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MINISTRY OF EDUCATION AND TRAINING MINISTRY OF DEFENCE 108 INSTITUTE OF CLINICAL MEDICAL AND PHARMACEUTICAL SCIENCES --- DAO DUY TUYEN RESEARCH OF CLINICAL PARACLINICAL CHARACTERISTICS

MINISTRY OF EDUCATION AND TRAINING MINISTRY OF DEFENCE

108 INSTITUTE OF CLINICAL MEDICAL AND PHARMACEUTICAL SCIENCES

-

DAO DUY TUYEN

RESEARCH OF CLINICAL PARACLINICAL CHARACTERISTICS AND MICROBIOLOGICAL PATHOGENS IN EXACERBATION OF CHRONIC OBSTRUCTIVE PULMONARY DISEASE WITH

THE THESIS WAS DONE IN: 108 INSTITUTE OF CLINICAL MEDICAL AND PHARMACEUTICAL SCIENCES

Supervisor:

1 Associate Professor Le Huu Song MD, PhD

2 Associate Professor Nguyen Dinh Tien MD, PhD

Day Month Year

The thesis can be found at:

1 National Library of Vietnam

2 Library of 108 Institute of Clinical Medical and Pharmaceutical Sciences

3 Central Institute for Medical Science Infomation and Tecnology

1

INTRODUCTION

Chronic obstructive pulmonary disease (COPD) is a heterogeneous lung condition characterized by chronic respiratory symptoms (dyspnea, cough, sputum production and/or exacerbations) due

to abnormalities of the airways (bronchitis, bronchiolitis) and/or alveoli (emphysema) that cause persistent, often progressive, airflow obstruction Recent studies show that patients with exacerbations of COPD have more than four times the risk of developing pneumonia compared to those without COPD [1] Furthermore, during exacerbations of COPD, the presence of pneumonia increases the number of patients with respiratory failure, necessitates mechanical ventilation, results in poorer treatment outcomes, and leads to higher mortality rates [2]

Semi-quantitative sputum cultures and antibiotic susceptibility testing are valuable in detecting bacterial causes and guiding the selection

of antibiotics for treating exacerbations of COPD with pneumonia However, many pathogens such as atypical bacteria and viruses, cannot be detected by this method Therefore, numerous researchers worldwide have applied multiplex real-time PCR to identify microbial pathogens Currently, in Vietnam as well as globally, there are few studies on microorganisms in exacerbations of COPD with pneumonia, particularly those combining sputum culture with multiplex real-time PCR On that basis, we implemented the topic “Research of clinical paraclinical characteristics and microbiological pathogens in exacerbation of chronic obstructive pulmonary disease with pneumonia” with the purposes:

1 Describe the clinical and paraclinical characteristics of patients during exacerbations of COPD with community-acquired pneumonia

2 Microbiological characteristics of pathogens in patients with exacerbations of COPD with community-acquired pneumonia and antibiotic susceptibility of isolated bacteria

3 Comparison of the value of multiplex real-time PCR with sputum culture in detecting microbial causes and its relation to certain clinical and paraclinical characteristics

2

Chapter 1 OVERVIEW 1.1 Characteristics of exacerbations of chronic obstructive pulmonary disease with pneumonia

1.1.1 Pneumonia in exacerbations of chronic obstructive pulmonary disease

According to Finney L.J et al (2019), the relationship between COPD exacerbations and pneumonia is quite complex due to differing perspectives Some authors consider pneumonia to be a cause of COPD exacerbations, while others view pneumonia as a clinical presentation and

a separate condition from COPD exacerbations [2] This lack of consensus may stem from inconsistent interpretations of the relationship between pneumonia and COPD exacerbations in various COPD guidelines The

2004 general guidelines from the American Thoracic Society for COPD listed pneumonia as one of several high-risk comorbid conditions commonly encountered during exacerbations The 2013 GOLD report included pneumonia in the list of conditions that can “mimic/worsen COPD exacerbations.” Meanwhile, the 2018 COPD guidelines from the UK's National Institute for Health and Care Excellence (NICE) stated that the presence of consolidation on chest X-rays is a risk factor for hospitalization, implying that pneumonia does not need to be ruled out in the diagnosis of a COPD exacerbation but is instead a clinical presentation

of exacerbations, which worsens their severity [2]

