Epitope Mapping of BmpA and BBK32 Borrelia burgdorferi Sensu Stricto Antigens for the Design of Chimeric Proteins with Potential Diagnostic Value.

Lyme disease is a tick-borne zoonosis caused by Gram-negative bacteria belonging to the Borrelia burgdorferi sensu lato (s.l.) group. In this study, IgM- and IgG-specific linear epitopes of two B. burgdorferi sensu stricto (s.s.) antigens BmpA and BBK32 were mapped using a polypeptide array. Subsequently, two chimeric proteins BmpA-BBK32-M and BmpA-BBK32-G were designed to validate the construction of chimeras using the identified epitopes for the detection of IgM and IgG, respectively, by ELISA. IgG-ELISA based on the BmpA-BBK32-G antigen showed 71% sensitivity and 95% specificity, whereas a slightly lower diagnostic utility was obtained for IgM-ELISA based on BmpA-BBK32-M, where the sensitivity was also 71% but the specificity decreased to 89%. The reactivity of chimeric proteins with nondedicated antibodies was much lower. These results suggest that the identified epitopes may be useful in the design of new forms of antigens to increase the effectiveness of Lyme disease serodiagnosis. It has also been proven that appropriate selection of epitopes enables the construction of chimeric proteins exhibiting reactivity with a specific antibody isotype.

Tick-borne pathogens Bartonella spp., Borrelia burgdorferi sensu lato, Coxiella burnetii and Rickettsia spp. may trigger endocarditis.

BACKGROUND: Infections caused by tick-borne pathogens such as Bartonella spp., Borrelia burgdorferi s.l., Coxiella burnetii, and Rickettsia spp. are capable of causing serious lesions of the mitral and aortic valves, leading to a need for valve replacement. OBJECTIVES: The aim of the study was to determine whether such cases are sporadic or frequent. An additional goal was to establish effective diagnostic methods to detect these infections. MATERIAL AND METHODS: The study involved 148 patients undergoing valve replacement. Blood samples were drawn for serological testing. Samples of the removed mitral and aortic valves were tested with polymerase chain reaction and immunohistochemical staining. RESULTS: Specific antibodies to Bartonella spp. were detected in 47 patients (31.7%) and in 1 of the healthy controls (1%) (p < 0.05). Antibodies to B. burgdorferi spirochetes were found in 18 of the patients (12.2%) and in 6 blood donors from the control group (5.8%) (p < 0.1). Antibodies to Rickettsia spp. were detected in 12 (8.1%) and to C. burnetii phase I and II antigens in the serum of 1 patient. All the participants in the control group were seronegative to C. burnetii and Rickettsia spp. antigens. Polymerase chain reaction (PCR) tests for detection of Bartonella spp., B. burgdorferi s.l., C. burnetii and Rickettsia spp. DNA in the valve samples were all negative. Inflammation foci with mononuclear lymphoid cells in the aortic and mitral valves were seen in sections stained with hematoxiline and eozine. In sections dyed using the indirect immunofluorescence method with hyperimmune sera, Bartonella spp. and Rickettsia spp. were found. CONCLUSIONS: The results obtained indicate that laboratory diagnostics for patients with heart disorders should be expanded to include tests detecting tick-borne zoonoses such as bartonelloses, Lyme borreliosis, rickettsioses and Q fever.

Leptospirosis in Poland in the years 2014-2017 ­ characteristics of infections and epidemiological surveillance data

OBJECTIVES: The aim of the study was the analysis of current Leptospira spp. infections in Poland on the basis of blood serum samples tests results and clinical data collected from clinicians in the Laboratory NIPH-NIH. METHODS: Clinical materials from 48 patients with clinical symptoms suggesting Leptospira spp suspected of leptospirosis from the years 2014-2017 were included to the study. Blood serum samples collected from patients were tested in Laboratory of Rickettsiae, Chlamydiae and Spirochaetes (currently Laboratory of Vector-borne Diseases) of NIPH-NIH. Levels of specific IgM and IgG antibodies to Leptospira spp. antigens were detected with the enzyme-linked immunosorbent assay (ELISA). RESULTS: Specific antibodies to Leptospira spp. were detected in 18 patients (37.5%). IgM antibodies were found in 6 patients (12.5%) and IgG antibodies were identified in 7 patients (14.6%). Both classes of antibodies of were detected in 5 patients (10.4%). The most samples for study were sent to laboratory from Masovian (13 samples) and Kuyavian-Pomeranian (11 samples) Voivodeships. Not any samples from the Lower Silesia, Lublin, Lódz, Podlaskie and Warmian-Masurian Voivodeships were received. In these patients the most common symptoms of disease were: fever, hepatitis with jaundice and renal failure. CONCLUSIONS: The number of diagnosed human leptospirosis in Poland is low in comparison to the number of cases in other countries, although the Leptospira spp. spirochetes occur in animals in the environment.

