1. bookVolume 71 (2022): Issue 1 (March 2022)
Journal Details
License
Format
Journal
eISSN
2544-4646
First Published
04 Mar 1952
Publication timeframe
4 times per year
Languages
English
access type Open Access

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

Published Online: 23 Feb 2022
Volume & Issue: Volume 71 (2022) - Issue 1 (March 2022)
Page range: 27 - 34
Received: 18 Sep 2021
Accepted: 15 Dec 2021
Journal Details
License
Format
Journal
eISSN
2544-4646
First Published
04 Mar 1952
Publication timeframe
4 times per year
Languages
English
Abstract

Pneumocystis jirovecii is an opportunistic fungus that can cause severe and potentially fatal Pneumocystis pneumonia (PCP) in immunodeficient patients. In this study, we investigated the genetic polymorphisms of P. jirovecii at eight different loci, including six nuclear genes (ITS, 26S rRNA, sod, dhps, dhfr and β-Tub) and two mitochondrial genes (mtLSU-rRNA and cyb) in three PCP cases, including two patients with HIV infection and one without HIV infection in Shanxi Province, P.R. China. The gene targets were amplified by PCR followed by sequencing of plasmid clones. The HIV-negative patient showed a coinfection with two genotypes of P. jirovecii at six of the eight loci sequenced. Of the two HIV-positive patients, one showed a coinfection with two genotypes of P. jirovecii at the same two of the six loci as in the HIV-negative patient, while the other showed a single infection at all eight loci sequenced. None of the three drug target genes (dhfr, dhps and cyb) showed mutations known to be potentially associated with drug resistance. This is the first report of genetic polymorphisms of P. jirovecii in PCP patients in Shanxi Province, China. Our findings expand our understanding of the genetic diversity of P. jirovecii in China.

Keywords

Introduction

Pneumocystis is a genus of atypical fungi demonstrating different degrees of genetic diversity between and within different species that infect mammals with high host specificity. The human-specific species, Pneumocystis jirovecii, causes life-threatening Pneumocystis pneumonia (PCP) in immunodeficient individuals, especially those with human immunodeficiency virus (HIV) infection (Ma et al. 2018). Recent studies have indicated a high prevalence of P. jirovecii colonization and infection in individuals with chronic obstructive pulmonary disease (COPD) (Wang et al. 2015; Cañas-Arboleda et al. 2019; Xue et al. 2020). However, the epidemiology and genetic diversity of P. jirovecii in different patient populations remain poorly understood.

Although genetic diversity of P. jirovecii has been reported in multiple studies from different regions in China (Li et al. 2013; Deng et al. 2014; Sun et al. 2015; Wang et al. 2019), all these studies are limited to only a few loci, and there is no such report from Shanxi Province in Northern China. In this study, we retrospectively investigated three confirmed cases of PCP, including two in HIV-positive patients and one in the HIV-negative patient from our hospital in Shanxi Province. Genetic polymorphisms of P. jirovecii in these patients were determined at eight different loci.

Experimental
Materials and Methods
Patients and samples

Three patients with PCP were included in this study, including two positive and one negative for HIV-1. Patients were admitted to the Department of Respiratory and Critical Care Medicine of First Affiliated Hospital of Shanxi Medical University between August 2019 and June 2020. The diagnosis of PCP was confirmed based on clinical manifestations and laboratory tests, including hematology, high-resolution computed tomography (HRCT), modified Gomori methenamine silver nitrate staining (GMS) of bronchoalveolar lavage fluid (BALF) samples. The two HIV-positive patients had a confirmed diagnosis of the acquired immune deficiency syndrome (AIDS) but did not receive highly active antiretroviral therapy. Based on the ELISA results, the HIV-negative patient was seronegative for HIV-1 and HIV-2 antibodies.

The Medical Ethics Committee approved this retrospective study of our hospital (2019-K051). In addition, written informed consent was obtained from all three patients.

DNA extraction

The BALF specimens were centrifuged at 350 g for 15 min, followed by washing the cell pellets with saline solution three times. DNA was extracted from washed cell pellets using the conventional phenol-chloroform extraction method. DNA extracts were quantified using a NanoDrop-UV-Vis spectrophotometer (Thermo Fisher Scientific, USA) and stored at −80°C until use.

