Scott A. Cunningham

Comparison of Bruker Biotyper Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometer to BD Phoenix Automated Microbiology System for Identification of Gram-Negative Bacilli

ABSTRACT We compared the BD Phoenix automated microbiology system to the Bruker Biotyper (version 2.0) matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry (MS) system for identification of Gram-negative bacilli, using biochemical testing and/or genetic sequencing to resolve discordant results. The BD Phoenix correctly identified 363 (83%) and 330 (75%) isolates to the genus and species level, respectively. The Bruker Biotyper correctly identified 408 (93%) and 360 (82%) isolates to the genus and species level, respectively. The 440 isolates were grouped into common (308) and infrequent (132) isolates in the clinical laboratory. For the 308 common isolates, the BD Phoenix and Bruker Biotyper correctly identified 294 (95%) and 296 (96%) of the isolates to the genus level, respectively. For species identification, the BD Phoenix and Bruker Biotyper correctly identified 93% of the common isolates (285 and 286, respectively). In contrast, for the 132 infrequent isolates, the Bruker Biotyper correctly identified 112 (85%) and 74 (56%) isolates to the genus and species level, respectively, compared to the BD Phoenix, which identified only 69 (52%) and 45 (34%) isolates to the genus and species level, respectively. Statistically, the Bruker Biotyper overall outperformed the BD Phoenix for identification of Gram-negative bacilli to the genus (P < 0.0001) and species (P = 0.0005) level in this sample set. When isolates were categorized as common or infrequent isolates, there was statistically no difference between the instruments for identification of common Gram-negative bacilli (P > 0.05). However, the Bruker Biotyper outperformed the BD Phoenix for identification of infrequently isolated Gram-negative bacilli (P < 0.0001).

Emergence of a New Pathogenic Ehrlichia Species, Wisconsin and Minnesota, 2009

BACKGROUND Ehrlichiosis is a clinically important, emerging zoonosis. Only Ehrlichia chaffeensis and E. ewingii have been thought to cause ehrlichiosis in humans in the United States. Patients with suspected ehrlichiosis routinely undergo testing to ensure proper diagnosis and to ascertain the cause. METHODS We used molecular methods, culturing, and serologic testing to diagnose and ascertain the cause of cases of ehrlichiosis. RESULTS On testing, four cases of ehrlichiosis in Minnesota or Wisconsin were found not to be from E. chaffeensis or E. ewingii and instead to be caused by a newly discovered ehrlichia species. All patients had fever, malaise, headache, and lymphopenia; three had thrombocytopenia; and two had elevated liver-enzyme levels. All recovered after receiving doxycycline treatment. At least 17 of 697 Ixodes scapularis ticks collected in Minnesota or Wisconsin were positive for the same ehrlichia species on polymerase-chain-reaction testing. Genetic analyses revealed that this new ehrlichia species is closely related to E. muris. CONCLUSIONS We report a new ehrlichia species in Minnesota and Wisconsin and provide supportive clinical, epidemiologic, culture, DNA-sequence, and vector data. Physicians need to be aware of this newly discovered close relative of E. muris to ensure appropriate testing, treatment, and regional surveillance. (Funded by the National Institutes of Health and the Centers for Disease Control and Prevention.).

Comparison of direct colony method versus extraction method for identification of gram-positive cocci by use of Bruker biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometry

ABSTRACT We evaluated Bruker Biotyper (version 2.0) matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry (MS) for the identification of 305 clinical isolates of staphylococci, streptococci, and related genera by comparing direct colony testing with preparatory extraction. Isolates were previously identified by use of phenotypic testing and/or 16S rRNA gene sequencing. Manufacturer-specified score cutoffs for genus- and species-level identification were used. After excluding 7 isolates not present in the Biotyper library, the Biotyper correctly identified 284 (95%) and 207 (69%) isolates to the genus and species levels, respectively, using extraction. By using direct colony testing, the Biotyper identified 168 (56%) and 60 (20%) isolates to the genus and species levels, respectively. Overall, more isolates were identified to the genus and species levels with preparatory extraction than with direct colony testing (P < 0.0001). The analysis was repeated after dividing the isolates into two subgroups, staphylococci, streptococci, and enterococci (n = 217) and “related genera” (n = 81). For the former subgroup, the extraction method resulted in the identification of 213 (98%) and 171 (79%) isolates to the genus and species levels, respectively, whereas the direct colony method identified 136 (63%) and 56 (26%) isolates to the genus and species levels, respectively. In contrast, for the subgroup of related genera, the extraction method identified 71 (88%) and 36 (44%) isolates to the genus and species levels, respectively, while the direct colony method identified 32 (40%) and 4 (5%) isolates to the genus and species levels, respectively. For both subgroups, preparatory extraction was superior to direct colony testing for the identification of isolates to the genus and species levels (P < 0.0001). Preparatory extraction is needed for the identification of a substantial proportion of Gram-positive cocci using the Biotyper method according to manufacturer-specified score cutoffs.

Comparative Evaluation of Two Commercial Multiplex Panels for Detection of Gastrointestinal Pathogens by Use of Clinical Stool Specimens

ABSTRACT The detection of pathogens associated with gastrointestinal disease may be important in certain patient populations, such as immunocompromised hosts, the critically ill, or individuals with prolonged disease that is refractory to treatment. In this study, we evaluated two commercially available multiplex panels (the FilmArray gastrointestinal [GI] panel [BioFire Diagnostics, Salt Lake City, UT] and the Luminex xTag gastrointestinal pathogen panel [GPP] [Luminex Corporation, Toronto, Canada]) using Cary-Blair stool samples (n = 500) submitted to our laboratory for routine GI testing (e.g., culture, antigen testing, microscopy, and individual real-time PCR). At the time of this study, the prototype (non-FDA-cleared) FilmArray GI panel targeted 23 pathogens (14 bacterial, 5 viral, and 4 parasitic), and testing of 200 μl of Cary-Blair stool was recommended. In contrast, the Luminex GPP assay was FDA cleared for the detection of 11 pathogens (7 bacterial, 2 viral, and 2 parasitic), but had the capacity to identify 4 additional pathogens using a research-use-only protocol. Importantly, the Luminex assay was FDA cleared for 100 μl raw stool; however, 100 μl Cary-Blair stool was tested by the Luminex assay in this study. Among 230 prospectively collected samples, routine testing was positive for one or more GI pathogens in 19 (8.3%) samples, compared to 76 (33.0%) by the FilmArray and 69 (30.3%) by the Luminex assay. Clostridium difficile (12.6 to 13.9% prevalence) and norovirus genogroup I (GI)/GII (5.7 to 13.9% prevalence) were two of the pathogens most commonly detected by both assays among prospective samples. Sapovirus was also commonly detected (5.7% positive rate) by the FilmArray assay. Among 270 additional previously characterized samples, both multiplex panels demonstrated high sensitivity (>90%) for the majority of targets, with the exception of several pathogens, notably Aeromonas sp. (23.8%) by FilmArray and Yersinia enterocolitica (48.1%) by the Luminex assay. Interestingly, the FilmArray and Luminex panels identified mixed infections in 21.1% and 13.0% of positive prospective samples, respectively, compared to only 8.3% by routine methods.

