epidemiology of nosocomial pneumonia in the intensive care unit of bach mai hospital, 2008-2009

baumannii AFLP Amplified Fragment Length Polymorphism CDC Centers for Disease Control and Prevention COPD Chronic Obstructive Pulmonary Disease HAI Healthcare - Associated Infection

A baumannii A baumannii

AFLP Amplified Fragment Length Polymorphism

CDC Centers for Disease Control and Prevention

COPD Chronic Obstructive Pulmonary Disease

HAI Healthcare - Associated Infection

HAP Healthcare-Associated Pneumonia

HCFs Health Care Facilities

MDRO Multidrug-Resistant Organism

MRSA Methicillin-Resistant Staphylococcus aureus

PCR Polymerase Chain Reaction

PFGE Pulse-Field Gel Electrophoresis

P.aeruginosa Pseudomonas aeruginosa

WHO World Health Organization

INTRODUCTION

Healthcare-associated pneumonia (HAP) is defined as pneumonia that occurs in patients that reside in or have resided in a long-term care facility, acute-care facility, or other healthcare facility HAP occurs for at least 48 hours after admission to the hospital/healthcare facility

HAP is currently the second most common nosocomial acquired) infection and is the leading infection among those detected in ICUs 83% of episodes of HAP were associated with mechanical ventilation Microorganisms colonize the stomach, upper airway and bronchi, and cause infection in the pneumonia They are often endogenous (digestive system or nose and throat), but may be exogenous, often from contaminated respiratory equipment According to the findings of some studies conducted in the United States and in the Europe, the mortality rate

(hospital-in the patients with of HAP caused by common multi-antibiotic resistant

bacteria such as A baumannii and P.aeruginosa, account for more than

70%; these pathogens are uncommon in non-ICU settings

HAP is the most common HAI in Vietnamese ICUs and it can extend hospitalization by an average of 7 to 14 days per patient and increase the costs of hospitalization Mechanical ventilation is found the important risk

factor associated with HAP A baumannii and P.aeruginosa are the

leading isolated microorganisms contributing to HAI More than 60% A baumannii and P.aeruginosa are resistant to commonly used antibiotics.The emergence and dissemination of resistant organisms is considered as one of the challenging problems in medical science with clinical, economical, and public health implications

There remains a paucity of information on the magnitude, risk factors, as well as antibiotic resistance and molecular characteristics of pathogens causing HAP in the Vietnamese ICUs The lack of such information represents a challenge for HAP control and prevention The objectives of this study were to determine: (1) The incidence of and risk factors for HAP in ICU of Bach Mai hospital; and (2) Pathogens, antibiotic resistance and molecular characteristics of common bacteria causing HAP

SUMMARY OF NEW CONTRIBUTIONS This is the first thesis in Vietnam aimed to describe comprehensively the incidence of, risk factors for HAP, as well as antibiotic resistance and molecular characteristics of pathogens causing HAP in the Intensive care

unit of Bach Mai Hospital which is the area with the highest incidence density of healthcare associated infections among Vietnamese HCFs

HAP was commonly observed in patients of Intensive care unit, Bach Mai hospital The incidence of HAP accounted for 18.9% The overall HAP density was 11.6 per 1,000 patient-days Higher densities found in patients with tracheotomy and endotracheal intubation The HAP rates per 1,000 tracheotomy and endotracheal intubation-days were 27.4 and 72.1, respectively Risk factors for nosocomial found by logistic regression analysis were: (1) Chronic respiratory diseases (OR = 1.9; p < 0.001), (2) Endotracheal intubation (OR = 3.9; p < 0.05), (3) Endotracheal intubation (OR = 6.3; p < 0.01), (4) Surgical procedure (OR = 2.5; p < 0.05) The

thesis not only confirmed the increased trend in HAP caused by A baumannii but also recognized the growth of antibiotic resistances of this pathogen Carbapenem is one of most effective antibiotics against A baumannii However, the percentages of A baumannii isolates resistant to

imipenem and meropenem were 84.9% and 86.8%, respectively, which were much higher than recent studies conducted in US and Europe

PFGE analysis revealed that a total of 46 (86.7%) among 53 A baumannii isolates belong to 6 major clusters (A, B, C, D, E and F) with

the high similarity index of DNA pattern ranged from 80.0% to 96.1% Clone D was the most predominant and had been detected through the entire 8-month study period These findings suggest a hypothesis that is

consistent with studies showing the cross-transmission of A baumannii

between patients The dissemination of this pathogen is facilitated by poor compliance with aseptic techniques and contributes to high colonization rates among hospitalized patients, healthcare workers and frequent

contamination of environments A baumannii colonizing might easily

transmitted to other patients via invasive procedures

The thesis shows scientific evidences on HAP, antibiotic resistance and molecular characteristics of pathogens causing HAP These findings suggest areas for intervention and for developing guideline for prevention and control of HAP in HCFs

