[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.

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.

IgA antibody response during acquired and congenital toxoplasmosis.

Toxoplasma gondii specific IgA and IgM antibodies were quantitated by an antibody capture agglutination assay in 260 patients with acquired toxoplasmosis and from 94 fetuses suspected of congenital toxoplasmosis and 30 infected children. In acquired toxoplasmosis, IgA antibodies to T gondii were found in 95% of the cases. In congenital toxoplasmosis IgA antibodies were more frequently detected (75%) in cord blood than IgM antibodies (61%). They persisted after birth, in some cases for up to 24 months. IgA antibodies were also detected in fetuses whose mothers had toxoplasmosis during their pregnancy. In infected fetuses IgM and IgA antibodies were detected in fetal blood as early as week 24 of pregnancy. Detection of IgA T gondii antibodies may be useful for the diagnosis of some recently acquired infection and for the diagnosis and follow up of the infection in the fetus and neonate.

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.

Immunological analysis of the aqueous humour in candida endophthalmitis. I: Experimental study.

An immunological study of aqueous humour and serum was carried out in experimental ocular candidosis in rabbits. Anti-candida antibody titres, immunoglobulin G, and albumin levels were evaluated in the aqueous humour and in the serum. A local synthesis of anti-candida antibodies was detected in numerous rabbits at the height of clinical activity of the lesions.

Immunological analysis of the aqueous humour in candida endophthalmitis. II: Clinical study.

An anterior chamber paracentesis was carried out on six patients with candida endophthalmitis and on control subjects. We endeavoured to find out if there was local production of antibodies by evaluating the anti-candida antibody titres, the immunoglobulin G, and the albumin levels in the aqueous humour and in the serum. A significant difference in the level of specific antibodies between patients and controls was found. Local synthesis of anti-candida antibodies was discovered in five out of six patients with candida endophthalmitis. There would seem to be a correlation between the severity of uveitis and antibody titres.

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.

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.

[Latex agglutination reaction for the diagnosis of toxoplasmosis]. / Etude d'une réaction d'agglutination au latex pour le diagnostic de la toxoplasmose.

Agglutination of latex-particles sensitized with toxoplasma-antigen is a simple, fast, inexpensive test for toxoplasmosis-diagnosis. This test has been compared with passive-hemagglutination (1091 sera) and immunofluorescence (1093 sera). From the results, this new test is showed to be closer to passive-hemagglutination than indirect immunofluorescence. After studying possible false results and biological qualities it is thought that this test has good qualities to render great services for the toxoplasmosis diagnosis, and specially for epidemiological investigations.

[Local production of specific antibodies in the aqueous humor in experimental Candida endophthalmia in rabbits]. / Etude de la production locale d'anticorps spécifiques dans l'humeur aqueuse dans l'endophtalmie expérimentale à Candida du lapin.

An increasing frequency of ocular lesions due to Candida albicans has been reported, but clinical detection of lesions, which may cause permanent blindness, remains difficult. Hematogenous endophthalmitis caused by Candida albicans in the rabbit is a useful model to study the pathogenesis of the disease, and perhaps to find new methods of biological diagnosis. A suspension of Candida albicans was injected in 15 rabbits, according to the method described by Edwards et al. In 1975, causing uni or bilateral chorioretinitis in 12 of them. Unaffected eyes were chosen as controls. After aspiration of the anterior chamber fluid, serum and aqueous humour titres of specific antibodies were determined. A local synthesis of IgG antibodies was detected, after comparison of the titres of specific antibodies in the aqueous humour and in the serum, related to the serum and aqueous humour concentrations of IgG antibodies. According to a formula proposed by Witmer and Desmonts in ocular lesions due to different pathological agents, we calculated the "C" coefficient: C = antibody titres aqueous humor/serum x IgG serum/aqueous humor. A local synthesis of specific antibodies in cases of experimental endophthalmitis leads us to believe that aqueous humour aspiration could be used in the diagnosis of endophthalmitis in humans.

