New Method Can Rapidly Detect Potential Bioterror Agent causing Q fever

New Method Can Rapidly Detect Potential Bioterror Agent causing Q fever
Modified Georgia Institute of Technology release
By Jane Sanders, Georgia Institute of Technology
September 6, 2005

Bacterial Bioterror: New Method Can Rapidly Detect Potential
Bioterror Agent and Pinpoint Bacterial Strain

A new combination of analytical chemistry and mathematical data analysis
techniques allows the rapid identification of the species, strain and
infectious phase of the potential biological terrorism agent Coxiella
burnetii. The bacterium causes the human disease Q
fever, which can cause serious illness and even death.

Research by the Georgia Institute of Technology and the Centers
for Disease Control and Prevention (CDC) has yielded a method that
proved to be 95.2 percent accurate in identifying and classifying Coxiella
burnetii. The laboratory test delivers results in about five minutes
compared to about two hours for the lab technique currently used to detect
this bacterium.

This micrograph shows the bacterium Coxiella burnetii up close. The highly infectious bacterial species causes the human disease Q fever, which can cause serious illness and even death. It is listed by the CDC as a potential bioterrorism agent. Q fever from the Centers for Disease Control and Prevention Q fever is a zoonotic disease caused by Coxiella burnetii, a species of bacteria that is distributed globally. In 1999, Q fever became a notifiable disease in the United States but reporting is not required in many other countries. Because the disease is underreported, scientists cannot reliably assess how many cases of Q fever have actually occurred worldwide. Many human infections are inapparent. Cattle, sheep, and goats are the primary reservoirs of C. burnetii. Infection has been noted in a wide variety of other animals, including other species of livestock and in domesticated pets. Coxiella burnetii does not usually cause clinical disease in these animals, although abortion in goats and sheep has been linked to C. burnetii infection. Organisms are excreted in milk, urine, and feces of infected animals. Most importantly, during birthing the organisms are shed in high numbers within the amniotic fluids and the placenta. The organisms are resistant to heat, drying, and many common disinfectants. These features enable the bacteria to survive for long periods in the environment. Infection of humans usually occurs by inhalation of these organisms from air that contains airborne barnyard dust contaminated by dried placental material, birth fluids, and excreta of infected herd animals. Humans are often very susceptible to the disease, and very few organisms may be required to cause infection. Ingestion of contaminated milk, followed by regurgitation and inspiration of the contaminated food, is a less common mode of transmission. Other modes of transmission to humans, including tick bites and human to human transmission, are rare. Signs and Symptoms in Humans Only about one-half of all people infected with C. burnetii show signs of clinical illness. Most acute cases of Q fever begin with sudden onset of one or more of the following: high fevers (up to 104-105° F), severe headache, general malaise, myalgia, confusion, sore throat, chills, sweats, non-productive cough, nausea, vomiting, diarrhea, abdominal pain, and chest pain. Fever usually lasts for 1 to 2 weeks. Weight loss can occur and persist for some time. Thirty to fifty percent of patients with a symptomatic infection will develop pneumonia. Additionally, a majority of patients have abnormal results on liver function tests and some will develop hepatitis. In general, most patients will recover to good health within several months without any treatment. Only 1%-2% of people with acute Q fever die of the disease. Chronic Q fever, characterized by infection that persists for more than 6 months is uncommon but is a much more serious disease. Patients who have had acute Q fever may develop the chronic form as soon as 1 year or as long as 20 years after initial infection. A serious complication of chronic Q fever is endocarditis, generally involving the aortic heart valves, less commonly the mitral valve. Most patients who develop chronic Q fever have pre-existing valvular heart disease or have a history of vascular graft. Transplant recipients, patients with cancer, and those with chronic kidney disease are also at risk of developing chronic Q fever. As many as 65% of persons with chronic Q fever may die of the disease. The incubation period for Q fever varies depending on the number of organisms that initially infect the patient. Infection with greater numbers of organisms will result in shorter incubation periods. Most patients become ill within 2-3 weeks after exposure. Those who recover fully from infection may possess lifelong immunity against re-infection. Treatment Doxycycline is the treatment of choice for acute Q fever. Antibiotic treatment is most effective when initiated within the first 3 days of illness. A dose of 100 mg of doxycycline taken orally twice daily for 15-21 days is a frequently prescribed therapy. Quinolone antibiotics have demonstrated good in vitro activity against C. burnetii and may be considered by the physician. Therapy should be started again if the disease relapses. Chronic Q fever endocarditis is much more difficult to treat effectively and often requires the use of multiple drugs. Two different treatment protocols have been evaluated: 1) doxycycline in combination with quinolones for at least 4 years and 2) doxycycline in combination with hydroxychloroquine for 1.5 to 3 years. The second therapy leads to fewer relapses, but requires routine eye exams to detect accumulation of chloroquine. Surgery to remove damaged valves may be required for some cases of C. burnetii endocarditis. Further information at http://www.cdc.gov/ncidod/dvrd/qfever/

“Because of its potential use as a bioweapon, we needed a method
to detect Coxiella burnetii at an early stage, and we needed
to be able to determine which strain is present so authorities can determine
the geographic area from which it came,” said Facundo
Fernandez, an assistant professor in the School
of Chemistry and Biochemistry at Georgia Tech. He presented the research
team’s findings Sept. 1 at the 230th American Chemical Society National
Meeting in Washington, D.C.

