Fungal Infections in Preterm Infants 

Introduction

The prevalence of invasive fungal infections is increasing in very low birth weight (VLBW) infants (< 1500 g), as more infants born at the youngest gestational ages survive past the immediate postnatal period.^[1] These immunocompromised infants usually require invasive therapies, such as central vascular catheters and endotracheal tubes, and are exposed to broad-spectrum antibiotics and parenteral nutrition. In addition, they occasionally receive postnatal steroids. All of these factors place them at high risk for fungal infection.

Most fungal infections in preterm neonates are due to Candida species; a much smaller number of infections may be attributed Malassezia, Zygomycetes, or Aspergillus pathogens. Candida species are commensal organisms that colonize the skin and mucosal surfaces and adhere to catheter surfaces. Candida can invade the bloodstream and disseminate in these infants because of their immature immune systems, complicated by the inevitable need to compromise their developing skin and mucosal membrane barrier defenses. For these reasons, fungal infections are often difficult to eradicate in the preterm infant.

Although these immunocompromised infants are at increased risk during most of their hospital stay, they are at the highest risk of acquiring invasive fungal infections during the first weeks of life, when the most invasive therapies are performed. Although an index of suspicion must always remain high, infection control, prophylaxis, and aggressive treatment (antifungal therapy and central catheter removal) during this period have the greatest potential to improve the outcome of this population.

Pathogenesis

The pathogenesis of fungal infections in preterm infants involves adherence, colonization, and dissemination (as is shown in the image below).

Adherence and the slow-growing nature of Candida facilitate its ability to colonize and disseminate into the bloodstream and body tissues before clinical signs and symptoms of infection become apparent. Surface glycoproteins play a role in fungal adherence. One such surface adherence glycoprotein is INT1p, which binds to beta-integrins present on the endothelium and WBCs. The absence of a functional INT1 gene diminishes adherence in yeast cells but not filamentous forms.

The preterm infant is immunocompromised and frequently exposed to broad-spectrum antibacterial medications. Investigators have studied the effect of steroids and antibiotics in mice orally inoculated with Candida albicans to mimic conditions in the preterm infant.^[2] Antibiotic treatment alone led to increased Candida colonization but did not affect dissemination. When dexamethasone was added to the antibiotic regimen (presumably amplifying the inherent immunoincompetence), both colonization and dissemination increased in these animal models. Dexamethasone plus antibiotics led to an increase in the percentage of filamentous forms in the GI tract compared with antibiotics alone. In addition, introduction of C albicans strains with 2 functional copies of the INT1 gene increased the number of fungi colonizing the cecum and disseminating to extraintestinal sites.

C albicans is dimorphic, having both yeast and filamentous forms (eg, hyphae, pseudohyphae, germ tubes), and is assumed to have increased virulence in immunocompromised patients because of the filamentous forms. Filamentous forms may contribute to colonization and infection, although species that do not form filaments, such as Candida glabrata, colonize and cause invasive disease in VLBW infants.

To further examine the role of yeast and filamentous forms, researchers intravenously or orally infected antibiotic-treated and dexamethasone-treated mice using 3 strains of C albicans: (1) a wild-type strain that had both yeast cell and filamentous forms, (2) a strain with only yeast cells, and (3) a strain that was constitutively filamentous.^[3] The mortality rate was significantly greater in both the wild-type (92%) and yeast-cell (56%) strains compared with the filamentous strain alone (0%). The filamentous strain had no dissemination, and cecal colonization was significantly less than that of the other 2 strains. The wild-type strain had diffuse hyphal invasion with increased tissue necrosis compared with the yeast-cell strain. The researchers speculated that the yeast forms are critically important for adherence and tissue dissemination and that hyphal formation in the tissues contributes to parenchymal destruction.

In preterm infants, vertical and horizontal transmission leads to colonization of the skin, mucosal membranes (GI and respiratory tracts), and central vascular catheters (as is shown in the image below).

After exposure, patient factors, such as degree of prematurity, skin condition, endotracheal intubation, central vascular access, diseases (eg, necrotizing enterocolitis [NEC], focal bowel perforation [FBP]), and abdominal surgery, can contribute to fungal infection. Fungal factors that contribute to infection include the size of the inoculum and factors that favor colonization and proliferation (eg, use of broad-spectrum antibiotics, postnatal steroids, histamine type-2 [H2] antagonists, parenteral nutrition, or lipid emulsions [Malassezia species]).

Invasive infection of the blood, urine, cerebrospinal fluid (CSF), or peritoneal fluid can lead to disseminated infection, which most commonly involves the heart, kidneys, CNS, eyes, and/or liver.

Invasive fungal infection risk factors are shown in the image below.

In the very low birth weight (VLBW) infant, colonization of the skin, mucosal membranes, and/or vascular catheters commonly precedes infection. Biofilm formation on catheters inhibits the host's defense mechanisms and the penetration of antifungal agents. Infusates may also become contaminated and directly seed the bloodstream.

