Received: 5 May 2009

Accepted: 1^st referee: 10 June 2009

2^nd referee: 8 July 2009

 

 

Comparison between Standard (NCCLS) Broth Microdilution and
Commercial Tests for In-vitro Antifungal Susceptibility
Testing of Candida Species

Noha El-Mashad and Mohamed Taha Mahmoud*

Departments of Clinical Pathology, Mansoura University & Microbiology (Mycology), Veterinary Medicine,
Zagazig University*, Egypt

Keywords: NCCS, E test, Candifast, antifungal drugs

                                                                                                                                                                                                         

 

 

 

Abstract

Background: Although the NCCLS M27-A reference method remains the standard, it is impossible for a modest-size laboratory to perform the test on a routine basis.

Aim: It is of value to evaluate the commercial susceptibility methods as possible alternatives to standard one for routine fungal susceptibility testing. So we aimed to compare the NCCLS microdilution method with each of commercial E-test and candifast kit.

Material and methods: A total of 30 Candida isolates were included and species identification was confirmed by morphological appearance on Sabouraud dextrose agar (SDA), gram stain, and candifast kit. The in vitro antifungal susceptibility testing of amphotericin B (amph. B) and fluconazole was performed by 3 methods (standard broth microdilution, E test & candifast) on the tested Candida species.

Results: The overall percentage of agreement of E test with standard method was 90% for each of amph. B and fluconazole. For amph. B, the agreement of E test with standard broth method was 100% in tested species except C. glabrata (85.7%) and C. parapsilosis (66.6%), while the % of agreement of candifast was100% in all species except C. glabrata (85.7%). For fluconazole the percentage of agreement of E-test, with the standard method, was 100% for each of C. glabrata and C. tropicalis and was 92.8%, 66.6% for C. albicans and C. parapsilosis respectively. The percentage of agreement of candifast method was 100% in all species except C. albicans, C. glabrata which was 92.8% and 85.7% respectively.

Conclusion: E test method is an alternative but cannot be considered a substitute for the NCCLS reference method. The antifungal susceptibility method is not greatly influenced by the type of tested antifungal agent. Candifast is a simple, rapid, method for simultaneous identification and susceptibility of Candida species with precise endpoint which is important in results interpretation.

Introduction

Antifungal drug susceptibility testing has become more important due to the increase in serious fungal infections and the concomitant emergence of resistance to antifungal agents⁽¹⁾.

In 1997, the National Committee for Clinical Laboratory standards (NCCLS) published an approved reference procedure (document M27-A) for the in vitro testing of five antifungal agents against Candida species and Cryptococcus neoformans. The NCCLS document describes a broth macrodilution method and its microdilution modifications, specifies a defined test medium as well as a standardized inoculum, and recommends the visual determination of the MIC end points after incubation at 35°C for 48 h for Candida species. By this method the end point is defined as the lowest drug concentration at which a “prominent decrease in turbidity” is observed compared with the growth in the control drug-free medium ⁽²⁾.

E-test is a susceptibility, agar-based, quantitative diffusion method which is based on the diffusion of a continuous concentration gradient of the antifungal agent tested from a plastic strip into an agar medium and provides MIC endpoints instead of inhibition zone diameters. The MIC by the E test is the lowest drug concentration at which the border of the elliptical inhibition zone intercepted the scale on the antifungal strip⁽³⁾.

Aim of Work

It is of value to evaluate the commercial susceptibility methods as possible alternatives for routine fungal susceptibility testing. So we aimed to compare the NCCLS microdilution method with each of commercial E-test and with candifast kit.

Material & Methods

Organisms used in this study were clinical isolates obtained from 6 blood cultures for patients with different hematological malignancies, and 24 patients with different fungal skin & nail infections. A total of 30 Candida isolates were included and species identification was confirmed by morphological appearance on SDA, gram stain, and candifast kit (International Microbio. Stago Group – Parc d’activities – Alleg d’Athenes 3870 Signes {France}). It is based on fermentation of seven sugars and change of color of the indicator to yellow due to medium acidification, or demonstration of urease activity which produces alkaline pH that changes color of indicator.

Preparation of antifungal agents: Stock solutions were prepared in dimethylsulfoxide (DMSO) for amphotericin B (amph. B) and in sterile water for fluconazole .They were 5120 µg/ml for each drug. On performing susceptibility testing, further dilutions of each antifungal agent were prepared with RPMI 1640 medium which had been buffered to pH 7.0 with 0.165 M of morpholinopropanesulfonic acid (MPOS). The final drug concentrations in two fold serial dilutions ranged from 0.03 – 16 mg/ml & 0.125 – 64 mg/ml for amph. B and fluconazole respectively^(4,5).

