The incidence of life threatening invasive fungal infections has been increasing with the increasing of the numbers of patients with deep and long lasting neutropenia, artificial pulmonary ventilation, having a central venous catheter, long-term administration of immunosuppressive drugs (e.g. corticosteroids), use of broad spectrum antibiotics, and other factors.1 However, the proportion of patients without neutropenia or severe immunosuppression also at risk of invasive fungal infections, which is higher than usually thought. In consequence, special attention should be paid to the exposure of vulnerable patients to conidia of some filamentous fungi, such as Aspergilli or, Fusarium species.2,3 Suitable preventive measures maybe an efficient way to decrease the incidence of these mycoses, including the control of potential endogenous and especially exogenous sources of infection by systematic monitoring of the fungal spore load in the patient's environment during hospitalization.4,5 Although the immune status of the host is thought to be the major contributor to the establishment of infection, air fungal load (AFL) and its fluctuations in the hospital environment are expected to indirectly influence the incidence of hospital-acquired fungal infections.6 Prevention of nosocomial fungal infections is also problematic. Efforts may be addressed to prevent acquisition of the infection or treatment of the pathogen before it causes disease. Little is known about the epidemiology of these opportunistic pathogens on a national level. The aim of this study was to evaluate the fungal load in environmental samples from air, surfaces and tap water during one year in two tertiary care hospitals which located in northern and southwest China respectively. The study was focused on the departments with high risk patients.
Sites and time of surveillance
The study was performed from November 2005 to October 2006 at a southwest China hospital and from January to December 2009 at a northern China hospital. Air, surfaces and tap water were sampled between 9:00 AM to 12:00 PM twice a month during a 12-month period. The Southwest China hospital is a 2200-bed universityaffiliated tertiary care hospital in Southwest China. The hospital has 3200 staff and consists of three inpatient department buildings of different ages. Three departments with high risk patients were selected for study. The Intensive central intensive care unit (ICUs) is on the second floor and air is mechanically imported into the room via an efficiency particulate air filter. The neurosurgery intensive care unit (NICU) is on the seventh floor and the live transplant department (LTD) is on the nineteenth floor. NICU and LTD have central air conditioning and air exchange occurred between the rooms and outdoor by windows. The three departments are all located in the surgery building, which was constructed in 2002.
The northern China hospital is a 1600-bed tertiary care teaching hospital, has 2800 staff and consists of four inpatient department buildings of different ages. Three departments with high risk patients were selected for study. The ICU is on the eleventh floor and the NICU is on the fifth floor, both have central air conditioning and air exchange occurred between the rooms and outdoors by windows. The bone marrow transplant (BMT) unit is on the forth floor and air is mechanically imported into the room via an efficiency particulate air filter. The three departments are all located in same building, which was constructed in 1997.
Sampling of air
Air sampling was performed with the LWC-I centrifugal sampler (Liaoyang Application Technology Corporation, China), which inoculated 1600 L of air onto Sabouraud's dextrose agar (SDA) culture media. Air samples were obtained approximately 1.5 m above the floor and lasting for about two minutes. The air in the ward, corridor, and an outdoor site were selected for sampling. One outdoor site, the entrance of the hospital building, was investigated to compare the measurements in the hospital with those outside the hospital. During each sampling we recorded the environmental conditions such as humidity, temperature, and special events, just like the presence of visitors, healthcare staff and cleaning crew.
Sampling of surface
Surface sampling was also performed with the LWC-I centrifugal sampler. Surface samples were taken along a surface for about one minute. Air conditioning units, nursing consoles and water tap surface were selected.
Sampling of water
Five hundred milliliters of tap water from the wards was collected in sterile containers and filtered through filter units with a porediameter of 0.45 μm (Sigma, USA). The filters were then cultured on SDA plates with 24 hours.
All samples were incubated at 26°C and inspected for growth (colony and colonial morphology) daily for three days. The environmental fungal load was calculated after three days. Colonies grown were then inoculated onto separate potato dextrose agar (PDA) plates and the isolates were identified according to their macroscopic and microscopic morphological characteristics. Blastomycetes were identified by germ tube positivity and API 20C AUX yeast identification kit (bioMérieux Corporation, France).
Statistical analysis was performed using SPSS 13.0 software (SPSS Inc., USA). Data had anormal distribution and were expressed as mean ± standard error (SE) and analyzed by least significant difference (LSD) tests. The correlation of AFL and temperature, humidity and personnel were analyzed statistically by a linear correlation test. Differences were considered statistically significant at P <0.05.
