Acute Q fever is a zoonotic disease caused by the intracellular bacterium Coxiella burnetii; it typically presents with a sudden onset of high fever, severe headache and myalgia, often accompanied by pneumonia or hepatitis. Transmission to humans primarily occurs through inhalation of contaminated aerosols from infected animals, though tick bites may also serve as a transmission route (1-4), albeit less commonly documented.

Recent studies suggest that C. burnetii may have originated from tick endosymbionts, specifically Coxiella-like endosymbionts (CLEs), which share similar 16S rRNA gene sequences with C. burnetii (1). This genetic similarity poses a significant challenge in accurately identifying the source of infection. When ticks are found to carry CLEs that closely resemble C. burnetii, it can lead to the erroneous conclusion that the infection is not tick-borne. This misinterpretation occurs as the genetic markers used to identify C. burnetii may also detect these symbiotic bacteria, leading to false-negative results for C. burnetii when it is, in fact, present (5).

Such complications in distinguishing between C. burnetii and its genetically similar endosymbionts can result in underestimating the role of ticks in transmitting Q fever. Consequently, patients who contract Q fever through tick bites might not be correctly identified, especially if the diagnostic methods fail to differentiate between these bacteria. This underscores the importance of using highly specific and sensitive diagnostic tools, such as targeted next-generation sequencing (tNGS), which can provide a more accurate identification of the pathogens involved. Although the probability of contracting Q fever through tick bites is lower compared with contracting the disease through airborne transmission, ticks are recognized by the scientific community as a potential vector (3,4). Documenting this case underscores the necessity for heightened clinical vigilance regarding the potential for tick-borne transmission of Q fever and demonstrates the effectiveness of tNGS in accurately diagnosing such infections. This case also serves as a reminder of the importance of considering tick-borne pathogens in patients with compatible clinical presentations and potential tick exposure.

A 26-year-old male internal medicine resident physician at Guang'anmen Hospital (Beijing, China) presented to the Emergency Department in May 2024 (day 0) with a severe headache, persistent high fever, chills and significant nausea. The patient reported that these symptoms/complaints began following a hiking trip to Baiguzha Mountain in the Xiaowutai Nature Reserve (Zhangjiakou, China) earlier that month. Approximately 3 days before the hospital visit (day-3), the patient's condition suddenly deteriorated with a severe headache, high fever of 39.5˚C, chills, sweating and nausea. The patient performed a self-initiated complete blood count and C-reactive protein (CRP) test, revealing a slightly elevated CRP of 13.66 mg/l, with otherwise normal parameters. Suspecting severe influenza, the patient began self-treatment with loxoprofen sodium, moxifloxacin (0.4 g) and oseltamivir (75 mg). Despite this, the symptoms persisted and the headache worsened significantly, prompting the patient's visit to the Emergency Department by 9 p.m.

At the Emergency Department, the patient reported persistent high fever, chills and nausea, and the patient's headache had become unmanageable. Initial laboratory tests 2 days post-admission (day 2) showed mildly elevated aspartate aminotransferase (AST) as well as direct and indirect bilirubin, decreased potassium and sodium levels, and elevated lactate (Table I). High-resolution chest CT and viral tests for influenza and COVID-19 were negative (data not shown), so the symptoms were initially attributed to stress and dehydration, considering the patient's own earlier negative tests. However, over the next three days, the patient's symptoms did not improve with fluid and electrolyte correction (Fig. 1A and B). The patient's condition continued to deteriorate, with fluctuating fevers between 38.6 and 40.4˚C and persistent headaches. The patient's body weight had dropped from 70.2 to 67.1 kg over the 3 days before admission to the hospital. Despite receiving daily intravenous fluids and electrolytes, the patient's liver enzymes continued to rise and electrolytes remained imbalanced. Urgent lab tests also supported this deterioration (Fig. 1C and D).

Upon presentation, while examining the patient due to unrelenting headaches and persistent high fever, the Emergency Department physician noticed significant conjunctival edema (Fig. 2). The physician learned that the patient had returned home from the hike and slept directly under an air conditioner set to 24˚C. The next morning, the patient developed a mild headache and a body temperature of 37.6˚C, which the patient attributed to possible cold exposure or air conditioning. Given the patient's recent exposure to air conditioning, the combination of respiratory and gastrointestinal symptoms and the presence of hyponatremia, the physician initially suspected Legionella infection. Legionella pneumophila, often associated with air conditioning systems, can present with these symptoms and is known to cause hyponatremia (6-8).

