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1.  Accuracy of Five Serologic Tests for the Follow up of Strongyloides stercoralis Infection 
PLoS Neglected Tropical Diseases  2015;9(2):e0003491.
Traditional faecal-based methods have poor sensitivity for the detection of S. stercoralis, therefore are inadequate for post-treatment evaluation of infected patients who should be carefully monitored to exclude the persistence of the infection. In a previous study, we demonstrated high accuracy of five serology tests for the screening and diagnosis of strongyloidiasis. Aim of this study is to evaluate the performance of the same five tests for the follow up of patients infected with S. stercoralis.
Retrospective study on anonymized, cryo-preserved samples available at the Centre for Tropical Diseases (Negrar, Verona, Italy). Samples were collected before and from 3 to 12 months after treatment. The samples were tested with two commercially-available ELISA tests (IVD, Bordier), two techniques based on a recombinant antigen (NIE-ELISA and NIE-LIPS) and one in-house IFAT. The results of each test were evaluated both in relation to the results of fecal examination and to those of a composite reference standard (classifying as positive a sample with positive stools and/or at least three positive serology tests). The associations between the independent variables age and time and the dependent variable value of serological test (for all five tests), were analyzed by linear mixed-effects regression model.
A high proportion of samples demonstrated for each test a seroreversion or a relevant decline (optical density/relative light units halved or decrease of at least two titers for IFAT) at follow up, results confirmed by the linear mixed effects model that showed a trend to seroreversion over time for all tests. In particular, IVD-ELISA (almost 90% samples demonstrated relevant decline) and IFAT (almost 87%) had the best performance. Considering only samples with a complete negativization, NIE-ELISA showed the best performance (72.5% seroreversion).
Serology is useful for the follow up of patients infected with S. stercoralis and determining test of cure.
Author Summary
Patients infected by S. stercoralis are at risk of fatal complications. It is therefore mandatory to demonstrate complete response to therapy. Post treatment evaluation should be done with highly sensitive diagnostic methods, which can exclude the persistence of the infection. Serology is more sensitive than fecal examination and coproculture. In this study, we compare the post-treatment performance of five serology tests, and suggest that they can be useful for the follow up of patients with S. stercoralis infection, especially in non-endemic areas, where there is no risk of reinfection. In fact, the results of the tests show a progressive decrease, towards negativization, of the values (expressed in different units, depending on the specific test) through time.
PMCID: PMC4323101  PMID: 25668740
2.  Trypanosoma cruzi Infection Is a Potent Risk Factor for Non-alcoholic Steatohepatitis Enhancing Local and Systemic Inflammation Associated with Strong Oxidative Stress and Metabolic Disorders 
PLoS Neglected Tropical Diseases  2015;9(2):e0003464.
The immune mechanisms underlying experimental non-alcoholic steatohepatitis (NASH), and more interestingly, the effect of T. cruzi chronic infection on the pathogenesis of this metabolic disorder are not completely understood.
Methodology/Principal Findings
We evaluated immunological parameters in male C57BL/6 wild type and TLR4 deficient mice fed with a standard, low fat diet, LFD (3% fat) as control group, or a medium fat diet, MFD (14% fat) in order to induce NASH, or mice infected intraperitoneally with 100 blood-derived trypomastigotes of Tulahuen strain and also fed with LFD (I+LFD) or MFD (I+MFD) for 24 weeks. We demonstrated that MFD by itself was able to induce NASH in WT mice and that parasitic infection induced marked metabolic changes with reduction of body weight and steatosis revealed by histological studies. The I+MFD group also improved insulin resistance, demonstrated by homeostasis model assessment of insulin resistance (HOMA-IR) analysis; although parasitic infection increased the triglycerides and cholesterol plasma levels. In addition, hepatic M1 inflammatory macrophages and cytotoxic T cells showed intracellular inflammatory cytokines which were associated with high levels of IL6, IFNγ and IL17 plasmatic cytokines and CCL2 chemokine. These findings correlated with an increase in hepatic parasite load in I+MFD group demonstrated by qPCR assays. The recruitment of hepatic B lymphocytes, NK and dendritic cells was enhanced by MFD, and it was intensified by parasitic infection. These results were TLR4 signaling dependent. Flow cytometry and confocal microscopy analysis demonstrated that the reactive oxygen species and peroxinitrites produced by liver inflammatory leukocytes of MFD group were also exacerbated by parasitic infection in our NASH model.
We highlight that a medium fat diet by itself is able to induce steatohepatitis. Our results also suggest a synergic effect between damage associated with molecular patterns generated during NASH and parasitic infection, revealing an intense cross-talk between metabolically active tissues, such as the liver, and the immune system. Thus, T. cruzi infection must be considered as an additional risk factor since exacerbates the inflammation and accelerates the development of hepatic injury.
Author Summary
Chagas disease caused by the protozoan parasite T. cruzi is a neglected tropical disease widespread in Latin America, and non-alcoholic steatohepatitis constitutes a prominent health concern with increasing incidence of obesity and diabetes worldwide. Parasitic infection induced marked metabolic changes, improved insulin resistance, but it increased the triglycerides and cholesterol plasma levels. Our findings demonstrate for the first time a synergic effect between the hepatic damage caused during the steatohepatitis process (generated by a medium fat diet) and the exacerbated inflammation triggered by the chronic parasitic infection, revealing an intense cross-talk between the liver, a metabolically active tissue, and the immune system. We propose that T. cruzi infection must be considered as an additional risk factor for this metabolic disorder. These findings may provide new insight for the American Trypanosomiasis associated with the steatohepatitis.
PMCID: PMC4323252  PMID: 25668433
3.  Surveillance of Aedes aegypti: Comparison of House Index with Four Alternative Traps 
PLoS Neglected Tropical Diseases  2015;9(2):e0003475.
The mosquito Aedes aegypti, vector of dengue, chikungunya and yellow fever viruses, is an important target of vector control programs in tropical countries. Most mosquito surveillance programs are still based on the traditional household larval surveys, despite the availability of new trapping devices. We report the results of a multicentric entomological survey using four types of traps, besides the larval survey, to compare the entomological indices generated by these different surveillance tools in terms of their sensitivity to detect mosquito density variation.
The study was conducted in five mid-sized cities, representing variations of tropical climate regimens. Surveillance schemes using traps for adults (BG-Sentinel, Adultrap and MosquiTRAP) or eggs (ovitraps) were applied monthly to three 1 km2 areas per city. Simultaneously, larval surveys were performed. Trap positivity and density indices in each area were calculated and regressed against meteorological variables to characterize the seasonal pattern of mosquito infestation in all cities, as measured by each of the four traps.
The House Index was consistently low in most cities, with median always 0. Traps rarely produced null indices, pointing to their greater sensitivity in detecting the presence of Ae. aegypti in comparison to the larval survey. Trap positivity indices tend to plateau at high mosquito densities. Despite this, both indices, positivity and density, agreed on the seasonality of mosquito abundance in all cities. Mosquito seasonality associated preferentially with temperature than with precipitation even in areas where temperature variation is small.
All investigated traps performed better than the House Index in measuring the seasonal variation in mosquito abundance and should be considered as complements or alternatives to larval surveys. Choice between traps should further consider differences of cost and ease-of-use.
Author Summary
Dengue vector surveillance programs regularly use household surveys for searching breeding sites that are positive for Ae. aegypti larvae. Infestation indices are calculated as the percentage of positive houses, or percentage of positive containers, and are used to guide control actions and to issue alerts. However, these indices are costly and prone to error due to variation in searching effort and the sometimes cryptic nature of the mosquito egg laying behavior. In the recent years, many devices for trapping mosquitoes were developed. One problem when deciding which trap to choose for surveillance is the lack of a gold standard to compare trap-based indices with. In this scenario, choice relies on the behavior of the trap indices, that is, how well they inform about mosquito population growth, decline, or spread. Our study compared infestation indices produced by four different trap schemes as well the immature survey. We observed that any trap is more sensitive in detecting the mosquito presence than the immature survey, and they are better in capturing the temporal variation in mosquito abundance as well. However, traps are very different in terms of cost, ease-of-use and sensitivity and trap choice should further consider these factors.
