Blood culture procedures and diagnosis of Malassezia furfur bloodstream infections: Strength and weakness

Blood culture procedures and diagnosis of Malassezia furfur bloodstream infections: Strength and... Abstract The occurrence of Malassezia spp. bloodstream infections (BSIs) in neonatal intensive care unit was evaluated by using pediatric Isolator, BacT/Alert systems and central venous catheter (CVC) culture. The efficacy of BacT/Alert system in detecting Malassezia was assessed by conventional procedures, culturing 1 ml of bottle content before incubation and by studying the survival of Malassezia spp. strains in BacT/Alert bottles. Of the 492 neonates enrolled, blood was collected by pediatric Isolator (290 patients; group I) or by BacT/Alert bottles (202 patients; group II). The survival of Malassezia furfur and Malassezia pachydermatis in BacT/Alert bottles was evaluated by culturing the inoculum suspension (from 106 to 10 colony-forming units, cfu/ml) and assessing the cfu/ml for 15 days. In total, 15 Malassezia BSIs were detected, of which six (2.1%) from both blood and CVC culture in Dixon agar (DixA) in patients belong to group I (blood collected by paediatric Isolator tube) and nine (4.4%) only from CVC culture in DixA in patients of group II (blood collected by BacT/Alert bottle). Only one patient (0.5%) from group II scored positive for M. furfur also by culturing in DixA 1 ml blood content of BacT/Alert bottle before incubation in BacT/Alert system.M. furfur population size in BacT/Alert bottles decreased during the incubation time, whereas that of M. pachydermatis increased. The BacT/Alert system detected M. pachydermatis even at very low concentration (i.e., 10 cfu/ml) but not any positive blood culture for M. furfur. For a correct diagnosis of Malassezia furfur BSI, the blood should be culture in lipid-enriched fungal medium, and the BacT/Alert system implemented by adding lipid substrates to increase the method sensibility. Finally, CVC cultures on lipid-supplemented media may be proposed as a routine procedure to diagnose the Malassezia fungemia. Malassezia furfur, bloodstream infections, diagnosis, BacT/Alert system Introduction Malassezia fungemia by Malassezia pachydermatis, Malassezia furfur and Malassezia sympodialis1–6 may be associated with nosocomial outbreaks in immunocompromised patients.7 Though occasionally reported, this infection has attracted the attention of scientists for the inherent difficulties in its diagnosis and treatment.8–14 A diagnostic survey on bloodstream infections (BSIs) resulted in a higher prevalence of M. furfur (2.1%) than Candida spp. (1.4%) suggesting that Malassezia BSIs might be underestimated, due to improper diagnosis.1 BSIs caused by lipid-dependent Malassezia species are detected either using systems pre-enriched with lipid substrates15,16 or by direct isolation using the Isolator system (DuPont Co., Wilmington, DE, USA).1,17,18 Although the automated blood culture system BacT/Alert (bioMérieux, Marcy l’Etoile, France) is worldwide used in many laboratories for the detection of bacterial and fungal pathogens, its failure to detect M. furfur has been reported through a retrospective clinical review.17 In this study, the occurrence of Malassezia spp. BSIs in a neonatal intensive care unit (NICU) has been evaluated by using two different blood culture procedures (i.e., pediatric Isolator and BacT/Alert systems) and by culturing blood and central venous catheter (CVC) tips onto specific medium (i.e., Dixon agar). The survival of Malassezia spp. strains in BacT/Alert bottles was also evaluated, and the efficacy of BacT/Alert system in detecting positive samples was correlated with the Malassezia spp. population size. Methods Human patients and Malassezia spp. BSIs diagnosis Blood and CVC were collected from 492 neonates, of which 290 (group I) enrolled during a 1-year survey on yeasts fungemia1 conducted from July 2011 to July 2012 and 202 (group II) enrolled from January 2016 to January 2017. All patients displayed at least one of the following clinical signs: respiratory distress, elevated or depressed leukocyte count, increased C-reactive protein levels (CRP ≥5 mg/l), abdominal distension, or thrombocytopenia. Blood samples were collected by lysis centrifugation tubes (pediatric Isolator 1.5-ml system, DuPont Co., Wilmington, DE, USA) from 290 patients of group I and by BacT/Alert pediatric (PF) bottles from patients of group II. For Malassezia spp. BSIs diagnosis, pediatric Isolator tubes were processed according to the manufacturer's recommendations by culturing 0.35 ml of Isolator tube content onto Dixon agar containing 0.5% chloramphenicol (DixA). BacT/Alert PF bottles were processed according to the manufacturer's recommendations with some modifications. In particular, before bottle incubation (i.e., T0), 1 ml of bottle content (i.e., sampled blood + culture medium) was aseptically collected, centrifuged, and the pellet inoculated onto DixA, then incubated at 32°C for 3 days. Bottles were incubated at 35°C into the BacT/Alert system and monitored for up to 15 days for fungal growth. When a positive culture was indicated by the system, 0.2 ml of BacT/Alert bottle content was subcultured