Preponderance of enterovirus C in RD-L20B-cell-culture-negative stool samples from children diagnosed with acute flaccid paralysis in Nigeria

Abstract

Recently, a reverse transcriptase semi-nested polymerase chain reaction (RT-snPCR) assay was recommended by the WHO for direct detection of enteroviruses in clinical specimens. In this study, we use this assay and a modification thereof to screen acute flaccid paralysis (AFP) samples that had previously tested negative for enteroviruses by the RD-L20B algorithm. Thirty paired stool suspensions collected in 2015 as part of the national AFP surveillance program in different states of Nigeria were analyzed in this study. The samples had previously tested negative for enteroviruses in the polio laboratory in accordance with the WHO-recommended RD-L20B-cell-culture-based algorithm. Two samples that had previously been found to contain enteroviruses were included as positive controls. All samples were subjected to RNA extraction, the RT-snPCR assay and a modified version of the RT-snPCR. All amplicons were sequenced, and enteroviruses were identified using the enterovirus genotyping tool and phylogenetic analysis. Amplicons were recovered from the two controls and 50% (15/30) of the samples screened. Fourteen were successfully typed, of which, 7.1% (1/14), 21.4% (3/14), 64.3% (9/14) and 7.1% (1/14) were enterovirus (EV) -A, EV-B, EV-C and a mixture of EV-B and C (EV-C99 and E25), respectively. The two controls were identified as EV-C99 and coxsackievirus (CV) -A1, both of which belong to the species Enterovirus C. In one sample, poliovirus serotype 2 was detected and found to have the VP1 143 variation and was therefore identified as a vaccine strain. The results of this study showed that significant proportion of enterovirus infections (including some with Sabin PV2) are being missed by the RD-L20B-cell-culture-based algorithm, thus highlighting the value of the RT-snPCR assay and its modifications. The circulation and preponderance of EV-C in Nigeria was also confirmed.

Molecular Characterization and Clinical Description of Non-Polio Enteroviruses Detected in Stool Samples from HIV-Positive and HIV-Negative Adults in Ghana

“…In our study, the majority of strains were assigned to Enterovirus C (29/39), which is often described as the most common group circulating in sub-Saharan Africa [43,47]. Within this species, CVA20 and EV-C99 were the most common serotypes identified in our cohort.…”

Comparison of algorithms for the detection of enteroviruses in stool specimens from children diagnosed with Acute Flaccid Paralysis

“…While those where the same assay was used directly on fecal suspensions that did not show CPE in RD cell line, showed an abundance of EV-Cs [12]. Hence, the predominance of EV-Bs as documented by the PE-VP1-PCR assay, in this study, might be due to the fact that only samples that had yielded isolate in RD cell lines were selected and analyzed in this study.…”

Abundance of Enterovirus C in RD-L20B cell culture negative stool samples from Acute Flaccid Paralysis cases in Nigeria is geographically defined

“…RNA extraction, cDNA synthesis and snPCR were done as recently described (Faleye et al, 2016;Adeniji et al, 2017). Briefly, RNA extraction and cDNA synthesis were done using Jena Bioscience kits for RNA extraction and cDNA synthesis kits (Jena Bioscience, Jena, Germany), respectively, according to manufacturer's recommendations.…”

“…This role of NPEV-Cs in VDPV emergence exemplifies their importance, and the need to carefully catalogue their diversity and geographical distribution. It has however been shown (Sadeuh-Mba 2013, Adeniji and Faleye, 2014b, Faleye and Adeniji, 2015b, Adeniji et al, 2017) that the RD-L20B cell culture based algorithm (WHO, 2003;2004) recommended for and largely in use by the Global Polio Laboratory Network (GPLN) under-reports the preponderance of EV-Cs. This is because the L20B cell line component of the algorithm is largely specific for poliovirus detection due to its expression of the human poliovirus receptor (Pipkin et al 1993),while the RD cell line component that is supposed to be more promiscuous for EV detection is EV-B bias (Sadeuh-Mba 2013, Adeniji and Faleye, 2014a, Faleye and Adeniji, 2015a & b, Adeniji et al, 2017) We recently showed that EVs were present in about 46.7% of faecal samples from children <15 years old diagnosed with AFP but declared to be EV free by the RD-L20B cell culture based algorithm (WHO, 2003(WHO, , 2004.…”