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Other research product . Other ORP type . 2022

Establishment of molecular assays for improved diagnostics of acute Lyme neuroborreliosis

Leth, Trine Andreasen;
Closed Access
English
Published: 11 May 2022
Publisher: Syddansk Universitet. Det Sundhedsvidenskabelige Fakultet
Country: Denmark
Abstract

Bakterien Borrelia burgdorferi sensu lato er den ætiologiske årsag til Lyme neuroborreliose (LNB). Borrelia bakterier overføres til mennesker efter bid fra skovflåten, og kan spredes fra bidestedet i huden til andre organer, herunder nervesystemet. Lymfocytisk meningoradiculitis (Bannwarths syndrom) er en typisk manifestation af tidlig LNB, som er defineret ved stærke, udstrålende nervesmerter (radikulitis), halvsidige ansigtslammelser og lymfocytær pleocytose i cerebrospinalvæske (CSF). Det må antages som sikkert, at der er levende Borrelia bakterier i centralnervesystemet (CNS) hos patienter med LNB, da de er blevet identificeret ved PCR-analyse og dyrkning af CSF, og da penicillin virker effektivt hos patienter med symptomer på LNB. Desværre er den kliniske sensitivitet af både Borrelia dyrkning og PCR ikke tilfredsstillende høj, hvilket har ført til brugen af serologiske test. LNB diagnosticeres derfor primært på en tilstedeværelse af neurologiske symptomer, CSF pleocytose, samt påvisning af intratekalt produceret Borrelia specifikke antistoffer bestemt ud fra et beregnet CSF/serum-antistofindeks. Hos nogle patienter, med kort sygdomsvarighed, kan antistofproduktionen være meget lav og ikkepåviselig, hvilket vanskeliggør en skelnen mellem LNB og andre sygdomme i CNS. Det er derfor ønskeligt at finde supplerende metoder til at forbedrer diagnostikken af LNB, særligt i sygdommens tidlige stadie. I denne PhD-afhandling vil jeg først give en introduktion til Borrelia burgdorferi sensu lato bakterien, og give et overblik over diagnosticeringen af LNB, samt de muligheder og udfordringer, der er knyttet dertil. Derefter vil de tre studiers formål, metoder og resultater blive gennemgået og diskuteret. Studie I " Discriminating between Lyme neuroborreliosis and other central nervous system infections by use of biomarkers CXCL13 and IL-6"Denne tværsnitsundersøgelse inkluderede patienter under udredning for neuroinfektioner. Mængden af CXCL13 og IL-6 i CSF blev bestemt samtidigt ved brug af et bead-baseret multiplex Bio-Plex 200 System. Patienterne blev grupperet i sikker LNB, mulig LNB, viral CNS-infektion, anden bakteriel CNS-infektion, anden CNS-sygdom (med pleocytose) og negativ (uden pleocytose) baseret på kliniske og parakliniske fund. En Principal Component Analyse (PCA) blev udført under anvendelse af resultater (CXCL13, IL-6, leukocytcelletal og proteinkoncentration) fra den laboratoriemæssige analyse af CSF. PCA afslørede tre distinkte klynger (sikker LNB, anden bakteriel CNS-infektion og negativ (uden pleocytose) baseret på leukocytcelletal, proteinkoncentration, CXCL13- og IL-6-mængder i CSF fra 380 inkluderede patienter. Mulige LNB-tilfælde viste sig at være fordelt på tværs af forskellige klynger, med forskellige sandsynligheder for at tilhøre LNB-klyngen. En ROC-analyse indikerede en optimal CXCL13-cut-off-værdi på 50,7 pg/ml, hvilket resulterede i en sensitivitet og en specificitet på henholdsvis 93,6 og 91,1%, når man sammenlignede patienter med sikker LNB-diagnose med ikke-LNB-tilstande med CSF-pleocytose. Studie II " Establishment of a droplet digital PCR protocol for detection of Borrelia burgdorferi sensu lato DNA in cerebrospinal fluid " Studie II er en systematisk validering af en digital droplet-PCR-analyse (ddPCR), samt optimering af præanalytiske parametre til påvisning af Borrelia bakterier. Protokollen blev optimeret ved hjælp af syntetiske DNA-gBlocks og dyrkede Borrelia referencestammer. Den optimerede ddPCR-protokol blev evalueret på kliniske prøver fra patienter undersøgt for LNB (n=59). Den optimerede ddPCR-protokol inkluderede et centrifugeringstrin til koncentrering af prøvematerialet før DNA-oprensningen. Den analytiske følsomhed blev bestemt til at være 100 % med mindst 4 kopier af gBlocks pr. PCR og 100 %, når mere end 10 Borrelia bakterier blev tilsat 1 ml CSF. Den kliniske sensitivitet og specificitet blev beregnet til at være henholdsvis 11,1 % og 100 %. Studie III " Detection of Borrelia burgdorferi sensu lato DNA in cerebrospinal fluid samples followed by pre-enrichment culture " I denne prospektive tværsnitsundersøgelse blev voksne patienter (> 17 år), som skulle lumbalpunkteres med henblik på undersøgelse for LNB, inviteret til at deltage. CSF prøver fra patienterne blev inokuleret direkte i prøvetagningsrør indeholdende Borrelia dyrkningsmedium, som efterfølgende blev inkuberet i mindst 8 uger og undersøgt for tilstedeværelsen af Borrelia bakterier ved PCR og mikroskopi. En opformering af Borrelia bakterier blev opnået hos 23 % af patienterne med LNB, og et positivt resultat blev bekræftet af to uafhængige PCR-analysemetoder. Konklusion Resultaterne fra studie I bekræfter, at CXCL13 er et værdifuldt supplement til diagnosticering af LNB, og at kombinationen af CXCL13 og IL-6 kan bruges differential diagnostisk til at evaluere patienter med mulig LNB med henblik på at underbygge den kliniske diagnose yderligere. De lave diagnostiske sensitiviteter opnået i studie II og III understøtter ikke brugen af Borrelia PCR som en rutinediagnostisk metode. Imidlertid kan præopformering af Borrelia bakterier fra CSF-prøver forbedre påvisningen af Borrelia DNA, hvilket kan vise sig at være af yderligere diagnostisk værdi hos klinisk