A Laboratory Medicine Best Practices Systematic Review and Meta-analysis of Nucleic Acid Amplification Tests (NAATs) and Algorithms Including NAATs for the Diagnosis of Clostridioides (Clostridium) difficile in Adults.

The evidence base for the optimal laboratory diagnosis of Clostridioides (Clostridium) difficile in adults is currently unresolved due to the uncertain performance characteristics and various combinations of tests. This systematic review evaluates the diagnostic accuracy of laboratory testing algorithms that include nucleic acid amplification tests (NAATs) to detect the presence of C. difficile The systematic review and meta-analysis included eligible studies (those that had PICO [population, intervention, comparison, outcome] elements) that assessed the diagnostic accuracy of NAAT alone or following glutamate dehydrogenase (GDH) enzyme immunoassays (EIAs) or GDH EIAs plus C. difficile toxin EIAs (toxin). The diagnostic yield of NAAT for repeat testing after an initial negative result was also assessed. Two hundred thirty-eight studies met inclusion criteria. Seventy-two of these studies had sufficient data for meta-analysis. The strength of evidence ranged from high to insufficient. The uses of NAAT only, GDH-positive EIA followed by NAAT, and GDH-positive/toxin-negative EIA followed by NAAT are all recommended as American Society for Microbiology (ASM) best practices for the detection of the C. difficile toxin gene or organism. Meta-analysis of published evidence supports the use of testing algorithms that use NAAT alone or in combination with GDH or GDH plus toxin EIA to detect the presence of C. difficile in adults. There is insufficient evidence to recommend against repeat testing of the sample using NAAT after an initial negative result due to a lack of evidence of harm (i.e., financial, length of stay, or delay of treatment) as specified by the Laboratory Medicine Best Practices (LMBP) systematic review method in making such an assessment. Findings from this systematic review provide clarity to diagnostic testing strategies and highlight gaps, such as low numbers of GDH/toxin/PCR studies, in existing evidence on diagnostic performance, which can be used to guide future clinical research studies.

Point-Counterpoint: Differences between the European Committee on Antimicrobial Susceptibility Testing and Clinical and Laboratory Standards Institute Recommendations for Reporting Antimicrobial Susceptibility Results.

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.

Direct-from-Blood-Culture Disk Diffusion To Determine Antimicrobial Susceptibility of Gram-Negative Bacteria: Preliminary Report from the Clinical and Laboratory Standards Institute Methods Development and Standardization Working Group.

The performance of a disk diffusion test using broth from positive blood cultures as inoculum (direct disk diffusion [dDD]) was evaluated for a collection of 20 challenge isolates of Enterobacteriaceae, Acinetobacter baumannii, and Pseudomonas aeruginosa Isolates seeded into human blood were inoculated into Bactec Plus Aerobic/F, VersaTREK Redox 1, and BacT/Alert FA Plus bottles and incubated in the respective automated blood culture systems. Disk diffusion results were compared to reference disk diffusion results. Categorical agreement (CA) values for dDD, after removal of random errors due to natural MIC variation, were 87.8%, 88.4%, and 92.2% for the BacT/Alert, Bactec, and VersaTREK systems, respectively. No very major errors (VME) were observed, and major error (ME) rates were 3.0%, 2.3%, and 1.7%, respectively. Incubation of the dDD test samples for 6 h compared to incubation for 16 to 18 h resulted in 19.9% of tests having too light of growth to allow reading of zones of inhibition. Among the evaluable dDD tests, CA values were 58.9%, 76.6%, and 73.2% for the isolates seeded into the BacT/Alert, Bactec, and VersaTREK systems, respectively. VME rates for isolates seeded into these systems were 2.2%, 1.8%, and 3.0%, respectively, and ME rates were 25.4%, 6.1%, and 2.8%, respectively, at the 6-h reading. The best performance of dDD was found for blood cultures with bacterial concentrations in the range of 7.6 × 107 to 5.0 × 108 CFU/ml; CA values ranged from 94.7 to 96.2% for these concentrations after 18 h of incubation and from 76.9 to 84.1% after 6 h of incubation. These preliminary data demonstrate the potential accuracy of dDD testing by the clinical laboratory.

Assessing Clinical Microbiology Practice Guidelines: American Society for Microbiology Ad Hoc Committee on Evidence-Based Laboratory Medicine Practice Guidelines Assessment.

