Download From, Dr. Swathi V Post Graduate in Department of Microbiology

From,
Dr. Swathi V
Post Graduate in Department of Microbiology,
A.J. Institute of Medical Sciences,
Mangalore- 575004
To,
The Registrar,
Rajiv Gandhi University of Health Sciences,
Bangalore
Respected Sir,
Sub: Submission of Synopsis of Dissertation
Herewith, I am submitting synopsis of my dissertation work: “Study of aerobic bacterial profile and
their antibiogram in endotracheal aspirates of mechanically ventilated patients in MICU’’ for
the registration in Rajiv Gandhi University of Health Sciences, Bangalore.
Kindly accept the same and oblige.
Thanking you,
Yours faithfully,
(Dr. Swathi V)
Place: Mangalore
Date: 23-11-2012
SYNOPSIS
Submission to Rajiv Gandhi University of Health Sciences
“STUDY OF AEROBIC BACTERIAL PROFILE AND THEIR ANTIBIOGRAM IN
ENDOTRACHEAL ASPIRATES OF MECHANICALLY VENTILATED PATIENTS IN
MICU”
Name of the candidate
:
Dr. SWATHI V
Guide
:
Dr. NARENDRA NAYAK
Course and Subject
:
M.D. (Microbiology)
A.J. Institute of Medical Sciences,
Mangalore – 575004
2012
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,
KARNATAKA, BANGALORE.
ANNEXURE II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION
1
Name of the candidate and
SWATHI V.
address
POST GRADUATE
( in block letters )
DEPARTMENT OF MICROBIOLOGY
A.J INSTITUTE OF MEDICAL SCIENCES
KUNTIKANA
MANGALORE-575004
2
Name of the Institution
A.J INSTITUTE OF MEDICAL SCIENCES
KUNTIKANA, MANGALORE.
3
Course of study and Subject
MD MICROBIOLOGY
4
Date of admission to course
31-05-2012
5
Title of the topic
STUDY OF AEROBIC BACTERIAL PROFILE AND THEIR ANTIBIOGRAM IN
ENDOTRACHEAL
ASPIRATES
OF
MECHANICALLY
VENTILATED
PATIENTS IN MICU
6
BRIEF RESUME OF THE WORK
6.1 Need for the study:
Ventilator associated pneumonia (VAP) is a major contributor for the increasing
morbidity in patients on ventilator. The incidence of ventilator associated pneumonia
increases with the duration of mechanical ventilation. Hence it requires a rapid
diagnosis and initiation of an appropriate antibiotic therapy for reducing the
morbidity and mortality in these patients. Inappropriate and inadequate antibiotic
treatment will lead to emergence of drug resistant pathogens and poor prognosis in
patients on ventilator.1,2
As there is very limited data available on VAP in this coastal area of Karnataka, this
study is undertaken to guide the clinicians in formulating an appropriate antibiotic
policy. This will in turn prevent the emergence of multidrug resistant strains in
MICU.3-5
6.2 Review of literature:
Ventilator associated pneumonia (VAP) is defined as pneumonia that occurs 48hours
or more after endotracheal intubation or within 48 hours of endotracheal tube
removal. VAP can be categorized into early onset VAP which occurs within first four
days of mechanical ventilation and late onset VAP occurring after four days of
mechanical ventilation.2
However, the diagnosis of VAP does not solely depend on the microbiological data
and also requires other parameters to be met. The Pugin’s Clinical Pulmonary
Infection Score (CPIS), which combines clinical, radiographic, physiological and
microbiological data into a single numerical result, is used as a diagnostic tool for
VAP. Other etiologies mimicking pneumonia such as atelectasis, pulmonary edema,
adult respiratory distress syndrome, pulmonary embolism, drugs-induced pneumonitis,
radiation pneumonitis, and pulmonary hemorrhage were excluded. When the CPIS
exceeds 6, a high possibility of the presence of VAP can be assumed as defined by
quantitative cultures of bronchoscopic and non-bronchoscopic bronchoalveolar lavage
(BAL) specimens.2
Recently CPIS was modified by Singh and his colleagues. They observed that the
empiric antibiotic treatment could be stopped on day 3 if the scoring on m-CPIS is <6
and can be continued for the entire course if m-CPIS is > 6.2,4
Components of modified clinical pulmonary infection scores (m-CPIS)2
Parameters
Points
1. Temperature (0C)
36.5–38.40 C
0
38.5–38.90 C
< 36.00 C or > 39.00 C
1
2
2. WBC (cells/mm3)
4000–11,000
0
