J. M. W. Chatterton

Simultaneous Diagnosis of Toxoplasmosis in Goats and Goatowner's Family

Four out of 6 goats from a small British goat herd gave birth to weak or stillborn kids. All were seropositive for Toxoplasma gondii. The parasite was isolated from the tissues of one kid and the milk of one goat. Concurrently, one of the goatowners sons developed a mononucleosis-like illness with serological evidence of current toxoplasma infection. Investigation of the family showed past infection in the other son, but both parents were seronegative. The source of infection for both boys appeared to be the consumption of unpasteurized goats milk.

Cell-culture system for continuous production of Toxoplasma gondii tachyzoites

Abstract The aim of this study was to identify a sustainable cell line and culture method that could continuously provide a sufficient quantity of Toxoplasma gondii tachyzoites to serve the needs of a general hospital laboratory. Three continuous cell lines (HeLa, LLC and Vero) and three cell-culture methods (culture in conventional flasks, culture in membrane-based flasks and an automated culture system) were investigated. In multiplicity-of-infection and time-course experiments, HeLa was the cell line of choice. Harvests from HeLa cells had significantly higher tachyzoite yields than those from LLC cells (P<0.00005) or Vero cells (P<0.05). Membrane-based flasks gave higher yields (6.15×106 tachyzoites/ml) than conventional flasks (1–2×106 tachyzoites/ml) initially, but these were not sustained. The automated cell-culture system was unsuitable for parasite culture. Continuous passage in 25 cm2 flasks was successful, yielding 1×106 tachyzoites/ml; viability exceeded 90% after 96–120 h of infection throughout 38 passes, during which time the viability improved and the time to harvest became more consistent. Toxoplasma gondii grown in continuous culture in HeLa cells can provide a regular supply of viable tachyzoites. Demonstration that HeLa-derived tachyzoites could be used for the dye test confirms the potential of this in vitro system for use in general hospital laboratories.

Toxoplasma polymerase chain reaction on experimental blood samples

A two-stage polymerase chain reaction (PCR) assay employing oligonucleotide primers from the B1 gene of Toxoplasma gondii was developed and assessed for sensitivity and specificity. It was able to detect T. gondii DNA from as little as one parasite/sample in mock-infected rat or mouse leucocyte preparations. Parasitaemia was also identified in animals at five stages between 16 and 66 h after infection with the virulent RH strain, and at 12 stages between 2 and 38 days after infection with the cyst-forming Beverley strain. In the latter case, PCR was more sensitive than animal culture. No cross-reactions were observed in samples containing various opportunist pathogens which may also be found in the blood of immunocompromised patients.

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.

The use of an IgM immunosorbent agglutination assay to diagnose congenital toxoplasmosis

An IgM immunosorbent agglutination assay (ISAGA) was compared with a standard ELISA IgM test for the diagnosis of congenital toxoplasmosis. It was more sensitive, detecting all of five mothers of infected babies whereas the IgM ELISA was positive in two of three mothers tested at delivery and neither of two mothers referred 10-12 months after delivery. Five women infected in a previous pregnancy had IgM detectable by ISAGA in a subsequent pregnancy. The assays were comparable when sera from patients with past infection were tested or following toxoplasma-associated miscarriage or abortion. Four cord sera from congenitally-infected babies were positive by the ISAGA but only three of these were positive by ELISA for IgM. The ISAGA also detected IgM in another four sera from congenitally-infected babies referred late (10-18 months old); none were IgM positive by ELISA. The increased sensitivity of the ISAGA is an improvement in the diagnosis of congenital toxoplasmosis.

Congenital toxoplasmosis: to screen or not to screen?

We have reviewed the present day quantifiable cost to society of the 73 cases of congenital toxoplasmosis which are estimated to occur annually in Scotland with the cost of preventing the disease by screening and treatment. Our analysis includes advances in laboratory techniques. The cost of screening would depend on its scale and if in-house or commercial tests are used. If only 2 specimens were screened, at booking and at delivery, the screening costs are estimated to be between 0.5-0.9 times the preventable costs. If a third specimen were tested in the second trimester, to maximise scope for remedial action during pregnancy, the screening costs are 0.7-1.2 times preventable costs. As likely screening costs in most of the schemes we consider are now less than the preventable costs, a screening programme should be adopted.

