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Summary: The maturation of genomic surveillance in the past decade has enabled tracking of the emergence and spread of epidemics at an unprecedented level. During the COVID-19 pandemic, for example, genomic data revealed that local epidemics varied considerably in the frequency of SARS-CoV-2 lineage importation and persistence, likely due to a combination of COVID-19 restrictions and changing connectivity. Here, we show that local COVID-19 epidemics are driven by regional transmission, including across international boundaries, but can become increasingly connected to distant locations following the relaxation of public health interventions. By integrating genomic, mobility, and epidemiological data, we find abundant transmission occurring between both adjacent and distant locations, supported by dynamic mobility patterns. We find that changing connectivity significantly influences local COVID-19 incidence. Our findings demonstrate a complex meaning of ‘local’ when investigating connected epidemics and emphasize the importance of collaborative interventions for pandemic prevention and mitigation.

As SARS-CoV-2 continues to spread and evolve, detecting emerging variants early is critical for public health interventions. Inferring lineage prevalence by clinical testing is infeasible at scale, especially in areas with limited resources, participation, or testing and/or sequencing capacity, which can also introduce biases1-3. SARS-CoV-2 RNA concentration in wastewater successfully tracks regional infection dynamics and provides less biased abundance estimates than clinical testing4,5. Tracking virus genomic sequences in wastewater would improve community prevalence estimates and detect emerging variants. However, two factors limit wastewater-based genomic surveillance: low-quality sequence data and inability to estimate relative lineage abundance in mixed samples. Here we resolve these critical issues to perform a high-resolution, 295-day wastewater and clinical sequencing effort, in the controlled environment of a large university campus and the broader context of the surrounding county. We developed and deployed improved virus concentration protocols and deconvolution software that fully resolve multiple virus strains from wastewater. We detected emerging variants of concern up to 14 days earlier in wastewater samples, and identified multiple instances of virus spread not captured by clinical genomic surveillance. Our study provides a scalable solution for wastewater genomic surveillance that allows early detection of SARS-CoV-2 variants and identification of cryptic transmission.

Abstract: Background: Chagas disease (CD) is a neglected parasitic disease that affects >6 million people in the Americas, including >200,000 people in the United States (US). Medical provider knowledge of CD is key to decreasing morbidity and transmission; however, few studies have assessed diagnostic practices in US health systems serving at-risk patients. Our study aimed to describe existing provider approaches to diagnosing CD in California and Texas. Methods: Site-based research teams at four hospital systems (the University of California [UC] San Francisco [UCSF], San Diego [UCSD], Irvine [UCI], and the Harris Health System [HHS] in Houston, TX) retrospectively identified patients ≥18 years old tested for CD between 2016-2019 and systematically extracted electronic medical record data using complementary electronic data entry forms. Specifically, eligible patients were identified using laboratory orders at UCSF and UCI, while the remaining sites employed SlicerDicer (Epic Systems). This study was approved by institutional review boards at each site. Results: We identified 333 patients tested for CD, including 109 from UCSF, 88 from UCSD, 25 from UCI, and 111 from HHS. These patients had 125, 99, 31, and 181 tests sent to commercial laboratories, respectively. Test reactivity varied by system with the greatest percent reactivity among tests ordered at UCI (23%) followed by UCSD (16%), HHS (15%), and UCSF (10%). Among patients who screened positive for CD by commercial assays, confirmatory testing through the Centers for Disease Control and Prevention was sought for 100% at UCI; 59% at HHS, 55% at UCSF, and 40% at UCSD. The medical specialty that most often ordered CD testing was Cardiology at all UC sites (UCSF, 50%; UCSF, 55%; UCI, 35%) and Internal Medicine at HHS (46%; Cardiology ordered 13%). Only one recorded CD test was ordered by an Obstetrics/Gynecology service at any site. Conclusion: These early results report positivity rates between our healthcare systems and demonstrate inconsistency in attaining recommended confirmatory testing, as well as a paucity of CD testing ordered through Obstetrics/Gynecology despite risk of congenital transmission. These findings suggest areas of opportunity to improve provider awareness and lay a foundation for standardizing CD diagnostic practices in the US. Disclosures: Caryn Bern, MD, MPH, UpToDate (Wolters Kluwer) (Other Financial or Material Support, Author Royalties)

BackgroundIt is unknown whether a positive serology result correlates with protective immunity against SARS-CoV-2. There are also concerns regarding the low positive predictive value of SARS-CoV-2 serology tests, especially when testing populations with low disease prevalence.MethodsA neutralization assay was validated in a set of PCR-confirmed positive specimens and in a negative cohort. In addition, 9530 specimens were screened using the Diazyme SARS-CoV-2 IgG serology assay and all positive results (N = 164 individuals) were reanalyzed using the neutralization assay, the Roche total immunoglobin assay, and the Abbott IgG assay. The relationship between the magnitude of a positive SARS-CoV-2 serology result and neutralizing activity was determined. Neutralizing antibody titers (50% inhibitory dilution, ID50) were also longitudinally monitored in patients confirmed to have SARS-CoV-2 by PCR.ResultsThe SARS-CoV-2 neutralization assay had a positive percentage agreement (PPA) of 96.6% with a SARS-CoV-2 PCR test and a negative percentage agreement (NPA) of 98.0% across 100 negative control individuals. ID50 neutralization titers positively correlated with all 3 clinical serology platforms. Longitudinal monitoring of hospitalized PCR-confirmed patients with COVID-19 demonstrated they made high neutralization titers against SARS-CoV-2. PPA between the Diazyme IgG assay alone and the neutralization assay was 50.6%, while combining the Diazyme IgG assay with either the Roche or Abbott platforms increased the PPA to 79.2 and 78.4%, respectively.ConclusionsThese 3 clinical serology assays positively correlate with SARS-CoV-2 neutralization activity observed in patients with COVID-19. All patients confirmed SARS-CoV-2 positive by PCR develop neutralizing antibodies.

