Central nervous system infections including Meningitis

• The first step in the diagnosis of a patient with suspected CNS infection is a lumbar puncture (spinal tap).

Specimen: Cerebrospinal fluid (CSF)

Collection and Transport of CSF:

• Aseptically CSF is collected.
• A needle is inserted into the subarachnoid space (lumbar puncture), at the lumbar spine region between L3, L4, or L5.
• In the sterile collection tubes, three or four tubes of CSF should be collected. It should not contain additives.
• Tube 1 is used for:
  • chemistry studies
  • glucose and protein count
  • immunology studies
• Tube 2 is used for culture.
• Tubes 3 and 4 are used for cell count and differential count.
• The amount of volume to be collected depends on the volume available in the patient which may differ between the adults and the neonates.
• When the needle first punctures the subarachnoid space, the opening pressure of the CSF is observed.
• In the high opening pressure, CSF should be collected slowly to prevent the collection of a larger volume of fluid.
• For the detection of mycobacteria and fungi, a minimum of 5 to 10 mL is recommended.
• Centrifugation and subsequent culture are done.
• The false-negative result may be seen if the sample is inadequate.
• CSF should be sent to the laboratory as soon as possible.
• In the case of delay after an hour or longer, agents such as Streptococcus pneumoniae, may not be detectable.
• CSF should not be refrigerated for microbiological studies.
• In the case of delay, it should be left at room temperature or incubated at the 35°C.
• For the viral study, CSF may be refrigerated, for as long as 23 hours after collection or frozen at −70°C.
• For hematology studies, CSF specimens can be refrigerated,
• For chemistry and serology, CSF can be frozen (−20° C).

Initial processing of CSF:

• All the CSF specimens for the bacterial, fungal, or parasitic studies should be centrifuged.
• Volume greater than 1 ml should be used.
• Centrifugation should be done at 1500× g for 15 minutes.
• Suspected specimens for cryptococci or mycobacteria should be handled carefully.
• When CSF fewer than 1 mL is available, Gram stain should be done and plated directly to the blood and chocolate agar plates.
• The supernatant is removed to a sterile tube, leaving approximately 0.5 mL of fluid.
• For visual examination and culture, the remaining fluid is used to suspend the sediment.
• The supernatant can be used:
• To test the presence of antigens
• rapid diagnostic test (vertical flow immunochromatography)
• for meningitidis
• For chemistry evaluations (e.g., protein, glucose, lactate, C-reactive protein).

Laboratory diagnosis:

• Communication between the physician and the microbiology laboratory is essential for the proper diagnosis and treatment of the patient.
• The diagnosis of acute bacterial meningitis can be excluded in patients with normal fluid parameters in almost all cases.
• Similar criteria have been used to exclude the performance of smear and culture for tuberculosis, as well as syphilis serology, on CSF specimens.

1. Visual Detection of Etiologic Agents in CSF:

• CSF sediment is examined for the presence of cells and organisms.

i) Stained Smear of Sediment:

• Gram staining should be performed on all the CSF sediments.
• The use of contaminated slides may give false-positive smears.
• The sediment should be thoroughly mixed and a heaped drop should be placed in the slide.
• The slide should be sterile or alcohol-cleaned.
• The sediment should never be spread out on the slide surface.
• It is because of the difficulty to find small numbers of microorganisms.
• The drop of sediment is allowed to air dry.
• Then it is heated or methanol fixed.
• Then it is stained by either Gram or acridine orange.
• A faster examination of the slide under high-power magnification (400×) can be done by the acridine orange fluorochrome stain.
• The brightly fluorescing bacteria can be visualized easily.
• Confirmation of the presence and the morphology of the organism can be done, using the Gram stain (directly over the acridine orange.
• The use of a cytospin centrifuge is an excellent alternative method for the preparation of slides for staining.
• It concentrates cellular material and bacterial cells up to 1000-fold.
• Centrifugation is done then the CSF is concentrated onto a circular area of a microscopic slide.
• It is then fixed, stained, and examined.
• Reporting should be done for the presence or absence of bacteria, inflammatory cells, and erythrocytes.

ii) Wet Preparation:

• Amoebas are best observed by this method.
• Sediment can be examined as wet preparation under phase-contrast microscopy.
• The light microscope can be used as an alternative, by slightly closing the condenser.
• Amoebas must be distinguished from motile macrophages, which occasionally occur in CSF.
• A trichrome stain can be used in the differentiation of amoebas from somatic cells.
• On the lawn of Klebsiella pneumoniae or Escherichia coli, the pathogenic amoebas can be cultured. Lawn.

iii) India Ink Stain:

• Cryptococcus neoformans consists of the large polysaccharide capsule which could be visualized by the India ink stain.
• For capsular antigen, latex agglutination testing is more sensitive and extremely specific.
• Antigen test is recommended than the India ink stain.
• Culture is essential in case of the AIDS patients because detectable capsules of neoformans may be absent.
• A drop of CSF sediment is mixed with one-third volume of India ink, for the India ink preparation.
• By the addition of 0.05 mL thimerosal, India ink can be protected from contamination.
• Smooth suspension is made by mixing the CSF and ink.
• Then a coverslip is applied to the drop.
• Then it is examined under high-power magnification (400×) for characteristic encapsulated yeast cells.
• Examination can be done under oil immersion.
• White blood cells must not be confused with yeasts.
• The presence of encapsulated buds, smaller than the mother cell, is diagnostic.

