Preponderance of Enterovirus Species C in RD-L20B cell culture negative stool samples from children diagnosed with Acute Flaccid Paralysis in Nigeria

AbstractsRecently, a reverse transcriptase seminested polymerase chain reaction (RT-snPCR) assay was recommended by the WHO for direct detection of enteroviruses from clinical specimen. In this study, we use the assay and its modification to screen acute flaccid paralysis (AFP) samples previously confirmed 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 were previously confirmed negative for enteroviruses by the polio laboratory in accordance with the WHO recommended RD-L20B cell culture based algorithm. Two samples previously confirmed to contain enteroviruses were included as positive controls. All samples were subjected to RNA extraction, and the RT-snPCR assay and its modifications. All amplicons were sequenced and enteroviruses identified using the enterovirus genotyping tool.Overall, amplicons were recovered from the two controls and 50% (15/30) of 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 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 CV-A1, both EV-Cs. The PV-2 detected had VP1 ILE143. Hence, a vaccine strain.The results of this study showed that about 50% of enterovirus infections (including some Sabin PV2s) are being missed by the RD-L20B cell culture based algorithm. This highlights the value of the RT-snPCR assay and its modifications. The circulation and preponderance of EV-Cs in Nigeria was also confirmed.

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

10.1101/222935 ◽

2017 ◽

Author(s):

E Donbraye ◽

O.I. Olasunkanmi ◽

B.A. Opabode ◽

T.R. Ishola ◽

T.O.C. Faleye ◽

...

Keyword(s):

Cell Culture ◽

Phylogenetic Analysis ◽

Acute Flaccid Paralysis ◽

Rna Extraction ◽

Geographical Region ◽

Flaccid Paralysis ◽

Cdna Synthesis ◽

Faecal Samples ◽

Stool Samples ◽

Culture Negative

ABSTRACTWe recently showed that Enteroviruses (EVs); majorly species Cs (EV-Cs) were present in about 46.7% of faecal samples from children <15 years old diagnosed with Acute Flaccid Paralysis (AFP) in Nigeria but declared to be EV free by the RD-L20B cell culture based algorithm. In this study, we investigated whether this observed preponderance of EVs (and EV-Cs) in such samples vary by geographical region.In all, 108 samples (i.e. 54 paired stool suspensions from 54 AFP cases) previously confirmed negative for EVs by the WHO recommended RD-L20B cell culture based algorithm were analyzed in this study. The 108 samples were made into 54 pools (27 each from Northwest [NW] and Southsouth [SS] Nigeria). All samples were subjected to RNA extraction, cDNA synthesis and the WHO recommended seminestedPCR (snPCR) assay and its modifications. All amplicons were sequenced, and enteroviruses identified using the enterovirus genotyping tool and phylogenetic analysis.Altogether, EVs were detected in 16 (29.63%) of the 54 samples screened but successfully identified in 14 (25.93%): 10 from NW- and 4 from SS-Nigeria. Precisely, one (7.14%), two (14.29%) and 11 (78.57%) of the strains detected were EV-A, EV-B and EV-C respectively. The 10 strains from NW-Nigeria are 7 EV types and include CV-A10, E29, CV-A13, CV-A17, CV-A19, CV-A24 and EV-C99. The four EV types recovered from SS-Nigeria include E31, CV-A1, EV-C99 and EV-C116. EV-C99 is the only EV type that was detected in both NW- and SS-Nigeria.The results of this study showed that the preponderance of EVs and consequently EV-Cs in AFP samples declared to be EV free by the RD-L20B cell culture based algorithm vary by geographical region in Nigeria. It further confirmed the EV-B bias of the RD-L20B cell culture based algorithm.

