1. Laboratory exams in the diagnosis of CNS infections Dr Paul Matthew Pasco June 7, 2008

2. Lab exams for bacterial meningitis CSF GS/CS CSF cytology (+) of bacterial antigens in CSF Neuroimaging Molecular techniques (PCR)

3. CSF culture & sensitivity Gonzaga (1967): (+) in 57/85 patients  Pneumococcus in 26%; G(-) bacilli in 33% Punsalan (1988) = (+) in 9/12 Handumon (2000) = (+) in 11/50 adults Reyes (1979): 82 children  Most common: G(-) bacilli in 53.7%  Others: S. pneumoniae, N. meningitidis Kho (1992): 50 culture-proven cases; G(+) in 62% (S. pneumoniae), G(-) in 38%

4. CSF cytology & GS (Reyes 1986)

5. SENS = 81% SPEC = 34% SENS = 85%SPEC = 51%

6. How do we use sensitivity & specificity? SnNout = for a test with high sensitivity, a negative result rules out the diagnosis SpPin = for a test with high specificity, a positive result rules in the diagnosis A perfect test is both a SpPin & SnNout A useless test: SENS + SPEC – 100 = 0

7. CSF cytology & GS (Reyes 1986) PPV = 44% NPV = 73% PPV = 63% NPV = 77%

8. Likelihood ratios LR(+) = probability of (+) test for a person with the disease probability of (+) test for a person without the disease LR(-) = probability of (-) test for a person with the disease probability of (-) test for a person without the disease

9. Likelihood ratios For cytology:  LR(+) = 22/27 = 1.23 27/41  LR(-) = 5/27 = 0.54 14/41 For gram stain:  LR(+) = 23/27 = 1.77 13/27  LR(-) = 4/27 = 0.29 14/27 Not very good!

10. A likelihood ratio nomogram

11. How do we estimate our patient’s pre-test probability of having the disease? Clinical experience Local prevalence statistics Information from databases Original studies to assess diagnostic tests Studies devoted specifically to determining pre-test probabilities

12. Etiology of CNS infections in 7 hospitals (Punsalan 1999) (892 cases) Bacterial meningitis – 29.9% TB meningitis – 28.9% Meningitis unspecified – 12.2% Viral meningitis – 10.5% Brain abscess – 8.1% Cryptococcal meningitis – 2.0% Tuberculoma – 1.6% Others – 3.3%

13. Local experience in bacterial meningitis (Handumon 2000) Typical clinical picture:  Drowsy, 50%  Meningismus, 85%  Seizure, 26%  Focal neurological deficit, 18%  Fever + headache + sensorial change, 85%

14. Bacterial antigens in CSF (Garcia 1988) Phadebact, with culture as gold standard:  Sensitivity = 83%  Specificity = 93%  PPV = 83%  NPV = 93%

15. Bacterial antigens in CSF (Coovadia 1985) *CSF culture as gold standard

16. Other tests on CSF CSF CRP: sensitivity of 61%, specificity of 100%, PPV of 100%, NPV of 80% (Changco 1987) CSF leukocyte esterase: sensitivity of 100%, specificity of 93%; CSF nitrite: specificity and NPV of 85% (Tan 1997) CSF pH: decreased in 10/11 cases of purulent meningitis (Espiritu 1986)

17. Neuroimaging CT scan of head:  Not routinely done  Only to rule out other causes of CNS infection Cranial ultrasound (Lee 2001): 95 culture- proven cases  Wide and highly echogenic sulci = 87%  Convexity leptomeningeal thickening = 86%  Hydrocephalus = 62%  Extra-axial fluid collection = 8-48%

18. Other tests GS/CS from throat and petechiae (esp. for meningococcal disease) and blood Serum CRP (Sutinen 1998): elevated CRP (>10 mg/ml) has 100% sensitivity in 19 cases of bacterial meningitis (but may be low in early stages of infection) Molecular techniques – not available locally  PCR for N. meningitidis & S. pneumoniae  Quantitative PCR to determine bacterial load?