PMCID: PMC4323346  PMID: 25668559
4.  The Emergence of Resistance to the Benzimidazole Anthlemintics in Parasitic Nematodes of Livestock Is Characterised by Multiple Independent Hard and Soft Selective Sweeps 
PLoS Neglected Tropical Diseases  2015;9(2):e0003494.
Anthelmintic resistance is a major problem for the control of parasitic nematodes of livestock and of growing concern for human parasite control. However, there is little understanding of how resistance arises and spreads or of the “genetic signature” of selection for this group of important pathogens. We have investigated these questions in the system for which anthelmintic resistance is most advanced; benzimidazole resistance in the sheep parasites Haemonchus contortus and Teladorsagia circumcincta. Population genetic analysis with neutral microsatellite markers reveals that T. circumcincta has higher genetic diversity but lower genetic differentiation between farms than H. contortus in the UK. We propose that this is due to epidemiological differences between the two parasites resulting in greater seasonal bottlenecking of H. contortus. There is a remarkably high level of resistance haplotype diversity in both parasites compared with drug resistance studies in other eukaryotic systems. Our analysis suggests a minimum of four independent origins of resistance mutations on just seven farms for H. contortus, and even more for T. circumincta. Both hard and soft selective sweeps have occurred with striking differences between individual farms. The sweeps are generally softer for T. circumcincta than H. contortus, consistent with its higher level of genetic diversity and consequent greater availability of new mutations. We propose a model in which multiple independent resistance mutations recurrently arise and spread by migration to explain the widespread occurrence of resistance in these parasites. Finally, in spite of the complex haplotypic diversity, we show that selection can be detected at the target locus using simple measures of genetic diversity and departures from neutrality. This work has important implications for the application of genome-wide approaches to identify new anthelmintic resistance loci and the likelihood of anthelmintic resistance emerging as selection pressure is increased in human soil-transmitted nematodes by community wide treatment programs.
Author Summary
Parasitic nematodes (roundworms) are major causes of disease in both domestic animals and humans. Strategic treatments with anthelmintic drugs have been used to control livestock parasites for several decades resulting in widespread drug resistance. Drug treatments have, until recently, been applied at a relatively low level to control human parasites. However, in recent years community wide treatment programs have been massively increased for the 1–2 billion people infected with roundworms. Hence, for both human and animal health, there is an urgent need to understand how resistance emerges and spreads and how we can detect resistance mutations in this important group of pathogens. In this study, we investigated how drug resistance mutations appear and spread in the two livestock parasites for which resistance is most widespread. We have found that resistance appears repeatedly and frequently in parasite populations, and propose a model to explain the high capacity of these pathogens to develop drug resistance. Our work suggests that anthelmintic resistance is likely to occur when repeated drug treatment is relied upon to control this group of pathogens. Our results also suggest that resistance mutations should be detectable when modern genome-wide approaches are used to scan the genomes of resistant parasites.
PMCID: PMC4319741  PMID: 25658086
5.  Mycobacterium Ulcerans Treatment – Can Antibiotic Duration Be Reduced in Selected Patients? 
PLoS Neglected Tropical Diseases  2015;9(2):e0003503.
Mycobacterium ulcerans (M. ulcerans) is a necrotizing skin infection endemic to the Bellarine Peninsula, Australia. Current treatment recommendations include 8 weeks of combination antibiotics, with adjuvant surgery if necessary. However, antibiotic toxicity often results in early treatment cessation and local experience suggests that shorter antibiotic courses may be effective with concurrent surgery. We report the outcomes of patients in the Barwon Health M. ulcerans cohort who received shorter courses of antibiotic therapy than 8 weeks.
Methodology / Principal findings
A retrospective analysis was performed of all M. ulcerans infections treated at Barwon Health from March 1, 1998 to July 31, 2013. Sixty-two patients, with a median age of 65 years, received < 56 days of antibiotics and 51 (82%) of these patients underwent concurrent surgical excision. Most received a two-drug regimen of rifampicin combined with either ciprofloxacin or clarithromycin for a median 29 days (IQR 21–41days). Cessation rates were 55% for adverse events and 36% based on clinician decision. The overall success rate was 95% (98% with concurrent surgery; 82% with antibiotics alone) with a 50% success rate for those who received < 14 days of antibiotics increasing to 94% if they received 14–27 days and 100% for 28–55 days (p<0.01). A 100% success rate was seen for concurrent surgery and 14–27 days of antibiotics versus 67% for concurrent surgery and < 14 days of antibiotics (p = 0.12). No previously identified risk factors for treatment failure with surgery alone were associated with reduced treatment success rates with < 56 days of antibiotics.
In selected patients, antibiotic treatment durations for M. ulcerans shorter than the current WHO recommended 8 weeks duration may be associated with successful outcomes.
Author Summary
Buruli ulcer is a necrotizing skin and subcutaneous tissue infection caused by Mycobacterium ulcerans and is the third most common mycobacterial infection, behind tuberculosis and leprosy, world-wide. In recent years, the World Health Organisation has modified its guidelines for M. ulcerans treatment, moving from predominantly surgical to predominantly medical based management. It now recommends the combination of oral rifampicin and intramuscular streptomycin for a period of eight weeks as first-line therapy, with surgery as adjunctive therapy if necessary. The Barwon Health experience from south-eastern Australia has demonstrated that the entirely oral combination of rifampicin with either ciprofloxacin or clarithromycin for eight weeks can be an effective treatment option. However, these antibiotics are often toxic leading to early cessation, especially in the elderly. In addition, clinicians have been using a shorter duration of therapy for smaller lesions that have also been surgically managed. This study reviews our experience treating M. ulcerans with antibiotic durations of less than 8 weeks and demonstrates that successful outcomes can be achieved in selected patients, with success rates influenced by the duration of treatment and the use of surgical excision. This finding needs confirmation in further studies, but could have significant benefits in terms of reducing toxicity and improving adherence associated with Buruli ulcer antibiotic treatment.
PMCID: PMC4319776  PMID: 25658304
6.  IL-33/ST2 Correlates with Severity of Haemorrhagic Fever with Renal Syndrome and Regulates the Inflammatory Response in Hantaan Virus-Infected Endothelial Cells 
PLoS Neglected Tropical Diseases  2015;9(2):e0003514.
Hantaan virus (HTNV) causes a severe lethal haemorrhagic fever with renal syndrome (HFRS) in humans. Despite a limited understanding of the pathogenesis of HFRS, the importance of the abundant production of pro-inflammatory cytokines has been widely recognized. Interleukin 33 (IL-33) has been demonstrated to play an important role in physiological and pathological immune responses. After binding to its receptor ST2L, IL-33 stimulates the Th2-type immune response and promotes cytokine production. Depending on the disease model, IL-33 either protects against infection or exacerbates inflammatory disease, but it is unknown how the IL-33/ST2 axis regulates the immune response during HTNV infection.
Methodology/Principal Findings
Blood samples were collected from 23 hospitalized patients and 28 healthy controls. The levels of IL-33 and soluble ST2 (sST2) in plasma were quantified by ELISA, and the relationship between IL-33, sST2 and the disease severity was analyzed. The role of IL-33/sST2 axis in the production of pro-inflammatory cytokines was studied on HTNV-infected endothelial cells. The results showed that the plasma IL-33 and sST2 were significantly higher in patients than in healthy controls. Spearman analysis showed that elevated IL-33 and sST2 levels were positively correlated with white blood cell count and viral load, while negatively correlated with platelet count. Furthermore, we found that IL-33 enhanced the production of pro-inflammatory cytokines in HTNV-infected endothelial cells through NF-κB pathway and that this process was inhibited by the recombinant sST2.