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Isolation and Identification of Enterovirus D111 in a Child with Acute Flaccid Paralysis in Nigeria

10.1101/479873 ◽

2018 ◽

Author(s):

T.O.C. Faleye ◽

M.O. Adewumi ◽

O.T. Olayinka ◽

J.A Adeniji

Keyword(s):

Cell Culture ◽

Cell Line ◽

Cell Lines ◽

Illumina Sequencing ◽

Acute Flaccid Paralysis ◽

Rna Extraction ◽

Flaccid Paralysis ◽

Isolation And Identification ◽

Test Kit ◽

Group A

ABSTRACTThe WHO recommended cell-culture-based algorithm requires enterovirus (EV) isolates to produce reproducible cytopathic effect (R-CPE) in RD and/or L20b cell lines. Samples with non-reproducible CPE (NR-CPE) are considered negative for EVs. We investigated whether there could be EVs lurking in samples with NR-CPE.Fifty-nine (59) cell culture supernatants (CCS) (collected between 2016 and 2017) recovered from RD and L20b cell culture tubes with NR-CPE, were analyzed in this study. The tubes had been previously inoculated with stool suspension from children (<15 years) in Nigeria with acute flaccid paralysis (AFP). All CCS were screened for Enteric Adenoviruses and group A Rotavirus using a rapid immunochromatographic test kit. Subsequently, they were passaged in HEp-2 cell line. All isolates were subjected to RNA extraction, cDNA synthesis, three (5l-UTR, VP1 and EV Species C [EV-C]) different PCR assays and sequencing of amplicons. EVs were further subjected to Illumina sequencing.All CCS were negative for Adenoviruses and group A Rotaviruses. Four CCS produced R-CPE in HEp-2 cell line, three of which were positive for the 5l-UTR assay. Of the 3 isolates two and none were positive for the VP1 and EV-C assays, respectively. One of the two VP1 amplicons was successfully sequenced and identified as Echovirus 1(E1). Illumina sequencing of the three 5l- UTR positive isolates confirmed the E1 isolate and typed the remaining two as EV-Ds (94 and 111).We describe the first EV-D94 and 111 isolates of Nigerian origin. We also show that NR-CPE could sometimes be caused by EVs that do not produce R-CPE in RD and L20b cell lines but do so in other cell lines like HEp-2.

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

Archives of Virology ◽

10.1007/s00705-017-3466-2 ◽

2017 ◽

Vol 162 (10) ◽

pp. 3089-3101 ◽

Cited By ~ 12

Author(s):

J. A. Adeniji ◽

A. O. Oragwa ◽

U. E. George ◽

U. I. Ibok ◽

T. O. C. Faleye ◽

...

Keyword(s):

Cell Culture ◽

Acute Flaccid Paralysis ◽

Flaccid Paralysis ◽

Stool Samples ◽

Culture Negative

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Protocol for the investigation of acute flaccid paralysis and suspected paralytic poliomyelitis

Paediatrics & Child Health ◽

10.1093/pch/2.6.409 ◽

1997 ◽

Vol 2 (6) ◽

pp. 409-412 ◽

Cited By ~ 1

Author(s):

Adwoa Bentsi-Enchill ◽

Keyword(s):

Acute Flaccid Paralysis ◽

Clinical Symptoms ◽

Incidental Finding ◽

Wild Strain ◽

Clinical Specimen ◽

Polio Eradication ◽

Flaccid Paralysis ◽

Paralytic Poliomyelitis ◽

Surveillance Program ◽

Wild Poliovirus

Abstract Despite the elimination of indigenous wild poliovirus in Canada, ongoing surveillance for poliomyelitis is important because of the risk of wild virus importation from endemic regions. Most recently, importation of wild poliovirus into Canada occurred in 1993 and 1996,inboth instances with no associated clinical illness. Since 1991, the active surveillance of acute flaccid paralysis (AFP) in children less than 15 years old has been carried out in Canada to monitor for suspected cases of paralytic poliomyelitis. AFP surveillance is currently implemented through the Canadian Paediatric Surveillance Program. All suspected cases of paralytic poliomyelitis reported to the Laboratory Centre for Disease Control are evaluated by the national Working Group on Polio Eradication. The proper laboratory and neurological investigation of all AFP cases younger than 15 years old and suspected cases of poliomyelitis of any age is essential for the rapid detection of paralytic poliomyelitis, the most important clinical specimen for laboratory investigation being a stool sample collected within two weeks after the onset of paralysis for viral studies. This paper provides guidelines for investigating all suspected cases of paralytic poliomyelitis, including AFP cases less than 15 years old. Guidelines are also provided for reporting confirmed or suspected cases of paralytic poliomyelitis as well as the incidental finding of wild strain poliovirus, with or without any clinical symptoms.