2. Direct Detection of Etiologic Agents:

Antigen detection:

• For the rapid detection of antigen in the CSF, commercial reagents and kits are available.
• By latex agglutination, rapid antigen detection can be done from CSF.
• An antibody-coated particle binds to a specific antigen which results in macroscopically visible agglutination.
• The soluble capsular polysaccharide, including the group B streptococcal polysaccharide, is well suited to serve as bridging antigens.
• Polyclonal or monoclonal antibody or an antigen from an infectious agent is present in the agglutination assay.
• Different commercial systems have been developed.
• Soluble antigens may concentrate in the urine from Streptococcus agalactiae and Haemophilus influenza.
• For the performance of antigen detection test systems, the manufacturers’ directions must be followed
• Some systems may also require the pretreatment of samples which is usually for 5 minutes.
• The pretreatment, called rapid extraction of antigen procedure (REAP), is recommended for laboratories that use commercial body fluid antigen detection kits.
• Only a limited number of clinically useful situations warrant bacterial antigen testing (BAT).
• Practice guidelines for the diagnosis and management of bacterial meningitis do not recommend routine use of BAT.

Bacteria involved in meningitis:

Cryptococcus neoformans:

• For the detection of polysaccharide capsular antigen of Cryptococcus neoformans, the reagents are available commercially.
• When the positive result for cryptococcal antigen is obtained in CSF specimens, a second latex agglutination test for rheumatoid factor should be done.
• Both latex agglutination assays (numerous commercial manufacturers) and enzyme immunoassays are available for the detection of Cryptococcus antigen.
• The false-negative reaction may be seen in the undiluted specimens which contain large amounts of capsular antigens.
• False-negative reaction is caused by a prozone phenomenon.
• Patients with AIDS may have an antigen titer over 100,000.
• It requires many dilutions to reach an endpoint.
• Parasites and Viruses are also involved in meningitis

  Conditions for the culture of free-living amoebae and viral agents should be maintained to detect viruses and parasites.

3. Molecular methods:

• PCR (Polymerase Chain Reaction )
• Real-time PCR

4. Other Tests

• the Limulus lysate test
• CSF lactate determinations,
• C-reactive protein
• mass spectrometry
• gas-liquid chromatography

5. Culture:

• Routine bacteriologic media: chocolate agar plate, 5% sheep blood agar plate, and an enrichment broth, usually thioglycolate without indicator.
• Blood agar plates help in the recognization of pneumoniae.
• For the isolation of influenzae and N.meningitidis, a chocolate agar plate is used.
• Plates should be incubated at 37° C in 5% to 10% carbon dioxide (CO2) for at least 72 hours.
• Candle jar can be used, if a CO2 incubator is not available.
• The broth should be incubated in the air at 37° C for at least 5-10 days.
• Anaerobic blood agar plate may also be inoculated, when Gram stain shows the morphologically resembling anaerobic bacteria.
• If a brain abscess is suspected then also anaerobic blood agar plate is used.
• For CSF fungal cultures, two drops of the well-mixed sediment should be inoculated onto:
• Sabouraud dextrose agar
• other non-blood containing medium
• brain-heart infusion with 5% sheep blood.
• Incubation of Fungal media should be done at 30° C for 4 weeks.
• If possible, two sets of media should be inoculated.
• One set should be incubated at 30° C and the other at 35° C.

Specimen: Brain Abscess/Biopsies samples

Collection, Transport, and Processing of brain absscess and biopsies

• Under anaerobic conditions, biopsy specimens or aspirates from brain abscesses should be submitted.
• Devices are commercially available too for transportation.
• Swabs are not considered an optimum specimen.
• If swabs are used to collect abscess material, during transportation, they should be maintained in an anaerobic environment.
• Before plating and smear preparation, biopsy specimens should be homogenized in sterile saline.
• processing should be kept to a minimum to reduce oxygenation.
• Inoculation should be done onto 5% sheep blood and chocolate agar plates, for the abscess and biopsy specimens.
• Incubation should be done in 5% to 10% CO2 for 72 hours at 35° C.
• In addition, an anaerobic agar plate and broth with an anaerobic indicator, vitamin K, and hemin should be inoculated and incubated in an anaerobic environment at 35° C.
• Incubation of the anaerobic culture plate is done at a minimum of 72 hours.
• It is examined after 48 hours of incubation.
• Anaerobic broths should be incubated for a minimum of 5 days.
• When fungi are suspected, fungal media, such as brain-heart infusion with blood and antibiotics or inhibitory mold agar, should be inoculated.

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