Showing papers in "Journal of Clinical Microbiology in 2019"
TL;DR: It is argued that standardization of WGS-AST by challenge with consistently phenotyped strain sets of defined genetic diversity is necessary to compare the efficacy of methods of prediction of antibiotic resistance based on genome sequences.
Abstract: Clinical microbiology has long relied on growing bacteria in culture to determine antimicrobial susceptibility profiles, but the use of whole-genome sequencing for antibiotic susceptibility testing (WGS-AST) is now a powerful alternative. This review discusses the technologies that made this possible and presents results from recent studies to predict resistance based on genome sequences. We examine differences between calling antibiotic resistance profiles by the simple presence or absence of previously known genes and single-nucleotide polymorphisms (SNPs) against approaches that deploy machine learning and statistical models. Often, the limitations to genome-based prediction arise from limitations of accuracy of culture-based AST in addition to an incomplete knowledge of the genetic basis of resistance. However, we need to maintain phenotypic testing even as genome-based prediction becomes more widespread to ensure that the results do not diverge over time. We argue that standardization of WGS-AST by challenge with consistently phenotyped strain sets of defined genetic diversity is necessary to compare the efficacy of methods of prediction of antibiotic resistance based on genome sequences.
199 citations
TL;DR: A collection of 5,278 nontyphoidal Salmonella genomes was used to generate extreme gradient boosting (XGBoost)-based machine learning models for predicting MICs for 15 antibiotics, showing that highly accurate MIC prediction models can be generated with less than 500 genomes.
Abstract: Nontyphoidal Salmonella species are the leading bacterial cause of foodborne disease in the United States. Whole-genome sequences and paired antimicrobial susceptibility data are available for Salmonella strains because of surveillance efforts from public health agencies. In this study, a collection of 5,278 nontyphoidal Salmonella genomes, collected over 15 years in the United States, was used to generate extreme gradient boosting (XGBoost)-based machine learning models for predicting MICs for 15 antibiotics. The MIC prediction models had an overall average accuracy of 95% within ±1 2-fold dilution step (confidence interval, 95% to 95%), an average very major error rate of 2.7% (confidence interval, 2.4% to 3.0%), and an average major error rate of 0.1% (confidence interval, 0.1% to 0.2%). The model predicted MICs with no a priori information about the underlying gene content or resistance phenotypes of the strains. By selecting diverse genomes for the training sets, we show that highly accurate MIC prediction models can be generated with less than 500 genomes. We also show that our approach for predicting MICs is stable over time, despite annual fluctuations in antimicrobial resistance gene content in the sampled genomes. Finally, using feature selection, we explore the important genomic regions identified by the models for predicting MICs. To date, this is one of the largest MIC modeling studies to be published. Our strategy for developing whole-genome sequence-based models for surveillance and clinical diagnostics can be readily applied to other important human pathogens.
168 citations
TL;DR: The general challenges of pathogen detection in clinical specimens by metagenomic sequencing, the advantages and disadvantages of the ONT platform, and how research to date supports the potential future use of nanopore sequencing in infectious disease diagnostics are highlighted.
Abstract: Metagenomic sequencing for infectious disease diagnostics is an important tool that holds promise for use in the clinical laboratory. Challenges for implementation so far include high cost, the length of time to results, and the need for technical and bioinformatics expertise. However, the recent technological innovation of nanopore sequencing from Oxford Nanopore Technologies (ONT) has the potential to address these challenges. ONT sequencing is an attractive platform for clinical laboratories to adopt due to its low cost, rapid turnaround time, and user-friendly bioinformatics pipelines. However, this method still faces the problem of base-calling accuracy compared to other platforms. This review highlights the general challenges of pathogen detection in clinical specimens by metagenomic sequencing, the advantages and disadvantages of the ONT platform, and how research to date supports the potential future use of nanopore sequencing in infectious disease diagnostics.
125 citations
TL;DR: While further optimization is required to improve sensitivity, this approach shows promise for the Nanopore platform to be used in the diagnosis and genetic analysis of influenza virus and other respiratory viruses.
Abstract: Influenza is a major global public health threat as a result of its highly pathogenic variants, large zoonotic reservoir, and pandemic potential. Metagenomic viral sequencing offers the potential for a diagnostic test for influenza virus which also provides insights on transmission, evolution, and drug resistance and simultaneously detects other viruses. We therefore set out to apply the Oxford Nanopore Technologies sequencing method to metagenomic sequencing of respiratory samples. We generated influenza virus reads down to a limit of detection of 102 to 103 genome copies/ml in pooled samples, observing a strong relationship between the viral titer and the proportion of influenza virus reads (P = 4.7 × 10−5). Applying our methods to clinical throat swabs, we generated influenza virus reads for 27/27 samples with mid-to-high viral titers (cycle threshold [CT] values, 99% complete sequences for all eight gene segments. We also detected a human coronavirus coinfection in one clinical sample. While further optimization is required to improve sensitivity, this approach shows promise for the Nanopore platform to be used in the diagnosis and genetic analysis of influenza virus and other respiratory viruses.
109 citations
TL;DR: The breakpoint revisions in the M100 supplement since 2010 are described and strategies for the laboratory on how to best adopt these in clinical testing are described.
