- Email: [email protected]
Clinically-diagnosed Mediterranean Spotted Fever in Malta
Accepted Manuscript Clinically-diagnosed Mediterranean Spotted Fever in Malta Paul Torpiano, David Pace PII:
S1477-8939(18)30016-4
DOI:
10.1016/j.tmaid.2018.02.005
Reference:
TMAID 1220
To appear in:
Travel Medicine and Infectious Disease
Received Date: 8 September 2017 Revised Date:
13 February 2018
Accepted Date: 16 February 2018
Please cite this article as: Torpiano P, Pace D, Clinically-diagnosed Mediterranean Spotted Fever in Malta, Travel Medicine and Infectious Disease (2018), doi: 10.1016/j.tmaid.2018.02.005. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Title page
Title: Clinically-diagnosed Mediterranean Spotted Fever in Malta
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Running title: MSF in Malta
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Authors: Paul Torpiano, David Pace
Affiliation: Department of Paediatrics, Mater Dei Hospital, Tal-Qroqq, Msida, MSD 2090,
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Malta
Corresponding Author: Paul Torpiano
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Department of Paediatrics, Mater Dei Hospital, Tal-Qroqq, Msida, MSD 2090, Malta Telephone: +356 2545 5567 Fax: +356 2545 4154
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Email: [email protected]
Key words Mediterranean spotted fever; children; Rickettsia conorii; diagnosis
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ACCEPTED MANUSCRIPT Abstract (194 words) Background Mediterranean Spotted Fever (MSF) is a tick-borne zoonosis caused by Rickettsia conorii which is endemic in Malta, an island in the South Mediterranean that is a popular tourist
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destination. Diagnosis is frequently based on clinical manifestations as laboratory results are often limited to a retrospective diagnosis. We describe the clinical presentation, diagnosis and
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treatment of children <16 years who presented with MSF from 2011 – 2016.
Method
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The demographics, clinical findings, laboratory results, management and outcome of all children hospitalised with suspected MSF based on the presence of fever and an eschar, were retrieved from their case notes.
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Results
Over the five-year study period six children, aged between 17 months and 15 years, were diagnosed with MSF. All children had contact with ticks and the majority presented in
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summer. Laboratory results were non-specific and included elevated inflammatory markers,
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lymphocytosis/lymphopenia and hyponatraemia. Serological and molecular techniques were used for diagnosis. Response to clarithromycin or doxycycline was immediate.
Conclusion
MSF should be included in the differential diagnosis of fever, rash and an eschar in children who travel to Malta. Despite advances in molecular diagnostics, clinical diagnosis remains important in the management of children with suspected MSF.
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ACCEPTED MANUSCRIPT Funding sources
The research work disclosed in this publication is partially funded by the ENDEAVOUR Scholarships Scheme. “The scholarship is part-financed by the European Union – European
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Social Fund”.
Acknowledgements
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The authors would like to thank Dr Matthew Snape for his help and guidance in writing this
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Word count: 3632
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manuscript.
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ACCEPTED MANUSCRIPT 1 Introduction Mediterranean spotted fever (MSF) is a tick-borne illness caused by Rickettsia conorii. It is endemic across southern Europe and North Africa, with the seroprevalence of anti-R.conorii antibodies being as high as 10% in some Mediterranean countries [1–5]. Rhipicephalus
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sanguineus (brown dog tick) acquires R. conorii by feeding on infected dogs, and can transmit rickettsiae to humans through feeding [6]. Following inoculation at the site of an eschar, R. conorii replicates in the cytoplasm of endothelial cells, releasing pro-inflammatory
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cytokines that lead to a widespread vasculitis [7]. MSF is characterised by fever, headache, rash and an eschar [8,9]. Diagnosis is usually serological, though sensitivity is limited early
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in the illness, providing only a retrospective diagnosis [10,11]. Molecular techniques provide a faster diagnosis, though with limited sensitivity [12]. Although MSF runs a milder course in paediatric patients, there is a need to diagnose and treat the disease early to avoid complications [1]. Diagnosing MSF on epidemiological and clinical features has an important
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role in its management.
Malta is an archipelago in the centre of the Mediterranean, and a popular destination amongst
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tourists: in 2016 alone, the Maltese islands received 2 million visitors [13]. Over half of these
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visited between June and September, when warm weather encourages outdoor activities as well as tick-biting behaviour in R. sanguineus [13,14]. We describe the manifestations, laboratory findings and management of all children hospitalised with MSF from 2011-2016 in order to make doctors in non-endemic countries aware of the persistence of this zoonosis in Malta.
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2 Methods MSF is a notifiable disease in Malta [4]. All patients under 16 years of age, admitted to Mater Dei Hospital, the only teaching hospital in Malta, for the period 2011-2016, and suspected to
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have MSF based on the presence of fever and an eschar were identified from the database kept by the Paediatric Infectious Diseases team and their hospital records were retrieved. For the literature review, we searched the MEDLINE, EMBASE, and NHS Evidence
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databases using the search terms ‘Mediterranean Spotted Fever’ and ‘Boutonneuse fever’. These searches gave a total of 1182 articles. We included articles written in English, Italian,
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and French. We excluded articles in other languages, individual case reports, articles focused on non-MSF rickettsiosis and other infections, articles describing rickettsial infection in non-
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human hosts, and papers irrelevant to the subject. This gave a total of 83 articles (Figure 1).
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EMBASE (n=130)
Records excluded due to nonhuman subjects (n=279)
Records screened (n=1180)
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573 records screened (title/abstract)
Eligibility
Records excluded as not in English/French/Italian (n=294)
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Records in English/French/Italian (n=607)
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Screening
901 records screened by language
Included
NHS evidence (n=13)
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MEDLINE (n=1037)
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Identification
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121 full-text articles assessed for eligibility
34 duplicate records excluded
Records excluded (n=452): • Case reports (n=314) • Full-text unavailable (n=32) • Non-MSF infections (n=81) • Irrelevant to subject (n=25)
Records excluded (n=38): • Reviews (n=12) • Duplicate information (n=16) • Irrelevant to subject (n=10)
83 articles included Fig 1. PRISMA flow diagram to describe literature search. (MSF = Mediterranean spotted fever)
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ACCEPTED MANUSCRIPT 3 Results 3.1 Demographics Six paediatric patients were diagnosed with MSF for the period 2011-2016 (Table 1). Four patients were female. The age at presentation varied between 17 months and 15 years. Five
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cases presented during the summer months (June to September), while one presented in November. All reported contact with ticks. Four cases denied direct contact with dogs, despite having tick bites. We cannot exclude the involvement of an animal reservoir other
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than the dog in transmission to these cases. Although a history of contact with dogs is part of
Epidemiological features
Case 1
Case 2
Case 3
Case 4
Case 5
Case 6
F
M
M
F
F
F
15y
9y
9y
1y 5m
13y
Gender
1y 10m
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Age
Month of the year
July
June
September
August
September
November
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
No
No
No
Beach*
Beach**
Rural village**
Suburban home✝
Crosscountry run✝
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Contact with ticks
Yes
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Contact with dogs
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the diagnostic scoring system described below, it is often absent [15].
Location of contact with dogs/ticks
Beach*
*South of Malta; **North of Malta; ✝ Central Malta Table 1. Epidemiological features.
3.2 Clinical features All cases presented with fever and one or more eschars, and 5/6 had a generalised maculopapular rash (Table 2, Figure 3). One patient presented with multiple eschars. Three patients also had non-purulent conjunctivitis. One patient, who developed neck stiffness, was
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ACCEPTED MANUSCRIPT investigated with a lumbar puncture. This revealed an elevated lymphocyte count and protein concentration of the cerebrospinal fluid. None of the patients developed any complications of rickettsial infection, though one patient developed transient synovitis of the right hip. In one
Duration of fever before presentation (days)
Rash
Case 2
Case 3
Case 4
Case 5
Case 6
39.4
40.9
38.5
40
40
38.8
5
6
3
5
5
1
Yes
Yes
Yes
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Fever (/0C)
Case 1
Yes
Yes
No
Yes
Yes
Yes
Yes
-
Yes
Yes
Yes
Yes
-
Yes
Yes
Yes
Yes
-
Yes
Yes
Yes
-
Yes
-
Papular
Yes
-
Involving palms and soles
No
-
Blanching
Yes
-
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Headache
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Clinical features
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case, the mother was simultaneously hospitalised with a suspected diagnosis of MSF.
No
No
Cervical
Cervical
No
No
No
Yes
No
No
No
No
Yes
Yes
No
No
No
Yes
-
Yes
No
No
-
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Right popliteal fossa, vertex of scalp
Right upper back
Left side of neck
Nape of neck
Left external auditory canal
Left shoulder
Meningism
No
Yes
No
No
No
No
Hepatomegaly
No
2cm
No
No
No
No
Lymphadenopathy
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Conjunctivitis
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Nausea and vomiting
Arthralgia
Eschar
-
Location of eschar
Tip of
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ACCEPTED MANUSCRIPT Clinical features Splenomegaly
Case 1
Case 2
Case 3
Case 4
Case 5
Case 6
No
No
spleen
No
No
No
Table 2. Clinical features.
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3.3 Laboratory investigations at presentation Two patients had an elevated white cell count with a predominant lymphocytosis (Table 3). One patient had leukopenia with lymphopenia. None developed thrombocytopenia, while 2/6
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patients had mild hyponatraemia. Four patients had an elevated C-reactive protein (CRP). One patient had an Erythrocyte Sedimentation Rate (ESR) taken, and this was elevated at
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32mm/hr. One patient had a mildly elevated bilirubin of 44.5µmol/l, and another had elevated liver enzymes. Blood investigations
Case 1
Case 2
Case 3
Case 4
Case 5
Case 6
White cell count (x109/L)
15.82 (↑)
8.06
5.45
3.2 (↓)
17.73 (↑)
7.9
Neutrophil count (x109/L)
5.78
4.89
3.15
1.76
7.77 (↑)
5.52
9.23 (↑)
1.66
1.64
1.12 (↓)
8.68 (↑)
1.62
192
242
277
175
311
276
11.3
14.2
11.8
12
11.1
14
140
133 (↓)
133 (↓)
136
135
138
4.39
4.4
3.92
4.3
4.53
4.47
25
80
40
52
17
61
2.1
5
3.5
2.5
3.1
3.5
48 (↑)
210 (↑)
12 (↑)
<6
67.4 (↑)
3.5
ESR (mm in 1st hour)
32
-
-
12
-
-
Bilirubin (µmol/L)
2.7
7
6.9
44.5 (↑)
-
9.8
Alanine aminotransferase (U/l)
61 (↑)
36
16
21
-
17
Aspartate aminotransferase (U/l)
73 (↑)
30
-
-
-
-
Alkaline phosphatase (U/l)
123
97
144
129
-
149
Gamma-glutarylaminotransferase (U/l)
22
11
11
34
-
16
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Lymphocytes (x109/L) Platelet count (x109/L) Haemoglobin (g/dL)
Potassium (mmol/L)
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Creatinine (µmol/L)
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Sodium (mmol/L)
Urea (mmol/L) CRP* (mg/L)
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ACCEPTED MANUSCRIPT Table 3. Laboratory investigations at presentation. *CRP values reflect the highest values recorded during the illness.
3.4 Diagnostic investigations
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All children had anti-rickettsial antibodies tested by enzyme-linked immunosorbent assay (ELISA) in the first week of the illness (Vircell, S.L., Granada, Spain), with all being negative for IgM and IgG against R. conorii, R. rickettsia and R. typhi (Table 4). Two
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patients were additionally tested quantitatively (Bioscientia Institute for Diagnostic Medicine, GmbH, Germany) for anti-rickettsial antibodies by means of immunofluorescence assay
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testing (Focus Diagnostics Inc., California, USA) in the first week of the illness. Both these children had low titres of anti-rickettsial antibodies at this point. Low anti-rickettsial antibody titres are well-reported in the first week of the illness [16]. Two patients were tested by ELISA during the second-to-third weeks of the illness, and these were confirmed to have
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developed anti-R. conorii IgM antibodies. These patients also had positive titres for anti-R. rickettsii IgG antibodies at week 2-3, while one had a positive titre for R. typhi IgG antibodies. R. rickettsiae has never been identified in Malta, while R. typhi, although thought
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to be endemic in Malta but never described, is associated with murine typhus, an illness in which an eschar is absent, being spread by flea faeces contaminating a flea-bite [17]. These
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results are therefore likely to be false-positive, secondary to cross-reactivity of ELISA [18]. None of the patients were tested during the convalescent phase at 4 weeks, as venepuncture was deemed inappropriate in children whose symptoms had completely resolved. Eschar biopsy was also deemed inappropriate because of the invasive nature of this procedure. Blood samples for real-time multiplex rickettsial polymerase chain reaction (PCR) with primers directed against the citrate synthase (gltA) gene (Fast Track Diagnostics Luxembourg S. á. r. l., Esch-sur-Alzette, Luxembourg) were taken at presentation in 4/6 patients, but were negative in all cases. This is well documented in PCR seeking the gltA rickettsial gene in 10
ACCEPTED MANUSCRIPT blood samples [19]. We are therefore unable to comment on the causative rickettsial species behind the cases of MSF. Rickettsial culture was not performed in our series. Applying the diagnostic scoring system for MSF to this series, 4/6 patients had a positive score of ≥29. One case had a score of 19 because the patient’s fever did not reach
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39oC, while another scored 9 because of the absence of the typical rash, a maximal fever of 38.8oC, and a presentation in November, instead of the typical period between May and October [20]. It should be noted, however, that warm weather in Malta may persist beyond
Diagnostic investigations
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October.