DNA amplification, cloning, and sequencing

We amplified eight different loci of the P. jirovecii genome using nested PCR with the Premix-Taq PCR kit (TaKaRa Biotechnology Co., Ltd., Dalian, China) following the manufacturer’s instructions. The loci included mitochondrial large-subunit rRNA (mtLSU-rRNA), cytochrome b (cyb), nuclear large rRNA subunit (26S), and the complete internal transcribed spacers 1 and 2 (ITS1 and ITS2) along with the 5.8S rRNA of the nuclear rRNA operon (referred to as ITS hereafter), superoxide dismutase (sod), dihydropteroate synthase (dhps), dihydrofolate reductase (dhfr), and β-tubulin (β-Tub). The primers used in this study are listed in Table I. The PCR amplification conditions for β-Tub and 26S were the same as those previously reported (Pasic et al. 2020), and the conditions for other genes were the same as described in previous studies (Lee et al. 1998; Wang et al. 2019; Xue et al. 2019). DNA from P. jirovecii-positive specimens stored in our laboratory was used as the positive control. A non-template control with ultrapure-distilled water was included in each PCR run. To prevent cross-contamination of the samples, separate rooms were used, and the PCR mixture from each step of nested PCR was covered with 40 µl of sterile liquid paraffin. All PCR products were separated by electrophoresis on 2% agarose gels, stained with 4S Green Plus Nucleic Acid Stain (Sangon Biotech Co., Ltd. Shanghai, China), and visualized under UV irradiation. The amplified DNA bands of the expected sizes were excised from the gel and extracted using an aga-rose gel DNA extraction kit (Tiangen Biotech Co., Ltd., Beijing, China). Following the manufacturer’s instructions, the extracted DNA fragment was cloned into the TA cloning vector pMD18-T (TaKaRa Biotechnology Co., Ltd., Dalian, China). Recombinant plasmid clones were selected by blue-white screening on agar plates containing ampicillin. For each PCR product, 8 to 13 plasmid clones were randomly selected for Sanger sequencing in the ABI 3730xl DNA analyzer (Thermo Fisher Scientific, USA).

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
Sequence analysis and genotyping

The nucleotide sequences obtained in this study were analyzed and aligned using ClustalW software (https://www.genome.jp/tools-bin/clustalw). At least two plasmid clones are required to define a nucleotide polymorphism. The genotypes were named based on previously published nomenclature (Table II). The reference sequence for each gene was obtained from GenBank, with its accession number listed as follows: ITS, MK300654; mtLSU-rRNA, M58605; cyb, AF320344; sod, AF146753; dhfr, AF090368; dhps, AF139132; β-Tub, MG208106 and 26S KT272445. Known P. jirovecii multi-locus sequence type (MLST) profiles at β-Tub, cyb, 26S, and sod genes were retrieved from the Fungal MLST Database at http://mlst.mycologylab.org.

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

ITS – internal transcribed spacer regions of rRNA operon, sod – superoxide dismutase, dhfr – dihydrofolate reductase, dhp – dihydropteroate synthase, WT – wild-type, cyb – cytochrome b, mt – mitochondrial large rRNA subunit, β-Tub – β-tubulin, 26S rRNA – 26S ribosomal RNA gene

– the genotype nomenclature based on previously published studies and

– the genotype locations according to the studies previously reported (Walker et al. 1998; Ma et al. 1999; Beard et al. 2000; Denis et al. 2000; Takahashi et al. 2002; Esteves et al. 2010b; Maitte et al. 2013; Xue et al. 2019; Pasic et al. 2020)

Results
General information on PCP patients

Clinical information of the patients involved in this study is summarized in Table III. The presence of P. jirovecii in all patients was confirmed by microscopic observation of P. jirovecii cysts in BALF samples stained with GMS (Fig. 1).

Multilocus sequence genotyping

All eight genetic loci P. jirovecii were successfully amplified and sequenced in the BALF specimen from all three patients. Table II shows the polymorphic nucleotide sites, and the number of plasmid clones sequenced for each PCR product from 8 loci. Genotype profiles are summarized in Table IV.

The HIV-negative patient (SX_0003) showed a co-infection with two genotypes of P. jirovecii at six of the eight loci sequenced. Of the two HIV-positive patients, one (SX_0001) showed a co-infection with two geno-types of P. jirovecii at two loci, while the other (SX_0002) showed a single infection at all eight loci sequenced.

Of note, the dhps gene (the target of sulfa drugs) in all three P. jirovecii specimens was present as a wild-type sequence. The dhfr gene (the target of trimetho-prim) in all three P. jirovecii specimens showed a single synonymous change in the same position (from T to C at nucleotide 312). The cyb gene (the target of atovaquone) in the three P. jirovecii specimens showed polymorphisms in three nucleotide positions (at 279, 566 and 838), resulting in 4 genotypes including cyb 1, cyb 2, cyb 7 and cyb 8 based on the nomenclature system described by Esteves and Maitte (Esteves et al. 2010b; Maitte et al. 2013). Genotypes cyb 2 and cyb 7 were presented only in patients SX_0002 and SX_0001, respectively, while genotypes cyb 1 and cyb 8 were present as a mixture in the patient SX_003. Of the three polymorphisms, one is synonymous (at 279 in genotype cyb 8) and the other two are nonsynonymous (at 566 in genotype cyb 7 and 838 in genotype cyb 2).

