Prenatal diagnosis of viral infections. A two year study in Strasbourg]

We report here the results of a 2-year study on the prenatal diagnosis of viral infections in Strasbourg. This screening was carried out by virus isolation, by PCR assay, or by detection of IgM fetal antibody for 98 pregnant women at risk of transmitting one of the viruses that causes fetal disease such as parvovirus B19 (B19), Herpesviruses [cytomegalovirus (CMV), varicella-zoster virus, herpes simplex virus] and rubella virus. A viral etiology was proven in 7 out 98 cases: PCR applied to B19 DNA detection was positive in 5 amniotic fluids (AF), 2 fetal serums and one ascitic liquid. The diagnosis of 2 cases of CMV infection was obtained by both PCR and virus isolation in AF from twins fetuses. The detection of specific IgM in maternal serum or fetal serum is useful to achieve the diagnosis but serological tests on other samples have no efficiency. No virus was found in any other specimen, but the genome of Toxoplasma gondii was detected by PCR in 1 of 17 AF samples analyzed at the Institut de Parasitologie. These findings show that PCR assay is a sensitive method for the positive diagnosis of intrauterine infection and promises to careful follow-up of the pregnancy.     

Assessment of IgM enzyme immunoassay and IgG avidity assay for distinguishing between primary and secondary immune response to rubella vaccine

The primary test for the laboratory confirmation of rubella is IgM serology. It is important to distinguish IgM reactivity caused by primary infection from that caused by reinfection or persistence, especially in pregnant women; as termination of pregnancy is considered when primary rubella is diagnosed during the first trimester.

In this study, the performance of rubella IgM enzyme immunoassay (IgM-EIA) and rubella IgG avidity assay were compared using well-defined panels of sera from persons vaccinated against rubella and commercial rubella IgM and IgG enzyme immunoassay kits (Dade Behring, Marburg, Germany).

The sensitivity and specificity of rubella IgM-EIA were found to be 77.4 and 97.9%, respectively, while the results for rubella IgG avidity assay were 100 and 100%. IgG avidity assay showed higher positive and negative predictive values than the IgM-EIA (100 and 100% compare to 96.9 and 82.9%).

In conclusion, the rubella IgG avidity assay is more sensitive and specific than IgM-EIA for differential detection of primary rubella infection from rubella reinfection.     

Prevalence of HI antibody titer against rubella virus to determine the effect of mass vaccination in Tehran.

BACKGROUND: Rubella is an infectious viral disease, has a worldwide distribution and is normally a mild childhood disease. Infection during early pregnancy may cause fetal death or congenital rubella syndrome. The highest risk of CRS is found in countries with high susceptibility rates among women of childbearing age. In many developed and some developing countries, large-scale rubella vaccination during the past decade has drastically reduced or practically eliminated rubella and CRS. Mass vaccination campaigns and Expanded Program of Immunization (EPI) have increased vaccine coverage in the world with a substantial impact on the reduction of rubella infections, such as CRS. OBJECTIVE: The present study was preformed to evaluate the immune status against rubella before and after the mass campaign vaccination on 22 December 2003. STUDY DESIGN: A total of 320 samples were collected from the healthy subjects before and after the vaccination and 80 paired sera were collected and tested for the presence of rubella antibody using HI test. RESULTS AND CONCLUSIONS: Based on the results, 98.1% of the population has gained anti-rubella antibody, compared with 92.2% before the vaccination. The data revealed that 98.75% of the paired subjects had rubella antibody after mass vaccination which is statistically significant.     

Antibody response to rubella virion (V) and soluble (S) antigens in rubella infection and following vaccination with live attenuated rubella virus

