Advance Publication by J-STAGE

Japanese Journal of Infectious Diseases

Possible Rickettsia massiliae infection in Greece: an imported case

Dimosthenis Chochlakis, Christine Bongiorni, Nikolaos Partalis, Yannis Tselentis, and Anna Psaroulaki

Received: April 28, 2015. Accepted: July 28, 2015 Published online: September 11, 2015 DOI: 10.7883/yoken.JJID.2015.195

Advance Publication articles have been accepted by JJID but have not been copyedited or formatted for publication.

Possible Rickettsia massiliae infection in Greece: an imported case.

Dimosthenis Chochlakis1, Christine Bongiorni2, Nikolaos Partalis3, Yannis Tselentis4, Anna

Psaroulaki1,4

1Regional Laboratory of Public Health, Heraklion-Crete, Greece

2Health Centre of Vamos, Chania, Crete, Greece

3A’ Unit of Internal Medicine, General Hospital of Chania, Chania, Crete, Greece

4Laboratory of Clinical Bacteriology, Parasitology, Zoonoses and Geographical Medicine,

Medical School, University of Crete, Heraklion, Greece.

Running title: Rickettsia massiliae infection.

Keywords: Rickettsia massiliae, Spotted Fever Group, Greece

Corresponding author: Dimosthenis Chochlakis, e-mail: [email protected]

Regional Laboratory of Public Health of Crete

School of Medicine

University of Crete – Greece

Staurakia, PC 71110 Tel: 00302810394624Accepted Manuscript

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Summary

Tick-borne rickettsioses are endemic in Greece, however until recently only Rickettsia typhi and

R. conorii were routinely tested in human samples arriving at the National Reference Centre.

During the last years, the identification of different rickettsia species in ticks led to the introduction of other spotted fever group rickettsiae in the routine analysis. Under the new scheme, R. massiliae is now routinely tested in human samples, hence the description of the current human case.

A 62 year old English man, permanent resident during the last years of Crete, was presented at the end of July of 2013 at the Health Centre of a small village in Chania suffering from headache, severe myalgia and weakness. No visual disturbance was observed. As he reported the symptoms began at the beginning of July and got gradually worsen with impact on daily activities. Since then, he suffered from daily fevers up to 38oC for which he was getting ibuprofen, with temporary improvement of the condition.

The patient reported a tick-bite on the 15th of June during camping at the Exmoor National Park in south west England; the removal of the tick from the arm proved to be difficult according to his report. He attributed the tick-bite to the presence of numerous deer at that part, with which he stated having close contact. The clinical examinationAccepted revealed an eschar at theManuscript arm, of about 5mm in diameter, and a macular rash of upper extremities. As regards the patient’s clinical signs, grade fever was recorded together with normal pulse (65 beats/minute) and respiratory rate (14 breaths/minute). No hepatomegaly, spleenomegaly or lymph enlargement was recorded. Laboratory evaluation showed a slight lymphopenia (White Blood Cells: 7.37, Lymphocytes: 1.39), while C-reactive 2 protein (0.28), Erythrocyte sedimentation Rate (11 mm/h) were within normal range and the rest of the tests did not reveal any worth mentioning findings.

He was started on doxycyclin 100mg twice per day at the 9th of August and he became afebrile within 48 hours. However, seven days later the patient was feverish, but myalgia still persisted.

He was therefore hospitalized at the General Hospital of Chania where ofloxacin was added on the antibiotic regimen and therapy was continued for a further week. Fifteen days past the end of treatment the patient showed a remarkable improvement with the exception of myalgias, which still persisted and for which he was prescribed painkillers.

Two serum and whole blood samples were drawn, the first at presentation at the Health Centre and the second one month later. No eschar was available at the time of clinical inspection. All samples were sent at the Regional Laboratory of Public Health of Crete - Laboratory of Clinical

Bacteriology, Parasitology, Zoonoses and Geographical Medicine, University of Crete for further testing. DNA was extracted by using the QIAamp DNA blood mini Kit (QIAGEN, Hilden,

Germany) according to the manufacturer’s instructions. Real-time PCR targeting the glta gene of

Rickettsia spp was performed as previously described (1). Immunoglobulin (Ig)G and IgM antibody titers were determined for the presence of antibodies against Rickettsia spp using a slide that could test against R. conorii, R. siberica mongolotimonae, R. slovaca, R. felis, R. massiliae and R. typhi as individual antigens (Fuller laboratories, California, USA), by an immunofluorescence antibody assay (IFA) that is based on the targeting of the rLPS. At a cut-off Acceptedof IgG ≥ 1/480 and IgM ≥ 1/200, Manuscript diagnostic titers only against R. massiliae were recorded for the first (IgG 1/3840, IgM -) and second (IgG 1/480, IgM -) serum samples. The absence of IgM antibodies may be due to the delayed presentation of patient at the Health Centre

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(almost a month past the tick bite). The titers revealed for each of the Rickettsia species tested, are given in Table 1.

