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Background: Diarrhea is the second leading cause of death in children < 5 years globally and the parasite genus Cryptosporidium is a leading cause of that diarrhea. The global disease burden attributable to cryptosporidiosis is substantial and the only approved chemotherapeutic, nitazoxanide, has poor efficacy in HIV positive children. Chemotherapeutic development is dependent on the calf model of cryptosporidiosis, which is the best approximation of human disease. However, the model is not consistently applied across research studies. Data collection commonly occurs using two different methods: Complete Fecal Collection (CFC), which requires use of confinement housing, and Interval Collection (IC), which permits use of box stalls. CFC mimics human challenge model methodology but it is unknown if confinement housing impacts study end-points and if data gathered via this method is suitable for generalization to human populations. Methods: Using a modified crossover study design we compared CFC and IC and evaluated the impact of housing on study end-points. At birth, calves were randomly assigned to confinement (n = 14) or box stall housing (n = 9), or were challenged with 5 x 107 C. parvum oocysts, and followed for 10 days. Study end-points included fecal oocyst shedding, severity of diarrhea, degree of dehydration, and plasma cortisol. Findings: Calves in confinement had no significant differences in mean log oocysts enumerated per gram of fecal dry matter between CFC and IC samples (P = 0.6), nor were there diurnal variations in oocyst shedding (P = 0.1). Confinement housed calves shed significantly more oocysts (P = 0.05), had higher plasma cortisol (P = 0.001), and required more supportive care (P = 0.0009) than calves in box stalls. Conclusion: Housing method confounds study end-points in the calf model of cryptosporidiosis. Due to increased stress data collected from calves in confinement housing may not accurately estimate the efficacy of chemotherapeutics targeting C. parvum. [ABSTRACT FROM AUTHOR]

Background: Emerging resistance to antibiotics renders therapy of Typhoid Fever (TF) increasingly challenging. The current single-drug regimens exhibit prolonged fever clearance time (FCT), imposing a great burden on both patients and health systems, and potentially contributing to the development of antibiotic resistance and the chronic carriage of the pathogens. The aim of our study was to assess the efficacy of combining third-generation cephalosporin therapy with azithromycin on the outcomes of TF in patients living in an endemic region. Methods: An open-label, comparative trial was conducted at Dhulikhel Hospital, Nepal, between October 2012 and October 2014. Only culture-confirmed TF cases were eligible. Patients were alternately allocated to one of four study arms: hospitalized patients received either intravenous ceftriaxone or a combination of ceftriaxone and oral azithromycin, while outpatients received either oral azithromycin or a combination of oral azithromycin and cefexime. The primary outcome evaluated was FCT and the secondary outcomes included duration of bacteremia. Results: 105 blood culture-confirmed patients, of whom 51 were treated as outpatients, were eligible for the study. Of the 88 patients who met the inclusion criteria for FCT analysis 41 patients received a single-agent regimen, while 47 patients received a combined regimen. Results showed that FCT was significantly shorter for the latter (95 versus 88 hours, respectively, p = 0·004), and this effect was exhibited in both the hospitalized and the outpatient sub-groups. Repeat blood cultures, drawn on day 3, were positive for 8/47 (17%) patients after monotherapy, versus 2/51 (4%) after combination therapy (p = 0·045). No severe complications or fatalities occurred in any of the groups. Conclusions: Combined therapy of third-generation cephalosporins and azithromycin for TF may surpass monotherapy in terms of FCT and time to elimination of bacteremia. Trial registration: Trial registration number: . [ABSTRACT FROM AUTHOR]

Background: Children are substantially affected by enteric fever in most settings with a high burden of the disease, including Nepal. However pathogen population structure and transmission dynamics are poorly delineated in young children, the proposed target group for immunization programs. Here we present whole genome sequencing and antimicrobial susceptibility data on 198 S. Typhi and 66 S. Paratyphi A isolated from children aged 2 months to 15 years of age during blood culture surveillance at Patan Hospital, Nepal, 2008–2016. Principal findings: S. Typhi was the dominant agent and comprised several distinct genotypes, dominated by 4.3.1 (H58). The heterogeneity of genotypes in children under five was reduced compared to data from 2005–2006, attributable to ongoing clonal expansion of H58. Most isolates (86%) were non-susceptible to fluoroquinolones, associated mainly with S. Typhi H58 Lineage II and S. Paratyphi A harbouring mutations in the quinolone resistance-determining region (QRDR); non-susceptible strains from these groups accounted for 50% and 25% of all isolates. Multi-drug resistance (MDR) was rare (3.5% of S. Typhi, 0 S. Paratyphi A) and restricted to chromosomal insertions of resistance genes in H58 lineage I strains. Temporal analyses revealed a shift in dominance from H58 Lineage I to H58 Lineage II, with the latter being significantly more common after 2010. Comparison to global data sets showed the local S. Typhi and S. Paratyphi A strains had close genetic relatives in other South Asian countries, indicating regional strain circulation. Multiple imports from India of ciprofloxacin-resistant H58 Lineage II strains were identified, but these were rare and showed no evidence of clonal replacement of local S. Typhi. Significance: These data indicate that enteric fever in Nepal continues to be a major public health issue with ongoing inter- and intra-country transmission, and highlights the need for regional coordination of intervention strategies. The absence of a S. Paratyphi A vaccine is cause for concern, given its prevalence as a fluoroquinolone resistant enteric fever agent in this setting. [ABSTRACT FROM AUTHOR]

Background: The mediators of protection against cholera, a severe dehydrating illness of humans caused by Vibrio cholerae, are unknown. We have previously shown that plasma IgA as well as memory B IgG cells targeting lipopolysaccharide (LPS) of Vibrio cholerae O1 correlate with protection against V. cholerae O1 infection among household contacts of cholera patients. Protection against cholera is serogroup specific, and serogroup specificity is defined by the O-specific polysaccharide (OSP) component of LPS. Therefore, we prospectively followed household contacts of cholera patients to determine whether OSP-specific immune responses present at the time of enrollment are associated with protection against V. cholerae infection. Methodology: In this study, we enrolled two hundred forty two household contacts of one hundred fifty index patients who were infected with Vibrio cholerae. We determined OSP-specific memory B cells and plasma IgA, IgG and IgM antibody responses on study entry (day 2). Principle findings: The presence of OSP-specific plasma IgA, IgM, and IgG antibody responses on study entry were associated with a decrease in the risk of infection in household contacts (IgA, p = 0.015; IgM, p = 0.01, and IgG, p = 0.024). In addition, the presence of OSP-specific IgG memory B cell responses in peripheral blood on study entry was also associated with a decreased risk of infection (44% reduction; 95% CI: 31.1 to 99.8) in contacts. No protection was associated with cholera toxin B subunit (CtxB)-specific memory B cell responses. Conclusion: These results suggest that immune responses that target OSP, both in plasma and memory responses, may be important in mediating protection against infection with V. cholerae O1. [ABSTRACT FROM AUTHOR]

Although flea-borne rickettsiosis is endemic in Los Angeles County, outbreaks are rare. In the spring of 2015 three human cases of flea-borne rickettsiosis among residents of a mobile home community (MHC) prompted an investigation. Fleas were ubiquitous in common areas due to presence of flea-infested opossums and overabundant outdoor cats and dogs. The MHC was summarily abated in June 2015, and within five months, flea control and removal of animals significantly reduced the flea population. Two additional epidemiologically-linked human cases of flea-borne rickettsiosis detected at the MHC were suspected to have occurred before control efforts began. Molecular testing of 106 individual and 85 pooled cat fleas, blood and ear tissue samples from three opossums and thirteen feral cats using PCR amplification and DNA sequencing detected rickettsial DNA in 18.8% of the fleas. Seventeen percent of these cat fleas tested positive for R. felis-specific DNA compared to under two (<2) percent for Candidatus R. senegalensis-specific DNA. In addition, serological testing of 13 cats using a group-specific IgG-ELISA detected antibodies against typhus group rickettsiae and spotted fever group rickettsiae in six (46.2%) and one (7.7%) cat, respectively. These results indicate that cats and their fleas may have played an active role in the epidemiology of the typhus group and/or spotted fever group rickettsial disease(s) in this outbreak. [ABSTRACT FROM AUTHOR]

Background: Chikungunya virus (CHIKV) is an emerging arbovirus, belonging to the Togaviridae family, Alphavirus genus, transmitted by Aedes spp. mosquitoes. Since 2007, two different CHIKV stains (E1-226A and E1-226V) have been responsible for outbreaks in European countries, including Italy, sustained by Ae. albopictus mosquitoes. Findings: In this study, we assessed the susceptibility to the CHIKV E1-226V, strain responsible for the Italian 2007 outbreak, of eight Ae. albopictus populations collected in Northern, Central, Southern, and Island Italy, by experimental infections. Vector competence was evaluated by estimating infection, dissemination, and transmission rates (IR, DR, TR), through detection of the virus in the bodies, legs plus wings, and saliva, respectively. Additionally, vertical transmission was evaluated by the detection of the virus in the offspring. The results of our study demonstrated that the Italian populations of Ae. albopictus tested were susceptible to CHIKV infection, and can disseminate the virus outside the midgut barrier with high values of IR and DR. Viral infectious RNA was detected in the saliva of three populations from Central, Southern, and Island Italy, also tested for TR and population transmission rate (PTR) values. No progeny of the first and second gonotrophic cycle were positive for CHIKV. Conclusions: This study strongly confirms the role of Ae. albopictus as a potential CHIKV vector in Italy. This may represent a threat, especially considering both the high density of this species, which is widespread throughout the country, and the increasing number of cases of imported arbovirus. [ABSTRACT FROM AUTHOR]

