© 2014 APMIS. Published by John Wiley & Sons Ltd. DOI 10.1111/apm.12253
APMIS
Increased prevalence and altered species composition of filamentous fungi in respiratory specimens from cystic fibrosis patients SIGNE M. NIELSEN,1 LISE KRISTENSEN,2 ANNETTE SØNDERGAARD,1 KURT J. HANDBERG,1 JØRGEN STENDERUP2 and NIELS NØRSKOV-LAURITSEN1 1
Department of Clinical Microbiology, Aarhus University Hospital, Aarhus; and 2Department of Microbiology, Viborg Hospital, Viborg, Denmark
Nielsen SM, Kristensen L, Søndergaard A, Handberg KJ, Stenderup J, Nørskov-Lauritsen N. Increased prevalence and altered species composition of filamentous fungi in respiratory specimens from cystic fibrosis patients. APMIS 2014. Filamentous fungi cultured from respiratory tract specimens submitted to the department of clinical microbiology, Aarhus University Hospital, during 2010 were identified by morphology and by internal transcribed spacer (ITS) sequencing. Of 343 fungal isolates, discrepancies between identification methods were observed for four isolates (1.2%), while identification to species was achieved only with ITS sequencing for 16 isolates (4.7%). Filamentous fungi were isolated from 15% of cystic fibrosis (CF) respiratory samples in contrast to 2% of non-CF samples. From CF patients, a total of nine different species were found in 188 samples from 48 patients, whereas from non-CF patients, 24 different species were found in 155 samples from 111 patients. CF was associated with a significant overrepresentation of Aspergillus fumigatus and Scedosporium species; in contrast, the frequency of Penicillium spp. and other putative contaminants were significantly increased in non-CF patients. The altered species variation of filamentous fungi in CF respiratory specimens is contradictory to a scenario of incidentally inhaled spores, trapped in the viscous airway mucus of these patients and subsequently expectorated; rather, our data most likely reflect both an increased prevalence and an increased proportion of truly colonizing fungi in this patient group. Key words: Cystic fibrosis; ITS sequencing; identification discrepancies; Scedosporium. Niels Nørskov-Lauritsen, Department of Clinical Microbiology, Aarhus Brendstrupgaardsvej, DK-8200 Aarhus, Denmark. e-mail: [email protected]
A wide variety of filamentous fungi are able to cause infections in humans. Some species are true pathogens capable of infecting healthy individuals, but most are opportunistic pathogens that occur naturally in the environment where they disperse large amounts of spores into the atmosphere. The spores may be inhaled or otherwise ingested by healthy humans without causing any disease. In some cases, however, opportunistic moulds may be the cause of superficial or invasive infections, or may cause allergic reactions (1–3). Invasive infections usually occur in immunocompromised hosts, such as cancer patients, transplant recipients and other patients receiving immunosuppressive
Received 17 December 2013. Accepted 20 December 2013
University
Hospital
Skejby,
100
treatment. In contrast, allergic broncho-pulmonary aspergillosis (ABPA) usually occurs in patients with underlying lung diseases, such as asthma or cystic fibrosis (CF) (2, 3). CF is a genetic disease characterized by increased viscosity of bronchial mucus and impaired mucocilliary clearance of inhaled aerosolized bacteria and fungal spores, resulting in a vicious cycle of colonization, infection and inflammation (4–8). Similar to other patient groups, Aspergillus fumigatus is by far the most commonly cultured mould from the sputum of CF patients. Scedosporium species have, however, recently attracted attention, partly because of the reduced possibilities for antifungal treatment (4, 5, 8, 9). The most common disorder in CF patients caused by A. fumigatus is ABPA, but A. fumigatus may also be associated with deteriorating lung function 1
NIELSEN et al.
