Clinical characteristics of Pneumocystis jirovecii pneumonia in 20 non-HIV-infected patients

Introduction

Pneumocystis jirovecii pneumonia (PCP) is an opportunistic infection caused by Pneumocystis jirovecii, commonly seen in patients with acquired immunodeficiency syndrome (AIDS) (1). With the widespread use of immunosuppressants and various biologics, the incidence of PCP is increasing among human immunodeficiency virus (HIV) negative patients. Significant differences exist between HIV-positive and HIV-negative PCP patients. Compared to their HIV-positive counterparts, HIV-negative PCP patients are typically older (mean age 62.9 vs. 44.7 years), include a higher proportion of females (46.6% vs. 26.4%), and exhibit higher rates of underlying diabetes (33.3% vs. 12.0%) and glucocorticoid use (32.7% vs. 6.4%) (2). Critically, their prognosis is worse: HIV-negative patients experience higher mortality rates (24.3% vs. 10.5%; 1-month mortality 21.9% vs. 5.4%, P<0.0001) (2,3).This disparity may stem from several factors: (I) delayed diagnosis due to lower clinical suspicion in non-HIV settings, resulting in more advanced disease at presentation (4,5); (II) the absence of prophylactic medication; and (III) an abrupt immune dysfunction in non-HIV immunocompromised hosts—contrasting with the gradual CD4+ T lymphocyte count decline in HIV patients—which triggers a more severe inflammatory lung injury (2). The timely and accurate diagnosis of PCP remains challenging. Conventional methods have limitations: Pneumocystis jirovecii cannot be routinely cultured; microscopy has low sensitivity, especially in non-HIV patients with lower organism burdens (6); β-1,3-D-glucan (BDG) lacks specificity (7); and while polymerase chain reaction (PCR) is more sensitive, it is targeted, misses co-infections, and cannot reliably distinguish colonization from disease (8). Metagenomic next-generation sequencing (mNGS) offers a promising solution to these diagnostic hurdles. Its advantages include unbiased, high-sensitivity detection of Pneumocystis jirovecii even at low loads, simultaneous identification of co-pathogens (common in immunocompromised hosts), and faster turnaround times compared to culture (9,10). Given the increasing burden and poorer outcomes of PCP in HIV-negative patients, coupled with diagnostic challenges, a comprehensive analysis of this population is crucial. This study therefore aimed to collect clinical data from non-HIV PCP patients diagnosed via bronchoalveolar lavage fluid (BALF) mNGS and to analyze their clinical characteristics, treatment, and prognosis. We present this article in accordance with the STROBE reporting checklist (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-499/rc).

Methods

The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethics Committee of Hangzhou TCM Hospital (No. 2024KLL214) and individual consent for this retrospective analysis was waived.

Clinical data

Clinical data were retrospectively collected from 20 patients with PCP admitted to Hangzhou TCM Hospital Affiliated to Zhejiang Chinese Medical University from June 2019 to May 2024. BALF mNGS was performed when initial empirical antibiotic therapy failed (defined as persistent fever or radiographic progression at 72 hours). The data included: (I) patients’ general information, including gender, age, underlying disease conditions, and previous use of glucocorticoid or immunosuppressant; (II) clinical symptoms; (III) pulmonary imaging manifestations of patients; (IV) laboratory tests, including peripheral blood leukocyte count, lymphocyte count, oxygen partial pressure, platelet count, blood creatinine, lactate dehydrogenase (LDH) level, CD4+ lymphocyte count, CD8+ lymphocyte count, and Pneumocystis jirovecii sequence count in BALF; (V) drug treatment regimen; (VI) patient outcome.

Inclusion and exclusion criteria

Diagnosis of PCP infection required all of the following (inclusion criteria): (I) patients presenting with at least two of the following clinical symptoms: persistent fever (≥38 ℃ for >72 h), non-productive cough, or progressive dyspnea; (II) characteristic pulmonary imaging findings on chest computed tomography (CT): bilateral ground-glass opacities (diffuse or patchy), with or without cystic changes or pneumatoceles, and absence of pleural effusion or lymphadenopathy (to exclude alternative diagnoses); (III) detection of Pneumocystis jirovecii in BALF through mNGS.

Exclusion criteria: (I) patients who are HIV-positive; (II) patients under the age of 18 years; (III) patients with incomplete clinical data.

