Silje Andreassen
Cognitive function in patients with neuroborreliosis
– a prospective follow-up from the acute phase to 12 months post-treatment
© Silje Andreassen, 2023
Series of dissertations submitted to the Faculty of Medicine, University of Oslo
ISBN 978-82-348-0175-4
All rights reserved. No part of this publication may be
reproduced or transmitted, in any form or by any means, without permission.
Cover: UiO.
Print production: Graphics Center, University of Oslo.
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Table of Contents
Acknowledgement ... 5
Summary ... 7
Sammendrag ... 9
List of papers ... 11
Abbreviations ... 12
Introduction ... 13
1.1 Topic of the thesis ... 13
1.2 Epidemiology ... 14
1.2.1 Lyme borreliosis, historical background ... 14
1.2.2 Borrelia burgdorferi (Bb) sensu lato and its vectors ... 16
1.2.3 The term Lyme borreliosis ... 17
1.2.4 Clinical manifestations of borreliosis ... 17
1.2.5 Neuroborreliosis ... 19
1.2.6 Post Lyme Disease Syndrome (PLDS) ... 20
1.3 Fatigue ... 21
1.4 MRI ... 22
1.5 Cognitive outcome – IQ ... 23
1.6 Cognitive outcome – Neuropsychology ... 24
1.6.1 Attention and executive functions ... 24
1.6.2 Processing speed ... 24
1.6.3 Memory functions ... 25
1.6.4 Methodological problems in neuropsychological assessment ... 26
2. Aims of the study ... 30
3. Materials and methods ... 30
3.1 Study design ... 30
3.2 Recruitment and participants ... 31
3.3 Cognitive testing ... 34
3.3.1 Screening in acute phase and 12 months follow-up (paper I and III) ... 34
3.3.2 Neuropsychological assessment at 6 months follow-up (paper II) ... 35
3.4 Additional outcome measures ... 38
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3.4.1 Fatigue ... 38
3.4.2 Cerebral MRI ... 38
3.4.3 Other variables ... 41
3.4.4 Socioeconomic status ... 41
3.5 Statistics ... 41
3.5.1 Sample size ... 41
3.5.2 Statistics used in paper I-III ... 42
3.6 Ethics ... 43
4. Main results... 44
5. Discussion ... 50
5.1 Discussion of methods ... 50
5.1.1 Recruitment and participants ... 50
5.1.2 Outcome measures paper I and III ... 52
5.1.3 Outcome measures paper II ... 54
5.2 Discussion of results ... 56
5.2.1 Attention and processing speed (paper I and III) ... 56
5.2.2 Extended neuropsychological assessment (paper II) ... 60
5.2.3 Fatigue (paper I, II and III) ... 61
5.2.4 Structural brain changes (paper I and II) ... 63
6. Conclusion ... 65
7. Future perspectives ... 66
8. References ... 67
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Acknowledgement
The South Eastern Health Authority and Sørlandet Hospital HF financially supported this dissertation. The work was performed mainly at Sørlandet Hospital, but also Oslo University Hospital contributed to the collection of data.
Several people have contributed to this work, and it is a pleasure for me to express my gratitude to them.
First, I want to thank all the patients and controls for your interest, time and effort.
Without you, I could not have written this thesis.
To Randi Eikeland, my main supervisor: I could not have asked for a more insightful and positive mentor. You have been thorough and effective, but also supportive and caring when I felt overwhelmed and defeated. I would have been lost without you!
To Gro Løhaugen, my co-supervisor: You introduced me to the world of psychology and neuropsychology, and I am forever grateful to you for that. You are a great motivator, a respected colleague and a dear friend to me.
To Hanne Harbo, my co-supervisor: Your experience and insightful comments have been reassuring and of great value.
Big thanks to my co-PhD students Anne Marit Solheim and Elisabeth Lindland. It has been a pleasure to work with you both! Anne Marit, thank you for your sharing your knowledge about neurology and for your support. Elisabeth, your attention to details is impressive. Thank you for trying to teach me about MRI, I imagine it has been a trial of patience.
I would like to thank the Research department at Sørlandet Hospital for practical assistance and advice. I am also grateful to all the members in BorrSci for sharing your knowledge about research in general and research on tick-borne diseases in particular with me.
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Great thanks to Jon Skranes, head of Habilitation at the Pediatric department (HABU) Sørlandet Hospital, Arendal. I would not have finished in time without your support and flexibility. Also great thanks to all my colleagues; your positive attitude means a lot to me.
To my dear friend and unofficial PhD mentor Marianne: Thank you for keeping my spirit up in my darkest hours!
Warm thanks to my beloved husband Kristian for always standing by my side and to Inger and Eivind for bringing joy into my life. I promise to make better dinners and not be as cranky in the future (at least I hope so).
Big thanks to my mother, for looking after Inger and Eivind when I have been busy working. I am so grateful for everything you have done for me! To my father, thank you for teaching me the importance of hard work and for always believing in me. To my brother, thank you for never letting me forget that deep down I am just a simple country-girl.
To Liv-Anna and all the members in Tromøy ultraløpeklubb: Thank you for friendship, laughter and for introducing me to ultra-running. From now on, I promise to be more serious and run at least 60K every week.
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Summary
Borreliosis is a tick-borne disease caused by the spirochete Borrelia burgdorferi (Bb) and is the most prevalent tick borne disease in the Northern hemisphere. The
infection usually affects the skin, but left untreated, the infection will disseminate to other organs in about 10 % of the cases. In Europe, borrelia usually affects the nervous system causing neuroborreliosis. Although neuroborreliosis is effectively treated with antibiotics, some patients report persistent symptoms like fatigue, subjective cognitive problems and pain. Remaining symptoms after treated borreliosis is commonly termed Post Lyme Disease Syndrome, of which etiology remains unknown. Today, there is no convincing scientific support for an ongoing infection, or effect of prolonged antibiotic treatment. Still there is a controversy regarding both the severity and prevalence of these symptoms. Several studies have investigated Post Lyme Disease Syndrome using subjective measurements like
questionnaires, but few studies have included a control group and examined patients prospectively from the acute phase of the infection with standardized
neuropsychological tests.
Aims
Our aim was to assess cognitive function in patients with well-characterized
neuroborreliosis, and compare the results with a control group. We explored brain structures with MRI and searched for associations between cognitive function and possible structural changes. Finally, we were interested in measuring patients’
subjective level of fatigue compared to controls, and see if level of fatigue decreased during the follow-up.
Methods
Between November 2015 and December 2018, patients with definite or possible neuroborreliosis were included at Sørlandet Hospital and Oslo University Hospital.
Serenity-two patients were tested with eight neuropsychological subtests assessing attention and processing speed, and underwent MRI scan within four weeks after treatment start with antibiotics. An age and gender matched control group (n=68)
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was included for comparison. We used Fatigue Severity Scale (FSS) to measure level of fatigue, and Fazekas score to grade white matter hyperintensities. After six
months, we assessed patients and controls with a comprehensive neuropsychological test protocol, including measures of general abilities, executive functions, verbal and visual memory. The participants also filled out FSS and were scanned with MRI again.
