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OBJECTIVES: Rapid and accurate mold identification is critical for guiding therapy for mold infections. MALDI-TOF MS has been widely adopted for bacterial and yeast identification; however, few clinical laboratories have applied this technology for routine mold identification due to limited database availability and lack of standardized processes. Here, we evaluated the versatility of the NIH Mold Database in a multicenter evaluation. METHODS: The NIH Mold Database was evaluated by eight US academic centers using a solid media extraction method and a challenge set of 80 clinical mold isolates. Multiple instrument parameters important for spectra optimization were evaluated, leading to the development of two specialized acquisition programs (NIH method and the Alternate-B method). RESULTS: A wide range in performance (33-77%) was initially observed across the eight centers when routine spectral acquisition parameters were applied. Use of the NIH or the Alternate-B specialized acquisition programs, which are different than those used routinely for bacterial and yeast spectral acquisition (MBT_AutoX), in combination with optimized instrument maintenance, improved performance, illustrating that acquisition parameters may be one of the key limiting variable in achieving successful performance. CONCLUSION: Successful mold identification using the NIH Database for MALDI-TOF MS on Biotyper systems was demonstrated across multiple institutions for the first time following identification of critical program parameters combined with instrument optimization. This significantly advances our potential to implement MALDI-TOF MS for mold identification across many institutions. Because instrument variability is inevitable, development of an instrument performance standard specific for mold spectral acquisition is suggested to improve reproducibility across instruments.
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Clostridium difficile is a Gram-positive bacterium commonly found in health care and long-term-care facilities and is the most common cause of antibiotic-associated diarrhea. Rapid detection of this bacterium can assist physicians in implementing contact precautions and appropriate antibiotic therapy in a timely manner. The purpose of this study was to compare the clinical performance of the Quidel Lyra Direct C. difficile assay (Lyra assay) (Quidel, San Diego, CA) to that of a direct cell culture cytotoxicity neutralization assay (CCNA) and enhanced toxigenic culture. This study was performed at three geographically diverse laboratories within the United States using residual stool specimens submitted for routine C. difficile testing. Residual samples were tested using the Lyra assay on three real-time PCR platforms, and results were compared to those for direct CCNA and enhanced toxigenic culture. The test results for all platforms were consistent across all three test sites. The sensitivity and specificity of the Lyra assay on the SmartCycler II, ABI 7500 Fast DX, and ABI QuantStudio DX instruments compared to CCNA were 90.0% and 93.3%, 95.0% and 94.2%, and 93.8% and 95.0%, respectively. Compared to enhanced toxigenic culture, the sensitivity and specificity of the Lyra assay on the SmartCycler II, ABI 7500, and QuantStudio instruments were 82.1% and 96.9%, 89.3% and 98.8%, and 85.7% and 99.0%, respectively. Overall, the Lyra assay is easy to use and versatile and compares well to C. difficile culture methods.
Assuntos
Clostridioides difficile/isolamento & purificação , Infecções por Clostridium/diagnóstico , Diarreia/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Toxinas Bacterianas/análise , Clostridioides difficile/genética , Infecções por Clostridium/microbiologia , Diarreia/microbiologia , Fezes/microbiologia , Humanos , Testes de Neutralização/métodos , Estudos Prospectivos , Sensibilidade e Especificidade , Estados UnidosRESUMO
OBJECTIVES: The genus Mycobacterium contains over 150 species including pathogenic and nonpathogenic strains. Accurate species level identification can aid in differentiating environmental contamination from true infection, and also can aid in selection of antimicrobial therapy. METHODS: We evaluated the performance of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the routine identification of clinical isolates of mycobacteria using 2 commercially available spectral reference libraries, and also assessed the impact of mycobacterial culture using solid medium and broth on MALDI-TOF MS-based identification. RESULTS: All results were compared with those obtained on high-pressure liquid chromatography and nucleic acid sequencing. Optimal results were obtained with a mycobacterium-specific reference library (Mycobacterium Library v1.0). The identification rate was 89.8% (79/88) for isolates cultured on solid medium and 98.8% (85/86) for isolates analyzed directly from broth. Among these, the proportion identified with a high confidence level was 50.0% (44/88) for isolates cultured on solid medium and 80.2% (69/86) for isolates cultured in broth. CONCLUSIONS: Agreement with nucleic acid sequencing for species present in Mycobacterium Library v1.0 was 97.6% (81/83) for isolates cultured on solid medium and 97.5% (79/81) for those cultured in broth.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Mycobacterium/classificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Técnicas de Tipagem Bacteriana/métodos , Meios de Cultura , Humanos , Mycobacterium/genética , Análise de Sequência de DNARESUMO
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.
