[A Rickettsia case caused by Rickettsia conorii]. / Rickettsia conorii'nin neden oldugu riketsiyal enfeksiyon olgusu.

Rickettsia species are gram-negative intracellular, small pleomorphic coccobacilli in the Rickettsiaceae family. This genus is serologically and genotypically divided into four groups as spotted fever group, typhus group, Rickettsia belli and Rickettsia canadensis. Rickettsia conorii (R.conorii subsp. conorii) in the spotted fever group was reported to cause mediterranean spotted fever in Europe, especially in mediterranean countries including Turkey. The major vectors of Rickettsia species are ticks, and in some species fleas or mites. In this report a case with R.conorii infection was presented. A 46-year-old female patient, who had anorexia, fatigue, muscle aches, chills and high fever was admitted to a health institution. The patient was diagnosed as influenza. There was no regression in the patient's complaints with the recommended treatment. The patient was examined in our infectious diseases clinic and had several symptoms like severe muscle and joint pain with significant headache, and rashes at her body including hands and feet. The patient had a single eschar in the upper midline of the belly that matched tick biting and pink small maculopapular scars on the trunk, arms, legs, feet, and hands. Considering a Rickettsia pre-diagnosis, liquid electrolyte and doxycycline 2 x 100 mg oral treatment was started. On the third day of treatment, high fever, muscle and joint pain were decreased. On the fifth day, active skin lesions were started to fade. R.conorii IgM and IgG were negative in the first serum sample of the patient. In the biopsy sample taken from eschar tissue, Rickettsia spp. was detected as positive with rt-PCR. PCR was used by using the specific regions of the genetically specific gltA and ompA genes in the biopsy specimens and then the PCR products were determined by DNA sequence analysis. The DNA sequence results were compA red with Genbank data and determined that the gltA sequence was 99%, similar to R.conorii with accession number JN182786 and the ompA sequence was 99%, similar to R.conorii with accession number KR401144. When the phylogenetic tree was created, it was observed that the etiological agent was R.conorii. A week after the treatment, in the second serum sample R.conorii IFA IgM 1/192 titer and IgG 1/320 titer were detected as positive. In this case report, we have presented a Rickettsia case, clinically diagnosed as Rickettsia, serologically negative in the acute phase, PCR positive, with post-treatment seroconversion and etiologic agent determined as R.conorii.

First detection of Rickettsia conorii ssp. caspia in Rhipicephalus sanguineus in Zambia.

Ticks are important vectors for Rickettsia spp. of the spotted fever group all around the world. Rickettsia conorii is the etiological agent of boutonneuse fever in the Mediterranean region and Africa. Tick identification was based on morphological features and further characterized using the 16S rRNA gene. The ticks were individually tested using pan-Rickettsia real-time-PCR for screening, and 23S-5S intergenic spacer region, 16S rDNA, gltA, sca4, ompB, and ompA genes were used to analyze the Rickettsia positive samples. Rickettsia conorii ssp. caspia was detected in tick collected in Zambia for the first time, thus demonstrating the possibility of the occurrence of human disease, namely Astrakhan fever, due to this Rickettsia ssp. in this region of Africa. The prevalence of R. conorii ssp. caspia was 0.06% (one positive tick out of 1465 tested ticks) and 0.07% (one positive tick out of 1254 tested Rh. sanguineus).

Genome sequence of Rickettsia conorii subsp. caspia, the agent of Astrakhan fever.

Rickettsia conorii subsp. caspia is the agent of Astrakhan fever, a spotted fever group rickettsiosis endemic to Astrakhan, Russia. The present study reports the draft genome of Rickettsia conorii subsp. caspia strain A-167.

Identification of rickettsial infections by using cutaneous swab specimens and PCR.

To determine the usefulness of noninvasive cutaneous swab specimens for detecting rickettsiae, we tested skin eschars from 6 guinea pigs and from 9 humans. Specimens from eschars in guinea pigs were positive for rickettsiae as long as lesions were present. Optimal storage temperature for specimens was 4 degrees C for 3 days.

Fatal Rickettsia conorii subsp. israelensis infection, Israel.