onto DixA. CVCs from both groups I and II were cultured by rolling the CVC tip19 onto DixA immediately after their sampling and the inoculated fungal medium was incubated at 32°C for 15 days and daily examined, as routine diagnostic method.20 Following preliminary yeast identification using an automated system (Vitek2; bioMérieux, Craponne, France), Malassezia spp. isolates were characterized based on morphological and biochemical features (e.g., catalase reaction, ability to assimilate Tweens (i.e., 20, 40, 60, 80), growth on PEG-35 castor oil, and β-glucosidase activity).20Malassezia spp. isolates were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) as previously reported.21 Because of the observational nature of this study a written informed consent from the parents or guardian of patients was obtained according to the current Italian legislation (Art. 81- D.Lgs.vo n.196/2003). Efficacy of the BacT/Alert system in detecting Malassezia BSI The efficacy of the BacT/Alert system in detecting Malassezia BSI was assessed testing 15 Malassezia strains (i.e., n. 7 from blood and n. 8 from CVC) from 15 patients, and one M. pachydermatis (ITS-1 GenBank Accession number DQ915503) from the skin of a dog with dermatitis, used as control being the only non–lipid-dependent species within the genus.22 For each strain, an inoculum suspension (IS), corresponding to 1–5 × 105 colony-forming units (cfu/ml) in 5 ml of sterile distilled water, was prepared using 5-day-old colonies and 1 ml transferred into a 20-ml culture medium BacT/Alert PF bottle containing 3 ml of blood obtained from healthy volunteers. Two bottles for each test were processed either by automated BacT/Alert system or by quantitative plate count of cfu/ml on DixA. For the control cultures, 1 ml of the IS was transferred into 23 ml of Dixon broth, incubated at 35°C in a shaking incubator and monitored for the Malassezia population size under the same conditions. The bottles were incubated in the BacT/Alert automated system at 35°C for 15 days, and cfu/ml was evaluated before incubation (i.e., T0), after 1 day and every 2 days up to 15 days of incubation (i.e., from T1 to T15). An IS corresponding to 1–5 × 108 cfu/ml and five consecutives 1:10 dilutions (i.e., D1-D5) in sterile distillated water for both M. furfur (GenBank Accession n. KF682274), and M. pachydermatis (GenBank Accession number DQ915503) strains were performed to assess the sensitivity of the BacT/Alert system in detecting Malassezia BSI in relationship to Malassezia population size. In particular, 1 ml of the IS and of each dilution was transferred into a 20-ml culture medium BacT/Alert PF bottle containing 3 ml of blood and processed as above. The experiment was also performed using BacT/Alert FA bottles (i.e., those employed for adult patients) under the same conditions. Statistical analysis All the experiments were performed in duplicate and repeated twice on different days. The results were plotted and reported as mean values of Log10 cfu/ml. The population size of Malassezia spp. in the control medium (i.e., Dixon broth) and in the BacT/Alert bottles, at each time point, were compared by using the t test for paired samples. Statistical significance was set at P ≤ 0.05. Results Of the 15 Malassezia BSI-positive samples (3.1%), M. furfur was isolated from blood and CVC cultures in six patients (2.1%) from group I (blood in pediatric Isolator tube) and from CVC cultures in nine patients (4.4%) from group II (blood in BacT/Alert bottle), of which one was also positive, at T0 for 1 ml blood culture from the BacT/Alert bottle (Table 1). The BacT/Alert instrument gave no positive signal. All Malassezia strains were identified by biochemical features and MALDI-TOF MS as M. furfur (data not shown). The M. furfur strains isolated from blood and CVC culture in six patients from group I showed the same MALDI-TOF MS profile.21 Table 1. Number and percentage of infants from Group I (blood collected by lysis centrifugation tube) and Group II (blood collected by BacT/Alert pediatric - PF bottles) scored positive for Malassezia bloodstream infections by using blood and central venous catheter (CVC) cultures.   Positive /Total (%)  Diagnosis  Group I  Group II  Total  Blood in BacT/Alert system  —  0/202 (0)  0/202 (0)  Blood in Dixon agar at T0  6/290 (2.1)  1/202 (0.5)  7/492 (1.6)  CVC in Dixon agar  6/290 (2.1)  9/202 (4.4)  15/492 (3.1)    Positive /Total (%)  Diagnosis  Group I  Group II  Total  Blood in BacT/Alert system  —  0/202 (0)  0/202 (0)  Blood in Dixon agar at T0  6/290 (2.1)  1/202 (0.5)  7/492 (1.6)  CVC in Dixon agar  6/290 (2.1)  9/202 (4.4)  15/492 (3.1)  View Large Table 1. Number and percentage of infants from Group I (blood collected by lysis centrifugation tube) and Group II (blood collected by BacT/Alert pediatric - PF bottles) scored positive for Malassezia bloodstream infections by using blood and central venous catheter (CVC) cultures.   