THESIS STRUCTURE

The thesis consists of 129 pages Background: 3 pages; Overview: 39 pages; Methods: 20 pages; Results: 33 pages; Discussion: 31 pages; Conclusions: 2 pages; Recommendations: 1 page; 45 tables ans 18

illustrations; 155 references including 37 Vietnamese and 118 English ones

Chapter 1 OVERVIEW

1.1 Epidemiology of HAP

nosocomial (hospital-acquired) infection and is the leading infection among those detected in ICUs More than 80% of episodes of HAP were associated with mechanical ventilation Previous multicenter cohort studies that consisted of more than 5 mixed ICUs of multidisciplinary hospitals had reported nearly comparable infection rates for ventilator associated pneumonia: 14.8 cases per 1,000 ventilator-days in Canadian ICUs, 13.3 cases per 1,000 ventilator-days in German ICUs, 9.4 cases per 1,000 ventilator-days in French ICUs, and 12.6 per 1,000 ventilator-days in Japan HAP is the most common HAI in Vietnamese ICUs and it can extend hospitalization by an average of 7 to 14 days per patient and increase the mortality rate by from 20% to 30% The highest rate of death

is found in patients with HAP which was caused by multi-antibiotic

resistant bacteria such as A baumannii and P.aeruginosa

1.1.2 HAP in Vietnam: A one day-prevalence study at 36 hospitals

across Vietnam in 2008 showed an overall HAI prevalence of 7.8% The most common infection was pneumonia, account for > 60% of the detected HAIs HAP rates ranged from 20% to 25% in recent years Respiratory invasive procedures (endotracheal intubation and tracheotomy) are the most important risk factors for HAP. The increased trend in HAP caused

by multi-antibiotic resistant negative bacteria is recognized The

percentages of A baumannii and P.aeruginosa isolates resistant to

imipenem in ICU, Bach Mai hospital increased from < 20% (2002) to > 40% (2006)

1.1.3 Etiology of HAP: Bacteria have been the most frequently isolated pathogens Among microorganisms isolated from oropharyngeal aspiration and sputum, anaerobes and fungi account for 73% and 4%, respectively HAP tends to be associated with multiple organisms and the common

bacteria are gram-negative bacilli However, MRSA and other positive cocci including Streptococcus pneumoniae has increased in

Gram-frequency in the last few years The data collected and reported by hospitals participating in the National Nosocomial Infections Surveillance

(NNIS) System showed that P.aeruginosa, Enterobacter sp., Klebsiella

pneumoniae, Escherichia coli, Serratia marcescens and Proteus sp

accounted for 50% isolates identified from respiratory specimens of patients with HAP

1.1.4 Source of Infectious Agents: HCWs, patiens, visitors and hospital

environment

1.1.5 Modes of transmission

1.1.5.1 Contact Transmission: The most common mode of transmission, contact transmission is divided into 2 subgroups: direct contact and indirect contact Indirect contact is important contributor to the pathogen transmission involving the transfer of an infectious agent through a contaminated intermediate object or person such as contaminated hands of HCWs, patient-care devices, food, water or contaminated infusion

1.1.5.2 Airborne transmission: Microorganisms from infectious individual

or person who is colonized with bacteria, may be inhaled by susceptible individuals through the dissemination of either airborne droplet

nuclei or small particles (< 5μm)in the respirable size range containing infectious agents that remain infective over time and distance (eg, spores of

Aspergillus spp and M tuberculosis)

1.1.5.3 Droplet transmission: Respiratory droplets (> 5 μm) carrying

infectious pathogens transmit infection when they travel directly from the respiratory tract of the infectious individual to susceptible mucosal surfaces of the recipient, generally over short distances

1.1.6 Susceptible individuals: age 65 years or more, underlying

conditions, especially chronic respiratory diseases, depressed level of consciousness, thoracic or chest surgery, continuous mechanical ventilation, use of paralytic agents, severe trauma, upper abdominal surgery, and recent bronchoscop

1.1.7 Pathogenesis of HAP: (1) HAP is caused by bacteria from remote

infection sites; (2) HAP is caused by bacteria from adjadcent aer subdiaphragmatic abscesseas of lungs (subdiaphragmatic abscess, mediastinal abscess, pleural infection v.v), (3) Aspiration of oropharyngeal

bacteria is the most common initiating event These bacteria come from

exogenous sources (environment, medical devices, HCWs) or from endogenous flora (hollow viscera such as respiaratory tract or gastrointestinal tract)

