Development and evaluation of a non-ribosomal random PCR and next-generation sequencing based assay for detection and sequencing of hand, foot and mouth disease pathogens.

BACKGROUND: Hand, foot and mouth disease (HFMD) has become a major public health problem across the Asia-Pacific region, and is commonly caused by enterovirus A71 (EV-A71) and coxsackievirus A6 (CV-A6), CV-A10 and CV-A16. Generating pathogen whole-genome sequences is essential for understanding their evolutionary biology. The frequent replacements among EV serotypes and a limited numbers of available whole-genome sequences hinder the development of overlapping PCRs for whole-genome sequencing. We developed and evaluated a non-ribosomal random PCR (rPCR) and next-generation sequencing based assay for sequence-independent whole-genome amplification and sequencing of HFMD pathogens. A total of 16 EV-A71/CV-A6/CV-A10/CV-A16 PCR positive rectal/throat swabs (Cp values: 20.9-33.3) were used for assay evaluation. RESULTS: Our assay evidently outperformed the conventional rPCR in terms of the total number of EV-A71 reads and the percentage of EV-A71 reads: 2.6 % (1275/50,000 reads) vs. 0.1 % (31/50,000) and 6 % (3008/50,000) vs. 0.9 % (433/50,000) for two samples with Cp values of 30 and 26, respectively. Additionally the assay could generate genome sequences with the percentages of coverage of 94-100 % of 4 different enterovirus serotypes in 73 % of the tested samples, representing the first whole-genome sequences of CV-A6/10/16 from Vietnam, and could assign correctly serotyping results in 100 % of 24 tested specimens. In all but three the obtained consensuses of two replicates from the same sample were 100 % identical, suggesting that our assay is highly reproducible. CONCLUSIONS: In conclusion, we have successfully developed a non-ribosomal rPCR and next-generation sequencing based assay for sensitive detection and direct whole-genome sequencing of HFMD pathogens from clinical samples.

Incidence and Epidemiology of Intussusception among Infants in Ho Chi Minh City, Vietnam.

OBJECTIVE: To evaluate rates of intussusception hospitalization among infants in Vietnam before the introduction of rotavirus vaccine. STUDY DESIGN: Between 2009 and 2011, we identified intussusception hospitalizations among infants using the International Statistical Classification of Diseases and Related Health Problems, 10th Revision, Clinical Modification code K56.1 at 2 large pediatric hospitals in Ho Chi Minh City, Vietnam that treat most of the intussusception cases in the city. We reviewed all medical records to confirm a Brighton level 1 case definition for intussusception. RESULTS: We identified a total of 869 intussusception cases in Ho Chi Minh City during the 3-year study period, for an annual rate of 296 per 100,000 infants. The mean age of intussusception was ∼ 37 weeks (8.6 months), with <2% of the cases occurring before age 15 weeks. Cases of intussusception were observed year-round in these hospitals with no evident seasonal pattern. Ultrasonography was used to diagnose most cases (97%), and reduction was performed by air enema in >95% of the cases, with only 1% of cases at 1 hospital and 5% at the other hospital requiring surgical intervention. Ultrasound diagnosis was confirmed by an independent radiologist in 94% of a randomly selected group of intussusception cases at 1 of the 2 hospitals. No mortality was reported. CONCLUSION: Vietnam has a substantially higher rate of intussusception in children aged >15 weeks compared with most other regions of the world. Most of our cases were diagnosed by ultrasound, and only a small proportion required surgical intervention with no fatalities, suggesting that the higher rates may be related in part to better and earlier detection of intussusception.