Articles

Treatment of erythema migrans with doxycycline for 7 days

versus 14 days in Slovenia: a randomised open-label
non-inferiority trial
Daša Stupica, Stefan Collinet-Adler, Rok Blagus, Anja Gomišček, Tjaša Cerar Kišek, Eva Ružić-Sabljić, Maša Velušček

Summary
Background Lyme borreliosis is the most prevalent vector-borne disease in Europe and the USA. Doxycycline for Lancet Infect Dis 2022
10 days is the primary treatment recommendation for erythema migrans. To reduce potentially harmful antibiotic Published Online
overuse by identifying shorter effective treatments, we aimed to assess whether oral doxycycline for 7 days is October 6, 2022
https://doi.org/10.1016/
non-inferior to 14 days in adults with solitary erythema migrans.
S1473-3099(22)00528-X
See Online/Comment
Methods In this randomised open-label non-inferiority trial, we enrolled patients with a solitary erythema at the https://doi.org/10.1016/
University Medical Centre in Ljubljana, Slovenia. Patients were excluded if they were pregnant or lactating, S1473-3099(22)00581-3
immunosuppressed, allergic to doxycycline, or had received antibiotics with anti-borrelial activity within 10 days Department of Infectious
preceding enrolment or had additional manifestations of Lyme borreliosis Adults were randomly allocated 1:1 to Diseases, University Medical
receive oral doxycycline 100 mg twice a day for 7 days or 14 days. The primary efficacy endpoint was the difference in Centre Ljubljana, Ljubljana,
Slovenia (D Stupica MD,
proportion of patients with treatment failure, defined as persistent erythema, new objective signs of Lyme borreliosis, M Velušček MD); Slovenia and
or borrelial isolation on skin re-biopsy at 2 months, in a per-protocol analysis (the population that completed the Faculty of Medicine (D Stupica)
assigned doxycycline regimen according to the study protocol and did not receive any other antibiotics with anti- and Institute for Biostatistics
and Medical Informatics,
borrelial activity until the 2-month visit). The non-inferiority margin was 6 percentage points. Safety was assessed in
Faculty of Medicine
all randomly assigned patients who followed the study protocol and were evaluable at the 14-day visit. This study is (R Blagus PhD) and Faculty of
registered with ClinicalTrials.gov, NCT03153267. Sports (R Blagus) and Institute
for Microbiology and
Immunology Ljubljana, Faculty
Findings Between July 3, 2017, and Oct 2, 2018, we enrolled 300 patients (150 per group: median age 56 years
of Medicine
[IQR 47–65]; 126 [45%] of 300 male; skin culture positive 72 [30%] of 239 assessed). 295 patients completed antibiotic (Prof E Ružić-Sabljić MD),
therapy as per protocol and 294 (98%) patients were evaluable 2 months post-enrolment. Five (3%) of 147 patients University of Ljubljana,
from the 7-day group versus 3 (2%) of 147 patients from the 14-day group (one patient did not attend the 2-month Ljubljana, Slovenia;
Department of Infectious
visit and was unreachable by telephone) had treatment failure manifesting as persistence of erythema (difference
Diseases, Park Nicollet/Health
1·4 percentage points; upper limit of one-sided 95% CI 5·2 percentage points; p=0·64). No patients developed new Partners, Methodist Hospital,
objective manifestations of Lyme borreliosis during follow-up or had positive repeat skin biopsies. Two (1%) of Saint Louis Park, MN, USA
150 patients in the 7-day and one (1%) of 150 patients in the 14-day group discontinued therapy due to adverse (S Collinet-Adler MD); Slovenia
and Faculty of Mathematics,
events.
Natural Sciences and
Information Technologies,
Interpretation Our data support 7 days of oral doxycycline for adult European patients with solitary erythema migrans, University of Primorska, Koper,
permitting less antibiotic exposure than current guideline-driven therapy. Slovenia (R Blagus); General
Hospital Jesenice, Jesenice,
Slovenia (Anja Gomišček, MD);
Funding Slovenian Research Agency and the University Medical Centre Ljubljana. National Laboratory of Health,
Environment and Food,
Copyright © 2022 Elsevier Ltd. All rights reserved. Maribor, Slovenia
(T Cerar Kišek PhD)

Introduction assessing doxycycline efficacy for treating erythema Correspondence to:

Dr Daša Stupica, Department for
To minimise side-effects and reduce bacterial resis­tance, migrans, similar outcomes were achieved using 10 days Infectious Diseases, University
it is important to optimise duration of antibiotic versus 20 days in the USA5 and 10 days versus 15 days in Medical Centre Ljubljana,
therapies.1 Lyme borreliosis is the most prevalent vector- Europe.6 1525 Ljubljana, Slovenia,
dasa.stupica@kclj.si
borne disease in Europe and the USA.2 The shortest We aimed to conduct a randomised study assessing
effective treatment for erythema migrans, the most non-inferiority of 7 days compared with 14 days of oral
frequent manifestation of early Lyme borreliosis, doxycycline in adult European patients with solitary
remains unclear. Antibiotic regimens ranging from erythema migrans to determine the shortest effective
3 days to 21 days have been evaluated for the treatment of treatment.
erythema migrans (appendix p 2). Doxycycline for 10 days
to 14 days, or 14 days of amoxicillin or cefuroxime axetil Methods
are the preferred recommended treatments for adult Study design and participants
patients with erythema migrans, with 5 days to 10 days of Our randomised, open-label non-inferiority trial eval­uated
azithromycin as an alternative.3,4 In clinical trials non-inferiority of 7 days versus 14 days of oral doxycycline

