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Nosocomial transmission of Saccharomyces cerevisiae in bone marrow transplant patients
Journal of Hospital Infection (2002) 52: 268±272 doi:10.1053/jhin.2002.1314, available online at http://www.idealibrary.com on
Nosocomial transmission of Saccharomyces cerevisiae in bone marrow transplant patients W. J. Olver*, S. A. Jamesz, A. Lennardx, A. Galloway{, I. N. Robertsz, T. C. Boswell* and N. H. Russelly Departments of *Microbiology and yHaematology, City Hospital, Nottingham; zNational Collection of Yeast Cultures, Institute of Food Research, Norwich; Departments of xHaematology and {Microbiology, Royal Victoria Infirmary, Newcastle upon Tyne, UK Summary: Saccharomyces cerevisiae is an unusual cause of clinical infection. We describe three bone marrow transplant patients on a haematology unit who developed possible invasive disease with the organism. Two patients died and both these patients appeared to have a related strain of S. cerevisiae. Screening for S. cerevisiae from throat and stool samples revealed four further patients who were carriers. Genotyping of the invasive and carriage strains demonstrated an indistinguishable strain from patients who had been on the unit at the same time, suggesting cross-infection. & 2002 The Hospital Infection Society
Keywords: Saccharomyces cerevisiae; nosocomial transmission; bone marrow transplantation. Introduction Saccharomyces cerevisiae, also known as brewer's or baker's yeast, is relatively azole-resistant.1 It is widespread in nature and can be found on various plants, fruits and in soil. It can constitute part of the normal flora of the gastrointestinal tract in humans. Although previously thought to be non-pathogenic, there is increasing evidence that it can cause invasive disease in immunosuppressed patients.2±4 Infection has also been described following ingestion of commercial preparations of Saccharomyces boulardii.5,6 Case reports Patient 1 A 24 year-old female with chronic myeloid leukaemia (CML) diagnosed one year previously, underwent Received 22 July 2002; revised manuscript accepted 2 August 2002; published online 18 October 2002. Author for correspondence: Dr William J. Olver, Department of Microbiology, City Hospital, Hucknall Road, Nottingham NG5 1PB. Tel.: 44-115-9691169 ext 45572; Fax: 44-115-9627766; E-mail: [email protected]
0195±6701/02/120268 05 $35.00/0
matched, unrelated, donor bone marrow transplantation (MUD BMT) at Nottingham City Hospital in October 1999. She received prophylactic penicillin V, ofloxacin, co-trimoxazole, colistin, metronidazole, fluconazole and acyclovir, as per protocol. Her post-transplant course was complicated by Clostridium difficile diarrhoea, treated with oral metronidazole. Engraftment was satisfactory and there were no signs of graft-versus-host disease (GVHD). She was discharged home on day 29. On day 43 she was admitted to the haematology unit at the Royal Victoria Infirmary, Newcastle. She was pyrexial but no localizing signs of infection were found. Staphylococcus epidermidis sensitive to teicoplanin was isolated from venous blood. Her temperature did not settle despite broad-spectrum antibiotics and liposomal amphotericin, and over the following seven days she developed painful cervical lymphadenopathy and hepatosplenomegaly. Extensive microbiological and virological investigations were negative. On day 55 she had an episode of acute left upper quadrant pain and developed renal failure. S. cerevisiae was isolated from sputum, faeces and a vulval swab. Despite intensive supportive measures her condition continued to deteriorate and & 2002 The Hospital Infection Society
S. cerevisiae transmission in bone marrow transplant patients
269
she died of multi-organ failure on day 59 posttransplant. A post-mortem sample of lymph node sent for culture grew a heavy growth of S. cerevisiae. Culture of a specimen of lung did not grow any yeasts. Histology of the liver, spleen and lymph nodes revealed a high grade non-Hodgkins lymphoma representing a post-transplant lymphoproliferative disorder.
developed fever with no obvious source of infection. He made a good response to treatment with meropenem and amphotericin B for two weeks, although suspicions of a fungal chest infection were not confirmed by microbiology, histology or imaging. Engraftment was satisfactory and there were no signs of GVHD. He was discharged home on day 34. On day 62 he presented with fever, shivering and a cough productive of yellow sputum and was admitted to the haematology unit at Royal Victoria Infirmary, Newcastle. S. cerevisiae was isolated from sputum, throat and rectal swabs. Antifungal treatment was begun with liposomal amphotericin and flucytosine. His symptoms and signs resolved gradually and he remains well one year posttransplant.
