Showing papers in "Journal of Clinical Microbiology in 2011"
TL;DR: Whereas the sensitivity of the Xpert assay with tissue specimens was 69.0% (20 out of 29 culture-positive cases detected), 100% sensitivity was found with the urine and stool specimens and the combined sensitivity and specificity of theXpert assay were calculated to be 77.3% and 98.2%, respectively.
Abstract: In total, 521 nonrespiratory specimens (91 urine, 30 gastric aspirate, 245 tissue, 113 pleural fluid, 19 cerebrospinal fluid [CSF], and 23 stool specimens) submitted to the German National Reference Laboratory for Mycobacteria (NRL) from May 2009 to August 2010 were comparatively investigated with the new molecular-based GeneXpert MTB/RIF (Xpert) assay system and conventional liquid and solid culture methods. Twenty (3.8%) of the 521 specimens gave no interpretable result. Whereas the sensitivity of the Xpert assay with tissue specimens was 69.0% (20 out of 29 culture-positive cases detected), 100% sensitivity was found with the urine and stool specimens. The combined sensitivity and specificity of the Xpert assay were calculated to be 77.3% and 98.2%, respectively.
382 citations
TL;DR: The first three cases of nosocomial fungemia caused by C. auris are described, which confirms that it is a causative agent of bloodstream infections and emphasizes the importance of accurately identifying this species.
Abstract: Candida auris is a newly described species whose clinical significance is not clear. Here, we describe the first three cases of nosocomial fungemia caused by C. auris, which confirms that it is a causative agent of bloodstream infections. All three patients presented persistent fungemia for 10 to 31 days. The isolates obtained from the three patients were misidentified as Candida haemulonii and Rhodotorula glutinis by the Vitek 2 and the API 20C systems, respectively. C. auris was confirmed by sequence analysis of the internal transcribed spacer region and D1/D2 regions of the 26S ribosomal DNA of the rRNA gene. The MIC ranges of amphotericin B (AMB), fluconazole (FLU), itraconazole, and voriconazole were 0.5 to 1, 2 to 128, 0.125 to 2, and 0.06 to 1 μg/ml, respectively. All isolates were susceptible to caspofungin (MIC = 0.06 μg/ml) and micafungin (MIC = 0.03 μg/ml). One patient developed breakthrough fungemia while receiving FLU therapy, and two patients who received FLU therapy followed by AMB showed therapeutic failure and fatal outcomes. Our cases show that C. auris fungemia can be persistent, despite FLU or AMB therapy, which emphasizes the importance of accurately identifying this species.
337 citations
TL;DR: Twenty-seven NDM-1-positive isolates of worldwide origin were included in this study to identify these strains as not only pathogens but also colonizers of normal flora for infection control screening.
Abstract: Enterobacterial isolates expressing the carbapenemase NDM-1 are emerging worldwide. Twenty-seven NDM-1-positive isolates of worldwide origin were included in this study to identify these strains as not only pathogens but also colonizers of normal flora for infection control screening. Although susceptibility to carbapenems varied, a combined test (IMP/IMP + EDTA), the Etest MBL, and automated susceptibility testing by Vitek2 (bioMerieux) identified those NDM-1 producers as verified by PCR using specific primers. Screening for carriers of NDM-1 producers may be based on media such as the ChromID ESBL culture medium routinely used to screen for extended-spectrum β-lactamase producers, which gives excellent detection levels with low limits of detection ranging from 8 × 100 to 5 × 102 CFU/ml. The CHROMagar KPC culture medium had higher limits of detection (1 × 101 to 5 × 105 CFU/ml) and may be proposed for the follow-up of outbreaks of infections with NDM-1 producers. Colonies growing on these screening media can be verified as NDM-1 producers with molecular methods as described herein.
329 citations
TL;DR: The GeneXpert MTB/RIF test appeared to be as sensitive as culture with smear-positive specimens but less sensitiveWith smear-negative pulmonary and extrapulmonary specimens that include low numbers of bacilli.
