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Rubella and congenital rubella syndrome (CRS) continue to be important health problems in many countries. In June 2004, the World Health Organization Steering Committee on Research Related to Measles and Rubella Vaccines and Vaccination met to evaluate data from research and operational activities and to identify critical scientific issues and gaps in knowledge that need to be addressed to improve the global control of rubella and CRS. Information about surveillance for rubella, natural and vaccine-induced immunity to rubella, laboratory diagnosis, the molecular epidemiological profile of rubella virus, and mathematical modeling to assess the burden of CRS and the impact of rubella vaccination was reviewed. This report summarizes the presentations and recommendations for future research.

Background: Until rubella is eradicated there will be a continuing need for rubella antibody surveillance. Antigen production using recombinant DNA technology may be a viable alternative to traditional techniques of producing antigens for enzyme immunoassays (EIAs). Objectives: To investigate the potential of bacterial fusion proteins containing rubella E1 protein sequences for use in EIAs to detect rubella antibodies. Study design: Purified bacterial fusion proteins containing rubella E1 sequences to be used as antigens in EIAs and compared to ‘traditional' assays using virus derived antigens for rubella antibody screening. Results: cDNA clones coding for the complete rubella E1 protein sequence and subfragments of E1 were modified for expression as carboxy terminal fusions with either β-galactosidase or glutathione-S-transferase. β-galactosidase fusions with the complete E1 coding sequence or amino acids 201 to 307, which contain known epitopes, resulted in the production of predominantly insoluble fusion proteins unsuitable for use in EIA. Nine glutathione-S-transferase-E1 fusion proteins were produced with individual fusion proteins exhibiting varying properties with regard to the levels of protein produced, apparent stability, solubility and the potential for affinity purification using glutathione agarose. Reduction of the E1 component to only 44 amino acids containing three B-cell epitopes (Terry et al. , 1988) produced an abundant soluble GST-E1 fusion protein (3.5 μg/ml), which could be affinity purified using glutathione agarose. This fusion protein has been successfully used in EIA to detect rubella antibodies. Conclusions: We have shown that GST-E1 fusions have potential as antigens in tests for rubella antibodies.

Without appropriately timed specimens, serological confirmation of congenital rubella infection may be a problem. We have compared the persistence of specific IgM and low avidity specific IgG1 in 141 sera from 120 cases of serologically confirmed congenital rubella infection with the known time scales for postnatal primary rubella. The results demonstrate that the maturation of the immune response to the rubella virus is abnormally slow in congenital rubella cases both in terms of the isotype switch and especially the development of high avidity specific IgG1. Thus avidity studies may permit serological confirmation of congenital rubella for longer than is possible with tests currently in use. The pathological implications of prolonged low avidity antibody production are discussed.

Persistence of specific IgM in sera following primary rubella infection was compared with the maturation of the specific IgG1 response. 206 sera, from 171 patients with primary rubella, taken 1 day to 2.5 years after onset of illness, were tested. Rubella-specific IgM was detected by M-antibody capture radioimmunoassay in 100% of sera taken 15–28 days after onset, but in only 9% taken 3–4 months after onset. However, using the diethylamine (DEA) shift value (DSV) method, low avidity specific IgG1 was detected in 91% sera taken at 3–4 months and at 5–7 months 21% of sera remained positive. Using an avidity index method, with urea in the wash buffer, none of the sera were positive for low avidity specific IgG1 beyond 3 months after onset. With DEA in the wash buffer, the number of sera positive rose to 38% at 3–4 months. Thus, the DSV method for detecting low avidity specific IgG1 is a useful additional test for confirming or refuting a diagnosis of primary rubella and is of particular value for assessing pregnant patients.

Synovial fluid samples and/or biopsies from 79 patients with various chronic inflammatory joint diseases or traumatic joint injury were tested for rubella virus (RV) in order to confirm or refute results from other studies that suggested RV as a cause of chronic inflammatory joint disease. Sixty-eight of the 72 patients tested had RV antibodies. RV RNA was detected by reverse transcription-PCR in the synovial fluid cells from two patients. RV was also isolated by cell culture from the synovial fluid of one of these two patients. This patient was a 42-year-old female with common variable immune deficiency and Mycoplasma hominis arthritis, while the other was a 68-year-old female with rheumatoid arthritis. While these results fail to confirm that RV is associated with chronic inflammatory joint disease, they suggest that RV may persist within a joint and be reactivated when cell-mediated immunity is suppressed.

