mushroom worker's lung resulting from indoor cultivation of

Occup. Med. Vol. 48, No. 7, pp. 465-468, 1998Copyright © 1998 Uppincott Williams & Wilkins for SOM

Printed in Great Britain. All rights reserved0962-7480/98

CASE REPORT Mushroom worker's lungresulting from indoorcultivation of Pleurotus osteatusS. Mori*, K. Nakagawa-Yoshida*, H. Tsuchihashi*,Y. Koreeda*, M. Kawabata*, Y. Nishiura*, M. Andot

and M. Osame**The Third Department of Internal Medicine, Kagoshima University Schoolof Medicine, 8-35-1 Sakuragaoka, Kagoshima 890, Japan; * The FirstDepartment of Internal Medicine, Kumamoto University School of Medicine,1-1-1 Honjo, Kumamoto 860, Japan

Indoor cultivation of oyster mushroom Pleurotus osteatus lead to an outbreak of extrinsicallergic alveolitis in two workers. High titer of indirect fluorescent antibody and positiveprecipitins against basidiospores of P. osteatus were demonstrated in sera of the patients.Mushroom workers should protect themselves from the basidiospores, being aware of theirpathogenicity.

Key words: Basidiospore; extrinsic allergic alveolitis; hypersensitivity pneumonitis; indirectfluorescent antibody; mushroom worker's lung; Pleurotus osteatus.

Occup. Med. Vol. 48, 465-468, 1998

Received 11 December 1997; accepted in final form 16 February 1998

INTRODUCTION

Edible oyster mushrooms, Pleurotus species, are grown allover the world.1 A cultivation technique of P. osteatus onartificial substrate in air-conditioned rooms rendered theproduction economical throughout the year. The indoorcultivation, however, regularly led to allergic symptoms inworkers.2"5 We present outbreaks of mushroom worker'slung (MWL) in a modern factory of P. osteatus anddiscuss the diagnosis and prevention of the disease.

CASE REPORTS

Case 1

A 33-year-old man, who had been working in a factory ofP. osteatus for a month, developed a productive cough, lowgrade fever and exertional dyspnoea. He repeatedly had thesymptoms on the evening of the day he worked in thegrowing room. He was a non-smoker and had no pasthistory of respiratory diseases. The physical examinationwas normal. C-reactive protein (CRP) was 8.1 mg/dl andwhite blood cell count (WBC) was 10,700/mm3.

Correspondence and reprint requests to: S. Mori, 4513 Akasegawa, AkuneCity, Kagoshima 899-16, Japan Tel: ( + 81) 996-73-1331; Fax: ( + 81)996-73-3708.

A chest radiograph and computed tomographic scanshowed diffuse bilateral ground glass appearanceand patchy infiltrates (Figure 1). Pulmonary functiontests showed a reduced diffusion capacity to 68.9%of the prediction.

Cultures of pathogenic microorganisms includingmycobacterium from sputa and bronchoalveolar lavage(BAL) fluid were negative. The BAL was performed at5 days after the last exposure. Of the 160 ml saline injec-ted, 111 ml of bronchoalveolar fluid was recovered. Totalcell yield was 23.8 x 106 (21.4 x 104/ml), and the dif-ferential cell count was 32% lymphocytes, 66% pul-monary alveolar macrophages, 1% polymorphonuclearleukocytes and 1% eosinophils with a CD4/CD8 ratio of1.36. A transbronchial lung biopsy specimen revealedgranulomatous alveolitis.

On suspicion of MWL, he was subjected to a naturalchallenge in the growing room for 2 h. Cough was repro-duced 3 h later and the maximum effect with a feverof 39.1 °C was observed 10 h later. The shadows on chestradiographs reappeared, and CRP and WBC had alsochanged from 0 mg/dl to 11.3 mg/dl and 5000/mm3 to13,400/mm3, respectively.

Case 2

A 65-year-old man, who had started working in the samefactory with his son (Case 1) in August 1996. He was

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466 Occup. Med. Vol. 48,1998

a current smoker but had no symptoms or past history ofrespiratory diseases. In late September, he developeda productive cough, low grade fever and night sweat,followed one month later by exertional dyspnoea. Thesymptoms became worse after work and disappearedwhen he was away from the factory. He had fine cracklesat both lung bases.

A chest radiograph and computed tomographic scanshowed diffuse bilateral fine nodular shadows par-tially accompanied by bullae and fibrosis. His diffu-sion capacity was 71.6% of the prediction and arterialoxygen tension was 79 mmHg.

