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
Eucaryotic and diploid (C. albicans)
Dimorphic yeast
Increasingly important nosocomial
pathogen
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
Numbers of Cases of Sepsis in the United States,
According to the Causative Organism, 19792000. Martin GL et al, N Engl J Med 348:1546-54, 2003
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
Frequency of Isolation of
Candida species
All patients
55% C. albicans
20% C. glabrata
9% C. tropicalis
7% C. krusei
15% C. parapsilosis
1% C. dubliniensis
2% others
Neonates
58% C. albicans
34% C. parapsilosis
2% C. glabrata
4% C. tropicalis
2% others
 Phaller, J Clin Micro 2001:39
 Fridkin, Pediatrics, 2006:117
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
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
Cutler. Ann Rev Microbiol 1991;45:187
Mitchell. Curr Opin Microbiol 1998; 1:687
Gale. Science 1998;279:1355
Corner. Curr Biol 1997; 7:R761
Calcofluor stained cecal contents from a mouse
colonized with C. albicans showing yeast and
filamentous forms in vivo
C. albicans CAG1 (+/-) incubated 3 h on Caco-2
enterocytes showing yeast cluster partially embedded
C. albicans CAF2 (+/+) incubated 3 h on Caco-2 enterocytes with
microvilli intimately associated with distal tip of germtube
C. albicans CAF2 (+/+) incubated 3 h on Caco-2 enterocytes
showing elongated enterocyte microvilli anchoring a C.
albicans filament to the enterocyte surface
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
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
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)
NEC or IP
 Strong association
 IP ~25% incidence of canididasis
 Empiric therapy
Incidence of Systemic Candidiasis associated
with TPO in infants with BW ≤ 1500 grams
Campbell JR, Zaccaria E, & Baker CJ, Pediatrics 2000;105:1041-1045.
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
Skin and GI tract colonization
C. albicans and
others
GI tract commensal
Acquired perinatally
Vertical transmission
from mother
Colonization within 710 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
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.
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)
Oral (107) or intravenous (105) inoculation of mice
with C. albicans CAF-2, HCL54 or BCa2-10
I.V.
C. albicans
oral
C. albicans
Antibiotics
3 days
Monitor for
death
Sacrifice 1,
7, 14, & 21
days later
C. albicans
3 days
I.P.
Dex
Sacrifice
Antibiotics in
drinking water:
• bacitracin
• streptomycin
• gentamicin
Dexamethasone:
• 2mg I.P. BID
6
6
*
4
3
-/-
-/-/+
3
*
+/+
3
-/-
4
-/-/+
4
5
-/-/+
5
*
+/+
5
Antibiotics
+ LPS
Antibiotics
-/-
No
6 antibiotics
+/+
Avg ±SE log10 viable
C. albicans /g cecum
Affect of antibiotic treatment on cecal
colonization in mice
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.
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
8
Cecal Colonization
* = p<0.05
% of mice with C. albicans in tissue
Avg±SE log10 viable C. albicans /g cecum
Cecal colonization and dissemination of C. albicans to
mesenteric lymph nodes (MLN) and kidneys of orally
inoculated mice
MLN
* = p<0.05
100
100
7
6
*
5
4
3
CAF2
HLC54 BCa2-10
C.albicans strain
Kidneys
* = p<0.01
*
21/24
75
75
*
*
12/24
11/22
50
25
50
25
3/22
0/21
0/21
0
0
CAF2
HLC54 BCa2-10
C.albicans strain
CAF2
HLC54 BCa2-10
C.albicans strain
% Cumulative Survival
Mortality in mice inoculated IV with C. albicans
wild type and mutant strains
“Filamentous” only
100
80
60
“Nonfilamentous”
40
20
“Wild Type”
0
0
5
10
15
20
25
30
Days after intravenous 105 C. albicans
Avg±SE log10 viable C. albicans. per gram
Persistence of C. albicans in the kidney and liver of
intravenously inoculated mice
Kidney
8
*
*
6
*
Liver
8
* = P<.01 &
† = P<.05
vs. BCa2-10
Day 1
Day 7
Day 14
Day 21
6
*
*
4
4
†
2
2
0
0
CAF2
HLC54 BCa2-10
C. albicans strain
CAF2
HLC54 BCa2-10
C. albicans strain
A
50 microns
B
20 microns
50 microns
A
20 microns
B
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.