< 4000 or > 11,000
1
Band forms > 50% WBC
2
3. Sputum
No sputum
0
Non-purulent sputum
1
Purulent sputum
2
4. Oxygenation: PaO2/FIO2 (mmHg)
> 240 or presence of ARDS (PaO2/FIO2 < 200 or
0
PAWP <18 mmHg plus new chest infiltrate)
</= 240 and no ARDS
2
5. CXR
No infiltrate
0
Diffuse or patchy infiltrate
1
Localized infiltrate
2
6. Progression of infiltration from CXR
No infiltrate progression
0
Infiltrate progression (no ARDS or CHF)
2
7. Culture from tracheal aspirate
No, light, or rare growth of pathogenic bacteria
0
Moderate or heavy growth of pathogenic bacteria
1
Growth of pathogenic bacteria similar to that from
Gram stain
2
[ARDS: adult respiratory distress syndrome, PAWP: pulmonary artery wedge
pressure, PaO2/FIO2: arterial oxygen pressure divided by fraction of inspired oxygen,
CXR: chest X-ray, CHF: congestive heart failure.]

In a study conducted by RP Jakribettu and colleagues, Klebsiella pneumoniae was the
most common pathogen followed by Pseudomonas aeruginosa, Acinetobacter
species, Citrobacter species, Escherichia coli, methicillin resistant Staphylococcus
aureus and the least was Serratia species. Cephalosporins were ineffective in > 80%
of the cases, most of the pathogens were susceptible to amikacin (82.6%),
carbapenems (82%) and levofloxacin (77.5%).1

Noyal Mariya Joseph et al, in their study observed that the incidence of VAP was
22.94 per 1000 ventilator-days. Most cases were caused by Gram negative bacteria,
which accounted for 80.9% of the causative organisms. Acinetobacter baumannii
(23.4%) and Pseudomonas aeruginosa (21.3%) were the predominant Gram-negative
bacteria, and Staphylococcus aureus (14.9%) was the most common Gram-positive
bacterium isolated. Many of the pathogenic microorganisms causing VAP were
initially present as colonizers in the respiratory tract followed by subsequent
development of VAP. Colonization rates were relatively higher with non-fermenters
and MRSA than Enterobacteriaceae. Acinetobacter and Pseudomonas species were
resistant to even the higher antibiotics like meropenem, piperacillin–tazobactam,
ceftazidime, gatifloxacin and amikacin.3

T Rajasekhar et al, in their study have considered modified CPIS for diagnosing VAP
and also found that the most frequently isolated organisms were Acinetobacter
baumanii and Pseudomonas aeruginosa. The predominant organisms in the earlyonset VAP group were Acinetobacter baumanii and Klebsiella species. In the late
onset group Pseudomonas aeruginosa was the most common organism isolated.
Other pathogens isolated were Stenotrophomonas maltophilia, Burkholderia cepacia,
Staphylococcus aureus and Streptococcus pneumoniae. Acinetobacter, Klebsiella and
Pseudomonas species were multi drug resistant. The emergence of drug resistant
organisms in this study was attributed to the prior use of broad-spectrum antibiotics.
The other drug resistant organisms were Stenotrophomonas maltophilia and
methicillin resistant Staphylococcus aureus (MRSA), both of which were also found
in the early-onset group.4

In a study by Pratirodh Koirala et al, 90% cases showed bacterial growth, out of
which 44.4% were poly-microbial. Pseudomonas aeruginosa and enteric gram
negative bacteria were predominant bacteria (40.3%) followed by Staphylococcus
aureus (10.4%), other Gram negative bacteria (5.9%) and Viridans Streptococci
(2.9%). Pseudomonas aeruginosa were most sensitive to amikacin (81.4%) and
ciprofloxacin (70.3%). All Pseudomonal isolates were resistant to cefotaxime. Enteric
Gram Negative bacteria were most sensitive to amikacin and chloramphenicol
(74.0%) and all were resistant to ampicillin and cephalexin. All the gram positive
bacteria isolated were sensitive to vancomycin. Among all the isolates, 88.8% of
Pseudomonas aeruginosa, 66.6% of enteric gram negative bacteria and 55.5% of
Gram positive bacteria were multidrug resistant.5

In a study by S Singh et al, the organisms most commonly isolated in VAP were
Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus and
Escherichia coli wherein the organisms were observed to be multi drug resistant.
