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A sensitive, rapid and specific diagnostic method is essential for the diagnosis of toxoplasmosis in immunocompromised hosts or in congenital infection. We report the development of a real-time PCR assay for quantitative diagnosis of toxoplasmosis with competitive internal amplification control. This PCR was applied after allogeneic stem cell transplantation to estimate the frequency of reactivation.Primers and Taqman probe (FAM-BHQ1) were designed to amplify the 529 bp element of T. gondii. The internal amplification control was developed by cloning a fragment of Arabidopsis thaliana DNA flanked by sequences specific for T. gondii 529 bp element. A Taqman probe specific for the competitive internal control was designed and tested (YY-BHQ1). We determined the repeatability and reproducibility of the method. A prospective study was performed on adults who received an allogeneic hematopoietic stem cell transplantation. After transplantation, patients were monitored once per week during the first 100 days; they were then monitored once every 2 weeks until day 180 after transplantation (i.e., day +180).A total of 451 samples from 40 patients were tested. Twenty-five patients had both positive toxoplasmosis serology and an adequate chemoprophylaxis. One sample from one patient was found positive. The rate of reactivation in the population of this study is 4%.A monitoring by T. gondii PCR should be realized weekly for patients receiving an allogeneic hematopoietic stem cell transplantation, without an adequate chemoprophylaxis.

The presence of Helicobacter pylori. in the oral cavity remains controversial and the most appropriate method for detection of oral H. pylori has yet to be established. The aim of the present study was to compare four different primer sets on the detection of H. pylori in gastric biopsies and oral samples using real-time PCR.Gastric biopsy and oral samples were collected from eight patients with gastric symptoms. DNA from clinical samples was extracted and analyzed for the presence of H. pylori by real-time PCR (LightCycler 2.0) using four pairs of primers which targeted 16S rRNA (16S rRNA#295; 16S rRNA#120) or glmM (glmM#294; glmM#722) DNA genes. Three H. pylori strains and three clinical isolates served as reference. The specificities of the four primer pairs were examined for seven oral microorganisms and two Helicobacter non-pylori species.Primer pair 16S rRNA#120 showed an acceptable specificity and a high sensitivity. Primer pairs glmM#294 and glmM#722 demonstrated a high specificity but a low sensitivity and primer pair 16S rRNA#295 demonstrated a poor specificity but acceptable sensitivity. Four H. pylori positive gastric biopsies were demonstrated by culture, histology and real-time PCR with primer pairs 16S rRNA#295 or 16S rRNA#120. No H. pylori was detected in oral samples, either by culture or by real-time PCR.Of the four different primer pairs examined, 16S rRNA#120 was the most appropriate to detect H. pylori in clinical samples using real-time PCR.

Fluorescent in situ hybridization (FISH) analysis is a molecular technique allowing the detection of recurrent translocations in cancer. Several hybridization protocols were assayed in order to evaluate their performances for interphase FISH analysis of histological sections and imprints using split probes. Adult and foetal lymphoid tissues were selected. Touch imprints of fresh (EF) or frozen (EC) tissues, sections (CF) and isolated nuclei (NI) of formol-fixed paraffin-embedded tissues were performed. The cut-off values of the IGH, IGlambda, BCL-2, BCL-6, CCND1 and MYC DNA FISH split signal probes were calculated for adult reactive lymph nodes on the different histological preparations (EC, CF, CC, NI) and on several tissues for the IGH and BCL-6 probes. In reactive lymph nodes, the cut-off values of the probes were between 3 and 13% and found independent of the preparation type. Conversely, slight but significant variations of the cut-off level were observed when different foetal control tissues were assayed with the same probe set. Finally, this study provided optimized-protocols for FISH analysis of either fresh/frozen imprints or formalin-fixed paraffin-embedded sections using split signal DNA probes.

Microscopic examination of sputum smears (search for acid-fast bacilli or AFB) is the most rapid procedure for diagnosis of contagious tuberculosis. Gene amplification is not yet reliable for direct detection of M. tuberculosis in clinical specimens that are AFB smear-negative. Culture (3 to 8 weeks on Lowenstein-Jensen medium or 1 to 4 weeks in liquid media) remains essential to identify AFB and conduct antibiotic susceptibility testing. AFB from culture can be identified in a few hours by molecular approaches with specific DNA probes. Results of susceptibility testing, even in liquid media, are not available until 2 to 4 weeks after the recovery of specimens, although mutations of the rpoB and katG 315 genes, which confer resistance to rifampin and isoniazid, can be detected within hours by molecular hybridization with specific probes fixed on strips. Immunologic tests that measure the interferon gamma produced by sensitized lymphocytes are promising tools for the diagnosis of latent tuberculosis.

