P554 IgG IgM Western Blot in early diagnosis of congenital toxoplasmosis

S124 Results: Persistent low avidity was found in some patients even after a median follow-up period of 6 years. Avidity index of IgG was significantly heterogeneous and ranged from 7.8 to 35.3% for 95% of the women 12 weeks after infection (p < 0.05). When plotted against time after logarithmic transformation, evolution of the avidity index displayed heterogeneous patterns with slopes between −0.017 and 0.051 for 95% of the women (p = 0.011). Maturation of avidity decreased when gestational age at infection increased (p = 0.03) and increased when the delay between infection and onset of treatment increased (p = 0.0003). In conclusion, we demonstrated that maturation of Toxoplasma IgG avidity in pregnant women is highly variable and that persistent low avidity can be observed. Avidity evolution over time is influenced by gestational age at maternal infection and delay in the onset of treatment. The results of this study clearly demonstrate that, in a pregnant woman, an acute toxoplasmic infection cannot be reliably diagnosed solely on the basis of low avidity of immunoglobulin G. P552 Improved performance of the automated toxoplasmosis IgG, IgM & IgG avidity assays on the Abbott ARCHITECT Instrument J. Schultess, E. Sickinger, J. Dhein, M. Hausmann, D. Smith, E. Frias, M. Palafox, J. Prostko, D. Pucci, Reno Stricker, Reto Stricker, P. Thulliez, H. Braun (Wiesbaden, DE; Abbott Park, US; Geneva, CH; Paris, FR) Objectives: Optimisation of IgG, IgM & IgG Avidity immunoassays as a complete screening panel for the acute infection and immunity status to Toxoplasma gondii. Methods: The Toxo-IgG assay uses recombinant Toxoplasma antigens (P30 and P35) coupled on the solid phase and a labeled anti-human IgG antibody. The Toxo-IgM assay employs a m-capture format with an anti-human IgM mouse monoclonal antibody bound to the solid phase and tachyzoite lysate complexed with a labeled Anti-P30 antibody as tracer. The Toxo IgG Avidity assay applies a comparative assay format suppressing high avidity IgG antibodies with soluble P30 antigen in an indirect anti-human IgG format. The avidity assay is run with automated, sample specific dilution into a defined concentration range. The specificity of the Toxo assay panel to detect primary Toxoplasma infections and to detect the correct immune status was determined by testing 988 samples from different populations (518 blood donors, 220 hospitalised patients and 250 pregnant women. 165 seroconversion panels were used to explore sensitivity. For resolution of discordant results, a set of additional Toxo assays was tested, including the respective AxSYM assays. Results: In early seroconversion, the Architect Toxo IgG ranked equivalent to AxSYM and more sensitive than other comparator assays. It showed an excellent ability to detect past infection low level IgG’s, equivalent or better than comparator assays, paired with high specificity to detect immunity even on challenging specimens. The Toxo IgM assay was found to have a relative specificity ranging from 99.7 to 100%, for populations of random blood donors, hospital patients or pregnant females. Its sensitivity to detect seroconversion in acute infection was equivalent to AxSYM Toxo-M. The avidity assay found no specimen drawn less than 4 months after infection as high avidity and showed faster kinetics to higher avidity. Conclusion: The fully automated Toxoplasmosis panel for the Architect instrument is, in terms of sensitivity and specificity values, comparable to the reference assays. In addition, a fully automated algorithm allows the evaluation of Toxo G and Toxo IgM positive results by Toxo IgG Avidity, to rule out acute infection with Toxoplasma in pregnant women. P553 Evaluation of the new Vidia® toxoplasmosis IgG and IgM assays in women of childbearing age J. Zufferey, C. Di Mito, R. Auckenthaler (Lausanne, Geneva, CH) Objectives: The aim of the present study was to evaluate the performance of the new VIDIA® Toxoplasmosis IgG and IgM assays (bioM´erieux, France), using the VIDIA® system easy to use with a high

17th ECCMID / 25th ICC, Posters level of traceability, with clinical specimens prospectively collected in women of childbearing age. Methods: A total of 1000 fresh serum samples consecutively obtained from 1000 women aged 18–44 years (median 30) during a period of 4 weeks were tested with ADVIA® CentaurTM Toxoplasma IgG and IgM assays according to routine conditions. Serum aliquots stored at + 4ºC were blindly rechecked within 24 hours with the VIDIA system for the same parameters. Discrepancies of results between both methods were resolved considering avidity test, direct agglutination (Toxo-Screen DA, bioM´erieux), ISAGA (bioM´erieux) and when available, the analysis of previous drawn serum samples. Results: Among the 1000 women screened with VIDIA, 89.4% had non detectable IgG and IgM (T. gondii seronegative) and 8.2% had a pattern of past acquired infection (positive IgG and no detectable IgM). Positive IgM were detected in 1.1% of them with VIDIA system versus 2% with ADVIA Centaur. Equivocal rate was 0.9% for VIDIA TOXO IgG and 0.5% for VIDIA TOXO IgM (versus 0.9% and 1.1%, respectively for the compared method). For VIDIA TOXO IgG the relative sensitivity and specificity were 96.7% and 99.7%, respectively. After the resolution of discrepancies, the sensitivity as well as the specificity was 100%. For VIDIA TOXO IgM the initial relative sensitivity and specificity were 64.7% and 100%, respectively. In fact, 4 of the 6 negative samples with VIDIA and positive with the compared method were found with high avidity index and the remaining two samples were negative with the reference test (ISAGA-IgM). Taking this into account, the absolute sensitivity was found 100%. Conclusion: The two evaluated assays VIDIA TOXO IgG and TOXO IgM have shown an excellent sensitivity and specificity and are well adapted to the routine screening of toxoplasmosis in pregnant women.

