B. Wildemann, H. K. Geiss, P. Schnitzler
The most important pathogens infecting the nervous system are bacteria, mycobacteria, and viruses—and, in persons with impaired cellular immune defenses (e. g., those with organ transplantation, HIV infection, or AIDS), also fungi and parasites. Rapid and reliable detection of infectious pathogens in the CSF is essential if antibiotic treatment is to be as prompt and specific as possible. In most pathogeninduced diseases, maximum diagnostic sensitivity is only achieved by a combination of detection methods.
Lumbar puncture. Sterility must be maintained during lumbar puncture so as to avoid contamination, which could lead to false-positive findings.
Transport. For reliable and successful direct detection of pathogens, it is absolutely essential that the CSF sample is transferred immediately to the CSF or microbiological laboratory that will undertake the analysis. The sample should be transported at room temperature in a sterile plastic tube with sterile screw cap. CSF samples for PAS stain should be processed in a special laboratory within 30 minutes after collection.
The following volumes of CSF are required for microbiological diagnosis:
• Bacteriological–mycological diagnosis: 5 mL.
• Tuberculosis diagnosis: 10–15 mL.
• Virological diagnosis: at least 2 mL (for virus isolation, 1 mL per species of virus).
Storage. If immediate transport is not possible, samples should be stored as follows:
• For bacteriological–mycological diagnosis: protected from light and at room temperature.
• For serological and virological tests: at 4°C.
• For molecular detection of pathogen-specific genome sequences: at 4°C.
If the period between sample collection and processing in the laboratory much exceeds 2 hours, a portion of the untreated CSF (2 mL) should be transferred into a blood culture flask.
Diagnosis of pathogens in CSF involves direct and indirect methods of detection:
• Direct identification of pathogens:
– Microscopic detection of pathogens.
– Detection of pathogen-specific antigens.
– Culture of pathogens.
– Molecular identification of pathogens by methods based on selective amplification of pathogen-specific genomic sequences by nucleic acid amplification techniques.
• Indirect detection by serological determination of pathogen-specific humoral immune responses. The most important diagnostic parameter here is the detection of intrathecal pathogen-specific antibody reactions by the calculation of antibody indices (AIs).
The diagnostic value of pathogen detection in the CSF is high; positive detection of a pathogen usually confirms a florid infection of the nervous system.
Microscopic Detection of Pathogens
Morphological detection of pathogens. Following appropriate differential staining of the CSF smear, microscopy enables morphological detection of pathogens and, thus, prompt confirmation of the diagnosis of the underlying infection.
Stains. The most important differential staining techniques are:
• Gram stain for bacteria.
• Ziehl-Neelsen stain, modified Ziehl-Neelsen stain (Kinyoun carbolfuchsin stain), or Truant auramine-rhodamine stain for mycobacteria.
• Indian ink stain for fungi (Table 5.14).
• Rarely, periodic acid–Schiff stain (PAS).
Indications. As a procedure, staining is suitable for laboratory diagnosis of bacterial infections and mycoses of the nervous system, but it does not have a role in the diagnosis of viral and parasitic diseases. Positive staining of CSF macrophages in the PAS stain is diagnostic of glycogen-containing degradation products of Tropheryma whipplei, the pathogen of Whipple's disease.
Microscopic Detection of Bacteria
Gram-positive and gram-negative pathogens. The Gram staining method consists of staining, destaining, and counterstaining. In bacteria with a cell wall made of several peptidoglycan layers, alcohol does not remove gentian violet during the destaining step, and the bacteria keep their blue-black or dark-violet stain (gram-positive). Bacteria with a thin cell wall becomes destained in alcohol and take up the red stain during counterstaining with safranin (gram-negative). Pathogens of bacterial meningitis can be identified in gram-stained preparations (Table 5.15).
Mycobacteria. Mycobacteria are very lipophilic and do not stain well by the usual methods. Once they have taken up a stain, they cannot be destained by exposure to acid. Mycobacteria appear as fine, red, acid-fast rods.