Summary As SARS-CoV-2 continues to spread and evolve, detecting emerging variants early is critical for public health interventions. Inferring lineage prevalence by clinical testing is infeasible at scale, especially in areas with limited resources, participation, or testing/sequencing capacity, which can also introduce biases. SARS-CoV-2 RNA concentration in wastewater successfully tracks regional infection dynamics and provides less biased abundance estimates than clinical testing. Tracking virus genomic sequences in wastewater would improve community prevalence estimates and detect emerging variants. However, two factors limit wastewater-based genomic surveillance: low-quality sequence data and inability to estimate relative lineage abundance in mixed samples. Here, we resolve these critical issues to perform a high-resolution, 295-day wastewater and clinical sequencing effort, in the controlled environment of a large university campus and the broader context of the surrounding county. We develop and deploy improved virus concentration protocols and deconvolution software that fully resolve multiple virus strains from wastewater. We detect emerging variants of concern up to 14 days earlier in wastewater samples, and identify multiple instances of virus spread not captured by clinical genomic surveillance. Our study provides a scalable solution for wastewater genomic surveillance that allows early detection of SARS-CoV-2 variants and identification of cryptic transmission.

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a novel beta-coronavirus that has recently emerged as the cause of the 2019 coronavirus pandemic (COVID-19). Polymerase chain reaction (PCR) based tests are optimal and recommended for the diagnosis of an acute SARS-CoV-2 infection. Serology tests for viral antibodies provide an important tool to diagnose previous exposure to the virus. Here we evaluate the analytical performance parameters of the Diazyme SARS-CoV-2 IgM/IgG serology assays and describe the kinetics of IgM and IgG seroconversion observed in patients with PCR-confirmed COVID-19 who were admitted to our hospital.MethodsWe validated the performance of the Diazyme assay in 235 presumed SARS-CoV-2 negative subjects to determine specificity. Subsequently, we evaluated the SARS-CoV-2 IgM and IgG seroconversion of 54 PCR-confirmed COVID-19 patients and determined sensitivity of the assay at three different timeframes.ResultSensitivity and specificity for detecting seropositivity at ≥15 days following a positive SARS-CoV-2 PCR result, was 100.0% and 98.7% when assaying for the panel of IgM and IgG. The median time to seropositivity observed for a reactive IgM and IgG result from the date of a positive PCR was 5 days (IQR: 2.75-9 days) and 4 days (IQR: 2.75-6.75 days), respectively.ConclusionsOur data demonstrate that the Diazyme IgM/IgG assays are suited for the purpose of detecting SARS-CoV-2 IgG and IgM in patients with suspected SARS-CoV-2 infections. For the first time, we report longitudinal data showing the evolution of seroconversion for both IgG and IgM in a cohort of acutely ill patients in the United States. We also demonstrate a low false positive rate in patients who were presumed to be disease free.

BackgroundPublic reporting of Clostridioides difficile infection (CDI) using laboratory-identified events has led some institutions to revert from molecular-based tests to less sensitive testing modalities. At one academic medical center, researchers chose to use nucleic acid amplification test alone in CDI diagnosis with institutional protocols aimed at diagnostic stewardship.MethodsA single-center, quasi-experimental study was conducted to introduce and analyze the effects of various diagnostic stewardship interventions. In April 2017 an order report was created to inform providers of patients' recent bowel movements, laxative use, and prior Clostridioides difficile (CD) testing (Intervention 1). In November 2017 nursing staff were empowered to not send nondiarrheal stools for testing (Intervention 2). In February 2019, an interruptive alert was implemented to prevent testing that was not indicated (Intervention 3). CD testing rates and healthcare facility-onset CDI (HO-CDI) rates were compared before and after the interventions using one-way analysis of variance (ANOVA).ResultsAt baseline, testing for CD after 3 days of admission was performed at mean ± standard deviation of 15.9 ± 1.7 tests/1,000 patient-days. After Intervention 1, it decreased to 12.1 ± 1.1 tests. This further decreased to 10.6 ± 0.8 after Intervention 2 and to 8.1 ± 0.1 after Intervention 3 (p < 0.001). HO-CDI cases per 10,000 patient-days declined from 12.7 ± 1.4 cases at baseline to 10.7 ± 1.2 after Intervention 1, to 8.7 ± 2.4 after Intervention 2, and to 5.8 ± 0.2 after Intervention 3 (p = 0.03).ConclusionA multidisciplinary approach optimizing electronic health record support tools and leveraging nursing education can reduce both testing and HO-CDI rates while using the most sensitive testing modality.

