[Serology of Lyme borreliosis and human granulocytic ehrlichiosis in 2005-2010]. / Sérologie lymeské borreliózy a humánní granulocytární ehrlichiózy v letech 2005-2010.

The subject of this study is serological screening of blood and CSF (cerebrospinal fluid) samples for the presence of borrelial and ehrlichial antibodies. A total of 165 patients suspected to be at risk of Lyme disease were tested. Indirect immunofluorescence and enzyme immunoassay were used as diagnostic methods. Ehrlichial antibodies were detected in 36 (21.8%) patients. Borrelial antibodies were found in 70 samples (42.4%). The widening range of tick-borne diseases brings about the need for more data on these zoonoses.

The role of electron microscopy in the rapid diagnosis of viral infections--review.

Electron microscopy (EM) allows fast visualization of viruses in a wide range of clinical specimens. Viruses are grouped into families based on their morphology. Viruses from various families look distinctly and these morphological variances are the basis for identification of viruses by EM. The identification to the family level is often sufficient for the clinician or recognition of an unknown infectious agent. Diagnostic EM has two advantages over enzyme-linked immunosorbent assay and nucleic acid amplification tests. After a simple and fast negative staining, EM allows fast morphological identification and differential diagnosis of infectious agents contained in the specimen without the need for special considerations and/or reagents. Nevertheless, EM has the disadvantage of being unsuitable as a screening method.

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.

Molecular and serological evidence of Borrelia burgdorferi sensu lato in wild rodents in the Czech Republic.

The aim of the present study was to determine the frequency and spatial distribution of the Borrelia species in wild rodents in the Czech Republic. In total, 293 muscle tissue samples and 106 sera from 293 wild rodents captured in North Bohemia and North-East and South Moravia were examined for the presence of Borrelia spp. and antibodies. Muscle samples were investigated with real-time polymerase chain reaction (PCR) with a recA primer set, with DNA quantification and melting curve analysis, and with restriction fragment length polymorphism (RFLP) analysis of the 5S-23S rDNA intergenic spacer. Infection with Borrelia burgdorferi sensu lato was found in 16.4% of the muscle samples. The most abundant genospecies was Borrelia afzelii (11.3%), followed by Borrelia burgdorferi sensu stricto (4.8%) and Borrelia garinii (0.7%). Borrelia infection was more frequently observed in Clethrionomys glareolus than in Apodemus spp. Sera were analyzed with an enzyme-linked immunosorbent assay (ELISA) test, yielding the total seropositivity rates of 24.5% for anti-Borrelia IgM antibodies and 25.5% for IgG antibodies. Total seroprevalence was higher in Apodemus spp. than in C. glareolus. In conclusion, our data indicate that in the Czech Republic small wild rodents can serve as hosts for B. burgdorferi s. s. as well as for B. afzelii.

Phenotypic and genotypic analysis of Borrelia spp. isolated from Ixodes ricinus ticks by using electrophoretic chips and real-time polymerase chain reaction.

The genotype of Borrelia burgdorferi sensu lato was detected in 371 out of 1244 ticks. Borrelia determination was based on partial sequencing of the 16S rRNA gene and real-time polymerase chain reactions for identification and quantitation of ospA and recA genes. Different Borrelia spp. were identified; B. garinii in 40% ticks followed by B. afzelii (36.3%), B. burgdorferi sensu stricto (12.9%), B. valaisiana (3.5%), B. lusitaniae (0.8%), B. bissettii (0.5%) and B. miyamotoi-like (0.5%). Cultivation of 30 borrelia strains in BSK-H medium, among them B. valaisiana, B. bissettii-like and B. miyamotoi-like strains was unique in Czechia. Calibrated microfluidic-based quantification showed differences in the concentration of the nucleic acids and molar mass of the outer surface proteins of different Borrelia spp. with standard sensitivity and specificity and was helpful for their identification. The outer surface protein OspA was absent in B. miyamotoi-like and the OspB protein in B. valaisiana, B. lusitaniae and in three subtypes of B. garinii.

[The information value of detection of borrelia antibodies in the healthy blood donors and in the population at risk]. / Informacní hodnota prukazu antiborreliových protilátek u zdravých dárcu krve a rizikové skupiny populace.

