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Background Dengue is re-emerging throughout the tropical world, causing frequent recurrent epidemics. The initial clinical manifestation of dengue often is confused with other febrile states confounding both clinical management and disease surveillance. Evidence-based triage strategies that identify individuals likely to be in the early stages of dengue illness can direct patient stratification for clinical investigations, management, and virological surveillance. Here we report the identification of algorithms that differentiate dengue from other febrile illnesses in the primary care setting and predict severe disease in adults. Methods and Findings A total of 1,200 patients presenting in the first 72 hours of acute febrile illness were recruited and followed up for up to a 4-week period prospectively; 1,012 of these were recruited from Singapore and 188 from Vietnam. Of these, 364 were dengue RT-PCR positive; 173 had dengue fever, 171 had dengue hemorrhagic fever, and 20 had dengue shock syndrome as final diagnosis. Using a C4.5 decision tree classifier for analysis of all clinical, haematological, and virological data, we obtained a diagnostic algorithm that differentiates dengue from non-dengue febrile illness with an accuracy of 84.7%. The algorithm can be used differently in different disease prevalence to yield clinically useful positive and negative predictive values. Furthermore, an algorithm using platelet count, crossover threshold value of a real-time RT-PCR for dengue viral RNA, and presence of pre-existing anti-dengue IgG antibodies in sequential order identified cases with sensitivity and specificity of 78.2% and 80.2%, respectively, that eventually developed thrombocytopenia of 50,000 platelet/mm3 or less, a level previously shown to be associated with haemorrhage and shock in adults with dengue fever. Conclusion This study shows a proof-of-concept that decision algorithms using simple clinical and haematological parameters can predict diagnosis and prognosis of dengue disease, a finding that could prove useful in disease management and surveillance., Author Summary Dengue illness appears similar to other febrile illness, particularly in the early stages of disease. Consequently, diagnosis is often delayed or confused with other illnesses, reducing the effectiveness of using clinical diagnosis for patient care and disease surveillance. To address this shortcoming, we have studied 1,200 patients who presented within 72 hours from onset of fever; 30.3% of these had dengue infection, while the remaining 69.7% had other causes of fever. Using body temperature and the results of simple laboratory tests on blood samples of these patients, we have constructed a decision algorithm that is able to distinguish patients with dengue illness from those with other causes of fever with an accuracy of 84.7%. Another decision algorithm is able to predict which of the dengue patients would go on to develop severe disease, as indicated by an eventual drop in the platelet count to 50,000/mm3 blood or below. Our study shows a proof-of-concept that simple decision algorithms can predict dengue diagnosis and the likelihood of developing severe disease, a finding that could prove useful in the management of dengue patients and to public health efforts in preventing virus transmission.

Malaria remains one of the world's greatest killers and a vaccine is urgently required. There are no established correlates of protection against malaria either for natural immunity to the disease or for immunity conferred by candidate malaria vaccines. The RTS,S/AS02A vaccine offers significant partial efficacy against malaria.mRNA expression of five key cytokines interferon-gamma (IFN-[gamma]), monokine induced by gamma (MIG), interleukin-10 (IL-10), transforming growth factor-[beta] (TGF-[beta]) and forkhead box P3 (FoxP3) in peripheral blood mononuclear cells were measured by real-time RT-PCR before and after vaccination with RTS,S/AS02A and Modified Vaccinia virus Ankara encoding the circumsporozoite protein (MVA-CS) in healthy malaria-naive adult volunteers. The only significant change was in IFN-[gamma] mRNA expression, which was increased seven days after vaccination (P = 0.04). Expression of MIG mRNA seven days after vaccination correlated inversely with time to detection of parasites by blood film in an experimental sporozoite challenge (r = 0.94 P = 0.005). An inverse relationship was seen between both TGF-[beta]1 and IL-10 mRNA at baseline and the anti-circumsporozoite IgG antibody response (r = -0.644 P = 0.022 and r = -0.554 P = 0.031 respectively). This study demonstrates the potential for MIG expression as a correlate of protection against malaria. Baseline levels of the regulatory cytokines TGF-[beta] and IL-10 inversely correlated with antibody levels post vaccination and warrant further studies to improve understanding of individual differences in response to vaccination.

BACKGROUND: Malaria and bacterial infections account for most infectious disease deaths in developing countries. Prompt treatment saves lives, but rapid deterioration often prevents the use of oral therapies; delays in reaching health facilities providing parenteral interventions are common. Rapidly and reliably absorbed antimalarial/antibacterial rectal formulations used in the community could prevent deaths and disabilities. Rectal antimalarial treatments are currently available; rectal antibacterial treatments are yet to be developed. Assessment of the likely cost-effectiveness of these interventions will inform research priorities and implementation. METHODS AND FINDINGS: The burden of malaria and bacterial infections worldwide and in Sub-Saharan and Southern Africa (SSA) and South and South-East Asia (SEA) was summarised using published data. The additional healthcare costs (USD) per death and per Disability Adjusted Life Year (DALY) avoided following pre-referral treatment of severe febrile illness with rectal antimalarials, antibacterials or combined antimalarial/antibacterials in populations at malaria risk in SSA/SEA were assessed. 46 million severe malaria and bacterial infections and 5 million deaths occur worldwide each year, mostly in SSA/SEA. At annual delivery costs of 0.02 dollars/capita and 100% coverage, rectal antimalarials (2 dollars per dose) would avert 240,000 deaths in SSA and 7,000 deaths in SEA at 5 and 177 dollars per DALY avoided, respectively; rectal antibacterials (2 dollars per dose) would avert 130,000 deaths in SSA and 27,000 deaths in SEA at 19 and 97 dollars per DALY avoided, respectively. Combined rectal formulations (2.50 dollars per dose) would avert 370,000 deaths in SSA and 33,000 deaths in SEA at 8 and 79 dollars per DALY avoided, respectively, and are a cost-effective alternative to rectal antimalarials or antibacterials alone. CONCLUSIONS: Antimalarial, antibacterial and combined rectal formulations are likely to be cost-effective interventions for severe febrile illness in the community. Attention should focus on developing effective rectal antibacterials and ensuring that these lifesaving treatments are used in a cost-effective manner.

Background The need for economical rabies post-exposure prophylaxis (PEP) is increasing in developing countries. Implementation of the two currently approved economical intradermal (ID) vaccine regimens is restricted due to confusion over different vaccines, regimens and dosages, lack of confidence in intradermal technique, and pharmaceutical regulations. We therefore compared a simplified 4-site economical PEP regimen with standard methods. Methods Two hundred and fifty-four volunteers were randomly allocated to a single blind controlled trial. Each received purified vero cell rabies vaccine by one of four PEP regimens: the currently accepted 2-site ID; the 8-site regimen using 0.05 ml per ID site; a new 4-site ID regimen (on day 0, approximately 0.1 ml at 4 ID sites, using the whole 0.5 ml ampoule of vaccine; on day 7, 0.1 ml ID at 2 sites and at one site on days 28 and 90); or the standard 5-dose intramuscular regimen. All ID regimens required the same total amount of vaccine, 60% less than the intramuscular method. Neutralising antibody responses were measured five times over a year in 229 people, for whom complete data were available. Findings All ID regimens showed similar immunogenicity. The intramuscular regimen gave the lowest geometric mean antibody titres. Using the rapid fluorescent focus inhibition test, some sera had unexpectedly high antibody levels that were not attributable to previous vaccination. The results were confirmed using the fluorescent antibody virus neutralisation method. Conclusions This 4-site PEP regimen proved as immunogenic as current regimens, and has the advantages of requiring fewer clinic visits, being more practicable, and having a wider margin of safety, especially in inexperienced hands, than the 2-site regimen. It is more convenient than the 8-site method, and can be used economically with vaccines formulated in 1.0 or 0.5 ml ampoules. The 4-site regimen now meets all requirements of immunogenicity for PEP and can be introduced without further studies. Trial Registration Controlled-Trials.com ISRCTN 30087513, Author Summary All human deaths from rabies result from failure to give adequate prophylaxis. After a rabid animal bite, immediate wound cleaning, rabies vaccine and immunoglobulin injections effectively prevent fatal infection. Immunoglobulin is very rarely available in developing countries, where prevention relies on efficacious vaccine. WHO approved vaccines are prohibitively expensive, but 2 economical regimens (injecting small amounts of vaccine intradermally, into the skin, at 2 or 8 sites on the first day of the course) have been used for many years in a few places. Practical or perceived difficulties have restricted widespread uptake of economical methods. These could largely be overcome by introducing a new, simpler regimen, involving 4 site injections on the first day. We vaccinated volunteers to compare the antibody levels induced by the 4-site intradermal regimen with those induced by the current 2-site and 8-site regimens and the “gold standard” intramuscular regimen favoured internationally. All the economical intradermal regimens were at least as immunogenic as the intramuscular method. The results provide sufficient evidence that the 4-site regimen meets the criteria necessary for its recommendation for use wherever the cost of vaccine is prohibitive and especially where 2 or more patients are treated on the same day.

Background Early diagnosis of dengue can assist patient triage and management and prevent unnecessary treatments and interventions. Commercially available assays that detect the dengue virus protein NS1 in the plasma/serum of patients offers the possibility of early and rapid diagnosis. Methodology/Principal Findings The sensitivity and specificity of the Pan-E Dengue Early ELISA and the Platelia™ Dengue NS1 Ag assays were compared against a reference diagnosis in 1385 patients in 6 countries in Asia and the Americas. Platelia was more sensitive (66%) than Pan-E (52%) in confirmed dengue cases. Sensitivity varied by geographic region, with both assays generally being more sensitive in patients from SE Asia than the Americas. Both kits were more sensitive for specimens collected within the first few days of illness onset relative to later time points. Pan-E and Platelia were both 100% specific in febrile patients without evidence of acute dengue. In patients with other confirmed diagnoses and healthy blood donors, Platelia was more specific (100%) than Pan-E (90%). For Platelia, when either the NS1 test or the IgM test on the acute sample was positive, the sensitivity versus the reference result was 82% in samples collected in the first four days of fever. NS1 sensitivity was not associated to disease severity (DF or DHF) in the Platelia test, whereas a trend for higher sensitivity in DHF cases was seen in the Pan-E test (however combined with lower overall sensitivity). Conclusions/Significance Collectively, this multi-country study suggests that the best performing NS1 assay (Platelia) had moderate sensitivity (median 64%, range 34–76%) and high specificity (100%) for the diagnosis of dengue. The poor sensitivity of the evaluated assays in some geographical regions suggests further assessments are needed. The combination of NS1 and IgM detection in samples collected in the first few days of fever increased the overall dengue diagnostic sensitivity., Author Summary Dengue is the most important mosquito-borne viral disease of humans and an enormous public health burden in affected countries. Early, sensitive and specific diagnosis of dengue is needed for appropriate patient management as well as for early epidemic detection. Commercially available assays that detect the dengue virus protein NS1 in the plasma/serum of patients offer the possibility of early and rapid diagnosis. Here we evaluated two commercially available ELISA kits for NS1 detection (Pan-E Dengue Early ELISA and the Platelia™ Dengue NS1 Ag). Results were compared against a reference diagnosis in 1385 patients in 6 countries in Asia and the Americas. Collectively, this multi-country study suggests that the best performing NS1 assay (Platelia) had moderate sensitivity (median 64%, range 34–76%) and high specificity (100%) for the diagnosis of dengue. The combination of NS1 and IgM detection in samples collected in the first few days of fever increased the overall dengue diagnostic sensitivity.

