Development of a PCR assay followed by nonradioactive hybridization using oligonucleotides covalently bound to CovaLink NH microwells for detection of four Plasmodium species in blood samples from humans.

We developed and evaluated a PCR-based assay to detect four Plasmodium species in 79 blood samples from 56 travelers returning from areas where malaria is endemic. DNA amplification targeting a small region of the 18S rRNA gene was performed with Plasmodium genus-specific primers. The biotinylated PCR products were then identified by PCR-colorimetric Covalink NH microwell plate hybridization (CMPH) using species-specific phosphorylated probes covalently bound to a pretreated polystyrene surface. The results from PCR-CMPH showed high specificity, and for 47 of the 56 patients (84%), microscopy and PCR-CMPH results were in agreement. Discordant results were reevaluated with microscopy examination, other molecular methods, and DNA sequencing. Except for one patient, discrepancies were resolved in favor of PCR-CMPH: three mixed infections were detected, four species identification errors were corrected, and two negative results were shown to be positive. Our results indicate that PCR-CMPH is a simple, rapid, and specific method for malaria diagnosis. It employs stable reagents and inexpensive equipment, making it suitable for routine epidemiological use.

Genetic identification of the main opportunistic Mucorales by PCR-restriction fragment length polymorphism.

Mucormycosis is a rare and opportunistic infection caused by fungi belonging to the order Mucorales. Recent reports have demonstrated an increasing incidence of mucormycosis, which is frequently lethal, especially in patients suffering from severe underlying conditions such as immunodeficiency. In addition, even though conventional mycology and histopathology assays allow for the identification of Mucorales, they often fail in offering a species-specific diagnosis. Due to the lack of other laboratory tests, a precise identification of these molds is thus notoriously difficult. In this study we aimed to develop a molecular biology tool to identify the main Mucorales involved in human pathology. A PCR strategy selectively amplifies genomic DNA from molds belonging to the genera Absidia, Mucor, Rhizopus, and Rhizomucor, excluding human DNA and DNA from other filamentous fungi and yeasts. A subsequent digestion step identified the Mucorales at genus and species level. This technique was validated using both fungal cultures and retrospective analyses of clinical samples. By enabling a rapid and precise identification of Mucorales strains in infected patients, this PCR-restriction fragment length polymorphism-based method should help clinicians to decide on the appropriate treatment, consequently decreasing the mortality of mucormycosis.

Unspecific reactivity of IgM directed against the low-molecular-weight antigen of Toxoplasma gondii.

During routine serological survey, eight patients (5 pregnant women, 3 grafted patients) were positive for Toxoplasma gondii-specific IgM by enzyme-linked immunoassay but negative by a simultaneously performed immunosorbent agglutination assay. No clinical or biological symptoms of toxoplasmosis were observed later, despite the absence of treatment. Only one IgM-reactive band, which corresponded to the low-molecular-weight antigen of Toxoplasma gondii, was observed by Western blotting of these patients' sera. Dot blotting of lipid extracts of Toxoplasma gondii demonstrated that this reactivity was directed against sphingolipids or ceramides. This IgM positivity, which is unrelated to acute toxoplasmosis, raises strong concerns about the possibility of misleading results of this test in the diagnosis of toxoplasmosis in humans.

Epidemiology of dermatophytoses in children living in northeast France: a 5-year study.

Dermatologic fungal infections are thought to occur less frequently in children than in adults. This study, performed over a 5-year period, emphasizes the interregional variability of dermatophytes that cause skin and cutaneous apprendageal diseases in children. In northeast France, two-thirds of dermatophytoses are due to zoophilic fungi, while they are most commonly caused by anthropophilic agents in the Paris region and in other countries. The clinical features of pediatric dermatophytoses vary with the age of the child: tinea capitis and tinea corporis are far more frequent before the age of 12 years. After the age of 12, even if these are still quite frequent, tinea pedis and onychomycosis become more common.

[Evaluation of the Immulite 2000 Toxoplasma quantitative IgG et Toxoplasma IgM for the diagnosis of human toxoplasmosis]. / Utilisation des trousses Immulite 2000 Toxoplasmose IgG et Toxoplasmose IgM pour le diagnostic de la toxoplasmose humaine.

