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Co-infections with Borrelia species, Anaplasma phagocytophilum and Babesia spp. in patients with tick-borne encephalitis

The purpose of this study was evaluation of the prevalence of co-infection with Borrelia species, A. phagocytophilum and Babesia spp. in patients with tick-borne encephalitis (TBE). At total of 110 patients with TBE were included in the study. Serological tests for tick-borne encephalitis virus (TBEV), PCR for Borrelia species, Anaplasma phagocytophilum and Babesia spp., blood smears for A. phagocytophilum and Babesia spp. and BLAST analysis for Babesia spp. were performed. Results showed a significant majority of patients co-infected with Borrelia species (30/110; 27 %), much less with A. phagocytophilum (12/110; 10.9 %) and with Babesia spp. (1/110; 0.9 %). The BLAST analysis of the 18S rDNA sequence obtained with the Babesia spp. specific primers indicated that the patient was infected with Babesia microti. Triple co-infections (TBEV-Borrelia species- A. phagocytophilum) were observed in three (3/110; 2.7 %) patients. Conclusions were such that differential diagnosis in patients after the tick bite, presenting with acute symptoms, should include not only TBE and Lyme disease, but also other diseases transmitted by ticks. In patients with low parasitemia in suspicion of Babesia spp. infection PCR seems to be a more sensitive method than blood smear. Co-infection with various tick-borne pathogens must be always considered, especially in endemic regions.


Introduction
It is known that certain tick species are able to transmit more than one pathogen, e.g. tick-borne encephalitis virus (TBEV), Borrelia burgdorferi sensu lato (Borrelia species), Anaplasma phagocytophilum (A. phagocytophilum), and Babesia species (Babesia spp.), which may be a reason for co-infections [1]. Co-infections are becoming a serious epidemiological and clinical problem, especially due to the fact that the methods of treatment of infectious diseases caused by various tickborne pathogens differ from each other. Therefore, this fact is more frequently considered in clinical research, especially in the areas endemic for tick-borne diseases. We suppose that positive results of PCR tests, especially in patients with nonspecific symptoms in an early phase of infection, when antibodies cannot be detected with the help of traditional methods, may be helpful in the diagnosis and introduction of treatment of zoonoses such as Lyme disease (LD), tick-borne encephalitis (TBE), anaplasmosis or babesiosis.

Objective
The objective of our study was to evaluate the prevalence of infection with Borrelia species, A. phagocytophilum and Babesia spp. in patients with TBE hospitalized between July 2009 and October 2012 in The Department of Infectious Diseases and Neuroinfections of Medical University of Bialystok, Poland.

Material
A group of 110 patients with TBE (47 female and 63 male) was included in the study. None of patients was vaccinated against TBE and all of them had a history of tick bites. Diagnosis was made on the basis of clinical manifestation, cerebrospinal fluid (CSF) examination and the presence of serum and CSF specific antibodies. Anti/TBEV antibodies titer was measured with SERION ELISA classic TBE Virus IgG/IgM (Institut Virion/Serion GmbH, Germany). Patients presented symptoms as shown in Table 1. None of them was immunocompromised. Laboratory tests and cerebrospinal tests results, as well as levels of TBEV antibodies are also presented in Table 1.
Serum was collected to search for anti/TBEV and anti/Borrelia burgdorferi antibodies. Whole blood in EDTA was used for PCR for Borrelia species, Babesia spp. and A. phagocytophilum. Blood smears were performed to search for Babesia spp. and A. phagocytophilum circulating stages. Cerebrospinal fluid was collected to perform biochemical, immunological and PCR examination.
A control group (CG) consisted of 20 healthy blood donors, in whom PCR for Borrelia species, Babesia spp. and A. phagocytophilum and blood smears were performed.
The study was approved by the Bioethical Commission of the Medical University of Bialystok.