BACKGROUND: A substantial decline in malaria was reported to have occurred over several years until 2007 in the western part of The Gambia, encouraging consideration of future elimination in this previously highly endemic region. Scale up of interventions has since increased with support from the Global Fund and other donors. METHODOLOGY/PRINCIPAL FINDINGS: We continued to examine laboratory records at four health facilities previously studied and investigated six additional facilities for a 7 year period, adding data from 243,707 slide examinations, to determine trends throughout the country until the end of 2009. We actively detected infections in a community cohort of 800 children living in rural villages throughout the 2008 malaria season, and assayed serological changes in another rural population between 2006 and 2009. Proportions of malaria positive slides declined significantly at all of the 10 health facilities between 2003 (annual mean across all sites, 38.7%) and 2009 (annual mean, 7.9%). Statistical modelling of trends confirmed significant seasonality and decline over time at each facility. Slide positivity was lowest in 2009 at all sites, except two where lowest levels were observed in 2006. Mapping households of cases presenting at the latter sites in 2007-2009 indicated that these were not restricted to a few residual foci. Only 2.8% (22/800) of a rural cohort of children had a malaria episode in the 2008 season, and there was substantial serological decline between 2006 and 2009 in a separate rural area. CONCLUSIONS: Malaria has continued to decline in The Gambia, as indicated by a downward trend in slide positivity at health facilities, and unprecedented low incidence and seroprevalence in community surveys. We recommend intensification of control interventions for several years to further reduce incidence, prior to considering an elimination programme.

Background Since the first recorded epidemic of syphilis in 1495, controversy has surrounded the origins of the bacterium Treponema pallidum subsp. pallidum and its relationship to the pathogens responsible for the other treponemal diseases: yaws, endemic syphilis, and pinta. Some researchers have argued that the syphilis-causing bacterium, or its progenitor, was brought from the New World to Europe by Christopher Columbus and his men, while others maintain that the treponematoses, including syphilis, have a much longer history on the European continent. Methodology/Principal Findings We applied phylogenetics to this problem, using data from 21 genetic regions examined in 26 geographically disparate strains of pathogenic Treponema. Of all the strains examined, the venereal syphilis-causing strains originated most recently and were more closely related to yaws-causing strains from South America than to other non-venereal strains. Old World yaws-causing strains occupied a basal position on the tree, indicating that they arose first in human history, and a simian strain of T. pallidum was found to be indistinguishable from them. Conclusions/Significance Our results lend support to the Columbian theory of syphilis's origin while suggesting that the non-sexually transmitted subspecies arose earlier in the Old World. This study represents the first attempt to address the problem of the origin of syphilis using molecular genetics, as well as the first source of information regarding the genetic make-up of non-venereal strains from the Western hemisphere., Author Summary For 500 years, controversy has raged around the origin of T. pallidum subsp. pallidum, the bacterium responsible for syphilis. Did Christopher Columbus and his men introduce this pathogen into Renaissance Europe, after contracting it during their voyage to the New World? Or does syphilis have a much older history in the Old World? This paper represents the first attempt to use a phylogenetic approach to solve this question. In addition, it clarifies the evolutionary relationships between the pathogen that causes syphilis and the other T. pallidum subspecies, which cause the neglected tropical diseases yaws and endemic syphilis. Using a collection of pathogenic Treponema strains that is unprecedented in size, we show that yaws appears to be an ancient infection in humans while venereal syphilis arose relatively recently in human history. In addition, the closest relatives of syphilis-causing strains identified in this study were found in South America, providing support for the Columbian theory of syphilis's origin.

Background Nematode polyprotein allergens (NPAs) are an unusual class of lipid-binding proteins found only in nematodes. They are synthesized as large, tandemly repetitive polyproteins that are post-translationally cleaved into multiple copies of small lipid binding proteins with virtually identical fatty acid and retinol (Vitamin A)-binding characteristics. They are probably central to transport and distribution of small hydrophobic compounds between the tissues of nematodes, and may play key roles in nutrient scavenging, immunomodulation, and IgE antibody-based responses in infection. In some species the repeating units are diverse in amino acid sequence, but, in ascarid and filarial nematodes, many of the units are identical or near-identical. ABA-1A is the most common repeating unit of the NPA of Ascaris suum, and is closely similar to that of Ascaris lumbricoides, the large intestinal roundworm of humans. Immune responses to NPAs have been associated with naturally-acquired resistance to infection in humans, and the immune repertoire to them is under strict genetic control. Methodology/Principal Findings The solution structure of ABA-1A was determined by protein nuclear magnetic resonance spectroscopy. The protein adopts a novel seven-helical fold comprising a long central helix that participates in two hollow four-helical bundles on either side. Discrete hydrophobic ligand-binding pockets are found in the N-terminal and C-terminal bundles, and the amino acid sidechains affected by ligand (fatty acid) binding were identified. Recombinant ABA-1A contains tightly-bound ligand(s) of bacterial culture origin in one of its binding sites. Conclusions/Significance This is the first mature, post-translationally processed, unit of a naturally-occurring tandemly-repetitive polyprotein to be structurally characterized from any source, and it belongs to a new structural class. NPAs have no counterparts in vertebrates, so represent potential targets for drug or immunological intervention. The nature of the (as yet) unidentified bacterial ligand(s) may be pertinent to this, as will our characterization of the unusual binding sites., Author Summary Parasitic nematode worms cause serious health problems in humans and other animals. They can induce allergic-type immune responses, which can be harmful but may at the same time protect against the infections. Allergens are proteins that trigger allergic reactions and these parasites produce a type that is confined to nematodes, the nematode polyprotein allergens (NPAs). These are synthesized as large precursor proteins comprising repeating units of similar amino acid sequence that are subsequently cleaved into multiple copies of the allergen protein. NPAs bind small lipids such as fatty acids and retinol (Vitamin A) and probably transport these sensitive and insoluble compounds between the tissues of the worms. Nematodes cannot synthesize these lipids, so NPAs may also be crucial for extracting nutrients from their hosts. They may also be involved in altering immune responses by controlling the lipids by which the immune and inflammatory cells communicate. We describe the molecular structure of one unit of an NPA, the well-known ABA-1 allergen of Ascaris, and find its structure to be of a type not previously found for lipid-binding proteins, and we describe the unusual sites where lipids bind within this structure.

Background Disease incidence data are needed to guide decision-making for public health interventions. Although dengue is a reportable disease in Thailand and Cambodia, the degree that reported incidence underrecognizes true disease burden is unknown. We utilized dengue incidence calculated from laboratory-confirmed outpatient and inpatient cases in prospective cohort studies to estimate the magnitude of dengue underrecognition and to establish more accurate disease burden estimates for these countries. Methods and Findings Cohort studies were conducted among children aged 95,000 children in Thailand and >58,000 children in Cambodia were estimated to be hospitalized due to dengue. Conclusion Calculating MFs by comparing prospective cohort study data to locally-reported national surveillance data is one approach to more accurately assess disease burden. These data indicate that although dengue is regularly reported in many countries, national surveillance data significantly underrecognize the true burden of disease., Author Summary Dengue is a major public health problem especially in tropical and subtropical countries of Asia and Latin-America. An effective dengue vaccine is not yet available, but several vaccine candidates are currently being evaluated in clinical trials. Accurate country-level incidence data are crucial to assess the cost-effectiveness of such vaccines and will assist policy-makers in making vaccine introduction decisions. Existing national surveillance systems are often passive and are designed to monitor trends and to detect disease outbreaks. Our analyses of data from prospectively followed cohorts with laboratory confirmation of dengue cases show that, in Thailand and Cambodia, dengue incidence is underrecognized by more than 8-fold. The magnitude of the outpatient burden caused by dengue is not assessed or reflected by the national surveillance data. We estimate that a median of more than 340,000 symptomatic dengue virus infections occurred annually in children less than 15 years of age in Thailand in Cambodia between 2003 and 2007.

Many pathogenic bacteria, fungi, and protozoa achieve chronic infection through an immune evasion strategy known as antigenic variation. In the human malaria parasite Plasmodium falciparum, this involves transcriptional switching among members of the var gene family, causing parasites with different antigenic and phenotypic characteristics to appear at different times within a population. Here we use a genome-wide approach to explore this process in vitro within a set of cloned parasite populations. Our analyses reveal a non-random, highly structured switch pathway where an initially dominant transcript switches via a set of switch-intermediates either to a new dominant transcript, or back to the original. We show that this specific pathway can arise through an evolutionary conflict in which the pathogen has to optimise between safeguarding its limited antigenic repertoire and remaining capable of establishing infections in non-naïve individuals. Our results thus demonstrate a crucial role for structured switching during the early phases of infections and provide a unifying theory of antigenic variation in P. falciparum malaria as a balanced process of parasite-intrinsic switching and immune-mediated selection., Author Summary The malaria parasite Plasmodium falciparum avoids recognition and clearance by the immune system by sequentially switching between members of the var multi-gene family which encode the immunodominant surface proteins PfEMP1. However, some mechanism must exist to prevent rapid exposure of the pathogen's entire antigenic repertoire as this would quickly terminate the infection. It has previously been shown that the immune system can play an important role in orchestrating the sequential display of variants once an infection is established; however this does not explain how repertoire exhaustion is avoided in the initial phases of infection before an immune response has been established. Here we show that P. falciparum has evolved a highly structured switching pattern to prevent repertoire exhaustion in the early stages of infection without compromising the ability to establish new infections among partially immune individuals.

Dengue virus (DENV) is a mosquito-borne flavivirus, and symptoms of infection range from asymptomatic to the severe dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). High viral loads correlate with disease severity, and both type I & II interferons (IFNs) are crucial for controlling viral replication. We have previously reported that signal transducer and activator of transcription (STAT) 1-deficient mice are resistant to DENV-induced disease, but little is known about this STAT1-independent mechanism of protection. To determine the molecular basis of the STAT1-independent pathway, mice lacking STAT1, STAT2, or both STAT1 and STAT2 were infected with a virulent mouse-adapted strain of DENV2. In the first 72 hours of infection, the single-deficient mice lacking STAT1 or STAT2 possessed 50–100 fold higher levels of viral RNA than wild type mice in the serum, spleen, and other visceral tissues, but remained resistant to DENV-induced death. In contrast, the double-deficient mice exhibited the early death phenotype previously observed in type I and II IFN receptor knockout mice (AG129), indicating that STAT2 is the mediator of the STAT1-independent host defense mechanism. Further studies demonstrated that this STAT2-dependent STAT1-independent mechanism requires the type I IFN receptor, and contributes to the autocrine amplification of type I IFN expression. Examination of gene expression in the spleen and bone marrow-derived macrophages following DENV infection revealed STAT2-dependent pathways can induce the transcription of a subset of interferon stimulated genes even in the absence of STAT1. Collectively, these results help elucidate the nature of the poorly understood STAT1-independent host defense mechanism against viruses by identifying a functional type I IFN/STAT2 signaling pathway following DENV infection in vivo., Author Summary Dengue virus (DENV) is a mosquito-borne pathogen present in the tropical and sub-tropical regions of the world, and an estimated 2.5 billion people are at risk of infection. Interferon (IFN) mediated innate responses in the host are critical for limiting viral spread following DENV infection. We have previously demonstrated that mice lacking STAT1, a key mediator of both type I and II IFN responses, are not susceptible to DENV-mediated disease. In this study, we sought to determine the mechanism responsible for protection against DENV disease in the absence of STAT1. Using knockout mice, we identify STAT2 as the protein that mediates type I IFN signaling during DENV infection in the absence of STAT1. The resulting antiviral response includes amplification of type I IFN and the expression of interferon stimulated genes. These data suggest DENV infection is especially sensitive to STAT2-mediated antiviral responses in vivo, and provide novel insights towards how IFNs protect against viral infections.

