NK Cell-Monocyte Cross-talk Underlies NK Cell Activation in Severe COVID-19.

NK cells in the peripheral blood of severe COVID-19 patients exhibit a unique profile characterized by activation and dysfunction. Previous studies have identified soluble factors, including type I IFN and TGF-ß, that underlie this dysregulation. However, the role of cell-cell interactions in modulating NK cell function during COVID-19 remains unclear. To address this question, we combined cell-cell communication analysis on existing single-cell RNA sequencing data with in vitro primary cell coculture experiments to dissect the mechanisms underlying NK cell dysfunction in COVID-19. We found that NK cells are predicted to interact most strongly with monocytes and that this occurs via both soluble factors and direct interactions. To validate these findings, we performed in vitro cocultures in which NK cells from healthy human donors were incubated with monocytes from COVID-19+ or healthy donors. Coculture of healthy NK cells with monocytes from COVID-19 patients recapitulated aspects of the NK cell phenotype observed in severe COVID-19, including decreased expression of NKG2D, increased expression of activation markers, and increased proliferation. When these experiments were performed in a Transwell setting, we found that only CD56bright CD16- NK cells were activated in the presence of severe COVID-19 patient monocytes. O-link analysis of supernatants from Transwell cocultures revealed that cultures containing severe COVID-19 patient monocytes had significantly elevated levels of proinflammatory cytokines and chemokines, as well as TGF-ß. Collectively, these results demonstrate that interactions between NK cells and monocytes in the peripheral blood of COVID-19 patients contribute to NK cell activation and dysfunction in severe COVID-19.

Breastfeeding in the United States Among Women With HIV: Con Viewpoint.

To breast feed or not has long been a difficult question for women with human immunodeficiency virus (HIV) in high-income countries, as undetectable HIV in maternal plasma does not translate to zero risk of transmission while breastfeeding, and clean water and formula are readily available. Recent, and more permissive, changes in US and other high-income-country guidelines regarding breastfeeding underscore this issue and acknowledge the information gaps that are essential for informed maternal choice and provider management. These include lack of guidance as to routine monitoring of mothers during lactation, type and length of prophylaxis for infants, and lack of data on factors associated with increased breast-milk viral load and risk of transmission. Ancillary to data are the education and staffing needs for providers participating in the management of breastfeeding individuals. Future studies of breast-milk transmission will need to evaluate these gaps so that we can move transmission to zero.

Autophagic UVRAG Promotes UV-Induced Photolesion Repair by Activation of the CRL4(DDB2) E3 Ligase.

UV-induced DNA damage, a major risk factor for skin cancers, is primarily repaired by nucleotide excision repair (NER). UV radiation resistance-associated gene (UVRAG) is a tumor suppressor involved in autophagy. It was initially isolated as a cDNA partially complementing UV sensitivity in xeroderma pigmentosum (XP), but this was not explored further. Here we show that UVRAG plays an integral role in UV-induced DNA damage repair. It localizes to photolesions and associates with DDB1 to promote the assembly and activity of the DDB2-DDB1-Cul4A-Roc1 (CRL4(DDB2)) ubiquitin ligase complex, leading to efficient XPC recruitment and global genomic NER. UVRAG depletion decreased substrate handover to XPC and conferred UV-damage hypersensitivity. We confirmed the importance of UVRAG for UV-damage tolerance using a Drosophila model. Furthermore, increased UV-signature mutations in melanoma correlate with reduced expression of UVRAG. Our results identify UVRAG as a regulator of CRL4(DDB2)-mediated NER and suggest that its expression levels may influence melanoma predisposition.

Longitudinal Evaluation of Antibody Persistence in Mother-Infant Dyads After Severe Acute Respiratory Syndrome Coronavirus 2 Infection in Pregnancy.