Twelve months after treatment, we re-tested the patents with the same test-protocol as in the acute phase and measured level of fatigue.
Results
Paper I: We found no differences in attention or processing speed between patients with acute neuroborreliosis and healthy controls. The patients reported a higher level of fatigue. We found no differences in Fazekas score between the groups.
Paper II: Six months after treatment, we found no differences between the groups in cognitive function, cortical thickness or brain volumes. The patients had higher level of fatigue, and more patients (25.4%) than controls (5 %) reported severe fatigue (p=.002), but neither mean score nor proportion of patients with severe fatigue differed from findings in the general Norwegian population.
Paper III: We found no changes in attention or processing speed from the acute phase to 12 months after treatment, but the level of fatigue was reduced compared with level of fatigue in the acute phase.
Conclusion
Overall, the prognosis regarding cognitive function after adequately treated
neuroborreliosis is favorable. The patients have a higher level of fatigue in the acute phase of the infection, but this tend to decrease. Regarding structural changes, we were not able to detect differences between patients and controls in the MRI analysis used here.
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Sammendrag
Borreliose en infeksjon forårsaket av spiroketen Borrelia burgdorferi, og er den mest utbredte flåttbårne sykdommen på den nordlige halvkule. Infeksjonen er vanligvis lokalisert i huden, men kan spre seg til andre organer i omtrent 10 % av tilfellene dersom den ikke behandles. I Europa er nevroborreliose den vanligste formen for systemisk borrelia infeksjon. Antibiotika er effektiv behandling mot nevroborreliose, men noen pasienter rapporterer likevel om vedvarende plager som fatigue,
subjektive kognitive plager og smerter i etterkant. Post Lyme Disease Syndrome brukes gjerne som betegnelse for vedvarende plager etter behandlet borreliose, og vi kjenner ikke til årsaken til denne tilstanden. Per i dag er det ingen vitenskapelige holdepunkter for at dette skyldes en pågående og aktiv infeksjon, eller at langvarig antibiotikabehandling har effekt. Flere studier på pasienter med vedvarende plager etter borreliose har inkludert subjektive kartleggingsverktøy som spørreskjema, men svært få har inkludert kontrollgruppe og fulgt pasientene prospektivt fra akuttfasen med standardiserte nevropsykologiske tester og MR. Målsetningen vår var å
undersøke kognitiv funksjon hos pasienter med nevroborreliose, diagnostisert etter gjeldende retningslinjer, og sammenligne resultatene med en kontrollgruppe. Vi ønsket også å undersøke strukturelle forhold i hjernen med MR og se om det var sammenheng mellom kognitiv funksjon og eventuelle strukturelle endringer.
Avslutningsvis ønsket vi å undersøke grad av fatigue og se hvorvidt pasientene opplevde mindre fatigue i løpet av oppfølgingsperioden.
Metode
I tidsrommet november 2015 til desember 2018 inkluderte vi pasienter med sikker eller mulig nevroborreliose ved Sørlandet sykehus og Oslo universitetssykehus. I alt inkluderte vi 72 pasienter som gjennomgikk en kort nevropsykologisk testing og MR innen fire uker etter oppstart med antibiotikabehandling. Vi inkluderte også en kontrollgruppe med lik alder (+/- 2 år) og kjønn (n=68). I akuttfasen var utfallsmålene resultat på nevropsykologiske tester som skulle måle oppmerksomhet og
prosesseringshastighet, grad av fatigue målt med kartleggingsskjemaet Fatigue
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Severity Scale (FSS) og Fazekas skåre for å gradere omfanget av hvit substans
lesjoner. Seks måneder etter avsluttet behandling ble pasienter og kontroller testet med en omfattende nevropsykologisk testprotokoll som inkluderte mål på generelt evnenivå, eksekutive funksjoner og verbal og visuell hukommelse. Deltakerne fylte også ut FSS på nytt, og det ble gjennomført en ny MR undersøkelse. Tolv måneder etter avsluttet behandling re-testet vi pasientene med den samme testprotokollen som i akuttfasen og målte grad av fatigue med FSS igjen.
Resultater
Artikkel 1: Vi fant ingen forskjell mellom gruppene på tester som målte
oppmerksomhet eller prosesseringshastighet, og det var ingen forskjell i grad av hvit substans lesjoner. Pasientene rapporterte om høyere grad av fatigue enn
kontrollgruppen.
Artikkel 2: Vi fant ikke forskjell mellom gruppene da vi sammenlignet
gjennomsnittskårer på kognitiv funksjon, kortikal tykkelse eller hjernevolumer.
Pasientene rapporterte om mer fatigue enn kontrollpersonene, og flere pasienter (25.4%) enn kontrollpersoner (5 %) rapporterte om svært høy grad av fatigue (p=.002). Både gjennomsnittlig fatigue eller andel som rapporterte om høy grad av fatigue i pasientgruppen var imidlertid sammenlignbare med nivå i den norske befolkningen.
Artikkel 3: Vi fant ingen endringer i oppmerksomhet eller prosesseringshastighet hos pasienter fra akuttfasen til 12 måneder etter avsluttet behandling. Pasientene hadde mindre fatigue 12 måneder etter avsluttet behandling, sammenlignet med
akuttfasen.
Konklusjon
Pasienter med behandlet nevroborreliose ser ikke ut til å ha mer kognitive
problemer. De har mer fatigue i akuttfasen, men dette bedres i løpet av 12 måneder.
Vi fant ikke strukturelle endringer i hjernen hos pasientene, hverken økt forekomst av hvit substans lesjoner, endringer i kortikal tykkelse eller endringer i hjernevolumer.
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List of papers
Paper 1
Andreassen S, Lindland EMS, Solheim AM, Beyer MK, Ljøstad U, Mygland Å, Lorentzen ÅR, Reiso H, Harbo HF, Løhaugen GCC, Eikeland R.
Cognitive function, fatigue and Fazekas score in patients with acute neuroborreliosis.
Ticks and Tick-borne Diseases. 2021 May;12(3):101678.
doi: 10.1016/j.ttbdis.2021.101678. Epub 2021 Jan 25.
Paper 2
Andreassen S, Lindland EMS, Beyer MK, Solheim AM, Ljøstad U, Mygland Å, Lorentzen ÅR, Reiso H, Bjuland KJ, Pripp AH, Harbo HF, Løhaugen GCC, Eikeland R.
Assessment of cognitive function, structural brain changes and fatigue six months after treatment of neuroborreliosis.
Accepted in Journal of Neurology 2022 October 27.
Paper 3
Andreassen S, Solheim AM, Ljøstad U, Mygland Å, Lorentzen ÅR, Reiso H, Beyer MK, Harbo HF, Løhaugen GCC, Eikeland R.
Cognitive function in patients with neuroborreliosis: A prospective cohort study from the acute phase to 12 months post treatment.
Brain and Behaviour. 2022 Jun;12(6):e2608.
doi: 10.1002/brb3.2608. Epub 2022 May 20.
