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.

Prenatal diagnosis of congenital cytomegalovirus infection.

OBJECTIVE: To assess prospectively the diagnostic reliability and prognostic significance of prenatal diagnosis of cytomegalovirus (CMV) infection. METHODS: One hundred ten pregnant women (four with twin pregnancies) with a risk of congenital CMV infection were investigated. Prenatal diagnosis was carried out by amniocentesis and fetal blood sampling (n = 75) or amniocentesis alone (n = 35). Serial ultrasonographic examinations were performed from time of referral until pregnancy end. All infected neonates were given long-term follow-up. Autopsy was performed in all cases of termination of pregnancy. RESULTS: Nearly 23% (26 of 114) of fetuses were infected and prenatal diagnosis was positive in 20 cases. Sensitivity of prenatal diagnosis was 77% and specificity 100%. In eight cases, parents requested termination of pregnancy on the basis of abnormal ultrasonographic findings and/or biologic abnormalities in fetal blood. In 12 cases, parents decided to proceed with the pregnancy. In this group, one intrauterine and one neonatal death were observed. In one case, prenatal diagnosis revealed an abnormal cerebral sonography and the infant had bilateral hearing loss at birth. In 15 cases (nine positive and six false-negative prenatal diagnoses), no apparent lesion was present at birth, nor did it develop during the follow-up period (mean 31 months). In 88 (77.2%) of 114 infants, no evidence of vertical transmission was found during the pre- or postnatal period. CONCLUSION: Prenatal diagnosis provides the optimal means for both diagnosing fetal infection (amniocentesis) and identifying fetuses at risk of severe sequelae (ultrasound examination, fetal blood sampling), thus allowing proper counseling.

Sources of toxoplasma infection in pregnant women: European multicentre case-control study. European Research Network on Congenital Toxoplasmosis.

OBJECTIVE: To determine the odds ratio and population attributable fraction associated with food and environmental risk factors for acute toxoplasmosis in pregnancy. DESIGN: Case-control study. SETTING: Six large European cities. PARTICIPANTS: Pregnant women with acute infection (cases) detected by seroconversion or positive for anti-Toxoplasma gondii IgM were compared with pregnant women seronegative for toxoplasma (controls). MAIN OUTCOME MEASURES: Odds ratios for acute infection adjusted for confounding variables; the population attributable fraction for risk factors. RESULTS: Risk factors most strongly predictive of acute infection in pregnant women were eating undercooked lamb, beef, or game, contact with soil, and travel outside Europe and the United States and Canada. Contact with cats was not a risk factor. Between 30% and 63% of infections in different centres were attributed to consumption of undercooked or cured meat products and 6% to 17% to soil contact. CONCLUSIONS: Inadequately cooked or cured meat is the main risk factor for infection with toxoplasma in all centres. Preventive strategies should aim to reduce prevalence of infection in meat, improve labelling of meat according to farming and processing methods, and improve the quality and consistency of health information given to pregnant women.

[Diagnosis of materno-fetal toxoplasmosis]. / Diagnostic de la toxoplasmose materno-foetale.

The antenatal diagnosis of congenital toxoplasmosis represents today an important application of molecular diagnostic methods such as PCR. Done directly in amniotic fluid, the PCR for T. gondii is able to detect in utero infected foetus with high probability. Doing so, it precludes to obtain foetal blood and to use more cumbersome and lengthy procedures such as inoculation to cell lines or to mice. Although it is use today in many centres, the molecular diagnosis of T. gondii by PCR is neither commercialized nor standardised. The objective of this report is to present the methodology used in our institution and to establish its degree of validation.

Value of the comparative enzyme-linked immunofiltration assay for early neonatal diagnosis of congenital Toxoplasma infection.

