Epidemiological and molecular characterization of rubella virus isolated in São Paulo,Brazil during 1997–2004

Rubella virus (RV) infection during the early stages of pregnancy can lead to serious birth defects, known as the congenital rubella syndrome (CRS). In 2003, the Pan American Health Organization (PAHO) adopted a resolution calling for the elimination of rubella and the congenital rubella syndrome (CRS) in the Americas by the year 2010. Brazil will have implemented the recommended PAHO strategy for elimination and interruption of endemic rubella virus transmission. The characterization of genotypes during the final stages of rubella elimination is important for determining whether new rubella isolates represent endemic transmission or importations. Samples (blood, urine, cerebrospinal fluid, and throat swabs) collected from patients with symptoms suggestive of rubella infection in 1997–2004 were isolated in cell culture and genotyped. Twenty‐eight sequences were analyzed and two genotypes were identified: 1a and 1G. The information reported in this paper will contribute to understanding the molecular epidemiology of RV in São Paulo, Brazil. J. Med. Virol. 84:1831–1838, 2012. © 2012 Wiley Periodicals, Inc.     

Isolation and genotyping of rubella virus from a case of congenital infection in Brazil

The incidence of CRS and CRI has decreased markedly worldwide with the implementation of efficient vaccination programs. We report a congenital rubella case with fetal death occurred at 29th week of gestation. RV was confirmed in placenta. The results of phylogenetic analysis showed that the RVs/SaoPaulo01.- BRA/08.CRI belongs to the genotype 2B of RV.     

Application of molecular and serological assays to case based investigations of rubella and congenital rubella syndrome

Rubella and congenital rubella syndrome continue to be important health problems worldwide. The detection of rubella RNA directly in clinical specimens is a critical factor in early laboratory diagnosis of recent or congenital infection, in addition to detection of rubella-specific IgM. In order to comply with recent WHO recommendations for establishing uniform genetic analysis protocols for rubella virus we have developed a new block based PCR assay (PCR-E317), which extends the sequence generated by the block based PCR-E592 currently in use, to cover the minimum acceptable 739 nucleotides (nt) window at the E1 gene. In addition, a real-time PCR assay has been developed to allow rapid detection of the virus in the laboratory. The assays were applied to a number of clinical specimens collected from patients including recent rubella incidences in the UK, Ethiopia and Turkey, two prenatal and two congenital rubella syndrome cases. Rubella RNA was detected in specimens from two patients that were collected too early for IgM detection, in two amniotic fluids for prenatal diagnosis and in the follow up specimens from the two infant with congenital rubella syndrome tested for viral secretion. At least four genotypes were identified among these patients. The results showed that molecular assays are important tools in the early diagnosis of rubella and congenital rubella syndrome, in the provision of molecular epidemiological information for tracking transmission pathways and in adding to the knowledge of rubella strain distribution worldwide.     

Seroepidemiological study of respiratory syncytial virus in São Paulo State,Brazil

Transmission of respiratory syncytial virus is thought to be highly seasonal based on reported clinical cases, although transmission resulting in mild disease in all age groups has been little studied. This has been investigated in a seroepidemiological survey using sera from São Paulo, Brazil. Seroprevalence was found to increase rapidly with age, reaching over 90% by three years of age. This is typical of viral infections, which produce life-long immunity following primary infection. One-hundred percent seropositivity was attained by five years of age and maintained throughout adulthood, whereas mean antibody titers continued to increase with age. The mean duration of maternal antibodies was estimated to be 3.3 months with antibody decay demonstrated in paired samples from infants. The results are discussed in relation to possible mechanisms generating such a profile. J. Med. Virol. 55:234–239, 1998. © 1998 Wiley-Liss, Inc.     

