Carbapenem-resistant Enterobacterales (CRE) and carbapenem-resistant Pseudomonas aeruginosa (CRPA) are significant public health threats, particularly when harboring carbapenemases. Literature describing the frequencies and phenotypic and genotypic characteristics of isolates harboring multiple carbapenemase genes is limited. Using data collected from the Antimicrobial Resistance Laboratory Network (AR Lab Network) in 2018-2022, we describe CRE and CRPA isolates that harbor multiple acquired carbapenemase genes. Clinical laboratories submitted CRE and CRPA isolates to AR Lab Network public health laboratories for additional characterization that included antimicrobial susceptibility testing and detection of five targeted carbapenemase genes. Isolates were classified as non-carbapenemase producing (non-CP) when negative for carbapenemase production and all targeted carbapenemase genes, or positive for a single-CP (SCP) or multiple-carbapenemase (MCP) targeted gene. Among 79,799 CREs tested, 27,599 (35%) were SCP and 611 (1%) were MCP. MCP-CRE most often carried bla(KPC)/bla(NDM) (n = 285, 47%). Both SCP-CRE and MCP-CRE were most commonly Klebsiella spp. Enterobacter spp. and Escherichia coli isolates harboring MCP were detected at slightly higher frequencies (18% and 15%; n = 109 and n = 88, respectively) than Enterobacter spp. and Escherichia coli isolates harboring SCP (13% and 13%; n = 3,653 and 3,471, respectively). The number of MCP-CRE detected increased from 54 of 5,105 (1%) in 2018 to 223 of 6,994 (3%) in 2022. Among 54,490 CRPA tested, 2% (n = 1,249) were SCP and 31 were MCP. MCP-CRPA most often carried bla(VIM)/bla(IMP) (n = 13, 42%). A higher proportion of MCP-CRE (97%, n = 330) isolates were categorized as resistant to meropenem, compared to SCP-CRE (79%; n = 11,227) and non-CP (13%; n = 2,683). Although MCP organisms represent a small proportion of total CP detected in the AR Lab Network, there is a need for continued monitoring and additional research.IMPORTANCECarbapenemase-producing organisms are of significant clinical and public health concerns, and rapid detection and containment of such threats are vital to preventing their spread. In this article, we used a collection of over 130,000 contemporary isolates to evaluate frequencies and phenotypic and genotypic properties of CRE and CRPA isolates harboring multiple carbapenemase genes across the United States, from 2018 to 2022. Of note, 95% and 100% of CRE and CRPA isolates co-harbored at least one metallo-β-lactamase gene, respectively, indicating a high proportion of isolates originating from patients with difficult-to-treat infections. Both clinical and public health professionals across the nation can use these data and key findings to better understand the molecular landscape of these isolates. Timely detection and control of these organisms are essential to combating the spread of antibiotic resistance and ensuring the availability of effective treatment options for patients.

BACKGROUND: Patients with ESKD on maintenance dialysis receive dialysis in common spaces with other patients and have a higher risk of severe SARS-CoV-2 infections. They may have persistently or intermittently positive SARS-CoV-2 RT-PCR tests after infection. We describe the clinical course of SARS-CoV-2 infection and the serologic response in a convenience sample of patients with ESKD to understand the duration of infectivity. METHODS: From August to November 2020, we enrolled patients on maintenance dialysis with SARS-CoV-2 infections from outpatient dialysis facilities in Atlanta, Georgia. We followed participants for approximately 42 days. We assessed COVID-19 symptoms and collected specimens. Oropharyngeal (OP), anterior nasal (AN), and saliva (SA) specimens were tested for the presence of SARS-CoV-2 RNA, using RT-PCR, and sent for viral culture. Serology, including neutralizing antibodies, was measured in blood specimens. RESULTS: Fifteen participants, with a median age of 58 (range, 37‒77) years, were enrolled. Median duration of RT-PCR positivity from diagnosis was 18 days (interquartile range [IQR], 8‒24 days). Ten participants had at least one, for a total of 41, positive RT-PCR specimens ≥10 days after symptoms onset. Of these 41 specimens, 21 underwent viral culture; one (5%) was positive 14 days after symptom onset. Thirteen participants developed SARS-CoV-2-specific antibodies, 11 of which included neutralizing antibodies. RT-PCRs remained positive after seroconversion in eight participants and after detection of neutralizing antibodies in four participants; however, all of these samples were culture negative. CONCLUSIONS: Patients with ESKD on maintenance dialysis remained persistently and intermittently SARS-CoV-2-RT-PCR positive. However, of the 15 participants, only one had infectious virus, on day 14 after symptom onset. Most participants mounted an antibody response, including neutralizing antibodies. Participants continued having RT-PCR-positive results in the presence of SARS-CoV-2-specific antibodies, but without replication-competent virus detected.