As part of the American Society for Microbiology (ASM) Evidence-Based Laboratory Medicine Practice Guidelines Committee of the Professional Practice Committee, an ad hoc committee was formed in 2014 to assess guidelines published by the committee using an assessment tool, Appraisal of Guidelines for Research Evaluation II (AGREE II). The AGREE II assessment helps reviewers determine whether published guidelines are robust, transparent, and clear in presenting practice recommendations in a standardized manner. Identifying strengths and weaknesses of practice guidelines by ad hoc assessments helps with improving future guidelines through the participation of key stakeholders. This minireview describes the development of the ad hoc committee and results from their review of several ASM best practices guidelines and a non-ASM practice guideline from the Emergency Nurses Association.

Automatic Digital Plate Reading for Surveillance Cultures.

The automation of specimen processing and culture workup has rapidly emerged in clinical microbiology laboratories throughout the world and more recently in the United States. While many U.S. laboratories have implemented some form of automated specimen processing and some have begun performing digital plate reading, automated colony analysis is just beginning to be utilized clinically. In this issue of the Journal of Clinical Microbiology, M. L. Faron et al. (J Clin Microbiol 54:2470-2475, 2016, http://dx.doi.org/10.1128/JCM.01040-16) report the results of their evaluation of the performance of the WASPLab Chromogenic Detection Module (CDM) for categorizing chromogenic agar plates as negative or "nonnegative" for vancomycin-resistant enterococci (VRE). Their major finding was 100% sensitivity for detection of "nonnegative" specimens using CDM compared to manual methods for specimens plated on two different types of VRE chromogenic agar plates. Additionally, utilization of digital plate reading in conjunction with automated colony analysis was predicted to result in significant savings based on greatly reduced labor costs.

Cost-Effectiveness of 30- Compared to 20-Milliliter Blood Cultures: a Retrospective Study.

The importance of blood culture (BC) volume for detection of bloodstream infections (BSIs) is documented. Recently, improved diagnostic sensitivity was demonstrated for 30- versus 20-ml BCs in adults (Cockerill FR, Wilson JW, Vetter EA, Goodman KM, Torgerson CA, Harmsen WS, Schleck CD, IIstrup DM, Washington JA, Wilson WR. Clin Infect Dis 38:1724-1730, 2004, http://dx.doi.org/10.1128/JCM.01314-11). Hospitals receive higher reimbursement for patients with documented septicemia. We determined the cost-effectiveness of 30-ml versus 20-ml BCs using results from our institution and previously published data. Positive BC results from 292 bacteremic episodes were reviewed. The costs of the reagents, equipment, phlebotomist, and technologist time were determined. The medical records department provided Medicare reimbursement (MR) data for patients with selected ICD-9 codes. These data provided an estimate of the annualized increase in MR versus costs associated with conversion to 30-ml BCs. MR for 464 annual primary BSIs was $24,808/episode. An expected 7.2% increase in BSIs detected using 30-ml BCs would add 34 additional cases annually and increase MR by $843,472. Comparative MR data for cases where septicemia complicated another diagnosis were available for 4 International Classification of Diseases, Ninth Revision (ICD-9) codes: laparoscopic cholecystectomy, biliary tract disorders, pneumonia, and cellulitis. The mean incremental MR was $9,667 per episode, which projected to a $483,350 revenue increase annually. The annual cost associated with conversion to 30-ml BCs was estimated to be $157,798. Thus, the potential net increase in hospital revenue would be $1,169,031 for 30-ml versus 20-ml BCs. Our results suggest that conversion to 30-ml BCs may not only improve patient care by detecting more BSIs but also increase hospital revenue substantially.

Point-Counterpoint: A Nucleic Acid Amplification Test for Streptococcus pyogenes Should Replace Antigen Detection and Culture for Detection of Bacterial Pharyngitis.

Nucleic acid amplification tests (NAATs) have frequently been the standard diagnostic approach when specific infectious agents are sought in a clinic specimen. They can be applied for specific agents such as S. pyogenes, or commercial multiplex NAATs for detection of a variety of pathogens in gastrointestinal, bloodstream, and respiratory infections may be used. NAATs are both rapid and sensitive. For many years, S. pyogenes testing algorithms used a rapid and specific group A streptococcal antigen test to screen throat specimens, followed, in some clinical settings, by a throat culture for S. pyogenes to increase the sensitivity of its detection. Now S. pyogenes NAATs are being used with increasing frequency. Given their accuracy, rapidity, and ease of use, should they replace antigen detection and culture for the detection of bacterial pharyngitis? Bobbi Pritt and Robin Patel of the Mayo Clinic, where S. pyogenes NAATs have been used for well over a decade with great success, will explain the advantages of this approach, while Richard (Tom) Thomson and Tom Kirn of the NorthShore University HealthSystem will discuss their concerns about this approach to diagnosing bacterial pharyngitis.