The endosymbiotic bacterium Wolbachia is being investigated as a potential control agent in several important vector insect species. Recent studies have shown that Wolbachia can protect the insect host against a wide variety of pathogens, resulting in reduced transmission of parasites and viruses. It has been proposed that compromised vector competence of Wolbachia-infected insects is due to up-regulation of the host innate immune system or metabolic competition. Anopheles mosquitoes, which transmit human malaria parasites, have never been found to harbor Wolbachia in nature. While transient somatic infections can be established in Anopheles, no stable artificially-transinfected Anopheles line has been developed despite numerous attempts. However, cultured Anopheles cells can be stably infected with multiple Wolbachia strains such as wAlbB from Aedes albopictus, wRi from Drosophila simulans and wMelPop from Drosophila melanogaster. Infected cell lines provide an amenable system to investigate Wolbachia-Anopheles interactions in the absence of an infected mosquito strain. We used Affymetrix GeneChip microarrays to investigate the effect of wAlbB and wRi infection on the transcriptome of cultured Anopheles Sua5B cells, and for a subset of genes used quantitative PCR to validate results in somatically-infected Anopheles mosquitoes. Wolbachia infection had a dramatic strain-specific effect on gene expression in this cell line, with almost 700 genes in total regulated representing a diverse array of functional classes. Very strikingly, infection resulted in a significant down-regulation of many immune, stress and detoxification-related transcripts. This is in stark contrast to the induction of immune genes observed in other insect hosts. We also identified genes that may be potentially involved in Wolbachia-induced reproductive and pathogenic phenotypes. Somatically-infected mosquitoes had similar responses to cultured cells. The data show that Wolbachia has a profound and unique effect on Anopheles gene expression in cultured cells, and has important implications for mechanistic understanding of Wolbachia-induced phenotypes and potential novel strategies to control malaria., Author Summary Wolbachia are bacteria that infect many insect species, but do not infect Anopheles mosquitoes. These mosquitoes transmit Plasmodium parasites, which cause malaria in humans. Wolbachia infection in Aedes aegypti mosquitoes reduces their ability to transmit diverse pathogens including viruses, nematodes and bird malaria parasites. Wolbachia-induced stimulation of the mosquito's innate immune system has been suggested as a mechanism conferring this pathogen interference. Since no Wolbachia-infected Anopheles mosquito strain exists, we used infected cultured Anopheles cells to examine the effect of infection on Anopheles gene expression. Wolbachia had a profound influence on Anopheles gene expression. Many of the genes regulated by Wolbachia have been seen in other studies to influence Plasmodium levels in mosquitoes, but interestingly and in contrast to other mosquitoes, many of the host genes were suppressed rather than induced.

Background Characterizing the evolutionary relationships and population structure of parasites can provide important insights into the epidemiology of human disease. Methodology/Principal Findings We examined 142 isolates of Trypanosoma brucei from all over sub-Saharan Africa using three distinct classes of genetic markers (kinetoplast CO1 sequence, nuclear SRA gene sequence, eight nuclear microsatellites) to clarify the evolutionary history of Trypanosoma brucei rhodesiense (Tbr) and T. b. gambiense (Tbg), the causative agents of human African trypanosomosis (sleeping sickness) in sub-Saharan Africa, and to examine the relationship between Tbr and the non-human infective parasite T. b. brucei (Tbb) in eastern and southern Africa. A Bayesian phylogeny and haplotype network based on CO1 sequences confirmed the taxonomic distinctness of Tbg group 1. Limited diversity combined with a wide geographical distribution suggested that this parasite has recently and rapidly colonized hosts across its current range. The more virulent Tbg group 2 exhibited diverse origins and was more closely allied with Tbb based on COI sequence and microsatellite genotypes. Four of five COI haplotypes obtained from Tbr were shared with isolates of Tbb, suggesting a close relationship between these taxa. Bayesian clustering of microsatellite genotypes confirmed this relationship and indicated that Tbr and Tbb isolates were often more closely related to each other than they were to other members of the same subspecies. Among isolates of Tbr for which data were available, we detected just two variants of the SRA gene responsible for human infectivity. These variants exhibited distinct geographical ranges, except in Tanzania, where both types co-occurred. Here, isolates possessing distinct SRA types were associated with identical COI haplotypes, but divergent microsatellite signatures. Conclusions/Significance Our data provide strong evidence that Tbr is only a phenotypic variant of Tbb; while relevant from a medical perspective, Tbr is not a reproductively isolated taxon. The wide distribution of the SRA gene across diverse trypanosome genetic backgrounds suggests that a large amount of genetic diversity is potentially available with which human-infective trypanosomes may respond to selective forces such as those exerted by drugs., Author Summary Trypanosoma brucei, the parasite causing human African trypanosomiasis (sleeping sickness) across sub-Saharan Africa is traditionally split into three subspecies: T. b. gambiense (Tbg), causing a chronic form of human disease in West and Central Africa; T. b. rhodesiense (Tbr), causing an acute form of human disease in East and Southern Africa; and T. b. brucei (Tbb), which is restricted to animals. Tbg is further split into Tbg group 1 and Tbg group 2. Better understanding the evolutionary relationships between these groups may help to shed light on the epidemiology of sleeping sickness. Here, we used three different types of genetic markers to investigate the phylogeographic relationships among the four groups across a large portion of their range. Our results confirm the distinctiveness of Tbg group 1 while highlighting the extremely close relationships among the other three taxa. In particular, Tbg group 2 was closely related to Tbb, while Tbr appeared to be a variant of Tbb, differing only in its phenotype of human infectivity. The wide geographic distribution of the gene conferring human infectivity (SRA) and the fact that it is readily exchanged among lineages of T. brucei in eastern Africa suggests that human-infective trypanosomes have access to an extensive gene pool with which to respond to selective pressures such as drugs.

Diseases transmitted by mosquitoes have a devastating impact on global health and this is worsening due to difficulties with existing control measures and climate change. Genetically modified mosquitoes that are refractory to disease transmission are seen as having great potential in the delivery of novel control strategies. Historically the genetic modification of insects has relied upon transposable elements which have many limitations despite their successful use. To circumvent these limitations the Streptomyces phage phiC31 integrase system has been successfully adapted for site-specific transgene integration in insects. Here, we present the first site-specific transformation of Anopheles gambiae, the principal vector of human malaria. Mosquitoes were initially engineered to incorporate the phiC31 targeting site at a defined genomic location. A second phase of genetic modification then achieved site-specific integration of Vida3, a synthetic anti-malarial gene. Expression of Vida3, specifically in the midgut of bloodfed females, offered consistent and significant protection against Plasmodium yoelii nigeriensis, reducing average parasite intensity by 85%. Similar protection was observed against Plasmodium falciparum in some experiments, although protection was inconsistent. In the fight against malaria, it is imperative to establish a broad repertoire of both anti-malarial effector genes and tissue-specific promoters for their expression, enabling those offering maximum effect with minimum fitness cost to be identified. In the future, this technology will allow effective comparisons and informed choices to be made, potentially leading to complete transmission blockade.

History and Epidemiology Historically, noma is a disease of extreme poverty and malnourishment, reported throughout history in Asia, Europe, South America, and Africa [1,3,4]. It was described by Hippocrates [5] and found in German and Japanese concentration camps during World War II [1,6]. Infection occurs mostly in children, although it has been described in neonates, adults, and the chronically ill [3,7]. The true prevalence and incidence of noma are not fully known, because it is believed that only ,15% of patients with acute cases of the disease seek medical care [1,8]. Noma is a disease of shame, and the condition often results in forced isolation from the community and family; many children are sent to live in isolation rather than being taken to medical care [1,8–10]. In the late 1990s, the incidence of acute childhood noma was placed at 25,600 in countries bordering the Sahara [11], and worldwide at between 100,000 and 140,000 per year, primarily in sub-Saharan Africa and Asia [12]. Peak incidence is among children aged 1–4 [3,4,7]. Worldwide prevalence of those living with the sequelae of noma was placed at 770,000 in 1997 [8].

Background Treatment of Mycobacterium ulcerans disease, or Buruli ulcer (BU), has shifted from surgery to treatment with streptomycin(STR)+rifampin(RIF) since 2004 based on studies in a mouse model and clinical trials. We tested two entirely oral regimens for BU treatment, rifampin(RIF)+clarithromycin(CLR) and rifapentine(RPT)+clarithromycin(CLR) in the mouse model. Methodology/Principal Findings BALB/c mice were infected in the right hind footpad with M. ulcerans strain 1059 and treated daily (5 days/week) for 4 weeks, beginning 11 days after infection. Treatment groups included an untreated control, STR+RIF as a positive control, and test regimens of RIF, RPT, STR and CLR given alone and the RIF+CLR and RPT+CLR combinations. The relative efficacy of the drug treatments was compared on the basis of footpad CFU counts and median time to footpad swelling. Except for CLR, which was bacteriostatic, treatment with all other drugs reduced CFU counts by approximately 2 or 3 log10. Median time to footpad swelling after infection was 5.5, 16, 17, 23.5 and 36.5 weeks in mice receiving no treatment, CLR alone, RIF+CLR, RIF alone, and STR alone, respectively. At the end of follow-up, 39 weeks after infection, only 48%, 26.4% and 16.3% of mice treated with RPT+CLR, RPT alone and STR+RIF had developed swollen footpads. An in vitro checkerboard assay showed the interaction of CLR and RIF to be indifferent. However, in mice, co-administration with CLR resulted in a roughly 25% decrease in the maximal serum concentration (Cmax) and area under the serum concentration-time curve (AUC) of each rifamycin. Delaying the administration of CLR by one hour restored Cmax and AUC values of RIF to levels obtained with RIF alone. Conclusions/Significance These results suggest that an entirely oral daily regimen of RPT+CLR may be at least as effective as the currently recommended combination of injected STR+oral RIF., Author Summary Buruli ulcer (BU) is found throughout the world but is particularly prevalent in West Africa. Until 2004, treatment for this disfiguring disease was surgical excision followed by skin grafting, procedures often requiring months of hospitalization. More recently, an 8-week regimen of oral rifampin and streptomycin administered by injection has become the standard of care recommended by the World Health Organization. However, daily injections require sterile needles and syringes to prevent spread of blood borne pathogens and streptomycin has potentially serious side effects, most notably hearing loss. We tested an entirely oral regimen, substituting the long acting rifapentine for rifampin and clarithromycin for streptomycin. We also evaluated each drug separately. We found that rifapentine alone is as good as rifampin plus streptomycin, but the simultaneous addition of effective clarithromycin doses, at least in the mouse, reduces the activity of both rifampin and rifapentine, making it difficult to assess the efficacy of the oral regimens in the model. Studies of serum drug concentrations indicated that separating treatment times by one hour or reducing the clarithromycin dose to one active in humans should overcome this issue in experimental and clinical BU treatment, respectively.

The International Symposium on Leishmaniasis Vaccines, held in Olinda, Brazil, on March 9–11, 2009, congregated international experts who conduct research on vaccines against the leishmaniases. The questions that were raised during that meeting and the ensuing discussions are compiled in this report and may assist in guiding a research agenda. A group to further discussion on issues raised in this policy platform has been set up at http://groups.google.com/group/leishvaccines-l.