Positive /Total (%)  Diagnosis  Group I  Group II  Total  Blood in BacT/Alert system  —  0/202 (0)  0/202 (0)  Blood in Dixon agar at T0  6/290 (2.1)  1/202 (0.5)  7/492 (1.6)  CVC in Dixon agar  6/290 (2.1)  9/202 (4.4)  15/492 (3.1)    Positive /Total (%)  Diagnosis  Group I  Group II  Total  Blood in BacT/Alert system  —  0/202 (0)  0/202 (0)  Blood in Dixon agar at T0  6/290 (2.1)  1/202 (0.5)  7/492 (1.6)  CVC in Dixon agar  6/290 (2.1)  9/202 (4.4)  15/492 (3.1)  View Large In control cultures, M. furfur and M. pachydermatis population size increased during the incubation from 2–9 × 103 cfu/ml (Log10 cfu/ml = 3.15–3.96) at T0 to the stationary phase (Log10 cfu/ml = 6.01–6.26) at T3 (Fig. 1). In BacT/Alert PF bottles, M. furfur population size decreased from 9 × 103 cfu/ml (i.e., Log10 cfu/ml = 3.96) at T0 to zero cfu/ml at T9 with values significantly lower in BacT/Alert bottles than that in control cultures after 1 day of incubation (P < 0.05) later on (Fig. 1). Conversely, at the same conditions, M. pachydermatis population size increased from 7.6 × 103 cfu/ml (Log10 cfu/ml = 3.88) at T0 to 2.5 × 106 cfu/ml (Log10 cfu/ml = 6.41) at T3 (Fig. 1). In the BacT/Alert bottles the population size of M. pachydermatis was significantly lower than that in control culture after T13 (i.e., Log10 cfu/ml = 4.51 in BacT/Alert bottles vs Log10 cfu/ml = 6.57 in Dixon broth) (Fig. 1). In the event of a population size of 7.6 × 103 cfu/ml (Log10 cfu/ml = 3.88), M. pachydermatis positive samples were revealed by the BacT/Alert system after 30 hours of incubation. Figure 1. View largeDownload slide Population size of Malassezia furfur and Malassezia pachydermatis expressed as Log10 of Colony Forming Units - CFU/ml in BacT/Alert PF bottles and in Dixon broth as control after different days of incubation. The statistically significant differences are indicated with the same letters. Figure 1. View largeDownload slide Population size of Malassezia furfur and Malassezia pachydermatis expressed as Log10 of Colony Forming Units - CFU/ml in BacT/Alert PF bottles and in Dixon broth as control after different days of incubation. The statistically significant differences are indicated with the same letters. M. furfur inoculum dilutions (i.e., from 106 to 10 cfu/ml) decreased during the incubation from 1.2 × 106 cfu/ml (Log10 cfu/ml = 6.12) at T0 to zero cfu/ml within 14 days (T14) (Fig. 2) and from ∼10 cfu/ml at T0 to zero cfu/ml after 3 days (data not shown), in both BacT/Alert FA and PF bottles. Conversely, all M. pachydermatis inoculum dilutions increased during the incubation reaching the stationary phase in both BacT/Alert FA and PF bottles and the BacT/Alert system revealed M. pachydermatis positive samples after 2 days of incubation regardless of the population size at T0. Figure 2. View largeDownload slide Malassezia furfur growth at different time points into BacT/Alert FA and PF bottles and in Dixon broth as control. Figure 2. View largeDownload slide Malassezia furfur growth at different time points into BacT/Alert FA and PF bottles and in Dixon broth as control. Discussion The results of this study indicate that Malassezia fungemia in preterm infants might occur with a prevalence ranging from 2.1% to 4.4%, according to the diagnostic procedures employed, therefore suggesting the relevance of a proper diagnosis. The Isolator system gave better results in detecting M. furfur fungemia than automated system in the case of lipid-supplemented medium (i.e., Dixon agar) used for culturing this yeast.17 However, even if the beneficial effect of the Isolator system for the detection of M. furfur or dimorphic fungi (i.e., Histoplasma spp.) is documented,23 the reduced use of this system for fungemia diagnosis, has allowed a reduction in costs, sample processing times and false-positive.17 In this study, we evaluated the efficacy of the BacT/Alert system in detecting M. furfur and M. pachydermatis BSIs, through a direct culturing procedure of bottle content at T0 and by incubation of BacT/Alert bottles in the automated system. However, though blood was immediately cultured on specific medium (i.e., DixA), M. furfur was detected only in one case probably as an effect of (i) the low Malassezia population size in blood (usually lower than 102 cfu/ml)24 and of (ii) yeast dilution in the automated BacT/Alert bottles containing at least 20 ml of tryptic soy broth as medium. Conversely, the finding of one blood sample positive for M. furfur might be due to the presence of detectable yeast population size in the blood of the hospitalized patient. Therefore, the absence of lipid supplementation in the medium of BacT/Alert bottles is most likely the cause of Malassezia growth failure. This was also confirmed by the fact that M. pachydermatis grows in the BacT/Alert bottles being easily detected by the system. The Malassezia population size does not affect the sensitivity of the system since at T0M. pachydermatis was detected at low density (i.e., 10 cfu/ml) but not M. furfur, even at high density (i.e., 106 cfu/ml). Therefore, since the instrument gave a positive signal after 2 days, regardless the initial load of M. pachydermatis, the increase of one logarithm cfu/ml could be enough for a positive diagnosis. Finally, the highest prevalence of Malassezia BSI detected using CVC culture in DixA suggests that this procedure should be used to diagnose Malassezia CVC related fungemia when other causes of sepsis have been ruled out.25,26 Although new methods might be used