BACKGROUND: There are limited data on how coronavirus disease 2019 (COVID-19) severity, timing of infection, and subsequent vaccination impact transplacental transfer and persistence of maternal and infant antibodies. METHODS: In a longitudinal cohort of pregnant women with polymerase chain reaction-confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, maternal/infant sera were collected at enrollment, delivery/birth, and 6 months. Anti-SARS-CoV-2 spike immunoglobulin (Ig)G, IgM, and IgA were measured by enzyme-linked immunosorbent assay. RESULTS: Two-hundred fifty-six pregnant women and 135 infants were enrolled; 148 maternal and 122 neonatal specimens were collected at delivery/birth; 45 maternal and 48 infant specimens were collected at 6 months. Sixty-eight percent of women produced all anti-SARS-CoV-2 isotypes at delivery (IgG, IgM, IgA); 96% had at least 1 isotype. Symptomatic disease and vaccination before delivery were associated with higher maternal IgG at labor and delivery. Detectable IgG in infants dropped from 78% at birth to 52% at 6 months. In the multivariate analysis evaluating factors associated with detectable IgG in infants at delivery, significant predictors were 3rd trimester infection (odds ratio [OR] = 4.0), mild/moderate disease (OR = 4.8), severe/critical disease (OR = 6.3), and maternal vaccination before delivery (OR = 18.8). No factors were significant in the multivariate analysis at 6 months postpartum. CONCLUSIONS: Vaccination in pregnancy post-COVID-19 recovery is a strategy for boosting antibodies in mother-infant dyads.

Longitudinal relationship between the gut microbiota variation and diversity and gut graft-versus-host disease (GVHD) following pediatric allogeneic hematopoietic cell transplantation (HCT) - Case series.

Allogeneic Hematopoietic Cell Transplantation (HCT) offers children with life-threatening diseases a chance at survival. Complications from graft-versus-host disease (GVHD, Stages 0-4) represent a significant cause of morbidity and mortality which has been recently associated with gut dysbiosis the adult HCT population. Here, our objective was to conduct a prospective, longitudinal cohort study in nine pediatric allogeneic HCT participants by collecting longitudinally post-HCT stool specimens up to 1 year. Stool microbiota analyses showed that allogeneic HCT and antibiotic therapy lead to acute shifts in the diversity of the gut microbiota with those experiencing stages 3-4 gut GVHD having significantly greater microbiota variation over time when compared to control participants (p = 0.007). Pre-HCT microbiota diversity trended towards an inverse relationship with gut microbiota stability over time, however, this did not reach statistical significance (p = 0.05). Future large prospective studies are necessary to elucidate the mechanisms underlying these dynamic changes in the gut microbiota following pediatric allogeneic HCT.

Metabolomic profiling of preterm birth in pregnant women living with HIV.

BACKGROUND: Preterm birth is a leading cause of death in children under the age of five. The risk of preterm birth is increased by maternal HIV infection as well as by certain antiretroviral regimens, leading to a disproportionate burden on low- and medium-income settings where HIV is most prevalent. Despite decades of research, the mechanisms underlying spontaneous preterm birth, particularly in resource limited areas with high HIV infection rates, are still poorly understood and accurate prediction and therapeutic intervention remain elusive. OBJECTIVES: Metabolomics was utilized to identify profiles of preterm birth among pregnant women living with HIV on two different antiretroviral therapy (ART) regimens. METHODS: This pilot study comprised 100 mother-infant dyads prior to antiretroviral initiation, on zidovudine monotherapy or on protease inhibitor-based antiretroviral therapy. Pregnancies that resulted in preterm births were matched 1:1 with controls by gestational age at time of sample collection. Maternal plasma and blood spots at 23-35 weeks gestation and infant dried blood spots at birth, were assayed using an untargeted metabolomics method. Linear regression and random forests classification models were used to identify shared and treatment-specific markers of preterm birth. RESULTS: Classification models for preterm birth achieved accuracies of 95.5%, 95.7%, and 80.7% in the untreated, zidovudine monotherapy, and protease inhibitor-based treatment groups, respectively. Urate, methionine sulfone, cortisone, and 17α-hydroxypregnanolone glucuronide were identified as shared markers of preterm birth. Other compounds including hippurate and N-acetyl-1-methylhistidine were found to be significantly altered in a treatment-specific context. CONCLUSION: This study identified previously known as well as novel metabolomic features of preterm birth in pregnant women living with HIV. Validation of these models in a larger, independent cohort is necessary to ascertain whether they can be utilized to predict preterm birth during a stage of gestation that allows for therapeutic intervention or more effective resource allocation.

