Feasibility of the Bag-Mediated Filtration System for Environmental Surveillance of Poliovirus in Kenya.

The bag-mediated filtration system (BMFS) was developed to facilitate poliovirus (PV) environmental surveillance, a supplement to acute flaccid paralysis surveillance in PV eradication efforts. From April to September 2015, environmental samples were collected from four sites in Nairobi, Kenya, and processed using two collection/concentration methodologies: BMFS (> 3 L filtered) and grab sample (1 L collected; 0.5 L concentrated) with two-phase separation. BMFS and two-phase samples were analyzed for PV by the standard World Health Organization poliovirus isolation algorithm followed by intratypic differentiation. BMFS samples were also analyzed by a cell culture independent real-time reverse transcription polymerase chain reaction (rRT-PCR) and an alternative cell culture method (integrated cell culture-rRT-PCR with PLC/PRF/5, L20B, and BGM cell lines). Sabin polioviruses were detected in a majority of samples using BMFS (37/42) and two-phase separation (32/42). There was statistically more frequent detection of Sabin-like PV type 3 in samples concentrated with BMFS (22/42) than by two-phase separation (14/42, p = 0.035), possibly due to greater effective volume assayed (870 mL vs. 150 mL). Despite this effective volume assayed, there was no statistical difference in Sabin-like PV type 1 and Sabin-like PV type 2 detection between these methods (9/42 vs. 8/42, p = 0.80 and 27/42 vs. 32/42, p = 0.18, respectively). This study demonstrated that BMFS can be used for PV environmental surveillance and established a feasible study design for future research.

Haiti Poliovirus Environmental Surveillance.

Poliovirus (PV) environmental surveillance was established in Haiti in three sites each in Port-au-Prince and Gonaïves, where sewage and fecal-influenced environmental open water channel samples were collected monthly from March 2016 to February 2017. The primary objective was to monitor for the emergence of vaccine-derived polioviruses (VDPVs) and the importation and transmission of wild polioviruses (WPVs). A secondary objective was to compare two environmental sample processing methods, the gold standard two-phase separation method and a filter method (bag-mediated filtration system [BMFS]). In addition, non-polio enteroviruses (NPEVs) were characterized by next-generation sequencing using Illumina MiSeq to provide insight on surrogates for PVs. No WPVs or VDPVs were detected at any site with either concentration method. Sabin (vaccine) strain PV type 2 and Sabin strain PV type 1 were found in Port-au-Prince, in March and April samples, respectively. Non-polio enteroviruses were isolated in 75-100% and 0-58% of samples, by either processing method during the reporting period in Port-au-Prince and Gonaïves, respectively. Further analysis of 24 paired Port-au-Prince samples confirmed the detection of a human NPEV and echovirus types E-3, E-6, E-7, E-11, E-19, E-20, and E-29. The comparison of the BMFS filtration method to the two-phase separation method found no significant difference in sensitivity between the two methods (mid-P-value = 0.55). The experience of one calendar year of sampling has informed the appropriateness of the initially chosen sampling sites, importance of an adequate PV surrogate, and robustness of two processing methods.

Field Performance of Two Methods for Detection of Poliovirus in Wastewater Samples, Mexico 2016-2017.

To enhance our ability to monitor poliovirus circulation and certify eradication, we evaluated the performance of the bag-mediated filtration system (BMFS) against the two-phase separation (TPS) method for concentrating wastewater samples for poliovirus detection. Sequential samples were collected at two sites in Mexico; one L was collected by grab and ~ 5 L were collected and filtered in situ with the BMFS. In the laboratory, 500 mL collected by grab were concentrated using TPS and the sample contained in the filter of the BMFS was eluted without secondary concentration. Concentrates were tested for the presence of poliovirus and non-poliovirus enterovirus (NPEV) using Global Poliovirus Laboratory Network standard procedures. Between February 16, 2016, and April 18, 2017, 125 pairs of samples were obtained. Collectors spent an average (± standard deviation) of 4.3 ± 2.2 min collecting the TPS sample versus 73.5 ± 30.5 min collecting and filtering the BMFS sample. Laboratory processing required an estimated 5 h for concentration by TPS and 3.5 h for elution. Sabin 1 poliovirus was detected in 37 [30%] samples with the TPS versus 24 [19%] samples with the BMFS (McNemar's mid p value = 0.004). Sabin 3 poliovirus was detected in 59 [47%] versus 49 (39%) samples (p = 0.043), and NPEV was detected in 67 [54%] versus 40 [32%] samples (p < 0.001). The BMFS method without secondary concentration did not perform as well as the TPS method for detecting Sabin poliovirus and NPEV. Further studies are needed to guide the selection of cost-effective environmental surveillance methods for the polio endgame.