for the diagnosis of Malassezia BSI,27 the blood culture BacT/Alert system remains one of the most common system used in many laboratories for the detection of bacterial and fungal pathogens. Therefore, for a correct diagnosis of M. furfur BSI the blood culture in lipid-enriched fungal medium, such as Dixon agar, should be used for the diagnosis and the BacT/Alert system implemented to increase the sensibility in the diagnosis of M. furfur BSIs. Finally, the prevalence of 4.4% of Malassezia infection registered in patients of group II and gained by CVC cultures on lipid-supplemented media might suggest that this approach may be proposed as a routine procedure to diagnose the infection. A proper diagnosis will further contribute to scientific knowledge about the clinical significance of M. furfur and its implication in the management of immunocompromised hosts. Acknowledgements This study was supported by Fondo di Sviluppo e Coesione 2007–2013 – APQ Ricerca Regione Puglia “Programma regionale a sostegno della specializzazione intelligente e della sostenibilità sociale ed ambientale – FutureInResearch”. Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper. References 1. Iatta R, Cafarchia C, Cuna T et al.   Bloodstream infections by Malassezia and Candida species in critical care patients. Med Mycol . 2014; 52: 264– 269. Google Scholar CrossRef Search ADS PubMed  2. Roman J, Bagla P, Ren P, Blanton LS, Berman MA. Malassezia pachydermatis fungemia in an adult with multibacillary leprosy. Med Mycol Case Rep . 2016; 12: 1– 3. Google Scholar CrossRef Search ADS PubMed  3. Chu CM, Lai RW. Malassezia furfur fungemia in a ventilator-dependent patient without known risk factors. Hong Kong Med J . 2002; 8: 212– 214. Google Scholar PubMed  4. Chryssanthou E, Broberger U, Petrini B. Malassezia pachydermatis fungaemia in a neonatal intensive care unit. Acta Paediatr . 2001; 90: 323– 327. Google Scholar CrossRef Search ADS PubMed  5. Chang HJ, Miller HL, Watkins N et al.   An epidemic of Malassezia pachydermatis in an intensive care nursery associated with colonization of health care workers pet dogs. N Engl J Med . 1998; 338: 706– 711. Google Scholar CrossRef Search ADS PubMed  6. van Belkum A, Boekhout T, Bosboom R. Monitoring spread of Malassezia infections in a neonatal intensive care unit by PCR-mediated genetic typing. J Clin Microbiol . 1994; 32: 2528– 2532. Google Scholar PubMed  7. Velegraki A, Cafarchia C, Gaitanis G, Iatta R, Boekhout T. Malassezia infections in humans and animals: pathophysiology, detection, and treatment. PLoS Pathog . 2015; 11: e1004523. Google Scholar CrossRef Search ADS PubMed  8. Iatta R, Immediato D, Montagna MT, Otranto D, Cafarchia C. In vitro activity of two amphotericin B formulations against Malassezia furfur strains recovered from patients with bloodstream infections. Med Mycol . 2015; 53: 269– 274. Google Scholar CrossRef Search ADS PubMed  9. Aguirre C, Euliarte C, Finquelievich J, Sosa Mde L, Giusiano G. Fungemia and interstitial lung compromise caused by Malassezia sympodialis in a pediatric patient. Rev Iberoam Micol . 2015; 32: 118– 121. Google Scholar CrossRef Search ADS PubMed  10. Al-Sweih N, Ahmad S, Joseph L, Khan S, Khan Z. Malassezia pachydermatis fungemia in a preterm neonate resistant to fluconazole and flucytosine. Med Mycol Case Rep . 2014; 5: 9– 11. Google Scholar CrossRef Search ADS PubMed  11. Choudhury S, Marte RL. Malassezia pachydermatis fungaemia in an adult on posaconazole prophylaxis for acute myeloid leukaemia. Pathology  2014; 46: 466– 467. Google Scholar CrossRef Search ADS PubMed  12. Iatta R, Figueredo LA, Montagna MT, Otranto D, Cafarchia C. In vitro antifungal susceptibility of Malassezia furfur from bloodstream infections. J Med Microbiol . 2014; 63: 1467– 1473. Google Scholar CrossRef Search ADS PubMed  13. Miceli MH, Díaz JA, Lee SA. Emerging opportunistic yeast infections. Lancet Infect Dis . 2011; 11: 142– 151. Google Scholar CrossRef Search ADS PubMed  14. Ashbee HR. Update on the genus Malassezia. Med Mycol . 2007; 45: 287– 303. Google Scholar CrossRef Search ADS PubMed  15. Kaneko T, Murotani M, Ohkusu K, Sugita T, Makimura K. Genetic and biological features of catheter-associated Malassezia furfur from hospitalized adults. Med Mycol . 2012; 50: 74– 80. Google Scholar CrossRef Search ADS PubMed  16. Nelson SC, Yau YC, Richardson SE, Matlow AG. Improved detection of Malassezia species in lipid-supplemented Peds Plus blood culture bottles. J Clin Microbiol . 1995; 33: 1005– 1007. Google Scholar PubMed  17. Campigotto A, Richardson SE, Sebert M et al.   Low utility of the pediatric Isolator blood culture system for the detection of fungemia in children: a ten-year review. J Clin Microbiol . 2016; 54: 2284– 2297. Google Scholar CrossRef Search ADS PubMed  18. Oliveri S, Trovato L, Betta P, Romeo MG, Nicoletti G. Malassezia furfur fungaemia in a neonatal patient detected by lysis-centrifugation blood culture method: first case reported in Italy. Mycoses  2011; 4: e638– 40. Google Scholar CrossRef Search ADS   19. Maki DG, Weise CE, Sarafin HW. A semiquantitative culture method for identifying intravenous-catheter-related infection. N Engl J Med . 