Multicenter Assessment of Gram Stain Error Rates.

Gram stains remain the cornerstone of diagnostic testing in the microbiology laboratory for the guidance of empirical treatment prior to availability of culture results. Incorrectly interpreted Gram stains may adversely impact patient care, and yet there are no comprehensive studies that have evaluated the reliability of the technique and there are no established standards for performance. In this study, clinical microbiology laboratories at four major tertiary medical care centers evaluated Gram stain error rates across all nonblood specimen types by using standardized criteria. The study focused on several factors that primarily contribute to errors in the process, including poor specimen quality, smear preparation, and interpretation of the smears. The number of specimens during the evaluation period ranged from 976 to 1,864 specimens per site, and there were a total of 6,115 specimens. Gram stain results were discrepant from culture for 5% of all specimens. Fifty-eight percent of discrepant results were specimens with no organisms reported on Gram stain but significant growth on culture, while 42% of discrepant results had reported organisms on Gram stain that were not recovered in culture. Upon review of available slides, 24% (63/263) of discrepant results were due to reader error, which varied significantly based on site (9% to 45%). The Gram stain error rate also varied between sites, ranging from 0.4% to 2.7%. The data demonstrate a significant variability between laboratories in Gram stain performance and affirm the need for ongoing quality assessment by laboratories. Standardized monitoring of Gram stains is an essential quality control tool for laboratories and is necessary for the establishment of a quality benchmark across laboratories.

Identification of Gram-Negative Bacteria and Genetic Resistance Determinants from Positive Blood Culture Broths by Use of the Verigene Gram-Negative Blood Culture Multiplex Microarray-Based Molecular Assay.

Bloodstream infection is a serious condition associated with significant morbidity and mortality. The outcome of these infections can be positively affected by the early implementation of effective antibiotic therapy based on the identification of the infecting organism and genetic markers associated with antibiotic resistance. In this study, we evaluated the microarray-based Verigene Gram-negative blood culture (BC-GN) assay in the identification of 8 genus or species targets and 6 genetic resistance determinants in positive blood culture broths. A total of 1,847 blood cultures containing Gram-negative organisms were tested using the BC-GN assay. This comprised 729 prospective fresh, 781 prospective or retrospective frozen, and 337 simulated cultures representing 7 types of aerobic culture media. The results were compared to those with standard bacterial culture and biochemical identification with nucleic acid sequence confirmation of the resistance determinants. Among monomicrobial cultures, the positive percent agreement (PPA) of the BC-GN assay with the reference method was as follows; Escherichia coli, 100%; Klebsiella pneumoniae, 92.9%; Klebsiella oxytoca, 95.5%; Enterobacter spp., 99.3%; Pseudomonas aeruginosa, 98.9%; Proteus spp., 100%; Acinetobacter spp., 98.4%; and Citrobacter spp., 100%. All organism identification targets demonstrated >99.5% negative percent agreement (NPA) with the reference method. Of note, 25/26 cultures containing K. pneumoniae that were reported as not detected by the BC-GN assay were subsequently identified as Klebsiella variicola. The PPA for identification of resistance determinants was as follows; blaCTX-M, 98.9%; blaKPC, 100%; blaNDM, 96.2%; blaOXA, 94.3%; blaVIM, 100%; and blaIMP, 100%. All resistance determinant targets demonstrated >99.9% NPA. Among polymicrobial specimens, the BC-GN assay correctly identified at least one organism in 95.4% of the broths and correctly identified all organisms present in 54.5% of the broths. The sample-to-result processing and automated reading of the detection microarray results enables results within 2 h of culture positivity.

Multicenter evaluation of the Verigene Clostridium difficile nucleic acid assay.

The Verigene Clostridium difficile Nucleic Acid test (Verigene CDF test) (Nanosphere, Northbrook, IL) is a multiplex qualitative PCR assay that utilizes a nanoparticle-based array hybridization method to detect C. difficile tcdA and tcdB in fecal specimens. In addition, the assay detects binary toxin gene sequences and the single base pair deletion at nucleotide 117 (Δ 117) in tcdC to provide a presumptive identification of the epidemic strain 027/NAP1/BI (referred to here as ribotype 027). This study compared the Verigene CDF test with anaerobic direct and enriched toxigenic culture on stool specimens from symptomatic patients among five geographically diverse laboratories within the United States. The Verigene CDF test was performed according to the manufacturer's instructions, and the reference methods performed by a central laboratory included direct culture onto cycloserine cefoxitin fructose agar (CCFA) and enriched culture using cycloserine cefoxitin mannitol broth with taurocholate and lysozyme. Recovered isolates were identified as C. difficile using gas liquid chromatography and were tested for toxin using a cell culture cytotoxicity neutralization assay. Strains belonging to ribotype 027 were determined by PCR ribotyping and bidirectional sequencing for Δ 117 in tcdC. A total of 1,875 specimens were evaluable. Of these, 275 specimens (14.7%) were culture positive by either direct or enriched culture methods. Compared to direct culture alone, the overall sensitivity, specificity, positive predictive value, and negative predictive value for the Verigene CDF test were 98.7%, 87.5%, 42%, and 99.9%, respectively. Compared to combined direct and enriched culture results, the sensitivity, specificity, positive predictive value, and negative predictive values of the Verigene CDF test were 90.9%, 92.5%, 67.6%, and 98.3%, respectively. Of the 250 concordantly culture-positive specimens, 59 (23.6%) were flagged as "hypervirulent"; 53 were confirmed as ribotype 027, and all 59 possessed Δ 117 in tcdC. Time to results was approximately 2.5 h per specimen. The Verigene CDF test is a novel nucleic acid microarray that reliably detects both C. difficile toxins A and B in unformed stool specimens and appears to adequately identify ribotype 027 isolates.