Air pollution particulate matter alters antimycobacterial respiratory epithelium innate immunity.

Inhalation exposure to indoor air pollutants and cigarette smoke increases the risk of developing tuberculosis (TB). Whether exposure to ambient air pollution particulate matter (PM) alters protective human host immune responses against Mycobacterium tuberculosis has been little studied. Here, we examined the effect of PM from Iztapalapa, a municipality of Mexico City, with aerodynamic diameters below 2.5 µm (PM2.5) and 10 µm (PM10) on innate antimycobacterial immune responses in human alveolar type II epithelial cells of the A549 cell line. Exposure to PM2.5 or PM10 deregulated the ability of the A549 cells to express the antimicrobial peptides human ß-defensin 2 (HBD-2) and HBD-3 upon infection with M. tuberculosis and increased intracellular M. tuberculosis growth (as measured by CFU count). The observed modulation of antibacterial responsiveness by PM exposure was associated with the induction of senescence in PM-exposed A549 cells and was unrelated to PM-mediated loss of cell viability. Thus, the induction of senescence and downregulation of HBD-2 and HBD-3 expression in respiratory PM-exposed epithelial cells leading to enhanced M. tuberculosis growth represent mechanisms by which exposure to air pollution PM may increase the risk of M. tuberculosis infection and the development of TB.

Systematic evaluation of commercial susceptibility testing methods for determining the in vitro activity of daptomycin versus Staphylococcus aureus and Enterococci.

We systematically evaluated 5 methods for testing daptomycin versus 48 Enterococcus faecalis, 51 Enterococcus faecium, and 50 Staphylococcus aureus isolates using (i and ii) broth microdilution (BMD) with 50-mg/liter calcium medium supplementation (reference method) and 30-mg/liter calcium medium supplementation (BMD30 method), (iii) Etest, and (iv and v) MicroScan panel 33 using 2 methods to prepare the bacterial inoculum (MicroScan turbidity and MicroScan Prompt). Isolates were categorized as susceptible (S) or nonsusceptible (NS) based on measured MICs. Essential (± 1 dilution) agreement (EA) and categorical (S/NS) agreement (CA) for each method were compared to the reference method. For E. faecium, categorical agreement was poor between the reference method and BMD30 as well as with the three commercial methods, with frequent false-NS results (30 for BMD30, 18 for Etest, 22 for MicroScan Prompt, and 25 for MicroScan turbidity). All E. faecalis isolates were judged to be S by the reference method; two of these isolates were categorized as NS using the BMD30 method, and one was categorized as NS by all three commercial methods. All S. aureus isolates were judged to be S using all five methods. MIC values determined by the comparator methods tended to be higher than those for the reference method, especially for E. faecium isolates. EAs between the reference BMD and BMD30, Etest, MicroScan Prompt, and MicroScan turbidity were 63%, 63%, 63%, and 56%, respectively, for E. faecium, 87%, 83%, 98%, and 80%, respectively, for E. faecalis, and all 100% for S. aureus.

Evidence of transmission of New Delhi metallo-ß-lactamase-producing Klebsiella pneumoniae through a gastrointestinal endoscope without an elevator channel.

OBJECTIVE: To investigate the source and transmission dynamics of an endoscope-associated New Delhi metallo-ß-lactamase-producing Klebsiella pneumonia (NDM-KP) outbreak. DESIGN: Epidemiological and genomic investigation. SETTING: Academic acute care hospital in New Jersey. PATIENTS: Five patients with active NDM-KP infection identified on clinical isolates, and four NDM-KP colonized patients identified via rectal swab screening. RESULTS: Over a twelve-month period, nine patients were identified with NDM-KP infection or colonization. Whole-genome sequencing (WGS) revealed that all of the identified cases were related by 25 mutational events or less. Seven of the cases were linked to gastrointestinal endoscopic procedures (four clinical cases and three positive screens among patients exposed to endoscopes suspected of transmission). Two cases demonstrated delayed transmission that occurred five months after the initial outbreak, likely through shared usage of a non-therapeutic gastroscope without an elevator channel. CONCLUSIONS: Although all endoscope cultures in our investigation were negative, the epidemiological link to gastrointestinal endoscopes, the high degree of relatedness via WGS, and the identification of asymptomatic NDM-KP colonization among patients exposed to shared endoscopes make the endoscopic mode of transmission most likely. This investigation highlights the probable transmission of NDM-KP via a gastroscope without an elevator channel, observed several months after an initial outbreak. We hypothesize that persistent mechanical defects may have contributed to the delayed device-related transmission of NDM-KP.

Laboratory diagnosis of HIV in adults: a review of current methods.

