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Detection of Vaccinia DNA in the Blood Following Smallpox Vaccination
To the Editor: Routine administration of the smallpox vaccine ended in the United States in 1972. With the reinitiation of the US smallpox vaccination program in 2002, the risk of transmission of vaccinia virus from a recently vaccinated person to a susceptible host is a concern. Secondary transmission is biologically plausible because of evidence of viral persistence in vaccinees. Vaccinia virus has been cultured from the oropharynx of vaccine recipients with a normal course following vaccination.1 In the 1960s and 1970s, it was isolated from the blood and urine of a limited number of vaccine recipients who had complications following vaccination.2 More sensitive molecular techniques are now available for detecting viruses in clinical specimens. We describe findings using real-time polymerase chain reaction (PCR) to detect vaccinia DNA in smallpox vaccine recipients.
Methods
Between April 2003 and April 2004, 431 persons who were to receive the smallpox vaccine at Travis Air Force Base, California, in accordance with Department of Defense and Centers for Disease Control and Prevention guidelines were invited to participate in this study. Participants were vaccinated with the New York City Board of Health strain of vaccinia (Dryvax; Wyeth Laboratories, Marieta, Pa). They were instructed to keep their vaccination sites covered with a semipermeable dressing until scab separation and to return for study visits 6 to 8 days and 20 to 22 days after vaccination.
At each visit, swab samples of the oropharynx, external surface of the dressing, and hands were collected, along with 7 mL of whole blood. Blood samples were centrifuged and separated into plasma and cells. DNA was extracted from 1-mL aliquots of plasma or transport media for oropharyngeal, dressing, and hand swab samples and purified using spin processing (QIAamp MinElute Virus Spin; QIAGEN, Hilden, Germany). PCR analysis with the LightCycler instrument (Roche, Basel, Switzerland) was performed by using TaqMan-based chemistry (PE Applied Biosystems, Foster City, Calif), targeting a 123–base pair segment of the pan-orthopox hemaglutinin gene.3 Samples were batch tested and each sample was run in duplicate along with positive and negative template controls. Amplification of hemaglutinin-specific products with PCR was confirmed using 7.5% polyacrylamide gel electrophoresis.
This study was approved by the institutional review board at the David Grant US Air Force Medical Center, Travis Air Force Base, California. All participants provided written informed consent. None received compensation for participating.
Results
A total of 97 persons enrolled in the study (mean age, 29 years; 62% vaccine naive). No data are available on nonparticipants. Fourteen patients were lost to follow-up. Of the remaining 83 patients, 77 had vesicle formation signifying major reaction to the vaccine and are described herein. Forty-seven patients (61%) were vaccine naive and 30 (39%) were repeat vaccinees; mean ages for these patients were 23 years and 40 years, respectively. None had been vaccinated within the past 10 years. A total of 74 of 77 patients returned for the study visit 6 to 8 days after vaccination, 51 returned for the study visit 20 to 22 days after vaccination, and 48 returned for both visits. Failure to return for visits was due to military deployment during the study. Mean ages and vaccination history were similar among all subgroups.
At the study visit 6 to 8 days after vaccination, vaccinia DNA was found in 15 samples from 12 (16%) of 74 patients (4 patients had vaccinia DNA in the blood, 8 in the oropharynx, 3 on the dressing, and 0 on the hands) (Table). At the study visit 20 to 22 days after vaccination, vaccinia DNA was found in 11 (22%) of 51 patients (1 patient had vaccinia DNA in the blood, 4 in the oropharynx, 6 on the dressing, and 0 on the hands). Three patients who were positive at 20 to 22 days had also been positive at 6 to 8 days. Twenty of 77 patients (26%) had at least 1 positive sample at a study visit. The single patient with vaccinia DNA in the blood at 20 to 22 days had undergone scab separation before 20 to 22 days and was previously vaccinated in 1960 and 1980. None of the patients had complications following vaccination. The positive blood samples had approximately 1000 genome copies per milliliter.
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Table. Positive Vaccinia DNA Samples Among 77 Participants With Smallpox Vaccination and Vesicle Formation*
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Comments
Our findings indicate the possibility of a significant transmission risk for patients after smallpox vaccination, even after an uncomplicated course. A recent in vitro study of the correlation between orthopox virus DNA quantity and infectivity showed that approximately 100 genome copies were detected by PCR for each infectious viral particle,4 which corresponds to 10 virions per milliliter in our study. The minimum infectious dose of vaccinia is not known.