During the examination, the physician discovered two small lesions on the patient's right hand with a 4-mm red mark between them, which the patient had not noticed (Fig. 3). Upon further questioning, the patient recalled brushing off a small insect during his hike, later identified as a longhorned tick (Haemaphysalis longicornis) based on online images and local reports (9) confirming the tick's presence in the area. Despite the rarity of tick-borne infections leading to acute illnesses in China (10), this finding led the physician to consider tick-borne infections and order both metagenomic NGS (mNGS) and tNGS to identify potential pathogens.

The tNGS assay (11), performed at Sanway Clinical Laboratories, used the tNGS 296 PLUS panel, which screens for 296 pathogens. DNA was extracted using the Pathogen Target Gene Detection Kit (cat. no. #sx0010; Sansure Biotech Inc.), and the quality was assessed by capillary electrophoresis on the Qsep400 system. Sequencing was carried out on a GenoLab M platform (GeneMind Biosciences, Co., Ltd.) with a single-end read length of 75 base pairs, using the GenoLab M Sequencing Kit V3.0 (cat. no. FCM-D SE075-D; GeneMind Biosciences, Co., Ltd.). The final library was loaded at 4000 pM, measured by a Qubit Fluorometer. Data analysis included fastp for quality control, BWA for alignment, Samtools for SNP calling and BLAST for pathogen identification.

The tNGS results, available later that day, identified 43 sequences of Coxiella burnetii, diagnosing acute Q fever (Table II). In addition to Coxiella burnetii, other microorganisms were also detected, including 44 reads of Burkholderia cepacia, 40 reads of Candida parapsilosis and 7 reads of SARS-CoV-2. However, these organisms were considered unlikely to be the causative agents of the patient's symptoms due to their lower pathogenic relevance in the given clinical context and epidemiological background. Specifically, Burkholderia cepacia is primarily associated with immunocompromised patients or those with chronic lung disease, neither of which applied to this patient, and it is often a colonizer rather than a true pathogen in non-immunocompromised hosts (12). Furthermore, Candida parapsilosis and SARS-CoV-2 had very low reads and were not consistent with the patient's presenting symptoms. To confirm the presence of Coxiella burnetii, the tNGS results were further supported by two genomic alignment maps (Fig. 4A and B), which illustrate the alignment of the patient's sequenced reads with the Coxiella burnetii reference genome. The genomic alignment maps (Fig. 4A and B) were generated using Integrative Genomics Viewer version 2.18.4 software (Broad Institute), which is commonly used for visualizing sequencing data aligned to a reference genome. These results provide strong evidence of Coxiella burnetii as the primary pathogen responsible for the patient's acute symptoms.

The mNGS results on day 1 confirmed the presence of Staphylococcus haemolyticus (relative abundance, 0.17%) and 9 sequences of Coxiella burnetii (relative abundance, 0.05%). Of note, the patient had undergone QFR-IgM [immunofluorescence assay (IFA)] testing and blood cultures the day before, both of which returned negative results after the tNGS results were obtained.

The timely tNGS results led to the initiation of doxycycline and glutathione for liver protection on the evening of day 0 (Table III). Upon retrospective examination, the patient reported the highest intensity of symptoms, including headache, chills and anorexia from day -3 to day 0, with gradual improvement following the initiation of doxycycline. The patient experienced the highest recorded temperatures of 40.4˚C on day -1 and 40.1˚C on day 0, which gradually decreased after day 1. The patient's treatment regimen evolved over time. Initially, the patient self-administered oral moxifloxacin for 3 days prior to admission (from day -3 to day -1). Upon presenting to the Emergency Department on day 0, the treatment was adjusted to intravenous moxifloxacin from days 0 to day 2 to enhance the anti-infective effect. The patient also received potassium citrate and electrolytes to correct imbalances, and pain was managed using loxoprofen and oxycodone-acetaminophen.

