Life-threatening angioedema of the tongue: the detection of the RNA of B henselae in the saliva of a male patient and his dog as well as of the DNA of three Bartonella species in the blood of the patient

Abstract

Non-hereditary angioedema is a common disease with a prevalence between 5% and 19% and approximately half of the patients experience a swelling of the tongue. We report a case of a 49-year-old Caucasian man with a gross life-threatening angioedema of the tongue, whose attacks occurred every 4 weeks. The most frequent causes of angioedema were excluded. We detected DNA and RNA from Bartonella henselae in the blood and saliva of the patient and in the saliva of the patient’s hunting dog. Treatment with azithromycin plus minocycline cleared the blood and saliva of RNA and DNA of Bartonella species, and the patient has been free from angioedema for 1 year. None of the therapy modalities used to treat the hereditary form or ACE or allergy-induced angioedema affect the detrimental course caused by Bartonella species. We therefore suggest that a molecular Bartonella test be included in the analysis of angioedema.

Background

Non-hereditary angioedema is a common disease with a prevalence between 5% and 19%, according to epidemiological studies, whereas hereditary angioedema has a prevalence of 0.0014%.¹ Angioedema presents with swelling of the cutaneous or mucous membranes and frequently involves the lips, eyes, neck, arms, legs and throat. Approximately 50% of patients with angioedema experience a swelling of the tongue. A frequent cause of the angioedema of the tongue is a quantitative or functional defect in the complement system of the C1 esterase inhibitor. To the best of our knowledge, no association between angioedema and Bartonella infection has been reported so far.

Bartonella species and Bartonella henselae have emerged as important infectious pathogens in many diseases. Classically, patients infected with B henselae present with cat scratch disease, typically accompanied by regionally swollen lymph nodes which is called lymphadenopathy. Atypical symptoms are granulomatous conjunctivitis, optic neuritis, bacillary angiomatosis (in immune-compromised patients) and acute encephalopathy. The life cycle, as well as the cellular targets of Bartonella species, indicates that Bartonella species can cause a variety of symptoms and diseases. Bartonella species are facultative intracellular bacteria that mainly infect erythrocytes and endothelial cells. In humans, transmission generally occurs through arthropod or animal bites and scratches. More than 30 different Bartonella species have been identified in small mammals.² At present, 12 Bartonella species are recognised or suspected to be human pathogens.³ B henselae is the most frequent pathogen in humans, and cats are the main zoonotic reservoir of B henselae, as shown in serological and PCR-based studies.^4–6 Vectors such as fleas and ticks commonly transmit the infection between cats, and it is less often transmitted to humans. We present an unusual case of gross angioedema of the tongue in a patient infected with B henselae and Bartonella elisabethae.

Case presentation

A healthy-looking 49-year-old man suffered from recurrent gross swelling of the tongue. During the angioedema attacks, he also noticed a swelling of his hands and arms. Two years prior to presentation, the attacks occurred every 4 weeks with decreasing intervals. At the time of presentation, the patient suffered from these attacks every second week and felt as if he was being suffocated. The efficiency of various medications (etoricoxib, gabapentin) has decreased over time.

The family history did not indicate a familial burden for angioedema. Both parents are alive and in reasonable condition. The patient's brother, his 7-year-old child and his wife are all healthy. He complained about frequent respiratory infections between January and July 2010.

The patient's only pet is a hunting dog. The patient noticed that he did not experience an attack during a 4-week vacation without this dog. In 2010 and 2011, hospital doctors carried out careful physical examinations as well as thorough biochemical, physiological and radiological screening tests. Only minimal deviations from the normal values were observed. All of the blood tests were normal, including tests for complement factors C1q and C4, and the quantitative and functional measurements of the C1q inhibitor because deficiencies in the complement system can be the cause of angioedema. ELISA IgG and ELISA IgM against Borrelia burgdorferi were negative.

Our screening aimed to identify an infectious agent, to determine the proper drug treatment and to explore the conditions for an adoptive cell therapy, which requires the absence of certain viral infections. Serological ELISA tests for hepatitis A, B and C, HIV, cytomegalovirus, and human herpesvirus-6 were negative, analyses of the herpes simplex virus (HSV1), HSV2 and Epstein-Barr virus antibodies showed no signs of an acute or active recurring infection. A low-grade IgG antibody titre against Toxoplasma gondii indicated a previous infection. No antibodies were detected against Chlamydia spp. The patient showed no antibody responses against B henselae.

Molecular analyses were performed on samples of blood, saliva and stool collected from the patient and his wife, blood and stool collected from the son, and stool and saliva collected from the dog. The molecular results for Bartonella spp. and Helicobacter spp. are shown in table 1.

Table 1.

Real-time PCR amplification of Bartonella species in a patient with angioedema and his family

	Patient		Wife		Son		Dog
	Blood	Saliva	Blood	Saliva	Blood	Saliva	Blood	Saliva
Bartonella henselae
DNA	+	+	+	−	−	n.t.	n.t.	+
RNA	+	+	+	n.t.	n.t.	n.t.	n.t.	+
Bartonella elisabethae
DNA	+	−	−	−	−	n.t.	n.t.	−
RNA	−	n.t.	n.t.	n.t.	n.t.	n.t.	n.t.	n.t.
Bartonella bacilliformis
DNA	+	−	−	−	−	n.t.	n.t.	−
RNA	−	n.t.	n.t.	n.t.	n.t.	n.t.	n.t.	n.t.

+, DNA or RNA of the respective Bartonella species was detected. Positive results of DNA samples were confirmed by sequence analysis.

−, no DNA or RNA was detected.

n.t., Not tested.

