Molecular strategy for the direct detection and identification of human enteroviruses in clinical specimens associated with hand, foot and mouth disease

Abstract

Background: Diseases caused by human enteroviruses (EVs) are a major global public health problem. Thus, the effective diagnosis of all human EVs infections and the monitoring of epidemiological and ecological dynamic changes are urgently needed.

Methods: Based on two comprehensive virological surveillance systems of hand, foot and mouth disease (HFMD), real-time PCR and nested RT-PCR (RT-snPCR) methods based on the enteroviral VP1, VP4-VP2 and VP4 regions were designed to directly detect all human EVs serotypes in clinical specimens.

Results: The results showed that the proposed serotyping strategy exhibit very high diagnostic efficiency (Study 1: 99.9%; Study 2: 89.5%), and the variance between the study was due to inclusion of the specific Coxsackie virus A6 (CVA6) real-time RT-PCR and VP4 RT-snPCR in Study 1 but not Study 2. Furthermore, only throat swabs were collected and analyzed in Study 2, whereas in Study 1, if a specific EV serotype was not identified in the primary stool sample, other sample types (rectal swab and throat swab) were further tested where available. During the study period from 2013 to 2018, CVA6 became one of the main HFMD causative agents, whereas the level of enterovirus A71 (EV-A71) declined in 2017.

Conclusion: The findings of this study demonstrate the appropriate application of PCR methods and the combination of biological sample types that are useful for etiological studies and propose a molecular strategy for the direct detection of human EVs in clinical specimens associated with HFMD.

Fig 1. Overview of the testing assay procedures and typing results in Anhua County (Study 1).

N.B: “*” indicates that the clinical samples satisfied the ideal selection criteria for typing, and the stool samples were selected first. If the stool samples were negative or untyped, rectal swabs were used for typing, and it the rectal swabs also yielded negative results, throat swabs were used for typing if available.

Fig 2. Overview of the testing assay procedures and typing results in Zhengzhou City (Study 2).

Fig 3. Methodology of the serotyping strategy for HFMD clinical specimens.

3A –Flowchart depicting the serotyping procedure. 3B –Flowchart depicting the serotyping procedure for samples identified using the ‘generic primers’ and unbale identified using the ‘specific primers’. Note: “+” indicates an EV-positive result; “-” indicates an EV-negative result.