Yvette J. Debets-Ossenkopp

Clinical implications of azole resistance in Aspergillus fumigatus, The Netherlands, 2007-2009.

The prevalence and spread of azole resistance in clinical Aspergillus fumigatus isolates in the Netherlands are currently unknown. Therefore, we performed a prospective nationwide multicenter surveillance study to determine the effects of resistance on patient management strategies and public health. From June 2007 through January 2009, all clinical Aspergillus spp. isolates were screened for itraconazole resistance. In total, 2,062 isolates from 1,385 patients were screened; the prevalence of itraconazole resistance in A. fumigatus in our patient cohort was 5.3% (range 0.8%-9.5%). Patients with a hematologic or oncologic disease were more likely to harbor an azole-resistant isolate than were other patient groups (p<0.05). Most patients (64.0%) from whom a resistant isolate was identified were azole naive, and the case-fatality rate of patients with azole-resistant invasive aspergillosis was 88.0%. Our study found that multiazole resistance in A. fumigatus is widespread in the Netherlands and is associated with a high death rate for patients with invasive aspergillosis.

Aspergillosis due to Voriconazole Highly Resistant Aspergillus fumigatus and Recovery of Genetically Related Resistant Isolates From Domiciles

BACKGROUND Azole resistance is an emerging problem in Aspergillus fumigatus and complicates the management of patients with Aspergillus-related diseases. Selection of azole resistance may occur through exposure to azole fungicides in the environment. In the Netherlands a surveillance network was used to investigate the epidemiology of resistance selection in A. fumigatus. METHODS Clinical A. fumigatus isolates were screened for azole resistance in 8 university hospitals using azole agar dilution plates. Patient information was collected using an online questionnaire and azole-resistant A. fumigatus isolates were analyzed using gene sequencing, susceptibility testing, and genotyping. Air sampling was performed to investigate the presence of resistant isolates in hospitals and domiciles. RESULTS Between December 2009 and January 2011, 1315 A. fumigatus isolates from 921 patients were screened. A new cyp51A-mediated resistance mechanism (TR46/Y121F/T289A) was observed in 21 azole-resistant isolates from 15 patients in 6 hospitals. TR46/Y121F/T289A isolates were highly resistant to voriconazole (minimum inhibitory concentration ≥16 mg/L). Eight patients presented with invasive aspergillosis due to TR46/Y121F/T289A, and treatment failed in all 5 patients receiving primary therapy with voriconazole. TR46/Y121F/T289A Aspergillus fumigatus was recovered from 6 of 10 sampled environmental sites. CONCLUSIONS We describe the emergence and geographical migration of a voriconazole highly resistant A. fumigatus that was associated with voriconazole treatment failure in patients with invasive aspergillosis. Recovery of TR46/Y121F/T289A from the environment suggests an environmental route of resistance selection. Exposure of A. fumigatus to azole fungicides may facilitate the emergence of new resistance mechanisms over time, thereby compromising the use of azoles in the management of Aspergillus-related diseases.

Nosocomial Spread of a Staphylococcus capitis Strain with Heteroresistance to Vancomycin in a Neonatal Intensive Care Unit

ABSTRACT A premature infant in a neonatal intensive care unit (NICU) developed a bloodstream infection caused by coagulase-negative staphylococci (CoNS) sensitive to vancomycin. The infection persisted for 3 weeks, despite therapy with vancomycin and replacement of all intravenous catheters. The neonate died due to necrotizing enterocolitis which developed during the ongoing sepsis. We screened this strain and 216 other strains of CoNS from cultures of blood obtained from neonates between 1997 and 2000 for heteroresistance to vancomycin. Forty-eight isolates, including the strain that caused ongoing sepsis, proved heteroresistant. All isolates were identified as Staphylococcus capitis and were identical, just as their resistant stable subcolonies were, when they were genetically fingerprinted by amplified-fragment length polymorphism analysis. The heteroresistant phenotype of this endemic strain was confirmed by population analysis. We conclude that heteroresistance to vancomycin occurs in S. capitis and might be the cause of therapeutic failures in NICUs. Moreover, heteroresistant strains can become endemic in such units.

