Differences in fungi present in induced sputum samples from asthma patients and non-atopic controls: a community based case control study

  • Hugo Cornelis van Woerden1Email author,

    Affiliated with

    • Clive Gregory1Email author,

      Affiliated with

      • Richard Brown2,

        Affiliated with

        • Julian Roberto Marchesi2,

          Affiliated with

          • Bastiaan Hoogendoorn1 and

            Affiliated with

            • Ian Price Matthews1

              Affiliated with

              BMC Infectious Diseases201313:69

              DOI: 10.1186/1471-2334-13-69

              Received: 10 August 2012

              Accepted: 28 January 2013

              Published: 5 February 2013

              Abstract

              Background

              There is emerging evidence for the presence of an extensive microbiota in human lungs. It is not known whether variations in the prevalence of species of microbiota in the lungs may have aetiological significance in respiratory conditions such as asthma. The aim of the study was to undertake semi-quantitative analysis of the differences in fungal species in pooled sputum samples from asthma patients and controls.

              Methods

              Induced sputum samples were collected in a case control study of asthma patients and control subjects drawn from the community in Wandsworth, London. Samples from both groups were pooled and then tested for eukaryotes. DNA was amplified using standard PCR techniques, followed by pyrosequencing and comparison of reads to databases of known sequences to determine in a semi-quantitative way the percentage of DNA from known species in each of the two pooled samples.

              Results

              A total of 136 fungal species were identified in the induced sputum samples, with 90 species more common in asthma patients and 46 species more common in control subjects. Psathyrella candolleana, Malassezia pachydermatis, Termitomyces clypeatus and Grifola sordulenta showed a higher percentage of reads in the sputum of asthma patients and Eremothecium sinecaudum, Systenostrema alba, Cladosporium cladosporioides and Vanderwaltozyma polyspora showed a higher percentage of reads in the sputum of control subjects. A statistically significant difference in the pattern of fungi that were present in the respective samples was demonstrated using the Phylogenetic (P) test (P < 0.0001).

              Conclusion

              This study is novel in providing evidence for the widespread nature of fungi in the sputum of healthy and asthmatic individuals. Differences in the pattern of fungi present in asthma patients and controls merit further investigation. Of particular interest was the presence of Malassezia pachydermatis, which is known to be associated with atopic dermatitis.

              Keywords

              Asthma Sputum Fungi Case–control study

              Background

              The human lung has a surface area of around 50 m2[1] and is in contact with more than 15,000 litres of air each day [2]. At each breath around 5,000 particles of dust are inhaled [3]. On average, the dust in the earth’s atmosphere contains 10,000 to 100,000 organisms per gram of dust [4], some of which is in the ‘respirable dust’ fraction, consisting of particles smaller than 5 μm [5]. This extensive exposure to the environment means that the lungs are a common portal for infection by viruses, bacteria, fungi, protozoa and other infectious agents.

              Historically, healthy lungs were believed to be free of bacteria and that during infection organisms “gain a foothold in the normally sterile lung tissue” [6]. However, there is increasing evidence that microbiota are present even in healthy lungs [7]. This finding raises the possibility of a potential overlap between pathogenic and commensal microbiota in the respiratory tract.

              There is relatively little literature examining microbiota in human lungs. Tunney et al. [8] showed that approximately 50% of healthy individuals harbour between 1000 and 10,000 culturable anaerobic bacteria per ml of induced sputum A range of microbial species were also found in induced sputum at low numbers in another study which examined sputum from healthy subjects [9]. One previous study has been identified which used metagenomic culture independent genomic techniques and demonstrated that microbial communities in asthmatic airways were disordered, with pathogenic Proteobacteria more frequently found in the bronchi of asthmatics patients than in controls [10].

              The current study further examines the role of atypical microbiota in respiratory disease. The study used molecular techniques to identify eukaryote species that were present in induced sputum samples taken from asthma patients and controls, living in Wandsworth, London. The aim of the study was to undertake semi-quantitative analysis of the differences in fungal species present in pooled sputum samples from asthma patients and controls.