Chromosomal aberrations are the first cause of mental impairment and dysmorphism. Rearrangements involving large chromosomal segments can be detected by standard chromosome analysis using GTG-banding, but this technique is not suited for the detection of small chromosome abnormalities. Array comparative genomic hybridisation (array-CGH) is a method used to detect segmental DNA copy number alterations. Recently, advances in this technology have enabled high-resolution examination for identifying genetic alterations and copy number variations on a genome-wide scale. This review describes the current genomic array platforms and CGH methodologies and highlights their applications for studying constitutional disease.

The HER2 proto-oncogene encodes a transmembrane protein, which is considered to function as a growth factor receptor. Overexpression of this protein found by immunohistochemistry in about 20% of infiltrating breast carcinomas, has a predictive value of response to treatment by trastuzumab, an anti-HER2 humanized monoclonal antibody. Search for HER2 gene amplification is necessary to adapt the immunohistochemical technique quality and also in the cases of delicate analysis or weak overexpression. It is usually carried out by Fluorescence In Situ Hybridization (FISH). A more recent hybridization technique, named CISH because of its chromogenic revelation is an alternative method, which gives highly correlated results with FISH. We present details of this technique, which may be more familiar for the pathologists than FISH, because reading analysis is similar to that of immunohistochemical staining.

This study reports on the evaluation of two L. infantum specific DNA probes for the diagnosis of canine leishmaniasis. The probes presented very satisfying performances in terms of specificity (100%) and predictive value of the positive result (100%). However, their sensitivity (35.3%) and the clinical complexity of canine infections render their use difficult in epidemiological surveys of visceral leishmaniasis aiming at measuring the prevalence of the dog infection by L. infantum. The sensitivity of these tools has improved (66.7%) when dogs presenting patent leishmaniasis were considered. Such probes constitute appropriate tools to confirm suspected cases of leishmaniasis. Unlike the classical parasitological and serological tools, this kind of tools allows a concomitant detection and identification of the causative agent. Therefore, despite their low sensitivity, these probes can still be of importance in epidemiological investigations.

Fetal cells exist in maternal blood and can be utilized for prenatal diagnosis. These cells are present from the sixth week of gestation, with frequency increasing as gestation advances, to many years after the birth. Enrichment of trophoblast cells, erythroblasts and lymphocytes was performed with various density gradient techniques and either magnetic activated or fluorescent activated sorting techniques. The abnormalities were detected by fluorescent in-situ hybridation (FISH) with chromosome-specific DNA probes for the detection of trisomy 21, trisomy 18, Klinefelter syndrome 47 XXY, or by polymerase chain reaction (PCR) for the detection of fetal sex, certain Mendelian disorders (as beta-globin mutations), HLA polymorphisms and fetal Rhesus D blood type. However others studies were necessary to determine the sensitivity and specificity of this technique as a noninvasive alternative to conventional methods of prenatal cytogenetic diagnosis.

A specific link between human papillomavirus (HPV) types 16, 18, 31, and 33 and genital carcinomas and between HPV type 5 and cutaneous extragenital carcinomas in patients with epidermodysplasia verruciformis and renal transplant has been previously found. The aim of this prospective study was to detect HPV in cases of cutaneous extragenital Bowen's disease (BD) from non-immunosuppressed patients.Twelve cases of cutaneous extragenital BD or Bowen's carcinoma (BC), seen in the period 1994-1996 and confirmed by histologic examination, were included in the study. Tissue sections were studied by in situ hybridization with a mixture of HPV DNA probes and specific HPV DNA probes. In addition, study on fresh materiel from 1995 included: Southern blot hybridization with various usual HPV probes (6, 11, 16, 18, 31, 33, 35, 39, 42), polymerase chain reaction (PCR) with hybridization using consensus HPV probes and probes specific for HPV types 6, 11, 16, 18 and 33. In positive samples with conventional PCR, in situ PCR with probes specific for HPV types 6/11 and 16 was performed on tissue sections.In situ hybridization was negative in all the cases. Southern blot hybridization was negative in our 9 studied cases. Three cases studied by consensus PCR were positive. PCR with specific HPV probes revealed positivity on two of these cases: HPV 6 in one, and HPV 16 in another. In situ PCR was positive with a mixed 6/11 HPV probe in the third positive consensus PCR case.Our study revealed the presence of HPV in 3 out of 12 cases of cutaneous extragenital BD and BC. HPV type 16, found in BC of skull, was the most usually found type in the literature. HPV types 6/11, detected in 2 cases, were rarely found in cutaneous extragenital BD and BC and these results are in favor of the oncogenic effect of these virus types. In our study, in situ hybridization and Southern blot hybridization were negative in all the cases; HPV was only found in 3 cases by conventional PCR and in 1 case by in situ PCR. The low range of detection of HPV in cutaneous extragenital BD may be due to the used methods, to difficulties related to sampling and/or to a low number of copies of the HPV genoma.