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.
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.
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.
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
Increased adhesion of C. albicans strains to
Int 407 neonatal enterocytes following
pretreatment with dexamethasone
*
*
% Change in Adhesion
100.00%
*
100uM de x
*
1000uM de x
*
*
50.00%
*
*
*
0.00%
CAF2
10uM de x
CAG3
C. a lbi ca nsstr a in
CAG5
* p< 0.015
Increased adhesion of C. albicans strains to
Caco-2 adult enterocytes following
pretreatment with dexamethasone
100uM de x
% Change in Adhesion
100.00%
1000uM de x
*
*
50.00%
*
0.00%
CAF2
CAG3
C. a lbi ca ns str ai n
CAG5
* p< 0.05
C. albicans CAF2 (+/+) incubated 3 h on Caco-2
enterocytes showing alignment of adherent
filamentous form along enterocyte cell borders
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.
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.
Adhesion of Candida strains to
human epithelial cell monolayers
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
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
Congenital Candidiasis - Preterm
 Age at onset : Birth or < 24 hours
 Presence of Risk Factors: None
 Skin involvement:
Classical candidal dermatitis
 Atypical diffuse erythematous dermatitis
 Respiratory involvement: Common
 Positive Blood culture: Uncommon
 Multiorgan involvement: Rare
 Treatment: Parenteral Antifungal, avoid
central catheters
 Prognosis: Guarded
Catheter-Related Fungemia
 Age at onset : > 7 Days
 Presence of Risk Factors: Necessary
 Skin involvement: Uncommon
 Respiratory involvement: Infrequent
 Positive Blood culture: Yes
 Multiorgan involvement: Rare
 Treatment:
Parenteral Antifungal
Catheter removal
 Prognosis: Good
Catheter-Related Fungemia:
Complications
Specific to site and type of catheter
Cystitis
Peritonitis
Pleuritis
Mediastinitis
Central venous catheters
Right atrial or other vascular fungal mass
Disseminated Candidiasis
 Age at onset : > 7 Days
 Presence of Risk Factors: Necessary
 Skin involvement: None / Uncommon
 Respiratory involvement: Frequent
 Positive Blood culture: Yes
 Multiorgan involvement: Frequent
 Treatment: Parenteral Antifungal
 Prognosis: Fair - Poor
Disseminated Candidiasis:
Common Complications
 Pneumonia
 Meningitis
< 500 WBC, lymphocytes
Normal glucose
Normal or increased protein
 Renal involvement
Cystitis
Multiple or single abcesses
Pelvic fungal balls
 Endophthalmitis
 Significant ROP
Drohan et al, Pediatrics 2002;110(5).
Disseminated Candidiasis:
Less Common Complications
 Endocarditis, pericarditis
 Bone or joint involvement
 Peritonitis ( with bowel perforation)
 Liver abscess
 Splenic abscess
 Cutaneous nodules/subcutaneous
abcesses
Diagnosis
 Nonspecific signs and symptoms of sepsis
Fever Uncommon
 Positive Culture — NO Rapid test available
Blood
Urine (catheterized or suprapubic tap)
CSF
Other fluids as indicated
 ETT, skin, mucous membrane, GI tract
cultures indicate colonization only
Diagnosis — Cultures
 Fungal isolater tube not usually helpful
 Cultures should be held for a minimum of 710 days: non-albicans species are slow
growing
 Monitor blood cultures daily until no growth
and then weekly while on therapy
 Specific Candida species should be
identified
 Sensitivities to antifungal drugs should be
determined
Schelonkam RL and Moser SA, J Pediatr 2003:142:564-5
Diagnosis — Other Studies
 CBC with differential and platelet count
Thrombocytopenia COMMON
WBC can be low, normal or high
 Serum glucose
 BUN and creatinine
 Ultrasound: renal, cranial, cardiac
 Ophthalmologic exam
 Bilirubin and liver enzymes
Treatment — Topical
Nystatin
Miconazole
Clotrimazole
Gentian Violet
Treatment — Parenteral
Amphotericin B - mainstay of
therapy
Daily dosage:
No “test dose” required in neonates
± Initial dose 0.5 mg/kg IV over 2-6 hours
Increase by 0.5 mg/kg/d to goal of
1.0-1.5 mg/kg/d
Adjust for renal insufficiency
Treatment — Ampho B
Treatment Course
10-14 days for uncomplicated line sepsis
 3- 6 weeks for disseminated or complicated
sepsis. Cumulative dose of 30-35 mg/kg or
clearance of disease — whichever comes
first!