Case 1
ELISA
Case 2
Case 3
Case 4
Case 5
Case 6
Week 1
Week 2-3
Week 1
Week 2-3
Week 1
Week 1
Week 1
Week 1
R. conorii IgM
Negative
Positive
Negative
Positive
Negative
Negative
Negative
Negative
-
R. conorii IgG
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
-
R. rickettsii IgM
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
-
R. rickettsii IgG
Negative
Positive
Negative
Positive
Negative
Negative
Negative
Negative
-
R. typhi IgM
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
-
R. typhi IgG
Negative
Negative
Negative
Positive
Negative
Negative
Negative
Negative
-
R. conorii/R. typhi IgM and IgG
PCR (Blood)
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(Week 1)
-
-
Negative
-
Negative
-
Negative
-
Negative
-
Negative
Negative
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IFAT
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-
Table 4. Diagnostic investigations. (ELISA=Enzyme-linked immunosorbent assay, IFAT=Immunofluorescence assay testing, PCR=polymerase chain reaction.)
3.5 Treatment and outcome Four children were treated with clarithromycin, and the two teenagers with doxycycline. Duration of treatment was 10 days. All patients were afebrile within 48 hours of starting appropriate antimicrobial therapy and recovered completely. None of the children developed
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ACCEPTED MANUSCRIPT significant complications of MSF, with similar outcomes reported in Barcelona and Sicily
4 Discussion and literature review
4.1 Epidemiology
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[9,21].
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Rickettsia conorii, an obligate intracellular organism, is the causative agent behind MSF [1]. Molecular techniques have allowed the identification of rickettsial subspecies, each responsible for a distinct spotted-fever syndrome. Rickettsia conorii conorii (type strain =
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Malish, ATCC VR-613) is the subspecies of R. conorii responsible for MSF [22]. MSF is
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endemic in most Mediterranean countries [4]. True incidence data for MSF are scarce because of inter-country variations in the criteria used for reporting. Spain, Algeria and Italy have a reported annual incidence of 5-10 cases per 100,000 inhabitants, while in Portugal it is >10 cases per 100,000 inhabitants [1]. In Malta, the incidence is lower, at 0.9 cases per 100,000 inhabitants [23]. However, this is likely an underestimation secondary to underrecognition and underreporting of cases. There are no animal studies to date that examine the prevalence of rickettsial diseases in Malta. Seroprevalence of antibodies against R. conorii in the Mediterranean depends on the region, varying between 5% in Spain and 10% in Israel
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ACCEPTED MANUSCRIPT [5,24]. Rhipicephalus sanguineus (Brown dog tick), the vector of MSF, acquires R. conorii by feeding on an infected canine host (Figure 2). R. sanguineus carriage amongst Maltese dogs was recently reported by Licari et al [25]. Aside from dogs, rabbits, hedgehogs and small rodents have all been postulated as the reservoir for the disease, as anti-R. conorii
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antibodies have been found in these animals [1,26].
4.2 Pathogenesis
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Following inoculation at the site of an eschar, R. conorii replicates in the cytoplasm of endothelial cells, releasing pro-inflammatory cytokines. This leads to widespread vasculitis,
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hypo-perfusion and multi-organ injury [7]. Injury to the vascular endothelium affects the liver, brain, lungs, coagulation pathway and skin, leading to the typical erythematous maculopapular rash (Figure 3a-c) [27]. Severe MSF is not associated with high loads of rickettsiae in the blood, suggesting this is an immunopathological phenomenon rather than
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overwhelming sepsis [7]. The eschar itself is characterized histologically by a central area of ischaemic necrosis (Figure 3d-f) with abundant lymphohistiocytic infiltration and
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Adult Brown Dog Tick (Rhipicephalus sanguineus)
Bites from ticks with infected salivary glands
Brown Dog Tick Nymph
Canine
Human host
Eggs
TRANSOVARIAL
TRANSSTADIAL
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surrounding dermal oedema, though relatively few rickettsial organisms [7,28].
Brown Dog Tick Larvae
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ACCEPTED MANUSCRIPT Fig 2. Transmission of Rickettsia conorii [29,30].
4.3 Clinical features MSF presents with fever (>39oC), a discrete maculopapular rash with palmo-plantar
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involvement, and an inoculation eschar at the site of the tick bite (Figure 3d-f) [15,31]. Other manifestations often include headache, myalgia, arthralgia, hepatomegaly, splenomegaly, gastrointestinal symptoms and conjunctivitis [15,31]. In Crete, 53% of patients in a case
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series had conjunctivitis [3]. Multiple eschars are rare but have been described in infections other than those due to R. conorii, such as R. aeschlimannii or R.sibirica. These pathogens
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are not endemic in Malta [1,32–34]. R. conorii MSF has been reported in patients with multiple eschars, and this atypical behaviour in Rhipicephalus sp. ticks may be attributable to
climate change in Southern Europe [14]. Mortality with untreated MSF may be as high as 32.3% in adults, though it is not usually severe in children [1,35,36]. Outcome is worse
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amongst patients with diabetes mellitus, immunosuppressive states, chronic cardiac
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pathology, chronic alcoholism, and glucose-6-phosphate dehydrogenase deficiency [35].
4.4 Laboratory investigations
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Leukopenia and thrombocytopenia are common in patients with MSF, being seen in 25% and 80% respectively of patients [12,19]. Hyponatraemia is present in 33% of patients [3]. Inflammatory markers such as the CRP, liver enzymes, and muscular enzymes such as creatine kinase, are typically elevated [37,38].
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Figure 3. Cutaneous manifestations of MSF (a) Maculopapular rash on the lower limb of an infant, involving the sole (Case 1) and (b) on the trunk, arms and (c) palms in an adolescent (Case 2). (d) Eschar on lower limb (Case 1), (e) posterior hairline (Case 4), and (f) close-up, with central necrosis and surrounding erythema with desquamation. (Source: Paediatric Infectious Disease services, Mater Dei Hospital, Malta)
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ACCEPTED MANUSCRIPT 4.5 Diagnostic investigations Serological analysis is the easiest and most widespread method of rickettsial diagnosis, but can only provide a retrospective diagnosis [17]. Seroconversion is detectable 7 to 15 days after the onset of disease [39]. Several serological techniques are available (Table 5). IF is the
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gold standard investigation for rickettsial diagnosis: a four-fold rise in anti-rickettsial antibody titres between acute and convalescent samples, IgM titres ≥64, or IgG titres of ≥128 are diagnostic [6,12,16]. Paired acute and convalescent samples are inconvenient in
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paediatric MSF, as venepuncture following clinical resolution may not be justified. Enzymelinked immunosorbent assay (ELISA) is highly sensitive, reproducible, and distinguishes
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between IgG and IgM antibodies [16,40].
Several rickettsial genetic sequences can be targeted by PCR (Table 5). Although R. conorii supsp. conorii is classically associated with MSF in Europe, R. massiliae, (acquired from bites by R. sanguineus or R. turpanicus ticks), R. conorii subsp. israelensis and R.
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conorii subsp. indica have also caused MSF in Sicily, Portugal and Spain respectively [41,42]. R. slovaca, seen in Hungary, France, Spain and Portugal, has been associated with Tick-borne lymphadenopathy (TIBOLA) or Dermacentor-borne necrosis, erythema and
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lymphadenopathy (DEBONEL). This is characterised by an eschar on the head and neck,
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surrounding erythema and painful cervical lymphadenopathy [42]. Although none of these pathogens have ever been identified in Malta, we cannot exclude causality. Yield from PCR could be improved by eschar biopsy, though this is invasive, and arguably inappropriate for paediatric patients when the clinical diagnosis is clear [19]. While species identification may not influence patient outcome, it is important for epidemiological investigation. Rickettsiae are difficult to grow in culture, but may be detectable in shell-vial culture 48-72 hours post-inoculation, prior to seroconversion [43]. However, the technique is less
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ACCEPTED MANUSCRIPT sensitive than serology or quantitative PCR. Specimens should be taken prior to treatment and early in the disease, and be inoculated immediately [44]. Raoult et al described a scoring system for the diagnosis of MSF that included clinical and epidemiological features, serology and culture (Table 6) [20]. This system was adapted
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score of 18/29 was used to diagnose MSF [45].
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for clinical diagnosis (Table 7), with a sensitivity of 60% and a specificity of 84.6% when a
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Investigation a.
Sensitivity
Specificity
Advantages -
Serology
Limitations
Easiest to use
-
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-
Retrospective diagnosis Unable to identify species/new pathogens Poor sensitivity/specifi city
References
Widespread
[17,19,39]
Largely obsolete. Restricted to low resource areas Restricted to seroepidemiological studies.
[46,47]
-
WF
33%
46%
-
Low-cost
-
CF
N/A
N/A
-
High specificity
-
Poor sensitivity
-
LA
93%
96%
Expensive kit
Not widespread
[16,48,49]
MIF
100% (day 29 of illness)
100%
Rapid Simple Sensitive/specific Widely available
-
-
-
-
Fails to identify aetiologic agent Retrospective diagnosis – poor early sensitivity Requires repeated blood sampling, expensive equipment, and expertise
Widespread. Gold standard
[3,6,10,12,16,46]
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-
Use
90% (day 20-29)
-
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46% (days 5-9)
-
-
ELISA
90-95%
-
WB
67% (before day 10)
100%
-
Reproducible
-
As for MIF
Widespread
[19,40]
100%
-
Species-specific Early diagnosis
-
Time-consuming Requires expensive equipment
Reference laboratories
[6,50,51]
97-100%
-
Rapid
-
As for WB
Endemic areas
[52]
90% (day 10-15)
-
LBA
100% (after day 35) 84-98%
[16]
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Advantages -
Tests numerous antigens simultaneously
-
Rapid Potentially speciesspecific
Allows identification of novel pathogenic species
[53–55]
-
Not SFGRspecific
Reference laboratories
[56,57]
Early diagnosis
-
Not SFGRspecific
Reference laboratories
[58]
-
Not SFGRspecific Poor yield from serum
Widespread
[19,59,60]
-
Real-time PCR can detect very low rickettsial counts Has been sequenced in almost all rickettsiae Quantitative
-
SFGR-specific
-
Fails to diagnose certain SFGR e.g. R. helvetica
Reference laboratories
[16,61]
100%
-
-
[62–64]
-
Not SFGRspecific: positive with TG Limited sensitivity Poor sensitivity
Widespread
100%
Early diagnosis Detects low rickettsial counts Early diagnosis SFGR-specific Early diagnosis
Reference laboratories Not widespread
[65]
16S ribosomal DNA
Not evaluated
Not evaluated
-
-
17kDa common antigen gene
14% (100% of fatal cases)
100%
-
-
gltA (citrate synthase) gene
77% (eschar biopsy)
100%
-
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-
0% (serum)
-
-
OmpB gene (Nested PCR)
71% (serum)
-
OmpB gene (LAMP) Immunodetection
73% (serum)
c.
Not evaluated
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Not evaluated
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OmpA gene
55% (eschar biopsy)
References
Widespread, though not standardised
PCR
100%
-
Use
Poor sensitivity, affected by antibiotic treatment
b.
-
Limitations
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Specificity
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Sensitivity
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Investigation
-
-
[66,67]
50-75% (circulating
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d.
Shell-vial culture
Sensitivity
Specificity
endothelial cells) 29.1% (59% prior to antibiotic therapy)
Advantages
100%
-
-
Limitations
Description of new pathogenic species/antibiotic susceptibility testing Early diagnosis
-
Difficult to perform Biopsies are painful Expensive
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Investigation
-
Use
Restricted to Biosafety level 3 laboratories.
