Serologic Prevalence of Ebola Virus in Equatorial Africa.

We conducted a serologic survey of 2,430 serum samples collected during 1997-2012 for various studies to determine the prevalence of the hemorrhagic fever virus Ebola virus (EBOV) in equatorial Africa. We screened serum samples for neutralizing antibodies by using a pseudotype microneutralization assay and a newly developed luciferase immunoprecipitation system assay. Specimens seroreactive for EBOV were confirmed by using an ELISA. Our results suggest a serologic prevalence of 2%-3.5% in the Republic of the Congo and the Democratic Republic of the Congo, which have reported outbreaks of infection with EBOV. In addition we detected a seroprevalence of 1.3% in southern Cameroon, which indicated a low risk for exposure in this region.

Inflammatory production of reactive oxygen species by Drosophila hemocytes activates cellular immune defenses.

The production of reactive oxygen species (ROS) is a prominent response to infection among innate immune cells such as macrophages and neutrophils. To better understand the relationship between antimicrobial and regulatory functions of blood cell ROS, we have characterized the ROS response to infection in Drosophila hemocytes. Using fluorescent probes, we find a biphasic hemocyte ROS response to bacterial infection. In the first hour, virtually all hemocytes generate a transient ROS signal, with nonphagocytic cells including prohemocytes and crystal cells displaying exceptionally strong responses. A distinct, and more delayed ROS response starting at 90 min is primarily within cells that have engulfed bacteria, and is sustained for several hours. The early response has a clear regulatory function, as dampening or intensifying the intracellular ROS level has profound effects on plasmatocyte activation. In addition, ROS are necessary and sufficient to activate JNK signalling in crystal cells, and to promote JNK-dependent crystal cell rupture. These findings indicate that Drosophila will be a promising model in which to dissect the mechanisms of ROS stimulation of immune activation.

Sensitive Next-Generation Sequencing Method Reveals Deep Genetic Diversity of HIV-1 in the Democratic Republic of the Congo.

As the epidemiological epicenter of the human immunodeficiency virus (HIV) pandemic, the Democratic Republic of the Congo (DRC) is a reservoir of circulating HIV strains exhibiting high levels of diversity and recombination. In this study, we characterized HIV specimens collected in two rural areas of the DRC between 2001 and 2003 to identify rare strains of HIV. The env gp41 region was sequenced and characterized for 172 HIV-positive specimens. The env sequences were predominantly subtype A (43.02%), but 7 other subtypes (33.14%), 20 circulating recombinant forms (CRFs; 11.63%), and 20 unclassified (11.63%) sequences were also found. Of the rare and unclassified subtypes, 18 specimens were selected for next-generation sequencing (NGS) by a modified HIV-switching mechanism at the 5' end of the RNA template (SMART) method to obtain full-genome sequences. NGS produced 14 new complete genomes, which included pure subtype C (n = 2), D (n = 1), F1 (n = 1), H (n = 3), and J (n = 1) genomes. The two subtype C genomes and one of the subtype H genomes branched basal to their respective subtype branches but had no evidence of recombination. The remaining 6 genomes were complex recombinants of 2 or more subtypes, including subtypes A1, F, G, H, J, and K and unclassified fragments, including one subtype CRF25 isolate, which branched basal to all CRF25 references. Notably, all recombinant subtype H fragments branched basal to the H clade. Spatial-geographical analysis indicated that the diverse sequences identified here did not expand globally. The full-genome and subgenomic sequences identified in our study population significantly increase the documented diversity of the strains involved in the continually evolving HIV-1 pandemic.IMPORTANCE Very little is known about the ancestral HIV-1 strains that founded the global pandemic, and very few complete genome sequences are available from patients in the Congo Basin, where HIV-1 expanded early in the global pandemic. By sequencing a subgenomic fragment of the HIV-1 envelope from study participants in the DRC, we identified rare variants for complete genome sequencing. The basal branching of some of the complete genome sequences that we recovered suggests that these strains are more closely related to ancestral HIV-1 strains than to previously reported strains and is evidence that the local diversification of HIV in the DRC continues to outpace the diversity of global strains decades after the emergence of the pandemic.

Discovery of a Novel Human Pegivirus in Blood Associated with Hepatitis C Virus Co-Infection.