Summary The antibody response to rubella virion antigen and rubella S antigen was studied in natural rubella infection and after vaccination with live attenuated rubella virus by the complement fixation (CF), platelet aggregation (PA), and hemagglutination inhibition (HI) techniques.In natural rubella infection CP and HI antibodies to rubella virion appeared early and rapidly reached high titers. The CP and PA antibody responses to rubella S antigen were more delayed and great individual variation was seen. Generally CF-S titers were several times lower than CF-V titers. The CF antibody response to rubella S antigen is thus different from the immune response to nucleoprotein S antigens of paramyxoviruses, supporting the concept that rubella S antigen is a subunit of virus envelope. Use of virus-free rubella S antigen preparations in routine CF test is recommended for detecting later rises of antibody titer.After rubella vaccination a 95% seroconversion rate was recorded in both HI and CF-V tests, but the titers were lower than after natural infection. The CF-S and PA antibody responses were weaker and measurable antibodies developed in only 50% and 25% of the cases respectively.Rubella-specific IgM antibodies could be detected in rubella infection by both sucrose gradient fractionation (followed by HI titration) and fluorescent antibody (FA) techniques. The former was a little more sensitive. In the seronegative vaccinees IgM antibodies became demonstrable in 50% of the cases between the 20th and 55th day after vaccination.     

Comparison of two commercial immunoassays for the detection of anti-rubella IgM and IgG in pediatric patients

The only threatening modality of rubella is the Congenital Rubella Syndrome that affects fetuses of women who acquire infection during early pregnancy. Laboratory diagnosis is based on serological parameters. We compared anti-rubella IgM and IgG detection of two commercial immunoassay kits (Abbott and Roche). Although we observed an agreement of 97.8% for IgM and 95.7% for IgG when the categories positive, negative and indeterminate were considered, mean titers of IgG and the absorbance/cut off of IgM were statistically different for both kits, thus corroborating the idea that serological results depend very much on the methodology and must be carefully interpreted.     

Diagnosis of parainfluenza virus infection in children and older patients by detection of specific IgM antibody

The significance of detecting specific antibody of the IgM class for the diagnosis of parainfluenza infections was examined. Paired sera from 763 children and adults admitted to the hospital for acute respiratory disease were tested for significant antibody titer rises in the hemagglutination inhibition (HI) test and for specific IgM antibody with the hemadsorption immunosorbent techniques (HIT). Sera were collected during two 6-month periods, January through June, 1982 and 1983. Evidence of parainfluenza infections was found in 122 patients (16%): 83 (25%) in 1982 and 39 (9.1%) in 1983. The HIT was superior to the HI test for detection of parainfluenza infection, in particular in infants and aged patients, 94 patients were positive only in the HIT test, 12 in the HI test, and 16 in both tests. In a control group of 120 persons (time- and age-matched to the patients of 1982) admitted for nonrespiratory illness, six (5%) showed parainfluenza IgM in their serum. Blocking experiments and retrospective clinical information indicated that the IgM antibody detected in these individuals is specific IgM acquired after a mild parainfluenza infection. Most (66%) patients showed IgM antibody titer rises or high titers (greater than 1,280) in both sera, and in 23%, a fall in IgM antibody titer was found. Detection of specific IgM antibody by HIT permitted early presumptive diagnosis in 71% of the patients with parainfluenza infection. IgM antibody persisted for 2-11 weeks. The HIT appears to be an important supplement for the diagnosis of parainfluenza infections.     

Rubella and pregnancy.

Maternal rubella infection during pregnancy can induce more or less severe congenital defects. In France in 1989, 43 rubella infections in pregnant women were recorded by the National Laboratory of Health. Diagnosis of primary rubella infection rests on detection of specific IgM antibodies. The most reliable technique for detection of IgM antibody is the antibody-capture immunoassay. Presence of rubella-specific IgM antibodies must be interpreted with caution: specific IgM antibodies may occur following reinfection and there have also been reports of rubella-specific reactivity in sera collected after infections with other viruses. In some cases, presence of rubella-specific IgM antibodies cannot be ascribed to any of the reasons above and remains completely unexplained. Fetal infection can be demonstrated by detecting rubella-specific IgM antibodies in fetal blood. However, very sensitive techniques must be used in order to obtain reliable results. Today, widespread immunization is still essential. Immunization of all seronegative pregnant women immediately after delivery is especially important since most mothers of babies with congenital rubella were multiparous. Women exposed to rubella may be offered normal human immunoglobulins as soon as possible after the exposure; if delayed, this treatment may be ineffective.     