HIV particles in the blood largely originate from activated lymphocytes and can overshadow variants which may be expressed from other cell types. Investigations of virus persistence must be able to distinguish cells refractory to viral clearance that serve as reservoirs. To investigate additional cell types that may be associated with in vivo HIV expression we developed a virus particle immunomagnetic capture method targeting several markers of cellular origin that become embedded within virion envelopes during budding. We evaluated the ability of markers to better distinguish cell lineage source subpopulations by assessing combinations of different antibodies with cell-sorted in vitro culture and clinical specimens. Various deductive algorithms were designed to discriminate source cell lineages and subsets. From the particle capture algorithms, we identified distinct variants expressed within individuals that were associated with disparate cellular markers. Among the variants uncovered were minority-level viruses with drug resistance mutations undetected by sequencing and often were associated with markers indicative of myeloid lineage (CD3-/CD10-/CD16+ or /CD14+, and CD3-/CD16-/CD14-/CD11c+ or /HLA-DR+) cell sources. The diverse HIV genetic sequences expressed from different cell types within individuals, further supported by the appearance of distinct drug-resistant variants, highlights the complexity of HIV reservoirs in vivo which must be considered for HIV cure strategies. This approach could also be helpful in examining in vivo host cell origins and genetic diversity in infections involving other families of budding viruses.

The Centers for Disease Control and Prevention has classified CRAB as an urgent public health threat. In this paper, we used a collection of >6,000 contemporary clinical isolates to evaluate the phenotypic and genotypic properties of CRAB detected in the United States. We describe the frequency of specific carbapenemase genes detected, antimicrobial susceptibility profiles, and the distribution of CRAB isolates categorized as multidrug resistant, extensively drug-resistant, or difficult to treat. We further discuss the proportion of isolates showing susceptibility to Food and Drug Administration-approved agents. Of note, 84% of CRAB tested harbored at least one class A, B, or D carbapenemase genes targeted for detection and 83% of these carbapenemase gene-positive CRAB were categorized as extensively drug resistant. Fifty-four percent of CRAB isolates without any of these carbapenemase genes detected were still extensively drug-resistant, indicating that infections caused by CRAB are highly resistant and pose a significant risk to patient safety regardless of the presence of one of these carbapenemase genes.

