Standardization of denaturing gradient gel electrophoresis (DGGE) for discrimination of Victoria and Yamagata lineages of influenza B.

In this study, a PCR-DGGE protocol was standardized in order to distinguish Victoria and Yamagata influenza B lineages directly from clinical samples. After routine multiplex PCR characterization, amplicons of the haemagglutinin gene bearing a 40bp-length GC clamp were generated by nested-PCR and analyzed by electrophoresis in 6% polyacrylamide gel with a 25-45% urea-formamide gradient. The results showed a perfect correlation between DGGE and phylogenetic analyses for all compared samples, besides some distinct profiles in Victoria and Yamagata groups that could be used to infer variability inside these groups. In summary, this DGGE protocol for the haemagglutinin gene is rapid, useful and efficient, being an alternative for discrimination between the influenza B lineages.

Prevalence and clinical aspects of respiratory syncytial virus A and B groups in children seen at Hospital de Clínicas of Uberlândia, MG, Brazil.

Respiratory syncytial virus (RSV) is well recognized as the most important pathogen causing acute respiratory disease in infants and young children, mainly in the form of bronchiolitis and pneumonia. Two major antigenic groups, A and B, have been identified; however, there is disagreement about the severity of the diseases caused by these two types. This study investigated a possible association between RSV groups and severity of disease. Reverse transcription-polymerase chain reaction was used to characterize 128 RSV nasopharyngeal specimens from children less than five years old experiencing acute respiratory disease. A total of 82 of 128 samples (64.1%) could be typed, and, of these, 78% were group A, and 22% were group B. Severity was measured by clinical evaluation associated with demographic factors: for RSV A-infected patients, 53.1% were hospitalized, whereas for RSV B patients, 27.8% were hospitalized (p = 0.07). Around 35.0% of the patients presented risk factors for severity (e.g., prematurity). For those without risk factors, the hospitalization occurred in 47.6% of patients infected with RSV A and in 18.2% infected with RSV B. There was a trend for RSV B infections to be milder than those of RSV A. Even though RSV A-infected patients, including cases without underlying condition and prematurity, were more likely to require hospitalization than those infected by RSV B, the disease severity could not to be attributed to the RSV groups.

Prevalence and clinical aspects of respiratory syncytial virus A and B groups in children seen at Hospital de Clínicas of Uberlândia, MG, Brazil

Respiratory syncytial virus (RSV) is well recognized as the most important pathogen causing acute respiratory disease in infants and young children, mainly in the form of bronchiolitis and pneumonia. Two major antigenic groups, A and B, have been identified; however, there is disagreement about the severity of the diseases caused by these two types. This study investigated a possible association between RSV groups and severity of disease. Reverse transcription-polymerase chain reaction was used to characterize 128 RSV nasopharyngeal specimens from children less than five years old experiencing acute respiratory disease. A total of 82 of 128 samples (64.1 percent) could be typed, and, of these, 78 percent were group A, and 22 percent were group B. Severity was measured by clinical evaluation associated with demographic factors: for RSV A-infected patients, 53.1 percent were hospitalized, whereas for RSV B patients, 27.8 percent were hospitalized (p = 0.07). Around 35.0 percent of the patients presented risk factors for severity (e.g., prematurity). For those without risk factors, the hospitalization occurred in 47.6 percent of patients infected with RSV A and in 18.2 percent infected with RSV B. There was a trend for RSV B infections to be milder than those of RSV A. Even though RSV A-infected patients, including cases without underlying condition and prematurity, were more likely to require hospitalization than those infected by RSV B, the disease severity could not to be attributed to the RSV groups.

Understanding the transmission dynamics of respiratory syncytial virus using multiple time series and nested models.

The nature and role of re-infection and partial immunity are likely to be important determinants of the transmission dynamics of human respiratory syncytial virus (hRSV). We propose a single model structure that captures four possible host responses to infection and subsequent reinfection: partial susceptibility, altered infection duration, reduced infectiousness and temporary immunity (which might be partial). The magnitude of these responses is determined by four homotopy parameters, and by setting some of these parameters to extreme values we generate a set of eight nested, deterministic transmission models. In order to investigate hRSV transmission dynamics, we applied these models to incidence data from eight international locations. Seasonality is included as cyclic variation in transmission. Parameters associated with the natural history of the infection were assumed to be independent of geographic location, while others, such as those associated with seasonality, were assumed location specific. Models incorporating either of the two extreme assumptions for immunity (none or solid and lifelong) were unable to reproduce the observed dynamics. Model fits with either waning or partial immunity to disease or both were visually comparable. The best fitting structure was a lifelong partial immunity to both disease and infection. Observed patterns were reproduced by stochastic simulations using the parameter values estimated from the deterministic models.

