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Comparison of Individual and Pooled Stool Samples for the Assessment of Soil-Transmitted Helminth Infection Intensity and Drug Efficacy
by Zeleke Mekonnen, Selima Meka, Mio Ayana, Johannes Bogers, Jozef Vercruysse, Bruno LeveckeBackground
In veterinary parasitology samples are often pooled for a rapid assessment of infection intensity and drug efficacy. Currently, studies evaluating this strategy in large-scale drug administration programs to control human soil-transmitted helminths (STHs; Ascaris lumbricoides, Trichuris trichiura, and hookworm), are absent. Therefore, we developed and evaluated a pooling strategy to assess intensity of STH infections and drug efficacy.Methods/Principal Findings
Stool samples from 840 children attending 14 primary schools in Jimma, Ethiopia were pooled (pool sizes of 10, 20, and 60) to evaluate the infection intensity of STHs. In addition, the efficacy of a single dose of mebendazole (500 mg) in terms of fecal egg count reduction (FECR; synonym of egg reduction rate) was evaluated in 600 children from two of these schools. Individual and pooled samples were examined with the McMaster egg counting method. For each of the three STHs, we found a significant positive correlation between mean fecal egg counts (FECs) of individual stool samples and FEC of pooled stool samples, ranging from 0.62 to 0.98. Only for A. lumbricoides was any significant difference in mean FEC of the individual and pooled samples found. For this STH species, pools of 60 samples resulted in significantly higher FECs. FECR for the different number of samples pooled was comparable in all pool sizes, except for hookworm. For this parasite, pools of 10 and 60 samples provided significantly higher FECR results.Conclusion/Significance
This study highlights that pooling stool samples holds promise as a strategy for rapidly assessing infection intensity and efficacy of administered drugs in programs to control human STHs. However, further research is required to determine when and how pooling of stool samples can be cost-effectively applied along a control program, and to verify whether this approach is also applicable to other NTDs.
by G. Sybren de Hoog, Sarah A. Ahmed, Mohammad J. Najafzadeh, Deanna A. Sutton, Maryam Saradeghi Keisari, Ahmed H. Fahal, Ursala Eberhardt, Gerard J. Verkleij, Lian Xin, Benjamin Stielow, Wendy W. J. van de SandeEumycetoma is a traumatic fungal infection in tropical and subtropical areas that may lead to severe disability. Madurella mycetomatis is one of the prevalent etiologic agents in arid Northeastern Africa. The source of infection has not been clarified. Subcutaneous inoculation from plant thorns has been hypothesized, but attempts to detect the fungus in relevant material have remained unsuccessful. The present study aims to find clues to reveal the natural habitat of Madurella species using a phylogenetic approach, i.e. by comparison of neighboring taxa with known ecology. Four species of Madurella were included in a large data set of species of Chaetomium, Chaetomidium, Thielavia, and Papulaspora (n = 128) using sequences of the universal fungal barcode gene rDNA ITS and the partial LSU gene sequence. Our study demonstrates that Madurella species are nested within the Chaetomiaceae, a family of fungi that mainly inhabit animal dung, enriched soil, and indoor environments. We hypothesize that cattle dung, ubiquitously present in rural East Africa, plays a significant role in the ecology of Madurella. If cow dung is an essential factor in inoculation by Madurella, preventative measures may involve the use of appropriate footwear in addition to restructuring of villages to reduce the frequency of contact with etiologic agents of mycetoma. On the other hand, the Chaetomiaceae possess a hidden clinical potential which needs to be explored.
Dual Targeting of Insulin and Venus Kinase Receptors of Schistosoma mansoni for Novel Anti-schistosome Therapy
by Mathieu Vanderstraete, Nadège Gouignard, Katia Cailliau, Marion Morel, Julien Lancelot, Jean-François Bodart, Colette DissousBackground
Chemotherapy of schistosomiasis relies on a single drug, Praziquantel (PZQ) and mass-use of this compound has led to emergence of resistant strains of Schistosoma mansoni, therefore pointing out the necessity to find alternative drugs. Through their essential functions in development and metabolism, receptor tyrosine kinases (RTK) could represent valuable drug targets for novel anti-schistosome chemotherapies. Taking advantage of the similarity between the catalytic domains of S. mansoni insulin receptors (SmIR1 and SmIR2) and Venus Kinase Receptors (SmVKR1 and SmVKR2), we studied the possibility to fight schistosomes by targeting simultaneously the four receptors with a single drug.Methodology/Principal Findings
Several commercial RTK inhibitors were tested for their potential to inhibit the kinase activities of SmIR1, SmIR2, SmVKR1 and SmVKR2 intracellular domains (ICD) expressed in Xenopus oocytes. We measured the inhibitory effect of chemicals on meiosis resumption induced by the active ICD of the schistosome kinases in oocytes. The IR inhibitor, tyrphostin AG1024, was the most potent inhibitory compound towards SmIR and SmVKR kinases. In vitro studies then allowed us to show that AG1024 affected the viability of both schistosomula and adult worms of S. mansoni. At micromolar doses, AG1024 induced apoptosis and caused schistosomula death in a dose-dependent manner. In adult worms, AG1024 provoked alterations of reproductive organs, as observed by confocal laser scanner microscopy. With 5 µM AG1024, parasites were no more feeding and laying eggs, and they died within 48 h with 10 µM.Conclusion/Significance
IRs and VKRs are essential in S. mansoni for key biological processes including glucose uptake, metabolism and reproduction. Our results demonstrate that inhibiting the kinase potential and function of these receptors by a single chemical compound AG1024 at low concentrations, leads to death of schistosomula and adult worms. Thus, AG1024 represents a valuable hit compound for further design of anti-kinase drugs applicable to anti-schistosome chemotherapy.