In 2017, the Centers for Disease Control and Prevention (CDC) established the Antimicrobial Resistance Laboratory Network to improve domestic detection of multidrug-resistant organisms. CDC and four laboratories evaluated a commercial broth microdilution panel. Antimicrobial susceptibility testing using the Sensititre GN7F (ThermoFisher Scientific, Lenexa, KS) was evaluated by testing 100 CDC and Food and Drug Administration AR Isolate Bank isolates [40 Enterobacterales (ENT), 30 Pseudomonas aeruginosa (PSA), and 30 Acinetobacter baumannii (ACB)]. We assessed multiple amounts of transfer volume (TV) between the inoculum and tubed 11-mL cation-adjusted Mueller-Hinton broth: 1 µL [tribe Proteeae (P-tribe) only] and 10, 30, and 50 µL, resulting in respective CFU per milliter of 1 × 10(4), 1 × 10(5), 3 × 10(5), and 5 × 10(5). Four TV combinations were analyzed: standard (STD) [1 µL (P-tribe) and 10 µL], enhanced standard (E-STD) [1 µL (P-tribe) and 30 µL], 30 µL, and 50 µL. Essential agreement (EA), categorical agreement, major error (ME), and very major error (VME) were analyzed by organism then TVs. For ENT, the average EA across laboratories was <90% for 7 of 15 β-lactams using STD and E-STD TVs. As TVs increased, EA increased (>90%), and VMEs decreased. For PSA, EA improved as TVs increased; however, MEs also increased. For ACB, increased TVs provided slight EA improvements; all TVs yielded multiple VMEs and MEs. For ENT and ACB, Minimum inhibitory concentrations (MICs) trended downward using a 1 or 10 µL TV; there were no obvious MIC trends by TV for PSA. The public health and clinical consequences of missing resistance warrant increased TV of 30 µL for the GN7F, particularly for P-tribe, despite being considered "off-label" use.

OBJECTIVES: Despite clinical relevance, commercially available molecular tools for accurate β-lactamase detection are limited. In this study, we evaluated the performance of the ARM-D® β-lactamase Kit, a commercially available multiplex PCR assay designed to detect nine β-lactamase genes, including the five major plasmid-mediated carbapenemases, ESBL, or AmpC genes circulating in the United States. METHODS: A diverse collection of 113 Gram-negative isolates, including 42 with multiple β-lactamases was selected from the U.S. Centers for Disease Control and Prevention (CDC) & Food and Drug Administration (FDA) Antibiotic Resistance Isolate Bank, to represent the most frequently detected bacterial species carrying plasmid-mediated β-lactam resistance genes. RESULTS: Results were compared with whole genome sequence data. Of 164 β-lactamase gene targets with 49 unique variants, all were detected correctly without any cross-reactivity. The sensitivity and specificity were 100% (164/164) and 99.9% (852/853), respectively. CONCLUSION: The ARM-D® β-lactamase Kit detected a wide range of β-lactamase genotypes at a low upfront cost. The Streck assay represents a suitable, comprehensive tool for the detection of key β-lactamase resistance genes of public health concern in the United States.

OBJECTIVE: Characterize and compare SARS-CoV-2-specific immune responses in plasma and gingival crevicular fluid (GCF) from nursing home residents during and after natural infection. DESIGN: Prospective cohort. SETTING: Nursing home. PARTICIPANTS: SARS-CoV-2-infected nursing home residents. METHODS: A convenience sample of 14 SARS-CoV-2-infected nursing home residents, enrolled 4-13 days after real-time reverse transcription polymerase chain reaction diagnosis, were followed for 42 days. Post diagnosis, plasma SARS-CoV-2-specific pan-Immunoglobulin (Ig), IgG, IgA, IgM, and neutralizing antibodies were measured at 5 timepoints and GCF SARS-CoV-2-specific IgG and IgA were measured at 4 timepoints. RESULTS: All participants demonstrated immune responses to SARS-CoV-2 infection. Among 12 phlebotomized participants, plasma was positive for pan-Ig and IgG in all 12, neutralizing antibodies in 11, IgM in 10, and IgA in 9. Among 14 participants with GCF specimens, GCF was positive for IgG in 13 and IgA in 12. Immunoglobulin responses in plasma and GCF had similar kinetics; median times to peak antibody response was similar across specimen types (4 weeks for IgG; 3 weeks for IgA). Participants with pan-Ig, IgG, and IgA detected in plasma and GCF IgG remained positive through this evaluation's end 46-55 days post-diagnosis. All participants were viral culture negative by the first detection of antibodies. CONCLUSIONS: Nursing home residents had detectable SARS-CoV-2 antibodies in plasma and GCF after infection. Kinetics of antibodies detected in GCF mirrored those from plasma. Non-invasive GCF may be useful for detecting and monitoring immunologic responses in populations unable or unwilling to be phlebotomized.

