Transcript Presentation

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)
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Rickettsia rickettsii
Causes the most severe rickettsiosis
Aerosolization studies conducted in 1970s
Select Agent B list
– 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).
In regards to the previous presentation all
opinions are mine and are not official policy
of the DoD nor have they been sanctioned
by any Admiral, General or SES , so don’t call
or email anyone, most likely they will deny
ever knowing who I am.
The views expressed in this article are those of the author and do not necessarily reflect the official policy or position of the Department of the Navy, Department of
Defense, nor the U.S. Government.
I am a military service member (or employee of the U.S. Government). This work was prepared as part of my official duties. Title 17 U.S.C. §105
provides that ‘Copyright protection under this title is not available for any work of the United States Government.’ Title 17 U.S.C. §101 defines a U.S.
Government work as a work prepared by a military service member or employee of the U.S. Government as part of that person’s official duties.