this study was based at the armed forces research institute of medical sciences ( afrims)kwai river christian hospital clinical center , sangkhlaburi district , kanchanaburi province , thailand .
( the protocol was approved by the human subjects research review board of the u.s .
army , ethical review committee for research in human subjects of the thai ministry of public health , and scientific review committee of afrims . )
patients were selected from those enrolled and sampled from june 1999 to february 2002 in an on - going  fever study ,  which focuses on the etiology of undifferentiated febrile illnesses ( oral temperature > 38c or history of fever within the past 48 h ) in local residents > 20 years of age .
criteria leading to the suspicion of rickettsioses included 1 ) a rash or eschar , 2 ) arthropod bites or recent exposure to the jungle , 3 ) a negative giemsa - stained malaria smear , and 4 ) serum specimens that tested positive by enzyme - linked immunosorbent assay ( elisa ) for sfg  specific immunoglobulin ( ig ) m ( panbio , brisbane , australia ) or dot - elisa for total ig of r. rickettsii or r. typhi ( panbio - indx , baltimore , md ) .
serum specimens were sent to the unit des rickettsies in marseille for specific diagnosis of rickettsioses .
serologic testing was performed by indirect immunofluorescence ( if ) on acute - phase ( day 0 ) and convalescent - phase ( approximately day 21 ) samples .
serum specimens were tested by using a panel of 13 rickettsial antigens , including sfg rickettsiae ( r. conorii indian , r. japonica , r. honei , r. helvetica , r. slovaca , at1 rickettsia , r. felis ,  r. heilongjiangii  ) typhus group rickettsiae ( r. typhi ) , orientia tsutsugamushi ( strain gilliam , kato , karp , and kawazaki ) , anaplasma phagocytophilum , ehrlichia chaffeensis , and coxiella burnetii .
the rationale for the antigen screening panel included the presence of the strains in asia and results of previous serosurveys for a. phagocytophilum and e. chaffeensis .
the standard procedure was followed for the use of western blot and cross - adsorption studies to complete the if assay at the unit des rickettsies ( 14,15 ) .
an immunofluorescence assay was considered positive for 1 ) igg with titers > 128 and/or igm titers > 64 for r. conorii ; and 2 ) for igg titers > 64 and/or igm titers > 32 for other rickettsial antigens .
when cross - reactions were noted between several rickettsial antigens , the standard procedure comprised three steps : 1 ) a rickettsial antigen was considered to represent the agent of infection when titers of igg and/or igm antibody against this antigen were at least two serial dilution higher than titers of igg and/or igm antibody against other rickettsial antigens .
2 ) when the difference in titers between several antigens was lower than two dilutions , western blot assays were performed .
a rickettsial antigen was considered to represent the agent of the infection when acute - phase or convalescent - phase sera reacted only with the specific proteins of this antigen .
3 ) when western blot assays were not diagnostic , cross - absorption studies were performed : igg / igm titers had to be > 128/32 .
specific diagnosis criteria after cross - absorption studies included a ) if serologic test results positive for a single antigen or b ) a western blot assay showing an exclusive reactivity with specific proteins of a sole agent . from june 1999 to february 2002 , 46 patients were selected to be specifically tested for rickettsioses .
these 46 patients were empirically treated by a 7-day doxycycline regimen ( 200 mg / d ) .
rickettsioses were serologically confirmed in 15 ( 33% ) patients by evidence of seroconversion , igm at significant titers , or both .
serum specimens from patients 1 and 3 provided the highest titers against gilliam and karp strains , and serum from patient 2 had titers against gilliam strain only .
two of these patients had returned from a trip into the jungle , and the third became sick several days after cutting grass in the fields .
one patient was initially thought to have bacterial meningitis and had been treated unsuccessfully by a broad - spectrum third - generation cephalosporin for 3 days before doxycycline was started .
one patient ( no.13 ) had an eschar at a tick bite site , and another had a rash ( no.15 ) .
one patient ( no . 8) with sfg rickettsioses seroconverted to r. felis , indicated by high level of antibody titers .
further , although igg titers were more than two serial dilutions higher than those for r. typhi , western blot assay was performed to confirm if findings .
two patients ( nos . 9 and 10 ) were shown to have the highest titers to r. conorii strain indian .