Detection of vaccine-derived polioviruses in Mexico using environmental surveillance.

BACKGROUND: Early detection and control of vaccine-derived poliovirus (VDPV) emergences are essential to secure the gains of polio eradication. METHODS: Serial sewage samples were collected in 4 towns of Mexico before, throughout, and after the May 2010 oral poliovirus vaccine (OPV) mass immunization campaign. Isolation and molecular analysis of polioviruses from sewage specimens monitored the duration of vaccine-related strains in the environment and emergence of vaccine-derived polioviruses in a population partially immunized with inactivated poliovirus vaccine (IPV). RESULTS: Sabin strains were identified up to 5-8 weeks after the campaign in all towns; in Aguascalientes, 1 Sabin 3 was isolated 16 weeks after the campaign, following 7 weeks with no Sabin strains detected. In Tuxtla Gutiérrez, type 2 VDPV was isolated from 4 samples collected before and during the campaign, and type 1 VDPV from 1 sample collected 19 weeks afterward. During 2009-2010, coverage in 4 OPV campaigns conducted averaged only 57% and surveillance for acute flaccid paralysis (AFP) was suboptimal (AFP rate<1 per 100,000 population<15 years of age) in Tuxtla Gutierrez. CONCLUSIONS: VDPVs may emerge and spread in settings with inadequate coverage with IPV/OPV vaccination. Environmental surveillance can facilitate early detection in these settings.

Diversity of picornaviruses in rural Bolivia.

The family Picornaviridae is a large and diverse group of viruses that infect humans and animals. Picornaviruses are among the most common infections of humans and cause a wide spectrum of acute human disease. This study began as an investigation of acute flaccid paralysis (AFP) in a small area of eastern Bolivia, where surveillance had identified a persistently high AFP rate in children. Stools were collected and diagnostic studies ruled out poliovirus. We tested stool specimens from 51 AFP cases and 34 healthy household or community contacts collected during 2002-2003 using real-time and semi-nested reverse transcription polymerase chain reaction assays for enterovirus, parechovirus, cardiovirus, kobuvirus, salivirus and cosavirus. Anecdotal reports suggested a temporal association with neurological disease in domestic pigs, so six porcine stools were also collected and tested with the same set of assays, with the addition of an assay for porcine teschovirus. A total of 126 picornaviruses were detected in 73 of 85 human individuals, consisting of 53 different picornavirus types encompassing five genera (all except Kobuvirus). All six porcine stools contained porcine and/or human picornaviruses. No single virus, or combination of viruses, specifically correlated with AFP; however, the study revealed a surprising complexity of enteric picornaviruses in a single community.

Acute febrile illness surveillance in a tertiary hospital emergency department: comparison of influenza and dengue virus infections.

In 2009, an increased proportion of suspected dengue cases reported to the surveillance system in Puerto Rico were laboratory negative. As a result, enhanced acute febrile illness (AFI) surveillance was initiated in a tertiary care hospital. Patients with fever of unknown origin for 2-7 days duration were tested for Leptospira, enteroviruses, influenza, and dengue virus. Among the 284 enrolled patients, 31 dengue, 136 influenza, and 3 enterovirus cases were confirmed. Nearly half (48%) of the confirmed dengue cases met clinical criteria for influenza. Dengue patients were more likely than influenza patients to have hemorrhage (81% versus 26%), rash (39% versus 9%), and a positive tourniquet test (52% versus 18%). Mean platelet and white blood cell count were lower among dengue patients. Clinical diagnosis can be particularly difficult when outbreaks of other AFI occur during dengue season. A complete blood count and tourniquet test may be useful to differentiate dengue from other AFIs.

Characteristics of young infants in whom human parechovirus, enterovirus or neither were detected in cerebrospinal fluid during sepsis evaluations.

