A Baker's Dozen of Top Antimicrobial Stewardship Intervention Publications in 2022.

Keeping abreast of the antimicrobial stewardship-related articles published each year is challenging. The Southeastern Research Group Endeavor identified antimicrobial stewardship-related, peer-reviewed literature that detailed an actionable intervention during 2022. The top 13 publications were selected using a modified Delphi technique. These manuscripts were reviewed to highlight actionable interventions used by antimicrobial stewardship programs to capture potentially effective strategies for local implementation.

Epidemiology and treatment of invasive Bartonella spp. infections in the United States.

OBJECTIVES: Bartonella spp., renowned for cat-scratch disease, has limited reports of dissemination. Tissue and blood cultures have limitations in detecting this fastidious pathogen. Molecular testing (polymerase chain reaction, PCR) and cell-free DNA have provided an avenue for diagnoses. This retrospective observational multicenter study describes the incidence of disseminated Bartonella spp. and treatment-related outcomes. METHODS: Inclusion criteria were diagnosis of bartonellosis via diagnosis code, serology testing of blood, polymerase chain reaction (PCR) of blood, 16/18S tests of blood or tissue, cultures of blood or tissue, or cell-free DNA of blood or tissue from January 1, 2014, through September 1, 2021. Exclusions were patients who did not receive treatment, insufficient data on treatment course, absence of dissemination, or retinitis as dissemination. RESULTS: Patients were primarily male (n = 25, 61.0%), white (n = 28, 68.3%), with mean age of 50 years (SD 14.4), and mean Charlson comorbidity index of 3.5 (SD 2.1). Diagnosis was primarily by serology (n = 34, 82.9%), with Bartonella henselae (n = 40, 97.6%) as the causative pathogen. Treatment was principally doxycycline with rifampin (n = 17, 41.5%). Treatment failure occurred in 16 (39.0%) patients, due to escalation of therapy during treatment (n = 5, 31.3%) or discontinuation of therapy due to an adverse event or tolerability (n = 5, 31.3%). CONCLUSIONS: In conclusion, this is the largest United States-based cohort of disseminated Bartonella spp. infections to date with a reported 39% treatment failure. This adds to literature supporting obtaining multiple diagnostic tests when Bartonella is suspected and describes treatment options.

Nephrotoxic Risk Associated With Combination Therapy of Vancomycin and Piperacillin-Tazobactam in Critically Ill Patients With Chronic Kidney Disease.

Background: The combination of vancomycin and piperacillin-tazobactam (VPT) has been associated with acute kidney injury (AKI) in hospitalized patients when compared to similar combinations. Additional studies examining this nephrotoxic risk in critically ill patients have not consistently demonstrated the aforementioned association. Furthermore, patients with baseline renal dysfunction have been excluded from almost all of these studies, creating a need to examine the risk in this patient population. Methods: This was a retrospective cohort analysis of critically ill adults with baseline chronic kidney disease (CKD) who received vancomycin plus an anti-pseudomonal beta-lactam at Emory University Hospital. The primary outcome was incidence of AKI. Secondary outcomes included stage of AKI, time to development of AKI, time to return to baseline renal function, new requirement for renal replacement therapy, intensive care unit and hospital length of stay, and in-hospital mortality. Results: A total of 109 patients were included. There was no difference observed in the primary outcome between the VPT (50%) and comparator (58%) group (P = .4), stage 2 or 3 AKI (15.9% vs 6%; P = .98), time to AKI development (1.7 vs 2 days; P = .5), time to return to baseline renal function (4 vs 3 days; P = .2), new requirement for RRT (4.5% vs 1.5%; P = .3), ICU length of stay (7.3 vs 7.4 days; P = .9), hospital length of stay (19.3 vs 20.1 days; P = .87), or in-hospital mortality (15.9% vs 10.8%; P = .4). A significant difference was observed in the duration of antibiotic exposure (3.32 vs 2.62 days; P = .045 days). Conclusion: VPT was not associated with an increased risk of AKI or adverse renal outcomes. Our findings suggest that the use of this antibiotic combination should not be avoided in this patient population. More robust prospective studies are warranted to confirm these findings.

Long-term Dalbavancin for Suppression of Gram-Positive Chronic Left Ventricular Assist Device Infections.

