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PubMed1999

Development of internal controls for probe-based nucleic acid diagnostic assays.

A method is described for the design, evaluation, and application of internal control targets and probes for use in probe-based nucleic acid diagnostic assays (i.e., PCR-ELISA). The technique is a modified version of oligonucleotide-directed mutagenesis in conjunction with PCR amplification to develop a novel probe-annealing sequence in a cloned IS1111a gene fragment of Coxiella burnetii. The internal control probe-recognition site with its complementary probe was identical to the wild-type-specific probe in length, base composition, location, and annealing temperature. Neither the internal control nor the wild-type probes annealed to the recognition sequence of the other. As both of the amplified nucleic acid fragments, internal control and wild type, were identical in length and base composition, the amplification conditions for the diagnostic assay were not affected. This allowed small copy numbers of the internal control clone to be loaded into a diagnostic assay without negatively affecting it. In a single reaction we were able to differentiate between an assay reporting a true or false-negative signal. A negative signal is defined as the absence of detectable pathogen genetic material (true) or inhibition/failure of the reaction (false).

Base Sequence↗

PubMed1993

[The dynamics of the metabolic and functional activities of the immunocompetent cells in mice following immunization with different vaccines against Q fever].

It was shown that the immunization of mice by the chemical Q fever vaccine prepared with the use of the strain "Coxiella burnetii, Nine Mile", phase I did not markedly activate the oxidative metabolism of the peritoneal exudate cells and did not induce the non-specific cytotoxicity in them. However, it resulted in a significant suppression of the mitogen-induced proliferation of splenocytes on days 3-5. The immunization of mice by the combined (corpuscular/chemical) vaccine prepared with the use of the strain "Apodemus flavicollis-Luga", phase I induced a significant activation of the oxidative metabolism of the peritoneal exudate cells and the development of the nonspecific cytotoxicity in them, whereas no suppression of the mitogen-induced proliferation of splenocytes was observed. The differences are likely associated with the presence of the corpuscular component in the combined vaccine and the strain antigenic specificity.

Animals↗

PubMed2004

Pseudoepidemic of Q fever at an animal research facility.

Serum samples from people exposed to sheep at a research facility were evaluated by a commercial laboratory and resulted in an overall Coxiella burnetii seroprevalence of 75%. We interviewed individuals to determine exposure history and compatible illness, and retested their sera. Analysis indicated that the commercial laboratory was misinterpreting its results; when corrected, the seroprevalence dropped to 27%. Test kits of the brand used by the commercial laboratory gave equivalent results to the in-house CDC assay when tested in parallel at CDC. Upon final analysis, only the attending veterinarian was confirmed as a Q fever case. This event resulted in increased risk reduction protocols at the research facility and improved public health communication among health authorities. This pseudoepidemic resulted from a lapse in laboratory quality control for testing. Similar errors can be avoided through standardization and improved review of laboratory procedures.

Animals↗

PubMed1998

Comparison of Q fever cellular and chloroform-methanol residue vaccines as skin test antigens in the sensitized guinea pig.

Coxiella burnetii phase I whole cell vaccine (WCV) is associated with risk of severe local delayed-type hypersensitivity (DTH) reactions in previously immunized individuals or those sensitized by natural exposure. We compared this vaccine to another investigational vaccine derived by chloroform-methanol extraction of phase I whole cells (chloroform-methanol residue vaccine, CMRV). Hairless guinea pigs, sensitized with either WCV or CMRV, were given 60,600 and 6,000 ng of WCV or CMRV in an intradermal (i.d.) skin test. The i.d. administration of WCV consistently caused more host reactions than comparable doses of CMRV in guinea pigs sensitized with either WCW or CMRV, suggesting that CMRV may be a safer vaccine. However, the CMRV was not innocuous and caused significant indurated lesions and micro-abscesses at the 600 ng and 6,000 ng skin test sites.

Animals↗

PubMed2003

Dysregulation of cytokines in acute Q fever: role of interleukin-10 and tumor necrosis factor in chronic evolution of Q fever.