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Cases of Acute Flaccid Paralysis Associated with Coxsackievirus A2: Findings of a 20-Year Surveillance in the Russian Federation

Microorganisms ◽

10.3390/microorganisms10010112 ◽

2022 ◽

Vol 10 (1) ◽

pp. 112

Author(s):

Olga E. Ivanova ◽

Armen K. Shakaryan ◽

Nadezhda S. Morozova ◽

Yulia A. Vakulenko ◽

Tatyana P. Eremeeva ◽

...

Keyword(s):

Acute Flaccid Paralysis ◽

Laboratory Data ◽

Foot And Mouth Disease ◽

Polio Eradication ◽

Vaccination Schedule ◽

Flaccid Paralysis ◽

Global Polio Eradication Initiative ◽

Afp Surveillance ◽

Stool Samples ◽

The Russian Federation

Surveillance for acute flaccid paralysis syndrome (AFP) in children under 15 is the backbone of the Global Polio Eradication Initiative. Laboratory examination of stool samples from AFP cases allows the detection of, along with polioviruses, a variety of non-polio enteroviruses (NPEV). The etiological significance of these viruses in the occurrence of AFP cases has been definitively established only for enteroviruses A71 and D68. Enterovirus Coxsackie A2 (CVA2) is most often associated with vesicular pharyngitis and hand, foot and mouth disease. Among 7280 AFP cases registered in Russia over 20 years (2001–2020), CVA2 was isolated only from five cases. However, these included three children aged 3 to 4 years, without overt immune deficiency, immunized with 4–5 doses of poliovirus vaccine in accordance with the National Vaccination Schedule. The disease resulted in persistent residual paralysis. Clinical and laboratory data corresponded to poliomyelitis developing during poliovirus infection. These findings are compatible with CVA2 being the cause of AFP. Molecular analysis of CVA2 from these patients and a number of AFP cases in other countries did not reveal association with a specific phylogenetic group, suggesting that virus genetics is unlikely to explain the pathogenic profile. The overall results highlight the value of AFP surveillance not just for polio control but for studies of uncommon AFP agents.

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Non-polio enteroviruses in faeces of children diagnosed with acute flaccid paralysis in Nigeria

10.1101/084350 ◽

2016 ◽

Author(s):

TOC Faleye ◽

MO Adewumi ◽

MO Japhet ◽

OM David ◽

AO Oluyege ◽

...

Keyword(s):

Cell Culture ◽

Acute Flaccid Paralysis ◽

Rna Extraction ◽

Flaccid Paralysis ◽

Free World ◽

Cdna Synthesis ◽

Nigerian Children

ABSTRACTBackgroundThe need to investigate the contribution of non-polio enteroviruses to acute flaccid paralysis (AFP) cannot be over emphasized as we move towards a poliovirus free world. Hence, we aim to identify non-polio enteroviruses recovered from the faeces of children diagnosed with AFP in Nigeria.MethodsNinety-six isolates, (95 unidentified and one previously confirmed Sabin poliovirus 3) recovered on RD cell culture from the stool of children <15 years old diagnosed with AFP in 2014 were analyzed. All isolates were subjected to RNA extraction, cDNA synthesis and three different PCR reactions (one panenterovirus 5′-UTR and two VP1 amplification assays). VP1 amplicons were then sequenced isolates identified.Results93.75% (90/96) of the isolates were detected by at least one of the three assays as an enterovirus. Precisely, 79.17% (76/96), 6.25% (6/96), 7.295% (7/96) and 6.25% (6/96) of the isolates were positive for both, positive and negative, negative and positive, as well as negative for both the 5′-UTR and VP1 assays, respectively. In this study, sixty-nine (69) of the 83 VP1 amplicons sequenced were identified as 27 different enterovirus types. The most commonly detected were CV-B3 (10 isolates) and EV-B75 (5 isolates). Specifically, one, twenty-four and two of the enterovirus types identified in this study belong to EV-A, EV-B and EV-C respectively.DiscussionThis study reports the circulating strains of 27 non-polio enterovirus types in Nigerian children with AFP in 2014 and Nigerian strains of CV-B2, CV-B4, E17, EV-B80, EV-B73, EV-B97, EV-B93, EV-C99 and EV-A120.

