Routine noninvasive prenatal screening for fetal RHD in plasma of RhD-negative pregnant women-2 years of screening experience from Denmark.

OBJECTIVE: Prenatal and postnatal RhD prophylaxis reduces the risk of RhD immunization in pregnancies of RhD-negative women. Based on the result from prenatal screening for the fetal RHD gene, prenatal RhD prophylaxis in Denmark is targeted to RhD-negative women who carry an RhD-positive fetus. Here, we present a 2-year evaluation of a nationwide prenatal RHD screening. METHODS: Blood samples were drawn from RhD-negative women in gestational week 25. DNA was extracted from maternal plasma and analyzed for the RHD gene. The prenatal RHD results were compared with the serological typing of newborns in 12,668 pregnancies. Early compliance was assessed for 690 pregnancies. RESULTS: The sensitivity for the detection of fetal RHD was 99.9% (95% CI: 99.7-99.9%). Unnecessary recommendation of prenatal RhD prophylaxis was avoided in 97.3% of the women carrying an RhD-negative fetus. Fetuses that were seropositive for RhD were not detected in 11 pregnancies (0.087%). The sample uptake percentage was 84.2%, and the compliance for prenatal anti-D administration was 93.2%. CONCLUSION: The high sensitivity, maintained over 2 years, underlines the reliability of routine prenatal fetal RHD screening in RhD-negative pregnant women, specifically at 25 weeks of gestation. The remaining challenges are logistical and are related to program compliance.

Direct detection of Mycoplasma pneumoniae antigen in clinical specimens by a monoclonal antibody immunoblot assay.

Throat swabs from patients with pharyngitis and sputum specimens from patients with atypical pneumonia were tested for the presence of a Mycoplasma pneumoniae polypeptide with a molecular weight of 43,000 with the use of an M. pneumoniae species-specific monoclonal antibody in an immunoblot assay. This 43,000-dalton polypeptide was detectable in 33 of 33 throat swabs from patients with pharyngitis that were positive for M. pneumoniae by conventional culture as well as a culture-amplified enzyme immunoassay. The 43,000-dalton polypeptide was also detected in three of three M. pneumoniae culture-positive sputum specimens. It was not detected in 3 sputum specimens culture-confirmed for Legionella pneumophila, 10 sputum specimens from normal persons, or 25 throat swabs also from normal persons. This immunoblot assay could be completed within five hours and may be an alternative method for detecting M. pneumoniae antigen directly in sputum or throat swab specimens.

The simultaneous direct detection of Mycoplasma pneumoniae and Legionella pneumophila antigens in sputum specimens by a monoclonal antibody immunoblot assay.

An immunoblot (Western) assay was developed employing a species-specific monoclonal antibody to a 43 kDa Mycoplasma pneumoniae membrane polypeptide and a species-specific monoclonal antibody to 29 kDa Legionella pneumophila outer membrane protein. This assay could simultaneously detect these two different antigens directly in sputum. The 43 kDa M. pneumoniae antigen was detected by this assay in each of three M. pneumoniae culture-confirmed sputum specimens. In addition, the 29 kDa L. pneumophila antigen was detected in three of three L. pneumophila culture-confirmed sputum specimens. Neither of these two specific antigens were detected in induced sputum specimens from ten normal individuals.

Species-specific monoclonal antibody to a 43,000-molecular-weight membrane protein of Mycoplasma pneumoniae.

A murine immunoglobulin G1 monoclonal antibody was produced that binds to a protease-sensitive, periodate-insensitive epitope on a 43,000-molecular-weight Mycoplasma pneumoniae membrane polypeptide. The 43,000-molecular-weight polypeptide appeared to be a major antigenic component of M. pneumoniae, as determined by immunoblot analysis. This monoclonal antibody reacted with 33 different clinical isolates of M. pneumoniae, but not with normal-flora Mycoplasma species or 18 other microorganisms potentially inhabiting the normal or diseased human respiratory tract. This apparent species-specific monoclonal antibody may have application for the detection of M. pneumoniae antigen in clinical specimens.

Reactivity of nonsensitized control erythrocytes in rubella passive hemagglutination assays.

Two commercially available, nonsensitized control erythrocytes for use in rubella antibody passive hemagglutination assays detected nonspecific reactions in 10 of 600 (1.7%) and 9 of 500 (1.8%) sera tested. Reactive sera were positive with one or the other cell type, but not both. The probability of obtaining a false-positive result owing to a nonspecific passive hemagglutination reaction was estimated to be 0.17% for either cell type.

