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Diagnosis of Q fever using indirect microimmunofluorescence.

A microimmunofluorescence technique for the diagnosis of Q fever is described. Although this method is useful for serological diagnosis of Q fever, some technical difficulties are associated with it. First, the test antigens must be produced by a cell culture method in a level-3 biohazard facility and, second, the antigen Coxiella burnetii, which is the causative agent of Q, is characterized by the presence of two phases. To obtain phase I antigen, mice must be inoculated with C. burnetii. The materials obtained from the spleens of the infected mice are then used for cell inoculation. After purification, the antigens of both phases are deposited and fixed on multiple-well microscope slides. The serially diluted sera to be tested are incubated on these slides, then rinsed and overlaid with anti-IgG, -IgM and/or IgA secondary antibodies. Finally, the slides are examined under a fluorescence microscope for presence of C. burnetii.

Animals↗

Guinea pig abscess/hypersensitivity model for study of adverse vaccination reactions induced by use of Q fever vaccines.

BACKGROUND AND PURPOSE: The Coxiella burnetii phase-I cellular vaccine is efficacious in humans, imparting nearly complete protection against Q fever. However, this vaccine can also induce sterile abscesses and granulomas at the inoculation site in humans previously sensitized by natural infection or vaccination. To decrease the possibility of vaccinating immune persons, vaccinees are currently screened by skin testing to detect pre-existing Q fever immunity. We developed a model of abscess hypersensitivity in Hartley guinea pigs to assess the likelihood that Q fever vaccines would induce adverse vaccination reactions in previously sensitized individuals. METHODS: Guinea pigs (4 to 6/group) were sensitized to C. burnetii by immunization and aerosol challenge, or by intraperitoneal inoculation. Eight weeks later, animals were then vaccinated SC with a Q fever cellular (WCI) or chloroform:methanol residue (CMR) vaccine. Development of adverse reactions at the vaccination site was assessed histologically and by observation of increases in erythema and/or induration. RESULTS: The WCI vaccine caused greater magnitude and duration of erythema and induration at the vaccination sites than did the CMR vaccine. In addition, non-immune guinea pigs developed induration when given WCI, but not CMR vaccine. CONCLUSIONS: The CMR vaccine may prove a safe alternative to WCI vaccines for use in individuals unscreened for prior immunity to C. burnetii.

Abscess↗

Q fever: a biological weapon in your backyard.

Coxiella burnetii, which causes Q fever, is a highly infectious agent that is widespread among livestock around the world. Although the culture process for coxiella is laborious, large amounts of infectious material can be produced. If used as an aerosolised biological weapon, coxiella may not cause high mortality, but could provoke acute disabling disease. In its late course, Q fever can be complicated by fatal (eg, endocarditis) or debilitating (eg, chronic fatigue syndrome) disorders. The diagnosis of Q fever might be delayed because of non-specific and protean presentations. Effective antibiotic treatment is available for the acute form of disease but not for the chronic complications. Vaccination and chemoprophylaxis in selected individuals may be used in the event of bioterrorism.

Animals↗

Clinical and serological analysis of a Q fever outbreak in western Slovakia with four-year follow-up.

In the spring of 1993, an outbreak of respiratory infection affected 113 persons (103 males) in Jedl'ové Kostol'any, a village located in a hilly area of western Slovakia. Q fever, manifested as a flu-like illness with atypical pneumonia and hepatic involvement, was diagnosed using four serological tests (microimmunofluorescence, microagglutination, complement fixation, and enzyme immunoassay). Aborting goats were proven as a source of infection. During a 4-year follow-up study, no chronic form of Q fever could be demonstrated, either clinically or by tests to detect phase I Coxiella burnetii antibodies.

Abortion, Veterinary↗

Developmental biology of Coxiella burnettii.

The obligate intracellular bacterial agent of human Q fever, Coxiella burnetii, has a remarkable ability to persist in the extracellular environment. It replicates only when phagocytosed and delivered to the phagolysosome, where it resists degradation. Different morphological forms of the bacterium have different resistance properties and appear to be stages of a developmental cycle. Despite the lack of genetic systems, the molecular events surrounding C. burnetii development are now being unraveled.

