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Invasive fungal infections

1.0 Introduction

Invasive fungal infections in Neonates appears to have increased over the past few decades this is partly due to an increase in neonates surviving preterm delivery. Invasive Candida infections have become the third most common late onset infection (>72 hour) in most neonatal intensive care units. The published incidence has ranged from 2.6% to 16.7% among very low birth weight infants (VLBW, =1500g) and from 5.5% to 20% among extremely low birth weight infants (ELBW, =1000g); with a mortality rate of around 30 %.( Chapman, 2007). Annually approximately 3000 infants develop Candidemia each year, infants who are most at risk are those born <28 weeks gestational age.

Candida exists in three morphological forms: Blastospores, Chlamydospores, and pseudomycelia or hyphae. They are small (4-6 mm), thin-walled, ovoid cells (blastospores) that reproduce by budding. There seems to be conflicting evidence in the literature that I have reviewed with Rao et al, 2005, reporting that Candida species cause the majority of fungal infections in neonates, particularly Candida albicans. Fenandez et al in 2000 reported that C.albicans was isolated from the blood or CSF in 96% (22/23) infants with Candidal meningitis. (see figure 1) However Levy et al and Kosoff et al, 1998 reported that in the past decade Candida parasilosis was the most prevalent. Candida tropicalis, Candida luistaniae and Candida parasilosis are other species that are also implicated in Neonatal intensive care outbreaks (NICU). (Fairchild et al, 2002).

1.1 Epidemiology and Pathogenesis of Infection

Epidemiological studies have demonstrated that the pattern of transmission is commonly vertical from mother to infant with C.albicans; however congenital candidiasis is uncommon and occurs due to ascending infection through the birth canal. These are normally localised to the skin but occasionally can cause disseminated disease. Studies using biotyping have demonstrated that infants with disseminated disease were infected with the same type of yeast that they were colonised. (Bendel, 2003). Horizontal transfer occurs with C.parapsilosis, this is normally due to the presence of a central catheter. (Waggoner et al, 1996). Formation of a thrombin sheath around the catheter promotes adhesion of Candida spp to the extracellular matrix. In addition to the protective effect from host immune defences provided by the thrombin sheath, Candida spp., such as C.albicans produce a biofilm that provides a barrier to drug activity and immunological attack.

The sources of candidiasis in NICU are often endogenous following colonisation of the babies with fungi. About 10% of babies become colonised in the first week of life and up to 64% by 4 weeks of hospital stay. (Hung et al, 1992). The gastrointestinal tract is usually the first site to become colonised although multiple sites can also be involved. The GI tract is an important reservoir for Candida infection. Administration of contaminated intravenous solutions, especially solutions for total parental nutrition may also cause NICU outbreaks. Spread can also occur through a colonised health worker or from baby to baby. (Robertson, 1992). Once colonisation occurs, the sequence of events which lead to serious infection include penetration of the epithelial barrier followed by haematogenous dissemination. (Bendel, 2003)

Most of the epidemiologic work in neonatal candidemia has occurred as single-centre studies. There have also been a number of retrospective evaluations of prospectively collected data for example Saiman and colleagues in 2000. These studies have suffered from similar statistical and epidemiologic pitfalls that seem to occur in all retrospective studies, which include multiple hypothesis testing and potential for residual confounding evidence despite multivariable analytic techniques.

1.2 Risk Factors

Many studies have been carried out outlining the risk factors associated with neonatal candidiasis (Saiman et al,2000 and 2001,El-Masry,2002).Development of neonatal candidiasis depends on several broad factors which include:

Gestational age <32 weeks
Apgar score <5 at 5 minutes
Prolonged umbilical or central venous catheter use
Mechanical ventilation
Total parenteral nutrition use
Prior use of intralipid
Prolonged antibiotic use (notably cephalosporins)
Corticosteroid use
Prior intubation
Use of H2 blockers
Prior fungal colonisation
Prolonged hospitalisation

A study that was carried out in 2005 by Shetty and colleagues in Baltimore city and County confirmed that a gestational age of <26 weeks and a history of abdominal surgery significantly increased the risk of candidemia as the intestinal lumen barrier is compromised. Several types of abdominal pathology are associated with infection risk; they include abdominal distension and feeding intolerance. (Fanaroff et al, 1998).

