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Congenital Viral Infections Slide Set

Laboratory Diagnosis of Rubella Infections

 

E. Laboratory Diagnosis

1. Serological diagnosis of rubella infection - Serology is the mainstay of diagnosis of rubella infection. A recent rubella infection can be diagnosed by (1) detection of rubella-specific IgM, (2) rising titres of antibody in HAI and ELISA tests, and (3) seroconversion. It is essential to obtain accurate information relating to the date and time of exposure, the date of onset of illness. A history of previous rubella vaccination as well as previous results of rubella screening tests. Blood should be collected from pregnant women with features of rubella-like illness as soon as possible after onset of symptoms. A significant rise in HAI antibodies can often be demonstrated. However rubella-specific IgM is the test of choice for demonstrating current infection. It has been shown though that low and transient levels of IgM can be detected in cases of reinfection. Furthermore, low levels of rubella IgM may persist for a few months to 4 years following rubella vaccination.
 

Typical serological events following acute rubella infection. Note that in reinfection, rubella-specific IgM is usually absent or present at a low level transiently

  1. Haemagglutination inhibition (HAI) assay remains the mainstay test for diagnosis of rubella infection. HAI Abs may be detected on the first day of the rash and rise rapidly to peak titres. It may be difficult to demonstrate a significant rise in titre unless the serum is obtained within the first few days of the onset of illness. Before testing by HAI, sera must be treated to remove red cell agglutininins and serum lipoproteins. It is current practice to confirm a serological diagnosis by HI with testing for rubella IgM.
  2. EIA and RIA have replaced HAI for the diagnosis of rubella in some laboratories. Occasionally SRH can be used for diagnostic purposes provided the wells are of consistent size and a measured volume of serum is added to each well.
  3. Detection of rubella-specific IgM by EIA or RIA. The most sensitive and reliable techniques in use are tM - antibody capture ELISA and radioimmunoassay). IgM antiglobulins such as Rheumatoid Factor can seldom cause false positive results as can heterophil antibodies. Indirect ELISA and RIA has also been developed but these are not as sensitive as direct ELISA.

2. Serological techniques used for rubella antibody screening;- Single Radial Haemolysis (SRH) and latex agglutination (LA), and .ELISA are used for screening for immunity against rubella. SRH is reckoned to be slightly less sensitive than LA or ELISA. False negative results may occur with SRH due to the interference with red cell lysis by a "blocking factor" which may be removed by absorbing the sera with erythrocytes from the same species used in SRH tests. ELISA is now the test of test in many laboratories but it is considerably more expensive than the SRH. An antibody titre of equal or greater than 15 IU/ml is regarded as being immune to rubella. However, there is some controversy as to the 15 IU/ml cutoff since it was arrived at empirically in the first place. It is quite clear that lower levels of antibody, such as 10 IU/ml would probably be protective as well. HAI is not used for rubella antibody screening because it is not sensitive enough.

3. Virus isolation and identification;- Virus isolation is now seldom used for diagnosing postnatally acquired rubella infection. It may still be useful in diagnosing congenital acquired disease and in determining the duration of virus excretion in these infants since they may transfer infection to susceptible adults. The specimen should minced or mixed with culture medium and the supernatant is then inoculated into culture medium. RK13 cell cultures are used exclusively in the UK for virus isolation. SIRC cells are used in Scandinavian countries. Rubella virus is fastidious and produces CPE in RK13 cells under carefully controlled conditions. The virus is more rapidly identified using IF techniques. Inoculation of specimens into Vero cells followed by passage into RK13 or SIRC cells, in which the virus can be identified by its characteristic CPE or by IF represents the most sensitive method for rubella isolation available.  

4. Diagnosis of congenital acquired infection

The diagnosis of congenitally acquired rubella is made by;

  1. The presence of rubella IgM in cord blood or serum samples taken in infancy.
  2. Detection of rubella antibodies at a time when maternal antibodies should have disappeared (approx.6 months of age)
  3. Isolation of rubella virus from infected infants in the first few months of life.

The detection of rubella-specific IgM in cord blood or infant sera is the method of choice for the diagnosis of congenital rubella. Specific IgM has been demonstrated in all confirmed cases to the age of 3 months, in 86% 3 to 6 months, 62% 6 months to 1 year and 42% 12 to 18 months and rarely over 18 months. If the IgM result is negative or equivocal and where there has been a history of rubella in pregnancy, a serum can be taken at 9-12 months to look for the presence of specific IgG. The detection of specific IgG may be of value where tests for IgM have not been conducted in early infancy. Since rubella is uncommon under the age of 2, IgG detected between 1 and 2 may been indicative of congenital infection. However each case must be assessed individually, taking into account factors such as age, maternal history, presence of clinical findings. Rubella virus may be recovered from nasopharyngeal secretions of most neonates with severe CRS. But by 3 months the proportion has declined to 50 - 60%.

5. Prenatal diagnosis of Congenital rubella infection

Prenatal diagnosis of congenital infection may be of value when maternal infection occurred after the first trimester, in cases of maternal reinfection and in cases where equivocal serology results from the mother were obtained. Possible methods include;-

  1. The testing of fetal blood samples obtained by fetoscopy for rubella specific IgM. However the fetus does not produce sufficient IgM for detection before 22 weeks.
  2. Virus may be isolated from amniotic fluid but the reliability of this technique has not been demonstrated.
  3. The detection of rubella RNA or viral proteins in chorionic villus biopsies and amniotic is currently being evaluated.

 

F. Prevention

The first vaccines were developed in the early 60's (HPV77.DE5 and Cendehill) and were licensed for use in 1969. In 1979 the HPV77.DE5 strain was replaced with RA27/3 and Cendehill is no longer available. RA 27/3 is now the most widely used vaccine strain and is made by 7 manufacturers. All vaccines are administered subcutaneously and are well tolerated and produced a response in 95% of recipients. Although the virus is excreted by vaccines, it is not transmitted to susceptible contacts.

