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Cryptosporidium is an enteric pathogen that is transmitted through animal-to-person or person-to-person contact or through ingestion of contaminated water or food. In the United States, Cryptosporidium affects an estimated 750,000 persons each year; however, only approximately 11,000 cases are reported nationally (1,2). Persons infected with Cryptosporidium typically develop symptoms within 2 to 10 days after exposure. Common symptoms include watery diarrhea, abdominal cramps, nausea, vomiting, or fever, which can last 1 to 2 weeks. Cryptosporidiosis is a nationally notifiable disease in the United States. Nebraska presents a unique setting for the evaluation of this pathogen because, compared with other states, Nebraska has a greater reliance on agriculture and a higher proportion of the population residing and working in rural communities. Cryptosporidium species and subtypes are generally indistinguishable using conventional diagnostic methods. Using molecular characterization, Nebraska evaluated the genetic diversity of Cryptosporidium and found a dichotomy in the distribution of cases of cryptosporidiosis caused by Cryptosporidium parvum and Cryptosporidium hominis among rural and urban settings. Characterizing clusters of C. hominis cases revealed that several child care facilities were affected by the same subtype, suggesting community-wide transmission and indicating a need for effective exclusion policies. Several cases of cryptosporidiosis caused by non-C. parvum or non-C. hominis species and genotypes indicated unique animal exposures that were previously unidentified. This study enhanced epidemiologic data by validating known Cryptosporidium sources, confirming outbreaks, and, through repeat interviews, providing additional information to inform cryptosporidiosis prevention and control efforts.

In March 2017, the Nebraska Department of Health and Human Services (NDHHS) and the Southwest Nebraska Public Health Department were notified of an apparent cluster of Campylobacter jejuni infections in city A and initiated an investigation. Overall, 39 cases were investigated, including six confirmed and 33 probable. Untreated, unboiled city A tap water (i.e., well water) was the only exposure significantly associated with illness (odds ratio [OR] = 7.84; 95% confidence interval [CI] = 1.69-36.36). City A is served by four untreated wells and an interconnected distribution system. Onsite investigations identified that a center pivot irrigation system intended to pump livestock wastewater from a nearby concentrated animal feeding operation onto adjacent farmland had malfunctioned, allowing excessive runoff to collect in a road ditch near two wells that supplied water to the city. These wells were promptly removed from service, after which no subsequent cases occurred. This coordinated response rapidly identified an important risk to city A's municipal water supply and provided the evidence needed to decommission the affected wells, with plans to build a new well to safely serve this community.

Congregate work and residential locations are at increased risk for infectious disease transmission including respiratory illness outbreaks. SARS-CoV-2, the virus that causes coronavirus disease 2019 (COVID-19), is primarily spread person to person through respiratory droplets. Nationwide, the meat and poultry processing industry, an essential component of the U.S. food infrastructure, employs approximately 500,000 persons, many of whom work in proximity to other workers (1). Because of reports of initial cases of COVID-19, in some meat processing facilities, states were asked to provide aggregated data concerning the number of meat and poultry processing facilities affected by COVID-19 and the number of workers with COVID-19 in these facilities, including COVID-19-related deaths. Qualitative data gathered by CDC during on-site and remote assessments were analyzed and summarized. During April 9-27, aggregate data on COVID-19 cases among 115 meat or poultry processing facilities in 19 states were reported to CDC. Among these facilities, COVID-19 was diagnosed in 4,913 (approximately 3%) workers, and 20 COVID-19-related deaths were reported. Facility barriers to effective prevention and control of COVID-19 included difficulty distancing workers at least 6 feet (2 meters) from one another (2) and in implementing COVID-19-specific disinfection guidelines.* Among workers, socioeconomic challenges might contribute to working while feeling ill, particularly if there are management practices such as bonuses that incentivize attendance. Methods to decrease transmission within the facility include worker symptom screening programs, policies to discourage working while experiencing symptoms compatible with COVID-19, and social distancing by workers. Source control measures (e.g., the use of cloth face covers) as well as increased disinfection of high-touch surfaces are also important means of preventing SARS-CoV-2 exposure. Mitigation efforts to reduce transmission in the community should also be considered. Many of these measures might also reduce asymptomatic and presymptomatic transmission (3). Implementation of these public health strategies will help protect workers from COVID-19 in this industry and assist in preserving the critical meat and poultry production infrastructure (4).

