OPEN Research Support
head

PhD Student
Trine Andreasen
Department of Clinical Microbiology, Vejle Hospital


Projekt styring
Projekt status    Sampling ongoing
 
Data indsamlingsdatoer
Start 01.02.2018  
Slut 31.10.2021  
 



Improved PCR diagnostics in Borrelia infections

Short summary

The PCR technique has revolutionized the diagnostics of diseases caused by microorganisms due to its high sensitivity and specificity. Studies using PCR in the diagnostics of Borrelia infections in the nervous system have shown variable results regarding sensitivity, supposedly because of a low number of Borrelia bacteria in the cerebrospinal fluid. In this prospective study, cultivation of Borrelia bacteria begins at bedside during lumbal puncture. This facilitates the most optimal pre-enrichment conditions prior to PCR analysis, thus aiming for improved PCR sensitivity.


Rationale

The bacteria Borrelia burgdorferi sensu lato (Bb) is the etiologic agent of acute Lyme Neuroborreliosis (LNB). Borrelia burgdorferi sensu lato is a collective name for at least 18 different species, where B. afzelli, B. burgdorferi sensu stricto and B. garinii are the major pathogens in humans. Bb is a gram-negative, spiral-shaped, motile, microaerophilic rod (spirochete). The genome of the Bb species is very segmented consisting of a relatively conserved linear chromosome of approximately 1 million base pairs and about 20 linear and circular plasmids. The genome has several genes encoding envelope lipoproteins. These proteins are differentially expressed, which helps the bacteria to adapt to changing environments in the host animal and tick. The alternating expression of surface antigens is essential for Bb, because it secures a lengthy survival in the tick gut and helps establish infection in humans and protects Bb from the immune system.

The tick-borne Bb bacteria enter humans through a tick bite on the skin and may disseminate from there to secondary organs, including the central nervous system (CNS). Cardinal neurological symptoms of LNB are radicular pains and paresis. The most common manifestation of acute LNB is a sub-acute painful meningo-radiculitis also called Bannwarth's syndrome, which is defined by, a painful radiculitis, peripheral motor paresis, and CNS inflammation. It is certain that there are living Bb in the CNS of patients with LNB, as they have been identified by polymerase chain reaction (PCR) and culture of cerebrospinal fluid (CSF), and since antibiotics e.g. penicillin, work effectively on symptoms. The histopathological reaction of CNS consists primarily of lymphocytic infiltration by T- and B-cells, but also of plasma cells and macrophages. Intrathecal synthesis of Bb-specific antibodies in the CSF is detectable in most patients at week 6 after the onset of neurological symptoms. A CSF to serum antibody index, assessing the intrathecal antibody production, can be calculated and used as diagnostic tool. However, the rate of the seroconversion from negative to positive may differ from individual to individual, which means that the result can be false-negative in up to two months and that it can be positive for years following successful treatment.

Today, when a patient is hospitalized with a suspected CNS infection, a diagnosis is based on a constellation of anamnestic, clinical, and laboratory findings. In the clinical laboratory, examinations are made for identification of the infectious agent in combination with methods examining immune responses. A lumbar puncture will be performed to examine the cerebrospinal fluid for i.e. changes in cell count, changes in glucose and protein levels, immunoglobulin level, microscopy and culture of bacteria and PCR for identification of microorganisms. The available laboratory techniques for the diagnosis of LNB are in 2 categories: direct methods to detect Bb or its DNA, and indirect methods that detect the immune response against it. Unfortunately, the positivity rates of Bb culture testing and specific PCR are unsatisfyingly low, leading to the use of indirect serological tests. 

The aim of this project is to improve the clinical PCR sensitivity for Borrelia identification by use of pre-enriched Borrelia samples. The cultivation of Borrelia bacteria will already begin at bedside during specimen collection by lumbal puncture.


Description of the cohort

The cohort will consist of men and women, adults and children (> five years) examined for neuroborreliosis. A written consent is an inclusion criteria. 


Data and biological material

Sample material will be cerebrospinal fluid. 

Clinical data such as; symptoms, disease duration, anamnesis, will be collected systematically from patient journals. 

Paraclinical data such as; CRP, csf cell count, csf protein concentration, csf CXCL13 concentration, microbiological culture and PCR results etc.


Collaborating researchers and departments

Department of Clinical Microbiology, University Hospital Lillebaelt

  • Professor Jens Kjølseth Møller, PhD
  • PhD student Trine Andreasen

Department of Infectious Diseases Q, Odense University Hospital

  • Consultant Sigurdur Skarphedinsson, PhD

Department of Clinical Microbiology, Odense University Hospital

  • Senior registrar, Nanna Skaarup Andersen, PhD