Patient 2 A 29 year-old female with CML diagnosed 18 months previously, underwent MUD BMT at Nottingham City Hospital in October 1999. She received antimicrobial prophylaxis as per protocol (see above). Her post-transplant course was complicated by reactivation of cytomegalovirus (CMV), which was successfully treated with ganciclovir. On day 38 she was discharged home, with satisfactory engraftment and no signs of GVHD. On day 55 she was admitted to the haematology unit at the Royal Victoria Hospital, Newcastle with a febrile illness. On day 61 CMV antigenaemia testing showed further reactivation, and treatment with ganciclovir and foscarnet was started. On day 63 she again became pyrexial and interstitial shadowing was seen on chest X-ray. Blood cultures yielded viridans streptococci, Staphylococcus epidermidis and diphtheroids. Her temperature failed to settle despite appropriate antibiotics and liposomal amphotericin. Hepatic and renal function began to deteriorate. Bronchoscopy on day 75 was normal and bronchial washings were negative for all pathogens. On day 79 she developed sudden onset chest pain, shortness of breath and increasing jaundice. A yeast was isolated from a throat swab and bronchoalveolar lavage (BAL), which was identified as S. cerevisiae. No other pathogens were identified in the BAL. CMV antigenaemia was resolving. She was transferred to the intensive care unit for ventilation and supportive treatment but continued to deteriorate and died on day 91 post-transplant. A post-mortem was performed and cause of death was certified as multi-organ failure. There was no histological or microbiological evidence of an infective aetiology. Patient 3 A 27 year-old male with CML diagnosed two years previously, underwent MUD BMT at Nottingham City Hospital in November 1999. At day 10 he
Materials and methods Point prevalence survey Due to the rare occurrence of colonization and infections due to S. cerevisiae (not previously detected on either unit), a point prevalence study was performed in the haematology units of Nottingham City Hospital (11 patients) and Royal Victoria Infirmary, Newcastle (20 patients), to detect carriage of the organism from throat swabs and stool samples. This was performed in January 2000. Throat swabs were processed by breaking off the end of the swab into 1 mL nutrient broth. The sample was then agitated on a vortex mixer for 15 s. One drop of suspension was inoculated on to one half of a Sabouraud±chloramphenicol agar plate. Stool samples were processed by emulsifying a pea-sized portion of the sample in 5 mL of Ringer's solution. One drop of suspension was inoculated on to one half of a Sabouraud±chloramphenicol agar plate. Plates were spread for discrete colonies, incubated at 37 C in air and read at 24 and 48 h. Colonies were identified using the germ tube test with horse serum, and read after 2±4 h at 37 C in air. Negative germ tube isolates were then identified using API 20C AUX (bioMeÂrieux, UK). Typing Isolates from the three clinical cases (Patients 1±3) and those subsequently found to be carriers were genotyped at the Institute of Food Research, Norwich.
270
Multiplex polymerase chain reaction (PCR) Yeasts were incubated on YM (0.3% yeast extract, 0.3% malt extract, 0.5% peptone, 1% glucose) agar overnight at 37 C. For each isolate, a small sample of confluent yeast growth was picked off the plate using a sterile Gilson pipette tip and mixed vigorously with 10 mL sterile distilled water in a 0.5 mL microcentrifuge tube, which was stored on ice. To this cell suspension was then added 90 mL of a mixture containing 78 mL distilled water, 10 mL 10X Taq polymerase buffer (Life Technologies), 0.8 mL dNTPs, 0.8 mL primers and 0.5 mL Taq polymerase (5 U/ mL). The PCR primers were designed using conserved DNA sequences from the S. cerevisiae transposons Ty1, Ty2 and Ty3 and these primers have been patented by the Institute of Food Research, Norwich. Each PCR sample was overlaid with approximately 50 mL paraffin oil (Sigma). The PCR cycling parameters used for DNA amplification were: 92 C for 2 min, followed by two cycles of 92 C for 2 min, 52 C for 3 min, 72 C for 2 min, followed by 28 cycles of 90 C for 2 min, 52 C for 3 min, 72 C for 2 min. DNA precipitation To improve the overall quality of the resulting DNA fingerprints, each PCR sample was first concentrated/purified by ethanol precipitation, before being analysed by agarose gel electrophoresis. For each sample, 200 mL of 95% ice-cold ethanol was mixed with 10 mL of 3 M sodium acetate in a 0.5 mL microcentrifuge tube, and to this was added the entire (100 mL) PCR sample. This mix was briefly vortexed and stored overnight at ÿ20 C, to allow DNA precipitation. After defrosting, centrifugation was performed at 14 000 rpm for 25±30 min, to pellet DNA. The supernatant was removed and the pellet washed with 70% ethanol. After removing residual ethanol, the sample was vacuum-dried.