Bacterial Proteins↗

Immune response genes in the post-Q-fever fatigue syndrome, Q fever endocarditis and uncomplicated acute primary Q fever.

BACKGROUND: The influence of immune response gene variations on the development of chronic complications of Q fever is presently unclear. AIM: To compare the frequencies of allelic polymorphisms in immune response genes in different Q fever patient groups. DESIGN: Genetic association study. METHODS: We measured the frequencies of immune response gene variants in: (i) an expanded group of 31 post-Q-fever fatigue patients (QFS); (ii) 22 Q fever endocarditis patients (QFE); and (iii) 22 patients who made an uncomplicated recovery from their initial attack of primary acute Q fever, comparing them with various standard control panels from the general population. RESULTS: There were significant differences between the three Q fever groups. QFS patients differed from both QFE and uncomplicated patients and controls in the frequency of carriage of HLA-DRB1*11 and of the 2/2 genotype of the interferon-gamma intron1 microsatellite. Carriage of the HLA DRB1*11 allele was associated with reduced interferon-gamma and IL-2 responses from PBMC stimulated with ligand in short-term culture. QFE showed differences in the IL-10 promoter microsatellites R and G and had higher frequencies of the TNF-alpha receptor II 196R polymorphism. Q fever patients who had made an uncomplicated recovery differed from those with QFS or QFE, but were not significantly different in allelic frequencies to the control panels. DISCUSSION: These immunogenetic differences support the concept of different immune states in chronic Q fever, determined by genetic variations in host immune responses, rather than by solely properties of Coxiella burnetii.

Coxiella burnetii↗

Modification of the diagnostic criteria proposed by the Duke Endocarditis Service to permit improved diagnosis of Q fever endocarditis.

BACKGROUND: Q fever endocarditis is a life-threatening disease for which the diagnosis is usually based on serology. The major microbiologic criterion for the diagnosis of infectious endocarditis (two separate positive blood cultures) cannot be achieved in most routine laboratories because of the biohazard associated with the culture of Coxiella burnetii, the etiological agent of Q fever. PURPOSE: Recently, new criteria for the diagnosis of infectious endocarditis have been proposed, and in this study we attempted to assess the suitability of these criteria specifically for the diagnosis of Q fever endocarditis. PATIENTS AND METHODS: To achieve this aim, we first selected from our series 20 recent cases in whom endocarditis had been confirmed following valvular pathological examination, and for whom microbiological evidence for the involvement of C burnetii was available. Then, we applied the criteria proposed by the Duke Endocarditis Service (ie, C burnetii positive serology being considered a minor criterion) to this cohort of patients but excluding pathological findings. Although the Duke Endocarditis Service criteria confirmed diagnosis in 16 of the patients, 4 were misclassified as "possible" cases (20%). However, when the Q fever serological results (using an 1/800 antiphase I immunoglobulin G cut off) and single blood culture results were changed from minor to major diagnostic criteria, endocarditis was confirmed in them all. A second time, prospectively, we applied the Duke Endocarditis Service criteria to a further 5 patients affected with Q fever endocarditis. Strict application of these criteria resulted in 1 of the 5 being misdiagnosed. Applying the suggested modification for C burnetii results, all 5 were confirmed as having infectious endocarditis. CONCLUSION: We propose that the modifications discussed in this study be applied to the Duke Endocarditis Service criteria in order that the diagnosis of C burnetii induced endocarditis is improved.

Coxiella burnetii↗

Q fever in Zimbabwe. A review of the disease and the results of a serosurvey of humans, cattle, goats and dogs.

Sera from 494 humans, 180 cattle, 180 goats and 27 dogs, collected from different regions of Zimbabwe, were examined by indirect fluorescence for antibodies reactive with phase II Coxiella burnetii antigen. Overall, 37% of humans were reactive at a titre of 1/40 or greater, and there was no evidence of age- or sex-related differences in seroprevalence. A review of clinical and epidemiological features of Q fever is presented in order to alert health workers to this infection, which apparently occurs frequently in Zimbabwe even though clinical cases have not been reported. In animals, serological evidence of Q fever infection was found in 39% of cattle, but only 15% of dogs and 10% of goats. These results suggest that cattle are important reservoirs of C. burnetii in Zimbabwe.