E-cigarette, or vaping, product use associated lung injury (EVALI) has become a recent concern among public health officials. Factors that contribute to the concern include an increasing number of cases over time, the severity of the illness, and an unknown understanding of the pathophysiology and etiology of the illness. We cared for three adolescent patients with acute respiratory failure secondary to EVALI. All three patients were treated with high-dose steroids in addition to antimicrobials, which resulted in clinical improvement and resolution of their respiratory failure. Pulmonary function testing was performed on these previously healthy patients both acutely and subacutely. Additionally, we report the results from the laboratory analysis of one vaping device fluid which revealed previously unpublished components within these products. EVALI is a recent public health concern without a known etiology which can cause life-threatening lung injury in patients without prior lung pathology. We hope these cases will highlight the importance of return precautions in adolescents with vague respiratory symptoms and provide a cautionary tale to providers while they counsel patients regarding the use of these products.

1. Lauren Hittson, MD* 2. Caitlin Pedati, MD, MPH* 3. Wendy Hoffner, MD* 1. *Pediatrics, Holy Cross Hospital, Silver Spring, MD; Hospitalist Division, Children’s National Medical Center, Washington, DC; Pediatrics, George Washington University School of Medicine and Health Sciences, Washington, DC. A previously healthy 8-year-old boy presents to the emergency department with a red, tender lump in his right proximal inner thigh that his mother noticed the day prior. According to the boy, the lump had been there for several months and has been causing pain with activities such as walking and running. He did not report these symptoms to his mother, so she had not sought care previously. The patient denies fevers, weight loss, rash, or trauma to the area, although he also reports a skin lesion on his right foot. Upon further questioning, he reveals an 8-month history of a hyperpigmented mass on the dorsal aspect of his right foot that has been gradually increasing in size. The mass began as a small skin-colored bump and has slowly grown and darkened over time. It has bled intermittently and blistered but has never been painful. The boy had presented to another health-care facility for evaluation of this mass several months ago but could not tolerate a biopsy attempt under local anesthesia. His family subsequently experienced a lapse in insurance coverage, which resulted in a failure to return for a repeat excision attempt. On physical examination today, the clinician finds a large, palpable, tender mass in the right groin measuring 4 cm with surrounding erythema. Also evident is a hyperpigmented, mobile nodule on his right dorsal foot that measures 2 × 2 cm with areas of darker hyperpigmentation and scaling (Figs 1 and 2). The nodule is only tender with deep pressure. An initial complete blood cell count shows a white blood cell count of 10,700/μL (10.7 × 109 …

Background Reported incidence of surgical site infections (SSIs) in the United States commonly ranges from approximately 0.5–20%. An infection preventionist at a critical access hospital (CAH) notified the Nebraska Department of Health Human Services (NDHHS) Healthcare-Associated Infection (HAI) program of a surgeon with a 6-month SSI rate of 29% (N = 7) and requested assistance. Methods NDHHS HAI program and local health department personnel conducted an investigation. Overall SSI rates were calculated, and seven reported SSI charts were abstracted using the Centers for Disease Control and Prevention’s HAI Outbreak Investigation Form. An additional nine-patient charts with similar characteristics were abstracted and used as the control group. These cases had the same procedures performed at the same facility, by the same surgeon, and during the same period of time but did not develop infections. Results In 2016, of the 452 procedures at this CAH, 17 developed SSIs (rate = 3.8%). SSIs occurred following the most invasive procedures being performed on the sickest patients at this CAH. Of the 17 SSIs, 15 (88.2%) were orthopedic and performed by three surgeons. Surgeon A performed 24 procedures with seven SSIs (rate = 29.2%). Surgeon B performed 171 procedures with five SSIs (rate = 2.9%) and Surgeon C performed 13 procedures with three SSIs (rate = 23.1%). The seven SSIs associated with Surgeon A used different operating room (OR) personnel, rooms, antibiotics, and durations. There were 0 deaths. The seven SSIs and nine controls were evaluated using a stepwise regression model. Using the variables for bone graft, hardware, OR location, and number of people in the OR, the only significant variable was the number of people in the OR. There was an average of 10 people in the OR among cases and seven among controls. Logistic regression yielded an odds ratio of 1.8 (95% CI: 0.99–3.26). Conclusion SSIs occurred primarily after orthopedic procedures, and two of three surgeons were found to have elevated rates. Analysis showed the number of people in the OR was potentially associated with SSIs. After following NDHHS recommendations to limit door openings and OR traffic, there were no additional cases. Additionally, we outlined our methodology in a publically-available response guideline posted to the NDHHS web page. Disclosures All authors: No reported disclosures.

