Laboratory Diagnosis of Meningitis

Laboratory Diagnosis of Meningitis  The most common cause of meningitis is viral infections that usually resolve without treatment.  There is an urgent need for the laboratory diagnosis of suspected meningitis, especially pyogenic as it is life threatening and can cause serious complications, such as brain damage, hearing loss, or learning disabilities. The pathogens that cause bacterial meningitis can also be associated with another serious illness like sepsis. Without timely treatment, sepsis can quickly lead to tissue damage ,organ failure ,and finally death. INTRODUCTION  Meningitis -Inflammation of the membranes surrounding the brain or spinal cord i.e. of subarachnoid space or the leptomeninges (arachnoid and piamater) but not duramater.  Encepalitis\ MeningoencephalitisEncephalitis is the inflammation of the brain parenchyma and is usually as a result of viral infection. Concomitant meningitis that occurs with encephalitis known as meningoencephalitis.  Brain abscess(localized collection of the pus in a cavity formed by the breakdown of tissue), it may occasionally cause changes in the CSF and clinical symptoms that mimic meningitis. Brain abscesses may also rupture into subarchnoid space, producing a severe meningitis with high mortality. Meningitis is divided into two major categories : Purulent Meningitis (pyogenic mengitis) and Aseptic Meningitis Aseptic mengitis – Purulent Meningitis It is characterized by elevated polymorphonuclear cells in CSF .  It is exclusively caused by bacterial agents.  According to age agents involved may vary.  Overall S. pneumoniae is the most common cause of pyogenic meningitis.  But in neonates common agents are group B streptococcus, Escherichia coli and listeria.  It is characterized by elevated lymphocytes in CSF.  The etiological agent include viral , tubercular ,fungal and parasite agent.  The term “aseptic” is frequently misnomer , implying lack of infection.  Lymphocytes are also found in the c.s.f.in trypanosomiasis meningoencephalitis , and neurosyphilis. Routes of infection  Haematogenous spread : most common route followed by entry into the subarachnoid space, choroid plexus or other blood vessels of the brain.  Direct spread : from an infected site present close to meninges; examples of such infection include otitis media, sinusitis and mastoiditis.  Anatomic defect in CNS : as a result of surgery, trauma, congenital defect can allow microorganisms easy and ready access to the CNS.  Shunt (venrticulo-peritoneal ).  Retrograde route : along the nerve: least common route, occurs in case of rabies virus or herpes simplex virus infection.  Itragenic implantaion of microorganism. Etiological Agents of Meningitis Bacterial meningitis  Neonates (<1 month)  GNB (Escherichia coli, Klebsiella sp., Enterobacter sp.)  Streptococcus agalactiae  Listeria monocytogenes Infants (1-23 months)  Streptococcus agalactiae  Escherichia coli  H. influenzae  Streptococcus pneumoniae  N. meningitidis  2-20 years  Neisseria meningitidis: Most common  Streptococcus pneumoniae  Haemophilus influenzae  >20 years (adult)  Streptococcus pneumoniae: Most common  Haemophilus influenzae  Neisseria meningitidis  Older adults( > 65 years)  Streptococcus pneumoniae  Neisseria meningitides  Listeria monocytogenes  Aerobic gram negative bacilli   Post traumatic /post surgical  Pyogenic Staphylococcus and Streptococcus  Staphylococcus epidermidis  Coliform bacilli  Anaerobic cocci  Bacteroides CSF- venous shunts   Staphylococcus epidermidis Associated with Brain abscess / contagious focus of infection  Anaerobic Streptococci  Bacteroides  Fusobacterium Viral meningitis  Enteroviruses (Coxsackie A & B , Echovirus, polio ): most common.  Adenovirus  Arbovirus (Flavivirus, Buniyavirus, West Nile virus )  Paramyxoviruses (Mumps ,Measles virus)  Herpes viruses (Herpes Simplex, Varicella Zoster, Epstein-Barr virus)  Influenzae virus A or B  Rabies virus  Lymphocytic choriomeningitis virus (rarely) Fungal meningitis  Cryptococcus neoformans- (major cause mainly in AIDS patients )  Coccidioides immitis  Histoplasma capsulatum  Blastomyces dermatitidis  Candida spp.  