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P555 Prevalence of Chagas' disease in pregnant women from Southamerica in Valencia (Spain)
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.
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.