# Association between mycophenolic acid treatment and infection with specific strains of Pneumocystis jirovecii in solid organ transplant recipients in France and Switzerland: a multicentre, retrospective, cross-sectional study

Claire V Hoffmann, Yannick Le Meur, Marie-Christine Moal, Anne Grall-Jézéquel, Nathan Le Nan, Nicolas Papon, Gilles Nevez, Solène Le Gal, Anne Totet, Hakim Mazouz, et al.

► To cite this version:

Claire V Hoffmann, Yannick Le Meur, Marie-Christine Moal, Anne Grall-Jézéquel, Nathan Le Nan, et al. Association between mycophenolic acid treatment and infection with specific strains of Pneumocystis jirovecii in solid organ transplant recipients in France and Switzerland: a multicentre, retrospective, cross-sectional study. The Lancet Microbe, 2025, 6 (9), pp.101146. 10.1016/j.lanmic.2025.101146. hal-05217817

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# Association between mycophenolic acid treatment and infection with specific strains of *Pneumocystis jirovecii* in solid organ transplant recipients in France and Switzerland: a multicentre, retrospective, cross-sectional study

Claire V Hoffmann, Yannick Le Meur, Marie-Christine Moal, Anne Grall-Jézéquel, Nathan Le Nan, Nicolas Papon, Gilles Nevez*, Solène Le Gal*, on behalf of the Pneumocystis IMPDH Study Group†

<figure><img src="image_2.png" /></figure>

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OPEN ACCESS

## Summary

**Background** *Pneumocystis* pneumonia (PCP) case clusters involving solid organ transplant (SOT) recipients have been reported worldwide. Mycophenolic acid, an immunosuppressant used to prevent rejection in SOT recipients that targets the inosine 5'-monophosphate dehydrogenase (IMPDH) protein, has been hypothesised to exert selective pressure on *Pneumocystis jirovecii*, with the missense mutation G1020A (Ala261Thr) in the *impdh* gene a possible marker of such selective pressure. The aim of this study was to test the hypothesis that SOT recipients harbour *P. jirovecii* with mutations in the *impdh* gene and are infected with specific *P. jirovecii* strains.

**Methods** In this retrospective, multicentre, cross-sectional study of nationally representative, individual-level data, we included SOT recipients, regardless of the organ transplanted, involved or not in a PCP case cluster, and non-SOT recipients (control group) without mycophenolic acid exposure, diagnosed with PCP from 26 French and one Swiss secondary health-care centres. We included patients aged 18 years or older for whom archival *P. jirovecii* DNA extracts were available in sufficient quantity and quality. *P. jirovecii* specimens were characterised using a multilocus sequence typing method including *impdh* gene analysis. The primary outcome of this study was the detection of the G1020A (Ala261Thr) mutation in the *impdh* gene. A multivariable logistic regression was done to assess the relation between this mutation and the following variables retrieved from medical records: age, mycophenolic acid exposure at the time of PCP diagnosis, involvement in a PCP case cluster, PCP prophylaxis, and clinical outcome.

**Findings** 58 SOT recipients (44 treated with mycophenolic acid) and 59 non-SOT recipients (control group; not treated with mycophenolic acid) diagnosed with PCP between Jan 1, 2001, and Dec 31, 2021, were enrolled. The G1020A (Ala261Thr) mutation was detected in *P. jirovecii* specimens from 40 (68.9%) SOT recipients (37 treated with mycophenolic acid) and in none of the *P. jirovecii* specimens from the patients in the control group. The multivariable analysis showed that the allele characterised by the G1020A mutation was associated with mycophenolic acid exposure at the time of PCP diagnosis (adjusted odds ratio 73.61 [95% CI 17.41–455.70]; p<0.0001) and involvement in a PCP case cluster (12.77 [1.58–171.90]; p=0.029), whereas it was not associated with age, PCP prophylaxis, and clinical deterioration. A second missense mutation, G1020T (Ala261Ser) was identified in *P. jirovecii* specimens from three SOT recipients (two treated with mycophenolic acid). Two specific multilocus genotypes (MLG-31 and MLG-34) of *P. jirovecii* associated with Ala261Thr and Ala261Ser substitutions in IMPDH, respectively, were detected only in SOT recipients (38 patients with MLG-31 and three patients with MLG-34).

**Interpretation** SOT recipients in this study were primarily infected with specific *P. jirovecii* strains with mutations in the *impdh* gene, which might confer a selective advantage as both the G1020A (Ala261Thr) and G1020T (Ala261Ser) are associated with mycophenolic acid resistance in other fungi. Mycophenolic acid selective pressure might explain the maintenance and circulation of these *P. jirovecii* strains within this patient population, and consequently their potential involvement in PCP case clusters.

**Funding** French Speaking Society for Transplantation, and Chiesi.

**Copyright** © 2025 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

## Introduction

*Pneumocystis jirovecii* is a transmissible micromycete causing pneumonia (*Pneumocystis pneumonia* [PCP]) in patients

who are immunosuppressed. PCP case clusters involving solid organ transplant (SOT) recipients have been reported worldwide.¹ These occurrences might be related to

Lancet Microbe 2025

Published Online
https://doi.org/10.1016/
j.lanmic.2025.101146

*Contributed equally
†Members listed at the end of
the Article

CHU de Brest, Laboratoire de
Parasitologie-Mycologie, Brest,
France (C V Hoffmann PhD,
Prof G Nevez PhD, S Le Gal PhD);

University of Brest, University of
Angers, Infections Respiratoires
Fongiques, Brest, France
(C V Hoffmann, N Le Nan,
Prof G Nevez, S Le Gal);

CHU de Brest, Service de néphrologie et
transplantations rénales, Brest,
France (Prof Y Le Meur PhD,
M-C Moal MD,

A Grall-Jézéquel MD);
Université de Brest, UMR1227,
Lymphocytes B et Autoimmunité,
Inserm, Labex IGO, Brest, France
(Prof Y Le Meur);

University of Brest,
Infections Respiratoires
Fongiques, SFR ICAT, Angers,
France (Prof N Papon PhD)

