Dataset Information

A Reverse Transcription-Polymerase Spiral Reaction (RT-PSR)-Based Rapid Coxsackievirus A16 Detection Method and Its Application in the Clinical Diagnosis of Hand, Foot, and Mouth Disease.

ABSTRACT: Hand, foot, and mouth disease (HFMD) is a common viral illness affecting infants and children that is usually caused by Coxsackievirus A16 (CVA-16). To diagnose HFMD, we developed a method for rapid detection of CVA-16 based on reverse transcription-polymerase spiral reaction (RT-PSR). We used two pairs of primers that specifically recognize the conserved sequences of VP1 coding region of CVA-16, and template RNA was reverse transcribed and amplified in a single tube under isothermal conditions, total reaction time could be reduced to less than 40 min. The detection limit of this method was between 2.4 × 10² and 2.4 × 10¹ copies/μl with excellent specificity. To test the clinical applicability of the method, 40 clinical stool samples were analyzed using RT-PSR and quantitative reverse transcription-polymerase chain reaction, and comparison showed that the coincidence rate was 100%. Compared with other similar detection methods, RT-PSR requires less time, simpler operation, and lower cost. These results prove that our novel, simple, and reliable isothermal nucleic acid testing assay has potential application for clinical detection of CVA-16.

SUBMITTER: He S

PROVIDER: S-EPMC7236501 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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A Reverse Transcription-Polymerase Spiral Reaction (RT-PSR)-Based Rapid Coxsackievirus A16 Detection Method and Its Application in the Clinical Diagnosis of Hand, Foot, and Mouth Disease.

He Shiyu S Huang Yanzhi Y Zhao Yanling Y Pang Bo B Wang Lixue L Sun Liwei L Yu Haoyan H Wang Juan J Li Juan J Song Xiuling X Li Hui H

Frontiers in microbiology 20200512

Hand, foot, and mouth disease (HFMD) is a common viral illness affecting infants and children that is usually caused by Coxsackievirus A16 (CVA-16). To diagnose HFMD, we developed a method for rapid detection of CVA-16 based on reverse transcription-polymerase spiral reaction (RT-PSR). We used two pairs of primers that specifically recognize the conserved sequences of VP1 coding region of CVA-16, and template RNA was reverse transcribed and amplified in a single tube under isothermal conditions, ...[more]

PMID: 32477283

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Project description:Introduction: Hand, foot, and mouth disease (HFMD) is a mild self-limited childhood infectious disease caused by a variety of enteroviruses (EVs). Aim: To investigate the molecular epidemiology of EVs associated with HFMD and their clinical presentation during the HFMD outbreak that occurred in the Jenin district, Palestine, from May to August 2024. Materials and Methods: Forty-four (44) throat and vesicular swabs were tested for enteroviral infections using two RT-PCR assays targeting both the 5'NTR and the VP1-2A regions of the enteroviral genome for the diagnosis and genotyping. Patients' demographic data and clinical history were used to create an epidemiological curve. EpiInfo free software was used to draw a cluster mapping. MEGA-X was used to construct a maximum likelihood (ML) tree. PopArt 1.7 software was used to construct neighbor-joining network. Results: The mean age of the study sample was 2.08 (0.25-12 years) with 95% (42/44) under five years old. The male/female ratio was 0.9. All cases presented with typical HFMD signs and symptoms with variable sites of signs. Of the 44 samples, 36 yield positive RT-PCR targeting the 5'NTR. Seven randomly selected positive RT-PCR-5'NTR samples were sequenced using Sanger sequencing for genotyping. It was shown that all were CV-A16 sub-genogroup B1c. Phylogenetic analysis of VP1-2A region sequences showed that all Palestinian CV-A16 isolates form a pure haplogroup of CV-A16 sub-genotype B1c. Furthermore, although haplotype network analysis showed high variation between the viral sequences, the haplotype analysis supported the ML phylogenetic tree in having them all in one haplogroup. Conclusion: CV-A16, sub-genotype B1c was the virus responsible for the HFMD outbreak in the Jenin district of Palestine in the summer of 2024. Phylogenetic and haplotype analysis showed that CV-A16 strains cluster closely with each other and very close to an Indian isolate (OR437338.1), indicating the monomorphic nature of this strain with low genetic variation and the probability of virus importation.