Background: Infection is the leading cause of morbidity and mortality in patients with left ventricular assist devices (LVADs). Prolonged suppressive therapy should be strongly considered and is often used in patients with recurrent infections when source control cannot be achieved. Dalbavancin is a promising option in patients with LVADs requiring prolonged durations of antibiotic therapy, especially when no oral alternatives are available. Methods: This case series included 8 patients receiving dalbavancin for the long-term suppression of gram-positive infections at Emory University Hospital and Emory St Joseph's Hospital. Results: The overall incidence of breakthrough infections occurred in 5 of the 8 patients included in the study. One patient experienced an early breakthrough infection within 1 month of dalbavancin initiation. Another experienced a breakthrough infection within 3 and 6 months of dalbavancin initiation, and the final 3 patients experienced a breakthrough infection within 6 and 12 months. The average duration of dalbavancin suppression therapy among all patients was 229 days, and no adverse effects were reported. Conclusions: Dalbavancin is a promising option in patients who require long-term suppression for chronic gram-positive LVAD infections, given its unique pharmacokinetic profile and excellent tissue penetration. The use of biweekly dalbavancin infusions in our 8 patients prevented infection for an extended period of time despite some of the patients not being able to consistently receive infusions. Larger studies are needed to determine the efficacy and safety of using dalbavancin for long-term suppression of gram-positive LVAD infections.

MAIRA: Protein-based Analysis of MinION Reads on a Laptop.

Third-generation sequencing technologies are being increasingly used in microbiome research and this has given rise to new challenges in computational microbiome analysis. Oxford Nanopore's MinION is a portable sequencer that streams data that can be basecalled on-the-fly. Here we give an introduction to the MAIRA software, which is designed to analyze MinION sequencing reads from a microbiome sample, as they are produced in real-time, on a laptop. The software processes reads in batches and updates the presented analysis after each batch. There are two analysis steps: First, protein alignments are calculated to determine which genera might be present in a sample. When strong evidence for a genus is found, then, in a second step, a more detailed analysis is performed by aligning the reads against the proteins of all species in the detected genus. The program presents a detailed analysis of species, antibiotic resistance genes, and virulence factors.

A Baker's Dozen of Top Antimicrobial Stewardship Intervention Publications in Non-Hospital Care Settings in 2021.

The scope of antimicrobial stewardship programs has expanded beyond the acute hospital setting. The need to optimize antimicrobial use in emergency departments, urgent, primary, and specialty care clinics, nursing homes, and long-term care facilities prompted the development of core elements of stewardship programs in these settings. Identifying the most innovative and well-designed stewardship literature in these novel stewardship areas can be challenging. The Southeastern Research Group Endeavor (SERGE-45) network evaluated antimicrobial stewardship-related, peer-reviewed literature published in 2021 that detailed actionable interventions specific to the nonhospital setting. The top 13 publications were summarized following identification using a modified Delphi technique. This article highlights the selected interventions and may serve as a key resource for expansion of antimicrobial stewardship programs beyond the acute hospital setting.

Efficacy of Noncarbapenem ß-Lactams Compared to Carbapenems for Extended-Spectrum ß-Lactamase-Producing Enterobacterales Urinary Tract Infections.

BACKGROUND: Extended-spectrum ß-lactamase (ESBL)-producing Enterobacterales are frequent causes of urinary tract infections (UTIs). Severe infections caused by ESBL Enterobacterales are often treated with carbapenems, but optimal treatment for less severe infections such as UTIs is unclear. METHODS: This retrospective cohort study included patients admitted to 4 hospitals in an academic healthcare system with an ESBL UTI treated with either a noncarbapenem ß-lactam (NCBL) or a carbapenem for at least 48 hours from 1 April 2014 to 30 April 2018. Those who received an NCBL were compared to those receiving a carbapenem, with a primary outcome of hospital length of stay (LOS) and secondary outcomes of clinical and microbiological response, days until transition to oral therapy, rate of relapsed infection, and rate of secondary infections with a multidrug-resistant organism. RESULTS: Characteristics were similar among patients who received carbapenems (n = 321) and NCBLs (n = 171). There was no difference in LOS for the NCBL group compared to the carbapenem group (13 days vs 15 days, P = .66). The NCBL group had higher rates of microbiologic eradication (98% vs 92%, P = .002), shorter time to transition to oral therapy (5 days vs 9 days, P < .001), shorter overall durations of therapy (7 days vs 10 days, P < .001), and lower rates of relapsed infections (5% vs 42%, P = .0003). CONCLUSIONS: Patients treated with NCBLs had similar LOS, higher rates of culture clearance, and shorter durations of antibiotic therapy compared to patients treated with carbapenems, suggesting that treatment for ESBL UTIs should not be selected solely based on phenotypic resistance.

