Molecular Techniques Compared for Diagnosis of Rickettsioses

The diagnosis and isolation of Rickettsia species from skin biopsies may be replaced by a real time polymerase chain reaction (rt-PCR).

Diagnosis of Rickettsia infection would benefit by use of the more rapid and sensitive method of quantitative rt-PCR than the time-intensive and less sensitive method of culturing Rickettsia species from skin biopsies.

Scientists at the University of the Mediterranean (Marseille, France) analyzed punch biopsies or scalpel incisions of eschars collected from patients with suspected rickettsial infections between January 2007 and January 2010. Patients were classified as definitely having rickettsiosis if there was direct evidence of infection with a Rickettsia species using culture or molecular assays or if serology was positive. Rickettsial diseases are zoonoses caused by obligate intracellular bacteria.

Total genomic DNA was extracted from samples using a QIAamp tissue kit (Qiagen, Hilden, Germany). Samples were screened for the presence of Rickettsia species using a previously developed rt-PCR assay targeting a 109 base pairs (bp) fragment of a hypothetical protein. Quantification of Rickettsia species were performed using serial ten-fold dilutions of R. africae, R. slovaca, R. raoultii, and R. helvetica. Bacteria were detected by indirect immunofluorescence using human serum and antiserum. All sera were tested by immunofluorescence (IF) for spotted fever group (SFG) rickettsial antigens. Samples were cultured in human embryonic lung (HEL) fibroblasts using the centrifugation-shell vial technique.

Rickettsia species infection was diagnosed in 47/145 (32%) skin biopsies from patients with suspected rickettsiosis. By rt-PCR a positive result was obtained for 41 skin biopsies (28.2%). Rickettsia africae was the most common detected species, followed by R. conorii conorii, R. slovaca, R. sibirica mongolitimonae, and R. raoultii. Comparison of culture and rt-PCR to serology was done for the 26 patients with suspected rickettsiosis with a skin biopsy that also had an acute serum and a convalescent-phase serum sample. The rt-PCR sensitivity was 82% as compared to serology whereas culture sensitivity was 29.4% as compared to serology.

The author concluded that for the diagnosis of Rickettsia infection the use of molecular and culture diagnostic tools, decreases the time of diagnosis and increases the sensitivity. However, a negative result using molecular assays does not exclude the diagnosis of Rickettsia infection. To increase the sensitivity of culture, skin biopsies should be sampled before treatment early in the course of the disease and should be inoculated as soon as possible. The study was published on March 6, 2012, in the journal Public Library of Science Neglected Tropical Diseases.

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