Accurate diagnosis of infection with the parasite Strongyloides stercoralis is hampered by the low concentration of larvae in stool, rendering parasitological diagnosis insensitive. Even if the more sensitive agar plate culture method is used repeated stool sampling is necessary to achieve satisfactory sensitivity. In this manuscript we describe the development of a coproantigen ELISA for diagnosis of infection. Polyclonal rabbit antiserum was raised against Strongyloides ratti excretory/secretory (E/S) antigen and utilized to develop an antigen capture ELISA. The assay enabled detection of subpatent rodent S. ratti and human S. stercoralis infection. No cross-reactivity was observed with purified E/S from Schistosoma japonicum, the hookworms Ancylostoma caninum, A. ceylanicum, nor with fecal samples collected from rodents harboring Trichuris muris or S. mansoni infection. Strongyloides coproantigens that appear stable when frozen as formalin-extracted fecal supernatants stored at −20°C remained positive up to 270 days of storage, whereas supernatants stored at 4°C tested negative. These results indicate that diagnosis of human strongyloidiasis by detection of coproantigen is an approach worthy of further development., Author Summary Strongyloides stercoralis is almost unique among human nematode infections in its ability to replicate within a patient's body, potentially leading to life-long infections if left untreated. Given the potential for severe life threatening Strongyloides infections and the unsatisfactory results of current parasitologic and antibody tests, there is a need for more efficient diagnostic tools. In this study we generated an assay to specifically detect proteins expelled by Strongyloides. Initially this assay for Strongyloides detection was not specific for the parasite; however, after developing a methodology using formaldehyde preservation of feces we specifically detected Strongyloides antigens in rodent and human stool. This methodology was then tested for cross-reactivity with purified proteins from closely related parasites and furthermore for cross-reactivity against faeces collected from animals harbouring single parasitic infections. Using this approach we found no non-specific reactivity with host or to various parasite antigens, suggesting that this assay is truly specific for Strongyloides detection.

Obligate intracellular Apicomplexa parasites share a unique invasion mechanism involving a tight interaction between the host cell and the parasite surfaces called the moving junction (MJ). The MJ, which is the anchoring structure for the invasion process, is formed by secretion of a macromolecular complex (RON2/4/5/8), derived from secretory organelles called rhoptries, into the host cell membrane. AMA1, a protein secreted from micronemes and associated with the parasite surface during invasion, has been shown in vitro to bind the MJ complex through a direct association with RON2. Here we show that RON2 is inserted as an integral membrane protein in the host cell and, using several interaction assays with native or recombinant proteins, we define the region that binds AMA1. Our studies were performed both in Toxoplasma gondii and Plasmodium falciparum and although AMA1 and RON2 proteins have diverged between Apicomplexa species, we show an intra-species conservation of their interaction. More importantly, invasion inhibition assays using recombinant proteins demonstrate that the RON2-AMA1 interaction is crucial for both T. gondii and P. falciparum entry into their host cells. This work provides the first evidence that AMA1 uses the rhoptry neck protein RON2 as a receptor to promote invasion by Apicomplexa parasites., Author Summary Apicomplexa parasites are obligate intracellular pathogens causing severe diseases such as the deadly malaria or toxoplasmosis. Host cell invasion by these parasites involves the formation of a structure between the apex of the parasite and the host cell membrane called the moving junction (MJ), which is built upon collaboration between secretory organelles from the parasite that insert microneme protein AMA1 in the parasite plasma membrane and a complex of four rhoptry neck (RON2/4/5/8) proteins at the host cell plasma membrane. We have now identified a strong interaction between AMA1 and a C-terminal region of RON2, which is crucial for invasion. In spite of sequence variations in both proteins orthologs from distinct Apicomplexa, we could show that this interaction is functionally conserved and equally important for the invasive process by Toxoplasma gondii and Plasmodium falciparum. These findings open the way for therapeutic approaches that could interfere with key functions of parasitic MJ.

Buruli ulcer is a neglected emerging disease that has recently been reported in some countries as the second most frequent mycobacterial disease in humans after tuberculosis. Cases have been reported from at least 32 countries in Africa (mainly west), Australia, Southeast Asia, China, Central and South America, and the Western Pacific. Large lesions often result in scarring, contractual deformities, amputations, and disabilities, and in Africa, most cases of the disease occur in children between the ages of 4–15 years. This environmental mycobacterium, Mycobacterium ulcerans, is found in communities associated with rivers, swamps, wetlands, and human-linked changes in the aquatic environment, particularly those created as a result of environmental disturbance such as deforestation, dam construction, and agriculture. Buruli ulcer disease is often referred to as the “mysterious disease” because the mode of transmission remains unclear, although several hypotheses have been proposed. The above review reveals that various routes of transmission may occur, varying amongst epidemiological setting and geographic region, and that there may be some role for living agents as reservoirs and as vectors of M. ulcerans, in particular aquatic insects, adult mosquitoes or other biting arthropods. We discuss traditional and non-traditional methods for indicting the roles of living agents as biologically significant reservoirs and/or vectors of pathogens, and suggest an intellectual framework for establishing criteria for transmission. The application of these criteria to the transmission of M. ulcerans presents a significant challenge., Author Summary Buruli ulcer (BU) is a serious necrotizing cutaneous infection caused by Mycobacterium ulcerans. It is a neglected emerging disease that has recently been reported in some countries as the second most frequent mycobacterial disease in humans after tuberculosis (TB). Cases have been reported from at least 32 countries in Africa (mainly west), Australia, Southeast Asia, China, Central and South America, and the Western Pacific. BU is a disease found in rural areas located near wetlands (ponds, swamps, marshes, impoundments, backwaters) and slow-moving rivers, especially in areas prone to human-made disturbance and flooding. Despite considerable research on this disease in recent years, the mode of transmission remains unclear, although several hypotheses have been proposed. In this article we review the current state of knowledge on the ecology and transmission of M. ulcerans in Africa and Australia, discuss traditional and non-traditional methods for investigating transmission, and suggest an intellectual framework for establishing criteria for transmission.

Enteric fever, caused by Salmonella enterica serovars Typhi and Paratyphi A (S. Typhi and S. Paratyphi A) remains a major public health problem in many settings. The disease is limited to locations with poor sanitation which facilitates the transmission of the infecting organisms. Efficacious and inexpensive vaccines are available for S. Typhi, yet are not commonly deployed to control the disease. Lack of vaccination is due partly to uncertainty of the disease burden arising from a paucity of epidemiological information in key locations. We have collected and analyzed data from 3,898 cases of blood culture-confirmed enteric fever from Patan Hospital in Lalitpur Sub-Metropolitan City (LSMC), between June 2005 and May 2009. Demographic data was available for a subset of these patients (n = 527) that were resident in LSMC and who were enrolled in trials. We show a considerable burden of enteric fever caused by S. Typhi (2,672; 68.5%) and S. Paratyphi A (1,226; 31.5%) at this Hospital over a four year period, which correlate with seasonal fluctuations in rainfall. We found that local population density was not related to incidence and we identified a focus of infections in the east of LSMC. With data from patients resident in LSMC we found that the median age of those with S. Typhi (16 years) was significantly less than S. Paratyphi A (20 years) and that males aged 15 to 25 were disproportionately infected. Our findings provide a snapshot into the epidemiological patterns of enteric fever in Kathmandu. The uneven distribution of enteric fever patients within the population suggests local variation in risk factors, such as contaminated drinking water. These findings are important for initiating a vaccination scheme and improvements in sanitation. We suggest any such intervention should be implemented throughout the LSMC area.

Background To date schistosomiasis control programmes based on chemotherapy have largely aimed at controlling morbidity in treated individuals rather than at suppressing transmission. In this study, a mathematical modelling approach was used to estimate reductions in the rate of Schistosoma mansoni reinfection following annual mass drug administration (MDA) with praziquantel in Uganda over four years (2003–2006). In doing this we aim to elucidate the benefits of MDA in reducing community transmission. Methods Age-structured models were fitted to a longitudinal cohort followed up across successive rounds of annual treatment for four years (Baseline: 2003, Treatment: 2004–2006; n = 1,764). Instead of modelling contamination, infection and immunity processes separately, these functions were combined in order to estimate a composite force of infection (FOI), i.e., the rate of parasite acquisition by hosts. Results MDA achieved substantial and statistically significant reductions in the FOI following one round of treatment in areas of low baseline infection intensity, and following two rounds in areas with high and medium intensities. In all areas, the FOI remained suppressed following a third round of treatment. Conclusions/Significance This study represents one of the first attempts to monitor reductions in the FOI within a large-scale MDA schistosomiasis morbidity control programme in sub-Saharan Africa. The results indicate that the Schistosomiasis Control Initiative, as a model for other MDA programmes, is likely exerting a significant ancillary impact on reducing transmission within the community, and may provide health benefits to those who do not receive treatment. The results obtained will have implications for evaluating the cost-effectiveness of schistosomiasis control programmes and the design of monitoring and evaluation approaches in general., Author Summary Schistosomiasis is a parasitic disease of enormous public health importance, infecting over 200 million people worldwide, of which the large majority live in sub-Saharan Africa. Control programmes based on the mass treatment of individuals in infected areas with the drug praziquantel have been shown to be successful in reducing the parasite burden and likelihood of developing morbidity in those individuals who receive treatment. Using data from an ongoing intestinal schistosomiasis control programme in Uganda and through the application of a mathematical model, we show that an additional benefit of mass treatment is a decrease in parasite acquisition, via a reduction in the number of transmission stages in the environment. This leads to a lower rate of infection and reinfection of individuals in those areas. We show that this result is valid in areas of differing average infection intensity. The importance of this finding is that this will benefit untreated as well as treated individuals, and will allow a fuller estimation of the benefits of schistosomiasis control programmes.

Background: Detailed analysis of the dynamic interactions among biological, environmental, social, and economic factors that favour the spread of certain diseases is extremely useful for designing effective control strategies. Diseases like tuberculosis that kills somebody every 15 seconds in the world, require methods that take into account the disease dynamics to design truly efficient control and surveillance strategies. The usual and well established statistical approaches provide insights into the cause-effect relationships that favour disease transmission but they only estimate risk areas, spatial or temporal trends. Here we introduce a novel approach that allows figuring out the dynamical behaviour of the disease spreading. This information can subsequently be used to validate mathematical models of the dissemination process from which the underlying mechanisms that are responsible for this spreading could be inferred. Methodology/Principal Findings: The method presented here is based on the analysis of the spread of tuberculosis in a Brazilian endemic city during five consecutive years. The detailed analysis of the spatio-temporal correlation of the yearly geo-referenced data, using different characteristic times of the disease evolution, allowed us to trace the temporal path of the aetiological agent, to locate the sources of infection, and to characterize the dynamics of disease spreading. Consequently, the method also allowed for the identification of socio-economic factors that influence the process. Conclusions/Significance: The information obtained can contribute to more effective budget allocation, drug distribution and recruitment of human skilled resources, as well as guiding the design of vaccination programs. We propose that this novel strategy can also be applied to the evaluation of other diseases as well as other social processes. © 2010 Zorzenon dos Santos et al. Fil: Zorzenon dos Santos, Rita M.. Universidade Federal de Pernambuco; Brasil Fil: Amador, Ana. Universidade Federal de Pernambuco; Brasil. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; Argentina Fil: de Souza, Wayner V.. Fundación Oswaldo Cruz; Brasil Fil: de Albuquerque, Maria Fatima P. M.. Universidade Federal de Pernambuco; Brasil. Fundación Oswaldo Cruz; Brasil Fil: Ponce Dawson, Silvina Martha. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; Argentina Fil: Ruffino-Netto, Antonio. Universidade de Sao Paulo; Brasil Fil: Zárate-Bladés, Carlos R.. Universidade de Sao Paulo; Brasil Fil: Silva, Celio L.. Universidade de Sao Paulo; Brasil