Microbiological analysis

Sputum and BALF samples were obtained via bronchoscopy and subjected to conventional bacterial cultures. Additionally, mNGS was performed on BALF to identify microbial pathogens. The sequencing results were analyzed to determine the predominant organisms and their relative abundance. mNGS was performed at Hangzhou Matridx Biotechnology Co., Ltd., Hangzhou, China, using clinically validated protocols: BALF samples underwent mechanical homogenization (BSP-100 Oscillator, Hangzhou Matridx Biotechnology Co., Ltd.) followed by co-extraction of total nucleic acids for DNA/RNA pathogens; dual libraries were constructed through fragmentation and adapter ligation, then sequenced on Illumina NextSeq 550 (2×75 bp). Bioinformatic analysis involved host DNA subtraction (GRCh38.p13) and microbial classification against combined GenBank NT/NR plus curated clinical databases (35,257 species) using KRAKEN2/Bracken. Pathogen significance required: (I) combined with either relative abundance ≥1% of microbial reads or RPM >5× negative control, (II) passing quality control (Q30 >80%, PhHV-1 recovery ≥80%).

Statistical analysis

Statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS) (Version 27, IBM Corp., Armonk, NY, USA). Continuous variables were analyzed using the independent t-test described by the mean if they conformed to the normal distribution. If they did not conform to the normal distribution, they were described by the median and analyzed using a non-parametric test. The correlation between the two variables was analyzed using Pearson correlation analysis. P value <0.05 was considered statistically significant.

Results

Case data

A total of 20 patients with PCP were included, and BALF mNGS confirmed the diagnosis in all patients. All the diagnoses were consistent with clinical presentations and pulmonary imaging findings. Two physicians independently confirmed the association of pneumonia with Pneumocystis. Table 1 demonstrates the basic clinical characteristics of these patients. There were 11 (55%) males and nine (45%) females. The median age of all patients was 66.5 years (range, 18–83 years). Among the 20 patients, 17 (85%) were immunosuppressed (having previously received glucocorticoid, immunosuppressive therapy or chemotherapy, etc.), while the remaining three (15%) had no immunosuppressive background. Among the three immunocompetent cases, Case 1 was a 66-year-old male with coronary heart disease and diabetes who presented with dyspnea. His CD4+ T lymphocyte count was 275 cells/µL, imaging showed bilateral ground-glass opacities, and he required compound sulfamethoxazole (SMZ-TMP) plus micafungin and mechanical ventilation but ultimately died. Case 2 involved a 75-year-old female with diabetes manifesting fever, cough and dyspnea. Her CD4+ T lymphocyte count was 508 cells/uL, and imaging showed ground-glass opacities. She survived with SMZ-TMP therapy. Case 3 is an 80-year-old female patient with no underlying diseases who presented with cough. Her CD4+ T lymphocyte count was 510 cells/uL, and imaging showed ground-glass opacities with consolidation. She survived with SMZ-TMP therapy. Among the 17 immunosuppressed patients, there were four with solid organ tumors, four with renal diseases, three with hematological malignancies, two with dermatological diseases, one with rheumatoid arthritis, one with thrombocytopenia, one with allergic purpura, and one with knee osteoarthritis. The median time from patient admission to diagnosis was 4 days (range, 2–12 days), and the median time from symptom onset to oral SMZ-TMP administration was 8 days (range, 2–31 days). None of the patients had received prophylaxis against PCP before the onset of symptoms. The median laboratory processing time for mNGS (from sample submission to result availability) was 20 hours (range, 16–28 hours).

Clinical symptoms and laboratory examination

The most common clinical symptoms among the patients were dyspnea in 17 cases (85%), fever in 16 cases (80%), and cough in 15 cases (75%). The median time from symptom onset to hospital admission was 5 days (range, 1–15 days). The median time from admission requiring supplemental oxygen was 3 days (range, 1–7 days). The median time from admission to requiring mechanical ventilation (for those who received it) was 10 days (range, 2–15 days). The median oxygenation index for all patients was 310 (range, 145.6–714.3). Laboratory findings included a mean leukocyte count of 6.48×10⁹/L, a mean lymphocyte count of 0.92×10⁹/L, and a mean platelet count of 173×10⁹/L. The mean CD4+ T cell count was 225 cells/µL, and the mean CD8+ T cell count was 292 cells/µL. The most common imaging manifestation was ground-glass shadow, present in all 20 patients (100%), followed by pulmonary consolidation in seven patients (35%), pleural effusion in four patients (20%), paving stone sign in three patients (15%), and pulmonary air sacs in one patient (5%).