Most of the isolates of Enterobacteriaceae group were extended spectrum beta
lactamase (ESBL) positive, Staphylococcus aureus was methicillin resistant and
Pseudomonas aeruginosa was sensitive only to imipenem.6
6.3
Objectives of the study:
 To assess the spectrum of aerobic bacteria in endotracheal aspirates of patients on
ventilator.
 To evaluate the antibiotic sensitivity pattern in the isolates and detection of
extended spectrum beta lactamase (ESBL) production in Escherichia coli and
Klebsiella species.
 To correlate the clinical findings with the bacterial isolates as a probable cause of
ventilator associated pneumonia.
7
MATERIALS AND METHODS
7.1 Source of Data
Patients on ventilator admitted in M.I.C.U of A.J.I.M.S, Mangalore during the period
from November 2012 to October 2013 will be included in the study.
7.2 Methods
A cross sectional study of 1 year duration involving 100 patients on ventilator will be
done, to ascertain the profile of the aerobic bacteria present in the endotracheal
aspirates7 and to study their antibiotic sensitivity pattern. Clinical assessment of the
patient will be done only after obtaining an informed consent regarding the nature of
the study from their relatives.
INCLUSION CRITERIA:
Patients >/= 18 years.
Ventilated for >/= 48hrs.
EXCLUSION CRITERIA:
Patients < 18years.
Ventilated for < 48hrs.
Severely immuno compromised states- AIDS, organ transplant patients, terminal
stages of malignancy etc.
Patients diagnosed to have pneumonia prior to intubation.
Clinical history will be taken according to the questionnaire and the pro forma is
mentioned in the later part of this synopsis.
Endotracheal aspirate will be collected using a sterile 21 inch Romsons’ 14FG
suction catheter with a sterile mucus extractor under aseptic precautions. The catheter
will be withdrawn and approximately 2-5 mL of sterile normal saline will be injected
into it with a sterile syringe to flush the exudate into a sterile container which will
immediately be transported to the Microbiology department for processing.1,2
A smear will be prepared from the sample and Gram staining will be done. The
sample will then be mechanically liquefied and homogenized by vortexing for a
minute. Using a standard wire loop (0.001 ml, semi-quantitative method8,9), it will be
inoculated onto MacConkey agar, Blood agar and Chocolate agar plates10-12 and
incubated at 370C for 24hrs to 48 hrs.
A positive Gram stain13 (>10 polymorphonuclear cells/low power field and >/= 1
bacteria/oil immersion field3) and a colony count of 105 CFUs/ml or more will be
considered significant.1,6
Identification of the organisms will be done by various biochemical tests as per the
Clinical Laboratory Standards Institute (CLSI) guidelines.14
Antibiotic susceptibility testing will be done by Kirby Bauer’s disc diffusion method
using commercially available discs (HiMedia Laboratories) on Mueller Hinton agar
and also ESBL production in E.coli and Klebsiella species will be detected by double
disc diffusion method using ceftazidime and ceftazidime - clavulanic acid discs
according to CLSI guidelines.14
ATCC strains of E.coli 25922 and K.pneumoniae 700603 will be used as quality
control strains for the detection of ESBL production.
Descriptive statistical analysis will be done at the end of the study.
Ventilator associated infection rate will be calculated by the following formula6
Number of patients developing VAP
Infection rate =
X 1000
Total number of ventilator days
Expressed as “number of ventilator associated infection per 1000 ventilator days”.
8
8.1 Does this study require any investigations or interventions to be conducted on patients
or other humans or animals? If so, please describe briefly.
