Introduction

Pneumocystis jirovecii is a fungal pathogen that causes opportunistic infections, particularly pneumocystis pneumonia (PJP), in individuals with compromised immune systems. Historically, these infections were most commonly seen in people with human immunodeficiency virus (HIV) infection, particularly in the context of acquired immunodeficiency syndrome (AIDS). However, advances in antiretroviral therapy and primary prophylactic strategies have significantly reduced the incidence of PJP. One such strategy involves administering trimethoprim–sulfamethoxazole (TMP/SMX) at a single dose of 80/400 mg once or twice daily for patients with a CD4⁺ T-cell count below 200 cells/mm³ [1,2]. The widespread availability of this intervention led to a dramatic drop in the annual incidence of PJP among AIDS patients in San Francisco from 95 to 8.4 cases per 1,000 individuals between 1993 and 2008 [3,4].

Despite this progress, the incidence of PJP has been increasing among patients not infected with HIV (non-HIV patients), particularly those receiving immunosuppressive drugs, corticosteroids, hematologic cancer treatments, or bone marrow transplants and those managing rheumatic diseases. In the absence of prophylaxis, the risk of PJP is significantly elevated in patients with lymphoma, particularly in those with pediatric acute lymphoblastic leukemia (22%–45%) and diffuse large B-cell lymphoma (2%–8%). This risk is also notable among organ transplant recipients (5%–15%) and individuals with rheumatic diseases (2%–4%) [5–11]. Patients in these categories frequently present with severe clinical symptoms and experience high mortality rates, ranging from 30% to 60% [12–14]. A study conducted in 2006 at our institution, Ramathibodi Hospital in Bangkok, Thailand, also found that the incidence of PJP among patients with connective tissue diseases undergoing immunosuppressive therapy was 7.6% [15].

Risk factors contributing to the increased incidence of PJP include the long-term use of corticosteroids at doses equivalent to >20 mg/day for at least 1 month. Certain immunosuppressive agents, such as vincristine, cyclophosphamide, and methotrexate, also increase the risk [16]. Another immunosuppressant, rituximab, a monoclonal antibody that specifically targets the CD20 antigen on the surface of B lymphocytes, has become a popular option for treating hematologic malignancies and autoimmune diseases due to its effectiveness in suppressing excessive antibody production. It is commonly used to treat conditions such as lymphoma, rheumatoid arthritis, and systemic lupus erythematosus. However, despite its therapeutic benefits, rituximab can produce adverse effects that include infusion-related reactions, rash, fever, and an increased risk of infections, including PJP [17]. Previous studies have shown that the incidence of PJP in lymphoma patients treated with rituximab ranged from 3% to 15% among those who did not receive primary prophylaxis [10,11,18–22]. Mortality rates at 30, 60, and 90 days after such infection were reported to be 12.1%, 16.6%, and 21.5%, respectively. Fortunately, the use of TMP/SMX for primary prophylaxis significantly improved 1-year survival rates, increasing from 38% to 73% [22]. PJP infections typically occurred between days 69 and 86 after starting lymphoma treatment, with a mean (SD) duration of primary prophylaxis lasting 153.8 (107.6) days [11,23].

According to the European Conference on Infections in Leukemia (ECIL) guidelines 5 (2016), the recommended options for TMP/SMX administration in acute lymphoblastic leukemia, prednisolone equivalent of > 20 mg/day for 4 weeks, fludarabine/cyclophosphamide/rituximab include one single-strength tablet (TMP/SMX 80/400 mg) per day, one double-strength tablet (TMP/SMX 160/800 mg) per day (Grade AII), or one double-strength tablet three times a week (Grade BII). This prophylactic treatment should be continued for at least 6 months following rituximab therapy. However, it is important to note that these recommendations have not been thoroughly validated in patients with other hematologic malignancies, as available data are primarily from small studies and pre–PCR era research. Therefore, the optimal dosage and duration of treatment for preventing PJP remain unclear [16,24].

As a preliminary analysis, we assessed prescribing practices at multiple academic centers in Thailand, which revealed various TMP/SMX dosing regimens used as primary prophylaxis in patients with non-Hodgkin lymphoma receiving rituximab. These regimens include daily administration of one single-strength tablet once daily, two single-strength tablets three times/week, one single-strength tablet three times/week, or two single-strength tablets twice daily two times/week. The attending physician determines the dosage choice, as there is no standardized protocol in Thai clinical practice.

