Cerebrospinal Fluid Analysis - Microbiology procedures, Fluid Analysis, Microbiology procedures, Graff's Textbook of Urinalysis and Body Fluids
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Among the most serious diagnoses made on CSF is meningitis. Detection of meningitis involves several microbiologic procedures. Of the three to four tubes of CSF normally collected, the most sterile is used for microbiology procedures. The tube least likely to be contaminated by the puncture site includes the second tube collected or any subsequent tube. If the volume of CSF collected is inadequate to fill more than one tube, microbiology procedures should be performed first and then any remaining specimen may be used for cell counts and chemistries.
STAINS
Several stains may be used on concentrated CSF. The specimen is concentrated using standard centrifugation or cytocentifugation. Cytocentrifugation results in a higher yield of microorganisms.
Figure 9-20. Comparison of Wright stained and Gram stained bacte-
ria in cerebral spinal fluid. A. Wright stain (notice bacteria stain blue).
B. Gram stain reveals that the bacteria are gram-negative (pink)
Initially, a Gram stain is performed, which demonstrates 60–90% sensitivity. Figure 9-20 compares a Wright stain of CSF with mircroorganisms with that of a Gram stain on the same CSF. Additional stains may be required for the detection of some microorganisms. Ziehl–Neelsen stain and fluorescent rhodamine stain are used to stain Mycobacteria tuberculosis. Cryptococcus neoformans is best detected by india ink (sensitivity of 25–50%) or nigrosin stain. Figure 9-21 illustrates a positive india ink stain.
CULTURES
Culture and sensitivity follow the staining procedures. Sediment of centrifuged CSF is inoculated into thioglycolate broth and plates of blood agar, chocolate, and MacConkey agar. Strips of X–V may be applied to the blood agar plate if Haemophilus is suspected. If a fungal meningitis is suspected, Sabouraud dextrose agar should be inoculated. Inoculate Middlebrook broth and agar if Mycobacteria is suspected.
Figure 9-21. Positive india ink stain.
Microorganisms most commonly responsible for causing meningitis include Haemophilus influenza, Neisseria meningitidis, Streptococcus pneumoniae, and Klebsiella species. Meningitis can be caused less frequently by staphylococci, other streptococci, Listeria monocytogenes, coliform bacteria, M. tuberculosis, C. neoformans, other fungi, leptospira, anaerobic bacteria, amebae, and parasites.
It is important to note that if antibiotic therapy was administered prior to the collection of CSF for culture, the recovery of microorganisms may be significantly reduced.
IMMUNOLOGIC TESTS
Immunologic tests for microorganisms provide a rapid method for detection of meningitis causing agents. However, sensitivity and specificity vary among assays; and the possibility of false positives and false negatives creates complications in interpretation. Immunologic tests do not replace microbiologic stains and cultures as standard procedures.
Various methods are used in immunologic testing. These methods include coagulation, counterimmunoelectrophoresis, enzyme-linked immunosorbence, fluorescent treponemal antibody test, latex agglutination, radioimmunoassay, and Venereal disease research laboratory (VDRL) test. In addition, molecular techniques such as polymerized chain reaction are beginning to be used for identification of infectious agents in body fluids. Detail of each of these procedures is not within the scope of this text.
CLINICAL CORRELATIONS
The importance of performing CSF analysis rapidly and correctly cannot be stressed enough. If the diagnosis of meningitis is overlooked, the results are often fatal. Meningitis is categorized as one of four types: bacterial, fungal, tubercular, and viral. Parasites also cause meningitis, but to a lesser extent.
Along with the presence of bacteria, bacterial meningitis is accompanied by elevated CSF protein levels and decreased CSF glucose. Increased numbers of leukocytes (pleocytosis) is present with neutrophils comprising the greatest percentage. Sometimes microorganisms can be seen on Wright stain, either intracellularly or extracellularly. These should be confirmed by Gram stain. Within a week of antibiotic therapy, the CSF leukocyte count decreases and the differential count shifts back to lymphocytes and monocytes. In addition, protein and glucose levels gradually return to normal. CSF results in meningitis caused by M. tuberculosis exhibit similar findings as bacterial meningitis.
Viral meningitis usually exhibits a lymphocytosis containing a variety of reactive lymphocyte morphology. However, atypical forms of lymphocytes in CSF must be differ entiated from those that may be seen in leukemia. The later stages of viral meningitis exhibit more monocytes and macrophages than lymphocytes. CSF glucose levels may remain normal in some forms of viral meningitis, although CSF protein is elevated.
Normal CSF glucose levels may also be seen in fungal meningitis, although CSF glucose can also be decreased. CSF protein is usually elevated in fungal meningitis. Leukocytes are increased in CSF during fungal meningitis with lymphocytes predominating. Meningitis may also be caused by parasites such as Acanthamoeba and Naegleria species.
Abnormal CSF results should be interpreted with consideration of side effects created by test procedures and interventions. The CSF leukocyte count and different may show a neutrophilic pleocytosis if a lumbar puncture is repeated within 12 hours of a previous lumbar puncture. Erythrophagocytosis may also be present. Similar findings of increased numbers of lymphocytes, neutrophils, monocytes, macrophages and eosinophils can be seen up to several weeks after either a pneumoencephalogram or a myelogram. Intracranial shunts may induce CSF monocytosis and allergic reactions resulting in eosinophilic pleocytosis.1
Malignant cells that appear in CSF may arise from primary CNS tumors or other sources. Primary CNS tumors include medulloblastoma, meningiomas, and gliomas. When some carcinomas and leukemias metastasize, the malignant cells may find their way into the CSF. A CSF examination provides valuable information in the diagnosis of these metastases. Tumors that most commonly metastasize to CSF include breast, lung gastrointestinal tract cancers, and melanoma. These cells readily proliferate in CSF because chemotherapy does not penetrate the blood–brain barrier.3 Malignant cells often occur in clumps of their own cell type. Care must be taken not to mistake macrophages and clumps of ependymal or choroid plexus cells for tumor cells.
REFERENCES
Lillian A. Mundt and Kristy Shanahan, Graff's Textbook of Urinalysis and Body Fluids, Second Edition 2011
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