Monitor systemic involvement for
improvement/clearance — serial ultrasounds,
repeat cx, etc.
Treatment — Ampho B
Complications of therapy:
Renal insufficiency
Monitor UOP, BUN,Cr qod initially; then q week if stable
Renal failure reversible, but dialysis may be required
Profound hypokalemia / hypomagnesemia
Monitor K and Mg levels closely
Hematologic - bone marrow suppression
Monitor CBC and platelets qod initially and then q week
Liver dysfunction
Adjunctive Therapy: 5-FC
5-fluorocytosine - Synergistic
Dosing:
50-100 mg/kg/day po divided q 12 hours
Monitor levels
Limitations:
Oral
Rapid resistance occurs if used alone
Alternative Therapy
 Liposomal Amphotericin B (Ambisome) or
Amphotericin B Lipid Complex (Ablecet) or
Amphotericin B colloidal dispersion
 Dosing: 3-5 mg/kg/day IV over 2hours
 Appears to be safe and effective, but not
superior to conventional Ampho B
 Diminished side effects, especially renal
 Limitations:
Decreased renal absorption
Comparison of Antifungal medications
Kicklighter SD. NeoReviews 3;12:249-255. 2002
Medication
Route Daily Dose Cost/500 mg
(mg/kg/24) (acquisition)
Amphotericin B
IV
Fluconazole
IV,PO 3 - 6
$66
Abelcet
IV
3-4
$93
Amph B Colloid Disp IV
5
$135
Ambisome
IV
3-5
$199
Flucytosine
PO
50 - 200
$7
0.5 - 1.5
$12
Alternative Therapy
Fluconazole — IV (vorconazole next generation)
Dosing, over 2-6 hours:
Preterm ≤ 29 weeks: 5-6 mg/kg/72 hours
Preterm 30-36 weeks: 3-6 mg/kg/48 hours
Term: 6-12 mg/kg/24-72 hours
Monitor levels
Side effects: renal, hepatic and
hematologic, but less than Ampho B
Yeast Susceptibilities
Fairview-University Medical Center – 2009
Candida
albicans
Candida
glabrata
Candida
krusei
Candida Candida
tropicalis parapsilosis
Ampho B
99% 99% 100% 100% 100%
5-FC
99% 98%
0%
93% 99%
Fluco nazole
97% 44% O%
79% 95%
Itraco nazole
92% 26% 39% 77% 95%
Alternative Therapy
Echinocandin
Caspofungin
Micafungin
? Prophylaxis ?
Key to preventing disease appears
to be diminishing colonization and
risk of dissemination.
Chemoprophylaxis ?
Nystatin / Mycostatin (?efficacy, benign)
Fluconazole (?efficacy, ? benign)
Molecularly generated inhibitory proteins?
Fluconazole Prophylaxis
 Kaufman et al, NEJM 200;345
 IV fluconazole for the first six weeks of life in
infants with BW ≤ 1000g
Decreased levels of colonization (20 v 60%)
Fewer invasive fungal infections (0 v 20%)
Safe, no change in sensitivity patterns
 Caveats
Extremely high rate of invasive infections
TPO/Aquaphor used
Unable to determine pressure for resistance
.
Many thanks to all who
have helped with the
research!
Adhesion studies: Mary
Keane & Beth Swanson
Animal studies: MaryAlice Johnson & Robb
Garni
Microscopy: Stanley
Erlandson & Michelle
Henry-Stanley
Everything: Carol Wells