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LDBIO TOXO II IGG & LDBIO TOXO II IGM: two new confirmation tests help discriminate between specific and non specific antibodies in pregnant women
III International Congress on Congenital Toxoplasmosis (ICOCT) 13 to 16 May 2007-D10
Genco F., Lanzarini P., Grosso A., Meroni V.
Infectious Diseases Department University of Pavia , Fondazione I.R.C.C.S. Policlinico " San Matteo " Via Taramelli 5 27100 Pavia Italy
E-Mail: v.meroni@smatteo.pv.it
INTRODUCTION
When Toxoplasma gondii infection is acquired during pregnancy, it may cause severe complications, such as miscarriage, stillbirths, congenital infections and late neonatal sequelae. The early and definitive detection of a recently acquired infection is critical for the clinical management of the mother and her fetus. IgG antibodies are often absent in early phases of infections, and IgM antibodies may be non specific and disappear at the end of pregnancy. Furthermore, therapy that is given as early as possible could affect antibody production, possibly as a consequence of a decrease in the parasitic load. Clearly, serological diagnosis needs to be improved if we have to distinguish early infections from non-specific antibody response. To this aim, we have evaluated LDBIO TOXO II IgG and LDBIO TOXO II IgM, two new confirmation immunoblots, in 24 pregnant women with suspected seroconversion.PATIENTS and METHODS
Twenty four pregnant women with suspected seroconversion were referred to the clinic of the Infectious Diseases Department for further diagnostic workup. All women were negative for anti-Toxoplasma IgG (Etitoxok IgG DIASORIN Saluggia Italy,VIDAS toxo IgG II BIOMERIEUX Marcy l'Etoile, France) and positive for IgM with at least one of the two tests routinely used in the laboratory (Etitoxok IgM DIASORIN Saluggia Italy, Toxo IgM ISAGA BIOMERIEUX Marcy l'Etoile, France). They were followed weekly to detect the production of anti toxoplasma IgG. Spiramicyn was given from the first positive IgM test. All samples were also tested with LDBIO TOXO II IgG and LDBIO TOXO II IgM - the latter being not commercially available at this date (LDBIO DIAGNOSTICS, Lyon France). In 9 out of 24 women, lymphocyte stimulation was performed too, and CD25 and Stimulation Index were evaluated.RESULTS
LDBIO TOXO II IgM was positive in twelve women. For 10 the seroconversion was proved with the appearance of specific anti-Toxoplasma IgG during the follow-up. In 4 they were already present in the first sample by using LDBIO TOXO II IgG. For the other 6, IgG antibodies were detected on subsequent samples earlier (at least two weeks) with LDBIO TOXO II IgG and later with traditional IgG serological tests. 6 of 10 were also positive in lymphocyte stimulation. Twelve women were LDBIO TOXO II IgM negative and no IgG was detected either with western blot nor with traditional tests on later samples, even after the treatment was discontinued based on this finding. In three cases, the lymphocyte stimulation confirmed these results. Two women resulted positive at LDBIO TOXO II IgM but constantly negative for IgG. One of them was also positive in lymphocyte stimulation.CONCLUSIONS
In 22 of 24 patients, an early and correct diagnosis was reached with LDBIO TOXO II IgM. In all
infected women LDBIO TOXO II IgG was positive earlier and confirmed the seroconversion several weeks before the other IgG serology tests. In all negative cases it was possible to stop safely the
therapy and to reassure the women. On the other hand, no infected woman was missed, all positive women were given the appropriate therapy and prenatal diagnosis was offered. We had two false
positive results at IgM (one also with lymphocyte stimulation), but the reliable evaluation of the
specificity of LDBIO TOXO II IgM was not possible because of the small sample size.
IgG IgM profile comparison by Western-Blot in early diagnosis of congenital toxoplasmosis
III International Congress on Congenital Toxoplasmosis (ICOCT) 13 to 16 May 2007-B3
Meroni V., Genco F., Piccoli L.,Grosso A., Bollani L.1,Stronati M.1
Infectious Diseases Department University of Pavia ,1 Unità Terapia Intensiva Neonatale
Fondazione I.R.C.C.S. Policlinico " " San Matteo " Via Taramelli 5 27100 Pavia Italy
E-Mail: v.meroni@smatteo.pv.it
Congenital toxoplasmosis does not usually produce recognizable signs of infection at birth, and passively transmitted maternal antibodies could interfere in serological diagnosis. Most infected newborns are undetected by routine clinical and serological examinations at birth and remain untreated for many months and can develop later serious clinical sequelae such as chorioretinitis. 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 profile comparison (TOXOPLASMA WB IgG-IgM - LDBIO DIAGNOSTICS, Lyon France) on 238 newborns at risk of congenital toxoplasmosis.
Two hundred thirty eight neonates born from mother with suspected or certain infection in pregnancy were evaluated retrospectively with TOXOPLASMA WB IgG-IgM (LDBIO Lyon France). Serum obtained from all the newborns at birth was compared with maternal sample and then with samples obtained monthly during their first three months of life.
Furthermore all the sample were analyzed with routine assays: ELISA IgG IgM, IgA(Diasorin Saluggia Italy), IgG ELFA, IgM ISAGA (Biomerieux 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 42 newborns were found infected. Thirty one were diagnosed at birth by the presence of IgM and/or IgA, in the other 11 diagnosis was made by antibody rebound or by IgG positivity at one year of age. The results of the test in comparison with traditional ISAGA IgM, plus ELISA IgA are shown in Tab 1.
CONCLUSIONS
IgG-IgM Western blot profile comparison showed a specificity (96,9) almost superimposable to the traditional tests (98,9). Sensitivity (95,2) was higher than the traditional tests (76,1) and the difference was statistically significative (P=0,02 Yates Corrected ? squared).
WB profile comparison let us find out 9 infected newborns not detectable with traditional tests that could undergone an early treatment, while 190 not infected newborns avoided
unnecessary therapy.
Diagnosis of congenital toxoplasmosis in Poland and its relation to the population tested
III International Congress on Congenital Toxoplasmosis (ICOCT) 13 to 16 May 2007-B4
Nowakowska D, Sobala W, Spiewak E, Wilczynski J
Department of Fetal Maternal Medicine, Research Institute Polish Mother's Memorial Hospital, Lodz Poland.
Email: dnowakowska@yahoo.comThe prevalence of anti-Toxoplasma gondii IgG is 41% among Polish pregnant woman. The incidence of congenital toxoplasmosis (CT) is 1.5 /1000. Due to the lack of routine testing in pregnancy and so-called "wild screening" performed by various laboratories with different methods, there is an urgent need for an additional confirmation tool.
Considering that the predictive values of a test depend critically on the incidence of disease in the patients being tested, the goal was to evaluate TOXOPLASMA WB IgG-IgM test (LDBIO DIAGNOSTICS, Lyon, France) in two groups.
GROUP 1. covered woman with IgG and IgM detected during pregnancy (independently on the time of infection) or tested only after delivery. In the GROUP 2. woman with recent infection in pregnancy based on evolution of IgG and IgM curves (Bessieres, 1999) were included. There were 136 couples mother - cord blood (M-CB) of sera in the group 1 with 24 CT cases and 91 couples M-CB of sera with 12 children born with CT in the group 2. All samples were tested for specific IgG and IgM with VIDAS Toxo-IgG and Toxo-IgM (bioMerieux) and for IgA with Platelia Toxo-IgA (Biorad). Serological follow-up during the first year of life was a "gold standard" method for CT diagnosis.RESULTS:
GROUP 1: The sensitivity (Se) of WB IgG was 75.3%, specificity (Sp) 100%, positive predictive value (PPV) 100% and negative predictive value (NPV) 97.2%. Se of WB IgM was 83.9%, Sp 98.5%, PPV 86.5% and NPV 98.1%. Se of WB IgG + WB IgM was 96.2%, Sp 98.2%, PPV 86.6% and NPV was 99.5%. Se of VIDAS IgM + Platelia IgA + WB IgG + WB IgM was 99.8%, Sp 97.0%, PPV 79.5% and NPV was 99.9%.
GROUP 2: Se of WB IgG was 54.6%, Sp 100.0%, PPV 100.0%, NPV, 91.7%. Se of WB IgM was 66.4%, Sp 98.3%, PPV 88.4%, NPV 93.6%.CONCLUSION:
The predictive values of WB M-CB vary between the two groups of women. The use of WB M-CB improves the final diagnosis especially when used as a confirmation test in unscreened population.
Toxoplasma gondii infection in pregnancy: opportunities and pitfalls of serological diagnosis
REVIEW Clin Microbiol Infect 2006; 12: 504-512
A. Sensini
University of Perugia, Experimental Medicine and Biochemical Sciences, Perugia, Italy
Because of its life cycle, the recovery of Toxoplasma gondii from biological samples is often impracticable. Consequently, a serological diagnosis represents the first and the most widely used approach to defining the stage of infection. The detection of IgG, IgM, IgA, IgE and IgG avidity by different methods offers this opportunity. However, the results may be affected by difficulties in interpretation, as the same antibody pattern may have a different valency, contingent upon subjects and clinical settings, e.g., pregnant women vs. neonates, and treated vs. untreated patients. This review describes the various factors that should be taken into account when performing serological tests for T. gondii, as well as the pitfalls that may be encountered during the interpretative process.
