Whipple’s disease, an uncommon systemic infectious ailment induced by Tropheryma whipplei (TW), was initially delineated by George H. Whipple in 1907. Predominantly targeting the small intestine, this condition has the potential to infiltrate various other organ systems, encompassing the heart, lungs, joints, and central nervous system (Lin et al., 2022; Cappellini et al., 2024). The clinical manifestations of Whipple’s disease are diverse, ranging from digestive system symptoms such as diarrhea, abdominal pain, and weight loss to joint pain, fever, and neurological symptoms (Holstein et al., 2023). On account of its rarity and non-specific clinical manifestations, Whipple’s disease frequently escapes initial diagnosis or is misidentified, resulting in treatment delays and adverse patient outcomes (Motwade and Osman Taha, 2023). Nonetheless, the existing diagnostic criteria mainly hinge significantly on the histopathological finding of periodic acid-Schiff (PAS) stain-positive macrophage infiltration, and its sensitivity strikingly decreases in non-intestinal samples.

In recent years, advancements in molecular biology techniques, particularly the integration of 16S rRNA gene sequencing and polymerase chain reaction (PCR) methodologies (Van Dong et al., 2025), have significantly enhanced the diagnostic precision of Whipple’s disease (Meyer et al., 2023). Nevertheless, these techniques may encounter multiple constraints in specific scenarios, such as sample quality, DNA extraction efficiency, and PCR inhibitors. Nanopore sequencing technology stands as an emerging high-throughput sequencing approach. As a consequence of its rapidity, simplicity, and high sensitivity, it presents enormous potential in the diagnosis of infectious diseases (Girgis et al., 2023; de Cesare et al., 2024). In comparison with traditional sequencing technologies, nanopore sequencing directly sequences the DNA or RNA in the sample without PCR amplification, thus avoiding the biases and errors that may be introduced throughout the PCR amplification process (Song et al., 2024a). Its single-molecule resolution can effectively identify low-abundance microbial signals and achieve high-precision species identification through the dynamic time correction algorithm. In the realm of clinical practice, bronchoalveolar lavage fluid (BALF) is a liquid collected after lung lavage via bronchoscopy. Originating directly from the affected lung area and typically containing a high concentration of pathogen DNA, BALF serves as a pivotal sample type for such diagnoses (Hogea et al., 2020). As a primary systemic infection targeting the small intestine, Whipple’s disease infrequently involves the lungs. Nevertheless, in certain instances, pulmonary symptoms may dominate the clinical manifestations of the disease. These symptoms may include persistent cough, dyspnea, chest pain, and pulmonary infiltration, which may occur alone or coexist with symptoms of other systems (Ladna et al., 2024). Furthermore, diagnosis is often challenging attributable to the non-specific nature demonstrated within the pulmonary symptoms of Whipple’s disease and their similarity to other respiratory diseases. Aside from that, individuals suffering from Whipple’s disease with pulmonary involvement may exhibit atypical features in imaging examinations, such as nodules, consolidation, or interstitial pneumonia, which further heightens the complexity of diagnosis (Hofmann et al., 2021). On that account, nanopore sequencing detection by employing BALF samples may improve early diagnosis and treatment for individuals enduring pulmonary symptoms, especially those whose etiology cannot be clearly identified by conventional diagnostic methods.

Nevertheless, to our knowledge, there is a notable lack of research exploring the utilization of this technology in diagnosing Whipple’s disease. On that account, this study endeavors to delve into the application value of nanopore sequencing technology in diagnosing Whipple’s disease by comprehensively analyzing the clinical manifestations, imaging characteristics, and treatment outcomes of 14 patients who tested positive for TW using nanopore sequencing. We hope that this study can furnish clinicians with insightful references and contribute to novel perspectives and approaches for the diagnosis and management of Whipple’s disease, ultimately enhancing patient care and medical services.

