Abstract
In 2002 it was decided to establish laboratory facilities in Denmark for diagnosing agents associated with bioterrorism in order to make an immediate appropriate response to the release of such agents possible. Molecular assays for detection of specific agents and molecular and proteomic techniques for identification of bacteria were introduced as part of the program. All assays and techniques were made accessible for use in diagnosing patients, even when an intentional release was not suspected. Medical expertise on different diseases was established at the department as an integrated part of the program. The analyses included PCR assays for specific bacteria, identification of isolated bacteria by DNA sequencing, detection and identification of bacteria in clinical sample material by universal bacterial PCR and DNA sequencing, and identification of bacteria by mass spectrometry. The established analyses formed a basis on which a series of further developments was built. In addition to reducing the time for obtaining diagnoses and improving the accuracy of diagnosis of individual infected patients, the analyses provided new knowledge on the frequency and distribution of some bacterial infections, including Q fever, tularemia, trench fever, brucellosis, and melioidosis. The implementation of an antibioterrorism program in a clinical diagnostic setting improved the diagnostic possibilities for patients in Denmark and provided new epidemiologic information. It also introduced a number of diagnostic assays for bacterial infections not associated with bioterrorism that are difficult to culture or identify.
Laboratory facilities were established in Denmark to diagnose agents associated with bioterrorism. These facilities were also used in patient diagnosis, with the result that diagnoses were obtained more quickly, accuracy was improved, and new knowledge about bacterial infections was brought to light, thus improving epidemiologic information as well.
The analyses for bacteria, viruses, and toxins were established in existing laboratories at SSI, using knowledge and laboratory facilities that were already present. Diagnosis of bacterial agents of bioterrorism was located in the department for clinical microbiology. The laboratories in this department had profound experience in identifying bacteria and treating bacterial infections, because it hosted both the National Reference Laboratory for Identification of Bacteria and a routine laboratory for clinical microbiology. The department provided local hospitals and general practitioners with in vitro diagnostics and counseling on the management of infections. Biosafety level 3 (BSL-3) laboratories were available at the institution. Other diagnostic laboratories in Denmark could refer isolated bacteria, including suspected bioterrorism agents, to the reference laboratory for urgent identification free of charge.
Because of the legal and political aspects of detecting an intentional release of biological agents, the program included a strategy for using several independent technologies to identify bacteria whenever possible. The assays needed to be fast, safe, and accurate. Culture under appropriate safety conditions was supplemented with polymerase chain reaction (PCR) and antigen detection tests. DNA sequencing was introduced for the identification of isolated bacteria and later also for direct identification of bacteria in sample material. Finally, recognition of bacteria by mass spectrometry was established. The experience of establishing the new assays was further used for improvements in other areas of diagnostic bacteriology, including introduction of new PCR assays for rare infectious diseases and new targets for DNA sequencing.
Using the assays, a number of patients were diagnosed with rare infections. Rarely encountered bacteria were accurately identified and linked to clinical presentations. New geographical locations where specific infections had been acquired were recognized. This article summarizes the use of the new analyses, the further developments they initiated, and new discoveries based on this work.
Specific PCR Analysis
A number of PCR assays were established for the detection of bioterrorism-associated bacteria in the environment and in patients. The focus was on demonstrating essential virulence factors in order to distinguish between pathogenic and avirulent strains. The performances of the analyses were regularly tested by participation in external quality assessment systems. Among the analyses that were established were specific PCR assays for botulinum toxin genes (botulism), 1 Francisella tularensis (tularemia), 2 Coxiella burnetii (Q fever), 3 Bartonella henselae (cat scratch disease) and B. quintana (trench fever), 4 Brucella melitensis B. abortus, B. suis (brucellosis), 5 and Burkholderia pseudomallei (melioidosis). 6
Shortly after the establishment of PCR assays for botulinum toxin genes, the assays were used to confirm type E botulinum toxin as the cause of botulism in a case in Greenland in which someone had eaten home-fermented seal meat. 1
Francisella tularensis is a potential bioterrorism agent. On rare occasions, tularemia is contracted in Denmark, in particular on the island Bornholm. By using specific PCR assays, it was also confirmed in other parts of the country, including Zealand and Jutland. 2 Specific PCR assays for Coxiella burnetii and Bartonella quintana were used to confirm infective endocarditis caused by these agents (see below).