Correspondence to:
Dr Solène Le Gal, CHU de Brest,
Laboratoire de Parasitologie-
Mycologie, Brest 29609, France

solene.legal@univ-brest.fr
Research in context

Evidence before this study

Solid organ transplant (SOT) recipients are exposed to complications such as rejection and infectious diseases. Fungal infections remain a major cause of morbidity and mortality in this setting, notably *Pneumocystis pneumonia* (PCP). To reduce such complications, SOT recipients receive immunosuppressive drugs to prevent organ or cell rejection and anti-infective drugs to prevent infections. PCP case clusters in transplantation units have been reported worldwide with an increased frequency in the past 15 years. Moreover, there is growing evidence that antibacterial and antifungal prophylactic regimens in SOT recipients trigger microbiota or mycobiota imbalances. These imbalances have the potential to promote the selection of less susceptible fungal isolates or species, which could lead to fatal infections. By contrast, the potential selective effect of immunosuppressants on microorganisms, particularly on fungi, in SOT recipients remains poorly investigated. We searched PubMed for articles published between database inception and Sept 1, 2023, using the terms (mycophenolic acid) OR (mycophenolate) AND (selection) AND ((fungi) OR (*Pneumocystis*)) AND (transplantation). This search retrieved 32 articles, only one of which was relevant and actually related to fungi, including *Pneumocystis jirovecii*. The article we identified was published by our own research team and described a missense mutation G1020A (Ala261Thr) in the gene encoding inosine 5'-monophosphate dehydrogenase (IMPDH) of *P. jirovecii* isolates from ten SOT recipients treated with mycophenolic acid (the main immunosuppressant used as organ anti-rejection in SOT recipients) at the time of PCP diagnosis. IMPDH is the target of mycophenolic acid and the mutation observed is analogous to mutations described in other fungi with intrinsic or acquired resistance to mycophenolic acid, such as *Penicillium brevicompactum* or *Candida albicans* strain EC3. This study was done in a single centre, in a small number of patients (ten SOT recipients and 16 control patients), and using only *impdh* gene analysis. An updated search using the same search terms conducted on

June 2, 2025, identified one additional study, which hypothesised that the resistance of *P. jirovecii* to mycophenolic acid has a role in a putative outbreak.

Added value of this study

This retrospective, cross-sectional study included 27 secondary health-care centres and recruited a larger number of patients (58 SOT recipients and 59 non-SOT recipients with PCP) than our previous study. Multilocus genotyping including *impdh* gene analysis was done on *P. jirovecii* specimens from these patients. This approach has a high discriminatory power, which allowed us to identify 44 multilocus genotypes (MLGs), two of which (MLG-31 and MLG-34) were associated with missense mutations (Ala261Thr and Ala261Ser, respectively) in the gene encoding IMPDH and were only detected in SOT recipients (ie, not in any control patients). In multivariable analysis, the Ala261Thr mutation was associated with mycophenolic acid exposure at the time of PCP diagnosis and with involvement in a PCP case cluster. This study shows that *P. jirovecii* organisms resistant to mycophenolic and specific strains are involved in PCP case clusters in SOT recipients.

Implications of all the available evidence

A mycophenolic acid-based immunosuppressive regimen might favour the selection of specific fungal strains in SOT recipients. The selective pressure of this immunosuppressant on fungal strains might explain their maintenance and circulation within this patient population, and consequently their involvement in PCP case clusters. This possibility must be considered to optimise PCP prophylaxis in SOT recipients receiving mycophenolic acid. Further studies conducted in a prospective way and involving centres from other European countries and from other continents would allow a better overview of *P. jirovecii* diversity in SOT recipients and, specifically, of the circulation of *P. jirovecii* strains potentially selected by mycophenolic acid or other immunosuppressants.

particular characteristics of the patients (eg, absence of PCP prophylaxis beyond 6–12 months after transplantation, absence of prevention measures in hospitals and, consequently, interindividual transmission of the fungus),² but might also be related to characteristics of *P. jirovecii* in SOT recipients. A survey done in France reported 16 PCP case clusters in transplantation units, involving 207 patients, over a 12-year period (October, 2006, to August, 2018), with 13 clusters concerning renal transplant recipients.³ A common *P. jirovecii* genotype was identified in four of these 16 case clusters, suggesting that specific strains were selected in renal transplant recipients and circulated in this patient population, as has been previously suggested.³⁻⁵ In 2021, we hypothesised that selection of *P. jirovecii* strains in SOT recipients might have occurred under mycophenolic acid pressure.⁶ Mycophenolic acid is an inhibitor of inosine 5'-monophosphate dehydrogenase

(IMPDH), an enzyme involved in de-novo purine biosynthesis. This drug is widely used in SOT recipients as an anti-rejection treatment. It inhibits the proliferation of B lymphocytes and T lymphocytes.⁷ Of note, mycophenolic acid incidentally also has an antifungal effect,⁸,⁹ and consequently also a potential effect on *P. jirovecii*.¹⁰,¹¹ However, its putative protective role against PCP¹⁰ is a subject of debate, as SOT recipients treated with mycophenolic acid have been observed to develop PCP.⁶ In a previous study, we identified a missense mutation (G1020A; Ala261Thr) in the *impdh* gene of *P. jirovecii* from ten SOT recipients treated with mycophenolic acid that was absent in *P. jirovecii* from 16 patients not treated with this drug.⁶ Considering that IMPDH of a mycophenolic acid-resistant *Candida albicans* strain (strain EC3) harbours threonine at the analogous position,⁸ we hypothesised the Ala261Thr mutation to be a signature of *P. jirovecii* exposure to mycophenolic acid and of
P *jirovecii* selection by mycophenolic acid.⁶ In the current study, we aimed to explore this hypothesis, arising from a single-centre study that only used *impdh* gene analysis, by obtaining additional data from multiple centres and using a more discriminatory multilocus sequence typing (MLST) approach, combined with the *impdh* gene analysis. Specifically, we aimed to identify SOT recipients who harbour *P jirovecii* carrying IMPDH mutations and who are infected with specific *P jirovecii* strains.

Methods

Study design and participants

Members of the French Society for Medical Mycology and the French-Speaking Society for Transplantation were asked to participate in this multicentre, retrospective, cross-sectional study of nationally representative, individual-level data. 26 secondary health-care centres (appendix p 2) in French cities took part in the study (Amiens, Angers, Besançon, Brest, Caen, Clermont-Ferrand, Dijon, Grenoble, Lille, Limoges, Lyon, Montpellier, Nancy, Nantes, Nice, Paris [two centres: Bichat and La Pitié-Salpêtrière], Créteil, Poitiers, Reims, Rennes, Rouen, Saint-Etienne, Strasbourg, Toulouse, and Tours) along with the Swiss city, Bern (appendix p 2). Each centre was requested to include SOT recipients, regardless of the organ transplanted, involved or not in a PCP case cluster, and non-SOT recipients (control patients) without mycophenolic acid exposure. Inclusion criteria were patients aged 18 years or older, diagnosed with PCP between 2001 and 2021, and for whom archival *P jirovecii* DNA extracts were available in sufficient quantity and quality. Exclusion criteria were patients younger than 18 years and adult patients under legal protection. The patients from the Brest health centre enrolled in our previous study⁶ were also enrolled in the present study.