Our results indicate that the IL-33 acts as an initiator of the “cytokine storm” during HTNV infection, while sST2 can inhibit this process. Our findings could provide a promising immunotherapeutic target for the disease control.
Author Summary
Hantaan virus (HTNV) causes human hemorrhagic fever with renal syndrome (HFRS) with a mortality rate of approximately 15% in Asia. At present, the primary treatment for HFRS is limited to critical care management and the use of anti-viral drugs, such as Ribavirin. However, the cytokine storm at the acute phase of HFRS, which is thought to contribute to the development of the disease, is still lacking an effective way to prevent. An alternative way to prevent the development of cytokine storm is of priority to overcome the problem. We found that IL-33 and sST2 levels were higher in the plasma of HFRS patients, especially in their acute phase. Although both of them were positively correlated with the severity of the diseases, they acted in different roles in the regulation of the immune response during HTNV infection. In vitro study showed that IL-33 acted as an initiator of the cytokine storm in HTNV-infected endothelial cells, while sST2 acted as an inhibitor of the process. For the first time, we defined the IL-33/ST2 axis as inflammatory regulators during HTNV infection. Our results may provide a novel therapeutic target of HTNV infections.
PMCID: PMC4319827  PMID: 25658420
7.  Genetic Diversity of Staphylococcus aureus in Buruli Ulcer 
PLoS Neglected Tropical Diseases  2015;9(2):e0003421.
Buruli ulcer (BU) is a necrotizing skin disease caused by Mycobacterium ulcerans. Previous studies have shown that wounds of BU patients are colonized with M. ulcerans and several other microorganisms, including Staphylococcus aureus, which may interfere with wound healing. The present study was therefore aimed at investigating the diversity and topography of S. aureus colonizing BU patients during treatment.
We investigated the presence, diversity, and spatio-temporal distribution of S. aureus in 30 confirmed BU patients from Ghana during treatment. S. aureus was isolated from nose and wound swabs, and by replica plating of wound dressings collected bi-weekly from patients. S. aureus isolates were characterized by multiple-locus variable number tandem repeat fingerprinting (MLVF) and spa-typing, and antibiotic susceptibility was tested.
Principal Findings
Nineteen (63%) of the 30 BU patients tested positive for S. aureus at least once during the sampling period, yielding 407 S. aureus isolates. Detailed analysis of 91 isolates grouped these isolates into 13 MLVF clusters and 13 spa-types. Five (26%) S. aureus-positive BU patients carried the same S. aureus genotype in their anterior nares and wounds. S. aureus isolates from the wounds of seven (37%) patients were distributed over two different MLVF clusters. Wounds of three (16%) patients were colonized with isolates belonging to two different genotypes at the same time, and five (26%) patients were colonized with different S. aureus types over time. Five (17%) of the 30 included BU patients tested positive for methicillin-resistant S. aureus (MRSA).
The present study showed that the wounds of many BU patients were contaminated with S. aureus, and that many BU patients from the different communities carried the same S. aureus genotype during treatment. This calls for improved wound care and hygiene.
Author Summary
Buruli ulcer (BU) is a disease of the skin and soft tissue caused by Mycobacterium ulcerans. The resulting skin lesions provide a niche for survival of other microorganisms such as Staphylococcus aureus, which may cause delayed wound healing. This study investigated the presence, diversity, and spatio-temporal distribution of S. aureus in BU patients from Ghana during treatment by isolating the bacteria from nose and wound swabs or wound dressings. S. aureus isolates were subsequently characterized by two complementary DNA typing approaches. This showed that 19 (63%) of the 30 investigated BU patients carried S. aureus. Five (26%) of these 19 BU patients carried the same S. aureus type in their anterior nares and wounds. Seven (37%) patients carried the same S. aureus type in their wounds, which is indicative of transmission. Three of them (16%) carried at least two different S. aureus types at the same time in their wounds, while five (26%) carried different S. aureus types over time. Notably, five (17%) BU patients tested positive for methicillin-resistant S. aureus (MRSA). These findings imply that the spatio-temporal diversity of S. aureus in BU is most likely related to factors such as antibiotic pressure, and insufficient wound care and hygiene.
PMCID: PMC4319846  PMID: 25658641
8.  High Rates of O’Nyong Nyong and Chikungunya Virus Transmission in Coastal Kenya 
PLoS Neglected Tropical Diseases  2015;9(2):e0003436.
Chikungunya virus (CHIKV) and o’nyong nyong virus (ONNV) are mosquito-borne alphaviruses endemic in East Africa that cause acute febrile illness and arthritis. The objectives of this study were to measure the seroprevalence of CHIKV and ONNV in coastal Kenya and link it to demographics and other risk factors.
Demographic and exposure questionnaires were administered to 1,848 participants recruited from two village clusters (Milalani-Nganja and Vuga) in 2009. Sera were tested for alphavirus exposure using standardized CHIKV IgG ELISA protocols and confirmed with plaque reduction neutralization tests (PRNT). Logistic regression models were used to determine the variables associated with seropositivity. Weighted K test for global clustering of houses with alphavirus positive participants was performed for distance ranges of 50–1,000 meters, and G* statistic and kernel density mapping were used to identify locations of higher seroprevalence.
Principal Findings
486 (26%) participants were seropositive by IgG ELISA. Of 443 PRNT confirmed positives, 25 samples (6%) were CHIKV+, 250 samples (56%) were ONNV+, and 168 samples (38%) had high titers for both. Age was significantly associated with seropositivity (OR 1.01 per year, 95% C.I. 1.00–1.01); however, younger adults were more likely to be seropositive than older adults. Males were less likely to be seropositive (p<0.05; OR 0.79, 95% C.I. 0.64–0.97). Adults who owned a bicycle (p<0.05; OR 1.37, 95% C.I. 1.00–1.85) or motor vehicle (p<0.05; OR 4.64, 95% C.I. 1.19–18.05) were more likely to be seropositive. Spatial analysis demonstrated hotspots of transmission within each village and clustering among local households in Milalani-Nganja, peaking at the 200–500m range.
Alphavirus exposure, particularly ONNV exposure, is common in coastal Kenya with ongoing interepidemic transmission of both ONNV and CHIKV. Women and adults were more likely to be seropositive. Household location may be a defining factor for the ecology of alphaviral transmission in this region.
Author Summary
Alphaviruses, such as chikungunya and o’nyong nyong viruses, are likely important causes of human disease in endemic regions, but are often misdiagnosed as malaria in the acute care setting. Our objective was to uncover the burden of alphavirus exposure in our study region, rural coastal Kenya. Of 1848 participants tested, 26% were seropositive by screening ELISA, demonstrating intense transmission to humans in this area. Surprisingly, confirmatory PRNT testing revealed that the majority of alphavirus exposures were due to o’nyong nyong virus, rather than chikungunya virus. Both CHIKV and ONNV antibodies were confirmed in young children, demonstrating undocumented and ongoing transmission in this region. Of the examined risk factors, older age and female gender were associated with alphavirus seropositivity.
PMCID: PMC4319898  PMID: 25658762
9.  Comparison of Vector Competence of Aedes mediovittatus and Aedes aegypti for Dengue Virus: Implications for Dengue Control in the Caribbean 
PLoS Neglected Tropical Diseases  2015;9(2):e0003462.