by Florian Gehre, Jacob Otu, Kathryn DeRiemer, Paola Florez de Sessions, Martin L. Hibberd, Wim Mulders, Tumani Corrah, Bouke C. de Jong, Martin AntonioBackground
Human tuberculosis (TB) in West Africa is not only caused by M. tuberculosis but also by bacteria of the two lineages of M. africanum. For instance, in The Gambia, 40% of TB is due to infections with M. africanum West African 2. This bacterial lineage is associated with HIV infection, reduced ESAT-6 immunogenicity and slower progression to active disease. Although these characteristics suggest an attenuated phenotype of M. africanum, no underlying mechanism has been described. From the first descriptions of M. africanum in the literature in 1969, the time to a positive culture of M. africanum on solid medium was known to be longer than the time to a positive culture of M. tuberculosis. However, the delayed growth of M. africanum, which may correlate with the less virulent phenotype in the human host, has not previously been studied in detail.Methodology/Principal Findings
We compared the growth rates of M. tuberculosis and M. africanum isolates from The Gambia in two liquid culture systems. M. africanum grows significantly slower than M. tuberculosis, not only when grown directly from sputa, but also in growth experiments under defined laboratory conditions. We also sequenced four M. africanum isolates and compared their whole genomes with the published M. tuberculosis H37Rv genome. M. africanum strains have several non-synonymous SNPs or frameshift mutations in genes that were previously associated with growth-attenuation. M. africanum strains also have a higher mutation frequency in genes crucial for transport of sulphur, ions and lipids/fatty acids across the cell membrane into the bacterial cell. Surprisingly, 5 of 7 operons, recently described as essential for intracellular survival of H37Rv in the host macrophage, showed at least one non-synonymously mutated gene in M. africanum.Conclusions/Significance
The altered growth behaviour of M. africanum might indicate a different survival strategy within host cells.
Fine Analysis of Genetic Diversity of the tpr Gene Family among Treponemal Species, Subspecies and Strains
by Arturo Centurion-Lara, Lorenzo Giacani, Charmie Godornes, Barbara J. Molini, Tara Brinck Reid, Sheila A. LukehartBackground
The pathogenic non-cultivable treponemes include three subspecies of Treponema pallidum (pallidum, pertenue, endemicum), T. carateum, T. paraluiscuniculi, and the unclassified Fribourg-Blanc treponeme (Simian isolate). These treponemes are morphologically indistinguishable and antigenically and genetically highly similar, yet cross-immunity is variable or non-existent. Although all of these organisms cause chronic, multistage skin and systemic disease, they have historically been classified by mode of transmission, clinical presentations and host ranges. Whole genome studies underscore the high degree of sequence identity among species, subspecies and strains, pinpointing a limited number of genomic regions for variation. Many of these “hot spots” include members of the tpr gene family, composed of 12 paralogs encoding candidate virulence factors. We hypothesize that the distinct clinical presentations, host specificity, and variable cross-immunity might reside on virulence factors such as the tpr genes.Methodology/Principal Findings
Sequence analysis of 11 tpr loci (excluding tprK) from 12 strains demonstrated an impressive heterogeneity, including SNPs, indels, chimeric genes, truncated gene products and large deletions. Comparative analyses of sequences and 3D models of predicted proteins in Subfamily I highlight the striking co-localization of discrete variable regions with predicted surface-exposed loops. A hallmark of Subfamily II is the presence of chimeric genes in the tprG and J loci. Diversity in Subfamily III is limited to tprA and tprL.Conclusions/Significance
An impressive sequence variability was found in tpr sequences among the Treponema isolates examined in this study, with most of the variation being consistent within subspecies or species, or between syphilis vs. non-syphilis strains. Variability was seen in the pallidum subspecies, which can be divided into 5 genogroups. These findings support a genetic basis for the classification of these organisms into their respective subspecies and species. Future functional studies will determine whether the identified genetic differences relate to cross-immunity, clinical differences, or host ranges.
