Exposure to biological agents in school

environments in Hungary

with special focus on fungi Donát MAGYAR1; Zsófia TISCHNER1,2, Tamás SZIGETI1, Péter RUDNAI1, Éva CSOBOD3, László KREDICS4

1National Public Health Center, Budapest, Hungary2Szent István University, Gödöllő, Hungary

3The Regional Environmental Center for Central and Eastern Europe, REC Country Office, Szentendre, Hungary4University of Szeged, Szeged, Hungary

Vulnerable age group

• Immune system

• Sensibility for respiratory

illnesses (asthma, allergy,…)

Children spend ~8 hours

in school buildings/day

6 schools/Hungary

3 classrooms/school

2 sampling/classrooms,

4 week between samplings

16 schools/Hungary

1 classrooms/school

2 sampling/classrooms

Materials & Methods

• Sampling sites• 11 towns in Hungary, 22 primary schools, 34 classrooms + outdoor reference

• Age group of children: 8-10 ys

• Sampling method• Air samples (100 l) were collected with a single-stage Andersen-type sampler

• The samples were taken during the lessons with closed windows and doors

• Sample processing• Bacteria have been incubated on Blood Agar at 37 °C for 3 days

• Allergenic fungi have been incubated on Malt Extract Agar with 2% chloramphenicol at 25 °C for 5 days

• morphological characterization

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CFU/m3

Concentration of bacteria in the air samples collected from classrooms (brown) and outdoors (green). Red line: threshold (500 CFU/m3, if above outdoor level)

Surprisingly high concentration!

School code

ResultsAirborne bacteria

Particles falling from the human body1.000.000 particulate/h (>0.5 µm) mostly bacteria

‘personal microbial cloud’ (Meadow et al. 2015)

No pathogenic bacteriawere detected.Mostly Bacillus and Micrococcus spp.

lipopolysacharides, the major components of the outer membrane of Gram-negative bacteria.Released from dead bacteria.Symptoms:Fever, accelerated breathing and low blood pressureThe effect is increased in the presence of tobacco smoke and NO2

Positive effects?!It can be protective against atopic / asthmatic diseasese.g. in airway remodeling (Liu 2002)

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HU

S1 R

1

HU

S2 R

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HU

S2 R

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HU

S2 R

3

HU

S3 R

1

HU

S3 R

2

HU

S3 R

3

HU

S4 R

1

HU

S5 R

1

HU

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3

HU

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HU

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HU

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EU/m

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CFU/m3

Total concentration of fungiin the air samples collected from

classrooms (blue) and outdoors (green).

School code

Fungi

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Cladosporium spp.

10930

050

100150200250300350400450500

Penicillium spp.

4800 1460

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U/m

3

Aspergillus spp.

CFU/m3

Concentration

of common

airborne fungi

collected from

classrooms

(blue) and

outdoors

(green).

In collaboration withUppsala University, Swenden - Dan NorbäckNational Institute for Health and Welfare, Finland Martin Täubel

Aspergillus+Penicillium spp.

Aspergillus versicolor

Trichoderma spp./ T. viridis

Cladosporium herbarum

Alternaria spp./A. alternata

classrooms

CFU

/m3

cell/

m2

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051015202530354045

0100200300400500600700

020406080

100120140160

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01000020000300004000050000600007000080000

01002003004005006007008009001000

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y = -0,0006x + 372,31R² = 0,0298

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Asp

/Pen

CFU

m-3

Asp/Pen cells mg-1

Aspergillus/Penicillium

y = -0,0053x + 3,602R² = 0,0063

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A.v

ersi

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FU m

-3

A. versicolor cells mg-1

Aspergillus versicolor

Common indoor fungi: no correlation between concentration of fungi in settled dust and air

y = -0,0045x + 0,4243R² = 0,0134

-1

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ATr

ich

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erm

a sp

p. C

FU m

-3

T. viridis cells mg-1

Trichoderma

A soilborne fungus: no correlation between concentration of fungi in settled dust and air

y = 0,1626x + 7,6518R² = 0,1195

y = 0,1792x + 7,0433R² = 0,6386

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Alt

ern

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sp

p. C

FU m

-3

Alternaria alternata cells mg-1

Alternaria

Common outdoor fungi: correlation between concentration of fungi in settled dust and air

Spearman’s rho: 0,693** p 0,004

y = -0,0005x + 137,74R² = 3E-05

y = 0,0107x + 108,92R² = 0,414

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C. herbarum cells mg-1

Cladosporium herbarum

Airborne levels of mouldsexceeded the threshold value in 41% (9) schools38% (13) classrooms.

Number of cases when theconcentration of fungal taxa exceeded the threshold level:

Alternaria; 2

Aspergillus; 2

Acremonium; 1

Penicillium; 5

Cladosporium; 4

Threshold levels were defined as the concentration (colony forming units/m3) higher than the corresponding outdoor concentration of a given fungal sp. by 50.

Although Cladosporium species are common outdoor fungi, they are able to grow in areas of condensation, where they are frequently associated with Acremonium spp.