PCP diagnoses were based on clinical criteria combined with *P* *jirovecii* detection using microscopic examination or a quantitative PCR (qPCR) assay, or both. *P* *jirovecii* specimens consisted of DNA extracted from pulmonary samples, stored at -20°C until further examination. The following patient data were collected from medical records by the participating centres: demographics, underlying diseases or conditions, the date of PCP diagnosis, *P* *jirovecii* prophylaxis, and clinical outcome for all participants and, for SOT recipients, interval between transplantation and PCP occurrence, immunosuppressive treatment (particularly mycophenolic acid treatment at the time of PCP diagnosis, whatever its duration), dose of mycophenolic acid, possible history of mycophenolic acid treatment for patients not submitted to mycophenolic acid at the time of PCP diagnosis, and involvement in PCP case clusters. Race or ethnicity data were not collected, in compliance with French law. A PCP case cluster was defined as the occurrence of at least three PCP cases in a transplantation centre over a 6-month period.

Additional patients diagnosed with pulmonary colonisa-
tion with *P. jirovecii* between 2001 and 2021 were included
from three centres (Brest University Hospital, Paris-Bichat

University Hospital, Reims University Hospital). These
patients were considered to be colonised since they had
alternative diagnoses of PCP, and the fungus was detected
by qPCR assays whereas the microscopic detection was
negative. Inclusion criteria were patients aged 18 years or
older, not under protection, and for whom archival *P* *jirovecii*
DNA extracts were available in sufficient quantity and
quality. Extracted DNA was stored at -20°C.

The protocol was approved by the ethics committee
of Brest University Hospital, Brest, France (approval
number B2022CE.16), which was the sponsor of the study
(study ID number 29BRC22.0106). In accordance with the
French Reference Methodology MR-004, applicable to the
retrospective use of health-related data, we individually
informed, by letter, participants who were alive at the time of
the present study of the research protocol and of the planned
use of their data and stored samples, and offered them the
opportunity to opt out of the study by including a non-
opposition form alongside the information note. Absence
of receipt of opposition within 15 days of the letter having
been dispatched confirmed permission to use the data and
samples. Also, in accordance with the MR-004 framework,
data and samples of participants who died before or during
the present study could be used provided that they had
not expressed refusal in writing during their lifetime. All
participant data were pseudonymised to protect their ano-
nymity. The study was registered with ClinicalTrials.gov
(NCT05452148).

Procedures

Extracted DNA was used for *P* *jirovecii* genotyping with an MLST method. Genotyping was done on specimens from SOT recipients and control patients with PCP, and from patients colonised by *P* *jirovecii*. Four genes were analysed: mitochondrial large subunit rRNA (*mtLSUrRNA*), cytochrome b (*cyb*), superoxide dismutase (*sod*), and *impdh*. The *impdh* gene was analysed focusing on a 682-base-pair region that includes the aforementioned nucleotide position 1020. The PCR-sequencing assay was done as previously described, using the primers IMPDH-1109F and IMPDH-1790R (appendix p 2).⁶ Consensus sequences were aligned with a reference sequence (GenBank accession number MZ272397.1) using the Clustal W programme from the BioEdit software (version 7.2.5).⁶ *Impdh* alleles were defined based on previously described single nucleotide polymorphisms (SNPs; G914A, G1020A, C1269T, and A1329T)⁶ and SNPs newly described in the present study.

*MtLSUrRNA, cyb, and sod* genes were analysed as previ-
ously described, using Sanger sequencing of PCR prod-
ucts.¹² Consensus sequences were aligned with reference
sequences (GenBank accession numbers: M58605
[*mtLSUrRNA*],¹³ AF074871 [*cyb*],¹⁴ and AF146753 [*sod*]¹⁵)
using the BioEdit software with Clustal W. *MtLSUrRNA*,
*cyb*, and *sod* alleles were named using the scoring positions
and nomenclature described elsewhere.¹⁶⁻¹⁸ The SNPs
identified for the first instance in only one specimen were
confirmed by two independent PCR and sequencing runs.

See Online for appendix
<figure><img src="image_3.png" /><figcaption>**Figure 1: Study profile**
SOT=solid organ transplant.</figcaption></figure>

Multilocus genotypes (MLGs) were defined combining *mtLSUrRNA*, *cyb*, *sod*, and *impdh* alleles.

In addition, an analysis of the locus encoding the dihydropteroate synthase, the target of sulphonamides, was done to search for potential cross-resistance between mycophenolic acid and sulphonamides in SOT recipients with PCP. The methods are detailed in the appendix (pp 2–3).

Mixed infections were defined by the presence of at least two strains of *P. jirovecii* characterised by different genotypes in a single sample. The visualisation of more than one peak on the sequence electropherograms of the two strands at, at least, one nucleotide position, indicated the presence of two strains or more in the sample.

Relatedness between MLGs was evaluated with the minimum spanning tree method using GrapeTree (version 1.5.0), a free web browser application implementing Edmonds' algorithm.¹⁹ Branch length corresponds to the number of allelic mismatches among MLG profiles. One minimum spanning tree was based on underlying condition and another on mycophenolic acid exposure.

The discriminatory power of the MLST scheme was assessed with the Hunter index. The Hunter index was determined based on genotyping results obtained from the control group of patients who were not epidemiologically linked.²⁰ An index value greater than 0.95 is necessary for an

efficient genotyping method (ie, there is a 95% chance that any two random unrelated strains will be distinguished based on the DNA sequences observed).¹⁶,²⁰

**Outcome**

The primary outcome was the detection of the G1020A mutation in the *impdh* gene. Secondary outcomes included the detection of a specific MLG associated with the G1020A mutation and of an association of this mutation with mycophenolic acid exposure at the time of PCP diagnosis.