Due to severe nausea and anorexia, the patient did not consume any oral nutrition for several days, relying on intravenous fluids for hydration and electrolyte balance. Oral intake resumed as the patient's symptoms improved. By day 6, the patient's AST and alanine aminotransferase (ALT) levels had peaked and then began to normalize gradually (Table I). Potassium and sodium levels continued to fluctuate, requiring ongoing supplementation. Imaging and diagnostic tests showed no significant findings, but the tNGS results on day 0 were pivotal in diagnosing acute Q fever. The diagnosis was confirmed by mNGS the following day, revealing Coxiella burnetii sequences. By day 11, as determined in a follow-up examination, the patient's laboratory indicators and symptoms had significantly improved. On that day, the IFA test reported positive results. The patient continued with oral doxycycline and liver protection medication for 24 days, while monitoring their condition independently.

Acute Q fever, caused by Coxiella burnetii, poses a diagnostic challenge due to its nonspecific symptoms and underreporting in certain regions, including China (13). This case underscores the vital role of tNGS in diagnosing Q fever, particularly when traditional methods are inconclusive.

The patient's initial presentation of mild headache and low-grade fever was nonspecific, contributing to a delay in identifying the underlying, more serious infection. Over the next few days, the symptoms progressed to severe headache, high fever (exceeding 40˚C) and gastrointestinal disturbances, including vomiting and chills. These clinical features, along with abnormal laboratory findings initially prompted differential diagnoses that included viral infections and systemic inflammatory responses. However, despite empirical treatments aimed at symptom control, the patient's high fever persisted and his headache remained severe, indicating the need for further diagnostic exploration.

The patient's symptom scores were highest between day-3 and day 0, particularly in terms of headache and fatigue. Despite initial symptomatic treatment, the severity of symptoms did not abate, suggesting that an infectious etiology was at play. By day 1, the persistence of these symptoms, combined with the patient's laboratory results and recent travel history, led to a reassessment of the differential diagnosis. Q fever, caused by Coxiella burnetii, frequently presents with a spectrum of non-specific symptoms, including flu-like illness, acute hepatitis and chronic fatigue syndrome (14,15), all of which were observed in the patient of the present study. The patient's symptom onset 10 days after potential exposure is consistent with the known median incubation period for acute Q fever, which ranges between 7 to 32 days post-exposure (16). Male gender and recent travel to rural areas, where tick exposure may occur, further supported the clinical suspicion of Q fever in this case (17).

Following the confirmation of the diagnosis using tNGS, doxycycline therapy was initiated immediately. The patient's maximum axillary temperature declined rapidly post-treatment, with the fever subsiding completely after 5 days. In addition, within 2 days, symptoms such as chills and vomiting had significantly improved, as evidenced by the reduced symptom severity reported by the patient. The patient's electrolyte balance was restored by day 6 and serum sodium levels returned to within the normal range (Fig. 1C). Furthermore, despite a transient increase in liver enzymes following treatment initiation, timely intervention with glutathione for liver protection resulted in a steady decline in both ALT and AST levels, as shown in Fig. 1D.

Given the patient's presentation, several potential causes for the key symptoms, including headache, elevated liver enzymes and overall clinical deterioration, needed to be considered. Below, the differential diagnoses for these symptoms are being discussed. Severe headache is a common symptom of Q fever, but oseltamivir, which the patient had self-administered, is also associated with headache as a mild side effect. However, oseltamivir-induced headaches are typically mild and transient (18). In the present case, the persistence and intensity of the headache, along with the patient's other systemic symptoms, made Q fever the more likely cause. This symptom, combined with the presence of fever and a known history of tick exposure, led to the prioritization of infectious causes over medication-induced effects.

Loxoprofen and oxycodone-acetaminophen are both known to cause hepatic injury and elevated liver enzymes; the typical onset of drug-induced liver damage occurs at least 24 h post-ingestion, often requiring sustained use to reach a critical threshold (19). However, in the patient of the present study, the elevated ALT and AST levels were detected before loxoprofen use. Therefore, the hepatic abnormalities were more likely due to the underlying Q fever infection.

Glutathione (GSH) plays a crucial role in cellular defense against oxidative stress, particularly during infection, but it is well-established that GSH may lower zinc levels by promoting the utilization of zinc in cellular repair and antioxidant processes. Given zinc's vital role in maintaining immune function, including T-cell activation, cytokine production and neutrophil activity, a reduction in zinc availability can impair the body's ability to fight infections (20). This connection suggests that during the acute phase of infections such as Q fever, early administration of GSH could theoretically exacerbate the condition by reducing zinc levels. However, in the present case, the rapid progression of liver damage and the patient's response to doxycycline were carefully weighted. Despite the potential risks, the preservation of liver function was prioritized by administering GSH early in the treatment to mitigate hepatic damage. The balance between infection control with doxycycline and hepatic protection with GSH appears to have been beneficial, as demonstrated by the patient's improved clinical course.