Recent Developments for Diagnosis of Toxoplasmosis
Journal of Clinical Microbiology, March 2004, p. 941-945, Vol. 42, No. 3 MINIREVIEW
Jack S. Remington,1,2* Philippe Thulliez,3 and Jose G. Montoya1,2
Department of Immunology and Infectious Diseases, Research Institute, Palo Alto Medical Foundation, Palo Alto,1 Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University School of Medicine, Stanford, California,2 Laboratoire de la Toxoplasmose, Institut de Puériculture, Paris, France3
Determinants of immunodiagnostic success in human ocular toxoplasmosis
Parasite Immunology 2005,27, 61-68 Review Article
J. G. Garweg
Department of Ophthalmology, University of Bern, Inselspital, 3010 Bern, SwitzerlandOcular toxoplasmosis is a local manifestation of systemic infection in which Toxoplasma spreads into the eye, affecting mainlythe posterior segment of the eye. Reactivation of the initial retinal condition presumably results from the rupture of quiescent parasitic cysts lying adjacent to pre-existing scars and may secondarily involve the choroid (leading to retinochoroiditis). Although the molecular mechanisms underlying host-parasite interaction are largely unknown, toxoplasmic retinochoroiditis usually remains a local event, and does not necessarily evoke a detectable systemic immune response. Local immunotolerance mechanisms may likewise confound attempts to confirm the clinical diagnosis by serology. Aqueous humour may be analysed for the presence of parasite DNA or of specific antibodies, but the DNA burden therein is low, and a more definite confirmation would require risky puncturing of the vitreous. Laboratory confirmation of the diagnosis is also frustrated by marked individual differences in the time elapsing between the onset of clinical symptoms and the activation of specific antibody production, resulting in a high proportion of false negative results. Whether a delay in the onset of local specific antibody production reflects immunotolerance in cases of congenital - but not obviously in those of acquired - infection remains an open question, but it could account for a relatively low confirmation rate in laboratory tests for local antibody production. Against this background, current diagnostic strategies need to be re-evaluated with a view to future improvements.
Aqueous Humor and Serum Immunoblotting for Immunoglobulin Types G, A, M, and E in Cases of Human Ocular Toxoplasmosis
Journal of Clinical Microbiology, October 2004, p. 4593-4598, Vol. 42, No. 10
Justus G. Garweg,1* Silvia-Daniela L. Garweg,1 Franziska Flueckiger,1 Patrick Jacquier,2 and Matthias Boehnke1,3
Department of Ophthalmology, University of Bern, Inselspital,1 ParaDiag, Laboratory for Clinical Parasitology, Bern, Switzerland,2 Institute for Eye Surgery, Hamburg, Germany3
The purpose of this study was to compare the local and systemic Toxoplasma-specific humoral immune responses in individuals with ocular toxoplasmosis (OT). To this end, paired aqueous humor and serum samples from 46 individuals with active OT and from 30 individuals without inflammatory eye disease (controls) were analyzed by immunoblotting for anti-Toxoplasma immunoglobulin G (IgG), IgA, IgM, and IgE directed against 20- to 120-kDa antigens. The presence in the aqueous humor of a unique band, or of at least three bands that were at least three times more intense in aqueous humor than in serum, was taken as evidence of local antibody production. IgG bands were detected in 98% of the aqueous humor samples, while IgA bands were detected in 76%, IgM bands were detected in 8%, and IgE bands were not detected in any. Evidence of local production of specific antibodies was found in 32 cases (70%) (IgG in 23 [50%]; IgA in 16 [35%]). In 10 instances (22%), routine laboratory tests were not indicative of OT. In 14 cases (30%), no local antibody production was detected by immunoblotting; 3 of these cases yielded evidence of local antibody production according to the Goldmann-Witmer coefficient. Local antibody production was revealed for 7 of the 30 controls (23%). Hence, the sensitivity of immunoblotting for IgG and IgA is 70%, and the specificity is 77%. We conclude that immunoblotting for local specific IgG and IgA supports the clinical diagnosis of OT in 70% of cases. In 22% of these, the diagnosis is not confirmed by other laboratory tests. Hence, immunoblotting increases the sensitivity of routine laboratory tests and should be considered for samples that register negative by such tests.
Problems and limitations of conventional and innovative methods for the diagnosis of Toxoplasmosis in humans and animals
Parassitologia 2004 Jun;46(1-2):177-81
Piergili Fioretti D
Dipartimento SBPV, Sezione di Parassitologia, Universita di Perugia.
Diagnosis of toxoplasmosis is useful for human and animal health. Several techniques are employed for the diagnosis in feline and canine population. Coprological tests for the detection of oocysts in cat faeces are of little significance owing to short patency (15 days). Histological examinations of biological samples show a lack of reliability when the animals are infected with few parasites; the mouse inoculation is the most reliable method even if the detection of cysts in mice brain require 40 days. However tachyzoites of virulent strains can be isolated from peritoneal exudate 3-4 days after inoculation. Samples inoculation in cell cultures (VERO, human fibroblasts) requires specialized laboratories and fails if non viable parasites are present due to tissutal autolysis. Serological tests are the most used diagnostic methods; Dye test and IFAT that require intact tachyzoites are more sensitive and specific compared to IHA, LA, ELISA because, during the infection, the first significant increase of IgM and IgG antibodies was observed against cuticolar antigens. A PCR to identify T. gondii DNA in canine and feline biological samples was developed. The B1 PCR performed on blood samples was less sensitive than when it was performed on other biological fluids requiring 100 tachyzoites, instead of 10. Aqueous humor PCR results could be negative if the infection is low grade or is restricted to the posterior segment or the animal was previously treated with anti-Toxoplasma drugs. SNC disease may be also difficult to diagnose because an high serum IgG titer may be associated with locally production or leakage from serum through a compromised blood-CSF barrier. AB1 PCR was successfully applied for the diagnosis of Toxoplasma abortion in ewes requiring only 10 parasites in placental cotyledon samples; the test compared with mouse inoculation showed similar sensitivity. Discrepancies may have been due to a low and focal distribution of parasites in the tissues or to the presence of non viable parasites if the tissues are autolysed. In regard to diagnostic methods adaptable to slaughter testing, several serological tests have been studied (IFAT, ELISA, IHA) for detection of IgG in sheep, pigs, cattle using also recombinant antigens (gene fragments H4 and H11) to lack the cross reactivity. The problem is the antibodies fall to near background levels as the infection became chronic (6-10 months p.-i.). A highly sensitive and specific method (Toxo Taq Man) has been developed to detect and quantitate T. gondii burden in animal tissue samples (0.1 pg of T. gondii genomic DNA, which is equivalent to 1 bradyzoite) using T. gondii ITS1-derived primers and a fluorogenic probe via Real-Time PCR. This assay is compatible with automation technology for potential slaughterhouse use. The diagnosis of acute infection in human pregnancy is difficult since IgM antibodies can be detected for a very long time after the acute phase; an IgA increase is of more diagnostic value because can be detected only for 6-7 months while the short kinetics of IgE can be useful only to date the infection precisely. In addition an IgG seroconversion is essential for the diagnosis. Among the most reliable tests, IgG avidity test is useful when a single serum sample, in the first months of gestation, is available, but low avidity results may persist for as long as 1 year. For this purpose a panel of serologic tests must be performed (ELISA, EIA, ISAGA, IgG avidity, IFAT, Dye test) for IgM, IgA, IgG and IgE. The serological diagnosis of prenatal infection is difficult since maternal IgG are passively transferred in utero to the foetus and caution must be exercised in interpretation of IgM or IgA results. A technique of Western blots of paired maternal and baby sera for evidencing different bands in the blots of two sera was developed for this purpose (specificity 97-100%, sensitivity 96-98%). The most reliable methods for prenatal diagnosis are PCR, mouse inoculation and cultural techniques performed on amniotic fluid, foetal blood and peripheral maternal blood in pregnants serologically positive. PCR (targets B1, SAG-1, rDNA) with amniotic fluid performed from 18 weeks of gestation is more sensitive and more rapid than conventional diagnostic procedures. PCR has been successfully used to diagnose Toxoplasma encephalitis in immunocompromised patients (cerebral biopsy is the only diagnostic method) and in ocular toxoplasmosis. In this evenience it is useful the study of IgG, IgM, IgA profile of paired serum and aqueous humor (Western blots).
Usefulness of immunoblotting and Goldmann-Witmer coefficient for biological diagnosis of toxoplasmic retinochoroiditis.
Eur J Clin Microbiol Infect Dis 2004 Jan;23(1):34-8
Robert-Gangneux F; Binisti P; Antonetti D; Brezin A; Yera H; Dupouy-Camet J
Laboratory of Parasitology and Mycology, University of Medicine Paris 5, Hospital Cochin-Port Royal, 27 rue du Faubourg Saint-Jacques, 75014 Paris, France. f.gangneux@ch-stmalo.fr.
Toxoplasmosis is a frequent cause of retinochoroiditis. Although the diagnosis relies mainly on ophthalmological examination, biological approaches are particularly useful in patients with atypical lesions. In a prospective study to determine the value of immunoblotting and immune load calculation in the diagnosis of active toxoplasmic retinochoroiditis, aqueous humor samples from 21 patients with retinochoroiditis and 5 control patients with cataracts were tested. Immune load was calculated on the basis of intraocular antibody production. The immune load ratio between aqueous humor and serum (Goldmann-Witmer coefficient) was significant (i.e. >2) in 9 of the 17 (53%) patients with retrospectively documented toxoplasmic retinochoroiditis. Immunoblotting suggested local antibody production in 10 of 17 (59%) patients. The combination of the two techniques gave a sensitivity of 71% (12/17). Both techniques were negative in the four patients in whom the final diagnosis of toxoplasmic retinochoroiditis was negative and in the five patients with cataracts. These results confirm the value of combining these two techniques. Moreover, immunoblotting has the advantages of being easy to perform and of requiring a very small sample.
IgG and IgM W.B. for diagnosis of congenital toxoplasmosis at birth
International Conference on Toxoplasmosis-Copenhagen 23-25 june 2003 POSTER 26
Meroni, A.Bemuzzi§, C.Vigano*, P.Lanzarini§, L.Bollani*
Department of Infectious Diseases, University of Pavia, IRCCS Policlinico San Matteo, Pavia.§Department of Infectious Diseases, IRCCS Policlinico San Matteo, Pavia. Department of Pediatrics, University of Pavia, IRCCS Policlinico San Matteo, Pavia.
