Page 1. Virus Infections in the Central Nervous System. Viral Encephalitis: Incidence. Herpes simplex virus

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1 Virus Infections in the Central Nervous System 2 main causes Herpes simplex: more encephalitis less meningitis Enterovirus: more meningitis less encephalitis sometimes the 2 combined Other causes: CMV, T. gondii, Mycoplasma pneumoniae some in relation to HIV or very rare Viral Encephalitis: Incidence Sweden: 2.3/10 6 people/yr Skoldenberg, Lancet 1984; ii:707 Denmark: 1.8/10 6 people/yr Fonsgaard, CDU 1998; 9: 45 Finland: 1.4/10 5 adults/yr: 16% due to HSV Rantalaiho, J. Neurol. Sci 2001; 184: 169 Vienna: 1/10 5 adults/yr: incidence of herpes encephalitis Puchhammer-Stöckl, J Med Virol 2001: 64: Most in children < 1 yr and adults > 65 yr, followed by yr Herpes simplex virus Structure of the virus - core, caspid, tegument and envelope Page 1

2 Herpes simplex Virus Infections in the Central Nervous System Neonatal HSV - mostly HSV-2 by retrograde spread secondary to maternal genital infection - incidence: 1/3500-1/5000 births in US HSV encephalitis 1.65/ births in UK* - most commonly caused by HSV-1 Recurrent aseptic meningitis (Mollaret s meninigitis) - mainly associated with HSV-2 Tang YW et al. J Clin Microbiol 1999;27: 2127 Tookey et al. Pediatr Perinat Epid 1996; 10: 432 HSV Encephalitis Leading cause of fatal encephalitis % of all viral encephalitis cases Mortality up to 70 % 2/3 of survivors neurological defectsl Herpes simplex Encephalitis: Diagnosis Imaging Techniques Focal inflammatory loci in the temporal lobes of the cerebral cortex - electro-encephalography (EEG) - computerized tomography (CTscan) - magnetic resonance imaging (MRI) helpful clinical direction but results lack sensitivity and specificity Page 2

3 HSV Encephalitis : Diagnosis Diagnosis is difficult - in early stage : % of encephalitis have normal cell counts - only 50 % elevated WBC - only 50 % elevated protein level - only 4 % with biopsy proven encephalitis : culture positive in CSF - serology is useless - CSF antibodies : elevated late in disease Herpes simplex Encephalitis: Diagnosis Virus isolation Isolation of HSV from brain tissue was considered as gold standard high efficiency of virus isolation ± 45% invasive procedure with serious complications including hemorrhage (2%) false negative results (4%) due to focal nature Isolation of HSV from CSF low sensitivity positive in maximum 4% of brain biopsy proven cases Tang YW et al. J Clin Microbiol 1999; 37: Herpes simplex Encephalitis: Diagnosis Serology by intrathecal antibodies: - of little clinical value since immune response in only few patients early; in most patients only after 2-3 weeks. - Standardization needed by concomitant detection of albumin to exclude passive diffusion of virusspecific antibodies into the CSF, thereby yielding false-positive results. Page 3

4 Molecular Diagnosis of HSV Encephalitis 1 st Molecular diagnosis: in situ hibridization with biotinylated cdna probe in cell preparations after cytocentrifugation 12 patients with HSE: 54 control cases 8/12: (67%) positive in methanol-fixed cells 3/12: (25%) in fresh acetone - fixed cells 11/12: (92%) POSITIVE sens. 92% 54/54: (controls) NEGATIVE spec. 100% Bamborschke S et al. J Neurol 1990; 237:73 PCR for Diagnosis of HSV Encephalitis Brain biopsy ± CSF in patients suspected for HSE PCR in CSF - 53/54 (98%): PCR positive in culture positive biopsies - 3/46 (6%):PCR positive in culture negative biopsies - all 18 CSF taken before brain biopsy: PCR positive - 4/19 CSF: PCR positive after 2 weeks of antiviral therapy PCR = GOLD STANDARD Lakeman F. J Infect Dis 1995; 172: 1641 Laboratory Techniques for Specific Diagnosis of HSV Infection in the CNS Test Ease of Turnaround Result interpretation performance a time Antigen detection h May indicate infection if correlated with symptoms Cell culture days Indicates active infection Serology h Indirect; probably indicates active infection PCR days Indicates active infection a Performance scores: 1, could be performed in most routine clinical laboratories; 2, could be performed in reference clinical laboratories; 3, could be performed in specialized research laboratories; 4, could be performed in laboratories with skilled technologists and space and equipment dedicated to performing molecular techniques. Tang YW et al. J Clin Microbiol 1999; 37: Page 4

