Hybrid capture shotgun sequencing detected unexpected viruses in the cerebrospinal fluid of children with acute meningitis and encephalitis.

PURPOSE: Investigation of undiagnosed cases of infectious neurological diseases, especially in the paediatric population, remains a challenge. This study aimed to enhance understanding of viruses in CSF from children with clinically diagnosed meningitis and/or encephalitis (M/ME) of unknown aetiology using shotgun sequencing enhanced by hybrid capture (HCSS). METHODS: A single-centre prospective study was conducted at Sant Joan de Déu University Hospital, Barcelona, involving 40 M/ME episodes of unknown aetiology, recruited from May 2021 to July 2022. All participants had previously tested negative with the FilmArray Meningitis/Encephalitis Panel. HCSS was used to detect viral nucleic acid in the patients' CSF. Sequencing was performed on Illumina NovaSeq platform. Raw sequence data were analysed using CZ ID metagenomics and PikaVirus bioinformatics pipelines. RESULTS: Forty episodes of M/ME of unknown aetiology in 39 children were analysed by HCSS. A significant viral detection in 30 CSF samples was obtained, including six parechovirus A, three enterovirus ACD, four polyomavirus 5, three HHV-7, two BKV, one HSV-1, one VZV, two CMV, one EBV, one influenza A virus, one rhinovirus, and 13 HERV-K113 detections. Of these, one sample with BKV, three with HHV-7, one with EBV, and all HERV-K113 were confirmed by specific PCR. The requirement for Intensive Care Unit admission was associated with HCSS detections. CONCLUSION: This study highlights HCSS as a powerful tool for the investigation of undiagnosed cases of M/ME. Data generated must be carefully analysed and reasonable precautions must be taken before establishing association of clinical features with unexpected or novel virus findings.

Untargeted metagenomic sequencing identifies Toscana virus in patients with idiopathic meningitis, southern Spain, 2015 to 2019.

BackgroundVarious pathogens, including bacteria, fungi, parasites, and viruses can lead to meningitis. Among viruses causing meningitis, Toscana virus (TOSV), a phlebovirus, is transmitted through sandfly bites. TOSV infection may be suspected if patients with enterovirus- and herpesvirus-negative aseptic (non-bacterial) meningitis recall recent insect bites. Other epidemiological factors (season, rural area) may be considered. The broad range of possible meningitis aetiologies poses considerable diagnosis challenges. Untargeted metagenomic next-generation sequencing (mNGS) can potentially identify pathogens, which are not considered or detected in routine diagnostic panels.AimIn this retrospective, single-centre observational study, we investigated mNGS usefulness to understand the cause of meningitis when conventional approaches fail.MethodsCerebrospinal fluid (CSF) samples from patients hospitalised in southern Spain in 2015-2019 with aseptic meningitis and no aetiology found by conventional testing, were subjected to mNGS. Patients' demographic characteristics had been recorded and physicians had asked them about recent insect bites. Obtained viral genome sequences were phylogenetically analysed.ResultsAmong 23 idiopathic cases, TOSV was identified in eight (all male; median age: 39 years, range: 15-78 years). Five cases lived in an urban setting, three occurred in autumn and only one recalled insect bites. Phylogenetic analysis of TOSV segment sequences supported one intra-genotype reassortment event.ConclusionsOur study highlights the usefulness of mNGS for identifying viral pathogens directly in CSF. In southern Spain, TOSV should be considered regardless of recalling of insect bites or other epidemiological criteria. Detection of a disease-associated reassortant TOSV emphasises the importance of monitoring the spread and evolution of phleboviruses in Mediterranean countries.

Prevalence and genetic characterization of noroviruses in children with acute gastroenteritis in Senegal, 2007-2010.

Norovirus is the leading cause of sporadic and epidemic acute gastroenteritis (AGE) in children and adults around the world. We investigated the molecular diversity of noroviruses in a pediatric population in Senegal between 2007 and 2010 before the rotavirus vaccine implementation. Stool samples were collected from 599 children under 5 years of age consulting for AGE in a hospital in Dakar. Specimens were screened for noroviruses using the Allplex™ GI-Virus Assay. Positive samples were genotyped after sequencing of conventional reverse transcription-polymerase chain reaction products. Noroviruses were detected in 79 (13.2%) of the children, with GII.4 (64%) and GII.6 (10%) as the most frequently identified genotypes. Our study describes the distribution of genotypes between 2007 and 2010 and should be a baseline for comparison with more contemporary studies. This could help decision-makers on possible choices of norovirus vaccines in the event of future introduction.

