Evaluation of serological tests for the diagnosis of Neospora caninum infection in dogs: optimization of cut off titers and inhibition studies of cross-reactivity with Toxoplasma gondii

Diagnosis of Neospora caninum infection in dogs is based on serological assays such as the indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assays (ELISA). This study evaluated two serological tests (IFAT and ELISA) for the detection of IgG antibodies to N. caninum in 300 serum samples of dogs through the optimization of cut off titers by using the two-graph receiver-operating characteristic (TG-ROC) curve. In addition, the identification of major cross-reactive antigens with Toxoplasma gondii was investigated by inhibition ELISA and immunoblotting (IB) assays. IFAT and ELISA results showed 74% agreement, with a good negative concordance (P(neg)=0.83), but a poor positive concordance (P(pos)=0.42). The great majority (86%) of sera with positive concordant results (IFAT+/ELISA+) recognized at least two out of three N. caninum immunodominant antigens, particularly the 29-32 and 35-37 kDa bands. Optimization of cut off titers in IFAT and ELISA was performed considering the reactivity to at least two out of three N. caninum immunodominant antigens as infection markers, obtaining a titer of 50 for IFAT and 200 for ELISA. Seropositivity to N. caninum was significantly associated with T. gondii-seropositive samples, particularly in ELISA (55.4%). Inhibition ELISA curves for N. caninum showed a partial heterologous inhibition, indicating some degree of cross-reactivity between N. caninum and T. gondii antigens. Inhibition IB assays showed a moderate heterologous inhibition for N. caninum antigens above 45-50 kDa. These results indicate that ELISA should be used critically when crude tachyzoite antigen preparations are employed, due to possible cross-reactivity with other related parasites as T. gondii. Also, the cut off dilution of 1:50 in IFAT showed to be the most appropriated for N. caninum serology in dogs. Therefore, we suggest that N. caninum immunodominant antigens, specially the 17 and 29-32 kDa proteins, should be selected markers in serological assays for canine neosporosis.     

The diagnosis of Neospora abortions in cattle

An indirect immunofluorescent antibody test (IFAT) and an enzyme-linked immunosorbent assay (ELISA) for specific anti-Neospora antibodies in bovine sera and foetal fluids were compared with histological examination results on aborted foetal material. The agreement between serological and histological examination results was poor, while the two serological tests showed a high degree of agreement. Serological testing of diagnostic serum samples and foetal fluids suggests that the prevalence of anti-Neospora antibodies in cattle which recently aborted is around 40%, in line with previous estimates of the number of abortions in dairy cattle caused by Neospora sp. A sero-epidemiological approach to the diagnosis of Neospora abortions in cattle may be suggested from these data.     

Evaluation of ovine abortion associated with Toxoplasma gondii in Spain by different diagnostic techniques

A total of 173 aborted ovine foetuses and seven aborted caprine foetuses, submitted from different points of north and central Spain, were analysed to determine the role of T. gondii in abortion and to compare the utility of the most widely used techniques in diagnosis of the congenital infection (histopathology, serology--IFAT and ELISA--and a nested-PCR). Parasite infection was diagnosed in 40 (23.1%; n = 173) ovine foetuses by at least one of the diagnostic techniques used. A higher percentage of foetuses were diagnosed using serological techniques (IFAT and ELISA) (28.3%; n = 106) than by histologic examination (8.7%; n = 173) or PCR (6.9%; n = 173). No significant association between infection and the foetal age categories was found (P > 0.05). In this study, 106 aborted foetuses were analysed by all of the three diagnostic techniques. When we compared serological results, perfect agreement between ELISA and IFAT was obtained. On the contrary, slight to fair agreements were observed when histology results were compared with those obtained by serology and PCR techniques. All the positive foetuses were aborted in the mid (60%) or last (40%) term of pregnancy, but no significant differences were found between ages of the infected and non-infected foetuses (P > 0.05). This report indicates that toxoplasmosis may be a common cause of small ruminant abortion and neonatal death in Spain and points out the necessity of using different and complementary techniques to increase the probability of detecting Toxoplasma infection in an aborted foetus.     