Abstract: The Clinical and Laboratory Standards Institute (CLSI) has revised several breakpoints since 2010 for bacteria that grow aerobically. In 2019, these revisions include changes to the ciprofloxacin and levofloxacin breakpoints for the Enterobacteriaceae and Pseudomonas aeruginosa, daptomycin breakpoints for Enterococcus spp., and ceftaroline breakpoints for Staphylococcus aureus Implementation of the revisions is a challenge for all laboratories, as not all systems have FDA clearance for the revised (current) breakpoints, compounded by the need for laboratories to perform validation studies and to make updates to laboratory information system/electronic medical record builds in the setting of limited information technology infrastructure. This minireview describes the breakpoint revisions in the M100 supplement since 2010 and strategies for the laboratory on how to best adopt these in clinical testing.
107 citations
TL;DR: CrAg titer predicts meningitis and death and could be used in the future to customize therapy according to burden of infection.
Abstract: Over the past ten years, standard diagnostics for cryptococcal meningitis in HIV-infected persons have evolved from culture to India ink to detection of cryptococcal antigen (CrAg), with the recent development and distribution of a point-of-care lateral flow assay. This assay is highly sensitive and specific in cerebrospinal fluid (CSF), but is also sensitive in the blood to detect CrAg prior to meningitis symptoms. CrAg screening of HIV-infected persons in the blood prior to development of fulminant meningitis and preemptive treatment for CrAg-positive persons are recommended by the World Health Organization and many national HIV guidelines. Thus, CrAg testing is occurring more widely, especially in resource-limited laboratory settings. CrAg titer predicts meningitis and death and could be used in the future to customize therapy according to burden of infection.
96 citations
TL;DR: Results show that MALDI-TOF MS and Vitek 2 antifungal susceptibility systems can be reliable diagnostic tools for testing C. auris isolates from Korean hospitals.
Abstract: Candida auris is an emerging worldwide fungal pathogen. Over the past 20 years, 61 patient isolates of C. auris (4 blood and 57 ear) have been obtained from 13 hospitals in Korea. Here, we reanalyzed those molecularly identified isolates using two matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) systems, including Biotyper and Vitek MS, followed by antifungal susceptibility testing, sequencing of the ERG11 gene, and genotyping. With a research-use-only (RUO) library, 83.6% and 93.4% of the isolates were correctly identified by Biotyper and Vitek MS, respectively. Using an in vitro diagnostic (IVD) library of Vitek MS, 96.7% of the isolates were correctly identified. Fluconazole-resistant isolates made up 62.3% of the isolates, while echinocandin- or multidrug-resistant isolates were not found. Excellent essential (within two dilutions, 96.7%) and categorical agreements (93.4%) between the Clinical and Laboratory Standards Institute (CLSI) and Vitek 2 (AST-YS07 card) methods were observed for fluconazole. Sequencing ERG11 for all 61 isolates revealed that only 3 fluconazole-resistant isolates showed the Erg11p amino acid substitution K143R. All 61 isolates showed identical multilocus sequence typing (MLST). Pulsed-field gel electrophoresis (PFGE) analyses revealed that both blood and ear isolates had the same or similar patterns. These results show that MALDI-TOF MS and Vitek 2 antifungal susceptibility systems can be reliable diagnostic tools for testing C. auris isolates from Korean hospitals. The Erg11p mutation was seldom found among Korean isolates of C. auris, and multidrug resistance was not found. Both MLST and PFGE analyses suggest that these isolates are genetically similar.
79 citations
TL;DR: Broad-range investigations on such critical issues in relation to polymyxins can be beneficial for the implementation of effective treatment against MDR Gram-negative bacterial infections, including chromosomally and plasmid-mediated mcr-related resistance.
Abstract: Polymyxins, including polymyxin B and polymyxin E (colistin), are now increasingly being used worldwide to treat patients with multidrug-resistant (MDR) Gram-negative bacterial infections. This necessitates that laboratories employ an accurate and reliable method for the routine performance of polymyxin susceptibility testing. A number of reasons have accounted for the difficulties with susceptibility testing for the polymyxins, including their multicomponent composition, poor diffusion in the agar medium, adsorption to microtiter plates, the lack of a reliable susceptibility test, the lack of a specific breakpoint from professional organizations, the synergistic effect of polysorbate 80, and the development of heteroresistance. This minireview discusses such problems that impact the results of currently available susceptibility testing methods. We also provide emerging concepts on mechanisms of polymyxin resistance, including chromosomally and plasmid-mediated mcr-related resistance. Broad-range investigations on such critical issues in relation to polymyxins can be beneficial for the implementation of effective treatment against MDR Gram-negative bacterial infections.
71 citations
TL;DR: The biology of CRISPR-Cas systems are summarized and existing and emerging applications to evaluate mechanisms of host-pathogen interactions, to develop accurate and portable diagnostic tests, and to advance the prevention and treatment of infectious diseases are discussed.
Abstract: Infectious diseases remain a global threat contributing to excess morbidity and death annually, with the persistent potential for destabilizing pandemics. Improved understanding of the pathogenesis of bacteria, viruses, fungi, and parasites, along with rapid diagnosis and treatment of human infections, is essential for improving infectious disease outcomes worldwide. Genomic loci in bacteria and archaea, termed clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins, function as an adaptive immune system for prokaryotes, protecting them against foreign invaders. CRISPR-Cas9 technology is now routinely applied for efficient gene editing, contributing to advances in biomedical science. In the past decade, improved understanding of other diverse CRISPR-Cas systems has expanded CRISPR applications, including in the field of infectious diseases. In this review, we summarize the biology of CRISPR-Cas systems and discuss existing and emerging applications to evaluate mechanisms of host-pathogen interactions, to develop accurate and portable diagnostic tests, and to advance the prevention and treatment of infectious diseases.
