Evaluating fungi indoor presence in homes through viable and non-viable sampling

It is widely accepted that moulds are common and important allergens. Although they are more abundant outdoors, patients affected by this problem stay indoors much more time. Therefore, properly indoor sampling is the best way to study their possible influence on allergic symptoms. The aim of this study was to assess the relative efficiencies of two air sampling methods, viable and not viable, for the quantification of airborne indoor fungi in the homes of patients sensitized to Alternaria, and compared them with outdoor levels. Material and Methods


ABSTRACT
Moulds are common and important allergens.They are more abundant outdoors but patients affected by mould allergy stay indoors much longer than outdoors.So, indoor sampling could help to assess the influence of the concentration of allergens in allergic symptoms.The aim of this study was to assess the relative efficiencies of two air sampling methods, viable and non viable, for the quantification of airborne indoor fungi in the homes of patients sensitized to Alternaria.Furthermore, outdoor sampling was carried out to compare results.Samples were taken over six months in Badajoz (SW Spain).Two houses were selected according to the presence of allergic patients to Alternaria.They were sampled once a month using both viable and non viable personal samplers at solar noon.A Burkard personal sampler was used to record spores and a Sampl'air AES Chemunex sampler was used for colonies.Three rooms were selected in each home: living room, kitchen and bathroom.Temperature and relative humidity were registered at each sample.Outdoor sampling was performed one day per week at the Faculty of Science, using a seven day Burkard sampler for spores and the same personal sampler for colonies.On average, 200-300 CFU/ m 3 were found from more than 40 taxa identified.The highest number of colonies was recorded in the kitchen, then in the bathroom and finally in the living room.Nevertheless, there were minor differences between rooms.The houses studied showed a similar temporal pattern, with maximum values in December and minimum in January.Cladosporium colonies showed statistical differences between homes, but these differences were not found with Alternaria, Aspergillus or Penicillium colonies.Differences between rooms appeared for Alternaria colonies and Cladosporium herbarium spores.Temperature was positively correlated in most cases and relative humidity negatively with Alternaria spores.The number of spores collected was as twice as that of colonies, with an average concentration between 600-700 colonies/m 3 , and nearly 1400 spores/m 3 as maximum concentration.The temporal pattern of spores was similar to that found for colonies.Comparing colonies to spores, Alternaria and Aspergillus-Penicillium showed similar values.Notwithstanding, Cladosporium spores were nearly five times more abundant than colonies.The advantage of viable methods is the identification to species level, but they have the disadvantage that spores from some ubiquitous species, as Cladosporium, do not always grow in those media, which raises the interest to use additional non-viable methods.
It is widely accepted that moulds are common and important allergens.Although they are more abundant outdoors, patients affected by mould allergy stay indoors much longer.Some authors suggested that proper indoor sampling is the best way to study their possible influence on allergic symptoms (Ammann et al., 2008).Moulds are not only important allergens, moreover, they are an important source of mycotoxins (Zain, 2011) and they are even considered responsible, to some extent, for the Sick Building Syndrome (Crook & Burton, 2010).
Although indoor airborne fungi may be found as a consequence of their outdoor presence, and accordingly the seasonal pattern may be followed indoors, in many cases this seasonal pattern disappears (Herbarth et al., 2003) or even changes.For instance, indoor concentration does not decrease during winter, as it frequently occurs outdoors; on the contrary, it may increase (Tuñón de Lara et al., 1990).In any case, indoor sources should be taken into account to explain fungus indoor presence.
A wide range of samplers were used to record indoor moulds.For non-viable airborne propagule capture, Burkard personal sampler was widely used either for limited period sampling (Escamilla et al., 1995;Emberlin et al., 1995;Garret et al., 1997;Pei-Chih et al., 2000) or for a continuous sampling (Fairs et al., 2010).For viable capture, Malt Extract Agar (MEA) is considered a suitable medium for growing most airborne fungi (Ren et al.;2001).If non-viable and viable methods are working simultaneously, a useful index is the culturability, which has been defined by Lee et al. (2006) as the rate between colonies and spores.As outdoor sampling is often performed in addition to indoor sampling, another useful index is the Indoor/Outdoor (I/O) ratio (Nevalainen et al., 1991;Lee et al., 2006).Furthermore, as the data provided by these techniques can show an important level of variability, it is useful to give general data as arithmetic average as well as geometric median (Lee et al., 2006;Pei-Chih et al., 2000;Nevalainen et al., 1991;Dassonville et al., 2008;Pasanen et al., 1992).
Identification of fungi from airborne samples may present some degree of difficulty, which is why some biochemical techniques have been performed, like PCR (Meklin et al., 2007).Nevertheless, sampling methods that involve viable and non-viable capture will continue to provide valuable information about organisms in indoor air (Flannigan, 1997).
The aim of this study was to assess the relative efficiencies of two air sampling methods, viable and non viable, for the quantification of airborne indoor fungi in the homes of patients sensitized to Alternaria and to compare this data with those from outdoor sampling, taking into account environmental factors to explain possible differences.Although the number of homes sampled is low, they were selected using medical criteria from a higher number of cases and it is only a preliminary study to assess the efficiency of the methodology.

