The third group includes inorganic materials with different primary components and distinct applications. Paint is a mixture of pigments, binders, solvents and additives for surface decoration. Plaster is composed of materials like gypsum, lime or cement and has usually a high pH value. Concrete is made with cement, water and aggregates to construct building elements. Finally, fibreglass wallpaper consists of woven fibreglass strands coated with a resinous binder for reinforcement or decoration of interior wall surfaces.
The last group contains insulation materials with different compositions. Bio-based insulation is made from renewable, organic resources. Foam-based insulation contains polymers and chemicals, which result in lightweight, rigid or flexible materials (e.g., Polyurethane, Polyisocyanurate, Polystyrene, Polyethylene, etc.). Mineral-based insulation materials are derived from naturally occurring minerals (e.g., Rockwool, Fibreglass, etc.). Each type of these materials has unique properties and is suitable for specific applications.
3.5. The Building-Associated Fungal Species
Materials, partly or totally organic, could support the growth of 102 different species, while on the inorganic materials, 70 different species were found. A total of 40 species were common to both organic and inorganic materials, with species like A. alternata, C. sphaerospermum, P. variotii and P. corylophilum being the most reported. Other species like A. glaucus and C. globosum were only found on organic materials, while A. charticola and M. spinosus only on inorganic materials.
4. Building Evaluation Process
Visible fungal growth on interior surfaces, furniture and other household effects is the most common reason for starting an investigation. However, often, an investigation is launched even in the absence of visible fungal growth because the occupants or building users experience mouldy odours and/or adverse health effects. An investigation can also be initiated before the renovation of a water-damaged building, e.g., due to flooding or another water-damage incident. Regardless, high humidity or water ingress is always the reason for the presence of fungal growth even though the source of water is not obvious or the building has dried out.
The purpose of an inspection is to ascertain the existence of fungal growth, to locate the source of humidity/water and to design a remediation plan. Knowing which fungal species are growing on a particular material and the preferred 𝑎𝑤
of the fungal species can ensure that all fungal growth is discovered and the correct renovation strategy is proposed
[17].
To assess the building-related fungal contamination risk and confirm any moisture problems, it is necessary to quantify the fungal load, identify the microbial diversity and determine the contamination source. The assessment procedure is performed in four phases: (1) physical inspection of the building, (2) sample collection, (3) fungal detection and identification and (4) evaluation report. Figure 1 depicts this process and potential steps.
Figure 1. Fungal contamination assessment process of damp buildings.
Fungal growth can be seen in buildings as discolouration, stains or blots on walls, floors and ceilings, especially on colder surfaces like thermal bridges, below windows, behind furniture, etc. When fungal growth is visible, the procedure is straightforward: to clean off/demolish the affected area, restore it and perform quality control. However, fungal growth can also be hidden in the building construction, cavities and behind the wallpaper or sit in plain sight but be colourless, thin and patchy, thus easily overlooked.
All fungal growth, visible or unseen, can release equally high concentrations of fungal particles in the indoor environment
[68], and it can be recognised through high humidity, musty odours or complaints of negative health symptoms by the occupants. Nonetheless, it can be challenging to find and sample, while restoration can be costly. Therefore, it is estimated that there is significant under-reporting of these cases. The microbial assessment of damp and mouldy buildings is an interdisciplinary challenge, spanning across the fields of mycology, building science and public health
[69].
There are various sampling techniques that can be used for sample collection, while different detection methods can be applied to the collected samples. Some samples can be analysed by several detection methods, while others are intended for specific analysis. In the following sections, the sampling techniques and detection and identification methods are first analysed independently. Subsequently, it is described how the sampling techniques can be paired with the commercially used detection and identification methods.
4.1. Physical Inspection
A thorough walk-through inspection of the building is pivotal. Through the visual inspection, the investigator can reveal evidence of current or past water ingress, detect critical/problematic areas with humid or mouldy spots and evaluate the mouldy odour, which can be indirect evidence of hidden fungal growth. For that, investigators need to have a broad knowledge of moisture transport in buildings, material properties and behaviour and be able to identify the potential areas for increased or concealed humidity
[17].
The inspection can be combined with a survey/questionnaire for the occupants about the experienced indoor air quality, possible symptoms or health problems related to fungal growth, their daily airing routines, cleaning practices, heating, ventilation and air conditioning (HVAC) system and the type and state of the building (e.g., past water damage incidents, insulation level, renovation works, etc.).
4.2. Sample Collection
4.2.1. Material Sampling
Sampling of visible fungal biomass directly where it grows is the first and most obvious choice to characterise the fungal contamination of a building. Material sampling techniques are normally used for genus or species identification. A surface sample can be taken to determine whether a stain is caused by fungal growth or another issue
[70][71] or the effectiveness of remediation measures
[64].