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 Articles
Research in context
Evidence before this study
We searched PubMed on June 29, 2022, with the key words
“erythema migrans”, “Lyme borreliosis”, Lyme disease”,
“therapy”, and “antibiotic”, and used guideline bibliographies to
find relevant studies published in English without any date
See Online for appendix restrictions and found 25 relevent publications (appendix p 2).
Antibiotic regimens ranging from 3 days to 21 days were
evaluated for treatment of erythema migrans. Doxycycline for
10 days or amoxicillin or cefuroxime axetil for 14 days are the
primary recommended treatments for adult patients with
erythema migrans. Only one study assessed efficacy of less than
10 days of doxycycline for treating erythema migrans, but it was
a retrospective cohort study. In prospective clinical trials
assessing treatment efficacy of doxycycline, similar outcomes
were achieved using 10 days versus 20 days in the USA, and
10 days versus 15 days in Europe.It is unclear if a treatment
course of less than 10 days of doxycycline would still be effective
for erythema migrans.
100 mg twice a day in adult patients with solitary erythema
migrans at the University Medical Centre in Ljubljana,
Slovenia. Solitary erythema migrans was defined
according to European criteria7 as an expanding erythema
with or without central clearing, developing days to weeks
after a tick bite or exposure to ticks in a Lyme borreliosis
area. The majority of the patients in our study resided in
the central region of Slovenia, which is highly endemic for
Lyme borreliosis. For a reliable diagnosis, erythema of
5 cm or more in diameter was generally required. If the
diameter was smaller, a history of tick bite, a delay in
appearance of at least 2 days, and an expanding erythema
at the bite site were deemed acceptable.7 Exclusion
criteria were pregnancy or lactation, immunosuppression
(ie, active malignant disease, HIV infection, and immuno­
suppressive therapy), allergy to doxycycline, inability to
avoid sun and treatment with warfarin due to potential
drug-drug interactions, receipt of antibiotics with anti-
borrelial activity within 10 days preceding enrolment,
presence of multiple erythema migrans, or extra­cutaneous
mani­festations of Lyme borreliosis. Data were analysed
from Feb 1, 2021, to Sept 30, 2021.
Patients were asked to refer an acquaintance of similar
age (±5 years), preferably a spouse, to serve as controls.
The controls were non-hospitalised individuals, not
chosen after or because they interacted with the health
system in some way. A history of Lyme borreliosis in
controls, as assessed by questionnaire, prevented their
participation in the study. Written informed consent was
obtained from all patients and no participant received a
stipend.
Principles of good clinical practice were followed. The
protocol was approved by the Medical Ethics Committee
of the Ministry of Health of the Republic of Slovenia
(0120-161/2017-5 and 0120-161/2017/23).
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Added value of this study
Our finding that 7-day treatment with twice-daily oral
doxycycline was non-inferior in terms of treatment failure to
14-day treatment suggests that 7 days of oral doxycycline
might be sufficient to treat patients effectively and safely
with early Lyme borreliosis manifesting as solitary erythema
migrans, thus reducing potentially harmful antibiotic overuse
and cost.
Implications of all the available evidence
The available evidence supports the use of a 7-day regimen of
oral doxycycline for treating adult European patients with
solitary erythema migrans. The 7-day regimen appears to
fulfil the fundamental treatment goal of preventing
progression to more advanced disease while limiting
antibiotic exposure. We propose similar studies in other Lyme
borreliosis endemic regions where disease characteristics and
response to treatment could differ.
Randomisation and masking
In this open-label trial, patients were randomly
assigned (1:1) using a computer-generated randomisation
schedule to receive oral doxycycline 100 mg twice a day
for 7 or 14 days. The sequence was generated before
enrollment by computer. Participants were enrolled by
medical doctors (MV and DS), who also assigned patients
to the trial groups (who prescribed antibiotics.
Procedures
History, medication reconciliation, and physical exa­mi­
nation (ie, assessment of general appearance, orientation
to time, place and person, vital signs [including
temperature, blood pressure, and respiratory rate],
inspection of skin, heart and lung auscultation, abdomen
palpation, inspection and palpation of extremities, and
evaluation for meningeal signs). were done for patients
at enrolment and at 14 days, 2 months, 6 months, and
12 months after enrolment. Health-related symptoms
that developed or worsened after onset of erythema
migrans and were not otherwise explained were
considered Lyme borreliosis-associated symptoms at
enrolment or post-Lyme borreliosis symptoms at
follow-up. For patients who missed follow-up visits
from 2 months onwards, information about post-Lyme
borreliosis symptoms, intercurrent antibiotics, and new
episodes of Lyme borreliosis were obtained by telephone.
Patients completed two written questionnaires at
enrolment and again at 12 months. The first questionnaire
evaluated eight non-specific symptoms (ie, fatigue,
arthralgias, headache, myalgias, paraesthesia, memory
difficulties, concen­ tration difficulties, and irritability)
within the preceding week, and the second questionnaire,
the RAND 36-Item Health Survey 1.0 (SF-36), measured
quality of life. Higher SF-36 scores reflect better quality of

