Screening of opportunistic Encephalitozoon intestinalis and Enterocytozoon bieneusi in immunocompromised patients in Slovakia.

OBJECTIVE: In recent years new infectious diseases, i.e. emerging or re-emerging diseases, have been coming to the forefront. Currently, microsporidia, considered to be a major cause of emerging and opportunistic infections particularly in immunocompromised individuals, are also included in this group. Therefore, the aim of our study was to map the prevalence of Encephalitozoon intestinalis and Enterocytozoon bieneusi infection in a group of patients and to compare it with the occurrence of specific antigens in immunocompetent people. METHODS: Detection of spores of both pathogens in faecal samples was performed by an immunofluorescence test using species-specific monoclonal antibodies. RESULTS: Positivity to E. intestinalis in 91 examined immunosuppressed patients reached 33% (30/91), while only 4.3% (3/70) of the control group samples were found to be positive (relative risk 7.7, p < 0.001). In case of E. bieneusi 14.3% (13/91) of immunocompromised patients were positive, as were 5.7% (4/70) of people from the control group (relative risk 2.5, p = 0.095). CONCLUSION: In case of development of any opportunistic infection, the infection is detected and removed in most cases at an early stage. The incidence of clinically manifested microsporidiosis in patients with immunodeficiency is rare as they are under constant medical supervision. However, we must not forget about opportunistic infections, and in case of any non-specific symptoms it is necessary to exclude or confirm the diagnosis for immediate treatment.

Chlamydiosis in farmed chickens in Slovakia and zoonotic risk for humans.

INTRODUCTION: Chlamydia psittaci is an obligate intracellular Gram-negative bacterium causing respiratory disease (chlamydiosis) or asymptomatic carriage in poultry. In humans, it is a zoonotic agent of ornithosis/psittacosis. Due to low awareness of the disease and variable clinical presentation, psittacosis is often remains unrecognised as such by general practitioners. Zoonotic transfer occurs through inhalation of contaminated aerosols, and originates from feathers, faecal material and respiratory tract exudates. OBJECTIVE: The aim of this study was to investigate chickens for the presence of Chlamydia sp. from pharyngeal and cloacal swabs and review the zoonotic risk for humans. MATERIAL AND METHODS: 138 clinically healthy chickens from farms in Slovakia were examined for the presence of Chlamydia sp. The age of the chickens was 6 months. Two different samples were used - pharyngeal swabs and cloacal swabs. Each sample was examined by the molecular PCR method, and in the case of a positive result the identity of the obtained sequences was examined by a BLAST search. RESULTS: Of the total number of 276 examined samples from 138 chickens, 19 (6.9%) showed positivity for C. psittaci infection, 12 (8.7%) which were positive from pharyngeal swabs and 7 (5.1%) from cloacal swabs. None of the chickens were positive in both samples. Phylogenetic examination of the 19 isolates identified in the study, based on the 23S rRNA gene sequence, revealed that the isolates obtained were identical with C. psittaci, and genetically very close to genotypes B and genotype E. CONCLUSIONS: C. psittaci infections are apparently emerging in chickens. Chicken-processing plant employees should be considered a risk group for human psittacosis. There is a need for higher awareness and for efficient risk assessment and management.

Prevalence of Chlamydia trachomatis Infection and Its Association with Sexual Behaviour and Alcohol Use in the Population Living in Separated and Segregated Roma Settlements in Eastern Slovakia.

The aim of the study was to explore sexual behaviour and the occurrence of Chlamydia trachomatis (CT) infection in the population living in Roma settlements compared to the majority population in Slovakia and to assess the association between alcohol use and sexual behaviour within both populations. A cross-sectional population-based Hepa-Meta study was conducted in Slovakia in 2011. The final sample comprised 452 Roma and 403 non-Roma respondents. The occurrence of CT was detected by direct proof of the pathogen by PCR. The association between alcohol use and the prevalence of risky sexual behaviour were assessed using a logistic regression. First intercourse at age 15 or younger was reported by 27.9% of Roma (vs. 4.5% of non-Roma); 93.4% of Roma (vs. 77.9% of non-Roma) used condom inconsistently, 22.8% of Roma (vs. 43.9% of non-Roma) used a condom for protection from unwanted pregnancies and only 8.8% of Roma (vs. 21.8% of non-Roma) due to protection against infectious diseases. However, Roma reported having had five or more sexual partners less often compared to the majority (11.5% of Roma vs. 20.6% of non-Roma). Binge drinking at least once a month was associated with a higher number of sexual partners in both groups, but not with condom non-use. The prevalence of CT infection in the Roma population was higher (3.8%) compared to non-Roma (2.7%); however, the difference was not statistically significant. Our study found no differences in the prevalence of CT infection between Roma and non-Roma despite differences in sexual behaviour. Roma begin their sexual life earlier and have unprotected sex more often, but on the other hand, they seem to be much more restrained in terms of the number of sexual partners compared to the majority population.