Prevalence and Genetic Diversity of Aichi Virus 1 from Urban Wastewater in Senegal.

Aichi virus 1 (AiV-1) has been proposed as a causative agent of human gastroenteritis. In this study, raw, decanted, and treated wastewater samples from a wastewater treatment plant in an urban area of Dakar, Senegal, were collected. AiV-1 was detected in raw (70%, 14/20), decanted (68.4%, 13/19), and treated (59.3%, 16/27) samples, revealing a noticeable resistance of AiV-1 to chlorine-based treatment. Phylogenetic analysis revealed that all sequences clustered within genotype B. Our study presents the first report on the detection of AiV-1 in the environment of Dakar and constitutes indirect evidence of virus circulation in the population.

Temporal and spatial analysis of the 2014-2015 Ebola virus outbreak in West Africa.

West Africa is currently witnessing the most extensive Ebola virus (EBOV) outbreak so far recorded. Until now, there have been 27,013 reported cases and 11,134 deaths. The origin of the virus is thought to have been a zoonotic transmission from a bat to a two-year-old boy in December 2013 (ref. 2). From this index case the virus was spread by human-to-human contact throughout Guinea, Sierra Leone and Liberia. However, the origin of the particular virus in each country and time of transmission is not known and currently relies on epidemiological analysis, which may be unreliable owing to the difficulties of obtaining patient information. Here we trace the genetic evolution of EBOV in the current outbreak that has resulted in multiple lineages. Deep sequencing of 179 patient samples processed by the European Mobile Laboratory, the first diagnostics unit to be deployed to the epicentre of the outbreak in Guinea, reveals an epidemiological and evolutionary history of the epidemic from March 2014 to January 2015. Analysis of EBOV genome evolution has also benefited from a similar sequencing effort of patient samples from Sierra Leone. Our results confirm that the EBOV from Guinea moved into Sierra Leone, most likely in April or early May. The viruses of the Guinea/Sierra Leone lineage mixed around June/July 2014. Viral sequences covering August, September and October 2014 indicate that this lineage evolved independently within Guinea. These data can be used in conjunction with epidemiological information to test retrospectively the effectiveness of control measures, and provides an unprecedented window into the evolution of an ongoing viral haemorrhagic fever outbreak.

An imported case of vaccine-derived poliovirus type 2, Spain in the context of the ongoing polio Public Health Emergency of International Concern, September 2021.

The monthly retrospective search for unreported acute flaccid paralysis (AFP) cases conducted as a complementary component of the Spanish AFP surveillance system identified a case of AFP in a child admitted in Spain from Senegal during August 2021. Vaccine-derived poliovirus 2 was identified in the stool in September 2021. We present public health implications and response undertaken within the framework of the National Action Plan for Polio Eradication and the Public Health Emergency of International Concern.

Emergence of Vaccine-Derived Polioviruses during Ebola Virus Disease Outbreak, Guinea, 2014-2015.

During the 2014-2015 outbreak of Ebola virus disease in Guinea, 13 type 2 circulating vaccine-derived polioviruses (cVDPVs) were isolated from 6 polio patients and 7 healthy contacts. To clarify the genetic properties of cVDPVs and their emergence, we combined epidemiologic and virologic data for polio cases in Guinea. Deviation of public health resources to the Ebola outbreak disrupted polio vaccination programs and surveillance activities, which fueled the spread of neurovirulent VDPVs in an area of low vaccination coverage and immunity. Genetic properties of cVDPVs were consistent with their capacity to cause paralytic disease in humans and capacity for sustained person-to-person transmission. Circulation ceased when coverage of oral polio vaccine increased. A polio outbreak in the context of the Ebola virus disease outbreak highlights the need to consider risks for polio emergence and spread during complex emergencies and urges awareness of the challenges in polio surveillance, vaccination, and diagnosis.

Genetic Characterization of Enterovirus A71 Circulating in Africa.

We analyzed whole-genome sequences of 8 enterovirus A71 isolates (EV-A71). We confirm the circulation of genogroup C and the new genogroup E in West Africa. Our analysis demonstrates wide geographic circulation and describes genetic exchanges between EV-A71 and autochthonous EV-A that might contribute to the emergence of pathogenic lineages.

Chikungunya virus infections among travellers returning to Spain, 2008 to 2014.