life.8 Controls completed the same questionnaires within
14 days of the 12-month visit of the corresponding patient
and self-reported basic demographics and comorbidities.
Blood counts and serum chemistries were done for
patients at enrolment and 14 days later. Serum IgM
antibodies to OspC and VlsE, and IgG antibodies to
VlsE borrelial antigens were determined by indirect
chemiluminescence immunoassay (LIAISON, DiaSorin,
Italy). Results were interpreted according to the
manufacturer’s instructions. At baseline, blood was
drawn and a skin biopsy in individuals who consented
was taken at the leading edge of erythema. Blood
samples were centrifuged and 1 mL of plasma and skin
samples were placed in 6 mL of an in-house modified
Kelly-Pettenkofer culture medium. If culture of the first
biopsy was positive for borreliae, a second biopsy was
collected from the same site 2–3 months after the first
biopsy. Skin and blood samples were cultivated at 33°C
and examined weekly by dark-field microscopy for the
presence of spirochetes for up to 9 weeks. Isolated
spirochetes were identified in-house to the species level
using MluI (Invitrogen, USA) restriction of genomic
DNA or PCR-based MseI (Biolab, USA), restriction
fragment length polymorphism of the intergenic region,
or PCR-based melting temperature deter­ mination of
hbb (U48676.1) gene. PCR primers were delivered by
TIB Molbiol, Germany, and reaction mix (LC TacqMan
master mix) was obtained from Roche, Germany.
Outcomes
Treatment outcomes were defined as complete response
(return to pre-Lyme borreliosis health status), partial
response (the presence of post-Lyme borreliosis
symptoms), or treatment failure (persistent erythema at
≥2 months post-enrolment or occurrence of new
objective dermatological, neurological, rheumatological
or cardiological manifestations of Lyme borreliosis or
isolation of borreliae on skin re-biopsy culture). Partial
responses and failures were categorised as unfavourable
responses.
The primary efficacy endpoint was the difference
between the proportion of patients with treatment failure
at the 2-month follow-up visit using the upper limit of
the 95% CI. Secondary efficacy endpoints were time to
resolution of erythema migrans (median [IQR]) and
unfavourable response beyond 2 months (percentage
points difference [95% CI]). Symptoms from the
questionnaires were compared between patients at
enrolment and 12 months, and between patients and
controls at 12 months (mean [SD]). Using interviews and
laboratory testing in patients following the study protocol
and attending the 14-day visit, antibiotic adherence was
assessed, and adverse events were graded according to
the Common Terminology Criteria for Adverse Events
(percentage points difference [95% CI]).9
The intention-to-treat population included all
ran­domly assigned patients. The per-protocol population
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Articles
repre­sented the population that completed the assigned
doxycycline regimen according to the study protocol and
did not receive any other antibiotics with anti-borrelial
activity until the 2-month visit. Analyses were done on
the per-protocol population, except for comparisons of
the study groups at enrolment, in which the intention-to-
treat population was used. Adverse events were analysed
using a modified intention-to-treat population including
all randomly assigned patients who followed the study
protocol and were evaluable at the 14-day visit, including
individuals who discontinued doxycycline early.
Statistical analysis
Sample size was calculated assuming 2% of patients
receiving 14 days of doxycycline would have treatment
failure.10 To obtain 90% statistical power with a one-sided
α equal to 0·05, 125 patients per group were necessary
for establishing the non-inferiority of 7 days compared
with 14 days of doxycycline, with a non-inferiority
margin of 6 percentage points. The original protocol
stated a margin of 10 percentage points, however the
relatively benign adverse event profile of doxycycline
prompted us to set a smaller and therefore more
stringent acceptable difference to assess our primary
efficacy endpoint in the final analysis. The test statistic
used was the one-sided Z test with continuity correction
(unpooled). To account for attrition, the required sample
size was estimated to be about 150 patients per group.
For the secondary efficacy outcomes, a sample size of
125 participants per group would have 95% power with a
one-sided α equal to 0·05 to show non-inferiority of
the 7-day regimen compared with 14-days using
10 per­centage points margin, assuming unfavourable
responses of approximately 6% at 12 months post-
enrolment.10 The sample size calculation was performed
using PASS (version 2019).
Categorical data were summarised as frequency (%)
and numerical data as median (IQR) or mean (SD) as
appropriate. Differences between the 7-day regimens and
14-day regimens were tested using the Mann–Whitney
test, independent sample t-test assuming unequal
variances for numeric variables or χ² test with Yates
continuity correction for categorical variables. The
difference between the proportions of patients with
treatment failure receiving the 7-day versus the 14-day
regimen was estimated using a one-sided 95% CI based
on normal approximation with continuity correction.
Clinically acceptable non-inferiority of the 7-day regimen
was established if the upper limit of the 95% CI did not
exceed 6 percentage points. Non-specific symptoms and
quality of life at enrolment and 12-month visit were
compared using the McNemar test with continuity
correction for categorical variables or paired sampled
t-test for numeric variables, adjusting the p values with
Holm’s method to control the family-wise error-rate.
The median duration of erythema migrans was
calculated using the Kaplan–Meier method; the log-rank
 Articles

