Multiple opportunistic lung infections pose significant diagnostic challenges in immunocompromised patients, particularly when involving rare pathogen combinations. Tropheryma whipplei, a Gram-positive opportunistic bacterium, can cause pulmonary manifestations including multiple nodules and patchy infiltrates with rapid radiographic progression (1). Penicillium digitatum, a pathogen usually infects plants, has been reported in only three global cases of human pulmonary infection. All cases mainly occurred in individuals with low immune function and citrus fruit exposure (2, 3). Conventional microbiological cultures have limited utility for diagnosing these fastidious organisms, while metagenomic next-generation sequencing (mNGS) offers a valuable diagnostic alternative (4). When such rare infections fail to respond to targeted antimicrobial therapy, an underlying malignant process should be suspected. Epstein–Barr virus-positive diffuse large B-cell lymphoma, not otherwise specified (EBV⁺DLBCL-NOS), is an aggressive B-cell lymphoma that can present with insidious onset and extranodal involvement. Patients often exhibit immune dysfunction, predisposing them to opportunistic infections. Rare co-infections, including cerebral cryptococcosis and toxoplasmosis, have been documented in these patients. Here, we present a unique case in which recurrent cytomegalovirus retinitis (CMVR) and a mixed pulmonary infection with Tropheryma whipplei and P. digitatum were the initial manifestations of an underlying EBV⁺DLBCL-NOS. While CMVR is a well-documented complication in lymphoma patients (5), pulmonary co-infection with Tropheryma whipplei and P. digitatum remains exceptionally rare. This highlights the critical importance of maintaining a high index of suspicion for underlying malignancy in adults presenting with unexplained severe immunodeficiency and refractory, multifocal infections.

Rare pathogen pneumonia refers to opportunistic pulmonary infections caused by atypical, uncommon, or environmentally sourced microorganisms, commonly occurring in immunocompromised patients or those with underlying pulmonary diseases. These pathogens include bacteria (non-tuberculous mycobacteria, Tropheryma spp., Chlamydia), fungi (Aspergillus spp., Mucor spp., Talaromyces spp., Penicillium spp., Pneumocystis), viruses (HHV, TTV) and other viruses (6, 7). Their clinical and radiographic presentations are often nonspecific. The diagnosis and treatment of rare pathogen pneumonia remain a therapeutic challenge in modern medicine.

Conventional diagnostic methods (standard culture, serology, and PCR), frequently fail to identify these pathogens due to their fastidious growth requirements, low microbial burden, or the sheer breadth of possible agents. The advent of mNGS has revolutionized the diagnosis of such infections (4). The incidence of such pneumonia is rising, paralleling the growing population of patients with compromised immunity due to hematologic malignancies, solid organ transplantation, long-term immunosuppressive therapy, or advanced HIV. In these individuals, impaired cellular and humoral defenses create a permissive environment for these unusual organisms to cause disease. CD4⁺ T cell count strongly predicts rare pathogen infection risk. a meta-analysis of BALF mNGS in 141 HIV patients showed increased rates of mixed and multiple pathogen infections when CD4⁺ T-cell counts were <200 cells/μL, with further increases observed when counts were <100 cells/μL (8). The management of these pneumonias is complex, and active identification of pathogens and immunodeficiency, anti-infection are the keys to treatment.

EBV⁺DLBCL-NOS is an aggressive B-cell lymphoma first described in 2003 by Oyama et al. in 22 elderly Japanese patients (9). The prognosis is poor, with a median overall survival of 36 months and a 5-year survival rate of 25% (10). EBV⁺DLBCL-NOS can involve rare or atypical anatomical sites, presenting with an occult onset and nonspecific manifestations. Documented cases in the literature include primary central nervous system involvement (11), gastrointestinal tract (12), trachea, and polyurethane-coated breast implants (13). The occurrence of these unusual sites indicates that the disease has a wide anatomical distribution, which poses challenges for early diagnosis. Lymphoma patients are at a significantly elevated risk for pulmonary infections, a leading cause of morbidity and mortality in this population. This susceptibility stems from a complex interplay of intrinsic immune dysfunction due to the underlying malignancy and extrinsic immunosuppression from cytotoxic chemotherapy, targeted agents, and immunomodulatory therapies. For endogenous immunosuppression, growing evidence links EBV⁺DLBCL-NOS to declining immune function, characterized by reduced CD4⁺ and CD8⁺ T cell counts (14, 15). This association may relate to specific EBV infection mechanisms.