              Methods

              Study population

              The study protocol was approved by Camden and Islington community local research ethics committee (ref 08/H0722/540). All patients participating in the study supplied informed consent. This case control study from which the induced sputum samples were drawn has previously been described. Further information on the characteristics of the subjects in this study has been provided in that paper [11]. In summary, participants were residents of Wandsworth, London, and were primarily identified from the patient registers of two GP practices. Asthma patients were defined as those individuals who had a current diagnosis of asthma, for example, by being on the GP practice asthma register. Most of the asthma patients were on inhaled corticosteroids. Non-atopic controls were defined as individuals who on questioning did not report having current or previous asthma, eczema or hay fever. All participants competed a published questionnaire [12] to assess the risk of mould in the home. The questionnaire contained four questions: Is there any visible mould growth on your house? Is there any odour of mould or cellar-like musty air in your house? Is there any moisture stains in your house? Is there any water/moisture damage in your house?

              Sputum collection and DNA extraction

              Participants inhaled isotonic saline via an ultrasonic nebuliser. Globules of sputum were coughed up into petri dishes, spread on microscope slides and stained for microscopic examination. Approximately 5 mm2 areas were excised from each microscope slide. The samples were combined to yield two pooled samples for subsequent DNA extraction and PCR: asthma patients and control subjects. (A sample from one asthma patient was inadvertently included in the control set). DNA was subsequently extracted using the Zymo research pinpoint system (Zymo Research, Irvine, Ca) in accordance with manufacturer’s instructions. The samples were taken from 30 asthma patients and 13 non-atopic control subjects involved in the case control study.

              Pyrosequencing of extracted DNA and statistical analysis

              Extracted DNA was amplified using a PCR protocol for the partial 18S rRNA gene using the primer pair (Euk1a (5’ CTG GTT GAT CCT GCC AG 3’) and Euk516r (5’ ACC AGA CTT GCC CTC C 3’)) in accordance with previously described protocols [13, 14]. The two pooled extract amplicons, from asthma patients and from controls, were sequenced using a 454 pyrosequencer by Research and Testing Inc, Lubbock, Texas, USA. DNA sequences were compared to the SILVA database of known eukaryotic 18S rRNA gene sequences to determine in a semi-quantitative way the proportional distribution in each of the two samples.

              The difference between the pattern of fungal species in each of the two pooled samples was compared using Unifrac [15, 16]. This online software uses phylogenetic information to test whether or not two environments are significantly different. The software estimates the similarity between communities by measuring the number of changes that would be required to explain the differences in the distribution of sequences between the two environments.

              Results

              Study population and presence of mould in the home

              Patients had a mean age of 41.6 years (SD 14.9, range 18–65 years) and control participants a mean age of 35.7 years (SD 12.8, range 24 – 58 years). The patient group was 40% male and the control group was 46% male.

              A positive answer to at least one of the four questions regarding possible mould in the home was recorded in 30% (9/30) asthma patients and 15.4% (2/13) of control subjects. Due to the small sample size, this relatively large difference was not statistically significant.

              Analysis of pyrosequencing data

              The differences based on the percent of total DNA reads of in the pooled samples from asthma patients and non-atopic controls are shown in Tables 1 and 2. A statistically significant difference in the pattern of fungi that were present in the respective samples was demonstrated using the Phylogenetic (P) test (P <0.0001).
              Table 1

              Fungi that were more common in asthma patients than in control participants (difference in percent of DNA reads in descending order)