Recent studies show that patients with exacerbations of COPD are more prone to pneumonia due to the impairment of the lungs' defense mechanisms, including mechanical, humoral, and cellular factors This makes these patients more susceptible to lower respiratory tract infections, increasing the risk and incidence of pneumonia According to Restrepo M.I et al (2018), the bronchial and pulmonary mucosal surfaces in COPD patients are frequently exposed to microbial agents capable of causing pneumonia The risk of pneumonia may be associated with host susceptibility factors or changes in the microbiota, allowing for an increased presence of pathogenic microorganisms Imbalance in the microbiome can contribute to pneumonia by disrupting the normal microenvironment, enabling pathogens to invade and grow in the lower respiratory tract [3] COPD patients are more susceptible to pneumonia due

to chronic bronchitis, which involves persistent mucus secretion and the regular presence of pathogenic bacteria in the airways, even during stable periods, with bacterial counts increasing during exacerbations [4] Additionally, the increased risk of pneumonia in COPD patients has been

3 shown to be related to the use of inhaled corticosteroids (ICS) in treatment [5]

1.1.2 Clinical characteristics of patients with exacerbations of chronic obstructive pulmonary disease with pneumonia

The clinical presentation of exacerbations of COPD with pneumonia does not differ significantly from that of COPD exacerbations without pneumonia Symptoms still include changes in respiratory symptoms (dyspnea, cough, and/or increased sputum production) beyond the normal daily variation However, COPD exacerbations with pneumonia tend to be more severe, with more pronounced symptoms and a higher risk

of mortality Patients often experience septic shock, rapid breathing, low PaO2, and low oxygen saturation, along with more purulent sputum and crackles at the site of the pneumonia Research by Restrepo M.I et al showed that COPD exacerbation patients with pneumonia treated in intensive care units had a high mortality rate (39%) The 30-day and 90-day mortality rates were also higher compared to COPD patients without pneumonia [6]

1.1.3 Paraclinical characteristics of patients with exacerbations of chronic obstructive pulmonary disease with pneumonia

* Imaging features of exacerbations of COPD with pneumonia on chest CT: In addition to the usual findings of COPD exacerbations, pneumonia can manifest on CT scans with the following characteristics [7]:

- Alveolar lesions: Homogeneous opacities affecting multiple segments or entire lobes of the lungs, with the air bronchogram sign (lobar pneumonia)

or diffuse consolidation in both lungs

- Bronchopulmonary lesions: Multiple nodular opacities that may cluster together or be scattered across lung segments, with an uneven distribution between healthy lung areas and damaged regions

- Interstitial lesions: Thickened bronchial walls, irregular nodular interstitial lesions, or a reticular pattern

* Blood test characteristics of COPD exacerbations with pneumonia: blood tests, including complete blood count and biochemical markers, show similar changes in patients with COPD exacerbations both with and without pneumonia However, in patients with pneumonia, more severe alterations may be observed in tests related to inflammation and respiratory failure

1.2 Microbiological characteristics of exacerbations of chronic obstructive pulmonary disease with pneumonia

Research by Huerta A et al (2013) found that Streptococcus

pneumoniae is the most common pathogen in patients with exacerbations

4

of COPD and pneumonia (24 out of 116 samples, accounting for 20.68%), significantly more than in patients with exacerbations without pneumonia

(6 out of 133 samples, accounting for 4.51%) In contrast, H influenzae

was more frequently found in patients with COPD exacerbations (11 out of

133 samples, 8.27%) than in those with pneumonia (3 out of 116 samples, 2.58%) [8] In another study by Finney L.J et al (2019), the authors compared the microbiological characteristics of isolates obtained from sputum cultures between two groups: 235 patients with exacerbations of COPD and pneumonia versus 706 patients with exacerbations of COPD without pneumonia They found that many bacterial species were isolated from both groups, with positive sputum cultures in 28% of patients with pneumonia and 15% of patients without pneumonia The most common

bacterium in the pneumonia group was Pseudomonas aeruginosa,

accounting for 7.23%, while in the non-pneumonia group, it was 4.98% [2] A limitation of these studies is that the identification of pathogens from sputum samples was conducted using culture methods, which may affect the reliability of the results This approach may overlook cases involving difficult-to-culture or non-culturable microorganisms, such as atypical bacteria or viruses

Chapter 2 SUBJECTS AND METHODS 2.1 Subjects

- A total of 138 patients with exacerbations of chronic obstructive pulmonary disease (COPD) were hospitalized in the Respiratory Department at 108 Military Central Hospital, divided into two groups: group I included 92 patients diagnosed with exacerbations of COPD with pneumonia, and Group II included 46 patients diagnosed with exacerbations of COPD without pneumonia for comparison

- Data collection period: from December 2019 to September 2023

- Inclusion criteria:

+ Patients diagnosed with COPD through pulmonary function tests after the exacerbation has stabilized Patients with community-acquired pneumonia during an exacerbation of COPD will be classified into Group

