Microbiology
C. burnetii is a short, pleomorphic rod that is a strict intracellular bacterium. While previously designated as a Rickettsia, C. burnetii has been re-classified as a Proteobacteria, which is closer to Legionella and Francisella (Stein et al., 1993). In mammals, the usual host cell of C. burnetii is the macrophage, which is unable to kill the bacterium. C. burnetii lives and multiplies in a single, large, acidic vacuole, which results from the fusion of cell lysosomes (Maurin et al., 1992). In addition, a sporulation-like process protects the organism from the external environment, where it can survive for long periods of time. An important characteristic of C. burnetii is its antigenic variation, called "phase variation". When C. burnetii express phase I antigen it is highly infectious and a single bacterium is sufficient to infect a human. This is the form that is isolated from animals or humans. After sub culturing C. burnetii in cells or embryonated eggs, modification of its lipopolysaccharide (LPS) capsule results in an antigenic shift to the phase II form, which is not infectious. This antigenic shift can be measured and forms the basis for differentiating acute from chronic Q fever (Raoult and Marrie, 1995).
Epidemiology of Q fever in Tunisia
Q fever in animals
Mammals, birds, and arthropods may be reservoirs for C. burnetii, but the main reservoir is ticks (Raoult and Marrie, 1995). The most commonly identified sources of human infection are farm animals, such as cattle, goats, and sheep. However, pets, including cats, rabbits, pigeons, hedgehogs, and dogs may serve as sources of urban outbreaks of human disease (Stein and Raoult, 1999; Balti et al., 2021). Q fever can cause abortions in sheep and goats and low birthweight in offspring of cattle. High concentrations of C. burnetii may occur in the placenta of infected animals. In Tunisia, a study conducted in 1997, showed that 40% of sheep from different regions were seropositive for C. burnetii, in addition, this bacteria was the abortive agent in 17% of sheep and goats (Rekiki et al., 2005). In recent study conducted during 2016-2017, among healthy camels, 44% of them were found to have positive C burnetii serology done by ELISA, a meaningful high sero-positivity was observed in female camels with a previous history of abortion (70%) (Selmi et al., 2018). In a study conducted by Selmi et al. (2018) among 327 partially engorged ticks collected from camels, C. burnetii, was detected in 3.6%, genotyping and phylogenetic analysis of obtained C. burnetii revealed 99 to 100% similarity to the pathogenic C. burnetii strains isolated from humans, this finding raises the possibility of the involvement of Hyalomma tick species in the active diffusion of these bacteria among camels, other domestic animals and humans (Selmi et al., 2019). Another recent study confirmed the high prevalence of Q fever in animals in Tunisian farms, authors concluded that Q fever increased when the intensive farm was exposed to carnivores and when the cleaning practices were not respected, while it decreased when a suitable quarantine was introduced for any introduction of a new animal. Good hygiene and sanitation practices on-farm should be handled as strategies to deal with this zoonotic pathogen in herds (Barkallah et al., 2018).
Q fever in human
Humans are incidental hosts for infection with C. burnetii. Occupational exposure to C. burnetii among veterinarians may occur through inhalation of contaminated aerosols arising from the placenta or parturient fluids of infected livestock. C. burnetii human infection has also resulted via the following routes: a) trans placental transmission to the fetus, b) intradermal inoculation, and c) blood transfusions. Consumption of raw milk is an additional cause of infection (Salifu et al., 2019; Shishido et al., 2016; Ghanem-Zoubi and Paul, 2020). Q fever can occur in any age group, but infection is most common between the ages of 30 and 70 years. Men are more frequently diagnosed with Q fever than women (Tissot et al., 1992). This can be explained by the fact that men are more exposed to the reservoir, and they have activities that generate aerosols infected by C. burnetii. Otherwise, women and children are more likely to have asymptomatic infection (Tissot et al., 1992). Since the clinical presentation is nonspecific, recognition of cases depends upon astute clinicians and the availability of a reference laboratory. Thus, incidence figures for the disease vary widely. Q fever is rampant in endemic mode with the possibility of epidemic outbreak.
