Mycopathologia DOI 10.1007/s11046-014-9760-6
Preliminary Study of the Fungal Ecology at the Haematology and Medical-Oncology Ward in Bamako, Mali Safiatou Niare´-Doumbo • Anne Ce´cile Normand • Yacouba Lazarre Diallo Abdoul Karim Dembele´ • Mahamadou A. Thera • Dapa Diallo • Renaud Piarroux • Ogobara Doumbo • Ste´phane Ranque
•
Received: 10 December 2013 / Accepted: 13 May 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Data on fungal epidemiology in subSaharan African countries are scarce. This exploratory study aimed to characterize the fungal flora at the Onco-Haematology ward of the National Teaching Hospital of Point G in Bamako, Mali. A crosssectional survey was conducted in the dry and in the rainy seasons. Nasal swab and sputum samples were collected from the hospitalized patients while airborne fungal spores were collected using electrostatic dustfall collectors. Fungi were identified by their morphological characteristics and MALDI-TOF mass spectrometry. Candida albicans was the most frequent S. Niare´-Doumbo M. A. Thera O. Doumbo De´partement d’Epide´miologie des Affections Parasitaires/ Malaria Research and Training Centre, Faculte´ de Me´decine et d’Odontostomatologie, USTTB, BP 1805, Bamako, Mali A. C. Normand R. Piarroux S. Ranque CHU Timone-Adultes, Assistance Publique-Hoˆpitaux de Marseille, 13005 Marseille Cedex, France A. C. Normand R. Piarroux S. Ranque IP-TPT UMR MD3, Aix-Marseille Universite´, 13885 Marseille Cedex, France Y. L. Diallo A. K. Dembele´ D. Diallo Service d’He´mato-Oncologie, CHU du Point G, Bamako, Mali S. Ranque (&) Laboratoire de Parasitologie-Mycologie, AP-HM Timone, 13385 Marseille Cedex, France e-mail: [email protected]
yeast species colonizing patients; Aspergillus species were isolated in 86 % of the patients and were the main airborne environmental contaminants. Overall, airborne fungal contamination rates increased from 33.8 % in the dry to 66.2 % in the rainy season (p \ 0.001). The most frequent Aspergillus species were Aspergillus niger (36.6 %) and Aspergillus flavus (32.92 %). In contrast, Aspergillus fumigatus (5.43 %) was relatively rare. This high level of fungal exposure raises concern regarding the management of at-risk patients in this Onco-Haematology ward and stresses the need for strengthening the mycological diagnostic capacities to accompany the implementation of adapted fungal infection prevention and management policies. Keywords Electrostatic dust-fall collectors Airborne fungal contamination Aspergillus Onco-Haematology Clinical samples Environmental samples
Introduction The risk of invasive fungal diseases in target hospital populations has been increasing in the recent years and a number of ecological and epidemiological studies have addressed this emerging public health concern [1, 2]. Candida spp. and Aspergillus spp. are the leading aetiological agents of invasive fungal diseases
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in immunocompromised patients with identified risk factors such as aggressive chemotherapies, organ or haematopoietic stem cell transplantation, corticosteroids, and other immunosuppressive treatments [1, 3–5]. Because of airborne transmission, the environment plays a critical role in aspergillosis and invasive fungal diseases caused by numerous other filamentous fungi [6]. Four major genera of filamentous airborne fungi are prevalent in the environment: Aspergillus, Penicillium, Cladosporium and Alternaria [4]. Growing evidence from studies conducted in developed countries indicates that preventive measures and environmental management in hospitals are effective in reducing the invasive fungal diseases burden [6]. Recent studies have shown that the epidemiology of these fungi in North Africa differs from the one in Northern countries where A. fumigatus is the most prevalent species. In Tunisia, for instance, A. flavus represented 79.2 % of the species isolated from patients with aspergillosis and from the environment of haematology wards and caused 18 % of invasive aspergillosis cases in the patients with haematological malignancies [7]. In contrast, similar data from subSaharan African countries are scarce. Particularly in Mali, to the best of our knowledge, the documented cases of human invasive aspergillosis are limited to two patients. In 2001, a case of aspergilloma has been reported in a Malian patient with pulmonary tuberculosis sequelae who presented with haemoptysia. The diagnostic was based only on radiological and serological findings, but the fungus was not cultured [8]. In 2011, a Malian patient with an acute myeloid leukaemia treated in the Onco-Haematology ward at the Point G National Teaching hospital had been diagnosed with invasive aspergillosis after being evacuated to France for treatment (Diallo et al. unpublished). Therefore, to gain more insights into the fungal ecology and epidemiology in a Malian hospital, we aimed to describe the inpatients’ upperairway fungal flora and the environmental airborne fungal contamination in the Onco-Haematology ward at the National Teaching hospital of Bamako, Mali.