Characterization of DIM-1, an Integron-Encoded Metallo-β-Lactamase from a Pseudomonas stutzeri Clinical Isolate in the Netherlands

ABSTRACT A carbapenem-resistant Pseudomonas stutzeri strain isolated from a Dutch patient was analyzed in detail. This isolate produced a metallo-β-lactamase (MBL) whose gene, with 43.5% GC content, was cloned and expressed in Escherichia coli. β-Lactamase DIM-1 (for Dutch imipenemase) was weakly related to other Ambler class B β-lactamases, sharing <52% amino acid identity with the most closely related MBL, GIM-1, and 45% identity with IMP-type MBLs. The β-Lactamase DIM-1 significantly hydrolyzed broad-spectrum cephalosporins and carbapenems and spared aztreonam. This MBL gene was embedded in a class 1 integron containing two other gene cassettes, encoding resistance to aminoglycosides and disinfectants, that was located on a 70-kb plasmid.

Extended-Spectrum-Beta-Lactamase Production in a Salmonella enterica Serotype Typhi Strain from the Philippines

ABSTRACT A Salmonella enterica serotype Typhi strain was cultured from blood and fecal samples from a 54-year-old man with fever and diarrhea. He had returned from travel to the Philippines a few days earlier. Phenotypic and genotypic analysis confirmed the production of the SHV-12 extended-spectrum beta-lactamase.

Enrichment broth improved detection of extended-spectrum-beta-lactamase-producing bacteria in throat and rectal surveillance cultures of samples from patients in intensive care units.

ABSTRACT We evaluated the use of a trypticase soy broth (TSB) for improving detection of extended-spectrum-beta-lactamase-producing (ESBL+) bacteria. Preenrichment of throat and rectal swabs in TSB prior to inoculation on solid medium doubled the number of ESBL+ bacteria detected in samples obtained from patients in our intensive care unit.

Zygomycete infection following voriconazole prophylaxis.

A 14-year-old boy was treated with chemotherapy for T-cell acute lymphoblastic leukaemia. Trimethoprimsulfamethoxazole and itraconazole were given for infection prophylaxis. In the course of this treatment he developed aplastic anaemia that required dose reduction of chemotherapy. During chemotherapy he developed bilateral upper lobe pneumonia. He was treated with meropenem; amphotericin B was added after 48 hours due to lack of improvement. A computed tomography scan showed lesions suspect for fungal infection. After 2 days, the patient remained febrile, and we switched amphotericin B to voriconazole. The lesions resolved almost completely in 10 days, suggesting an infection with Aspergillus spp. We continued voriconazole as prophylaxis instead of itraconazole. 6 months later, while still on voriconazole, he again developed pneumonia, refractory to meropenem, and was treated with amphotericin B. Culture and histology findings of bronchoalveolar lavage fluid disclosed no microbial pathogens. Persisting fever led to resection of the infected lobe and discontinuation of chemotherapy. Microscopy of pulmonary tissue showed necrosis and broad hyphae suspect for zygomycetes. The dose of amphotericin B was doubled but nonetheless the infection disseminated. He suffered from convulsions, spondylodiscitis, and a thromboembolic mass in his left cardiac ventricle. Because of cardiac decompensation and high risk of embolism, the mass was resected. Cultures of the thrombus showed growth of Rhizopus microsporus (figure), susceptible in vitro to amphotericin B and posaconazole, an experimental triazole agent. The thrombus reappeared and posaconazole was added to the amphotericin B treatment. Unfortunately, the boy died of progressive cardiac obstruction. Voriconazole, a broad-spectrum triazole antifungal, is increasingly used as prophylaxis in patients with haematological malignancies. As this case illustrates, prolonged use of this agent may lead to an increase of infections with zygomycetes, all resistant to voriconazole.

