During the 2022 multinational outbreak of monkeypox virus (MPXV) infection, the antiviral drug tecovirimat (TPOXX; SIGA Technologies, Inc., https://www.siga.com) was deployed in the United States on a large scale for the first time. The MPXV F13L gene homologue encodes the target of tecovirimat, and single amino acid changes in F13 are known to cause resistance to tecovirimat. Genomic sequencing identified 11 mutations previously reported to cause resistance, along with 13 novel mutations. Resistant phenotype was determined using a viral cytopathic effect assay. We tested 124 isolates from 68 patients; 96 isolates from 46 patients were found to have a resistant phenotype. Most resistant isolates were associated with severely immunocompromised mpox patients on multiple courses of tecovirimat treatment, whereas most isolates identified by routine surveillance of patients not treated with tecovirimat remained sensitive. The frequency of resistant viruses remains relatively low (<1%) compared with the total number of patients treated with tecovirimat.

Background: During the 2022 multinational outbreak of monkeypox virus (MPXV) clade IIb, the antiviral drug tecovirimat (TPOXX) was deployed in the US on a large scale for the first time ever. The MPXV F13L gene homolog encodes the target of tecovirimat, and single amino acid changes in the F13 protein are known to cause resistance to tecovirimat in orthopoxviruses (OPXV). Method(s): Whole genome metagenomic sequencing and amplicon-based sequencing targeting the F13L gene was used to identify nine mutations previously reported to cause resistance in other OPXV along with ten novel mutations that have been identified from the 2022 mpox outbreak. A cytopathic effect assay, previously established at CDC as part of WHO smallpox research, was adapted to MPXV for tecovirimat phenotype testing of virus isolated from mpox patients. Result(s): As of March 2023, in total, 70 isolates from 40 patients were tested, and 50 of these isolates from 26 patients were found to have a resistant phenotype. Most resistant isolates were associated with severely immunocompromised mpox patients on multiple courses of TPOXX treatment; while isolates with F13 mutations identified by routine surveillance of patients not treated with TPOXX have remained sensitive. Conclusion(s): These data indicate that tecovirimat resistance is developing in immunocompromised patients treated with TPOXX and that for isolates that we have analyzed, the frequency of resistant viruses remain relatively low (< 1%) compared to the total number of patients treated with TPOXX. These findings inform our understanding of when tecovirimat resistance is likely to occur and highlight the need for additional OPXV therapeutics. Copyright The copyright holder for this preprint is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license.

INTRODUCTION: Hazardous alcohol use (HAU), defined as a pattern of alcohol consumption that increases the risk of harmful consequences for the user or others, is associated with an elevated risk of human immunodeficiency virus (HIV) infection and poor health outcomes. We describe the association between people living with HIV (PLHIV) who report HAU and key HIV indicators. Gaps in current literature in estimating HAU on HIV outcomes at the regional level of Eastern and Southern Africa still exist and our analysis aims to address this issue. METHODS: We used weighted pooled data (2015-2017) from the nationally representative Population-based HIV Impact Assessments among adults who provided written consent aged 18-59 years from Eswatini, Malawi, Namibia, Tanzania, Zambia and Zimbabwe. We estimated differences in the prevalence of HIV infection and The Joint United Nations Programme on HIV and AIDS (UNAIDS) 90-90-90 indicators between PLHIV by HAU status using log-binomial regression, stratified by sex. HAU was determined using the Alcohol Use Identification Test-Consumption. RESULTS: Among the 9755 women and 4444 men who tested HIV positive, 6.6% of women and 21.8% of men engaged in HAU. Women who reported HAU were more likely to be HIV positive (adjusted prevalence ratio [aPR] = 1.31, 95% CI: 1.18-1.46) compared to those who did not report HAU. For the UNAIDS 90-90-90 targets, women who engaged in HAU were more likely to be unaware of their HIV-positive status (aPR = 1.22, 95% CI: 1.01-1.47) and not on antiretroviral therapy (ART) (aPR = 1.73, 95% CI: 1.26-2.37). Men who engaged in HAU were more likely to be unaware of their HIV-positive status (aPR = 1.56, 95% CI 1.39-1.76) and not on ART (aPR = 1.72, 95% CI: 1.30-2.29). No difference in viral load suppression, defined as <1000 copies/ml of HIV RNA, was seen by sex. CONCLUSIONS: PLHIV who engage in HAU were more likely to have suboptimal outcomes along the HIV care continuum when compared to those who did not engage in HAU. Targeted interventions, such as alcohol screening for HAU in HIV testing and treatment settings and HIV prevention efforts in alcohol-based venues, may help countries reach HIV epidemic control by 2030.