Abstract: Mycobacterium tuberculosis remains one of the most significant causes of death from an infectious agent. The rapid diagnosis of tuberculosis and detection of rifampin (RIF) resistance are essential for early disease management. The GeneXpert MTB/RIF assay is a novel integrated diagnostic device for the diagnosis of tuberculosis and rapid detection of RIF resistance in clinical specimens. We determined the performance of the MTB/RIF assay for rapid diagnosis of tuberculosis and detection of rifampin resistance in smear-positive and smear-negative pulmonary and extrapulmonary specimens obtained from possible tuberculosis patients. Two hundred fifty-three pulmonary and 176 extrapulmonary specimens obtained from 429 patients were included in the study. One hundred ten (89 culture positive and 21 culture negative for M. tuberculosis) of the 429 patients were considered to have tuberculosis. In pulmonary specimens, sensitivities were 100% (27/27) and 68.6% (24/35) for smear-positive and smear-negative specimens, respectively. It had a lower sensitivity with extrapulmonary specimens: 100% for smear-positive specimens (4/4) and 47.7% for smear-negative specimens (21/44). The test accurately detected the absence of tuberculosis in all 319 patients without tuberculosis studied. The MTB/RIF assay also detected 1 RIF-resistant specimen and 88 RIF-susceptible specimens, and the results were confirmed by drug susceptibility testing. We concluded that the MTB/RIF test is a simple method, and routine staff with minimal training can use the system. The test appeared to be as sensitive as culture with smear-positive specimens but less sensitive with smear-negative pulmonary and extrapulmonary specimens that include low numbers of bacilli.
316 citations
TL;DR: The results of this study suggest that the Xpert test also shows good potential for the diagnosis of extrapulmonary TB and that its ease of use makes it applicable for countries where TB is endemic.
Abstract: Approximately 10 to 15% of tuberculosis (TB) cases in India are estimated to have extrapulmonary disease, and due to a lack of diagnostic means, they often remain untreated. The early detection of Mycobacterium tuberculosis and multidrug resistance is a priority in TB diagnosis to improve the successful treatment rate of TB and reduce transmission. The Xpert MTB/RIF (Xpert) test, recently endorsed by the World Health Organization for the detection of pulmonary TB, was evaluated to test its utility in 547 patients with suspected extrapulmonary tuberculosis. Five hundred forty-seven extrapulmonary specimens were split and processed simultaneously for both culture (solid and liquid) and Xpert testing. For culture, the sensitivity was low, 53% (150/283 specimens). Xpert sensitivity and specificity results were assessed in comparison to a composite reference standard made up of smear and culture results and clinical, radiological, and histological findings. The sensitivity of the Xpert assay was 81% (228/283 specimens) (64% [89/138] for smear-negative cases and 96% [139/145] for smear-positive cases), with a specificity of 99.6%. The sensitivity was found to be high for the majority of specimen types (63 to 100%) except for cerebrospinal fluid, the sensitivity of which was 29% (2/7 specimens). The Xpert test correctly identified 98% of phenotypic rifampin (RIF)-resistant cases and 94% of phenotypic RIF-susceptible cases. Sequencing of the 6 discrepant samples resolved 3 of them, resulting in an increased specificity of 98%. In conclusion, the results of this study suggest that the Xpert test also shows good potential for the diagnosis of extrapulmonary TB and that its ease of use makes it applicable for countries where TB is endemic.
313 citations
TL;DR: This work has shown that the detection of carbapenem degradation using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) works for strains carrying NDM-1, VIMs, and different IMP enzymes.
Abstract: In recent years, the percentage of carbapenem-resistant bacteria has increased at an alarming pace and become a major threat for patient survival. Carbapenemase-induced carbapenem resistance can be confirmed through the detection of carbapenem degradation using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). This method works for strains carrying NDM-1, VIM-1, VIM-2, KPC-2, and different IMP enzymes.
307 citations
TL;DR: Direct PCR using blood samples had good sensitivity and specificity for the diagnosis of IC and offers an attractive method for early diagnosis of specific Candida spp.
Abstract: Invasive candidiasis (IC) is a significant cause of morbidity and mortality. Diagnosis relies on culture-based methods, which lack sensitivity and delay diagnosis. We conducted a systematic review assessing the diagnostic accuracy of PCR-based methods to detect Candida spp. directly in blood samples. We searched electronic databases for prospective or retrospective cohort and case-control studies. Two reviewers abstracted data independently. Meta-analysis was performed using a hierarchical logistic regression model. Random-effects metaregression was performed to assess the effects of study methods and infection characteristics on sensitivity or specificity values. We included 54 studies with 4,694 patients, 963 of whom had proven/probable or possible IC. Perfect (100%) sensitivity and specificity for PCR in whole-blood samples was observed when patients with cases had candidemia and controls were healthy people. When PCR was performed to evaluate patients with suspected invasive candidiasis, the pooled sensitivity for the diagnosis of candidemia was 0.95 (confidence interval, 0.88 to 0.98) and the pooled specificity was 0.92 (0.88 to 0.95). A specificity of >90% was maintained in several analyses considering different control groups. The use of whole-blood samples, rRNA, or P450 gene targets and a PCR detection limit of ≤ 10 CFU/ml were associated with improved test performance. PCR positivity rates among patients with proven or probable IC were 85% (78 to 91%), while blood cultures were positive for 38% (29 to 46%). We conclude that direct PCR using blood samples had good sensitivity and specificity for the diagnosis of IC and offers an attractive method for early diagnosis of specific Candida spp. Its effects on clinical outcomes should be investigated.
