Rhizomucor pusillus. Mucor.sp. Absidia corymbifera. Aspergillus sp. A. niger. Correct identification. The different genera may be distinguished based on the size ...
NRMM
The Nordic Reference group on Methods in medical Mycology
Nordic External Quality Assessment Program In Medical Mycology (NEQAMM)
NEQAMM 2009
Results and Comments Erja Chryssanthou, Victor Fernandez, Peter Gaustad, Pirkko Koukila-Kähkölä Per Sandven, Maiken Cavling Arendrup
NEQAMM 2009
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Nordic Reference Group On Methods In Medical Mycology The Nordic Reference Group on Methods in Medical Mycology (NRMM) has representatives from Denmark, Finland, Iceland, Norway and Sweden. It is organised by the Nordic Society of Medical Mycology (NSMM: http://www.nsmm.nu ), but the representatives are appointed by respective national society of clinical microbiology or a corresponding national body. The present members of NRMM are: Maiken Cavling Arendrup (chairman), Denmark, Erja Chryssanthou (secretary), Sweden, Pirkko Koukila-Kähkölä, Finland, Victor Fernandez, Sweden and Peter Gaustad, Norway. Per Sandven (Norway) is affiliated the NRMM board in relation to the EQA scheme. The main task of NRMM is to harmonise methods used for species identification and antifungal susceptibility testing in Nordic clinical mycology. It is also addressing antifungal resistance development in the Nordic countries and organise Nordic external quality assessment programs focusing on clinical mycology issues. NEQAMM 2009 is the fourth EQA organised by NRMM and the specimens were this year prepared by Victor Fernandez at SMI in Stockholm.
External Quality Assessments (EQA) – 2009 The fourth Nordic EQA was organized in spring 2009. Clinical microbiology laboratories in the five Nordic countries were invited to participate at no cost. The EQA had four simulated specimens, one with a zygomycetes, one without fungi, and two with several microorganisms: one contained Aspergillus, yeast and bacteria and the other two different yeast species. The specimens were freeze-dried to ensure that each laboratory received identical specimens. Information on specimen type and clinical information was given for each specimen. Sixty-seven laboratories accepted this offer: Sweden (SE) 21 of 29 possible, Norway (NO) 18 of 22 possible, Denmark (DK) 13 of 15 possible, Finland (FIN) 14 of 14 possible, and Iceland 1 of 1 possible laboratory. The number of laboratories submitting results differed with the type of specimen. Out of 67 laboratories, 60 submitted results from specimen 1, 66 from specimen 2, 62 from specimen 3, and 65 from specimen 4. All participating Danish and Finnish laboratories reported results from all the four specimens. The laboratories were asked to handle the specimens in exactly the same way as an ordinary, routine specimen and the results should reflect current mycological practice in the Nordic countries. The specimen type and clinical information should be decisive for how the specimen was handled and not the fact that the specimens were part of a mycological EQA. The laboratories were asked to report clinical important micro organisms (both fungi and bacteria) as in the routine work. They were also asked to adhere to routine methods (choice of media for primary plating, tests used for identification and susceptibility testing) and to report on methods used. To what degree the laboratories comply with these guidelines we are not able to determine, but the fact that a high number of laboratories used fungal chromogenic agars for primary plating of urine and that a significant number of laboratories especially in Finland did not include general microbiological media like blood agar, chocolate agar or selective gram negative media for urine, sinus aspirate and airway samples and might indicate that a number of laboratories treated these samples differently than routine samples. We can only emphasise that in our view this limits the value of the EQA since the results may reflect the best performance rather than performance in the routine setting. Considering the number of other EQA schemes that most laboratories participate showing that best performance is high when testing monocultures. We hope that the participating laboratories will refrain from handling these NRMM EQA samples with special attention in the future. Only in this way we will be able to have a clear picture of potential areas for further development.
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Media used for primary plating The media used for primary plating of the four specimens are shown in Table 1. Tabel 1. Media used for primary plating of various specimens.
1 5
R. microsporus
2.