Baley et al in 1984 also reported hyperglycaemia as a risk factor as high glucose levels provide increased substrate for fungal growth and potentially lead to up regulation of genes for adhesion proteins produced by Candida spp. Hyperglycaemia can also increase production of a fungal protein that binds the iC3b component of complement in a manner similar to neutrophils, this reduces the ability of complement to aid in opsonisation of the Candida.

2.0 Neonatal Infections caused by Candida

Candida spp can cause a wide range of infections in neonates one of them being congenital cutaneous candidiasis, this is an uncommon clinical condition. The disease is acquired by vertical transmission during delivery. Infants usually present with a diffusely erythematous popular rash that develops within the first 24 hours after birth. This rash often develops as a red dermatitis and is likely to be associated with positive urine, blood and CSF cultures. (Darmstadt, et al, 2000).

Invasive fungal dermatitis is also caused by Candida. Risk factors include extreme prematurity, low birth weight, hyperglycaemia, vaginal birth and post natal steroid administration. The entry point for this infection is the skin and lesions usually develop within the first two weeks of life (Rowen, 2003).

Candidal meningitis also referred to as meningoencephalitis is found in approximately 15% of disseminated candidiasis cases Candida infections of the central nervous system (CNS) often result in granulomas, parenchymal abscesses and vasculitis. Benjamin et al, in 2003 carried out a study that suggested that meningitis cases associated with candidemia have decreased over 30 years this could be due to empirical antifungal therapy becoming more widely used and clinicians viewing positive blood cultures as CNS infections rather than contaminants.

Moylett and Caplan in 2003 reported that isolation of Candida species from an appropriately obtained CSF sample is suggestive of Candidal meningitis and should never be dismissed as a contaminant. However Arisoy and co-workers in 1994 reported on 2 neonatal patients in whom Candida species was considered a CSF contaminant, they recovered without antifungal therapy being administered. Repeat CSF samples also proved to be negative in both these patients. Given the significant morbidity and mortality that misdiagnosis can lead to it is recommended that all neonates in which Candida species are isolated from the CSF serious consideration and evaluation is given to the diagnosis.

Candidemia signs of infection in the neonate are non-specific and can include lethargy, temperature instability, apnoea, abdominal distension, feeding intolerance and respiratory distress. Candidemia in an infant is usually due to the presence of a central vascular catheter prompt removal (or replacement) and antifungal therapy are essential if candidemia is suspected. End organ damage is more common and severe in systemic fungal infections and can involve the kidneys, brain, lungs, eyes, liver, spleen, bones and joints. (Benjamin et al, 2000).

Candida spp can also cause renal and urinary tract infections (UTI's), they can range in severity from isolated candiduria to involvement of the renal parenchyma or presence of fungal debris which is also referred to as 'Fungus balls' within the renal collecting system. (Karlowicz, 2003). Urinary infections caused by Candida in neonates can present with nonspecific symptoms which can include lethargy, fever, abdominal distension, apnoea or large gastric residuals. A common clinical presentation can also be renal failure which can be due to rising serum creatinine levels with normal urine output or obstruction of the urinary tract caused by 'fungus ball'.(Eckstein and Kass 1982). If a neonate is found to have a UTI it has been found that an associated bloodstream infection is more likely. Bryant et al reported renal candidiasis in 27% of infants with candidemia, whilst Benjamin et al reported an occurrence of 33%.

Urine culture which is normally obtained by catheterisation or suprapubic aspiration is used if candiduria is suspected. Suprapubic aspiration is seen as the 'gold standard' method. Renal ultrasonography should be performed if the urine culture is found to be positive for Candida spp.