P554 IgG IgM Western Blot in early diagnosis of congenital toxoplasmosis V. Meroni, F. Genco, L. Piccoli, A. Grosso, P. Lanzarini, L. Bollani, M. Stronati (Pavia, IT) Introduction: Toxoplasmosis is one of the most frequent congenital infections. Because congenital Toxoplasma infection does not usually produce recognizable signs of infection at birth, most infected newborns are undetected by routine clinical examinations and remain untreated. But serious clinical sequelae such as chorioretinitis can develope later. So the infected children must be identified and treated as early as possible. The aim of this study was to evaluate diagnostic accuracy of IgG IgM Western-blot (IgG IgM-WB LDBIO Lyon France) on 224 newborns at risk of congenital toxoplasmosis. Methods: 224 neonates born from mother with suspected or certain infection in pregnancy were evaluated retrospectively with IgG IgM WB (LDBIO Lyon France). Serum obtained from all the newborns at birth was compared with maternal sample and then with sample obtained monthly during their first three months of life. Furthermore all the sample were analysed with routine assays: ELISA IgG IgM, IgA(Diasorin Saluggia Italy), IgG ELFA, IgM ISAGA (bioM´erieux Marcy L’Etoile France). The patients were tested with all these routine assays monthly until seronegative and then at one year of age. Results: At the end of the study 40 newborns were found infected. Thirty were diagnosed at birth by the presence of IgM and/or IgA, in the other 10 diagnosis was made by antibody rebound or by IgG positivity at one year of age. Conclusions: IgG IgM Western blot showed a specificity (96.7) almost superimposable to the traditional tests (98.9). Sensitivity (95) was higher than the traditional tests (75) and the difference was statistically significative (P = 0.028 Yates Corrected c squared). WB let us find out 8 infected newborns not detectable with traditional tests that could undergone an early treatment, while 178 not infected newborns avoided unnecessary therapy.

Parasitology

S125

Table 1

Infected Not infected Tot

IgM ISAGA+IgA ELISA

IgG IGM Western blot

Pos

Pos

Neg

Tot

30 2 32 10 182 192 40 184 224 Sens. 75.0 (95% CI: 58.8−87.3) Spec. 98.9 (95% CI: 96.1−99.3)

Neg

Tot

38 6 44 2 178 180 40 184 224 Sens. 95.0 (95% CI: 83.1−99.4)* Spec. 96.7 (95% CI: 93.0−98.8)

Sens., sensitivity; Spec., specificity.

P555 Prevalence of Chagas’ disease in pregnant women from Southamerica in Valencia (Spain) A. Gil Brusola, M.J. Gimenez, Y. Garc´ıa, M.D. Gomez, M. Gobernado (Valencia, ES) Objectives: Chagas’ disease causes high morbidity in some countries in Southamerica. In Europe, where the triatomine vector is not found, its way of transmission can be either mother-to-child or through organ transplant or blood transfusion. The aim of this study was to determine the seroprevalence of this infection in immigrants from Southamerica to our city. Methods: 354 sera of pregnant women from Southamerica who attended our hospital between September 2003 and September 2005 were tested for anti-Trypanosoma cruzi antibodies (IgG) using an enzyme-linked immunosorbent assay (ELISA). Positive sera were then confirmed with either another ELISA, a particle gel immunoassay or an immunofluorescent assay (IFA). Results: The mean age of this group was 28. Most of them came from Ecuador (51%), Colombia (20%) or Bolivia (17%). Infection was confirmed in nine women (2.5%). Their mean age was 29. All of them were from Bolivia, resulting in a prevalence of 15% for women from Bolivia. Analysis of the vertical transmission couldn’t be conducted due to either miscarriage or lack of sera from the newborns. Conclusions: Prevalence of Chagas’ infection is high in pregnant women from Bolivia. Screening should be carried out in this group of patients before delivery, for follow-up of their children in order to determine the relevance of the disease, and to reduce the risk of transmission in case of organ transplant or blood transfusion. P556 Optimisation of a flow cytometry protocol for detection of Cryptosporidium parvum in hospital tap water and human stools J. Barbosa, S. Costa-de-Oliveira, A. Gon¸calves Rodrigues, C. Pina-Vaz (Porto, PT) Cryptosporidium parvum is a waterborne agent, causing diarrhoea in immunocompromised patients. Its diagnosis is based upon microscopic detection of oocysts in faeces following alcohol-acid staining. This conventional procedure presents low sensibility and specificity, being highly dependent of the observe expertise. Our main objective was to optimise a specific Flow Cytometric (FC) protocol for detection of C. parvum in water and stools and to establish its sensibility limit. C. parvum oocysts (Waterborne Inc, USA) were used for protocol optimisation. FC analysis was performed using oocyst suspensions stained with different concentrations of a specific monoclonal antibody conjugated with R-phycoerytrin (Crypt-a-Glo, Waterbone). Serial concentrations (2×105, 2×104, 2×103, 2×102 oocysts/mL) were later stained with an optimised antibody concentration and analysed by FC. Specificity and the sensibility limit of the method were established using both prokaryotic (Escherichia coli, Sthaphylococcus aureus) and eukaryotic microorganisms (Candida albicans, Giardia lamblia cysts). FC analysis