Zika virus (ZIKV) is primarily transmitted by Aedes mosquitoes in the subgenus Stegomyia but can also be transmitted sexually and vertically in humans. STAT1 is an important downstream factor that mediates type I and II interferon signaling. In the current study, we showed that mice with STAT1 knockout (Stat1-/-) were highly susceptible to ZIKV infection. As low as 5 plaque-forming units of ZIKV could cause viremia and death in Stat1-/- mice. ZIKV replication was initially detected in the spleen but subsequently spread to the brain with concomitant reduction of the virus in the spleen in the infected mice. Furthermore, ZIKV could be transmitted from mosquitoes to Stat1-/- mice back to mosquitoes and then to naïve Stat1-/- mice. The 50% mosquito infectious dose of viremic Stat1-/- mouse blood was close to 810 focus-forming units (ffu)/ml. Our further studies indicated that the activation of macrophages and conventional dendritic cells were likely critical for the resolution of ZIKV infection. The newly developed mouse and mosquito transmission models for ZIKV infection will be useful for the evaluation of antiviral drugs targeting the virus, vector, and host. [ABSTRACT FROM AUTHOR]

Background: Evidence from recent studies assessing the impact of school water, sanitation and hygiene (WASH) interventions on child health has been mixed. Self-reports of disease are subject to bias, and few WASH impact evaluations employ objective health measures to assess reductions in disease and exposure to pathogens. We utilized antibody responses from dried blood spots (DBS) to measure the impact of a school WASH intervention on infectious disease among pupils in Mali. Methodology/Principal findings: We randomly selected 21 beneficiary primary schools and their 21 matched comparison schools participating in a matched-control trial of a comprehensive school-based WASH intervention in Mali. DBS were collected from 20 randomly selected pupils in each school (n = 807). We analyzed eluted IgG from the DBS using a Luminex multiplex bead assay to 28 antigens from 17 different pathogens. Factor analysis identified three distinct latent variables representing vector-transmitted disease (driven primarily by dengue), food/water-transmitted enteric disease (driven primarily by Escherichia coli and Vibrio cholerae), and person-to-person transmitted enteric disease (driven primarily by norovirus). Data were analyzed using a linear latent variable model. Antibody evidence of food/water-transmitted enteric disease (change in latent variable mean (β) = -0.24; 95% CI: -0.53, -0.13) and person-to-person transmitted enteric disease (β = -0.17; 95% CI: -0.42, -0.04) was lower among pupils attending beneficiary schools. There was no difference in antibody evidence of vector-transmitted disease (β = 0.11; 95% CI: -0.05, 0.33). Conclusions/Significance: Evidence of enteric disease was lower among pupils attending schools benefitting from school WASH improvements than students attending comparison schools. These findings support results from the parent study, which also found reduced incidence of self-reported diarrhea among pupils of beneficiary schools. DBS collection was feasible in this resource-poor field setting and provided objective evidence of disease at a low cost per antigen analyzed, making it an effective measurement tool for the WASH field. Trial registration: The trial was registered at ClinicalTrials.gov () [ABSTRACT FROM AUTHOR]

Background: Lymphocytic choriomeningitis virus (LCMV) causes a variety of diseases, including asymptomatic infections, meningitis, and congenital infections in the fetus of infected mother. The development of a safe and effective vaccine against LCMV is imperative. This study aims to develop a new candidate vaccine against LCMV using a recombinant replication-incompetent rabies virus (RV) vector. Methodology/Principal findings: In this study, we have generated a recombinant deficient RV expressing the LCMV glycoprotein precursor (GPC) (RVΔP-LCMV/GPC) which is lacking the RV-P gene. RVΔP-LCMV/GPC is able to propagate only in cells expressing the RV-P protein. In contrast, the LCMV-GPC can be expressed in general cells, which do not express RV-P protein. The ability of RVΔP-LCMV/GPC to protect mice from LCMV infection and induce cellular immunity was assessed. Mice inoculated intraperitoneally with RVΔP-LCMV/GPC showed higher survival rates (88.2%) than those inoculated with the parental recombinant RV-P gene-deficient RV (RVΔP) (7.7%) following a LCMV challenge. Neutralizing antibody (NAb) against LCMV was not induced, even in the sera of surviving mice. CD8+ T-cell depletion significantly reduced the survival rates of RVΔP-LCMV/GPC-inoculated mice after the LCMV challenge. These results suggest that CD8+ T cells play a major role in the observed protection against LCMV. In contrast, NAbs against RV were strongly induced in sera of mice inoculated with either RVΔP-LCMV/GPC or RVΔP. In safety tests, suckling mice inoculated intracerebrally with RVΔP-LCMV/GPC showed no symptoms. Conclusions/Significance: These results show RVΔP-LCMV/GPC might be a promising candidate vaccine with dual efficacy, protecting against both RV and LCMV. [ABSTRACT FROM AUTHOR]

Background: The reduced efficacy of current Anopheline mosquito control methods underscores the need to develop new methods of control that exploit unique target sites and/or utilizes novel deployment methods. Autodissemination methodologies using insect growth regulators (IGRs) is growing in interest and has been shown to be effective at controlling Aedes mosquitoes in semi-field and field environments, yet little information exists for Anopheline mosquitoes. Therefore, we tested the hypothesis that female-driven autodissemination of an IGR combined with a new mechanism of action insecticide (Kir channel inhibitor) could be employed to reduce Anopheline populations. Methodology: We studied the ability of three IGRs to be transferred to the larval habitat during oviposition in laboratory and semi-field environments. Adult mosquitoes were exposed to the chemicals for 4 hours immediately after blood feeding and efficacy was tested using classical methodologies, including adult emergence inhibition and High Performance Liquid Chromatography (HPLC). A complete autodissemination design was tested in a semi-field environment. Principal findings: Larval survivability and adult emergence were significantly reduced in habitats that were visited by novaluron treated adults, but no statistical differences were observed with pyriproxyfen or triflumuron. These data suggested novaluron, but not pyriproxyfen or triflumuron, was horizontally transferred from the adult mosquito to the larval habitat during oviposition. HPLC studies supported the toxicity data and showed that novaluron was present in the majority of larval habitats, suggesting that novaluron can be horizontally transferred by Anopheles quadrimaculatus. Importantly, the combination of novaluron and the Kir channel inhibitor, VU041, was capable of reducing adult and larval populations in semi-field environments. Conclusions: Novaluron can be transferred to the adult at a greater efficacy and/or is not degraded as quickly during the gonotropic cycle when compared to pyriproxyfen or triflumuron. Pending field confirmation, autodissemination approaches with novaluron may be a suitable tool to manage Anopheles populations. [ABSTRACT FROM AUTHOR]

Brucellosis is an important zoonotic disease causing huge economic losses worldwide. Currently no effective immunotherapy for Brucellosis or any biomarker to monitor the efficacy of therapy is available. Treatment is ineffective and animals remain carrier lifelong. S19 and RB51 are live attenuated vaccine strains of Brucella abortus. However, S19 induces only antibody, ineffective for intracellular pathogen. RB51 induces cell mediated immunity (CMI) but it is Rifampicin resistant. Both organisms are secreted in milk and can infect humans and cause abortions in animals. Phage lysed bacteria (lysates) retain maximum immunogenicity as opposed to killing by heat or chemicals. We report here the successful immunotherapy of bovine Brucellosis by phage lysates of RB51 (RL) and S19 (SL). The SL induced strong antibody response and RL stimulated CMI. In vitro restimulation of leukocytes from RL immunized cattle induced interferon gamma production. A single subcutaneous dose of 2 ml of cocktail lysate (both RL and SL), eliminated live virulent Brucella from Brucellosis affected cattle with plasma level of Brucella specific 223 bp amplicon undetectable by RT-PCR and blood negative for live Brucella by culture in 3 monthspost-immunization. This is the first report on minimally invasive monitoring of the efficacy of antibacterial therapy employing plasma RNA specific for live bacteria as a biomarker as well as on the use of RB51 phage lysate for successful immunotherapy of Brucellosis in cattle. [ABSTRACT FROM AUTHOR]

West Nile virus (WNV), a zoonotic pathogen naturally transmitted by mosquitoes whose natural hosts are birds, has spread worldwide during the last few decades. Resident birds play an important role in flavivirus epidemiology, since they can serve as reservoirs and facilitate overwintering of the virus. Herein, we report the first experimental infection of magpie (Pica pica) with two strains of West Nile virus, lineages 1 (NY-99) and 2 (SRB Novi-Sad/12), which are currently circulating in Europe. Magpies were highly susceptible to WNV infection, with similar low survival rates (30% and 42.8%) for both lineages. All infected magpies developed viremia detectable at 3 days post-infection with titers above those necessary for successful transmission of WNV to a mosquito. Neutralizing antibodies were detected at all time points analyzed (from 7 to 17 days post-infection). WNV genome was detected in the brains and hearts of all magpies that succumbed to the infection, and, in some of the surviving birds. WNV-RNA was amplified from swabs (oral and cloacal) at 3, 6 and 7 days post-infection and feather pulps, from 3 to 17 days post-infection, of infected animals. Even more, infectious virus was recovered from swabs up to 7 days post-infection and from feather pulps up to 10 days post infection. Sham-infected control animals were negative for viremia, viral RNA, and antibodies. These results suggest that the magpie, which is one of the most abundant corvid species in Europe, could represent a source of WNV transmission for birds and humans. Our observations shed light on the pathogenesis, transmission, and ecology of WNV and can benefit the implementation of surveillance and control programs. [ABSTRACT FROM AUTHOR]