Detection of CHLAMYDIA PSITTACI in feral pigeons (COLUMBA LIVIA DOMESTICA) in Slovakia and their characterisation.

INTRODUCTION AND OBJECTIVES: Chlamydia psittaci, an obligate intracellular bacterium, which is the etiologic agent of avian chlamydiosis in birds and ornithosis/psittacosis in humans, has been reported to be one of the most common pathogens found in feral pigeons worldwide, and thus constitutes a zoonotic risk. The aim of the study was to investigate pigeons in Slovakia living in areas in close proximity to humans for the presence of C. psittaci, using pharyngeal and cloacal swabs. MATERIAL AND METHODS: 122 clinically healthy pigeons from different geographical regions of Slovakia were examined for the presence of C. psittaci. The adult pigeons of both genders were captured during the summer period in the urban centres of Slovakian towns. Each sample was examined by molecular method PCR, and in the case of positive result the identity of the obtained sequence was examined by a BLAST search. RESULTS: Of the total number of 244 examined samples, 14 (5.7%) showed positivity for C. psittaci infection, 5 of which were from pharyngeal swabs (4.1%) and 9 from cloacal swabs (7.4%). A positive result was detected in 13 pigeons (10.7%). Phylogenetic analysis showed that all the positive samples are genetically very close to genotypes B and genotype E. CONCLUSION: Phylogenetic examination of the 14 isolates of C. psittaci identified in the presented study, based on 23S rRNA gene sequence, revealed their close relationship with C. psittaci genotypes B and E. Both genotypes are predominantly prevalent in pigeons and both can be transmitted to humans. Therefore, it is necessary to perform screening examinations of animals and analyse the epidemiological factors affecting the way of transmission and circulation of pathogen.

Occurrence of antibodies to Anaplasma phagocytophilum in patients with suspected tick-borne encephalitis.

INTRODUCTION AND OBJECTIVES: Human granulocytic anaplasmosis (HGA) is an emerging tick-borne infectious disease caused by Anaplasma phagocytophilum. In Europe, the first serological evidence of HGA was described in 1995 in Switzerland, and the first clinical case was confirmed in 1997 in Slovenia. Since then, many European countries, including Slovakia, have reported the occurrence of HGA. The aim of this study was to examine the occurrence of IgG antibodies against A. phagocytophilum in blood sera of humans with suspected tick-borne encephalitis. MATERIAL AND METHODS: 181 people were examined for the presence of anti-A. phagocytophilum IgG antibodies; 113 were patients with suspected TBE (65 males, 48 females), and 68 from the control group (18 males, 50 females). Respondents were aged 2-80 years (mean age: 31.39; STD: 17.1). Anti-A. phagocytophilum IgG antibodies were detected by the IFA IgG test. Relative risk (RR) and their 95% confidence intervals (95% CI) were estimated for the occurrence of IgG A. phagocytophilum antibodies. RESULTS: Of the total number of 181 people examined, 32 (17.7%) showed positive for IgG antibodies against A. phagocytophilum, 22 of whom were patients with suspected TBE (19.5%) and 10 people from control group (14.7%). The RR of occurrence of IgG A. phagocytophilum was 1.3-times higher in the patients with suspected TBE than in the control group. CONCLUSION: None of the examined patients with suspected TBE had the disease confirmed. Hoever, as shown by the results, the relative risk of occurrence of anaplasmosis is higher in people examined for some another vector-borne disease (in this case TBE). Therefore, the performance of screening examinations in patients suspected of having any tick-borne disease is very important.