The use of tNGS in the present case was pivotal. Unlike traditional methods such as serology and culture, which are often time-consuming and may lack sensitivity, high-throughput sequencing technologies such as tNGS and mNGS offer rapid, comprehensive and unbiased pathogen identification (21-23). tNGS in particular provides several advantages over mNGS and traditional methods. Firstly, tNGS focuses on specific pathogens, allowing for higher sensitivity and faster results compared to mNGS, which screens for all potential pathogens and requires extensive data analysis (24-28). The high sensitivity and specificity of tNGS make it an invaluable tool in the diagnosis of Q fever, particularly given its exceptional ability to detect atypical pathogens with high accuracy (29-32). This is particularly crucial in regions where the disease is rare and clinicians may not readily consider it in their differential diagnoses (33). In the patient of the present study, tNGS identified 43 sequences of C. burnetii within a short turnaround time, leading to a prompt diagnosis of acute Q fever. The tNGS296 PLUS panel, used in the present case, is designed to detect 296 pathogens that are suitable for emergency screening. This targeted approach facilitated the rapid identification of C. burnetii and demonstrated the efficiency of tNGS in an acute clinical setting. mNGS results, which corroborated the presence of C. burnetii (9 sequences) and also detected Staphylococcus haemolyticus (relative abundance, 0.17%), took longer to process and analyze. Secondly, the rapid identification of C. burnetii through tNGS allowed for the timely initiation of doxycycline therapy, which was pivotal in the patient's recovery. Traditional diagnostic methods, which often take several days to weeks to yield results, would have delayed the initiation of appropriate treatment. This case illustrates how tNGS can directly impact clinical decision-making by providing fast and accurate pathogen identification, thereby improving patient outcomes in acute infectious diseases. Thirdly, tNGS has demonstrated higher accuracy in detecting low-abundance pathogens due to its targeted approach (34-36). This is particularly important in cases of rare or emerging infections where pathogen loads may be low or when patients have been pre-treated with antibiotics, which can reduce the pathogen load detectable by traditional methods. Furthermore, this case also highlights a less common route of Q fever transmission, emphasizing the need for clinicians to consider tick-borne transmission in patients with compatible symptoms and exposure history. The identification of C. burnetii in a patient with a recent tick bite, a less documented transmission route in China, adds to the growing body of evidence supporting the role of ticks in Q fever epidemiology. Furthermore, tNGS can provide insights into the genetic diversity of pathogens, such as C. burnetii, revealing information on virulence factors, resistance patterns and epidemiological trends (28,37). These insights are valuable for understanding disease outbreaks and tailoring appropriate public health responses. The use of tNGS296 PLUS in this case not only facilitated a rapid and accurate diagnosis but also underscored the potential of tNGS panels in enhancing the clinical management of infectious diseases.

Traditional diagnostic methods such as indirect IFA and ELISA, while useful, have notable limitations. These methods can take several days to weeks to yield results and may not detect early-stage infections due to the reliance on antibody presence, which may not be detectable in the initial stages of the disease (38,39). IFA, although considered the primary method for diagnosing acute Q fever, was outperformed by tNGS in terms of both sensitivity and speed in this case (40-42). PCR, another common diagnostic tool, offers rapid and specific pathogen detection but is limited to known targets and may have lower sensitivity in blood samples (43). tNGS, on the other hand, provides a targeted approach that ensures high sensitivity and quick diagnosis, crucial for timely clinical decision-making (33). By contrast, mNGS offers broad pathogen detection without pre-set targets, making it useful for identifying unknown or unexpected pathogens. However, mNGS is expensive, requires complex data analysis and may not consistently outperform traditional methods in certain contexts, such as diagnosing suspected pneumonia in immunocompromised patients (35,44). Compared to mNGS, tNGS focuses on specific pathogens, offering high sensitivity and rapid diagnosis, making it a highly effective tool for timely clinical decision-making (Table IV). mNGS offers broad pathogen detection without the need for pre-set targets, making it highly versatile, but it requires expensive equipment, complex data analysis and specialized expertise. On the other hand, tNGS is designed for high sensitivity and rapid diagnosis, focusing on specific pathogens, which makes it advantageous for time-sensitive clinical applications. However, the limitations of tNGS include narrower coverage and a dependency on pre-set targets, which restricts its ability to detect unexpected pathogens. Importantly, the diagnostic performance of NGS is not compromised by the empirical use of antibiotics prior to sampling (45,46). In addition, high-throughput sequencing technologies like tNGS and mNGS provide comprehensive and precise insights into the virulence, resistance patterns and epidemiological trends of C. burnetii (47). In fact, preliminary successes have already been achieved in using NGS to diagnose Q fever in China (48-52).

