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.

Acute haemorrhagic conjunctivitis outbreak in the city of Fortaleza, northeast Brazil.

BACKGROUND/AIM: Between February and May 2003 an epidemic of acute haemorrhagic conjunctivitis affected more than 200 000 people in all five geographic regions of Brazil (north, south, midwestern, southeast, and northeast). The aim was to identify the aetiological agent and to describe clinical aspects of this outbreak in a group of patients treated at the ophthalmology department of the Hospital Walter Cantídio (OD-HWC) at the Universidade Federal do Ceará, in the city of Fortaleza, capital of the state of Ceará, northeastern Brazil. METHODS: Conjunctival swabs were collected from patients who spontaneously went to the laboratory of virology. Specimens were inoculated in HEp-2 and RD cell lines. The viral isolation was confirmed by performing reverse transcriptase polymerase chain reaction and indirect immunofluorescence assay. RESULTS: Viral conjunctivitis was diagnosed in 56 patients but only 24 of them allowed the collection of samples. Of 24 conjunctival swabs tested, 11 were positive for a variant of coxsackie virus A24 (CA24v) and one of the isolates reacted with anti-adenovirus monoclonal antibodies. CONCLUSION: CA24v was confirmed as the aetiological agent of this outbreak of acute haemorrhagic conjunctivitis in the city of Fortaleza.

Starc II, a multicenter randomized placebo-controlled double-blind clinical trial of trapidil for 1-year clinical events and angiographic restenosis reduction after coronary angioplasty and stenting.

The objective of this study was to investigate the effect of trapidil 200 mg t.i.d. in preventing the occurrence of death, of myocardial infarction and the need for repeat revascularization at 12 months after balloon PTCA with or without stenting. Coronary restenosis after stenting is still a major drawback of percutaneous coronary interventions (PCI) for 30-40% of patients. Trapidil has been shown to prevent restenosis after PTCA. Eligible patients were randomized to placebo or oral trapidil 200 mg t.i.d. at least 48 hr before PCI and continuing 6 months after a successful balloon angioplasty or stent implantation. Aspirin was given to all patients, and ticlopidine 250 mg b.i.d. to those who received a stent for 4 weeks. In a randomized subgroup of 216 patients, quantitative coronary angiography was performed also at 6-month follow-up. Out of the 933 patients enrolled, primary endpoint incidence was 20.3% in trapidil and 18.0% in placebo (P = 0.37). When recurrence or deterioration of angina was added to the combined endpoint, incidence was 27.4% in trapidil and 23.0% in placebo (P = 0.12). Restenosis rate in patients with 6-month angiography was 25.0% in trapidil arm vs. 30.1% in placebo (P = 0.43). Stent restenosis rate was similar in patients randomized to trapidil or placebo (30.2% vs. 23.8%, respectively; P = 0.44), while in patients treated with balloon angioplasty, it was lower in trapidil (17.1%) than in placebo (40.0%; P = 0.03). Oral trapidil 200 mg t.i.d. for 6 months in addition to aspirin did not influence the occurrence of major cardiac events after coronary angioplasty with or without stenting. In a prespecified subgroup of 191 patients treated with balloon angioplasty only, trapidil reduced angiographic restenosis.

[Search of antimeasles antibodies in HIV-infected children after basic immunization] / Pesquisa de anticorpos contra o sarampo em crianças infectadas pelo HIV, após a imunização básica.

OBJECTIVE: To determine the presence of antimeasles antibodies in children perinatally infected with HIV and properly immunized. METHODS: A retrospective cohort study conducted in Belo Horizonte by the Universidade Federal de Minas Gerais, between 1995 and 1996. Twenty one children perinatally infected with HIV and 29 immunocompetent noninfected children were included in the study. Information about measles vaccination was obtained from patients immunization charts. The presence of neutralizing antibodies against the measles was determined by the plaque reduction neutralization test and IgM was measured by ELISA. The level of significance was set at 5% in all the performed statistical analyses. RESULTS: Median age was 44.5 months for HIV-infected patients and 62.0 months for noninfected children (P=0.64). Both groups received on average two doses of antimeasles vaccine. All HIV-seronegative patients presented antimeasles antibody titers greater than 50 mIU/ml, whereas 57.1% of infected children presented titers above this value (P=0.0001). The geometric mean titer of neutralizing antibodies was significantly lower in the group of HIV-infected children (433.5 mIU/ml) than in noninfected children (1,668.1 mIU/ml), P=0.001. All patients in both groups were negative for antimeasles IgM. CONCLUSION: In the present study, HIV-infected children showed a lower seroprevalence of antimeasles antibody after immunization than noninfected children. These results emphasize the risk of acquisition of measles virus and the need to evaluate alternatives to the vaccination of HIV-infected children in an attempt to maximize the protection against the measles in this group of patients.