The BAL was performed the next day of the lastexposure. Of 160 ml saline injected, 85 ml was recovered.Total cell yield was 15.3 x 106 (18xlO4/ml), and thedifferential cell count was 24% lymphocytes, 73%pulmonary alveolar macrophages, 2% polymorphonuc-lear leukocytes, and 1% eosinophils with a CD4/CD8ratio of 1: 52.

Environmental study. A pilot indoor cultivation of P. os-teatus was started in 1991, and the new factory was builtin August 1996. Every worker takes part in the wholeprocess, that is to place sawdust in plastic bottles, tosterilize the substrate, to inoculate the fungus into bottles,

Figure 1. A chest radiograph and computed tomographic scan onadmission show diffuse bilateral ground glass appearance and patchyinfiltrates.

to keep them at 20°C and 90% of relative air humidity for20 days and 17-18°C and 95% for 4 days in spawningrooms and then grow mushrooms at 16-18°C and 95%for 4 days in a growing room. Workers are exposed tobasidiospores for 4 hours per day when they work in thegrowing room, and when they pick and pack the mush-rooms in a shipping room. Most dominant particles ofdust on a mask worn in the room were basidiospores of P.osteatus, 3-4 x 7.5-11 \im in size. A few species of bac-teria and fungi were cultured from the dust but they werenot significant.

Indirect fluorescent antibody (IFA) test. The method ofVogel, slightly modified, was performed as reported pre-viously.6 Basidiospore of P. osteatus or the dust wassmeared on a glass slide and fixed by drying, then incu-bated with serum of the patients, eight co-workers or 10healthy non-exposed controls at 37°C for 30 min. Theslide was stained with fluorescent-labelled antiserum tohuman IgG, IgA and IgM and examined under an ultra-violet microscope. The antibody titer was determined bythe end point of serum dilution for the positive staining.The basidiospores alone reacted to the patients' sera atthe titer of 1:512, while it did not react to control sera

Gel double diffusion test. The method of Gerber andJones was performed with a slight modification, using1% agar in veronal buffer (pH 8.6) containing 1%sodium citrate and 0.1% NaN3.

6 This is well known asa standard method in the detection of specific antibodyactivities in EAA. Antigen was extracted from basidio-caps of P. osteatus with NaHCO3-buffered saline bya modified version of Santilli's method.7 The sera of thepatients showed precipitins against the extracted antigen(10 mg of dry weight/ml) but not against Thermoac-tinomyces vulgaris, Micropolyspora faeni or Aspergillus-related antigens (Hollister-Steir, Spokane, Washington).

Symptoms and antibodies of co-workers. We interviewed alleight co-workers in the factory and examined their serumantibodies to P. osteatus. The results are given in Table 1.

We advised patients and co-workers to wear a mech-anical filter respirator that filtrates 97% or more particleslarger than 0.44 u.m in size. All workers continue workingin the factory with minimum complaints since using themask.

DISCUSSION

The diagnosis of extrinsic allergic alveolitis (EAA)/hy-persensitivity pneumonia's (HP) involves documentingsymptoms related to the antigen exposure, and clinical,radiological and pathological findings compatible withEAA.8 The establishment of antigen exposure througha careful history-taking is a cornerstone of the diagnosis.Serum antibody determination to relevant antigens mayserve to confirm the exposure, though it must be evalu-ated in light of a patient's clinical feature. Provocationtesting of the suspected environment or antigen may

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S. Mori et al.: Mushroom worker's lung resulting from indoor cultivation of Pleurotus osteatus 467

Table 1.

Subject

Basidiospore antibodies in Pleurotus osteatus factory workers

Duration of engagement Symptoms

IFA

Antibodies

Precipitins

1 33 yr, male (case 1)2 65 yr, male (case 2)3 33 yr, female4 65 yr, male5 67 yr, female6 61 yr, female7 51 yr, male8 57 yr, female9 46 yr, male

10 29 yr, male

1 month3 months4 months5 years

4 months4 months4 months5 years

4 months4 months

Cough, fever, dyspnoeaCough, fever, dyspnoeaCough, sputumCoughCoughCough, sputumCough, sputumNilNilNil

1:5121:5121:5121:5121:1281:2561:1281:5121:2561:128

IFA = indirect fluorescent antibody.

confirm the diagnosis. In Japan, criteria for EAAproposed by the research committee on diffuse pul-monary diseases organized by the Japanese Ministry ofWelfare are standard.9'10 The present cases were definiteMWL, a well-known type of EAA, according to thecriteria.