BACKGROUND: Human parechovirus (HPeV) causes central nervous system (CNS) infection in infants. To further understand HPeV CNS infection, we describe its clinical, laboratory and epidemiologic characteristics from a Midwestern US tertiary care center. Because HPeV CNS infections have appeared clinically and seasonally similar to enterovirus (EV) infections, we retrospectively compared characteristics of young infants undergoing sepsis evaluations in whom HPeV, EV or neither were detected in cerebrospinal fluid (CSF). METHODS: HPeV real-time reverse-transcription polymerase chain reaction (RT-PCR) assay was performed on frozen nucleic acid extracts of CSF specimens submitted for EV RT-PCR assay from children seen at our hospital in 2009. HPeV genotyping was performed by sequencing of the viral protein 1 region. Clinical data were abstracted from medical records retrospectively for EV-positive, HPeV-positive and age-matched controls in whom neither virus was detected from CSF testing. RESULTS: HPeV was detected in 66 of the 388 (17%) CSF specimens whereas EV was detected in 54 of the 388 (14%) from June through October 2009. Genotyping identified HPeV3 in 51 of the 66 (77%) positive CSF specimens. Males predominated (61%) with the most common presenting symptoms (91%) being fever and irritability. All HPeV-positive patients were <5 months of age. Eight required admission to the pediatric intensive care unit. In multivariate analysis, lower peripheral white blood cell counts with lower absolute lymphocyte count values, higher maximum temperatures, longer fever duration, absence of pleocytosis and longer hospitalization were independently associated with HPeV patients compared with patients with EV or patients negative for both HPeV and EV. CONCLUSIONS: Our data indicate that HPeV3, an emerging CNS pathogen of infants in the United States, should be considered in sepsis-like presentation even without CSF pleocytosis. Addition of HPeV RT-PCR to EV RT-PCR assay for CSF specimens of patients <6 months of age could reduce hospital stay and costs while improving clinical management.

Detection of human parechovirus (HPeV)-3 in spinal fluid specimens from pediatric patients in the Chicago area.

BACKGROUND: The human parechoviruses (HPeV) have recently been recognized as important viral pathogens causing various illnesses including sepsis and meningitis in children. However, data from the United States is limited. OBJECTIVES: To better understand the epidemiology of HPeV in the United States and its role in pediatric disease through detection and typing of the virus in cerebrospinal fluid specimens. STUDY DESIGN: Four hundred and twenty-one spinal fluid samples collected from 373 patients ranging in age from 1 day to 18 years were tested using a real-time reverse transcription-PCR assay. The specimens were originally collected for routine viral and bacterial testing to assist in the diagnosis of meningitis or sepsis. Amplification products of the VP1 region in the virus genome were sequenced to identify the parechovirus type. RESULTS: Ten positive specimens were identified from 10 different patients. All ten samples were typed as HPeV3 and were negative for bacteria by culture, and for enterovirus and herpes simplex virus by PCR. All of the HPeV3-infected patients were young infants ranging in age from 6 to 59 days. Infants in whom HPeV3 was detected had significantly decreased peripheral white blood cell counts. Positive specimens were all from the summer and early fall. CONCLUSIONS: HPeV3 infection of the central nervous system is found in very young infants in certain years during the summer and early fall, and is associated with leukopenia. Real-time RT-PCR is an effective tool for rapid detection of these infections, and could help prevent unnecessary hospitalization and antibiotic use in HPeV infected infants. More widespread use of this tool in diagnosing HPeV infection would aid in further clarifying the prevalence of this disease in the United States.

Comparative evaluation of Taqman real-time PCR and semi-nested VP1 PCR for detection of enteroviruses in clinical specimens.

BACKGROUND: Molecular diagnostic tests to detect enterovirus in clinical specimens usually target highly conserved sites in the 5'-non-translated region, allowing detection of all members of the genus. The sequences in the 5'-NTR do not correlate with serotype, but PCR and sequencing of the VP1 region can be used for typing; this system has largely replaced traditional antigenic typing. OBJECTIVE: To investigate the relative performance of two common enterovirus assays. STUDY DESIGN: We compared the relative sensitivities of Taqman real-time RT-PCR (rRT-PCR) and a VP1 semi-nested PCR (RT-snPCR) assay in which sequencing the VP1 amplicon also provides typing information. RESULTS: There was 89% concordance between the two methods among the 371 clinical specimens tested (74 positive in both assays and 257 negative in both assays). Twenty-seven rRT-PCR-negative/VP1-positive specimens were confirmed positive by sequencing; 13 specimens were rRT-PCR-positive/VP1-negative. CONCLUSIONS: The results suggest that either assay can produce satisfactory results, but that using both assays in parallel should provide the highest sensitivity for clinical diagnostic testing.

