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1 Department of Otorhinolaryngology, Helsinki University Central Hospital, Helsinki, Finland
2 Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki, Helsinki, Finland
3 Department of Medical Microbiology, University of Turku, Turku, Finland
4 Department of Clinical Microbiology, Turku University Central Hospital, Turku, Finland
5 Department of Occupational Medicine, Section of Otorhinolaryngology, Finnish Institute of Occupational Health, Helsinki, Finland
6 Department of Otorhinolaryngology, Kymenlaakso Central Hospital, Kotka, Finland
Correspondence
Katriina Kostamo
katriina.kostamo{at}kymshp.fi
Received 29 November 2006
Accepted 30 May 2007
Abbreviations: CRS, chronic rhinosinusitis; CRSwNP, chronic rhinosinusitis with nasal polyposis; CT, threshold cycle; GM, galactomannan; NAL, nasal lavage.
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INTRODUCTION |
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 TOP INTRODUCTION METHODSRESULTS AND DISCUSSIONREFERENCES |
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The prognosis of patients with invasive fungal rhinosinusitis depends upon early diagnosis. In recent years, rapid, non-culture-based methods to detect fungi have been developed, such as ELISA for Aspergillus galactomannan antigen (GM ELISA) and fungal PCR. The clinical utility of GM ELISA has been demonstrated in several studies, mainly in patients with haematological malignancies, and the test has been shown to cross-react with a number of hyaline moulds (Ascioglu et al., 2002; Mennink-Kersten et al., 2004). The GM ELISA has been evaluated in one study of 23 rhinosinusitis patients (Kauffmann-Lacroix et al., 2001). Here the histology compared favourably with antigen detection while direct microscopic examination and culture were less sensitive. Also, PCR has proven to be more sensitive compared to fungal cultures, but results obtained by pan-fungal PCR have differed from those obtained by genus-specific primers, and genus-specific PCR results have been different from fungal culture results (Kim et al., 2005; Polzehl et al., 2005). Moreover, pan-fungal PCR has been reported to detect fungal DNA in comparable levels in nasal samples from CRS patients and controls (Catten et al., 2001; Kim et al., 2005). Similar to fungal culture results, the presence of fungal DNA in the nasal/sinus specimen is not enough to explain the role of fungi in the disease pathogenesis. However, PCR may have value as an adjunctive tool in diagnosing fungal rhinosinusitis and identifying the organism present in the microbiological/histological specimen (Buzina et al., 2003; Willinger et al., 2003).
With this background, we wished to evaluate the clinical usefulness of the Aspergillus GM ELISA and Aspergillus fumigatus PCR in detecting Aspergillus in chronic rhinosinusitis with nasal polyposis (CRSwNP). CRSwNP was chosen because it is a disease that is most often related to fungal infection of the paranasal sinuses (deShazo, 1998). Because fungal colonization in the nose is common, we also evaluated these assays in healthy volunteers.
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METHODS |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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The study was approved by the Ethical Committee of Helsinki University Central Hospital. Written consent was obtained from each patient.
Specimen collection. Patients' specimens were collected during the operation. Mucus from the paranasal sinus was placed into an empty sample tube for fungal examination. A specimen for fungal staining was taken with a sterile cotton swab and spread directly onto a microscope slide. The slide was then air-dried. The biopsy for histological analysis was obtained from the same paranasal sinus as the mucus.
The nasal lavage (NAL) method earlier described by Hirvonen et al. (1999) was used with some modifications for collecting samples from the controls. In brief, each nostril was flushed through with 5 ml PBS using a 5 ml sterile syringe and a sterile butterfly cannula of about 2 cm in length. The patient closed the nostrils by pinching them firmly together and leaned forward. PBS was pushed back and forth twice and finally collected into the syringe. Any residual PBS remaining in the nostril was collected in a pan placed underneath the nose and collected into the syringe.
Fungal staining and culture. The standard methods used in the diagnostic laboratories of Helsinki University Central Hospital to culture and identify fungi were used. The specimen was first vortexed to mechanically disperse the mucus. Calcofluor white fluorescence staining was carried out using the processed NAL sample or the sample placed directly onto a microscope slide and the preparation was examined using a fluorescence microscope with 40-fold magnification. The remaining sample was plated out on Sabouraud dextrose agar containing 6 mg penicillin l–1 and 26 mg streptomycin l–1. The cultures were incubated at 28 and 37 °C and examined at 7 and 10 days.