Results were presented as odds ratios (OR) with 95% CI. A

609 patients assessed for eligibility p value of less than 0·05 was considered significant,
unless otherwise stated. R statistical language was used
309 excluded
for the analyses (version 3.4.1). The random-effects
99 prior antibiotic therapy regression models were fitted using the lme4 R package.
72 multiple erythema migrans No data monitoring safety board was associated with
38 absence of study investigator
38 extracutaneous manifestations the study. The study is registered with ClinicalTrails.gov,
30 unable to avoid sun exposure or taking warfarin† NCT03153267.
17 pregnant or lactating
15 declined to participate
Role of the funding source
The funders of the study had no role in the study design,
300 randomly assigned data collection, data analysis, data interpretation, the
writing of the report, or the decision to submit the paper
for publication.
150 assigned to receive 7-day 150 assigned to receive
doxycycline 14-day doxycycline Results
Between July 3, 2017, and Oct 2, 2018, 150 patients were
3 discontinued treatment
randomly assigned to doxycycline for 7 days and
2 discontinued treatment
2 discontinued doxycycline 1 discontinued doxycycline 150 patients to doxycycline for 14 days (figure, table 1).
due to adverse event due to adverse event 147 (98%) of 150 patients completed per-protocol
1 discontinued owing to not 1 discontinued owing to not
adhering to treatment adhering to treatment treatment with doxycycline for 7 days and 148 (99%) of
150 patients completed per-protocol treatment with
doxycycline for 14 days (figure). Median time to resolution
147 completed antibiotic 148 completed antibiotic
per-protocol per-protocol
of erythema migrans after starting treatment was
comparable between the 7-day and 14-day treatment
groups (IQR 7–14 days vs 11 days, IQR 7–16 days; p=0·90).
3 did not attend follow-up* 3 did not attend follow-up* Five (3·4%) of 147 evaluable patients in the 7-day group
and three (2·0%) of 147 patients in the 14-day group (one
144 attended 14-day visit 145 attended 14-day visit
of the 148 patients did not attend the 2-month visit and
could not be reached by telephone) had treatment failure
manifesting as persistent erythema at the 2-month visit
7 did not attend follow-up 8 did not attend follow-up (difference 1·4 percentage points; upper limit of one-sided
95% CI 5·2 percentage points; p=0·64). Results of the
137 attended 2-month visit
intention-to-treat analysis (five [3%] of 150 vs three [2%]
137 attended 2-month visit
of 150; difference 1·3 percentage points; upper limit of
one-sided 95% CI 5·1 percentage points; p=0·64) and the
19 did not attend follow-up 10 did not attend follow-up analysis including only patients who attended the
2-month follow-up visit (five [4%] of 137 vs three [2%]
of 137; difference 1·5 percentage points; upper limit of
118 attended 6-month visit 127 attended 6-month visit
one-sided 95% CI 5·5 percentage points; p=0·64) closely
paralleled per-protocol results. Six (75%) of eight treatment
14 did not attend follow-up 24 did not attend follow-up failures had a skin biopsy performed at enrolment;
only one of these yielded a positive culture, which
104 attended 12-month visit 103 attended 12-month visit
subsequently was negative on re-biopsy. Four treatment
failures from the 7-day group and none from the 14-day
group received further antibiotics. All four retreated
Figure: Trial profile
*Patients were invited to follow-up, but if they did not attend follow-up visits, the reasons for their absence were patients recovered completely. Of the four non-retreated
not further investigated. †Taking warfarin was an exclusion due to potential drug-drug interactions. patients, two reported post-Lyme borreliosis symptoms at
6 months and all four were asymptomatic at 12 months.
test was used to test the difference between the duration Initial skin biopsy was performed in two of the four
curves of the two treatment groups. non-retreated patients, and both were Borrelia culture
The association between unfavourable response and negative. In all eight patients, residual erythema resolved
other covariates was estimated using mixed-effects logistic by the 6-month visit. Six (75%) of these eight patients
regression, adjusting the analysis for treatment and time were seropositive at enrolment and two (29%) of
from enrolment as categorical variables. To account for seven patients evaluated at the 12-month visit remained
multiple measurements in each patient, random intercept seropositive. Patients with residual erythema reported
by patient identification was included in the model. post-Lyme borreliosis symptoms at 2 months numerically