The fluctuation of the ALF in two hospitals as records at the 12 monthly sets of samples is shown in Figure 1. From January to March in the two hospitals, AFL outside the hospital was generally low, ranging from 93.75 to 115.63 cfu/m3 in the southwest China hospital and from 103.75 to 200.13 cfu/m3 in the northern China hospital. After March, a rise was noted in both hospitals. The highest AFL outside the hospital was recorded in the Southwest China hospital in June (384.75 cfu/m3) and in Northern China hospital in July (259.38 cfu/m3). Similarly to the AFL outside, from January to April in the AFL inside the hospital had a generally low range, from 26.04 to 80.21 cfu/m3 in the southwest China hospital and from 29.17 to 72.88 cfu/m3 in the northern China hospital. After April, a rise was noted in the two hospitals. The highest indoor AFL was recorded in the southwest China hospital in September (167.21 cfu/m3) and in the northern China hospital in July (133.23 cfu/m3). The median indoor AFL was 91.94 cfu/m3 (range 26.04 to 167.21) in the Southwest China hospital and 71.02 cfu/m3 (range 29.17 to 133.23) in the northern China hospital.
Our finding demonstrated that fungi were found in the air of the hospital and levels fluctuate over the year. The highest values were recorded during May to August in the northern China hospital and May to June and September to October in the southwest China hospital. AFL was lower in the winter and higher in spring and summer. AFL tended to be higher in the Southwest China hospital, but this difference was not statistically significant. Among all the departments studied in the two hospitals, the ICUs (7 cfu/m3) in the southwest China hospital was the least contaminated department, the NICU were the highest contaminated department; 139.90 cfu/m3 in the southwest China hospital and 115.77 cfu/m3 in the northern China hospital (Table 1). The five most prevalent fungi collected from the air in the northern China hospital was Penicillium spp. 13.875 cfu/m3, Fusarium spp. 12.5 cfu/m3, Aspergillus spp. 7.542 cfu/m3, Alternaria spp. 5.75 cfu/m3 and Cladospcrium spp. 3.625 cfu/m3. From the air in the southwest China hospital the five most often collected fungi were: Penicillium spp. 20 cfu/m3, Cladospcrium spp. 12.167 fu/m3, Alternaria spp. 9.875 cfu/m3, Aspergillus spp. 9.542 cfu/m3 and Saccharomyces spp. 2.208 cfu/m3.
In the northern China hospital, the AFL in the ICUs was correlate with the average humidity (Pearson correlation=0.703, P=0.011) and personnel (Pearson correlation=0.605, P=0.037), but the no correlation between AFL and the average temperature was observed (Pearson correlation=0.486, P=0.109). The AFL in the NICU was correlate with the average humidity (Pearson correlation=0.688, P=0.013), but no correlation between AFL and the average temperature (Pearson correlation=0.473, P=0.12), or personnel (Pearson correlation=0.287, P=0.366) was observed. There was no correlation between AFL in the BMT unit and the average temperature (Pearson correlation=0.509, P=0.091), the average humidity (Pearson correlation=0.388, P=0.212), or personnel (Pearson correlation=0.069, P=0.832).
In the southwest China hospital, the AFL in the LTD was correlate with the average temperature (Pearson correlation=0.520, P=0.008) and the average humidity (Pearson correlation=0.637, P=0.001), but not with the personnel (Pearson correlation=0.123, P=0.557). The AFL in the NICU was correlate with the average temperature (Pearson correlation=0.534, P=0.006) and the average humidity (Pearson correlation=0.573, P=0.003), but no correlation between AFL and personnel (Pearson correlation=0.352, P=0.084) was observed. No correlation between the AFL in the ICUs and the average temperature (Pearson correlation=0.203, P=0.329), the average humidity (Pearson correlation=0.329, P=0.108), or personnel (Pearson correlation=0.069, P=0.832) was observed.
From a total of 478 samples taken from surfaces in the northern China hospital, approximately 73% were contaminated. The concentration on surface was 0.104, 0.275 and 0.203 cfu/cm2 in the ICUs, the BMT units and the NICU of the northern China hospital. The concentrations on the nursing console, air-conditioning and water tap of the northern China hospital were 0.039, 0.317 and 0.158 cfu/cm2. In the northern China hospital, the concentration on the surface tended to be higher on the air-conditioning compared with the nursing console and water tap, but this difference was not statistically significant (P >0.05). The five most prevalent fungi collected from surface at the northern China hospital were Penicillium spp. 0.031 cfu/cm2, Aspergillus spp. 0.027 cfu/cm2, Cladospcrium spp. 0.014 cfu/cm2, Fusarium spp. 0.009 cfu/cm2 and Saccharomyces spp. 0.007 cfu/cm2.
