Anatomic Structure	Relative Location	Key Features
Epidural space	Outside the dura mater yet inside the skull	Cushion of fat and connective tissues
Dura mater	Outermost membrane	Membrane that adheres to the skull; white fibrous tissue
Subdural space	Between the dura mater and the arachnoid membrane	Cushion of lubricating serous fluid
Arachnoid membrane	Between the dura mater and pia mater	Delicate, cobweb-like membrane covering the brain and spinal cord
Subarachnoid space	Beneath the arachnoid membrane	Contains a significant amount of CSF in an adult (~125-150 mL)
Pia mater	Beneath the subarachnoid space	Adheres to the outer surface of the brain and spinal cord; contains blood vessels

Figure 71-1 Cross section of the brain shows the important membrane coverings and spacing and other key structures.

Cerebrospinal Fluid

Cerebrospinal fluid (CSF) surrounds the brain and spinal cord and has several functions. The CSF provides cushioning and buoyancy for the bulk of the brain, reducing the effective weight of the brain by a factor of 30. CSF carries essential metabolites into the neural tissue and cleanses the tissues of wastes as it circulates around the brain, ventricles, and spinal cord. Every 3 to 4 hours, the entire volume of CSF is exchanged. In addition to these functions, CSF provides a means by which the brain monitors changes in the internal environment.

CSF is found in the subarachnoid space (see Table 71-1) and within cavities and canals of the brain and spinal cord. There are four large, fluid-filled spaces within the brain referred to as ventricles. Specialized secretory cells, called the choroid plexus, produce CSF. The choroid plexus is located centrally within the brain in the third and fourth ventricles. Approximately 23 mL of CSF are contained within these ventricles in an adult. The fluid travels around the outside areas of the brain within the subarachnoid space, driven primarily by the pressure produced initially at the choroid plexus (Figure 71-2). By virtue of its circulation, chemical and cellular changes in the CSF may provide valuable information about infections within the subarachnoid space.

Figure 71-2 Flow of CSF through the brain. CSF originates in the choroid plexus and then flows through the ventricles and subarachnoid space and into the bloodstream.

Routes of Infection

One of the most important defense mechanisms of the CNS is the blood-brain barrier. The blood-brain barrier functions to maintain homeostasis in the brain through restricting the flow of chemical constituents from the blood to the CNS. In order for the CNS to become infected with bacteria, parasite, or virus, the blood-brain barrier must be penetrated.

Organisms may gain access to the CNS through several primary routes:

• Hematogenous spread: followed by entry into the subarachnoid space through the choroid plexus or through other blood vessels of the brain. This is the most common route of infection for the CNS.

• Direct spread from an infected site: the extension of an infection close to or contiguous with the CNS can occasionally occur; examples of such infections include otitis media (infection of middle ear), sinusitis, and mastoiditis.

• Anatomic defects in CNS structures: anatomic defects as a result of surgery, trauma, or congenital abnormalities can allow microorganisms easy and ready access to the CNS.

• Travel along nerves leading to the brain (direct intraneural): the least common route of CNS infection caused by organisms such as rabies virus, which travels along peripheral sensory nerves, and herpes simplex virus.

Diseases of the Central Nervous System

TABLE 71-2

Guidelines for Interpretation of Results Following Hematologic and Chemical Analysis of Cerebrospinal Fluid (CSF) from Children and Adults (Excluding Neonates)

Clinical Setting	Leukocytes/mm³	Predominant Cell Type	Protein	Glucose*
Normal	0-5	None	15-50 mg/dL	45-100 mg/dL
Viral infection	2-2000 (mean of 80)	Mononuclear^†	Slightly elevated (50-100 mg/dL) or normal	Normal
Purulent infection	5-20,000 (mean of 800)	PMN	Elevated (>100 mg/dL)	Low (<45 mg/dL), but may be normal early in the course of disease
Tuberculosis and fungi	5-2000 (mean of 100)	Mononuclear	Elevated (>50 mg/dL)	Normal or often low (>45 mg/dL)

^*Must consider CSF glucose level in relation to blood glucose level. Normally, the CSF glucose serum ratio is 0.6, or 50% to 70% of the blood glucose normal value.