**Statistical analysis**

Statistical analyses were done with the Prism software (version 9.4.0, GraphPad Software). Categorical variables were compared between SOT recipient group and control group using Fisher's exact test or $\chi^2$ test as appropriate (appendix p 3). A multivariable logistic regression was done to assess the relationship between the G1020A mutation in the *impdh* gene and the following variables: age, involvement in a cluster, mycophenolic acid exposure at the time of PCP diagnosis, PCP prophylaxis, and clinical outcome. A complete case analysis was decided since the levels of missingness were low, with 5.13% of cases dropped out from the overall sample size due to missingness in at least one variable included in the multivariable regression model. Odds ratio (OR) with 95% CIs were reported. p<0.05 was considered statistically significant. Patients colonised by *P. jirovecii* were not included in statistical analyses.

**Role of the funding source**

The funders of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report.

**Results**

Of the 120 patients diagnosed with PCP over a 21-year period (Jan 1, 2001, to Dec 31, 2021), 117 were included (figure 1). Patients' characteristics are described in table 1 and in the appendix (pp 5–9). A first patient group consisted of 58 SOT recipients (T1–T58), 44 (75.9%) of whom were treated with mycophenolic acid at the time of PCP diagnosis. 21 patients from nine centres (Amiens, Brest, Dijon, Paris-Bichat, Poitiers, Reims, Rennes, Strasbourg, and Tours) were involved in clusters (figure 2). We included one to five patients involved in each cluster reported by our French colleagues at Amiens, Dijon, Paris-Bichat, Poitiers, Reims, Rennes, Strasbourg, and Tours since *P. jirovecii* DNA extracts were not available for all patients or their amount or quality were insufficient, and since *P. jirovecii* genotyping done as a preliminary exploration to explore the clusters found a unique genotype in all patients involved in a cluster from a given centre.³,¹²,²¹,²² Another group consisted of 59 non-SOT recipients not treated with mycophenolic acid at the time of PCP diagnosis and not involved in clusters
<table><thead><tr><th></th><th>SOT recipients<br>(n=58)</th><th>Control<br>(n=59)</th></tr></thead><tbody><tr><td><strong>Age, years</strong></td><td>63.0 (54.0-68.0)</td><td>53.0 (39.0-62.5)</td></tr><tr><td><strong>Sex</strong></td><td></td><td></td></tr><tr><td>Male</td><td>37 (64%)</td><td>41 (69%)</td></tr><tr><td>Female</td><td>21 (36%)</td><td>18 (31%)</td></tr><tr><td><strong>Pulmonary specimens</strong></td><td></td><td></td></tr><tr><td>Bronchoalveolar lavage</td><td>51 (88%)</td><td>50 (85%)</td></tr><tr><td>Sputum</td><td>4 (7%)</td><td>6 (10%)</td></tr><tr><td>Bronchial aspiration</td><td>2 (3%)</td><td>3 (5%)</td></tr><tr><td>Oropharyngeal lavage</td><td>1 (2%)</td><td>0</td></tr><tr><td><strong>Period of PCP diagnosis</strong></td><td>2001-20</td><td>2007-21</td></tr><tr><td><strong>Risk factors for PCP</strong></td><td></td><td></td></tr><tr><td>Renal transplant recipient</td><td>47 (81%)</td><td>0</td></tr><tr><td>Heart transplant recipient</td><td>5 (9%)</td><td>0</td></tr><tr><td>Liver transplant recipient</td><td>5 (9%)</td><td>0</td></tr><tr><td>Renal transplant recipient plus<br>heart transplant recipient</td><td>1 (2%)</td><td>0</td></tr><tr><td>HIV</td><td>0</td><td>31 (53%)</td></tr><tr><td>Haematological malignancy</td><td>0</td><td>9 (15%)</td></tr><tr><td>Solid cancer</td><td>0</td><td>7 (12%)</td></tr><tr><td>Immunosuppressive therapy*</td><td>0</td><td>9 (15%)</td></tr><tr><td>Haematological stem-cell<br>transplant</td><td>0</td><td>2 (3%)</td></tr><tr><td>Other</td><td>0</td><td>1 (2%)†</td></tr><tr><td><strong>Interval between<br>transplantation and PCP<br>diagnosis, months</strong></td><td>45 (14-128)</td><td>Not applicable</td></tr><tr><td><strong>MPA exposure at the time of PCP diagnosis</strong></td><td></td><td></td></tr><tr><td>Yes</td><td>44 (76%)</td><td>0</td></tr><tr><td>No</td><td>14 (24%)‡</td><td>59 (100%)§</td></tr><tr><td><strong>PCP prophylaxis</strong></td><td></td><td></td></tr><tr><td>Yes</td><td>4 (7%)¶</td><td>3 (5%)||</td></tr><tr><td>No</td><td>50 (86%)</td><td>54 (92%)</td></tr><tr><td>Unknown</td><td>4 (7%)</td><td>2 (3%)</td></tr><tr><td><strong>First-line PCP treatment**</strong></td><td></td><td></td></tr><tr><td>Cotrimoxazole</td><td>52 (90%)</td><td>54 (92%)</td></tr><tr><td>Atovaquone</td><td>4 (7%)</td><td>3 (5%)</td></tr><tr><td>Atovaquone plus caspofungin</td><td>0 (0%)</td><td>1 (2%)</td></tr><tr><td>Pentamidine</td><td>1 (2%)</td><td>0</td></tr><tr><td>None</td><td>0</td><td>1 (2%)††</td></tr><tr><td>Unknown</td><td>1 (2%)</td><td>0 (0%)</td></tr><tr><td><strong>Outcome</strong></td><td></td><td></td></tr><tr><td>Improvement</td><td>43 (74%)</td><td>52 (88%)</td></tr><tr><td>Deterioration</td><td>15 (26%)</td><td>7 (12%)</td></tr><tr><td><strong>Centres that included patients</strong></td><td></td><td></td></tr><tr><td>Amiens</td><td>3 (5%)</td><td>3 (5%)</td></tr><tr><td>Angers</td><td>1 (2%)</td><td>1 (2%)</td></tr><tr><td>Besançon</td><td>1 (2%)</td><td>1 (2%)</td></tr><tr><td>Brest</td><td>15 (26%)</td><td>16 (27%)</td></tr><tr><td>Caen</td><td>2 (3%)</td><td>2 (3%)</td></tr><tr><td>Clermont-Ferrand</td><td>4 (7%)</td><td>4 (7%)</td></tr><tr><td>Paris-Créteil</td><td>1 (2%)</td><td>1 (2%)</td></tr><tr><td>Dijon</td><td>2 (3%)</td><td>1 (2%)</td></tr><tr><td>Grenoble</td><td>1 (2%)</td><td>0</td></tr><tr><td>Lille</td><td>2 (3%)</td><td>2 (3%)</td></tr><tr><td>Limoges</td><td>1 (2%)</td><td>1 (2%)</td></tr><tr><td>Lyon</td><td>1 (2%)</td><td>1 (2%)</td></tr></tbody></table>