W. J. Olver et al.
for 2.5±3.0 h, stained in ethidium bromide (0.5 mg/mL) for 25 min and visualized using a UV transilluminator. Environmental investigations Potential routes of cross-infection were investigated by taking environmental samples and looking at hand carriage of yeasts by healthcare workers. Swabs premoistened in sterile saline were taken from a variety of environmental sites, including air vents, door handles, bedclothes and curtains (sweep plates), bathroom surfaces, sinks, commodes, telephones, computer keyboards and other equipment. They were processed in the same way as the throat swabs. A wide variety of medicines were sampled to see if any were contaminated with yeasts. Ten members of staff had fingerprint agar plates performed. All cultures were incubated for 72 h at 37 C. Results In the Nottingham unit, four of 11 patients were positive for S. cerevisiae from throat and/or stool samples, compared to none of the 20 in the Newcastle unit (P < 0.05). Genotyping results of the isolates from the three clinical cases (Patients 1±3) and the four carriers (Patients 4±7) are shown in Figure 1.
DNA rehydration The DNA pellet was re-suspended in 20 mL distilled water and incubated at 37 C for 10 min to aid rehydration. Gel analysis Fifteen microlitres of the purified PCR product was added to 5 mL loading dye (bromophenol blue), and 18 mL of this mixture was loaded on to a 15 20 cm 1.5% agarose±TBE gel. The gel was run at 125 V
Figure 1 Genotyping of S. cerevisiae isolates. Lane 1 is DNA marker, then (left to right) Patient 2, Patient 3, Patient 1, Patient 6, Patient 5, Patient 7 and Patient 4 (twice).
S. cerevisiae transmission in bone marrow transplant patients
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1 * 2 * 3 Patient 4 * 5 * 6 7 † Figure 2 Inpatient stays on Nottingham haematology unit 1999/2000. * Genotypically indistinguishable. y Genotypically closely related.
Isolates from Patients 1, 2, 4 and 5 were indistinguishable, suggesting possible cross-infection. Patient 7 had a closely related strain. The temporal relationship of the inpatient episodes for Patients 1±7 on the haematology/BMT unit is shown in Figure 2. There was a temporal overlap between Patients 1, 2, 4 and 5. Patient 4 had received an allogeneic peripheral blood stem-cell transplant for peripheral T-cell lymphoma in October 1999, and was being given penicillin V and ofloxacin prophylaxis. Patient 5 had undergone MUD BMT for myelodysplasia in October 1999. He had been treated for possible fungal chest infection one week prior to screening. Patient 6 had relapsed high-grade non-Hodgkin's lymphoma and was being treated with palliative chemotherapy. Prior to screening she had received 10 days of meropenem and amphotericin B for neutropenic fever. Patient 7 was diagnosed with acute myeloid leukaemia in December 1999 and treated conservatively with hydroxyurea. Prior to screening she had not received any antibiotics or antifungals. Two of the rooms where curtains were sampled (using a `sweep' directly with the plate) yielded very scanty yeasts, which were identified as Candida parapsilosis. On the hands of one member of staff two fingers grew a single yeast colony, also identified as C. parapsilosis. All other environmental samples were negative after 72 h incubation. Discussion S. cerevisiae is an uncommon but increasingly recognized cause of infection in immunosuppressed patients.2±4 Although the three cases described here did not provide conclusive (histological) evidence of invasive disease, S. cerevisiae was nevertheless grown
in pure culture from each patient in the absence of any other pathogens. Because it has been longconsidered non-pathogenic, the epidemiology of the organism in the clinical setting has not been extensively studied. However, genotyping has been undertaken on a collection of 76 surveillance isolates collected from a bone marrow transplant unit.7 Pulsed-field gel electrophoresis (PFGE) typing revealed 62 distinct genotypes, but clusters of indistinguishable isolates were identified among different patients hospitalized at the same time, indicating possible nosocomial transmission. The source of the organism in cases of infection due to S. cerevisiae is not usually identified, but there have been reports of infection secondary to ingestion of S. boulardii, commercial preparations of which are marketed as Ultra-Levure in France (Biocodex, Montrouge) and Perenterol in the USA (Biocodex, Seattle) for the treatment of antibiotic-associated diarrhoea.5,6 Although there has been (taxonomic) controversy over whether S. cerevisiae is synonymous with S. boulardii,8 a recent study showed that commercial strains of S. boulardii (UltraLevure) were genotypically indistinguishable from S. cerevisiae.9 Nosocomial infection with S. cerevisiae was first confirmed in a report of a neonate who was treated with S. boulardii and subsequently developed infection (positive blood culture).6 A second neonate in a nearby cot then also developed bacteraemia with S. cerevisiae. Mitochondrial DNA restriction analysis and chromosomal DNA profiles [using CHEF (contour-clamped homogeneous electric-field) gel electrophoresis] revealed that the two clinical strains were indistinguishable. In the possible invasive cases of infection that we have described, none of the patients had been treated with S. boulardii. Interestingly, in the case of
