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1 Advance Publication by J-STAGE Japanese Journal of Infectious Diseases A case of human infection by Rickettsia slovaca in Greece Vasiliki Kostopoulou, Dimosthenis Chochlakis, Chrysoula Kanta, Andromachi Katsanou, Konstantina Rossiou, Aidonis Rammos, Spyridon-Filippos Papadopoulos, Theodora Katsarou, Yannis Tselentis, Anna Psaroulaki, and Chrysostomos Boukas Received: April 28, Accepted: August 24, 2015 Published online: September 11, 2015 DOI: /yoken.JJID Advance Publication articles have been accepted by JJID but have not been copyedited or formatted for publication.

2 Title page A case of human infection by Rickettsia slovaca in Greece Vasiliki Kostopoulou 1, Dimosthenis Chochlakis 2, Chrysoula Kanta 1, Andromachi Katsanou 1, Konstantina Rossiou 1, Aidonis Rammos 1, Spyridon-Filippos Papadopoulos 1, Theodora Katsarou 1, Yannis Tselentis 3, Anna Psaroulaki 2,3, Chrysostomos Boukas st Department of Internal Medicine, Corfu General Hospital, Corfu, Greece 2 Regional Laboratory of Public Health of Crete, Greece 3 Laboratory of Clinical Bacteriology, Parasitology, Zoonoses and Geographical Medicine, School of Medicine, University of Crete Running title: Human infection by R. slovaca, Greece Keywords: Rickettsia slovaca, IFA, Greece Corresponding author: Dimosthenis Chochlakis, surreydimos@hotmail.com Regional Laboratory of Public Health of Crete School of Medicine University of Crete Greece Staurakia, PC Tel:

3 Summary Although tick-borne rickettsiosis is endemic in Greece, until recently only Rickettsia typhi and R. conorii were routinely tested in human samples arriving at the National Reference Centre under the suspicion of rickettsial infection. However, the identification of more than these two rickettsia species in ticks, urged towards the change of the so far followed protocol in 2010; up until that year all human samples received by the laboratory were tested for antibodies against R. conorii and R. typhi only. Since then, R. slovaca, R. felis, R. mongolotimonae were added into the routine analysis. Under the new scheme, R. slovaca is now routinely tested in human samples, hence the description of the current human case. A 42 year-old woman from the island of Corfu, Greece was admitted in November 2011 suffering from high grade fever (up to C) and malaise. The patient had visited a physician two days before admission and she was administered clarythromycin (500 mg twice daily) with no improvement. Routine blood tests revealed moderately elevated transaminases: AST: 116 IU/L (NR < 33 IU/L), ALT: 99 IU/L (NR < 31 IU/L), γgt: 143 IU/L (NR < 32 IU/L), ALP: 141 IU/L (NR < 125 IU/L), LDH: 532 IU/L (NR < 480 IU/L) (Table 1). The patient reported living in a rural area of the island but no contact with animals. A careful clinical examination did not reveal any signs of a tick-bite such as an eschar or lymphadenopathy, however, two days after admission she developed a maculopapular rash on the face, thorax, and extremities, involving also the palms and soles. She also complained of myalgias, arthralgias and a non productive cough while on clinical examination conjunctivitis was also noticed. A summary of palient s clinical data is shown in Table 2. New blood tests four days after admission showed mild leucopenia (WBC: 3.3 x 10 6 /L) and 2

4 further increase of transaminase levels (AST: 210 IU/L, ALT: 243 IU/L, γgt: 300 IU/L, ALP: 296 IU/L, LDH: 829 IU/L) (Table 1). The patient received doxycycline (100 mg twice daily for seven days) as soon as the rash developed, one week later she became afebrile and the rash faded but malaise still persisted. She returned home and recovered eventually within one month at which follow-up there was complete resolution of her symptoms and normalization of blood tests. Blood samples, whole and serum, were drawn ten days after hospitalization. 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). As confirmation, the ompa gene was also amplified by PCR (2). Furthermore, DNA was tested for Anaplasma phagocytophilum, Borrelia spp and Bartonella spp by Real-time PCR. Serum was tested by IFA for the presence of antibodies against Rickettsia spp using a slide that could test for R. conorii, R. siberica, R. slovaca, R. felis, R. massiliae and R. typhi as individual antigens (Fuller laboratories, California, USA). IFA was also conducted for antibodies against A. phagocytophilum (Focus Diagnostics, California, USA), B. henselae and B. quintana (Focus Diagnostics, California, USA). Antibodies against Borrelia spp were tested by Western Blot analysis (Mikrogen Diagnostik, Neuried, Germany). All tests were performed at the Regional Laboratory of Public Health of Crete, Greece. IgM antibodies against R. slovaca were detected (1/512); no antibodies were detected against any the rest of Rickettsia spp, Borrelia spp, Bartonella spp and A. phagocytophilum. Both the Real-time PCR and the PCR targeting the ompa gene of Rickettsia spp were positive. All Real-time PCRs for the rest of the pathogens were negative. The PCR product corresponding to the ompa gene was sequenced revealing 3