The primary objective of this study was to evaluate the effectiveness of different dosages of TMP/SMX in preventing pneumocystis infections over a one-year period in patients with non-Hodgkin lymphoma who were receiving rituximab for the first time. The secondary objectives included assessing the incidence of other opportunistic infections that are susceptible to TMP/SMX, such as toxoplasmosis and nocardiosis. Additionally, we aimed to examine the incidence of adverse effects associated with TMP/SMX use in lymphoma patients receiving initial rituximab therapy. Specifically, we tested our null hypotheses, which stated that there would be no significant differences in the rates of opportunistic infections, adverse drug events, and overall clinical outcomes among participants receiving various prophylactic dosing regimens.

Materials and methods

Statistical analysis

Data were analyzed using STATA version 18.0. Descriptive statistics are presented as means and standard deviations (mean ± SD) for data with a normal distribution. For non-normally distributed data, percentages and medians with minimum and maximum values are reported. For inferential statistics, we compared baseline characteristics (Table 1) between groups using ANOVA for normally distributed data and the Kruskal-Wallis test for non-normally distributed data. Time-to-event outcomes were analyzed using Cox proportional hazards regression to estimate hazard ratios. Incidence outcomes were summarized using descriptive statistics. A p-value of <0.05 was considered statistically significant.

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Table 1. Baseline clinical and laboratory characteristics of patients in the standard, the alternative, and the non-prophylaxis groups.

https://doi.org/10.1371/journal.pone.0344273.t001

Results

Baseline characteristics

A search in the Ramathibodi Hospital medical records covering the period from January 1, 2013, to December 31, 2022, identified 833 patients newly diagnosed with non-Hodgkin lymphoma who received chemotherapy combined with rituximab, with or without TMP/SMX for prophylaxis against pneumocystis infection. Among these, 143 patients did not meet the inclusion criteria. Therefore, 690 patients treated with rituximab were analyzed and divided into three groups, namely, 1) ST group (n = 147), 2) AT group (n = 475 cases), and 3) NP group (n = 68 cases), as shown in Fig 1.

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Fig 1. Protocol flow chart.

https://doi.org/10.1371/journal.pone.0344273.g001

The majority of the patients were female (55%). The mean ages for the ST, AT, and NP groups were 63.67 (SD 13.39), 61.43 (+13.86), and 61.57 (+13.62) years, respectively. The most common diagnosis among patients was diffuse large B-cell lymphoma (n = 465/690; 67.4%), followed by follicular lymphoma (n = 88/690; 12.8%) and marginal zone lymphoma (n = 57/690; 8.3%). The baseline characteristics of all participants are presented in Table 1. Among those who received TMP/SMX prophylaxis, the mean time to initiation of the prophylaxis was 1.65 days, with a mean duration of 265.7 days (SD 85.66), starting from the first dose of rituximab. During the study period, 29 patients died after receiving the first dose of rituximab. The prevalence of non-PJP-related deaths, categorized based on available clinical documentation and those without evidence of PJP, was 5.44% (8 out of 147) in the ST group, 3.58% (17 out of 475) in the AT group, and 5.88% (4 out of 68) in the NP group.

Incidence of opportunistic infections

By the end of the study, seven patients had developed PJP, resulting in an overall incidence rate of 1%. Among these patients, four (5.8%) were in the NP group, which included three definite cases and one probable case. The remaining three patients were in the AT group, with two definite cases and one probable case, yielding an incidence rate of 0.6%. All patients in the AT group received the DS BID regimen on two days of the week. Notably, none of the patients in the ST group or those who received the SS OD regimen, every other day, three days of the week developed PJP.

The incidence of PJP per 10,000 person-years was as follows: 0.296 across all groups, 0.184 in the AT group, and 1.761 in the NP group (see Fig 2). The median time from the onset of PJP to the first dose of rituximab was 217 days, with a range of 31–355 days. The overall mortality rate associated with PJP was 14.3%, representing one death.