Sensitivity. In patients with untreated bacterial meningitis, bacterioscopy has a 65–85% sensitivity. Detection of bacteria in patients who have been treated with antibiotics is much less often successful. Ziehl-Neelsen staining is suitable for detecting mycobacteria (red), which do not stain with the Gram method because of their lipid-rich cell wall. The sensitivity of microscopy for detecting Mycobacterium tuberculosis is only 10–15% because of the low density of these bacteria.
Microscopic Detection of Fungi
Cryptococci. Cryptococci are the most common opportunistic pathogens of meningitis in patients with cellular immune deficiency. They are encapsulated yeast fungi (Table 5.16) and are identified with a sensitivity of 80–90% by negative staining of their mucous capsule with Indian ink (Fig. 5.27 j).
Candida. Candida albicans occasionally causes meningitis in the context of systemic candidiasis in immunocompromised persons. Gram-stained CSF smears reveal gram-positive yeast cells and hyphae.
Rapid Antigen Tests
Antibody-coated latex particles. The principle of these tests is to detect pathogen-specific antigens by means of antibody-coated latex particles. If the pathogen is present in the CSF, its contact with the latex particles triggers agglutination. To detect bacterial and fungal antigens, the sample material must be broken up either thermally (in a water bath) or by adding reagents for lysis or extraction.
Indications. Direct detection of pathogens with rapid antigen tests supplements the diagnosis of bacterial infections and mycoses of the nervous system (see also Pitfalls box). The advantages of antigen detection in fresh material are that it is quick to carry out and results are immediate.
Table 5.15 Classification of bacteria according to Gram stain (see also Table 5.12)
• Pneumococci (Streptococcus pneumoniae)
• Listeria monocytogenes
• Meningococci (Neisseria meningitidis)
• Haemophilus influenzae
• Enterobacteriaceae (E. coli, Klebsiella, Enterobacter, Proteus mirabilis)
• Pseudomonas aeruginosa
Fig. 5.27 a–j Microscopic detection of important pathogens in the CSF.
a Pneumococci (Streptococcus pneumoniae), Gram stain.
b Meningococci (Neisseria meningitidis), Gram stain.
c Staphylococci, Gram stain.
d Streptococci, Gram stain.
e Haemophilus influenzae, Gram stain.
f Enterobacteriaceae, Gram stain.
g Listeria, Gram stain.
h Mycobacterium tuberculosis, Kinyoun carbolfuchsin stain.
i–j Microscopic detection of the most important pathogens in the CSF. Tropheryma whipplei, PAS stain (i). Cryptococcus, India ink capsule stain (j).
Diagnosis of an infection on the sole basis of this test—without taking into account the microscopic CSF preparation and microbial culture—is indefensible.
Detection of Bacteria Using Rapid Antigen Tests
Rapid antigen tests for the direct detection of bacteria are available for N. meningitidis (serogroups A, B, C, Y, W135), Streptococcus pneumoniae, B-streptococci, Haemophilus influenzae, and E. coli.
Pitfalls with Rapid Antigen Tests for Detecting Bacterial Meningitis
With regard to the diagnostic sensitivity of this method, it should be borne in mind that a positive agglutination reaction can only be expected when the bacterial count in the CSF is high. Rapid antigen tests are therefore certainly no more sensitive than the detection of bacteria by Gram staining; their primary use is for confirmation of positive microscopic detection of pathogens. They may give a negative result despite positive pathogen detection under the microscope. Test sensitivity is particularly low for the identification of N. meningitidis. Routine performance of antigen detection tests when microscopic findings are normal is therefore neither cost-effective nor useful. Similarly, the use of urine in diagnosing meningitis is considered poor practice, since it can yield false-positive results. Indications for the use of rapid antigen tests to detect classic bacterial pathogens are:
Confirmation of a microscopically suspected diagnosis.
Suspected bacterial meningitis, markedly increased cell counts (> 50/μL), and negative microscopic findings.
Patients already treated with antibiotics.