Diagnosis of congenital infection is difficult because of presence of maternal antibodies that can mask and inhibit the immune response of the newborn. Western Blot assay can distinguish antigenic specificity of IgG and IgM antibodies and discriminate between mother and newborn antibodies production. We evaluated anti-toxoplasma Western Blot IgG and IgM (LDBIO Diagnostic-Lyon-France) on 70 mother-newborn coupled sera at birth and compared them with traditional tests (IgM ISAGA-Biomerieux, IgA ELISA Diasorin). All enrolled mothers had confirmed or suspected infection during pregnancy and all newborns were followed-up until one year old in order to confirm or exclude congenital infection. Of the 20 infected neonates, 19 had positive WB for IgG and/or IgM, 14 had also ISAGA IgM and/or IgA ELISA positive. 1 newborn was completely negative at birth and became positive at VVB in 15 days and with traditional test in 1 month. 50 uninfected newborns were negative with VVB but 1 was positive with IgM ISAGA test (false positive). In this study VVB shows higher sensibility (95%) in early diagnosis of congenital toxoplasmosis than other traditional tests ISAGA IgM and Elisa IgA (sensibility 70%) with almost the same specificity (p: 0,048 Fisher test).
Intraocular synthesis of specific antibodies in patients with active toxoplasmic retinochoroiditis demonstrated by Western blotting
International Conference on Toxoplasmosis-Copenhagen 23-25 june 2003 POSTER 32
M. Paul1, J. Stefaniak1, K. Pecold2, H. Twardosz-Pawlik2
1 Department and Clinic of Tropical and Parasitic Diseases, 2 Department of Ophthalmology, University of Medical Sciences, Poznan, Poland
Obiective: The aim of the study was to describe the clinical characteristics and classical laboratory findings in relation to the analysis of comparative immunological profiles of Toxoplasma-specific antibodies in intraocular fluid and serum samples in patients with active retinochoroiditis.
Materials and methods: Sixteen immunocompetent patients (male/female ratio 1: 1) aged from 5 to 62 years (median 20 years) were strongly suspected of ocular toxoplasmosis based on ophthalmic assessment and admitted to the dinic for anti-parasitic treatment. The patients complained of a sudden loss of visual acuity and vision troubles for 2 weeks to 4 months duration (mean 4 weeks). All the patients were characterized by a history of eye lesions. Results: The retinal inflammation was accompanied by anterior uveitis with vitreous opacities in 6 cases. Fresh macular lesions were observed in 8 patients. In all the patients, a puncture of anterior eye chamber was recommended for a final confirmation of the clinical diagnosis of ocular toxoplasmosis. The detection of Toxoplasma-specific IgG, IgM and IgA antibodies in serum samples was performed by classic enzyme immunoassays. The immunological profile of Toxoplasma-specific IgG and IgM antibodies in intraocular fluid and peripheral blood was analysed by immunoblotting. Results: In 15 of 16 examined patients, the traditional serology showed an immunological profile of the chronic Toxoplasma infection documented by low or moderate levels of specific IgG with high avidity values and an absence of IgM. Recently acquired T gondii infection with an increasing IgG titre of a low avidity and a presence of Toxoplasma-specific IgM and IgA antibodies in peripheral blood was only observed in one patient. In 9 of 16 patients (56%) the local production of T. gondii-specific IgG antibodies of a different antigenic specificity than those in serum samples was found in anterior eye chamber fluid. There was no evidence of intraocular synthesis of specific IgM. In two patients, including one with the coexisting primary T gondiiinfection, streptococcal tonsillitis was diagnosed which was probably a source of a secondary eye inflammation. In 5 cases, a concomitant positive serology for toxocarosis was detected. Conclusion: (i) Active chorioretinal lesions with coexisting positive serology in children and adults are not sufficient to define ocular T gondii infection and they require a profound differential diagnosis followed by an appropriate treatment in order to avoid a supradiagnosis of ocular toxoplasmosis. (ii) The evidence of the specific antibodies synthesis in aqueous humor shown by the Western blot seems to be a valuable immunodiagnostic tool for a final confirmation of eye lesions of toxoplasmic origin.
Usefulness of immunoblot and PCR in the diagnosis of toxoplasmic chorioretinitis
International Conference on Toxoplasmosis-Copenhagen 23-25 june 2003 POSTER 46
Odile Villard, Florence Roch-Deries, Denis Filisetti, Jacques Flament, Justus Garweg and Ermanno Candolfi. Institut de Parasitologie, Strasbourg, France.
Ocular toxoplasmosis is the major cause of posterior uvetis in Europe. The contribution of biology in etiological diagnosis is a valuable tool, especially for atypical forms. The specificity of the techniques was determined on a control group of 68 subjects unaffected by toxoplasmosis, and is 89% for ELISA and immunoblot and 100% PCR. Sensitivity on the other hand was calculated taking a population of 19 patients with a decline in visual acuity and fundus abnormalities were analyzed by immunoblot (IB) and PCR for evidence of Toxoplasma gondii infection. Most of the patients (89%) had serologic evidence of past exposure to the parasite. Comparative ELISA analysis of paired serum and aqueous humor samples showed local ocular production of anti- Toxoplasma gondii IgG in 63% of patients (12/19). Comparative immunoblot analysis of specific IgG patterns in serum and aqueous humor confirmed local IgG synthesis in 52,6% of cases (10/19). The sensitivity of Immunoblot in this series is less than that of ELISA (52,6% versus 63%), but IB methods may be conclusive when the blood-eye barrier is damaged. PCR demonstrated the toxoplasmic origin of chorioretinitis in three patients with negative or borderline local antibody production, permitting early specific treatment. Toxoplasma gondii DNA PCR analysis of aqueous humor was positive in 27,7% of cases. Combined ELISA, IB and PCR testing confirmed that the ocular lesions were due to Toxoplasma gondii in 83% of cases (15/18).
Congenital toxoplasmosis: value of prenatal and neonatal diagnosis
International Conference on Toxoplasmosis-Copenhagen 23-25 june 2003 POSTER 3
M.H. Bessières', R. Lelong, S. Cassaing', A. Berrebi", J. P. Séguéla' *Laboratoire de Parasitologie - Mycologie CHU Toulouse Hôpital Rangueil I Avenue du Professeur Jean Poulhès 31403 Toulouse Cedex. ' * Services de Gynécologie Obstétrique CHU Toulouse H6pital La Grave Place Lange 31052 Toulouse cedex (France).
We evaluated the performances of methods used for the prenatal and neonatal diagnosis of congenital toxoplasmosis. 128 women with infection during pregnancy were included in this study. When prenatal diagnosis showed signs of infection, mothers were treated with pyrimethamine and sulphonamide. We studied the influence of this treatment on the sensitivity of neonatal methods. Parasitological diagnosis included detection of Toxoplasma gondii on amniotic fluid, placenta and cord blood by mouse inoculation and PCR. Serological tests were performed on cord blood using immunocapture methods for detection of IgM and IgA antibodies and Westernblotting (WB) for comparison of maternal and newborn antibodies. 95 infants had a complete serological follow up during the first year. In the group of 65 infants with a prenatal diagnosis, the sensitivity of tests was 96 % (24/25) and the specificity 97,5 %. For the 30 infants who had only a neonatal diagnosis, sensitivity of tests was 92% (12/13). The combination of prenatal and neonatal screening diagnosed 97 % of infected infants (37/38). For the neonatal diagnosis, the sensitivity of parasitological tests was 48% and the specificity of 98%. The sensitivity of immunocapture tests was 63 % for IgA, 51 % for IgM antibodies and combined of 66 %. The sensitivity of WB was 51%. Specificity was 91% for IgM, 96% for IgA and 100% for WB. A similar sensitivity was obtained with serological methods including WB (77%) or with combination of parasitological methods and immunocapture tests (79%). Prenatal treatment decreased sensitivity of immunocapture tests and parasitological methods.
Evaluation of a Commercial IgG/IgM Western Blot Assay for Early Postnatal Diagnosis of Congenital Toxoplasmosis.
Eur J Clin Microbiol Infect Dis 2003 Mar;22(3):174-80
Rilling V; Dietz K; Krczal D; Knotek F; Enders G
Labor Prof. G. Enders und Partner und Institut fur Virologie, Infektiologie und Epidemiologie e.V., Rosenbergstrasse 85, 70193, Stuttgart, Germany.
The aim of this study was to evaluate a commercial Western blot IgG/IgM assay for use in the early serological diagnosis of congenital toxoplasmosis. This assay compares the immunological profile of mother and infant and allows differentiation between passive transmitted maternal antibodies and newly synthesized antibodies of the infant within the first 3 months of life. Over a 6-year period (1995-2001), the sera from 169 mothers and their 175 offspring (6 had twins) were examined for specific anti- Toxoplasma gondii IgG, IgM and IgA antibodies with an enzyme-linked immunosorbent assay or an immunosorbent agglutination assay. All mothers had primary Toxoplasma infection during pregnancy. Serological and clinical follow-up of the infants during the first year of life confirmed 36 cases of congenital toxoplasmosis. In 139 cases, infection could be ruled out. Three hundred fifty-one paired samples from 175 mother-child pairs were tested retrospectively for IgG and IgM patterns by Toxoplasma Western blot IgG/IgM (LDBIO Diagnostics, France). The results of conventional serological analysis (immunosorbent agglutination assay or enzyme-linked immunosorbent assay) to detect IgM or IgA were compared with the results of the Toxoplasma Western blot IgG/IgM on samples obtained within the first 3 months of life. The performance of the combination of the two methods was also assessed. At birth, the sensitivity values of conventional serological analysis and the Toxoplasma Western blot were 52% and 67%, with specificity values being 99% and 96%, respectively. Combination of the Western blot and conventional serological analysis increased the sensitivity at birth to 78% and within the first 3 months of life to 85%. Overall, the combination of both methods detected 94% of congenital infections. Therefore, this commercial Western blot represents a useful tool for early postnatal diagnosis of congenital toxoplasmosis.
Retinochoroiditis associated with congenital toxoplasmosis in children: IgG antibody profiles demonstrating the synthesis of local antibodies.
Eur J Ophthalmol 2003 Jan-Feb;13(1):74-9
De Marco R; Ceccarelli R; Frulio R; Palmero C; Vittone P
Department of Infectious Diseases, University of Genova, Genova, Italy.
