Clinical Management and Outcomes of Chlamydia pneumoniae Pneumonia Diagnosed by Targeted Next-Generation Sequencing: A Case Report and Insights

Summary

This report describes targeted NGS-enabled precise diagnosis of C. pneumoniae pneumonia in an adolescent, facilitating successful macrolide therapy and highlighting its utility in atypical respiratory infections.

Abstract

Chlamydia pneumoniae is a significant cause of community-acquired pneumonia, yet its diagnosis remains challenging due to non-specific symptoms and the limitations of traditional methods like serology, which can cause delays. We present the case of a 14-year-old female admitted with cough and right-sided chest pain. Initial laboratory tests were unremarkable, except for a positive C. pneumoniae IgM result. A chest CT scan revealed right upper lobe infiltrates. Despite empirical azithromycin, her improvement was limited. Bronchoscopy was performed, and bronchoalveolar lavage fluid was analyzed by targeted next-generation sequencing (tNGS), which identified C. pneumoniae as the dominant pathogen with a high sequence count (9886), providing strong evidence for the etiology. Targeted macrolide therapy was continued (transitioned to oral doxycycline), leading to marked clinical improvement within 72 h. Longitudinal lab monitoring revealed a self-resolving transaminitis and a rise in Serum Amyloid A post-treatment. This case suggests that tNGS can serve as a valuable adjunctive tool in providing a rapid, culture-independent etiological diagnosis for atypical pneumonias, helping to guide effective therapy in selected patients with complex respiratory infections.

Introduction

Community-acquired pneumonia (CAP) represents a significant global health burden, with a complex and diverse spectrum of causative pathogens that complicates empirical treatment¹. Among the atypical bacteria, Chlamydia pneumoniae is a well-established etiological agent, responsible for an estimated 10%–15% of CAP cases across all age groups². Since its identification as a distinct species in 1986, C. pneumoniae has been recognized not only for causing acute respiratory infections but also for its potential epidemiological links to chronic conditions such as asthma and atherosclerosis^3,4.

Despite its clinical significance, the accurate and timely diagnosis of C. pneumoniae pneumonia remains a formidable challenge for clinicians. The historically established serological method, microimmunofluorescence (MIF), requires paired acute and convalescent sera, precluding early diagnosis, and suffers from poor standardization and significant cross-reactivity among Chlamydia species^5,6. Although foundational studies have successfully isolated C. pneumoniae from lower respiratory specimens such as bronchoalveolar lavage fluid⁷, culture of this obligate intracellular pathogen is technically demanding, time-consuming, and frequently insensitive, rendering it impractical for routine clinical use⁸. While commercially available multiplex respiratory PCR panels offer rapid turnaround times and can detect multiple common mixed infections, their reliance on predefined targets limits their ability to screen for broad, unexpected, or rarer pathogens simultaneously.

The advent of high-throughput sequencing technologies has begun to revolutionize pathogen diagnosis in infectious diseases⁹. Among these, targeted next-generation sequencing (tNGS) represents a significant advancement by combining broad-spectrum detection with enhanced sensitivity and cost-effectiveness. Unlike metagenomic NGS (mNGS), which sequences all nucleic acids in a sample, tNGS utilizes targeted enrichment panels to deeply sequence specific pathogen groups, thereby improving the depth of coverage for relevant targets, reducing host background noise, and streamlining bioinformatic analysis^10. This makes it particularly suitable for clinical settings where rapid and actionable results are paramount. Previous studies have demonstrated the superior sensitivity of NGS-based methods (>90%) for detecting respiratory pathogens compared to traditional cultures, with the added advantage of identifying mixed infections in up to 30%–50% more cases¹¹. Furthermore, tNGS has proven valuable in complex respiratory infections where traditional diagnostics fail to identify an etiology in a substantial proportion (40%–60%) of cases¹¹.

However, reports on the specific application and clinical impact of tNGS for diagnosing C. pneumoniae pneumonia, particularly in the pediatric and adolescent population, remain scarce. There is a notable gap in the literature regarding standardized workflows that integrate tNGS from patient selection through to result interpretation and therapeutic decision-making. The overall goal of this case report is to illustrate the potential role of tNGS in the diagnostic algorithm for suspected atypical pneumonia and to provide practical clinical insights into its application. We present the case of an adolescent female with C. pneumoniae pneumonia, supported by BALF tNGS after an inconclusive initial workup. This report aims to delineate the advantages of tNGS over alternative diagnostic techniques, place its utility within the wider body of infectious disease diagnostics literature, and provide clear information to help clinicians determine when such an advanced diagnostic approach is appropriate for their patients.

