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Nghiên cứu đặc điểm lâm sàng, cận lâm sàng, kiểu gen của pneumocystis jirovecii gây viêm phổi trên bệnh nhân HIV AIDS tt tiếng anh

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MINISTRY OF
AND TRAINING
DEFENSE
VIET NAM MILIRATY MEDICAL UNIVERSITY

MINISTRY OF EDUCATION

NGUYỄN TUẤN ANH

STUDY ON CLINICAL, PARACLINICAL FEATURES,
GENOTYPE OF PNEUMOCYSTIS JIROVECII CAUSING
PNEUMONIA IN HIV/AIDS PATIENTS

Specilize: Internal medicine
Indentification number: 9720107


SUMMARY OF MEDICAL THESIS

HA NOI – 2020


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THE RESEARCH WAS PERFORMED AT:
VIET NAM MILITARY MEDICAL UNIVERSITY
Scientific instructors:
1. Prof. Ph.D. Do Quyet
2. Assoc.Prof. PhD. Thai Khac Chau

Judge 1: Assoc.Prof. PhD. Vu Van Giap
Judge 2: Assoc.Prof. PhD. Tran Van Khanh
Judge 3: Prof. Ph.D. Tran Viet Tien

The thesis will be defended before the Thesis Assessment Concil at
Institute level
At , date …… month……. year 2020

Be able to search the thesis at:
1. National library
2. Viet Nam Military Medical University library


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LIST OF WORKS RELATED TO THE THESIS
HAS BEEN PUBLISHED

1. Nguyen Tuan Anh, Do Quyet, Nguyen Huy Luc (2019). Clinical
characteristics and laboratory findings of PCP pneumonia in
HIV/AIDS patients at the National Hospital for Tropical
Diseases from 2014-2017. Vietnam Medical Journal, Vol. 2,
Issue 481: 64-68
2. Nguyen Tuan Anh (2019). Molecular characteristics and genotypes
of Pneumocystis jirovecii Pneumonia in HIV/AIDS patients in
National Hospital for Tropical Diseases from 2014 – 2017. Journal
of Military Medicine. Vol. 7: 28-34



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RATIONALE
HIV/AIDS infection often leads to opportunistic infections due to
weakened immune system. Common opportunistic infections are
infections of the lungs, nervous system, digestive tract, skin and mucous
membranes. The lungs are the most vulnerable organ in HIV/AIDS patients, of
which Pneumocytis jirovecii (PJ) is one of the leading causes of abnormal
opportunistic disease and a major cause of severe pneumonia with high
mortality in HIV/AIDS patients. The clinical symptoms of PJ pneumonia are
gradual, dull, accompanied by a dry cough, fever, fatigue, lose weight,
increased shortness of breath, dry rales, pulmonary x-ray images and
microscopic tomography image of heterogeneous infiltrates, sporadic over two
lungs, severe cases of pneumonia can be seen infiltrating each drive, testing
reduces both erythrocytes, leukocytes, platelets, CD4 cells is reduced
significantly (<200 cells/ml).
With the emergence of the HIV/AIDS epidemic, PJ emerged as a
common cause of disease among HIV/AIDS patients. In the absence of
specific prophylaxis regimens, this etiology was found in more than 60% of
HIV-infected people and about 80% of people with CD4 counts <200
cells/ml. After the prophylactic drug prevention used for PJ, the incidence
of infection has decreased significantly, and continues to sharply decline
when applying highly active ARV (HAART). Even so, PJ continues to be
one of the causes of pneumonia with high morbidity and mortality among
HIV/AIDS patients.
Molecular biology techniques such as gene amplification (PCR) and
nucleotide sequencing techniques have been applied to accurately
diagnose PJ pathogen in respiratory specimens and analyze their
molecular features without using other invasive techniques. Thereby
helping to improve the efficiency of diagnosis and knowledge of
molecular epidemiology of this microorganism. In Vietnam, there has
been less research on opportunistic infections caused by PJ in patients
with HIV/AIDS, especially there have been no studies on molecular
features of this fungus. Therefore, we conducted the project "Study on
clinical, paraclinical features, genotype of PJ causing pneumonia in
HIV/AIDS patients”.


