1. ¹Q Fever Research Group, IMVS, Adelaide, South Australia, Australia
2. ²Haematology and Transplant Service, Australian Red Cross Blood Service, Adelaide, South Australia, Australia

Correspondence: BP Marmion, Institute of Medical and Veterinary Science, Infectious Diseases Laboratories, Frome Rd, Adelaide, South Australia 5000, Australia. E-mail: kjhelbig@hotmail.com

Abstract

Acute primary Q fever is followed by various chronic sequelae. These include subacute Q fever endocarditis, granulomatous reactions in various organs or a prolonged debilitating post-infection fatigue syndrome (QFS). The causative organism, Coxiella burnetii, persists after an initial infection. The differing chronic outcomes may reflect variations within cytokine and accessory immune control genes which affect regulation of the level of persistence. As a preliminary test of the concept we have genotyped QFS patients and controls for gene variants spanning 15 genes and also examined HLA-B and DR frequencies. QFS patients exhibited a significantly increased frequency of HLA-DR-11 compared with controls and also significant differences in allelic variant frequencies within the NRAMP, and IFN genes. These results indicate a possible genetic role in the expression of overt chronic Q fever. Further studies will be undertaken to increase sample sizes, to survey other forms of chronic Q fever and to examine Q fever patients who have recovered without sequelae.

Q fever is Australia's commonest and most expensive occupational zoonosis, both in terms of patient morbidity and cost to industry and health services. The causative organism (Coxiella burnetii) multiples in the macrophage phagolysosome. In cultured cells it is not directly cytopathic and grows unchecked to form large cytoplasmic microcolonies. In vivo the cellular immune response (CMI) destroys infected cells; effector mechanisms involve generation of IFN, TNF and activation of the apoptosis pathway.¹

After the acute primary infection, the coxiella persists at low level in humans as evidenced, for example, by persistence of antibody and detection of C. burnetii genomic DNA in bone marrow cells from a majority of patients for 5 years or more after the initial illness.² Mostly, the persistent infection produces no overt illness, presumably because of a balanced regulation of the infection by the cellular and humoral immune systems. In a minority, balance is not achieved or maintained and chronic illness results. This may take the form of either a prolonged incapacitating post-infection fatigue syndrome (QFS),³ 8-15% of acute cases, or of recrudescent granulomatous lesions in liver, testis, bone, lung, pleura or soft tissues, or of infection of a heart valve with surface or intra-stromal vegetations. A hypothetical paradigm⁴ for the study of the chronic Q fever sequelae, derived by analogy from mycobacterial host-parasite relationships in which long-term, low-level bacterial persistence is also a feature, is one of anergic or hyper-sensitive polar disease states with some intermediate 'reactive' stages.

At the anergic pole is subacute Q fever endocarditis with low indices of cell-mediated immunity (CMI), such a lymphocyte mitogenic responses to Q fever antigens.^5,6 Scanty inflammatory infiltrates but large numbers of coxiellas are seen in valve vegetations, and generally there are very high levels of serum antibody. The coxiellas in valves are readily detected by animal inoculation, cell culture or PCR gene amplification. Studies by French workers^7,8 of cytokine response or release patterns from monocytes or PBMC from endocarditis patients have shown upregulated IL-10 and TNF production, downregulated IFN responses with increased TNF-R 75 production.

Intermediate stages are recrudescent granulomatous lesions in organs (testis, lung, etc), bone or in soft tissues. In contrast to endocarditis, there is marked lymphocytic infiltration of tissue, with activated macrophages (polykaryocytes/giant cells) and even cell necrosis. There are few coxiellas on microscopy although PCR detection of genomic sequences is positive. There is little information on attempts to culture the coxiella although positive results have been reported from liver.⁹ Medium levels of IgG class and, variably, some IgM antibody to Q fever antigens are present. Probably recrudescent infection at the end of pregnancy has a pattern between the anergic and intermediate stages with cellular infiltration and necrosis of placental cotyledons, easily demonstrable coxiellas, and infection of the foetus or newborn. The granulomatous state may be a 'clearing reaction' for the coxiella. There appear to be no reports of cytokine response patterns associated with granulomatous disease but on theoretical grounds upregulation of IFN and TNF might be expected.

