- Email: [email protected]
Kitchens as a source of Aspergillus niger infection
Journal
o.f Hospital
Kitchens
Infection
(1996)
32,
as a source
191-198
of Aspergitlus
K. W. Loudon”, A. P. Coke*, B. A. Oppenheim-f
niger
infection
J. P. Burnie”, A. J. Shaw-f, and C. Q. Morrisj:
“Department of Medical Microbiology, 2nd Floor Clinical Sciences Building, Manchester Healthcare Trust, Oxford Road, Manchester Ml3 9WL, UK; Withington Hospital, West Disdbury, j-Department of Microbiology, Manchester M20 8LR, UK; and $Department of Medical Oncology, Christie Hospital NHS Trust, Wilmslow Road, Withington, Manchester M20 9BM, UK Received
16 August
1995;
revised manuscript
accepted
21 November
1995
Summary: In this study we investigated the epidemiology of a cluster of cutaneous infections owing to Aspergillus niger, which occurred in neutropenic patients in a bone marrow transplant unit. Heavy environmental contamination with the mould was found in the ward kitchen adjacent to the unit. The clinical and environmental isolates were typed by random amplification of polymorphic DNA (RAPD), which showed one of the patients was infected with the same strain as that isolated repeatedly from the kitchen area. In another case, contaminated stockinette material was implicated as the source of infection. Thorough cleaning of the ward kitchen resulted in no further cases on the unit. This highlights the fact that aspergilli may spread to patients by air, food or other vehicles, and underlines the importance of searching for a source and ensuring high levels of hospital hygiene are maintained. Keywords: morphic
Aspergillus DNA (R.4PD).
niger;
epidemiology;
random
amplification
of
poly-
Introduction Despite advances in antifungal therapy, aspergilli are being increasingly reported as an important cause of mortality and morbidity among neutropenic patients.’ This has h eightened interest in prevention, either by chemoprophylaxis or the introduction of filtered air systems.2 The latter concept is supported by the observations that 90”/0 of cases of invasive aspergillosis either start in, or are confined to, the lung, spores occur in the environment, and cases occur in clusters around hospital building works.‘,“-” The weaknesses of this approach are that when patients are outside the filtered air unit, they are exposed to spores. The absolute elimination of spores is costly and difficult to achieve, and the filters Correspondence
to: K. UT. Loudon
191
192
K. W. Loudon
et at.
required may become contaminated and act as a source of infection.*a6 However, there can be no doubt that this aggressive approach, when introduced into an individual unit can lead to a reduction in the number of cases.7 Against this background, the Christie Hospital noted a cluster of three cases of cutaneous aspergillosis owing to Aspergillus niger. The dominant mould within the hospital had been Aspergillusfumigatus. A. niger is most commonly implicated in otomycosis and otitis externa in immunocompetent individuals’ and is only rarely reported to cause severe infections, such as fungal peritonitis.’ It has been isolated from invasive skin infections,” and has also been associated with the application of contaminated adhesive tapes or arm boards in neutropenic patients.” Aspergillus spp. have been isolated from numerous food sources, including pepper.12 The occurrence of a cluster of A. niger infection, thus prompted an intensive search for a source. In order to establish a link between isolates from individual patients and a potential source, it is necessary to type the micro-organism. Early phenotypic based systems such as immunoblotting’3 have, in the case of A. fumigatus, been replaced by genotypic based systems such as restriction fragment length polymorphisms (RFLPs),‘~“~ Southern blotting with defined probes16 and the random amplification of polymorphic DNA (RAPD).17,‘s The RFLP analysis of 23 isolates of A. niger generated only two types and these were proposed as being distinct species.” RAPD has been used in our laboratory to establish the genetic heterogeneity of isolates from within a single aspergilloma and the existence of cross-infection within a cluster of cases of invasive aspergillosis due to A. fumigatus.‘s,20 The primers developed for this were applied to A. niger to establish the epidemiology of the disease and facilitate the introduction of suitable crossinfection control measures. Material
and methods
Isolates Isolates of A. niger were identified by their standard cultural characteristics and appearance under the microscope. They were obtained from the skin lesions of the patients and the environment. A. niger NCPF 2022 was the control isolate. DNA was prepared from the isolates as described previously.i4 Typing method The isolates were typed by RAPD with two separate (5’-3’) primers: GCG CAC GG and GCT GGT GG. The reactions were carried out at a magnesium ion concentration of 3mM with Amplitaq DNA polymerase (Roche) as described in detail elsewhere.‘8,20 Each isolate was examined three times.