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1. The objectives
1.1. Describe the clinical and paraclinical features of PJ
pneumonia in HIV/AIDS patients.
1.2. Determination of PJ genotype and its correlation with clinical
and paraclinical features of PJ pneumonia in HIV/AIDS
patients.
2. New contributions of the thesis
- This study is one of the few studies done on the problem of
opportunistic infections caused by PJ in patients with HIV/AIDS.
Especially in this study, bronchoscopy of soft tubes was performed in
309 patients as well as summarized important information such as
clinical features, paraclinical, image of lung lesions of PJ infected
patients, In addition, the research has successfully used PCR, real-time
PCR and gene sequencing techniques to analyze molecular biology of
PJ strains. This data provides clinicians as well as the health sector a
general picture of the features of opportunistic infections caused by PJ
in HIV/AIDS patients so that they can provide solutions for diagnosis,
prevention and better treatment.
- The first study to use molecular biology techniques to analyze the
genetic features of pathogenic PJ in Vietnam. This is the basis for
further studies, thereby helping to shed light on the features and
mechanisms of pathogenesis of PJ in HIV/AIDS patients.
- PCR techniques, real-time PCR, genetic sequencing, diagnosis and
identification of genetic variants of pathogenic PJ strains in Vietnam are
very important in quickly identifying the cause of the disease as well as
improving understanding knowledge about the molecular
epidemiological features of this fungal pathogen. Therefore, these
techniques can be applied in practice to identify the cause early, making
treatment and prevention more effective.
- The entire data of this study is a reference for further studies on PJrelated pneumonia in particular and opportunistic infections in general
for HIV/AIDS patients.
3. The layout of the thesis
The thesis consists of 135 pages, with 4 chapters: Introduction 02
pages, Chapter 1 - Overview: 40 pages, Chapter 2 - Objects and research
methods 25 pages, Chapter 3 - Results 35 pages, Chapter 4 - Discussion
30 pages, Conclusions and Pettion 03 pages.


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The thesis has 34 tables, 05 charts, 03 figures, 124 references
including 13 Vietnamese references and 111 English references.
CHAPTER 1. OVERVIEW
1.1. HIV/AIDS infection in Vietnam
Data from the Ministry of Health recorded that in nine months of
2017, the all tested for new detection of 6,883 cases of HIV infection, the
number of patients turning to AIDS was 3,484, the number of patients
died 1,260 cases. The number of new HIV infections is concentrated
mainly in the ages of 20-29 (30%) and 30-39 (40%). The main route of
transmission is unsafe sex (58%) and through blood (32%). Surveillance
results in 2016 noted that the proportion of HIV infection was 9.53%
among injecting drug users, 2.39 among female sex workers, and men
(MSM) was 7.36%, HIV prevalence among MSM increased from 5.1% in
2015 to 7.36 in 2016. The HIV/AIDS epidemic continues to decrease but
still has high potential for HIV infection in the community.
1.2. Opportunistic infections in HIV/AIDS patients
Opportunistic infections in patients with HIV are caused by
pathogens that usually do not cause illness in hosts with normal healthy
immune systems but only when the immune system of the host is
weakened. The strength of the human immune system is assessed by the
number of CD4 cells, the lower the number of these cells, the higher the
risk of opportunistic infections. Common opportunistic infections in
HIV/AIDS patients in Vietnam include thrush, Tuberculosis, Penicillium
marneffei, Cryptococcus neoformans, meningitis, PJ infection, brain
toxoplasmosis, sagging due to Cytomegalovirus, Mycobacterium avium,
Cryptosporidium, Isospora and Cyclospora.
1.3. Clinical manifestations of PJ's pneumonia in HIV/AIDS patients
Pneumonia due to PJ usually starts slowly, thereafter progresses
within a few weeks, severe cases present with shortness of breath,
intermittent speech, cyanosis, head and mucosa. In HIV/AIDS patients, PJ
pneumonia tends to be more acute and has a milder disease presentation
than PJ infection in immunocompromised patients. Data from studies
show that the average incubation period for PJ infection in HIV/AIDS
patients is about 28 days, while that of other patients is only about 5 days.
The statistics show that the mortality rate of HIV-infected patients with PJ