(Table 1 continues in next column)

<table><thead><tr><th></th><th>SOT recipients<br>(n=58)</th><th>Control<br>(n=59)</th></tr></thead><tbody><tr><td>(Continued from previous column)</td><td></td><td></td></tr><tr><td>Montpellier</td><td>1 (2%)</td><td>1 (2%)</td></tr><tr><td>Nancy</td><td>0</td><td>2 (3%)</td></tr><tr><td>Nantes</td><td>1 (2%)</td><td>1 (2%)</td></tr><tr><td>Nice</td><td>1 (2%)</td><td>1 (2%)</td></tr><tr><td>Paris-Bichat</td><td>3 (5%)</td><td>1 (2%)</td></tr><tr><td>Paris-La Pitié-Salpêtrière</td><td>2 (3%)</td><td>2 (3%)</td></tr><tr><td>Poitiers</td><td>2 (3%)</td><td>1 (2%)</td></tr><tr><td>Reims</td><td>2 (3%)</td><td>1 (2%)</td></tr><tr><td>Rennes</td><td>3 (5%)</td><td>7 (12%)</td></tr><tr><td>Rouen</td><td>1 (2%)</td><td>2 (3%)</td></tr><tr><td>Saint-Etienne</td><td>1 (2%)</td><td>2 (3%)</td></tr><tr><td>Strasbourg</td><td>2 (3%)</td><td>1 (2%)</td></tr><tr><td>Toulouse</td><td>2 (3%)</td><td>2 (3%)</td></tr><tr><td>Tours</td><td>2 (3%)</td><td>1 (2%)</td></tr><tr><td>Bern</td><td>1 (2%)</td><td>1 (2%)</td></tr></tbody></table><p>Data are median (IQR) or n (%). Individual patient data are reported in the appendix (pp 5-9). MPA=mycophenolic acid. PCP=Pneumocystis pneumonia. SOT=solid organ transplant. *Immunosuppressants other than MPA. †Bronchial aspiration in a haemorrhagic stroke patient. ‡Eight patients had a history of MPA exposure, MPA was discontinued before PCP diagnosis (median 11-5 months [IQR 6-75-25-25]); four patients did not have any history of MPA exposure; and data are missing for two patients. §No patient had a history of exposure to MPA. ¶Two patients received atovaquone; one patient received cotrimoxazole; and one patient received pentamidine aerosols. ||Two patients received cotrimoxazole; and one patient received atovaquone. **Treatment doses for the first-line PCP treatment were not available. ††This patient died of PCP before treatment began.</p><p><strong>Table 1: Characteristics of 58 SOT recipients and 59 non-SOT recipients (control group) for whom Pneumocystis jirovecii specimens were examined by multilocus sequence typing analysis</strong></p>

(control group, C1–C59). The 26 patients (ten SOT recipients and 16 control patients) from Brest who were enrolled in our previous study on *impdh* gene analysis⁶ were also included in the present study.

In addition, eight patients colonised by *P. jirovecii* (seven SOT recipients and one patient with a nephrotic syndrome [median age 56 years; IQR 53–71]; six men and two women) monitored in three centres (Brest, Paris-Bichat, and Reims) were included. Individual patients’ characteristics are described in the appendix (p 10). Seven of eight patients were treated with mycophenolic acid at the time of *P. jirovecii* detection.

The results of genotyping are shown in table 2 and in the appendix (pp 3–4, 13–25, 31–32). Polymorphism analysis of the *impdh* locus rendered it possible to identify nine SNPs (table 2; appendix pp 18–22). Four SNPs, G914A, G1020A, C1269T, and A1329T, have already been identified in our previous study.⁶ G1020A was the missense mutation corresponding to the Ala261Thr change that we previously described in ten mycophenolic acid-treated SOT recipients.⁶ This SNP was identified herein in specimens from a total of 40 SOT recipients (including the aforementioned ten mycophenolic acid-treated SOT recipients), 37 (84.1%) of whom were treated with mycophenolic acid at the time of PCP diagnosis. Five other SNPs were newly identified: G1020T, C1092A, C1092G, A1266G, and C1386A. C1092A, C1092G, A1266G and C1386A are silent mutations,
<figure><img src="image_4.png" /><figcaption>**Figure 2:** Distribution of *Pneumocystis MLG-31* and *MLG-34* in the centres that participated in the study
MLG=multilocus genotype. PCP=Pneumocystis pneumonia. SOT=solid organ transplant.</figcaption></figure>

whereas G1020T corresponds to a missense mutation Ala261Ser and was identified in *Pjirovecii* specimens from three SOT recipients, two of whom were treated with mycophenolic acid at the time of PCP diagnosis.

Ten *impdh* alleles, named from *IMPDH-A* to *IMPDH-J*, were defined based on these polymorphisms (table 2), *IMPDH-B* being the most frequent allele in the SOT recipient group (40 [68.9%] of 58) and *IMPDH-A* being the most frequent allele in the control group (38 [64.4%] of 58; table 2). Only two alleles (*IMPDH-A* and *IMPDH-C*) were shared by the two groups (table 2). Alleles *IMPDH-D*, *IMPDH-E*, *IMPDH-G*, and *IMPDH-J* were identified only in the control group. Alleles *IMPDH-B*, *IMPDH-F*, *IMPDH-H*, and *IMPDH-I* were identified only in the SOT recipient

group. The presence of more than one allele indicating mixed infections was observed in 24 patients: four (6.9%) of 58 SOT recipients and 20 (33.9%) of 59 patients in the control group (p<0.0003, $\chi^2$ test; appendix pp 18–22).

*IMPDH-B* allele is characterised by the missense mutation G1020A (Ala261Thr). *IMPDH-F* is characterised by the missense mutation G1020T (Ala261Ser). These two alleles were only detected in the SOT recipient group; specifically, *IMPDH-B* was detected in 40 patients (37 of whom were treated with mycophenolic acid at the time of PCP diagnosis) from 15 of 26 centres across France and *IMPDH-F* was detected in three patients (two of whom were treated with mycophenolic acid at the time of PCP diagnosis) from two of 26 centres, respectively (figure 2).
IMPDH-B allele was detected in 37 (84.1%) of 44 SOT recipients treated with mycophenolic acid, whereas it was detected in only three (21.4%) of 14 SOT recipients not treated with mycophenolic acid at the time of PCP diagnosis, and in none of the control patients. Two of these three patients who were not treated with mycophenolic acid at the time of PCP diagnosis had a previous exposure to this drug, which had been discontinued 2 and 15 months, respectively, before PCP diagnosis; the third patient had no history of mycophenolic acid exposure. Moreover, the IMPDH-B allele was identified in 19 (90.5%) of 21 SOT recipients involved in clusters, whereas it was identified in 21 (56.7%) of 37 SOT recipients not involved in clusters (appendix pp 5–9, 18–22). The two SOT recipients involved in clusters who did not harbour the IMPDH-B allele harboured the IMPDH-F allele (appendix pp 5–9, 18–22).