Risk factors for Candida colonization and sepsis are similar.^{[4, 5]} Central vascular catheters, vaginal delivery, use of third-generation cephalosporins, and high acuity are risk factors for C albicans infection. H2 antagonists, third-generation cephalosporins, central vascular catheters, parental nutrition and lipid emulsions, and high acuity are risk factors for Candida parapsilosis infection. GI disease (eg, necrotizing enterocolitis [NEC], focal bowel perforation [FBP]), exposure to fluconazole or antibiotics, prolonged hospitalization, and infection with other fungi increase the risk of sepsis due to C glabrata. GI mucosal injury, antibiotic suppression of bacterial flora, neutropenia, and parenteral nutrition increase risk of sepsis due to Candida tropicalis.

Patient risk factors and odds ratios (ORs) summarized from 2 multicenter studies are as follows:^{[6, 4]}

• Gestational age: For patients born at less than 25 weeks' gestation, the OR was 4.2. For patients born at less than 25-27 weeks' gestation, the OR was 2. For patients born at less than 32 weeks' gestation, the OR was 4.
• Antibiotics: For patients who received third-generation cephalosporin or carbapenem treatment within 7 days prior to infection, the OR was 1.8. For patients who received 2 or more antibiotics prior to infection, the OR was 3.8.
• Invasive therapies: For patients who received mechanical ventilation therapy, the OR was 10.7. For patients with a central venous catheter, the OR was 3.9.
• Intravenous nutrition: For patients who received parenteral nutrition for longer than 5 days, the OR was 2.9. For patients who received lipid emulsion longer than 7 days, the OR was 2.9.
• Medications: For patients using H2 antagonists, the OR was 2.4.

Diseases that increase risk for fungal sepsis are as follows:

• Prior bloodstream infection: Patients with prior bloodstream infection may be more susceptible to infections and/or the effect of antibiotics on skin and mucosal microflora (OR, 8.02).^[7]
• NEC: Studies have found that as many as 16.5% of VLBW infants with NEC developed candidemia at presentation or during treatment for NEC.^{[8, 9]}
• FBP: If the GI tract is colonized with Candida species, Candida peritonitis and sepsis can complicate bowel perforation in affected infants.^[10]
• GI disease: Complicated GI disease in which infants receive nothing by mouth (not enterally feed) and/or antibiotics for longer than 7 days increases the risk for fungal sepsis. Examples include gastroschisis, omphalocele, intestinal atresias, tracheoesophageal fistula, and Hirschsprung disease. Complicated GI disease increases risk in both preterm and term infants (OR, 4.57).^[7]
• Candida dermatitis: As discussed above, skin inflammation caused by Candida species may precede invasion into the bloodstream in preterm infants because of the immature host defenses. Treatment with systemic antifungal therapy in preterm infants who weigh less than 1000 g has been suggested.^{[11, 12]}

Candida species

Any Candida species may cause disease in neonates.

C albicans remains the most frequently isolated yeast species in infected neonates, followed by C parapsilosis infections, which have exponentially increased over the past decade.^{[13, 14]}C glabrata and C tropicalis have also increased in frequency, and a small percentage of infections are due to Candida lusitaniae, Candida guilliermondii, or Candida dubliniensis.

Candidal infections

• Congenital candidiasis: This may manifest with pustules, vesicles, skin abscesses, and/or an erythematous maculopapular rash of the trunk and/or extremities. These lesions occasionally lead to desquamation.^{[15, 11]} Very low birth weight (VLBW) infants with congenital Candida infection are more likely to present with severe infection, such as pneumonia and widespread dermatitis with focal areas of superficial erosion and desquamation.^{[16, 17, 12]} This infection is invasive, with dissemination in infants who weigh less than 1000 g, and should be treated with systemic antifungals for a minimum of 14 days.
• Congenital cutaneous candidiasis