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is much higher than that of other patients. Therefore, the diagnosis,
prevention, and treatment of PJ infection in patients with HIV/AIDS is
extremely urgent and important to improve the life expectancy and quality
of life for patients.
1.4. Paraclinical pneumonia of PJ in HIV/AIDS patients
X-ray image: For PJ infection in HIV/AIDS patients, the classic
image commonly seen on pulmonary X-ray film is diffuse interstitial
infiltrate intermittent translucency, usually the area near the lung helium,
the interstitial blurs may be in the form of mesh, granular tissue or frosted
glass. If not treated in time, these lesions can progress and cause alveolar
coagulation within 3-4 days, alveolar coagulation phenomenon makes it
difficult for patients to breathe. Infiltrates are evident within 2 weeks but
there is a rate of PJ infection progressing to grid lesions and pulmonary
fibrosis, notably with 5-30% of PJ infections having normal chest X-ray
images but still have clinical manifestations of PJ pneumonia.
Chest CT scan: A CT scan can detect a sign of opaque glass on most
PJ cases. The appearance of opaque glass on CT images is associated with
parenchymal infiltrates, the lesions often have bilateral, symmetric,
umbilical, and morphological distribution in diffuse or mosaic form. The
image of solidification and thickening of intermittent walls is often
observed in the subacute stage of disease due to the organizational
inflammation. Cocoon disease is a common infection in pneumonia due to
PJ, usually in large numbers in both sides but different in size, shape and
extent of distribution in the lung, this disease can account for up to 1/cases
of PJ infection.
1.5. Genetic features of PJ
PJ’s genome is about 8.1 Mb in size, coding for 3,878 genes and a
gene density of 480 genes/Mb (or 1 gene/2,029 bp). The study showed
that the diversity of PJ's genotype variants, genetic alterations that
changed the epidemiology, mode of transmission and efficacy of PJ
infection. The genetic diversity of PJ is mainly due to gene mutations
caused by the presence of SNPs. Certain polymorphisms are thought to be
highly associated with clinical pathological manifestations and
epidemiological features. The current molecular techniques allow us to
analyze the relationship between genotype and clinical features of PJ
infection, thereby facilitating treatment decisions and prevention.
1.6. The diagnostic methods for PJ
Chest X-ray: Chest X-ray image, PJ pneumonia patients have opaque
glass on both sides or scatter. Some cases of pulmonary nodules, lobar
infiltrates, and normal chest X-ray images may also occur in about onethird of PJ pneumonia. High-resolution computerized tomography is also


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used to diagnose PJ-induced pneumonia, often seeing translucent or
patchy glasses on the lung's surface, which indicates an accumulation of
debris and fibrin of the alveolar and fungal cells.
Microscopy: The classic diagnostic method is often based on the
morphology of the etiologic cause in respiratory specimens such as sputum,
bronchial fluid or lung tissue. Standard staining methods include
methenamine silver, toluidine blue-O, Giemsa or Diff-Quik staining,
monoclonal antibodies are also used to detect PJ by rapid
immunofluorescence assay, which is very sensitive and easy to conduct.
Immunological method: PJ cell wall contains many components of
β-D-glucan, when infectious and developing in the patient's body, this
substance is usually released outside the patient's serum, but it is not specific
for the diagnosis of PJ infection. Despite this, they are still used as a useful
tool in the diagnosis of PJ pneumonia or at least in disease screening.
However, it should be noted that the rate of false-positive results may occur
due to a number of factors such as sepsis, hemodialysis, use of treatment
drugs, immunoglobulin infusion.
Molecular biology method: Using PCR technique to diagnose PJ
infection has been applied to improve the sensitivity in the diagnosis
of bronchial fluid samples and sputum collected by non-invasive
methods. The test is based on the principle of PJ-DNA presence
detection by amplifying specific gene segments of PJ on different loci.
The sensitivity of the technique has been significantly increased by
selecting polymorphic target genes (Msg gene or gene encoding the
large subunit mitochondrial rRNA - mtLSU) or by using nested PCR
techniques. The most commonly used assay is PCR detecting
polymorphic mtLSU gene
CHAPTER 2. SUBJECTS AND METHODS
2.1. Subjects
All HIV/AIDS patients > 18 years old diagnosed with pneumonia
were admitted to hospital and inpatient treatment at the Department of
Parasite - Virus - National Hospital of Tropical Diseases from 1/1/2014 to
31/12/2017.
2.2. Criteria for selecting subjects
Patients with HIV/AIDS > 18 years old, tested for HIV (+)
(according to HIV/AIDS diagnosis and treatment guidelines of the
Ministry of Health 2017). Clinical presentation of respiratory lesions:
fever, cough, chest pain, shortness of breath or rales. Have chest X-ray or
computerized tomography. Have bronchoscopy and bronchial lavage
samples taken for PCR test to identify PJ. There is a positive PCR test


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result for positive PJ in bronchial lavage swabs of study patients. Patients
agree to participate in the study.