PURPOSE: Retinochoroiditis is generally diagnosed after the first year of life and the association with congenital toxoplasmosis presents a diagnostic dilemma. The detection of local intraocular specific antibodies could be useful for diagnosis. METHODS: We studied six patients (mean age 7 +/- 5 years) with retinochoroiditis which appeared after the first year of life. Aqueous and serum were analysed by immunoblotting for anti T.
gondii IgG to diagnose toxoplasmosis. RESULTS: All serum samples were positive only for anti T. gondii IgG. The retinochoroiditis was active in three patients and inactive in the others. Immunoblot analysis of serum and aqueous from the patients with active lesions showed IgG versus the specific antigen of T. gondii. In the patients with inactive lesions the pattern was the same in the two compartments. In active forms, aqueous and serum Western blot patterns differed in proteins lower than 16kDa and higher than 116kDa: in aqueous the findings were typically positive for 30kDa. CONCLUSIONS: Aqueous humour analysis by the Western blot technique may be useful in the diagnosis of congenital toxoplasmosis. In the present small series, we nevertheless detected different patterns for inactive and active retinochoroiditis, confirming the diagnosis in the latter. Aqueous humour paracentesis may be indicated in a child with active retinochoroiditis with unusual clinical features, appearing after the first year of life, and with no clinical or serological evidence of congenital infection.
Usefulness of Western blot in serological follow-up of newborns suspected of congenital toxoplasmosis.
Eur J Clin Microbiol Infect Dis 2003 Feb;22(2):122-5
Tissot Dupont D; Fricker-Hidalgo H; Brenier-Pinchart MP; Bost-Bru C; Ambroise-Thomas P; Pelloux H
Service de Parasitologie-Mycologie, Centre Hospitalier Universitaire, BP217, 38043, Grenoble, France.
The goal of the study reported here was to compare the results of Western blot with other serological methods for testing newborns suspected of having congenital toxoplasmosis. Western blot, enzyme-linked immunosorbent assay, immunoglobulin (Ig)M immunosorbent agglutination assay, and indirect immunofluorescence assay were performed on the sera of 126 neonates collected at birth and at 1 and 3 months of life. Western blot was more sensitive than IgM detection with the immunosorbent agglutination assay (82.6% vs. 69.6%), and the specificity of the two methods was 96.1% and 92.2%, respectively. Among the serological techniques tested, the combination of Western blot (IgG and IgM) with IgM immunosorbent agglutination assay achieved the greatest improvement in the sensitivity of early (postpartum) diagnosis of congenital toxoplasmosis.
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.
J Clin Microbiol 2001 Jun;39(6):2267-71
Pinon JM; Dumon H; Chemla C; Franck J; Petersen E; Lebech M; Zufferey J; Bessieres MH; Marty P; Holliman R; Johnson J; Luyasu V; Lecolier B; Guy E; Joynson DH; Decoster A; Enders G; Pelloux H; Candolfi E
Service de Parasitologie-Mycologie, CHU Hopital Maison Blanche, UPRES EA 2070, IFR53, 51092, Reims, France.
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.
Prevalence of congenital Toxoplasma gondii infection among newborns from the Poznan region of Poland: validation of a new combined enzyme immunoassay for Toxoplasma gondii-specific immunoglobulin A and immunoglobulin M antibodies.
J Clin Microbiol 2001 May;39(5):1912-6
Paul M; Petersen E; Szczapa J
Department of Parasitic and Tropical Diseases, Institute of Microbiology and Infectious Diseases, Karol Marcinkowski University of Medical Sciences, Przybyszewskiego 49, 60-355 Poznan, Poland.
We determined the value of a new serological assay detecting Toxoplasma-specific immunoglobulin M (IgM) and IgA antibodies at birth for use in mass neonatal screening. The incidence of congenital infection in newborns was compared with data from an epidemiological investigation on the seroprevalence of Toxoplasma in the studied population. Peripheral blood was collected on Guthrie cards during the first 3 days of life and tested for anti-Toxoplasma IgA and IgM using a noncommercial immunocapture enzyme-linked immunosorbent assay (ELISA). When the screening assay was positive, serum samples from the child and the mother were collected for use in Western blotting comparative immunological profile analysis and traditional serological tests for determination of specific IgG, IgM, and IgA antibodies. From December 1998 to April 2000, 17,653 filter paper samples from live-born neonates were successively screened. Congenital T. gondii infection was finally confirmed in 19 newborns. In traditional assays, 13 of 19 infants were IgM and IgA positive using filter paper eluates at birth, 1 child was positive only for IgM, 1 patient was positive for IgM and borderline for IgA, 1 had an equivocal level of IgA, and 3 cases were confirmed only by the Western blot assay. The prevalence of Toxoplasma-specific IgA and/or IgM in filter paper samples at birth was 1 per 929 live-born neonates (1.08/1,000) or about 1 per 523 children (1.9/1,000) born to nonimmune women with a potential risk of primary T. gondii infection during pregnancy, compared to the actual seropositivity rate of 43.7%. The diagnostic sensitivity of the combined IgA-IgM ELISA using neonatal filter paper specimens was not more than 95%, the positive predictive value of the test was 82.6%, and the diagnostic specificity was calculated to be 99.9%. The combined IgA-IgM ELISA is a valuable method for the diagnosis of congenital toxoplasmosis at birth and fulfills criteria for neonatal screening programs. The method showed a good diagnostic sensitivity in neonates untreated prenatally who were born in an area of high seroprevalence of T. gondii infection.
Comparative immunoglobulin G antibody profiles between mother and child (CGMC test) for early diagnosis of congenital toxoplasmosis.
J Clin Microbiol 2000 Oct;38(10):3619-22
Gross U; Luder CG; Hendgen V; Heeg C; Sauer I; Weidner A; Krczal D; Enders G
Department of Bacteriology, University of Gottingen, D-37075 Gottingen, Germany.
Early diagnosis of congenital toxoplasmosis is rendered difficult when specific immunoglobulin M (IgM) and/or IgA antibodies are absent in the blood of the newborn infant. Since maternal IgG antibodies can cross the placenta, determination of IgG antibodies in newborn infants has hitherto not been used routinely for the diagnosis of congenital infection. The aim of this study was to assess the diagnostic usefulness of an immunoblot assay which compares the early IgG profiles between the mother and her child (comparative IgG profile between mother and child; CGMC test) directed against a total cell lysate of Toxoplasma gondii tachyzoites. Serum samples from 97 newborn infants at risk of toxoplasma infection were obtained from umbilical cord blood at birth or postnatally until 3 months of life and were directly compared with serum samples from the respective mothers. Congenital toxoplasmosis was diagnosed only when IgG-reactive protein bands that were present in any newborn serum samples were absent in the corresponding maternal serum sample. Congenital infection was defined by conventional serological assays when IgM and/or IgA antibodies were present in newborn infant blood or when IgG titers rose within the first 12 months or were persistently stable for more than 8 months. Using these criteria, congenital infection was definitely confirmed in 11 cases. Three additional cases were diagnosed based on indicative data. The CGMC test, which was performed without knowledge of the results of conventional serologal assays, had sensitivity and specificity of 82.4 and 93.0%, respectively, and positive and negative predictive values of 73.7 and 95.7%, respectively. When true positives and true negatives were considered, the comparative IgG profile had a ratio of 90.9% true results. The CGMC test thus is useful as an additional assay for the rapid diagnosis of congenital toxoplasmosis when paired serum samples from mother and child are available.
VIII European Multicolloquium of Parasitology, Poznan, 10-14 Sept. 2000
Congenital toxoplasmosis diagnosis by immunoblot; a prospective study using a new commercial immunobloting kit
J. Franck, F. Lanza-Silhol, L. Vuillemot, V. Pelissier, H. Dumon
Hôpital de la Timone, Marseille, France.
Objectives: Congenital toxoplasmosis is often subclinical and mainly in countries using screening programs. Without treatment, ocular sequels are frequent at short or long term and an earliest diagnosis is very important. In this context, we evaluated the performance of the kit Toxoplasma WB IgG-IgM (LDBIO DIAGNOSTICS 19A rue Louis Loucheur, 69009 Lyon). Methods: 106 mother-child couples were included in the study. All mothers had acquired toxoplasmosis during pregnancy. Children were tested at birth, D20, D60, and D90. Specific IgM and IgA were detected by ISAGA and IgG by EIA. 30/106 new-borns were congenitaly infected. The noninfected children had a follow up until the complete disappearence of maternal IgG. Results: At birth, the Positive Predictive Value (PPV) for WB G+M is 100%, the Negative Predictive Value (NPV) for WB G+M is 89.3%. PPV for Isaga is 95.2% and NPV 88.2% At D90, PPV for WB G+M is 100% and NPV 98.7%. For Isaga, PPV is 100% and NPV is 89.3%. False negative results in western blot assay at birth are mostly observed in new-borns treated by pyrimethaminesulfonamides in utero. Conclusions: The kit Toxoplasma WB Western Blot IgG-IgM greatly improves the performances of congenital toxoplasmosis serodiagnosis.
VIII European Multicolloquium of Parasitology, Poznan, 10-14 Sept. 2000
Comparative immunological profiles analysis for the early postnatal diagnosis of congenital toxoplasmosis
M. Paul
Karol Marcinkowski University of Medical Sciences, Poznan, Poland.
Objectives: The inconstant synthesis of specific IgM or IgA antibodies at birth and the passive transmission of maternal IgG antibody make the diagnosis of congenital toxoplasmosis quite difficult in the newborn. The diagnostic reliability of the comparative mother and infant immunological profile analysis by the new IgG-IgM Western blot assay (LDBIO DIAGNOSTICS, Lyon, France) was evaluated in the prospective study.