Candida spp can also cause optic complications in neonates they include Candida endophthalmitis which results from haematogenous seeding of the eye in preterm infants with candidiasis. The literature reports incidence rates of 6%-50%. (Baley et al, 1981 and Chen, 1994). Noyola et al in 2001 reported endophthalmitis in infants have all been due to Candidal species and the majority of infections have been caused by Candida albicans, however there have also been documented infections with Candida tropicalis and Candida glabrata. Reports have also associated Candida bloodstream infections with severe retinopathy of prematurity (ROP). The associated ROP may reach threshold criteria and require laser surgery. (Mittal et al, 1998). However other studies carried out by Karlowicz et al, in 2000 failed to show Candida sepsis as a major risk factor for ROP. Therefore infants with candidemia should be monitored carefully with indirect opthalmoscopy so prompt and appropriate care can be administered if required.

Endocarditis has only been documented in 5% or fewer cases of candidemia. It is thought that the low and variable reported cumulative incidence may relate to frequency of echocardiography following candidemia. Mecrow and Ladusans in 1994 reported that central vascular catheters are a risk factor for the development of this complication. However Levy et al in 2005 reported that the number of neonatal endocarditis cases caused by Candida spp will increase due to the increasing occurrence of Candida bloodstream infections. The main anatomic site differs in adults and neonates, in adults it effects the aortic valve followed by the mitral valve whereas in neonates the right atrium and the right tricuspid and pulmonic valves are involved. Fungal endocarditis differs in neonates and other age groups in terms of risk factors, underlying disease, sites of cardiac involvement, outcome and prognosis. In adults surgery along with antifungal therapy is recommended whereas neonates are often poor candidates for cardiac surgery.

3.0 DIAGNOSIS

3.1 Microbiology Culture

The current standard for diagnosis of invasive Candida infections is culture of normally sterile body fluids, for example CSF and blood. Isolation can also be carried out from non-sterile sites (stool, skin, endotracheal aspirate) but this may indicate colonization rather than true infection.

Sensitivity of blood cultures has increased due to the use of modern automated blood culture systems. In 1993 a 40-60% detection rate of invasive candidiasis was reported by Berenguer et al, however exact data to compare this with is limited as recent autopsy and comparative studies are scarce. In neonates it has been found that a single culture of 1ml of blood is as sensitive in diagnosing fungal infections as two separate peripheral blood cultures. However in contrast to bacterial pathogens, Candida spp exhibit a tissue tropism that leads to invasive disease despite a lack of organism in the bloodstream.

Berenguer et al also found that sensitivity of blood cultures varies with the number of deep organs involved in the infection. In adults <30% sensitivity was reported in single organ involvement and sensitivity improved to 80% when 4 or more organs were involved. In adults 10ml or more of blood is inoculated into blood culture bottles, however in neonates the total blood volume of a 500g baby is 40ml, therefore multiple 10ml blood culture samples are not feasible. Most neonatal blood culture bottles are inoculated with 0.5-1ml which may provide even lower sensitivity than adult patients. Schelonka and Moser in 2003, reported blood culture turned positive within 72 hours in 91% of patients with prior antifungal treatment and 97% in infants with no previous antifungal exposure.

If systemic candidiasis is suspected culture and analysis of CSF is always indicated. However Fernandez and colleagues in 2000 reported that CSF and blood cultures may be sterile in as many as 25-35% of Candidal CNS infections. Also Lenoir et al reported an unexpected case of C.albicans meningitis in an otherwise healthy 44 day old premature infant. The infant was born at 30 weeks gestation, except for mild jaundice the infant was doing well clinically and neurologically and had satisfactory weight gain. However on the 30th day of life cultures from anal and oral cavities grew Candida albicans, blood, CSF and urine cultures all proved to be negative; the baby was treated for the oral and anal C.albicans. The daily urine samples and arterial blood samples all remained negative. On the 44th day of life a neurological examination showed an abnormal encephalogram and asymmetry with decreased voltage and slow waves in the left temporal region, this finding led to a CSF sample being taken and C.albicans meningitis was suspected by direct examination of the CSF, this was confirmed after 4 days by culture. Lenoir et al concluded that the site of entry of C.albicans for this patient could have been the skin or gut, or even contamination during the first lumbar puncture at 30 days old.