Increased activity of Coxsackievirus B1 strains associated with severe disease among young infants in the United States, 2007-2008.

BACKGROUND: Enterovirus infections are very common and typically cause mild illness, although neonates are at higher risk for severe illness. In 2007, the Centers for Disease Control and Prevention (CDC) received multiple reports of severe neonatal illness and death associated with coxsackievirus B1 (CVB1), a less common enterovirus serotype not previously associated with death in surveillance reports to the CDC. METHODS: This report includes clinical, epidemiologic, and virologic data from cases of severe neonatal illness associated with CVB1 reported during the period from 2007 through 2008 to the National Enterovirus Surveillance System (NESS), a voluntary, passive surveillance system. Also included are data on additional cases reported to the CDC outside of the NESS. Virus isolates or original specimens obtained from patients from 25 states were referred to the CDC picornavirus laboratory for molecular typing or characterization. RESULTS: During 2007-2008, the NESS received 1079 reports of enterovirus infection. CVB1 accounted for 176 (23%) of 775 reported cases with known serotype, making it the most commonly reported serotype for the first time ever in the NESS. Six neonatal deaths due to CVB1 infection were also reported to the CDC during that time. Phylogenetic analysis of the 2007 and 2008 CVB1 strains indicated that the increase in cases resulted from widespread circulation of a single genetic lineage that had been present in the United States since at least 2001. CONCLUSIONS: Healthcare providers and public health departments should be vigilant to the possibility of continuing CVB1-associated neonatal illness, and testing and continued reporting of enterovirus infections should be encouraged.

Detection of echovirus 18 in human breast milk.

We detected enteroviral RNA and cultured infectious virus from a series of banked breast milk samples from the mother of an infant with neonatal sepsis; sequencing of the enterovirus isolate identified it as echovirus type 18. In this case, it is possible that enterovirus transmission occurred through the breast milk.

Histopathologic, immunohistochemical, and polymerase chain reaction assays in the study of cases with fatal sporadic myocarditis.

Paraffin tissue blocks from 27 cases with sporadic myocarditis were collected during a 12-year period at a single medical examiner's office. Blocks were studied by using histopathology; immunohistochemistry for viruses (adenovirus, enterovirus, influenza A and B, and human herpes types 4 and 5), bacteria (Neisseria meningitidis, Ehrlichia sp, spotted fever group Rickettsia) and parasites (Toxoplasma gondii and Trypanosoma cruzi); and polymerase chain reaction (PCR)/RT-PCR for adenovirus and enterovirus. We identified enterovirus in 5 (18.5%) cases and Sarcocystis in a 36-year-old woman who had focal inflammation and myocyte necrosis. Immunohistochemical evidence of enteroviruses was found in the myocytes of 2 patients less than 6 months old who had diffuse mononuclear myocardial inflammation, interstitial pneumonitis; one also had encephalitis. In these 2 patients, the presence of enterovirus was confirmed by RT-PCR targeting the 5' nontranslated region and was serotyped as coxsackievirus B2 by sequencing the VP1 capsid region. In another 3 cases (ages 12, 47, and 54), enterovirus was detected by the 5' nontranslated region region; VP1 sequencing identified these as echoviruses 6, 13, and 7, respectively. Accurately identifying an infectious agent is the foundation for clinical and public health interventions. Despite using multiple diagnostic methods, an organism could only be detected in a small proportion of sporadic myocarditis cases.

Species-specific RT-PCR amplification of human enteroviruses: a tool for rapid species identification of uncharacterized enteroviruses.