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2.3. Exclusion criteria
Patients with HIV/AIDS infection <18 years old, PCR tests negative
for PJ, patients without X-rays or and computerized tomography. The
patient did not undergo bronchoscopy and did not agree to participate in
the study.
2.4. Methods
2.4.1. Study design: prospective, cross-sectional description
2.4.2. Patient selection method
Sample collection: Select all patients to be treated at the Department of
Parasite - Virus - National Hospital of Tropical Diseases that meet the criteria
for patient selection as mentioned above.
2.4.3. Content and procedures
2.4.3.1. The clinical, paraclinical features of PJ's pneumonia in
HIV/AIDS patients
+ Information collection: Collecting information about patients such
as age, gender, time of HIV infection, transmission route, ARV
treatment….
+ Clinical features:
- Body status: Body condition, Glasgow score, fever, shortness of
breath, properties ...
- Respiratory: Symptoms of cough, shortness of breath, cyanosis,
sputum, rales, spO2 ...
- Cardiovascular: Heart rate, blood pressure, chest pain ....
+ Paraclinical features:
- Blood indices: Test indicators include: Red blood cells, white
blood cells, platelets, hemoglobin, prothrombin, fibrinogen ...
- Biochemistry: Laboratory parameters include: Na +, Cl-, K+, liver
enzymes AST - ALT, urea, creatinine, albumin, CRP ..
- CD4 concentration: Performed on Biomerieux's BD FACSPresto
™ system using 1ml of EDTA patient blood with EDTA according to the
flow principle.
- Chest computerized tomography: Evaluation criteria: bronchitis,
cloudiness, triangular fuzzy mass, nodular infiltration, reticular
infiltration,
cavernous
lesions,
and bronchial-lesion lesions
- Bronchoscopy of soft tubes: Evaluation criteria: Convex masses in the
heart of the bronchus, shallow slippery in the heart of the bronchus,
congestion congestion, stenosis of the top lobes, opacity in the bronchi,
increased foam secretion, bronchial lymphadenopathy, carina edema,
purulent discharge, laryngeal pseudomembranous.


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- HIV load: According to the real-time RT PCR principle, the viral
load is expressed in copies/ml.
2.4.3.2. Molecular features of PJ and clinical, paraclinical in
correlation with pneumonia in HIV AIDS patients
+ Molecular features of PJ: Using PCR technique and sequencing of
08 loci belonging to PJ's mitochondrial genome to determine polymorphic
polymorphisms, locus include: mt26S, 26S rDNA, ITS1, β -TUB, SOD,
CYB, DHPS, DHFR. The sequences of 08 locus were compared with the
original sequences to look for variants, the original sequences have
Genbank code: U07220 (ITS1), AF320344 (CYB), M58605 (mt26S),
L13615 (26S), AF146753 (SOD), AF170964 (β-TUB), AY628435
(DHPS), and AF090368 (DHFR).
+ The correlation with PJ's molecular features and clinical and
paraclinical pneumonia in HIV/AIDS patients:
- Factors related to clinical features: fever, respiratory failure, lung
injury.
- Factors related to clinical features: features of CD4 concentration,
CRP concentration, ARV treatment.
2.5. Enter, manage and process data
The collected data was entered, managed by Epidata 3.1
software, and processed using specialized software STATA 12,
imported and managed references using Endnote X7.
CHAPTER 3.RESULTS
3.1. Clinical and paraclinical features of PJ pneumonia in HIV/AIDS
patients
3.1.1. Features of subjects
Table 3.1. Age distribution of subjects
Age group
Male (n,%)
Female (n,%)
Total (n,%)
<30
4(17,39)
1(12,5)
5(16,13)
30 - 40
13(56,52)
5(62,5)
18(58,06)
Age
>40
6(26,09)
2(25,0)
8(25,81)
P
0.938
Median (SD)
38,1(10,43)
37,3(9,0)
37,9(9,94)
Lowest
20
26
20
Highest
59
56
59
The data show that patients in the age group below 30 accounts for
the lowest proportion, while the age group from 30-40 accounts for the


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highest percentage, the age over 40 has the rate of 25.81%. The average
age of male is 38.1 years (the oldest is 59 and the youngest is 20), the
average age of female is 37.3 years (the highest is 56 and the lowest is
26). The average age of both sexes is 37.9 years.
Table 3.2. Route of HIV tranmission
Male
Female
Total
Route
n
%
n
%
n
%
Sex
17
73,91
4
50,0
21
67,74
Drug injection
1
4,35
0
0,0
1
3,23
Sex+injection
2
8,70
0
0,0
2
6,46
Unknown
3
13,04
4
50,0
7
22,58
P
0,162
HIV transmission among sexually transmitted patients was most
common 67.74%, injecting drug user 3.22%, Sexual transmission and
drug injection 6.46%, unknown 22.58% transmission route.
3.1.2. Clinical features of PJ's pneumonia in HIV/AIDS patients
Table 3.3. Fever features of patient
Fever features
Patients (n)
Proportion (%)
Yes
27
87,09
Fever
No
4
12,90
< 5 days
2
7,41
Fever before
5-10 days
8
29,63
admission
>10 days
17
62.96
o
< 38 C
8
25,81
>38 oC
23
74,19
Fever
Median
38,9 ± 0,9
temperature
Lowest
37,5
Highest
41,0
Fever
7
25,92
Fever, chills
2
7,41
Fever
Constant
4
14,82
properties
Hot fever
10
37,04
Cold fever
4
14,81
≤ 7 days
6
22,22
No fever after
8-21 days
14
51,85
admission
> 21 days
7
25,92