BackgroundGastrointestinal pathogen panels (GPPs) are increasingly used to identify stool pathogens, but their impact in people with HIV (PWH) is unknown. We performed a retrospective cohort study comparing GPP and conventional stool evaluation in PWH.MethodsWe included all PWH who underwent GPP (Biofire Diagnostics; implemented September 15, 2015) or conventional testing, including stool culture, Clostridium difficile polymerase chain reaction testing, fluorescent smears for Cryptosporidium or Giardia, and ova and parasite exams (O&P) from 2013 to 2017. A total of 1941 specimens were tested, with 169 positive specimens detected in 144 patients. We compared result turnaround time, pathogen co-infection, antibiotic treatment, and treatment outcomes between positive specimens detected by conventional testing vs GPP.ResultsOverall, 124 patient samples tested positive by GPP, compared with 45 patient specimens by conventional testing. The GPP group demonstrated a higher co-infection rate (48.4% vs 13.3%; P

Universal HIV and HCV screening in emergency departments (ED) can reach populations who are less likely to get tested otherwise. The objective of this analysis was to evaluate universal opt-out HIV and HCV screening in two EDs in San Diego. HIV screening for persons aged 13-64 years (excluding persons known to be HIV+ or reporting HIV testing within last 12 months) was implemented using a 4th generation HIV antigen/antibody assay; HCV screening was offered to persons born between 1945 and 1965. Over a period of 16 months, 12,575 individuals were tested for HIV, resulting in 33 (0.26%) new HIV diagnoses, of whom 30 (90%) were successfully linked to care. Universal screening also identified 74 out-of-care for >12-months HIV+ individuals of whom 50 (68%) were successfully relinked to care. Over a one-month period, HCV antibody tests were conducted in 905 individuals with a seropositivity rate of 9.9% (90/905); 61 seropositives who were newly identified or never treated for HCV had HCV RNA testing, of which 31 (51%) resulted positive (3.4% of all participants, including 18 newly identified RNA positives representing 2% of all participants), and 13/31 individuals (42%) were linked to care. The rate of newly diagnosed HCV infections exceeded the rate of newly diagnosed HIV infections by >7-fold, underlining the importance of HCV screening in EDs.

This study aimed to determine the presence of giardiasis among HIV patients in San Diego, the rate of failure of metronidazole treatment, and factors associated with treatment failure. We used a 7 year retrospective single-center case series of HIV-infected individuals with giardiasis at University of California San Diego Medical Center. Data were analyzed for the changes in the hematological, biochemical, and immunologic results at pre- and at-diagnosis levels. We also compared the changes at the diagnosis level among patients who were treated successfully and those who experienced treatment failure as defined by retreatment with a second course of antibiotics. In 29 Giardia lamblia-infected HIV patients, following diagnosis of G. lamblia, there was a non-significant decrement in cluster of differentiation 4 (CD4), but a statistically significant increase in the number of white blood cell (WBC). Other indices did not differ between pre- and at-diagnosis levels. Twenty patients (69%) were treated with a single course of metronidazole or tinidazole and seven patients (24.1%) were treated with more than one course of metronidazole. These seven patients had statistically significant higher hemoglobin at the time of diagnosis, but further studies are required to confirm if this is a consistent finding and if this can predict failure from primary therapy.

This study aimed to determine the presence of giardiasis among HIV patients in San Diego, the rate of failure of metronidazole treatment, and factors associated with treatment failure. We used a 7 year retrospective single-center case series of HIV-infected individuals with giardiasis at University of California San Diego Medical Center. Data were analyzed for the changes in the hematological, biochemical, and immunologic results at pre- and at-diagnosis levels. We also compared the changes at the diagnosis level among patients who were treated successfully and those who experienced treatment failure as defined by retreatment with a second course of antibiotics. In 29 Giardia lamblia-infected HIV patients, following diagnosis of G. lamblia, there was a non-significant decrement in cluster of differentiation 4 (CD4), but a statistically significant increase in the number of white blood cell (WBC). Other indices did not differ between pre- and at-diagnosis levels. Twenty patients (69%) were treated with a single course of metronidazole or tinidazole and seven patients (24.1%) were treated with more than one course of metronidazole. These seven patients had statistically significant higher hemoglobin at the time of diagnosis, but further studies are required to confirm if this is a consistent finding and if this can predict failure from primary therapy.

BackgroundWe compared new Aspergillus Galactomannan Lateral Flow Assay with the newly formatted Aspergillus-specific Lateral Flow device tests for the diagnosis of invasive pulmonary aspergillosis (IPA) in non-neutropenic patients.MethodsWe performed both tests in 82 bronchoalveolar lavage fluid samples from 82 patients at risk for IPA but without underlying haematologic malignancy. Samples were collected between September 2016 and September 2018 at the University of California San Diego, United States. IPA was classified following two published consensus criteria.ResultsClassification of cases varied widely between the two consensus criteria. When using criteria established for the intensive care unit, 26/82 patients (32%) met criteria for proven or putative IPA. Both point-of-care assays showed sensitivities ranging between 58% and 69%, with specificities between 68% and 75%. Sensitivity increased up to 81% when both tests were combined.ConclusionThe study outlines the need for updated, unified and more broadly applicable consensus definitions for classifying IPA in non-neutropenic patients, a work that is currently in progress. Both point-of-care tests showed comparable performance, with sensitivities and specificities in the 60%-70% range when used alone and increasing to 80% when used in combination. The new point-of-care tests may serve a role at the bedside in those with clinical suspicion of IPA.