Methods: Thirty-four paired serum samples from congenitally infected infants and their mothers were tested for the pattern of IgG and IgM antibodies responses. Eight samples from women suspected of recent T. gondii infections during pregnancy and those from their healthy children constituted a control group. All patients originated from the area of regional neonatal screening detecting anti-Toxoplasma IgM and IgA from filter-paper eluates at birth. Specific IgM and IgA antibodies were simultaneously re-tested in serum samples between the 1st day to 12 weeks of infants' age (mean 29 days) by immunocapture Enzyme-Linked Immunosorbent Assays (ELISA) and/or Immunosorbent Agglutination Assays, and IgG was measured by a classic ELISA. Paediatric, ophthalmologic and imaging examinations as well as the serologic follow-up were conducted.Results: In an absence of prenatal anti-parasitic therapy, anti-Toxoplasma IgM and IgA were found by traditional techniques in 27 (79.4%) and 23 (67.6%) infected infants, respectively. During the first months of life, 21 children were positive for IgM and IgA antibodies (61.8%), 6 infants had only positive values of IgM (17.6%) and 2 patients had only detectable IgA antibody (5.9%). A significant increase in a level of specific IgG was observed in 4 infants (11.8%). The comparative Western blot assay allowed the detection of actively synthesized IgM in 33 cases (97.1%); 28 children produced IgG of other antigenic specificity and/or of higher concentration than the mothers (82.4%) (P<0.00001). In one premature infant without antibodies neosynthesized at birth, the final diagnosis of congenital toxoplasmosis was possible to be confirmed by Western blotting at the 28th day of life.
Conclusion: In an absence of screening during pregnancy followed by prenatal testing, the IgGIgM Western blot assay improves significantly the reliability of the early postnatal diagnosis of congenital Toxoplasma infection.
Value of prenatal diagnosis and early postnatal diagnosis of congenital toxoplasmosis: retrospective study of 110 cases.
J Clin Microbiol 1999 Sep;37(9):2893-8
Robert-Gangneux F; Gavinet MF; Ancelle T; Raymond J; Tourte-Schaefer C; Dupouy-Camet J
Laboratoire de Parasitologie, Centre Hospitalier Universitaire Cochin-Port Royal, Paris, France.
We reviewed the files of 110 women with Toxoplasma seroconversion during pregnancy. Prenatal diagnosis was attempted for 94 women by amniotic fluid sampling. Toxoplasma gondii was detected by PCR, with or without tissue culture and mouse inoculation. The early neonatal diagnostic procedure included placental testing by PCR and/or mouse inoculation, cord blood serological testing, and comparison of maternal and newborn antibodies by Western blotting (WB). Serological follow-up of the infants was conducted during the first year of life or until the diagnosis of congenital toxoplasmosis (CT) could be ruled out. Congenital infection was diagnosed in 27 individuals (20 live births) in the prenatal and/or neonatal period. The sensitivity and specificity of prenatal diagnosis were 81 and 100%, respectively. Placental examination was positive for 66.7% of individuals with CT and was always negative for neonates without CT. Cord blood serology detected immunoglobulin M (IgM) and/or IgA in 80% of infected newborns, with respective specificities of 91.2 and 87.7%. By WB we detected bands on IgG and IgM blots recognized by the newborn serum but not by the maternal serum (neosynthesized IgG and/or IgM) for 88.2% of infected infants within the first 2 months of life with a specificity of 100%. Early postnatal diagnosis was negative for 2 of the 20 neonates with CT. Both of these newborns had a negative prenatal diagnosis and were asymptomatic, suggesting a very low parasite load. In conclusion, despite the use of advanced methods, some cases of congenital toxoplasmosis cannot be detected early, which underlines the importance of careful follow-up of newborns who are at risk.
Neosynthesized IgG detected by Western blotting in Toxoplasma-seropositive heart or lung transplant recipients.
Transpl Int 2000;13(6):448-52
Robert-Gangneux F; Amrein C; Lavarde V; Botterel F; Dupouy-Camet J
Laboratoire de Parasitologie-Mycologie, Universite Paris 5, Centre Hospitalier Universitaire Cochin-Port Royal, 27 Rue du Faubourg Saint-Jacques, 75014 Paris, France.
Toxoplasmosis is a life-threatening disease in heart- or lung transplant recipients that can result either from the reactivation of a latent infection or from an organ-transmitted infection. The diagnosis of acute toxoplasmosis is easy in cases of seroconversion following a mismatch. However, when the recipient is Toxoplasma-seropositive before transplantation, usual serological techniques do not allow the differentiation between endogenous and organ-related reinfection. The aim of this study was to determine whether western blotting could contribute to this differentiation. Sequential sera from two heart- and one liver- and lung transplant patients whose anti-Toxoplasma antibody titers strongly increased after transplantation, were analyzed by western blotting. Neosynthesized IgG were observed on blots incubated with the sera from two patients who had received transplants from Toxoplasma-seropositive donors, whereas no neosynthesized IgG was detected on blots from the patient who had received a transplant from a Toxoplasma-seronegative donor. Our results suggest that the detection of neosynthesized IgG in the recipient may be related to the recognition of a new parasite strain possibly brought by the transplant from a Toxoplasma-seropositive donor.
Eosinophilic meningomyelitis in toxocariasis: case report and review of the literature.
Clin Neurol Neurosurg. 2005 Aug;107(5):432-8. Epub 2004 Nov 18.
Eberhardt O, Bialek R, Nagele T, Dichgans J.
Department of Neurology, University of Tuebingen, Hoppe-Seyler-Strasse 3, 72076 Tuebingen, Germany.
oeberhardt@excite.comToxocariasis is a worldwide-occurring parasitic infection leading to tissue damage in various organs due to wandering Toxocara larvae (visceral larva migrans). More than 40 cases of CNS involvement in children and immunocompetent adults have been documented in detail to date. Here, we present evidence of eosinophilic meningomyelitis in an adult without known risk factors and with positive Toxocara antibody response in CSF, but not in blood. Toxocariasis has to remain among the differential diagnosis in patients with eosinophilic CNS infection even if serological tests in blood are negative. Adult cases seem to be more frequent than previously thought (about 60%).
Evaluation of immunodiagnostics for toxocarosis in experimental porcine cysticercosis.
Trop Med Int Health. 2007 Jan;12(1):107-10.
Garcia HH, Cancrini G, Bartalesi F, Rodriguez S, Jimenez JA, Roldan W,
Mantella A, Nicoletti A, Bartoloni A.
Department of Microbiology, Universidad Peruana Cayetano Heredia, San Martin de Porres, Lima, Peru.
We assessed whether immunodiagnostic tests for cysticercosis can cross-react with the currently available immunodiagnostic tests for Toxocara canis in an established animal model for cysticercosis infection in pigs, known host for Toxocara. We examined by TES-enzyme-linked immunosorbent test and immunoblot assay for toxocarosis and cysticercosis the baseline and final follow-up sera of 10 pigs, before and after (3 months) infection with Taenia solium. After successful cysticercosis infection, the nine evaluable pigs became seropositive to T. solium (enzyme-linked immunoelectrotransfer blot assay), but did remain seronegative for Toxocara in both assays, documenting the lack of cross-reactivity with anti-T. solium antibodies in both T. canis assays. These findings should help clinicians better interpret serology for toxocariosis and cysticercosis in endemic areas for both helminth infections.
Seroprevalence of Toxocara antibodies among patients
suspected of ocular toxocariasis in Slovenia
Korean J Parasitol 2004 Sept;42(3):137-140
Jernej LOGAR 1) *, Barbara SOBA 1) , Aleksandra KRAUT 2) and Branka STIRN-KRANJC 2)
1) Department of Parasitology, Institute of Microbiology and Immunology, Medical Faculty, University of Ljubljana,
Zaloska 4, 1000 Ljubljana, Slovenia, and
2) Department of Ophthalmology, University Medical Centre Ljubljana, Slovenia
Ocular toxocariasis named also ocular larva migrans is caused by larvae of the roundworm Toxocara spp. The purpose of this study was to find out the seroprevalence of Toxocara antibodies in patients suspected of ocular toxocariasis. Between January 2001 and December 2003, sera from 239 ocular patients, aged 3 to 80 years, were examined by ELISA and confirmed by Western blot test. Out of the 239 patients, 172 (72%) were seronegative and 67 (28%) were Toxocara seropositive; 95% CI (22-34%). The median age of Toxocara seropositive patients was 37.6 years. There was no significant difference in the number of Toxocara positive sera between the younger age group (=14 years) and the older age group (>14 years), p>0.05. A high rate of Toxocara seropositivity in ocular patients should alert the ophthalmologists in Slovenia to include toxocariasis in the differential diagnosis of eye diseases more frequently.
A study on some epidemiologic and paediatric aspects of toxocarosis in Hungary
Journal of Thrombosis and Haemostasis 2003; 1 Supplement 1 July:
Szénási Z.
Department of Clinical Microbiology, Albert Szent-Gyorgyi Medical University, Szeged, Hungary
Objectives:Toxocarosis is the most frequent human helminthosis in Hungary. Most of the clinical symptoms are nonspecific, thus, its diagnosis depends considerably on laboratory methods, such as ELISA and Western blot (WB) techniques. The objectives of our study was to determine the frequency and distribution of Toxocara seroprevalence in the different regions and the different age groups of Hungary. Special reference will be given to children in whom anti-Toxocara antibodies were determined for differential diagnostic reasons, such as exanthemata and/or asthmatic symptoms.
Methods: Sera were obtained from 6985 asymptomatic individuals representing all age groups of the population of Hungary distributed to 20 different regions. Sera of 1427 symptomatic children aged <1-14 years were also obtained from different regions of Hungary. Seroprevalence of the symptomatic children were compared with 1605 asymptomatic children. Toxocara seroprevalence was measured with NOVATEC Toxocara ELISA IgG kit. In case of negative or low-positive values obtained by ELISA, the results were confirmed by WB method (LDBIO).
Results: The sera of 1977 persons of 6985 asymptomatic individuals were found to be positive for Toxocara IgG antibody (28.3%). The lowest prevalence (17.6%) was found in Budapest, the highest ones were found in Szabolcs-Szatmár-Bereg (39.4%) and Hajdú-Bihar (38.2%) countries. Seropositivity rapidly increases by age reaching 37.0% at the age of 10-14 years. A significantly higher percentage (31%, P = 0.01) of Toxocara seropositivity was found in children suffering from bronchial asthma when compared with the 17% seropositivity measured in asymptomatic control groups of children. The borderline anti-Toxocara IgG ELISA results were found to be positive with WB method.
