Although genetic diversity of
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.
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.
We amplified eight different loci of the
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′ |
578 |
MnSOD_Fw 5′-GGGTTTAATTAGTCTTTTTAGGCAC-3′ |
560 | |
FR208 5′-GCAGAAAGTAGGTACATTATTACGAGA-3′ |
798 | |
DHPS1 5′-CAAATTAGCGTATCGAATGACC-3′ |
278 | |
CytbFw 5′-CCCAGAATTCTCGTTTGGTCTATT-3′ |
590 | |
mtLSU rRNA (Wakefield 1996) | pAZ102_E 5′-GATGGCTGTTTCCAAGCCCA-3′ |
252 |
β-Tub (Pasic et al. 2020) | Pneumo |
303 |
26S rRNA (Pasic et al. 2020) | PneumoLSU_F 5′-TCAGGTCGAACTGGTGTACG-3′ |
297 |
The nucleotide sequences obtained in this study were analyzed and aligned using ClustalW software (
Nucleotide polymorphic sites and number of plasmid clones sequenced at eight distinct loci of
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 | |
110C/215T | 11 | 13 | 8 | ||
110T/215C | 0 | 0 | 2 | ||
165A (55Thr) / 171C (57Pro) | 12 | 12 | 12 | ||
312C (117Gly) | 12 | 11 | 11 | ||
279C/348A/516C/547C/566C/838C | 0 | 0 | 6 | ||
279C/348A/516C/547C/566C/838T | 0 | 8 | 0 | ||
279C/348A/516C/547C/566T/838C | 9 | 0 | 0 | ||
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,
– 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)
Clinical information of the patients involved in this study is summarized in Table III. The presence of
All eight genetic loci
The HIV-negative patient (SX_0003) showed a co-infection with two genotypes of
Of note, the
Clinical characteristics of patients with
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 | – | – | |
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
–
Genotypes of
Patient No. | HIV1/2 anti-body | Genotypes at 8 loci | |||||||
---|---|---|---|---|---|---|---|---|---|
ITS | mtLSU rRNA | β-Tub | 26S rRNA | ||||||
SX_0001 | +/− | ITS2 + ITS59 | mt3 | β-Tub1 + β-Tub2 | 26S2 | ||||
SX_0002 | +/− | ITS4 | mt3 | β-Tub2 | 26S2 | ||||
SX_0003 | −/− | ITS10 + ITS16 | mt1 + mt 2 | β-Tub1 + β-Tub2 | 26S3 + 26S4 |
ITS – internal transcribed spacer regions of rRNA operon,
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).
Despite having been recognized as an important human pathogen for many years, strain variation of
Multi-locus sequence type (MLST) profiles of
MLST types* | β-Tub | 26S rRNA | 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
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 (
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
In this study, we examined genetic polymorphisms of three drug target genes, including
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.
In conclusion, we assessed and analyzed the genetic polymorphisms of