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

NA – not available; ILD – interstitial lung disease; HRCT – high-resolution computed tomography; GGO – ground-glass opacity; HIV – human immunodeficiency virus; HAART – highly active antiretroviral therapy

+ – positive, − – negative

– Anti-PCP therapy, TMP-SMZ prophylaxis for P. jirovecii pneumonia

Candida norvegensis and Burkholderia cepacia

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

ITS – internal transcribed spacer regions of rRNA operon, sod – superoxide dismutase, dhfr – dihydrofolate reductase, dhps – dihydropteroate synthase, WT – wild-type, cyb – cytochrome b, mt – mitochondrial large rRNA subunit, β-Tub – β-tubulin, 26S rRNA – 26S rRNA gene

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.

Due to the presence of coinfection with two geno-types at 2 or 6 loci in two of the three patients (SX_0001 and SX_0003), we could not determine the exact MLST types in either patient (Table V).

Discussion

Despite having been recognized as an important human pathogen for many years, strain variation of P. jirovecii remains poorly understood due largely to the absence of a reliable in vitro culture system. To date, P. jirovecii strain typing has relied primarily on analyzing genetic markers after PCR amplification. While there have been about a dozen genetic markers reported (Ma et al. 2018), most studies have used only a small number of genetic markers in epidemiological investigations, potentially limiting the discriminatory power for strain differentiation. In this study, we performed strain typing of P. jirovecii using a total of eight genetic markers, including six nuclear genes (ITS, 26S rRNA, sod, dhps, dhfr and β-Tub) and two mitochondrial genes (mtLSU-rRNA and cyb).

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

– The first 11 MLST types (numbered 3 to 52) identified in this study correspond to those in the Fungal MLST Database at http://mlst.mycologylab.org

NA – types identified in this study and not available from the Fungal MLST Database

In both patients SX_0001 and SX_0003 (with co-infection of two genotypes at 2 or 6 loci, respectively), there were a total of four and 64 potential MLST profiles, respectively. Only two and 16 of those profiles are listed in this table while the true profiles could not be determined in this study.

While only three clinical specimens were examined including two from HIV-infected patients and one from a non-HIV patient, we identified complex genotype profiles (Table II). Multiple unique genotypes (from 2 to 5) were identified at all these eight loci except for two (dhps and dhfr), which showed a single genotype. Two of three clinical specimens showed a mixture of multiple genotypes at two or six loci, suggesting a coinfection with multiple P. jirovecii strains, without any strains shared between the three patients. This represents the first report of genetic polymorphisms in PCP patients in Shanxi Province, China. Our findings expand our understanding of the genetic diversity of P. jirovecii in China.

The ITS locus involved in this study includes ITS1 and ITS2, and 5.8S rRNA of the nuclear rRNA operon was amplified in one fragment of approximately 490 bp and is also known as ITS1-5.8S-ITS2 (Xue et al. 2019). Sequence analysis of this locus in this study identified five unique genotypes (nos. 4, 10, 16, 22, and 59) based on the genotype nomenclature system in our earlier report (Xue et al. 2019), which is more than genotypes identified from all other seven loci examined. This is consistent with many previous studies showing this locus to be the most polymorphic genetic marker for P. jirovecii genotyping (Ma et al. 2018). All ITS genotypes identified in this study have also been reported from previous studies conducted by our group (Xue et al. 2019) and others in China (Li et al. 2013; Sun et al. 2015) as well as studies from other countries (Atzori et al. 1998; Miller and Wakefield 1999; Matsumura et al. 2011).

In this study, we examined genetic polymorphisms of three drug target genes, including dhfr, dhps and cyb, which are the targets of trimethoprim, sulfa, and atovaquone drugs, respectively. No nonsynonymous mutation was found at dhfr or dhps in any specimens in this study, while a single synonymous change in the same position at dhfr (from T to C at nucleotide 312) was present in all three specimens. This change has been reported in previous studies from China (Deng et al. 2014; Wang et al. 2019) and other countries (Esteves et al. 2010b; Muñoz et al. 2012; Suárez et al. 2017; Singh et al. 2019). As for the cyb gene, we identified nucleotide changes at three positions (at 279, 566 and 838), which gave rise to 4 unique genotypes (cyb 1, cyb 2, cyb 7 and cyb 8). All these genotypes have also been reported from China (Deng et al. 2016; Wang et al. 2019) and other countries (Esteves et al. 2010b; Maitte et al. 2013; Sokulska et al. 2018; Szydlowicz et al. 2019; Le Gal et al. 2020; Goterris et al. 2022). The nucleotide changes at two positions (566 and 838) are synonymous (S189L and L180F) but do not correspond to any of the seven mutations that are suggested to be associated with atovaquone resistance in previous studies (Kessl et al. 2004). The absence of mutations in all these three drug targets potentially associated with resistance is consistent with no known use of the respective drugs in the history of the patients.

The major limitation of this study is the small sample size, which precludes the generalization of the results to a larger population and the assessment of correlation of genotypes with clinical characteristics and treatment outcomes. Further studies are required using more samples from different patient populations.