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Identification of previously untypable RD cell line isolates and detection of EV-A71 genotype C1 in a child with AFP in Nigeria

10.1101/334094 ◽

2018 ◽

Cited By ~ 1

Author(s):

M.O. Adewumi ◽

T.O.C. Faleye ◽

C.O. Okeowo ◽

A.M. Oladapo ◽

J. Oyathelemhi ◽

...

Keyword(s):

Cell Culture ◽

Phylogenetic Analysis ◽

Cell Line ◽

Acute Flaccid Paralysis ◽

Rna Extraction ◽

Sub Saharan Africa ◽

Flaccid Paralysis ◽

Cdna Synthesis ◽

Pcr Assay ◽

Sub Saharan

AbstractWe previously attempted to identify 96 nonpolio enteroviruses (EVs) recovered on RD cell culture from children <15 years with acute flaccid paralysis (AFP) in Nigeria. We succeeded in identifying 69 of the isolates. Here, we describe an attempt to identify the remaining 27 isolates.Twenty-six (the 27thisolate was exhausted) isolates that could not be typed previously were further analyzed. All were subjected to RNA extraction, cDNA synthesis, enterovirus 5‟-UTR– VP2 PCR assay and a modified VP1 snPCR assay. Both the 5’-UTR – VP2 and VP1 amplicons were sequenced, isolates identified and subjected to phylogenetic analysis.Twenty of the 26 isolates analyzed were successfully identified. Altogether, 23 EV strains were recovered. Thesebelong to 11 EV (one EVA, nine EVB and one EVC) types which were EVA71 genotype C1 (1 strain), CVB3 (7 strains), CVB5 (1 strain), E5 (2 strain), E11 (3 strains), E13 (2 strain), E19 (1 strain), E20 (1 strain), E24 (2 strains), EVB75 (1 strain) and EVC99 (2 strains). Of the 11 EV types, the 5’-UTR-VP2 assay identified seven while the VP1 assay identified 10. Both assays simultaneously detected 7 of the 11 EV types identified in this study with 100% congruence.In this study we identified 20 of 26 samples that were previously untypable. In addition, we provided evidence that suggests that a clade of EVA71 genotype C1 might have been circulating in sub-Saharan Africa since 2008. Finally, we showed that the 5’-UTR-VP2 assay might be as valuable as the VP1 assay in EV identification.

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Implementation of Acute Flaccid Paralysis Surveillance in Emilia-Romagna, Italy: 2018-2019 analysis

European Journal of Public Health ◽

10.1093/eurpub/ckaa166.1340 ◽

2020 ◽

Vol 30 (Supplement_5) ◽

Author(s):

M Morgado ◽

L Palandri ◽

M E Colucci ◽

P Affanni ◽

R Zoni ◽

...

