To improve understanding of the aetiology and epidemiology of human cryptosporidiosis, over 8,000 Cryptosporidium isolates were submitted for typing to the species level over a four year period. The majority were either Cryptosporidium parvum (45.9%) or Cryptosporidium hominis (49.2%). Dual infection occurred in 40 (0.5%) cases and six other known Cryptosporidium species or genotypes were found in 67 (0.9%) cases. These were Cryptosporidium meleagridis, Cryptosporidium felis, Cryptosporidium canis, and the Cryptosporidium cervine, horse and skunk genotypes. The remaining 3.5% were not typable. Epidemiology differed between infecting species. C. parvum cases were younger, although C. hominis was more prevalent in infants under one year and in females aged 15 to 44 years. Spring peaks in cases reported to national surveillance were due to C. parvum, while C. hominis was more prevalent during the late summer and early autumn as well as in patients reporting recent foreign travel. Temporal and geographical differences were observed and a decline in C. parvum cases persisted from 2001. Typing of isolates allowed outbreaks to be more clearly delineated, and demonstrated anthroponotic spread of C. parvum as well as C. hominis. Our findings suggest that national surveillance for Cryptosporidium should be conducted at the species level.

Recent work has confirmed our earlier assertion that cases of human toxoplasmosis can involve more than one parasite type. This possibility might not have been previously observed because parasite samples studied by earlier workers had been subjected to extended passage through mice. We thus agree with the suggestion that future work examining the relationship between Toxoplasma strain type and human disease should be confined to the analysis of primary clinical material.

We sought to determine the SAG2 genotypes of Toxoplasma gondii associated with cases of acute human toxoplasmosis in England and Wales. The samples examined were collected from a wide range of cases including congenital infections, AIDS and immunosuppressed patients and were derived from a number of different tissues. Parasite DNA was detected by PCR amplification without the need for prior template purification, and SAG2 genotype was determined by both restriction enzyme analysis and direct DNA sequencing of the PCR amplification products. Parasites of both SAG2 type I and type II genotypes were seen with approximately equal frequency amongst the samples examined. Neither of these genotypes was found to be more frequently associated with a particular clinical presentation or sample tissue. Unexpectedly, we found clear evidence of mixed (SAG2 type I+type II) infections in approximately the same number of samples as were seen to be associated with either type I or II alone. Our use of direct DNA sequencing rather than simple restriction analysis was essential for the detection of mixed infections since incomplete restriction digestion of samples containing a single parasite type was occasionally observed. It is possible that the presence of more than one type of parasite in single samples might be related to our recent demonstration that mixtures of SAG2 type I and type II parasites are present in a significant proportion of commercial meat preparations. Moreover, the presence of mixed infections in single patients might offer a direct molecular method of assessing risk factors for infection.

Immunofluorescence (IFA) and polymerase chain reaction (PCR) were examined as methods for detecting Borrelia burgdorferi sensu lato spirochaetes in unfed Ixodes ricinus nymphs. Although similar results were produced in some cases, a great deal of variation occurred. Furthermore, in both the highly controlled initial laboratory study, involving 252 shared samples, and the study on field-collected ticks (n = 460), the IFA tended to detect more infected ticks than the PCR. The basis for these findings are as yet undetermined. The development of a quality assurance scheme is recommended so that laboratories can validate their methods and a preliminary feasibility study suggested that such a scheme is practical.

At a series of meetings, involving 27 clinicians from 11 countries, case definitions for the diagnosis of Lyme borreliosis in Europe were agreed and are presented here, with appropriate serological criteria, as a diagnostic guide. In a separate study questionnaires directed to clinicians were used to collect information on clinical aspects and risk factors of Lyme borreliosis. Data on the number of Lyme borreliosis patients seen by physicians indicated a low prevalence of the disease in western Europe and a relatively high prevalence in eastern Europe. The most commonly encountered symptom was erythema migrans, followed by neurological manifestations. Cardiac problems were rare. Tick bite was strongly associated with Lyme borreliosis, but the only other significantly associated risk factor was the pastime of gardening.

This web site (URL http://www.dis.strath.ac.uk/vie/LymeEU/) provides information on Lyme borreliosis for physicians, scientists, health care workers, veterinarians and students. It consists of a review of the spirochaetes, vectors, reservoir hosts, diagnosis, treatment, epidemiology and prevention of the disease, as well as an account of the activities of EUCALB.

Serological testing for Lyme borreliosis was compared in 5 European reference laboratories with a total of 79 sera in order to determine variations in laboratory performance. A considerable range of methods were used and several laboratories employed 2 or 3 genomospecies of Borrelia burgdorferi sensu lato. No laboratory relied routinely on a single test and each weighted the significance of the findings of the various tests differently. A difference in strategy between laboratories in high and low prevalence areas was apparent in that laboratories in low prevalence areas emphasised specificity more than sensitivity and therefore produced fewer false positives, but also missed some cases. Overall agreement between the laboratories was poor and it was concluded that there is a need for a quality assurance scheme within Europe.