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Abbreviations
Bb: Borrelia burgdorferi CNS: Central nervous system CSF: Cerebrospinal fluid
CVLT-II: California Verbal Learning Test 2nd edition D-KEFS: Delis-Kaplan Executive Function System EFNS: European Federation of Neurological Societies eICV: estimated intracranial volume
FDR: False discovery rate FSS: Fatigue Severity Scale IQ: Intelligence quotient
MDD: Major depressive disorder ME: Myalgic encephalopathy
MMSE: Mini Mental Scale Examination
MPRAGE: Magnetization-prepared rapid gradient-echo MRI: Magnetic resonance imaging
MSIS: Norwegian Surveillance System for Communicable Diseases PLDS: Post Lyme Disease Syndrome
ROI: Region of interest SD: Standard deviation SES: Socioeconomic status
WAIS-IV: Wechsler Adult Intelligence Scale 4th edition WMH: White matter hyperintensities
WMS-III: Wechsler Memory Scale 3rd edition
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Introduction
1.1 Topic of the thesis
Neuroborreliosis is an infection affecting the nervous system caused by tick-bites from Ixodes Ricinus ticks infected by the bacterium Borrelia burgdorferi (Bb).
Although the objective manifestations of neuroborreliosis typically resolve after recommended antibiotic course, some patients report persistent symptoms like fatigue, malaise and subjective cognitive problems after treatment. Based upon different studies the percentage of patients reporting persistent symptoms vary greatly from 10 to nearly 50% (1-4). Currently there is a controversy between researchers, clinicians and patients regarding the etiology and treatment of these symptoms. Many studies on outcome after treated neuroborreliosis are based upon subjective measures like self-filling questionnaires and few have used more objective and standardized measurements. The severity and even presence of cognitive
sequela after well-treated neuroborreliosis are one of the questions that is yet to be answered. Some of the earlier studies described a condition with subjective
complaints including fatigue and memory problems of amnestic character, called
“Lyme encephalopathy”. Changes in cerebral white matter on imaging, similar to changes seen in multiple sclerosis, was hypnotized to have a connection with ”Lyme encephalopathy” (5-7). The hypothesis of a chronic infection affecting the brain was long debated, but until now, there has not been any convincing research supporting this (8, 9). Autoimmune and immunological mechanisms have been suggested as possible explanations for persistent symptoms, but until now, there are no convincing findings to support this either (9). Still, a proportion of the patients report subjective cognitive problems and fatigue after treated neuroborreliosis (10-12). In studies attempting to assess these cognitive problems, the results are diverging. While some researchers find none, or only modest, cognitive problems when using standardized tests (4, 13, 14), others report reduced function in some cognitive domains (15-17).
Another approach to search for objective signs of subjective symptoms like fatigue and cognitive complaints has been imaging studies. Patients with acute
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neuroborreliosis often have meningeal, leptomeningeal and cranial nerve
enhancement (18), but otherwise parenchymal abnormalities in the central nervous system that might have explained the subjective cognitive complaints and fatigue after treated neuroborreliosis on conventional MRI seems to be unusual (19, 20).
Previous studies on cognitive function and MRI after treated borreliosis with low sample size or heterogeneous patient groups have presented diverging findings. This make it difficult to compare results from different studies.
Although the conception of “Lyme encephalopathy” or “chronic Lyme” caused by an active infection no longer have scientific support, unanswered questions regarding cognitive function after adequately treated neuroborreliosis remain. Patients do worry about persistent symptoms, and especially the perspective of reduced memory or other cognitive problems is frightening. The main topic for this thesis was to
investigate cognitive function in patients with neuroborreliosis using standardized neuropsychological assessment. Furthermore, to search for possible associations between cognitive function and structural brain changes in patients with well characterized and treated neuroborreliosis from treatment start until 12 months afterwards.
Do the patients need to worry?
1.2 Epidemiology
1.2.1 Lyme borreliosis, historical background
The story of Lyme disease is intriguing, and has been held up as a successful example of how observant researchers’ systematic work and interest in public health can yield new knowledge. It is also an interesting story about how a disease can evoke strong feelings, contradictive views and heated discussion about diagnostics, treatment and long-term effects.
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Lyme disease, or Lyme borreliosis, is named after the region of Lyme located in Connecticut, where Allan Steere and coworkers reported several cases of arthritis among children and young adults in the mid-seventies. The high prevalence of arthritis in children within a geographically defined area, led them to suspect infection as a causative factor. Out of 51 patients being examined by Steere, a quarter reported about a rash, identified as erythema migrans, prior to the onset of arthritis (21). Later observations and field studies connected erythema migrans with tick bites (22). A “new” illness, Lyme arthritis, later named Lyme borreliosis was born.
Tick as a vector for infection was already an established knowledge, first proposed as a hypothesis in the forties and early fifties. The Swedish dermatologist Afzelius has later been credited as the first to describe erythema migrans back in 1910 (23).
While Garin and Bujadoux described the first known case of neurological Lyme borreliosis, presented as radiculitis in 1922. Their report describes a 58 year-old man who three weeks after a tick bite developed rash spreading gradually from the tick bite with concurrent severe pain located around his thorax and radiating out to his arm. The pain was accompanied by paresis in his right shoulder, making it impossible for him to lift the arm. Five months later, the patient seemed to have gradually recovered, but still had some persistent symptoms as reduced movement in his right arm and pain around his thorax. The authors concluded that the tick must have transferred some kind of microorganism (24). In later years, it has been questioned whether the patient in Garin and Bujadoux’s now classical report did indeed had neuroborreliosis, as both the type of tick described in the report, the clinical
symptoms and their timely occurrence are not typical for neuroborreliosis (25). Garin and Bujadoux correctly identified a tick as the vector, while their suspected causal microorganism of infection was identified 60 years later. Borrelia burgdorferi sensu lato, the spirochetes causing borreliosis were first described by Burgdorferi (26).
Since then, the research including the diverse aspects of borrelia-related topics has expanded to include divergent disciplines in medicine, in veterinary medicine, in zoology and in later years, psychology.
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1.2.2 Borrelia burgdorferi (Bb) sensu lato and its vectors
Vector borne diseases are infections transferred by the bite of an organism carrying a pathogen to a new host. The pathogen, in this case Bb, is dependent of its vector and cannot survive outside the host. The tick Ixodes Ricinus is the preferred vector in Europe, but like the distribution of Bb varies from different countries and regions, so does the species of ticks. The ticks are mainly active in the summer and autumn, but if the temperature stays above zero, they can be active during the winter and spring as well. The ticks are infected with Bb when they feed blood as larvae or nymph, mainly from small rodents or birds, and can afterward transmit Bb to humans.