Microbiological culture involves innoculating the sample onto a suitable growth medium at the Royal Liverpool University Hospital (RLUH) we use Sabouraud agar which seems to be the most widely used for the isolation of Candida. It is a general purpose medium that supports the growth of most pathogenic fungi. Sabouraud agar is not a differential medium and colonies of different pathogenic yeast species cannot be easily distinguished from each other. At RLUH if a yeast is isolated on a sabouraud plate we perform a germ tube test if this proves positive the yeast is reported as C.albicans,(see figure 4) if it is negative an API 32C or VITEK identification is carried out to confirm identification of the isolate.

A relatively new medium called CHROMagar Candida is currently being trialled at the RLUH at the moment, it claims to facilitate the isolation and presumptive identification of some clinically important yeast species. Previous studies by Odds and Bernaets in 1994 showed that the medium supported the growth of clinically isolated yeasts and of particular value was the recognition of mixtures of yeast species on a single isolation plate. A study carried out in 1998 by Willinger and Manafi also produced similar results with both studies comparing CHROMagar Candida with Sabouraud agar. CHROMagar Candida identified colonies of C.albicans, C.glabrata, C.tropicalis, and C.krusei in both studies. Advantages of CHROMagar Candida are it can provide a presumptive identification without the need for germ tube tests and it provides better detection of mixed cultures. Koehler et al in 1998 compared Chromagar Candida with corn meal-Tween 80 agar and found that C.albicans, C.tropicalis and C.glabrata could all be identified with CHROMagar, C.parapsilosis however was not always identified as the colonies appeared to have a variable appearance. The photos below illustrate the appearance of colonies on CHROMagar Candida (left hand side) compared to corn meal-Tween 80 agar (right hand side) after 48 hours incubation.

3.2 Fungal Antigen Tests

There are currently two antigens that are used to detect Candida the first of them being (1,3)ß-D Glucan which is a cell wall component found in most Candida infections causing neonatal infections, however this component has not been studied specifically in the paediatric population. (Arendrup et al,2009)

The Cand-Tec latex agglutination test (Ramco Laboratories,Houston ,Tex) was one of the first commercially available antigen detection tests, Bailey et al evaluated this kit in 1985 along with Lemieux et al in 1990, specificity and sensitivity of the assay varied in the reports and it was also found to give false-positives due to rheumatoid factor. The unknown nature and function of the target antigen impeded further development of this assay.

There are a number of Latex agglutination kits that are available which allow a species identification of C.albicans, C.dubliniensis and C.krusei within minutes. Freydiere et al carried out an evaluation of one of these kits in 1997, the kit that was tested were Bichro-latex albicans latex test. A total of 322 yeast strains that included organisms belonging to the genera Candida, Cryptococcus and Saccharomyces were tested with the monoclonal antibody-based latex kit. The results found the Bichro-latex albicans test to be 100% specific and sensitive. There have been several monoclonal antibodies proposed for the identification of C.albicans for example Brawner and Cutler, 1984 and Hopwood et al, 1986 both performed studies on a monoclonal antibody to a cell wall component of C.albicans, however the expression of their antigenic epitopes at the surface of the yeast cell was proven to be extremely variable. The Bichro-latex albicans test was the first immunological reagent that was rapid, easy to perform and gave reliable identification of C.albicans. It also has the advantage of being able to be performed directly on blood culture bottles without the need for subculture. Freydiere et al concluded that the Bio-latex albicans test may be used in the clinical laboratory for rapid identification of C.albicans especially from blood culture bottles.

The second antigen that is used for the detection of systemic candidiasis is Mannan antigenemia, this was suggested in 1979 by Weiner and Coats-Stephen, however it wasn't until the 1990's that this phenomenom was looked at in greater detail as previous studies had been hampered by the use of insensitive methods that resulted in poor sensitivity/specificity. (Repentigny, 1992). Improvement in the immunological detection of mannan involved the use of immune complex dissociation by heating serum before the test was performed, the use of a more sensitive test, and the use of monoclonal antibodies that react with particular epitopes. (Herent et al,1992). Two assays that use this monoclonal antibody are Pastorex Candida latex agglutination test (Bio-Rad) and Platelia Candida antigen test (Bio-Rad), this is a double sandwich enzyme immunoassay. The specificities of both tests were proven to be similar by Sendid et al in 1999, however the Platelia Candida antigen test was found to be more sensisitve. This is thought to be due to the rapid clearance of antigen from patients' sera especially when only a single sample is tested. Sendid et al, suggested that multiple serum samples were processed to improve the sensitivity for detection of the Candida mannan antigen.