As a rare chronic multi-system disorder, Whipple’s disease is characterized by a distinct array of clinical manifestations, encompassing arthritis, persistent diarrhea, significant weight loss, as well as extrapulmonary symptoms like fever, fatigue, and neurological involvement. In recent years, multiple case reports have revealed the potential pathogenicity of TW in respiratory tract infections, especially in individuals enduring immunosuppression or structural lung diseases. Nonetheless, as evidenced by the scarcity of systematic research in this area (Zhang and Xu, 2021; Chen et al., 2024; Li et al., 2024; Shi et al., 2024), a comprehensive understanding remains elusive with regard to the clinical features, imaging findings, and therapeutic approaches tailored to its pulmonary manifestations. When it comes to laboratory diagnosis, in vitro culture of TW has always confronted a multitude of significant challenges. TW exhibits insensitivity to conventional culture conditions and sluggish growth, posing challenges for prompt detection through standard microbiological assessments. Despite some literature documenting the successful cultivation and isolation of TW within human macrophages inactivated by Interleukin-4 (IL-4) and human fibroblast cell lines, this culture method approach harbors certain constraints. The average detection and growth time of TW spans an extensive 30 days, rendering it impractical for clinical contexts that necessitate swift diagnosis and intervention (Dolmans et al., 2017). Nonetheless, the clinical diagnosis of TW may be redefined as molecular diagnostic techniques move ahead speedily, especially the application of nanopore sequencing. In 2012, researchers successfully isolated and cultured TW for the first time by utilizing the BALF sample of a patient with acute pulmonary infection. This groundbreaking accomplishment not only reconfirms the presence of TW in pulmonary infections but also underscores the paramount significance of BALF samples in diagnosing pulmonary manifestations of TW (Fenollar et al., 2012).

By utilizing nanopore sequencing technology, this study examined BALF samples and conducted a comprehensive analysis of the clinical characteristics, radiological manifestations, and treatment outcomes of TW-positive patients. As an emerging sequencing technology, nanopore sequencing boasts rapidness, high throughput, and extensive read lengths, thereby enabling more precise detection of TW gene sequences. When compared to mNGS, nanopore sequencing may offer superior advantages in regard to detection speed and accuracy, especially in the detection of low-abundance pathogens in clinical samples (Song et al., 2024b; Zhao et al., 2025a, 2025). For this reason, this study adopted nanopore sequencing technology with the objective of gaining a comprehensive insight into the role of TW in pulmonary infections and digging into its impact on the prognosis of patients. This study is the pioneer in conducting a stratified analysis of patients infected with TW in accordance with the number of sequences obtained by nanopore sequencing (100 vs. ≤ 100). As evidently demonstrated by our research findings, the levels of CRP and LDH in the high-sequence patient group exhibit conspicuously elevated levels, and the difference is statistically significant. This finding suggests that the pathogen load of TW may be tightly correlated with the intensity of the host’s systemic inflammatory response. As an acute-phase reaction protein induced by IL-6, the conspicuous augment in the level of CRP may mirror the activation degree of macrophages by TW (Ngwa et al., 2022). As a facultative intracellular pathogen, TW can escape the degradation of lysosomes and therefore survive for a long time within macrophages, thus continuously releasing pro-inflammatory signals (Ayona et al., 2021; Boumaza et al., 2021; Munjal et al., 2021). Upon activation, macrophages further release a variety of pro-inflammatory cytokines, which subsequently trigger a series of cascading reactions, thereby exacerbating the inflammatory process. Simultaneously, endothelial cells also participate in this process, as they can generate IL-6, which further activates immune cells and drives their involvement in the inflammatory response (Cosenza et al., 2021). This persistent inflammatory provocation could result in the sustained generation of CRP, thereby intensifying the host’s systemic inflammatory response. Prior studies have similarly demonstrated that routine laboratory assessments of individuals with Whipple’s disease often yield nonspecific findings, notably including elevated CRP (Fenollar et al., 2012). On top of that, the increase in the level of LDH may reflect the enhancement of the metabolic activity of immune cells (Gupta, 2022). LDH is an enzyme present in a variety of cells, and its release increases when cells are damaged or metabolically active (Diederichs et al., 1979). As a consequence, the dramatic elevation in the level of LDH may indicate cell damage or metabolic disorders stemmed from TW infection, and it may become a potential biomarker for evaluating the disease activity. As previously demonstrated, the level of LDH is conspicuously increased in individuals suffering from co-infection of TW and other pathogens (Lu et al., 2022). This phenomenon further supports the potential mechanism of action of LDH in TW infection. To put it another way, TW infection may give rise to a conspicuous augment in the release of LDH by activating immune cells and inducing changes in cell metabolism (Lai et al., 2024). It is noteworthy that despite the heightened inflammatory response exhibited by the high-sequence group, no significant disparities were observed in routine infection indicators, including white blood cell count and lymphocyte subsets, between these two groups. This characteristic may result in a limited sensitivity of traditional inflammatory indicators, and CRP and LDH may become more reliable monitoring targets. On that account, it is imperative for future studies to further verify the correlation between the sequence load and clinical severity, as well as to dig into the feasibility of guiding the treatment course through dynamic monitoring of sequence counts.

