Pneumocystis jirovecii pneumonia (PjP) is an acute and potentially fatal infection caused by the opportunistic fungal pathogen pneumocystis jirovecii, which predominantly affects immunocompromised individuals globally (Grønseth et al., 2021), with a particularly high prevalence among people living with HIV (PLWH) (Thomas and Limper, 2004). The incidence of PjP in PLWH has gradually decreased due to highly effective antiretroviral therapy and prophylactic treatment against P. jirovecii (Crothers et al., 2011; Salzer et al., 2018). However, with the extensive use of glucocorticoids, immunosuppressants, radiotherapy and chemotherapy for tumors and organ transplantation, the incidence of PjP has risen significantly in non-HIV-infected populations (Bienvenu et al., 2016; Dunbar et al., 2020). Notably, non-HIV-infected immunocompromised individuals with PjP often exhibit a poor prognosis, characterized by severe clinical manifestations, an acute disease course, and rapid progression (Kanj et al., 2021). It has been reported that the mortality rate of PjP ranges from 10% to 20% in PLWH, whereas it significantly increases to 30%–60% in non-HIV-infected individuals (Morris and Norris, 2012; Stern et al., 2014; Kotani et al., 2017).

Traditionally, in the presence of clinical suspicion, PjP is diagnosed by the microscopic presence of pneumocysts in respiratory specimens by the use of staining methods, such as Giemsa, Gomori methenamine silver (GMS), calcofluor white, toluidine blue, and/or immunofluorescence antibody assay (Calderón et al., 2010). However, these methods are limited by suboptimal sensitivity and specificity, as well as prolonged turnaround times (TAT).

BDG, a highly conserved polysaccharide component of fungal cell walls, is synthesized during the biogenesis of the fungal cell wall in numerous pathogenic species. This immunogenic molecule has been established as a reliable biomarker for fungal infections, particularly pulmonary aspergillosis and invasive candidiasis (Obayashi et al., 1995). Although the assay lacks specificity for Pneumocystis jirovecii, quantitative detection of serum BDG levels has been established as an adjunctive tool for the diagnosis of PjP (Tasaka et al., 2007; Watanabe et al., 2009; Matsumura et al., 2012; Onishi et al., 2012). Previous studies have established varying diagnostic thresholds for serum BDG in Pneumocystis jirovecii detection. Tasaka et al (Tasaka et al., 2007). determined an optimal cut-off value of 31.1 pg/mL, demonstrating a negative predictive value of 0.980 and positive predictive value of 0.610. Notably, Matsumura et al (Matsumura et al., 2012). achieved a better area under the receiver operator characteristic curve (AUROC) of 0.91 and a lower threshold of 15.6 pg/mL, maintaining 100% sensitivity while providing 80.0% specificity for definitive PjP diagnosis. Meta-analysis by Del Corpo et al (Del Corpo et al., 2020). revealed that serum BDG assay demonstrated limited diagnostic utility for PjP, with insufficient pooled sensitivity to exclude infection and inadequate specificity to confirm PjP diagnosis, particularly in high-prevalence populations. Consequently, we recommend that a comprehensive diagnostic strategy integrating serum BDG assay with complementary microbiological or molecular diagnostic modalities is essential for establishing reliable PjP diagnosis, especially in high-risk populations.

With the rapid advancements in molecular biology, techniques such as metagenomic next-generation sequencing (mNGS) and multiplex PCR-based targeted next-generation sequencing (mp-tNGS) are now widely employed to directly detect target organisms in respiratory samples (Sun et al., 2024; Yin et al., 2024; Hsu et al., 2025). A literature search conducted by X. Li et al (Li et al., 2023). identified 9 studies encompassing 1,343 patients, demonstrating that the pooled sensitivity and specificity of mNGS for diagnosing PjP were 0.974 (95% CI: 0.953–0.987) and 0.943 (95% CI: 0.926–0.957), respectively. A large-scale prospective study by Yin et al (Yin et al., 2024). demonstrated that tNGS outperformed mNGS in detecting pneumocystis jirovecii in BALF samples. Moreover, compared to mp-tNGS, mNGS has the limitations of high costs, long TAT, human gene interference, and deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) must be separately detected (Miller et al., 2019). However, distinguishing colonization from active infection remains a challenge when using mNGS or tNGS, as positive results may be fungal colonization rather than infection; importantly, PjP is a life-threatening infection in the absence of specific treatment, whereas PjC is a less-severe presentation of asymptomatic colonization (Damiani et al., 2013).

However, the diagnostic performance of combining serum BDG assay and BALF mp-tNGS for differentiating between colonization and infection of pneumocystis jirovecii remains unexplored in current literature. In this study, we evaluated the diagnostic performance of BALF mp-tNGS combing with serum BDG for differentiating P. jirovecii infection between colonization.

