Breakthrough fungaemia in neonates and infants caused by Candida albicans and Candida parapsilosis susceptible to fluconazole in vitro

Vladimir Krcmerya,b,*, Maria Huttovaa,c, Frantisek Mateickaa, Ladislav Lahod, Ludovit Jurgaa, Adriana Ondrusovaa, Zuzana Tarekovaa, Karol Kralinskyd, Juraj Hanzene, Anna Liskovaf, Mariana Mrazovaa, Alex Saboa, Maria Pisarcikovag, Gabriela Kovacicovaa, Darina Chovancovac and Zuzana Szovenyiovaa

a Department of Paediatrics and Department of Pharmacology, University of Trnava, School of Public Health, Bratislava 812 50, Slovak Republic; b Department of Health Management, School of Health, University of Scranton, Scranton, PA, USA; c Department of Neonatology, St Cyrillus Hospital, Bratislava 833 03; d Department of Pediatrics, FD Roosvelt Hospital, 971 01 Banska Bystrica; e Department of Microbiology, HPL Laboratory of the Children's Faculty Hospital, Bratislava; f Departments of Neonatology and Microbiology, University Hospital, Nitra 997 02; g Departments of Neonatology and Microbiology,Faculty Hospital, Kosice, Slovak Republic


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Breakthrough fungaemias due to Candida albicans and Candida parapsilosis appearing during fluconazole therapy in neonates and infants were assessed for risk factors and outcome. Forty fungaemias occurred during therapy with fluconazole within a 12 year national survey and were compared with 161 cases of non-breakthrough paediatric fungaemias. The agar disc diffusion test method was used for antifungal susceptibility testing and the Vitek system for species identification. Univariate and multivariate analysis for risk factors for breakthrough fungaemia were carried out. All the fungaemias were a result of strains susceptible to fluconazole at 0.25–4 mg/L in vitro [C. albicans (85%) and C. parapsilosis (15%)]. The mean number of positive blood cultures per episode was 2.2. Sixteen children had ‘early’ breakthrough fungaemias (within 4–5 days) and 24 fungaemias appeared on day 6 and later. Mean fluconazole MICs in the ‘early’ group were 1.2, and 2.8 mg/L in the ‘late’ group (P < 0.03, t-test). However, no difference was observed in the average dose of fluconazole used in the two groups. Neonatal age, total parenteral nutrition, very low birth weight, before surgery, central or umbilical venous catheterization and artificial ventilation were all significantly related to breakthrough fungaemia in univariate analysis but only central or umbilical venous catheterization were significant in multivariate analysis. The outcome of breakthrough fungaemia was better overall and attributable mortalities in non-breakthrough fungaemia was significantly higher in comparison with breakthrough fungaemia.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Breakthrough fungaemias appear, despite antifungal prophylaxis or empirical antifungal therapy, and occur mainly in cancer patients. There have been several reports of fungaemia appearing during antifungal prophylaxis16 but none of them analysed the paediatric patient population separately. The fungaemias were usually caused by fungi other than Candida albicans (Candida krusei, Candida glabrata, Candida tropicalis, Trichosporon spp., Candida lusitaniae)711 and appeared mainly in cancer patients (particularly acute leukaemia and bone marrow transplant recipients). C. albicans was observed less frequently, as antifungals currently used for prophylaxis have shown sufficient in vitro activity against C. albicans.1 In our previous three papers we summarized our 10 year experience with breakthrough fungaemia in cancer and mixed non-cancer adult populations.5,11,12 However, none of our studies published findings for neonates and infants separately from those for adults.

Here we report 40 breakthrough fungaemias appearing during therapy and prophylaxis with fluconazole in infants and neonates within the last 12 years.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Forty neonates and infants (22 females and 18 males, 0–340 days old) with breakthrough fungaemia from six childrens' university clinics in the Slovak Republic were analysed for aetiology, risk factors and outcome. Breakthrough fungaemia was defined as at least one positive blood culture for a Candida sp. appearing after at least 3 days of onset of antifungal therapy with fluconazole (on day 4 and later), caused by an organism known to be susceptible to fluconazole in vitro (C. albicans, Candida parapsilosis, Candida guillermondii, C. tropicalis, C. lusitaniae). Fungaemias due to C. krusei and C. glabrata were not enrolled. Children were selected with the help of a protocol from a national 12 year survey from all university hospitals in Slovakia.

Fungi isolated from a routine blood culturing process with the BACTEC Signal System (BBL, Becton Dickinson, Cockeysville, MD, USA) were identified by the Vitek Jr. identification system (VITEK; bioMérieux, Hazelwood, MO, USA). Breakthrough cases (40 neonates and infants) were compared with a group of 161 children with non-breakthrough fungaemias, e.g. fungaemias that occurred when a patient was not receiving an antifungal prophylaxis or treatment.

