in february 2001 , a 39-year - old man was hospitalized in marseilles , france , with fever and disorientation . this truck driver lived in portugal and had been driving in spain for 2 weeks before entering france . the day of admission , his co - workers found him confused and febrile after sleeping in his truck . his temperature was 40c with tachycardia , and his blood pressure was 120/80 mm hg . clinical examination showed a drowsy , confused man with a bilateral headache , provoked myalgia of the legs , hepatalgia , and conjunctivitis . neurologic examination showed signs of meningeal irritation , including cervical rigidity , brudzinski 's sign , hyperesthesia , and photophobia . g / l ) , and c - reactive protein ( 273 mg / l ) , associated with hypoglycemia ( 2.9 mmol / l ) , hypoalbuminemia ( 26 the kaolin cephalin time was 58 s ( control 34 s ) , and the prothrombin rate was 42% . analysis of the csf on day 1 showed 4 leukocytes / mm , 50,000 erythrocytes / mm , and elevated protein levels ( 1.48 another csf analysis showed 75 leukocytes / mm , with 70% polymorphonuclear forms , 5,500 erythrocytes / mm , and increased protein ( 0.64 g / l ) . direct examination by dark - field microscopy of the csf from day 2 was performed and controlled by two experimental investigators , who observed many spirochetes ( 6 ) . the patient had been traveling recently in spain and portugal but had no apparent exposure to sources of leptospires . single 0.1 ml- and 0.01 ml - aliquots of lithium - heparin anticoagulated whole blood were spread onto 10 ml of leptospira medium , a polysorbate medium similar to ellinghausen and mccullough modified johnson and harris medium ( emjh ) ( bio - rad laboratories , inc . , aulnay / bois , france ) , and was incubated at 30c . the same procedure was carried out for csf . for urine samples , the specimens were first filtered on 0.45- and then 0.22-m filters before inoculation . once a week , 10 l of culture medium was examined by dark - field microscopy . cultures from both blood and csf were positive and yielded leptospira after 1 week of incubation . no agglutination of the isolated strain was obtained with any reference sera , except a weak agglutination ( titer 400 ) with serovar hurstbridge antiserum . the genomic dna of the spirochete was extracted from the blood culture by the qiamp blood kit procedure ( qiagen gmbh , hilden , germany ) . for polymerase chain reaction ( pcr ) , universal 16s rdna primers fd1 and rp2 ( 7 ) were used , and sequences of the pcr products were obtained as previously described ( 8) . the 16s rdna sequence of the isolate had 100% identity with the prototype strain sequence of l. fainei ( genbank accession no pulsed - field gel electrophoresis of noti macrorestriction fragments of leptospira dnas was performed as previously described ( 9 ) . however , the noti macrorestriction pattern was quite different from all patterns previously recorded for a large collection of leptospira strains belonging to different species and serovars ( figure 2 ) . sera collected on days 4 , 8 , 10 , and 45 were tested by a microimmunofluorescent antibody assay ( mifa ) with l. biflexa serovar patoc , l. fainei , l. interrogans , and own strain as antigens ( cut - off titer 200 ) ; western immunoblot with l. biflexa patoc and l. fainei as antigens ; and a microagglutination test ( mat ) ( 10,11 ) . for mat , sera were incubated with suspensions of live leptospires belonging to 17 distinct serogroups , including a strain of l. fainei serovar hurstbridge . the presence of immunoglobulin ( ig ) m was investigated by an enzyme - linked immunosorbent assay ( elisa ) developed at the institut pasteur , with serovar patoc as an antigen ( 12 ) . all sera were negative by mifa , mat , and igm elisa , even when l. fainei was used as an antigen . for western blot , the spirochetes were grown on leptospira medium centrifuged and used at a concentration of 2 mg / ml . after blocking , the first two sera were negative by western blot , but a reaction with l. fainei was observed in igg and igm at days 10 and 45 . sera reacted with protein bands of 28 , 25 , 24 , and 19 kda from l. fainei , while no reaction was obtained with serovar patoc ( figure 3 ) . the tree was derived from a 1,295-bp fragment of the 16s rrna gene and was constructed by the neighbor - joining method . bootstrap values , expressed as a percentage of 1,000 replications , are given at the branching point . noti restriction patterns of leptospira strains obtained with the bio - rad apparatus ( richmond , ca ) with a pulse time ramped from 5 to 90 s for 36 h. the lanes contain lambda concatemers ( lanes 1 and 10 ) and dna from isolates : patient strain ( lane 2 ) ; l. fainei hurstbridge , strain but 6 ( lane 3 ) ; l. inadai lyme , strain 10 ( lane 4 ) ; l. inadai biflexa , strain lt430 ( lane 5 ) ; l. biflexa patoc , strain patoc i ( lane 6 ) ; l. meyeri semaranga , strain vs173 ( lane 7 ) ; l. kirschneri grippotyphosa , strain moskvav ( lane 8) ; and l. interrogans icterohaemorrhagiae , strain verdun ( lane 9 ) . 