Margaret K. S. Ridehalgh

Outcome of asymptomatic infection with rubella virus during pregnancy

We have tried to detect prenatal infection in 34 infants whose mothers were re-infected with rubella virus during pregnancy and in six infants whose mothers had primary subclinical rubella during pregnancy. Two methods of assessment were used: first, serum obtained soon after birth was tested for IgM antibody; secondly, serum obtained after the age of 8 months was tested for specific IgG. The 34 women with re-infections had increases in IgG antibody titre but no IgM response. No evidence of prenatal infection was found in 33 of their 34 infants. One infant was found to have IgG antibody at the age of 11 months. This infant was IgM-negative at birth and had a rubelliform rash at the age of 5 1/2 months; it therefore probably contracted post- rather than pre-natal infection. Fetal infection from maternal re-infection during pregnancy is probably rare. The six women with primary subclinical rubella produced both IgG and IgM classes of antibody. Three of their six infants showed serological evidence of intrauterine infection. One, infected when its mother was 8 weeks pregnant, had clinical evidence of congenital rubella. Primary subclinical rubella during pregnancy therefore carries a significant risk of fetal infection. Because of the difference in outcome, great care should be taken to distinguish between primary infection and re-infection when investigating symptomless increases in antibody titre after contact with rubella during pregnancy.

Specific immunoglobulin responses after varicella and herpes zoster

The indirect immunofluorescence technique has been used to titrate the specific immunoglobulins in 200 sera from 64 patients with varicella, and 195 sera from 67 patients with herpes zoster. IgG and IgM antibodies were detected in all patients with varicella, and IgA in 59 (92%). All three classes of antibody appeared 2--5 days after the onset of the rash, increased virtually simultaneously and reached maximum titres during the second and third weeks. IgG then declined slowly, but never became undetectable and was still present in five subjects who were retested after 2--4 years; it was present in 88 out of 100 healthy young adults and probably persists indefinitely after varicella. IgA and IgM antibodies declined more rapidly and were not detected in specimens taken more than a year after the illness. IgA, however, may possibly persist in some cases since low titres were found in 8 out of 88 young adults who possessed IgG antibody and had presumably had varicella in the past. IgA responses were significantly weaker in children under the age of 6 years than in older children and adults. Six out of 67 patients with zoster were tested at various times before the onset of the rash: IgG antibody was detected in all. IgG was present in all sera taken after the onset of the rash, increased rapidly after 2--5 days, reached maximum titres during the second and third weeks and then declined slowly. IgA antibody was detected in 66 patients (99%) and IgM in 52 (78%); both types of antibody followed transient courses, as in varicella. Maximum titres of IgG and complement-fixing antibodies were greater after zoster than after varicella, but the differences were not significant. IgA and IgM titres in young adults with zoster were significantly lower than in older patients, and also lower than in young adults with varicella. Increases in varicella-zoster antibody in patients with herpes simplex virus infections consisted mainly of IgG, sometimes IgA, but never IgM.

Specific immunoglobulins in infants with the congenital rubella syndrome

The indirect immunofluorescent technique has been used to detect and titrate the specific immunoglobulins in serum specimens from 154 infants with confirmed or suspected congenital rubella. IgM antibody was stained more efficiently in sucrose density gradient fractions than in whole serum and was detected in this way in 27 out of 40 patients with confirmed congenital rubella at ages ranging from birth to 2 years. It was present in 48 out of 50 serum specimens during the first 6 months of life and in 11 out of 38 specimens obtained at ages between 6 1/2 months and 2 years. IgM antibody was therefore estimated to persist for about 6 months in the majority of cases and up to 2 years in a few individuals. IgM antibody was also detected by this method in 11 out of 114 infants with suspected but unconfirmed congenital rubella at ages up to 5 months. The total concentrations of IgM were above the normal range in nearly all sera taken from confirmed cases during the first 3 months of life and in half the specimens obtained between the ages of 3 and 6 months. IgG antibody was detected by fluorescent staining of whole serum in all patients with congenital rubella. Geometric mean titres increased during the first 3 months of life and then declined slowly. IgA antibody was not detected, except in two patients in whom traces were present at the age of 6 months, and the total concentrations of IgA were usually within normal limits. Fluorescent staining of fractions showed that the sedimentation characteristics of rubella IgG and IgM antibodies were the same in infants as in adults. The peak IgM fractions never contained IgG antibody, and the presence of specific IgM in these fractions could usually have been safely inferred from their HAI titres. Fluorescent staining, however, was more sensitive and frequently detected IgM antibody in fractions which had no definite HAI activity. Fluorescent staining of whole serum for IgM antibody was less distinct, and often unsuccessful, even in specimens in which specific IgM was detected in the fractions. The addition of IgG- to IgM-containing fractions caused depression of IgM staining and suggested that failure to detect IgM antibody in whole serum was partly due to competitive inhibition by specific IgG.