BACKGROUND: The transplacental transfer of specific maternal IgG antibodies makes the diagnosis of congenital Toxoplasma infection quite difficult in the neonate. The enzyme-linked immunofiltration assay (ELIFA), comparing at delivery the immunologic profile of the mother's antibody response and that of her child, allows discrimination between IgG antibodies of maternal origin and IgGs synthesized by the fetus. OBJECTIVE: To evaluate the diagnostic reliability of the comparative ELIFA for diagnosing congenital Toxoplasma infection as well as the reliability of testing for IgM- and IgA-specific antibodies in cord blood. METHODS: From November, 1991, to December, 1995, an ELIFA was prospectively performed at delivery on blood samples obtained from 227 women with primary Toxoplasma infection during pregnancy and from their infants. For each child the ELIFA result was evaluated in relation to the serologic follow-up: disappearance of specific anti-Toxoplasma gondii IgG antibodies in the absence of treatment before 12 months of age indicating an uninfected child, as opposed to persistence beyond 12 months of age indicative of a congenital infection. RESULTS: Of 227 children 139 were lost to follow-up. Among the 88 children available for follow up, the ELIFA was negative in 70 infants, 69 of whom were confirmed to be uninfected. Thirteen of these 69 cord blood ELIFA-negative samples were positive for anti-T. gondii IgM and/or IgA detected by means of a conventional immunosorbent agglutination assay. Of the remaining 18 children (representing 75% of all new cases of congenital toxoplasmosis diagnosed during the study period at our institution), the ELIFA was positive in 16, negative in 1 and inconclusive in 1. CONCLUSIONS: The ELIFA test is a valuable tool for diagnosing congenital T. gondii infection and in differentiating between true neonatal infection and cord blood contamination. In our experience the diagnostic sensitivity of the ELIFA test was 94.1% and the specificity was 98.6%. The cord blood was contaminated by specific maternal anti-T. gondii IgA and/or IgM in as many as 20% of the cases.

The nasolabial fold: an attempt at synthesis.

There is now enough information on the nasolabial fold to try to synthesize it with other well-known structures, such as the dermal terminations of the facial muscles, the superficial musculoaponeurotic system, and the fat pad. Rest dynamic equilibrium is a good concept to use to understand the nasolabial fold, because the nasolabial fold is not a passive, definitive structure, but an evolutive border whose limits depend on the absence or presence of fibromuscular terminations crossing the superficial musculoaponeurotic system of the cheek. A simple photograph of two men will help illustrate the difference between the convex and the concave nasolabial fold.

Persistence of parvovirus B19 DNA in testis of patients with testicular germ cell tumours.

Germ cell tumours (GCT) of the testis are the most common malignant tumours occurring in young adults. In view of the young age of patients, the increasing incidence of GCT and the overexpression of wild-type p53 observed in a majority of tumours, the possibility of the involvement of a virus in the development of this cancer was considered. Testicular GCT were analysed for the presence of cytomegalovirus and Epstein-Barr virus (EBV), which are known to cause overexpression of wild-type p53 protein, and parvovirus B19. The testicular tissue of 39 patients with testicular GCT and 12 patients with healthy testicular tissues was tested for presence of viral DNA by PCR. Neither cytomegalovirus nor EBV DNAs were detected in the 39 tumours analysed, but parvovirus B19 DNA sequences were demonstrated in the testicular tissue of 85% (33/39 cases) of patients with GCT. The sera of 16 of the 39 patients with GCT were tested for the presence of parvovirus B19 IgM and IgG. B19-specific IgG was detected in the sera of 11 patients (69%). Only one case was positive for parvovirus B19 IgM, which was also shown to have B19 genome sequences in the serum by PCR, indicating that in a majority of cases an acute B19 infection can be excluded as being the source of the B19 DNA sequences in the testis. B19 DNA could not be detected in normal testicular tissue and thus parvovirus B19 could play a role, direct or indirect, in the development of testicular GCT or have tropism for the tumour cells.

Seroprevalence of rubella among women of childbearing age in Switzerland.

A seroepidemiological study was carried out in Switzerland to define the population susceptible to rubella among women of childbearing age. IgG antibodies to rubella virus were determined in 9,046 women giving birth between 1 August 1990 and 30 September 1991 in 23 of 26 Swiss cantons. These sera represented 10-20% of the yearly total number of births in each Swiss canton. Anti-rubella IgG was measured by an automated enzyme-linked fluorescent assay for use with a commercial system (Vidas Rub IgG, bio-Mérieux, France). Before the study population was screened, the commercial system was compared to the traditional hemagglutination-inhibition (HAI) test using 500 consecutive samples from parturient women. The sensitivity was 97.7%, the specificity was 100%, and agreement between the two tests was 97.8%. The discrepancies corresponded to very low titres of antibodies as measured by HAI. The seroprevalence of rubella nationwide in women of childbearing age in Switzerland was 94.3%. The seroprevalence was higher (96.5%) in the 5,677 women of Swiss nationality than in the 3,090 women of a different nationality (90.4%) (p < 0.001). In Swiss women the seroprevalence of rubella did not increase significantly with age and was identical in primiparous and in multiparous women, thus indicating that women of childbearing age are probably not sufficiently immunised.

[Biological diagnosis of toxoplasmosis in the course of pregnancy: methods, interpretations and practical recommendations]. / Diagnostic biologique de la toxoplasmose au cours de la grossesse: méthodes, interprétations et recommandations pratiques.