Background: Yellow fever (Yf) outbreak was recently reported in South Omo of Southern Ethiopia. This area was also highly affected by Yf outbreak in the 1960s. However, there is no reliable information on the level of community knowledge attitudes and practices about the disease in the area. The objective of the current study was to assess level of community knowledge, attitudes and practices about Yf. Methods: Between March and May 2017, a community-based cross-sectional survey was conducted in two districts of the South Omo area. During the survey, 612 randomly selected adults were interviewed about Yf using structured questionnaire. Results: Out of the 612 study participants, 508 (83.0%) reported that they heard about Yf which is locally known as “a disease that causes vomiting blood”. Most (90.4%) of the study participants also said that Yf is different from malaria. Two hundred thirteen (41.9%) participants said that Yf can be transmitted from a patient to another person, while only 80 (37.6%) mentioned that the disease is transmitted through mosquitoes bite. Out of 333 (65.7%) study participants who believed that Yf is a preventable disease, 280 (84.1%) mentioned vaccine as a preventive method. The majority believed that the disease is a killer (97.2%) and a newly emerging (69.4%). Among the total of 612 study participants, 221(36.1%) were considered as having a high level of overall knowledge of Yf. Having educational level above 7th grade (AOR = 3.25, 95% CI: 1.39, 7.57, p = 0.006) and being resident of Bena-Tsemay district (AOR = 1.77, 95% CI: 1.12, 2.78, P = 0.014) were significantly associated with having a high level of overall knowledge of Yf. Agro-pastoralism as an occupation compared to farming was associated with having a low level of overall knowledge of Yf (AOR = 0.51, 95% CI, 0.33, 0.79, P = 0.003). Conclusion: The findings indicate that most of the study community members had a low level of overall knowledge of Yf, especially about its cause, mode of transmission and preventive methods. Thus, there is a need to increase people’s knowledge and practices regarding the cause, mode of transmission and preventive methods like avoiding mosquitoe breeding sites beside vaccination through various strategies like disseminating information through community health extension workers and community leaders in the study area. [ABSTRACT FROM AUTHOR]

Background: Cholera is an acute voluminous dehydrating diarrheal disease caused by toxigenic strains of Vibrio cholerae O1 and occasionally O139. A growing body of evidence indicates that immune responses targeting the O-specific polysaccharide (OSP) of V. cholerae are involved in mediating protection against cholera. We therefore assessed whether antibody responses against OSP occur after vaccination with live attenuated oral cholera vaccine CVD 103-HgR, and whether such responses correlate with protection against cholera. Methodology: We assessed adult North American volunteers (n = 46) who were vaccinated with 5 × 108 colony-forming units (CFU) of oral cholera vaccine CVD 103-HgR and then orally challenged with approximately 1 × 105 CFU of wild-type V. cholerae O1 El Tor Inaba strain N16961, either 10 or 90 days post-vaccination. Principal findings: Vaccination was associated with induction of significant serum IgM and IgA anti-OSP and vibriocidal antibody responses within 10 days of vaccination. There was significant correlation between anti-OSP and vibriocidal antibody responses. IgM and IgA anti-OSP responses on day 10 following vaccination were associated with lower post-challenge stool volume (r = −0.44, P = 0.002; r = −0.36, P = 0.01; respectively), and none of 27 vaccinees who developed a ≥1.5 fold increase in any antibody isotype targeting OSP on day 10 following vaccination compared to baseline developed moderate or severe cholera following experimental challenge, while 5 of 19 who did not develop such anti-OSP responses did (P = 0.01). Conclusion: Oral vaccination with live attenuated cholera vaccine CVD 103-HgR induces antibodies that target V. cholerae OSP, and these anti-OSP responses correlate with protection against diarrhea following experimental challenge with V. cholerae O1. Trial registration: ClinicalTrials.gov [ABSTRACT FROM AUTHOR]

Nutrition is a key factor in host–pathogen defense. Malnutrition can increase both host susceptibility and severity of infection through a number of pathways, and infection itself can promote nutritional deterioration and further susceptibility. Nutritional status can also strongly influence response to vaccination or therapeutic pharmaceuticals. Arthropod-borne viruses (arboviruses) have a long history of infecting humans, resulting in regular pandemics as well as an increasing frequency of autochthonous transmission. Interestingly, aside from host-related factors, nutrition could also play a role in the competence of vectors required for transmission of these viruses. Nutritional status of the host and vector could even influence viral evolution itself. Therefore, it is vital to understand the role of nutrition in the arbovirus lifecycle. This Review will focus on nutritional factors that could influence susceptibility and severity of infection in the host, response to prophylactic and therapeutic strategies, vector competence, and viral evolution. [ABSTRACT FROM AUTHOR]

Background: The lack of effective short-course therapies for treatment of the adult stage of filarial worms is a major limitation in the global effort to eliminate lymphatic filariasis. Studies using current small mammal models of lymphatic filariasis are limited by difficulties in quantifying adult worm numbers and in assessing lymphatic anatomy and function. Methodology/Principal findings: Here, we re-established Brugia malayi infection of ferrets as a model for lymphatic filariasis and demonstrated parasitological, immunological, and histological parallels with human infection. Subcutaneous injection of L3 larvae into a hind-footpad resulted in a mean of 18 adult worms recovered 16 weeks post-infection, primarily from the draining inguinal and femoral lymphatics of the injected limb. Infected ferrets developed microfilaremia, with patency lasting from 12–26 weeks post-infection. Quantitative PCR assessing cytokine transcription by antigen-stimulated lymph node cells demonstrated a mixed Th1/Th2 response occurring during early infection. Immunoregulation with production of down-regulatory cytokine IL-10 occurred just prior to peak microfilaremia. Histological analysis revealed progressive inflammation of the lymphatic vessel walls, with intimal thickening and disorganization of collagen fibers. Inflammation was observed as early as 8 weeks post-infection and extended into the perivascular and subcutaneous tissues by 16 weeks post-infection. Finally, we developed a novel ferret PET/CT lymphoscintigraphy method demonstrating substantial changes in lymphatic anatomy and function as early as 3 weeks post-infection, with progression over the course of infection. Conclusions/Significance: B. malayi infection of ferrets is a robust model of human lymphatic filariasis that can be utilized to study efficacy of novel antifilarial agents against adult worms residing within lymphatic vessels. In conjunction with PET/CT lymphoscintigraphy, this model can also be used to investigate pathogenesis of lymphatic dysfunction in lymphatic filariasis and efficacy of medications aimed at reversing lymphatic dysfunction after clearance of adult worms. [ABSTRACT FROM AUTHOR]

Background: The classical method for detection of Lassa virus-specific antibodies is the immunofluorescence assay (IFA) using virus-infected cells as antigen. However, IFA requires laboratories of biosafety level 4 for assay production and an experienced investigator to interpret the fluorescence signals. Therefore, we aimed to establish and evaluate enzyme-linked immunosorbent assays (ELISA) using recombinant Lassa virus nucleoprotein (NP) as antigen. Methodology/Principal findings: The IgM ELISA is based on capturing IgM antibodies using anti-IgM, and the IgG ELISA is based on capturing IgG antibody–antigen complexes using rheumatoid factor or Fc gamma receptor CD32a. Analytical and clinical evaluation was performed with 880 sera from Lassa fever endemic (Nigeria) and non-endemic (Ghana and Germany) areas. Using the IFA as reference method, we observed 91.5–94.3% analytical accuracy of the ELISAs in detecting Lassa virus-specific antibodies. Evaluation of the ELISAs for diagnosis of Lassa fever on admission to hospital in an endemic area revealed a clinical sensitivity for the stand-alone IgM ELISA of 31% (95% CI 25–37) and for combined IgM/IgG detection of 26% (95% CI 21–32) compared to RT-PCR. The specificity of IgM and IgG ELISA was estimated at 96% (95% CI 93–98) and 100% (95% CI 99–100), respectively, in non-Lassa fever patients from non-endemic areas. In patients who seroconverted during follow-up, Lassa virus-specific IgM and IgG developed simultaneously rather than sequentially. Consistent with this finding, isolated IgM reactivity, i.e. IgM in the absence of IgG, had no diagnostic value. Conclusions/Significance: The ELISAs are not equivalent to RT-PCR for early diagnosis of Lassa fever; however, they are of value in diagnosing patients at later stage. The IgG ELISA may be useful for epidemiological studies and clinical trials due its high specificity, and the higher throughput rate and easier operation compared to IFA. [ABSTRACT FROM AUTHOR]

The article describes a rare case of isolated skeletal cryptococcosis in a previously healthy 46-year-old women who presented with left hip pain of undetermined origin for two months. Topics discussed include clinical background on Cryptococcal osteomyelitis, the manifestation of the infection from both pathogen and host perspectives by in vitro and in vivo assays, and accurate diagnosis of bone cryptococcosis and treatment regimen.

Background: Novel surveillance strategies are needed to detect the rapid and continuous emergence of infectious disease agents. Ideally, new sampling strategies should be simple to implement, technologically uncomplicated, and applicable to areas where emergence events are known to occur. To this end, xenosurveillance is a technique that makes use of blood collected by hematophagous arthropods to monitor and identify vertebrate pathogens. Mosquitoes are largely ubiquitous animals that often exist in sizable populations. As well, many domestic or peridomestic species of mosquitoes will preferentially take blood-meals from humans, making them a unique and largely untapped reservoir to collect human blood. Methodology/Principal findings: We sought to take advantage of this phenomenon by systematically collecting blood-fed mosquitoes during a field trail in Northern Liberia to determine whether pathogen sequences from blood engorged mosquitoes accurately mirror those obtained directly from humans. Specifically, blood was collected from humans via finger-stick and by aspirating bloodfed mosquitoes from the inside of houses. Shotgun metagenomic sequencing of RNA and DNA derived from these specimens was performed to detect pathogen sequences. Samples obtained from xenosurveillance and from finger-stick blood collection produced a similar number and quality of reads aligning to two human viruses, GB virus C and hepatitis B virus. Conclusions/Significance: This study represents the first systematic comparison between xenosurveillance and more traditional sampling methodologies, while also demonstrating the viability of xenosurveillance as a tool to sample human blood for circulating pathogens. [ABSTRACT FROM AUTHOR]

