Rapid Detection of Candida albicans in Clinical Samples by DNA Amplification of Common Regions from C. albicans-Secreted Aspartic Proteinase Genes

Laboratory diagnosis based on genomic amplification methods such as PCR may provide an alternative and more sensitive method than conventional culture for the early detection of deep-seated candidiasis, an increasing cause of morbidity and mortality among immunocompromised patients. A novel method of DNA extraction from clinical samples based on treatment with proteinase K and isolation of DNA on a silica membrane was developed. The targets used for DNA amplification were the Candida albicans-secreted aspartic proteinase (SAP) genes, a multiple-gene family of at least seven members in C. albicans. A single pair of primers was designed in order to detect six of these SAP genes and, subsequently, to increase the sensitivity of the test. Detection of the PCR product by enzyme-linked immunosorbent assay was found to be as sensitive as Southern blotting with an SAP-labeled probe. The sensitivity of the assay was 1 cell/ml from serially diluted Candida cultures and 1 to 4 cells/ml from seeded blood specimens. The sensitivity and specificity of the present assay were tested in a retrospective study performed blindly with 156 clinical samples and were 100 and 98%, respectively, compared with the results of culture. For the subset of blood culture samples (n = 124), the sensitivity and the specificity were 100%. The two false-positive PCR samples came from patients treated with azole antifungal agents, indicating that PCR was probably able to detect damaged organisms that could not be recovered by culture.     

Cobas-Bact system for identification of members of the family Enterobacteriaceae in 4 h 20 min

The Cobas-Bact (Roche Diagnostics, Basel, Switzerland) new rotor for the identification (ID) in 4 h 20 min of 33 members of the family Enterobacteriaceae to genus and species level was evaluated by testing 444 strains of which 398 belonged to common species and 46 belonged to rare species of Enterobacteriaceae. Each strain was identified by the API 20E system (Analytab Products, Plainview, N.Y.), and additional discriminating tests were set up if necessary. Only first-choice ID were considered in this study and were classified either as high-confidence ID (normalized likelihood, greater than or equal to 80%) or as low-confidence ID (normalized likelihood, less than 80%) requiring additional tests for confirmation. The data were analyzed by two versions of Cobas-Bact software. With the first version of the software (SW8446), the overall accuracy of Cobas-Bact was 95.5% (424 correct ID of 444). When restricted to high-confidence ID it rose to 99.4% (350 of 352) for the common species and 96.9% (31 of 32) for the rare species. Only three strains of the high-confidence group were misidentified. Sixty ID were considered unacceptable because of their low confidence. Using the first software version (SW8446) they represented 12% (46 of 398) of the common species (17 typical strains, 10 Shigella species, 10 inactive Escherichia coli strains, and 9 rare biotypes) and 30% (14 of 46) of the rare species. The same data analyzed by the new version (SW8524) of the Cobas-Bact software resulted in an overall accuracy of 93.9% (417 correct ID). The number of high-confidence ID rose to 401, of which 392 (97.7%) were accurate. The decrease in low-confidence ID (43 versus 60) was mainly due to the Shigella species. In conclusion the accuracy of Cobas-Bact identification system was very good when restricted to high-confidence ID. The Cobas-Bact performance for rare species ID was poorer, but the small number of strains tested does not allow definitive conclusions.     

Cytogenetic and comparative genomic hybridization findings in four cases of breast cancer after neoadjuvant chemotherapy

To assess a potential common pattern of genetic alterations in chemotherapy-resistant tumors we analyzed four tumors from breast cancer patients (patients 1-4) after neoadjuvant chemotherapy, by comparative genome hybridization (CGH) and conventional chromosome banding analysis. All patients showed structural aberrations involving chromosomes 1, 5, 11, 16, and 17. In CGH analysis, the patients showed typical imbalances for ductal breast cancer: gains of 1q (3 patients), 5q (2 patients), 8q (3 patients), and X (4 patients) and losses of 1p33 approximately p36 (3 patients), 16q (3 patients), 17p (3 patients), 19 (4 patients), and 22q (4 patients). Other recurrent imbalances of atypical pattern for ductal breast cancer were gain of 4q21 approximately q32 (2 patients), 20q21 approximately q22 (2 patients), and 21 (2 patients) and loss of 20p (3 patients). Three patients showed involvement of several regions bearing genes of drug resistance (MDR1 [HUGO symbol: ABCB1], BCRP [HUGO symbol: ABCG2], MRP1 [HUGO symbol: ABCC1], RFC1); the fourth patient displayed an amplification in the region of MYC (alias c-myc), thus providing--at the level of the light microscope--an explanatory background for the ability of their tumors to survive anthracycline-, taxane- and cyclophosphamide-based chemotherapy. Conventional cytogenetic analysis and CGH displayed highly coincidental findings in the tumors of four patients after neoadjuvant chemotherapy for breast cancer.     

Relationship between neutrophil-mediated oxidative injury during acute experimental pyelonephritis and chronic renal scarring.

