| Abstract: | In the Netherlands, there is an ongoing and unparalleled outbreak of Q fever. Rapid and reliable methods to identify patients infected with Coxiella burnetii, the causative agent of Q fever, are urgently needed. We evaluated the performance of different DNA extraction methods and real-time PCR assays that are in use in seven diagnostic or reference laboratories in the Netherlands. A low degree of variation in the sensitivities of most of the developed real-time PCR assays was observed. However, PCR assays amplifying short DNA fragments yielded better results than those producing large DNA fragments. With regard to DNA extraction, the automated MagNA Pure Compact system and the manual QIAamp DNA mini kit consistently yielded better results than either the MagNA Pure LC system and NucliSens EasyMag (both automated) or the High Pure viral nucleic acid kit (manual). The present study shows that multiple combinations of DNA extraction kits and real-time PCR assays offer equivalent solutions to detect C. burnetii DNA in serum samples from patients suspected to have Q fever.Q fever is a worldwide zoonosis caused by Coxiella burnetii, an obligate intracellular bacterium (11). Whereas animals such as sheep and goats are generally asymptomatic carriers, infection with C. burnetii in these animals may become manifest by abortion. Although asymptomatic in ∼60% of infected persons, C. burnetii can cause serious illness in humans. Q fever can cause acute or chronic infection depending on the patient''s condition or immune status. Acute Q fever may present as a self-limiting flu-like atypical pneumonia accompanied by severe headache and sometimes hepatitis. Approximately 5% of all Q fever cases may progress in a chronic infection leading to life-threatening endocarditis (1, 3, 5, 7-9). C. burnetii is highly infectious and can survive for long periods in the environment. Human outbreaks have been associated with farms, slaughterhouses, and wind dispersion from farms where infected animals were kept. Ticks and pets, including cats and dogs, have also been demonstrated to be potential sources of Q fever (1, 4, 10).Laboratory diagnosis of Q fever is usually performed by serological methods such as the indirect immunofluorescence assay (IFA), complement fixation test (CFT), or enzyme-linked immunosorbent assay (ELISA), but these tests are of limited use in the early phase of the disease, as it may take up to 2 weeks for a detectable immune response to develop. Several PCR-based diagnostic methods, such as conventional PCR, nested PCR, or real-time PCR, have successfully been applied for the direct detection of C. burnetii DNA in clinical samples. The sequences targeted by these tests varied from plasmids (QpH1 or QpRS) to chromosomal genes, such as the isocitrate dehydrogenase gene (NADP) or the transposase gene of the C. burnetii IS1111a insertion element (3, 4, 14-16). The multicopy IS1111a insertion element is present in 20 copies in the genome of the C. burnetii Nine Mile RSA493 strain. Copy numbers per isolate vary and can reach up to ∼100 copies per genome (7). Due to the multicopy nature of this DNA element, it provides a highly sensitive target for detection of C. burnetii DNA in serum samples. Furthermore, real-time PCR can be useful for diagnosis of chronic Q fever, since in these patients C. burnetii DNA can be detected in serum over long periods of time (3).In the Netherlands, as of 2007, there is an unprecedented and ongoing outbreak of Q fever (12, 17). At present, more than 3,000 cases have been reported in the Netherlands. In order to improve diagnosis for Q fever, medical microbiology laboratories have implemented molecular methods to close the diagnostic gap between onset of the disease and the presence of specific antibodies in serum. The aim of this study was to compare the performances of different DNA extraction methods and real-time PCR assays, all targeting the C. burnetii IS1111a insertion element, that are being used in seven diagnostic or public health laboratories in the Netherlands. |