Conclusions

In conclusion, we assessed and analyzed the genetic polymorphisms of P. jirovecii genotypes at eight loci and identified complex genotype profiles, including the presence of coinfection with up to 5 genotypes at six loci. This is the first report of genetic polymorphisms in PCP patients in Shanxi Province, China. Our findings expand our understanding of the genetic diversity of P. jirovecii in China. However, a large-scale collection of clinical isolates of P. jirovecii from different patient populations is required for more detailed studies and the correlation of genotypes with clinical characteristics and outcomes.

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

Atzori C, Angeli E, Agostoni F, Mainini A, Micheli V, Cargnel A. Biomolecular techniques to detect Pneumocystis carinii f. sp. hominis pneumonia in patients with acquired immunodeficiency syndrome. Int J Infect Dis. 1998;3(2):76–81. https://doi.org/10.1016/s1201-9712(99)90013-9 AtzoriC AngeliE AgostoniF MaininiA MicheliV CargnelA Biomolecular techniques to detect Pneumocystis carinii f. sp. hominis pneumonia in patients with acquired immunodeficiency syndrome Int J Infect Dis 1998 3 2 76 81 https://doi.org/10.1016/s1201-9712(99)90013-9 10.1016/S1201-9712(99)90013-9 Search in Google Scholar

Beard CB, Carter JL, Keely SP, Huang L, Pieniazek NJ, Moura IN, Roberts JM, Hightower AW, Bens MS, Freeman AR, et al. Genetic variation in Pneumocystis carinii isolates from different geographic regions: implications for transmission. Emerg Infect Dis. 2000;6(3): 265–272. https://doi.org/10.3201/eid0603.000306 BeardCB CarterJL KeelySP HuangL PieniazekNJ MouraIN RobertsJM HightowerAW BensMS FreemanAR Genetic variation in Pneumocystis carinii isolates from different geographic regions: implications for transmission Emerg Infect Dis 2000 6 3 265 272 https://doi.org/10.3201/eid0603.000306 10.3201/eid0603.000306264087710827116 Search in Google Scholar

Cañas-Arboleda A, Hernández-Flórez C, Garzón J, Parra-Giraldo CM, Burbano JF, Cita-Pardo JE. Colonization by Pneumocystis jirovecii in patients with chronic obstructive pulmonary disease: association with exacerbations and lung function status. Braz J Infect Dis. 2019;23(5):352–357. https://doi.org/10.1016/j.bjid.2019.08.008 Cañas-ArboledaA Hernández-FlórezC GarzónJ Parra-GiraldoCM BurbanoJF Cita-PardoJE Colonization by Pneumocystis jirovecii in patients with chronic obstructive pulmonary disease: association with exacerbations and lung function status Braz J Infect Dis 2019 23 5 352 357 https://doi.org/10.1016/j.bjid.2019.08.008 10.1016/j.bjid.2019.08.00831545952 Search in Google Scholar

Deng X, Xiong M, Lan Y, Zhuo L, Chen W, Tang X. [The gene polymorphisms of drug targets in Pneumocystis jirovecii isolates] (in Chinese). Chin J Infect Dis. 2016;34(7):395–399. https://doi.org/10.3760/cma.j.issn.1000-6680.2016.07.002 DengX XiongM LanY ZhuoL ChenW TangX [The gene polymorphisms of drug targets in Pneumocystis jirovecii isolates] (in Chinese) Chin J Infect Dis 2016 34 7 395 399 https://doi.org/10.3760/cma.j.issn.1000-6680.2016.07.002 Search in Google Scholar

Deng X, Zhuo L, Lan Y, Dai Z, Chen WS, Cai W, Kovacs JA, Ma L, Tang X. Mutational analysis of Pneumocystis jirovecii dihydropteroate synthase and dihydrofolate reductase genes in HIV-infected patients in China. J Clin Microbiol. 2014;52(11):4017–4019. https://doi.org/10.1128/JCM.01848-14 DengX ZhuoL LanY DaiZ ChenWS CaiW KovacsJA MaL TangX Mutational analysis of Pneumocystis jirovecii dihydropteroate synthase and dihydrofolate reductase genes in HIV-infected patients in China J Clin Microbiol 2014 52 11 4017 4019 https://doi.org/10.1128/JCM.01848-14 10.1128/JCM.01848-14431324125122865 Search in Google Scholar

Denis CM, Mazars E, Guyot K, Odberg-Ferragut C, Viscogliosi E, Dei-Cas E, Wakefield AE. Genetic divergence at the SODA locus of six different formae speciales of Pneumocystis carinii. Med Mycol. 2000;38(4):289–300. https://doi.org/10.1080/mmy.38.4.289.300 DenisCM MazarsE GuyotK Odberg-FerragutC ViscogliosiE Dei-CasE WakefieldAE Genetic divergence at the SODA locus of six different formae speciales of Pneumocystis carinii Med Mycol 2000 38 4 289 300 https://doi.org/10.1080/mmy.38.4.289.300 10.1080/714030952 Search in Google Scholar