Aedes mediovittatus mosquitoes are found throughout the Greater Antilles in the Caribbean and often share the same larval habitats with Ae. Aegypti, the primary vector for dengue virus (DENV). Implementation of vector control measures to control dengue that specifically target Ae. Aegypti may not control DENV transmission in Puerto Rico (PR). Even if Ae. Aegypti is eliminated or DENV refractory mosquitoes are released, DENV transmission may not cease when other competent mosquito species like Ae. Mediovittatus are present. To compare vector competence of Ae. Mediovittatus and Ae. Aegypti mosquitoes, we studied relative infection and transmission rates for all four DENV serotypes.
To compare the vector competence of Ae. Mediovittatus and Ae. Aegypti, mosquitoes were exposed to DENV 1–4 per os at viral titers of 5–6 logs plaque-forming unit (pfu) equivalents. At 14 days post infectious bloodmeal, viral RNA was extracted and tested by qRT-PCR to determine infection and transmission rates. Infection and transmission rates were analyzed with a generalized linear model assuming a binomial distribution.
Ae. Aegypti had significantly higher DENV-4 infection and transmission rates than Ae. mediovittatus.
This study determined that Ae. Mediovittatus is a competent DENV vector. Therefore dengue prevention programs in PR and the Caribbean should consider both Ae. Mediovittatus and Ae. Aegypti mosquitoes in their vector control programs.
Author Summary
Dengue is a potentially life-threatening tropical disease caused by four serotypes of virus, dengue virus 1, -2, -3, and -4. Worldwide, as many as 390 million people become infected with dengue virus each year after being bitten by infectious Aedes mosquitoes. Unfortunately, there is no commercially available vaccine to prevent dengue; so, dengue prevention is attempted by controlling Aedes mosquitoes. Since the Aedes aegypti mosquito is responsible for most dengue virus infections worldwide, most dengue control efforts target this mosquito. However, Aedes mediovittatus, a common mosquito in the Caribbean, may also transmit dengue virus in Puerto Rico. Our goal was to compare dengue virus transmission by Aedes mediovittatus and Aedes aegypti mosquitoes for four serotypes of dengue virus. In the laboratory, we exposed Aedes mediovittatus and Aedes aegypti mosquitoes with dengue virus-1–4. We found that similar numbers of Aedes mediovittatus and Aedes aegypti mosquitoes became infected with dengue virus-1–3, but differed in dengue virus 4 infection rates.
PMCID: PMC4319915  PMID: 25658951
10.  Detection of Rickettsia spp in Ticks by MALDI-TOF MS 
PLoS Neglected Tropical Diseases  2015;9(2):e0003473.
Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) has been shown to be an effective tool for the rapid identification of arthropods, including tick vectors of human diseases.
Methodology/Principal Findings
The objective of the present study was to evaluate the use of MALDI-TOF MS to identify tick species, and to determine the presence of rickettsia pathogens in the infected Ticks. Rhipicephalus sanguineus and Dermacentor marginatus Ticks infected or not by R. conorii conorii or R. slovaca, respectively, were used as experimental models. The MS profiles generated from protein extracts prepared from tick legs exhibited mass peaks that distinguished the infected and uninfected Ticks, and successfully discriminated the Rickettsia spp. A blind test was performed using Ticks that were laboratory-reared, collected in the field or removed from patients and infected or not by Rickettsia spp. A query against our in-lab arthropod MS reference database revealed that the species and infection status of all Ticks were correctly identified at the species and infection status levels.
Taken together, the present work demonstrates the utility of MALDI-TOF MS for a dual identification of tick species and intracellular bacteria. Therefore, MALDI-TOF MS is a relevant tool for the accurate detection of Rickettsia spp in Ticks for both field monitoring and entomological diagnosis. The present work offers new perspectives for the monitoring of other vector borne diseases that present public health concerns.
Author Summary
Tick-borne rickettsioses include mild to life-threatening diseases in humans worldwide. When removing an attached tick from the human body, patients and physicians may have two questions: 1) is the tick a known vector of a human infectious disease, and 2) is the tick infected by a pathogenic agent that could have been transmitted during the attachment period? The morphological identification of Ticks is difficult, and requires expertise and specific documentation. The use of Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) has recently emerged as an effective, rapid and inexpensive tool to identify arthropods including Ticks. Here, we show the utility of MALDI-TOF MS for the dual identification of tick species and the rapid detection of Rickettsia spp in Ticks. Such results can be used to guide decisions related to specific patient monitoring or the administration of preventive treatment. Additionally, the low consumable costs, the minimum time required for sample preparation and the rapid availability of the results of MALDI-TOF MS could be useful for epidemiological studies and tick-borne disease monitoring via the dual identification of vectors and the pathogens they carry in one step. These results present new opportunities for the management of other vector-borne diseases that are of importance to public health.
PMCID: PMC4319929  PMID: 25659152
11.  Spatio-temporal Transmission and Environmental Determinants of Schistosomiasis Japonica in Anhui Province, China 
PLoS Neglected Tropical Diseases  2015;9(2):e0003470.
Schistosomiasis japonica still remains of public health and economic significance in China, especially in the lake and marshland areas along the Yangtze River Basin, where the control of transmission has proven difficult. In the study, we investigated spatio-temporal variations of S. japonicum infection risk in Anhui Province and assessed the associations of the disease with key environmental factors with the aim of understanding the mechanism of the disease and seeking clues to effective and sustainable schistosomiasis control.
Methodology/Principal Findings
Infection data of schistosomiasis from annual conventional surveys were obtained at the village level in Anhui Province, China, from 2000 to 2010 and used in combination with environmental data. The spatio-temporal kriging model was used to assess how these environmental factors affected the spatio-temporal pattern of schistosomiasis risk. Our results suggested that seasonal variation of the normalized difference vegetation index (NDVI), seasonal variation of land surface temperature at daytime (LSTD), and distance to the Yangtze River were negatively significantly associated with risk of schistosomiasis. Predictive maps showed that schistosomiasis prevalence remained at a low level and schistosomiasis risk mainly evolved along the Yangtze River. Schistosomiasis risk also followed a focal spatial pattern, fluctuating temporally with a peak (the largest spatial extent) in 2005 and then contracting gradually but with a scattered distribution until 2010.
The fitted spatio-temporal kriging model can capture variations of schistosomiasis risk over space and time. Combined with techniques of geographic information system (GIS) and remote sensing (RS), this approach facilitates and enriches risk modeling of schistosomiasis, which in turn helps to identify prior areas for effective and sustainable control of schistosomiasis in Anhui Province and perhaps elsewhere in China.
Author Summary
Schistosomiasis japonica is one of the most serious parasitic diseases in China. It is estimated that more than 50 million people are still at risk, especially those living in the lake and marshland areas along the Yangtze River Basin. The Chinese government has made great efforts to implement schistosomiasis control programs since 1950s. The latest, major two programs are the 10-year World Bank Loan Project (WBLP) terminated in 2001, which was based on large-scale chemotherapy, and the national integrated control strategy implemented since 2005, which was aimed at reducing the roles of bovines and humans as infection sources. Based on spatio-temporal analyses of the S. japonicum infection prevalence data during 2000–2010 in Anhui Province, we found schistosomiasis prevalence remained at a low level but the spatial distribution of the disease became widely scattered at the later stage of the study period, suggesting that the integrated program could not fully effectively reduce the spatial extent of schistosomiasis risk. To achieve an effective and sustainable control strategy, we emphasize the need to control snail habitats within areas of high schistosomiasis risk.
PMCID: PMC4319937  PMID: 25659112
12.  Description of a Nanobody-based Competitive Immunoassay to Detect Tsetse Fly Exposure 
PLoS Neglected Tropical Diseases  2015;9(2):e0003456.
