Rickettsia parkeri infection detected by polymerase chain reaction amplification from buffy coat and eschar swab specimens: a novel diagnostic approach for a rare condition. Todd Myers, PhD, HCLD(ABB), MB(ASCP) LCDR, USPHS Clinical Laboratory Director Armed Forces Health Surveillance Center Naval Medical Research Center

Rickettsiae Gram negative coccobacillary bacteria Obligate intracellular organisms Arthropod-borne Host for rickettsiae Vectors for human disease Cause febrile diseases (mild to life threatening)

June 23 1812

Grande Armée June 500K cross December less than 40K cross Est. 220,000 may have died solely from disease In the first month alone 80K had died from typhus and dysentery Rampant typhus and dysentery

Military importance Epidemic Typhus: Peloponnesian war, Napoleon, WWI, WWII Rickettsia prowazekii Biowarfare agent produced by USSR and Japan Select Agent B list Rocky Mountain spotted fever (RMSF) Rickettsia rickettsii Causes the most severe rickettsiosis Aerosolization studies conducted in 1970s Q Fever: Iraq & Afghanistan Wars Coxiella burnetii Naturally spread by aerosol Non-spore former that is very resistant to environment Scrub typhus: WWII, Vietnam, Camp Fuji Orientia tsutsugamushi Not a select agent

Rickettsia parkeri R. parkeri was first isolated in 1937 from Gulf Coast ticks (Amblyomma maculatum). R. parkeri remained a relatively obscure rickettsia for the next several decades. Was viewed as non-pathogenic. The maculatum tick , has a distribution in the United States that extends across all states bordering the Gulf of Mexico as well as several other southern, mid-Atlantic, and central states, including Georgia, Kansas, Kentucky, Maryland, North Carolina, Oklahoma, South Carolina, Tennessee, and Virginia

Rickettsia parkeri In 2004, an otherwise healthy US serviceman living in Virginia presented to an acute care clinic with fever, mild headache, malaise, and multiple eschars on his lower extremities. He reported frequent tick and flea exposures. Rickettsia parkeri, a tick-associated rickettsia was subsequently isolated, documenting the first recognized case of Tidewater spotted fever.

Rickettsia parkeri In 2006, another US serviceman presented with similar symptoms. He had recently returned from a vacation in the Virginia Beach area. To date there has been over 25 cases of Tidewater spotted fever diagnosed in the United States as well as South America

Clinical signs and symptoms of Rickettsioses The clinical signs and symptoms of spotted fever group rickettsioses begin 6 to 10 days after the bite of an infected arthropod and typically include fever, headache, myalgias, a characteristic inoculation eschar at the bite site , a macular or maculopapular rash. Spotted fever group rickettsioses in humans range from mild to sometimes life threatening. Rocky Mountain spotted fever is considered the most severe spotted fever group rickettsiosis, with more than 50% mortality without adequate antibiotic treatment . Death from the rickettsioses can be mostly avoided if timely diagnosed and properly treated.

The Study As part of ongoing prospective, clinical rickettsial disease study in Virginia and Florida, one of the primary objectives is to identify the prevalence for R. parkeri among individuals presenting for care with tick bite eschars or clinically diagnosed rickettsial illness.

Subjects Subject 1: a 43 year-old man, presented to his primary care clinic in early June 2011 after developing an eschar on the lateral aspect of his left knee, where he had removed an embedded tick 8 days previously Whole blood and a swab of the unroofed eschar specimens were collected at the time of presentation with the acute febrile illness prior to doxycycline administration. DNA preparations were applied to the Rickettsia genus-specific quantitative real-time polymerase chain reaction (qPCR) as well as the R.parkeri species-specific qPCR assay Positive reactions were obtained from all three qPCR assays for the swab sample, and Rick17b assay for the buffy coat. ELISA was performed with the acute and convalescent plasma samples placed side by side in 4-fold dilutions from 1:100 to 1:6400

Subjects Subject 2: was a 36 year-old man, otherwise healthy, who developed a pain less eschar on the inner aspect of his left lower extremity approximately 4 days following exposure to the tick. Serum sample was collected at the time of his initial clinic visit, prior to initiation of antibiotic therapy. He returned to the clinic and remained asymptomatic after 14 days treatment. Whole blood and a swab of his healing eschar –the crust was unroofed when sampling- were taken at his return. The DNA of the acute serum obtained from his initial visit was negative by all qPCR assays Swab of a healing eschar was obtained upon his return visit and yielded positive results. ELISA show four fold rise

Conclusion Both swabs from both patients were positive for R.parkeri In first patient CT values for the qPCR were 37.52 for the blood and 31.10 for the swab indicating more DNA form swab In the second patient acute blood was negative however a healing swab was positive To our knowledge this represents the first report of obtaining positive reaction results from a healing eschar 14 days post antibiotic treatment

Conclusion Swabs from eschars appear to yield better results then blood draws and are less invasive than blood draws. To date 79 patients screened: 27 enrolled and 3 positive results. 11% positivity rate of enrolled patients. R. parkeri should be considered a causative agent of a rickettsiosis where single or multiple eschars are present. In addition those eshcars can be tested for the presence of rickettsial DNA prior to presentation of illness all the way through convalescence (at least 14 days following acute illness).

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