Servicio de Micología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Ctra Majadahonda-Pozuelo Km 2, 28220 Majadahonda, Madrid, Spain
Received 5 April 2002; returned 18 June 2002; revised 11 July 2002; accepted 26 July 2002
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Abstract |
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Introduction |
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The present study compares the inoculum sizes obtained by spectrophotometric adjustment and haemocytometer counting of a wide range of filamentous fungi and a large number of isolates, analysing the species-dependent features of these two methods.
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Materials and methods |
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A panel of 267 clinical isolates belonging to 22 different species of filamentous fungi was included. Strains were received from 45 different hospitals over a 13 month period from June 2000 to July 2001. Each isolate was obtained from a different patient and was sent to the laboratory for identification or antifungal susceptibility testing. Aspergillus fumigatus ATCC9197 and Paecilomyces variotii ATCC22319 were included as control isolates in each set of experiments. Species distribution is shown in Table 1. The isolates were maintained as a suspension in sterile distilled water at 4°C until testing was performed.
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The isolates were subcultured (from stock water suspensions or from Petri plates used for morphological identification) on potato dextrose agar slants and incubated at 35°C. Inoculum suspensions were prepared from fresh, mature (3- to 5-day-old) cultures. In some cases an extended incubation was required for proper sporulation of the isolate. For hydrophobic genera, such as Aspergillus spp., Paecilomyces spp., Penicillium spp., Scopulariopsis spp. and Trichoderma spp., the colonies were covered with 5 mL of distilled sterile water containing 1% Tween 20. For hydrophilic genera, such as Fusarium spp. and Scedosporium spp., the colonies were covered with only 5 mL of distilled sterile water. Then, the conidia were rubbed carefully with a sterile cotton swab (Collection swab; EUROTUBO, Madrid, Spain) and transferred to a sterile tube; the resulting suspensions were homogenized for 15 s with a gyratory vortex mixer at 2000 rpm (MS 1 Minishaker; IFA, Cultek, Madrid, Spain). Appropriate dilutions were performed in order to get the right concentration for counting in a cell-counting haemocytometer (Neubauer chamber; Merck S.A., Madrid, Spain). All inoculum preparations were checked for the presence of hyphae or clumps by a previous examination in the cell-counting haemocytometer chamber. If a significant number of hyphae was detected (>5% of fungal structures), the 5 mL suspension was transferred to a sterile syringe attached to a sterile filter with a pore diameter of 11 µm (Millipore, Madrid, Spain) and filtered and collected in a sterile tube. This step removes hyphae and yields a suspension composed of spores. If clumps were detected, the inoculum was shaken again in the gyratory vortex mixer at 2000 rpm for a further 15 s. This step was repeated as many times as necessary if clumps were visualized again.
Inoculum adjustment
The final inoculum size was adjusted to a range of 1.0 x 1065.0 x 106 spores/mL by microscopic enumeration with a cell-counting haemocytometer. Five millilitres of this suspension was transferred to a 1/2-inch crystal tube (KIMAX; Labcenter, Madrid, Spain). The tube was shaken for 10 s with a gyratory vortex mixer at 2000 rpm (MS 1 Minishaker), and the optical density at 530 nm (OD530) of the suspensions was measured in a single-beam spectrophotometer (SPECTRONIC 20D; Milton Roy Company, Pacisa, Madrid, Spain). All adjusted suspensions were quantified by plating on Sabouraud agar plates. Volumes of 100, 50 and 25 µL were spread onto the Sabouraud agar plates. The plates were incubated at 35°C and were observed daily for the presence of growth. The colonies were counted as soon as possible after the observation of visible growth.
Statistical analysis
The target inoculum size range was established to be between 1.0 x 106 and 5.0 x 106 cfu/mL. The percentage of agreement between inoculum sizes determined by counting with a haemocytometer and colony counting was calculated by taking into account that both systems of measurement produced colony counts in a range between 1.0 x 106 and 5.0 x 106 cfu/mL.
The correlation between the results obtained by counting with a haemocytometer and the colony counting data was evaluated by using the intraclass correlation coefficient (ICC). The ICC assesses reliability as an internal consistency statistic by means of inter-item correlations. A two-way mixed effect model was used to calculate the ICC, which was expressed to a maximum value of 1 and with a confidence interval of 95% (95% CI). When appropriate, the variables were transformed to log10 data. In addition, the correlation between colony counts and the OD530 was also determined with Pearsons coefficient (r). Correlation indices and ICCs were calculated only for those species for which the number of strains tested was five or more.
All statistical analyses were done with the Statistical Package for the Social Sciences (version 11.0; SPSS, S.L., Madrid, Spain).
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Results |
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As summarized in Table 1, a total of 250 of 267 colony counts fell in the range 1.0 x 1065.0 x 106 cfu/mL, giving an overall agreement of 93.6%. Inoculum preparations for the A. fumigatus control isolate ATCC9197 were performed 61 times, with an agreement between colony counts and haemocytometer counts of 93.4%. For the P. variotii control isolate ATCC22319, inoculum preparations were performed 42 times, with 97.6% agreement between colony counts and haemocytometer counts. For Aspergillus spp., agreements between colony counts and haemocytometer counts ranged between 75% and 100%, with an overall agreement of 95.7%. Scopulariopsis brevicaulis, Scedosporium prolificans and Paecilomyces lilacinus exhibited lower rates of agreement with values of 76.5%, 83.3% and 66.6%, respectively.
Correlation between colony counts and haemocytometer counts
For all species tested, reproducibility between colony counting and haemocytometer counting was evaluated by an ICC. Overall, the ICC obtained throughout this study was 0.71 (95% CI 0.670.75). This value was statistically significant (P < 0.05). For Aspergillus isolates, ICCs ranged between 0.64 and 0.94, with the highest for Aspergillus niger. For the remaining species, ICCs ranged between 0.45 and 0.93, showing the best reproducibility for S. prolificans and the worst for P. variotii (Table 2).
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Overall, Pearsons correlation index (r) between spectrophotometric adjustment and the colony counts was 0.059, a value without statistical significance (P > 0.05). For the Aspergillus isolates, the OD530 range was wide: for A. fumigatus, OD530 ranged between 0.01 and 0.5 absorbance units, whereas the ranges for Aspergillus terreus, Aspergillus flavus and A. niger were 0.050.51, 0.050.85 and 0.10.42 absorbance units, respectively. For Scedosporium spp., the OD530 95% CI was 0.240.53, and Pearsons correlation coefficient between colony counts and optical density values was 0.39 (P = 0.09).
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Discussion |
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Acknowledgements |
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The EUROFUNG Network (EC-TMR-EUROFUNG network; ERBFMXR-CT970145) consists of the following participants: Emmanuel Roilides (co-ordinator) and Nicos Maglaveras, Aristotle University, Thessaloniki, Greece; Tore Abrahamsen and Peter Gaustad, Rikshospitalet National Hospital, Oslo, Norway; David W. Denning, University of Manchester, Manchester, UK; Paul E. Verweij and Jacques F. G. M. Meis, University of Nijmegen, Nijmegen, The Netherlands; Juan L. Rodriguez-Tudela, Instituto de Salud Carlos III, Madrid, Spain; and George Petrikkos, Athens University, Athens, Greece.
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Footnotes |
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The Eurofung Network participants are listed in the Acknowledgements.
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References |
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