Software for the frontiers of quantum chemistry: An overview of developments in the Q-Chem 5 package.

This article summarizes technical advances contained in the fifth major release of the Q-Chem quantum chemistry program package, covering developments since 2015. A comprehensive library of exchange-correlation functionals, along with a suite of correlated many-body methods, continues to be a hallmark of the Q-Chem software. The many-body methods include novel variants of both coupled-cluster and configuration-interaction approaches along with methods based on the algebraic diagrammatic construction and variational reduced density-matrix methods. Methods highlighted in Q-Chem 5 include a suite of tools for modeling core-level spectroscopy, methods for describing metastable resonances, methods for computing vibronic spectra, the nuclear-electronic orbital method, and several different energy decomposition analysis techniques. High-performance capabilities including multithreaded parallelism and support for calculations on graphics processing units are described. Q-Chem boasts a community of well over 100 active academic developers, and the continuing evolution of the software is supported by an "open teamware" model and an increasingly modular design.

Reductions in inpatient fluoroquinolone use and postdischarge Clostridioides difficile infection (CDI) from a systemwide antimicrobial stewardship intervention.

Objective: To determine the impact of an inpatient stewardship intervention targeting fluoroquinolone use on inpatient and postdischarge Clostridioides difficile infection (CDI). Design: We used an interrupted time series study design to evaluate the rate of hospital-onset CDI (HO-CDI), postdischarge CDI (PD-CDI) within 12 weeks, and inpatient fluoroquinolone use from 2 years prior to 1 year after a stewardship intervention. Setting: An academic healthcare system with 4 hospitals. Patients: All inpatients hospitalized between January 2017 and September 2020, excluding those discharged from locations caring for oncology, bone marrow transplant, or solid-organ transplant patients. Intervention: Introduction of electronic order sets designed to reduce inpatient fluoroquinolone prescribing. Results: Among 163,117 admissions, there were 683 cases of HO-CDI and 1,104 cases of PD-CDI. In the context of a 2% month-to-month decline starting in the preintervention period (P < .01), we observed a reduction in fluoroquinolone days of therapy per 1,000 patient days of 21% after the intervention (level change, P < .05). HO-CDI rates were stable throughout the study period. In contrast, we also detected a change in the trend of PD-CDI rates from a stable monthly rate in the preintervention period to a monthly decrease of 2.5% in the postintervention period (P < .01). Conclusions: Our systemwide intervention reduced inpatient fluoroquinolone use immediately, but not HO-CDI. However, a downward trend in PD-CDI occurred. Relying on outcome measures limited to the inpatient setting may not reflect the full impact of inpatient stewardship efforts.

A young adult with COVID-19 and multisystem inflammatory syndrome in children (MIS-C)-like illness: a case report.

BACKGROUND: A healthy 25-year-old woman developed COVID-19 disease with clinical characteristics resembling Multisystem Inflammatory Syndrome in Children (MIS-C), a rare form of COVID-19 described primarily in children under 21 years of age. CASE PRESENTATION: The patient presented with 1 week of weakness, dyspnea, and low-grade fevers, followed by mild cough, sore throat, vomiting, diarrhea, and lymph node swelling. She was otherwise healthy, with no prior medical history. Her hospital course was notable for profound acute kidney injury, leukocytosis, hypotension, and cardiac dysfunction requiring ICU admission and vasopressor support. MIS-C-like illness secondary to COVID-19 was suspected due to physical exam findings of conjunctivitis, mucositis, and shock. She improved following IVIG, aspirin, and supportive care, and was discharged on hospital day 5. CONCLUSION: MIS-C-like illness should be considered in adults presenting with atypical clinical findings and concern for COVID-19. Further research is needed to support the role of IVIG and aspirin in this patient population.

MAIRA- real-time taxonomic and functional analysis of long reads on a laptop.