Clinical risk factors of adverse outcomes among women with COVID-19 in the pregnancy and postpartum period: a sequential, prospective meta-analysis.

OBJECTIVE: This sequential, prospective meta-analysis sought to identify risk factors among pregnant and postpartum women with COVID-19 for adverse outcomes related to disease severity, maternal morbidities, neonatal mortality and morbidity, and adverse birth outcomes. DATA SOURCES: We prospectively invited study investigators to join the sequential, prospective meta-analysis via professional research networks beginning in March 2020. STUDY ELIGIBILITY CRITERIA: Eligible studies included those recruiting at least 25 consecutive cases of COVID-19 in pregnancy within a defined catchment area. METHODS: We included individual patient data from 21 participating studies. Data quality was assessed, and harmonized variables for risk factors and outcomes were constructed. Duplicate cases were removed. Pooled estimates for the absolute and relative risk of adverse outcomes comparing those with and without each risk factor were generated using a 2-stage meta-analysis. RESULTS: We collected data from 33 countries and territories, including 21,977 cases of SARS-CoV-2 infection in pregnancy or postpartum. We found that women with comorbidities (preexisting diabetes mellitus, hypertension, cardiovascular disease) vs those without were at higher risk for COVID-19 severity and adverse pregnancy outcomes (fetal death, preterm birth, low birthweight). Participants with COVID-19 and HIV were 1.74 times (95% confidence interval, 1.12-2.71) more likely to be admitted to the intensive care unit. Pregnant women who were underweight before pregnancy were at higher risk of intensive care unit admission (relative risk, 5.53; 95% confidence interval, 2.27-13.44), ventilation (relative risk, 9.36; 95% confidence interval, 3.87-22.63), and pregnancy-related death (relative risk, 14.10; 95% confidence interval, 2.83-70.36). Prepregnancy obesity was also a risk factor for severe COVID-19 outcomes including intensive care unit admission (relative risk, 1.81; 95% confidence interval, 1.26-2.60), ventilation (relative risk, 2.05; 95% confidence interval, 1.20-3.51), any critical care (relative risk, 1.89; 95% confidence interval, 1.28-2.77), and pneumonia (relative risk, 1.66; 95% confidence interval, 1.18-2.33). Anemic pregnant women with COVID-19 also had increased risk of intensive care unit admission (relative risk, 1.63; 95% confidence interval, 1.25-2.11) and death (relative risk, 2.36; 95% confidence interval, 1.15-4.81). CONCLUSION: We found that pregnant women with comorbidities including diabetes mellitus, hypertension, and cardiovascular disease were at increased risk for severe COVID-19-related outcomes, maternal morbidities, and adverse birth outcomes. We also identified several less commonly known risk factors, including HIV infection, prepregnancy underweight, and anemia. Although pregnant women are already considered a high-risk population, special priority for prevention and treatment should be given to pregnant women with these additional risk factors.

Longitudinal Assessment of Coronavirus Disease 2019 Vaccine Acceptance and Uptake Among Frontline Medical Workers in Los Angeles, California.

BACKGROUND: Sentiments of vaccine hesitancy and distrust in public health institutions have complicated the government-led coronavirus disease 2019 (COVID-19) vaccine control strategy in the United States. As the first to receive the vaccine, COVID-19 vaccine attitudes among frontline workers are consequential for COVID-19 control and public opinion of the vaccine. METHODS: In this study, we used a repeated cross-sectional survey administered at 3 time points between 24 September 2020 and 6 February 2021 to a cohort of employees of the University of California, Los Angeles Health and the Los Angeles County Fire Department. The primary outcome of interest was COVID-19 vaccination intent and vaccine uptake. RESULTS: Confidence in COVID-19 vaccines and vaccine uptake rose significantly over time. At survey 1, confidence in vaccine protection was 46.4% among healthcare workers (HCWs) and 34.6% among first responders (FRs); by survey 3, this had risen to 90.0% and 75.7%, respectively. At survey 1, about one-third of participants intended to receive a vaccine as soon as possible. By survey 3, 96.0% of HCWs and 87.5% of FRs had received a COVID-19 vaccine. CONCLUSIONS: Attitudes toward vaccine uptake increased over the study period, likely a result of increased public confidence in COVID-19 vaccines, targeted communications, a COVID-19 winter surge in Los Angeles County, and ease of access from employer-sponsored vaccine distribution.