Kitchens
as a source
of A. niger
193
Patient details Patient I was a 38-year-old woman with multiple myeloma who was admitted for an allogeneic bone marrow transplant in September 1992. While undergoing conditioning treatment, she sustained an uncomplicated pathological fracture of the left humeral shaft, which was stabilized in a plaster cast. When the plaster was removed, there was a necrotic ulcer from which Rhizopus oryzae and A. niger (isolates 1 and 2) were isolated (October 1992). A, niger was also isolated from a nose swab. This case has previously been reported in detail elsewhere.*’ Patient 2 was a 74-year-old man with acute myeloid leukaemia who developed a lesion on his left cheek whilst undergoing chemotherapy. It increased gradually in size until it had a 1 cm diameter black central area surrounded by erythema. Skin scrapings grew A. niger (isolate 3) and amphotericin B was started (5th October 1993). Liposomal amphotericin B was substituted 48 h later because of electrolyte and renal problems. l‘he lesion improved during the next seven days and the patient was discharged on oral itraconazole. At outpatient review, one-month later, the lesion had resolved. Patient 3 was a 55-year-old man with acute myeloid leukaemia diagnosed in 1988. This relapsed in November 1993 and he restarted chemotherapy. In January 1994 he received high-dose cytosine arabinoside followed by granulocyte colony stimulating factor (G-CSF). During the neutropenic phase he developed a blackish papule on the left cheek. Skin scrapings grew A. niger (isolates 4 and 5). He was commenced on amphotericin B with the rapid resolution of the lesion and was later discharged home on oral itraconazole. Unfortunately, his leukaemia failed to remit and he died in February 1994. Hospital setting The three patients were treated on the lo-bed adult leukaemia and bone marrow transplant unit at the Christie Hospital, Manchester between September 1992 and January 1994. Patient 1 was nursed in an isolation suite with high efficiency particulate air (HEPA) filtered air under positive pressure. Patients 2 and 3 were nursed in the same single room, which did not have filtered air. Both developed lesions on the left cheek, which was the side facing the windows. Epidemiological
inaestigations
Air Routine air sampling was carried out monthly with a Biotest RCS centrifugal air sampler (280 L/ min) at 4096 rpm for 8 min. Culture was performed on Rose Bengal agar strips (code 941-200) at 30°C for 48 h. Settle plates (Sabouraud’s dextrose agar containing chloramphenicol) (Oxoid code
194
K. W. Loudon
et al.
CM41) were placed on selected areas on the unit for 1 h and incubated at 30°C for 48 h. This was done in October 1992 (after case l), in September and October 1993 (after case 2) and in January 1994 (after case 3). Fomites and food Numerous samples from fomites were taken from the inanimate surfaces from within the rooms used by the patients, the ward and adjoining kitchen areas. This included the food items found in the kitchens including stocks of tea in a caddy. The stockinette of the type used on patient one was also tested. All swabs were dry, charcoal covered cotton wool swabs on a wooden stick (Medical Wire and Equipment Company MWllO). They were transported in Stuart’s transport medium (Oxoid code CMlll) and inoculated directly onto Sabouraud’s dextrose agar (containing chloramphenicol) plates (Oxoid code CM41). These were incubated for 48 h at 30°C.
Results
A. niger and Rhizopus sp. were isolated from the stockinette and Rhixopus sp. from the environment samples and settle plates from the room. In September 1993, settle plates grew two colonies of A. niger from one of the isolation rooms. This was the first time that it had been found in the unit. A swab from the air vent between the air lock of one of the isolation rooms also grew a single colony. None of these isolates were available for typing. In October 1993, following a systematic cleaning of the ward, further environmental swabs were taken. This time the kitchen adjacent to the ward was also sampled. A. niger was isolated from the top of a small fridge (isolate 6), a large fridge (isolates 7 and S), the side vent of an ice making machine (isolate 9), the large socket of a microwave door (isolate 10) and a tea caddy (isolate 11). A week later, after further cleaning a repeat swabbing of the kitchen yielded A. niger from a small ceiling airvent (isolate 12), the rear of the microwave (isolate 13) and the fire blanket holder (isolate 14). Two months later, following further cleaning, only a single isolate was obtained from the side vent of the ice maker. Nine swabs from the bedroom of patient 3, taken simultaneously, failed to isolate A. niger. The typing results demonstrated that all isolates typed, and reproducibility was excellent. Primer GCG CAC GG distinguished four types. All the environmental isolates and isolates 4 and 5 from Case 3 were identical in sharing bright bands at 1,082, 0.975, 0.872, O-737, O-704 and O-603 kb (Figure 1, isolates 4-l 1, tracks 5-l 2 respectively). Separate types were shown by A. niger NCPF 2022 (Figure 1, track 13), isolates 1 and 2 from case 1 (Figure 1, track 3 and 4) and isolate 3 from case 2 (Figure 1, track 2). Isolates 1 and 2 had bright bands at 1.590, 1.370, 1.353, 1.082 kb, a cluster of five bands above 0.737 kb, and a double band at 0.603 kb. Isolate 3 had bright bands at 1.590, 1.380, 1.353, 1.082, 0.923, 0.737kb,
Kitchens
as a source
of A. niger
195
Figure 1. RAPD fingerprints generated by primer GCG CAC GG (5’-3’) of isolates from Case 1 (tracks 3 and 4), Case 2 (track 2), Case 3 (tracks 11 and 12) and environmental isolates (tracks S-10). A. niger NCPF 2022 (track 13). Molecular mass markers Hae IIIdigested cp x 174 (track 14) and h Hind III/EcoRI (track 1). Sizes of the RAPD products (kilobases) are given on the left.