Aspergillus (rarely ) Parasitic infections  Protozoafree living Amoeba Naegleria fowleri (primary amoebic meningoencephalitis)  Acanthamoeba  Balamuthia mandrillaris (granulomatous amoebic encephalitis)  Trypanosoma gambiens- chronic meningoencephalitis .  Gnathostoma spinigerum- eosinophlic meningoencephalitis  Helminths Rarely the larvae of Angiostrongylus cantonensis & Dirofilaria immitis (eosinophlic meningitis).  Toxoplasma gondii (mainly in AIDS patients).  CSF Specimen collection (Obtained by subdural tap, ventricular aspiration or lumbar puncture ) -Must be collected by an experienced medical officer or health care worker. -C.S.F. must be collected aseptically inserting a spinal needle 22 (adult) or 23 gauge (children) into the subarachnoid space, usually at the level of lumbar spine, using all universal precaution to prevent organisms being introduced into the central nervous system. - Only 3-5 ml fluid should be collected in a fresh sterile screw capped tubes (1 ml each). - Rate of collection should be 4-5 drops/sec. -Large volume collected – headache   3-5 ml CSF collected in fresh, sterile , screw capped container or sterile tubes (1 ml each). C.S.F. is Collected into 3 tubes: Tube 1-for chemistry (for protein & glucose , lactate , C-reactive protein ).  Tube 2- for Microbiology (Gram’s stain & culture), special studies like VDRL, Cryptococcal Ag.  Tube 3- for cytology (cell count & differential count). Spinal tap technique for CSF Collection  Obtain patient consent.  Prepare the CSF collection kit and a puncture –resistant autoclavable container for used needle.  Wear surgical mask and sterile latex gloves that are impermeable to liquids.  Label the collection tubes with appropriate information as patient name, age, date and time of specimen collection and unique identification Number. Be sure this number matches the number on both the request and report forms.  Position the patient, by lying on his side, with knees flexed & back arched to separate lumbar vertebrae.  Disinfect the skin along a line drawn between the both iliac creastswith 70% alcohol and povidone –iodine to clean the surface and remove debries and oil. Allow to dry completely.  Position the spinal needle between two vertebral spine at L3 &L4 level, carefully directed between spinous processes, through intraspinous ligaments into spinal canal.  Accurate placement of the needle is checked by flow of fluid, which is normally clear and colorless.  Remove CSF and collect into sterile screw-cap tubes  Withdraw the needle and cover the insertion site with an adhesive bandage. Discard the needle in a puncture –resistant , autoclavable container.  Remove mask and gloves and discard in an autoclavable container.  Wash hands with antibacterial soap and water immediately after removing gloves.  Immediately transport the CSF with request form to laboratory Tube 1 Tube 2 Tube 3  Blood culture- is particularly useful in meningitis due to N.  Serum – for viral serology  Septic spots on skin- meningococcal meningitis- scraped & fluid  meningitidis, H. influenzae and Str. Pneumoniae. About 50 percent of these cases have positive blood culture. expressed on to a slide for gram stain. Faeces specimen & Throat swab- for Polio virus & other Enteroviruses.  Nasopharyngeal swab – mainly for viruses  CT –guided Aspiration of pus sample – for Brain abscess.  Urine – for Antigen detection of S. pneumoniae,C. neoformans. Transportation & storage of CSF  The CSF being the most precious specimen should be transported to lab as quickly as possible, delay may result in death of delicate pathogens and disintegration of leukocytes.  Specimen should never be refrigerated. Certain agents, such as S. pneumoniae , H. influenzae and N. meningitidis may not be detectable after an hour or longer unless antigen detection methods are used. If not rapidly processed CSF should be incubated at (35 ֯C) or left at room temperature.  Exception to this rule involves CSF For viral studies, specimens may be refrigerated for as long as 23 hrs after collection or frozen at -70 ֯C if a longer delay is anticipated until they are inoculated.  