Pauline Ernault

Comparison of two DNA targets for the diagnosis of Toxoplasmosis by real-time PCR using fluorescence resonance energy transfer hybridization probes

BackgroundToxoplasmosis is an infectious disease caused by the parasitic protozoan Toxoplasma gondii. It is endemic worldwide and, depending on the geographic location, 15 to 85% of the human population are asymptomatically infected. Routine diagnosis is based on serology. The parasite has emerged as a major opportunistic pathogen for immunocompromised patients, in whom it can cause life-threatening disease. Moreover, when a pregnant woman develops a primary Toxoplasma gondii infection, the parasite may be transmitted to the fetus and cause serious damnage. For these two subpopulations, a rapid and accurate diagnosis is required to initiate treatment. Serological diagnosis of active infection is unreliable because reactivation is not always accompanied by changes in antibody levels, and the presence of IgM does not necessarily indicate recent infection. Application of quantitative PCR has evolved as a sensitive, specific, and rapid method for the detection of Toxoplasma gondii DNA in amniotic fluid, blood, tissue samples, and cerebrospinal fluid.MethodsTwo separate, real-time fluorescence PCR assays were designed and evaluated with clinical samples. The first, targeting the 35-fold repeated B1 gene, and a second, targeting a newly described multicopy genomic fragment of Toxoplasma gondii. Amplicons of different intragenic copies were analyzed for sequence heterogeneity.ResultsComparative LightCycler experiments were conducted with a dilution series of Toxoplasma gondii genomic DNA, 5 reference strains, and 51 Toxoplasma gondii-positive amniotic fluid samples revealing a 10 to 100-fold higher sensitivity for the PCR assay targeting the newly described 529-bp repeat element of Toxoplasma gondii.ConclusionWe have developed a quantitative LightCycler PCR protocol which offer rapid cycling with real-time, sequence-specific detection of amplicons. Results of quantitative PCR demonstrate that the 529-bp repeat element is repeated more than 300-fold in the genome of Toxoplasma gondii. Since individual intragenic copies of the target are conserved on sequence level, the high copy number leads to an ultimate level of analytical sensitivity in routine practice. This newly described 529-bp repeat element should be preferred to less repeated or more divergent target sequences in order to improve the sensitivity of PCR tests for the diagnosis of toxoplasmosis.

Fetal RHD genotyping in maternal serum during the first trimester of pregnancy

Summary.  Fetal RHD genotype determination is useful in the management of sensitized RhD‐negative pregnant women. It can be ascertained early during pregnancy by chorionic villus sampling (CVS) or amniocentesis. However, these procedures are invasive, resulting both in an increased risk of fetal loss and in an increased severity of immunization due to fetomaternal haemorrhage. A reliable determination of RHD genotype by fetal DNA analysis in maternal serum during the first trimester of pregnancy is reported in this study. One hundred and six sera from RhD‐negative pregnant women were obtained during the first trimester of pregnancy. These sera were tested for the presence of RHD gene using a new real‐time polymerase chain reaction assay and the results compared with those obtained later in pregnancy on amniotic fluid cells and by RHD serology of the new‐born. All sera from women carrying a RhD‐positive fetus (n = 62) gave positive results for RHD gene detection and sera from women carrying a RhD‐negative fetus (n = 40) were negative. The high level of accuracy of fetal RHD genotyping obtained in this study could enable this technique to be offered on a routine basis for the management of RhD‐negative patients during the first trimester of pregnancy.

Prenatal diagnosis of congenital toxoplasmosis by duplex real-time PCR using fluorescence resonance energy transfer hybridization probes.