Background: Healing times following treatment with antibiotics, and factors that influence healing, have not been reported in Australian patients with Mycobacterium ulcerans. Methodology/Principal findings: Healing times were determined for all M. ulcerans cases treated by a single physician with antibiotics at Barwon Health, Victoria, from 1/1/13-31/12/16. Lesions were categorised by induration size: category A ≤ 400mm2, Category B 401-1600mm2 and Category C ≥1601mm2. A logistic regression analysis was performed to determine risk factors for prolonged wound healing (>150 days from antibiotic commencement). 163 patients were included; 92 (56.4%) were male and median age was 58 years (IQR 39–73 years). Baseline lesion size [available in 145 (89.0%) patients] was categorised as A in 46 (31.7%), B in 67 (46.2%) and C in 32 (22.1%) patients. Fifty (30.7%) patients had surgery. In those treated with antibiotics alone, 83.0% experienced a reduction in induration size after 2 weeks, then 70.9% experienced an increase in induration size from the lowest point, and 71.7% experienced an increase in ulceration size. A linear relationship existed between the time induration resolved and wound healing began. Median time to heal was 91 days (IQR 70–148 days) for category A lesions; significantly shorter than for category B lesions (128 days, IQR 91–181 days, p = 0.05) and category C lesions (169 days, IQR 159–214 days, p<0.001). Fifty-seven (35.0%) patients experienced a paradoxical reaction. Of those treated with antibiotics alone, lesions experiencing a paradoxical reaction had longer healing times [median time to heal 177 days (IQR 154–224 days) compared to 107 days (IQR 79–153 days), p<0.001]. On multivariable logistic regression, lesion size at baseline (p<0.001) and paradoxical reactions (p<0.001) were independently associated with prolonged healing times. For category A and B lesions, healing time was significantly shorter with antibiotics plus excision and direct closure compared with antibiotics alone [Category A lesions median 55 days (IQR 21–63 days) compared with 91 days (IQR 70–148 days), p<0.001; Category B lesions median 74 days (IQR 21–121 days) compared to 128 days (IQR 97–181 days), p<0.001]. Conclusions: In Australian patients treated with antibiotics M. ulcerans lesions usually initially improve, then clinically deteriorate with increased induration and ulceration, before healing after the inflammation associated with lesions resolves. The time to complete healing of lesions is generally long, and is further prolonged in those with larger initial lesion size or who develop paradoxical reactions. For small lesions (<4cm2), excisional surgery may reduce healing times. [ABSTRACT FROM AUTHOR]

Zika virus (ZIKV) is an emerging arbovirus belonging to the genus flavivirus that comprises other important public health viruses, such as dengue (DENV) and yellow fever (YFV). In general, ZIKV infection is a self-limiting disease, however cases of Guillain-Barré syndrome and congenital brain abnormalities in newborn infants have been reported. Diagnosing ZIKV infection remains a challenge, as viral RNA detection is only applicable until a few days after the onset of symptoms. After that, serological tests must be applied, and, as expected, high cross-reactivity between ZIKV and other flavivirus serology is observed. Plaque reduction neutralization test (PRNT) is indicated to confirm positive samples for being more specific, however it is laborious intensive and time consuming, representing a major bottleneck for patient diagnosis. To overcome this limitation, we developed a high-throughput image-based fluorescent neutralization test for ZIKV infection by serological detection. Using 226 human specimens, we showed that the new test presented higher throughput than traditional PRNT, maintaining the correlation between results. Furthermore, when tested with dengue virus samples, it showed 50.53% less cross reactivity than MAC-ELISA. This fluorescent neutralization test could be used for clinical diagnosis confirmation of ZIKV infection, as well as for vaccine clinical trials and seroprevalence studies. [ABSTRACT FROM AUTHOR]

Recognizing cholera cases early, especially in the initial phase of an outbreak and in areas where cholera has not previously circulated, is a high public health priority. Laboratory capacity in such settings is often limited. To address this, we have developed a rapid diagnostic test (RDT) termed Cholkit that is based on an immunochromatographic lateral flow assay for the diagnosis of cholera cases using stool. Cholkit contains a monoclonal antibody (ICL-33) to the O-specific polysaccharide (OSP) component of V. cholerae O1 lipopolysaccharide, and recognizes both Inaba and Ogawa serotypes. We tested the Cholkit dipstick using fresh stool specimens of 76 adults and children presenting with acute watery diarrhea at the icddr,b hospital in Dhaka, Bangladesh. We compared Cholkit’s performance with those of microbial culture, PCR (targeting the rfb and ctxA genes of V. cholerae) and the commercially available RDT, Crystal VC (Span Diagnostics; Surat, India). We found that all stool specimens with a positive culture for V. cholerae O1 (n = 19) were positive by Cholkit as well as Crystal VC. We then used Bayesian latent class modeling to estimate the sensitivity and specificity of each diagnostic assay. The sensitivity of Cholkit, microbiological culture, PCR and Crystal VC was 98% (95% CI: 88–100), 71% (95% CI: 59–81), 74% (95% CI: 59–86) and 98% (95% CI: 88–100), respectively. The specificity for V. cholerae O1 was 97% (95% CI: 89–100), 100%, 97% (95% CI: 93–99) and 98% (95% CI: 92–100), respectively. Of note, two Crystal VC dipsticks were positive for V. cholerae O139 but negative by culture and PCR in this area without known circulating epidemic V. cholerae O139. In conclusion, the Cholkit dipstick is simple to use, requires no dedicated laboratory capacity, and has a sensitivity and specificity for V. cholerae O1 of 98% and 97%, respectively. Cholkit warrants further evaluation in other settings. [ABSTRACT FROM AUTHOR]

Background: The Human T-Lymphotropic Virus type 1c subtype (HTLV-1c) is highly endemic to central Australia where the most frequent complication of HTLV-1 infection in Indigenous Australians is bronchiectasis. We carried out a prospective study to quantify the prognosis of HTLV-1c infection and chronic lung disease and the risk of death according to the HTLV-1c proviral load (pVL). Methodology/Principal findings: 840 Indigenous adults (discharge diagnosis of bronchiectasis, 154) were recruited to a hospital-based prospective cohort. Baseline HTLV-1c pVL were determined and the results of chest computed tomography and clinical details reviewed. The odds of an association between HTLV-1 infection and bronchiectasis or bronchitis/bronchiolitis were calculated, and the impact of HTLV-1c pVL on the risk of death was measured. Radiologically defined bronchiectasis and bronchitis/bronchiolitis were significantly more common among HTLV-1-infected subjects (adjusted odds ratio = 2.9; 95% CI, 2.0, 4.3). Median HTLV-1c pVL for subjects with airways inflammation was 16-fold higher than that of asymptomatic subjects. There were 151 deaths during 2,140 person-years of follow-up (maximum follow-up 8.13 years). Mortality rates were higher among subjects with HTLV-1c pVL ≥1000 copies per 105 peripheral blood leukocytes (log-rank χ2 (2df) = 6.63, p = 0.036) compared to those with lower HTLV-1c pVL or uninfected subjects. Excess mortality was largely due to bronchiectasis-related deaths (adjusted HR 4.31; 95% CI, 1.78, 10.42 versus uninfected). Conclusion/Significance: Higher HTLV-1c pVL was strongly associated with radiologically defined airways inflammation and with death due to complications of bronchiectasis. An increased risk of death due to an HTLV-1 associated inflammatory disease has not been demonstrated previously. Our findings indicate that mortality associated with HTLV-1c infection may be higher than has been previously appreciated. Further prospective studies are needed to determine whether these results can be generalized to other HTLV-1 endemic areas. [ABSTRACT FROM AUTHOR]

The article discusses the case of a healthy 35-year-old Swiss woman who was admitted in hospital for persistent fever, headache, and myalgia for three days. The patient further shows symptoms of thrombocytopenia, Abdominal ultrasound showed acute acalculous cholecystitis, cough, dyspnea, and hypoxemia which is further diagnosed as leptospirosis.

Few animal models of Zika virus (ZIKV) infection have incorporated arthropod-borne transmission. Here, we establish an Aedes aegypti mosquito model of ZIKV infection of mice, and demonstrate altered vector competency among three strains, (Orlando, ORL, Ho Chi Minh, HCM, and Patilas, PAT). All strains acquired ZIKV in their midguts after a blood meal from infected mice, but ZIKV transmission only occurred in mice fed upon by HCM, and to a lesser extent PAT, but not ORL, mosquitoes. This defect in transmission from ORL or PAT mosquitoes was overcome by intrathoracic injection of ZIKV into mosquito. Genetic analysis revealed significant diversity among these strains, suggesting a genetic basis for differences in ability for mosquito strains to transmit ZIKV. The intrathoracic injection mosquito-mouse transmission model is critical to understanding the influence of mosquitoes on ZIKV transmission, infectivity and pathogenesis in the vertebrate host, and represents a natural transmission route for testing vaccines and therapeutics. (152 words) [ABSTRACT FROM AUTHOR]

