Application of mechanistic modelling in membrane and fiber chromatography for purification of biotherapeutics - A review.

Mechanistic modelling is a simulation tool which has been effectively applied in downstream bioprocessing to model resin chromatography. Membrane and fiber chromatography are newer approaches that offer higher rates of mass transfer and consequently higher flow rates and reduced processing times. This review describes the key considerations in the development of mechanistic models for these unit operations. Mass transfer is less complex than in resin columns, but internal housing volumes can make modelling difficult, particularly for laboratory-scale devices. Flow paths are often non-linear and the dead volume is often a larger fraction of the overall volume, which may require more complex hydrodynamic models to capture residence time distributions accurately. In this respect, the combination of computational fluid dynamics with appropriate protein binding models is emerging as an ideal approach.

Sustained Transmission of Neisseria gonorrhoeae with High-Level Resistance to Azithromycin, in Indianapolis, Indiana, 2017-2018.

BACKGROUND: Since 2014, Neisseria gonorrhoeae azithromycin (AZM) susceptibility has declined in the United States, but high-level AZM resistance (HL-AZMR) has been infrequent and sporadic. We describe a cluster of 14 N. gonorrhoeae isolates with HL-AZMR identified in Indianapolis over 13 months. METHODS: N. gonorrhoeae culture specimens (genital and extragenital) were collected from attendees of the Bell Flower Clinic. Isolates underwent antimicrobial susceptibility testing (AST) using Etest. AZM minimum inhibitory concentrations ≥256 µg/mL were classified as HL-AZMR. Local disease intervention specialists interviewed patients whose isolates demonstrated HL-AZMR and conducted partner services. Relatedness of isolates was investigated by genomic analyses. RESULTS: During 2017-2018, AST was performed in 1016 N. gonorrhoeae isolates collected at the Bell Flower Clinic. Fourteen isolates (1.4%) from 12 men collected over 13 months demonstrated HL-AZMR; all were cephalosporin susceptible. Of the 12 men, 9 were white and reported male sex partners. Nine of the men were able to be retested; all were cured with 250-mg ceftriaxone plus 1-g AZM. Two men named each other as partners; no other partners in common were reported. Genomic analysis demonstrated close relatedness of the HL-AZMR isolates and a novel combination of a mosaic-mtrR promoter along with 23S ribosomal RNA mutations that appear to have emerged from circulating strains. CONCLUSIONS: The close genetic relatedness with limited epidemiologic linkages between patients highlights the challenges of gonorrhea partner investigations and suggests undetected local transmission. Local AST, rapid public health action, and epidemiologic investigations combined with genomic analysis provides a multipronged approach to understanding an outbreak of sexually transmitted disease.

The Implementation of Strengthening the US Response to Resistant Gonorrhea in the Emergency Department Setting: Successes and Lessons Learned in 2 Jurisdictions.

BACKGROUND: Neisseria gonorrhoeae (NG) continues to develop antimicrobial resistance (AR), and treatment options are limited. ARNG surveillance aids in identifying threats and guiding treatment recommendations but has traditionally been limited to sexually transmitted infection (STI) clinics. Large portions of STI care is delivered outside of STI clinics, such as emergency departments (EDs). These facilities might provide additional venues to expand surveillance and outbreak preparedness. METHODS: Through the Strengthening the US Response to Resistant Gonorrhea program, Greensboro, NC, and Indianapolis, IN, identified 4 EDs in high-morbidity areas to expand culture collection. Patient demographics, culture recovery rates, and antimicrobial susceptibility results between EDs and local STI clinics were compared along with lessons learned from reviewing programmatic policies and discussions with key personnel. RESULTS: During the period 2018-2019, non-Hispanic Black patients were the most represented group at all 6 sites (73.6%). Age was also similar across sites (median range, 23-27 years). Greensboro isolated 1039 cultures (STI clinic [women, 141; men, 612; transwomen, 3]; EDs, 283 [women, 164; men, 119]). Indianapolis isolated 1278 cultures (STI clinic, 1265 [women, 125; men, 1139; transwomen, 1]; ED, 13 all male). Reduced azithromycin susceptibility was found at the Indianapolis (n = 86) and Greensboro (n = 25) STI clinics, and one Greensboro ED (n = 8).Implementation successes included identifying an on-site "champion," integrating with electronic medical records, and creating an online training hub. Barriers included cumbersome data collection tools, time constraints, and hesitancy from clinical staff. CONCLUSIONS: Partnering with EDs for ARNG surveillance poses both challenges and opportunities. Program success can be improved by engaging a local champion to help lead efforts.

Enhancing U.S. Local, State, and Federal Preparedness Through Simulated Interactive Tabletop Exercises of a Mock Antibiotic-Resistant Gonorrhea Outbreak, 2018-2019.

