A total of 2456 CFUs were counted. The outdoor mean concentration was of 107CFU/m3, and that indoors was of 24CFU/m3. The highest indoor mean values were for the third-floor CR (31CFU/m3) and the lowest for the ground-floor CR (14CFU/m3). The mean values for the two OWs were very similar (25 and 27CFU/m3 for the ground and the third floors, respectively).

A total of 35,330 propagules were counted. The outdoor mean was of 2473 propagules/m3 and that indoors of 790 propagules/m3. The highest values: peaks were for the third-floor OW (1053 propagules/m3), and the lowest were again for the ground floor CR (505 propagules/m3). For the two OWs, the means were likewise similar (959 and 1053 propagules/m3 for the ground and the third floors, respectively).

Since neither the CFU nor the propagule data followed a normal distribution [Shapiro–Wilk test (W=0.605, p-value<0.001 for CFU; and W=0.610, p-value<0.001 for propagules)], non-parametric tests were applied. No statistically significant difference was found between the CFU and propagule data when both years were compared. The outdoor sampling results showed the lowest values in winter and the highest in summer, with those of spring and autumn being similar. For the indoor sampling, the differences in the CFU results were greater between the four sampling points than between seasons (Friedman test, p-value 0.0824) (Fig. 2A) but, for propagules, there were statistically significant differences between seasons (p-value 0.0194): the values were lowest in winter, highest in spring and autumn, and intermediate in summer (Fig. 2B). The Kruskal–Wallis test showed statistically significant seasonal differences for the indoor data (CFU K-W 31.2971, value 0.0006E−3 and propagules K-W 48.9005, p-value 0.0001E−6) and for outdoor propagules (K-W 11.9472, p-value 0.0076), but not for outdoor CFUs.

Fig. 2.

(A) Mean indoor CFU values in the four seasons and for the four sampling sites. (B) Mean indoor propagule values in the four seasons and for the four sampling sites. Bars represent standard errors.

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With regard to the meteorological data, only for the outdoor data were there statistically significant correlations for mean temperature (positive) and for relative humidity (negative) (temperature: Rho 0.370, p-value 0.004 for CFU; and Rho 0.486, p-value 0.000 for propagules; relative humidity: Rho −0.281, p-value 0.030 for CFU; and Rho −0.357, p-value 0.005 for propagules) (Fig. 3).

Fig. 3.

Annual variation of temperature (Temp) and outdoor CFU and propagule concentrations.

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The mean number of people present in the OWs was 25.2 (2–79, s 16.1) for the ground floor and 24.7 (2–57, s 11.2) for the third floor. The Spearman correlation coefficient was only statistically significant (positive) for propagules on the third floor OW (Rho 0.348, p-value 0.011).

Comparing the outdoor results with those indoors, the Spearman correlation coefficients were statistically significant for propagules at the four indoor sampling points (Rho 0.407, p-value 0.001, for the ground floor CR; Rho 0.644, p-value 0.000 for the ground floor OW; Rho 0.422, p-value for the third floor CR; Rho 0.548, p-value 0.000 for the third floor OW) and CFUs at the three indoor sampling points (Rho 0.300, p-value 0.020, for the ground floor CR; Rho 0.258, p-value 0.046 for the ground floor OW; Rho 0.408, p-value 0.001 for the third floor OW).

During the outdoor construction period (only studied in the first year), the mean outdoor fungal concentrations (62CFU and 1140 propagules/m3) were lower than for the rest of the sampling period (106CFU/m3 and 3083 propagules/m3). Only for one of the four indoor sampling points and for one set of measurements (CFU counts on the ground-floor OW) was the concentration higher during this period. The Friedman test applied to the data for the five sampling points comparing days with and days without construction works showed no difference for CFU values (p-value 0.1797), but there was a significant difference for propagules (p-value 0.0253). These differences apply to data from outdoors and from the ground floor (Fig. 4A and B).

Fig. 4.

(A) Mean CFU concentration for the four indoor sampling sites during days in the presence or absence of construction work. (B) Mean propagules/m3 for the four indoor sampling sites in the presence or absence of construction work. Bars represent standard errors.

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The data for the third-floor CR relating to the eight days of the first year studied, when the window found open before the sampling period gave means of 32CFUs and 894 propagules/m3, and for the days when the window was closed, 25CFU/m3 and 544 propagules/m3. During the renovation activities on this floor, the means were higher (42 CFUs and 1271 propagules/m3). In both cases there were no statistically significant differences using the Kruskal–Wallis test (Fig. 5).

Fig. 5.

Mean CFU concentration and propagules/m3 in the insulated room on the third floor, either on normal days, on days when the window was open prior to sampling, and in the presence of construction work. Bars represent standard errors.

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Comparing results for the same floor, we found statistically significant differences only for the CFU data between the CR and OW on the ground floor, even if the data corresponding to the renovation period in the third floor were ignored.

