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

AND TRAINING DEFENSE VIET NAM MILIRATY MEDICAL UNIVERSITYNGUYỄN TUẤN ANH STUDY ON CLINICAL, PARACLINICAL FEATURES, GENOTYPE OF PNEUMOCYSTIS JIROVECII CAUSING PNEUMONIA IN HIV/AIDS PATIEN

AND TRAINING DEFENSE VIET NAM MILIRATY MEDICAL UNIVERSITY

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

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

LIST OF WORKS RELATED TO THE THESIS

HAS BEEN PUBLISHED

1 Nguyen Tuan Anh, Do Quyet, Nguyen Huy Luc (2019) Clinicalcharacteristics and laboratory findings of PCP pneumonia inHIV/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

HIV/AIDS infection often leads to opportunistic infections due toweakened immune system Common opportunistic infections areinfections of the lungs, nervous system, digestive tract, skin and mucousmembranes 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 highmortality in HIV/AIDS patients The clinical symptoms of PJ pneumonia aregradual, dull, accompanied by a dry cough, fever, fatigue, lose weight,increased shortness of breath, dry rales, pulmonary x-ray images andmicroscopic tomography image of heterogeneous infiltrates, sporadic over twolungs, severe cases of pneumonia can be seen infiltrating each drive, testingreduces both erythrocytes, leukocytes, platelets, CD4 cells is reducedsignificantly (<200 cells/ml)

With the emergence of the HIV/AIDS epidemic, PJ emerged as acommon cause of disease among HIV/AIDS patients In the absence ofspecific prophylaxis regimens, this etiology was found in more than 60% ofHIV-infected people and about 80% of people with CD4 counts <200 cells/

ml After the prophylactic drug prevention used for PJ, the incidence ofinfection has decreased significantly, and continues to sharply decline whenapplying highly active ARV (HAART) Even so, PJ continues to be one ofthe causes of pneumonia with high morbidity and mortality amongHIV/AIDS patients

Molecular biology techniques such as gene amplification (PCR) andnucleotide sequencing techniques have been applied to accuratelydiagnose PJ pathogen in respiratory specimens and analyze theirmolecular features without using other invasive techniques Therebyhelping to improve the efficiency of diagnosis and knowledge ofmolecular epidemiology of this microorganism In Vietnam, there hasbeen less research on opportunistic infections caused by PJ in patientswith 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”.

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 opportunisticinfections caused by PJ in patients with HIV/AIDS Especially in thisstudy, 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 genesequencing techniques to analyze molecular biology of PJ strains Thisdata provides clinicians as well as the health sector a general picture ofthe features of opportunistic infections caused by PJ in HIV/AIDSpatients so that they can provide solutions for diagnosis, prevention andbetter treatment

- The first study to use molecular biology techniques to analyze thegenetic features of pathogenic PJ in Vietnam This is the basis forfurther studies, thereby helping to shed light on the features andmechanisms of pathogenesis of PJ in HIV/AIDS patients

- PCR techniques, real-time PCR, genetic sequencing, diagnosis andidentification of genetic variants of pathogenic PJ strains in Vietnam arevery important in quickly identifying the cause of the disease as well asimproving understanding knowledge about the molecularepidemiological features of this fungal pathogen Therefore, thesetechniques can be applied in practice to identify the cause early, makingtreatment and prevention more effective

- The entire data of this study is a reference for further studies on related pneumonia in particular and opportunistic infections in generalfor HIV/AIDS patients

PJ-3 The layout of the thesis

The thesis consists of 135 pages, with 4 chapters: Introduction 02pages, Chapter 1 - Overview: 40 pages, Chapter 2 - Objects and researchmethods 25 pages, Chapter 3 - Results 35 pages, Chapter 4 - Discussion

30 pages, Conclusions and Pettion 03 pages

The thesis has 34 tables, 05 charts, 03 figures, 124 referencesincluding 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, thenumber of patients turning to AIDS was 3,484, the number of patientsdied 1,260 cases The number of new HIV infections is concentratedmainly in the ages of 20-29 (30%) and 30-39 (40%) The main route oftransmission is unsafe sex (58%) and through blood (32%) Surveillanceresults 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 butstill 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 bypathogens that usually do not cause illness in hosts with normal healthyimmune systems but only when the immune system of the host isweakened The strength of the human immune system is assessed by thenumber of CD4 cells, the lower the number of these cells, the higher therisk 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 progresseswithin a few weeks, severe cases present with shortness of breath,intermittent speech, cyanosis, head and mucosa In HIV/AIDS patients, PJpneumonia tends to be more acute and has a milder disease presentationthan PJ infection in immunocompromised patients Data from studiesshow that the average incubation period for PJ infection in HIV/AIDSpatients 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