69 citations
TL;DR: Comparison of phenotypic characteristics and antifungal susceptibilities of isolates representative of the three geographic clades circulating in the United Kingdom found aggregation could be reversibly induced in isolates of the Southern Asian lineage by exposure to triazole and echinocandin ant ifungals but not by Exposure to amphotericin B or flucytosine.
Abstract: Candida auris is a serious nosocomial health risk, with widespread outbreaks occurring in hospitals worldwide. Sequence analyses of outbreak isolates revealed that C. auris has simultaneously emerged as four distinct continentally restricted clonal lineages. We previously reported multiple independent introductions of C. auris isolates from at least three of these lineages (the Southern Asia, South African, and Japanese/Korean lineages) into hospitals across the United Kingdom and that isolates circulating in the United Kingdom displayed two different cell phenotypes which correlated with differences in virulence in Galleria mellonella wax moths. Here, we compared the phenotypic characteristics and antifungal susceptibilities of isolates representative of the three geographic clades circulating in the United Kingdom. Isolates of the South African and Japanese/Korean lineages, but not those of the Southern Asian lineage, grew well on media containing actidione. However, unlike Southern Asian lineage isolates, they were unable to produce even rudimentary pseudohyphae in culture. Importantly, although all isolates were fluconazole resistant in vitro, fluconazole and voriconazole exhibited significantly higher MICs against isolates of the South African lineage than against isolates of the Southern Asian lineage. A similar trend was seen with minimum fungicidal concentrations (MFCs), with higher MFCs of the triazole antifungal agents being seen for the South African lineage isolates. Finally, the formation of large cellular aggregates was seen only with isolates of the South African and Japanese/Korean lineages, which correlates with the reduced virulence observed previously in Galleria wax moths inoculated with such isolates. Intriguingly, aggregation could be reversibly induced in isolates of the Southern Asian lineage by exposure to triazole and echinocandin antifungals but not by exposure to amphotericin B or flucytosine.
68 citations
TL;DR: The eCIM had an overall sensitivity and specificity of 100% and was adopted by the Clinical and Laboratory Standards Institute as a method to use in combination with the mCIM to identify MBL-producing Enterobacteriaceae.
Abstract: The increase in the prevalence and impact of infections caused by carbapenemase-producing Enterobacteriaceae is a global health concern. Therefore, rapid and accurate methods to detect these organisms in any clinical microbiology laboratory, including those in resource-limited settings, are essential to prevent and contain their spread. It is also important to differentiate between serine- and metal-dependent carbapenemases elaborated by carbapenemase-producing isolates for epidemiologic, infection control and prevention, and therapeutic purposes. Here, we describe the development and evaluation of the EDTA-modified carbapenem inactivation method (eCIM), an assay for discriminating between serine- and metal-dependent (i.e., metallo-β-lactamases [MBLs]) carbapenemases when used in conjunction with the modified carbapenem inactivation method (mCIM). The eCIM had an overall sensitivity and specificity of 100% and was adopted by the Clinical and Laboratory Standards Institute as a method to use in combination with the mCIM to identify MBL-producing Enterobacteriaceae.
TL;DR: Two leading organizations that set standards for antimicrobial susceptibility testing, the European Committee on Antimicrobial Susceptibility Testing (EUCAST and the Clinical and Laboratory Standards Institute (CLSI), have taken different strategies to deal with these challenges.
Abstract: INTRODUCTIONAntibiotic susceptibility test results are among the most important results issued by clinical microbiology laboratories because they routinely guide critical treatment decisions. Interpretations of MIC or disk diffusion test results, such as "susceptible" or "resistant," are easily understood. Clinical laboratories also need to determine whether and how their reports will reflect more complex situations. Such situations include, first, whether there is need to administer higher or more frequent doses of antibiotic than usual for clinical efficacy; second, whether an antimicrobial is likely to be effective at a body site where it concentrates; and third, whether there is some uncertainty in the test results due to technical variability that cannot be eliminated. Two leading organizations that set standards for antimicrobial susceptibility testing, the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the Clinical and Laboratory Standards Institute (CLSI), have taken different strategies to deal with these challenges. In this Point-Counterpoint, Gunnar Kahlmeter and Christian Giske discuss how EUCAST is addressing these issues, and Thomas Kirn and Susan Sharp discuss the CLSI approach.
TL;DR: Stool Xpert could be a noninvasive method of ruling in PTB in children, particularly those with HIV, and generalizability of the evidence is limited by the lack of standardized stool preparation and testing protocols.
Abstract: Invasive collection methods are often required to obtain samples for the microbiologic evaluation of children with presumptive pulmonary tuberculosis (PTB). Nucleic-acid amplification testing of easier to collect stool samples could be a non-invasive method of diagnosing PTB. We conducted a systematic review and meta-analysis to evaluate the diagnostic accuracy of testing stool with the Xpert MTB/RIF assay (‘stool Xpert’) for childhood PTB. Four databases were searched for publications from January 2008 to June 2018. Studies assessing the diagnostic accuracy amongst children of stool Xpert compared to a microbiological reference standard of conventional specimens tested by mycobacterial culture or Xpert were eligible. Bivariate random-effects meta-analyses were performed to calculate pooled sensitivity and specificity of stool Xpert against the reference standard. From 1589 citations, 9 studies (n=1681) were included. Median participant ages ranged from 1.3 to 10.6 years. Protocols for stool processing and testing varied substantially, with differences in reagents and methods of homogenization and filtering. Against the microbiological reference standard, pooled sensitivity and specificity of stool Xpert were 67% (95%CI:52-79) and 99% (95%CI:98-99), respectively. Sensitivity was higher among children with HIV (79%; 95%CI:68-87; versus 60%; 95%CI:44-74 among HIV-uninfected). Heterogeneity was high. Data were insufficient for subgroup analyses amongst children under age 5, the most relevant target population. Stool Xpert could be a non-invasive method of ruling-in PTB in children, particularly those with HIV. However, studies focused on children under 5 are needed, and generalizability of the evidence is limited by the lack of a standardized stool preparation and testing protocol.