The human immunodeficiency virus type 1 (HIV-1) Tat is a 14-kDa viral protein that acts as a potent transactivator by binding to the transactivation-responsive region, a structured RNA element located at the 5′ end of all HIV-1 transcripts. Tat transactivates viral gene expression by inducing the phosphorylation of the C-terminal domain of RNA polymerase II through several Tat-activated kinases and by recruiting chromatin-remodeling complexes and histone-modifying enzymes to the HIV-1 long terminal repeat. Histone acetyltransferases, including p300 and hGCN5, not only acetylate histones but also acetylate Tat at lysine positions 50 and 51 in the arginine-rich motif. Acetylated Tat at positions 50 and 51 interacts with a specialized protein module, the bromodomain, and recruits novel factors having this particular domain, such as P/CAF and SWI/SNF. In addition to having its effect on transcription, Tat has been shown to be involved in splicing. In this study, we demonstrate that Tat interacts with cyclin-dependent kinase 13 (CDK13) both in vivo and in vitro. We also found that CDK13 increases HIV-1 mRNA splicing and favors the production of the doubly spliced protein Nef. In addition, we demonstrate that CDK13 acts as a possible restriction factor, in that its overexpression decreases the production of the viral proteins Gag and Env and subsequently suppresses virus production. Using small interfering RNA against CDK13, we show that silencing of CDK13 leads to a significant increase in virus production. Finally, we demonstrate that CDK13 mediates its effect on splicing through the phosphorylation of ASF/SF2.

Tularemia is a rare, often serious disease caused by a gram-negative coccobacillus, Francisella tularensis, which infects humans and animals in the Northern Hemisphere. Approximately 125 cases have been reported annually in the United States during the last two decades. As of September 30, a total of 100 tularemia cases were reported in 2015 among residents of Colorado (n = 43), Nebraska (n = 21), South Dakota (n = 20), and Wyoming (n = 16) (Figure). This represents a substantial increase in the annual mean number of four (975% increase), seven (200%), seven (186%) and two (70%) cases, respectively, reported in each state during 2004-2014.

Background In 2016 all acute care hospitals, inpatient rehab facilities, and PPS-exempt cancer facilities in Nebraska were required to report laboratory identified (LabID) Clostridium difficile infections (CDIs) to the National Healthcare Safety Network (NHSN). Test results indicating CDIs must be reported to the Nebraska Department of Health and Human Services (NDHHS) via the National Electronic Disease Surveillance System (NEDSS). NHSN and NEDSS represent unique sources of CDI reports in Nebraska. Methods The NHSN Nebraska database was queried for CDIs reported in 2016. All lab tests indicating a CDI in 2016 were extracted from NEDSS. These extracts were analyzed to assess descriptive epidemiologic variables and compared for differences. Results In 2016 there were 1,546 CDI LabID events reported to NHSN Nebraska from 28 facilities. There were 249 outpatient CDIs and 1,297 inpatient CDIs. Infections were further characterized as community-onset (N = 773), community-onset, healthcare facility associated (N = 206), and hospital onset (N = 567). An average of 128 CDIs were reported per month (range: 111–155). In 2016 there were 2,177 lab results indicating a CDI reported to NEDSS among Nebraska residents from 42 facilities. Patient ages ranged from 4 months to 104 years (mean = 58 years). An average of 181 CDIs were reported per month (range: 151–218). Comparison of the two data sources found 781 reports among 591 unique patients at 11 facilities that were made to NHSN and were not in NEDSS. Additionally, there were 1,092 reports from 931 unique patients at 12 facilities that were made to NEDSS and should have been made to NHSN but were not. There were 9 shared facilities that accounted for the majority of these discrepancies. Conclusion NHSN and NEDSS represent two unique data sources that allow for a more comprehensive assessment of CDIs. The number and type of facility that report to each system is slightly different but there is some overlap. Therefore, this comparison allows for detection of a greater number of reports overall and also provides an opportunity for data validation. This assessment identified discrepancies in reporting among 9 facilities that can be targeted for further collaborative efforts to improve CDI reporting and management in Nebraska. Disclosures All authors: No reported disclosures.