The post-infection fatigue syndrome after Q fever (QFS) is hypothesised to be the CMI hypersensitivity pole of chronic Q fever. There is no overt organ involvement as in the granulomatous states; levels of coxiella in tissues (bone marrow or PBMC) are very low; medium levels of antibody in the IgG class to the two Q fever phase antigens are present. Cytokine response studies with PBMC in short-term culture have shown cytokine dysregulation with high IL-6, variable IFN, low IL-10 and IL-2 values.⁴

Variations in human immune response and other genes are well recognised to influence the course of infection, particularly by obligate intracellular pathogens which have a similar cell dependency to that of C. burnetii. The relation between immune response gene variation and disease outcomes may be clarified by the analysis of polymorphic variations in individual 'candidate genes' deduced to be of direct importance in disease pathogenesis.^10,11,12 This approach has been applied extensively to mycobacterium spp. infections and has linked disease severity either in individuals, or susceptibility of subpopulations to infection, with polymorphic variants within a number of genes, including natural resistance associated macrophage protein (NRAMP1), mannose-binding lectin (MBL), vitamin D receptor (VDR) and various cytokine genes.¹³

Given the broad similarities in the host–parasite relationships between C. burnetii and pathogenic mycobacteria taken together with the observed variations in cytokine response patterns between the various chronic sequelae of Q fever, we postulated that polymorphic variations in cytokine and accessory immune control genes may determine symptomless recovery or chronic illness after Q fever.

As a preliminary test of this concept, we have compared variability in phenotype distribution among a range of cytokine and accessory immune response genes in patients with post-Q fever fatigue syndrome and control subjects. This was done as an initial broad-based screening method to identify possibly significant polymorphic gene variants for further detailed and functional studies and as a prelude to examination of other chronic sequelae in Q fever. Twenty-three patients, described previously,⁴ and fulfilling the criteria for active or recovered QFS were recruited, informed consent obtained, and a blood donation taken; PBMC samples were separated on Lymphoprep gradients and stored in liquid nitrogen. DNA extraction was performed on PBMC samples.¹⁴ All patients and healthy controls were Caucasian.

Genotyping for all known polymorphic variants within the NRAMP1 gene was performed as described by Liu et al.¹⁵ Although no statistically significant variation was seen between the frequency of the individual single nucleotide polymorphisms (SNP) between patients and controls, Table 1 illustrates that in total there were statistically more variants differing from the wild type in the patient population than in the controls.

Table 1 - NRAMP1 gene variants in patients and control subjects.

Full table

HLA typing was performed for HLA-DR and HLA-B alleles. Differences between control subjects and patients were observed between allelic frequencies of HLA-DR alleles, and reached statistical significance with HLA-DR11 (see Table 2) but not with HLA-B alleles. The HLA region has been studied extensively with respect to disease susceptibility to infective agents, with a few associations being linked to the HLA-DR11 region (see Discussion).

Table 2 - Distribution of HLA-DR frequencies.

Full table

Sequence specific priming polymerase chain reaction (SSP-PCR) was utilised to phenotype 23 patients for a large panel of single nucleotide polymorphic (SNP) variants present in cytokine and other accessory genes of cellular immune function. SSP-PCR is a useful technique for initial typing of large numbers of SNP within candidate genes. The technique involves varying the terminal 3' nucleotide to gain allelic specificity. When two or more polymorphic sites are present within the one gene, forward and reverse allelic specific primers are used.¹⁷ In total, 25 polymorphic variants were analysed spanning 14 genes. Genes examined for polymorphic variants included MBL, VDR, IL-1RA, IL-1, IL1-R1, IL-4, IL-6, IL-10, TGF, INF, ICAM1, TNF, E-selectin and L-selectin. It was found to be essential to confirm potentially positive results by sequencing products to confirm base changes. Overall, phenotype frequencies did not depart significantly from control population values.

Functionally significant CA microsatellite variants in the IL-10 promoter and in intron 1 of the IFN gene were also typed utilizing a combination of sequencing and Gene Scan technologies (Applied Biosystems). Significant variation from published control data was not observed for IL-10 G and R microsatellite variants in the QFS group. However, variations in allele distribution were observed between QFS and control groups for the INF dinucleotide repeat ( Table 3a and b).

Table 3 - (a) Comparison of QFS patients and a group of Caucasian control subjects for the frequency distribution of allele #2 homozygotes and heterozygotes in the intron 1 INF microsatellite(b) INF microsatellite genotype distributions in QFS patients vs a group of Caucasian controls.

Full table

Comparison of the expected values for allele 2 homozygotes and heterozygotes in the QFS group predicted from the distribution in the control group, with the values observed in the QFS patients, revealed that the latter were more often homozygous (2/2) and less often heterozygous (2/3) at the gene allele 2 locus than control subjects (², df 3, P=0.0053).