1977; 296: 1305– 1309. Google Scholar CrossRef Search ADS PubMed  20. Cafarchia C, Gasser RB, Figueredo LA, Latrofa MS, Otranto D. Advances in the identification of Malassezia. Mol Cell Probes . 2011; 25: 1– 7. Google Scholar CrossRef Search ADS PubMed  21. Kolecka A, Khayhan K, Groenewald M et al.   Identification of medically relevant species of arthroconidial yeasts by use of matrix-assisted laser desorption ionization-time of flight mass spectrometry. J Clin Microbiol . 2013; 51: 2491– 2500. Google Scholar CrossRef Search ADS PubMed  22. Cafarchia C, Iatta R, Immediato D, Puttilli MR, Otranto D. Azole susceptibility of Malassezia pachydermatis and Malassezia furfur and tentative epidemiological cut-off values. Med Mycol . 2015; 53: 743– 748. Google Scholar CrossRef Search ADS PubMed  23. Lyon R, Woods G. Comparison of the BacT/Alert and Isolator blood culture systems for recovery of fungi. Am J Clin Pathol . 1995; 103: 660– 662. Google Scholar CrossRef Search ADS PubMed  24. Marcon MJ, Powell DA. Human infections due to Malassezia spp. Clin Microbiol Rev . 1992; 5: 101– 119. Google Scholar CrossRef Search ADS PubMed  25. Curvale-Fauchet N, Botterel F, Legrand P, Guillot J, Bretagne S. Frequency of intravascular catheter colonization by Malassezia spp. in adult patients. Mycoses  2004; 47: 491– 494. Google Scholar CrossRef Search ADS PubMed  26. Barber GR, Brown AE, Kiehn TE, Edwards FF, Armstrong D. Catheter-related Malassezia furfur fungemia in immunocompromised patients. Am J Med . 1993; 95: 365– 370. Google Scholar CrossRef Search ADS PubMed  27. Cornu M, Goudjil S, Kongolo G et al.   Evaluation of the (1,3)-β-D-glucan assay for the diagnosis of neonatal invasive yeast infections. Med Mycol . 2017. doi: 10.1093/mmy/myx021. © The Author 2017. 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Blood culture procedures and diagnosis of Malassezia furfur bloodstream infections: Strength and weakness

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Abstract

Abstract The occurrence of Malassezia spp. bloodstream infections (BSIs) in neonatal intensive care unit was evaluated by using pediatric Isolator, BacT/Alert systems and central venous catheter (CVC) culture. The efficacy of BacT/Alert system in detecting Malassezia was assessed by conventional procedures, culturing 1 ml of bottle content before incubation and by studying the survival of Malassezia spp. strains in BacT/Alert bottles. Of the 492 neonates enrolled, blood was collected by pediatric Isolator (290 patients; group I) or by BacT/Alert bottles (202 patients; group II). The survival of Malassezia furfur and Malassezia pachydermatis in BacT/Alert bottles was evaluated by culturing the inoculum suspension (from 106 to 10 colony-forming units, cfu/ml) and assessing the cfu/ml for 15 days. In total, 15 Malassezia BSIs were detected, of which six (2.1%) from both blood and CVC culture in Dixon agar (DixA) in patients belong to group I (blood collected by paediatric Isolator tube) and nine (4.4%) only from CVC culture in DixA in patients of group II (blood collected by BacT/Alert bottle). Only one patient (0.5%) from group II scored positive for M. furfur also by culturing in DixA 1 ml blood content of BacT/Alert bottle before incubation in BacT/Alert system.M. furfur population size in BacT/Alert bottles decreased during the incubation time, whereas that of M. pachydermatis increased. The BacT/Alert system detected M. pachydermatis even at very low concentration (i.e., 10 cfu/ml) but not any positive blood culture for M. furfur. For a correct diagnosis of Malassezia furfur BSI, the blood should be culture in lipid-enriched fungal medium, and the BacT/Alert system implemented by adding lipid substrates to increase the method sensibility. Finally, CVC cultures on lipid-supplemented media may be proposed as a routine procedure to diagnose the Malassezia fungemia. Malassezia furfur, bloodstream infections, diagnosis, BacT/Alert system Introduction Malassezia fungemia by Malassezia pachydermatis, Malassezia furfur and Malassezia sympodialis1–6 may be associated with nosocomial outbreaks in immunocompromised patients.7 Though occasionally reported, this infection has attracted the attention of scientists for the inherent difficulties in its diagnosis and treatment.8–14 A diagnostic survey on bloodstream infections (BSIs) resulted in a higher prevalence of M. furfur (2.1%) than Candida spp. (1.4%) suggesting that Malassezia BSIs might be underestimated, due to improper diagnosis.1 BSIs caused by lipid-dependent Malassezia species are detected either using systems pre-enriched with lipid substrates15,16 or by direct isolation using the Isolator system (DuPont Co., Wilmington, DE, USA).1,17,18 Although the automated blood culture system BacT/Alert (bioMérieux, Marcy l’Etoile, France) is worldwide used in many laboratories for the detection of bacterial and fungal pathogens, its failure to detect M. furfur has been reported through a retrospective clinical review.17 In this study, the occurrence of Malassezia spp. BSIs in a neonatal intensive care unit (NICU) has been evaluated by using two different blood culture procedures (i.e., pediatric Isolator and BacT/Alert systems) and by culturing blood and central venous catheter (CVC) tips onto