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Neonatal Candidiasis

Neonatal Candidiasis. Catherine M. Bendel, M.D. University of Minnesota Medical School, Minneapolis. Neonatal Candidiasis Outline. Epidemiology Risk Factors -- Yeast and Host Research correlates Clinical Presentation Diagnosis and Treatment Prophylaxis?. Candida. Commensal organism

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Neonatal Candidiasis

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  1. Neonatal Candidiasis Catherine M. Bendel, M.D. University of Minnesota Medical School, Minneapolis

  2. Neonatal Candidiasis Outline • Epidemiology • Risk Factors -- Yeast and Host • Research correlates • Clinical Presentation • Diagnosis and Treatment • Prophylaxis?

  3. Candida • Commensal organism • Eucaryotic and diploid (C. albicans) • Dimorphic yeast • Increasingly important nosocomial pathogen

  4. Nosocomial Candidiasis • 6th most common nosocomial pathogen • 4th most common cause of nosocomial septicemia (7.8% of all bloodstream infections) • 2nd most common nosocomial pathogen in NICU - incidence 2-20% in ELBW • 24% to 54% mortality in those who receive therapy • High morbidity Pfaller. Clin Infect Dis 1996;22(Suppl 2):S89 Pittet & Wenzel. Arch Intern Med 1995;155:1177 Baltimore. Sem in Perinat 1998;22: 25 Benjamin. Pediatrics 2000;106:712

  5. Numbers of Cases of Sepsis in the United States, According to the Causative Organism, 1979-2000. Martin GL et al, N Engl J Med 348:1546-54, 2003

  6. Virulence Factors • C.albicans accounts for the majority of Candida spp isolated • Multiple intrinsic C. albicans virulence factors have been identified • Systemic infections associated with specific host risk factors • The gastrointestinal tract is considered a major portal of entry for C. albicans

  7. All patients 55% C. albicans 20% C. glabrata 9% C. tropicalis 7% C. krusei 15% C. parapsilosis 1% C. dubliniensis 2% others Phaller, J Clin Micro 2001:39 Neonates 58% C. albicans 34% C. parapsilosis 2% C. glabrata 4% C. tropicalis 2% others Fridkin, Pediatrics, 2006:117 Frequency of Isolation of Candida species

  8. Virulence Factors • C.albicans accounts for the majority of Candida spp isolated • Multiple intrinsic C. albicans virulence factors have been identified • Systemic infections associated with specific host risk factors • The gastrointestinal tract is considered a major portal of entry for C. albicans

  9. C. albicans virulence factors • Adherence to host tissues (epithelium/endothelium) • Receptor or adhesin mediated • Cytolytic enzymes (proteinases, phospholipases) • Hydrophobicity • Variable phenotypic expression • Morphologic switching • Filamentous forms believed to facilitate adhesion and penetration of epithelium Andrutis. J Clin Microbiol 2000; 38: 2317 Gale. Science 1998;279:1355 Cutler. Ann Rev Microbiol 1991;45:187 Corner. Curr Biol 1997; 7:R761 Mitchell. Curr Opin Microbiol 1998; 1:687

  10. Calcofluor stained cecal contents from a mouse colonized with C. albicans showing yeast and filamentous forms in vivo

  11. C. albicans CAG1 (+/-) incubated 3 h on Caco-2 enterocytes showing yeast cluster partially embedded

  12. C. albicans CAF2 (+/+) incubated 3 h on Caco-2 enterocytes with microvilli intimately associated with distal tip of germtube

  13. C. albicans CAF2 (+/+) incubated 3 h on Caco-2 enterocytes showing elongated enterocyte microvilli anchoring a C. albicans filament to the enterocyte surface

  14. Virulence Factors • C.albicans accounts for the majority of Candida spp isolated • Multiple intrinsic C. albicans virulence factors have been identified • Systemic infections associated with specific host risk factors • The gastrointestinal tract is considered a major portal of entry for C. albicans

  15. Host Risk Factors for Systemic Candidiasis • Colonization with Candida species • Prematurity, especially GA < 28 weeks • Very low birth weight • Prolonged hospitalization • Indwelling catheters • Neutropenia • Broad spectrum antibiotic therapy • Third generation cephalosporin exposure • Steroids

