Philipp P. Bosshard

Etiologic Diagnosis of Infective Endocarditis by Broad-Range Polymerase Chain Reaction: A 3-Year Experience

We analyzed surgically resected endocardial specimens from 49 patients by broad-range PCR. PCR results were compared with (1) results of previous blood cultures, (2) results of culture and Gram staining of resected specimens, and (3) clinical data (Duke criteria). Molecular analyses of resected specimens and previous blood cultures showed good overall agreement. However, in 18% of patients with sterile blood cultures, bacterial DNA was found in the resected materials. When data from patients with definite or rejected cases of infective endocarditis (IE) were included, the sensitivity, specificity, and positive and negative predictive values of broad-range PCR were 82.6%, 100%, 100%, and 76.5%, respectively, overall, and 94.1%, 100%, 100%, and 90%, for cases of native valve endocarditis. The sensitivity, specificity, and positive and negative predictive values of culture of resected specimens from patients with native valve endocarditis were 17.6%, 88.9%, 75%, and 36.4%. We recommend broad-range PCR of surgically resected endocardial material in cases of possible IE, in cases of suspected IE in which blood cultures are sterile, and in cases in which organisms grow in blood cultures but only Duke minor criteria are met. We propose to add molecular techniques to the pathologic criteria of the Duke classification scheme.

Ribosomal DNA Sequencing for Identification of Aerobic Gram-Positive Rods in the Clinical Laboratory (an 18-Month Evaluation)

ABSTRACT We have evaluated over a period of 18 months the use of 16S ribosomal DNA (rDNA) sequence analysis as a means of identifying aerobic gram-positive rods in the clinical laboratory. Two collections of strains were studied: (i) 37 clinical strains of gram-positive rods well identified by phenotypic tests, and (ii) 136 clinical isolates difficult to identify by standard microbiological investigations, i.e., identification at the species level was impossible. Results of molecular analyses were compared with those of conventional phenotypic identification procedures. Good overall agreement between phenotypic and molecular identification procedures was found for the collection of 37 clinical strains well identified by conventional means. For the 136 clinical strains which were difficult to identify by standard microbiological investigations, phenotypic characterization identified 71 of 136 (52.2%) isolates at the genus level; 65 of 136 (47.8%) isolates could not be discriminated at any taxonomic level. In comparison, 16S rDNA sequencing identified 89 of 136 (65.4%) isolates at the species level, 43 of 136 (31.6%) isolates at the genus level, and 4 of 136 (2.9%) isolates at the family level. We conclude that (i) rDNA sequencing is an effective means for the identification of aerobic gram-positive rods which are difficult to identify by conventional techniques, and (ii) molecular identification procedures are not required for isolates well identified by phenotypic investigations.

Quantitative Detection of Streptococcus pneumoniae in Nasopharyngeal Secretions by Real-Time PCR

ABSTRACT Streptococcus pneumoniae is an important cause of community-acquired pneumonia. However, in this setting the diagnostic sensitivity of blood cultures is below 30%. Since during such infections changes in the amounts of S. pneumoniae may also occur in the upper respiratory tract, quantification of these bacteria in nasopharnygeal secretions (NPSs) may offer a suitable diagnostic approach. Real-time PCR offers a sensitive, efficient, and routinely reproducible approach to quantification. Using primers and a fluorescent probe specific for the pneumolysin gene, we were able to detect DNA from serial dilutions of S. pneumoniae cells in which the quantities of DNA ranged from the amounts extracted from 1 to 106 cells. No difference was noted when the same DNA was mixed with DNA extracted from NPSs shown to be deficient ofS. pneumoniae following culture, suggesting that this bacterium can be detected and accurately quantitated in clinical samples. DNAs from Haemophilus influenzae,Moraxella catarrhalis, or alpha-hemolytic streptococci other than S. pneumoniae were not amplified or were only weakly amplified when there were ≥106 cells per reaction mixture. When the assay was applied to NPSs from patients with respiratory tract infections, the assay performed with a sensitivity of 100% and a specificity of up to 96% compared to the culture results. The numbers of S. pneumoniae organisms detected by real-time PCR correlated with the numbers detected by semiquantitative cultures. A real-time PCR that targeted the pneumolysin gene provided a sensitive and reliable means for routine rapid detection and quantification of S. pneumoniae present in NPSs. This assay may serve as a tool to study changes in the amounts of S. pneumoniae during lower respiratory tract infections.

Internal Transcribed Spacer Sequencing versus Biochemical Profiling for Identification of Medically Important Yeasts

ABSTRACT 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.

Turicibacter sanguinis gen. nov., sp. nov., a novel anaerobic, Gram-positive bacterium

An unknown, strictly anaerobic, Gram-positive, rod-shaped bacterium (strain MOL361T) was isolated from a blood culture of a febrile patient with acute appendicitis and characterized using phenotypic and molecular methods. Fatty acid analysis and biochemical examination indicated that the isolate most closely resembles members of the Gram-positive bacteria with low DNA G+C content. 16S rDNA sequencing revealed a relatively high overall similarity (97%) to an uncultured bacterium, but these two strains both exhibit low (<87%) 16S rDNA similarity to other bacteria. Phylogenetic analysis with different treeing methods showed that this strain forms a novel line of descent within the Gram-positive bacteria with low G+C content. Strain MOL361T is described as the type strain of a novel species within a new genus, Turicibacter sanguinis gen. nov., sp. nov.