and a single band at 0.603 kb. The NCPF strain had bright bands at 1.380, 1.217, 0.737, and a double band at 0.975 kb. Primer GCT GGT GG confirmed the same picture with a single pattern for the environmental isolates and isolates 4 and 5 from patient 3. There was a triple band above 1.590 kb, bright bands at 1.215, 1.146, 1.010 kb and a double band at 0.737 kb (Figure 2, isolates 4-11, tracks 5-12). Isolates 1 and 2 were identical (Figure 2, tracks 3 and 4) with a double band at 1.353 kb and bright bands at 1.146, 1 .OlO, 0.872 and 0.776 kb. Isolate 3 had only a bright double band at 1.353 kb (Figure 2, track 2). The A. niger NCPF 2022 was again unique (Figure 2, track 13), with bright bands at 1.400, 1.353, 1.143, 1.010 and 0.935 kb. The molecular weight markers used were Hae III-digested ox174 and h Hind III/EcoRI. Discussion
This paper reports a cluster of three cases of infection due to A. niger occurring in a hospital where infection due to this pathogen had not previously been seen. Typing by RAPD was equally effective with both primers and showed that each individual case was due to a different strain of A. niger. Where two isolates were available from the same patient, these were indistinguishable. In patient 1, the lesion occurred on a non-exposed area of skin and A. niger and Rhizopus sp. were isolated from a sample of stockinette material similar to that covering the wound. This, combined with the timing of the case before A. niger was isolated from the unit, suggests that it was a separate discrete event.
196
K. W. Loudon
I
2
3
a
5
2
7
et al.
2
9
10
II
12
73
ia
Figure 2. RAPD fingerprints generated by primer GCT GGT GG (5’-3’) of isolates from Case 1 (tracks 3 and 4), Case 2 (track 2), Case 3 (tracks 11 and 12) and environmental isolates (tracks S-10). A. niger NCPF 2022 (track 13). Molecular mass markers Hae IIIdigested cp x 174 (track 14) and h Hind III/EcoRI (track 1). Sizes of the RAPD products (kilobases) are given on the left.
In the majority of outbreaks of invasive aspergillosis the pathogen is A. fumigatus or Aspergillus$avus, the route of transmission is via the air and the environmental source, if identified, is associated with the air ~upply.‘,~* It has been shown using RAPD that, for A. funzigatus, clusters of cases can be due to the same strain.” Typing using Southern blotting has shown fomite sources occur within a unit.23 In the present episode the environmental isolates were indistinguishable by RAPD suggesting that a single strain had colonized multiple sources within the kitchen area. The source could have been food because A. niger was isolated from the tea caddy and has been repeatedly isolated from pepper and other food stuffs.‘2*24*25 The isolates obtained from Case 3 were different from the isolate from Case 2. This eliminated the possibility of direct transmission from Case 2 to Case 3 despite both patients being nursed in the same room. Environmental isolates similar to that found in Case 2 may have existed in the unit and been missed in the limited samples taken for testing. In both cases the lesion involved the cheek and there was no evidence of pulmonary involvement. In a neutropenic patient, spore inhalation usually leads to a primary lesion in the lung, with secondary dissemination to skin and central nervous system.3 The confinement of the lesions to the skin suggests local inoculation. It is possible that the Aspergillus may have been carried from the ward kitchen area to the patient via contaminated food or fomites. Local inoculation may have occurred directly onto the surface of the skin or from the inside of the mouth onto the buccal mucosa via contaminated food or utensils. Mucositis is a common complication of chemotherapy in these patients,
Kitchens
as a source
of A. niger
197
and a compromised oral mucosa would provide an ideal portal of entry for the mould. However, neither of the patients was noted to have lesions inside the mouth making it more likely that direct inoculation had occurred onto the skin surface. A. niger was isolated from the air within the unit but this was associated with Case 2 and not Case 3. These observations, in conjuction with the heavy contamination of the kitchen area, suggest that Case 3 may have received food or fomites contaminated by A. Niger, which acted as the source for the localized cutaneous infection. Since the realization that the ward kitchen may have been the source of the infection and the tightening of the cross-infection control measures to include this area, there have been no further cases. This episode underlines the importance of perceiving aspergilli as not solely airborne pathogens. It also emphasizes the importance of rigorously monitoring the small kitchen areas commonly found adjacent to wards as these may, inadvertently, act as a source of infection. We would like to thank Morgenstern, Consultant K. W. Louden received
Dr J. H. Scarffe, Consultant Medical Haematologist for giving us permission a grant from the Peel Trust.
