The use of tNGS in the diagnosis of Q fever: A case report and review of cases of liver injury

Abstract

This report describes a case of Q fever with predominantly liver injury presentation. We used targeted next generation sequencing (tNGS) and immunologic methods to identify a case of acute stage infection of Q fever with liver injury; prompt diagnosis and treatment significantly improved the patient's prognosis and life quality. Further, to predict the homology and genetic diversity, the patient's sample was partially sequenced for insertion sequence (IS)1111 gene. Additionally, we reviewed similar cases in the past five years, aiming to provide evidence-based evidence for subsequent accurate diagnosis and treatment.

Graphical abstract

1. Introduction

Q fever (query fever), a globally distributed zoonosis, results from infection with the pathogen Coxiella burnetii (C. burnetii). Primary reservoirs comprise domestic ruminants, notably goats, cattle, and sheep,^1,2 and ticks serve as the main transmission vector.³ Q fever with insidious onset, ranging from asymptomatic infection to critical condition, with variable clinical presentations categorized into acute infection and persistent focalized infections.⁴ Nearly all patients exhibit elevated liver enzyme levels, with severe cases often involving multi-organ damage. Without prompt treatment, 1%–5% of patients progress to persistent focalized infections, which carry a poorer prognosis.⁵ This article documents a Q fever case characterized by fever and liver injury from Jiangxi Province, China. The diagnosis was confirmed using targeted next generation sequencing (tNGS), indirect immunofluorescence (IFA) and verify with nested polymerase chain reaction (PCR), which identified C. burnetii infection. Meanwhile, this article provides a review of the relevant literature with the aim of increasing the vigilance of clinicians to Q fever and to provide reference for the early detection and diagnosis of this disease.

2. Clinical data

Fig. 1A summarizes the contents of the report of the case in chronological sequence. A 47-year-old man presented four days earlier with high fever and generalized muscle aches without apparent cause. He was admitted to local Hospital on May 27, 2024. Complete blood count revealed white blood cell (WBC) count of 3.77 × 10⁹/L and neutrophil percentage of 76.4%. After administering antipyretic and anti-infective (specifics unspecified) treatments, the condition did not satisfactory. Therefore, he was admitted to the Second Affiliated Hospital of Nanchang University on May 29, 2024. Four days prior to admission, his highest temperature was 39.6°C, and previous health. The results of admission examination were elevated temperature (38.1°C), decreased WBC and platelets (WBC: 2.66 × 10⁹/L; platelet: 92 × 10⁹/L), abnormally elevated inflammatory indexes (CRP: 47.17 mg/L; PCT: 2.77 ng/mL), liver dysfunction (AST: 202.34 U/L; ALT: 202.36 UL; γ-GT: 136.51 U/L), abnormal coagulation function (D-dimer: 55.03 mg/L). There was scattered bronchitis and few inflammatory lesions in both lungs and possible intrahepatic cysts. Blood cultures, cardiac function, renal function and other tests showed no significant abnormalities. The patient was considered to have unexplained fever with multi-system involvement and infectious disease was suspected. To clarify the pathogen, a whole blood sample was taken from the patient on the day of admission (May 29, 2024) and sent for tNGS. Doxycycline hydrochloride 0.1 g quaque 12 hora (q12h) was given empirically for anti-infective treatment and monoammonium cysteine glycyrrhizinate combined with bicyclic alcohol for hepatoprotective and enzyme-lowering symptomatic treatment. The patient's temperature dropped from 38.1 to 37.5°C in the afternoon of the same day, with a gradual decrease to the normal range. On May 31 2024, the laboratory tests showed the patient's temperature was 36.8°C, blood indicators (WBC: 3.75 × 10⁹/L; PLT: 132 × 10⁹/L), inflammation indicators (CRP: 36.43 mg/L), liver function indicators (AST: 156.57 U/L; ALT: 177.71 U/L; γ-GT: 127.81 U/L), coagulation indicators (D-dimer: 2.91 mg/L) were significantly improved. The tNGS sequencing results confirmed an infection with C. burnetii, with 26,514 sequence reads detected at a high level of confidence. All drug resistance gene tests were negative. Subsequently, IFA was used to detect the specific antibodies (IgG and IgM). The results showed that the IgG titer in phase I was 1∶32 and in phase II was 1∶1,024 (Table 1); the IgM titer in phase I was 1∶128 and in phase II was 1∶2,048. This suggests acute stage infection of Q fever. Table 2 summarizes the patient's laboratory test results. Pursuing the patient's medical history, he has been a long-term resident in a rural area with more than 80 poultry (chickens, ducks, geese). Combined with the clear pathogenic evidence provided by tNGS, the patient's clinical manifestations such as persistent fever with significant liver injury, and epidemiological history, the diagnosis of infectious fever (Q fever rickettsial infection) and toxic hepatitis (Q fever-related) was confirmed, confirming that the initial empirical anti-infective regimen was effective, and the patient was continued to be maintained on doxycycline hydrochloride 0.1 g q12h anti-infective and monoammonium cysteine glycyrrhetinate combined with bisabolol to protect the liver and lower the symptomatic treatment. The patient's temperature continued to be normal, clinical symptoms were significantly relieved, liver function and other indicators steadily recovered. He was discharged from the hospital on June 4, 2024, with no complaints of special discomfort and good general condition at the time of discharge.