Tropheryma whipplei is a Gram-positive, rod-shaped, obligate intracellular bacterium belonging to the Actinobacteria phylum. While gastrointestinal tract and joints involvement is classic, pulmonary infection caused by Tropheryma whipplei is increasingly recognized, particularly in immunocompromised individuals or those with structural lung disease. At present, only 4 cases of lymphoma patients with pulmonary infection have been reported (16–19) (Table 3). Their clinical features are nonspecific, while radiological findings such as ground-glass opacities, interstitial infiltrates, and pulmonary nodules mimic lymphoma relapse, drug-induced pneumonitis, or other opportunistic infections. Therefore, the diagnosis of Tropheryma whipplei pneumonia in lymphoma patients remains exceptionally challenging. Periodic acid–Schiff (PAS) staining lacks sensitivity and specificity, targeted PCR may miss atypical strains, and culture is notoriously difficult. In immunosuppressed individuals, serological responses are often blunted. mNGS is the key to diagnosis.

Initially, the patient presented only with cough and pulmonary nodules and consolidations, which were attributed to Tropheryma whipplei infection based on the BALF mNGS results. Antibiotics generally effective against Tropheryma whipplei include penicillin, ceftriaxone, meropenem, streptomycin, tetracycline, SMZ-TMP, doxycycline, and hydroxychloroquine (20). Current main recommendations are ceftriaxone or meropenem for 14 days, followed by SMZ-TMP for one year (21). This patient had immunodeficiency and CMVR. Monotherapy might be ineffective, and hydroxychloroquine should to be avoided due to potential retinal toxicity. Therefore, initial treatment with ceftriaxone combined with SMZ-TMP was started. However, high fever persisted during treatment, and pulmonary progression was observed, potentially related to SMZ-TMP resistance (22). Therapy was then changed to meropenem combined with doxycycline, but efficacy remained poor and pleural effusion and progressive lung exudative lesions were present.

Penicillium spp. are ubiquitous environmental molds, and human infections are rare. Although often regarded as contaminants in clinical settings, invasive infections should be considered in immunocompromised hosts (23). Pulmonary infections caused by P. chrysogenum (24), P. citrinum (25), P. janthinellum (26), P. digitatum (2), P. oxalicum (27), P. notatum (28), and P. capsulatum (29) have been reported, with manifestations including disseminated parenchymal infiltration, lobar pneumonia, bronchopneumonia, fungal ball, and pleural effusion (30). To date, only 3 global cases of P. digitatum infection have been documented, with risk factors including corticosteroid use, COVID-19, and exposure to citrus fruits (2, 3, 31).

In this case, the patient exhibited progressive pneumonia despite broad-spectrum antibiotic therapy, in the context of underlying immunodeficiency. A detailed history revealed long-term citrus consumption. mNGS of BALF detected P. digitatum-specific sequences with high relative abundance (36.73%), supporting the diagnosis of co-infection. Although both serum galactomannan and (1,3)-β-D-glucan assays were negative, this is consistent with prior reports by Isabel Iturrieta-González et al. (2), in which P. digitatum infection also yielded negative biomarkers The limited diagnostic value of these serological tests in this context is likely due to the high specificity of galactomannan for Aspergillus spp., as well as severely impaired host immune responses in profoundly immunocompromised individuals, resulting in minimal release of fungal components into the bloodstream.