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invasive infection where the S. cerevisiae genotype was different (Patient 3), the patient later reported to be very fond of a locally brewed beer. Although there is no direct evidence, it is possible that this strain of S. cerevisiae originated from beer, as it is known that ingestion of yeast can produce fungaemia and funguria.10 Microsatellite genotyping has been used to show that a cluster of clinical isolates shared their genotype with a bakery strain of S. cerevisiae, suggesting intestinal colonization following ingestion.11 The four patients whose isolates of S. cerevisiae were genotypically indistinguishable were on the haematology unit at the same time. The mode of transmission is unclear but is may be faecal±oral. Sampling results did not show an obvious environmental source for S. cerevisiae. C. parapsilosis was isolated from two sets of curtains and the hand of a member of staff. This organism has been found previously on the hands of hospital staff, including an instance where nosocomial spread occurred and caused invasive infection in a neonatal intensive care unit.12 However, there are no reports of S. cerevisiae being isolated from the hands of staff or the hospital environment. Although food on the unit was not sampled, this is potentially a source of infection. S. cerevisiae can survive in sugary foodstuffs such as jam, fruit and fruit drinks. Bread can become contaminated after baking (i.e., from the baker's hands), and beer can contain the yeast even though it is normally pasteurized. However, a study of a variety of food in a French haematology unit did not yield any S. cerevisiae,13 with mainly filamentous fungi being isolated. Length of stay in hospital has been identified as a risk factor for acquisition of S. cerevisiae in patients with haematological malignancy.14 Isolates were found to be highly resistant to azole antifungals, and it was postulated that this might have been due to extensive use of these antifungals. However, the isolates from the invasive cases discussed in this study were sensitive to fluconazole and had only intermediate resistance to itraconazole, as previously reported.15 Further epidemiological studies are now planned to examine the carriage of S. cerevisiae over time. Isolates will be genotyped and patient factors recorded, to assess whether cross-infection has occurred, and what patient factors increase the risk of acquisition of this yeast species.
W. J. Olver et al.
References 1. Barchiesi F, Arzeni D, Compagnucci P, Di Francesco LF, Giacometti A, Scalise G. In vitro activity of five antifungal agents against clinical isolates of Saccharomyces cerevisiae. Med Mycol 1998; 36: 437±440. 2. Aucott JN, Fayen J, Grossnicklas H, Morrissey A, Lederman MM, Salata RA. Invasive infection with Saccharomyces cerevisiae: report of three cases and review. Rev Infect Dis 1990; 12: 406±411. 3. Cairoli R, Marenco P, Perego R, de Cataldo F. Saccharomyces cerevisiae fungemia with granulomas in the bone marrow in a patient undergoing BMT. Bone Marrow Transplant 1995; 15: 785±786. 4. Tawfik OW, Papasian CJ, Dixon AY, Potter LM. Saccharomyces cerevisiae pneumonia in a patient with acquired immune deficiency syndrome. J Clin Microbiol 1989; 27: 1689±1691. 5. Bassetti S, Frei R, Zimmerli W. Fungemia with Saccharomyces cerevisiae after treatment with Saccharomyces boulardii. Am J Med 1998; 105: 71±72. 6. Perapoch J, Planes AM, Querol A et al. Fungemia with Saccharomyces cerevisiae in two newborns, only one of whom had been treated with ultra-levura. Eur J Clin Microbiol Infect Dis 2000; 19: 468±470. 7. Zerva L, Hollis RJ, Pfaller MA. In vitro susceptibility testing and DNA typing of Saccharomyces cerevisiae clinical isolates. J Clin Microbiol 1996; 34: 3031±3034. 8. McFarland LV. Saccharomyces boulardii is not Saccharomyces cerevisiae. Clin Infect Dis 1996; 22: 200±201. 9. McCullough MJ, Clemons KV, McCusker JH, Stevens DA. Species identification and virulence attributes of Saccharomyces boulardii (nom. inval.). J Clin Microbiol 1998; 36: 2613±2617. 