From a total of 375 samples taken from surfaces at the Southwest China hospital approximately 86% were contaminated. The concentration on surfaces was 0.221, 0.181 and 0.110 cfu/cm2 in the ICUs, the LTD and the NICU at the southwest China hospital. The concentration of the nursing console, air-conditioning and water tap at the southwest China hospital were 0.035, 0.431 and 0.145 cfu/cm2. In the southwest China hospital, the concentration on surfaces tended to be higher on the air-conditioning compared with in the nursing console and water tap and this difference was statistically significant (P <0.01). The five most prevalent fungi collected from surfaces in the northern China hospital were Penicillium spp. 0.055 cfu/cm2, Aspergillus spp. 0.045 cfu/cm2, Cladospcrium spp. 0.021 cfu/cm2, Saccharomyces spp. 0.014 cfu/cm2 and Alternaria spp. 0.013 cfu/cm2.
A total of 168 water samples were examined and 54% samples yielded fungi in the northern China hospital. The concentration in tap water was 4.39, 3.76 and 4.02 cfu/500 ml in the BMT unit, the NICU and the ICUs, respectively. This difference between units was not statistically significant (P >0.05). The five most prevalent fungi collected from water in the northern China hospital were Saccharomyces spp. 1.04 cfu/500 ml, Candida spp. 0.813 cfu/500 ml, Penicillium spp. 0.647 cfu/500 ml, Fusarium spp. 0.55 cfu/500 ml and Aspergillus spp. 0.387 cfu/500 ml. The water samples yielded a rate of yeast to filamentous fungus approximately 1.13:1, and of Aspergillus spp. to non Aspergillus spp. approximately 1:10.78 (Figure 2).
A total of 150 water samples were examined and 49% of samples yielded fungi in the southwest China hospital. The concentration in tap water was 2.90, 2.86 and 2.94 cfu/500 ml in the LTD, NICU and ICU, respectively, and differences between sites were not statistically significant (P >0.05). The five most prevalent fungi collected from water in southwest China were Saccharomyces spp. 0.787 cfu/500 ml, Candida spp. 0.713 cfu/500 ml, Aspergillus spp. 0.527 cfu/500 ml, Penicillium spp. 0.367 cfu/500 ml and Rhodotorula spp. 0.127 cfu/500 ml. The water samples yielded a rate of yeast to filamentous fungus approximately 1.6:1, Aspergillus spp. to non Aspergillus spp. approximately 1:4.14 (Figure 3).
The steady world-wide increase in the number of severely immunocompromised patients in most hospitals has made the control and prevention of nosocomial systemic fungal infections a critical quality-of-care standard. Early diagnosis and antifungal prophylaxis of these infections are complicated, so avoiding the acquisition of the pathogen and minimizing the predisposing risk factors are more effective approaches. The maintenance of good air quality in critical areas in hospitals is mandatory to reduce the incidence of invasive fungal infections. During recent years, research has focused on the epidemiology of invasive fungal infections and the prevalence of fungi in the hospital environment.7 Alberti et al8 found the contamination of hospital environments by fungal spores was frequently implicated as the cause of aspergillosis in haematological wards. Heinemann et al9 performed mycological surveys of air and surfaces throughout the surgical ward and in other areas of the hospital after an outbreak of sternal surgical-site infections with Aspergillus flavus (A. flavus) following cardiac surgery. Results showed massive contamination by A. flavus in some areas of the surgical ward. Strains from patients and from the hospital environment selected showed the same genotype when typed by random amplification of polymorphic DNA, proving the clonal single-source of the environmental contamination and the intra-operative acquisition of A. flavus in the outbreak. In this study, we examined the environmental fungal load of two tertiary care hospitals in China. This is first study carried out in China hospitals and first comparing hospital fungal loads and species distribution in the air, on surfaces and in the water in representative regions of China. It demonstrated that the fungi are found in the environment of the hospital including in the air, on surfaces and in the water. The AFL fluctuates over the year and the species most commonly isolated from the air belonged to Penicillium spp., Aspergillus spp., Cladospcrium spp., Alternaria spp., and Fusarium spp. The highest values were recorded during May to August in the northern China hospital and May to June and September to October in the southwest China hospital. AFL showed a seasonal variation; higher densities were measured during the summer and spring, while lower densities were found during the winter. The finding of species and seasonal variation of AFL is in agreement with findings from other countries, which, however, show different seasonal patterns.10,11 The highest AFL was recorded in the southwest China hospital in September (167.21 cfu/m3) and in the northern China hospital in July (133.23 cfu/m3). A theoretical estimation of 10 cfu/m3 is considered an acceptable level of fungal spores for a non-protective environment.12 In the southwest China hospital, the AFL in the ICUs was <10 cfu/m3 for nine months, while the AFL in the BMT unit of northern China hospital was <10 cfu/m3 for seven months. We found decreasing numbers of fungi when comparing air from outside the hospital to the air inside the hospital and when comparing open areas within the hospital to the closed departments; such as the ICUs in the southwest China hospital and the BMT unit in the northern China hospital. The differences could be attributed to better protection measures such as high-efficiency particulate air (HEPA) filtration and laminar airflow with frequent air changes that were used in the closed departments. However, air exchanges by influx from the outside to refurnish and air conditioning were implemented in open departments. Ensuring the filter ventilation system is working properly together with strict hygiene measures seems to be sufficient for the control of the fungal conidia load.
