Assessment of PCR real time for quantification of human enterovirus in children with acute gastroenteritis in Italy

Aim: The aim of the present study was to detect the prevalence of norovirus and genotypes determination by real-time PCR among children below 18 years as an etiology of acute gastroenteritis and to compare rapid detection of norovirus by Enzyme-Linked Immunoassay (ELISA) to virus detection by real-time PCR. Methods: The research was a cross-sectional study conducted on children below 18 years complaining of community-acquired acute gastroenteritis. A stool sample was subjected to direct-antigen detection by ELISA for norovirus and molecular study by real-time polymerase chain reaction. Results: The study included 200 children with acute gastroenteritis with a mean age of 6.7±3.8 years. Norovirus antigen was detected by EIA in 34.5% and by real-time PCR in 30.5% of studied children with genotype GII, the predominant detected genotype (80.97%). Both real-time PCR and antigen detection of norovirus were positive in 43 (70.5%) of the children and negative in 113(81.3%) of the studied children. The sensitivity, specificity, positive predictive value, negative predictive value and accuracy for antigen detection by ELISA were 70.5%, 81.3%, 62.3%, 86.3% and 78%, respectively. Comparison between patients positive for norovirus and those negative for norovirus by real-time PCR revealed non-significant difference as regards age, sex, the season of occurrence and residence. Conclusion: The present study highlights that norovirus prevalence is common among pediatric patients with gastroenteritis above 5 years with GII genotype as the prevalent genotype. There was a significant correlation between positive and negative results of antigen detection of norovirus by ELISA and detection of RNA of norovirus by real-time PCR in stool samples. However, the screening for norovirus by ELISA has limited sensitivity and needs to be associated with a molecular method for accurate diagnosis of sporadic cases of gastroenteritis.

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Comparative Evaluation of Norovirus Infection in Children with Acute Gastroenteritis by Rapid Immunochromatographic Test, RT-PCR and Real-time RT-PCR

Journal of Tropical Pediatrics ◽

10.1093/tropej/fmx014 ◽

2017 ◽

Vol 63 (6) ◽

pp. 468-475 ◽

Cited By ~ 5

Author(s):

Kattareeya Kumthip ◽

Pattara Khamrin ◽

Wilaiporn Saikruang ◽

Kanittapon Supadej ◽

Hiroshi Ushijima ◽

...

Keyword(s):

Real Time ◽

Acute Gastroenteritis ◽

Comparative Evaluation ◽

Immunochromatographic Test ◽

Rt Pcr

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A New Real-Time Reverse Transcription-PCR Assay for Detection of Human Enterovirus 68 in Respiratory Samples

Journal of Clinical Microbiology ◽

10.1128/jcm.03691-14 ◽

2015 ◽

Vol 53 (5) ◽

pp. 1725-1726 ◽

Cited By ~ 15

Author(s):

Antonio Piralla ◽

Alessia Girello ◽

Marta Premoli ◽

Fausto Baldanti

Keyword(s):

Real Time ◽

Reverse Transcription ◽

Human Enterovirus ◽

Pcr Assay ◽

Reverse Transcription Pcr

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

10.1101/830281 ◽

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.

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Evaluation of RIDA®GENE norovirus GI/GII real time RT-PCR using stool specimens collected from children and adults with acute gastroenteritis

Journal of Clinical Virology ◽

10.1016/j.jcv.2018.04.006 ◽

2018 ◽

Vol 104 ◽

pp. 1-4 ◽

Cited By ~ 5

Author(s):

N. Kanwar ◽

F. Hassan ◽

L. Barclay ◽

C. Langley ◽

J. Vinjé ◽

...

Keyword(s):

Real Time ◽

Acute Gastroenteritis ◽

Rt Pcr ◽

Stool Specimens

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Screening and detection of human enterovirus 71 infection by a real-time RT-PCR assay in Marseille, France, 2009–2011

Clinical Microbiology and Infection ◽

10.1111/j.1469-0691.2012.03769.x ◽

2012 ◽

Vol 18 (4) ◽

pp. E77-E80 ◽

Cited By ~ 9

Author(s):

C.Y.Q. Tan ◽

G. Gonfrier ◽

L. Ninove ◽

C. Zandotti ◽

A. Dubot-Pérès ◽

...

Keyword(s):

Real Time ◽

Enterovirus 71 ◽

Human Enterovirus ◽

Rt Pcr ◽

Pcr Assay ◽

Human Enterovirus 71 ◽

Enterovirus 71 Infection

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Diagnostic Accuracy of Seven Commercial Assays for Rapid Detection of Group A Rotavirus Antigens

Journal of Clinical Microbiology ◽

10.1128/jcm.01984-15 ◽

2015 ◽

Vol 53 (11) ◽

pp. 3670-3673 ◽

Cited By ~ 15

Author(s):

Jérôme Kaplon ◽

Céline Fremy ◽

Sylvie Pillet ◽

Lucile Mendes Martins ◽

Katia Ambert-Balay ◽

...