The reappearance of Victoria lineage influenza B virus in Brazil, antigenic and molecular analysis.

BACKGROUND: In contrast to influenza A, minor influenza B viruses can co-circulate with the dominant strain during an epidemic allowing the re-emergence of old strains and reassortment between those different strains. The 2001-2002 influenza season in the northern hemisphere was distinguished by the re-emergence of the Victoria-lineage viruses, which replaced the Yamagata-lineage, after being restricted to East Asia throughout the 1990s. OBJECTIVES: To describe the antigenic and genetic characteristics of influenza B viruses detected in South and South East Brazil and determine their lineages. STUDY DESIGN: Influenza samples collected during epidemics between 1999 and 2002 were analyzed by indirect immunofluorescence assay (IFA). Positive results were confirmed through multiplex PCR and isolation in cell culture. Isolated viruses were antigenically characterized by hemagglutination inhibition. Fourteen hemagglutinin (HA) gene sequences obtained in this work were used for phylogenetic analysis. RESULTS: Brazilian isolates from 2002 were associated with the Victoria-lineage, diverging from the vaccine used throughout that influenza season in Brazil. CONCLUSIONS: These results indicate the reappearance of Sichuan/7/97-like samples in South and South East Brazilian Regions simultaneously. They indicate the need for neuraminidase gene evaluation and demonstrate the importance of influenza laboratory surveillance to establish which strains should be included in the influenza vaccine.

Comparison of PCR, enzyme immunoassay and conventional culture for adenovirus detection in bone marrow transplant patients with hemorrhagic cystitis.

BACKGROUND: Adenovirus-associated hemorrhagic cystitis (HC) has become a recognized sequel of immunosuppression. The diagnosis of viral infection is usually determined by viral cultures. OBJECTIVES: Analysis of different diagnostic methods for adenovirus (AdV) detection in bone marrow transplant patients with hemorrhagic cystitis. STUDY DESIGN: We describe a prospective study for AdV detection in the urine of patients with hematuria in the first 100 days after bone marrow transplant (BMT), comparing different laboratory techniques, PCR, enzyme immunoassay (EIA) and conventional culture. RESULTS: A total of 143 urine samples were analyzed, 75 collected in the pre-transplant period with and without hematuria and 68 post-transplant, only with microscopic or macroscopic hematuria. After BMT, hematuria occurred in 38.9% of patients, being more frequent in unrelated donor transplants. AdV was isolated in one pre-transplant patient without symptoms and in three post-transplant patients with HC grades 3 and 4 (severe), who were in month 2 or 3 post-transplant. Compared to culture as the gold standard, the accuracy, specificity and sensitivity of EIA were 95, 30 and 100% and for PCR were 63, 100 and 60%, respectively. CONCLUSIONS: We concluded that despite technical difficulties and the long time that elapsed before results were obtained, cell culture still remains the best method for adenovirus detection in the urine of patients with hemorrhagic cystitis.

Use of RT-PCR on oral fluid samples to assist the identification of measles cases during an outbreak.

This study investigated the occurrence of mild modified measles cases during an outbreak in Niterói, RJ, Brazil by using RT-PCR on oral fluid samples. From August to December 1997 a total of 76 patients with rash were seen at the study sites. Confirmed diagnosis by serology was achieved in 47 cases: measles (39.5%), rubella (13.2%), HHV-6 (3.9%), human parvovirus B19 (3.9%), dengue fever (3%). For 19 of the 29 patients without a conclusive diagnosis paired serum and saliva samples were available for further tests. In four of them, measles virus RNA was detected by RT-PCR in saliva samples in the absence of specific IgM in serum samples. Vaccination histories obtained from three of the RT-PCR positive cases showed that individuals previously immunized can still be infected and contribute to the circulation of measles virus. This study demonstrated the usefulness of RT-PCR on non-invasive clinical samples for the investigation of measles cases.

Occurrence and severity of infections caused by subgroup A and B respiratory syncytial virus in children in southeast Brazil.

The frequency and severity of infections caused by respiratory syncytial virus (RSV) were assessed in children <2 years of age seen at the emergency department. The frequency of RSV detection in the clinical virology laboratory during the past 3 years was also analyzed retrospectively. RSV was found in 21.6% (188/869) of the samples collected from children seen at the emergency department and was found to be more frequent during the autumn, being less frequent or negligible by midwinter. RSV subgroups A and B co-circulated within the same time period in children seen at the emergency department, with varying predominance of either subgroup. There was no significant association of RSV subgroup with disease severity, but only a trend for RSV subgroup B being more frequent in children with risk factors for severe disease.

Respiratory syncytial virus groups A and B in Porto Alegre, Brazil, from 1990 to 1995 and 1998.