Blood samples (1888) from febrile and non-febrile patients from six African countries were investigated retrospectively for Q fever infection by molecular assays showed a prevalence ranging from 0.3 to 0.5% (Angelakis et al., 2014). In Tunisia, Q fever is an endemic disease, but rarely diagnosed and confirmed by physicians. In fact, in 1983, Kennou and Edlinger (1984) found that prevalence of C. burnetii antibodies among healthy population was 8.7% (3). Another study conducted in 1993, noted that 26% of blood donors in the Tunisia eastern central region (Sousse, Monastir) had antibodies to C. burnetii, majority of them were male (Letaief et al., 1995). During the same period, among 300 inpatients presented with acute fever, C. burnetii serology was done for all patients, showed a typical profile of acute Q fever among 2% of patients and old contact with C. burnetii in 29% of cases (Omezzine-Letaief et al., 1997). In addition, a serological survey carried out during 2004, among 47 inpatients with unexplained fever, acute Q fever had been confirmed in 8 cases (17%) (Kaabia et al., 2006).
Clinical manifestations
Patients with Q fever present with a wide spectrum of disease manifestations. While for some patients the clinical manifestations of acute or chronic infection are severe, the clinical signs and symptoms of Q fever are mild or absent in others.
Acute Q fever (AQF)
The incubation period for acute infection is approximately 20 days (range 14 to 39) (Raoult et al., 2005). Patients with acute Q fever can present with any of the following manifestations.
Flu-like illness: A self-limited flu-like syndrome is the most common manifestation of acute infection. The onset is typically abrupt, with high-grade fevers (40°C), fatigue, headache, and myalgia being the most frequent associated symptoms. In such patients, the headaches are usually severe and can be associated with photophobia. Febrile episodes usually last from one to three weeks.
Pneumonia: Most cases of Q fever pneumonia are mild, and patients present with a non-productive cough and fever. However, acute respiratory distress may occur in some patients. Findings on the chest radiograph are not specific and resemble a viral pneumonia. Pleural effusion may also occur, but it is uncommon. In addition to respiratory symptoms, patients often have extra pulmonary manifestations including severe headaches, myalgia, and arthralgia. Symptoms can last from 10 to 90 days. Mortality rates are low (ranging 0.5 to 1.5%) (Raoult and Marrie, 1995). Among 240 Tunisian patients admitted for acute exacerbations of chronic obstructive pulmonary disease, acute Q fever was diagnosed in 6.6% (Messous et al., 2018).
Hepatitis: Patients with hepatic involvement present with high liver enzymes and can also have: hepatomegaly, which usually occurs without jaundice, an acute febrile episode. Prolonged fever of unknown origin with characteristic granulomas on liver biopsy. The granulomas appear to be "doughnut-like" because they contain a lipidic vacuole surrounded by a fibrinoid ring (Dauby et al., 2020).
Other manifestations of acute Q fever: They include maculopapular or purpuric rash (10%), pericarditis and/or myocarditis (1%), myocarditis can be a particularly severe (Fournier et al., 2001), aseptic meningitis and/or encephalitis (1%) (Bernit et al. 2002). Other rare features were described like neuritis, hemolytic anemia, thyroiditis, gastroenteritis, pancreatitis, lymphadenopathy mimicking lymphoma, erythema nodosum, orchitis, and acute acalculous cholecystitis (Rolain et al., 2003).
In Tunisian studies, the main clinical presentations of acute Q fever were interstitial pneumonia, isolated fever or associated with cytolysis (Bellazreg et al., 2009; Kaabia and Letaief, 2009), and one case of chronic fever of unknown origin with granulomatous in liver biopsy (Omezzine-Letaief et al., 1997). Recently two cases of acute Q fever were published, the first case, was a 19 year-old healthy patient with myocarditis (Hammami et al., 2021), the second, an acute fever in patient treated by Etanercept for Ankylosing Spondyloarthritis (Guiga et al., 2021), both patient treated by doxycycline and their outcome was favorable.