Differences between Roma and non-Roma in how social support from family and friends helps to overcome health care accessibility problems.

BACKGROUND: Roma are the most deprived ethnic minority in Slovakia, suffering from discrimination, poverty and social exclusion. Problematic access to good quality health care as result of institutional and interpersonal discrimination affects their health; therefore, factors which affect health care accessibility of Roma are of high importance for public health and policy makers. The aim of this study was to explore the association between health care accessibility problems and ethnicity and how different levels of social support from family and friends affect this association. METHODS: We used data from the cross-sectional HepaMeta study conducted in 2011 in Slovakia. The final sample comprised 452 Roma (mean age = 34.7; 35.2% men) and 403 (mean age = 33.5; 45.9% men) non-Roma respondents. RESULTS: Roma in comparison with non-Roma have a more than 3-times higher chance of reporting health care accessibility problems. Social support from family and friends significantly decreases the likelihood of reporting health care accessibility problems in both Roma and non-Roma, while the family seems to be the more important factor. CONCLUSION: The worse access to health care of Roma living in so-called settlements seems to be partially mediated by social support. Interventions should focus on Roma health mediators and community workers who can identify influential individuals who are able to change a community's fear and distrust and persuade and teach Roma to seek and appropriately use health care services.

The prevalence of Chlamydia trachomatis in the population living in Roma settlements: a comparison with the majority population.

BACKGROUND: We aimed to study the occurrence of Chlamydia trachomatis infection in the population living in Roma settlements and to compare the obtained results with the prevalence in the majority population. METHODS: We examined 340 people for the presence of bacterium C. trachomatis, 208 of them were Roma (66 men, 142 women) and 132 were from the majority population (75 men, 57 women). Respondents were aged 18-55 years (mean age = 33.44, STD = 9.57). The occurence of C. trachomatis was detected by direct proof of the pathogen by polymerase chain reaction (PCR). RESULTS: Of 340 respondents included in the study, 22 (6.5%) showed positivity for C. trachomatis infection, 15 of them were Roma (7.2%) and 7 non-Roma (5.3%). The highest positivity was detected in Roma women (8.5%), while positivity in both non-Roma women and men was 5.3%, and in Roma men 4.5%. We did not confirm any significant contribution of age, gender or ethnicity to the occurrence of C. trachomatis infection. CONCLUSIONS: Despite the increased number of people with risk factors in the Roma community, no significant difference in the occurrence of C. trachomatis infection was found. Neither age nor gender contributes to the probability of C. trachomatis infection. Nevertheless, there are other health consequences which might be more pronounced among the population living in Roma settlements due to barriers to the health care and their lower ability to benefit from health care services provided.

Serological screening of selected microsporidia in HPV-positive women.

Microsporidiosis is considered to be emerging opportunistic infection in immunocompromised individuals worldwide. The aim of this study was to identify the specific serum antibodies to intestinal microsporidia Encephalitozoon cuniculi and Encephalitozoon intestinalis in women with Human Papillomavirus HPV and without HPV by the indirect immunofluorescence (IFA). From total number of 669 examined women, 225 were HPV positive and 444 women HPV negative. Overall the study comprised of 10.8% women with positive result for presence of E. cuniculi antibodies. In group 1 (HPV-positive women) it was more than 28% and in group 2 (HPV-negative women) it was less than 2% (p<0.001). E. intestinalis infection was found in total of 4.48% women, in group 1 it was present in less than 6% and in group 2 in less than 4% of women.

Occurrence of microsporidia as emerging pathogens in Slovak Roma children and their impact on public health.

