Polymerase chain reaction (PCR) technology was used to detect Toxoplasma gondii DNA in 253 immunodeficient subjects, 179 of whom were infected with the human immunodeficiency virus (HIV). The incidence of toxoplasmosis was 12.3% (22/179) in the HIV-infected subjects and 2.7% (2/74) in the remainder. The sensitivity of the PCR during episodes of toxoplasmosis in HIV-infected subjects not on antiparasitic treatment was 86.6% on peripheral blood and 60% on cerebrospinal fluid (CSF), but was only 25% and 16.7%, respectively, in subjects receiving specific treatment or prophylaxis against Pneumocystis carinii. Among the HIV-seronegative population, six patients undergoing anticancer chemotherapy were PCR positive on bronchoalveolar lavage fluid but did not develop pulmonary toxoplasmosis, suggesting transient carriage.

Diagnostic strategies for congenital toxoplasmosis have changed profoundly in recent years. Immunological diagnostic methods, long considered disappointing, can now be used at a very early stage. Over a 3-year period, 1,050 infants at risk of congenital toxoplasmosis (born to 1,048 mothers infected during pregnancy) were monitored for a minimum of 12 months and a maximum of 7 years. More than 6,000 serum specimens were analyzed by comparative mother-infant immunological profiles (CIPs) based on an enzyme-linked immunofiltration assay (ELIFA) and an immunocapture method for the detection of specific immunoglobulin M (IgM) and IgA. IgG antibodies were also titrated. One hundred three cases of congenital toxoplasmosis were demonstrated. The CIP-ELIFA method had a better diagnostic yield (sensitivity, 90%) than specific IgM and/or IgA detection by immunocapture assay (sensitivity, 77%). By using a combination of these tests, congenital infection was diagnosed in the first month and the first 3 months of life in 90 and 94% of infants with toxoplasmosis, respectively, with a specificity of 99.8% and a positive predictive value of 99% at 8 months of age. This dual diagnostic approach (ELIFA and IgM-IgA immunocapture) is highly efficient and has important implications for therapy. Indeed, early postnatal diagnosis based on objective evidence enables therapy with pyrimethamine-sulfadoxine to be started immediately for 24 months, while spiramycin (which used to be given preventively for 9 to 12 months to all infants at risk) can be stopped after the first 3 months of life.

Enzyme-linked immunofiltration assay (ELIFA) for labeling transferred proteins is an interesting and powerful technique for the rapid specific detection (15 min) of proteins immobilized on nitrocellulose or nylon membranes (0.20 and 0.45 micron). ELIFA does not require fastidious handling of the membranes. Saturation, specific labeling and washing procedures are achieved by filtration, controlled by a monitoring unit which regulates the flow rate and ensures excellent specificity, repetition and reproducibility. The recycling by closed circuit or by repetitive inversion of the flow direction offers the advantage of reducing the volumes of expensive reagents while simultaneously increasing the sensitivity of the technique. The detection limit is at least as low as 1-5 ng using directly or indirectly enzymatically labelled probes. ELIFA may be extended to the identification of glycoproteins using specific ligands such as lectins or to the immunocapture of an antigen using specific antibodies immobilized on an activated membrane. ELIFA complements fast separation, by e.g., isoelectric focusing, polyacrylamide gel electrophoresis, or sodium dodecyl sulfate-polyacrylamide gel electrophoresis and accelerated electrotransfer to membranes with rapid detection reducing the total time for separation transfer and detection to less than 2 h.

No abstract

The proposed serological diagnosis of systemic Candida infections is based on a microplate immunocapture technique detecting IgM, IgA and IgE anti-Candida antibodies. Activity is revealed with a suspension of human erythrocytes sensitized with somatic antigen of Candida albicans, and is quantified on an automated plate reader. The sera were obtained from patients with deep-seated (n = 56) and superficial (n = 193) candidosis. We compared this immunological method with a combination of indirect immunofluorescence and co-immunoelectrodiffusion. The immunocapture method was more sensitive (80.4% vs. 48.2% with indirect immunofluorescence and 58.9% with co-immunoelectrodiffusion), and often provided the diagnosis at an earlier stage, with clear therapeutic advantages. The IgA isotype was a particularly valuable marker of deep-seated Candida infections.

In order to define transmission routes of cryptosporidiosis and develop markers that distinguish Cryptosporidium parvum isolates, we have identified 2 polymorphic restriction enzyme sites in a C. parvum repetitive DNA sequence. The target sequence was amplified by polymerase chain reaction from 100 to 500 oocysts and the amplified product was subjected to restriction enzyme digestion. Typing of 23 isolates showed that 10/10 calf isolates had the same profile. In contrast, 2 patterns were observed among human isolates: 7/13 displayed the calf profile, and 6/13 presented another pattern. The PCR-RFLP assay described here is a sensitive tool to distinguish C. parvum isolates.

A monoclonal antibody directed against the oocyst wall of Cryptosporidium parvum was produced which strongly reacted with 25 strains of the parasite. This antibody was assessed for use in a diagnostic immunoperoxidase assay performed on tissue sections. It permitted an intense and specific staining of the parasite in five biopsies positive for Cryptosporidium parvum fixed with routine techniques. The procedure described may be useful in screening mucosal biopsies from immunosuppressed patients.

No abstract