Case presentation
A 14-year-old female student with no significant past medical history was admitted to the Respiratory and Critical Care Medicine department in June 2025. She presented with a seven-day history of cough and expectoration of yellow-green purulent sputum. The cough intensified with sharp, needle-like right-sided chest pain upon inspiration one day prior to admission. She also reported concomitant symptoms of a sore throat, nasal congestion, and purulent nasal discharge. Notably, she denied any fever, chills, wheezing, shortness of breath, hemoptysis, night sweats, joint pain, or rash. She had self-medicated with a compound Houttuynia cordata mixture for five days without symptomatic relief. An outpatient chest CT scan revealed shadows in the right upper lobe, suggestive of infection, prompting her admission for suspected community-acquired pneumonia.

On physical examination at the time of admission, her vital signs were stable: temperature was 36.4 °C, pulse was 88 beats/min, respiratory rate was 18 breaths/min, and blood pressure was 87/57 mmHg. She was alert and conscious. Examination of the head and neck revealed no lip cyanosis, with mild pharyngeal congestion present. Her chest wall was symmetrical, percussion was clear bilaterally, and auscultation revealed clear breath sounds without any audible rales. Cardiac examination demonstrated a regular rhythm at 88 beats/min with no pathological murmurs. Abdominal examination was soft and non-tender. Neurological examination was unremarkable.

Diagnosis, assessment, and plan
The initial diagnosis upon admission was community-acquired pneumonia (Right upper lobe). Auxiliary examinations were performed to elucidate the etiology. Laboratory findings showed a normal complete blood count (WBC 7.05 × 10⁹/L, neutrophils 56.6%) and unremarkable inflammatory markers (high-sensitivity CRP 0.55 mg/L, serum amyloid A <5 mg/L, procalcitonin <0.04 ng/mL). A critical clue was a positive Chlamydia pneumoniae IgM serology (22.9 COI, reference <10). However, a comprehensive respiratory virus nucleic acid panel (including adenovirus, influenza A/B, RSV, Mycoplasma pneumoniae, human Rhinovirus, and SARS-CoV-2) returned negative results, as did tests for tuberculosis (sputum acid-fast stain, TB DNA, recombinant Mycobacterium tuberculosis fusion protein skin test) and cryptococcal infection. Chest CT imaging confirmed patchy high-density shadows with blurred margins in the right upper lobe (Figure 1).

A differential diagnosis was carefully considered. Bacterial pneumonia was deemed less likely due to the absence of high fever and the normal inflammatory markers. Pulmonary tuberculosis was considered unlikely given the negative supportive tests and lack of constitutional symptoms. Mycoplasma pneumoniae pneumonia was ruled out by the negative serum IgM. The primary diagnostic challenge lay in interpreting the positive C. pneumoniae IgM. While suggestive of recent infection, a single positive IgM result can reflect past infection, cross-reactivity, or a non-specific immune response, making it an unreliable sole indicator for committing to a prolonged course of targeted therapy⁶. This diagnostic uncertainty, coupled with the clear radiographic evidence of pneumonia, created a compelling rationale for pursuing a definitive microbiological diagnosis.

The management plan was therefore formulated to resolve this diagnostic impasse. After discussion with the patient’s family, a decision was made to proceed with fiberoptic bronchoscopy to obtain a lower respiratory tract sample directly from the site of infection. The specific plan was: (1) to perform bronchoscopy with bronchoalveolar lavage (BAL) in the affected right upper lobe; (2) to submit the BAL fluid (BALF) for comprehensive pathogen analysis using targeted next-generation sequencing (tNGS); (3) to adjust the anti-infective therapy based on the tNGS results; and (4) to closely monitor the patient’s clinical symptoms and signs during treatment. This approach was designed to move beyond the limitations of serology and obtain unambiguous, pathogen-specific data to guide a precision medicine approach to her care.

Protocol

This study is approved by the Ethics Committee of Yueqing Third People’s Hospital (Approval No. 2026003), ensuring adherence to ethical standards for human research. The primary objective is to obtain a high-quality lower respiratory tract specimen for comprehensive pathogen analysis. Written informed consent was obtained from the patient’s legal guardian for the clinical procedure and the publication of this case report. The following protocol is implemented for the diagnostic evaluation and management of patients with suspected atypical pneumonia, utilizing targeted next-generation sequencing (tNGS) to achieve an etiological diagnosis. The reagents and the equipment used are listed in the Table of Materials.

1. Patient selection and clinical indication for tNGS

1. Patient selection was based on clinical and radiographic evidence of community-acquired pneumonia (CAP).
2. The decision to proceed with advanced molecular diagnostics was triggered by the convergence of several factors: positive but potentially non-specific C. pneumoniae IgM serology, a lack of significant systemic inflammatory response, and the clinical necessity to exclude other pathogens such as Mycobacterium tuberculosis.