specific medium (i.e., Dixon agar). The survival of Malassezia spp. strains in BacT/Alert bottles was also evaluated, and the efficacy of BacT/Alert system in detecting positive samples was correlated with the Malassezia spp. population size. Methods Human patients and Malassezia spp. BSIs diagnosis Blood and CVC were collected from 492 neonates, of which 290 (group I) enrolled during a 1-year survey on yeasts fungemia1 conducted from July 2011 to July 2012 and 202 (group II) enrolled from January 2016 to January 2017. All patients displayed at least one of the following clinical signs: respiratory distress, elevated or depressed leukocyte count, increased C-reactive protein levels (CRP ≥5 mg/l), abdominal distension, or thrombocytopenia. Blood samples were collected by lysis centrifugation tubes (pediatric Isolator 1.5-ml system, DuPont Co., Wilmington, DE, USA) from 290 patients of group I and by BacT/Alert pediatric (PF) bottles from patients of group II. For Malassezia spp. BSIs diagnosis, pediatric Isolator tubes were processed according to the manufacturer's recommendations by culturing 0.35 ml of Isolator tube content onto Dixon agar containing 0.5% chloramphenicol (DixA). BacT/Alert PF bottles were processed according to the manufacturer's recommendations with some modifications. In particular, before bottle incubation (i.e., T0), 1 ml of bottle content (i.e., sampled blood + culture medium) was aseptically collected, centrifuged, and the pellet inoculated onto DixA, then incubated at 32°C for 3 days. Bottles were incubated at 35°C into the BacT/Alert system and monitored for up to 15 days for fungal growth. When a positive culture was indicated by the system, 0.2 ml of BacT/Alert bottle content was subcultured onto DixA. CVCs from both groups I and II were cultured by rolling the CVC tip19 onto DixA immediately after their sampling and the inoculated fungal medium was incubated at 32°C for 15 days and daily examined, as routine diagnostic method.20 Following preliminary yeast identification using an automated system (Vitek2; bioMérieux, Craponne, France), Malassezia spp. isolates were characterized based on morphological and biochemical features (e.g., catalase reaction, ability to assimilate Tweens (i.e., 20, 40, 60, 80), growth on PEG-35 castor oil, and β-glucosidase activity).20Malassezia spp. isolates were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) as previously reported.21 Because of the observational nature of this study a written informed consent from the parents or guardian of patients was obtained according to the current Italian legislation (Art. 81- D.Lgs.vo n.196/2003). Efficacy of the BacT/Alert system in detecting Malassezia BSI The efficacy of the BacT/Alert system in detecting Malassezia BSI was assessed testing 15 Malassezia strains (i.e., n. 7 from blood and n. 8 from CVC) from 15 patients, and one M. pachydermatis (ITS-1 GenBank Accession number DQ915503) from the skin of a dog with dermatitis, used as control being the only non–lipid-dependent species within the genus.22 For each strain, an inoculum suspension (IS), corresponding to 1–5 × 105 colony-forming units (cfu/ml) in 5 ml of sterile distilled water, was prepared using 5-day-old colonies and 1 ml transferred into a 20-ml culture medium BacT/Alert PF bottle containing 3 ml of blood obtained from healthy volunteers. Two bottles for each test were processed either by automated BacT/Alert system or by quantitative plate count of cfu/ml on DixA. For the control cultures, 1 ml of the IS was transferred into 23 ml of Dixon broth, incubated at 35°C in a shaking incubator and monitored for the Malassezia population size under the same conditions. The bottles were incubated in the BacT/Alert automated system at 35°C for 15 days, and cfu/ml was evaluated before incubation (i.e., T0), after 1 day and every 2 days up to 15 days of incubation (i.e., from T1 to T15). An IS corresponding to 1–5 × 108 cfu/ml and five consecutives 1:10 dilutions (i.e., D1-D5) in sterile distillated water for both M. furfur (GenBank Accession n. KF682274), and M. pachydermatis (GenBank Accession number DQ915503) strains were performed to assess the sensitivity of the BacT/Alert system in detecting Malassezia BSI in relationship to Malassezia population size. In particular, 1 ml of the IS and of each dilution was transferred into a 20-ml culture medium BacT/Alert PF bottle containing 3 ml of blood and processed as above. The experiment was also performed using BacT/Alert FA bottles (i.e., those employed for adult patients) under the same conditions. Statistical analysis All the experiments were performed in duplicate and repeated twice on different days. The results were plotted and reported as mean values of Log10 cfu/ml. The population size of Malassezia spp. in the control medium (i.e., Dixon broth) and in the BacT/Alert bottles, at each time point, were compared by using the t test for paired samples. Statistical significance was set at P ≤ 0.05. Results Of the 15 Malassezia BSI-positive samples (3.1%), M. furfur was isolated from blood and CVC cultures in six patients (2.1%) from group I (blood in pediatric Isolator tube) and from CVC cultures in nine patients (4.4%) from group II (blood in BacT/Alert bottle), of which one was also positive, at T0 for 1 ml blood culture from the BacT/Alert bottle (Table 1). The BacT/Alert instrument gave no positive signal. All Malassezia strains were identified by biochemical features and MALDI-TOF MS as M. furfur (data not shown). The M. furfur strains isolated from blood and CVC culture in six patients from group I showed the same MALDI-TOF MS profile.21 Table 1. Number and percentage of infants from Group I (blood collected by lysis centrifugation tube) and Group II (blood collected by BacT/Alert pediatric - PF bottles) scored positive for Malassezia bloodstream infections by using blood and central venous catheter (CVC) cultures.   