Borrelia miyamotoi DNA in a patient suspected of Lyme borreliosis

Introduction and Objective . Manifestations of infection caused by Borrelia miyamotoi can mimic highly variable symptoms of Lyme borreliosis. The aim of the study was to detect DNA from B. miyamotoi samples from patients with suspected neuroborreliosis. Materials and Method . Samples of blood serum and cerebrospinal fluid (CSF) were collected from 133 patients. Diagnosis was established by the detection of specific antibodies to Borrelia burgdorferi sensu lato (s.l.) with ELISA and immunoblot. All Borrelia-positive samples were tested by nested PCR for the B. miyamotoi and B. burgdorferi s.l. DNA. Results . B. miyamotoi DNA was detected in the CSF of one (0.8%) patient. DNA of B. burgdorferi s.l. was not found in any samples. Conclusions. Detection of the B. miyamotoi in patients with central nervous system infections expand the development of knowledge on infections caused by Borrelia spirochetes.


INTRODUCTION
The spirochetes of Borrelia miyamotoi belong to the group of bacteria that cause relapsing fever (Borrelia Relapsing Fever Group).These bacteria were first isolated from Ixodes persulcatus in Japan in 1995 [1,2].Subsequently, B. miyamotoi DNA has been found in Ixodes ticks in Asia, North America and Europe [3,4].They were considered non-pathogenic bacteria until the first human cases of B. miyamotoi disease were diagnosed in Russia in 2009 [2,5].The status of B. miyamotoi as a pathogen was established only recently; subsequently, cases have been described in the United States, Europe and Asia.There have been 561 total diagnosed cases: 367 in Russia, 101 in the United States, 57 in France, 30 elsewhere in Asia, and 6 elsewhere in Europe [2,6].
Both in the USA and in Europe, it has been reported that BMD in people causes flu-like symptoms and neurological abnormalities [2,6].Routine serological test C6 ELISA in the confirmation of B. burgdorferi s.l.can detect B. miyamotoi antibodies in 50-80% of samples [10].
Recent serological studies on reactivity to GlpQ and Vmp proteins ('in-house' test) have revealed maximum sensitivities of 79% for IgM and 86.7% for IgG and a specificity of 100% for IgM antibodies, and 98.3% for IgG.[11].Molecular tests (PCR or RT-PCR) are currently more appropriate and reliable methods for routine diagnostics [12].The genes detected most frequently in the molecular diagnostics of B. miyamotoi are glpQ, p66, and fla genes [2].The glpQ gene is present in relapsing fever Borrelia but not in B. burgdorferi s.l. and therefore can discriminate between the two types [13].

OBJECTIVE
The aim of the study is to detect DNA from B. miyamotoi samples from patients with suspected neuroborreliosis.

MATERIALS AND METHOD
Only patients with meningitis, neck stiffness, facial nerve palsy, and cerebrovascular diseases were included in the study.All had a tick bite history within the last six months.
No information was available on the history of erythema migrans in the study subjects.Blood and cerebrospinal fluid (CSF) samples from 133 patients (72 women and 61 men) were taken from the patients within one or two days after the onset of symptoms.A total of 266 samples (serum and CSF from each patient) were tested with Borrelia burgdorferi s.l.ELISA (DRG MedTec, Germany) and immunoblot (Euroimmun, Germany) [14].DNA was extracted from CSF and serum samples with a Syngen Tissue DNA kit (Syngen Biotech).The presence of B. miyamotoi in DNA isolates was detected by the amplification of three loci: the glycerophosphodiester phosphodiesterase (glpQ) gene, the p66 gene, and the flagellin (fla) gene.Nested PCR targeting a fragment of glpQ gene and p66 gene was performed with Gold Hot Start PCR MIX LOAD (Syngen Biotech, Poland) [13].Two sets of primers were used to amplify a fragment of fla gen [13,15].
A positive sample was analyzed with primers targeting a 723 bp fragment of the glpQ gene [16].Each run of the PCR included positive (B.miyamotoi DNA, concentration 5×10 4 organisms/mL) and negative controls (water).The PCR products of glpQ (425 bp and 700 bp), p66 (569 bp), and fla (411 bp) genes were sequenced and identified using BLAST software.
The presence of B. burgdorferi s.l.DNA was investigated with PCR in CSF and serum samples to rule out the cases of co-infection [17].
DNA of B. miyamotoi (glpQ, p66 and fla genes) was detected in the CSF of one patient.DNA of B. burgdorferi s.l. was not found in any samples.