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 classicimage commonly seen on pulmonary X-ray film is diffuse interstitialinfiltrate intermittent translucency, usually the area near the lung helium,the interstitial blurs may be in the form of mesh, granular tissue or frostedglass If not treated in time, these lesions can progress and cause alveolarcoagulation within 3-4 days, alveolar coagulation phenomenon makes itdifficult for patients to breathe Infiltrates are evident within 2 weeks butthere is a rate of PJ infection progressing to grid lesions and pulmonaryfibrosis, notably with 5-30% of PJ infections having normal chest X-rayimages 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 withparenchymal infiltrates, the lesions often have bilateral, symmetric,umbilical, and morphological distribution in diffuse or mosaic form Theimage of solidification and thickening of intermittent walls is oftenobserved in the subacute stage of disease due to the organizationalinflammation Cocoon disease is a common infection in pneumonia due to

PJ, usually in large numbers in both sides but different in size, shape andextent 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 agene density of 480 genes/Mb (or 1 gene/2,029 bp) The study showedthat the diversity of PJ's genotype variants, genetic alterations thatchanged the epidemiology, mode of transmission and efficacy of PJinfection The genetic diversity of PJ is mainly due to gene mutationscaused by the presence of SNPs Certain polymorphisms are thought to behighly associated with clinical pathological manifestations andepidemiological features The current molecular techniques allow us toanalyze the relationship between genotype and clinical features of PJinfection, thereby facilitating treatment decisions and prevention

1.6 The diagnostic methods for PJ

Chest X-ray: Chest X-ray image, PJ pneumonia patients have opaqueglass on both sides or scatter Some cases of pulmonary nodules, lobarinfiltrates, and normal chest X-ray images may also occur in about one-third of PJ pneumonia High-resolution computerized tomography is alsoused to diagnose PJ-induced pneumonia, often seeing translucent or

patchy glasses on the lung's surface, which indicates an accumulation ofdebris and fibrin of the alveolar and fungal cells.

Microscopy: The classic diagnostic method is often based on themorphology of the etiologic cause in respiratory specimens such as sputum,bronchial fluid or lung tissue Standard staining methods includemethenamine silver, toluidine blue-O, Giemsa or Diff-Quik staining,monoclonal antibodies are also used to detect PJ by rapidimmunofluorescence assay, which is very sensitive and easy to conduct.Immunological method: PJ cell wall contains many components ofβ-D-glucan, when infectious and developing in the patient's body, thissubstance is usually released outside the patient's serum, but it is not specificfor the diagnosis of PJ infection Despite this, they are still used as a usefultool 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 occurdue to a number of factors such as sepsis, hemodialysis, use of treatmentdrugs, 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 pneumoniawere admitted to hospital and inpatient treatment at the Department ofParasite - Virus - National Hospital of Tropical Diseases from 1/1/2014 to31/12/2017

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 theMinistry of Health 2017) Clinical presentation of respiratory lesions:fever, cough, chest pain, shortness of breath or rales Have chest X-ray orcomputerized tomography Have bronchoscopy and bronchial lavagesamples taken for PCR test to identify PJ There is a positive PCR test

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

2.3 Exclusion criteria

Patients with HIV/AIDS infection <18 years old, PCR tests negativefor PJ, patients without X-rays or and computerized tomography Thepatient did not undergo bronchoscopy and did not agree to participate inthe 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 ofParasite - Virus - National Hospital of Tropical Diseases that meet the criteriafor 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, ARVtreatment…

- CD4 concentration: Performed on Biomerieux's BD FACSPresto

™ system using 1ml of EDTA patient blood with EDTA according to theflow principle

- Chest computerized tomography: Evaluation criteria: bronchitis,cloudiness, triangular fuzzy mass, nodular infiltration, reticularinfiltration, cavernous lesions, and bronchial-lesion lesions

- Bronchoscopy of soft tubes: Evaluation criteria: Convex masses in theheart 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

- HIV load: According to the real-time RT PCR principle, the viralload 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,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, SOD,TUB), AY628435 (DHPS), and AF090368 (DHFR).

+ The correlation with PJ's molecular features and clinical andparaclinical pneumonia in HIV/AIDS patients:

- Factors related to clinical features: fever, respiratory failure, lunginjury

- 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,%)

highest percentage, the age over 40 has the rate of 25.81% The averageage of male is 38.1 years (the oldest is 59 and the youngest is 20), theaverage age of female is 37.3 years (the highest is 56 and the lowest is26) The average age of both sexes is 37.9 years.

Table 3.2 Route of HIV tranmission

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 (%)

The majority of patients had a fever, of which more than 62.96% ofcases were over 10 days before admission, 29.63% were 5-10 days and7.41% were less than 5 days The average fever temperature is 38.9degrees, the lowest is 37.5 degrees and the highest is 41 degrees Thereare many different forms of fever, such as fever, constant fever, chills, andfever + chills The hospitalization time is up to 8-21 days, with caseslasting 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 (%)

Most conscious patients had a Glasgow index of > 15, while thespO2 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,