TL;DR: The largest collection of clinical isolates from Clade II is described, which is also the longest-running span of clinical cases, 20 years, from any single region to date, which has important implications for future surveillance.
Abstract: The emerging yeast Candida auris can be highly drug resistant, causing invasive infections, and large outbreaks. C. auris went from an unknown pathogen a decade ago to being reported in over thirty countries on six continents. C. auris consists of four discrete clades, based on where the first isolates of the clade were reported, South Asian (clade I), East Asian (clade II), African (clade III), and South American (clade IV). These clades have unique genetic and biochemical characteristics that are important to understand and inform the global response to C. auris. Clade II has been underrepresented in the literature despite being the first one discovered. In this issue of the Journal of Clinical Microbiology, Y. J. Kwon et al. (J Clin Microbiol 57:e01624-18, 2019, https://doi.org/10.1128/JCM.01624-18) describe the largest collection of clinical isolates from Clade II, which is also the longest-running span of clinical cases, 20 years, from any single region to date. Clade II appears to have a propensity for the ear that is uncharacteristic of the other clades, which typically cause invasive infections and large-scale outbreaks. This study provides new information on an understudied lineage of C. auris and has important implications for future surveillance.
TL;DR: Clinical and molecular characteristics of K. pneumoniae bloodstream infections from hospitals across Japan were analyzed by a review of the medical records and revealed that patients with hvKp infections had higher proportions of diabetes mellitus, and their infections had significantly higher propensity to involve pneumonia.
Abstract: Although hypervirulent Klebsiella pneumoniae (hvKp) has been associated with severe community-acquired infections that occur among relatively healthy individuals, information about hvKp infections in health care settings remains limited. Here, we systematically analyzed the clinical and molecular characteristics of K. pneumoniae isolates causing bloodstream infections in a cross-sectional study. Clinical characteristics of K. pneumoniae bloodstream infections from hospitals across Japan were analyzed by a review of the medical records. Whole-genome sequencing of the causative isolates was performed. Bacterial species were confirmed and hvKp were identified using whole-genome sequencing data. Clinical characteristics of hvKp infections were compared with those of non-hvKp infections by bivariate analyses. Of 140 cases of K. pneumoniae bloodstream infections, 26 cases (18.6%) were caused by various clones of hvKp defined by the carriage of cardinal virulence genes. Molecular identification revealed that 24 (17.1%) and 14 (10%) cases were caused by Klebsiella variicola and Klebsiella quasipneumoniae, respectively. Patients with hvKp infections had higher proportions of diabetes mellitus (risk ratio [RR], 1.75; 95% confidence interval [CI], 1.05 to 2.94), and their infections had significantly higher propensity to involve pneumonia (RR, 5.85; 95% CI, 1.39 to 24.6), liver abscess (RR, 5.85; 95% CI, 1.39 to 24.6), and disseminated infections (RR, 6.58; 95% CI, 1.16 to 37.4) than infections by other isolates. More than one-half of hvKp infections were health care associated or hospital acquired, and a probable event of health care-associated transmission of hvKp was documented. hvKp isolates, which are significantly associated with severe and disseminated infections, are frequently involved in health care-associated and hospital-acquired infections in Japan.
TL;DR: Recommendations for first-line and second-line drug testing and organism group, specific methodologies, and reporting recommendations have been addressed by the Clinical and Laboratory Standards Institute and are important in the selection of appropriate antimicrobial treatment regimens for nontuberculous mycobacteria (NTM) disease.
Abstract: Recommendations for first-line and second-line drug testing and organism group, specific methodologies, and reporting recommendations have been addressed by the Clinical and Laboratory Standards Institute (CLSI) and are important in the selection of appropriate antimicrobial treatment regimens for nontuberculous mycobacteria (NTM) disease. This review also includes recent information on new antimicrobials proposed for the treatment of NTM but not yet addressed by the CLSI and molecular (gene sequencing) methods associated with the detection of antimicrobial resistance of two major therapeutic antimicrobials, clarithromycin and amikacin.
TL;DR: The data support an increased sensitivity of Ultra compared to Xpert in a low-prevalence setting and correlation between the Ultra semiquantitative result and AFB burden can help in evaluating a patient’s transmission potential.
