Urban and rural risks of Lyme disease in the Scottish Highlands.

BACKGROUND: This paper investigates the pattern of Lyme disease testing and infection within the Highland region of Scotland. METHODS: Data from all Highland samples tested during 2004-2006 were analysed according to result and patient's residence in relation to the eight fold Scottish Executive's urban/rural classification, and distance from woodland. RESULTS: In total, 1602 patients were tested for Lyme disease, 0.71% of the Highland population. From these, 104 (6.5%) were seropositive. There were more patients tested, and seropositive patients from rural than urban locations, 1113 vs 489, and 79 vs 25 respectively. There were also significantly more seropositive patients per patients tested from rural locations (chi2, p<0.0001). The number of patients tested and seropositive patients increased as the rural areas become more remote. The likelihood of being tested for Lyme disease also increased as the distance between a patient's residence and woodland decreased. The relative risk of being tested elevated by 74% for those patients living within 200 metres of woodland. CONCLUSIONS: Those living in the most rural areas of Highland and those living closest to woodland have an increased risk of being tested and having Lyme disease.

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 10(5)-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.

The use of local isolates in Western blots improves serological diagnosis of Lyme disease in Scotland.

Nine Scottish Borrelia burgdorferi isolates were investigated in IgG Western blot tests. Sera previously found to be positive and negative when tested by routine Western blots prepared from reference strain B. burgdorferi sensu stricto antigen had different outcomes with these isolates. Two isolates, E5 (Borrelia afzelii) and G4 (B. burgdorferi sensu stricto) performed well, reproducing Western blot-positive results in 90 and 95% of tests, respectively. When antigens from both isolates were incorporated into a single IgG Western blot, the results of a panel of sera were improved when compared to the routine reference strain IgG Western blot. All of the sera positive by the routine Western blot remained positive using the Scottish isolate antigen mix. Twenty-three of the 25 negative sera remained negative and two produced an equivocal result. Of the 15 samples that tested IgG Western blot equivocal with the B. burgdorferi sensu stricto reference strain, 11 (73%) became weak or strong positive when tested with the B. afzelii/B. burgdorferi sensu stricto antigen mix (chi(2)=14.35, Yates' correction, P<0.001). In seven of these, a clinical picture of Lyme disease was consistent with the new results. The use of Scottish strains of B. afzelii and B. burgdorferi sensu stricto to provide antigen for the IgG Western blot improves the diagnosis of Lyme disease for patients in Scotland.

Do Toxoplasma gondii RH strain tachyzoites evolve during continuous passage?

AIM: To examine three lineages of Toxoplasma gondii RH strain in terms of performance in the dye test, culture, and gene expression. METHODS: Historical data (culture growth and performance in the dye test) from three lineages of RH strain tachyzoites (B, J, and Q) that had been continuously cultured in HeLa cells was assessed. Tachyzoite harvests obtained during continuous cell culture were retrieved from liquid nitrogen and cultured in HeLa cells, providing mRNA that was extracted and used to study gene expression using random amplified polymorphic DNA analysis at different stages of lineage adaptation to continuous culture. RESULTS: The B and Q lineages consistently produced tachyzoites that were successfully used in the dye test and their gene expression was stable after multiple passages. The J lineage had unpredictable growth, tachyzoites unsuitable for use in the dye test, and changing gene expression with multiple passage. CONCLUSION: This study has explained some anomalies in the performance of different stocks of T gondii, and suggests that lineages that are still evolving in cell culture should be avoided.

Strategies for detecting toxoplasma immunity.

A strategy for identifying toxoplasma immunity in pregnancy must provide good evidence of immunity but not falsely reassure; that for immunocompromised patients should identify immunity and also the risk of reactivated toxoplasmosis. Using sera from both of these patient groups, the performance of an in-house IgG EIA and two commonly used commercial assays (Abbott AxSYM Toxo-G and Eiken latex test) were compared with the dye test. False-positive results were obtained using the IgG enzyme immunoassay (EIA) and AxSYM Toxo-G, and false negatives using all three screen tests. During pregnancy, positive results may falsely reassure, and patients should be tested for toxoplasma-specific IgM to differentiate between current infection and immunity. In immunocompromised patients, positive results indicate immunity but negative results do not exclude it; these should be tested by dye test. Despite these reservations, we have demonstrated that immunity screening can be performed within a district general hospital.

Toxoplasma gondii from liquid nitrogen for continuous cell culture: methods to maximise efficient retrieval.

This study aims to increase the efficiency of continuous growth of Toxoplasma gondii in HeLa cells from tachyzoite stocks frozen in liquid nitrogen. Freezing and retrieval of tachyzoites for continuous cell culture requires more stringent protocols than those published for animal culture. The freezing and retrieval conditions are optimised so that a quality harvest (> or = 1 x 10(6) tachyzoites/mL, > or = 90% viability) can be produced using T. gondii recovered from liquid nitrogen as fast and reliably as possible. Retrieval success rate increased from 36% to 100%. An improved freezing procedure using chilled reagents and freshly harvested parasites, and adoption of an effective recovery protocol with retrieval of 3 x 10(7) tachyzoites into 75 cm2 flasks, change of maintenance media after six hours and subsequent blind passage all contributed to this success. The result is faster and more dependable production of T. gondii for diagnostic and experimental use.

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.

Isolation of Borrelia burgdorferi from ticks in the Highlands of Scotland.

Borrelia burgdorferi, the causative agent of Lyme disease, was first isolated in 1982 and since then has been regularly isolated from ticks and clinical material in both Continental Europe and the USA. However, only three isolations have been reported in Britain. During the summer of 1997, 128 ticks were collected from two sites in the Highlands of Scotland and examined by the polymerase chain reaction (PCR) and culture. Eleven fresh isolates were obtained from culture and passed up to 22 times. Seven of the tick emulsions were also positive by flagellin gene PCR, and a further one was positive by PCR but negative on culture. All 11 isolate cultures were positive by the flagellin gene PCR. Further studies on four of these isolates confirmed their identity by immunofluorescence, but also detected possible differences between them and B. burgdorferi ACA-1 by enzyme profiles and by PCR with OspA gene primers. Culture of these new strains provides antigens that should improve diagnostic serological tests in Britain.