Fluconazole susceptibility was tested with an agar disc diffusion test (DDT) carried out with 25 mg fluconazole discs according to Bille & Glauser,13 which showed close correlation13,14 (95%) with the NCCLS recommended method.15 Notwithstanding this correlation, we understand that fluconazole disc diffusion testing is not a standardized method in Europe. Inhibition zones <19 mm were interpreted as indicating susceptibility of fluconazole, 13–18 mm as susceptible dose-dependent and <=2 mm as resistant.

Quality control strains of C. albicans (ATCC 90028) and C. parapsilosis (ATCC 22019) were included with each batch of yeasts tested. The agar DDT was carried out exactly as described by Bille & Glauser13 on 4 mm deep high resolution agar medium in 90 mm Petri plates. After incubation for 48 h at 35°C, the diameter of the zone of inhibition of yeast growth around the disc was measured.

Univariate analysis was carried out with the {chi}2 test in Mantel–Haenzsel modification with Yates' correction (Fisher's exact test was used in members <5). P < 0.05 was considered statistically significant (EPI INFO computerized package manufactured by CDC, Atlanta, GA, USA). Student's t-test was used for the comparison of the mean APACHE II score, mean MICs and mean doses of fluconazole. In addition, a logistic regression model was used for multivariate analysis. Cases were enrolled to the model and only those variables that showed statistical significance in the univariate analysis with P < 0.05 were compared. They were included into the logistic regression model looking for independence while predicting breakthrough versus non-breakthrough fungaemia (STAT-ADV 23 B computerized package of the Postgraduate Medical School, Bratislava).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Fluconazole was given to children on the basis of non-responding fever and/or infection in children who were colonized with Candida spp. and had other risk factors for fungaemia, for example central venous catheters or antibiotic therapy. All children had blood cultures drawn before they were positive for Candida as all of them had suspicious or confirmed bacterial infection and 95% of them also had broad-spectrum antibiotics. Sixteen children had ‘early’ breakthrough fungaemias (within 4–5 days) and 24 ‘later’ breakthrough fungaemias (appearing on day 6 and later). The mean fluconazole MICs were 1.2 mg/L for isolates from the ‘early’ group and 2.8 mg/L for the ‘late’ group of breakthrough fungaemias (P < 0.03). However, there was no significant difference in the average dose of fluconazole used in the ‘early’ (7.2 mg/kg/day) and ‘late’ groups (9 mg/kg/day). As all 40 children had central or umbilical catheters inserted, the relationship between fluconazole MICs and catheter insertion could not be assessed. The mean number of positive blood cultures per episode was 2.2.

The MIC of fluconazole for all Candida spp. isolated was 0.25–4 mg/mL, indicating that all isolates were susceptible in vitro. In eight patients (20%) the same organism was isolated from blood cultures and catheter tips (catheter-related fungaemia). Thirty patients were cured (75%), six (15%) died due to fungal infections (fungaemia) and four (10%) died due to underlying diseases, which were relatively severe in most patients with breakthrough fungaemia (mean APACHE score was 15.9). Concerning the underlying disease, 28 of 40 cases (70%) appeared in neonates with a very low birth weight, all had central vascular catheters (in eight the catheter was also infected with the same species), 34 (85%) had ventilatory support and 24 (60%) received total parenteral nutrition. All but two received broad-spectrum antibiotic therapy for 3–22 days. All children received fluconazole for 4–17 days at doses of 6–10 mg/kg/day before the first positive blood culture for a Candida sp. appeared. In 14 children, the dose was 10 mg/kg/day, and in 26 it was 6–9 mg/kg/day in an od infusion.

The TableGo presents results of a univariate and multivariate analysis of risk factors and outcome between breakthrough and non-breakthrough fungaemias: neonatal age (P < 0.0001), very low birth weight (P < 0.0001), before surgery (P < 0.04), central or umbilical catheter insertion (P < 0.008), artificial ventilation (P < 0.0001), total parenteral nutrition (P < 0.0001), C. albicans (P < 0.002) and C. parapsilosis (P < 0.005) were significantly related to breakthrough fungaemia. Only central or umbilical catheter insertion was a significant independent risk factor, a predictor for breakthrough fungaemia in a multivariate analysis (P < 0.02, OR 3.06). Surprisingly, the outcome of breakthrough fungaemia was better than that of non-breakthrough fungaemia—overall and attributable mortality in the latter group were significantly higher (P < 0.04 and P < 0.03) in comparison with the former.