Urine
SE (no 21) DK (no 13) NO (no 18) FIN (no14) IC (no 1) Total no 67 %
13 4 12 12 1 42 62.7
15 4 6 5 3 1 31 3 46.3 4.5
No fungus
SE (no 21)
16
DK (no 13) NO (no 18) FIN (no 14 ) IC (no 1) Total no 67 %
4 18 11 1 50 74.6
6 9 7 3 1 37 3 55.2 4.5
SE (no 21) DK (no 13) NO (no 18) FIN (no 14 ) IC (no 1) Total no 67 %
14 7 17 12 1 51 76.1
16 12 9 7 6 1 45 6 67.2 9.0
3.
Endotracheal aspirate A. niger, C. albicans (P. aeruginosa)
4.
Blood culture C. dubliniensis C. parapsilosis
14
6 13 19.4 4
14 3 11 1 11 1 1 4 4 1 7 41 9 10.4 61.1 13.4
Anaerobic
Chocolate
3
Blood
14 14 12 3 5 12 9 9 1 7 16 17 10 4 8 5 5 2 2 1 1 1 1 1 6 48 46 34 10 22 9.0 71.6 68.7 50.7 14.9 32.8 4
6
5 9 13.4 6
13 6
6 12 17.9 5
13
4 6.0
7 8 12 1 1 1 1 29 1.5 43.3
6
11 9 1 16 17 2 6 6 2 1 1 6 47 46 11 9.0 70.1 68.7 16.4
3
1
12 12 12 4 3 13 8 11 8 15 15 10 2 10 5 5 3 1 1 1 1 1 6 46 41 37 7 22 9.0 68.7 61.2 55.2 10.4 32.8
1 2
5 5 2 4
3 16 4.5 23.9 2
3 1
1
3 4.5
4 6.0
2
4
2 3.0
4 6.0
6
5 10 14.9
* Res-plate: In Denmark an agar plate with antibacterial disks is used for antibacterial screening and for detecting of yeasts. ** Numbers in parentheses indicate the total number of laboratories which reported their choice of media.
It is generally accepted that a selective fungal medium such as Sabouraud agar supplemented with antibiotics and/or a yeast selective chromogenic medium should be used NEQAMM 2009
4 2 1
1 5 7 7.5 10.4
5
6 12 2 1 1 5 25 7.5 37.3
Other selective bacterial media
6 1
Selective G pos
Sinus aspirate
Selective G neg
Primary Resistance plate*
15 4 6 5 4 1 31 4 46.3 6.0
Country
1.
BHI & other liquid media
Other selective fungal media
17 5 18 13 1 54 80.6
Chromogenic
SE (no 21) DK (no 13) NO (no 18) FIN (no 14 ) IC (no 1) Total no 67 %
Sabouraud
Specimen
Other Media
Dixon agar or other lipid
Fungal media
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for the cultivation of yeasts from clinical specimens, because the growth of yeasts may be suppressed by concomitant bacteria. Most of the laboratories included a Sabouraud agar plate (63-81%; last year 79-85%) and a yeast selective chromogenic medium (46-67%; last year 47-64%) for all the specimens. Dixon agar for isolation of Malassezia spp. was used by 3-6 Finnish laboratories. We consider that Dixon plate is not needed unless Malassezia is a relevant pathogen – that is for a blood culture from neonatal or haematological neutropenic patients especially if yeasts is observed by microscopy but not grown on regular agars. It may also be relevant to use Dixon or a comparable agar for surveillance cultures from premature and neonatal patients.
Methods used for identification Identification of micro-organisms to species or genus level was done on the specimens by most of the participating laboratories. Specimens 1 and 3 containing moulds were identified by colony morphology and/or microscopy though few laboratories specified the methods used. One Danish and one Swedish laboratory used sequencing. Only 32/67 (48%) reported the identification procedures for specimen 1 containing R. microsporus and for A. niger the data was mostly lacking. The identification methods for the Candida species in specimens 3 and 4 are shown in Table 2. Chromogenic agars (66% and 75%) and germ tube test (28% and 34%) were the most common methods used for presumptive species identification of C. albicans (specimen 3), C. dubliniensis and C. parapsilosis (specimen 4). The commercial agglutination test (Fumouze®, France) for rapid identification of C. albicans was used by 16% (specimen 3) and 24% (specimen 4) of the laboratories. Vitek was the most widely applied system for final identification of C. albicans (24% of the laboratories), C. parapsilosis and C. dubliniensis (48%). The majority of Danish laboratories have access to Vitek identification but of the other Nordic countries less than 40% of the laboratories are users today.