Q fever manifests as primary infection or acute Q fever and may become chronic in patients with underlying valvulopathy. Because Coxiella burnetii infection depends on host response, we measured tumor necrosis factor (TNF), interleukin (IL)-6, IL-12, and IL-10 in patients with different clinical presentations of acute Q fever. Compared with control subjects, patients with uncomplicated acute Q fever exhibited increased release of the 4 cytokines. Their amounts were higher in patients with hepatitis than in patients with fever or pneumonia. In patients with valvulopathy, who exhibited the highest risk of chronic evolution, the amounts of TNF and IL-10 were higher than in patients without valvulopathy. TNF production was specifically enhanced in patients who developed Q fever endocarditis. These results show that acute Q fever is associated with cytokine overproduction. Persistent TNF amounts were associated with the occurrence of endocarditis in patients with valvulopathy, and that may be a marker of chronic evolution of Q fever.

Adolescent↗

PubMed2006

Autoimmunohistochemistry: a new method for the histologic diagnosis of infective endocarditis.

BACKGROUND: Although the pathologic examination of cardiac valves remains the reference standard for the diagnosis of infective endocarditis, the detection of microorganisms often poses a challenge for pathologists. This can be done by use of nonspecific histochemical stains or by immunohistochemical analysis, but specific antibodies are often not available. We describe a novel method for the detection of microorganisms in valve specimens from patients with infective endocarditis. METHODS: Detection of microorganisms was performed in valve specimens from patients with endocarditis caused by gram-positive cocci (25 specimens), blood culture-negative endocarditis (15 specimens: 6 cases caused by Coxiella burnetii, 5 caused by Tropheryma whipplei, and 4 caused by Bartonella species), or noninfective degenerative damage (30 specimens, used as negative controls), using the patients' own serum. This technique, called "autoimmunohistochemistry," is an immunohistochemical peroxidase-based method that we compared with results of culture and polymerase chain reaction (PCR) assay. RESULTS: Bacteria were detected by autoimmunohistochemistry in 20 (80%) specimens from patients with endocarditis caused by gram-positive cocci and in 15 (100%) specimens from patients with blood culture-negative endocarditis but in no control specimens. The rate of detection of bacteria by autoimmunohistochemistry was significantly higher than that by culture but was similar to that by PCR. CONCLUSIONS: Autoimmunohistochemistry may be useful for the detection of microorganisms in samples of valves from patients with infective endocarditis. This new diagnostic tool may be particularly useful in cases of blood culture-negative endocarditis.

Actinomycetales↗

PubMed1994

Q fever in Taiwan.

BACKGROUND: Although Q fever, a rickettsial disease caused by Coxiella burnetii, is supposed to occur sporadically worldwide, there is only one case report of this disease in Taiwan. We examined the antibody of Q fever to evaluate the status of Q fever in Taiwan. METHODS: In total, 579 paired serum specimens of suspected febrile patients sent by several hospitals and sanitary stations to this Institute in 1991 and 1992 were tested according to the technique of indirect fluorescent antibody (IFA). RESULTS: In total 48 serologically acute Q fever cases were found, mostly from Tainan, Pingtung and Kaohsiung. There was only one female, and the age of most patients was 40-50 years. The high season is in June and July. CONCLUSIONS: The antibody titer of all 48 positive cases reached 1:320 or higher on the 13th day after onset of the disease. IFA as a diagnostic tool to detect acute and convalescent serum antibody titer is discussed.

Adult↗

PubMed1996

[A Q-fever pneumonia epidemic in Dusseldorf].

There was an unusually high incidence of atypical pneumonias in the catchment area of the Rhine river near the university of Düsseldorf in July 1994 during a long period of hot and dry weather. The 18 patients described in this paper (5 women and 13 men) complained of sudden onset of fever up to over 40 degrees C, often associated with severe headache and dry cough. Almost all of these patients had previously been healthy and active and of young to middle age (average 38 years) without any bronchopulmonary anamnesis. Radiology revealed that all the patients had in most cases defined pulmonary infiltrates without any specific preference for a particular site. Serology was initially negative, but four weeks later the complement fixation reaction titre was positive for Coxiella burnetii antibodies in 14 patients (78%). All patients became symptom-free within a few days'time when treated with a combination of antibiotics which included doxycycline, whereas the infiltrates receded completely only after several weeks. The occurrence of pulmonary Q-fever in a large northern German conurbation had been rare at that time. Such epidemics, however, were also noted in Berlin (1992) and in Dortmund (1993). The epidemic reported in this article probably originated from one of the frequent flocks of sheep grazing along the banks of the Rhine river near Düsseldorf. The infections were probably acquired by inhalation of airborne organisms in infected aerosols derived from infected sheep, promoted by the long-term very hot and dry weather which was at the same time very windy, leading to an unusually extensive spreading of the pathogens throughout a very large infected aerosol area.