Detection of Fungi with Rapid Antigen Tests
A latex agglutination test is available for detecting Cryptococcus-specific capsule polysaccharides. Antigen detection in CSF and serum is almost always successful with cryptococcal meningitis, and the sensitivity of the test is very close to that of pathogen detection in culture. Aspergillus fumigatus can also be detected by the latex agglutination test. Aspergillus infection of the nervous system is rare; it is seen only as a complication of disseminated septic aspergillosis in severely immunocompromised persons.
Propagation of Pathogens in Culture
Advantages and disadvantages. Propagation of pathogens from the CSF in a culture medium is a standard microbiological procedure in pathogen diagnosis. It is used for:
• Isolation of pathogens.
• Accurate phenotyping of pathogens.
• Testing pathogens for their susceptibility to pharmaceuticals.
The disadvantage is that it takes some time.
Indications. The diagnostic value of isolating pathogens from the CSF varies: it is
• Highly important in bacterial infections.
• Suitable for the detection of rare mycoses and parasitoses in immunocompromised persons.
• Less important in viral diseases.
Procedure for Detecting Microorganisms in Culture
Centrifuge the fresh CSF for 10 minutes at 3000g.
Apply at least 20–50 μL of the sediment with a sterile pipette to solid and liquid media.
If bacterial meningitis is suspected, inoculate at least one plate each of blood agar and chocolate agar, and also one tube with brain–heart infusion (BHI) broth or thioglycolate broth.
Incubate solid culture media for at least 72 hours at 35–37°C and increased CO2 pressure; check for growth at least once daily.
Extend the incubation period to up to 14 days if microscopy of the original material has been positive, since, e. g., Actinomycetes and Brucella grow very slowly.
Differentiate the pathogens using standard procedures.
Always test for sensitivity to antibiotics when dealing with pure cultures.
Blood cultures. In addition to culturing pathogens from the CSF, it is essential that two or three blood samples for culture are collected at the same time and before the start of antibiotic treatment; and perhaps also several more sets at further intervals. The blood culture bottles are stored and prepared at room temperature and shipped to a laboratory. Isolation of pathogens of bacterial meningitis from blood is successful in up to 50% of cases.
Large volumes of CSF (at least 5 mL) are required for detection of mycobacteria in culture, and at least two solid media and one liquid medium are inoculated. A positive result for the Mycobacterium tuberculosis complex cannot be expected before 14 days are up, and it may take up to 6–8 weeks. Mycobacteria are differentiated using special biochemical tests or, increasingly, with less time-consuming molecular biology methods (gene probes, sequencing). The same holds for the sensitivity testing, which with the rise in resistant strains has become extremely important.
Culture of Fungi and Parasites
Fungi. For fungal diagnosis, two additional plates of a secondary medium (e. g., Sabouraud agar) are inoculated and incubated at 22°C and 37°C over at least 1 week. If dimorphic fungi are suspected (Cryptococcus neoformans), the incubation time may be extended to 4 weeks. Fungi are differentiated by morphological characterization and biochemical tests.
Parasites. Detection of parasites by culture methods is extremely difficult and is undertaken at only a few specialized laboratories. For this reason, diagnosis of cerebral parasitoses is normally limited to serological and possibly molecular biological methods.
Indication and importance. Culture of viruses from CSF is not always successful and has become less important with the availability of molecular detection of pathogens by means of nucleic acid amplification techniques. Isolation of viruses is worthwhile, so long as specific virostatics are available for the suspected viral infection of the nervous system and antiviral susceptibility testing is indicated.
Principle. Isolation of viruses requires propagation of the pathogens in susceptible cell cultures. Viral multiplication in cell culture causes morphological changes in the cells, known as the cytopathic effect (CPE). In many cases the features of the CPE allow a preliminary diagnostic categorization of the virus detected. The time needed to evaluate a typical CPE is 1–2 days for herpes simplex virus (HSV) and 1–3 weeks for cytomegalovirus (CMV). If necessary, the virus is further differentiated by antibody-mediated detection of virus-specific antigens, using the following methods:
• Immunofluorescent or immunoperoxidase staining.