The etiology of Nodding Syndrome remains unclear, and comprehensive genotyping and phenotyping data from patients remain sparse. Our objectives were to characterize the phenotype of patients with Nodding Syndrome, investigate potential contributors to disease etiology, and evaluate response to immunotherapy. This cohort study investigated members of a single-family unit from Lamwo District, Uganda. The participants for this study were selected by the Ugandan Ministry of Health as representative for Nodding Syndrome and with a conducive family structure for genomic analyses. Of the eight family members who participated in the study at the National Institutes of Health (NIH) Clinical Center, three had Nodding Syndrome. The three affected patients were extensively evaluated with metagenomic sequencing for infectious pathogens, exome sequencing, spinal fluid immune analyses, neurometabolic and toxicology testing, continuous electroencephalography, and neuroimaging. Five unaffected family members underwent a subset of testing for comparison. A distinctive interictal pattern of sleep-activated bursts of generalized and multifocal epileptiform discharges and slowing was observed in two patients. Brain imaging showed two patients had mild generalized cerebral atrophy, and both patients and unaffected family members had excessive metal deposition in the basal ganglia. Trace metal biochemical evaluation was normal. Cerebrospinal fluid (CSF) was non-inflammatory, and one patient had CSF-restricted oligoclonal bands. Onchocerca volvulus specific antibodies were present in all patients and skin snips were negative for active onchocerciasis. Metagenomic sequencing of serum and CSF revealed hepatitis B virus in the serum of one patient. Vitamin B6 metabolites were borderline low in all family members, and CSF pyridoxine metabolites were normal. Mitochondrial DNA testing was normal. Exome sequencing did not identify potentially causal candidate gene variants. Nodding Syndrome is characterized by a distinctive pattern of sleep-activated epileptiform activity. The associated growth stunting may be due to hypothalamic dysfunction. Extensive testing years after disease onset did not clarify a causal etiology. A trial of immunomodulation (plasmapheresis in two patients and intravenous immunoglobulin in one patient) was given without short-term effect, but longer-term follow-up was not possible to fully assess any benefit of this intervention.

Human leukocyte antigen (HLA) alleles have been linked to HIV disease progression and attributed to differences in cytotoxic T lymphocyte (CTL) epitope representation. These findings are largely based on treatment-naive individuals of European and African ancestry. We assessed HLA associations with HIV-1 outcomes in 1,318 individuals from Thailand and found HLA-B∗46:01 (B∗46) associated with accelerated disease in three independent cohorts. B∗46 had no detectable effect on HIV-specific T cell responses, but this allele is unusual in containing an HLA-C epitope that binds inhibitory receptors on natural killer (NK) cells. Unbiased transcriptomic screens showed increased NK cell activation in people with HIV, without B∗46, and simultaneous single-cell profiling of surface proteins and transcriptomes revealed a NK cell subset primed for increased responses in the absence of B∗46. These findings support a role for NK cells in HIV pathogenesis, revealed by the unique properties of the B∗46 allele common only in Asia.

The most common causes of eosinophilia globally are helminth parasites. Refugees from high endemic areas are at increased risk of infection compared with the general U.S. population. It is widely accepted that eosinophilia is a good marker for helminth infection in this population, yet its absence has little predictive value for excluding infection. During an enhanced premigration health program, the CDC offered voluntary testing and management of intestinal parasites, among other conditions, to U.S.-bound refugees in Thailand. Stool specimens were tested for Ascaris lumbricoides, Strongyloides stercoralis, Trichuris trichiura, hookworms, Giardia lamblia, Cryptosporidium spp., and Entamoeba histolytica using quantitative polymerase chain reaction. Complete blood counts were performed to identify eosinophilia. Predictive values of eosinophilia for parasitic infections were calculated within nematode groups. Between July 9, 2012 and November 29, 2013, 2,004 participants were enrolled. About 73% were infected with at least one parasite. The overall median eosinophil count was 483 cells/L (interquartile range [IQR] = 235-876 cells/L). Compared with participants who did not test positive for any infection, higher eosinophil counts were observed in those infected with A. lumbricoides (RR = 1.3, 95% CI = 1.1-1.4), S. stercoralis (RR = 1.8, 95% CI = 1.4-2.4), Necator americanus (RR = 1.2, 95% CI = 1.1-1.4), and Ancylostoma ceylanicum (RR = 1.8, 95% CI = 1.5-2.2). Eosinophil counts were higher in younger participants (2-4 years versus 65+ years: RR = 4.2, 95% CI = 2.5-6.9), and lower in female participants (RR = 0.9, 95% CI = 0.8-0.9). Sensitivities ranged from 51% to 73%, specificities from 48% to 65%, and predictive values from 4% to 98%. The predictive value of eosinophilia is poor for the most common parasitic infections, and it should not be used alone for screening refugees.