References
[12,44,68]
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Table 5. Laboratory diagnosis of MSF. (WF=Weil-Felix; CF=complement fixation; LA=latex agglutination; MIF=microimmunofluorescence; ELISA=enzyme-linked immunofluorescence assay; WB=western blot; LBA=line blot assay; PCR=polymerase chain reaction; SFGR=spotted fever-group rickettsiae; TG=typhus-group rickettsiae; LAMP=loop-mediated isothermal amplification)
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Criteria
Score
Stay in endemic area
2
2.
Occurrence in May-October
2
3.
Contact (certain/possible) with dog ticks
2
Clinical criteria Fever>39oC
5
2.
Eschar
5
3.
Maculopapular/purpuric rash
5
4.
Two clinical criteria
3
5.
Three clinical criteria
5
Non-specific laboratory findings 1.
Platelets<150G/L
2.
SGOT/SGPT>50U/L
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1.
1
1
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Bacteriological criteria 1.
Blood culture positive for Rickettsia conorii
25
2.
Detection of Rickettsia conorii in skin biopsy
25
Serological criteria (IF)
SC
1.
RI PT
Epidemiological criteria
Single serum IgG>1/128
5
2.
Single serum IgG>1/128 and IgM>1/64
10
3.
Four-fold increase in two sera obtained within 2-
20
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EP
1.
week interval
Table 6. Diagnostic criteria for MSF. Score ≥25 signifies positive diagnosis [12]. (SGOT = serum glutamateoxaloacetate transaminase, SGPT = serum glutamate-pyruvate transaminase.)
21
ACCEPTED MANUSCRIPT Epidemiologic criteria
Score
1.
Endemic area
2
2.
Hot season (May–September)
2
3.
Tick bite
2
1.
Fever>39oC
5
2.
Eschar
5
3.
Maculopapular/purpuric rash
5
4.
Two clinical criteria
3
5.
Three clinical criteria
5
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Score
SC
Clinical criteria
4.6 Treatment and outcome
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Table 7. Modified version of Raoult et al scoring system to allow clinical diagnosis [45].
There are no placebo-controlled trials of treatment in MSF, so the need for treatment is based on observational studies and experience with other rickettsial illnesses [36]. Tetracyclines
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have good anti-rickettsial activity [39,69,70], while randomised controlled trials have shown macrolides to be effective and safe in children [71–76]. A recent study has revealed a worse
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4.7 Prevention
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outcome with the use of fluoroquinolones [77].
There are few specific public health recommendations regarding the prevention of MSF when traveling to Malta. The risk of travelers contracting tick-borne rickettsioses such as MSF remains low [17]. Prevention relies on preventing tick bites, as there is no licensed vaccine for MSF [78]. Travelers to Malta should avoid contact with local dogs, while those hiking in rural areas should check for and remove any ticks from clothing or skin [17]. The application of insect repellents to exposed skin provides little protection against ticks, so treating clothing with permethrin is recommended in endemic areas [79]. Travelers who bring their own dogs
22
ACCEPTED MANUSCRIPT should consider treating them with acaricides [80]. There is no evidence that antimicrobial prophylaxis is beneficial to prevent MSF after a tick bite, while serological testing is not recommended because of the poor sensitivity and the cost of the tests. Clinicians should
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SC
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observe the patient and treat only if MSF occurs [79].
23
ACCEPTED MANUSCRIPT 5 Conclusions The incidence of rickettsiosis in Europe has increased [81]. Outdoor activities among Europeans has increased contact with ticks and tick-borne diseases [82]. Climate change might contribute to changing behaviour in Rhipicephalus sp. ticks, and thus influence MSF
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incidence [83]. Widespread international travel to endemic areas, including Malta, makes MSF part of the differential diagnosis of fever in a returning traveller [8]. Despite improvements in MSF diagnosis, clinical diagnosis remains important in the management of
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children with suspected MSF.
24
ACCEPTED MANUSCRIPT References
[1]
Rovery C, Brouqui P, Raoult D. Questions on Mediterranean spotted fever a century after its discovery. Emerg Infect Dis 2008;14:1360–7. doi:10.3201/eid1409.071133. Tonna I, Mallia Azzopardi C, Piscopo T, Cuschieri P, Fenollar F, Raoult D.
RI PT
[2]
Characterisation of rickettsial diseases in a hospital-based population in Malta. J Infect 2006;53:394–402. doi:10.1016/j.jinf.2005.12.024.
Germanakis A, Psaroulaki A, Gikas A, Tselentis Y. Mediterranean spotted fever in
SC
[3]
Crete, Greece: Clinical and therapeutic data of 15 consecutive patients. Ann N Y Acad
[4]
Herve Z, Wim VB. Epidemiological situation of rickettsioses in EU / EFTA countriesTechnical Report. 2013.
[5]
M AN U
Sci 2006;1078:263–9. doi:10.1196/annals.1374.049.
Harrus S, Lior Y, Ephros M, Grisaru-Soen G, Keysary A, Strenger C, et al. Rickettsia
TE D
conorii in humans and dogs: a seroepidemiologic survey of two rural villages in Israel. Am J Trop Med Hyg 2007;77:133–5. [6]
Brouqui P, Parola P, Fournier PE, Raoult D. Spotted fever rickettsioses in southern and
EP
eastern Europe. FEMS Immunol Med Microbiol 2007;49:2–12. doi:10.1111/j.1574-
[7]
AC C
695X.2006.00138.x.
de Sousa R, Ismail N, Nobrega SD, Franca A, Amaro M, Anes M, et al. Intralesional expression of mRNA of interferon- gamma , tumor necrosis factor- alpha , interleukin10, nitric oxide synthase, indoleamine-2,3-dioxygenase, and RANTES is a major immune effector in Mediterranean spotted fever rickettsiosis. J Infect Dis 2007;196:770–81. doi:10.1086/519739.
[8]
Laurent M, Voet A, Libeer C, Lambrechts M, Van Wijngaerden E. Mediterranean spotted fever, a diagnostic challenge in travellers. Acta Clin Belg 2009;64:513–6.
25
ACCEPTED MANUSCRIPT doi:10.1179/acb.2009.087. [9]
Colomba C, Saporito L, Polara VF, Rubino R, Titone L. Mediterranean spotted fever: clinical and laboratory characteristics of 415 Sicilian children. BMC Infect Dis 2006;6:60. doi:10.1186/1471-2334-6-60. RN,
Casper
EA,
Ormsbee
RA,
Peacock
MG,
Burgdorfer
RI PT
[10] Philip
W.
Microimmunofluorescence test for the serological study of Rocky Mountain spotted fever and typhus. J Clin Microbiol 1976;3:51–61.
SC
[11] Raoult D. current approaches to diagnosis of old and new rickettsial diseases . Laboratory Diagnosis of Rickettsioses : Current Approaches to Diagnosis of Old and
M AN U
New Rickettsial Diseases 1997;35:2715–27.
[12] Brouqui P, Bacellar F, Baranton G, Birtles RJ, Bjoërsdorff A, Blanco JR, et al. Guidelines for the diagnosis of tick-borne bacterial diseases in Europe. Clin Microbiol Infect 2004;10:1108–32. doi:10.1111/j.1469-0691.2004.01019.x.
TE D
[13] Rome FI. Tourism in 1975:206–7.
[14] Parola P, Socolovschi C, Jeanjean L, Bitam I, Fournier P-E, Sotto A, et al. Warmer weather linked to tick attack and emergence of severe rickettsioses. PLoS Negl Trop
EP
Dis 2008;2:e338. doi:10.1371/journal.pntd.0000338.
AC C
[15] Crespo P, Seixas D, Marques N, Oliveira J, da Cunha S, Meliço-Silvestre A. Mediterranean spotted fever: case series of 24 years (1989-2012). Springerplus 2015;4:272. doi:10.1186/s40064-015-1042-3.
[16] La Scola B, Raoult D. Laboratory diagnosis of rickettsioses: current approaches to diagnosis of old and new rickettsial diseases. J Clin Microbiol 1997;35:2715–27. [17] Jensenius M, Fournier P-E, Raoult D. Rickettsioses and the international traveler. Clin Infect Dis 2004;39:1493–9. doi:10.1086/425365. [18] Eisen RJ, Gage KL. Transmission of flea-borne zoonotic agents. Annu Rev Entomol
26
ACCEPTED MANUSCRIPT 2012;57:61–82. doi:10.1146/annurev-ento-120710-100717. [19] Madeddu G, Fiore V, Mancini F, Caddeo A, Ciervo A, Babudieri S, et al. Mediterranean spotted fever-like illness in Sardinia, Italy: a clinical and microbiological study. Infection 2016;44:733–8. doi:10.1007/s15010-016-0921-z.
RI PT
[20] Raoult D, Roux V. Rickettsioses as paradigms of new or emerging infectious diseases. Clin Microbiol Rev 1997;10:694–719.
[21] Moraga FA, Martinez-Roig A, Alonso JL, Boronat M, Domingo F. Boutonneuse fever.
SC
Arch Dis Child 1982;57:149–51.
[22] Zhu Y, Fournier P-E, Eremeeva M, Raoult D. Proposal to create subspecies of
M AN U
Rickettsia conorii based on multi-locus sequence typing and an emended description of Rickettsia conorii. BMC Microbiol 2005;5:11. doi:10.1186/1471-2180-5-11. [23] Unit MS. The estimated total population of Malta and Gozo at the end of 2016 stood at World Population Day : 11 July 2017 2017:1–12.
TE D
[24] Espejo E, Andres M, Perez J, Prat J, Guerrero C, Munoz MT, et al. Prevalence of antibodies to Rickettsia conorii in human beings and dogs from Catalonia: a 20-year perspective. Epidemiol Infect 2016;144:1889–94. doi:10.1017/S0950268816000261.
from
Malta.
Ticks
Tick
Borne
Dis
2017;8:396–9.
AC C
dogs
EP
[25] Licari E, Takács N, Solymosi N, Farkas R. First detection of tick-borne pathogens of
doi:10.1016/j.ttbdis.2017.01.002.
[26] Le Gac P. [Repercussions of myxomatosis on Mediterranean boutonneuse exanthematic fever]. Bull World Health Organ 1966;35:143–7.
[27] Sousa R de, Franca A, Doria Nobrega S, Belo A, Amaro M, Abreu T, et al. Host- and microbe-related risk factors for and pathophysiology of fatal Rickettsia conorii infection in Portuguese patients. J Infect Dis 2008;198:576–85. doi:10.1086/590211. [28] Walker DH, Occhino C, Tringali GR, Di Rosa S, Mansueto S. Pathogenesis of
27
ACCEPTED MANUSCRIPT rickettsial eschars: the tache noire of boutonneuse fever. Hum Pathol 1988;19:1449– 54. [29] Gray J, Dantas-Torres F, Estrada-Pena A, Levin M. Systematics and ecology of the
doi:10.1016/j.ttbdis.2012.12.003.
RI PT
brown dog tick, Rhipicephalus sanguineus. Ticks Tick Borne Dis 2013;4:171–80.
[30] Parola P, Socolovschi C, Raoult D. Deciphering the relationships between Rickettsia conorii conorii and Rhipicephalus sanguineus in the ecology and epidemiology of spotted
fever.
Ann
doi:10.1111/j.1749-6632.2009.04518.x.
N
Y
Acad
Sci
2009;1166:49–54.
SC
Mediterranean
M AN U
[31] Segura-Porta F, Font-Creus B, Espejo-Arenas E, Bella-Cueto F. New trends in Mediterranean spotted fever. Eur J Epidemiol 1989;5:438–43. [32] Fournier P-E, Gouriet F, Brouqui P, Lucht F, Raoult D. Lymphangitis-associated rickettsiosis, a new rickettsiosis caused by Rickettsia sibirica mongolotimonae: seven
TE D
new cases and review of the literature. Clin Infect Dis 2005;40:1435–44. doi:10.1086/429625.
[33] de Sousa R, Barata C, Vitorino L, Santos-Silva M, Carrapato C, Torgal J, et al.
EP
Rickettsia sibirica isolation from a patient and detection in ticks, Portugal. Emerg
AC C
Infect Dis 2006;12:1103–8. doi:10.3201/eid1207.051494. [34] Fernandez-Soto P, Perez-Sanchez R, Alamo-Sanz R, Encinas-Grandes A. Spotted fever group rickettsiae in ticks feeding on humans in northwestern Spain: is Rickettsia conorii vanishing? Ann N Y Acad Sci 2006;1078:331–3. doi:10.1196/annals.1374.063.
