15. CNS

Bangar Raju Dept of Microbiology KMC-IC INFECTIONS OF THE CENTRAL NERVOUS SYTEM 9/29/2008 BANGAR RAJU- 2008 2 USF student Rachel Futterman, radiant and energetic 9/29/2008 BANGAR RAJU- 2008 3 • Thursday, when she left her restaurant job. She was excited about her recent promotion from host to server. • That night, she attended a campus wide sorority dinner and went out with friends. When she woke the next morning, she thought she had the flu. • Her Delta Gamma sorority sisters nursed her. Roommate Amber Powers brought her Gatorade to settle her stomach. • Then Saturday came. • At 7 a.m. Powers was awakened by Futterman kicking in what appeared to be a seizure, so she called 911. • Two days later, USF honored the energetic student during an outdoor memorial service on campus. 9/29/2008 BANGAR RAJU- 2008 4 INTRODUCTION: • Infections of CNS are rare but life threatening • Presents special milieu for bacterial, fungal, viral, and parasitic infections • Brain and spinal cord are protected by bone and meningieal coverings • Lack intrinsic immune system • Unique compact structure 9/29/2008 BANGAR RAJU- 2008 5 Gross Anatomy • Protected by bony covering • Meningieal layers give some kind of protection • Piamater and arachnoid layers are not adherent to each other • Infection can spread rapidly over cerebral hemisphere • CSF is present in subarachnoid space- true space • CSF contains little antibody or complement and few phagocytic cells • Bacteria in logarithmic phase- acute onset of bacterial meningitis 9/29/2008 BANGAR RAJU- 2008 6 9/29/2008 BANGAR RAJU- 2008 7 Blood-Brain barrier • This barrier excludes most microbes, immunocompetent cells and antibodies. • cellular configuration of cerebral capillaries, the choriod plexus and arachnoid cells. • Therefore, although the barrier deters invasion of infectious agents, it hampers their clearance once it is penetrated. • Dye-trypan blue injected into systemic circulation • Stain all tissues except the brain and spinal cord – blood brain barrier 9/29/2008 BANGAR RAJU- 2008 8 9/29/2008 BANGAR RAJU- 2008 9 9/29/2008 BANGAR RAJU- 2008 10 9/29/2008 BANGAR RAJU- 2008 12 Immune system: • Lacks antibodies • Antibodies found are derived from serum • IgA & IgG are found in low levels • IgM not possible due to its large size • Very few phagocytic cells found during infection- special microglial cells • Complement is almost absent • Hence clearing of pathogens difficult once entered 9/29/2008 BANGAR RAJU- 2008 13 • During trauma and inflammation BBB gets disrupted and inflow of antibodies seen along with serum proteins • Sometimes when inflammatory reactions sets in B cells leak into CSF and generate antibodies • PMNs are major cells found in bacterial infections • Attracted by chemotactic factors • Mononuclear cells are dominant in viral infections, sub acute infections such as tuberculosis and fungal infections. • These cells matain the infection in dormant stage and gets reactivated due to HIV, drugs and immunosupression • Organism choose specific receptors on neural cells to enter CNS 9/29/2008 BANGAR RAJU- 2008 14 • Blood borne invasion- Polio, Neisseria • Through peripheral nerves- Herpes simplex, Varicella-Zoster, Rabies- less common • Local invasion- infected ears, sinuses, local injury, congenital defects (spinal bifida) • Through olfactory tract- Amoebic meningitis. 