Rubella is associated with an 80% risk of congenital abnormalities if acquired in the first 12 weeks of pregnancy. Reinfection in early pregnancy presents a much smaller risk. Prenatal diagnosis may be useful to assess the risk to the fetus. Congenital rubella is a progressive disease and some abnormalities will not be present at birth. Rubella and congenital rubella are usually diagnosed by detection of rubella-specific IgM; it may be difficult to confirm a diagnosis of congenital rubella in children over 3 months of age. Rubella vaccines are usually combined with measles and mumps vaccines. Their use has enabled some industrialised countries to eliminate rubella and congenital rubella. Countries should ensure that susceptible women of child-bearing age and health care workers are offered a rubella-containing vaccine. Rubella vaccine is contraindicated during pregnancy, but if a pregnant woman is inadvertently vaccinated it is not an indication for termination or prenatal diagnosis.

International audience; The WHO Steering Committee reviewed and evaluated the progress towards global control of measles and rubella and provided guidelines for future research activities concerning both diseases during its meeting in New Delhi, in April 2005. Global measles vaccination coverage increased from 71% in 1999 to 76% in 2004 and indigenous transmission was interrupted or kept at very low levels in many countries. However, Africa and Southeast Asia continue to experience endemic transmission and high mortality rates, despite a global mortality reduction of 39% between 1999 and 2003. On the basis of reports from countries with continued indigenous measles virus transmission, future control strategies as well as advantages and potential drawbacks of global measles eradication were discussed. Similarly the burden of rubella and congenital rubella syndrome (CRS) as well as the cost-effectiveness of rubella vaccination was assessed using different methods in several countries without vaccination programs. As measles and rubella viruses continue to circulate surveillance and control strategies need further optimization. RT-PCR was considered as an alternative method for laboratory diagnosis of CRS. The value of dried blood spots and oral fluid as alternative samples for measles and rubella IgG and IgM detection and genotype determination was evaluated. However further validation of these methods in different settings is required before their routine use can be recommended.

Publisher Summary This chapter presents a range of serological methods for the diagnosis of Rubella and congenital Rubella. Detection of rubella-specific IgM remains the method of choice for the diagnosis of both postnatally and congenitally acquired rubella, but isolation of virus and detection of viral RNA by nested reverse transcription-polymerase chain reaction (RT-nPCR) are useful for the diagnosis of congenital rubella in the fetus and newborn infant. Detection of rubella IgM in a serum taken 3–6 days after onset of rash is the method of choice for diagnosis of acute rubella. Although specific IgM may be detected earlier in some patients, this will depend on the assay used. If no specific IgM is detected in a serum taken

Prevalence of antibodies to measles and rubella was tested in sera collected from 1368 subjects in urban and rural Sana'a. Overall, 11.7% had no antibodies to measles and 14.6% had no antibodies to rubella, despite the fact that measles but not rubella vaccine is included in the vaccination program in Yemen. Of 89 children

E1 gene nucleotide sequences of 63 rubella virus isolates from North America, Europe, and Asia isolated between 1961 and 1997 were compared phylogenetically. Two genotypes were evident: Genotype I contained 60 viruses from North America, Europe, and Japan, and genotype II contained 3 viruses from China and India. The genotype I isolates prior to 1970 grouped into a single diffuse clade, indicating intercontinental circulation, while most post-1975 viruses segregated into geographic clades from each continent, indicating evolution in response to vaccination programs. The E1 amino acid sequences differed by no more than 3%; thus, no major antigenic variation was apparent. Among 4 viruses from congenital rubella syndrome that occurred following reinfection, only one amino acid substitution occurred in several important epitopes, indicating that antigenic drift is not important in this phenomenon. However, 2 viruses isolated from chronic arthritis exhibited changes in these epitopes. Isolates of the RA 27/3 vaccine strain were readily identifiable by nucleotide sequence.

A reverse transcription-nested PCR assay (RT-PCR) was evaluated for diagnosis of congenitally acquired rubella in utero and during infancy. RT-PCR was compared with virus isolation for retrospective detection of rubella virus in placental and fetal tissues obtained after termination of pregnancy following primary rubella or rubella virus reinfection. Concordant results were obtained for 85% of samples; rubella virus RNA was detected by RT-PCR alone in four samples, and rubella virus was detected by isolation alone in two samples. Samples were also obtained for prenatal diagnosis of congenital infection; rubella virus RNA was detected in three of seven chorionic villus samples and one of three amniotic fluid samples by RT-PCR, while rubella virus was isolated in only one chorionic villus sample. To demonstrate that the RNA extracted from chorionic villus samples contained amplifiable RNA, a nested RT-PCR was used to detect keratin mRNA. Rubella virus was detected in placenta in two cases in which the fetus was uninfected, and there was no evidence of rubella virus in the placenta from one case in which the fetus was infected. Thus, detection of rubella virus in chorionic villus samples by RT-PCR may not always correctly predict fetal rubella virus infection. RT-PCR was successfully used for the diagnosis of congenitally acquired rubella in infancy. Rubella virus RNA was detected in cyropreserved or formalin-fixed lens aspirates obtained from infants in India with serologically confirmed congenital rubella but not in samples from controls with inherited cataract.