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The majority of patients had a fever, of which more than 62.96% of
cases were over 10 days before admission, 29.63% were 5-10 days and
7.41% were less than 5 days. The average fever temperature is 38.9
degrees, the lowest is 37.5 degrees and the highest is 41 degrees. There
are many different forms of fever, such as fever, constant fever, chills, and
fever + chills. The hospitalization time is up to 8-21 days, with cases
lasting more than 21 days and a few cases less than 7 days.
Table 3.4. Respiratory function features of the patients
Features
Patients (n)
Proportion (%)
Coma
1
3,23
Alert
27
87,09
Status
Alert, tired
2
6,45
drowsy
1
3,23
<15
2
6,45
Glasgow
>15
29
93,55
Normal (>95%)
4
12,90
Respiratory distress
spO2
grade I (90-95%)
15
48,38
Respiratory distress
grade II (< 90%)
12
38,71
<18
0
0
Breathing
18-30
28
90,32
rate
>30
3
9,68
No
17
54,84
Fine crackles
5
16,13
Coarse crackles
3
9,68
Rales
Coarse, fine,
rhonchus
1
3,23
Coarse + fine
5
16,13
No
18
58,06
Two lungs
9
29,03
Right lung
1
3,22
Position of
rales
Scatter
1
3,22
Base of the lungs
1
3,22
Base of left lungs
1
3,22
Most conscious patients had a Glasgow index of > 15, while the
spO2 in the blood of patients had 12 (38.71%) cases < 90%, 15 (48.38%)
90-95 % and only 4 (12,90%) > 95%. Types of rales include fine, coarse,


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rhonchus rales. The most common lung position is in two lungs, 29.03%,
followed by the right lung, scattered, the base of the lung and left lung
3.22%.
3.1.3. Clinical features of PJ pneumonia in HIV/AIDS patients
Table 3.5. Hematological test index of the patient group
Hematological index
Patient (n)
Proportion
(%)
≥ 10.000
6
19,35
White blood cells
(106/L)
< 10.000
25
80,65
≥ 70%
18
58,06
Neutrophils
< 70%
13
41,94
≥ 20%
11
35,48
Lymphocytes
< 20%
20
64,52
> 110
17
54,84
Hemoglobin (g/L)
90 - 110
8
25,81
< 90
6
19,35
< 150
2
6,45
Platelet (10^9/L)
150 – 300
18
58,06
> 300
11
35,48
The majority of patients have normal leukocytosis (<10,000), a small
number of cases of leukocytosis (≥ 10,000). Increased neutrophils (>70%)
accounted for 58.06% and lymphocytes increased (>20%) 35.48%. There
were 54.84% of patients with hemoglobin >110 (g/L), only 19.35% <90 (g/L)
and 25.81% at 90-110 (g/L). There were 58.06% of patients with normal
platelet levels (150-300), 35.48% above normal (> 300 (10 9/L)) and 6.45%
below normal (<150 ( 109/L).
Biochemical indices show that 48.39% of patients have normal Na +
levels and 51.61% of cases show hyponatremia, no increase in Na + index.
There were 61.29% of patients at the normal K + threshold, 3.22% of cases
increased and 35.48% of cases decreased K +, while in Cl- index, 64.52%
of patients decreased, 35,48% of cases were normal and no patients had a
Cl- increase. Patients had a high rate of liver enzyme increase, in which
80.65% increased AST and 61.29% increased ALT, but 77.42% had a
normal urea. There were 93.55% and 74.19% of patients had normal
creatinine and %PT, but up to 64.52% of patients showed signs of
increased fibrinogen, only 32.26% of cases in normal fibrinogen level.
Only 41.94% of patients had normal albumin levels from 35-50 g/l while
58.06 cases showed signs of hypoalbuminemia (<35g/l), no patients
showed an increase in albumin at > 50g/l.


