Erja Chryssanthou

Interlaboratory variability of caspofungin MICs for Candida spp. using CLSI and EUCAST methods: Should the clinical laboratory Be testing this agent?

ABSTRACT Although Clinical and Laboratory Standards Institute (CLSI) clinical breakpoints (CBPs) are available for interpreting echinocandin MICs for Candida spp., epidemiologic cutoff values (ECVs) based on collective MIC data from multiple laboratories have not been defined. While collating CLSI caspofungin MICs for 145 to 11,550 Candida isolates from 17 laboratories (Brazil, Canada, Europe, Mexico, Peru, and the United States), we observed an extraordinary amount of modal variability (wide ranges) among laboratories as well as truncated and bimodal MIC distributions. The species-specific modes across different laboratories ranged from 0.016 to 0.5 μg/ml for C. albicans and C. tropicalis, 0.031 to 0.5 μg/ml for C. glabrata, and 0.063 to 1 μg/ml for C. krusei. Variability was also similar among MIC distributions for C. dubliniensis and C. lusitaniae. The exceptions were C. parapsilosis and C. guilliermondii MIC distributions, where most modes were within one 2-fold dilution of each other. These findings were consistent with available data from the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (403 to 2,556 MICs) for C. albicans, C. glabrata, C. krusei, and C. tropicalis. Although many factors (caspofungin powder source, stock solution solvent, powder storage time length and temperature, and MIC determination testing parameters) were examined as a potential cause of such unprecedented variability, a single specific cause was not identified. Therefore, it seems highly likely that the use of the CLSI species-specific caspofungin CBPs could lead to reporting an excessive number of wild-type (WT) isolates (e.g., C. glabrata and C. krusei) as either non-WT or resistant isolates. Until this problem is resolved, routine testing or reporting of CLSI caspofungin MICs for Candida is not recommended; micafungin or anidulafungin data could be used instead.

In Vitro Susceptibility of Respiratory Isolates of Aspergillus species to Itraconazole and Amphotericin B. Acquired Resistance to Itraconazole

122 Aspergillus strains were isolated from respiratory specimens from 80 patients. Aspergillus fumigatus was the most common species, constituting 88% of the isolates. Susceptibility testing by the NCCLS broth macrodilution procedure revealed that the minimal inhibitory concentration for 50% of the strains (MIC50) was 0.25 mg/l for itraconazole and 0.5 mg/l for amphotericin B. The MIC90 was 1 mg/l for both drugs. To our knowledge, no cases of in vitro resistance during long-term itraconazole use in treatment of Aspergillus infection have been documented. We identified 3 patients infected with A. fumigatus strains that acquired in vitro resistance to itraconazole during prolonged therapy. This finding supports the importance of susceptibility testing.

Detection of Candida albicans DNA in serum by polymerase chain reaction.

Polymerase chain reaction (PCR) was applied in order to improve the diagnosis of disseminated candidosis. A nested PCR technique with 2 primer pairs was used to increase the sensitivity. We were able to detect Candida DNA in serum and tissue samples from experimentally infected mice as well as in serum samples from candidemia patients and patients with deep-seated Candida infection. Our PCR could detect as little as 1 pg Candida albicans DNA. The PCR method was more sensitive than culture in both the mouse experiments and the patients with deep candidosis (5/7 were PCR positive and 0/7 blood culture positive), and of similar sensitivity in candidemia patients (11/17 were 15/17 blood culture positive). The relatively short processing time of PCR, when compared with culturing, its sensitivity, as well as the possibility of using serum samples for analysis, all helped improve the diagnostics in deep-seated candidosis and candidemia.

Histoplasmosis in Europe: Report on an epidemiological survey from the European Confederation of Medical Mycology Working Group

The purpose of this survey was to systematically collect data on individuals with histoplasmosis in Europe over a 5-year period (from January 1995 to December 1999). This included information on where and how the infection was acquired, the patients risk factors, the causative organism, how the infection was diagnosed and what therapy the patients received. Data were sent on a standardized survey form via a national convenor to the coordinator. During the survey, 118 cases were reported, with 62 patients having disseminated disease, 31 acute pulmonary infection, chronic pulmonary infection in 6 and localized disease in 2 patients. For 17 patients, the diagnosis of histoplasmosis was incidental, usually secondary to investigations for lung cancer. Most patients had travelled to known endemic areas, but 8 patients (from Italy, Germany and Turkey) indicated that they had not been outside their countries of origin and hence these cases appear to be autochthonous. Notable observations during the survey were the reactivation of the disease up to 50 years after the initial infection in some patients and transmission of the infection by a transplanted liver. Itraconazole was the most commonly used therapy in both pulmonary and disseminated disease. The observation of autochthonous cases of disease suggests that the endemic area of histoplasmosis is wider than classically reported and supports continued surveillance of the disease throughout Europe.