The methods currently available for genotype-specific diagnosis of T. cruzi infection still present relevant limitations, especially to identify mixed infection. In the present investigation, we have evaluated the performance of Chagas-Flow ATE-IgG2a test for early and late differential diagnosis of single and dual genotype-specific T. cruzi infections. Serum samples from Swiss mice at early and late stages of T. cruzi infection were assayed in parallel batches for genotype-specific diagnosis of single (TcI, TcVI or TcII) and dual (TcI+TcVI, TcVI+TcII or TcII+TcI) infections. The intrinsic reactivity to TcI, TcVI and TcII target antigens, including amastigote (AI/AVI/AII), trypomastigote-(TI/TVI/TII) and epimastigote (EI/EVI/EII), at specific reverse of serum dilutions (500 to 64,000), was employed to provide reliable decision-trees for “early” vs “late”, “single vs “dual” and “genotype-specific” serology. The results demonstrated that selective set of attributes “EII 500/EI 2,000/AII 500” were able to provide high-quality accuracy (81%) to segregate early and late stages of T. cruzi infection. The sets “TI 2,000/AI 1,000/EII 1,000” and “TI 8,000/AII 32,000” presented expressive scores to discriminate single from dual T. cruzi infections at early (85%) and late stages (84%), respectively. Moreover, the attributes “TI 4,000/TVI 500/TII 1,000”, “TI 16,000/EI 2,000/EII 2,000/AI 500/TVI 500” showed good performance for genotype-specific diagnosis at early stage of single (72%) and dual (80%) T. cruzi infections, respectively. In addition, the attributes “TI 4,000/AII 1,000/EVI 1,000”, “TI 64,000/AVI 500/AI 2,000/AII 1,000/EII 4,000” showed moderate performance for genotype-specific diagnosis at late stage of single (69%) and dual (76%) T. cruzi infections, respectively. The sets of decision-trees were assembled to construct a sequential algorithm with expressive accuracy (81%) for serological diagnosis of T. cruzi infection. These findings engender new perspectives for the application of Chagas-Flow ATE-IgG2a method for genotype-specific diagnosis in humans, with relevant contributions for epidemiological surveys as well as clinical and post-therapeutic monitoring of Chagas disease. [ABSTRACT FROM AUTHOR]

Early detection of leptospirosis with field-ready diagnostics may improve clinical management and mitigate outbreaks. We previously validated the point-of-care Dual Path Platform (DPP) for leptospirosis with sera in the laboratory. This prospective study compares the diagnostic accuracy and clinical utility of the DPP using finger stick blood (FSB) against the serum DPP, venous whole blood (VWB) DPP, IgM-ELISA, and clinical impression. We sequentially enrolled 98 patients hospitalized for acute febrile illnesses, of which we confirmed 32 by leptospirosis reference tests. Among syndromes consistent with classic leptospirosis, the FSB DPP showed similar sensitivity and specificity (Se 93% and Sp 80%), and positive and negative predictive values (PPV 74% and NPV 95%), to VWB DPP (Se 96%, Sp 75%, PPV 68%, and NPV 97%), serum DPP (Se 85%, Sp 87%, PPV 79%, and NPV 91%) and IgM-ELISA (Se 81%, Sp 100%, PPV 100%, and NPV 90%). The FSB DPP provided a favorable likelihood ratio profile (positive LR 4.73, negative LR 0.09) in comparison to other assays and clinical impression alone. Additionally, we identified four of five leptospirosis-associated meningitis patients by whole blood DPP, none of which clinicians suspected. This demonstrates potential for the DPP in routine detection of this less common syndrome. The FSB DPP demonstrated similar discrimination for severe human leptospirosis compared with serum assays, and it is a simpler option for diagnosing leptospirosis. Its performance in other epidemiological settings and geographic regions, and for detecting atypical presentations, demands further evaluation. [ABSTRACT FROM AUTHOR]

Background: Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) has recently emerged in the field of entomology as a promising method for the identification of arthropods and the detection of associated pathogens. Methodology/Principal findings: An experimental model of Ctenocephalides felis (cat fleas) infected with Bartonella quintana and Bartonella henselae was developed to evaluate the efficacy of MALDI-TOF MS in distinguishing infected from uninfected fleas, and its ability to distinguish fleas infected with Bartonella quintana from fleas infected with Bartonella henselae. For B. quintana, two groups of fleas received three successive blood meals, infected or not. A total of 140 fleas (100 exposed fleas and 40 control fleas) were engorged on human blood, infected or uninfected with B. quintana. Regarding the second pathogen, two groups of fleas (200 exposed fleas and 40 control fleas) were fed in the same manner with human blood, infected or not with Bartonella henselae. Fleas were dissected longitudinally; one-half was used for assessment of B. quintana and B. henselae infectious status by real-time PCR, and the second half was subjected to MALDI-TOF MS analysis. Comparison of MS spectra from infected fleas and uninfected fleas revealed distinct MS profiles. Blind queries against our MALDI-TOF MS arthropod database, upgraded with reference spectra from B. quintana and B. henselae and infected but also non-infected fleas, provided the correct classification for 100% of the different categories of specimens tested on the first model of flea infection with Bartonella quintana. As for Bartonella henselae, 81% of exposed positive fleas, 96% of exposed negative fleas and 100% of control fleas were correctly identified on the second model of flea infection. MALDI-TOF MS successfully differentiated Bartonella spp.-infected and uninfected fleas and was also able to correctly differentiate fleas infected with Bartonella quintana and fleas infected with Bartonella henselae. MALDI-TOF MS correctly identified flea species as well as their infectious status, consistent with the results of real-time PCR. Conclusions/Significance: MALDI-TOF is a promising tool for identification of the infection status of fleas infected with Bartonella spp., which allows new possibilities for fast and accurate diagnosis in medical entomology and vector surveillance. [ABSTRACT FROM AUTHOR]

West Nile virus (WNV) and St. Louis encephalitis (SLEV) virus are enzootically maintained in North America in cycles involving the same mosquito vectors and similar avian hosts. However, these viruses exhibit dissimilar viremia and virulence phenotypes in birds: WNV is associated with high magnitude viremias that can result in mortality in certain species such as American crows (AMCRs, Corvus brachyrhynchos) whereas SLEV infection yields lower viremias that have not been associated with avian mortality. Cross-neutralization of these viruses in avian sera has been proposed to explain the reduced circulation of SLEV since the introduction of WNV in North America; however, in 2015, both viruses were the etiologic agents of concurrent human encephalitis outbreaks in Arizona, indicating the need to re-evaluate host factors and cross-neutralization responses as factors potentially affecting viral co-circulation. Reciprocal chimeric WNV and SLEV viruses were constructed by interchanging the pre-membrane (prM)-envelope (E) genes, and viruses subsequently generated were utilized herein for the inoculation of three different avian species: house sparrows (HOSPs; Passer domesticus), house finches (Haemorhous mexicanus) and AMCRs. Cross-protective immunity between parental and chimeric viruses were also assessed in HOSPs. Results indicated that the prM-E genes did not modulate avian replication or virulence differences between WNV and SLEV in any of the three avian species. However, WNV-prME proteins did dictate cross-protective immunity between these antigenically heterologous viruses. Our data provides further evidence of the important role that the WNV / SLEV viral non-structural genetic elements play in viral replication, avian host competence and virulence. [ABSTRACT FROM AUTHOR]

Background: During 2015–16 Brazil experienced the largest epidemic of Zika virus ever reported. This arthropod-borne virus (arbovirus) has been linked to Guillain-Barré syndrome (GBS) in adults but other neurological associations are uncertain. Chikungunya virus has caused outbreaks in Brazil since 2014 but associated neurological disease has rarely been reported here. We investigated adults with acute neurological disorders for Zika, chikungunya and dengue, another arbovirus circulating in Brazil. Methods: We studied adults who had developed a new neurological condition following suspected Zika virus infection between 1st November 2015 and 1st June 2016. Cerebrospinal fluid (CSF), serum, and urine were tested for evidence of Zika, chikungunya, and dengue viruses. Results: Of 35 patients studied, 22 had evidence of recent arboviral infection. Twelve had positive PCR or IgM for Zika, five of whom also had evidence for chikungunya, three for dengue, and one for all three viruses. Five of them presented with GBS; seven had presentations other than GBS, including meningoencephalitis, myelitis, radiculitis or combinations of these syndromes. Additionally, ten patients positive for chikungunya virus, two of whom also had evidence for dengue virus, presented with a similar range of neurological conditions. Conclusions: Zika virus is associated with a wide range of neurological manifestations, including central nervous system disease. Chikungunya virus appears to have an equally important association with neurological disease in Brazil, and many patients had dual infection. To understand fully the burden of Zika we must look beyond GBS, and also investigate for other co-circulating arboviruses, particularly chikungunya. [ABSTRACT FROM AUTHOR]

Background: Trypanosoma cruzi parasite, the causative agent of Chagas disease, infects about six million individuals in more than 20 countries. Monitoring parasite persistence in infected individuals is of utmost importance to develop and evaluate treatments to control the disease. Routine screening for infected human individuals is achieved by serological assays; PCR testing to monitor spontaneous or therapy-induced parasitological cure has limitations due to the low and fluctuating parasitic load in circulating blood. The aim of the present study is to evaluate a newly developed antibody profiling assay as an indirect method to assess parasite persistence based on waning of antibody following spontaneous or therapy-induced clearance of the infection. Methodology/Principal findings: We designed a multiplex serology assay, an array of fifteen optimized T. cruzi antigens, to evaluate antibody diversity in 1654 serum samples from chronic Chagas patients. One specific antibody response (antibody 3, Ab3) showed a strong correlation with T. cruzi parasite persistence as determined by T. cruzi PCR positive results. High and sustained Ab3 signal was strongly associated with PCR positivity in untreated patients, whereas significant and progressive decline in Ab3 signals was observed in BZN-treated patients who cleared parasitemia based on blood PCR results. Conclusion/Significance: Ab3 is a new surrogate biomarker that strongly correlates with parasite persistence in chronic and benznidazole-treated Chagas patients. We hypothesize that Ab3 is induced and maintained by incessant stimulation of the immune system by tissue-based and shed parasites that are not consistently detectable by blood based PCR techniques. Hence, a simple immunoassay measurement of Ab3 could be beneficial for monitoring the infectious status of seropositive patients. [ABSTRACT FROM AUTHOR]