BACKGROUND: Geographic differences of particular species of ticks and variant strains of Borrelias impede the comparison of local and foreign experience. The authors tried to support some literary observations with their own results and designed a study aimed at determination of anti-Borrelia antibodies (AB) in the selected population samples. The goal of the study was to find and statistically evaluate: a) difference in distribution of values of AB in the sample of probands from the risk and non-risk environment (foresters in Sumava and Prague blood donors, respectively) by means of ELISA method, b) tick bite frequency in the risk and non-risk groups, c) to determine and statistically evaluate the prevalence of probands with increased levels of antiborrelial antibodies in the local study population by ELISA method and the prevalence of probands with non-normal values found by Western blot (WB) method. METHODS AND RESULTS: In the group of Prague blood donors (control group), in total 200 probands were examined; in the risk group, 71 probands from Sumava forest were examined. Blood specimens were examined in the National Reference Laboratory for Lyme Borreliosis in Prague. In all specimens, ELISA NRLB KC90 was used. Western blot assay was used for confirmation. CONCLUSIONS: On the basis of literature review from Europe and USA, it is possible to generalize the following, despite some equivocations: 1. regional differences are not significant, with the exception of high-risk territories or "occupational exposure"; 2. there is no significant difference between males and females; 3. considerable caution is necessary in comparing the control and risk groups; prerequisite for such comparison is defining the characteristics of both groups.

Significance of Borrelia infection in development of dilated cardiomypathy (a pilot study).

A heart involvement known as Lyme carditis (LC), a consequence of Lyme borreliosis (LB), is relatively rare in contrast to the involvement of skin, joints and nervous system; it accounts for < 4% of all these patients in European countries. However, the diagnosis of the disease belongs to the most difficult challenges. While various forms of AV blocks dominate in the USA as confirmed by the literature, there is a clear predominance of arrhythmias of various incidence in the Czech Republic. The authors of this article focused on the form belonging to the rarest manifestations of LC, namely dilated cardiomyopathy (DCMP). The goal was to elucidate the etiological participation of Borrelia infection in the development of DCMP, which has attracted controversial opinions so far. In total, 33 patients with DCMP were enrolled in the study, 23 males and 10 females, with mean age 57.7 years (range 24-76 years). ELISA NRLB KC 90 method was used in all blood samples for detection of Borrelia infection (BI), Western blot method was used for confirmation, followed by identification of DNA of pathogenic Borreliae using PCR method. Bioptic material was examined by electronmicroscopy with an attempt to detect Spirochaetae in myocardium. 16 patients were excluded from the study owing to the absence of signs of LB. The study group included 17 patients (3 females, 13 males) with mean age 58 years (range 43-76 years), in whom the presence of Bb was proved by identification of DNA of pathogenic Borreliae or by electronmicroscopic detection of Spirochetae in myocardial bioptic sample. The findings obtained during the study confirmed that BI very probably participated in the development of dilated cardiomyopathy. It may be concluded that most of cases were either unapparent forms of LB or insufficiently treated cutaneous forms of this disease.

Lyme borreliosis infection as a cause of dilated cardiomyopathy.

The relatively low percent of patients affected with the cardiac form of Lyme borreliosis is difficult to diagnose, especially if the disease manifests itself in ways other than atrio-ventricular blockade. The advanced stage of Lyme carditis manifesting as dilated cardiomyopathy is a special case of this affliction. The authors of this report present clinical experience with an attempt to support the working hypothesis about involvement of Lyme borreliosis infection in the development of dilated cardiomyopathy. The patients were clinically examined thoroughly with special attention to the cardiovascular system. In addition to the basic clinical methods, the following procedures have been employed: dynamic Holter's electrocardiography, exercise ECG test, coronarography, and myocardial biopsy. From laboratory methods pertaining to the detection of Borrelia, ELISA method, Western blot, PCR, electron microscopy and histopathological analysis were used. In all three cases, clinical and laboratory findings provided the evidence of the borreliosis infection involvement in the development of dilated cardiomyopathy.

Lyme borreliosis--waiting for Lyme carditis? A long-term prospective study.