Positive /Total (%)  Diagnosis  Group I  Group II  Total  Blood in BacT/Alert system  —  0/202 (0)  0/202 (0)  Blood in Dixon agar at T0  6/290 (2.1)  1/202 (0.5)  7/492 (1.6)  CVC in Dixon agar  6/290 (2.1)  9/202 (4.4)  15/492 (3.1)    Positive /Total (%)  Diagnosis  Group I  Group II  Total  Blood in BacT/Alert system  —  0/202 (0)  0/202 (0)  Blood in Dixon agar at T0  6/290 (2.1)  1/202 (0.5)  7/492 (1.6)  CVC in Dixon agar  6/290 (2.1)  9/202 (4.4)  15/492 (3.1)  View Large Table 1. Number and percentage of infants from Group I (blood collected by lysis centrifugation tube) and Group II (blood collected by BacT/Alert pediatric - PF bottles) scored positive for Malassezia bloodstream infections by using blood and central venous catheter (CVC) cultures.   Positive /Total (%)  Diagnosis  Group I  Group II  Total  Blood in BacT/Alert system  —  0/202 (0)  0/202 (0)  Blood in Dixon agar at T0  6/290 (2.1)  1/202 (0.5)  7/492 (1.6)  CVC in Dixon agar  6/290 (2.1)  9/202 (4.4)  15/492 (3.1)    Positive /Total (%)  Diagnosis  Group I  Group II  Total  Blood in BacT/Alert system  —  0/202 (0)  0/202 (0)  Blood in Dixon agar at T0  6/290 (2.1)  1/202 (0.5)  7/492 (1.6)  CVC in Dixon agar  6/290 (2.1)  9/202 (4.4)  15/492 (3.1)  View Large In control cultures, M. furfur and M. pachydermatis population size increased during the incubation from 2–9 × 103 cfu/ml (Log10 cfu/ml = 3.15–3.96) at T0 to the stationary phase (Log10 cfu/ml = 6.01–6.26) at T3 (Fig. 1). In BacT/Alert PF bottles, M. furfur population size decreased from 9 × 103 cfu/ml (i.e., Log10 cfu/ml = 3.96) at T0 to zero cfu/ml at T9 with values significantly lower in BacT/Alert bottles than that in control cultures after 1 day of incubation (P < 0.05) later on (Fig. 1). Conversely, at the same conditions, M. pachydermatis population size increased from 7.6 × 103 cfu/ml (Log10 cfu/ml = 3.88) at T0 to 2.5 × 106 cfu/ml (Log10 cfu/ml = 6.41) at T3 (Fig. 1). In the BacT/Alert bottles the population size of M. pachydermatis was significantly lower than that in control culture after T13 (i.e., Log10 cfu/ml = 4.51 in BacT/Alert bottles vs Log10 cfu/ml = 6.57 in Dixon broth) (Fig. 1). In the event of a population size of 7.6 × 103 cfu/ml (Log10 cfu/ml = 3.88), M. pachydermatis positive samples were revealed by the BacT/Alert system after 30 hours of incubation. Figure 1. View largeDownload slide Population size of Malassezia furfur and Malassezia pachydermatis expressed as Log10 of Colony Forming Units - CFU/ml in BacT/Alert PF bottles and in Dixon broth as control after different days of incubation. The statistically significant differences are indicated with the same letters. Figure 1. View largeDownload slide Population size of Malassezia furfur and Malassezia pachydermatis expressed as Log10 of Colony Forming Units - CFU/ml in BacT/Alert PF bottles and in Dixon broth as control after different days of incubation. The statistically significant differences are indicated with the same letters. M. furfur inoculum dilutions (i.e., from 106 to 10 cfu/ml) decreased during the incubation from 1.2 × 106 cfu/ml (Log10 cfu/ml = 6.12) at T0 to zero cfu/ml within 14 days (T14) (Fig. 2) and from ∼10 cfu/ml at T0 to zero cfu/ml after 3 days (data not shown), in both BacT/Alert FA and PF bottles. Conversely, all M. pachydermatis inoculum dilutions increased during the incubation reaching the stationary phase in both BacT/Alert FA and PF bottles and the BacT/Alert system revealed M. pachydermatis positive samples after 2 days of incubation regardless of the population size at T0. Figure 2. View largeDownload slide Malassezia furfur growth at different time points into BacT/Alert FA and PF bottles and in Dixon broth as control. Figure 2. View largeDownload slide Malassezia furfur growth at different time points into BacT/Alert FA and PF bottles and in Dixon broth as control. Discussion The results of this study indicate that Malassezia fungemia in preterm infants might occur with a prevalence ranging from 2.1% to 4.4%, according to the diagnostic procedures employed, therefore suggesting the relevance of a proper diagnosis. The Isolator system gave better results in detecting M. furfur fungemia than automated system in the case of lipid-supplemented medium (i.e., Dixon agar) used for culturing this yeast.17 However, even if the beneficial effect of the Isolator system for the detection of M. furfur or dimorphic fungi (i.e., Histoplasma spp.) is documented,23 the reduced use of this system for fungemia diagnosis, has allowed a reduction in