For Ag detection tests- specimen stored at < 4 ֯C as bacterial polysaccharide Ag breakdown faster at RT & 37 ֯C Specimen Requisition  Proper labelling of specimen should be done and requisition form should be filled completely.  A complete requisition should include following :  The patient’s name  Hospital name or number  Age or date of birth  Sex  Collection date and time  Exact nature and source of the specimen  A short history about patient’s clinical signs and symptoms,  Diagnosis  Immunization history  Current antimicrobial therapy This information helps the microbiologist to work up the Rejection Criteria  The information on the label does not match the information on the requisition (patient name or source of specimen is different).  The specimen has been transported at the improper temperature.  The quantity is insufficient for testing.  The specimen is leaking.  The specimen transported time exceeds 2 hours post collection .  The specimen is dried up.  Processing the specimen, having any of these rejection criteria would produce information of questionable medical value. Processing of CSF  Must be done immediately .  Probabilities of detecting organisms by staining & culture depend upon volume of specimen.  1-2 ml sufficient to detect bacteria.  For fungi & mycobacteria 5-10 ml needed.  Probability of detecting organism increased by concentrating specimen. Examination of C.S.F. DAY 1 1. 2. Report appearance Test C.S.F. Purulent or cloudy C.S.F. Suspect pyogenic bacterial meningitis  Gram smear Report: - Number of pus cells -Bacteria  Culture C.S.F. -Blood agar ,chocolate agar(incubate in CO2) - If neonate: also MacConkey agar. Incubate aerobically  Describe whether C.S.F.  Clear, slightly turbid, purulent  Contains blood  Contains clots Slightly turbid or clear C.S.F. Perform cell count: Note whether pus cells or lymphs Pus cells • Gram smear • Culture C.S.F. Additional tests • Wet preparation: For motile amoebae • • • • • • • Lymphs Measure protiens Measure glucose ZN for AFB Silver impregnation stain India Ink: for encapsulated yeasts Dark ground microscopy Wet preparation and Giemsa smear DAY 2 and Onwards 3.  Examine and Report Cultures: Chocolate agar and Blood agar cultures: Look particularly for: N.meningitidis S.pneumoniae H.influenzae(chocolate agar)  MacConkey agar culture: look especially for bacteria that cause neonatal meningitis Additional  Perform AST (antimicrobial susceptibility test ) as indicated  Beta lactamase test. H.influenzae isolates Microscopic Examination of CSF    Direct wet preparation  Examine thoroughly mixed sediment under phase contrast microscope/ light microscope with condenser low.  Amoeba recognized by typical slow movement in one direction by pseudopodia. Remain motile for several hrs at room temp. & up to 24 hrs at 35-37 ֯C. Following a suspicious wet preparation, a Trichrome stain can be done.  Trypanosomes begin to loss motility within 15 min of withdrawal of CSF & are rapidly lysed, so fresh CSF required to detect them.  Motile leptospires may be seen. Gram stain – Make thick film about 10 mm diameter from centrifuged deposit. Microscopic examination of Gram stained smear may give a preliminary clue about the etiological agent of pyogenic meningitis based on the morphology of the bacteria. It should be reported immediately This helps in early initiation of appropriate antimicrobial agents(empirical therapy). Morphological clue suggesting bacterial pathogens in CSF causing pyogenic meningitis Morphology of bacteria Suggestive of-  Gram positive cocci in pair, lanceolate shaped. Streptococcus pneumoniae  Gram positive cocci in short chain(in neonate) Streptococcus agalactiae  Gram negative cocci in pair, intracellular, inside the pus cell  Pleomorphic gramnegative bacilli Neisseria meningitidis Haemophilus influenzae Escherichia coil or other gram- negative bacillis Gram stain of N. meningitidis in CSF with associated PMNs Pneumococci in gram stain Acridine orange stain  To screen smears of CSF for bacteria.  Fluorochrome stain that can intercalate into nucleic acid.  