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Table 3.6. Lung features on computerized tomography
Types of injury
Patients Proportion
(n)
(%)
No
27
87,10
Lung injury
Yes
4
12,90
No
4
12,90
Diffuse
23
74,19
Right upper lobe
1
3,22
Position of lung injury
Left upper lobe
1
3,22
Right lung
0
0
Left lung
1
3,22
Scatter
1
3,22
Yes
23
74,19
Blurred
Opacity
2
6,45
nodules
No
6
19,35
Properties
of lung
Yes
27
87,10
Riticular
injury
No
4
12,90
Yes
21
67,74
Opaque
glass
No
10
32,26
Yes
5
16,13
Freezing lung lobes
No
26
83,87
Computerized tomography of patients showed that 87.10% of
patients showed lung injury and 12.90% of patients showed no signs of
lung injury. The position of lung lesions is mainly diffuse form (74.19%),
followed by the right upper lobe, the left upper lobe, scattered and the left
lung each position has 1 (3.22%) patients. Regarding the nature of the
lesions, there are 74.19% of patients with blurred nodules, 6.45% of cases
with opacity, of which 87.10% of patients have grid lesions. The image of
opaque glass appeared in 67.74% of patients and only 16.13% of cases
showed pulmonary solidification, while 83.87% of patients had no signs
of lung lobation.

Chart 3.1. Results of bronchoscopy of flexible tubes


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64.51% of patients had no signs of bronchial injury, of which up to
16.12% of patients had congestion edema, opaque manifestations,
increased secretion of foam, purulent fluid and carina edema each. Of the
9.67% of patients with other endoscopy results, including 3.22% of
patients with multiple convex lumps in the heart of the bronchus, 3.22%
of patients with superficial truncations in the lumen and 3.22 % of
patients with left root lymph nodes.
3.2. Molecular features of PJ and clinical, paraclinical in correlation
with pneumonia in HIV/AIDS patients
3.2.1. Molecular features of PJ
Table 3.7. Results of identifying genotypes of PJ based on nucleotide
sequence alreation on 08 locus
Genotype of each locus
Sam speci
mt26S 26S β-TUB ITS1 CYB SOD DHFR DHPS
1 DPQ
7
1 β-TUB 1 A3 CYB1 SOD 1 Wt
Wt
2 DPQ
7
1 β-TUB 1 A3 CYB1 SOD 1 Wt
Wt
3 DPQ
7
11 β-TUB 1 A3 CYB1 SOD 1 Wt
Wt
4 DPQ
7
1 β-TUB 1 A2 CYB1 SOD 1 Wt
Wt
5 DPQ
2
1 β-TUB 1 A6 CYB1 SOD 1 Wt
Wt
6 DPQ
2
1 β-TUB 1 A1 CYB1 SOD 1 Wt
Wt
7 DPQ
7
1 β-TUB 1 A1 CYB1 SOD 1 Wt
Wt
8 DPQ
2
12 β-TUB 1 A1 CYB1 SOD 1 Wt
Wt
9 DPQ
2
12 β-TUB 1 B7 CYB1 SOD 1 Wt
Wt
10 DPQ 15
13 β-TUB 1 A2 CYB1 SOD 1 Wt
Wt
11 DPQ 16
11 β-TUB 1 A2 CYB1 SOD 1 Wt
Wt
12 DPQ 17
1 β-TUB 1 A5 CYB1 SOD 1 Wt
Wt
13 DPQ 18
1 β-TUB 1 A2 CYB1 SOD 1 Wt
Wt
14 DPQ 19
1 β-TUB 1 B8 CYB1 SOD 1 Wt
Wt
15 DPQ
8
1 β-TUB 1 ND* CYB1 SOD 1 Wt
Wt
16 DPQ
8
11 β-TUB 1 B9 CYB1 SOD 1 Wt
Wt
17 DPQ 20
12 β-TUB 1 B2 CYB1 SOD 1 Wt
Wt
18 DPQ 17
12 β-TUB 1 A1 CYB1 SOD 1 Wt
Wt
19 DPQ 21
1 β-TUB 1 B CYB1 SOD 1 Wt
Wt
20 DPQ
11
12 β-TUB 1 B CYB1 SOD 1 Wt
Wt
21 DPQ 21
1 β-TUB 1 B1 CYB1 SOD 1 Wt
Wt
22 DPQ
7
1 β-TUB 1 A4 CYB1 SOD 1 Wt
Wt
23 DPQ 22
1 β-TUB 1 A2 CYB1 SOD 1 Wt
Wt