Randomized Trial of Rapid Multiplex Polymerase Chain Reaction–Based Blood Culture Identification and Susceptibility Testing

BACKGROUND The value of rapid, panel-based molecular diagnostics for positive blood culture bottles (BCBs) has not been rigorously assessed. We performed a prospective randomized controlled trial evaluating outcomes associated with rapid multiplex PCR (rmPCR) detection of bacteria, fungi, and resistance genes directly from positive BCBs. METHODS A total of 617 patients with positive BCBs underwent stratified randomization into 3 arms: standard BCB processing (control, n = 207), rmPCR reported with templated comments (rmPCR, n = 198), or rmPCR reported with templated comments and real-time audit and feedback of antimicrobial orders by an antimicrobial stewardship team (rmPCR/AS, n = 212). The primary outcome was antimicrobial therapy duration. Secondary outcomes were time to antimicrobial de-escalation or escalation, length of stay (LOS), mortality, and cost. RESULTS Time from BCB Gram stain to microorganism identification was shorter in the intervention group (1.3 hours) vs control (22.3 hours) (P < .001). Compared to the control group, both intervention groups had decreased broad-spectrum piperacillin-tazobactam (control 56 hours, rmPCR 44 hours, rmPCR/AS 45 hours; P = .01) and increased narrow-spectrum β-lactam (control 42 hours, rmPCR 71 hours, rmPCR/AS 85 hours; P = .04) use, and less treatment of contaminants (control 25%, rmPCR 11%, rmPCR/AS 8%; P = .015). Time from Gram stain to appropriate antimicrobial de-escalation or escalation was shortest in the rmPCR/AS group (de-escalation: rmPCR/AS 21 hours, control 34 hours, rmPCR 38 hours, P < .001; escalation: rmPCR/AS 5 hours, control 24 hours, rmPCR 6 hours, P = .04). Groups did not differ in mortality, LOS, or cost. CONCLUSIONS rmPCR reported with templated comments reduced treatment of contaminants and use of broad-spectrum antimicrobials. Addition of antimicrobial stewardship enhanced antimicrobial de-escalation. CLINICAL TRIALS REGISTRATION NCT01898208.

Prosthetic Joint Infection Diagnosis Using Broad-Range PCR of Biofilms Dislodged from Knee and Hip Arthroplasty Surfaces Using Sonication

ABSTRACT Periprosthetic tissue and/or synovial fluid PCR has been previously studied for prosthetic joint infection (PJI) diagnosis; however, few studies have assessed the utility of PCR on biofilms dislodged from the surface of explanted arthroplasties using vortexing and sonication (i.e., sonicate fluid PCR). We compared sonicate fluid 16S rRNA gene real-time PCR and sequencing to culture of synovial fluid, tissue, and sonicate fluid for the microbiologic diagnosis of PJI. PCR sequences generating mixed chromatograms were decatenated using RipSeq Mixed. We studied sonicate fluids from 135 and 231 subjects with PJI and aseptic failure, respectively. Synovial fluid, tissue, and sonicate fluid culture and sonicate fluid PCR had similar sensitivities (64.7, 70.4, 72.6, and 70.4%, respectively; P > 0.05) and specificities (96.9, 98.7, 98.3, and 97.8%, respectively; P > 0.05). Combining sonicate fluid culture and PCR, the sensitivity was higher (78.5%, P < 0.05) than those of individual tests, with similar specificity (97.0%). Thirteen subjects had positive sonicate fluid culture but negative PCR, and 11 had negative sonicate fluid culture but positive PCR (among which 7 had prior use of antimicrobials). Broad-range PCR and culture of sonicate fluid have equivalent performance for PJI diagnosis.

Evaluation of the Bruker Biotyper and Vitek MS Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Systems for Identification of Nonfermenting Gram-Negative Bacilli Isolated from Cultures from Cystic Fibrosis Patients