Hepatitis C virus (HCV) and human pegivirus (HPgV), formerly GBV-C, are the only known human viruses in the Hepacivirus and Pegivirus genera, respectively, of the family Flaviviridae. We present the discovery of a second pegivirus, provisionally designated human pegivirus 2 (HPgV-2), by next-generation sequencing of plasma from an HCV-infected patient with multiple bloodborne exposures who died from sepsis of unknown etiology. HPgV-2 is highly divergent, situated on a deep phylogenetic branch in a clade that includes rodent and bat pegiviruses, with which it shares <32% amino acid identity. Molecular and serological tools were developed and validated for high-throughput screening of plasma samples, and a panel of 3 independent serological markers strongly correlated antibody responses with viral RNA positivity (99.9% negative predictive value). Discovery of 11 additional RNA-positive samples from a total of 2440 screened (0.45%) revealed 93-94% nucleotide identity between HPgV-2 strains. All 12 HPgV-2 RNA-positive cases were identified in individuals also testing positive for HCV RNA (12 of 983; 1.22%), including 2 samples co-infected with HIV, but HPgV-2 RNA was not detected in non-HCV-infected individuals (p<0.0001), including those singly infected by HIV (p = 0.0075) or HBV (p = 0.0077), nor in volunteer blood donors (p = 0.0082). Nine of the 12 (75%) HPgV-2 RNA positive samples were reactive for antibodies to viral serologic markers, whereas only 28 of 2,429 (1.15%) HPgV-2 RNA negative samples were seropositive. Longitudinal sampling in two individuals revealed that active HPgV-2 infection can persist in blood for at least 7 weeks, despite the presence of virus-specific antibodies. One individual harboring both HPgV-2 and HCV RNA was found to be seronegative for both viruses, suggesting a high likelihood of simultaneous acquisition of HCV and HPgV-2 infection from an acute co-transmission event. Taken together, our results indicate that HPgV-2 is a novel bloodborne infectious virus of humans and likely transmitted via the parenteral route.

Enhanced Recovery in Orthopedics: A Prospective Audit of an Enhanced Recovery Program for Patients Undergoing Hip or Knee Arthroplasty.

Hip and knee arthroplasty are common 'surgical procedures. Enhanced recovery programs help reduce length of stay with good patient outcomes and satisfaction. An audit is described showing improved pain management, reduced nausea and vomiting, and quicker recovery through an interprofessional approach.

A Pan-HIV Strategy for Complete Genome Sequencing.

Molecular surveillance is essential to monitor HIV diversity and track emerging strains. We have developed a universal library preparation method (HIV-SMART [i.e.,switchingmechanismat 5' end ofRNAtranscript]) for next-generation sequencing that harnesses the specificity of HIV-directed priming to enable full genome characterization of all HIV-1 groups (M, N, O, and P) and HIV-2. Broad application of the HIV-SMART approach was demonstrated using a panel of diverse cell-cultured virus isolates. HIV-1 non-subtype B-infected clinical specimens from Cameroon were then used to optimize the protocol to sequence directly from plasma. When multiplexing 8 or more libraries per MiSeq run, full genome coverage at a median ∼2,000× depth was routinely obtained for either sample type. The method reproducibly generated the same consensus sequence, consistently identified viral sequence heterogeneity present in specimens, and at viral loads of ≤4.5 log copies/ml yielded sufficient coverage to permit strain classification. HIV-SMART provides an unparalleled opportunity to identify diverse HIV strains in patient specimens and to determine phylogenetic classification based on the entire viral genome. Easily adapted to sequence any RNA virus, this technology illustrates the utility of next-generation sequencing (NGS) for viral characterization and surveillance.

Identifying and Responding to Lead in Drinking Water in a University Setting.