Tsetse flies are the main vectors of human and animal African trypanosomes. The Tsal proteins in tsetse fly saliva were previously identified as suitable biomarkers of bite exposure. A new competitive assay was conceived based on nanobody (Nb) technology to ameliorate the detection of anti-Tsal antibodies in mammalian hosts.
Methodology/Principal Findings
A camelid-derived Nb library was generated against the Glossina morsitans morsitans sialome and exploited to select Tsal specific Nbs. One of the three identified Nb families (family III, TsalNb-05 and TsalNb-11) was found suitable for anti-Tsal antibody detection in a competitive ELISA format. The competitive ELISA was able to detect exposure to a broad range of tsetse species (G. morsitans morsitans, G. pallidipes, G. palpalis gambiensis and G. fuscipes) and did not cross-react with the other hematophagous insects (Stomoxys calcitrans and Tabanus yao). Using a collection of plasmas from tsetse-exposed pigs, the new test characteristics were compared with those of the previously described G. m. moristans and rTsal1 indirect ELISAs, revealing equally good specificities (> 95%) and positive predictive values (> 98%) but higher negative predictive values and hence increased sensitivity (> 95%) and accuracy (> 95%).
We have developed a highly accurate Nb-based competitive immunoassay to detect specific anti-Tsal antibodies induced by various tsetse fly species in a range of hosts. We propose that this competitive assay provides a simple serological indicator of tsetse fly presence without the requirement of test adaptation to the vertebrate host species. In addition, the use of monoclonal Nbs for antibody detection is innovative and could be applied to other tsetse fly salivary biomarkers in order to achieve a multi-target immunoprofiling of hosts. In addition, this approach could be broadened to other pathogenic organisms for which accurate serological diagnosis remains a bottleneck.
Author Summary
Our previous studies have revealed that the saliva of the savannah tsetse fly (Glossina morsitans morsitans) and the main constituting Tsal proteins are sensitive immunological probes to detect contact with tsetse flies. A nanobody (Nb) library was generated against tsetse salivary gland proteins and used to select Nbs against the highly immunogenic Tsal proteins by a procedure of phage display and selection for binding onto the recombinant Tsal proteins. One Nb family was identified with the appropriate characteristics for the development of a competitive assay to detect Tsal-specific antibodies raised by the mammalian host when exposed to tsetse fly bites. In this immunoassay, exposure was detected by the inhibition of Nb binding by tsetse fly saliva induced antibodies in plasma. Evaluation of the competitive ELISA test using a set of porcine plasmas revealed an improved accuracy as compared to previously described tests. Moreover, the advantage of this assay is that it does not require adaptation to the sampled host species. We propose the Nb-based competitive ELISA as an additional tool to the indirect ELISA to serologically detect tsetse bite exposure and to monitor the impact of vector control programs and to detect re-invasion of cleared areas by tsetse flies on the African continent. In addition, the concept of using Nbs for the development of competitive antibody detection tests is innovative and broadens the scope of medical diagnostic applications of Nbs.
PMCID: PMC4320081  PMID: 25658871
13.  Neutralizing Dengue Antibody in Pregnant Thai Women and Cord Blood 
PLoS Neglected Tropical Diseases  2015;9(2):e0003396.
The WHO ‘Global Strategy for Dengue Prevention and Control, 2012–2020’ addresses the growing need for the treatment of dengue, and targets a 25% reduction in morbidity and 50% in mortality (using 2010 estimates as baseline). Achieving these goals requires future dengue prevention strategies that will employ both potential vaccines and sustainable vector-control measures. Maternally transferred dengue antibody is an important factor in determining the optimal age for dengue vaccination.
To estimate the seroprevalence of dengue antibodies among mothers living in an area of high endemicity – Ban Pong, Ratchaburi Province – and to assess maternal dengue antibodies transferred to cord blood.
Materials & Methods
A cross-sectional study was conducted with 141 pregnant women who delivered at Ban Pong Hospital, Ratchaburi, Thailand. Maternal-cord paired sera were tested for dengue neutralizing (NT) antibody by PRNT50 assay. A ratio of ≥ 1:10 NT titer to dengue serotype was considered seropositive.
Most mothers (137/141, 97.2%) had NT antibodies to at least one dengue serotype in their sera. At birth, the proportion of cord sera with NT antibodies to DEN-1, DEN-2, DEN-3, and DEN-4, were high and similar to the sera of their mothers, at 93.6%, 97.2%, 97.9%, and 92.2%, respectively. The dengue geometric mean titers (GMT) in cord blood were significantly higher than the maternal antibodies (p<0.001): highest in DEN-2, followed by DEN-3, and then DEN-1. The GMT of DEN-4 was the lowest among all four serotypes.
Dengue infection is highly prevalent among pregnant women in this dengue-endemic area. Most of the cord blood had transferred dengue antibodies, which may have an impact on the disease burden in this population.
Author Summary
Dengue is the fastest spreading mosquito-borne viral infection. Infections cause mild to severe diseases, including dengue hemorrhagic fever (DHF), a severe form that may kill infants and young children. Dengue virus antibody transfer from mother to fetus in pregnancy confers protection at birth, thereafter subsiding to a lower level that may cause DHF in infants. Infant dengue antibodies levels also influence the optimal age for dengue vaccination because of neutralization of the proposed live virus vaccine by the protective antibody levels in the newborn. To establish the optimal age, we identified mother-child pairs in which maternal dengue antibodies were transferred from mother to fetus in this study. Then a follow-up study would measure the infant antibody levels. Our study found that 97.2% of pregnant women giving birth in a dengue-endemic area had evidence of previous dengue infection. All umbilical cord blood from fetuses had the same proportion of positive tests for the presence of dengue antibodies, but had a higher dengue antibody levels compared to their mothers. The period of protection provided by maternally transferred dengue antibodies might affect the disease burden among infants and offer a better understanding of the optimal age for dengue vaccination.
PMCID: PMC4320096  PMID: 25658481
14.  Is Strongyloidiasis Endemic in Spain? 
PLoS Neglected Tropical Diseases  2015;9(2):e0003482.
PMCID: PMC4318575  PMID: 25654324
16.  A Literature Review of Economic Evaluations for a Neglected Tropical Disease: Human African Trypanosomiasis (“Sleeping Sickness”) 
PLoS Neglected Tropical Diseases  2015;9(2):e0003397.
Human African trypanosomiasis (HAT) is a disease caused by infection with the parasite Trypanosoma brucei gambiense or T. b. rhodesiense. It is transmitted to humans via the tsetse fly. Approximately 70 million people worldwide were at risk of infection in 1995, and approximately 20,000 people across Africa are infected with HAT. The objective of this review was to identify existing economic evaluations in order to summarise cost-effective interventions to reduce, control, or eliminate the burden of HAT. The studies included in the review were compared and critically appraised in order to determine if there were existing standardised methods that could be used for economic evaluation of HAT interventions or if innovative methodological approaches are warranted. A search strategy was developed using keywords and was implemented in January 2014 in several databases. The search returned a total of 2,283 articles. After two levels of screening, a total of seven economic evaluations were included and underwent critical appraisal using the Scottish Intercollegiate Guidelines Network (SIGN) Methodology Checklist 6: Economic Evaluations. Results from the existing studies focused on the cost-effectiveness of interventions for the control and reduction of disease transmission. Modelling was a common method to forecast long-term results, and publications focused on interventions by category, such as case detection, diagnostics, drug treatments, and vector control. Most interventions were considered cost-effective based on the thresholds described; however, the current treatment, nifurtomix-eflornithine combination therapy (NECT), has not been evaluated for cost-effectiveness, and considerations for cost-effective strategies for elimination have yet to be completed. Overall, the current evidence highlights the main components that play a role in control; however, economic evaluations of HAT elimination strategies are needed to assist national decision makers, stakeholders, and key funders. These analyses would be of use, as HAT is currently being prioritized as a neglected tropical disease (NTD) to reach elimination by 2020.