Adult↗

PubMed2006

Q fever in the Southern California desert: epidemiology, clinical presentation and treatment.

Despite the absence of a natural reservoir for Q fever in the desert of Southern California, six cases have been identified during the past 32 years. During that period of time, two areas have been used by northern sheep ranchers from Idaho and Wyoming to import sheep to an area in the Coachella Valley through 1985. Thereafter, because of housing development, the sheep area was moved to Blythe along the Colorado River. All but two of these patients probably acquired infection by Coxiella burnetii by living or working in close proximity to these grazing areas but not directly involved with the sheep. The shift of infected patients from the Coachella Valley to Blythe (100 miles distant) seems to support that supposition. All patients with acute Q fever developed antibodies primarily to phase II antigen, whereas the only person with chronic Q fever developed phase I antibodies. All patients presented with granulomatous hepatitis. One also had a pulmonary infiltrate, and the single individual with chronic Q fever also had a mitral valve prosthesis, although echocardiography could not define endocarditis. All patients with acute infections responded to 3-5 weeks of therapy with doxycycline, whereas the patient with chronic disease failed 3 years of therapy with combination regimens. Further studies at the Eisenhower Medical Center on the prevalence of infection in Blythe, CA, and elsewhere are anticipated.

Adult↗

PubMed2003

Q fever as a biological weapon.

Q fever is a bacterial zoonosis caused by Coxiella burnetii, a unique intracellular coccobacillus, adapted to live within the phagolysosomes of macrophages and monocytes. It is highly infectious, with as little as one organism needed to cause clinical infection, making it an attractive organism for use in biowarfare. Despite its high infectivity, it has low virulence, and most patients undergo only asymptomatic seroconversion. Acute clinical manifestations are a nonspecific febrile illness, pneumonia, hepatitis, and neurologic abnormalities ranging from headache to meningoencephalitis. Chronic Q fever can result in endocarditis, hepatitis, or a chronic fatigue syndrome. Diagnosis usually is made by serology because culture of the highly contagious organism is potentially hazardous. Tetracyclines are the antibiotics of choice. When individualized therapy is possible, a 14- to 21-day course of doxycycline usually is used. In a mass casualty situation, a 5- to 7-day course of doxycycline is recommended, both for therapy and prophylaxis. For chronic infections such as endocarditis, 18 months of doxycycline supplemented with hydroxychloroquine is currently the best therapy.

Anti-Infective Agents↗

PubMed1996

Seroepidemiological survey of Q fever in León province, Spain.

A seroepidemiological survey, using an indirect immunofluorescence test against Coxiella burnetii (antigenic phase II), was carried out in León province, north-western Spain. A total of 406 serum samples was collected from people (from 15 to more than 65 years old) living in a rural environment during the winter and spring of 1994. The overall prevalence was 40.6%, titres ranged from 1:80 to 1:640, and a titre of 1:80 was encountered among 60.6% of positive samples. A significant higher prevalence was observed among males globally, as well as among those aged 15-44 and 45-64 years old; however, no difference was encountered among males and females older than 64 years. In the same way, a significant higher Q fever prevalence was observed among individuals having occupations related to agriculture and among those having close relationship with animals.

Adolescent↗

PubMed2003

Comparison of PCR and serology assays for early diagnosis of acute Q fever.

Q fever is a potentially severe disease which can occur in large outbreaks of acute infections and is a possible bioterrorism agent. In order to lessen the delay in diagnosing acute Q fever, we compared LightCycler Nested PCR (LCN-PCR), a rapid nested PCR assay that uses serum sampled early during the disease as a specimen and the LightCycler as a thermal cycler, to serology by indirect immunofluorescence. We used the 20-copy htpAB-associated element as the DNA target. The detection sensitivity of this method was one Coxiella burnetii DNA copy. We applied this method to the first serum samples taken from 100 patients diagnosed in our laboratory as having acute Q fever on the basis of clinical manifestations and serology and to 80 controls. The LCN-PCR had a specificity of 100%. The sensitivity was 26% when no antibodies were detected but only 5% with seropositive patients (P < 10(-2)). The technique was most efficient in the first 2 weeks following the onset of symptoms (P = 0.02), when its sensitivity was 24% compared with 14% for serology. With combined use of LCN-PCR and serology within the first 2 weeks, the sensitivity was significantly increased over that with serology alone (P < 10(-2)). Thus, we propose a strategy for improving the early diagnosis of acute Q fever where LCN-PCR should be performed together with serology in the first 2 weeks of the disease but should be reserved for seronegative patients in the next 2 weeks and not used later than 4 weeks following onset, when serology is highly sensitive.

