Synopsis for PG DISSERTATION for MD/MS

SYNOPSIS FOR PG DISSERTATION FOR MD/MS,

UNDER RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, BENGALURU.

NAME OF THE CANDIDATE
AND ADDRESS
(IN BLOCK LETTERS) / DR SYEDA MISBAH UL KHAIR
DEPARTMENT OF MICROBIOLOGY
KEMPEGOWDA INSTITUTE OF MEDICAL SCIENCES.
BANGALORE
NAME OF THE INSTITUTION / KEMPEGOWDA INSTITUTE OF MEDICAL SCIENCES (KIMS)
COURSE OF THE STUDY AND SUBJECT / M. D.MICROBIOLOGY

SYNOPSIS FOR PG DISSERTATION FOR MD/MS,

UNDER RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, BENGALURU.

1. / Name and
Address of the candidate
(in block letters) / DR. SYEDA MISBAH UL KHAIR
DEPARTMENT OF MICROBIOLOGY,
KEMPEGOWDAINSTITUTE OF MEDICAL SCIENCES (KIMS), BANGALORE
Permanent Address / 1051,R.H.ROAD
BANGARAPET.
PIN CODE-563114.
2. / Name of the institution / KEMPEGOWDA INSTITUTE OF MEDICAL SCIENCES (KIMS), BANGALORE
3. / Course of study and subject / M.D. MICROBIOLOGY
4. / Date of admission to course / 28-05-2013
5. / Title of the Topic:
STUDY OF BACTERIAL AND FUNGAL PROFILE IN BRONCHOALVEOLAR LAVAGE FLUID AMONG PATIENTS WITH CHRONIC RESPIRATORY DISEASES.
6. / Brief resume of the intended work:
6.1 :Need for the study:
• According to latest WHO estimates (2004), chronic respiratory diseases account for 4 million deaths annually,contributing to 5% of global deaths1.Measured in Disability Adjusted Life Years (DALY), in 2005 the burden of chronic respiratory diseases was projected to account for 4% of the global burden and 8.3% of the burden of chronic diseases1.
•In India, chronic respiratory disease accounted 7% deaths and 3% DALYs lost.(2005)2
• The course of these diseases is punctuated by exacerbations. Exacerbations areassociated with greater and irreversible decline in lung function, significant mortality and morbidity.(Mortality is 40% at 1 year for those requiring hospitalisation).Infections are most frequent cause of exacerbations.3
• Early diagnosis and proper choice of antimicrobials is crucial for management of these patients.
• Sputum culture yields diagnosis in fewer than 50% of patients with pneumonia.4
• The advent of bronchoscopy and quantitative invasive techniques like Bronchoalveolar lavage have improved sensitivity and specificity of diagnostic techniques in diagnosis of pulmonary infections.5
6.2Review of literature:
Chronic respiratory diseases are a group of chronic disease affecting the airways and other structures of lungs.
Common chronic respiratory diseases are-
Asthma / Pulmonary eosinophilia
Bronchiectasis / Pulmonary heart diseases and disease of pulmonary embolism,pulmonary hypertension,corpulmonale.
Chronic obstructive lung diseases, including chronic obstructive pulmonary disease, emphysema and bronchitis) / Sarcoidosis
Chronic pleural disease / Sleep apnoea syndrome.1
Pneumoconiosis
Other diseases like Cystic fibrosis, pulmonary fibrosis and occupational lung diseases are included.6
They present with symptoms of cough ,pain in throat or chest, abnormalities of breathing,hemorrhages of respiratory passages, signs involving respiratory and circulatory system(asphyxia ,pleurisy, cardio respiratory arrest, abnormal sputum)1.
Most common causes of infections in these patients are viruses and bacteria (75-80%).Most frequent bacteria involved in exacerbations include Haemophilus influenzae, Streptococcus pneumoniae,Moraxella catarrhalis.3
About 25% of patients with chronic respiratory diseases have upper airway colonisation with pathogenic bacteria making it difficult to diagnose the pathogen.4
In a study conducted in Spain among patients with chronic lung diseases like Bronchogenic carcinoma,COPD, bronchiectasis) found most frequent potentially pathogenic bacteria colonising were H.infleunzae,Pneumococcus,Moraxella and Stapylococcus aureus.Gram negative rods were found in severely ill patients suggesting a relationship between their presence and exacerbations . Exacerbations are also associated with increase in burden of these bacteria.7
A bronchoscopic study conducted in Himalayan region in patients with chronic lung disease showed isolation of fungus, in which 46.7% were BAL culture positive.Most frequent fungus isolated were Candida species(50%),Aspergillus(46.4%).8
Bronchoalveolar lavage of a lung sub segment samples a large area of alveolar surface and sensitive tool in diagnosing pulmonary infections.4
BAL refers to sequential instillation and aspiration of physiologic solutions into lungs through a bronchoscope wedged in an airway. It provides better reflection of lung microbiology.4
Quantitative culture of BAL allows exclusion of even low level of contamination of virtue of culture growth below diagnostic threshold(>104 colony forming units/ml) .low colony counts suggest oropharyngeal contaminants and high colony counts suggest pathogen.9
A prospective study conducted to determine usefulness of quantitative bacterial culture as an aid to diagnose pneumonia over a period of 61/2 month demonstrated a sensitivity and specificity of 90% and 97% with threshold of 103cfu/ml.10
A review of 23 studies evaluating accuracy of BAL culture to diagnose Ventilator associated pneumonia(VAP), reported sensitivities of quantitative BAL culture ranges from 42-93% with mean of 73%,specificity of 45-100% with mean of 82%.11
A study conducted in Brazil in 62 patients with pneumonia, BAL fluid was positive 45 out of 62 patients(72.6%) with 58 of 62 BAL performed under antibiotics5.
In a study conducted in intensive care unit of a teaching hospital in Brazil between 1992-1997 to validate the quantitative culture and cellularity of BAL fluid for diagnosis of VAP ,quantitative culture of BAL effluent showed 90% sensitivity and 94.1% specificity.12
Isolation of a systemic, dimorphic fungus from any respiratory secretion is
clinically significant.8
In a study conducted by Kahn and Jones in 94 immunocompromised patients to
analyze the laboratory protocol of BAL fluid analysis found BAL microscopy and
culture both were diagnostic in 12 out of 18 cases(66%).13
A study conducted in Belguim on 99 patients with hematologic diseases found BAL
fluid culture and microscopy showed sensitivity and specificity of 50% and 53.3%
respectively in diagnosis of invasive pulmonary aspergillosis.14
6.3Objectives of the study:
1. To detect pathogenic organism by microscopy of BAL fluid.
2. To Isolate and identify aerobic bacteria and fungi from BAL
Fluid specimen.
3. To determine antimicrobial susceptibility pattern of the isolates.
7. / Materials and Methods:
7.1Source of data:
BAL fluid specimens from patients undergoing bronchoscopy in KIMS
Hospital, Bangalore
7.2 Method of data collection:

Duration of the study:18 months
Sample size-100
Study design-prospective study
Sampling method-purposive sampling.

E. Methods :
BAL fluid specimens will be collected under aseptic precautions and immediately transported to the laboratory for further processing.
The sample will be inoculated for quantitative bacterial culture using standard laboratory techniques on Blood agar, chocolate agar and Macconkey agar using a sterile 4mm nichrome loop (0.01ml), and incubated at 370C for 72hours .The colony forming units will then be calculated for growth positive plates15.Sample will also be inoculated in brain heart infusion broth.
The sample is subjected for direct microscopy with wet mount preparation (10% potassium hydroxide) for ruling out fungal filaments, and remaining part of sample is centrifuged at 3000rpm for 15-20 minutes16, supernatant is decanted and pellet is resuspended in phosphate buffer saline or 1-5ml of the sample itself and subjected to Gram’s stain for Bacteria or fungus, acid fast staining techniques like Ziehl-Neelsen stain to rule out Mycobacteria, Kinyoun’s technique for Nocardia13,May-Grunwald- Giemsa stain to rule out cysts of parasites like Pneumocystis17 and fungus18.
A part of centrifuged sample is also inoculated on Sabouraud’s dextrose agar for fungal culture and incubated for 4 weeks at 370C.
Antibiotic susceptibility testing will be done for bacterial isolates by Kirby-Bauer’s disc diffusion method and Antifungal susceptibility testing will be done for yeast like fungal
Isolates by E-test for fluconazole using commercially available MIC test strips(HiMedia Laboratories).

Inclusion criteria

Adult patients with chronic respiratory diseases undergoing Bronchoalveolar lavage.

Exclusion criteria

Patients with unstable cardiac conditions (recent myocardial infarction,cardiac arrhythmias etc) .
Pregnant women.
Patients who do not give consent for the procedure