Paternity testing is based on biological analyses that have drastically developed during the past 20 years. According to scientific developments, paternity testing was based on red blood groups studies, the analysis of red cell enzymes and plasma proteins polymorphisms, the typing of the HLA-A and B antigens, and the DNA polymorphism in its various forms. This study aims at comparing the various analyses so that each becomes a reference for the others, in order to determine (i) the acceptable level of the paternity probability evaluation, and (ii) the exclusion rules according to new technologies. The relative performances of the various combinations of tests is analyzed in this report, based on a study of 877 cases. Thanks to studies based on the gene amplification of micro-satellites, the efficiency of this technique has been proved: it is however necessary to identify the limits of the tests and to know how to deal with those cases which require further analysis. Beyond the most efficient biological analysis, it is very important to think about paternity testing as a process in which biological tests are only one step.

Five different methods for identification of pneumococci (optochine susceptibility, bile solubility, Slidex Pneumo-kit, Phadebact Pneumococcus test, AccuProbe DNA test) were evaluated with a total of 280 Streptococcus pneumoniae non typable strains. 189 strains were identified as pneumococci according to the AccuProbe test results. Among these, 180 strains (95.2%) were optochine sensitive (dor = 12 mm). Bile solubility was seen in 125 (66.1%) of the pneumococci. Immunological identifications were respectively positive for 67 and 56 among 140 strains. By comparison with the DNA/RNA reassociation method, the poor sensitivities and specificities of the presumptive identification tests are actually demonstrated for pneumococcal non typable strains. Thus, the AccuProbe DNA test is seen as the only adequate method for identification of such strains.

Before the 1980s, the only available method for control of onchocerciasis was elimination of blackfly vector populations. This strategy was used with considerable success in the Onchocerciasis Control Programme in West Africa (OCP). The discovery of ivermectin, the first effective drug suitable for mass treatment of onchocerciasis, has revived international interest not only in fundamental research but also in development of new strategies to control onchocerciasis in the countries outside the OCP area. This report gives an overview of current parasitological, clinical, epidemiological and diagnostic data about onchocerciasis. Although little is known about the early development of Onchocerca volvulus in the human host, significant insight has been gained into the population dynamics of the parasite. The pathogenesis of cutaneous and ocular manifestations in onchocerciasis is now better understood. Epidemiological studies are under way to evaluate the extent of systemic manifestations. Recently developed diagnostic methods are more sensitive than conventional parasitological techniques. A new method for rapid assessment of endemic level has provided a detailed picture of the distribution of onchocerciasis. Species- and strain-specific DNA probes have been developed for identification of parasites in West Africa. New methods for quantifying disability allow evaluation of the socio-economic impact of the cutaneous and ocular complications of onchocerciasis.

The Philadelphia chromosome (Ph) resulting from translocation t(9;22)(q34;q11) is observed in more than 90% of patients with chronic myeloid leukemia (CML). Its molecular consequence is the genesis of a fusion gene BCR-ABL between the 5' sequences of the BCR gene (chromosome 22) and the 3' end of the ABL gene (chromosome 9). Fluorescence in situ hybridization (FISH) using specific DNA probes provides a useful tool for the detection of t(9;22) and BCR-ABL rearrangement. We report our results using the FISH technique for t(9;22) assessment in the hematopoietic cells of patients with Ph-positive CML. The DNA libraries pBS 9 and pBS 22 containing multiple sequences derived from chromosomes 9 and 22 have been used to identify t(9;22) in metaphase cells. The cos bcr-51 and cos abl-18 probes that hybridize to unique sequences specific to the BCR and ABL genes have the ability to detect the BCR-ABL rearrangement in metaphase cells as well as in interphase nuclei. FISH is a sensitive and specific technique that represents a valuable complement to conventional cytogenetics. The BCR-ABL rearrangement can be detected in metaphase spreads of insufficient quality or from interphase nuclei in the case of terminally differentiated cells or of cells which do not divide in vitro. When the efficiency of hybridization and detection is good, a large number of cells can be analyzed. This is of major significance in assessment of response to treatment and definition of a cytogenetic remission. However, interphase cytogenetics may be difficult due to variations in signal resolution and background level. The FISH technique can also be used to detect the BCR-ABL rearrangement in cases of Ph negative BCR-ABL positive CML.