Kinetics of rubella-specific IgM antibody response in postnatal rubella infection

The serological diagnosis of primary postnatal rubella infection is based on detection of rubella-virus-specific IgM antibody or a four-fold rise in rubella-specific IgG antibody. Although there are several different methods of enzyme immunoassays that are commercially available, the cost benefit evaluation makes them impractical for use in developing countries. For this reason, we have standardized the measurement of rubella IgM antibody by HAI following serum fractionation by ion-exchange chromatography. The sera samples obtained from pregnant women infected with rubella virus at different times during gestation were fractionated and tested by HAI. Seven out of nine sera collected within the first two days after onset of rash showed detectable levels of rubella IgM antibody. All 57 sera collected between 3 and 30 days after the onset of rash contained rubella IgM antibody. After 30 days, only 1 of 5, or 20%, of sera contained IgM antibody. The HAI testing method was rapid and specific and the cost was not prohibitive. HAI-IgM testing could be used to diagnose primary rubella infections in developing countries where expensive EIAs are unaffordable.     

TORCH infection in women with bad obstetric history--a pilot study in Kumaon region

TORCH infections in the mother are transmissible to fetus in the womb or during the birth process and cause a cluster of symptomatic birth defects. In mother they are inapparent or asymptomatic and hence difficult to diagnose clinically. Over a nine months period 20 pregnant women with bad obstetric history were-studied. Seropositivity of Toxoplasma, rubella, CMV, and HSV infections (TORCH) were demonstrated by the presence of IgM and IgG antibodies by ELISA method. It was found that, IgM antibodies were positive in 4 cases (20%) for Toxoplasma, 4 cases (28.6%) for rubella and 4 cases (26.7%) for CMV and HSV each. IgG antibodies were positive in 11cases (55%) for Toxoplasma, 10 cases (66.6%) for rubella, 14 cases (93%) for CMV and 11 (73%) for HSV. Therefore all antenatal cases with BOH should be routinely screened for TORCH as early diagnosis and appropriate intervention will help in proper management and fetal outcome.     

Simultaneous IgM reactivity by EIA against more than one virus in measles, parvovirus B19 and rubella infection.

BACKGROUND: A clinical diagnosis of rash-causing infections is not always possible and reliance has to be placed on serological evidence of infection, especially on the presence of specific immunoglobulin (Ig)M. However, despite the use of modern serological methods and validated commercial kits, reports appear in the literature of simultaneous IgM reactivity against more than one virus in cases of Epstein Barr virus, rubella, cytomegalovirus, human parvovirus B19 (HPV B19) and measles infections, all with implications for the pregnant woman. OBJECTIVES: We decided to evaluate the extent of the problem in rubella, measles and HPV B19 infections in a routine diagnostic laboratory. STUDY DESIGN: We tested sera from cases with initial clinical and serological evidence of infection with measles, HPV B19 or rubella for evidence of simultaneous IgM reactivity against more than one virus. We confirmed primary infections with specific-IgG antibody avidity tests, and subjected sera with IgM reactivity against more than one virus to avidity tests to identify which, if any, of the three viruses was the cause of the primary infection. Groups of monoreactive IgM sera were randomly selected from the presented sera to demonstrate that the avidity of the IgG specific for the other two viruses would be of high avidity compared with the low avidity of the IgG specific for the virus against which specific IgM had been detected. RESULTS: Our results confirm that simultaneous IgM reactivity against more than one virus does occur in these three infections, and that this is unlikely to be caused by the presence of rheumatoid factor. CONCLUSIONS: In the absence of seroconversion, reliance on specific IgM results alone for diagnosis of these infections should be avoided and tests such as specific IgG antibody avidity should also be employed. The simultaneous occurrence of IgM reactivity against more than one virus is also important for epidemiological and surveillance reasons as the widespread use of the mumps, measles and rubella vaccine makes its impact on the population. Falsely diagnosed cases of apparent measles or rubella could throw into question the efficacy of the vaccine.     

