To investigate the molecular characteristics and genotype distribution of Pneumocystis jirovecii in HIV/AIDS patients who were admitted to the National Hospital for Tropical Diseases. Subjects and methods: Prospective and cross-sectional description. 31 HIV/AIDS patients infected with P. jirovecii and confirmed by bronchoscopy and realtime PCR from 01 - 2014 to 12 - 2017 were included for this study. Results and conclusions: Nucleotide analysis revealed that no nucleotide alteration occured in the loci β-TUB, CYB, DHFR, DHPS, whereas minor variable positions were observed in the SOD locus. The most frequent nucleotide variation was found in three loci: mt26S, 26S and ITS1. Based on the nucleotide variation, the SOD locus was classified into two genotypes (SOD and SOD6), the mt26S locus was classified into 14 genotypes (2, 7, 8, 11, 12, 15, 16, 17, 18, 19, 20, 21, 22, and 23), the ITS1 locus was classified into 14 genotypes (A1, A2, A3, A4, A5, A6, B1, B2, B3, B7, B8, B9 and B10), and the 26S locus was classified into 4 genotypes (1, 11, 12, and 13). Several new genotypes were only distributed in the P. jirovecii strains circulating in Vietnam.
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MOLECULAR CHARACTERISTICS AND GENOTYPE
DISTRIBUTION OF Pneumocystis jirovecii IN HIV/AIDS
PATIENTS IN NATIONAL HOSPITAL FOR TROPICAL DISEASES
FROM 2014 - 2017
Nguyen Tuan Anh 1 ; Do Quyet 2 ; Nguyen Huy Luc 3
SUMMARY
Objectives: To investigate the molecular characteristics and genotype distribution of Pneumocystis jirovecii in HIV/AIDS patients who were admitted to the National Hospital for Tropical Diseases Subjects and methods: Prospective and cross-sectional description 31 HIV/AIDS patients infected with P jirovecii and confirmed by bronchoscopy and realtime PCR from 01 - 2014 to 12 - 2017 were included for this study Results and conclusions: Nucleotide analysis revealed that no nucleotide alteration occured in the loci β-TUB, CYB, DHFR, DHPS, whereas minor variable positions were observed in the SOD locus The most frequent nucleotide variation was found in three loci: mt26S, 26S and ITS1 Based on the nucleotide variation, the SOD locus was classified into two genotypes (SOD and SOD6), the mt26S locus was classified into 14 genotypes (2, 7, 8, 11, 12, 15, 16, 17, 18, 19, 20, 21, 22, and 23), the ITS1 locus was classified into 14 genotypes (A1, A2, A3, A4, A5, A6, B1, B2, B3, B7, B8, B9 and B10), and the 26S locus was classified into 4 genotypes (1, 11, 12, and 13) Several new genotypes were only distributed in the P jirovecii strains circulating in Vietnam
* Keywords: Pneumonia; Pneumocystis jirovecii; HIV/AIDS; Genotype; Locus
INTRODUCTION
Pneumocystis jiroveci is an abnormal
opportunistic pathogen that causes severe
pneumonia in immunocompromised people
with high mortality rates [1] Until the 1980s,
P jirovecii was not common and mainly
infected people with immunodeficiency
syndrome or individuals using prolonged
immunosuppressive drugs, especially
cancer chemotherapy [2] However, in the
regions with HIV/AIDS epidemic, P jirovecii
emerged as the most common infectious pathogen in HIV/AIDS patients In the past, there was no specific preventive regimen
for P jirovecii, this infectious pathogen
was found in more than 60% of HIV-infected patients and over 80% of patients with less than 200 CD4 cells were
infected by P jirovecii [11, 12] After the
introduction of the first- and the
second-line prophylaxis for P jirovecii in the early 1990s, the incidence of P jirovecii in
HIV/AIDS patients decreased significantly,
1 National Hospital for Tropical Diseases
2 Vietnam Military Medical University
3 103 Military Hospital
Corresponding author: Nguyen Tuan Anh (dranhnhtd@gmail.com)
Date received: 22/07/2019
Date accepted: 27/08/2019
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and continued to decline after treatment
with high activity antiretroviral drugs
(HAART) in the mid-1990s [9, 10]
However, P jirovecii continues to be a
common opportunistic pathogen with high
morbidity and mortality rates in the era of
industrialization P jirovecii is still one of
the common threats to HIV/AIDS-infected
patients [9], especially in patients who did
not know they were infected with HIV or
did not receive medical check regularly
Because P jirovecii is difficult to detect,
the infection with this fungus is almost
undiagnosed in developing countries [3]
Therefore, molecular characterization and
genotyping of P jirovecii pathogen circling
in Vietnam are highly necessary in order
to improve understanding of pathogenic characteristics of this fungus In this study,
we aim: To molecularly characterize and
to determine the genotype distribution of
P jirovecii in HIV/AIDS patients in the National Hospital for Tropical Diseases during the period from January 2014 to December 2017
SUBJECTS AND METHODS
1 Subjects
A total of 31 bronchial lavage specimens
from HIV/AIDS patients infected with P
jirovecii confirmed by realtime PCR were
collected from 1 - 2014 to 12 - 2017
2 Methods
* Study design: Prospective and cross-sectional description