304 citations
TL;DR: There are few activities in the clinical microbiology laboratory that utilize more technologist time and laboratory resources than antimicrobial susceptibility testing (AST); it has been suggested that, at least in terms of direct relevance to the care of patients with infection, AST may be the most relevant.
Abstract: There are few activities in the clinical microbiology laboratory that utilize more technologist time and laboratory resources than antimicrobial susceptibility testing (AST). It has been suggested that, at least in terms of direct relevance to the care of patients with infection, AST may be the
299 citations
TL;DR: The results support the need for a minimum culture incubation period of 13 days to be applied to both aerobic and anaerobic culture media for all periprosthetic specimens.
Abstract: Propionibacterium acnes is increasingly recognized as an important agent of prosthetic joint infection (PJI). However, the optimum culture conditions for recovery of this organism from PJI specimens have not been determined. By applying a prolonged 28-day culture incubation to all periprosthetic specimens received for bacterial culture from 198 revision arthroplasty procedures, we retrospectively determined that a 13-day culture incubation period is necessary for the recovery of P. acnes from patients with PJI. Incubation beyond this period was associated with increasing recovery of nondiagnostic isolates: 21.7% of P. acnes isolates believed to be clinically unimportant were recovered after 13 days of incubation. Importantly, a diagnosis of P. acnes PJI would have been missed in 29.4% of patients had extended culture incubation been applied only to anaerobic culture media. Although specimens from P. acnes PJIs were more commonly associated with the presence of ≥2 culture media positive for growth, acute inflammation (≥5 neutrophils/high-power field) was observed in only 40% of patients with PJIs that had more than one specimen submitted for bacterial culture. These results support the need for a minimum culture incubation period of 13 days to be applied to both aerobic and anaerobic culture media for all periprosthetic specimens. Optimal recovery of infecting organisms from PJI specimens will be an important component in generating a universal definition for PJI due to indolent agents of infection, such as P. acnes.
292 citations
TL;DR: The ability of this method to routinely detect carbapenemases in Enterobacteriaceae and Pseudomonas spp.
Abstract: Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry is used for the determination of molecular weights of different chemical compounds. We describe here the use of MALDI-TOF mass spectrometry to detect a carbapenem antibiotic, meropenem, and its degradation products. Buffered meropenem solution (0.1 mM Tris-HCl, pH 6.8) was mixed with an overnight culture of bacteria. After 3-h incubation, the reaction mixture was centrifuged, and the supernatant was analyzed by MALDI-TOF mass spectrometry. The presence or absence of peaks representing meropenem and its sodium salts was crucial. The average turnaround time of this test, considering the use of overnight culture, is 4 h. We validated this method for the detection of resistance to carbapenems in Enterobacteriaceae and Pseudomonas aeruginosa mediated by carbapenemase production. A total of 124 strains, including 30 carbapenemase-producing strains, were used in the study. The sensitivity of this method is 96.67%, with a specificity of 97.87%. Our results demonstrate the ability of this method to routinely detect carbapenemases in Enterobacteriaceae and Pseudomonas spp. in laboratories. This assay is comparable with a labor-intensive imipenem-hydrolyzing spectrophotometric assay that is a reference method for the detection of carbapenemase. As demonstrated here, MALDI-TOF mass spectrometry may be used in microbiological laboratories not only for microbial identification but also for other applications, such as studies of mechanisms of antibiotic resistance.
284 citations
TL;DR: In this paper, a matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry was compared to phenotypic testing for yeast identification.
Abstract: Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry was compared to phenotypic testing for yeast identification MALDI-TOF mass spectrometry yielded 963% and 845% accurate species level identifications (spectral scores, ≥ 18) for 138 common and 103 archived strains of yeast MALDI-TOF mass spectrometry is accurate, rapid (51 min of hands-on time/identification), and cost-effective ($050/sample) for yeast identification in the clinical laboratory
TL;DR: Asymptomatic carriage of a respiratory virus occurs frequently in young children, and significant differences in the amount of virus present were observed between cases and controls, suggesting that defining cutoff levels should be feasible and represents the next necessary step for diagnosing viral respiratory infections using molecular tests.
