© Borgis - New Medicine 3/2010, s. 84-87
*Małgorzata Tokarska-Rodak1, Hanna Fota-Markowska2, Maria Kozioł-Montewka1, Filip Śmiechowicz2, Roma Modrzewska2
The detection of specific antibodies against B. burgdorferi s.s., B. afzelii , B. garinii, and B. spielmani antigens in patients with Lyme disease in eastern Poland
1Department of Medical Microbiology, Medical University, Poland
Head of Department of Medical Microbiology: prof. dr hab. Maria Kozioł-Montewka
2Department of Infectious Diseases, Medical University, Lublin, Poland
Head of Department of Infectious Diseases: prof. dr hab. n. med. Roma Modrzewska
Summary
Aim. The purpose of the investigation was to determine the presence of antibodies for B. burgdorferi s.s., B. afzelii , B. garinii , and B. spielmani in persons with Lyme disease from the Lubelski region (eastern Poland) in the aspect of existing disease symptoms.
Materials and methods. The study was conducted in a group of 45 patients with the second stage of Lyme disease hospitalized in 2007-2008. The presence of anti- B. burgdorferi s.l. antibodies IgM and IgG was determined in the patients' serum (Western blot test, Mikrogen).
Results. The antibodies (IgM/IgG) for four gene-species, B. burgdorferi s.s., B. afzelii , B. garinii , and B. spielmani, were present in 39 patients from 45 tested. The presence of IgM anti-OspC antibodies for 1 gene-species, B. spielmani, was found only in 1 person.
Symptoms involving bone/joint structure in the tested group of patients were present among 41 persons, and symptoms of muscle pain among 21. Patients also complained of headaches and attention deficit disorder (6 persons). Erythema migrans appeared in 2 persons.
Conclusions. The results of the study suggest the possibility of co-infections with several gene-species of B. burgdorferi s.l. evidenced by the presence of IgM antibodies to OspC of B. burgdorferi s.s., B. afzelii , B. garinii , and B. spielmani.The detection of antibodies directed against specific B. spielmani antigens suggests that this microorganism may be responsible for triggering Lyme disease both as a single etiologic agent and in co-infection with other Borrelia gene-species. A relation between the types of symptoms present among patients with Lyme disease and gene-species responsible for causing diseases was not found.
Introduction
The complex of Borrelia burgdorferi sensu lato encompasses at least 12 species: B. burgdorferi sensu stricto, B. garinii , B. afzelii , B. spielmani , B. valaisiana , B. lusitaniae , B. japonica , B. andersonii , B. tanukii , B. turdae , B. bissettii and B. sinica. The main pathogenic genomic species responsible for human Lyme disease in Central Europe and Eastern Europe are: Borrelia burgdorferi sensu stricto, Borrelia garinii and Borrelia afzelii. However, the DNA of B. valaisiana, B. lusitaniae , and B. spielmani detected in samples of human origin or the spirochetes were isolated from patients with symptoms of Lyme disease (1, 2). The genetic variability of Borrelia species has a considerable impact on the pathogenesis and clinical picture (3, 4, 5, 6). The heterogeneous clinical manifestation observed in Lyme disease inclined researchers to analyze the relation of infection by particular gene-species of B. burgdorferi s.l. with clinical symptoms that appeared. Borrelia afzelii seems to be associated with acrodermatitis chronica atrophicans (ACA) and with borrelia lymphocytoma, B. garinii with neuroborreliosis and B. burgdorferi s.s with arthritis (2, 3). Patients with EM reveal infection with all three gene-species: B. burgdorferi s.s., Borrelia afzelii and Borrelia garinii. Examinations of cerebrospinal fluid in patients with neurological symptoms of Lyme disease have revealed the possibility of a relation of neuroborreliosis with the presence of each of those three gene-species (7). DNA of B. valaisiana was detected in the cerebrospinal fluid of a patient with chronic neuroborreliosis in Greece and in a patient with erythema migrans. B. lusitaniae was isolated from a patient with suspected Lyme disease in Portugal (3). A direct association of skin changes of erythema migrans (EM) type with an infection of B. spielmani was revealed in some European countries (Netherlands, Germany, Hungary, Slovenia). Thus, the association of these gene-species with Lyme disease has been proven (8). The number of substantiated infections of B. spielmani is still insufficient in order to present the differences in Lyme disease manifestations caused by B. spielmani in comparison with Lyme disease caused by other gene-species. To date, an investigation which could explicitly affirm the relation of Lyme infection morbidity with infection by B. spielmani within Poland has not been performed yet.