1.1.8 Risk factors for HAP

1.1.8.1 Host related: age 65 years or more, underlying conditions,

especially chronic respiratory diseases, depressed level of consciousness,

thoracic or chest surgery, use of paralytic agents, severe trauma, upper abdominal surgery, and recent bronchoscopy

1.1.8.2 Device related: Tracheal intubation, continuous mechanical

ventilation, orogastric or nasogastric tube placement, and frequent (e.g., every 24 hours) ventilator circuit changes

1.1.8.3 Increased colonization: Admission to the ICU, administration of

broad-spectrum antibiotics, prophylaxis for stress ulcer bleeding with antacids or H2 blocker, exposure to contaminated medical equipment, and inadequate hand hygiene

1.1.8.4 MDRO: Hospitalization for more than 7 days before the diagnosis

of HAP, transferred from another care facility, ventilation for more than 3 days before the diagnosis of HAP, active malignancy, AIDS, end-stage liver or renal disease, steroids (e.g., prednisone 10 mg/day or more for more than 7 days), active chemotherapy or radiotherapy, and bronchiectasis Prior antibiotic use for more than 3 days within the previous 14 days of the diagnosis of HAP also is considered a risk factor for resistant organisms

1.1.8.5 Treatment related: No recommendation can be made for routinely

acidifying gastric feeding It creates favorable conditions for the growth of bacteria

1.1.8.6 Others: Inhalation of contaminated aerosols or airborne microbes

can also introduce bacteria into the lower respiratory tract This can occur during events such as utility interruptions, remodeling, or construction where infection control recommendations have not been followed

1.1.9 Control and prevention of HAP

1.1.9.1 Prevention of person-to-person transmission of bacteria:

Respiratory equipment must be properly cleaned and sterilized Ventilator tubing must be handled appropriately to avoid spilling condensate into the patient’s airway Sandard and transmission based precautions must be applied in patient care

1.1.9.2 Pneumococcal vaccination: Vaccinate patients at high risk for severe pneumococcal infections

1.1.9.3 Precautions for prevention of aspiration: rinsing oral for patients,

positioning the patient supine with the head elevated 30–45 degrees, suctioning measures including continuous suctioning of subglottic secretions, and minimizing the use of sedating or paralytic agents

1.1.9.4 Precautions for MDRO: Selection of appropriate therapy is

essential to avoid the detrimental effects of antibiotic overuse and the production of selective pressure for resistant organisms

1.1.9.5 Other precautions: Educate HCWs about measures to prevent and

control HAP, conduct surveillance for HAP in ICU patients and provide feedback on HAP rates and the compliance of HCWs with aseptic techniques

1.2 Antibiotic resistance characteristics of bacteria causing HAP

Growing cause of HAP including Acinetobacter baumannii, P.aeruginosa which are associated with increasing antibiotic resistance in

HCFs Exposure to any antibiotic active against GNB has been associated with the emergence of multidrug-resistant 3 classes of antibiotics have been most frequently implicated The use of third-generation cephalosporins has been implicated in numerous case-control studies In addition, Landman found that aggregate use of cephalosporins plus aztreonam, but not other antibiotic classes, was associated with the presence of multidrug-resistant (including carbapenem-resistant)

Acinetobacter isolates Numerous subsequent studies have shown that the

higher rate of multiantibiotic resistance in the strains can be responsible for oligoclonal outbreaks Therefore, MDROs illustrate the potential for outbreak isolates

Four major mechanisms of antimicrobial resistance include: (1) Drug inactivation: Occurs when a bacterium produces an enzyme that can destroy or inactivate the antimicrobial; (2) Alteration in target site: Drug receptor or target sites may undergo alteration; (3) Decreased permeability

or efflux: Changes in drug permeability or an efflux of drug may be observed as in the case of P aeruginosa that has developed resistance to

the carbapenem; (4) Bypass of a metabolic pathway: bacteria may develop alternative metabolic pathways to bypass the pathway that was inhibited by the antimicrobial; resistance to trimethoprim-sulfamethoxazole commonly occurs in this manner

1.3 Molecular epidemiological profile of infection with

multidrug-resistant A baumannii and Pseudomonas species

Early studies of panresistance in Pseudomonas aeruginosa showed

little evidence of clonality among these strains However, numerous subsequent studies have shown that these strains can be responsible for

oligoclonal outbreaks, particularly in ICU, clearly illustrating the potential for person-to-person spread It is not clear whether transmission occurred via common environmental sources or the hands of health care workers