Conclusions: In Hungary, 28.3% of the population has anti-Toxocara IgG antibodies. The highest positivity (39.4%, 38.2%) were found in the most under-developed regions of Hungary, the lowest one (17.6%) was found in the capital. Probably, this reflects the differences in the hygienic conditions. The Toxocara seroprevalence rapidly increases by the age. This can be explained by the frequent contact of children with contaminated soil. A significantly higher percentage of Toxocara seropositivity was found in asthmatic children compared with the asymptomatic children. The Western blot technique is very useful in confirming the borderline and negative anti- Toxocara IgG values obtained by ELISA method.
Immunodiagnosis of ocular toxocariasis using Western-blot for the detection of specific anti-Toxocara IgG and CAP for the measurement of specific anti-Toxocara IgE.
J Helminthol 2002 Dec;76(4):335-9
Magnaval JF; Malard L; Morassin B; Fabre R
Service de Parasitologie, CHU Rangueil, 31403 Toulouse 4, France.
A prospective multicentric study was carried out to assess both the performance of Western-blot (WB) detecting specific anti-Toxocara IgG and that of CAP measuring specific IgE titre for the immunodiagnosis of ocular toxocariasis. For 14 outpatients presenting ophthalmic symptoms (choroiditis, chorioretinitis, papillar oedema, hyalitis, retinal detachment and/or uveitis), samples of serum and aqueous fluid (AF) were sent to the Department of Parasitology, University Hospitals, Toulouse, France. All patients but two tested positive with WB on the serum; 13 WB tests were performed on the AF, 12 of which were positive. The two patients who had a negative WB serum result tested positive for the AF. Specific IgE detection was considered as a complementary test of WB. Two patients showed a greater specific IgE titre in the AF than in the serum, and one had a positive result in the AF, but not in the serum. These six patients were considered as clear cases of ocular toxocariasis. Western-blot coupled with specific anti-Toxocara IgE detection appeared therefore to be an accurate procedure for the immunodiagnosis of ocular toxocariasis, provided the testing was simultaneously performed on the serum and AF.
Highlights of human toxocariasis.
Korean J Parasitol (Korea 2001 Mar;39(1):1-11
Magnaval JF; Glickman LT; Dorchies P; Morassin B
Service de Parasitologie, Centre Hospitalier Universitaire Rangueil 31403 Toulouse 4, France.
Human toxocariasis is a helminthozoonosis due to the migration of Toxocara species larvae through human organism. Humans become infected by ingesting either embryonated eggs from soil (geophagia, pica), dirty hands or raw vegetables, or larvae from undercooked giblets. The diagnosis relies upon sensitive immunological methods (ELISA or western-blot) which use Toxocara excretory-secretory antigens. Seroprevalence is high in developed countries, especially in rural areas, and also in some tropical islands. The clinical spectrum of the disease comprises four syndromes, namely visceral larva migrans, ocular larva migrans, and the more recently recognized "common" (in adults) and "covert" (in children) pictures. Therapy of ocular toxocariasis is primarily based upon corticosteroids use, when visceral larva migrans and few cases of common or covert toxocariasis can be treated by anthelmintics whose the most efficient appeared to be diethylcarbamazine. When diagnosed, all of these syndromes require thorough prevention of recontamination (especially by deworming pets) and sanitary education.
VIII European Multicolloquium of Parasitology, Poznan, 10-14 Sept. 2000
Is toxocariasis an etiology of unexplained chronic prurigo, pruritus and urticaria?
R. Piarroux1, P. Humbert2, V. Janin1, A. Degouy2, S. Clair1, F. Aubin2
1 : Santé et Environnement Rural, Université de Franche-Comté, France.
Fax (33) 3 81 66 89 14, e-mail renaud.piarroux@ufc-chu.univ-fcomte.fr
2 : Department of Dermatology, Hôpital Saint Jacques, Besançon, France.
Objectives: Evaluation of the prevalence of toxocarosis infection in patients suffering from chronic prurigo, pruritus or urticaria and in a control population living in the same area (Franche-Comté, France).
Methods: 128 inpatients (Department of dermatology, Besançon) were included from March 1999 to December 1999 : 8 suffered from chronic prurigo, 30 had chronic pruritus, and 90 presented with chronic urticaria.
All patients had a clinical and biological screening to detect any disturbance known to be associatedwith these conditions, such as alimentary allergy, autoimmune disease, ectoparasitosis etc.. 60 sera, initially collected for a systematic screening for toxoplasmosis performed before marriage, during pregnancy or before corneal graft, were selected as a control group. This group had identical sex ratio and age distribution than the dermatological patients group. Toxocarosis serological diagnosis were performed using an immunoblot assay (LDBio Diagnostics, Lyon, France).
Results: Clinical examination and biological tests led to an explanation of symptoms in 22 cases of urticaria, 13 cases of pruritis and 1 case of prurigo. Other patients are considered to have unexplained cause of their condition. Immunoblot test for toxocarosis was positive in 22% (13 / 60) of control group versus 41% (28 / 68) of unexplained urticaria cases (p = 0.02, Chi2 test), 76% (13 / 17) of unexplained pruritus cases (p = 0.00003, Chi2 test), and 57% (4 / 7) of unexplained prurigo cases (not significant).
Conclusions: Our results strongly support the involvement of toxocarosis as an etiology of unexplained chronic pruritus and chronic urticaria. A prospective case-control study is ongoing to confirm these findings.
Ocular toxocariasis in Austria.
Dtsch Med Wochenschr 1998 May 15;123(20):626-30
Dietrich A; Auer H; Tittl M; Barisani-Asenbauer T
Universitatsklinik fur Augenheilkunde und Optometrie, Universitat Wien.
HISTORY AND CLINICAL FINDINGS: Two women (aged 21 and 44 years) were referred because of a suspect retinal lesion. An ophthalmological examination in both revealed prominent retinal granulomatous foci, probably ocular toxocariasis. Both women were otherwise well; both reported close contact with dogs. INVESTIGATIONS: Among a full array of laboratory tests the only major pathologic findings were high antibody titres against Toxocara canis (patient 1: 70 antibody units [AU]; patient 2: > 100 AU), specific antibodies in the ELISA and Western blot tests confirming the diagnosis of T. canis infection. DIAGNOSIS, TREATMENT AND COURSE: Both patients were treated with prednisolone (initially 75 mg/d, gradually decreasing over 4 months) and albendazole (2 x 800 mg/d for 6 days), with complete healing of the chorioretinal foci. CONCLUSION: General physicians as well as ophthalmologists should more often include Toxocara canis infection in the differential diagnosis, because the larvae, in their migration through the body, can infest various organs where they can cause inflammatory or allergic reactions.
Human alveolar echinococcosis in Slovenia
Clin Microbiol Infect 2007; 13: 544-546
J. Logar1, B. Šoba1, T. Lejko-Zupanc2, T. Kotar2
Department of Parasitology, Institute of Microbiology and Immunology, Medical Faculty, University of Ljubljana, Slovenia1,
Department of Infectious Diseases and Febrile Illnesses, University Medical Centre Ljubljana, Ljubljana, Slovenia2
E-mail: jernej.logar@mf.uni-lj.siBetween January 2001 and December 2005, 1263 patients suspected of having echinococcosis were screened serologically by indirect haemagglutination assay (IHA). IHA-positive patient sera were then retested by western blot for confirmation and differentiation between Echinococcus granulosus and Echinococcus multilocularis infection. Of 43 sera confirmed as Echinococcus-positive, nine appeared to be specific for alveolar echinococcosis (AE) caused by E. multilocularis. AE-positive serological results corresponded to the clinical and/or imaging findings concerning the patients' liver cysts. The detected incidence of AE was 0.45/105 inhabitants, which suggests that clinicians and health authorities in Slovenia should give greater attention to AE in the future.
The immunodiagnosis of Echinococcus multilocularis infection
REVIEW: Clinical Microbiology and Infection (OnlineEarly Articles). doi:10.1111/j.1469-0691.2007.01665.x
D. Carmena, A. Benito, E. Eraso
Department of Immunology, Microbiology and Parasitology, Faculty of Pharmacy, University of the Basque Country, Vitoria, Spain. E-mail: d.carmena@imperial.ac.uk Alveolar echinococcosis (AE) is a severe zoonotic disease caused by the metacestode stage of Echinococcus multilocularis. The infection can have fatal consequences in humans if treatment is not provided, so early diagnosis is fundamental for initiating treatment and reducing morbidity and mortality. In addition, detection of the parasite in the definitive host plays a central role in epidemiological studies and surveillance programmes for control of AE. This review presents an overview of the present situation regarding the immunodiagnosis of E. multilocularis infection. Special attention is given to the description of the native, partially purified and recombinant antigens available currently for immunodiagnostic purposes. Recent advances in the primary serodiagnosis and follow-up of AE patients are highlighted, including the detection of specific cytokine profiles. Progress in the immunodiagnosis of intestinal E. multilocularis infection in definitive hosts, particularly the detection of excretory-secretory and integument products of the worm in faeces (copro-antigens) by ELISA, is also discussed.
Active Alveolar Hydatidosis with Sero-Negativity for Antibody to the 18 kDa Antigen
Jpn J Infect Dis 2005 Apr;58(2):122-4 (ISSN: 1344-6304)
Yamano K; Yagi K; Furuya K; Sawada Y; Honma H; Sato N
Department of Biological Science, Hokkaido Institute of Public Health, Sapporo 060-0819, Japan. yamano@iph.pref.hokkaido.jp.
Laboratory evaluation of commercial immunoblot assay kit for serodiagnosis of Echinococcus infections using sera from patients with alveolar hydatidosis in Hokkaido
Kansenshogaku Zasshi 2004 Apr;78(4):320-6
Furuya K; Kawanaka M; Yamano K; Sato N; Honma H
Hokkaido Institute of Public Health.