INTRODUCTION AND OBJECTIVE: Microsporidia are identified as ubiquitous organisms of almost every animal group and are now recognized as emerging opportunistic pathogens of human. The risk factors include immunodeficiency, lack of sanitation, and exposure to contaminated water and infected animals. In Slovakia, the places with an increased risk of infection due to the presence of risk factors and routes of transmission are represented by Roma settlements. Therefore, the aim of this work was to study the occurrence of Encephalitozoon spp. and E. bieneusi in children living in Roma settlements. MATERIALS AND METHODS: Stool samples were examined of 72 clinically healthy children coming from a group of the non-integrated Roma minority for the presence of microsporidia Encephalitozoon spp. and E. bieneusi. Microsporidian spores were detected by standard Rylux D, staining and by PCR and DNA sequencing. RESULTS: Of the total number of 72 stool smears examined, 22 were positive, which represented 30.6%. By the Real Time PCR, E. bieneusi was detected in 3 samples (4.2 %) and E. cuniculi in 19 samples (26.4 %). By comparing the sequences with sequences in the GenBank, E. cuniculi genotype I (Accession No. AJ005581.1) and E. bieneusi genotype A (Accession No. AF101197.1). CONCLUSIONS: Microsporidia, as newly emerging pathogens of humans and animals, are characterised by the production of spores which are environmentally resistant. Diseases caused by them have a cosmopolitan occurrence. Although E. bieneusi and E. cuniculi belong to the most frequently diagnosed species of microsporidia in humans, in Slovakia, this is the first confirmed evidence of E. bieneusi genotype A, as well as E. cuniculi genotype I in humans by the molecular method.

Changes in surveillance of acute respiratory infections including influenza in the Slovak Republic during 1993-2008.

The authors evaluated surveillance of acute respiratory infections (ARI), influenza and influenza-like illnesses (ILI) in the Slovak Republic (SR). They analyze morbidity, age-specific morbidity, complications, mortality, number of influenza viruses isolations and vaccination coverage rates in the SR in the years 1993-2008. They focus mainly on the analysis during the epidemic. Most epidemics have been caused by influenza virus A subtype H3N2. The age group mostly affected by morbidity during the year were children at the age of 0-5, while during the epidemic, the highest morbidity was recorded among school children at the age of 6-14. A complicated clinical course of the disease was reported in 1,422,836 patients (5.1%). Since the 2002/2003 influenza season, the sentinel physicians have participated in taking biological material, which ensures monitoring of influenza viruses circulating in the SR. As of the 2006/2007 season, the ARI/ILI have been reported separately in the SR in accordance with the monitoring requirements set by the European Influenza Surveillance Scheme (EISS) project, and the calculation of morbidity is done from the number of persons, who are in care of the reporting physicians: Vaccination coverage in SR is still very low in comparison with other European Union (EU) countries.

Rare positive laboratory tests for the presence of influenza virus A/H1N1--2009 in May, June, July, 2011, in the districts of Kosice I-IV and surroundings of Kosice in the Slovak Republic.

Influenza illnesses and positive laboratory tests for the presence of influenza virus in recent years in the districts of Kosice I-IV and surroundings have only occurred during the winter season. In May to July 2010 only one positive laboratory test for the presence of influenza virus A/H1N1-2009 was reported. In 2011, during the same period, a total of 29 positive laboratory tests recorded the presence of influenza virus A/ H1N1-2009 in individuals with typical clinical symptoms of influenza. Of 29 clinical cases, 27 were diagnosed as influenza and 2 as SARI; 4 cases involved children.

Occurrence of IgG antibodies to Anaplasma phagocytophilum in humans suspected of Lyme borreliosis in eastern Slovakia.

Human granulocytic anaplasmosis (HGA) and Lyme borreliosis (LB) are tick-borne and emerging infectious diseases caused by the Anaplasma phagocytophilum and Borrelia burgdorferi species. In Europe, including Slovakia, the principal vector of both pathogens is the common tick - Ixodes ricinus, in which double infections with these pathogens have been reported. The aim of our study was evidence of IgG antibodies against A. phagocytophilum in blood sera of humans with suspects LB from several Clinics of University Hospitals, and the evaluation of the possibility of B. burgdorferi and A. phagocytophilum co-infection in examined patients. The serological method ELISA was used to detect IgM and IgG antibodies against B. burgdorferi. Anti-A. phagocytophilum IgG antibodies were analyzed by the A. phagocytophilum Indirect Immunofluorescence Antibody (IFA) IgG test. A total of 214 human samples (91 men, 123 women) were obtained from patients living in Kosice town and in villages around Kosice (Eastern Slovakia). IgG antibodies against A. phagocytophilum were detected in 15 cases (6 men, 9 women), which represented 7.0 % positivity. Two cases of the co-infection B. burgdorferi with A. phagocytophilum, which equals 0.93 % of the total number, were found.

[Ehrlichiosis/Anaplasmosis]. / Ehrlichióza/Anaplazmóza.

