Heidrun Peltroche-Llacsahuanga

Multicenter evaluation of the Candida albicans/Candida glabrata peptide nucleic acid fluorescent in situ hybridization method for simultaneous dual-color identification of C. albicans and C. glabrata directly from blood culture bottles

ABSTRACT We evaluated the performance of the Candida albicans/Candida glabrata peptide nucleic acid fluorescent in situ hybridization (PNA FISH) method, a rapid two-color assay for detection of C. albicans and C. glabrata, in a multicenter study. The assay is designed for use directly from positive blood culture bottles in a FISH format. Intact, fixed cells are labeled fluorescent green (C. albicans) or fluorescent red (C. glabrata) by rRNA hybridization of fluorophore-labeled PNA probes. Results are available <3 h after cultures signal positive. An evaluation of 197 routine blood culture bottles newly positive for yeast by Gram staining was performed at five hospitals. The sensitivities of detection for C. albicans, and C. glabrata were 98.7% (78/79) and 100% (37/37), respectively, and the specificity for both components of the assay was 100% (82/82). The assay was also evaluated with 70 fungal reference strains and was challenged in the BacT/ALERT microbiological detection system with spiked blood culture bottles. These results support the use of the assay for rapid, simultaneous identification of C. albicans and C. glabrata in positive blood culture bottles. This rapid assay may aid in the selection of initial antifungal drugs, leading to improved patient outcomes.

Direct Identification of Staphylococcus aureus from Positive Blood Culture Bottles

ABSTRACT Fluorescence in situ hybridization (FISH) using peptide nucleic acid (PNA) probes targeting Staphylococcus aureus 16S rRNA is a novel method for direct identification of S. aureus from positive blood culture bottles. The test (S. aureus PNA FISH) is performed on smears made directly from positive blood culture bottles with gram-positive cocci in clusters (GPCC) and provides results within 2.5 h. A blinded comparison of S. aureus PNA FISH with standard identification methods was performed in collaboration with eight clinical microbiology laboratories. A total of 564 routine blood culture bottles positive for GPCC recovered from both aerobic and anaerobic media from three different manufacturers (ESP, BACTEC, and BacT/Alert) were included in the study. The sensitivity and specificity of S. aureus PNA FISH were 100% (57 of 57) and 99.2% (116 of 117), respectively, with 174 GPCC-positive ESP blood culture bottles, 98.5% (67 of 68) and 98.5% (129 of 131), respectively, with 200 GPCC-positive BACTEC blood culture bottles, and 100% (74 of 74) and 99.1% (115 of 116), respectively, with 190 GPCC-positive BacT/Alert blood culture bottles. It is concluded that S. aureus PNA FISH performs well with commonly used continuously monitoring blood culture systems.

Recovery of Candida dubliniensis from sputum of cystic fibrosis patients

Summary.  Yeasts have been cultivated in a high percentage of sputum samples from patients with cystic fibrosis (CF). In cases of Candida dubliniensis a high recovery rate (15–25%) has so far only been reported from the oral cavity of HIV‐infected persons. When studying sputum samples of patients suffering from cystic fibrosis (n = 54) we could repeatedly observe C. dubliniensis in 6 patients (11.1%) by using a complex isolation procedure involving the use of Staib agar. This revealed that the prevalence rate was significantly higher than the estimated overall prevalence for all non‐HIV infected patients (0.8%) currently reported.

Investigation of infectious organisms causing pericoronitis of the mandibular third molar

PURPOSE The purpose of the study was to identify the most frequently encountered pyogenic organisms involved in pericoronitis to permit more targeted antibiotic therapy. PATIENTS AND METHODS Pericoronal pockets of mandibular third molars from 37 patients showing symptoms of acute, severe pericoronitis were sampled and subjected to microbiologic analysis, including primary evaluation by phase-contrast microscopy. To avoid overgrowth with faster-growing, less fastidious organisms, specimens were cultured on a wide variety of selective media (supporting growth of fastidious bacteria, protozoa, and fungi). RESULTS Microscopic examination indicated spirochetes in 55% and fusiform bacteria in 84% of the samples. A total of 441 microorganisms were isolated and identified from the 37 cultured samples. Besides obligate anaerobic bacteria, including various Actinomyces and Prevotella species, a predominantly facultative anaerobic microflora was cultivated, that is, Streptococcus milleri group (78% of samples), Stomatococcus mucilaginosus (71%), and Rothia dentocariosa (57%). CONCLUSION It was concluded that the Streptococci milleri group bacteria, well-known for their ability to cause suppurative infections, are most likely involved in the pathogenesis of acute severe pericoronitis of the lower third molar.