Aim of the study
The aim of the study was to determine the presence of antibodies for four gene-species of Borrelia burgdorferi s.l. - B. burgdorferi s.s., B. afzelii , B. garinii , and B. spielmani - in persons with Lyme disease from the Lubelski region (eastern Poland) in the aspect of existing disease symptoms.
Materials and methods
The study was conducted in a group of 45 patients: 23 men (age 20-66 yrs) and 22 women (age 20-60 yrs) with the second stage of Lyme disease hospitalized in the Department of Infectious Diseases, Medical University of Lublin in 2007-2008.
The study group involved patients demonstrating manifestations of joint involvement including arthralgia and/or arthritis treated symptomatically and receiving etiotropic antimicrobial therapy. The diagnosis of borreliosis was established on the basis of the patient's medical history, physical examination, clinical picture, and serologic investigation (ELISA and Western blot).
Testing for antibodies IgG and IgM to Borrelia burgdorferi sensu lato was performed using the Western blot test (recomLine Borrelia IgG and IgM, Mikrogen) which included antigens for IgM and IgG: OspC ( B. burgdorferi s.s., B. afzelii , B. garinii , B. spielmani), p18 ( B. burgdorferi s.s., B. afzelii , B. garinii , B. spielmani), VlsE, p100, p58, p41, p39, OspA. The presence of anti- B. burgdorferi sensu lato antibodies IgM and IgG was determined in the patients' serum.
Results
IgM antibodies alone against specific Borrelia burgdorferi s.l. antigens were detected in 17 of 45 patients (38%). The IgM antibodies anti-OspC and anti-p18 B. burgdorferi s.l. were observed with varying frequency in this group. Table 1 shows IgM antibodies anti-OspC and anti-p18 B. burgdorferi s.s., B. afzelii , B. garinii, and B. spielmani in persons with Lyme disease.
Table 1. IgM antibodies against B. burgdorferi s.l. specific antigens in patients with Lyme borreliosis.
| N=17 | Presence of IgM anti-B. burgdorferi s.l. antigens |
| anti-OspC | anti-p-18 | anti-p41 |
| 12 | b, a, g, s | - | + |
| 1 | b, a, g, s | b, s | + |
| 2 | a, g, s | - | + |
| 1 | b, a, s | - | + |
| 1 | a, s | - | + |
b – Borrelia burgdorferi sensu stricto
a – Borrelia afzelii
g – Borrelia garinii
s – Borrelia spielmani
+ presence of antibodies
IgM and IgG antibodies against specific Borrelia burgdorferi s.l. antigens were detected in 28 of 45 patients (62%). Table 2 shows IgM and IgG antibodies anti-OspC and anti-p18 for B. burgdorferi s.s., B. afzelii , B. garinii , and B. spielmani in persons with Lyme borreliosis.
Table 2. IgG antibodies against specific Borrelia burgdorferi s.l. antigens in patients with Lyme borreliosis.
| N=28 | Presence of IgM
anti-B. burgdorferi s.l. antigens | Presence of IgG
anti-B. burgdorferi s.l. antigens | Presence of IgM/IgG |
| anti-OspC | anti-p-18 | anti-OspC | anti-p18 | against other specific antigenic
B. burgdorferi s.l protein |
| 8 | b, a, g, s | - | b, a, g, s | - | + |
| 4 | b, a, g, s | - | - | - | + |
| 1 | b, a, g, s | - | a | a | + |
| 3 | b, a, g, s | - | - | a | + |
| 1 | b, a, g, s | - | - | - | + |
| 1 | b, a, g, s | | b, g, s | a, g1, g2 | + |
| 1 | b, a, g, s | - | b, a | - | + |
| 1 | b, a, g, s | s | b, a, g, s | - | + |
| 1 | b, a, g, s | s | - | g2 | + |
| 1 | b, a, g, s | - | g, s | - | + |
| 1 | b, a, g, s | - | b | - | + |
| 1 | b, a, g, s | - | b, g, s | - | + |
| 1 | a, s | s | - | a | + |
| 1 | b, a, g, s | - | a | b | + |
| 1 | s | - | - | - | + |
| 1 | b, a, s | s | g, s | - | + |
b – Borrelia burgdorferi sensu stricto
a – Borrelia afzelii
g – Borrelia garinii
s – Borrelia spielmani
+ presence of antibodies
The IgM and IgG antibodies anti-VlsE, p100, p58, p41, p39 and p18 were observed in this group with various frequency. IgM anti-p39 antibodies were observed in 6 patients, anti-p100 in 5 patients, and anti-OspA in 1 patient. The most frequently detected were IgG anti-in-vivo VlsE antigens in 27 patients. Less frequently were detected: IgG anti-p39 in 11 patients, anti-p100 in 11 patients, and anti-p58 in 6 patients.