Lyme borreliosis is the most common tick borne disease in the northern hemisphere, and the incidence is increasing (27). Spirochetes within the Borrelia Burgdorferi (Bb) sensu lato complex cause the infection, and are transferred by tick bite from the Ixodes ticks. After the discovery of Bb in 1982, several genotypes have been
identified, were some are considered pathogenic to humans, while others not. The most common pathogenic species accountable for borreliosis in humans are Bb sensu stricto, Bb afzelii and Bb garinii. The distribution of these genotypes varies in different countries and regions. While Bb sensu stricto is almost completely dominant in the United States, the distribution in Europe is more heterogeneous. Bb sensu stricto exists in Europe as well, but Bb afzelii and Bb garinii are far more prevalent here. (28).
Along the coast in South of Norway, were this study is carried out, the distribution of Bb in ticks are mainly Bb afzelii (61.6%), followed by Bb garinii (23.4%), Bb burgdorferi sensu stricto (10.6%) and Bb valaisiana (4.5%) (29). In addition to rash around the tick bite, the different genotypes of Bb have different affection to organ tissue: Bb sensu stricto often presents as arthritis, Bb afzelii is associated with the chronic skin infection Acrodermatitis chronica atrophicans (ACA), while Bb Garinii more often will spread to the nervous system and cause neuroborreliosis.
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1.2.3 The term Lyme borreliosis
Lyme borreliosis includes all the above manifestations, which might explain why some regard Lyme disease as a mysterious and unpredictable disease. The
connection between borreliosis and the geographical name Lyme has been debated in Europe and Norway. The Norwegian National Advisory Unit on Tick-borne
diseases does not use the term Lyme borreliosis anymore, and Lyme is mentioned only once in their website (30). In the Journal of the Norwegian Medical Association, Mysterud recommended excluding Lyme, and instead using the term borreliosis, neuroborreliosis or borrelia induced arthritis and meningitis in cases where a specification was needed. The term Lyme is not commonly used in the Norwegian population. It is also strongly related to Lyme disease in the US where the genotype sensu stricto predominates, while the genotypes in Europe are more heterogeneous.
As different genotypes are associated with different clinical pictures, research from US can be difficult to compare directly to research from Europe. Although Lyme might not be commonly used in everyday language, Lyme borreliosis or Lyme disease is widely used in international research, which has been the main argument to keep Lyme as a term, also in Norway (31). Currently, there is no agreement whether it should be called Lyme borreliosis or borreliosis. Regarding neuroborreliosis, the term Lyme neuroborreliosis is used side by side with neuroborreliosis. Throughout this manuscript, I will use borreliosis and neuroborreliosis.
1.2.4 Clinical manifestations of borreliosis
The conception of different and clearly divided stages of borreliosis has been
questioned in later years, and nowadays we seldom describe the development of the infection in a patient as stages. A stage-like conception implicates a progression of the infection that starts at a specific stage and follow a specific pattern, however this is not always the case with neuroborreliosis. It seems to be individual differences between patients regarding how the symptoms start and how the infection develops.
Asymptomatic courses are usual, and in a high endemic region in South of Norway
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22% of the population was positive for Bb antibodies in serum (32). For practical reasons, we classify the clinical features of borreliosis into localized infection and disseminated infection. If borrelia disseminates from the site of inoculation in the skin to other organs, it usually happens within few weeks after the tick bite. In Europe, dissemination to the nervous system is common, but joints can also be affected causing arthritis. Dissemination to other organs like heart and eyes are rare, but do occur. There are exceptions, ACA, a chronic skin-condition, can develop several weeks to months after the tick bite and appears like a paper-thin skin and bluish red discoloration.
Table 1. Clinical manifestation of borreliosis
Localized infection < 21 days after tick bite Erythema migrans
Disseminated 4-8 weeks after tick bite
Weeks to months after tick bite
Neuroborreliosis Arthritis
Other organ manifestations Acrodermatitis Chronica Atrophicans (ACA)
Early-localized infection
Solitary erythema migrans is the most common early-localized manifestation of infection with borrelia. Erythema migrans, a rash localized around the tick bite, usually develops within few days and is gradually expanding. Traditionally, erythema migrans has a characteristic red border and a central clearing with a “bull’s eye” in the middle. This view has later been adjusted, and erythema migrans does not always have a clear border or a bull’s eye, but can instead appear as a red rash around the
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tick bite (33). As erythema migrans is considered a local infection, cases are not reported to the Norwegian Surveillance System for Communicable Diseases (MSIS).
The incidence of erythema migrans in Norway is approximately 7000 each year (34).
Left untreated erythema migrans usually resolves within few weeks, but left untreated the infection can disseminate to other organs, leading to a more severe outcome. To prevent dissemination, erythema migrans should be treated with penicillin for 10 days according to Norwegian guidelines (35).
1.2.5 Neuroborreliosis
In Europe, borrelia usually disseminates to the nervous system, causing
neuroborreliosis. Disseminated borreliosis is notifiable and registered in MSIS (36).
Table 2. Registered cases of disseminated borreliosis in Norway last five years in adults >20 years of age
Year 2017 2018 2019 2020 2021
Registered cases
295 277 338 347 352
(from MSIS retrieved 03.08.22)
In 2010, The European Federation of Neurological Societies (EFNS) reviewed the guideline on diagnosis and treatment for neuroborreliosis. The EFNS guidelines define neuroborreliosis as either possible or definite based on the following three criteria:
1. Neurological symptoms indicating neuroborreliosis without any other obvious reasons
2. Cerebrospinal fluid pleocytosis 3. Intrathecal Bb antibody production
Neuroborreliosis is regarded as definite if all three criteria are fulfilled, and possible if two out of three are fulfilled (37).
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Bannwarth syndrome, a triad of lymphocytic meningitis, cranial neuritis, and
radiculitis is a typical manifestation of neuroborreliosis. The most common symptoms are intense radicular pain radiating from the neck and back into the thorax, arms or legs, often accompanied by peripheral paresis in the same regions as the radiating pain. Facial palsy, paresthesia and headache are also frequent (3). Meningitis, vasculitis and encephalitis are uncommon, but do occur (38, 39). Neuroborreliosis is treated with two weeks of antibiotics and complete recovery is expected (37)
1.2.6 Post Lyme Disease Syndrome (PLDS)
Although complete recovery after well-treated neuroborreliosis is expected, some patients report persistent symptoms like fatigue, malaise and subjective cognitive complaints function (1, 2, 12, 40, 41). The term PLDS has been used to describe symptoms that persist for six months or more after treatment, or new symptoms which develop within six months after treatment. Infectious Diseases Society of America has proposed the following definition (42):
1. A documented episode of borreliosis
2. After treatment with a generally accepted treatment regime, objective symptoms of borreliosis should improve
3. Any of the following symptoms must occur within six months after the episode of borreliosis or persist for at least six months after completion of antibiotic therapy: Fatigue, widespread musculoskeletal pain or cognitive problems 4. The severity of the symptoms negatively affect previous levels of occupational,
educational, social and personal activity
The etiology behind PLDS is uncertain, but there is no convincing biologic evidence of an ongoing or chronic borrelia infection in patients after recommended antibiotic treatment (9). Clinical signs of inflammation in the nervous system vanish quickly during, or shortly after, treatment in nearly all patients. Several randomized studies have shown no beneficial effect of prolonged antibiotic treatment (43-46). They
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found no reduction in long-term symptoms like fatigue, subjective cognitive complaints, cognitive function or quality of life. Furthermore, there were no difference between short- and long-term treatment regarding serologic and
cerebrospinal fluid findings. On the contrary, patients are at risk of having negative side effects from prolonged antibiotic treatment. Systemic inflammation and immune responses leading to impaired CNS function have been presented as possible
hypotheses (47-49). Furthermore, treatment delay from symptom start and severity of the disease at treatment start might affect the long-term prognosis negatively (38, 50). It has been estimated that about 5-10 % of patients with adequately treated neuroborreliosis have symptoms consistent with PLDS (33, 42), but there is no consensus of how many patients have symptoms indicating PLDS or if PLDS even exists at all (9).