MATERIAL AND METHODS
Sampling was performed over six months in Badajoz (SW Spain), from October 2010 to April 2011.Two houses were selected according to the presence of allergic patients to Alternaria, one in the centre of the city (home A) and the other one in outskirts (home B).The former was a flat in the 7 th floor of a nine floor building, and the latter was a two floor house with a small garden (home B).They were sampled once a month, between 11:00 to 13:00 h in the morning, using both viable and non-viable personal samplers at solar noon.A Burkard personal sampler was used to record spores for 5 minutes at 10 litres/minute flow rate, using white petrolatum as adhesive (CAS number 8009-03-8).A Sampl'air AES Chemunex sampler was used for viable colonies for 1 minute at 100 litres/minute, using MEA as culture media.Indoor samples were taken by placing the sampler at ground level in the center of the rooms, keeping doors and windows closed.
Three rooms were selected in each home: living room, kitchen and bathroom.Temperature and relative humidity were registered at each sample.Non-viable samples were analysed at x1000 magnification optical microscope, and data were given as spores per cubic meter (spores/m 3 ).Petri dishes were cultivated at 27ºC for 5-7 days to identify colonies and data were given as colonies forming units per cubic meter (CFU/m 3 ).Outdoor sampling was performed one day per week at the Faculty of Science (University of Extremadura), 2980 m and 3520 m away from the homes studied, respectively, using a seven day Burkard sampler for spores and the same personal AES Chemunex sampler for colonies following the same sampling procedures.Monthly data from this sampling was compared to indoor sampling data.Data were given as average concentrations per cubic meter and geometric mean (GM) in order to compare them with the literature.Two ratio values have been calculated, indoor/outdoor (I/O) ratio and colonies/spores (C/S) ratio, the former to express the level of fungi infiltration and the latter to express in some way the viability of propagules.
Non parametric statistics were used: Spearman correlation and Kruskall-Wallis test were performed in order to compare the data from outdoor-indoor homes and rooms.For the former, correlation coefficient (r) and p-value (p) were given and chi-square value (cs) and p-value (p)were calculated for the later.

RESULTS
Table 1 shows date and meteorological data from sampling.Indoor temperature was 6-7ºC higher than outdoors, and indoor relative humidity was 24-26% lower than outdoors.However, both indoor and outdoor temperature and relative humidity had a strong negative correlation (r 0.533, p < 0.001)

