Echovirus 30 detection in an outbreak of acute myalgia and rhabdomyolysis, Brazil 2016-2017.

OBJECTIVES: To describe an outbreak of acute myalgia accompanied by elevated levels of muscle enzymes that occurred in the northeast region of Brazil from December 2016 through to May 2017. METHODS: Clinical data were analysed and laboratory tests were performed in 86 specimens obtained from 52 individuals with suspected acute myalgia. A broader reactive enterovirus real-time RT-PCR followed by a semi-nested PCR amplification of partial VP1 gene were performed to identify the causative agent. RESULTS: Eighty-six clinical samples were received in our laboratory during the myalgia outbreak. Median age of individuals was 39 years. Sudden acute myalgia and dark urine were the most common symptoms. Creatine phosphokinase levels were elevated with mean value ∼16 893 U/L. Human enterovirus was detected in 67% (58/86) of the patient's specimens (urine, serum, faeces and rectal swab). The enterovirus positivity per patient was 82.7% (43/52). Echovirus 30 (E-30) (82% of the typed specimens, 18/22; 76.4% (13/17) of the typed specimens per patient) was the main enterovirus identified. In addition to E-30, CV-A16 (1/22) and E-6 (3/22) were detected in 4% and 14% of the typed specimens, respectively. No deaths occurred. CONCLUSION: The 2016-2017 outbreak of acute myalgia that occurred in the northeast region of Brazil can be associated with E-30. Despite the clinical manifestations, a favourable outcome was observed for all patients.

Rubella virus genotype 1G and echovirus 9 as etiologic agents of exanthematous diseases in Brazil: insights from phylogenetic analysis.

The aim of the present study was to identify the rubella virus (RV) and enterovirus (EV) genotypes detected during the Epidemiological Surveillance on Exanthematic Febrile Diseases (VIGIFEX) study and to perform phylogenetic analysis. Ten RV- and four EV-positive oropharyngeal samples isolated from cell culture were subjected to RT-PCR and sequencing. Genotype 1G and echovirus 9 (E-9) was identified in RV- and EV-positive samples, respectively. The RV 1G genotype has been persisting in Brazil since 2000-2001. No evidence of E-9 being involved in exanthematic illness in Brazil has been reported previously. Differential laboratory diagnosis is essential for management of rash and fever disease.

Expression of innate immunity genes and damage of primary human pancreatic islets by epidemic strains of Echovirus: implication for post-virus islet autoimmunity.

Three large-scale Echovirus (E) epidemics (E4,E16,E30), each differently associated to the acute development of diabetes related autoantibodies, have been documented in Cuba. The prevalence of islet cell autoantibodies was moderate during the E4 epidemic but high in the E16 and E30 epidemic. The aim of this study was to evaluate the effect of epidemic strains of echovirus on beta-cell lysis, beta-cell function and innate immunity gene expression in primary human pancreatic islets. Human islets from non-diabetic donors (n = 7) were infected with the virus strains E4, E16 and E30, all isolated from patients with aseptic meningitis who seroconverted to islet cell antibody positivity. Viral replication, degree of cytolysis, insulin release in response to high glucose as well as mRNA expression of innate immunity genes (IFN-b, RANTES, RIG-I, MDA5, TLR3 and OAS) were measured. The strains of E16 and E30 did replicate well in all islets examined, resulting in marked cytotoxic effects. E4 did not cause any effects on cell lysis, however it was able to replicate in 2 out of 7 islet donors. Beta-cell function was hampered in all infected islets (P<0.05); however the effect of E16 and E30 on insulin secretion appeared to be higher than the strain of E4. TLR3 and IFN-beta mRNA expression increased significantly following infection with E16 and E30 (P<0.033 and P<0.039 respectively). In contrast, the expression of none of the innate immunity genes studied was altered in E4-infected islets. These findings suggest that the extent of the epidemic-associated islet autoimmunity may depend on the ability of the viral strains to damage islet cells and induce pro-inflammatory innate immune responses within the infected islets.

Molecular diagnosis of echovirus 30 as the etiological agent in an outbreak of aseptic meningitis in Panama: May-June 2008.