The screening of genomic microdeletions is a difficult challenge when these deletions are too small for an efficient FISH analysis, and to large for a classic molecular detection. The DIRVISH technique appears as an attractive alternative in this situation.

Hepatitis C virus (HCV) has been discovered in 1989 and is probably the most common cause of chronic hepatitis, cirrhosis and hepatocellular carcinoma. HCV is a single-stranded, positive-sense RNA virus, 9.4 kilobases in length. The genetic organisation and the properties of viral proteins have been characterized. At least 50 HCV genotypes or subtypes have been identified. Genotypes 1, 2 and 3 are the most commonly observed in patients from Europe and USA. Genotype 1 is more resistant to interferon treatment. The hypervariability of HCV is responsible, within a single patient, of the existence of a spectrum of very closely-related genomes reffered as quasispecies that may be a mechanism of escape from the immune response and may explain chronicity. Virological diagnosis of HCV infection is based on the detection of anti-HCV antibodies by ELISA. In some cases (acute hepatitis, problems in the interpretation of ELISA tests, or in immunosuppressed patients), it is necessary to search for HCV RNA using genomic amplification or amplification of hybridization. These technics can also be useful to predict the response to interferon, as it has been demonstrated that patients with low viremia are better responders than others. HCV RNA detection or quantification could also be useful to follow the efficiency of anti-viral drugs.

Probes and amplification systems represent new technologic developments in the domain of clinical microbiology. In spite of their high specificity, simple applicability and high velocity the DNA probes show only little sensitivity especially for the direct detection of microorganisms in clinical samples. This sensitivity lead to the development of DNA-amplification technics. PCR is the most famous of them and the most often used one. Next to the direct detection of microorganisms in clinical samples without culture these technics also allow the direct isolation of bacterial toxins, of genes for resistance to antibiotics as well as a systematic search for pathogens in diseases with unknown cause. Different problems of these methods in daily clinical practice are illustrated by four applications 1. direct detection of mycobacterium tuberculosis complex (M. tbc) in clinical samples, 2. rapid identification of mycobacterial species, 3. their typing for epidemiologic intentions, 4. rapid detection of resistance to antibiotics.

The recognition and the identification of previously unrecognized infectious agents require a multidisciplinary approach to specify the nosologic entity of the disease and the epidemiological data, especially the modes of transmission and the risk factors, as well as to discover the microorganism in the laboratory. In the past 20 years, significant breakthroughs have been achieved in cellular cultures (growth factors), in immunology (monoclonal antibodies), and moreover in molecular biology, which have been widely used in the field of infectious diseases. Whereas the classical methods used to grow microorganisms remain of major interest in many cases, innovating strategies have been recently designed to identify previously unknown pathogens. The genomic amplification by polymerase chain reaction (PCR) of highly conserved bacterial genes (as those coding for ribosomal RNA), from tissue biopsies for example, allow to recognize unknown bacteria. The evolutionary distance between a newly recognized pathogen and known microorganisms can be calculated through sequencing of these genes, as described for Rochalimaea henselae or Tropheryma whipplelii. The constitution of cDNA banks from infected tissues is also a novel approach allowing to clone and sequence viral genes, such as those from hepatitis C or from hepatitis E. In the near future, noteworthy improvements will be achieved to rapidly detect microorganisms with highly sensitive and specific tests using monoclonal antibodies, molecular probes (including branched DNA) and with PCR (including Q beta replicase and ligase chain reaction), and to determine the genetic diversity of microbial pathogens by new methods as pulse field gel electrophoresis or arbitrarily primed PCR. This will result in a better knowledge of the pathophysiology of infectious diseases, in a better recognition of a typical, previously unrecognized clinical expression of pathogenicity, and also in a more precise assessment of the actual impact of a given pathogen in human populations by highly sophisticated diagnosis tests.