The multivariable analysis showed that IMPDH-B was associated with mycophenolic acid exposure at the time of PCP diagnosis (OR 73.61 [95% CI 17.41–455.70]; p<0.0001), and involvement in a cluster (OR 12.77 [1.58–171.90]; p=0.029) whereas it was not associated with age, PCP prophylaxis, and clinical deterioration (table 3). IMPDH-B allele was identified in the seven patients colonised by *P. jirovecii* and treated with mycophenolic acid, but not in the patient colonised by *P. jirovecii* and not treated with mycophenolic acid (appendix pp 10, 31–32).

44 MLGs combining *mtLSUrRNA*, *cyb*, *sod*, and *impdh* alleles (numbered from MLG-1 to MLG-44) were identified. MLGs were not identified in 31 patients since they harboured a mix of alleles for two or more loci. The Hunter index was evaluated at 0.993, showing the high discriminatory power (>0.95) of this MLST scheme (appendix p 27).

A higher proportion of mixed infections was observed in the control group (29 [49.1%] of 59) than in the SOT recipient group (six [10.3%] of 58; p<0.0001; $\chi^2$ test). Only one MLG, MLG-8, was shared by both groups, suggesting that the two groups were infected with distinct *P. jirovecii* populations (appendix p 27). Fifteen MLGs were identified in the SOT recipient group. MLG-31, corresponding to the association of alleles *mtLSU4*, *CYB2*, *SOD1*, and *IMPDH-B*, was the most frequent, was exclusively identified in the SOT recipient group (38 [65.5%] of 58 vs none of 59 in the control group; p<0.0001, $\chi^2$ test), and was identified in 19 (90.5%) of 21 patients involved in clusters, whereas it was identified in 19 (51.4%) of 37 patients not involved in clusters (p=0.0026; $\chi^2$ test). Each of the other MLGs was identified in three patients at most. Only two MLGs were identified in the patients involved in clusters: the aforementioned MLG-31 (19 patients) and MLG-34, corresponding to the association of the *mtLSU2*, *CYB2*, *SOD1*, and *IMPDH-F* alleles (two patients).

The MLG distribution was heterogeneous in the control group, with 30 different MLGs identified, each observed in one or two patients. These results are consistent with greater diversity in the control group than in the SOT recipient group. Minimum spanning trees were used to depict the MLG distribution and highlight a major group consisting of

<table><caption>Table 2: Pneumocystis *impdh* alleles and their distribution in the solid organ transplant recipient group and the control group</caption><thead><tr><th rowspan="2"></th><th colspan="7">Nucleotide position (base pairs)</th><th rowspan="2">Recipients of solid organ transplant (n=58)</th><th rowspan="2">Patients in the control group (n=59)</th></tr><tr><th>914</th><th>1020</th><th>1092</th><th>1266</th><th>1269</th><th>1329</th><th>1386</th></tr></thead><tbody><tr><td>IMPDH-A</td><td>G</td><td>G</td><td>C</td><td>A</td><td>C</td><td>A</td><td>C</td><td>12 (21%)</td><td>38 (64%)</td></tr><tr><td>IMPDH-B</td><td>G</td><td>A</td><td>C</td><td>A</td><td>C</td><td>A</td><td>C</td><td>40 (69%)</td><td>0</td></tr><tr><td>IMPDH-C</td><td>G</td><td>G</td><td>C</td><td>A</td><td>C</td><td>T</td><td>C</td><td>4 (7%)</td><td>21 (36%)</td></tr><tr><td>IMPDH-D</td><td>G</td><td>G</td><td>C</td><td>A</td><td>T</td><td>A</td><td>C</td><td>0</td><td>6 (10%)</td></tr><tr><td>IMPDH-E</td><td>G</td><td>G</td><td>C</td><td>A</td><td>C</td><td>A</td><td>A</td><td>0</td><td>3 (5%)</td></tr><tr><td>IMPDH-F</td><td>G</td><td>T</td><td>C</td><td>A</td><td>C</td><td>A</td><td>C</td><td>3 (5%)</td><td>0</td></tr><tr><td>IMPDH-G</td><td>G</td><td>G</td><td>C</td><td>G</td><td>C</td><td>A</td><td>C</td><td>0</td><td>5 (8%)</td></tr><tr><td>IMPDH-H</td><td>G</td><td>G</td><td>A</td><td>A</td><td>C</td><td>A</td><td>A</td><td>1 (2%)</td><td>0</td></tr><tr><td>IMPDH-I</td><td>G</td><td>G</td><td>G</td><td>A</td><td>C</td><td>T</td><td>C</td><td>2 (3%)</td><td>0</td></tr><tr><td>IMPDH-J</td><td>A</td><td>G</td><td>C</td><td>A</td><td>C</td><td>T</td><td>C</td><td>0</td><td>6 (10%)</td></tr></tbody></table>

<table><caption>Table 3: Multivariable analysis of factors associated with the IMPDH-B allele</caption><thead><tr><th></th><th>Adjusted odds ratio (95% CI)</th><th>p value</th></tr></thead><tbody><tr><td>Age group, years</td><td></td><td></td></tr><tr><td>20-39</td><td>0.25 (&lt;0.01 to 7.67)</td><td>0.48</td></tr><tr><td>40-49</td><td>0.12 (&lt;0.01 to 2.65)</td><td>0.21</td></tr><tr><td>50-59</td><td>1.00 (0.11 to 9.34)</td><td>&gt;0.99</td></tr><tr><td>60-69</td><td>1.69 (0.23 to 14.01)</td><td>0.61</td></tr><tr><td>Involved in case cluster</td><td>12.77 (1.58 to 171.90)</td><td>0.029</td></tr><tr><td>Clinical deterioration as an outcome</td><td>1.26 (0.19 to 8.05)</td><td>0.80</td></tr><tr><td>Mycophenolic acid exposure at the time of Pneumocystis pneumonia diagnosis</td><td>73.61 (17.41 to 455.70)</td><td>&lt;0.0001</td></tr><tr><td>Prophylaxis</td><td>0.42 (0.02 to 9.25)</td><td>0.59</td></tr></tbody></table>

SOT recipients infected with MLG-31 (figure 3A) and treated with mycophenolic acid (figure 3B).

