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.     

Molecular epidemiology of rubella viruses involved in congenital rubella infections in São Paulo,Brazil, between 1996 and 2009

Rubella virus (RV) infection during the early stages of pregnancy can lead to serious birth defects, known as congenital rubella syndrome (CRS). This retrospective study was conducted between 1996 and 2009 with surveillance specimens collected from patients suspected of congenital rubella infection (CRI) and CRS. The clinical samples (nine amminiotic fluid, eight urine, eight blood, one conception product, and one placenta) were sent for viral isolation and genotyping. Twenty‐seven sequences were analysed and four genotypes (1a, 1B, 1G, and 2B) were identified in São Paulo that were involved in congenital infection. To our knowledge, this study is the first report that describes genetic diversity of the circulating rubella strains involved in CRI. J Med. Virol. 85:2034–2041, 2013. © 2013 Wiley Periodicals, Inc.

     

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.     

Application of new assays for rapid confirmation and genotyping of isolates of rubella virus

Rubella virus (RV) isolation is recommended by the WHO Measles and Rubella Labnet for studying the etiology and epidemiology of rubella. However, the absence of cytopathologic effects (CPE) in many of the cell lines used commonly makes it difficult to confirm RV growth. In this study, two assays amplifying RV cDNA were developed and validated in order to confirm and genotype RV isolates after cell culture. A SYBR Green I-based real-time PCR (Rtime-SGE317) was established for initial rapid detection of RV in Vero cells and a nested PCR (PCR-E860) was used for amplifying further the 739 nt window of the E1 gene for the identification of RV genotype as recommended by the WHO. Sensitivities of the two assays were evaluated using eight RV isolates, two from infants with the congenital rubella syndrome (CRS) and six from patients with acute rubella. All the isolates had cycle threshold (C(t)) values <37 after the third passage, which is recommended as the cut-off for the confirmation of a viable RV isolate. Phylogenetic analysis based on the 739 nt window generated by the PCR-E860 showed that the eight RV isolates belonged to genotypes 1E, 1G, and 2B. The Rtime-SGE317 assay can be carried out in local public health laboratories, which would extend the molecular surveillance of rubella and contribute to the WHO goal of eradicating rubella worldwide.     

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.     

The role of rubella-immunoblot and rubella-peptide-EIA for the diagnosis of the congenital rubella syndrome during the prenatal and newborn periods

Rubella infection during the first trimester of pregnancy can cause the congenital rubella syndrome (CRS). Patients with CRS were shown to have a decreased humoral and cellular immunity. It is not known whether asymptomatic newborns who had experienced intrauterine infection with rubella virus (RV) differ in their antibody response from newborns with CRS. In this study we compared both groups for a difference which might be a useful diagnostic criterion for CRS during the prenatal and newborn periods. We used the nonreducing Rubella-Immunoblot and the Rubella-IgG-Peptide-Enzyme Immunoassay (EIA) to determine the antibodies directed to rubella proteins E1, E2 and C. The results showed that only newborns with CRS who had experienced RV infection during the first 12 weeks of gestation showed significantly reduced levels of antibodies directed to both the linear RV E1 epitope (SP 15) and the topographic RV E2 epitope. Asymptomatic newborns infected mostly later than week 10 of gestation showed normal levels of antibodies. These data suggest that the lack of antibody response in CRS is linked to the immaturity of the fetal immune system during the first trimester of gestation. Rubella-IgG-Peptide-EIA and Rubella-Immunoblot should be used additionally for CRS diagnosis in the prenatal/newborn periods. These results may have an impact on the early treatment of late-onset symptoms of CRS patients. J. Med. Virol. 51:280–283, 1997. © 1997 Wiley-Liss, Inc.     

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.     

Humoral beta-cell autoimmunity is rare in patients with the congenital rubella syndrome