2. Specimen collection and processing

1. Bronchoscopy was performed under intravenous anesthesia using propofol (220 mg) and alfentanil (0.4 mg). The bronchoscope was advanced to the affected lobe.
   1. To ensure sample consistency, bronchoalveolar lavage (BAL) was performed specifically in the mid-segment of the lung, and fluid from the anterior segment was systematically discarded. Sterile saline was instilled, and approximately 5–10 mL of bronchoalveolar lavage fluid (BALF) was aspirated using sterile techniques.
2. The BALF sample was immediately separated into two aliquots under sterile conditions. The first aliquot was sent for routine microbiological workup. The second aliquot, designated for tNGS analysis, was placed in a sterile screw-cap tube and transported under controlled cold-chain conditions (≤20 °C) to the laboratory to preserve nucleic acid integrity.

3. Laboratory analysis via targeted next-generation sequencing

1. Viscous bronchoalveolar lavage fluid (BALF) samples were liquefied using a digestive solution and vortexing.
   1. The processed fluid was mixed with a nucleic acid preservative, and mechanical lysis was performed using a bead-beating apparatus to ensure efficient cell wall disruption. Total nucleic acids were extracted using an automated co-extraction platform according to the manufacturer’s instructions.
2. Sequencing libraries were constructed using a respiratory pathogen-targeted enrichment kit. Following purification and quantification via fluorometric analysis, the libraries were subjected to targeted enrichment of respiratory pathogens. The pooled libraries were converted into nanoballs and sequenced on a high-throughput sequencing platform.
3. Raw sequencing data were processed automatically using pathogen analysis software, which performed quality control, read filtering, and alignment against a curated reference database to identify and semi-quantify detected microorganisms.

4. Interpretation of results and therapeutic intervention

1. The tNGS report was reviewed within 48 h. The threshold for clinically relevant pathogen loads was established dynamically based on the background noise of negative controls (NC) within the specific experimental batch.
2. Based on the results, the anti-infective therapy was adjusted. For confirmed C. pneumoniae, intravenous macrolide therapy was continued, followed by oral sequential therapy with a tetracycline-class antibiotic (100 mg twice daily for 10 days).

Results

The implementation of this tNGS-based diagnostic protocol provided critical evidence identifying Chlamydia pneumoniae as the dominant pathogen, guiding precise antimicrobial therapy and favorable clinical outcomes. As detailed in the comprehensive pathogen profile (Figure 2), tNGS analysis of BALF confirmed C. pneumoniae infection with 9,886 sequences, while also identifying Rhinovirus A as a significant co-infection (6,977 sequences). The threshold for clinically relevant ...

Discussion

This case demonstrates the potential utility of targeted next-generation sequencing (tNGS) in assisting the diagnosis of Chlamydia pneumoniae pneumonia. When serological findings prove inconclusive and empirical therapy yields a suboptimal response, tNGS analysis of lower respiratory specimens provides comprehensive pathogen identification, enabling targeted therapeutic intervention.

Compared to traditional methods, tNGS offers significant diagnostic benefits. Unlike microimmunofluore...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
–80°C Ultra-Low Temperature Freezer	Thermo Scientific	905	For long-term storage of specimens and nucleic acids.
Biological Safety Cabinet	Thermo Scientific	1300 Series A2	Provides sterile environment for sample processing.
Bronchoalveolar Lavage (BAL) Catheter	Teleflex	22-160	For instillation and recovery of sterile saline during BAL procedure.
Computer Workstation	Dell	Precision 5860 Tower	High-performance computing for data analysis.
Cryogenic Vial (2.0 mL)	Thermo Scientific	375418	For storage of extracted DNA at controlled temperatures.
DNA/RNA Shield	Zymo Research	R1100	Nucleic acid preservation solution used during sample processing.
Doxycycline Hyclate Capsules	(Local supplier)	100 mg	Tetracycline-class antibiotic for sequential oral therapy.
Fiberoptic Bronchoscope	Olympus	BF-XP190	Used for bronchoalveolar lavage (BAL) under conscious sedation.
High-Speed Centrifuge	Eppendorf	5425 R	For sample preparation and processing.
High-Throughput Sequencer	MGI	MGISEQ-200	Genetic sequencer platform for targeted NGS analysis.
Microcentrifuge Tubes (1.5 mL)	Eppendorf	22431021	Sterile, DNase-/RNase-free tubes for sample handling.
Pathogen Analysis Software	Genseq	Genseq-PM	Bioinformatics pipeline for species-level identification.
Qubit 4.0 Fluorometer	Thermo Fisher Scientific	Q33238	Used for precise library quantification.
Sequencing Reaction Kit	Dian Diagnostics	No. 20240005	Universal kit for sequencing reaction on MGI platform.
Serological Test Kit (C. pneumoniae IgM)	Euroimmun	EI 2390-9601 G	Used for initial serological screening.
Sterile Normal Saline (0.9%)	Baxter	2B2324	Used as lavage fluid during bronchoscopy.
Targeted NGS Enrichment Panel	Dian Diagnostics	Respiratory Pathogen 200+ Panel	Custom panel (Genseq RTI) covering 200+ pathogens.