• Immunoelectron microscopy.
Alternative methods are in-situ hybridization and filter hybridization using labeled DNA or RNA probes complementary to the viral genome.
Modified Propagation in Culture
Shell vial culture. This method considerably shortens the observation time required for virus detection in cell culture, for example, from several weeks to 2–5 days for the detection of CMV. The method involves centrifugation of the CSF sample onto a monolayer of cells adhering to a coverslip. After brief incubation for 1–2 days, virus-specific proteins synthesized early during the viral infection cycle are detected by means of immunofluorescence microscopy. The technique can be used for rapid isolation of HSV, varicellazoster virus (VZV), CMV, and enteroviruses.
Genetically manipulated cell lines. Alternatively, genetically manipulated cell lines can be used for cell culture. Indicator cell lines undergo transfection with genes mediating the integration of viral receptors into cell membranes, or with genes mediating the expression of promoters that react with virus-specific proteins. Presence of the virus in the cell culture is indicated colorimetrically after activation of the promoter and reaction with a reporter enzyme (e. g., β-galactosidase).
Detection of Pathogen-Specific Genome Sequences Using Nucleic Acid Amplification Techniques
The development of nucleic acid amplification techniques, particularly the introduction of the polymerase chain reaction (PCR) (Saiki et al., 1988), has greatly improved the diagnosis of infectious diseases of the nervous system. These methods identify pathogens directly by detecting microbial nucleic acid sequences. Compared to conventional direct methods (microscopy, rapid antigen test, propagation in culture) and indirect procedures (serology), they have several advantages:
• They are extremely sensitive and can be performed within a minimum of time.
• They allow detection of the pathogen during the early stage of the infection—much earlier than serological or culture methods.
Polymerase Chain Reaction
Direct pathogen detection by PCR-based methods is the gold standard in diagnosing numerous viral infections of the nervous system, and supplements the laboratory diagnosis of bacterial diseases and parasitoses.
Various PCR methods are available for the detection of pathogens in the CSF. PCR can be used for qualitative and quantitative detection of DNA, and also for detection of RNA.
Indications. DNA PCR is used to detect DNA viruses—such as HSV-1, HSV-2, VZV, Epstein-Barr virus (EBV), CMV, Jamestown Canyon virus (JCV), and proviral HIV-1 DNA—as well as Mycobacteriumand Borrelia spp. and Toxoplasma gondii.
Procedure. PCR works like an automated amplification system and is used to multiply a specific DNA sequence (Fig. 5.28). To identify the target sequence during the reaction and initiate DNA synthesis, two oligonucleotide primers are synthesized that are exactly complementary to the nucleotide sequences at the beginning and end of the target sequence.
For PCR analysis, DNA is isolated from CSF cells or whole CSF. The DNA double helix is first denatured at high temperature to form single strands (denaturation step). Next, at lower temperature the two oligonucleotide primers each hybridize with one of the target single strands (annealing step). After addition of a heat-stable DNA polymerase (Taq polymerase), the synthesis of new complementary strands is initiated using the oligonucleotides as primers and the DNA single strands as templates (elongation step). Through multiple cycles of DNA denaturation, primer annealing, and DNA elongation, the DNA fragment flanked by the primers is exponentially amplified. The PCR reaction is carried out in an automated thermocycler for about 30 cycles. This way, about 106 copies of the target sequence are generated within a few hours. The length of the amplified sequence is exactly defined by the distance between the two oligonucleotide primers. The PCR products (amplicons) are subjected to agarose gel electrophoresis and visualized on the gel by staining with ethidium bromide; their exact size is determined by reference to DNA fragments of known size run in the same gel.
Fig. 5.28 Flow chart of polymerase chain reaction (PCR).
Nested PCR. The specificity and sensitivity of the method can be significantly increased by nested PCR, which includes a second round of amplification with an internal primer pair. This method facilitates the detection of single copies of the target sequence and thus a single pathogen genome.