Rapid, Simple and Sensitive Detection of Q Fever by Loop-Mediated Isothermal Amplification of the htpAB Gene
by Lei Pan, Lijuan Zhang, Desheng Fan, Xiuchun Zhang, Hong Liu, Qunying Lu, Qiyi XuBackground
Q fever is the most widespread zoonosis, and domestic animals are the most common sources of transmission. It is not only difficult to distinguish from other febrile diseases because of the lack of specific clinical manifestations in humans, but it is also difficult to identify the disease in C. burnetii-carrying animals because of the lack of identifiable features. Conventional serodiagnosis requires sera from the acute and convalescent stages of infection, which are unavailable at early diagnosis. Nested PCR and real-time PCR require equipment. In this study, we developed a Loop-Mediated Isothermal Amplification (LAMP) assay to identify C. burnetii rapidly and sensitively.Methods
A universal LAMP primer set was designed to detect the repeated sequence IS1111a of the htpAB gene of C. burnetii using PrimerExplorer V4 software. The sensitivity of the LAMP assay was evaluated using known quantities of recombined reference plasmids containing the targeted genes. The specificity of the developed LAMP assay was determined using 26 members of order Rickettsiae and 18 other common pathogens. The utility of the LAMP assay was further compared with real time PCR by the examination 24 blood samples including 6 confirmed and 18 probable Q fever cases, which diagnosed by IFA serological assessment and real time PCR. In addition, 126 animal samples from 4 provinces including 97 goats, 7 cattle, 18 horses, 3 marmots and 1 deer were compared by these two methods.Results
The limits of detection of the LAMP assay for the htpAB gene were 1 copy per reaction. The specificity of the LAMP assay was 100%, and no cross-reaction was observed among the bacteria used in the study. The positive rate of unknown febrile patients was 33.3%(95%CI 30.2%–36.4%) for the LAMP assay and 8.3%(95%CI 7.4%–9.2%) for the real time PCR(P<0.05). Similarly, the total positive rate of animals was 7.9%(95%CI 7.1%–8.7%) for the LAMP assay and 0.8%(95%CI 0.7%–0.9%)for the real time PCR(P<0.01). Using the developed LAMP assay, Q fever in the Yi Li area, Xinjiang Province, was confirmed.Conclusions
The LAMP assay is a potential tool to support the diagnosis of Q fever in humans and domestic animals in the field, especially in the rural areas of China, because of its rapid and sensitive detection without the aid of sophisticated equipment or a complicated protocol.
The Iron Distribution and Magnetic Properties of Schistosome Eggshells: Implications for Improved Diagnostics
by Stephan Karl, Lucía Gutiérrez, Rafael Lucyk-Maurer, Roland Kerr, Renata R. F. Candido, Shu Q. Toh, Martin Saunders, Jeremy A. Shaw, Alexandra Suvorova, Andreas Hofmann, Michael J. House, Robert C. Woodward, Carlos Graeff-Teixera, Timothy G. St. Pierre, Malcolm K. JonesBackground
Schistosoma mansoni and Schistosoma japonicum are the most frequent causative agents of human intestinal schistosomiasis. Approximately 200 million people in the world are infected with schistosomes. Diagnosis of schistosomiasis is often difficult. High percentages of low level infections are missed in routine fecal smear analysis and current diagnostic methodologies are inadequate to monitor the progress of parasite control, especially in areas with low transmission. Improved diagnostic methods are urgently needed to evaluate the success of elimination programs. Recently, a magnetic fractionation method for isolation of parasite eggs from feces was described, which uses magnetic microspheres to form parasite egg – magnetic microsphere conjugates. This approach enables screening of larger sample volumes and thus increased diagnostic sensitivity. The mechanism of formation of the conjugates remains unexplained and may either be related to specific surface characteristics of eggs and microspheres or to their magnetic properties.Methods/Principal Findings
Here, we investigated iron localization in parasite eggs, specifically in the eggshells. We determined the magnetic properties of the eggs, studied the motion of eggs and egg-microsphere conjugates in magnetic fields and determined species specific affinity of parasite eggs to magnetic microspheres. Our study shows that iron is predominantly localized in pores in the eggshell. Parasite eggs showed distinct paramagnetic behaviour but they did not move in a magnetic field. Magnetic microspheres spontaneously bound to parasite eggs without the presence of a magnetic field. S. japonicum eggs had a significantly higher affinity to bind microspheres than S. mansoni eggs.Conclusions/Significance
Our results suggest that the interaction of magnetic microspheres and parasite eggs is unlikely to be magnetic in origin. Instead, the filamentous surface of the eggshells may be important in facilitating the binding. Modification of microsphere surface properties may therefore be a way to optimize magnetic fractionation of parasite eggs.