Esteves F, Gaspar J, Marques T, Leite R, Antunes F, Mansinho K, Matos O. Identification of relevant single-nucleotide polymorphisms in Pneumocystis jirovecii: relationship with clinical data. Clin Microbiol Infect. 2010a;16(7):878–884. https://doi.org/10.1111/j.1469-0691.2009.03030.x EstevesF GasparJ MarquesT LeiteR AntunesF MansinhoK MatosO Identification of relevant single-nucleotide polymorphisms in Pneumocystis jirovecii: relationship with clinical data Clin Microbiol Infect 2010a 16 7 878 884 https://doi.org/10.1111/j.1469-0691.2009.03030.x 10.1111/j.1469-0691.2009.03030.x19719744 Search in Google Scholar

Esteves F, Gaspar J, Tavares A, Moser I, Antunes F, Mansinho K, Matos O. Population structure of Pneumocystis jirovecii isolated from immunodeficiency virus-positive patients. Infect Genet Evol. 2010b;10(2):192–199. https://doi.org/10.1016/j.meegid.2009.12.007 EstevesF GasparJ TavaresA MoserI AntunesF MansinhoK MatosO Population structure of Pneumocystis jirovecii isolated from immunodeficiency virus-positive patients Infect Genet Evol 2010b 10 2 192 199 https://doi.org/10.1016/j.meegid.2009.12.007 10.1016/j.meegid.2009.12.00720060502 Search in Google Scholar

Goterris L, Pasic L, Murillo MG, Kan A, Anton A, Company JA, Ruiz-Camps I, Meyer W, Martin-Gomez MT. Pneumocystis jirovecii genetic diversity in a Spanish tertiary hospital. Med Mycol. 2022; 60(1):myab065. https://doi.org/10.1093/mmy/myab065 GoterrisL PasicL MurilloMG KanA AntonA CompanyJA Ruiz-CampsI MeyerW Martin-GomezMT Pneumocystis jirovecii genetic diversity in a Spanish tertiary hospital Med Mycol 2022 60 1 myab065 https://doi.org/10.1093/mmy/myab065 10.1093/mmy/myab06534698858 Search in Google Scholar

Kessl JJ, Hill P, Lange BB, Meshnick SR, Meunier B, Trumpower BL. Molecular basis for atovaquone resistance in Pneumocystis jirovecii modeled in the cytochrome bc(1) complex of Saccharomyces cerevisiae. J Biol Chem. 2004;279(4):2817–2824. https://doi.org/10.1074/jbc.M309984200 KesslJJ HillP LangeBB MeshnickSR MeunierB TrumpowerBL Molecular basis for atovaquone resistance in Pneumocystis jirovecii modeled in the cytochrome bc(1) complex of Saccharomyces cerevisiae J Biol Chem 2004 279 4 2817 2824 https://doi.org/10.1074/jbc.M309984200 10.1074/jbc.M30998420014576156 Search in Google Scholar

Lane BR, Ast JC, Hossler PA, Mindell DP, Bartlett MS, Smith JW, Meshnick SR. Dihydropteroate synthase polymorphisms in Pneumocystis carinii. J Infect Dis. 1997;175(2):482–485. https://doi.org/10.1093/infdis/175.2.482 LaneBR AstJC HosslerPA MindellDP BartlettMS SmithJW MeshnickSR Dihydropteroate synthase polymorphisms in Pneumocystis carinii J Infect Dis 1997 175 2 482 485 https://doi.org/10.1093/infdis/175.2.482 10.1093/infdis/175.2.4829203679 Search in Google Scholar

Le Gal S, Hoarau G, Bertolotti A, Negri S, Le Nan N, Bouchara JP, Papon N, Blanchet D, Demar M, Nevez G. Pneumocystis jirovecii diversity in Réunion, an overseas French Island in Indian Ocean. Front Microbiol. 2020;11:127. https://doi.org/10.3389/fmicb.2020.00127 Le GalS HoarauG BertolottiA NegriS Le NanN BoucharaJP PaponN BlanchetD DemarM NevezG Pneumocystis jirovecii diversity in Réunion, an overseas French Island in Indian Ocean Front Microbiol 2020 11 127 https://doi.org/10.3389/fmicb.2020.00127 10.3389/fmicb.2020.00127 Search in Google Scholar