Keyword(s):

Active Surveillance ◽

Acute Flaccid Paralysis ◽

Flaccid Paralysis ◽

Surveillance Program ◽

Local Health ◽

Case Investigation ◽

Acute Flaccid Paralysis Surveillance ◽

Afp Surveillance ◽

E Mail

Abstract Problem Acute flaccid paralysis (AFP) active surveillance is the gold standard in polio eradication process. Italy was classified in 2017 at intermediate risk of poliovirus reintroduction based on suboptimal poliovirus surveillance. The aim of this study was to restore an adequate level of AFP surveillance in Emilia-Romagna Region. Description of the Problem The Emilia-Romagna's Reference Centre for AFP surveillance, in collaboration with the regional Public Health Service identified a collaborative network for AFP Surveillance by analysing the 2015-2017 Hospital Discharge Registers in all region's hospitals. The surveillance protocol was reviewed with the development of a computerized system of Active Surveillance-Zero Reporting sent by e-mail to doctors in the network every 15 days since October 2018. The goal was to restore the AFP notification system to meet WHO requirements sensitivity, completeness of case investigation, completeness of follow-up and to monitor the active surveillance program adherence. Results The active surveillance network was composed by 49 doctors from both hospital administrations and clinical wards from 4 University Hospitals and 7 Local Health Authorities throughout the region. By the end of 2019, the mean response to each e-mail was 48.5% (SD 7.5%); 7 AFP cases have been reported; 85.7% received a full clinical and virological investigation and 83.3% completed the 60 day's follow-up. The final diagnosis of the cases was: 3 Guillain-Barre, 1 transient polyneuritis after HAV vaccination, 1 neuromyopathy from chronic disease, 1 acute myelitis in patient with DADA2. In 2 cases the paralysis persisted after 60 days. Lessons In 2019, the active surveillance system reached sensitivity, completeness of case investigation and follow-up required. Intervention had no expenses, is easily reproducible, created a direct collaboration between clinical colleagues in the network and the reference center which restored adherence to AFP surveillance. Key messages This work proposes how to improve AFP surveillance in a setting with low polio risk perception due to the absence of cases. Additionally, it reinforces the importance of direct collaboration/teamwork between institutions, regional reference centres and clinicians.

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Molecular Characterization of Enteroviruses Isolated from Acute Flaccid Paralysis Cases in Poland, 1999–2014

Polish Journal of Microbiology ◽

10.5604/17331331.1227670 ◽

2016 ◽

Vol 65 (4) ◽

pp. 443-450 ◽

Cited By ~ 3

Author(s):

Magdalena Wieczorek ◽

Arleta Krzysztoszek

Keyword(s):

Acute Flaccid Paralysis ◽

Wide Distribution ◽

Flaccid Paralysis ◽

Viral Pathogens ◽

The Past ◽

Different Types ◽

Afp Surveillance ◽

Stool Samples ◽

Healthy Contact

Enteroviruses (EVs) are among viral pathogens that can cause acute flaccid paralysis (AFP). This study represents an overview of EVs isolated through AFP surveillance in Poland between 1999 and 2014. The presence of enteroviruses was studied in stool samples that were collected from 747 AFP cases and their asymptomatic contacts. Fifty five (6.12%) cases of AFP were associated with enterovirus isolation. Out of the 55 positive cases, 40 were associated with detection of enterovirus in patient, and 15 with detection of EV in healthy contact, without positive detection in paralytic patient. Polioviruses were isolated from 35 AFP cases. The results of this study showed that about 43.6% of positive AFP cases were found in association with the isolation of non-polio enteroviruses (NPEV). A total of 12 different types of the species B were detected (CVA9, CVB1, CVB3, CVB4, CVB5, E3, E4, E9, E11, E13, E30), and one additional isolate represented the species enterovirus A (EV71). Among the 12 serotypes of species B, CVB3 and CVB5 were more frequently detected than others, representing 40% of the characterized isolates, followed by CVB4 (16%), E4 (8%), and E11(8%). Phylogenetic analysis revealed that strains from Poland had the closest genetic relationship with isolates previously identified in Europe (France, Finland, Denmark, Moldova) but also in other parts of the world (Tunisia, China, USA), suggesting wide distribution of these lineages. The paper provides information about NPEV circulation in Poland in the past 16 years, about its association with the AFP and it indicates the need for monitoring NPEV circulation even after the eradication of poliomyelitis.

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