Investigation of Toxoplasma gondii presence in farmed shellfish by nested-PCR and real-time PCR fluorescent amplicon generation assay (FLAG).

To evaluate the presence of Toxoplasma gondii in edible farmed shellfish, 1734 shellfish specimens i.e., 109 Crassostrea gigas (6 pools), 660 Mytilus galloprovincialis (22 pools), 804 Tapes decussatus (28 pools) and 161 Tapes philippinarum (6 pools), were collected from the Varano Lagoon (Apulia, Italy). Shellfish from 62 pools were subjected to two molecular techniques: a nested-PCR assay, and a fluorescent amplicon generation (FLAG) real-time PCR assay, both based on the multi-copy B1 target, were performed. One pooled sample of gills from C. gigas and one pooled sample of haemolymphs from T. decussatus were assessed as positive for T. gondii DNA by both techniques. The results demonstrated the presence of T. gondii in edible farmed C. gigas and T. decussatus and indicate that there may be a considerable health threat involved in eating contaminated raw shellfish.

Space distribution of harmonic mode vibration amplitudes in nonlinear finite piezoelectric transducer.

Nonlinear elastic vibrations of cylindrical piezoelectric transducers are investigated both experimentally and theoretically. A particular behaviour, that relates the space distribution of the fundamental mode vibration to those of the second and third harmonic components, is observed. A simplified physical interpretation of the phenomenon is given.

[Prevention and treatment of congenital toxoplasmosis: organization protocol]. / Prevenzione e trattamento della toxoplasmosi congenita. Modello organizzativo.

AIM: Prevention and treatment of congenital toxoplasmosis are still a matter of debate among obstetricians, pediatricians and epidemiologists. There is no consensus about antenatal screening and diagnostic tests, nor there is about treatment for presumed infection in pregnancy. As an example of this type of organisation for health care delivery, a regional model has been promoted as a multidisciplinary approach for prenatal diagnosis of congenital toxoplasmosis. The model had been designed on the national guidelines of the National Health Institute (Istituto Superiore di Sanità, ISS). METHODS: Suspected maternal infections are referred and seen as outpatients at our centre on a specific day of the week; maternal investigation (specific IgG, IgM, IgA and IgG avidity titres) are performed at the Institute of Virology of the University of Bari, and patients are started on spiramycin. All cases of true or presumed seroconversion are counselled for amniotic fluid sampling and the sample is sent to ISS. In cases of late seroconversion and positive amniotic fluid results, patients are prescribed pyrimethamine+sulphonamide+folinic acid and alternate spiramycin until the end of pregnancy. A fetal-neonatal follow-up is performed in all cases. RESULTS: During the period 1999-2001, 180 cases of presumed toxoplasmosis infection have been referred (average 60 cases per year). We have been able to reclute, since the adoption of the national network protocol, 1/3 of presumed regional cases with a positive increasing trend. CONCLUSION: The service for prenatal diagnosis of toxoplasma gondii infection has definitely benefitted from the adoption of this protocol, which combines adherence to a national network and pays respect to regional requirements.

Self-interference between forward and backward propagating parts of a single acoustic plate mode.

Near and far fields of a particular (S(1)) Lamb mode, generated on a steel plate by means of a wedge transducer, are investigated. These show an oscillating behavior of the radial profile of the acoustic field amplitude that can be interpreted and modelled as interference phenomenon between forward and backward propagating parts of the Lamb mode, simultaneously generated at the interface between transducer and plate.

[Risk assessment of vertical transmission of Toxoplasma infections]. / Valutazione del rischio di trasmissione verticale dell'infezione da Toxoplasma.

BACKGROUND: It is important to estimate the risk of congenital toxoplasmosis infection and its clinical sequelae. This information will assist the clinical counselling of pregnant women with acute toxoplasmosis and may guide individual decisions on investigative and therapeutic options. METHODS AND PATIENTS: Since January 1998 to July 2000 we collected data on 63 pregnant women referred to our Prenatal Diagnosis Centre for suspicion of acute maternal toxoplasmosis. In case of positive screening tests for IgG and IgM or documented seroconversion, we sent samples of maternal serum to a reference laboratory to detect specific IgM, IgG, IgA antibodies and IgG avidity. We estimated the risk of mother-to-child transmission as high or low in relation to gestational age at the time of maternal seroconversion. Antiparasitic treatment with spiramycin since before confirmation of infection and evaluation of fetal biometry were done. In high risk women we advised amniocentesis for polymerase chain reaction (PCR). If infection was diagnosed in the fetus, the regimen of treatment was changed. Live-born children of all these mothers were followed up serologically for at least 1 year. RESULTS: In 13/63 cases (20%) we excluded maternal infection. Thirty-six out of 63 cases (57%) with high transmission risk underwent an amniocentesis, except one case requesting termination of pregnancy. Four out of the remaining 14 cases estimated at low risk had an amniocentesis for maternal anxiety. Among high risk women, PCR on amniotic fluid was positive in 4 cases (10%): 2 of these requested pregnancy termination, while the other 2 decided to proceed with the pregnancy and the therapy was shifted to 3 weeks courses of pyrimethamine and sulphadiazine with folinic acid alternating with spiramycin. Five infants were lost at follow-up, 20 babies resulted not infected and the remaining 21 are still followed-up. CONCLUSION: It is possible to select by specific serological tests, high risk cases for mother-to-child transmission of infection and to decide a specific approach.

