Twenty-two molecular diagnostic laboratories from 14 countries participated in a consortium study to estimate the impact of Factor VIII gene inversions in severe hemophilia A. A total of 2,093 patients with severe hemophilia A were studied; of those, 740 (35%) had a type 1 (distal) factor VIII inversion, and 140 (7%) showed a type 2 (proximal) inversion. In 25 cases, the molecular analysis showed additional abnormal or polymorphic patterns. Ninety-eight percent of 532 mothers of patients with inversions were carriers of the abnormal factor VIII gene; when only mothers of nonfamilial cases were studied, 9 de novo inversions in maternal germ cells were observed among 225 cases (approximately 1 de novo maternal origin of the inversion in 25 mothers of sporadic cases). When the maternal grandparental origin was examined, the inversions occurred de novo in male germ cells in 69 cases and female germ cells in 1 case. The presence of factor VIII inversions is not a major predisposing factor for the development of factor VIII inhibitors; however, slightly more patients with severe hemophilia A and factor VIII inversions develop inhibitors (130 of 642 [20%]) than patients with severe hemophilia A without inversions (131 of 821 [16%]).

To improve the diagnosis of invasive aspergillosis (IA), we retrospectively compared competitive polymerase chain reaction (PCR) and sandwich ELISA for detection of serum galactomannan (GM) antigen. We studied 281 serum samples collected weekly during the period at risk for IA from 41 selected hematology patients. Twenty-two patients had confirmed, probable, or suspected IA, according to clinical and mycologic data. Fifteen of them had positive GM titers (87 samples) and 12 had positive PCRs (20 samples). Nineteen of the 20 PCR-positive samples were also GM-positive. Of the 19 patients without IA (83 samples), one had 3 GM-false-positive samples. Neither test anticipated the initiation of antifungal therapy on the basis of clinical suspicion. Both tests were more likely to be positive before death. This study suggests that PCR on serum samples is not more sensitive than GM detection. However, PCR can improve the specificity of the GM test. Together, these noninvasive tests should improve the diagnosis of IA.

To improve the diagnosis of invasive aspergillosis (IA), we developed a LightCycler PCR assay targeted to Aspergillus fumigatus and A. flavus mitochondrial DNA. To avoid contamination, fully automated nucleic acid extraction with the MagNA Pure LC apparatus was used. The linearity of the results was achieved over a 6-log range of input A. fumigatus DNA, from 0.3 ng to 3 fg/10 l of water. We retrospectively compared the LightCycler PCR and an enzyme-linked immunosorbent assay for the detection of galactomannan (GM) in serum from 14 patients with IA. The GM assay was more frequently positive (57 of 109; 52%) than the PCR assay (49 of 109; 45%). The LightCycler PCR assay, combined with automated DNA extraction, could be used in association with the GM assay to improve the reliability of IA diagnosis.The incidence of invasive aspergillosis (IA) is difficult to assess, as at least 30% of the cases that occur remain undiagnosed and untreated at death (7). Two approaches have been investigated to improve the sensitivity of aspergillosis detection by noninvasive techniques; these are antigen detection and DNA detection (13). Among the antigen tests, detection of galactomannan (GM) by an enzyme-linked immunosorbent assay (ELISA) has been shown to have good sensitivity, but a false-positivity rate of 10 to 15% has been reported (5, 16). Nevertheless, this method is now included in the IA diagnosis criteria (1).PCR technology is not as acceptable a diagnostic tool as the GM assay because very divergent results have been reported (3, 9). These discrepancies are most likely due to technical reasons because the PCR techniques used are not standardized. Real-time PCR assays dramatically decrease the risk of false-positive results. Indeed, contamination with previously amplified products is reduced because the reaction tubes need not be opened following amplification, thus avoiding potential contamination of the environment with amplicons (6, 15). We therefore developed a PCR assay based on the LightCycler technology (19). We also employed a fully automated nucleic acid extraction technique with the MagNA Pure LC apparatus (12). This apparatus can purify DNA from a broad variety of samples by magnetic bead technology, thus eliminating the need for vacuum pumps, centrifugation, or other manual steps that may result in cross contamination. We subsequently compared the performance of the GM assay and the real-time PCR test with samples from selected patients.LightCycler PCR test development. The LightCycler PCR test was targeted at the Aspergillus fumigatus mitochondrial gene (GenBank accession number L37095) (4). The amplicon comprises a 91-bp fragment. The two hybridization probes (TibMolBiol, Berlin, Germany) used are the 24-mer 5Ј-CTGT TAGTGCGGGAGTTCAAAXTCT-3Ј, where X represents the internal labeling of the T with LC-Red 640, and the 28-mer 5Ј-CTGAGCTAATTTCTTTCAACCCAAGGGA-3Ј, which is labeled at the 3Ј end with fluorescein isothiocyanate. Because of the thermodynamic constraints on the choice of the primers and probes, the LC...

Circulating cell-free fetal DNA in maternal serum offers an early and non-invasive method for prenatal diagnosis, but the origin of this DNA is still unknown. We report the absence of the SRY gene in maternal serum of a pregnant woman despite male genitalia at ultrasound. The karyotype was 45,X after direct trophoblast analysis and 45,X/46,Xidic(Yp) after culture and in all fetal tissues studied. Due to the absence of the SRY sequence in maternal blood and in the cytotrophoblast, we presume that free fetal DNA in this case originates from trophoblastic cells. As the case presented here is exceptional, it only has a minor impact on the accuracy of fetal sex determination by maternal serum analysis, but highlights the importance of and the necessity for the complementary ultrasonographic control.