  • An example of congenital cutaneous candidiasis is shown in the image below.Congenital cutaneous candidiasis in a 26-week-old infant.
  • This usually manifests as an erythematous papulopustular rash. A diffuse, burnlike, erythematous, macular dermatitis with skin exfoliation is more likely to manifest with blood, urine, or cerebrospinal fluid (CSF) involvement (75%) than with a papulopustular rash in patients of any gestational age.^[11]
  • Extremely low birth weight (ELBW) infants (< 1000 g) with congenital cutaneous candidiasis are at greater risk of developing invasive fungal infection (66%) than LBW or term infants and need to be treated with systemic antifungal therapy for a minimum of 14 days.
• Mucocutaneous candidiasis: This manifests postnatally with an erythematous papulopustular rash similar to congenital cutaneous candidiasis. As many as 70% of ELBW infants with candidal dermatitis develop bloodstream dissemination.^{[17, 18, 19]} Skin isolates include C albicans, C parapsilosis, and Curvularia, Aspergillus, and Trichosporon species. ELBW infants with mucocutaneous candidiasis in the first weeks of life should be treated with systemic antifungal therapy for a minimum of 14 days.
• Bloodstream infection: Bloodstream infection with fungal species demonstrates clinical signs and symptoms similar to bacterial sepsis. The incidence of candidemia is reported as 2-6.8% among VLBW infants.^{[20, 21, 22, 13, 23, 24, 25, 12, 4]} The incidence is higher in ELBW infants, ranging from 4-16%.^{[5, 13, 26, 27]} The incidence increases in an inverse linear pattern, from around 3% at 28 weeks' gestation to 24% at 23 weeks' gestation.^[27] Most importantly, candidemia becomes disseminated disease in approximately one third of VLBW infants. Evaluation of cardiac, renal, ophthalmologic, and central nervous systems is warranted.
• Urinary tract infection: This infection is extremely common. Evaluation of late-onset sepsis should include a urine culture obtained via sterile catheterization or suprapubic bladder aspiration. If the urine culture is positive for fungus, renal ultrasonography should be performed to detect fungus in the collecting system.^[28] Candiduria develops in approximately 2.4% of VLBW infants and 6% of ELBW infants.^[29]
• Meningitis: The reported frequency of fungal meningitis among VLBW infants is 1.6%.^[30] The true incidence is likely higher because lumbar punctures are not obtained in many VLBW infants at the onset of sepsis. Cell counts in preterm infants may not always be helpful because the results may not be abnormal in the presence of meningitis.

Disseminated infection

Patients with disseminated infection may present with several entities.^{[8, 31, 32]}

• Endocarditis: Endocarditis has been reported in 5-15% of candidemia cases.
• Renal abscess: Renal abscess is detected in 5% of patients with candidemia. It may occur in as many as 36.6% of VLBW infants with fungal urinary tract infections.
• CNS abscess/ventriculitis: CNS abscess occurs in 4% of patients with candidemia.^[31] This may be a complication in as many as one third of infants with fungal meningitis.^[33]
• Endophthalmitis: This occurs in 3-6% of patients with candidemia. Endophthalmitis occurs as multiple or single, yellow-white, raised lesions with indistinct (fluffy) or circular edges located in the posterior fundus or vitreous. It may affect one or both eyes.^[34]
• Liver abscess

  • Liver abscess occurs in 3% of patients with candidemia.
  • Liver ultrasonography is recommended when candidemia first manifests but is particularly indicated if hepatomegaly or significant change in liver enzymes results or in patients with persistent candidemia (>5 d).^{[8, 9]}
  • Hepatic candidiasis is shown in the image below.Hepatic candidiasis. Note the white pustules of Candida albicans on the surface of the liver.
• Splenic abscess: Splenic ultrasonography is recommended in patients with candidemia and should be performed if splenomegaly occurs or candidemia persists longer than 5 days.
• Cutaneous abscess: Skin abscesses should be cultured and drained, if indicated.
• Osteomyelitis: Evaluation should be considered in infants with infection who are not moving, have limited range of motion, or have swelling of an extremity.
• Septic arthritis: Septic arthritis manifests with joint swelling. It may also occur several months after antifungal treatment.^[35]
• Peritonitis: Peritonitis may occur with any bowel perforation, focal bowel perforation (FBP), and necrotizing enterocolitis (NEC). It can be a complication of any abdominal surgery.

Clinical presentation

Although, the very low birth weight (VLBW) infant with candidiasis can present with many of the nonspecific signs and symptoms associated with invasive bacterial infection, symptoms are often more subtle and indolent. Cultures should be obtained whenever sepsis is suspected. Cultures should be repeated after the initial evaluation if the infant does not clinically improve within 48 hours or if the infant's condition worsens. New-onset thrombocytopenia (< 100 X 10⁹/L [< 100 X 10³/µL, or < 100,000/µL]) is present in most cases of fungal sepsis and decrease an additional 50% to a mean platelet count of less than 50 X 10⁹/L (< 100 X 10³/µL, or < 100,000/µL).^[36] Persistent thrombocytopenia may indicate therapeutic failure.

Signs and symptoms in VLBW infants with candidemia are summarized according to incidence, as follows:^[37]

• Thrombocytopenia with WBC count of less than 100 X 10⁹/L (< 100 X 10³/µL, or < 100,000/µL) - 84%
• Immature-to-total neutrophil ratio of 0.2 or higher - 77%
• Increase in apnea and/or bradycardia - 63%
• Increase in oxygen requirement - 56%
• Increase in assisted ventilation - 52%
• Lethargy and/or hypotonia - 39%
• GI symptoms (eg, gastric aspirates, distention, bloody stools) - 30%
• Hypotension - 15%
• Glucose concentration of more than 140 mg/dL - 13%
• WBC count of more than 20 X 10⁹/L (>20 X 10³/µL, or >20,000/µL) - 12%
• Metabolic acidosis - 11%
• Absolute neutrophil count less than 1.5 X 10⁹/L (< 1500/µL) - 3%

Differential diagnosis

• Gram-positive or gram-negative sepsis
• Necrotizing enterocolitis (NEC)
• Focal bowel perforation (FBP)
• Abscess
• Intracranial hemorrhage
• Central vascular catheter thrombosis

Morbidity

Neurodevelopmental impairment (NDI) is more common in infants with fungal sepsis who weigh less than 1000 g than in extremely low birth weight (ELBW) infants without infection.^{[38, 39, 33]} NDI does not appear to be more common in infants who weigh 1000-1500 g, but a more detailed study of this subgroup is needed.