1 ) and convalescent - phase ( no . 2 ) sera on leptospira serovar patoc and l. fainei . mwm indicates molecular weight markers . this clinical picture was highly suggestive of leptospirosis , with the association of meningeal syndrome , provoked leg myalgias , and conjunctivitis ; nonspecific laboratory findings included hepatic enzyme elevation , hyperleukocytosis , thrombocytopenia , and low prothrombin rate ( 1 ) . the isolated leptospira was identified as closely related to l. fainei on the basis of 16rrna amplification and sequencing . the strain has been isolated in our laboratory in marseille , where this species had never been cultivated . thus , laboratory contamination is unlikely , and the isolated strain of l. fainei , which is close to the serovar hurstbridge , can be considered the causative agent of the patient s meningitis . moreover , a spirochete resembling a leptospire was seen by well - trained investigators at the dark - field microscope . l. fainei has been only recently been considered an emerging pathogen . in australia , a sample of 723 human sera from patients from a dairy and pig - producing area of victoria , all of whom had symptoms consistent with leptospirosis , was submitted for leptospirosis serologic testing . furthermore , a leptospirosis surveillance program was conducted for 12 months on the entire population of the seychelles ; the incidence of leptospirosis was 10/100,000 , with a 20 % seroprevalence of l. fainei . this organism was suspected to be involved in severe forms such as acute renal failure , pulmonary hemorrhage , and possibly death ( 4 ) . the first human isolation of l. fainei occurred in denmark , from two patients ( 5 ) . the first patient had chronic disease with increasing jaundice for 6 months before admission to the hospital , and test results showed elevated hepatic enzymes . the second patient had abdominal and lower back pain for 5 months and severe headaches and dizziness for 2 months before admission . thus , both patients had atypical chronic disease , unlike the typical case of leptospirosis that we describe . in our case , we tested sera 4 , 8 , 10 , and 45 days after onset of illness . no reactivity was detected by mif and mat tests , although a reaction in igg and igm at day 10 and 45 was detected by western blot . the two danish patients from whom l. fainei was isolated were tested by mat and showed no substantial serologic reaction ( 5 ) . however , published serologic studies have shown that an immunologic response may be observed , in some cases at a high level ( 4 ) . as reported here antibiotic treatment by amoxicillin ( 12 g / day for 10 days ) was effective . in the seychelles , for 8 patients among 75 with confirmed leptospirosis , a 5-day treatment with penicillin was insufficient to eradicate the bacteria , as indicated by positive pcr results ( 4 ) . treatment for longer than 1 week may be necessary , because l. fainei could be still isolated from danish patients after a 1-week treatment with intravenous penicillin , but not after 4 weeks of amoxicillin treatment ( 3 ) . this case confirms the pathogenic role of l. fainei in humans and extends its geographic distribution to southern europe . the clinical finding of our case did not differ from that of other leptospira infections , but the route of exposure remains unknown . although the usual procedures of direct detection , isolation , and identification of leptospires are effective for l. fainei , we did not observe substantial serologic reactivity . further studies , including western blot , are needed to explain the weak immune response and to evaluate the prevalence of infection by this newly discovered leptospira species .
we report a human case of leptospirosis in which the spirochete was detected by dark - field microscopy examination of cerebrospinal fluid ( csf ) and isolated from both csf and blood . 
 leptospira fainei was identified by sequencing the 16s rdna gene , which had been amplified by polymerase chain reaction . 
 this case confirms the role of l. fainei as a human pathogen and extends its distribution to southern europe .