Welkom bij het Lymeforum

On April 1 2012, a 70-year-old patient came to our clinic reporting slow cognitive processing, memory deficits, and a disturbed gait, all of which had gradually developed over several months and progressed during the last few weeks before the patient’s initial visit. He did not report fever, and he had not been outside the country for several years. He had recently been treated with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisolone), poly chemotherapy, and rituximab (last dose on Aug 2, 2011) for a stage 4 diffuse large B cell lymphoma. His medical history also included Pneumocystis jirovecii pneumonia, un explained chronic diarrhoea, a splenectomy, extensive tick exposure, and two tick bites in the summer and fall before onset of symptoms. On neurological examination there was a distinct bradyphrenia, and on cognitive assessment, the patient scored 26 of 30 points on the mini mental state examination. Vital signs were normal and body temperature was 36·4°C. Cranial MRI showed no abnormalities, but two lumbar punctures showed cerebrospinal fluid pleocytosis with raised protein values. The cause of this chronic meningitis was not identified by wide-ranging micro biological, pathological, and haematological diagnostic testing (appendix). A C6-immunofluorescence assay for Borrelia burgdorferi in serum, but not in cerebrospinal fluid, was weakly positive (index 1·8). However, a B burgdorferi IgG and IgM immunoblot were non-conclusive and negative, respectively. A B burgdorferi sl qPCR in cerebrospinal fluid was negative. Nonetheless, because of the absence of an alternative diagnosis and the progression of symptoms, on April 17, 2012, the patient was treated for a possible Lyme neuroborreliosis with once daily 2000 mg ceftriaxone intravenously for 2 weeks.

Supported by the recent evidence of the presence of B miyamotoi in Ixodes ricinus ticks across Europe,1,2 the relation in time of the patient’s symptoms with the tick bites, and his immunocompromised status, we retrospectively considered B miyamotoi as the causative agent. We identified motile spirochaetes in stored pre-treatment cerebrospinal fluid by dark-field microscopy (appendix). Additionally, a 16S rDNA pan-relapsing fever Borrelia quantitative (q)PCR and a qPCR targeting the B miyamotoi flagellin gene was positive in two separate pre-treatment cerebrospinal fluid samples and one pre-treatment blood sample (appendix). Notably, 2·2% of 352 I ricinus nymphal ticks from the vicinity of the patient’s recreational house in the dunes of Zandvoort, the Netherlands, proved to be positive for B miyamotoi by qPCR (appendix). Amplification and sequencing of the glpQ and p66 genes confirmed B miyamotoi as the causative agent and showed 100% identical sequences in ticks and the patient’s clinical samples

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1. Introduction

Reliable diagnosis of Lyme disease caused by Borrelia burgdorferi at its early stage of infection is important for the timely implementation of appropriate treatments to prevent tissue damages and to achieve a “cure” of the disease [1]. However, the diagnosis of Lyme disease is primarily based on evaluating the highly variable symptoms, physical findings (e.g., rash), and the possibility of exposure to infected ticks [2,3]. The commonly used 2-tier serology laboratory test which usually only turns positive during convalescence of the infection is reported to be negative or non-diagnostic in 75% of the “clinically confirmed” cases of early Lyme disease [4]. Blood culture at the stage of bacteremia has met only limited success in specialized laboratories and offers little help to the timely management of patients suffering from early Lyme disease bacterial infection due to the slow growth rate of the spirochetes in artificial media [5]. Conventional polymerase chain reaction (PCR) amplification of the bacterial DNA for detection is not sensitive enough for routine diagnostic purpose because the copies of the target DNA extracted from the very low number of spirochetes in the patient blood samples are often below the limit of detection [6,7]. False positive PCR results have also been reported, and may be due to non-specific PCR amplification of irrelevant DNA in the patient samples [8,9]. Using species-specific primers, such as the LD1 and LD2 [10,11], and TEC1 [12] primers to amplify a highly conserved segment of the 16S ribosomal RNA gene DNA (16S rDNA) of B. burgdorferi sensu lato for detection, followed by direct DNA sequencing of the nested PCR amplicon for validation, has been used to provide sensitive and specific molecular diagnosis of Lyme arthritis [13] and early Lyme disease at the spirochetemic stage [14].

Recently, human infections by Borrelia miyamotoi—a spirochete distantly related to B. burgdorferi, but classified in the relapsing fever Borrelia group—have been reported in the United States [15,16]. Since B. miyamotoi is transmitted by the same Ixodes vector as B. burgdorferi sensu stricto [17], and may cause clinical symptoms similar to those of Lyme disease, including skin rashes [18], B. miyamotoi infection should be included in the differential diagnosis of patients presenting with unexplained fever, headache and myalgia with or without skin rash in Lyme disease-endemic areas during the seasons when ticks are active. Molecular assays are the only option to test for B. miyamotoi infection because this spirochete is hard to culture in modified Barbour-Stoenner-Kelly medium and the currently available two-tier serology test offers little help for its diagnosis [15,18].

In this paper, we introduce a pair of common PCR primers for same-nested PCR amplification of a highly conserved segment with hypervariable regions of the 16S rDNA of B. burgdorferi and B. miyamotoi followed by direct DNA sequencing of the PCR amplicon for the molecular diagnosis of these two borrelial infections in patients with spirochetemia. This highly sensitive, DNA sequencing-based test can even diagnose off-season spirochetemias with low bacterial density in the deep winter months in the Northeast of the United States when human exposure to tick bites was very limited.

2. Results and Discussion

Nested PCR amplification of a 600-nucleotide fragment of the 16S rDNA followed by direct DNA sequencing of the PCR amplicon has been used for construction of the phylogenetic tree of various Borrelia species [18,19]. One obstacle in transferring this methodology to diagnosing Lyme disease for patient care is the very low number of spirochetes in the blood of the patients even at the spirochetemic stage of the infection [6,20], and B. burgdorferi sensu stricto contains only one copy of 16S rDNA per cell [21]. PCR amplification of a 600-bp target DNA for direct DNA sequencing is technically challenging in a routine diagnostic laboratory because the sensitivity of 16S rDNA PCR amplification in diagnostic clinical microbiology is inversely related to the size of the PCR amplicon [22]. In addition, to construct the phylogenetic tree it is desirable to perform DNA sequencing on a segment of the gene with highly variable regions for its discriminatory power to distinguish different closely related species. The 600-nucleotide fragment of 16S rDNA selected for borrelial speciation [18,19] encompasses the highly discriminatory V-regions 1, 2, 3 and 4 of the 16S rDNA [23]. However, for molecular diagnostic nucleic acid amplification tests it is more beneficial to target less species-specific regions for amplification so that one fixed set of primers may detect as many clinically relevant pathogens of the same group as possible for timely diagnosis and patient management. For the latter reason, clinical laboratory diagnosticians are most interested in developing general PCR primers capable of amplifying a highly conserved segment of the 16S rDNA for all of the three tick-borne pathogenic borrelial species known to be prevalent in Europe and in the U.S.A., namely the species of B. burgdorferi sensu stricto, B. garinii and B. afzelii, often collectively referred to as B. burgdorferi sensu lato [24].

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