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Table 2. Methods used for presumptive and final identification of yeast species.
3.
SE (no 21)
17
C. albicans
DK (no 11)
9
NO (no 18)
9
1
6
FIN (no 14)
8
3
6
IC (no 1)
1
Total (no 67 )
44
8
%
65.7
11.9
SE (no 21)
19
6
4
DK (no 11)
11
2
4
NO (no 18)
12
2
8
FIN (no 14)
7
3
6
IC (no 1)
1
Total (no 67)
50
13
%
74.6
19.4
4. C. dubliniensis
C. parapsilosis
3
3
5
3
4
3
1
Sequencing/fungal PCR
Fermentation
1
5 4
3
2
1
1
2
4
6
4
1
11
16
11
6
2
1.5
16.4
9.0
3.0
2
9
9
7
2
2
11
1
1
2
3
9
5
3
1
2
3
6
6
1 19 28.4
23.9 16.4
23 34.3
2
1
1
1
1 5
16
7.5
23.9
32
19
Specimen 1 Specimen type:
Sinus aspirate
Clinical information:
49-year old male diabetic patient
Microorganism:
Rhizopus microsporus
13
2
47.8 28.4 19.4 3.0
Detection and identification of the microorganisms
NEQAMM 2009
API 20C
ID32C
Final identification (1-3-7 days)
Vitek
Rapid Trehalose test (glabrata) Latex agglutination (albicans, dubliniensis and/or krusei)
Germ Tube test
Rapid identification tests (Same day)
Corn Meal Agar or similar agars
Specimen
Chromogenic yeast agar
Country
Morphology (2-3 days)
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2 3.0
Zygomycetes are mainly associated with sinus and pulmonary disease, especially in patients with predisposition such as immunosuppression, poorly controlled diabetes, burns, malnourishment or intravenous drug abuse. Zygomycota is a large group of lower fungi with generally unseptate hyphae. It has 3 orders and one of them is Mucorales which in turn is composed of several genus as Rhizopus, Rhizomucor, Mucor and Absidia to name the most common encountered in clinical specimens. Sixty (90%) of the 67 laboratories succeeded to isolate a filamentous fungus in this specimen. Identification was difficult for many of the laboratories, only 13% succeeded with species identification and additionally 27% reported correct genus Rhizopus sp. (fig 1). Some laboratories reported Zygomycetes sp. but it is important to point out that zygomycetes is not a genus but a group of fungi and thus that this term is not taxonomically correct although the information that the fungus belongs to the zygomycetes group is important. Of all laboratories 11 would have referred this isolate to a reference laboratory for final identification. Six laboratories reported mould and 4 of them would have referred the isolate to a reference laboratory. For the choice of correct antifungal therapy it is important to identify a mould isolate from this type of specimen and patient category at least to genus (e.g. Rhizopus) or group (Zygomycetes) since the treatment of zygomycetes infection differs from e.g Aspergillus. Amphotericin B or its lipid formulation is the drug of choice followed by posaconazole if the isolate is susceptible in vitro.
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Fig.1. Results of the final identification of the isolate in specimen 1.
1; 2% 1; 2% 1; 2% 1; 2%
3; 5%
13; 22%
2; 3% 3; 5%
Correct identification
8; 13%
17; 27%
6; 10%
R. microsporus Rhizopus sp. Zygomycetes Mould R. arrhizus R. oryzae Rhizomucor sp. Rhizomucor pusillus Mucor.sp Absidia corymbifera Aspergillus sp. A. niger
4; 7%
The different genera may be distinguished based on the size and shape of the sporangia, the presence or absence of rhizoids and their location, presence or absence of branched sporangiophores, shape of the columella, and the shape of the apophysis. Typical characteristics for the genus Rhizopus are expanding hairy colonies and rhizoids (fig 2). R. microsporus has greyish-brown colonies and sporangiophores are seldom higher than 0.8 mm. It has good growth at 45°C which discriminates it from other clinically relevant Rhizopus spp. (R. oryzae grows at 37°C but not at 45°C and R. stolonifer doesn’t even grow at 37°C). In addition to observation of morphological characteristics the identification may be aided by assimilation pattern as recently described (Schwarz et al. J Clin Microbiol 2007, 45:1433-9) Fig.2. Micro and macro morphology of R. microsporus
Micro morphology
Colony morphology
columella
sporangiophore
rhizoids
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As final identification we recommend that clinical microbiology laboratories should be able to detect and identify zygomycetes to a genus level e.g Rhizopus sp. or refer such isolates to reference laboratories for species identification. The laboratories needed 2-20 days (mean 6.7 days) to give their final identification and report. It is important and should be possible to give final identification and the susceptibility testing in 3-5 days.