Evaluation by different diagnostic techniques of bovine abortion associated with Neospora caninum in Spain

Eighty foetuses from some of the main cattle-producing regions in Spain were analysed to investigate the participation of Neospora caninum in cases of bovine abortion. Diagnosis of the infection was determined by histopathological analysis complemented with immunohistochemistry, serology (IFAT and ELISA) and PCR tests. A total of 38.8% of the bovine foetuses analysed were considered to be infected by at least one of the diagnostic techniques used. Microscopic lesions consistent with Neospora infection in brain were identified in 31.3% of the samples, whereas only 10.7 and 15.3% were positive using serological and PCR analysis, respectively. Perfect agreement was shown between IFAT and ELISA, although there was little agreement among results of the other diagnostic techniques. Gestational age of aborted foetuses checked ranged from <3 to 9 months, with a mean of 5.9 months, and no difference in age was found between infected and non-infected foetuses (P>0.05). This study confirms the importance of N. caninum as a cause of abortion in Spain and underlines the need to use different diagnostic techniques to increase the chance to detect the infection in aborted foetuses.     

Molecular and immunodiagnostic studies of bovine neosporosis in Switzerland

Cyst-forming coccidia may cause significant losses in livestock, primarily due to abortion, loss of young animals and neuromuscular diseases. Rather recently, Neospora caninum has been recognized as one of the major protozoal abortion-inducing parasites in cattle. The present study addressed the performance of different diagnostic tools (in vitro-cultivation; histology; immunohistochemistry; serology; PCR) suitable for the direct or indirect detection of N. caninum. By PCR, Neospora-DNA was detected in 24 brains (29%) from 83 bovine abortion, many of these brains were simultaneously characterized by histopathological findings typical for a protozoal, cerebral parasitosis. The diagnostic methods were furthermore assessed using samples of different tissues and body fluids from three experimentally Neospora-infected pregnant cows and their foetuses. The diaplacental passage of N. caninum to the foetus was successful in two of the three cases. In these two cases, PCR was positive for different foetal organs and, additionally, for the abomasal and amniotic fluid. The successfully infected cows developed anti-Neospora serum antibodies between 10 and 17 days post infection, foetuses remained serologically negative in all cases. The results obtained in the present study demonstrated the usefulness of PCR, complemented by serology, for the specific diagnosis of bovine neosporosis. Such tests may prove suitable to perform epidemiological investigations. Taken together, our data indicated that prenatal neosporosis may be an important cause of infectious bovine abortion in Switzerland.     

Potential involvement of Neospora caninum in naturally occurring ovine abortions in New Zealand

Neospora caninum is an obligate intracellular parasite and is recognised as the leading cause of bovine abortion worldwide. Natural infection with N. caninum has been described in sheep but it has generally not been regarded as a significant cause of abortion. Recently, there have been several New Zealand cases of foetal abortions where N. caninum was detected which strongly suggested the involvement of Neospora in these abortions. However, there is minimal information about the prevalence of N. caninum infection naturally occurring in New Zealand sheep flocks and particularly its impact on reproduction success. Thus, this present study provides preliminary data on the role that Neospora is playing in ovine reproductive failure by establishing the prevalence of N. caninum antibodies and DNA in ewe blood and foetal material present in 21 New Zealand sheep farms with ongoing unexplained abortion problems and 10 farms with consistently high fertility levels. The results of this study demonstrated an overall seroprevalence of 1.4% which varied between Aborting/non-pregnant (1.8%), age-matched pregnant controls (0.6%) and high fertility (2.1%) ewes. However, despite the variation observed, there was no statistical difference between the three groups. In addition, Neospora DNA was detected by PCR in 13% of submitted foetal brains and in ewe blood from aborting/non-pregnant (6.9%), age-matched pregnant controls (3.6%) and high fertility pregnant (2.1%) ewes. When the PCR results were considered with the IFAT and IDEXX ELISA results, there was no correlation between serology positive and PCR positive blood samples. Taken together, these results reveal that reliance on ELISA-based serology or PCR alone may underestimate the involvement of Neospora. Furthermore, determining the involvement of Neospora appears to require a multi-facetted approach where diagnostic methods and serological cut-off values may need to be adjusted as further information about the effect of natural infections with N. caninum in the ovine host is elucidated.     

