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Two novel real-time PCR assays were developed for the detection of Rickettsia spp. One assay detects all tested Rickettsia spp.; the other is specific for Rickettsia rickettsii. Evaluation using DNA from human blood and tissue samples showed both assays to be more sensitive than nested PCR assays currently in use at the CDC.
Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever (RMSF), is a tick-borne bacterial agent that is a member of the spotted fever group (SFG) rickettsiae. Symptoms of this disease may include, but are not limited to, high fever, headache, and rash, with the potential for a fatal outcome (1). National case fatality rates are <1% but can approach 7% in some regions to which it is highly endemic; a fatal outcome may be averted with timely administration of doxycycline (2, 3).
In the United States, serologic tests are most often used to diagnose RMSF with seroconversion (4-fold titer increase from acute to convalescent phase) by immunofluorescence assay (IFA), the gold standard. However, serologic tests are frequently negative during the acute phase of illness, and currently available molecular tests are not reliable for use in patient management when applied to acute-phase blood samples, which may have very few organisms (3, 4). Clinical management of suspected RMSF cases based on test results is not recommended, partly because of diagnostic assay limitations, and physicians must treat suspected cases empirically (4). While empirical treatment of suspected cases will always be recommended, the development of more sensitive molecular tests that could be applied to patient specimens during the acute phase of illness will enhance the identification of nonfatal cases, improve the timeliness of public health actions following identification of a case, and aid national surveillance efforts by better defining the spectrum and burden of tick-borne rickettsial infections.
At the Centers for Disease Control and Prevention (CDC), nested PCR assays have been the standard molecular diagnostic method for testing of blood and fresh tissue specimens. PCR methods for detection of R. rickettsii in clinical samples include nested assays for two SFG target genes, the 17-kDa-protein-encoding gene and the ompA outer membrane protein gene (5–7). The ompA nested amplicon may then be sequenced for species identification (8–15). However, this methodology is time-consuming (1 to 2 days, minimum) and has not proven highly sensitive for the detection of Rickettsia spp. in blood samples during acute disease, except in cases of advanced or fatal illness (4, 16). A 2000-2007 national surveillance summary noted that <0.5% of reported cases were diagnosed using nested PCR methodology (3).
Several real-time PCR assays have been developed for rickettsial agent detection, including Rickettsia genus-specific (panrickettsia) and R. rickettsii-specific assays (17–20). Panrickettsia assays have utilized conserved sites in the 17-kDa, outer membrane protein (ompB), 16S rRNA, and citrate synthase (gltA) genes, while species-discriminating assays have targeted the 16S rRNA, sca4, ompB, gltA, and ompA genes (18–20). However, using recently obtained Rickettsia genome sequences to identify new conserved and specific sites suitable for TaqMan assays, we have designed panrickettsia and R. rickettsii-specific assays. The assays were optimized on the Applied Biosystems 7500 FastDX (Life Technologies Corp., NY) instrument, which is widely available in U.S. public health laboratories, and their utility was validated with a large set of clinical samples submitted to the CDC for Rocky Mountain spotted fever testing. The primers for the standard nested PCR assays previously in use at the CDC and the primers, probes, and cycling parameters for the new real-time PCR assays for panrickettsia (PanR8) and R. rickettsii (RRi6) validated in this study are shown in Table 1. Real-time PCRs utilized the “fast 7500” ramp rate. The analytical sensitivity (range, 10 to 104 fg; limit, 8 to 9 genome copies for both PanR8 and RRi6 with 95% reproducibility) of the PanR8 assay was determined using serial dilutions of genomic DNA of R. rickettsii, R. prowazekii, and R. typhi (1 isolate each), and that for the RRi6 assay was determined using genomic DNA from 2 isolates of R. rickettsii. The number of cycles (n = 40) was determined by testing serial dilutions of genomic DNA with the lower limit of detection producing a threshold cycle (CT) value of <40. All clinical specimens, with the exception of 1 tissue sample with limited DNA available (see Table 3, sample 38), were run in duplicate, at minimum; each set of reactions included 1 positive control and 1 to 3 nontemplate, negative controls. A clinical specimen was considered positive only if the amplification reaction produced a CT value of <40, the reaction showed logarithmic amplification, and all controls performed as expected.
The specificities of the PanR8 and RRi6 assays for Rickettsia spp. and R. rickettsii, respectively, were confirmed as shown in Table 2. The diagnostic utility of the PanR8 and RRi6 assays was assessed by examining banked DNA extracts (n = 223) from blood and skin biopsy specimens previously tested for Rickettsia by nested PCR at the CDC between 2004 and 2011. Nested PCR had identified 35 (15.7%) samples as positive for Rickettsia. Thirteen of these 35 were determined to be SFG by nested PCR but were not further characterized to the species level. The remaining 22 nested PCR positives were identified by DNA sequencing as R. rickettsii (14), R. parkerii (3), R. akari (2), R. africae (1), and R. typhi (2) (Table 3).
In comparison, the PanR8 assay identified 41 (18.4%; 95% confidence interval [CI], 13.8 to 24.0%) Rickettsia species positives from the 223 banked DNA specimens (including all samples positive by nested PCR), or 6 additional specimens compared to the nested results (Table 3). The RMSF-specific RRi6 assay identified R. rickettsii in 28 (12.6%; 95% CI, 8.8 to 17.5%) of the DNA extracts, 14 more than previous results that involved nested PCR and sequencing of amplicons. The exact confidence interval for the difference of correlated proportions (StatXact v. 9.0.0 software program; Cytel Corp., Cambridge, MA) was used to compare the new assays with the corresponding nested PCR and sequencing results, showing that both real-time assays had detection results that were statistically significantly different from the corresponding nested PCR and sequencing assays (RRi6, 2.7%; 95% CI, 0.7 to 5.8%; PanR8, 6.3%; 95% CI, 3.5 to 10.3%) (21).
The 41 real-time assay positive samples (Table 3) represented 29 patients (samples 2 to 7, 3 and 33, 17 to 19, and 20 to 24 are multiple specimens from 4 individuals). Nested results identified a total of 23 of these patients as having rickettsial infections: 8 with RMSF, 7 with SFG Rickettsia, and 8 with other rickettsioses. Real-time PCR results identified rickettsial infections in all 29 patients, 16 with RMSF and 13 with other rickettsioses. This comparison showed that 20.7% (6/29) of cases were not identified using nested PCR assays, clearly demonstrating the higher clinical sensitivity of the real-time assays than of the traditional nested PCR assays. A potential limitation of the real-time assays as described is that no internal control was used. As a result, specimens with PCR inhibitors could be incorrectly reported as negative. Inclusion of an internal control will serve to reduce false negatives and may further improve the clinical sensitivity of the assays.
In summary, the real-time assays described significantly improve the detection of R. rickettsii and other Rickettsia spp. The assays are rapid and specific, taking <1 h to complete with RMSF species identification, compared to 1 to 2 days for traditional nested PCR and DNA sequencing. While empirical treatment of suspected RMSF cases is critical for preventing severe and fatal outcomes, the assays described will be useful for the diagnosis of RMSF, particularly during the acute stage of illness, and for patient management by directing appropriate treatment for those patients not empirically treated.
We thank Leslie Dauphin, Michael Bowen, William Nicholson, and Marina Eremeeva for reference materials and assistance, Brad Biggerstaff for his help with statistical analysis, Jennifer McQuiston for her thoughtful review of the manuscript, and Aubree Kelly, Joseph Singleton, and James Son for their valuable laboratory support.
The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the CDC or the Department of Health and Human Services.
Published ahead of print 7 November 2012