Determining the analytical specificity of PCR-based assays for the diagnosis of IA: What is Aspergillus?

Abstract A wide array of PCR tests has been developed to aid the diagnosis of invasive aspergillosis (IA), providing technical diversity but limiting standardisation and acceptance. Methodological recommendations for testing blood samples using PCR exist, based on achieving optimal assay sensitivity to help exclude IA. Conversely, when testing more invasive samples (BAL, biopsy, CSF) emphasis is placed on confirming disease, so analytical specificity is paramount. This multicenter study examined the analytical specificity of PCR methods for detecting IA by blind testing a panel of DNA extracted from a various fungal species to explore the range of Aspergillus species that could be detected, but also potential cross reactivity with other fungal species. Positivity rates were calculated and regression analysis was performed to determine any associations between technical specifications and performance. The accuracy of Aspergillus genus specific assays was 71.8%, significantly greater (P < .0001) than assays specific for individual Aspergillus species (47.2%). For genus specific assays the most often missed species were A. lentulus (25.0%), A. versicolor (24.1%), A. terreus (16.1%), A. flavus (15.2%), A. niger (13.4%), and A. fumigatus (6.2%). There was a significant positive association between accuracy and using an Aspergillus genus PCR assay targeting the rRNA genes (P = .0011). Conversely, there was a significant association between rRNA PCR targets and false positivity (P = .0032). To conclude current Aspergillus PCR assays are better suited for detecting A. fumigatus, with inferior detection of most other Aspergillus species. The use of an Aspergillus genus specific PCR assay targeting the rRNA genes is preferential.

Rapid selection of carbapenem-resistant Pseudomonas aeruginosa by clinical concentrations of ertapenem

1] McDonnell G, Russell AD. Antiseptics and disinfectants: activity, action, and resistance. Clin Microbiol Rev 1999;12:147–79. 2] Alam MM, Ishino M, Kobayashi N. Analysis of genomic diversity and evolution of the low-level antiseptic resistance gene smr in Staphylococcus aureus. Microb Drug Resist 2003;9(Suppl. 1):S1–7. 3] Bjorland J, Sunde M, Waage S. Plasmid-borne smr gene causes resistance to quaternary ammonium compounds in bovine Staphylococcus aureus. J Clin Microbiol 2001;39:3999–4004. 4] Costa SS, Falcão C, Viveiros M, Machado D, Martins M, Melo-Cristino J, et al. Exploring the contribution of efflux on the resistance to fluoroquinolones in clinical isolates of Staphylococcus aureus. BMC Microbiol 2011;11:e241. 5] Leelaporn A, Firth N, Paulsen IT, Hettiaratchi A, Skurray RA. Multidrug resistance plasmid pSK108 from coagulase-negative staphylococci; relationships to Staphylococcus aureus qacC plasmids. Plasmid 1995;34:62–7.

Characterization of fluoroquinolone resistance in a clinical isolate of Pseudomonas stutzeri

common mechanisms of fluoroquinolone resistance in Gramnegative organisms, whole-cell DNA of isolate 13 was extracted and used as a template for PCR amplification using primers able to amplify the corresponding quinolone resistance determining regions (‘QRDRs’) of the gyrA and parC genes of P. stutzeri ATCC 17588 (as control) and of P. stutzeri 13, and subsequently sequenced. The primers used were: GyrA-PstF (5 0 -CAT GGG CGA ACT GGC CAA AG-3 0 ) and GyrA-PstR (5 0 -CTG CCG GAA TCT GCT CGG TG-3 0 ) for gyrA (expected amplicon of 473 bp); and ParC-PstF (5 0 -ATG AGC GAA TCC CTC GAC CTG-3 0 ) and ParC-PstR (5 0 -GGT TCG TCC AGG GTG CCG TCG A-3 0 ) for parC (expected amplicon of