[Predictive value of non-specific signs of fetal infection in congenital toxoplasmosis]. / Valeur prédictive des signes non spécifiques d'infection foetale dans la toxoplasmose congénitale.

AIMS: To study the predictive value of 9 non-specific signs for congenital toxoplasmosis. METHOD: The work was carried out as a prospective study comparing non-specific signs in 22 fetuses infected with toxoplasmosis and 59 fetuses free of the disease. RESULTS: Four of the nine parameters were found to be statistically higher in the group of infected fetuses. These are the level of leucocytes (p < 0.05) gamma glutamyl transferase (p < 0.001) total M immunoglobulin (p < 0.0001) and C4 fraction of complement (p < 0.0001). This last sign which is non-specific for infection has never been looked at until now in congenital toxoplasmosis but is seems to be of great importance because its sensitivity is much greater than of all the other parameters that have been used up till now (81%) and the positive predictive value is excellent (72%). On the other hand the value of a raised leucocyte count is a matter for discussion. CONCLUSION: The presence of even one abnormally raised feature in the fetal blood of these non-specific signs is enough to screen for a population which is of high risk for congenital toxoplasmosis and sufficient to change our attitude to treating the condition.

[Antenatal diagnosis of toxoplasmosis. 176 case reports]. / Diagnostic anténatal de la toxoplasmose. A propos de 176 observations.

OBJECTIVE: To determine the value of antenatal diagnosis of congenital toxoplasmosis by ultrasound guided aspiration of cord blood for testing. MATERIAL: This is a prospective study of 176 cases. As well as obtaining fetal blood and amniotic fluid the searched for specific IgM and A as well as culturing for the parasites on human fibroblasts and inoculation of mice, as well as researching them for non-specific signs of fetal infection. 149 children were able to be followed up one year after birth. RESULTS: 15% of the children (22/149) were infected with toxoplasmosis. 11 out of these were diagnosed positive antenatally. For the 11 others the diagnosis of fetal infection could only be made after birth, but the non-specific signs made it possible to expect early that they had been contaminated. 59% (13/22) had latent toxoplasmosis which only showed up after a mean interval of 34 months after birth. 41% (9/22) had clinical and/or paraclinical signs of toxoplasmosis (mainly unilateral non-macular chorioretinitis and intracranial calcifications) but they are well after a follow-up period averaging 30 months. COMMENTARY: Ultrasound alone, when it shows up fetal abnormalities, can make the diagnosis of the severity of the condition. The role of taking fetal specimens is to make clear those infants that are infected because of specific signs, and to find those fetuses which are at high risk because of non-specific signs in order to improve the management of the cases. This development has made it possible to avoid carrying out a large number of unnecessary terminations of pregnancy and has resulted in the birth of affected infants that had no functional sequelae from the infection.

[Biological reference values in the human fetus. 106 cord blood sampling in utero]. / Valeurs biologiques de référence chez le foetus humain. A propos de 106 ponctions de cordon in utero.

The authors give biological reference figures obtained from 106 fetuses that were sampled in utero between the 20th and 34th week of amenorrhoea. These fetuses were considered to be normal because there was no clinical or ultrasound evidence of an abnormality. Furthermore the biological values sought in antenatal testing and the absence of all pathology in the first year of life, confirmed that these were normal fetuses. The result has been expressed as a global figure for all 106 fetuses; then they have been divided up according to the gestational age groups (20-23, 24-27, and 28-34 weeks of amenorrhoea). These biological reference values and their changes as the age of the fetuses advanced are discussed and compared with the figures reported in the literature.

[Fetal blood sampling under ultrasonic control]. / Prélèvement de sang du cordon foetal sous contrôle échographique.

Withdrawing foetal blood from the umbilical cord under ultrasonic control requires close co-ordination between obstetricians and echographists. The most important indications for this procedure are infections and foetal karyotype determination.

[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.