Underdiagnosis of fatal spotted fever may be attributed to nonspecific clinical features and insensitive acute-phase serologic studies. We describe the importance of molecular and immunohistochemical methods in establishing the postmortem diagnosis of locally acquired Israeli spotted fever due to Rickettsia conorii subsp. israelensis in a traveler returning to Israel from India.

Evaluation of conventional and real-time PCR assays for detection and differentiation of Spotted Fever Group Rickettsia in dog blood.

Spotted Fever Group Rickettsia is important cause of emerging and re-emerging infectious disease in people and dogs. Importantly, dogs can serve as sentinels for disease in people. Sensitive and specific diagnostic tests that differentiate among species of infecting Rickettsia are needed. The objective of this study was to develop a sensitive and specific PCR that differentiates SFG Rickettsia infecting dog blood. Conventional and real-time PCR assays were developed using primers that targeted a small region of the ompA gene. Their sensitivity, determined by testing a cloned target sequence in the presence of host DNA, was 15-30 and 5 copies of DNA, respectively. Testing of Rickettsia cultures and analysis of Rickettsia gene sequences deposited in GenBank verified DNA could be amplified and used to differentiate species. DNA from the blood of infected dogs was also tested. Importantly, Rickettsia DNA was detected before seroconversion in some dogs. The species of infecting Rickettsia was also identified. We conclude these assays may assist in the timely diagnosis of infection with SFG Rickettsia. They may also facilitate the discovery of novel SFG Rickettsia infecting dogs, and in the investigation of dogs as sentinels for emerging rickettsioses.

Ten years' experience of isolation of Rickettsia spp. from blood samples using the shell-vial cell culture assay.

Two strategies to improve the efficacy of the shell-vial method for Rickettsia were analyzed. Blood samples from 59 patients with Mediterranean spotted fever (MSF) were examined using the shell-vial technique. (i) DNA from positive lenses was obtained when they were contaminated. (ii) Blood sample from one patient was cultured in 17 shell-vials. R. conorii was identified in four cases by polymerase chain reaction (PCR)-RFLP. Three of these were obtained from cells adherent to lenses and the fourth one by using total patient blood sample. Rickettsia isolation using all blood samples as well as DNA from shell-vial lenses could be useful in the study of rickettsial infections.

Presence of Rickettsia conorii subsp. israelensis, the causative agent of Israeli spotted fever, in Sicily, Italy, ascertained in a retrospective study.

A retrospective analysis by molecular-sequence-based techniques was performed to correctly identify the etiological agent of 24 Mediterranean spotted fever cases occurring in Western Sicily, Italy, from 1987 to 2001. Restriction analysis of a 632-bp PCR-amplified portion of the ompA gene allowed presumptive identification of five clinical isolates as belonging to Rickettsia conorii subsp. israelensis, the etiological agent of Israeli spotted fever (ISF). The remaining 19 rickettsial isolates were Rickettsia conorii subsp. conorii, the only pathogenic rickettsia of the spotted fever group reported in Italy until the present. Sequence analysis of the ompA gene confirmed the identification of all the R. conorii subsp. israelensis isolates and demonstrated that rickettsiosis caused by R. conorii subsp. israelensis can be traced back to 1991 in Sicily. The recorded clinical data of the five ISF patients support the idea that these strains could correlate to more-severe forms of human disease. Three of five patients experienced severe disease, and one of them died.

Proposal to create subspecies of Rickettsia conorii based on multi-locus sequence typing and an emended description of Rickettsia conorii.