The fungal pathogen Cryptococcus neoformans is a major cause of illness in immunocompromised individuals such as AIDS patients. The ability of the fungus to acquire nutrients during proliferation in host tissue and the ability to elaborate a polysaccharide capsule are critical determinants of disease outcome. We previously showed that the GATA factor, Cir1, is a major regulator both of the iron uptake functions needed for growth in host tissue and the key virulence factors such as capsule, melanin and growth at 37°C. We are interested in further defining the mechanisms of iron acquisition from inorganic and host-derived iron sources with the goal of understanding the nutritional adaptation of C. neoformans to the host environment. In this study, we investigated the roles of the HAP3 and HAPX genes in iron utilization and virulence. As in other fungi, the C. neoformans Hap proteins negatively influence the expression of genes encoding respiratory and TCA cycle functions under low-iron conditions. However, we also found that HapX plays both positive and negative roles in the regulation of gene expression, including a positive regulatory role in siderophore transporter expression. In addition, HapX also positively regulated the expression of the CIR1 transcript. This situation is in contrast to the negative regulation by HapX of genes encoding GATA iron regulatory factors in Aspergillus nidulans and Schizosaccharomyces pombe. Although both hapX and hap3 mutants were defective in heme utilization in culture, only HapX made a contribution to virulence, and loss of HapX in a strain lacking the high-affinity iron uptake system did not cause further attenuation of disease. Therefore, HapX appears to have a minimal role during infection of mammalian hosts and instead may be an important regulator of environmental iron uptake functions. Overall, these results indicated that C. neoformans employs multiple strategies for iron acquisition during infection., Author Summary Cryptococcus neoformans causes life-threatening central nervous system infections in immunocompromised people such as AIDS patients. The competition for iron between pathogens such as C. neoformans and mammalian hosts is a key aspect of disease outcome. We previously identified and characterized the major iron regulatory protein Cir1 in C. neoformans, as well as proteins for the transport of iron-binding molecules (siderophores) and for high-affinity iron uptake. In this study, we examined the roles of additional regulatory proteins (Hap proteins) in the response to low-iron conditions and the use of host iron sources such as heme and transferrin. We discovered that the HapX protein has a conserved regulatory function to repress iron-dependent functions during iron deprivation, as well as a positive regulatory role for the expression of putative siderophore transporters. A hapX mutant was defective in the use of heme as an iron source in culture but was only modestly attenuated for virulence in mice. This result suggests that additional mechanisms for iron uptake must be available to support C. neoformans proliferation in the host, and that HapX may play an important role in environmental iron acquisition.

Background Molecular diagnostic methods can complement existing tools to improve the diagnosis of malaria. However, they require good laboratory infrastructure thereby restricting their use to reference laboratories and research studies. Therefore, adopting molecular tools for routine use in malaria endemic countries will require simpler molecular platforms. The recently developed loop-mediated isothermal amplification (LAMP) method is relatively simple and can be improved for better use in endemic countries. In this study, we attempted to improve this method for malaria diagnosis by using a simple and portable device capable of performing both the amplification and detection (by fluorescence) of LAMP in one platform. We refer to this as the RealAmp method. Methodology and Significant Findings Published genus-specific primers were used to test the utility of this method. DNA derived from different species of malaria parasites was used for the initial characterization. Clinical samples of P. falciparum were used to determine the sensitivity and specificity of this system compared to microscopy and a nested PCR method. Additionally, directly boiled parasite preparations were compared with a conventional DNA isolation method. The RealAmp method was found to be simple and allowed real-time detection of DNA amplification. The time to amplification varied but was generally less than 60 minutes. All human-infecting Plasmodium species were detected. The sensitivity and specificity of RealAmp in detecting P. falciparum was 96.7% and 91.7% respectively, compared to microscopy and 98.9% and 100% respectively, compared to a standard nested PCR method. In addition, this method consistently detected P. falciparum from directly boiled blood samples. Conclusion This RealAmp method has great potential as a field usable molecular tool for diagnosis of malaria. This tool can provide an alternative to conventional PCR based diagnostic methods for field use in clinical and operational programs.

Background: Pneumonia is the major cause of mortality and morbidity in children worldwide. Procalcitonin (PCT) and C-reactive protein (CRP) are used in developed countries to differentiate between viral and bacterial causes of pneumonia. Validity of these markers needs to be further explored in Africa. Methodology and Principal Findings: We assessed the utility of PCT and CRP to differentiate viral from invasive bacterial pneumonia in children

Mycobacterium africanum consists of two phylogenetically distinct lineages within the Mycobacterium tuberculosis complex, known as M. africanum West African 1 and M. africanum West African 2. These lineages are restricted to West Africa, where they cause up to half of human pulmonary tuberculosis. In this review we discuss the definition of M. africanum, describe the prevalence and restricted geographical distribution of M. africanum West African 1 and 2, review the occurrence of M. africanum in animals, and summarize the phenotypic differences described thus far between M. africanum and M. tuberculosis sensu stricto.

Understanding the role of humans in the dispersal of predominately animal pathogens is essential for their control. We used newly developed Bayesian phylogeographic methods to unravel the dynamics and determinants of the spread of dog rabies virus (RABV) in North Africa. Each of the countries studied exhibited largely disconnected spatial dynamics with major geo-political boundaries acting as barriers to gene flow. Road distances proved to be better predictors of the movement of dog RABV than accessibility or raw geographical distance, with occasional long distance and rapid spread within each of these countries. Using simulations that bridge phylodynamics and spatial epidemiology, we demonstrate that the contemporary viral distribution extends beyond that expected for RABV transmission in African dog populations. These results are strongly supportive of human-mediated dispersal, and demonstrate how an integrated phylogeographic approach will turn viral genetic data into a powerful asset for characterizing, predicting, and potentially controlling the spatial spread of pathogens., Author Summary At least 15 million doses of anti-rabies post-exposure prophylaxis are administered annually worldwide, and an estimated 55,000 people die of rabies every year. Over 99% of these deaths occur in developing countries, predominantly in Asia and in Africa where rabies is endemic in domestic dogs. Despite the global health burden due to rabies, little is known about the patterns of the spread of dog rabies in these endemic regions. We used recently developed Bayesian analytical methods to unravel the dynamics and determinants of the spatial diffusion of dog rabies viruses in North Africa based on viral genetic data. Our analysis reveals a combination of restricted spread across administrative borders, the occasional long-distance movement of rabies viruses, and a strong fit between spatial spread of the virus and road distances between localities. Together, these data indicate that by transporting dogs, humans have played a key role in the dispersal of a major animal pathogen. Our studies therefore provide essential new information on the transmission dynamics of rabies in Africa, and in doing so will greatly assist in future intervention strategies.

Background Cerebral malaria (CM) is a neurological syndrome that includes coma and seizures following malaria parasite infection. The pathophysiology is not fully understood and cannot be accounted for by infection alone: patients still succumb to CM, even if the underlying parasite infection has resolved. To that effect, there is no known adjuvant therapy for CM. Current murine CM (MCM) models do not allow for rapid clinical identification of affected animals following infection. An animal model that more closely mimics the clinical features of human CM would be helpful in elucidating potential mechanisms of disease pathogenesis and evaluating new adjuvant therapies. Methodology/Principal Findings A quantitative, rapid murine coma and behavior scale (RMCBS) comprised of 10 parameters was developed to assess MCM manifested in C57BL/6 mice infected with Plasmodium berghei ANKA (PbA). Using this method a single mouse can be completely assessed within 3 minutes. The RMCBS enables the operator to follow the evolution of the clinical syndrome, validated here by correlations with intracerebral hemorrhages. It provides a tool by which subjects can be identified as symptomatic prior to the initiation of trial treatment. Conclusions/Significance Since the RMCBS enables an operator to rapidly follow the course of disease, label a subject as affected or not, and correlate the level of illness with neuropathologic injury, it can ultimately be used to guide the initiation of treatment after the onset of cerebral disease (thus emulating the situation in the field). The RMCBS is a tool by which an adjuvant therapy can be objectively assessed.

Background Nontyphoidal Salmonellae frequently cause life-threatening bacteremia in sub-Saharan Africa. Young children and HIV-infected adults are particularly susceptible. High case-fatality rates and increasing antibiotic resistance require new approaches to the management of this disease. Impaired cellular immunity caused by defects in the T helper 1 pathway lead to intracellular disease with Salmonella that can be countered by IFNγ administration. This report identifies the lymphocyte subsets that produce IFNγ early in Salmonella infection. Methodology Intracellular cytokine staining was used to identify IFNγ production in blood lymphocyte subsets of ten healthy adults with antibodies to Salmonella (as evidence of immunity to Salmonella), in response to stimulation with live and heat-killed preparations of the D23580 invasive African isolate of Salmonella Typhimurium. The absolute number of IFNγ-producing cells in innate, innate-like and adaptive lymphocyte subpopulations was determined. Principal Findings Early IFNγ production was found in the innate/innate-like lymphocyte subsets: γδ-T cells, NK cells and NK-like T cells. Significantly higher percentages of such cells produced IFNγ compared to adaptive αβ-T cells (Student's t test, P

Mosquitoes rely on RNA interference (RNAi) as their primary defense against viral infections. To this end, the combination of RNAi and invertebrate cell culture systems has become an invaluable tool in studying virus-vector interactions. Nevertheless, a recent study failed to detect an active RNAi response to West Nile virus (WNV) infection in C6/36 (Aedes albopictus) cells, a mosquito cell line frequently used to study arthropod-borne viruses (arboviruses). Therefore, we sought to determine if WNV actively evades the host's RNAi response or if C6/36 cells have a dysfunctional RNAi pathway. C6/36 and Drosophila melanogaster S2 cells were infected with WNV (Flaviviridae), Sindbis virus (SINV, Togaviridae) and La Crosse virus (LACV, Bunyaviridae) and total RNA recovered from cell lysates. Small RNA (sRNA) libraries were constructed and subjected to high-throughput sequencing. In S2 cells, virus-derived small interfering RNAs (viRNAs) from all three viruses were predominantly 21 nt in length, a hallmark of the RNAi pathway. However, in C6/36 cells, viRNAs were primarily 17 nt in length from WNV infected cells and 26–27 nt in length in SINV and LACV infected cells. Furthermore, the origin (positive or negative viral strand) and distribution (position along viral genome) of S2 cell generated viRNA populations was consistent with previously published studies, but the profile of sRNAs isolated from C6/36 cells was altered. In total, these results suggest that C6/36 cells lack a functional antiviral RNAi response. These findings are analogous to the type-I interferon deficiency described in Vero (African green monkey kidney) cells and suggest that C6/36 cells may fail to accurately model mosquito-arbovirus interactions at the molecular level., Author Summary Cell culture systems are invaluable tools for studying virus-host interactions. These systems are typically easy to maintain and manipulate; however, they can fail to accurately mimic the host environment encountered by viruses. Therefore, defining the limitations of each system is critical to properly interpreting the results. C6/36 Aedes albopictus cells are commonly used to study arthropod-borne viruses (arboviruses), such as West Nile virus (WNV). Recent evidence suggests that the RNA interference (RNAi) pathway, a critical aspect of the cellular innate antiviral immune response in invertebrates, may not actively target WNV in C6/36 cells. However, it is unknown whether this observation is limited to WNV. Therefore, we examined small RNA populations from C6/36 and Drosophila melanogastor S2 cells infected with WNV, Sindbis virus and La Crosse virus by high-throughput sequencing. We demonstrate that the RNAi pathway actively targets each of the three viruses in S2 cells, but does not in C6/36 cells. These findings suggest that C6/36 cells may fail to accurately model mosquito-arbovirus interactions.