ABSTRACT The Bruker Biotyper and Vitek MS matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry (MS) instruments were evaluated for the identification of nonfermenting Gram-negative bacilli (NFGNB) by a blinded comparison to conventional biochemical or molecular methods. Two hundred NFGNB that were recovered from cultures from cystic fibrosis patients in the University of Iowa Health Care (UIHC) Microbiology Laboratory between 1 January 2006 and 31 October 2010 were sent to Mayo Clinic for analysis with the Bruker Biotyper (software version 3.0) and to bioMérieux for testing with Vitek MS (SARAMIS database version 3.62). If two attempts at direct colony testing failed to provide an acceptable MALDI-TOF identification, an extraction procedure was performed. The MS identifications from both of these systems were provided to UIHC for comparison to the biochemical or molecular identification that had been reported in the patient record. Isolates with discordant results were analyzed by 16S rRNA gene sequencing at UIHC. After discrepancy testing, the Bruker Biotyper result agreed with the biochemical or molecular method, with 72.5% of isolates to the species level, 5.5% to the complex level, and 19% to the genus level (3% not identified). The level of agreement for Vitek MS was 80% species, 3.5% complex, 6% genus, and 3.5% family (7% not identified). Both MS systems provided rapid (≤3 min per isolate) and reliable identifications. The agreement of combined species/complex/genus-level identification with the reference method was higher for the Bruker Biotyper (97% versus 89.5%, P = 0.004) but required an extraction step more often. Species-level agreement with the reference method was similar for both MS systems (72.5% and 80%, P = 0.099).

Three-Hour Molecular Detection of Campylobacter, Salmonella, Yersinia, and Shigella Species in Feces with Accuracy as High as That of Culture

ABSTRACT Campylobacter jejuni and Salmonella, Shigella, and Yersinia species (along with Shiga toxin-producing Escherichia coli) are the most common causes of acute bacterial diarrheal disease in the United States. Current detection techniques are time-consuming, limiting usefulness for patient care. We developed and validated a panel of rapid PCR assays for the detection and identification of C. jejuni, C. coli, Salmonella, and Yersinia species and Shigella and enteroinvasive E. coli in stool samples. A total of 392 archived stool specimens, previously cultured for enteric pathogens, were evaluated by PCR. Overall, 104 stool specimens had been culture positive (C. jejuni/coli [n = 51], Salmonella species [n = 42], Shigella species [n = 6], and Yersinia species [n = 5]). Compared to culture, the overall sensitivity and specificity of PCR detection of these organisms were 92 and 98% (96/104 and 283/288), respectively, from fresh or Cary Blair stool (P = 0.41); 87 and 98% (41/47 and 242/246), respectively, from fresh stool (P = 0.53); and 96 and 98% (55/57 and 41/42), respectively, from Cary Blair stool (P = 0.56). For individual genera, PCR was as sensitive as the culture method, with the exception of Salmonella culture using selenite enrichment for which PCR was less sensitive than culture from fresh, but not Cary Blair (P = 0.03 and 1.00, respectively) stools. This PCR assay panel for the rapid diagnosis of acute infectious bacterial diarrheal pathogens has a sensitivity and specificity equivalent to that of culture for stools in Cary Blair transport medium. Paired with reflexive culture of stools testing positive, this should provide an improvement in care for patients with acute infectious diarrheal disease.

Comparison of a Novel, Rapid Chromogenic Biochemical Assay, the Carba NP Test, with the Modified Hodge Test for Detection of Carbapenemase-Producing Gram-Negative Bacilli

ABSTRACT We compared carbapenemase detection among 271 Gram-negative bacilli (of which 131 were carbapenemase producers) using a novel chromogenic rapid test—the Carba NP test (CNP)—and the modified Hodge test (MHT). Sensitivities were comparable (CNP, 100%, versus MHT, 98%; P = 0.08), but CNP was more specific (100% versus 80%; P < 0.0001) and faster.

Identification of Non-diphtheriae Corynebacterium by Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

ABSTRACT We evaluated the Bruker Biotyper matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry for identification of 92 clinical isolates of Corynebacterium species in comparison to identification using rpoB or 16S rRNA gene sequencing. Eighty isolates (87%) yielded a score of ≥1.700, and all of these were correctly identified to the species level with the exception of Corynebacterium aurimucosum being misidentified as the closely related Corynebacterium minutissimum.