Oncologist, to report
and these
Dr G. R. cases. Dr
References
3. 4. 5. 6. 7. 8.
9.
10. 11. 12. 13. 14. 15.
Bodey GP, Vartivarian S. Aspergillosis. EurJ Clin Microbial Infect Dis 1989; 8: 413-437. Rhame FS. Prevention of nosocomial aspergillosis. J Hosp Infect 1991; 18: 466-472. Young RC, Bennet JE, Vogel CL, Carbone PP, de Vita VT. Aspergillosis. The spectrum of disease in 98 patients. Medicine. 1970; 49: 147-173. Goodley JM, Clayton YM, Hay RJ. Environmental sampling for aspergilli during building construction on a hospital site. r Hosp Infect 1994; 26: 27-35. Walsh TJ, Dixon DM. Nosocomial aspergillosis environmental microbiology, hospital epidemiology, diagnosis and treatment. Eur J Epidemiol 1989; 5: 131-142. Wadowsy RM, Benner SM. Brief report: distribution of the genus Aspergillus in hospital room air conditioners. Infect Control 1987; 8: 516-518. Barnes RA, Rogers TR. Control of an outbreak of nosocomial aspergillosis by laminar air-flow isolation. J Hasp Infect 1989; 14: 89-94. Talwar P, Chakrabarti A, Poonamji Kaur, Pahwa RK, Ashak Mitall, Mehra YN. Fungal infections of ear with special reference to chronic suppurative otitis media Mycopathologia 1988; 104: 47-50. Bibashi E, Papagianni A, Kelesidis A, Antoniadou R, Papadimitriou M. Peritonitis due to Aspergillus nigev in a patient on continuous ambulatory peritoneal dialysis shortly after kidney graft rejection. Nephrol Dial Transplant 1993; 8: 185-187. Panke TW, McManus AT, Spebar lVIJ. Infection of a burn wound by Aspergillus niger. Am J Clin Path01 1979; 72: 230-232. McCarty JM, Flam MS, Pullen G, Jones R, Kassel SH. Outbreak of primary cutaneous aspergillosis related to intravenous arm boards. J Pediatr 1986; 108: 721-724. DeBock R, Gyssens I, Peeterman M, Nolard N. Aspergillus in pepper. Lancet 1989; ii: 331-332. Burnie JP, Matthews RC, Clark I, Milne LJR. Immunoblot fingerprinting Aspergillus fumigatus. J Immunol Methods 1989; 118: 179-186. Burnie JP, Coke A, Matthews RC. Restriction endonuclease analysis of Aspergillus fumigatus DNA. J Clin Path01 1992; 45: 324-327. Denning DW, Clemons KV, Hanson LH, Stevens DA. Restriction endonuclease analysis of total cellular DNA of Aspergillus fumigatus isolates of geographically and epidemiologically diverse origin. J Infect Dis 1990; 162: 1151-l 158.
198 16. 17.
18.
19.
20. 21.
22. 23. 24. 25.
K. W. Loudon
et al.
Girardin H, Latge JP, Srikantha T, Morrow B, Sol1 DR. Development of DNA probes to fingerprinting Aspergillus fumigatus. J Clin Microbial 1993; 31: 1547-1554. Aufauvre-Brown A, Cohen J, Holden DW. Use of randomly amplified polymorphic DNA markers to distinguish isolates of Aspergillus fumigatus. J Clin Microbial 1992; 30: 2991-2993. Loudon KW, Burnie JP, Coke AP, Matthews RC. Application of polymerase chain reaction to fingerprinting Aspergillus fumigatus by random amplification of polymorphic DNA. J Clin Microbial 1993; 31: 1117-1121. Margo A, Kusters-van Someren, SRA, Visser J. The use of RFLP analysis in classification of the black Aspergilli: reinterpretation of the Aspergillus niger aggregate. Curr Genet 1991; 19: 21-26. Loudon KW, Coke AP, Burnie JP, Lucas GS, Liu Yin JA. Invasive aspergillosis: clusters and sources? J Med Vet Mycol 1994; 32: 217-224. Johnson AS, Ranson M, Scarffe JH, Morgenstern GR, Shaw AJ, Oppenheim BA. Cutaneous infection with Rhizopus oryzae and Aspergillus niger following bone marrow transplantation. J Hosp Infect 1993; 25: 293-296. Solomon WR, Burge HP, Boise JR. Airborne Aspergillus fumigatus levels outside and within a large clinical centre. J Allergy Clin Immunol 1978; 62: 56-60. Girardin H, Sarfati J, Traore F, Dupouy Camet J, Derouin F, Latge JP. Molecular epidemiology of nosocomial invasive Aspergillosis. J Clin Microbial 1994; 32: 684-690. Vargas S, Hughes WT, Giannini M. Aspergillus in pepper. Lancet 1989; ii: 881. Eccles NK, Scott GM. Aspergillus in pepper. Lancet 1992; 399: 618.