Evaluation of wild-type MIC distributions as a tool for determination of clinical breakpoints for Mycobacterium tuberculosis

OBJECTIVES The aim of this study was to establish wild-type MIC distributions of first-line drugs for Mycobacterium tuberculosis, as well as to explore the usefulness of such distributions when setting clinical breakpoints. METHODS We determined the MICs of rifampicin, isoniazid and ethambutol for M. tuberculosis using a Middlebrook 7H10 dilution method for 90 consecutive clinical isolates, 8 resistant strains and 16 isolates from the WHO proficiency test panel. M. tuberculosis H37Rv was used for quality control and susceptibility results using 7H10 were compared with the results obtained with BACTEC460. RESULTS The agreement with BACTEC460 was very high for isoniazid (99.1%) and rifampicin (99.1%) but lower for ethambutol (94.7%). Intra- and inter-assay variation was below one MIC dilution. The MIC distributions for isoniazid and rifampicin provided a clear separation between susceptible and resistant strains. Regarding ethambutol, the current breakpoint for 7H10 (5 mg/L) is close to the wild-type and all strains (n = 6) showing a disagreement between BACTEC460 and 7H10 were distributed very close to the breakpoint (MIC 4-8 mg/L). This problematic relation was confirmed by investigating isolates from the WHO panel with an agreement <95% (64%-88% among 26 laboratories, n = 4) for which the MICs were 4-8 mg/L. CONCLUSIONS Utilizing the wild-type MIC distribution was found to be as useful in M. tuberculosis as in other bacteria when setting clinical breakpoints. We suggest that the present clinical breakpoints should be re-evaluated, taking into account wild-type MIC distributions and available pharmacokinetic data.

Epidemiology of dermatophyte infections in Stockholm, Sweden: a retrospective study from 2005–2009

Dermatophytic infections are common worldwide but the distribution of dermatophyte species varies among geographical areas and changes over time. The aim of this study was to determine the epidemiologic profile of dermatophytosis in Stockholm, Sweden. Laboratory records comprising direct microscopy and culture results of 37,503 specimens from skin, hair and nail scrapings collected from January 2005 through December 2009 were retrospectively analyzed in the mycology laboratory at Karolinska University Hospital. Onychomycosis had, over time, the highest overall prevalence of 14.1%, followed by tinea pedis (4.4%). Trichophyton rubrum was the predominant pathogen isolated from these cases (83.2%), followed by T. mentagrophytes (7.4 %). In contrast, T. violaceum and T. soudanense accounted for 81.6% of the isolates from patients with tinea capitis.

PCR and other non-culture methods for diagnosis of invasive Candida infections in allogeneic bone marrow and solid organ transplant recipients.

In this prospective study 197 serum and 152 urine samples were collected from 40 bone marrow and solid organ transplant recipients with clinically suspected invasive fungal infection before, during and after empirical treatment with lipid formulation of amphotericin B or fluconazole. Serum was analysed by Candida polymerase chain reaction (PCR) and urine by measurement of d/l‐arabinitol ratio. One serum from each patient was also tested for concentration of (1→3)‐β‐glucan and two commercial Candida antigens. Invasive fungal infection was diagnosed in four candidosis and one aspergillosis patients (13%). Positive PCR, elevated d/l‐arabinitol ratio, (1→3)‐β‐glucan concentration and antigens were detected in nine, 15, 17, and seven patients, respectively. The agreement between PCR and d/l‐arabinitol assays was poor. However, 56% agreement was observed between positive PCR and β‐glucan and/or antigen assays, and 60% agreement between positive d/l‐arabinitol and β‐glucan and/or antigen assays. Combination of several non‐culture assays is needed to diagnose invasive fungal infection in high‐risk transplant recipients. No single test was sufficient for diagnosis.

