In this study, we established an in-house database of yeast internal transcribed spacer (ITS) sequences. This database includes medically important as well as colonizing yeasts that frequently occur in the diagnostic laboratory. In a prospective study, we compared molecular identification with phenotypic identification by using the ID32C system (bioMérieux) for yeast strains that could not be identified by a combination of CHROMagar Candida and morphology on rice agar. In total, 113 yeast strains were included in the study. By sequence analysis, 98% of all strains were identified correctly to the species level. With the ID32C, 87% of all strains were identified correctly to the species or genus level, 7% of the isolates could not be identified, and 6% of the isolates were misidentified, most of them as Candida rugosa or Candida utilis. For a diagnostic algorithm, we suggest a three-step procedure which integrates morphological criteria, biochemical investigation, and sequence analysis of the ITS region.Yeast infections are increasing due to the growing number of immunocompromised and severely ill patients (5, 9). In addition, widespread use of antibiotics and invasive procedures facilitate infections with yeasts (18). Although Candida albicans is still the most frequently encountered yeast species, others have gained increasing importance in the last few years (1). Some species, such as Candida krusei (resistance to fluconazole) or Trichosporon sp. (reduced susceptibility to amphotericin B), may show inherent resistance to antimycotics (13). Rapid and accurate identification is thus essential for proper treatment. Various identification methods have been proposed in the past, including morphology, physiological properties, nucleic acid amplification, restriction fragment length polymorphism analysis, and sequencing (2).For molecular identification, we have chosen sequence analysis since this procedure is simple and can be fully automated. In addition, interpretation of nucleic acid sequences is straightforward and does not depend on too much expertise compared to morphological analyses. As target, we have chosen the internal transcribed spacer (ITS) region, which is located between the highly conserved genes coding for 18S and 28S rRNA. The ITS encompasses the two noncoding regions ITS1 and ITS2, which are separated by the highly conserved 5.8S rRNA gene (20). The ITS1 and ITS2 regions are more variable than the adjacent rRNA gene sequences and thus promise a better separation of closely related species. As the inspection of yeast ITS sequences which are available in the public database GenBank (NCBI) suggested that some entries are incorrect and because certain medically relevant species are not included, we decided to establish an in-house database. Since the number of known yeast species is enormous, we restricted our database to species occurring in the medical diagnostic laboratory.In this study, we compared sequence-based identification with conventional identification. Based on these results, we establish...

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The implementation of internal transcribed spacer (ITS) sequencing for routine identification of molds in the diagnostic mycology laboratory was analyzed in a 5-year study. All mold isolates (n ‫؍‬ 6,900) recovered in our laboratory from 2005 to 2009 were included in this study. According to a defined work flow, which in addition to troublesome phenotypic identification takes clinical relevance into account, 233 isolates were subjected to ITS sequence analysis. Sequencing resulted in successful identification for 78.6% of the analyzed isolates (57.1% at species level, 21.5% at genus level). In comparison, extended in-depth phenotypic characterization of the isolates subjected to sequencing achieved taxonomic assignment for 47.6% of these, with a mere 13.3% at species level. Optimization of DNA extraction further improved the efficacy of molecular identification. This study is the first of its kind to testify to the systematic implementation of sequence-based identification procedures in the routine workup of mold isolates in the diagnostic mycology laboratory.

c Xpert-MTB/Rif is one of the most frequently used molecular screening tests for multidrug-resistant tuberculosis worldwide. We report false-negative assay results in the presence of rpoB Leu533Pro, which is associated with low-level phenotypic rifampin resistance. Accurate and timely confirmation of rifampin susceptibility results obtained with Xpert-MTB/Rif is imperative.T he accurate diagnosis of drug-resistant and multidrug-resistant (MDR) tuberculosis (TB) is imperative to initiate adequate treatment, to avoid transmission of the disease, and to prevent the development of further drug resistance. Because of its worldwide rollout and rapid implementation, the Xpert-MTB/Rif assay (Cepheid) has become one of the most frequently used molecular screening tests for TB and MDR TB in both resource-poor and resource-rich countries (1). Recently, a 67-year-old Swissborn male patient was admitted to a secondary-care hospital in Switzerland with clinical and radiologic suspicion of pulmonary TB. Rapid testing by Xpert-MTB/Rif showed the presence of Mycobacterium tuberculosis complex in two sputum samples (Table 1; samples 1 and 2). In addition, these specimens showed indeterminate and definite rifampin (RMP) resistance, respectively (Table 1, samples 1 and 2). Since the patient had no history of TB and was from Switzerland (a low MDR TB incidence setting), based on previous reports of false RMP resistance assay results (2, 3), the clinician doubted the accuracy of the Xpert-MTB/ Rif RMP results. Therefore, an additional four sputum samples were submitted to the Swiss National Reference Center for Mycobacteria for rapid confirmation before initiation of MDR TB therapy. Further patient testing revealed HIV positivity.The four additional sputum specimens (Table 1, samples 3 to 6) were acid-fast smear positive by the Ziehl-Neelsen method (4), and Xpert-MTB/Rif testing detected the presence of M. tuberculosis complex. Surprisingly, all of the samples were scored RMP susceptible by the molecular assay (Table 1). In order to resolve the discrepant Xpert-MTB/Rif results and to rapidly confirm or rule out MDR TB, direct rpoB sequencing of the 81-bp core region and additional molecular screening for isoniazid (INH) resistance were performed as described previously (3, 5) and revealed rpoB Leu(CTG)533Pro(CCG) and katG Ser(AGC)315Thr(ACC) mutations, respectively, in all four specimens. Growth detection and quantitative phenotypic drug susceptibility testing (DST) for firstand second-line antituberculosis drugs with the MGIT 960 system and EpiCenter software with the TB eXiST module (Becton, Dickinson Microbiology Systems, Sparks, MD) were performed as described earlier (6). Quantitative DST identified resistance to RMP at 0.5 g/ml and susceptibility at 1.0, 4.0, and 20 g/ml, and resistance to rifabutin at 0.1 g/ml and susceptibility at 0.4 and 2.0 g/ml. DST for INH showed resistance at 0.1 and 1.0 g/ml and intermediate resistance at 3.0 and 10.0 g/ml. No drug resistance was identified by conventional DST for other first-and second...