Plants produce various secondary metabolites that offer a potential source of novel insecticides and repellents for the control of mosquito vectors. Plants of the genus Cinnamosma are endemic to, and widely-distributed throughout, the island of Madagascar. The barks of these species are commonly used in traditional medicines for treating a wide range of maladies. The therapeutic nature of the bark is thought to be associated with its enrichment of pungent drimane sesquiterpenes, which elicit antifeedant and toxic effects in some insects. Here we test the hypothesis that a bark extract of Cinnamosma fragrans (CINEX) and its major drimane sesquiterpenes are insecticidal, antifeedant, and repellent to Aedes aegypti, the principal mosquito vector of chikungunya, dengue, yellow fever, and Zika viruses. We demonstrate that CINEX is 1) toxic to larval and adult female mosquitoes, and 2) antifeedant and repellent to adult female mosquitoes. Moreover, we show that cinnamodial (CDIAL), a sesquiterpene dialdehyde isolated from CINEX, duplicates these bioactivities and exhibits similar toxic potency against pyrethroid-susceptible and -resistant strains of Ae. aegypti. Importantly, we show that CDIAL is an agonist of heterologously-expressed mosquito Transient Receptor Potential A1 (TRPA1) channels, and the antifeedant activity of CDIAL is dampened in a TRPA1-deficient strain of Ae. aegypti (TRPA1-/-). Intriguingly, TRPA1-/- mosquitoes do not exhibit toxic resistance to CDIAL. The data indicate that modulation of TRPA1 is required for the sensory detection and avoidance of CDIAL by mosquitoes, but not for inducing the molecule’s toxicity. Our study suggests that CDIAL may serve as a novel chemical platform for the development of natural product-based insecticides and repellents for controlling mosquito vectors. [ABSTRACT FROM AUTHOR]

Dengue virus (DENV) infection imposes enormous health and economic burden worldwide with no approved treatment. Several small molecules, including lovastatin, celgosivir, balapiravir and chloroquine have been tested for potential anti-dengue activity in clinical trials; none of these have demonstrated a protective effect. Recently, based on identification and characterization of cross-serotype neutralizing antibodies, there is increasing attention on the potential for dengue immunotherapy. Here, we tested the ability of VIS513, an engineered cross-neutralizing humanized antibody targeting the DENV E protein domain III, to overcome antibody-enhanced infection and high but brief viremia, which are commonly encountered in dengue patients, in various in vitro and in vivo models. We observed that VIS513 efficiently neutralizes DENV at clinically relevant viral loads or in the presence of enhancing levels of DENV immune sera. Single therapeutic administration of VIS513 in mouse models of primary infection or lethal secondary antibody-enhanced infection, reduces DENV titers and protects from lethal infection. Finally, VIS513 administration does not readily lead to resistance, either in cell culture systems or in animal models of dengue infection. The findings suggest that rapid viral reduction during acute DENV infection with a monoclonal antibody is feasible. [ABSTRACT FROM AUTHOR]

Background: Entamoeba nuttalli is an intestinal protozoan with pathogenic potential that can cause amoebic liver abscess. It is highly prevalent in wild and captive macaques. Recently, cysts were detected in a caretaker of nonhuman primates in a zoo, indicating that E. nuttalli may be a zoonotic pathogen. Therefore, it is important to evaluate the pathogenicity of E. nuttalli in detail and in comparison with that of E. histolytica. Methodology/Principal findings: Trophozoites of E. nuttalli GY4 and E. histolytica SAW755 strains were inoculated into liver of hamsters. Expression levels of proinflammatory factors of hamsters and virulence factors from E. histolytica and E. nuttalli were compared between the two parasites. Inoculations with trophozoites of E. nuttalli resulted in an average necrotic area of 24% in liver tissue in 7 days, whereas this area produced by E. histolytica was nearly 50%. Along with the mild liver tissue damage induced by E. nuttalli, expression levels of proinflammatory factors (TNF-α, IL-6 and IL-1β) and amebic virulence protein genes (lectins, cysteine proteases and amoeba pores) in local tissues were lower with E. nuttalli in comparison with E. histolytica. In addition, M2 type macrophages were increased in E. nuttalli-induced amebic liver abscesses in the late stage of disease progression and lysate of E. nuttalli trophozoites induced higher arginase expression than E. histolytica in vitro. Conclusions/Significance: The results show that differential secretion of amebic virulence proteins during E. nuttalli infection triggered lower levels of secretion of various cytokines and had an impact on polarization of macrophages towards a M1/M2 balance. However, regardless of the degree of macrophage polarization, there is unambiguous evidence of an intense acute inflammatory reaction in liver of hamsters after infection by both Entamoeba species. [ABSTRACT FROM AUTHOR]

Background: Campylobacter is one of the main causes of gastroenteritis worldwide. Most of the current knowledge about the epidemiology of this food-borne infection concerns two species, C. coli and C. jejuni. Recent studies conducted in developing countries and using novel diagnostic techniques have generated evidence of the increasing burden and importance of other Campylobacter species, i.e. non-C. coli/jejuni. We performed a nested case-control study to compare the prevalence of C. coli/jejuni and other Campylobacter in children with clinical dysentery and severe diarrhea as well as without diarrhea to better understand the clinical importance of infections with Campylobacter species other than C. coli/jejuni. Methodology/Principal findings: Our nested case-control study of 439 stool samples included dysenteric stools, stools collected during severe diarrhea episodes, and asymptomatic stools which were systematically selected to be representative of clinical phenotypes from 9,160 stools collected during a birth cohort study of 201 children followed until two years of age. Other Campylobacter accounted for 76.4% of the 216 Campylobacter detections by qPCR and were more prevalent than C. coli/jejuni across all clinical groups. Other Campylobacter were also more prevalent than C. coli/jejuni across all age groups, with older children bearing a higher burden of other Campylobacter. Biomarkers of intestinal inflammation and injury (methylene blue, fecal occult test, myeloperoxidase or MPO) showed a strong association with dysentery, but mixed results with infection. MPO levels were generally higher among children infected with C. coli/jejuni, but Shigella-infected children suffering from dysentery recorded the highest levels (26,224 ng/mL); the lowest levels (10,625 ng/mL) were among asymptomatic children infected with other Campylobacter. Adjusting for age, sex, and Shigella infection, dysentery was significantly associated with C. coli/jejuni but not with other Campylobacter, whereas severe diarrhea was significantly associated with both C. coli/jejuni and other Campylobacter. Compared to asymptomatic children, children suffering from dysentery had a 14.6 odds of C. coli/jejuni infection (p-value < 0.001, 95% CI 5.5–38.7) but were equally likely to have other Campylobacter infections–odds ratio of 1.3 (0.434, 0.7–2.4). Children suffering from severe diarrhea were more likely than asymptomatic children to test positive for both C. coli/jejuni and other Campylobacter–OR of 2.8 (0.034, 1.1–7.1) and 1.9 (0.018, 1.1–3.1), respectively. Compared to the Campylobacter-free group, the odds of all diarrhea given C. coli/jejuni infection and other Campylobacter infection were 8.8 (<0.001, 3.0–25.7) and 2.4 (0.002, 1.4–4.2), respectively. Eliminating other Campylobacter in this population would eliminate 24.9% of the diarrhea cases, which is almost twice the population attributable fraction of 15.1% due to C. coli/jejuni. Conclusions/Significance: Eighty-seven of the dysentery and 59.5% of the severe diarrhea samples were positive for Campylobacter, Shigella, or both, emphasizing the importance of targeting these pathogens to limit the impact of dysentery and severe diarrhea in children. Notably, the higher prevalence of other Campylobacter compared to C. coli/jejuni, their increasing burden during early childhood, and their association with severe diarrhea highlight the importance of these non-C. coli/jejuni Campylobacter species and suggest a need to clarify their importance in the etiology of clinical disease across different epidemiological contexts. [ABSTRACT FROM AUTHOR]

Mosquito-borne Zika virus (ZIKV) typically causes a mild and self-limiting illness known as Zika fever. Since its recent emergence in 2014 in the American continent, ZIKV infection during pregnancy has been closely associated with a wide range of congenital abnormalities. To date, no vaccines or antivirals are publicly available. We developed Zika virus-like particles (VLPs) and evaluated their immunogenicity and protective efficacy in mouse models. ZIKV VLPs (ZIKVLPs) formulated with alum were injected into 6-8-week-old interferon deficient AG129 mice as well as wild type BALB/c mice. Control mice received PBS/alum. Animals were challenged with 200 PFU (>1000 AG129 LD50s) of ZIKV strain H/PF/2013. All vaccinated mice survived with no morbidity or weight loss while control animals either died at 9 days post challenge (AG129) or had increased viremia (BALB/c). Neutralizing antibodies were observed in all ZIKVLP vaccinated mice. The role of neutralizing antibodies in protecting mice was demonstrated by passive transfer. Our findings demonstrate the protective efficacy of the ZIKVLP vaccine and highlight the important role that neutralizing antibodies play in protection against ZIKV infection. [ABSTRACT FROM AUTHOR]

Rhinoscleroma is a human specific chronic granulomatous infection of the nose and upper airways caused by the gram-negative bacterium Klebsiella pneumoniae subsp. rhinoscleromatis. Although considered a rare disease, it is endemic in low-income countries where hygienic conditions are poor. A hallmark of this pathology is the appearance of atypical foamy monocytes called Mikulicz cells. However, the pathogenesis of rhinoscleroma remains poorly investigated. Capsule polysaccharide (CPS) is a prominent virulence factor in bacteria. All K. rhinoscleromatis strains are of K3 serotype, suggesting that CPS can be an important driver of rhinoscleroma disease. In this study, we describe the creation of the first mutant of K. rhinoscleromatis, inactivated in its capsule export machinery. Using a murine model recapitulating the formation of Mikulicz cells in lungs, we observed that a K. rhinoscleromatis CPS mutant (KR cps-) is strongly attenuated and that mice infected with a high dose of KR cps- are still able to induce Mikulicz cells formation, unlike a K. pneumoniae capsule mutant, and to partially recapitulate the characteristic strong production of IL-10. Altogether, the results of this study show that CPS is a virulence factor of K. rhinoscleromatis not involved in the specific appearance of Mikulicz cells. [ABSTRACT FROM AUTHOR]

