We identified Rickettsia monacensis as a cause of acute tickborne rickettsiosis in 2 humans. Its pathogenic role was assessed by culture and detection of the organism in patients’ blood samples. This finding increases the number of recognized human rickettsial pathogens and expands the known geographic distribution of Mediterranean spotted fever–like cases.

dSeveral real-time PCR approaches to develop field detection for Francisella tularensis, the infectious agent causing tularemia, have been explored. We report the development of a novel qualitative real-time isothermal recombinase polymerase amplification (RPA) assay for use on a small ESEQuant Tube Scanner device. The analytical sensitivity and specificity were tested using a plasmid standard and DNA extracts from infected rabbit tissues. The assay showed a performance comparable to real-time PCR but reduced the assay time to 10 min. The rapid RPA method has great application potential for field use or point-of-care diagnostics. Because of its extraordinary infectiousness, the zoonotic pathogen Francisella tularensis causing tularemia was in the past the subject of state-run biowarfare research programs and therefore is included on the CDC category A list of biothreat agents. It causes disease in a vast range of animals, with relevant disease transmission to humans by direct contact or via vectors such as deer flies, horse flies, mosquitoes, and hard ticks. Infection due to inhalation of aerosols can occur through contact with infected hares. There are three F. tularensis subspecies, F. tularensis subsp. tularensis, F. tularensis subsp. holarctica, and F. tularensis subsp. mediasiatica, which can be found in several environments and geographical regions. The first two subspecies and Francisella novicida cause the bulk of human infections (7,19). Infection by Francisella hispaniensis has also been described, and results of phylogenetic analysis suggest that F. novicida should be regrouped as a fourth subspecies of F. tularensis (3,12).In all scenarios dealing with intentional release of biothreat agents, timely diagnosis is regarded as essential to identifying and containing outbreaks of infectious disease (4). Many efforts have been made to reduce the assay time of PCR-based nucleic acid detection. In spite of engineering constraints regarding temperature cycling needed for the PCR assays, short protocols and miniaturized cyclers or chip platforms are being developed (5, 6, 18).In recent years a variety of isothermal amplification methods have been developed which offer the possibility of developing even simpler point-of-care systems. One example is the ESEQuant Tube Scanner device (Qiagen Lake Constance GmbH, Stockach, Germany). This device contains a sophisticated fluorescence sensor which slides back and forth under a set of eight tubes, collecting fluorescence signals over time and allowing for real-time documentation of increasing fluorescence signals. A combined threshold and signal slope analysis is used for signal interpretation, which can be confirmed by second-derivative analysis (11; also ESEQuant Tube Scanner software [Qiagen]). The recombinase polymerase amplification (RPA) assay is an isothermal amplification method which can be combined with a sequencespecific fluorescent probe for real-time detection. In RPA the phage-derived recombinase UvsX, assisted by its cofactor UvsY, aggregates with oligo...

Description of Francisella hispaniensis sp. nov., isolated from human blood, reclassification of Francisella novicida (Larson et al. 1955 T exhibited 91.6-91.7 % similarity to strains of F. tularensis subspecies, 91.2 % to F. novicida U112 T and 84 % to F. philomiragia ATCC 25017. The genus affiliation was supported by a quinone system typical of Francisella (Q-8 as the major component), a complex polar lipid profile similar to that of F. tularensis with the major components diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine and an unknown aminophospholipid (APL4) and a fatty acid profile consisting mainly of C 10 : 0 (17.2 %), C 14 : 0 (11.2 %), C 16 : 0 (13.1 %), C 18 : 0 3-OH (14.2 %) and C 18 : 1 v9c (7.1 %). DNA-DNA hybridization, which showed unambiguously that FhSp1 T represents a novel species, and the results of biochemical tests allowed genotypic and phenotypic differentiation of the isolate from all hithertodescribed Francisella species. A multiplex PCR developed in the course of this study discriminated FhSp1 T from representatives of all other Francisella species and subspecies, clades A.I and A.II of F.tularensis subsp. tularensis and F. tularensis subsp. holarctica biovar japonica and also between these representatives of the genus. Therefore, we propose the name Francisella hispaniensis sp. nov., with the type strain FhSp1 . We also present an emended description of the genus Francisella.Francisella tularensis is a facultatively intracellular bacterium, and all its subspecies cause the zoonotic disease tularaemia in humans and animals. Francisella philomiragia and Francisella novicida strains, although generally less virulent than strains of F. tularensis, may cause a tularaemia-like disease in immunocompromised patients (Hollis et al., 1989).The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene and recA sequences of strain FhSp1 T are FN252413 and FN252412, respectively, and the accession number of the recA sequence of F. tularensis subsp. tularensis ATCC 6223T is FN545355.