CANDIDA-RELATED MORBIDITY IN PRETERM NEONATES
Fungi (particularly Candida species) have become important causative agents of infections in preterm neonates. The various Candida species are now the third most frequent cause of late-onset sepsis in very low birth weight (VLBW) preterm neonates (birth weight <1500 g) in neonatal intensive care units (NICU) (1–3). The estimated incidence of systemic fungal infection (SFI) is between 1.6% and 9% and reaches 15% in extremely low birth weight (ELBW) neonates (birth weight <1000 g). Neonatal systemic (or invasive) fungal infections are associated with high severity, substantial morbidity, and neurodevelopmental impairment. Candida species–attributable mortality ranges from 25% to 55%, and according to recent reports, SFIs produce as many as 73% of deaths or neurodevelopmental impairments at 18 months of life (4).
CANDIDA COLONIZATION AND GUT RESERVOIR IN PRETERM NEONATES
Colonization by Candida species, detected from all of the sites that can be monitored with serial cultures, is the condition most frequently associated with the development of SFI (5,6). A thorough search will nearly always disclose previous colonization. Approximately 60% of VLBW neonates are thought to be colonized during their first month in an NICU, with 85% of them acquiring colonization horizontally (7). Many of the colonized neonates are eventually infected. Baley et al (8) calculated that among 100 VLBW neonates, 33% were colonized and 7 of them experienced progression to SFI.
Examination of the digestive tract (cultures from pharyngeal swabs, gastric aspirate, feces, and rectal swabs) shows it to be the colonization site with the greatest predictivity for subsequent dissemination. Its colonization at various levels is a well-known risk factor for dissemination and SFI in preterm neonates, and rectal swab positivity is seen as an expression of colonization tout-court(9).
Preterm neonates in intensive care are, in any event, at high risk of intestinal disorders with proliferation of a pathogenic microflora (including fungi). In the preterm infant, the methods used in neonatal intensive care, such as treatment with antibiotics, total parenteral nutrition, or nursing in incubators, may delay or impair the intestinal colonization process. Preterm neonates are thus slower to acquire commensals such as bifidobacteria and are more susceptible to pathogenic colonization (10). In addition, their oral feeding cannot always be started and maintained. It is also known that the intestine of the premature infant has poor motility and is predisposed to bacterial overgrowth (11). The digestive tract is thus regarded as the most important reservoir and site for colonization by all kinds of pathogens and subsequent sepsis, including fungal.
CURRENT PROPHYLACTIC STRATEGIES
Research has focused on devising an optimum prophylactic strategy to reduce fungal colonization rates and thereby reduce the incidence of SFI (12). Antifungal systemic drugs, mostly fluconazole, have been tested with promising results (9,13–20). In all studies of fluconazole, it was consistently found to decrease the incidence rates of Candida species colonization in preterm neonates in NICU. However, it must be admitted that what should be done and how, and for which groups of preterm neonates, is far from clear. Some by no means secondary questions, in fact, are still open, and further prospective clinical investigations are needed. Some workers believe that the onset of adverse reactions to fluconazole and the selection of drug-resistant strains are subjects that remain to be clarified. The cost:benefit ratio of fluconazole prophylaxis and the type of population at risk for which it could best be used are other doubtful issues, along with the best outcome parameters for assessment of its effectiveness: colonization, incidence of SFI, the colonization-to-infection switch rate, mortality, length of hospital stay, and sequelae. For all of these reasons, the use of antifungal agents still raises questions and has not yet been established as the standard of care (21–24). Probiotics could provide an innovative and less invasive approach because they modify the bowel flora by colonizing the gastrointestinal tract.
MIGHT PROBIOTICS WORK?
The background for the efficacy of probiotics in preterm neonates comes from the observation that premature infants have fewer episodes of late-onset sepsis, necrotizing enterocolitis, diarrhea, and urinary tract infection, and they need less antibiotic therapy when fed their own mothers' milk in comparison with similar infants who are fed formula (25–27). Breast-feeding, in fact, allows close contact between the mother and her infant, and mammary milk has biofactors that help establish bifidobacteria and lactobacilli (the so-called “bifidus flora”) as predominant intestinal bacteria (28). Not only maternal milk nutrients but also probiotic bacteria and/or prebiotics (nutritional substrates that promote the growth of probiotic bacteria) through their stimulation of a normal commensal microflora may also play an important role in the regulation of interleukin-8 and neutrophil infiltration into the intestine development, in enhancement of the barrier function, and in the composition of the neonatal gut microbiota (29–36).