Laboratory confirmation of congenital rubella syndrome in infants: an eye hospital based investigation

A total of 190 specimens from South Indian children aged 0-59 months with ocular anomalies consistent with suspected congenital rubella syndrome (CRS) were investigated. Twenty-six of the 65 infants (40%) were confirmed as CRS by detection of rubella specific IgM. Rubella RNA was detected in 41 samples from 26 infants by both real-time and block based PCR. The PCR results correlated well with the presence of anti-rubella IgM/IgG (23/27 cases with rubella IgM were PCR positive). Whereas, only 17 of 26 infants met the WHO CRS case definition. Amongst the various specimens tested from the sero-confirmed cases (n = 27), a high percentage of positives were detected in lens (92%) and oral fluid (60%) specimens, when compared to other samples. The quantification of viral load by real-time PCR demonstrated higher copy number of virus in lens samples of 0-11 months infants. The rubella viruses were characterized and revealed the circulation of genotype 2B in three South Indian states. The integrated analysis of clinical manifestations, serological and molecular data in the study has generated baseline information of rubella infection and CRS in infants with ocular anomalies.     

MICA polymorphism in a sample of the São Paulo population,Brazil

The major histocompatibility complex (MHC) class I chain‐related A (MICA) gene, located near HLA‐B, codes for protein products with structural similarities to those of classical MHC class I genes, but which neither bind β₂‐microglobulin nor present peptide. Expressed predominantly on gastrointestinal and tumour epithelial cells, they are stress‐induced and interact with C‐type lectin like receptor (NKG2D) on γδ, αβ CD8+ T cells and natural killer (NK) cells. MICA is highly polymorphic, with 54 extracellular allelic sequences described. We typed 200 healthy subjects in a sample of the São Paulo population by extended polymerase chain reaction–sequence‐specific primers (PCR‐SSP) to characterize the MICA polymorphism and analysed MICA/HLA‐B linkage disequilibrium. The MICA*008 group (g) was predominant (47%), with several HLA‐B associations. Rare combinations MICA*008g‐HLA‐B37, MICA*008g‐B72 and MICA*010‐HLA‐B52 were detected. Given the extent of this polymorphism and its possible relevance for disease association, we determined MICA and HLA‐B alleles in 33 Behçet's patients, in an attempt to clarify the associated genetic marker. Our results showed an increase of MICA*006, but not MICA*009, in the patient group (6/33) compared with controls (3/200) (18.2% vs. 1.5%; P_c = 0.005). Both alleles were always in association with HLA‐B51, suggesting that HLA‐B is indeed the primary susceptibility locus (P = 0.00008) and that MICA*006 may be an additional risk factor.     

Assessment of RNA amplification by multiplex RT‐PCR and IgM detection by indirect and capture ELISAs for the diagnosis of measles and rubella

Sanz JC, Mosquera M, Ramos B, Ramírez R, de Ory F, Echevarria JE. Assessment of RNA amplification by multiplex RT‐PCR and IgM detection by indirect and capture ELISAs for the diagnosis of measles and rubella. APMIS 2010; 118: 203–9. The aim of the study was to compare RNA amplification using multiplex RT‐PCR and IgM detection by means of indirect and capture ELISAs for the diagnosis of measles and rubella. A total of 229 cases of maculopapular rash with serum and throat swab samples were included. Specific serological IgM to measles and rubella was determined by Enzygnost^® (Siemens) and Platelia^TM (Bio‐Rad). Both viruses were researched using multiplex RT‐PCR performed on throat samples. Criteria for inclusion of measles or rubella cases were a positive RT‐PCR result for one virus and negative for the other; and/or a positive IgM result for one virus by both ELISAs and negative RT‐PCR for the other virus. A total of 74 cases were classified as measles and 54 as rubella. In measles, sensitivity and specificity were 93.2% and 100% for RT‐PCR, 97.3% and 98.1% for Enzygnost^®, and 90.5% and 95.5% for Platelia^TM. For rubella, these values were 42.6% and 100% for RT‐PCR, 100% and 97.1% for Enzygnost^®, and 94.4% and 98.3% for Platelia^TM. Enzygnost^® and Platelia^TM are useful techniques for detecting IgM against measles and rubella. RNA amplification by RT‐PCR was both sensitive and specific for the diagnosis of measles; however, for rubella, the sensitivity of this technique must be improved.     