Keyword(s):

Diagnostic Accuracy ◽

Real Time ◽

Reverse Transcription ◽

Acute Gastroenteritis ◽

Rapid Detection ◽

Group A Rotavirus ◽

Reverse Transcription Pcr ◽

Group A ◽

Stool Samples ◽

Human Stool

Seven commercial immunochromatographic assays intended for the detection of group A rotavirus antigens in human stool samples were evaluated. These assays showed similar levels of diagnostic accuracy and were suitable for the detection of rotavirus in patients with acute gastroenteritis but missed some asymptomatic rotavirus shedding identified by real-time reverse transcription-PCR.

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Determination of cut-off cycle threshold values in routine RT–PCR assays to assist differential diagnosis of norovirus in children hospitalized for acute gastroenteritis

Epidemiology and Infection ◽

10.1017/s095026881500059x ◽

2015 ◽

Vol 143 (15) ◽

pp. 3292-3299 ◽

Cited By ~ 21

Author(s):

N. V. TRANG ◽

M. CHOISY ◽

T. NAKAGOMI ◽

N. T. M. CHINH ◽

Y. H. DOAN ◽

...

Keyword(s):

Differential Diagnosis ◽

Real Time ◽

Acute Gastroenteritis ◽

Bimodal Distribution ◽

Clinical Samples ◽

Rt Pcr ◽

Prevalence Studies ◽

Cycle Threshold ◽

Asymptomatic Children ◽

Enteric Infections

SUMMARYNorovirus (NV) is an important cause of acute gastroenteritis in children, but is also frequently detected in asymptomatic children, which complicates the interpretation of NV detection results in both the clinical setting and population prevalence studies. A total of 807 faecal samples from children aged <5 years hospitalized for acute gastroenteritis were collected in Thai Binh, Vietnam, from January 2011 to September 2012. Real-time RT–PCR was used to detect and quantify NV-RNA in clinical samples. A bimodal distribution of cycle threshold (Ct) values was observed in which the lower peak was assumed to represent cases for which NV was the causal agent of diarrhoea, whereas the higher peak was assumed to represent cases involving an alternative pathogen other than NV. Under these assumptions, we applied finite-mixture modelling to estimate a threshold of Ct <21·36 (95% confidence interval 20·29–22·46) to distinguish NV-positive patients for which NV was the likely cause of diarrhoea. We evaluated the validity of the threshold through comparisons with NV antigen ELISA results, and comparisons of Ct values in patients co-infected with rotavirus. We conclude that the use of an appropriate cut-off value in the interpretation of NV real-time RT–PCR results may improve differential diagnosis of enteric infections, and could contribute to improved estimates of the burden of NV disease.

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Development and Evaluation of an Enterovirus D68 Real-Time Reverse Transcriptase PCR Assay

Journal of Clinical Microbiology ◽

10.1128/jcm.00923-15 ◽

2015 ◽

Vol 53 (8) ◽

pp. 2641-2647 ◽

Cited By ~ 28

Author(s):

Todd N. Wylie ◽

Kristine M. Wylie ◽

Richard S. Buller ◽

Maria Cannella ◽

Gregory A. Storch

Keyword(s):

Reverse Transcriptase ◽

Real Time ◽

Limit Of Detection ◽

Respiratory Illness ◽

The United States ◽

Human Enterovirus ◽

Rt Pcr ◽

Pcr Assay ◽

Reverse Transcriptase Pcr ◽

Enterovirus D68

We have developed and evaluated a real-time reverse transcriptase PCR (RT-PCR) assay for the detection of human enterovirus D68 (EV-D68) in clinical specimens. This assay was developed in response to the unprecedented 2014 nationwide EV-D68 outbreak in the United States associated with severe respiratory illness. As part of our evaluation of the outbreak, we sequenced and published the genome sequence of the EV-D68 virus circulating in St. Louis, MO. This sequence, along with other GenBank sequences from past EV-D68 occurrences, was used to computationally select a region of EV-D68 appropriate for targeting in a strain-specific RT-PCR assay. The RT-PCR assay amplifies a segment of the VP1 gene, with an analytic limit of detection of 4 copies per reaction, and it was more sensitive than commercially available assays that detect enteroviruses and rhinoviruses without distinguishing between the two, including three multiplex respiratory panels approved for clinical use by the FDA. The assay did not detect any other enteroviruses or rhinoviruses tested and did detect divergent strains of EV-D68, including the first EV-D68 strain (Fermon) identified in California in 1962. This assay should be useful for identifying and studying current and future outbreaks of EV-D68 viruses.

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