costs, sample processing times and false-positive.17 In this study, we evaluated the efficacy of the BacT/Alert system in detecting M. furfur and M. pachydermatis BSIs, through a direct culturing procedure of bottle content at T0 and by incubation of BacT/Alert bottles in the automated system. However, though blood was immediately cultured on specific medium (i.e., DixA), M. furfur was detected only in one case probably as an effect of (i) the low Malassezia population size in blood (usually lower than 102 cfu/ml)24 and of (ii) yeast dilution in the automated BacT/Alert bottles containing at least 20 ml of tryptic soy broth as medium. Conversely, the finding of one blood sample positive for M. furfur might be due to the presence of detectable yeast population size in the blood of the hospitalized patient. Therefore, the absence of lipid supplementation in the medium of BacT/Alert bottles is most likely the cause of Malassezia growth failure. This was also confirmed by the fact that M. pachydermatis grows in the BacT/Alert bottles being easily detected by the system. The Malassezia population size does not affect the sensitivity of the system since at T0M. pachydermatis was detected at low density (i.e., 10 cfu/ml) but not M. furfur, even at high density (i.e., 106 cfu/ml). Therefore, since the instrument gave a positive signal after 2 days, regardless the initial load of M. pachydermatis, the increase of one logarithm cfu/ml could be enough for a positive diagnosis. Finally, the highest prevalence of Malassezia BSI detected using CVC culture in DixA suggests that this procedure should be used to diagnose Malassezia CVC related fungemia when other causes of sepsis have been ruled out.25,26 Although new methods might be used for the diagnosis of Malassezia BSI,27 the blood culture BacT/Alert system remains one of the most common system used in many laboratories for the detection of bacterial and fungal pathogens. Therefore, for a correct diagnosis of M. furfur BSI the blood culture in lipid-enriched fungal medium, such as Dixon agar, should be used for the diagnosis and the BacT/Alert system implemented to increase the sensibility in the diagnosis of M. furfur BSIs. Finally, the prevalence of 4.4% of Malassezia infection registered in patients of group II and gained by CVC cultures on lipid-supplemented media might suggest that this approach may be proposed as a routine procedure to diagnose the infection. A proper diagnosis will further contribute to scientific knowledge about the clinical significance of M. furfur and its implication in the management of immunocompromised hosts. Acknowledgements This study was supported by Fondo di Sviluppo e Coesione 2007–2013 – APQ Ricerca Regione Puglia “Programma regionale a sostegno della specializzazione intelligente e della sostenibilità sociale ed ambientale – FutureInResearch”. Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper. References 1. Iatta R, Cafarchia C, Cuna T et al.   Bloodstream infections by Malassezia and Candida species in critical care patients. Med Mycol . 2014; 52: 264– 269. Google Scholar CrossRef Search ADS PubMed  2. Roman J, Bagla P, Ren P, Blanton LS, Berman MA. Malassezia pachydermatis fungemia in an adult with multibacillary leprosy. Med Mycol Case Rep . 2016; 12: 1– 3. Google Scholar CrossRef Search ADS PubMed  3. Chu CM, Lai RW. Malassezia furfur fungemia in a ventilator-dependent patient without known risk factors. Hong Kong Med J . 2002; 8: 212– 214. Google Scholar PubMed  4. Chryssanthou E, Broberger U, Petrini B. Malassezia pachydermatis fungaemia in a neonatal intensive care unit. Acta Paediatr . 2001; 90: 323– 327. Google Scholar CrossRef Search ADS PubMed  5. Chang HJ, Miller HL, Watkins N et al.   An epidemic of Malassezia pachydermatis in an intensive care nursery associated with colonization of health care workers pet dogs. N Engl J Med . 1998; 338: 706– 711. Google Scholar CrossRef Search ADS PubMed  6. van Belkum A, Boekhout T, Bosboom R. Monitoring spread of Malassezia infections in a neonatal intensive care unit by PCR-mediated genetic typing. J Clin Microbiol . 1994; 32: 2528– 2532. Google Scholar PubMed  7. Velegraki A, Cafarchia C, Gaitanis G, Iatta R, Boekhout T. Malassezia infections in humans and animals: pathophysiology, detection, and treatment. PLoS Pathog . 2015; 11: e1004523. Google Scholar CrossRef Search ADS PubMed  8. Iatta R, Immediato D, Montagna MT, Otranto D, Cafarchia C. In vitro activity of two amphotericin B formulations against Malassezia furfur strains recovered from patients with bloodstream infections. Med Mycol . 2015; 53: 269– 274. Google Scholar CrossRef Search ADS PubMed  9. Aguirre C, Euliarte C, Finquelievich J, Sosa Mde L, Giusiano G. Fungemia and interstitial lung compromise caused by Malassezia sympodialis in a pediatric patient. Rev Iberoam Micol . 2015; 32: 118– 121. Google Scholar CrossRef Search ADS PubMed  10. Al-Sweih N, Ahmad S, Joseph L, Khan S, Khan Z. 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Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

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Medical MycologyOxford University Press

Published: Dec 27, 2017

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