BACKGROUND: Responding effectively to outbreaks of antibiotic-resistant gonorrhea (ARGC) in the future will likely prove challenging. Tabletop exercises (TTXs) may assist local, state, and federal public health officials evaluate existing ARGC outbreak response plans, strengthen preparedness and response effectiveness, and identify critical gaps to address before an outbreak. METHODS: In 2018 to 2019, Centers for Disease Control and Prevention (CDC) collaborated with state partners to develop and implement TTXs to simulate a public health emergency involving an ARGC outbreak. Before the TTXs, 2 state-local health department pairs developed ARGC outbreak response plans. During each 1-day exercise (in Indiana and Illinois), participants discussed roles, clinical management, public health response, and communication based on predeveloped response plans. Observers identified outbreak response strengths and gaps, and participants completed feedback forms. RESULTS: Forty-one (Illinois) and 48 people (Indiana) participated in each TTX, including sexually transmitted disease clinical staff, laboratorians, public health infectious disease program staff, and CDC observers. Strengths and gaps varied by jurisdiction, but identified gaps included: (1) local access to gonorrhea culture and timely antimicrobial susceptibility testing, (2) protocols for clinical management of suspected treatment failures, (3) communication plans, and (4) clarity regarding state and local responsibilities. The CDC observers identified opportunities to provide national-level technical assistance, foster local antimicrobial susceptibility testing, and develop further response guidance. Tabletop exercises summary reports were used to guide modifications to local response plans to address gaps. CONCLUSIONS: The TTXs allowed participants to practice responding to a simulated public health emergency and may have enhanced local response capacity. Centers for Disease Control and Prevention made TTX implementation materials publicly available.

Test of Cure Return Rate and Test Positivity, Strengthening the US Response to Resistant Gonorrhea, United States, 2018-2019.

BACKGROUND: Reduced antibiotic susceptibility (RS) in Neisseria gonorrhoeae (GC) may increase treatment failure. Conducting tests of cure (TOC) for patients with RS-GC may facilitate identification of treatment failures. METHODS: We examined 2018 to 2019 data from 8 jurisdictions participating in the US Centers for Disease Control and Prevention's Strengthening US Response to Resistant Gonorrhea project. Jurisdictions collected GC isolates and epidemiological data from patients and performed antimicrobial susceptibility testing. Minimum inhibitory concentrations of ceftriaxone, 0.125 µg/mL or greater; cefixime, 0.250 µg/mL or greater; or azithromycin, 2.0 µg/mL or greater were defined as RS. Patients with RS infections were asked to return for a TOC 8 to 10 days posttreatment. We calculated a weighted TOC return rate and described time to TOC and suspected reasons for any positive TOC results. RESULTS: Overall, 1165 patients were diagnosed with RS infections. Over half returned for TOC (weighted TOC, 61%; 95% confidence interval, 50.1%-72.6%; range by jurisdiction, 32%-80%). Test of cure rates were higher among asymptomatic (68%) than symptomatic patients (53%, P = 0.001), and men who have sex with men (62%) compared with men who have sex with women (50%; P < 0.001). Median time between treatment and TOC was 12 days (interquartile range, 9-16). Of the 31 (4.5%) TOC patients with positive results, 13 (42%) were suspected because of reinfection and 11 (36%) because of false-positive results. There were no treatment failures suspected to be due to RS-GC. CONCLUSIONS: Most patients with a RS infection returned for a TOC, though return rates varied by jurisdiction and patient characteristics. Test of cure can identify and facilitate treatment of reinfections, but false-positive TOC results may complicate interpretation and clinical management.

Impact of Anatomic Site, Specimen Collection Timing, and Patient Symptom Status on Neisseria gonorrhoeae Culture Recovery.

BACKGROUND: Neisseria gonorrhoeae culture is required for antimicrobial susceptibility testing, but recovering isolates from clinical specimens is challenging. Although many variables influence culture recovery, studies evaluating the impact of culture specimen collection timing and patient symptom status are limited. This study analyzed urogenital and extragenital culture recovery data from Centers for Disease Control and Prevention's Strengthening the US Response to Resistant Gonorrhea (SURRG) program, a multisite project, which enhances local N. gonorrhoeae culture and antimicrobial susceptibility testing capacity. METHODS: Eight SURRG jurisdictions collected gonococcal cultures from patients with N. gonorrhoeae-positive nucleic acid amplification test (NAAT) results attending sexually transmitted disease and community clinics. Matched NAAT and culture specimens from the same anatomic site were collected, and culture recovery was assessed. Time between NAAT and culture specimen collection was categorized as same day, 1 to 7 days, 8 to 14 days, or ≥15 days, and patient symptoms were matched to the anatomic site where culture specimens were collected. RESULTS: From 2018 to 2019, among persons with N. gonorrhoeae-positive NAAT, urethral infections resulted in the highest culture recovery (5927 of 6515 [91.0%]), followed by endocervical (222 of 363 [61.2%]), vaginal (63 of 133 [47.4%]), rectal (1117 of 2805 [39.8%]), and pharyngeal (1019 of 3678 [27.7%]) infections. Culture recovery was highest when specimens were collected on the same day as NAAT specimens and significantly decreased after 7 days. Symptoms were significantly associated with culture recovery at urethral (P = <0.0001) and rectal (P = <0.0001) sites of infection but not endocervical, vaginal, or pharyngeal sites. CONCLUSIONS: Culture specimen collection timing and patient symptomatic status can impact culture recovery. These findings can guide decisions about culture collection protocols to maximize culture recovery and strengthen detection of antimicrobial-resistant infections.