Abstract: Xpert MTB/RIF (Xpert) for direct molecular detection of Mycobacterium tuberculosis and rifampin resistance from clinical specimens has dramatically improved the diagnosis of tuberculosis (TB). Xpert MTB/RIF Ultra (Ultra) is proposed as a substitute of Xpert with increased sensitivity and improved rifampin resistance detection. We evaluated the diagnostic performance of Ultra and Xpert for pulmonary TB diagnosis in a low-TB-burden setting. Performance of Ultra and Xpert were compared to culture on respiratory specimens from patients with suspected pulmonary TB (November 2016 to August 2018; n = 196) in Lausanne (Switzerland). Clinical data were used to investigate discrepant results. Correlation between semiquantitative result of Ultra and smear microscopy status for the detection of acid-fast bacilli (AFB) was established. The sensitivities of Xpert and Ultra were 82.9% (39/47) and 95.8% (45/47), respectively, when considering all culture-positive specimens, 100% (23/23) for both assays on smear-positive specimens, and 66.7% (16/24) and 91.7% (22/24) on smear-negative specimens. Using culture as gold standard, the specificities of Xpert and Ultra were 97.3% (145/149) and 96.64% (144/149), respectively. All the patients with Ultra-positive results with the new category "trace" were diagnosed with active TB based on clinical findings and microbiological culture. The semiquantitative results of both Xpert and of Ultra positively correlated with the semiquantitative result of AFB detection. Our data support an increased sensitivity of Ultra compared to Xpert in a low-prevalence setting. Correlation between the Ultra semiquantitative result and AFB burden can help in evaluating a patient's transmission potential.
TL;DR: Systematic application of genomics has uncovered the emergence of polyclonal CPE as a significant threat in Australia, providing important insights to inform local public health guidelines and interventions.
Abstract: Carbapenemase-producing Enterobacterales (CPE) are being increasingly reported in Australia, and integrated clinical and genomic surveillance is critical to effectively manage this threat. We sought to systematically characterize CPE in Victoria, Australia, from 2012 to 2016. Suspected CPE were referred to the state public health laboratory in Victoria, Australia, from 2012 to 2016 and examined using phenotypic, multiplex PCR and whole-genome sequencing (WGS) methods and compared with epidemiological metadata. Carbapenemase genes were detected in 361 isolates from 291 patients (30.8% of suspected CPE isolates), mostly from urine (42.1%) or screening samples (34.8%). IMP-4 (28.0% of patients), KPC-2 (25.3%), NDM (24.1%), and OXA carbapenemases (22.0%) were most common. Klebsiella pneumoniae (48.8% of patients) and Escherichia coli (26.1%) were the dominant species. Carbapenemase-inactivation method (CIM) testing reliably detected carbapenemase-positive isolates (100% sensitivity, 96.9% specificity), identifying an additional five CPE among 159 PCR-negative isolates (IMI and SME carbapenemases). When epidemiologic investigations were performed, all pairs of patients designated "highly likely" or "possible" local transmission had ≤23 pairwise single-nucleotide polymorphisms (SNPs) by genomic transmission analysis; conversely, all patient pairs designated "highly unlikely" local transmission had ≥26 pairwise SNPs. Using this proposed threshold, possible local transmission was identified involving a further 16 patients for whom epidemiologic data were unavailable. Systematic application of genomics has uncovered the emergence of polyclonal CPE as a significant threat in Australia, providing important insights to inform local public health guidelines and interventions. Using our workflow, pairwise SNP distances between CPE isolates of ≤23 SNPs suggest local transmission.
TL;DR: The implementation of multiplex PCR stool testing was associated with a reduction in the utilization of endoscopy, abdominal radiology, and antibiotic prescribing.
Abstract: PCR-based multiplex gastrointestinal (GI) pathogen panels have started to replace stool culture and ova and parasite exam as a rapid and accurate means of diagnosing acute gastroenteritis. However, there are limited data on the impact of panel testing on patient outcomes. The objective of this study was to evaluate the management and health care utilization of patients following GI panel compared with conventional stool testing. We performed a retrospective comparative analysis of 9,402 patients who underwent testing with the FilmArray GI panel from March 2015 through May 2017 and 5,986 patients who underwent conventional stool testing from December 2012 through February 2015. GI panel was positive in 2,746 exams (29.2%) compared with 246 exams (4.1%) with conventional testing. Within 30 days following stool testing, compared with patients who received a conventional stool test, patients who received a GI panel were less likely to undergo any endoscopic procedure (8.4% GI panel versus 9.6% stool culture, P = 0.008) or any abdominal radiology (29.4% GI panel versus 31.7%, P = 0.002). Within 14 days following stool testing, patients who received a GI panel were less likely to be prescribed any antibiotic (36.2% GI panel versus 40.9%, P < 0.001). The implementation of multiplex PCR stool testing was associated with a reduction in the utilization of endoscopy, abdominal radiology, and antibiotic prescribing.
TL;DR: Several elements favor self-testing for large-scale implementation, including ease of use, convenience, potential for integration with mobile health (mHealth), and availability for various modes of distribution.
Abstract: HIV self-testing (HIVST) provides an at-home option to counter the barriers that patients face with testing performed in health care settings. HIVST has gradually increased in popularity in a time when social media and technology-based solutions are preferred. In this paper, we consider the aspects of self-testing that merit its integration into HIV testing and prevention systems in the United States. Several elements favor self-testing for large-scale implementation, including ease of use, convenience, potential for integration with mobile health (mHealth), and availability for various modes of distribution. HIVST has a demonstrated ability to reach at-risk individuals who otherwise rarely test. The paradigm of self-testing, however, introduces new challenges, including lesser test performance relative to that in health care settings, nonstandard counseling following receipt of test results, and difficulty in providing linkage to care. After discussing the performance of oral fluid versus blood-based HIVST, we review data regarding acceptability of HIVST, offer insights into counseling and linkage to care for HIVST, and provide examples of novel applications of and future research directions for HIVST.
TL;DR: The results highlight the potential role of dogs as a reservoir of K. pneumoniae to humans and vice versa and to the best knowledge, this is the first report of healthy humans and dogs sharing K. influenza pneumoniae strains that were undistinguishable by PFGE/MLST.