I Patients with exacerbations of COPD without pneumonia will be classified into Group II

+ Patients must be alert and capable of answering research questions + Patients must consent to participate in the study

- Diagnosis criteria for COPD: according to the GOLD 2019 standards [9]

5 + Clinical signs: presence of risk factors (age > 40, smoking, living and/or working in polluted air), chronic cough, sputum production, and shortness

- Diagnosis criteria for pneumonia: according to CDC/NHSN 2014 [11] + Abnormalities on chest X-ray or CT showing consolidation

+ And at least one of the following major criteria: fever over 38°C not explained by other causes; leukocytosis ≥ 12 G/L or leukopenia < 4 G/L; mental changes not explained by other causes in patients ≥ 70 years old + And two of the following minor symptoms: new onset of purulent sputum; change in sputum characteristics; increased respiratory secretions; increased frequency of suctioning sputum; new or progressively worsening cough; shortness of breath, or respiratory rate > 25 breaths/min; lung auscultation revealing crackles or wheezing; blood gas changes: decreased oxygen saturation (PaO2/FiO2 < 240), increased oxygen demand, or increased ventilation needs

- Exclusion criteria:

+ Patients with other chronic lung diseases such as lung tumors, pulmonary tuberculosis, bronchiectasis, or pulmonary fibrosis (based on clinical examination, sputum tests, X-ray, or CT scan)

+ Patients with combined heart failure or severe respiratory complications who cannot undergo chest CT or pulmonary function tests

+ Patients who do not consent to participate in the study

6 Anthonisen, BAP65, Burge, severity of pneumonia using PSI, SMART-COP, and CURB-65

- Paraclinical characteristics: Assessment of blood count, blood biochemistry, CRP and PCT levels, pulmonary function tests, arterial blood gases, characteristics of lung lesions on chest X-ray and CT scan

2.2.2 Microbiological characteristics of pathogens in patients with exacerbations of COPD with community-acquired pneumonia and antibiotic susceptibility of isolated bacteria

- Determine the rates of various bacterial species through semi-quantitative sputum culture and assess their antimicrobial susceptibility

- Identify the rates of certain bacteria, atypical bacteria, and viruses using multiplex real-time PCR

2.2.3 Comparison of the value of multiplex real-time PCR with sputum culture in detecting microbial causes and its relation to certain clinical and paraclinical characteristics

- The ability to detect microbial pathogens, the number of agents identified through semi-quantitative cultures, and multiplex real-time PCR

- Characteristics of microbial pathogens, the ability to identify subtypes of microbes, and the concordance between semi-quantitative cultures and multiplex real-time PCR

- The relationship between microbiological results and certain clinical and paraclinical characteristics

2.3 Research Methods

- Study design: Cross-sectional descriptive, prospective, controlled study

- Sample size: The number of patients in group I was calculated based on the sample size formula for estimating proportions [12], resulting in a requirement of n ≥ 82 patients In this study, we included 92 patients in group I, while group II was composed of half the size of group I, totaling

46 patients

- Ethics of the study: The research protocol was approved by the ethics

committee of 108 institute of clinical medical and pharmaceutical sciences Patients and their relatives voluntarily participated

- Data analysis: Data was processed using medical statistical methods, with the SPSS 20.0 statistical software Qualitative results are expressed as percentages, while quantitative results are expressed as mean ± standard deviation (SD) A p-value < 0.05 is considered statistically significant

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Chapter 3 RESULTS 3.1 Clinical and paraclinical characteristics of patients with

exacerbation of chronic obstructive pulmonary disease with

community-acquired pneumonia

3.1.1 Clinical characteristics

Table 3.8 Functional symptoms of the two patient groups in the study

Symptoms Group I (n=92) Group II (n=46) p

Number Percentage Number Percentage

Comments: In group I, a majority of patients (57.61%) had fever, while

group II had a significantly lower rate of fever (21.74%), with this

difference being statistically significant (p < 0.05) The proportion of

patients with green sputum and chest pain in Group I was higher and

statistically significant compared to group II (p < 0.05)

Table 3.10 Physical symptoms of the two patient groups in the study

Symptoms

Group I (n=92)

8 Comments: The physical examination symptoms such as crackles and moist rales were found in group I at rates of 86.95% and 39.13%, respectively, which are significantly higher than those in group II (17.39% and 19.56%) with p<0.05 However, the rates of other physical examination symptoms, such as wheezing and decreased vesicular breath sounds, were lower in group I compared to group II, with a statistically significant difference (p<0.05)