Ultrastructural and immunocytochemical study on the infection of enterovirus 71 (EV 71) in rhabdomyosarcoma (RD) cells.

Rhabdomyosarcoma monolayers were inoculated with enterovirus 71 (EV 71) at 73 degrees C, sampled at intervals during the replicative cycle, and examined in thin sections by electron microscopy, using routine and immunoelectronmicroscopy with polyclonal antibodies against EV 71. The location of EV 71 or its precursors was followed during the viral replicative cycle. The earliest samples (3 h postinoculation) showed a cell shape change, from elongated to rounded. At 6 h postinoculation, the presence of early virus-induced vesicles developing within the cytoplasm was pointed out, although no virus particles were observed at these stages. At 12 and 20 h postinoculation, virus particles appeared in the cytoplasm. They were found free or in clusters in the cytoplasmic matrix, between the virus-induced vesicles. EV 71 particles were also occasionally observed in the form of paracrystalline arrays situated in the vesiculated areas. The immunolabel (gold beads) was found, initially, over dense cytoplasmic areas and in more advanced process at the vesiculated area and over the virus particles. Therefore the main cellular alterations observed in this infection were the shape change of the cell and the appearance of electron-dense areas (viroplasm) and smooth walled vesicles which are probably the site of the virus replication.

Enteroviruses isolated from patients with acute respiratory infections during seven years in Rio de Janeiro (1985-1991).

Enteroviruses were investigated in respiratory secretions collected from patients with acute respiratory infections (ARI) over a seven year period (1985-1991), as part of a longitudinal study of ARI aetiology. All the viruses that are most commonly associated with ARI were found in this study. Among the virus isolates, enteroviruses were only less frequent than respiratory syncytial viruses, adenoviruses and influenzaviruses. Forty five enterovirus samples were isolated from patients with either upper respiratory tract infections (URTI) or lower respiratory tract infections (LRTI). From these enterovirus isolates, thirty one samples were identified as poliovirus (n = 18) and non polio enterovirus (n = 13) by serum neutralization. Poliovirus were identified as type 1 and 2 and all of them were vaccinal strains. From thirteen non polio enterovirus, twelve were identified as echovirus serotypes 1, 2, 7, 11, 19 and 31. The remainder was identified as coxsackievirus B4.

Poliovirus type 1 isolated from a vaccine-associated case of paralytic poliomyelitis in Brazil.

This study reports a type 1 poliovirus strain isolated in Brazil from a case classified as vaccine-associated paralytic poliomyelitis (VAPP). After serotyping of the viral isolate with hyperimmune equine sera, PCR and molecular hybridization techniques characterized the strain as P1/Sabin-derived. The isolate was partially sequenced to identify mutations at nucleotides 480, 525 and 6203, which are important for reversion of the P1/Sabin strain to neurovirulence. In a recent study, a P1/Sabin-derived strain isolated from the central nervous system of a VAPP case did not mutate at these positions, but maintained 480-G and 525-U (and 6203-C), suggesting that these mutations are not essential for the occurrence of disease (Georgescu et al., (1994), Journal of Virology, 68: 8089-8101). Although the Brazilian strain also maintained 480-G and 525-U (and 6203-C) and was isolated from the stool, the possibility that this isolate invaded the central nervous system after replicating in the gut, causing the paralysis, cannot be ruled out. This is the first report of a type 1 VAPP case in Brazil, although some cases caused by type 2 and type 3 strains have been described.

Genomic characterization of type 2 polioviruses isolated from vaccine-associated cases in Brazil.