Amphotericin B was supplied as a lyophilized powder for intravenous administration by Bristol – Myers Squib (Squib – Egypt). Fluconazole was supplied as capsule for oral administration by Global Napi (Global Napi pharmaceutical Egypt).

Prior to antifungal susceptibility testing, each isolate was subcultured on SDA plates to insure purity. Five colonies were suspendedin 0.9% saline and adjusted to 0.5 McFarland standard (correspondsto 1 × 10⁶ to 5 × 10⁶ CFU/ml). This stock solution was diluted 1:100 in RPMI 1640 medium buffered to pH 7.0 obtain a 2× test concentration.

1)  MIC by broth microdilution (National
Committee for Clinical Laboratory Standards M27-A method 1997)

One hundred microlitersof the 2× inoculum was pipetted in the wells of sterile microtitration plate  to which 100 ul of the each drug dilution were added to the corresponding well. 10 wells were used for each test and additional control well was used containing 100 ml of organism suspension & 100 ml of drug free medium. This achieves a final concentration of 0.5×10³ to 2.5×10³ CFU/ml in a final test volume of 200 µl.

Microwell plates wereincubated at 35°C for 48 h. TheMICs were determined visually. The MICs values were defined as the lowest drug concentration which resulted in reduction of 80% in turbidity in comparison with the drug free growth control well for fluconazole, while for amphotericin B the MIC value was defined as the lowest drug concentration for which the well was optically clear. Break points for fluconazole were introduced and validated by Pfaller et al.⁽⁶⁾ and Ostrosky – Zeichner et al.⁽⁷⁾. These breakpoints for fluconazole are susceptible <16μg/ml; susceptible dose- dependent, 16-32 μg/ml; resistant ≥64 μg/ml. The susceptibility for amphotericin B is <2.0 μg/ml and resistance is ≤2.0 μg/ml.

2)  E-test (AB BIODISK, Solana, Sweden)

Medium for E – test was prepared by RPMI 1640 , buffered  to pH 7.0 by MOPS buffer,  supplemented with 20 g/L 0f glucose and 15 g/L of agar base according to E test technical guide. It was poured into 15 cm diameter sterile plates. The prepared 0.5 Mc Farland suspension of each isolate was applied on the agar surface with cotton swab. Plates were allowed to dry for 15 min before application of E test strips and then incubated at 35^oC for 48h. The MIC was taken as the lowest drug concentration at which the border of the elliptical inhibition zone intercepted the scale on the antifungal strip.

3)  Candifast

Principally the determination of susceptibility of Candida to antifungal agent is based on presence or absence of growth of inoculated Candida.100 ul of inoculated standardized reagent 1 (dilution identification reagent) were inoculated into reagent 2 (susceptibility reagent), from which 100 ul were dispensed into each well of corresponding susceptibility row and covered with 2 drops of paraffin oil, sealed and incubated at 35^oC for 48 h. It was read visually. If the well was red, orange or orange – red, the strain was inhibited by the drug in that well. The organism was considered resistant to the antifungal in the well when the color changed to yellow, presence of turbidity, or even a sediment.

Results

Identification of Candida isolates (30) by candifast revealed the flowing distribution: C. albicans 46.7% (n=14), followed by C. glabrata 23.3% (n=7), C. parapsilosis 20% (n=6) and C. tropicalis 10% (n=3).

Table (1) summarizes the in vitro antifungal susceptibility of amph. B and fluconazole on different Candida species (30 isolates) by 3 methods (Standard broth dilution, E test and Candifast). We will comment on amphotericin B and fluconazole only in candifast plate.

The overall percentage of agreement of E test with standard method was 90% for each of amph. B and fluconazole. The overall percentage of agreement of candifast with standard broth method was 96.6% for amph. B and 93.3% for fluconazole.

For amph. B the agreement of E test with standard method was 100% in tested spcies except C. glabrata (85.7%) and C. parapsilosis (66.6%). The number of susceptible isolates in those 2 species were lower when tested by E test method than standard method. The % of agreement of candifast was 100% in all species except C. glabrata (85.7%). The overall modal MICs obtained was 1.0 µg/ml for both standard method and E test.