Simultaneous serological screening for congenital cytomegalovirus and toxoplasma infection

Sera from mother and baby matched by computer, were examined for evidence of infection with toxoplasma and cytomegalovirus (CMV) in a serological screening programme, based on simultaneous enzyme-linked immunosorbent assays of immunoglobulin G (IgG-ELISA) from a single master dilution of serum. There was evidence of primary CMV infection in 23 of 4717 pregnancies (13 by seroconversion in the last two trimesters: 10 by detection of specific IgM at antenatal booking) and primary toxoplasma infection in 10 of 4548 pregnancies (4 by seroconversion; 6 by detection of specific IgM). Detection of seroconversion depended on matching antenatal booking and cord specimen data. The best matching efficiency that could be achieved was 83%. The effectiveness of this approach to identifying neonates at risk of developmental abnormalities is discussed.

Comparison of relative uses of commercial assays for Toxoplasma gondii IgM antibodies.

AIMS: To compare the sensitivity and user friendliness of seven commercially available enzyme linked immunoabsorbent assay (ELISA) kits for toxoplasma specific IgM. METHODS: Five antibody capture assays supplied by Abbott, Mercia, Northumbria, Organon and Sorin, and two indirect ELISA assays from Biostat and Mast, were assessed. Using defined dilutions of Toxoplasma gondii specific IgM, the performance and sensitivity of each assay was established. They were further assessed on a panel of 27 sera with a range of dye test and IgM results (as determined by the Scottish Toxoplasma Reference Laboratory). All of the assays were performed by three experienced operators and assessed for user satisfaction. RESULTS: The Mast, Organon, and Abbott assays were of low sensitivity; the Mercia and Northumbria were of high sensitivity; and the Biostat and Sorin assays produced too many false positive results. The Mercia kit provided most user satisfaction; the Mast and Abbott assays were most difficult to use. CONCLUSIONS: Local laboratories investigating toxoplasma infection should have three tests: one IgG and two IgM (high and low sensitivity) to help in the timing of infection. Alternatively, one sensitive IgM assay, such as that of Mercia, could be used by selecting appropriate high and low thresholds.

Biotin-labelled antigen screening test for toxoplasma IgM antibody.

A method for the simple preparation of biotin-labelled toxoplasma antigen was used with avidin peroxidase in an IgM-capture enzyme linked immunosorbent assay (BAM-ELISA). Although the overall predictive value of a positive result was only 38%, its low cost and 100% sensitivity makes it a very suitable screening test. Positive results can be confirmed by an alternative assay, thus providing a more economical and effective diagnostic service than either screening all sera by a commercial test or selecting sera for IgM testing.

Development of real time PCR to detect Toxoplasma gondii and Borrelia burgdorferi infections in postal samples

Background: Most of the samples sent to reference laboratories are delivered by post. Thus, diagnostic PCR tests on blood samples have to be performed using methods which are optimised and validated for such conditions. There is a low probability that the organisms Toxoplasma gondii and Borrelia burgdorferi will be present. Aim: To confirm that robotic extraction methods followed by real time PCR will detect as little as one organism/test sample in postal specimens. Methods: Human blood samples spiked with decreasing numbers of each organism (range 105–1/per extract) were extracted using two commercial kits on a Qiagen BioRobot EZ1 Workstation. Extracts of whole blood and blood fractions were tested by real time PCR. The effect of storage of blood for 1–6 days at room temperature was also investigated. Results: Maximum sensitivity (1 organism/test sample) was achieved for T gondii with either extraction method; the sensitivity for B burgdorferi was between 1 and 10 organisms/test. Whole blood was the most suitable sample to extract, as both organisms were as likely to be detectable in the red cell as the white cell fraction. Sensitivity was not reduced by storing spiked samples at room temperature for up to 6 days. Inhibitory effects on PCR were not a significant problem provided that samples were extracted using the blood extraction kit. Conclusions: Using appropriate robotic extraction methods, both T gondii and B burgdorferi can be detected by real time PCR with near maximum possible sensitivity in whole blood samples. Blood samples can be transferred to reference laboratories by post without loss of sensitivity over the likely transit period.