^†About 20% to 75% of cases may have PMN leukocytosis early in the course of infection.

Epidemiology/Etiologic Agents-Acute Meningitis.

The etiology of acute meningitis depends on the age of the patient. Most cases in the United States occur in children younger than 5 years of age. Before 1985, H. influenzae type b H. influenzae type b was the most common infectious agent in children between 1 month and 6 years of age within the United States. Ninety-five percent of all cases were due to H. influenzae type b, Neisseria meningitidis, and Streptococcus pneumoniae. In 1985, the first Hib vaccine, a polysaccharide vaccine, was licensed for use in children 18 months of age or older but was not efficacious in children younger than 18 months. However, the widespread use of conjugate vaccine, Hib polysaccharide-protein conjugate, in children as young as 2 months of age has significantly affected the incidence of invasive H. influenzae type b disease; the total number of annual cases of H. influenzae disease in the United States have been reduced by 55% and the number of cases of H. influenzae meningitis by 94%. However, the risks for meningococcal and pneumococcal diseases resulting from agents other than H. influenzae have remained level. Children older than 6 years of age are less likely to develop meningitis, but the risk for meningitis infection increases when the child reaches early adulthood. As previously mentioned, neonates have the highest incidence of acute meningitis, with a concomitant increased mortality rate (as high as 20%). Organisms causing disease in the newborn are different from those that affect other age groups; many of them are acquired by the newborn during passage through the mother’s vaginal vault. Neonates are likely to be infected with, in order of incidence, group B streptococci, Escherichia coli, other gram-negative bacilli, and Listeria monocytogenes; occasionally other organisms may be involved. For example, Elizabethkingia meningoseptica has been associated with nursery outbreaks of meningitis. This organism is a normal inhabitant of water in the environment and is presumably acquired as a nosocomial infection.

Important causes of meningitis in the adult, in addition to the meningococcus in young adults, include pneumococci, Listeria monocytogenes, and, less commonly, Staphylococcus aureus and various gram-negative bacilli. Meningitis caused by the latter organisms results from hematogenous seeding from various sources, including urinary tract infections. The percentage of adults with nosocomial bacterial meningitis at large urban hospitals has been increasing. The various etiologic agents of chronic meningitis are listed in Box 71-1.

Aseptic Meningitis.

Aseptic meningitis is usually viral and characterized by an increase of lymphocytes and other mononuclear cells (pleocytosis) in the CSF; bacterial and fungal cultures are negative. (This is in contrast to bacterial meningitis, which is characterized by purulence and the polymorphonuclear [PMN] cell response in the CSF.) Aseptic meningitis is usually self-limiting with symptoms that may include fever, headache, stiff neck, nausea, and vomiting.

In addition to the increase of lymphocytes and other mononuclear cells in the CSF, the glucose level remains normal, whereas the protein CSF level may remain normal or be slightly elevated. Aseptic meningitis can also be a symptom for syphilis and some other spirochete diseases (e.g., leptospirosis and Lyme borreliosis). Stiff neck and CSF pleocytosis may also be associated with other disease processes, such as malignancy.

Encephalitis/Meningoencephalitis

Encephalitis is an acute inflammation of the brain parenchyma and is usually caused by direct viral invasion. Concomitant meningitis occurring with encephalitis is known as meningoencephalitis, and the cellular infiltrate present in the CSF is typically lymphocytic rather than polymorphonuclear cells.

The host response to these CNS infections can differ somewhat from those associated with purulent or aseptic meningitis. Early in the course of viral encephalitis, or when considerable tissue damage occurs as a part of encephalitis, the nature of the inflammatory cells found in the CSF may be no different from that associated with bacterial meningitis; cell counts, however, are typically much lower.