Fig. 1. Outdoor and indoor concentrations for total spores and colonies.
On average, for the whole samples, we recorded 741 spores/m 3 and 317 CFU/m 3 (GM: 267 spores/m 3 and 536 CFU/ m 3 ) indoors, whereas 1890 spores/m 3 and 487 CFU/m 3 (GM: 1032 spores/m 3 and 402 CFU/m 3 ) were recorded outdoors (Table 2).Monthly total values are shown in Fig. 1.Minimum values were found usually in February and maximum values recorded depended on sampling type and home.Maximum concentrations of spores were recorded in April, both indoors and outdoors.For indoor colonies, maximum concentrations were recorded in December (home A) and April (home B), and maximum outdoor colonies were recorded in November.
Figs. 2-4 show monthly values for indoor and outdoor Cladosporium, Alternaria, Aspergillus and Penicillium spores and colonies.Outdoor Cladosporium spores were not always more abundant than indoor, even with lower values in some cases, as in December for Cladosporium cladosporioides.Colonies of Cladosporium showed a great variation, home A showed lower values than home B outdoors, and home B showed some higher values than outdoors in two occasions (Fig. 2).Alternaria showed on average higher values outdoors than indoors, mainly in colonies, except in one case, which is April in home A (Fig. 3).Aspergillus-   Penicillium spores showed higher concentrations indoors except in November.Penicillium colonies showed higher values indoors than outdoors, and Aspergillus colonies were less frequent indoors than outdoors (Fig. 4).
Table 2 shows that I/O ratios were below zero except in Penicillium colonies (1.6) and Aspergillus-Penicillium spores (1.3).Colonies/Spores ratios were always below zero except in Alternaria (1.21).
Total differences between rooms are shown in Table 3.On average, we recorded 347 CFU/m 3 and 1070 spores/m 3    in the kitchen, 306 CFU/m 3 and 635 spores/m 3 in the bathroom, and finally 296 CFU/m 3 and 518 spores/m 3 in the living room.
The differences between the rooms of the two homes studied are showed in Figs.5-8.There were no statistically significant differences between the two homes, except for Cladosporium colonies (cs 15.658, p<0.001) with higher values in the house B (Fig. 6).Comparisons between rooms showed that Alternaria colonies had statistically significant Evaluating fungi indoor presence in homes -S.Fernández et al.  differences (cs 9.998, p 0.007), see Fig. 7, and Cladosporium herbarium (cs 6.366, p 0.041), see Fig. 6.Both of them with the lowest concentrations in the bathroom.
The indoor/outdoor ratio is always below 1 except for Penicillium colonies and Aspergillus-Penicillium spores.This could mean that, at least for Penicillium, indoor environment would be more suitable for growth and that there could be other sources inside that would increase their concentrations.This data agree with those found by other authors (Medrela-Kuder, 2003;Lee et al., 2006;Gómez de Ana et al., 2006;Docampo et al., 2011).These fungi seem to be more abundant in the bathroom and in the kitchen than in the living room (Fig. 8), probably because there are more suitable media or environmental conditions for proliferation.
Regarding the ratio between colonies and spores, about half of the spores were able to grow in the culture media provided.Nevertheless, there were differences between fungi type.Cladosporium spores seem to be the least able to grow, while Alternaria colonies outnumber spores.This could be possible because some colonies of Alternaria grew from hyphal fragments, or because some conidia grow in chains with the apical conidia so different and smaller than the basal ones that they could not be counted under light microscope as Alternaria type.Other possible explanation could be that multicellular conidia of Alternaria are more efficient than unicellular conidia of Cladosporium to develop colonies.
There are no clear differences between both houses analysed.They follow a similar monthly pattern with similar concentrations (Figs.1-4), even the pattern for the three rooms shows similarities.However, home B showed a statistically significant concentration of Cladosporium colonies higher than that of home A. This difference could be attributed to a closer contact with rural environment (Pasanen, 1992;Mitakakis & McGee, 2000, Rodríguez-Rajo et al., 2005).Nevertheless, the number of Cladosporium spores is only slightly higher in home B with no statistically significant difference.Perhaps home B could provide better conditions for the growth of Cladosporium colonies, but temperature and relative humidity are quite similar in both places.One difference that could explain these differences is that home A is higher than home B, nevertheless this should affect only Cladosporium colonies.Other possible explanations could be that fungi may be introduced into buildings through the dust and dirt swept away by the shoes and clothing or that the environmental conditions of house B may influence the growth of fungi, including heating and ventilation systems, furniture etc.
The indoor and outdoor viabilities of fungi, ratio between colonies and spores (C/S), were on average 0.43 and 0.26, respectively, which indicates that indoor environment provides more favourable survival conditions for airborne fungi.These values are quite similar to those found by Lee et al. (2006).Nevertheless, this ratio was higher for Alternaria indoors and Aspergillus-Penicillium outdoors.In the former case we can assume better conditions indoors for Alternaria and in the later better condition for Aspergillus-Penicillium. Nevertheless, as low Evaluating fungi indoor presence in homes -S.Fernández et al.
values of concentrations have been recorded in some cases, this should be taken with care when calculating the ratio, as in the case for both fungi types, since small figures could lead to great differences in ratio calculation.

CONCLUSIONS
As the differences between outdoor and indoor colonies and spores, on average, are not quite relevant, and the monthly pattern is similar too, we can conclude that most indoor fungi comes from outdoors, and, only a small proportion depends on indoor sources, as in Penicillium.After using simultaneously viable and non-viable sampling we consider that the only advantage of viable methods is the identification to species level, but they have the disadvantage that spores from some ubiquitous species, as Cladosporium, do not always grow in those media, so their presence is often underestimated and the interest to use additionally non-viable methods is proved.
Evaluating fungi indoor presence in homes -S.Fernández et al.

Fig
Fig. 2. Outdoor and indoor concentrations for Cladosporium spores and colonies.

Fig
Fig. 3. Outdoor and indoor concentrations for Alternaria spores and colonies.

Fig. 4 .
Fig. 4. Outdoor and indoor concentrations for Aspergillus and Penicillium spores and colonies.

Fig. 5 .
Fig. 5. Average total concentration indoors for spores and CFU depending on the room, bars represent errors.

Fig. 6 .
Fig. 6.Average Cladosporium concentration indoors for spores and CFU depending on the room, bars represent errors.

Fig. 7 .
Fig. 7. Average Alternaria concentration indoors for spores and CFU depending on the room, bars represent errors.

Fig. 8 .
Fig. 8. Average Aspergillus and Penicillium concentration indoors for spores and CFU depending on the room, bars represent errors.