* Procedure of the study:
- Materials: Qiagen Kit (USA) was used for DNA extraction and PCR amplification and Applied Biosystem Kit (USA) was use nucleotide sequencing
- Primers for PCR and sequencing [13] (table 1):
mt26S
347
26S rDNA
426
ITS1
204
β-TUB
309
SOD
652
CYB
638
DHPS
318
DHFR
610
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- Equipment: ProLex PCR machine
(Applied Biosystem, USA), MuPis
(Japan), 3130 sequencer (Applied
Biosystem, USA)
- PCR amplification was done
according to the method of Céline Maitte
et al [13], PCR component to amplify 08
target loci includes 0.5 µL of primers, 1 X
Qiagen Multiplex PCR master mix and 5
µL of DNA template The thermal cycle
begins by activating HotStarTaq DNA
Polymerase at 95°C for 15 minutes,
repeating 35 cycles of denaturation at
94°C for 30 seconds, 60°C for 45
seconds and 72°C for 1 minute PCR
products were then run on 1.5% agarose
gel
- The PCR products were then purified
by using Qiagen PCR purification kit and purified products were used as template for sequencing PCR using BigDye™ Terminator v.3.1 (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions The forward
and reverse primers for the mt26S, 26S,
ITS1, SOD, β-TUB, DHPS, and DHFR
genes were used for sequencing PCR
- Genotyping: The DNA sequences of the loci were compared with the reference
sequences of the mt26S, 26S, ITS1,
SOD, β-TUB, DHPS, and DHFR genes
from GenBank (GenBank - NCBI) using ATGC software 7.2 (Japan) to identify
genetic variations of P jirovecii
RESULTS AND DISCUSSION
1 Genetic variations of 08 loci
Table 2: The determination of nucleotide variation of 08 loci from 31 P jirovecii strains
Genotype of each locus Sample
ID
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We analyzed sequences the 08 loci of
31 P jirovecii strains and the results
revealed that no nucleotide alteration in
the β-TUB, CYB, DHFR, and DHPS loci
was observed, whereas SOD locus had
two changes at C/110 and A/215 and was
classified into SOD6 genotype The data
indicated that the most frequent nucleotide
alterations were in the mt26S, 26S and
ITS1 loci and these changes were
grouped into different genotypes In this
study, we not only found major mutations (genotypes) reported previously by Maitte
et al [13], Hauser et al [6], Esteves et al [5], Ma et al [8], Kazanjian et al [7], and Costa et al [4], but also identified several new genotypes (mutations) that only
found in P jirovecii strains circulating in Vietnam This result indicated that P
jirovecii in the study had different variants
in the genome suggesting a high genetic diversity
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2 New genotype found in the P jirovecii circulating in Vietnam
Table 3: New nucleotide variation identified in the P jirovecii strains
Mt26S
TTA/111-113
TTA/111-113
TTA/111-113
TTA/111-113
ITS1
TTA/111-113
Genotyping was based on previous
studies of Maitte et al [13], Hauser et al
[6], Esteves et al [5], Ma et al [8],
Kazanjian et al [7], and Costa et al [4]
The nucleotide variants of the mt26S,
ITS1 and SOD were consistent with several
new genotypes that only distributed in
Vietnamese P jirovecii strains The reason
for the difference between these pathogenic
P jirovecii strains may be derived from
various geographical and climate regions, host, and exposure to different drugs In addition, the preventive treatment for HIV/AIDS patients by HAART and other infectious pathogens can be also created nucleotide alterations, which may be associated with disease symptoms and the risk of mortality
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3 Genotype distribution of P jirovecii circulating in Vietnam
Table 4: Genotype distribution of P jirovecii circulating in Vietnam
A1 (4) A2 (6) A3 (4) A4 (1) A5 (1) A6 (1)
B (2) B1 (1) B2 (1) B3 (3) B7 (1) B8 (1) B9 (1) B10 (1)
ND (3)
2 (5)
7 (6)
8 (2)
11 (3)
12 (2)
15 (1)
16 (1)
17 (2)
18 (1)
19 (1)
20 (1)
21 (3)
22 (2)
23 (1)
1 (20)
11 (3)
12 (6)
13 (2)
SOD6 (1)
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Data showed that ITS1 locus was
classified into 14 genotypes, of which the
major strains were in A6, A1, A3 B, and
B3, the minor strains were distributed in
the rest of genotypes Genetic variation in
the mt26S locus was also divided into 14
genotypes, of these the genotypes 15 - 23
were only found in Vietnamese P jirovecii
strains The 26S locus was classified into
four genotypes, the majority of strains were
genotype 1 The nucleotide variation in
the SOD locus was classified into 2 main
genotypes, SOD1 genotype and SOD6
The CYB, β-TUB, DHPS, DHFR loci
revealed no mutation Thus, 31 P jirovecii
strains circulating in Vietnam indicate a
high level of genetic variation, mainly at
the ITS1, mt26S, 26S and SOD loci
CONCLUSIONS
The most frequent nucleotide variation
of 31 P jirovecii strains was found in
ITS1, mt26S, 26S and SOD loci Several
mutations were only distributed in the P
jirovecii strains circulating in Vietnam No
nucleotide alterations were found in the
CYB, β-TUB, DHPS, DHFR loci of the 31
P jirovecii strains
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