DNA isolation
DNA isolation was performed with the 200 μl of fresh or kept in +4°C whole blood according to QIAamp DNA Mini Kit (Qiagen, Germany). DNA extracts in 100 μl volume were received from doubled elution by the mini spin column.
Amplification of A. phagocytophilum genetic material was performed with the diagnostic kit PCR Anaplasma (Blirt-DNA Gdańsk, Poland) coding a fragment of 16S rDNA gene encoding small ribosomal 16S RNA subunit. Analyses were conducted in accordance with the manufacturers instruction, in the period from 2009 to 2011 in single PCR and in 2012 in a nested type of PCR. In conventional, single course PCR in 2011, 1 μl of the template DNA isolate was added to 43.7 μl of the Master Mix with 5 μl of dNTPs and 0.3 μl of Hypernova polymerase for a final reaction mix volume of 50 μl. Amplification was performed in the following PCR program: initial denaturation at 94°C for 5 min, 35 cycles (denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 3 s) and final extension at 72°C for 2 min. Positive results were 227 bp long fragments of the 16S rDNA gene. Nested PCR for A. phagocytophilum DNA detection was performed in two amplifications. In the first, PCR-OUT 2 μl of the template DNA isolates was added to 42 μl of the Master Mix with 5 μl of dNTPs and 1 μl of Taq nova polymerase for a final reaction mix volume of 50 μl. First amplification was performed in the following PCR program: For A. phagocytophilum the specificity and selectivity were determined by bioinformatic method using the NCBI database. Lack of specificity of tick DNA template kit was confirmed experimentally. Results of analysis showed that the system selectively detects only A. phagocytophilum DNA. In addition, the integrated package does not show homology to DNA templates of other prokaryotes or eukaryotes.
As positive controls we used DNA extracted from a deer spleen infected with Babesia spp.; in Borrelia species and in A. phagocytophilum cases a positive control was included in the particular kits. In a negative control redistilled water replaced DNA isolates. The size of amplification products for fla gene of Borrelia species was 276 base pairs (bp), for Babesia spp. Nucleotide sequencing of the Babesia 18 rDNA amplicon Amplification products of the Babesia spp. 18S rDNA were purified using the QiaAmp PCR purification kit (Qiagen). Sequencing reactions of both strands were performed using Big Dye Terminator cycle sequencing kit (Applied Biosystems) with the F2 and R2 primers under the same conditions as above. Cycle sequencing reactions were purified using the ExTerminator Kit (A&A Biotechnology, Gdynia, Poland) and sequenced with the ABI3500 automated

Immunoserology diagnostic
The detection of TBE virus infection was performed with SERION ELISA classic TBE Virus IgG/IgM (Institut Virion/ Serion GmbH, Germany) diagnostic kit twice in patients with meningitis or encephalitis, namely, at the time of admission to the hospital and 2 weeks later. Anti/TBEV antibody level dynamics were observed. A level of viral specific IgM and IgG antibodies was marked according to manufacturer's recommendations.
Anti/B. burgdorferi IgM and IgG antibodies in enzymelinked immunosorbent assay (ELISA; Borrelia recombinant IgG and IgM High Sensitivity, Biomedica, Austria) and intrathecal production of anti/B. burgdorferi antibodies (EcoLine test, Virotech Classic Kits, Germany) in cases with suspicion of neuroborreliosis were performed.

Blood smears
Obligatory, from full blood collected on EDTA, blood smears stained with May-Grunwald and Giemsa (MGG) were performed twice. Piroplasm forms in erythrocytes were searched for Babesia spp. infection and morulae in neutrophiles for A. phagocytophilum infection.

Statistical analysis
Statistical analysis to compare patients with only TBE infection and co-infections was performed using Statistica 10. Groups were compared by Mann-Whitney and Pearson's chi-squared tests. P value <0.05 was considered statistically significant.

Results
In our study, conducted in the northeastern part of Poland on a group of 110 patients with TBE, a significant majority was coinfected with Borrelia species (30/110; 27 %), much less with A. phagocytophilum (12/110; 10.9 %) and with Babesia spp. (1/110; 0.9 %). The BLAST analysis of the 18S rDNA sequence obtained with the Babesia spp. specific primers indicated that the patient was infected with Babesia microti. One patient was diagnosed as neuroborreliosis, based on anti/B. burgdorferi IgM and IgG serum antibodies and intrathecal production of anti/B. burgdorferi antibodies in CSF.
In blood smear we observed no piroplasm forms in erythrocytes typical of Babesia spp. infection or morulae in neutrophiles typical of A. phagocytophilum infection.
Analysis of patients infected only with TBEV and patients co-infected with other pathogens showed no significant difference in the presented symptoms, which indicates overlapping of symptoms in cases of TBEV infection. However, there were significant differences in the laboratory parameter values such as the erythrocyte sedimentation rate (ESR) (p=0.028) and alanine aminotransferase activity (p=0.006) (Tables 2 and 3).