  16. Host Risk Factors for Systemic Candidiasis • Hyperalimentation • Hyperglycemia • Indomethacin • Intestinal perforation > NEC/IP • Abdominal or cardiac surgery • Compromised anatomic barriers • Apgar score <5 at 5 min • Topical Petrolatum Ointment (Aquaphor)

  17. NEC or IP • Strong association • IP ~25% incidence of canididasis • Empiric therapy

  18. Incidence of Systemic Candidiasis associated with TPO in infants with BW ≤ 1500 grams Campbell JR, Zaccaria E, & Baker CJ, Pediatrics 2000;105:1041-1045.

  19. Virulence Factors • C.albicans accounts for the majority of Candida spp isolated • Multiple intrinsic C. albicans virulence factors have been identified • Systemic infections associated with specific host risk factors • The gastrointestinal tract is considered a major portal of entry for C. albicans

  20. C. albicans and others GI tract commensal Acquired perinatally Vertical transmission from mother Colonization within 7-10 days (skin & GI) Higher levels of colonization associated with increased risk of systemic disease C. parapsilosis Skin colonization in adults (tinea versicolor) Skin or GI tract colonization in neonates Acquired postnatally Transmission from caregivers Colonization later, usually >14 days: rate higher with increased LOS Skin and GI tract colonization

  21. Research Correlates • Mouse models of IV infection and GI tract colonization developed. • Models then used to evaluate: • The role of yeast filamentation in colonization and infection. • The role of antibiotics and steroids in colonization and dissemination.

  22. Candida albicans strains • Comparisons were made using three C. albicans strains (all URA3/ura3): • CAF-2: “wild type”/parent strain; exhibiting a normal phenotype forming blastoconidia, germ tubes, hyphae and pseudohyphae.(Fonzi, Genetics 1993;134:717) • HLC4: cph1/cph1,efg1/efg1; a nonfilamentous mutant described as growing exclusively as blastoconidida. (Lo, Cell 1997;90:939) • BCa2-10: tup1/tup1; grows exclusively in filamentous form.(Braun, Science 1997;277:105)

  23. Oral (107) or intravenous (105) inoculation of mice with C. albicans CAF-2, HCL54 or BCa2-10 I.V. oral C. albicans Monitor for death C. albicans Sacrifice 1, 7, 14, & 21 days later Antibiotics 3 days C. albicans 3 days I.P. Dex Sacrifice • Antibiotics in drinking water: • bacitracin • streptomycin • gentamicin • Dexamethasone: • 2mg I.P. BID

  24. Affect of antibiotic treatment on cecal colonization in mice Antibiotics + LPS No antibiotics Antibiotics 6 6 6 /g cecum * 5 * 5 5 Avg ±SE log10 viable * 4 4 4 C. albicans 3 3 3 -/- -/- +/+ -/- -/-/+ -/-/+ -/-/+ +/+ +/+ INT1 Genotype NOTE:C. albicans was recovered from the mesenteric lymph nodes of only an occasional mouse and never from the kidneys, with all treatments. Bendel et al. SHOCK. 2000; 13:453-8.

  25. Dexamethasone administration • Colonization facilitated by dexamethasone - increased to 107.5 • C. albicans recovered from the mesenteric lymph nodes in 56% of mice • C. albicans disseminated to the kidneys in 83% of mice Bendel et al, Pediatric Research 51:290;2002

  26. * 21/24 Cecal colonization and dissemination of C. albicans to mesenteric lymph nodes (MLN) and kidneys of orally inoculated mice Cecal Colonization * = p<0.05 MLN * = p<0.05 Kidneys * = p<0.01 8 100 100 7 75 75 * Avg±SE log10 viable C. albicans /g cecum * 6 12/24 11/22 % of mice with C. albicans in tissue 50 50 * 5 25 25 4 3/22 0/21 0/21 3 0 0 CAF2 HLC54 BCa2-10 CAF2 HLC54 BCa2-10 CAF2 HLC54 BCa2-10 C.albicans strain C.albicans strain C.albicans strain