Prevalence of tinea capitis in Ethiopian schoolchildren

The prevalence of dermatophytosis and the spectrum of dermatophyte species were determined in children attending two schools in Addis Ababa, Ethiopia. Demographic and clinico‐dermatological data were collected. Specimens were taken for microscopy and culture from all suspected lesions. Dermatophyte species were identified by morphology and biochemical tests, supplemented by sequencing of the rDNA ITS 2 region in selected isolates. From the Biruh Tesfa Elementary School (BTES) 824 students, and from Mount Olive Academy (MOA) all 124 students, were included. In BTES 513 (62.3%) students were clinically diagnosed with dermatophytosis, 463 (90.3 %) of them with tinea capitis. In 200 consecutive samples from BTES, and in 66 from MOA, 75 and 62%, respectively, contained fungal elements at microscopy. From BTES, 163/496 (33%) samples were culture‐positive, of which 149 (91.4%) grew with dark purple colonies identified as Trichophyton violaceum, while 244 (49.4%) samples were contaminated. A few strains grew slowly developing white to cream colonies, two were identified as T. verrucosum, and 12 as white T. violaceum. From MOA 44 (66.7%) of samples were culture‐positive, 38 (87%) were identified as T. violaceum, and one (2.3%) as T. verrucosum, while 33% showed no growth. Four white isolates of T.violaceum were confirmed by DNA‐sequencing. Dermatophytosis was thus diagnosed in 55–62% of children screened at two schools of different socioeconomic standards in the Ethiopian capital. Trichophyton violaceum constituted 87–90% of all isolates. White variants of T. violaceum were diagnosed in 16 cases.

Wild-type MIC distributions of four fluoroquinolones active against Mycobacterium tuberculosis in relation to current critical concentrations and available pharmacokinetic and pharmacodynamic data

OBJECTIVES To describe wild-type distributions of the MIC of fluoroquinolones for Mycobacterium tuberculosis in relation to current critical concentrations used for drug susceptibility testing and pharmacokinetic/pharmacodynamic (PK/PD) data. METHODS A 96-stick replicator on Middlebrook 7H10 medium was used to define the MICs of ciprofloxacin, ofloxacin, moxifloxacin and levofloxacin for 90 consecutive clinical strains and 24 drug-resistant strains. The MICs were compared with routine BACTEC 460 susceptibility results and with MIC determinations in the BACTEC MGIT 960 system in a subset of strains using ofloxacin as a class representative. PK/PD data for each drug were reviewed in relation to the wild-type MIC distribution. RESULTS The wild-type MICs of ciprofloxacin, ofloxacin, moxifloxacin and levofloxacin were distributed from 0.125 to 1, 0.25 to 1, 0.032 to 0.5 and 0.125 to 0.5 mg/L, respectively. The MIC data correlated well with the BACTEC 960 MGIT and BACTEC 460 results. PD indices were the most favourable for levofloxacin, followed by moxifloxacin, ofloxacin and ciprofloxacin. CONCLUSIONS We propose S (susceptible) <or= 1.0 mg/L as the tentative epidemiological cut-off (ECOFF) for ofloxacin and ciprofloxacin, and S <or= 0.5 mg/L for levofloxacin and moxifloxacin, although it is possible that adding more MIC data could shift the ECOFFs for ofloxacin and levofloxacin one dilution upwards. The proposed ECOFFs may be more appropriate if used as clinical breakpoints on Middlebrook 7H10 agar than the current critical concentrations of S <or= 2.0 mg/L for ciprofloxacin, ofloxacin and levofloxacin, and S <or= 0.5 mg/L could be considered as a clinical breakpoint for moxifloxacin, provided other investigators can confirm our findings.

Candidaemia in Sweden: a nationwide prospective observational survey

A prospective observational nationwide investigation was performed from September 2005 to August 2006 to study the epidemiology of candidaemia in Sweden. From 385 patients, 403 isolates were recovered, yielding an incidence of 4.2 cases per 100 000 inhabitants. Candida albicans was the most common species (61%), followed by Candida glabrata (20%) and Candida parapsilosis (9%). The rates of resistance to fluconazole were ≤ 1% in C. albicans and 6-29% in non-albicans species other than C. glabrata and Candida krusei. Resistance to voriconazole was rare, except for C. glabrata and C. krusei. Only three isolates had reduced susceptibility to amphotericin B, and one had reduced susceptibility to caspofungin.