In the largest study of NDI to date in ELBW infants with infection, Stoll et al examined mental and psychomotor developmental indexes, cerebral palsy (CP), and hearing or visual impairment.^[39] Forty-one percent of infected infants (any clinical sepsis, bloodstream infection, or meningitis) and 57% of infants with fungal sepsis had at least one adverse neurodevelopmental outcome. The prevalence of adverse neurodevelopmental outcomes in infants with fungal sepsis were as follows:

• Mental developmental index of less than 70 - 34%
• Psychomotor developmental index of less than 70 - 24%
• CP - 18%
• Visual impairment - 14%
• Hearing impairment - 5%

The rate of NDI in infants with fungal sepsis did not differ significantly from those with bloodstream infection with other microorganisms (coagulase-negative staphylococcus [CONS], non-CONS, and gram-positive and gram-negative organisms).

The effect of invasive fungal infection on other morbidities is still being studied. In ELBW infants, several studies have described an association with retinopathy of prematurity. One study demonstrated an increased incidence of bronchopulmonary dysplasia.^[33]

Because of the infants' maturing immune systems, outcomes may better correlate with corrected gestational age at the time of infection. Infants who develop infection later in their hospital stay (ie, after 6 wk) may have better outcomes, but this requires further study.

Mortality

In very low birth weight infants (VLBW) infants, candidemia is associated with a mortality rate of 21-32% in multicenter studies.^{[40, 41, 42, 43]} Less than 26 weeks' gestation and a birth weight of less than 1000 g correlate with increased mortality rates (40-50%). The mortality rate is significantly higher when sepsis is due to C albicans (nearly 44%) than when it is due to C parapsilosis (15%) or other Candida species.^[41] Results vary from center to center, and several single-center studies have reported no mortality; thus, intensive care management of the septic infant and other factors may play an important role in survival.^{[24, 29]}

Follow-up

All preterm infants with infection should receive neurodevelopmental follow-up in the first few years of life and early intervention services, if needed.

Workup and evaluation

Workup and evaluation for fungal infections in preterm infants includes the following tests: blood, urine, and cerebrospinal fluid (CSF) cultures. Clearance of blood stream infection should be documented with 3 or more negative blood culture results. Each negative culture result should be obtained at least 24 hours apart.

• Laboratory studies at presentation

  • Obtain a CBC count with manual differential and platelet count.
  • To assess liver function, aspartate amino transferase (AST), alanine aminotransferase (ALT), alkaline phosphatase, and total and direct bilirubin levels should be measured. Triglyceride levels should be included because lipid metabolism is impaired during active infections. Gamma-glutamyltransferase (GGT) levels may change with bile duct inflammation and cholestasis.
  • Measure BUN and creatinine levels to assess renal function.
• Laboratory studies during the infection

  • Thrombocytopenia is extremely common and can persist until clearance of Candida infection and should be closely monitored during treatment until resolution.
  • Liver and renal function should be evaluated at the time of diagnosis (or if candidemia is persistent) because they may suggest liver or renal dissemination and the need for ultrasonography.
  • Antifungal treatment can affect serum electrolytes and the hematologic, hepatic, and renal systems and should be closely monitored during treatment.
• Screening tests for dissemination

  • Screening for end-organ dissemination should be performed at the time of diagnosis in all sepsis cases and repeated if fungemia persists for longer than 5 days. Dissemination affects length of treatment.
  • The optimal timing of surveillance is not well defined, but persistent fungemia (>5 d) is associated with increased dissemination.^{[9, 8]} Consequently, appropriate cultures and surveillance for end-organ dissemination should occur at onset and after 5-7 days if persistent fungemia is present.
  • The screening for dissemination includes the following:
    • Echocardiography
    • Renal ultrasonography
    • Head ultrasonography
    • Indirect ophthalmoscopy
    • Peritoneal cultures if laparotomy is performed to manage necrotizing enterocolitis (NEC) or focal bowel perforation (FBP)
• Laboratory testing in patients with persistent candidemia