Symptoms of Lyme disease in the aspect of infection with different gene-species of B. burgdorferi s.l.
Among 45 persons with the presence of IgM or IgM and IgG anti- B. burgdorferi antibodies, 23 reported being bitten in 2007 whereas the others reported being bitten in 1980-2006. Among tested persons, 21 reported multiple bites and 24 single bites.
All of the tested persons underwent antibiotic treatment in relation to the symptoms of Lyme disease (in 38 of them due to EM).
Symptoms involving the bone/joint structure (back pain, hip joint, talocrural joint, knee joint and shoulder joint syndromes) in the tested group of patients were present among 41 persons, while muscle pains occurred among 21. Patients also complained of headaches and attention deficit disorder (6 persons). Erythema migrans appeared in 2 persons.
The occurrence of mixed infections among the tested group of patients with Lyme disease symptoms can be assumed on the basis of the presence of IgM and IgG antibodies for specific B. burgdorferi s.s., B. afzelii , B. garinii, and B. spielmani antigens. Antibodies for the four mentioned gene-species of Borrelia were present in 39 patients from 45 tested. It was observed that 1 patient had antibodies against specific B. burgdorferi s.s., B. afzelii, and B. spielmani antigens, and 2 patients had antibodies against antigens of B. afzelii, B. garinii , and B. spielmani gene-species. Antibodies against specific B. afzelii and B. spielmani antigens were observed in 2 patients. The presence of IgM anti-OspC antibodies for 1 gene-species ( B. spielmani) was found only in 1 person.
Discussion
Lyme disease is of a worldwide scope and it has become a significant problem both diagnostically and therapeutically in contemporary medicine more than once. The dissemination of Lyme disease is dependent on geographical, environmental and climate factors and on the pathogenicity of species of B. burgdorferi sensu lato. Additionally, the distribution of gene-species can vary locally. The largest risk of this disease is present in the north-eastern regions of Poland (Puszcza Białowieska, Mazury, Podlasie, Roztocze), but it is also reported across the whole country (Pomorze Zachodnie, area near Wrocław, area near Góry Tarnowskie) (4, 9, 10, 11).
In the eastern side of Poland the percentage participation of separate gene-species of B. burgdorferi s.l. in infection of ticks is estimated as follows: B. burgdorferi s.s 37%, B. afzelii 44%, B. garinii 31%. Slightly different frequencies of participation of these gene-species were noted in north-west Poland: B. burgdorferi s.s 41%, B. afzelii 8%, B. garinii 23%, B. valaisiana 24%, B. lusitaniae 5% (7).
In the Lubelski region of Poland (eastern Poland) in 81.4% of infected ticks only single infection with 1 gene-species was observed, while co-infections with 2 or 3 gene-species were detected respectively in 16.8% and 1.8% of infected ticks. The authors of this work found Borrelia burgdorferi sensu stricto the dominant gene-species in the evolution of all examined ticks. It was present as the single infectious microorganism or together with other gene-species (in co-infections) and was found in a total of 62.8% of I. ricinus ticks infected with B. burgdorferi s.s. Other gene-species such as Borrelia afzelii and Borrelia garinii were detected in respectively 39.8% and 17.8% of the infected ticks (12).
All species of B. burgdorferi s.l. complex present in Europe are pathogenic for humans. They were isolated from bodily fluids and tissues of patients with Lyme disease symptoms. However, only three of them, B. burgdorferi s.s., B. afzelii , and B. garinii, cause more severe symptoms and complications in the event of lack of or improper therapy (13, 14, 15).
According to conducted research among examined patients, anti-OspC B. afzelii antibodies were the most frequently detected (44 persons); more seldom were B. burgdorferi s.s. (40 persons) and B. garinii (40 persons). The association of these species with Lyme disease is commonly acknowledged within European countries. The presence of antibodies IgM and IgG directed against specific B. spielmani antigens, which are usually accompanied by IgM or IgG anti-OspC of other Borrelia gene-species (in 44 persons from 45 examined), is quite controversial and surprising.