Q fever in pregnancy
Pregnant women presenting with acute Q fever are significantly more likely to be asymptomatic than other patients, however, Q fever may result in obstetrical complications such as spontaneous abortion, intrauterine growth retardation, intrauterine fetal death, oligoamnios, and premature delivery (Ghanem-Zoubi and Paul, 2020), in Tunisian pregnant woman, Q fever studies are rare, its prevalence is unknown, only one study investigated the relationship between miscarriage in humans and infections caused by zoonotic bacteria and genital pathogens, did not find any link between C. burnetii and abortion (Smaoui et al., 2019).
Chronic Q fever (CQF)
It is rare; reported in less than 5% after acute illness, occurred within few months, years or decades after AQF. Pregnancy, cardiac and valve diseases and immunosuppression are high risks for later CQF.
Infective endocarditis with negative blood cultures on prosthetic valve or preexisting lesions is the most common form of CQF (Kampschreur et al., 2015), followed by infection on aneurysm or vascular prosthesis (Botelho-Nevers et al., 2007). The prognosis of CQF is pejorative with high mortality.
In a recent systematic review of C. burnetii epidemiology in Africa, C. burnetii accounted for 1 to 3% of infective endocarditis in Tunisia (Ben et al., 2014). Rekik et al. (2009) in their series of 48 patients with prosthesis valve endocarditis, collected during 10 years (1997-2006) from tertiary care hospital in central Tunisia, one patient had chronic Q fever. Others cases reports were published; all were endocarditis with negative blood culture in immunocompetent patient. The first case, a 35-year-old man, who had recurrent endocarditis (4 episodes) on mitral prosthetic valve. Q fever serology requested only in the fourth episode, showed profile of chronic Q fever. Despite appropriate antibiotics, the patient died after prosthetic valve disinsertion (Ameur et al., 1997). The second case, a 42-year-old patient, with history of mitral and aortic rheumatic disease, presented shortness of breath and fever, since more 3 months, he had splenomegaly. Cardiac echocardiography showed abscess of the mitro-aortic trigone. Blood culture was sterile, C. burnetii serology confirms the diagnosis of chronic Q fever with high titer of antibodies phase I. Patient underwent mitral and aortic valves replacement. He received doxycycline, hydroxychloroquine and ofloxacine for 18 months, with favorable outcome (Fradi et al., 2006). The third case, 48-year-old male patient, presented native aortic valve endocarditis with negative blood culture. Biological prosthesis insertion was done because of hemodynamic instability due to acute heart failure. Unfortunately, patient experienced postoperative bleeding with disseminated intravascular coagulation and died few days after cardiac surgery. Serology for C. burnetii was positive, with high titer for IgG phase I (1/6400). Culture of the valve remained sterile. Quantitative real-time PCR (qPCR) analysis of the valvular sample was strongly positive (355, 395, and 350 copies/mL) for C. burnetii. Whole genome sequencing of the strain was performed directly on the valvular sample to test whether direct sequencing would be feasible for such highly positive sample and to provide genomic data on a Tunisian strain (Delaloye et al., 2017)
Diagnosis
Nonspecific laboratory findings
In AQF, leucocytes count is often normal whereas thrombocytopenia is found in 25 to 30% of cases. Increased liver enzymes could be found up to 71% of cases, cholestasis is less common. In Tunisian series cases, normal WBC or leucopenia, and high liver enzymes (ALT/AST) were the most frequent laboratory findings (71.5%), followed by thrombocytopenia noted in57% (Bellazreg et al., 2009).
In CQF, an increase inflammatory markers is frequently seen sometimes along with circulating immune complexes, rheumatoid factors, microscopic hematuria and cryoglobulinemia.
Confirmatory diagnostic tests
Serologic Testing (Immunofluorescence Assay): The diagnosis of Q fever is confirmed serologically in majority of cases. In AQF, a fourfold rise in titer of phase II IgG between acute and convalescence samples is diagnostic of Q fever. A single serum specimen could be used for the diagnosis of AQF, a titer of phase II IgG > 1/128 in patient with prolonged febrile illness and clinical manifestations of Q fever is suggestive of AQF. IgM antibodies have limited diagnosis value. In CQF, continued increasing phase I IgG antibodies with a titer ≥ 1/1024 along with identifiable site of chronic infection such as endocarditis or endovascular infection is diagnostic of CQF (Kampschreur et al., 2015).