Since the first documented autochthonous transmission of chikungunya virus in the Caribbean island of Saint Martin in 2013, the infection has been reported within the Caribbean region as well as North, Central and South America. The risk of autochthonous transmission of chikungunya virus becoming established in Spain may be elevated due to the large numbers of travellers returning to Spain from countries affected by the 2013 epidemic in the Caribbean and South America, as well as the existence of the Aedes albopictus vector in certain parts of Spain. We retrospectively analysed the laboratory diagnostic database of the National Centre for Microbiology, Institute of Health Carlos III (CNM-ISCIII) from 2008 to 2014. During the study period, 264 confirmed cases, of 1,371 suspected cases, were diagnosed at the CNM-ISCIII. In 2014 alone, there were 234 confirmed cases. The highest number of confirmed cases were reported from the Dominican Republic (n = 136), Venezuela (n = 30) and Haiti (n = 11). Six cases were viraemic in areas of Spain where the vector is present. This report highlights the need for integrated active case and vector surveillance in Spain and other parts of Europe where chikungunya virus may be introduced by returning travellers.

Aetiology of non-malaria acute febrile illness fever in children in rural Guinea-Bissau: a prospective cross-sectional investigation.

Background: With growing use of parasitological tests to detect malaria and decreasing incidence of the disease in Africa; it becomes necessary to increase the understanding of causes of non-malaria acute febrile illness (NMAFI) towards providing appropriate case management. This research investigates causes of NMAFI in pediatric out-patients in rural Guinea-Bissau. Methods: Children 0-5 years presenting acute fever (≥38°) or history of fever, negative malaria rapid diagnostic test (mRDT) and no signs of specific disease were recruited at the out-patient clinic of 3 health facilities in Bafatá province during 54 consecutive weeks (dry and rainy season). Medical history was recorded and blood, nasopharyngeal, stool and urine samples were collected and tested for the presence of 38 different potential aetiological causes of fever. Results: Samples from 741 children were analysed, the protocol was successful in determining a probable aetiological cause of acute fever in 544 (73.61%) cases. Respiratory viruses were the most frequently identified pathogens, present in the nasopharynx samples of 435 (58.86%) cases, followed by bacteria detected in 167 (22.60%) samples. Despite presenting negative mRDTs, P. falciparum was identified in samples of 24 (3.25%) patients. Conclusions: This research provides a description of the aetiological causes of NMAFI in West African context. Evidence of viral infections were more commonly found than bacteria or parasites.

Shotgun metagenomics to investigate unknown viral etiologies of pediatric meningoencephalitis.

INTRODUCTION: Meningoencephalitis in children poses a diagnostic challenge, as etiology remains unknown for most of patients. Viral metagenomics by shotgun sequencing represents a powerful tool for investigating unknown viral infections related to these cases. PATIENTS AND METHODS: In a two-year, reference-centre, retrospective study, we investigated the usefulness of viral metagenomics of cerebrospinal fluid (CSF) for the diagnosis of viral infectious meningoencephalitis in forty seven pediatric patients, forty of them previously tested negative with a routine neurologic panel of viral targets that included herpesvirus 1-3 and enterovirus. We enhanced the detection by targeting viral sequences by hybrid capture. Raw sequence data was analysed using three bioinformatics pipelines. RESULTS: Out of forty remaining children with meningoencephalitis of unknown viral etiology, a significant detection of viral nucleic acid by shotgun sequencing was found in twenty one, which was confirmed in ten of them by specific PCR: seven human endogenous retrovirus K113 (HER K113), one parechovirus 3, one human herpesvirus 5 (HHV5); one enterovirus B (Echovirus 9). The remaining eleven CSF were not confirmed by PCR: three rotavirus, one human herpesvirus 7 (HHV7), one influenza A, one mastadenovirus C, one sindbis virus, one torque teno virus, one human immunodeficiency virus 1 (HIV-1), one human alphaherpesvirus 3 (HHV3), one human alphaherpesvirus 2 (HHV2). CONCLUSIONS: Underutilization of currently available meningitis-encephalitis diagnostic techniques such as BioFire® FilmArray® is the main cause of undiagnosed cases of meningoencephalitis. However, in this study we detected uncommon viruses that should be considered, including virus, rotavirus, sindbis virus, influenza A virus and HHV7. No other viral sequences that could be readily linked to CNS inflammation were detected. Some findings may stem from reagent or sample contamination, as seen with papillomavirus; for others, the clinical relevance of the virus remains uncertain and should be substantiated by further studies, as is the case with endogenous retrovirus K113 virus. Online bioinformatics pipeline CZID represents a valuable tool for analysing shotgun sequencing data in cases of neurological conditions with unknown etiology. Altogether, this study highlights the potential of shotgun sequencing in identifying previously unknown viral neuropathogens and sheds light on the interpretation issues related to its application in clinical microbiology.