Application of a recombinant protein for the serological diagnosis of canine leishmaniasis

This work reports the results obtained by a new enzyme-linked immunosorbent assay (ELISA) test developed for the serological diagnosis of canine leishmaniasis.The new ELISA is based on a recombinant protein obtained by joining different antigens of Leishmania infantum.Test performances have been evaluated through the screening 227 sera of dogs, infected and uninfected by L. infantum. The new ELISA test has been compared to the indirect immunofluorescent-antibody test (IFAT) as a reference assay of canine leishmaniasis, and to a commercial ELISA.Excluding from the total number of IFAT positive sera the 27 sera with IFAT titre 1:40 (considered doubtful), the recombinant ELISA showed 97.0% specificity, 93.9% sensitivity and 95.5% agreement with IFAT. The commercial ELISA showed 78.2% specificity, 94.9% sensitivity and 86.5% agreement with IFAT.The results demonstrate a higher performance of the new recombinant ELISA test for the detection of negative samples, with a greater agreement with the reference test (IFAT).     

Neosporosis in a pup

CASE: A 13-week-old female boxer pup was found to be suffering from rigidity of the left hindleg. Antibiotic and anti-inflammatory treatment over a 3-week period failed to improve the condition and the pup was humanely killed. METHODS: Serological examination for Neospora antibodies was carried out by the indirect fluorescent antibody test and for Toxoplasma gondii antibodies with a latex agglutination test. A variety of tissues were examined histologically, and the central nervous system by immunohistochemistry and the polymerase chain reaction. RESULTS: The IFAT for anti-Neospora antibodies showed a titre of 1:51 200 in the clinically affected pup while the latex agglutination test for Toxoplasma antibodies was negative. The dam and one of two tested litter-mates had anti-Neospora IFAT titres of 1:1600, the other litter mate was negative. All three were not clinically affected. Histological, immunohistochemical and polymerase chain reaction examinations of the affected pup confirmed the diagnosis of Neospora infection. CONCLUSION: In the live animal, serological examination is thought to be the most useful specific test. Post-mortem examination by traditional histology, immunohistochemistry and the polymerase chain reaction confirmed the diagnosis. The case is discussed in the context of present knowledge about Neospora infection in New Zealand.     

Evaluation of an in-house TgSAG1 (P30) IgG ELISA for diagnosis of naturally acquired Toxoplasma gondii infection in pigs

Toxoplasma gondii is an apicomplexan protozoan parasite which is able to infect a large variety of warm-blooded animals. Raw or undercooked pork has been regarded as an important source of infection for humans. The aim of this study was to evaluate an in-house enzyme-linked immunosorbent assay to diagnose natural T. gondii infection in swine using native affinity chromatography-purified T. gondii surface protein-1 (TgSAG1-ELISA) as antigen, comparing its performance to that of indirect fluorescent antibody test (IFAT) and immunoblotting (IB). To obtain a panel of sera showing the evolution of the antibody response in the time course 12 pigs were experimentally inoculated intravenously (iv) with tachyzoites of the T. gondii strains RH (clonal type I), ME49 (clonal type II) and NED (clonal type III) and serologically monitored for a period of 11 weeks. Both IFAT and ELISA showed a similar time course of antibody response to T. gondii; but by IFAT this response was characterized by rapidly rising titers with peaks at two weeks post inoculation (wpi), while the ELISA indices increased slowly and reached a maximum in most animals at five wpi. Three-hundred randomly selected sera from a total of 602 pigs of different ages derived from outdoor and indoor farms from Argentina were analyzed. Serum samples testing either positive or negative by both IFAT and IB were considered as "relative standards of comparison" (RSC). Sensitivity and specificity of TgSAG1-ELISA were obtained by a Receiver Operating Characteristics (ROC) analysis and statistical agreement among serological tests was evaluated. Antibodies to T. gondii were detected in 160 of 300 sera (53.3%) by IB, in 133 of 300 (44.3%) by IFAT and in 123 of 300 sera (41%) by TgSAG1-ELISA. One hundred and eleven sera tested positive and 118 sera tested negative by both IFAT and IB (RSC); 103 of 111 positive RSC sera tested positive by TgSAG1-ELISA, and 116 of 118 negative RSC sera tested negative by TgSAG1-ELISA. Agreement observed between RSC and TgSAG1-ELISA was almost perfect (κ=0.9124, p≥0.05) and between IFAT and IB was moderate (κ=0.53, p≥0.05). Relative sensitivity and specificity of the TgSAG1-ELISA using a cut-off index of 0.204 were of 92.8% and 98.3%, respectively. ROC analysis revealed that TgSAG1-ELISA was highly accurate (AUC=0.983) relative to the RSC. According to the results in this study, the ELISA based on affinity purified T. gondii surface antigen TgSAG1 was useful for the specific and sensitive detection of antibodies to this protozoan parasite in naturally infected pigs.     

