[Quality controls in medical mycology]. / Les contrôles de qualité en mycologie médicale.

The Quality Management System in medical mycology refers to the systematic monitoring with internal and external quality controls: it needs to be organized in the laboratory. ISO 15189 standard is not precise in how to demonstrate the correctness of tests, in terms of frequency and requirements for quality controls QC. That's why the COFRAC, the French Accreditation Committee has published guides to which we should refer. The laboratory has to apply internal Quality Control Programs. They consist of various tests to check the reagents including the culture media. Reference strains have to be provided and preparations of homemade reagents are needed, because few are commercialized. Maintaining the competence of the technical staff through identification of unknown strains is also required. In the fungal serology field, home made antibodies with pooled sera or antigen controls are needed. This monitoring has to follow the recommandations from the Cofrac technical guide LAB GTA 06. For quantitative analysis, the Levey-Jennings chart is a graph with quality control data plotted on to give a visual indication. Some external QC references, besides the national quality control AFSSAPS, are available. Data evaluation, corrective actions in case of out of range results and preventive actions have to be determined in the Quality System documents and presented in the annual management review.

Diagnosis of congenital toxoplasmosis: prenatal and neonatal evaluation of methods used in Toulouse University Hospital and incidence of congenital toxoplasmosis.

The aim of this study was to determine the incidence of congenital toxoplasmosis (CT) and to assess the performances of prenatal and neonatal diagnoses. From 1994-2005, in Toulouse University Hospital, France, amniocentesis was performed on 352 pregnant women who were infected during pregnancy. All women were treated with spiramycin and pyrimethamine-sulfadoxine when prenatal diagnosis was positive. Among the 275 foetuses with follow-up, 66 (24%) were infected. The transmission rates of Toxoplasma gondii were 7%, 24% and 59% in the first, second and third trimesters, respectively. The sensitivity and specificity of PCR on amniotic fluid (AF) were 91% and 99.5%, respectively. One case was diagnosed by mouse inoculation with AF and six cases were diagnosed by neonatal or postnatal screening. The sensitivity and specificity of PCR on placentas were 52% and 99%, respectively. The sensitivity of tests for the detection of specific IgA and IgM in cord blood was 53% and 64%, respectively, and specificity values were 91% and 92%. In conclusion, PCR performed on AF had the highest levels of sensitivity and specificity for the diagnosis of CT. This permits an early diagnosis of most cases and should be recommended.

Comparison between two amplification sets for molecular diagnosis of toxoplasmosis by real-time PCR.

PCR is now commonly applied to the diagnosis of toxoplasmosis. Although several methods are available, comparative studies are few, making it difficult to compare the performance of each technique. We compared the sensitivities of two real-time PCR assays through a prospective study on fetuses, neonates, and immunocompromised patients and on the ocular diagnosis of toxoplasmosis. The first system targeted the widely used B1 gene (GenBank accession number AF179871) while the second (RE) targeted a more recently described sequence repeated roughly 200 to 300 times (GenBank accession number AF146527). We demonstrated that molecular diagnosis requires the duplication of PCR assays, especially with the B1 system, as only one PCR was positive in 33.3% of cases. Our study showed that the RE target was more sensitive for all biological samples (amniotic fluid, placenta, aqueous humor, whole blood, and cerebrospinal and bronchoalveolar fluids) and significantly improved the performance of the diagnosis of toxoplasmosis. Taking into consideration all clinical samples, the mean gain in the crossing point value was 4.2 +/- 1.7 cycles and was even more significant for amniotic fluid (5.8 +/- 1.7 cycles).

Prediction of congenital toxoplasmosis by polymerase chain reaction analysis of amniotic fluid.

OBJECTIVE: To determine the accuracy of polymerase chain reaction (PCR) analysis of amniotic fluid for fetal toxoplasmosis according to clinical predictors of outcome and study centre. DESIGN: Prospective cohort study. SETTING: Nine European centres. POPULATION: Women with suspected toxoplasma infection identified by prenatal screening. METHODS: Logistic regression was used to examine the effects of gestational age at maternal seroconversion, treatment and timing of amniocentesis, on PCR accuracy, and to calculate the post-test probability of congenital toxoplasmosis. MAIN OUTCOME MEASURES: Infants had congenital toxoplasmosis if specific IgG persisted beyond 11.5 months. Uninfected infants had undetectable IgG in the absence of anti-toxoplasma treatment. RESULTS: Of 593 PCR results, 64 were positive (57 confirmed infected), and 529 were negative (23 confirmed infected). The likelihood ratio for a positive PCR result decreased significantly with trimester at seroconversion, but did not change significantly for a negative result. Weak associations were detected between sensitivity and, inversely, with specificity, and gestational age at maternal seroconversion. There was no significant association between sensitivity and centre, type or duration of treatment, or timing of amniocentesis. Specificity differed significantly between centres (P < 0.001). The change in pre- to post-test probability of infection was maximal for a positive PCR after first trimester seroconversion, affecting 1% of women tested, and a negative PCR after third trimester seroconversion, affecting half the women tested. CONCLUSIONS: Prediction of the risk of congenital toxoplasmosis should combine estimates of test accuracy and maternal-fetal transmission, which take account of the gestational age at which the mother seroconverted. Local laboratory standards will affect the generalisability of these results.