The congenital rubella syndrome (CRS) is associated with increased risk for diabetes and thyroid disease. However, the mechanisms by which the rubella virus may cause these diseases are poorly characterized. Previous studies were carried out before modern immunological methods were available. The present study aimed at evaluating whether autoimmune mechanisms are involved in the pathogenesis by analysing antibodies to biochemically characterized autoantigens. The incidence of clinical diabetes, thyroid disease, coeliac disease and related antibodies (islet cell antibodies, ICA; insulin autoantibodies, IAA; antibodies to the tyrosine phosphatase related IA-2 molecule, IA-2 A and glutamic acid decarboxylase, GADA; thyroid peroxidase, TPO; tissue transglutaminase, TTGA; and gliadin, AGA) and HLA risk genotypes were analysed in 37 subjects affected by or exposed to rubella during fetal life (mean age 22.5 years). One patient had diabetes and four patients had clinical hypothyroidism at the time of the examination. ICA, IAA, GADA or IA-2 A were not detected in any of the patients, while five patients tested positive for TPO antibodies. Coeliac disease or TTGA were not observed. Eight patients carried the HLA-DR3-associated HLA-DQB1*02-DQA1*05 haplotype. These results provide no evidence of an increased frequency of markers for humoral beta-cell autoimmunity in patients with CRS suggesting that diabetes in CRS may be caused by other than autoimmune mechanisms.     

The present situation and limitation of antibody assays for diagnosis of rubella virus infection in pregnant women]

Rubella virus infection during early stages of pregnancy often results in a number of developmental disorders referred to as congenital rubella syndrome(CRS). Both clinical and laboratory diagnosis of suspect cases of CRS can be made with relative ease, particularly when expectant mothers show the typical rubella-specific rash. Serological diagnosis of CRS is accomplished using hemagglutination inhibition (HI) and enzyme-linked immunosorbent(IgM-EIA) assays. Antibody titers as determined by these assays are generally very high following acute apparent rubella infections, thus making serological diagnosis relatively easy in most cases. However, the detection of possible CRS cases can be hampered by clinically inapparent rubella infections during early pregnancy. As much as 30 percent of all acute rubella cases are inapparent infections, and there is the very real potential for such inapparent infections to occur during pregnancy, to result in fetal infections, and consequently to cause CRS. Detection of CRS becomes extremely difficult in such settings. Complicating CRS detection even more are rare rubella re-infections that might occur in early pregnancy, and unknown risk of fetal infection and CRS. In re-infection cases, HI antibody titer becomes elevated due to a secondary immune response, and IgM antibody is produced in a significant number of cases. To determine directly the fetal infection, virus genome detection was developed and applied clinically for the past decade. Using a combination of serological and genomic detection methods, the results of the investigation suggest that when rubella infection during early pregnancy occurs 1) there is a significant risk of fetal infection that results from acute apparent rubella infection, 2) there is a measurable risk of fetal infection resulting from inapparent infections as defined by HI antibody titers > or = 256 and with and IgM-EIA index > or = 7, and 3) high HI antibody titers with low IgM-EIA indices or no detectable IgM antibody in cases of inapparent rubella infections may represent rubella re-infections and result in a low risk of fetal infections.     

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.     

Molecular surveillance of rubella viruses in Taiwan from 2005 to 2011

Rubella has been listed as a mandatory notifiable disease in Taiwan since 1988. Because of high coverage rates with an effective vaccine, rubella cases have decreased dramatically in Taiwan since 1994. However, rubella outbreaks still occur due to imported transmission. Five large clusters were detected in Taiwan from 2007 to 2011. In 2007, one cluster was caused by rubella genotype 1E viruses that were imported from Vietnam, whereas another cluster was caused by genotype 2B viruses and was untraceable. In 2008, two clusters were caused by different lineages of genotype 1E viruses that were imported from Malaysia. In 2009, a cluster that was caused by genotype 2B viruses was associated with imported cases from Vietnam. The rubella viruses from 124 confirmed cases from 2005 to 2011 were characterized, and the data revealed that these viruses were distributed in the following four genotypes: 1E (n = 56), 1h (n = 1), 1j (n = 4), and 2B (n = 63). Of these viruses, 93 (75%) were associated with imported cases, and 43 of 56 genotype 1E viruses were associated with imported cases from China, Vietnam, Malaysia, and Indonesia. One genotype 1h virus was imported from Belarus, and three of four genotype 1j viruses were imported from the Philippines. Of 63 rubella genotype 2B viruses, 46 were imported from Vietnam, Thailand, Malaysia, China, Germany, and South Africa. Molecular surveillance allows for the differentiation of circulating rubella viruses and can be used to investigate transmission pathways, which are important to identify the interruption of endemic virus transmission. J. Med. Virol. 85:745–753, 2013. © 2013 Wiley Periodicals, Inc.     