1.3 Fatigue
Fatigue is as an overwhelming feeling of tiredness or lack of energy, either physical, mental or a combination of both. Fatigue is not the same as feeling drowsy or sleepy, and a proper rest or a good night’s sleep does not resolve fatigue. Fatigue is common in many neurological illnesses, like multiple sclerosis, Parkinson’s disease, after
traumatic brain injury and stroke. In these, and other, neurological illnesses and conditions fatigue is considered a primary symptom and such distinguishable from related symptoms like sleepiness, depression, apathy and medication (51).
Furthermore, in patients with multiple sclerosis and Parkinson’s disease, 30 – 40 % of the patients report fatigue to be their most disabling symptom, making fatigue a genuine health problem (51). Fatigue is common in patients with acute
neuroborreliosis (3, 52), but also after treatment (1, 2, 38, 41). In the general population, fatigue seems to be quite common as well. In a British study including patients from general practitioners, as much as 10 % of the patients had substantial fatigue for one month or more, and they attributed fatigue to both physical and social factors (53). The prevalence in Norway seems to be even higher, and 20 % of
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the respondents reported severe fatigue a study by Lerdal et.al (54). Difficulties in assessing a subjective experience like fatigue can explain the differences in
prevalence. Although fatigue has grown to be a common expression in everyday language, there is no consensus of how it should be defined. Fatigue includes physical lack of energy, but it also includes mental or cognitive aspects (51). Different
definitions of fatigue can affect which items have been included in the
questionnaires. This make it hard to compare studies where different questionnaires have been used. As for neuroborreliosis, there is another catch when comparing results from different studies. In addition to the variations in questionnaires meant to measure the level of fatigue, there are variations in inclusion criteria and design as well. These factors make it challenging to compare results from different studies directly.
1.4 MRI
Imaging studies on patients with neuroborreliosis are limited. High intensity white matter lesions in the brain were early on considered to be related to
neuroborreliosis. The description of these lesions resembled the ones seen in multiple sclerosis, often combined with reduced cognitive function (7, 55-58).
However, later studies do not confirm these findings and instead suggest these lesions are unspecific and not typical for patients with neuroborreliosis (19, 59, 60).
The varying results from earlier to later studies could have different explanations. The diagnostic criteria in earlier reports varied, and not all of them would meet the EFNS guidelines today. Not all of them included a control group, and some were hampered with a low sample size. Furthermore, higher resolution MRI and knowledge about age-related white matter hyperintensities has evolved throughout this time.
Consequently, the scientific evidence to support that neuroborreliosis causes high intensity matter lesions is weak. Apart from meningeal, leptomeningeal and cranial nerve enhancement in acute neuroborreliosis, positive findings on conventional MRI
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after treatment are unusual, but do occur (18, 20). Cases of vasculitis, myelitis and encephalitis are rare, but can cause sequelae seen on MRI (61).
1.5 Cognitive outcome – IQ
Intelligence, or general cognitive ability, can be defined as the ability to acquire knowledge, learn from experiences and adapt to, shape and select environments (62). The field of testing intelligence is well over 100 years old, and the outcome on intelligence tests is called intelligence quotient or IQ. The Wechsler tests are widely used throughout the world (62), they are standardized, validated and have a high reliability. IQ testing is an important part of a neuropsychological exam to establish the general cognitive ability. In a clinical setting, this provide the basis for interpreting neuropsychological test results, but strengths and weaknesses in the IQ profile can also influence the selection of tests. IQ correlates with different neuropsychological tests in varying degree, which is an argument for including IQ-testing in an
assessment, even when IQ not is the primary outcome. Full scale IQ is the most reliable index score with a split-half reliability coefficient of 0.98 across the age groups (63). Short forms of estimating IQ where some subtests are excluded are common in research, and have been found to correlate well with Full Scale IQ, although leaving out several subtests do affect the reliability of estimated IQ (64).
Only one study on patients with neuroborreliosis has included Wechsler Adult
Intelligence Scale with the all subtests needed to calculate index scores in addition to full scale IQ (16). Both the patients and the control group had full-scale IQ above average, but the patients scored significantly lower compared with the control group due to lower scores on processing speed and information.
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1.6 Cognitive outcome – Neuropsychology
1.6.1 Attention and executive functions
Executive functions are higher order cognitive functions involved in self-regulation and goal directed behavior. These mental processes enable us to plan, focus attention, organize and work with different tasks simultaneously or shift between them in an effective manner. Being such a broad concept, executive functions have been defined in numerous ways, but today most investigators see executive function processes as part of a system that supervise and coordinate other cognitive processes in the brain (64). Executive functions have traditionally been linked to the frontal lobes, and the prefrontal lobes in particular (65). However, executive functions involve associations between different brain areas as well, and not the frontal lobes exclusively (64, 66). There is limited amount of research on executive functions in patients with neuroborreliosis. From 2010 until today, six studies have included one or more subtest to measure attention and/ or executive functions (13-15, 67-70). The results have varied from no reduction (13, 67) to modest reduction (15, 69, 70) and lastly a small subgroup of patients showing deficits in attention, executive functions or both (15, 68).
1.6.2 Processing speed
Processing speed refers to the time required to respond or process information.
Processing speed is sensitive to several illnesses and injuries, and different measures on processing speed have been included in several earlier studies on patients with borreliosis, where reduction has been reported in varying degree (71). In recent studies, some find reduced processing speed (13, 15-17), while others find no differences between patients and controls (67, 68).
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1.6.3 Memory functions
Memory refers to a complex process of encoding, consolidating and retrieval of information. Different models of memory have been presented, but today there is a general consent regarding the major components (64) (figure 1).
Figure 1.
Hypothesized structure of memory from “A Compendium of Neuropsychological Tests”, Strauss, Sherman & Spreen, 2006 (64)
One of these components can be impaired, while other functions are intact. Explicit memory refers to intentional or conscious recollection. Semantic memory is general knowledge about the world we have acquired during life. Episodic memory on the other hand, is specific recollection of personal events, context or specific information you try to learn, like reading for an exam. It is explicit episodic memory we measure on traditional memory tests like California Verbal Learning Test 2nd edition (CVLT-II) or Family pictures from Wechsler Memory Scale 3rd edition (WMS-III).
