Yves Brossard

Frequency of early in utero HIV-1 infection : a blind DNA polymerase chain reaction study on 100 fetal thymuses

Objective: To estimate the prevalence of in utero transmission of HIV‐1 through the second trimester. Material and methods: One hundred consecutive, unselected, intact fetuses, beyond 15 weeks gestational age (mean, 22.4 weeks) were studied. These were obtained following spontaneous intrauterine deaths (n = 4), miscarriages (n = 4), and elective mid‐trimester terminations (n = 92), eight of which were fetuses with malformations from HIV‐1‐positive pregnancies. Coded DNA extracts from the fetal thymuses were tested blindly by polymerase chain reaction in three laboratories using a total of six different primer pairs. Results: Two thymuses tested positive [95% confidence interval (Cl), 0.2‐7]. Results from the three laboratories were consistent in all 100 cases. The two fetuses with HIV in the thymus both tested positive in other organs, demonstrating systemic HIV infection. The first fetus, whose mother had advanced AIDS, had died in utero and had diffuse toxoplasmosis. The second died following extremely premature delivery in a pregnancy complicated by repeated bleeding. HIV infection was observed in none of the 92 fetuses that resulted from elective mid‐trimester terminations (95% Cl, 0‐4). Conclusion: The frequency of early in utero HIV infection appears to be low, compared with transmission rates in infants born to HIV‐1‐infected mothers, suggesting that transmission occurs mostly later in pregnancy and/or at delivery. Specific risk factors may have implications in the occurrence of early as opposed to late transmission. AIDS 1995, 9:359‐366

Large-Scale Pre-Diagnosis Study of Fetal RHD Genotyping by PCR on Plasma DNA from RhD-Negative Pregnant Women

AbstractBackground: The routine prenatal determination of fetal RhD blood group would be very useful in the management of pregnancies in RhD-negative women, as up to 40% of these pregnancies bear a RhD-negative fetus. The fetal DNA present in maternal plasma offers an opportunity for risk-free prenatal diagnosis. Aim: This study focused on the feasibility and accuracy of large-scale RhD fetal diagnosis in non-immunized and anti-D immunized RhD-negative women. Methods: Plasma DNA was extracted from 893 RhD-negative pregnant women and amplified in exons 7 and 10 of the RHD gene using conventional and real-time PCR. The results were then compared with the RHD fetal genotype determined on amniotic cells and/or the RhD phenotype of the red blood cells of the infants at birth. Results: After exclusion of 42 samples from women exhibiting a nonfunctional or rearranged RHD gene, fetal RhD status was predicted with a 99.5% accuracy. A strategy is also proposed to avoid the small number of false-positive and -negative results. Conclusion: Fetal RHD genotyping from maternal plasma DNA in different clinical situations may be used with confidence.

Two hundred intrauterine exchange transfusions in severe blood incompatibilities

Two hundred intrauterine exchange transfusions were performed under local anesthesia in 107 cases of blood incompatibilities (60 fetuses with severe anemia and 47 with hydrops). Under sonographic guidance, depending on fetal and placental position, an optimal puncturing site was selected along the umbilical vein: placental insertion, fetal insertion, or fetal intraabdominal segment. Tests were immediately performed to confirm fetal origin of blood obtained and estimate hemoglobin level. Blood used for exchange transfusion was compatible with maternal blood and had a hematocrit value of 75%. Exchange transfusion was continued until a hemoglobin level of 16 gm/dl was reached. This procedure was first associated with intraperitoneal transfusions and was subsequently used independently once a month to maintain an adequate hemoglobin level. In 4 fetuses with hydrops, antenatal regression of this sign was observed in 33 cases (70.2%). Overall outcome of 107 fetuses after exchanges was 84 living neonates (78.5%), 15 deaths in utero, and eight neonatal deaths. The survival rate was 91.6% for fetuses without hydrops and 61.7% for those with hydrops. The advantage of exchange transfusion appears to be rapid and efficient correction of anemia with elimination of incompatible fetal red blood cells.