A prerequisite for correct interpretation of PCR findings is the inclusion of adequate positive and negative controls. The specificity of the amplified material is confirmed either by hybridization of the PCR product with a DNA probe specific for the microbial nucleic acid sequence in question, or by nucleotide sequence analysis of the amplified fragments.
Pitfalls in the Evaluation of PCR Results
Since PCR is extremely sensitive, it is also very susceptible to contamination. Any contaminating DNA or RNA fragments become target sequences for further primer annealing and thus yield false-positive results. The risk of contamination is particularly high when nested PCR is used, since this requires transfer of PCR products from the first amplification reaction. Strict separation of the rooms where samples are prepared for amplification and for detection is therefore absolutely essential if PCR is to be carried out correctly.
RNA PCR (Reverse Transcriptase PCR, RT-PCR)
Indications. RNA PCR is used for detecting RNA viruses (enteroviruses, HIV-1, HIV-2), bacteria in patients pretreated with antibiotics, and Tropheryma whipplei.
Procedure. It is relatively easy to modify PCR for the detection of RNA. For PCR analysis, total RNA is isolated from cells or tissue samples. The isolated RNA molecules are subsequently transcribed into a DNA copy (cDNA) by RNA-dependent DNA polymerase (reverse transcriptase, RT). The cDNA is then used as starting material in a standard PCR. By using suitable oligonucleotide primers, specific DNA sequences reflecting the target RNA can be amplified.
Potential indications. Multiplex PCR can be used for detecting many viral pathogens, and for differentiation of cerebral round lesions in AIDS patients (EBV-associated primary CNS lymphoma vs. cerebral toxoplasmosis) (Roberts and Storch, 1997; Casas et al., 1999).
Procedure. Cost-intensive multiplex PCR kits for virus detection are often available commercially and allow the detection of several pathogens in a single amplification reaction.
Real-Time Quantitative DNA/RNA PCR
Indications. Real-time quantitative PCR is suitable for the detection and quantification of nearly all pathogens.
Procedure. Modern PCR detection systems work with optical devices that excite fluorescent dyes and detect fluorescent emissions (e. g., Light Cycler, Roche; GeneAMP 5700 Sequence Detection System, Applied Biosystems, Foster City, California, USA). They confirm the specificity of the emerging PCR products even while the automated amplification of the target sequence is still in process, and allow a quantitative evaluation at the same time. The combination of amplification and signal detection saves time (the amplification reaction is completed within 30–60 minutes) and the closed system considerably reduces the risk of contamination.
Other Nucleic Acid Amplification Techniques
Various other methods may be used for quantitative analysis of the amount of pathogen in CSF. They amplify either the target sequence or the signal that detects a probe annealed to the target sequence.
Potential indication. A commercially available nucleic-acidsequence-based amplification (NASBA) test kit (Organon Teknika, Eppelheim, Germany) may be used for quantitative determination of the number of HIV-1 copies in CSF.
Procedure. NASBA is a method of primer-directed enzymatic, isothermal amplification of target sequences (mainly RNA). The advantage of this technique is easy distinction between RNA and DNA target sequences, thus permitting direct mRNA amplification without the time-consuming removal of DNA. In the first step, a cDNA molecule complementary to the target RNA is synthesized. Attached to the primer for this step is an RNA promoter sequence. The target RNA is digested by RNase H, and a DNA double strand is produced from the cDNA as template, using reverse transcriptase (which has also polymerase activity). From the RNA promoter sequence attached to the DNA double strand, new anti-sense RNA copies are synthesized by RNA polymerase. From the anti-sense RNA, cDNA copies are repeatedly transcribed and converted to DNA double strands.
Branched DNA (bDNA) Technology
Potential indication. Commercially available bDNA test kits (Chiron Diagnostics, Dietlikon, Switzerland) may be used for quantitative determination of the number of HIV-1 copies in the CSF.