5 Laboratory Techniques for Specific Diagnosis of HSV Infection in the CNS Test Advantage(s) Disadvantage(s) Antigen detection Rapid Poor sensitivity and specificity Cell culture Isolate available for Very poor sensitivity; phenotypic timing of early antiviral specimen susceptibility collection critical testing Serology Potential for Results generally automation retrospective PCR High sensitivity and Facility requirement; specificity false positive due to carryover contamination and false negative due to inhibitors in specimen Tang YW et al. J Clin Microbiol 1999; 37: PCR for HSV Encephalitis : Sample Transport and Processing Transport to lab at 4 C in sterile vial Stable for days up to weeks at 4 C Multiple freeze-thawing to be avoided Avoid contamination between samples (e.g. by aliquoting) Tang YW et al. J Clin Microbiol 1999; 37: 2127 CSF Preparation Prior to Nucleic Acid Amplification Principle Method (examples) CSF cell lysis CSF cell lysis-protein digestion Nucleic acid concentration Nucleic acid extraction Heating to 95 C, freezing thawing Detergents (SDS), proteases (protease K), chaotropioc agents (guanidiniun thiocyanate) a Ultracentrifugation Ethanol precipitation of nucleic acid Phenol-chloroform, spin column, silicate absorption, magnetic separation Cinque P et al. J Clin Virol 2003; 26: Page 5

6 PCR Methods for HSV Encephalitis Mono reaction with agarose gel electrophoresis or EIA detection Multiplex reaction: - up to 6 viruses: HSV 1-2, VZV, CMV, EBV, HHV6 PCR with consensus primers Real time PCR Cinque P et al. J Clin Virol 2003; 26: Example of Multiplex PCR M HSV-1 (138 bp) HSV-2 (101 bp) VZV (266 bp) Cinque P et al. J Clin Virol 2003; 26: Example of PCR Assay with Consensus Primers Cinque P et al. J Clin Virol 2003; 26: Page 6

7 Indications for molecular amplification techniques for the detection of Herpes Simplex Virus (HSV1-HSV2) 1. Patients with neurological symptoms: encephalitis, meningo-encephalitis, meningitis, myelitis 2. Patients with ophthalmological symptoms: keratitis, uveïtis, acute retinitis 3. Neonatal herpes infections 4. Imunocompromised patients with oesophageal and intestinal lesions HSV Encephalitis : Diagnosis by PCR More sensitive than culture - 53/54 biopsy proven : positive (Lakeman) No commercial kits available All methods are in house methods HSV PCR = not standardized PCR results may be different from different labs Types of NATs in use in 2002 Types of NATs in use in 2003 Commercial In-house Conventional In-house Real-time Commercial In-house Conventional In-house Real-time 100% 100% 80% 60% 40% 80% 60% 40% 20% 20% 0% Ct Mtb HCV HIV HSV CMV EV 0% Ct Mtb HCV HIV HSV CMV EV Forde C. ECCMID 2004, P831 Page 7

8 Molecular Diagnostic Tests : Proficiency Testing To confirm skill of lab in test performance To ensure reproducibility To validate amplification methods Frequency : testing events / year - 5 test samples / testing event covering full range : non reactive highly reactive Samples : - whole organisms or isolated nucleic acids - previously characterized specimens - or duplicate, blinded specimens (internal consistency) NCCLS MM3-A., 1995 Quality Control for Molecular Diagnostics Past, present trends. Program *No Participants QCCA **No Part3cipants 2003 *QCCA % false Positive **2003 % false Positive QCCA % false Negative **2003 % false Negative *QCCA % Commercial Assays **2003 % commercial Assays $ %$ &$ '#$!"# Quality Control for the Detection of HSV Techniques for NA extraction, amplification and target sequences are heterogeneous All labs use in-house developed methods Application of real-time PCR increased from 7/12 (58%) labs in 2002 to 11/13 (85%) in 2004 The use of inhibition control also increased from 7/12 (58%) labs in 2002 to 10/13 (77%) Sensitivity and specificity of all methods used were excellent No false positive results were reported in 2002; in % of negative samples were reported false positive Page 8