DNA Topoisomerase II Is Involved in Regulation of Cyst Wall Protein Genes and Differentiation in Giardia lamblia
by Bo-Chi Lin, Li-Hsin Su, Shih-Che Weng, Yu-Jiao Pan, Nei-Li Chan, Tsai-Kun Li, Hsin-Chih Wang, Chin-Hung SunThe protozoan Giardia lamblia differentiates into infectious cysts within the human intestinal tract for disease transmission. Expression of the cyst wall protein (cwp) genes increases with similar kinetics during encystation. However, little is known how their gene regulation shares common mechanisms. DNA topoisomerases maintain normal topology of genomic DNA. They are necessary for cell proliferation and tissue development as they are involved in transcription, DNA replication, and chromosome condensation. A putative topoisomerase II (topo II) gene has been identified in the G. lamblia genome. We asked whether Topo II could regulate Giardia encystation. We found that Topo II was present in cell nuclei and its gene was up-regulated during encystation. Topo II has typical ATPase and DNA cleavage activity of type II topoisomerases. Mutation analysis revealed that the catalytic important Tyr residue and cleavage domain are important for Topo II function. We used etoposide-mediated topoisomerase immunoprecipitation assays to confirm the binding of Topo II to the cwp promoters in vivo. Interestingly, Topo II overexpression increased the levels of cwp gene expression and cyst formation. Microarray analysis identified up-regulation of cwp and specific vsp genes by Topo II. We also found that the type II topoisomerase inhibitor etoposide has growth inhibition effect on Giardia. Addition of etoposide significantly decreased the levels of cwp gene expression and cyst formation. Our results suggest that Topo II has been functionally conserved during evolution and that Topo II plays important roles in induction of the cwp genes, which is key to Giardia differentiation into cysts.
Disruption of Aedes aegypti Olfactory System Development through Chitosan/siRNA Nanoparticle Targeting of semaphorin-1a
by Keshava Mysore, Ellen M. Flannery, Michael Tomchaney, David W. Severson, Molly Duman-ScheelDespite the devastating impact of mosquito-borne illnesses on human health, surprisingly little is known about mosquito developmental biology, including development of the olfactory system, a tissue of vector importance. Analysis of mosquito olfactory developmental genetics has been hindered by a lack of means to target specific genes during the development of this sensory system. In this investigation, chitosan/siRNA nanoparticles were used to target semaphorin-1a (sema1a) during olfactory system development in the dengue and yellow fever vector mosquito Aedes aegypti. Immunohistochemical analyses and anterograde tracing of antennal sensory neurons, which were used to track the progression of olfactory development in this species, revealed antennal lobe defects in sema1a knockdown fourth instar larvae. These findings, which correlated with a larval odorant tracking behavioral phenotype, identified previously unreported roles for Sema1a in the developing insect larval olfactory system. Analysis of sema1a knockdown pupae also revealed a number of olfactory phenotypes, including olfactory receptor neuron targeting and projection neuron defects coincident with a collapse in the structure and shape of the antennal lobe and individual glomeruli. This study, which is to our knowledge the first functional genetic analysis of insect olfactory development outside of D. melanogaster, identified critical roles for Sema1a during Ae. aegypti larval and pupal olfactory development and advocates the use of chitosan/siRNA nanoparticles as an effective means of targeting genes during post-embryonic Ae. aegypti development. Use of siRNA nanoparticle methodology to understand sensory developmental genetics in mosquitoes will provide insight into the evolutionary conservation and divergence of key developmental genes which could be exploited in the development of both common and species-specific means for intervention.