Seasonal and spatial community dynamics in the meromictic Lake Cadagno.

Abstract. The seasonal and spatial variations in the community structure of bacterioplankton in the meromictic alpine Lake Cadagno were examined by temporal temperature gradient gel electrophoresis (TTGE) of PCR-amplified 16S rDNA fragments. Two different amplifications were performed, one specific for the domain Bacteria (Escherichia coli positions 8–536) and another specific for the family Chromatiaceae (E. coli positions 8–1005). The latter was followed by semi-nested reamplification with the bacterial primer set, allowing comparison of the two PCR approaches by TTGE. The TTGE patterns of samples from the chemocline and the anoxic monimolimnion were essentially identical, whereas the oxic mixolimnion displayed distinctively different banding patterns. For samples from the chemocline and the monimolimnion, dominant bands in the Bacteria-specific TTGE profiles comigrated with bands obtained by the semi-nested PCR approach specific for Chromatiaceae. This observation suggested that Chromatiaceae are in high abundance in the anoxic water layer. All dominant bands were excised and sequenced. Changes in the community structure, as indicated by changes in the TTGE profiles, were observed in samples taken at different times of the year. In the chemocline, Chromatium okenii was dominant in the summer months, whereas Amoebobacter purpureus populations dominated in autumn and winter. This change was confirmed by fluorescent in situ hybridization.

Identification of dermatophytes by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

In this study we evaluated the suitability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the identification of dermatophytes in diagnostic laboratories. First, a spectral database was built with 108 reference strains belonging to 18 species of the anamorphic genera Epidermophyton, Microsporum and Trichophyton. All strains were well characterized by morphological criteria and ITS sequencing (gold standard). The dendrogram resulting from MALDI-TOF mass spectra was almost identical with the phylogenetic tree based on ITS sequencing. Subsequently, MALDI-TOF MS SuperSpectra were created for the identification of Epidermophyton floccosum, Microsporium audouinii, M. canis, M. gypseum (teleomorph: Arthroderma gypseum), M. gypseum (teleomorph: A. incurvatum), M. persicolor, A. benhamiae (Tax. Entity 3 and Am-Eur. race), T. erinacei, T. interdigitale (anthropophilic and zoophilic populations), T. rubrum/T. violaceum, T. tonsurans and T. terrestre. Because T. rubrum and T. violaceum did not present enough mismatches, a SuperSpectrum covering both species was created, and differentiation between them was done by comparison of eight specific peptide masses. In the second part of this study, MALDI-TOF MS with the newly created SuperSpectra was tested using 141 clinical isolates representing nine species. Analyses were done with 3-day-old cultures. Results were compared to morphological identification and ITS sequencing; 135/141 (95.8%) strains were correctly identified by MALDI-TOF MS compared to 128/141 (90.8%) by morphology. Therefore, MALDI-TOF MS has proven to be a useful and rapid identification method for dermatophytes.

Serological Response to Treatment of Syphilis According to Disease Stage and HIV Status

The serological response to treatment was studied in 264 syphilis patients; it was influenced by syphilis stage but not by human immunodeficiency virus infection and reinfection. Some of the recommendations of current guidelines are critically discussed, and amendments are proposed.

Evaluation of the Colorimetric VITEK 2 Card for Identification of Gram-Negative Nonfermentative Rods: Comparison to 16S rRNA Gene Sequencing

ABSTRACT Ninety strains of a collection of well-identified clinical isolates of gram-negative nonfermentative rods collected over a period of 5 years were evaluated using the new colorimetric VITEK 2 card. The VITEK 2 colorimetric system identified 53 (59%) of the isolates to the species level and 9 (10%) to the genus level; 28 (31%) isolates were misidentified. An algorithm combining the colorimetric VITEK 2 card and 16S rRNA gene sequencing for adequate identification of gram-negative nonfermentative rods was developed. According to this algorithm, any identification by the colorimetric VITEK 2 card other than Achromobacter xylosoxidans, Acinetobacter sp., Burkholderia cepacia complex, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia should be subjected to 16S rRNA gene sequencing when accurate identification of nonfermentative rods is of concern.

Paenibacillus turicensis sp. nov., a novel bacterium harbouring heterogeneities between 16S rRNA genes

A Gram-positive, facultatively anaerobic, rod-shaped, endospore-forming bacterium (strain MOL722(T)) was characterized using phenotypic and molecular methods. Fatty acid analysis and biochemical examination indicated that the isolate belongs to the Gram-positive bacteria with low DNA G+C content, probably to the genus Paenibacillus. Direct sequencing of the 16S rRNA gene showed ambiguities, suggesting heterogeneity. Cloned 16S rDNA yielded seven different sequences varying at 15 positions, with one being an insertion. The isolate shares 98.5% sequence similarity with Paenibacillus sp. P15-9, but less than 94% similarity with other paenibacilli and bacilli. Phylogenetic analysis with different treeing methods revealed that strain MOL722(T), together with Paenibacillus sp. P15-9, forms a novel monophyletic clade within the genus Paenibacillus. Based on phenotypic and phylogenetic evidence, it is proposed that the unknown bacterium is classified as the novel species Paenibacillus turicensis sp. nov., the type strain of which is strain MOL722(T) (= DSM 14349(T) = NCCB 100011(T)).