9 Influence of Prevalence on Predictive Values for given test : Se = 99%, Sp = 98% Prevalence PPV NPV 1 / 4.9 % % 1 / 4.7 % % 1 % 33.3 % % 2 % 50.0 % % 3 % 60.0 % % 4 % 67.0 % % 5 % 72.0 % % 10 % 84.0 % % 20 % 92.0 % % 30 % 95.0 % % Goldberg M, 1990; L epidémiologie sans peine Algorithm for Specimen Processing and Reporting Results Specimen type / volume adequate Yes No specimen preparation Reject Control amplification - + dilute, reamplify Control amplification - Qiagen extraction - Report : unable to process Target amplification - Product analysis + Report as - Repeat testing + - Report as + Report as - Utility of Amplification Methods for Virus Detection in CSF HSV: PCR was shown to be the reference method Lakeman et al, J. Infect. Dis. 1995; 171:857 Extended to herpes virus group Extended to enterovirus detection in cases of meningitis Tanel et al., Arch. Pediatr. Adolesc. Med. 1996; 150: 919 Ahmed A et al, J. Pediatr. 1997; 131: 393 Van Vliet et al, J. Clin. Microbiol. 1998; 36: 2652 Enormous increase of requests for PCR on CSF Page 9

10 Molecular Diagnostic Methods in Meningo- encephalitis Variety of possible etiologic agents Stepwise approach, each step aimed at a combination of agents Multiplex approach Regional epidemiologic situation e.g. LCM, Coxiella burnetii, Borrelia burgdorferi : reference centers Clinical condition : immunocompromised patient : Toxoplasma gondii, CMV Molecular Diagnostics for Meningo-encephalitis pos HSV neg VZV M. pneumoniae pos Repeat to confirm neg pos pos CMV T. gondii neg Report result Report result Detection of HSV DNA from CSF Specimens Collected at the Mayo Clinic from August 1993 through December 1997 Yr No. of No. of % No. of subjects positive HSV-positive specimens positive positive Male Female Unknown male/female tested specimens gender ratio , , , Total 6, Tang YW et al. J Clin Microbiol 1999; 37: Page 10

11 Effective Use of PCR for Diagnosis of CNS Infections No. (%) of tests with indicated result/no. of tests performed Both protein Protein level Leukocyte Both protein Organism level and normal, count normal, level and detected leukocyte leukocyte protein level leukocyte count count abnormal count normal abnormal abnormal Total Herpesvirus* 0/209 (0) 1/33 (3.0) 5/317 (1.6) 18/173 (10.4) 24/732 (3.3) T. whippelii 0/56 (0) 0/3 (0) 1/101 (1.0) 0/30 (0) 1/190 (0.5) B. burgdorferi 0/149 (0) 0/18 (0) 0/215 (0) 0/89 (0) 0/471 (0) * Including HSV, EBV, VZV, and CMV Tang et al. Clin Infect Dis 1999; 29: Restriction Rules for HSV Detection in CSF Reference N cases / specimens Criterium Tang (1999) 24 / 723 WBC > 5 cells / mm 3 workload reduction 29% and / or > 45 mg/dl protein Simko (2002) 10 / 406 WBC > 5 cells / mm 3 and / or > 55 mg/dl protein workload reduction 38% increase of positivity rate: 1.9% 4% 2-fold Tang et al. Clin Infect Dis 1999; 29: 803 Simko et al. Clin Infect Dis 2002; 35: 414 Results of HSV DNA Detection in CSF by PCR and of HSVspecific Antibody Measurement in CSF and Sera in Patients with Clinical Suspicion of Encephalitis Method/detection Number of Positive Negative Interpretation patients results results PCR/HSV-1 DNA in CSF (1.3%) 623 Direct confirmation of CNS infection by PCR PCR/HSV-2 DNA in CSF (1.1%) 624 IFAT/intrathecal HSV IgM (2.1%) 611 Serological evidence of CNS infection IFAT/intrathecal HSV IgG (1.9%) 612 IFAT/HSV IgM, 4 fold (11.3%) 2099 Serological evidence of increase in IgG titres, active infection seroconversion Sauerbrei A et al. J Clin Virol 2000; 17: 31 Page 11