Methods Hospital-air and patient sampling was carried out in the Onco-Haematology ward at the National Teaching Hospital of Point G, in Bamako, Mali, where long-stay
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(mean duration of hospital stay: 25 days) patients, who are often immunocompromised and at risk of invasive fungal diseases, are hospitalized. About 130 patients with solid or haematological malignancies or HIV/AIDS infection are hospitalized each year in eleven rooms hosting each two to four beds [9–11]. Two cross-sectional observational studies were conducted in 2012: the first in March, during the dry season, and the second in September, during the rainy season. All who were present the day of the survey were included in the study. One sputum and nasal swab sample were collected for fungal culture from each inpatients present the day of the survey. Airborne fungal spores were collected by passive sampling using electrostatic dust-fall collectors, referred to as EDC [12]. EDC were placed in each room in the ward during the two surveys in March and September 2012. EDC were deposited horizontally on a clean surface exposed to air, allowing the collection of settling dust and fungal spores for 15 days. All biological samples were kept at ?4 °C and send to the Parasitology-Mycology laboratory in Marseille, where they were processed within 72 h. Airborne fungal spores were harvested from EDC by following these steps: (1) EDC were suspended in plastic bags containing 30 ml of Tween 80 at 0.1 % in NaCl 0.9 %; (2) the suspension was homogenized in a homogenius (Mayo, France) homogenizer for 10 min and then transferred to two 15-ml tubes (Falcon, France). After centrifugation (3,500 rpm for 15 min), the supernatant was discarded. Then, the pellet was resuspended two times in 1 ml of Tween 80 at 0.1 % in NaCl 0.9 %, 200 ll of which was used for culture, the remaining was stored at -20 °C. The sputa, nasal swabs and EDCs’ suspension were inoculated onto Sabouraud gentamicinchloramphenicol agar plates (Oxoı¨d, France) and incubated up to 5 days at 30 °C. Yeast colonies from clinical samples were sub-cultured onto a chromogenic medium (ChromIDTM Candida, bioMe´rieux, France) to check for purity. Yeast species identification relied on the colour of the colonies on the chromogenic medium and MALDITOF mass spectrometry (MS) [13]. Filamentous fungi colonies identification was based on both morphological criteria [14] and MALDI-TOF MS, with a Microflex LTTM instrument and the MALDI Biotyper v3.0 software (Bruker Daltonics, Germany) as described by Cassagne et al. [15]. The criterion for accurate yeast species identification was a LogScore C 2 as recommended by the manufacturer.
Mycopathologia Table 1 Frequencies of the primary diagnoses categories of the patients included in the two cross-sectional surveys conducted in the Onco-Haematology ward at the Point G University Hospital in Bamako in 2012 Diagnoses n (%)
March
Solid cancersa
16 (84.2)
6 (35.3)
22 (61.1)
Haematological malignanciesb Othersc
1 (5.3)
5 (29.4)
6 (16.7)
2 (10.5)
6 (35.3)
8 (22.2)
19 (52.8)
17 (47.2)
36 (100)
Total
September
Total
a
Choriocarcinoma; Brain cancer; Breast cancer; Oesophageal cancer; Gastric cancer; Colon cancer; Cervix cancer; Ovarian cancer
b
Lymphoma; Myeloma
c
Thrombophlebitis; HIV; Anaemia; Sickle cell disease
MALDI-TOF MS identification of filamentous fungi was performed following the procedure described in [15]. Spectra were compared to a large in-house filamentous fungi reference spectra library, which characteristics were described in [16]. Accurate filamentous fungi MS identification criteria were: at least three of the four spectra issued from each isolate match a reference spectrum of the same species with a LogScore C 1.9 in at least one of them. When identification criteria were not met, the colony was subjected to a second MALDI-TOF MS identification assay. If this second assay did not yield validation criteria, identification was performed by DNA sequence analysis as described in [17].