Single tube nested PCR for the detection of Toxoplasma gondii in fetal tissues from naturally aborted ewes.

A single tube nested polymerase chain reaction (PCR) assay targeting the multicopy 18S-5.8S rRNA internal transcribed spacer (ITS1) region has been developed for the diagnosis of Toxoplasma gondii-induced abortion in ovine fetal tissues. In all, 145 ovine fetal samples including brain, spleen, lung, liver, kidney, placenta and fetal fluids from 53 fetuses and stillborns of 32 farms in Northern Spain were analyzed. Thirty-six samples belonging to nine fetuses and one stillborn lamb were T. gondii PCR-positive. Although T. gondii DNA was amplified from different types of tissues, brain was the tissue with the highest detection rate. All animals that had histopathological lesions associated to T. gondii infection were positive by PCR. In addition, four fetuses whose histological examination was hindered by autolysis were PCR-positive. Results obtained by PCR and indirect fluorescent antibody test (IFAT) showed good correspondence, demonstrating the diagnostic value of the two techniques. However, PCR has the advantage over serology in its ability to diagnose T. gondii infection at earlier stages of gestation when the fetus is not yet immunocompetent and in lambs that have taken colostrum. Once other abortifacient agents are ruled out, PCR detection of the ITS1 region in fetal tissues is a valuable and relatively rapid technique for the diagnosis of ovine abortion caused by T. gondii.     

The use of polymerase chain reaction assay to diagnose proliferative gill disease in channel catfish (Ictalurus punctatus).

To assess the potential of a polymerase chain reaction (PCR) assay as a diagnostic tool in the detection of proliferative gill disease (PGD) in channel catfish (Ictalurus punctatus), PCR assays were compared with the traditional diagnostic methods of gill wet mounts and histology. A PCR assay using primers for Aurantiactinomyxon ictaluri, the actinospore associated with PGD, was performed with tissues from fish from commercial ponds. Using histology as the "gold standard," the sensitivity, specificity, and accuracy of the PCR assay were all >90%. In comparison, the wet mount examinations had a lower sensitivity and specificity. Using the chi-square test and a test for strength of association, there was a significant, strong association between results obtained by PCR and those obtained by the other 2 methods. These results demonstrate that the PCR assay is a good diagnostic tool for the detection of PGD.     

The sensitivity of PCR and serology in different Theileria parva epidemiological situations in Rwanda

Theileria parva is the causative agent of a lethal tick-borne disease of cattle occurring in eastern, central and southern Africa. Variations in the sensitivity of the serological and molecular tests with seasonal vector occurrence and discrepancies between low PCR prevalence and high T. parva vector density are a setback to estimate true prevalences. Therefore, the objectives of the present studies were to evaluate (1) the sensitivity of three serological tests (IFAT, ELISA and SELISA) and one molecular test (PCR) in the diagnosis of chronic T. parva infections in four different agro-ecological zones of Rwanda and (2) the effect of tick challenge and animal's age on the sensitivity of PCR. Blood samples from 635 bovines were collected in four agro-ecological zones of Rwanda. All sera were screened using the IFAT, ELISA, SELISA and PCR. The binary results of the four diagnostic tests were introduced separately for each agro-ecological zone in a Bayesian model to estimate the prevalence of T. parva infections and the sensitivity of the four diagnostic tests. All test specificities were set to 100%. The estimated T. parva prevalence was much higher (83–85%) than estimations based on single diagnostic tests. The estimated sensitivity of serological tests was relatively constant and ranged from 57 to 75% in the various areas. The sensitivity of PCR showed more pronounced variations, ranging from 66% in the low T. parva transmission (high land) zones compared to 24% in the highly vector infested (low land) zones. Calves and adult cattle (n = 194) were also sampled in regularly and irregularly dipped herds in the low land region. The apparent T. parva prevalence detected by PCR was significantly higher in calves than in adult cattle and in herds regularly treated with acaricides, while no significant differences were found with IFAT. The conditional probability that a sample was positive at PCR while it was positive at IFAT was significantly lower in adults. The implication of these findings in the use of diagnostic assays for epidemiological studies is discussed.     