Viral.

Viral encephalitis, which cannot always be distinguished clinically from meningitis, is common in the warmer months. The primary agents are enteroviruses (coxsackie viruses A and B, echoviruses), mumps virus, herpes simplex virus, and arboviruses (West Nile virus, togavirus, bunyavirus, equine encephalitis, St. Louis encephalitis, and other encephalitis viruses). Other viruses—such as measles, cytomegalovirus, lymphocytic choriomeningitis, Epstein-Barr virus, hepatitis, varicella-zoster virus, rabies virus, myxoviruses, and paramyxoviruses—are less commonly encountered. Any preceding viral illness and exposure history are important considerations in establishing a cause by clinical means. Since 1999, with the first debut of West Nile in the United States, the West Nile virus has been an important consideration in the diagnosis of viral encephalitis. The Centers for Disease Control and Prevention (CDC) reports that the incidence of West Nile infection peaked in 2003 with 9862 cases of West Nile infection; 2860 were reported cases of meningitis and encephalitis, resulting in 264 deaths. Since then the rates of infection have dropped: human cases reported to the CDC in 2010 were significantly lower with 1021 total reported cases of West Nile; 629 were neuroinvasive cases resulting in 57 deaths; a state-by-state breakdown of the disease incidence is outlined in Table 71-3. In 2012, a deadly resurgence of West Nile virus occurred, including neuroinvasive and non-neuroinvasive, for a total of 4531 cases through mid-October, according to the CDC.

TABLE 71-3

Final 2010 West Nile Virus Human Infections in the United States *

State	Neuroinvasive Disease Cases	Non-neuroinvasive Disease Cases	Total Cases	Deaths	Presumptive Viremic Donors*
Alabama	1	2	3	0	0
Arizona	107	60	167	15	31
Arkansas	6	1	7	1	0
California	72	39	111	6	24
Colorado	26	55	81	4	1
Connecticut	7	4	11	0	5
District of Columbia	3	3	6	0	0
Florida	9	3	12	2	1
Georgia	4	9	13	0	1
Idaho	0	1	1	0	0
Illinois	45	16	61	4	5
Indiana	6	7	13	1	0
Iowa	5	4	9	2	1
Kansas	4	15	19	0	1
Kentucky	2	1	3	1	7
Louisiana	20	7	27	0	7
Maryland	17	6	23	2	0
Massachusetts	6	1	7	0	1
Michigan	25	4	29	3	2
Minnesota	4	4	8	0	1
Mississippi	3	5	8	0	2
Missouri	3	0	3	0	0
Nebraska	10	29	39	2	10
Nevada	0	2	2	0	0
New Hampshire	1	0	1	0	0
New Jersey	15	15	30	2	0
New Mexico	21	4	25	1	6
New York	89	39	128	4	16
North Dakota	2	7	9	0	0
Ohio	4	1	5	0	0
Oklahoma	1	0	1	0	1
Pennsylvania	19	9	28	0	0
South Carolina	1	0	1	0	0
South Dakota	4	16	20	0	0
Tennessee	2	2	4	0	1
Texas	77	12	89	6	14
Utah	1	1	2	0	3
Virginia	4	1	5	1	2
Washington	1	1	2	0	0
Wisconsin	0	2	2	0	1
Wyoming	2	4	6	0	0
Totals	629	392	1021	57	144

Neuroinvasive disease refers to severe cases of disease that affect a person’s nervous system. These include encephalitis, meningitis, and acute flaccid paralysis that is an inflammation of the spinal cord that can cause a sudden onset of weakness in the limbs or breathing muscles.

^*Human Cases Reported to CDC.