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Fig 2. Cumulative incidence of pneumocystis pneumonia in the standard, the alternative, and the non-prophylaxis groups.

https://doi.org/10.1371/journal.pone.0344273.g002

In the DS BID two days of the week group, two out of three cases of PJP were diagnosed after the discontinuation of TMP/SMX, with intervals of 162 and 184 days, respectively. Additionally, one patient had received high-dose corticosteroids due to coronavirus infection (COVID-19) 1 month prior to the PJP diagnosis. Further characteristics of the patients diagnosed with PJP are summarized in Table 2. An intention-to-treat analysis documented four cases of PJP in the NP group and three cases in the TMP/SMX group. Prophylaxis significantly reduced the 1-year incidence of PJP, with a hazard ratio (comparison between AT and NP) of 0.105 (95% confidence interval: 0.023–0.469). No other opportunistic infections sensitive to TMP/SMX were recorded in this study.

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Table 2. PJP patients in the alternative and the non-prophylaxis groups.

https://doi.org/10.1371/journal.pone.0344273.t002

Discussion

The incidence of PJP is increasing globally among non-HIV patients, particularly those on immunosuppressive therapies, including among lymphoma patients on rituximab and cyclophosphamide, doxorubicin, vincristine, and prednisolone (CHOP-14) chemotherapy without prophylaxis [5,7]. According to research by Elsegeiny et al., B cells play a critical role in supporting T-cell proliferation and differentiation, emphasizing the cooperative function of B and T cells in controlling pneumocystis infections [25]. The fact that rituximab depletes B cells and inactivates/depletes T cells is suggestive of the mechanism by which it increases the risk of PJP. At present, no standardized TMP/SMX prophylactic regimen for non-HIV patients has been established in Thailand, leading to variations in prescribing practices.

A study conducted by Park et al in 2022 involved 3,524 patients (hematologic diseases, rheumatic diseases, and solid organ transplantation) who were treated with rituximab between 2002 and 2018. The patients were divided into two groups: Group 1 included 1,001 patients who received TMP/SMX to prevent primary infection, while Group 2 consisted of 2,523 patients who did not receive this treatment. The results indicated that the incidence of PJP was significantly lower in Group 1. Most participants in the study were administered either an SS OD dose or a DS OD three times a week. The mean duration of TMP/SMX treatment was 153.8 days, with PJP infection occurring on day 86 on average. In both groups, the rate of infection started to plateau approximately 4–6 months after the initiation of treatment [23].

Several studies have compared the use of low-dose TMP/SMX with standard dosing regimens, showing fewer side effects in patients receiving lower doses after 6 months of follow-up [26,27]. For instance, a study in Japan by Utsunomiya et al., published in 2017, evaluated TMP/SMX prophylaxis in patients with systemic rheumatic disease receiving prednisolone at doses of 0.6 mg/kg/day or higher. The participants were divided into three groups based on the TMP/SMX regimen: a single-strength group (TMP/SMX of 80/400 mg daily), half-strength group (40/200 mg daily), and escalation group (started with 8/40 mg daily, increasing incrementally to 40/200 mg daily). The incidence of PJP-free survival at 24 weeks did not differ among these groups. Moreover, the rate of discontinuation due to adverse effects was lower in the half-strength group than in the single-strength group [26].

The current study compared the effectiveness of primary prophylaxis for PJP and evaluated the safety of TMP/SMX at different dosages to determine the optimal regimen for preventing PJP in non-Hodgkin lymphoma patients receiving rituximab. The data revealed that the baseline characteristics, including age, sex, comorbidities, hematologic conditions, and the duration of rituximab administration, were comparable across the three patient groups. The majority of patients were female, diagnosed with DLBCL, and received R-CHOP (rituximab-CHOP) as their initial treatment regimen. Notably, PJP infections were more prevalent among patients over 65 years of age, which was above the average age of the study population.