A majority of the Plasmodium falciparum genome codes for genes with unknown functions, which presents a major challenge to understanding the parasite's biology. Large-scale functional analysis of the parasite genome is essential to pave the way for novel therapeutic intervention strategies against the disease and yet difficulties in genetic manipulation of this deadly human malaria parasite have been a major hindrance for functional analysis of its genome. Here, we used a forward functional genomic approach to study P. falciparum and identify genes important for optimal parasite development in the disease-causing, intraerythrocytic stages. We analyzed 123 piggyBac insertion mutants of P. falciparum for proliferation efficiency in the intraerythrocytic stages, in vitro. Almost 50% of the analyzed mutants showed significant reduction in proliferation efficiency, with 20% displaying severe defects. Functional categorization of genes in the severely attenuated mutants revealed significant enrichment for RNA binding proteins, suggesting the significance of post-transcriptional gene regulation in parasite development and emphasizing its importance as an antimalarial target. This study demonstrates the feasibility of much needed forward genetics approaches for P. falciparum to better characterize its genome and accelerate drug and vaccine development.

Background The possible emergence of resistance to the only available drug for schistosomiasis spurs drug discovery that has been recently incentivized by the availability of improved transcriptome and genome sequence information. Transient RNAi has emerged as a straightforward and important technique to interrogate that information through decreased or loss of gene function and identify potential drug targets. To date, RNAi studies in schistosome stages infecting humans have focused on single (or up to 3) genes of interest. Therefore, in the context of standardizing larger RNAi screens, data are limited on the extent of possible off-targeting effects, gene-to-gene variability in RNAi efficiency and the operational capabilities and limits of RNAi. Methodology/Principal Findings We investigated in vitro the sensitivity and selectivity of RNAi using double-stranded (ds)RNA (approximately 500 bp) designed to target 11 Schistosoma mansoni genes that are expressed in different tissues; the gut, tegument and otherwise. Among the genes investigated were 5 that had been previously predicted to be essential for parasite survival. We employed mechanically transformed schistosomula that are relevant to parasitism in humans, amenable to screen automation and easier to obtain in greater numbers than adult parasites. The operational parameters investigated included defined culture media for optimal parasite maintenance, transfection strategy, time- and dose- dependency of RNAi, and dosing limits. Of 7 defined culture media tested, Basch Medium 169 was optimal for parasite maintenance. RNAi was best achieved by co-incubating parasites and dsRNA (standardized to 30 µg/ml for 6 days); electroporation provided no added benefit. RNAi, including interference of more than one transcript, was selective to the gene target(s) within the pools of transcripts representative of each tissue. Concentrations of dsRNA above 90 µg/ml were directly toxic. RNAi efficiency was transcript-dependent (from 40 to >75% knockdown relative to controls) and this may have contributed to the lack of obvious phenotypes observed, even after prolonged incubations of 3 weeks. Within minutes of their mechanical preparation from cercariae, schistosomula accumulated fluorescent macromolecules in the gut indicating that the gut is an important route through which RNAi is expedited in the developing parasite. Conclusions Transient RNAi operates gene-selectively in S. mansoni newly transformed schistosomula yet the sensitivity of individual gene targets varies. These findings and the operational parameters defined will facilitate larger RNAi screens., Author Summary RNA interference (RNAi) is a technique to selectively suppress mRNA of individual genes and, consequently, their cognate proteins. RNAi using double-stranded (ds) RNA has been used to interrogate the function of mainly single genes in the flatworm, Schistosoma mansoni, one of a number of schistosome species causing schistosomiasis. In consideration of large-scale screens to identify candidate drug targets, we examined the selectivity and sensitivity (the degree of suppression) of RNAi for 11 genes produced in different tissues of the parasite: the gut, tegument (surface) and otherwise. We used the schistosomulum stage prepared from infective cercariae larvae which are accessible in large numbers and adaptable to automated screening platforms. We found that RNAi suppresses transcripts selectively, however, the sensitivity of suppression varies (40%–>75%). No obvious changes in the parasite occurred post-RNAi, including after targeting the mRNA of genes that had been computationally predicted to be essential for survival. Additionally, we defined operational parameters to facilitate large-scale RNAi, including choice of culture medium, transfection strategy to deliver dsRNA, dose- and time-dependency, and dosing limits. Finally, using fluorescent probes, we show that the developing gut allows rapid entrance of dsRNA into the parasite to initiate RNAi.

Background The binding of Leishmania promastigotes to the midgut epithelium is regarded as an essential part of the life-cycle in the sand fly vector, enabling the parasites to persist beyond the initial blood meal phase and establish the infection. However, the precise nature of the promastigote stage(s) that mediate binding is not fully understood. Methodology/Principal Findings To address this issue we have developed an in vitro gut binding assay in which two promastigote populations are labelled with different fluorescent dyes and compete for binding to dissected sand fly midguts. Binding of procyclic, nectomonad, leptomonad and metacyclic promastigotes of Leishmania infantum and L. mexicana to the midguts of blood-fed, female Lutzomyia longipalpis was investigated. The results show that procyclic and metacyclic promastigotes do not bind to the midgut epithelium in significant numbers, whereas nectomonad and leptomonad promastigotes both bind strongly and in similar numbers. The assay was then used to compare the binding of a range of different parasite species (L. infantum, L. mexicana, L. braziliensis, L. major, L. tropica) to guts dissected from various sand flies (Lu. longipalpis, Phlebotomus papatasi, P. sergenti). The results of these comparisons were in many cases in line with expectations, the natural parasite binding most effectively to its natural vector, and no examples were found where a parasite was unable to bind to its natural vector. However, there were interesting exceptions: L. major and L. tropica being able to bind to Lu. longipalpis better than L. infantum; L. braziliensis was able to bind to P. papatasi as well as L. major; and significant binding of L. major to P. sergenti and L. tropica to P. papatasi was observed. Conclusions/Significance The results demonstrate that Leishmania gut binding is strictly stage-dependent, is a property of those forms found in the middle phase of development (nectomonad and leptomonad forms), but is absent in the early blood meal and final stages (procyclic and metacyclic forms). Further they show that although gut binding may be necessary for parasite establishment, in several vector-parasite pairs the specificity of such in vitro binding alone is insufficient to explain overall vector specificity. Other significant barriers to development must exist in certain refractory Leishmania parasite-sand fly vector combinations. A re-appraisal of the specificity of the Leishmania-sand fly relationship is required., Author Summary Many infectious diseases such as leishmaniasis are transmitted to people by biting insects, in this case by female sand flies. To control this and similar diseases we need to understand why particular species of sand fly transmit particular species of Leishmania. One important feature of the Leishmania parasite-sand fly interaction is the ability of the parasite to bind to the midgut wall of the fly, as it is within the gut that the parasite lives. Here we have studied the specificity of this interaction and report two main findings. The first is that only specific stages in the parasite life-cycle are capable of binding to the gut. The second is that, providing these life-cycle stages are analysed, parasite species that can be transmitted by particular sand flies are always capable of binding to their guts, but in some cases they are also capable of binding to non-transmitting sand fly species. This shows that gut binding by parasites is necessary but not sufficient to explain transmission. This research advances our understanding of Leishmania biology, but also shows us that there are further aspects that need to be investigated before we can fully understand the Leishmania-sand fly relationship.

The cycle inhibiting factors (Cif), produced by pathogenic bacteria isolated from vertebrates and invertebrates, belong to a family of molecules called cyclomodulins that interfere with the eukaryotic cell cycle. Cif blocks the cell cycle at both the G1/S and G2/M transitions by inducing the stabilization of cyclin-dependent kinase inhibitors p21waf1 and p27kip1. Using yeast two-hybrid screens, we identified the ubiquitin-like protein NEDD8 as a target of Cif. Cif co-compartmentalized with NEDD8 in the host cell nucleus and induced accumulation of NEDD8-conjugated cullins. This accumulation occurred early after cell infection and correlated with that of p21 and p27. Co-immunoprecipitation revealed that Cif interacted with cullin-RING ubiquitin ligase complexes (CRLs) through binding with the neddylated forms of cullins 1, 2, 3, 4A and 4B subunits of CRL. Using an in vitro ubiquitylation assay, we demonstrate that Cif directly inhibits the neddylated CUL1-associated ubiquitin ligase activity. Consistent with this inhibition and the interaction of Cif with several neddylated cullins, we further observed that Cif modulates the cellular half-lives of various CRL targets, which might contribute to the pathogenic potential of diverse bacteria., Author Summary Among the arsenal of virulence factors used by bacterial pathogens to infect and manipulate their hosts, cyclomodulins are a growing family of bacterial toxins that interfere with the eukaryotic cell-cycle. Cif is one of these cyclomodulins produced by both mammalian and invertebrate pathogenic bacteria. Cif blocks the host cell cycle by inducing the accumulation of two regulators of cell cycle progression: the cyclin-dependent kinase inhibitors p21 and p27. To decipher the mode of action of Cif, we performed yeast two-hybrid screenings. We show that Cif binds to NEDD8 and induce accumulation of neddylated cullins early after infection. Cullins are scaffold components of cullin-RING ubiquitin ligases (CRLs), which ubiquitinate proteins and target them for degradation by the 26S proteasome. We demonstrate that Cif directly inhibits the ubiquitin ligase activity of these CRLs and consequently the targeting of p21 and p27 for ubiquitin-dependent degradation. Targeting at NEDD8 represents a novel strategy for modulation of host cell functions by bacterial pathogens. By inhibiting the most prominent class of ubiquitin-ligases, Cif controls the stability of a cohort of key regulators and impinge on not only cell cycle progression but also on many cellular and biological processes such as immunity, development, transcription, and cell signaling.

Infection by Leptospira interrogans has been causally associated with human and equine uveitis. Studies in our laboratories have demonstrated that leptospiral lipoprotein LruA and LruB are expressed in the eyes of uveitic horses, and that antibodies directed against LruA and LruB react with equine lenticular and retinal extracts, respectively. These reactivities were investigated further by performing immunofluorescent assays on lenticular and retinal tissue sections. Incubation of lens tissue sections with LruA-antiserum and retinal sections with LruB-antiserum resulted in positive fluorescence. By employing two-dimensional gel analyses followed by immunoblotting and mass spectrometry, lens proteins cross-reacting with LruA antiserum were identified to be α-crystallin B and vimentin. Similarly, mass spectrometric analyses identified β-crystallin B2 as the retinal protein cross-reacting with LruB-antiserum. Purified recombinant human α-crystallin B and vimentin were recognized by LruA-directed antiserum, but not by control pre-immune serum. Recombinant β-crystallin B2 was likewise recognized by LruB-directed antiserum, but not by pre-immune serum. Moreover, uveitic eye fluids contained significantly higher levels of antiibodies that recognized α-crystallin B, β-crystallin B2 and vimentin than did normal eye fluids. Our results indicate that LruA and LruB share immuno-relevant epitopes with eye proteins, suggesting that cross-reactive antibody interactions with eye antigens may contribute to immunopathogenesis of Leptospira-associated recurrent uveitis., Author Summary Leptospira is the most common infectious cause of uveitis, a potentially debilitating inflammation of the eye. In our earlier work, we discovered that eye fluids of uveitic horses contain high levels of antibodies directed against novel leptospiral proteins, which we named LruA and LruB (Leptospiral recurrent uveitis associated proteins A and B). Significantly, antibodies raised against LruA and LruB also recognize lens and retinal tissue. We have now identified the cross-reactive eye proteins as alpha-crystallin B, vimentin and beta-crystallin B2. We also demonstrated that ocular fluids from uveitic horses contain high levels of antibodies recognizing alpha-crystallin B, vimentin and beta-crystallin B2. These data suggest that antibodies directed against leptospiral LruA and LruB during infection can also react with eye proteins, alpha-crystallin B, vimentin and beta-crystallin B2, potentially contributing to the severity of this eye disease.