Mannan antigen as a surrogate marker for the detection of invasive candidiasis was investigated by Oliveri et al, in 2008 using the Platelia Candida Elisa in a neonatal intensive care setting. The sensitivity and specificity of the assay were 94.4% and 94.2%. They suggested that the inclusion of regular serological surveillance for mannan antigen could complement blood cultures for the early detection of invasive candidiasis.

3.3 Fungal Metabolites

C.albicans, C.parapsilosis and C.tropicalis all produce the metabolite D-arabinitol, which is present in urine and serum and is detected using gas chromatography or enzyme chromogenic assays.. In paediatric patients with invasive candidiasis, the D-arabinitol (DA) and L-arabinitol (LA) have been found to be elevated in urine. (Yeo and Wong,2002). A study by Walsh et al in 1995 found an elevated urinary DA/LA ratio in six infants that were infected with C.albicans, five of these infants also had levels taken during therapy which demonstrated a decrease in DA/LA ratio. Disadvantages of DA/LA detection include low specificity as colonised patients are known to have elevated DA/LA levels and C.glabrata and C.krusei produce little or no D-arabinitol.

3.4 Molecular Tests

PCR amplification of a genetic region common to C.albicans, C.parapsilosis and other Candida species has been the target of major interest because of the possibility of detecting low amounts of DNA and non-viable fungi. Arendrup et al, 2009 referred to 3 common challenging problems (i) the high degree of homology between human and fungal DNA; (ii) the effective release of DNA from the fungi despite the fungal cell wall; and (iii) the risk of contamination of samples, reagents or equipment. Only one commercial PCR blood test has been marketed that detects the five most common Candida spp this was reviewed by Louie et al in 2008. At the moment no data is available on the performance of this test in the neonatal setting. Tirodker et al, in 2003 performed a study on detection of fungemia by PCR in critically ill neonates and children, Candida was diagnosed in nine out of nine cases and in seven of 13 additional PCR positive cases there were other signs of invasive candidiasis for example positive blood cultures which suggests that PCR may be a useful tool in the addition to culture in the high-risk NICU setting. A number of studies have been carried out on paediatric patients with onco-haematological diseases for example Bialek et al, 2002 found a high rate of false-positives and El-Mahal-lawy et al in 2006 found a sensitivity of 75%. These studies show that PCR has got potential in the future but further investigation needs to be carried out especially in the neonatal population.

4.0 TREATMENT

4.1 Empirical Therapy

There are no dedicated guidelines for empirical therapy in neonates, guidelines however do exist for invasive fungal infections in adults (Pappas et al,2009) which comment on the treatment of fungal infections in children. Friedman et al, in 2000 stated that antifungal therapy initiated 3 days earlier to the first positive culture in infected ELBW infants has been associated with improved mortality and morbidity.

Empirical therapy is initiated in adults based on a predictive model of risk, there is no validated model for neonates, in 2003 Benjamin et al performed a multicentre cohort study to develop a similar model, they examined risk factors and subsequent candidiasis from blood cultures taken from 6172 premature infants. The study concluded that thrombocytopenia, gestational age < 25 or 25-27 weeks and cephalosporin/carbapenem use 7 days prior to blood culture were independently associated with candidiasis when controlling other variables in the analysis. Scores were assigned to each variable and a combined score of =2 was found to be 85% sensitive and 47% specific in predicting candidemia. It was concluded that data from this study should be interpreted with caution and further multicentre studies should be carried out before guidelines for empirical therapy in neonates can be recommended.

4.2 Antifungal Drugs

4.2.1 Ampthotericin B

The standard therapy for invasive fungal infections has traditionally been Amphotericin B, the drug binds to ergosterol in the fungal cell membrane which leads to cell leakage and death. It is generally well tolerated in full and preterm infants as reported by Bliss et al in 2003, patients have to be monitored closely as side effects can include renal insufficiency, however this was reported to be reversible by Kingo et al in 1997. Measurement of renal function and serum electrolytes whilst on therapy is important.