  • In patients with persistent candidemia that lasts more than 2 days, central catheters should be removed if they still remain (central catheters should be removed when bloodstream infection is diagnosed).
  • In patients with persistent candidemia of longer than 5 days, repeat screening tests for vegetation or abscess, including the following:
    • Echocardiography and renal and head ultrasonography
    • Liver and spleen ultrasonography
    • Lumbar puncture
    • Bone scan or joint aspiration (if clinical symptoms warrant)
    • Ultrasonography or laparotomy of the abdomen (in patients with a history of abdominal surgery, NEC, or FBP, to evaluate for abscesses)
    • Ultrasonography, venography, or magnetic resonance venography (MRV) of the previous location of the catheter tip (if the patient had any vascular catheters prior to or at the time of diagnosis, to evaluate for a thrombus)

Future diagnostic tests

Investigators are studying molecular techniques to identify fungi and other microorganisms and to reveal the diagnosis more rapidly and with higher sensitivity than with blood cultures. Examples include polymerase chain reaction (PCR) and DNA microarray technology. These techniques will hopefully allow for the rapid detection of small numbers of organisms in minute volumes of blood, even after antimicrobial treatment is started.

Fungal PCR to detect the gene for 18S ribosomal RNA (rRNA) in very low birth weight (VLBW) infants has yielded promising results but requires additional study.^[44] PCR results detect a broader number of infections as they not only detect patients with candidemia but are also positive in those with Candida peritonitis and those with candiduria.^[45]

In addition, investigators are examining the role of monitoring markers of fungal disease to diagnose and evaluate responses to antifungal therapy. These markers include beta-glucan of the cell wall, anti-Candida antibodies, D-arabinitol (candidal metabolite), and fungal chitin synthase (assessed with PCR).

Microarray technology and gene chips are being studied to rapidly determine susceptibility and resistance patterns at the time of diagnosis. These will facilitate the initiation of therapy with an appropriate antifungal agent when resistance occurs and, hopefully, improve outcomes.

Prompt initiation of systemic antifungal therapy and central vascular catheter removal (in cases of sepsis) at the time of diagnosis are needed to optimize successful eradication, prevent dissemination, and improve outcomes. The image below shows antifungal mechanisms.

Antifungal therapy

Amphotericin B deoxycholate (Fungizone) remains the primary antimicrobial medication for invasive fungal infection. This drug binds to the sterol component (ergosterol) of the cell membrane, creating a pore that leads to cell death. Although test doses have preceded administration in the past based on pediatric and adult responses to the drug, this is not necessary and delays appropriate treatment. Enough safety data now support initiating administration with a starting dose of 1 mg/kg/d without need for lower test doses.^{[46, 47]} Poor outcomes may be related to the delay in reaching appropriate antifungal dosing. It should be intravenously administered once daily over 2-6 hours.

Several studies have examined lipid formulations of amphotericin B.^{[48, 47]} Lipid formulations distribute to the mononuclear phagocytic system, and doses of 5 mg/kg are required for efficacy similar to that of amphotericin B deoxycholate. One study examined doses of 5-7 mg/kg of lipid amphotericin B formulations in 36 very low birth weight (VLBW) infants and reported no adverse effects.^[48] Lipid formulations include liposomal amphotericin, amphotericin B colloidal dispersion (ABCD), and amphotericin B lipid complex (ABLC).

One special circumstance is worth discussing. In patients with urinary tract infections or renal abscesses, amphotericin B deoxycholate has higher renal penetration compared with the lipid preparations and may be more effective.

Amphotericin resistance is extremely rare. Most C lusitaniae strains are susceptible, and infections due to these organisms clear with amphotericin. However, C lusitaniae resistance has been reported. Susceptibility testing can help guide therapeutic choices.

Fluconazole, an azole that inhibits the enzyme C-14 lanosterol demethylase in the formation of ergosterol, has demonstrated similar efficacy to amphotericin B deoxycholate. Fluconazole has excellent tissue penetration.^[49] The dose is 6 mg/kg/d. Fluconazole is available as a parenteral for intravenous infusion or as a powder for oral suspension. The oral products are 100% bioavailable; therefore, the same dose may be used for oral or intravenous administration. The frequency of dosing varies with the patient's gestational and postnatal age. Resistance can occur, and susceptibility testing should be performed if resistance is a concern. Most C krusei isolates are intrinsically resistant to fluconazole. As with all azoles, the drug has many potential interactions and should not be concomitantly administered with cisapride, cotrimoxazole, cyclosporine, phenytoin, rifampin, or macrolides.

Voriconazole is an azole derived from fluconazole with a broader spectrum of antifungal activity. To date, it has not been studied in neonates. Unlike fluconazole, voriconazole is 58% protein bound and contains a cyclodextrin carrier that is cleared by the kidney and can accumulate in infants with renal insufficiency. A rare complication is torsades de pointes, and 13% of pediatric patients have reported visual disturbances (ie, photophobia, blurred vision, color changes). Until further study is completed, administration should be considered only in patients with aspergillosis. Similar to all azoles, voriconazole should not be concomitantly administered with cisapride or macrolides. Drug levels should be monitored because pharmacokinetic studies in this population are currently lacking and exact dosing may vary by gestational and postconceptional age and birth weight (4-6 mg/kg/dose every 12 h).^{[50, 51]}

A new class of antifungals is the echinocandins, which inhibit 1,3-beta-glucan synthesis of the cell wall. Caspofungin acetate (Cancidas) and micafungin sodium (Mycamine) are now approved in the United States. Caspofungin is approved to treat aspergillosis and infection with Candida species. Micafungin is approved for prophylaxis of Candida infections in patients undergoing hematopoietic stem cell transplantation and for treatment of esophageal candidiasis. Another drug in this class undergoing study is anidulafungin (Eraxis). Because these agents inhibit an enzyme, resistance and safety need to be studied along with efficacy.