The biological diagnosis of infection with Toxoplasma gondii during pregnancy can be performed: either (1.) with direct methods (microscopic examination, in vitro isolation on cell cultures, histology, detection of T. gondii DNA) or (2.) by indirect serological methods in order to demonstrate specific antibodies in serum. An appropriate combination of complementary techniques (antibody detection in serum and demonstration of the parasite) must lead, in the majority of cases, to a precise diagnosis of congenital toxoplasmosis. Serological examination of a woman before or at the beginning of pregnancy serves to distinguish between women with or without specific immune status. The diagnosis of a seroconversion during pregnancy causes few difficulties particularly if carefully followed up. However, it is rather difficult to date an infection during pregnancy based only on a initial positive sample with the presence of anti-T. gondii IgM. Thus, around 1 to 5% of serological examinations during pregnancy cause practical problems of interpretation. A certain number of recommendations are suggested to the laboratory supervisor: 1. Pregnant women should be systematically tested for specific anti-T. gondii IgG or total Ig and IgM. The choice of the techniques serves to reliably distinguish women without anti-T. gondii antibodies (women at risk for primary infection) from those with anti-T. gondii antibodies (immune women). 2. Interpretations of results should be well formulated to be understandable to the practitioner who must adequately inform the patient and determine the proper medical action on the basis of the serological results. 3. Sera should be preserved systematically for a minimum of 12 months (whatever the results may be). 4. Cases or situations causing problems should be referred to a specialized laboratory for expert advice.

Prevalence of latent toxoplasmosis and serological diagnosis of active infection in HIV-positive patients.

The seroprevalence of latent Toxoplasma gondii infection was determined in a cohort of 715 HIV-positive patients followed up at an HIV outpatient clinic. Using indirect immunofluorescence and direct agglutination assays for detecting IgG, the prevalence of anti-Toxoplasma gondii antibodies was shown to be 50%. During a four-year period, clinically apparent acute toxoplasmosis occurred in 47 patients (43 with cerebral, 3 with ocular and 1 with bone marrow toxoplasmosis) among the 360 patients positive for anti-Toxoplasma gondii IgG and in one patient (with cerebral toxoplasmosis) among the 355 patients who were serologically negative. A significant rise in IgG levels could be shown during acute toxoplasmosis episodes in only 30% of patients, compared with 3% of patients without active toxoplasmosis. During acute toxoplasmosis, IgM antibodies were detected in only two patients (6%) by an immunosorbent agglutination assay and in one (3%) by an enzymatic immunocapture assay. Specific IgA was detected by a non-enzymatic immunocapture assay in six patients (18%) during acute episodes. The very high predictive value (99.7%) of a negative IgG test remains the best serological parameter for excluding an acute episode of toxoplasmosis in HIV-positive patients.

Anatomic variations of the nasolabial fold.

The nasolabial fold varies considerably from person to person. Three main groups may be distinguished: convex, concave, and straight. It is the muscles of smiling that are directly responsible for the shape and depth of the fold, and in their absence of function, as in facial palsy, the nasolabial fold disappears. Cadavers were selected in accordance with the nasolabial fold they presented and were dissected to analyze the difference in underlying anatomy between one fold shape in one cadaver and another fold shape in another. The study demonstrates that the nasolabial fold is the result of a conflict between soft and dynamic tissues of the middle face or an interaction between the skin and fat envelope on one side and the underlying muscles on the other. The greater this conflict, the more excess there is of cheek skin and the more pronounced a nasolabial fold. The mechanism that creates the nasolabial fold and the anatomy of the fold are described in this paper.

Simple method for rapid diagnosis of catheter-associated infection by direct acridine orange staining of catheter tips.

Direct acridine orange (AO) staining was used to detect bacteria adherent to intravascular catheters (IVC). Samples from 710 IVC tips were first cultured on blood agar plates by a semiquantitative technique and then independently colored with AO and screened dry at a magnification of x100 for 3 min. In the absence of fluorescence, they were considered negative. When fluorescence was present, they were further examined for the presence of microorganisms at x1,000 with immersion oil. Of 710 IVC tips, 37 (5.2%) were positive upon culture (greater than or equal to 15 colonies) and 673 were negative (640 were sterile and 33 [4.6%] had 1 to 14 colonies). The AO sensitivity was 84%, and the AO specificity was 99%. When restricted to the 212 long IVC, AO sensitivity rose to 94%. AO staining was positive in all cases of catheter-associated bacteremia. The negative predictive value of the preliminary screening at x100 was 99.5%. The direct examination of IVC tips stained by AO appears to be a simple and rapid method for diagnosing IVC-associated infections. In addition, AO staining is easier to perform than Gram staining.