Volume 61, no. 10, e00954-17, 2017, https://doi.org/10.1128/AAC.00954-17. Materials and Methods, 2nd paragraph, 2nd sentence: “Phages provided by the Biological Defense Research Directorate (BDRD) of the Naval Medical Research Center (AB-Navy1, AB-Navy4, AB-Navy71, AB-Navy97, and AbTP31) were isolated from various environmental samples by using routine isolation techniques, as previously described (9)” should read “Phages provided by the Biological Defense Research Directorate (BDRD) of the Naval Medical Research Center (AB-Navy1, AB-Navy4, AB-Navy97, and AbTP31) were isolated from various environmental samples by using routine isolation techniques, as previously described (9), except for one phage (AB-Navy71, also known as phage vB_Ab-M-G7, DSM strain no. 25639), which was purchased from Liebniz Institute DSMZ (Germany).”

Non-Aspergillus invasive mould infections (IMIs) are associated with devastating morbidity and mortality rates and are increasingly diagnosed in immunocompromised hosts. The aim of this study was to describe the epidemiology and outcomes of non-Aspergillus IMIs at a university hospital in San Diego, California, USA. A retrospective chart review of the medical records of all patients with cultures growing non-Aspergillus moulds at the microbiology laboratory in the Center for Academic Laboratory Medicine, Department of Pathology, University of California, San Diego (UCSD) Health between mid-2014 and mid-2017 (3-year period) was performed. A total of 23 cases of non-Aspergillus IMI were identified, including 10 cases of mucormycosis, 8 cases of lomentosporiosis and 5 cases of fusariosis. Antifungal susceptibility testing was performed for 14 isolates, and 10/11 Fusarium and Lomentospora isolates had minimum inhibitory concentrations (MICs) of >16 µg/mL for voriconazole and/or posaconazole. Overall 180-day mortality was significantly lower among those who received combination antifungal therapy than among those who received single-agent therapy [3/13 (23%) vs. 9/10 (90%); P = 0.003]. In conclusion, Lomentospora prolificans (35% of non-Aspergillus IMIs) and Fusarium spp. (22%) accounted for high proportions of non-Aspergillus IMIs during the study period. Non-Aspergillus IMIs were detected in patients with various underlying diseases and were associated with high mortality rates, which was significantly lower in those who received antifungal combination therapy.

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Cysteine proteases (CPs) play key roles in the pathogenesis of protozoan parasites, including cell/tissue penetration, hydrolysis of host or parasite proteins, autophagy, and evasion or modulation of the host immune response, making them attractive chemotherapeutic and vaccine targets. This review highlights current knowledge on clan CA cysteine proteases, the best-characterized group of cysteine proteases, from 7 protozoan organisms causing human diseases with significant impact: Entamoeba histolytica, Leishmania species (sp.), Trypanosoma brucei, T. cruzi, Cryptosporidium sp., Plasmodium sp., and Toxoplasma gondii. Clan CA proteases from three organisms (T. brucei, T. cruzi, and Plasmodium sp.) are well characterized as druggable targets based on in vitro and in vivo models. A number of candidate inhibitors are under development. CPs from these organisms and from other protozoan parasites should be further characterized to improve our understanding of their biological functions and identify novel targets for chemotherapy.

Neglected tropical diseases affect >1 billion of the world's poorest persons. Control programs range from near-elimination (dracunculiasis) to increasing prevalence (dengue and cutaneous leishmaniasis). These are some of the most cost-effective public health interventions and should be a global priority.

Although toxoplasmosis is one of the most common parasitic infections worldwide, therapeutic options remain limited. Cathepsins, proteases that play key roles in the pathogenesis of toxoplasmosis and many other protozoan infections, are important potential therapeutic targets. Because both TgCPB and TgCPL play a role in T. gondii invasion, we evaluated the efficacy of the potent, irreversible vinyl sulfone inhibitor, K11777 (N-methyl-piperazine-Phe-homoPhe-vinylsulfone-phenyl). The inhibitor's toxicity and pharmacokinetic profile have been well-studied because of its in vitro and in vivo activity against a number of parasites. We found that it inhibited both TgCPB (EC50 = 114 nM) and TgCPL (EC50 = 71 nM) in vitro. K11777 also inhibited invasion of human fibroblasts by RH tachyzoites by 71% (p = 0.003) and intracellular replication by >99% (p

Macrophages are specialized phagocytic cells involved in clearing invading pathogens. Previously we reported that engulfment and cell motility protein 1 (ELMO1) in macrophages mediates bacterial internalization and intestinal inflammation. Here we studied the role of ELMO1 in the fate of internalized targets. ELMO1 is present in the intracellular vesicles and enhances accumulation of the protein LC3B following engulfment of Salmonella or treatment with autophagy-inducing rapamycin. The protein ATG5 and the kinase ULK1 are involved in classical autophagy, while LC3-associated phagocytosis is ULK1 independent. ATG5 but not ULK1 cooperated with ELMO1 in LC3 accumulation after infection, suggesting the ELMO1 preferentially regulated LC3-associated phagocytosis. Because LC3-associated phagocytosis delivers cargo for degradation, the contribution of ELMO1 to the lysosome degradation pathways was evaluated by studying pH and cathepsin B activity. ELMO1-depleted macrophages showed a time-dependent increase in pH and a decrease in cathepsin B activity associated with bacterial survival. Together, ELMO1 regulates LC3B accumulation and antimicrobial responses involved in the clearance of enteric pathogens. This paper investigated how innate immune pathways involving ELMO1 work in a coordinated fashion to eliminate bacterial threats. ELMO1 is present in the phagosome and enhances bacterial clearance by differential regulation of lysosomal acidification and enzymatic activity.