The most persistent finding in patients with neuroborreliosis has been mild reduction on verbal list-learning tasks (13, 15, 71), while others find that either a small
subgroup has reduced verbal memory or no differences between patients and controls (4, 67, 68, 70).
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1.6.4 Methodological problems in neuropsychological assessment
In clinical neuropsychology, cognitive functions are classified into domains, which include different abilities and processes. Neuropsychological tests aim to measure one or more of these abilities. The domains are generally agreed upon, but some functions are challenging to place within a certain domain. Working memory for instance, is by many regarded to be a part of the executive function domain (72), but is also an important part of learning and memory (64). The origins of these domains were earlier linked to specific brain regions based on lesion studies. Although this still is an important view, newer research indicates a complex activation of different brain regions and circuits (73). Consequently, many neuropsychological tests are
multifunctional, meaning the test relies on more than the single function the test is meant to measure. One example is Trail Making Test (TMT) number-letter
sequencing, or TMT B, where flexibility, attention, processing speed and fine motor function all are involved. Many will call TMT B an executive test, while other place it under processing speed. Another challenge both in clinical setting, but also in follow- up studies where participant are tested repeatedly, is the re-test effect. The re-test effect is commonly associated with practice effect, where results tend to improve on repetition because of practice. Some tests, like learning and memory tests are
sensitive to practice effect, and a way to solve this is by including alternative versions.
In CVLT-II there is a standard version, and an alternative version which can be used when retesting (74). Other tests are not prone to practice effect in the same degree (65). The use of a control group will also help to control the practice effect in
research.
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Table 3. Studies including cognitive testing in patients with borreliosis. Studies after publication of EFNS guideline (2012) are included.
Study Material Functions Results
Eikeland et.al. 2012 (15)
50 patients (EFNS guidelines)/
50 matched controls
Processing speed Executive function Verbal memory
Patients had lower score on 1
executive subtest, 2 subtests on
processing speed and 1 condition on the verbal memory test. The majority of patients had normal scores, while a subgroup had low scores.
Chandra et.al 2013 (69)
37 patients with history of borrelia and memory impairment No control group
Motor and processing speed
Attention
Executive functions Verbal memory Visual memory
Z-scores used to obtain a global cognitive index score.
Mild, global cognitive
impairment, which did not contribute to perceived functional impairment.
Dersch et.al 2015 (4)
30 definite NB patients (EFNS guidelines) / 35 controls
Case-control study
Verbal memory Cognitive screening (MMSE)
No difference in cognitive function between groups
Patients with persistent symptoms had lower quality of life Schmidt 2015 (70) 60 NB patients /
30 matched controls
Working memory Verbal memory Visual memory Executive functions Processing speed Visuospatial function
Patients lower executive function, but within normal range. No
difference between groups in other domains.
28 Oczko-Grzesik 2017
(75)
121 patients with borreliosis (46 arthritis/ 75 NB) No control group Retrospective
Cognitive screening (MMSE and clock draw)
Cognitive problems (MMSE and clock draw combined), and depression were more
frequent in patients with NB compared to patients with arthritis.
Bechtold et.al 2017 (67)
107 patients with early borreliosis/
26 controls
6 month follow-up
Premorbid function Working memory Processing speed Verbal memory Executive function
No differences I cognitive function between the groups in the early phase or at 6 months follow-up.
A small subgroup (n=6) reported persistent health problems after 6 months
Keilp JG et.al 2018(17)
81 PLDS patients/
92 patients with major depression (MDD) /
91 controls
General abilities (IQ) working memory Processing speed Verbal memory Visual memory
Patients with PLDS had reduced verbal function and working memory compared with patients with MDD and controls.
Patients with PLDS and MDD had reduced processing speed compared with controls.
Touradij 2018 (13) 124 PLDS patients No control group
Premorbid function Working memory Processing speed Executive function Malingering
92 % reported subjective cognitive problems, of which cognitive decline was evident in 26 % and suboptimal engagement in 24%. Cognitive decline group had reduced score on verbal memory and processing speed.
Berende 2019 (68) 280 patients with symptoms
Verbal memory Working memory Processing speed
Small percentage (2.7%) of patients with optimal test-
29 attributed to
borrelia.
Executive functions Malingering
Subjective cognitive function
performance had cognitive
impairment, primarily verbal memory. No association
between subjective cognitive
complaints and test results.
Berende 2019 (14) Netherland
280 patients with symptoms
attributed to borrelia, and sufficient test- validity
Part of clinical trial comparing 12 weeks of antibiotic and placebo.
Verbal memory Working memory Processing speed Executive functions
Long-term
antibiotic treatment did not lead to better cognitive performance. All groups performed better at week 14, 26 and 40 – not specific to
treatment group.
Sigurdadottir et.al 2022 (76)
88 NB patients No control group
Processing speed (1 subtest)
Subjective
concentration and memory difficulties
31.4 % had
impaired scores on processing speed 1 month after
treatment start.
12 months after treatment start 14.3 % had impaired scores.
Nearly 50 %
reported subjective cognitive problems 1 month after treatment start.
Subjective cognitive problems improved during the follow- up.
Abbreviations: EFNS, European Federation of Neurological Societies; NB,
neuroborreliosis; MMSE, Mini Mental State Examination; PLDS, Post Lyme Disease Syndrome; MDD, major depressive disorder; IQ, intelligence quotient.
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2. Aims of the study
The overall aim of this thesis was to investigate cognitive function and explore the association between cognition, fatigue and structural brain changes in patients with neuroborreliosis. This was sought through neuropsychological testing and MRI examinations of a patient cohort with newly diagnosed neuroborreliosis over a 12- month period.
Specific aims were:
1. To investigate if patients with acute neuroborreliosis have reduced attention or processing speed, a higher level of fatigue or more white matter
hyperintensities compared with controls. (Paper I)
2. To compare cognitive function in patients with neuroborreliosis six months post treatment to a matched control group. Furthermore, to measure fatigue and assess whether neuropsychological test results or fatigue correlate with cortical thickness or brain volumes (Paper II)
3. To investigate long-term outcome regarding attention, processing speed and fatigue in patients with neuroborreliosis 12 months post treatment (Paper III)
3. Materials and methods
3.1 Study design
This study is part of a Norwegian multicenter treatment trial comparing two weeks and six weeks of doxycycline treatment for neuroborreliosis (77). In this prospective cohort study of patients with neuroborreliosis, we followed the patients from the acute phase of the infection until 12 months post treatment. A control group was included and assessed with neuropsychological tests and MRI at baseline and after six months for comparison (table 4).