12 Virological Diagnosis of Herpes simplex Encephalitis PCR versus serological diagnosis Study design: 624 CSF samples: PCR + virus-specific antibodies 2409 serum samples: virus-specific antibodies CONCLUSIONS: No intrathecal antibodies in patients with positive PCR Intrathecal immune response when CSF negative for PCR PCR: method of choice in early phase of disease Intrathecal antibodies: in later stage of disease Sauerbrei A et al. J Clin Virol 2000; 17: Limits of Early Diagnosis of HSV Encephalitis in Children Prognosis depends on early and appropriate administration of specific antiviral therapy 38 children with proven HSV initial negative results: 8/33 CSF before day 3 associated with low level of protein < 10 WBC/mm3 De Tiege X et al. Clin Infect Dis 2003; 36: 1335 Quantitative PCR for Diagnosis of HSE No correlation between quantity of virus genomes and severity of disease or prognosis Revello M. Clin Diagn Virol 1997; 7: 183 Patients with >100 copies DNA/µl - older - brain lesions by CT scan - poorer outcomes than patients with <100 copies Dominguez R. J Clin Microbiol 1998; 36: 2229 Page 12

13 Quantitative Real-Time PCR for the Detection of VZV in CSF Methods Quantitative PCR on LightCycler with Real Art VZV LCkit DNA isolation by High Pure Viral Nucleic Acid Kit Results CSF viral load: - 10 x 10 2 copies/ml: meningitis - 5 x 10 4 copies/ml: facial nerve paresis - viral load in vesicular fluid: 3x10 6 copies/ml correlation between viral load and severity of disease remains uncertain! Zampachova E et al. ECCMID 2004, P840 Example of NA Quantification in the CSF Virus Quantitative techniques Significance of NA quantitation in CSF HSV-1 Competitive PCR, Wide range of level variation (up to 10 7 copies/ml). real-time PCR Association of high DNA levels with bad HSE outcome? Decline of DNA levels following aciclovir therapy in HSE HSV-2 Real-time PCR Narrower range of level variation in patients with HSV-2 meningitis than in patients with HSV-1 encephalitis. Highest levels found in children with congenital infection (up to 10 6 copies/ml) VZV Semiquantitative PCR, Higher levels in patients with herpes zoster complireal-time PCR cations than in those with varicella Cinque P et al. J Clin Virol 2003; 26: PCR Results following Completion of Antiviral Therapy PCR Infant characteristic Negative a Positive b P value Disease classification CNS 4 (36.4%) 14 (73.7%) <0.001 Disseminated 0 (0.0%) 5 (26.3 %) SEM 7 (63.6%) 0 (0.0%) Morbidity and mortality after 12 mo Normal 6 (54.5%) 1 (5.3%) <0.001 Mild 0 (0.0%) 0 (0.0%) Moderate 1 (9.1%) 3 (15.8%) Severe 2 (18.2%) 10 (52.6%) Dead 0 (0.0%) 5 (26.3%) a All samples were negative after treatment b one positive result Romero JR, Kimberlin DW. Clin Lab Med 2003; 23: Page 13

14 Etiology of Viral Meningitis Retrospective analysis of 43 causecutive cases of aseptic meningitis 43 cases : 19 (44%) enterovirus 1 (2%) HIV 2 (5%) VZV 5 (12%) HSV (2%) CEE 15 (35%) unknown Acyclovir initially administered to all cases Hospitalization time : days Nowak A et al. Eur J Neurol 2003; 10: Enterovirus Types and Characteristics of Human Enterovirus 66 serotypes known Group Virus types CPE in cell cultures Pathology in Monkey Human newborn Kidney cells mice Poliovirus 3 types: Coxsacke A 23 types/ A1-22, A24 - or ± - or ± + Coxsackie B 6 types : B1-B Echovirus 31 types (1-9, 11-27, + ± ) Enteroviruses 4 types (68-71) Page 14