Widespread antibiotic use in clinical medicine and the livestock industry has contributed to the global spread of multidrug-resistant (MDR) bacterial pathogens, including Acinetobacter baumannii We report on a method used to produce a personalized bacteriophage-based therapeutic treatment for a 68-year-old diabetic patient with necrotizing pancreatitis complicated by an MDR A. baumannii infection. Despite multiple antibiotic courses and efforts at percutaneous drainage of a pancreatic pseudocyst, the patient deteriorated over a 4-month period. In the absence of effective antibiotics, two laboratories identified nine different bacteriophages with lytic activity for an A. baumannii isolate from the patient. Administration of these bacteriophages intravenously and percutaneously into the abscess cavities was associated with reversal of the patient's downward clinical trajectory, clearance of the A. baumannii infection, and a return to health. The outcome of this case suggests that the methods described here for the production of bacteriophage therapeutics could be applied to similar cases and that more concerted efforts to investigate the use of therapeutic bacteriophages for MDR bacterial infections are warranted.

This study evaluated opt-out inpatient HIV screening delivered by admitting physicians, and compared number of HIV tests and diagnoses to signs and symptoms-directed HIV testing (based on physician orders) in the emergency department (ED). The opt-out inpatient HIV screening program was conducted over a one year period in patients who were admitted to the 386-bed University of California San Diego (UCSD) teaching hospital. Numbers of HIV tests and diagnoses were compared to those observed among ED patients who underwent physician-directed HIV testing during the same time period. Survey data were collected from a convenience sample of patients and providers regarding the opt-out testing program. Among 8488 eligible inpatients, opt-out HIV testing was offered to 3017 (36%) patients, and rapid antibody testing was performed in 1389 (16.4%) inpatients, resulting in 6 (0.4% of all tests) newly identified HIV infections (5/6 were admitted through the ED). Among 27,893 ED patients, rapid antibody testing was performed in 88 (0.3%), with 7 (8.0% of all tests) new HIV infections identified. HIV diagnoses in the ED were more likely to be men who have sex with men (MSM) (p = 0.029) and tended to have AIDS-related opportunistic infections (p = 0.103) when compared to HIV diagnoses among inpatients. While 85% of the 150 physicians who completed the survey were aware of the HIV opt-out screening program, 44% of physicians felt that they did not have adequate time to consent patients for the program, and only 30% agreed that a physician is best-suited to consent patients. In conclusion, the yield of opt-out HIV rapid antibody screening in inpatients was comparable to the national HIV prevalence average. However, uptake of screening was markedly limited in this setting where opt-out screening was delivered by physicians during routine care, with limited time resources being the major barrier.

Under an NIH priority to identify new drugs to treat class B parasitic agents, we performed high-throughput screens, which identified the activity of auranofin (Ridaura) against Entamoeba histolytica and Giardia intestinalis, major causes of water- and foodborne outbreaks. Auranofin, an orally administered, gold (Au)-containing compound that was approved by the FDA in 1985 for treatment of rheumatoid arthritis, was effective in vitro and in vivo against E. histolytica and both metronidazole-sensitive and -resistant strains of Giardia We now report the results of an NIH-sponsored phase I trial to characterize the pharmacokinetics (PK) and safety of auranofin in healthy volunteers using modern techniques to measure gold levels. Subjects received orally 6 mg (p.o.) of auranofin daily, the recommended dose for rheumatoid arthritis, for 7 days and were followed for 126 days. Treatment-associated adverse events were reported by 47% of the subjects, but all were mild and resolved without treatment. The mean gold maximum concentration in plasma (Cmax) at day 7 was 0.312 μg/ml and the half-life (t1/2) 35 days, so steady-state blood levels would not be reached in short-term therapy. The highest concentration of gold, 13 μM (auranofin equivalent), or more than 25× the 50% inhibitory concentration (IC50) for E. histolytica and 4× that for Giardia, was in feces at 7 days. Modeling of higher doses (9 and 21 mg/day) was performed for systemic parasitic infections, and plasma gold levels of 0.4 to 1.0 μg/ml were reached after 14 days of treatment at 21 mg/day. This phase I trial supports the idea of the safety of auranofin and provides important PK data to support its potential use as a broad-spectrum antiparasitic drug. (This study has been registered at ClinicalTrials.gov under identifier NCT02089048.).

BackgroundCytomegalovirus (CMV) infection can be asymptomatic in healthy individuals but may cause serious complications in immunocompromised patients. We investigated the clinicopathological correlation of CMV in gastrointestinal (GI) biopsies at our institute between January 1, 2013 and December 31, 2015.MethodsA total of 105 non-neoplastic GI biopsies tested positive for CMV by immunohistochemistry (IHC). The IHC results were stratified as "true positive" if > 2 cells stained, or "rare positive" if only 1 - 2 cells stained. Clinical information including comorbidities, serum CMV viral loads, and treatment was reviewed and correlated.ResultsOverall 1% of all GI biopsies were positive for CMV by immunostaining. The most frequently involved organ was colon, followed by esophagus, stomach, ileum and duodenum. When > 2 cells were stained positive, serum CMV viral loads were positive in 52.2%, negative in 17.2%, and not tested in 27.6% of cases. When only 1 - 2 cells stained positive, CMV viral loads were positive in 23.4%, negative in 25.5%, and not tested in 51.1% of cases. We further showed that clinical management of CMV differs based on both pathological findings and underlying diseases.ConclusionsThe role of CMV in GI biopsies remains controversial. We propose an algorithm of performing CMV immunostaining based on clinicopathological correlation.