Project description:Hand, foot and mouth disease (HFMD) is a common childhood infectious disease, caused by enteroviruses (EVs), which can present with typical or atypical lesions. The illness is self-limiting, but it can also have serious complications. Since 1997, HFMD infections have become endemic and have increased to epidemic proportions across the Asia Pacific region, including India. Coxsackievirus-A16 (CV-A16) outbreaks occurred in India from 2005 onwards, although the clinical symptoms were noticeably different during this period. Understanding the population dynamics of enteroviruses that cause HFMD is crucial in the post-polio era because one of the circulating strain may replace another as the dominant strain. The aim of this study is to describe the genetic features of the CV-A16 strains isolated from hand, foot and mouth disease (HFMD) patients in India. Reverse transcription PCR (RT-PCR) and cell-culture-based isolation of CV-A16 was done from the 55 clinical samples. The entire genome of the CV-A16 isolate was performed from the seven isolates. After the sequences were analysed, a phylogenetic tree was created using bioinformatics tools. The total genomic length obtained was 7411 base pairs (bp). Nucleotide similarity across various regions, including 5'UTR, P1, P2 and 3'UTR, ranged from 87.0-97.9 %, 77.0-95.4 %, 80.3-96.9 %, and 77.9-96.2 %, respectively. Correspondingly, similarities in the VP1 region's nucleotide and amino acid sequences were 91.4-96.4 % and 99.3-99.7 %, respectively. Phylogenetic analysis highlighted that CV-A16 strains identified in Pune, Maharashtra, were grouped within the same cluster. The analysed CV-A16 isolates in this study aligned with subgenotype B1c. These findings have far-reaching implications for the surveillance, prevention and management of HFMD and CV-A16. Monitoring the dynamics of CV-A16 strains, informed by the genetic characteristics identified here, will significantly impact strategies aimed at tackling HFMD and its associated public health challenges.

Project description:BackgroundIn 2024, mainland China witnessed a significant upsurge in Hand, Foot, and Mouth Disease (HFMD) cases. Coxsackievirus A16 (CVA16) is one of the primary causative agents of HFMD. Long-term monitoring of theCVA16 infection rate and genotype changes is crucial for the prevention and control of HFMD.MethodsA total of 40,673 clinical specimens were collected from suspected HFMD cases in Guangdong province from 2018 to 2024, including rectal swabs (n = 27,954), throat swabs (n = 6791), stool (n = 5923), cerebrospinal fluid (n = 3), and herpes fluid (n = 2). A total of 24,410 samples were detected as EV-positive and further typed by RT-PCR. A total of 872 CVA16-positive samples were isolated and further sequenced to obtain the full-length VP1 sequence. Phylogenetic analysis was performed based on viral protein 1 gene (VP1).ResultsIn the first 25 weeks of 2024, reported cases of HFMD were 1.36 times higher than the mean rates of 2023. In 2024, CVA16 predominated at 75.42%, contrasting with the past etiological pattern in which the CVA6 was predominant with the detection rate ranging from 32.85 to 77.61% from 2019 to 2023. Phylogenetic analysis based on the VP1 gene revealed that the B1a and B1b subtypes co-circulated in Guangdong from 2018 to 2022. The B1c outbreak clade, detected in Guangdong in 2023, constituted 68.24% of the 148 strains of CVA16 collected in 2024, suggesting a subtype shift in the CVA16 virus. There were three specific amino acid variations (P3S, I235V, and T240A) in the VP1 sequence of B1c.ConclusionsThe new emergence of the CVA16 B1c outbreak clade in Guangdong during 2023-2024 highlights the necessity for the enhanced surveillance of the virus evolution epidemiological dynamic in this region. Furthermore, it is imperative to closely monitor the etiological pattern changes in Hand, Foot, and Mouth Disease (HFMD) in other regions as well. Such vigilance will be instrumental in guiding future vaccination strategies for HFMD.

Dataset Information

A Reverse Transcription-Polymerase Spiral Reaction (RT-PSR)-Based Rapid Coxsackievirus A16 Detection Method and Its Application in the Clinical Diagnosis of Hand, Foot, and Mouth Disease.

Publications

A Reverse Transcription-Polymerase Spiral Reaction (RT-PSR)-Based Rapid Coxsackievirus A16 Detection Method and Its Application in the Clinical Diagnosis of Hand, Foot, and Mouth Disease.

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