Twenty strains of P2/Sabin-related polioviruses isolated in Brazil were analyzed; ten from persistent paralytic poliomyelitis cases, three from suspected polio cases with transient paralysis, and seven from healthy contacts. The serotypes of the viral isolates were identified by the neutralization test with hyperimmune equine sera. The relationship of the isolates to the P2/Sabin strain was demonstrated by molecular hybridization and polymerase chain reaction (PCR). Partial sequencing demonstrated mutations at nucleotide 481 in the 5' noncoding region and at amino acid 143 of the capsid protein VP1 in most of these isolates from vaccine-associated cases in Brazil. These data support previous studies on the importance of mutations at these attenuated determinants in the establishment of the disease. However, the existence of isolates without mutations at these positions suggests that they are not essential. The results also strengthen the possibility of the participation of a mutation at nucleotide 398 in the establishment of the disease, and suggest that a mutation at nucleotide 491 or 500 may also be involved in this process. The isolates from healthy contacts presented the same mutations as the isolates from vaccine-associated cases with which they were in contact. This strengthens the observation that, although mutations in the genome of the P2/Sabin strain are important for the establishment of the disease, host factors are also involved.

Genomic characterization of type 1 Sabin-related polioviruses isolated in Brazil.

Eight strains of P1/Sabin-derived polioviruses isolated in Brazil from paralysis cases were analyzed. The serotypes of the viral isolates were identified by neutralization test with hyperimmune equine sera. The relationship of the isolates to the P1/Sabin strain was demonstrated by molecular hybridization and PCR. The isolates were partially sequenced with the objective of finding mutations at nucleotides (nt) 480 and 525 of the 5'-noncoding region (5' NCR) and at nt 6203 of the 3Dpol coding region (3Dpol), which are important for reversion towards neurovirulence. Four isolates from paralysis cases classified as Guillain Barré Syndrome (GBS; three with sequels) were analyzed; one presented G-->A (480) and C-->U (6203) mutations, one G-->A (480) mutation, one G-->A (480) and U-->C (525) mutations, and one did not mutate at the analyzed positions. Two isolates from transient facial paralysis cases were analyzed; one presented U-->C (525) mutation and the other G-->A (480) mutation. One isolate from a transient paralysis case classified as a neuroviral disease and one isolate from a paralysis case with sequels were analyzed and none mutated at the analyzed positions. Although the isolates may not be the causative agent of the disease, a temporal association between the isolation of the P1/Sabin-derived isolates and the disease was observed. The possibility that GBS and the facial paralysis were caused by these isolates could not be excluded.

Genomic characterization of type 3 polioviruses isolated from vaccine-associated poliomyelitis cases in Brazil.

Eight strains of P3/Sabin-related polioviruses were analyzed; four from persistent paralytic poliomyelitis cases classified as vaccine associated, one from a transient paralysis case classified as transverse myelitis, one from a transient paralysis case classified as Guillain-Barré syndrome, one from a transient facial paralysis case, and one from a healthy vaccine. The serotypes of the viral isolates were identified by the neutralization test with hyperimmune equine sera and the relationship of the isolates with the P3/Sabin strain was demonstrated by molecular hybridization of the viral RNA of the isolates with a P3/Sabin-specific probe. The P3/Sabin relationship was confirmed by PCR, using a pair of specific primers for P3/Sabin-related isolates. The available data indicate that a U-->C mutation at nucleotide 472 in the 5' noncoding region of the genome of the type 3 Sabin strain increases the neurovirulence of this strain and this mutation was observed in all type 3 isolates from vaccine-associated cases. These eight P3/Sabin-related isolates were partially sequenced in the 5' noncoding region and seven presented a U-->C mutation at nucleotide 472, except the isolate from a transient paralysis case classified as transverse myelitis, that maintained a U at nucleotide 472. Although this virus maintaining U at nucleotide 472 may not be the etiological agent of the disease, the possibility that the virus was the causative agent of the disease could not be ruled out.

Intratypic differentiation of polioviruses isolated from suspected cases of poliomyelitis in Brazil during the period of 1990 to 1993.

This study analyzed 3129 fecal samples derived from 1626 patients with sudden onset acute flaccid paralysis clinically compatible with poliomyelitis. The samples were collected in the period ranging from January 1990 to September 1993 in all regions of Brazil. Among the 1626 cases studied, 196 had isolation of poliovirus. Nevertheless, it was observed that some factors influenced the isolation rate and the intratypic characterization of these polioviruses. No cases of acute flaccid paralysis has been found to be etiologically related with wild polioviruses.

Application of the polymerase chain reaction (PCR) to poliomyelitis surveillance through the analyses of sewage samples.