Specimen 2 Specimen type:
Urine
Clinical information:
23-year old woman with recurrent UTI.
Microorganism:
No fungus (Uropathogenic Escherichia coli in specimen)
Specimen 2 contained only uropathogenic E. coli and no yeasts or fungi. The isolate is resistant against cephalosporins, quinolones and aminoglycosides. All the laboratories reporting bacterial growth made a correct species identification. Only 11 laboratories reported no growth of fungi/yeasts with or without bacterial finding. It is important to report to clinicians that the urine specimen has been examined for yeast. Two of the 66 laboratories reported a fault organism (C. kefyr, A. fumigatus) which is most probably due to a laboratory contamination.
Fig.2. Results of the final identification of the isolate in specimen 2.
E. coli E. coli, no growth of fungi/yeasts Correct report
bacteria, no growth of fungi/yeasts
1; 2%
2; 3%
3; 5%
6; 8%
gram negative rods bacteria
1; 2% 1; 2%
no growth of fungi/yeasts negative C. kefyr
7; 10%
A. fumigatus
3; 5%
42; 63%
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Specimen 3 Specimen type:
Endotracheal aspirate
Clinical information:
59- year old male ICU patient with chronic obstructive pulmonary disease. Patient is receiving corticosteroid treatment and broad spectrum antibiotics. Aspergillus niger, Candida albicans (Pseudomonas aeruginosa)
Microorganism:
Patients with chronic obstructive pulmonary disease (COPD) are often colonized with different filamentous fungi and/or Candida species. Patients requiring ICU ward and receiving corticosteroids are at risk to develop an invasive Aspergillus infection due to the impaired architecture of the lung tissue, the decreased number of neutrophiles and macrophages to eradicate aspergillus spores and hyphae, and the growth stimulating activity of corticosteroids on the aspergillus itself. Pulmonary infection in COPD patients is associated with a notably high mortality. Growth of Aspergillus in specimens from lower respiratory tract may represent contamination but should not be discarded as such. It is important, therefore, to perform fungal cultures on specimens from this patient category by including a Sabouraud agar plate/slope, while the use of a chromogenic agar is less needed as yeasts are rarely true lung pathogens. Sixty two (93%) of 67 laboratories reported fungi in this specimen. Fifty two (84%) reported correctly C. albicans and A. niger/tubingensis or mould and additionally five laboratories (8%) reported only A. niger but not the C. albicans which may have been motivated by the fact that this yeast was regarded as a contaminant deriving from the oral cavity. Five laboratories missed A. niger but reported C. albicans (± P. aeruginosa) and thus in total 8% of the laboratories failed to detect the aspergillus isolate in this sample. The species identification of A. niger is quite easy based on its specific black colony and micro-morphology. One laboratory had identified the A. niger isolate as A. tubingensis by sequencing. A. niger and A. tubingensis have a high morphological similarity. Classical taxonomy does not allow the discrimination of these two species. With the development of molecular methods and their application to fungal identification, the section Nigri has been defined including 3 clades: A. niger, A. tubingensis and A. foetidus. Their classification as species, subspecies or varieties has not yet reached a consensus and clinical significance of differentiating these new species from A. niger is not yet determined. It has recently been reported that compared to A. niger some A. tubingensis isolates had higher MICs to itraconazole (Alcazar-Fuoli et al. 2009, doi:10.1128/AAC.00585-09).
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Fig.3. Results of the final identification of the isolate in specimen 3.