We report four epidemiologically unrelated cases of KPC-carrying Klebsiella pneumoniae identified in Switzerland between May 2009 and November 2010. Three cases were transferred from Italy (two KPC-3, one KPC-2) and one from Greece (KPC-2). Resistance to colistin and doxycycline emerged in one KPC-3carrying K. pneumoniae strain during therapy. These results demonstrate ongoing dissemination of KPC throughout Europe. Rapid and reliable identification of KPC and implementation of control measures is essential to limit spread.

Hepatitis E virus genotype 3 (HEV-3) is the causal pathogen of chronic hepatitis E. We report here the full-length genome sequence of an HEV-3 strain, isolated from a kidney transplant recipient in Switzerland (SW/16-0282). This HEV-3 strain showed less than 88% homology compared to known strains, suggesting a new HEV-3 strain.

BackgroundDetection of HIV-1 p24 antigen permits early identification of primary HIV infection and timely intervention to limit further spread of the infection. Principally, HIV screening should equally detect all viral variants, but reagents for a standardised test evaluation are limited. Therefore, we aimed to create an inexhaustible panel of diverse HIV-1 p24 antigens.MethodsWe generated a panel of 43 recombinantly expressed virus-like particles (VLPs), containing the structural Gag proteins of HIV-1 subtypes A-H and circulating recombinant forms (CRF) CRF01_AE, CRF02_AG, CRF12_BF, CRF20_BG and group O. Eleven 4th generation antigen/antibody tests and five antigen-only tests were evaluated for their ability to detect VLPs diluted in human plasma to p24 concentrations equivalent to 50, 10 and 2 IU/ml of the WHO p24 standard. Three tests were also evaluated for their ability to detect p24 after heat-denaturation for immune-complex disruption, a pre-requisite for ultrasensitive p24 detection.ResultsOur VLP panel exhibited an average intra-clade p24 diversity of 6.7%. Among the 4th generation tests, the Abbott Architect and Siemens Enzygnost Integral 4 had the highest sensitivity of 97.7% and 93%, respectively. Alere Determine Combo and BioRad Access were least sensitive with 10.1% and 40.3%, respectively. Antigen-only tests were slightly more sensitive than combination tests. Almost all tests detected the WHO HIV-1 p24 standard at a concentration of 2 IU/ml, but their ability to detect this input for different subtypes varied greatly. Heat-treatment lowered overall detectability of HIV-1 p24 in two of the three tests, but only few VLPs had a more than 3-fold loss in p24 detection.ConclusionsThe HIV-1 Gag subtype panel has a broad diversity and proved useful for a standardised evaluation of the detection limit and breadth of subtype detection of p24 antigen-detecting tests. Several tests exhibited problems, particularly with non-B subtypes.

SummaryPhaeohyphomycosis caused by Cladophialophora bantiana was diagnosed in a 5-month-old snow leopard with spastic paralysis of the hind legs and inability to defaecate or urinate. At post-mortem examination, a greenish soft mass resembling an abscess was found on one side of the epidural space at the fourth lumbar vertebral body. Histological examination revealed a purulent meningitis with myelomalacia. Dematiaceous fungal hyphae, present within the inflammatory infiltrate, were identified as C. bantiana by culture and sequence analysis of the 18S ribosomal RNA gene. This neurotropic fungus rarely affects organs other than the brain in human beings and cats, and has been reported only occasionally in Europe. The case described suggests that phaeohyphomycosis due to C. bantiana infection may be recognized more frequently in the future and the possible involvement of organs other than the brain should be borne in mind.q