Fig. 1.

Disease course schedule and genetic evolution tree. (A) Timeline of medication administration. (B) A phylogenetic tree was constructed using the maximum likelihood method in MEGA-X software based on 30 IS1111 insertion sequences from NCBI database, along with the sequence from this case. (Note: the red triangle [] indicates the partial transposase gene of IS element IS1111 detected in the blood sample of this case.)

Table 1.

Laboratory confirmation of Coxiella burnetii infection by tNGS and IFA.

Term	tNGS				IFA
	Genus	Species	Confidence	Special sequence number	C. Burnetii IgM phase Ⅰ/II	C. Burnetii IgG Phase I/II
Result	Coxiella	Coxiella burnetii	High	26514	1∶128/1∶2,048	1∶32/1∶1,024

Abbreviations: tNGS, targeted next generation sequencing; IFA, indirect immunofluorescence.

Table 2.

Results of laboratory tests.

Term	Reference range	May 27	May 29 (First day in hospital)	May 31 (Third day in hospital)
Blood test
WBC (109/L)	4.0–10.0	3.77	2.66	3.75
RBC (109/L)	4.3–5.9	NA	4.96	NA
PLT (109/L)	150–400	NA	92	132
HGB (g/L)	130–175	NA	153	135
NEUT (%)	40–75	76.4	76.3	NA
LYMPH (%)	20–50	NA	18.8	NA
NEUT (109/L)	2.0–7.5	NA	2.03	1.97
LYM (109/L)	1.1–3.2	NA	0.5	1.2
Inflammation indicators
CRP (mg/L)	< 10	NA	47.14	36.43
PCT	≤ 0.05	NA	2.77	2.17
ESR	< 15	NA	12	NA
Liver function
ALB (g/L)	35–51	NA	36.14	33.86
TBIL (μmol/L)	5.13–22.24	NA	16.37	10.10
DBIL (μmol/L)	0–6.8	NA	4.65	2.42
IBIL (μmol/L)	1.7–10.2	NA	11.72	7.72
AST (U/L)	8–28	NA	202.34	156.57
ALT (U/L)	0–35	NA	202.36	177.71
γ-GT (U/L)	5–54	NA	136.51	127.81
Coagulation indicators
D-dimer (mg/L)	< 0.5	NA	55.03	2.91
Kidney function
K+ (mmol/L)	3.5–5.0	NA	3.85	NA
Na+ (mmol/L)	136–145	NA	134.36	NA
Ca2+ (mmol/L)	2.10–2.55	NA	2.09	NA

Abbreviations: WBC, white blood cell; RBC, red blood cell; PLT, platelet; HGB, hemoglobin; NEUT, neutrophil; LYMPH, lymphocyte; LYM, lymphocyte; CRP, C-reactive protein; PCT, procalcitonin; ESR, erythrocyte sedimentation rate; ALB, albumin; TBIL, total bilirubin; DBIL, direct bilirubin; IBIL, indirect bilirubin; AST, aspartate aminotransferase;ALT, alanine aminotransferase; γ-GT, γ-glutamyl transferase.