Lee CH, Helweg-Larsen J, Tang X, Jin S, Li B, Bartlett MS, Lu JJ, Lundgren B, Lundgren JD, Olsson M, et al. Update on Pneumocystis carinii f. sp. hominis typing based on nucleotide sequence variations in internal transcribed spacer regions of rRNA genes. J Clin Microbiol. 1998;36(3):734–741. https://doi.org/10.1128/JCM.36.3.734-741.1998 LeeCH Helweg-LarsenJ TangX JinS LiB BartlettMS LuJJ LundgrenB LundgrenJD OlssonM Update on Pneumocystis carinii f. sp. hominis typing based on nucleotide sequence variations in internal transcribed spacer regions of rRNA genes J Clin Microbiol 1998 36 3 734 741 https://doi.org/10.1128/JCM.36.3.734-741.1998 10.1128/JCM.36.3.734-741.1998 Search in Google Scholar

Li K, He A, Cai WP, Tang XP, Zheng XY, Li ZY, Zhan XM. Geno-typing of Pneumocystis jirovecii isolates from Chinese HIV-infected patients based on nucleotide sequence variations in the internal transcribed spacer regions of rRNA genes. Med Mycol. 2013; 51(1):108–112. https://doi.org/10.3109/13693786.2012.695458 LiK HeA CaiWP TangXP ZhengXY LiZY ZhanXM Geno-typing of Pneumocystis jirovecii isolates from Chinese HIV-infected patients based on nucleotide sequence variations in the internal transcribed spacer regions of rRNA genes Med Mycol 2013 51 1 108 112 https://doi.org/10.3109/13693786.2012.695458 10.3109/13693786.2012.695458 Search in Google Scholar

Ma L, Borio L, Masur H, Kovacs JA. Pneumocystis carinii dihydropteroate synthase but not dihydrofolate reductase gene mutations correlate with prior trimethoprim-sulfamethoxazole or dapsone use. J Infect Dis. 1999;180(6):1969–1978. https://doi.org/10.1086/315148 MaL BorioL MasurH KovacsJA Pneumocystis carinii dihydropteroate synthase but not dihydrofolate reductase gene mutations correlate with prior trimethoprim-sulfamethoxazole or dapsone use J Infect Dis 1999 180 6 1969 1978 https://doi.org/10.1086/315148 10.1086/315148 Search in Google Scholar

Ma L, Cisse OH, Kovacs JA. A molecular window into the biology and epidemiology of Pneumocystis spp. Clin Microbiol Rev. 201813;31(3):e00009–18. https://doi.org/10.1128/CMR.00009-18 MaL CisseOH KovacsJA A molecular window into the biology and epidemiology of Pneumocystis spp. Clin Microbiol Rev 2018 13 31 3 e00009 18 https://doi.org/10.1128/CMR.00009-18 10.1128/CMR.00009-18 Search in Google Scholar

Maitte C, Leterrier M, Le Pape P, Miegeville M, Morio F. Multi-locus sequence typing of Pneumocystis jirovecii from clinical samples: how many and which loci should be used? J Clin Microbiol. 2013;51(9):2843–2849. https://doi.org/10.1128/JCM.01073-13 MaitteC LeterrierM Le PapeP MiegevilleM MorioF Multi-locus sequence typing of Pneumocystis jirovecii from clinical samples: how many and which loci should be used? J Clin Microbiol 2013 51 9 2843 2849 https://doi.org/10.1128/JCM.01073-13 10.1128/JCM.01073-13 Search in Google Scholar

Matsumura Y, Shindo Y, Iinuma Y, Yamamoto M, Shirano M, Matsushima A, Nagao M, Ito Y, Takakura S, Hasegawa Y, et al. Clinical characteristics of Pneumocystis pneumonia in non-HIV patients and prognostic factors including microbiological genotypes. BMC Infect Dis. 2011;11:76. https://doi.org/10.1186/1471-2334-11-76 MatsumuraY ShindoY IinumaY YamamotoM ShiranoM MatsushimaA NagaoM ItoY TakakuraS HasegawaY Clinical characteristics of Pneumocystis pneumonia in non-HIV patients and prognostic factors including microbiological genotypes BMC Infect Dis 2011 11 76 https://doi.org/10.1186/1471-2334-11-76 10.1186/1471-2334-11-76 Search in Google Scholar

Miller RF, Wakefield AE. Pneumocystis carinii genotypes and severity of pneumonia. Lancet. 1999;353(9169):2039–2040. https://doi.org/10.1016/S0140-6736(99)01690-6 MillerRF WakefieldAE Pneumocystis carinii genotypes and severity of pneumonia Lancet 1999 353 9169 2039 2040 https://doi.org/10.1016/S0140-6736(99)01690-6 10.1016/S0140-6736(99)01690-6 Search in Google Scholar

Muñoz C, Zuluaga A, Restrepo A, Tobon A, Cano LE, Gonzalez A. Molecular diagnosis and detection of Pneumocystis jirovecii DHPS and DHFR genotypes in respiratory specimens from Colombian patients. Diagn Microbiol Infect Dis. 2012;72(3):204–213. https://doi.org/10.1016/j.diagmicrobio.2011.11.015 MuñozC ZuluagaA RestrepoA TobonA CanoLE GonzalezA Molecular diagnosis and detection of Pneumocystis jirovecii DHPS and DHFR genotypes in respiratory specimens from Colombian patients Diagn Microbiol Infect Dis 2012 72 3 204 213 https://doi.org/10.1016/j.diagmicrobio.2011.11.015 10.1016/j.diagmicrobio.2011.11.01522321995 Search in Google Scholar