With advances in nanotechnology, engineered nanomaterial applications are a rapidly growing sector of the economy. Some nanomaterials can reach the brain through nose-to-brain transport. This transport creates concern for potential neurotoxicity of insoluble nanomaterials and a need for toxicity screening tests that detect nose-to-brain transport. Such tests can involve intranasal instillation of aqueous suspensions of nanomaterials in dispersion media that limit particle agglomeration. Unfortunately, protein and some elements in existing dispersion media are suboptimal for potential nose-to-brain transport of nanomaterials because olfactory transport has size- and ion-composition requirements. Therefore, we designed a protein-free dispersion media containing phospholipids and amino acids in an isotonic balanced electrolyte solution, a solution for nasal and olfactory transport (SNOT). SNOT disperses hexagonal boron nitride nanomaterials with a peak particle diameter below 100 nm. In addition, multiwalled carbon nanotubes (MWCNTs) in an established dispersion medium, when diluted with SNOT, maintain dispersion with reduced albumin concentration. Using stereomicroscopy and microscopic examination of plastic sections, dextran dyes dispersed in SNOT are demonstrated in the neuroepithelium of the nose and olfactory bulb of B6;129P2-Omp(tm3Mom)/MomJ mice after intranasal instillation in SNOT. These findings support the potential for SNOT to disperse nanomaterials in a manner permitting nose-to-brain transport for neurotoxicity studies.

BACKGROUND: Chlamydia trachomatis (Ct) infection ascending to the upper genital tract can cause infertility. Direct association of genetic variants as contributors is challenging because infertility may not be diagnosed until years after infection. Investigating the intermediate trait of ascension bridges this gap. METHODS: We identified infertility genome-wide association study (GWAS) loci using deoxyribonucleic acid from Ct-seropositive cisgender women in a tubal factor infertility study and Ct-infected cisgender women from a longitudinal pelvic inflammatory disease cohort with known fertility status. Deoxyribonucleic acid and blood messenger ribonucleic acid from 2 additional female cohorts with active Ct infection and known endometrial infection status were used to investigate the impact of infertility single-nucleotide polymorphisms (SNPs) on Ct ascension. A statistical mediation test examined whether multiple infertility SNPs jointly influenced ascension risk by modulating expression of mediator genes. RESULTS: We identified 112 candidate infertility GWAS loci, and 31 associated with Ct ascension. The SNPs altered chlamydial ascension by modulating expression of 40 mediator genes. Mediator genes identified are involved in innate immune responses including type I interferon production, T-cell function, fibrosis, female reproductive tract health, and protein synthesis and degradation. CONCLUSIONS: We identified Ct-related infertility loci and their potential functional effects on Ct ascension.

Flavorings-related lung disease is a potentially disabling and sometimes fatal lung disease of workers making or using flavorings. First identified almost 20 years ago in microwave popcorn workers exposed to butter-flavoring vapors, flavorings-related lung disease remains a concern today. In some cases, workers develop bronchiolitis obliterans, a severe form of fixed airways disease. Affected workers have been reported in microwave popcorn, flavorings, and coffee production workplaces. Volatile alpha-dicarbonyl compounds, particularly diacetyl (2,3-butanedione) and 2,3-pentanedione, are implicated in the etiology. Published studies on diacetyl and 2,3-pentanedione document their ability to cause airway epithelial necrosis, damage biological molecules, and perturb protein homeostasis. With chronic exposure in rats, they produce airway fibrosis resembling bronchiolitis obliterans. To add to this knowledge, we recently evaluated airway toxicity of the 3-carbon alpha-dicarbonyl compound, methylglyoxal. Methylglyoxal inhalation causes epithelial necrosis at even lower concentrations than diacetyl. In addition, we investigated airway toxicity of mixtures of diacetyl, acetoin, and acetic acid, common volatiles in butter flavoring. At ratios comparable to workplace scenarios, the mixtures or diacetyl alone, but not acetic acid or acetoin, cause airway epithelial necrosis. These new findings add to existing data to implicate alpha-dicarbonyl compounds in airway injury and flavorings-related lung disease.

During 2012-2015, US-bound refugees living in Myanmar-Thailand border camps (n = 1,839) were surveyed for hookworm infection and treatment response by using quantitative PCR. Samples were collected at 3 time points: after each of 2 treatments with albendazole and after resettlement in the United States. Baseline prevalence of Necator americanus hookworm was 25.4%, Ancylostoma duodenale 0%, and Ancylostoma ceylanicum (a zoonosis) 5.4%. Compared with N. americanus prevalence, A. ceylanicum hookworm prevalence peaked in younger age groups, and blood eosinophil concentrations during A. ceylanicum infection were higher than those for N. americanus infection. Female sex was associated with a lower risk for either hookworm infection. Cure rates after 1 dose of albendazole were greater for A. ceylanicum (93.3%) than N. americanus (65.9%) hookworm (p<0.001). Lower N. americanus hookworm cure rates were unrelated to beta-tubulin single-nucleotide polymorphisms at codons 200 or 167. A. ceylanicum hookworm infection might be more common in humans than previously recognized.