Four hundred and ninety five human sera with clinical and biological data were tested for the evaluation of Immulite 2000 Toxoplasma Quantitative IgG and Immulite 2000 Toxoplasma IgM produced by Diagnostic Products Corporation (Los Angeles, USA) for the diagnosis of human toxoplasmosis. The results of these kits were compared to those of the University Hospital of Nancy where the reference assays were Enzygnost Toxoplasmosis IgG and Enzygnost Toxoplasmosis IgM (Berhing-Dade, Germany), Toxoscreen (bioMérieux, France), ISAgA Plus (IgM et IgA) (bioMérieux, France). The sensitivity and the specificity of IgG detection by Immulite 2000 Toxoplasma Quantitative IgG were 98% and 100%, respectively. The high sensitivity of IgM detection by Immulite 2000 Toxoplasma IgM was adapted to the early diagnosis of toxoplasmic primo-infection and to the pediatric diagnosis or follow-up of congenital toxoplasmosis but could reveal IgM a long time after primary infection.

Antidermatophytic properties of extracts from the leaves of Aristolochia paucinervis Pomel.

Several fractions of a methanol extract from the leaves of Aristolochia paucinervis Pomel (Aristolochiaceae) were screened for their antidermatophytic efficiency against different human pathogenic fungi responsible for tinea and other skin infections. The antifungal study was carried out by the macrodilution agar method and the results showed that, with the exception of the aqueous fraction, all the fractions exhibited antifungal activities against the dermatophytic fungi tested. The hexane fraction was found to be the most effective (MIC range: 64-2048 microg/mL), whereas the butanol fraction was the least active (MIC range: 1024 microg/mL to more than 2048 microg/mL). The most susceptible fungi were Epidermophyton floccosum and Trichophyton violaceum in contrast to Trichophyton mentagrophytes and Trychophyton rubrum which were less sensitive to the fractions tested. The effects were compared with those of ketoconazole, amphotericin B and griseofulvin, for which MIC ranges were, respectively, 0.12-4 microg/mL, 0.5-4 microg/mL and 0.5-2 microg/mL.

Laboratory diagnosis of cystic hydatic disease.

The main purpose of this article is to answer the questions about which test to perform for hydatic diagnosis and when. Several techniques for biologic diagnosis and follow-up of human cystic hydatidosis are reviewed. The specificity and sensitivity of immunologic reactions are reported. The differential diagnosis between Echinococcus granulosus and E. multilocularis is examined. The characteristics of the immunologic diagnosis according to the stage and the treatment of hydatidosis disease is discussed. Laboratory diagnosis of cystic hydatic disease is complementary to the clinical data. A judicious association of the usual techniques (indirect immunofluorescence assay, indirect hemagglutination assay, immunoelectrophoresis, co-electrophoresis with antigen 5 identification) confirms the diagnosis in 80% to 94% of hepatic hydatidosis cases and in 65% of pulmonary hydatidosis cases. Special techniques (enzyme-linked immunosorbent assay, Western blot, polymerase chain reaction) must be used for other localizations or when cysts are calcified. A serologic survey is necessary for the follow-up of operated medically treated patients. Despite poor standardization, purified antigens can distinguish between E. granulosus and E. multilocularis infections, although false-positive results are observed during other helminthiases, such as cysticerocosis.

Successful reinfection of chronically infected mice by a different Toxoplasma gondii genotype.

It is generally assumed that primary infection by Toxoplasma gondii protects from reinfection. A recent study using a murine model has questioned this dogma using indirect procedures to detect the reinfecting strain. We have reinvestigated this issue using a transfected strain of T. gondii (Prugniaud beta galactosidase: Pru beta gal) which expresses Escherichia coli beta-galactosidase. Detection of enzyme activity on fixed parasites allows a direct distinction between transfected and untransfected strains. We have found that in OF1 mice primary infection with the 76 K strain of T. gondii fully protects mice against tissue cyst production upon reinfection with the Pru beta gal T. gondii strain whereas primary infection with the Pru beta gal T. gondii strain does not impair tissue cyst formation upon reinfection with the Ned strain of T. gondii, which belongs to another T. gondii genotype. These results suggest that the immune protection conferred by one strain of T. gondii can be breached by reinfection with a strain belonging to another genotype; which can have significant consequences in human or veterinary medicine.

[Application from chemiluminescence to serological diagnosis of human toxoplasmosis]. / Application de la chimiluminescence au diagnostic sérologique des toxoplasmoses humaines.

Diagnostic Products Corporation has chosen chemiluminescent for the new kit of quantitative measurement of IgG and qualitative detection of IgM antibodies to Toxoplasma gondii, 878 human sera of principal diagnosis situations were tested, and the results obtained with the IMMULITE Toxoplasmosis kit were compared with those of the Parasitology and Mycology Laboratory of the University of Lille. Chemiluminescent allows a sensitive and specific determination of immunity. In the same ways, this method is able to detect earlier specific IgM and IgG during seroconversion. The kit of quantitative measurement of IgG and qualitative detection of IgM is reproducible and sensitive; this confirms the interest for the pediatric diagnosis of congenital toxoplasmosis.

