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.

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 sub arachnoid 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 An elevated pressure requires the CSF fluid to be withdrawn more slowly, which may prevent the collection of a larger volume.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. 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 1.

Table1. Guidelines for Interpretation of Results Following Hematologic and Chemical Analysis of Cerebrospinal Fluid (CSF) from  Children and Adults (Excluding Neonates)

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 per formed 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 1) 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.

Fig1. 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 2), fixed, stained, and then examined.

Fig2.  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 micros copy with the condenser closed slightly can be used as an alternative. Amoebas are identifiable by their typical slow, methodical movement in one direction via pseudo podia. (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.

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 (Merthio late, 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. 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 per formed with latex particle reagents. This pretreatment, called rapid extraction of antigen procedure (REAP; see Procedure 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.

PROCEDURE1.

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 crypto coccus 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.

Parasites and Viruses. Conditions for the culture of free-living amoebae and viral agents are discussed in Chapters 47 and 65, respectively. The physician must notify the laboratory to culture these agents.

Brain Abscess/Biopsies

Specimen Collection, Transport and Processing. Whenever possible, biopsy specimens or aspirates from brain abscesses should be submitted to the laboratory under anaerobic conditions. Several devices are commercially available to transport biopsy specimens under anaerobic conditions. Swabs are not considered an optimum specimen, but if used to collect abscess material they should be sent in a transport device that maintains an anaerobic environment.

Biopsy specimens should be homogenized in sterile saline before plating and smear preparation. This processing should be kept to a minimum to reduce oxygenation.

Abscess and biopsy specimens submitted for culture should be inoculated onto 5% sheep blood and chocolate agar plates. Plates should be incubated 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. Anaerobic culture plates are incubated for a minimum of 72 hours but are examined after 48 hours of incubation. Anaerobic broths should be incubated for a minimum of 5 days. If a fungal etiology is suspected, fungal media, such as brain-heart infusion with blood and antibiotics or inhibitory mold agar, should be inoculated.

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مواضيع ذات صلة

Laboratory Diagnosis of Infections of the Eyes

Diagnosis and Differential Diagnosis of Paroxysmal Nocturnal Hemoglobinuria

Differential Diagnosis of Pancytopenia

Diagnosis of Aplastic anemia

Diagnosis of Upper Respiratory Tract Infections

tuberculosis testing (TB testing, Interferon gamma release assay [IGRA, T-Spot.TB], QuantiFERON-TB Gold [QFT, QFT-G, TB Gold test, TB blood test], Nucleic acid amplification for TB [NAAT], TB antibody)

thyrotropin-releasing hormone stimulation test (TRH stimulation test, Thyrotropin-releasing factor stimulation test [TRF stimulation test])

Laboratory Diagnosis of Lower Respiratory Tract Infections: Specimen Processing

Laboratory Diagnosis of Lower Respiratory Tract Infections: Specimen Collection and Transport

thyroid scanning (Thyroid scintiscan)

thyroid fine needle aspiration biopsy (FNAB, Skinny needle thyroid biopsy, Fine-needle aspiration [FNA])

thoracentesis and pleural fluid analysis (Pleural tap)