Atovaquone exposure and Pneumocystis jirovecii cytochrome b mutations: French data and review of the literature.

Pneumocystis jirovecii is a transmissible fungus responsible for severe pneumonia (Pneumocystis pneumonia [PCP]) in immunocompromised patients. Missense mutations due to atovaquone selective pressure have been identified on cytochrome b (CYB) gene of P. jirovecii. It was recently shown that atovaquone prophylaxis can lead to the selection of specific P. jirovecii CYB mutants potentially resistant to atovaquone among organ transplant recipients. In this context, our objectives were to provide data on P. jirovecii CYB mutants and the putative selective pressure exerted by atovaquone on P. jirovecii organisms in France. A total of 123 patients (124 P. jirovecii specimens) from four metropolitan hospitals and two overseas hospitals were retrospectively enrolled. Fourteen patients had prior exposure to atovaquone, whereas 109 patients did not at the time of P. jirovecii detection. A 638 base-pair fragment of the CYB gene of P. jirovecii was amplified and sequenced. A total of 10 single nucleotide polymorphisms (SNPs) were identified. Both missense mutations C431T (Ala144Val) and C823T (Leu275Phe), located at the Qo active site of the enzyme, were significantly associated with prior atovaquone exposure, these mutations being conversely incidental in the absence of prior atovaquone exposure (P < 0.001). Considering that the aforementioned hospitals may be representative of the national territory, these findings suggest that the overall presence of P. jirovecii CYB mutants remains low in France.

The mutations C431T (Ala144Val) and C823T (Leu275Phe) at the cytochrome b (CYB) active site of Pneumocystis jirovecii are associated with patient prior exposure to atovaquone. Conversely, these mutations are incidental in the absence of exposure. Overall, the presence of P. jirovecii CYB mutants remains low in France.

Apparent Absence of Selective Pressure on Pneumocystis jirovecii Organisms in Patients with Prior Methotrexate Exposure.

Pneumocystis jirovecii infections occur in patients treated with methotrexate (MTX) because of immunosuppressive effects of this highly potent dihydrofolate reductase (DHFR) inhibitor. Conversely, MTX may act as an anti-P. jirovecii drug and consequently may exert a selective pressure on this fungus. In this context, we compared the sequences of the dhfr gene of P. jirovecii isolates obtained from two groups of patients with P. jirovecii infections. The first group, with systemic diseases or malignancies, had prior exposure to MTX (21 patients), whereas the second group (22 patients), the control group, did not. Three single nucleotide polymorphisms (SNPs) were observed at positions 278, 312, and 381. The first one was located in the intronic region and the two others were synonymous. Based on these SNPs, three P. jirovecii dhfr alleles, named A, B, and C, were specified. Allele A was the most frequent, as it was observed in 18 patients (85.7%) and in 16 patients (72.7%) of the first and second groups, respectively. No significant difference in P. jirovecii dhfr gene diversity in the two patient groups was observed. In conclusion, these original results suggest that MTX does not exert an overt selective pressure on P. jirovecii organisms.

Selection of Pneumocystis jirovecii Inosine 5'-Monophosphate Dehydrogenase Mutants in Solid Organ Transplant Recipients: Implication of Mycophenolic Acid.

Mycophenolic acid (MPA) targets the inosine 5'-monophosphate dehydrogenase (IMPDH) of human lymphocytes. It is widely used as an immunosuppressant to prevent rejection in solid organ transplant (SOT) recipients who, incidentally, are at risk for Pneumocystis pneumonia (PCP). We hypothesized that MPA exerts selective pressure on P. jirovecii microorganisms considering its in vitro antifungal activity on other fungi. Thus, we analysed impdh gene in P. jirovecii isolates from SOT recipients. P. jirovecii specimens from 26 patients diagnosed with PCP from 2010 to 2020 were retrospectively examined: 10 SOT recipients treated with MPA and 16 non-SOT patients without prior exposure to MPA. The P. jirovecii impdh gene was amplified and sequenced. Nucleotide sequences were aligned with the reference sequences retrieved from available P. jirovecii whole genomes. The deduced IMPDH protein sequences were aligned with available IMPDH proteins from Pneumocystis spp. and other fungal species known to be in vitro sensitive or resistant to MPA. A total of nine SNPs was identified. One SNP (G1020A) that results in an Ala261Thr substitution was identified in all SOT recipients and in none of the non-SOT patients. Considering that IMPDHs of other fungi, resistant to MPA, harbour Thr (or Ser) at the analogous position, the Ala261Thr mutation observed in MPA-treated patients was considered to represent the signature of P. jirovecii exposure to MPA. These results suggest that MPA may be involved in the selection of specific P. jirovecii strains that circulate in the SOT recipient population.

Pneumocystis jirovecii Diversity in Réunion, an Overseas French Island in Indian Ocean.

Data on Pneumocystis jirovecii characteristics from the overseas French territories are still scarce whereas numerous data on P. jirovecii genotypes are available for metropolitan France. The main objective of the present study was to identify P. jirovecii multilocus genotypes in patients living in Réunion and to compare them with those identified using the same method in metropolitan France and in French Guiana. Archival P. jirovecii specimens from immunosuppressed patients, 16 living in Réunion (a French island of the Indian ocean), six living in French Guiana (a South-American French territory), and 24 living in Brest (Brittany, metropolitan France) were examined at the large subunit rRNA (mtLSUrRNA) genes, cytochrome b (CYB), and superoxide dismutase (SOD) genes using PCR assays and direct sequencing. A total of 23 multi-locus genotypes (MLG) were identified combining mtLSUrRNA, CYB, and SOD alleles, i.e., six in Reunionese patients, three in Guianese patients, and 15 in Brest patients. Only one MLG (mtLSU1-CYB1-SOD2) was shared by Reunionese and Guianese patients (one patient from each region) whereas none of the 22 remaining MLG were shared by the 3 patient groups. A total of eight MLG were newly identified, three in Réunion and five in Brest. These results that were obtained through a retrospective investigation of a relatively low number of P. jirovecii specimens, provides original and first data on genetic diversity of P. jirovecii in Réunion island. The results suggest that P. jirovecii organisms from Réunion present specific characteristics compared to other P. jirovecii organisms from metropolitan France and French Guiana.