o.f Hospital
Kitchens
Infection
(1996)
32,
as a source
191-198
of Aspergitlus
K. W. Loudon”, A. P. Coke*, B. A. Oppenheim-f
niger
infection
J. P. Burnie”, A. J. Shaw-f, and C. Q. Morrisj:
“Department of Medical Microbiology, 2nd Floor Clinical Sciences Building, Manchester Healthcare Trust, Oxford Road, Manchester Ml3 9WL, UK; Withington Hospital, West Disdbury, j-Department of Microbiology, Manchester M20 8LR, UK; and $Department of Medical Oncology, Christie Hospital NHS Trust, Wilmslow Road, Withington, Manchester M20 9BM, UK Received
16 August
1995;
revised manuscript
accepted
21 November
1995
Summary: In this study we investigated the epidemiology of a cluster of cutaneous infections owing to Aspergillus niger, which occurred in neutropenic patients in a bone marrow transplant unit. Heavy environmental contamination with the mould was found in the ward kitchen adjacent to the unit. The clinical and environmental isolates were typed by random amplification of polymorphic DNA (RAPD), which showed one of the patients was infected with the same strain as that isolated repeatedly from the kitchen area. In another case, contaminated stockinette material was implicated as the source of infection. Thorough cleaning of the ward kitchen resulted in no further cases on the unit. This highlights the fact that aspergilli may spread to patients by air, food or other vehicles, and underlines the importance of searching for a source and ensuring high levels of hospital hygiene are maintained. Keywords: morphic
Aspergillus DNA (R.4PD).
niger;
epidemiology;
random
amplification
of
poly-
Introduction Despite advances in antifungal therapy, aspergilli are being increasingly reported as an important cause of mortality and morbidity among neutropenic patients.’ This has h eightened interest in prevention, either by chemoprophylaxis or the introduction of filtered air systems.2 The latter concept is supported by the observations that 90”/0 of cases of invasive aspergillosis either start in, or are confined to, the lung, spores occur in the environment, and cases occur in clusters around hospital building works.‘,“-” The weaknesses of this approach are that when patients are outside the filtered air unit, they are exposed to spores. The absolute elimination of spores is costly and difficult to achieve, and the filters Correspondence
to: K. UT. Loudon
191
192
K. W. Loudon
et at.
required may become contaminated and act as a source of infection.*a6 However, there can be no doubt that this aggressive approach, when introduced into an individual unit can lead to a reduction in the number of cases.7 Against this background, the Christie Hospital noted a cluster of three cases of cutaneous aspergillosis owing to Aspergillus niger. The dominant mould within the hospital had been Aspergillusfumigatus. A. niger is most commonly implicated in otomycosis and otitis externa in immunocompetent individuals’ and is only rarely reported to cause severe infections, such as fungal peritonitis.’ It has been isolated from invasive skin infections,” and has also been associated with the application of contaminated adhesive tapes or arm boards in neutropenic patients.” Aspergillus spp. have been isolated from numerous food sources, including pepper.12 The occurrence of a cluster of A. niger infection, thus prompted an intensive search for a source. In order to establish a link between isolates from individual patients and a potential source, it is necessary to type the micro-organism. Early phenotypic based systems such as immunoblotting’3 have, in the case of A. fumigatus, been replaced by genotypic based systems such as restriction fragment length polymorphisms (RFLPs),‘~“~ Southern blotting with defined probes16 and the random amplification of polymorphic DNA (RAPD).17,‘s The RFLP analysis of 23 isolates of A. niger generated only two types and these were proposed as being distinct species.” RAPD has been used in our laboratory to establish the genetic heterogeneity of isolates from within a single aspergilloma and the existence of cross-infection within a cluster of cases of invasive aspergillosis due to A. fumigatus.‘s,20 The primers developed for this were applied to A. niger to establish the epidemiology of the disease and facilitate the introduction of suitable crossinfection control measures. Material
and methods
Isolates Isolates of A. niger were identified by their standard cultural characteristics and appearance under the microscope. They were obtained from the skin lesions of the patients and the environment. A. niger NCPF 2022 was the control isolate. DNA was prepared from the isolates as described previously.i4 Typing method The isolates were typed by RAPD with two separate (5’-3’) primers: GCG CAC GG and GCT GGT GG. The reactions were carried out at a magnesium ion concentration of 3mM with Amplitaq DNA polymerase (Roche) as described in detail elsewhere.‘8,20 Each isolate was examined three times.
