1 microplate alamar blue assay for paracoccidioides susceptibility
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Microplate Alamar Blue Assay for Paracoccidioides susceptibility test. 1
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de Paula e Silva, A. C. A.1; 3
OLIVEIRA, H.C. 1; 4
SILVA, J. F. 1; 5
LEITE, F. S. 1; 6
SCORZONI, L. 1; 7
FUSCO-ALMEIDA, A. M. 1; 8
MENDES-GIANNINI, M. J. S.1 9
1 Faculdade de Ciências Farmacêuticas, UNESP – Univ. Estadual Paulista, Araraquara, 10
Departamento de Analises Clinicas, Laboratório de Micologia Clínica, SP, Brazil 11
Correspondence to: 12
Dra. Maria José Soares Giannini, Faculdade de Ciências Farmacêuticas, UNESP – Univ. 13
Estadual Paulista, Araraquara, Departamento de Analises Clinicas, Laboratório de Micologia 14
Clínica, SP, Brazil, Rua Expedicionários do Brasil, 1621, CEP: 14801-901, Araraquara, São 15
Paulo, Brasil. 16
Tel: +55 – 16 – 3301 – 5715 17
Fax: +55 – 16 – 3301 – 5715 18
e-mail: [email protected] 19
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Copyright © 2013, American Society for Microbiology. All Rights Reserved.J. Clin. Microbiol. doi:10.1128/JCM.02914-12 JCM Accepts, published online ahead of print on 23 January 2013
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ABSTRACT 22
The M27-A3 method is not available for use with dimorphic fungi, such as those of the 23
Paracoccidioides genus. In this study, we developed a microdilution method and added the 24
alamarBlue® reagent to test the responses of P. brasiliensis and P. lutzii against amphotericin B 25
and itraconazole antifungals. The test proved to be sensitive, practical and inexpensive and can 26
be used to monitor the activity of low-growth microorganisms and their response to various 27
drugs. 28
Keys words: Paracoccidioides, MABA, antifungal susceptibility test. 29
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Paracoccidioides brasiliensis and P. lutzii are dimorphic fungi and the etiologic agents of 32
paracoccidioidomycosis, a disease with multiple clinical presentations, prolonged evolution, and 33
high rates of mortality and morbidity (1, 2, 14, 25). Until now, a lack of routine tests has made it 34
difficult to determine whether patients, who did not respond to treatment, were infected with 35
resistant strains (4, 13, 19, 22). Amphotericin B (AMB) (Sigma) is still the first-choice drug 36
despite its high toxicity, and itraconazole (ITZ) (Sigma) is effective for the mild and moderate 37
forms of the disease (7, 23). A long course of therapy and sometimes even lifelong secondary 38
prophylaxis are required, especially in AIDS patients with a high incidence of relapse (17). A 39
more effective drug with a better safety profile than the currently available drugs would greatly 40
improve the treatment of dimorphic fungal infections (18, 23). 41
The CLSI (Clinical Laboratory Standard Institute), M27-A3 document, has proposed a 42
method for determining the in vitro susceptibility of yeasts to different drugs. However, no 43
standardised method is available for dimorphic fungi, because of its difficult culture procedure. 44
For these reasons, more reliable assays are needed for use with the genus Paracoccidioides. The 45
ability to determine the MIC (minimum inhibitory concentration) of different compounds would 46
inform clinicians about the choice of the antifungal agent and therapy management, as well as 47
facilitating screens of new molecules for their antifungal activity. 48
However, the microdilution method used today presents, however, difficulties in visually 49
reading the MIC because there is a lack of significant fungal growth. Nevertheless, this test has 50
been used in the literature (6, 11, 12, 18). In contrast, the MABA (Microplate Alamar Blue 51
Assay) has been evaluated by several authors in Aspergillus fumigatus (16), Mycobacterium sp 52
(3, 5, 26) and Trypanosoma cruzi (21). Resazurin, the active ingredient in the MABA 53
(alamarBlue® BIOSOURCE), is permeable, blue, and virtually non-fluorescent, but after entry 54
into viable cells, resazurin is continuously reduced by the cells to resorufin, a highly fluorescent 55
red compound. 56
The aim of our study was to compare the reference broth macrodilution (MAM) and 57
microdilution (MIM) method with the MABA to measure the presence of fungi of the 58
Paracoccidioides genus and the activity of two drugs: AMB and ITZ. 59
The MIM and MAM were performed according to document M27-A3, and ITZ and 60
AMB were prepared with RPMI-1640 medium (GIBCOTM) supplemented with 2% glucose 61
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(RPMI-1640-2%) (25). Final concentrations of ITZ and AMB varied from 1 to 0.002 mg/L and 62
0.8 to 0.0015 mg/L, respectively, after addition of the inoculums. The antifungal activity was 63
analysed against the P. brasiliensis phylogenetic species S1 isolate 18 (São Paulo), D03 and 339 64
(São Paulo), S2 isolate 02 (Venezuela), and PS3 isolate Epm83 (Colombia), and the P. lutzii 65
strain 01 (ATCC MYA-826/Goiânia) and the isolates EE (Mato Grosso) and 8334MMT 66