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Table. Breakthrough fungaemia in neonates and infants appearing during fluconazole therapy
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
To our knowledge, no studies on breakthrough fungaemia in neonates or children have been published. The current study reviewed breakthrough fungaemias in neonates and infants, which is a patient population generally less pre-exposed to prophylactic antifungals (in comparison with HIV and cancer patients). However, our group published three studies on breakthrough fungaemia in a cancer or non-HIV adult patient population.5,11,12 In contrast to our previously published studies, all the fungaemias in children were due to species susceptible to fluconazole in vitro (C. albicans and C. parapsilosis). All Candida isolates were inhibited by fluconazole at MICs of 0.25–4 mg/L. Only two were susceptible at 4 mg/L, and for six the MIC was 2 mg/L; therefore, the majority of isolates were highly susceptible to fluconazole. However, in cases with ‘late’ breakthrough fungaemia, occurring after day 6 of therapy, the mean MICs were slightly higher: 2.8 versus 1.2 mg/L (P < 0.03 in comparison with ‘early’ breakthrough fungaemia). This may be because of a stepwise increase in MICs when lower doses of antimicrobials are used10 and is frequently observed in bacteria.

An interesting fact concerning mortality was observed in our study: children with breakthrough fungaemia had significantly lower overall and attributable mortality than those with fungaemia that arose before antifungal therapy. The only explanation we have for this observation may be that when fungaemia broke through during treatment the patients were already receiving at least partially effective antifungal therapy.

Previous studies in cancer patients410 found at least three risk factors for the development of breakthrough fungaemia. The first risk factor is decreased in vitro susceptibility or resistance in the infecting organism. Our previous study in cancer patients12 found that c. 80% of breakthrough fungaemias were caused by fungi that were less susceptible to antifungals in vitro (non-Candida spp. and Candida spp. other than C. albicans). Breakthrough fungaemias caused by C. krusei and C. glabrata during prophylaxis with fluconazole were also described.8,9

The second risk factor is insufficient antifungal dosing. Low doses of amphotericin B resulted in 12 cases of breakthrough fungaemia due to C. albicans in at least two studies.5,10 Because several fungaemias have been seen in adults receiving fluconazole in the dose range 50–200 mg/day, higher doses for therapy of fungaemia (400–800 mg) are recommended.16

The third risk factor may be an infected foreign body resulting in persistent/breakthrough fungaemia. Three other studies6,17,18 observed breakthrough fungaemia due to organisms resistant in vitro and in patients with infected vascular catheters. Lecciones et al.17 underlined the necessity of catheter removal in fungaemia, as was recommended by the advisory panel of the Infectious Diseases Society of America in 1997.16

Analysis of the reasons for breakthrough of fungaemia in our 40 cases found that none were caused by species such as C. glabrata, C. krusei or Candida dubliniensis with reduced susceptibility to fluconazole, and none of the C. albicans isolates was resistant in vitro, but eight cases were catheter related. In the remaining 32 cases, we have no explanation for fungaemia breakthrough apart from the fluconazole dose that was administered. Twenty-six children received 6–9 mg/kg/day and 14 received 10 mg/kg/day despite a current recommended dose in neonates of 10–19 mg/kg/day of fluconazole.16,19 In addition, bd dosing in neonates has been recommended.19 The reason for underdosing of the paediatric patients in our study was possibly the absence of knowledge of published guidelines,16 and the absence of guidelines for antifungal therapy in the Slovak Republic (National Clinical and Laboratory Standards are currently in press and may appear in late 2001). The cost of fluconazole in neonates is minimal and the drug has been available for 10 years in all hospitals over the country, so we do not expect that insufficient dosing occurs for economic reasons. Therefore, in neonates and infants, at least 10 mg/kg/day of fluconazole divided into two daily doses should be administered when fungaemia appears. All foreign bodies, if present (vascular catheters, shunts), should be removed. Antifungal susceptibility testing for all isolates should be carried out (i) if organisms other than C. albicans are present, (ii) in breakthrough fungaemias and (iii) in neonates/infants not responding to appropriate doses of antifungal therapy after 3–5 days.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This study was supported by grants PL 930366 and 1/6064-3233 of the Ministry of Education and the EC.