Studies are underway to determine the effectiveness of echinocandins in pediatric and neonatal patients. In 2 small studies, the drug has shown promise and some efficacy, but its optimal dosage and safety needs further study. Caspofungin therapy was studied in 10 neonates with candidemia that persisted 13-49 days despite treatment with amphotericin.^[52] Nine infants survived, including 1 who had a relapse after 15 days of treatment that cleared after caspofungin was administered for another 15 days. Central venous catheters were removed as soon as blood-culture results were known. The dosage was 1 mg/kg/d for 2 days then 2 mg/kg/d. The limitations of the study included small size, lack of pharmacokinetic data, and lack of attempted combination therapy. Another study of 13 patients had a much lower success rate, with 1 mg/kg/d of caspofungin combined with other antifungals.^[53] This study was complicated by delayed catheter removal.

A randomized study to compare micafungin with amphotericin B deoxycholate is currently underway. In the future, agents such as nikkomycins, which inhibit chitin synthase of the cell wall, may be added to the antifungal armamentarium.

Central catheter removal

Central catheter removal is critical in the treatment of neonatal candidemia. The catheter should be removed upon the first positive blood culture result. Prompt removal, within 24 hours of documented positive blood culture results, is associated with lowered mortality rates, reduced end-organ dissemination, improved neurodevelopmental outcomes, and increased scores on the Bayley scale.^{[9, 8, 38]} The most recent study demonstrated decreased mortality with prompt catheter removal and candidemia (21% vs 37%, P = 0.024) and a trend toward decreased neurodevelopmental impairment (NDI) alone (45% vs 63%, P = 0.08).^[38]

Combination therapy

Amphotericin B with the addition of flucytosine (Ancobon) has been used to treat meningitis in infants who can tolerate the oral formulation of flucytosine. However, efficacy of this regimen has not been shown to be superior to that of amphotericin B alone. Flucytosine is a fluorine analog of cytosine that is converted to 5-fluorouracil, leading to inhibition of thymidylate synthetase and disruption of DNA synthesis. Flucytosine monotherapy rapidly leads to resistance, so flucytosine cannot be used alone. For meningitis in patients with CNS abscess or persistent cerebrospinal fluid (CSF) cultures, the addition of fluconazole (because of its excellent CSF penetration) is a therapeutic option.

One study examined the use of a second antifungal agent (fluconazole) in combination with amphotericin B in patients with fungal sepsis. The second agent was administered immediately upon discovery of an abscess or a positive urine culture result and also administered in patients with a persistent culture-positive infection for longer than 10 days.^[47] Infants received 1 mg/kg of amphotericin B deoxycholate (n=34) if their creatinine level was less than 1.2. If the creatinine level was more than 1.2, they received 5 mg/kg of liposomal amphotericin B (n=6) or ABCD (n=14). Patients were treated for 14 days after negative culture result or until radiographic resolution of abscess. Sterilization occurred in 36 patients (67%) with monotherapy and increased to 52 patients (96%) with polytherapy.

Another issue is the treatment of presumed invasive fungal infection in the absence of positive fungus culture results. Although postmortem diagnosis of invasive candidiasis was common in the past, 2 recent studies demonstrated that only 2.7% of cases were diagnosed at autopsy.^{[8, 42]}

Empiric treatment

In the VLBW infant, an evaluation for signs and symptoms of late-onset sepsis is typically accompanied by antibacterial treatment for at least 48 hours. Some studies have reported on the use of empiric antifungals pending culture results. Fungal cultures generally take 2-3 days to demonstrate positive results; therefore, empiric therapy may need to be longer than its antibacterial counterpart.^[54] Some authors propose that starting empiric antifungal therapy while culture results are pending may decrease the high mortality rate associated with candidemia in VLBW infants, especially those born at less than 28 weeks' gestation.^{[24, 6]} In other studies, empiric therapy may have improved outcomes in VLBW infants.^{[24, 54]} Neither approach has been treated in a randomized controlled trial.

A scoring system has been proposed that includes thrombocytopenia, a gestational age of less than 28 weeks, and broad-spectrum antibiotic treatment; however, this system has not been prospectively studied for safety or efficacy.^[6] Infants with necrotizing enterocolitis (NEC) or focal bowel perforation (FBP) are also at increased risk. Further study is needed to investigate the efficacy and safety of empiric antifungal therapy.