C. albicans, C. albicans, A. niger C. albicans, C. albicans, C. albicans, C. albicans, C. albicans, C. albicans
Correct identification
2; 3%
3; 5%
3; 5%
2; 3%
3; 5% 1; 2%
A. niger A. niger, P. aer. A. tubing. A. sp., P. aer./Pseud mould, P. aer. mould P. aer.
30; 48%
5; 8%
13; 21%
Specimen 4 Specimen type: Clinical information:
Microorganism:
Blood culture (blood sample drawn from CVC) 68-year old man in intensive care. Patient was treated with caspofungin after positive blood culture (Candida glabrata) two months ago. Candida dubliniensis and Candida parapsilosis
Blood cultures with mixed yeasts constitute 3-5% of fungaemia cases in most studies. It is important to be able to identify them for correct and timely treatment of the patient. The use of chromogenic media has improved the detection of mixed cultures in clinical laboratory practice and in agreement with this 90% of the laboratories using such plates for primary plating of the positive blood culture detected the polyfungal nature of this sample in contrast to 44% which did not use chromogenic agars. Of the 65 laboratories 47% succeeded to isolate and identify both C. parapsilosis and C. dubliniensis correctly, additional 5% identified C. parapsilosis and Candida sp. Fifty eight (89%) and 33 (51%) laboratories, respectively, detected and identified C. parapsilosis and/or C. dubliniensis as a single isolate or together with an other Candida species. One laboratory identified C. parapsilosis and both C. dubliniensis and C. albicans. C. parapsilosis and C. albicans were reported by 19% of the laboratories. If only chromogenic agar and/or a germ-tube test are used for final identification of C. albicans, the correct species identification of C. dubliniensis is missed, since it is also green on Chromagar Candida and positive in germ-tube test. We recommend C. dubliniensis agglutination test (Fumouze®, France), Vitek, lack of growth at 43 ˚C or ID 32C for identification of C. dubliniensis. We also recommend that laboratories should perform species identification of all yeasts from blood cultures for the timely and efficient treatment of the NEQAMM 2009
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patient. However the need for differentiation of C. albicans from C. dubliniensis from a therapeutic point of view can be debated since the susceptibility patterns are very similar. Fig.4. Results of the final identification of the isolate in specimen 4. Correct identification C. fam.; 1; 2% C. alb., C. gl.; 1; 2% C. sp/yeast species; 2; 3% C. alb., yeast nonalb./unspec; 2; 3% C. dubl.; 1; 2% C. para.; 10; 15%
C. para., C. dubl. C. para., C. sp. C. para., C. dubl., C. alb. C. para., C. alb. C. para. C. dubl. C. alb., yeast non-alb./unspec C. sp/yeast species C. alb., C. gl. C. fam.
C. para., C. dubl.; 31; 47%
C. para., C. alb.; 13; 19% C. para., C. dubl., C. alb.; 1; 2%
C. para., C. sp.; 3; 5%
Susceptibility data The number of laboratories performing susceptibility testing is shown in Table. The number varied with the fungus isolated in the specimen. Very few laboratories provided susceptibility testing results for R. microsporus (specimen 1) and A. niger (specimen 3) in agreement with the fact that susceptibility testing of moulds is a specialist task. Out of the 67 laboratories 54% reported susceptibility results for C. albicans, 51% for C. dubliniensis and 63% for C. parapsilosis. Laboratories not doing susceptibility testing of moulds should send relevant clinical isolates to a reference laboratory. Table 3. Number of the participating laboratories reporting susceptibility testing results on the different specimens.