Multiplex identification of gram-positive bacteria and resistance determinants directly from positive blood culture broths: evaluation of an automated microarray-based nucleic acid test.

BACKGROUND: A multicenter study was conducted to evaluate the diagnostic accuracy (sensitivity and specificity) of the Verigene Gram-Positive Blood Culture Test (BC-GP) test to identify 12 Gram-positive bacterial gene targets and three genetic resistance determinants directly from positive blood culture broths containing Gram-positive bacteria. METHODS AND FINDINGS: 1,252 blood cultures containing Gram-positive bacteria were prospectively collected and tested at five clinical centers between April, 2011 and January, 2012. An additional 387 contrived blood cultures containing uncommon targets (e.g., Listeria spp., S. lugdunensis, vanB-positive Enterococci) were included to fully evaluate the performance of the BC-GP test. Sensitivity and specificity for the 12 specific genus or species targets identified by the BC-GP test ranged from 92.6%-100% and 95.4%-100%, respectively. Identification of the mecA gene in 599 cultures containing S. aureus or S. epidermidis was 98.6% sensitive and 94.3% specific compared to cefoxitin disk method. Identification of the vanA gene in 81 cultures containing Enterococcus faecium or E. faecalis was 100% sensitive and specific. Approximately 7.5% (87/1,157) of single-organism cultures contained Gram-positive bacteria not present on the BC-GP test panel. In 95 cultures containing multiple organisms the BC-GP test was in 71.6% (68/95) agreement with culture results. Retrospective analysis of 107 separate blood cultures demonstrated that identification of methicillin resistant S. aureus and vancomycin resistant Enterococcus spp. was completed an average of 41.8 to 42.4 h earlier using the BC-GP test compared to routine culture methods. The BC-GP test was unable to assign mecA to a specific organism in cultures containing more than one Staphylococcus isolate and does not identify common blood culture contaminants such as Micrococcus, Corynebacterium, and Bacillus. CONCLUSIONS: The BC-GP test is a multiplex test capable of detecting most leading causes of Gram-positive bacterial blood stream infections as well as genetic markers of methicillin and vancomycin resistance directly from positive blood cultures.

Pilot trial of genetically modified, attenuated Salmonella expressing the E. coli cytosine deaminase gene in refractory cancer patients.

We performed a pilot trial in refractory cancer patients to investigate the feasibility of intratumoral injection of TAPET-CD, an attenuated Salmonella bacterium expressing the E. coli cytosine deaminase gene. A total of three patients received three dose levels of TAPET-CD (3 x 10(6)-3 x 10(7) CFU/m(2)) via intratumoral injection once every 28 days as long as progression of disease or intolerable toxicity was not observed. From days 4 to 14 of each 28 day cycle, patients also received 5-fluorocytosine (5-FC) at a dose of 100 mg/kg/day p.o. divided three times daily. Six cycles of treatment were administered. No significant adverse events clearly attributable to TAPET-CD were demonstrated. Two patients had intratumor evidence of bacterial colonization with TAPET-CD, which persisted for at least 15 days after initial injection. Conversion of 5-FC to 5-fluorouracil (5-FU) as a result of cytosine deaminase expression was demonstrated in these two patients. The tumor to plasma ratio of 5-FU for these two colonized patients was 3.0, demonstrating significantly increased levels of 5-FU at the site of TAPET-CD colonization and insignificant systemic spread of the bacteria. In contrast, the tumor to plasma ratio of 5-FU of the patient who did not show colonization of TAPET-CD was less than 1.0. These results support the principle that a Salmonella bacterium can be utilized as a delivery vehicle of the cytosine deaminase gene to malignant tissue and that the delivered gene is functional (i.e. able to convert 5-FC to 5-FU) at doses at or below 3 x 10(7) CFU/m(2).