Abstract: This study aimed to characterize the fecal colonization and sharing of Klebsiella pneumoniae strains between companion animals and humans living in close contact. Fecal samples were collected from 50 healthy participants (24 humans, 18 dogs, and 8 cats) belonging to 18 households. Samples were plated onto MacConkey agar (MCK) plates with and without cefotaxime or meropenem supplementation. Up to five K. pneumoniae colonies per participant were compared by pulsed-field gel electrophoresis (PFGE) after XbaI restriction. K. pneumoniae strains with unique pulse types from each participant were characterized for antimicrobial susceptibility, virulence genes, and multilocus sequence type (MLST). Fecal K. pneumoniae pulse types were compared to those of clinical K. pneumoniae strains from animal and human patients with urinary tract infections (n = 104). K. pneumoniae colonization was detected in nonsupplemented MCK in around 38% of dogs (n = 7) and humans (n = 9). K. pneumoniae strains isolated from dogs belonged to sequence type 17 (ST17), ST188, ST252, ST281, ST423, ST1093, ST1241, ST3398, and ST3399. None of the K. pneumoniae strains were multidrug resistant or hypervirulent. Two households included multiple colonized participants. Notably, two colonized dogs within household 15 (H15) shared a strain each (ST252 and ST1241) with one coliving human. One dog from H16 shared one PFGE-undistinguishable K. pneumoniae ST17 strain with two humans from different households; however, the antimicrobial susceptibility phenotypes of these three strains differed. Two main virulence genotypes were detected, namely fimH-1 mrkD ycfM entB kfu and fimH-1 mrkD ycfM entB kpn. These results highlight the potential role of dogs as a reservoir of K. pneumoniae to humans and vice versa. Furthermore, to our best knowledge, this is the first report of healthy humans and dogs sharing K. pneumoniae strains that were undistinguishable by PFGE/MLST.
TL;DR: Compared to currently used techniques, CBDE is an easy and practical method to perform colistin MIC testing.
Abstract: Limited methods for colistin MIC determination are available to clinical microbiology laboratories. The purpose of this study was to evaluate the accuracy of the colistin broth disk elution (CBDE) test compared to that of broth microdilution (BMD) for identifying colistin MICs. CBDE was compared to colistin BMD using a collection of Gram-negative bacilli tested at two U.S. microbiology laboratories. The isolates tested included 121 retrospective clinical isolates, 45 prospective clinical isolates, and 6 mcr-1-positive Escherichia coli isolates. CBDE was performed with four 10-ml cation-adjusted Mueller-Hinton broth tubes per isolate, to which 0, 1, 2, and 4 colistin 10-µg disks were added, generating final concentrations in the tubes of 0 (growth control), 1, 2, and 4 µg/ml, respectively. MICs were evaluated visually and interpreted using Clinical and Laboratory Standards Institute breakpoints. Site 2 also compared CBDE to the reference broth macrodilution (BMAD) method (n = 110 isolates). Overall, CBDE yielded a categorical agreement (CA) and essential agreement (EA) of 98% and 99%, respectively, compared to the results of colistin BMD. Very major errors occurred for mcr-1-producing strains, with MICs fluctuating from 2 to 4 µg/ml on repeat testing. The results for all other isolates were in CA with those of BMD. CBDE versus BMAD had an EA of 100% and a CA of 100%. Compared to currently used techniques, CBDE is an easy and practical method to perform colistin MIC testing. Some mcr-1-producing isolates yielded MICs of 2 µg/ml by CBDE and 4 µg/ml by BMD. As such, the results for isolates with colistin MICs of 2 µg/ml by CBDE should be confirmed by the reference BMD method, and isolates with MICs of ≥2 µg/ml should be evaluated for the presence of mcr genes.
TL;DR: Based on data, the CLSI antimicrobial susceptibility testing (AST) subcommittee endorsed the CBDE and CAT-10 methods for colistin testing of Enterobacterales and P. aeruginosa.
Abstract: Susceptibility testing of the polymyxins (colistin and polymyxin B) is challenging for clinical laboratories. The Clinical and Laboratory Standards Institute (CLSI) Antimicrobial Susceptibility Testing Subcommittee evaluated two methods to enable accurate testing of these agents. These methods were a colistin broth disk elution (CBDE) and a colistin agar test (CAT), the latter of which was evaluated using two inoculum volumes, 1 μl (CAT-1) and 10 μl (CAT-10). The methods were evaluated using a collection of 270 isolates of Enterobacterales, 122 Pseudomonas aeruginosa isolates, and 106 Acinetobacter spp. isolates. Overall, 94.4% of CBDE results were in essential agreement and 97.9% in categorical agreement (CA) with reference broth microdilution MICs. Nine very major errors (VME; 3.2%) and 3 major errors (ME; 0.9%) were observed. With the CBDE, 98.6% CA was observed for Enterobacterales (2.5% VME, 0% ME), 99.3% CA was observed for P. aeruginosa (0% VME, 0.7% ME), and 93.1% CA was observed for Acinetobacter spp. (5.6% VME, 3.3% ME). Overall, CA was 94.9% with 6.8% VME using CAT-1 and improved to 98.3% with 3.9% VME using CAT-10. No ME were observed using either CAT-1 or CAT-10. Using the CAT-1/CAT-10, the CA observed was 99.4%/99.7% for Enterobacterales (1%/0.5% VME), 98.7%/100% for P. aeruginosa (8.3%/0% VME), and 88.5%/92.3% for Acinetobacter spp. (21.4%/14.3% VME). Based on these data, the CLSI antimicrobial susceptibility testing (AST) subcommittee endorsed the CBDE and CAT-10 methods for colistin testing of Enterobacterales and P. aeruginosa.