The birth of an extremely preterm infant interrupts early nutrition with maternal milk that occurs when the neonate is healthy and at term. Preterm infants cannot have close maternal contact, cannot leave the hospital shortly after birth, are often subject to delayed breast-feeding, and thus acquire intestinal flora from the NICU. In some studies, probiotics have been demonstrated to be effective in reducing the overgrowth of pathogens in the preterm neonate's gut and reducing the incidence of necrotizing enterocolitis and bacterial sepsis (37–47). In the light of these findings, may we hypothesize any relationships between probiotics and Candida species in the preterm neonate in the NICU?
In humans, Candida species is a commensal microorganism, and the enteric reservoir is a major site of fungal colonization inasmuch as Candida species adhere to gut. As mentioned above, colonization by Candida species is the most important predictor of invasive fungal disease, and the gastrointestinal tract is the site of colonization that is most frequently implicated in subsequent systemic dissemination. It is thus obvious that strategies affecting the fungal enteric reservoir may presumably lead to the avoidance of many potential systemic disseminations of fungi from the gut.
Studies in mice have shown that probiotics may interfere with fungi in the enteric reservoir. Lactobacillus casei subsp Rhamnosus GG (LGG) reduces both enteric colonization and SFI (48,49). Various mechanisms could be responsible for this protection. In the aforementioned studies, LGG was found to act at several levels simultaneously to reduce both Candida species colonization and systemic infection: exclusion of fungi by competition, prevention of adhesion, reduction of their ability to colonize the mucosa through an enhanced IgA response, changes in mucosal permeability enhancing the barrier effect, and immunomodulation with modification of the immune response to their products.
Also, in humans, it is feasible that colonization by benign flora such as LGG can afford protection against the proliferation of fungi in the gastrointestinal tract. An in vivo model of these interactions is the use of probiotics to prevent recurrences of vulvovaginal candidiasis (49–53). In vitro studies have shown that lactobacilli can inhibit the growth of Candida albicans and/or its adherence on the vaginal epithelium. The results of some clinical trials support the effectiveness of lactobacilli, among them Lactobacillus rhamnosus GG, administered either orally or intravaginally, in colonizing the vagina and/or preventing the colonization and infection of the vagina by C albicans. Because of this promising evidence, some practitioners use probiotics empirically for the prevention of such diseases.
A PILOT STUDY
The first evidence of a positive effect of probiotics in the prevention of fungal enteric colonization in human neonates comes from a prospective, randomized, double-blind clinical trial performed in 2006 in Italy (54). In this study, 80 preterm VLBW infants were randomly assigned during the first 3 days of life to receive either LGG at a dosage of 6 × 109 colony-forming units with human milk (LGG group, n = 39) or human milk alone (control group, n = 41) for 6 weeks or until discharge if earlier than 6 weeks. The neonates were all older than 3 days and had started oral feeding with human milk (either from their own mothers or from donors) before randomization. None had baseline fungal colonization at enrollment, and they were not undergoing any other form of antifungal prophylaxis. Enteric fungal colonization was monitored by surveillance (oropharyngeal, stool, gastric aspirate, and rectal cultures) performed weekly.
The primary efficacy endpoint was the overall incidence of enteric fungal colonization, expressed as incidence of colonization (at least 1 positive result from the cultures) during the first 6 weeks of stay in the NICU. The secondary outcomes were the intensity of fungal colonization; the ratio of non-albicans to albicans Candida species in the 2 groups; the incidence of bacterial sepsis (caused by both gram-positive and gram-negative microorganisms); the incidence of sepsis by LGG; necrotizing enterocolitis, both surgical and stage III; and the incidence of SFI. The results showed that the total number of positive cultures, the mean number of fungal isolates obtained from each patient, and the mean number of fungal isolates obtained from each colonized patient were significantly lower in the LGG group (P = 0.009, P = 0.005, and P = 0.005, respectively). The overall gastrointestinal colonization rate was 23.1% in the infants receiving LGG and 48.8% in those with placebo (P = 0.01). Clustering the analysis for weight range, the reduction of overall gastrointestinal colonization was significant in neonates with birth weight 1001 to 1500 g (from 42.9% to 14.8%; P = 0.02) but not in neonates with birth weight below 1000 g (P = 0.27).
The distribution of fungal species, in particular the ratio of C albicans to C non-albicans, was similar, with C albicans representing 78% of the isolates in the LGG group and 70% in the placebo group (P = 0.32). In no case was LGG isolated from blood cultures or other specimens. Similarly, LGG was never the suspected causal agent of sepsis that was not microbiologically documented in any neonate studied. No adverse effects occurred. The incidence of SFI before discharge was slightly, but not significantly, lower in the LGG group (10.3% vs 12.2%; P = 0.53), as was the incidence of surgical necrotizing enterocolitis (0% vs 2.5%; P = 0.51).