PMCID: PMC4318581  PMID: 25654605
17.  Chemotherapy of Second Stage Human African Trypanosomiasis: Comparison between the Parenteral Diamidine DB829 and Its Oral Prodrug DB868 in Vervet Monkeys 
PLoS Neglected Tropical Diseases  2015;9(2):e0003409.
Human African trypanosomiasis (HAT, sleeping sickness) ranks among the most neglected tropical diseases based on limited availability of drugs that are safe and efficacious, particularly against the second stage (central nervous system [CNS]) of infection. In response to this largely unmet need for new treatments, the Consortium for Parasitic Drug Development developed novel parenteral diamidines and corresponding oral prodrugs that have shown cure of a murine model of second stage HAT. As a rationale for selection of one of these compounds for further development, the pharmacokinetics and efficacy of intramuscular (IM) active diamidine 2,5-bis(5-amidino-2-pyridyl)furan (DB829; CPD-0802) and oral prodrug2,5-bis[5-(N-methoxyamidino)-2-pyridyl]furan (DB868) were compared in the vervet monkey model of second stage HAT. Treatment was initiated 28 days post-infection of monkeys with T. b. rhodesiense KETRI 2537. Results showed that IM DB829 at 5 mg/kg/day for 5 consecutive days, 5 mg/kg/day every other day for 5 doses, or 2.5 mg/kg/day for 5 consecutive days cured all monkeys (5/5). Oral DB868 was less successful, with no cures (0/2) at 3 mg/kg/day for 10 days and cure rates of 1/4 at 10 mg/kg/day for 10 days and 20 mg/kg/day for 10 days; in total, only 2/10 monkeys were cured with DB868 dose regimens. The geometric mean plasma Cmax of IM DB829 at 5 mg/kg following the last of 5 doses was 25-fold greater than that after 10 daily oral doses of DB868 at 20 mg/kg. These data suggest that the active diamidine DB829, administered IM, should be considered for further development as a potential new treatment for second stage HAT.
Author Summary
Treatment of human African trypanosomiasis (HAT, sleeping sickness) suffers from a shortage of medicines that are both effective, especially against the second (late) stage of the disease, and safe for patients. The development of new HAT medicines also has been significantly influenced by the perceived need for easily administered oral medicines to reduce the need for hospitalization of patients in resource-poor settings where HAT typically occurs. However, the clinical status of second stage patients is likely to dictate the need for their hospitalization, thus both oral and parenterally administered medicines would be utilised effectively. Therefore, in an effort to develop new medicines that meet efficacy and safety requirements, we evaluated a novel injectable diamidine 2,5-bis(5-amidino-2-pyridyl)furan (DB829; CPD-0802) and its oral prodrug formulation 2,5-bis[5-(N-methoxyamidino)-2-pyridyl]furan (DB868) in the vervet monkey model of second stage HAT. Treatment with either compound was initiated 28 days post-infection of monkeys with T. b. rhodesiense KETRI 2537. DB829 was dosed at 5 mg/kg/day for 5 consecutive days, 5 mg/kg/day every other day for 5 doses or 2.5 mg/kg/day for 5 consecutive days intramuscularly (IM) while DB868 was administered at 20, 10 or 3 mg/kg/day for 10 consecutive days orally. Clinical and parasitological monitoring was carried out for at least 300 days before the monkeys were declared cured. All IM DB829 and oral DB868 dose regimens were well tolerated. In addition, all monkeys (5/5) treated with IM DB829 were confirmed cured. In contrast, oral DB868 cured only 1/4 monkeys at either 10 or 20 mg/kg and did not cure any monkey when dosed at 3 mg/kg. These results indicate that IM DB829 is a suitable compound for further development as treatment for second stage HAT.
PMCID: PMC4318582  PMID: 25654243
18.  High Prevalence of the Liver Fluke Amphimerus sp. in Domestic Cats and Dogs in an Area for Human Amphimeriasis in Ecuador 
PLoS Neglected Tropical Diseases  2015;10(2):e0003526.
Amphimerus sp. is a liver fluke which recently has been shown to have a high prevalence of infection among an indigenous group, Chachi, who reside in a tropical rainforest in the northwestern region of Ecuador. Since it is unknown which animals can act as a reservoir and/or definitive hosts for Amphimerus sp. in this endemic area, a study was done to determine the prevalence of infection in domestic cats and dogs. This information is important to understand the epidemiology, life cycle and control of this parasite.
In July 2012, three Chachi communities located on Rio Cayapas, province of Esmeraldas, were surveyed. A total of 89 of the 109 registered households participated in the study. Of the 27 cats and 43 dogs found residing in the communities, stool samples were collected from 14 cats and 31 dogs (total of 45 animals) and examined microscopically for the presence of Amphimerus eggs. The prevalence of infection was 71.4% in cats and 38.7% in dogs, with similar rates of infection in all three communities. Significantly more cats were infected than dogs (p = 0.042).
The data show a high rate of Amphimerus sp. infection in domestic cats and dogs residing in Chachi communities. It can be concluded that these animals act as definitive and reservoir hosts for this liver fluke and that amphimeriasis is a zoonotic disease. These findings provide important epidemiological data which will aid in the development and implementation of control strategies against the transmission of Amphimerus.
Author Summary
Amphimerus sp. is a fluke that infects the bile ducts of its definitive hosts. Recently, it has been shown that an indigenous Amerindian group, the Chachi, living in a rural and remote tropical area of Ecuador, are infected with this parasite. The epidemiology and life cycle of this parasite remains elusive, and research is needed to understand the mode of transmission and zoonotic potential of the parasite. It was hypothesized that the domestic animals of the Chachi households may act as definitive and reservoir hosts for Amphimerus infection. Hence, the presence and prevalence of infection in these animals residing in communities endemic for human amphimeriasis was investigated. Some 45 animal stool samples were examined microscopically for the presence of Amphimerus eggs. The results showed an infection rate of 71.4% in cats and 38.7% in dogs. The data provided evidence that these domestic animals act as both definitive and reservoir hosts for the parasite and that amphimeriasis is a zoonotic disease. The implementation of a mass treatment/control program must target both humans and animals in order to minimize the transmission of this liver fluke.
PMCID: PMC4315407  PMID: 25647171
19.  Severity of Old World Cutaneous Leishmaniasis Is Influenced by Previous Exposure to Sandfly Bites in Saudi Arabia 
PLoS Neglected Tropical Diseases  2015;9(2):e0003449.
The sandfly Phlebotomus papatasi is the vector of Leishmania major, the main causative agent of Old World cutaneous leishmaniasis (CL) in Saudi Arabia. Sandflies inject saliva while feeding and the salivary protein PpSP32 was previously shown to be a biomarker for bite exposure. Here we used recombinant PpSP32 to evaluate human exposure to Ph. papatasi bites, and study the association between antibody response to saliva and CL in endemic areas in Saudi Arabia.
Methodology/Principal Findings
In this observational study, anti-PpSP32 antibodies, as indicators of exposure to sandfly bites, were measured in sera from healthy individuals and patients from endemic regions in Saudi Arabia with active and cured CL. Ph. papatasi was identified as the primary CL vector in the study area. Anti-PpSP32 antibody levels were significantly higher in CL patients presenting active infections from all geographical regions compared to CL cured and healthy individuals. Furthermore, higher anti-PpSP32 antibody levels correlated with the prevalence and type of CL lesions (nodular vs. papular) observed in patients, especially non-local construction workers.
Our findings suggest a possible correlation between the type of immunity generated by the exposure to sandfly bites and disease outcome.