A 58-year-old Korean-born woman with a history of seizures and psychiatric issues was found dead at home. Autopsy was notable for large, calcified nodules that had nearly replaced her right temporal lobe. Histologic examination revealed the presence of Paragonimus eggs. This case demonstrates a rare manifestation of an aberrantly migrated lung fluke that resulted in epilepsy and sudden death years after the initial infection.

Bacillus cereus is typically considered a blood culture contaminant; however, its presence in blood cultures can indicate true bacteremia. We report 4 episodes of B. cereus bacteremia in 3 persons who inject drugs. Multilocus sequence typing showed that the temporally associated infections were caused by unrelated clones.

Rapid diagnosis and treatment of infectious meningitis and encephalitis are critical to minimize morbidity and mortality. Comprehensive testing of cerebrospinal fluid (CSF) often includes Gram stain, culture, antigen detection, and molecular methods, paired with chemical and cellular analyses. These methods may lack sensitivity or specificity, can take several days, and require significant volume for complete analysis. The FilmArray Meningitis/Encephalitis (ME) Panel is a multiplexed in vitro diagnostic test for the simultaneous, rapid (∼1-h) detection of 14 pathogens directly from CSF specimens: Escherichia coli K1, Haemophilus influenzae, Listeria monocytogenes, Neisseria meningitidis, Streptococcus pneumoniae, Streptococcus agalactiae, cytomegalovirus, enterovirus, herpes simplex virus 1 and 2, human herpesvirus 6, human parechovirus, varicella-zoster virus, and Cryptococcus neoformans/Cryptococcus gattii We describe a multicenter evaluation of 1,560 prospectively collected CSF specimens with performance compared to culture (bacterial analytes) and PCR (all other analytes). The FilmArray ME Panel demonstrated a sensitivity or positive percentage of agreement of 100% for 9 of 14 analytes. Enterovirus and human herpesvirus type 6 had agreements of 95.7% and 85.7%, and L. monocytogenes and N. meningitidis were not observed in the study. For S. agalactiae, there was a single false-positive and false-negative result each, for a sensitivity and specificity of 0 and 99.9%, respectively. The specificity or negative percentage of agreement was 99.2% or greater for all other analytes. The FilmArray ME Panel is a sensitive and specific test to aid in diagnosis of ME. With use of this comprehensive and rapid test, improved patient outcomes and antimicrobial stewardship are anticipated.

Amebiasis causes approximately 70,000 deaths annually and is the third cause of death due to parasites worldwide. It is treated primarily with metronidazole, which has adverse side effects, is mutagenic and carcinogenic, and emergence of resistance is an increasing concern. Unfortunately, better therapeutic alternatives are lacking. Re-purposing of older FDA approved drugs is advantageous to drug discovery since safety and pharmacokinetic effects in humans are already known. In high throughput screening studies, we recently demonstrated that auranofin, a gold containing compound originally approved to treat rheumatoid arthritis, has activity against trophozoites of E. histolytica, the causative agent of amebiasis. Auranofin's anti-parasitic activity is attributed to its monovalent gold molecule that readily inhibits E. histolytica thioredoxin reductase. This anti-oxidant enzyme is the only thiol-dependent flavo-reductase present in E. histolytica. Auranofin has also shown promising activity against other protozoans of significant public health importance. Altogether, this evidence suggests that auranofin has the potential to become a broad spectrum alternative therapeutic agent for diseases with a large global burden.

Entamoeba histolytica and Giardia lamblia are anaerobic protozoan parasites that cause amebiasis and giardiasis, two of the most common diarrheal diseases worldwide. Current therapy relies on metronidazole, but resistance has been reported and the drug has significant adverse effects. Therefore, it is critical to search for effective, better-tolerated antiamebic and antigiardial drugs. We synthesized several examples of a recently reported class of Hsp90 inhibitors and evaluated these compounds as potential leads for antiparasitic chemotherapy. Several of these inhibitors showed strong in vitro activity against both E. histolytica and G. lamblia trophozoites. The inhibitors were rescreened to discriminate between amebicidal and giardicidal activity and general cytotoxicity toward a mammalian cell line. No mammalian cytotoxicity was found at >100 μM for 48 h for any of the inhibitors. To understand the mechanism of action, a competitive binding assay was performed using the fluorescent ATP analogue bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid dipotassium salt) and recombinant E. histolytica Hsp90 preincubated in both the presence and absence of Hsp90 inhibitors. There was significant reduction in fluorescence compared to the level in the control, suggesting that E. histolytica Hsp90 is a selective target. The in vivo efficacy and safety of one Hsp90 inhibitor in a mouse model of amebic colitis and giardiasis was demonstrated by significant inhibition of parasite growth at a single oral dose of 5 mg/kg of body weight/day for 7 days and 10 mg/kg/day for 3 days. Considering the results for in vitro activity and in vivo efficacy, Hsp90 inhibitors represent a promising therapeutic option for amebiasis and giardiasis.