A reverse transcription nested PCR (RT-PCR) assay for the detection of rubella virus RNA using primers from the E1 open reading frame was established. This assay was found to be sensitive (detecting approximately two synthetic RNA copies and RNA extracted from 0.1 50% tissue culture infective dose of rubella virus) and specific; five wild-type rubella strains and four vaccine strains were detected, and no nonspecific amplification of 16 other RNA viruses or RNAs from seven cell types occurred. Rubella virus RNA was detected in 12 pharyngeal swabs from patients with serologically confirmed rubella; these RT-PCR results were in complete agreement with virus isolation. Analysis of products of conception obtained after confirmed primary maternal rubella infection by RT-PCR gave 92% agreement (12 of 13 samples) with virus isolation. No false-positive results were obtained. The potential use of this assay for prenatal diagnosis of congenital rubella infection and for investigating aspects of the pathogenesis of chronic disease is discussed.

Background: Maternal rubella infection in early pregnancy has a high probability of causing congenital rubella infection. In some cases it may be difficult to establish the risk of congenital infection and polymerase chain reaction (PCR) based techniques are therefore being applied to prenatal diagnosis. Objectives: To investigate whether the non-structural region of the rubella virus (RV) genome can be detected in clinical samples using PCR, thereby providing a prenatal assay independent of those currently used to detect the structural protein coding region. Study Design: Oligonucleotide primers coding for RV nucleotides 1–17 and 541–558 from the non-structural protein coding region of the RV genome were used in a reverse transcription polymerase chain reaction (NS RT-PCR) to amplify 558 nucleotides of RV cDNA. Amplification of RV specific sequences was confirmed by Southern hybridization. Results: The specificity of the assay was confirmed by the detection of RV RNA from both wild-type and vaccine strains of RV, pharyngeal swabs from two adult males with acute rubella and products of conception from three women with serologically confirmed primary rubella in pregnancy. RV RNA was not detected in uninfected HEL and Vero cells or peripheral blood mononuclear cells. The results were concordant with those of an RT-PCR directed to the E1 protein coding region and with virus isolation. Conclusions: Detection of the non-structural coding region of the RV genome in clinical samples suggests that NS RT-PCR could be used as a confirmatory assay for prenatal diagnosis of congenital rubella, and that it will be of value for the identification of nucleotide changes in the 5′ region of the RV genome.

Two glutathione S-transferase fusion proteins containing 44 (p1503) and 75 (p1509) amino acid residues of the rubella virus E1 glycoprotein were expressed in Escherichia coli with the aim of producing a recombinant rubella virus antigen for use in serological assays. p1503 contained three neutralizing and hemagglutinating epitopes (G. M. Terry, L. M. Ho-Terry, P. Londesborough, and K. R. Rees, Arch. Virol. 98:189-197, 1988); p1509 contained the putative neutralization domain described by Mitchell et al. (L. A. Mitchell, T. Zhang, M. Ho, D. Decarie, A. Tingle, M. Zrein, and M. Lacroix, J. Clin. Microbiol. 30:1841-1847, 1992) in addition to the three epitopes present in p1503. Both fusion proteins were soluble and affinity purified on glutathione-Sepharose 4B. In Western blots (immunoblots), p1503 and p1509 reacted with human sera containing rubella virus-specific immunoglobulin G. When used as antigens in indirect enzyme immunoassays to detect rubella virus-specific immunoglobulin G, p1503 correctly identified the rubella virus antibody status of 43 (76.8%) and p1509 correctly identified that of 48 (85.7%) of 56 serum samples received for routine rubella virus antibody screening. The results obtained with p1509 compare well with those obtained with a latex agglutination assay.

