Q Fever Endocarditis in Romania: The First Cases Confirmed by Direct Sequencing

Ani Ioana Cotar,Otilia Banu,Sorin Dinu,Daniela Badescu,Monica Straut,Minodora Dobreanu,Dan Dobreanu,Mihaela Oprea,Adina Ionac,Mirela Flonta

doi:10.3390/ijms12129504

Abstract

Infective endocarditis (IE) is a serious, life-threatening disease with highly variable clinical signs, making its diagnostic a real challenge. A diagnosis is readily made if blood cultures are positive, but in 2.5 to 31% of all infective endocarditis cases, routine blood cultures are negative. In such situations, alternative diagnostic approaches are necessary. Coxiella burnetii and Bartonella spp. are the etiological agents of blood culture-negative endocarditis (BCNE) most frequently identified by serology. The purpose of this study is to investigate the usefulness of molecular assays, as complementary methods to the conventional serologic methods for the rapid confirmatory diagnostic of Q fever endocarditis in patients with BCNE. Currently, detection of C. burnetii by culture or an antiphase I IgG antibody titers >800 represents a major Duke criterion for defining IE, while a titers of >800 for IgG antibodies to either B. henselae or B. quintana is used for the diagnosis of endocarditis due to Bartonella spp. We used indirect immunofluorescence assays for the detection of IgG titers for C. burnetii, B. henselae and B. quintana in 57 serum samples from patients with clinical suspicion of IE. Thirty three samples originated from BCNE patients, whereas 24 were tested before obtaining the blood cultures results, which finally were positive. The results of serologic testing showed that nine out of 33 BCNE cases exhibited antiphase I C. burnetii IgG antibody titer >800, whereas none has IgG for B. henselae or B. quintana. Subsequently, we used nested-PCR assay for the amplification of C. burnetii DNA in the nine positive serum samples, and we obtained positive PCR results for all analyzed cases. Afterwards we used the DNA sequencing of amplicons for the repetitive element associated to htpAB gene to confirm the results of nested-PCR. The results of sequencing allowed us to confirm that C. burnetii is the causative microorganism responsible for BCNE. In conclusion, the nested PCR amplification followed by direct sequencing is a reliable and accurate method when applied to serum samples, and it may be used as an additional test to the serological methods for the confirmatory diagnosis of BCNE cases determined by C. burnetii.

Highlights

Infective endocarditis is a serious, life-threatening disease with highly variable clinical signs that are making the condition a diagnostic challenge
1 to 5% of patients progress to chronic infection, which can develop after months to several years after acute Q fever infection, the longest interval being 20 years after infection [12,13]
IgG to C. burnetii, B. quintana and B. henselae originated from patients with clinical suspicion of Blood culture negative endocarditis (BCNE)

Summary

Introduction

Infective endocarditis is a serious, life-threatening disease with highly variable clinical signs that are making the condition a diagnostic challenge. A diagnosis is readily made if blood cultures are positive, but in 2.5 to 31% of all infective endocarditis cases, routine blood cultures are negative [1,2,3]. This variation in incidence could be explained by several factors, including: (i) differences in the diagnostic criteria used; (ii) specific epidemiological factors, as for fastidious zoonotic agents;. Q fever is characterized by its clinical polymorphism and the presentation of the disease is variable, with both acute and chronic manifestations [10,11]. 1 to 5% of patients progress to chronic infection, which can develop after months to several years after acute Q fever infection, the longest interval being 20 years after infection [12,13]

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Conclusion