Francisella tularensis/Rickettsia spp. co-infections in patients with skin changes and lymphadenopathy.

INTRODUCTION: Tularemia and spotted fever group rickettsioses (SFG) can be transmitted by ticks and have a number of common clinical symptoms. Most characteristic are a maculopapular or vesicular rash or an eschar at the site of the tick or insect bite accompanied by painful lymph nodes. The aim of this study was to determine whether Rickettsia spp./Francisella tularensis mixed infections occurred in patients with similar symptoms who were diagnosed with either Rickettsia spp. or F. tularensis infection. MATERIAL AND METHODS: Thirty-six cases from 2011-2014, including 15 individuals with clinically and serologically recognized SFG and 21 with tularemia, were analyzed retrospectively using immunofluorescence for detection of Rickettsia spp. or ELISA for detection of F. tularensis. RESULTS: Of the 36 cases examined, specific high titers of antibodies to Rickettsia spp. were found in 1 (4.4%) patient with tularemia and specific high titers of antibodies to F. tularensis were detected in 1 (6.7%) patient with SFG. CONCLUSIONS: The results of our study show that in infections with fever, enlarged lymph nodes and skin lesions after tick and insect bites, laboratory testing of both diseases - SFG rickettsiosis and tularemia - should be implemented. Identification of F. tularensis and Rickettsia spp. mixed infections is crucial in order to administer appropriate antibiotics and to avoid treatment failure and relapse.

An outbreak of leptospirosis imported from Germany to Poland.

BACKGROUND: Leptospirosis is a zoonotic disease caused by spirochetes of the Leptospiraceae family. In both humans and animals the main route of infection is indirect contact - through water or other products contaminated with urine containing spirochetes. Infection most commonly occurs through ingestion of water or food contaminated with Leptospira spp. OBJECTIVES: The aim of the study was to characterize cases of leptospirosis imported to Poland from Germany in 2014 and to analyze methods that are helpful for making a diagnosis. MATERIAL AND METHODS: The 10 patients examined were reported as suspected leptospirosis cases on the basis of clinical symptoms and epidemiological investigations. They originated from different regions of Poland and had been working together at a strawberry plantation in the Cloppenburg district of Lower Saxony in Germany. Blood and urine samples were tested by PCR and serum samples by serology. All ELISA positive and negative cases were examined using a reference microscopic agglutination test (MAT). RESULTS: In the tested group, 6 individuals (60%) were seropositive according to the ELISA, and 2 of them were confirmed by the MAT. The PCR results for the blood and urine samples were negative. CONCLUSIONS: Using the ELISA in the diagnosis of leptospirosis allowed the disease to be identified much faster, differentiating classes of antibodies and recognizing levels of them that are too low to be detectable by the MAT.

Seroprevalence of Bartonella Species in Patients with Ocular Inflammation.

Bartonella species, vector-borne etiologic agents of many systemic or self-limited infections, are responsible for a widening spectrum of diseases in humans, including inflammatory conditions of the eye. The aim of this study was to determine whether there is any relationship between uveitis and the evidence of Bartonella spp. infection in the serum, ocular fluid, and cataract mass in patients with intraocular inflammation. Polymerase chain reaction (PCR)-based tests and DNA sequencing were performed on surgery-extracted specimens of intraocular fluid and lens mass of 33 patients. Sera from 51 patients and 101 control subjects were tested for the presence of specific antibodies against Bartonella spp. Neither IgM-class antibodies against Bartonella spp. nor Bartonella spp. DNA were detected. A specific IgG-class antibody was found in 33.3% of the patients with uveitis. The rate of positive Bartonella serology was higher among the uveitis patients than that in control subjects. This high rate may in part result from unrecognized indirect mechanisms rather than the immediate presence and multiplication of Bartonella spp. in the eyeball. Nonetheless we believe that screening for Bartonella spp. should become part of the diagnostic workup in uveitis.