SPECIMEN 1 R. microsporus SPECIMEN 3.1 A. niger SPECIMEN 3.2 C. albicans SPECIMEN 4.1 C. dubliniensis SPECIMEN 4.2 C. parapsilosis
SE (no 21)
DK (no 13)
NO (no 18)
FIN (no 14)
ICE (no 1)
3
1
1
0
0
5
0
3
0
0
14
6
9
9
0
12
6
9
6
1
14
8
10
9
1
The results from this EQA show again that there is a wide variation of MIC values between the laboratories performing susceptibility testing, especially caspofungin and voriconazole NEQAMM 2009
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MICs for C. albicans covered a wide range. Standardisation and harmonization of susceptibility testing and reading of MIC end points is urgently needed. In this context the regular use of control strains with known MICs is valuable. The MICs for the NEQAMM isolates from the Nordic reference laboratories is given in table 4. Methods used for susceptibility testing. Most laboratories use E-test but also Vitek is increasingly used in the Nordic countries. One Finnish laboratory reported results on disk diffusion test for specimen 3.2 C. albicans and 4.1 C. dubliniensis. Results. E-test endpoints have been rounded up to the next 2-fold dilution endpoints (e.g 0.38 to 0.5 mg/L and >32 to 64 mg/L) in order to ease comparison. Susceptibility testing results obtained by Vitek were transformed as follows: 16 >4 0.25-0.5 2 0.25-1
Caspofungin Fluconazole Voriconazole Posaconazole Itraconazole Amphotericin B Anidulafungin
A. niger 0.032-0.125 >16 0.5-1 0.25 1 0.25-5
C. albicans 0.032-0.125 0.125 0.016-0.03 0.016-0.06 0.016-0.03 0.25-0.5 0.03
C. dubliniensis* 16-32 0.064-0.125 0.016-0.03 0.016-0.03 0.016-0.03 0.5-1 1-4
C. parapsilosis 1 1-2 0.03-0.06 0.016-0.03 0.06 0.25-1 1-2
* this isolate has acquired echinocandin resistance due to a mutation in the target gene Table 5. Breakpoints used by Nordic reference laboratories for Candida spp. Non-species dependent breakpoints AMB
ANI*
CAS*
5-FC
S (< X)
R (> X)
S (< X)
Denmark
1
1
0.125
Finland
1
1
2
Norway
1
1
1
1
4
Sweden
1
1
2
2
4
0.5
R (> X)
S (< X)
R (> X)
1
0.5
FLU
ITR
R (> X)
S (< X)
R (> X)
S (< X)
R (> X)
S (< X)
4
16
2
4
0.125
0.5
0.125
2
4
0.125
0.5
0.125
8
2
4
0.125
0.5
0.125
16
2
4
0.125
0.5
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0.125
R (> X)
VOR
S (< X)
*Breakpoints are pending. The suggested limits for susceptibility are based on the national wildtype distributions.
NEQAMM 2009
POS*
S (< X)
R (> X)
0.125
0.125
0.125
Table 6. MIC-distributions for specimen 1: R. microsporus
MIC (mg/L)
No of observations reported by the participating laboratories AmB Caspo Fluco Itra Vori Posa Anidula
0.004 0.008 0.016 0.032 1 0.064 1 0.125 1 1 1 0.25 0.5 2 1 1 1 1 2 1 4 1 1 8 1 1 16 32 3 1 64 128 256 1 Total 4 3 1 3 3 4 1 MIC range obtained at the Nordic reference laboratories are indicated in green, when this was truncated (ex caspo >32) all values above this MIC is coloured green Very few laboratories reported MIC determinations for this specimen and we in general recommend that mould isolates are referred to reference labs for susceptibility testing unless special mycology expertise is present. Only eleven laboratories would have sent this isolate to a reference laboratory which is quite disappointing considering the potential pathogenicity of zygomycetes in the specified patient population. Echinocandins and azoles with the exception of posaconazole are not active against zygomycetes. According to the current knowledge amphotericin B and posaconazole are the treatment options for infection caused by Rhizopus spp. but susceptibility of zygomycetes to amphotericin B and posaconazole is not uniform.