Results A total of 70 samples were collected from 36 inpatients during the two surveys: Both a sputum sample and a nasal swab were collected from all the 19 patients hospitalized in March and from 15 of the 17 patients hospitalized in September because sputum collection failed in two. The patients’ primary diagnoses are described in Table 1. Their mean age was 42 ± 13 (range 25–67) years and 43 ± 19 (range 18–75) years in March and September, respectively. The male/female ratio was 6/13 = 0.46 and 7/10 = 0.70 (p = 0.730) in March and September, respectively. The frequency of sample with at least one filamentous fungus isolate increased from 20 % in the dry
season to 48.6 % in the rainy season. Overall, 35 filamentous fungi were isolated, of which 85.7 % (30/ 35) belonged to the Aspergillus genus. Aspergillus niger and A. tubingensis 29 % (10/35) were the most frequent species isolated, followed by Aspergillus flavus 14 % (5/35). The frequencies of the other species are detailed in Table 2. Aspergillus spp. colonization frequency varied significantly (p \ 0.01) depending on the season with a proportion of Aspergillus positive samples that increased from 34.2 % (13/38) in the dry season to 53.12 % (17/32) in the rainy season. Among the 42 identified yeast isolates, Candida albicans (42.85 %) was the most frequent species colonizing the patients’ airways whatever the season (Table 2). Yeast was isolated in none (0/19) and 43.7 % (7/16) of the patients in the dry and rainy seasons, respectively. Infrequent yeast species were identified in the rainy season, including three Kodamaea ohmeri and one Hanseniaspora opuntiae isolates. The frequencies of the airborne fungal species recovered from the EDC at the Onco-Haematology ward are tabulated in Table 3. One hundred forty-six filamentous fungi colony forming units were isolated from 33 EDC. The genus Aspergillus accounted for 56.16 % of the 146 filamentous fungi and was present in all air samples. The most frequent Aspergillus species (n = 82) were: A. niger 36.58 %, A. flavus 32.92 % and A. fumigatus 5.43 %. Overall, airborne fungal contamination was significantly higher in the rainy season (66.43 %) than in the dry season (33.56 %). In particular, Aspergillus spp. were isolated from 66.22 % versus 33.78 % (p \ 10-3) of the air samples in the rainy and dry seasons, respectively.
Discussion This is the first study describing the upper-airways fungal flora of hospital patients in the Sahel region. With respect to the filamentous fungi cultured from clinical samples, 85.7 % were Aspergillus species and the most common species were A. niger (29 %), followed by A. flavus (14 %). Our results differed from those of Hadrich et al. in Sfax, Tunisia, who found that Aspergillus species were isolated in 8 % of 1,680 clinical samples, with the following species pattern: 79 % A. flavus, 10 % A. niger, 2 % A. ochraceus, 2 %
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Mycopathologia Table 2 Frequencies of filamentous fungi and yeast airways colonization in the patient hospitalized in the Onco-Haematology ward in Bamako according to the dry (March) or rainy (September) season
Species
March
September
Total (%)
Nasal
Sputum
Nasal
Sputum
A. niger and A. tubingensis
3
2
3
2
10 (29)
A. flavus A. terreus
1 1
2 1
2 2
0 0
5 (14) 4 (11)
A. sydowii
0
0
4
0
4 (11)
Eurotium chevalieri
0
0
2
1
3 (08)
A. versicolor
1
0
0
1
2 (06)
Penicillium
0
2
0
0
2 (06)
Syncephalastrum ? Rhizopus
0
0
2
0
2 (06)
A. fumigatus
0
1
0
0
1 (03)
A. nidulans
1
0
0
0
1 (03)
Curvularia sp.
0
0
1
0
7 (20)
8 (23)
17 (49)
3 (8)
35 (100)
Candida albicans
0
9
3
6
18 (43)
C. kefyr
0
2
0
0
2 (4.8)
C. glabrata
0
2
0
0
2 (4.8)
C. tropicalis
0
4
0
3
7 (17)
C. krusei C. sorboxylosa
0 0
3 1
1 0
0 0
4 (9) 1 (2.4)
C. parapsilosis
0
0
0
1
1 (2.4)
Geotrichum capitatum
0
0
0
2
2 (4.8)
Kodamaea ohmeri
0
0
1
2
3 (7)
Hanseniaspora opuntiae
0
0
1
0
1 (2.4)
Trichosporon asahii
0
0
1
0
0
21 (50)
7 (17)
14 (33)
Filamentous fungi
Total
1 (03)
Yeast
Sub-total
A. fumigatus and 6 % of other species. This difference may, at least in part, be explained by the differences between the Mediterranean climate in Sfax and the Sahelian climate in Bamako. There was a statistically significant 19 % increase in the patients’ Aspergillus colonization frequency in the rainy season compared with the dry season. With respect to yeast, C. albicans was the most common species, colonizing the airways of 42.9 % of the patients, whatever the season. It should be noted that relatively infrequent yeast species, among others, K. ohmeri and H. opuntiae were identified during the rainy season. Both were isolated from a 54-year-old patient with multiple myeloma, and K. ohmeri was isolated from a patient with severe anaemia. Relatively rare K. ohmeri human infections have been reported, including nine cases presenting with fungemia and two heath care-associated
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1 (2.4) 42 (100)
infections [18–21]. In contrast, H. opuntiae has never been involved in a human infection. Along with other yeast species such as Hanseniaspora uvarum and Saccharomyces cerevisiae, it has been found to be associated with the cocoa bean fermentation processes [22]. The parallel environmental study shows that airborne fungal contamination varied according to the season and that A. niger and A. flavus were the most common species in this Onco-Haematology ward. Interestingly, A. fumigatus was cultured in only 6 % of the environment samples. Many studies that have evaluated hospital environment airborne Aspergillus contamination pointed to contaminated surfaces and air as potential invasive aspergillosis sources [23–28]. Most of these studies reported the predominance of airborne Aspergillus spp. spores in hospital wards
Mycopathologia Table 3 Comparison of airborne filamentous fungal contamination burden of the haematology ward at the Point G hospital in Bamako during the dry (March) or rainy (September) season
a
Phaeohyphomycetes: non-identified melanized fungi that lacked characteristic morphological features
b
Hyalohyphomycetes: non-identified white (nonmelanized) fungi that lacked characteristic morphological features
Species
March
September
Total (%)
Aspergillus niger
11
19
30 (20)
Aspergillus flavus
11
16
27 (18)
Penicillium spp.