udvalgte patienter, som undersøges i den tidlige fase af deres neuroborreliose. Tick-borne spirochetes of the Borrelia burgdorferi sensu lato complex are the causes of various disease manifestations collectively termed Lyme borreliosis. The Borrelia spirochetes infect humans through a tick bite on the skin and can spread from there to other organs, including the central nervous system (CNS). In Europe, typical manifestations of Lyme neuroborreliosis (LNB) are intense pain from the nerve roots (radicular neuritis), facial nerve palsy, and CNS inflammation by mononuclear cells. It is certain that there are living Borrelia spirochetes in the CNS of patients with LNB, as they have been identified by culture and by polymerase chain reaction (PCR) analysis of cerebrospinal fluid (CSF), and since penicillin works effectively in patients with symptoms of LNB. Unfortunately, the positivity rates of Borrelia culture testing and specific PCR are unsatisfyingly low, leading to the use of serological tests. LNB diagnosis is therefore primarily based on symptomology, CSF pleocytosis and intrathecal synthesis of Borrelia specific antibodies determined by the CSF/serum antibody index. However, there are limitations to the intrathecal antibody index analyses because antibody production may be absent at a detectable level in early LNB infection, making it challenging to discriminate LNB from other CNS diseases. The CXCL13 chemokine has been investigated as a biomarker for LNB with the aim of improving the diagnostics of LNB. Supplementary tools improving LNB diagnostics in the early stage of the disease are thus warranted. This PhD thesis provides an introduction to the Borrelia burgdorferi sensu lato spirochetes, together with an overview of some of the diagnostic challenges concerning LNB. Subsequently, the included manuscripts' aims, methods, and results are presented and discussed.Study I “Discriminating between Lyme neuroborreliosis and other central nervous system infections by use of biomarkers CXCL13 and IL-6”This cross-sectional study consecutively included patients examined for neuroinfections. We quantified the levels of CXCL13 and IL-6 in CSF simultaneously using a bead-based dupleximmunoassay and the Bio-Plex 200 System. Patients were grouped into definite LNB, possible LNB, viral CNS infection, bacterial CNS infection, Other CNS disease (with pleocytosis) and Negative (without pleocytosis) based on clinical and paraclinical findings. We performed a Principal component analysis (PCA) using results from CSF analysis (CXCL13, IL-6, leukocyte cell countsand protein concentration). We visually inspected the PCA-plot and found three distinct clusters (definite LNB, bacterial CNSinfection and negative) based on leukocyte cell counts, protein concentration, CXCL13 and IL-6concentrations from 380 included patients. In addition, we showed that possible LNB cases weredispersed across multiple clusters and had different probabilities of belonging to the LNB cluster.ROC calculation combined with a Youden index score indicated an optimal CXCL13 cut-off valueof 50.7 pg/ml, resulting in a sensitivity and a specificity of 93.6 and 91.1%, respectively, whencomparing definite LNB patients to non-LNB conditions with CSF pleocytosis. Study II “Establishment of a droplet digital PCR protocol for detection of Borrelia burgdorferi sensulato DNA in cerebrospinal fluid” In Study II, a thorough validation of a digital droplet PCR (ddPCR) assay and pre-analyticalparameters for detection of Borrelia spirochetes were performed. The ddPCR protocol was optimizedusing synthetic DNA gBlocks and cultured Borrelia species. The optimized ddPCR protocol wasevaluated on clinical samples from patients examined for LNB (n=59). PCR-plate incubation at 4 °C before droplet reading and a centrifugation step for concentrating thesample prior to DNA purification was shown to have a significant effect on the ddPCR performance.The analytical sensitivities were determined to be 100% with at least 4 copies of gBlocks per PCRand 100% when more than 10 Borrelia spirochetes were spiked in 1 mL CSF. The clinical sensitivityand specificity were calculated to be 11.1% and 100%, respectively. Study III “Detection of Borrelia burgdorferi sensu lato DNA in cerebrospinal fluid samples usingpre-enrichment culture” Study III was a prospective cross-sectional study inviting adult patients to participate beforeundertaking a lumbar puncture on suspicion of LNB. CSF specimens were inoculated directly intosample tubes containing Borrelia culture medium. Cultures were incubated for at least 8 weeks andexamined for the presence of Borrelia spirochetes by three separate PCR protocols in twoindependent laboratories. Pre-enrichment culture of Borrelia spirochetes in CSF was accomplished in 23% of patients withLNB. The presence of Borrelia was confirmed by the three independent PCR analyses employed. Conclusion The results from Study I confirms that CXCL13 is a valuable supplement for the diagnosis of LNBpatients and that the combination of CXCL13 and IL-6 may be used to evaluate patients with possibleLNB to substantiate the clinical diagnosis further. The low diagnostic yield in studies II and III donot support the use of Borrelia-specific PCR in CSF as a supplemental routine diagnostic tool.However, pre-enrichment of Borrelia spirochetes from CSF specimens can improve the detection ofBorrelia DNA and may prove of additional value in clinically selected patients who are admitted inthe early phase of their neuroborreliosis. 

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