Multicenter evaluation of strategies for serodiagnosis of primary infection with Toxoplasma gondii.

The diagnostic performance of single-serum assays for toxoplasma-specific immunoglobulin (Ig)M. IgA. IgG, and IgE antibodies and of different combinations of such antibody assays in 20 European reference centers was assessed. A panel of 276 sera, of which 73 came from patients who seroconverted within 3 months (acute infection), 49 from patients who had seroconverted 3-12 months earlier (convalescence), and 154 from subjects who had two IgG-positive samples obtained 12 months apart (past infection), was tested with 20 toxoplasma-antibody assays and 195 combinations. In general, every assay with high diagnostic sensitivity showed low diagnostic specificity, i.e. no assay performed alone could reliably distinguish acute from past infection. Furthermore, no single assay (or combination) could separate convalescence from the other stages of toxoplasma infection. However, excellent diagnostic performances were reached by sequential use of highly sensitive IgM assays and methods examining IgG avidity or stage specificity. IgA or IgM assays were less suitable for confirmation of toxoplasma-IgM positivity. This study documents the strength of test combinations in assessing the stage of toxoplasma infection.

Strategy for diagnosis of congenital toxoplasmosis: evaluation of methods comparing mothers and newborns and standard methods for postnatal detection of immunoglobulin G, M, and A antibodies.

In a study involving 14 laboratories supported by the European Community Biomed 2 program, we evaluated immunologic methods for the postnatal diagnosis of congenital toxoplasmosis (CT). Among babies born to mothers who seroconverted to positivity for toxoplasmosis during pregnancy, we analyzed 55 babies with CT on the basis of persistent anti-Toxoplasma immunoglobulin G (IgG) at 1 year of life and 50 control babies without anti-Toxoplasma IgG at 1 year of life in the absence of curative treatment with pyrimethamine-sulfonamides. We tested in-house methods such as the enzyme-linked immunofiltration assay (ELIFA) or Immunoblotting (IB) for the detection of IgG or IgM; these methods allowed comparison of the immunologic profiles of the mothers and the infants. We compared ELIFA and IB with a commercial enzyme immunoassay (EIA) or in-house immunosorbent agglutination assay (ISAGA) for the detection of IgM or IgA. The performances of combinations of methods were also assessed. A cumulative sensitivity of 98% during a 1-year follow-up was obtained with the ELIFA plus ISAGA combination. Only one case of CT was missed by the ELIFA plus ISAGA combination, whereas three cases were missed by the IB plus ISAGA combination, even though 48% of patients with CT were treated with pyrimethamine-sulfonamides, which are known to inhibit antibody neosynthesis. A similar performance was obtained with either ELIFA or IB in combination with EIA. The difference in performance between ELIFA plus ISAGA and IB plus ISAGA was not statistically significant (P = 0.31), and we conclude that both combinations of tests can be used for the diagnosis of CT in newborns.

Neonatal screening for congenital toxoplasmosis in a cohort of 165 women infected during pregnancy and influence of in utero treatment on the results of neonatal tests.