Pharmokinetic studies in neonates by Bayley et al in 1990 have demonstrated therapeutic levels of amphotericin using an intravenous dosing regime of 0.5 to 1mg/kg/d. There are no guidelines on the duration of treatment but a survey of neonatologists revealed that most neonates are treated for 14 days after the infected body fluid becomes sterile. (Rowen,1998).

4.2.2 Flucytosine

Flucytosine is a fluorine analog of cytosine that causes disruption of DNA synthesis. It is sometimes given with ampthotericin B for infections of the central nervous system. It is not recommended as a single agent as Candida spp can develop resistance as reported by Fanaroff and Martin in 2002. Flucytosine has been used in the neonatal population to treat patients with persistent Candida meningitis. (Francis and Walsh,1992). Smego et al in 1984 conducted a study that included 17 cases of which 11 patients were <12months old, improvement was seen in 15 patients using a combined ampthotericin B and flucytosine combination. In contrast to this study Baley et al, in 1990 reported that amphotericin B used as a monotherapy showed good penetration into the CSF of neonates and efficacy was demonstrated in neonatal Candida meningitis.

4.2.3 Triazoles

Fluconazole and Itraconazole are first generation triazoles that act by inhibiting fungal- 14- -sterol demethylase an enzyme that is necessary for the production of ergosterol, a major component of the cell membrane. The triazoles are fungistatic they inhibit cell growth but are not fungicidal, they may interfere with the pharmokinetics of other medication and may be associated with hepatotoxicity.

Itraconazole has not been widely investigated for the treatment of invasive Candida infections, Fluconazole is currently the main alternative to amphotericin B in the treatment of neonates, (Chapman,2007) it can be given orally or intravenously. Saxen et al, in 1993 reported that fluconazole is ideal for fungal urinary infections as it is concentrated and is excreted unchanged in the urine. Huttova et al, in 1998 carried out a prospective study of 40 neonates that had been treated with fluconazole, 5% of them had transient increases in liver function tests after 3 weeks of therapy. Other side effects included thrombocytopenia, anaemia and Stevens-Johnson syndrome.

A second generation triazole called Voriconazole has been developed with the goal of expanding the spectrum of activity. Chapman, 2007 reported that the drug had improved activity against non-albicans species, for example C.krusei and C.glabrata that are becoming increasingly resistant to fluconazole. No pharmokinetic studies have been carried out as yet on the neonatal population. However, Muldrew et al, in 2005 reported on a case of a premature infant that was treated with voriconazole in combination with lipid amphotericin B for persistent disseminated fluconazole-resistant C.albicans infection. The infection cleared successfully whereas previous treatment with monotherapy amphotericin B had failed. Voriconazole could therefore prove effective in the treatment of resistant Candida infections.

4.2.4 Echinocandins

Echinocandins (caspofungin,micafungin,anidulafungin) are the newest class of anti-fungal drugs, they target the cell wall by inhibition of (1,3)-ß-D-glucan synthase enzyme complex that forms glucan polymers which are a major component of the fungal cell wall. Toxicity is minimised as there is no mammalian equivalent to the cell wall. Pfaller et al,2003 reported that resistance to these drugs is uncommon even in isolates that are resistant to amphotericin B and fluconazole. A report of 10 neonates with candidiasis treated with caspofungin after failure of therapy with amphotericin B demonstrated survival in 9/10 (90%) of the neonates with no adverse clinical events observed. (Odio et al,2004). An international non comparitive trial in 2005 by Ostrosky et al included 11 neonates, ten out of the 11 had failed prior antifungal therapy, however 8 out of 11 had a complete response with micafungin used as a primary or combination treatment therapy.

Anidulafungin a third echinocandin has yet to be tested in the neonatal population, but was shown to be well tolerated in the paediatric population. (Benjamin et al,2004).

The results of studies carried out look promising for the use of echinocandins in the treatment of antifungal resistant isolates, however further investigation of the pharmokinetics is required before these drugs can be used widely in the neonatal population.

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