title: Validation and utilization of an internally controlled multiplex Real-time RT-PCR assay for simultaneous detection of enteroviruses and enterovirus A71 associated with hand foot and mouth disease.
abstract: BACKGROUND
  Hand foot and mouth disease (HFMD) is a disease of public health importance across the Asia-Pacific region. The disease is caused by enteroviruses (EVs), in particular enterovirus A71 (EV-A71). In EV-A71-associated HFMD, the infection is sometimes associated with severe manifestations including neurological involvement and fatal outcome. The availability of a robust diagnostic assay to distinguish EV-A71 from other EVs is important for patient management and outbreak response.
  
  
  METHODS
  We developed and validated an internally controlled one-step single-tube real-time RT-PCR in terms of sensitivity, linearity, precision, and specificity for simultaneous detection of EVs and EV-A71. Subsequently, the assay was then applied on throat and rectal swabs sampled from 434 HFMD patients.
  
  
  RESULTS
  The assay was evaluated using both plasmid DNA and viral RNA and has shown to be reproducible with a maximum assay variation of 4.41 % and sensitive with a limit of detection less than 10 copies of target template per reaction, while cross-reactivity with other EV serotypes was not observed. When compared against a published VP1 nested RT-PCR using 112 diagnostic throat and rectal swabs from 112 children with a clinical diagnosis of HFMD during 2014, the multiplex assay had a higher sensitivity and 100 % concordance with sequencing results which showed EVs in 77/112 (68.8 %) and EV-A71 in 7/112 (6.3 %). When applied to clinical diagnostics for 322 children, the assay detected EVs in throat swabs of 257/322 (79.8 %) of which EV-A71 was detected in 36/322 (11.2 %) children. The detection rate increased to 93.5 % (301/322) and 13.4 % (43/322) for EVs and EV-A71, respectively, when rectal swabs from 65 throat-negative children were further analyzed.
  