EDITOR, - Those responsible for students' health (for example, occupational health departments) frequently seek advice about immunity to viral infections among medical students. We therefore tested serum obtained between 1988 and 1990 from 199 first year students at the United Medical and Dental Schools of Guy's and St Thomas's Hospitals (St Thomas's campus) for antibodies to measles, mumps, rubella, herpes simplex, varicella zoster, and hepatitis A viruses and to cytomegalovirus. The table gives the techniques used and results obtained. The prevalence of antibodies to rubella virus among our female students (99%) confirms the success of …

Clinical and laboratory expertise is essenrial for evaluating rubella specific IgM test results in pregnancy

Background: Rubella virus may be transmitted to the fetus following rubella reinfection in early pregnancy. The mechanisms responsible for maternal-fetal transmission in women with pre-existing rubella antibodies are, however, unclear. Objectives: To evaluate the protective role of rubella specific neutralising antibodies and cell-mediated immunity in rubella reinfection. Study design: Rubella Nt antibodies were measured in sera obtained from: women who experienced rubella reinfection during pregnancy, volunteers experimentally challenged with rubella vaccine and women attending for routine rubella antibody screening. Cell-mediated immunity was assessed by measuring rubella specific LT responses among women who had experienced rubella reinfection during pregnancy. Results: Some women experienced rubella reinfection during pregnancy despite the presence of Nt antibodies in sera obtained before contact. Only 2 of 13 (15.3%) volunteers with low levels (< 15 IU/ml) of rubella antibody had detectable Nt antibodies prior to experimental challenge. Despite this, they did not develop rubella viraemia following challenge and virus excretion was detected in only one case, although the majority (1113, 84.6%) demonstrated a significant rise in antibody titre. Among pregnant women screened as having a low level of rubella antibody antenally, only 37% had detectable Nt antibodies. Among 23 women who experienced rubella reinfection during pregnancy 4 months to 6 years (mean 32 months) previously, 20 (86.9%) had a positive rubella-specific LT response. Only one of 4 cases where rubella virus was transmitted to the fetus failed to produce a a specific LT response. Conclusions: This study indicates that rubella reinfection is not associated with a lack of Nt antibodies or persistent impairment of rubella-specific LT responses.

A rubella-specific lymphocyte transformation assay, using cryopreserved mononuclear cells, has been developed and used to evaluate specific responses among 21 children with congenitally acquired rubella (CAR), 25 healthy control children and 10 children with sensorineural deafness of unknown aetiology. Although all 21 children with CAR were seropositive, 12 (57.1%) failed to respond to rubella antigen in the transformation assay. Negative in vitro lymphocyte transformation responses were detected significantly more frequently among congenitally infected children below 3 years of age. Thirteen of the 25 (52%) control children were seropositive; only one of these seropositive children (7.6%) gave a negative transformation response. A negative rubella-specific lymphocyte transformation response in a seropositive child, particularly when aged 3 years or younger, is therefore suggestive of CAR. Four of the 10 children with deafness of unknown aetiology were rubella seropositive but gave negative responses in the transformation assay, suggesting that these children had CAR. Our assay may provide a very useful test for retrospective diagnosis of CAR, particularly in children under the age of 3.

The E1 and E2 glycoprotein coding regions of the rubella genome have been cloned, sequenced and modified for expression in mammalian cells as both membrane bound and secreted proteins.

The association between rubella in pregnancy and congenital anomalies was first reported 50 years ago, by N. McAlister Gregg, an Australian ophthalmologist [1]. During the next 20 years his findings were confirmed by others (reviewed in [2]). However, the first reports of the isolation of rubella virus in cell cultures and development of tests for neutralizing antibodies were not published until 1962 [3, 4]. Subsequent studies conducted in the UK and North America during a pandemic of rubella in 1963–4, were therefore able to make a more accurate estimate of the risks of maternal rubella at different stages of pregnancy. It was estimated that about 30000 rubella-damaged babies were born in the USA alone in 1963–4 [5]. This emphasized the importance of developing a vaccine to prevent infection in pregnancy and thereby, the birth of babies with rubella-induced congenital defects.

Five cases of asymptomatic maternal reinfection with rubella are described that occurred in England and Wales during 1985-8 and resulted in intrauterine infection. The criteria for diagnosing reinfection are described. In four cases the rubella contact was with the woman's own children. Two women had therapeutic abortions, rubella virus being recovered from the products of conception, and three were delivered of infants with congenitally acquired disease. Though the risks associated with maternal reinfection with rubella are very small and being measured in a prospective study, it is hoped that the recently introduced augmented programme of rubella vaccination will reduce rubella in the community and therefore this small risk still further.