Although BAL is not essential for the diagnosis ofEAA, it has the advantage of sensitive detection of al-veolitis in EAA and distinguishing it from other dis-eases.10'" For clinicians, a positive BAL finding (i.e., thecharacteristic profile) in a patient with interstitial lungdisease of unknown origin should direct them towards theprobable diagnosis of EAA. A careful re-examination ofthe occupational environmental history and the screeningof serum antibody might then reveal previously unknownsources of relevant antigen exposure and confirm thediagnosis. For researchers, information on phenotypes ofBAL lymphocytes is necessary to investigate immunolo-gical etiology of EAA; this is because the phenotypes ofBAL lymphocytes vary with the type of EAA, probablydepending on factors such as causative agent, smoking orstaging of the diseases.10 This is, to our best knowledge,the first report of BAL findings in EAA caused byP. osteatus.

Basidiomycetes consist of approximately 25,000 spe-cies and include mushrooms, puffballs, smuts, rustsand bracket fungi. Recent studies have demonstrated thatbasidiospores are important aeroallergens and P. osteatusis one of the most reactive species among patients withatopic respiratory diseases in the USA and Europe.12

In spite of the significance in type I and III/IV allergy,basidiospore antigens for diagnostic or therapeutic useare not available commercially. A few groups have extrac-ted and purified the antigens from Pleurotus or othermushrooms and detected the specific IgE or IgG antibod-ies in sera from patients.2"5112 However, the preparation ofspore extracts requires many mushrooms and the proced-ure is too complicated and time-consuming to follow inclinical laboratories.

The IFA assay is simple, semi-quantitative and applic-able to a small amount of particles collected from theenvironment directly, without preparation of antigen.6 Inprevious studies, we demonstrated the high specificityand sensitivity of the assay in 262 patients with summer-

type HP,9 and the practicality in cases of EAA caused bynew antigens.14"17 In the present study, the IFA titeragainst basidiospores of P. osteatus was significantly high-er in patients than controls. The higher IFA titer tendedto correlate to positive precipitins, but did not reflect theduration of engagement or symptoms.

Mushroom worker's lung was originally reported 40years ago.18'19 The original and common type of MWL iscaused by the inhalation of large numbers of thermophilicactinomycetes from compost used for the cultivation ofAgaricus bisporus.8'18'20 The white button mushroomA. bisporus (Lange) Imbach (= A. brunnescens Peck) hasbeen the most popular mushroom cultivated worldwide.1

Recently, however, alternative mushrooms such asP. osteatus and Shii-take Lentinus edodes are becomingimportant crops for a developing market.1"5 Conse-quently, MWL in P. osteatus or L. edodes farms is emerg-ing and the cause has proved to be basidiospores of themushroom itself.2"5

The pathogenicity of P. osteatus and L. edodes should berecognized as distinct from A. bisporus.2'4'5'18'20 It is be-cause the former has no veil and releases a tremendousamount of basidiospores during cap growth before attain-ing a marketable size, while the latter is harvested beforethe veil is broken and thus minimum spores are released.A cross-sectional study in a farm of A. bisporus revealedthat approximately 20% of the more heavily exposedworkers reported symptoms consistent with MWL and10% had impaired pulmonary function. Seven cases(2-3% of total workers) of MWL were diagnosed but nospecific antigens were identified as the cause.20 In thepresent study, two cases of MWL were diagnosed and70% of participants were symptomatic. It has also beenreported that all workers of a Shii-take farm developedrespiratory symptoms consistent with MWL caused bybasidiospores of L. edodes} The high incidence of MWLor respiratory symptoms may result from not only thehigh antigenicity but also the high concentration of thebasidiospores in indoor cultivation of these mushrooms.

Mushroom workers should become aware thatbasidiospores themselves are pathogenic. Instructions forthe indoor cultivation of P. osteatus need to containsufficient information of health risks and the protec-tive measures. Occupational physicians are responsible

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468 Occup. Med. Vol. 48, 1998

for advising workers on effective mechanical filterrespirators and to monitor workers health via question-naires, pulmonary function tests and specific antibodies,since an old problem is recurring in a new workenvironment.

ACKNOWLEDGEMENT

We thank Dr H. Neda for mycological advice.

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