Neuroinvasive infection with West Nile presents with symptoms of headache, fever, and a change in consciousness along with altered mental status. The examination of the CSF shows an increase in leukocytes with a marked increase in lymphocytes. Chemistries demonstrate an elevated protein count and normal glucose levels. Definitive diagnosis requires testing for the presence of the IgM antibody to West Nile in the serum or CSF, and because IgM does not cross the blood-brain barrier, presence of IgM antibody to West Nile in the CSF is a strong indicator for CNS infection. Polymerase chain reaction (PCR) can also be used to test for West Nile infection, but because West Nile infections have a transient and low viremia, results must be interpreted with caution. A negative result does not necessarily rule out West Nile infection.

Involvement of the nervous system in patients who are infected with the human immunodeficiency virus (HIV) is common. HIV is a neurotropic (attracted to nerve cells) virus capable of entering the CNS by macrophage transport and the cause of various neurologic syndromes. As HIV-infected individuals become progressively more immunosuppressed, the CNS becomes a target for opportunistic pathogens, such as cytomegalovirus, BK virus, and JC (John Cunningham) virus, which can produce meningitis or encephalitis. BK virus is named after the initials of the first renal transplant patient where the virus was identified in association with clinical disease.

Parasitic

Parasites can cause meningoencephalitis, brain abscess (see the following discussion), or other CNS infection via two routes. A rare but devastating meningoencephalitis is caused by the free-living amebae, Naegleria fowleri and Acanthamoeba spp., which invade the brain via direct extension from the nasal mucosa. These organisms are acquired during swimming or diving in natural, stagnating freshwater ponds and lakes.

Other parasites reach the brain via hematogenous spread. Toxoplasmosis, caused by an intracellular parasite that destroys brain parenchyma, is a common CNS affliction in HIV-infected patients with acquired immunodeficiency syndrome (AIDS). Entamoeba histolytica and Strongyloides stercoralis have been identified in brain tissue, and the larval form of Taenia solium (the pork tapeworm), called a cysticercus, can travel to the brain via the bloodstream and encyst within the brain tissue. Amebic brain infection and cysticercosis cause changes in the CSF similar to meningitis.

Brain Abscess

Brain abscesses (localized collections of pus in a cavity formed by the breakdown of tissue) may occasionally cause changes in the CSF and clinical symptoms similar to meningitis. Brain abscesses result from contiguous infection of the sinuses, middle ear, or mastoids (25%-50%), hematogeneously (15%-30%), or through direct inoculation as a result of trauma or surgery (8%-19%). Brain abscesses may rupture into the subarachnoid space, producing severe meningitis with a high mortality rate. If anaerobic organisms or viridans streptococci are recovered from CSF cultures, the diagnosis of brain abscess should be considered; however, CSF culture is typically negative in brain abscess. Patients who are immunosuppressed or who have diabetes with ketoacidosis may present with a rapid progressive fungal infection (phycomycosis) of the nasal sinuses or palatal region capable of traveling directly to the brain. The complex polymicrobial infections isolated from brain abscesses are far too extensive to list.

Shunt Infections

Information and studies related to infections involving CSF shunts is limited. The organisms reported most frequently associated with infections include coagulase-negative staphylococcus, Staphylococcus aureus, Propionibacterium acnes, and viridians group streptococci, A few Gram-negative rods have been identified, including Pseudomonas aeruginosa, Klebsiella spp., Escherichia coli, and Serratia marcescens. Positive cultures were most often associated with shunt tip cultures, shunt valves, and cerebral ventricle fluid.

Laboratory Diagnosis of Central Nervous System Infections

Meningitis

Except in unusual circumstances, a lumbar puncture (spinal tap) is one of the first steps in the diagnosis of a patient with suspected CNS infection, in particular, meningitis. Refer to Table 5-1 to review the procedure for collecting, transporting, and processing specimens obtained from the central nervous system.