Differential Activation of Diverse Glutathione Transferases of Clonorchis sinensis in Response to the Host Bile and Oxidative Stressors
by Young-An Bae, Do-Whan Ahn, Eung-Goo Lee, Seon-Hee Kim, Guo-Bin Cai, Insug Kang, Woon-Mok Sohn, Yoon KongBackground
Clonorchis sinensis causes chronic cumulative infections in the human hepatobiliary tract and is intimately associated with cholangiocarcinoma. Approximately 35 million people are infected and 600 million people are at risk of infections worldwide. C. sinensis excretory-secretory products (ESP) constitute the first-line effector system affecting the host-parasite interrelationship by interacting with bile fluids and ductal epithelium. However, the secretory behavior of C. sinensis in an environment close to natural host conditions is unclear. C. sinensis differs from Fasciola hepatica in migration to, and maturation in, the hepatic bile duct, implying that protein profile of the ESP of these two trematodes might be different from each other.Methodology/Principal Findings
We conducted systemic approaches to analyze the C. sinensis ESP proteome and the biological reactivity of C. sinensis glutathione transferases (GSTs), such as global expression patterns and induction profiles under oxidative stress and host bile. When we observed ex host excretion behavior of C. sinensis in the presence of 10% host bile, the global proteome pattern was not significantly altered, but the amount of secretory proteins was increased by approximately 3.5-fold. Bioactive molecules secreted by C. sinensis revealed universal/unique features in relation to its intraluminal hydrophobic residing niche. A total of 38 protein spots identified abundantly included enzymes involved in glucose metabolism (11 spots, 28.9%) and diverse-classes of glutathione transferases (GSTs; 10 spots, 26.3%). Cathepsin L/F (four spots, 10.5%) and transporter molecules (three spots, 7.9%) were also recognized. The universal secretory proteins found in other parasites, such as several enzymes involved in glucose metabolism and oxygen transporters, were commonly detected. C. sinensis secreted less cysteine proteases and fatty acid binding proteins compared to other tissue-invading or intravascular trematodes. Interestingly, secretion of a 28 kDa σ-class GST (Cs28σGST3) was significantly affected by the host bile, involving reduced secretion of the 28 kDa species and augmented secretion of Cs28σGST3-related high-molecular-weight 85 kDa protein. Oxidative stressors induced upregulated secretion of 28 kDa Cs28σGST3, but not an 85 kDa species. A secretory 26 kDa μ-class GST (Cs26μGST2) was increased upon treatment with oxidative stressors and bile juice, while another 28 kDa σ-class GST (Cs28σGST1) showed negligible responses.Conclusions/Significance
Our results represent the first analysis of the genuine nature of the C. sinensis ESP proteome in the presence of host bile mimicking the natural host environments. The behavioral patterns of migration and maturation of C. sinensis in the bile ducts might contribute to the secretion of copious amounts of diverse GSTs, but a smaller quantity and fewer kinds of cysteine proteases. The Cs28σGST1 and its paralog(s) detoxify endogenous oxidative molecules, while Cs28σGST3 and Cs26μGST2 conjugate xenobiotics/hydrophobic substances in the extracellular environments, which imply that diverse C. sinensis GSTs might have evolved for each of the multiple specialized functions.
by Ronnie G. Gavilan, Maria L. Zamudio, Jaime Martinez-UrtazaVibrio parahaemolyticus is a foodborne pathogen that has become a public health concern at the global scale. The epidemiological significance of V. parahaemolyticus infections in Latin America received little attention until the winter of 1997 when cases related to the pandemic clone were detected in the region, changing the epidemic dynamics of this pathogen in Peru. With the aim to assess the impact of the arrival of the pandemic clone on local populations of pathogenic V. parahaemolyticus in Peru, we investigated the population genetics and genomic variation in a complete collection of non-pandemic strains recovered from clinical sources in Peru during the pre- and post-emergence periods of the pandemic clone. A total of 56 clinical strains isolated in Peru during the period 1994 to 2007, 13 strains from Chile and 20 strains from Asia were characterized by Multilocus Sequence Typing (MLST) and checked for the presence of Variable Genomic Regions (VGRs). The emergence of O3:K6 cases in Peru implied a drastic disruption of the seasonal dynamics of infections and a shift in the serotype dominance of pathogenic V. parahaemolyticus. After the arrival of the pandemic clone, a great diversity of serovars not previously reported was detected in the country, which supports the introduction of additional populations cohabitating with the pandemic group. Moreover, the presence of genomic regions characteristic of the pandemic clone in other non-pandemic strains may represent early evidence of genetic transfer from the introduced population to the local communities. Finally, the results of this study stress the importance of population admixture, horizontal genetic transfer and homologous recombination as major events shaping the structure and diversity of pathogenic V. parahaemolyticus.