INTRODUCTION: Aseptic meningitis outbreaks are commonly caused by viral pathogens with enterovirus a common etiological agent. Between May and June of 2008, an outbreak of 173 cases of aseptic meningitis occurred in the Chiriqui Province of Panama. Molecular techniques were used to identify the etiological agent. METHODOLOGY: Cerebrospinal fluid (CSF) samples from 75 patients were received at the Gorgas Memorial Institute for Health Studies.  RNA extraction and one-step RT-PCR were performed on each sample to determine the presence of enterovirus.  Thirty-four samples which were positive for enterovirus were subject to group-specific PCR, sequencing, and phylogenetic analysis to identify the etiological agent of the outbreak. RESULTS: The CSF of 58 subjects was found positive for the enterovirus family using RT-PCR. Thirty-four samples were found to belong to the enterovirus B group. Phylogenetic analysis of four successfully sequenced samples revealed echovirus 30 as the etiological agent. CONCLUSION: Echovirus 30 is reported as the likely cause of an outbreak of aseptic meningitis in Panama, the first since the 1980s.

Description of a widespread outbreak of aseptic meningitis due to echovirus 30 in Rio de Janeiro state, Brazil

Echovirus 30 belongs to the genus Enterovirus and is widely associated with aseptic meningitis (AM) outbreaks. In Brazil epidemics due to this serotype were reported in several states but in Rio de Janeiro, before this study, it was only involved in sporadic episodes. We retrospectively collected data from AM notifications charts and enterovirus isolation database from Rio de Janeiro State Health Department (RJSHD) and Enterovirus Reference Laboratory in the year of 2005. An outbreak of AM was detected during March, April and May associated with a high cell culture isolation rate for echovirus 30 (17.4 percent). Male children with ages varying from 1 to 9 years were more affected. Of the 22 patients with confirmed echovirus 30 disease, clinical information was available in eight; fever, headache and vomiting were the most common manifestations. CSF analysis showed a typical pattern of viral infection with median of cellularity of 100 cells/mm³ and mononuclear cell predominance in 64.7 percent of the cases. The median of protein and glucose levels of 49 mg/dL and 56.5 mg/dL. The fatality rate was null. Despite its benign course and the lack of treatment options, aseptic meningitis surveillance is crucial for early identification of causative agents of outbreaks, which helps to avoid additional testing and inappropriate use of antimicrobials.

Echovirus 30 associated with cases of aseptic meningitis in state of Pará, Northern Brazil.

Investigation of the aetiology of viral meningitis in Brazil is most often restricted to cases that occur in the Southern and Southeastern Regions; therefore, the purpose of this study is to describe the viral meningitis cases that occurred in state of Pará, Northern Brazil, from January 2005-December 2006. The detection of enterovirus (EV) in cerebrospinal fluid was performed using cell culture techniques, RT-PCR, nested PCR and nucleotide sequencing. The ages of the 91 patients ranged from < one year old to > 60 years old (median age 15.90 years). Fever (87.1%), headache (77.0%), vomiting (61.5%) and stiffness (61.5%) were the most frequent symptoms. Of 91 samples analyzed, 18 (19.8%) were positive for EV. Twelve were detected only by RT- PCR followed by nested PCR, whereas six were found by both cell culture and RT-PCR. From the last group, five were sequenced and classified as echovirus 30 (Echo 30). Phylogenetic analyses revealed that Echo 30 detected in Northern Brazil clustered within a unique group with a bootstrap value of 100% and could constitute a new subgroup (4c) according to the phylogenetic tree described by Oberste et al. (1999). This study described the first molecular characterization of Echo 30 in Brazil and this will certainly contribute to future molecular analyses involving strains detected in other regions of Brazil.

Echovirus 30 associated with cases of aseptic meningitis in state of Pará, Northern Brazil

Investigation of the aetiology of viral meningitis in Brazil is most often restricted to cases that occur in the Southern and Southeastern Regions; therefore, the purpose of this study is to describe the viral meningitis cases that occurred in state of Pará, Northern Brazil, from January 2005-December 2006. The detection of enterovirus (EV) in cerebrospinal fluid was performed using cell culture techniques, RT-PCR, nested PCR and nucleotide sequencing. The ages of the 91 patients ranged from < one year old to > 60 years old (median age 15.90 years). Fever (87.1 percent), headache (77.0 percent), vomiting (61.5 percent) and stiffness (61.5 percent) were the most frequent symptoms. Of 91 samples analyzed, 18 (19.8 percent) were positive for EV. Twelve were detected only by RT- PCR followed by nested PCR, whereas six were found by both cell culture and RT-PCR. From the last group, five were sequenced and classified as echovirus 30 (Echo 30). Phylogenetic analyses revealed that Echo 30 detected in Northern Brazil clustered within a unique group with a bootstrap value of 100 percent and could constitute a new subgroup (4c) according to the phylogenetic tree described by Oberste et al. (1999). This study described the first molecular characterization of Echo 30 in Brazil and this will certainly contribute to future molecular analyses involving strains detected in other regions of Brazil.