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Genotype of each locus
mt26S 26S β-TUB ITS1 CYB SOD DHFR DHPS
24 DPQ 23
1 β-TUB 1 B3 CYB1 SOD 1 Wt
Wt
*
25 DPQ 12
1 β-TUB 1 ND CYB1 SOD 1 Wt
Wt
26 DPQ 21
1 β-TUB 1 B10 CYB1 SOD 6 Wt
Wt
27 DPQ
2
13 β-TUB 1 ND* CYB1 SOD 1 Wt
Wt
28 DPQ 22
1 β-TUB 1 B3 CYB1 SOD 1 Wt
Wt
29 DPQ
11
1 β-TUB 1 A2 CYB1 SOD 1 Wt
Wt
30 DPQ
11
1 β-TUB 1 B3 CYB1 SOD 1 Wt
Wt
31 DPQ 12
12 β-TUB 1 A3 CYB1 SOD 1 Wt
Wt
By analyzing the alteration of nucleotide sequence on 08 locus of 31
pathogenic PJ samples in Vietnam, in addition to identifying the known
mutations (genotypes) (published in previous studies), also identify some new
genotypes (variants) that are specific to PJ strains distributed in Vietnam.
Through this result, PJ found that there are many different variants and
therefore their genetic diversity is very large.
Table 3.8. New genotypes found in PJ strains
Locus
Genotype
Position of nucleotide alterations
15
CGAA/54-57, C/85, C/248, A/288
16
GAT/54-57, A/85, C/248, A/288
17
AAAA/54-57, A/85, T/248, A/288
18
AGTG/54-57, A/85, C/248, A/288
Mt26S
19
GAAA/54-57, C/85, C/248, A/288
20
GCG/54-57, T/85, C/248, A/288
21
GAA/54-57, A/85, C/248, A/288
22
GCAA/54-57, T/85,C /248, A/288
23
GAAA/54-57, A/85, T/248, A/288
T/2, TTT/8-10, A/11, T/17, T/22, TC/46-47,
A6
10T/54-62, GAGG/71-72, TTA/111-113
T/2, TT/8-10, A/11, T/17, T/22, TC/46-47,
B7
10T/54-62, GAGG/71-72, TTA/111-113
C/2, TT/8-10, C/11, T/17, C/22, TC/46-47,
ITS1
B8
9T/54-62, GAGG/71-72, TTA/111-113
C/2, TT/8-10, A/11, T/17, T/22, TC/46-47,
B9
10T/54-62, GAGG/71-72, TTA/111-113
T/2, TT/8-10, C/11, T/17, C/22, TC/46-47,
B10
10T/54-62, GAGG/71-72, TTA/111-113
SOD
6
C/110, A/215

Sam speci


19
Analysis of variants on mt26S locus found nine new genotypes to
appear in PJ samples in this study including genotypes 15, 16, 17, 18, 19,
20, 21, 22 and 23. Locus analysis of ITS1 found five new genotypes, A6,
B7, B8, B9 and B10, while SOD analysis found a new genotype, SOD6.
3.2.2. The correlation between PJ's molecular features and clinical,
paraclinical pneumonia in HIV/AIDS patients
Table 3.9. The correlation between PJ's genotype and patient's fever
features
Fever
Locus Genotype
Yes (%) No (%)
OR
95%CI
p
A
14(56)
3(50)
1
B
9(36)
2(33,33) 0.96 0.13-6.95 0.971
ITS1
0.03ND
2(8)
1(16,67) 0.43
0.539
6.41
2
5(20)
0
Mt26S 7
3(12)
3(50)
Others
17(68)
3(50)
1
15(60)
5(83,33)
1
11
3(12)
0
1
0.1526S
12
5(20)
1(16,67) 1.67
17.8 0.673
9
13
2(8)
0
1
SOD1
24(96)
6(100)
SOD
SOD6
1(40)
0(0)
β-TUB Β-TUB
25(100)
6(100)
DHPS
DHPSwt
25(100)
6(100)
DHFR DHFRwt
25(100)
6(100)
CYB
CYB1
25(100)
6(100)


20
Analysis of the correlation between PJ's genotypic features and
patient's fever expression showed that there was no difference in the
risk of fever expression with infection of ITS1 A and B genotype due
to the distribution of genotypes in locus mt26S is too small, so the risk
of these genotypes cannot be calculated. For genotypes in the 26S
locus, it was found that infection with genotype 12 had a 1.67 times
higher risk of infection than infection with genotypes 1, 11, and 13.
The SOD, β-TUB, DHPS, DHFR, CYB loci were no mutations that are
mostly wild-type, could not calculate the OR and P index.