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Table 4. Timeline and assessments Baseline
Patients and controls
6 months Patients and controls
12 months Patients only
Attention
Digit span Spatial span
Color word inhibition Processing speed
Color word read TMT motor speed
General abilities WAIS-IV Attention/ executive
Color word Tower
Verbal fluency Verbal memory
CVLT-II Visual memory
Family pictures
Attention
Digit span Spatial span
Color word inhibition Processing speed
Color word read TMT motor speed
Fatigue FSS
Fatigue FSS
Fatigue FSS Brain structure
MRI
Brain structure MRI
Abbreviations: FSS, Fatigue Severity Scale; MRI, magnetic resonance imaging; TMT, Trail making test, WAIS-IV, Wechsler Adult Intelligence Scale 4th edition; CVLT-II, California Verbal Learning Test 2nd edition.
3.2 Recruitment and participants
Patients aged ≥ 18 years with neuroborreliosis classified as probable or definite according to the EFNS guidelines were invited to participate (37). Patients excluded from the treatment study due to treatment with other antibiotic agents than doxycycline, were invited if they fulfilled the other inclusion criteria (table 5). We aimed to include and assess the patients as soon as possible, and within four weeks after diagnosis/ treatment start. In addition, patients who were prevented or unable
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to participate at treatment start were invited to participate at six months follow up if they fulfilled the inclusion criteria. Patients, who were not able to carry out MRI scanning due to contraindications, were invited to participate in neuropsychological testing only. From November 2015 to December 2018, we included 72 patients diagnosed with neuroborreliosis from Sørlandet Hospital and Oslo University Hospital, of which 63 patients were recruited from the treatment trial.
A flowchart of the inclusion is shown in figure 2.
Table 5. Inclusion/ exclusion criteria
Inclusion criteria Exclusion criteria
1. Inclusion in treatment study or 1. Contraindication for MRI (MRI only)
2. Possible / definite neuroborreliosis
a. Neurological symptoms indicative of neuroborreliosis without other obvious reasons
b. Lymphocytic pleocytosis (>5 leucocytes/mm3)
c. Intrathecal Bb antibody production Definite all three criteria, possible two out of three criteria
3. Adequate antibiotic treatment
2. Not speaking Norwegian (NP only)
Abbreviations: Bb, Borrelia burgdorferi; MRI, magnetic resonance imaging; NP, neuropsychology.
Control persons with the same sex and same age +/- 2 years as the patients were included by asking the patients to bring someone they knew and by advertising in the
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local newspapers. In total 68 control persons were included at baseline for
neuropsychological testing, of whom 64 carried out both neuropsychological testing and MRI.
Both patients and controls signed informed consent at inclusion.
Figure 2. Flowchart inclusion
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3.3 Cognitive testing
The same neuropsychologist (Silje Andreassen) carried out all neuropsychological testing in patients and controls. All tests are standardized, validated and were administered in a fixed order. If needed, breaks were given during testing.
3.3.1 Screening in acute phase and 12 months follow-up (paper I and III)
We chose eight subtests from three different test batteries to assess attention and processing speed in patients with acute neuroborreliosis and healthy controls.
Patients were retested 12 months after treatment with the exact same test protocol as in the acute phase. The aim was so see if patients’ achievements on the
neuropsychological tests of attention or processing speed changed compared to their results in the acute phase. Controls were not re-tested, as we did not expect changes in test scores in a healthy control group.
Attention
Digit span forward and backward are two subtests from WAIS-IV. Digit span forward is a sequence of numbers in random order from 1 to 9 to be repeated by the
participant. The sequence starts with two numbers and gradually increases until the patient is unable to repeat correctly any longer. In Digit Span backward, the patient is asked to repeat the examiner’s number sequence backwards, starting with the last presented number. One point is given for each correct sequence, and the raw score is the total number of points (78).
Spatial span forward and backward is a subtest in WMS-III and consists of a 10 block tapping board with blocks placed in random order. The examiner taps the blocks in a prearranged order, and the patient is asked to repeat this tapping pattern. The sequences increase gradually from two blocks, until the patient is no longer able to keep track of it. In Spatial span backward, the patient tap the blocks backward, starting with the last block the examiner tapped. One point is given for each correct sequence, and the raw score is the total number of points (79).
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In Color word interference test- inhibition from Delis-Kaplan Executive Function System (D-KEFS), the patient must inhibit the automatic reading response, in order to name the dissonant ink color on the written color words. Raw score is the amount of time measured in seconds used to complete the task (80).
Processing speed
Color word interference test- read consists of color words that the patient reads as fast and correctly as possible. Raw score is the amount of time, measured in seconds to complete the task (80).
TMT motor speed from D-KEFS is a motor-function speed test. The patient draws along a dotted line as fast as possible. Raw score is the time used to complete the task (80).
3.3.2 Neuropsychological assessment at 6 months follow-up (paper II) General cognitive ability
We used WAIS-IV which is the most widely used intelligence test for adults to assess general cognitive ability (78). The test consist of 15 subtests of which 10 are core subtests while five are supplemental. Supplemental tests are typically used either to provide additional clinical information or used as substitutes for core subtests. WAIS- IV assess verbal comprehension, perceptual reasoning, working memory and
processing speed in addition to full scale IQ. We chose eight subtests to get an estimated full-scale IQ. Selected subtests are underlined (see figure 3).
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Figure 3. Structure of WAIS-IV, with indexes and subtests
Verbal learning and memory
We used CVLT-II to assess verbal learning and memory (74). This test measures recall and recognition by presenting two lists of words over several immediate and delayed memory trials. Immediate recall is a list composed of 16 words from four sematic categories, and words from the same semantic category are not presented in consecutive order. The same list is presented five times, and the patient is asked to repeat as many words as possible after each trail. After trial one to five, an
interference list is presented, before the patient is asked to retrieve the original list after a short delay and then after a long delay (20-30 minutes afterwards).
Visual learning and memory
We used the subtest Family pictures from WMS-III to assess visual learning and memory (79). In this subtest the examinee looks at four different pictures of a family in four social situations. Directly after presentation of the pictures, the examinee is asked to recall which of the family member they saw in each picture, where they
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were placed and what they did. After a delay of 30 minutes, the examinee is asked to recall the same information again.
Executive function
Color word interference test has four conditions, color naming, read, inhibition and inhibition switch. Color naming is a card with colored squares (red, green or blue) presented as rows and the examinee names the colors. Read and inhibition have already been described above. The last condition, inhibition switch, is a combination of read and inhibition. Similar to inhibition, the task is still to name the ink color instead of reading the words, but the examinee has to read the words when the word is placed within a square. The last condition requires focused attention and ability to inhibit the automatic reading response, in addition to flexibility in order to switch between the rules. The raw score is the amount of time needed to complete the task measured in seconds
Verbal fluency consists of three different conditions; letter fluency, category fluency and category switching. In letter fluency the task is to produce as many words as possible beginning with the letter F, A and S. In categories the examinee is to name as many different words as possible based on the semantic category animals and boys’
names. The last condition is to name different fruits and furniture, alternating between the two semantic categories. All the tasks has a time limit of 60 seconds, and the raw score is the total number of words. Executive functions tapped by this test is initiation, simultaneous processing and processing speed. A limited vocabulary can affect performance in this task (81).