Of the estimated 1.2 million people infected with human immunodeficiency virus (HIV) in the United States, 20% are unaware of their diagnosis. Improved methods of HIV testing could decrease this number, as well as identify those who have recently acquired HIV infection and are at the most critical stage of infectivity. People with acute HIV infection have demonstrated enhanced transmission of HIV in multiple epidemiologic and pathogenetic studies. More than 50 000 HIV infections occur annually in the United States, and 30%-50% have been attributed to persons with recent infection. The original HIV diagnostic testing algorithm was developed by the Centers for Disease Control and Prevention in 1989. Recently proposed alterations to the algorithm would incorporate advancements made in HIV diagnostic testing, thereby increasing sensitivity while reducing turnaround time and cost. Improved diagnosis of acute HIV, and HIV type 2 in particular, would be expected. Knowledge of the available laboratory methods for HIV diagnosis is essential in the fight against the spread of HIV.

Impact of phenotypic rapid diagnostic assay on duration of empiric antibiotics for gram-negative bacteremia.

Objective: Rapid diagnostic tests (RDTs) are increasingly being implemented as antimicrobial stewardship tools to facilitate antibiotic modification and reduce complications related to their overutilization. We measured the clinical impact of a phenotypic RDT with antimicrobial stewardship (AMS) in the setting of gram-negative bacteremia. Setting and participants: In this single-center retrospective cohort study, we evaluated adult patients with gram-negative bacteremia who received at least 72 hours of an antibiotic. Methods: The primary outcome was the duration of empiric antibiotic therapy for gram-negative bacteremia. Secondary outcomes included time-to-directed therapy, proportion of modifications, hospital length of stay (LOS), and subsequent infection with a multidrug-resistant organism (MDRO) or C. difficile infection (CDI). Results: The duration of empiric antibiotics decreased in the RDT+AMS group (4 days vs 2 days; P < .01). Time to directed therapy decreased from 75.0 to 27.9 hours (P < .01). Conclusions: The clinical outcomes of LOS, MDRO, and CDI were reduced. The phenotypic RDT demonstrated an improvement in stewardship measures and clinical outcomes.

Molecular and Clinical Epidemiology of SARS-CoV-2 Infection among Vaccinated and Unvaccinated Individuals in a Large Healthcare Organization from New Jersey.

New Jersey was among the first states impacted by the COVID-19 pandemic, with one of the highest overall death rates in the nation. Nevertheless, relatively few reports have been published focusing specifically on New Jersey. Here we report on molecular, clinical, and epidemiologic observations, from the largest healthcare network in the state, in a cohort of vaccinated and unvaccinated individuals with laboratory-confirmed SARS-CoV-2 infection. We conducted molecular surveillance of SARS-CoV-2-positive nasopharyngeal swabs collected in nine hospitals from December 2020 through June 2022, using both whole genome sequencing (WGS) and a real-time RT-PCR screening assay targeting spike protein mutations found in variants of concern (VOCs) within our region. De-identified clinical data were obtained retrospectively, including demographics, COVID-19 vaccination status, ICU admission, ventilator support, mortality, and medical history. Statistical analyses were performed to identify associations between SARS-CoV-2 variants, vaccination status, clinical outcomes, and medical risk factors. A total of 5007 SARS-CoV-2-positive nasopharyngeal swabs were successfully screened and/or sequenced. Variant screening identified three predominant VOCs, including Alpha (n = 714), Delta (n = 1877), and Omicron (n = 1802). Omicron isolates were further sub-typed as BA.1 (n = 899), BA.2 (n = 853), or BA.4/BA.5 (n = 50); the remaining 614 isolates were classified as "Other". Approximately 31.5% (1577/5007) of the samples were associated with vaccine breakthrough infections, which increased in frequency following the emergence of Delta and Omicron. Severe clinical outcomes included ICU admission (336/5007 = 6.7%), ventilator support (236/5007 = 4.7%), and mortality (430/5007 = 8.6%), with increasing age being the most significant contributor to each (p < 0.001). Unvaccinated individuals accounted for 79.7% (268/336) of ICU admissions, 78.3% (185/236) of ventilator cases, and 74.4% (320/430) of deaths. Highly significant (p < 0.001) increases in mortality were observed in individuals with cardiovascular disease, hypertension, cancer, diabetes, and hyperlipidemia, but not with obesity, thyroid disease, or respiratory disease. Significant differences (p < 0.001) in clinical outcomes were also noted between SARS-CoV-2 variants, including Delta, Omicron BA.1, and Omicron BA.2. Vaccination was associated with significantly improved clinical outcomes in our study, despite an increase in breakthrough infections associated with waning immunity, greater antigenic variability, or both. Underlying comorbidities contributed significantly to mortality in both vaccinated and unvaccinated individuals, with increasing risk based on the total number of comorbidities. Real-time RT-PCR-based screening facilitated timely identification of predominant variants using a minimal number of spike protein mutations, with faster turnaround time and reduced cost compared to WGS. Continued evolution of SARS-CoV-2 variants will likely require ongoing surveillance for new VOCs, with real-time assessment of clinical impact.