Sera was obtained from patients with squamous carcinoma of the cervix from Great Britain (29), Sri Lanka (32), Malawi (27), and Sudan (27), and from controls from all countries except Sudan. Controls were matched for ethnic origin, age and social class. Sera were tested by indirect immunofluorescence (IF) for IgG and IgA antibodies to membrane antigens (MA) and IgA antibodies to VCA of HSV-2 infected cells. Compared with controls, IgA antibodies to MA (IgA anti-MA) were detected more frequently and at higher titres in all groups of patients. However, there was no significant difference in prevalence of these antibodies at titres greater than or equal to 1:4 between Malawian patients and controls, although a significantly higher proportion of patients had IgA anti-MA titres of greater than or equal to 1:16. In contrast, IgA anti-VCA did not distinguish patients from controls. More than 90% of both patients and controls from all countries had IgG antibodies to MA (IgA anti-MA). Malawian patients had a significantly higher geometric mean titre (GMT) of IgG anti-MA than controls and both patients and controls had significantly higher GMTs than their counterparts from other countries. The variation between herpes IgG anti-MA titres in subjects from different countries did not reflect differences in serum immunoglobulin levels and similar variations in titre were not seen in rubella and measles HAI titres. Among the patients there was a geographical variation in the prevalence of HSV-2 specific antibodies detected by ELISA, which varied from 52% among British and Sudanese patients to 73% among Malawian patients. Even when adjustment was made for possible false negative results, there were between 10 and 31% patients without HSV-2-specific antibodies, although only 2 of 103 (1.9%) patients had neither HSV-1 nor HSV-2 antibodies. The association of HSV-2 with cervical carcinoma appeared to vary with age.

Radioimmunoassay (RIA) and single radial hemolysis (SRH) were used to measure the persistence of rubella antibodies in 123 women who had received one of four rubella vaccines (Cendehill, HPV-77 DE-5, RA27/3, and To-336) six to 16 years earlier. Only two (1.6%) vaccinees had no antibodies detectable by RIA, although 19 (15.4%) had very low levels (less than 15 IU/ml) detectable by SRH. Rubella-specific IgA persisted in sera for seven to nine years in the majority of 43 vaccinees from whom serial samples were obtained, and rubella-specific nasopharyngeal IgA could be detected up to two years after natural infection and vaccination in all vaccinees. Rubella-specific serum IgM responses were detected in 41 of 43 vaccinees and persisted at low levels for up to three years in seven of them, four of whom had received HPV-77 DE-5 vaccine. After 31 volunteers with antibody levels of less than 15 IU were challenged intranasally with RA27/3, viremia was detected in one, a girl who had a history of vaccination at school.

Attempts were made to correlate virus excretion, joint symptoms and antibody response with human leukocyte antigens (HLA) in seronegative adult women given attenuated rubella vaccine. No association was shown between HLA antigens of the A and B loci and excretion of either high or low titres of RA27/3 vaccine among 26 volunteers. However, virus excretion was influenced by such factors as the time of day at which specimens were collected and the method of virus isolation. Our study therefore failed to confirm the hypothesis that certain persons are good ‘spreaders’ of rubella virus and that this capacity is associated with HLA-A1 and B8.The study of joint symptoms following vaccination with Cendehill, HPV77. DE-5, RA27/3 or To-336 vaccines showed no association between such symptoms and HLA antigens. However, joint symptoms occurred within 7 days of the onset of menstruation in 33 of 47 (70%) vaccinees (P < 0.01) and it is therefore suggested that hormonal factors must play a role. No association between HLA antigens and haemagglutination inhibition (HAI) antibody titres, 8 weeks after vaccination with RA27/3, was found amongst 34 volunteers.

A total of 142 seronegative volunteers were given one of the following rubella vaccines: Cendehill, HPV77. DE-5, RA27/3, or a new Japanese vaccine, To-336. To-336 vaccine produced a slightly higher geometric mean antibody titre (G.M.T.) (65.7) than did the HPV77. DE-5 (63.1) or RA27/3 vaccine (61.9) but the G.M.T. induced by Cendehill vaccine was much lower (39.3).Reactions, particularly joint symptoms, occurred least commonly after vaccination with To-336 vaccine. Joint symptoms occurred within seven days of menstruation in 30 out of 37 (81%) vaccines (P

Serial samples of serum and nasopharyngeal washings were obtained from 43 volunteers given one of four rubella vaccines (HPV77.DE5, RA27/3, To-336, and Cendehill) and from nine naturally infected volunteers. Rubella-specific serum IgG was detected by radioimmunoassay for up to 12 years in all but one vaccinee, and booster responses occurred in 23.3% of vaccinees. Rubella-specific serum IgA was detected in 37 (90.2%) of 41 vaccinees one year after vaccination but in only five (45.5%) of 11 vaccinees tested 10-12 years after vaccination. Low levels of rubella-specific IgM detected by M-antibody capture radioimmunoassay persisted in seven volunteers--four of them HPV77.DE5 vaccinees--four more than one year after vaccination. Rubella-specific nasopharyngeal IgA was detected for up to five years after natural infection or vaccination with RA27/3 but for no longer than three years among Cendehill, HPV77.DE5, and To-336 vaccinees. Nasopharyngeal IgG antibodies were detected less frequently and at lower levels.