Genomic characterization of a persistent rubella virus from a case of Fuch’ uveitis syndrome in a 73 year old man

BackgroundMany cases of Fuchs’ uveitis have been associated with persistent rubella virus infection. A 73-year-old male patient with typical Fuchs’ Uveitis Syndrome (FUS) first experienced heterochromia of the left eye at the age fourteen, when rubella was endemic in the US.ObjectivesThe purposes of this report are to describe the patient’s FUS clinical presentations and to characterize the virus detected in the vitreous fluid.Study designThe patient underwent a therapeutic pars plana vitrectomy in May 2013. A real-time RT-PCR assay for rubella virus was performed on the vitreous fluid by Focus Diagnostics. Additional real-time RT-PCR assays for rubella virus detection and RT-PCR assays for generation of templates for sequencing were performed at the Centers for Disease Control and Prevention (CDC).ResultsThe results from Focus Diagnostics were positive for rubella virus RNA. Real-time RT-PCR assays at CDC were also positive for rubella virus. A rubella virus sequence of 739 nucleotides was determined and phylogenetic analysis showed that the virus was the sole member of a new phylogenetic group when compared to reference virus sequences.ConclusionsWhile FUS remains a clinical diagnosis, findings in this case support the association between rubella virus and the disease. Phylogenetic analysis provided evidence that this rubella virus was likely a previously undetected genotype which is no longer circulating. Since the patient had rubella prior to 1955, this sequence is from the earliest rubella virus yet characterized.     

Detection of measles,mumps, and rubella antibodies in saliva using antibody capture radioimmunoassay

Antibody capture radioimmunoassays were developed for detecting virus specific IgM (MAC-RIA) and IgG (GACRIA) to measles, mumps, and rubella and used to investigate saliva as an alternative specimen to serum for diagnosis. Saliva was collected from 63 patients with measles, 19 with mumps, and 150 with rubella, which were all clinically diagnosed and serologically confirmed. Virus specific IgM was detected in 92% of measles, 75% of mumps, and 100% of rubella saliva samples collected during the first week of illness. Between 1 and 5 weeks after onset virus specific IgM was detected in 100% of saliva specimens. After the 5th week the proportion of reactive specimens declined. The specificity of the MACRIA tests was established by testing saliva samples collected from blood donors for measles (88), mumps (88), and rubella IgM (91). All of the saliva specimens tested for measles and rubella specific IgM were unreactive, 1/88 specimens tested for mumps specific IgM contained significant reactivity. Saliva specimens collected from acute cases of MMR were tested in all 3 MACRIAs. A small proportion of saliva samples contained detectable IgM of more than one virus infection. Rubella and measles specific IgG was detected in the saliva of all cases from the 4th or 5th day of illness, respectively. Detection of mumps specific IgG was less successful. We have demonstrated that virus specific IgM can be reliably detected in saliva samples collected from acute cases of measles, mumps, and rubella and identified 1–5 weeks after onset of illness as the optimum time for collection of samples. © 1993 Wiley-Liss, Inc.     

Measurement of avidity of specific IgG for verification of recent primary rubella

Sixty-four subjects' serum samples, positive or equivocal by rubella IgM assays and containing rubella IgG, were examined for the avidity of rubella IgG. Four of the sera originated from rubella reinfections; others had false-positive IgM results due to interference by parvovirus infection or by other mechanisms; and the remaining were sera from the acute phase or convalescence of primary rubella. A novel IgG avidity test, avidity-ELISA, and a semiquantitative haemolysis typing assay were used. According to the avidity-ELISA, 29 subjects had recent primary rubella (low IgG avidity), and another 29 had previous rubella immunity (high IgG avidity), whereas 6 serum samples gave borderline avidity values. Comparison of these results with pre-existing clinical records and laboratory data showed that all samples with low IgG avidity were obtained during or shortly after acute primary rubella. All sera with high IgG avidity originated from the previously immune subjects; the rubella reinfections were confined within this group. Five of the six sera with borderline avidity values were obtained within 2 months from primary rubella. In conclusion, the measurement of IgG avidity is a powerful tool for the distinction of acute or recent primary rubella from pre-existing rubella immunity, including rubella reinfections.     

Clinical evaluation of a rubella passive hemagglutination test system.