  
  CONCLUSIONS
  We have successfully developed and validated a sensitive internally controlled multiplex assay for rapid detection of EVs and EV-A71, which is useful for clinical management and outbreak control of HFMD.
authors.affiliation: Oxford University Clinical Research Unit in partnership with the Hospital for Tropical Diseases, Wellcome Trust Major Overseas Programme, Ho Chi Minh City, Vietnam. thanhtt@oucru.org.
authors.forename: Tran Tan
authors.initials: TT
authors.lastname: Thanh
authors.affiliation: Oxford University Clinical Research Unit in partnership with the Hospital for Tropical Diseases, Wellcome Trust Major Overseas Programme, Ho Chi Minh City, Vietnam.
authors.forename: Nguyen To
authors.initials: NT
authors.lastname: Anh
authors.affiliation: Oxford University Clinical Research Unit in partnership with the Hospital for Tropical Diseases, Wellcome Trust Major Overseas Programme, Ho Chi Minh City, Vietnam.
authors.forename: Nguyen Thi
authors.initials: NT
authors.lastname: Tham
authors.affiliation: Children Hospital Number Two, Ho Chi Minh City, Vietnam.
authors.forename: Hoang Minh Tu
authors.initials: HM
authors.lastname: Van
authors.affiliation: Oxford University Clinical Research Unit in partnership with the Hospital for Tropical Diseases, Wellcome Trust Major Overseas Programme, Ho Chi Minh City, Vietnam.
authors.forename: Saraswathy
authors.initials: S
authors.lastname: Sabanathan
authors.affiliation: Hospital for Tropical Diseases, Ho Chi Minh City, Vietnam.
authors.forename: Phan Tu
authors.initials: PT
authors.lastname: Qui
authors.affiliation: Oxford University Clinical Research Unit in partnership with the Hospital for Tropical Diseases, Wellcome Trust Major Overseas Programme, Ho Chi Minh City, Vietnam.
authors.forename: Tran Thuy
authors.initials: TT
authors.lastname: Ngan
authors.affiliation: Hospital for Tropical Diseases, Ho Chi Minh City, Vietnam.
authors.forename: Tran Thi My
authors.initials: TT
authors.lastname: Van
authors.affiliation: Oxford University Clinical Research Unit in partnership with the Hospital for Tropical Diseases, Wellcome Trust Major Overseas Programme, Ho Chi Minh City, Vietnam.
authors.forename: Lam Anh
authors.initials: LA
authors.lastname: Nguyet
authors.affiliation: Oxford University Clinical Research Unit in partnership with the Hospital for Tropical Diseases, Wellcome Trust Major Overseas Programme, Ho Chi Minh City, Vietnam.
authors.forename: Nguyen Thi Han
authors.initials: NT
authors.lastname: Ny
authors.affiliation: Hospital for Tropical Diseases, Ho Chi Minh City, Vietnam.
authors.forename: Le Thi My
authors.initials: le TM
authors.lastname: Thanh
authors.affiliation: Facuty of Medicine, University of Malaya, Lumpur, Malaysia.
authors.forename: Ong Kien
authors.initials: OK
authors.lastname: Chai
authors.affiliation: Institute of Health and Community Medicine, Universiti Malaysia Sarawak, Sarawak, Malaysia.
authors.forename: David
authors.initials: D
authors.lastname: Perera
authors.affiliation: Children Hospital Number Two, Ho Chi Minh City, Vietnam.
authors.forename: Do Chau
authors.initials: do C
authors.lastname: Viet
authors.affiliation: Children Hospital Number One, Ho Chi Minh City, Vietnam.
authors.forename: Truong Huu
authors.initials: TH
authors.lastname: Khanh
authors.affiliation: Oxford University Clinical Research Unit in partnership with the Hospital for Tropical Diseases, Wellcome Trust Major Overseas Programme, Ho Chi Minh City, Vietnam.
authors.forename: Do Quang
authors.initials: do Q
authors.lastname: Ha
authors.affiliation: Children Hospital Number Two, Ho Chi Minh City, Vietnam.
authors.forename: Ha Manh
authors.initials: HM
authors.lastname: Tuan
authors.affiliation: Facuty of Medicine, University of Malaya, Lumpur, Malaysia.
authors.forename: Kum Thong
authors.initials: KT
authors.lastname: Wong
authors.affiliation: Children Hospital Number One, Ho Chi Minh City, Vietnam.
authors.forename: Nguyen Thanh
authors.initials: NT
authors.lastname: Hung
authors.affiliation: Hospital for Tropical Diseases, Ho Chi Minh City, Vietnam.
authors.forename: Nguyen Van Vinh
authors.initials: NV
authors.lastname: Chau
authors.affiliation: Oxford University Clinical Research Unit in partnership with the Hospital for Tropical Diseases, Wellcome Trust Major Overseas Programme, Ho Chi Minh City, Vietnam.|Centre for Tropical Medicine, Nuffield Department of Medicine, University of Oxford, Oxford, UK.
authors.forename: Guy
authors.initials: G
authors.lastname: Thwaites
authors.affiliation: Oxford University Clinical Research Unit in partnership with the Hospital for Tropical Diseases, Wellcome Trust Major Overseas Programme, Ho Chi Minh City, Vietnam.|Centre for Tropical Medicine, Nuffield Department of Medicine, University of Oxford, Oxford, UK.
authors.forename: H Rogier
authors.initials: HR
authors.lastname: van Doorn
authors.affiliation: Oxford University Clinical Research Unit in partnership with the Hospital for Tropical Diseases, Wellcome Trust Major Overseas Programme, Ho Chi Minh City, Vietnam.
authors.forename: Le
authors.initials: L
authors.lastname: Van Tan
mesh_terms: D000818:Animals; D001208:Asia; D002648:Child; D002675:Child, Preschool; D004770:Enterovirus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D004769:Enterovirus Infections / Q000175:diagnosis*; D005260:Female; D006801:Humans; D007223:Infant; D008297:Male; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D010614:Pharynx / Q000821:virology; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012007:Rectum / Q000821:virology; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012680:Sensitivity and Specificity