Most fungi are isolated from cultures within 48 hours.^[54] Therefore, some experts do not recommend empiric antifungal therapy. They recommend prompt initiation of antifungal treatment and removal of any central venous catheters upon positive culture results. In a study by Noyola et al, the start of antifungal therapy and the removal of central vascular catheters within 2 days after blood cultures were obtained was not associated with increased morbidity or mortality in episodes of fungal sepsis.^[8]

In certain circumstances, empiric antifungal therapy for 48-72 hours may be warranted in infants with negative initial culture results who still have signs and symptoms of sepsis after 48 hours of antibacterial treatment and who are recultured. In addition, the infants must have one of the following criteria:

• Thrombocytopenia (< 100 X 10⁹/L [< 100 X 10³/µL, or < 100,000/µL])
• NEC or FBP
• Weight of less than 750 g or a gestational age of less than 26 weeks

Prophylaxis

Because of the high mortality rate and NDI associated with fungal sepsis in VLBW infants, prevention with nystatin, miconazole, and fluconazole has been studied in the highest-risk patients. In randomized placebo-controlled trials, oral prophylaxis with both nystatin and miconazole decreased fungal colonization and nystatin decreased candiduria, but neither decreased candidemia.

One retrospective study of nystatin prophylaxis (administered orally or via nasogastric tube 3 times daily) reported a lower frequency of candidemia in infants who weighed less than 1500 g during the treatment period; however, few infants in the study group weighed less than 750 g.^[55] In that study, prophylaxis initiated after birth was more effective in preventing infection than treatment begun after colonization was detected. A randomized controlled trial of nystatin prophylaxis is needed in high-risk infants because other studies have reported conflicting results.^[29] To date, studies of human milk, intravenous immunoglobulins, and granulocyte-macrophage colony-stimulating factor have not demonstrated a decrease in fungal sepsis.

In a randomized controlled trial, prophylaxis with intravenous fluconazole in high-risk infants who weighed less than 1000 g and had an endotracheal tube or central vascular catheter was effective in preventing invasive fungal infection.^[26] Investigators studied prophylaxis using 3 mg/kg of intravenous fluconazole every 72 hours on days 1-14, every 48 hours on days 15-28, and then daily administration on days 29-42 for as long as 6 weeks if intravenous access is not required. No adverse effect or fungal resistance was detected during the 30-month study period. The same authors examined dosing with 3 mg/kg twice a week compared with the regimen described above and found similar efficacy.^[56]

Fluconazole is an excellent drug for prophylaxis because of its long half-life, high tissue concentration, low lipophilicity, and low protein binding. One concern with fluconazole prophylaxis is the potential for the emergence of resistance over time, and this issue is under further study.

Several observational studies have been completed to examine fluconazole prophylaxis in both extremely low birth weight (ELBW) and VLBW infants.^{[57, 58, 59, 60]} A meta-analysis of randomized and observational studies with control subjects using Mantel-Haenszel methods demonstrated an 84% reduction in invasive fungal infections among 2111 preterm infants (odds ratio [OR], 0.16; 95% confidence interval [CI], 0.08-0.31; P < .001).^{[26, 23, 57, 58, 59, 60]} For high-risk ELBW infants, the studies demonstrated an 88% reduction in invasive fungal infections (OR, 0.12; 95% CI, 0.05-0.29; P < .001).

Dosing with 3 mg/kg twice weekly is effective and limits exposure, cost, and potential adverse effects. When initiated around birth, prophylaxis should be administered for 6 weeks or less in patients with a birth weight of less than 1000 g or less than 6 weeks if intravenous access is no longer needed. For patients with a birth weight of more than 1000 g, continue prophylaxis until intravenous access is no longer needed and until adequate enteral feedings are achieved.

If antifungal prophylaxis with fluconazole is administered, using a different antifungal agent (eg, amphotericin B) for primary treatment of an invasive fungal infection is important. This ensures treatment with a susceptible antifungal agent and possibly decreases the risk of fungal resistance. Surveillance cultures for fungal resistance are recommended when fluconazole prophylaxis is completed.

Patients may have a combination of risk factors for fungemia and associated mortality. For some neonatal ICUs (NICUs) with high acuity, those with a high rate of infection, and those with large populations of ELBW infants, prophylaxis may be appropriate. For NICUs with a low incidence of candidemia, the relatively select group of infants who weigh less than 750 g and were born at less than 27 weeks' gestation are likely to be at high risk and may benefit from prophylaxis.^{[6, 27]}

Prophylaxis should be administered only while high-risk infants require intravenous access during the first 6 weeks of life. When intravenous access is no longer required, the risk for invasive fungal infection decreases because the additional risk factors for fungemia (eg, parenteral nutrition, use of lipid emulsions or broad-spectrum antibiotics, central venous access) are no longer present. After 6 weeks of life, the frequency of candidemia is reduced in preterm infants, and the disease may be associated with better survival rates.^{[13, 26]} Further study is needed in other high-risk preterm and full-term infants with complicated GI disease who require prolonged periods without enteral feedings.