Animals↗

Identification of the Coxiella sp. detected from Haemaphysalis longicornis ticks in Korea.

Two Haemaphysalis longicornis ticks were found positive in PCR assay of com-1 gene to detect Coxiella burnetii DNA from 100 ticks. The nucleotide sequences of com-1 and 16S rRNA gene were determined from 2 ticks and compared with those of other C. burnetii strains. The results suggest that H. longicornis harbor Coxiella sp. bacteria in Korea. Furthermore, icd, cbhE', and cbbE' genes are C. burnetii specific genes whereas com-1 gene is Coxiella genus specific gene. This study gives the first documentation to prove the existence of Coxiella sp. in tick collected in Korea.

Animals↗

[Q fever prevention and vaccination.].

The simpliest way how to avoid the development and spreading of any infection, including Q fever in extenso, is prevention. The main principles of prevention are: educate the public on sources of infection, appropriately dispose of placenta, birth products, fetal membranes, and aborted fetuses at facilities housing sheep and goats, restrict access to barns and laboratories used in housing potentially infected animals, use only pasteurized milk and milk products, use appropriate procedures for bagging, autoclaving, and washing of laboratory clothing, quarantine imported animals, periodical monitoring of seroprevalence in cattle, sheeps and goats, vaccination of the risk groups etc. Four different basic types of Q fever vaccines have been developed: 1) live attenuated vaccines were derived from the Coxiella burnetii strain Grita in phase I of a highly reduced virulence but preserved immunogenicity, 2) highly purified corpuscular formalin-inactivated vaccine derived from the C. burnetii Henzerling strain in phase I, 3) a soluble vaccine prepared from the C. burnetii strain Nine Mile corpuscules in phase I by extraction with trichloracetic acid, and 4) in the USA, the use of a chloroform-methanol extraction residue subunit vaccine of C. burnetii strain Nine Mile in phase I of a low reactogenicity has been recommended.

Animals↗

[Q fever with endocarditis: clinical presentation and serologic follow-up of 21 patients].

AIM OF THE STUDY: The purpose of this study was to analyse the clinical and serological follow-up in 21 patients with Q fever endocarditis in Switzerland from 1981 to 1993. PATIENTS AND METHODS: Criteria for Q fever endocarditis were the following: Coxiella burnetii phase I IgG > 1 : 2560 and IgA > 1 : 20 by indirect immunofluorescence. Methods to confirm the diagnosis include immunohistochemical demonstration of C. burnetii by microscopy in valvular material (1 case) and inoculation of this material in experimental animals (10 cases). Information on clinical course of the disease, laboratory abnormalities and treatment were obtained by chart review and a questionnaire sent to physicians who requested the serological tests for Q fever. RESULTS: The average age of the patients was 47 years (15 men and 6 women). 64% of patients had a history of environmental exposure to C. burnetii. The median time of symptomatology before diagnosis was 5 months (1-108). 19/21 patients had valvular lesions, and 2/21 vascular Dacron prosthesis. Most patients presented with fever (18/21), congestive cardiac failure (14/21), weight loss (12/21), anemia (6/19), or thrombocytopenia (6/19). All the patients required antibiotic treatment. Cardiac surgery was performed in 15/21 patients. For 10 patients the geometric mean serological follow-up included at least titers at time of diagnosis (IgG anti-phase I antibodies 1 : 27024, IgA anti-phase I antibodies 1 : 685), at the end of therapy (IgG anti-phase I antibodies 1 : 2941, IgA anti-phase I antibodies 1 : 153) and 6 months after the end of therapy (IgG anti-phase I antibodies 1 : 368, IgA anti-phase I antibodies 1 : 40). The fall in anti-phase I titers was significant. During the clinical and serological outcome (median of 60 months and 69 months respectively) there was no recurrence of endocarditis and antibody titers to C. burnetii phase I remained low. Two patients died during the observation period, one from lung cancer, while the cause of death in the other was unknown. CONCLUSIONS: Serology is the key to Q fever diagnosis. The duration of treatment, and the values to be used to establish cure of endocarditis, are not clearly defined. During the clinical and serological outcome (median of 60 months and 69 months respectively) there was no recurrence of endocarditis and antibody titers to C. burnetii phase I remained low.