With an unprecedented number of displaced persons worldwide, strategies for improving the health of migrating populations are critical. United States-bound refugees undergo a required overseas medical examination to identify inadmissible conditions (e.g., tuberculosis) 2-6 months before resettlement, but it is limited in scope and may miss important, preventable infectious, chronic, or nutritional causes of morbidity. We sought to evaluate the feasibility and health impact of diagnosis and management of such conditions before travel. We offered voluntary testing for intestinal parasites, anemia, and hepatitis B virus infection, to U.S.-bound refugees from three Thailand-Burma border camps. Treatment and preventive measures (e.g., anemia and parasite treatment, vaccination) were initiated before resettlement. United States refugee health partners received overseas results and provided post-arrival medical examination findings. During July 9, 2012 to November 29, 2013, 2,004 refugees aged 0.5-89 years enrolled. Among 463 participants screened for seven intestinal parasites overseas and after arrival, helminthic infections decreased from 67% to 12%. Among 118 with positive Strongyloides-specific antibody responses, the median fluorescent intensity decreased by an average of 81% after treatment. The prevalence of moderate-to-severe anemia (hemoglobin < 10 g/dL) was halved from 14% at baseline to 7% at departure (McNemar P = 0.001). All 191 (10%) hepatitis B-infected participants received counseling and evaluation; uninfected participants were offered vaccination. This evaluation demonstrates that targeted screening, treatment, and prevention services can be conducted during the migration process to improve the health of refugees before resettlement. With more than 250 million migrants globally, this model may offer insights into healthier migration strategies.

Literature suggests that early exposure to the farming environment protects against atopy and asthma; few studies have examined pulmonary function. We evaluated associations between early-life farming exposures and pulmonary function in 3061 adults (mean age=63) from a US farming population using linear regression. Childhood raw milk consumption was associated with higher FEV1 (beta=49.5 mL, 95% CI 2.8 to 96.1 mL, p=0.04) and FVC (beta=66.2 mL, 95% CI 13.2 to 119.1 mL, p=0.01). We did not find appreciable associations with other early-life farming exposures. We report a novel association between raw milk consumption and higher pulmonary function that lasts into older adulthood.

OBJECTIVE: Develop methodology to estimate the annual cost of resources used by school sealant programs (SSPs) and demonstrate its use. METHODS: We used existing literature and expert opinion to identify SSP cost components and the most appropriate units for their measurement (e.g., per operator) and collection frequency (e.g., per day). For equipment and reusable instruments, costs were sufficiently homogenous across SSPs that we could provide default per unit cost estimates (2016 US$) that SSPs can use in lieu of collecting their own data. We also provide default costs for supply items such that SSPs can estimate total supply costs with program-specific information on sealant material used, as well as number of: sealant stations, operators, service delivery days, children screened/sealed, and number of teeth sealed. For the remaining three categories (labor, mileage, and administrative), costs varied substantially by SSP and required us to develop and pilot collection logs for program-specific data. RESULTS: The annual cost per sealant station ranged from $584 to $797 depending on program characteristics. For a hypothetical SSP that staffed each of two stations with two operators (hygienist and assistant) compensated at the national rate, hourly labor costs would equal $77.97. Assuming this SSP used disposable instruments, light-cured sealants and delivered sealants (3 per child) to 60 percent of the 3,390 children screened over 100 service days, infection control/supply costs per child would equal $5.30. CONCLUSION: This methodology allows SSPs to estimate costs with minimal data collection and time.

RATIONALE: Endotoxin initiates a pro-inflammatory response from the innate immune system. Studies in children suggest endotoxin exposure from house dust may be an important risk factor for asthma, but few studies have been conducted in adult populations. OBJECTIVES: To investigate the association of house dust endotoxin levels with asthma and related phenotypes (wheeze, atopy, and pulmonary function) in a large U.S. farming population. METHODS: Dust was collected from the bedrooms (n=2,485) of participants enrolled in a case-control study of current asthma (927 cases) nested within the Agricultural Health Study. Dust endotoxin was measured by Limulus amebocyte lysate assay. Outcomes were measured by questionnaire, spirometry, and blood draw. We evaluated associations using linear and logistic regression. MEASUREMENTS AND MAIN RESULTS: Endotoxin was significantly associated with current asthma (Odds Ratio (OR)=1.30, 95% confidence interval (CI)=1.14-1.47), and this relationship was modified by early life farm exposure (born on a farm: OR=1.18, 95% CI=1.02-1.37; not born on a farm: OR=1.67, 95% CI=1.26-2.20, Interaction p-value=0.05). Significant positive associations were seen with both atopic and nonatopic asthma. Endotoxin was not related to either atopy or wheeze. Higher endotoxin was related to lower FEV1/FVC in asthma cases only (Interaction p=0.01). For asthma, there was suggestive evidence of a gene by environment interaction for the CD14 variant, rs2569190 (Interaction p=0.16), but not for the TLR4 variants, rs4986790 or rs4986791. CONCLUSIONS: House dust endotoxin was associated with current atopic and nonatopic asthma in a U.S. farming population. The degree of the association with asthma depended on early life farm exposures. Furthermore, endotoxin was associated with lower pulmonary function in asthmatics.

