The detection of Coxiella burnetii (Q fever) in clinical and environmental samples
Lockhart, Michelle (2010) The detection of Coxiella burnetii (Q fever) in clinical and environmental samples. PhD thesis, Murdoch University.
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The zoonotic intracellular bacterium Coxiella burnetii is the cause of the human disease Q fever. Coxiella burnetii can be shed by infected animals, can survive harsh environments and has been shown to persist within the human host. The detection and isolation of this bacterium is difficult due to its intracellular nature. In order to detect minimal concentrations of this bacterium in various clinical and environmental samples, highly sensitive assays were needed. A duplex real-time polymerase chain reaction (qPCR) assay was developed to detect C. burnetii DNA (targeting the Com1 gene and the IS1111a gene). This assay was then tested on a variety of environmental and clinical sample types.
Samples (such as water, soil, aerosols, blood and bone marrow) were spiked with C. burnetii (either living cell cultures or formalin killed cells) to determine the optimal method for extracting and detecting C. burnetii DNA. The silica column method followed by qPCR assay of the Com1 gene was shown to have a sensitivity of approximately 1100 copies/litre in water, 1900 copies/kg in soil, 870 copies/litre in milk, and seven copies/litre of air. When the same technique was applied to clinical samples the silica column method proved to be the most effective in purifying DNA from the small cell variant of C. burnetii and effectively removed potential PCR inhibitors from mock clinical samples of blood, plasma, serum and bone marrow. However, because the qPCR cannot differentiate between viable and non-viable C. burnetii DNA it was important to establish a sensitive assay for the detection of viable C. burnetii in order to investigate persistent infections and to obtain isolates of the bacteria from cases of Q fever for further studies.
As isolation of Coxiella can be achieved using cell culture or animal inoculation these methods were compared for their sensitivity for C. burnetii detection. Vero and DH82 cell lines were the most sensitive for cell culture isolation of the Arandale and Henzerling isolates of C. burnetii respectively. When cell culture was compared to PCR and inoculation of severely combined immunodeficient (SCID) mice it was found that inoculation of SCID mice followed by euthanasia (at day 42) and removal and analysis of the spleen was the most sensitive method for the detection of viable C. burnetii.
It has recently been hypothesised that genetic differences between isolates of C. burnetii are responsible for differences in pathogenicity and disease outcomes. Hence the differences between Australian isolates were investigated. Seven new Australian isolates of C. burnetii were genetically analysed by conventional PCR of insertion sequences and detection of the acute disease antigen A (adaA) gene. Six Australian isolates of C. burnetii were placed in geno-group III but were negative for the adaA gene. One new Australian isolate (Poowong) was placed in geno-group II and was positive for the adaA gene. The Poowong isolate was from a seronegative asymptomatic patient, with bacteraemia detected by PCR in four initial samples as well as all 12 blood samples taken over a one month period. Through sequencing of 468bp of the ankyrin gene (ankH sequenced in triplicate) it was shown that the Poowong isolate had two base pair differences compared to the Henzerling isolate (also genogroup II) and the Nine Mile isolate (geno-group I). This demonstrates that the Poowong isolate can be distinguished from the other isolates within the laboratory.
The optimal methods of detection as determined in this study were used to analyse and evaluate clinical specimens. Blood samples (serum, plasma and peripheral blood mononuclear cells) from 12 patients infected during an outbreak of Q fever in Newport UK in 2002 were examined. Cell culture of the peripheral blood mononuclear cells (PBMC) demonstrated that no viable C. burnetii cells were present. In contrast, six of the spleens from SCID mice inoculated with the PBMCs were positive for C. burnetii DNA (by Com1 qPCR) and six were positive for C. burnetii antigen (by IFA). However, only two were positive for both. This suggests that in some patients low numbers of viable C. burnetii cells persist and in others C. burnetii persist as nonviable antigen.
In conclusion, this study demonstrated sensitive and specific optimal methods for the detection of C. burnetii in clinical and environmental samples, the optimal method for isolation of C. burnetii, the application of these methods on a number of clinical samples and the characterisation of seven new isolates, including an isolate from a highly unusual asymptomatic case that is genetically unique from the others. This study has also shown that the pathogenesis of C. burnetii infection in humans and the effect of genetic differences in isolates on pathogenesis are far from adequately understood. The optimal methods of detection, isolation and grouping determined in this study will have an effect on future studies and will allow a greater understanding of C. burnetii and its persistence, both in the environment and in Q fever infections.
|Publication Type:||Thesis (PhD)|
|Murdoch Affiliation:||School of Veterinary and Biomedical Sciences|
|Supervisor:||Graves, Stephen, Stenos, John and Fenwick, Stan|
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