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Técnicas Bacteriológicas/métodos , Clostridioides difficile/isolamento & purificação , Infecções por Clostridium/diagnóstico , Enterite/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Toxinas Bacterianas/análise , Técnicas de Cultura de Células , Cromatografia Gasosa , Clostridioides difficile/química , Clostridioides difficile/genética , Infecções por Clostridium/microbiologia , DNA Bacteriano/genética , Enterite/microbiologia , Fezes/microbiologia , Humanos , Valor Preditivo dos Testes , Estudos Prospectivos , Ribotipagem , Sensibilidade e Especificidade , Estados UnidosRESUMO
Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a rapid method for the identification of bacteria. Factors that may alter protein profiles, including growth conditions and presence of exogenous substances, could hinder identification. Bacterial isolates identified by conventional methods were grown on various media and identified using the MALDI Biotyper (Bruker Daltonics, Billerica, MA) using a direct smear method and an acid extraction method. Specimens included 23 Pseudomonas isolates grown on blood agar, Pseudocel (CET), and MacConkey agar (MAC); 20 Staphylococcus isolates grown on blood agar, colistin-nalidixic acid agar (CNA), and mannitol salt agar (MSA); and 25 enteric isolates grown on blood agar, xylose lysine deoxycholate agar (XLD), Hektoen enteric agar (HE), salmonella-shigella agar (SS), and MAC. For Pseudomonas spp., the identification rate to genus using the direct method was 83% from blood, 78% from MAC, and 94% from CET. For Staphylococcus isolates, the identification rate to genus using the direct method was 95% from blood, 75% from CNA, and 95% from MSA. For enteric isolates, the identification rate to genus using the direct method was 100% from blood, 100% from MAC, 100% from XLD, 92% from HE, and 87% from SS. Extraction enhanced identification rates. The direct method of MALDI-TOF analysis of bacteria from selective and differential media yields identifications of varied confidence. Notably, Staphylococci spp. from CNA exhibit low identification rates. Extraction enhances identification rates and is recommended for colonies from this medium.
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Bactérias/classificação , Bactérias/isolamento & purificação , Técnicas Bacteriológicas/métodos , Meios de Cultura/química , Manejo de Espécimes/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Bactérias/química , Bactérias/crescimento & desenvolvimento , Humanos , Sensibilidade e EspecificidadeRESUMO
Bloodstream infections are a leading cause of admissions to hospital intensive care units and carry a high mortality rate. Clinical outcome can be greatly improved by early effective antibiotic therapy; therefore, broad-spectrum antimicrobial therapy is often initiated when there is a clinical suspicion of bloodstream infection. Unfortunately, this method may not always be effective when dealing with inherently resistant organisms and can also result in iatrogenic infection and the development of resistant isolates. Rapid identification of the infecting organism may aid in choosing appropriate antimicrobial therapy, thereby reducing these potential adverse events. We compared the matrix-assisted laser desorption ionization (MALDI) Biotyper system with Sepsityper specimen processing (Bruker Daltonics, Billerica, MA) to routine methods for the identification of microorganisms from 164 positive blood cultures. The MALDI Biotyper/Sepsityper identified 85.5% of bacterial isolates directly from positive monomicrobial blood cultures with 97.6% concordance to genus and 94.1% concordance to species with routine identification methods. Gram-negative isolates were more likely to produce acceptable confidence scores (97.8%) than gram-positive isolates (80.0%); however, genus and species concordance with routine identification methods were similar in both groups. Reanalysis of collected spectra using modified blood culture-specific parameters resulted in an improved overall identification rate for gram-positive bacteria (89.0%). Median times to identification using the MALDI Biotyper/Sepsityper were 23 to 83 h faster than routine methods for gram-positive isolates and 34 to 51 h faster for gram-negative isolates.