in CF patients in the absence of ABPA (1, 3), and CF patients colonized with A. fumigatus are more frequently hospitalized than non-colonized patients (10). Identification of medically important fungi is important for correct diagnosis and for prescription of optimal antifungal chemotherapy. Species identification by microscopic and macroscopic characterization is inexpensive and reliable, but the morphological structures used for identification can be atypical, slowly presented, or even absent (11, 12). Molecular identification methods have, in recent years, gained a foothold in many clinical laboratories. Especially, sequencing of the internal transcribed spacer (ITS) regions, located between the 18S and 28S rRNA gene subunits, has emerged as a general method for identification of fungal species. This region is chosen because primers are available in the adjacent, highly conserved rRNA genes, while the ITS region includes enough sequence variation to distinguish between species or species groups (11, 13–15). In this study, we have determined the prevalence and species variation of filamentous fungi cultured from clinical respiratory tract specimens from CF and non-CF patients. Identification was performed by both morphological criteria and ITS sequencing. MATERIALS AND METHODS Samples and culture Routine sputum samples were inoculated on 5% blood agar and chocolate agar without pre-treatment of the sputum, and incubated for 48 h at 35 °C. A M€ uller-Hinton agar (Oxoid, Denmark) with selected antibiotic discs was also inoculated; this agar was not used for antibiotic susceptibility testing, but as a selective medium aiding the initial differentiation of microorganisms. If special culture for fungi was requested, an additional 5% blood agar and a Sabouraud-glucose agar were inoculated and incubated at 30 °C for 7 days. All filamentous fungi cultured from respiratory specimens submitted to the department of clinical microbiology, Aarhus University Hospital, during 2010 were included in the study, irrespective of whether special fungal culture was requested. Morphological identification was conducted at the Department of Microbiology, Viborg Hospital, and isolates were stored in 10% glycerol at 80 °C for later sequencing of ITS. If discrepant results were obtained with the two identification methods, the isolates were thawed and reinvestigated by both methods.
Conventional identification Samples were inoculated on V8 Juice agar and/or Sabouraud dextrose agar and incubated at 25 °C up to 12–14 days. Morphological identification was based on colonial form, surface colour and growth rate, and
2
microscopic assessment of hyphae (size and branching, septate or aseptate), conidia and conidia-bearing structures. For some species, the maximum growth temperature was determined. Identification was done according to de Hoog et al. (14).
PCR and identification by ITS sequence DNA was extracted using PrepMan Ultra Sample Preparation Reagent (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. The ITS2 region between the 58S and 28S rRNA genes was amplified by PCR using the primers ITS 3 and ITS 4 (16). The amplified PCR products were visualized by UV light after gel electrophoresis on a 1% agarose gel stained with GelRedR. The remaining product was treated with SAP/Exo (Shrimp Alkaline Phosphatase/Exonuclease I) (Medinova and Fermentas respectively) and Sanger sequenced at an extern facility (GATC Biotech AB, Konstanz, Germany) by use of the ITS 4 primer. In approximately 4% of reactions, sequences with ambiguously determined nucleotides were obtained. For these isolates, the ITS PCR was repeated and sequencing was performed on both DNA strands using the ITS 3 and ITS 4 primer, respectively. The ITS sequences were edited and searched against the ITS Sequence database from the Molecular Mycology Research Laboratory (MYC), University of Sydney (http://www.mycologylab.org). For 19 isolates, identification was not possible using the MYC database; this was the case for some species of Penicillium spp., Rhizomucor spp., Fusarium spp. and for S. apiospermum and A. nomius, probably due to a lack of reference sequences in the database. These isolates were identified by comparison with deposited sequences in GenBank (http://www.ncbi. nlm.nih.gov/BLAST). PCR amplification of Aspergillus b-tubulin (17) was carried out to confirm the identity of a single isolate which had no morphological characteristics to support the identification of A. fumigatus obtained by ITS (Table 1, specimen 5).