[35] de Sousa R, Nobrega SD, Bacellar F, Torgal J. Mediterranean spotted fever in Portugal: risk factors for fatal outcome in 105 hospitalized patients. Ann N Y Acad Sci 2003;990:285–94. [36] Demeester R, Claus M, Hildebrand M, Vlieghe E, Bottieau E. Diversity of life-
28
ACCEPTED MANUSCRIPT threatening complications due to mediterranean spotted fever in returning travelers. J Travel Med 2010;17:100–4. doi:10.1111/j.1708-8305.2009.00391.x. [37] Micalizzi A, La Spada E, Corsale S, Arculeo A, La Spada M, Ouartararo P, et al. Abnormal liver function in Mediterranean spotted fever | Interessamento epatico in
RI PT
corso di febbre bottonosa del Mediterraneo. Infez Med 2007;15:105–10.
[38] Mert A, Ozaras R, Tabak F, Bilir M, Ozturk R. Mediterranean spotted fever: A review of fifteen cases. J Dermatol 2006;33:103–7. doi:10.1111/j.1346-8138.2006.00021.x.
SC
[39] Parola P, Paddock CD, Socolovschi C, Labruna MB, Mediannikov O, Kernif T, et al. Update on tick-borne rickettsioses around the world: A geographic approach. Clin
M AN U
Microbiol Rev 2013;26:657–702. doi:10.1128/CMR.00032-13.
[40] Do E-J, Kim J-E, Park J-M, Lee K-M, Jung M-Y, Lee H-J, et al. Development of recombinant OmpA and OmpB proteins as diagnostic antigens for rickettsial disease. Microbiol Immunol 2009;53:368–74. doi:10.1111/j.1348-0421.2009.00142.x.
TE D
[41] Vitale G, Mansuelo S, Rolain J-M, Raoult D. Rickettsia massiliae human isolation. Emerg Infect Dis 2006;12:174–5. doi:10.3201/eid1201.050850. [42] Portillo A, Santibáñez S, García-Álvarez L, Palomar AM, Oteo JA. Rickettsioses in
EP
Europe. Microbes Infect 2015;17:834–8. doi:10.1016/j.micinf.2015.09.009.
AC C
[43] Marrero M, Raoult D. Centrifugation-shell vial technique for rapid detection of Mediterranean spotted
fever rickettsia in blood culture. Am J Trop Med Hyg
1989;40:197–9.
[44] Angelakis E, Richet H, Rolain J-M, La Scola B, Raoult D. Comparison of real-time quantitative PCR and culture for the diagnosis of emerging Rickettsioses. PLoS Negl Trop Dis 2012;6:e1540. doi:10.1371/journal.pntd.0001540. [45] Letaief A, Souissi J, Trabelsi H, Ghannem H, Jemni L. Evaluation of clinical diagnosis scores for Boutonneuse fever 2003;990:327–30.
29
ACCEPTED MANUSCRIPT [46] Raoult D, Rousseau S, Toga B, Tamalet C, Gallais H, De Micco P, et al. [Serological diagnosis of Mediterranean boutonneuse fever]. Pathol Biol (Paris) 1984;32:791–4. [47] Kularatne SAM, Gawarammana IB. Validity of the Weil-Felix test in the diagnosis of
doi:10.1016/j.trstmh.2008.11.020.
RI PT
acute rickettsial infections in Sri Lanka. Trans R Soc Trop Med Hyg 2009;103:423–4.
[48] Hechemy KE, Raoult D, Eisemann C, Han YS, Fox JA. Detection of antibodies to Rickettsia conorii with a latex agglutination test in
patients with Mediterranean
SC
spotted fever. J Infect Dis 1986;153:132–5.
[49] De la fuente L, Anda P, Rodriguez I, Hechemy KE, Raoult D, Casal J. Evaluation of a
M AN U
latex agglutination test for Rickettsia conorii antibodies in seropositive patients. J Med Microbiol 1989;28:69–72. doi:10.1099/00222615-28-1-69. [50] Teysseire
N,
Raoult
D.
Comparison
of
western
immunoblotting
and
microimmunofluorescence for diagnosis of Mediterranean spotted fever. J Clin
TE D
Microbiol 1992;30:455–60.
[51] Babalis T, Dupont HT, Tselentis Y, Chatzichristodoulou C, Raoult D. Rickettsia conorii in Greece: comparison of a microimmunofluorescence assay and western
EP
blotting for seroepidemiology. Am J Trop Med Hyg 1993;48:784–92.
AC C
[52] Raoult D, Dasch GA. Line blot and western blot immunoassays for diagnosis of mediterranean spotted fever. J Clin Microbiol 1989;27:2073–9.
[53] Wang JM, Hudson BJ, Watts MR, Karagiannis T, Fisher NJ, Anderson C, et al. Diagnosis of Queensland tick typhus and African tick bite fever by PCR of lesion swabs. Emerg Infect Dis 2009;15:963–5. doi:10.3201/eid1506.080855. [54] Mouffok N, Socolovschi C, Benabdellah A, Renvoise A, Parola P, Raoult D. Diagnosis of rickettsioses from eschar swab samples, Algeria. Emerg Infect Dis 2011;17:1968–9. doi:10.3201/eid1710.110332.
30
ACCEPTED MANUSCRIPT [55] Khrouf F, Sellami H, Elleuch E, Hattab Z, Ammari L, Khalfaoui M, et al. Molecular diagnosis of Rickettsia infection in patients from Tunisia. Ticks Tick Borne Dis 2016;7:653–6. doi:10.1016/j.ttbdis.2016.02.010.
sequencing. Res Microbiol 1995;146:385–96.
RI PT
[56] Roux V, Raoult D. Phylogenetic analysis of the genus Rickettsia by 16S rDNA
[57] Woo PCY, Lau SKP, Teng JLL, Tse H, Yuen K-Y. Then and now: use of 16S rDNA gene sequencing for bacterial identification and discovery of novel bacteria in clinical
SC
microbiology laboratories. Clin Microbiol Infect 2008;14:908–34. doi:10.1111/j.14690691.2008.02070.x.
M AN U
[58] Leitner M, Yitzhaki S, Rzotkiewicz S, Keysary A. Polymerase chain reaction-based diagnosis of Mediterranean spotted fever in serum and tissue samples. Am J Trop Med Hyg 2002;67:166–9.
[59] Stenos J, Graves SR, Unsworth NB. A highly sensitive and specific real-time PCR
TE D
assay for the detection of spotted fever and typhus group Rickettsiae. Am J Trop Med Hyg 2005;73:1083–5.
[60] Roux V, Rydkina E, Eremeeva M, Raoult D. Citrate synthase gene comparison, a new
EP
tool for phylogenetic analysis, and its application for the rickettsiae. Int J Syst
AC C
Bacteriol 1997;47:252–61. doi:10.1099/00207713-47-2-252. [61] Roux V, Fournier P, Raoult D. Differentiation of spotted fever group rickettsiae by sequencing and analysis of restriction fragment length polymorphism of PCRamplified DNA of the gene encoding. J Clin Microbiol 1996;34:2058–65.
[62] Fournier PE, Roux V, Raoult D. Phylogenetic analysis of spotted fever group rickettsiae by study of the outer surface protein rOmpA. Int J Syst Bacteriol 1998;48 Pt 3:839–49. doi:10.1099/00207713-48-3-839. [63] Roux V, Raoult D. Phylogenetic analysis of members of the genus Rickettsia using the
31
ACCEPTED MANUSCRIPT gene encoding the outer-membrane protein rOmpB (ompB). Int J Syst Evol Microbiol 2000;50 Pt 4:1449–55. doi:10.1099/00207713-50-4-1449. [64] Choi Y, Lee S, Park K, Koh Y, Lee K, Baik H, et al. Evaluation of PCR-Based Assay for Diagnosis of Spotted Fever Group Rickettsiosis in Human Serum Samples. Clin
RI PT
Diagn Lab Immunol 2005;12:759–63. doi:10.1128/CDLI.12.6.759.
[65] Pan L, Zhang L, Wang G, Liu Q. Rapid, simple, and sensitive detection of the ompB gene of spotted fever group rickettsiae by loop-mediated isothermal amplification.
SC
BMC Infect Dis 2012;12:254. doi:10.1186/1471-2334-12-254.
[66] Raoult D, de Micco C, Gallais H, Toga M. Laboratory diagnosis of Mediterranean
M AN U
spotted fever by immunofluorescent demonstration of Rickettsia conorii in cutaneous lesions. J Infect Dis 1984;150:145–8.
[67] Drancourt M, George F, Brouqui P, Sampol J, Raoult D. Diagnosis of Mediterranean spotted fever by indirect immunofluorescence of Rickettsia conorii in circulating
TE D
endothelial cells isolated with monoclonal antibody-coated immunomagnetic beads. J Infect Dis 1992;166:660–3.
[68] Quesada M, Sanfeliu I, Cardeñosa N, Segura F. Ten years’ experience of isolation of
EP
Rickettsia spp. from blood samples using the shell-vial cell culture assay. Ann N Y
AC C
Acad Sci 2006;1078:578–81. doi:10.1196/annals.1374.115. [69] Rovery C, Raoult D. Mediterranean Spotted Fever. Infect Dis Clin North Am 2008;22:515–30. doi:10.1016/j.idc.2008.03.003.
[70] Rolain JM, Maurin M, Vestris G, Raoult D. In vitro susceptibilities of 27 rickettsiae to 13 antimicrobials. Antimicrob Agents Chemother 1998;42:1537–41. [71] Anton E, Muñoz T, Travería FJ, Navarro G, Font B, Sanfeliu I, et al. Clarithromycin for Mediterranean Spotted Fever: a Randomized Trial. Antimicrob Agents Chemother 2015;60:1–22. doi:10.1128/AAC.01814-15.
32
ACCEPTED MANUSCRIPT [72] Cascio A, Colomba C, Antinori S, Paterson DL, Titone L. Clarithromycin versus azithromycin in the treatment of Mediterranean spotted fever in children: a randomized controlled trial. Clin Infect Dis 2002;34:154–8. doi:10.1086/338068. [73] Cascio A, Colomba C, Di Rosa D, Salsa L, di Martino L, Titone L. Efficacy and safety
RI PT
of clarithromycin as treatment for Mediterranean spotted fever in children: a randomized controlled trial. Clin Infect Dis 2001;33:409–11. doi:10.1086/321864. [74] Cascio A, Colomba C. [Macrolides in the treatment of children with Mediterranean
SC
spotted fever]. Infez Med 2002;10:145–50.
Eur J Epidemiol 1991;7:276–81.
M AN U
[75] Raoult D. Antibiotic treatment of rickettsiosis, recent advances and current concepts.
[76] Bella F, Font B, Uriz S, Munoz T, Espejo E, Traveria J, et al. Randomized trial of doxycycline versus josamycin for Mediterranean spotted fever. Antimicrob Agents Chemother 1990;34:937–8.
TE D
[77] Botelho-Nevers E, Rovery C, Richet H, Raoult D. Analysis of risk factors for malignant Mediterranean spotted fever indicates that fluoroquinolone treatment has a deleterious effect. J Antimicrob Chemother 2011;66:1821–30. doi:10.1093/jac/dkr218.
EP
[78] Richards AL. Rickettsial vaccines: the old and the new. Expert Rev Vaccines
AC C
2004;3:541–55. doi:10.1586/14760584.3.5.541. [79] Parola P, Raoult D. Ticks and tickborne bacterial diseases in humans: an emerging infectious threat. Clin Infect Dis 2001;32:897–928. doi:10.1086/319347.
[80] Estrada-Peña A, Venzal Bianchi JM. Efficacy of several anti-tick treatments to prevent the transmission of Rickettsia conorii under natural conditions. Ann N Y Acad Sci 2006;1078:506–8. doi:10.1196/annals.1374.097. [81] Blanco JR, Oteo JA. Rickettsiosis in Europe. Ann N Y Acad Sci 2006;1078:26–33. doi:10.1196/annals.1374.003.
33
ACCEPTED MANUSCRIPT [82] Parola P, Raoult D. Tick-borne bacterial diseases emerging in Europe. Clin Microbiol Infect 2001;7:80–3. doi:10.1046/j.1469-0691.2001.00200.x. [83] De Sousa R, Luz T, Parreira P, Santos-Silva M, Bacellar F. Boutonneuse fever and climate
variability.
Ann
N
Acad
Sci
2006;1078:162–9.
AC C
EP
TE D
M AN U
SC
RI PT
doi:10.1196/annals.1374.029.