Background The recent outbreak of M. chimaera has focused attention on NTM species as the potential source of other clusters of infection. Since 2010 NTMs have been reportable in Nebraska. Methods The NEDSS data base for Nebraska was searched for all NTM infections reported from May 1, 2013 through April 30, 2017. These entries were reviewed for species or group type and source of material. All pulmonary isolates were excluded. Results Over 4 years, 421 lab reports of NTMs from non-pulmonary sites were received by the DPH. The most common group isolated was M. avium intracellulare group consisting of 246 specimens. Next was M. chelonae with 57 specimens, followed by M. fortuitum with 32, and M. abscessus and M. gordonae with 17 each. Three or fewer isolates of M. kansasii, M. marinum, M. mucogenicum or M. arupense were reported. Eight isolates were not speciated. The sources of those isolates were included in the report 46% of the time. Source MAI M.chelonae M. fortuitum M. abscessus M. gordonae Source Reported 40% 63% 50% 71% 94% % of those cases reported by site Abscess, wound 26% 14% 25% 33.3% (1 ear) 19% Tissue, mass, skin, or lesion 22 11 (1 sinus) 0 0 12.5 Neck or LN 12 11 0 0 0 Bone or Joint 11 14 19 0 25 Blood 3 22 25 25 0 Fluid Non Specified 24 5.5 12.5 16.7 12.5 Abdomen 0 11 6 0 12.5 Peritoneal fluid 0 3 0 8.3 0 Stool 0 14 6 8.3 CSF 2 0 0 0 0 Other Urine-3 Eye-5.5 Cervix-8.3 Urine 12.5 Conclusion The majority of NTM reported in Nebraska over 4 years came from an abscess, wound, mass, bone, joint or skin source. Interesting results included: the number of cases detected by blood culture (between 15 and 20%) in non-MAI NTMS, the number of abdominal sources or stool (5–10% each) in non-MAI NTM, and the unusual sites discovered such as the CSF (MAI only), peritoneal fluid, ear, eye, and cervix. These results suggest that looking for NTM in the blood or stool could be an effective way of making the diagnosis, that it is important to consider NTM in abdominal infections and infections of peritoneal dialysis fluid of unclear etiology. Lastly, NTM may play a role in infections in unusual sites such as the eye, the ear or cervix. Disclosures All authors: No reported disclosures.

Tularemia is a rare, often serious disease caused by a gram-negative coccobacillus, Francisella tularensis, which infects humans and animals in the Northern Hemisphere. Approximately 125 cases have been reported annually in the United States during the last two decades. As of September 30, a total of 100 tularemia cases were reported in 2015 among residents of Colorado (n = 43), Nebraska (n = 21), South Dakota (n = 20), and Wyoming (n = 16) (Figure). This represents a substantial increase in the annual mean number of four (975% increase), seven (200%), seven (186%) and two (70%) cases, respectively, reported in each state during 2004-2014.