Pneumocystis jirovecii is a fungal pathogen that causes opportunistic pulmonary infections, particularly in immunocompromised individuals (Thomas and Limper, 2004; Grønseth et al., 2021). Notably, PjP is a life-threatening infection in the absence of specific treatment, whereas PjC is a less-severe presentation of Pneumocystis infection (Damiani et al., 2013). Therefore, there is an urgent need to develop novel diagnostic approaches to accurately distinguish between Pneumocystis jirovecii colonization and active infection. mp-tNGS is a highly sensitive method for detecting Pneumocystis jirovecii (Liu et al., 2024; Yin et al., 2024). However, it remains challenging to differentiate between colonization and true infection. Therefore, based on a comprehensive assessment of clinical features, the established cut-off value of mp-tNGS can be utilized to guide the initiation or discontinuation of anti-Pneumocystis jirovecii therapy.

In our study, 36.9% (n=27) of Pneumocystis jirovecii-positive patients were identified as colonization. This finding was consistent with previous reports, including a 40% colonization rate observed by Y. Jiang et al (Jiang et al., 2025). and 37.5% (24/64) documented by S. Tasaka et al (Tasaka et al., 2014); but differed from 11.7% (15/128) reported by Matsumura and colleagues (Matsumura et al., 2012), this differ is likely due to the inclusion of 60 pneumonia patients without colonization (non-colonization) in their overall sample, which expanded the sample size and consequently resulted in a lower colonization rate. Several populations have been considered as being colonized by Pneumocystis jirovecii, including immunocompromised individuals with varying degrees of immunodeficiency, patients with acute or chronic pulmonary diseases, pregnant women experiencing immunological changes, and healthcare workers frequently exposed to patients with PjP (Morris and Norris, 2012). In our study, the underlying diseases align with the reported risk factors predisposing individuals to colonization.

Various studies have investigated serum BDG as a noninvasive adjunct biomarker for diagnosing PjP in both PLWHand non-HIV-infected individuals, consistently demonstrating an elevated serum BDG levels in individuals with PjP (Tasaka et al., 2007; Cuétara et al., 2008; Pisculli and Sax, 2008; Watanabe et al., 2009; Held et al., 2011). And further studies have demonstrated that patients with active Pneumocystis jirovecii infection exhibit significantly higher serum BDG levels compared to those with P. jirovecii colonization (Matsumura et al., 2012; Damiani et al., 2013; Tasaka et al., 2014; Liu et al., 2021). Consistent with their observations, our study also found that serum BDG levels were significantly higher in the PjP group compared to the PjC group. Furthermore, we observed that PLWH with PjP had a higher serum BDG level than non-HIV-infected individuals with PjP, which is compatible with the observation by Tasaka and colleagues (Tasaka et al., 2014). This finding may be attributed to the higher fungal burden of Pneumocystis jirovecii in the lungs of PLWHwith PjP. Despite the high sensitivity of the BDG assay, false-positive results may occur in the presence of certain confounding factors, including other fungal infections, hemodialysis, Gram-negative bacteremia, severe mucositis, or the administration of intravenous immunoglobulin and the use of certain antibiotics (Onishi et al., 2012; Karageorgopoulos et al., 2013; Del Corpo et al., 2020). However, the high negative predictive value (NPV) of 96% for serum BDG suggests that levels below 80 pg/mL can almost rule out PjP, as previously concluded by Alanio et al (Alanio et al., 2016). Our findings are consistent with those of previous studies and indicate that the BDG assay is useful for excluding PjP. In our study, we established a serum BDG cut-off value of 84.5 pg/mL for distinguishing colonization from active infection; this threshold demonstrated a sensitivity of 85.2% and a specificity of 80.4%. Our findings are consistent with the results reported by Zhang et al (Zhang et al., 2025), who concluded that serum BDG showed high pooled sensitivity (0.83, 95% CI 0.77-0.88) but lower specificity (0.78, 95% CI 0.69-0.85).