Carbapenemase gene-positive (CP) Gram-negative bacilli are of significant clinical and public health concern. Their rapid detection and containment are critical to preventing their spread and additional infections they can cause. To this end, CDC developed the Antibiotic Resistance Laboratory Network (AR Lab Network), in which public health laboratories across all 50 states, several cities, and Puerto Rico characterize clinical isolates of carbapenem-resistant Enterobacterales (CRE), Pseudomonas aeruginosa (CRPA), and Acinetobacter baumannii (CRAB), and conduct colonization screens to detect the presence of mobile carbapenemase genes. In its first three years, the AR Lab Network tested 76,887 isolates and 31,001 rectal swab colonization screens. Targeted carbapenemase genes (bla(KPC), bla(NDM), bla(OXA-48-like), bla(VIM), or bla(IMP)) were detected by PCR in 35% of CRE, 2% of CRPA, <1% of CRAB, and 8% of colonization screens tested, respectively. bla(KPC) and bla(VIM) were the most common CP-CRE and CP-CRPA, respectively, but regional differences in the frequency of carbapenemase genes detected were apparent. In CRE and CRPA isolates tested for carbapenemase production and the presence of the targeted genes, 97% had concordant results; 3% of CRE and 2% of CRPA were carbapenemase production-positive but PCR-negative for those genes. Isolates harboring bla(NDM) showed the highest frequency of resistance across the carbapenems tested and those harboring bla(IMP) and bla(OXA-48-like) genes showed the lowest frequency of carbapenem resistance. The AR Lab Network provides a national snapshot of rare and emerging carbapenemase genes, delivering data to inform public health actions to limit the spread of these antibiotic resistance threats.

Facility-wide testing performed at four outpatient hemodialysis facilities in the absence of an outbreak or escalating community incidence did not identify new SARS-CoV-2 infections and illustrated key logistical considerations essential to successful implementation of SARS-CoV-2 screening. Facilities could consider prioritizing facility-wide SARS-CoV-2 testing during suspicion of an outbreak in the facility or escalating community spread without robust infection control strategies in place. Being prepared to address operational considerations will enhance implementation of facility-wide testing in the outpatient dialysis setting.

Aztreonam-avibactam is a drug combination pending phase 3 clinical trials and is suggested for treatment of severe infections caused by metallo-beta-lactamase (MBL)-producing Enterobacterales by combining ceftazidime-avibactam and aztreonam. Beginning in 2019, four Antibiotic Resistance Laboratory Network regional laboratories offered aztreonam-avibactam susceptibility testing by broth microdilution. For 64 clinical isolates tested, the MIC(50) and MIC(90) of aztreonam-avibactam were 0.5/4 μg/mL and 8/4 μg/mL, respectively. Aztreonam-avibactam displayed potent in vitro activity against the MBL-producing Enterobacterales tested.

BACKGROUND: SARS-CoV-2 testing remains essential for early identification and clinical management of cases. We compared the diagnostic performance of three specimen types for characterizing SARS-CoV-2 in infected nursing home residents. METHODS: A convenience sample of 17 residents were enrolled within 15 days of first positive SARS-CoV-2 result by real-time reverse transcription polymerase chain reaction (RT-PCR) and prospectively followed for 42 days. Anterior nasal swabs (AN), oropharyngeal swabs (OP), and saliva specimens (SA) were collected on the day of enrollment, every 3 days for the first 21 days, then weekly for 21 days. Specimens were tested for presence of SARS-CoV-2 RNA using RT-PCR and replication-competent virus by viral culture. RESULTS: Comparing the three specimen types collected from each participant at each time point, the concordance of paired RT-PCR results ranged from 80-88%. After the first positive result, SA and OP were RT-PCR-positive for ≤48 days; AN were RT-PCR-positive for ≤33 days. AN had the highest percentage of RT-PCR-positive results (81%; 21/26) when collected ≤10 days of participants' first positive result. Eleven specimens were positive by viral culture: nine AN collected ≤19 days following first positive result and two OP collected ≤5 days following first positive result. CONCLUSIONS: AN, OP, and SA were effective methods for repeated testing in this population. More AN than OP were positive by viral culture. SA and OP remained RT-PCR-positive longer than AN, which could lead to unnecessary interventions if RT-PCR detection occurred after viral shedding has likely ceased.