Abstract: Highly sensitive techniques, such as PCR, have greatly improved the detection of respiratory viruses. However, the sensitivity of PCR tests also complicates clinical interpretation, as the presence of small amounts of viral targets may not necessarily have clinical relevance. We performed a prospective case-control study in asymptomatic and symptomatic young children. PCR detection of 14 respiratory viruses was performed in nasal washes, and results were quantified in copies per milliliter. A total of 141 cases and 157 controls were included. In 72% of the cases and 28% of the controls, at least one virus was identified. When stratified for age, at least one virus was identified in 47% of the controls younger than 1 year old. Rhinovirus (RV) was frequently detected in both symptomatic and asymptomatic individuals. Receiver operating characteristic analysis for quantitative rhinovirus detection showed that cutoff values for clinical relevance are feasible for RV. In contrast to rhinovirus, respiratory syncytial virus (RSV) was rarely detected in controls, suggesting that a positive RSV test result is almost always of clinical relevance, independent of viral quantity. In conclusion, our study shows that asymptomatic carriage of a respiratory virus occurs frequently in young children. However, significant differences in the amount of virus present were observed between cases and controls. This suggests that defining cutoff levels should be feasible and represents the next necessary step for diagnosing viral respiratory infections using molecular tests.
TL;DR: It is concluded that anaerobic culture detected as wide a diversity of species in ECC as that observed using cloning approaches.
Abstract: Severe early childhood caries (ECC), while strongly associated with Streptococcus mutans using selective detection (culture, PCR), has also been associated with a widely diverse microbiota using molecular cloning approaches. The aim of this study was to evaluate the microbiota of severe ECC using anaerobic culture. The microbial composition of dental plaque from 42 severe ECC children was compared with that of 40 caries-free children. Bacterial samples were cultured anaerobically on blood and acid (pH 5) agars. Isolates were purified, and partial sequences for the 16S rRNA gene were obtained from 5,608 isolates. Sequence-based analysis of the 16S rRNA isolate libraries from blood and acid agars of severe ECC and caries-free children had >90% population coverage, with greater diversity occurring in the blood isolate library. Isolate sequences were compared with taxon sequences in the Human Oral Microbiome Database (HOMD), and 198 HOMD taxa were identified, including 45 previously uncultivated taxa, 29 extended HOMD taxa, and 45 potential novel groups. The major species associated with severe ECC included Streptococcus mutans, Scardovia wiggsiae, Veillonella parvula, Streptococcus cristatus, and Actinomyces gerensceriae. S. wiggsiae was significantly associated with severe ECC children in the presence and absence of S. mutans detection. We conclude that anaerobic culture detected as wide a diversity of species in ECC as that observed using cloning approaches. Culture coupled with 16S rRNA identification identified over 74 isolates for human oral taxa without previously cultivated representatives. The major caries-associated species were S. mutans and S. wiggsiae, the latter of which is a candidate as a newly recognized caries pathogen.
TL;DR: Overall agreement compared to culture was 89% (98% for smear positives and 72% for smears negatives) for detection of Mycobacterium tuberculosis in the GeneXpert MTB/RIF assay.
Abstract: A total of 217 specimens submitted for routine smear and culture from three different sites within the western United States were used to evaluate the GeneXpert MTB/RIF assay (for research use only) (Cepheid, Sunnyvale, CA). Overall agreement compared to culture was 89% (98% for smear positives and 72% for smear negatives) for detection of Mycobacterium tuberculosis.
TL;DR: It is concluded that MALDI-TOF MS analysis can be incorporated into the work flow of the microbiology laboratory for rapid and accurate identification of most strains of mycobacteria isolated from solid growth media.
Abstract: Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has recently been introduced into the clinical microbiology laboratory as a rapid and accurate method to identify bacteria and yeasts. In this paper we describe our work on the use of MALDI-TOF MS for the identification of mycobacterial isolates. We developed a protocol for protein extraction from mycobacteria and utilized it to construct a database containing 42 clinically relevant type and reference strains of mycobacteria. The database was used to identify 104 clinical isolates of mycobacteria. All members of the Mycobacterium tuberculosis complex were identified accurately at the complex level but could not be separated at the species level. All other organisms were identified at the species level, with the exception of one strain of M. kansasii (accurately identified but with a low spectral score) and three pairs of closely related strains: M. abscessus and M. massiliense, M. mucogenicum and M. phocaicum, and M. chimaera and M. intracellulare. These pairs of organisms can currently be identified only by multilocus gene sequence analysis. We conclude that MALDI-TOF MS analysis can be incorporated into the work flow of the microbiology laboratory for rapid and accurate identification of most strains of mycobacteria isolated from solid growth media.
TL;DR: The study confirmed an incidence rate of fungemia in Denmark three times higher than those in other Nordic countries and identified marked differences related to age and gender.