To date, only two studies have analyzed the use of mp-tNGS for detecting pathogens in pulmonary infections, highlighting its superior performance in identifying Pneumocystis jiroveci (Liu et al., 2024; Yin et al., 2024). As reported by YIN Y et al (Yin et al., 2024). in a prospective cohort study comparing the detection rate of pathogens in 251 BALF samples between tNGS and mNGS, and found that mNGS performed better in detecting rare pathogens (such as Rhizobium budding, Aspergillus Niger complex, etc.), but missed a lot of P. jirovecii; while tNGS detected more viruses and P. jirovecii. Moreover, they demonstrated that the TAT for mp-tNGS was 10.3 hours, which was significantly shorter than the TAT of 16–24 h for mNGS. Importantly, compared to mp-tNGS, mNGS is limited by higher costs, interference from human genetic material, and the requirement for separate detection of DNA and RNA (Miller et al., 2019). Our results suggest that the combination of the mp-tNGS tests, applying cut-off values of 355 reads, and serum BDG detection, applying an 84.5 pg/ml threshold, can effectively differentiate Pneumocystis jirovecii pneumonia from colonization. However, five patients diagnosed with PjP revealed mp-tNGS reads below 355 (51, 205, 229, 256 and 349, respectively). Therefore, when mp-tNGS reads are fewer than 355, the results must be interpreted cautiously in conjunction with clinical manifestations and radiological features. The observed results—specifically, five non-HIV-infected immunocompromised patients with PjP showing mp-tNGS reads below 355 and four PjC case with read above 355—can be attributed to fungal burdens of P. jirovecii. As previously reported, the expression of PjP occurs with a lower fungal load in non-HIV-infected immunocompromised patients than PLWH (Limper et al., 1989; Fauchier et al., 2016). In our study, microscopic examination with GMS staining confirmed three cases of PjP, two of which were in PLWH, while none of the colonized or non-colonized patients had HIV infection. Consistent with previous reports, non-HIV-infected patients often develop PjP with a lower fungal load compared to PLWH (Limper et al., 1989), which can lead to false-negative results on microscopic examination (Azoulay et al., 2009).

Previous studies have suggested that serum BDG levels can be used to evaluate fungal load. However, Held et al (Held et al., 2011). demonstrated that BDG levels do not correlate with Pneumocystis jirovecii load. Consistent with these findings, our analysis revealed no correlation between mp-tNGS reads and BDG levels in mp-tNGS-positive patients, further supporting the notion that BDG may not reliably reflect P. jirovecii burden. A multicenter retrospective study by H. Sun et al (Sun et al., 2022). revealed that BALF-mNGS achieved a sensitivity of 97.40% and specificity of 85.12% for diagnosing PjP, which significantly outperforming blood BDG/LDH and BALF microscopy in sensitivity and BDG/LDH in specificity (p<0.05). Despite demonstrating good sensitivity, a notable limitation of their study is the lack of validation in distinguishing between colonization and infection. In this study, when using a combined diagnostic approach with a BDG threshold of 84.5 pg/mL and 355 mp-tNGS reads for the diagnosis of PjP, the sensitivity reached 84.2%, the PPV was 97.0%, and the diagnostic accuracy was 83.7%, which strongly support the diagnosis of PjP. Furthermore, the combination of BDG<84.5 pg/mL and mp-tNGS<355 reads for the diagnosis of PjC achieved a sensitivity of 86.9% and a PPV of 86.9%. These findings demonstrate that the combined use of serum BDG and BALF mp-tNGS provides excellent performance for distinguishing between colonization and infection with P jirovecii.

As previously reported, patients with PjP showed a high prevalence of mixed pulmonary infections (Jiang et al., 2021), we hypothesize that this may be attributed to varying degrees of immunosuppression in PjP patients. mp-tNGS has advantages in the identification of multiple microorganisms in respiratory samples, especially in the lower respiratory tract (Yin et al., 2024). In our study, BALF mp-tNGS detected one or more mixed microorganisms in 36 patients with PjP. Among these, cytomegalovirus (CMV) was the most frequently identified, followed by Epstein-Barr virus (EBV), Streptococcus mitis, and influenza A virus (H1N1) and herpes simplex virus (HSV). Clinically, we consider these microorganisms less likely to be primary pathogens but rather potential colonizers. In this study, we did not perform a detailed evaluation of these patients, as the primary focus was to differentiate between colonization and infection by P jirovecii.

The application of tNGS encounters several substantial challenges, particularly in resource-limited settings. One of the primary barriers is the high upfront investment required for tNGS platforms, reagents, and bioinformatics infrastructure, which can be prohibitively expensive for many laboratories. Additionally, the tNGS workflow is inherently complex, involving multiple steps such as nucleic acid extraction, library preparation, sequencing, and data analysis (Yin et al., 2024); each of these steps introduces potential sources of error or variability, further complicating the process. Moreover, many laboratories, especially those in low- and middle-income countries (LMICs), often lack the necessary infrastructure and resources to effectively implement tNGS, limiting its accessibility and utility in these regions. Our study has several limitations, including its retrospective, single-center design and the relatively small sample size. In the near future, we will provide a detailed report on the other detected mixed microorganisms in PjP.

In conclusion, the combination of BALF mp-tNGS results and serum BDG levels can effectively differentiate between P jirovecii. infection and colonization, potentially providing valuable guidance for clinical decision-making and therapeutic strategies.