Metagenomic sequencing, molecular characterization, and Bayesian phylogenetics of imported type 2 vaccine-derived poliovirus, Spain, 2021.

Introduction: In 2021, a type 2 vaccine-derived poliovirus (VDPV2) was isolated from the stool of a patient with acute flaccid paralysis (AFP) admitted to Spain from Senegal. A virological investigation was conducted to characterize and trace the origin of VDPV2. Methods: We used an unbiased metagenomic approach for the whole-genome sequencing of VDPV2 from the stool (pre-treated with chloroform) and from the poliovirus-positive supernatant. Phylogenetic analyses and molecular epidemiological analyses relying on the Bayesian Markov Chain Monte Carlo methodology were used to determine the geographical origin and estimate the date of the initiating dose of the oral poliovirus vaccine for the imported VDPV2. Results: We obtained a high percentage of viral reads per total reads mapped to the poliovirus genome (69.5% for pre-treated stool and 75.8% for isolate) with a great depth of sequencing coverage (5,931 and 11,581, respectively) and complete genome coverage (100%). The two key attenuating mutations in the Sabin 2 strain had reverted (A481G in the 5'UTR and Ile143Thr in VP1). In addition, the genome had a recombinant structure between type-2 poliovirus and an unidentified non-polio enterovirus-C (NPEV-C) strain with a crossover point in the protease-2A genomic region. VP1 phylogenetic analysis revealed that this strain is closely related to VDPV2 strains circulating in Senegal in 2021. According to Bayesian phylogenetics, the most recent common ancestor of the imported VDPV2 could date back 2.6 years (95% HPD: 1.7-3.7) in Senegal. We suggest that all VDPV2s circulating in 2020-21 in Senegal, Guinea, Gambia, and Mauritania have an ancestral origin in Senegal estimated around 2015. All 50 stool samples from healthy case contacts collected in Spain (n = 25) and Senegal (n = 25) and four wastewater samples collected in Spain were poliovirus negative. Discussion: By using a whole-genome sequencing protocol with unbiased metagenomics from the clinical sample and viral isolate with high sequence coverage, efficiency, and throughput, we confirmed the classification of VDPV as a circulating type. The close genomic linkage with strains from Senegal was consistent with their classification as imported. Given the scarce number of complete genome sequences for NPEV-C in public databases, this protocol could help expand poliovirus and NPEV-C sequencing capacity worldwide.

Appraising the roles of CBLL1 and the ubiquitin/proteasome system for flavivirus entry and replication.

The ubiquitin ligase CBLL1 (also known as HAKAI) has been proposed to be a critical cellular factor exploited by West Nile virus (WNV) for productive infection. CBLL1 has emerged as a major hit in a recent RNA interference screen designed to identify cellular factors required for the early stages of the WNV life cycle. Follow-up experiments showed that HeLa cells knocked down for CBLL1 by a small interfering RNA (siRNA) failed to internalize WNV particles and resisted infection. Furthermore, depletion of a free-ubiquitin pool by the proteasome inhibitor MG132 abolished WNV endocytosis, suggesting that CBLL1 acts in concert with the ubiquitin proteasome system to mediate virus internalization. Here, we examined the effect of CBLL1 knockdown and proteasome inhibitors on infection by WNV and other flaviviruses. We identified new siRNAs that repress the CBLL1 protein and strongly inhibit the endocytosis of Listeria monocytogenes, a bacterial pathogen known to require CBLL1 to invade host cells. Strikingly, however, we detected efficient WNV, dengue virus, and yellow fever virus infection of human cells, despite potent downregulation of CBLL1 by RNA interference. In addition, we found that the proteasome inhibitors MG132 and lactacystin did not affect WNV internalization but strongly repressed flavivirus RNA translation and replication. Together, these data do not support a requirement for CBLL1 during flavivirus entry and rather suggest an essential role of the ubiquitin/proteasome pathway for flavivirus genome amplification.

Norovirus GII.3[P12] Outbreak Associated with the Drinking Water Supply in a Rural Area in Galicia, Spain, 2021.

