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.

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. CONCLUSION: 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.

A birth cohort study of viral infections in Vietnamese infants and children: study design, methods and characteristics of the cohort.

BACKGROUND: In Ho Chi Minh City, Vietnam, more than one-third of admissions to the two paediatric hospitals are attributable to four infectious syndromes: dengue, diarrhoeal disease, acute respiratory infection, and hand, foot and mouth disease. We have established a large prospective birth cohort study to investigate individual, environmental, virological, and immunological determinants of infection and disease in infants. Specific research questions are focused on the role of maternal antibody in protection against infection in infancy, and the adaptive immune response to vaccination and natural infection. This paper presents the cohort design, methods, and baseline characteristics of the participants enrolled in the first two years. METHODS/DESIGN: Women are enrolled prior to delivery at one hospital in each of two catchment areas: an urban district in central HCMC, and a mixed urban/rural district in the Mekong Delta 150 km southwest of HCMC. Infants are enrolled within 3 days of birth, and maternal and cord blood samples are collected. Routine blood samples and data on growth, health status and vaccinations are collected from infants at scheduled visits at 4, 9 and 12 months. Clinical data and specimens are collected from infants presenting at a study clinic, or admitted to hospital, with any of the the four infectious syndromes of interest. DISCUSSION: In four years since since the study began in July 2009, >6400 infants have been enrolled, and enrolment is ongoing. Attrition is low: 84% of participants have completed the full 12-month follow-up period. Baseline characteristics of the first 4300 enrollees are presented here. We have demonstrated the feasibility of establishing a large prospective study of infectious diseases in infancy in a resource-limited setting, with minimal loss to follow-up. Our linked socio-demographic, clinical and laboratory data will help elucidate the viral aetiology and epidemiology of common infectious diseases of infancy, and can inform the implemention of existing and future vaccines. This study furthermore provides a platform to which additional endpoints could be added in the future.