Due to the nonspecific clinical presentation of acute Q fever, which includes high fever, severe headache, chills and gastrointestinal disturbances, its differential diagnosis encompasses various infectious and non-infectious diseases, such as bacterial and viral infections, and systemic inflammatory responses, particularly Legionnaires disease. The causative agent of Q fever, Coxiella burnetii, is closely related to Legionella species and both can cause severe atypical pneumonia, making clinical differentiation particularly challenging (1,53,54).

The present case underscores the significant advantages of integrating tNGS into emergency clinical practice, particularly for diagnosing rare infectious diseases such as Q fever. By providing rapid and accurate pathogen identification, tNGS facilitates timely clinical decision-making, allowing for earlier and more targeted therapeutic interventions. We advocate for the broader application of tNGS in emergency settings, particularly in regions where rare infectious diseases may not be readily considered.

This case also raises several questions for future research. One area of interest is the role of ticks in the epidemiology of Q fever. While aerosol transmission from livestock is well documented, the contribution of tick-borne transmission to the overall incidence of Q fever remains less clear. In China, there is no sufficient evidence to suggest that Q fever is commonly transmitted to humans through tick bites (55). However, individual case reports have documented instances of Q fever following tick bites in China (36). In addition, numerous other infectious diseases are transmitted through tick bites (56-60), making them a significant infection factor that cannot be ignored. Further studies are needed to elucidate the prevalence of C. burnetii in tick populations and the risk factors associated with tick bites in endemic areas.

Another area for research is the optimization of tNGS protocols for the detection of C. burnetii and other intracellular pathogens. Current studies indicate that the design of primers is critical to the sensitivity and specificity of tNGS assays, yet significant advancements are still needed in this area (61-63). Improving these aspects could enhance the utility of tNGS in clinical diagnostics and epidemiological surveillance. In addition, exploring the genetic diversity of C. burnetii strains through tNGS could provide insight into the pathogen's virulence, resistance patterns and epidemiological trends (44). While tNGS shows promise, mNGS has not consistently demonstrated statistical superiority over traditional methods in certain contexts, such as in diagnosing suspected pneumonia in immunocompromised patients, and may have lower sensitivity for specific pathogens such as Aspergillus species (41). Therefore, focused research on tNGS protocol optimization is essential to maximize its clinical application.

Further research into the pathophysiology of acute Q fever is also warranted, particularly its association with hyponatremia. Although reliable evidence to definitively link acute Q fever to hyponatremia appears to be lacking, this patient exhibited persistent hyponatremia before doxycycline treatment. A 1998 study reported hyponatremia in 28.2% of Q fever cases (64), suggesting a potential underlying mechanism worth exploring. It may be hypothesized that, similar to Legionella infections (65,66), C. burnetii may induce increased secretion of antidiuretic hormone, leading to enhanced sodium excretion. The close phylogenetic relationship between C. burnetii and Legionella supports this speculation. However, the mechanism of hyponatremia was not further investigated in this case.

In conclusion, the present case of acute Q fever diagnosed through tNGS underscores the diagnostic challenges posed by the disease and the potential of advanced molecular techniques to overcome these challenges. The integration of tNGS into clinical practice can facilitate the rapid and accurate diagnosis of infectious diseases, leading to timely and effective treatment. Further research into the epidemiology of Q fever and the optimization of tNGS protocols will enhance our understanding and management of this complex disease. Further research and clinical awareness are needed to improve the recognition and management of Q fever, particularly in endemic regions. Future studies should also focus on the cost-effectiveness and feasibility of implementing tNGS in routine clinical practice, particularly in resource-limited settings.