Description of a widespread outbreak of aseptic meningitis due to echovirus 30 in Rio de Janeiro state, Brazil.

Echovirus 30 belongs to the genus Enterovirus and is widely associated with aseptic meningitis (AM) outbreaks. In Brazil epidemics due to this serotype were reported in several states but in Rio de Janeiro, before this study, it was only involved in sporadic episodes. We retrospectively collected data from AM notifications charts and enterovirus isolation database from Rio de Janeiro State Health Department (RJSHD) and Enterovirus Reference Laboratory in the year of 2005. An outbreak of AM was detected during March, April and May associated with a high cell culture isolation rate for echovirus 30 (17.4%). Male children with ages varying from 1 to 9 years were more affected. Of the 22 patients with confirmed echovirus 30 disease, clinical information was available in eight; fever, headache and vomiting were the most common manifestations. CSF analysis showed a typical pattern of viral infection with median of cellularity of 100 cells/mm(3) and mononuclear cell predominance in 64.7% of the cases. The median of protein and glucose levels of 49 mg/dL and 56.5 mg/dL. The fatality rate was null. Despite its benign course and the lack of treatment options, aseptic meningitis surveillance is crucial for early identification of causative agents of outbreaks, which helps to avoid additional testing and inappropriate use of antimicrobials.

An outbreak of concurrent echovirus 30 and coxsackievirus A1 infections associated with sea swimming among a group of travelers to Mexico.

BACKGROUND: Enteroviruses are shed in human stool and can cause a wide spectrum of illness. They are the leading cause of aseptic meningitis. METHODS: In 2004, the Connecticut Department of Public Health investigated a meningitis cluster among persons returning from a school-organized trip to Mexico. RESULTS: Among 29 travelers (25 teenagers and 4 adult chaperones), 21 became acutely ill. Viral culture and nucleic acid amplification testing of stool (n=27) and cerebrospinal fluid (n=4) specimens identified enteroviral infection in 20 of 28 travelers from whom any specimen was obtained; 4 had echovirus 30 only, 11 had coxsackievirus (CV) A1 only, 4 had both echovirus 30 and CVA1, and 1 had CVA5 only. Illness onset dates were tightly clustered 4 days after a prolonged swim in the Gulf of Mexico. Time spent swimming was significantly associated with the odds of enteroviral infection (univariate odds ratio for each additional hour swimming, 14.3; 95% confidence interval, 1.3-154.3). Headache, fever, vomiting, and nausea occurred more frequently among the echovirus 30-infected travelers than among the uninfected control subjects (P< .05). The most frequent symptoms among travelers infected with only CVA1 identified were nausea and diarrhea (36% each), but neither was significantly associated with CVA1 infection; 5 patients with CVA1 infection were asymptomatic. CONCLUSIONS: We identified multiple enteroviruses among the travelers. Clustered illness onsets suggest point-source exposure, which likely was a sea swim in sewage-contaminated seawater. Novel molecular amplification and sequencing methodologies were required to recognize the rarely identified CVA1, but it is ambiguous whether CVA1 infection caused illness. Travelers should be aware of risks associated with swimming in natural waters when visiting areas where there is limited sewage treatment.

RT-PCR for confirmation of echovirus 30 isolated in Belém, Brazil.