We analyzed the respiratory syncytial virus (RSV) groups and their epidemiological pattern that were detected over the course of seven years in southern Brazil. The two RSV groups co-circulated each year, but frequencies of groups A and B varied both between and within yearly outbreaks. In 1991, group A predominated over group B (p=0.0016). RSV outbreaks analyzed showed a temperature-dependent pattern and no association with rainfall, similarly to other countries from southern South America. Knowledge of the variants is important in terms of both diagnosis and definition of a vaccine composition.

Genomic characterization of wild-type measles viruses that circulated in different states in Brazil during the 1997 measles epidemic.

Despite the marked reduction in the incidence of measles in Brazil, a measles epidemic occurred in this country in 1997. The measles cases observed during this epidemic began to reappear in large numbers in São Paulo, and spread to Rio de Janeiro and other Brazilian states. In the present study molecular biology techniques were used for the detection and genomic characterization of measles viruses from clinical samples such as urine and nasopharyngeal secretions collected in the states of Rio de Janeiro, Minas Gerais and Paraná, during the 1997 epidemic. RT-PCR and nucleotide sequence analysis of part of the carboxyl-terminal region of the nucleoprotein gene of measles viruses obtained directly from clinical samples or from infected cell cultures during this epidemic classified all as wild-type of genotype D6. As the genotype D6 was identified in different Brazilian states, this study demonstrated that this genotype was circulating in Brazil during the 1997 epidemic.

[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.

The aetiology of maculopapular rash diseases in Niterói, State of Rio de Janeiro, Brazil: implications for measles surveillance.

A study investigating the causes of rash diseases using systematic laboratory testing was conducted in Niterói, Rio de Janeiro, between January 1994 to April 1998. Sera from 327 patients were tested for evidence of anti-rubella virus, measles virus, human parvovirus B19 and dengue fever virus specific immunoglobulin IgM and anti-human herpes virus type 6 (HHV-6) IgG antibodies. A laboratory confirmed diagnosis was achieved in 71.3% of the cases investigated: dengue fever (33.0%), rubella (20.2%), parvovirus B19 (9.2%), measles (6.7%) and HHV-6 (2.1%). No diagnosis was established for 94 cases (28.7%). An outbreak of measles was detected during 1997, with a peak in September and October. All of the diseases studied here presented with clinical features similar to measles and classical symptoms were found in all measles confirmed cases. The large overlap of combinations of signs and symptoms seen in this study highlights the difficulties of diagnosing a rash illness on clinical grounds alone.

Recent measles viral activity in Uruguai: serological and genetic approaches.

In the summer 1999, a measles outbreak occurred in Uruguai. During this outbreak 58 cases were recorded, 36 of which were laboratory confirmed as positive for measles virus (MV) IgM. The cases occurred in touristic places (Montevideo and Maldonado) predominantly among health facilities and tourist service personnel. Urine specimens collected between days 1 and 4 after the onset of the rash from seven cases were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and nested PCR with primers specific for the carboxyl-terminal region of the nucleoprotein (N) gene. Three of these specimens/cases were positive for MV. Sequencing of 300 nucleotides (nt) of PCR products corresponding to a part of the carboxyl-terminal region of the MV N gene detected in these specimens MV of D6 genotype. The same nucleotide sequences and the same genotype were also previously observed for MV isolates from the 1997 epidemic in Brazil and the 1998 epidemic in Argentina, demonstrating that the D6 genotype was, and may be still circulating in South America.

Diagnosis of rubella infection by detecting specific immunoglobulin M antibodies in saliva samples: a clinic-based study in Niterói, RJ, Brazil.

This study was designed to investigate whether saliva could be a feasible alternative to serum for the diagnosis of recent rubella infection in a clinic setting. Forty-five paired blood and saliva samples collected 1 to 29 days after onset of illness were tested for specific immunoglobulin (Ig) M by antibody-capture radioimmunoassay (MACRIA). Rubella IgM was detected in all serum samples and in 38 (84.4%) saliva specimens. Forty-six serum and saliva samples from other patients with rash diseases were tested by MACRIA for control purposes and two saliva specimens were reactive. The saliva test had specificity of 96%. These results indicate that salivary IgM detection may be a convenient non-invasive alternative to serum for investigation of recent rubella cases, especially for disease surveillance and control programmes.

Genetic characterization of wild-type measles viruses isolated during the 1998 measles epidemic in Argentina.

This study reports on genomic characterization of six measles virus (MV) isolates obtained from a measles epidemic in Argentina in 1998. Reverse transcription-polymerase chain reaction (RT-PCR) and nucleotide sequence analysis of the carboxyl-terminal region of the nucleoprotein (N) gene of these isolates classified all of them as wild type MV of D6 genotype. MVs of D6 genotype with identical nucleotide sequences in the region analyzed were also identified during the 1997 measles epidemic in Brazil and the 1999 measles outbreak in Uruguai. These results suggest that the MVs associated with the 1998 measles epidemic in Argentina might have originated from Brazil. As the D6 genotype is also widely distributed in Europe, it is possible that this genotype was brought to South America from Europe.