1.4 Application of Molecular Techniques to the Study of HAI

1.4.1 Characteristics of typing methods

1.4.1.1 PFGE: The principle of this technique is to digest chromosomal

DNA with restriction enzymes, resulting in a series of fragments of different sizes that form different patterns when analyzed by agarose gel electrophoresis By periodically changing the direction of the electrical field in which the DNA is separated, PFGE allows the separation of DNA molecules of over 50 kbp in length In general PFGE is one of most reproducible and highly discriminatory typing methods available, and it generally is the method of choice for many hospital epidemiologic evaluations

1.4.1.2 Southern blot analysis: The bacterial DNA is digested using a

frequent cutting restriction enzyme, the DNA fragments are separated by agarose gel electrophoresis, and then the fragments are transferred (blotted) onto a nitrocellulose or nylon membrane Next, a labeled (colorimetric or radioactive) piece of homologous DNA is used to probe the membrane The discriminatory power of this method is related to the copy numbers of the targeted genetic elements in the bacterial genome and their distribution among the restriction fragments following electrophoresis

1.4.1.3 Plasmid Analysis: Typing is performed through the isolation of

plasmid DNA and comparison of the numbers and sizes of the plasmids by agarose gel electrophoresis.Evaluation of plasmid content is not generally useful in delineation of strain relatedness Plasmid analysis has been applied in clinical situations to determine the evolution and spread of antibiotic resistance among isolates with different PFGE profiles or among different species of organisms within hospitals

1.4.1.4 Typing Methods Using PCR: This technique is a biochemical in

vitro reaction that permits the synthesis of large quantities of a targeted nucleic acid sequence The procedure requires template DNA from the organism being typed, two complementary oligonucleotide primers that are designed to flank the sequence on the template DNA to be amplified, and a heat-stable DNA polymerase A growing number of organisms have been studied using this approach

1.4.1.5 AFLP: This is a typing method that utilizes a combination of

restriction enzyme digestion and PCR The method utilizes the benefits of restriction fragment length polymorphisms analysis with the increased sensitivity of PCR to generate profiles that are reproducible and relatively easy to interpret and compare to those for other isolates from a nosocomial outbreak

1.4.2 Cost-effective application of typing methods in HAI study

Understanding pathogen distribution and relatedness is essential for determining the epidemiology of HAIs Molecular techniques can be very effective in tracking the spread of nosocomial infections due to genetically related pathogens, which would allow infection control personnel to more rationally identify potential sources of pathogens and aid infectious disease physicians in the development of treatment regimens to manage patients affected by related organisms In addition, the incorporation of molecular testing in the infection control program for endemic HAIs is associated with the ability to enact early interventions following the identification of pathogen clonality, which could be an early indication of an outbreak.Conversely, the determination of the unrelatedness of isolates (sporadic infections), avoids triggering unneeded and costly epidemic investigations Cost reduction was also accomplished by earlier recognition of person-to-person spread of isolates compared to that with traditional surveillance, thus potentially preventing the spread to additional patients

Chapter 2 STUDY Population, matERIALS and METHODS

2.1 Study population: Patients admitted to the ICU of Bach Mai hospital

for at least 48 hoursand microbial isolates identified from clinical samples

of suspected/confirmed patients with HAP

2.2 Study design: Descriptive and molecular study

2.2.1 Selection of study sample

- Study patients: Patients admitted to the ICU for at least 48 hours were assessed during the study period from September 2008 to April 2009

- Bacteria and fungi were isolated from patients with HAP

2.2.2 Sample size: The caculation was based on WHO formula for

estimating a population proportion with specified relative precision The details are as follows:

2.3 Methods: Data collection techniques include clinical/paraclinical

examination, microbiologic evaluation and PFGE analyses of common bacteria causing HAP

2.3.1 Study indicators:

2.3.1.1 HAP characteristics:(1) Incidence and density of HAP; (2)

Etiology of HAP: The distribution of HAP pathogens and their antibiotic resistant level, (3) Risk factors for HAP and (4) Outcomes of HAP: length

of stay in ICU, patient outcomes, and hospital cost

2.3.1.2 Molecular characteristics of common bacteria causing HAP:

Number of bacteria, number of clusters, the similarity index of each cluster and between clusters

2.3.2 Determination of study indicators

2.3.2.1 HAP surveillance: surveillance team included IC practitioners (an

IC nurse and doctor) and 1 representative physician and nurse from the ICU who had been trained on the study objectives, surveillance methodology, HAP definition, clinical sample and data collection