Procedure. The method amplifies a detection signal rather than a target sequence. The target sequence is annealed simultaneously to an immobilized capture probe and a detection probe. In subsequent hybridization steps, multiple synthetically branched and chemically labeled oligonucleotide probes (amplifier DNA) are annealed to the detection probe. Quantification is done colorimetrically after addition of a substrate.
Detection of Viral Genomes
Importance. PCR is of special importance for the diagnosis of viral infections of the nervous system (Table 5.17). Qualitative detection of viral nucleic acids in CSF indicates a florid infection in nearly all cases. The probability of definite viral infection of the central nervous system is 88 times higher when viral DNA or RNA is detected in the CSF than when the PCR is negative (Jeffrey et al., 1997).
HSV, VZV, and enteroviruses. PCR is the diagnostic method of choice for early diagnosis of herpes simplex encephalitis. Furthermore, PCR diagnosis reliably detects infections with HSV-2 (Mollaret's meningitis), VZV, and enteroviruses (Darnell, 1993; Weber et al., 1996).
Opportunistic infections. PCR is of high diagnostic value and has important implications for the diagnostic regimen in opportunistic infections associated with acquired immunodeficiency (AIDS, organ transplantation). Examples include neurological manifestations caused by the reactivation of CMV, progressive multifocal leukoencephalopathy (PML) induced by papovaviruses of the JCV type, and EBVassociated primary central nervous system lymphoma (de Luca et al., 1995; Weber et al., 1996).
Detection of Bacterial and Parasite Genomes
Importance. PCR is a useful supplementary diagnostic test in bacterial infections of the central nervous system. However, compared to its use for viral diseases, it is less sensitive and/or not universally established for routine diagnosis of bacterial disease (Table 5.18).
Mycobacteria. Detection of mycobacterial DNA sequences supplements the CSF-based diagnosis of tuberculous meningitis. Its sensitivity varies considerably, depending on the protocol used.
Neuroborreliosis and neurosyphilis. PCR has not much relevance for the diagnosis of neuroborreliosis and neurosyphilis; molecular detection of pathogens is far less sensitive than serological detection of infections by calculating the antibody index (see above, “Oligoclonal IgG”).
Bacterial meningitis. In bacterial meningitis, PCR detects microbial DNA in the CSF with high sensitivity and specificity when conventional methods have shown negative results, even in patients treated with antibiotics (Saravolatz et al., 2003). Amplification of trans-specific ribosomal RNA genes (16 S rRNA) and subsequent sequencing of the PCR amplicons, or reamplification of species-specific regions of the gene sequence, permit identification of the most important pathogens of meningitis. This diagnostic test is only available in special laboratories.
Tropheryma whipplei. PCR with specificity for 16S or 23S rRNA of the pathogen reliably also detects Tropheryma whipplei in CSF (A. von Herbay, www.whipplesdisease.net).
Toxoplasmosis. PCR has little to contribute to the diagnosis of cerebral toxoplasmosis.
Antibody detection is performed either quantitatively using the antibody index, or qualitatively using Western blots. The principle and calculation of the antibody index have been explained above (“Oligoclonal IgG”). Detection in Western blots is described for neuroborreliosis in Chap. 4, “Methods of Immune Complex Analysis.” The relative importance of each method is discussed above (“Antibody Index”). Basically, the antibody index is always superior to the blot.
Antibody index. A raised antibody index—i. e., indirect detection of an infection of the nervous system via the pathogen-specific intrathecal immune response—plays an important role as an extremely sensitive and specific laboratory parameter in many pathogen-induced diseases, being comparable or even superior to PCR diagnosis. A disadvantage for prompt diagnosis of acute infections is the latency period of up to several weeks before the humoral immune response in serum and CSF can be successfully detected.
Neuroborreliosis and neurosyphilis. Calculation of the antibody index is the gold standard for the diagnosis of neuroborreliosis and neurosyphilis. In neuroborreliosis, pathogen-specific intrathecal antibody production is successfully detected within 6 weeks after the onset of symptoms; it is highly specific with a sensitivity of 100%. The antibody index has a similar sensitivity in the diagnosis of neurosyphilis (Table 5.19).