[Control of congenital toxoplasmosis in Italy: The project of the Istituto Superiore di Sanità]. / Il controllo della toxoplasmosi congenita in Italia: il progetto dell'Istituto Superiore di Sanità.

Toxoplasma gondii infection during pregnancy is a public health concern; many resources are used in diagnostic and therapeutic activities, sometime with a low benefit/cost due to lack of standardization in practices. In the lack of suitable epidemiological knowledge at national level regarding the congenital toxoplasmosis trend in Italy, an evaluation of the public health impact of this pathology is required. The Istituto Superiore di Sanità (ISS) has worked out a project on a prevalence-incidence study at national level, performing standard reference methods for diagnosis and both case definition and case management. Furthermore, the ISS urges all the involved centers on congenital toxoplasmosis to set up a network for an active collaboration to this project.

A second European collaborative study on polymerase chain reaction for Toxoplasma gondii, involving 15 teams.

In order to investigate the accuracy and practicability of the polymerase chain reaction (PCR) in the antenatal diagnosis of congenital toxoplasmosis, a collaborative study involving 15 European laboratories was performed under the auspices of the Biomed 2 Programme of the European Community. Each team received 12 aliquots (four negative, eight positive) of 'artificial samples' made of amniotic fluid spiked with tachyzoites of the RH strain of Toxoplasma gondii. Each team performed its own PCR protocol (all were different). Nine of the 15 laboratories were able to detect a single parasite, but two of the 15 found all samples negative. Four of the 15 laboratories found one or more control samples to be falsely positive. This study highlights the lack of homogeneity between PCR protocols and performance and underlines the need for an external quality assurance scheme which could provide 'reference' samples that could be used by any laboratory wanting to establish and maintain an accurate diagnostic test based on PCR.

Induction of interleukin 1 alpha in murine macrophages infected in vitro with different species and strains of Leishmania.

It is now generally agreed that several cytokines released by immunocompetent cells such as macrophages play a crucial role in the outcome of infections caused by protozoa belonging to the genus Leishmania. In particular, tumor necrosis factor (TNF) induction during the course of cutaneous leishmaniasis has been related to resistance to L. major infection in mice. However, the role played by interleukin 1 (IL-1) in the host response to leishmaniasis has yet to be completely elucidated. The aim of this work was to study whether different species and strains of Leishmania could induce IL-1 alpha in murine macrophages in vitro. Resident peritoneal macrophages of BALB/c and C3H/HeN mice were infected with L. donovani, L. major, or different strains of L. infantum. It was found that L. donovani did not induce IL-1 alpha in macrophages from either mice strain. Infection with L. major or with three out of six strains of L. infantum induced consistent amounts of IL-1 alpha, but only in macrophages from genetically resistant C3H/HeN mice. No relationship was found between the rate of infection of macrophages and the amount of IL-1 alpha detected in the supernatants of infected macrophages. Data obtained confirm that the release of IL-1 alpha by murine macrophages infected in vitro with Leishmania is influenced by the genetic background of the cells as well as by the parasite species.

A kinetoplast DNA probe diagnostic for Leishmania infantum.

A 196 bp fragment of cloned kinetoplast (k) DNA of Leishmania infantum has been sequenced and shown to be diagnostic for this species. The DNA from 10(4)-10(5) promastigotes was routinely and specifically detected in crude dot blots of whole cells lysed in situ, and there was no cross-hybridization with 10(6) cells of L. major, L. tropica, L. killicki, L. aethiopica, L. braziliensis and L. mexicana. A low degree of hybridization was found with L. donovani and L. chagasi. This probe will be of particular use in Mediterranean countries, such as Italy, in which dermotropic zymodemes of L. infantum can be difficult to isolate and grow in numbers sufficient for isoenzyme typing.

Study on genetic polymorphism of Leishmania infantum through the analysis of restriction enzyme digestion patterns of kinetoplast DNA.

Twenty-nine Leishmania infantum strains characterized by different host source, tropism and belonging to 6 zymodemes, were examined by restriction enzyme analysis of kinetoplast DNA (kDNA) using 15 endonucleases. The enzymes which produced only one fragment revealed full identity between all the strains examined, while those producing many bands gave different electrophoretic patterns. They were interpreted with the aid of numerical analyses (cluster and multifactorial analysis). The results show a cline of genetic variability among the strains, the highest similarity being observed between most of the viscerotropic strains isolated from man, dog, black rat and sandflies. The strain agents of human cutaneous leishmaniasis show a varying degree of genetic divergence from this group, which appears more evident when characters from isoenzymes are considered.