8
8
16 (11)
Phaeohyphomycetesa
4
6
10 (7)
Alternaria ? Curvularia ? Cladosporium
0
10
10 (7)
Chrysonilia sp.
0
9
9 (6)
Hyalohyphomycetesb
3
5
8 (5)
Rhizopus ? Mucor ? Syncephalastrum Aspergillus fumigatus
2 2
5 3
7 (5) 5 (3)
Aspergillus tamarii
1
3
4 (3)
Aspergillus nidulans
1
3
4 (3)
Aspergillus sydowii
0
3
3 (2)
Aspergillus ochraceus
2
1
3 (2)
Aspergillus tubingensis
1
2
3 (2)
Aspergillus terreus
2
0
2 (1)
Trichoderma longibrachiatum
1
1
2 (1)
Yeast sp.
1
1
2 (1)
Aspergillus versicolor
0
1
1 (1)
Paecillomyces sp.
0
1
1 (1)
Beauveria sp.
0
1
Total
50 (34)
98 (66)
[26–28]. Similarly to the patients’ colonization pattern, the total airborne fungal contamination increased by 33 % in the rainy season; and in particular the Aspergillus spp. airborne contamination significantly increased by 32 %. A study in Poland [29] described the predominance of Aspergillus fumigatus that accounted for 77 % of the fungal flora in a pneumonology ward. A. fumigatus airborne contamination was relatively high during winter and spring. The predominance of A. fumigatus was also reported in France and the Netherlands, where, in contrast, fungal airborne contamination peaked in summer and decreased in winter [23, 30]. Although a study in France [23] found no significant airborne spores concentration variation according to rainfall, atmospheric humidity or wind, our finding that both airborne environmental contamination and patients colonization with filamentous fungi increased during the rainy season, compared to the dry season, is likely to be correlated with the dramatic changes in atmospheric humidity that characterize the Sahelian climate in this region of Mali. The main limitation of this study lies in its crosssectional design that allows only a snapshot picture of the fungal eco-epidemiology in this ward. Furthermore,
1 (1) 148 (100)
none of the patients included in this survey was diagnosed with an invasive fungal disease. However, this study’s strengths were that, on the one hand, the fungal flora in both the patients and their environment was monitored via simple and robust methods and, on the other hand, the fungal identification was done via highly performing method based on MALDI-TOF MS. The EDC method has been successfully adapted from methods used to assess environmental exposure to endotoxin, fungi [12] and to cultivable microorganisms in farms [31, 32]. Here, the use of EDC for collecting environmental sample proved well suited for studying airborne fungal exposure in this Sahelian African hospital setting. Our findings raise concern regarding heavy exposition to fungi in this Onco-Haematology ward and stress the need for strengthening the mycological diagnostic capacities as immunocompromised patients are at high risk of developing an invasive fungal disease. In this respect, we advocate for a wider availability of MALDI-TOF mass spectrometry technology in Mali and in comparable developing countries, because building on technical progress should be a successful strategy for them to gain the capacity of performing a fast and accurate identification of clinical
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as well as environmental fungi, without having to train highly skilled specialists mycology. Indeed, clinical microbiology laboratory techniques should keep up with the improvement of treatment modalities of haematologic malignancies available in developing countries.
10.
11.
Acknowledgments This study was funded by a training grant to SDN from the Universite´ des Sciences, des Techniques et des Technologies de Bamako, Mali. We would like to thank, the Director of the National Teaching Hospital of Point G, all the workers in the oncology-hematology ward and the technicians of the Parasitology-Mycology laboratory at APHM CHU Timone, Marseille, France. These results were presented in part as an oral communication at the International Conference of the ‘‘West African Society of Parasitology’’ in December 2012, Dakar, Senegal.
12.
The authors declare no conflict of interest.
15.