Specimen Collection and Transport

CSF is collected by aseptically inserting a needle into the subarachnoid space (lumbar puncture), at the lumbar spine region between L3, L4, or L5. Three or four tubes of CSF should be collected into sterile collection tubes that contain no additives. The tubes are numbered sequentially in the order in which they were collected along with the patient’s name. When processing the CSF collection tubes in the laboratory, tube 1 is used for chemistry studies, glucose and protein count, as well as immunology studies, as these tests are least affected by the presence of blood cells or bacteria introduced as a result of the spinal tap procedure; tube 2 is used for culture, allowing a larger proportion of the total fluid to be concentrated, which can facilitate the detection of infectious agents present in low numbers; tubes 3 and 4 are used for cell count and differential, as these tubes are least likely to contain cells introduced by the collection procedure. If a small capillary blood vessel is inadvertently broken during the spinal tap, blood cells picked up from this source will usually be absent from the last tube collected; comparison of counts between tubes 1 and 3 (4) is occasionally needed if a traumatic tap is suspected as well as to differentiate a traumatic bloody tap from a true subarachnoid hemorrhage. In a traumatic tap, the red blood cells will be unevenly distributed among the three tubes, with the heaviest concentration of red blood cells being in tube 1 and diminishing amounts in tubes 2 and 3. In an intracranial hemorrhage, the red blood cells will be evenly distributed among the three tubes. The volume of CSF that can be collected is based on the volume available in the patient (adult versus neonate) and the opening pressure of the CSF when the needle first punctures the subarachnoid space. An elevated pressure requires the CSF fluid to be withdrawn more slowly, which may prevent the collection of a larger volume. The volume of CSF is critical for detecting certain microorganisms, such as mycobacteria and fungi. A minimum of 5 to 10 mL is recommended for detecting these agents by centrifugation and subsequent culture. When the laboratory receives an inadequate volume of CSF, the physician should be consulted regarding the order of priority for laboratory tests. Processing too little specimen lowers the sensitivity of laboratory tests, which may lead to false-negative results. This is potentially more harmful to patient care than performing an additional lumbar puncture to obtain the necessary or required amount of sample.

CSF should be hand-delivered immediately to the laboratory. Certain agents, such as Streptococcus pneumoniae, may not be detectable after an hour or longer. Specimens for microbiology studies should never be refrigerated; if not rapidly processed, CSF should be incubated (35° C) or left at room temperature. One exception to this rule involves CSF for viral studies. These specimens may be refrigerated for as long as 23 hours after collection or frozen at −70° C if a longer delay is anticipated until they are processed and inoculated into culture media. CSF for viral studies should never be frozen at temperatures above −70° C. If not processed immediately, CSF specimen for hematology studies can be refrigerated, whereas the CSF for chemistry and serology can be frozen (−20° C).

Information gathered from specimen analysis should be promptly relayed to the clinician who can directly affect therapeutic outcome. Such specimens should be processed immediately upon receipt in the laboratory (STAT) and results reported to the physician as soon as possible.

Initial Processing

Initial processing of CSF for bacterial, fungal, or parasitic studies includes centrifugation of all specimens with a volume greater than 1 mL for at least 15 minutes at 1500× g. Specimens in which cryptococci or mycobacteria are suspected require special handling. (Discussions of techniques for culturing CSF for mycobacteria and fungi are found in Chapters 43 and 59, respectively.) If fewer than 1 mL of CSF is available, the specimens should be gram stained and plated directly to blood and chocolate agar plates. The supernatant is removed to a sterile tube, leaving approximately 0.5 mL of fluid. The remaining fluid is used to suspend the sediment for visual examination or culture. Mixing of the sediment after the supernatant has been removed is critical. Forcefully aspirating the sediment up and down into a sterile pipette several times will adequately disperse the organisms that remained adherent to the bottom of the tube after centrifugation. Laboratories that use a sterile pipette to remove portions of the sediment from underneath the supernatant will miss a significant number of positive specimens. The supernatant can be used to test for the presence of antigens, rapid diagnostic test (vertical flow immunochromatography), for N. meningitidis, or for chemistry evaluations (e.g., protein, glucose, lactate, C-reactive protein). As a safeguard, keep the supernatant even if it has no immediate use.