Y
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Conflict of Interest Statement
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The authors declare that there are no conflicts of interest.
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S1477-8939(18)30016-4
DOI:
10.1016/j.tmaid.2018.02.005
Reference:
TMAID 1220
To appear in:
Travel Medicine and Infectious Disease
Received Date: 8 September 2017 Revised Date:
13 February 2018
Accepted Date: 16 February 2018
Please cite this article as: Torpiano P, Pace D, Clinically-diagnosed Mediterranean Spotted Fever in Malta, Travel Medicine and Infectious Disease (2018), doi: 10.1016/j.tmaid.2018.02.005. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Title page
Title: Clinically-diagnosed Mediterranean Spotted Fever in Malta
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Running title: MSF in Malta
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Authors: Paul Torpiano, David Pace
Affiliation: Department of Paediatrics, Mater Dei Hospital, Tal-Qroqq, Msida, MSD 2090,
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Malta
Corresponding Author: Paul Torpiano
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Department of Paediatrics, Mater Dei Hospital, Tal-Qroqq, Msida, MSD 2090, Malta Telephone: +356 2545 5567 Fax: +356 2545 4154
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Email: [email protected]
Key words Mediterranean spotted fever; children; Rickettsia conorii; diagnosis
1
ACCEPTED MANUSCRIPT Abstract (194 words) Background Mediterranean Spotted Fever (MSF) is a tick-borne zoonosis caused by Rickettsia conorii which is endemic in Malta, an island in the South Mediterranean that is a popular tourist
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destination. Diagnosis is frequently based on clinical manifestations as laboratory results are often limited to a retrospective diagnosis. We describe the clinical presentation, diagnosis and
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treatment of children <16 years who presented with MSF from 2011 – 2016.
Method
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The demographics, clinical findings, laboratory results, management and outcome of all children hospitalised with suspected MSF based on the presence of fever and an eschar, were retrieved from their case notes.
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Results
Over the five-year study period six children, aged between 17 months and 15 years, were diagnosed with MSF. All children had contact with ticks and the majority presented in
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summer. Laboratory results were non-specific and included elevated inflammatory markers,
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lymphocytosis/lymphopenia and hyponatraemia. Serological and molecular techniques were used for diagnosis. Response to clarithromycin or doxycycline was immediate.
Conclusion
MSF should be included in the differential diagnosis of fever, rash and an eschar in children who travel to Malta. Despite advances in molecular diagnostics, clinical diagnosis remains important in the management of children with suspected MSF.
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ACCEPTED MANUSCRIPT Funding sources
The research work disclosed in this publication is partially funded by the ENDEAVOUR Scholarships Scheme. “The scholarship is part-financed by the European Union – European
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Social Fund”.
Acknowledgements
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The authors would like to thank Dr Matthew Snape for his help and guidance in writing this
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Word count: 3632
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manuscript.
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ACCEPTED MANUSCRIPT 1 Introduction Mediterranean spotted fever (MSF) is a tick-borne illness caused by Rickettsia conorii. It is endemic across southern Europe and North Africa, with the seroprevalence of anti-R.conorii antibodies being as high as 10% in some Mediterranean countries [1–5]. Rhipicephalus
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sanguineus (brown dog tick) acquires R. conorii by feeding on infected dogs, and can transmit rickettsiae to humans through feeding [6]. Following inoculation at the site of an eschar, R. conorii replicates in the cytoplasm of endothelial cells, releasing pro-inflammatory
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cytokines that lead to a widespread vasculitis [7]. MSF is characterised by fever, headache, rash and an eschar [8,9]. Diagnosis is usually serological, though sensitivity is limited early
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in the illness, providing only a retrospective diagnosis [10,11]. Molecular techniques provide a faster diagnosis, though with limited sensitivity [12]. Although MSF runs a milder course in paediatric patients, there is a need to diagnose and treat the disease early to avoid complications [1]. Diagnosing MSF on epidemiological and clinical features has an important
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role in its management.
Malta is an archipelago in the centre of the Mediterranean, and a popular destination amongst
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tourists: in 2016 alone, the Maltese islands received 2 million visitors [13]. Over half of these
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visited between June and September, when warm weather encourages outdoor activities as well as tick-biting behaviour in R. sanguineus [13,14]. We describe the manifestations, laboratory findings and management of all children hospitalised with MSF from 2011-2016 in order to make doctors in non-endemic countries aware of the persistence of this zoonosis in Malta.
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2 Methods MSF is a notifiable disease in Malta [4]. All patients under 16 years of age, admitted to Mater Dei Hospital, the only teaching hospital in Malta, for the period 2011-2016, and suspected to
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have MSF based on the presence of fever and an eschar were identified from the database kept by the Paediatric Infectious Diseases team and their hospital records were retrieved. For the literature review, we searched the MEDLINE, EMBASE, and NHS Evidence
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databases using the search terms ‘Mediterranean Spotted Fever’ and ‘Boutonneuse fever’. These searches gave a total of 1182 articles. We included articles written in English, Italian,
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and French. We excluded articles in other languages, individual case reports, articles focused on non-MSF rickettsiosis and other infections, articles describing rickettsial infection in non-
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human hosts, and papers irrelevant to the subject. This gave a total of 83 articles (Figure 1).
5
EMBASE (n=130)
Records excluded due to nonhuman subjects (n=279)
Records screened (n=1180)
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573 records screened (title/abstract)
Eligibility
Records excluded as not in English/French/Italian (n=294)
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Records in English/French/Italian (n=607)
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Screening
901 records screened by language
Included
NHS evidence (n=13)
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MEDLINE (n=1037)
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Identification
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121 full-text articles assessed for eligibility
34 duplicate records excluded
Records excluded (n=452): • Case reports (n=314) • Full-text unavailable (n=32) • Non-MSF infections (n=81) • Irrelevant to subject (n=25)
Records excluded (n=38): • Reviews (n=12) • Duplicate information (n=16) • Irrelevant to subject (n=10)
83 articles included Fig 1. PRISMA flow diagram to describe literature search. (MSF = Mediterranean spotted fever)
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ACCEPTED MANUSCRIPT 3 Results 3.1 Demographics Six paediatric patients were diagnosed with MSF for the period 2011-2016 (Table 1). Four patients were female. The age at presentation varied between 17 months and 15 years. Five
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cases presented during the summer months (June to September), while one presented in November. All reported contact with ticks. Four cases denied direct contact with dogs, despite having tick bites. We cannot exclude the involvement of an animal reservoir other
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than the dog in transmission to these cases. Although a history of contact with dogs is part of
Epidemiological features
Case 1
Case 2
Case 3
Case 4
Case 5
Case 6
F
M
M
F
F
F
15y
9y
9y
1y 5m
13y
Gender
1y 10m
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Age
Month of the year
July
June
September
August
September
November
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
No
No
No
Beach*
Beach**
Rural village**
Suburban home✝
Crosscountry run✝
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Contact with ticks
Yes
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Contact with dogs
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the diagnostic scoring system described below, it is often absent [15].
Location of contact with dogs/ticks
Beach*
*South of Malta; **North of Malta; ✝ Central Malta Table 1. Epidemiological features.
3.2 Clinical features All cases presented with fever and one or more eschars, and 5/6 had a generalised maculopapular rash (Table 2, Figure 3). One patient presented with multiple eschars. Three patients also had non-purulent conjunctivitis. One patient, who developed neck stiffness, was
7
ACCEPTED MANUSCRIPT investigated with a lumbar puncture. This revealed an elevated lymphocyte count and protein concentration of the cerebrospinal fluid. None of the patients developed any complications of rickettsial infection, though one patient developed transient synovitis of the right hip. In one
Duration of fever before presentation (days)
Rash
Case 2
Case 3
Case 4
Case 5
Case 6
39.4
40.9
38.5
40
40
38.8
5
6
3
5
5
1
Yes
Yes
Yes
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Fever (/0C)
Case 1
Yes
Yes
No
Yes
Yes
Yes
Yes
-
Yes
Yes
Yes
Yes
-
Yes
Yes
Yes
Yes
-
Yes
Yes
Yes
-
Yes
-
Papular
Yes
-
Involving palms and soles
No
-
Blanching
Yes
-
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Headache
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Clinical features
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case, the mother was simultaneously hospitalised with a suspected diagnosis of MSF.
No
No
Cervical
Cervical
No
No
No
Yes
No
No
No
No
Yes
Yes
No
No
No
Yes
-
Yes
No
No
-
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Right popliteal fossa, vertex of scalp
Right upper back
Left side of neck
Nape of neck
Left external auditory canal
Left shoulder
Meningism
No
Yes
No
No
No
No
Hepatomegaly
No
2cm
No
No
No
No
Lymphadenopathy
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Conjunctivitis
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Nausea and vomiting
Arthralgia
Eschar
-
Location of eschar
Tip of
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ACCEPTED MANUSCRIPT Clinical features Splenomegaly
Case 1
Case 2
Case 3
Case 4
Case 5
Case 6
No
No
spleen
No
No
No
Table 2. Clinical features.
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3.3 Laboratory investigations at presentation Two patients had an elevated white cell count with a predominant lymphocytosis (Table 3). One patient had leukopenia with lymphopenia. None developed thrombocytopenia, while 2/6
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patients had mild hyponatraemia. Four patients had an elevated C-reactive protein (CRP). One patient had an Erythrocyte Sedimentation Rate (ESR) taken, and this was elevated at
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32mm/hr. One patient had a mildly elevated bilirubin of 44.5µmol/l, and another had elevated liver enzymes. Blood investigations
Case 1
Case 2
Case 3
Case 4
Case 5
Case 6
White cell count (x109/L)
15.82 (↑)
8.06
5.45
3.2 (↓)
17.73 (↑)
7.9
Neutrophil count (x109/L)
5.78
4.89
3.15
1.76
7.77 (↑)
5.52
9.23 (↑)
1.66
1.64
1.12 (↓)
8.68 (↑)
1.62
192
242
277
175
311
276
11.3
14.2
11.8
12
11.1
14
140
133 (↓)
133 (↓)
136
135
138
4.39
4.4
3.92
4.3
4.53
4.47
25
80
40
52
17
61
2.1
5
3.5
2.5
3.1
3.5
48 (↑)
210 (↑)
12 (↑)
<6
67.4 (↑)
3.5
ESR (mm in 1st hour)
32
-
-
12
-
-
Bilirubin (µmol/L)
2.7
7
6.9
44.5 (↑)
-
9.8
Alanine aminotransferase (U/l)
61 (↑)
36
16
21
-
17
Aspartate aminotransferase (U/l)
73 (↑)
30
-
-
-
-
Alkaline phosphatase (U/l)
123
97
144
129
-
149
Gamma-glutarylaminotransferase (U/l)
22
11
11
34
-
16
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Lymphocytes (x109/L) Platelet count (x109/L) Haemoglobin (g/dL)
Potassium (mmol/L)
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Creatinine (µmol/L)
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Sodium (mmol/L)
Urea (mmol/L) CRP* (mg/L)
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ACCEPTED MANUSCRIPT Table 3. Laboratory investigations at presentation. *CRP values reflect the highest values recorded during the illness.
3.4 Diagnostic investigations
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All children had anti-rickettsial antibodies tested by enzyme-linked immunosorbent assay (ELISA) in the first week of the illness (Vircell, S.L., Granada, Spain), with all being negative for IgM and IgG against R. conorii, R. rickettsia and R. typhi (Table 4). Two
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patients were additionally tested quantitatively (Bioscientia Institute for Diagnostic Medicine, GmbH, Germany) for anti-rickettsial antibodies by means of immunofluorescence assay
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testing (Focus Diagnostics Inc., California, USA) in the first week of the illness. Both these children had low titres of anti-rickettsial antibodies at this point. Low anti-rickettsial antibody titres are well-reported in the first week of the illness [16]. Two patients were tested by ELISA during the second-to-third weeks of the illness, and these were confirmed to have
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developed anti-R. conorii IgM antibodies. These patients also had positive titres for anti-R. rickettsii IgG antibodies at week 2-3, while one had a positive titre for R. typhi IgG antibodies. R. rickettsiae has never been identified in Malta, while R. typhi, although thought
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to be endemic in Malta but never described, is associated with murine typhus, an illness in which an eschar is absent, being spread by flea faeces contaminating a flea-bite [17]. These
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results are therefore likely to be false-positive, secondary to cross-reactivity of ELISA [18]. None of the patients were tested during the convalescent phase at 4 weeks, as venepuncture was deemed inappropriate in children whose symptoms had completely resolved. Eschar biopsy was also deemed inappropriate because of the invasive nature of this procedure. Blood samples for real-time multiplex rickettsial polymerase chain reaction (PCR) with primers directed against the citrate synthase (gltA) gene (Fast Track Diagnostics Luxembourg S. á. r. l., Esch-sur-Alzette, Luxembourg) were taken at presentation in 4/6 patients, but were negative in all cases. This is well documented in PCR seeking the gltA rickettsial gene in 10
ACCEPTED MANUSCRIPT blood samples [19]. We are therefore unable to comment on the causative rickettsial species behind the cases of MSF. Rickettsial culture was not performed in our series. Applying the diagnostic scoring system for MSF to this series, 4/6 patients had a positive score of ≥29. One case had a score of 19 because the patient’s fever did not reach
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39oC, while another scored 9 because of the absence of the typical rash, a maximal fever of 38.8oC, and a presentation in November, instead of the typical period between May and October [20]. It should be noted, however, that warm weather in Malta may persist beyond
Diagnostic investigations
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October.
