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Genetic Polymorphisms of Pneumocystis jirovecii in HIV-Positive and HIV-Negative Patients in Northern China

Ting Xue
Ting Xue
, 
Wei-Qin Du
Wei-Qin Du
, 
Wen-Juan Dai
Wen-Juan Dai
, 
Yi-Shan Li
Yi-Shan Li
, 
Shu-Feng Wang
Shu-Feng Wang
, 
Jun-Ling Wang
Jun-Ling Wang
 y 
Xin-Ri Zhang
Xin-Ri Zhang
   | 23 feb 2022
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Article Category: Original Paper
Publicado en línea: 23 feb 2022
Páginas: 27 - 34
Recibido: 18 sept 2021
Aceptado: 15 dic 2021
DOI: https://doi.org/10.33073/pjm-2022-002
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© 2022 Ting Xue et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Fig. 1

Identification of Pneumocystis jirovecii using GMS staining methods. Cysts appear as brown or puce spheres or ovoids with a small black stick inside (arrows). The reddish background instead of the typical greenish background is most likely due to periodic acid treatment and without light green counterstaining in our staining method.
Identification of Pneumocystis jirovecii using GMS staining methods. Cysts appear as brown or puce spheres or ovoids with a small black stick inside (arrows). The reddish background instead of the typical greenish background is most likely due to periodic acid treatment and without light green counterstaining in our staining method.

Genotypes of Pneumocystis jirovecii detected at eight genetic loci.

Patient No. HIV1/2 anti-body Genotypes at 8 loci
ITS sod dhfr dhps cyb mtLSU rRNA β-Tub 26S rRNA
SX_0001 +/− ITS2 + ITS59 sod1 dhfr312 WT cyb7 mt3 β-Tub1 + β-Tub2 26S2
SX_0002 +/− ITS4 sod1 dhfr312 WT cyb2 mt3 β-Tub2 26S2
SX_0003 −/− ITS10 + ITS16 sod1 + sod2 dhfr312 WT cyb1 + cyb8 mt1 + mt 2 β-Tub1 + β-Tub2 26S3 + 26S4

Nucleotide polymorphic sites and number of plasmid clones sequenced at eight distinct loci of Pneumocystis jirovecii.

Locus Genotypesa Locationb No. of plasmid clones sequenced
SX_0001 SX_0002 SX_0003
ITS ITS 4 KC470776 0 12 0
ITS 10 JQ365725 0 0 4
ITS 16 AB469817 0 0 8
ITS 22 KC470795 6 0 0
ITS 59 MK300661 10 0 0
sod sod 1 110C/215T 11 13 8
sod 2 110T/215C 0 0 2
dhps dhps WT 165A (55Thr) / 171C (57Pro) 12 12 12
dhps dhfr312 312C (117Gly) 12 11 11
cyb cyb 1 279C/348A/516C/547C/566C/838C 0 0 6
cyb 2 279C/348A/516C/547C/566C/838T 0 8 0
cyb 7 279C/348A/516C/547C/566T/838C 9 0 0
cyb 8 279T/348A/516C/547C/566C/838C 0 0 3
mt LSU rRNA mt1 85C/248C 0 0 2
mt2 85A/248C 0 0 8
mt3 85T/248C 10 10 0
β-Tub β-Tub 1 86G/281A 8 0 6
β-Tub 2 86G/281G 4 12 5
26S rRNA 26S 2 86T/290A 12 11 0
26S 3 86C/290A 0 0 5
26S 4 86A/290A 0 0 6

Multi-locus sequence type (MLST) profiles of P. jirovecii from PCP patients in this study in comparison with known P. jirovecii MLST profiles in Fungal MLST Database.

MLST types* β-Tub cyb 26S rRNA sod Patient no.
3 1 1 4 2 SX_0003
8 2 8 4 1 SX_0003
13 1 1 4 1 SX_0003
15 1 8 4 1 SX_0003
19 1 8 3 2 SX_0003
21 2 1 3 1 SX_0003
22 2 1 3 2 SX_0003
23 2 1 4 1 SX_0003
35 2 7 2 1 SX_0001
51 1 7 2 1 SX_0001
52 2 2 2 1 SX_0002
NA 1 1 3 2 SX_0003
NA 2 8 3 1 SX_0003
NA 2 8 3 2 SX_0003
NA 2 1 4 2 SX_0003
NA 2 8 4 2 SX_0003
NA 1 1 3 1 SX_0003
NA 1 8 3 1 SX_0003
NA 1 8 4 2 SX_0003

PCR primers used in this study.