5 a 100% similarity to an already published sequence (U43808), which has also found to be identical to the strain detected in ticks in Northern Greece (3). Tick-borne rickettsiosis is endemic in Greece; various Rickettsia species have been identified in humans and ticks in the past ten years (4-10). Recently, Rickettsia slovaca was detected for the first time in infected ticks in Northern Greece (3). The pathogen was first isolated in 1968 in Dermacentor marginatus in Slovakia (11). The first proven case of infection was reported in France in 1997 (12). Within the last years, human infection by R. slovaca has been increasingly reported in France, Hungary, Spain, Italy, Germany, Bulgaria (13-16). Clinical signs of infection consist of a skin lesion (eschar) at the site of inoculation (usually on the scalp) and regional lymphadenopathy. This is a recently described syndrome called SENLAT (scalp eschar associated with neck lymphadenopathy after a tick bite) also known as TIBOLA/DEBONEL (tick-borne lymphadenopathy/dermacentor-borne necrosis, erythema, lymphadenopathy) caused often by R.slovaca and characterized as a local infection controlled by the immune system which is neither pathogen-specific nor vector-specific (17, 18). Fever and rash are not as common as in other Rickettsial infections. The clinical presentation in our case included high grade fever and a maculopapular rash whereas other main signs (eschar, lymphadenopathy) were missing. There are other reports of R.slovaca infections without skin lesions (19) or presenting a maculopapular rash (14) indicating that R.slovaca can be associated with other clinical forms. Moreover, our patient presented with moderately elevated transaminase levels indicating hepatic involvement in the course of infection. To our knowledge this is the first reported case of human infection by R. slovaca in Greece. This case is also associated with atypical clinical signs. Clinicians 4

6 should bear in mind the presence of other possible infections apart from the well known caused by R. conorii and R. typhi. Conflict of interest The authors declare no conflict of interest. References 1. Stenos J, Graves SR, Unsworth NB. A highly sensitive and specific real-time PCR assay for the detection of spotted fever and typhus group Rickettsiae. Am J Trop Med Hyg Dec;73(6): Regnery RL, Spruill CL, Plikaytis BD. Genotypic identification of rickettsiae and estimation of intraspecies sequence divergence for portions of two rickettsial genes. J Bacteriol Mar;173(5): Kachrimanidou M, Souliou E, Pavlidou V, Antoniadis A, Papa A. First detection of Rickettsia slovaca in Greece. Exp Appl Acarol Jan;50(1): Antoniou M, Economou I, Wang X, Psaroulaki A, Spyridaki I, Papadopoulos B, et al. Fourteen-year seroepidemiological study of zoonoses in a Greek village. Am J Trop Med Hyg Jan;66(1): Gikas A, Doukakis S, Pediaditis J, Kastanakis S, Psaroulaki A, Tselentis Y. Murine typhus in Greece: epidemiological, clinical, and therapeutic data from 83 cases. Trans R Soc Trop Med Hyg May-Jun;96(3): Gikas A, Kokkini S, Tsioutis C, Athenessopoulos D, Balomenaki E, Blasak S, et al. Murine typhus in children: clinical and laboratory features from 41 cases in Crete, Greece. Clin Microbiol Infect Dec;15 Suppl 2:

7 7. Psaroulaki A, Germanakis A, Gikas A, Scoulica E, Tselentis Y. Simultaneous detection of "Rickettsia mongolotimonae" in a patient and in a tick in Greece. J Clin Microbiol Jul;43(7): Psaroulaki A, Germanakis A, Gikas A, Scoulica E, Tselentis Y. First isolation and genotypic identification of Rickettsia conorii Malish 7 from a patient in Greece. Eur J Clin Microbiol Infect Dis Apr;24(4): Psaroulaki A, Ragiadakou D, Kouris G, Papadopoulos B, Chaniotis B, Tselentis Y. Ticks, tick-borne rickettsiae, and Coxiella burnetii in the Greek Island of Cephalonia. Ann N Y Acad Sci Oct;1078: Psaroulaki A, Spyridaki I, Ioannidis A, Babalis T, Gikas A, Tselentis Y. First isolation and identification of Rickettsia conorii from ticks collected in the region of Fokida in Central Greece. J Clin Microbiol Jul;41(7): Řeháček J. Rickettsia Slovaca, the Organism and Its Ecology: Academia; Raoult D, Berbis P, Roux V, Xu W, Maurin M. A new tick-transmitted disease due to Rickettsia slovaca. Lancet Jul 12;350(9071): Ibarra V, Oteo JA, Portillo A, Santibanez S, Blanco JR, Metola L, et al. Rickettsia slovaca infection: DEBONEL/TIBOLA. Ann N Y Acad Sci Oct;1078: Raoult D, Lakos A, Fenollar F, Beytout J, Brouqui P, Fournier PE. Spotless rickettsiosis caused by Rickettsia slovaca and associated with Dermacentor ticks. Clin Infect Dis May 15;34(10): Rieg S, Schmoldt S, Theilacker C, de With K, Wolfel S, Kern WV, et al. Tickborne lymphadenopathy (TIBOLA) acquired in Southwestern Germany. BMC Infect Dis. 2011;11:167. 6

8 16. Selmi M, Bertolotti L, Tomassone L, Mannelli A. Rickettsia slovaca in Dermacentor marginatus and tick-borne lymphadenopathy, Tuscany, Italy. Emerg Infect Dis May;14(5): Dubourg G, Socolovschi C, Del Giudice P, Fournier PE, Raoult D. Scalp eschar and neck lymphadenopathy after tick bite: an emerging syndrome with multiple causes. Eur J Clin Microbiol Infect Dis Aug;33(8): Foissac M, Socolovschi C, Raoult D. [Update on SENLAT syndrome: scalp eschar and neck lymph adenopathy after a tick bite]. Ann Dermatol Venereol Oct;140(10): Komitova R, Lakos A, Aleksandrov A, Christova I, Murdjeva M. A case of tick-transmitted lymphadenopathy in Bulgaria associated with Rickettsia slovaca. Scand J Infect Dis. 2003;35(3):213. 7

9 Symptoms and signs 3 days before admission On admission Table 1: Summary of patient s clinical data 2 nd day 6 th day 10 th day Fever Malaise Eschar Lymphadenopathy Conjunctivitis Maculopapular rash /- Myalgias /- Arthralgias /- Cough

10 Variable Reference On 3 nd day 6 th day 10 th day Range, Adults admission Hematocrit Hemoglobin White-cell count (per mm 3 ) 4,500-11,000 6,150 3,390 6,080 5,720 Differential count (%) Neutrophils Lymphocytes Monocytes Eosinophils Basophils Platelet count (per mm 3 ) 150, , , , , ,000 Erythrocyte sedimentation rate (mm/h) Sodium (mmol/lt) Potassium (mmol/lt) Urea nitrogen (mg/dl) Creatinine (mg/dl) Glucose (mg/dl) Alkaline phopsphatase (U/lt) Aspartate aminotransferase (U/lt) Alanine aminotransferase (U/lt) Lactate dehydrogenase (U/lt) Creatine kinase (U/lt) γ-glutanyltranferase (U/lt) Bilirubin (mg/dl) Total Direct Table 2: Summary of laboratory data

Detection and Identification of Rickettsia helvetica and Rickettsia sp. IRS3/IRS4 in Ixodes ricinus Ticks found on humans in Spain.

1 Title Detection and Identification of Rickettsia helvetica and Rickettsia sp. IRS3/IRS4 in Ixodes ricinus Ticks found on humans in Spain. Authors P. Fernández-Soto, R. Pérez-Sánchez, A. Encinas-Grandes,