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Background: Rickettsioses are caused by pathogenic species of the genus Rickettsia and play an important role as emerging diseases. The bacteria are transmitted to mammal hosts including humans by arthropod vectors. Since detection, especially in tick vectors, is usually based on PCR with genus-specific primers to include different occurring Rickettsia species, subsequent species identification is mainly achieved by Sanger sequencing. In the present study a real-time pyrosequencing approach was established with the objective to differentiate between species occurring in German Ixodes ticks, which are R. helvetica, R. monacensis, R. massiliae, and R. felis. Tick material from a quantitative real-time PCR (qPCR) based study on Rickettsia-infections in I. ricinus allowed direct comparison of both sequencing techniques, Sanger and real-time pyrosequencing. Methods: A sequence stretch of rickettsial citrate synthase (gltA) gene was identified to contain divergent single nucleotide polymorphism (SNP) sites suitable for Rickettsia species differentiation. Positive control plasmids inserting the respective target sequence of each Rickettsia species of interest were constructed for initial establishment of the real-time pyrosequencing approach using Qiagen's PSQ 96MA Pyrosequencing System operating in a 96-well format. The approach included an initial amplification reaction followed by the actual pyrosequencing, which is traceable by pyrograms in real-time. Afterwards, real-time pyrosequencing was applied to 263 Ixodes tick samples already detected Rickettsia-positive in previous qPCR experiments. Results: Establishment of real-time pyrosequencing using positive control plasmids resulted in accurate detection of all SNPs in all included Rickettsia species. The method was then applied to 263 Rickettsia-positive Ixodes ricinus samples, of which 153 (58.2%) could be identified for their species (151 R. helvetica and 2 R. monacensis) by previous custom Sanger sequencing. Real-time pyrosequencing identified all Sanger-determined ticks as well as 35 previously undifferentiated ticks resulting in a total number of 188 (71.5%) identified samples. Pyrosequencing sensitivity was found to be strongly dependent on gltA copy numbers in the reaction setup. Whereas less than 101 copies in the initial amplification reaction resulted in identification of 15.1% of the samples only, the percentage increased to 54.2% at 101-102 copies, to 95.6% at >102-103 copies and reached 100% samples identified for their Rickettsia species if more than 103 copies were present in the template. Conclusions: The established real-time pyrosequencing approach represents a reliable method for detection and differentiation of Rickettsia spp. present in I. ricinus diagnostic material and prevalence studies. Furthermore, the method proved to be faster, more cost-effective as well as more sensitive than custom Sanger sequencing with simultaneous high specificity. [ABSTRACT FROM AUTHOR]

Background In the Pacific islands countries and territories, very little is known about the incidence of infectious diseases due to zoonotic pathogens. To our knowledge, human infections due to Rickettsia spp., Coxiella burnetii, Ehrlichia spp. and Bartonella spp. have never been reported in French Polynesia; and infections due to C. burnetti have been reported worldwide except in New Zealand. To evaluate the prevalence of this disease, we conducted a serosurvey among French Polynesian blood donors. Methods The presence of immunoglobulin G antibodies against R. felis, R. typhi, R. conorii, C. burnetii, B. henselae, B. quintana, and E. chaffeensis was evaluated by indirect immunofluorescence assay in sera from 472 French Polynesian blood donors collected from 2011 to 2013. In addition, 178 ticks and 36 cat fleas collected in French Polynesia were also collected and tested by polymerase chain reaction to detect Rickettsia spp., B. henselae and Ehrlichia spp. Results None of the blood donors had antibodies at a significant level against Rickettsia spp., Coxiella burnetii, Ehrlichia spp. and Bartonella spp. All tested ticks and cat fleas were PCRnegative for Rickettsia spp., B. henselae, and Ehrlichia spp. Conclusion We cannot conclude that these pathogens are absent in French Polynesia but, if present, their prevalence is probably very low. C. burnetii has been reported worldwide except in New Zealand. It may also be absent from French Polynesia. [ABSTRACT FROM AUTHOR]

Background: Rickettsia felis is a recently described flea-borne spotted fever group Rickettsia that is an emerging human pathogen. Although there is information on the organism from around the world, there is no information on the organism in China. Methods: We used a commercial ELISA to detect antibodies reactive against R. felis in blood samples and developed a PCR to detect the gltA of the organism in blood samples and external parasites. Results: We found reactive antibodies in people (16%; 28/180), dogs (47%; 128/271) and cats (21%; 19/90) and positive PCRs with DNA from people (0.1%; 1/822), dogs (0.8%; 8/1,059), mice (10%; 1/10), ticks (Rhipicephalus sanguineus; 10%; 15/146), lice (Linognathus setosus; 16%; 6/37), fleas (Ctenocephalides felis felis; 95%; 57/60) and mosquitoes (Anopheles sinensis, Culex pipiens pallens; 6%; 25/428), but not from cats (0/135) or canine fecal swabs (0/43). Conclusions: This is the first report of R. felis in China where there is serological and/ or PCR evidence of the organism in previously reported [people, dogs, cats, ticks (Rhipicephalus sanguineus), fleas (Ctenocephalides felis felis) and mosquitoes (Anopheles sinensis, Culex pipiens pallens)] and novel species [mice and lice (Linognathus setosus)]. [ABSTRACT FROM AUTHOR]

Background: Increasing numbers of tick-borne pathogens are being discovered, including those that infect humans. However, reports on co-infections caused by two or more tick-borne pathogens are scarce.Case Presentation: A 38-year-old male farmer was bitten by a hard tick, presented with fever (37.7 °C), severe headache and ejection vomiting. Lumbar puncture was performed in the lateral decubitus. The cerebrospinal fluid (CSF) was clear, and analysis showed severe increased pressure (320 mm H2O), mild leukocytosis (126.0 × 106/L, mononuclear cells accounting for 73%) and elevated total protein concentration (0.92 g/L). Bacterial cultures of CSF and blood were negative. The diagnosis of Rickettsia raoultii and Tacheng tick virus 1 (TcTV-1) co-infection was confirmed by amplifying four rickettsial genetic markers and the partial small (S) RNA segment of TcTV-1 from the patient's blood. The patient gradually recovered after treatment with levofloxacin and ribavirin.Conclusions: This is the first reported co-infection case with fever and meningitis caused by R. raoultii and TcTV-1. It is vital to screen for multiple pathogens in tick-bitten patients, especially in those with severe complex symptoms. [ABSTRACT FROM AUTHOR]