SummaryTwo strains of rubella virus multiplied in organ cultures of human embryonic trachea, nasal epithelium, pharynx, larynx, skin and brain derived from foetuses aged 15–28 weeks. Growth curve experiments conducted on cultures of nasal epithelium and trachea showed that virus appeared in the culture fluid 72 hr. after inoculation and thereafter rose to titres varying from 10 to 103·7TCD50/ml. These titres persisted for periods up to 34 days after inoculation. Intracelmlar and organ culture fluid virus titres were shown to be similar in specimens tested in both the early and late stages of the growth curve. No degenerative changes or loss of ciliary activity was observed in these cultures.We are grateful to Dr H. E. M. Kay and his staff (Royal Marsden Hospital), for the supply of human embryos; Dr D. A. J. Tyrrell (M.R.C. Common Cold Research Unit, Salisbury) for his encouragement, and Miss G. E. Fairbairn (St Thomas’s Hospital) for the histological preparations.This research was supported by a grant from the Medical Research Council.

Summary Kinetic haemagglutination-inhibition tests were used to investigate possible antigenic variation between four strains of rubella virus which had been isolated in different years and in different geographical areas. No significant differences were detected using hyperimmune rabbit sera and human sera obtained following both naturally acquired infection and vaccination with an attenuated rubella vaccine. These results suggest that major antigenic variation is unlikely to constitute a problem with rubella.

SUMMARYOrgan cultures prepared from 15 different organs obtained from 43 fetuses were consistently found to support the growth of rubella virus, irrespective of the gestational age of the fetus or the strain of rubella virus inoculated. Although rubella virus replicated in fetal lenses, adult lenses did not support the growth of rubella virus. Organs obtained from four fetuses between 8 and 17 weeks gestational age produced similar titres of an inhibitor which had the characteristics of interferon. The use of Trowell T8 medium and incubation in a mixture of 5% CO2 in oxygen provided the most suitable conditions for the maintenance of most organ cultures. Under these circumstances it was possible to obtain adequate histological preparations from these organs, but light microscopy studies revealed no significant differences in sections of rubella inoculated and control organ cultures.

Sera from 123 volunteers vaccinated six to 16 years previously with one of four rubella vaccines (Cendehill, RA27/3, HPV77-DE5, and To-336) were tested for rubella antibodies by single radial haemolysis and radioimmunoassay. By radioimmunoassay 110 (89.4%) of the vaccinees had antibody concentrations greater than the minimum immune titre (that is, greater than 15,000 IU/1), 11 (8.9%) were seropositive but had concentrations less than or equal to 15,000 IU/1, and two (1.6%) were seronegative. Eight (6.5%) were seronegative by single radial haemolysis, of whom five had received Cendehill vaccine. Six to eight years after vaccination subjects who had received Cendehill vaccine had the lowest geometric mean titre of antibody by radioimmunoassay while the subjects who had received HPV77-DE5 vaccine had the highest. Although antibody concentrations less than or equal to 15,000 IU/1 were not detected among subjects given RA27/3 vaccine six to eight years previously, such low levels were detected in two (15.4%) vaccinated 11-16 years previously. These results emphasise the importance of long-term surveillance programmes so that vaccination policies may be reviewed.

Japanese strains of rubella virus have been claimed not to be teratogenic, and tests on three Japanese strains showed that they induced high levels of interferon in human placental cell cultures obtained from conceptuses ranging from 13 to 24 weeks' gestational age, whereas two strains derived from the U.S.A. induced low levels. Both Japanese and U.S. strains induced similar but low levels in fetal lung cell cultures and leucocyte preparations. A representative Japanese strain and a U.S. strain were both interferon-sensitive. If indeed a strain can be shown to be non-teratogenic it could lead to an alternative, safer rubella vaccine.

When 42 sera with low or inconsistent levels of haemagglutination-inhibiting (HAI) antibodies were tested by single radial haemolysis (SRH) radioimmunoassay (RIA), and enzyme-linked immunoassay (ELISA), RIA was shown to be the most reliable test for detecting low levels of antibody. SRH, however, was found to be an acceptable alternative screening test for rubella antibodies and was more reliable than HAI. Although SRH plates prepared in our own laboratory failed to detect antibodies in six sera, in five of the six, antibodies were only at a low level (RIA titre 1:20-1:80). OriVir plates (Orion Diagnostica, Finland) failed to detect low levels of antibody in only three sera. There were six (14.3%) sera which were false positives in the HAI test. These women were shown to be seronegative by radioimmunoassay and, when three of these six volunteers were vaccinated, they developed a typical primary immune response which resembled that developed by 43 seronegative women following vaccination. Fifteen of the young women with consistently low HAI titres and one woman who was seronegative by HAI but seropositive by RIA and ELISA were subsequently vaccinated. Six (37.5%) of these women showed no significant rise in titre by any of the tests employed, while ten had a significant rise in titre, detected by at least one test, with a low level of rubella-specific IgM detectable in one.