9/29/2008 BANGAR RAJU- 2008 15 Pathways of spread to the central nervous system • Direct: skull fracture, from adjacent sinus, congenital ectodermal defect, and surgical shunt Staphylococcus aureus and members of Enterobacteriacae • Neural ( via axon): tetanus toxin, rabies, Herpes • Olfactory: Herpes simplex, Naegleria fowleri • Hematogenous: H.inflenzae, streptococcus pneumoniae, Neisseria, mycobacterium, Cryptococcus, Rickettsia, Enteroviruses, plasmodium , HIV, tape worm embryos 9/29/2008 BANGAR RAJU- 2008 16 Terminology: • Encephalitis: Inflammation of the brain • Myelitis: Inflammation of spinal cord • Encephalomyelitis: Inflammation of brain and spinal cord • Meningitis: Inflammation of membranous covering of the brain and spinal cord. • Meningoencephalitis: Inflammation of brain and meninges. • Epidural abscess: Inf of soft tissue • Osteomyelitis: Inf of the bones 9/29/2008 BANGAR RAJU- 2008 17 • • • • Encephalitis Myelitis Encephalomyelitis Meningitis 9/29/2008 BANGAR RAJU- 2008 18 Meningitis 9/29/2008 BANGAR RAJU- 2008 19 Types of Meningitis • • • • • • Bacterial Meningitis Viral Meningitis Fungal Meningitis Syphilitic Meningitis Carcinomatous Meningitis Drug Induced Meningitis 9/29/2008 BANGAR RAJU- 2008 20 Categorizing of CNS infections and etiologic agents Acute meningitis: bacterial- Streptococcus pneumoniae Neisseria meningitides Haemophilus influenzae type B Group B streptococci Escherichia coli viruses- Arbo viruses Entero viruses 9/29/2008 BANGAR RAJU- 2008 21 Chronic meningitis: Bacterial - mycobacterium tuberculosis Fungi - Cryptococcus neoformans others Acute encephalomyelitis Viruses - Arboviruses Enteroviruses Herpes viruses Acute brain abscess staphylococcus aureus mixed anaerobic & aerobic flora group A & D streptococci 9/29/2008 BANGAR RAJU- 2008 22 Chronic brain abscess Bacteria – mycobacterium tuberculosis Fungi – Cryptococcus neoformans Parasites – Taenia solium Toxoplasmosis 9/29/2008 BANGAR RAJU- 2008 23 Risk Factors • Immunodeficiency states, respiratory tract infections, malnutrition, anaemia, smoking • Male > Female • Age distribution of meningitis varies from area to area • Dry season • Low socio-economic status 9/29/2008 BANGAR RAJU- 2008 24 ENTRY, REPLICATION AND SPREAD Infectious agents can invade the CNS using one of three routes: • Hematogenous spread • Neural pathways - less common • Direct inoculation 9/29/2008 BANGAR RAJU- 2008 25 Hematogenous spread of organisms • Most of the viruses and bacteria invade by this route • Gain access to blood stream following colonization and penetration mucosal surfaces- respiratory • Sometimes the bite of the arthropod, bite of the mammal or transplacentally • Penetrates the blood brain barrier elaborating different virulence factors • Can enter even through places where tight junctions between endothelial cells are less significant 9/29/2008 BANGAR RAJU- 2008 26 • Direct entry through choriod plexus- mumps virus • Arboviruses directly infect endothelial cells including the microvascular circulation the brain • Symptoms or manifestations depend on the microbial load in blood stream meningococcal meningitis arboviruses meningitis rabies , mumps viruses staphylococcus aureus etc……… 9/29/2008 BANGAR RAJU- 2008 27 Spread by neural route: • Organisms use special neural pathways Rabies HSV Varicella- Zoster • Some use only olfactory nerves endings to reach the brain Naegleria fowleri- Meningoencephalitis 9/29/2008 BANGAR RAJU- 2008 28 Bacterial evasion mechanisms to host responses Pathogenic Event Colonization and mucosal invasion, Host Defense Bacterial Evasion Mechanism IgA protease secretion Ciliostasis Adhesive pili Blockage of Alternative Compliment Pathway by Mechanisms on the cell surface Passage through tight junctions between cells, mechanism unknown Rapid bacterial replication 1. Secretory IgA 2. Cellular cilia activity 3. Mucosal epithelium Survival in the blood stream Activation of Complement Pathways Crossing the bloodbrain-barrier Cerebral endothelium Survival within the CSF Poor opsonic activity 9/29/2008 BANGAR RAJU- 2008 29 Damage: • Death of CNS cells is due to action of bacterial toxins, lytic cycles of viral replication, intracellular growth of bacteria immune response of the host Multiplication and spread – elicits inflammatory response, less intense that other