Enzygnost-Rubella: an enzyme immunoassay for screening and quantitation of antibodies to rubella virus.

Calbiochem-Behring, Enzygnost-Rubella is an enzyme-linked immunosorbent assay (ELISA) for the determination of IgG antibodies to rubella virus. Two procedures, screening and quantitative, were used for the measurement of rubella antibodies in a characterized panel of sera and in random, premarital serum specimens. Using the screening procedure, no false-positive or false-negative results were observed in testing a panel of 40 sera previously characterized by human "O" cell and chick cell hemagglutination-inhibition (HI) tests. Additional testing of 323 random, premarital serum samples resulted in 99.4% agreement with HI tests in identifying positive and negative specimens. In the evaluation of the quantitative procedure, a coefficient of correlation (r) of 0.93 between ELISA titers and the geometric mean HI titers was obtained when testing the panel of 40 characterized sera. Furthermore, 100% agreement with HI test was obtained in the detection of titer rises in 20 pairs of acute and convalescent sera. The average within-run coefficients of variation for the screening and quantitative procedures were determined to be 11.2% and 15.6%, respectively. It is concluded that the Enzygnost-Rubella reagents are sensitive, specific, and provide objective means for assessing immunestatus and detecting rises in antibody titers to rubella virus.

New passive hemagglutination assay kit that uses hemagglutinin-sensitized erythrocytes for detection of rubella antibodies.

A new passive hemagglutination assay for the detection of antibodies to rubella virus hemagglutinin (PHAST-Rubella) was compared with the hemagglutination inhibition (HI) test and another passive hemagglutination test that uses a soluble rubella virus antigen (SA-PHA). When the immune responses of vaccinated individuals were monitored, similar rises in antibody titer were detected by HI or PHAST-Rubella, whereas the rise in titer detected by SA-PHA was delayed. Early-phase vaccine-induced immunoglobulin M antibody analyzed by sucrose gradient fractionation was detected to the same degree by HI and PHAST-Rubella, but early-phase immunoglobulin G antibody reacted more strongly in the HI test. When acute and convalescent serum pairs from rubella-infected individuals were evaluated, a fourfold rise in titer was detected by PHAST-Rubella and HI in 15 of 15 pairs, whereas SA-PHA, which is not intended for detecting antibody titer rises in acute infections, detected a rise in titer in only 3 of 15 pairs. In studies to determine rubella immune status, testing of 1,078 premarital and random serum specimens resulted in 98.6% agreement among the three methods in identifying rubella antibody-positive and -negative individuals. For the quantitative PHAST-Rubella procedure, a coefficient of correlation of 0.93 was obtained, in comparison with HI, when a panel of 40 characterized sera were tested. These results indicate that PHAST-Rubella reagents can detect rubella antibodies as well as HI reagents and thus may be used as a fast and accurate means of determining rubella immune status and for the quantitation of rubella antibodies.

Synthesis and antiviral activity of certain 9-beta-D-ribofuranosylpurine-6-carboxamides.

To examine the structural parameters necessary for antiviral efficacy of certain purine nucleosides, several 9-beta-D-ribofuranosylpurine-6-carboxamides have been synthesized. Glycosylation of the Me3Si derivative of purine--6-carboxamide with protected ribofuranose in the presence of a Lewis acid gave the blocked nucleoside which on deprotection furnished 9-beta-D-ribofuranosyl-6-iodopurine with cyanide ion. Certain 2-amino- and 2-methyl-9-beta-D-ribofuranosylpurine-6-carboxamides have also been prepared. 8-Carbamoylguanosine (16) has been prepared by homolytic acylation of the parent nucleoside. These compounds were tested against several RNA and DNA viruses in cell culture. 9-beta-D-Ribofuranosylpurine-6-carboxamide (6a), the corresponding 6-thiocarboxamide (7b), and 4-amino-8-(beta-D-ribofuranosylamino)pyrimido[5,4-d]pyrimidine (8) showed significant in vitro antiviral activity at nontoxic dosage levels. 6a employed in the treatment of Rift Valley fever virus infected mice at 50 (mg/kg)/day gave a 55% survival rate on day 21 compared to a 30% survival in the controls.