BACKGROUND: Previous studies, mostly from Europe, suggest that early-life farming exposures protect against childhood asthma and allergy; few data exist on asthma and allergy in adults. OBJECTIVE: To examine associations between early-life farming exposures and current asthma and atopy in an older adult US farming population. METHODS: We analyzed data from 1,746 farmers and 1,555 spouses (mean age=63) from a case-control study nested within the Agricultural Health Study. Current asthma and early-life farming exposures were assessed via questionnaires. We defined atopy based on specific IgE>0.70 IU/ml to at least one of ten allergens measured in blood. We used logistic regression, adjusted for age, sex, race, state (Iowa or North Carolina), and smoking (pack-years), to estimate associations between early-life exposures and asthma (1,198 cases and 2,031 non-cases) or atopy (578 cases and 2,526 non-cases). RESULTS: Exposure to the farming environment in utero and in early childhood had little or no association with asthma but was associated with reduced odds of atopy. The strongest association was seen for having a mother who performed farm activities while pregnant (Odds Ratio=0.60, 95% Confidence Interval=0.48-0.74) and remained significant in models with correlated early-life exposures including early childhood farm animal contact and raw milk consumption. CONCLUSIONS: In a large US farming population, early-life farm exposures, particularly maternal farming activities while pregnant, were strongly associated with reduced risk of atopy in adults. These results extend previous work done primarily on childhood outcomes and suggest that protective associations of early-life farming exposures on atopy endure across the life-course.

BACKGROUND: Mucormycosis is an invasive fungal infection with a high fatality rate. We investigated an outbreak of mucormycosis in a pediatric hospital to determine routes of pathogen transmission from the environment and prevent additional infections. METHODS: A case was defined as a hospital-onset illness consistent with mucormycosis, confirmed by culture or histopathology. Case-patient medical records were reviewed for clinical course and exposure to items and locations within the hospital. Environmental samples were collected from air and surfaces. Fungal isolates collected from case-patients and the environment were identified using DNA sequencing. RESULTS: Five case-patients had hospital-associated cutaneous mucormycosis over an eleven month period; all subsequently died. Three case-patients had conditions known to be associated with susceptibility to mucormycosis, while two had cardiac conditions with persistent acidosis. The cases occurred on several different wards throughout the hospital, and hospital linens were the only exposure identified as common to the case-patients. Rhizopus species were recovered from 26 (42%) of 62 environmental samples from clean linens and associated areas, and from one (4%) of 25 samples from non-linen-related items. Case-patients were infected with Rhizopusdelemar, which was also isolated from cultures of clean linens and clean linen delivery bins from the off-site laundry facility. CONCLUSIONS: Hospital linens were identified as a vehicle that carried Rhizopusdelemar into contact with susceptible patients. Fungal species identification using DNA-based methods is useful for corroborating epidemiologic links in hospital outbreak investigations. Hospital linens should be laundered, packaged, shipped, and stored in a manner that minimizes exposure to environmental contaminants.

BACKGROUND AND PURPOSE: Although the prothrombin G20210A mutation has been implicated as a risk factor for venous thrombosis, its role in arterial ischemic stroke is unclear, particularly among young adults. To address this issue, we examined the association between prothrombin G20210A and ischemic stroke in a white case-control population and additionally performed a meta-analysis. METHODS: From the population-based Genetics of Early Onset Stroke (GEOS) study, we identified 397 individuals of European ancestry aged 15 to 49 years with first-ever ischemic stroke and 426 matched controls. Logistic regression was used to calculate odds ratios (ORs) in the entire population and for subgroups stratified by sex, age, oral contraceptive use, migraine, and smoking status. A meta-analysis of 17 case-control studies (n=2305 cases <55 years) was also performed with and without GEOS data. RESULTS: Within GEOS, the association of the prothrombin G20210A mutation with ischemic stroke did not achieve statistical significance (OR=2.5; 95% confidence interval [CI]=0.9-6.5; P=0.07). However, among adults aged 15 to 42 years (younger than median age), cases were significantly more likely than controls to have the mutation (OR=5.9; 95% CI=1.2-28.1; P=0.03), whereas adults aged 42 to 49 years were not (OR=1.4; 95% CI=0.4-5.1; P=0.94). In our meta-analysis, the mutation was associated with significantly increased stroke risk in adults ≤55 years (OR=1.4; 95% CI=1.1-1.9; P=0.02), with significance increasing with addition of the GEOS results (OR=1.5; 95% CI=1.1-2.0; P=0.005). CONCLUSIONS: The prothrombin G20210A mutation is associated with ischemic stroke in young adults and may have an even stronger association among those with earlier onset strokes. Our finding of a stronger association in the younger young adult population requires replication.