Background: Detection of carbapenem-resistant Pseudomonas aeruginosa (CRPA) with carbapenamase-producing (CP) genes is critical for preventing transmission. Our objective was to assess whether certain antimicrobial susceptibility testing (AST) profiles can efficiently identify CP-CRPA.Methods: We defined CRPA as P. aeruginosa with imipenem or meropenem MICs of ≥8μg/ml; CP-CRPA were CRPA with CP genes (bla (KPC)/bla (IMP)/bla (NDM)/bla (VIM)). We assessed the sensitivity and specificity of AST profiles to detect CP-CRPA among CRPA collected by CDC's Antibiotic Resistance Laboratory Network (AR Lab Network) and the Emerging Infections Program (EIP) during 2017-2019.Results: Three percent (195/6192) of AR Lab Network CRPA were CP-CRPA. Among CRPA, adding not susceptible (NS) to cefepime or ceftazidime to the definition had 91% sensitivity and 50% specificity for identifying CP-CRPA; NS to ceftolozane-tazobactam had 100% sensitivity and 86% specificity. Of 965 EIP CRPA evaluated for CP genes, seven CP-CRPA were identified; 6 of 7 were NS to cefepime and ceftazidime, and all 7 were NS to ceftolozane-tazobactam. Among 4182 EIP isolates, clinical laboratory AST results were available for 96% for cefepime, 80% for ceftazidime, and 4% for ceftolozane-tazobactam. The number of CRPA needed to test (NNT) to identify one CP-CRPA decreased from 138 to 64 if the definition of NS to cefepime or ceftazidime was used and to 7 with NS to ceftolozane-tazobactam.Conclusion: Adding not susceptible to cefepime or ceftazidime to CRPA carbapenemase testing criteria would reduce the NNT by half and can be implemented in most clinical laboratories; adding not susceptible to ceftolozane-tazobactam could be even more predictive once AST for this drug is more widely available.

BACKGROUND: To estimate the infectious period of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in older adults with underlying conditions, we assessed duration of coronavirus disease 2019 (COVID-19) symptoms, reverse-transcription polymerase chain reaction (RT-PCR) positivity, and culture positivity among nursing home residents. METHODS: We enrolled residents within 15 days of their first positive SARS-CoV-2 test (diagnosis) at an Arkansas facility from July 7 to 15, 2020 and instead them for 42 days. Every 3 days for 21 days and then weekly, we assessed COVID-19 symptoms, collected specimens (oropharyngeal, anterior nares, and saliva), and reviewed medical charts. Blood for serology was collected on days 0, 6, 12, 21, and 42. Infectivity was defined by positive culture. Duration of culture positivity was compared with duration of COVID-19 symptoms and RT-PCR positivity. Data were summarized using measures of central tendency, frequencies, and proportions. RESULTS: We enrolled 17 of 39 (44%) eligible residents. Median participant age was 82 years (range, 58-97 years). All had ≥3 underlying conditions. Median duration of RT-PCR positivity was 22 days (interquartile range [IQR], 8-31 days) from diagnosis; median duration of symptoms was 42 days (IQR, 28-49 days). Of 9 (53%) participants with any culture-positive specimens, 1 (11%) severely immunocompromised participant remained culture-positive 19 days from diagnosis; 8 of 9 (89%) were culture-positive ≤8 days from diagnosis. Seroconversion occurred in 12 of 12 (100%) surviving participants with ≥1 blood specimen; all participants were culture-negative before seroconversion. CONCLUSIONS: Duration of infectivity was considerably shorter than duration of symptoms and RT-PCR positivity. Severe immunocompromise may prolong SARS-CoV-2 infectivity. Seroconversion indicated noninfectivity in this cohort.