Until further information is available, prophylaxis in the highest-risk patients in NICUs is still being studied for safety and optimal patient selection.^{[61, 62]} The highest-risk patients who may benefit include the following:

• All infants with a birth weight of less than 750 g
• Patients with a birth weight of 750-1000 g and 2 or more additional risk factors (eg, presence of a central venous catheter, not receiving full enteral feedings, receiving antibiotics for a bloodstream infection, receiving a third- or fourth-generation cephalosporins or carbapenems, steroid therapy)
• Preterm and full-term infants with GI disease, such as NEC, FBP, or complicated cases of gastroschisis, omphalocele, intestinal atresia, and Hirschsprung disease (eg, not receiving or expected to receive enteral feedings for >7 d).

For patients who receive fluconazole prophylaxis, the dose should be 3 mg/kg twice weekly until intravenous access is no longer required (≤ 6 wk). In patients with suspected or documented fungal infection who receive prophylaxis, amphotericin B should be administered as the initial antifungal therapy until Candida species infection and fungal susceptibility is determined.

Malassezia infections

Presentation of infection with Malassezia organisms is similar to that seen with invasive candidiasis. Infection does not routinely disseminate; therefore, end-organ surveillance is needed only if species are persistently isolated from several cultures.

Malassezia furfur is a lipid-dependent fungus that may colonize central venous catheters when lipid emulsions are infused. It can also colonize the skin and GI tract. Horizontal transmission is common. These fungi readily grow in Sabouraud medium coated with sterile olive oil. Treatment can include any one of the following measures:

• Stopping lipid infusions for 48-72 hours
• Stopping lipid infusions for 48-72 hours and administering amphotericin B for 7 days
• Stopping lipid infusion for 48-72 hours and removing the central venous catheter
• Removing a central venous catheter

Malassezia pachydermatis is not an obligate lipophilic organism. It has been reported to cause sepsis, urinary tract infection, and meningitis in very low birth weight (VLBW) infants but not in other neonates. Horizontal transmission occurs and can be prevented with handwashing.

Aspergillosis

Aspergillus infections are rare in neonates but are associated with a high morbidity and mortality rate. Aspergillus species are ubiquitous filamentous fungi (eg, molds) that form spores in the air, soil, decaying vegetation, and dust. For the neonate, transmission usually involves airborne spores. The site of entry may be the respiratory tract, skin, or central vascular catheter. Infection is usually due to exposure to contaminated dust. Invasive aspergillosis in infants can be cutaneous, pulmonary, or systemic infections, with occasional dissemination to the CNS.

Diagnosis is difficult, and a high index of suspicion is needed. Any culture that is positive for Aspergillus must be considered serious in preterm infants. Any skin or oral rashes or lesions should be cultured. Pulmonary presentation should be considered if infection is suspected with negative culture results and persistent signs despite antibacterial treatment.

One presentation involves injured skin areas that rapidly (over 24 h) progress to necrotic eschars. Diagnosis is made by demonstrating septate hyphae with 45° angles characteristic of Aspergillus species. Spores do not readily grow in blood cultures. The organism can be isolated from lung or skin samples when they are cultured on Sabouraud dextrose agar. Bronchoalveolar lavage fluid can also be microscopically examined and cultured.

Treatment has routinely involved amphotericin B, but with little success. Studies in adults have shown that newer antifungals, including voriconazole and echinocandins (eg, caspofungin or micafungin), are more effective than amphotericin B. Because the efficacy, safety, and optimal dosing of these antifungals is currently being determined, consultation with a pediatric infectious disease specialist and pharmacologist is important.

Prevention is crucial and involves filtration of NICU ventilation systems and containment of dust, especially during hospital renovation and construction. High-efficiency particulate air (HEPA) filters are excellent in clearing almost all of these fungi. NICUs should have continuous surveillance programs for mold, especially in and around windows, which can lead to good preventative measures. Ensuring that all ceiling tiles are in proper alignment is critical because all ceilings have some degree of mold.

Any construction in the surrounding area outside of the hospital can also increase the air spore count. During renovation or construction, the air should be tested for Aspergillus with the aid of infection control and microbiology services. HEPA filters should be used to prevent infection if significant levels of Aspergillus are detected.

Zygomycosis

Zygomycotic infections initially present as a black eschar at a site of local trauma or intravenous catheter insertion or infiltrate and progress to a necrotizing soft tissue infection.^{[25, 44]} Early diagnosis, treatment with amphotericin B, and surgical debridement are needed to prevent ulceration, necrosis, and rapidly fatal dissemination. A high degree of suspicion is needed, and tissue biopsy must be performed to identify the nonseptate hyphae with right-angled branches. The mortality rate associated with these infections is reported to be 61%.^{[25, 44]}