CASE STUDY
A 47-year-old alcoholic male from the Warsaw area was admitted to hospital in 2011 after suffering for three months from blurred vision in the left eye.No history of fever, recurrent fever, erythema migrans (EM) was found in the medical interview.Ophthalmoscopy examination revealed a mildly oedematous optic disc.The vessels, macula lutea, and retina were normal.Extraocular optic neuritis of the left eye was recognized.Routine laboratory investigations did not show abnormalities.Magnetic resonance imaging (MRI) revealed abnormalities in hyperintense signal in the white matter of the brain hemispheres (FLAIR-T2 images).The optic nerve was thinned and obliterated, which was indicative of fibrosis of the nerve and its sheath.In addition, some demyelinating changes were found in both hemispheres.The parameters of the CSF were as follows: elevated total protein 107 mg/dL ( No. MK674170) revealed 100% homology to the sequences: LC164098, KU749386, and KJ950108.The 723 bp fragment of the glpQ gene (Acc.No. MK674171) revealed 100% homology to B. miyamotoi sequences: AP024399, CP036914, and CP037471 from Ixodes persulcatus or human blood (Fig. 1).The omp66 gene fragment (Acc.No. OP946656) revealed 100% homology to B. miyamotoi sequences: MN689815, AP024396, and CP024351 from I. persulcatus and human blood.The results are summarized in Figure 2.
The fla gene fragment (Acc.No OP946657) revealed 100% homology to B. miyamotoi sequences: CP037471, CP037215, and KU749379 from I. persulcatus and human blood (Fig. 3).All sequences obtained were identical to those of B. miyamotoi obtained from Asia.

DISCUSSION
Molecular results confirmed the presence of an etiological agent of BMD in the CSF of one patient.In serological results, antibodies to B. miyamotoi infection probably cross-reacted with B. burgdorferi antigens in the serological tests for B. burgdorferi.
However, from obtained results, it cannot be unequivocally excluded that co-infection with B. burgdorferi s.l. and B. miyamotoi due to the low sensitivity of B. burgdorferi s.l.PCR -B.miyamotoi spirochetes often co-exist with B. burgdorferi s.l. in ticks [13,19].Mixed infections with B. afzelii, B. burgdorferi sensu stricto or B. garinii have also been recognized.This indicates the possibility of mixed infections of this etiology in humans.In the co-infections, B. burgdorferi s.l. is responsible for the development of erythema migrans, while B. miyamotoi can cause meningoencephalitis, mainly in immunosuppressed persons, including alcoholics [2,20].
Optic neuritis has not been reported in patients infected with B. miyamotoi so far [21].B. miyamotoi infection may    cause pathological changes, including erythrocyte aggregates and obstructed sinuous capillaries [22].In 79% of patients with B. miyamotoi, organ dysfunctions were found by microscopic examination of the eye capillary blood flow.Patients with neurological symptoms and questionable serological findings pose a serious diagnostic problem due to the failure to meet the criteria for neuroboreliosis.In Poland, 25,293 cases of Lyme borreliosis have been registered, including 471 (1.9%) cases of neuroborreliosis in 2023 [23].Detection of this bacterium in patients with central nervous system infections expand the development of knowledge on infections caused by Borrelia spirochetes, allow diagnosis in severe neurological cases of infections caused by spirochetes, and reduce the time to initiate treatment.

CONCLUSIONS
Patients with neurological symptoms and questionable serological findings are a serious diagnostic problem due to failure to meet the criteria for neuroboreliosis.This indicates the need for further studies in patients with signs of the central nervous system (CSN CNS) infection.In the current study, B. miyamotoi infection in a patient with extraocular optic neuritis was confirmed by sequencing the amplified products of PCR (fragments of fla, omp66 and glpQ genes).The influence of detected transversion within the qlpQ B. miyamotoi gene on function and changes in the structure of the encoded protein was not determined, and further research is necessary.
This study is a commentary on the question of whether patients with specific B. burgdorferi s.l.antibodies in blood serum only (negative CSF result) can also be regarded confirmed cases of neuroborreliosis, and whether the criteria of EFNS (neurological symptoms, cerebrospinal fluid pleocytosis, specific antibodies of B. burgdorferi s.l.produced intrathecally), should be modified [18].

Figure 1 .
Figure 1.Phylogenetic tree of glpQ gene of Borrelia spp., constructed by MEGA (ME) analysis using MEGA version 11.For ME analysis (nucmodel=codon), the T93+G model was chosen based on jModelTest version 2.1.4[24, 25] using Akaike Information Criterion.Hosts, country and GenBank accession numbers of origin are shown.Nodal support is indicated as MEGA posterior probabilities.Sequences generated are show in bold.

Figure 2 .
Figure 2. Phylogenetic tree of omp66 gene of Borrelia spp., constructed by MEGA (ME) analysis using MEGA version 11.For ME analysis (nucmodel=codon), the T92+G+I model was chosen based on jModelTest version 2.1.4[24, 25] using Akaike Information Criterion.Hosts, country and GenBank Accession Numbers of origin are shown.Nodal support is indicated as MEGA posterior probabilities.Sequences generated are show in bold

Figure 3 .
Figure 3. Phylogenetic tree of flaB gene of Borrelia spp., constructed by MEGA (ME) analysis using MEGA version 11.For ME analysis (nucmodel=codon), the T92+G model was chosen based on jModelTest version 2.1.4[24, 25] using Akaike Information Criterion.Hosts, country and GenBank accession numbers of origin are shown.Nodal support is indicated as MEGA posterior probabilities.Sequences generated are show in bold.

Table 1 .
ELISA test results of patients with clinical symptoms of Lyme borreliosis