Ehrlichiosis and anaplasmosis are zoonoses caused by bacteria from the family Anaplasmataceae, including human and animal pathogens. The human pathogens are Ehrlichia chaffeensis, the causative agent of human monocytic ehrlichiosis (HME), Anaplasma phagocytophilum, the pathogen causing human granulocytic anaplasmosis (HGA), E. ewingii and Neorickettsia sennetsu, granulocytotropic and monocytotropic Ehrlichia species, respectively. Ehrlichia spp. are small, gram-negative, obligate intracellular bacteria. They replicate in the cytoplasmic vacuoles of host cells, especially granulocytes and monocytes, to form microcolonies called morulae. These agents are transmitted through the bite of infected tick. In the United States, the vectors are Amblyomma americanum, Ixodes scapularis and Ixodes pacificus ticks. The primary vector in Europe is Ixodes ricinus. Rodents, deer, roe deer, foxes, cattle, sheep, goats, horses and dogs are reservoirs of these bacteria in Europe. Peromyscus leucopus, the white-footed mouse, and Odocoileus virginianus, the white-tailed deer, are the most important reservoirs in the United States. Infection in humans is manifested as a nonspecific flu-like illness. The laboratory diagnosis is most frequently serological--evidence of antibody by indirect immunofluorescence assay (IFA) and detection of DNA by polymerase chain reaction (PCR), or microscopy evidence--Giemsa stain of blood smears (morulae in granulocytes or monocytes). Doxycycline is the drug of choice in therapy. Avoiding exposure to ticks is the best method of prevention of infection.

[Q fever]. / Q horúcka.

Coxiella burnetii, the causative agent of Q fever, is a Gram negative coccobacillus. It resides and replicates in the host s monocytes and macrophages. The developmental cycle of C. burnetii includes macrocellular and microcellular forms and the formation of spore-like bodies. It undergoes a phase variation of outer cell surface antigens from virulent phase I to avirulent phase II after passaging in the yolk sac of embryonated chicken eggs or in cell cultures. C. burnetii belongs to the most resistant bacteria. The main reservoirs of C. burnetii are cattle, sheep and goats. Human Q fever usually results from inhalation of contaminated aerosols. Acute infection mostly takes the course of a flu-like disease, atypical pneumonia or hepatitis, the chronic form resembles endocarditis. Laboratory examinations are based on the presence of antibodies. The drugs of choice are broad-spectrum antibiotics.

Occurrence of antibodies against Chlamydophila abortus in sheep and goats in the Slovak Republic.

Chlamydophila abortus is one of the most important causative agents of enzootic abortion and other chlamydial infections of sheep and goats. The presence of specific serum antibodies to Chlamydophila abortus was studied in sheep and goats breeding in the Slovak Republic by the complement fixation test. 22,040 sheep and goats were examined during 5 years. Specific anti-Ch. abortus antibodies were found in 2,360 out of 20,878 sheep sera examined (11.7%), and in 85 out of 1,162 examined goats (7.7%). The occurrence of antichlamydial antibodies indicates the importance of performing screening examinations in commercial breeding with the aim of reducing the spread of this disease between animals, and also interrupting the spread and transmission from animals to human.

[Present possibilities of Q fever therapy.].

The first-line therapy for acute Q fever is 14 to 21 days of doxycycline, or fluoroquinolones such as ofloxacin or pefloxacin. Clarithromycin is a potential effective alternative. Chronic Q fever, especially endocarditis, requires treatment for at least 18 months, with the preferred combination of doxycycline and hydroxychloroquine. An alternative treatment for chronic Q fever, when adverse effects of hydroxychloroquine are a problem, is the combination of doxycycline and fluoroquinolones with extended therapy for 3 years or more. Serologic tests may be used to monitor the response to therapy.

[Q fever prevention and vaccination.].