Furthermore, DNA was also tested for Anaplasma species, by Real-time PCR and for antibodies against A. phagocytophilum (Focus Diagnostics, California, USA) by IFA; none of the PCR amplifications was positive either for Rickettsia or for Anaplasma species, while IFA against A. phagocytophilum gave negative results as well.

Rickettsia massiliae is a spotted fever group rickettsia first described in 1993 (2), while its first isolation from human dates back to 1985 although it was fully identified in 2005 (3). It is mostly associated with ticks of the Rhipicephalus species, while lately there have been reports for the presence of the pathogen in Ixodid tick species (4) and possibly in Haemaphysalis punctata (5).

The impact presence of the pathogen in these later species in terms of human infection needs to be clarified, although both these species are known as capable of biting humans as well.

To date, four cases of R. massiliae infection in humans have been described, three of which were reported in Europe (3, 6, 7) and one in Argentina (8). In Greece, three Rickettsia species of the

Spotted Fever Group have been implicated in human disease, R. conorii (9), R. sibirica mongolotimonae (10) and R. aeschlimanii (11) so far.

Contrary to the case described herein, all four cases (Table 2) (3, 6-8) described so far have been diagnosed by molecular means. It should be pointed out that whenever diagnosis is based on IFA, the possibilityAccepted of cross-reactions among species Manuscript should be taken under consideration; that is why we cannot definitely exclude the possibility of an infection from a different species in our case.

In such cases the use of Western blotting could be proven of great assistance in order to come to a definitive outcome at the species level, which is important especially in epidemiological studies.

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All patients recorded tick bite, as was the case in our patient as well. Although we did not manage to collect ticks from the Exmoor National Park, in the past there has been a study investigating the presence of Ixodes ricinus at the surrounding area of the Park (12).

Although R. massiliae has been detected in Rhipicephalus species in the past in Greece (13, 14), the current case perhaps corresponds to an imported one in Crete; nevertheless, it still counts for the 5th bibliographically described case.

Introducing the testing of more SFG Rickettsiae in routine diagnosis may prove of great usefulness where PCR may not be available and/or conclusive. In any case, the possible limitation of potential cross-reactions among different Rickettsia species may be overcome through the use of Western blotting, wherever of course this is possible.

Conflict of interest

The authors declare no conflict of interest.

Accepted Manuscript

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Rickettsia species Antibodies R. R. sibirica R. R. R. typhi R. felis tested conorii mongolotimonae massiliae slovaca IgG (1/) 240 240 480 960 3840 240 1st IgM (1/) ------sample IgG (1/) 60 - - - 480 120 2nd IgM (1/) ------sample

Table 1: Antibody titers regarding G and M immunoglobulins tested against Rickettsia species by indirect immunofluorescence. “-” indicates a negative result.

Accepted Manuscript Disturbed Month/ Night Head- Necrotic Vision Occipital Hepato- Lymph- Country oC Chills Malaise Myalgia Rash hepatic Therapy Ref. year sweats ache eschar loss swelling megaly adenopathy enzymes June/ Maculopapular (palms; Tetracycline Italy y Right ankle Slight 3 1985 soles) (13d) July/ Palpable purpuric Doxycycline, Argentina y y y Tache noir y 8 2005 (trunk; palms; soles) prednisolone Doxycycline, Bilateral May/ Maculopapular (palms; Buttocks, ofloxacin, France y y y chorio- 7 2007 soles) thighs methyl- retinitis prednisolone May/ Italy y y Scalp y y Doxycycline 6 2012 Ibuprofen, July/ Macular (upper Greece* y y y y Arm Doxycycline, 2013 extremities) ofloxacin Table 2: Clinical characteristics of the human cases due to R. massiliae infection described so far. *: probable imported case from the

United Kingdom. y: present.

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