At low pH bacteria & yeast appear bright red & WBC pale apple green.  More sensitive than Gram stain.  Advantage- Gram staining can be done over it India ink stain  Used to detect large polysaccharide capsule of Cryptococcus neoformans .  A drop of sediment CSF mixed with 1/3 volume of India ink make smooth suspension, apply coverslip and observe under 40x.  Characteristic encapsulated yeast cells seen.  Quellung Reaction  Used to confirm organisms with a morphology typical of S. pneumoniae, N. meningitides or H. influenzae type b. Culture  Routine Media for bacteria  5% sheep blood agar  Enriched chocolate agar  Thioglycolate enrichment broth  Culture plates – incubation at 37 ֯C for 72 hr in 5-10 % CO2  Enrichment broth- incubated with loose cap at 37 ֯C days. for 5-10  If Gram stain show – GNB - MacConkey agar  If Gram stain show- anaerobic bacterial morphology or patient with a condition predisposing to anaerobic infection, an anaerobic blood agar plate also be inoculated. Culture for fungi  Handled in a biosafety cabinet.  Inoculate on non-blood containing medium- 1. BHI with Gentamicin or chloramphenicol slant/plate. 2. Bird seed agar is used for Cryptococcus species. 3. Myocel/ mycobiotic agar(selective agar).  Deposit material without spreading.  Incubate in air at 30 ֯C for 4 weeks. Cultivation of amoeba On artificial media supplied with living nutrient pneumonia) (K.  Viral cultureis done using tissue culture , egg inoculation or animal inoculation   0.25 ml CSF / tube inoculate into tubes of Primary Monkey kidney cells,HEp-2 cell lines , human fibroblast cell culture.  Incubate at 35 ֯C aerobically.  Cytopathic effects are noted. Tubercle bacilli   Cultured on slope of L-J media Leptospira  CSF or blood inoculated on Korthof’s medium AST Should be performed on all clinically relevant isolated bacteria to confirm resistance.  Serology:  Latex agglutination for antigen detection , done for S. pneumoniae, H. Influenzae and N. meningitidis. In neonates , the test for antigens of group B streptococcus and Listeria monocytogenes may also be performed.  Serological test for antibodies to different virus depending on clinical diagnosis.  Test to detect microbial antigen in urine: test are available to detect S.pneumoniae antigen(immunochromatography based) and Cryptococcus neoformans antigen (latexagglutination based) in Urine.  Detection of bacterial endotoxin: The Limulus lysate test is extremely sensitive for the detection of bacterial endotoxin. The principle of the test is based on coagulation of horse shoe crab (Limulus polypemus) amebocytes (blood cells) with blood containing endotoxin.  Molecular method : To detect microbial nucleic acid-- For suspected infections due to M.tuberculosis and viral infections, PCR based tests are increasingly used. Characteristic CSF findings in Meningitis Normal Bacterial fungal T.B. Syphilis Viral Parasitic CSF Pressure (mm of water) 50-150 Highly elevated (>180) Moderately elevated Moderately elevated normal Slightly elevate d/ normal normal Cell count (leukocy te/ ml) Adult= 0-5 (L) Neonat e=upto 30 (N) 10010000 (N) Normal 500 (L) 50-500 (L) 100750 (L) Normal- Normal200 (L) 200 lym. &/ or (E) Protein (mg/dl) 15-50 100-500 Markedly increase. Normal-250 Normal-150 50-250 frequen t normal Usually increase Glucose (mg/dl) 40-80 Usually decrease Normaldecrease. Usually decrease. normal normal Normal to decrease. Bacterial Ag test Calcoflou white stain, latexagglutinatio PCR, Auramine Rhodamine & ZN stain VDRL Serolog y, cell culture, PCR Serology, Biopsy Addition al findings Treatment Pathogen T/t Prevention Meningococci Pn(or chloramphenicol ) Rifampicin or ciprofloxacin prophylaxis for close contacts,pecaution to prevent inhalation of organism. Polysaccharide vaccine (gp B) H. influenzae Ampicillin/ ceftriaxone/ cefotaxime Hib vaccine Pneumococci Pn/ceftriaxone/chloramphenicol Prompt t/t of OM & RTI Polyvalent vaccine E. coli, other coliform GM+ cefotaxime/ceftriaxone Mycobacteria INH + R + Z +- S Cryptococcus neoformans Amphotericin B + flucytosine Protozoa Naegleria Amph.B + miconozole + rifampicin Listeria Pn/Amp + GM BCG INH prophylaxis for contacts