Cutoff levels of immunoglobulin M antibody against viral core antigen for differentiation of acute, chronic, and past hepatitis B virus infections

The titer of antibody against core antigen of hepatitis B virus in the immunoglobulin M class (IgM anti-HBc) was determined by an IgM capture assay of reduced sensitivity (30 arbitrary units). The distribution of titers among 235 acute hepatitis patients who were hepatitis B surface antigen (HBsAg) positive suggested that 600 U forms a lower cutoff value for acute hepatitis B. Clinically apparent cases of acute hepatitis with high IgM anti-HBc and without HBsAg were rare (2.6%). Acute, non-B hepatitis in HBsAg carriers was more frequent (9.4%). In chronic hepatitis B, 39% of 174 biopsy-proven cases had moderate titers of 30 to 600 U, whereas healthy HBsAg carriers were rarely (4/84) positive. In mild or inapparent infections without HBsAg, titers were between 50 and 400 U. Thus, sufficiently accurate and sensitive quantitation of IgM anti-HBc allows for differentiation of acute and nonacute hepatitis B virus infection in acute hepatitis, partial differentiation between clinically symptomatic and asymptomatic chronic infections, and identification of recent subclinical infections.     

Hemagglutination inhibition,single radial hemolysis,and ELISA tests for the detection of IgG and IgM to rubella virus

Hemagglutination inhibition (HI), single radial hemolysis (SRH) and enzyme-linked immuno sorbent assay (ELISA), performed with commercial antigen and reagents are described and were compared in the three distinct situations that require rubella antibody detection. Determination of immunity status was carried out on 156 sera. A degree of correlation > 0.9 was found when comparing the three methods. Analysis of a further 74 sera, from 31 primary infections and three congenital syndromes, was performed to compare the occurrence of the various classes of antibodies in the three tests: HI test and IgM-ELISA become positive the day after the rash, whereas SRH test is not positive before the sixth day. From our limited study bearing on a total of 230 sera, each test has a precise assignment. For the determination of immunity status, SRH is simpler, faster, and inexpensive; absence or evidence of past infection can be unequivocally obtained especially in cases of low (1:10, 1:20) residual immunity. In the seriodiagnosis of a rubella rash, SRH alone, due to the delayed rise in antibody titers, will demonstrate a complete seroconversion with a first serum collected up to the fifth day of the eruption. In case of absence of an early serum, of primary infection in a pregnant woman, of a newborn with suspicion of congenital syndrome, the measurement of rubella specific IgM is best obtained with ELISA, a procedure less time-consuming than HI following centrifugal, chromatographic, or electrophoretic separation. And “light” (8 S) RF with SRH test is discussed. Interference of IgM Rheumatoid Factor (RF) with IgM ELISA and IgG RF with SRH test is discussed.     

Hemolysis-in-gel test in immunity surveys and diagnosis of rubella

The hemolysis-in-gel (HIG) technique was adapted for rubella antibody determinations. Use of sucrose gradient purified virus and its coupling with CrCl₃ to chicken erythrocytes resulted in gel plates that could be stored for several weeks and were suitable for reproducible antibody determinations. In a serological survey of young healthy adults the HIG values (range > 2–13 mm) were in close correlation to those obtained by the HI test (> 10 to 320). The HIG test seems well suited for screening the need of vaccination. Seronegative sera (HIG > 2, HI> 10) gave without heat inactivation hemolysis zones ranging from 4 to 6.5 mm. Although the present rubella HIG test did not measure IgM antibodies, the test, by virtue of its accuracy and sensitivity — extending to antibody levels corresponding to HI titers 2–10 — provides a simpler and more rapid means for diagnosis of rubella infections than the conventional HI and CF tests.     

Detection of rubella virus immunoglobulin G (IgG) and IgM antibodies in whole blood on Whatman paper: comparison with detection in sera.

We compared detection of rubella virus hemagglutination inhibition (HI) antibody and rubella virus-specific immunoglobulin M (IgM) in dried whole blood spotted onto Whatman filter paper and serum samples, both of which were obtained from the same subject by venipuncture. Of 1,000 paired serum samples obtained to study HI antibodies, 807 dried blood samples had HI titers identical to those of the corresponding serum samples, and 193 dried blood samples showed 1 dilution difference. Storage of dried blood at room temperature for 28 days did not affect the HI antibodies. In a study of specific IgM by a solid-phase immunosorbent HI test done with blood from healthy subjects and patients with rubella, the result of the presence, positive or negative, of specific IgM from both blood sample sources corresponded when the dried blood samples were stored at room temperature from 5 h to 38 days. This study demonstrated that the use of Whatman filter paper as a transport medium for blood samples for the determination of rubella virus immunity and the diagnosis of rubella virus infection is possible.     

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.     

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.     