The aim of this study was to determine the performances of methods used for the neonatal diagnosis of congenital toxoplasmosis. We included 165 pregnant women infected during pregnancy over a 10-year period. Fifty-seven cases of congenital toxoplasmosis were demonstrated (34.5%). Neonatal diagnosis gave positive results in 50 cases (88%). Parasites were isolated from placenta or cord blood in 61% of the infected newborns, more frequently from placenta (60%) than from cord blood (43%). This method was the only criterion of infection in 18% of these infected infants. The detection of specific IgM and IgA antibodies performed on 42 sera of infected infants allowed the diagnosis of congenital infection in 34 cases (81%). IgA antibodies were more frequently detected (60%) than specific IgM (50%). Neonatal and prenatal screening were carried out for 143 pregnant women. This combination diagnosed 39 of 40 infected infants (98%). Prenatal diagnosis identified 30 of 40 cases (75%). Nine cases were diagnosed through neonatal screening and one case with the postnatal follow-up. When prenatal diagnosis was positive, pyrimethamine and sulfadoxine were administered to the mothers (25 cases) in addition to spiramycin. Toxoplasma gondii was less frequently isolated in the placenta and the cord blood of these women (32% and 19%, respectively) than in women treated by spiramycin alone (83% and 63%) proving the antiparasitic action of these drugs. In conclusion, neonatal screening combining parasite detection in placenta and immunological methods on cord blood is essential particularly when prenatal diagnosis is negative. Therefore, when this diagnosis is positive, a treatment with pyrimethamine and sulfamide can be started in the first month of life.

Toxoplasma gondii secretes a calcium-independent phospholipase A(2).

Phospholipases A(2) (PLA(2)) play an important role in Toxoplasma gondii host cell penetration. They are also key enzymes in the host cell response to the parasite invasion. PLA(2) hydrolyse cellular phospholipids, releasing multiple inflammatory lipidic mediators. We have investigated the biochemical characterisation of T. gondii PLA(2) activity in a mouse-cultured tachyzoite homogenate and in the peritoneal exudate from infected mice, using the hydrolysis of a fluorescent phosphatidylglycerol labelled at the sn-2 position. Spectrofluorimetry and thin-layer chromatography showed a PLA(2) activity (about 0.5-2 nmol/min per mg), calcium-independent, secreted into infected mice peritoneal exudate, with a broad pH activity ranging between 6.5 and 9.5 and resistant to a great number of potential PLA(2) inhibitors except dithio-nitrobenzoic acid (1 mM). An associated phospholipase A(1) activity was also displayed. These results suggest that Toxoplasma gondii displays specific phospholipases different from host enzymes and probably involved at critical steps of infectious cycle.

Inhibitory activity of anti-interleukin-4 and anti-interleukin-10 antibodies on Toxoplasma gondii proliferation in mouse peritoneal macrophages cocultured with splenocytes from infected mice.

Cocultures of splenocytes from Toxoplasma gondii-immunized mice or from naive mice, separated by a transwell membrane from naive macrophage layers, induced a decrease in T. gondii proliferation in macrophages in comparison with cultures without splenocytes or cocultures with splenocytes from infected mice. Interleukin 4 (IL-4) and IL-10 levels increased in cocultures of splenocytes from infected mice with naive macrophages. In contrast, the levels of these cytokines decreased in cocultures with splenocytes from immunized mice. No correlation was found between the release of interferon-gamma (IFN-gamma) and the inhibition of parasite multiplication. Cocultures with splenocytes from immunized mice induced an increase in tumor necrosis factor-alpha (TNF-alpha) levels. In contrast, in cocultures with splenocytes from infected mice, TNF-alpha production decreased. In cocultures with splenocytes from infected mice, T. gondii proliferation in macrophages was neutralized by anti-IL4 or anti-IL10 antibodies and was associated with increased TNF-alpha production. Moreover, this study demonstrates the significant combined effect of IL-4 and IL-10 on the down-regulation of macrophage-effector functions. A soluble positive signal was given by splenocytes to induce the production of TNF-alpha by macrophages. This signal was inhibited by IL4 and IL10. This process is biologically relevant in the regulation of T. gondii proliferation.

[A new case of subcutaneous dirofilariasis]. / A propos d'un nouveau cas de dirofilariose sous-cutanée.

Authors describe another case of subcutaneous dirofilariasis due to Dirofilaria (Nochtiella) repens in France. A 26-year-old woman was infected while on vacation in Cap d'Agde on the Mediterranean coast. The patient presented with a subcutaneous nodule in the right subclavicular region. Examination of the nodule after surgical excision revealed the presence of a worm identified as an immature female Dirofilaria repens. Dirofilariasis is a rare anthroponotic disease encountered only in the Old World, particularly in Southeastern France including Corsica which has the second highest number of reported cases after Italy. Since man is a dead-end for the parasite, Dirofilaria repens does not mature and hence most human infections present as isolated subcutaneous nodules. Nodules are usually located in areas exposed to bites by the dipteres, i.e. the face (46 p. 100 of cases reported in the world) and the periocular and palpebral region (30 p. 100). Diagnosis is based mainly on morphological examination of the worm after surgical excision. However promising results in diagnosis of ocular and visceral forms of Dirofilaria repens and understanding of helminthiasis have been achieved thanks to progress in immunological techniques, i.e., ELISA and western blot, and DNA analysis based on polymerase chain reaction.