We investigated a cluster of 10 Burkholderia cepacia complex-positive cultures among ventilated patients and those with a tracheostomy in an acute care hospital. Isolates from 5 patients had outbreak-strain-related Burkholderia contaminans. Isolates of B. cepacia complex unrelated to the outbreak strain were cultured from a sink drain. The investigation identified practices that might have led to contamination of patient respiratory care supplies with tap water, which might have contributed to the cluster.

The effect of packaging, shipping temperatures and storage times on recovery of Bacillus anthracis. Sterne spores from swabs was investigated. Macrofoam swabs were pre-moistened, inoculated with Bacillus anthracis spores, and packaged in primary containment or secondary containment before storage at -15°C, 5°C, 21°C, or 35°C for 0-7 days. Swabs were processed according to validated Centers for Disease Control/Laboratory Response Network culture protocols, and the percent recovery relative to a reference sample (T(0)) was determined for each variable. No differences were observed in recovery between swabs held at -15° and 5°C, (p ≥ 0.23). These two temperatures provided significantly better recovery than swabs held at 21°C or 35°C (all 7 days pooled, p ≤ 0.04). The percent recovery at 5°C was not significantly different if processed on days 1, 2 or 4, but was significantly lower on day 7 (day 2 vs. 7, 5°C, 10(2), p=0.03). Secondary containment provided significantly better percent recovery than primary containment, regardless of storage time (5°C data, p ≤ 0.008). The integrity of environmental swab samples containing Bacillus anthracis spores shipped in secondary containment was maintained when stored at -15°C or 5°C and processed within 4 days to yield the optimum percent recovery of spores.

We investigated an increase in Trichosporon asahii isolates among inpatients. We identified 63 cases; 4 involved disseminated disease. Trichosporon species was recovered from equipment cleaning rooms, washbasins, and fomites, which suggests transmission through washbasins. Patient washbasins should be single-patient use only; adherence to appropriate hospital disinfection guidelines was recommended.

BACKGROUND: In January 2012, on the basis of an initial report from a dermatologist, we began to investigate an outbreak of tattoo-associated Mycobacterium chelonae skin and soft-tissue infections in Rochester, New York. The main goals were to identify the extent, cause, and form of transmission of the outbreak and to prevent further cases of infection. METHODS: We analyzed data from structured interviews with the patients, histopathological testing of skin-biopsy specimens, acid-fast bacilli smears, and microbial cultures and antimicrobial susceptibility testing. We also performed DNA sequencing, pulsed-field gel electrophoresis (PFGE), cultures of the ink and ingredients used in the preparation and packaging of the ink, assessment of source water and faucets at tattoo parlors, and investigation of the ink manufacturer. RESULTS: Between October and December 2011, a persistent, raised, erythematous rash in the tattoo area developed in 19 persons (13 men and 6 women) within 3 weeks after they received a tattoo from a single artist who used premixed gray ink; the highest occurrence of tattooing and rash onset was in November (accounting for 15 and 12 patients, respectively). The average age of the patients was 35 years (range, 18 to 48). Skin-biopsy specimens, obtained from 17 patients, showed abnormalities in all 17, with M. chelonae isolated from 14 and confirmed by means of DNA sequencing. PFGE analysis showed indistinguishable patterns in 11 clinical isolates and one of three unopened bottles of premixed ink. Eighteen of the 19 patients were treated with appropriate antibiotics, and their condition improved. CONCLUSIONS: The premixed ink was the common source of infection in this outbreak. These findings led to a recall by the manufacturer.

PURPOSE: To investigate an outbreak of bacterial meningitis at an outpatient radiology clinic (clinic A) and to determine the source and implement measures to prevent additional infections. METHODS: A case was defined as bacterial meningitis in a patient undergoing myelography at clinic A from October 11 to 25, 2010. Patients who underwent myelography and other procedures at clinic A during that period were interviewed, medical records were reviewed, and infection prevention practices were assessed. Case-patient cerebrospinal fluid (CSF) specimens, oral specimens from health care personnel (HCP), and opened iohexol vials were tested for bacteria. Bacterial isolates were compared using pulsed-field gel electrophoresis. A culture-negative CSF specimen was tested using a real-time polymerase chain reaction assay. RESULTS: Three cases were identified among 35 clinic A patients who underwent procedures from October 11 to 25, 2010. All case-patients required hospitalization, 2 in an intensive care unit. Case-patients had myelography performed by the same radiology physician assistant and technician on October 25; all patients who underwent myelography on October 25 were affected. HCP did not wear facemasks and reused single-dose iohexol vials for multiple patients. Streptococcus salivarius (a bacteria commonly found in oral flora) was detected in the CSF of 2 case-patients (1 by culture, 1 using real-time polymerase chain reaction) and in HCP oral specimens; 1 opened iohexol vial contained Staphylococcus epidermidis. Pulsed-field gel electrophoresis profiles from the case-patient S salivarius and the radiology physician assistant were indistinguishable. CONCLUSIONS: Bacterial meningitis likely occurred because HCP performing myelography did not wear facemasks; lapses in injection practices may have contributed to transmission. Targeted education regarding mask use and safe injection practices is needed among radiology HCP.