Aspergillus section Versicolores(A. versicolor, A. sydowii)are frequent in damp buildings.

Their high concentration could be regarded as an indicator of fungal growth in schools.

Aspergillus fumigatusin composts

Penicillium spp.

common indoor and food-borne fungi.

Remnants of mouldy foods in schoolbags might be a major source of this fungus in classrooms.

uzsi

Modern buildingsdropped ceilingairductsgipsum board walls…

Visual inspection(suggested by guidelines) is not effective anymore.

But…sampling-baseddetection methodshave limitations-expensive, -time-consuming-needs expertise-etc…

hidden mold

mold colonies behind a gipsum board wall in a school

Conclusion

• It is important to improve microbial air quality in schools. • Modern buildings are new challange for microbiologists. • Research should focus on-the development of a new, simple methodto detect hidden mold

-risk assessment of mold exposure-following new technologies in architecture and keepenvironmental health guidelines updated

Thank you for your attention!

Acknowledgements: This work was implemented within the projects SINPHONIE (Schools Indoor Pollution and Health, initiated and funded by the European Parliament) funded by DG Sanco, Health and Consumer Protection Directorate and InAirQ(Transnational adaption actions for integrated indoor air quality management) funded by Interreg CENTRAL EUROPE.

Window vent (air intake)

A tapétarétegek alatt Chaetomium gomba telepei és háziporatkák.

A spórák kiszóródtak.

A csoportszoba fala az ereszcsatorna rossz bekötése és az alap mentén történő beázások miatt vizesedik.

4. eset

Penészgombák

(Hyphomycetes, Zygomycetes):

80 faj!

szenzitizálás

allergiás rhinitis

atópiás dermatitis

asztma

tüdőmikózis, aspergilloma,

hyperszenzitív tüdőgyulladás

humidifier fever,

SBS

BRI

A természetes szellőzés akkor hatékony, ha

egyoldali szellőzésnél a tér mélysége a belmagasság 2,5-szeresénél nem nagyobb

átszellőzésnél a tér mélysége a belmagasság 5-szörösénél nem nagyobb.

Horn V. 2017

3. eset

-Padlásajtó nyílik a

lakótérbe

-A padlásról MMVF

törmelék szóródik,

-kimutatható a

gyerekszobában

Reményné Nagy Z, Magyar D, Varró M, Nagy L, Mácsik A, Szentmihályi R, Rudnai P, Beregszászi T (2012) Iskolás gyerekek endotoxin-terhelésének vizsgálata passzív pormintavételimódszerrel. TOX'2012 konferencia, Hévíz, 2012. október 17-19.

Total concentration of airborne fungi

in Hungarian schools

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CFU/m3

School code

Fungal taxa having concentration above threshold level

Threshold levels were defined as the concentration (colony forming units/m3) higher than the corresponding outdoor concentration of a given fungal sp. by 50.

• Isolated fungal taxa:– Acremonium sp.

– Aspergillus sp.

– A. clavatus

– A. flavus

– A. sydowii

– A. sect. Versicolores

– Beauveria sp.

– Bipolaris sp.

– Cladosporium sp.

– Eurotium sp.

– Penicillium sp.

– P. digitatum

– Phoma sp.

– Rhodotorula sp.

– Sporothrix sp.

– Talaromyces sp.

– Trichotecium roseum

– Ulocladium sp.

Long-term exposure to indoor air pollution could lead to respiratory diseases, such as allergy and asthma. Children aged 6 to 14 represent one of the most sensitive groups, spending 6-8 h daily in classrooms. Air samples were collected in 22 primary schools (34 classrooms and outdoors) in 11 towns in Hungary with a single-stage Andersen device. Two samples per classroom were taken during the lessons with closed windows and doors onto malt extract agar with 10% chloramphenicol and incubated at 25 °C for 5 days. Threshold levels were defined as the concentration (colony forming units/m3) higher than the corresponding outdoor concentration of a given fungal sp. by 50. Sporulating filamentous fungi were identified at the genus level with a Carl Zeiss Jenaval light microscope at 300×. Airborne levels of mouldsexceeded the threshold value in 9 schools (13 classrooms). Penicillium (36%), Cladosporium (29%), Alternaria (14%), Aspergillus (14%) and Acremonium (7%) spp. reached high concentrations. Many species of Penicillium are common indoors and are food-borne fungi. Remnants of mouldy foods in schoolbags might be a major source of this fungus in classrooms. Alternaria and Cladosporium are common outdoor fungi, but the latter one often grows in areas of condensation, where it is frequently associated with Acremonium spp. Aspergillus spp. (especially spp. of the section Versicolores) are frequent in damp buildings. Their high concentration could be regarded as an indicator of fungal growth in schools. The above-mentioned fungi can trigger respiratory diseases such as allergy and asthma. Other biological agents, such as bacteria in Hungarian schools are also reviewed. Based on these results it is important to improve microbial air quality in schools.

LAL- Limulus amoebocyte lysate teszt