Adolescent↗

Epidemics of Q fever in Poland in 1992-1994.

From 1992 till the end of 1994, three epidemics of Q fever among people have been recognised in Poland. In 1992 the outbreak of Q fever was detected at the farm near Jawor in Legnica district. Coxiella burnetii infection was recognized serologically in 25 persons (27 tested). Titres of serum antibodies ranged from 8 to 256. It has been found that cattle were the source of human infection. C. burnetii strain has been isolated from the bull semen. In the same year, Q fever was recognized among 18 workers of the tannery in Myślenice, in Kraków district. Antibodies to C. burnetii, in titres from 8 to 2048, were detected in all 18 persons who had a contract with imported skins of wild animals. Serological tests performed again in 1994 showed that elevated titres of serum antibodies persisted in 10 of 14 persons and 9 of them developed phase I antibodies also. Since 1993 several cases of Q fever have been observed among people living in various regions of the country. Epidemiological investigations have revealed that infected patients are seasonal workers employed during the shearing time in Spain. Specific serum antibodies reached titre 512. The strains of C. burnetii were isolated from urine and semen of 2 patients.

Adult↗

Q fever.

An outbreak of Q fever occurred in Scotland during this summer and was reported in news headlines. Despite these newsworthy headlines, Q fever remains poorly understood. The causative organism, Coxiella burnetii, has a worldwide distribution, with the notable exception of New Zealand. Even with its ubiquitous nature, Q fever is rarely reported. We explore some of the underlying reasons for this apparent under diagnosis together with some of the diagnostic challenges posed by this obligate intracellular pathogen. The host range for this microbe spans arthropods, through to birds and a diverse range of mammals including livestock, companion animals and man. In most, infection remains sub-clinical, however, in some, infection can cause severe and life-threatening complications. Furthermore, possible long-term persistence within those infected, may result in long-term sequelae disassociated from initial risk factors or acute clinical presentation. We review current thinking on C. burnetii, and identify some of our current knowledge gaps.

Adult↗

Myocarditis, a rare but severe manifestation of Q fever: report of 8 cases and review of the literature.

Myocarditis has only rarely been described as a manifestation of acute Q fever. Among our series of 1276 patients in whom acute Q fever was diagnosed during 1985--1999, myocarditis was diagnosed in 8. Two patients (25.0%) developed cardiac symptoms during the course of interstitial pneumonia, 2 (25.0%) initially presented with unexplained fever, and 1 (12.5%) presented with febrile cutaneous rash. In 3 patients, cardiac symptoms were inaugural: 1 patient experienced heart failure, and 2 experienced precordial pain. Dilated cardiomyopathy was documented in 7 patients, and 2 (1 of whom had undergone heart transplantation) died despite therapy. In addition, 1 patient was scheduled for heart transplantation because of cardiac insufficiency. When the patients in this study were compared with 32 control patients with acute Q fever, no specific epidemiological or clinical features were associated with this disease except worse prognosis (P=.006). Moreover, among the 12 patients from our series who died as a result of acute Q fever, 2 patients, who were significantly younger than the other 9 patients (P=.03), had myocarditis. Our study highlights the severity of Coxiella burnetii myocarditis.

Adolescent↗

[Q fever, a potential biowarfare agent].