A rapid and sensitive method for the detection of wild poliovirus from sewage samples using the polymerase chain reaction (PCR) technique was investigated. To eliminate the toxicity of sample concentrates to the enzymatic system used in PCR, a methodology was developed for the purification of these concentrates, consisting of treatment with trichlorofluoroethane and Sephadex column chromatography. The viral RNA was extracted from the purified concentrates, submitted to PCR with primers specific for Brazilian wild poliovirus type 1 and for Sabin types 1, 2 and 3. The amplified products were detected by electrophoresis in vertical polyacrylamide gels and stained with ethidium bromide. The results suggest that sewage sampling for environmental surveillance, combined with the rapid and precise PCR technology, provides a powerful tool for assessment of the success of the poliovirus eradication programme.

Randomized trial of late elective angioplasty versus conservative management for patients with residual stenoses after thrombolytic treatment of myocardial infarction. Treatment of Post-Thrombolytic Stenoses (TOPS) Study Group.

BACKGROUND: After thrombolytic therapy for patients with acute myocardial infarction (MI), percutaneous transluminal coronary angioplasty (PTCA) is frequently performed because of the presence of a "significant" infarct vessel stenosis demonstrated at predischarge coronary angiography. Several studies have shown PTCA performed early after thrombolysis to be unnecessary or even harmful. However, PTCA in these trials was generally performed 1-3 days after MI, when the milieu in the infarct artery may be unsuited for PTCA, and the incidence of major ischemic complications was high. To date, no trial has assessed whether delayed PTCA (4-14 days) should be performed in patients without evidence of ischemia on stress testing. METHODS AND RESULTS: To test the hypothesis that delayed PTCA might provide clinical benefit compared with medical therapy alone, 87 patients treated within 6 hours of chest pain onset with thrombolytic therapy and with negative functional test were randomized between PTCA to be performed 4-14 days after MI versus no PTCA. Both groups received medical therapy. Patients with postinfarct angina or prior Q wave infarction in the infarct distribution were excluded. The primary study end point was increase in left ventricular ejection fraction with exercise measured by radionuclide studies 6 weeks after MI, a parameter known from other studies to correlate inversely with future ischemic events. Clinical outcome was also monitored for 12 months. There were no differences between the study groups for any prerandomization variable recorded. Mean age was 57 +/- 10 years, 84% of patients were male, 21% had prior MI, 36% had anterior MI, 7% had multivessel disease, and the infarct stenosis measured 70 +/- 17% before randomization. PTCA was successful in 38 of 42 patients (88%) but resulted in non-Q wave MI due to acute closure of the treated site in three of 42 (9.5%). There was no difference in 6-week resting ejection fraction or increase in ejection fraction with exercise between the two groups (47 +/- 12% and 6 +/- 8%, respectively, in the PTCA group; 49 +/- 10% and 5 +/- 9% in the no-PTCA group; p = NS for both.) There were no deaths in either group. Actuarial 12-month infarct-free survival was 97.8% in the no-PTCA group and 90.5% in the PTCA group (p = 0.07). CONCLUSIONS: There was no functional or clinical benefit from routine late PTCA after MI treated with thrombolytic therapy in this relatively low-risk cohort of patients. These data strongly suggest that patients with an uncomplicated MI after thrombolytic therapy, even if they have a "significant" residual stenosis of the infarct vessel, should be treated medically if they are without evidence of ischemia on stress testing before hospital discharge.

Oligonucleotide probes for the specific detection of the wild poliovirus types 1 and 3 endemic to Brazil.

Synthetic oligodeoxynucleotide probes, 21-23 nucleotides in length, were prepared which specifically hybridize to the genomes of the wild type 1 and 3 polioviruses currently endemic to the northeastern region of Brazil. The probes are complementary to sequences near the 5'-terminus of the VP1 gene that differ substantially among genetically distant polioviruses but are largely conserved among related isolates. The probes have been routinely used in the laboratory surveillance of poliomyelitis cases in Brazil, permitting direct, rapid identification of the indigenous wild polioviruses by dot-blot hybridization.

Nucleotide sequences of the VP1 capsid proteins of wild poliovirus types 1 and 3 from epidemic areas of Brazil.

The nucleotide sequences encoding the capsid protein VP1 were determined for the wild polioviruses of serotypes 1 and 3 endemic to the northeastern region of Brazil. Compared with the corresponding Sabin vaccine strain sequences, the wild isolates differed at 20% (type 1) and 22% (type 3) of their nucleotide positions, and in 7% (type 1) and 11% (type 3) of their amino acid residues. The highest degree of amino acid heterogeneity occurred within the amino-terminal residues of the VP1 proteins. Intratypic amino acid differences also occurred in VP1 surface residues that form parts of antigenic sites for neutralizing antibodies.