A cost-effective protocol for screening patients for methicillin-resistant Staphylococcus aureus.

The incidence of hospital-acquired infection with methicillin-resistant Staphylococcus aureus (MRSA) is rising worldwide. Rapid identification of MRSA carriers is an important step in reducing the risk of transmission to other patients. Molecular methods are increasingly popular but are technically demanding and expensive. This study assesses the modification of one of the commercially available latex agglutination tests (Mastalex-MRSA) for the identification of penicillin-binding protein 2' on known strains of MRSA as well as other organisms identified from chromogenic agar plates. A total of 3050 patients with unknown MRSA status were processed through the routine laboratory during the investigation period and 73 of these were presumptive positive following overnight incubation. Of 70 patients who could be evaluated, 32 (43.8%) specimens would be suitable for use with the kit directly from overnight incubation on chromogenic agar, and the other 38 (52.1%) would be suitable following four hours' incubation on blood agar. The cost of one positive MRSA test with the inclusion of this test is Euro 15.15 compared with published reports of Euro 35.00 for a commercial polymerase chain reaction (PCR) test. This protocol would allow the reporting of presumptive positive MRSA results approximately 24 hours earlier than currently achieved.

Interpretation criteria in Western blot diagnosis of Lyme borreliosis.

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.

Is Tayside becoming a Scottish hotspot for Lyme borreliosis?

The epidemiology of Lyme borreliosis (LB) in Tayside was studied and compared with Highland (an area of high endemicity) and the rest of Scotland. From April 2001 to March 2008 the incidence of LB in Tayside rose from an estimated 2.57 to 5.84 per 100,000 population. In 2008/09 the incidence of LB in Tayside increased further to an estimated 13.85 per 100,000 population. This rise was significant and, although numerically less than that in Highland (37.24 to 49.69 per 100,000 population), it was proportionally much larger (137% vs 33%) and confirmed that LB in Tayside has diverged from that in non-endemic Scottish regions. The dramatic rise of LB in Tayside cannot be accounted for by changes in laboratory protocol or changes in the number or demographics of patients tested. However, changes in climatic conditions and alterations in clinical presentations may have contributed to this significant rise.

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.

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.

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.

Chlamydia infection among pregnant women and those seeking termination.

A total of 60 women tested positive for chlamydia during the study period. Of these, 75% (n = 45) were pregnant and were divided into those seeking termination (38%) and the antenatal group (62%). Of all pregnant chlamydia-positive women, 35% were teenagers. Most were aged <25 years, but if all test-positive women up to 30 years were included, 100% infections among the termination group and 92% infection among the antenatal group were detected. Of all chlamydia-positive pregnant women, 69% were nulliparous and 15.6% had multiple sexual partners. While 28.6% of the women in the antenatal group were married, in the termination group, none were married (p = 0.02). All women in the termination group were asymptomatic, while 75% of the antenatal chlamydia-positive women were emergency admissions (p < 0.0001). To conclude, opportunistic screening should be offered to pregnant women aged 25 - 29 years, to enhance detection. Testing strategies should focus on testing pregnant women of <19 years.

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.

Lyme disease--what is the cost for Scotland?

This paper analyses likely Lyme disease costs incurred by patients tested in the authors' laboratory over an 18 month period, based on patient histories and test results relating to 2110 samples submitted from laboratories serving 59% of the Scottish population. Cost analysis takes account of the direct costs of consultation, laboratory tests, antibiotic treatment and management of any sequelae, as well as indirect costs of the loss of healthy time through illness. Standard costs for each element are derived from published information, and the proportions applied to each patient category are estimated from studies described elsewhere in the literature. Of the sample, 295 patients had evidence of early Lyme disease and 31 had late Lyme disease symptoms. Based on these figures, the total annual cost for Lyme disease, when projected to the whole of Scotland, is estimated to be significant at 331,000 Pounds (range 47,000-615,000 Pounds). The range is inevitably wide because it was not possible to document complete clinical and management histories on individual patients. In addition, some late Lyme disease sequelae will require management for more than 1 year, and costs are also identified that could justifiably be included for all the other patients who tested negative for Lyme disease. These data raise the question of whether there is sufficient focus on prevention and the best management of this disease.

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.

Morphoea and Borrelia burgdorferi: results from the Scottish Highlands in the context of the world literature.

AIMS: Previous studies investigating the link between infection with Borrelia burgdorferi and morphoea have produced conflicting results. Often, these studies have been undertaken in patients from different regions or countries, and using methods of varying sensitivity for detecting Borrelia burgdorferi infection. This study aimed to establish whether a relation could be demonstrated in the Highlands of Scotland, an area with endemic Lyme disease, with the use of a sensitive method for detecting the organism. METHODS: The study was performed on biopsies of lesional skin taken from 16 patients from the Highlands of Scotland with typical clinical features of morphoea. After histological confirmation of the diagnosis, a nested polymerase chain reaction (PCR) using primers to a unique conserved region of the Borrelia burgdorferi flagellin gene was performed on DNA extracts from each biopsy. A literature search was also performed for comparable studies. RESULTS: None of the 16 patients had documented clinical evidence of previous infection with B burgdorferi. DNA was successfully extracted from 14 of the 16 cases but all of these were negative using PCR for B burgdorferi specific DNA, despite successful amplification of appropriate positive controls in every test. The results were compared with those of other documented studies. CONCLUSIONS: Examination of the literature suggests that there is a strong geographical relation between B burgdorferi and morphoea. These results, in which no such association was found, indicate that morphoea may not be associated with the subspecies of B burgdorferi found in the Highlands of Scotland.

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.

Success in the toxoplasma dye test.

OBJECTIVES: To compare the success rate of the toxoplasma dye test using different accessory factors (human serum as a source of complement) and different batches of tachyzoites produced in vivo and in vitro. METHODS: Twenty-five accessory factors were used in the dye test to assess both types of tachyzoite. Batches of tachyzoites were produced in vivo (n = 49) and in vitro (n = 23) and their performance assessed against panels of accessory factors. Performance was recorded as success or failure (incorrect results, total killing or no killing). RESULTS: With in vivo tachyzoites 21/25 accessory factors were successful in P > or = 1 dye test runs, whereas with in vitro tachyzoites all 25 were successful. One or more failure was recorded for 19/25 and 12/25 accessory factors using in vivo and in vitro tachyzoites, respectively (P < 0.05). The number of successful dye test runs was less for in vivo (92/141, 65%) than in vitro (140/163, 86%) tachyzoites (P < 0.001). This was due to a higher success rate in citrated accessory factors used for in vitro tachyzoites compared to the corresponding uncitrated accessory factors used for in vivo tachyzoites (P < 0.001). Success in the dye test was recorded for 48/49 and 23/23 batches of in vivo and in vitro tachyzoites, respectively. The number of successful dye test runs was lower with in vivo (156/234, 67%) than in vitro (116/142, 82%) tachyzoites (P < 0.01). CONCLUSIONS: Success in the dye test may be due to the accessory factor, tachyzoites, or a combination of both. Problems due to the accessory factor can be minimized by careful quality control or use of modification procedures. Tachyzoites produced in vitro may also increase success in the dye test. Careful selection of accessory factor/tachyzoite combination makes it possible to use the dye test in a district general hospital.