2.3.2.2 HAP ascertainment: HAIs were diagnosed and ascertained using

surveillance criteria established by the Bach Mai hospital This criteria was adjusted from criteria of CDC, USA (1988) HAI case definitions were based on the combination of objective clinical findings and supportive data (eg, radiographs, ultrasound scans, endoscopy findings, and pathology reports) Isolates from any patients with suspected or confirmed HAIs were identified, speciated, and tested for susceptibility to a panel of antimicrobials susceptibility commonly used at the Bach Mai hospital

review of nursing and medical charts Data were collected by recording

study variables (age, gender, primary admission diagnosis, conditions associated with increased risk of APACHE II index scores, operation,

invasive procedures) in patients diagnosed with HAI and comparing with

similar data for patients without HAIs during the same study period

2.3.2.4 Microbiologic evaluation: Microbiologic technique was performed

according to standard guidelines of Bach Mai hospital to determine the HAP pathogens Samples were sent to Microbiology Department, Bach Mai hospital during 2 hours after sampling and incubated at 37oC under aerobic conditions Colony-forming units (CFUs) were subsequently counted after 48 h Potential pathogenic CFUs were cultured and identified

at Microbiology Department, Bach Mai hospital

2.3.2.5 Antimicrobial susceptibility testing

- Paper disc diffusion method: Susceptibilities of bacteria are categorized into: (1) Sensitive (S), (2) Intermediate (I) and (3) Resistance (R) The results were interpreted following the Clinical and Laboratory Standards Institute (NCCLS-National Committee for Clinical Laboratory Standards)

- Broth microdilution method: Antimicrobial agent are diluted at various concentrations The concentration range used may vary with the drug, the organism tested, and the site of the infection The antimicrobial dilutions are in 0.1 volumes in wells of a microdilution tray (usually 96 well trays The results were interpreted following the Clinical and Laboratory Standards Institute (NCCLS-National Committee for

Clinical Laboratory Standards) A baumannii isolates were considered

multidrug-resistant when they showed resistance to more than 3 of the following 5 drug classes: cephalosporin, carbapenems, piperacillin-tazobactam, fluoroquinolone, aminoglycoside and polymixin

2.3.2.6 Molecular typing: PFGE was performed at the Department of

Infectious Diseases, Research Institute, National Center for Global Health and Medicine, Tokyo Images of ethidium-bromide-stained isolates were converted into TIFFTM formats by ChemDoc (BIO-RAD) with which a cluster analysis was performed using Figerprinting Ⅱ software (Bio-Rad Laboratories) to construct a dendrogram Isolates with a similarity index of more than 80% were considered to be closely related

2.3.3 Statistical analyses

- Analyses were performed using SPSS 12.0 at the Infection control Department, Bach Mai hospital

- The differences in proportions were compared by using χ2 tests Continuous variables were compared by means of the Student t test P-value <0.05 was considered statistically significant

- To study risk factors, univariate analyses were first performed All risk factors with a univariate P value of less than 0.05 were included in a multivariate analysis using a stepwise forward multivariable logistic regression model

- Isolates that differ by three fragments in PFGE analysis were considered epidemiologically related subtypes of the same strain Conversely, isolates differing in the positions of more than three restriction fragments may represent a more tenuous epidemiologic relation These isolates were not included in a cluster

2.3.4 Study ethics

- Study activities and data collection methods were approved by the Ethics and Health Research Review Committee of Bach Mai hospital and Ministry of Health, Vietnam

- All prospective research participants were fully informed about the study objectives, research activities The principle of voluntary participation requires that people not be coerced into participating in research In the case of the patients who were unable to discuss study issues and poor decision making, their relatives were explained about the study

- Study results were timely notified director board and physicians, and aid them in development appropriate treatments and interventions

Chapter 3 STUDY RESULTS

3.1 Incidence and risk factors for HAP

Figure 3.1 shows that from September 2008 to April 2009, a total of

477 patients were included in this study Of them, 90 acquired HAP The

overall HAP incidence was 18.9%

Table 3.11 Incidence density rate of HAP (n = 477)

Exposure factors No of

HAP

No of exposure days

No of HAP per/1.000 exposure days

Hospitalization days

90

7.748 11,6 Endotracheal

3.1.2 Risk factors for HAP

Table 3.25 Risk factors for HAP per logistic regression analysis

Indwelling urinary catheter 1.4 0.8 – 2.5 > 0.05

Central venous catheter 1.5 0.9 – 2.7 > 0.05