SYTTY
DETECTION OF PREVIOUS EXPOSURE OF WET-DAMAGE MICROBES
WITH AN IgG-AVIDITY,
ENDOTOXIN AND TOXICITY ASSAY
Subproject: Mould and moisture transfer in building
structures and buildings with particular regard to the prevention of health
hazards
Osa projektia: Homeen ja kosteuden kulku rakenteissa
ja rakennuksissa erityisesti terveydellisten haittojen torjunnan kannalta
Researchers:
Helena Mussalo-Rauhamaa1, Peter Elg2, Arpo Orpana3, Timo Helin1,
Lea Paloheimo2, Outi Itkonen2
1 Departments of Dermatology and Allergic Diseases, Helsinki University
Central Hospital, P.O. Box 160, 00029 HUS, Finland, tel. +358-9-47186526
e-mail: Helena.Mussalo-Rauhamaa@helsinki.fi
2 HUCH-Laboratorydiagnostics, Helsinki University Central Hospital,
P.O. Box 160, 00029 HUS, Finland, tel. +358 -9-47186423, e-mail Peter.Elg@hus.fi
3 HUCH-Laboratorydiagnostics, Helsinki University Central Hospital,
P.O. Box 140, 00029 HUS, Finland, tel. +358-9-47174309, e-mail Arto.Orpana@hus.fi
Financing SYTTY organisation: Tekes
KEY WORDS: mould, health, avidity, endotoxin, toxicity
TIIVISTELMÄ
Tässä tutkimuksessa on kehitetty kolme eri menetelmää,
joilla voidaan tutkia yksilön mahdollista altistumista homeille tai
bakteereille ja tästä aiheutuvaa terveyshaittaa. Tutkimus on
osa ‘Homeen ja kosteuden kulku rakenteissa ja rakennuksissa erityisesti
terveydellisten haittojen torjunnan kannalta’ projektia. Homeille altistumisen
toteamismenetelmä perustuu seerumin IgG-luokan homevasta-aineiden
aviditeetin määrittämiseen. Projektissa kehitettiin myös
menetelmät aktinobakteerien (ent. aktinomykeettien) toksisuuden toteamiseksi
in vitro potilasnäytteistä ja sovellettiin kirjallisuudessa julkaistua
menetelmää huonepölyn endotoksiinipitoisuuden määrittämiseksi.
Endotoksiini on lipopolysakkaridia, joka on peräisin gram-negatiivisten
bakteerien kuorikerroksesta. Endotoksiinien runsas esiintyminen viittaa
bakteerikertymään rakennuksessa.
Soveltamalla viruksille (rokotevasteelle) kehitettyä aviditeettimenetelmää
potilaiden seeruminäytteisiin vastaavanlainen IgG-vasta-aineiden pitoisuuden
nousu ja matala aviditeetti oli nähtävissä tuoreen uuden
altistumisen jälkeen. Poikkeavaa oli kuitenkin se että
aviditeettitaso saattoi laskea altistumisvaiheessa. Matala aviditeettitaso,
20% tai vähemmän osoittaa uutta altistumista ja tasoa 20-30%
voidaan pitää raja-arvoisena tuoreelle, muutaman viime kuukauden
aikana tapahtuneelle altistumiselle.
Suomalaisista asunnoista ja työpaikoista löytyneet endotoksiinipitoisuudet
vaihtelivat tasolla 6,5 - 125 EU/mg. Suurimmat pitoisuudet löytyivät
rakennuksista, joissa oli koti-eläimiä ja/ tai kosteusvaurio.
Ryhmämme kehitti myös testin, jolla tutkia bakteeritoksiini
valinomysiinin vaikutusta koehenkilöiden valkosoluihin in vitro. Menetelmää
ei tämän tutkimuksen yhteydessä sovellettu
potilaiden materiaalinäytteisiin.
EXTENDED ABSTRACT
1 Introduction
This study forms independent part of the project 'Mould and moisture transfer in building structures and buildings with particular regard to prevention of health hazards' which is a co-operation between Helsinki University of Technology, HVAC Laboratory, and University of Kuopio. The aim of our study was to develop a new serum IgG avidity test that could be used to estimate exposure to moulds in cross-sectional and follow-up studies. The development of methods suitable for use in research and clinical practice is essential as there is a shortage of good laboratory parameters that can be used to confirm patients' exposure to moulds and bacteria in houses damaged by damp. In addition, a in vitro toxicity test was developed to test for Actinobacteria toxin in furnishing materials and dust. We also modified an endotoxin assay reported in the literature to detect cell components of gram-negative bacteria in house-dust.