As revealed by profound studies on the clinical baseline characteristics of TW-positive patients, the proportions of patients in the high-sequence group with clinical manifestations such as fever, cough, and expectoration are higher than those in the low-sequence group. Although these distinctions were not statistically significant, they suggest that the TW sequence counts may be bound up with the clinical characteristics of patients. This may be associated with the pathophysiological mechanism of TW infection. TW infection may trigger the body’s immune response, ultimately bringing about the release of inflammatory factors, thus causing symptoms such as fever, cough, and expectoration. Nevertheless, it remains essential to verify this association by further research. Future studies can not only expand the sample size but also delve deep into the potential mechanism between the TW sequence counts and clinical characteristics. As already demonstrated by imaging studies, the major outlooks of TW pneumonia comprise patchy consolidation, ground-glass opacities, and bronchiectasis, which highly overlap with tuberculosis or nonspecific interstitial pneumonia. This phenomenon thereby gives rise to a comparatively high misdiagnosis rate, which demonstrates consistency with previous research reports (Zhang and Xu, 2021). Nonetheless, some literature has put forth a viewpoint that patients with Whipple’s disease pneumonia exhibit unique radiological manifestations. For example, micronodules can be observed gathering in the upper lungs to form a “galaxy sign”, and the pulmonary infiltrates show a certain degree of mobility before, during, and after treatment, which necessitates further validation by more cases in the future (Li et al., 2024).

With regard to treatment, there is currently no consensus on the treatment regimen for Whipple’s disease. In typical Whipple’s disease, it is recommended to use doxycycline (200 mg/d) and hydroxychloroquine (600 mg/d) for 12 months and then use doxycycline (200 mg/d) for life to avoid reinfection (Lagier et al., 2014). Nonetheless, there is no standard treatment regimen for the special infection type of pulmonary Whipple’s disease. Although moxifloxacin is universally employed in many individuals suffering from Whipple’s disease pneumonia, most of the evidence for its efficacy is on the basis of case reports or small retrospective studies (Lai et al., 2024). Among the 14 TW-positive patients enrolled in this study, the majority of patients received antibiotic treatment, with fluoroquinolones, particularly moxifloxacin monotherapy or combination regimens, which emerged as the most prevalent choice. Efficacy analysis revealed that 85.7% of the patients exhibited clinical improvement post-treatment, underscoring the clinical merit of fluoroquinolones and combined regimens in the management of Whipple’s disease. Nevertheless, the patient’s symptoms in case 7 were relieved after being treated with a triple regimen for 23 days, which also indicates that patients characterized by severe conditions or advanced age may need an extended treatment course. On top of that, although case 13 received combined treatment, the symptoms persisted, which may be bound up with central nervous system involvement or antibiotic resistance.

There are several limitations in the design and implementation of this study. The number of cases included in this case series is less abundant, thereby bringing about a small sample size. This, to some extent, restricts the generalizability and universality of our research findings. The small sample size may also give rise to insufficient statistical power, thereby affecting the ability to detect certain potential associations. In consideration of the above limitations, this study centers around nothing more than the detailed analysis with reference to the clinical features of these cases, aiming to provide foundational data and preliminary insights for subsequent research. With an aim to further validate and expand upon the findings of this study, we will strive to collect more cases, conduct prospective studies, plan to include a negative control group, and carry out multivariate modeling analysis in future research. By increasing the sample size and introducing a control group, we hope to more comprehensively evaluate the clinical features, pathological mechanisms, and potential therapeutic strategies of respiratory diseases caused by Whipple’s disease, thereby providing more instructive research outcomes for clinical practice.

To sum up, pulmonary TW infection, as an emerging clinical entity, necessitates urgent refinement of its diagnostic and therapeutic framework. This study endeavors to systematically reveal the clinical-microbiological association of TW pneumonia, evaluate the prognostic value of the pathogen load, and lay an evidence-based foundation for optimizing the anti-infection strategy by integrating the nanopore sequencing data of BALF, imaging features, and treatment follow-up results. This exploration not only helps to facilitate early recognition of TW infection but also provides a unique model for understanding the interaction between intracellular pathogens and the host.

Based on the analysis of 14 patients with TW-positive bronchoalveolar lavage fluid via nanopore sequencing, this study demonstrates that TW pneumonia lacks specific imaging features, frequently presenting as patchy shadows, ground-glass opacities, or bronchiectasis, which overlap significantly with tuberculosis or interstitial pneumonia. Higher TW sequence counts (100) were associated with significantly elevated levels of CRP and LDH, indicating a correlation between pathogen load and systemic inflammation. Antibiotic therapy, particularly fluoroquinolones (e.g., moxifloxacin) and combination regimens, showed clinical efficacy, with 85.7% of patients improving symptomatically. However, severe or elderly cases may require extended treatment durations. These findings underscore the importance of nanopore sequencing for accurate diagnosis and support tailored therapeutic strategies based on disease severity and sequence burden.