Kitchens
as a source
of A. niger
193
Patient details Patient I was a 38-year-old woman with multiple myeloma who was admitted for an allogeneic bone marrow transplant in September 1992. While undergoing conditioning treatment, she sustained an uncomplicated pathological fracture of the left humeral shaft, which was stabilized in a plaster cast. When the plaster was removed, there was a necrotic ulcer from which Rhizopus oryzae and A. niger (isolates 1 and 2) were isolated (October 1992). A, niger was also isolated from a nose swab. This case has previously been reported in detail elsewhere.*’ Patient 2 was a 74-year-old man with acute myeloid leukaemia who developed a lesion on his left cheek whilst undergoing chemotherapy. It increased gradually in size until it had a 1 cm diameter black central area surrounded by erythema. Skin scrapings grew A. niger (isolate 3) and amphotericin B was started (5th October 1993). Liposomal amphotericin B was substituted 48 h later because of electrolyte and renal problems. l‘he lesion improved during the next seven days and the patient was discharged on oral itraconazole. At outpatient review, one-month later, the lesion had resolved. Patient 3 was a 55-year-old man with acute myeloid leukaemia diagnosed in 1988. This relapsed in November 1993 and he restarted chemotherapy. In January 1994 he received high-dose cytosine arabinoside followed by granulocyte colony stimulating factor (G-CSF). During the neutropenic phase he developed a blackish papule on the left cheek. Skin scrapings grew A. niger (isolates 4 and 5). He was commenced on amphotericin B with the rapid resolution of the lesion and was later discharged home on oral itraconazole. Unfortunately, his leukaemia failed to remit and he died in February 1994. Hospital setting The three patients were treated on the lo-bed adult leukaemia and bone marrow transplant unit at the Christie Hospital, Manchester between September 1992 and January 1994. Patient 1 was nursed in an isolation suite with high efficiency particulate air (HEPA) filtered air under positive pressure. Patients 2 and 3 were nursed in the same single room, which did not have filtered air. Both developed lesions on the left cheek, which was the side facing the windows. Epidemiological
inaestigations
Air Routine air sampling was carried out monthly with a Biotest RCS centrifugal air sampler (280 L/ min) at 4096 rpm for 8 min. Culture was performed on Rose Bengal agar strips (code 941-200) at 30°C for 48 h. Settle plates (Sabouraud’s dextrose agar containing chloramphenicol) (Oxoid code
194
K. W. Loudon
et al.
CM41) were placed on selected areas on the unit for 1 h and incubated at 30°C for 48 h. This was done in October 1992 (after case l), in September and October 1993 (after case 2) and in January 1994 (after case 3). Fomites and food Numerous samples from fomites were taken from the inanimate surfaces from within the rooms used by the patients, the ward and adjoining kitchen areas. This included the food items found in the kitchens including stocks of tea in a caddy. The stockinette of the type used on patient one was also tested. All swabs were dry, charcoal covered cotton wool swabs on a wooden stick (Medical Wire and Equipment Company MWllO). They were transported in Stuart’s transport medium (Oxoid code CMlll) and inoculated directly onto Sabouraud’s dextrose agar (containing chloramphenicol) plates (Oxoid code CM41). These were incubated for 48 h at 30°C.
Results
A. niger and Rhizopus sp. were isolated from the stockinette and Rhixopus sp. from the environment samples and settle plates from the room. In September 1993, settle plates grew two colonies of A. niger from one of the isolation rooms. This was the first time that it had been found in the unit. A swab from the air vent between the air lock of one of the isolation rooms also grew a single colony. None of these isolates were available for typing. In October 1993, following a systematic cleaning of the ward, further environmental swabs were taken. This time the kitchen adjacent to the ward was also sampled. A. niger was isolated from the top of a small fridge (isolate 6), a large fridge (isolates 7 and S), the side vent of an ice making machine (isolate 9), the large socket of a microwave door (isolate 10) and a tea caddy (isolate 11). A week later, after further cleaning a repeat swabbing of the kitchen yielded A. niger from a small ceiling airvent (isolate 12), the rear of the microwave (isolate 13) and the fire blanket holder (isolate 14). Two months later, following further cleaning, only a single isolate was obtained from the side vent of the ice maker. Nine swabs from the bedroom of patient 3, taken simultaneously, failed to isolate A. niger. The typing results demonstrated that all isolates typed, and reproducibility was excellent. Primer GCG CAC GG distinguished four types. All the environmental isolates and isolates 4 and 5 from Case 3 were identical in sharing bright bands at 1,082, 0.975, 0.872, O-737, O-704 and O-603 kb (Figure 1, isolates 4-l 1, tracks 5-l 2 respectively). Separate types were shown by A. niger NCPF 2022 (Figure 1, track 13), isolates 1 and 2 from case 1 (Figure 1, track 3 and 4) and isolate 3 from case 2 (Figure 1, track 2). Isolates 1 and 2 had bright bands at 1.590, 1.370, 1.353, 1.082 kb, a cluster of five bands above 0.737 kb, and a double band at 0.603 kb. Isolate 3 had bright bands at 1.590, 1.380, 1.353, 1.082, 0.923, 0.737kb,
Kitchens
as a source
of A. niger
195
Figure 1. RAPD fingerprints generated by primer GCG CAC GG (5’-3’) of isolates from Case 1 (tracks 3 and 4), Case 2 (track 2), Case 3 (tracks 11 and 12) and environmental isolates (tracks S-10). A. niger NCPF 2022 (track 13). Molecular mass markers Hae IIIdigested cp x 174 (track 14) and h Hind III/EcoRI (track 1). Sizes of the RAPD products (kilobases) are given on the left.