Lead is an established neurotoxicant, and it has known associations with adverse neurodevelopmental and reproductive outcomes. Exposure to lead at any level is unsafe, and the United States (US) has enacted various federal and state legislations to regulate lead levels in drinking water in K-12 schools and childcare facilities; however, no regulations exist for higher education settings. Upon the discovery of lead in drinking water fixtures in the University of North Carolina at Chapel Hill (UNC-CH) campus, a cross-campus water testing network and sampling plan was developed and deployed. The campaign was based on the US Environmental Protection Agency's (EPA) 3Ts (Training, Testing, and Taking Action) guidance. The seven-month campaign involved 5954 tests on 3825 drinking water fixtures across 265 buildings. A total of 502 (8.43%) tests showed lead above the limit of detection (1 part per billion, ppb), which represented 422 (11.03%) fixtures. Fewer than 1.5% of the tests were above the EPA action level for public water systems (15 ppb). In conclusion, systematic testing of all the fixtures across campus was required to identify localized contamination, and each entity in the cross-campus network undertook necessary roles to generate a successful testing campaign. UNC-CH established preventative measures to test drinking water fixtures every three years, which provide a framework for other higher education institutions in responding to lead contamination.

Early HLA-B*57-restricted CD8+ T lymphocyte responses predict HIV-1 disease progression.

Although HLA-B*57 (B57) is associated with slow progression to disease following HIV-1 infection, B57 heterozygotes display a wide spectrum of outcomes, including rapid progression, viremic slow progression, and elite control. Efforts to identify differences between B57-positive (B57(+)) slow progressors and B57(+) rapid progressors have largely focused on cytotoxic T lymphocyte (CTL) phenotypes and specificities during chronic stages of infection. Although CTL responses in the early months of infection are likely to be the most important for the long-term rate of HIV-1 disease progression, few data on the early CTL responses of eventual slow progressors have been available. Utilizing the Multicenter AIDS Cohort Study (MACS), we retrospectively examined the early HIV-1-specific CTL responses of 14 B57(+) individuals whose time to development of disease ranged from 3.5 years to longer than 25 years after infection. In general, a greater breadth of targeting of epitopes from structural proteins, especially Gag, as well as of highly conserved epitopes from any HIV-1 protein, correlated with longer times until disease. The single elite controller in the cohort was an outlier on several correlations of CTL targeting and time until disease, consistent with reports that elite control is typically not achieved solely by protective HLA-mediated CTLs. When targeting of individual epitopes was analyzed, we found that early CTL responses to the IW9 (ISPRTLNAW) epitope of Gag, while generally subdominant, correlated with delayed progression to disease. This is the first study to identify early CTL responses to IW9 as a correlate of protection in persons with HLA-B*57.

Training general practitioners in remote Western Australia in a method of screening and brief intervention for harmful alcohol use: a pilot study.

OBJECTIVE: High levels of alcohol-related harm are a salient feature of many rural communities in Australia. General practitioners (GPs) are uniquely placed to identify and treat patients with harmful alcohol use in remote settings, yet corresponding opportunities for education in effective brief psychological interventions for harmful alcohol use are limited. This study piloted a training model for alcohol screening and brief intervention for GPs working in Kalgoorlie-Boulder, a remote Western Australian community facing significant alcohol-related problems. DESIGN: Observational pilot study. SETTING: Primary care. MAIN OUTCOME MEASURE(S): Perceived role in responding to harmful alcohol use, and confidence and knowledge of alcohol screening and brief intervention; satisfaction with a short training session focused on alcohol screening and brief intervention; and impact of training on implementation of screening and brief intervention for harmful alcohol use. RESULTS: Fifty per cent of GPs took up the training opportunity. GPs recognised their professional responsibility for conducting brief intervention but reported comparatively lower confidence and skills in implementing screening and intervention prior to training. The training improved knowledge and confidence in conducting alcohol screening and brief intervention. All GPs increased their frequency of alcohol screening, and 88% of GPs reported increasing the frequency of brief intervention at 6 months. CONCLUSIONS: Preliminary findings suggest that among participating GPs, subsequent compliance with identification and management of harmful alcohol use was improved. Further work examining methods to improve rural and remote GP participation in alcohol-related harm prevention training is required, as the potential impact on communities with disproportionately high alcohol-related difficulties is significant.

Confirmation of putative HIV-1 group P in Cameroon.

We report the second human immunodeficiency virus (HIV) belonging to the new HIV type 1 (HIV-1) group P lineage that is closely related to the simian immunodeficiency virus found in gorillas. This virus was identified in an HIV-seropositive male hospital patient in Cameroon, confirming that the group P virus is circulating in humans. Results from screening 1,736 HIV-seropositive specimens collected in Cameroon indicate that HIV-1 group P infections are rare, accounting for only 0.06% of HIV infections. Despite its rarity, group P shows evidence of adaptation to humans.