On 30 September 2021, the city council of Muxia, Spain (population of 4,564 inhabitants), reported an unusual increase of patients with acute gastroenteritis (AGE). Because geographically widespread villages belonging to the same water supply were affected, a waterborne outbreak was suspected. Overall, 115 probable cases were ascertained during epidemiological investigations carried out by the local health authority (attack rate, 5.7%); the age range was 0 to 92 years, and 54% were female. The main symptoms were vomiting (78.1%) and diarrhea (67.5%). Primary cases peaked on 29 September and subsided on 1 October, compatible with a point-source outbreak followed by possible secondary cases until 7 October. We conducted an unmatched case-control study using phone surveys. The case-control study included 62 cases and 46 controls. Univariate analysis showed that cases had a higher exposure to tap water through direct consumption (odds ratio [OR] = 86; 95% confidence interval [CI], 18 to 409) or vegetable washing (OR = 27; 95% CI, 7 to 98). Norovirus GII was detected in two terminal points of the water supply system, and 14 cases were laboratory confirmed after detection of GII in stool samples. A unique genotype (GII.3[P12]) was identified in stool samples. On 1 October, a tap water ban was put in place and the water was purged and chlorinated. The rapid increase in the number of cases and its decline after implementing control measures suggested a waterborne point-source outbreak among the residents of Muxia sharing the same water distribution system. IMPORTANCE Noroviruses are likely to be underrecognized in most suspected waterborne outbreaks. Therefore, effective norovirus detection and the early recognition of water as a possible source of infection are important to reduce morbidity as appropriate steps are taken to control the source. In our study, we combined epidemiological, environmental, and microbiological investigations to demonstrate that it was a waterborne outbreak caused by norovirus. Metagenomic sequencing in one norovirus-positive stool sample confirmed norovirus etiology and the absence of other potential pathogens. Detection of fecal indicator bacteria and the fact that the drinking water was not chlorinated suggest a breakdown in chlorination as the cause of the outbreak. This outbreak investigation also demonstrated the importance of timely communication to the public about the risk linked to tap water consumption.

Epidemiology of Echovirus 30 Infections Detected in a University Hospital in Catalonia, Spain, in 1995-2020.

There is a growing interest in echovirus 30 (E30), an enterovirus responsible for neurological disease and hospitalization. There are multiple studies of outbreaks, but few that study the epidemiology over long periods of time. Our study aims to describe the clinical, epidemiological and microbiological characteristics of a series of E30 infections detected over 26 years. Data were retrospectively collected from a database of all enterovirus infections identified in our laboratory. They were detected by viral isolation or nucleic acid detection in patients presenting with respiratory or neurological infections, rash, sepsis-like syndrome, or gastroenteritis. Enterovirus genotyping was performed by amplification of the VP1 gene using RT-nested PCR, followed by sequencing and BLAST analysis. Of the 2402 enterovirus infections detected, 1619 were linked to at least one genotype and 173 were caused by E30. Clinical information was available for 158 (91.3%) patients. E30 was associated with neurological infection in 107 (67.8%) cases and it was detected almost every year. Phylogenetic analysis was performed with 67 sequences. We observed that E30 strains circulating in Catalonia from 1996 to 2016 belong to two lineages (E and F), although the majority cluster was in F. In 2018, lineage I emerged as the dominant lineage.

Genomic surveillance of enterovirus associated with aseptic meningitis cases in southern Spain, 2015-2018.

New circulating Enterovirus (EV) strains often emerge through recombination. Upsurges of recombinant non-polio enteroviruses (NPEVs) associated with neurologic manifestations such as EVA71 or Echovirus 30 (E30) are a growing public health concern in Europe. Only a few complete genomes of EVs circulating in Spain are available in public databases, making it difficult to address the emergence of recombinant EVs, understand their evolutionary relatedness and the possible implication in human disease. We have used metagenomic (untargeted) NGS to generate full-length EV genomes from CSF samples of EV-positive aseptic meningitis cases in Southern Spain between 2015 and 2018. Our analyses reveal the co-circulation of multiple Enterovirus B (EV-B) types (E6, E11, E13 and E30), including a novel E13 recombinant form. We observed a genetic turnover where emergent lineages (C1 for E6 and I [tentatively proposed in this study] for E30) replaced previous lineages circulating in Spain, some concomitant with outbreaks in other parts of Europe. Metagenomic sequencing provides an effective approach for the analysis of EV genomes directly from PCR-positive CSF samples. The detection of a novel, disease-associated, recombinant form emphasizes the importance of genomic surveillance to monitor spread and evolution of EVs.

