PCR diagnosis of rickettsia infections

Polymerase Chain Reaction (PCR) assays are the primary method to detect rickettsial pathogens, especially for the early detection of infection before the development of detectable antibodies. PCR assays, which selectively amplify a specific region of the organisms DNA to detectable levels, may target a number of different genes to enable a variety of levels of detection, from broad Rickettsia genus- or SFGR-level of detection, to species-specific detection (Rickettsia typhi and Orientia tsutsugamushi for example). Assays which target outer membrane proteins such as 17kDa, ompB and 47kDa, generally confirms the presence or absence of Rickettsia spp, R. typhi or Orientia tsutsugamushi (respectively). With other gene targets, down-stream sequencing of the PCR product will, in most cases, identify the specific species. It is strongly recommended that multiple gene targets are used to gain an accurate identification. Different formats of PCR assays are available, all with varying levels of sensitivity and specificity, including conventional, nested and quantitative (real-time) PCRs. In addition a number of LAMP-assays (loop-mediated isothermal amplification) have been developed, allowing relatively sensitive and specific rapid detection at a constant temperature (minimizing the need for expensive thermocyclers).

The sample type used for a PCR assay is very important. Whole blood or buffy coat is the preferred sample to be used for a number of reasons, including that Rickettsia are intracellular (the cellular component being concentrated in the buffy coat fraction, increasing the sensitivity of detection) and the blood sample being easily collected. Serum or plasma may be used but these samples are less than optimal as there will be fewer patient cells (meaning lower rickettsiae concentration). In addition, serum may have increased concentrations of blood fibrinogen and fibrin materials which can bind to rickettsia DNA decreasing availability of DNA target for PCRs. Eschars are a suitable sample type for PCR and may be sampled as either scrapings, swabs or biopsied specimens.