Tower test includes five disks varying in size from small to large, which are placed on a board with three pegs in a prearranged order. The examinee’s task is to move the disks across the pegs to build a tower in as few moves as possible. It is not allowed to move more than one disk at a time, and a large disk cannot be placed over a smaller
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one. Points are given based on correct tower and total number of moves within a time limit. The Tower test measures key executive functions such as planning, spatial organizing and inhibition of preservative responding (81).
3.4 Additional outcome measures
3.4.1 FatigueWe used FSS to measure level of fatigue. FSS is a nine-item self-filling questionnaire, initially developed to measure level of fatigue in patients with multiple sclerosis and lupus erythematosus (82). The respondents consider statements regarding fatigue and answer using a Likert scale ranging from 1 (strongly disagree) to 7 (strongly
agree). FSS has been translated and validated in the Norwegian population. A score of
≥5 is regarded as severe fatigue (54) as opposed to the original threshold (mean FSS
≥4) to define severe fatigue.
3.4.2 Cerebral MRI
The whole brain scan for the study was a sagital 3D T1 weighted MPRAGE
magnetization-prepared rapid gradient-echo (MPRAGE) sequence. MRI scannings were performed at two sites, Sørlandet Hospital Kristiansand and Oslo University Hospital. The scanners were a Siemens Skyra and a General Electric Signa, and the head coil was used with 64 and 32 channels, respectively. Protocols were as similar as possible with slice thickness 1,1/1,0 mm, field of view read 256 mm and phase
96,9%/256 mm, repetition time 2300 ms, echo time 2,98 ms/minimum and inversion time 900/943 ms.
3.4.2.1 Fazekas score (paper I)
In paper one, we used Fazekas scale to grade white matter hyperintensities in patients and controls (83). Fazekas scale ranges from zero to three. Fazekas 0, none or a single punctate lesion; Fazekas 1, multiple punctate lesions; Fazekas 2, beginning confluence of lesions (bridging) and Fazekas 3, large confluent lesions. In elderly, Fazekas grade 1 is considered normal. Fazekas grade 2 is considered normal in
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individuals above 71 years old, while grade 3 is considered pathological (84). The same neuroradiologist graded white matter hyperintensities with Fazekas score in all patients and controls.
3.4.2.2 Image analysis
Analysis of cortical thickness and brain volumes were done using the freely available FreeSurfer (85) software package (https://surfer.nmr.mgh.harvard.edu/). All analysis was done by an experienced neuroscientist. FreeSurfer calculates various
morphometry metrics based on high resolution T1 weighted images. T1 weighted images present high signal for fat content, like the white matter, and these areas will appear lighter. Areas with more water content, like the grey matter and
cerebrospinal fluid will appear darker (86). Surface based analysis: FreeSurfer transforms the cortex into a 2D surface based on models of cortical surface and identification of boundaries between different tissues (87). These surfaces are then inflated into a sphere. The cortex is inflated with a mesh built up by triangles. Each of the triangles have a meeting point, vertices. Each vertex contains numbers indicating gray matter volume, thickness and other surface measurements. Cortical thickness is the distance between pial surface and white matter surface (figure 4).
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Figure 4. Cortical thickness
The cortex is parcellated into different Regions of Interest (ROI), based on anatomical structures in a predefined atlas, Desikan-Killiany, which comes with FreeSurfer (88).
Desikan-Killiany generate 36 ROI in each hemisphere, each giving an estimation of thickness in that specific region. To avoid an excessive number of variables, we chose to combine ROI into left and right frontal, temporal, parietal and occipital lobes, reducing the number of output variables to eight. The location of ROI are described in Desikan-Killiany (88). In addition to ROI based analysis, we also performed a vertex- wise analysis given the explorative nature of the study. Vertex-wise analysis fits a general-linear model between subjects at each vertex to compare values of cortical thickness, and might be useful in studies without a priori hypotheses of specific brain regions might be affected (86). FreeSurfer’s automated algorithm performed cortical and subcortical segmentation, without manual adjustment (89, 90). Cortical and cerebellar grey matter, cerebral and cerebellar white matter, corpus callossum and estimated intracranial volume (eICV) were included. Subcortical structures in each hemisphere were combined resulting in the following dependent variables;
Illustration: smart.elsevier
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hippocampus, amygdala, thalamus, caudate nucleus, putamen, globus pallidius and nucleus accumbens. Each image was visually inspected to avoid segmentation errors, and one patient and five controls were excluded from further analysis because of this.
3.4.3 Other variables
In the treatment trial, a clinical composite score based on in total 32 items was calculated in all patients at inclusion. Two of the items were subjective memory and/or concentration problems and fatigue, which were rated as 0 = no problems, 1 = mild problems without daily influence and 2 = severe problems, with daily influence.
We used the item subjective memory/ concentration problem to estimate patients’
subjective cognitive problems at treatment start, and six months after treatment. We used the item subjective fatigue from clinical composite score at treatment start, but not six months after treatment.
We registered medication with fatigue as possible side effects in the patient group at baseline, and divided them into non-opioids, weak opioids, strong opioids,
benzodiazepines, non-benzodiazepines (sleep aid), neuroleptics and anti-neuralgias.
We were unable to retrieve information about medication in three patients.
3.4.4 Socioeconomic status
Socioeconomic status (SES) was calculated using Hollingshead’s index of educational and occupational position, scaled from 1 (low) to 5 (high) (91). For retired persons, the status score is calculated based on the occupation he or she had before
retirement.
3.5 Statistics
3.5.1 Sample sizeCalculation of sample size is based on three executive function tests 6 months after treatment, Color word, Verbal fluency and Tower. Estimated sample size for a two-
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sample mean test with 95 % confidence level showed 64 subjects in each group were needed to detect effect size (Cohen’s d) of 0.5 standard deviation, meaning a
difference of 1.5 scaled score between the groups would be a significant difference.
3.5.2 Statistics used in paper I-III
The Statistical Package for the Social Sciences (SPSS) version 25-28 was used in the analysis.
Paper I
We used independent samples t-test to compare mean scores in the two groups, and Mann-Whitney U when variables not were normally distributed. As FSS scores did not meet the criteria for using parametric correlation, we used Spearman rho to analyze associations between neuropsychological test results and FSS scores. We used age- corrected scaled scores based on normative data to determine whether a subject had a pathological low score (defined as -2 SD). Chi-square test was used to compare proportions. Missing data occurred randomly and were handled by pairwise deletion in analysis. Bonferroni correction was used to adjust for multiple comparison.
Paper II
Differences between the groups on neuropsychological tests and ROI based differences in cortical thickness were analyzed with Independent samples t-test.
Mann-Whitney U was chosen to compare level of fatigue, as FSS scores were not normally distributed. For the surface-based analysis we fitted a general linear model at each vertex, with cortical thickness as dependent variable and age and gender as covariates. We used general linear model with eICV as covariate to compare group differences in brain volumes between patients and controls. We used Spearman rho to investigate correlation between FSS scores and cortical thickness, brain volumes and neuropsychological tests. Chi square test was used to inspect proportions. We did not adjust for multiple comparison when analyzing correlation or proportions. To