Abstract: A 6-year nationwide study of fungemia in Denmark was performed using data from an active fungemia surveillance program and from laboratory information systems in nonparticipating regions. A total of 2,820 episodes of fungemia were recorded. The incidence increased from 2004 to 2007 (7.7 to 9.6/100,000) and decreased slightly from 2008 to 2009 (8.7 to 8.6/100,000). The highest incidences were seen at the extremes of age (i.e., 11.3 and 37.1/100,000 for those 50 years of age. The species distribution varied significantly by both age and gender. Candida species accounted for 98% of the pathogens, and C. albicans was predominant, although the proportion decreased (64.4% to 53.2%, P 4 μg/ml) occurred in C. albicans (7/1,183 [0.6%]), C. dubliniensis (2/65 [3.1%]), C. parapsilosis (5/83 [6.0%]), and C. tropicalis (7/104 [6.7%]). Overall, 70.8% of fungemia isolates were fully fluconazole susceptible, but the proportion decreased (79.7% to 68.9%, P = 0.02). The study confirmed an incidence rate of fungemia in Denmark three times higher than those in other Nordic countries and identified marked differences related to age and gender. Decreased susceptibility to fluconazole was frequent and increasing.
TL;DR: High crude mortality and a high proportion of nBSIs due to antibiotic-resistant organisms are found in this multicenter study at 16 Brazilian hospitals.
Abstract: Nosocomial bloodstream infections (nBSIs) are an important cause of morbidity and mortality. Data from a nationwide, concurrent surveillance study, Brazilian SCOPE (Surveillance and Control of Pathogens of Epidemiological Importance), were used to examine the epidemiology and microbiology of nBSIs at 16 Brazilian hospitals. In our study 2,563 patients with nBSIs were included from 12 June 2007 to 31 March 2010. Ninety-five percent of BSIs were monomicrobial. Gram-negative organisms caused 58.5% of these BSIs, Gram-positive organisms caused 35.4%, and fungi caused 6.1%. The most common pathogens (monomicrobial) were Staphylococcus aureus (14.0%), coagulase-negative staphylococci (CoNS) (12.6%), Klebsiella spp. (12.0%), and Acinetobacter spp. (11.4%). The crude mortality was 40.0%. Forty-nine percent of nBSIs occurred in the intensive-care unit (ICU). The most frequent underlying conditions were malignancy, in 622 patients (24.3%). Among the potential factors predisposing patients to BSI, central venous catheters were the most frequent (70.3%). Methicillin resistance was detected in 157 S. aureus isolates (43.7%). Of the Klebsiella sp. isolates, 54.9% were resistant to third-generation cephalosporins. Of the Acinetobacter spp. and Pseudomonas aeruginosa isolates, 55.9% and 36.8%, respectively, were resistant to imipenem. In our multicenter study, we found high crude mortality and a high proportion of nBSIs due to antibiotic-resistant organisms.
TL;DR: For the first time, a number of miRNAs were differentially expressed during active pulmonary tuberculosis infection, and circulating miR-29a has great potential to serve as a marker for the detection of active pulmonaryculosis infection.
Abstract: Emerging evidence shows that microRNAs (miRNAs) play an important role in pathogen-host interactions. Circulating miRNAs have been repeatedly and stably detected in blood and hold promise to serve as molecular markers for diverse physiological and pathological conditions. To date, the relationship between circulating miRNAs and active pulmonary tuberculosis (TB) has not been reported. Using microarray-based expression profiling followed by real-time quantitative PCR validation, the levels of circulating miRNAs were compared between patients with active pulmonary tuberculosis and matched healthy controls. The receiver operating characteristic curve was used to evaluate the diagnostic effect of selected miRNA. Bioinformatic analysis was used to explore the potential roles of these circulating miRNAs in active pulmonary tuberculosis infection. Among 92 miRNAs significantly detected, 59 miRNAs were downregulated and 33 miRNAs were upregulated in the TB serum compared to their levels in the control serum. Interestingly, only two differentially expressed miRNAs were increased not only in the serum but also in the sputum of patients with active pulmonary tuberculosis compared to the levels for the healthy controls. Upregulated miR-29a could discriminate TB patients from healthy controls with reasonable sensitivity and specificity. A number of significantly enriched pathways regulated by these circulating miRNAs were predicted, and most of them were involved in acute-phase response, inflammatory response, and the regulation of the cytoskeleton. In all, for the first time our results revealed that a number of miRNAs were differentially expressed during active pulmonary tuberculosis infection, and circulating miR-29a has great potential to serve as a marker for the detection of active pulmonary tuberculosis infection.