  27. 100 80 60 40 20 0 0 5 10 15 20 25 30 Mortality in mice inoculated IV with C. albicanswild type andmutant strains “Filamentous” only “Nonfilamentous” % Cumulative Survival “Wild Type” Days after intravenous 105C. albicans

  28. Day 1 Day 7 Day 14 Day 21 Persistence of C. albicans in the kidney and liver of intravenously inoculated mice Kidney Liver 8 8 * * * = P<.01 & † = P<.05 vs. BCa2-10 * 6 6 * * 4 4 Avg±SE log10 viable C. albicans. per gram † 2 2 0 0 CAF2 HLC54 BCa2-10 CAF2 HLC54 BCa2-10 C. albicans strain C. albicans strain

  29. A B 50 microns 20 microns

  30. B A 50 microns 20 microns

  31. PCR Results • Using specific primers for the INT1 locus of CAF-2, the EFG-A and EFG-D fragments of HLC54, and the altered TUP1 locus of BCa2-10, PCR was performed on two C. albicans colonies recovered from each cecum and one colony from each MLN, liver and kidney that was positive for yeast. • Results confirmed that mice were colonized with the inoculated strain.

  32. Summary I • All three C.albicans strains persisted in the GI tract. • “Wild type” and “Nonfilamentous” strains colonized in similarly high numbers, while the numbers of cecal “Filamentous only” present were at least 100-fold lower.

  33. Summary II • Of the three C.albicans strains studied: • CAF-2 (wild type) was most virulent in IV inoculated mice as assessed by mortality • HLC54 (defective in filamentation) was most virulent in orally inoculated mice as assessed by extraintestinal dissemination of C. albicans. • BCa2-10 (constitutively filamentous) was avirulent in both models.

  34. Summary III • Both yeast cell and filamentous forms of wild type and “nonfilamentous” strains were found in kidney sections, but tissue necrosis was associated only with filamentous forms. • Therefore, “morphologic switching” provides candida with an advantage for persistence throughout the host under a variety of conditions.

  35. C. albicans Adherence to Cultured Epithelium - ELISA wash, lightly fix, wash Incubate C. albicans with enterocytes (pretreated for 24o with dexamethasone) Incubate with primary antibody Remove enterocytes from plastic dish Color development and O.D. at 450 nm Secondary antibody conjugated to HRP

  36. Increased adhesion of C. albicans strains to Int 407 neonatal enterocytes following pretreatment with dexamethasone

  37. Increased adhesion of C. albicans strains to Caco-2 adult enterocytes following pretreatment with dexamethasone

  38. C. albicans CAF2 (+/+) incubated 3 h on Caco-2 enterocytes showing alignment of adherent filamentous form along enterocyte cell borders

  39. Summary I • Dexamethasone pretreatment of enterocytes resulted in a significant, dose dependent increase in the adhesion of all three C. albicans strains to both neonatal and adult cell lines. • Neonatal enterocytes (Int407) were more sensitive to the effects of dexamethasone therapy. Greater increases in adhesion were observed for all C. albicans strains, at all concentrations, when compared to the adult Caco-2 cell line.

  40. Summary II • No differences were seen in the dexamethasone effect on the adhesion of CAG3 and CAG5, compared to CAF2 — equivalent increases were noted to both cell lines at each concentration tested. • Following dexamethasone treatment, preferential adhesion of the C. albicans along enterocyte cellular junctions was observed, compared to more homogeneous adhesion in the untreated cell lines.

  41. Adhesion of Candida strains to human epithelial cell monolayers

  42. Clinical Candidiasis • Candida species are responsible for a wide variety of clinical disease with variable presentation and degree of severity • “Benign” colonization • Locally invasive disease • Catheter-related fungemia • Widespread systemic disease • The specific disease process depends on the interplay of both host risk and yeast virulence factors

  43. Congenital Candidiasis - Term • Age at onset : Birth or < 24 hours • Presence of Risk Factors: None • Skin involvement: Hallmark • Respiratory involvement: Occasionally • Positive Blood culture: No • Multiorgan involvement: Never • Treatment: Topical Antifungals • Prognosis: Excellent

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