BACKGROUND: Rickettsiae closely related to the Malish strain, the reference Rickettsia conorii strain, include Indian tick typhus rickettsia (ITTR), Israeli spotted fever rickettsia (ISFR), and Astrakhan fever rickettsia (AFR). Although closely related genotypically, they are distinct serotypically. Using multilocus sequence typing (MLST), we have recently found that distinct serotypes may not always represent distinct species within the Rickettsia genus. We investigated the possibility of classifying rickettsiae closely related to R. conorii as R. conorii subspecies as proposed by the ad hoc committee on reconciliation of approaches to bacterial systematics. For this, we first estimated their genotypic variability by using MLST including the sequencing of 5 genes, of 31 rickettsial isolates closely related to R. conorii strain Malish, 1 ITTR isolate, 2 isolates and 3 tick amplicons of AFR, and 2 ISFR isolates. Then, we selected a representative of each MLST genotype and used multi-spacer typing (MST) and mouse serotyping to estimate their degree of taxonomic relatedness. RESULTS: Among the 39 isolates or tick amplicons studied, four MLST genotypes were identified: i) the Malish type; ii) the ITTR type; iii) the AFR type; and iv) the ISFR type. Among these four MLST genotypes, the pairwise similarity in nucleotide sequence varied from 99.8 to 100%, 99.4 to 100%, 98.2 to 99.8%, 98.4 to 99.8%, and 99.2 to 99.9% for 16S rDNA, gltA, ompA, ompB, and sca4 genes, respectively. Representatives of the 4 MLST types were also classified within four types using MST genotyping as well as mouse serotyping. CONCLUSION: Although homogeneous genotypically, strains within the R. conorii species show MST genotypic, serotypic, and epidemio-clinical dissimilarities. We, therefore, propose to modify the nomenclature of the R. conorii species through the creation of subspecies. We propose the names R. conorii subsp. conorii subsp. nov. (type strain = Malish, ATCC VR-613), R. conorii subspecies indica subsp. nov. (type strain = ATCC VR-597), R. conorii subspecies caspia subsp. nov. (type strain = A-167), and R. conorii subspecies israelensis subsp. nov. (type strain = ISTT CDC1). The description of R. conorii is emended to accomodate the four subspecies.

The presence of eschars, but not greater severity, in Portuguese patients infected with Israeli spotted fever.

In Portugal, Mediterranean spotted fever (MSF) is caused by R. conorii Malish and Israeli spotted fever (ISF) strains. It has been suggested that the ISF strain isolated from patients with MSF causes different clinical manifestations compared to those caused by Malish strain, namely the absence of eschar and greater severity. The aim of this study was to analyze the presence or absence of eschar and of fatality in Portuguese patients infected with either Malish or ISF strain. Of 94 patients with a clinical diagnosis of MSF between 1994 to 2004, 47 were infected with Malish strain and 47 with ISF strain. Eschars were reported in 20 patients (49%) infected with Malish strain, and in 17 (39%) with ISF strain. The presence of eschar is not statistically associated to a greater extent with either R. conorii strain (P=0.346). A total of 22 patients died, 9 infected with Malish strain and 13 infected with ISF strain, and no statistically significant difference was found (P=0.330). This study showed that the concepts of absence of the eschar and greater severity in Israeli spotted fever infection should be revised.

Use of highly variable intergenic spacer sequences for multispacer typing of Rickettsia conorii strains.

By use of the nearly perfectly colinear genomes of Rickettsia conorii and Rickettsia prowazekii, we compared the usefulness of three types of sequences for typing of R. conorii isolates: (i) 5 variable coding genes comprising the 16S ribosomal DNA, gltA, ompB, and sca4 (gene D) genes, which are present in both genomes, and the ompA gene, which is degraded in R. prowazekii; (ii) 28 genes degraded in R. conorii but intact in R. prowazekii, including 23 split and 5 remnant genes; and (iii) 27 conserved and 25 variable intergenic spacers. The 4 conserved and 23 split genes as well as the 27 conserved intergenic spacers each had identical sequences in 34 human and 5 tick isolates of R. conorii. Analysis of the ompA sequences identified three genotypes of R. conorii. The variable intergenic spacers were significantly more variable than conserved genes, split genes, remnant genes, and conserved spacers (P < 10(-2) in all cases). Four of the variable intergenic spacers (dksA-xerC, mppA-purC, rpmE-tRNA(fMet), and tRNA(Gly)-tRNA(Tyr)) had highly variable sequences; when they were combined for typing, multispacer typing (MST) identified 27 different genotypes in the 39 R. conorii isolates. Two batches from the same R. conorii strain, Malish (Seven), with different culture passage histories were found to exhibit the same MST type. MST was more discriminatory for strain genotyping than multiple gene sequencing (P < 10(-2)). Phylogenetic analysis based on MST sequences was concordant with the geographic origins of R. conorii isolates. Our study supports the usefulness of MST for strain genotyping. This tool may be useful for tracing a strain and identifying its source during outbreaks, including those resulting from bioterrorism.

