Identification of Anaplasma phagocytophilum and Borrelia burgdorferi sensu lato in patients with erythema migrans.

Anaplasma phagocytophilum has been first isolated from the blood of two Czech patients simultaneously with a cultivation of Borrelia burgdorferi sensu lato from their erythema migrans lesions. Cultivation of different Borrelia spp. from 12 erythema migrans biopsies, from 2 blood, one liquor and one placenta sample in BSK-H medium was successful. Adapted conventional methods targeting 16S rRNA and OspA genes for real-time polymerase chain reaction (PCR) and partial sequencing of these genes together with microscopical examinations of the blood smears provided a direct detection of the B. afzelii, B. burgdorferi, B. garinii, B. valaisiana and B. bissettii in the skin, B. garinii in the blood, placenta and liquor in 24 (36.3 %) patients, and A. phagocytophilum in 10 (15 %) patients with erythema migrans. Positive indirect IgM immunofluorescence against Anaplasma sp. was obtained in 7 cases, specific IgG antibodies were detected in 12 patients. Three women suffering from erythema migrans in the first trimester had positive PCR for Anaplasma and/or for Borrelia in the blood and two of them, later, in the placenta. Interpretation of laboratory data can bring important contribution to establishing the role of Anaplasma sp. in erythema migrans and forming the principle of precaution with laboratory diagnosis during pregnancy which always should be reflected in the resistance of Anaplasma sp. toward penicillins.

[Prevalence of Borrelia burgdorferi sensu lato species among patients in the Czech Republic; direct sequencing analysis and real-time polymerase chain reaction]. / Prevalence druhu Borrelia burgdorferi sensu lato u pacientu v Ceské republice; prímá sekvenacní analýza a polymerázová retezová reakce v reálném case.

The spread of borreliosis depends on geographical, environmental and climatic factors as well as on the pathogenesis of the causative agent of the group of Borrelia burgdorferi sensu lato. The rise in the incidence of the disease and emergence of new symptoms are of concern. Relationships between genospecies and symptoms, their geographical spread and possible interference of other pathogens are the subject of the present study. Eighty-seven patients with borreliosis from Central and Eastern Bohemia and Moravia were enrolled in the study. Forty-nine patients of group 1 showed clinical positivity, 21 patients of group 2 tested positive at PCR screening and 17 patients of group 3 were culture positive. Forty-eight patients and 17 isolated strains showed positivity for plasmids and the Borrelia burgdorferi sensu lato genome in conventional nested PCR. Borrelial genotypes and subtypes were detected by direct sequencing of OspA and OspC products. Quantitative data were determined from specific product melting temperature curves for real time PCR. Based on sequencing of the OspA gene, B. garinii (subtypes 6, 5, 4 and 3), B. burgdorferi s.s. and B. afzelii were detected in 14 (51.8%), 8 (29.6%) and 5 (18.5%) out of 27 Central Bohemian patients, respectively. Eastern Bohemian patients showed predominance of B. garinii subtype 5 and co-infection with Anaplasmaphagocytophilum in 7.6%. The predominant causative agent in 25 Moravian patients was B. afzelii (11 patients, i.e. 44%), followed by B. burgdorferi s.s. (9 patients, 36%) and B. garinii 5 patients, i.e. 20%). Sequences of two hypervariable regions of the OspA and OspC genes and distances in phylogenetic trees showed differences not only between genospecies and subtypes but also between wild strains detected by direct sequencing from patient specimens and in vitro cultured strains. The greatest differences were found for patients with long-term borrelial infection.

Study of awareness of tick-borne diseases among children and young people in the Czech Republic.