15 Types and Characteristics of Human Enterovirus Group Virus types Major disease associations Poliovirus 3 types (poliovirus 1-3) Paralytic poliomyelitis; aseptic meningitis; febrile illness Coxsackie virus 23 types (A1-A22, A24) Aseptic meningitis; herpangina; febrile group A illness; conjunctivitus (A24); hand, foot and mouth disease Coxsackie virus 6 types (B1-B6) Aseptic meningitis; severe generalised group B neonatal disease; myopericarditis;: encephalitis; pleurodynia (Bornholm disease); fibrile illness Echovirus 31 types (types1-9, Aseptic meningitis, rash, febrile illness 11-27, 29-33) conjunctivitis; severe generalised neonatal disease Enterovirus 4 types (types 68-71) Polio-like illness (E71): aseptic meningitis (E71); hand, foot and mouth disease (E71); epidemic conjunctivitis (E70) Enterovirus: Epidemiology Distribution of the 15 most commonly reported nonpolio enterovirus, serotypes, by rank - National Enterovirus Surveillance System, United States, (n=577) 2001 (n=1,285) (n=1,862) Rank Serotype % Serotype % Serotype % 1 Coxsackie B echo echo echo echo echo coxsackie A9 8.7 coxsackie B2 7.6 coxsackie B Coxsackie B4 8.3 echo coxsackie B echo echo echo echo echo echo coxsackie B2 3.5 coxsackie B3 3.0 coxsackie A echo coxsackie B1 2.7 echo echo echo coxsackie B enterovirus coxsackie A9 2.0 echo echo coxsackie B5 1.7 coxsackie B echo echo coxsackie B echo coxsackie B4 0.9 echo echo echo echo parecho 1* 1.4 enterovirus enterovirus Total * Formerly echo 22. For all other serotypes, percentages were 7.8% in 2000, 4.7% in 2001, and 6.5% during Belgie 2000: echo 30; echo 6, coxsackie B 5 (M.Van Ranst) MMWR 2002;51: Enteroviral Meningitis Incidence: 219/10 5 children < 1 yr of age 19/10 5 children 1-4 yrs of age Rantakallia Sc. J Inf Dis. 1986; 18: 286 Responsible for % of meningitis with known etiology in USA % of encephalitis cases in USA : estimate of cases annualy Underreported especially in adults Page 15

16 Enteroviral meningitis in Adults: Underestimated Retrospective analysis of 30 cases Characteristics symptoms: inconstant CSF showed pleocytosis in 29/30 cases but predominance of lymphocytes in only 44% of patients Management of patient varied markedly - CT scan : 33% - acyclovir: 20% - antibiotics: 53% Laboratory tests requested on admission: - PCR herpes simplex: 9/30 (30%): all negative after 4 days PCR for enterovirus : 9/30 : alle positive - PCR enterovirus: 21/30 (70%) : all positive Rapid PCR results may avoid considerable medical expenditure Evidence for syndromic approach Peigue-La feuille H et al. Pathologie Biologie 2002; 50: Diagnosis of Enteroviral Meningitis by Virus Culture Insensitive: especially for coxsackie A viruses Serotyping necessary for identification and epidemiology Turnaround time : 4-8 days No cell type supports replication of all EV types Even with use of several cell types: - 25%-35% negative specimens - coxsackie on none of cell lines (suckling mice) Virus : CPE of Enterovirus in Cell Culture CPE after 4-8 days Page 16

17 Diagnosis of Enteroviral Meningitis by Culture Total number of isolates: 73 Number RD cells MRC5 Vero Hep o o 25 + o o o 5 o o o o + o 4 o + o o 1 o o o + Verstrepen et al., 2002 Diagnosis of Enteroviral Meningitis by Culture Interpretation of results CSF: very specific but low sensitivity blood: very specific but low sensitivity stool and pharynx : sensitive but low specificity - excretion of virus in pharynx : 1-2 weeks in faeces : 7-11 weeks Chang et al. J Microbiol Infect 2001; 34: Diagnosis of Enteroviral Meningitis by Serology Neutralization tests: for seroepidemiological purposes - determining exposure and immunity of population group - responses to polio vaccination - tests are labour intensive, TAT 3-4 days, not widely availabe Seroconversion or significant increase in antibody titres - detected only occasionally Elevated titres - frequently occur in normal individuals NOT RELEVANT FOR INDIVIDUAL DIAGNOSIS Page 17