10. Krause W, Matheis H, Wulf K. Fungaemia and funguria after oral administration of Candida albicans. Lancet 1969; 1: 598±599. 11. Hennequin C, Thierry A, Richard GF et al. Microsatellite typing as a new tool for identification of Saccharomyces cerevisiae strains. J Clin Microbiol 2001; 39: 551±559. 12. Saxen H, Virtanen M, Carlson P et al. Neonatal Candida parapsilosis outbreak with a high case fatality rate. Pediatr Infect Dis J 1995; 14: 776±781. 13. Bouakline A, Lacroix C, Roux N, Gangneux JP, Derouin F. Fungal contamination of food in hematology units. J Clin Microbiol 2000; 38: 4272± 4273. 14. Salonen JH, Richardson MD, Gallacher K et al. Fungal colonization of haematological patients receiving cytotoxic chemotherapy: emergence of azole-resistant Saccharomyces cerevisiae. J Hosp Infect 2000; 45: 293±301. 15. Olver WJ, Galloway A, Boswell TC, Russell NH. Sensitivity of S. cerevisiae in haematology patients. J Hosp Infect 2001; 47: 75.
Nosocomial transmission of Saccharomyces cerevisiae in bone marrow transplant patients W. J. Olver*, S. A. Jamesz, A. Lennardx, A. Galloway{, I. N. Robertsz, T. C. Boswell* and N. H. Russelly Departments of *Microbiology and yHaematology, City Hospital, Nottingham; zNational Collection of Yeast Cultures, Institute of Food Research, Norwich; Departments of xHaematology and {Microbiology, Royal Victoria Infirmary, Newcastle upon Tyne, UK Summary: Saccharomyces cerevisiae is an unusual cause of clinical infection. We describe three bone marrow transplant patients on a haematology unit who developed possible invasive disease with the organism. Two patients died and both these patients appeared to have a related strain of S. cerevisiae. Screening for S. cerevisiae from throat and stool samples revealed four further patients who were carriers. Genotyping of the invasive and carriage strains demonstrated an indistinguishable strain from patients who had been on the unit at the same time, suggesting cross-infection. & 2002 The Hospital Infection Society
Keywords: Saccharomyces cerevisiae; nosocomial transmission; bone marrow transplantation. Introduction Saccharomyces cerevisiae, also known as brewer's or baker's yeast, is relatively azole-resistant.1 It is widespread in nature and can be found on various plants, fruits and in soil. It can constitute part of the normal flora of the gastrointestinal tract in humans. Although previously thought to be non-pathogenic, there is increasing evidence that it can cause invasive disease in immunosuppressed patients.2±4 Infection has also been described following ingestion of commercial preparations of Saccharomyces boulardii.5,6 Case reports Patient 1 A 24 year-old female with chronic myeloid leukaemia (CML) diagnosed one year previously, underwent Received 22 July 2002; revised manuscript accepted 2 August 2002; published online 18 October 2002. Author for correspondence: Dr William J. Olver, Department of Microbiology, City Hospital, Hucknall Road, Nottingham NG5 1PB. Tel.: 44-115-9691169 ext 45572; Fax: 44-115-9627766; E-mail: [email protected]
0195±6701/02/120268 05 $35.00/0
matched, unrelated, donor bone marrow transplantation (MUD BMT) at Nottingham City Hospital in October 1999. She received prophylactic penicillin V, ofloxacin, co-trimoxazole, colistin, metronidazole, fluconazole and acyclovir, as per protocol. Her post-transplant course was complicated by Clostridium difficile diarrhoea, treated with oral metronidazole. Engraftment was satisfactory and there were no signs of graft-versus-host disease (GVHD). She was discharged home on day 29. On day 43 she was admitted to the haematology unit at the Royal Victoria Infirmary, Newcastle. She was pyrexial but no localizing signs of infection were found. Staphylococcus epidermidis sensitive to teicoplanin was isolated from venous blood. Her temperature did not settle despite broad-spectrum antibiotics and liposomal amphotericin, and over the following seven days she developed painful cervical lymphadenopathy and hepatosplenomegaly. Extensive microbiological and virological investigations were negative. On day 55 she had an episode of acute left upper quadrant pain and developed renal failure. S. cerevisiae was isolated from sputum, faeces and a vulval swab. Despite intensive supportive measures her condition continued to deteriorate and & 2002 The Hospital Infection Society
S. cerevisiae transmission in bone marrow transplant patients
269
she died of multi-organ failure on day 59 posttransplant. A post-mortem sample of lymph node sent for culture grew a heavy growth of S. cerevisiae. Culture of a specimen of lung did not grow any yeasts. Histology of the liver, spleen and lymph nodes revealed a high grade non-Hodgkins lymphoma representing a post-transplant lymphoproliferative disorder.
