WASHINGTON — Several rapid diagnostic tools for detecting the unique profiles of both common pathogens and bioterrorism pathogens in exhaled breath and body fluids may soon be tested on patients in emergency departments, speakers said at a biodefense research meeting sponsored by the American Society for Microbiology.
In a real bioterrorism (BT) event, physicians in multiple disciplines will likely collaborate, but in particular, it will be emergency department, hospital, and research laboratory personnel who will use such diagnostic equipment in real clinical settings, said Dr. Richard E. Rothman, of the emergency medicine department at Johns Hopkins University, Baltimore.
“The current diagnostic tools that we use in clinical practice are really limited” in terms of a prolonged wait time for results and sampling problems such as multiple blood draws and tedious steps, he said.
From the perspective of an emergency physician, he said, the key diagnostic questions that physicians need to ask routinely when patients come to the ED include, “Is there an infection? What is the organism? Is it uncommon? Could it be a BT agent?”
Dr. Rothman and his associates have developed several large-scale institutional review board protocols to sample breath condensates initially in animals, then in volunteers, and ultimately in ED patients. Other protocols have been developed to collect excess sera, cerebrospinal fluid, or nasopharyngeal aspirates from large numbers of ED patients and compare the results of standard microbiologic assays with those of new assays under development.
The rationale for analyzing breath condensates is based on evidence that pathogens in the respiratory system may elicit a host response that can be detected in the breath of an infected patient. Analyses of the patient's immune response could potentially discriminate between bacterial and viral infections and possibly determine what microbe is causing the symptoms.
In vitro studies have shown that specific cytokines and other inflammatory mediators from murine macrophages are activated in a time-dependent manner in the presence of bacterial lipopolysaccharides.
Researchers collaborating with Dr. Rothman have used a mask device called the ECoScreen to hold condensate for analysis by a mass spectrometer. The investigators also have developed their own mask to collect exhaled breath condensate.
In one study, collections of breath condensates from a group of 60 piglets that were exposed to bacteria showed a peak in interleukin-2 as early as 1 hour after exposure to Staphylococcus aureus enterotoxin B—long before the onset of symptoms, Dr. Rothman said.
The work has already shown that animals have unique cytokine profiles to different pathogens, and “now we're moving on to try to define baseline mass spectrometry profiles in healthy humans” and choose an optimal device for sampling, he said. From there, Dr. Rothman and his colleagues plan on sampling ED patients who are healthy or have acute respiratory symptoms.
An ideal diagnostic assay for detecting microorganisms would be sensitive and specific, applicable to multiple types of specimens, able to detect common organisms and BT agents, and useful as a triage tool, Dr. Rothman said.
Several years ago, Dr. Rothman conducted a pilot study with Charlotte A. Gaydos, Dr.P.H., to determine if a group of 51 febrile intravenous drug users were bacteremic when they arrived at the ED. At the time, most of these patients were hospitalized while diagnostic tests were run. The study findings suggested that it might be possible to detect bacteremia by running polymerase chain reaction (PCR) assays for 16S ribosomal RNA, which is highly conserved among bacteria. Different species of bacteria have differences in the 16S nucleotide sequence.
The PCR assay had 87% sensitivity and 87% specificity for bacteremia, compared with blood culture; all eight patients who had culture-positive infective endocarditis also were determined to be positive by PCR (J. Infect. Dis. 2002;186:1677–81).
Real-time PCR assays that detect single category A BT agents already exist. But in real BT events the particular agent isn't known. It is better to be able to detect multiple pathogens in a single reaction, Dr. Rothman said.
Dr. Rothman and Dr. Gaydos have developed universal primers and probes to use in their quantitative, real-time PCR assay for 16S ribosomal RNA. They also designed species-specific primers and probes to detect agents commonly seen in ED and infectious disease settings or in BT events.
The assay was able to detect 25 common bacterial pathogens (such as Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, Listeria monocytogenes, Streptococcus agalactiae, and Staphylococcus aureus) and inactivated BT agents (such as Bacillus anthracis, Francisella tularensis, Yersinia pestis, and Brucella species) in about 3 hours and 45 minutes, compared with 24 or more hours for detection by culture, said Dr. Gaydos, of the school of public health at Johns Hopkins.