A Cluster Randomized Study of The Safety of Integrated Treatment of Trachoma and Lymphatic Filariasis in Children and Adults in Sikasso, Mali
by Yaya Ibrahim Coulibaly, Ilo Dicko, Modibo Keita, Mahamadou Minamba Keita, Moussa Doumbia, Adama Daou, Fadima Cheick Haidara, Moussa Hama Sankare, John Horton, Caroline Whately-Smith, Samba Ousmane SowBackground
Neglected tropical diseases are co-endemic in many areas of the world, including sub Saharan Africa. Currently lymphatic filariasis (albendazole/ivermectin) and trachoma (azithromycin) are treated separately. Consequently, financial and logistical benefit can be gained from integration of preventive chemotherapy programs in such areas.Methodology/Findings
4 villages in two co-endemic districts (Kolondièba and Bougouni) of Sikasso, Mali, were randomly assigned to coadministered treatment (ivermectin/albendazole/azithromycin) or standard therapy (ivermectin/albendazole with azithromycin 1 week later). These villages had previously undergone 4 annual MDA campaigns with ivermectin/albendazole and 2 with azithromycin. One village was randomly assigned to each treatment arm in each district. There were 7515 eligible individuals in the 4 villages, 3011(40.1%) of whom participated in the study. No serious adverse events occurred, and the majority of adverse events were mild in intensity (mainly headache, abdominal pain, diarrhoea and “other signs/symptoms”). The median time to the onset of the first event, of any type, was later (8 days) in the two standard treatment villages than in the co-administration villages. Overall the number of subjects reporting any event was similar in the co-administration group compared to the standard treatment group [18.7% (281/1501) vs. 15.8% (239/1510)]. However, the event frequency was higher in the coadministration group (30.4%) than in the standard treatment group (11.0%) in Kolondièba, while the opposite was observed in Bougouni (7.1% and 20.9% respectively). Additionally, the overall frequency of adverse events in the co-administration group (18.7%) was comparable to or lower than published frequencies for ivermectin+albendazole alone.Conclusions
These data suggest that co-administration of ivermectin+albendazole and azithromycin is safe; however the small number of villages studied and the large differences between them resulted in an inability to calculate a meaningful overall estimate of the difference in adverse event rates between the regimens. Further work is therefore needed before co-administration can be definitively recommended.Trial Registration
Leishmania mexicana Infection Induces IgG to Parasite Surface Glycoinositol Phospholipids that Can Induce IL-10 in Mice and Humans
by Laurence U. Buxbaum
Infection with the intracellular protozoan parasite Leishmania mexicana causes chronic disease in C57BL/6 mice, in which cutaneous lesions persist for many months with high parasite burdens (107–108 parasites). This chronic disease process requires host IL-10 and FcγRIII. When Leishmania amastigotes are released from cells, surface-bound IgG can induce IL-10 and suppress IL-12 production from macrophages. These changes decrease IFN-γ from T cells and nitric oxide production in infected cells, which are both required for Leishmania control. However, antibodies targets and the kinetics of antibody production are unknown. Several groups have been unsuccessful in identifying amastigote surface proteins that bind IgG. We now show that glycoinositol phospholipids (GIPLs) of L. mexicana are recognized by mouse IgG1 by 6 weeks of infection, with a rapid increase between 12 and 16 weeks, consistent with the timing of chronic disease in C57BL/6 mice vs. healing in FcγRIII-deficient mice. A single prominent spot on TLC is recognized by IgG, and the glycolipid is a glycosyl phosphatidylinositol containing a branched mannose structure. We show that the lipid structure of the GIPL (the sn-2 fatty acid) is required for antibody recognition. This GIPL is abundant in L. mexicana amastigotes, rare in stationary-phase promastigotes, and absent in L. major, consistent with a role for antibodies to GIPLs in chronic disease. A mouse monoclonal anti-GIPL IgG recognizes GIPLs on the parasite surface, and induces IL-10 from macrophages. The current work also extends this mouse analysis to humans, finding that L. mexicana-infected humans with localized and diffuse cutaneous leishmaniasis have antibodies that recognize GIPLs, can bind to the surface of amastigotes, and can induce IL-10 from human monocytes. Further characterization of the target glycolipids will have important implications for drug and vaccine development and will elucidate the poorly understood role of glycolipids in the immunology of infections.
by Zeno Bisoffi, Dora Buonfrate, Antonio Montresor, Ana Requena-Méndez, Jose Muñoz, Alejandro J. Krolewiecki, Eduardo Gotuzzo, Maria Alejandra Mena, Peter L. Chiodini, Mariella Anselmi, Juan Moreira, Marco Albonico
Is Pregnancy Associated with Severe Dengue? A Review of Data from the Rio de Janeiro Surveillance Information System
by Carolina Romero Machado, Elizabeth Stankiewicz Machado, Roger Denis Rohloff, Marina Azevedo, Dayse Pereira Campos, Robson Bruniera de Oliveira, Patrícia BrasilBackground
Dengue is a reportable disease in Brazil; however, pregnancy has been included in the application form of the Brazilian notification information system only after 2006. To estimate the severity of maternal dengue infection, the available data that were compiled from January 2007 to December 2008 by the official surveillance information system of the city of Rio de Janeiro were reviewed.Methods and Principal Findings
During the study period, 151,604 cases of suspected dengue infection were reported. Five hundred sixty-one women in their reproductive age (15–49 years) presented with dengue infection; 99 (18.1%) pregnant and 447 (81.9%) non-pregnant women were analyzed. Dengue cases were categorized using the 1997 WHO classification system, and DHF/DSS were considered severe disease. The Mann-Whitney test was used to compare maternal age, according to gestational period, and severity of disease. A chi-square test was utilized to evaluate the differences in the proportion of dengue severity between pregnant and non-pregnant women. Univariate analysis was performed to compare outcome variables (severe dengue and non-severe dengue) and explanatory variables (pregnancy, gestational age and trimester) using the Wald test. A multivariate analysis was performed to assess the independence of statistically significant variables in the univariate analysis. A p-value<0.05 was considered statistically significant.A higher percentage of severe dengue infection among pregnant women was found, p = 0.0001. Final analysis demonstrated that pregnant women are 3.4 times more prone to developing severe dengue (OR: 3.38; CI: 2.10–5.42). Mortality among pregnant women was superior to non-pregnant women.Conclusion
Pregnant women have an increased risk of developing severe dengue infection and dying of dengue.