Echovirus (Echo) 30 or human enterovirus B is the most frequent enterovirus associated with meningitis cases. Epidemics and outbreaks of this disease caused by Echo 30 have occurred in several countries. In Brazil, Echo 30 has been isolated from sporadic cases and outbreaks that occurred mainly in the south and southeast regions. We used RT-PCR to examine Echo 30 isolates from meningitis cases detected from March 2002 to December 2003 in Belém, state of Pará, in northern Brazil. The patients were attended in a Basic Health Unit (State Health Secretary of Pará), where cerebrospinal fluid (CSF) was collected and stored in liquid nitrogen. Weekly visits were made by technicians from Evandro Chagas Institute to the health unit and samples were stored at -70 degrees C in the laboratory until use. HEp-2 and RD cell lines were used for viral isolation and neutralization with specific antisera for viral identification. RNA extraction was made using Trizol reagent. The RT-PCR was made in one step, and the total mixture (50 microL) was composed of: RNA, reaction buffer, dNTP, primers, Rnase inhibitor, reverse transcriptase, Taq polymerase and water. The products were visualized in agarose gel stained with ethidium bromide, visualized under UV light. Among the 279 CSF samples examined, 30 (10.7%) were EV positive, 29 being Echo 30 and one was Cox B. Nineteen Echo 30 were examined with RT-PCR; 18 tested positive (762 and 494 base pairs). The use of this technique permitted viral identification in less time than usual, which benefits the patient and is of importance for public-health interventions.

RT-PCR for confirmation of echovirus 30 isolated in Belém, Brazil

Echovirus (Echo) 30 or human enterovirus B is the most frequent enterovirus associated with meningitis cases. Epidemics and outbreaks of this disease caused by Echo 30 have occurred in several countries. In Brazil, Echo 30 has been isolated from sporadic cases and outbreaks that occurred mainly in the south and southeast regions. We used RT-PCR to examine Echo 30 isolates from meningitis cases detected from March 2002 to December 2003 in Belém, state of Pará, in northern Brazil. The patients were attended in a Basic Health Unit (State Health Secretary of Pará), where cerebrospinal fluid (CSF) was collected and stored in liquid nitrogen. Weekly visits were made by technicians from Evandro Chagas Institute to the health unit and samples were stored at -70°C in the laboratory until use. HEp-2 and RD cell lines were used for viral isolation and neutralization with specific antisera for viral identification. RNA extraction was made using Trizol reagent. The RT-PCR was made in one step, and the total mixture (50 æL) was composed of: RNA, reaction buffer, dNTP, primers, Rnase inhibitor, reverse transcriptase, Taq polymerase and water. The products were visualized in agarose gel stained with ethidium bromide, visualized under UV light. Among the 279 CSF samples examined, 30 (10.7 percent) were EV positive, 29 being Echo 30 and one was Cox B. Nineteen Echo 30 were examined with RT-PCR; 18 tested positive (762 and 494 base pairs). The use of this technique permitted viral identification in less time than usual, which benefits the patient and is of importance for public-health interventions.

First epidemic of echovirus 16 meningitis in Cuba.

From April to September 2000, an epidemic of aseptic meningitis spread throughout Cuba, with 16,943 reported cases. Virologic studies identified echovirus 16 as the cause of this epidemic. This is the first reported isolate of echovirus 16 from patients with viral meningitis in Cuba.

[Viral meningoencephalitis caused by enterovirus in Cuba from 1990-1995]. / Meningoencefalitis virales por enterovirus en Cuba en el período 1990-1995.

The results of a study on enterovirus as causal agents of viral meningoencephalitis from 1990 to 1995 are described. From the 1458 samples of patients studied, 586 were from feces, 108 from cerebrospinal fluid and 1095 were paired sera. The samples for viral isolation were inoculated in two different cellular systems. A total of 225 samples (32.42%) were found to be positive; the highest number of isolations (n = 217) was found in feces and in diploid human lung fibroblast cells (PHuE-1). Antibody determination were carried out using neutralization by micromethod test with 10 enterovirus antigens (Echovirus 4, 6, 9, 11 and Coxsackievirus B1-6). The isolated virus was also used as antigen during epidemic periods. There were 3 epidemic outbreaks: Coxsackievirus A9 (1990-1991), Echovirus 30 (1994), and Coxsackievirus B5 (1995). Coxsackievirus A9 and Echovirus 30 have been related to viral meningoencephalitis epidemics in Cuba since 1970. In paired sera 66.84% were positive, Echovirus 6 and 11 exhibited higher positivity. When positive results by isolation and serology were considered together, more than 80% of the studied cases were associated to enterovirus infection. These results show the importance of enteroviruses as cause of viral meningoencephalitis in our country.