TL;DR: ST1193-H64 (where “H64” refers to a phylogenetic subdivision within ST1193 that is characterized by the fimH64 allele), which was uniformly fluoroquinolone resistant, appeared to emerge in the United States in a geographically staggered fashion beginning around 2011.
Abstract: Escherichia coli sequence type 1193 (ST1193) is an emerging multidrug-resistant pathogen. We performed longitudinal and cross-sectional surveillance for ST1193 among clinical and fecal E. coli isolates from Minneapolis Veterans Affairs Medical Center (VAMC) patients and their household members, other Minnesota centers, and national VAMCs and compared these ST1193 isolates with archival human and canine ST1193 isolates from Australia (2008). We also developed and extensively validated a novel multiplex PCR assay for ST1193 and its characteristic fimH64 (type 1 fimbrial adhesin) allele. We found that ST1193-H64 (where “H64” refers to a phylogenetic subdivision within ST1193 that is characterized by the fimH64 allele), which was uniformly fluoroquinolone resistant, appeared to emerge in the United States in a geographically staggered fashion beginning around 2011. Its prevalence among clinical and fecal E. coli isolates at the Minneapolis VAMC rose rapidly beginning in 2013, peaked in early 2017, and then plateaued or declined. In comparison with other ST14 complex (STc14) isolates, ST1193-H64 isolates were more extensively multidrug resistant, whereas their virulence genotypes were less extensive but included (uniquely) K1 capsule and fimH64. Pulsed-field gel electrophoresis separated ST1193-H64 isolates from other STc14 isolates and showed genetic commonality between archival Australian versus recent U.S. isolates, fecal versus clinical isolates, and human versus canine isolates. Three main ST1193 pulsotypes differed significantly in resistance profiles and capsular types; emergent pulsotype 2123 was associated with trimethoprim-sulfamethoxazole resistance and K1 (versus K5) capsule. These findings clarify ST1193-H64’s temporal prevalence trends as a fluoroquinolone-resistant pathogen and commensal; document clonal subsets with distinctive geographic, temporal, resistance, and virulence gene associations; and establish a new laboratory tool for rapid and simple detection of ST1193-H64.
TL;DR: The ePlex panels provided highly accurate results and proved to be an excellent diagnostic tool for the rapid identification of pathogens causing bloodstream infections.
Abstract: Rapid identification and susceptibility testing results are of importance for the early appropriate therapy of bloodstream infections. The ePlex (GenMark Diagnostics) blood culture identification (BCID) panels are fully automated PCR-based assays designed to identify Gram-positive and Gram-negative bacteria, fungi, and bacterial resistance genes within 1.5 h from positive blood culture. Consecutive non-duplicate positive blood culture episodes were tested by the ePlex system prospectively. The choice of panel(s) (Gram-positive, Gram-negative, and/or fungal pathogens) was defined by Gram-stained microscopy of blood culture-positive bottles (BacT/Alert; bioMerieux). Results with the ePlex panels were compared to the identification results obtained by standard culture-based workflow. In total, 216 positive blood culture episodes were evaluable, yielding 263 identification results. The sensitivity/positive predictive value for detection by the ePlex panels of targeted cultured isolates were 97% and 99% for the Gram-positive panel and 99% and 96% for the Gram-negative panel, resulting in overall agreement rates of 96% and 94% for the Gram-positive and Gram-negative panel, respectively. All 26 samples with targeted resistance results were correctly detected by the ePlex panels. The ePlex panels provided highly accurate results and proved to be an excellent diagnostic tool for the rapid identification of pathogens causing bloodstream infections. The short time to results may be of added value for optimizing the clinical management of patients with sepsis.
TL;DR: The filtered dried plasma spot (FDPS), created using the newly developed, instrument-free VLPlasma device, outperformed that with the DBS in identifying treatment failure at a threshold of 1,000 copies/ml and compared well with the quantification of VL in plasma.
Abstract: HIV viral load (VL) testing is the recommended method for monitoring the response of people living with HIV and receiving antiretroviral therapy (ART). The availability of standard plasma VL testing in low- and middle-income countries (LMICs), and access to this testing, are limited by the need to use fresh plasma. Good specimen collection methods for HIV VL testing that are applicable to resource-constrained settings are needed. We assessed the diagnostic performance of the filtered dried plasma spot (FDPS), created using the newly developed, instrument-free VLPlasma device, in identifying treatment failure at a VL threshold of 1,000 copies/ml in fresh plasma. Performance was compared with that of the conventional dried blood spot (DBS). Venous blood samples from 201 people living with HIV and attending an infectious disease clinic in Malaysia were collected, and HIV VL was quantified using fresh plasma (the reference standard), FDPS, and DBS specimens. VL testing was done using the Roche Cobas AmpliPrep/Cobas TaqMan v2.0 assay. At a threshold of 1,000 copies/ml, the diagnostic performance of the FDPS was superior (sensitivity, 100% [95% confidence interval {CI}, 89.1 to 100%]; specificity, 100% [95% CI, 97.8 to 100%]) to that of the DBS (sensitivity, 100% [95% CI, 89.4 to 100%]; specificity, 36.8% [95% CI, 29.4 to 44.7%]) (P
TL;DR: Patients with MRMp were more likely to have received a macrolide prior to presentation, and their treatment was morelikely to have been changed to a fluoroquinolone after presentation, in the first national surveillance program for M. pneumoniae in the United States.