Objectives. We measured HIV testing and seropositivity among veterans in Veterans Affairs (VA) care for calendar years 2009 through 2011 and analyzed 2011 results by patient demographics. Methods. We performed a repeated-measures cross-sectional study using standardized electronic data extraction from the VA electronic health records for all veterans with at least 1 outpatient visit during 2009 through 2011. We analyzed testing rates and seropositivity by demographic characteristics for 2011. Results. Of veterans with an outpatient visit, 20.0% had an HIV test in 2011, compared with 9.2% in 2009. Documented HIV testing rates were highest in women and Blacks. Of confirmed positive test results, 67.0% were in outpatients older than 50 years. Seropositivity was highest among men aged 30 to 49 years, women aged 50 to 69 years, and Black outpatients of both genders. Implementation of an electronic clinical reminder was associated with higher testing rates. Conclusions. The significant effect of an electronic clinical reminder suggests that such decision support tools can substantially increase testing rates. The frequency of positive test results in older individuals suggests the need for additional work to define optimum approaches to HIV testing in this population.

Background Approximately half of hereditary breast cancers have mutations in either BRCA1 or BRCA2. BRCA1 is a multifaceted tumor suppressor protein that has implications in processes such as cell cycle, transcription, DNA damage response and chromatin remodeling. This multifunctional nature of BRCA1 is achieved by exerting its many effects through modulation of transcription. Many cellular events are dictated by covalent modification of proteins, an important mechanism in regulating protein and genome function; of which protein methylation is an important posttranslational modification with activating or repressive effects. Methods/Principal Findings Here we demonstrate for the first time that BRCA1 is methylated both in breast cancer cell lines and breast cancer tumor samples at arginine and lysine residues through immunoprecipitation and western blot analysis. Arginine methylation by PRMT1 was observed in vitro and the region of BRCA1 504–802 shown to be highly methylated. PRMT1 was detected in complex with BRCA1 504–802 through in vitro binding assays and co-immunoprecipitated with BRCA1. Inhibition of methylation resulted in decreased BRCA1 methylation and alteration of BRCA1 binding to promoters in vivo as shown through chromatin immunoprecipitation assays. Knockdown of PRMT1 also resulted in increased BRCA1 binding to particular promoters in vivo. Finally, following methylation inhibition, Sp1 was found to preferentially associate with hypo-methylated BRCA1 and STAT1 was found to preferentially associate with hyper-methylated BRCA1. Conclusions/Significance These results suggest that methylation may influence either the ability of BRCA1 to bind to specific promoters or protein-protein interactions which alters the recruitment of BRCA1 to these promoters. Thus, given the importance of BRCA1 to genomic stability, methylation of BRCA1 may ultimately affect the tumor suppressor ability of BRCA1.

The development of novel techniques and systems to study human infectious diseases in both anin vitroandin vivosettings is always in high demand. Ideally, small animal models are the most efficient method of studying human afflictions. This is especially evident in the study of the human retroviruses, HIV-1 and HTLV-1, in that current simian animal models, though robust, are often expensive and difficult to maintain. Over the past two decades, the construction of humanized animal models through the transplantation and engraftment of human tissues or progenitor cells into immunocompromised mouse strains has allowed for the development of a reconstituted human tissue scaffold in a small animal system. The utilization of small animal models for retroviral studies required expansion of the early CB-17scid/scidmouse resulting in animals demonstrating improved engraftment efficiency and infectivity. The implantation of uneducated human immune cells and associated tissue provided the basis for the SCID-hu Thy/Liv and hu-PBL-SCID models. Engraftment efficiency of these tissues was further improved through the integration of the non-obese diabetic (NOD) mutation leading to the creation of NODSCID, NOD/Shi-scid IL2rγ-/-, and NOD/SCID β2-microglobulinnullanimals. Further efforts at minimizing the response of the innate murine immune system produced the Rag2-/-γc-/-model which marked an important advancement in the use of human CD34+ hematopoietic stem cells. Together, these animal models have revolutionized the investigation of retroviral infectionsin vivo.