The simpliest way how to avoid the development and spreading of any infection, including Q fever in extenso, is prevention. The main principles of prevention are: educate the public on sources of infection, appropriately dispose of placenta, birth products, fetal membranes, and aborted fetuses at facilities housing sheep and goats, restrict access to barns and laboratories used in housing potentially infected animals, use only pasteurized milk and milk products, use appropriate procedures for bagging, autoclaving, and washing of laboratory clothing, quarantine imported animals, periodical monitoring of seroprevalence in cattle, sheeps and goats, vaccination of the risk groups etc. Four different basic types of Q fever vaccines have been developed: 1) live attenuated vaccines were derived from the Coxiella burnetii strain Grita in phase I of a highly reduced virulence but preserved immunogenicity, 2) highly purified corpuscular formalin-inactivated vaccine derived from the C. burnetii Henzerling strain in phase I, 3) a soluble vaccine prepared from the C. burnetii strain Nine Mile corpuscules in phase I by extraction with trichloracetic acid, and 4) in the USA, the use of a chloroform-methanol extraction residue subunit vaccine of C. burnetii strain Nine Mile in phase I of a low reactogenicity has been recommended.

Heterogeneity of Borrelia burgdorferi sensu lato and their reflection on immune response.

Geographically different strains of Borrelia burgdorferi sensu lato (B. burgdorferi sensu stricto Ir 105, B. burgdorferi s.s. + B. afzelii V 123, B. garinii Ir 112 - isolates from eastern Slovakia, B. garinii K24 - isolate from western Slovakia and B. burgdorferi s.s. B 31 - American strain) were compared as antigens for serological study of Lyme borreliosis by IgG ELISA on a group of horses from eastern Slovakia. In a set of 101 horse serum samples, positivity with the use of Ir 105 strain was 53 (52.4%), with V 123 51 (51.49%), with Ir 112 48 (47.5%), with K 24 47 (46.5%) and with B 31 only 25 (24.7%). The seroprevalence between strains B 31 and Ir 105, B 31 and V 123, B 31 and Ir 112, B 31 and K 24 differed statistically significantly (test chi2, p<0.05); however, the differences between strains Ir 105, V 123, Ir 112 and K24 were insignificant. Consistency of positive and negative findings between American and Slovak strains ranged from 50.5-62.4%. Comparison of Slovak strains (Ir105, V 123, Ir 112 and K 24) consistency of positive and negative findings was higher from 79.2-95.04%. The highest consistency of findings was reached comparing strains Ir 112 and K 24, and the same high agreement of results was observed between the strains Ir 105 and V 123 and also Ir 112 and Ir 105. Higher consistency of findings of serologically examined horses with geographically close trains is in accordance with greater similarity of protein profiles of Slovak strains compared to the American strain.

Application of specific primers in the diagnosis of Encephalitozoon spp.

In our experiment, 3 species-specific primer pairs cultivated in cell lines were used: Encephalitozoon cuniculi -specific primer pairs (ECUNF and ECUNR), Encephalitozoon hellem -specific primer pairs (EHELF and EHELR), and Encephalitozoon intestinalis -specific primer pairs (SINTF and SINTR). The PCR products were estimated to be 550 bp in E. cuniculi , 547 bp in E. hellem and 545 bp in E. intestinalis respectively, which can prove the precision and reliability of this method in the species identification of the genus Encephalitozoon. All 3 primer pairs were species-specific and none of them amplified gene sequences from other Encephalitozoon spp.

Small mammals (Insectivora, Rodentia) as a potential source of chlamydial infection in East Slovakia.

The presence of antibodies against Chlamydophila (Chlamydia) psittaci in small mammals (Insectivora, Rodentia) in the region of East Slovakia are presented. The hosts were caught in several areas of Slovakia in habitats with different levels of anthropogenic disturbance. Research was carried out during 2000-2002. The authors examined 1,947 sera coming from 4 insectivore and 10 rodent species. Each serum was examined by micromethod of complement binding reactions using antigen Chlamydophila (Chlamydia) psittaci. Chlamydial infections were found in 251 individuals (prevalence 12.9 %) of 8 mammal species. The antichlamydial antibodies were proved at levels ranging from 1:32-1:1024. The highest prevalence of antibodies was detected in the most abundant rodent species Apodemus microps (14.8 %), Apodemus agrarius (13.9 %), Apodemus flavicolis (12.4 %), Microtus arvalis (12 %), and Clethrionomys glareolus (10.9 %). Positive hosts were registered in all studied localities. Testing of prevalence values in the individual research years confirmed significant changes. Our results showed that small mammals probably play an important role in the circulation of chlamydiae in nature.