Evaluation of four serological techniques to determine the seroprevalence of Neospora caninum in foxes (Vulpes vulpes) and coyotes (Canis latrans) on Prince Edward Island, Canada

The objectives of this study were (1) to evaluate the performance and agreement of serological assays (ELISA, IFAT, Neospora caninum agglutination test and immunoblot) using reference sera and field sera from foxes and coyotes and (2) to estimate the N. caninum seroprevalence in foxes and coyotes on Prince Edward Island, Canada. With fox and coyote reference sera the test performance of the ELISA, IFAT and IB was excellent (100% sensitivity and specificity). NAT showed a low sensitivity (50%). Serum was collected from 201 coyotes and 271 foxes. The seroprevalence observed in the different assays ranged from 0.5 to 14.0% in coyotes and 1.1 to 34.8% in foxes. The seroprevalence, when taking more than one test positive as cut-off value was 3.3 and 1.1% for coyotes and foxes, respectively. From the N. caninum-positive group, all coyotes were older than 3 years. Agreement among assays (measured as prevalence-adjusted bias-adjusted kappa) using the field sera ranged from 0.17 to 0.97. Best agreement was observed between ELISA and IFAT, poor agreement was observed between NAT and the other assays. Positive agreement was moderate to poor among all assays utilized in this study. Although the seroprevalence observed was low, N. caninum antibodies are present in foxes and coyotes on Prince Edward Island (PEI) and their role in the N. caninum epidemiology needs further study.     

A field evaluation of an indirect immunofluorescent antibody test developed to diagnose plasmacytoid leukemia in chinook salmon (Oncorhynchus tshawytscha).

An immunofluorescent antibody test (IFAT) developed for the diagnosis for plasmacytoid leukemia was evaluated against histology under field conditions. Previously published results from a laboratory evaluation indicated that the IFAT had a much higher sensitivity than did histology. One hundred seventy-seven moribund chinook salmon from 3 farms located in British Columbia were sampled. Sensitivity, specificity and their respective quality indices were estimated for the IFAT relative to histology. The IFAT was shown to be unreliable, particularly with respect to sensitivity. Cohen's kappa was also calculated and revealed that the agreement between the 2 tests was no better than random. In contrast to previously published results the IFAT did not perform better than histology in the presence of bacterial kidney disease. The results emphasize the importance of evaluating tests in the field conditions in which they are to be used. The possible reasons for the shortcomings of the IFAT are discussed.     

Detection of Neospora caninum in dog organs using real time PCR systems

Neospora caninum is a parasite responsible for paresis in dogs. The dog can harbour enkysted parasites in several organs. The detection of N. caninum was performed using 3 different real time PCR systems all amplifying the NC5 DNA region. One system was based on Sybrgreen, one on Plexor technology and the last on Taqman probe. Comparison of the three methods indicated that the detection limit was 1 equivalent genome on pure DNA but that this detection limit increased in the presence of foreign DNA using the Sybrgreen and Plexor systems. Therefore, the Taqman system was chosen to detect N. caninum in liver and spleen of naturally infected dogs. The overall prevalence was 32.2%. Comparison between PCR results and serological results using IFAT showed that among the 28 PCR positive dogs only 9 were seropositive and that 8 seropositive dogs were PCR negative. Therefore serology can underestimate the real carriage in dogs. However, PCR methods must be improved in terms of sensitivity and inhibition problems.     

Comparison of diagnostic methods to detect piroplasms in asymptomatic cattle

This study was carried out to compare different diagnostic techniques to reveal the presence of piroplasms in asymptomatic cattle kept at pasture. Nineteen blood samples were collected from animals of two different areas of Emilia Romagna Region of Italy and processed for microscopic observation, PCR, serological test (IFAT) for Babesia bovis and Babesia bigemina antibodies and in vitro cultivation. The cultures were performed on both bovine and ovine erythrocytes. Seventeen blood smears (89%) were positive for piroplasms, while PCR was positive on 18 samples (95%). DNA sequencing of 18S rRNA identified the piroplasms as Theileria spp. In vitro cultures were successful for 6 samples (32%) cultured on bovine blood and subsequent identified these as Babesia major by PCR. On IFAT analyses of 16 samples, 36.8% resulted positive for B. bovis and 31.6% positive for B. bigemina. These results show, in the same animals, the co-infection with Babesia spp. and Theileria spp.; the detection of B. major was possible only using the in vitro cultures.     