7.3Statistical analysis
Data collected will be analyzed using descriptive statistical methods by computing percentage,mean and standard deviation. Appropriate tests will be employed for categorical, parametric or non parametric data. Wherever necessary the results will be
depicted in the form of graphs and percentages.
7.4 Does the study require any investigation or interventions to be conducted on patients or other humans or animals? If so, please describe briefly.
Yes. The study requires bronchoscopy to be done on patientswhich is a part of routine diagnostic work up and management, for which informed consent will be taken.
The study doesnot involve animal experiments.
7.5Has ethical clearance been obtained from your institution in case of 7.3?
Yes, and the certificate has been enclosed.
8. / List of references:
1. WHO. Global surveillance, prevention and control of CHRONIC RESPIRATORY DISEASES.A comprehensive approach;2007:5-14.
2.Koul PA.Chronic obstructive pulmonary disease: Indian guidelines and the road ahead. Lung India 2013;30:175-177.
3. Gupta Dheeraj, Agarwal Ritesh,Aggarwal Ashutosh,Maturu VN,Dhooria sahajal,Jindal SK.et al. Guidelines for diagnosis and management of chronic obstructive pulmonary disease: Joint ICS/NCCP (I) recommendations. Lung India 2013 jul-sep;30(3).
4. Meduri G Umberto,Beals H David,Maijub G Amado,Baselski Vickie. Protected Bronchoalveolar lavage.AM REV RESPIR DIS 1991;143;855-864.
5.Joao Carlos Pereira Gomes, Wilson L Pedreira Jr,Evangelina M P, Araujo A,
Francisco G Soriano,Elnara M Negri et al.Impact of BAL in the Management of
Pneumonia With Treatment failure-Positivity of BAL Culture Under Antibiotic
Therapy.CHEST 2000;118:1739–46.
6. Australian Institute of Health and Welfare.chronic respiratory conditions including asthma
and COPD [internet].http:/
conditions/.last accessed on Nov 2013.
7.Cabello H, Torres A, Celis R,El-Ebiary M, Puig de la Bellacasa J,Xaubet A, González J.
Bacterial colonization of distal airways in healthy subjects and
chronic lung disease: a bronchoscopic study. Eur Respir J 1997;10:1137–1144.
8. Biswas Debasis,Agarwal Sonal,Sindhwani Girish,Rawat Jagdish .Fungal Colonisation in patients with chronic respiratory diseases from Himalayan region of India .Annuals of clinical microbiology and Antimicrobials 2010;9:28.
9. Baselski VS and Wunderink RG. Bronchoscopic diagnosis of pneumonia.Clin.Microbiol. Rev 1994;7(4):533.
10. Cantral David E, Tape G Thomas,Reed C Elizabeth,Rennard I Stephen,Thompson B Austin.Quantitative culture of BAL fluid for the diagnosis of bacterial pneumonia. The Am J of Med1993 Dec;95(6):601-607.
http:/ accessed on Nov 2013.
11. Carroll Karen. Laboratory Diagnosis of Lower Respiratory Infections: Controversy and
Conundrums.J of Clin. Microbiol 2002;40(9):3115-3120.
12. Balthazar AB, Nowakonski Von A,De Capitani EM,Bottini PV,Terzi RGG,
Aruajo S.Diagnostic investigation of Ventilator associated pneumonia,comparative
study with a post mortem lung biopsy.Brazilian journal of medical and biological
research 2001;34:993-1001.
13.Kahn Frederick and Jones J Jefrey.Analysis of Bronchoalveolar lavage specimens
from immunocompromised patients with protocol applicable in microbiology
Laboratory.J of Clin Microbiology 1988 june;1150-1155.
14. Maertens Johan, Maertens Vincent.Theunissen Koen,Meersseman Wouter,
Meersseman Phileppe,Meers Stef et al.Bronchoalveolar Lavage Fluid Galactomannan
for the Diagnosis of Invasive Pulmonary Aspergillosisin Patients with Hematologic
Diseases. ClinicalInfectious Diseases2009;49:1688–93.
15. Mary K. York,Peter Gilligan, and Deirdre L Church.Processing and Interpretation of
Lower respiratory tract specimens.In:Lynne S Garcia,Henry D Isenberg(eds).
Clinical Microbiology Procedure Handbook.2nd ed.ASM PRESS,Washington
DC 2007;1:3.11.2.
16.Initial processing, inoculation and incubation of aerobic bacteriology
specimens. In:Henry D Isenberg, editor. Clinical Microbiology Procedure Handbook.1st
Ed.ASM PRESS,Washington DC.1992;1:1.4.12.
17.Rasmussen TR,Korsgaard J,Moller JK,Sommer T,Kilian M.Quantitative culture of
Bronchoalveolar Lavage Fluid in community acquired lower respiratory tract infections.
Respiratory medicine2001;95:885-890.
18. Knox.S.kenneth, Meinke Laura. Role of Bronchoalveolar lavage diagnostics in fungal
infections. Clin chest Med 2009;30:355-365.
9. / Signature of the candidate
10. / Remark of the guide / The study is essential to study the bacterial and fungal profile of BAL fluid in patients with chronic respiratory diseases to help in early detection of infectious agent and initiation of appropriate treatment, thereby helping in reducing the mortality and morbidity.
11. / 11.1 Name and
designation of Guide
11.2 Signature / Dr. ANJANA GOPI
ASSOCIATE PROFESSOR,
Department of Microbiology,
Kempegowda Institute of Medical Sciences,
Bangalore.
11.3 Co guide
11.4 Signature
11.5Head of theDepartment
11.4 Signature / DR HULIRAJ N
PROFESSOR AND HOD
Department of Pulmonary Medicine,
Kempegowda Institute of Medical Sciences,
Bangalore.
DR JAGADEEESH
PROFESSOR AND HOD
Department of Microbiology.
Kempegowda Institute of Medical Sciences,
Bangalore.
12 / 12.1Remarks of the Principal
12.2Signature