Pasic L, Goterris L, Guerrero-Murillo M, Irinyi L, Kan A, Ponce CA, Vargas SL, Martin-Gomez MT, Meyer W. Consensus multilocus sequence typing scheme for Pneumocystis jirovecii. J Fungi (Basel). 2020;6(4):259. https://doi.org/10.3390/jof6040259 PasicL GoterrisL Guerrero-MurilloM IrinyiL KanA PonceCA VargasSL Martin-GomezMT MeyerW Consensus multilocus sequence typing scheme for Pneumocystis jirovecii J Fungi (Basel) 2020 6 4 259 https://doi.org/10.3390/jof6040259 10.3390/jof6040259771198833143112 Search in Google Scholar

Singh Y, Mirdha BR, Guleria R, Kabra SK, Mohan A, Chaudhry R, Kumar L, Dwivedi SN, Agarwal SK. Genetic polymorphisms associated with treatment failure and mortality in pediatric Pneumocystosis. Sci Rep. 2019;9(1):1192. https://doi.org/10.1038/s41598-018-38052-x SinghY MirdhaBR GuleriaR KabraSK MohanA ChaudhryR KumarL DwivediSN AgarwalSK Genetic polymorphisms associated with treatment failure and mortality in pediatric Pneumocystosis Sci Rep 2019 9 1 1192 https://doi.org/10.1038/s41598-018-38052-x 10.1038/s41598-018-38052-x636194330718779 Search in Google Scholar

Sokulska M, Kicia M, Wesolowska M, Piesiak P, Kowal A, Lobo ML, Kopacz Z, Hendrich AB, Matos O. Genotyping of Pneumocystis jirovecii in colonized patients with various pulmonary diseases. Med Mycol. 2018;56(7):809–815. https://doi.org/10.1093/mmy/myx121 SokulskaM KiciaM WesolowskaM PiesiakP KowalA LoboML KopaczZ HendrichAB MatosO Genotyping of Pneumocystis jirovecii in colonized patients with various pulmonary diseases Med Mycol 2018 56 7 809 815 https://doi.org/10.1093/mmy/myx121 10.1093/mmy/myx12129228377 Search in Google Scholar

Suárez I, Roderus L, van Gumpel E, Jung N, Lehmann C, Fätkenheuer G, Hartmann P, Plum G, Rybniker J. Low prevalence of DHFR and DHPS mutations in Pneumocystis jirovecii strains obtained from a German cohort. Infection. 2017;45(3):341–347. https://doi.org/10.1007/s15010-017-1005-4 SuárezI RoderusL van GumpelE JungN LehmannC FätkenheuerG HartmannP PlumG RybnikerJ Low prevalence of DHFR and DHPS mutations in Pneumocystis jirovecii strains obtained from a German cohort Infection 2017 45 3 341 347 https://doi.org/10.1007/s15010-017-1005-4 10.1007/s15010-017-1005-428303545 Search in Google Scholar

Sun L, Huang M, Wang J, Xue F, Hong C, Guo Z, Gu J. Genotyping of Pneumocystis jirovecii isolates from human immunodeficiency virus-negative patients in China. Infect Genet Evol. 2015;31:209–215. https://doi.org/10.1016/j.meegid.2015.01.021 SunL HuangM WangJ XueF HongC GuoZ GuJ Genotyping of Pneumocystis jirovecii isolates from human immunodeficiency virus-negative patients in China Infect Genet Evol 2015 31 209 215 https://doi.org/10.1016/j.meegid.2015.01.021 10.1016/j.meegid.2015.01.02125653130 Search in Google Scholar

Szydlowicz M, Jakuszko K, Szymczak A, Piesiak P, Kowal A, Kopacz Z, Wesolowska M, Lobo ML, Matos O, Hendrich AB, et al. Prevalence and genotyping of Pneumocystis jirovecii in renal transplant recipients-preliminary report. Parasitol Res. 2019;118(1):181–189. https://doi.org/10.1007/s00436-018-6131-0 SzydlowiczM JakuszkoK SzymczakA PiesiakP KowalA KopaczZ WesolowskaM LoboML MatosO HendrichAB Prevalence and genotyping of Pneumocystis jirovecii in renal transplant recipients-preliminary report Parasitol Res 2019 118 1 181 189 https://doi.org/10.1007/s00436-018-6131-0 10.1007/s00436-018-6131-0632973030392033 Search in Google Scholar