Table 2 shows the results and sequence counts of mNGS in the BALF of all patients. Pneumocystis jirovecii, the causative agent of PCP, was detected in BALF in all patients. Among them, 14 patients had co-infections, including five with Epstein-Barr virus (EBV), two with cytomegalovirus, two with Candida albicans, one with human herpesvirus type one, and one with Cryptococcus. There was also one case with Mycobacterium tuberculosis, one with Streptococcus parasanguis, one with Corynebacterium diphtheriae, one with Veillonella parvula, one with Streptococcus salivarius, one with Haemophilus influenzae, one with Enterococcus faecium, one with human Boca virus, one with rhinovirus type A, and one with Streptococcus pneumoniae.

Treatment and outcomes

Of the 20 patients, a total of six patients (30%) were treated with SMZ-TMP alone, 12 (60%) received a combination of SMZ-TMP and caspofungin, and two (10%) were treated with SMZ-TMP and micafungin. Glucocorticoids were administered to 16 patients (80%) during their treatment. Nasal high-flow oxygen therapy was utilized in four patients (20%), and mechanical ventilation was required by seven patients (35%). In total, six patients (30%) died, while 14 patients (70%) recovered. The mean duration of hospitalization was 17 days. The clinical characteristics of patients in the death group and the survival group are shown in Table 3.

Correlation analysis

Bivariate Pearson’s test showed a negative correlation between patients’ oxygenation index and age (r=−0.493, P=0.03), indicating that the older the patient, the lower the oxygenation index. Serum LDH levels were positively correlated with serum creatinine levels (r=0.557, P=0.01). Additionally, LDH levels were positively correlated with the time between symptom onset and oral administration of SMZ-TMP (r=0.477, P=0.03), suggesting that delayed treatment is associated with higher LDH levels. There was also a positive correlation between serum creatinine levels and the time to oral SMZ-TMP administration (r=0.607, P=0.005), indicating that later treatment initiation is associated with higher serum creatinine levels.

Discussion

Early diagnosis of PCP is crucial for initiating timely pharmacological intervention, which significantly improves the prognosis of patients with PCP. Traditional laboratory methods for diagnosing PCP mainly include in vitro culture, BDG test, and PCR. However, culturing Pneumocystis jirovecii in vitro is challenging, and traditional staining microscopy has a low detection rate (6,11). BDG, a main component of various fungi’s cell walls, correlates with the fungal load in the bloodstream, but its specificity for diagnosing PCP is low (7,12). PCR is another commonly used method for diagnosing PCP, but it has limited value in detecting mixed infections (8). With the development of molecular biology, mNGS technology has been widely used in clinical practice. All patients in this study were diagnosed using BALF mNGS. mNGS is a molecular technique for nucleic acid sequencing using high-throughput sequencing and is considered a promising technology for microbial identification (9). mNGS technology is used to sequence multiple individual DNA or RNA molecules in parallel (10). Zhu et al. found that, compared with PCR and the BDG test, mNGS had higher sensitivity (97.8%) and specificity (95.2%) in diagnosing non-HIV-infected PCP, and the detection rate of mNGS in mixed infections was significantly higher than that of traditional etiological detection (13). In addition, one of the major advantages of mNGS technology over traditional microbiological culture is the shortened detection time, approximately 24–48 hours (10), which is helpful for early diagnosis. In this study, mNGS provided diagnoses with a median turnaround time of 4 days post-admission (range, 2–12 days), significantly faster than traditional methods like fungal culture (typically 5–14 days) (6,12). However, mNGS still cannot completely distinguish infection from colonization and requires comprehensive analysis and judgment by clinicians in combination with patients’ clinical symptoms and lung imaging features.