Author Summary
Leishmania is transmitted by the bite of infected female sandflies. When a sandfly bites a vertebrate host, it injects a cocktail of salivary proteins meant to facilitate blood feeding. The constant exposure to sandfly bites in endemic areas triggers a humoral response against the major antigenic components in the saliva. These antibodies can be then exploited to measure exposure to vector sandflies, which is useful for surveillance in leishmaniasis control programmes. In Saudi Arabia, cutaneous leishmaniasis (CL) is mainly transmitted by the Phlebotomus papatasi sandfly. Here we study the recognition of the main antigenic salivary protein from Ph. papatasi, PpSP32, in leishmaniasis patients and healthy individuals from three CL endemic areas in Saudi Arabia. Anti-PpSP32 antibody levels were significantly higher in CL patients presenting active infections from all geographical regions compared to the CL-cured and healthy individuals. Furthermore, higher anti-PpSP32 antibody levels correlated with the prevalence and type of CL lesions observed in patients. Our results suggest that previous long-term exposure to sandfly saliva can have a role in modulating the severity of leishmaniasis infection, resulting in a milder form of the disease.
PMCID: PMC4315490  PMID: 25646796
20.  Review on Dog Rabies Vaccination Coverage in Africa: A Question of Dog Accessibility or Cost Recovery? 
PLoS Neglected Tropical Diseases  2015;9(2):e0003447.
Rabies still poses a significant human health problem throughout most of Africa, where the majority of the human cases results from dog bites. Mass dog vaccination is considered to be the most effective method to prevent rabies in humans. Our objective was to systematically review research articles on dog rabies parenteral vaccination coverage in Africa in relation to dog accessibility and vaccination cost recovery arrangement ( of charge or owner charged).
Methodology/Principal Findings
A systematic literature search was made in the databases of CAB abstracts (EBSCOhost and OvidSP), Scopus, Web of Science, PubMed, Medline (EBSCOhost and OvidSP) and AJOL (African Journal Online) for peer reviewed articles on 1) rabies control, 2) dog rabies vaccination coverage and 3) dog demography in Africa. Identified articles were subsequently screened and selected using predefined selection criteria like year of publication (viz. ≥ 1990), type of study (cross sectional), objective(s) of the study (i.e. vaccination coverage rates, dog demographics and financial arrangements of vaccination costs), language of publication (English) and geographical focus (Africa). The selection process resulted in sixteen peer reviewed articles which were used to review dog demography and dog ownership status, and dog rabies vaccination coverage throughout Africa. The main review findings indicate that 1) the majority (up to 98.1%) of dogs in African countries are owned (and as such accessible), 2) puppies younger than 3 months of age constitute a considerable proportion (up to 30%) of the dog population and 3) male dogs are dominating in numbers (up to 3.6 times the female dog population). Dog rabies parenteral vaccination coverage was compared between “free of charge” and “owner charged” vaccination schemes by the technique of Meta-analysis. Results indicate that the rabies vaccination coverage following a free of charge vaccination scheme (68%) is closer to the World Health Organization recommended coverage rate (70%) than the achieved coverage rate in owner-charged dog rabies vaccination schemes (18%).
Most dogs in Africa are owned and accessible for parenteral vaccination against rabies if the campaign is performed “free of charge”.
Author Summary
Rabies is one of the most fatal diseases in both humans and animals. A bite by a rabid dog is the main cause of human rabies in Africa. Parenteral mass dog vaccination is the most cost-effective tool to prevent rabies in humans. Our main objective was to review research articles on the parenteral dog rabies vaccination coverage in Africa. We aimed to review published research articles on percentage of dogs owned and percentage of dogs vaccinated against rabies, and on the relation between vaccination coverage and cost recovery. We followed the standard procedures of a systematic literature review resulting in a final review of 16 scientific articles. Our review results indicate that only a small percentage of African dogs is ownerless. Puppies younger than 3 months of age constitute a considerable proportion of the African dog population. There are considerably more male dogs than female dogs present within the dog population. The dog rabies parenteral vaccination coverage following a “free of charge” vaccination scheme (68%) is closer to World Health Organization recommended threshold coverage rate (70%) compared to the coverage rate achieved in “owner-charged” dog rabies vaccination schemes (18%). In conclusion, most dogs in Africa are owned and accessible for vaccination once the necessary financial arrangements have been made.
PMCID: PMC4315526  PMID: 25646774
21.  Platelet Activating Factor Contributes to Vascular Leak in Acute Dengue Infection 
PLoS Neglected Tropical Diseases  2015;9(2):e0003459.
Although plasma leakage is the hallmark of severe dengue infections, the factors that cause increased vascular permeability have not been identified. As platelet activating factor (PAF) is associated with an increase in vascular permeability in other diseases, we set out to investigate its role in acute dengue infection.
Materials and Methods
PAF levels were initially assessed in 25 patients with acute dengue infection to determine if they were increased in acute dengue. For investigation of the kinetics of PAF, serial PAF values were assessed in 36 patients. The effect of dengue serum on tight junction protein ZO-1 was determined by using human endothelial cell lines (HUVECs). The effect of dengue serum on and trans-endothelial resistance (TEER) was also measured on HUVECs.
PAF levels were significantly higher in patients with acute dengue (n = 25; p = 0.001) when compared to healthy individuals (n = 12). In further investigation of the kinetics of PAF in serial blood samples of patients (n = 36), PAF levels rose just before the onset of the critical phase. PAF levels were significantly higher in patients with evidence of vascular leak throughout the course of the illness when compared to those with milder disease. Serum from patients with dengue significantly down-regulated expression of tight junction protein, ZO-1 (p = 0.004), HUVECs. This was significantly inhibited (p = 0.004) by use of a PAF receptor (PAFR) blocker. Serum from dengue patients also significantly reduced TEER and this reduction was also significantly (p = 0.02) inhibited by prior incubation with the PAFR blocker.
Our results suggest the PAF is likely to be playing a significant role in inducing vascular leak in acute dengue infection which offers a potential target for therapeutic intervention.
Author Summary
Although plasma leakage is the hallmark of severe dengue infections, the factors that cause increased vascular permeability have not been identified. As platelet activating factor (PAF) is associated with an increase in vascular permeability in other diseases, we set out to investigate its role in acute dengue infection. In this study, we found that PAF was significantly increased in patients with DHF, and the PAF levels rose just before the onset of the critical phase of dengue, during which vascular leak is thought to occur. PAF in serum of dengue patients was associated with reduced expression of tight junction proteins (ZO-1) and reduction in trans-endothelial resistance (TEER) of human endothelial cells. Use of PAFR blockers significantly reduced the down regulation of ZO-1 by serum of dengue patients and also the reduction of TEER, suggesting that PAF plays a significant role in inducing vascular leak in acute dengue infections.
PMCID: PMC4315531  PMID: 25646838
22.  Describing the Breakbone Fever: IDODEN, an Ontology for Dengue Fever 
PLoS Neglected Tropical Diseases  2015;9(2):e0003479.
Ontologies represent powerful tools in information technology because they enhance interoperability and facilitate, among other things, the construction of optimized search engines. To address the need to expand the toolbox available for the control and prevention of vector-borne diseases we embarked on the construction of specific ontologies. We present here IDODEN, an ontology that describes dengue fever, one of the globally most important diseases that are transmitted by mosquitoes.
Methodology/Principal Findings
We constructed IDODEN using open source software, and modeled it on IDOMAL, the malaria ontology developed previously. IDODEN covers all aspects of dengue fever, such as disease biology, epidemiology and clinical features. Moreover, it covers all facets of dengue entomology. IDODEN, which is freely available, can now be used for the annotation of dengue-related data and, in addition to its use for modeling, it can be utilized for the construction of other dedicated IT tools such as decision support systems.
