Diagnosis of a Rare Rickettsia felis Infection Complicated with Unusual Pericardial Effusion and Cardiac Tamponade Using an mNGS Test

The occurrence of sporadic rickettsial infections has been consistently undervalued and overlooked, primarily owing to a limited emphasis on routine examinations for rickettsioses in clinical practice. At present, the immunofluorescence assay is the prevailing diagnostic method for suspected rickettsioses that enables the detection of specific antibodies against rickettsia in human serum. Herein, we present an exceptional instance of rickettsial infection that was characterized by a rare manifestation of extensive pericardial effusion leading to dyspnea and cardiac tamponade. A diagnosis of chronic fibrosing pericarditis was established based on pericardium tissue obtained through pericardiotomy, and a conclusive metagenomic next-generation sequencing test confirmed the presence of Rickettsia felis infection. The cat flea, scientifically known as Ctenocephalides felis, is the predominant carrier of R. felis. An escalating incidence of human R. felis infections has raised concerns, particularly in light of the burgeoning population of domesticated animals in many contemporary societies.


Introduction
Among the expansive array of arthropods, including feas, ticks, mites, and lice, the cat fea (Ctenocephalides felis) is widely acknowledged as the principal reservoir of Rickettsia felis bacteria [1].A recent report highlighted the emergence of mosquitoes as carriers of R. felis in the United States [2].
Te clinical manifestations of human R. felis infection, commonly referred to as fea-borne spotted fever or cat fea typhus, are similar to those of other rickettsial diseases.Tese similarities manifest as symptoms including fever, rash, myalgia, and headache.Furthermore, in more severe cases, individuals may exhibit additional manifestations such as hepatomegaly, myocarditis, meningoencephalitis, and acute respiratory distress syndrome [3].
Although it was initially recognized as a pathogenic agent in humans in the United States in 1994 [4], R. felis (an intracellular gram-negative bacterium) continues to be underestimated, despite advancements in serological and molecular diagnostic techniques such as immunofuorescence assay (IFA) and polymerase chain reaction (PCR).Tis lack of recognition can be attributed to the limited emphasis placed on the routine examination of rickettsioses in clinical practice.Consequently, it is possible that a signifcant proportion of human cases are not accurately diagnosed.
In this report, we present a unique case of R. felis infection that was characterized by an uncommon clinical manifestation of dyspnea and cardiac tamponade caused by extensive pericardial efusion.Te diagnosis was validated through an examination of the collected pericardial fuid using a novel metagenomic next-generation sequencing (mNGS) test, which provided conclusive results.

Case Presentation
A 79-year-old female patient was admitted to the intensive care unit with dyspnea lasting one week and cardiac tamponade, which was determined through echocardiography to be caused by substantial pericardial efusion of unclear origin.Her medical history included the following: (1) a history of breast cancer with a left mastectomy in 2008 and a partial right mastectomy in 2015; (2) prior instances of ventricular fbrillation and long QT syndrome, which prompted the implantation of an implantable cardioverterdefbrillator (ICD) in 2013; (3) paroxysmal atrial fbrillation under direct oral anticoagulants (DOAC) since 2013; (4) type 2 diabetes mellitus; and (5) hyperlipidemia.
Computed tomography revealed bilateral pleural efusion and suggested the presence of cardiac tamponade caused by a substantial pericardial efusion (Figure 1).Initially, a pericardiocentesis procedure extracted 1030 cc of dark red drainage.Te patient was additionally administered 4.5 g of tazocin (sodium piperacillin 2.0 g + sodium tazobactam 0.25 g) intravenously every 8 hours for 14 days, as part of a routine antibiotic treatment for suspected lung infection.
In response to the recurrent accumulation of pericardial and pleural efusions, a surgical pericardial-pleural window and pleurodesis were performed.To test for potential tuberculosis, the collected bloody pericardial fuid was subjected to routine analysis, cytology, and culture.Additionally, two pieces of pericardial tissue (measuring 3 × 4 cm and 2 × 6 cm) were excised anterior to the phrenic nerve for pathological examination.
Conclusive pathological fndings from the pericardial fuid and tissue demonstrated an absence of malignant cells.A subsequent mNGS test was performed to examine the pericardial fuid and serum blood samples for the presence of other infectious pathogens, owing to negative results obtained on a tuberculosis test.Nucleic acids were extracted from the collected samples using a TIANamp Micro DNA Kit (Tiangen Biotech Co., Ltd, Beijing, China) and then used for library construction with a MGIEasy Cell-free DNA Library Prep Kit (MGI Tech Co., Ltd, Shenzhen, China) and high-throughput sequencing on the MGISEQ-200 platform (MGI Tech Co., Ltd, Shenzhen, China).High-quality sequencing data were generated by removing short (<35 bp), low-quality, and low-complexity reads.Human reads were removed by mapping to the human reference genome hg38 (GRCh38, December 2017) using the Burrows-Wheeler Aligner.Te remaining data were compared to the Microbial Genome Database (https://ftp.ncbi.nlm.nih.gov/genomes/) using the Burrows-Wheeler Alignment tool v0.7.10-r789.Our mNGS results indicated the presence of Rickettsia felis (275 reads) that accounted for 2.18% of nucleotide sequence coverage and showed 62.29% relative abundance in the pericardial fuid (Table 1, Figure 2) but was not detected in the blood.Tis prompted the addition of oral doxycycline 100 mg twice daily to the treatment regimen for 10 days, in accordance with relevant treatment guidelines [5].A thorough investigation of the patient's history, as well as that of her family, revealed that the potential pathogen originated from a domesticated cat that lived in the patient's household.
Te patient recovered from the illness, showing regression of the lung infection on chest radiography, and a decrease in pleural and pericardial efusions.Te patient was discharged after completing the doxycycline treatment.
IFA has become the standard and most commonly used method to detect specifc antibodies against R. felis in the serum.However, a notable limitation arises because of crossreactivity among various Rickettsiae species.Higher concentrations of antibodies against R. felis may potentially aid in distinguishing R. felis infections from other rickettsial diseases [7].An additional diagnostic technique used to detect R. felis infection involves the use of PCR to amplify specifc gene fragments of R. felis-including gltA, ompA, ompB, and 17-kDa antigen [6][7][8].In contrast to conventional pathogen detection techniques, novel mNGS analysis yields fast and precise pathogen detection and identifcation [9][10][11].
R. felis infection is a global health concern in the human population, exhibiting a correlation with febrile ailments that can induce severe or even lethal complications.Its presenting symptoms resemble those observed in other rickettsial diseases-ranging from mild fever, skin rash, cutaneous eschar at the bite site, myalgia, and headache; to less frequent yet more intricate manifestations involving the visceral organs, neurological complications, and the heart [8].
Although relatively rare, a range of neurological manifestations associated with R. felis infection-including hearing loss, photophobia, meningitis, meningoencephalitis, and symptoms resembling polyneuropathy-were comprehensively summarized in a literature review conducted by Zeng et al., spanning the period between 2000 and 2020 [12].
While myocarditis and pericarditis have been extensively documented as the primary cardiac complications associated with various rickettsial infections-including Rickettsia rickettsii, Rickettsia conorii, Rickettsia africae, Rickettsia japonica, and Rickettsia tsutsugamushi [13][14][15]-there remains a dearth of case reports that have specifcally documented cardiac complications related to R. felis infection.Pericardial efusion has been reported to be a manifestation of Rickettsia tsutsugamushi and Rickettsia typhi [16][17][18].Additionally, a previous case study reported pericarditis and pericardial efusion associated with Bartonella quintana [19].