Assuntos
Bacteriemia/diagnóstico , Bactérias/classificação , Bactérias/isolamento & purificação , Sangue/microbiologia , Técnicas de Laboratório Clínico/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Bactérias/química , Humanos , Fatores de TempoRESUMO
The performance of the automated Vitek 2 (bioMérieux, Inc., Marcy l'Etoile, France) antifungal susceptibility system was compared to that of broth microdilution (BMD) for the determination of MICs of various antifungal drugs. A total of 112 challenge strains and 755 clinical isolates of Candida spp. were tested against caspofungin and micafungin. An additional 452 clinical isolates of Candida albicans were tested against posaconazole. Reference BMD MIC endpoints were established after 24 h of incubation for caspofungin and micafungin and after 48 h of incubation for posaconazole. Essential agreements (EAs) between the Vitek 2 and BMD methods for caspofungin and micafungin were 99.5% and 98.6%, respectively. EA between the Vitek 2 and BMD methods was 95.6% for posaconazole. The overall categorical agreements (CAs) between the Vitek 2 system and BMD were 99.8% for caspofungin, 98.2% for micafungin, and 98.1% for posaconazole. The Vitek 2 system reliably determined caspofungin and micafungin MICs among Candida spp. and posaconazole MICs among C. albicans isolates and demonstrated excellent quantitative and qualitative agreement with the reference BMD method.
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Antifúngicos/farmacologia , Candida/efeitos dos fármacos , Equinocandinas/farmacologia , Lipopeptídeos/farmacologia , Triazóis/farmacologia , Automação/métodos , Caspofungina , Humanos , Micafungina , Testes de Sensibilidade Microbiana/métodos , Estados UnidosRESUMO
Spectra VRE (Remel, Lenexa, KS) is a chromogenic medium designed to recover and differentiate vancomycin-resistant Enterococcus faecium and Enterococcus faecalis (VRE). This medium was compared to bile esculin azide agar (BEAV) and was 98.2% sensitive and 99.3% specific compared to BEAV, which was 87.6% sensitive and 87.1% specific at 24 h.
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Técnicas Bacteriológicas/métodos , Meios de Cultura/química , Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecium/efeitos dos fármacos , Infecções por Bactérias Gram-Positivas/microbiologia , Resistência a Vancomicina , Ágar , Compostos Cromogênicos/metabolismo , Enterococcus faecalis/isolamento & purificação , Enterococcus faecium/isolamento & purificação , Humanos , Sensibilidade e EspecificidadeRESUMO
Spectra MRSA agar (Remel, Lenexa, KS), a novel chromogenic medium originally developed to detect methicillin-resistant Staphylococcus aureus (MRSA) from nasal swabs, was evaluated in this multicenter study for the detection of MRSA from positive blood cultures exhibiting Gram-positive cocci upon initial Gram staining.
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Técnicas Bacteriológicas/métodos , Sangue/microbiologia , Meios de Cultura/química , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Infecções Estafilocócicas/diagnóstico , Ágar , Humanos , Sensibilidade e EspecificidadeRESUMO
A novel chromogenic medium, Spectra MRSA (Remel, Lenexa, KS), was designed to detect methicillin-resistant Staphylococcus aureus (MRSA) rapidly and more efficiently than traditional media (i.e., tryptic soy agar with 5% sheep blood [SBA] and mannitol salt agar [MSA]). A multicenter study (including four clinical trial sites and the Medical College of Wisconsin [MCW] Milwaukee, WI) compared the performance characteristics of Spectra MRSA to those of the traditional media for the detection of MRSA. For this study, 767 nasal swab specimens from the multicenter study (traditional medium used, SBA) and 667 nasal swab specimens from MCW (traditional medium used, MSA) were plated on each test medium and examined after 24 and 48 h of incubation. At 24 h, the sensitivity and the specificity of each medium were as follows: in the multicenter study, 95.4% and 99.7%, respectively, for Spectra MRSA and 93.6% and 100%, respectively, for SBA; at MCW, 95.2% and 99.5%, respectively, for Spectra MRSA and 88.7% and 94.0%, respectively, for MSA. The positive predictive values of each medium at 24 h were as follows: in the multicenter study, 98.1% for Spectra MRSA and 100% for SBA; at MCW, 95.2% for Spectra MRSA and 60.4% for MSA. In our evaluation, we found that Spectra MRSA was able to rapidly identify and differentiate methicillin-resistant S. aureus from methicillin-susceptible S. aureus on the basis of the utilization of chromogens that result in denim blue colonies, thus eliminating the need for biochemical analysis and antimicrobial susceptibility testing. Extending the incubation beyond 24 h did not significantly improve the recovery of MRSA and resulted in decreased specificity.