Case 1
ELISA
Case 2
Case 3
Case 4
Case 5
Case 6
Week 1
Week 2-3
Week 1
Week 2-3
Week 1
Week 1
Week 1
Week 1
R. conorii IgM
Negative
Positive
Negative
Positive
Negative
Negative
Negative
Negative
-
R. conorii IgG
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
-
R. rickettsii IgM
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
-
R. rickettsii IgG
Negative
Positive
Negative
Positive
Negative
Negative
Negative
Negative
-
R. typhi IgM
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
-
R. typhi IgG
Negative
Negative
Negative
Positive
Negative
Negative
Negative
Negative
-
R. conorii/R. typhi IgM and IgG
PCR (Blood)
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(Week 1)
-
-
Negative
-
Negative
-
Negative
-
Negative
-
Negative
Negative
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IFAT
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-
Table 4. Diagnostic investigations. (ELISA=Enzyme-linked immunosorbent assay, IFAT=Immunofluorescence assay testing, PCR=polymerase chain reaction.)
3.5 Treatment and outcome Four children were treated with clarithromycin, and the two teenagers with doxycycline. Duration of treatment was 10 days. All patients were afebrile within 48 hours of starting appropriate antimicrobial therapy and recovered completely. None of the children developed
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ACCEPTED MANUSCRIPT significant complications of MSF, with similar outcomes reported in Barcelona and Sicily
4 Discussion and literature review
4.1 Epidemiology
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[9,21].
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Rickettsia conorii, an obligate intracellular organism, is the causative agent behind MSF [1]. Molecular techniques have allowed the identification of rickettsial subspecies, each responsible for a distinct spotted-fever syndrome. Rickettsia conorii conorii (type strain =
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Malish, ATCC VR-613) is the subspecies of R. conorii responsible for MSF [22]. MSF is
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endemic in most Mediterranean countries [4]. True incidence data for MSF are scarce because of inter-country variations in the criteria used for reporting. Spain, Algeria and Italy have a reported annual incidence of 5-10 cases per 100,000 inhabitants, while in Portugal it is >10 cases per 100,000 inhabitants [1]. In Malta, the incidence is lower, at 0.9 cases per 100,000 inhabitants [23]. However, this is likely an underestimation secondary to underrecognition and underreporting of cases. There are no animal studies to date that examine the prevalence of rickettsial diseases in Malta. Seroprevalence of antibodies against R. conorii in the Mediterranean depends on the region, varying between 5% in Spain and 10% in Israel
12
ACCEPTED MANUSCRIPT [5,24]. Rhipicephalus sanguineus (Brown dog tick), the vector of MSF, acquires R. conorii by feeding on an infected canine host (Figure 2). R. sanguineus carriage amongst Maltese dogs was recently reported by Licari et al [25]. Aside from dogs, rabbits, hedgehogs and small rodents have all been postulated as the reservoir for the disease, as anti-R. conorii
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antibodies have been found in these animals [1,26].
4.2 Pathogenesis
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Following inoculation at the site of an eschar, R. conorii replicates in the cytoplasm of endothelial cells, releasing pro-inflammatory cytokines. This leads to widespread vasculitis,
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hypo-perfusion and multi-organ injury [7]. Injury to the vascular endothelium affects the liver, brain, lungs, coagulation pathway and skin, leading to the typical erythematous maculopapular rash (Figure 3a-c) [27]. Severe MSF is not associated with high loads of rickettsiae in the blood, suggesting this is an immunopathological phenomenon rather than
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overwhelming sepsis [7]. The eschar itself is characterized histologically by a central area of ischaemic necrosis (Figure 3d-f) with abundant lymphohistiocytic infiltration and
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Adult Brown Dog Tick (Rhipicephalus sanguineus)
Bites from ticks with infected salivary glands
Brown Dog Tick Nymph
Canine
Human host
Eggs
TRANSOVARIAL
TRANSSTADIAL
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surrounding dermal oedema, though relatively few rickettsial organisms [7,28].
Brown Dog Tick Larvae
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ACCEPTED MANUSCRIPT Fig 2. Transmission of Rickettsia conorii [29,30].
4.3 Clinical features MSF presents with fever (>39oC), a discrete maculopapular rash with palmo-plantar
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involvement, and an inoculation eschar at the site of the tick bite (Figure 3d-f) [15,31]. Other manifestations often include headache, myalgia, arthralgia, hepatomegaly, splenomegaly, gastrointestinal symptoms and conjunctivitis [15,31]. In Crete, 53% of patients in a case
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series had conjunctivitis [3]. Multiple eschars are rare but have been described in infections other than those due to R. conorii, such as R. aeschlimannii or R.sibirica. These pathogens
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are not endemic in Malta [1,32–34]. R. conorii MSF has been reported in patients with multiple eschars, and this atypical behaviour in Rhipicephalus sp. ticks may be attributable to
climate change in Southern Europe [14]. Mortality with untreated MSF may be as high as 32.3% in adults, though it is not usually severe in children [1,35,36]. Outcome is worse
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amongst patients with diabetes mellitus, immunosuppressive states, chronic cardiac
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pathology, chronic alcoholism, and glucose-6-phosphate dehydrogenase deficiency [35].
4.4 Laboratory investigations
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Leukopenia and thrombocytopenia are common in patients with MSF, being seen in 25% and 80% respectively of patients [12,19]. Hyponatraemia is present in 33% of patients [3]. Inflammatory markers such as the CRP, liver enzymes, and muscular enzymes such as creatine kinase, are typically elevated [37,38].
14
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Figure 3. Cutaneous manifestations of MSF (a) Maculopapular rash on the lower limb of an infant, involving the sole (Case 1) and (b) on the trunk, arms and (c) palms in an adolescent (Case 2). (d) Eschar on lower limb (Case 1), (e) posterior hairline (Case 4), and (f) close-up, with central necrosis and surrounding erythema with desquamation. (Source: Paediatric Infectious Disease services, Mater Dei Hospital, Malta)
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ACCEPTED MANUSCRIPT 4.5 Diagnostic investigations Serological analysis is the easiest and most widespread method of rickettsial diagnosis, but can only provide a retrospective diagnosis [17]. Seroconversion is detectable 7 to 15 days after the onset of disease [39]. Several serological techniques are available (Table 5). IF is the
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gold standard investigation for rickettsial diagnosis: a four-fold rise in anti-rickettsial antibody titres between acute and convalescent samples, IgM titres ≥64, or IgG titres of ≥128 are diagnostic [6,12,16]. Paired acute and convalescent samples are inconvenient in
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paediatric MSF, as venepuncture following clinical resolution may not be justified. Enzymelinked immunosorbent assay (ELISA) is highly sensitive, reproducible, and distinguishes
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between IgG and IgM antibodies [16,40].
Several rickettsial genetic sequences can be targeted by PCR (Table 5). Although R. conorii supsp. conorii is classically associated with MSF in Europe, R. massiliae, (acquired from bites by R. sanguineus or R. turpanicus ticks), R. conorii subsp. israelensis and R.
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conorii subsp. indica have also caused MSF in Sicily, Portugal and Spain respectively [41,42]. R. slovaca, seen in Hungary, France, Spain and Portugal, has been associated with Tick-borne lymphadenopathy (TIBOLA) or Dermacentor-borne necrosis, erythema and
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lymphadenopathy (DEBONEL). This is characterised by an eschar on the head and neck,
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surrounding erythema and painful cervical lymphadenopathy [42]. Although none of these pathogens have ever been identified in Malta, we cannot exclude causality. Yield from PCR could be improved by eschar biopsy, though this is invasive, and arguably inappropriate for paediatric patients when the clinical diagnosis is clear [19]. While species identification may not influence patient outcome, it is important for epidemiological investigation. Rickettsiae are difficult to grow in culture, but may be detectable in shell-vial culture 48-72 hours post-inoculation, prior to seroconversion [43]. However, the technique is less
16
ACCEPTED MANUSCRIPT sensitive than serology or quantitative PCR. Specimens should be taken prior to treatment and early in the disease, and be inoculated immediately [44]. Raoult et al described a scoring system for the diagnosis of MSF that included clinical and epidemiological features, serology and culture (Table 6) [20]. This system was adapted
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score of 18/29 was used to diagnose MSF [45].
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for clinical diagnosis (Table 7), with a sensitivity of 60% and a specificity of 84.6% when a
17
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Investigation a.
Sensitivity
Specificity
Advantages -
Serology
Limitations
Easiest to use
-
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-
Retrospective diagnosis Unable to identify species/new pathogens Poor sensitivity/specifi city
References
Widespread
[17,19,39]
Largely obsolete. Restricted to low resource areas Restricted to seroepidemiological studies.
[46,47]
-
WF
33%
46%
-
Low-cost
-
CF
N/A
N/A
-
High specificity
-
Poor sensitivity
-
LA
93%
96%
Expensive kit
Not widespread
[16,48,49]
MIF
100% (day 29 of illness)
100%
Rapid Simple Sensitive/specific Widely available
-
-
-
-
Fails to identify aetiologic agent Retrospective diagnosis – poor early sensitivity Requires repeated blood sampling, expensive equipment, and expertise
Widespread. Gold standard
[3,6,10,12,16,46]
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-
Use
90% (day 20-29)
-
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46% (days 5-9)
-
-
ELISA
90-95%
-
WB
67% (before day 10)
100%
-
Reproducible
-
As for MIF
Widespread
[19,40]
100%
-
Species-specific Early diagnosis
-
Time-consuming Requires expensive equipment
Reference laboratories
[6,50,51]
97-100%
-
Rapid
-
As for WB
Endemic areas
[52]
90% (day 10-15)
-
LBA
100% (after day 35) 84-98%
[16]
18
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Advantages -
Tests numerous antigens simultaneously
-
Rapid Potentially speciesspecific
Allows identification of novel pathogenic species
[53–55]
-
Not SFGRspecific
Reference laboratories
[56,57]
Early diagnosis
-
Not SFGRspecific
Reference laboratories
[58]
-
Not SFGRspecific Poor yield from serum
Widespread
[19,59,60]
-
Real-time PCR can detect very low rickettsial counts Has been sequenced in almost all rickettsiae Quantitative
-
SFGR-specific
-
Fails to diagnose certain SFGR e.g. R. helvetica
Reference laboratories
[16,61]
100%
-
-
[62–64]
-
Not SFGRspecific: positive with TG Limited sensitivity Poor sensitivity
Widespread
100%
Early diagnosis Detects low rickettsial counts Early diagnosis SFGR-specific Early diagnosis
Reference laboratories Not widespread
[65]
16S ribosomal DNA
Not evaluated
Not evaluated
-
-
17kDa common antigen gene
14% (100% of fatal cases)
100%
-
-
gltA (citrate synthase) gene
77% (eschar biopsy)
100%
-
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-
0% (serum)
-
-
OmpB gene (Nested PCR)
71% (serum)
-
OmpB gene (LAMP) Immunodetection
73% (serum)
c.
Not evaluated
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Not evaluated
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OmpA gene
55% (eschar biopsy)
References
Widespread, though not standardised
PCR
100%
-
Use
Poor sensitivity, affected by antibiotic treatment
b.
-
Limitations
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Specificity
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Sensitivity
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Investigation
-
-
[66,67]
50-75% (circulating
19
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d.
Shell-vial culture
Sensitivity
Specificity
endothelial cells) 29.1% (59% prior to antibiotic therapy)
Advantages
100%
-
-
Limitations
Description of new pathogenic species/antibiotic susceptibility testing Early diagnosis
-
Difficult to perform Biopsies are painful Expensive
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Investigation
-
Use
Restricted to Biosafety level 3 laboratories.