BackgroundThe mainstay of toxoplasmosis treatment targets the folate biosynthetic pathways and has not changed for the last 50 years. The activity of these chemotherapeutic agents is restricted to one lifecycle stage of Toxoplasma gondii, they have significant toxicity, and the impending threat of emerging resistance to these agents makes the discovery of new therapies a priority. We now demonstrate that auranofin, an orally administered gold containing compound that was FDA approved for treatment of rheumatoid arthritis, has activity against Toxoplasma gondii in vitro (IC50 = 0.28 µM) and in vivo (1 mg/kg).Methods/principal findingsReplication within human foreskin fibroblasts of RH tachyzoites was inhibited by auranofin. At 0.4 µM, auranofin inhibited replication, as measured by percent infected fibroblasts at 24 hrs, (10.94% vs. 24.66% of controls; p = 0.0003) with no effect on parasite invasion (16.95% vs. 12.91% p = 0.4331). After 18 hrs, 62% of extracellular parasites treated with auranofin were non-viable compared to control using an ATP viability assay (p = 0.0003). In vivo, a previously standardized chicken embryo model of acute toxoplasmosis was used. Fourteen day old chicken embryos were injected through the chorioallantoic vein with 1×104 tachyzoites of the virulent RH strain. The treatment group received one dose of auranofin at the time of inoculation (1 mg/kg estimated body weight). On day 5, auranofin-treated chicken embryos were 100% protected against death (p = 0.0002) and had a significantly reduced parasite load as determined by histopathology, immunohistochemistry and by the number of parasites quantified by real-time PCR.ConclusionsThese results reveal in vitro and in vivo activity of auranofin against T. gondii, suggesting that it may be an effective alternative treatment for toxoplasmosis.

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Background Chagas disease (CD) is a parasitic disease that affects ∼300 000 people living in the United States. CD leads to cardiac and/or gastrointestinal disease in up to 30% of untreated people. However, end-organ damage can be prevented with early diagnosis and antiparasitic therapy. Methods We reviewed electronic health records of patients who underwent testing for CD at four hospital systems in California and Texas between 2016 and 2020. Descriptive analyses were performed as a needs assessment for improving CD diagnosis. Results In total, 470 patients were tested for CD. Cardiac indications made up more than half (60%) of all testing, and the most frequently cited cardiac condition was heart failure. Fewer than 1% of tests were ordered by obstetric and gynecologic services. Fewer than half (47%) of patients had confirmatory testing performed at the Centers for Disease Control and Prevention. Discussion Four major hospitals systems in California and Texas demonstrated low overall rates of CD diagnostic testing, testing primarily among older patients with end-organ damage, and incomplete confirmatory testing. This suggests missed opportunities to diagnose CD in at-risk individuals early in the course of infection when antiparasitic treatment can reduce the risk of disease progression and prevent vertical transmission. [ABSTRACT FROM AUTHOR]

Giardiasis is one of the most common causes of diarrheal disease worldwide. Treatment is primarily with 5-nitro antimicrobials, particularly metronidazole. Resistance to metronidazole has been described, and treatment failures can occur in up to 20% of cases, making development of alternative antigiardials an important goal. To this end, we have screened a chemical library of 746 approved human drugs and 164 additional bioactive compounds for activity against Giardia lamblia. We identified 56 compounds that caused significant inhibition of G. lamblia growth and attachment. Of these, 15 were previously reported to have antigiardial activity, 20 were bioactive but not approved for human use, and 21 were drugs approved for human use for other indications. One notable compound of the last group was the antirheumatic drug auranofin. Further testing revealed that auranofin was active in the low (4 to 6)-micromolar range against a range of divergent G. lamblia isolates representing both human-pathogenic assemblages A and B. Most importantly, auranofin was active against multiple metronidazole-resistant strains. Mechanistically, auranofin blocked the activity of giardial thioredoxin oxidoreductase, a critical enzyme involved in maintaining normal protein function and combating oxidative damage, suggesting that this inhibition contributes to the antigiardial activity. Furthermore, auranofin was efficacious in vivo, as it eradicated infection with different G. lamblia isolates in different rodent models. These results indicate that the approved human drug auranofin could be developed as a novel agent in the armamentarium of antigiardial drugs, particularly against metronidazole-resistant strains.

Human milk oligosaccharides (HMO), complex sugars that are highly abundant in breast milk, block viral and bacterial attachment to the infant's intestinal epithelium and lower the risk of infections. We hypothesised that HMO also prevent infections with the protozoan parasite Entamoeba histolytica, as its major virulence factor is a lectin that facilitates parasite attachment and cytotoxicity and binds galactose (Gal) and N-acetyl-galactosamine. HMO contain Gal, are only minimally digested in the small intestine and reach the colon, the site of E. histolytica infection. The objective of the present study was to investigate whether HMO reduce E. histolytica attachment and cytotoxicity. Our in vitro results show that physiological concentrations of isolated, pooled HMO detach E. histolytica by more than 80 %. In addition, HMO rescue E. histolytica-induced destruction of human intestinal epithelial HT-29 cells in a dose-dependent manner. The cytoprotective effects were structure-specific. Lacto-N-tetraose with its terminal Gal rescued up to 80 % of the HT-29 cells, while HMO with fucose α1-2-linked to the terminal Gal had no effect. Galacto-oligosaccharides (GOS), which also contain terminal Gal and are currently added to infant formula to mimic some of the beneficial effects of HMO, completely abolished E. histolytica attachment and cytotoxicity at 8 mg/ml. Although our results need to be confirmed in vivo, they may provide one explanation for why breast-fed infants are at lower risk of E. histolytica infections. HMO and GOS are heat tolerant, stable, safe and in the case of GOS, inexpensive, which could make them valuable candidates as alternative preventive and therapeutic anti-amoebic agents.