Incidence of Rabies in Humans and Domestic Animals and People's Awareness in North Gondar Zone, Ethiopia
by Wudu Temesgen Jemberu, Wassie Molla, Gizat Almaw, Sefinew AlemuBackground
Rabies is a zoonotic disease that has been prevalent in humans and animals for centuries in Ethiopia and it is often dealt with using traditional practices. There is lack of accurate quantitative information on rabies both in humans and animals in Ethiopia and little is known about the awareness of the people about the disease. In this study, we estimated the incidence of rabies in humans and domestic animals, and assessed the people's awareness about the disease in North Gondar zone, Ethiopia.Methodology/Principal Findings
The incidence of rabies in humans and domestic animals was prospectively followed up for one year period based on clinical observation. A questionnaire was also administered to 120 randomly selected dog owners and 5 traditional healers to assess the knowledge and practices about the disease. We found an annual estimated rabies incidence of 2.33 cases per 100,000 in humans, 412.83 cases per 100,000 in dogs, 19.89 cases per 100,000 in cattle, 67.68 cases per 100,000 in equines, and 14.45 cases per 100,000 in goats. Dog bite was the source of infection for all fatal rabies cases. Ninety eight percent of the questionnaire respondents were familiar with rabies and mentioned dog bite as a means of transmission. But discordant with current scientific knowledge, 84% and 32% of the respondents respectively mentioned any type of contact (irrespective of skin condition) with saliva, and inhalation as a means of transmission of rabies. Eighty four percent of the respondents relied on traditional healers for management of rabies.Conclusions
The study shows high canine rabies burden, and lack of sufficient awareness about the disease and high reliance on traditional treatment that interfere with timely post exposure management. Vaccination of dogs, proper post exposure management, and increasing the awareness of the community are suggested to reduce the disease burden.
Prospective Study on the Incidence and Progression of Clinical Signs in Naïve Dogs Naturally Infected by Leishmania infantum
by Valentina Foglia Manzillo, Trentina Di Muccio, Sivia Cappiello, Aldo Scalone, Rosa Paparcone, Eleonora Fiorentino, Manuela Gizzarelli, Marina Gramiccia, Luigi Gradoni, Gaetano OlivaThe incidence of clinical and clinicopathological signs associated with the progression of infection was evaluated prospectively in 329 naïve young dogs exposed to Leishmania infantum transmission and examined periodically during 22 months (M). The dogs were part of Leishmania vaccine investigations performed under natural conditions. Vaccinated groups were considered in the evaluation when the vaccine resulted non-protective and the appearance and progression of signs did not differ statistically from controls at each time point, otherwise only control groups were included. 115 beagles were part of 3 studies (A to C) performed in the same kennel; 214 owned dogs (29 breeds, 2.3% beagles) were included in a study (D) performed in 45 endemic sites. At M22 the prevalence of any Leishmania infection stage classified as subpatent, active asymptomatic, or symptomatic was 59.8% in studies A–C and 29.2% in study D. Despite different breed composition and infection incidence, the relative proportion of active infections and the progression and type of clinical and clinicopathological signs have been similar in both study sets. All asymptomatic active infections recorded have invariably progressed to full-blown disease, resulting in 56 sick dogs at M22. In these dogs, lymph nodes enlargement and weight loss — recorded from M12 — were the most common signs. Cutaneous signs were seen late (M18) and less frequently. Ocular signs appeared even later, being sporadically recorded at M22. Most clinicopathological alterations became evident from M12, although a few cases of thrombocytopenia or mild non-regenerative anemia were already observed at M6. Albumin/globulin inversions were recorded from M12 and urea/creatinine increase appeared mostly from M18. Altogether our findings indicate that any susceptible young dogs naturally infected by L. infantum present a common pattern of progression of signs during 2 years post infection, providing clues for medical and epidemiological applied aspects.