1. Vaccination Policies

Two main policies were initially used:-

  1. Universal childhood immunization; The aim was to eradicate rubella infection by vaccinating all preschool children. This policy a high uptake of vaccine (at least 85%) to be effective.
  2. Selective vaccination; The aim is to protect the population at risk. All prepubertal schoolgirls in the UK were selectively vaccinated. This policy is more suitable for countries which are unlikely to attain the necessary 85% uptake.

Universal childhood vaccination was adopted by the USA in 1969 with great success. It was offered as part of the MMR (Mumps, Measles, Rubella) vaccine. The uptake was very high as proof of immunization of measles is mandatory before school entry. Since 1978 there has been a steady decline in CRS as well as postnatally acquired rubella and CRS is on verge of being eliminated in the USA. The USA policy results in financial saving: cost-benefit analysis shows that the cost of rubella in an unvaccinated population is approx. 11 times more than the cost of vaccination policy.

In the UK selective vaccination of 11 - 14 yr old girls was introduced in 1970. The vaccine was also offered to susceptible people eg.nurses, doctors and schoolteachers and to women who were found to be seronegative when they attended antenatal clinics. Seronegative women attending antenatal clinics were offered vaccination in the immediate postpartum period. It appears that overall 90% of schoolgirls have been vaccinated. However there are districts where the uptake rates are lower. The proportion of susceptible women attending antenatal clinics between 1984 to 1986 varied between 2.3% and 5.8%. Although there has been some decline in cases of reported CRS, maternal rubella is still relatively common. In 1986, 173 cases of laboratory confirmed rubella were reported during the first 16 weeks of pregnancy. A high proportion of these pregnancies were terminated.

Since it became apparent that complete vaccination of the target population was an unrealistic goal and because despite high uptake rates.rubella still infects pregnant women, the rubella vaccination programme in the UK was augmented in 1988 by offering rubella vaccination to preschool children of both sexes. Rubella vaccine is given as part of the MMR vaccine. As part of the " catch up" programme MMR is also given to children age 4 - 5 yrs. The vaccination of 10 to 14 yr olds and seronegative women is to continue until it can be demonstrated that rubella is no longer circulating in the community and that serological surveillance shows that 90-95% of adolescents are already immune. The augmented programme is designed to eradicate rubella. However in the USA this has been harder to achieve than expected. Mathematical models show that poor uptake amongst preschool children may actually increase the proportion of rubella susceptibles in older age groups. Therefore high vaccine uptake is totally essential.  

2. Immune responses

Rubella vaccination induces an immune response in 95% of recipients, but antibody concentrations are generally lower after vaccination than after naturally acquired infection. Testing for antibodies should wait until 8 weeks after immunization. Of the 5% who fail to seroconvert, the majority will respond if revaccinated. A few may fail to respond or respond poorly due to concurrent infection or a low level of preexisting antibodies, which may be undetectable by HAI or SRH. Antibodies persists at levels >15 IU/ml in the majority of vaccinees for at least 21 years. In approx. 10% , the Ab levels fall below 15 IU/ml within 5 to 8 years and a small number may become completely seronegative. Rubella specific IgG, IgM and IgG can be detected in the patient's serum. IgM may persists in 73% of vaccinees after 6 mths and occasionally been shown to persist up to 4 years.

Virus excretion can be detected in the majority of vaccinees between 6 to 28 days after vaccination. However transmission of the virus to susceptible contacts rarely occur. It is therefore safe to vaccinate persons who may come into pregnant women. Vaccine virus may also be shed in the breast milk of women who were vaccinated postpartum. However even though some infants are infected, they develop no clinical features. Rubella vaccines are well tolerated. Lymphadenopathy, rash and arthropathy may occur between 10 days and 4 weeks after vaccination. These reactions are less severe than the natural infection. Postpurbertal females are more likely to develop symptoms than children. Like other live vaccines, rubella vaccine should not be given to immunocompromised patients, as a result of disease or treatment.

Pregnancy is an absolute contraindication and pregnancy should be avoided for 1 month after vaccination. Where inadvertent rubella vaccination had occurred just before or during pregnancy, there had no been a single case of fetal damage reported. Even though it has been shown vaccine virus does cross the placenta and establishes a persistent fetal infection. In a series of 486 babies delivered by women who were inadvertently vaccinated during the first trimester, no congenital abnormalities consistent with CRS were reported. However, there was serological evidence of infection in 8 babies

3. Reinfection

Reinfection with rubella may occur. It is more likely to occur in those whose immunity is induced by vaccination rather than natural infection. The IgG response is highly elevated. A slight and transient IgM response may be present. It has been suggested that IgG avidity assays may be useful in distinguishing between primary and reinfection.

4. Passive Immunization

Post-exposure prophylaxis with immunoglobulins does not prevent infection in non-immune contacts and is therefore not recommended for protection of women exposed to rubella. However it may reduce the likelihood of clinical symptoms which may reduce the level of maternal viraemia and the risk to the fetus. Women who contract rubella during the first trimester of pregnancy but are determined to proceed with the pregnancy may be offered HNIG or rubella immunoglobulin. There is evidence to suggest that infants of women who experienced subclinical rubella in early pregnancy following administration of HNIG is less likely to be infected in utero, or if infected, less likely to be less severely affected. Possible mode of action seems to be decreased maternal viraemia in the presence of HNIG. Dudgeon advocates the administration of HNIG to women who are determined to proceed to term. He suggests a dose of 1500 mg i.m. as soon as possible after exposure and 3 to 4 days later.

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Congenital Viral Infections Slide Set