Sally Mavin

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.

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.

Local Borrelia burgdorferi sensu stricto and Borrelia afzelii strains in a single mixed antigen improves Western blot sensitivity.

Aims: This study evaluates the use of local Borrelia burgdorferi sensu stricto and Borrelia afzelii strains in a single mixed antigen for in-house IgG western blots in the routine diagnostic setting by comparing it with the current in-house protocol. Methods: Sera from 233 patients from areas of Scotland with low and high prevalence for Lyme borreliosis were tested by western blots prepared from reference strain antigen (B burgdorferi sensu stricto) and mixed antigen (B burgdorferi sensu stricto and B afzelii). Results were scored using original and revised criteria, and results were compared. Results: The mixed antigen produced significantly more bands than the reference antigen. Using the original interpretation criteria the mixed antigen produced more positive results than the reference antigen (90 versus 85). When the revised criteria were applied to the mixed antigen there were 14 more patients with positive results than with the reference antigen (99 versus 85); this difference was significant. Although 22 patients were positive with the mixed antigen and revised criteria, but negative/equivocal with the reference antigen, eight patients who were positive with reference antigen remained negative with the mixed antigen. The positive predictive value of the two antigen preparations was the same (96%). The negative predictive value of the mixed antigen with revised criteria was higher than the reference antigen (96% versus 88%), but the specificity was similar (97% versus 98%). Conclusions: The mixed antigen and revised interpretation criteria have been successfully incorporated into the routine diagnostic testing service, increasing the sensitivity of the in-house IgG western blot test for Scottish patients.

Interpretation criteria in Western blot diagnosis of Lyme borreliosis.

Abstract This study reviews the Lyme borreliosis Western blot interpretation process, including what bands are classed as specific, the number of bands needed for a positive result, the role of band intensity and the use of clinical information. In 2008, 3688 patients (4223 serum samples) were tested by enzyme immunoassay (EIA), with 832 patients tested by confirmatory in-house IgG Western blot: 272 patients were Western blot-positive, 170 were weak positive, 156 were equivocal and 234 were negative. These results were assessed, and a review of interpretation criteria from both the USA and Europe was carried out. New interpretation criteria and a testing algorithm were developed. The revised criteria changed the results in 109/3688 (3%) patients and produced significantly more Western blot-positive and weak-positive patients than with the current criteria (485 vs. 442, P<0.0001). In total, 76 patients who were negative/equivocal became positive, which may have led to a change in their management. Conversely, 33 patients who were weak-positive became equivocal but their management may not have been affected. The authors believe that the revised criteria have simplified blot interpretation and improved the sensitivity and robustness of their Western blot method. Using a protocol tailored to patients that incorporates clinical characteristics means that the entire process will be easier and will aid the management of patients

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

Abstract 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 (≥ 1 x 106 tachyzoites/mL, ≥ 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 107 tachyzoites into 75cm2 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.

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.

Increased incidence of Lyme borreliosis following mild winters and during warm, humid summers

It was with great interest that we read the article by Bennet et al. [1], in which they analysed the influence of climatic features on the incidence of Lyme borreliosis during 1997– 2003 in a defined area of southern Sweden. We carried out a similar analysis in Scotland for the years 1999–2003 and published our results in 2005 [2]. The remarkable finding is that although the two study designs were significantly different, but in overlapping years, similar conclusions were drawn. Bennet et al. [1] derived the incidence of Lyme borreliosis from the cases of erythema migrans recorded in the patients’ medical records, whereas we based it on laboratory-confirmed cases. As erythema migrans (EM) has a relatively close temporal relationship with the onset of infection and tick bite, Bennet et al. [1] analysed their incidence rates at monthly intervals and expressed them as cases per 100,000 population; in this way they effectively analysed the whole study population. In our study, the temporal relationship was less tightly defined, since only a portion of our cases demonstrated early Lyme disease with EM. Also, because we were aware that the rates of samples referred to our laboratory were not comparable in different years and from different parts of the country, we measured infection rates as cases/100 samples. We divided the country into three areas with distinctly different climatic conditions and compared the infection rates. As in the Swedish study, we were able to identify that colder winters depressed the incidence of Lyme disease in the succeeding summer. This effect was most evident in 2001, in the northern and eastern regions of Scotland. The effect of excess summer rainfall was also noted. The western region, which usually has wetter summers, especially compared to the east, had a significantly lower incidence than the other two regions in all years studied. However, in 2000, a significantly drier summer coincided with a significantly higher infection rate. Like Bennet et al. [1], we suggested this peak could be attributed to increased tick exposure as a result of increased outdoor activity. However, in our country we had an additional factor supporting the contribution of human activity, which would not apply to the Swedish study. In 2001, a major foot-andmouth disease epidemic led to countryside access restrictions and depressed visitor numbers, particularly in the eastern part of the country in June and July, as reported in the Scottish Visitor Attraction Barometer for 2000 and 2001 (http://www.scotexchange.net). The resulting reduction in tick exposure, combined with the effects of the preceding cold winter on the tick population, may explain the reduced infection rate in this region. Identifying the factors that most influence the incidence of Lyme borreliosis is very difficult, so the additional evidence provided by the Swedish study in support of rational explanations is welcomed.

Potentially misleading Western blot results in Lyme disease diagnosis.

P. aeruginosa is associated with significant morbidity and mortality among CF patients. However, they also suggest that first acquisition of P. aeruginosa does not appear to cause an immediate and rapid decline in lung function, as early isolates are generally non-mucoid, antibiotic-sensitive and present at low densities. This suggests a possible window of opportunity for early intervention. To date, there have been no reports on the bacterial composition of cosmetic products. Furthermore, the survival dynamics of problem Gram-negative pathogens in cosmetics are not known. Thus, further work is required to determine the survival and persistence of Gram-negative pathogens, including P. aeruginosa and B. cepacia, in these matrices. Until such studies report, it is recommended that patients with CF avoid sharing their cosmetic products among each other, as a precautionary measure, to help eliminate the potential for cross-infection with these pathogens.