Human leucocyte interferon preparation (6·5 × 10 5 units) was administered systemically in two doses, seven and three hours before giving attenuated rubella vaccine to four seronegative volunteers. A further four seronegative volunteers acted as controls and were given rubella vaccine alone. The interferon produced some delay in virus excretion and in the production of maximum haemagglutination inhibition antibody titres. Mild side effects were experienced within 24 hours of administration of interferon in all four volunteers.

Eleven 4-13 year old schoolgirls, who were seronegative by haemagglutination inhibition (HI) and radioimmunoassay (RIA) tests despite having been given HPV77-DE5 vaccine 3-9 years previously, were revaccinated with RA27/3. They showed evidence of residual immunity since they had accelerated immune responses, little or no rubella-specific IgM, no viraemia, and no vaccine-induced reactions. In contrast, all but one of the five adult women who were primary vaccinees showed a more delayed immune response. Three of four women tested had viraemia and two had vaccine-induced reactions. Enhanced HI and enhanced RIA showed that many of the schoolgirls had antibody before challenge, as did a fifth adult, who also showed an accelerated immune response, yet became viraemic.

After intranasal challenge of volunteers with rubella virus vaccine, viremia was assayed by inoculation of lymphocytes and whole blood from vaccinees into Vero cell cultures. Viremia was detected in one of 19 volunteers with low levels ( 15 IU), and in none of 12 volunteers with low levels of preexisting naturally acquired antibody. Excretion of the virus was detected in four volunteers with preexisting vaccine-induced antibody but in none with naturally acquired antibody; eight of 10 seronegative volunteers excreted virus. After challenge, all volunteers with low levels of preexisting vaccine-induced antibody developed booster antibody responses that were measured by radioimmunoassay, and low levels of rubella-specific IgM were detected in four volunteers by M-antibody capture radioimmunoassay. One seronegative, one seropositive, and five low-titer volunteers developed arthralgia. The risk of viremia after challenge in individuals with low levels of rubella antibody appears to be low but may be higher than usual when immunity is induced by rubella vaccine. Rubella virus vaccines have now been licensed for more than 13 years in the United Kingdom and the United States. The success of the vaccination programs in these countries in eliminating congenitally acquired rubella will depend on the persistence of antibody throughout the child-bearing years of vaccinees, a period of perhaps 30 years in vaccinees in the United Kingdom and an even

Thirty-nine volunteers, who were either naturally immune to rubella virus or immune as a result of vaccination with RA 27/3, Cendehill, or To-336 vaccines, were challenged intranasally with high-titered RA 27/3 virus. Before and after challenge, rubella-specific IgG and IgA in serum and nasopharyngeal washings were measured by hemagglutination inhibition, neutralization, and radioimmunoassay. The reinfection rate (at least a fourfold rise in titer of serum antibody by one or more tests) was highest among recipients of Cendehill vaccine. Significant rises in titer were most frequently detected by radioimmunoassay for rubella-specific IgG. After challenge of immune volunteers, rubella-specific IgM was detected in six of the 29 with vaccine-induced immunity. Although high levels of rubella-specific serum and nasopharyngeal IgA before challenge appeared to be associated with protection in recipients of RA 27/3 vaccine, no level of any antibody tested was invariably associated with protection. For comparison, volunteers with vaccine-induced immunity challenged intranasally with the same dose of vaccine after inactivation did not show evidence of reinfection.

One-hundred and eighty-six carefully selected sera were tested for rubella-specific IgM by Rubazyme-M (Abbott Diagnostics) and an M-antibody capture radioimmunoassay (MACRIA). Eleven of these sera were from cases of infectious mononucleosis, six of which gave positive results in MACRIA, while one gave a positive result in Rubazyme-M. Of the remaining 175 sera, 158 gave concordant results whilst 17 sera gave discordant results; these 17 were also tested by serum fractionation. Problems were encountered with all assay systems used. It is therefore recommended that the results of all tests for rubella-specific IgM should be interpreted with caution.