A number of infectious diseases associated with bioterrorism are infrequently brought into Denmark from other countries. Specific PCR assays were used to diagnose cases of melioidosis, 7 mostly imported from Thailand, and brucellosis from Asia and Africa. 5
Safety, speed, and accuracy are major advances obtained by using PCR. In cases of brucellosis, bacteria are typically isolated from blood at local laboratories. Handling of cultures of Brucella species represents a risk to laboratory staff and should be avoided in ordinary biosafety level 2 (BSL-2) laboratories. For confirmation of identity by PCR, cultures may be inactivated before DNA extraction, completely removing any risk of laboratory infection. The time before conclusive results are available is important, not only for rapid initiation of optimal treatment of the patient, but also to secure appropriate follow up on accidentally exposed laboratory staff members handling the isolated bacteria prior to identification; timeliness is also important for identifying other people who may have been exposed to food products suspected as the source of the infection. In contrast to agglutination by antisera, which was used earlier, species-specific PCR assays could accurately identify the Brucella species causing brucellosis in Denmark as B. melitensis. 5
Besides providing accurate identification of the causes in selected cases, the new assays for bioterrorism-associated bacteria assays proved useful in a number of other cases where suspicion of specific infections could be rejected when negative test results were obtained. Based on the experiences from establishing PCR assays for bacteria associated with bioterrorism, a number of new PCR assays were introduced for the detection of clinically relevant bacteria that are difficult to culture. These included Tropheryma whipplei (Whipple's disease), Rickettsia species (various rickettsioses; in Denmark, mostly African tick bite fever imported from South Africa), and Haemophilus ducreyi (chancroid). 8
It was essential to the department that the diagnostic assays should be supplemented by medical expertise on the diseases caused by the bacteria. Therefore, a unit for emerging bacterial diseases was established. The unit provided expert advice to clinicians and health authorities on diseases for which diagnostic tests were established. Thus, members of the unit participated in a work group under the National Health Agency when it was discovered that Danish cattle were infected by Coxiella burnetii, a bacterium that had not previously been found in Denmark. 9
Identification of Bacteria by DNA Sequencing
Identification of bacteria by DNA sequencing was introduced as a part of the program, and it was soon found to be a very valuable tool in the National Reference Laboratory for Identification of Bacteria.10–12 DNA sequencing can identify an isolated bacterium with high accuracy in 1 to 2 days. Traditional identification by biochemical tests takes longer—for some species, several weeks. By using DNA sequencing in combination with other techniques, bacteria not previously (or only rarely) found as causes of infections in humans were demonstrated to be true pathogens. These included Caulobacter species and Actinobaculum schaalii.13,14 For some rarely encountered bacteria, accurate identification by DNA sequencing helped to link them to a clinical presentation of infection.15–20 Caulobacter species, which was previously not regarded as a human pathogen, was found to be a cause of peritonitis as a complication to peritoneal dialysis; Granulicatella elegans in blood was associated with abdominal infections; and Actinobaculum schaalii had a very strong association to urinary tract infections, including urosepsis.