The 65 serotypes of human enteroviruses are classified into four species, Human enterovirus (HEV) A to D, based largely on phylogenetic relationships in multiple genome regions. The 3'-non-translated region of enteroviruses is highly conserved within a species but highly divergent between species. From this information, species-specific RT-PCR primers were developed that can be used to rapidly screen collections of enterovirus isolates to identify species of interest. The four primer pairs were 100 % specific when tested against enterovirus prototype strains and panels of isolates of known serotype (a total of 193 isolates). For evaluation in a typical application, the species-specific primers were used to screen 186 previously uncharacterized non-polio enterovirus isolates. The HEV-B primers amplified 68.3 % of isolates, while the HEV-A and HEV-C primers accounted for 9.7 and 11.3 % of isolates, respectively; no isolates were amplified with the HEV-D primers. Twelve isolates (6.5 %) were amplified by more than one primer set and eight isolates (4.3 %) were not amplified by any of the four primer pairs. Serotypes were identified by partial sequencing of the VP1 capsid gene, and in every case sequencing confirmed that the species-specific PCR result was correct; the isolates that were amplified by more than one species-specific primer pair were mixtures of two (11 isolates) or three (one isolate) species of viruses. The eight isolates that were not amplified by the species-specific primers comprised four new serotypes (EV76, EV89, EV90 and EV91) that appear to be unique members of HEV-A based on VP1, 3D and 3'-non-translated region sequences.

Complete genome sequences of all members of the species Human enterovirus A.

The species Human enterovirus A (HEV-A) in the family Picornaviridae consists of coxsackieviruses (CV) A2-A8, A10, A12, A14 and A16 and enterovirus 71. Complete genome sequences for the prototype strains of the 10 serotypes whose sequences were not represented in public databases have been determined and analysed in conjunction with previously available complete sequences in GenBank. Members of HEV-A are monophyletic relative to all other human enterovirus species in all regions of the genome except in the 5' non-translated region (NTR), where they are known to cluster with members of HEV-B. The HEV-A prototype strains were about 66 to 86 % identical to one another in deduced capsid amino acid sequence. Antigenic cross-reactivity has been reported between CVA3-Olson and CVA8-Donovan, between CVA5-Swartz and CVA12-Texas-12 and between CVA16-G-10 and EV71-BrCr. Similarity plots, individual sequence comparisons and phylogenetic analyses demonstrate a high degree of capsid sequence similarity within each of these three pairs of prototype strains, providing a molecular basis for the observed antigenic relationships. In several cases, phylogenies constructed from the structural (P1) and non-structural regions of the genome (P2 and P3) are incongruent. The incongruent phylogenies and the similarity plot analyses imply that recombination has played a role in the evolution of the HEV-A prototype strains. CVA6-Gdula clearly contains sequences that are also present in CVA10-Kowalik and CVA12-Texas-12, suggesting that these three strains have a shared evolutionary history despite their lack of similarity in the capsid region.

RNA recombination plays a major role in genomic change during circulation of coxsackie B viruses.

RNA recombination has been shown to occur during circulation of enteroviruses, but most studies have focused on poliovirus. To examine the role of recombination in the evolution of the coxsackie B viruses (CVB), we determined the partial sequences of four genomic intervals for multiple clinical isolates of each of the six CVB serotypes isolated from 1970 to 1996. The regions sequenced were the 5'-nontranslated region (5'-NTR) (350 nucleotides [nt]), capsid (VP4-VP2, 416 nt, and VP1, approximately 320 nt), and polymerase (3D, 491 nt). Phylogenetic trees were constructed for each genome region, using the clinical isolate sequences and those of the prototype strains of all 65 enterovirus serotypes. The partial VP1 sequences of each CVB serotype were monophyletic with respect to serotype, as were the VP4-VP2 sequences, in agreement with previously published studies. In some cases, however, incongruent tree topologies suggested that intraserotypic recombination had occurred between the sequenced portions of VP2 and VP1. Outside the capsid region, however, isolates of the same serotype were not monophyletic, indicating that recombination had occurred between the 5'-NTR and capsid, the capsid and 3D, or both. Almost all clinical isolates were recombinant relative to the prototype strain of the same serotype. All of the recombination partners appear to be members of human enterovirus species B. These results suggest that recombination is a frequent event during enterovirus evolution but that there are genetic restrictions that may influence recombinational compatibility.

Characterization of a novel coronavirus associated with severe acute respiratory syndrome.

In March 2003, a novel coronavirus (SARS-CoV) was discovered in association with cases of severe acute respiratory syndrome (SARS). The sequence of the complete genome of SARS-CoV was determined, and the initial characterization of the viral genome is presented in this report. The genome of SARS-CoV is 29,727 nucleotides in length and has 11 open reading frames, and its genome organization is similar to that of other coronaviruses. Phylogenetic analyses and sequence comparisons showed that SARS-CoV is not closely related to any of the previously characterized coronaviruses.