Smaller parts of building material/construction (bulk samples) or larger parts of surfaces (scrapings and shavings) can be removed for analyses in the laboratory
[17]. Surfaces can also be sampled and tested for fungal growth using contact plates (for cultivation), sterile swabs (for cultivation or enzymatic analysis)
[72] or tape lifts (for microscopy), which is relatively economical and quick
[73]. The tape-lift method can be used to complement culture or enzymatic methods.
4.2.2. Dust Sampling
Settled dust, 3 to 6 months old, can be a good proxy for either hidden fungal growth or for evaluation of long-term exposure of occupants to fungal particles. Dust sampling can be performed using sterile swabs, dust fall collectors (DFCs) or electrostatic dust fall collectors (EDCs) for long-term collection. Swab samples (usually 10 cm
2) are obtained from horizontal surfaces 1.5 m or more above floor level on places that are not cleaned regularly, like on top of doors or picture frames, curtain rails, bookcases or cupboards
[14]. As DFCs (usually 60–100 cm
2), an empty, sterile Petri dish without medium can be used
[69] or even a cardboard box with aluminium foil-covered inner surfaces
[74]. Using the DFC method, airborne dust and fungal particles can be sampled over hours, days, weeks or even months, depending on the aim of the study
[75]. EDCs
[17][69][74][76] have been mostly used for exposure studies to endotoxins
[76]. Floor dust, 1 to 3 weeks old, can also be sampled using a nozzle with micro-vacuum cassettes attached to an ordinary vacuum cleaner
[71][77] or by analysing directly the dust collected from an ordinary vacuum cleaner bag (usually the whole living area)
[78] and used to identify the present fungal particles
[47][79].
4.2.3. Air Sampling
Air sampling provides a short-term exposure assessment through the collection of airborne fungal biomass. It can be done either passively (sampling over time) or actively (volumetric sampling). Passive air sampling is normally performed using Petri dishes containing growth medium, exposing the agar surface to the air for 30–60 min
[65]. Active air sampling is carried out by using a device (sampler) drawing in a predefined volume of air. The most commonly used air sampling devices are (1) impactors and sieve samplers, (2) impingers, (3) filter samplers and (4) centrifugal and cyclonic samplers
[17][80][81]. Impactors and sieve samplers collect a fixed volume of air impacted onto a Petri dish with growth medium or an adhesive surface (i.e., glass slides or membranes coated with a transparent, sticky substance). Centrifugal and cyclonic samplers use circular flow patterns to increase the airflow and deposit the airborne particles into a liquid, semi-solid or solid growth medium
[80].
4.2.4. Choice of Sampling Techniques
During the sampling process, several parameters and choices can affect the outcome of the investigation, and they must be considered in advance. In most cases, the purpose of the inspection dictates this decision-making process. Figure 2 shows the growth of C. globosum in the interface between OSB and gypsum wallboard (which has been removed) following a basement flood together with tape lifts, air samples on Petri dishes and pure cultures for identification.
Figure 2. Growth of C. globosum in the interface between OSB and gypsum wallboard (A,B). Tape lifts from the OSB and direct microscopy mostly reveal C. globosum (D), but some Penicillium conidia in chains are also present (C). Active air sampling onto DG18 (E) and V8 (F) show mostly Penicillium spp. because the conidia of C. globosum do not become as airborne as Penicillium conidia. Pure cultures of C. globosum on DG18 (G) and V8 (H) also show its hydrophilic nature by better growing on V8 than DG18.
The air sample volume determines the concentration of biomass that can be detected and is dictated by the sampling time and airflow rate
[17][82][83]. The sampling time can vary greatly, from minutes to months, based on the selected sampling method and the needs of the investigation. The exact sampling location is important, especially for dust sampling, as the proximity to the source of fungal growth affects the concentration of spores
[14].
4.3. Fungal Detection and Identification
Detection and identification methods concern the laboratory analyses of the collected samples to confirm the presence of fungal contaminants, estimate the fungal load and/or perform species identification. The analysis can be quantitative, assessing the amount of fungal biomass, or qualitative, listing the identity of the different fungal species. Samples can be analysed using microscopy, cultivation or molecular methods for identification and chemical/enzymatic methods for biomass determination.
4.3.1. Direct Microscopy
Using a dissecting or stereo microscope (×40 magnification), fungal growth can be observed directly on bulk materials, scrapings or shavings. For tape lifts, either directly from the fungal-infested materials or the bulk materials, scrapings or shavings, a light microscope (×400 magnification) is used. When performing microscopy analysis directly on the material, identification can typically be carried out to genus level only, while its use is limited in highly contaminated sites or samples due to overloading
[84]. There is no need for an incubation period, and samples can be analysed directly, making this method low-cost and fast. On the other hand, there are no protocols and guidelines for the analysis, and identification demands a skilled mycologist. Therefore, it is not possible to standardise the processes between different laboratories
[17].