*P. jirovecii* MLST was successful in four colonised patients (two patients from Reims, one patient from Brest, and one patient from Paris-Bichat). These patients harboured MLG-31, the same major genotype identified in SOT recipients with PCP (appendix p 32).

Dihydropteroate synthase locus analysis of *P. jirovecii* specimens was successful in 47 (81.0%) of 58 SOT recipients (appendix p 4). A wild type was identified in these 47 patients, 38 (80.9%) of whom harboured MLG-31, three (6.4%) harboured MLG-34, and six (12.8%) harboured other different MLGs.

Discussion

The G1020A (Ala261Thr) missense mutation previously described in *P. jirovecii* specimens from only ten mycophenolic acid-treated SOT recipients from a single centre,⁶ was observed here in 30 additional SOT recipients (27 treated with mycophenolic acid) from 15 of 26 participating French health centres. This mutation was associated with exposure to mycophenolic acid. Another missense mutation, G1020T (Ala261Ser), was identified in the *impdh* gene of *P. jirovecii* from three SOT recipients, two of whom had been treated with mycophenolic acid at the time of PCP diagnosis. Interestingly, the
<figure><img src="image_5.png" /></figure>

(Figure 3 continues on next page)

IMPDH of *Penicillium brevicompactum* (PbIMPDHA), *Saccharomyces cerevisiae* (ScIMD2), and *Meyerozyma guilliermondii* (MgIMD3.2), which have intrinsic resistance to mycophenolic acid, harbours the same Ala to Ser substitution at the analogous position as the substitutions we observed in *P. jirovecii*.⁹,²³,²⁴ Consequently, the Ala261Ser substitution could represent, like the Ala261Thr substitution, a signature of mycophenolic acid exposure.

The major MLG-31 was exclusively identified in SOT recipients. The MLST approach provided molecular evidence for infection occurrence in SOT recipients caused by a specific *P. jirovecii* strain. The presence of this specific strain is unlikely to result from a casual event since it was maintained in the SOT recipient population for at least 15 years (ie, from 2006, when PCP was diagnosed in patient T27, to 2020, when PCP was diagnosed in patient T52; appendix pp 8–9). The hypothesis that specific *P. jirovecii* strains infected SOT recipients, specifically renal transplant

recipients, was previously postulated based on the results of different genotyping methods, which did not include *impdh* gene analysis (ie, analysis of six Short Tandem Repeat markers²⁵ and restriction fragment length polymorphism analysis of the multicopy major surface glycoprotein [msg] family).⁴

We showed that 19 of 21 patients involved in eight PCP case clusters in France (Amiens, Brest, Dijon, Paris, Poitiers, Reims, Rennes, and Tours) included patients infected with MLG-31 (figure 2; appendix pp 5–9, 23–24). These findings provide evidence for a link between solid organ transplantation, specific *P. jirovecii* strains, and PCP case clusters.

Similarly, Sassi and colleagues⁴ showed that two clusters that occurred among renal transplant recipients in Munich (Germany) and Zurich (Switzerland) were effectively due to an identical common strain of *P. jirovecii* harbouring the same msg-restriction fragment length polymorphism genotype. These two clusters were initially investigated with an MLST method by Schmoldt and colleagues²⁶ and
<figure><img src="image_6.png" /><figcaption>**Figure 3:** Minimum spanning tree analysis of 44 MLGs from 86 *Pneumocystis jirovecii* specimens (86 patients) harbouring a unique genotype (one allele per marker) or multiple genotypes (multiple alleles in one marker)</figcaption></figure>

(A) Underlying conditions of patients. (B) Exposure to MPA. Each circle corresponds to one MLG. The size of the circles is correlated with the number of specimens which have the indicated MLG. The branch lengths between each circle correspond to the number of allelic mismatches between the two linked MLGs: the longer the branch, the more different the genotypes. The colour of the circles indicates the underlying conditions of patients (A) or the exposure to MPA (B). The number of allelic mismatches among MLGs was used as distance. MLG=multilocus genotype. MPA=mycophenolic acid.

Gianella and colleagues,²⁷ respectively. Combining the results of Sassi and colleagues⁴ with those of Schmoldt and colleagues²⁶ and of Gianella and colleagues,²⁷ Hauser and colleagues⁵ showed that the main *P. jirovecii* genotype involved in both Munich and Zurich was the same as another cluster in Frankfurt (Germany). Conversely, they showed that the main genotype involved in a cluster that occurred from 1994 to 1996 in Lyon (France)²⁸ was different.⁵

We identified two MLGs, MLG-31 and MLG-34, in patients involved in clusters, with MLG-31 being the most widespread (identified in eight French cities; figure 2; appendix p 30). MLG-34, characterised by the mutation G1020T (Ala261Ser), was identified in a cluster in Strasbourg, eastern France. Although the typing method was not strictly identical, it is noteworthy that the MLG identified in

Strasbourg was characterised by the same *mtLSUrRNA* allele (*mtLSU2*) as the MLG identified in Germany (Munich and Frankfurt) and Switzerland (Zurich).⁴,⁵,²⁶,²⁷ Thus, the strain characterised by MLG-34 might also be involved in the German and Swiss clusters. The relative geographical proximity between Munich, Frankfurt, Zurich, and Strasbourg, combined with the *P. jirovecii* MLGs identified in these cities, is compatible with a potential relationship between geographical location of the patients and the *P. jirovecii* strains that they carried.

Our results combined with literature analysis suggest that at least the two main *impdh* mutants (MLG-31 and MLG-34) might be circulating in SOT recipients.⁴,⁵,²⁶,²⁷

The presence of an identical *P. jirovecii* strain involved in a cluster can be explained by airborne interindividual transmission. However, the presence of identical strains in
SOT recipients in 15 centres was unexpected but might be partly explained by effective mycophenolic acid pressure, which favoured the circulation of specific strains characterised by mutations in the *impdh* gene. The possibility of spread of a specific strain initially selected by mycophenolic acid treatment from a hospital centre or a region, possibly from Dijon (Burgundy) to hospital centres in other regions (appendix p 29), is difficult to verify.

