Enterovirus D68 and other enterovirus serotypes identified in South African patients with severe acute respiratory illness, 2009–2011

Background Human enteroviruses (EV) have been associated with severe acute respiratory illness (SARI) in South Africa. Objectives We aimed to describe the molecular epidemiology of EV serotypes among patients hospitalized with SARI during 2009‐2011. Patients/Methods Study samples from patients were tested for the presence of enterovirus using a polymerase chain reaction assay. Results 8.2% (842/10 260) of SARI cases tested positive for enterovirus; 16% (7/45) were species EV‐A, 44% (20/45) EV‐B, 18% (8/45) EV‐C and 22% (10/45) EV‐D. Seventeen different EV serotypes were identified within EV‐A to EV‐D, of which EV‐D68 (22%; 10/45) and Echovirus 3 (11%; 5/45) were the most prevalent. Conclusions EV‐D68 should be monitored in South Africa to assess the emergence of highly pathogenic strains.

USA renewed interest in EV as cause of severe acute respiratory illness. 8 EV-D68 infects primary target tissues of the respiratory tract directly and occasionally infects the central nervous system. 3 Bayesian phylogenetic analysis of EV-D68 strains globally revealed the presence of three primary clades (A, B and C) 7,9,10 although other literature has separated EV-D68 into clusters (1, 2 and 3) 11 and lineages (1 and 2). 12 It is unclear if the recent upsurge in EV-D68-associated cases of pneumonia and flaccid paralysis-like illness are due to true changes in EV-D68 disease pathogenesis or improved molecular diagnostics. 13 A previous study from South Africa, using real-time polymerase chain reaction (PCR) assays, identified EVs in 6% (515/8173) of patients that were hospitalized with pneumonia in South Africa. 14

| Study design and population
Study samples were obtained from participants enrolled in a prospective hospital-based surveillance programme for SARI initiated in February 2009, which aimed to describe the aetiology and risk factors for acute lower respiratory tract infection in all age groups in South Africa. The methodology of this study has been described. 14,15

| Sample selection and detection of EV infection
Respiratory specimens (ie nasopharyngeal aspirates for children

| Amplification and sequencing of Enteroviruses
The 5′ proximal part of VP1 region was amplified and sequenced,

| Sequencing analysis
Sequence alignments were performed using MAFFT multiple sequence alignment programme. 17 Reference sequences included in the final data set were obtained from GenBank. The Kimura-2 parameter nucleotide substitution model determined as the optimal substitution model using jModelTest was used for the neighbour-joining (NJ) analysis, 18,19 and the NJ trees were generated using MEGA 5.2 software. 20

| Statistical analysis
Differences in characteristics of EV serotypes were assessed using the Fisher's exact test. P-values <.05 were considered to be statistically significant. Analysis was performed using STATA 13 (Stata Corporation, Texas USA).

| Ethical considerations
The SARI protocol was reviewed and approved by the University of   with EV-B strains (Table 1, Figure 1).
Here EV-D68 strains are grouped according to phylogenetic clades A, B and C. The majority of EV-D68 strains (90%, 9/10)  No neurological disease or paralysis symptoms were reported or available for patients from this study who were positive for EV-D68.
Demographic and clinical characteristics of patients in whom EV-D68 were isolated are described in Table 2. Ages ranged from 3 to 54 months and respiratory symptoms included cough, chest indrawing, breathing difficulty and tachycardia. No amino acid changes in the VP1 region that could lead to increased virulence 21 were observed in the South African samples (results not shown).

| DISCUSSION
We described the EV species circulating among patients from all age groups with SARI in South Africa. During 2009-2011, EV was detected in 8.2% of hospitalized SARI patients, which is within the range reported in other studies (3-25%). [22][23][24] Seventeen EV serotypes were identified representing all four EV species. EV-D68 was detected in 22% of EV cases characterized, which compares to frequencies reported in studies from the Netherlands (25%) and Germany  Further investigation is needed to determine the disease association of EVs and specifically EV-D68 infection with non-respiratory illness as well as full genome sequencing to determine possible recombination of EV-D68. As this study did not include an asymptomatic control group, we were limited interpreting the attributing causality to enteroviruses in the presence of high-frequency co-infections as well as determining concurrent prevalence in the healthy population.
In conclusion, we showed enteroviruses frequently contributed to SARI in South Africa. A high diversity in the EV species that circulated in South Africa during 2009-2011, EV-D68 specifically circulated at high frequencies among children <5 years of age. EV should be closely monitored to assess the emergence of highly pathogenic strains. There is no vaccine or antiviral available for non-polio EVs. Understanding the contribution of EV serotypes to severe illness will allow for informed decision-making on potential candidate vaccines and development of therapeutic interventions. personal fees from BMGF, outside the submitted work.

DISCLAIMER
The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the US Centers for Disease Control and Prevention.