Zoster Meningitis in an Immunocompetent Child after COVID-19 Vaccination, California, USA.

Varicella zoster virus reactivation after COVID-19 vaccination has been reported in older or immunocompromised adults. We report zoster meningitis from live-attenuated varicella vaccine reactivation in an immunocompetent child after COVID-19 vaccination. This type of case is rare; COVID-19 and varicella vaccines remain safe and effective for appropriate recipients in the pediatric population.

No infectious SARS-CoV-2 in breast milk from a cohort of 110 lactating women.

BACKGROUND: Genomic RNA of severe acute respiratory syndrome-associated coronavirus type 2 (SARS-CoV-2) has been detected in the breast milk of lactating women, but its pathological significance has remained uncertain due to the small size of prior studies. METHODS: Breast milk from 110 lactating women was analyzed by reverse transcription-polymerase chain reaction (285 samples) and viral culture (160 samples). Those containing SARS-CoV-2 viral RNA (vRNA) were examined for the presence of subgenomic RNA (sgRNA), a putative marker of infectivity. RESULTS: Sixty-five women had a positive SARS-CoV-2 diagnostic test, 9 had symptoms but negative diagnostic tests, and 36 symptomatic women were not tested. SARS-CoV-2 vRNA was detected in the milk of 7 (6%) women with either a confirmed infection or symptomatic illness, including 6 of 65 (9%) women with a positive SARS-CoV-2 diagnostic test. Infectious virus was not detected in any culture and none had detectable sgRNA. In control experiments, infectious SARS-CoV-2 could be cultured after addition to breastmilk despite several freeze-thaw cycles, as it occurs in the storage and usage of human milk. CONCLUSIONS: SARS-CoV-2 RNA can be found infrequently in the breastmilk after recent infection, but we found no evidence that breastmilk contains an infectious virus or that breastfeeding represents a risk factor for transmission of infection to infants. IMPACT: This article goes beyond prior small studies to provide evidence that infectious SARS-CoV-2 is not present in the milk of lactating women with recent infection, even when SARS-CoV-2 RNA is detected. Recent SARS-CoV-2 infection or detection of its RNA in human milk is not a contraindication to breastfeeding.

An HIV Diagnostic Testing Algorithm Using the cobas HIV-1/HIV-2 Qualitative Assay for HIV Type Differentiation and Confirmation.

Human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) diagnostic testing algorithms recommended by the Centers for Disease Control involve up to three tests and rely mostly on detection of viral antigen and host antibody responses. HIV-1 p24 antigen/HIV-1/HIV-2 antibody-reactive specimens are confirmed with an immunochromatographic HIV-1/HIV-2 antibody differentiation assay, and negative or indeterminate results from the differentiation assay are resolved by an HIV-1-specific nucleic acid amplification test (NAT). The performance of a proposed alternative algorithm using the cobas HIV-1/HIV-2 qualitative NAT as the differentiation assay was evaluated in subjects known to be infected with HIV-1 (n = 876) or HIV-2 (n = 139), at low (n = 6,017) or high (n = 1,020) risk of HIV-1 infection, or at high-risk for HIV-2 infection (n = 498) (study A). The performance of the cobas HIV-1/HIV-2 qualitative test was also evaluated by comparison to an HIV-1 or HIV-2 alternative NAT (study B). The HIV-1 and HIV-2 overall percent agreements (OPA) in study A ranged from 95% to 100% in all groups. The positive percent agreements (PPA) for HIV-1 and HIV-2 were 100% (876/876) and 99.4% (167/168), respectively, for known positive groups. The negative percent agreement in the HIV low-risk group was 100% for both HIV-1 and HIV-2. In study B, the HIV-1 and HIV-2 OPA ranged from 99% to 100% in all groups evaluated (n = 183 to 1,030), and the PPA for HIV-1 and HIV-2 were 100% and 99.5%, respectively, for known positive groups. The cobas HIV-1/HIV-2 qualitative assay can discriminate between HIV-1 and HIV-2 based on HIV RNA and can be included in an alternative diagnostic algorithm for HIV.