Atypical measles in a patient twice vaccinated against measles: transmission from an unvaccinated household contact.

Described are two cases within the same household that were involved in an outbreak of measles in Niterói, RJ. Measles diagnosis was confirmed serologically by specific IgM detection in Case 1 (classic measles) who was unvaccinated, and rising measles specific IgG in the absence of IgM in Case 2 (mild modified measles) who had a history of two vaccinations with measles-containing vaccines. Measles virus was detected by reverse transcriptase polymerase chain reaction (RT-PCR) in saliva samples from both cases. The nucleic acid amplified by RT-PCR was sequenced and showed identical measles sequence in the two cases. This study highlights the difficulty of diagnosing nonclassical measles infection on clinical and serological grounds, and the usefulness of PCR for viral RNA sequencing from noninvasive specimens for confirming epidemiologic links.

Genome type analysis of Brazilian adenovirus strains of serotypes 1,2,3,5, and 7 collected between 1976 and 1995.

A collection of 92 epidemiologically unrelated isolates of Ad1 (n = 14), Ad2 (n = 29), Ad3 (n = 19), Ad5 (n = 16), and Ad7 (n = 14) collected in the cities of Belem do Pará (1 degree S 48 degrees W) and Rio de Janeiro (23 degrees S 43 degrees W) between 1976 and 1995 from patients with respiratory disease and conjunctivitis were characterized by restriction enzyme analysis of genomic DNA. Among the strains of subgenus B, two different genome types of serotype 7, 7b and 7e, were identified. The analysis of their temporal distribution throughout the study period suggested an alternating appearance of these two DNA variants. Only one genome type of Ad3, 3p, was detected during the sampling period. Further analysis with Xba I, Bcl I, and Hpa I indicated that it is a p1-like genome type. Both previously described and new genomic variants were identified among subgenus C strains. Genome types D1, D7, D10, and one not previously described were identified among the 14 Ad1 strains analyzed. Genome types D2, D5, D25, and 13 new DNA variants were identified among the 29 Ad2 isolates. Genome type D38 and 5 new variants were found among the 16 strains of Ad5. In spite of the relatively small size of the sample analyzed, the results of this study confirm the important genetic variability previously observed for members of subgenus C by other authors.

[The joint manifestations of exanthematous viroses]. / Manifestações articulares nas viroses exantemáticas.

The frequency of arthropathy was evaluated in 251 patients with clinical and serological diagnosis (specific IgM detection by enzyme immunoassay) of exanthematous disease. Arthropathy (arthralgia and/or arthritis) was more frequent in dengue fever (49%) and rubella (38.2%) cases than in human parvovirus (30%) and measles (28.1%) cases. Except for measles cases, joint complaints prevailed in adults (> or = 15 years of age) and this difference was significant. The higher frequency of arthropathy in adults was more evident in human parvovirus (75%), rubella (65%) and dengue fever (57.7%) cases than in measles cases (31%). Arthropathy was also more frequent in females for all rash diseases studied. The results of this study showed the high occurrence of joint complaints in the disease described here and the importance of laboratory confirmation for their differential diagnosis.

Measles virus antigen in macrophage/microglial cells and astrocytes of subacute sclerosing panencephalitis.

In two patients with subacute sclerosing panencephalitis (SSPE) of 10 and 25 months duration we demonstrated by immunohistochemistry the presence of measles-virus nucleocapsid antigen (MVNA) in CD68+ cells and astrocytes of brain tissues. In both cases, CD68+ hematogenous monocyte/ macrophages and perivascular microglial cells (Mphi) were found infiltrating the brain parenchyma, and often partially or completely invested by perivascular reactive astrocytes expressing glial fibrillary acidic protein (GFAP). Mphi with cytoplasmic MVNA were often seen in the Virchow-Robin spaces and in close association with perivascular astrocytes, which often also contained MVNA+ intracytoplasmic inclusions. Reactive astrocytosis was more severe in the patient with long-standing illness, and a correspondingly elevated number of strongly GFAP+ MVNA+ or MVNA- perivascular binucleated astrocytes was observed. An uptake of MVNA+ cell debris by reactive astrocytes was evident in areas of white matter displaying extensive demyelination and necrosis. Taken together, these observations seem to indicate that the brain infiltration by Mphi carrying measles virus could represent one pathway of virus entry and dissemination in the central nervous system. Virus transfer to perivascular astrocytes via cell-to-cell contacts with infected macrophages is also suggested.