Adolescent↗

PubMed2003

Brucellosis and Q-fever seroprevalences of nomadic pastoralists and their livestock in Chad.

As a part of a research-and-action partnership between public health and veterinary medicine, the relationships between the seroprevalences of brucellosis and Q-fever in humans and livestock were evaluated in three nomadic communities of Chad (Fulani cattle breeders, and Arab camel and cattle breeders). Nomad camps were visited between April 1999 and April 2000. A total of 860 human and 1637 animal sera were tested for antibodies against Brucella spp., and 368 human and 613 animal sera for Coxiella burnetii. The same indirect ELISA was used for livestock and human sera, and the test characteristics for its use on human sera were evaluated. Twenty-eight people were seropositive for brucellosis (seroprevalence 3.8%). Brucella seroprevalence was higher in cattle (7%) than other livestock, and brucellosis seropositivity was a significant factor for abortion in cattle (OR=2.8). No correlation was found between human brucellosis serostatus and camp proportions of seropositive animals. Q-fever-seropositive blood samples were taken from 11 Arab camel and 4 Arab cattle breeders (seroprevalence 1%). Being a camel breeder was associated with Q-fever seropositivity in humans (OR=9). Camels had the highest Q-fever seroprevalence (80%) among livestock species. Although there was high-risk human behaviour for the acquisition of brucellosis and Q-fever from livestock through raw-milk consumption (98%) and contact with placentas of livestock (62%), we concluded that seroprevalences in humans were relatively low (likely due to limited active foci in livestock).

Adolescent↗

PubMed1991

Coxiella and Rickettsiella: comparison of ultrastructure with special reference to their envelope.

Ultrastructure of Rickettsiella phytoseiuli (R.p.) multiplying in female ticks Dermacentor reticulatus was compared with that of Coxiella burnetii (C.b.) in the same ticks and in mice. C.b. in ticks and mice were always represented by 2 main cell types: small dense round or rod-like cells (DC) and larger bacteria-like cells (BC). DC were surrounded with a characteristic five-layered 20 nm thick envelope. Under the envelope DC had a stack of parallel intracytoplasmic membranes with a periodicity 5-6 nm. R.p. in tick fat body and synganglion were also inside phagosomes and formed 6 sequentially developing cell forms: dense (elementary), intermediate, bacterial, giant, and crystal-forming in which small dark bodies (initial particles) condensed. Two of them--dense and bacterial--corresponded to DC and BC of C.b. The DC envelope structure of R.p. was strikingly similar to that of some C.b. DC in mouse. We confirmed the general morphologic similarities in the structure of C.b. and R.p. DC and that of C.b. BC and intermediate cells of R.p. The envelope structure of DC type was found in other gracilicute bacteria and is supposed to have no taxonomic value but to be a reflection of population heterogeneity.

Animals↗

PubMed1992

Enzyme immunoassay for Q fever: comparison with complement fixation and immunofluorescence tests and dot immunoblotting.

Enzyme-linked immunosorbent assays (ELISA) for the detection of specific immunoglobulin G (IgG) and IgM antibodies were developed by using purified Coxiella burnetii cells. Variables, including type of microtiter plate, blocking agent, incubation conditions, antigen stability, and substrate type, were examined to achieve optimal ELISA performance. The reliabilities of the assay systems were compared with those of complement fixation (CF) and enhanced immunofluorescence (EIF) tests with 600 human serum samples from defined clinical cases of Q fever, routine samples, and serum specimens from farmers. ELISA and EIF test results agreed in all cases. Dot immunoblotting was also used to test some of these sera and gave a rapid, qualitative result, which agreed with ELISA and EIF test results in all cases. No instances were found in which both ELISA and EIF test results were negative and the CF test results was positive. However approximately 5% of the sera were positive by ELISA and the EIF test while the CF test result was either negative or unreadable because of serum anticomplementary activity. We conclude that dot immunoblotting is a useful screening test, whereas ELISA and the EIF test are both rapid and sensitive tests when used for the serodiagnosis of Q fever and should be considered to be replacements for the CF test.

Antibodies, Bacterial↗

PubMed1994

'Atypical' bacteria are a common cause of community-acquired pneumonia in hospitalised adults.