BACKGROUND: Advances in mobile sequencing devices and laptop performance make metagenomic sequencing and analysis in the field a technologically feasible prospect. However, metagenomic analysis pipelines are usually designed to run on servers and in the cloud. RESULTS: MAIRA is a new standalone program for interactive taxonomic and functional analysis of long read metagenomic sequencing data on a laptop, without requiring external resources. The program performs fast, online, genus-level analysis, and on-demand, detailed taxonomic and functional analysis. It uses two levels of frame-shift-aware alignment of DNA reads against protein reference sequences, and then performs detailed analysis using a protein synteny graph. CONCLUSIONS: We envision this software being used by researchers in the field, when access to servers or cloud facilities is difficult, or by individuals that do not routinely access such facilities, such as medical researchers, crop scientists, or teachers.

Pursuing Experimental Reproducibility: An Efficient Protocol for the Preparation of Cerebrospinal Fluid Samples for NMR-based Metabolomics and Analysis of Sample Degradation.

NMR-based metabolomics investigations of human biofluids offer great potential to uncover new biomarkers. In contrast to protocols for sample collection and biobanking, procedures for sample preparation prior to NMR measurements are still heterogeneous, thus compromising the comparability of the resulting data. Herein, we present results of an investigation of the handling of cerebrospinal fluid (CSF) samples for NMR metabolomics research. Origins of commonly observed problems when conducting NMR experiments on this type of sample are addressed, and suitable experimental conditions in terms of sample preparation and pH control are discussed. Sample stability was assessed by monitoring the degradation of CSF samples by NMR, hereby identifying metabolite candidates, which are potentially affected by sample storage. A protocol was devised yielding consistent spectroscopic data as well as achieving overall sample stability for robust analysis. We present easy to adopt standard operating procedures with the aim to establish a shared sample handling strategy that facilitates and promotes inter-laboratory comparison, and the analysis of sample degradation provides new insights into sample stability.

The impact of an electronic medical record nudge on reducing testing for hospital-onset Clostridioides difficile infection.

OBJECTIVE: To determine the effect of an electronic medical record (EMR) nudge at reducing total and inappropriate orders testing for hospital-onset Clostridioides difficile infection (HO-CDI). DESIGN: An interrupted time series analysis of HO-CDI orders 2 years before and 2 years after the implementation of an EMR intervention designed to reduce inappropriate HO-CDI testing. Orders for C. difficile testing were considered inappropriate if the patient had received a laxative or stool softener in the previous 24 hours. SETTING: Four hospitals in an academic healthcare network. PATIENTS: All patients with a C. difficile order after hospital day 3. INTERVENTION: Orders for C. difficile testing in patients administered a laxative or stool softener in <24 hours triggered an EMR alert defaulting to cancellation of the order ("nudge"). RESULTS: Of the 17,694 HO-CDI orders, 7% were inappropriate (8% prentervention vs 6% postintervention; P < .001). Monthly HO-CDI orders decreased by 21% postintervention (level-change rate ratio [RR], 0.79; 95% confidence interval [CI], 0.73-0.86), and the rate continued to decrease (postintervention trend change RR, 0.99; 95% CI, 0.98-1.00). The intervention was not associated with a level change in inappropriate HO-CDI orders (RR, 0.80; 95% CI, 0.61-1.05), but the postintervention inappropriate order rate decreased over time (RR, 0.95; 95% CI, 0.93-0.97). CONCLUSION: An EMR nudge to minimize inappropriate ordering for C. difficile was effective at reducing HO-CDI orders, and likely contributed to decreasing the inappropriate HO-CDI order rate after the intervention.

Impact of fluoroquinolone prophylaxis on infectious-related outcomes after hematopoietic cell transplantation.