Borrelia miyamotoi - new etiologic agent of neuroborreliosis?

Borrelia miyamotoi spirochetes discovered in Ixodes persulcatus ticks in Japan, in 1994 and documented in ticks and rodents in moderate climate zone of northern hemisphere. They belong to tick-borne relapsing fever group spirochetes. Borrelia miyamotoi is an etiologic agent of B.miyamotoi disease with acute febrile illness, including fever, headache, dizziness, fatigue, chills, and muscle and joint pain. Recurrence of fever has been observer in more than 10% patients. In some patients meningoencephalitis, encephalitis and cranial neuritis were observed. Laboratory recognition is based mainly upon PCR testing. Serological testing is limited due to inaccessibility of tests.

Bacterial tick-borne diseases caused by Bartonella spp., Borrelia burgdorferi sensu lato, Coxiella burnetii, and Rickettsia spp. among patients with cataract surgery.

BACKGROUND: Clinical data have shown that tick-borne diseases caused by Borrelia burgdorferi sensu lato, Bartonella spp., Coxiella burnetii, and Rickettsia spp. can affect the central nervous system, including the eye. The aim of this study was to establish a relationship between the incidence of cataract and evidence of bacterial infections transmitted by ticks. MATERIAL AND METHODS: Fluid with lenticular masses from inside of the eye and blood from 109 patients were tested by PCR and sequencing. Sera from patients and the control group were subjected to serological tests to search specific antibodies to the bacteria. RESULTS: Microbiological analysis revealed the presence of Bartonella sp. DNA in intraoperative specimens from the eye in 1.8% of patients. Serological studies have shown that infections caused by B. burgdorferi sensu lato and Bartonella sp. were detected in 34.8% and 4.6% of patients with cataract surgery, respectively. CONCLUSIONS: Presence of DNA of yet uncultured and undescribed species of Bartonella in eye liquid indicates past infection with this pathogen. Specific antibodies to B. burgdorferi sensu lato and Bartonella sp. are detected more frequently in patients with cataract compared to the control group. This could indicate a possible role of these organisms in the pathological processes within the eyeball, leading to changes in the lens. Further studies are needed to identify Bartonella species, as well as to recognize the infectious mechanisms involved in cataract development.

Case report of African tick-bite fever from Poland.

A confirmed case of rickettsiosis acquired in South Africa and recognized in Poland was described. The patient fulfilled clinical criteria highly suggestive of African tick bite fever, such as eschars, regional lymphadenitis, cutaneous rash within 10 days after his return from sub-Saharan Africa. Infection with Rickettsia africae was confirmed by polymerase chain reaction and sequencing.

Characteristics of Bartonella spp. infections in Poland in the years 2009-2012 identified in the laboratory of National Institute of Public Health--National Institute of Hygiene.

INTRODUCTION: Various Bartonella species, (Gram-negative aerobic bacilli) are etiologic agents ofzoonotic diseases called bartonelloses, which manifest with different symptoms depending on the bacterial species, reservoir and vector. In Poland and Europe, the most common bacterial species of the genus Bartonella is Bartonella henselae. MATERIAL AND METHODS: [corrected] Serum samples derived from patients with clinical symptoms suggesting Bartonella spp. infection, sent in 2009-2012 to the Laboratory of Rickettsiae, Chlamydiae and Spirochaetes of National Institute of Public Health--National Institute of Hygiene in Warsaw were tested. Levels of specific IgM and IgG antibodies to B. henselae and B. quintana antigens were detected with indirect immunofluorescence method (IFA). RESULTS: Six hundred sixty three serum samples were examined from humans with clinical symptoms suggestive bartonellosis, in 2009-2012. Specific antibodies for B. henselae were detected in 435 patients (65.6%). IgM antibodies were found in 93 patients (21.4%) including 11 patients (2.5%) with IgM only. IgG antibodies were identified in 424 people (78.6%) of whom 342 had IgG antibodies only. The antibodies of both classes were detected in 82 people (18.9%). B. quintana infections were not found. The majority of samples for study of bartonellosis were submitted in the autumn. In patients with confirmed bartonellosis, the most common symptoms of disease were lymphadenopathy (86 people, 13%), fever (13 patients, 2%) and nodular changes in various organs (13 patients, 2%). CONCLUSIONS: Infections caused by Bartonella spp. in Poland should be monitored to acquire the information on the frequency and distribution of disease in the country and their clinical course.