For fluconazole the percentage of agreement of E-test with the standard method, was 100% for each of C. glabrata and C. tropicalis and 92.8%, 66.6% for C. albicans and C. parapsilosis respectively. The number of susceptible isolates in those 2 species was lower when tested by E test than standard method. The percentage of agreement of candifast method was 100% in all species except C. albicans, C. glabrata which was 92.8% and 85.7% respectively. The overall modal MICs obtained for fluconazole were 0.25 μg/ml for the standard methods and 0.5 µg/ml for E test.

 

Table 1.    Comparison of susceptibility results by different methods for 30 Candida isolates for amphotericin B and fluconazole.

 

 

Discussion

In the past, in vitro testing of antifungal agents was regarded as problematic. The development of reliable and reproducible broth dilution reference procedures against Candida species has, however, enabled MICs to be correlated with clinical outcomes and has permitted interpretive breakpoints to proposed for the drugs^(1,8).

Although the NCCLS M27-A reference method remains the standard by which all other methods are Judged, it is impossible for a modest-size laboratory to perform the test on a routine basis⁽⁹⁾. There have been many alternatives developed over the past several years including flow cytometic⁽¹⁰⁾ and MIC diffusion strips (E test)^(11,12).

In this study, (Table 1) on examining amph. B on different Candida species, by E test compared with the reference NCCLS, the number of susceptible species was the same except in C. glabrata and C. parapsilosis which were lower in E test.

The overall agreement percentage among E test and standard MIC method was 90% according to the MIC breakpoints recommended by M27-A method for each amph. B and fluconazole.

E test has introduced as an easier testing procedure and an alternative for the NCCLS method^(13,14). The great advantage of E test is the simplicity of the methodology. However not all antifungal agents are available in E test and there is difficulty associated with endpoint interpretation⁽¹⁵⁾.

In a similar study by Matsmoto et al.⁽¹⁶⁾ for comparison of E test and standard microdilution method susceptibility of bloodstream yeasts, they recorded that results presented a greater agreement between E test MICs and the standard NCCLS. The percentage of agreement was 98% for fluconazole for C. albicans and C. parasilosis. However for amphotericin B, the agreement between methods was low for all species. However, in another study Koga- Ito et al.⁽¹⁵⁾ on fluconazole and itraconazole, they reported poor agreement for fluconazole (53.33%) at 24h. In addition, agreement observed at 48 h. was even worst.

Pfaller et al.⁽¹²⁾ obtained the best agreement between the NCCLS reference method and the E test using RPMI agar with 2% glucose. for amphotericic B susceptibility testing , good Correlation was observed between the tested method at 24h. (66.6%) and 48 h (71.6). Interestingly, best agreement percentage was observed after 48 h⁽¹⁵⁾.

By comparing candifast susceptibility method to the reference method for amphotericin B in this study, the percentage of nagreement was 100% in all tested C. species except C. glabrata (85.7%) and the overall percentage of agreement was 96.6%. For fluconazole, the overall percentage of agreement was.  We could report that candifast is a rapid, easy, reproducible method for simultaneous identification and susceptibility testing.

Candifast method was used previously by Waller et al.⁽¹⁷⁾ who assayed the susceptibility of Candida albicans to different antifungals .

The poor agreement of E test than candifast could be explained on the basis that the determination of endpoints in E test is a significant factor in the variability of MIC results with fluconazole, the E test often produces inhibitory zones with diffuse edges. In addition, the MICs of an isolate of C. albicans and an isolate of C. parapsilosis were 16 μg/ml in reference method (dose-dependent susceptibility strain), while they were 64 μg/ml by E test (resistant). So the MIC near the resistant endpoint should be reported cautiously and further validation of the data is important.

In a similar study by Claudino et al.⁽¹⁸⁾ they reported that agreement between MICs provided by the E test and reference Clinical laboratory Standard Institute (CLSI) method was 100% for amphotericicn B and 96.6% for fluconazole. Their study demonstrated the adequacy of E test method using Muller Hinton agar to evaluate amphotericin B and fluconazole susceptibility of clinical isolates of Candida species.

Conclusion

·   E test method is an alternative but cannot be considered a substitute for the NCCLS reference method.

·   The antifungal susceptibility method not greatly influenced by the type of tested antifungal.

·   Candifast is a simple, rapid, method for simultaneous identification and susceptibility of Candida species. It has also a precise endpoint which is important in results interpretation.

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