Takahashi T, Endo T, Nakamura T, Sakashita H, Kimura K, Ohnishi K, Kitamura Y, Iwamoto A. Dihydrofolate reductase gene polymorphisms in Pneumocystis carinii f. sp. hominis in Japan. J Med Microbiol. 2002;51(6):510–515. https://doi.org/10.1099/0022-1317-51-6-510 TakahashiT EndoT NakamuraT SakashitaH KimuraK OhnishiK KitamuraY IwamotoA Dihydrofolate reductase gene polymorphisms in Pneumocystis carinii f. sp. hominis in Japan J Med Microbiol 2002 51 6 510 515 https://doi.org/10.1099/0022-1317-51-6-510 10.1099/0022-1317-51-6-51012018659 Search in Google Scholar

Wakefield AE. DNA sequences identical to Pneumocystis carinii f. sp. carinii and Pneumocystis carinii f. sp. hominis in samples of air spora. J Clin Microbiol. 1996;34(7):1754–1759. https://doi.org/10.1128/JCM.34.7.1754-1759.1996 WakefieldAE DNA sequences identical to Pneumocystis carinii f. sp. carinii and Pneumocystis carinii f. sp. hominis in samples of air spora J Clin Microbiol 1996 34 7 1754 1759 https://doi.org/10.1128/JCM.34.7.1754-1759.1996 10.1128/jcm.34.7.1754-1759.1996 Search in Google Scholar

Walker DJ, Wakefield AE, Dohn MN, Miller RF, Baughman RP, Hossler PA, Bartlett MS, Smith JW, Kazanjian P, Meshnick SR. Sequence polymorphisms in the Pneumocystis carinii cytochrome b gene and their association with atovaquone prophylaxis failure. J Infect Dis. 1998;178(6):1767–1775. https://doi.org/10.1086/314509 WalkerDJ WakefieldAE DohnMN MillerRF BaughmanRP HosslerPA BartlettMS SmithJW KazanjianP MeshnickSR Sequence polymorphisms in the Pneumocystis carinii cytochrome b gene and their association with atovaquone prophylaxis failure J Infect Dis 1998 178 6 1767 1775 https://doi.org/10.1086/314509 10.1086/3145099815231 Search in Google Scholar

Wang DD, Zheng MQ, Zhang N, An CL. Investigation of Pneumocystis jirovecii colonization in patients with chronic pulmonary diseases in the People’s Republic of China. Int J Chron Obstruct Pulmon Dis. 2015;10:2079–2085. https://doi.org/10.2147/COPD.S89666 WangDD ZhengMQ ZhangN AnCL Investigation of Pneumocystis jirovecii colonization in patients with chronic pulmonary diseases in the People’s Republic of China Int J Chron Obstruct Pulmon Dis 2015 10 2079 2085 https://doi.org/10.2147/COPD.S89666 10.2147/COPD.S89666459822126491278 Search in Google Scholar

Wang M, Xu X, Guo Y, Tao R, Hu C, Dong X, Huang Y, Zhu B. Polymorphisms involving the Pneumocystis jirovecii-related genes in AIDS patients in eastern China. Infect Genet Evol. 2019;75:103955. https://doi.org/10.1016/j.meegid.2019.103955 WangM XuX GuoY TaoR HuC DongX HuangY ZhuB Polymorphisms involving the Pneumocystis jirovecii-related genes in AIDS patients in eastern China Infect Genet Evol 2019 75 103955. https://doi.org/10.1016/j.meegid.2019.103955 10.1016/j.meegid.2019.10395531284044 Search in Google Scholar

Xue T, Ma Z, Liu F, Du W, He L, Wang J, An C. Pneumocystis jirovecii colonization and its association with pulmonary diseases: a multicenter study based on a modified loop-mediated isothermal amplification assay. BMC Pulm Med. 2020;20(1):70. https://doi.org/10.1186/s12890-020-1111-4 XueT MaZ LiuF DuW HeL WangJ AnC Pneumocystis jirovecii colonization and its association with pulmonary diseases: a multicenter study based on a modified loop-mediated isothermal amplification assay BMC Pulm Med 2020 20 1 70 https://doi.org/10.1186/s12890-020-1111-4 10.1186/s12890-020-1111-4708514432197601 Search in Google Scholar

Xue T, Ma Z, Liu F, Du WQ, He L, Ma L, An CL. Genotyping of Pneumocystis jirovecii by use of a new simplified nomenclature system based on the internal transcribed spacer regions and 5.8S rRNA gene of the rRNA operon. Clin Microbiol. 2019;57(6):e02012–18. https://doi.org/10.1128/JCM.02012-18 XueT MaZ LiuF DuWQ HeL MaL AnCL Genotyping of Pneumocystis jirovecii by use of a new simplified nomenclature system based on the internal transcribed spacer regions and 5.8S rRNA gene of the rRNA operon Clin Microbiol 2019 57 6 e02012 18 https://doi.org/10.1128/JCM.02012-18 10.1128/JCM.02012-18653558830918046 Search in Google Scholar

Recommended articles from Trend MD

Plan your remote conference with Sciendo