Most PCP patients are immunosuppressed, and we often overlook those who are immunocompetent. Since PCP patients with normal immune function are rare, only scattered cases have been reported thus far (14,15). A study comparing the clinical course and prognosis of PCP patients with different immune status (16), which included seven PCP patients with normal immune function and 16 patients with PCP after kidney transplantation, found that patients with PCP after kidney transplantation had a relatively longer average hospitalization time and a higher mortality rate. However, another study found that (17), compared with immunosuppressed patients, PCP patients with normal immune function had a higher in-hospital mortality. The most significant factor is the profound delay in diagnosis and subsequent treatment initiation. This article also found that the median interval from admission to the initiation of anti-PCP treatment was significantly longer for immunocompetent patients than for immunocompromised patients (7 vs. 2 days), and a longer interval between admission and anti-PCP treatment was associated with an increased 90-day mortality rate in PCP patients (17). The rarity of PCP in immunocompetent individuals may leads to low clinical suspicion, resulting in delayed performance of definitive diagnostic tests like bronchoscopy with BALF analysis (e.g., mNGS). In our study, there were three patients with PCP without an immunosuppressive background, and among these three patients, the last one died. The pathogenesis of PCP in our three patients without classic immunosuppression warrants exploration. Notably, Case 1 (a 66-year-old male with diabetes and coronary heart disease) presented with a markedly low CD4+ T lymphocyte count (275 cells/µL), which suggests an underlying, unidentified cellular immune defect. Among these patients, two (Cases 1 and 2) had diabetes mellitus. Diabetes has detrimental effects on neutrophil function, T-cell responsiveness, and alveolar macrophage activity, creating a state of relative immunodeficiency (18). Furthermore, age-related immunosenescence may have contributed significantly in Cases 2 and 3 (aged 75 and 80 years), given that aging can lead to a reduction in the number of naive T cells and functional limitations of memory T cells, thereby increasing susceptibility to opportunistic infections (19). However, due to the small sample size, this study did not compare immunocompetent patients with immunocompromised patients. Currently, the pathogenesis and clinical characteristics of PCP patients with normal immune function are not well understood. A recent case report by Kawame et al. (20) described an immunocompetent PCP patient who presented with subacute symptoms and bilateral central consolidation on imaging, contrasting with typical ground-glass opacities in immunocompromised hosts. This suggests that preserved host immunity may alter disease manifestations, potentially leading to granulomatous inflammation and atypical radiological features (e.g., consolidation). Importantly, their patient recovered fully with SMZ-TMP monotherapy. In our cohort, however, one of three immunocompetent patients (Case 1, with diabetes and CD4+ lymphopenia) died despite combination therapy, while the other two survived with SMZ-TMP alone. Notably, our immunocompetent cases exhibited typical ground-glass opacities rather than consolidation, suggesting that radiological patterns in this population may vary. This discrepancy highlights the heterogeneous clinical course of PCP even in immunocompetent individuals, possibly influenced by undetected immune variations or diagnostic delays. In the future, more clinical studies are needed to explore the manifestations and prognostic characteristics of PCP patients with different immune states.

The clinical manifestations of PCP patients in this study lacked specificity, mainly including fever, cough, and dyspnea. Classically, PCP in HIV-infected patients presents with an insidious onset of progressive dyspnea, non-productive cough, and fever, developing over weeks (21). In contrast, the clinical presentation observed in our cohort of HIV-negative patients was characterized by a more rapid progression. As shown in the results, the median time from symptom onset to hospital admission was only 5 days, and the median time from admission to requiring supplemental oxygen was 3 days. The median time from admission to requiring mechanical ventilation (for those who received it) was 10 days. This pattern of acute onset and swift clinical deterioration aligns with previous reports describing PCP in non-HIV immunocompromised populations (22). This difference in the tempo of disease progression may contribute to the diagnostic challenges and higher mortality rates observed in this population. Patients with PCP were often accompanied by decreased CD4+ and CD8+ T lymphocyte counts (23), and the median CD4+ T lymphocyte count in patients in this study was 225 cells/µL. A study has found that (24), CD4+ T lymphocyte count in PCP patients is correlated with patient prognosis, and patients with CD4+ T lymphocyte <200 cells/µL have a higher mortality rate. Another study found that CD4+ T lymphocyte levels were lower in the death group than in the survival group (112 vs. 255, P=0.046) (25). This suggests that CD4+ T lymphocyte levels may reflect the severity of the disease. In our study, the median CD4+ T lymphocytes of patients in the death group were significantly lower than those in the survival group (94.64 vs. 247.16), but a statistical analysis was not performed due to the small sample size. Formal group comparisons (e.g., survival vs. death) were avoided given the death cohort size (n=6) would produce unreliable statistical inferences. LDH levels in the peripheral blood of PCP patients are often elevated to varying degrees (23). In our study, the median LDH of patients was 343 U/L, which was higher than the normal level. Several studies have found that LDH levels in patients who died were higher than those who survived (25,26). These findings suggest that serum LDH levels are helpful for diagnosis and may also be an independent predictor of survival. In our study, the median LDH of patients in the death group was also higher than that of patients in the survival group (448.0 vs. 304.5). Due to the small sample size, the effect of CD4+ T lymphocytes and LDH on patients’ prognosis were not analyzed in this study. Our study revealed a significant positive correlation between delayed SMZ-TMP initiation and elevated LDH levels (r=0.477, P=0.03). We also observed higher mortality in patients with higher LDH (death group median: 448.0 U/L vs. survival: 304.5 U/L). Although limited statistical power prevented direct analysis of the ‘delayed treatment + high LDH’ mortality link, the convergence of these findings—alongside established evidence that LDH elevation predicts PCP mortality (25,26)—implies that delayed treatment may exacerbate biological severity (reflected by LDH rise), potentially worsening prognosis. This pathway requires validation in larger studies.