In the 2000 and 2001 years, there was performed a study which has given some insight on the knowledge of youths about ticks and tick-borne diseases. Two thousand seven hundred and sixty-three respondents from 6 to 26 years of age took part in this investigation. More than 98% of respondents knew about the existence of ticks. Almost 93% of children and 97% of adolescents reported that ticks feed on blood. Although the majority of respondents aged 10 to 26 years is convinced that ticks live on vegetation, 23% of them supposed that ticks jump on humans from trees. As many as 93.5% of youths knew that Lyme disease is transmitted by ticks. The main sources of information about Lyme disease for students and pupils older than 10 years of age are television and radio (40.9%) and the press (37.5%). The frequency of contact of young people with ticks is high--90.4% of children younger than 12 years, and 93.7% of youths from 10 to 26 years of age had at least once an attached tick. 56.1% of youths older than 10 years use oil to remove an attached tick. Almost 23.7% remove ticks with bare hands. People used those methods of tick removal although even children younger than 12 years of age knew that it was an incorrect habit. After removing, a tick is most frequently burned.

[First detection of Ehrlichiosis detected serologically and with the polymerase chain reaction in patients with borreliosis in the Czech Republic]. / První poznatky o ehrlichióze, prokázané sérologicky a polymerázovou retezovou reakcí u pacientu s borreliózou v Ceské republice.

Infectious complications caused by genus Ehrlichia were confirmed when samples taken from patients with Lyme borreliosis with non-characteristic development and those sent directly by physician who treated the patient were examined. Laboratory diagnostics of ehrlichiosis was based on hematological examination of blood, on anamnestic and epidemiological data, on indirect immunofluorescence serology verified using Western blot test with specific antigen and by determination of DNA agents using polymerase chain reaction analysis (PCR). From 37 goal-directed examinations done in years 1998-2000, bacteria Ehrlichia chaffeensis was found to be the cause of human monocytic ehrlichiosis (HME) in two patients who had stayed in Bulgaria, USA and Thailand. DNA of E. chaffeensis was confirmed by PCR with subsequent product sequentiation (determination of the order of pairs of the oligonucleotide bases). In both patients monocytosis and positive antibodies in indirect immunofluorescence were found. The agent of human granulocytic ehrlichiosis (HGE) caused by group Ehrlichia phagocytophila was determined in three serologically positive patients using PCR and Western blot test with specific antigen P44. Cocobacils were identified electronmicroscopically in intracytoplasmatic vacuoles of isolated leukocytes. Coincidence of Borrelia afzelii and Ehrlichia sp. was found in the blood of a woman patient with erythema migrans formed in the place where from the tick's nymph was removed one week earlier. Cutaneous manifestations were accompanied by subfebrilia, myalgia and gastroenteritis. The patient was positive for both agents in Western blood and PCR. DNA of Borrelia and Ehrlichia was isolated from the patient's blood and urine in the first week of illness. Further study and an increase of sensitivity of the g. Borrelia and Ehrlichia cultivation become necessary to clarify the role of ehrlichias in the disease in our conditions.

[Culture of Borrelia burgdorferi sensu lato from patients in the Czech Republic]. / Kultivace Borrelia burgdorferi sensu lato z pacientu v Ceské republice.

Spirochete of Borrelia burgdorferi sensu lato were isolated in a modified BSK medium from 26 patients of 275 investigated suffering from early and late borreliosis. The isolates were specified by phenotype and genotype analysis using monoclonal antibodies, by immunochemical methods and by including species specific 16S rRNA, OspA and OspC primers and probes in a polymerase chain reaction with hybridization. Borrelia afzelii was found in 9 patients, Borrelia garinii in 16 and B. burgdorferi sensu stricto in one instance. B. afzelii was isolated from 5 biopsies with erythema migrans. 2 with acrodermatitis chronica atrophicans and from blood of 2 patients with arthritis. B. garinii cultures were prepared from 6 cerebrospinal fluids, 3 blood samples, 1 placenta, 1 liver biopsy and 5 skin samples. Unique was B. burgdorferi detected in a bioptic sample of the heart muscle which was positive on immunohistochemical examination. Monoclonal antibodies against OspA and genotype analysis provided evidence that Borrelia garinii isolates from cerebrospinal fluid are close to serotype IV. One CSF isolate was resistant to antibiotics; the tropism of B. garinii to nervous tissue is contemplated.