TL;DR: B Bruker Biotyper (version 2.0) matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry for the identification of 305 clinical isolates of staphylococci, streptococci and related genera was evaluated by comparing direct colony testing with preparatory extraction.
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.
TL;DR: In this article, Matrix-assisted laser desorption ionization ionization-time of flight mass spectrometry (MALDI-TOF MS) was used to identify human bacterial pathogens.
Abstract: Conventional methods are sometimes insufficient to identify human bacterial pathogens, and alternative techniques, often molecular, are required. Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) identified with a valid score 45.9% of 410 clinical isolates from 207 different difficult-to-identify species having required 16S rRNA gene sequencing. MALDI-TOF MS might represent an alternative to 16S rRNA gene sequencing.
TL;DR: The TaqMan low-density array (TLDA) cards approach offers promise for rapid and simultaneous identification of multiple respiratory pathogens for outbreak investigations and disease surveillance.
Abstract: The large and growing number of viral and bacterial pathogens responsible for respiratory infections poses a challenge for laboratories seeking to provide rapid and comprehensive pathogen identification. We evaluated a novel application of the TaqMan low-density array (TLDA) cards for real-time PCR detection of 21 respiratory-pathogen targets. The performance of the TLDA was compared to that of individual real-time PCR (IRTP) assays with the same primers and probes using (i) nucleic acids extracted from the 21 pathogen strains and 66 closely related viruses and bacteria and (ii) 292 clinical respiratory specimens. With spiked samples, TLDA cards were about 10-fold less sensitive than IRTP assays. By using 292 clinical specimens to generate 2,238 paired individual assays, the TLDA card exhibited 89% sensitivity (95% confidence interval [CI], 86 to 92%; range per target, 47 to 100%) and 98% specificity (95% CI, 97 to 99%; range per target, 85 to 100%) overall compared to IRTP assays as the gold standard with a threshold cycle (CT) cutoff of 43. The TLDA card approach offers promise for rapid and simultaneous identification of multiple respiratory pathogens for outbreak investigations and disease surveillance.
TL;DR: The widespread use of Haemophilus influenzae type b (Hib) conjugate vaccines has nearly eradicated invasive Hib disease where the vaccines are used, but there is no convincing evidence of a true increase in the incidence of non-serotype b invasive infections.
Abstract: The widespread use of Haemophilus influenzae type b (Hib) conjugate vaccines has nearly eradicated invasive Hib disease where the vaccines are used. This success was accompanied by a shift in capsular serotypes of invasive H. influenzae disease, with nontypeable strains replacing type b strains as the most common bloodstream isolate, but there is no convincing evidence of a true increase in the incidence of non-serotype b invasive infections. H. influenzae causes predominantly mucosal infections. The introduction of vaccines for otitis media and global shifts in antimicrobial susceptibility emphasize the importance of continued surveillance of H. influenzae colonization and disease patterns.
TL;DR: Results from the SENTRY Antimicrobial Surveillance Program were analyzed for regional variations of invasive Candida species infections and resistance to echinocandins was most prevalent among C. glabrata isolates, as determined using recently established CLSI breakpoint criteria.
Abstract: Antifungal testing results from the SENTRY Antimicrobial Surveillance Program (2008 to 2009) were analyzed for regional variations of invasive Candida species infections. Among 2,085 cases from the Asian-Pacific (APAC) (51 cases), Latin American (LAM) (348 cases), European (EU) (750 cases), and North American (NAM) (936 cases) regions, Candida albicans predominated (48.4%), followed by C. glabrata (18.0%), C. parapsilosis (17.2%), C. tropicalis (10.5%), and C. krusei (1.9%). Resistance to echinocandins (anidulafungin [2.4%] and micafungin [1.9%]) and azoles (3.5 to 5.6%) was most prevalent among C. glabrata isolates, as determined using recently established CLSI breakpoint criteria. C. glabrata isolates were more common in NAM (23.5%), and C. albicans isolates were more common in APAC (56.9%), with C. parapsilosis (25.6%) and C. tropicalis (17.0%) being more prominent in LAM. Emerging resistance patterns among C. glabrata cases in NAM require focused surveillance.
TL;DR: 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.
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 0.05). However, the Bruker Biotyper outperformed the BD Phoenix for identification of infrequently isolated gram-negative bacilli (P < 0.0001).
TL;DR: In this article, the authors evaluated the utility of matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) for bacterial identification.