areas Infiltration of microglial cells and astrocytes - vascular edema due to increased vascular permeability Neutrophils and macrophages infiltrate and phagocytize the organisms. Lysis of infiltrating neutrophils releases enzymes, leading to further tissue destruction Focal lesions are important to diagnose the CNS diseases BANGAR RAJU- 2008 30 • • • • • 9/29/2008 Anatomical basis of localization of symptoms • Frontal lobe: frontal sinusitis headache memory defects attention loss diminished intellectual performances • Temporal lobe: personality changes visual field defects focal seizures 9/29/2008 BANGAR RAJU- 2008 31 • Basilar (base of the brain): sub occipital headache neck stiffness diplopia cranial nerve palsies 9/29/2008 BANGAR RAJU- 2008 32 Viral Meningitis • Acute onset • Meningeal signs and symptoms • CSF abnormalities typical of meningitis • Absence of bacteria on smear/culture of CSF • Self limiting, benign course 9/29/2008 BANGAR RAJU- 2008 33 Fungal Meningitis • Most common in the immunosupperssed due to: – AIDS – Organ transplantation – Immunosuppressive chemotherapy – Chronic corticosteroid therapy • Meningeal signs and symptoms may occur up to 3 – 6 months after initial infection 9/29/2008 BANGAR RAJU- 2008 34 Clinical Features of Fungal Meningitis • Usually mild meningeal symptoms – – – – – – Headache Fever Photophobia Nausea / Vomiting Skin lesions Cranial nerve palsies 9/29/2008 BANGAR RAJU- 2008 35 Syphilitic Meningitis • 6 types of neurosyphilitic syndromes • Acute syphilitic meningitis one of them – Meningeal symptoms typically develop within 1 year – Usual symptoms: • Headache • Nausea / Vomiting • Stiff neck • Papilloedema 9/29/2008 BANGAR RAJU- 2008 36 Carcinomatous Meningitis • Dissemination of malignant cells throughout the leptomeninges • Adenocarcinoma and malignant melanoma are the most common solid tumours to metastasize to leptomeninges • Mechanism for how tumour cells reach the leptomeninges is unknown 9/29/2008 BANGAR RAJU- 2008 37 Bacterial meningitis: • Also called as septic or pyogenic meningitis • Virus meningitis is a self-limiting condition • Bacterial meningitis far less common but lifethreatening • Rare causes - spirochetes, fungi • Early acute-influenza-like syndrome, (fever, severe head ache, stiff neck, lethargy.) • In elderly immunocompromised, confusion, agitation, disorientation, coma • Bacterial meningitis is a severe life threatening infection that requires prompt diagnosis and treatment –uncommon • Viral meningitis more common – less serious • Agent of bact meningitis varies with age 9/29/2008 BANGAR RAJU- 2008 38 9/29/2008 BANGAR RAJU- 2008 39 Etiology of bacterial meningitis by patient category age Birth to 3 months Most common Strep agalactiae ( group B) others E coli Listeria monocytogenes N. meningitidis H. influenzae type B 3- 60 months Strep pneumoniae >60 MONTHS Strep pneumoniae N. meningitidis L. monocytogenes Staphylococcus epidermidis Pseudomonas and GNB GNB & pseudomonas Any age Staphylococcus aureus Any age L. monocytogenes 9/29/2008 BANGAR RAJU- 2008 40 9/29/2008 BANGAR RAJU- 2008 41 Neisseria meningitidis Streptococcus pneumoniae Haemophilus influenzae 9/29/2008 BANGAR RAJU- 2008 42 Predisposing Conditions Age: 18-50 - S. pneumo, N. mening >50 - S. pneumo, N. mening + Listeria Trauma/Surgery: – S. aureus, coag neg staph, P. acnes, gram neg bacilli CSF Leak: H. influenza Immunocompromised : • ↓Immunoglobulins - S. pneumo • Splenectomy: S. pneumo, H.influenza • Steroids- nocardia, cryptococcus • Neutropenic –gram neg bacilli, molds • AIDS – Cryptococcus, histo, TB • Alcoholic: S. pneumo, H.influenza, Listeria 9/29/2008 BANGAR RAJU- 2008 43 Meningitis - At birth to 3 months • 20,000 cases of bacterial meningitis in US yearly. • 70% of these in children < than 10 yrs • Reason:- predisposition to bact infections, lower