R. Evans

Testing by Sysmex UF-100 flow cytometer and with bacterial culture in a diagnostic laboratory: a comparison

A large proportion of the samples tested in routine diagnostic microbiology laboratory are urine samples. The gold standard is bacterial culture, but a high proportion of samples cultured are negative. Unnecessary testing can be reduced and an improved service provided by an effective screening test. The Sysmex UF-100 flow cytometer has been developed to count cells and casts accurately in urine samples. Its performance in a screening test was compared with bacterial culture by using 1005 consecutive urine samples, and cut-off criteria were established. Cut-off values of 3000 bacteria/μl and 111 WBC/μl provided the best discrimination. Of 1005 samples, 606 (60%) would be cultured. Sixteen samples that were not selected according to these criteria were culture positive. This was considered acceptable for our routine use. The use of a testing algorithm incorporating the Sysmex UF-100 flow cytometer has improved the quality and efficiency of urine testing within the routine microbiology laboratory.

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.

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.

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.

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.

Strategies for detecting toxoplasma immunity.

Abstract 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.

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