developed fever with no obvious source of infection. He made a good response to treatment with meropenem and amphotericin B for two weeks, although suspicions of a fungal chest infection were not confirmed by microbiology, histology or imaging. Engraftment was satisfactory and there were no signs of GVHD. He was discharged home on day 34. On day 62 he presented with fever, shivering and a cough productive of yellow sputum and was admitted to the haematology unit at Royal Victoria Infirmary, Newcastle. S. cerevisiae was isolated from sputum, throat and rectal swabs. Antifungal treatment was begun with liposomal amphotericin and flucytosine. His symptoms and signs resolved gradually and he remains well one year posttransplant.
Patient 2 A 29 year-old female with CML diagnosed 18 months previously, underwent MUD BMT at Nottingham City Hospital in October 1999. She received antimicrobial prophylaxis as per protocol (see above). Her post-transplant course was complicated by reactivation of cytomegalovirus (CMV), which was successfully treated with ganciclovir. On day 38 she was discharged home, with satisfactory engraftment and no signs of GVHD. On day 55 she was admitted to the haematology unit at the Royal Victoria Hospital, Newcastle with a febrile illness. On day 61 CMV antigenaemia testing showed further reactivation, and treatment with ganciclovir and foscarnet was started. On day 63 she again became pyrexial and interstitial shadowing was seen on chest X-ray. Blood cultures yielded viridans streptococci, Staphylococcus epidermidis and diphtheroids. Her temperature failed to settle despite appropriate antibiotics and liposomal amphotericin. Hepatic and renal function began to deteriorate. Bronchoscopy on day 75 was normal and bronchial washings were negative for all pathogens. On day 79 she developed sudden onset chest pain, shortness of breath and increasing jaundice. A yeast was isolated from a throat swab and bronchoalveolar lavage (BAL), which was identified as S. cerevisiae. No other pathogens were identified in the BAL. CMV antigenaemia was resolving. She was transferred to the intensive care unit for ventilation and supportive treatment but continued to deteriorate and died on day 91 post-transplant. A post-mortem was performed and cause of death was certified as multi-organ failure. There was no histological or microbiological evidence of an infective aetiology. Patient 3 A 27 year-old male with CML diagnosed two years previously, underwent MUD BMT at Nottingham City Hospital in November 1999. At day 10 he
Materials and methods Point prevalence survey Due to the rare occurrence of colonization and infections due to S. cerevisiae (not previously detected on either unit), a point prevalence study was performed in the haematology units of Nottingham City Hospital (11 patients) and Royal Victoria Infirmary, Newcastle (20 patients), to detect carriage of the organism from throat swabs and stool samples. This was performed in January 2000. Throat swabs were processed by breaking off the end of the swab into 1 mL nutrient broth. The sample was then agitated on a vortex mixer for 15 s. One drop of suspension was inoculated on to one half of a Sabouraud±chloramphenicol agar plate. Stool samples were processed by emulsifying a pea-sized portion of the sample in 5 mL of Ringer's solution. One drop of suspension was inoculated on to one half of a Sabouraud±chloramphenicol agar plate. Plates were spread for discrete colonies, incubated at 37 C in air and read at 24 and 48 h. Colonies were identified using the germ tube test with horse serum, and read after 2±4 h at 37 C in air. Negative germ tube isolates were then identified using API 20C AUX (bioMeÂrieux, UK). Typing Isolates from the three clinical cases (Patients 1±3) and those subsequently found to be carriers were genotyped at the Institute of Food Research, Norwich.