Background There is currently no licensed antiviral drug for treatment of dengue. Chloroquine (CQ) inhibits the replication of dengue virus (DENV) in vitro. Methods and Findings A double-blind, randomized, placebo-controlled trial of CQ in 307 adults hospitalized for suspected DENV infection was conducted at the Hospital for Tropical Diseases (Ho Chi Minh City, Vietnam) between May 2007 and July 2008. Patients with illness histories of 72 hours or less were randomized to a 3-day course of CQ (n = 153) or placebo (n = 154). Laboratory-confirmation of DENV infection was made in 257 (84%) patients. The primary endpoints were time to resolution of DENV viraemia and time to resolution of DENV NS1 antigenaemia. In patients treated with CQ there was a trend toward a longer duration of DENV viraemia (hazard ratio (HR) = 0.80, 95% CI 0.62–1.05), but we did not find any difference for the time to resolution of NS1 antigenaemia (HR = 1.07, 95% CI 0.76–1.51). Interestingly, CQ was associated with a significant reduction in fever clearance time in the intention-to-treat population (HR = 1.37, 95% CI 1.08–1.74) but not in the per-protocol population. There was also a trend towards a lower incidence of dengue hemorrhagic fever (odds ratio = 0.60, PP 95% CI 0.34–1.04) in patients treated with CQ. Differences in levels of T cell activation or pro- or anti-inflammatory plasma cytokine concentrations between CQ- and placebo-treated patients did not explain the trend towards less dengue hemorrhagic fever in the CQ arm. CQ was associated with significantly more adverse events, primarily vomiting. Conclusions CQ does not reduce the durations of viraemia and NS1 antigenaemia in dengue patients. Further trials, with appropriate endpoints, would be required to determine if CQ treatment has any clinical benefit in dengue. Trial Registration Current Controlled Trials number ISRCTN38002730., Author Summary There is no available drug or vaccine against dengue, an acute viral disease that affects ∼50 million people annually in tropical and sub-tropical countries. Chloroquine (CQ), a cheap and well-tolerated drug, inhibits the growth of dengue viruses in the laboratory with concentrations achievable in the body. To measure the antiviral efficacy of CQ in dengue, we conducted a study involving 307 adults with suspected dengue. Patients received a 3-day oral dosage of placebo or CQ early in their illness. Unfortunately, we did not see an effect of CQ on the duration of viral infection. We did, however, observe that CQ had a modest anti-fever effect. In patients treated with CQ, we observed a trend towards a lower incidence of dengue hemorrhagic fever, a severe form of dengue. We did not find any differences in the immune response that can explain this trend. We also found more adverse events, primarily vomiting, with CQ. This trial provides valuable new information on how to perform trials of antiviral drugs for dengue.

Background Travelers who acquire dengue infection are often routes for virus transmission to other regions. Nevertheless, the interplay between infected travelers, climate, vectors, and indigenous dengue incidence remains unclear. The role of foreign-origin cases on local dengue epidemics thus has been largely neglected by research. This study investigated the effect of both imported dengue and local meteorological factors on the occurrence of indigenous dengue in Taiwan. Methods and Principal Findings Using logistic and Poisson regression models, we analyzed bi-weekly, laboratory-confirmed dengue cases at their onset dates of illness from 1998 to 2007 to identify correlations between indigenous dengue and imported dengue cases (in the context of local meteorological factors) across different time lags. Our results revealed that the occurrence of indigenous dengue was significantly correlated with temporally-lagged cases of imported dengue (2–14 weeks), higher temperatures (6–14 weeks), and lower relative humidity (6–20 weeks). In addition, imported and indigenous dengue cases had a significant quantitative relationship in the onset of local epidemics. However, this relationship became less significant once indigenous epidemics progressed past the initial stage. Conclusions These findings imply that imported dengue cases are able to initiate indigenous epidemics when appropriate weather conditions are present. Early detection and case management of imported cases through rapid diagnosis may avert large-scale epidemics of dengue/dengue hemorrhagic fever. The deployment of an early-warning surveillance system, with the capacity to integrate meteorological data, will be an invaluable tool for successful prevention and control of dengue, particularly in non-endemic countries., Author Summary Dengue/dengue hemorrhagic fever is the world's most widely spread mosquito-borne arboviral disease and threatens more than two-thirds of the world's population. Cases are mainly distributed in tropical and subtropical areas in accordance with vector habitats for Aedes aegypti and Ae. albopictus. However, the role of imported cases and favorable meteorological conditions has not yet been quantitatively assessed. This study verified the correlation between the occurrence of indigenous dengue and imported cases in the context of weather variables (temperature, rainfall, relative humidity, etc.) for different time lags in southern Taiwan. Our findings imply that imported cases have a role in igniting indigenous outbreaks, in non-endemics areas, when favorable weather conditions are present. This relationship becomes insignificant in the late phase of local dengue epidemics. Therefore, early detection and case management of imported cases through timely surveillance and rapid laboratory-diagnosis may avert large scale epidemics of dengue/dengue hemorrhagic fever. An early-warning surveillance system integrating meteorological data will be an invaluable tool for successful prevention and control of dengue, particularly in non-endemic countries.

Ebolaviruses (EBOV) (family Filoviridae) cause viral hemorrhagic fevers in humans and non-human primates when they spill over from their wildlife reservoir hosts with case fatality rates of up to 90%. Fruit bats may act as reservoirs of the Filoviridae. The migratory fruit bat, Eidolon helvum, is common across sub-Saharan Africa and lives in large colonies, often situated in cities. We screened sera from 262 E. helvum using indirect fluorescent tests for antibodies against EBOV subtype Zaire. We detected a seropositive bat from Accra, Ghana, and confirmed this using western blot analysis. The bat was also seropositive for Lagos bat virus, a Lyssavirus, by virus neutralization test. The bat was fitted with a radio transmitter and was last detected in Accra 13 months after release post-sampling, demonstrating long-term survival. Antibodies to filoviruses have not been previously demonstrated in E. helvum. Radio-telemetry data demonstrates long-term survival of an individual bat following exposure to viruses of families that can be highly pathogenic to other mammal species. Because E. helvum typically lives in large urban colonies and is a source of bushmeat in some regions, further studies should determine if this species forms a reservoir for EBOV from which spillover infections into the human population may occur.

Background: Association studies are a promising way to uncover the genetic basis of complex traits in wild populations. Data on population stratification, linkage disequilibrium and distribution of variant effect-sizes for different trait-types are required to predict study success but are lacking for most taxa. We quantified and investigated the impacts of these key variables in a large-scale association study of a strongly selected trait of medical importance: pyrethroid resistance in the African malaria vector Anopheles gambiae.\ud \ud Methodology/Principal Findings: We genotyped

The transmission dynamics of Schistosoma japonicum remain poorly understood, as over forty species of mammals are suspected of serving as reservoir hosts. However, knowledge of the population genetic structure and of the full-sibship structuring of parasites at two larval stages will be useful in defining and tracking the transmission pattern between intermediate and definitive hosts. S. japonicum larvae were therefore collected in three marshland and three hilly villages in Anhui Province of China across three time points: April and September-October 2006, and April 2007, and then genotyped with six microsatellite markers. Results from the population genetic and sibling relationship analyses of the parasites across two larval stages demonstrated that, within the marshland, parasites from cattle showed higher genetic diversity than from other species; whereas within the hilly region, parasites from dogs and humans displayed higher genetic diversity than those from rodents. Both the extent of gene flow and the estimated proportion of full-sib relationships of parasites between two larval stages indicated that the cercariae identified within intermediate hosts in the marshlands mostly came from cattle, whereas in the hilly areas, they were varied between villages, coming primarily from rodents, dogs or humans. Such results suggest a different transmission process within the hilly region from within the marshlands. Moreover, this is the first time that the sibling relationship analysis was applied to the transmission dynamics for S. japonicum., Author Summary Schistosoma japonicum involves two obligatory host stages, with asexual reproduction within a molluscan host and sexual reproduction within a mammalian host. Having over 40 species of mammals suspected of being potential reservoirs complicates the transmission patterns. Understanding the complex transmission patterns is further hampered by the ethical and logistical difficulty in sampling adult worms from mammalian hosts. However, the two free-swimming larval stages, cercariae (released from a mollusc and then infective to a mammal) and miracidia (hatched from eggs passed in a mammal's faeces, and then infective to a mollusc), are available, and elucidating the genetic composition of parasites at theses two stages could provide information of infection processes. Here we sampled cercariae during April 2006, miracidia during September-October 2006, and cercariae during April 2007 in three marshland and three hilly villages in Anhui Province of China, and, using microsatellite markers, analyzed the population genetic structure and, for the first time, the familial relationships of parasites at different stages. We found contrasting population structures of parasites, and host species-associated diversities and transmission patterns of parasites between and within two regions. Moreover, we demonstrate that the successful application of sibship analyses to infection process provides an alternative approach to the dissection of transmission dynamics.

Background Combination therapies are now recommended to treat uncomplicated malaria. We used a longitudinal design to assess the incidence of malaria and compare the efficacies of 3 combination regimens in Kampala, Uganda. Methodology/Principal Findings Children aged 1–10 years were enrolled from randomly selected households in 2004–05 and 2007, and were followed at least monthly through 2008. Insecticide-treated bednets (ITNs) were provided in 2006. Children were randomized upon their first episode, and then treated for all episodes of uncomplicated malaria with amodiaquine/sulfadoxine-pyrimethamine (AQ/SP), artesunate/amodiaquine (AS/AQ), or artemether/lumefantrine (AL). Risks of parasitological failure were determined for each episode of uncomplicated malaria and clinical parameters were followed. A total of 690 children experienced 1464 episodes of malaria. 96% of these episodes were uncomplicated malaria and treated with study drugs; 94% were due to Plasmodium falciparum. The rank order of treatment efficacy was AL > AS/AQ > AQ/SP. Failure rates increased over time for AQ/SP, but not the artemisinin-based regimens. Over the 4-year course of the study the prevalence of asymptomatic parasitemia decreased from 11.8% to 1.4%, the incidence of malaria decreased from 1.55 to 0.32 per person year, and the prevalence of anemia (hemoglobin

Cerebral malaria, a major cause of death during malaria infection, is characterised by the sequestration of infected red blood cells (IRBC) in brain microvessels. Most of the molecules implicated in the adhesion of IRBC on endothelial cells (EC) are already described; however, the structure of the IRBC/EC junction and the impact of this adhesion on the EC are poorly understood. We analysed this interaction using human brain microvascular EC monolayers co-cultured with IRBC. Our study demonstrates the transfer of material from the IRBC to the brain EC plasma membrane in a trogocytosis-like process, followed by a TNF-enhanced IRBC engulfing process. Upon IRBC/EC binding, parasite antigens are transferred to early endosomes in the EC, in a cytoskeleton-dependent process. This is associated with the opening of the intercellular junctions. The transfer of IRBC antigens can thus transform EC into a target for the immune response and contribute to the profound EC alterations, including peri-vascular oedema, associated with cerebral malaria., Author Summary Cerebral malaria, a major cause of death during malaria infection, is characterised by the sequestration of infected red blood cells (IRBC) in brain microvessels. This study describes the interactions between plasmodium infected red blood cell and human brain endothelial cells. It highlights the activation of a trogocytosis-like mechanism followed by an engulfment of the infected red blood cells by endothelial cells (EC). This transfer concerns up to 20% of the IRBC cocultured with EC. This means that the parasite infected erythrocyte can mimic the leukocytes interaction with endothelial cells. This process is associated with i) a transfer of malaria antigens to the EC which can inappropriately activate the immune system and ii) an opening of the intercellular junctions, which can trigger blood-brain-barrier leakage during cerebral malaria. This transfer of IRBC antigens can thus transform EC into a target for the immune response and contribute to cerebral malaria pathogenesis.

