A new combination of analytical chemistry and mathematical dataanalysis techniques allows the rapid identification of the species,strain and infectious phase of the potential biological terrorism agentCoxiella 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 forDisease Control and Prevention (CDC) has yielded a method that provedto be 95.2 percent accurate in identifying and classifying Coxiellaburnetii. The laboratory test delivers results in about five minutescompared to about two hours for the lab technique currently used todetect this bacterium.
“Because of its potential use as a bioweapon, we needed a method todetect Coxiella burnetii at an early stage, and we needed to be able todetermine which strain is present so authorities can determine thegeographic area from which it came,” said Facundo Fernandez, anassistant professor in the School of Chemistry and Biochemistry atGeorgia Tech. He presented the research team’s findings Sept. 1 at the230th American Chemical Society National Meeting in Washington, D.C.
Fernandez and his Ph.D. student Carrie Young, a chemist in the CDC'sEnvironmental Health Lab, collaborated with CDC researchers in theNational Center for Environmental Health and the National Center forInfectious Diseases. They combined mass spectrometry -- an analyticaltechnique to study ionized molecules in the gas phase -- and amathematical data analysis technique called partial least squaresanalysis.
Mass spectrometry allows researchers to look at the profiles ofdifferent proteins expressed in a microorganism. Partial least squaresanalysis lets researchers separate important information from “noise”-- or biological baseline shifts caused by sample preparationvariations -- that could corrupt a predictive model.
Not only is the combination of these techniques into one method anovel concept, this research also represents the first time thatCoxiella burnetii has been detected at the strain level with a rapiddetection process, Fernandez noted. Such classification is achallenging task with bacteria, he added. Researchers believe thetechnique also will work with other pathogens, which they expect tobegin studying this fall.
Coxiella burnetii is a species of concern because it causes thehighly infective human disease Q fever, which is transmitted primarilyby cattle, sheep and goats. A human can be infected by as few as onebacterium. The disease can be manifested as a chronic or acute case,depending on the strain. Symptoms can include high fever, severeheadache, vomiting, diarrhea, abdominal pain and chest pain. Q fevercan also lead to pneumonia and hepatitis. The chronic form of thedisease can cause endocarditis, an infection of a heart valve, and evenlead to death.
In addition to being a public health threat, Coxiella burnetii islisted as a Category B bioterrorism agent because of its long-termenvironmental stability, resistance to heat and drying, extremely lowinfectious dose, aerosol infectious route and history of weaponizationby various countries, according to the CDC.
To date, Georgia Tech and CDC researchers can differentiate betweenseven Coxiella burnetii strains, which come from Australia, the UnitedStates and Europe. Some strains are more infective than others, and theresearchers’ method determines not only the strain, but whether it’s aPhase I or II strain depending on its ability to infect, Fernandezexplained.
“The next step is to fine tune our model and increase the number ofstrains we can identify,” Fernandez said. “There is a library of strainsamples available to us, though the samples are sanitized with gammaradiation 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 abiomarker. And sometimes we see the same biomarker proteins, but atvarying levels, in different strains,” Fernandez noted.
The researchers’ detection technique is highly sensitive, meaning itcan detect Coxiella burnetii strains at very low concentrations –specifically at the attomole level, which is equivalent to 1 X 10-17moles. (Moles measure the actual number of atoms or molecules in asample.)
Until now, the best method to differentiate between strains ofCoxiella burnetii was a laboratory technique called polymerase chainreaction (PCR), which analyzes the genes of a bacterium and yieldsresults in one to two hours. The new method, which analyzes theproteins of a bacterium, can yield results in five minutes. For now, itis also a laboratory test, though separate research involving Fernandezand other Georgia Tech researchers is pursuing development of afield-testing instrument.
“In a bioterrorism event, you want more than one method to determinethe strain you are dealing with,” Fernandez noted. “So you would useour technique first and then use PCR as a second method toindependently confirm your results. Also, our method using massspectrometry, allows you to quickly replicate your analysis – even 10times if you want to. That gives you an added degree of statisticalsignificance.”
Fernandez and his colleagues began the research in June 2004 withfunding from the CDC and a Georgia Tech Research Corporation seedgrant. 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 colleaguesAdrian Woolfitt and Hercules Moura of the National Center forEnvironmental Health, and Edward Shaw (now at Oklahoma StateUniversity) and Herbert Thompson of the National Center for InfectiousDiseases.
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