Statistical analysis Filamentous fungi were combined into four groups: A. fumigatus; A. non-fumigatus; Scedosporium spp. and putative contaminants (Penicillium spp., Trichoderma viride and Cladosporium sphaerospermum). The groups were compared between CF and non-CF patients (Table 4). Statistical analysis comparing samples are based on group comparisons using logistic regression with robust variance estimation to allow for the clustered data structure, where single patients may contribute multiple observations. Ninety-five per cent confidence intervals are based on binomial regression with robust variance estimation. However, comparison of the group Scedosporium is not possible using this statistical method due to the low number of observations in this group. Statistical analysis comparing patients (one sample of each species of fungi from each patient was included in this group) was based on group comparisons using Fishers exact test. All statistical analyses were carried out using Stata Statistical Software Release 12 (StataCorp LP, College Station, TX, USA). A p-value of
APMIS
Increased prevalence and altered species composition of filamentous fungi in respiratory specimens from cystic fibrosis patients SIGNE M. NIELSEN,1 LISE KRISTENSEN,2 ANNETTE SØNDERGAARD,1 KURT J. HANDBERG,1 JØRGEN STENDERUP2 and NIELS NØRSKOV-LAURITSEN1 1
Department of Clinical Microbiology, Aarhus University Hospital, Aarhus; and 2Department of Microbiology, Viborg Hospital, Viborg, Denmark
Nielsen SM, Kristensen L, Søndergaard A, Handberg KJ, Stenderup J, Nørskov-Lauritsen N. Increased prevalence and altered species composition of filamentous fungi in respiratory specimens from cystic fibrosis patients. APMIS 2014. Filamentous fungi cultured from respiratory tract specimens submitted to the department of clinical microbiology, Aarhus University Hospital, during 2010 were identified by morphology and by internal transcribed spacer (ITS) sequencing. Of 343 fungal isolates, discrepancies between identification methods were observed for four isolates (1.2%), while identification to species was achieved only with ITS sequencing for 16 isolates (4.7%). Filamentous fungi were isolated from 15% of cystic fibrosis (CF) respiratory samples in contrast to 2% of non-CF samples. From CF patients, a total of nine different species were found in 188 samples from 48 patients, whereas from non-CF patients, 24 different species were found in 155 samples from 111 patients. CF was associated with a significant overrepresentation of Aspergillus fumigatus and Scedosporium species; in contrast, the frequency of Penicillium spp. and other putative contaminants were significantly increased in non-CF patients. The altered species variation of filamentous fungi in CF respiratory specimens is contradictory to a scenario of incidentally inhaled spores, trapped in the viscous airway mucus of these patients and subsequently expectorated; rather, our data most likely reflect both an increased prevalence and an increased proportion of truly colonizing fungi in this patient group. Key words: Cystic fibrosis; ITS sequencing; identification discrepancies; Scedosporium. Niels Nørskov-Lauritsen, Department of Clinical Microbiology, Aarhus Brendstrupgaardsvej, DK-8200 Aarhus, Denmark. e-mail: [email protected]
A wide variety of filamentous fungi are able to cause infections in humans. Some species are true pathogens capable of infecting healthy individuals, but most are opportunistic pathogens that occur naturally in the environment where they disperse large amounts of spores into the atmosphere. The spores may be inhaled or otherwise ingested by healthy humans without causing any disease. In some cases, however, opportunistic moulds may be the cause of superficial or invasive infections, or may cause allergic reactions (1–3). Invasive infections usually occur in immunocompromised hosts, such as cancer patients, transplant recipients and other patients receiving immunosuppressive
Received 17 December 2013. Accepted 20 December 2013
University
Hospital
Skejby,
100
treatment. In contrast, allergic broncho-pulmonary aspergillosis (ABPA) usually occurs in patients with underlying lung diseases, such as asthma or cystic fibrosis (CF) (2, 3). CF is a genetic disease characterized by increased viscosity of bronchial mucus and impaired mucocilliary clearance of inhaled aerosolized bacteria and fungal spores, resulting in a vicious cycle of colonization, infection and inflammation (4–8). Similar to other patient groups, Aspergillus fumigatus is by far the most commonly cultured mould from the sputum of CF patients. Scedosporium species have, however, recently attracted attention, partly because of the reduced possibilities for antifungal treatment (4, 5, 8, 9). The most common disorder in CF patients caused by A. fumigatus is ABPA, but A. fumigatus may also be associated with deteriorating lung function 1
NIELSEN et al.
