Comparison of cell culture with RT-PCR for enterovirus detection in stool specimens from patients with acute flaccid paralysis

Introduction: Guillain-Barré Syndrome (GBS) is an autoimmune disease characterized by acute or subacute symmetrical ascending motor weakness, areflexia, and mild-to-moderate sensory abnormalities. Campylobacter jejuni is reported to be the most common bacterium associated with GBS cases. Despite the eradication of polio, the number of reported GBS cases remains considerably high in South Africa with the causative agents not being well described. Methodology: The aim of the study was to investigate the proportion of Campylobacter spp. detected in stool specimens from patients with symptoms of acute flaccid paralysis (AFP). Stool specimens from patients presenting with AFP, that were negative for polio and non-polio enteroviruses (NPENT), were processed and screened for the presence of Campylobacter spp. using quantitative PCR (qPCR). Results: Of the 512 stool specimens screened between October 2014 to December 2015, 12% (62/512) were positive for Campylobacter spp. Of these 62 Campylobacter infections: 77.4% (48/62) was C. jejuni; 19.4% (12/62) was Campylobacter coli; 3.2% (2/62) was mixed infections of C. jejuni and C. coli. Conclusions: True association of the disease with Campylobacter spp. will enable the proportion of Campylobacter-induced GBS to be better described in South Africa; this can only be done through systematic studies that include bacterial culture and serology together with molecular methodologies.

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Surveillance during an era of rapidly changing poliovirus epidemiology in India: the role of one vs. two stool specimens in poliovirus detection, 2000–2010

Epidemiology and Infection ◽

10.1017/s0950268813000800 ◽

2013 ◽

Vol 142 (1) ◽

pp. 163-171 ◽

Cited By ~ 4

Author(s):

C. V. CARDEMIL ◽

M. RATHEE ◽

H. GARY ◽

K. WANNEMUEHLER ◽

A. ANAND ◽

...

Keyword(s):

Maximum Likelihood ◽

Maximum Likelihood Estimation ◽

Stool Specimen ◽

Acute Flaccid Paralysis ◽

Likelihood Estimation ◽

Polio Eradication ◽

Flaccid Paralysis ◽

Stool Specimens

SUMMARYSince 2004, efforts to improve poliovirus detection have significantly increased the volume of specimen testing from acute flaccid paralysis (AFP) patients in India. One option to decrease collection and testing burden would be collecting only a single stool specimen instead of two. We investigated stool specimen sensitivity for poliovirus detection in India to estimate the contribution of the second specimen. We reviewed poliovirus isolation data for 303984 children aged <15 years with AFP during 2000–2010. Using maximum-likelihood estimation, we determined specimen sensitivity of each stool specimen, combined sensitivity of both specimens, and sensitivity added by the second specimen. Of 5184 AFP patients with poliovirus isolates, 382 (7·4%) were identified only by the second specimen. Sensitivity was 91·4% for the first specimen and 84·5% for the second specimen; the second specimen added 7·3% sensitivity, giving a combined sensitivity of 98·7%. Combined sensitivity declined, and added sensitivity increased, as the time from paralysis onset to stool collection increased (P = 0·032). The sensitivity added by the second specimen is important to detect the last chains of poliovirus transmission and to achieve certification of polio eradication. For sensitive surveillance, two stool specimens should continue to be collected from each AFP patient in India.

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

10.1101/116004 ◽

2017 ◽

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 ◽

Direct Detection ◽

Rna Extraction ◽

Clinical Specimen ◽

Flaccid Paralysis ◽

Surveillance Program ◽

Two Samples ◽

Afp Surveillance ◽

Stool Samples

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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Whole-Genome Sequences of Enteroviruses D94 and D111 Isolated from Stool Specimens in Angola

Microbiology Resource Announcements ◽

10.1128/mra.00728-21 ◽

2021 ◽

Vol 10 (40) ◽

Author(s):

Shur-Wern W. Chern ◽

Nicksy Gumede ◽

Christina J. Castro ◽

W. Allan Nix ◽

Terry Fei Fan Ng

Keyword(s):

Acute Flaccid Paralysis ◽

Flaccid Paralysis ◽

Whole Genome ◽

Genome Sequences ◽

Stool Specimens

We report the whole-genome sequences of new enterovirus D94 and D111 strains, isolated from a culture from stool specimens collected from acute flaccid paralysis (AFP) cases for poliovirus surveillance in Angola during 2010.