and a single band at 0.603 kb. The NCPF strain had bright bands at 1.380, 1.217, 0.737, and a double band at 0.975 kb. Primer GCT GGT GG confirmed the same picture with a single pattern for the environmental isolates and isolates 4 and 5 from patient 3. There was a triple band above 1.590 kb, bright bands at 1.215, 1.146, 1.010 kb and a double band at 0.737 kb (Figure 2, isolates 4-11, tracks 5-12). Isolates 1 and 2 were identical (Figure 2, tracks 3 and 4) with a double band at 1.353 kb and bright bands at 1.146, 1 .OlO, 0.872 and 0.776 kb. Isolate 3 had only a bright double band at 1.353 kb (Figure 2, track 2). The A. niger NCPF 2022 was again unique (Figure 2, track 13), with bright bands at 1.400, 1.353, 1.143, 1.010 and 0.935 kb. The molecular weight markers used were Hae III-digested ox174 and h Hind III/EcoRI. Discussion
This paper reports a cluster of three cases of infection due to A. niger occurring in a hospital where infection due to this pathogen had not previously been seen. Typing by RAPD was equally effective with both primers and showed that each individual case was due to a different strain of A. niger. Where two isolates were available from the same patient, these were indistinguishable. In patient 1, the lesion occurred on a non-exposed area of skin and A. niger and Rhizopus sp. were isolated from a sample of stockinette material similar to that covering the wound. This, combined with the timing of the case before A. niger was isolated from the unit, suggests that it was a separate discrete event.
196
K. W. Loudon
I
2
3
a
5
2
7
et al.
2
9
10
II
12
73
ia
Figure 2. RAPD fingerprints generated by primer GCT GGT GG (5’-3’) of isolates from Case 1 (tracks 3 and 4), Case 2 (track 2), Case 3 (tracks 11 and 12) and environmental isolates (tracks S-10). A. niger NCPF 2022 (track 13). Molecular mass markers Hae IIIdigested cp x 174 (track 14) and h Hind III/EcoRI (track 1). Sizes of the RAPD products (kilobases) are given on the left.
In the majority of outbreaks of invasive aspergillosis the pathogen is A. fumigatus or Aspergillus$avus, the route of transmission is via the air and the environmental source, if identified, is associated with the air ~upply.‘,~* It has been shown using RAPD that, for A. funzigatus, clusters of cases can be due to the same strain.” Typing using Southern blotting has shown fomite sources occur within a unit.23 In the present episode the environmental isolates were indistinguishable by RAPD suggesting that a single strain had colonized multiple sources within the kitchen area. The source could have been food because A. niger was isolated from the tea caddy and has been repeatedly isolated from pepper and other food stuffs.‘2*24*25 The isolates obtained from Case 3 were different from the isolate from Case 2. This eliminated the possibility of direct transmission from Case 2 to Case 3 despite both patients being nursed in the same room. Environmental isolates similar to that found in Case 2 may have existed in the unit and been missed in the limited samples taken for testing. In both cases the lesion involved the cheek and there was no evidence of pulmonary involvement. In a neutropenic patient, spore inhalation usually leads to a primary lesion in the lung, with secondary dissemination to skin and central nervous system.3 The confinement of the lesions to the skin suggests local inoculation. It is possible that the Aspergillus may have been carried from the ward kitchen area to the patient via contaminated food or fomites. Local inoculation may have occurred directly onto the surface of the skin or from the inside of the mouth onto the buccal mucosa via contaminated food or utensils. Mucositis is a common complication of chemotherapy in these patients,
Kitchens
as a source
of A. niger
197
and a compromised oral mucosa would provide an ideal portal of entry for the mould. However, neither of the patients was noted to have lesions inside the mouth making it more likely that direct inoculation had occurred onto the skin surface. A. niger was isolated from the air within the unit but this was associated with Case 2 and not Case 3. These observations, in conjuction with the heavy contamination of the kitchen area, suggest that Case 3 may have received food or fomites contaminated by A. Niger, which acted as the source for the localized cutaneous infection. Since the realization that the ward kitchen may have been the source of the infection and the tightening of the cross-infection control measures to include this area, there have been no further cases. This episode underlines the importance of perceiving aspergilli as not solely airborne pathogens. It also emphasizes the importance of rigorously monitoring the small kitchen areas commonly found adjacent to wards as these may, inadvertently, act as a source of infection. We would like to thank Morgenstern, Consultant K. W. Louden received
Dr J. H. Scarffe, Consultant Medical Haematologist for giving us permission a grant from the Peel Trust.