    Notes
 
* Corresponding author. Tel/Fax: +421-2-52924308; E-mail: krcmery{at}spamba.sk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
1 . Wingard, J. R., Merz, W. G., Rinaldi, M. G., Johnson, T. R., Karp, J. E. & Saral, R. (1991). Increase in Candida krusei infection among patients with bone marrow transplantation and neutropenia treated prophylactically with fluconazole. New England Journal of Medicine 325, 1274–7.[Abstract]

2 . Wingard, J. R., Merz, W. G., Rinaldi, M. G., Miller, C. B., Karp, J. E. & Saral, R. (1993). Association of Torulopsis glabrata infections with fluconazole prophylaxis in neutropenic bone marrow transplant patients. Antimicrobial Agents and Chemotherapy 37, 1847–9.[Abstract]

3 . Hansen, J. A., Goodey, T., Martin, P. J., Appelbaum, F. A. & Anaselti, C. (1998). Bone marrow transplants from unrelated donors for patients with chronic myeloid leukemia. New England Journal of Medicine 338, 962–5.[Abstract/Free Full Text]

4 . Krupova, I., Trupl, J., Kunova, A. & Krcmery, V., Jr (1999). Bacteremia and fungemia during antimicrobial prophylaxis in cancer patients. International Journal of Antimicrobial Agents 8, 336–8.

5 . Krcmery, V., Spanik, S., Kunova, A. & Trupl, J. (1997). Breakthrough fungaemia appearing during empiric therapy with amphotericin B. Chemotherapy 43, 367–70.[ISI][Medline]

6 . Krcmery, V., Kaiserova, E., Sejnova, D. & Trupl, J. (1994). Breakthrough candidaemia to C. tropicalis. Acta Paediatrica 22, 113–5.

7 . Rex, J., Rinaldi, M. G. & Pfaller, M. A. (1995). Resistance of Candida spp. to fluconazole. Antimicrobial Agents and Chemotherapy 39, 1–8.[Free Full Text]

8 . Goodman, J. L., Winston, D. J., Greenfield, R. A., Chandrasekar, P. H., Fox, B., Kaizer, H. et al. (1992). A controlled trial of fluconazole to prevent fungal infections in patients undergoing bone marrow transplantation. New England Journal of Medicine 326, 845–51.[Abstract]

9 . Slavin, M., Bowden, R., Osborne, B., Adams, R., Levenstein, M., Feldman, A. et al. (1995). Efficiency and safety of fluconazole prophylaxis for fungal infection after marrow transplantation. Journal of Infectious Diseases 171, 1545–52.[ISI][Medline]

10 . Blomberg, E. & Rebioli, A. (1996). Failure of systemic empiric therapy with amphotericin B. Clinical Infectious Diseases 22, 462–6.[ISI][Medline]

11 . Kovacicova, G. & the Fungaemia Study Group. (2001). Breakthrough candidaemias during empirical therapy with fluconazole in non-cancer and non-HIV patients caused by in vitro-susceptible Candida spp. Report of 33 cases. Scandinavian Journal of Infectious Diseases 31, 554–8.

12 . Krcmery, V., Oravcova, E., Spanik, S., Studena-Mrazova, M., Trupl, J., Kunova, A. et al. (1998). Breakthrough fungaemia during antifungal prophylaxis or empiric therapy in 41 cancer patients receiving antineoplastic chemotherapy. Journal of Antimicrobial Chemotherapy 41, 373–80.[Abstract]

13 . Bille, J. & Glauser, M. P. (1997). Evaluation of the susceptibility of pathogenic Candida species to fluconazole. European Journal of Clinical Microbiology and Infectious Diseases 16, 924–8.[ISI][Medline]

14 . Ghannoum, M. A., Rex, H. J. & Gulgiani, J. N. (1996). Susceptibility testing of fungi: current status of correlation of in vitro data with clinical outcome. Journal of Clinical Microbiology 34, 489–95.[Abstract]

15 . National Committee for Clinical Laboratory Standards. (1997). Reference Method for Broth Dilution Antifungal Susceptibility of Yeasts: Approved Standard M27-A. NCCLS, Wayne, PA.

16 . Edwards, S. J. & the Expert Panel on Management of Candidiasis. (1997). International conference for the development of a course on the management and prevention of severe candidal infections. Clinical Infectious Diseases 25, 43–59.[ISI][Medline]

17 . Lecciones, J., Walsh, T. & Pizzo, F. (1995). Catheter associated fungemia: report of 155 cases from a single institution. Clinical Infectious Diseases 21, 735–41.

18 . Levin, S. A., Costa, I. N. S., Mussi, M., Basso, S. I., Sinto, C. & Machado, D. (1998). Candida parapsilosis fungaemia associated with inplantable central venous catheters. Diagnostic Microbiology and Infectious Diseases 30, 243–9.[ISI][Medline]

19 . Huttova, M., Filka, J., Kurak, J., Kralinsky, K. & Krcmery, V. (1998). Candida fungaemia in neonates treated with fluconazole. Pediatric Infectious Diseases Journal 17, 1112–6.

Received 20 December 2000; returned 6 March 2001; revised 23 April 2001; accepted 5 July 2000