Previous experiments with rats have suggested that pyelonephritic scarring after acute ascending Escherichia coli pyelonephritis partly results from excessive polymorphonuclear leukocyte (PMN) infiltration and activation in the kidney parenchyma. We have studied the role of PMN oxidative metabolism in generating tissue injury during acute pyelonephritis. Rats with acute pyelonephritis were treated with dapsone (25 mg/kg twice daily for 3 days), a compound known to prevent PMN oxidant damage. In vitro, levels of dapsone easily achieved in vivo inhibited myeloperoxidase (MPO)-mediated reactions involving the oxidation of halides to reactive cytotoxic hypohalites (such as MPO-mediated iodination and luminol-enhanced chemiluminescence). In contrast, dapsone had no effect on superoxide production, lysosomal enzyme release, or bacterial killing by activated PMN. In vivo, dapsone treatment had no significant effect on acute pyelonephritis with respect to (i) bacterial counts, (ii) inflammatory swelling, and (iii) PMN infiltration. However, dapsone-treated animals sacrificed 2 months after acute pyelonephritis had a 65% reduction of renal scars when compared with controls. Since dapsone had no antibacterial effect, this protection is compatible with the hypothesis that dapsone prevented oxidant-generated tissue injury due to the extracellular release of the MPO system by activated PMN during acute suppurative pyelonephritis.     

Cluster of oral atypical Candida albicans isolates in a group of human immunodeficiency virus-positive drug users.

Twenty-one chlamydospore-forming and germ tube-positive Candida albicans clinical isolates from 15 human immunodeficiency virus (HIV)-positive and 3 HIV-negative patients were examined by two different genetic methods. Multilocus enzyme electrophoresis and hybridization with the C. albicans-specific Ca3 probe showed that such isolates can be split into two genetically distinct groups that can be clearly distinguished. One group mainly contained strains with atypical sugar assimilation patterns and could be distinguished from the other group by the absence of intracellular beta-glucosidase activity. All 13 strains belonging to this group were isolated from the oral cavities of asymptomatic HIV-positive drug users and may be less pathogenic than the eight strains from the other group isolated either from HIV-positive patients with oropharyngeal candidiasis or from HIV-negative patients with invasive candidiasis.     

Evaluation of a rapid latex agglutination test (Directigen®) for the direct detection of group A streptococci from throat swabs

Summary A rapid group A beta-hemolytic streptococci (GAS) antigen detection test using latex agglutination (Directigen^®, Becton Dickinson) was compared with a conventional culture method for the direct detection of GAS from throat swabs. One throat specimen was collected from each of 229 patients. After standard inoculation onto sheep blood agar plates, all swabs were tested for GAS antigen. Of the 229 specimens tested, 46 (20%) were GAS-positive by culture. Direct latex agglutination agreed with culture in 222 of them (97%). The sensitivity of the test was 93% (43/46), the specificity 98% (179/183), the positive predictive value 91%, and the negative predictive value 98%. The detection of GAS antigen by coagglutination (Phadebact^®, Pharmacia) was also carried out. 81% (35/43) of the cultures and Directigen^®-positive specimens gave a positive coagglutination for GAS. 100% (179/179) of the cultures and Directigen^®-negative specimens were also negative by the coagglutination test. We conclude that the Directigen^® rapid latex agglutination test for the direct detection of GAS from throat swabs compares favorably with culture and has the advantage of providing same-day results.

---

Zuverlässigkeit eines Schnelltests mittels Latexagglutination (Directigen^®) zum direkten Nachweis von Streptokokken der Gruppe A (SGA) im Rachenabstrich

Zusammenfassung In dieser Studie haben wir die Zuverlässigkeit eines Schnelltests mittels Latexagglutination (Directigen^®, Becton Dickinson) zum direkten Nachweis von -hämolysierenden Streptokokken der Gruppe A (SGA) im Rachenabstrich mit der konventionellen Kulturmethode verglichen. Bei 229 zufällig ausgewählten Patienten wurde jeweils ein Rachenabstrich entnommen. Alle Rachenabstriche wurden zuerst auf eine Schafblutagarplatte ausgestrichen und mittels Latex-Schnellagglutination auf das Vorhandensein von Antigenen von SGA getestet. 46 (20%) der 229 getesteten Rachenabstriche enthielten in der Kultur SGA. Das Resultat der direkten Latexagglutination stimmte in 222 Fällen (97%) mit der Kultur überein. Die Sensitivität des Tests betrug 93% (43/46), die Spezifität 98% (179/183), die positive Voraussagekraft des Tests 91%, die negative 98%. Wir haben zudem den Nachweis von SGA-Antigenen mittels Koagglutination (Phadebact^®, Pharmacia) durchgeführt. 81% (35/43) der Rachenabstriche, welche in der Kultur und im Directigen^®-Test positiv waren für SGA-Antigene, waren dies auch im Koagglutinationstest. 100% (179/179) der Rachenabstriche, welche keine SGA-Antigene in der Kultur und mittels Directigen^® aufgewiesen haben, waren mittels Koagglutinationstest ebenfalls negativ. Der Directigen^®-Schnelltest mittels Latexagglutination zum direkten Nachweis von SGA in Rachenabstrichen ist in seiner Zuverlässigkeit mit der Kulturmethode vergleichbar. Dieser Schnelltest hat den Vorteil, daß das Ergebnis am selben Tag vorliegt.

     

Comparison of the three-level and the five-level versions of the EQ-5D

The European Journal of Health Economics - EQ-5D is a generic instrument to measure health-related quality of life. In 2009, a new version, EQ-5D-5L, was introduced as an attempt to reduce ceiling...