Alteration of chromosome 1 is the most consistent cytogenetic abnormality found in human breast carcinoma. Cytogenetic studies have shown independent alterations on the two arms of chromosome 1; increased copy number of the long arm and loss of the short arm of chromosome 1. We carried out deletion analysis of the 1p region by using restriction fragment length polymorphism markers mapping to the long (six markers) and short arm (22 markers). Thirty-five of the 74 (47%) human breast tumors tested showed somatic loss of heterozygosity at one or more loci on the short arm. Two commonly deleted regions, 1p13-p21 and 1p32-pter, were identified. Our findings suggest that two tumor suppressor genes involved in the development of human breast carcinoma may occur on the short arm of the chromosome 1.

Hybridization in situ of chlamydia trachomatis allows identification of the bacteria. Moreover exact chlamydia trachomatis localization at tissue level can be demonstrated. It seems to us very important to know for instance in conjunctiva were chlamydia trachomatis are located. We used DNA DIGOXIGENIN labelled plasmidic probe of 503 b.p. Detection of chlamydia trachomatis on paraffin embedded cells becomes possible. Specificity is 100% as plasmidic sequence is not found in other bacteria. Sensitivity seems to be good quality on experimental model. At present time, it is under clinical evaluation. This method could complete PCR for chlamydia detection in case of controversial diagnosis.

The authors briefly review the clinical forms of equine rhinopneumonitis and indicate changes in the nomenclature of equine herpesviral infections. The value of restriction profiles for epidemiological studies is described, taking as an example the strains of virus isolated in France. A technique is given for preparing molecular probes, as well as the application of these probes in direct diagnosis from biological specimens.

The course of bladder tumours is difficult to predict. The most reliable prognostic factor at the present time is histological grade. Cytogenetic subclasses of bladder tumours can be distinguished on the basis of the demonstration of karyotype anomalies in bladder tumour cells. Eighteen patients underwent cytogenetic examination of their bladder tumour and were followed for an average of 35 months. Multivariate analysis of the clinical and laboratory parameters studied revealed the importance of age and the absence of trisomy 7 in the tumour on patient survival. The presence of trisomy 7 in a bladder tumour may therefore constitute a factor of poor prognosis. This hypothesis needs to be confirmed by further studies in larger populations. The search for this anomaly can be performed by fluorescent in situ chromosomal hybridisation, a technique which transforms cytogenetics from an experimental procedure into a routine complementary investigation. These techniques can be performed on urine samples, suggesting the possibility of their application to screening or follow-up of bladder tumours.

Tracheobronchial lavage has proven useful to research. The technique has been performed in anaesthetized swine using a catheter introduced by oral or intratracheal routes. The risk of mortality during anaesthesia is not negligible. In our trial, tracheobronchial lavages were realized after introducing a catheter into the lung via the nasal route. The analysis of the tracheobronchial fluid samples using a nonradioactive DNA-specific probe showed the presence of Mycoplasma hyopneumoniae, one of the major contaminants of swine lungs. This technique does not cause any traumatism to the animal and should be an interesting method for the study of some contaminants of the lower respiratory tract.

We evaluated an alkaline phosphatase-labeled oligonucleotide probe for the rapid identification of Mycobacterium tuberculosis and mycobacteria belonging to the M avium and M intracellulare complex (MAIS). Sixty-two strains of mycobacteria and eight strains belonging to related genera were studied. All M tuberculosis strains hybridized with the tuberculosis probe. All M avium and M intracellulare gave a strong signal with their probes. However the 3 M xenopi strains tested hybridized with all probes for MAIS complex.

Human alloantibodies specific of some Rh antigens cross-react with non human primates red blood cells. These crossreactions demonstrated that only African apes express equivalents of Rho (D) and hr' (c). The antigenic resemblance between these two human antigens and their primate homologues is confirmed by the reactivities of human anti-D and anti-c monoclonal antibodies. The use of a human Rh cDNA probe allowed to confirm by Southern blot hybridization that nonhuman primates possess Rh-like genes. The number of Rh-like genes per haploid genome was deduced from the results obtained with exon-specific probes.