Genes (reference) Primer names and sequences (5′-3′) Size of nested PCR products (bp)
ITS (Lee et al. 1998) 1724F 5′-AAGTTGATCAAATTTGGTC-3′ITS2R 5′-CTCGGACGAGGATCCTCGCC-3′ITS1F 5′-CGTAGGTGAACCTGCGGAAGGATC-3′ITS2R1 5′-GTTCAGCGGGTGATCCTGCCTG-3′ 578
sod (Esteves et al. 2010b) MnSOD_Fw 5′-GGGTTTAATTAGTCTTTTTAGGCAC-3′MnSOD_Rw 5′-CATGTTCCCACGCATCCTAT-3′SODF3 5′-AGTCTTTTTAGGCACTTGAACCT-3′SODR4 5′-TCCAAGAATAACTTTGCCTTGAGT-3′ 560
dhfr (Lane et al. 1997) FR208 5′-GCAGAAAGTAGGTACATTATTACGAGA-3′FR1018 5′-AAGCTTGCTTCAAACCTTGTGTAACGCG-3′FR242 5′-GTTTGGAATAGATTATGTTCATGGTGTACG-3′FR1038 5′-GCTTCAAACCTTGTGTAACGCG-3′ 798
dhps (Ma et al. 1999) DHPS1 5′-CAAATTAGCGTATCGAATGACC-3′DHPS2 5′-GCAAAATTACAATCAACCAAAGTA-3′DHPS3 5′-AGCGCCTACACATATTATGG-3′DHPS4 5′-GTTCTGCAACCTCAGAACG-3′ 278
cyb (Esteves et al. 2010a) CytbFw 5′-CCCAGAATTCTCGTTTGGTCTATT-3′CytbRw 5′-AAGAGGTCTAAAAGCAGAACCTCAA-3′CytbF3 5′-TCTCGTTTGGTCTATTGGTG-3′CytbR4 5′-AAGCAGAACCTCAAATTCAAGATA-3′ 590
mtLSU rRNA (Wakefield 1996) pAZ102_E 5′-GATGGCTGTTTCCAAGCCCA-3′pAZ102_H 5′-GTGTACGTTGCAAAGTACTC-3′pAZ102_X 5′-GTGAAATACAAATCGGACTAGG-3′pAZ102_Y 5′-TCACTTAATATTAATTGGGGACC-3′ 252
β-Tub (Pasic et al. 2020) PneumoTub_F 5′-TCATTAGGTGGTGGAACGGG-3′PneumoTub_R 5′-ATCACCATATCCTGGATCCG-3′ 303
26S rRNA (Pasic et al. 2020) PneumoLSU_F 5′-TCAGGTCGAACTGGTGTACG-3′PneumoLSU_R 5′-TGTTCCAAGCCCACTTCTT-3′ 297

Clinical characteristics of patients with Pneumocystis jirovecii pneumonia.

Clinical information Patient No.
SX_0001 SX_0002 SX_0003
Age (years) 65 51 65
Sex Male Male Male
Underlying conditions NAa Hepatic cysts ILD
Thoracic HRCT findings GGO d + GGO + GGO +
HIV 1/2 antibody +/– +/– –/–
CD4 T-lymphocyte count (cells/µl) 232 176 NA
Serum parameters
1,3-β-D-glucan, normal < 10 pg/ml > 600 NA > 600
Lactate dehydrogenase, normal 120–250 U/l 432 699 9,734
C-reactive protein, normal 0–6 mg/l 73.63 129.17 340.00
Procalcitonin, normal 0–0.05 ng/ml 0.975 0.161 11.26
Partial pressure of oxygen, normal 80–110 mmHg 80 65 59.70
Erythrocyte sedimentation rate, normal 0–15 mm/h 61.10 60.80 47.30
Concurrent infection – – C.n. and B.c.b
Anti-PCP therapya – – –
HAART before PCP – – –
Clinical outcomes survived survived died
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