Further evaluation of a rubella passive hemagglutination test

Clinical experience with the Rubacell passive hemagglutination (PHA) test over a one-year period has shown the test to be a rapid, reliable, and economical method for determining antibody to rubella. The data from two separately administered rubella proficiency surveys showed 100% correlation between the PHA and the hemagglutination inhibition (HI) qualitative results with 24 reference specimens. Also, the PHA titers appeared to be generally higher than the HI in these specimens and in the sera of immune individuals. The efficacy of detecting HI antibody in the absence of PHA antibody as an indication of recent infection was compared to the HI paired sera method and to a rubella-specific immunoglobulin M (IgM) test based on protein A absorption. From the results obtained with the sera of 76 rubella patients, the efficacy of the three diagnostic methods was of the following order: protein A IgM test > positive HI/negative PHA > HI paired sera method.     

Gestational and fetal outcomes in B19 maternal infection: a problem of diagnosis

Parvovirus B19 infection during pregnancy is a potential hazard to the fetus because of the virus' ability to infect fetal erythroid precursor cells and fetal tissues. Fetal complications range from transitory fetal anemia and nonimmune fetal hydrops to miscarriage and intrauterine fetal death. In the present study, 72 pregnancies complicated by parvovirus B19 infection were followed up: fetal and neonatal specimens were investigated by serological and/or virological assays to detect fetal/congenital infection, and fetuses and neonates were clinically evaluated to monitor pregnancy outcomes following maternal infection. Analysis of serological and virological maternal B19 markers of infection demonstrated that neither B19 IgM nor B19 DNA detected all maternal infections. IgM serology correctly diagnosed 94.1% of the B19 infections, while DNA testing correctly diagnosed 96.3%. The maximum sensitivity was achieved with the combined detection of both parameters. B19 vertical transmission was observed in 39% of the pregnancies, with an overall 10.2% rate of fetal deaths. The highest rates of congenital infections and B19-related fatal outcomes were observed when maternal infections occurred by the gestational week 20. B19 fetal hydrops occurred in 11.9% of the fetuses, and 28.6% resolved the hydrops with a normal neurodevelopment outcome at 1- to 5-year follow-up. In conclusion, maternal screening based on the concurrent analysis of B19 IgM and DNA should be encouraged to reliably diagnose maternal B19 infection and correctly manage pregnancies at risk.     

Single-serum diagnosis of rubella by combined use of the hemagglutination inhibition and passive hemagglutination tests.

We explored the feasibility of using the passive hemagglutination (PHA) test in combination with the hemagglutination inhibition (HI) test for the single-serum diagnosis of rubella. We found by sedimentation analysis of the serum that early 7S antibody produced within 1 month after the onset of the rash had high HI but much lower PHA titers, whereas the PHA titers of antibody produced later were slightly higher than the HI titers. (19S antibody in the early serum had some PHA activity.) This disparity between the HI and PHA activities of the early 7S antibody could be used for the routine diagnosis of rubella. A comparison of the HI titers of the test serum with the PHA titers of mercaptoethanol-treated serum constitutes a simple method for determining whether the serum sample was taken shortly or remotely after the infection.     

Analysis of specific IgM responses in secondary dengue virus infections: levels and positive rates in comparison with primary infections.

BACKGROUND: Dengue viruses are a serious cause of illness in tropical and subtropical areas of the world. Laboratory diagnosis is essential for confirmation of dengue virus infections. Detection of specific IgM by IgM-capture enzymed-linked immunoassay (ELISA) has been widely used as a main serological diagnostic technique. OBJECTIVES: The levels of specific IgM in secondary dengue virus infections were compared with those in primary infections. STUDY DESIGN: A total of 1780 samples collected from 924 confirmed dengue cases were tested for anti-dengue IgM by IgM-capture ELISA. RESULTS AND CONCLUSIONS: Specific IgM was detected in all the cases with primary dengue virus infection on disease day 9 or later. However, specific IgM cannot be detected in 28% (204/716) of the cases in secondary infections. The average titers of IgM were higher in primary infections than in secondary infections. The results confirmed that IgM detection is a reliable serological diagnostic test in primary dengue virus infections. Although IgM detection is also a useful test, other serological diagnostic tests or tests for dengue virus detection are necessary for confirmation of all the secondary dengue virus infections.