Jul 3, 1989 - Rickettsia rickettsia, the etiologic agent of Rocky. Mountain spotted fever (RMSF), is found primarily in the. United States; Rickettsia conorii,a ...
PART XIII. POLYMERASE CHAIN REACTION TECHNIQUE FOR THE IDENTIFICATION OF RICKETTSIAE
Detection of Rickettsia rickettsii DNA in Clinical Specimens by Enzymatic Amplification Using Polymerase Chain Reaction Technology THEODORE TZIANABOS, BURT E. ANDERSON, AND JOSEPH E. McDADE Viral and Rickettsia1 Zoonoses Branch Center for Infectious Diseases Centers for Disease Control Public Health Service United States Department of Health and Human Services Atlanta, Georgia 30333
INTRODUCTION A laboratory test for rapid diagnosis of rickettsial diseases has been sought for many years. The most widely used procedure for serologic diagnosis is the indirect fluorescent antibody test (IFA). Isolation procedures are laborious and potentially dangerous, albeit specific when coupled with serologic and fluorescent antibody testing. Direct fluorescent antibody staining of properly taken skin biopsies also offers a specific method for the early diagnosis of Rocky Mountain spotted fever (RMSF), but the procedure lacks sensitivity and is potentially haza r d ~Advances ~ ~ . ~in molecular biology have provided us with new tools for the rapid detection of disease-causing organisms. For example the development of the Thermal Cycler and the discovery of a heat-stable polymerase (Taq) have greatly facilitated the application of gene amplification for detecting DNA of disease-causing organisms. DNA of agents such as HIV-1,4 cytomegalovir~s,~ papilloma virus,6 and hepatitis B virus7 has been detected in infected tissue or fluids. Similar experiments were performed in our laboratory for the early detection of RMSF. This report reviews the data obtained.* 's2
MATERIALS AND METHODS We applied polymerase chain reaction (PCR) technology to the detection of RMSF DNA in human blood specimens. This has been made possible by the characterization of a 17-kDa antigen found in all rickettsial species tested. The gene responsible for directing the synthesis of the 17-kDa antigen has been cloned and s e q ~ e n c e dThis . ~ information allowed the construction of two oligonucleotide primers that flank the DNA segment to be amplified. The primers define a 246-bp fragment of the coding region of the 17-kDa antigen gene from Rickettsia rickettsii and were chosen because they contain sequences at the 3' end that are unique to R. rickettsii and R. conorii. The primers were tested and found to be specific to R. 553
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Date of Onset
Sex
Age
Patients Tested for R . rickettsii by PCR
Patient No.
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Date of Specimen Penicillin Ampicillin Ampicillin/ cephalosporin Cleocin/ gentamycinl tetracycline Chloromycetin Ampicillin/ chloromycetin/ penicillin Tetracycline Tetracycline Tetracycline
Antibiotic Therapy
Fatal Recovered Recovered
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TZIANABOS et al.: R. rickettsii DNA IN CLINICAL SPECIMENS
555
rickettsii and R . conorii but not reactive to R. akari or rickettsial species in the typhus group. This was not unexpected, since others have shown that the DNA of R. conorii is 96% homologous to the DNA of R. rickettsii, whereas the DNA of R. akari is 46% homologous. Nine human blood clot specimens from which RMSF had been isolated were processed in an effort to detect rickettsial DNA by the use of PCR technology. Three hundred microliters from 10% suspensions of blood clots from RMSF patients and six specimens of normal human blood were treated with 40 p1 of 10% sodium dodecyl sulfate and 20 p l of proteinase K, mixed gently, and heated at 50°C for 1 h. They were then phenol-chloroform extracted; the DNA was precipitated, dried, and resuspended in 22 p1 of PCR diluent (10 mM Tris, pH 8.0; 1 m M EDTA; 10 m M NaCl). These were the templates used for DNA amplification.
RESULTS AND DISCUSSION TABLE 1 shows the salient features of the patients from whom RMSF had been isolated and the results of the gene amplification assays of their blood specimens. Of the nine specimens tested, rickettsial gene amplification was successful in seven. Failure to detect rickettsial DNA from two isolation-positive blood specimens may be because the rickettsemia in those patients was below the limits of detection for this test. The sensitivity of this test can be improved. Properly obtained specimens and reamplification of PCR products with an additional pair of nested primers will undoubtedly raise the limits of detection. This should help reduce the number of false-negative results from patients in the early stages of RMSF infection. Specific primers to other rickettsial species will enable us to differentiate species, which will aid both in diagnostic and epidemiologic studies.
REFERENCES 1 . GOLDWASSER, R. A. & C. C. SHEPARD.1959. Fluorescent antibody methods in differentiation of murine and epidemic typhus sera: Specificity changes resulting from previous immunization. J. Immunol. 82: 373-380. 2. PHILIP,R. N., E. A. CASPER,R. A. ORMSBEE, M. G . PEACOCK & W. BURGDORFER. 1976. Microimmunofluorescence test for the serologic study of Rocky Mountain spotted fever and typhus. J. Clin. Microbiol. 3: 51-61. T. E., C. E. PEDERSON, C. N. OSTER,L. R. BAGLEY,J. ROMBERGER & 3. WOODWARD, M. J. SNYDER. 1976. Prompt confirmation of Rocky Mountain spotted fever: Identification of rickettsiae in skin tissues. J. Infect. Dis. 13: 297-301. 4. Ou, C.-Y., S. KWOK,S. W. MITCHELL, D. H. MACK,J. J. SNINSKY, J. W. KREBS,P. FEORINO, D. WARFIELD& G. SCHOCHETMAN. 1988. DNA amplification for direct detection of HIV-1 in DNA of peripheral blood mononuclear cells. Science 239: 487491. 5. DEMMLER, G. J., G. J. BUFFONE,C. M. SCHIMBOR & R. A. MAY.1988. Detection of cytomegalovirus in urine from newborns by using polymerase chain reaction DNA amplification. J. Infect. Dis. 158 1177-1192. D. K., N. ARNHEIM & W. J. MARTIN.1988. Detection of human papilloma 6. SHIBATA, virus in paraffin-imbedded tissue using the polymerase chain reaction. J. Exp. Med. 167: 225-230.
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& G. N. VYAS.1989. 7. ULRICH,P. P., R. A. BHAT,B. SETO,D. MACK,J. SNINSKY Enzymatic amplification of hepatitis B virus DNA in serum compared with infectivity testing in chimpanzees. J. Infect. Dis. 160: 37-43. 8. TZIANABOS, T., B. E. ANDERSON & J. E. MCDADE.1989. Detection of Rickettsia rickertsii DNA in clinical specimens by using polymerase chain reaction technology. J. Clin. Microbiol. 27: 2866-2868. 9. ANDERSON, B. E., R. L. REGNERY, G. M. CARLONE, T. TZIANABOS, J. E. MCDADE,Z. Y. Fu 62 W.J. BELLINI.1987. Sequence analysis of the 17-kilodalton-antigengene from R. rickettsii. J. Bacteriol. 169: 2385-2390.