Specificity of low anti-Toxoplasma IgG titers with IMx and AxSYM Toxo IgG assays.

Low antibody titers (3 to 6 IU/mL) detected by IMx and AxSYM Toxo IgG assays in serum samples from 264 pregnant women were confirmed by the dye test and a high-sensitivity agglutination test in, respectively, 98.5% and 95.6% of cases, attesting a premunition of these patients.

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.

Parasitemia and parasitic loads in acute infection and after anti-gamma-interferon treatment in a toxoplasmic mouse model.

Toxoplasma gondii infection was induced in Swiss-Webster mice by intraperitoneal inoculation of avirulent Beverley strain cysts. We studied parasitemia and parasitic loads first in acute toxoplasmosis, then in the chronic stage of the disease. In the latter stage a group of mice received weekly administration of a rabbit antiserum directed against mouse gamma-interferon. Parasitemia, sequentially determined by amplification of the B1 gene using polymerase chain reaction, persisted for more than 1 month in acute toxoplasmosis. Brain and lung parasitic loads, assessed by a tissue-culture method, were significantly increased in mice treated with anti-interferon. Moreover, this increase was prevented by concomitant administration of pyrimethamine and sulfadiazine, suggesting that early prophylaxis would be suitable. Surprisingly, the anti-interferon treatment induced neither abnormal clinical signs nor a significant rise in the parasitemia level. Such a model seems to be particularly appropriate for the comparison of different strains of Toxoplasma gondii in a moderately immunodeficient state.

The evolution of ocular toxoplasmosis in anti-interferon gamma treated mice.

PURPOSE: A clinico-histopathological cross correlation was made to study the mechanism of tissue damage in toxoplasmic retino-choroiditis during an experimental reactivation of chronic toxoplasmosis and to compare the influence of treatment by sulfadiazine on the retinal lesions. METHODS: Chronically infected Swiss-Webster mice were treated, six weeks after infection, with an avirulent strain of Toxoplasma gondii (Beverley strain) with polyclonal rabbit antibody directed against murine interferon gamma. RESULTS: Mice treated by anti-interferon gamma developed clinical lesions between day 5 and day 30 (lesions including single foci of retinochoroiditis, multifocal lesions or diffuse areas of retinal necrosis). These lesions did not arise from borders of pre-existing scars. The retina was photographed with an operating microscope fitted with a 90 diopter lens. Biological study showed a significant rise of parasitic loads in the eye and brain. Histological examination is in favour of free organism dissemination via retinal vessels; the lesions are restricted to the inner retina and ciliary body, the parasites migrated from extra-ocular cysts via the vasculature. No cysts were seen at the beginning of the study; they were found at the scar phase and appeared in mice treated with sulfadiazine. The clinical lesions were not caused by cysts but by coagulated necrosis in the retinal tissue. Parasite migration may have played a trigger role. CONCLUSIONS: The retinal damage was constituted either as a result of a toxic effect of the organisms or as a hypertensive reaction to the toxoplasma organism. The results of this study showed that the treatment with anti interferon gamma was sufficient to reactivate chronic infection.

Termination of pregnancy for maternal toxoplasmosis.

Termination of pregnancy is usually recommended to pregnant women who have infection with Toxoplasma gondii before 26 weeks of pregnancy if the fetus is infected. No prospective studies are available on the outcome if such pregnancies are allowed to continue with anti-parasitic treatment. We prospectively studied 163 mothers with acute toxoplasma infection before 28 weeks of amenorrhoea. All received anti-parasitic treatment with 9 million IU spiramycin orally. 23 also received pyrimethamine and sulphadiazine. All had cordocentesis and regular obstetric ultrasound examinations. The 162 liveborn infants were followed up for 15 to 71 months. 3 fetuses died in utero. 27 of 162 liveborn infants had proven congenital toxoplasmosis: 10 had one or more clinical signs of congenital toxoplasmosis; 5 had isolated or multiple intracranial calcifications; 7 had peripheral chorioretinitis; and 2 had moderate ventricular dilations. All 27 are free from symptoms and have normal neurological development at 15 to 71 months of age. We conclude that in first and second trimester pregnancies with acute fetal toxoplasma infection, the pregnancy need not be interrupted if repeated fetal ultrasound is normal, and antiparasitic treatment is given.