Comparison of dried blood spot and plasma sampling for untargeted metabolomics.

INTRODUCTION: Untargeted metabolomics holds significant promise for biomarker detection and development. In resource-limited settings, a dried blood spot (DBS)-based platform would offer significant advantages over plasma-based approaches that require a cold supply chain. OBJECTIVES: The primary goal of this study was to compare the ability of DBS- and plasma-based assays to characterize maternal metabolites. Utility of the two assays was also assessed in the context of a case-control predictive model in pregnant women living with HIV. METHODS: Untargeted metabolomics was performed on archived paired maternal plasma and DBS from n = 79 women enrolled in a large clinical trial. RESULTS: A total of 984 named biochemicals were detected across both plasma and DBS samples, of which 627 (63.7%), 260 (26.4%), and 97 (9.9%) were detected in both plasma and DBS, plasma alone, and DBS alone, respectively. Variation attributable to study individual (R2 = 0.54, p < 0.001) exceeded that of the sample type (R2 = 0.21, p < 0.001), suggesting that both plasma and DBS were capable of differentiating individual metabolomic profiles. Log-transformed metabolite abundances were strongly correlated (mean Spearman rho = 0.51) but showed low agreement (mean intraclass correlation of 0.15). However, following standardization, DBS and plasma metabolite profiles were strongly concordant (mean intraclass correlation of 0.52). Random forests classification models for cases versus controls identified distinct feature sets with comparable performance in plasma and DBS (86.5% versus 91.2% mean accuracy, respectively). CONCLUSION: Maternal plasma and DBS samples yield distinct metabolite profiles highly predictive of the individual subject. In our case study, classification models showed similar performance albeit with distinct feature sets. Appropriate normalization and standardization methods are critical to leverage data from both sample types. Ultimately, the choice of sample type will likely depend on the compounds of interest as well as logistical demands.

Microbiome is not linked to clinical disease severity of familial Mediterranean fever in an international cohort of children.

OBJECTIVES: The severity of familial Mediterranean fever (FMF) may vary in different areas, suggesting a role for environmental factors. We analysed the composition of gut microbiota among children with FMF and healthy controls from Turkey and the USA and determined its effect on disease severity. METHODS: Children with FMF with pathogenic MEFV mutations and healthy controls from Turkey and the USA were enrolled. FMF disease activity was evaluated with the Autoinflammatory Disease Activity Index (AIDAI). Gut bacterial diversity was assessed by sequencing 16S rRNA gene libraries. RESULTS: We included 36 children from Turkey (28 patients with FMF, 8 healthy controls), and 21 patients and 6 controls from the USA. In the Turkish group, 28.6% of patients had severe disease, while 13.3% of US group patients had severe disease. As expected, we observed substantial differences between the gut microbiota of children from the two geographic regions, with Turkish patients and controls exhibiting higher relative abundances of Bacteriodia, while US patients and controls exhibited higher relative abundances of Clostridia. Alpha- and betadiversity did not differ significantly between FMF patients and controls, and neither was predictive of disease severity within each geographic region. We observed differences between FMF patients and controls in the relative abundance of some bacterial taxa at the amplicon sequence variant (ASV) level, but these differences received mixed statistical support. CONCLUSIONS: Among an international cohort of children with FMF, we did not find a strong effect of gut microbiota composition on disease severity. Other environmental or epigenetic factors may be operative.

Complicated Monochorionic-Diamniotic Twins in a Pregnant Woman with COVID-19 in the Second Trimester.