Yes. In mechanically ventilated patients suctioning of endotracheal aspirates is done
after obtaining an informed consent by the patient or patient’s attendant under
supervision of the Intensivist.
8.2 Has the ethical clearance been obtained from your Institution?
Attached with the synopsis (hard copy)
9. REFERENCES:
1. Jakribettu RP, Boloor R. Characterisation of aerobic bacteria isolated from
endotracheal aspirate in adult patients suspected ventilator associated pneumonia in a
tertiary care center in Mangalore. Saudi J Anaesth 2012 Apr;6(2):115-9.
2. Clinical Practice Guidelines for Prevention and Management of Adults with Hospitalacquired and Ventilator–associated Pneumonia 2007 Apr.
http://www.idthai.org/Guidelines/CPG%20For%20HAP-VAP%20English-23-Apr2007.pdf
3. Joseph NM, Sistla S, Dutta TK, Badhe AS, Parija SC. Ventilator-associated
pneumonia: role of colonizers and value of routine endotracheal aspirate cultures. Int J
Infect Dis. 2010 Aug;14(8):e723–9.
4. Rajasekhar T, Anuradha K, Suhasini T, Lakshmi V. The role of quantitative cultures of
non-bronchoscopic samples in ventilator associated pneumonia. Indian J Med
Microbiol. 2006 Apr;24(2):107-13.
5. Koirala P, Bhatta DR, Ghimire P, Pokhrel BM, Devkota U. Bacteriological Profile of
Tracheal Aspirates of the Patients Attending a Neuro-hospital of Nepal. Int J Life Sci.
2010;4:60-5.
6. Singh S, Pandya Y, Patel R, Paliwal M, Wilson A, Trivedi S. Surveillance of device
associated infections at a teaching hospital in rural Gujarat-India. Indian J Med
Microbiol. 2010;28(4):342-7.
7. Ioanas M, Ferrer R, Angrill J, Ferrer M, Torres A. Microbial investigation in ventilator
associated pneumonia. Eur Respir J. 2001 Apr;17(4):791-801.
8. Fujitani S, Cohen-Melamed MH, Tuttle RP, Delgado E, Taira Y, Darby JM.
Comparison of Semi- Quantitative Endotracheal Aspirates to Quantitative Non-
Bronchoscopic
Bronchoalveolar
Lavage
in
Diagnosing
Ventilator-Associated
Pneumonia. Respir care. 2009 Nov;54(11):1453-61.
9. Koenig SM, Truwit JD. Ventilator-Associated Pneumonia: Diagnosis, Treatment, and
Prevention. American Society for Microbiology. 2006 Oct;19(4):637-57.
10. Shin YM, Oh YM, Kim MN, Shim TS, Lim CM, Lee SD, Koh Y, Kim WS, Kim DS,
Hong SB. Usefulness of quantitative endotracheal aspirate cultures in Intensive Care
Unit patients with suspected Pneumonia. J Korean Med Sci. 2011 Jul;26:865-9.
11. El Solh AA, Akinnusi ME, Pineda LA, Mankowski CR. Diagnostic yield of
quantitative endotracheal aspirates in patients with severe nursing home-acquired
pneumonia. Crit Care. 2007;11(3):R57.
12. Flanagan PG, Paull A, Barnes RA. A novel simple method for quantifying bacteria
from endotracheal aspirate. J Med Microbiol. 1996;45:153-4.
13. Cook D, Mandell L. Endotracheal aspiration in the diagnosis of ventilator-associated
pneumonia. Chest 2000 Apr;117:195S–197S.
14. Clinical and laboratory standard Institute, Performance standards for antimicrobial
susceptibility testing; 21st informational supplement. M100-S21,2011;31(1):42-107.
9
Signature of the candidate
10
Remarks of the Guide
11
Name and designation of (in
block letters)
11.1 Guide
Dr. Narendra Nayak
Professor
MD Microbiology
A.J. Institute of Medical Sciences
Mangalore.
11.2 Signature
11.3 Head of the department
Dr. Ronald Roche
Head of the department
Department of Microbiology
A.J. Institute of Medical Sciences
Mangalore
11.4 Signature
12
12.1 Remarks of the Chairman
and Principal
12.2 Signature