Subsequently, the DNA from patient's blood was extracted using DNeasy blood and tissue kit (Qiagen, USA). A nested PCR assay targeting the partial transposase gene of insertion sequence (IS) element IS1111 of C. burnetii was performed, and the PCR products of were sequenced. The sequence was subjected to Basic Local Alignment Search Tool (BLAST) search using the blast program and from the top hits, representative nucleotide sequences were chosen for phylogenetic analysis. We downloaded 30 C. burnetii IS1111 insertion gene sequences from the National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov/) database and constructed a phylogenetic tree (Fig. 1B) using the maximum likelihood method in MEGA-X software (University of Texas at Arlington, USA) to assessed the genetic similarity of the sequence from this case. The results revealed that this case clustered within a branch exhibiting high support (bootstrap = 65%) alongside a strain derived from human in the Commonwealth of Australia (Australia) (JF968204.1) and a cloned strain from sheep in the Sultanate of Oman (Oman) (MN084854.1). Furthermore, tick-derived strains from various countries were dispersed across different branches.

3. Discussion

Q fever is a globally distributed zoonotic disease caused by C. burnetii, which infects humans through four main mechanisms: inhalation of aerosols containing the pathogen, skin or mucous membrane contact with infected animal feces, consumption of contaminated dairy products, and tick-mediated transmission.¹² The patient in this case resided long-term in a rural area and maintained a poultry flock exceeding 80 birds. Based on the patient's epidemiologic history and major transmission routes of Q fever, we hypothesize that infection likely originated from exposure to contaminated environmental aerosols or animal excreta. However, C. burnetii testing was not performed on the patient's poultry or surrounding environment to confirm the infection source. Future studies could trace transmission sources more precisely through systematic environmental sampling and animal serologic surveys.

The PubMed database was searched from January 1, 2020, to October 1, 2024, using the search terms “Q fever” and “case report”, and 68 relevant articles were retrieved to review Q fever-related cases (Table S1). In the past five years, 83 cases of Q fever have been diagnosed. Except for 9 confirmed cases in children under 12 years of age, the majority of the patients were men between 30 and 60 years of age, and there were only 2 cases in women. Men who engaged in outdoor-related work with a higher risk of being bitten by ticks or inhaling aerosols infected by C. burnetii. Also, the children were predilected for outdoor activities, are also at high risk. Since clinical cases of Q fever are very rare, the clinical symptoms are not specific, and the inability of routine laboratory culture techniques to cultivate C. burnetii, this disease is highly susceptible to misdiagnosis and underdiagnosis.¹³ Currently, serological testing remains the primary reference standard. Indirect immunofluorescence is considered the gold standard of diagnosis.¹⁴ In the last five years, 71.08% of the confirmed cases of Q fever were diagnosed by serologic testing, which has a good detection effect. However, the use of serological methods can affect the accuracy of diagnostic results due to limitations in antigen acquisition techniques or inconsistencies in analytical standards, making them difficult to use on a large scale.¹⁵ And there is a high risk of false negativity as some patients with Q fever can't be diagnosed by serologic methods.¹⁶ Patients whose first serologic tests were negative were supplemented with pathogenetic testing by PCR or next generation sequencing (NGS) in conjunction with clinical presentation and history of outdoor activity, and were also successfully tested positive for C. burnetii. The tNGS is a next-generation sequencing technology that integrates ultra-multiplex PCR amplification with high-throughput sequencing. The multiplex PCR amplification method enriches target pathogen nucleotide sequences through the design of specific primers for ultra-multiplex PCR amplification, followed by high-throughput sequencing. The technology can sequence all nucleic acids in the specimen by virtue of the advantages of unbiased, large sequencing throughput and fast sequencing speed, which led to a significant increase in the diagnostic rate of Q fever. Studies have shown that using NGS technology as a first-line screening method increases diagnostic rates by up to 10.9% and provides greater diagnostic sensitivity than other traditional methods.¹⁷