In our study, the majority of patients had lymphoma, which likely minimized the potential confounding effect of pneumonia caused by PJP related to lymphoma. PJP risk is generally higher in patients with acute lymphoblastic leukemia (ALL), whereas the incidence is relatively low in other lymphoma subtypes [5–11].Specifically, the incidence of PJP in patients treated with rituximab was only 1%, which is considerably lower than previous reports that indicated rates between 2.56% and 2.95% [10,23]. In the NP group, the incidence of PJP was 5.8%, while earlier studies reported rates ranging from 3% to 15% [10,11,18–22]. Meanwhile, the incidence in the AT group was 0.6%, while no PJP cases were observed in the ST group. These findings support existing evidence that primary prophylaxis with TMP/SMX significantly reduces the risk of PJP, emphasizing the protective effect of prophylaxis. Additionally, the calculated HR of 0.105 further underscores the substantial protective benefit of TMP/SMX prophylaxis in patients treated with rituximab, supporting its continued use in this high-risk population.

At the end of the current study, TMP/SMX prophylaxis was associated with a reduced incidence of PJP after 1 year among the study population. There was also no significant difference in this regard between the AT group and the ST group. Notably, no cases of PJP were reported in the low-dose group (SS OD, administered three times a week). In those patients receiving TMP/SMX prophylaxis, all patients diagnosed with PJP received the DS BID twice-weekly regimen. Additionally, one patient with PJP was concurrently using immunosuppressive agents, such as methotrexate, leflunomide, and azathioprine, which may have heightened the risk for PJP. Another patient had been on high doses of corticosteroids (over 20 mg per day) due to COVID-19-related pneumonia one month prior to the PJP diagnosis, further increasing the susceptibility to the infection. The twice-weekly dosing regimen of DS tablets may have led to subtherapeutic drug levels, as sulfamethoxazole and trimethoprim have half-lives of 6–12 hours and 8–10 hours, respectively [28]. To maintain adequate drug levels, a shorter dosing interval of approximately every two days may be more effective.

Regarding adverse side effects, patients receiving DS BID two times a week experienced a higher incidence of hematologic abnormalities. ADRs related to TMP/SMX were generally mild, classified as grade 1–2, with an overall incidence of 3.05%. Additionally, 2.25% of patients had to discontinue TMP/SMX due to these effects. These findings are consistent with previous research, which indicates that TMP/SMX is well-tolerated and has manageable side effects. Considering the significant reduction in the incidence of PJP and associated mortality, our findings suggest that the benefits of prophylaxis outweigh the risks.

The strengths of this study include the use of a database from a large patient population and the stratification of groups based on different TMP/SMX dosage regimens to assess primary prophylaxis efficacy in a real-world setting. However, several limitations of this work should be acknowledged. First, this cohort study was retrospective in nature and conducted at a single center, so caution should be taken when attempting to extrapolate the findings to other contexts. Additionally, patient selection bias may have influenced the results, as the treating physician determined TMP/SMX prophylaxis administration. Second, the distribution of participants was unequal across groups; the NP and ST groups had relatively small sample sizes, which may have led to underestimation of the incidence of PJP, particularly in the ST group, where no PJP cases were observed. In contrast, those receiving the regimen of DS BID two times a week constituted the largest proportion of participants (62.90%), resulting in more PJP cases and TMP/SMX-related adverse effects. Future studies should aim for larger, more balanced sample sizes across groups.

Another limitation of this work is that the follow-up period in this study was relatively short. Among all participants, 46.37% had relapsed or refractory disease, potentially requiring prolonged administration of rituximab and TMP/SMX. The short follow-up period may not have been sufficient to fully assess the long-term outcomes of opportunistic infections. Future studies with extended follow-up beyond 12 months would provide more comprehensive insights. Finally, in addition to evaluating the various chemotherapy regimens among participants, the total dosage of corticosteroids was not specifically examined. This oversight could be significant, as a higher corticosteroid dose may independently increase the risk of PJP. Future studies should include a more thorough assessment of this potential confounding factor to provide a clearer understanding of how pneumocystis infections develop during lymphoma treatment.

Conclusions

All dosing regimens of TMP/SMX prophylaxis were associated with a reduced incidence of PJP in non-Hodgkin lymphoma patients receiving rituximab in this study. Low-dose regimens provided clinical outcomes comparable to those of higher-dosing regimens in terms of preventing PJP infections, while also having fewer adverse effects at 1-year follow-up. Further study is required to verify the impact of a lower dose of TMP/SMX on opportunistic infections in this population.