was repeated according of the optimised conditions, using human stools and hospital tap water, simulating clinical and environmental settings. Several procedures were also assayed to reduce the loss of oocysts and improve its detection: different filters (gauze, paper filters), centrifugation time and velocity, and distinct flotation solutions (NaCl, ZnSO4.7H2O). As the antibody concentration decreased, a decline of peak intensity was registered. The optimal concentration of antibody was 3.0 mg/mL for 105 oocysts/mL. We established a threshold of detection of 2×103 oocysts/mL. The staining procedure was shown to be specific, no cross-reaction occurring with bacteria, fungi or parasites. However, a decrease in staining was seen especially when Giardia was present, but it did not interfere with the result. Bellow threshold limit, fluorescence was not enough to allow the discrimination of oocysts. Interference of debris was more frequently observed in faeces than in water samples. A prolonged incubation of faeces in a ZnSO4 solution followed by centrifugation for 10 minutes allowed a clear separation of oocysts from debris. With the use of specific antibodies, a distinct cellular population corresponding to oocysts could be represented in the FC histogram. This study describes the first optimised FC protocol for detection of C. parvum in hospital tap water and human faeces.

P557 A multiplex microsphere-based assay for the simultaneous detection of C. parvum, E. histolytica and G. lamblia antigen in human faecal sample A. Huwe, X. Su, S. Cox, H. Truong, P. Nguyen, E. Laderman, J. Groen (Cypress, US) Background: Cryptosporidium, Entamoeba, and Giardia are protozoan parasites that infect the gastrointestinal tract of animals and humans by oral-faecal transmission or through contaminated water sources. Infections may be asymptomatic or may cause a range of symptoms including diarrhoea, fever, and vomiting. Immunocompromised individuals are often unable to clear the parasites from their systems and ultimately suffer severe illness and possible death. Current methods of diagnosis include microscopy (O&P), and ELISA; these “single-plex” methods prove to be labour intensive, and require special skills in addition to other limitations. A high performance, multiplexed assay with the ability to simultaneously detect the presence of all three parasites in a single faecal sample is highly desirable. This study describes the development of the PlexusTM Parasitic Multi-Analyte Diagnostics assay based on the Luminex xMAPTM system (Austin, TX). Methods: Samples: A retrospective panel of 248 human stool samples submitted for parasite testing was collected. In addition, stool samples, and culture supernatants of common enteric pathogens were collected for cross-reactivity testing. PLEXUSTM Parasitic Multi-Analyte Multiplex Assay: Monoclonal antibodies specific for each parasite were covalently linked to microspheres. The capture microspheres were mixed with extracted human faecal samples, washed, and incubated with polyclonal detection antibodies specific for each parasite antigen. Finally, the microspheres were incubated with fluorescent Phycoerythrin (PE) conjugate. The median fluorescence intensity of PE measured by the Luminex 100 System indicates the amount of antigen captured. Reference Assay: Samples were tested by microscopy or with the TechLabTM Cryptosporidium II, E. histolytica II, and Giardia II ELISA systems per the manufacturer’s suggested protocol. Results: The sensitivity of the PLEXUS Parasitic Panel compared to ELISA was 100% (62/62) for C. parvum, 95.5% (21/22) for E. histolytica, and 98.4% for G. lamblia. The specificity was determined to be 100% (176/176) for all three parasites. In addition, the system does not display cross-reactivity with any of the common enteric pathogens, bacteria, or viruses. Conclusion: The multiplex capability of the PLEXUSTM Parasitic MultiAnalyte Diagnostics system offers a high performance, time-saving alternative to microscopy and ELISA for the qualitative detection of C. parvum, E. histolytica and G. lamblia.