(Goiânia). All the strains were isolated from human patients and were maintained in Fava-Netto 67
medium at 37°C, and subcultures were performed every 4 days. 68
The cells were suspended in sterile saline, allowed to settle for several minutes to 69
eliminate large aggregates, and then the supernatants were collected. The number of viable cells 70
was estimated by staining with Trypan blue and counting with a haemocytometer, and the final 71
concentration was 106 cells/mL. This suspension was diluted 1:50 in sterile saline and 1:20 in 72
RPMI-1640-2% for the MIM test. For the MAM test, the same dilutions were performed, but the 73
first dilution was 1:100. The final inoculum used was 0.5x103 to 2.5x103 cells/mL after the 74
addition of antifungal. The plates were incubated at 35°C at 150 rpm for 48 hours. After this 75
period, the MABA was employed (according the manufacturer´s instructions), and the plates 76
were incubated for an additional 24 hours, totalizing 72 hours for the MIC final reading. The 77
lowest antifungal agent concentration that substantially inhibited the growth of the organism was 78
visually determined at the point at which there was no change in the original blue colour of the 79
reagent. 80
Similar results were obtained when the MAM and MIM were compared for the 81
P.brasiliensis 18 (Figure 1) isolate and P. lutzii 01 strain (Table 1). The MICs ranged from one 82
to two dilutions, values acceptable according to the document M27-A3 (CLSI). 83
The visual readings of MICs are easily performed, and the MIC values obtained by both 84
methods are equivalent. Additionally, these values are similar to those described in the literature 85
(6, 8-12, 15, 18, 20, 24). 86
There are several studies using MAM with the Paracoccidioides genus, and these 87
generally follow the method suggested by Shadomy et al. (1987) (8-10) or that suggested by the 88
NCCLS-standardised document M27-A (15, 20), the problem of this method is the high cost due 89
to the need of higher amount of culture medium and other reagents and materials. Also, there are 90
in the literature studies (6, 11, 12, 18) of MIM with the Paracoccidioides genus, however with 91
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higher final inoculums than the recommended in document M27-A3. This approach improved 92
the ability to identify the MIC in the slow-growing fungus but is a significant modification in 93
relation to the recommended document. Therefore, we followed the CLSI-standardised document 94
M27-A3, and the use of MABA makes the MIM more sensitive and reliable, allowing detection 95
of fewer than 50 viable cells per well. 96
The use of MABA can replace the MFC (minimum fungicide concentration) test that is 97
inconclusive in most cases because the long incubation of the plate results in dehydration of the 98
solid medium and the consequent death of cells which might have been viable, leading to false 99
negative results. Similarly, the MIC could be considered equivalent to the MFC because it is a 100
sensitive method to assay the viability of fungal cells. 101
Thus, the MIM/MABA has become a reliable and reproducible test for this genus. The 102
test was extended to different isolates of Paracoccidioides genus, confirming that this 103
methodology can be applied for a wide range of isolates (Table 2). Comparing the determined 104
MIC values of antifungal against different isolates shows that MIC values for ITZ and AMB 105
were within the expected ranges found in the literature (6, 11, 12, 18). The MIC values varied 106
independently of Paracoccidioides species from 0.008 to 0.25 mg/L for AMB and 0.008 to 0.03 107
mg/L to ITZ. 108
Thus, the Paracoccidioides MABA test is reliable, reproducible, quick and an accurate 109
tool for routine laboratory. 110
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ACKNOWLEDGMENT 112
This work was financially supported by the Brazilian organizations FAPESP, Conselho 113
Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and PADCFCF-UNESP. Ana 114
Carolina Alves de Paula e Silva has a fellowship from CAPES. We are very grateful to Prof Dr. 115
Eduardo Bagagli, from Biosciences Institute of Botucatu, UNESP - Univ Estadual Paulista, for 116
kindly provide the Paracoccidiodes isolates used in this study. 117
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FIGURE LEGEND 211
Figure 1. MAM and MIM tests developed with P.brasiliensi 18 against AMB and ITZ. The 212
MIC values were determined after addition of alamarBlue®. 213
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Table 1: Comparison of MAM and MIM from two Paracoccidioides species.
Isolate 18
(mg/L)
Strain 01
(mg/L)
MAM MIM MAM MIM
AMB 0.05 0.06 0.1 0.06
ITZ 0.003 0.008 0.001 0.004
Table 2: Paracoccidioides MABA test: MIC values of AMB and ITZ.
P. lutzii mg/L P. brasiliensis mg/L
01-like S1 S2 PS3
EE 8334MMT 01 18 D03 339 02 Epm83
AMB 0.25 0.25 0.06 0.12 0.008 0.06 0.12 0.06
ITZ 0.008 0.03 0.008 0.015 0.015 0.015 0.008 0.015
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