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7-day doxycycline 14-day doxycycline Treatment group Difference Upper bound p value*
group (n=150) group (n=150) (percentage of 95% CI*
points)
Age, years 57 (47–64) 56 (47–66)
Sex 7-day 14-day
doxycycline doxycycline
Male 66 (44%) 60 (40%) group group
Female 84 (56%) 90 (60%)
14 days post-enrolment 25/144 (17%) 29/145 (20%) –2·6 5·6 0·34
History of previous Lyme 46 (31%) 34 (23%)
borreliosis 2 months post-enrolment

Comorbidities* 72 (48%) 75 (50%) Obtained from patients 28/137 (20%) 24/137 (18%) 2·9 11·4 0·68
attending follow-up
Tick bite† 69 (46%) 61 (41%) visits†
Days since erythema migrans 14 (6–30) 14 (7–30) Obtained by telephone‡ 28/147 (19%) 24/147 (16%) 2·7 10·7 0·68
first observed
6 months post-enrolment
Diameter of erythema 15 (10–23) 16 (10–24)
migrans, cm Obtained from patients 17/118 (14%) 14/127 (11%) 3·4 11·2 0·73
attending follow-up visits
Erythema migrans with 85 (57%) 84 (56%)
central clearing Obtained by telephone 17/144 (12%) 15/147 (10%) 1·6 8·3 0·60

Lyme borreliosis-associated 39 (26%) 41 (27%) 12 months post-enrolment

symptoms‡ Obtained from patients 10/104 (10%) 10/103 (10%) –0·1 6·8 0·50
Fatigue 15 (10%) 17 (11%) attending follow-up visits

Arthralgia 14 (9%) 19 (13%) Obtained by telephone 10/141 (7%) 11/146 (8%) –0·4 5·1 0·50

Headache 12 (8%) 19 (13%) Last evaluable visit

Myalgia 7 (5%) 6 (4%) Obtained from patients 13/146 (9%) 18/148 (12%) –3·3 3·3 0·24
attending follow-up visits
Seropositive§ 109 (73%) 116 (77%)
Obtained by telephone 10/147 (7%) 11/148 (7%) –0·6 4·9 0·50
IgM 63 68
Data are n/n (%). *Estimated using a one-sided 95% CI based on the normal approximation with continuity correction.
IgG 89 92
p<0·01 was considered significant. †Treatment failure: 5/137 (4%) versus 3/137 (2%); difference 1·5 percentage points;
Skin culture positive¶ 34/115 (30%) 38/124 (31%) upper limit of one-sided 95% CI 5·5 percentage points; p=0·64. ‡Treatment failure: 5/147 (3%) versus 3/147 (2%);
Borrelia afzelii 32 33 difference 1·4 percentage points; upper limit of one-sided 95% CI 5·2 percentage point; p=0·64.
Borrelia burgdorferi sensu 2 0
Table 2: Patients with erythema migrans who had an unfavourable response at follow-up according to
stricto
treatment group (per protocol population)
Borrelia garinii 0 3
Borrelia spielmanii 0 2
Blood culture positive 1/147 (1%) 1/149 (1%) Rates of Borrelia seropositivity in patients from both
Data are median (IQR), n (%), or n/n (%). *Patients with an underlying chronic groups were similar at enrolment (109 [73%] of 150 in the
illness (eg, arterial hypertension, hyperlipidaemia, osteoporosis, diabetes, 7-day group vs 116 [77%] of 150 in the 14-day group;
thyroid disease, cardiac rhythm abnormality, psychiatric illness, ischaemic heart
disease, osteoarthritis, or asthma). †Patients with a history of tick bite at the site p=0·42; table 1) and at the 6-month visit (64 [54%]
of the erythema migrans. ‡Patients who reported Lyme borreliosis-associated of 118 vs 84 [67%] of 126; p=0·05) and 12-month visit
symptoms at enrolment (some patients had more than one symptom). (51 [49%] of 104 vs 54 [53%] of 102; p=0·58). At the
§Positive and borderline test result for IgM and/or IgG to Borrelia burgdorferi
sensu lato at enrolment. ¶Comparison between Borrelia afzelii and other
2-month visit, the proportion of seropositive patients was
identified Borrelia species lower in the 7-day group (90 [67%] of 134 vs 110 [81%]
of 136; p=0·01). Borrelia burgdorferi sensu lato was
Table 1: Baseline characteristics of the intention-to-treat population
isolated from 72 (30%) of 239 pre-treatment skin biopsies
and from two (1%) of 296 blood specimens (table 1).
more often than the patients with resolved erythema Among the skin isolates, 65 (90%) of 72 were B afzelii.
(three [38%] of eight vs 45 [16%] of 289; p=0·12), but the Repeat biopsies were performed in 63 (88%) of 72 patients
difference was not statistically significant. who were initially culture positive. All repeat biopsies
The frequency of unfavourable responses decreased with were culture negative.
time from enrolment (table 2). The odds for unfavourable Six patients were treated for a new erythema migrans
response were increased for patients with Lyme borreliosis- at least 4 months after enrolment and all had a complete
associated symptoms at enrolment and for women response at the 12-month visit (one patient [1%] of 147 in
compared with men but were not associated with treatment the 7-day group and five patients [3%] of 148 in the
duration (table 3). Results were similar when including 14-day group). Some patients received antibiotics with
information obtained by telephone (tables 2, 3). No patients anti-borrelial activity for unrelated conditions during
from either group developed new objective manifestations follow-up: four (3%) of 147 in the 7-day group and
of Lyme borreliosis during follow-up. eight (5%) of 148 in the 14-day group. The proportion of
Doxycycline-related adverse events were generally mild patients with intercurrent antibiotics during follow-up
(grade 1)9 and comparably frequent between the treatment was similar between the 7-day group and 14-day group
groups (table 4). (nine [6%] of 147 vs 13 (9%) of 148; difference