Psidin is required in Drosophila blood cells for both phagocytic degradation and immune activation of the fat body.

Phagocytic blood cells are critical to innate immune defense: They internalize and destroy microbial invaders and produce signals that trigger other immune responses. Despite this central role, the in vivo contributions of phagocytosis to systemic immune activation are not well understood. Drosophila has proven a fruitful model for the investigation of evolutionarily conserved innate immune mechanisms, including NF-kappaB-dependent transcriptional induction, RNAi in antiviral responses, and phagocytosis. The phagocytes of Drosophila encounter bacterial invaders early in infection and contribute to survival of infection. Phagocytosis in flies and mammals is highly homologous: Both rely on scavenger receptors, opsonins, and actin rearrangements for engulfment; have phagosomal cysteine proteases active at low pH; and can be subverted by similar intracellular pathogens. Although the role of Drosophila phagocytes in the activation of other immune tissues has not been clear, we show that induction of the antibacterial-peptide gene Defensin in the fat body during infection requires blood-cell contributions. We identify a gene, psidin, that encodes a lysosomal protein required in the blood cells for both degradation of engulfed bacteria and activation of fat-body Defensin. These data establish a role for the phagocytic blood cells of Drosophila in detection of infection and activation of the humoral immune response.

Expanded evaluation of blood donors in the United States for human immunodeficiency virus type 1 non-B subtypes and antiretroviral drug-resistant strains: 2005 through 2007.

BACKGROUND: In a previous study of 66 human immunodeficiency virus (HIV)-infected US blood donors from 1999 to 2005, HIV-1 non-B and antiretroviral drug-resistant strains accounted for 4.7 and 6.5% of HIV infections, respectively. This study was expanded to include an additional 11 recently acquired infections and 197 established infections collected from January 2005 through December 2007. STUDY DESIGN AND METHODS: HIV-infected donors were detected using FDA-licensed assays. Drug resistance profiles for protease and reverse transcriptase (RT) genes were determined using a genotyping system (ViroSeq, Celera Diagnostics); genetic subtype was determined by phylogenetic analysis of these sequences. RESULTS: Drug resistance profiles were obtained for 203 of 208 specimens; 9.9% had mutations that confer drug resistance. Ten showed resistance to a single drug class: nine to nonnucleoside RT inhibitors (NNRTIs) and one to nucleoside RT inhibitors (NRTIs). Eight showed two drug class resistance: five NRTI plus NNRTI, two NRTI plus protease inhibitor (PI), and one NNRTI plus PI. Two showed three drug class resistance. Non-B strains were identified in 2.5% of donors and consisted of subtypes A1 and D, CRF02_AG, CRF43-02G, and URF_BF. CONCLUSIONS: Data from this and the previous study show that antiretroviral drug-resistant HIV-1 is present in 9.1% of HIV-infected donors from 1999 through 2007; 9.3% of established infections and 6.9% of recent infections. Diverse HIV-1 non-B strains presently account for 3.0% of HIV infections in US donors.

Seroreactivity against Marburg or related filoviruses in West and Central Africa.

A serological survey of 2,430 archived serum samples collected between 1997 and 2012 was conducted to retrospectively determine the prevalence of Marburg virus in five African countries. Serum samples were screened for neutralizing antibodies in a pseudotype micro-neutralization assay and confirmed by enzyme-linked immunosorbent assay (ELISA). Surprisingly, a seroprevalence for Marburg virus of 7.5 and 6.3% was found in Cameroon and Ghana, respectively, suggesting the circulation of filoviruses or related viruses outside of known endemic areas that remain undetected by current surveillance efforts. However, due to the lack of validated assays and appropriate positive controls, these results must be considered preliminary.

HIV testing in a high-incidence population: is antibody testing alone good enough?