All histological samples were stained with haematoxylin and eosin and with periodic acid–Schiff. If the periodic acid–Schiff staining was negative for fungi, the Gomori methenamine silver staining method was done.
GM ELISA. The Aspergillus GM antigen was detected in sinus mucus/NAL fluid by a direct double-sandwich ELISA (Platelia) according to the manufacturer's instructions. Positive and negative controls were included in each assay. Results were interpreted from the relationship of the absorbance of the sample to that of the threshold control (GM index=absorbance sample/absorbance threshold control).
A. fumigatus PCR. The samples were concentrated for DNA purification by centrifugation for 5 min at 3000 g, the supernatant was discarded, and 200 µl of the pellet was processed further. The release of fungal DNA was enhanced by lyticase treatment (7 U) at 30 °C for 1 h and homogenization with 0.3 g of zirconia–silica beads and a Mini-Beadbeater. The DNA purification was done with a High Pure PCR template preparation kit (Roche Diagnostic) following the instructions of the kit.
The PCR primers and probes used have been described by Rantakokko-Jalava et al. (2003). Briefly, the PCR amplified a 134 bp fragment from the A. fumigatus mtDNA encoding mitochondrial tRNA gene with primers 5'-GAA AGC TCA GGT GTT CGA GTC A-3' and 5'-CTT GGT TGC GGG TTT AGG GAT T. The Aspergillus tRNA gene was detected by fluorescence resonance energy transfer using the pair of probes tRNA FL (5'-TTC TTA TTT ATA TGC GGG TTG ATG TAA TAG TAA CA-3') and tRNA LC (5'-AGA TGG CTC ATG ACC ATA ATA TTT AGG TGC p).
The DNA amplification was done using the LightCycler instrument as described by Rantakokko-Jalava et al. (2003). Forty-five cycles were performed, and the fluorescence was measured at the end of the annealing phase of each cycle. All runs contained distilled water as negative isolations and reaction controls and A. fumigatus DNA in four concentrations (20 ng, 2 ng, 200 pg and 20 pg per reaction mixture) as amplification standards. Samples with detectable exponential amplification on the fluorescence versus cycle plot were regarded as positive. The threshold cycle (CT), i.e. the cycle at which the exponential amplification becomes detectable over the background fluorescence, has an inverse linear relation to the amount of target DNA in the original sample. All samples were tested for PCR inhibition by amplification of the human growth hormone gene as described previously (Rantakokko-Jalava et al., 2000).
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RESULTS AND DISCUSSION |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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The results for GM ELISA, A. fumigatus PCR, fungal staining and culture of the sinus mucus and fungal staining of tissue specimens in CRSwNP patients together with the clinical diagnosis are summarized in Table 1
. A total of seven patients (28 %) were diagnosed as having fungal rhinosinusitis according to the presence of filaments in histological specimens or direct microscopic examination, whereas in the control group no sample was positive for fungi by either fungal staining or culture. The clinical diagnosis was mycetoma in two patients (patient nos 2 and 9). They had fungal hyphae morphologically consistent with Aspergillus visible in both sinus mucus and tissue specimens. The remaining five patients were considered to have allergic fungal rhinosinusitis-like syndrome. No invasive fungal infection was discovered. The occurrence of fungal rhinosinusitis in the present study (28 %) exceeds the estimated prevalence of fungal infection among rhinosinusitis patients in the literature (Granville et al., 2004). Vennewald et al. (1999) reported an equal prevalence of fungal positive findings in 117 CRSwNP patients who also had granulomatous material within the sinus by endoscopy. The authors interpreted these results to represent fungal colonization of the paranasal sinuses instead of a fungal rhinosinusitis. However, these results may indicate that when the typical findings of fungal rhinosinusitis are present, there is a strong possibility of fungal infection (deShazo, 1998).