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.

Fetal infection resulting from maternal rubella after the first trimester of pregnancy.

We have tried to measure the incidence of prenatal infection in 304 infants whose mothers had had rubella at various times after the first 12 weeks of pregnancy. Two methods of assessment were used: first, serum obtained soon after birth was tested for specific IgM antibody; secondly, serum obtained after the age of eight months was tested for specific IgG. When maternal rubella occurred 12-16 weeks after the last menstrual period specific IgM antibody was detected in 28 out of 50 infants (56%). The proportion fell progressively to 12% after maternal rubella at 24-28 weeks, rose to 19% after rubella at 28-36 weeks and then to 58% when the illness occurred during the last month of pregnancy. In all, IgM antibody was detected in 77 out of 260 infants (29%). The fetus can thus be infected at any time during the second and third trimesters of pregnancy, but the risk varies at different stages.The figures for the prevalence of IgG antibody were greater throughout, because some infants had IgG who had previously lacked specific IgM. After maternal rubella at 12-16 weeks IgG antibody persisted in 22 out of 31 infants (71%). The proportion fell to 28% after rubella at 24-28 weeks and then increased progressively to 94% after rubella during the last month. In all, IgG antibody persisted in 94 out of 190 infants (49%). The true rate of fetal infection probably lies between the rates estimated from the presence of IgM antibody and the subsequent prevalence of IgG.Infants whose mothers had rubella at any time during pregnancy should be examined regularly for possible evidence of damage.

Detection of specific IgM in varicella and herpes zoster by antibody-capture radioimmunoassay.

A simple and sensitive M antibody-capture radioimmunoassay (MACRIA) is described which utilizes crude commercial VZV antigen and a single monoclonal anti-VZV antibody. This was compared to the immunofluorescence (IF) test for IgM antibody and was used to study IgM responses in sera from 261 patients with varicella and 220 patients with herpes zoster. With MACRIA, IgM antibodies were detected in all patients with varicella. The IgM antibodies appeared shortly after onset of rash, reached peak levels between 1 and 4 weeks after onset and then declined to low or undetectable levels in most, though not all, patients after 3 months. IgM antibodies were also detected in 98.2% of patients with herpes zoster, but the levels of IgM were significantly lower than after varicella and there was wider individual variation both in magnitude and duration of the IgM responses, in some cases only lasting 2-3 weeks. Comparison between MACRIA and IF showed good agreement in the detection of IgM antibodies following varicella. Discordant results were obtained with 13% of sera, of which 81% were taken either early or late after onset of rash and contained very low IgM levels. In contrast, 62 (28%) of the 220 sera from patients with zoster gave discordant results in the two tests, all except five being MACRIA-positive but IF-negative. The largest proportion of discordant results were obtained with sera taken more than 3 months after onset of rash, but 18 (29%) contained high IgM levels and were taken during the period of peak IgM responses. The diagnostic applications of the VZV MACRIA are discussed.

Specific immunoglobulin responses in serum and nasal secretions after the administration of attenuated rubella vaccine.