Conflict of interest
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Preliminary Study of the Fungal Ecology at the Haematology and Medical-Oncology Ward in Bamako, Mali Safiatou Niare´-Doumbo • Anne Ce´cile Normand • Yacouba Lazarre Diallo Abdoul Karim Dembele´ • Mahamadou A. Thera • Dapa Diallo • Renaud Piarroux • Ogobara Doumbo • Ste´phane Ranque
•
Received: 10 December 2013 / Accepted: 13 May 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Data on fungal epidemiology in subSaharan African countries are scarce. This exploratory study aimed to characterize the fungal flora at the Onco-Haematology ward of the National Teaching Hospital of Point G in Bamako, Mali. A crosssectional survey was conducted in the dry and in the rainy seasons. Nasal swab and sputum samples were collected from the hospitalized patients while airborne fungal spores were collected using electrostatic dustfall collectors. Fungi were identified by their morphological characteristics and MALDI-TOF mass spectrometry. Candida albicans was the most frequent S. Niare´-Doumbo M. A. Thera O. Doumbo De´partement d’Epide´miologie des Affections Parasitaires/ Malaria Research and Training Centre, Faculte´ de Me´decine et d’Odontostomatologie, USTTB, BP 1805, Bamako, Mali A. C. Normand R. Piarroux S. Ranque CHU Timone-Adultes, Assistance Publique-Hoˆpitaux de Marseille, 13005 Marseille Cedex, France A. C. Normand R. Piarroux S. Ranque IP-TPT UMR MD3, Aix-Marseille Universite´, 13885 Marseille Cedex, France Y. L. Diallo A. K. Dembele´ D. Diallo Service d’He´mato-Oncologie, CHU du Point G, Bamako, Mali S. Ranque (&) Laboratoire de Parasitologie-Mycologie, AP-HM Timone, 13385 Marseille Cedex, France e-mail: [email protected]
yeast species colonizing patients; Aspergillus species were isolated in 86 % of the patients and were the main airborne environmental contaminants. Overall, airborne fungal contamination rates increased from 33.8 % in the dry to 66.2 % in the rainy season (p \ 0.001). The most frequent Aspergillus species were Aspergillus niger (36.6 %) and Aspergillus flavus (32.92 %). In contrast, Aspergillus fumigatus (5.43 %) was relatively rare. This high level of fungal exposure raises concern regarding the management of at-risk patients in this Onco-Haematology ward and stresses the need for strengthening the mycological diagnostic capacities to accompany the implementation of adapted fungal infection prevention and management policies. Keywords Electrostatic dust-fall collectors Airborne fungal contamination Aspergillus Onco-Haematology Clinical samples Environmental samples
Introduction The risk of invasive fungal diseases in target hospital populations has been increasing in the recent years and a number of ecological and epidemiological studies have addressed this emerging public health concern [1, 2]. Candida spp. and Aspergillus spp. are the leading aetiological agents of invasive fungal diseases
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in immunocompromised patients with identified risk factors such as aggressive chemotherapies, organ or haematopoietic stem cell transplantation, corticosteroids, and other immunosuppressive treatments [1, 3–5]. Because of airborne transmission, the environment plays a critical role in aspergillosis and invasive fungal diseases caused by numerous other filamentous fungi [6]. Four major genera of filamentous airborne fungi are prevalent in the environment: Aspergillus, Penicillium, Cladosporium and Alternaria [4]. Growing evidence from studies conducted in developed countries indicates that preventive measures and environmental management in hospitals are effective in reducing the invasive fungal diseases burden [6]. Recent studies have shown that the epidemiology of these fungi in North Africa differs from the one in Northern countries where A. fumigatus is the most prevalent species. In Tunisia, for instance, A. flavus represented 79.2 % of the species isolated from patients with aspergillosis and from the environment of haematology wards and caused 18 % of invasive aspergillosis cases in the patients with haematological malignancies [7]. In contrast, similar data from subSaharan African countries are scarce. Particularly in Mali, to the best of our knowledge, the documented cases of human invasive aspergillosis are limited to two patients. In 2001, a case of aspergilloma has been reported in a Malian patient with pulmonary tuberculosis sequelae who presented with haemoptysia. The diagnostic was based only on radiological and serological findings, but the fungus was not cultured [8]. In 2011, a Malian patient with an acute myeloid leukaemia treated in the Onco-Haematology ward at the Point G National Teaching hospital had been diagnosed with invasive aspergillosis after being evacuated to France for treatment (Diallo et al. unpublished). Therefore, to gain more insights into the fungal ecology and epidemiology in a Malian hospital, we aimed to describe the inpatients’ upperairway fungal flora and the environmental airborne fungal contamination in the Onco-Haematology ward at the National Teaching hospital of Bamako, Mali.