Abstract: There are sparse data to indicate the extent that macrolide-resistant Mycoplasma pneumoniae (MRMp) occurs in the United States or its clinical significance. Between 2015 and 2018, hospitals in 8 states collected and stored respiratory specimens that tested positive for M. pneumoniae and sent them to the University of Alabama at Birmingham, where real-time PCR was performed for detection of 23S rRNA mutations known to confer macrolide resistance. MRMp was detected in 27 of 360 specimens (7.5%). MRMp prevalence was significantly higher in the South and East (18.3%) than in the West (2.1%). A2063G was the predominant 23S rRNA mutation detected. MICs for macrolide-susceptible M. pneumoniae (MSMp) were ≤0.008 μg/ml, whereas MICs for MRMp were 16 to 32 μg/ml. Patients with MRMp infection were more likely to have a history of immunodeficiency or malignancy. Otherwise, there were no other significant differences in the clinical features between patients infected with MRMp and those infected with MSMp, nor were there any differences in radiographic findings, hospitalization rates, viral coinfections, the mean duration of antimicrobial treatment, or clinical outcomes. There was no significant change in MRMp incidence over time or according to age, sex, race/ethnicity, or status as an inpatient or an outpatient. Patients with MRMp were more likely to have received a macrolide prior to presentation, and their treatment was more likely to have been changed to a fluoroquinolone after presentation. This is the first national surveillance program for M. pneumoniae in the United States. Additional surveillance is needed to assess the clinical significance of MRMp and to monitor changes in MRMp prevalence.
TL;DR: The results show that the Vitek 2 (version 8.01) yeast identification system has a limited ability to correctly identify C. auris, and suggest that an identification result for C. duobushaemulonii should warrant further testing to rule out C. Auris.
Abstract: Candida auris is an emerging multidrug-resistant yeast that has been systematically incorrectly identified by phenotypic methods in clinical microbiology laboratories. The Vitek 2 automated identification system (bioMerieux) recently included C. auris in its database (version 8.01). We evaluated the performance of the Vitek 2 YST ID card to identify C. auris and related species. A panel of 44 isolates of Candida species (C. auris, n = 35; Candida haemulonii, n = 5; Candida duobushaemulonii, n = 4) were tested by three different hospital-based microbiology laboratories. Among 35 isolates of C. auris, Vitek 2 yielded correct identification in an average of 52% of tested samples. Low-discrimination (LD) results with an inability to distinguish between C. auris, C. duobushaemulonii, and Candida famata were obtained in an average of 27% of samples. Incorrect identification results were obtained in an average of 21% of samples, the majority (91%) of which were reported as C. duobushaemulonii and the remaining 9% of which were reported as Candida lusitaniae/C. duobushaemulonii. The proportion of correct identification was not statistically different across different centers (P = 0.78). Stratification by genetic clades demonstrated that 100% (n = 8) of the strains of the South American clade were correctly identified compared to 7% (n = 10) and 0% (n = 4) from the African and East Asian clades, respectively. None of the non-auris Candida strains (n = 9) were incorrectly identified as C. auris. Our results show that the Vitek 2 (version 8.01) yeast identification system has a limited ability to correctly identify C. auris. These data suggest that an identification result for C. duobushaemulonii should warrant further testing to rule out C. auris. The overall performance of the Vitek 2 seems to differ according to C. auris genetic clade, with the South American isolates yielding the most accurate results.
TL;DR: The current stage of research about antimicrobial compounds based on bacteriophages and endolysins against MRSA infections is shown and discussed and a brief overview of the latest options in antibacterial therapies, mainly focusing on phage therapy is provided.
Abstract: Even though antibiotic resistance in bacteria is a natural phenomenon, the alarming increase in pathogenic bacteria refractory to a wide range of antimicrobials is attracting attention worldwide. Indeed, the World Health Organization (WHO) has recently published a list of priority pathogens for which new antimicrobial alternatives are urgently needed. Among these pathogens, methicillin-resistant Staphylococcus aureus (MRSA) strains are perhaps the best known by the general public. In addition to its potential to acquire antibiotic resistance, S. aureus can produce a large number of virulence factors, such as hemolysins, enterotoxins, and proteases, and exhibits the ability to form biofilms as well as to evolve into different clones that can spread and colonize new environments. This review provides a brief overview of the latest options in antibacterial therapies, mainly focusing on phage therapy. In this regard, the current stage of research about antimicrobial compounds based on bacteriophages and endolysins against MRSA infections is shown and discussed.
TL;DR: The Rapid ResaPolymyxin Acinetobacter/Pseudomonas NP test is inexpensive, easy to perform, highly sensitive and specific, and can be completed in 4 hours and could be useful in countries facing endemic spread of colistin-resistant nonfermenters.
Abstract: A rapid test was developed for identification of polymyxin resistance in nonfermenting bacteria. This test detects viable cells after growth in a medium containing a defined concentration of colistin. The principle of this test is based on the visual detection of the reduction of the resazurin reagent, a viability colorant, as observed by its color change (blue to purple or pink). Its evaluation was performed by using 92 colistin-resistant and colistin-susceptible Acinetobacter baumannii and Pseudomonas aeruginosa isolates. Sensitivity and specificity were found to be 100% and 95%, respectively, by comparison with the standard broth microdilution method. The Rapid ResaPolymyxin Acinetobacter/Pseudomonas NP test is inexpensive, easy to perform, highly sensitive and specific, and can be completed in 4 hours. It could be useful in countries facing endemic spread of colistin-resistant nonfermenters.