Demographic and Epidemiological Characteristics Associated With Reduced Antimicrobial Susceptibility to Neisseria gonorrhoeae in the United States, Strengthening the US Response to Resistant Gonorrhea, 2018 to 2019.

BACKGROUND: Jurisdictions participating in Strengthening the US Response to Resistant Gonorrhea (SURRG) implemented specimen collection for culture and antimicrobial susceptibility testing from a sample of persons of all genders (at multiple anatomic sites) attending sexually transmitted disease clinics and community clinics. We describe the percentage and characteristics of patients whose isolates demonstrated reduced susceptibility (RS) to azithromycin, ceftriaxone, or cefixime. METHODS: We included patients from clinics that participated in SURRG whose isolates underwent antimicrobial susceptibility testing by Etest. We defined RS as azithromycin minimum inhibitory concentrations (MICs) ≥2 µg/mL (AZM-RS), ceftriaxone MICs ≥0.125 µg/mL (CRO-RS), or cefixime MICs ≥0.25 µg/mL (CFX-RS). Patients with repeated infections appeared >1 time in the data. We calculated the frequency and percentage of patients with an isolate demonstrating RS by epidemiological characteristics. RESULTS: During the period 2018-2019, 10,013 patients from 8 jurisdictions provided 10,735 isolates. Among 10,013 patients, 11.0% (n = 1099) had ≥1 isolate with AZM-RS (range by jurisdiction, 2.5%-18.0%). Approximately 11.3% of 8771 of patients visiting sexually transmitted disease clinics and approximately 8.8% of 1242 patients visiting community clinics had an AZM-RS isolate. Nearly 6% of 1013 females had an AZM-RS isolate; among males, the percents of patients with an AZM-RS isolate were 17.7% among 4177 men who have sex only with men and 6.1% among 3581 men who have sex only with women. Few (0.4%) patients had isolates with CFX-RS (n = 40) or CRO-RS (n = 43). CONCLUSIONS: Although infections with reduced cephalosporin susceptibility were rare, AZM-RS infections were prevalent in this sample of patients in multiple jurisdictions and across gender and gender of sex partner categories.

Exploring and Comparing the Structure of Sexual Networks Affected by Neisseria gonorrhoeae Using Sexual Partner Services Investigation and Genomic Data.

BACKGROUND: Sexual networks are difficult to construct because of incomplete sexual partner data. The proximity of people within a network may be inferred from genetically similar infections. We explored genomic data combined with partner services investigation (PSI) data to extend our understanding of sexual networks affected by Neisseria gonorrhoeae (NG). METHODS: We used 2017-2019 PSI and whole-genome sequencing (WGS) data from 8 jurisdictions participating in Centers for Disease Control and Prevention's Strengthening the US Response to Resistant Gonorrhea (SURRG) project. Clusters were identified from sexual contacts and through genetically similar NG isolates. Sexual mixing patterns were characterized by describing the clusters by the individual's gender and gender of their sex partners. RESULTS: Our study included 4627 diagnoses of NG infection (81% sequenced), 2455 people received a PSI, 393 people were negative contacts of cases, and 495 were contacts with an unknown NG status. We identified 823 distinct clusters using PSI data combined with WGS data. Of cases that were not linked to any other case using PSI data, 37% were linked when using WGS data. Overall, 40% of PSI cases were allocated to a larger cluster when PSI and WGS data were combined compared with PSI data alone. Mixed clusters containing women, men who report sex with women, and men who report sex with men were common when using the WGS data either alone or in combination with the PSI data. CONCLUSIONS: Combining PSI and WGS data improves our understanding of sexual network connectivity.

Cellular localisation of adenylyl cyclase: a post-genome perspective.

The intracellular messenger cAMP is essential for vital processes ranging from ovulation to cognition. There are 10 genes for adenylyl cyclase (AC), the biosynthetic enzyme of cAMP. Nine of these encode membrane-bound proteins and one gives rise to soluble AC. The understanding of the biological significance of this molecular diversity is incomplete. Membrane-bound ACs conform to the same structural blueprint but have markedly different regulatory characteristics. AC mRNAs are differentially distributed in the body suggesting non-redundant physiological functions. The subcellular localisation of AC isoforms has not been examined in detail. Here we discuss the current knowledge on the intracellular targeting of AC isoforms, and highlight the technical problems of AC detection, some of which appear to be caused by the poor quality-control of commercially supplied antibodies. The principal message is that intracellular targeting of ACs may be isoform-specific and also dependent on the cellular context of expression.