A long-term prospective study of patients with confirmed non-cardiac form of Lyme disease (n=221) over a mean follow-up period of 40.6 months is reported. The study revealed no case of Borrelia-related cardiac involvement developed after several years in patients who had received antibiotic therapy in the early period. Therefore, these patients do not need follow-up by a cardiologist.

Long term and repeated electron microscopy and PCR detection of Borrelia burgdorferi sensu lato after an antibiotic treatment.

The diagnosis of Lyme disease in 18 patients has been proved by detection of Borrelia burgdorferi sensu lato when using immunoelectron microscopy or detecting its nucleic acid by PCR in the plasma or the cerebrospinal fluid. The positive results occurred in the plasma or in the cerebrospinal fluid in the period of 4-68 months after an antibiotic treatment. The typical clinical manifestations of Lyme disease were observed in 9 patients and non-specific symptoms in another 9 patients. According to presented results we can recommend repeated examination using PCR of the plasma and other biological specimens in the individuals with persistent or recurring complaints after an acute form of Lyme disease and its antibiotic treatment. Also examination of the cerebrospinal fluid with non-specific symptoms and simultaneously displayed pathology electroencephalogram and/or magnetic resonance imaging findings can be advantageous.

[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.

Interaction of Borrelia burgdorferi sensu lato with Epstein-Barr virus in lymphoblastoid cells.

Since the possibility of interruption of latent EBV infection has been suggested by the induction of the lytic virus cycle with chemical substances, other viruses, and by immunosuppression, we hypothesized that the same effect might happen in B. burgdorferi sensu lato infection as happens in Lyme disease patients with positive serology for both agents. We have observed EBV replication in lymphoblastoid cells after superinfection with B. garinii and B. afzelii strains after 1 and 4 h of their interaction. We found that viral and borrelial antigens persisted in the lymphoblasts for 3 and 4 days. Morphological and functional transformation of both agents facilitate their transfer to daughter cells. Association with lymphoblasts and internalization of B. garinii by tube phagocytosis increased replication of viruses more successfully than B. afzelii and chemical inductors. Demonstration of such findings must be interpreted cautiously, but may prove a mixed borrelial and viral cause of severe neurological disease.

Molecular and microscopical evidence of Ehrlichia spp. and Borrelia burgdorferi sensu lato in patients, animals and ticks in the Czech Republic.

We report moderately severe cases of human ehrlichiosis and a lethal one caused by human granulocytic Ehrlichia, the HGE agent, closely related to Ehrlichia phagocytophila and Ehrlichia equi. Their vector is the Ixodes ricinus tick, which also transmits Borrelia burgorferi sensu lato in central, west and east regions of the Czech Republic. The diagnosis was established by PCR with sequence analysis of the genes encoding 16S rRNA of Ehrlichia and with reverse hybridization by using enzyme linked immunosorbent assay with different covalently coupled probes to the activated plate. Ten out of 47 patients and 10 huntsmen were PCR positive and 7 of them seroconverted to the HGE. Coinfection of Ehrlichia phagocytophila with Borrelia burgdorferi sensu lato was detected in 3 patients. Ehrlichia spp., the HGE agent, was isolated and propagated only from one patient in the HL-60 promyelocytic cell line. The maintenance of Ehrlichia in culture and in patients was assayed also by immunocytological staining and electron microscopy. Sequence or hybridization analysis of PCR results in different wild mammals and birds showed significant sources of Ehrlichia fagocytophila in nature. Three variants of E. phagocytophila in wild roe deer and boars, as well as for the first time in birds, have been described. Cultures from the blood of horses, and from the spleen and kidney specimens of roes and boars, PCR positive for Ehrlichia spp., displayed a disappearing level of the pathogen or contamination with other bacteria.

Seronegative Lyme arthritis caused by Borrelia garinii.

A case of a female patient suffering from Lyme arthritis (LA) without elevated antibody levels to Borrelia burgdorferi sensu lato is reported. Seronegative Lyme arthritis was diagnosed based on the classic clinical manifestations and DNA-detected Borrelia garinii in blood and synovial fluid of the patient, after all other possible causes of the disease had been ruled out. The disease was resistant to the first treatment with antibacterial agents. Six months after the therapy, arthritis still persisted and DNA of Borrelia garinii was repeatedly detected in the synovial fluid and the tissue of the patient. At the same time, antigens or parts of spirochaetes were detected by electron microscopy in the synovial fluid, the tissue and the blood of the patient. The patient was then repeatedly treated by antibiotics and synovectomy has been performed.