The indirect immunofluorescent technique has been used to study the specific immunoglobulin responses in the sera of 63 non-immune adult women who received either Cendehill rubella vaccine subcutaneously, RA27/3 rubella vaccine subcutaneously, or RA27/3 vaccine intranasally. IgG, IgA and IgM antibodies increased virtually simultaneously, starting about 2 weeks after vaccination. IgG antibody appeared in all subjects and reached maximum titres 4-6 weeks after vaccination. The mean IgG titres elicited by the three different methods of vaccination did not differ significantly. IgA and IgM antibodies reached their highest titres between 21 and 28 days after vaccination and then declined to low or undetectable titres within about 9 weeks. The maximum IgA titres observed after intranasal administration of RA27/3 vaccine were significantly higher than those which occurred when the same vaccine was given subcutaneously, but no significant difference in IgM titres was observed. When unfractionated sera were examined IgA antibody was detected in 57 cases (91%) and IgM in 51 (81%). Fluorescent examination of fractions obtained by centrifugation on sucrose density gradients frequently revealed small amounts of IgA and IgM antibody which could not be detected by staining unfractionated serum, and with the inclusion of these results IgA antibody was detected in 61 cases (97%) and IgM in 59 (94%).When 39 adults with pre-existing serum antibody were challenged with vaccine a definite IgA response was detected in only one subject and in no case was there any evidence of the appearance of IgM antibody.Nasal antibody, consisting of IgG or IgA or both, was detected in 17 out of 23 non-immune subjects (74%) who received RA27/3 vaccine, either subcutaneously or intranasally. Titres were much lower than those which occur in the natural disease and there was no evidence that nasal antibody was elicited more readily by intranasal than by subcutaneous vaccination.

Nasal immunoglobulin responses in acute rubella determined by the immunofluorescent technique

The indirect immunofluorescent technique has been used to study the specific immunoglobulin responses in nasal secretions from ten adults with acute rubella. Titres of IgA antibody in nasal washings usually exceeded those of IgG, but both types of antibody were detected in all patients. They appeared a few days after the rash, reached maximum titres during the second week and then declined. IgA antibody was no longer detectable after 47 days and was not detected at all in nasal washings from adults who had experienced rubella in the past. Low titres of IgG antibody persisted in some patients for longer than IgA and traces of IgG were found in nasal washings from a minority of adults with a past history of rubella. Nasal antibodies in acute rubella are therefore transient and unlikely to take part in resistance to reinfection.In sucrose-density gradients nasal IgA antibody sedimented more rapidly than IgG and there was little overlap between these two types of antibody. IgA antibody in serum was more heterogeneous; it was found in nearly all the fractions which contained IgG antibody and in many of those which contained IgM.

A comparison of indirect immunofluorescence and radioimmunoassay for detecting antibody to varicellaoster virus

Immunofluorescence (IF) and radioimmunoassay (RIA) were found to be more sensitive methods than complement fixation (CF) for detecting antibody to varicella-zoster (V-Z) virus. RIA yielded titres about 30 times greater than those obtained by IF, but for screening purposes RIA was only about six times more sensitive since the minimum serum dilutions that could be tested were 1/100 and 1/16 respectively. When 539 sera from subjects of different ages were screened for V-Z antibody, IF and RIA gave concordant results with 527 specimens (98%). When 19 patients were tested who had not previously had varicella but were experiencing primary infection with herpes simplex (HS) virus, crossreacting antibodies to V-Z antigens were detected in six patients by IF but in only two of these by RIA. IF and RIA are preferable to CF as tests for immune status because of their greater sensitivity, but weak positive reactions caused by presumptive low titres of homologous antibody or by higher titres of heterologous antibody can occur in one or both tests. Such reactions could cause difficulty in assessing the need for vaccine or for specific immune globulin, and in interpreting the response to vaccination.

IMMUNOGLOBULIN RESPONSES AFTER RUBELLA INFECTION

The indirect immunofluorescence technique has been used to study specific IgG, IgA, and IgM antibodies in adults with acute rubella, volunteers who receive attentuated rubella vaccine, and infants with suspected congenital infection. In acute rubella, IgA and IgM antibodies reached peak titers during the second week after the rash and then declined, but specific IgG persisted. A similar pattern of response occurred after rubella vaccination, but titers were lower. Occasionally, fluorescent staining failed to detect specific IgM in whole serum but demonstrated it clearly in sucrose density gradient fractions. The improvement in fluorescence obtained by staining serum fractions was particularly striking in samples obtained from congenitally infected infants. In these cases, IgM staining with whole serum was often poor, but when fractions were tested, specific IgM was demonstrated in nearly all samples obtained within 9 months of birth. IgM antibody was seldom detected after the first year of life, but IgG antibody persisted. IgA antibody was not detected in congenital rubella.