Current guidelines recommend that vaccinees defer blood donation for 21 days after vaccination or until the scab separates, whichever is later.5 We detected vaccinia DNA in the blood of 5 smallpox vaccine recipients, including 1 vaccinee 21 days after vaccination. Whether detection of vaccinia DNA in blood correlates with infectivity is uncertain and a larger study with more prolonged and extensive sampling and concomitant viral culture is warranted to understand the natural history of viremia following vaccination. Our estimate of rates of positive samples is limited by incomplete follow-up and possible selection bias. However, our results suggest that until more data are available, extending the duration of deferral for blood donation would be appropriate.
Author Contributions: Drs Danaher and Savona had full access to all of the data in the study and take full responsibility for the integrity of the data and the accuracy of the data analysis.
Study concept and design: Dela Cruz, Jones, Thornton, Hadfield, Danaher.
Acquisition of data: Savona, Dela Cruz, Danaher.
Analysis and interpretation of data: Savona, Dela Cruz, Jones, Thornton, Xia, Hadfield, Danaher.
Drafting of the manuscript: Savona, Dela Cruz, Hadfield, Danaher.
Critical revision of the manuscript for important intellectual content: Savona, Dela Cruz, Jones, Thornton, Xia, Hadfield, Danaher.
Statistical analysis: Savona, Thornton.
Obtained funding: Dela Cruz, Danaher.
Administrative, technical, or material support: Savona, Dela Cruz, Xia, Danaher.
Study supervision: Savona, Dela Cruz, Jones, Danaher.
Provided technical oversight: Hadfield.
Financial Disclosures: None reported.
Funding/Support: This study was funded by the US Air Force Surgeon General and performed under Clinical Investigation No. FDG20030013H.
Role of the Sponsor: The sponsor had no role in the design and conduct of the study; in the collection, management, analysis, or interpretation of the data; and in the preparation of the manuscript. The director of the Clinical Investigation Facility at the David Grant US Air Force Medical Center reviewed and approved the manuscript prior to submission.
Disclaimer: The opinions and assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the official policy of the Department of Defense or other departments of the US Government. The voluntary and fully informed consent of the participants described in this study was obtained as required by 32 CFR 219 and AFI 40-402, Protection of Human Subjects in Biomedical and Behavioral Research.
Acknowledgment: We are grateful to the many military members who volunteered for this study. We also thank Sarah Stassen for her voluntary technical assistance in the laboratory, Dana Wallace and Robert Duck of the immunizations clinic for their voluntary assistance with vaccination, and Regina Rowell, MLS, medical librarian, for her voluntary assistance.
Michael R. Savona, MD
msavona{at}med.umich.edu
Department of Internal Medicine
University of Michigan
Ann Arbor
Wilfred P. Dela Cruz, PhD;
Morris S. Jones, PhD;
Jennifer A. Thornton, PhD
Clinical Investigation Facility
David Grant US Air Force Medical Center
Travis Air Force Base, Calif
Dongxiang Xia, MD, PhD
Norfolk Public Health Laboratory
Commonwealth of Virginia
Norfolk
Ted L. Hadfield, PhD
Midwest Research Institute
Palm Bay, Fla
Patrick J. Danaher, MD
Department of Infectious Disease
David Grant US Air Force Medical Center
Travis Air Force Base, Calif
1. Blattner RJ, Norman JO, Heys FM, Aksu I. Antibody response to cutaneous inoculation with vaccinia virus: viremia and viruria in vaccinated children. J Pediatr. 1964;64:839-852.
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2. Gurvich EB, Braginskaya VP, Shenkman LS, Sokolova AF, Davydova AV. Isolation of vaccinia virus from the pharynx of children vaccinated against smallpox. J Hyg Epidemiol Microbiol Immunol. 1974;18:69-76.
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3. Xia D, Gagni C, Ravizee A, Dempsy M, Cooper L, Hadfield TL. Rapid detection of orthopoxvirus DNA by LightCycler RTPCR with TaqMan primer probe set. Abstr Gen Meet Am Soc Microbiol. 2003:175-176.4. Panning M, Asper M, Kramme S, Schmitz H, Drosten C. Rapid detection and differentiation of human pathogenic orthopox viruses by a fluorescence resonance energy transfer real-time PCR assay. Clin Chem. 2004;50:702-708.
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5. Food and Drug Administration; Center for Biologics Evaluation and Research Guidance for industry: recommendations for deferral of donors and quarantine and retrieval of blood and blood products in recent recipients of smallpox vaccine (vaccinia virus) and certain contacts of smallpox vaccine recipients. Available at: http://www.fda.gov/cber/gdlns/smpoxdefquar.pdf. Accessed July 4, 2005.
Letters Section Editor: Robert M. Golub, MD, Senior Editor.
JAMA. 2006;295:1898-1900.
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