OBJECTIVE: A majority of studies evaluating the risk of vertical transmission and adverse outcomes in pregnancies with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are mostly based on third-trimester infections. There is limited data available on pregnancy sequelae of maternal infection in the first or second trimester. STUDY DESIGN: We present a patient with monochorionic-diamniotic twins that develops coronavirus disease 2019 infection at 15 weeks of gestation. The pregnancy is further complicated by stage II twin-twin transfusion syndrome. She undergoes laser ablation, which is complicated by development of a subchorionic hematoma. The patient then develops Escherichia coli bacteremia, resulting in septic shock and preterm labor followed by previable delivery at 21 weeks of gestation. Amniotic fluid and placenta were negative for SARS-CoV-2 by real-time polymerase chain reaction. CONCLUSION: This case of SARS-CoV-2 argues against transplacental transmission after a second-trimester infection but brings attention to the possible downstream complications that may arise following early infection. KEY POINTS: · Vertical transmission of SARS-CoV-2 is not evident after a second-trimester infection.. · Antepartum coronavirus disease 2019 may cause vascular placental changes and placental insufficiency.. · SARS-CoV-2 is associated with a maternal hypercoagulable state with adverse perinatal outcomes..

Coinfections of Two Strains of NDM-1- and OXA-232-Coproducing Klebsiella pneumoniae in a Kidney Transplant Patient.

We report here a fatal case of carbapenem-resistant Klebsiella pneumoniae (CRKP) infections in a renal transplant patient without a travel history in the prior year, from whom 2 genetically different CRKP (sequence type 14 [ST14] and ST2497) strains carrying the same plasmids and antimicrobial resistance genes, including blaNDM-1, blaOXA-232, blaCTX-M-15, armA, and tet(D), were isolated from blood and the abdominal cavity. The isolates were susceptible to colistin, tigecycline, eravacycline, and cefiderocol, which was used to treat the CRKP in combination with ceftazidime-avibactam and polymyxin B and resulted in bacterial clearance. Despite the aggressive treatment, the patient died of ischemic colitis and multiorgan failure.

Effects of Substance Use and Sex Practices on the Intestinal Microbiome During HIV-1 Infection.

Background: Human immunodeficiency virus type 1 (HIV-1) infection alters the human intestinal microbiome; however, behavioral factors driving these changes remain poorly defined. Here we examine the effects of substance use and sex behavior on the microbiome during HIV-1 infection. Methods: Archival rectal swab specimens, urine drug test results, and responses to substance use and sex behavior questionnaires were obtained from 37 HIV-positive participants at 2 time points, separated by 6 months, in a cohort examining the effects of substance use in men who have sex with men (MSM). Microbiome profiling was performed using 16S ribosomal RNA gene sequencing, and associations with behavioral factors were examined using 0-inflated negative binomial regression. Further analysis of selected variables of interest was performed using propensity scores to account for multiple confounders. Results: Using permutational multivariate analysis of variance, we found that receptive anal intercourse, methamphetamine use, and marijuana use were among the most important drivers of microbiome variation. Propensity score-adjusted analyses revealed that methamphetamine use and marijuana use displayed unique associations; methamphetamine use was associated with an increased abundance of Porphyromonas and Granulicatella organisms and a decreased abundance of Ruminococcus, Collinsella, and Parabacteroides organisms, whereas marijuana use was associated with an increased abundance of Ruminococcus, Clostridium cluster IV, Solobacterium, and Fusobacterium organisms and a decreased abundance of Acidaminococcus, Prevotella, Dialister, Anaerostipes, and Dorea organisms. Conclusions: Drug use and sex behavior are important factors associated with intestinal dysbiosis during chronic HIV-1 infection among young MSM.

The importance of the microbiome in pediatrics and pediatric infectious diseases.

