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Paul E. Verweij, MD Department of Medical Microbiology

Advances in the diagnosis of invasive aspergillosis. Paul E. Verweij, MD Department of Medical Microbiology Nijmegen University Center of Infectious Diseases, UMC St Radboud. Advances………. Understanding release and kinetics of surrogate markers Comparison of surrogate markers

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Paul E. Verweij, MD Department of Medical Microbiology

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  1. Advances in the diagnosis of invasive aspergillosis Paul E. Verweij, MD Department of Medical Microbiology Nijmegen University Center of Infectious Diseases, UMC St Radboud

  2. Advances……… Understanding release and kinetics of surrogate markers Comparison of surrogate markers Comparison of strategies

  3. specificity sensitivity Performance of GM detection in published studies

  4. BG in invasive fungal infection AML, MDS Receiving antifungal prophylaxis Clin Infect Dis 2004;39:199-205

  5. Prospective monitoring of blood by PCR Study design : prospective screening Study population : 165 hematology patients + HSCT Samples : 1522 whole blood (3-5 ml) Strategy : 6.4 – 17 samples per neutropenic episode Method : nested PCR Aspergillus sp. specific (18 S rRNA) positive samples analyzed by Light Cycler PCR galactomannan ELISA Br J Haematol 2004;125:196-202

  6. Prospective monitoring of blood by PCR Performance sensitivityspecificity Single + 7 of 11 (63.6%) 66/104 (63.5%)  2 + 4 of 11 (36.4%) 96/104 (92.3%) GM 33.3% 98.9% Br J Haematol 2004;125:196-202

  7. “false” positives “false” negatives EORTC/MSG Proven Prob. Poss. No IA Samples67 46 781 681 Patients8 3 90 104 PCR+8 5 56 50 PCR-59 41 725 576 Br J Haematol 2004;125:196-202

  8. colonisation EORTC/MSG definitions colonisation infection disease

  9. Invasive opportunistic mycoses (IM) Host: high risk colonisation infection disease positive CT-scan negative negative Possible/probable/ proven IM Class.: No IM No IM/possible -/+ -/+ - PCR

  10. Clinical studies 117 nested PCR + -> 25 (21.4%) LightCycler + LightCycler Copies/ml Proven IA 0 - Probable IA 2 5,270 – 1,902,099 Possible IA 16 52 – 410, 667 No IA 7 37 – 90,909 Br J Haematol 2004;125:196-202

  11. Galactomannan variability: Published factors: False positive reactivity……….. Piperacillin-tazobactam LTA of bifidobacteria False negative reactivity………… N Engl J Med 2003;349:24-5 Lancet 2004;363:325-7

  12. Factors that influence performance Biological factors Site of infection Aspergillus species Microenvironment at site of infection (nutrients, pH, etc) Molecule structure of released GM Underlying condition / immune suppression Exposure to antifungals Renal clearance, hepatic metabolism Presence of GM antibodies Lancet Infect Dis 2004;4:349-57

  13. Factors that influence performance-continued…. Biological factors Storage of sample Pre-treatment procedure Epidemiological factors Patient population Prevalence of infection Sampling strategy Definitions (positive result (cut-off), positive patient) Lancet Infect Dis 2004;4:349-57

  14. GM cut-off 986 serum samples from 67 patients with hematological malignancy J Infect Dis 2004;190:641-9

  15. Effect of exposure to mould-active antifungals J Infect Dis 2004;190:641-9

  16. Release of surrogate markers is a dynamic process

  17. Growth phases of A. fumigatus in vitro Release of markers by the fungus I II III Carbon source (glucose) Excretion of organic acids Decrease of pH Logaritmic growth No glucose Re-use of organic acids Increase of pH Decreased growth No glucose No acids Stable pH Lysis and cannibalism Arch Aller Appl Immun 1980;62:252-64

  18. hours 0 24 48 72 144 168 192 96 120 Release of surrogate markers 7 6 5 4 3 2 1 I II III Concentration glucose Dry weight GM

  19. Release of surrogate markers 7 6 5 4 3 2 1 hours 0 24 48 72 144 168 192 96 120 Concentration - - - - - - - - + PCR (supernatant) Need damage of fungus by host defences?

  20. Comparative studies GM versus PCR versus BG Adult hematology patients on mould active antifungal prophylaxis Prospective study, 149 treatment episodes, 96 patients Weekly sampling ROC analysis: GM > PCR and GM > BG J Clin Microbiol 2004;42:2733-41

  21. PCR Antigen Both? Need for strategic studies: which test to use

  22. PCR,GM and BG may be useful for the early detection of invasive mycoses when intensive sampling is achieved. Some progress has been made in understanding the kinetics of surrogate markers Correlation with other diagnostics and optimal sequence of testing needs to be determined Conclusions

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