We recently investigated three cases of bacterial meningitis that were reported from a midwestern radiology clinic where facemasks were not worn during spinal injection of contrast agent during myelography procedures. Using pulsed field gel electrophoresis we linked a case strain of S. salivarius to an oral specimen of a radiology physician assistant (RPA). We also used a real-time PCR assay to detect S. salivarius DNA within a culture-negative cerebrospinal fluid (CSF) specimen. Here we extend this investigation through using a nested PCR/sequencing strategy to link the culture-negative CSF specimen to the case strain. We also provide validation of the real-time PCR assay used, demonstrating that it is not solely specific for Streptococcus salivarius, but is also highly sensitive for detection of the closely related oral species Streptococcus vestibularis. Through using multilocus sequence typing and 16S rDNA sequencing we further strengthen the link between the CSF case isolate and the RPA carriage isolate. We also demonstrate that the newly characterized strains from this study are distinct from previously characterized S. salivarius strains associated with carriage and meningitis.

This work was initiated to address the gaps identified by Congress regarding validated biothreat environmental sampling and processing methods. Nine Laboratory Response Network (LRN) affiliated laboratories participated in a validation study of a cellulose sponge-wipe processing protocol for the recovery, detection, and quantification of viable Bacillus anthracis Sterne spores from steel surfaces. Steel coupons (645.16 cm(2)) were inoculated with 1-to 4-log(10) spores and then sampled with cellulose sponges (3M Sponge-Stick, St Paul, MN). Surrogate dust and background organisms were added to the sponges to mimic environmental conditions. Labs processed the sponges according to the provided protocol. Sensitivity, specificity, and mean percent recovery (%R), between-lab, within-lab and total %CV were calculated. The mean %R (SE) of spores from the surface was 32.4 (4.4), 24.4 (2.8) and 30.1 (2.3) for 1-, 2- and 4-log(10) inoculum levels, respectively. Sensitivities for colony counts were 84.1%, 100% and 100% for 1-, 2- and 4-log(10)inocula, respectively. These data help to characterize the variability of the processing method and thereby enhance confidence in the interpretation of the results of environmental sampling conducted during a B. anthracis contamination investigation.

AIMS: To evaluate the efficacy of chlorine dioxide (ClO(2) ) against seven species of bacterial threat (BT) agents in water. METHODS AND RESULTS: Two strains of Bacillus anthracis spores, Yersinia pestis, Francisella tularensis, Burkholderia pseudomallei, Burkholderia mallei, and Brucella species were each inoculated into a ClO(2) solution with an initial concentration of 2.0 mg l(-1) (spores only) and 0.25 mg l(-1) (all other bacteria) at pH 7 or 8, 5 degrees C or 25 degrees C. At 0.25 mg l(-1) in potable water, six species were inactivated by at least 3 orders of magnitude within 10 min. B. anthracis spores required up to 7 h at 5 degrees C for the same inactivation with 2.0 mg l(-1) ClO(2) . CONCLUSIONS: Typical ClO(2) doses used in water treatment facilities would be effective against all bacteria tested except B. anthracis spores which would require up to 7 h with the largest allowable dose of 2 mg l(-1) ClO(2) . Other water treatment processes may be required in addition to ClO(2) disinfection for effective spore removal or inactivation. SIGNIFICANCE AND IMPACT OF STUDY: The data obtained from this study provides valuable information for water treatment facilities and public health officials in the event that a potable water supply is contaminated with these BT agents.

AIMS: To determine the range of free available chlorine (FAC) required for disinfection of the live vaccine strain (LVS) and wild-type strains of Francisella tularensis. METHODS AND RESULTS: Seven strains of planktonic F. tularensis were exposed to 0.5 mg.l(-1) FAC for two pH values, 7 and 8, at 5 and 25 degrees C. LVS was inactivated 2 to 4 times more quickly than any of the wild-type F. tularensis strains at pH 8 and 5 degrees C. CONCLUSIONS: Free available chlorine residual concentrations routinely maintained in drinking water distribution systems would require up to two hours to reduce all F. tularensis strains by 4 log10. LVS was inactivated most quickly of the tested strains. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides contact time (CT) values that are useful for drinking water risk assessment and also suggests that LVS may not be a good surrogate in disinfection studies.