Adenovirus genome types isolated from stools of children with gastroenteritis in São Paulo,Brazil

In a prospective one-year study of acute gastroenteritis in hospitalized children less than 2 years of age, in São Paulo (Brazil), adenoviruses were detected by specific enzyme immunoassay (El-ARA) in 7 of 67 (10%) ill children and in 9 of 79 (11.4%) controls. They were the sole recognizable agent of diarrhea in 6 ill children. In another child these viruses were detected in a dual infection with astrovirus. Enteric adenoviruses (Ad40/41) were the most common serotypes detected in children with diarrhea (3/7) and Ad7 the sero-type most detected in the controls (5/9), associated with lower respiratory tract infection. Thirteen adenovirus strains, isolated in HEp2 or HEK-293 cells, were characterized by seroneutralization and restriction enzyme analysis. The established adenoviruses were typed as AV-7-D5 (five associated to lower respiratory tract infection and one to diarrhea), AV-1-D10 (one diarrhea case), AV-31-D2 (two controls with respiratory infection), and two isolates as AV-12-D7, a new genome type. One subgenus D isolate, sero-type 28, with restriction patterns different from those of the prototype, remained untyped. Only one enteric adenovirus could be typed. The restriction patterns of this isolated were similar to those of the prototype AV-41-D1. The genome type of the other three enteric adenoviruses could not be determined. © 1995 wiley-Liss, inc.     

Seroprevalence and incidence of rubella in and around Delhi (1988-2002)

National Institute of Communicable Diseases (NICD) has been engaged in rubella testing for serodiagnosis of the infection and screening for immunity status. The compiled and evaluated data of the work done on rubella testing for the past fifteen years has been presented here to show the trend and changing scenario of the disease in Delhi. Blood samples were from 7424 patients referred to NICD, Delhi for serodiagnosis of congenital Rubella syndrome (CRS) in malformed babies, in utero rubella infection in women and immunity status of pregnant women and women with bad obstetric history. They were tested for rubella IgG and/or rubella IgM antibodies using commercially available reagents and kits. The data from the 15 years of testing was then compiled and evaluated. From the available data it was seen that immunity status against rubella in childbearing age group of women increased steadily from 49% in 1988 to 87% in 2002. Reported cases of CRS at NICD are also on the decline over the time period. There is periodic indication of high incidence of rubella in the year 1988; 1991 and 1998 as the reported cases of acute rubella infection in childbearing age group is high during these years.     

Comparison of various serological methods and diagnostic kits for the detection of acute, recent, and previous rubella infection, vaccination, and congenital infections

The antibody development after natural rubella infection and rubella vaccination has been followed in 802 sera from 493 patients and 71 sera from 22 vaccinees. Also examined were 67 sera from 28 infants with rubella embryopathy and sera from 50 children with presumed prenatal infection. In addition, 777 sera from 641 patients tested for routine rubella diagnosis were studied. Anamnestic information was available from all these patients. These sera were assayed for IgM antibody detection by sucrose density gradient (SDG), the commercial ELISAs (Enzygnost IgM and Rubazyme M), and the non-commercial anti-my-hemadsorption immunosorbent technique (HIT). For the determination of IgG antibodies the hemagglutination inhibition test (HAI), the commercial ELISAs (Enzygnost IgG, Rubazyme), and a single radial hemolysis test (SRH) were used. The SDG and HIT were less sensitive for IgM antibody detection than the two ELISAs, particularly when IgM concentrations were low. In total 26.5% of the IgM results with the newer tests were discordant with SDG, but only 0.5-1.3% of these results were not explicable when the clinical data was considered. Problems were encountered with all IgM assay systems used. For the detection of rubella antibodies after acute infection and vaccination the ELISA Enzygnost IgG was as sensitive as the HAI whereas the ELISA Rubazyme and SRH detected antibodies with some delay. Corresponding results with all tests were found more than 25 days after acute infection and more than 50 days after vaccination. All methods can be used for detection of antibodies in infants with rubella embryopathy. The results of this study suggest that certain combinations of tests can be used for the reliable detection of rubella infection.