BACKGROUND: The Centers for Disease Control and Prevention recently recommended the expansion of human immunodeficiency virus (HIV) antibody testing. However, antibody tests have longer "window periods" after HIV acquisition than do nucleic acid amplification tests (NAATs). METHODS: Public Health-Seattle & King County offered HIV antibody testing to men who have sex with men (MSM) using the OraQuick Advance Rapid HIV-1/2 Antibody Test (OraQuick; OraSure Technologies) on oral fluid or finger-stick blood specimens or using a first- or second-generation enzyme immunoassay. The enzyme immunoassay was also used to confirm reactive rapid test results and to screen specimens from OraQuick-negative MSM prior to pooling for HIV NAAT. Serum specimens obtained from subsets of HIV-infected persons were retrospectively evaluated by use of other HIV tests, including a fourth-generation antigen-antibody combination assay. RESULTS: From September 2003 through June 2008, a total of 328 (2.3%) of 14,005 specimens were HIV antibody positive, and 36 (0.3%) of 13,677 antibody-negative specimens were NAAT positive (indicating acute HIV infection). Among 6811 specimens obtained from MSM who were initially screened by rapid testing, OraQuick detected only 153 (91%) of 169 antibody-positive MSM and 80% of the 192 HIV-infected MSM detected by the HIV NAAT program. HIV was detected in serum samples obtained from 15 of 16 MSM with acute HIV infection that were retrospectively tested using the antigen-antibody combination assay. CONCLUSIONS: OraQuick may be less sensitive than enzyme immunoassays during early HIV infection. NAAT should be integrated into HIV testing programs that serve populations that undergo frequent testing and that have high rates of HIV acquisition, particularly if rapid HIV antibody testing is employed. Antigen-antibody combination assays may be a reasonably sensitive alternative to HIV NAAT.

Identification of human immunodeficiency virus type 1 non-B subtypes and antiretroviral drug-resistant strains in United States blood donors.

BACKGROUND: In this study, human immunodeficiency virus type 1 (HIV-1)-infected blood donors were evaluated for genetic subtype and drug resistance to determine the prevalence of divergent HIV strains in the US donor population. STUDY DESIGN AND METHODS: Subtype was determined by phylogenetic analysis of viral sequences amplified by reverse transcription-polymerase chain reaction. The drug resistance profile of the protease and reverse transcriptase (RT) genes was determined using an HIV-1 genotyping system (ViroSeq). RESULTS: From 1999 through 2005, 26 recently infected donors, defined as HIV-1 RNA-positive, antibody-negative (RNA+/Ab-), were identified (yield, 1:1.61 million). Over the same period, the frequency of anti-HIV-positive donors was 1:34,700. Twenty RNA+/Ab- specimens were evaluated; all were infected with HIV-1 subtype B. Drug resistance profiles obtained for 18 donors identified one strain with protease mutation L90M that confers resistance to nelfinavir and one with RT mutation Y188H that confers resistance to nevirapine. Genetic subtype was determined for 44 of 46 HIV antibody-reactive and confirmed-positive (Ab+) specimens. Three infections (6.8%) were due to circulating recombinant forms: 2 CRF01_AE and 1 CRF02_AG. In the Ab+ group, one strain was resistant to all nucleoside RT inhibitors and one had mutations that confer resistance to protease inhibitors. CONCLUSION: The data show that antiretroviral drug-resistant HIV strains are being transmitted in the United States. Overall 6.5 percent (4 of 62) of HIV-1-infected donors harbored drug-resistant strains. HIV-1 non-B strains accounted for 4.7 percent (3 of 64) of the infections in donors. HIV-1 subtype B is still the predominant strain in the United States; however, non-B strains are increasing.

Human Pleural Fluid Elicits Pyruvate and Phenylalanine Metabolism in Acinetobacter baumannii to Enhance Cytotoxicity and Immune Evasion.