Methods Hospital-air and patient sampling was carried out in the Onco-Haematology ward at the National Teaching Hospital of Point G, in Bamako, Mali, where long-stay
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(mean duration of hospital stay: 25 days) patients, who are often immunocompromised and at risk of invasive fungal diseases, are hospitalized. About 130 patients with solid or haematological malignancies or HIV/AIDS infection are hospitalized each year in eleven rooms hosting each two to four beds [9–11]. Two cross-sectional observational studies were conducted in 2012: the first in March, during the dry season, and the second in September, during the rainy season. All who were present the day of the survey were included in the study. One sputum and nasal swab sample were collected for fungal culture from each inpatients present the day of the survey. Airborne fungal spores were collected by passive sampling using electrostatic dust-fall collectors, referred to as EDC [12]. EDC were placed in each room in the ward during the two surveys in March and September 2012. EDC were deposited horizontally on a clean surface exposed to air, allowing the collection of settling dust and fungal spores for 15 days. All biological samples were kept at ?4 °C and send to the Parasitology-Mycology laboratory in Marseille, where they were processed within 72 h. Airborne fungal spores were harvested from EDC by following these steps: (1) EDC were suspended in plastic bags containing 30 ml of Tween 80 at 0.1 % in NaCl 0.9 %; (2) the suspension was homogenized in a homogenius (Mayo, France) homogenizer for 10 min and then transferred to two 15-ml tubes (Falcon, France). After centrifugation (3,500 rpm for 15 min), the supernatant was discarded. Then, the pellet was resuspended two times in 1 ml of Tween 80 at 0.1 % in NaCl 0.9 %, 200 ll of which was used for culture, the remaining was stored at -20 °C. The sputa, nasal swabs and EDCs’ suspension were inoculated onto Sabouraud gentamicinchloramphenicol agar plates (Oxoı¨d, France) and incubated up to 5 days at 30 °C. Yeast colonies from clinical samples were sub-cultured onto a chromogenic medium (ChromIDTM Candida, bioMe´rieux, France) to check for purity. Yeast species identification relied on the colour of the colonies on the chromogenic medium and MALDITOF mass spectrometry (MS) [13]. Filamentous fungi colonies identification was based on both morphological criteria [14] and MALDI-TOF MS, with a Microflex LTTM instrument and the MALDI Biotyper v3.0 software (Bruker Daltonics, Germany) as described by Cassagne et al. [15]. The criterion for accurate yeast species identification was a LogScore C 2 as recommended by the manufacturer.
Mycopathologia Table 1 Frequencies of the primary diagnoses categories of the patients included in the two cross-sectional surveys conducted in the Onco-Haematology ward at the Point G University Hospital in Bamako in 2012 Diagnoses n (%)
March
Solid cancersa
16 (84.2)
6 (35.3)
22 (61.1)
Haematological malignanciesb Othersc
1 (5.3)
5 (29.4)
6 (16.7)
2 (10.5)
6 (35.3)
8 (22.2)
19 (52.8)
17 (47.2)
36 (100)
Total
September
Total
a
Choriocarcinoma; Brain cancer; Breast cancer; Oesophageal cancer; Gastric cancer; Colon cancer; Cervix cancer; Ovarian cancer
b
Lymphoma; Myeloma
c
Thrombophlebitis; HIV; Anaemia; Sickle cell disease
MALDI-TOF MS identification of filamentous fungi was performed following the procedure described in [15]. Spectra were compared to a large in-house filamentous fungi reference spectra library, which characteristics were described in [16]. Accurate filamentous fungi MS identification criteria were: at least three of the four spectra issued from each isolate match a reference spectrum of the same species with a LogScore C 1.9 in at least one of them. When identification criteria were not met, the colony was subjected to a second MALDI-TOF MS identification assay. If this second assay did not yield validation criteria, identification was performed by DNA sequence analysis as described in [17].