Currently, there is no standardized guideline for the treatment of P. digitatum infection. However, case reports by Isabel Iturrieta-González et al. (2) and Xiaojuan Shi et al. (31) demonstrated susceptibility to voriconazole. In addition, an in vitro antifungal susceptibility study of various Penicillium isolates showed favorable activity of triazoles and echinocandins (30). Unfortunately, fungal culture was negative in this case, which is not uncommon given the fastidious growth requirements of Penicillium spp. Empirical voriconazole therapy was initiated and resulted in significant clinical improvement.

The pulmonary lesions were complex. Initial presentation was characterized by marked elevation of inflammatory markers and rapid progression of pulmonary infiltrates, consistent with active co-infection involving Tropheryma whipplei and P. digitatum. Following treatment with meropenem, doxycycline, and voriconazole, inflammatory markers and exudative lesions improved, though persistent nodular opacities remained. Although PET-CT is crucial for evaluating systemic tumor burden, its utility is limited in differentiating between pulmonary atypical pathogen infection lesions and lymphoma, as infection by Tropheryma whipple or fungi can exhibit high SUV_max (32, 33). Therefore, early percutaneous lung biopsy was necessary. Unfortunately, the patient initially refused, which may have partially delayed interpretation of the complex condition. Eventually, percutaneous lung nodule biopsy confirmed lymphoma, explaining the primary cause of immunodeficiency and atypical pathogen infection. Ultimately, percutaneous lung biopsy confirmed the diagnosis of lymphoma, which explained the basis of the patient’s immunodeficiency and susceptibility to opportunistic infections. We infer that the early phase was predominantly infectious with superimposed lymphomatous involvement, evolving into a lymphoma-dominant proliferative process during follow-up.

Furthermore, while CMVR is most associated with HIV infection, it can also occur in patients with hematologic malignancies (5) or solid organ tumors (34). According to HIV opportunistic infection guidelines, discontinuing therapy is safe when CMVR is quiescent and CD4⁺ T cells remain >100 cells/μL for 3–6 months (35). The patient received 5 months of oral ganciclovir plus intravitreal injections, with subsequent undetectable CMV DNA in blood and aqueous humor and CD4⁺ T cells stably >100 cells/μL for 3 months (Supplementary Figures S5B–D). We consider the prior treatment to have been appropriate and sufficient. CMV DNA load is inversely correlated with CD4⁺ T cell counts, a relationship observed in this patient. Despite prolonged and appropriate ganciclovir therapy, CMV DNA levels surged as CD4⁺ counts progressively declined, consistent with lymphoma-associated immunosuppression leading to CMV reactivation (36), suggesting occult progression of lymphoma. This underscores the limited efficacy of antiviral monotherapy in the setting of profound immunosuppression, highlighting the need for immune reconstitution alongside targeted antiviral treatment.

This case illustrates a rare presentation of EBV⁺DLBCL-NOS, initially manifesting as CMVR and a complex pulmonary co-infection with Tropheryma whipplei and P. digitatum in a patient with severe, unexplained immunodeficiency. The diagnostic journey underscores several critical lessons. First, mNGS proved invaluable for identifying fastidious pathogens that eluded conventional microbiology, enabling targeted antimicrobial therapy. However, persistent radiographic progression despite appropriate treatment warranted further investigation. Second, PET-CT imaging, while helpful in assessing systemic disease burden, has limited specificity in distinguishing between infectious and malignant pulmonary processes, as both can exhibit high metabolic activity. Third, and most importantly, refractory, multifocal opportunistic infections in adults with profound immunosuppression, particularly when not attributable to HIV/AIDS or iatrogenic causes, should prompt urgent evaluation for an underlying occult hematologic malignancy. The definitive diagnosis ultimately required histopathological confirmation. We advocate early consideration of tissue biopsy when clinical suspicion persists despite antimicrobial therapy, to prevent critical delays in initiating appropriate oncologic management. This case reinforces the complex interplay between malignancy-associated immunosuppression and opportunistic pathogens, and highlights the necessity of a multidisciplinary approach in diagnosing and managing such challenging clinical presentations.