Acinetobacter baumannii (Ab) is one of the most treacherous pathogens among those causing hospital-acquired pneumonia (HAP). A. baumannii possesses an adaptable physiology, seen not only in its antibiotic resistance and virulence phenotypes but also in its metabolic versatility. In this study, we observed that A. baumannii undergoes global transcriptional changes in response to human pleural fluid (PF), a key host-derived environmental signal. Differential gene expression analyses combined with experimental approaches revealed changes in A. baumannii metabolism, affecting cytotoxicity, persistence, bacterial killing, and chemotaxis. Over 1,220 genes representing 55% of the differentially expressed transcriptomic data corresponded to metabolic processes, including the upregulation of glutamate, short chain fatty acid, and styrene metabolism. We observed an upregulation by 1.83- and 2.61-fold of the pyruvate dehydrogenase complex subunits E3 and E2, respectively. We also found that pyruvate (PYR), in conjunction with PF, triggers an A. baumannii pathogenic behavior that adversely impacts human epithelial cell viability. Interestingly, PF also amplified A. baumannii cytotoxicity against murine macrophages, suggesting an immune evasion strategy implemented by A. baumannii. Moreover, we uncovered opposing metabolic strategies dependent on the degree of pathogenicity of the strains, where less pathogenic strains demonstrated greater utilization of PYR to promote persister formation in the presence of PF. Additionally, our transcriptomic analysis and growth studies of A. baumannii suggest the existence of an alternative phenylalanine (PA) catabolic route independent of the phenylacetic acid pathway, which converts PA to phenylpyruvate (PP) and shuttles intermediates into styrene metabolism. This alternative route promoted a neutrophil-evasive state, as PF-induced degradation of PP significantly reduced overall human neutrophil chemotaxis in ex vivo chemotactic assays. Taken together, these data highlight A. baumannii pathoadaptabililty in response to host signals and provide further insight into the role of bacterial metabolism in virulence traits, antibiotic persistence strategies, and host innate immune evasion.

HIV type 2 intergroup recombinant identified in Cameroon.

A unique HIV-2 intergroup recombinant strain was identified in Cameroon. The virus, CM-03-510-03, was amplified from blood collected from a 47-year-old female patient in Douala, Cameroon in 2003 who was seroreactive for HIV-2. A near full-length genome 9089 nucleotides in length was amplified from proviral DNA. The genome for CM-03-510-03 is composed of segments of HIV-2 groups A and B with four recombination break-points and has open reading frames for all the structural and regulatory genes. A comparison of CM-03-510-03 to the only previously reported HIV-2 intergroup recombinant shows that the two strains share one recombination breakpoint but are otherwise distinct from each other. Similar to HIV-1, HIV-2 intergroup recombination is an indication that coinfection with more than one strain has occurred in individuals and is a mechanism that increases strain genetic diversity.

Requirement for and polarized localization of integrin proteins during Drosophila wound closure.

Wound reepithelialization is an evolutionarily conserved process in which skin cells migrate as sheets to heal the breach and is critical to prevent infection but impaired in chronic wounds. Integrin heterodimers mediate attachment between epithelia and underlying extracellular matrix and also act in large signaling complexes. The complexity of the mammalian wound environment and evident redundancy among integrins has impeded determination of their specific contributions to reepithelialization. Taking advantage of the genetic tools and smaller number of integrins in Drosophila, we undertook a systematic in vivo analysis of integrin requirements in the reepithelialization of skin wounds in the larva. We identify αPS2-ßPS and αPS3-ßPS as the crucial integrin dimers and talin as the only integrin adhesion component required for reepithelialization. The integrins rapidly accumulate in a JNK-dependent manner in a few rows of cells surrounding a wound. Intriguingly, the integrins localize to the distal margin in these cells, instead of the frontal or lamellipodial distribution expected for proteins providing traction and recruit nonmuscle myosin II to the same location. These findings indicate that signaling roles of integrins may be important for epithelial polarization around wounds and lay the groundwork for using Drosophila to better understand integrin contributions to reepithelialization.

HIV-1 strains identified in Brazilian blood donors: significant prevalence of B/F1 recombinants.

In the Brazilian HIV-1 epidemic subtypes B, C, and F1 are cocirculating in the high risk population groups, and there is a high prevalence of intersubtype recombinant forms. The dynamic nature of the HIV epidemic in Brazil led us to study HIV-1 subtypes present in HIV-infected blood donations collected from 2001 to 2003. Donations from 91 seropositive donors were evaluated. Genetic subtype was obtained for 88 specimens based on sequence analysis of gag p24, pol IN, and env gp41 IDR. HIV-1 subtype B was the predominant strain present in the donor population (73.9%). A significant prevalence of intersubtype recombinants of subtypes B and F1 was found (22.7%). Subtype C (1.1%) and F1 (2.3%) were rare. None of the B/F1 recombinants is CRF28_BF or CRF29_BF. The high level of unique B/F1 recombinant strains in this population demonstrates the dynamic and complex nature of the HIV epidemic in Brazil.