in CF patients in the absence of ABPA (1, 3), and CF patients colonized with A. fumigatus are more frequently hospitalized than non-colonized patients (10). Identification of medically important fungi is important for correct diagnosis and for prescription of optimal antifungal chemotherapy. Species identification by microscopic and macroscopic characterization is inexpensive and reliable, but the morphological structures used for identification can be atypical, slowly presented, or even absent (11, 12). Molecular identification methods have, in recent years, gained a foothold in many clinical laboratories. Especially, sequencing of the internal transcribed spacer (ITS) regions, located between the 18S and 28S rRNA gene subunits, has emerged as a general method for identification of fungal species. This region is chosen because primers are available in the adjacent, highly conserved rRNA genes, while the ITS region includes enough sequence variation to distinguish between species or species groups (11, 13–15). In this study, we have determined the prevalence and species variation of filamentous fungi cultured from clinical respiratory tract specimens from CF and non-CF patients. Identification was performed by both morphological criteria and ITS sequencing. MATERIALS AND METHODS Samples and culture Routine sputum samples were inoculated on 5% blood agar and chocolate agar without pre-treatment of the sputum, and incubated for 48 h at 35 °C. A M€ uller-Hinton agar (Oxoid, Denmark) with selected antibiotic discs was also inoculated; this agar was not used for antibiotic susceptibility testing, but as a selective medium aiding the initial differentiation of microorganisms. If special culture for fungi was requested, an additional 5% blood agar and a Sabouraud-glucose agar were inoculated and incubated at 30 °C for 7 days. All filamentous fungi cultured from respiratory specimens submitted to the department of clinical microbiology, Aarhus University Hospital, during 2010 were included in the study, irrespective of whether special fungal culture was requested. Morphological identification was conducted at the Department of Microbiology, Viborg Hospital, and isolates were stored in 10% glycerol at 80 °C for later sequencing of ITS. If discrepant results were obtained with the two identification methods, the isolates were thawed and reinvestigated by both methods.
Conventional identification Samples were inoculated on V8 Juice agar and/or Sabouraud dextrose agar and incubated at 25 °C up to 12–14 days. Morphological identification was based on colonial form, surface colour and growth rate, and
2
microscopic assessment of hyphae (size and branching, septate or aseptate), conidia and conidia-bearing structures. For some species, the maximum growth temperature was determined. Identification was done according to de Hoog et al. (14).
PCR and identification by ITS sequence DNA was extracted using PrepMan Ultra Sample Preparation Reagent (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. The ITS2 region between the 58S and 28S rRNA genes was amplified by PCR using the primers ITS 3 and ITS 4 (16). The amplified PCR products were visualized by UV light after gel electrophoresis on a 1% agarose gel stained with GelRedR. The remaining product was treated with SAP/Exo (Shrimp Alkaline Phosphatase/Exonuclease I) (Medinova and Fermentas respectively) and Sanger sequenced at an extern facility (GATC Biotech AB, Konstanz, Germany) by use of the ITS 4 primer. In approximately 4% of reactions, sequences with ambiguously determined nucleotides were obtained. For these isolates, the ITS PCR was repeated and sequencing was performed on both DNA strands using the ITS 3 and ITS 4 primer, respectively. The ITS sequences were edited and searched against the ITS Sequence database from the Molecular Mycology Research Laboratory (MYC), University of Sydney (http://www.mycologylab.org). For 19 isolates, identification was not possible using the MYC database; this was the case for some species of Penicillium spp., Rhizomucor spp., Fusarium spp. and for S. apiospermum and A. nomius, probably due to a lack of reference sequences in the database. These isolates were identified by comparison with deposited sequences in GenBank (http://www.ncbi. nlm.nih.gov/BLAST). PCR amplification of Aspergillus b-tubulin (17) was carried out to confirm the identity of a single isolate which had no morphological characteristics to support the identification of A. fumigatus obtained by ITS (Table 1, specimen 5).
Statistical analysis Filamentous fungi were combined into four groups: A. fumigatus; A. non-fumigatus; Scedosporium spp. and putative contaminants (Penicillium spp., Trichoderma viride and Cladosporium sphaerospermum). The groups were compared between CF and non-CF patients (Table 4). Statistical analysis comparing samples are based on group comparisons using logistic regression with robust variance estimation to allow for the clustered data structure, where single patients may contribute multiple observations. Ninety-five per cent confidence intervals are based on binomial regression with robust variance estimation. However, comparison of the group Scedosporium is not possible using this statistical method due to the low number of observations in this group. Statistical analysis comparing patients (one sample of each species of fungi from each patient was included in this group) was based on group comparisons using Fishers exact test. All statistical analyses were carried out using Stata Statistical Software Release 12 (StataCorp LP, College Station, TX, USA). A p-value of