Fungal burden, and genus or species spread, depend on various factors such as season, temperature and humidity.13 In our study, environmental conditions (humidity and temperature) and events, such as the presence of many people including visitors, healthcare staff or cleaning crew, were also recorded and correlated with the air fungal load. Although the temperature and personnel in departments were similar, the AFL was different. Air influx from outside to replace air is simple and available air sterilization because it can contraflow and dilute pathogenic microorganism. But in our results the ward air sampling results correlated with those from outdoors showing that measures, such as air influx from outside to refurnish air supply, are not always effective if not combined with air filtration. We conclude that it is important to control fungal contamination by air filtration, controlling temperature, humidity and personnel.
Air sampling gives only an estimation of environmental fungal load because spores settle after a period of time.14 So surface sampling is an alternative way of assessing environment qualitatively. A total of 853 samples taken from treatment units, air-conditioning and water taps found that 82.2% of samples yielded fungi. Of note, no surface remained sterile during the entire study period. Although the air fungal load is the lowest in the BMT units of the Northern China hospital and the ICUs of the Southwest China hospital, high numbers of colony-forming units were isolated from air-conditioning units in those departments. These results illustrate fungus concentrates on air-conditioning units that should be cleaned and sterilized regularly. The five most prevalent fungi collected from surfaces were Penicillium spp., Aspergillus spp., Cladospcriumspp., Fusarium spp., Saccharomyces spp. and Alternaria spp. Surface sampling results correlated with those from the air showing that prophylactic measures such as night-long use of ultraviolet light and damp cleaning with an antiseptic are not always effective if not combined with prevention of air influx from outside and efficient air filtration.
Water is another hidden source of nosocomiumfungal infections. But data about the presence of filamentous fungi in water are controversial and the importance of their presence as a potential threat to immunosuppressed patients is not fully understood. Opportunistic fungal pathogens have been recovered from sinks and shower heads in several hospitals in the USA. Fusarium spp. and Aspergillus spp. were cultured from the shower heads. Sampling before and after showering revealed a significant increase in spore counts in the air.15–17A. fumigutus, which is by far the most frequent causative pathogen of invasive aspergillosis (IA), has not been recovered from water in any hospital in the USA, but it has been found in tap water samples in a Norwegian hospital.18 Arvanitidou et al19 found a high level of filamentous fungi and blastomycetes in community and hospital potable water as well as in the water of some haemodialysis units. The clinical significance of this finding remains unclear. Some authorities feel that fungi proliferation around sink outlets may represent another environmental reservoir, so water leaks should be cleaned up and repaired. In our study, a total of 318 water samples were examined and 51.8% of the samples yielded fungi. The concentration of tap water was 2.90 and 4.06 cfu/500 ml in the southwest China and the northern China hospital. The five most prevalent fungi collected from water were Saccharomyces spp., Candida spp., Penicillium spp., Aspergillus spp. and Fusarium spp. In contrast to air and surface samples, high numbers of yeast were isolated from water. Our findings suggest that, in hospitals with adequate air precautions and water chlorination, water may still be another important source of opportunistic fungal infection. An effective and inexpensive approach to prevent patient exposure to waterborne fungi in the hospital setting is to provide high-risk patients with sterile (boiled) water for drinking and sterile sponges for bathing. These variable findings of Norwegian hospital and USA hospital and our results may be due to different sources of water, different methods of storage or other differences including subtle methodological variations, geographic setting and climate. Nevertheless, the role of water in the transmission of fungi needs further study.
In conclusion, the finding of this study suggested that air, surface and water fungal load fluctuate over the year. From this study, we known the air fungal load, species and correlating factors in the environment in two tertiary care hospitals located in China. These results may add our knowledge about the prophylaxis and treatment of nosocomial fungal infection. But the variation among hospitals of fungal contamination of the hospital environment as well as its role in hospital environment and appropriate measures to eliminate it need further study.
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