Comparison of Two Chromogenic Media for Selective Isolation of Vancomycin-Resistant Enterococci from Stool Specimens

ABSTRACT Two chromogenic media (Chromagar VRE and chromID VRE [C-ID]) performed equally well in the direct detection of vancomycin-resistant enterococci (VRE) in stool specimens after an overnight enrichment step and a 48-h incubation period, with a sensitivity of 98.2% (56/57) for both and specificities of 96.5% (195/202) and 97.5% (197/202), respectively. However, assigning discriminatory colony color was sometimes difficult, especially on C-ID. In order to facilitate simple species identification, biochemical key reactions were implemented.

Simultaneous measurement of biopolymer-mediated Mac-1 up-regulation and adherence of neutrophils: a novel flow cytometric approach for predicting initial inflammatory interaction with foreign materials

Implantation of any medical device normally causes an inflammatory cell interaction with the foreign material. In vitro cell activation of human neutrophils (Mac-1 upregulation) has been taken as one measure to assess the attributable risk of inflammation due to biopolymers before their clinical application. Mac-1 expression has generally been measured by flow cytometric assays, whereas quantification of neutrophil adhesion to the biopolymer surfaces has been performed by separate and time-consuming assays, e.g. microscopically by differential cell counting. However, due to an increasing number of surface-modified novel biopolymers entering clinical usage, effective testing of their inflammatory potential is now mandatory. To facilitate these analyses, we have developed a novel flow cytometric assay permitting simultaneous measurement of biopolymer-mediated neutrophil activation and adhesion. The biopolymers were used as beads (diameter 25+/-10 microm), and were demonstrated to be non-phagocytosable and non-fluorescent before being co-incubated with whole human blood (range of ratio granulocytes/beads from 5:1 to 1:1). Besides flow cytometric measurement of Mac-1 up-regulated neutrophils as fluorescing events, a fluorescence of the bead population indicates the adherence of activated neutrophils to the biopolymer surface.After establishing this assay, we evaluated it by comparing six different biopolymers. We observed high levels of Mac-1 expression (>70% of positive control) accompanied by increased adhesiveness (>60% of neutrophils) for polyurethane (PUR), polymethylmetacrylate (PMMA), and poly-DL-lactide (PDLLA) beads. Low Mac-1 expression levels (<10%) accompanied by a low percentage of adhering neutrophils (<10%) were observed for polyethylene (PE), polyisoprene (PI), and silicone (SI) beads.

Discriminative power of fatty acid methyl ester (FAME) analysis using the Microbial Identification System (MIS) for Candida (Torulopsis) glabrata and Saccharomyces cerevisiae

Candida (Torulopsis) glabrata is frequently isolated in cases of fungal infection and commonly shows acquired or innate fluconazole resistance. Saccharomyces cerevisiae, an emerging opportunistic yeast pathogen, causes serious systemic infections in immunocompromised, and vaginitis and superficial infections in immunocompetent patients. For both species reliable identification in the routine laboratory is mandatory, but species identification of strains, e.g. trehalose-negative C. glabrata, may be difficult. Therefore, gas-liquid chromatography (GLC) of whole cell fatty acid methyl ester (FAME) profiles, that is independent of assimilation profiles of strains and suitable for reliable and rapid identification of clinically important yeasts, was applied. However, frequent misidentification of C. glabrata as S. cerevisiae has been reported when using the Yeast Clinical Database of MIS. Accuracy of MIS identification may be strongly influenced by the amounts of cell mass analyzed. Therefore, the present study compared the MIS results of these two yeasts achieved with different cell masses. Primarily we optimized, especially with respect to cost-effectiveness, the recommended streaking technique yielding a maximal recovery of 90-130 mg of cell mass from one plate, enabling testing of poor growing strains of C. glabrata. For all C. glabrata strains tested (n = 10) the highest identification scores (SI [Similarity Index] range 0.525-0.963, median 0.832) were achieved with 30 to 45 mg of cell mass. Only 5 of 10 S. cerevisiae strains revealed good library comparisons (SI > or = 0.5) when using 30 mg of cell mass, whereas with 45 mg all strains but two revealed this SI-level. For S. cerevisiae a higher amount of cell mass processed (up to 90 mg) was correlated with better identification scores (SI range using 90 mg: 0.464-0.870, median, 0.737). Several passages prior to FAME analysis of C. glabrata strains on recommended media revealed narrowing of SI ranges, but differences in SI values were not statistically significant.