The pathogenesis of the various periodontal diseases cannot only be explained on the basis of the relationship of specific bacteria to the clinical features of the disease. The microbiologic examination of various members of the same family, either by culturing or by the use of the DNA probe often showed similarities among several individuals in the same family. This could be accounted for on the basis of bacterial transmission or by contamination. These observations suggest the incidence of familial and genetic factors in the etiology of some forms of periodontitis.

Salmonella serotype Typhimurium is a facultative intracellular pathogen that causes a systemic infection in naturally, or experimentally, infected mice. After oral contamination, Typhimurium colonizes the ileal mucosa and Peyer's patches and invades draining mesenteric lymph nodes. From these primary sites of infection, bacteria dissiminate to the reticuloendothelial system and proliferate rapidly in spleen and liver. Several virulence factors are encoded by chromosomal genes. The ability of Typhimurium to adhere to and invade epithelial cells has been associated with flagella, pili of type I and mannose-resistant haemagglutinating activity. By comparing the virulence of isogenic strains, it appeared that these traits played a marginal role and were not essential for full virulence expression. It is now clear that other surface structures are important for the invasiness capacity of Typhimurium. To multiply in the reticuloendothelial system, a complete lipopolysaccharide is necessary for the bacteria in resisting serum bactericidal activity and producing tissue damage. Salmonella have evolved a specialized iron-binding ligand, termed enterobactin, to acquire iron necessary for their multiplication. Enterobactin competes with the host iron-binding proteins (transferrin or lactoferrin) to secure the iron required by the bacteria. Though the presence of an enterotoxin in Salmonella is still controversial, there is now substantial evidence to support this concept. Recently, a gene encoding an enterotoxin has been cloned from Typhimurium and expressed in E. coli. Typhimurium strains harbour a 90 kilobases (kb) plasmid which is essential for virulence. This plasmid encodes virulence factors required for replication of Salmonella in liver and spleen. It was postulated that the plasmid allowed Typhimurium to multiply in Kupffer cells and in splenic macrophages. The virulence-associated region of the plasmid restored full virulence to plasmidless strains. Transposon insertion mutagenesis demonstrated the existence of two DNA sequences, designated Vir A and Vir B, which are essential for virulence expression. The Vir A region has been sequenced; it encodes four polypeptides with apparent molecular mass of 27,000, 28,000, 33,000 and 70,000. The Vir B region encodes two polypeptides of 38,000 and 43,000. In an attempt to identify bacterial components contributing to invasion of HeLa cells by Salmonella serovar Typhi, we cloned a 30 kb DNA sequence necessary for entry of bacteria into epithelial cells. However, this sequence is not sufficient for conferring an invasive phenotype to E. coli strains. From this DNA fragment, a short segment of 487 bp was subcloned, sequenced and used as probe to detect Salmonella.(ABSTRACT TRUNCATED AT 400 WORDS)

Reverse Genetics consists in identifying the gene responsible for a hereditary condition, the biochemical mechanism of which is unknown. We have applied this approach to families recruited according to the following criteria: 1) at least two members of the family must be affected; 2) one of the two subjects must have an abnormality of thoracic and/or abdominal lateralization. This abnormality of situs can form part of a syndrome: asplesia, polysplenia, heterotaxia, dextrocardia, situs inversus, Kartagener' or Ivemark's syndrome; 3) the other affected subject(s) must have some form of congenital heart disease and/or one of the phenotypes above described in 2); 4) in consanguinous marriages, only one child fulfilling the criteria in 2) above described is required. Eight families conforming to the above criteria are presented; lymphocytic transformation was successful in 90% of cases when the blood sample was received within 48 hours. The segregation of the alleles is in favour of a linkage between the tested probe and the syndrome(s) studied. In order to identify the gene responsible, we will have to increase the number of families studied and extend our research to sporadic cases.

DNAS from 325 individuals representing 2 different populations (234 Brazilians and 91 Asiatics) were analysed for Restriction Fragment Length Polymorphisms (RFLPs). These DNAs were digested with Pvu II enzyme and successively hybridized to two HVR probes: alpha globin-3'HVR and Mucin-HVR. An allele frequency distribution was determined for each couple probe/enzyme and each ethnic groupe studied. The results were compared with frequencies observed in french population and we showed that there is no statistic significant difference between allele frequencies obtained with the couple probe/enzyme 3'HVR/Pvu II in Brazilian and French populations. We also showed that both populations studied (Brazilian and Asiatic) do not follow a Hardy-Weinberg balance.