Background: Vibrio cholerae causes over 2 million cases of cholera and 90,000 deaths each year. Serosurveillance can be a useful tool for estimating the intensity of cholera transmission and prioritizing populations for cholera control interventions. Current methods involving venous blood draws and downstream specimen storage and transport methods pose logistical challenges in most settings where cholera strikes. To overcome these challenges, we developed methods for determining cholera-specific immune responses from dried blood spots (DBS). Methodology/principal findings: As conventional vibriocidal assay methods were unsuitable for DBS eluates from filter paper, we adopted a drop-plate culture method. We show that DBS collected from volunteers in South Sudan, and stored for prolonged periods in field conditions, retained functional vibriocidal antibodies, the titers of which correlated with paired serum titers determined by conventional spectrophotometric methods (r = 0.94, p = 0.00012). We also showed that eluates from DBS Serum Separator cards could be used with conventional spectrophotometric vibriocidal methods, and that they correlated with paired serum at a wide range of titers (r = 0.96, p<0.0001). Similarly, we used ELISA methods to show that V. cholerae O-specific polysaccharide antibody responses from DBS eluates correlated with results from paired serum for IgG (r = 0.85, p = 0.00006), IgM (r = 0.79, p = 0.00049) and IgA (r = 0.73, p = 0.0019), highlighting its potential for use in determination of isotype-specific responses. Storage of DBS cards at a range of temperatures did not change antibody responses. Conclusion: In conclusion, we have developed and demonstrated a proof-of-concept for assays utilizing DBS for assessing cholera-specific immune responses. [ABSTRACT FROM AUTHOR]

The mosquito Aedes aegypti is the primary vector of human arboviral diseases caused by dengue, chikungunya and Zika viruses. Many studies have shown the potential roles of small RNA molecules such as microRNA, small interfering RNA and PIWI-interacting RNA in vector mosquitoes. The function of tRNA fragments (tRF), the newly discovered class of small RNAs, in mosquitoes is not known. In this study, we show that specific tRFs are expressed in significantly differential manner between males and females of Ae. aegypti strains. Specific tRFs also show differential response during developmental transition from larvae to adults, as well as after blood feeding of adult females. The expression pattern of tRFs upon blood feeding varied depending upon if the blood contained dengue virus, and also if the females were treated with antibiotic prior to feeding to cleanse of the gut bacteria. Our findings show that a single tRF derived from the precursor sequences of a tRNA-Gly was differentially expressed between males and females, developmental transitions and also upon blood feeding by females of two laboratory strains that vary in midgut susceptibility to dengue virus infection. The multifaceted functional implications of this specific tRF suggest that biogenesis of small regulatory molecules from a tRNA can have wide ranging effects on key aspects of Ae. aegypti vector biology. [ABSTRACT FROM AUTHOR]

Non-typhoidal Salmonella (NTS) is a leading cause of bloodstream infections in Africa, but the various contributions of host susceptibility versus unique pathogen virulence factors are unclear. We used data from a population-based surveillance platform (population ~25,000) between 2007–2014 and NTS genome-sequencing to compare host and pathogen-specific factors between individuals presenting with NTS bacteremia and those presenting with NTS diarrhea. Salmonella Typhimurium ST313 and Salmonella Enteritidis ST11 were the most common isolates. Multi-drug resistant strains of NTS were more commonly isolated from patients presenting with NTS bacteremia compared to NTS diarrhea. This relationship was observed in patients under age five [aOR = 15.16, 95% CI (2.84–81.05), P = 0.001], in patients five years and older, [aOR = 6.70 95% CI (2.25–19.89), P = 0.001], in HIV-uninfected patients, [aOR = 21.61, 95% CI (2.53–185.0), P = 0.005], and in patients infected with Salmonella serogroup B [aOR = 5.96, 95% CI (2.28–15.56), P < 0.001] and serogroup D [aOR = 14.15, 95% CI (1.10–182.7), P = 0.042]. Thus, multi-drug-resistant NTS was strongly associated with bacteremia compared to diarrhea among children and adults. This association was seen in HIV-uninfected individuals infected with either S. Typhimurium or S. Enteritidis. Risk of developing bacteremia from NTS infection may be driven by virulence properties of the Salmonella pathogen. [ABSTRACT FROM AUTHOR]

Although neurological manifestations associated with dengue viruses (DENV) infection have been reported, there is very limited information on the genetic characteristics of neurotropic DENV. Here we describe the isolation and complete genome analysis of DENV serotype 3 (DENV-3) from cerebrospinal fluid of an encephalitis paediatric patient in Jakarta, Indonesia. Next-generation sequencing was employed to deduce the complete genome of the neurotropic DENV-3 isolate. Based on complete genome analysis, two unique and nine uncommon amino acid changes in the protein coding region were observed in the virus. A phylogenetic tree and molecular clock analysis revealed that the neurotropic virus was a member of Sumatran-Javan clade of DENV-3 genotype I and shared a common ancestor with other isolates from Jakarta around 1998. This is the first report of neurotropic DENV-3 complete genome analysis, providing detailed information on the genetic characteristics of this virus. [ABSTRACT FROM AUTHOR]

Background: Podoconiosis is a non-filarial elephantiasis, which causes massive swelling of the lower legs. It was identified as a neglected tropical disease by WHO in 2011. Understanding of the geographical distribution of the disease is incomplete. As part of a global mapping of podoconiosis, this study was conducted in Cameroon to map the distribution of the disease. This mapping work will help to generate data on the geographical distribution of podoconiosis in Cameroon and contribute to the global atlas of podoconiosis. Methods: We used a multi‐stage sampling design with stratification of the country by environmental risk of podoconiosis. We sampled 76 villages from 40 health districts from the ten Regions of Cameroon. All individuals of 15-years old or older in the village were surveyed house-to-house and screened for lymphedema. A clinical algorithm was used to reliably diagnose podoconiosis, excluding filarial-associated lymphedema. Individuals with lymphoedema were tested for circulating Wuchereria bancrofti antigen and specific IgG4 using the Alere Filariasis Test Strips (FTS) test and the Standard Diagnostics (SD) BIOLINE lymphatic filariasis IgG4 test (Wb123) respectively, in addition to thick blood films. Presence of DNA specific to W. bancrofti was checked on night blood using a qPCR technique. Principal findings: Overall, 10,178 individuals from 4,603 households participated in the study. In total, 83 individuals with lymphedema were identified. Of the 83 individuals with lymphedema, two were found to be FTS positive and all were negative using the Wb123 test. No microfilaria of W. bancrofti were found in the night blood of any individual with clinical lymphedema. None were found to be positive for W. bancrofti using qPCR. Of the two FTS positive cases, one was positive for Mansonella perstans DNA, while the other harbored Loa loa microfilaria. Overall, 52 people with podoconiosis were identified after applying the clinical algorithm. The overall prevalence of podoconiosis was found to be 0.5% (95% [confidence interval] CI; 0.4–0.7). At least one case of podoconiosis was found in every region of Cameroon except the two surveyed villages in Adamawa. Of the 40 health districts surveyed, 17 districts had no cases of podoconiosis; in 15 districts, mean prevalence was between 0.2% and 1.0%; and in the remaining eight, mean prevalence was between 1.2% and 2.7%. Conclusions: Our investigation has demonstrated low prevalence but almost nationwide distribution of podoconiosis in Cameroon. Designing a podoconiosis control program is a vital next step. A health system response to the burden of podoconiosis is important, through case surveillance and morbidity management services. [ABSTRACT FROM AUTHOR]

West Nile virus (WNV) and Rift Valley fever virus (RVFV) are two emerging arboviruses transmitted by Culex pipiens species that includes two biotypes: pipiens and molestus. In Lebanon, human cases caused by WNV and RVFV have never been reported. However, the introduction of these viruses in the country is likely to occur through the migratory birds and animal trades. In this study, we evaluated the ability of Cx. pipiens, a predominant mosquito species in urban and rural regions in Lebanon, to transmit WNV and RVFV. Culex egg rafts were collected in the West Bekaa district, east of Lebanon and adult females of Cx. pipiens were experimentally infected with WNV and RVFV Clone 13 strain at titers of 1.6×108 and 1.33×107 plaque forming units (PFU)/mL, respectively. We estimated viral infection, dissemination and transmission at 3, 7, 14 and 19 days post infection (dpi). Results showed that infection was higher for WNV than for RVFV from 3 dpi to 19 dpi. Viral dissemination and transmission started from 3 dpi for WNV; and only from 19 dpi for RVFV. Moreover, Cx. pipiens were able to excrete in saliva a higher number of viral particles of WNV (1028 ± 405 PFU/saliva at 19 dpi) than RVFV (42 PFU/saliva at 19 dpi). Cx. pipiens from Lebanon are efficient experimental vectors of WNV and to a lower extent, RVFV. These findings should stimulate local authorities to establish an active entomological surveillance in addition to animal surveys for both viruses in the country. [ABSTRACT FROM AUTHOR]

Cryptosporidiosis causes life-threatening diarrhea in infants, but the best available treatment is only modestly efficacious. Rodents infected with relevant Cryptosporidium species do not develop diarrhea, which complicates drug development. Cryptosporidium parvum infection of dairy calves, however, causes an illness like that seen in infants. Here, the clinical and microbiologic anti-Cryptosporidium efficacy of the piperazine-based compound MMV665917 was demonstrated in neonatal calves. Oral administration of MMV665917 (22 mg/kg once daily) was begun two days after the onset of severe diarrhea and continued for seven days. Treatment resulted in prompt resolution of diarrhea, and reduced total fecal oocyst shedding by ~94%. MMV665917 was useful for treatment, rather than just prophylaxis, since it was safe and effective when administered well after the onset of diarrhea. Furthermore, even though all animals received intensive supportive care, there was a strong trend towards improved secondary health outcomes, including general health, appetite, and dehydration measures amongst treated animals. These data establish MMV665917 as an outstanding lead compound for Cryptosporidium drug development. [ABSTRACT FROM AUTHOR]