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The Acute Flaccid Paralysis (AFP) Surveillance System in Yemen, 2010-2015: Descriptive Study Based on Secondary Data Analysis (Preprint)

10.2196/preprints.14413 ◽

2019 ◽

Author(s):

Khaled Abdullah Almoayed ◽

Ali Bin Break ◽

Mutahar Al-Qassimi ◽

Ali Assabri ◽

Yousef Khader

Keyword(s):

Surveillance System ◽

Acute Flaccid Paralysis ◽

Descriptive Study ◽

Flaccid Paralysis ◽

World Health ◽

Reference Laboratory ◽

Sample Collection ◽

Afp Surveillance ◽

Stool Specimens ◽

Central Level

BACKGROUND Acute flaccid paralysis (AFP) surveillance is an essential strategy for poliovirus eradication. OBJECTIVE This study aimed to evaluate the performance of the AFP surveillance system in Yemen from 2010 to 2015, identify components that require strengthening, and compare the indicators by year and governorates. METHODS This descriptive study was based on secondary analysis of AFP surveillance data reported during 2010-2015 from all Yemeni governorates. The World Health Organization (WHO) minimum performance standards were used to evaluate the performance of the AFP surveillance system. RESULTS A total of 3019 AFP cases were reported between January 2010 and December 2015. At the national level, AFP surveillance achieved WHO targets throughout the evaluating period for the nonpolio AFP rate of cases per 100,000 members of the population younger than 15 years of age, proportion of AFP cases reported within 7 days, proportion of AFP cases investigated within 48 hours of notification, proportion of AFP cases with two adequate stool specimens, and proportion of stool specimens from which nonpolio enterovirus was isolated. However, the proportion of specimens that arrived at the central level within 3 days of the first sample collection and the proportion of stool specimens with results sent from the reference laboratory within 28 days of receipt did not reach targets in 2011 and 2015, respectively. CONCLUSIONS The AFP surveillance system in Yemen has met most of the WHO indicator levels. Nevertheless, the evaluation showed areas of weakness regarding the arrival of specimens at the central level within 3 days of the first sample collection and delays in processing of the results and submitting feedback by the laboratory. Therefore, there is a need to strengthen the follow-up of specimens submitted to the laboratory.

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Etiologic Involvement of Enterovirus and Human Bocavirus in Acute Flaccid Paralysis Cases in India

Open Forum Infectious Diseases ◽

10.1093/ofid/ofx163.731 ◽

2017 ◽

Vol 4 (suppl_1) ◽

pp. S313-S313

Author(s):

Manjari Baluni ◽

Dharamveer Singh ◽

Sneha Ghildiyal ◽

Tanzeem Fatima ◽

Amreen Zia ◽

...

Keyword(s):