Seventeen suspension of Legionella pneumophila and ten of Legionella bozemanii in saline or bronchoalveolar lavage (BAL) fluid were tested using the Gen Probe technique. The detection threshold was found to be 10(3)-10(4) CFU/ml. Specificity and sensitivity were evaluated by using the probe on 8 suspensions of bacteria other than Legionella and by performing a comparative study of the probe test, direct immunofluorescence and culture with 103 specimens (BAL fluid in most instances) from 92 patients with possible legionellosis. Sensitivity was found to be acceptable (3 of the 4 culture-positive specimens were positive by the probe test) and specificity was 100% despite the fact that most (80/99) BAL specimens were not sterile and regardless of the cutoff level used to define positivity. The advantages of the DNA probe test, including rapidity, simplicity and objectivity, should be weighed against its disadvantage, i.e., only acceptable sensitivity and use of radioactivity.

Homologous sequences of the acute RNA tumor virus oncogenes have been found to be highly conserved within vertebrates, insects and yeasts. In the present work, seven different oncogene DNA sequences have been used as probes to search for homologous sequences in the DNA of the protozoan Plasmodium falciparum. Both the v-fms v-Ha ras probes hybridized P. falciparum DNA. The oncogene study will allow an understanding of the biology of the parasite and particularly the host-parasite relationships which allow P. falciparum to develop, keeping the established harmony between the parasite and his host.

We have tested in adult rat the specificity and the sensitivity of two cDNA probes derived from recombinant plasmids pJG19 and pJG418. The Northern blot analysis of total RNA extracted from small bowel reveals 0.9 and 0.7 kb mRNA corresponding respectively to the I and L-FABPc mRNA. A linear relation occurs between the amounts of input total RNA and the quantities of specific mRNA found by hybridization with cDNA probes (r = 0.991 and r = 0.998 for I and L-FABPc respectively). We have also evaluated the effect of drastic nutritional status: a high fat diet containing 45% of lipids and a starvation of three days. A two fold increase in I and L-FABPc mRNA occurs in starved rats. By contrast, only the I-FABPc mRNA are slightly enhanced after the hyperlipidic diet (50%). These preliminary results, which must be confirmed by further experiments, suggest a differential nutritional regulation of I and L-FABPc gene expression in rat intestine.

Human skeletal tissues fixed in Bouin's solution and embedded in paraffin were studied in order to identify cells responsible for production of types I and II collagens by in situ hybridization. The probes used were a double stranded cDNA fragment (type I collagen) and a synthetic oligonucleotide (type II collagen). The specificity of these probes labeled with 32P was proven in hybridizations to sections of human fetal femoral heads: fibroblasts and chondrocytes, known to produce respectively type I and type II collagen were only recognized by the corresponding probes. These results suggest that paraffin blocks available in pathology departments could be reexamined by in situ hybridization. Furthermore, the use of synthetic oligonucleotides without cloning the gene of interest could increase the usefulness of the technique in studies on human cartilage diseases and possibly on other diseases.

Recent studies using hybridization histochemistry have demonstrated the presence of cholecystokinin (CCK) gene expression in thalamocortical and thalamo-striatal neurons. To further understand the chemical anatomy of the thalamus, we used the same method to study the coexpression of CCK and vasoactive intestinal peptide (VIP) genes in the same neurons. Most of the neurons expressing the VIP gene in the rat dorsal thalamus (found especially in the ventrolateral nucleus) also expressed the CCK gene, as demonstrated by autoradiography on emulsion-coated adjacent sections studied with probes recognizing either mRNA. Some further neurons, located in the reticular nucleus, had VIP mRNA at lower levels of expression but did not appear to contain CCK mRNA.

Familial growth hormone deficiency has been often associated to homozygous gene deletions. In this work we have looked for the possible absence of this gene in patients with isolated GH deficiency. The patient genomic DNAs have been digested with two restriction enzymes and hybridized with a 32P labelled growth hormone cDNA. The presence of the growth hormone gene has been proved in the patients. This situation, in which the gene is present but not expressed, might be due to changes in gene regulation or to punctual gene deletions or mutations.