270
Multiplex polymerase chain reaction (PCR) Yeasts were incubated on YM (0.3% yeast extract, 0.3% malt extract, 0.5% peptone, 1% glucose) agar overnight at 37 C. For each isolate, a small sample of confluent yeast growth was picked off the plate using a sterile Gilson pipette tip and mixed vigorously with 10 mL sterile distilled water in a 0.5 mL microcentrifuge tube, which was stored on ice. To this cell suspension was then added 90 mL of a mixture containing 78 mL distilled water, 10 mL 10X Taq polymerase buffer (Life Technologies), 0.8 mL dNTPs, 0.8 mL primers and 0.5 mL Taq polymerase (5 U/ mL). The PCR primers were designed using conserved DNA sequences from the S. cerevisiae transposons Ty1, Ty2 and Ty3 and these primers have been patented by the Institute of Food Research, Norwich. Each PCR sample was overlaid with approximately 50 mL paraffin oil (Sigma). The PCR cycling parameters used for DNA amplification were: 92 C for 2 min, followed by two cycles of 92 C for 2 min, 52 C for 3 min, 72 C for 2 min, followed by 28 cycles of 90 C for 2 min, 52 C for 3 min, 72 C for 2 min. DNA precipitation To improve the overall quality of the resulting DNA fingerprints, each PCR sample was first concentrated/purified by ethanol precipitation, before being analysed by agarose gel electrophoresis. For each sample, 200 mL of 95% ice-cold ethanol was mixed with 10 mL of 3 M sodium acetate in a 0.5 mL microcentrifuge tube, and to this was added the entire (100 mL) PCR sample. This mix was briefly vortexed and stored overnight at ÿ20 C, to allow DNA precipitation. After defrosting, centrifugation was performed at 14 000 rpm for 25±30 min, to pellet DNA. The supernatant was removed and the pellet washed with 70% ethanol. After removing residual ethanol, the sample was vacuum-dried.
W. J. Olver et al.
for 2.5±3.0 h, stained in ethidium bromide (0.5 mg/mL) for 25 min and visualized using a UV transilluminator. Environmental investigations Potential routes of cross-infection were investigated by taking environmental samples and looking at hand carriage of yeasts by healthcare workers. Swabs premoistened in sterile saline were taken from a variety of environmental sites, including air vents, door handles, bedclothes and curtains (sweep plates), bathroom surfaces, sinks, commodes, telephones, computer keyboards and other equipment. They were processed in the same way as the throat swabs. A wide variety of medicines were sampled to see if any were contaminated with yeasts. Ten members of staff had fingerprint agar plates performed. All cultures were incubated for 72 h at 37 C. Results In the Nottingham unit, four of 11 patients were positive for S. cerevisiae from throat and/or stool samples, compared to none of the 20 in the Newcastle unit (P < 0.05). Genotyping results of the isolates from the three clinical cases (Patients 1±3) and the four carriers (Patients 4±7) are shown in Figure 1.
DNA rehydration The DNA pellet was re-suspended in 20 mL distilled water and incubated at 37 C for 10 min to aid rehydration. Gel analysis Fifteen microlitres of the purified PCR product was added to 5 mL loading dye (bromophenol blue), and 18 mL of this mixture was loaded on to a 15 20 cm 1.5% agarose±TBE gel. The gel was run at 125 V
Figure 1 Genotyping of S. cerevisiae isolates. Lane 1 is DNA marker, then (left to right) Patient 2, Patient 3, Patient 1, Patient 6, Patient 5, Patient 7 and Patient 4 (twice).
S. cerevisiae transmission in bone marrow transplant patients
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1 * 2 * 3 Patient 4 * 5 * 6 7 † Figure 2 Inpatient stays on Nottingham haematology unit 1999/2000. * Genotypically indistinguishable. y Genotypically closely related.