Fernandez and his Ph.D. student Carrie Young, a chemist in the CDC’s
Environmental Health Lab, collaborated with CDC researchers in the National
Center for Environmental Health and the National
Center for Infectious Diseases. They combined mass spectrometry —
an analytical technique to study ionized molecules in the gas phase —
and a mathematical data analysis technique called partial least squares
analysis.

Mass spectrometry allows researchers to look at the profiles of different
proteins expressed in a microorganism. Partial least squares analysis
lets researchers separate important information from “noise”
— or biological baseline shifts caused by sample preparation variations
— that could corrupt a predictive model.

Not only is the combination of these techniques into one method a novel
concept, this research also represents the first time that Coxiella
burnetii has been detected at the strain level with a rapid detection
process, Fernandez noted. Such classification is a challenging task with
bacteria, he added. Researchers believe the technique also will work with
other pathogens, which they expect to begin studying this fall.

Coxiella burnetii is a species of concern because it causes
the highly infective human disease Q fever, which is transmitted primarily
by cattle, sheep and goats. A human can be infected by as few as one bacterium.
The disease can be manifested as a chronic or acute case, depending on
the strain. Symptoms can include high fever, severe headache, vomiting,
diarrhea, abdominal pain and chest pain. Q fever can also lead to pneumonia
and hepatitis. The chronic form of the disease can cause endocarditis,
an infection of a heart valve, and even lead to death.

In addition to being a public health threat, Coxiella burnetii
is listed as a Category B bioterrorism agent because of its long-term
environmental stability, resistance to heat and drying, extremely low
infectious dose, aerosol infectious route and history of weaponization
by various countries, according to the CDC.

To date, Georgia Tech and CDC researchers can differentiate between
seven Coxiella burnetii strains, which come from Australia, the
United States and Europe. Some strains are more infective than others,
and the researchers’ method determines not only the strain, but
whether it’s a Phase I or II strain depending on its ability to
infect, Fernandez explained.

Research by Georgia Tech and the CDC has yielded a rapid testing method that is 95.2 percent accurate in identifying and classifying the potential bioterrorism agent Coxiella burnetii. The method combines mass spectrometry, being done here by Ph.D. student Carrie Young, and mathematical data analysis techniques.

“The next step is to fine tune our model and increase the number
of strains we can identify,” Fernandez said. “There is a library
of strain samples available to us, though the samples are sanitized with
gamma radiation and rendered inactive before analysis.” To identify
strains, researchers examine the appearance of biomarker proteins in samples.

“In some cases, we classify a strain by the presence or absence
of a biomarker. And sometimes we see the same biomarker proteins, but
at varying levels, in different strains,” Fernandez noted.

The researchers’ detection technique is highly sensitive, meaning
it can detect Coxiella burnetii strains at very low concentrations
– specifically at the attomole level, which is equivalent to 1 X
10-17 moles. (Moles measure the actual number of atoms or molecules in
a sample.)

Until now, the best method to differentiate between strains of Coxiella
burnetii was a laboratory technique called polymerase chain reaction
(PCR), which analyzes the genes of a bacterium and yields results in one
to two hours. The new method, which analyzes the proteins of a bacterium,
can yield results in five minutes. For now, it is also a laboratory test,
though separate research involving Fernandez and other Georgia Tech researchers
is pursuing development of a field-testing instrument.

Georgia Tech Assistant Professor of Chemistry and Biochemistry Facundo Fernandez, center, explains a mass spectrometry procedure to graduate students in his lab. Fernandez and Ph.D. student Carrie Young, second from left, use mass spectrometry and mathematical data analysis techniques to rapidly identify strains of the potential bioterrorism agent Coxiella burnetii.

“In a bioterrorism event, you want more than one method to determine
the strain you are dealing with,” Fernandez noted. “So you
would use our technique first and then use PCR as a second method to independently
confirm your results. Also, our method using mass spectrometry, allows
you to quickly replicate your analysis – even 10 times if you want
to. That gives you an added degree of statistical significance.”

Fernandez and his colleagues began the research in June 2004 with funding
from the CDC and a Georgia Tech Research Corporation seed grant. With
their encouraging results about the method’s capability, they plan
to apply for additional federal funds in the near future.

Working with Fernandez and Young are John Barr and his colleagues Adrian
Woolfitt and Hercules Moura of the National Center for Environmental Health,
and Edward Shaw (now at Oklahoma State University) and Herbert Thompson
of the National Center for Infectious Diseases.

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MEDIA RELATIONS CONTACTS:
1) Jane Sanders (404-894-2214); E-mail: jane.sanders[AT] edi.gatech.edu);
Fax (404-894-4545)
2) John Toon (404-894-6986); E-mail: (john.toon [AT] edi.gatech.edu)
3) National Center for Environmental Health, CDC (404-498-0070); E-mail:
(atsdric [AT] cdc.gov)

TECHNICAL CONTACTS:
1. Facundo Fernandez, Georgia Tech (404-385-4432); E-mail: (facundo.fernandez [AT] chemistry.gatech.edu)
2. John Barr, CDC (770-488-7848); E-mail: (JBarr [AT] cdc.gov)

WRITER: Jane Sanders

This is a modified news release from the Georgia Institute of Technology. The original can be found at Bacterial Bioterror: New Method Can Rapidly Detect Potential Bioterror Agent and Pinpoint Bacterial Strain.