Clostridium difficile MazF toxin exhibits selective, not global, mRNA cleavage.

Clostridium difficile is an important, emerging nosocomial pathogen. The transition from harmless colonization to disease is typically preceded by antimicrobial therapy, which alters the balance of the intestinal flora, enabling C. difficile to proliferate in the colon. One of the most perplexing aspects of the C. difficile infectious cycle is its ability to survive antimicrobial therapy and transition from inert colonization to active infection. Toxin-antitoxin (TA) systems have been implicated in facilitating persistence after antibiotic treatment. We identified only one TA system in C. difficile strain 630 (epidemic type X), designated MazE-cd and MazF-cd, a counterpart of the well-characterized Escherichia coli MazEF TA system. This E. coli MazF toxin cleaves mRNA at ACA sequences, leading to global mRNA degradation, growth arrest, and death. Likewise, MazF-cd expression in E. coli or Clostridium perfringens resulted in growth arrest. Primer extension analysis revealed that MazF-cd cleaved RNA at the five-base consensus sequence UACAU, suggesting that the mRNAs susceptible to cleavage comprise a subset of total mRNAs. In agreement, we observed differential cleavage of several mRNAs by MazF-cd in vivo, revealing a direct correlation between the number of cleavage recognition sites within a given transcript and its susceptibility to degradation by MazF-cd. Interestingly, upon detailed statistical analyses of the C. difficile transcriptome, the major C. difficile virulence factor toxin B (TcdB) and CwpV, a cell wall protein involved in aggregation, were predicted to be significantly resistant to MazF-cd cleavage.

Effective utilization of evolving methods for the laboratory diagnosis of Clostridium difficile infection.

Physicians should understand the performance characteristics of evolving laboratory tests used to diagnose Clostridium difficile infection if they are to correctly integrate test results with clinical information and formulate an appropriate therapeutic intervention for patients with antibiotic-associated diarrhea.

Mycobacterium chelonae-abscessus complex associated with sinopulmonary disease, Northeastern USA.

Members of the Mycobacterium chelonae-abscessus complex represent Mycobacterium species that cause invasive infections in immunocompetent and immunocompromised hosts. We report the detection of a new pathogen that had been misidentified as M. chelonae with an atypical antimicrobial drug susceptibility profile. The discovery prompted a multicenter investigation of 26 patients. Almost all patients were from the northeastern United States, and most had underlying sinus or pulmonary disease. Infected patients had clinical features similar to those with M. abscessus infections. Taxonomically, the new pathogen shared molecular identity with members of the M. chelonae-abscessus complex. Multilocus DNA target sequencing, DNA-DNA hybridization, and deep multilocus sequencing (43 full-length genes) support a new taxon for these microorganisms. Because most isolates originated in Pennsylvania, we propose the name M. franklinii sp. nov. This investigation underscores the need for accurate identification of Mycobacterium spp. to detect new pathogens implicated in human disease.

A colonization factor links Vibrio cholerae environmental survival and human infection.

Many bacteria that cause diseases must be able to survive inside and outside the host. Attachment to and colonization of abiotic or biotic surfaces is a common mechanism by which various microorganisms enhance their ability to survive in diverse environments. Vibrio cholerae is a Gram-negative aquatic bacillus that is often found in the environment attached to the chitinous exoskeletons of zooplankton. It has been suggested that attachment to zooplankton enhances environmental survival of Vibrio spp., probably by providing both an abundant source of carbon and nitrogen and protection from numerous environmental challenges. On ingestion by humans, some serogroups of V. cholerae cause the diarrhoeal disease cholera. The pathophysiology of cholera is a result of the effects of cholera toxin on intestinal epithelial cells. For sufficient quantities of cholera toxin to reach the intestinal epithelium and to produce clinical symptoms, colonization of the small bowel must occur. Because most V. cholerae do not colonize humans, but all probably require strategies for survival in the environment, we considered that colonization factors selected for in the environment may be the same as those required for intestinal colonization of humans. In support of this hypothesis, here we have identified a single protein required for efficient intestinal colonization that mediates attachment to both zooplankton and human epithelial cells by binding to a sugar present on both surfaces.