Changes in Toxoplasma diagnosis.

The serological laboratory workload in detecting toxoplasma infection may be expected to change with changes in the clinical profile of patient populations. We have examined the clinical information and laboratory results for patients referred to the Scottish Toxoplasma Reference Laboratory in April-March 1999-2000 and 2009-2010. Numbers of patient sera submitted for testing were similar (1624 and 1552) but there was a change in the clinical profile, with a significant fall in patients with symptoms of current infection (612 versus 335; P<0.0001) and a significant rise in immunocompromised patients (275 versus 531; P<0.0001). Although the percentage of patient samples with toxoplasma antibody decreased (53.9% versus 37.5%; P<0.0001), the number of positives increased with age, demonstrating the continuing risk of toxoplasma infection. More cases of current infection were identified in 2009-2010 than in 1999-2000 (48 versus 35). This increase was significant both for all females with current infection (P=0.0253) and also for those in the childbearing 20-39 years age group (P=0.0388). Our literature search did not find any published information on toxoplasma workload in the last decade. In summary, we have shown that there have been significant changes in the laboratory diagnosis of toxoplasma infection but it is as important as ever that effective diagnostic strategies are maintained.

More specific bands in the IgG western blot in sera from Scottish patients with suspected Lyme borreliosis.

AIMS: To identify further Western blot bands that may be specific in the diagnosis of Lyme borreliosis. METHODS: The Borrelia burgdorferi antibody profiles of 270 western blot positive patients and 241 western blot negative patients from 2008 were examined. RESULTS: 27 different non-specific bands were detected in both groups. Six of 27 (22%) of the non-specific bands were detected significantly more in the western blot positive patients compared to the western blot negative patients (20 kDa, p<0.0001; 28 kDa, p<0.002; 36 kDa, p<0.002; 37 kDa, p<0.007; 48 kDa, p<0.023; 56 kDa, p<0.028; two-tailed F test). CONCLUSION: Results suggest that the 20, 28 and 48 kDa bands should be regarded as specific.

Toxoplasma tachyzoites from cell culture are more appropriate in some situations.

BACKGROUND: Laboratories traditionally culture toxoplasma tachyzoites in animals for testing and experimental use. This article considers why available cell culture methods are not used more often. AIM: To compare HeLa cell culture and animal culture for production of toxoplasma tachyzoites. METHODS: In 2000 HeLa culture replaced animal culture for continuous production of toxoplasma tachyzoites in the Scottish Toxoplasma Reference Laboratory. The performance of animal culture (1994-1998) was compared with HeLa culture (2004-2008). A PubMed search was carried out for 1998 and 2008 to assess the culture methods used in laboratories. RESULTS: Animal culture was able to produce higher yields of tachyzoites (10(9) from a cotton rat peritoneal harvest compared to 10(7) from a 75 cm(2) cell culture flask) but significantly more HeLa cultures were successful (93% versus 84%; p=0.025). There was no difference in the quality of tachyzoites from animal and HeLa cultures as demonstrated by the high levels of success in the dye test. HeLa culture offered significant advantages in flexibility and control. A review of the literature showed no significant change in the method of culture used in laboratories between 1998 and 2008 (p=0.36). CONCLUSION: The availability of cell culture methods and the increasingly stringent regulations on the use of animals have not resulted in a decline in the use of animal culture. Animals are necessary for certain experiments but many studies could use cell-culture-derived parasites.

Toxoplasma gondii in vitro culture for experimentation.

The aim of this study was to develop a culture method for Toxoplasma gondii that could provide fresh viable tachyzoites as and when required. When T. gondii was continuously maintained in HeLa cell cultures at 37 degrees C, the time to harvest varied from 48 to 144 h. Tachyzoite yields of > or = 1 x 10(6)/ml and > or = 90% viable were obtained from 519/882 (58.8%) cultures and 120/155 (77.4%) harvests were successfully used in the dye test. When cultures were transferred from 37 to 25 degrees C when maximally infected (48-54-h post-infection), they could be stabilised and tachyzoites could be harvested as required, up to 168 h later. When harvested from 25 degrees C, significantly more cultures 783/811 (96.5%) produced tachyzoite yields > or = 1 x 10(6)/ml > or = 90% viable (p < 0.001). Tachyzoite quality also significantly improved and 206/224 harvests (91.9%) (p < 0.001) were successfully used in the dye test. We have demonstrated that tachyzoites can be maintained at dye test quality for at least 7 days in HeLa cultures at 25 degrees C. The system is flexible and robust and provides a means whereby tachyzoites of standard quality can be stored for use in experimental models as and when required.

Usefulness of rat-derived antigen in the serodiagnosis of Pneumocystis carinii infection.

Sera from patients with likely and possible Pneumocystis carinii pneumonia (PCP) on the basis of clinical information and laboratory investigations were tested by immunoblotting to assess the usefulness of trophozoites in the serodiagnosis of PCP. IgG antibodies to 50-60- kDa proteins were demonstrated with cyst antigen, but antibodies to additional proteins of 61, 70, 82, 95, 99 and 116 kDa were found with trophozoite antigen. These bands were not demonstrated with control sera. IgG antibody to the 116-kDa protein was found in 18 (46%) of 39 sera from patients with possible PCP compared with 5 (17%) of 30 sera from patients with likely PCP. There was no other significant difference between the two patient groups in detection of these proteins. Sera with higher indirect immunofluorescence assay (IFA) IgG titres were more likely to be immunoblot positive. Only 4 of 16 patients with likely PCP were IgG negative in the IFA; three of these were IgG immunoblot positive. In 4 of 10 patients with likely PCP and 6 of 15 patients with possible PCP, demonstration of IgM or IgA, or both, by IFA or immunoblotting provided evidence suggestive of current infection. This study confirms the usefulness of rat-derived antigen, especially trophozoite antigen, in PCP serology. The IgG IFA remains the most useful test, but IgM and IgA testing and immunoblotting can support the diagnosis.

Absorption of IgG does not enhance toxoplasma IgM and IgA immunoblotting.

Total IgG, IgM and IgA levels and toxoplasma IgG, IgM and IgA immunoblotting patterns were assayed in 10 sera before and after IgG absorption with Protein G-Sepharose 4. Removal of IgG (mean reduction 96%) was accompanied by a significant reduction in the level of IgM (mean reduction 56%) and IgA (mean reduction 53%) in nine of the 10 sera. The absorbed supernates showed fewer and weaker IgM bands in five sera, but IgA immunoblotting patterns were unaffected by absorption. There was no benefit in removing IgG in toxoplasma IgM and IgA immunoblotting.