Spotted fever group rickettsiae from ticks captured in Sudan.

Ticks were collected from ruminants in various areas of Sudan in 1998 and 2000. Primer pairs of rickettsial citrate synthase gene (gltA) and a spotted fever group (SFG) rickettsial 190-kDa surface antigen gene (rompA), respectively, were used for identification. Polymerase chain reaction (PCR)-positive products were used for DNA sequencing. The gltA gene was detected in 55% of the ticks examined (57/104). Among the 57 ticks studied, 19 were positive for the rompA gene. Thus, 18% of the ticks examined were found to be infected with SFG rickettsiae. The nucleotide sequences of the amplified rompA gene fragment of Hyalomma spp. and Amblyomma spp. were similar to those of Rickettsia aeschlimannii and Rickettsia africae, respectively. In this study, we succeeded in detecting the SFG rickettsiae gene in ticks, and established that there were at least two species of SFG rickettsiae in field ticks in Sudan.

Detection of Astrakhan fever rickettsia from ticks in Kosovo.

Astrakhan fever is a summer spotted fever resembling Mediterranean spotted fever, endemic in Astrakhan, a region of Russia located by the Caspian sea. Its agent is a spotted fever group rickettsia, member of the Rickettsia conorii complex, transmitted to humans by Rhipicephalus sanguineus and Rhipicephalus pumilio ticks. In Summer 2001, French United Nations troops in Kosovo collected 2 ticks on asymptomatic soldiers (1 R. sanguineus and 1 Hyalomma marginatum) and 10 ticks on dogs (7 R. sanguineus, 2 Ixodes ricinus, and 1 H. marginatum) in the Morina region. By PCR amplification of both the gltA and ompA genes, we detected a rickettsia in 4 R. sanguineus, i.e., 3 of those collected on dogs and those taken from military personnel. As ticks were preserved in alcohol, culture was not possible. The sequences obtained from these PCR products identified, with a 100% homology, Astrakhan fever rickettsia. None of the other collected tick species was positive. The patient with the positive tick remained asymptomatic. Our study demonstrates, for the first time, the presence of Astrakhan fever rickettsia in ticks outside Russia. We suspect that the area of distribution of this rickettsia could be wider than initially suspected. Moreover, as R. sanguineus ticks bite humans, Astrakhan fever might be a cause of spotted fever in Kosovo.

Isolation of a rickettsia related to Astrakhan fever rickettsia from a patient in Chad.

We isolated a novel spotted fever group rickettsia from a patient coming back from Chad with fever and a maculopapulous rash. In Africa, only six pathogenic spotted fever group rickettsiae have been identified, R. conorii, R. africae, R. akari, R. aeschlimannii, "R. mongolotimonae," and R. felis. Our isolate was identified by PCR amplification and sequencing of the 16S rRNA (16S rDNA), citrate synthase (gltA), and rOmpA (ompA) encoding genes. The 16S rDNA, gltA, and ompA sequences of the isolate were found to be 99.7, 99.6, and 99.5% identical with that of Astrakhan fever rickettsia, respectively. This rickettsia is endemic in the Caspian sea area and has also recently been identified in Kosovo. Using mouse serotyping, the currently accepted method for the identification of spotted fever group rickettsiae, the Chad isolate exhibited a specificity difference of 2 when compared to Astrakhan fever rickettsia and at least 4 when compared with other members of the R. conorii complex. The Chad isolate should be considered a variant of Astrakhan fever rickettsia. This is the first description of Astrakhan fever rickettsia outside Europe and the bacterium may be responsible for cases of spotted fever in Chad. Although Astrakhan fever rickettsia is transmitted by Rhipicephalus ticks in Europe, further studies are indicated to identify its vector in Africa where these ticks are also prevalent.