Laboratory diagnosis of rickettsioses: current approaches to diagnosis of old and new rickettsial diseases

Members of the genera Rickettsia and Orientia are morphologically and biochemically similar to other gram-negative bacteria. They are, however, fastidious bacterial organisms that are obligate intracellular parasites. Although rickettsial species are arthropod-associated bacteria, they are also frequently capable of infecting vertebrates, including humans, usually as accidental hosts. They are short, rod-shaped, or coccobacillary organisms, usually 0.8 to 2.0 mm long and 0.3 to 0.5 mm in diameter. The order Rickettsiales has historically been divided into three families: Rickettsiaceae, Bartonellaceae, and Anaplasmataceae. Rickettsiae belong to the Rickettsiae tribe within the family Rickettsiaceae (180) and have long been subdivided into three genera: Coxiella, Rickettsia, and Rochalimaea. The advent of 16S rRNA gene analysis has enabled the determination of phylogenetic relationships among members of the order Rickettsiales (Fig. 1). Coxiella burnetii has been shown to be quite distinct from other rickettsiae, lying in the g subgroup of Proteobacteria, whereas Rickettsia belongs to the a1 subgroup (178). Furthermore, the genus Rochalimaea has recently been united with the genus Bartonella, and the unified genus has been removed from the order Rickettsiales because phylogenetically, its members are in the a2 subgroup of the Proteobacteria (26). The species of the genus Rickettsia have been subdivided into three groups of antigenically related microorganisms, namely, the spotted fever, typhus, and scrub typhus groups (Table 1). This minireview is restricted to these organisms. Phylogenetic data have recently been used to support the reclassification of the agent of scrub typhus into a new genus, because it belongs to a unique and distinct clade within the rickettsia radius. Rickettsia tsutsugamushi is therefore now named Orientia tsutsugamushi (156). Rickettsiae are transmitted to humans by infected arthropod bites or feces. Several rickettsiae are considered to be nonpathogenic in humans because they have been isolated only from arthropods. This opinion may well be contradicted in the future, as in the case of Rickettsia africae, which was first isolated from ticks and subsequently from a patient’s blood. We believe that every rickettsial species may have pathogenic potential, provided that its reservoir arthropod is capable of biting humans. The main symptoms of infection consist of fever and headache. Cutaneous eruption, which is sometimes associated with inoculation eschar, is reported in most cases. The pathogenesis of these diseases is vasculitis caused by the proliferation of organisms in the endothelial lining of small arteries, veins, and capillaries. New culture isolation techniques with shell vials have led to the isolation of an increasing number of strains over recent years. Such isolation is a prerequisite for the characterization of a new species and for the delineation of new rickettsial diseases, since serologic testing of sera from infected patients is unable to distinguish between different rickettsial species. Prior to 1984, only six spotted fever group rickettsioses were recognized, whereas in the last 12 years a further seven have been reported, including six since 1991. These new emerging diseases, with unique clinical manifestations and epidemiologic conditions (Table 2), include Japanese spotted fever due to Rickettsia japonica, first reported in 1984 (90); Flinders island spotted fever caused by Rickettsia honei, described in 1991 (147); Astrakhan spotted fever, reported in 1991 (159); African tick bite fever caused by R. africae, described in 1992 (85); the pseudotyphus of California due to Rickettsia felis, described in a patient in 1994 (78); and two new spotted fevers, the first due to “Rickettsia mongolotimonae,” reported in 1996 (121), and the other due to Rickettsia slovaca, reported in 1997 (120). We suspect that infections due to several spotted fever group (SFG) rickettsiae, at present classified as nonhuman pathogens (Table 1), will increase the size of this group of new emerging diseases. Isolation of etiologic agents also allows for the recognition of rickettsial diseases in regions where they were not previously identified (antibodies against “new” rickettsiae are likely to cross-react with antigens from strains from areas where rickettsial diseases are endemic), as demonstrated by the isolation of Rickettsia akari in Croatia, an area where Mediterranean spotted fever (MSF) is endemic (117). Furthermore, the development of PCR amplification-based approaches and techniques for the analysis of amplified fragments, especially automatic DNA sequencing, allows convenient and rapid identification of rickettsiae, even in nonreference laboratories. To date, the diagnosis of a rickettsial illness has most often been confirmed by serologic testing. Serologic evidence of infection occurs no earlier than the second week of illness for any of the rickettsial diseases; thus, a specific diagnosis may not be available until after the patient has recovered or died. Severe forms of MSF and Rocky Mountain spotted fever (RMSF), with high mortality rates, have been described in patients with underlying infections such as diabetes mellitus, alcoholism, chronic liver diseases, or glucose-6phosphate dehydrogenase deficiency (130, 167). Higher mortality rates are also correlated with delays in consulting a physician and delays in the administration of appropriate antibiotic therapy. In order to reduce the delay in diagnosis, new * Corresponding author. Mailing address: Unité des Rickettsies, UPRESA 6020, Faculté de Médecine, Université de la Méditerrannée, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 05, France. Phone: 33.91. 38.55. 17. Fax: 33.91. 83.03. 90. E-mail: Didier.Raoult@medecine . univ-mrs.fr.