18 Tissue Culture Versus RT-Polymerase Chain Reaction for the Detection of Enterovirus From Cerebrospinal Fluid Source, Y RT-PCR assay Tissue culture* (%) RT-PCR* (%) Rotbart, 1990 In-house 9/13 (69) 13/13 (100) Sawyer et al, 1994 In-house 112/217 (52) 135/217 (62) Riding et al, 1996 In-house 6/140 (4) 35/140 (25) Rotbart et al, 1997 Amplicor 36/209 (17) 51/209 (24) Ahmed et al, 1997 Amplicor 5/61 (8) 18/61 (30) Kessler et al, 1997 Amplicor 27/103 (26) 34/103 (33) Pozo et al, 1998 In-house 26/50 (52) 46/50 (92) Amplicor 26/50 (52) 43/50 (86) In-house 1/29 (3) 4/29 (14) Amplicor 1/29 (3) 3/29 (10) Gorgievski-Hrisoho et al, 1998 Amplicor 16/68 (24) 58/68 (85) * Values presented as number positive/number tested. Romero J. Arch Pathol Lab Med 1999; 123: PCR for diagnosis of Enteroviral Meningitis Conventional PCR Real-Time PCR TAT : 2-3 days risk for contamination qualitative TAT: 3-4 hours single tube reaction: minimal carry-over risk quantitative results possible Real-time tests are only technique allowing immediate impact on therapeutic decisions Indications for PCR for Enteroviruses Viral meningitis or meningo-encephalitis (CSF) Acute pericarditis or myocarditis (pericard fluid, blood, myocardial biopsy) Prenatal diagnosis of congenital infections in case of echographic abnormalities (amniotic fluid, faetal blood) Page 18

19 Amplification Methods for the Diagnosis of Enteroviral Meningitis Organization of the enterovirus RNA genome. NTR inidicates nontranslated region. 5 NTR : - critical role in enteroviral life cycle - conserved regions of high nucleotide identity among EV - ideal for development of primers and probes for the detection of enteroviruses : most serotypes detected Romero J. Arch Pathol Lab Med 1999; 123: Selection Criteria for PCR on EV in CSF WBC: increase with increasing age children: 15% < 10WBC / mm 3 Henquell et al. J Clin Virol 2001; 21: Adults: - 29/30 (97%) pleocytosis : > 5/mm 3-44%: predominance of lymphocytes - 37%: predominance of polymorphonuclear leucocytes - mainly during first days after onset of symptoms - protein concentration: normal or slightly increased - glucose concentration: generally within normal limits A NORMAL CSF DOES NOT EXCLUDE EV INFECTION. Peigue-Lafeuille H et al. Pathologie Biologie 2002; 50: Impact of Enteroviral PCR on Patient Management Comparison of management in two groups of patients: N=95 : positive PCR results N=95: negative EV-PCR+ EV-PCR- P values (N=95) (N=92) Additional laboratory tests 26% 72% <0.01 IV antibiotics 2 d 3.5 d <0.01 hospitalization time 42 hours 71.5 hours <0.01 Ramers C et al. JAMA 2000; 283: Page 19

20 Impact of EV PCR on Adult Patient Management Nationwide outbreak of EV meningitis due to echo 30 Objective: evaluation of management strategy including early PCR on hospitalization Methods: - N=21: before implementation of early PCR - N=27: after implementation of early PCR Results: significant reduction of - hospital stay: 103 hrs 80 hrs P= mean duration of antibiotic treatment: 115 hrs 69 hrs : P=0.02 Conclusion: systematic testing of CSF in cases of aseptic meningitis by PCR may be cost-effective Tattevin P et al. Scand J Infect Dis 2002; 34: Impact of PCR on Management of Pediatric Patients with Enteroviral Meningitis Objectives : - Comparison of antibiotic use and hospital stay in children with EV meningitis after PCR results available < 24 h or > 24 h after collection Methods: - EV PCR performed 5d/week - CSF from 113 patients with suspected meningitis Results: 50/113 (44%) of patients positive 17/50 (34%) results < 24 h 33/50 (66%) results > 24 h difference in antibiotic use: 20 hrs less (P=0.006) difference in hospital charges: 2798 $ less (P=0.001) Rapid reporting of PCR resutls can have significant impact Robinson CC et al. Pediatr Infect Dis 2002; 21: Diagnosis of viral encephalitis: CONCLUSIONS Amplification methods are a major advance for the detection of both herpes viruses and enteroviruses. Conventional PCR s are gradually replaced by real-time techniques. Rapid PCR results allow immediate impact on therapeutic decisions and may be cost-effective. Page 20