PCR-based determination of Rhc and RhE status of fetuses at risk of Rhc and RhE haemolytic disease

Summary. After anti‐RhD, anti‐Rhc is the most important red cell alloantibody which can cause haemolytic disease of the newborn (HDN) when the mother is Rhc‐negative and the fetus Rhc‐positive. We report here the development of polymerase chain reaction (PCR) assays which detect the presence of the Rhc alleles in amniotic cells by the use of allele‐specific primers (ASP). It is expected that such determination will help in the management of pregnancies at risk of Rhc haemolytic disease. In the course of this study we have similarly performed PCR‐ASP experiments to detect fetal RHE alleles since, in rare cases, anti‐RhE can also cause HDN.

Noninvasive fetal RHD genotyping from maternal plasma: Use of a new developed Free DNA Fetal Kit RhD®

Fetal RHD genotyping from maternal plasma was performed by real-time PCR amplification of exons 7 and 10 of the RHD gene and the amplified products were detected either with SYBR Green I dye according to our previously published method [Mol Diagn 8 (2004) 23-31] or with hydrolysis probes in a new Free DNA Fetal Kit RhD((R)). Plasma specimen from 300 RhD-negative pregnant women (between 10 to 34 weeks of gestation) were analysed and validation of the results was ascertained either by RHD genotyping on amniotic cells or by blood typing of the neonate at birth. We found 100% concordant results when comparing the two methods. Two false-positive but no false-negative results were found. Thus, the sensitivity of the assay was 100% and the specificity superior than 99%. These data confirm the accuracy of fetal RHD genotyping on maternal plasma using the Free DNA Fetal Kit RhD, thus allowing to propose non invasive PCR-based fetal RHD genotyping for all RhD-negative pregnant women and to restrict the use of anti-D immunoglobulins only to those bearing an RhD-positive fetus.

Prenatal diagnosis of anoxic cerebral lesions caused by profound fetal anemia secondary to maternal red blood cell alloimmunization.

BACKGROUND: The long-term neurological prognosis of severe fetal anemia is usually considered favorable, especially when fetal hydrops regresses after successful in utero transfusion. CASES: We report two cases of prenatally diagnosed fetal cerebral anoxic lesions associated with severe fetal anemia despite appropriate and successful treatment by in utero transfusion. The two pregnancies were terminated. CONCLUSION: Profound fetal anemia may cause anoxic lesions of the fetal brain that may be diagnosed prenatally. If new onset ventriculomegaly is observed on ultrasonography after in utero transfusion for severe fetal anemia, anoxic lesions could be suspected.

Le point sur le suivi des allo-immunisations érythrocytaires

Anti-RhD allo-immunisation has become rare since anti-D prophylaxis was introduced in the seventies; however, it remains the first cause of fetal anemia. It may cause severe fetal complications such as fetal hydrops, cerebral anoxic lesions and fetal death. In the neonatal period, severe jaundices and anemias requiring transfusion or exsanguino-transfusion are still common in case of severe allo-immunisation. Neonatal death and sequellae due to bilirubin encephalopathy have not fully disappeared. Follow-up of pregnancies with maternal allo-immunisation requires identification of the antibody (anti-RhD, anti-Kell and anti-c are the most frequently responsible for fetal complications), dosage and titration. In RhD allo-immunization, feto-maternal incompatibility may be confirmed by non-invasive RHD genotyping of the fetus in maternal blood. In cases at risk for fetal anemia, weekly Doppler assessment of middle cerebral artery peak systolic velocity (MCA-PSV) allows identification of fetal anemia before the occurrence of fetal hydrops. The reference treatment of fetal anemia is in utero fetal transfusion. The risk of fetal loss due to in utero transfusion (IUT) is 3% per procedure. The cumulated risk of fetal loss can thus exceed 10% in case of early occurrence of fetal anemia requiring up to five or six IUTs in a single pregnancy.