2 Patients and Methods
Study of changes in specific mould IgG avidity
The Clinic for Indoor Air Health Problems began its work at the Helsinki
University Central Hospital, Departments of Dermatology and Allergic Diseases,
in 1995. Patients were selected on the basis of symptoms attributed
to dampness and examined by the staff physician (HMR). A trained environmental
inspector from the clinic visited the homes of these patients and
collected material samples from water damaged structures and surfaces.
The mycological laboratory of Helsinki University Central Hospital
isolated and identified different mould genera from these samples.
In total, 11 houses inhabited by the patients from the
clinic were examined. Nineteen employees of a day-care
center in Helsinki which was investigated by researchers from the University
of Technology also participated in this study. Thirteen laboratory
workers from Helsinki University Central Hospital acted as a reference
group.
The amount of specific mould allergen IgE-antibody in serum was determined by the Pharmacia CAP System FEIA method against 12 moulds (Aspergillus fumigatus, Aspergillus niger, Aspergillus versicolor, Cephalosporium acremonium, Cladosporium herbarum, Fusarium moniliforme, Stachybotrys atra, Trichoderma viridae, Penicillium species (6 different Penicillium species) and against one bacteria Thermoactinomyces vulgaris. The concentrations of specific IgG-antibodies against the above mentioned microbes were determined using the Pharmacia IgG CAP System Specific IgG FEIA method. The avidity of the circulating antibodies was measured by modifying the Pharmacia IgG CAPFEIA methods. IgG avidity test has previously been used for viral infections (Klaus Hedman et al. 1989). In the current project, we measured the IgG avidity in 18 adult patients with a known history of exposure to moulds. For comparison, the change in avidity was followed in a group of patients during hyposensitization treatment for birch allergy and three persons with fresh exposure to moulds. The basic avidity level in a population was estimated using a non-randomly selected reference group of same range of ages with no known history of exposure to moulds in damp or wet buildings.
Measurement of endotoxin content in house dust
In total 20 patients attending the Clinic for Indoor Air Health
Problems, with symptoms suspected to be related to exposure to moulds from
buildings damaged by damp, collected house-dust into a new vacuum cleaner
bag from surfaces in the rooms of their home or workplace.
The dust collected in the vacuum bag was emptied into a clean
PVC bag and kept frozen until the analyses. After thawing, the dust was
sieved through a 200 µm steel filter to remove sand and large particles.
Fine dust was extracted by a physiological NaCl (0.9 %)-phosphate buffer
(pH 7.4) in the concentration 5 g/l. Eluted endotoxin was assayed
with a quantitative Limulus test (Limulus Amebocyte Lysate QCL-1000 Quantitative
Chromogenic LAL method n:o 50-650 U; BioWhittaker,USA) at 37 oC as described
by Douwes et al. 1995). Escherichia coli O55:B5 endotoxin (BioWhittaker)
was used as standard endotoxin. Serially diluted samples were analyzed
in duplicate in two independent test runs. Endotoxin concentrations were
expressed per g house dust. The CV of the method was 6.6 %.
In vitro measurement of mithocondrial toxicity caused by Actinobacteries
Actinobacterium species (e.g. Streptomyces griseus) has been shown
to produce valinomycin and other related depsipeptides with toxic effects
on the mitochondrion of eucharyotic cells (Andersson et al. 1988). By incubating
isolated leukocytes from whole blood of healthy volunteers with various
doses of valinomycin and using the Cell Proliferation Reagent WST-1 (Roche),
a dose response was obtained in the range of 1-1000 ng valinomycin/l.
The use of the patient’s own blood cells in vitro will facilitate
the further development of methods of testing the influence of Actinobacteria,
and certain gram-positive bacteria found in damaged building, on the development
and persistence of symptoms in those exposed.
Health examination
The general health of participants whose houses or workplace
were investigated was examined. Clinical assessment included medical
history and current symptoms, physical examination, pulmonary function
tests, chest and maxillary sinus radiographs, complete blood cell count,
skin prick tests to common allergens and moulds, and sputum tests
(concentration of eosinophil cationic protein (ECP) and myeloperoxidase
MPO, as an indicator of inflammation process in the mucous membranes) and
determinations of IgG and IgE antibodies to moulds. In some cases,
examinations like high-resolution computer tomography, flexible bronchoscopy
and/ or diffusing capacity determination were also performed.