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Case Reports in Infectious Diseases However, to date, no published reports have described pericarditis or pericardial efusion associated with R. felis infection.
To the best of our knowledge, this is the frst case report to document R. felis infection accompanied by pericarditis and pericardial efusion substantiated by direct pathological evidence obtained from the pericardial fuid, which was confrmed through mNGS.
Misdiagnosis of this type of infection is frequent, owing to inadequate awareness of it and the limited availability of specifc laboratory testing required to confrm R. felis involvement.A recent review hypothesized that ∼33% of cat feas collected from companion dogs and cats in Taiwan are infected with R. felis (although the available data were limited) [6].
Nevertheless, Lai et al. emphasized the long-standing underappreciation and neglect of spotted fever group rickettsioses in southern Taiwan, particularly focusing on species that are closely associated with R. felis [20].Likewise, Yang et al. conducted a retrospective seroepidemiological analysis in Taiwan involving 122 patients who were suspected of having rickettsioses but tested negative for scrub typhus, murine typhus, or Q fever [21].Tis study revealed a seropositivity rate of 19%, indicating exposure to rickettsia.Among these cases, eight individuals had antibodies that were responsive to R. felis-of whom four showed evidence of ongoing R. felis infection and one, whose doxycycline treatment was discontinued because negative results for   scrub typhus, Q fever, and murine typhus experienced a fatal outcome.

Conclusion
In contrast to other rickettsial diseases in Taiwan, which are primarily transmitted by arthropods in natural habitats, the emergence of Rickettsia felis infection is thought to be associated with the expanding population of companion animals in contemporary society [6,22,23].Terefore, it is imperative to engage in proactive surveillance of patients with unidentifed causes of pericardial efusion.Furthermore, beyond conventional antibody detection using IFA, mNGS provides a new method for surveying rare infectious pathogens.

Table 1 :*
Results of mNGS on our patient's pericardial fuid.Relative abundance at the genus level refers to the proportion of detected microorganisms within the genus in the entire sample.* * Relative abundance at the species level refers to the proportion of detected microorganisms within the species in the entire sample.

Figure 2 :
Figure2: Results of mNGS analysis on the patient's pericardial fuid.Te sequenced gene fragment regions were widely distributed across diferent locations on the Rickettsia felis genome.Tis indicated that the detected reads did not originate from singlefragmented DNA pieces through repeated sequencing but rather came from the entire genome, thus proving the presence of Rickettsia felis in the sample.

Figure 1 :
Figure 1: Transverse plane (a, b) and coronal plane (c) computed tomography (CT): observed pericardial efusion (red arrows) with relative hyperdensity of the collections and suspected cardiac tamponade.