References
[12,44,68]
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Table 5. Laboratory diagnosis of MSF. (WF=Weil-Felix; CF=complement fixation; LA=latex agglutination; MIF=microimmunofluorescence; ELISA=enzyme-linked immunofluorescence assay; WB=western blot; LBA=line blot assay; PCR=polymerase chain reaction; SFGR=spotted fever-group rickettsiae; TG=typhus-group rickettsiae; LAMP=loop-mediated isothermal amplification)
20
ACCEPTED MANUSCRIPT
Criteria
Score
Stay in endemic area
2
2.
Occurrence in May-October
2
3.
Contact (certain/possible) with dog ticks
2
Clinical criteria Fever>39oC
5
2.
Eschar
5
3.
Maculopapular/purpuric rash
5
4.
Two clinical criteria
3
5.
Three clinical criteria
5
Non-specific laboratory findings 1.
Platelets<150G/L
2.
SGOT/SGPT>50U/L
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1.
1
1
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Bacteriological criteria 1.
Blood culture positive for Rickettsia conorii
25
2.
Detection of Rickettsia conorii in skin biopsy
25
Serological criteria (IF)
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1.
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Epidemiological criteria
Single serum IgG>1/128
5
2.
Single serum IgG>1/128 and IgM>1/64
10
3.
Four-fold increase in two sera obtained within 2-
20
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1.
week interval
Table 6. Diagnostic criteria for MSF. Score ≥25 signifies positive diagnosis [12]. (SGOT = serum glutamateoxaloacetate transaminase, SGPT = serum glutamate-pyruvate transaminase.)
21
ACCEPTED MANUSCRIPT Epidemiologic criteria
Score
1.
Endemic area
2
2.
Hot season (May–September)
2
3.
Tick bite
2
1.
Fever>39oC
5
2.
Eschar
5
3.
Maculopapular/purpuric rash
5
4.
Two clinical criteria
3
5.
Three clinical criteria
5
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Score
SC
Clinical criteria
4.6 Treatment and outcome
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Table 7. Modified version of Raoult et al scoring system to allow clinical diagnosis [45].
There are no placebo-controlled trials of treatment in MSF, so the need for treatment is based on observational studies and experience with other rickettsial illnesses [36]. Tetracyclines
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have good anti-rickettsial activity [39,69,70], while randomised controlled trials have shown macrolides to be effective and safe in children [71–76]. A recent study has revealed a worse
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4.7 Prevention
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outcome with the use of fluoroquinolones [77].
There are few specific public health recommendations regarding the prevention of MSF when traveling to Malta. The risk of travelers contracting tick-borne rickettsioses such as MSF remains low [17]. Prevention relies on preventing tick bites, as there is no licensed vaccine for MSF [78]. Travelers to Malta should avoid contact with local dogs, while those hiking in rural areas should check for and remove any ticks from clothing or skin [17]. The application of insect repellents to exposed skin provides little protection against ticks, so treating clothing with permethrin is recommended in endemic areas [79]. Travelers who bring their own dogs
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ACCEPTED MANUSCRIPT should consider treating them with acaricides [80]. There is no evidence that antimicrobial prophylaxis is beneficial to prevent MSF after a tick bite, while serological testing is not recommended because of the poor sensitivity and the cost of the tests. Clinicians should
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observe the patient and treat only if MSF occurs [79].
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ACCEPTED MANUSCRIPT 5 Conclusions The incidence of rickettsiosis in Europe has increased [81]. Outdoor activities among Europeans has increased contact with ticks and tick-borne diseases [82]. Climate change might contribute to changing behaviour in Rhipicephalus sp. ticks, and thus influence MSF
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incidence [83]. Widespread international travel to endemic areas, including Malta, makes MSF part of the differential diagnosis of fever in a returning traveller [8]. Despite improvements in MSF diagnosis, clinical diagnosis remains important in the management of
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children with suspected MSF.
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ACCEPTED MANUSCRIPT References
[1]
Rovery C, Brouqui P, Raoult D. Questions on Mediterranean spotted fever a century after its discovery. Emerg Infect Dis 2008;14:1360–7. doi:10.3201/eid1409.071133. Tonna I, Mallia Azzopardi C, Piscopo T, Cuschieri P, Fenollar F, Raoult D.
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[2]
Characterisation of rickettsial diseases in a hospital-based population in Malta. J Infect 2006;53:394–402. doi:10.1016/j.jinf.2005.12.024.
Germanakis A, Psaroulaki A, Gikas A, Tselentis Y. Mediterranean spotted fever in
SC
[3]
Crete, Greece: Clinical and therapeutic data of 15 consecutive patients. Ann N Y Acad
[4]
Herve Z, Wim VB. Epidemiological situation of rickettsioses in EU / EFTA countriesTechnical Report. 2013.
[5]
M AN U
Sci 2006;1078:263–9. doi:10.1196/annals.1374.049.
Harrus S, Lior Y, Ephros M, Grisaru-Soen G, Keysary A, Strenger C, et al. Rickettsia
TE D
conorii in humans and dogs: a seroepidemiologic survey of two rural villages in Israel. Am J Trop Med Hyg 2007;77:133–5. [6]
Brouqui P, Parola P, Fournier PE, Raoult D. Spotted fever rickettsioses in southern and
EP
eastern Europe. FEMS Immunol Med Microbiol 2007;49:2–12. doi:10.1111/j.1574-
[7]
AC C
695X.2006.00138.x.
de Sousa R, Ismail N, Nobrega SD, Franca A, Amaro M, Anes M, et al. Intralesional expression of mRNA of interferon- gamma , tumor necrosis factor- alpha , interleukin10, nitric oxide synthase, indoleamine-2,3-dioxygenase, and RANTES is a major immune effector in Mediterranean spotted fever rickettsiosis. J Infect Dis 2007;196:770–81. doi:10.1086/519739.
[8]
Laurent M, Voet A, Libeer C, Lambrechts M, Van Wijngaerden E. Mediterranean spotted fever, a diagnostic challenge in travellers. Acta Clin Belg 2009;64:513–6.
25
ACCEPTED MANUSCRIPT doi:10.1179/acb.2009.087. [9]
Colomba C, Saporito L, Polara VF, Rubino R, Titone L. Mediterranean spotted fever: clinical and laboratory characteristics of 415 Sicilian children. BMC Infect Dis 2006;6:60. doi:10.1186/1471-2334-6-60. RN,
Casper
EA,
Ormsbee
RA,
Peacock
MG,
Burgdorfer
RI PT
[10] Philip
W.
Microimmunofluorescence test for the serological study of Rocky Mountain spotted fever and typhus. J Clin Microbiol 1976;3:51–61.
SC
[11] Raoult D. current approaches to diagnosis of old and new rickettsial diseases . Laboratory Diagnosis of Rickettsioses : Current Approaches to Diagnosis of Old and
M AN U
New Rickettsial Diseases 1997;35:2715–27.
[12] Brouqui P, Bacellar F, Baranton G, Birtles RJ, Bjoërsdorff A, Blanco JR, et al. Guidelines for the diagnosis of tick-borne bacterial diseases in Europe. Clin Microbiol Infect 2004;10:1108–32. doi:10.1111/j.1469-0691.2004.01019.x.
TE D
[13] Rome FI. Tourism in 1975:206–7.
[14] Parola P, Socolovschi C, Jeanjean L, Bitam I, Fournier P-E, Sotto A, et al. Warmer weather linked to tick attack and emergence of severe rickettsioses. PLoS Negl Trop
EP
Dis 2008;2:e338. doi:10.1371/journal.pntd.0000338.
AC C
[15] Crespo P, Seixas D, Marques N, Oliveira J, da Cunha S, Meliço-Silvestre A. Mediterranean spotted fever: case series of 24 years (1989-2012). Springerplus 2015;4:272. doi:10.1186/s40064-015-1042-3.
[16] La Scola B, Raoult D. Laboratory diagnosis of rickettsioses: current approaches to diagnosis of old and new rickettsial diseases. J Clin Microbiol 1997;35:2715–27. [17] Jensenius M, Fournier P-E, Raoult D. Rickettsioses and the international traveler. Clin Infect Dis 2004;39:1493–9. doi:10.1086/425365. [18] Eisen RJ, Gage KL. Transmission of flea-borne zoonotic agents. Annu Rev Entomol
26
ACCEPTED MANUSCRIPT 2012;57:61–82. doi:10.1146/annurev-ento-120710-100717. [19] Madeddu G, Fiore V, Mancini F, Caddeo A, Ciervo A, Babudieri S, et al. Mediterranean spotted fever-like illness in Sardinia, Italy: a clinical and microbiological study. Infection 2016;44:733–8. doi:10.1007/s15010-016-0921-z.
RI PT
[20] Raoult D, Roux V. Rickettsioses as paradigms of new or emerging infectious diseases. Clin Microbiol Rev 1997;10:694–719.
[21] Moraga FA, Martinez-Roig A, Alonso JL, Boronat M, Domingo F. Boutonneuse fever.
SC
Arch Dis Child 1982;57:149–51.
[22] Zhu Y, Fournier P-E, Eremeeva M, Raoult D. Proposal to create subspecies of
M AN U
Rickettsia conorii based on multi-locus sequence typing and an emended description of Rickettsia conorii. BMC Microbiol 2005;5:11. doi:10.1186/1471-2180-5-11. [23] Unit MS. The estimated total population of Malta and Gozo at the end of 2016 stood at World Population Day : 11 July 2017 2017:1–12.
TE D
[24] Espejo E, Andres M, Perez J, Prat J, Guerrero C, Munoz MT, et al. Prevalence of antibodies to Rickettsia conorii in human beings and dogs from Catalonia: a 20-year perspective. Epidemiol Infect 2016;144:1889–94. doi:10.1017/S0950268816000261.
from
Malta.
Ticks
Tick
Borne
Dis
2017;8:396–9.
AC C
dogs
EP
[25] Licari E, Takács N, Solymosi N, Farkas R. First detection of tick-borne pathogens of
doi:10.1016/j.ttbdis.2017.01.002.
[26] Le Gac P. [Repercussions of myxomatosis on Mediterranean boutonneuse exanthematic fever]. Bull World Health Organ 1966;35:143–7.
[27] Sousa R de, Franca A, Doria Nobrega S, Belo A, Amaro M, Abreu T, et al. Host- and microbe-related risk factors for and pathophysiology of fatal Rickettsia conorii infection in Portuguese patients. J Infect Dis 2008;198:576–85. doi:10.1086/590211. [28] Walker DH, Occhino C, Tringali GR, Di Rosa S, Mansueto S. Pathogenesis of
27
ACCEPTED MANUSCRIPT rickettsial eschars: the tache noire of boutonneuse fever. Hum Pathol 1988;19:1449– 54. [29] Gray J, Dantas-Torres F, Estrada-Pena A, Levin M. Systematics and ecology of the
doi:10.1016/j.ttbdis.2012.12.003.
RI PT
brown dog tick, Rhipicephalus sanguineus. Ticks Tick Borne Dis 2013;4:171–80.
[30] Parola P, Socolovschi C, Raoult D. Deciphering the relationships between Rickettsia conorii conorii and Rhipicephalus sanguineus in the ecology and epidemiology of spotted
fever.
Ann
doi:10.1111/j.1749-6632.2009.04518.x.
N
Y
Acad
Sci
2009;1166:49–54.
SC
Mediterranean
M AN U
[31] Segura-Porta F, Font-Creus B, Espejo-Arenas E, Bella-Cueto F. New trends in Mediterranean spotted fever. Eur J Epidemiol 1989;5:438–43. [32] Fournier P-E, Gouriet F, Brouqui P, Lucht F, Raoult D. Lymphangitis-associated rickettsiosis, a new rickettsiosis caused by Rickettsia sibirica mongolotimonae: seven
TE D
new cases and review of the literature. Clin Infect Dis 2005;40:1435–44. doi:10.1086/429625.
[33] de Sousa R, Barata C, Vitorino L, Santos-Silva M, Carrapato C, Torgal J, et al.
EP
Rickettsia sibirica isolation from a patient and detection in ticks, Portugal. Emerg
AC C
Infect Dis 2006;12:1103–8. doi:10.3201/eid1207.051494. [34] Fernandez-Soto P, Perez-Sanchez R, Alamo-Sanz R, Encinas-Grandes A. Spotted fever group rickettsiae in ticks feeding on humans in northwestern Spain: is Rickettsia conorii vanishing? Ann N Y Acad Sci 2006;1078:331–3. doi:10.1196/annals.1374.063.