CSF Laboratory Results

As previously mentioned, CSF is also removed for analysis of cells, protein, and glucose. Ideally, the glucose content of the peripheral blood is determined simultaneously for comparison to CSF levels. General guidelines for the interpretation of results are shown in Table 71-2.

Because the results of hematologic and chemical tests directly relate to the probability of infection, communication between the physician and the microbiology laboratory is essential. Among 555 cerebrospinal fluid samples from patients older than 4 months of age tested at the University of California–Los Angeles, only 2 showed normal cell count and protein in the presence of bacterial meningitis. Thus, the diagnosis of acute bacterial meningitis can be excluded in patients with normal fluid parameters in almost all cases, precluding further expensive and labor-intensive microbiologic processing beyond a standard smear and culture (which must be included in all cases). Similar criteria have been used to exclude performance of smear and culture for tuberculosis, as well as syphilis serology, on CSF specimens.

Visual Detection of Etiologic Agents

Following centrifugation, the resulting CSF sediment may be visually examined for the presence of cells and organisms.

Stained Smear of Sediment.

Gram stain must be performed on all CSF sediments. False-positive smears have resulted from inadvertent use of contaminated slides. Therefore, use of alcohol-dipped and flamed or autoclaved slides is recommended. After thoroughly mixing the sediment, a heaped drop is placed on the surface of a sterile or alcohol-cleaned slide. The sediment should never be spread out on the slide surface, because this increases the difficulty of finding small numbers of microorganisms. The drop of sediment is allowed to air dry, is heat or methanol fixed, and is stained by either Gram (Figure 71-3) or acridine orange. The acridine orange fluorochrome stain may allow faster examination of the slide under high-power magnification (400×) and thus a more thorough examination. The brightly fluorescing bacteria will be easily visible. All suspicious smears can be stained using the Gram stain (directly over the acridine orange) to confirm the presence and morphology of organisms.

Figure 71-3 Gram stain of cerebrospinal fluid showing white blood cells and many gram-positive diplococci. This specimen subsequently grew *Streptococcus pneumoniae*.

Using a cytospin centrifuge to prepare slides for staining has also been found to be an excellent alternative procedure. This method for preparing smears for staining concentrates cellular material and bacterial cells up to a 1000-fold. By centrifugation, a small amount of CSF (or other body fluid) is concentrated onto a circular area of a microscopic slide (Figure 71-4), fixed, stained, and then examined.

Figure 71-4 A, Cytocentrifuge. B, Device used to prepare the concentrated smears of material from body fluid specimens such as CSF by cytocentrifugation. (A courtesy Cytospin 2, Shandon, Inc., Pittsburgh, Pa.)

The presence or absence of bacteria, inflammatory cells, and erythrocytes should be reported following examination. Based on demographic and clinical patient data and Gram stain morphology, the etiology of the majority of bacterial meningitis cases can be presumptively determined within the first 30 minutes following receipt of the specimen.

Wet Preparation.

Amoebas are best observed by examining thoroughly mixed sediment as a wet preparation under phase-contrast microscopy. If a phase-contrast microscope is not available, observing under light microscopy with the condenser closed slightly can be used as an alternative. Amoebas are identifiable by their typical slow, methodical movement in one direction via pseudopodia. (The organisms may require a little time under the warm light of the microscope before they begin to move.) Organisms must be distinguished from motile macrophages, which occasionally occur in CSF. Following a suspicious wet preparation, a trichrome stain can assist in the differentiation of amoebas from somatic cells. The pathogenic amoebas can be cultured on a lawn of Klebsiella pneumoniae or Escherichia coli (see Chapter 47).

India Ink Stain.