[Leptospira spp. and Coxiella burnetii infections occuring in Radomskie District in people of selected professional groups]. / Zakazenia Leptospira spp. i Coxiella burnetii wystepujace w powiecie radomskim u ludzi z wybranych grup zawodowych.

INTRODUCTION: Leptospirosis and Q fever are a zoonotic diseases with global occurring. OBJECTIVES: The aim of this study was to determine the prevalence of Leptospira spp. and C. burnetii in humans, who have contacts with infected animals or are exposed to an environment potentially contaminated with these bacteria. MATERIAL AND METHODS. Blood serum samples originating from 177 veterinarians and farmers and 134 garbage collectors (blood samples) were examined. Control group consisted 43 blood samples derived from blood donors. For the detection of specific IgM and IgG antibodies of Leptospira spp., enzyme-linked immunosorbent assay (ELISA) was used. Indirect immunofluorescence method (IFA) was used for detection of specific IgG C. burnetii antibodies. DNA of Leptospira spp. and C. burnetii was detected by PCR method with appropriate pairs of primers. RESULTS: Specific IgG C. burnetii antibodies of phase II were detected in sera of 4.4% of the farmers and veterinarians, and in 12% of garbage men. Antibodies in blood donors was not found. Antibodies of Leptospira spp. were present in the serum of 23.6% of farmers and veterinarians, 26.2% of garbage men and 14% of blood donors. C. burnetii DNA was detected in one sample derived from the veterinarian (1.1%). Leptospira spp. DNA was not detected in tested material. Blood samples from farmers, veterinarians and garbage collectors showed the higher prevalence of antibodies of Leptospira spp. and C. burnetii as compared to the control group (blood donors). CONCLUSIONS: Beside farmers and veterinarians, garbage collectors should be consider as high risk group of contracting leptospirosis and Q fever. Both leptospirosis as well as Q fever should be considered in the differential diagnosis in humans with animals and animals' material contact when they reveal flu-like symptoms.

[Ticks infected with bacteria pathogenic to humans in municipal parks in Warsaw]. / Kleszcze zakazone bakteriami chorobotwórczymi dla czlowieka na terenach parków miejskich warszawy.

The aim of the study was to detect bacteria pathogenic to humans, such as Borrelia burgdorferi sensu lato, Rickettsia spp, and Coxiella burnetii in ticks found in municipal parks in Warsaw, including The Royal Lazienki Park, Mokotów Fields, Józef Sowinski Park, Ujazdów Park and Fort Bema Park. To detect microorganisms PCR technique was used. It was shown that 6.1% of the ticks in Warsaw's parks are infected with B. burgdorferi sensu lato, and 2.9% with Rickettsia spp. Analysis of sequencing results revealed in examined ticks the presence of B. burgdorferi sensu stricto and R. helvetica.

[Case of TIBOLA/DEBONEL (tick - borne lymphadenopathy/Dermacentor spp. - borne necrosis - erythema - lymphadenopathy) in Poland]. / Przypadek TIBOLA/DEBONEL (tick - borne lymphadenopathy/Dermacentor spp. - borne necrosis - erythema - lymphadenopathy) w Polsce.

Rickettsia slovaca was recognized as the causative agent of the disease named tick - borne lymphadenopathy (TIBOLA) or Dermacentor spp.- borne necrosis - erythema lymphadenopathy (DEBONEL). Generally, this ricketsiosis is considered a mild disease but the disease is considered a mild rickettsiosis, but severe symptoms can appeare, especially in untreated patients. Described infection is the first case of TIBOLA/DEBONEL found in Poland. The patient had typical symptoms, such as enlarged painful cervical lymph nodes and maculo - papular rash. Milium on the scalp behind the ear concha, was a probably scar formation associated with typical eschar. The diagnosis was confirmed by detection of 128 titer in MIF tests with R. rickettsii and R. slovaca antigens and four-fold decrease after treatment.