methods ◽  
Economic Losses ◽  
Rt Pcr ◽  
Pcr Assays ◽  
Prunus Spp

Viruses can cause economic losses in fruit trees, including Prunus spp., by reducing yield and marketable fruit. Given the genetic diversity of viruses, reliable diagnostic methods relying on PCR are critical in determining viral infection in fruit trees. This study evaluated the broad-range detection capacity of currently available real-time RT-PCR assays for Prunus-infecting viruses and developed new assays when current tests were inadequate or absent. Available assays for 15 different viruses were exhaustively evaluated in silico to determine their capacity to detect virus isolates deposited in GenBank. During this evaluation, several isolates deposited since the assay was designed exhibited nucleotide mismatches in relation to the existing assay’s primer sequences. In cases where updating an existing assay was impractical, we performed a redesign with the dual goals of assay compactness and comprehensive inclusion of genetic diversity. The efficiency of each developed assay was determined by a standard curve. To validate the assay designs, we tested them against a comprehensive set of 87 positive and negative Prunus samples independently analyzed by high throughput sequencing. As a result, all the real-time RT-PCR assays described herein successfully detected the different viruses and their corresponding isolates. To further validate the new and updated assays a Prunus germplasm collection was surveyed. The sensitive and reliable detection methods described here will be used for the large-scale pathogen testing required to maintain the highest quality nursery stock.

scholarly journals Evaluation of immunochromatography tests for detection of novel GII.17 norovirus in stool samples

2015 ◽  
Vol 20 (28) ◽  
Author(s):  
P Khamrin ◽  
A Thongprachum ◽  
S Takanashi ◽  
S Okitsu ◽  
N Maneekarn ◽  
...  
Keyword(s):  
Positive Result ◽  
Real Time ◽  
Acute Gastroenteritis ◽  
Rt Pcr ◽  
Stool Samples

A novel GII.17 norovirus has emerged as a major cause of epidemic and endemic acute gastroenteritis in several countries in Asia. We used a small panel of stool samples in which GII.17 virus had been quantified by real-time RT-PCR to evaluate four commercially available norovirus immunochromatography (IC) kits. At least 108 copies/mL of GII.17 virus were required by each IC kit for a positive result, which is 1,000-fold more than that reported for these assays for GII.4 viruses.

DEVELOPMENT OF MULTIPLEX REAL-TIME PCR ASSAY FOR SIX PATHOGENS: RAPID TOOL FOR DIAGNOSIS OF BACTERIAL MENINGITIS

2019 ◽  
pp. 60-66
Author(s):  
Viet Quynh Tram Ngo ◽  
Thi Ti Na Nguyen ◽  
Hoang Bach Nguyen ◽  
Thi Tuyet Ngoc Tran ◽  
Thi Nam Lien Nguyen ◽  
...  
Keyword(s):  
Bacterial Meningitis ◽  
Real Time ◽  
Real Time Pcr ◽  
Diagnostic Methods ◽  
Type B ◽  
E Coli ◽  
Pcr Assays ◽  
Set Up ◽  
Etiological Agents ◽  
Suis Serotype

Introduction: Bacterial meningitis is an acute central nervous infection with high mortality or permanent neurological sequelae if remained undiagnosed. However, traditional diagnostic methods for bacterial meningitis pose challenge in prompt and precise identification of causative agents. Aims: The present study will therefore aim to set up in-house PCR assays for diagnosis of six pathogens causing the disease including H. influenzae type b, S. pneumoniae, N. meningitidis, S. suis serotype 2, E. coli and S. aureus. Methods: inhouse PCR assays for detecting six above-mentioned bacteria were optimized after specific pairs of primers and probes collected from the reliable literature resources and then were performed for cerebrospinal fluid (CSF) samples from patients with suspected meningitis in Hue Hospitals. Results: The set of four PCR assays was developed including a multiplex real-time PCR for S. suis serotype 2, H. influenzae type b and N. meningitides; three monoplex real-time PCRs for E. coli, S. aureus and S. pneumoniae. Application of the in-house PCRs for 116 CSF samples, the results indicated that 48 (39.7%) cases were positive with S. suis serotype 2; one case was positive with H. influenzae type b; 4 cases were positive with E. coli; pneumococcal meningitis were 19 (16.4%) cases, meningitis with S. aureus and N. meningitidis were not observed in any CSF samples in this study. Conclusion: our in-house real-time PCR assays are rapid, sensitive and specific tools for routine diagnosis to detect six mentioned above meningitis etiological agents. Key words: Bacterial meningitis, etiological agents, multiplex real-time PCR

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 Comparison of nine different commercially available molecular assays for detection of SARS-CoV-2 RNA

2021 ◽  
Author(s):  
Ute Eberle ◽  
◽  
Clara Wimmer ◽  
Ingrid Huber ◽  
Antonie Neubauer-Juric ◽  
...  
Keyword(s):  
Real Time ◽  
Rt Pcr ◽  
Diagnostic Assays ◽  
Molecular Assays ◽  
Pcr Assays ◽  
Laboratory Experiences

AbstractTo face the COVID-19 pandemic, the need for fast and reliable diagnostic assays for the detection of SARS-CoV-2 is immense. We describe our laboratory experiences evaluating nine commercially available real-time RT-PCR assays. We found that assays differed considerably in performance and validation before routine use is mandatory.

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