during july 2007july 2011 , six patients from the mediterranean coast city of elche , spain , who had high fever and inoculation eschars received a diagnosis of infection with r. sibirica mongolitimonae ( table ) . for laboratory confirmation
, dna was extracted from eschars , lymph nodes ( fine - needle aspiration ) , and blood samples by using the qiaamp tissue kit ( qiagen , hilden , germany ) , according to the manufacturer s instructions . for molecular detection , 200400 ng of dna from each sample
was subjected to pcr targeting the 23s-5s rrna intergenic spacer , followed by hybridization with specific probes by reverse line blotting , as described ( 15 ) .
when using the probe for r. sibirica mongolitimonae , a positive hybridization signal was obtained from eschar samples from all 6 patients ; this result was confirmed by sequencing ( 100% similarity to a reference r. sibirica mongolitimonae strain [ genbank accession no .
hq710799 ] in all cases in the 357 bp sequenced ) . to further confirm this result ,
nested pcr targeting the gene for outer membrane protein a was performed as described ( 15 ) ; these sequences ( 514 bp ) also showed 100% similarity to a reference r. sibirica mongolitimonae strain ( genbank accession no .
* na , not available ; ast , aspartate aminotransferase ; alt , alanine aminotransferase . 
serologic response was analyzed by using an in - house microimmunofluorescence assay for igg and igm , performed as described ( 4 ) ; r. conorii and r. sibirica mongolitimonae were used as antigens , and cutoff values were 1:40 for igg and 1:20 for igm .
acute- and convalescent - phase serum samples were obtained from 3 case - patients and single serum samples from the other 3 case - patients .
results for samples from 2 case - patients were negative , but results for the remaining 4 samples showed low to medium titers .
these results are consistent with previous reports ( 6 ) , in which 30% of cases had a positive igm result and 50% had negative or near - cutoff igg results .
all 6 case - patients lived in elche and its surroundings ( 230,112 inhabitants ) .
three of the cases occurred during the spring , which is when 10/18 cases reported in the literature occurred ( 414 ) .
all 6 case - patients had fever ( 38.5c39.5c ) , myalgia , and headache ; in the cases from the literature , 18/18 patients had fever , 13/18 myalgia , and 11/18 headache . in our study , 1 case - patient was confused and drowsy on arrival at the emergency department .  
all 6 case - patients had a single inoculation eschar develop : 2 on the neck , 2 on a lower limb ( figure ) , 1 on the scalp , and 1 on an upper limb .
five ( 83% ) case - patients had enlarged lymph nodes in the region from which the eschar drained , as reported for 10/18 ( 55% ) cases from the literature .
three case - patients ( 50% ) had lymphangitis extending from the eschar to the draining lymph nodes ( figure ) , compared with 6/18 ( 33% ) in cases from the literature .  
inoculation eschar on popliteal area and discrete maculopapular elements in patient with lymphangitis infected with rickettsia sibirica mongolitimonae , spain , 2011 .   for 4/6 ( 67% ) case - patients , a generalized maculopapular rash developed on the palms and soles but not the face ; for 2 case - patients
these findings are consistent with our review of the literature , which indicated rash occurring in 13/18 ( 72% ) cases .
all 6 case - patients recovered without sequelae after antimicrobial drug treatment using doxycycline or azithromycin .
fournier et al . ( 4 ) , who reported 7 cases of r. sibirica mongolitimonae infection in 2005 , proposed the name lymphangitis - associated rickettsiosis for the disease , on the basis of associated clinical features .
however , for the case - patients reported here , the most common clinical signs and symptoms were fever and skin eschar , similar to those from previously reported case series ; 5 of the case - patients reported here showed regional lymph node enlargement , 4 rash , and 3 lymphangitis . because only 24 total cases have been reported and other rickettsioses produce lymphadenopathy and lymphangitis , the term lympangitis - associated rickettsiosis may be unwarranted for this disease . in our case series , 1 case - patient had mental confusion after 10 days of a febrile disease before hospitalization and was found to be hyponatremic . in this patient , the eschar was located on the scalp , and neither rash nor other clinical clues were suggestive of rickettsiosis .
the patient had increased c - reactive protein plasma levels and the highest serologic antibody titers for r. sibirica mongolitimonae of the 6 case - patients ( table , patient 1 ) .  
since r. sibirica mongolitimonae was isolated from h. asiaticum ticks in 1991 ( 1 ) , it has been recovered from h. truncatum ticks in sub - saharan africa ( 6 ) and h. anatolicum excavatum ticks in greece ( 7 ) .
however , in spain , r. sibirica mongolitimonae has been detected in 2/8 tick species tested ( rhipicephalus pusillus and rh .
bursa ) at similar percentages ( 3.7% and 3.6% , respectively ) but not from hyalomma spp .
pusillus ticks from portugal was positive for r. sibirica mongolitimonae ( 8) , while testing of other tick species , including h. lusitanicum , rh .
( h. lusitanicum and h. marginatum ) ticks have not been found to be infected in spain does not mean that these ticks are not vectors for r. sibirica mongolitimonae .
ticks are a vector for this rickettsia on the iberian peninsula , and our findings confirm that this rickettsia species is circulating in spain , where specific vectors have yet to be described . in summary ,
we report 6 cases of human infection with r. sibirica mongolitimonae that occurred in the same geographic area of spain .
our results indicate that pcr of eschar samples is the most useful diagnostic procedure for this pathogen ; samples from all 6 case - patients had positive results , while test results for 1 whole blood sample and 2 lymph node samples were negative
. however , the limited number of samples does not make it possible to infer specific diagnostic sensitivities . the epidemiology and pathogenicity of illness caused by r. sibirica mongolitimonae infection require further investigation .
an active search for the vector of r. sibirica mongolitimonae in countries of the mediterranean region is necessary to complete the epidemiology of this rickettsiosis , which is likely to be more widespread than originally assumed . in particular
, clinicians caring for patients who have traveled to the mediterranean coast of spain should consider this rickettsiosis in the differential diagnosis .
 human infection with rickettsia sibirica mongolitimonae was initially reported in 1996 , and reports of a total of 18 cases have been published . 
 we describe 6 additional cases that occurred in the mediterranean coast region of spain during 20072011 . 
 clinicians should consider this infection in patients who have traveled to this area . 
The Study
Conclusions