21
Table 3.10. The correlation between PJ genotype and lung injury
Lung injury
genotyp
e

Yes (n,
%)

No (n,
%
)

OR

A

14(51,85)

3(75,0)

1

B

11(40,74

0

1

ND

2(7,41)

1(25)

0.43

2

5(18,52)

0

1

7

4(14,81)

2(50)

0.22

Others

18(66,67)

2(50)

1

1

17(62,96)

3(75)

1

11

3(11,11)

0

1

12

5(18,52)

1(25)

0.88

13

2(7,41)

0

1

SOD1
SOD6

26(96,3)
1(3,7)

4(100)
0

Β-TUB

27(100)

4(100)

DHPS

DHPSwt

27(100)

4(100)

DHF
R

DHFRwt

27(100)

4(100)

CYB

CYB1

27(100)

4(100)

Locus

ITS1

Mt26S

26S

SOD

95%CI

p

0.03-6.41

0.539

0.02-2.08

0.188

0.07-10.46

0.921

βT
U
B

The risk of no lung injury in unspecified ITS1 genotype was lower
than for A and B genotypes. Meanwhile, there was no lung injury in PJ
infection with mt26S 7 genotype was 0.22 times lower than in other
genotypes. PJ infection with the genotype 26S 12 also was not lower the


22
risk of lung injury compared to other genotypes. The remaining loci have
only one genotype so the risk of lung injury cannot be compared.


23
Table 3.11. The correlation between PJ genotype and CD4
concentration
CD4 (cell/ml)

Locus

ITS1

Mt26S

26S

Genotype

< 100 (n,
%)

> 100
(
n
,
%
)

A

14(53,85)

3(60)

1

B

9(34,62)

2(40)

1.03

ND

3(11,54)

0(0)

2

5(19,23)

0

1

7

4(15,38)

2(40)

2.83

Others

17(65,38)

3(60)

1

1

16(61,54)

4(80)

1

11

3(11,54)

0

1

12

5(19,23)

1(20)

0.8

13

2(7,69)

0

1

SOD

SOD1
SOD6

25(96,15)
1(3,85)

5(100)

β-TUB

Β-TUB

26(100)

5(100)

DHPS

DHPSwt

26(100)

5(100)

DHFR

DHFRwt

26(100)

5(100)

CYB

CYB1

26(100)

5(100)

OR

95%CI

p

0.14 -7.48

0.971

0.35-23.02

0.330

0.07-8.91

0.856

Similarly, the analysis of the risk of CD4 cell decline when infected
with different genotypes shows that there was no difference between
genotypes A and B in ITS1 locus. The risk of advanced CD cell decline
<100 cells/ml is 2.83 times higher than that of genotype 2 and other


24
genotypes. Meanwhile, locus 26S, genotype 12, has the lowest risk
compared to genotypes 1, 11, and 13. The remaining loci have no
mutation, most of them are wild genotypes so there is no difference in
their risk of CD4 reduction.


25
CHAPTER 4. DISCUSSION
4.1. Clinical and paraclinical features of PJ pneumonia in patients
with HIV/AIDS
4.1.1. Clinical features
Reasons for admission and fever features
Of the 31 patients, the main reason for hospitalization was high fever
and shortness of breath, only a small number of patients were hospitalized
for unknown reasons. According to many studies, the lung is the most
vulnerable organ and infection in HIV-infected people. In our study, the
prevalence of PJ is about ~ 10% mainly in patients who first discovered
HIV, have not been on ARV treatment, or have been treated infrequently
or in patients with very low CD4 count < 50 cells/ml, and HIV patients
who are on ARV prophylaxis and have a high CD4 count are less likely to
get the disease. This is also the result of a changing HIV management and
treatment program when it is detected that HIV is managed and treated with
ARV, previously having a test with a CD4 cell count lower than 250 cells to
be treated. This disease is caused by the fungus Pneumocytis jirovecii with
clinical manifestations such as dry cough, fever, tiredness, weight loss,
increasing shortness of breath, these symptoms progress from dull.
The initial clinical manifestation of PJ's pneumonia is a dry cough
and fever, systemic symptoms with prolonged fever are a valuable sign of
importance in the diagnosis of PJ pneumonia in HIV/AIDS patients. In
this study we recorded more than 87% of patients were hospitalized with
fever symptoms, in which many cases (nearly 63%) had manifested fever
more than 10 days before admission and the fever temperature averaged
to nearly 39 degrees. Patients present with various forms of fever,
including fever, malaria, constant fever, and patients continue to have
fever for a long time after admission, especially nearly 26% of the
patients lasting more than 21 days, whereas fever after being admitted
from 8-21 days has nearly 52% of patients and only about 22% of patients
with fever less than 7 days after admission. A number of previous studies
have also recorded a high proportion of patients with fever symptoms
such as that of Le Manh Hung et al. (fever - 93.7%), Meng et al. (fever 100 %), Fei Guo et al implemented from 2008-2012 (fever - 90%), Rego
de Figueiredo et al. (fever - 77%). Thus, the features of PJ's fever
symptoms have similarities between these studies, this is the body's
natural inflammatory response when an attack of pathogenic
microorganisms occurs fever, therefore, is considered one of the most
typical signs of infection diagnosis.


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