Quantitative correlation of parasitological and serological techniques for the diagnosis of Trypanosoma congolense infection in cattle

A comparison was made between serological and parasitological techniques for the diagnosis of bovine trypanosomiasis in Zambia. Overall sero-prevalence rates as determined by IFAT and ELISA were respectively 2.7-fold and 2.9-fold greater then the percentage of samples found positive with the dark ground/phase contrast buffy coat technique (DG). The results obtained by the two serological techniques were found to be closely correlated (94.2%) agreement) and titres obtained by ELISA tended to be slightly higher than those obtained by IFAT. Linear regression analysis of the results obtained by the IFAT and DG techniques revealed a highly significant correlation. This finding would permit the use of only one of the techniques in an epidemiological survey and to extrapolate the results from the regression line.     

Detection of Toxoplasma gondii antigens reactive with antibodies from serum, amniotic, and allantoic fluids from experimentally infected pregnant ewes

Toxoplasma gondii, an intracellular protozoan parasite, is one of the major causes of infectious abortion in sheep. To further understand the pathogenesis of toxoplasmosis, serum, amniotic and allantoic fluids and foetal stomach contents were collected from experimentally infected pregnant ewes to determine pathogen numbers and other markers of infection. Fifteen pregnant ewes (90 days of gestation) were each orally inoculated with 3000 sporulated oocysts of T. gondii. Serum samples were collected weekly following challenge. Amniotic and allantoic fluids and foetal stomach contents were collected at 21, 25, 28, 33 and 35 days post-infection. Characteristic placental lesions were detected in 1 of 4 challenged ewes at day 25, 3 of 4 challenged ewes at day 28 and in all challenged ewes at days 33 and 35 post-infection. T. gondii was detected only sporadically in amniotic and allantoic fluids before 35 days of infection, by real-time PCR, and only in ewes with placental lesions. At 35 days post-infection, high numbers of parasite were detected in both amniotic and allantoic fluids. An increase in the number of fluids from challenged animals with IgM and IgG was detected over time, except for IgG in allantoic fluid, which was detected in all samples from day 21 post-infection. IgG in amniotic and allantoic fluids was shown to be specific for T. gondii, and reacted with antigens with an apparent molecular mass of approximately 22 kDa and 30 kDa. Results suggest a maternal source of immunoglobulin in the allantoic fluid and a foetal source of immunoglobulin in the amniotic fluid early in infection but that both sources may contribute immunoglobulin to both fluids at a later stage.     

Serology of experimental toxoplasmosis in pregnant ewes and their foetuses

Ewes were inoculated orally with 1500 Toxoplasma gondii oocysts at 6 to 14 weeks of pregnancy. Abortions occurred at 26 to 55 days after inoculation, and maternal serum T. gondii antibody titres did not peak/plateau until 20 to 103 days after inoculation, usually after abortion occurred. Serums or body fluids from unautolysed foetuses aborted at 35 days or more after maternal inoculation, contained significant levels of Toxoplasma antibodies as determined by the indirect fluorescent-antibody test. It was considered that foetal serology was a quick and efficient method of diagnosing congenital toxoplasmosis in sheep on a flock basis.     

An interlaboratory comparison of immunohistochemistry and PCR methods for detection of Neospora caninum in bovine foetal tissues

Seven European laboratories contributed to a multi-centre evaluation of detection techniques for Neospora caninum in bovine foetuses. Six laboratories participated in immunohistochemistry (IHC) testing. All seven laboratories participated in PCR testing, but the results from one laboratory were not included in the analysis, because of contamination problems in the preparation of the samples. A coded panel of tissue sections from 36 infected and non-infected foetuses was used to evaluate the IHC detection of parasites. A coded panel consisting of 44 homogenized foetal brain samples from natural bovine abortion cases and 32 spiked samples were used to evaluate the PCR methods. Inclusion of a duplicate dilution series of spiked samples was used to evaluate detection limits and repeatability. IHC methods had a relatively low sensitivity, but a high specificity. There was considerable variation in IHC results between participating laboratories, which may be partly explained by examination practices that depended on the experience of the operator. In addition, the use of different antibody reagents, different antibody dilutions, and different enzymatic treatments of tissues may have contributed to the observed variation. PCR methods generally had a higher sensitivity than IHC methods and also a high specificity. The agreement between the majority scores of IHC and PCR methods was low. False positive PCR results indicated contamination problems in some instances. Agreement between the PCR results of the various laboratories was better, compared with the IHC results. There appeared to be no clear relationship between the PCR format (i.e. single or nested) and diagnostic sensitivity. Consequently, an improvement of diagnostic performance of PCR might possibly be achieved by optimizing DNA extraction methods.