[Study of developing clinical outbreak and serological rebounds in children with congenital toxoplasmosis and follow-up during the first 2 years of life]. / Etude des poussées cliniques évolutives et des rebonds sérologiques d'enfants atteints de toxoplasmose congénitale et suivis durant les 2 premières années de vie.

BACKGROUND: The survival of T gondii bradyzoites in cysts explains clinical recurrences and serological rebounds after birth in children with congenital toxoplasmosis. At the present time, management of such manifestations is not well defined. PATIENTS AND METHODS: Sixty-three infants with congenital toxoplasmosis were followed-up at the University Hospital of Lille (France) during the first two years of life. For each child, the treatment before and after birth was well defined. Clinical, ophthalmological, radiological and serological data were collected every third month. Serological assays specially adapted to this age bracket were used for the quantification of specific IgG, or for the detection of T gondii specific IgM and IgA. RESULTS: Seventy-six serological rebounds were reported in 55 of the 63 children (87%). They concerned essentially IgG (96%) and less frequently IgM (47%) or IgA (60%). At the same time, only five clinical recurrences were observed, four of them being preceded by a serological rebound. DISCUSSION: Treatment of fetuses or children with pyrimethamine and sulfonamides versus spiramycin alone was associated with a decrease in the frequency of serological rebounds during the first year of life (P < 0.001). Such a therapeutic regimen during the second year of life decreases the appearance of serological rebounds in children without rebound antecedent (P < 0.001). CONCLUSION: The increase in number of rebounds after the end of a course of pyrimethamine and sulfonamides necessitates the evaluation of such a long term treatment without interruption.

[Acute myocarditis caused by toxoplasmosis simulating infarction. Apropos of a case]. / Myocardite aiguë à toxoplasme simulant un infarctus. A propos d'un cas.

Acute myocarditis due to toxoplasmosis infection has been previously reported, usually in patients suffering from immuno-depression. Cardiac involvement by toxoplasmosis is rare in subjects with a normal immunological status. The authors report the case of a 16-year-old patient without immuno-depression with acute myocarditis caused by toxoplasmosis simulating myocardial infarction.

[Structure and biology of Toxoplasma gondii bradyzoites]. / Structure et biologie des bradyzoïtes de Toxoplasma gondii.

Tachyzoite-bradyzoite interconversion is one characteristic feature of Toxoplasma gondii. Although highly similar in structure, tachyzoite and bradyzoite differ by the relative amount of certain organelles and by specific surface or cytoplasmic molecules. Differences in structure and contents also exist between parasitophorous vacuoles and cysts. Using stage specific markers, it was shown the quickness of stage switching in vivo as well as in vitro, together with the occurrence of intermediate stages. Regulatory mechanisms of interconversion remain unknown. However, stress or inhibition of the mitochondrial metabolism of the parasite trigger bradyzoite formation.

Stage conversion of Toxoplasma gondii in mouse brain during infection and immunodepression.

The kinetics and pattern of expression of bradyzoite-specific proteins were studied in mouse brain during infection with Toxoplasma gondii. Parasites found in the brain 6 days after ingestion of cysts were expressing only tachyzoite-specific proteins (anti-SAG1 antibodies being used as a marker). Bradyzoite-specific protein (Pb36) expression was first found after 9 days in vacuoles containing mixed parasites simultaneously expressing SAG1 and Pb36 or cysts containing parasites expressing only the bradyzoite marker. Reactivation of toxoplasmosis was studied in mouse brain using corticosteroids for immunosuppression. Parasites expressing SAG1 were first found 6 days after the beginning of treatment, but a very heterogeneous pattern was found throughout the study. We simultaneously found vacuoles containing parasites expressing only SAG1 or containing intermediate stages or cysts containing parasites expressing only bradyzoite proteins. A striking observation was the multiplication of cysts in foci, suggesting that the immune suppression triggered the release of parasites from preexisting cysts but that the factors inducing bradyzoite development remained fully effective in driving parasites into this pathway.

Laser photocoagulation around the foci of toxoplasma retinochoroiditis: a descriptive statistical analysis of 35 patients with long-term follow-up.