The diagnosis of congenital toxoplasmosis frequently relies on PCR tests of amniotic fluid (AF). A duplex real‐time quantitative PCR test based on fluorescence resonance energy transfer was developed to quantify the parasite load and to decrease the risk of contamination. An internal control based on the detection of 10 pg mouse DNA added to the AF was included to check for PCR efficiency. The relationship between the parasite load and the occurrence of ultrasonographic abnormalities in 87 samples of AF was analyzed. Seven AF (8%) had a parasitic load >103; 14 (16%) had >102–≤103; 26 (30%) had >10–≤102; and 40 (46%) had ≤10 parasites/ml. Four of the six AF with cerebral ventriculomegaly had >103 parasites/ml. The other two had 130 and 24 parasites/ml, respectively. No parasitic loads of >103 parasites/ml and no ultrasonographic abnormalities were observed in the 11 AF with maternal toxoplasmosis in the third trimester. Therefore, there is a trend to associate high parasite count with ultrasonographic abnormality, but the main concern remains early maternal infection. The importance of quantification should be better evaluated with postnatal studies. The duplex LightCycler PCR test currently provides rapid and safe results. Copyright

Detection and Quantification of Mycoplasma genitalium in Male Patients with Urethritis

Detection and quantification of Mycoplasma genitalium were evaluated in 83 patients with urethritis (group 1), 60 patients with urethral symptoms but no urethritis (group 2), and 50 asymptomatic men (group 3). Quantification of M. genitalium was carried out using real-time polymerase chain reaction (PCR) analysis of first-pass urine samples. The rate of detection of M. genitalium was significantly higher in group 1 than in groups 2 and 3 (P<.0001). The mean observed concentration of M. genitalium was 1.2x10(4) equivalent genomes/mL of urine (range, 50 to 8x10(4) equivalent genomes/mL). Analysis of M. genitalium load in serial urine samples collected before and after the administration of antibacterial treatment showed an association between clinical and microbiological responses, with a shift to negative PCR results in symptom-free patients. Our results illustrate the usefulness of monitoring the M. genitalium load in evaluating the susceptibility of M. genitalium to antibacterial treatment.

OP01.22: Very early prenatal diagnosis of genetic diseases based on celomic fluid analysis: a feasibility study

noted, the initial risk, which was based on maternal age and/or maternal serum screening, was multiplied by the LR of that marker. When more than one marker was found, then the initial risk was multiplied by the highest LR or 6.2. The absence of a marker had a LR of 0.5. After URAD, the patients were assigned into two groups depending upon whether their final calculated risk was greater or less than 1/270. The at risk group was further divided into an intermediate risk and high risk group (> 1/100 risk). Results: Group

Rapid and Quantitative Detection of Toxoplasma gondii by PCR — A LightCycler Application in Prenatal Diagnosis

Toxoplasmosis is an infectious disease that is distributed worldwide and caused by the protozoan parasite Toxoplasma gondii (T. gondii). The infection is usually asymptomatic and harmless in immunocompetent patients but can be life-threatening or responsible for severe sequelae in immunocompromised individuals such as the fetus and HIV and transplant patients. In these situations, early treatment significantly reduces the extent of the damage [1]. However, the classical diagnosis of toxoplasmosis based on serological tests is inefficient and inadequate in these patients. Therefore, the diagnosis is based on the direct demonstration of the parasite in tissues or biological fluids. This can be achieved by using tissue-culturing or mouse inoculation. However, tissue-culturing has a low sensitivity and inoculation into mice requires waiting more than 3 weeks for the results [2]. PCR overcomes these shortcomings [3]. Indeed, the PCR is sensitive and the diagnosis can be given in 1 day. The use of PCR has been particularly useful in facilitating the greatly improved and simplified prenatal diagnosis of toxoplasmosis, by making it faster and more sensitive [4]. However, the main risk is that of false positive results due to contamination with previously amplified products. The recent development of a real-time, on-line PCR procedure gives the opportunity to control this risk, because it uses a closed tube format for amplification and detection. We have, therefore, tested the feasibility of this technique in a routine laboratory and compared the results to PCR with ELISA detection of biotin-labeled probes (conventional PCR).