Meningitis and mycosis. The antibody index is not an important laboratory parameter in the routine diagnosis of bacterial meningitis, tuberculous meningitis, or mycosis of the nervous system.
Viruses. A positive antibody index is diagnostic proof of infection of the nervous system by herpesviruses (HSV-1, VZV) and polyomaviruses (JCV) (Reiber and Lange, 1991; Weber et al., 1997). In cases of extremely acute infection, such as HSV-1 encephalitis, the pathogen-specific intrathecal antibody production is detected after 1–2 weeks at the earliest. By contrast, in acute VZV infections and chronic PML (JCV), an increased antibody index is often detected in the very first CSF sample collected for diagnostic purposes (Table 5.19). The relative sensitivity of these methods is discussed above (“Antibody Index”).
Casas I, Pozo F, Trallero G, et al. Viral diagnosis of neurological infection by RT multiplex PCR: a search for entero- and herpesviruses in a prospective study. J Med Virol 1999;57:145–151
Darnell RB. The polymerase chain reaction: application to nervous system disease. Ann Neurol 1993;34:513–523
De Luca A, Antinori A, Cingolani A, et al. Evaluation of cerebrospinal fluid EBV-DNA and IL-10 as markers for in vivo diagnosis of AIDS-related primary central nervous system lymphoma. Br J Haematol 1995;90:844–849
Jeffrey KJM, Read SJ, Peto TEA, et al. Diagnosis of viral infections of the central nervous system: clinical interpretation of PCR results. Lancet 1997;349:313–317
Reiber H, Lange P. Quantification of virus-specific antibodies in cerebrospinal fluid and serum: sensitive and specific detection of antibody synthesis in brain. Clin Chem 1991;37:1153–1160
Roberts TC, Storch GA. Multiplex polymerase chain reaction for diagnosis of AIDS-related lymphoma and toxoplasmosis. J Clin Microbiol 1997;35:268–269
Saiki RK, Gelfand DH, Stoffel S, et al. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 1988;239:487–491
Saravolatz LD, Manzor O, VanderVelde N, et al. Broad-range bacterial polymerase chain reaction for early detection of bacterial meningitis. Clin Infect Dis 2003;36:40–45
Weber T, Frye S, Bodemer M, et al. Clinical implications of nucleic acid amplification methods for the diagnosis of viral infections of the nervous system. J Neurovirol 1996;2:175–190
Weber T, Trebst C, Frye S, et al. Analysis of the systemic and intrathecal humoral immune response in progressive multifocal leukoencephalopathy. J Infect Dis 1997;176:250–254
Felgenhauer K, Reiber H. The diagnostic significance of antibody specificity indices in multiple sclerosis and herpes virus induced diseases of the nervous system. Clin Investig 1992;70:28–37
Fredricks DN, Relman DA. Application of polymerase chain reaction to the diagnosis of infectious diseases. Clin Infect Dis 1999;29:475–488
Innis MA, Gelfand DH, Sninsky JJ, White TJ, eds. PCR protocols. A guide to methods and applications. San Diego: Academic Press; 1990
Kayser FH, Bienz KA, Eckert J, Zinkernagel RM, eds. Medizinische Mikrobiologie. 9th ed. Stuttgart: Thieme; 1998
Kniehl E, Dörries R, Geis HK, et al. Infektionen des Nervensystems. MIO 17 Qualitätsstandards in der mikrobiologisch-infektiologischen Diagnostik. Munich, Urban & Fischer; 2001
Lu HZ, Bloch KC, Tang YW, et al. Molecular techniques in the diagnosis of central nervous system infections. Curr Infect Dis Rep 2002; 4:339–350
Roos KL. Pearls and pitfalls in the diagnosis and management of central nervous system infectious diseases. Semin Neurol 1998;18:185–196
Storch GA. Diagnostic virology. Clin Infect Dis 2000;31:739–751
Thomas L (eds). Labor und Diagnose. Frankfurt am Main: TH-Books; 1998