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.     

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.     

Evaluation of a commercial rubella IgM assay for use on oral fluid samples for diagnosis and surveillance of congenital rubella syndrome and postnatal rubella.

BACKGROUND: Clinical diagnosis (surveillance) of rubella is unreliable and laboratory confirmation is essential. Detection of virus specific IgM in serum is the most commonly used method. However, the use of serum necessitates the drawing of blood, either through venipuncture or finger/heel prick, which can be difficult in young babies. Oral fluid samples have proved useful as an alternative, less invasive sample for virus specific IgM detection however until recently no commercial rubella IgM tests were available, restricting the usefulness of this approach. OBJECTIVES: To evaluate the performance of the Microimmune Rubella IgM capture EIA using oral fluid samples from outbreaks as well as in cases of suspected congenital rubella syndrome (CRS). STUDY DESIGN: Paired serum and oral fluids were collected from cases during a rubella outbreak in three provinces in Turkey. Matched serum and oral fluid samples were collected from children with suspected CRS in an active surveillance programme at the Aravind Eye Hospital in South India. Serum samples were collected as part of the measles surveillance programme in Ethiopia. RESULTS: On serum samples the sensitivity and specificity of the Microimmune Rubella IgM capture EIA compared to Behring Enzygnost rubella IgM test was 96.9% (62/64; 95% CI 94.2-100%) and 100% (53/53; 95% CI 93.2-100%). On oral fluids compared to matched Behring results on serum the sensitivity was 95.5% (42/44; 95% CI 84.5-99.4%). The sensitivity and specificity of Microimmune Rubella IgM capture EIA on oral fluids from suspected CRS cases compared to serum results using Behring Enzygnost IgM assay was 100% (95% CI 84.5-100%) and 100% (95% CI 95.8-100.0%) respectively. CONCLUSION: Microimmune Rubella IgM capture EIA has adequate performance for diagnosis and surveillance of rubella in outbreak using either serum or oral fluid specimens.     

Primary investigation of 31 infants with suspected congenital rubella syndrome in Sudan

Between 2005 and 2006, clinical specimens were collected from 31 infants with suspected congenital rubella syndrome (CRS) who presented at six hospitals in Khartoum, Sudan. Eleven (35.5%) were laboratory confirmed as CRS cases by testing for anti-rubella IgM, IgG and viral genome. For the first time in Sudan, the rubella virus genome was directly detected in clinical specimens of six CRS cases and two viruses were isolated in cell culture. Phylogenetic analysis suggested that three genotypes of rubella virus (RV; 1E, 2B and 1G) were co-circulating in Sudan. The study introduced the methodology for CRS confirmation and surveillance in Sudan and provides preliminary data.     

Evaluation of rubella IgM enzyme immunoassays.

BACKGROUND: Rubella virus generally causes a mild fever, rash illness similar in clinical presentation to infections by other viruses including measles and parvovirus B19. Rubella infections in pregnant women in the first trimester carry a high risk of congenital rubella syndrome (CRS) which can result in severe congenital defects in the infants. The goal of rubella immunization programs is therefore to eliminate CRS. The primary test for the laboratory confirmation of rubella is IgM serology. It is therefore important to evaluate currently available commercial rubella IgM immunoassays to ensure high quality rubella diagnostic testing. STUDY DESIGN: In this study, we compared the performance of seven commercial rubella IgM enzyme immunoassays (EIA) (Meddens, Denka Seiken, Behring, Wampole, Captia, Sigma and Abbott Axsym) using well-defined panels of sera from rubella and non-rubella/rash-illness cases. RESULTS: The Meddens, Denka Seiken, Behring and Wampole rubella IgM EIAs all performed similarly for sensitivity (range of 74.1-76.8%) and specificity (range of 93.9-96.1%). Relative to the other assays, the Axsym had a higher sensitivity (78.9%) but lower specificity (86.5%). The Captia assay had the lowest overall sensitivity (66.4%), while the Sigma assay had a lower specificity (85.6%) in relation to the other assays. CONCLUSIONS: In conclusion, the Meddens, Denka Seiken, Behring and Wampole rubella IgM EIAs are comparable in their overall performance with respect to sensitivity and specificity.     

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.     

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.     

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.

     

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.     

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.     

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.     

孕前及孕期妇女风疹免疫力调查和风疹近期感染检测

目的了解孕前妇女及孕期妇女风疹易感水平和近期感染状况，为预防先天风疹感染做好优生优育工作提供依据。方法用生物蛋白芯片技术对2986例孕前妇女及863例孕期妇女（包括725例正常妊娠和138例异常妊娠）进行风疹特异性抗体IgM、IgG检测。结果80．27％的孕前妇女具有免疫力，4．99％孕前妇女为风疹近期感染，14．03％妇女对风疹易感。89．00％的孕期妇女具有免疫力，1．96％孕期妇女为风疹近期感染，7．76％妇女对风疹易感。孕前妇女及孕期妇女的风疹近期感染率和易感率比较有显著性差异（χ^2=14．797、23．846，P〈0．001）；不良妊娠与正常妊娠之间风疹近期感染率和易感率比较有显著性差异（X。=30．635、4．174，P〈0．05）；市区与农村的孕前妇女及孕期妇女在风疹近期感染率和易感率比较无明显不同；孕前妇女及孕期妇女风疹近期感染月份分布均以5月最高。结论本地区孕前妇女风疹感染率高；风疹感染与不良妊娠密切相关：市区与农村妇女的风疹易感水平和近期感染状况相当。对孕前妇女及孕期妇女同时检测风疹特异性抗体IgM、IgG可以正确判定妇女的免疫状况，有效预防、主动发现并合理处理孕妇感染，从而降低风疹感染率提高优生优育水平。     

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.     

Assessment of the diagnostic value of RT-PCR on amniotic fluid for prenatal diagnosis of congenital rubella infection

AIM OF THE STUDY: To assess the diagnostic value of RT-PCR on amniotic fluid (AF) for prenatal diagnosis of congenital rubella infection. MATERIAL AND METHODS: RT-PCR on AF was compared to specific IgM antibody detection in foetuses and/or newborns in 45 pregnant women with confirmed primary infection. RESULTS: specificity of RT-PCR was 100% and sensitivity ranged between 83 and 95%. CONCLUSION: RT PCR may be considered as a valuable tool for prenatal diagnosis of foetal rubella infection.     

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.     

Evaluation of the new ARCHITECT Rubella IgM assay.

BACKGROUND: Rubella infections are usually characterized by mild self-limiting courses in immunocompetent individuals. However, infections in pregnant women during the first trimester of pregnancy pose a high risk of congenital rubella syndrome possibly resulting in severe defects in the unborn child. Rubella serology of a primary rubella infection is mainly determined by diagnostic confirmation of levels of specific IgM. STUDY DESIGN: Here, we report on the performance of the Rubella IgM assay in development on the ARCHITECT instrument, a fully automated high throughput chemiluminescent microparticle immunoassay platform. Sensitivity was examined using commercially available seroconversion panels from vaccinated individuals; specificity was addressed by testing populations of pregnant women, blood donors and hospitalized patients. In addition, the potential for assay interference was evaluated by testing samples of several disease states. As methods of comparison AxSYM, BioMérieux VIDAS and Behring Rubella IgM assays were used. RESULTS: The study demonstrates that the ARCHITECT Rubella IgM assay shows improved specificity compared to AxSYM and Behring. Seroconversion sensitivity is equivalent on all assays evaluated. CONCLUSION: Together with high throughput, optimized specificity and suppression of rheumatoid factor (RF) interference the ARCHITECT assay provides a useful improvement for the diagnosis of rubella serology.