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In-person follow up visit Telephone visit 7-day doxycycline 14-day doxycycline

group (n=146) group (n=146)
OR (95% CI) p value OR (95% CI) p value
Photosensitivity 0 2 (1%)
Intercept 0·03 (0·00–0·29) 0·0 0·02 (0·00–0·16) 0·0
Allergic reaction 1 (1%) 1 (1%)
Antibiotic regimen (7 days vs 14 days) 0·96 (0·38–2·40) 0·93 0·83 (0·35–1·93) 0·66
Gastrointestinal 20 (14%) 17 (12%)
Age, years 1·00 (0·96–1·04) 0·88 1·01 (0·97–1·04) 0·75
Dyspepsia* 9 (6%) 8 (6%)
Sex (male vs female) 0·12 (0·04–0·36) 0·0 0·13 (0·05–0·36) 0·0
Nausea 5 (3%) 8 (6%)
Presence of comorbidities (yes vs no) 2·18 (0·80–5·97) 0·13 1·95 (0·78–4·90) 0·15
Vomiting* 1 (1%) 0
History of Lyme borreliosis (yes vs no) 0·55 (0·18–1·62) 0·28 0·62 (0·23–1·67) 0·34
Diarrhoea 4 (3%) 4 (3%)
Time
Obstipation 2 (1%) 0
2 months vs 14 days 0·23 (0·10–0·51) 0·0 0·18 (0·09–0·37) <0·0001
Other 12 (8%) 19 (13%)
6 months vs 2 months 1·80 (0·80–4·04) 0·15 1·90 (0·91–3·98) 0·01
Headache* 4 (3%) 6 (4%)
12 months vs 6 months 0·56 (0·25–1·25) 0·16 0·49 (0·23–1·03) 0·01
Dizziness 1 (1%) 6 (4%)
LB-associated symptoms at enrolment 5·15 (1·81–14·65) 0·0 3·81 (1·47–9·86) 0·01
(yes vs no) Fatigue or malaise 5 (3%) 8 (6%)
Vaginitis or thrush 2 (1%) 1 (1%)
OR=odds ratio. *Estimated from a multiple logistic regression model with unfavourable response as the dependent
variable. Each OR is adjusted for all other variables in the table. p<0·05 was considered significant. Anxiety 0 1 (1%)
Hair loss 1 (1%) 0
Table 3: Factors associated with unfavourable response (per protocol population)
Laboratory abnormality
White blood cell decreased 0/141 1/142 (1%)
–2·7 percentage points; upper limit of one-sided 95% CI Bilirubin increased 3/142 (2%) 9/139 (7%)
3·0 percentage points; p=0·26). Intercurrent antibiotics Aspartate Transferase 8/142 (6%) 6/142 (4%)
with anti-borrelial activity during follow-up did not increased

lessen the odds for unfavourable response (appendix p 4).
Subanalysis of the per-protocol population excluding
the 22 patients who received intercurrent antibiotics
with anti-borrelial activity, led to similar outcomes in
terms of proportion of patients with unfavourable
response at follow-up and the odds for unfavourable
response (appendix pp 5–6).
Frequencies of non-specific symptoms and health-
related quality of life were similar in patients at
enrolment and at the 12-month visit (appendix p 7). We
observed no statistical differences in the frequency of
non-specific symptoms or quality of life between patients
and controls at 12 months after enrolment. These last
two groups were well matched in terms of basic
demographics (table 5).
Alanine Transaminase 8/142 (6%) 4/142 (3%)
increased
Any adverse event 41 (28%) 49 (34%)
Data are n (%) or n/n assessed (%). Treatment-related adverse events were defined
according to the Common Terminology Criteria for Adverse Events.9 Some patients
had more than one adverse event. The modified intention-to-treat population
included all randomly assigned patients who followed the study protocol and were
evaluable at the 14-day visit, including those who discontinued doxycycline early.
*Side-effects that led to a change in treatment. Two patients in the 7 day group
discontinued doxycycline due to an adverse event (azithromycin was introduced in
one patient due to vomiting after the first dose of doxycycline, and in another,
cefuroxime axetil was prescribed due to a headache after 2 days of doxycycline) as
did one patient in the 14 day group (discontinued doxycycline due to dyspepsia
after 11 days of therapy).
Table 4: Treatment-related adverse events by day 14 (modified
intention-to-treat population)