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Table 7. MIC-distributions for specimen 2 A. niger
MIC (mg/L)
0.002 0.004 0.008 0.016 0.032 0.064 0.125 0.25 0.5 1 2 4 8 16 Total
No of observations reported by the participating laboratories AmB Caspo Fluco Itra Vori Posa Anidula 1 1 1 1 1 1 1 2 1 2 2 1 2 1 3 1 1 1 1 1 5 3
1 7
4
1
7
8
5
3
The MIC values reported by the laboratories were all low (≤1 mg/L) for the tested antifungals except fluconazole as was expected since A. niger is normally susceptible to amphotericin B, echinocandins, voriconazole, itraconazole and posaconazole but as other moulds resistant to fluconazole. However, resistance to itra-, with or without cross resistance to vori- and posaconazole has been reported in clinical failures as well as in azole naïve patients in The Netherlands. We recommend that Aspergillus isolates considered of clinical significance are susceptibility tested or referred to reference lab for testing. Susceptibility testing for azoles can be performed by an initial screening for itraconazole resistance as resistance to voriconazole and posaconazole has not been detected in isolates susceptible to itraconazole. Only four laboratories indicated they would send this isolate to a reference laboratory. Table 8. MIC-distributions for specimen 3.2 C. albicans No. of observations reported by participating laboratories MIC (mg/L) AmB Caspo Fluco Vori 0.002 1 0.004 1 1 0.008 1 12 0.016 1 7 0.032 1 0.064 1 1 1 0.125 5 16 18 62 0.25 8 3 6 1 0.5 15 3 1 2 1 8 Total 31 25 35 29 1 2 According to Vitek ≤1 mg/L , Vitek ≤0.125 mg/L NEQAMM 2009
Itra
5-FC
2 1 2 1 1
7
Anidula 1 5
Posa
1
1 1
1
1
2 61 6
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6
3
50
5-FC
45
Itra Vori
40
Fluco
35
Caspo
30
AmB
Number of 25 observations 20 15 10 5 0 0,002 0,004 0,008 0,016 0,032 0,064 0,125
0,25
0,5
1
2
4
MIC (mg/L)
MIC distributions for fluconazole and amphotericin B showed better performance of the laboratories with less variation this year covering only 4 and 5 dilutions, respectively. The other antifungal, especially caspofungin, showed more variation in MIC distributions. Voriconazole and 5-fluorocytosin distributions show some bias because the lowest MIC values by Vitek are reported as ≤0.125 and ≤1 mg/L respectively. Table 9. MIC-distributions for specimen 4.1 C. dubliniensis No of observations reported by participating laboratories MIC (mg/L) AmB Caspo Fluco Vori Itra 5-FC 0,002 10 0,004 9 2 1 0,008 1 3 0,016 3 2 2 2 0,032 14 1 1 2 0,064 10 1 0,125 2 1 2 1 0,25 0 1 0,5 8 1 1 10 3 21 2 9 4 1 1 8 3 16 4 32 1 9 64 12 Total 31 29 34 28 6 7 1 According to Vitek ≤1 mg/L
NEQAMM 2009
Anidula
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Posa
1 1 2
1 1 1 1 3 7
4
Vori
16
Fluco Caspo
14
AmB
Number of observations
12
10
8
6
4
2
0
0,002 0,004 0,008 0,016 0,032 0,064 0,125
0,25
0,5
1
2
4
8
32
16
64
MIC (mg/L)
This C. dubliniensis isolate is echinocandin resistant (R) and twenty-five (74%) laboratories reported MIC ≥16 mg/L. Echinocandin resistance is still rare but it can emerge during treatment. Eleven laboratories (8 from Norway) reported this isolate as amphotericin B resistant and 9 as susceptible. Amphotericin B resistance is very uncommon in Candida species. According to the reference laboratories the isolate was susceptible to amphotericin B. If resistance is suspected the isolate should be send to a reference laboratory for confirmation of the MIC result. Validation of E-test MIC endpoint reading is warranted in laboratories with erroneous MICs. Ten laboratories (7 Norwegian and 3 Danish) reported that they would have sent this isolate to a reference laboratory. Table 10. MIC-distributions for specimen 4.2 C. parapsilosis No of observations reported by participating laboratories MIC (mg/L) 0,004 0,008 0,016 0,032 0,064 0,125 0,25 0,5 1 2 4 8 16 32 64
AmB
Caspo
Fluco
1 2
1 7 17 6 1 1 1
1 15 11 2 1
2 Total 37 32 1 According to Vitek ≤1 mg/L NEQAMM 2009
1 1 6 13 14 7
42
Vori 2 2 8 12 6 6
Itra
5-FC
1
1 1
2 3 1
7
2
9
Posa
1 1 2
51
36
Anidula
1 2 2 4
9
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4
40
Vori Fluco
35
Caspo AmB
Number of observations
30
25
20
15
10
5
0 0,002 0,004 0,008 0,016 0,032 0,064 0,125 0,25
0,5
1
2
4
8
16
32
64
MIC (mg/L)
