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RAJIVGANDHI UNIVERSITY OF HEALTH SCIENCES, BANGALORE KARNATAKA PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERTATION 1.Name of the Candidate and Address : DR JAGADEVI # 7/5 F VINODA NILAYA 2ND FLOOR, 3RD MAIN 9TH CROSS, CHAMRAJPET, BANGALORE-560018. 2.Name of Institution : KEMPEGOWDA INSTITUTE OF MEDICAL SCIENCES, BANGALORE-560070. . 3.Course of the study and subject : M.D MICROBIOLOGY 4.Date of admission to the course : 31st MAY 2011 5.Title of the Dissertation : EVALUATION OF CONVENTIONAL CULTURE METHOD WITH LATEX AGGLUTINATION TEST FOR DIAGNOSIS OF BACTERIAL MENINGITIS AMONG PEDIATRIC AGE GROUP. 1 6. 6.1 BRIEF RESUME OF THE INTENDED WORK NEED FOR THE STUDY Acute bacterial meningitis is one of the most sever infectious disease in infants and children. It is associated with high rate of mortality and morbidity in pediatric population.1 As acute bacterial meningitis is a medical emergency, early and accurate etiologic diagnosis and appropriate antimicrobial treatment remains the cornerstone in the management of patients and helps in improving the outcome of the disease. Cerebrospinal fluid (CSF) culture is considered to be gold standard for diagnosis of bacterial meningitis, but it is time consuming (24-48 hours) and can give false negative results if the sample is not properly collected, transported and processed. Although CSF Gram’s stain may provide immediate information about causative microorganisms, it depends on factors like the number of organisms , prior use of antibiotics, technique and observer’s skill.2 Hence there is a definite need for rapid ,sensitive and specific method for accurate identification of bacteria causing meningitis to help in diagnosis. 6.2 REVIEW OF LITERATURE Bacterial meningitis is an inflammatory reaction of pia and arachnoid membranes surrounding the brain and spinal cord with a definitive or presumptive evidence of bacteria and inflammatory cells in the CSF. Most common route of spread being haematogenous from distant site infection such as, lower respiratory tract infection, osteomyelitis, pyoderma. contagious spread occurs from otitis media, infections of paranasal sinuses. Direct spread from compound fracture of skull is the less common route. The advent and widespread use of antibacterial agents in the treatment of meningitis have drastically reduced the mortality caused by this disease. However, untreated and inappropriately treated bacterial meningitis is highly fatal and leaving serious neurologic sequel in survivors.3 MICROBIOLOGY Etiology of bacterial meningitis may vary in different parts of the world and common pathogens of bacterial meningitis also vary with different age.4 Neonate (0-28 days) – Group B streptococci, Escherichia coli, Listeria monocytogenes, Klebsiella and Enterococcus species. 4-12 weeks – Haemophilus influengae, streptococcus pneumoniae, Group B streptococci and Listeria monocytogenes. 12 weeks and older - Neisseria meningitidis , streptococcus pneumonia and Haemophilus influenae. 2 Alteration of host defence due to immune deficits or anatomical defects also increase the risk of bacterial meningitis from less common pathogens such as, Staphylococcus aureua , Coagulase negative staphylococci,Pseudomonas, Proteus and Salmonella species.5 EPIDEMIOLOGY Bacterial meningitis is much more common in developing countries than developed.3 It occurs in both epidemic and sporadic patterns. The exact incidence is not known. 4 The average incidence in India works out to be 1% of the total admissions to pediatric ward and 0.8% of the total number of new pediatric cases seen in all out patient departments.6 More than two third of cases meningitis occur in first two years of life owing to decreased immunity and high vascularity of the brain.7 PATHOGENESIS The subarachnoid space and its CSF are relatively defenseless in stopping invasion by bacterial pathogens because of the CSF’s paucity of phagocytic cells and low concentration of complement and immunoglobulin. 3 Inflammation of meninges is initiated by the presence of bacterial lipopolysaccharide, teichoic acid and/or other bacterial cell wall components in subarachnoid space. The bacterial antigens stimulate monocytes to produce the cytokine interleukin-1 and stimulate macrophages, astrocytes, microglial cells, ependymal cells and endothelial cells in the central nervous system to produce the tumor necrosis factor. Interleukin-1and tumor necrosis factor probably act synergistically to elicit inflammatory responses which manifest clinically as meningitis.3 sequence of such responses are structural changes in blood brain barrier; cytotoxic parancymal edema; increased intracranial pressure; decreased intracranial perfusion; cerebral infarction; and focal or diffuse brain damage. 6.3 OBJECTIVE OF THE STUDY 1. To compare the sensitivity and specificity of CSF culture, Gram’s stain and latex agglutination test. 2. To evaluate the diagnostic technique for early diagnosis of the cases. 