PURPOSE OF REVIEW: Emerging research on the pediatric microbiome implicates the importance of the microbiome on the development of the immune system, nervous system, and growth. Changes to the microbiome during infancy are associated with the development of chronic illnesses such as asthma and inflammatory bowel disease. Additionally, the microbiome provides protection against certain pathogens, affects vaccine responses, and alters drug metabolism. This review highlights what is known about the microbiome, the establishment of a healthy microbiome and the significance that changes to the microbiome composition have on growth and health of children and adolescents. RECENT FINDINGS: Vaginal delivery, breastfeeding, maternal health, and nutrition help shape a healthy microbiome. Caesarian delivery, formula feeding, and antibiotic use perturb the microbiome and are associated with the development of type II diabetes, asthma, allergic diseases, and obesity later in life. Specific interventions using pre and probiotics in multiple settings are under investigation with limited success. SUMMARY: A better understanding of the microbiome and the interaction with the immune system may help guide interventions to alter the microbiome toward a state of lifelong health.

Evolution and Transmission of Carbapenem-Resistant Klebsiella pneumoniae Expressing the blaOXA-232 Gene During an Institutional Outbreak Associated With Endoscopic Retrograde Cholangiopancreatography.

BACKGROUND: Whole-genome sequencing (WGS) is an emerging and powerful technique by which to perform epidemiological studies in outbreak situations. METHODS: WGS was used to identify and evaluate an outbreak of OXA-232-expressing carbapenem-resistant Klebsiella pneumoniae (CRKP) transmitted to 16 patients over the course of 40 weeks via endoscopic retrograde cholangiopancreatography procedures at a single institution. WGS was performed on 32 OXA-232 CRKP isolates (1-7 per patient) and single-nucleotide variants (SNVs) were analyzed, with reference to the index patient's isolate. RESULTS: Interhost genetic diversity of isolates was between 0 and 15 SNVs during the outbreak; molecular clock calculations estimated 12.31 substitutions per genome per year (95% credibility interval, 7.81-17.05). Both intra- and interpatient diversification at the plasmid and transposon level was observed, significantly impacting the antibiogram of outbreak isolates. The majority of isolates evaluated (n = 27) harbored a blaCTX-M-15 gene, but some (n = 5) lacked the transposon carrying this gene, which resulted in susceptibility to aztreonam and third- and fourth-generation cephalosporins. Similarly, an isolate from a colonized patient lacked the transposon carrying rmtF and aac(6')lb genes, resulting in susceptibility to aminoglycosides. CONCLUSIONS: This study broadens the understanding of how bacteria diversify at the genomic level over the course of a defined outbreak and provides reference for future outbreak investigations.

Duodenoscope-Related Outbreak of a Carbapenem-Resistant Klebsiella pneumoniae Identified Using Advanced Molecular Diagnostics.

Background: Carbapenem-resistant Klebsiella pneumoniae infections are increasingly prevalent in North American hospitals. We describe an outbreak of carbapenem-resistant K. pneumoniae containing the blaOXA-232 gene transmitted by contaminated duodenoscopes during endoscopic retrograde cholangiopancreatography (ERCP) procedures. Methods: An outbreak investigation was performed when 9 patients with blaOXA-232 carbapenem-resistant K. pneumoniae infections were identified at a tertiary care hospital. The investigation included 2 case-control studies, review of duodenoscope reprocessing procedures, and culture of devices. Carbapenem-resistant Enterobacteriacieae (CRE) isolates were evaluated with polymerase chain reaction analysis for carbapenemase genes, and isolates with the blaOXA-232 gene were subjected to whole-genome sequencing and chromosome single-nucleotide polymorphism analysis. On recognition of ERCP as a key risk factor for infection, targeted patient notification and CRE screening cultures were performed. Results: Molecular testing ultimately identified 17 patients with blaOxa-232 carbapenem-resistant K. pneumoniae isolates, including 9 with infections, 7 asymptomatic carriers who had undergone ERCP, and 1 additional patient who had been hospitalized in India and was probably the initial carrier. Two case-control studies established a point-source outbreak associated with 2 specific duodenoscopes. A field investigation of the use, reprocessing, and storage of deuodenoscopes did not identify deviations from US Food and Drug Administration or manufacturer recommendations for reprocessing. Conclusions: This outbreak demonstrated the previously underappreciated potential for duodenoscopes to transmit disease, even after undergoing high-level disinfection according to manufacturers' guidelines.