Discussion Intermittent or persistent subjective post-Lyme

This open-label, randomised trial showed that 7 days of
doxycycline in adult European patients with solitary
erythema migrans was safe, effective, and non-inferior to
a 14-day regimen for all efficacy endpoints: treatment
failure at 2 months, time to resolution of erythema
migrans, and unfavourable response beyond 2 months.
Furthermore, the frequency of unfavourable responses
decreased similarly from time of enrolment in both
treatment groups.
Treatment failure rates between the two therapeutic
groups at 2 months showed that 14 days of treatment
was associated with a small advantage (at most
5·2 percentage points when considering one-sided 95%
CI) compared with the 7-day regimen. We believe that a
minimal decrease in efficacy of the 7-day group is
acceptable because no patients in either group developed
objective signs of persistent or progressive infection.
borreliosis symptoms were not incapacitating and did
not excessively compromise quality of life. Our non-
inferiority margin was more stringent than those used
in previous trials exploring therapeutic efficacy in early
Lyme borreliosis.3,11 The benign adverse event profile of
doxycycline12 prompted us to set a smaller acceptable
difference than used previously.6 Adverse events were
generally mild and comparably frequent in both groups
in our study. Nevertheless, reducing duration of
antibiotic exposure is an important consideration. There
is a substantial body of evidence describing the
disadvantages of unnecessarily long antibiotic courses in
terms of incrementally decreasing benefits on patient
outcomes and the risks of adverse events, super­
infections, increased cost, and selection of resistance.13
Although doxycycline resistance in borreliae has not
been identified as a major clinical issue,14 doxycycline

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negatively effects microbiota, increasing selection of

Patients (n=186) Controls (n=142) p value
resistance in other micro-organisms.15
A fundamental goal of treatment of erythema migrans Age, years 57 (48–65·8) 60 (49·3–65) 1·00

is to ensure that later stages of Lyme borreliosis do not Sex 1·00

develop after treatment. No cases of later stage Lyme Male 69 (37%) 60 (42%)
borreliosis developed up to 12 months after treatment in Female 117 (63%) 82 (58%)
either study arm. Although one study showed that Comorbidities 92 (50%) 66 (46%) 1·00
recurrence of Lyme arthritis can be seen at intervals of up Any non-specific 156/185 (84%) 121/131 (92%) 0·97
to 23 months after untreated erythema migrans, onset symptom

of the first episode of arthritis was 1–24 weeks from Fatigue 126 (68%) 99 (76%) 1·00
erythema migrans.16 Other studies of treated Lyme Arthralgia 109 (59%) 84 (64%) 1·00
borreliosis suggest that progression to later mani­ Headache 90 (49%) 64 (49%9) 1·00
festations of Lyme borreliosis is rare, usually occurring Myalgia 88 (48%) 67 (51%) 1·00
within days to weeks, but no later than 9 months after Paraesthesia 79 (42%) 57 (44%) 1·00
treatment.5,17–20 Following patients for 12 months therefore Memory difficulties 89 (48%) 76 (58%) 1·00
appears to be a reasonable time period to assess Concentration 91 (49%) 80 (61%) 0·97
difficulties
protection from progressive infection.
At the 12-month visit, 10% of the patients in the 7-day Irritability 99 (53%) 88 (67%) 0·43

group and 10% of the patients in the 14-day group were Physical functioning 82 (22) 86 (18) 1·00
score
classified as unfavourable responders, which is largely
Limitations due to 73 (39) 75 (37) 1·00
comparable with previous studies.5,6,21,22 These pro­ physical health score
portions were even lower when information obtained by Limitations due to 79 (37) 86 (31) 1·00
telephone was assessed compared with the evaluation emotional health
in which only patients attending follow-up visits were score
included (7% and 8%). Demographic, clinical, and Vitality score 65 (23) 64 (20) 1·00
microbiological characteristics of participants in our Mental health score 76 (18) 77 (17) 1·00
study were similar to patients from previous European Social functioning 79 (23) 79 (21) 1·00
treatment trials of erythema migrans.6,10,11,23 The score