              Fungal species

              Control participants

              Asthma patients

              Difference

              Psathyrella candolleana

              0.000

              27.294

              27.294

              Malassezia pachydermatis

              0.000

              21.651

              21.651

              Termitomyces clypeatus

              0.000

              7.071

              7.071

              Grifola sordulenta

              0.000

              4.489

              4.489

              Pycnoporus sp

              0.000

              2.938

              2.938

              Phlebiopsis gigantean

              0.000

              2.932

              2.932

              Dichostereum pallescens

              0.000

              2.746

              2.746

              Peniophorella praetermissa

              0.168

              2.617

              2.449

              Aspergillus zonatus

              0.016

              1.959

              1.942

              Acanthophysium cerussatum

              0.000

              1.825

              1.825

              Pleurotus ostreatus

              0.000

              1.615

              1.615

              Candelabrochaete africana

              0.016

              1.556

              1.540

              Basidiobolus ranarum

              0.000

              1.510

              1.510

              Tapinella atrotomentosa

              0.000

              1.487

              1.487

              Pleurocybella porrigens

              0.000

              1.399

              1.399

              Debaryomyces hansenii

              0.000

              1.294

              1.294

              Collybia tuberosa

              0.000

              1.282

              1.282

              Galerina atkinsoniana

              0.000

              1.230

              1.230

              Punctularia strigosozonata

              0.000

              1.148

              1.148

              Elderia arenivaga

              0.000

              1.143

              1.143

              Pseudoarmillariella ectypoides

              0.000

              0.834

              0.834

              Pulcherricium caeruleum

              0.000

              0.665

              0.665

              Tilletia goloskokovii

              0.000

              0.618

              0.618

              Cerrena sp

              0.000

              0.600

              0.600

              Serpula lacrymans

              0.000

              0.571

              0.571

              Bondarcevomyces taxi

              0.000

              0.554

              0.554

              Resinicium bicolor

              0.000

              0.519

              0.519

              Cortinarius sodagnitus

              0.000

              0.414

              0.414

              Trichaptum abietinum

              0.000

              0.315

              0.315

              Chamaeota sinica

              0.000

              0.262

              0.262

              Peziza vesiculosa

              0.000

              0.239

              0.239

              Pterula echo

              0.000

              0.204

              0.204

              Laccocephalum mylittae

              0.000

              0.198

              0.198

              Coprinopsis cinerea

              0.000

              0.198

              0.198

              Exidiopsis calcea

              0.000

              0.187

              0.187

              Dioszegia fristingensis

              0.000

              0.157

              0.157

              Inonotus baumii

              0.060

              0.210

              0.150

              Hydnochaete olivacea

              0.000

              0.134

              0.134

              Derxomyces boekhoutii

              0.000

              0.128

              0.128

              Xeromphalina campanella

              0.000

              0.117

              0.117

              Pulchromyces fimicola

              0.000

              0.111

              0.111

              Aspergillus oryzae

              0.000

              0.099

              0.099

              Entoloma prunuloides

              0.000

              0.099

              0.099

              Dioszegia zsoltii

              0.000

              0.093

              0.093

              Basidiobolus haptosporus

              0.000

              0.087

              0.087

              Saccharomycopsis fibuligera

              0.000

              0.087

              0.087

              Galiella rufa

              0.000

              0.082

              0.082

              Derxomyces simaoensis

              0.000

              0.070

              0.070

              Mycoclelandia arenacea

              0.000

              0.064

              0.064

              Steccherinum fimbriatum

              0.000

              0.064

              0.064

              Austropaxillus sp

              0.000

              0.058

              0.058

              Meyerozyma guilliermondii

              0.000

              0.058

              0.058

              Volvariella caesiotincta

              0.000

              0.058

              0.058

              Galerina marginata

              0.000

              0.052

              0.052

              Occultifur externus

              0.000

              0.052

              0.052

              Hericium americanum

              0.000

              0.047

              0.047

              Penicillium commune

              0.000

              0.041

              0.041

              Volvopluteus earlei

              0.000

              0.041

              0.041

              Gymnopus dryophilus

              0.000

              0.029

              0.029

              Aspergillus terreus

              0.000

              0.023

              0.023

              Tritirachium sp

              0.000

              0.023

              0.023

              Gloiocephala aquatic

              0.000

              0.023

              0.023

              Tricholoma matsutake

              0.000

              0.023

              0.023

              Exidia uvapsassa

              0.000

              0.017

              0.017

              Rhodocollybia maculate

              0.000

              0.017

              0.017

              Lentinus sp

              0.000

              0.017

              0.017

              Teratosphaeria acidotherma

              0.076

              0.093

              0.017

              Taphrina deformans

              0.000

              0.012

              0.012

              Antrodia vaillantii

              0.000

              0.012

              0.012

              Aspergillus penicillioides

              0.000

              0.012

              0.012

              Passalora vaginae

              0.000

              0.012

              0.012

              Malassezia furfur

              0.000

              0.012

              0.012

              Candida sp

              0.000

              0.012

              0.012

              Piromyces sp

              0.000

              0.012

              0.012

              Paxillus vernalis

              0.000

              0.012

              0.012

              Derxomyces mrakii

              0.000

              0.012

              0.012

              Lasiodiplodia gonubiensis

              0.000

              0.012

              0.012

              Teratosphaeria ohnowa

              0.005

              0.012

              0.006

              Diversispora celata

              0.000

              0.006

              0.006

              Geomyces destructans

              0.000

              0.006

              0.006

              Coprinopsis sp

              0.000

              0.006

              0.006

              Chlamydosauromyces punctatus

              0.000

              0.006

              0.006

              Mortierella minutissima

              0.000

              0.006

              0.006

              Saccobolus dilutellus

              0.000

              0.006

              0.006

              Thanatephorus fusisporus

              0.000

              0.006

              0.006

              Boletellus shichianus

              0.000

              0.006

              0.006

              Puccinia poarum

              0.000

              0.006

              0.006

              Mallocybe dulcamara

              0.000

              0.006

              0.006

              Coniophora marmorata

              0.000

              0.006

              0.006

              Trichosporon sp

              0.000

              0.006

              0.006

              Table 2

              Fungi that were more common in control participants than in asthma patients (difference in percent of DNA reads in descending order)