Results A total of 70 samples were collected from 36 inpatients during the two surveys: Both a sputum sample and a nasal swab were collected from all the 19 patients hospitalized in March and from 15 of the 17 patients hospitalized in September because sputum collection failed in two. The patients’ primary diagnoses are described in Table 1. Their mean age was 42 ± 13 (range 25–67) years and 43 ± 19 (range 18–75) years in March and September, respectively. The male/female ratio was 6/13 = 0.46 and 7/10 = 0.70 (p = 0.730) in March and September, respectively. The frequency of sample with at least one filamentous fungus isolate increased from 20 % in the dry
season to 48.6 % in the rainy season. Overall, 35 filamentous fungi were isolated, of which 85.7 % (30/ 35) belonged to the Aspergillus genus. Aspergillus niger and A. tubingensis 29 % (10/35) were the most frequent species isolated, followed by Aspergillus flavus 14 % (5/35). The frequencies of the other species are detailed in Table 2. Aspergillus spp. colonization frequency varied significantly (p \ 0.01) depending on the season with a proportion of Aspergillus positive samples that increased from 34.2 % (13/38) in the dry season to 53.12 % (17/32) in the rainy season. Among the 42 identified yeast isolates, Candida albicans (42.85 %) was the most frequent species colonizing the patients’ airways whatever the season (Table 2). Yeast was isolated in none (0/19) and 43.7 % (7/16) of the patients in the dry and rainy seasons, respectively. Infrequent yeast species were identified in the rainy season, including three Kodamaea ohmeri and one Hanseniaspora opuntiae isolates. The frequencies of the airborne fungal species recovered from the EDC at the Onco-Haematology ward are tabulated in Table 3. One hundred forty-six filamentous fungi colony forming units were isolated from 33 EDC. The genus Aspergillus accounted for 56.16 % of the 146 filamentous fungi and was present in all air samples. The most frequent Aspergillus species (n = 82) were: A. niger 36.58 %, A. flavus 32.92 % and A. fumigatus 5.43 %. Overall, airborne fungal contamination was significantly higher in the rainy season (66.43 %) than in the dry season (33.56 %). In particular, Aspergillus spp. were isolated from 66.22 % versus 33.78 % (p \ 10-3) of the air samples in the rainy and dry seasons, respectively.
Discussion This is the first study describing the upper-airways fungal flora of hospital patients in the Sahel region. With respect to the filamentous fungi cultured from clinical samples, 85.7 % were Aspergillus species and the most common species were A. niger (29 %), followed by A. flavus (14 %). Our results differed from those of Hadrich et al. in Sfax, Tunisia, who found that Aspergillus species were isolated in 8 % of 1,680 clinical samples, with the following species pattern: 79 % A. flavus, 10 % A. niger, 2 % A. ochraceus, 2 %
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Mycopathologia Table 2 Frequencies of filamentous fungi and yeast airways colonization in the patient hospitalized in the Onco-Haematology ward in Bamako according to the dry (March) or rainy (September) season
Species
March
September
Total (%)
Nasal
Sputum
Nasal
Sputum
A. niger and A. tubingensis
3
2
3
2
10 (29)
A. flavus A. terreus
1 1
2 1
2 2
0 0
5 (14) 4 (11)
A. sydowii
0
0
4
0
4 (11)
Eurotium chevalieri
0
0
2
1
3 (08)
A. versicolor
1
0
0
1
2 (06)
Penicillium
0
2
0
0
2 (06)
Syncephalastrum ? Rhizopus
0
0
2
0
2 (06)
A. fumigatus
0
1
0
0
1 (03)
A. nidulans
1
0
0
0
1 (03)
Curvularia sp.
0
0
1
0
7 (20)
8 (23)
17 (49)
3 (8)
35 (100)
Candida albicans
0
9
3
6
18 (43)
C. kefyr
0
2
0
0
2 (4.8)
C. glabrata
0
2
0
0
2 (4.8)
C. tropicalis
0
4
0
3
7 (17)
C. krusei C. sorboxylosa
0 0
3 1
1 0
0 0
4 (9) 1 (2.4)
C. parapsilosis
0
0
0
1
1 (2.4)
Geotrichum capitatum
0
0
0
2
2 (4.8)
Kodamaea ohmeri
0
0
1
2
3 (7)
Hanseniaspora opuntiae
0
0
1
0
1 (2.4)
Trichosporon asahii
0
0
1
0
0
21 (50)
7 (17)
14 (33)
Filamentous fungi
Total
1 (03)
Yeast
Sub-total
A. fumigatus and 6 % of other species. This difference may, at least in part, be explained by the differences between the Mediterranean climate in Sfax and the Sahelian climate in Bamako. There was a statistically significant 19 % increase in the patients’ Aspergillus colonization frequency in the rainy season compared with the dry season. With respect to yeast, C. albicans was the most common species, colonizing the airways of 42.9 % of the patients, whatever the season. It should be noted that relatively infrequent yeast species, among others, K. ohmeri and H. opuntiae were identified during the rainy season. Both were isolated from a 54-year-old patient with multiple myeloma, and K. ohmeri was isolated from a patient with severe anaemia. Relatively rare K. ohmeri human infections have been reported, including nine cases presenting with fungemia and two heath care-associated
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1 (2.4) 42 (100)
infections [18–21]. In contrast, H. opuntiae has never been involved in a human infection. Along with other yeast species such as Hanseniaspora uvarum and Saccharomyces cerevisiae, it has been found to be associated with the cocoa bean fermentation processes [22]. The parallel environmental study shows that airborne fungal contamination varied according to the season and that A. niger and A. flavus were the most common species in this Onco-Haematology ward. Interestingly, A. fumigatus was cultured in only 6 % of the environment samples. Many studies that have evaluated hospital environment airborne Aspergillus contamination pointed to contaminated surfaces and air as potential invasive aspergillosis sources [23–28]. Most of these studies reported the predominance of airborne Aspergillus spp. spores in hospital wards
Mycopathologia Table 3 Comparison of airborne filamentous fungal contamination burden of the haematology ward at the Point G hospital in Bamako during the dry (March) or rainy (September) season
a
Phaeohyphomycetes: non-identified melanized fungi that lacked characteristic morphological features
b
Hyalohyphomycetes: non-identified white (nonmelanized) fungi that lacked characteristic morphological features
Species
March
September
Total (%)
Aspergillus niger
11
19
30 (20)
Aspergillus flavus
11
16
27 (18)
Penicillium spp.