4.3.2. Culture-Based Analysis
Traditionally, the most-used method has been culture-based analysis. It can be applied to most sample sources and types, while it can be used for species identification. On the other hand, it is time- and labour-intensive and requires skilled mycologists for correct species identification. For culture analysis, spores, fungal fragments or microparticles are collected and cultivated in different media in the laboratory under controlled conditions. The media selection and growing conditions are of great importance to the outcome of the analysis. Each cultivation medium favours specific genera and species, and it is, therefore, necessary to use a variety of media (e.g., DG18, V8, MEA) to cover the whole spectrum of indoor fungi
[43]. Even the selected technique to introduce the sampled organic matter in the Petri dish (e.g., scattering, shaking, direct or dilution plating) can influence the growth rate and detected species
[17][85].
4.3.3. Molecular Analysis
Molecular analysis of fungal biomass by quantitative polymerase chain reaction (qPCR) or next-generation sequencing (NGS) has been gaining popularity in recent years, as it can provide quantitative results of high specificity, precision and sensitivity
[72][84][86]. Culture-independent methods can detect both viable and most non-viable fungal fragments. The method has a fast analysis turnaround, and identification does not require highly trained mycologists. There are two approaches to molecular diagnostics, qPCR assays (commercial use) are designed to detect targeted, known species, while NGS (research use) provides higher discovery power to identify any species present
[87].
4.3.4. Enzymatic/Chemical Analysis
Finally, chemical tests use surrogate markers
[88] detecting specific proteins, enzymes or other organic compounds. Usually, these tests provide an assessment of the indoor microbial load. A widely used commercial method for the built environment is the
𝛽-N-acetylhexosaminidase (NAHA) enzyme test, which assesses the indoor microbial load
[89]. The test has been developed for both surface and air sampling and lies in the detection of the NAHA enzyme
[90].
4.3.5. Other Methods
There is a plethora of studies that have investigated the use of MVOCs and ergosterol, which could be used as biological markers
[22][23][67][77][91]. For example, the particulate (1→3)-
𝛽-D-Glucan is a carbohydrate that has been extensively researched as a measure of fungal biomass
[17][22][75][92]. However, no commercial methods are available yet for assessing indoor environmental contamination due to the difficulty of determining the emission source
[77].
4.3.6. Choice of Analytical Methods
All methods have strengths and weaknesses, and at present, no single method can be used to reliably confirm whether there is moisture or microbial damage
[52][69][86][93]. Culture-dependent methods are highly selective due to the growing conditions, medium properties and the fact that heavy spore-producing, tolerant or general species are overestimated as they outgrow predominantly mycelial taxa and slow-developing, more fragile or specialised fungi
[17][85]. In addition, spores’ viability is species-specific
[17]. Molecular analysis can detect non-viable spores and fragments that cannot grow in a culture. That information can provide long-term insight, for example, about older water damage incidents that might have dried out or dead spores coated in toxins that might still be present, as spores and fragments can be allergenic despite their culturability
[14][17]. On the other hand, culture methods do not require special equipment; they are widely used, well characterised and extensively researched and reliable reference data are available
[84].
4.4. Evaluation Report
After the completion of the investigation, the outcome of the inquiry needs to be reported and communicated to the building owner and occupants. The results must be interpreted, and their significance explained in layman’s terms. It is fundamental that the report states the source and cause of moisture, a plan for repair and a risk assessment. The report should also contain all investigation steps with photo documentation, a description of the performed analyses and procedures, analyses’ results and conclusions. Finally, an action plan for the renovation of the building should be provided, including means to remove the fungal growth, a cleaning scheme and quality control of fungal removal.
5. Conclusions and Perspectives
Overall, the literature highlights the challenges of investigating the existence of fungal growth. At the same time, a targeted approach is often needed where the inspector knows which species to look for
[52][69]. Focusing on specific fungal groups and species likely to grow in damp indoor environments and on the present, specific building materials can help, for example, to choose the suitable medium for isolation or adequate detection method. In addition, research has shown that the production of mycotoxins is species-specific
[65] and the substrate and its characteristics influence their production
[66]. Therefore, a better understanding of the associations between fungal species and various construction materials can be employed to limit adverse health effects stemming from exposure to building-related fungal species, as well as reduce material decay
[15]. Nevertheless, standardised, widely accepted protocols and guidelines are missing
[69][71][80][88], making it difficult to obtain reproducible and comparable results, as well as definite recommendations on fungal contamination problems.