3 Results and Discussion
Most of the patients exposed to houses damaged by damp reported blocked nose, shortness of breath, sore throat, cough, increased mucus secretion, and irritation of nose and eyes. They often also report lethargy and headache. Only one person exposured to organic dusts experienced pulmonary disease such as allergic alveolitis (hypersensitivity pneumonitis) or ODTS (organic dust toxic syndrome), i.e., ’flu-like’ episodes with chills, fever, malaise, dyspnea, cough and chest tightness occurring 4 to 6 hours after exposure to the bioaerosol. Asthma was also rarely diagnosed, but patients often had asthma-like-symptoms. Increased concentration of myeloperoxidase (MPO) was found in the sputum of several persons. We have shown previously that high MPO content in sputum may also indicate an exposure to indoor air pollutants (Mussalo-Rauhamaa et al. 2000).
Study of changes in specific mould IgG avidity
In none of the patients, day-care workers or reference group
was the level of specific IgE response towards the mould species
studied increased. No significant differences were found in IgG concentrations
between these three groups except in the case of those against Thermoactinomyces
vulgaris where highest concentrations were found in patients' group, Table
1. Specific IgG antibody response with a lower avidity was
found in serum from several of the mould exposed persons from wet-damaged
houses, Table 2. The moulds in question were the same as those isolated
from the surfaces sampled. Thus these results may be interpreted
as showing, that these patient have undergone recent exposure to the moulds
in question.
Table 1. IgG antibody concentrations against certain moulds (mg/l) among adults
| PATIENTS | PATIENTS | DAY-CARE WORKERS | CONTROLS
(Laboratory) |
REFERENCE VALUES* | |
| Microbe
|
IgG antibody
concentration in sera N=16 Median N)** |
Range
|
IgG antibody
concentration in sera N=19 Median N)** |
IgG antibody
concentration in sera N=13 Median N)** |
Median
90% percentile mg/l |
| Aspergillus fumigatus | 24.1 (14) | <2.0 - 93.7 | 11.1 (19) | 16.3 (13) | 11.1 2.6 |
| Aspergillus niger | 15.9 (16) | 4.7 - 91.2 | 17.1 (19) | 25.9 (13) | 17.1 69 |
| Aureobasidium pullulans | 6.7 (14) | <2.0 - 24.9 | 5.1 (17) | 5.1 (12) | 5.1 2.8 |
| Cephalosporium acremonium | 11.2 (15) | <2.0 - 33.1 | 8.1 (17) | 12.1 (13) | 8.1 8.7 |
| Chaetomium globosum | 4.6 (13) | <2.0 - 12.6 | 3.7 (14) | 5.4 (13 ) | 3.7 8.7 |
| Cladosporium herbarum | 20.5 (14) | <2.0 - 78.4 | 14.3 (19) | 15.8 (13) | 14.3 4.6 |
| Fusarium moniliformis | 9.4 (14) | <2.0 - 109 | 7.8 (17) | 6.5 (12) | 7.9 5.6 |
| Phoma betae | 3.1 (9 ) | <2.0 - 5.5 | 3.1 (13) | 2.0 (7 ) | 1.8 7.5 |
| Stachybotrys atra | 5.2 (14) | <2.0 - 15.8 | 4.5 (17) | 4.2 (6 ) | 4.4 0.9 |
| Trichoderma viridae | 6.2 (12) | <2.0 - 15.7 | 2.6 (14) | 3.9 (10) | 2.6 2.3 |
| Penicillium sp. | 20.4 (15) | <2.0 - 190 | 15.7 (19) | 25.8 (13) | 15.7 6.7 |
| Aspergillus versicolor | 22.2 (11) | <2.0 - 135 | 20.0 (19) | 32.7 (13) | 29.7 100 |
| Thermoactinomyces vulgaris | 11.0 (16) | 3.6 - 29.8 | 5.6 (16) | 4.7 (10) | 14.9 34.4 |
Table 2. Results of the avidity assays. (*Only results over the detectable level 2 mg/l taken into the consideration in the avidity results.)
