Bacteriology
Diagnosis of acute Q fever with emphasis on enzyme-linked immunosorbent assay and nested polymerase chain reaction regarding the time of serum collection

https://doi.org/10.1016/j.diagmicrobio.2010.06.001Get rights and content

Abstract

A commercially available enzyme-linked immunosorbent assay (ELISA) (Virion/Serion [Wuerzburg, Germany]), an indirect fluorescent antibody test (IFAT) (BIOS/Focus [Cypress, CA]), and a nested polymerase chain reaction (PCR) were explored for diagnosis of acute Q fever in reference to time of serum collection. Serum samples of 22 patients with acute Q fever collected around the fifth day of illness were included. A sensitivity of 30% by ELISA and 80% by IFAT (P = 0.1) was found for the first 5 days of illness and 92% by ELISA and 83% by IFAT during the sixth and eleventh day. PCR revealed a positive result in 8 cases (36%) with 6 cases deriving from the first 5 days of illness. We conclude that ELISA aids especially in the diagnosis of Q fever after 5 days of illness. The benefit of PCR as an additional tool to ELISA was especially evident in the early days of serum sampling.

Introduction

The awareness of Q fever has increased in recent years due to several large Q fever outbreaks and the tremendous situation in the Netherlands with 2357 reported cases just in 2009 (van der Hoek et al., 2010). Q fever is a worldwide zoonotic disease caused by Coxiella burnetii, an obligate intracellular, small, Gram-negative bacterium (0.2- to 0.4-μm wide, 0.4- to 1-μm long). It is extremely infectious, is weather resistant, and may survive for several weeks in the environment. Transmission to humans is usually airborne and associated with contact to birth products like amniotic fluid and placenta of infected animals or contaminated dust. The most commonly identified sources of human cases are infected ruminants. In humans, Q fever presents after an incubation period of 2 to 5 weeks mainly as a self-limited respiratory infection, sometimes complicated by atypical pneumonia, hepatitis, or myopericarditis. Only 30% of the infected individuals develop clinical signs. In a few cases, symptomatic and asymptomatic infections can ultimately lead to chronic Q fever presenting as culture-negative endocarditis (Maurin and Raoult, 1999). Besides sporadic cases, Q fever mainly causes large outbreaks (including metropolitan areas) by windborne spread (Carrieri et al., 2002, Hawker et al., 1998).

Such an outbreak occurred in Jena, a city of 100 000 inhabitants in Thuringia (Germany), during the summer months of 2005. Within a period of 7 weeks, 331 cases were reported in a residential district with a population of 11 500 (Gilsdorf et al., 2008). A flock of sheep was identified as the source of the outbreak. One vaginal swab from a ewe was soon tested positive by polymerase chain reaction (PCR) for C. burnetii, but confirmation of a human Q fever case by serology took 9 days after the occurrence of the first clinical case. This delay in diagnosing human Q fever is a known challenge. Unlike the essential role of culture in most bacterial infections, their impact on the diagnoses of human Q fever is insignificant, as it needs several weeks, is difficult, and requires a biosafety level 3 laboratory.

Thus, serology is the diagnostic method used. Thereby, another feature of C. burnetii, the so-called phase variation, is employed. The wild-type C. burnetii strains possess a typical Gram-negative outer membrane, with a complex structure of the lipopolysaccharide (LPS). Under different circumstances like serial passages in cell cultures, a degenerative mutation of the LPS is found and was traditionally referred to phase variation. Thereby, the complete LPS named phase I LPS (ph I) was distinguished from the truncated one termed phase II LPS (ph II). During Q fever infection, both antigenic features appear and antibodies specific for them are generated. The detection of these antibodies is used to diagnose and differentiate between acute, past, and chronic infections. Acute Q fever is diagnosed by antibodies against phase II LPS. At first, ph II IgM antibodies appear followed by ph II IgG antibodies. Past infections are characterized by ph II and low ph I IgG antibody levels. For chronic infections, ph I IgG antibodies are crucial. A diagnostic titer was set at ≥1:800 for the IgG response to phase I (Fournier et al., 1996).

To determine these antibody levels, the indirect immunofluorescent antibody test (IFAT), the reference technique for Q fever is used (Maurin and Raoult, 1999). But the IFAT is expensive and needs experience. As a commercially available test, the complement fixation test (CFT) was extensively used in the past but is no longer recommended due to low sensitivity. The commercially available enzyme-linked immunosorbent assay (ELISA) seems to be a good opportunity for most diagnostic laboratories. But the late appearance of antibodies against C. burnetii around 1 to 3 weeks after the onset of clinical signs makes the diagnostic still unsatisfactory. To solve this problem, specialized laboratories implemented the PCR as an additional tool in recent years. It has been found that examination of serum samples, the most easily obtained specimen, by PCR for C. burnetii DNA may decrease the time for a positive result in cases of acute Q fever (Turra et al., 2006). The combined use of PCR and IFAT showed a significant increase in sensitivity over that of IFAT alone within the first 2 weeks of illness (Fournier and Raoult, 2003).

In the following study, we wish to present our experience with a commercially available ELISA (Virion/Serion, Wuerzburg, Germany) as an easy technique to implement in combination with nested PCR targeting the IS1111 element in diagnosing the early stage of acute Q fever. Only patients with acute Q fever as proven by X-ray, C-reactive protein (CRP), and confirmation of C. burnetii infection were included. We focused especially on the time of serum collection during the course of illness. IFAT (BIOS/Focus, Cypress, CA) was used as a reference method in comparison.

Section snippets

Patients

All patients with acute Q fever diagnosed between June and October 2005 at the University Hospital, Jena, Germany, were included in the study. Acute Q fever was defined as the association of a C. burnetii infection with clinical and radiologic evidence of pneumonia, elevated CRP (CRP threshold, 7.5 mg/L), and residence in the outbreak area. The C. burnetii infection was confirmed by seroconversion or by the detection of high ph II IgM antibodies combined either with a positive PCR result or

Results

Our laboratory received sera from 41 patients with the request of Q fever diagnostic between June and October 2005. Out of those, 22 patients fulfilled our case definition of acute Q fever. In 15 patients, a C. burnetii infection was confirmed by the development of ph II IgG antibodies in the follow-up serum. In 1 patient, it was confirmed by a high ph II IgM antibody level (titer of ≥1:64) together with a positive PCR result. The remaining 6 patients were included based on their high ph II IgM

Discussion

In the present study, we confirmed the value of a commercially available ELISA for the diagnosis of acute Q fever and determined its lack of sensitivity regarding the duration of illness. A benefit combining a nested PCR with serology was seen.

During an outbreak in Jena in 2005, sera from 41 patients with the request of Q fever diagnostic were sent to our laboratory. Out of them, 22 patients fulfilled our criteria of an acute Q fever illness: confirmation of a C. burnetii infection, clinical

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    Address of the institution at which the work was performed: Institute of Medical Microbiology, Jena University Hospital, Erlanger Allee 101, 07747 Jena.

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