Thirty-five patients with toxoplasma retinochoroiditis, receiving medical treatment and then treated with laser photocoagulation around the foci, were retrospectively evaluated for the risk of recurrence of the retinochoroiditis with a Kaplan-Meier representation. The recurrence rates with 95% symmetric confidence intervals were: at 1 year, 12.7 + or - 13%, at 2 years, 19.8 + or - 15%; at 3 years, 24.0 + or - 16%; at 4 years, 33.3 + or - 19%; at 5,6 and 7 years, 53.5 + or - 21%; at 8 and 9 years 66. 8 + or - 22%. With the data provided by our series, it is not possible to show the efficacy of laser photocoagulation as a prevention of recurrence in toxoplasma retinochoroiditis. Moreover, because of their heterogeneity, the recurrence rates from the literature cannot provide precise data for a comparison. Concerning the laser-induced thermal damage, the potential therapeutic mechanism of the laser procedure is discussed.

Immunolocalization and characterization of the low molecular weight antigen (4-5 kDa) of Toxoplasma gondii that elicits an early IgM response upon primary infection.

A striking feature of toxoplasmic seroconversion is the prominent and early IgM response to a low molecular weight antigen of 4-5 kDa. Two different monoclonal antibodies directed against the 4-5 kDa antigen have been generated and used to characterize this molecule. Using these monoclonal antibodies, we could demonstrate the surface localization of the low M(r) antigen by immunofluorescence and immuno-electron microscopy assays. By immunoblotting, we observed that one of the monoclonal antibodies was unable to recognize the 4-5 kDa antigen in tachyzoites propagated in cell culture, indicating an epitope variability between Toxoplasma gondii tachyzoites grown in vivo and in vitro. We discuss the implications of this latter finding in the design of diagnostic reagents.

Toxoplasma gondii: kinetics of bradyzoite-tachyzoite interconversion in vitro.

Stage-specific monoclonal antibodies have been used to investigate the bradyzoite-tachyzoite interconversion of Toxoplasma gondii in vitro. The differentiation of bradyzoites isolated from brain cysts (strains 76K and BQNC2) and grown in culture proceeded through intermediate stages which expressed both the specific markers of bradyzoites (P36, P34, P21, and P18) and a specific marker of tachyzoites (SAG1-P30). Differentiation started before parasite division, but large vacuoles containing intermediate stages were also found, suggesting that these were able to multiply. Intermediate stages were also observed during the differentiation of peritoneal tachyzoites (strain Prugniaud) into bradyzoites in vitro. Triggering of bradyzoite protein synthesis is not a single event since one of the four bradyzoite-specific proteins (P21) always appeared later than the others during bradyzoite differentiation. During interconversion, heterogeneous vacuoles containing parasites expressing different levels of bradyzoite or tachyzoite proteins were observed. This observation together with the fact that differentiation is not synchronous within a culture or within a host cell suggest a complex triggering process.

[Medullary aplasia during treatment for congenital toxoplasmosis in a twin pregnancy]. / Aplasie médullaire au cours du traitement d'une toxoplasmose congénitale lors d'une grossesse gémellaire.

The authors report a case of a patient who in the 24th week of a twin pregnancy became sero-positive for toxoplasmosis. This was diagnosed by cordocentesis as being infected, and the treatment was therefore started with pyrimethamine and sulfadiazine and folic acid at the 28th week of pregnancy. At 35 weeks, the patient had an acute medullary aplasia due to the absence of the folates. The mother's state was improved rapidly by giving her folinic acid and the twins were normal haematologically. In this case, the authors point out how important the folates are in a pregnancy, especially in twin pregnancies, and point out the precautions that have to be taken when treatment with pyrimethamine and sulfadiazine is started for congenital toxoplasmosis.

[Malaria and pregnancy. Therapeutic problems, a case report]. / Paludisme et grossesse. Problèmes thérapeutiques, à propos d'un cas.

Malaria is an old but still current parasitosis. Transmitted by the bite of the female anopheles, it can be revealed by more or less serious symptoms according to the species of Plasmodium. Plasmodium falciparum is responsible for the serious forms. It is the most frequent species, resistant to 4 amino-quinolein and sulfamides in some areas. P. vivax and P. ovale, usually harmless, are not so widely geographically spread as P. falciparum; and apparently are not drug resistant but they expose patients to the risk of relapses. During pregnancy, the choice of a curative and prophylactic therapy must take into account the supposed or confirmed species, the place of the stay and the duration of the exposure. On this case the authors call to mind the epidemiological criteria necessary for early diagnosis, the antimalarial drugs that can be used in pregnant women and draw attention to immuno-allergic and the classical secondary effects of quinine, which can also occur with chloroquine.