For patients with suspected PCP, anti-PCP therapy should be initiated immediately, and SMZ-TMP is the drug of first choice. In addition, echinocandins, a class of antifungal medicines, have been investigated as adjunctive therapy for PCP. Among echinocandins, caspofungin is the most studied in this context. These drugs target Pneumocystis jirovecii by inhibiting beta-(1,3)-D-glucan synthesis, primarily affecting cyst wall formation (27). Recent clinical evidence robustly supports two echinocandin-based strategies tailored to distinct scenarios. Specifically, two recent meta-analyses, based primarily on retrospective case-control and cohort studies, have demonstrated that the combination of caspofungin and SMZ-TMP can reduce mortality and improve treatment response rates compared to SMZ-TMP monotherapy (28,29). For patients with SMZ-TMP intolerance, Bilbao et al. reported a case series of 14 non-HIV PCP patients treated with an echinocandin-clindamycin combination (without SMZ-TMP), primarily for cotrimoxazole intolerance (30). Their regimen achieved clinical cure in 12 patients (85.7%), with only one PCP-attributed mortality. Notably, no adverse effects were observed with this combination, reinforcing the safety profile of echinocandins in immunocompromised hosts. However, it is important to note that the current clinical evidence stems largely from retrospective analyses. Future prospective, randomized controlled trials are needed to definitively investigate the efficacy of caspofungin (and potentially other echinocandins) for the treatment of PCP. Studies have shown that in HIV-positive PCP patients, the combined use of glucocorticoids is helpful to improve the patient prognosis (31,32). However, the use of glucocorticoids in HIV-negative PCP patients is controversial. Adjunctive glucocorticoids were administered to 16 patients (80%) in this cohort. This therapeutic decision was primarily guided by the severity of illness observed: significant hypoxemia (median Oxygenation index: 310) and respiratory failure requiring mechanical ventilation in 7 patients (35%). Our institutional practice aligns with recommendations extrapolated from meta-analyses in non-HIV populations (33), where corticosteroids aim to mitigate inflammatory lung injury triggered by Pneumocystis jirovecii clearance. We acknowledge the ongoing controversy regarding glucocorticoid efficacy in non-HIV PCP. While Ding et al. (33) suggested potential benefit in patients with respiratory failure, other studies found no mortality improvement (34,35), and Lemiale et al. (36) even associated high-dose steroids with increased risk. In our cohort, 10 of 16 steroid-treated patients (62.5%) survived, while all 4 non-steroid-treated patients survived. However, the non-steroid group had higher baseline oxygenation indices (median: 412 vs. 292 mmHg in steroid group), suggesting less severe initial presentation. Due to significant confounding by indication, small sample size, and retrospective design, no causal inferences regarding steroid efficacy can be drawn from these observations. Prospective randomized trials are urgently needed to definitively establish the role of adjunctive corticosteroids in non-HIV PCP.

This research has several limitations. One notable limitation is the relatively small sample size, which may lead to bias. Additionally, some cases in this study had co-infections. Therefore, further research is needed to explore the differences between co-infections and single bacterial infections, as well as their respective clinical characteristics. It is also important to determine whether these co-infections have an impact on disease severity or clinical outcomes. Another limitation of this study is the lack of confirmatory testing with conventional methods (e.g., direct fluorescent antibody test or specific stains) for Pneumocystis jirovecii in cases identified by mNGS.

Conclusions

HIV-negative PCP patients frequently present with acute respiratory symptoms and demonstrate rapid clinical progression, differing from the more insidious course typically seen in HIV-infected individuals. This atypical presentation, coupled with a lack of clinical suspicion in non-HIV settings, contributes to diagnostic delays. HIV-negative PCP patients have atypical clinical manifestations and a high mortality rate. mNGS testing of BALF may represent a valuable tool for early diagnosis in this population, given its rapid median laboratory turnaround time (20 hours) and high sensitivity. To reduce mortality, it is imperative to initiate SMZ-TMP drug treatment as soon as PCP is clinically suspected. In the future, more multi-center clinical studies are needed to further improve our understanding of PCP in HIV-negative patients.

Acknowledgments

The authors would like to acknowledge all the staff at the Department of Respiratory and Critical Care Medicine, Hangzhou TCM Hospital Affiliated to Zhejiang Chinese Medical University for their work.

Footnote

Reporting Checklist: The authors have completed the STROBE reporting checklist. Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-499/rc

Data Sharing Statement: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-499/dss

Peer Review File: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-499/prf

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-499/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethics Committee of Hangzhou TCM Hospital (No. 2024KLL214) and individual consent for this retrospective analysis was waived.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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