Abstract: Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) was evaluated prospectively in a diagnostic laboratory. Nine hundred twenty-seven organisms were tested in triplicate; 2,351/2,781 (85%) species and 2,681/2,781 (96%) genus identifications were correct. Known issues such as the misidentification of alpha-hemolytic streptococci as Streptococcus pneumoniae were easily corrected. Identifications cost AUD$0.45 per isolate and were available in minutes. MALDI-TOF MS is rapid, accurate, and inexpensive. Matrix-assisted laser desorption–ionization time of flight mass spectrometry (MALDI-TOF MS) has been shown to be both accurate in the identification of bacteria (1, 8, 9) and rapid (5, 10, 11, 13, 14), which is of proven benefit to patient care (2, 4). New technology is never able to completely replace conventional methods; rather, these tests form part of the overall diagnostic algorithms. We therefore undertook this prospective study to determine the utility of the MALDI-TOF MS in a routine diagnostic laboratory for bacterial identification. All bacteria isolated within one calendar month from any site or specimen type that would normally undergo identification were tested in parallel with our routine methods and in triplicate (to assess the reproducibility of the results) using the microflex MALDI Biotyper 2.0 (Bruker Daltonics, GmbH, Bremen, Germany) according to the manufacturer’s instructions (software version 3.1.1.0). As specified by the manufacturer, identification scores of 2 and between 1.7 and 1.9 were required for a reliable identification to the species and genus level, respectively, while identification scores of 1.7 were considered unreliable. MALDI-TOF MS identifications were compared to identifications by current methods, which in
TL;DR: The Xpert MTB/RIF test is a simple rapid method well adapted to a routine laboratory that appeared to be as sensitive as the IS6110-TaqMan assay with respiratory specimens but less sensitive with paucibacillary specimens, such as smear-negative nonrespiratory specimens.
Abstract: The sensitivities of the Xpert MTB/RIF test and an in-house IS6110-based real-time PCR using TaqMan probes (IS6110-TaqMan assay) for the detection of Mycobacterium tuberculosis complex (MTBC) DNA were compared by use of 117 clinical specimens (97 culture positive and 20 culture negative for MTBC) that were frozen in sediment. The 97 clinical specimens included 60 respiratory and 37 nonrespiratory specimens distributed into 36 smear-positive and 61 smear-negative specimens. Among the 97 culture-positive specimens, 4 had rifampin-resistant isolates. Both methods were highly specific and exhibited excellent sensitivity (100%) with smear-positive specimens. The sensitivity of the Xpert MTB/RIF test with the whole smear-negative specimens was more reduced than that of the IS6110-TaqMan assay (48 versus 69%, P = 0.005). Both methods exhibited similar sensitivities with smear-negative respiratory specimens, but the Xpert MTB/RIF test had lower sensitivity with smear-negative nonrespiratory specimens than the IS6110-TaqMan assay (37 versus 71%, P = 0.013). Finally, the sensitivities of the Xpert MTB/RIF test and the IS6110-TaqMan assay were 79% and 84%, respectively, with respiratory specimens and 53% and 78%, respectively (P = 0.013), with nonrespiratory specimens. The Xpert MTB/RIF test correctly detected the rifampin resistance in smear-positive specimens but not in the one smear-negative specimen. The Xpert MTB/RIF test is a simple rapid method well adapted to a routine laboratory that appeared to be as sensitive as the IS6110-TaqMan assay with respiratory specimens but less sensitive with paucibacillary specimens, such as smear-negative nonrespiratory specimens.
TL;DR: In this paper, the authors measured how including patient presentation with the C. difficile assay result impacted assay performance to diagnose CDI and found that clinical presentation is important when interpreting C.difficile diagnostic assays.
Abstract: Asymptomatic Clostridium difficile colonization is common in hospitalized patients. Existing C. difficile assay comparisons lack data on severity of diarrhea or patient outcomes, limiting the ability to interpret their results in regard to the diagnosis of C. difficile infection (CDI). The objective of this study was to measure how including patient presentation with the C. difficile assay result impacted assay performance to diagnose CDI. Stool specimens from 150 patients that met inclusion and exclusion criteria were selected. Nine methods to detect C. difficile in stool were evaluated. All patients were interviewed prospectively to assess diarrhea severity. We then assessed how different reference standards, with and without the inclusion of patient presentation, impact the sensitivity, specificity, and positive and negative predictive values of the assays to diagnose CDI. There were minimal changes in sensitivity; however, specificity was significantly lower for the assays Tox A/B II, C. diff Chek-60, BD GeneOhm Cdiff, Xpert C. difficile, and Illumigene C. difficile and for toxigenic culture (P was <0.01 for all except Tox A/B II from fresh stool, for which the P value was 0.016) when the reference standard was recovery of toxigenic C. difficile from stool plus the presence of clinically significant diarrhea compared to when the reference standard was having at least four assays positive while ignoring diarrhea severity. There were 15 patients whose assay result was reported as negative but subsequently found to be positive by at least four assays in the comparison. None suffered from any CDI-related adverse events. In conclusion, clinical presentation is important when interpreting C. difficile diagnostic assays.