High Diversity of Human Non-Polio Enterovirus Serotypes Identified in Contaminated Water in Nigeria.

Human enteroviruses (EVs) are highly prevalent in sewage and have been associated with human diseases with complications leading to severe neurological syndromes. We have used a recently developed molecular method to investigate the presence of EVs in eight samples collected in 2017-2018 from water streams contaminated by drainage channels in three different locations in Nigeria. A total of 93 human EV strains belonging to 45 different serotypes were identified, far exceeding the number of strains and serotypes found in similar samples in previous studies. Next generation sequencing analysis retrieved whole-capsid genomic nucleotide sequences of EV strains belonging to all four A, B, C, and D species. Our results further demonstrate the value of environmental surveillance for the detection of EV transmission of both serotypes commonly associated with clinical syndromes, such as EV-A71, and those that appear to circulate silently but could eventually cause outbreaks and disease. Several uncommon serotypes, rarely reported elsewhere, were detected such as EV-A119, EV-B87, EV-C116, and EV-D111. Ten EV serotypes were detected in Nigeria for the first time and two of them, CV-A12 and EV-B86, firstly described in Africa. This method can be expanded to generate whole-genome EV sequences as we show here for one EV-D111 strain. Our data revealed phylogenetic relationships of Nigerian sewage strains with EV strains reported elsewhere, mostly from African origin, and provided new insights into the whole-genome structure of emerging serotype EV-D111 and recombination events among EV-D serotypes.

Identification and molecular characterization of the first complete genome sequence of Human Parechovirus type 15.

Using a metagenomics approach, we have determined the first full-length genome sequence of a human parechovirus type 15 (HPeV15) strain, isolated from a child with acute flaccid paralysis and co-infected with EV-A71. HPeV15 is a rarely reported type. To date, no full-length genome sequence of HPeV15 is available in the GenBank database, where only limited VP1 sequences of this virus are available. Pairwise comparisons of the complete VP1 nucleotide and deduced amino acid sequences revealed that the study strain belongs to type 15 as it displayed 79.6% nucleotide and 93.4% amino acid identity with the HPeV15 prototype strain. Comparative analysis of available genomic regions and phylogenetic analysis using the P2 and P3 coding regions revealed low nucleotide identity to HPeV reference genomes. Phylogenetic and similarity plot analyses showed that genomic recombination events might have occurred in the UTRs and nonstructural region during HPeV15 evolution. The study strain has high similarity features with different variants of HPeV3 suggesting intertypic recombination. Our data contributes to the scarce data available on HPeVs in Africa and provides valuable information for future studies that aim to understand the evolutionary history, molecular epidemiology or biological and pathogenic properties of HPeV15.

Development of a New Internally Controlled One-Step Real-Time RT-PCR for the Molecular Detection of Enterovirus A71 in Africa and Madagascar.

Enterovirus A71 (EV-A71) is a leading cause of hand-foot-and-mouth disease (HFMD) and can be associated with severe neurological complications. EV-A71 strains can be classified into seven genogroups, A-H, on the basis of the VP1 capsid protein gene sequence. Genogroup A includes the prototype strain; genogroups B and C are responsible of major outbreaks worldwide, but little is known about the others, particularly genogroups E and F, which have been recently identified in Africa and Madagascar, respectively. The circulation of EV-A71 in the African region is poorly known and probably underestimated. A rapid and specific assay for detecting all genogroups of EV-A71 is required. In this study, we developed a real-time RT-PCR assay with a competitive internal control (IC). The primers and TaqMan probe specifically target the genomic region encoding the VP1 capsid protein. Diverse EV-A71 RNAs were successfully amplified from the genogroups A, B, C, D, E, and F, with similar sensitivity and robust reproducibility. Neither cross reaction with other EVs nor major interference with the competitive IC was detected. Experimentally spiked stool and plasma specimens provided consistent and reproducible results, and validated the usefulness of the IC for demonstrating the presence of PCR inhibitors in samples. The analysis in an African laboratories network of 1889 untyped enterovirus isolates detected 15 EV-A71 of different genogroups. This specific real-time RT-PCR assay provides a robust and sensitive method for the detection of EV-A71 in biological specimens and for the epidemiological monitoring of EV-A71 including its recently discovered genogroups.