Summary In foetal cell cultures and leucocytes, Sendai virus induced considerably higher levels of interferon than rubella, whereas in placental cultures this was reversed. A low passage strain of rubella virus, ko-1, which was originally isolated in Japan and is non-teratogenic in rabbits, produced particularly high levels of interferon in placental cultures, this being most marked at an m.o.i. of 1 and 15. Although the number of experiments conducted with brain, spleen, heart and amnion cultures was far fewer than with lung cultures, it appeared that comparable levels of interferon were produced by different cell cultures derived from the same foetus. Foetal cultures, leucocytes and placental cultures, derived from foetuses varying in gestational age from 10 to 23 weeks, were capable of producing interferon when infected by Sendai or rubella virus, but levels were unrelated to gestational age.

RK 13 and primary PMK and VMK cell cultures were compared for the isolation of RV by means of the simultaneous inoculation of original specimens and early passage material. RK 13 was found to be the most sensitive and reliable and provided a result for both isolation and neutralization in the shortest time. As the interpretation of CPE and the propagation of these cultures is sometimes difficult, the simultaneous use of a second system in which RV is easy to identify, e.g. VMK or PMK cell cultures, is recommended. Both PMK cell cultures and LLC-RK1 were suitable for isolating RV from clinical specimens. Preliminary studies with LLC-RK 1 indicate that it may provide an even more sensitive alternative to RK 13, but further studies employing clinical material require to be carried out before firm conclusions can be reached.

In 45 patients with rubella-like illnesses during pregnancy serological tests showed that the clinical diagnosis had been accurate in only 20. Since only 16 of these patients had presented for laboratory investigations within a week of the onset of symptoms, the value of haemagglutination-inhibition tests was considerably reduced; the diagnosis in these cases was confirmed by complement-fixation and rubella-specific IgM tests. Of 172 patients exposed to a rubella-like illness, only 17 were seronegative; 105 sought advice within two weeks of exposure, and therefore the haemagglutination-inhibition antibody tests were useful in determining immunity. Since the clinical diagnosis of rubella was proved incorrect in a number of cases, these pregnancies were saved. Hence both doctors and patients should report both exposure to and rubella-like illnesses as early as possible, so that laboratory investigations may be carried out without delay.

The United Kingdom is in the grip of a nationwide mumps epidemic with almost 5000 notifications in the first month of 2005 alone.1 Most patients are aged between 19 and 23, and there is now the threat of outbreaks among under-immunised children. As a result of the measles, mumps, and rubella (MMR) vaccine, which was introduced in 1988, the current generation of practising doctors have little experience of mumps infection. Mumps may have permanent sequelae, and not all cases can be diagnosed clinically. Here we explain the basis of the current epidemic and review the epidemiology, clinical presentation, complications, laboratory confirmation, and treatment of mumps. We searched Medline for evidence based information on the internet, using a range of search terms. Other internet based resources included the websites of the Health Protection Agency (HPA), the World Health Organization (WHO), and the US Centers for Disease Control (CDC). We also used various formal texts. Mumps is an enveloped, single stranded RNA virus belonging to the family paramyxoviridae, which causes an acute infectious disease mainly in children and young adults.2 Transmission is by droplet spread, and humans are the only known host. Mumps is highly infectious and spreads rapidly in susceptible people living in close proximity. The number of secondary cases of infection expected to result from an index case of mumps in a fully susceptible population (R or basic reproduction number) is 10-12. By comparison, measles—a notoriously infectious virus—has an R of 15-17.3 The incubation period from infection to appearance of the characteristic swelling of the parotid glands is 15-24 days.4 The infectious period starts several days before the onset of parotitis and continues for several days afterwards.4 w1Infection control guidance for schools and nurseries advises that children stay away from school for …

Sera from 123 volunteers vaccinated six to 16 years pre? viously with one of four rubella vaccines (Cendehill, RA27/3, HPV77-DE5, and To-336) were tested for rubella antibodies by single radial haemolysis and radioimmuno assay. By radioimmunoassay 110 (89 4%) of the vaccin?es had antibody concentrations greater than the minimum immune titre (that is, > 15 000 IU/1), 11 (8 9%) were seropositive but had concentrations ^15 000 IU/1, and two (1-6%) were seronegative. Eight (6-5%) were sero? negative by single radial haemolysis, of whom five had received Cendehill vaccine. Six to eight years after vaccination subjects who had received Cendehill vaccine had the lowest geometric mean titre of antibody by radio? immunoassay while the subjects who had received HPV77-DE5 vaccine had the highest. Although antibody concentrations ^15 000 IU/1 were not detected among subjects given RA27/3 vaccine six to eight years pre? viously, such low levels were detected in two (15-4%) vaccinated 11-16 years previously. These results emphasise the importance of long-term surveillance programmes so that vaccination policies may be reviewed.