by Saja Asakrah, Wildaliz Nieves, Zaid Mahdi, Mallory Agard, Arnold H. Zea, Chad J. Roy, Lisa A. MoriciBurkholderia pseudomallei is a Gram-negative, facultative intracellular bacillus and the etiologic agent of melioidosis, a severe disease in Southeast Asia and Northern Australia. Like other multidrug-resistant pathogens, the inherent antibiotic resistance of B. pseudomallei impedes treatment and highlights the need for alternative therapeutic strategies that can circumvent antimicrobial resistance mechanisms. In this work, we demonstrate that host prostaglandin E2 (PGE2) production plays a regulatory role in the pathogenesis of B. pseudomallei. PGE2 promotes B. pseudomallei intracellular survival within macrophages and bacterial virulence in a mouse model of pneumonic melioidosis. PGE2-mediated immunosuppression of macrophage bactericidal effector functions is associated with increased arginase 2 (Arg2) expression and decreased nitric oxide (NO) production. Treatment with a commercially-available COX-2 inhibitor suppresses the growth of B. pseudomallei in macrophages and affords significant protection against rapidly lethal pneumonic melioidosis when administered post-exposure to B. pseudomallei-infected mice. COX-2 inhibition may represent a novel immunotherapeutic strategy to control infection with B. pseudomallei and other intracellular pathogens.
by Dimitrios-Alexios Karagiannis-Voules, Ronaldo G. C. Scholte, Luiz H. Guimarães, Jürg Utzinger, Penelope VounatsouBackground
Leishmaniasis is endemic in 98 countries with an estimated 350 million people at risk and approximately 2 million cases annually. Brazil is one of the most severely affected countries.Methodology
We applied Bayesian geostatistical negative binomial models to analyze reported incidence data of cutaneous and visceral leishmaniasis in Brazil covering a 10-year period (2001–2010). Particular emphasis was placed on spatial and temporal patterns. The models were fitted using integrated nested Laplace approximations to perform fast approximate Bayesian inference. Bayesian variable selection was employed to determine the most important climatic, environmental, and socioeconomic predictors of cutaneous and visceral leishmaniasis.Principal Findings
For both types of leishmaniasis, precipitation and socioeconomic proxies were identified as important risk factors. The predicted number of cases in 2010 were 30,189 (standard deviation [SD]: 7,676) for cutaneous leishmaniasis and 4,889 (SD: 288) for visceral leishmaniasis. Our risk maps predicted the highest numbers of infected people in the states of Minas Gerais and Pará for visceral and cutaneous leishmaniasis, respectively.Conclusions/Significance
Our spatially explicit, high-resolution incidence maps identified priority areas where leishmaniasis control efforts should be targeted with the ultimate goal to reduce disease incidence.
A Real-Time ITS1-PCR Based Method in the Diagnosis and Species Identification of Leishmania Parasite from Human and Dog Clinical Samples in Turkey
by Seray Ozensoy Toz, Gulnaz Culha, Fadile Yıldız Zeyrek, Hatice Ertabaklar, M. Ziya Alkan, Aslı Tetik Vardarlı, Cumhur Gunduz, Yusuf Ozbel
Human visceral leishmaniasis (VL) caused by L. infantum and cutaneous leishmaniasis (CL) caused by L. tropica and L. infantum have been reported in Turkey. L. infantum is also responsible for canine leishmaniasis (CanL) and it is widely common in the country. The main aim of the present study was to design a real-time PCR method based on the internal transcribed spacer 1 (ITS1) region in the diagnosis of all clinical forms of leishmaniasis in Mediterranean, and to identify the species directly from clinical samples. Totally, 315 clinical specimens, human/canine visceral (blood, bone marrow, lymph node) and cutaneous (lesion aspiration) samples, and 51 Turkish Leishmania isolates typed by isoenzymatic method were included in the study. For optimization, DNA samples of the 34 strains were amplified by conventional ITS1-PCR and then sequenced for designing the primers and probes, allowing the species identification. Following the validation with the isolates, the test was applied on clinical samples and melting temperatures were used for genotyping. A group of PCR products were further sequenced for confirmation and assigning the inter- and intraspecies heterogeneity. The diagnosis of leishmaniasis is successfully achieved by the new real-time PCR method, and the test identified 80.43% of human and canine VL samples as L.infantum and 6.52% as L.tropica; 52.46% of CL samples as L. infantum and 26.90% as L. tropica. In 13.04% of visceral and 20.62% of cutaneous samples, two peaks were observed. Hovewer, the higher peak was found to be concordant with the sequencing results in 96.96%, in terms of species identification. The real-time ITS1 PCR assay clearly identified the leishmanial species in 81.58% of all clinical samples. Genotypic variations of Leishmania parasites in Turkey within species and intraspecies were observed, and L. tropica is also found as causative agent of human and canine VL in Turkey.