Using serum specimens from patients with alveolar hydatidosis (AH) in Hokkaido, we assessed the usefulness of "Echinococcus Western Blot IgG" (the French immunoblot assay, FIA), which has recently been launched from Ldbio Diagnostics (Lyon, France) as new commercial immunoblot assay kit of immunodiagnosis of Echinococcus infections. Eighty serum specimens were used for the present study: 64 preoperative sera and nine postoperative sera, which were taken from AH patients in Hokkaido, and seven sera from persons who were ELISA (enzyme-linked immunosorbent assay)--positive in mass screening which was conducted for checking on Echinococcus infections in Hokkaido since 1982. When the 64 preoperative sera were examined by the Western blotting method (the Hokkaido method of Western blotting, HWB) which had been carried out at Hokkaido Institute of Public Health between 1987 and 1993, it was found that 53 cases were positive and six cases were quasi-positive, i.e. the rate of the positive cases including quasi-positive cases was 92.2%. From immunostaining patterns, HWB-positive sera could be grouped in two types: the complete type, which showed a pattern of multiple bands containing the 55 and 66 kDa bands, and the incomplete type, which showed patterns of only few bands containing the AH-specific polysaccharide antigen named C antigen. Forty-three of the 53 HWB-positive sera were of the complete type and the residue was of the incomplete type. On the other hand, when the 64 preoperative sera were examined by FIA, 60 sera (93.8%) were judged to be positive and the others as negative sera. On the basis of the interpretation of immunostaining patterns described in the instruction manual, 47 (78.3%) of the 60 positive sera were regarded as pattern P3, five (8.3%) as pattern P4, and eight (13.3%) as pattern P5. All of the complete-type sera were regarded as P3, indicating high antibody titers. Contrarily, most of the incomplete-type or quasi-positive sera resulted in other patterns such as P4 and P5, indicating low antibody titers. Of 5 HWB-negative sera, two were FIA-positive (which showed P3 and P5 patterns respectively), however their immunoreactions were significantly low. Therefore, apart from interpretation of pathological conditions of cases with exceedingly low antibody titers, FIA may be able to give a serologically clear interpretation to HWB-quasi-positive cases, indicating that it is a highly sensitive and useful method for immunodiagnosis of Echinococcus infections.
Molecular confirmation of human alveolar echinococcosis in Poland.
Clin Infect Dis 2003 Oct 15;37(8):121-5
Myjak P; Nahorski W; Pietkiewicz H; von Nickisch-Rosenegk M; Stolarczyk J; Kacprzak E; Felczak-Korzybska I; Szostakowska B; Lucius R
Institute of Maritime and Tropical Medicine, Gdynia, Poland. pemyjak@immt.gdynia.pl.
Infections of humans with Echinococcus multilocularis, the causative agent of alveolar echinococcosis (AE), a zoonosis, have been described with increasing frequency in Poland since 1994. In the attempt to verify these reports, we analyzed specimens obtained from a representative group of Polish patients. Liver lesions in patients with AE that was diagnosed on the basis of results of histological and serological tests contained E. multilocularis DNA, as shown by the presence of specific microsatellite sequences and mitochondrial 12S rDNA. The same tests clearly distinguished between AE and cystic echinococcosis, which is caused by Echinococcus granulosus. These data are unequivocal proof that human infections with E. multilocularis occur in Poland.
Evaluation of an enzyme-linked immunosorbent assay (ELISA) with affinity-purified Em18 and an ELISA with recombinant Em18 for differential diagnosis of alveolar echinococcosis: results of a blind test.
J Clin Microbiol 2002 Nov;40(11):4161-5
Ito A; Xiao N; Liance M; Sato MO; Sako Y; Mamuti W; Ishikawa Y; Nakao M; Yamasaki H; Nakaya K; Bardonnet K; Bresson-Hadni S; Vuitton DA.
Department of Parasitology. Animal Laboratory for Medical Research, Asahikawa Medical College, Asahikawa, Japan.
Alveolar echinococcosis (AE) is the most potentially lethal parasitic zoonosis of the nontropical areas in the northern hemisphere, where cystic echinococcosis (CE) is also endemic. Both AE and CE are highly endemic in China, and both serologic detection of echinococcosis, either AE or CE, and differentiation of AE from CE are crucial problems. Evaluation of Western blot analysis (WB) and enzyme-linked immunosorbent assay (ELISA) for the Em18 antigen, using affinity-purified and recombinant Em18, was carried out "blindly" using 60 human sera from patients diagnosed in France. The results were compared with those obtained using a commercially available Echinococcus WB immunoglobulin G (IgG) kit developed in France. The Em18 WB and Echinococcus WB IgG showed very similar results for detection of AE. Both affinity-purified Em18 or a recombinant Em18 WB and Echinococcus WB IgG seem useful for identification of AE, and the latter seems appropriate for both AE and CE, whereas affinity-purified Em18 ELISA and the newly developed recombinant Em18 ELISA appear to be suitable for detection of AE, especially for epidemiological surveys.
Laboratory diagnosis of cystic hydatic disease.
World J Surg 2001 Jan;25(1):10-4
Biava MF; Dao A; Fortier B
Service de Parasitologie-Mycologie, H pital de Brabois, CHU de Nancy, France.
The main purpose of this article is to answer the questions about which test to perform for hydatic diagnosis and when. Several techniques for biologic diagnosis and follow-up of human cystic hydatidosis are reviewed. The specificity and sensitivity of immunologic reactions are reported. The differential diagnosis between Echinococcus granulosus and E. multilocularis is examined. The characteristics of the immunologic diagnosis according to the stage and the treatment of hydatidosis disease is discussed. Laboratory diagnosis of cystic hydatic disease is complementary to the clinical data. A judicious association of the usual techniques (indirect immunofluorescence assay, indirect hemagglutination assay, immunoelectrophoresis, co-electrophoresis with antigen 5 identification) confirms the diagnosis in 80% to 94% of hepatic hydatidosis cases and in 65% of pulmonary hydatidosis cases. Special techniques (enzyme-linked immunosorbent assay, Western blot, polymerase chain reaction) must be used for other localizations or when cysts are calcified. A serologic survey is necessary for the follow-up of operated medically treated patients. Despite poor standardization, purified antigens can distinguish between E. granulosus and E. multilocularis infections, although false-positive results are observed during other helminthiases, such as cysticerocosis.
Immunodiagnosis of Echinococcus infections:
confirmatory testing and species differentiation by a new commercial Western Blot.
J Clin Microbiol 2000 Oct;38(10):3718-21
Liance M; Janin V; Bresson-Hadni S; Vuitton DA; Houin R; Piarroux R
Laboratoire de Parasitologie-Mycologie, Hopital Henri Mondor AP-HP, et Universite Paris XII, 94010 Creteil, France.
The Echinococcus Western Blot IgG (LDBIO Diagnostics, Lyon, France), using a whole larval antigen from Echinococcus multilocularis, was evaluated for serodiagnosis and differentiation between two human parasitic infections of worldwide importance: cystic echinococcosis, due to Echinococcus granulosus, and alveolar echinococcosis, due to E. multilocularis. Fifty and 61 serum samples from patients with cystic and alveolar echinococcosis, respectively, were used for assessing diagnostic sensitivity. The sensitivity of the assay was compared with those of screening tests used for these applications. Sera used for assessing cross-reactivities were from 154 patients with other diseases, either parasitic or not. The assay allowed the detection of serum immunoglobulin G antibodies in 97% of Echinococcus-infected patients. It had a higher sensitivity than screening assays for the detection for each echinococcosis. The assay allowed us to correctly distinguish between E. granulosus- and E. multilocularis-infected patients in 76% of cases. It did not allow us to distinguish active from inactive forms of both echinococcoses. The occurrence of cross-reactivities with neurocysticercosis indicates the necessity for retesting sera with species-specific antigens, for rare patients with neurologic disorders. This study shows the usefulness of the commercially available Echinococcus Western Blot IgG for the serological confirmation of human echinococcosis.
Sensitivity and specificity of ELISA and immunoblot for diagnosing neurocysticercosis.
Eur J Clin Microbiol Infect Dis 2002 Mar;21(3):227-9
Gekeler F; Eichenlaub S; Mendoza EG; Sotelo J; Hoelscher M; Loscher T
Neurologische Klinik Grosshadern, Klinikum der Ludwig-Maximilians-Universitat Munchen, Germany.
In patients with neurocysticercosis (NCC), clinical manifestations and the results of neuroimaging procedures vary widely and often do not facilitate a definite diagnosis. In order to determine the value of immunodiagnosis for NCC, 222 serum and cerebrospinal fluid samples from patients with NCC and healthy subjects were examined. The samples represented patients from various endemic regions, those with other neurological disorders from an endemic area (Mexico), persons with various helminth infections other than NCC, and a group of healthy volunteers. All specimens were tested by enzyme-linked immunosorbent assay and immunoblot for the presence of Taenia solium-specific antibodies. The sensitivities of the enzyme-linked immunosorbent assay and the immunoblot test in NCC patients were almost identical (80% and 81.7%, respectively). For both tests, the sensitivity was higher when cerebrospinal fluid (86%) was tested compared with serum (75%). The overall specificity of enzyme-linked immunosorbent assay was only 75.3% because of frequent false-positive results in patients with other helminth infections, especially in those with echinococcosis. The specificity (99.4%) of the immunoblot test was clearly superior. It is concluded that enzyme-linked immunosorbent assay as a screening method and immunoblot as a confirmatory test contribute considerably to the diagnosis of NCC.
Development and evaluation of a Western blot kit for diagnosis of human trichinellosis.
Clin Diagn Lab Immunol 2003 Sep;10(5):793-6
Yera H; Andiva S; Perret C; Limonne D; Boireau P; Dupouy-Camet J
Laboratoire de Parasitologie-Mycologie, Centre National de Reference des Trichinella Hopital Cochin, Universite R. Descartes, Assistance Publique-Hopitaux de Paris, 75014 Paris, France.
