Rickettsia monacensis and Human Disease, Spain

We identified Rickettsia monacensis as a cause of acute tickborne rickettsiosis in 2 humans. Its pathogenic role was assessed by culture and detection of the organism in patients’ blood samples. This finding increases the number of recognized human rickettsial pathogens and expands the known geographic distribution of Mediterranean spotted fever–like cases.


The Study
Patient 1 was an 84-year-old man from La Rioja, who sought medical attention on June 19, 2003, 7 days after onset of fever (39.5ºC), general discomfort, headache, and joint pain. At the time of the physical examination, he had a nonpruritic, disseminated maculopapular rash, with no inoculation eschar, of the trunk and lower extremities, including palms and soles. Other than a slightly low platelet count (82,000/mm 3 ), examination fi ndings were within normal limits. MSF was diagnosed, and serum and defibrinated blood samples were taken before a course of oral doxycycline (100 mg/12 h for 10 d) was initiated. Three days later, fever and rash were gone without sequelae. Additional serial serum samples were taken during weeks 4, 13, and 26 after onset and reserved for serologic analysis (Table).
Patient 2 was a 59-year-old woman from Basque Country, who sought medical attention on September 20, 2003, 4 days after onset of fever (38ºC), headache, and an erythematous rash, with no inoculation eschar, at the site of a tick bite. The patient reported a history of tick bites, most recently 1 week before symptom onset. Blood cell counts and other blood chemistry values were normal. MSF was diagnosed, and oral doxycycline (100 mg/12 h for 10 d) was prescribed. Serial serum samples were taken the day of the visit and weeks 4 and 6 after onset and were reserved for serologic analysis (Table). Defi brinated blood was also taken 2 days after treatment was initiated. The patient recovered without sequelae.
DNA was extracted with the QIAGEN Tissue kit (IZASA S.A., Barcelona, Spain), and an ompA-nested PCR was designed. The fi rst set of primers (Rr190.70p and Rr190.602n) have been described (10). Those used for the nested amplifi cation were designed in this study: Nom-pA-F (5′-AGC GAT AAT GCT GAG TAG TAG-3′) and NompA-R (5′-TAT ATT TCC TAA ACC TGT ATA A-3′) nucleotide positions 150-170 and 576-555, respectively, were numbered according to Regnery et al. (10). Amplification conditions were as described, except annealing temperature was 40ºC for the second PCR and AmpliTaq Gold DNA Polymerase (Applied Biosystems, Branchburg, NJ, USA) was used. The specifi city of the method was tested against DNA obtained from Vero cells and Coxiella burnetii, and fragments of the expected sizes (532 and 427 bp) were obtained from different rickettsia species (data not shown). The amplicons obtained from blood samples were run in 1% low-melt agarose gels (Pronadisa, Barcelona, Spain), and the bands of interest were excised, purifi ed with QIAquick Gel Extraction kit (IZASA S.A), and sequenced as described (9).
Blood samples from each patient were cultured by using shell vial technique (11). Giménez stain and PCR, performed after 7 days of incubation, confi rmed the growth of a Rickettsia-like organism (strain Rp-Sp1) from patient 1. The sequences of ompA and gltA of this isolate (GenBank accession nos. DQ157778 and DQ517498, respectively) were identical to those obtained from the blood samples of each patient and to that of R. monacensis (6) (GenBank accession nos. AF201329 and DQ100163). The sequences generated in this study were subjected to phylogenetic analyses as described (9) and belonged to the same clade as R. monacensis and other related strains that have been detected in I. ricinus (3,4,12) (Figure).
In-house microimmunofl uorescence assay (IFA) ([1] and references therein) that used R. monacensis, R. conorii, R. helvetica, R. akari, and R. australis as antigens was performed in serial serum samples from each patient (Table). The isolate Rp-Sp1 from patient 1 could not be used as antigen because of poor adaptation of this isolate to culture in Vero cell monolayers; R. monacensis slides for IFA were obtained from the Department of Entomology, University of Minnesota, Minneapolis, MN, USA. Seroconversion against the 5 rickettsia species was observed from patient 1's second serum sample (day 30 after the onset). Patient 2's fi rst serum sample also had high titers against the 5 antigens. Although the reactivity against the 5 rickettsial antigens was similar, the titers observed were slightly higher against R. monacensis and R. helvetica, which are phylogenetically closer to each other than to the other species tested. However, because the serologic results may only loosely implicate a given rickettsia species, isolation of R. monacensis from patient 1 and its detection by PCR for both patients confi rm it as the etiologic agent.

Conclusions
We describe a new, to our knowledge, rickettsia species that caused human disease. R. monacensis was the etiologic agent of MSF-like illness in northern Spain. Strain Rp-Sp1 was obtained from 1 patient. Because the sequences of ompA and gltA were identical to this rickettsia species and also amplifi ed from blood samples of each patient studied, we conclude that this rickettsia is responsible for the symptoms observed in these patients. Therefore, R. monacensis joins the list of autochthonous rickettsia species (R. conorii [13], R. slovaca [14], R. typhi [15]) confi rmed as human pathogens in Spain.
We were not able to study the vectors involved; however, each patient contracted the disease in areas where I. ricinus is the most prevalent tick species (9), and strains close to R. monacensis have been recently detected in I. ricinus in Spain (2,12). Thus, I. ricinus may eventually be shown to be the vector. Studies of R. monacensis incidence in autochthonous I. ricinus specimens are in progress to evaluate the risk of its transmission to humans.