As a review of the last five years of Q fever reports shows, a total of 24.10% of patients had combined infective endocarditis, 21.69% had combined pneumonia, 13.25% had combined bone and joint infections, and 7.23% had combined hepatitis or liver injury.6, 7, 8, 9, 10, 11 To understand the characteristics of Q fever presenting primarily with liver injury, we have retrieved and discussed six cases of Q fever with clearly documented liver injury reported over the past five years (Table 3). Comprehensive analysis of reviewed cases revealed universal significant liver injury, primarily manifested by elevated transaminases (AST, ALT). Some cases demonstrated jaundice or hepatomegaly, with fever consistently present. Doxycycline proved effective as first-line therapy post-diagnosis; patients receiving prompt treatment typically showed rapid clinical improvement, liver function recovery, and favorable prognoses. However, diagnostic delays directly impacted outcomes. Case 2 progressed to endocarditis due to reliance on traditional serologic testing, while Case 4 required bone marrow biopsy revealing characteristic fibrin ring granulomas for diagnosis after seronegativity. Conversely, cases employing molecular testing (Cases 1, 5, 6, and the present case) achieved early, accurate diagnosis, establishing a rapid diagnostic pathway for febrile patients with unexplained liver injury.

Table 3.

Diagnostic characteristics of patients with Q fever with liver injury in the last 5 years.

No.	Age (years)	Sex	Country	Diagnosis	Clinical data	Complications	References
1	34	Male	China	mNGS (mapping sequence number 79, relative abundance 15.96%)	ALT: 167 U/L; AST: 143 U/L	Hepatitis	6
2	35	Male	USA	Serological Testing (IgG phase II titer 1∶8,192; IgM phase II titer 1∶2,048)	ALT: 281 U/L; AST: 111 U/L	Hepatitis progressing to chronic endocarditis	7
3	59	Male	USA	Serological Testing (IgG phase II titer 1∶131,072; IgM phase II titer 1∶4,096)	ALT: 100 U/L; AST: 142 U/L	Alcohol associated hepatitis	8
4	53	Male	The Socialist Republic of Viet Nam	Bone marrow biopsy (fibrin ring granulomas)	ALT: 208 U/L; AST: 369 U/L	Hepatitis	9
5	55	Male	China	NGS (2404 sequence reads)	ALT: 285 U/L; AST: 235 U/L	Hepatitis and pneumonia	10
6	52	Male	China	NGS (A total of 21 contigs targeting the genome of C. burnetii)	ALT: 114.1 U/L; AST: 118 U/L; GGT: 470.3 U/L	Hepatitis, and pneumonia	11

Abbreviations: mNGS, metagenomic next-generation sequencing; ALT, alanine aminotransferase; AST, aspartate aminotransferase.

As a global zoonotic infectious disease, Q fever is not categorized as a legally reported diseases in China, medical professionals may have shortcomings in their diagnosis and treatment, leading to misdiagnosis and delayed diagnosis, which underestimates the actual incidence of Q fever, making it difficult to detect and control outbreaks in a timely manner, and posing a potential risk of an outbreak. Furthermore, the clinical manifestations of Q fever lack specificity. If Q fever is not promptly and accurately identified, inappropriate treatment may be administered, which not only delays the progression of the disease but may also exacerbate the patient's condition and increase their economic burden. If not treated promptly during the acute phase, some patients may develop chronic Q fever, which is difficult to treat and has a poor prognosis. Additionally, as a zoonotic disease, Q fever has a high infection rate in animals, leading to issues such as abortion and stillbirths, causing significant losses to livestock production and impacting the economic development of related industries. Therefore, it is crucial to increase awareness of Q fever and the need for a well-established Q fever control system.

Ruminants serve as primary reservoirs for C. burnetii, while household pets represent an overlooked transmission source. In patients presenting with nonspecific febrile illness and hepatitis of unclear etiology, characteristic laboratory findings-such as marked transaminase elevation observed in this and prior cases-offer key diagnostic clues. Q fever deserves to be included in the differential diagnosis in the case of negative routine test results. Molecular techniques like tNGS can overcome diagnostic bottlenecks and should be prioritized, supplemented by serology, immunology, and imaging to optimize ancillary testing. tNGS is an accurate, effective tool for detecting atypical pathogens, particularly those that are clinically rare or uncultivable. It holds significant promise for future diagnosis of such diseases. In this study, Q fever was diagnosed by the combined use of tNGS and IFA, which greatly improved the accuracy of diagnosis. However, there were also certain limitations, such as the lack of comparative with other cases of infection of Q fever with liver injury. Further research should include systematic analysis of the pathogen or further genomic characterization (genotyping, phylogenetic analysis or resistance determinants), and national epidemiological surveys to clarify Q fever's disease burden and validate tNGS utility in primary care settings.