integrity of barriers, immature defense mechanisms. • Neonatal meningitis - <10% of cases but >50% meningitis deaths. • Infants suffer more common from Haemophilus influenzae meningitis 9/29/2008 BANGAR RAJU- 2008 44 • In babies - diagnosis of life threatening infs is difficult • They do not display clinical signs • Symptoms - failure to thrive, off their feeds, fever, diarrhea. • Group B Streptococci in female genital tract, infects babies during passage through birth canal. • Penicillin + Gentamicin 9/29/2008 BANGAR RAJU- 2008 45 • Meningitis in postnatal period is common but now very rare due to capsular polysaccharide protein -Conjugate vaccine during infancy. • A vaccine has been developed to protect infants---Hib – children from 2 months of age. Non vaccinated children: Permanent neurological damage-risk hearing loss, delayed language development, mental retardation, seizures. 3rd generation cephalosporin or chloramphenicol . close contacts- rifampicin prophylaxis 9/29/2008 BANGAR RAJU- 2008 46 • • • • • Encapsulated GNB- pathogens of RT. Spread from RT via blood to brain. Infants 3-4months protected by maternal abs, By age 3-4 yrs will have their own abs. Window of opportunity between these ages. 9/29/2008 BANGAR RAJU- 2008 47 • Listeria – GPB found in high • • • • numbers in cheese. Consumption of these foods during pregnancy is discouraged They might cause fetal infections. In immunocompromised – renal transplant & cancer too Newborn babies also at risk- healthy carriers not uncommon 1st week of life- septicemia ---> meningitis BANGAR RAJU- 2008 48 • 9/29/2008 9/29/2008 BANGAR RAJU- 2008 49 Meningitis in children & young adults N meningitidis- most common • S pneumoniae cause bacterial meningitis at any age • 80% -N meningitidis, S pneumoniae, H influenzae. • N meningitidis – epidemic disease • All others are sporadic. 9/29/2008 BANGAR RAJU- 2008 50 Meningococcal meningitis • GN diplococcus found within PMN. • 20% population carry in oropharynx • Carriage rate higher in crowded areasmilitary barracks, boarding school dormitories. • Spread by inhalation of infected droplets. • Serotypes in developed countries- B,C,Y • Less developed areas-A,C ,Y and W-135 9/29/2008 BANGAR RAJU- 2008 51 • UK –first to introduce-conjugate C vaccine • Part of routine childhood immunization since November 1999. • Incub period 1-3 days, sore throat, head ache, fever, neck stiffness, photophobia • In 80% cases, petechial rash-evidence of septicemia. • In 35% , disseminated intravascular coagulation (DIC), endotoxiemia, shock, renal failure • Severe cases, bleeding in brain and adrenal glands 9/29/2008 BANGAR RAJU- 2008 52 9/29/2008 BANGAR RAJU- 2008 53 Severe toxic symptoms of N meningitidis 9/29/2008 BANGAR RAJU- 2008 54 • Untreated-mortality 100%- 10% even if treated, • Hearing loss in some survivors. • Penicillin or 3rd generation cephalosporin 9/29/2008 BANGAR RAJU- 2008 55 Differential diagnosis of fever, altered sensorium and petechial rash • • • • Meningococcal disease Rickettesial infection (RMSF etc.) S. aureus endocarditis S. pneumoniae, H. influenzae (especially with Splenectomy) Septic shock Viral meningitis Viral hemorrhagic fevers HUS, vasculitis, etc. • • • • 9/29/2008 BANGAR RAJU- 2008 56 Streptococcus pneumoniae meningitis: • Capsulated GP Diplococcus • Children less than two yrs and elderly. • Sickle cell anemia, alcoholics, splenectomized patients, head trauma- more at risk. • Abs to capsular ags opsonises the bacteria and promotes phagocytosis but protection is type specific-there are more than 85 capsular types. • High mortality (20-30%)even when treated • In 15-20% permanent neurological damage. • CDC-heptavalent protein conjugate vaccine • For children from 2-23 months of age 9/29/2008 BANGAR RAJU- 2008 57 9/29/2008 BANGAR RAJU- 2008 58 9/29/2008 BANGAR RAJU- 2008 59 • Older children from 24-59 months are at high risk (sickle cell disease, HIV inf, chronic illness etc) • Penicillin - in some countries strains are resistant to penicillin. 