We evaluated industrially prepared Western blot strips designed to avoid the cross-reactions observed with indirect immunofluorescence and enzyme-linked immunosorbent assays used for the serodiagnosis of trichinellosis. The antigen preparations were crude extracts of Trichinella spiralis. The Western blot profile characteristic of trichinellosis was characterized by comparing 60 sera from patients infected by Trichinella to 11 sera from healthy subjects, 51 sera from patients with other proven parasitic diseases (cysticercosis, schistosomiasis, strongyloidosis, fascioliasis, toxocariasis, liver amebiasis, anisakiasis, filariasis, toxoplasmosis, hydatidosis, or malaria), and 23 sera from patients with autoantibodies. Specific 43- to 44-kDa and 64-kDa bands were obtained with all of the sera from 51 patients with acute trichinellosis, in 4 out of 9 patients at the early stages of the disease, and in only 1 control patient, who had suspected anisakiasis and in whom trichinellosis could not be ruled out by muscle biopsy.
Comparative evaluation of a latex agglutination test, two Elisa tests and a Western blot test for the serodiagnosis of human trichinellosis
Ann Biol Clin (Paris) 2002 Jan-Feb;60(1):79-83
Andiva S; Yera H; Haeghebaert S; Tourte-Schaefer C; Magnaval JF; Dupouy-Camet J
Laboratoire de parasitologie-mycologie, Hopital Cochin, 27, rue du Faubourg-Saint-Jacques, 75014 Paris.
Serology is a helpful test for the diagnosis of human trichinellosis because clinical signs of this disease (fever and myalgias) are non specific. A lot of techniques have been studied but very few are commercialized and have been comparatively evaluated. We report here the performances of 4 commercially available kits: two Elisa tests, one western blot test & one latex agglutination test. The specificity and sensitivity of the different tests were assayed on 60 sera from patients with a proven trichinellosis and on 70 controls. The four tests had a 100% sensitivity. The specificity was of 98.6% for the western blot, of 93% for the latex agglutination test and of 87 & 77% for the two Elisa tests. Cross reactions are mainly observed in patients with other helminthic infections.
Xth International Conference on Trichinellosis, Fontainebleau, 20-24 August 2000
Evaluation of " Trichinella western blot IgG " kit in the diagnosis of human trichinellosis
Shakir Andiva1, Catherine Perret2, Sylvie Haeghebaert3, Claudine Tourte-Schaefer 1, Jean François Magnaval4, Sun Xin1,5, Pascal Boireau2, Jean Dupouy-Camet1
Parasitology, Hopital Cochin, Descartes University, 27 Fbrg St Jacques, 75014 Paris, tel/fax 33 1 584 12 251/245 e-mail:jean.dupouy-camet@cch.ap-hopparis.fr
UMR BIPAR INRA-AFSSA-ENVA , AFSSA-Alfort, 94703, Maisons-Alfort
Institut de la Veille Sanitaire, 94415, Saint Maurice
Parasitology, Hopital Rangueil, 31403, Toulouse, France
Bengbu Medical College, Bengbu, China
Key words: western-blot, human, diagnosis
Objective: Serodiagnosis of trichinellosis, based on indirect immunofluorescence or ELISA may be subject to cross-reactions making it difficult to interpret. Immunoblot analysis of such sera was proven to be an efficient method to eliminate such crossreactions (de la Rosa et al., 1995; Robert et al., 1996). In this study, we evaluated the performance of a new western blot kit, recently manufactured (Trichinella WB IgG, LDBIO DIAGNOSTICS, 19A rue Louis Loucheur, 69009 Lyon, France).
Methods: The antigenic preparation was obtained from Trichinella spiralis larvae, grounded in liquid nitrogen and sonicated. The WB strips were incubated with sera diluted 1:50. Protein fractions recognised by the sera were revealed by incubation with a rabbit anti-human immunoglobulin alkaline phosphatase conjugate and then with an appropriate substrate-chromogen solution, following the manufacturer instructions.
Results: Sensitivity of the kit was tested on 51 cases of trichinellosis (including 39 due to T. spiralis, 4 to T. pseudospiralis, 2 to T. nelsoni, 2 to T. murrelli, one to T. nativa and 3 to T. britovi). Bands were observed with all these samples at 37, 38, 39, 43, 44, 46, 49, 64, 96, 110 and 127 kDa. Specificity was tested on sera of 23 patients with autoimmune disorders (anti DNA, anti-mitochondrial, anti-stomach antibodies, rheumatoid factor), of 51 patients with different parasitosis (bilharziasis, fasciolasis, filariasis, amoebiasis, malaria, hydatidosis, anisakiasis, toxocarosis, toxoplasmosis, strongyloidosis, cysticercosis) and 11 negative controls. Bands were observed in 42 patients: 2 with anti-stomach antibodies, 7 with rheumatoid factors, 5 with anti-nuclei antibodies, 3 with bilharziasis, 4 with fasciolasis, 5 with filariasis, 4 with anisakiasis, 5 with cysticercosis and 6 with toxocarosis, 2 with the negative controls. The comparison of these profiles with those obtained with sera from cases of trichinellosis allowed defining three specific bands at 43, 44 and 64 kDa. These bands appeared precociously as they were observed with sera from 5 out of 18 patients recently infected, but with an ELISA test still negative.
Conclusion: This new kit appears to be 100 % sensitive and 97.65 % specific.
Development and Evaluation of a Western Blot Kit for Diagnosis of Schistosomiasis
Clinical and Diagnostic Laboratory Immunology, Apr. 2005, p. 548-551 Vol. 12, No. 4
Annie Sulahian,1* Yves Jean François Garin,1 Arezki Izri,2 Caroline Verret,1
Pascal Delaunay,3 Tom van Gool,4 and Francis Derouin1
Laboratory of Parasitology, St Louis Hospital, Assistance Publique-Hoˆpitaux de Paris, Paris,1 Laboratory of Parasitology, Avicenne Hospital, Bobigny,2 and Laboratory of Parasitology, Archet 2 Hospital, Nice,3 France, and Department of Microbiology, Parasitology Section, Academic Medical Center, Amsterdam, The Netherlands4We evaluated the performance of Western blot (WB) analysis using commercially available antigen strips and compared the results with those of indirect hemagglutination (IHA) and indirect immunofluorescence (IFAT) for the serodiagnosis of human schistosomiasis. The antigen preparation was a crude extract of Schistosoma mansoni. The WB profile characteristics of schistosomiasis were characterized by comparing the results for 58 serum samples from patients with parasitologically proven S. mansoni (n = 12) and S. haematobium (n = 46) infections and 37 individuals with probable cases of schistosomiasis but with only positive serology results. The specificity of WB analysis was assessed by testing 12 serum samples from healthy subjects, 67 serum samples from patients with other proven helminthic and protozoan infections, and 16 serum samples from patients with autoantibodies. Six immunodominant bands (65, 70, 80, 95, 110, and 120 kDa) were revealed with sera from patients with schistosomiasis. The presence of three or more bands in the range 65 to 120 kDa, with the exception of the 100-kDa band, was considered diagnostic for Schistosoma infection and had a specificity of 100% in our series. In patients with proven schistosomiasis, the sensitivity of WB analysis was 84.5%, whereas those of IFAT and IHA were 65.5 and 72.9%, respectively. For serologically proven cases, the sensitivity of WB analysis was 97.3%. The overall sensitivity and specificity for both groups of patients were 89.5 and 100%, respectively, with positive and negative predictive values of 100 and 91.3%, respectively. We conclude that WB analysis is a useful technique for the immunological diagnosis of schistosomiasis.
Molecular Study of Microsporidiosis Due to Enterocytozoon bieneusi and Encephalitozoon intestinalis among Human Immunodeficiency Virus-Infected Patients from Two Geographical Areas: Niamey, Niger, and Hanoi, Vietnam.
J Clin Microbiol , Sept. 2007, p. 2999-3002 Vol. 45, No. 9
Anne Espern,1 Florent Morio,1 Michel Miegeville,1 Hachimou Illa,2 Moustapha Abdoulaye,2 Vanina Meyssonnier,3 Eric Adehossi,2 Anne Lejeune,1,4 Phung Dac Cam,4 Bernard Besse,5 and Francoise Gay-Andrieu,1,2* Laboratory of Parasitology and Mycology, Nantes university Hospital, Nantes, France 1; National Hospital of Niamey, Niamey, Niger 2; SOLTHIS, Niamey, Niger 3; Institute of Hygiene and Epidemiology of Hanoi, Hanoi, Vietnam 4; and Virology Laboratory, Nantes University Hospital, Nantes, France 5
* Corresponding author. Mailing address: Laboratoire de Parasitologie-Mycologie, Hôtel Dieu, 9 quai Moncousu, 44093 Nantes cedex 1, France. Phone: 33 2 40 08 40 79. Fax: 33 2 40 08 42 49. E-mail:francoise.gay-andrieu@chu-nantes.fr.Microsporidiosis cases due to Enterocytozoon bieneusi and Encephalitozoon intestinalis are emerging opportunistic infections associated with a wide range of clinical syndromes in humans. The aim of this study was to specify microsporidial epidemiology in two different geographical areas. From November 2004 to August 2005, 228 and 42 stool samples were collected in Niamey, Niger, and Hanoi, Vietnam, respectively. Screening for microsporidia was performed using UV-light microscopy. Detection was confirmed by molecular biology using two methods specific for E. bieneusi and E. intestinalis. All samples positive for E. bieneusi were subjected to genotyping. In this study, we found high prevalences of microsporidiosis among human immunodeficiency virus-infected patients, 10.5% and 9.5%, respectively, in Niamey and Hanoi. These levels of prevalence are similar to those recorded in European countries before highly active antiretroviral therapy was introduced. In the samples positive for E. bieneusi, we found seven distinct genotypes, including two genotypes not previously described. The E. bieneusi genotype distributions in the two geographical areas suggest different routes of infection transmission, person-to-person in Niger and zoonotic in Vietnam.