19. How should lab results help us in management of CNS infections? *Lab results should help us cross a threshold; *We may have to perform several tests to cross a threshold.

20. Viral encephalitis Standard cell culture Brain biopsy Serologic diagnosis: detect a 3-fold or more increase in specific antibody production CSF ELISA & PCR – how to determine sensitivity and specificity?

21. Problem: no single lab test or clinical feature can distinguish between different types of CNS infections Solution: propose clinical decision rules which combine clinical and simple laboratory features

22. Clinical decision rules to distinguish between bacterial and viral meningitis (Dubos 2006)

23. Decision rule by Nigrovic (2002) *BMS > 2 predicts bacterial meningitis with 100% sensitivity

24. Lab exams for tuberculous meningitis CSF AFB smear and TB culture CSF qualitative & quantitative exams ELISA – to detect IgG antibodies to mycobacterial antigens in CSF PCR – to detect mycobacterial DNA elements Neuroimaging

25. CSF TB culture Montoya (1991) – (+) in 4/17 clinically presumptive cases of TBM Pasco (2007) – (+) in 3/63 probable TBM De Guzman (2005) – MGIT mycobacterial culture system: using a surrogate gold standard, 75% sensitive and 31% specific

26. ELISA for TB meningitis Montoya (1991) – 30 kDa native antigen: (+) in 3 of 4 definite TBM, (-) in all normal & non-TBM cases Valenzuela (2000) – 38 kDa antigen: (+) in 1 of 1 definite TBM; specificity of 72% Montoya (2000) – antigen A60: 3 definite cases; 100% sensitive and 94% specific

27. The Polymerase Chain Reaction (PCR) Technique

28. PCR for TB Meningitis Montoya (1997) – (+) in 7/8 culture-proven TB Meningitis; no data in non-TBM Pasco (2007) – 63 probable TBM: 3/63 (+) by smear or culture, 14/63 (+) by PCR; 2/3 definite TBM also (+) by PCR Udarbe-Agustin (2004) – 3/6 definite TBM (+) by PCR Montoya (2001) – 9 definite TBM: 1 (+) by Amplicor, 2 (+) by nested PCR Meta-analysis by Pai (2003) – sensitivity is 56%, specificity is 98%

29. CT scan in TB Meningitis Malazo (1995) – 30 children with TBM: 28 had hydrocephalus, 14 had basal exudates, 2 were normal Kumar (1996) – compared CT scans of 94 children with TBM and 52 with pyogenic meningitis: basal meningeal enhancement, tuberculoma, or both, were 89% sensitive and 100% specific for TBM

30. Clinical decision rules in TBM Kumar (1994) – 110 Indian children with TBM and 94 with non-TBM; predictive of TBM:  Symptoms > 6 days  Optic atrophy  Focal neurological deficit  Abnormal movements  Neutrophils < 50% of CSF WBC count Thwaites (2002) – 143 Vietnamese adults with TBM & 108 with non-TBM; predictive of TBM:  Age > 36  Blood WBC < 15,000  Symptoms > 6 days  CSF WBC < 750  CSF neutrophils < 90% Pasco (200?) – 300+ Filipino adults with TBM  focal deficit  (+) PTB on CXR  CSF WBC > 50, lymphocytes predominant  CSF < 50% serum RBS  Increased CSF protein

31. Cryptococcal meningitis India Ink & Sabouraud’s culture CALAS titers Lokin (2000) – 8 cases of cryptococcal meningitis: 8 (+) by India Ink and mucicarmine; after 24h, still (+) by mucicarmine

32. Summary Lab results should help us move across a testing or treatment threshold Use clinical decision rules that combine clinical and laboratory exam results  These should not replace the clinician’s skills and perceptions;  They should only be applied after a complete validation process.