BACKGROUND: Patients immediately post-hematopoietic cell transplantation are at high risk for bacteremia. Judicious prophylactic antimicrobial utilization must balance anticipated benefits (reduction infections) versus risk (bacterial resistance, Clostridium difficile) . OBJECTIVE: To compare infectious outcomes (primary: incidence bacteremia; secondary: febrile neutropenia, C. difficile, susceptibility of bacteremia, time to discharge and 30-day mortality) between hematopoietic cell transplantation who received fluoroquinolone prophylaxis to those who did not. METHODS: A local institutional review board-approved retrospective study was conducted on all hematopoietic cell transplantation patients ( n = 171) comparing those who received fluoroquinolone prophylaxis ( n = 105) to those who did not ( n = 66). Data included infectious outcomes and mortality for the first 30 days post-hematopoietic cell transplantation. Chi-squared was performed for categorical variables (GraphPad Software Inc., 2015). Secondary analysis compared outcomes within autologous and allogeneic sub-groups. RESULTS: Bacteremia was significantly lower for the overall cohort receiving fluoroquinolone (median duration eight days) versus those without fluoroquinolone (15.2% vs. 31.8%; P < 0.01). No difference was seen in C. difficile infection ( P = 0.81) or 30-day mortality (2.9% vs. 4.5%; P = 0.67). In the autologous sub-group ( n = 115), bacteremia was significantly lower in the fluoroquinolone cohort (8.5% vs. 27.3%; P = 0.0069), while no differences were seen in C. difficile infection ( P = 1) or 30-day mortality ( P = 1). In the allogeneic sub-group ( n = 56), there was no difference between those with and without fluoroquinolone in bacteremia (29.4% vs. 40.9%; P = 0.4) or C. difficile ( P = 0.72); however, there was a trend toward improved 30-day mortality (2.9% vs. 9.1%; P = 0.55). CONCLUSIONS: Fluoroquinolone prophylaxis reduces incidence of bacteremia in autologous hematopoietic cell transplantation without increasing C. difficile after hematopoietic cell transplantation.

MEGAN-LR: new algorithms allow accurate binning and easy interactive exploration of metagenomic long reads and contigs.

BACKGROUND: There are numerous computational tools for taxonomic or functional analysis of microbiome samples, optimized to run on hundreds of millions of short, high quality sequencing reads. Programs such as MEGAN allow the user to interactively navigate these large datasets. Long read sequencing technologies continue to improve and produce increasing numbers of longer reads (of varying lengths in the range of 10k-1M bps, say), but of low quality. There is an increasing interest in using long reads in microbiome sequencing, and there is a need to adapt short read tools to long read datasets. METHODS: We describe a new LCA-based algorithm for taxonomic binning, and an interval-tree based algorithm for functional binning, that are explicitly designed for long reads and assembled contigs. We provide a new interactive tool for investigating the alignment of long reads against reference sequences. For taxonomic and functional binning, we propose to use LAST to compare long reads against the NCBI-nr protein reference database so as to obtain frame-shift aware alignments, and then to process the results using our new methods. RESULTS: All presented methods are implemented in the open source edition of MEGAN, and we refer to this new extension as MEGAN-LR (MEGAN long read). We evaluate the LAST+MEGAN-LR approach in a simulation study, and on a number of mock community datasets consisting of Nanopore reads, PacBio reads and assembled PacBio reads. We also illustrate the practical application on a Nanopore dataset that we sequenced from an anammox bio-rector community. REVIEWERS: This article was reviewed by Nicola Segata together with Moreno Zolfo, Pete James Lockhart and Serghei Mangul. CONCLUSION: This work extends the applicability of the widely-used metagenomic analysis software MEGAN to long reads. Our study suggests that the presented LAST+MEGAN-LR pipeline is sufficiently fast and accurate.

Exploring the Role of Solvent on Carbohydrate-Aryl Interactions by Diffusion NMR-Based Studies.

Carbohydrate-protein interactions play an important role in many molecular recognition processes. An exquisite combination of multiple factors favors the interaction of the receptor with one specific type of sugar, whereas others are excluded. Stacking CH-aromatic interactions within the binding site provide a relevant contribution to the stabilization of the resulting sugar-protein complex. Being experimentally difficult to detect and analyze, the key CH-π interaction features have been very often dissected using a variety of techniques and simple model systems. In the present work, diffusion NMR spectroscopy has been employed to separate the components of sugar mixtures in different solvents on the basis of their differential ability to interact through CH-π interactions with one particular aromatic cosolute in solution. The experimental data show that the properties of the solvent did also influence the diffusion behavior of the sugars present in the mixture, inhibiting or improving their separation. Overall, the results showed that, for the considered monosaccharide derivatives, their diffusion coefficient values and, consequently, their apparent molecular sizes and/or shapes depend on the balance between solute/cosolute as well as solute/solvent interactions. Thus, in certain media and in the presence of the aromatic cosolute, the studied saccharides that are more suited to display CH-π interactions exhibited a lower diffusion coefficient than the noncomplexing sugars in the mixture. However, when dissolved in another medium, the interaction with the solvent strongly competes with that of the aromatic cosolute.