Nucleic acid detection by PCR: PCR has been successfully employed to detect DNA in both cell cultures and clinical samples (Stein et al., 1993). PCR testing can be performed on excised heart valve tissue from the site of active infection (even if frozen or embedded in paraffin), serum, cerebrospinal fluid, pleural fluid, bone marrow, bone and liver biopsies, breast milk, placenta, and fetal tissue. PCR testing was used to diagnose patients whom were suspected of having acute infection but initial serologic testing reveals no or low levels of antibodies. The PCR generally remains positive for 7 to 10 days in acute infection. PCR testing is also helpful in confirming the serologic diagnosis of endocarditis or vascular infection in patients who have persistent elevations of IgG anti-phase I titers.
Culture
Although C. burnetii does not grow in routine blood cultures, culture of this organism can be performed on blood, bone biopsies, cardiac valves, and vascular samples.
Isolation of the bacteria by culture is not recommended for routine diagnosis. It is difficult, time consuming and dangerous requiring a biosafety level 3 laboratory.
In Tunisia, few referent laboratories, from academic hospitals, perform Q fever serology, they look for total Immunoglobulins against C burnetii, without differentiating between Phase I and II.
Treatment
AQF
Majority of AQF resolve spontaneously within 2 to 3 weeks. However, symptomatic or suspected AQF should be treated whereas the treatment is not routinely recommended for asymptomatic form or resolved symptoms if there is no risk factors to develop CQF. The first line antibiotic is doxycycline with a duration of 2 weeks. If this antibiotic is contraindicated, fluoroquinolones, macrolides, rifampicin and trimethoprim/sulfamethoxazol (cotrimoxazol) could be used. Thorough clinical assessment for all patients with AQF is recommended to look for any immunosuppression, pregnancy and cardiac valve defect or valve disease.
Clinical and serologic follow up monitoring is mandatory for all patients after AQF. This monitoring would be closer and longer in patients with cardiovascular risk factors (Melenotte et al., 2020).
CQF
The presence of a nidus of infection with increase of phase I IgG titer (≥ 1/1024) is an indicator to start treatment. The regimen of choice is the combination of Doxycycline with Hydroxychloroquine. The duration of the treatment is 18 to 24 months in case of infective endocarditis or vascular infection. For other CQF sites the duration depends on the clinical and serologic response. Surgical treatment might be necessary if no response to antibiotics. The hydroxychloroquine is contra-indicated in case of G6PD deficiency and in case of retinal or visual field deficits. In such situations, fluoroquinolones, rifampicin, macrolides and cotrimoxazol could be an alternative for the treatment.
Cured case is defined by decrease of phase I IgG (≤1/200) with recovery of clinical symptoms. After the treatment serologic monitoring every 6 months should be done for at least 5 years, lifelong if sever valvular disease (Melenotte et al., 2020).
Pregnant woman
Treatment of AQF in pregnant woman reduces risks of adverse consequences for the fetus, and conversion to CQF. Cotrimoxazol is the antibiotic of choice; it should be given throughout pregnancy except the last two weeks. Macrolides would be alternative for treatment of CQF in pregnant women (Ghanem-Zoubi and Paul, 2020).
Perspective
Actually, based on the recent knowledge on Q fever there is tendency to change the old classification of acute and chronic Q fever to: a) acute infection including flu like illness, pneumonia, hepatitis, acute endocarditis, and other rare manifestation, and b) persistent localized infection, including chronic endocarditis, vascular infection, bone and joint infection and other forms of persistent infection. Many retrospective studies highlighted the role of anticardiolipin antibodies as a useful biological predictive marker for acute Q fever complications, and relationship between persistent C. burnetii infection and non-Hodgkin lymphoma (Melenotte et al., 2020).
