Comparison of PCR, immunofluorescence assay, and pathogen isolation for diagnosis of q fever in humans with spontaneous abortions

Abstract

Coxiella burnetii, an obligate intracellular parasite with a worldwide distribution, is the causative agent of Q fever in humans. We tested a total of 368 samples (placental bits, genital swabs, fecal swabs, and urine and serum samples) collected from women (n = 74) with spontaneous abortions for C. burnetii by a PCR assay targeting IS1111, the repetitive transposon-like region of C. burnetii (trans-PCR); real-time PCR; an indirect immunofluorescence assay (IFA); and the isolation of the pathogen. The IFA showed seropositivity for 25.68% of the women with spontaneous abortions, whereas trans-PCR and real-time PCR each detected the pathogen in 21.62% of cases. Overall, 25.68% of the subjects were positive by one or more assays. Real-time PCR showed a slightly higher level of sensitivity than trans-PCR. With the IFA as the reference, the two PCR assays showed a higher level of sensitivity (84.21%) than pathogen isolation (26.31%), while both the PCR assays and pathogen isolation were specific (100%). The detection of high numbers of C. burnetii cells in clinical samples and the frequent association of the pathogen with cases of spontaneous abortions observed in this study revealed that Q fever remains underdiagnosed and that the prevalence in India is underestimated.

FIG. 1.

Sensitivity of trans-PCR for the detection of various amounts of the C. burnetii standard Nine Mile, phase I, strain (RSA 493) DNA template (687 bp). Lanes: 1 to 6, different dilutions of the DNA template (undiluted [70 ng/μl], 10⁻¹, 10⁻², 10⁻³, 10⁻⁴, and 10⁻⁵); 7, negative control (no DNA); 8, 100-bp DNA ladder.

FIG. 2.

Trans-PCR detection of C. burnetii in clinical samples from women with spontaneous abortions. Lanes: 1, 100-bp DNA ladder as a marker; 2, DNA template from the standard C. burnetii Nine Mile, phase I, strain; 3 to 7, DNA of C. burnetii isolates from clinical samples.

FIG. 3.

Trans-PCR detection of C. burnetii DNA in clinical samples from women with spontaneous abortions. Lanes; 1, 100-bp DNA ladder as a marker; 2 to 6, DNA of C. burnetii isolates from clinical samples; 7, DNA template from the standard C. burnetii Nine Mile, phase I, strain; 8, negative control (no DNA).