9/29/2008 BANGAR RAJU- 2008 60 Mycobacterium tuberculosis meningitis: (Tubercular meningitis) • Rare-difficult to diagnose and so delay in treatment • Patients here have a focus of My. tuberculosis elsewhere in the body. • In > than 50% cases, meningitis is associated with acute miliary tuberculosis • In high prevalent TB areas, meningitis seen from 0-4 yrs of age• less prevalent areas –meningitis seen in adults. 9/29/2008 BANGAR RAJU- 2008 61 • • • • • • • Gradual onset over a few weeks. Malaise, apathy, anorexia within few weeks to photophobia. Neck stiffness and impairment of consciousness. Delay in diagnosis, treatment – serious neurological damage. Spinal TB found only in developing countries The bacteria in the vertebrae destroy intervertebral disks to form epidural abscesses. They compress spinal cord and cause paraplegia. 9/29/2008 BANGAR RAJU- 2008 62 • Treatment- isoniazid and rifampicin both cross blood-brain barrier. • Conventional culture of samples take 8 weeks. • PCR based identification is done in a day. 9/29/2008 BANGAR RAJU- 2008 63 HOST RESPONSES TO INVASION • • • • • • Normal CSF: water clear < 5 lymphocytes/ml glucose- 45-100mg/dl Protein – 14-45mg/dl sterile opening pressure 70-180mm H2O 9/29/2008 BANGAR RAJU- 2008 64 Bacteria infections: • increase in polymorphonuclear leucocytes, • increase in proteins, • CSF visibly turbid- SEPTIC MENINGITIS slow growing or les pyogenic- TB or Listeria less changes Viral infections: • increase in lymphocytes, • slight increase in proteins, • CSF clear- ASEPTIC MENINGITIS 9/29/2008 BANGAR RAJU- 2008 65 origin bacteria viruses Partial treated bact meningitis Leptospirosis WBC count 1001000 10-100 10-100 Predominant cell type Neutrophils Neutrophils Neutrophils & lymphocytes Neutrophils & lymphocytes Neutrophils & lymphocytes monocytes& lymphocytes monocytes& lymphocytes Neutrophils Glucose level Decreased Normal to slightly decreased Normal to slightly decreased Protein level increased Normal to slightly increased increased 100500 100500 10-100 normal increased Lyme disease normal increased Myco-bacteria Decreased increased fungi 10-100 Decreased increased Autoimmune diseases drugs 9/29/2008 10-100 Normal to slightly decreased Normal to slightly increased 100500 normal Neutrophils BANGAR RAJU- 2008 increased 66 Clinical diagnosis Clinical Presentations of patients: Pathophysiology Systemic infection Clinical features Fever, myalgia rash Meningieal inflammation Neck stiffness, briduzinskis, kerning's sign, headache Cerebral vasculitis Focal neurological abnormalities, seizures Elevated intracranial pressure Change in mental status, headache, cranial nerve palacies, seizures 67 9/29/2008 BANGAR RAJU- 2008 Symptoms and Signs of Bacterial Meningitis Rapid Onset (25%) hospitalized < 24h Respiratory Symptoms days – weeks Headache 90% Fever >90% consciousness 80% Meningismus 85% Focal neurosigns 25% Ocular palsies – 10% Vomiting – 30% Seizures – 30% Petechiae/purpura 50% (meningo.) Hemiparesis Myalgias 20% Papilledema 1% 9/29/2008 BANGAR RAJU- 2008 68 Pictorial depiction of symptoms of meningitis 9/29/2008 BANGAR RAJU- 2008 69 Approach to suspicious patient • History • Clinical Exam • LP • Empirical Treatment • Does H& PE help in determining when to perform an LP? 9/29/2008 BANGAR RAJU- 2008 70 Signs of meningeal irritation: • Brudzinski’s sign: resistance to passive neck flexion. • Kernig’s sign: inability to extend leg when thigh is flexed at 90º with the trunk. Subacute/chronic: TB/Fungal infections increased cranial pressure (papilledema, nausea, vomiting), disorientation, confusion, personality change. 