The large polysaccharide capsule of Cryptococcus neoformans allows these organisms to be visualized by the India ink stain. However, latex agglutination testing for capsular antigen is more sensitive and extremely specific. Antigen testing is recommended over the use of an India ink stain. Furthermore, strains of C. neoformans that infect patients with AIDS may not possess detectable capsules making culture essential. To perform the India ink preparation, a drop of CSF sediment is mixed with one-third volume of India ink (Pelikan Drawing Ink, Block, Gunther, and Wagner; available at art supply stores). The India ink can be protected against contamination by adding 0.05 mL thimerosal (Merthiolate, Sigma Chemical Co., St. Louis, Missouri) to the stain. After mixing the CSF and ink to make a smooth suspension, a coverslip is applied to the drop and the preparation is examined under high-power magnification (400×) for characteristic encapsulated yeast cells, which can be confirmed by examination under oil immersion. The inexperienced microbiologist must be careful not to confuse white blood cells with yeast. The presence of encapsulated buds, smaller than the mother cell, is diagnostic.

Direct Detection of Etiologic Agents

Antigen.

Commercial reagents and kits are available for the rapid detection of antigen in the CSF; a review of the methodologies used will be discussed in the following sections; for more detailed specifics, please refer back to Chapter 9.

Bacteria.

Rapid antigen detection from CSF has been largely accomplished by the techniques of latex agglutination (see Chapter 9). All commercial agglutination systems use the principle of an antibody-coated particle capable of binding to specific antigen, resulting in macroscopically visible agglutination. The soluble capsular polysaccharide found in the common etiologic agents of meningitis, including the group B streptococcal polysaccharide, are well suited to serve as bridging antigens. The agglutination assays may contain either a polyclonal or monoclonal antibody or an antigen from an infectious agent.

In general, the commercial systems have been developed for use with CSF, urine, or serum, although results with serum have not been as diagnostically useful as those with CSF. Soluble antigens from Streptococcus agalactiae and Haemophilus influenzae may concentrate in the urine. Urine, however, seems to produce a higher incidence of nonspecific reactions than either serum or CSF. The manufacturers’ directions must be followed for performance of antigen detection test systems for different specimen types. Although some of the systems require pretreatment of samples (usually heating for 5 minutes), not all manufacturers recommend such a step. The reagents, however, may yield false positive or cross-reactions unless the specimen is pretreated. Interference by rheumatoid factor and other substances, more often present in body fluids other than CSF, has also been reported. The method of Smith and colleagues has been shown to effectively reduce a substantial portion of nonspecific and false-positive reactions, at least for tests performed with latex particle reagents. This pretreatment, called rapid extraction of antigen procedure (REAP; see Procedure 71-1 on the Evolve site), is recommended for laboratories that use commercial body fluid antigen detection kits. Certain commercial systems have an extraction procedure included in the protocol.

Procedure 71-1 Rapid Extraction of Antigen Procedure (REAP)

Principle

Removal of nonspecific cross-reactive material can improve the specificity of direct antigen detection particle agglutination tests. Ethylenediaminetetraacetic acid (EDTA) forms complexes with cross-reactive materials, and they are removed from the reaction mixture by centrifugation.

Method

1. Pipette 0.05 mL fluid to be tested (CSF, serum, or urine) into a 1.5-mL plastic, conical microcentrifuge tube.

2. Add 0.15 mL of 0.1 M EDTA (Sigma Chemical Co.) to the microcentrifuge tube, close the cap tightly, and vortex the tube.

3. Heat in a dry bath (available from instrument supply companies) for 3 minutes at 100° C.

4. Centrifuge the tubes for 5 minutes at 13,000× g in a tabletop microcentrifuge. Be certain that the instrument achieves the required centrifugal force.

5. Remove the supernatant with a capillary pipette and use 1 drop of this solution as the test sample in the antigen detection test, following the manufacturer’s instructions for performance of the test.

Expected Results

Nonspecific agglutination should not occur.