Partial or whole gene DNA sequencing of the 16S rRNA gene was found sufficient to identify many but not all clinically relevant bacteria, 11 and other targets were investigated for those that were not satisfactorily classified. For nonhemolytic streptococci, especially of the mitis group, a combination of partial sequencing of the 16S-23S rRNA intergene segment and the gdh gene were found to result in more accurate identifications. 21 This discovery will be helpful for the exact identification of the cause of infection in cases of infective endocarditis 22 and pleural empyemas. 23
Culture-Independent Identification of Bacteria in Clinical Samples
With slight modifications, the technology used for identification of bacteria by DNA sequencing can be used to directly identify bacteria in clinical samples. 24 Because the assay detects DNA from any bacterium present in the sample, it is best suited for samples from sites that are normally sterile. Results obtained from nonsterile sites, such as skin and mucosal surfaces, are difficult to interpret because detection of bacteria in these places does not necessarily represent an abnormal condition. The limitations and possibilities of the method have been discussed in detail elsewhere. 24
A 16S rRNA gene–based assay was offered as a routine diagnostic service for culture-negative samples. The usefulness of the assay was documented by a steadily increasing use by clinicians 25 and by identification of bacteria in selected patient populations such as children. 26 The technique allowed demonstration of anaerobic bacteria overlooked by culture 27 and was also used to establish cultured bacteria assumed to be skin contaminants as a real cause of infection. 28
It was found that by far most of the infections were due to well-known cultivable bacteria and that the reason for negative culture results was often administration of antibiotics prior to sampling. Occasionally, however, noncultivable bacteria and bacteria with special requirements for in vitro culture were demonstrated. These included osteo-articular infections by the sexually transmitted bacteria Treponema pallidum (syphilis) and Neisseria gonorrhoeae (gonorrhea) in patients in whom these pathogens were not expected. Also, a few cases of extrapulmonary tuberculosis were found by use of DNA sequencing of bacterial DNA PCR-amplified directly from clinical sample material. Kingella kinga is a fastidious bacterium that often primary fails to grow unless the sample material is inoculated directly into blood culture bottles. On several occasions this bacterium was demonstrated by the method in culture negative samples from small children suffering from infective arthritis.
Mycoplasma species are usually not detected by culture using standard conditions. DNA sequencing of bacterial DNA from patient material revealed Mycoplasma species in some infections, including a case of seal finger, a condition largely unknown in Denmark outside Greenland. 4
Other infections by noncultivable bacteria identified by DNA sequencing of PCR-amplified bacterial DNA included cases of infective endocarditis caused by B. quintana and C. burnetii. 3 The findings were immediately confirmed by the new species-specific PCR assays. These cases of infective endocarditis were acquired in Greenland, where the bacteria had not been detected before. Thus, the techniques provided new discoveries on the geographical distribution of Q fever and trench fever.
In each case of unexpected finding, substantial efforts were put into providing confirmation by use of specific PCR assays and other techniques, such as antibody and antigen detection or culture under appropriate conditions (eg, special media or atmosphere, propagation in cultured cell lines).
Targets other than the 16S rRNA gene were also tested and found useful in analyzing clinical samples. Thus, PCR and DNA sequencing of part of the 23S rRNA gene worked well in establishing the cause of infectious arthritis. 29
Mass Spectrometry
To speed up bacterial identification, matrix-assisted laser desorption/ionization time of flight mass spectrometry (Maldi Tof MS) was introduced with financial support from the antibioterrorism program. Mass spectrometry proved its value as it was used for fast, safe, and accurate identification of naturally acquired brucellosis. 30 Also, matrix-assisted laser desorption/ionization time of flight mass spectrometry was used for identification of non-terror-associated bacteria and eventually implemented for routine use in the laboratory.
Conclusion
In conclusion, the bioterrorism preparedness program was the driving force in a significant technical development in diagnostic bacteriology for the benefit of the health system in Denmark. The availability of specific PCR assays for agents associated with bioterrorism was used for rapid identification of cases of botulism, brucellosis, melioidosis, tularemia, Q fever, and trench fever. The latter 3 diseases had been acquired in areas where they were not expected, thus providing new insights into the geographical distribution of these infections.
Using the technology introduced by the program, assays for bacteria that are not associated with bioterrorism were established in the laboratory and used for diagnosing patients suffering from rickettsiosis, chancroid, and Whipple's disease. DNA sequencing was introduced in the laboratory as a method of identifying bacteria as a part of the program and was soon applied to isolated bacteria referred from other laboratories for identification. The accuracy obtained by DNA sequencing made identification of bacteria not (or rarely) associated with human infection possible. Identified bacteria, including Caulobacter species, Actinobaculum schaalii, Granulicatella elegans, and Aerococcus sanguinicola, could be linked to clinical presentation of infection, thus giving new informtion on the nature of these infections. Matrix-assisted laser desorption/ionization time of flight mass spectrometry was established as a very fast method of identifying baceria and was used for confirmation of brucellosis and other diseases.
The extension of the program into clinical usefulness and use of the experience obtained from the program to further development in clinical diagnostics was obtained by placing the program in a clinical setting and by close collaboration between molecular biologists and experienced medical doctors specialized in clinical microbiology.