| PATIENTS | PATIENTS | DAY-CARE WORKERS | CONTROLS
(Laboratory) |
CONTROLS | |
| Microbe
|
Avidity
Median (%) (N)* |
Range
(%)
|
Avidity
Median (%) (N)* |
Avidity
Median (%) (N)* |
Range
(%)
|
| Aspergillus fumigatus | 59 (13) | 43 - 70 | 45 (17) | 60 (12) | 49 - 75 |
| Aspergillus niger | 53 (16) | 23 - 78 | 53 (18) | 55 (11) | 36 - 75 |
| Aureobasidium pullulans | 54 (11) | 40 - 69 | 42 (8 ) | 60 (8 ) | 46 - 65 |
| Cephalosporium acremonium | 65 (15) | 44 - 85 | 66 (15) | 70 (11 ) | 54 - 87 |
| Chaetomium globosum | 47 (9 ) | 36 - 64 | 54 (9 ) | 57 (7 ) | 51 - 72 |
| Cladoporium herbarum | 57 (15) | 17 - 65 | 52 (17) | 55 (13) | 41 - 72 |
| Fusarium moniliformis | 76 (14) | 50 - 90 | 70 (15) | 83 (11 ) | 67 - 111 |
| Phoma betae | 56 (4 ) | 47 - 64 | 53 (5 ) | 54 (3 ) | 53 - 59 |
| Stachybotrys atra | 57 (12) | 41 - 75 | 58 (10) | 68 (6 ) | 52 - 85 |
| Trichoderma viridae | 71 (11 ) | 55 - 77 | 64 (9 ) | 67 (8 ) | 51 - 83 |
| Penicillium sp. | 60 (14) | 41 - 70 | 65 (17) | 59 (12) | 51 - 89 |
| Aspergillus versicolor | 57 (14) | 39 - 66 | 49 (18) | 56 (13) | 52 - 77 |
| Thermoactinomyces vulgaris | 57 (14) | 30 - 77 | 63 (13 ) | 54 (10 ) | 48 - 78 |
The endotoxin study
Results of the endotoxin assays are summarized in Table 3. High
endotoxin concentrations in house-dust were found in rooms
where moisture problems were identified and/ or in dwellings
where pets especially dogs, were kept.
Table 3. Occurrence of endotoxin in house-dust
| Sampling place | No of samples | Endotoxin (EU/ml)
median range |
Endotoxin (EU/mg)
median range |
| Home | 19 | 47,5 7,3-625 | 9,5 0,9-125 |
| Workplace | 11 | 43,8 16,3-406 | 8,8 3,3-81 |
4 Conclusion
The assays developed here for the serum IgG-avidity and house-dust endotoxin determinations seem to be promising new laboratory parameters to help clinicians in their work to detect possible mould and bacteria exposure. Further work should test the robustness of these findings in larger study populations.
5 References
Andersson MA, Mikkola R, Helin J, Andersson MC, Salkinoja-Salonen MS. A novel sensitivity bioassay for the derection of Bacillus cereus emetic toxin and related depsipeptide ionophores. Applied and Environmental Microbiology 1988;64, 4767-4773.
Gereda JE, Leung DYM, Thatayatikum A, Streib JE, Price MR, Klinnert MD, Liu AH. Relation between house-dust endotoxin exposure, type 1 T-cell development, and allergen sensitisation in infants at high risk or asthma. Lancet 2000; 355, 1680-1683.
Douwes J, Doekes G, Heinrich J, Koch A, Bischof W, Brunekreef B. Endotoxin and beta1->3)-glucan in house dust and the relation with home characteristics: A pilot study in 25 German houses. Indoor Air 1998, 255-263.
Hedman K, Lappalainen M, Seppälä I. Recent primary toxoplasma infection indicated by a low avidity of specific IgG. Journal of Infectious Diseases 1989; 159,736- 740.
Mussalo-Rauhamaa H, Hakala K, Kiviranta K, Metso T, Haahtela T. Increased
myeloperoxidase (MPO) in induced sputum in patients with a history of indoor
air health problems. Proceedings of Healthy Buildings 2000, Exposure,
Human Responses and Building Investigations, vol. 1., Eds. O. Seppänen,
J. Säteri, SIY Indoor Air, Information Oy, Helsinki, Finland/ Gummerus
Kirjapaino Oy, Jyväskylä, pp. 263- 266