Based on the findings of several studies, only a limited number of clinically useful situations warrant bacterial antigen testing (BAT). Examples include CSF specimens from previously treated patients and Gram stain–negative CSF specimens with abnormal parameters (elevated protein, decreased glucose, or an abnormal white blood cell count). The assays are not substitutes for properly performed smears and cultures. Some of the assays demonstrate a decreased sensitivity and specificity. In light of these limitations, practice guidelines for the diagnosis and management of bacterial meningitis do not recommend routine use of BAT.

Cryptococcus neoformans.

Reagents for the detection of the polysaccharide capsular antigen of Cryptococcus neoformans are available commercially. CSF specimens that yield positive results for cryptococcal antigen should be tested with a second latex agglutination test for rheumatoid factor. The commercial test systems incorporate rheumatoid factor testing in the protocol. A positive rheumatoid factor test renders the cryptococcal latex test unable to interpret, and the results should be reported as such, unless the rheumatoid factor antibodies have been inactivated. Both latex agglutination assays (numerous commercial manufacturers) and enzyme immunoassays are available for the detection of cryptococcus antigen. Undiluted specimens containing large amounts of capsular antigen may yield a false-negative reaction caused by a prozone phenomenon. Patients with AIDS may have an antigen titer in excess of 100,000 requiring many dilutions to reach an end point. Serial dilution protocols are useful for monitoring a patient’s response to treatment, as well as for initial diagnosis.

Molecular Methods.

With the introduction of amplification technologies, such as polymerase chain reaction (PCR), many reports in the literature recommend the application of molecular technologies for the diagnosis of CNS infections caused by various microorganisms. Published data indicate that molecular assays demonstrate increased sensitivity and specificity compared with presently available techniques, particularly of CNS infections caused by herpes simplex virus and enteroviruses. PCR testing for HSV, EBV, CMV, and enterovirus in CNS infections has a sensitivity nearing 100%. Reagents for some of these amplification assays are commercially available for both conventional and real-time PCR assays.

Miscellaneous Tests

Other tests—such as the limulus lysate test, CSF lactate determinations, C-reactive protein, mass spectrometry, and gas-liquid chromatography—have been evaluated for use in the diagnosis of CNS infections. However, the utility and value of these tests are either controversial or remain to be defined, or these tests are impractical for routine use in the clinical laboratory.

Culture

The majority of cases of bacterial meningitis is usually caused by a single organism and requires a limited number of culture media.

Bacteria and Fungi.

Routine bacteriologic media should include a chocolate agar plate, 5% sheep blood agar plate, and an enrichment broth, usually thioglycolate without indicator. The chocolate agar plate is needed to recover fastidious organisms, most notably H. influenzae and isolates of N. meningitidis, which are unable to grow on blood agar plates; the use of the blood agar plate aids in the recognition of S. pneumoniae. After vortexing the sediment and preparing smears, several drops of the sediment should be inoculated to each medium. Plates should be incubated at 37° C in 5% to 10% carbon dioxide (CO₂) for at least 72 hours. If a CO₂ incubator is not available, a candle jar can be used. The broth should be incubated in air at 37° C for at least 5-10 days. The broth cap must be loose to allow free exchange of air. If organisms morphologically resembling anaerobic bacteria are seen on the Gram stain or if a brain abscess is suspected, an anaerobic blood agar plate may also be inoculated. These media will support the growth of almost all bacterial pathogens and several fungi.

The symptoms of chronic meningitis that prompt a physician to request fungal cultures are the same as those for tuberculous meningitis. Cultures for mycobacteria are addressed in Chapter 43. For CSF fungal cultures, two drops of the well-mixed sediment should be inoculated onto Sabouraud dextrose agar or other non-blood-containing medium and brain-heart infusion with 5% sheep blood. Fungal media should be incubated in air at 30° C for 4 weeks. If possible, two sets of media should be inoculated, with one set incubated at 30° C and the other at 35° C.