Nucleic acid hybridization techniques have been used for several decades in basic research to isolate genes, to determine their structure or analyse their mechanisms. The possibility of using them in clinical biology has been considered for several years. The transfer from basic to applied research can now take place due to the new technology of non-radioactive probes (cold probes). Hybridization features the use of a probe nucleic acid molecule and of a target nucleic acid molecule. It leads to the formation of a double-stranded molecule, also called duplex, which can be detected with great sensitivity. For this to be done, the probe can be tagged with a variety of labels such as a radio-isotope or a non-isotopic marker which can be detected for its fluorescence, luminescence or enzyme activity or by immuno-reaction(s). Hybridization can be performed either in a liquid solution or on a solid support. In the first case, hybridization is generally followed by a separation step which makes use of solid phase to isolate the hybrid (duplex) formed. This separation can be achieved by biochemical means (adsorption chromatography, differential precipitation, electrophoresis, etc.) or by immunological means (affinity chromatography, immuno-capture). In the second case, the support can be used in different ways according to the assay format employed to perform hybridization. The target DNA can be immobilized by contacting the sample to be tested with the support: simple specimen deposit with a pipette (for low volumes), or by aspiration under vacuum or by (capillary or electrophoretic) transfer from an agarose gel. In the sandwich assay format, probe DNA is immobilized onto the support to specifically extract the target DNA from the medium under assay. Support to be employed for hybridization purpose can be selected from the group consisting of: nylon (N), nitrocellulose (NC) or polyvinylidene difluoride (PVDF) membranes; N+NC mixed membranes; cellulose and its derivatives; polyoside supports (Sephacryl, Sepharose, etc.), latex particles, polystyrene microplates. The coupling procedures and capacity vary according to the type of support used.

Molecular biology techniques have contributed, in the last ten years, to a better understanding of parasitic diseases. DNA probes, for example, have been successfully used not only for taxonomic purposes, but also for the diagnosis, the epidemiology and the pathogenicity of these infections. Due to their high sensitivity and specificity, the DNA probes allow, from a diagnostic point of view, the detection of very few of parasites in a given sample. This detection is also valid for any parasitic stage considered. It can be specially used to detect the infective stage in the vector, which, in epidemiological studies, is very important. The identification of parasitic species or sub-species indicates the human infectivity of species considered in the past as zoophilic. Finally, by allowing the specific identification of strains, isolates or even particular clones, the DNA probes also show differences which can be related to pathogenicity, to particular biological characteristics, or even to drug sensitivity. In this review, I relate the main results obtained in malaria and toxoplasmosis in our laboratory in Grenoble. I will also consider some recent data on amoebiasis, leishmaniasis, trypanosomiasis, filariasis, schistosomiasis and echinococcosis.

Huntington disease (HD) is a neurodegenerative disorder caused by an autosomal dominantly inherited defect. The discovery of DNA polymorphisms genetically linked to the HD locus provided the possibility of an early presymptomatic test. The first marker locus described (G-8) had an approximately 5% recombination rate with the HD locus, and the subsequent discovery of some more tightly linked marker loci, notably D 495, has greatly improved the accuracy of presymptomatic testing. We describe here the preliminary results obtained and the difficulties encountered in a French predictive testing program on presymptomatic subjects belonging to choreic families.

A technique of detection of human papillomaviruses by in situ hybridization in paraffin-embedded formalin-fixed sections of genito-anal lesions is described. The probes are labeled by incorporation of digoxigenin-labeled nucleotides. The hybrids are revealed by immunohistochemistry with an anti-digoxigenin antibody coupled with alkaline phosphatase. This technique is sensitive, cheap and may be used in routine in laboratories of pathology.

After a brief recall on the difficulties of serological and parasitological diagnosis of acute forms of Toxoplasmosis, we present advantages of a new type of detection by highly reiterated genetic probes. Previous reports on genetic probes application in Mycology and Parasitology support our own research of toxoplasma diagnosis.

Thanks to a bovine genomic library enriched with Y chromosome-specific sequences, a probe specific to this chromosome of genera Bos and Bison and repeated on about 2,000 copies in the male genome was isolated. To be compatible with embryo transfer and/or freezing, sex determination has to be done on a 10-20 cell embryo biopsy from a day-7 bovine blastocyst. The in situ hybridization with biotinylated BC 1.2 probe and immunocytochemical revelation permitted the visualization of the hybridization signal on the nucleus of each cell. Because of the numerous steps required, this technique was difficult to apply routinely. For that reason we worked out the polymerase chain reaction technique. From the BC 1.2 sequence, we determined several oligonucleotide primers and probes with the aim of amplifying the Y chromosome-specific sequences. This technique, which is easy to perform and to automate, enabled us to obtain a strong male-specific hybridization signal on genomic DNA and embryo cells.