The availability of the dengue ontology will enable databases hosting dengue-associated data and decision-support systems for that disease to perform most efficiently and to link their own data to those stored in other independent repositories, in an architecture- and software-independent manner.
Author Summary
The need for the construction of a dengue ontology arose through the fact that the incidence of dengue fever is on the rise across the world; the number of cases may be three to four times higher than the 100 million estimated by the WHO and a vaccine is still not available in spite of the significant efforts undertaken. Thus, control of dengue fever still relies mostly on controlling its mosquito vectors. Large amounts of entomological, epidemiological and clinical data are generated; these need to be efficiently organized in order to further our comprehension of the disease and its control. IDODEN aims to cover the different aspects and intricacies of dengue fever and syndromes caused by dengue virus(es). It contains more than 5000 terms describing epidemiological data, vaccine development, clinical features, the disease course, and more. We show here that it can be a helpful tool for researchers and that, in addition to allowing sophisticated search strategies, it is also useful for tasks such as modeling.
PMCID: PMC4315569  PMID: 25646954
23.  Evidence of Reversible Bradycardia and Arrhythmias Caused by Immunogenic Proteins Secreted by T. cruzi in Isolated Rat Hearts 
PLoS Neglected Tropical Diseases  2015;9(2):e0003512.
Chagas cardiomyopathy, caused by the protozoan Trypanosoma cruzi, is characterized by alterations in intracellular ion, heart failure and arrhythmias. Arrhythmias have been related to sudden death, even in asymptomatic patients, and their molecular mechanisms have not been fully elucidated.
The aim of this study is to demonstrate the effect of proteins secreted by T. cruzi on healthy, isolated beating rat heart model under a non-damage-inducing protocol.
Methods and Results
We established a non-damage-inducing recirculation-reoxygenation model where ultrafiltrate fractions of conditioned medium control or conditioned infected medium were perfused at a standard flow rate and under partial oxygenation. Western blotting with chagasic patient serum was performed to determine the antigenicity of the conditioned infected medium fractions. We observed bradycardia, ventricular fibrillation and complete atrioventricular block in hearts during perfusion with >50 kDa conditioned infected culture medium. The preincubation of conditioned infected medium with chagasic serum abolished the bradycardia and arrhythmias. The proteins present in the conditioned infected culture medium of >50 kDa fractions were recognized by the chagasic patient sera associated with arrhythmias.
These results suggest that proteins secreted by T. cruzi are involved in Chagas disease arrhythmias and may be a potential biomarker in chagasic patients.
Author Summary
Chagas disease, caused by the parasite Trypanosoma cruzi, is an endemic disease of Latin-American countries, affecting an estimated 8 million people in 21 countries. It is spread by the bite of triatomine reduvid bug. Due to immigration towards non-endemic regions, the disease can spread and affect people around the world via blood transfusions. Infection usually occurs in childhood, and some patients may develop acute myocarditis; however, most remain asymptomatic for many years before chronic cardiac and/or gastrointestinal manifestations appear. Chagas disease is characterized by an acute phase, which is generally asymptomatic, or oligosymptomatic, an indeterminate phase, which may persist for several years, and a chronic phase in which dilated cardiomyopathy and arrhythmias are primarily observed and sudden death may occur. Once heart failure develops, death usually occurs within several years. In this work, we demonstrate the pathophysiological role of proteins secreted by T. cruzi on cardiac arrhythmias. The antigenicity of these fractions was tested by an immunological test using chagasic patients’ sera associated with arrhythmias. We showed that perfusion of the proteins secreted by T. cruzi, in an isolated beating rat heart model, induced cardiac arrhythmias such as bradycardia and complete atrioventricular block.
PMCID: PMC4315581  PMID: 25647069
24.  Population Structure of the Chagas Disease Vector Triatoma infestans in an Urban Environment 
PLoS Neglected Tropical Diseases  2015;9(2):e0003425.
Chagas disease is a vector-borne disease endemic in Latin America. Triatoma infestans, a common vector of this disease, has recently expanded its range into rapidly developing cities of Latin America. We aim to identify the environmental features that affect the colonization and dispersal of T. infestans in an urban environment. We amplified 13 commonly used microsatellites from 180 T. infestans samples collected from a sampled transect in the city of Arequipa, Peru, in 2007 and 2011. We assessed the clustering of subpopulations and the effect of distance, sampling year, and city block location on genetic distance among pairs of insects. Despite evidence of genetic similarity, the majority of city blocks are characterized by one dominant insect genotype, suggesting the existence of barriers to dispersal. Our analyses show that streets represent an important barrier to the colonization and dispersion of T. infestans in Arequipa. The genetic data describe a T. infestans infestation history characterized by persistent local dispersal and occasional long-distance migration events that partially parallels the history of urban development.
Author Summary
The colonization and dispersal of disease vectors in new and expanding urban areas pose important health risks. The population and demographic dynamics of these vectors are often unclear, and their temporal and spatial associations with urbanization are unknown. Here, we use molecular markers to describe the genetic structure of populations of T. infestans, an important vector of the etiologic agent of Chagas disease, in an expanding urban environment. Samples were obtained along a transect in Arequipa, Peru, that includes old, well-developed communities and new communities characterized by rudimentarily constructed houses. We assessed the clustering of subpopulations and the effect of distance, sampling year, and city block on genetic distance among pairs of insects. Our analyses show that streets represent an important barrier to the colonization and dispersion of T. infestans in Arequipa. The genetic data describe a T. infestans infestation history characterized by persistent local dispersal and occasional long-distance migration events that partially parallels the history of urban development.
PMCID: PMC4315598  PMID: 25646757
25.  High Prevalence of Schistosoma japonicum and Fasciola gigantica in Bovines from Northern Samar, the Philippines 
PLoS Neglected Tropical Diseases  2015;9(2):e0003108.
The cause of zoonotic schistosomiasis in the Philippines is Schistosoma japonicum, which infects up to 46 mammalian hosts, including humans and bovines. In China, water buffaloes have been identified as major reservoir hosts for schistosomiasis japonica, contributing up to 75% of human transmission. In the Philippines, water buffaloes (carabao; Bubalus bubalis carabanesis) have, historically, been considered unimportant reservoirs. We therefore revisited the possible role of bovines in schistosome transmission in the Philippines, using the recently described formalin-ethyl acetate sedimentation (FEA-SD) technique and a qPCR assay to examine fecal samples from 153 bovines (both carabao and cattle) from six barangays in Northern Samar. A high prevalence of S. japonicum was found using qPCR and FEA-SD in both cattle (87.50% and 77.08%, respectively) and carabao (80.00% and 55.24%, respectively). The average daily egg output for each bovine was calculated at 195,000. High prevalence and infection intensity of F. gigantica was also found in the bovines by qPCR and FEA-SD (95.33% and 96.00%, respectively). The identification of bovines as major reservoir hosts for S. japonicum transmission suggests that bovine treatment and/or vaccination, as one becomes available, should be included in any future control program that aims to reduce the disease burden due to schistosomiasis in the Philippines.
Author Summary
Schistosomiasis japonica, a zoonosis of over 40 different mammalian species, is endemic to China, the Philippines and Indonesia. In China, water buffaloes have been shown to be major reservoir hosts, while in the Philippines, the smaller sub species (carabao) has been been considered unimportant in transmission, possibly due to the lack of sensitive copro-parasitological techniques employed. We used an exhaustive microscopic technique, the FEA-SD, and a sensitive qPCR assay on a cohort of bovines to assess their potential role in transmission in the Philippines. Both cattle and carabao were highly infected with Schistosoma japonicum and Fasciola gigantic and co-infection was common. The high prevalence and intensity of bovine infection with S. japonicum suggest their heavy involvement in human transmission and that future control programs should target these reservoirs to reduce human infection.
PMCID: PMC4313937  PMID: 25643317

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