3. To determine the antibiotic sensitivity pattern of pathogens isolated in bacterial meningitis 3 7. MATERIALS AND METHOD 7.1 SOURCE OF DATA The study will comprise of clinically suspected cases of meningitis attending inpatient and outpatient Department of Pediatrics KIMS, Bangalore. PERIOD OF STUDY: 1 year 6 months SAMPLE SIZE: Around 60 INCLUSION CRITERIA Clinically suspected meningitis patients with the fallowing altered parameters in CSF : Protein - >80 mg/dl Glucose -< 40 mg/dl Leucocyte neutrophils - >5cells.cumm EXCLUSION CRITERIA : 1. No alteration of CSF parameters in clinically suspected meningitis patients. 2. Consent refusal. STUDY DESIGN Comparative study. SAMPLE DESIGN Purposive sampling. METHODOLOGY The data for the study will be collected from all those who fulfill the inclusion and exclusion criteria by purposing sampling. STATISTICAL ANALYSIS Data collected will be analyzed statistically by computing percentage, mean, Standard deviation. Appropriate tests will be employed for categorical, parametric or nonparametric data. Wherever necessary the results will be depicted in the form of graphs and percentage. 4 7.2 METHOD OF COLLECTION OF DATA SPECIMEN COLLECTION CSF samples will be collected from suspected meningitis patients under aseptic precautions. A sterile lumbar puncture needle is introduced into subarachnoid space between the third and fourth lumbar vertebrae and the sample is collected into a sterile container and transported to the laboratory immediately for processing. PROCEDURE CSF collected from the patients is first analyzed for cell count and type, protein and glucose. The same sample is subjected to centrifugation at 1500rpm for 15minutes.sediment is used for Gram’s stain and culture and the supernatant is used for antigen detection. Microscopic examination – comprising of Gram’s staining. Two drops of sediment is placed on glass slide, air dried, heat fixed and used for gram staining. Culture – 1-2 drops of sediment is added on to Blood Agar, Chocolate Agar, Mac Conkey Agar and Brain heart infusion (BHI)Broth. The solid media are streaked. The Blood Agar and Chocolate Agar are incubated in carbon dioxide jar at 37⁰C for 48 hours. The Mac Conkey Agar is incubated at 37⁰C for 48 hours. BHI Broth is incubated at 37⁰C for 7days. The Agar plates and Broth are examined after every 24 hours, any growth present is identified appropriately and processed further by biochemical tests and antibiotic sensitivity. Antigen detection – By using WELLCOGEN bacterial antigen kit ,a latex agglutination test to detect antigens of 5 organisms; Ecoli k1,N.meningitidis ABCYW135,Streptococcus pneumoniae, group B streptococcus and H.influenzae type b is performed according to the manufacturer’s instructions. 7.3 DOES THE STUDY REQUIRE ANY INVESTIGATION OR INTERVENTIONS TO BE CONDUCTED ON PATIENTS OR OTHER HUMANS OR ANIMALS?IF SO,PLEASE DISCRIBE BRIEFLY. The study involves investigating CSF samples from suspected meningitis cases . The study does not involve animal experiments. 7.4 HAS THE ETHICAL CLEARANCE BEEN OBTAINED FROM YOUR INSTITTION IN CASE OF 7.3? yes. 5 8. LIST OF REFERENCES (ABOUT 4-6) 1. Das BK, Gurubacharya RL, Mohapatra M, Mishra OP. Bacterial antigen detection test in Meningitis. Indian Journal of Pediatrics 2003;70(10):799-801. 2.Surinder K, Bineeta K, Megha M. Latex particle agglutination test as an adjunct to the diagnosis of bacterial meningitis. Indian J Med Microbiol 2007;25:395-7. 3.Larry D. Gray and Daniel P. Fedorko. Laboratory Diagnosis of Bacterial Meningitis.Clinical Microbiology Review,Apr.1992.p.130-145. 4. Nandhita Chinchankar, Meenakshi Mane, Sheila Bhave, Swate Bapat, Ashish Bavdekar, Anand Pandit et al. Diagnosis and outcome of Acute Bacterial Meningitis in early childhood. Indian pediatrics 2002; 39: 914-921. 5. Prober CG.Central nervous system infections.In:Nelson Textbook of Pediatrics,18th ed. In : Kliegman, Behrman , Jenson. 2007.p.2512-2521.` 6. Athavale.V.B .Pyogenic meningitis.In:Textbook of Pediatrics,Vol 3, Rev 1ST ed. Udani PM, Nelson Waleto E, Koko.W. 1998.p.2107-2116. 7. Xavier Saez-Llorens and George H. Mc cracken Jr. Infectious disease of children. 11th ed. Philadelphia: Mosby publications; 2004. 9. SIGNATURE OF CANDIDATE 10. REMARKS OF GUIDE : : Bacterial meningitis is commonly seen in Pediatric age group and more than two third of meningitis cases occurs in first 2 years of life due to decreased immunity, and also carries high mortality and morbidity .Hence this study is taken up to early and specific diagnosis and to reduce the complications. 11. NAME AND DESIGNATION OF (In block letters) 11.1 GUIDE : DR.JAGADEESH, PROFESSOR, DEPARTMENT OF MICROBIOLOGY, KEMPEGOWDA INSTITUTE OF MEDICAL SCIENCES, BENGALURU. SIGNATURE : 6 11.2 CO GUIDE : DR.YASHODA. H.T , PROFESSOR, DEPARTMENT OF PEDIATRICS, KEMPEGOWDA INSTITUTE OF MEDICAL SCIENCES, BENGALURU. SIGNATURE : 11.3 HEAD OF DEPARTMENT : DR.K.L.RAVIKUMAR, PROFESSOR& HEAD, DEPARTMENT OF MICROBIOLOGY, KEMPEGOWDA INSTITUTE OF MEDICAL SCIENCES, BENGALURU. SIGNATURE : 12. REMARKS OF CHAIRMAN AND PRINCIPAL: SIGNATURE : . 7