Genomic DNA from 1×10⁶ peripheral blood mononucleated cells and saliva samples from the patient and his relatives was obtained using the Quick Gene DNA Whole Blood Kit (Kurabo, Japan). Using the PSP Spin Stool DNA Plus Kit of Stratec Molecular (Germany), DNA was extracted from the stool samples according to manufacturer's instructions. Complete RNA was isolated using the Qiagen RNeasy Mini Kit (Qiagen, Germany), cDNA was generated using random hexamer primers and the RevertAid First Strand Synthesis Kit (Thermo Scientific, Germany). The DNA and cDNA were analysed using the non-quantitative real-time PCR-based detection methods published by Pantchev et al,⁷ Miyashita et al,⁸ and Chen et al.⁹ The results were verified using the published nested PCR methods to identify current Chlamydiaceae infection.¹⁰ In parallel, we used DNA from Chlamydia trachomatis, Chlamydia pneumoniae and Chlamydia psittaci as control samples, and we also included a non-template control. We investigated the samples for the presence of Bartonella spp. infection using the non-quantitative real-time PCR-based method of Maggi et al¹¹ and Breitschwerdt et al.¹² Positive results are verified using the method published by Jensen et al,¹³ which allows for discrimination, but not quantification, of Bartonella clarridgeiae, Bartonella quintana, B henselae, Bartonella bacilliformis, Bartonella elizabethae, Bartonella vinsonii subsp. berkhoffii and Bartonella grahamii. In parallel, we used the DNA of B henselae as a control sample, and we included a non-template control. Our efforts to minimise cross-contamination included the physical separation of all PCR steps and the use of interspersed negative controls. For the investigation of Helicobacter hepaticus we used the method of Ge et al¹⁴ and Ito et al¹⁵ that was established to detect Helicobacter pylori. For the detection of Helicobacter felis, we followed the method of Begue et al.¹⁶ To verify the positive results, we performed a second round of investigation using the method published by Chisholm et al,^{17 18} which allows for the discrimination, but not quantification, of H pylori-related and Helicobacter heilmannii-related organisms (such as H heilmannii and H felis). In parallel, we used DNA from H pylori, H hepaticus and H felis as control samples, and we included a non-template control. To confirm the results after the first PCR analysis, the PCR was repeated at least once. All samples were verified as positive by the second PCR. To obtain PCR amplification for sequencing, individual rather than multiplex PCRs were used. The Sequencing Service (SeqService) of the Department of Biology, University of Munich, Germany, sequenced all of the amplification products for the positive samples (defined as the presence of detectable amounts of DNA) to verify the specificity of the PCR products.

Treatment

Treatment with azithromycin (500 mg daily for 3 days, repeated after 7 days) plus minocycline (50 mg daily for 2 weeks) cleared the blood and saliva of the RNA and DNA of all three Bartonella species.

Outcome and follow-up

Until publication of this article, the patient stayed in good condition and showed no sign of recurrence.

Discussion

To the best of our knowledge, this is the first report associating Bartonella species with a gross angioedema of the tongue. Bartonella species have been described as cause of a variety of diseases but so far not of angioedema of the tongue, neither in animals nor in men. Dogs are less frequently infected with B henselae than cats. A PCR-based study in California of 103 shelter cats and 120 dogs detected Bartonella in 21 cats but only in 2 dogs.¹⁹ A study in North Carolina detected Bartonella DNA in 61 of 663 dogs, and B henselae DNA was identified in 30 of the 61 dogs.²⁰ There is considerable regional variability in the prevalence of fleas infected with Bartonella spp.²¹ In 952 fleas collected from 148 cats and 133 dogs, 9.5% of the French dog fleas, but none of the German dog fleas, were positive for Bartonella DNA.²¹

A frequent cause of the angioedema of the tongue is a quantitative or functional defect in the complement system of the C1 esterase inhibitor. Eradication of Helicobacter can essentially improve the symptoms of the hereditary angioedema.^{22 23} In our patient, no defect could be detected in the complement system of the C1 esterase inhibitor. Furthermore, the patient did not receive drugs that may have caused angioedema of the tongue, such as the antihypertensive modality ACE inhibitor, or antiepileptic drugs, such as phenytoin.²⁴ Helicobacter, which can induce angioedema, was detected in the blood and the stool of the patient's wife and his son, but it was not detected in the patient’s blood or stool (data not shown).

The blood sample from the patient’s son was free of B henselae RNA and DNA, and his saliva was not tested. B henselae RNA was detected in the blood of the patient's wife, but not in her saliva (table 1). The patient's wife and son had no symptoms of angioedema. Taken together, the Helicobacter tests of the patient were negative, but the RNA of B henselae was detected in the patient's blood and saliva (table 1). This finding strongly suggests that B henselae is responsible for the angioedema of the tongue. It is unclear whether the DNA of B elisabethae and B bacilliformis interfered with the immune response, either by suppressing the antibody response or enhancing the inflammatory angioedema reaction by B henselae. Only one report exists of B elizabethae as a human pathogen causing bacteraemia and endocarditis.²⁵ One can speculate whether B elizabethae caused or contributed as a pathogenic factor to the development of the patient’s hypermobile interatrial septum.

Learning points.

• Bartonella species can be relevant pathogens in angioedema of the tongue.

• Therefore, and since serological analyses can be misleading, a molecular Bartonella test should be included in the analysis of angioedema (we recommend real-time PCR and reverse transcription real-time PCR to detect bacterial DNA and RNA).

• Treatment with azithromycin (500 mg daily for 3 days, repeated after 7 days) plus minocycline (50 mg daily for 2 weeks) should be sufficient to clear the Bartonella species infection.