1115 ) had the highest titers to r. helvetica . for patients 4 , 9 , 10 , 13 , and 15 , if results showed differences lower than two dilutions in igg titers , igm titers , or both , between several antigens .
thus , if assays were completed by western blot and with cross - absorption studies for patient 4 ( table ) .
no cases of infection due to c. burnetii or ehrlichioses were diagnosed in the 46 tested patients .
abbreviations : a. , arthropod ; ig , immunoglobulin ; r , rash ; e , eschar ; n , nodes ; rh , rickettsia helvetica ; rc , r. conorii indian ; rjap , r. japonica ; rhon , r. honei ; rslo , r. slovaca ; at1 , rickettsia strain at1 from japan ; rheil ,  r. heilongjiangii  ; rfel , r. felis ; g , strain gilliam ; kw , strain kawazaki ; kp , strain karp ; ko , strain kato . 
immunofluorescence assay was completed by western blot and cross - adsorption as described in the text . for typhus and
spotted fever group antigens , the rickettsia considered potentially responsible for the infection are in bold type . 
in this study , we report rickettsioses in sangkhlaburi , including the first case of r. felis infection reported in asia .
r. felis was likely first detected ( as r. ctenocephali ) in european cat fleas ( ctenocephalides felis ) in 1918 ( 16 ) , then rediscovered in 1990 in the united states ( 17 ) .
r. felis was then cultivated and characterized as a unique sfg rickettsia ( 18 ) .
its pathogenic role was recently demonstrated in patients with serologic evidence of infection in brazil , france , and germany .
r. felis dna has also been detected in sera in texas , mexico , brazil , and germany ( 19 ) .
this rickettsia has also been recently detected in fleas in brazil , africa , spain , and france ( 20 ) .
further , during an entomologic survey , r. felis  like rickettsiae were detected in fleas collected in sanghklaburi ( p. parola , unpub .
murine typhus , a mild disease with nonspecific signs ( 21 ) , was found in four of our patients .
although this disease has a worldwide distribution , it is often unrecognised , and documented cases are rarely reported .
the classic triad of fever , headache , and skin rash is observed in < 15% of cases ( 22 ) .
arthralgia , myalgia , and respiratory and gastrointestinal symptoms ( as demonstrated by one of our patients ) are frequent ( 21,22 ) .
regarding disease transmission , although rats and mice are very common within and around houses in the villages , our patients did not report contact with rat fleas or a flea bite . in this study ,
seven patients with sfg rickettsioses may have been infected by r. helvetica ( five patients ) or r. conorii indian strain ( two patients ) , according to if assays completed for some cases by western blot and cross - adsorption studies .
r. helvetica is an emerging pathogen known to be prevalent in europe ( 23 ) and japan ( 13 ) . in both areas ,
r. helvetica is associated with ixodes ticks , which are also found in thailand , although they have not previously been reported in sangkhlaburi ( 24 ) .
r. conorii indian is known as an agent of tick - borne rickettsioses prevalent in india , where it is associated with the dog tick ( rhipicephalus sanguineus ) ( 25 ) , which is found worldwide .
however , an unknown rickettsia sp . that is cross - reactive with r. conorii indian and r. helvetica could also be responsible for the cases reported here .
in particular , we have recently detected , by polymerase chain reaction , rickettsia spp . from ticks that have bitten people in the sangkhlaburi area , including dermacentor auratus and dermacentor sp .
larvae ( 9 ) . the pathogenic role of these rickettsiae has yet to be demonstrated .
scrub typhus is essentially an occupational disease among rural residents in the asia - pacific region ( 2 ) .
this disease is often underdiagnosed or misdiagnosed when the classic eschar at the chigger bite sites and the rash are absent , as reported for two of our three patients ( 2 ) .
the severity of the disease varies from asymptomatic to fatal ( up to 30% ) . delayed or inappropriate treatment such as with third - generation cephalosporins , as reported for one of our patients ,
the four major serotypes studied here have been shown to have sufficient cross - reactivity with antigens from other strains to be used for serologic diagnostic testing . in our patients , although the highest titers were obtained by using o. tsutsugamushi strain gilliam antigens , other strains that share common epitopes and cross - react with this strain could be involved .
patients with rickettsioses may have isolated fever or fever with nonspecific clinical and laboratory findings .
these diseases are easily misdiagnosed because rash or eschar ( the hallmark for rickettsial diseases ) is absent , the diseases are not recognized by local physicians , or the diseases have never been reported in the area .
more studies are needed on tropical rickettsioses , in particular , molecular detection or rickettsial isolation from patient samples , complemented by detailed case reports .
studying possible vectors and animal reservoirs would provide estimates of the degree of zoonotic potential .
ultimately , such studies will provide the basis for determining prevalence of rickettsiosis in the tropics and their effects on public health .
 to investigate the presence of rickettsioses in rural residents of the central thai - myanmar border , we tested the blood of 46 patients with fever . 
 four patients had murine typhus , three patients had scrub typhus , and eight patients had spotted fever group rickettsioses , including the first case of rickettsia felis infection reported in asia . 
The Study
Conclusions