[Prevalence of antiborrelia antibodies in two populations: various interpretations of the same data]. / Prevalence antiborreliových protilátek u dvou populací: ruzná interpretace stejných dat.

OBJECTIVE: The objective of the work was to test whether differently formulated hypotheses, when using correct statistical methods, and the same data lead to the same results. The work pertains to the interpretation of the prevalence of antiborrelia antibody levels in more or less high risk populations. GROUP: A group of 200 blood donors from Prague (100 men, mean age 33.5 years and 100 women mean age 24.4 years) and 71 subjects with a higher risk of contact with ticks infected with Borrelia burgdorferi--forestry workers from the Sumava mountains (39 men, mean age 40.6 years and 32 women, mean age 44.5 years). METHOD: In all participants antiborrelia antibodies class IgM and IgG were assessed and using the Western blot method the presence of specific antigens was examined. The blood samples of all probands were analyzed in the national reference laboratory for Lyme borreliosis CEM in the National Institute of Public Health in Prague. To test the hypothesis on the difference of groups as regards the frequency of positive antibody levels the chi square test was used. For testing the hypothesis on the difference of the groups as regards antibodies irrespective of normal laboratory values the non-parametric method--the Mann-Whitney test was used. RESULTS: The group of Prague blood donors and forestry workers from the Sumava mountains do not differ as to the individual frequency of positive antibody values assessed by the ELISA method in class IgM and IgG but they differ in class IgM assessed by the Western blot method. Both groups differ moreover markedly in the general distribution of antibodies unrelated to normal laboratory values assessed by the ELISA method in class IgM, in class IgG the distribution does not differ. CONCLUSION: We provided evidence that when using a different formulation of the hypothesis while using correct statistical methods identical data can be differently interpreted. Thus in the interpretation of the prevalence of antiborrelia antibody levels in healthy populations with a different risk--even if the two groups do not differs as to the frequency of positive findings, there is a significant statistical difference in their distribution.

[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.

[Persistence of Borrelia burgdorferi sensu lato in patients with Lyme borreliosis]. / Perzistence Borrelia burgdorferi sensu lato u pacientu s lymeskou borreliózou.

In 18 patients with Lyme borreliosis the authors proved the persistence of Borrelia burgdorferi sensu lato by detection of the causal agent by immune electron microscopy or of its DNA by PCR in plasma or cerebrospinal fluid after an interval of 4-68 months. Clinical manifestations common in Lyme borreliosis were present in only half the patients, in the remainder non-specific symptoms were found. In nine subjects with confirmed Borrelia burgdorferi sensu lato in the cerebrospinal fluid the cytological and biochemical finding was normal. Examination of antibodies by the ELISA method was negative in 7 of 18 patients during the first examination and in 12 of 18 during the second examination. In all negative examinations the specific antibodies were assessed by the Western blot or ELISA method after liberation from the immunocomplexes. In the authors' opinion it is advisable to examine repeatedly plasma and other biological material from potentially affected organs by PCR and subjects with persisting or relapsing complaints after the acute form of Lyme borreliosis as well as to examine cerebrospinal fluid in case on non-specific symptoms and concurrent pathic EEG or MR findings.

Sensitivity of Borrelia burgdorferi strains isolated in the Czech Republic.

We tested in vitro susceptibility of 5 strains of Borrelia burgdorferi sensu lato, isolated from patients in the Czech Republic, to penicillin G, ceftriaxone, cefotaxime, doxycycline and amoxycillin combined with clavulanic acid by broth macrodilution method on BSK medium. Results confirmed high efficacy of the tested drugs, particularly on B. afzelii and B. burgdorferi sensu stricto. A strain of B. garinii, isolated from cerebrospinal fluid, required a longer period of antibiotic activity before inhibitory effects were achieved. Penicillin G was effective only in higher concentrations, attainable by i. v. application. Ceftriaxone had the strongest borreliacidal activity.