from march through november 2015 , at the people s liberation army 154 hospital in xinyang city , henan province , china , patients who were acutely symptomatic with fever and had a history of tick bites or animal contact within the past month were screened for sfg rickettsiae infection . at admission ,
dna was extracted by using a qiaamp dna blood mini kit ( qiagen , germantown , md , usa ) .
nested pcrs selective for outer membrane protein a ( ompa ) and citrate synthase ( glta ) genes were concurrently performed to detect sfg rickettsial dna ( technical appendix table 1 ) .
positive amplicons were purified and then sequenced in both directions . acute - phase ( < 7 days after illness onset ) and convalescent - phase ( > 14 days after illness onset ) serum samples were tested by indirect immunofluorescence assay ( ifa ) for igg against r. rickettsii by using a commercially available ifa kit ( focus diagnostics inc . , cypress , ca , usa ) .
positive amplification of ompa and glta genes was found for 5 patients , and the obtained sequences for each of the 2 genes from all 5 patients were identical .
ku853020 ) from each of the 5 patients showed 10-bp differences from that of r. massiliae strain azt80 ( genbank accession no .
cp003319 ) and 12-bp differences from that of r. rhipicephali strain hj#5 ( genbank accession no .
ku853022 ) from each of the 5 patients differed from that of r. massiliae strain azt80 by 4 bp and from that of r. rhipicephali strain hj#5 by 5 bp ( technical appendix table 2 ) . according to phylogenetic analysis , the novel sfg rickettsiae genotype , here designated as rickettsia sp .
xy99 , seems to represent a distinct lineage and could constitute a new species ( figure 1 ) .
for all 5 patients , seroconversion or a 4-fold increase of igg against r. rickettsii was found between the acute- and convalescent - phase samples , and the patients were determined to have acute infection with sfg rickettsiae ( technical appendix table 3 ) .
subsequent testing of the 5 patients for infection with severe fever with thrombocytopenia syndrome virus , anaplasma phagocytophilum , 
a. capra ,  and babesia microti by molecular ( real - time pcr or nested pcr ) and serologic tests ( elisa or ifa ) produced no positive results .
phylogenetic analyses based on nucleotide sequences of the outer member protein a ( 307-bp ) ( a ) and citrate synthase ( 1,150-bp ) ( b ) genes of rickettsia .
asterisks after taxon names indicate that the sequence of rickettsia species was found in china .
neighbor - joining trees were conducted by using the maximum composite likelihood method by means of mega version 5.0 ( http://www.megasoftware.net ) .
bootstrap analysis of 1,000 replicates was applied to assess the reliability of the reconstructed phylogenies .
patient median age was 65 ( range 6280 ) years , and 3 were male ( table ) .
two patients had a history of tick exposure , and the other 3 had had contact with livestock .
the median time from illness onset to admission was 4 ( range 36 ) days , and the median duration of hospitalization was 10 ( range 812 ) days . all patients experienced fever ( highest 38.4c 40.0c ) , asthenia , anorexia , and nausea ; 4 had cough , 3 vomiting , 2 myalgia , 1 headache , and 1 dizziness . of note , all 5 patients had lymphadenopathy , but none had rash or eschar . at admission , all 5 patients had leukopenia , thrombocytopenia , and elevated hepatic aminotransferase levels ; 4 had elevated lactate dehydrogenase levels , and 2 had elevated creatine kinase levels ( figure 2 ) .
dynamic changes of 6 laboratory parameters ( with 2-day intervals ) during hospitalization of 5 patients with rickettsia sp . xy99 infection , china , 2015 .
red , patient 1 ; yellow , patient 2 ; green , patient 3 ; purple , patient 4 ; gray , patient 5 .
alt , alanine aminotransferase , reference range 040 u / l ; ast , aspartate aminotransferase , reference range 040 u / l ; ck , creatine kinase , reference range 25200 u / l ; ldh , lactate dehydrogenase , reference range 109245 u / l ; platelets , reference range 100300  10/l ; leukocytes , reference range 4.010.5
complications included pneumonia ( 3 patients ) , hemorrhagic signs ( 3 ) , hydrothorax ( 2 ) , and neurologic syndromes ( 1 ) . for 1 patient ,
severe complications progressively emerged 6 days after disease onset and included pneumonia and hydrothorax ( technical appendix figure ) , confusion , meningeal irritation sign , ecchymosis , and hematuria .
laboratory indicators were substantially more out of range 7 days after disease onset , indicative of severe multiorgan dysfunction ( figure 2 ) .
the other 4 patients were discharged after 812 days hospitalization ; at that time , all clinical signs and symptoms had resolved , but for certain patients , laboratory values remained out of reference range , suggesting slow recovery of organ dysfunction ( figure 2 ) .
xy99 dna in blood samples collected during the acute period of illness ( days 36 after onset ) from 5 patients in the same region of china suggests that this organism was the etiologic agent of the infection .
seroconversion or a 4-fold increase in titers of igg against r. rickettsii provided supportive evidence of sfg rickettsia infection .
in contrast to humans with r. massiliae infection and many other sfg rickettsioses reported previously ( 4,10 ) , none of the 5 patients infected with rickettsia sp . xy99 had rash or eschar , and only 1 had headache . in recent years , the concept of the classic triad of fever , rash , and headache suggesting infection with sfg rickettsiae has been increasingly challenged .
several emerging sfg rickettsiae species , such as r. slovaca ( 2 ) , r. raoultii ( 11 ) , r. africae ( 12 ) , and r. helvetica ( 13 ) , can infect humans , but such infections lack these traditional features , which were also lacking in the cases reported here . therefore ,
absence of rash and tick - bite history should not exclude suspicion of sfg rickettsiae infection .
similar to r. slovaca and r. raoultii infections , which can be associated with tickborne lymphadenopathy and dermacentor - borne necrosis - erythema - lymphadenopathy ( 14 ) , lymphadenopathy was also observed in all 5 patients with rickettsia sp .
thus , lymphadenopathy might be a typical sign useful for clinical diagnosis of rickettsia sp .
all 5 patients had gastrointestinal syndromes , indicating potential tissue lesions or vascular injury of the gastrointestinal tract .
the hydrothorax and multiple hemorrhagic signs in 4 patients is possibly suggestive of vascular invasion or damage caused by this novel rickettsia species .
confirmation of the novel rickettsia genotype was achieved only by sequencing the ompa and glta genes .
although differences based on 2 gene segments support its designation as a novel species , isolation efforts and characterization of all 5 genes ( rrs , glta , ompa , ompb , and gened ) are warranted , according to the guidelines for classification of a new rickettsia species ( 15 ) .
physicians in this area of china should be aware of human infections with rickettsia sp .
it should be included in differential diagnoses with severe fever with thrombocytopenia syndrome , which causes similar clinical illness and is also endemic to the same area in eastern central china .
additional materials and methods used in study of human infection with novel spotted fever group rickettsia genotype , china , 2015 .
 only 4 species of spotted fever group rickettsiae have been detected in humans in china . however , phylogenetic analysis of samples from 5 ill patients in china indicated infection with a novel spotted fever group rickettsia , designated rickettsia sp . 
 xy99 . 
 clinical signs resembled those of severe fever with thrombocytopenia syndrome . 
The Study
Conclusions
None