Methicillin-resistente Staphylococcus aureus (MRSA) – Klinische Implikationen

Summary. The isolation rate of MRSA from Staphylococcus aureus infections rose to 8.7 % in German hospitals between 1990 and 1995. Patients undergoing surgical treatment or physiotherapy showed a threefold increase in the risk of being colonized or infected with MRSA. Surgical wound infection is the most frequent among MRSA-induced infections (28 %). The main transmission path of MRSA inside a hospital is bacterial spread from one patient to another through contact with the hands of nursing staff. Therefore, the crucial strategy in avoiding the spread of bacteria is strict hygiene management through hand disinfection. The most widespread therapeutic regimen for simultaneous eradication of nasal colonization and treatment of infection on other body sites is mupirocin nasal ointment combined with parenteral vancomycin application. The non-indicated use of vancomycin, e. g., for perioperative prophylaxis or prevention of catheter-induced infections, should be avoided, especially after the appearance of vancomycin-intermediately sensitive S. aureus (VISA) strains that have been reported recently from Japan and the USA.Zusammenfassung. Die Isolationsrate von MRSA bei Staphylococcus-aureus-Infektionen ist in deutschen Krankenhäusern im Zeitraum 1990 bis 1995 auf 8,7 % angestiegen. Patienten mit chirurgischen Behandlungen und Physiotherapie wiesen ein dreifach erhöhtes Risiko für MRSA-Kolonisation bzw. -Infektionen auf. Die häufigste durch MRSA verursachte Infektion ist die chirurgische Wundinfektion (28 %). Der Hauptübertragungsweg von MRSA innerhalb eines Krankenhauses ist die Weiterverbreitung des Keims von einem Patienten zum anderen über Kontakt mit den Händen des behandelnden Personals. Daher ist die entscheidende Strategie zur Vermeidung von Übertragungen die Einhaltung einer strikten Händehygiene. Bei gleichzeitig nasaler Besiedlung eines Patienten und Infektion an anderer Körperstelle ist das derzeit verbreitetste Behandlungsregime die lokale Gabe von Mupirocin-Nasensalbe kombiniert mit parenteraler Vancomycin-Therapie. Der nicht indizierte Einsatz von Vancomycin, wie beispielsweise zur perioperativen Prophylaxe oder zur Prävention von Katheterinfektionen, sollte vermieden werden, insbesondere nach den kürzlich erschienenen ersten Berichten über das Auftreten Vancomycin-intermediär empfindlicher S.-aureus-Stämme (VISA) in Japan und den USA.

Rapid detection of Streptococcus agalactiae from swabs by peptide nucleic acid fluorescence in situ hybridization

The applicability of the PNA FISH (peptide nucleic acid fluorescence in situ hybridization) method for detection of Streptococcus agalactiae [group B streptococci (GBS)] from swab samples was evaluated. Three swab-sample-processing protocols with different time-to-result (TTR) values were compared: (i) direct smearing of fresh swabs onto microscope slides (n=153, TTR 2.5 h), (ii) further extraction and concentration of cells from these same swabs (n=153, TTR 2.7 h), and (iii) short-term LIM broth enrichment culture incubation (7 h, 37 degrees C) of fresh swabs (n=120, TTR 9.5 h). The sensitivity, specificity, positive predictive value and negative predictive value for GBS PNA FISH for sample processing procedures, with TTR values of 2.5, 2.7 and 9.5 h, were 68, 100, 100 and 95 %; 91, 100, 100 and 98 %; and 100, 100, 100 and 100 %; respectively. Improved test results were achieved by subjecting swabs to an extraction procedure or abbreviated LIM broth enrichment culture incubation prior to performing GBS PNA FISH.

Case Report. Pathohistological findings in a clinical case of disseminated infection with Fusarium oxysporum

Despite appropriate antimicrobial and antifungal therapy (amphotericin B), a disseminated infection with Fusarium oxysporum in a 75‐year‐old immunocompromised patient (acute myeloid leukaemia, minimal leucocyte count of 0.5 giga l−1) led rapidly to death. A similarly fatal course of an F. oxysporum infection has been reported in several cases. Fusarium oxysporum could be isolated shortly before death from blood cultures and from a swab taken from skin efflorescences. An autopsy revealed histopathologically typical fungal infiltrates in the mucosa of the pharynx, epiglottis, trachea, and oesophagus and in the parenchyma of the spleen, the lung and both kidneys. Because of the high risk of a fatal outcome of this infection, the clinician should aim at maximum diagnostic enforcement. We propose both analysis of blood cultures and immediate skin biopsy—with PAS‐staining—of suspicious dermal efflorescences for microscopic examination. The treatment of choice is discussed controversially but a beneficial effect has been reported from granulocyte transfusion, subcutaneous administration of GM‐CSF and concomitant treatment with amphotericin B.