8
8
16 (11)
Phaeohyphomycetesa
4
6
10 (7)
Alternaria ? Curvularia ? Cladosporium
0
10
10 (7)
Chrysonilia sp.
0
9
9 (6)
Hyalohyphomycetesb
3
5
8 (5)
Rhizopus ? Mucor ? Syncephalastrum Aspergillus fumigatus
2 2
5 3
7 (5) 5 (3)
Aspergillus tamarii
1
3
4 (3)
Aspergillus nidulans
1
3
4 (3)
Aspergillus sydowii
0
3
3 (2)
Aspergillus ochraceus
2
1
3 (2)
Aspergillus tubingensis
1
2
3 (2)
Aspergillus terreus
2
0
2 (1)
Trichoderma longibrachiatum
1
1
2 (1)
Yeast sp.
1
1
2 (1)
Aspergillus versicolor
0
1
1 (1)
Paecillomyces sp.
0
1
1 (1)
Beauveria sp.
0
1
Total
50 (34)
98 (66)
[26–28]. Similarly to the patients’ colonization pattern, the total airborne fungal contamination increased by 33 % in the rainy season; and in particular the Aspergillus spp. airborne contamination significantly increased by 32 %. A study in Poland [29] described the predominance of Aspergillus fumigatus that accounted for 77 % of the fungal flora in a pneumonology ward. A. fumigatus airborne contamination was relatively high during winter and spring. The predominance of A. fumigatus was also reported in France and the Netherlands, where, in contrast, fungal airborne contamination peaked in summer and decreased in winter [23, 30]. Although a study in France [23] found no significant airborne spores concentration variation according to rainfall, atmospheric humidity or wind, our finding that both airborne environmental contamination and patients colonization with filamentous fungi increased during the rainy season, compared to the dry season, is likely to be correlated with the dramatic changes in atmospheric humidity that characterize the Sahelian climate in this region of Mali. The main limitation of this study lies in its crosssectional design that allows only a snapshot picture of the fungal eco-epidemiology in this ward. Furthermore,
1 (1) 148 (100)
none of the patients included in this survey was diagnosed with an invasive fungal disease. However, this study’s strengths were that, on the one hand, the fungal flora in both the patients and their environment was monitored via simple and robust methods and, on the other hand, the fungal identification was done via highly performing method based on MALDI-TOF MS. The EDC method has been successfully adapted from methods used to assess environmental exposure to endotoxin, fungi [12] and to cultivable microorganisms in farms [31, 32]. Here, the use of EDC for collecting environmental sample proved well suited for studying airborne fungal exposure in this Sahelian African hospital setting. Our findings raise concern regarding heavy exposition to fungi in this Onco-Haematology ward and stress the need for strengthening the mycological diagnostic capacities as immunocompromised patients are at high risk of developing an invasive fungal disease. In this respect, we advocate for a wider availability of MALDI-TOF mass spectrometry technology in Mali and in comparable developing countries, because building on technical progress should be a successful strategy for them to gain the capacity of performing a fast and accurate identification of clinical
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as well as environmental fungi, without having to train highly skilled specialists mycology. Indeed, clinical microbiology laboratory techniques should keep up with the improvement of treatment modalities of haematologic malignancies available in developing countries.
10.
11.
Acknowledgments This study was funded by a training grant to SDN from the Universite´ des Sciences, des Techniques et des Technologies de Bamako, Mali. We would like to thank, the Director of the National Teaching Hospital of Point G, all the workers in the oncology-hematology ward and the technicians of the Parasitology-Mycology laboratory at APHM CHU Timone, Marseille, France. These results were presented in part as an oral communication at the International Conference of the ‘‘West African Society of Parasitology’’ in December 2012, Dakar, Senegal.
12.
The authors declare no conflict of interest.
15.
Conflict of interest
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![[Cutaneous tuberculosis in Bamako, Mali].](/assets/img/hold.png)