              Fungal species

              Control participants

              Asthma patients

              Difference

              Eremothecium sinecaudum

              41.319

              1.026

              40.293

              Systenostrema alba

              23.587

              0.000

              23.587

              Cladosporium cladosporioides

              14.484

              0.111

              14.374

              Vanderwaltozyma polyspora

              6.778

              0.140

              6.638

              Entophlyctis helioformis

              2.976

              0.064

              2.912

              Rozella allomycis

              3.009

              0.198

              2.811

              Protomyces macrosporus

              1.971

              0.082

              1.890

              Mortierella verticillata

              1.135

              0.000

              1.135

              Pseudotaeniolina globosa

              1.086

              0.210

              0.876

              Dothidea ribesia

              0.701

              0.000

              0.701

              Sporobolomyces yunnanensis

              0.549

              0.006

              0.543

              Teratosphaeria mexicana

              0.261

              0.000

              0.261

              Myriangium duriaei

              0.179

              0.000

              0.179

              Phaeobotryosphaeria visci

              0.174

              0.000

              0.174

              Kionochaeta sp

              0.152

              0.012

              0.140

              Catenulostroma chromoblastomycosum

              0.157

              0.017

              0.140

              Phaeobotryon mamane

              0.125

              0.000

              0.125

              Allomyces arbuscula

              0.125

              0.000

              0.125

              Schizothyrium pomi

              0.109

              0.000

              0.109

              Mycosphaerella endophytica

              0.103

              0.000

              0.103

              Penidiella columbiana

              0.098

              0.000

              0.098

              Aspergillus fumigatus

              0.174

              0.087

              0.086

              Cladosporium sp

              0.081

              0.000

              0.081

              Aleuria aurantia

              0.054

              0.000

              0.054

              Ascodesmis sphaerospora

              0.049

              0.000

              0.049

              Penicillium decumbens

              0.043

              0.000

              0.043

              Metschnikowia bicuspidata

              0.027

              0.000

              0.027

              Dothidea insculpta

              0.016

              0.000

              0.016

              Dendryphiella arenaria

              0.016

              0.000

              0.016

              Aigialus rhizophorae

              0.011

              0.000

              0.011

              Scutellospora spinosissima

              0.011

              0.000

              0.011

              Volvariella volvacea

              0.011

              0.000

              0.011

              Flammulina velutipes

              0.011

              0.000

              0.011

              Candida bituminiphila

              0.005

              0.000

              0.005

              Ascobolus carbonarius

              0.005

              0.000

              0.005

              Cyttaria sp

              0.005

              0.000

              0.005

              Halosarpheia japonica

              0.005

              0.000

              0.005

              Phymatotrichopsis omnivora

              0.005

              0.000

              0.005

              Sporobolomyces sp

              0.005

              0.000

              0.005

              Orphella haysii

              0.005

              0.000

              0.005

              Coccocarpia erythroxyli

              0.005

              0.000

              0.005

              Termitaria sp

              0.005

              0.000

              0.005

              Candida glabrata

              0.005

              0.000

              0.005

              Schizosaccharomyces japonicus

              0.005

              0.000

              0.005

              Cladochytrium sp

              0.005

              0.000

              0.005

              Cyttaria hookeri

              0.011

              0.006

              0.005

              A total of 136 fungal species were identified in the induced sputum samples, with 90 species more common in asthma patients and 46 species more common in control subjects, based on the percent of total DNA reads (see Figure 1). Psathyrella candolleana, Malassezia pachydermatis, Termitomyces clypeatus and Grifola sordulenta were particularly prevalent in the sputum of asthma patients and Eremothecium sinecaudum, Systenostrema alba, Cladosporium cladosporioides and Vanderwaltozyma polyspora were particularly prevalent in the sputum of control subjects. No other eukaryote species were identified in the sputum samples.
              http://static-content.springer.com/image/art%3A10.1186%2F1471-2334-13-69/MediaObjects/12879_2012_2243_Fig1_HTML.jpg
              Figure 1

              Graph showing the percentage of pyrosequencing reads for fungal species identified in the Asthma patient ( A ) or control participant ( C ) samples (species identified for reads greater than 1%).