The 2-fold reduction in gut colonization by fungi in the VLBW neonates given LGG in this study is in line with most of the studies performed in preterm infants given a prophylactic antifungal drug, thus allowing the authors to consider LGG supplementation as a promising minimally invasive prophylactic strategy in such settings. The authors remark that these results were obtained with no adverse effect, particularly with no onset of sepsis caused by LGG. Moreover, the results did not seem to be attributable to the type of feeding (human milk might positively influence the gut microflora), inasmuch as human milk was the main nutritional policy in both randomized groups. The relative frequencies of Candida subspecies were unmodified in the 2 groups, meaning that the beneficial effect of LGG was unspecific (ie, evenly distributed over all fungal colonies).
In this study, the infants weighing 1001 to 1500 g benefited more from the use of probiotics than did those weighing less than 1000 g: the paradox that a greater per-kilogram dosage had a lesser effect was possibly due to the even greater increase in the overall risk for colonization with the decrease in birth weight. We suggest that combinations of drugs and probiotics may be beneficial in all VLBW neonates, with greater reliance on probiotics in the bigger neonates and vice versa in the smaller ones. Such combinations should allow a reduction in the dosage of drugs needed to suppress colonization. Clearly, the optimal combination in each case should not be a strict reflection of the current separation between infants with birth weight below 1000 g and those weighing between 1001 and 1500 g, and may be adjusted as required; for example, more reliance could be placed on antifungal drugs in NICUs with higher frequencies of colonization and invasive fungal disease.
It now remains to be determined, in a sufficiently large population, whether the decrease in gastrointestinal fungal colonization produced by LGG also results in a corresponding reduction of SFI, as may logically be expected. The pilot study by Manzoni et al (54) was designed and powered to test the effect of LGG on colonization, not on SFI. In this study, a slight decrease in the SFI rates occurred in the LGG group, but it was not significant. LGG dosages higher than those used in that study may enhance the direct positive effect of the probiotic on immunity in the infants who receive lesser benefit. However, it is noteworthy that the per-kilogram dosages used were considerably higher than those recommended by the producing company, and the possibility that the probiotic will be carried indefinitely should raise a safety concern.
In conclusion, although the results on colonization are encouraging, the hypothesis that LGG or other probiotics may be effective in preventing SFI must be tested in ad hoc studies. These studies must be not only adequately powered (a few hundred randomized VLBW neonates are needed) but also dose finding, to better address the question whether the impact of LGG on invasive disease may depend on changes in the administered dose.
Lactoferrin (LF) is a protein of the wide array of biologically active components of human milk. It acts in vivo together with many other probiotics and prebiotics or symbiotic nutrients, inhibiting the growth of pathogenic bacteria and viruses in the enteric tract. Recent data from basic research suggest that the combination of LGG and LF could boost the defenses of an immature intestine. Sherman et al (55), in fact, has shown that LGG plus LF administered orally limited the intestinal growth of Escherichia coli in newborn rats more effectively than did LGG alone, and without increasing the number of LGG colonies. Administration of LF has thus been proposed as a way of reducing the incidence of necrotizing enterocolitis and sepsis originating in the intestine (56). Moreover, Tanida et al (57) recently demonstrated a synergic antifungal effect of several peptides, including LF, used in combination with antifungal drugs.
On the basis of these background data, the Neonatal Fungal Infections Task Force of the Collaborative Neonatal Infections Study Group (affiliated with the Italian Society of Neonatology) has designed a blind, multicenter, international, randomized study to test the effectiveness of an antifungal prophylaxis with LGG plus LF (Dicoflor 60 + LF100; Dicofarm, Rome, Italy) in VLBW neonates in the NICU. Thanks to the large number of centers involved in recruiting (15 to 20 NICUs in Italy and the United States, with an estimated 500 to 800 VLBW infants enrolled), the primary endpoint of this study will be not only the incidence of intestinal fungal colonization but also the incidence of SFI, of bacterial sepsis, and of necrotizing enterocolitis. Neonates will be randomized in 3 groups (LGG + LF, LF alone, placebo), and oral administration of the nutrients in the study will last 6 weeks, regardless of the type of milk used. It is hoped that the results of this study will shed a clearer light on the effectiveness of the strategy based on probiotic oral supplementation in the prevention of fungal-related morbidity in preterm infants.
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