[35] de Sousa R, Nobrega SD, Bacellar F, Torgal J. Mediterranean spotted fever in Portugal: risk factors for fatal outcome in 105 hospitalized patients. Ann N Y Acad Sci 2003;990:285–94. [36] Demeester R, Claus M, Hildebrand M, Vlieghe E, Bottieau E. Diversity of life-
28
ACCEPTED MANUSCRIPT threatening complications due to mediterranean spotted fever in returning travelers. J Travel Med 2010;17:100–4. doi:10.1111/j.1708-8305.2009.00391.x. [37] Micalizzi A, La Spada E, Corsale S, Arculeo A, La Spada M, Ouartararo P, et al. Abnormal liver function in Mediterranean spotted fever | Interessamento epatico in
RI PT
corso di febbre bottonosa del Mediterraneo. Infez Med 2007;15:105–10.
[38] Mert A, Ozaras R, Tabak F, Bilir M, Ozturk R. Mediterranean spotted fever: A review of fifteen cases. J Dermatol 2006;33:103–7. doi:10.1111/j.1346-8138.2006.00021.x.
SC
[39] Parola P, Paddock CD, Socolovschi C, Labruna MB, Mediannikov O, Kernif T, et al. Update on tick-borne rickettsioses around the world: A geographic approach. Clin
M AN U
Microbiol Rev 2013;26:657–702. doi:10.1128/CMR.00032-13.
[40] Do E-J, Kim J-E, Park J-M, Lee K-M, Jung M-Y, Lee H-J, et al. Development of recombinant OmpA and OmpB proteins as diagnostic antigens for rickettsial disease. Microbiol Immunol 2009;53:368–74. doi:10.1111/j.1348-0421.2009.00142.x.
TE D
[41] Vitale G, Mansuelo S, Rolain J-M, Raoult D. Rickettsia massiliae human isolation. Emerg Infect Dis 2006;12:174–5. doi:10.3201/eid1201.050850. [42] Portillo A, Santibáñez S, García-Álvarez L, Palomar AM, Oteo JA. Rickettsioses in
EP
Europe. Microbes Infect 2015;17:834–8. doi:10.1016/j.micinf.2015.09.009.
AC C
[43] Marrero M, Raoult D. Centrifugation-shell vial technique for rapid detection of Mediterranean spotted
fever rickettsia in blood culture. Am J Trop Med Hyg
1989;40:197–9.
[44] Angelakis E, Richet H, Rolain J-M, La Scola B, Raoult D. Comparison of real-time quantitative PCR and culture for the diagnosis of emerging Rickettsioses. PLoS Negl Trop Dis 2012;6:e1540. doi:10.1371/journal.pntd.0001540. [45] Letaief A, Souissi J, Trabelsi H, Ghannem H, Jemni L. Evaluation of clinical diagnosis scores for Boutonneuse fever 2003;990:327–30.
29
ACCEPTED MANUSCRIPT [46] Raoult D, Rousseau S, Toga B, Tamalet C, Gallais H, De Micco P, et al. [Serological diagnosis of Mediterranean boutonneuse fever]. Pathol Biol (Paris) 1984;32:791–4. [47] Kularatne SAM, Gawarammana IB. Validity of the Weil-Felix test in the diagnosis of
doi:10.1016/j.trstmh.2008.11.020.
RI PT
acute rickettsial infections in Sri Lanka. Trans R Soc Trop Med Hyg 2009;103:423–4.
[48] Hechemy KE, Raoult D, Eisemann C, Han YS, Fox JA. Detection of antibodies to Rickettsia conorii with a latex agglutination test in
patients with Mediterranean
SC
spotted fever. J Infect Dis 1986;153:132–5.
[49] De la fuente L, Anda P, Rodriguez I, Hechemy KE, Raoult D, Casal J. Evaluation of a
M AN U
latex agglutination test for Rickettsia conorii antibodies in seropositive patients. J Med Microbiol 1989;28:69–72. doi:10.1099/00222615-28-1-69. [50] Teysseire
N,
Raoult
D.
Comparison
of
western
immunoblotting
and
microimmunofluorescence for diagnosis of Mediterranean spotted fever. J Clin
TE D
Microbiol 1992;30:455–60.
[51] Babalis T, Dupont HT, Tselentis Y, Chatzichristodoulou C, Raoult D. Rickettsia conorii in Greece: comparison of a microimmunofluorescence assay and western
EP
blotting for seroepidemiology. Am J Trop Med Hyg 1993;48:784–92.
AC C
[52] Raoult D, Dasch GA. Line blot and western blot immunoassays for diagnosis of mediterranean spotted fever. J Clin Microbiol 1989;27:2073–9.
[53] Wang JM, Hudson BJ, Watts MR, Karagiannis T, Fisher NJ, Anderson C, et al. Diagnosis of Queensland tick typhus and African tick bite fever by PCR of lesion swabs. Emerg Infect Dis 2009;15:963–5. doi:10.3201/eid1506.080855. [54] Mouffok N, Socolovschi C, Benabdellah A, Renvoise A, Parola P, Raoult D. Diagnosis of rickettsioses from eschar swab samples, Algeria. Emerg Infect Dis 2011;17:1968–9. doi:10.3201/eid1710.110332.
30
ACCEPTED MANUSCRIPT [55] Khrouf F, Sellami H, Elleuch E, Hattab Z, Ammari L, Khalfaoui M, et al. Molecular diagnosis of Rickettsia infection in patients from Tunisia. Ticks Tick Borne Dis 2016;7:653–6. doi:10.1016/j.ttbdis.2016.02.010.
sequencing. Res Microbiol 1995;146:385–96.
RI PT
[56] Roux V, Raoult D. Phylogenetic analysis of the genus Rickettsia by 16S rDNA
[57] Woo PCY, Lau SKP, Teng JLL, Tse H, Yuen K-Y. Then and now: use of 16S rDNA gene sequencing for bacterial identification and discovery of novel bacteria in clinical
SC
microbiology laboratories. Clin Microbiol Infect 2008;14:908–34. doi:10.1111/j.14690691.2008.02070.x.
M AN U
[58] Leitner M, Yitzhaki S, Rzotkiewicz S, Keysary A. Polymerase chain reaction-based diagnosis of Mediterranean spotted fever in serum and tissue samples. Am J Trop Med Hyg 2002;67:166–9.
[59] Stenos J, Graves SR, Unsworth NB. A highly sensitive and specific real-time PCR
TE D
assay for the detection of spotted fever and typhus group Rickettsiae. Am J Trop Med Hyg 2005;73:1083–5.
[60] Roux V, Rydkina E, Eremeeva M, Raoult D. Citrate synthase gene comparison, a new
EP
tool for phylogenetic analysis, and its application for the rickettsiae. Int J Syst
AC C
Bacteriol 1997;47:252–61. doi:10.1099/00207713-47-2-252. [61] Roux V, Fournier P, Raoult D. Differentiation of spotted fever group rickettsiae by sequencing and analysis of restriction fragment length polymorphism of PCRamplified DNA of the gene encoding. J Clin Microbiol 1996;34:2058–65.
[62] Fournier PE, Roux V, Raoult D. Phylogenetic analysis of spotted fever group rickettsiae by study of the outer surface protein rOmpA. Int J Syst Bacteriol 1998;48 Pt 3:839–49. doi:10.1099/00207713-48-3-839. [63] Roux V, Raoult D. Phylogenetic analysis of members of the genus Rickettsia using the
31
ACCEPTED MANUSCRIPT gene encoding the outer-membrane protein rOmpB (ompB). Int J Syst Evol Microbiol 2000;50 Pt 4:1449–55. doi:10.1099/00207713-50-4-1449. [64] Choi Y, Lee S, Park K, Koh Y, Lee K, Baik H, et al. Evaluation of PCR-Based Assay for Diagnosis of Spotted Fever Group Rickettsiosis in Human Serum Samples. Clin
RI PT
Diagn Lab Immunol 2005;12:759–63. doi:10.1128/CDLI.12.6.759.
[65] Pan L, Zhang L, Wang G, Liu Q. Rapid, simple, and sensitive detection of the ompB gene of spotted fever group rickettsiae by loop-mediated isothermal amplification.
SC
BMC Infect Dis 2012;12:254. doi:10.1186/1471-2334-12-254.
[66] Raoult D, de Micco C, Gallais H, Toga M. Laboratory diagnosis of Mediterranean
M AN U
spotted fever by immunofluorescent demonstration of Rickettsia conorii in cutaneous lesions. J Infect Dis 1984;150:145–8.
[67] Drancourt M, George F, Brouqui P, Sampol J, Raoult D. Diagnosis of Mediterranean spotted fever by indirect immunofluorescence of Rickettsia conorii in circulating
TE D
endothelial cells isolated with monoclonal antibody-coated immunomagnetic beads. J Infect Dis 1992;166:660–3.
[68] Quesada M, Sanfeliu I, Cardeñosa N, Segura F. Ten years’ experience of isolation of
EP
Rickettsia spp. from blood samples using the shell-vial cell culture assay. Ann N Y
AC C
Acad Sci 2006;1078:578–81. doi:10.1196/annals.1374.115. [69] Rovery C, Raoult D. Mediterranean Spotted Fever. Infect Dis Clin North Am 2008;22:515–30. doi:10.1016/j.idc.2008.03.003.
[70] Rolain JM, Maurin M, Vestris G, Raoult D. In vitro susceptibilities of 27 rickettsiae to 13 antimicrobials. Antimicrob Agents Chemother 1998;42:1537–41. [71] Anton E, Muñoz T, Travería FJ, Navarro G, Font B, Sanfeliu I, et al. Clarithromycin for Mediterranean Spotted Fever: a Randomized Trial. Antimicrob Agents Chemother 2015;60:1–22. doi:10.1128/AAC.01814-15.
32
ACCEPTED MANUSCRIPT [72] Cascio A, Colomba C, Antinori S, Paterson DL, Titone L. Clarithromycin versus azithromycin in the treatment of Mediterranean spotted fever in children: a randomized controlled trial. Clin Infect Dis 2002;34:154–8. doi:10.1086/338068. [73] Cascio A, Colomba C, Di Rosa D, Salsa L, di Martino L, Titone L. Efficacy and safety
RI PT
of clarithromycin as treatment for Mediterranean spotted fever in children: a randomized controlled trial. Clin Infect Dis 2001;33:409–11. doi:10.1086/321864. [74] Cascio A, Colomba C. [Macrolides in the treatment of children with Mediterranean
SC
spotted fever]. Infez Med 2002;10:145–50.
Eur J Epidemiol 1991;7:276–81.
M AN U
[75] Raoult D. Antibiotic treatment of rickettsiosis, recent advances and current concepts.
[76] Bella F, Font B, Uriz S, Munoz T, Espejo E, Traveria J, et al. Randomized trial of doxycycline versus josamycin for Mediterranean spotted fever. Antimicrob Agents Chemother 1990;34:937–8.
TE D
[77] Botelho-Nevers E, Rovery C, Richet H, Raoult D. Analysis of risk factors for malignant Mediterranean spotted fever indicates that fluoroquinolone treatment has a deleterious effect. J Antimicrob Chemother 2011;66:1821–30. doi:10.1093/jac/dkr218.
EP
[78] Richards AL. Rickettsial vaccines: the old and the new. Expert Rev Vaccines
AC C
2004;3:541–55. doi:10.1586/14760584.3.5.541. [79] Parola P, Raoult D. Ticks and tickborne bacterial diseases in humans: an emerging infectious threat. Clin Infect Dis 2001;32:897–928. doi:10.1086/319347.
[80] Estrada-Peña A, Venzal Bianchi JM. Efficacy of several anti-tick treatments to prevent the transmission of Rickettsia conorii under natural conditions. Ann N Y Acad Sci 2006;1078:506–8. doi:10.1196/annals.1374.097. [81] Blanco JR, Oteo JA. Rickettsiosis in Europe. Ann N Y Acad Sci 2006;1078:26–33. doi:10.1196/annals.1374.003.
33
ACCEPTED MANUSCRIPT [82] Parola P, Raoult D. Tick-borne bacterial diseases emerging in Europe. Clin Microbiol Infect 2001;7:80–3. doi:10.1046/j.1469-0691.2001.00200.x. [83] De Sousa R, Luz T, Parreira P, Santos-Silva M, Bacellar F. Boutonneuse fever and climate
variability.
Ann
N
Acad
Sci
2006;1078:162–9.
AC C
EP
TE D
M AN U
SC
RI PT
doi:10.1196/annals.1374.029.
Y
34
ACCEPTED MANUSCRIPT
Conflict of Interest Statement
AC C
EP
TE D
M AN U
SC
RI PT
The authors declare that there are no conflicts of interest.
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