The fact that in the examined patients the presence of antibodies against specific antigens of B. spielmani was affirmed suggests the presence of this gene-species in the animal reservoir within Poland. Currently, data on the subject of the B. spielmani reservoir concern the garden dormouse ( Eliomys quercinus). These rodents also occur locally in Poland and are still considered as the only reservoir of B. spielmani, which in this case reduces the risk of transmission of bacteria by ticks from rodents to humans. The presence of other reservoir species is not definitely excluded (5).
The confirmation of the presence of B. spielmani in the animal reservoir would suggest the ability of this gene-species to participate in the induction of Lyme disease both separately and with other gene-species of Borrelia recognized as pathogenic in Poland.
Diagnostic tests used routinely in Lyme disease diagnosis in Poland (ELISA, Western blot) usually have antigen extracts of B. burgdorferi s.s., B. afzelii , B. garinii or electrophoretically separated antigen extracts of Borrelia afzelii enriched with recombinant VlsE antigen. According to researchers, the antigens of B. spielmani gene-species, which researchers mentioned as the fourth along with B. burgdorferi s.s., B. afzelii , and B. garinii, should be additionally used in the tests and be considered in the diagnosis of Lyme disease within Europe (5, 7, 8).
It has been confirmed in studies that in 39 persons from 45 examined, the IgM anti-OspC antibodies for four gene-species - B. burgdorferi s.s., B. afzelii , B. garinii, and B. spielmani - were present simultaneously. On this basis, it can be assumed that co-infections with a few gene-species of B. burgdorferi s.l. are not rare and PCR tests are essential in order to confirm or exclude this hypothesis.
It is significant from the serological diagnosis point of view whether IgM/IgG antibodies, which are produced in an immunological response to an infection caused only by B. spielmani, can be detected with diagnostic tests used conventionally in the diagnosis of Lyme disease. From the examined 45-person group with symptoms of Lyme disease, the simultaneous presence of the antibodies IgM anti-OspC B. spielmani and anti-p41 along with IgG anti-VlsE antibodies was affirmed in 1 person. The identification of anti-OspC B. spielmani antibodies seems to be extremely important considering the previous stage of immunological response when only IgM anti-OspC antibodies are frequently present without antibodies of IgG class. In the case of infection with only one gene-species, the identification of IgM anti-OspC antibodies may be the only chance in the early identification of an infection with a lack of skin symptoms such as erythema migrans (EM) (2, 3, 7).
According to reports, differences in sensitivity to antibiotics within the three gene-species B. burgdorferi s.s., B. afzelii , and B. garinii are possible. Sicklinger et al. found the highest susceptibility of B. burgdorferi s.s. of the three species to amoxicillin. Borrelia afzelii proved to be especially susceptible to ceftriaxone and B. garinii demonstrated excellent sensitivity to azithromycin (2).
Preac-Mursic et al. reported that the susceptibilities of B. garinii to amoxicillin, doxycycline, cefotaxime, ceftriaxone, azithromycin and penicillin G were higher than those of B. afzelii (16). The differentiation of gene-species of B. burgdorferi s.l. detected in the collected material from patients has a serious justification.
The affirmation of the presence of antibodies for specific B. burgdorferi s.l. antigens is only indirect proof suggesting the simultaneous participation of all four gene-species ( B. burgdorferi s.s., B. afzelii , B. garinii , B. spielmani) in triggering Lyme disease. However, on this basis we note the necessity to undertake further studies with the use of PCR to ultimately confirm or exclude the participation of B. spielmani in triggering clinical symptoms of Lyme disease in persons from the Lubelski region.
Conclusions:
1. The results of the study suggest the possibility of co-infections with several gene-species of B. burgdorferi s.l. evidenced by the presence of IgM/IgG antibodies to OspC and p18 of B. burgdorferi s.s., B. afzelii , B. garinii , and B. spielmani.
2. The fact that in the examined patients the antibodies against specific antigens OspC and p18 of B. spielmani are present suggests that this gene-species is also present in the animal reservoir within eastern Poland and can be responsible for participation in the induction of Lyme disease both separately and with other gene-species of Borrelia recognized as pathogenic within this region.
3. An association between the types of symptoms present among patients with Lyme disease and gene-species responsible for causing diseases was not found.
Piśmiennictwo
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