Malaria, caused by the apicomplexan parasite Plasmodium, still represents a major threat to human health and welfare and leads to about one million human deaths annually. Plasmodium is a rapidly multiplying unicellular organism undergoing a complex developmental cycle in man and mosquito – a life style that requires rapid adaptation to various environments. In order to deal with high fluxes of reactive oxygen species and maintain redox regulatory processes and pathogenicity, Plasmodium depends upon an adequate redox balance. By systematically studying the subcellular localization of the major antioxidant and redox regulatory proteins, we obtained the first complete map of redox compartmentation in Plasmodium falciparum. We demonstrate the targeting of two plasmodial peroxiredoxins and a putative glyoxalase system to the apicoplast, a non-photosynthetic plastid. We furthermore obtained a complete picture of the compartmentation of thioredoxin- and glutaredoxin-like proteins. Notably, for the two major antioxidant redox-enzymes – glutathione reductase and thioredoxin reductase – Plasmodium makes use of alternative-translation-initiation (ATI) to achieve differential targeting. Dual localization of proteins effected by ATI is likely to occur also in other Apicomplexa and might open new avenues for therapeutic intervention., Author Summary The unicellular parasite Plasmodium falciparum is the causative agent of tropical malaria, which represents a global health burden. In order to survive in its human host and the malaria vector Anopheles, malaria parasites depend on adequate antioxidant defense and efficient redox regulation. Furthermore, as shown by glucose-6 phosphate dehydrogenase deficiency, a genetic variation protecting from malaria, redox equilibrium plays a vital role in parasite pathogenicity. Using a green fluorescent protein reporter gene, we systematically studied the subcellular compartmentation of redox networks in Plasmodium falciparum. Based on our results and data from literature, we provide the first thorough map of redox compartmentation. Most interestingly, for the two major antioxidant redox-enzymes – glutathione reductase (GR) and thioredoxin reductase (TrxR) – Plasmodium falciparum makes use of alternative translation initiation to translate protein isoforms with differing subcellular localization from one gene. Dual localization of proteins due to alternative translation initiation might occur frequently in Apicomplexa and identification of further genes that have evolved alternative translation initiation is likely to offer new therapeutic strategies against this devastating disease.

Background High throughput screening (HTS) is one of the primary tools used to identify novel enzyme inhibitors. However, its applicability is generally restricted to targets that can either be expressed recombinantly or purified in large quantities. Methodology and Principal Findings Here, we described a method to use activity-based probes (ABPs) to identify substrates that are sufficiently selective to allow HTS in complex biological samples. Because ABPs label their target enzymes through the formation of a permanent covalent bond, we can correlate labeling of target enzymes in a complex mixture with inhibition of turnover of a substrate in that same mixture. Thus, substrate specificity can be determined and substrates with sufficiently high selectivity for HTS can be identified. In this study, we demonstrate this method by using an ABP for dipeptidyl aminopeptidases to identify (Pro-Arg)2-Rhodamine as a specific substrate for DPAP1 in Plasmodium falciparum lysates and Cathepsin C in rat liver extracts. We then used this substrate to develop highly sensitive HTS assays (Z’>0.8) that are suitable for use in screening large collections of small molecules (i.e >300,000) for inhibitors of these proteases. Finally, we demonstrate that it is possible to use broad-spectrum ABPs to identify target-specific substrates. Conclusions We believe that this approach will have value for many enzymatic systems where access to large amounts of active enzyme is problematic. Published version

Protection against malaria often decays in the absence of infection, suggesting that protective immunological memory depends on stimulation. Here we have used CD4+ T cells from a transgenic mouse carrying a T cell receptor specific for a malaria protein, Merozoite Surface Protein-1, to investigate memory in a Plasmodium chabaudi infection. CD4+ memory T cells (CD44hiIL-7Rα+) developed during the chronic infection, and were readily distinguishable from effector (CD62LloIL-7Rα−) cells in acute infection. On the basis of cell surface phenotype, we classified memory CD4+ T cells into three subsets: central memory, and early and late effector memory cells, and found that early effector memory cells (CD62LloCD27+) dominated the chronic infection. We demonstrate a linear pathway of differentiation from central memory to early and then late effector memory cells. In adoptive transfer, CD44hi memory cells from chronically infected mice were more effective at delaying and reducing parasitemia and pathology than memory cells from drug-treated mice without chronic infection, and contained a greater proportion of effector cells producing IFN-γ and TNFα, which may have contributed to the enhanced protection. These findings may explain the observation that in humans with chronic malaria, activated effector memory cells are best maintained in conditions of repeated exposure., Author Summary Protective immunity against malaria develops only after several infections and can be lost on leaving an area in which malaria is transmitted. This suggests that the chronic infection may maintain the protective immune response. In this paper we have used a mouse model of a blood-stage malaria infection to examine the memory response of CD4+ T cells during chronic infection. These T cells are required for protective immunity, and also play a part in the inflammatory response that gives rise to malaria disease. Understanding what constitutes a protective CD4+ T cell may help us design more protective vaccines. We show that these memory CD4+ T cells persist in an activated state, produce the inflammatory cytokines TNFα and IFN-γ, and are more protective than “resting” memory CD4+ T cells obtained from mice in which the infection has been eliminated. This may explain why people are better protected against malaria disease when they are infected frequently.

Background Infection with dengue viruses (DENV) leads to a spectrum of disease outcomes. The pathophysiology of severe versus non-severe manifestations of DENV infection may be driven by host responses, which could be reflected in the transcriptional profiles of peripheral blood immune cells. Methodology/Principal Findings We conducted genome-wide microarray analysis of whole blood RNA from 34 DENV-infected children in Nicaragua collected on days 3–6 of illness, with different disease manifestations. Gene expression analysis identified genes that are differentially regulated between clinical subgroups. The most striking transcriptional differences were observed between dengue patients with and without shock, especially in the expression of mitochondrial ribosomal proteins associated with protein biosynthesis. In the dengue hemorrhagic fever patients, one subset of differentially expressed genes encode neutrophil-derived anti-microbial peptides associated with innate immunity. By performing a meta-analysis of our dataset in conjunction with previously published datasets, we confirmed that DENV infection in vivo is associated with large changes to protein and nucleic acid metabolism. Additionally, whereas in vitro infection leads to an increased interferon signature, this was not consistently observed from in vivo patient samples, suggesting that the interferon response in vivo is relatively transient and was no longer observed by days 3–6 of illness. Conclusions/Significance These data highlight important differences between different manifestations of severity during DENV infection as well as identify some commonalities. Compilation of larger datasets in the future across multiple studies, as we have initiated in this report, may well lead to better prediction of disease manifestation via a systems biology approach., Author Summary Dengue is a widespread viral disease for which over 3 billion people are at risk. There are no drug treatments or vaccines available for this disease. It is also difficult for physicians to predict which patients are at highest risk for the severe manifestations known as dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). We used genome-wide transcriptional profiling analysis to study peripheral blood responses to dengue among patients from Nicaragua. We found that patients with severe manifestations involving shock had very different transcriptional profiles from dengue patients with mild and moderate illness. We then compared our results with other microarray experiments on dengue patients available from public databases and confirmed that dengue is often associated with large changes to the metabolic processes within cells. This approach could identify prognostic markers for severe dengue as well as provide a better understanding of the pathophysiology associated with different grades of disease severity.

Plasmodium falciparum is a highly lethal malaria parasite of humans. A major portion of its life cycle is dedicated to invading and multiplying inside erythrocytes. The molecular mechanisms of erythrocyte invasion are incompletely understood. P. falciparum depends heavily on sialic acid present on glycophorins to invade erythrocytes. However, a significant proportion of laboratory and field isolates are also able to invade erythrocytes in a sialic acid-independent manner. The identity of the erythrocyte sialic acid-independent receptor has been a mystery for decades. We report here that the complement receptor 1 (CR1) is a sialic acid-independent receptor for the invasion of erythrocytes by P. falciparum. We show that soluble CR1 (sCR1) as well as polyclonal and monoclonal antibodies against CR1 inhibit sialic acid-independent invasion in a variety of laboratory strains and wild isolates, and that merozoites interact directly with CR1 on the erythrocyte surface and with sCR1-coated microspheres. Also, the invasion of neuraminidase-treated erythrocytes correlates with the level of CR1 expression. Finally, both sialic acid-independent and dependent strains invade CR1 transgenic mouse erythrocytes preferentially over wild-type erythrocytes but invasion by the latter is more sensitive to neuraminidase. These results suggest that both sialic acid-dependent and independent strains interact with CR1 in the normal red cell during the invasion process. However, only sialic acid-independent strains can do so without the presence of glycophorin sialic acid. Our results close a longstanding and important gap in the understanding of the mechanism of erythrocyte invasion by P. falciparum that will eventually make possible the development of an effective blood stage vaccine., Author Summary Plasmodium falciparum malaria is a blood parasite that lives for the most part inside red cells. It is responsible for the death of 1-2 million people every year. The mechanisms by which the parasite invades red cells are complex and not completely understood. For many years it has been known that proteins called glycophorins are used by the parasite to gain entry into the red cell. However, the existence of another protein that allows entry independent of glycophorins has been suspected for nearly as long. The identity of the alternative protein has been a mystery difficult to solve. In this article we present strong evidence that the alternative protein is the complement receptor 1. The complement receptor 1 is a well-studied protein that is known to be important in protecting red cells from attack by the host immune system as well as suspected of having other roles in the development of malaria complications. The recognition of the additional role of complement receptor 1 in red cell invasion will allow the definitive identification of malaria proteins that interact with it and that could be used in a future vaccine cocktail to block red cell invasion.

Background: Even with the advent of nucleic acid (NA) amplification technologies the culture of mycobacteria for diagnostic and other applications remains of critical importance. Notably microscopic observed drug susceptibility testing (MODS), as opposed to traditional culture on solid media or automated liquid culture, has shown potential to both speed up and increase the provision of mycobacterial culture in high burden settings. Methods: Here we explore the growth of Mycobacterial tuberculosis microcolonies, imaged by automated digital microscopy, cultured on a porous aluminium oxide (PAO) supports. Repeated imaging during colony growth greatly simplifies ‘‘computer vision’’ and presumptive identification of microcolonies was achieved here using existing publically available algorithms. Our system thus allows the growth of individual microcolonies to be monitored and critically, also to change the media during the growth phase without disrupting the microcolonies. Transfer of identified microcolonies onto selective media allowed us, within 1-2 bacterial generations, to rapidly detect the drug susceptibility of individual microcolonies, eliminating the need for time consuming subculturing or the inoculation of multiple parallel cultures. Significance: Monitoring the phenotype of individual microcolonies as they grow has immense potential for research, screening, and ultimately M. tuberculosis diagnostic applications. The method described is particularly appealing with respect to speed and automation.

Background Periodic outbreaks of dengue fever occur in the United States Virgin Islands. In June 2005, an outbreak of dengue virus (DENV) serotype-2 with cases of dengue hemorrhagic fever (DHF) was detected in St. Croix, US Virgin Islands. The objective of this report is to describe this outbreak of DENV-2 and the findings of a case-control study examining risk factors for DHF. Methodology/Principal Findings This is the largest dengue outbreak ever recorded in St. Croix, with 331 suspected dengue cases reported island-wide during 2005 (62.2 cases/10,000 population); 54% were hospitalized, 21% had at least one hemorrhagic manifestation, 28% had thrombocytopenia, 5% had DHF and 1 patient died. Eighty-nine laboratory-positive hospitalized patients were identified. Of these, there were 15 (17%) who met the WHO criteria for DHF (cases) and 74 (83%) who did not (controls). The only variable significantly associated with DHF on bivariate or multivariable analysis was age, with an adjusted odds ratio (95% confidence interval) of 1.033 (1.003,1.064). Conclusions/Significance During this outbreak of DENV-2, a high proportion of cases developed DHF and increasing age was significantly associated with DHF.