OBJECTIVES: To assess the proportion of cases of community-acquired pneumonia caused by 'atypical' bacteria, including the recently discovered Chlamydia pneumoniae, and to compare the clinical, radiographic and laboratory features of patients with and without 'atypical' bacteria. METHODS: A prospective serological study was carried out on consecutive adult pneumonia patients from July 1987 to July 1988. Acute and convalescent sera were tested in batches for antibodies against Legionella pneumophila serogroup 1, C. pneumoniae, Chlamydia psittaci, Coxiella burnetii (phase-2 antigen) and Mycoplasma pneumoniae (IgG and IgM). Records and chest radiographs were examined retrospectively. RESULTS: Acute and convalescent sera were available from 113 patients. The records of 4 patients could not be traced and 17 patients did not fulfil the inclusion criteria. Thirty-two of these 92 patients (35.9%) were found to be infected with 'atypical' bacteria. The two most common organisms were C. pneumoniae (20.7%) and L. pneumophila (8.7%). There were no differences in the clinical and radiographic features of patients with and without 'atypical' bacteria. Clinicians prescribed erythromycin or tetracyclines with equal frequency in the two groups. CONCLUSIONS: 'Atypical' bacteria, especially C. pneumoniae, are a common cause of community-acquired pneumonia in adults in South Africa. This is the first demonstration of an aetiological role of C. pneumoniae in this country. We confirmed the finding of other studies that there are no clinical, radiographic or laboratory features characteristic of 'atypical' bacterial infection in hospitalised patients. This has major implications for therapy, as these organisms respond to erythromycin and tetracyclines, but not to beta-lactam antibiotics.

Adult↗

PubMed1995

Seroepidemiology of Q fever in Nova Scotia: evidence for age dependent cohorts and geographical distribution.

To determine the incidence of Q fever in Nova Scotia, a randomly selected sample of 492 volunteers aged 18-70 years was recruited by mail from all 18 urban and rural counties in this province. Volunteers were followed from 1988 to 1991 for antibody titres to Coxiella burnetii antigens. Analysis of seroprevalence by age revealed two statistically different cohorts. Those younger than 35 years had a low, stable seroprevalence while those 35 years and older had a seroprevalence statistically correlated with increasing age. The finding of age related cohorts suggests a sporadic or cyclical infection rate. An age-independent geographical clustering was also noted, with higher seroprevalence in a major agricultural area of the province. No seroconversions were observed, but four subjects had seroreversions. At baseline, 72 (14.6%) of the 492 subjects were considered seropositive (> or = 1:8 C. burnetti phase II antibody titre by microimmunofluorescence). Of 47 seropositive subjects who provided yearly serum samples, two (4.3%) showed > or = 4-fold increase in antibody titre and did not react to antigens of the 11 other respiratory pathogens tested, suggesting a reactivation of Q fever. All 22 subjects positive to phase I antigen were positive to phase II antigen. This study suggests that the rate of infection in Nova Scotia has been low since the 1960's and that risk of infection is associated more with a geographically clustered reservoir of infection than with occupation or gender.

Adolescent↗

PubMed1995

The partition region of plasmid QpH1 is a member of a family of two trans-acting factors as implied by sequence analysis.

Sequencing analysis revealed that the partition region of the Coxiella burnetii plasmid QpH1 contains a putative operon, designated qsopAB. The two open reading frames (ORFs), qsopA and qsopB, specify the QsopA and QsopB proteins, with deduced molecular masses of 45.7 and 37.6 kDa, respectively. Maxicell analysis demonstrated that although qsopB was located downstream from qsopA, it had its own promoter that was active in Escherichia coli. Several direct or inverted repeats were found around this operon. The most distinct was a 20-bp long imperfect palindrome in the promoter region of qsopA, with homology to a palindrome in the promoter region of P1 parA. Structurally qsopAB was similar to parAB of the P1 plasmid. However, at the amino acid (aa) sequence level, QsopA and QsopB were closest to the F plasmid SopA and SopB proteins, respectively. QsopA shared 58.0% homology and 32.7% identity with SopA, but only 45-50% homology and 22-26% identity with other members of the protein A partition family. QsoB had even lower (41-45%) homology to other members of the protein B partition family, with the highest homology and identity to SopB. Despite lower homologies, both QsopA and QsopB did share conserved aa sequence regions and invariant residues with other members within each family.

Amino Acid Sequence↗