Real Time ◽

Viral Infections ◽

Acute Flaccid Paralysis ◽

Clinical Specimen ◽

Clinical Manifestations ◽

Etiologic Agent ◽

Flaccid Paralysis ◽

Human Bocavirus ◽

Stool Specimens ◽

Pcr Targeting

Abstract Background Acute flaccid paralysis (AFP), characterized by the rapid onset of asymmetric paralysis, can be caused by a variety of viral infections or coinfections. Besides wild-type and revertant vaccine strains of polioviruses, several nonpolio enteroviruses, have also been associated with AFP. Enteroviruses (EVs) are RNA viruses in the family Picornaviridae comprising more than 100 serotypes that are divided into four species, human enteroviruses A to D. The clinical manifestations of EVs range from conjunctivitis, respiratory tract infection, myocarditis, meningitis, encephalitis, and neonatal sepsis, like illness. Human Bocavirus (HBoV), a newly classified member of the Parvoviridae family, has been detected frequently in feces of diarrhoeic children suggesting its possible etiological involvement in the disease. Methods Total 586 stool specimens were collected in 2016 from children suspected for AFP. Molecular method for targeting 5’ untranslated region (UTR) and VP1 capsid region was used for detection of human enteroviruses (HEV), human boca viruses (HBoV) and saffold viruses in direct clinical specimen. Results EV RNA was detected in 103 (17.6%) of 586 stool specimens by real-time RT-PCR targeting the highly conserved 5’ UTR region. Out of them, 71 (12.11%) were NPEV, partially sequenced by VP1 which revealed the prevalence of echovirus (ECV) 19 (n = 6), ECV 11 (n = 7), ECV 18 (n = 4), ECV 33 (n = 5), ECV 29 (n = 1), ECV 25 (n = 2), ECV 24 (n = 3), ECV 3 (n = 3), ECV 14 (n = 2), ECV 13 (n = 1), ECV 2 (n = 1), ECV 20 (n = 2), ECV 27 (n = 4), ECV 6 (n = 2), CV A10 (n = 2), CV A9 (n = 1), CV A6 (n = 2), CV B4 (n = 1), CV B5 (n = 3), CV B6 (n = 3), EV 80 (n = 1), EV 83 (n = 1), EV 97 (n = 2). Total 63 (10.75%) HBoVs were detected by real-time PCR which were further sequenced by VP1, consists of HBoV-1 (n = 8), HBoV-2 (n = 15), HBoV-3 (n = 9) and HBoV-4 (n = 5). Out of them 9 (1.5%) were detected as co infection with NPEVs. Phylogenetic analysis showed 0.9 - 5.6% divergence at nucleotide level among HBoVs. Total 9 (1.5%) saffold viruses was detected and characterized by VP1 sequencing. Conclusion ECV and HBoV were found the main etiologic agent in children suspected with AFP. Molecular typing of these viruses is useful for characterizing emerging serotypes and their epidemiological investigation. Disclosures All authors: No reported disclosures.

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Using Nonpolio Enterovirus Detection to Assess the Integrity of Stool Specimens Collected From Acute Flaccid Paralysis Cases in Somalia During 2014–2017

Open Forum Infectious Diseases ◽

10.1093/ofid/ofaa135 ◽

2020 ◽

Vol 7 (5) ◽

Author(s):

Amen Ben Hamida ◽

Kamil Mohamed Ali ◽

Rennatus Mdodo ◽

Abdinoor Mohamed ◽

Kumlachew Mengistu ◽

...

Keyword(s):

Odds Ratio ◽

Surveillance System ◽

Virus Isolation ◽

Acute Flaccid Paralysis ◽

Flaccid Paralysis ◽

System Quality ◽

Multivariable Model ◽

Afp Surveillance ◽

Stool Specimens ◽

Key Indicators

Abstract Background Despite insecurity challenges in Somalia, key indicators for acute flaccid paralysis (AFP) surveillance have met recommended targets. However, recent outbreaks of vaccine-derived polioviruses have raised concerns about possible gaps. We analyzed nonpolio enterovirus (NPEV) and Sabin poliovirus isolation rates to investigate whether comparing these rates can inform about the integrity of stool specimens from inaccessible areas and the likelihood of detecting circulating polioviruses. Methods Using logistic regression, we analyzed case-based AFP surveillance data for 1348 cases with onset during 2014−2017. We assessed the adjusted impacts of variables including age, accessibility, and Sabin-like virus isolation on NPEV detection. Results NPEVs were more likely to be isolated from AFP case patients reported from inaccessible areas than accessible areas (23% vs 15%; P = .01). In a multivariable model, inaccessibility and detection of Sabin-like virus were positively associated with NPEV detection (adjusted odds ratio [AOR], 1.75; 95% confidence interval [CI], 1.14–2.65; and AOR, 1.79; 95% CI, 1.07–2.90; respectively), while being aged ≥5 years was negatively associated (AOR, 0.42; 95% CI, 0.20–0.85). Conclusions Rates of NPEV and Sabin poliovirus detection in inaccessible areas suggest that the integrity of fecal specimens tested for AFP surveillance in Somalia can generate useful AFP data, but uncertainties remain about surveillance system quality.

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