Behavioural factors of SOT recipients colonised by *P. jirovecii* might also contribute to the spread of particular strains or genotypes in different geographical regions. Patients colonised by *P. jirovecii* are assumed to be paucisymptomatic, and widely present in the community. These patients colonised by *P. jirovecii* have been identified as possible infectious sources in the context of PCP case clusters in hospitals. However, they are usually undiagnosed in the community.²¹,²² Moreover, patients colonised by *P. jirovecii* might represent a part of the human reservoir of the fungus, and *P. jirovecii* acquisition and transmission might mainly occur in the community.²⁹ The overall number of SOT recipients in France in 2021 was about 65 952 including 42 117 renal transplant recipients.³⁰ SOT recipients are supported by associations that organise cultural, sporting, and social events at local, regional, and national levels. Moreover, the total number of patients with chronic renal failure is about 50 501 (latest updated data in 2019),³¹ 1200 of whom are renal transplant recipients. There are about 400 dialysis centres in France and patients receive dialysis weekly and are also organised in associations. Thus, there are networks that promote encounters between SOT, particularly renal transplant recipients, and these encounters might facilitate *P. jirovecii* transmission, as preventive measures such as mask wearing, physical distancing, and chemoprophylaxis beyond the 6–12 months after the transplant are rarely applied; however, this possibility remains an hypothesis that needs to be explored.² In this study, we examined *P. jirovecii* specimens from eight colonised patients (seven SOT recipients and one dialysis patient) and found that four patients harboured MLG-31, the same major genotype identified in SOT recipients with PCP. This result is consistent with a possible contribution of colonised SOT recipients in the transmission of specific *P. jirovecii* strains selected under mycophenolic acid pressure.

Our study has some limitations. First, we did not do an exhaustive analysis of *P. jirovecii* specimens over the study period due to the retrospective design and the lack of availability of specimens of adequate quantity and quality for all cases of PCP in France and Switzerland. Therefore, we might have missed centres where the mutant strains could have circulated. Second, centres from only two countries participated to this study. A prospective study involving centres from other countries in Europe or in other continents would enable a more comprehensive characterisation of the circulation of specific *P. jirovecii* *impdh* mutant strains in SOT recipients. Third, the fact that we did not

identify *impdh* mutant strains in control patients might be due to the lower sensitivity of Sanger sequencing compared with ultradeep sequencing to detect minor genotypes.²⁵,³² However, we later developed a melting-qPCR assay that is capable of detecting and identifying *impdh* mutant strains at a high resolution³³ and is more sensitive than Sanger sequencing for the detection of co-infections. The approach can distinguish mutant strains in a mixture consisting of 10% mutant and 90% wild strains (unpublished data). Fourth, despite the excellent discriminatory power of our genotyping method, we cannot rule out that the 38 strains that we identified as MLG-31 might differ in other genomic markers that we did not examine. A whole nuclear or mitochondrial genome sequencing approach could provide additional information. However, whole-genome sequencing is time-consuming, expensive, and difficult to implement for an uncultivable microorganism, and requires a large amount of DNA. Finally, we cannot rule out that the differences observed in MLG distribution between groups might be attributable to patient or clinical characteristics, such as race, ethnicity, or other factors that we did not consider.

Despite these limitations, our results suggest that SOT recipients in France are primarily infected with two *P. jirovecii* strains selected under mycophenolic acid pressure and presenting a selective advantage. This selection pressure and the role of colonised SOT recipients might explain their persistence, and their involvement in PCP case clusters in transplantation units.

**Pneumocystis IMPDH Study Group**

Anne Totet, Hakim Mazouz, Jean-Philippe Bouchara, Virginie Besson, Emeline Scherer, Didier Ducloz, Konrad Mühlethaler, Pierre L Bonnet, Julie Bonhomme, Thierry Lobbedez, Valérie Chatelet, Céline Nourrisson, Cyril Garrouste, Françoise Botterel, Marie Matignon, Frédéric Dalle, Claire Tinel, Danièle Maubon, Johan Noble, Boualem Sendid, Jordan Leroy, François Provot, Marie-Fleur Durieux, Clément Danthu, Jean Menotti, François Bailly, Christophe Ravel, Moglie Le Quintrec, Anne Debourgogne, Marc Ladrière, Florent Morio, Magali Giral, Lilia Hasseine, Laetitia Albano, Nicolas Argy, Richard Dorent, Arnaud Fekkar, Valérie Pourcher, Estelle Cateau, Antoine Thierry, Antoine Huguenin, Alain Wynckel, Florence Robert-Gangneux, Elsa Vabret, Damien Costa, Dominique Bertrand, Pierre Flori, Emmanuelle Tavernier, Julie Brunet, Laura Braun, Xavier Iriart, Arnaud Del Bello, Guillaume Desoubeaux, Matthias Buchler, Natalja Kulagina, Vincent Courdavault.

**Contributors**

SLG, GN, and CVH conceived and designed the trial. SLG, GN, and CVH verified, and oversaw analysis of the data. NLN contributed to laboratory processing. SLG, GN, and CVH wrote the manuscript. SLG and GN contributed equally. All other authors contributed equally to the implementation of the study, inclusion of patients, or data collection. All authors critically reviewed and approved the final version and had final responsibility for the decision to submit for publication. According to ethics committee recommendations, the scientific research manager (GN) and people responsible for data collection, processing, and analysis (SLG and CVH) had full access to all the data in the study.

**Declaration of interests**

We declare no competing interests. This study was supported by the French Speaking Society for Transplantation and Chiesi. GN and SLG are presently supported by the European Commission (COST Action CA23142 - Delve-into-Pneumocystis). NP are presently supported by the Agence Nationale pour la Recherche (Projet-ANR-24-CE15-2995).
Data sharing

All *impdh* sequence data are available on the GenBank database (accession numbers OP020933 to OP021049). The partial *mtLSUrRNA, cyb*, and *sod* sequences have been deposited in GenBank (accession numbers: PP740517 to PP740633, PP736202 to PP736318, and PP719700 to PP719816, respectively). Associated metadata can be found in the appendix (pp 13–22).

Acknowledgments

We thank Emmanuel Nowak for helpful discussion. This study was
presented in part during the American Transplant Congress in San Diego,
California, on June 3–7, 2023 (abstract number 7943, DLB011), and the 16th
International Workshop on Opportunistic Protists in Lausanne, Switzerland,
on Aug 25–26, 2023 (https://doi.org/10.5281/zenodo.8421509).

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