TL;DR: A large proportion of Danish fungemia patients were severely ill and received suboptimal initial antifungal treatment, and Optimization of diagnosis and therapy is possible.
Abstract: This study investigated microbiological, clinical, and management issues and outcomes for Danish fungemia patients. Isolates and clinical information were collected at six centers. A total of 334 isolates, 316 episodes, and 305 patients were included, corresponding to 2/3 of the national episodes. Blood culture positivity varied by system, species, and procedure. Thus, cases with concomitant bacteremia were reported less commonly by BacT/Alert than by the Bactec system (9% [11/124 cases] versus 28% [53/192 cases]; P < 0.0001), and cultures with Candida glabrata or those drawn via arterial lines needed longer incubation. Species distribution varied by age, prior antifungal treatment (57% occurrence of C. glabrata, Saccharomyces cerevisiae, or C. krusei in patients with prior antifungal treatment versus 28% occurrence in those without it; P = 0.007), and clinical specialty (61% occurrence of C. glabrata or C. krusei in hematology wards versus 27% occurrence in other wards; P = 0.002). Colonization samples were not predictive for the invasive species in 11/100 cases. Fifty-six percent of the patients had undergone surgery, 51% were intensive care unit (ICU) patients, and 33% had malignant disease. Mortality increased by age (P = 0.009) and varied by species (36% for C. krusei, 25% for C. parapsilosis, and 14% for other Candida species), severity of underlying disease (47% for ICU patients versus 24% for others; P = 0.0001), and choice but not timing of initial therapy (12% versus 48% for patients with C. glabrata infection receiving caspofungin versus fluconazole; P = 0.023). The initial antifungal agent was deemed suboptimal upon species identification in 15% of the cases, which would have been 6.5% if current guidelines had been followed. A large proportion of Danish fungemia patients were severely ill and received suboptimal initial antifungal treatment. Optimization of diagnosis and therapy is possible.
TL;DR: A 20-year-old man died following the ingestion of pasta contaminated with Bacillus cereus in Brussels, Belgium, and high levels of cereulide were found in the spaghetti meal.
Abstract: A lethal intoxication case, which occurred in Brussels, Belgium, is described. A 20-year-old man died following the ingestion of pasta contaminated with Bacillus cereus. Emetic strains of B. cereus were isolated, and high levels of cereulide (14.8 μg/g) were found in the spaghetti meal.
TL;DR: SeptiFast served as a highly valuable adjunct to conventional blood culture in children, adding diagnostic value and shortening the time to result (TTR) to 6 h.
Abstract: Sepsis is a major health problem in newborns and children. Early detection of pathogens allows initiation of appropriate antimicrobial therapy that strongly correlates with positive outcomes. Multiplex PCR has the potential to rapidly identify bloodstream infections, compensating for the loss of blood culture sensitivity. In an Italian pediatric hospital, multiplex PCR (the LightCycler SeptiFast test) was compared to routine blood culture with 1,673 samples obtained from 803 children with suspected sepsis; clinical and laboratory information was used to determine the patient infection status. Excluding results attributable to contaminants, SeptiFast showed a sensitivity of 85.0% (95% confidence interval [CI] = 78.7 to 89.7%) and a specificity of 93.5% (95% CI = 92.1 to 94.7%) compared to blood culture. The rate of positive results was significantly higher with SeptiFast (14.6%) than blood culture (10.3%) (P < 0.0001), and the overall positivity rate was 16.1% when the results of both tests were combined. Staphylococcus aureus (11.6%), coagulase-negative staphylococci (CoNS) (29.6%), Pseudomonas aeruginosa (16.5%), and Klebsiella spp. (10.1%) were the most frequently detected. SeptiFast identified 97 additional isolates that blood culture failed to detect (24.7% P. aeruginosa, 23.7% CoNS, 14.4% Klebsiella spp., 14.4% Candida spp.). Among specimens taken from patients receiving antibiotic therapy, we also observed a significantly higher rate of positivity of SeptiFast than blood culture (14.1% versus 6.5%, respectively; P < 0.0001). On the contrary, contaminants were significantly more frequent among blood cultures than SeptiFast (n = 97 [5.8%] versus n = 26 [1.6%]), respectively; P < 0.0001). SeptiFast served as a highly valuable adjunct to conventional blood culture in children, adding diagnostic value and shortening the time to result (TTR) to 6 h.