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Three definite false-negative GM ELISA results (GM index cut-off value 1.0) were obtained in the present study: two patients with morphologically suspected Aspergillus mycetoma and the patient with Aspergillus grown in culture. One explanation may be that the environment inside the paranasal sinuses is lacking in essential nutrients, especially glucose and oxygen, leading to a reduced amount of antigen released (Mennink-Kersten et al., 2004). Antifungal drug therapy is known to reduce mycelial growth, resulting in suppression of GM expression, but none of the patients received antifungal drugs (Mennink-Kersten et al., 2004). Another factor influencing the assay performance is the optimal index cut-off value to define positivity of GM ELISA. When the Platelia ELISA kit was first introduced a decade ago, the recommended index cut-off value for serum samples was 1.5. Since then, the cut-off value 1.0 has been applied in several studies. The US Food and Drug Administration have now approved the ELISA kit with a cut-off index of 0.5 when applied to serum. Musher et al. (2004) achieved the optimal performance of GM ELISA performed on bronchoalveolar lavage fluid by also using an index cut-off of 0.5. If the cut-off value of the GM index was lowered to 0.5 in the present study, three more patient samples and two more control samples were positive for GM. Only one of these three patients had positive fungal staining of sinus mucus, and the patient had yeast cells, not hyphae typical of Aspergillus species. Thus lowering the cut-off value did not improve the assay performance in this study.
The poor performance of GM ELISA in this study differs considerably from the results obtained by Kauffmann-Lacroix et al. (2001). In their study of 23 rhinosinusitis patients, out of which 16 had fungal rhinosinusitis, 20 patients had concordant GM ELISA and mycological and/or histological results in sinus mucus samples obtained during endoscopic operation. The sensitivity and specificity of Platelia Aspergillus ELISA were 87 % and 88 %, respectively. Perhaps the lower prevalence of fungal rhinosinusitis among the patients explains partly the much poorer performance of GM ELISA seen in the present study. However, recent studies have shown significant variation in the performance of the GM assay with the overall sensitivity being as low as 29.4 % (Herbrecht et al., 2002; Weisser et al., 2005). The importance of serial screening has been highlighted because of false-positive results, whereas we tested a single sample only. However, in the recent study even the serial screening of GM in serum from patients at risk for invasive aspergillosis did not improve the performance of the test, as the positive GM ELISA result, determined as GM index of 
0.5 in two consecutive measurements, did not precede detection of major lesions by pulmonary CT (Weisser et al., 2005).
The A. fumigatus PCR identified the patient with fungal rhinosinusitis caused by A. fumigatus (patient no. 10). Also the mycetoma patient (patient no. 2 in Table 1
) most likely had a true-positive PCR result, because the hyphae present in histology were morphologically consistent with Aspergillus, although culture remained negative. The third patient (no. 17) with a positive A. fumigatus PCR had otherwise negative fungal results and did not meet the criteria of fungal rhinosinusitis. The CT of this sample was >37.70, indicating that the amount of A. fumigatus DNA in the sample was low and may be derived from fungal colonization of the nose. Unfortunately we were not able to test the other mycetoma patient (no. 9) with probable Aspergillus infection by A. fumigatus PCR because of the limited volume of the sample. The PCR assay used in the present study is designed to be specific for A. fumigatus (Rantakokko-Jalava et al., 2003). In fact, the one patient whose fungal culture grew Aspergillus niger had a negative PCR result. Rantakokko-Jalava et al. (2003) in their study of patients with invasive aspergillosis found the sensitivity and specificity of the same A. fumigatus PCR method applied to bronchoalveolar lavage and tissue biopsy specimens to be 72 % and 93 %, respectively. All the patients with positive PCR and CT <35 had a proven or probable invasive pulmonary aspergillosis, whereas samples with higher CT values were obtained both from patients with invasive disease and from those who were at risk, but had no other evidence of invasive disease. The respiratory tracts of these patients were probably colonized by A. fumigatus. In the present study, every control sample tested negative by A. fumigatus PCR, although the GM ELISA was positive in 21 %, casting more doubt that fungal colonization would explain the positive GM ELISA results in controls. Detection of GM by ELISA has been suggested to be more sensitive than amplification of Aspergillus mitochondrial DNA from serum mainly due to the higher concentration of GM (Costa et al., 2002). However, the lack of any concordance between GM ELISA results and results obtained by other methods used in the patient group makes it impossible to compare these assays in the control group either.
In conclusion, fungal staining and culture of sinus mucus together with histology using specific fungal stains remain pivotal in defining diagnosis and differentiating invasive from non-invasive forms of fungal rhinosinusitis. A. fumigatus PCR did not increase the accuracy of fungal rhinosinusitis diagnosis, but it may accelerate the detection of fungi in nasal/sinus samples and thus it may have additional value in diagnosing Aspergillus rhinosinusitis. Conversely, the GM ELISA, whether the result is either positive or negative, is not reliable in the diagnosis of Aspergillus infection of the paranasal sinuses.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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