SEVERAL POSSIBLE METHODS OF TRANSFUSION: Q fever is a zoonosis due to Coxiella burnetii. Its interest as a potential biowarfare agent is in its possible transmission by inhalation of sprayed particles. This form of transmission would probably be used: the inhalation of 1 to 10 bacteria could provoke the development of an infection in humans. Another possible method of transmission in the context of a terrorist act would be the intentional introduction of the bacteria into foodstuff. A DISABLING WEAPON: However, C. burnetii has never been used as a biological weapon. The probability that this germ could be used is very low: indeed, the incubation of Q fever is very long, and the majority of the infections is asymptomatic and mortality is low. In fact C. burnetii would more likely be used as a disabling weapon.

Air Microbiology↗

Autophagy induction favours the generation and maturation of the Coxiella-replicative vacuoles.

Pathogens evolved mechanisms to invade host cells and to multiply in the cytosol or in compositionally and functionally customized membrane-bound compartments. Coxiella burnetii, the agent of Q fever in man is a Gram-negative gamma-proteobacterium which multiplies in large, acidified, hydrolase-rich and fusogenic vacuoles with phagolysosomal-like characteristics. We reported previously that C. burnetii phase II replicative compartments are labelled by LC3, a protein specifically localized to autophagic vesicles. We show here that autophagy in Chinese hamster ovary cells, induced by amino acid deprivation prior to infection with Coxiella increased the number of infected cells, the size of the vacuoles, and their bacterial load. Furthermore, overexpression of GFP-LC3 or of GFP-Rab24 - a protein also localized to autophagic vacuoles - likewise accelerated the development of Coxiella-vacuoles at early times after infection. However, overexpression of mutants of those proteins that cannot be targeted to autophagosomes dramatically decreased the number and size of the vacuoles in the first hours of infection, although by 48 h the infection was similar to that of non-transfected controls. Overall, the results suggest that transit through the autophagic pathway increases the infection with Coxiella by providing a niche more favourable to their initial survival and multiplication.

Animals↗

Autophagosomes: a fast-food joint for unexpected guests.

Coxiella burnetii is a Gram-negative obligate intracellular bacterium that infects a wide range of hosts including humans, causing Q fever, a disease characterized by high fever and flu-like symptoms. After its internalization the Coxiella-containing phagosomes interact with intracellular compartments and generate a large replicative vacuole that displays certain characteristics of a phagolysosome. We have shown that this bacterially-customized replicative vacuole also has the hallmarks of an autophagosomal compartment. Furthermore, in a recent publication we have reported that induction of autophagy is beneficial for the replication and survival of Coxiella. Different morphological forms of this bacterium have been described during its developmental cycle. Here we present additional data and discuss a model indicating that induction of autophagy favors the differentiation of the Coxiella small cell variants to the metabolically active large cells variants. We postulate that nutrient acquisition, likely by fusion with the nutrient-rich autophagic vacuoles, triggers the development of the large cell variants which actively multiply in the host cell.

Autophagy↗

Molecular genetic methods for the diagnosis of fastidious microorganisms.

Technological innovations in the detection and identification of microorganisms using molecular techniques such as polymerase chain reaction (PCR) have ushered in a new era with respect to diagnostic microbiology. PCR using universal or specific primers followed by identification of amplified product, mainly by sequencing, has enabled the rapid identification of cultured or uncultured bacteria. Thus, PCR may allow quick diagnosis of infections caused by fastidious pathogens for which culture could be extremely difficult. However, several pitfalls, such as false positives, have been observed with PCR, underlining the necessity to interpret the results obtained with caution. At present, certain improvements in the molecular genetic methods may be helpful for the diagnosis of infectious diseases. Indeed, the recent development of bacterial genome sequencing has provided an important source of potential targets for PCR, allowing rational choice of primers for diagnosis and genotyping. In addition, the development of new techniques such as real-time PCR offers several advantages in comparison to conventional PCR, including speed, simplicity, reproducibility, quantitative capability and low risk of contamination. Herein, we review the general principles of PCR-based diagnosis and molecular genetic methods for the diagnosis of several hard-to-culture bacteria, such as Rickettsia spp., Ehrlichia spp., Coxiella burnetii, Bartonella spp., Tropheryma whipplei and Yersinia pestis.

Actinomycetales↗