Oncologist, to report
and these
Dr G. R. cases. Dr
References
3. 4. 5. 6. 7. 8.
9.
10. 11. 12. 13. 14. 15.
Bodey GP, Vartivarian S. Aspergillosis. EurJ Clin Microbial Infect Dis 1989; 8: 413-437. Rhame FS. Prevention of nosocomial aspergillosis. J Hosp Infect 1991; 18: 466-472. Young RC, Bennet JE, Vogel CL, Carbone PP, de Vita VT. Aspergillosis. The spectrum of disease in 98 patients. Medicine. 1970; 49: 147-173. Goodley JM, Clayton YM, Hay RJ. Environmental sampling for aspergilli during building construction on a hospital site. r Hosp Infect 1994; 26: 27-35. Walsh TJ, Dixon DM. Nosocomial aspergillosis environmental microbiology, hospital epidemiology, diagnosis and treatment. Eur J Epidemiol 1989; 5: 131-142. Wadowsy RM, Benner SM. Brief report: distribution of the genus Aspergillus in hospital room air conditioners. Infect Control 1987; 8: 516-518. Barnes RA, Rogers TR. Control of an outbreak of nosocomial aspergillosis by laminar air-flow isolation. J Hasp Infect 1989; 14: 89-94. Talwar P, Chakrabarti A, Poonamji Kaur, Pahwa RK, Ashak Mitall, Mehra YN. Fungal infections of ear with special reference to chronic suppurative otitis media Mycopathologia 1988; 104: 47-50. Bibashi E, Papagianni A, Kelesidis A, Antoniadou R, Papadimitriou M. Peritonitis due to Aspergillus nigev in a patient on continuous ambulatory peritoneal dialysis shortly after kidney graft rejection. Nephrol Dial Transplant 1993; 8: 185-187. Panke TW, McManus AT, Spebar lVIJ. Infection of a burn wound by Aspergillus niger. Am J Clin Path01 1979; 72: 230-232. McCarty JM, Flam MS, Pullen G, Jones R, Kassel SH. Outbreak of primary cutaneous aspergillosis related to intravenous arm boards. J Pediatr 1986; 108: 721-724. DeBock R, Gyssens I, Peeterman M, Nolard N. Aspergillus in pepper. Lancet 1989; ii: 331-332. Burnie JP, Matthews RC, Clark I, Milne LJR. Immunoblot fingerprinting Aspergillus fumigatus. J Immunol Methods 1989; 118: 179-186. Burnie JP, Coke A, Matthews RC. Restriction endonuclease analysis of Aspergillus fumigatus DNA. J Clin Path01 1992; 45: 324-327. Denning DW, Clemons KV, Hanson LH, Stevens DA. Restriction endonuclease analysis of total cellular DNA of Aspergillus fumigatus isolates of geographically and epidemiologically diverse origin. J Infect Dis 1990; 162: 1151-l 158.
198 16. 17.
18.
19.
20. 21.
22. 23. 24. 25.
K. W. Loudon
et al.
Girardin H, Latge JP, Srikantha T, Morrow B, Sol1 DR. Development of DNA probes to fingerprinting Aspergillus fumigatus. J Clin Microbial 1993; 31: 1547-1554. Aufauvre-Brown A, Cohen J, Holden DW. Use of randomly amplified polymorphic DNA markers to distinguish isolates of Aspergillus fumigatus. J Clin Microbial 1992; 30: 2991-2993. Loudon KW, Burnie JP, Coke AP, Matthews RC. Application of polymerase chain reaction to fingerprinting Aspergillus fumigatus by random amplification of polymorphic DNA. J Clin Microbial 1993; 31: 1117-1121. Margo A, Kusters-van Someren, SRA, Visser J. The use of RFLP analysis in classification of the black Aspergilli: reinterpretation of the Aspergillus niger aggregate. Curr Genet 1991; 19: 21-26. Loudon KW, Coke AP, Burnie JP, Lucas GS, Liu Yin JA. Invasive aspergillosis: clusters and sources? J Med Vet Mycol 1994; 32: 217-224. Johnson AS, Ranson M, Scarffe JH, Morgenstern GR, Shaw AJ, Oppenheim BA. Cutaneous infection with Rhizopus oryzae and Aspergillus niger following bone marrow transplantation. J Hosp Infect 1993; 25: 293-296. Solomon WR, Burge HP, Boise JR. Airborne Aspergillus fumigatus levels outside and within a large clinical centre. J Allergy Clin Immunol 1978; 62: 56-60. Girardin H, Sarfati J, Traore F, Dupouy Camet J, Derouin F, Latge JP. Molecular epidemiology of nosocomial invasive Aspergillosis. J Clin Microbial 1994; 32: 684-690. Vargas S, Hughes WT, Giannini M. Aspergillus in pepper. Lancet 1989; ii: 881. Eccles NK, Scott GM. Aspergillus in pepper. Lancet 1992; 399: 618.