A recently marketed passive hemagglutination (PHA) test (Rubacell, Abbott Laboratories) was compared to the hemagglutination inhibition (HI) test for the detection of antibody to rubella virus. The performance of the PHA system in determining immunity to rubella was evaluated by comparing the PHA results and HI results on 1, 086 randomly selected sera submitted for routine premarital and prenatal testing. Of the 1, 079 specimens assayable by both procedures, 1, 053 of the results (97.6%) were in agreement on initial testing. When the 26 initially discrepant specimens were retested for clarification, there was final agreement in 1, 067 of the specimens (98.9%). Twelve specimens were classified as persistently discrepant (five were not retested by PHA) and seven were unassayable by HI.Seven of the specimens with discrepant results were PHA positive and HI negative, and five were PHA negative and HI positive. Discussion of the two tests with respect to technical difficulty, cost, and controls is also included.     

Laboratory-based investigation of fever with rash cases in the Maharashtra State - India, 2014 to 2017

The Maharashtra State (MS), India, launched measles surveillance in the year 2013. From 2014 to 2017, specimens from the patients presenting with fever and skin rashes were received at the National Institute of Virology, Pune. From 36 districts of the MS, 2795 cases (1428 males and 1367 females) were referred for the laboratory diagnosis of measles and rubella using immunoglobulin M enzyme immunoassay and/or RT-PCRs. The majority of the cases (93.3%, n = 2609) were under 15 years of age. About 17.7% (494) cases had a history of measles immunization (one dose) during their childhood. Virus isolation was attempted from 107 throat swabs and 84 urine samples obtained from 191 cases using Vero hSLAM cells. The results confirmed measles in 1756 and rubella in 282 fever with rash cases by serological and molecular tools. Precisely, 170 of 382 and 35 of 149 specimens were positive for measles and rubella RT-PCRs, respectively. Sequencing of the representative PCR products showed the circulation of measles virus genotypes D4 (n = 26) and D8 (n = 107), and rubella virus genotype 2B (n = 1). Twenty-three measles viruses were isolated and genotyped, of which, 6 were D4 and 17 were D8 genotypes. Amongst the measles-immunized individuals, 51.2% (253/494) had laboratory-confirmed measles. Overall, 72.9% fever with skin rash cases (n = 2038) from the MS was laboratory confirmed for either measles or rubella. The contribution of measles was higher than rubella in the fever with rash cases. As expected, more fever with rash cases were documented in children compared with adults and highlighted the need to increase measles-rubella immunization coverage.     

Institutional outbreak of rubella in a healthcare center in Chandigarh,North India

Rubella is traditionally considered a childhood disease but it has the potential to cause outbreaks in closed communities when a susceptible population accumulates. The present study reports an outbreak of rubella among healthcare workers in the pediatric center of a tertiary care North Indian hospital. The cases of rubella were identified by clinical features and confirmed by the detection of anti‐rubella IgM antibodies in blood by enzyme linked immunosorbent assay. A total of 23 cases of rubella occurred over a period of one and a half month, out of which 9 (39%) were males. All the patients were in the age group of 21–35 years. None of the patients gave a history of rubella vaccination. This outbreak of rubella occurred due to the accumulation of a susceptible population in a closed hospital environment. There is need for the introduction of rubella vaccination in healthcare workers to prevent outbreaks at work place. J. Med. Virol. 82:341–344, 2010. © 2009 Wiley‐Liss, Inc.     

A comparison of antibody capture radio- and enzyme immunoassays with immunofluorescence for detecting IgM antibody in infants with congenital rubella

IgM antibody capture radioimmunoassay (MACRIA) and enzyme immunoassay (MACEIA) were compared with immunofluorescence (IF) for detecting specific IgM antibody in 99 sera from 76 infants with confirmed congenital rubella, and 61 sera from a comparative group of 59 infants who had miscellaneous abnormalities but in whom congenital rubella was not confirmed.All of 35 specimens collected from confirmed cases within 12 weeks of birth were positive by all three methods and all but one of 17 specimens collected after the age of 18 months were uniformly negative. At intermediate ages discrepancies occurred in 18 specimens, of which eight were positive and 10 negative by IF. Three of these 18 specimens were negative by both antibody capture procedures but showed weak fluorescence; the other 15 were negative by MACEIA, but positive by MACRIA which appears to be the more sensitive of the antibody capture methods. Sera from five infants in the comparative group were clearly positive by all three methods. These five infants were probably congenitally infected with rubella. Sera from the other 54 infants were negative, except for one that gave a weakly positive result by MACRIA alone.Antibody capture procedures offer several advantages over previous methods for detecting IgM antibody. Although MACRIA was found to be slightly more sensitive than MACEIA, the greater stability of the enzyme label, together with the possibility of both visual and quantitative assessment, could make MACEIA the method of choice for detecting rubella-specific IgM.