Q J Med 2002; 95: 832-833
© 2002 Association of Physicians
Correspondence |
Q fever: still a mysterious disease
Q Fever Research Group, Adelaide, Australia
Sir,
In the concluding paragraph of his editorial,1 Dr Raoult makes a statement about the dangers of false-positive PCR assays arising from DNA contamination during the detection of Coxiella burnetii. The caution references a study2 from our Q fever Research Group. The general reader is likely to conclude that the artifacts mentioned by Raoult invalidate our observations. We urge the reader to study the actualities of our report before deciding whether Raoult's comments have any validity for our paper.
In relation to long-term carriage of C. burnetii, we make two main points. First, there is ample evidence from the 1940s onwards, based on the isolation of the organism from humans and animals, that C. burnetii may persist in the host after an initial infection. Persistence of infection is necessary for recrudescence: e.g. some years later as Q-fever endocarditis or in late pregnancy after an infection up to 3 years previously.3 Harris et al. addressed the question of how often infection persists and where in the body; the report did not claim that persistence is the sole and sufficient cause for the post-Q-fever fatigue syndrome (QFS), because at that stage we had not examined enough people who had had acute Q fever but who had not developed a chronic sequel.
Second, under Methods and in the Discussion we made it abundantly clear (based on experience dating from the late 1980s) that we are well aware of the dangers of DNA cross-contamination of PCR reactions from amplicon, control positive suspensions or positive specimens. We used primer sets directed at two different targets in the IS1111a insertion sequence and one in the superoxide dismutase gene—thereby in fact meeting Raoult's stipulations. We do not use ‘water’ controls but non-template controls—i.e. the complete reaction mix without added target DNA. Typically, a third of the tubes under test were/are non-template controls, distributed among the test samples, and left open while the tests are set up. All must be negative for a test result to be accepted. PCR products were identified by probing in Taqman or Rotor Gene instruments, and in some instances by sequencing. The use of a positive control target with an inserted sequence not present in wild strains, or a strain (e.g. Henzerling Q Vax) with a natural unique polymorphism, are other checks on cross-contamination. ‘Multiroom containment’ was described.
The post-Q-fever fatigue syndrome was described as long ago as 1996.4,5 The time is well past for sceptical opinion from the sidelines based on experience in unrelated Q fever research. We submit that it is now time for Dr Raoult's group to follow accepted scientific process and to attempt to confirm our results locally now that they have identified the fatigue syndrome (QFS=‘asthenia Q fever’) in French patients. It is necessary to follow patients systematically for more than two years after the initial acute infection. We agree (and have reported earlier) that a substantial proportion of primary acute Q fever patients have similar symptoms to QFS—essentially a downsized version of the acute phase symptoms without the fever—for 6 to 9 months after the acute attack, and then recover. It is the small proportion, some 8–10%, who exhibit similar symptoms but do not reach immune or other homeostasis after one year or longer that constitute the serious social and medical problem. They are the proper group for comparative studies.
Finally, in turn, we caution Dr Raoult that PCR detection of small numbers of C. burnetii in the presence of highly inhibitory human DNA is considerably more difficult than the simple examination of endocarditis vegetations or placenta which have large target numbers. Despite its theoretical sensitivity, the IS1111a PCR gives variable results with the tissue samples. Currently, we find the nested PCR with a target in the com1 gene described by Zhang et al.6 and used by Kato et al.7 for blood or serum appears to offer a way round the inhibition problem with tissue extracts. Sequencing of amplicon is mandatory for final verification of the presence of C. burnetii genomic sequences.
References
1. Raoult D. Q fever: still a mysterious disease.Q J Med2002; 95:491–2.
2. Harris RJ, Storm PA, Lloyd A, Arens M, Marmion BP. Long-term persistence of Coxiella burnetii in the host after primary Q fever. Epidemiol Infect2000; 124:543–9.[Medline]
3. Syrucek L, Sobeslawsky O, Gutwick I. Isolation of Coxiella burnetii from human placentas. J Hyg Epidemiol Microbiol Immunol1958; 2:29–35.[Medline]
4. Marmion BP, Shannon M, Maddocks I, Storm PA, Penttila IA. Protracted debility and fatigue after acute Q fever. Lancet1996; 347:977–8.[Medline]
5. Ayres JG, Flint N, Smith EG. Chronic fatigue syndrome after acute Q fever. Lancet1996; 347:978–9.[Web of Science][Medline]
6. Zhang GQ, Nguyen H, To H, Ogawa M, Hotta A, Yamaguchi T, Kim HJ, Fukushi H, Hirai K. Clinical evaluation of a new PCR assay for detection of Coxiella burnetii in human serum samples. J Clin Microbiol1998; 36:77–80.
7. Kato K, Arashima Y, Asai S, Furuya Y, Yoshida Y, Murakami M, et al. Detection of Coxiella burnetii specific DNA in blood samples from Japanese patients with chronic nonspecific symptoms by nested polymerase chain reaction. FEMS Immunol Med Microbiol1998; 21:139–44.[Web of Science][Medline]
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