Table 3.15 Results of blood PCT level test

I (50%) is also higher than in group II (23.91%), with these differences being statistically significant (p < 0.05)

9

Figure 3.1 ROC curve of leukocytes (WBC), neutrophils (N), CRP, and PCT levels in blood for the diagnosis of community-acquired

pneumonia

Comment: The area under the curve (AUC) for evaluating the ability to

diagnose community-acquired pneumonia in the exacerbation of COPD patients is 0.67 for leukocytes, 0.68 for neutrophils, 0.67 for PCT, and 0.78 for CRP CRP is a marker with fairly good reliability in predicting community-acquired pneumonia in patients with exacerbation of COPD, with a Youden index of 0.43 at a CRP cutoff of ≥ 40.8 mg/L, yielding a sensitivity of 56% and a specificity of 87%

3.2 Microbiological characteristics of pathogens in patients with exacerbations of COPD with community-acquired pneumonia and antibiotic susceptibility of isolated bacteria

3.2.1 Results of sputum bacterial culture and antibiotic sensitivity of isolated sputum bacteria

Chart 3.2 Results of sputum culture in the two studied patient groups

Comment: The rate of positive sputum cultures in group I was 50/92

(54.35%), and in group II was 30/46 (65.22%); however, there was no

10 statistically significant difference between the two groups in terms of the positive sputum culture rate, with p>0.05

Table 3.24 Bacterial species isolated from the two studied patient

groups with positive sputum culture results

Comments: Among the gram-negative bacteria isolated from sputum

cultures, K pneumoniae was the most frequently encountered bacterium in Group I, accounting for 20.00%, followed by M catarrhalis at 16.00% and

A baumannii at 14.00% Among the gram-positive bacteria, S mitis had

the highest proportion (6.00%) in group I There was no statistically significant difference in the proportions of bacterial species cultured from sputum samples between group I and group II, with p>0.05

84.62 84.62 71.43 28.57

28.57 18.18

15.38 7.69 7.14 7.14

7.14 9.09

0 7.69 21.43 64.29

64.29 72.73

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Chart 3.4 Antibiotic susceptibility results of Moraxella catarrhalis

Comments: M catarrhalis exhibited a high antibiotic sensitivity rate to

Chloramphenicol (84.62%), Amoxicillin/Clavulanic acid (84.62%), and Cefotaxime (71.43%) However, it was resistant to Trimethoprim/Sulfamethoxazole (72.73%), as well as to Levofloxacin and Ciprofloxacin, both with a resistance rate of 64.29%

Chart 3.5 Antibiotic susceptibility results of Klebsiella pneumoniae

Comments: The antibiotic susceptibility results for K pneumoniae

indicate that the highest sensitivity rate is to Amikacin (87.50%), followed

by Fosfomycin (76.92%), Ertapenem, and Meropenem (both at 75.00%), and Gentamicin (73.33%) A high resistance rate is observed for Ampicillin (93.75%) and Nitrofurantoin (60.00%)

3.2.2 Microbiological results in sputum using multiplex real-time PCR

Chart 3.7 Microbiological results by multiplex real-time

87.5 76.92 75 75 73.33 62.5 62.5 62.5 62.5 53.85 50 20 0

12.5 0

0 0 0 0 0 0 18.75 7.69 12.5 20

6.25

0 23.08 25 25 26.67 37.5 37.5 37.5 18.75 38.46 37.5 60 93.75

12 Comments: The rate of positive sputum real-time PCR tests in group I was 79.35%, and in group II was 67.39% There was no statistically significant difference between the two groups in terms of positive sputum rates, with p>0.05

Table 3.25 Characteristics of microbial species in sputum using

multiplex real-time PCR

Comments: Among the bacteria, K pneumoniae was the most common gram-negative bacterium found in sputum samples from both group I patients (67.12%) and group II patients (77.42%) Among the gram-

positive bacteria, S pneumoniae was detected at the highest rate, with 21.92% in group I and 22.58% in group II Regarding atypical bacteria, M

pneumoniae and L pneumophila were found in 3 group I patients (4.11%)

and in 1 group II patient (3.23%) As for viruses, influenza A and B were

detected 5 in group I patients (6.85%) and 3 in group II patients (9.68%) There was no statistically significant difference in the detection rates of the various agents in sputum samples between the two groups using multiplex

real-time PCR, with p>0.05

3.3 Comparison of the value of multiplex real-time PCR with sputum culture in detecting microbial causes and its relation to certain clinical and paraclinical characteristics

3.3.1 Comparison of the value of the multiplex real-time PCR method with sputum culture