Fetal sex prediction can be achieved using PCR targeted at the SRY gene by analysing cell-free fetal DNA in maternal serum. Unfortunately, the results reported to date show a lack of sensitivity, especially during the first trimester of pregnancy. Therefore, determination of fetal sex by maternal serum analysis could not replace karyotype analysis following chorionic villus sampling. A new highly sensitive real-time PCR was developed to detect an SRY gene sequence in maternal serum. Analysis was performed on 121 pregnant women during the first trimester of pregnancy (mean gestational age: 11.8 weeks). Among them, 51 had at least one previous male-bearing pregnancy. Results were compared with fetal sex. SRY PCR analysis of maternal serum was in complete concordance with fetal sex. Among the 121 pregnant women, 61 were bearing a male fetus and 60 a female fetus. No false-negative results were observed. Furthermore, no false-positive results occurred, even though 27 women carrying a female fetus during the current pregnancy had at least one previous male-bearing pregnancy. This study demonstrates that a reliable, non-invasive sex determination can be achieved by PCR analysis of maternal serum during the first trimester of pregnancy. This non-invasive approach for fetal sex prediction should have great implications in the management of pregnant women who are carriers of an X-linked genetic disorder. Prenatal diagnosis might thus be performed for male fetuses only, avoiding invasive procedures and the risk of the loss of female fetuses.

No abstract

The human testis-determining factor resides within a 35-kilobase (kb) region of the Y chromosome immediately adjacent to the pseudoautosomal buary. A candidate gene for human sex determination (SRY) was isolated in this region. Here, we describe a study of 25 cases of XY females with pure gonadal dysgenesis for mutations on the Y chromosome short arm, including SRY. Southern blotting revealed a sex-reversed female harboring a deletion extending from -8 kb from the pseudoautosomal boundary of the Y chromosome to at least 33 kb and no more than 60 kb upstream, toward the centromere. The deletion begins no more than 1.8 kb upstream from the first ATG of the SRY open reading frame present in the clone pY53.3. To our knowledge, no mutation has been described previously outside the SRY "HMG box" on the short arm of the Y chromosome, which is assoiated with sex reversal. Since the 5' extent of the SRY tnswriptional unit has not been defined, the deletion may remove up m exons of SRY and/or transcriptional regulatory motifs, either situation resulting in lack of ticular development. It cannot be formally excluded that the mutation removes a second locus, independent of SRY, that is critical for sex deternmnation. Denaturant gradient gel electrophoresis analysis of the SRY open reading frame in the remaining 24 cases revealed de novo single base-pair transitions in the SRY conserved domain in 4 cases.

Rapid identification of patients infected with clarithromycin-resistant Helicobacter pylori without the need for culture can help to avoid useless prescriptions of clarithromycin. We developed and tested a routine real-time quantitative PCR assay dedicated to that purpose. One hundred ninety-six consecutive gastric biopsy specimens were examined by culture, histology performed by a trained physician, and rapid PCR with the LightCycler apparatus. Infection was defined as (i) positivity of culture, (ii) positivity of histology, or (iii) positivity of PCR if confirmed by positivity of a concomitant indirect test (serology or urea breath test). Susceptibility to clarithromycin was tested by E-test and PCR. The prevalence of infection was 33.7% (66 of 196 samples). The sensitivities of culture, histology, and PCR were 90.9% (60 of 66 samples), 87.9% (58 of 66 samples), and 97.0% (64 of 66 samples), respectively. The specificity of PCR was 94.6% (123 of 130 samples). The linearity of the PCR results was achieved over a 6-log range of input DNA, and we were able to accurately quantify as few as 300 bacteria and to qualitatively detect as few as 30 bacteria per DNA sample. For clarithromycin susceptibility testing, there was 98.2% (55 of 56 samples) concordance between E-test and PCR. Forty-eight strains were clarithromycin susceptible, and 9 strains were clarithromycin resistant. The single discrepancy concerned a culture which was a mixture of mutant and wild type, with a susceptible-to-resistant ratio of 11.5: the resistant population was detected by E-test but not by PCR. Our PCR assay is accurate for fast detection of H. pylori as well as of clarithromycin resistance and is also able to objectively determine bacterial density.Many diagnostic assays have been developed for Helicobacter pylori: culture, histology, rapid urease test, urea breath test, serology, stool antigen test, and molecular-based tests (24). Culture has the great advantage of permitting subsequent determination of the antimicrobial susceptibility of the strain isolated, in particular to macrolides. Indeed, the macrolide drug clarithromycin is the key component of many combination therapies used to eradicate H. pylori, and macrolide resistance is the most important cause of treatment failure (13, 22). However, disadvantages of culture include special conditions for specimen transportation, the use of complicated media with special conditions for maintenance, the need for special incubation conditions, and the length of time necessary to obtain a result (20).Clarithromycin resistance in H. pylori is due to the lack of binding of the macrolides to the 23S rRNA components of the bacterial ribosome due to modification of the target site by occurrence of a single spontaneous point mutation in the peptidyltransferase region of the 23S rRNA gene (17,25). Mutations A2142G and A2143G are the most often observed, with the A2142C mutation being much rarer (25). Other mutations (A2115G, G2141A, and T2717C) have been described but appear to be exceptional (10,1...