Suicide PCR on skin biopsy specimens for diagnosis of rickettsioses

Abstract

As rickettsioses may be severe diseases and Rickettsia prowazekii is a potential agent of bioterrorism, highly efficient diagnostic techniques are required to detect rickettsiae in patients. We developed a nested PCR assay using single-use primers targeting single-use gene fragments present in the genomes of both Rickettsia conorii and R. prowazekii. We used this "suicide" PCR with DNA from 103 skin biopsy specimens from patients who definitely had a rickettiosis, 109 skin biopsy specimens from patients who possibly had a rickettsiosis, and 50 skin biopsy specimens from patients with nonrickettsial diseases. The suicide PCR detected "R. conorii conorii" in 38 biopsy specimens, R. africae in 28 biopsy specimens, R. slovaca in 12 biopsy specimens, "R. sibirica mongolotimonae" in 5 biopsy specimens, R. aeschlimannii in 2 biopsy specimens, and "R. conorii caspia" and "R. sibirica sibirica" in 1 biopsy specimen each. The technique had a specificity of 100% and a sensitivity of 68%. It was 2.2 times more sensitive than culture (P < 10(-2)) and 1.5 times more sensitive than regular PCR (P < 10(-2)). The efficacy of the suicide PCR was reduced by antibiotic therapy prior to biopsy (P < 10(-2)) and was increased when it was performed with eschar biopsy specimens (P = 0.03). We propose the use of the suicide PCR as a sensitive, specific, and versatile technique for improving the diagnosis of rickettsioses, especially when it is used on eschar biopsy specimens taken prior to antibiotic therapy.