Characterisation at the species level of 142 Borrelia isolates obtained from ticks, humans and rodents in Western Europe was carried out and their geographical distribution was described. Borrelia garinii was the predominant species representing 44% of the isolates and B. afzelii and B. burgdorferi sensu stricto constituted 27% and 19% of isolates respectively. B. valaisiana, (formerly group VS116) constituted 10.5% of isolates. Some differences in the Borrelia species distribution were observed from one country to another, possibly linked to different sources of samples. In the human samples, which were mostly collected in Austria, B. afzelii was preferentially isolated from skin and B. garinii from CSE B. afzelii was consistently isolated from rodents captured in Switzerland, but one isolate of B. garinii was obtained from a rodent in Austria. B. garinii was by far the most abundant species isolated from Ixodes ricinus ticks in all studied countries. B. valaisiana was isolated from I. ricinus ticks collected from vegetation and from I. ricinus engorged on birds.

To investigate the accuracy of the polymerase chain reaction (PCR) method for the detection of Toxoplasma gondii in clinical specimens, aliquots of amniotic fluid to which known amounts of Toxoplasma gondii DNA had been added were tested by five European Centres, Four laboratories were able to detect DNA at levels equivalent to ten tachyzoites or less, including two that detected DNA equivalent to a single parasite, Two laboratories erroneously found one of eight negative control samples to be positive, These findings confirm that the high level of sensitivity associated with the PCR method can be readily achieved under routine laboratory conditions, but they also underscore the potential for both false-positive and false-negative findings to occur, Furthermore, the results confirm the urgent need for an external quality assurance scheme to support laboratories employing PCR in a clinical context for the detection of Toxoplasma gondii.

Blood samples from 54 patients presenting with acute toxoplasmic lymphadenopathy were tested for the presence of Toxoplasma gondii DNA using a nested polymerase chain reaction (PCR). PCR test results of a single blood sample obtained 2-23 weeks after onset of illness were positive for 19 (35%) of the 54 patients. Nine (53%) of 17 patients were positive by PCR when the initial blood sample was collected within the first 5 weeks of illness. In 7 of the 19 patients found positive, further blood samples were available, and subsequent clearance of T. gondii DNA from the blood was demonstrated. On the basis of positive findings among patients with acute toxoplasmosis and the absence of positive findings among 10 uninfected persons and 43 with past Toxoplasma infection, a positive PCR result appears to be a helpful indicator of active disease. However, since only 53% of patients with lymphadenopathy persisting < or = 5 weeks were positive, a negative PCR result does not exclude recent infection.

Gene amplification by the polymerase chain reaction (PCR) is useful in ophthalmic diagnosis since it enables exceedingly small amounts of nucleic acid to be detected in ocular samples. Studies have indicated a potential for the use of PCR on ocular specimens to assist the clinical diagnosis of ocular toxoplasmosis.' We describe a case in which the technique was used in the diagnosis of acquired ocular toxoplasmosis in an immunocompetent adult.

We report the case of an apparently immunocompetent woman whose symptoms and signs have persisted for 8 years following a serologically and histologically confirmed diagnosis of toxoplasmosis. During this period she had two successful pregnancies despite persistently increased anti-toxoplasma IgM antibodies. Neither child is infected.

Borrelia burgdorferi, the causative agent of Lyme disease, was detected in patients' serum by DNA amplification using the polymerase chain reaction (PCR). B burgdorferi was pelleted from serum samples by centrifugation (10,000 x g for 10 minutes) and lysed by treatment with ammonium hydroxide (100 degrees C for 15 minutes). Two pairs of "nested" PCR primers complementary to the gene encoding a major outer surface protein (OSP A) of B burgdorferi were used in DNA amplification under standard PCR conditions (Perkin-Elmer Cetus). Two out of five patients with erythema migrans, the characteristic primary skin lesion associated with early Lyme disease, were positive by the PCR. This method could form the basis of a useful routine laboratory test in those cases of early Lyme disease where conventional serological testing commonly yields equivocal or false negative results.

41 forestry workers, who had a high occupational risk of tick-bites, were screened for antibodies to Borrelia burgdorferi by ELISA and western blotting techniques, and questioned about possible symptoms of Lyme disease. Antibodies were detected in 10 of the 40 men who had been bitten by ticks. Definite symptoms of Lyme disease, in the form of erythema migrans, were reported by only 2 workers and none had a history of neurological illness.