JAC Advance Access originally published online on September 20, 2002
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Journal of Antimicrobial Chemotherapy (2002) 50, 719-722
© 2002 The British Society for Antimicrobial Chemotherapy
Comparative evaluation of two different methods of inoculum preparation for antifungal susceptibility testing of filamentous fungi
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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Two different methods of inoculum preparation for susceptibility testing were analysed. The first method was adjustment of inoculum size by haemocytometer counting. The second method was spectrophotometric adjustment at 530 nm. The reliability of both methods was assessed by colony counting. The overall agreement between colony counting and haemocytometer counts was 93.6%, and the intraclass coefficient correlation was 0.71 (P < 0.05). Pearson’s correlation index between colony counts and optical density values was –0.059 (P > 0.05). Optical densities ranged between 0.01 and 1.2, showing less reproducibility than expected by the NCCLS M38-P standard. Haemocytometer counting is a more reliable method of inoculum preparation for antifungal susceptibility testing.
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Introduction |
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In recent years, filamentous fungi have gained importance as common pathogens in patients receiving immunosuppressive therapy or suffering from AIDS.1,2 The most frequently identified moulds are Aspergillus species, but other species such as Scedosporium and Fusarium spp. are becoming more relevant because of their higher frequency and degree of resistance to antifungal drugs.3,4 This increase in the prevalence of fungal infections has generated interest in reliable susceptibility testing procedures for filamentous fungi.5 One of the most important aspects of antifungal susceptibility testing is inoculum size, which can have an influence on the susceptibility results.6,7 The proposed standard for susceptibility testing of conidium-forming moulds of the NCCLS recommends spectrophotometric adjustment to yield a reliable and appropriate inoculum size.8 In contrast, however, other reports have indicated that variables such as size and colour of spores can have an influence on optical density, and that the spectrophotometric method for inoculum preparation requires a species-dependent standardization process.9 In addition, previous studies suggest that inoculum preparation by counting spores with a haemocytometer is a more accurate procedure, independent of spore colour and size.1,4,9
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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Isolates
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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Inoculum preparation
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 106–5.0 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 Pearson’s 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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Agreement between colony counting and haemocytometer counting
As summarized in Table 1, a total of 250 of 267 colony counts fell in the range 1.0 x 106–5.0 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 0.67–0.75). 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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Correlation between colony counting and spectrophotometric adjustment
Overall, Pearson’s 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 0.05–0.51, 0.05–0.85 and 0.1–0.42 absorbance units, respectively. For Scedosporium spp., the OD530 95% CI was 0.24–0.53, and Pearson’s correlation coefficient between colony counts and optical density values was 0.39 (P = 0.09).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The present work compares two methods for inoculum preparation of filamentous fungi. The first method was haemocytometer counting, which yielded a high percentage of agreement (93.6%) and a statistically significant correlation coefficient (ICC = 0.71) with colony counting. The other method was spectrophotometric adjustment, as proposed by the NCCLS document M38-P.8 The Pearson’s correlation coefficient obtained by this method in relation to colony counts was not statistically significant (r = –0.059). This could be explained by the differences in size of the spores (i.e. A. fumigatus, 2.5–3.0 µm; A. niger, 3.5–5.0 µm) and the colour of the species (green versus black, respectively). The NCCLS document M38-P8 proposes that the inoculum size for Aspergillus species should be adjusted spectrophotometrically to OD530s ranging between 0.09 and 0.11. However, this range seems to be too narrow, as the results of the present study show. On the other hand, Pearson’s correlation indices between colony counting and spectrophotometric adjustment for Scedosporium apiospermum, S. prolificans, S. brevicaulis, P. variotii, Fusarium solani and Penicillium spp. ranged between 0.012 and 0.66, suggesting that the use of spectrophotometric adjustment for inoculum preparation requires separate standardization for each species, to avoid variability due to the size and colour of the spores. Haemocytometer counting could be a more appropriate method for evaluating the inoculum size of filamentous fungi, providing more accuracy to antifungal susceptibility testing.
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Acknowledgements |
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This work was partially supported by a grant from the EC-TMR-EUROFUNG network (grant ERBFMXR-CT970145), by a grant from the Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III (grant 99/0198) and by research project 99/1199 from the Instituto de Salud Carlos III. A.G.-L. is a fellow of the Instituto de Salud Carlos III (grant 99/0198).
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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* Corresponding author. Tel: +34-91-5097961; Fax: +34-91-5097966; E-mail: juanl.rodriguez-tudela{at}isciii.es
The Eurofung Network participants are listed in the Acknowledgements.
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References |
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