9/29/2008 BANGAR RAJU- 2008 71 9/29/2008 BANGAR RAJU- 2008 72 9/29/2008 BANGAR RAJU- 2008 73 9/29/2008 BANGAR RAJU- 2008 74 9/29/2008 BANGAR RAJU- 2008 75 Complications of meningitis in infants and children - Hearing loss is the most encountered sequelae; it occurs * in 30% cases of S. pneumoniae meningitis, * in 20% of H. influenzae meningitis, * in 10% of N. meningitidis meningitis - Mental retardation, seizures, delay in language acquisition, visual impairment, behavioural problems and hydrocephalus. 9/29/2008 BANGAR RAJU- 2008 76 • Blood stained CSF- bad take or subarachnoid haemorrhage. • TB meningitis- spider’s web clot-high protein content Biochemical tests on CSF: • Viral, Mycobacterium, Cryptococcus meningitis-- glucose normal or just subnormal, protein slightly raised • Bacterial--– glucose very low due to metabolism, protein significantly raised 9/29/2008 BANGAR RAJU- 2008 77 LABORATORY DIAGNOSIS: SAMPLE: • CSF IN A STERILE CONTAINER • DIVIDED INTO THREE PARTS 1. STAINING— gram staining, AFB 2. CULTURE- selective medium 3. OTHER REACTIONS & TESTS 9/29/2008 BANGAR RAJU- 2008 78 Laboratory methods of diagnosis * CSF Gram staining * CSF biochemistry -(protein, glucose, chlore) and culture which may take 24 to 48 hours, * Latex agglutination test detects antigens of common pathogens (H. influenzae type b, S. pneumoniae, N. meningitidis, E. coli K1 and group B Streptococcus)  results are available within 20 – 30 minutes, but the sensitivity ranges from 50 to 90% and specificity can be variable. * Blood culture is of great help. 9/29/2008 BANGAR RAJU- 2008 79 Are CSF cytokine/chemokines concentrations useful in the differential diagnosis of meningitis? Higher in bacterial IL-1 TNF IL-8 (81% sens. 92% spec) Higher in viral IP-10 Mig I-TAC MIP-1a 9/29/2008 BANGAR RAJU- 2008 80 ANTIBIOTIC RECOMMENDATIONS BASED ON GRAM STAINING FINDING GRAM POSITIVE COCCI ANTIBIOTIC VANCOMYCIN + BROAD SPECTRUM CEPHALOSPORIN GRAM POSITIVE RODS AMPICILLIN+/- AMINOGLYCOCIDE GRAM NEGATIVE RODS ANTIPEUDOMONAL CEPAHLOSPORIN + AMINOGLYCOSIDE BROAD SPECTRUM CEPHALOSPORINS GRAM NEGATIVE DIPLOCOCCI 9/29/2008 BANGAR RAJU- 2008 81 9/29/2008 BANGAR RAJU- 2008 82 Acute therapy for acute bacterial meningitis Organism Step. pneumoniae Treatment Vancomycin + 3rd cephalosporin N.meningitidis L.Monocytogenes Grp B streptococci H.Influenzae Enterobacteriaceae Pseudomonas aeruginosa Ceftriaxone Ampicillin + gentamicin Penicillin G Ceftriaxone 3rd cephalosporin + aminoglycoside Cephalosporin + aminoglycoside 9/29/2008 BANGAR RAJU- 2008 83 Empiric Rx for purulent meningitis Predisposing Factor Age 18-50 years Age > 50 years Immunocompromised Basilar skull fx Head trauma/ neurosurgery CSF fluid shunt 9/29/2008 BANGAR RAJU- 2008 Antimicrobial Therapy Vanco + Ceftriaxone Ceftriaxone + Amp Vanco + Amp + Cefepime Vanco + Ceftriaxone Vanco + Cefepime Vanco + Cefepime 84 Specific antibiotic treatment N. meningitidis Antibiotic Penicillin Ceftriaxone Ampicillin Ceftriaxone Penicillin Ceftriaxone Vanco +Ceftriaxone + Rifampin Alternative Chloramphenicol Bactrim or Chloramphenicol Vanco Vanco Vanco and Rifampin H. influenzae B lactamase neg B lactamase pos S. pneumoniae PCN MIC< 0.06 PCN MIC 0.1-1.0 PCN MIC >2.0 Duration of antibiotic Based on tradition more than scientific evidence Pneumococcal 10-14 days Meningococcal 7 days H influenza 7-10days Listeria 3 weeks • • • • Many of life's failures are men who did not realize how close they were to success when they CSF: • Microscopy & culture ofgave up. Microscopy: cell types - Thomas PMN • acute bacterial meningitis—excess of Edison • Viral,Cryptococcal,TB meningitis-excess of lymphocytes. • India-ink for C neoformans –capsule • Gram stain , Ziehl-Neelsen stain-rapid provisional diagnosis • Culture difficult- antibiotics • Ag detection kits –before culture results. • Blood culture & CSF samples for isolation 9/29/2008 BANGAR RAJU- 2008 87