Total colonies and propagules found appear in Tables 1 and 2. The five most frequent fungal taxa identified appear in Table 3.

The presence of Cladosporium showed the same seasonality as that of the total airborne fungi, with lowest values in winter (both indoors and outdoors), and peaks in summer for outdoors and in spring and autumn for indoors (Fig. 6). The mean loads during the outdoor construction works were lower than during the rest of the period, for both the indoor and the outdoor sampling. For indoors, differences between seasons were higher (Friedman p-value for CFU 0.0192 and propagules 0.0169) than between sites (Friedman p-value for CFU 0.0314 and propagules 0.1272).

Fig. 6.

Mean annual variation of Cladosporium indoor concentrations measured as CFUs or propagules/m3. The shaded area represents the periods with outdoor construction work.

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For either CFUs or propagules of Aspergillus and Penicillium, the differences between seasons were greater than between sites when using the Friedman test, although none of them was statistically significant (Fig. 7). In the third-floor CR, the Kruskal–Wallis test showed no statistically significant differences when the window was open or when renovations were taking place.

Fig. 7.

Mean annual variation of Aspergillus-Penicillium indoor concentrations measured as CFU or propagules/m3. The shaded area represents the periods with outdoor building work.

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The two sampling methods could distinguish presumably between viable and non-viable fungi. Another useful index is culturability, which has been defined as the rate between colonies and spores (C/S).19Fig. 8 shows average concentrations for both colonies and spores of the three main fungal types, Alternaria, Aspergillus-Penicillium and Cladosporium at the five sites studied. The highest values of C/S were for Alternaria (0.12–0.23), followed successively by Aspergillus-Penicillium (0.04–0.07) and Cladosporium (0.01–0.03).

Fig. 8.

Average concentrations of colonies and spores at the five sites sampled for the main fungal types: Alternaria (A), Aspergillus-Penicillium (B), and Cladosporium (C). Bars represent standard errors.

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Overall, our mean concentration of indoor airborne fungi, with less than 25U/m3, is similar to values reported in other sampling areas accessed by many people.2,22,27,28,30 The lower values found in other studies27,28 can be explained because they were performed in special, restricted-access areas, such as haematology units.

The seasonal presence of airborne fungi outdoors was evident, temperature being the main meteorological factor involved. Thus, the highest values corresponded to summer, and the lowest to winter. But for the indoor data, the values in summer were lower than in spring and autumn. Indoor summer increases have been detected in some studies,9,11,27,29,35,36 while others have not detected seasonality.28 In the present study, the decrease observed in summer could be because main doors or even some windows are kept closed for more time in the air-conditioned rooms, thus reducing the access of fungal propagules.

The presence of people during the sampling period seems to affect propagule levels, although the evidence is not very strong. This could mean that people act as vectors of propagules. Nevertheless, we only counted the people present during the sampling period, not those present beforehand, who could have also affected the results. We cannot discard this potential influence as it has been previously adduced.21

Propagule data from the four indoor sampling points were correlated with those from outdoors. The same was found for the CFUs, except those from the third floor CR. This is congruent with the idea that the main door is one of the main avenues of propagule entry, as observed by other workers,32 although other sources have been suggested, as water.43 The insulation of the ground floor closed room was only reflected in the CFU values. This could be attributed to the fact that many propagules may be of fungi that do not usually grow on the media supplied, for example most Basidiomycetes and Myxomycetes, and many Ascomycetes as teleomorphs.

Studies on the presence of airborne fungi during construction works have had contradictory results. While some workers find an increase,4,23,24,34 others do not.6,15,39 In many cases, the increase was tested outdoors but not indoors because the insulation measures applied were sufficiently effective as it seems that occurred in the present study. Nevertheless seasonal differences could be strong enough to mask the effects of construction works.

When using culture media as traps,10,28,32Cladosporium conidia are considered to be the most frequent airborne spores in nearly all environments, but some workers have found Aspergillus more so. This would support that only using culture media is insufficient for estimating the overall presence of fungi in indoor air, as many airborne micro-organisms cannot be cultured, with the result that they are not detected.3

In conclusion, culturing fungus spores only detects a small fraction of the total airborne spora. It is thus necessary to also count and identify spores on glass slides for a good estimate of the indoor spora concentration.13 The five most frequent groups of CFU identified were Cladosporium, yeasts, Alternaria, Penicillium and Aspergillus. For the propagules, they were Cladosporium, Ustilago, Aspergillus-Penicillium, Leptosphaeria, and hyphae. The indoor fungus occurrence in our hospital was independent of meteorological conditions and of the insulation of the indoor sites selected, but was correlated with the season and number of people in one of the wards. Outdoor construction works affected the propagule data although seasonal differences could mask this evidence.

The authors have no conflict of interest to declare.