Background: Dengue is one of the fastest spreading vector-borne diseases, caused by four antigenically distinct dengue viruses (DENVs). Antibodies against DENVs are responsible for both protection as well as pathogenesis. A vaccine that is safe for and efficacious in all people irrespective of their age and domicile is still an unmet need. It is becoming increasingly apparent that vaccine design must eliminate epitopes implicated in the induction of infection-enhancing antibodies. Methodology/principal findings: We report a Pichia pastoris-expressed dengue immunogen, DSV4, based on DENV envelope protein domain III (EDIII), which contains well-characterized serotype-specific and cross-reactive epitopes. In natural infection, <10% of the total neutralizing antibody response is EDIII-directed. Yet, this is a functionally relevant domain which interacts with the host cell surface receptor. DSV4 was designed by in-frame fusion of EDIII of all four DENV serotypes and hepatitis B surface (S) antigen and co-expressed with unfused S antigen to form mosaic virus-like particles (VLPs). These VLPs displayed EDIIIs of all four DENV serotypes based on probing with a battery of serotype-specific anti-EDIII monoclonal antibodies. The DSV4 VLPs were highly immunogenic, inducing potent and durable neutralizing antibodies against all four DENV serotypes encompassing multiple genotypes, in mice and macaques. DSV4-induced murine antibodies suppressed viremia in AG129 mice and conferred protection against lethal DENV-4 virus challenge. Further, neither murine nor macaque anti-DSV4 antibodies promoted mortality or inflammatory cytokine production when passively transferred and tested in an in vivo dengue disease enhancement model of AG129 mice. Conclusions/significance: Directing the immune response to a non-immunodominant but functionally relevant serotype-specific dengue epitope of the four DENV serotypes, displayed on a VLP platform, can help minimize the risk of inducing disease-enhancing antibodies while eliciting effective tetravalent seroconversion. DSV4 has a significant potential to emerge as a safe, efficacious and inexpensive subunit dengue vaccine candidate. [ABSTRACT FROM AUTHOR]

The article covers the implementation of the Xpert Ebola Assay, a rapid molecular diagnostic test using the GeneXpert platform, in Liberia in response to the Ebola Virus Disease (EVD) outbreak in West Africa between 2014 and 2016. It discusses the effectiveness of coordination between surveillance and laboratory teams before noting the costs and methodology associated with the GeneXpert platform.

Background: Rift Valley fever virus (RVFV) is a mosquito-borne bunyavirus of the genus Phlebovirus that is highly pathogenic to ruminants and humans. The disease is currently confined to Africa and the Arabian Peninsula, but globalization and climate change may facilitate introductions of the virus into currently unaffected areas via infected animals or mosquitoes. The consequences of such an introduction will depend on environmental factors, the availability of susceptible ruminants and the capacity of local mosquitoes to transmit the virus. We have previously demonstrated that lambs native to the Netherlands are highly susceptible to RVFV and we here report the vector competence of Culex (Cx.) pipiens, the most abundant and widespread mosquito species in the country. Vector competence was first determined after artificial blood feeding of laboratory-reared mosquitoes using the attenuated Clone 13 strain. Subsequently, experiments with wild-type RVFV and mosquitoes hatched from field-collected eggs were performed. Finally, the transmission of RVFV from viremic lambs to mosquitoes was studied. Principal findings: Artificial feeding experiments using Clone 13 demonstrated that indigenous, laboratory-reared Cx. pipiens mosquitoes are susceptible to RVFV and that the virus can be transmitted via their saliva. Experiments with wild-type RVFV and mosquitoes hatched from field-collected eggs confirmed the vector competence of Cx. pipiens mosquitoes from the Netherlands. To subsequently investigate transmission of the virus under more natural conditions, mosquitoes were allowed to feed on RVFV-infected lambs during the viremic period. We found that RVFV is efficiently transmitted from lambs to mosquitoes, although transmission was restricted to peak viremia. Interestingly, in the mosquito-exposed skin samples, replication of RVFV was detected in previously unrecognized target cells. Significance: We here report the vector competence of Cx. pipiens mosquitoes from the Netherlands for RVFV. Both laboratory-reared mosquitoes and well as those hatched from field-collected eggs were found to be competent vectors. Moreover, RVFV was transmitted efficiently from indigenous lambs to mosquitoes, although the duration of host infectivity was found to be shorter than previously assumed. Interestingly, analysis of mosquito-exposed skin samples revealed previously unidentified target cells of the virus. Our findings underscore the value of including natural target species in vector competence experiments. [ABSTRACT FROM AUTHOR]

Objectives: We determined the seroprevalence of chikungunya virus (CHIKV) infection in the adult resident population in Singapore following local outbreaks of chikungunya fever (CHIKF) in 2008–2009. Methods: Our cross-sectional study involved residual sera from 3,293 adults aged 18–79 years who had participated in the National Health Survey in 2010. Sera were tested for IgG antibodies against CHIKV and dengue virus (DENV) and neutralizing antibodies against CHIKV. Results: The prevalence of CHIKV-neutralizing antibodies among Singapore residents aged 18–79 years was 1.9% (95% confidence interval: 1.4%– 2.3%). The CHIKV seroprevalence was highest in the elderly aged 70–79 years at 11.5%, followed by those aged 30–39 years at 3.1%. Men had significantly higher CHIKV seroprevalence than women (2.5% versus 1.3%, p = 0.01). Among the three main ethnic groups, Indians had the highest seroprevalence (3.5%) compared to Chinese (1.6%) and Malays (0.7%) (p = 0.02 and p = 0.01, respectively). Multivariable logistic regression identified adults aged 30–39 years and 70–79 years, men, those of Indian ethnicity and ethnic minor groups, and residence on ground floor of public and private housing apartments as factors that were significantly associated with a higher likelihood of exposure to CHIKV. The overall prevalence of anti-DENV IgG antibodies was 56.8% (95% CI: 55.1%– 58.5%), while 1.5% (95% CI: 1.1%– 2.0%) of adults possessed both neutralizing antibodies against CHIKV and IgG antibodies against DENV. Conclusions: Singapore remains highly susceptible to CHIKV infection. There is a need to maintain a high degree of vigilance through disease surveillance and vector control. Findings from such serological study, when conducted on a regular periodic basis, could supplement surveillance to provide insights on CHIKV circulation in at-risk population. [ABSTRACT FROM AUTHOR]

Zika virus (ZIKV) is a mosquito-borne flavivirus of significant public health concern. In the summer of 2016, ZIKV was first detected in the contiguous United States. Here we present one of the first cases of a locally acquired ZIKV infection in a dengue-naïve individual. We collected blood from a female with a maculopapular rash at day (D) 5 and D7 post onset of symptoms (POS) and we continued weekly blood draws out to D148 POS. To establish the ontogeny of the immune response against ZIKV, lymphocytes and plasma were analyzed in a longitudinal fashion. The plasmablast response peaked at D7 POS (19.6% of CD19+ B-cells) and was undetectable by D15 POS. ZIKV-specific IgM was present at D5 POS, peaked between D15 and D21 POS, and subsequently decreased. The ZIKV-specific IgG response, however, was not detected until D15 POS and continued to increase after that. Interestingly, even though the patient had never been infected with dengue virus (DENV), cross-reactive IgM and IgG binding against each of the four DENV serotypes could be detected. The highest plasma neutralization activity against ZIKV peaked between D15 and D21 POS, and even though DENV binding antibodies were present in the plasma of the patient, there was neither neutralization nor antibody dependent enhancement (ADE) of DENV. Interestingly, ADE against ZIKV arose at D48 POS and continued until the end of the study. CD4+ and CD8+ T-cells recognized ZIKV-NS2A and ZIKV-E, respectively. The tetramer positive CD8+ T-cell response peaked at D21 POS with elevated levels persisting for months. In summary, this is the first study to establish the timing of the ontogeny of the immune response against ZIKV. [ABSTRACT FROM AUTHOR]

Background: Rabies is a major public health problem with a fatality rate close to 100%; however, complete prevention can be achieved through pre- or post-exposure prophylaxis. The rapid fluorescent focus inhibition test (RFFIT) is one of the recommended testing methods to determine the production of neutralizing antibodies after vaccination. Here, we report the development of a new monoclonal antibody (mAb) designed to react specifically with Rabies virus (RABV) phosphoprotein (P protein), and the evaluation of its applicability to the RFFIT and its effectiveness as a diagnostic reagent for human rabies. Methodology/principal findings: The mAb KGH P 16B8 was produced to target the P protein of the Korean KGH RABV strain. An indirect immunofluorescence assay (IFA) was conducted to detect various strains of RABV in various cell lines. Alexa-conjugated KGH P 16B8 (16B8-Alexa) was developed for the RFFIT. The IFA test could detect RABV up to a 1:2,500 dilution, with a detection limit comparable to that of a commercial diagnostic reagent. The sensitivity, specificity, positive predictive value, and negative predictive value of the RFFIT using 16B8-Alexa in 414 clinical specimens were 98.67%, 99.47%, 99.55%, and 98.42%, respectively. The results of the RFFIT with 16B8-Alexa were strongly correlated with those obtained using an existing commercial diagnostic reagent (r = 0.995, p<0.001). Conclusions/significance: The mAb developed in this study shows high sensitivity and specificity, confirming its clinical utility with the RFFIT to measure the rabies neutralizing antibody titer and establish a diagnosis in human. Thus, 16B8-Alexa is expected to serve as an alternative diagnostic reagent that is widely accessible, with potentially broad applications beyond those of the RFFIT in Korea. Further studies with 16B8-Alexa should provide insight into the immunological mechanism of the P protein of Korean RABV. [ABSTRACT FROM AUTHOR]