Twelve Laboratory Response Network (LRN) affiliated laboratories participated in a validation study of a macrofoam swab protocol for the recovery, detection, and quantification of viable B. anthracis (BA) Sterne spores from steel surfaces. CDC personnel inoculated steel coupons (26cm(2)) with 1-4 log(10) BA spores and recovered them by sampling with pre-moistened macrofoam swabs. Phase 1 (P1) of the study evaluated swabs containing BA only, while dust and background organisms were added to swabs in Phase 2 (P2) to mimic environmental conditions. Laboratories processed swabs and enumerated spores by culturing eluted swab suspensions and counting colonies with morphology consistent with BA. Processed swabs were placed in enrichment broth, incubated 24h, and cultured by streaking for isolation. Real-time PCR was performed on selected colonies from P2 samples to confirm the identity of BA. Mean percent recovery (%R) of spores from the surface ranged from 15.8-31.0% (P1) and 27.9-55.0% (P2). The highest mean percent recovery was 31.0% (sd 10.9%) for P1 (4 log(10) inoculum) and 55.0% (sd 27.6%) for P2 (1 log(10) inoculum). The overall %R was higher for P2 (44.6%) than P1 (24.1%), but the overall reproducibility (between lab variability) was lower in P2 than in P1 (25.0 vs 16.5 %CV, respectively). The overall precision (within lab variability) was close to identical for P1 and P2 (44.0 and 44.1, respectively), but varied greatly between inoculum levels. The protocol demonstrated linearity in %R over the three inoculum levels and is able to detect between 26 and 5x10(6) spores/26cm(2). Sensitivity as determined by culture was >98.3% for both phases and all inocula, suggesting the culture method maintains sensitivity in the presence of contaminants. The enrichment broth method alone was less sensitive for sampled swabs (66.4%) during P2, suggesting that the presence of background organisms inhibited growth or isolation of BA from the broth. The addition of real-time PCR testing to the assay increased specificity from >85.4% to >95.0% in P2. Although the precision was low at the 1 log(10) inoculum level in both phases (59.0 and 50.2%), this swab processing protocol, was sensitive, specific, precise, and reproducible at 2-4 log(10) /26cm(2) spore concentrations.

Burkholderia pseudomallei is a select agent and the causative agent of melioidosis. Variations in previously reported chlorine and monochloramine concentration time (Ct) values for disinfection of this organism make decisions regarding the appropriate levels of chlorine in water treatment systems difficult. This study identified the variation in Ct values for 2-, 3-, and 4-log(10) reductions of eight environmental and clinical isolates of B. pseudomallei in phosphate-buffered water. The greatest calculated Ct values for a 4-log(10) inactivation were 7.8 mg.min/liter for free available chlorine (FAC) at pH 8 and 5 degrees C and 550 mg.min/liter for monochloramine at pH 8 and 5 degrees C. Ionic strength of test solutions, culture hold times in water, and cell washing were ruled out as sources of the differences in prior observations. Tolerance to FAC was correlated with the relative amount of extracellular material produced by each isolate. Solid-phase cytometry analysis using an esterase-cleaved fluorochrome assay detected a 2-log(10)-higher level of organisms based upon metabolic activity than did culture, which in some cases increased Ct values by fivefold. Despite strain-to-strain variations in Ct values of 17-fold for FAC and 2.5-fold for monochloramine, standard FAC disinfection practices utilized in the United States should disinfect planktonic populations of these B. pseudomallei strains by 4 orders of magnitude in less than 10 min at the tested temperatures and pH levels.

BACKGROUND AND PURPOSE: Migraine with aura is a risk factor for ischemic stroke, but the mechanism by which these disorders are associated remains unclear. Both disorders exhibit familial clustering, which may imply a genetic influence on migraine and stroke risk. Genes encoding for endothelial function are promising candidate genes for migraine and stroke susceptibility because of the importance of endothelial function in regulating vascular tone and cerebral blood flow. METHODS: Using data from the Stroke Prevention in Young Women study, a population-based case-control study including 297 women aged 15 to 49 years with ischemic stroke and 422 women without stroke, we evaluated whether polymorphisms in genes regulating endothelial function, including endothelin-1 (EDN), endothelin receptor type B (EDNRB), and nitric oxide synthase-3 (NOS3), confer susceptibility to migraine and stroke. RESULTS: EDN SNP rs1800542 and rs10478723 were associated with increased stroke susceptibility in whites (OR, 2.1; 95% CI, 1.1-4.2 and OR, 2.2; 95% CI, 1.1-4.4; P=0.02 and 0.02, respectively), as were EDNRB SNP rs4885493 and rs10507875, (OR, 1.7; 95% CI, 1.1-2.7 and OR, 2.4; 95% CI, 1.4-4.3; P=0.01 and 0.002, respectively). Only 1 of the tested SNP (NOS3 rs3918166) was associated with both migraine and stroke. CONCLUSIONS: In our study population, variants in EDN and EDNRB were associated with stroke susceptibility in white but not in black women. We found no evidence that these genes mediate the association between migraine and stroke.