Discussion
In Poland, similarly to other European countries, tick-borne diseases are an increasing epidemiological and clinical problem. Annual incidence of LD has also been increasing systematically. The number of registered cases in 2005 and 2009 was 4.406 (incidence 11.5/100.000 inhabitants) and 10,333 cases (incidence 27.1/100.000 inhabitants), respectively. During a period of 5 years a 2-fold increase in LD incidence has been observed. The regions considered to be endemic are the Podlaskie and Warminsko-Mazurskie regions (northeastern Poland) with annual 5-fold higher incidence in comparison to the whole country. In 2012, a total of 9.159 cases were reported (incidence 24/100.000 inhabitants) [6]. TBE incidence has also been increasing for years. The number of registered cases in 2005 and 2009 was 174 (incidence 0.46/100.000 inhabitants) and 344 (incidence 0.9/ 100.000 inhabitants), respectively. In 2012, 188 cases were reported (incidence 0.49/100.000 inhabitants). Similarly to LD most cases have been observed in the Podlaskie and Warminsko-Mazurskie regions [6].
At the moment the exact number of cases suffering from anaplasmosis and babesiosis is unknown. At the same time, it is known that from 1956, only in Europe, more than 50 cases of human babesiosis have been confirmed [7][8][9]. Human Granulocytic Ehrlichiosis (HGE, anaplasmosis) has been It is known that more than one pathogen may co-exist in one vector. According to various European sources, coinfection of I. ricinus ticks with different pathogens appears to be quite common [5,[10][11][12][13]. In Poland, the prevalence of co-infected I. ricinus ticks with at least two pathogens varies from 0.12-8.30 % and depends on the area of tick sampling [1,[14][15][16].
Human co-infection with various pathogens may be the result of a single tick bite by the tick infected with more than one pathogen or the result of multiple bites by ticks infected with one pathogen. Both situations may result in a coinfection, often difficult to diagnose and differentiate [17].    [7]. In our study conducted in northeastern Poland we observed a rate of co-infections of 2.7 %: B. burgdorferi/ A. phagocytophilum/TBEV. However, we observed a quite high rate of co-infection with TBEV and Borrelia species (27 % of patients with TBEV were positive for Borrelia species), which was not observed in other studies. It may be explained by pre-selections of patients, who were admitted to hospital due to suspected meningitis. We also noticed a patient with Babesia microti infection, which was asymptomatic or with mild course. This observation may suggest significantly higher importance of Babesia infection than it has been considered previously.
Various methods may be used to diagnose infections caused by tick-borne agents. Among these are serological examinations (TBE, LD, babesiosis, anaplasmosis), PCR (LD, TBE, babesiosis, anaplasmosis) and blood smear (babesiosis, anaplasmosis) [22]. Each of these methods might be used separately and it is not necessary to have positive results of all of them simultaneously. The ideal situation would be to obtain the same results from all available methods; however, none of them has 100 % specificity and sensitivity. Blood smear, which in some cases may be negative, is the best example of problems in the diagnostic process. In general, the analysis of blood smears is a fairly subjective process. The need to discriminate the subtleties of babesial/anaplasmal morphology and possible low parasitemias may result in inaccurate diagnoses, which might require further analysis.
In cases of anaplasmosis suspicion 25-75 % of patients have morulae in peripheral blood smear examinations, with the highest sensitivity during the first week of infection [23]. Only one-third of patients with babesiosis have piroplasm forms in erythrocytes in microscopic study [21]. Additionally, Aktas et al. observed in sheep blood samples that only in four out of 98 piroplasms B. ovis forms were present in thin blood smears, whereas in PCR 21 samples were positive [24]. If there is a strong suspicion of anaplasmosis or babesiosis and parasitemia is low, detection of parasites may by difficult on a thin blood smear, and molecular techniques are recommended [22].
From the clinical point of view the influence of coinfections on the course of disease and treatment administration is the most important issue. Logina et al. analyzed 51 patients with double infection-TBEV and Borrelia species-and concluded that the clinical occurrence of both LD and TBE varies after exposure to tick bite, and the neurological manifestations of each disorder differ significantly, with appreciable overlap [25]. However, their study provided no proof that co-infection manifested with extraordinary symptoms due to unexpected interaction between these two pathogens. Nevertheless, they suggest that all the patients from endemic areas presenting with acute neurological symptoms after the tick bite should be investigated for both LD and TBE and simultaneous treatment of both conditions should be introduced as quickly as possible.
In another study of 687 patients with TBE 2 % were diagnosed with neuroborreliosis. They more frequently had pleocytosis of over 300 cells/mm³ and statistically higher concentration of protein (88.2 mg/dl vs 67.4 mg/dl). In the group without neuroborreliosis, symptoms like headaches, vertigo, nausea and vomiting were more frequent than neurological symptoms in comparison to the group with neuroborreliosis [26].
On the other hand, Alekseev et al. suggested that Borrelia species might suppress viral replication in ticks and in TBEsusceptible individuals, hence it is still a matter of controversy [27]. In our study no differences in clinical picture between patients with only TBE infection and co-infection with other tick-borne pathogens were stated.
Grab et al. suggested that A. phagocytophilum co-infection contributes to the severity, dissemination and possible sequelae of LD. They showed that co-infection enhanced reductions in transendothelial electrical resistance and enhanced or synergistically increased production of metalloproteinases, cytokines and chemokines, which are known to affect vascular permeability and inflammatory responses [28].
Co-infection with Babesia and Borrelia species may influence the clinical course, especially in non-immunocompetent patients, and might be difficult to diagnose due to the fact that both diseases cause nonspecific symptoms, such as fever, fatigue, and flu-like illness. Patients with an inadequate response to appropriate therapy for proven or suspected LD following a tick bite should be examined for infections with other tick-borne agents, including Babesia species [28]. It is also worth remembering during travel to endemic areas for tick borne diseases, as babesiosis is starting to appear as a travel-related disease [29].
We assume that administration of antibiotic therapy in cases suspected of co-infection should be introduced as soon as possible and should be verified after the advanced laboratory test results, such as molecular biology methods (e.g. PCR). We also should be aware that number of pathogens transmitted on humans by ticks is constantly increasing (e.g. Fransicella tularensis, Bartonella spp., spotted fever rickettsiae group and many others) and further studies are necessary [30].