Isolates from Patients 1, 2, 4 and 5 were indistinguishable, suggesting possible cross-infection. Patient 7 had a closely related strain. The temporal relationship of the inpatient episodes for Patients 1±7 on the haematology/BMT unit is shown in Figure 2. There was a temporal overlap between Patients 1, 2, 4 and 5. Patient 4 had received an allogeneic peripheral blood stem-cell transplant for peripheral T-cell lymphoma in October 1999, and was being given penicillin V and ofloxacin prophylaxis. Patient 5 had undergone MUD BMT for myelodysplasia in October 1999. He had been treated for possible fungal chest infection one week prior to screening. Patient 6 had relapsed high-grade non-Hodgkin's lymphoma and was being treated with palliative chemotherapy. Prior to screening she had received 10 days of meropenem and amphotericin B for neutropenic fever. Patient 7 was diagnosed with acute myeloid leukaemia in December 1999 and treated conservatively with hydroxyurea. Prior to screening she had not received any antibiotics or antifungals. Two of the rooms where curtains were sampled (using a `sweep' directly with the plate) yielded very scanty yeasts, which were identified as Candida parapsilosis. On the hands of one member of staff two fingers grew a single yeast colony, also identified as C. parapsilosis. All other environmental samples were negative after 72 h incubation. Discussion S. cerevisiae is an uncommon but increasingly recognized cause of infection in immunosuppressed patients.2±4 Although the three cases described here did not provide conclusive (histological) evidence of invasive disease, S. cerevisiae was nevertheless grown
in pure culture from each patient in the absence of any other pathogens. Because it has been longconsidered non-pathogenic, the epidemiology of the organism in the clinical setting has not been extensively studied. However, genotyping has been undertaken on a collection of 76 surveillance isolates collected from a bone marrow transplant unit.7 Pulsed-field gel electrophoresis (PFGE) typing revealed 62 distinct genotypes, but clusters of indistinguishable isolates were identified among different patients hospitalized at the same time, indicating possible nosocomial transmission. The source of the organism in cases of infection due to S. cerevisiae is not usually identified, but there have been reports of infection secondary to ingestion of S. boulardii, commercial preparations of which are marketed as Ultra-Levure in France (Biocodex, Montrouge) and Perenterol in the USA (Biocodex, Seattle) for the treatment of antibiotic-associated diarrhoea.5,6 Although there has been (taxonomic) controversy over whether S. cerevisiae is synonymous with S. boulardii,8 a recent study showed that commercial strains of S. boulardii (UltraLevure) were genotypically indistinguishable from S. cerevisiae.9 Nosocomial infection with S. cerevisiae was first confirmed in a report of a neonate who was treated with S. boulardii and subsequently developed infection (positive blood culture).6 A second neonate in a nearby cot then also developed bacteraemia with S. cerevisiae. Mitochondrial DNA restriction analysis and chromosomal DNA profiles [using CHEF (contour-clamped homogeneous electric-field) gel electrophoresis] revealed that the two clinical strains were indistinguishable. In the possible invasive cases of infection that we have described, none of the patients had been treated with S. boulardii. Interestingly, in the case of
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invasive infection where the S. cerevisiae genotype was different (Patient 3), the patient later reported to be very fond of a locally brewed beer. Although there is no direct evidence, it is possible that this strain of S. cerevisiae originated from beer, as it is known that ingestion of yeast can produce fungaemia and funguria.10 Microsatellite genotyping has been used to show that a cluster of clinical isolates shared their genotype with a bakery strain of S. cerevisiae, suggesting intestinal colonization following ingestion.11 The four patients whose isolates of S. cerevisiae were genotypically indistinguishable were on the haematology unit at the same time. The mode of transmission is unclear but is may be faecal±oral. Sampling results did not show an obvious environmental source for S. cerevisiae. C. parapsilosis was isolated from two sets of curtains and the hand of a member of staff. This organism has been found previously on the hands of hospital staff, including an instance where nosocomial spread occurred and caused invasive infection in a neonatal intensive care unit.12 However, there are no reports of S. cerevisiae being isolated from the hands of staff or the hospital environment. Although food on the unit was not sampled, this is potentially a source of infection. S. cerevisiae can survive in sugary foodstuffs such as jam, fruit and fruit drinks. Bread can become contaminated after baking (i.e., from the baker's hands), and beer can contain the yeast even though it is normally pasteurized. However, a study of a variety of food in a French haematology unit did not yield any S. cerevisiae,13 with mainly filamentous fungi being isolated. Length of stay in hospital has been identified as a risk factor for acquisition of S. cerevisiae in patients with haematological malignancy.14 Isolates were found to be highly resistant to azole antifungals, and it was postulated that this might have been due to extensive use of these antifungals. However, the isolates from the invasive cases discussed in this study were sensitive to fluconazole and had only intermediate resistance to itraconazole, as previously reported.15 Further epidemiological studies are now planned to examine the carriage of S. cerevisiae over time. Isolates will be genotyped and patient factors recorded, to assess whether cross-infection has occurred, and what patient factors increase the risk of acquisition of this yeast species.
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