Intravenous fetal exchange transfusion before 22 weeks of gestation in early and severe red-cell fetomaternal alloimmunization.

Objectives: To determine the perinatal outcome in severe red-cell fetomaternal alloimmunization. Methods: Retrospective series of 32 affected fetuses treated with intravenous fetal exchange transfusion (IFET) before 22 weeks of gestation. The main outcome measures were the degree of fetal anemia, fetal transfusions and perinatal outcome. Results: The first IFET was performed at 19.8 ± 1.8 weeks of gestation. All fetuses were severely anemic and hemoglobin levels were not different between 20 hydropic and 12 nonhydropic fetuses (4.1 ± 2.5 vs. 5.6 ± 2.8 g/dl, p = 0.33). The initial maternal anti-D level ranged from 4 to 76 µg/l and was not correlated to fetal anemia (r = –0.07). Conclusion: The overall perinatal survival rate was 78% compared to a previous perinatal loss rate excluding first pregnancies of 55.5%.

Trends in HIV infection among sexually transmitted disease patients in Paris.

ObjectiveTo assess trends in HIV infection among sexually transmitted disease (STD) patients. DesignRepeated unlinked anonymous survey, 1991–1993. SettingSTD clinics in Paris, France. SubjectsPatients (n = 4354) with a new suspected STD. MethodsHIV antibody testing, using blood from syphilis samples. ResultsHIV prevalences were stable over time in all transmission groups. One-third of homo-/bisexual men were HIV-positive. Prevalence was 2.5 times higher among heterosexual patients from Africa or the Caribbean than among those from other countries. Among patients under 25 years of age prevalence significantly decreased from 4.3% in 1991 to 0.8%, in 1993 (P= 0.01). Among homo-/bisexual men, despite a 50% reduction in the incidence of STD, the absolute number of those newly HIV-infected remained stable; median age increased from 28 years in 1991 to 32 years in 1993 (P=0.02). Among heterosexuals, trends in HIV incidence were difficult to assess: recently infected patients were more likely to be identified in 1993 than in 1991, since the proportion of patients who reported a recent HIV-negative test increased over time. ConclusionPrevalence studies contribute to define specific subgroups which should be targeted for prevention (HIV-positive or older homosexuals, heterosexuals from Africa and the Caribbean). Despite a decrease in both overall STD incidence and HIV prevalence among patients aged under 25 years, overall HIV incidence has not decreased, at least among homo-/bisexual men in whom recent HIV infections occurred at a high rate overall, and increased in those aged 35 years or more. Sentinel site-based HIV seroprevalence studies are best interpreted in the light of results obtained from different populations and through routine surveillance of STD.

Testing for in utero human immunodeficiency virus infection with fetal blood sampling

OBJECTIVE Our purpose was to evaluate the potential of midtrimester fetal blood sampling to detect in utero human immunodeficiency virus type 1 infection. STUDY DESIGN Ultrasonographically guided fetal blood sampling was performed before pregnancy termination in 28 women infected with human immunodeficiency virus type 1. Mean gestational age was 22 weeks (range 15 to 29 weeks). Samples were tested for human immunodeficiency virus with undissociated p24 antigen and deoxyribonucleic acid polymerase chain reaction or viral isolation by cell culture. After terminations fetal thymuses were also tested for human immunodeficiency virus infection by polymerase chain reaction. Polymerase chain reaction was also performed on maternal cells to confirm that the primer pairs used were able to detect human immunodeficiency virus type 1 strains present. RESULTS All fetal blood samples had negative results by p24 antigen and polymerase chain reaction or virus culture and all fetal thymuses were negative by polymerase chain reaction. CONCLUSION Early in utero human immunodeficiency virus infection appears infrequent, supporting the hypothesis that mother-to-child transmission predominantly occurs late in pregnancy. Therefore midtrimester prenatal diagnosis is not currently of use to women in making reproductive decisions.