Entamoeba histolytica, a protozoan intestinal parasite, is the causative agent of human amebiasis. Amebiasis is the fourth leading cause of death and the third leading cause of morbidity due to protozoan infections worldwide(1), resulting in ~70,000 deaths annually. E. histolytica has been listed by the National Institutes of Health as a category B priority biodefense pathogen in the United States. Treatment relies on metronidazole(2), which has adverse effects(3), and potential resistance of E. histolytica to the drug is an increasing concern(4,5). To facilitate drug screening for this anaerobic protozoan, we developed and validated an automated, high-throughput screen (HTS). This screen identified auranofin, a US Food and Drug Administration (FDA)-approved drug used therapeutically for rheumatoid arthritis, as active against E. histolytica in culture. Auranofin was ten times more potent against E. histolytica than metronidazole. Transcriptional profiling and thioredoxin reductase assays suggested that auranofin targets the E. histolytica thioredoxin reductase, preventing the reduction of thioredoxin and enhancing sensitivity of trophozoites to reactive oxygen-mediated killing. In a mouse model of amebic colitis and a hamster model of amebic liver abscess, oral auranofin markedly decreased the number of parasites, the detrimental host inflammatory response and hepatic damage. This new use of auranofin represents a promising therapy for amebiasis, and the drug has been granted orphan-drug status from the FDA.

Tritrichomonas foetus is a sexually transmitted protozoon that causes genital inflammation and adverse pregnancy outcomes in cattle. Cysteine proteinases (CPs) released by T. foetus degrade immunoglobulin G (IgG) antibodies, complement component 3 and matrix proteins as well as inducing apoptosis of bovine genital epithelial cells. In this study, the efficacies of the vinyl sulfone CP inhibitors K11777 and WRR-483 were tested against CPs of T. foetus. The activity of secreted T. foetus CPs in culture supernatants was decreased in the presence of vinyl sulfone inhibitors. Inhibitor K11777 reduced the in vitro cytopathogenic effects of T. foetus in bovine foetal trophoblast cells, which are relevant target cells since this pathogen interferes with pregnancy. Pre-treatment of T. foetus prior to intravaginal inoculation diminished genital infection in a murine model. Therefore, vinyl sulfone CP inhibitors reduce several effects of T. foetus-secreted CPs, including cytotoxicity on relevant target host cells and genital infection in a murine model. These inhibitors have potential as chemotherapeutic agents against bovine trichomoniasis. Generalisation to human trichomoniasis requires further study.

We tested the hypothesis that cathepsins and specifically toxopain-1, a cathepsin B, play a critical role in the pathogenesis of toxoplasmosis. We found that inhibiting the expression of toxopain-1-specific mRNA and protein by >60% significantly decreased the capacity of the parasites to multiply and invade in vitro. To relate these in vitro results to the role of toxopain-1 in pathogenesis in vivo, we developed a novel chicken embryo model of congenital toxoplasmosis. Inhibiting either toxopain-1 expression or specific cysteine proteinase activity significantly reduced congenital infection of chicken embryos, as determined by histopathology and by the number of parasites quantified by real-time PCR. Our new model provides key in vivo validation for the hypothesis that toxopain-1 is a potential drug target in Toxoplasma gondii and also provides a new animal model for rapid, inexpensive screening of antiparasitic compounds.

Amebiasis is a major cause of morbidity and mortality worldwide. Invasion by Entamoeba histolytica trophozoites causes secretion of proinflammatory cytokines from host epithelial cells, leading to a local acute inflammatory response, followed by lysis of colonic cells. Extracellular cysteine proteinases from amebic trophozoites are key virulence factors and have a number of important interactions with host defenses, including cleavage of immunoglobulin G (IgG), IgA, and complement components C3 and C5. Amebic lysates have also been shown to activate the precursor to interleukin I-beta (proIL-1beta), mimicking the action of caspase-1. IL-18 is also a central cytokine, which induces gamma interferon (IFN-gamma) and activates macrophages, one of the main host defenses against invading trophozoites. Because proIL-18 is also activated by caspase-1, we evaluated whether amebic proteinases had a similar effect. Instead, we found that recombinant proIL-18 was cleaved into smaller fragments by the complex of surface-associated and released amebic proteinases. To evaluate the function of an individual proteinase from the complex pool, we expressed an active surface proteinase, EhCP5, which is functional only in E. histolytica. Recombinant EhCP5 expressed in Pichia pastoris had kinetic properties similar to those of the native enzyme with respect to substrate specificity and sensitivity to proteinase inhibitors. In contrast to the activation of proIL-1beta by amebic lysates, the purified proteinase cleaved proIL-18 and mature IL-18 to biologically inactive fragments. These studies suggest that the acute host response and amebic invasion result from a complex interplay of parasite virulence factors and host defenses. E. histolytica may block the host inflammatory response by a novel mechanism, inactivation of IL-18.

Copyright of Forestry Chronicle is the property of Canadian Institute of Forestry and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Nitidulidae trapping performed from 2018 to 2021 to characterize flight behaviors of potential vectors of the oak wilt pathogen yielded three new species records for Canada, six new species records for Ontario, and three new species records for Manitoba. The new records for Canada include Carpophilus (Ecnomorphus) corticinus reported from Ontario, C. (Myothorax) nepos reported from Ontario and Manitoba, and Glischrochilus (Librodor) obtusus reported from Ontario. In addition, the following species are first recorded in Ontario: Carpophilus (Ecnomorphus) antiquus, C. (Megacarpolus) sayi, Stelidota coenosa; and also in Manitoba: Carpophilus (Megacarpolus) lugubris and Cychramus adustus. Collection data is provided for the two provinces and national records. [ABSTRACT FROM AUTHOR]