The isolate was a wild-type isolate fully susceptible to azoles, 5-FC and amphotericin B and with elevated MICs to the echinocandins when compared to the other Candida spp. This is characteristic for C. parapsilosis and due to intrinsic mutation in the target gene known to confer resistance (Garcia Effron et al Antimicrob. Agents Chemother 2008, 52:2305–12). C. parapsilosis is not clinically truly echinocandin resistant as outcomes in patients treated with these compounds are comparable to the outcome for patients with other species. This is likely due to this organism being significantly less virulent than the other common human pathogenic Candida spp. However, numerically the outcome for C. parapsilosis was better on fluconazole (Reboli et al. N Engl J Med 2007;356:2472-82.) and breakthrough infections in caspofungin treated patients has been significantly linked to C. parapsilosis (Sipsas et al Cancer 2009 e-pub ahead of print). Thus other drug classes are preferable in patients with invasive C. parapsilosis infections. Three Norwegian laboratories reported this isolate as amphotericin B resistant. Two laboratories found the isolate highly resistant to caspofungin (MIC=64 mg/L) which might be explained by mixed culture with C. dubliniensis though chromogenic agar was used in both laboratories. It is recommended that the laboratories make sure that pure cultures are used for susceptibility testing. Ten laboratories (6 Norwegian, 3 Danish and 1 Swedish) reported that they would have sent this isolate to a reference laboratory.
Recommendations - The laboratories should adhere to routine methods (choice of media for primary plating, tests used for identification and susceptibility testing) and to report on methods used when participating in the Nordic EQAs. The laboratories should not treat these samples differently from routine samples. - For correct antifungal treatment it is important to identify a mould isolate from sinus aspirate specimens at least to genus (e.g. Rhizopus) or group (Zygomycetes). Those laboratories not performing species identification should send the isolate to a reference laboratory.
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- The laboratories should be able to isolate and identify the most common clinically relevant Aspergillus species from lower respiratory specimens. Those laboratories not performing species identification should send the isolate to a reference laboratory. - The laboratories should use chromogenic media for improved detection of mixed yeast cultures which was nicely shown for specimen 4: 90% of the laboratories using such plates for primary plating of the positive blood culture detected both yeasts in contrast to 44% which did not. - The wide MIC ranges of many antifungals for the yeasts highlights the need in the laboratories to continuously validate the performance of the used methods and the reading of MIC endpoints. - The laboratories should send clinically relevant mould isolates to a reference laboratory for susceptibility testing as this requires specialist training and an expertise based on experience with reference methods and a large number of mould isolates which will not be available in most laboratories.
References
Epidemiological cutoffs and cross-resistance to azole drugs in Aspergillus fumigatus. RodriguezTudela JL, Alcazar-Fuoli L, Mellado E, Alastruey-Izquierdo A, Monzon A, Cuenca-Estrella M. Antimicrob Agents Chemother. 2008; 52:2468-72. EUCAST Technical Note on the method for the determination of broth dilution minimum inhibitory concentrations of antifungal agents for conidia-forming moulds. Subcommittee on Antifungal Susceptibility Testing of the ESCMID European Committee for Antimicrobial Susceptibility Testing. Clin Microbiol Infect. 2008;14:982-4. EUCAST definitive document EDef 7.1: method for the determination of broth dilution MICs of antifungal agents for fermentative yeasts. Subcommittee on Antifungal Susceptibility Testing (AFST) of the ESCMID European Committee for Antimicrobial Susceptibility Testing (EUCAST). Clin Microbiol Infect. 2008;14:398-405. Reboli AC, Rotstein C, Pappas PG, Chapman SW, Kett DH, Kumar D, Betts R, Wible M, Goldstein BP, Schranz J, Krause DS, Walsh TJ. Anidulafungin versus fluconazole for invasive candidiasis. N Engl J Med 2007;356:2472-82. Sipsas NV, Lewis RE, Tarrand J, Hachem R, Rolston KV, Raad II, Kontoyiannis DP. Candidemia in patients with hematologic malignancies in the era of new antifungal agents (2001-2007): stable incidence but changing epidemiology of a still frequently lethal infection. Cancer 2009. Epub ahead of print.
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