Enterovirus D68 Subclade B3 in Children with Acute Flaccid Paralysis in West Africa, 2016

We tested for enterovirus D68 in fecal samples collected during June–September 2016 from 567 patients with acute flaccid paralysis in 7 West Africa nations. Children <5 years old comprised 64.3% of enterovirus D68 positive patients. Our findings emphasize the need for active surveillance for acute flaccid myelitis.

We tested for enterovirus D68 in fecal samples collected during June-September 2016 from 567 patients with acute flaccid paralysis in 7 West Africa nations. Children <5 years old comprised 64.3% of enterovirus D68 positive patients. Our findings emphasize the need for active surveillance for acute flaccid myelitis.  Phylogenetic relationships among EV-D68 strains detected in Guinea (red), Niger (green), and Senegal (blue), June-September 2016. We used the maximum-likelihood method based on the Tamura-Nei model method in MEGA7 (http://www. megasoftware.net) to generate the phylogenetic tree constructed on the viral protein 1 region of EV-D68 strains. Sequences are identified by GenBank accession number, country, and period of detection. The phylogenetic tree is rooted by the oldest EV-D68 sequence in GenBank, the Fermon strain. We performed 1,000 bootstrap replications to determine the consensus tree; support for nodes present in >70% of the trees are annotated. EV-D68, enterovirus D68. chloroform. After RNA extraction, we screened all samples for EV-D68 by real time reverse transcription PCR as described elsewhere (2). For molecular characterization, the viral protein 1 region was amplified by a nested PCR and sequenced as described elsewhere (2). The alignment and phylogenic analyses of sequences obtained after cleaning were performed using MEGA 7.0 software (https://www. megasoftware.net).
The first EV-D68-associated AFP case was detected in Guinea in June 2016. Most cases (10/16) in West Africa in 2016 were detected during July (Figure 1), similar to the seasonality that has been observed in several other countries, including the United States (12), the Netherlands (13), and Senegal (3). Most EV-D68-positive patients (64.3%) were children <5 years old, consistent with our previous report from Senegal (2,3). BLAST analysis (https:// blast.ncbi.nlm.nih.gov/Blast.cgi) showed that all sequenced EV-D68 strains shared >98% homology with strains detected in Spain, Sweden, Germany, Japan, and China. Phylogenetic analysis of the viral protein 1 fragment revealed that all sequences from West Africa belonged to clade B, subclade B3 (Figure 2). Indeed, EV-D68 subclade B3 was the predominant strain reported in several global regions during the same period (9)(10)(11)(12)(13)(14). Moreover, results from phylogenetic testing showed that EV-D68 strains in West Africa clustered with strains circulating in Spain (GenBank accession no. MH307403) and Sweden (accession no. MH674138), with a bootstrap value of 97.
Our study has some limitations. One ongoing issue is the inability to accurately describe the flaccid paralysis syndrome. Radiography imaging will probably help distinguish AFM from other AFP conditions. In addition, detecting EV-D68 in feces does not prove a causal relationship with AFM, although in this study all of the fecal samples tested negative for poliovirus and other enteroviruses, ruling out those possible alternative diagnoses.
The absence of EV-D68 positive patients from the other West Africa countries may be due to the small number of samples collected and screened during the study period. EV-D68 prevalence in West Africa might be higher if respiratory samples, known to yield higher EV-D68 counts than fecal samples, were used for screening (6,14). Recently, the Pan American Health Organization and the World Health Organization provided updated recommendations to include respiratory sampling in suspected AFP cases (15).

Conclusions
This study provides evidence of more widespread EV-D68 circulation in West Africa in 2016 than previously reported. Enhanced surveillance for EV-D68, including collecting respiratory specimens from patients with confirmed cases of AFM, is needed to improve our understanding of this disease and its burden. Phylogeographic and phylodynamic studies based on full genomes are needed to better understand the introduction of EV-D68 in Africa during these different outbreaks.