Human T-cell leukemia virus type-1 (HTLV-1) induces adult T-cell leukemia/lymphoma (ATL/L), a fatal lymphoproliferative disorder, and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a chronic progressive disease of the central nervous system after a long period of latent infection. Although the mechanism of transformation and leukemogenesis is not fully elucidated, there is evidence to suggest that the viral oncoprotein Tax plays a crucial role in these processes through the regulation of several pathways including NF-κB and the cell cycle pathways. The observation that NF-κB, which is strongly induced by Tax, is indispensable for the maintenance of the malignant phenotype of HTLV-1 by regulating the expression of various genes involved in cell cycle regulation and inhibition of apoptosis provides a possible molecular target for these infected cells. To develop potential new therapeutic strategies for HTLV-1 infected cells, in this present study, we initially screened a battery of NF-κB and CDK inhibitors (total of 35 compounds) to examine their effects on the growth and survival of infected T-cell lines. Two drugs namely BMS-345541 and Purvalanol A exhibited higher levels of growth inhibition and apoptosis in infected cell as compared to uninfected cells. BMS-345541 inhibited IKKβ kinase activity from HTLV-1 infected cells with an IC50 (the 50% of inhibitory concentration) value of 50 nM compared to 500 nM from control cells as measured by in vitro kinase assays. The effects of Purvalanol A were associated with suppression of CDK2/cyclin E complex activity as previously shown by us. Combination of both BMS-345541 and Purvalanol A showed a reduced level of HTLV-1 p19 Gag production in cell culture. The apparent apoptosis in these infected cells were associated with increased caspase-3 activity and PARP cleavage. The potent and selective apoptotic effects of these drugs suggest that both BMS-345541 and Purvalanol A, which target both NF-κB and CDK complex and the G1/S border, might be promising new agents in the treatment of these infected patients.

Background The rate of transcription of the HIV-1 viral genome is mediated by the interaction of the viral protein Tat with the LTR and other transcriptional machinery. These specific interactions can be affected by the state of post-translational modifications on Tat. Previously, we have shown that Tat can be phosphorylated and acetylated in vivo resulting in an increase in the rate of transcription. In the present study, we investigated whether Tat could be methylated on lysine residues, specifically on lysine 50 and 51, and whether this modification resulted in a decrease of viral transcription from the LTR. Results We analyzed the association of Tat with histone methyltransferases of the SUV39-family of SET domain containing proteins in vitro. Tat was found to associate with both SETDB1 and SETDB2, two enzymes which exhibit methyltransferase activity. siRNA against SETDB1 transfected into cell systems with both transient and integrated LTR reporter genes resulted in an increase in transcription of the HIV-LTR in the presence of suboptimal levels of Tat. In vitro methylation assays with Tat peptides containing point mutations at lysines 50 and 51 showed an increased incorporation of methyl groups on lysine 51, however, both residues indicated susceptibility for methylation. Conclusion The association of Tat with histone methyltransferases and the ability for Tat to be methylated suggests an interesting mechanism of transcriptional regulation through the recruitment of chromatin remodeling proteins to the HIV-1 promoter.

It has been demonstrated that the p53 pathway plays an important role in HIV-1 infection. Previous work from our lab has established a model demonstrating how p53 could become inactivated in HIV-1 infected cells through binding to Tat. Subsequently, p53 was inactivated and lost its ability to transactivate its downstream target gene p21/waf1. P21/waf1 is a well-known cdk inhibitor (CKI) that can lead to cell cycle arrest upon DNA damage. Most recently, the p21/waf1 function was further investigated as a molecular barrier for HIV-1 infection of stem cells. Therefore, we reason that the restoration of the p53 and p21/waf1 pathways could be a possible theraputical arsenal for combating HIV-1 infection. In this current study, we show that a small chemical molecule, 9-aminoacridine (9AA) at low concentrations, could efficiently reactivate p53 pathway and thereby restoring the p21/waf1 function. Further, we show that the 9AA could significantly inhibit virus replication in activated PBMCs, likely through a mechanism of inhibiting the viral replication machinery. A mechanism study reveals that the phosphorylated p53ser15 may be dissociated from binding to HIV-1 Tat protein, thereby activating the p21/waf1 gene. Finally, we also show that the 9AA-activated p21/waf1 is recruited to HIV-1 preintegration complex, through a mechanism yet to be elucidated.