              Discussion

              The range of fungal species present in both asthma patients and control subjects was larger than expected. There were also clear differences in the pattern of fungal species between asthma patients and control subjects. The fungi Malassezia pachydermatis, was found in patients with asthma and not the control group. This organism has a known association with atopic conditions including atopic dermatitis [17]. However, there were no other obvious associations were identified in the published literature between asthma and the other fungi found in the pooled samples from the asthma patients. Two of the fungi most commonly found in the sputum of asthma patients (Termitomyces clypeatus and Psathyrella candolleana) represent members of the basidiomycete family [18]. The latter has been found in indoor dust [19] and one can speculate that fungal spores may have been inhaled within the home. It is possible that most of the fungi identified could have come from a single individual, or a small number of individuals, whose samples were heavily colonised by fungi.

              Except for Cladosporium, the species identified in induced sputum are not commonly found in air samples examined using standard culture techniques [20]. Analysis of air samples using molecular techniques may demonstrate that these species are commonly present in the air, but this research has not been undertaken so far. Three out of four species detected in the sputum of asthma patients were from the macromycetes group (commonly known as mushrooms). Although asthma is associated with damp environments that are affected by mould growth, we are unaware of any study that has identified an association between macromycetes and asthma. Future studies should consider analysing air samples from the homes of participants using molecular techniques, so as to take into account the presence of fungi in the ambient environment of participants.

              We used universal primers for the eukaryotic 18S rRNA gene and were surprised that no eukaryotes other than fungi were identified in cases or controls. We have considered a number of potential reasons why this may be the case and the most likely was that levels of non-fungal eukaryotic DNA, present in the samples, was below the limits of detection. The PCR primers were chosen after considerable deliberation and a probeCheck test showed that they were universal and matched Homo sapiens’ 18S rRNA gene 100% [21]. However other potential reasons include: a genuine absence of other eukaryotes and unintended removal of DNA from other eukaryotes as part of the processing of the samples.

              Individual level analysis of samples was considered, but rejected as it was anticipated that, if the samples from each individual were analysed separately, the number of eukaryotes in each sample would be below the threshold of detection. Samples for this study were therefore pooled to maximise the number of copies of each species in the pooled samples and consequently maximise the probability of detecting all the species that were present.

              The study has a number of weaknesses. The sputum was not fresh when it was examined and although every effort was made to prevent contamination of samples by spores in the air, this is a possibility. The sample size is small and therefore may not be representative of asthma patients. Unfortunately, information on pets was not collected in this study and therefore could not be correlated with the presence of absence of particular fungi. It is possible to speculate that the presence of a pet (particularly a dog) in the subjects' house might be associated with the presence of Malassezia pachydermatis in the sputum of the research subjects, as this organism has been identified as a commensal on the skin of dogs and have could contaminated air in the homes of some of the research participants.

              The potential significance of these fungi is unclear. There is tentative emerging evidence that microbiota may form part of a complex causal web that results in disease, for example, by their effects on the immune system, without becoming pathogenic in the classical sense. For example, microbial compounds present in sputum may play a role as adjuvant factors and encourage a Th2-biased allergic response [22, 23].

              Conclusion

              This study provides emerging evidence for the widespread presence of fungi in the sputum of asthma patients and control subjects. Significant differences have been identified in the pattern of fungi present in asthma patients and control subjects drawn from the same community. Although this method demonstrates the possibility of using microscopy samples, further investigation is warranted which applies these techniques to fresh sputum samples. This method may in itself be applicable to analysis of historical samples and may in turn prove of interest in evaluating the microbiome of the lung demographically and between generations.

              Declarations

              Acknowledgments

              Funding was by Wandsworth PCT and Cardiff University. We are grateful to a range of colleagues who have contributed to the previously reported study based on this case control study and to the two GP practices that provided access to patients.

              The authors would like to acknowledge the assistance of Research and Testing Inc, Lubbock, Texas, USA in carrying out the final stage pyrosequencing.

              Authors’ Affiliations

              (1)
              Institute of Primary Care & Public Health, Cardiff University School of Medicine, Neuadd Meirionnydd
              (2)
              Cardiff School of Bioscience

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              24. Pre-publication history

                1. The pre-publication history for this paper can be accessed here:http://​www.​biomedcentral.​com/​1471-2334/​13/​69/​prepub

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              © van Woerden et al.; licensee BioMed Central Ltd. 2013

              This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.