Influence of Hofmeister Ions on the Structure of Proline-Based Peptide Models: A Combined Experimental and Molecular Modeling Study.

The influence of three sodium salts, covering a wide range of the Hofmeister series, on the conformation of three proline-based peptide models in aqueous solution is examined using a combination of nuclear magnetic resonance spectroscopy and molecular dynamics simulations. The anions preferentially interact with the cis conformers of the peptide models, which is rationalized by the respective electrostatic potential surfaces. These preferred interactions have a strong impact on the thermodynamics of the cis/trans equilibria, leading to a higher population of the cis conformers. In distinct cases, these equilibria are nearly independent of temperature, showing that the salts are also able to stabilize the conformers over wide temperature ranges.

Computing all hybridization networks for multiple binary phylogenetic input trees.

BACKGROUND: The computation of phylogenetic trees on the same set of species that are based on different orthologous genes can lead to incongruent trees. One possible explanation for this behavior are interspecific hybridization events recombining genes of different species. An important approach to analyze such events is the computation of hybridization networks. RESULTS: This work presents the first algorithm computing the hybridization number as well as a set of representative hybridization networks for multiple binary phylogenetic input trees on the same set of taxa. To improve its practical runtime, we show how this algorithm can be parallelized. Moreover, we demonstrate the efficiency of the software Hybroscale, containing an implementation of our algorithm, by comparing it to PIRNv2.0, which is so far the best available software computing the exact hybridization number for multiple binary phylogenetic trees on the same set of taxa. The algorithm is part of the software Hybroscale, which was developed specifically for the investigation of hybridization networks including their computation and visualization. Hybroscale is freely available(1) and runs on all three major operating systems. CONCLUSION: Our simulation study indicates that our approach is on average 100 times faster than PIRNv2.0. Moreover, we show how Hybroscale improves the interpretation of the reported hybridization networks by adding certain features to its graphical representation.

A rapid analytical method for the quantification of paclitaxel in rat plasma and brain tissue by high-performance liquid chromatography and tandem mass spectrometry.

RATIONALE: Paclitaxel, an antitumor agent for the treatment of several types of cancers, has recently been reported to cause impaired cognitive function and neuropathic pain in humans. To assess the effects of paclitaxel on the central nervous system, a sensitive and accurate method is required to quantify paclitaxel concentrations in plasma and brain tissue obtained from rodents receiving paclitaxel. METHODS: The biological samples were prepared by liquid-liquid extraction and separated by a 3.5 min reversed-phase liquid chromatography (RPLC) method using a BDS Hypersil C8 column under isocratic conditions. Paclitaxel was quantified using multiple reaction monitoring (MRM) with a triple quadrupole tandem mass spectrometer working in the positive electrospray ionization (ESI+) mode. A stable isotope labeled analogue of paclitaxel was used as the internal standard (IS). RESULTS: The method was validated to be precise and accurate within the dynamic range of 0.5-100 ng/mL based on 100 µL plasma and 1.5-300 ng/g based on 33 mg of brain tissue in homogenate. This method was applied to samples from 2 mg/kg intravenously dosed rats. The plasma concentrations were observed to be 26.62 ± 8.93 ng/mL and brain concentrations 11.08 ± 4.18 ng/g when measured 4 h post-dose. CONCLUSIONS: This rapid LC/MS/MS method was validated to be sensitive, specific, precise and accurate for the quantification of paclitaxel in rat plasma and brain tissue homogenate. Application of the method to study samples provided sufficient proof of blood-brain barrier penetration of paclitaxel, allowing further investigation of its influence on the central nervous system.

A first step toward computing all hybridization networks for two rooted binary phylogenetic trees.

Recently, considerable effort has been put into developing fast algorithms to reconstruct a rooted phylogenetic network that explains two rooted phylogenetic trees and has a minimum number of hybridization vertices. With the standard app1235roach to tackle this problem being combinatorial, the reconstructed network is rarely unique. From a biological point of view, it is therefore of importance to not only compute one network, but all possible networks. In this article, we make a first step toward approaching this goal by presenting the first algorithm--called ALLMAAFs--that calculates all maximum-acyclic-agreement forests for two rooted binary phylogenetic trees on the same set of taxa.