seropositivity rate to borreliae was similar to previous
studies enrolling patients with solitary erythema
migrans in Slovenia.10,17,24
The non-significant higher proportion of retreatment in
the 7-day group might have been due to lack of blinding
and reflects a cautious and conservative approach from
the physician. Residual erythema at 2 months post-
enrolment could merely represent post-inflammatory
consequences but was defined as treatment failure as we
often retreat these patients.
Histopathology was not performed. Cultures to
confirm or reject persistent infection were not available
for all patients, but no repeat biopsies were culture
positive. The difference in frequency of post-Lyme
borreliosis symptoms in patients with and without
residual erythema was not statistically significant.
Residual erythema might not require re-treatment.
The frequency of intercurrent antibiotics with anti-
borrelial efficacy was similar in both treatment groups,
and probably did not introduce bias. To properly assess
the secondary outcomes, we performed an exploratory
analysis excluding patients who received intercurrent
antibiotics and obtained comparable results.
At 12 months, the eight non-specific symptoms from
questionnaires were seen with similar frequencies in
cases and controls, consistent with some previous
European and US studies,6,10,24,25 which could suggest that
these non-specific symptoms might not necessarily be
related to previous Lyme borreliosis. Also, quality of life
Pain score 71 (27) 68 (24) 1·00
General health score 66 (22) 64 (18) 1·00
Data are median (IQR), n (%), n/n (%), or mean (SD). p values were adjusted with
Holm’s method to control family-wise error-rate. p<0·05 was considered
significant.
Table 5: Comparison of patients and controls regarding non-specific
symptoms and quality of life assessed by the RAND 36-Item Health
Survey 1.0 at the 12-month visit
was high in patients at enrolment and at 12 months
suggesting that Lyme borreliosis-associated symptoms or
persistent post-Lyme borreliosis symptoms might not
interfere substantially with quality of life. However,
contrary to our results, studies from both the Netherlands
and the USA found that a subset of patients treated for
erythema migrans reported increased frequency and
severity of non-specific symptoms on quality of life
metrics compared with those without prior Lyme
borreliosis.26,27 These discordant results could be due to
differences in study design, power, enrolment criteria,
and infecting Borrelial species.
Post-Lyme borreliosis symptoms can be non-specific
and challenging to differentiate from symptoms
unrelated to Lyme borreliosis.24 Post-Lyme borreliosis
symptoms might not be a reliable measure of treatment
efficacy in patients treated for erythema migrans. More
objective measures are desirable. Progression to other
manifestations of Lyme borreliosis clearly reflects
treatment failure and persistent culture positivity for

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borreliae suggests possible inadequacy of therapy
whereas residual erythema 2 months after starting
antibiotics does not prove persistent or progressive
borrelial infection.
Compared with North American Lyme borreliosis,
symptoms are often less frequent and milder in Europe,
which is likely due to increased inflammatory responses
to Borrelia burgdorferi sensu stricto (in North America),
which is rarely implicated in European Lyme
borreliosis.3,28–30 Given the differences between Lyme
borreliosis in Europe and North America, a similar study
to ours could be considered in the USA and might better
evaluate the speed and durability of short course
doxycycline on Lyme borreliosis-associated or erythema
migrans-associated manifestations such as fevers,
lymphadenopathy, elevated liver function tests, and
inflammatory markers.29,30
Our study has several limitations. First, the study
was not blinded, which could have influenced
symptom reporting by patients and decisions to
retreat, but the multivariate analysis was adjusted for
the latter potential confounder. Second, some post-
Lyme borreliosis symptoms could have been
misclassified to be Lyme borreliosis-associated.
Exhaustive evaluations for other causes were not
performed. Conversely, a third limitation is that
potential borrelial infections in controls were not
rigorously excluded and could have been more
aggressively investigated, for example, by performing
a full clinical examination and serological testing.
Perhaps, a proportion of non-specific symptoms in
controls might have been associated with Lyme
borreliosis. Fourth, our results might not be valid for
other antibiotics used to treat erythema migrans, for
multiple erythema migrans, and other clinical
manifestations of Lyme borreliosis or in regions with
different proportions of borrelial species causing
erythema migrans.
To our knowledge, this study is the first evaluation of a
7-day doxycycline regimen for early Lyme borreliosis
manifesting as solitary erythema migrans. Our results
showed that 7 days of doxycycline was non-inferior to a
14-day regimen for treatment of solitary erythema
migrans in adult patients in Slovenia. Regardless of
regimen, unfavourable responses were rare and no cases
with objective progression of Lyme borreliosis were seen.
The frequency of non-specific symptoms and quality of
life scores in patients were comparable to those in
controls without a history of Lyme borreliosis. This
finding suggests that clinicians must remain circumspect
when interpreting non-specific symptoms thought to be
associated with Lyme borreliosis as these symptoms
could be unrelated to Lyme borreliosis. Similar studies
should be carried out in other Lyme borreliosis-endemic
regions where disease characteristics and response to
treatment could differ, to investigate the generalisability
of our findings.
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Contributors
DS conceptualised and designed the study. DS, AG, and MV gathered
clinical data and did the data interpretation. RB performed the statistical
analyses and participated in data interpretation. TCK and ER-S did the
microbiological analyses. DS and SC-A searched the literature,
interpreted data, and wrote the manuscript. DS and MV had full access
to all the data in the study and verified the underlying data. All authors
had final responsibility for the decision to submit for publication.
All authors read and approved the final version of the manuscript.
Declaration of interests
DS received support to speak at educational events sponsored by Merck
& Co, Sanofi, and Madison Pharma. All other authors declare no
competing interests.
Data sharing
Deidentified data generated or analysed during this study and the full
trial protocol are available from the corresponding author upon
reasonable request.
Acknowledgments
This work was supported by the Slovenian Research Agency (grant
numbers P3-0296 and J3-6788) and the University Medical Centre
Ljubljana, Ljubljana, Slovenia (TP20170076).
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