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2.  Low Levels of Awareness Despite High Prevalence of Schistosomiasis among Communities in Nyalenda Informal Settlement, Kisumu City, Western Kenya 
Introduction
Intestinal schistosomiasis is widely distributed around Lake Victoria in Kenya where about 16 million people in 56 districts are at risk of the infection with over 9.1 million infected. Its existence in rural settings has been extensively studied compared to urban settings where there is limited information about the disease coupled with low level of awareness. This study therefore assessed community awareness on existence, signs and symptoms, causes, transmission, control and risk factors for contracting schistosomiasis as well as attitudes, health seeking behaviour and environmental antecedents that affect its control so as to identify knowledge gaps that need to be addressed in order to strengthen schistosomiasis control interventions in informal urban settings.
Methods
The study was carried out in an informal urban settlement where the prevalence of intestinal schistosomiasis was previously reported to be the highest (36%) among the eight informal settlements of Kisumu city. The study adopted cross-sectional design and purposive sampling technique. Eight focus group discussions were conducted with adult community members and eight key informant interviews with opinion leaders. Data was audio recorded transcribed, coded and thematically analyzed using ATLAS.ti version 6 software.
Results
Most respondents stated having heard about schistosomiasis but very few had the correct knowledge of signs and symptoms, causes, transmission and control of schistosomiasis. However, there was moderate knowledge of risk factors and at high risk groups. Their attitudes towards schistosomiasis and its control were generally indifferent with a general belief that they had no control over their environmental circumstances to reduce transmission.
Discussion/Conclusion
Although schistosomiasis was prevalent in the study area, majority of the people in the community had low awareness. This study, therefore, stresses the need for health education to raise community's awareness on schistosomiasis in such settings in order to augment prevention, control and elimination efforts.
Author Summary
Bilharzia also known as schistosomiasis is one of the neglected tropical diseases found in western part of Kenya. The major source of infection is Lake Victoria; however, there is evidence of inland transmission especially within the informal settlements of Kisumu city. Schistosomiasis can be controlled using three key approaches which include improved sanitation, health education and mass treatment with praziquantel. Additional interventions for infection prevention include: promotion of hygiene, access to safe water, and sanitation improvement and environmental management. However, the success of control initiatives involving the community depend on the level of the communities' uptake of the program, which is hinged upon understanding the community knowledge and practices towards the disease. This study therefore collected information from the community to assess level of awareness of schistosomiasis. The findings revealed a low level of awareness in spite of a high prevalence of schistosomiasis. These findings are invaluable in the designing of appropriate education messages targeted at raising community awareness on schistosomiasis and relevant behavioural change required for a successful control programme.
doi:10.1371/journal.pntd.0002784
PMCID: PMC3974654  PMID: 24699502
3.  Transmission Dynamics of Borrelia turicatae from the Arthropod Vector 
Background
With the global distribution, morbidity, and mortality associated with tick and louse-borne relapsing fever spirochetes, it is important to understand the dynamics of vector colonization by the bacteria and transmission to the host. Tick-borne relapsing fever spirochetes are blood-borne pathogens transmitted through the saliva of soft ticks, yet little is known about the transmission capability of these pathogens during the relatively short bloodmeal. This study was therefore initiated to understand the transmission dynamics of the relapsing fever spirochete Borrelia turicatae from the vector Ornithodoros turicata, and the subsequent dissemination of the bacteria upon entry into murine blood.
Methodology/Principal Findings
To determine the minimum number of ticks required to transmit spirochetes, one to three infected O. turicata were allowed to feed to repletion on individual mice. Murine infection and dissemination of the spirochetes was evaluated by dark field microscopy of blood, quantitative PCR, and immunoblotting against B. turicatae protein lysates and a recombinant antigen, the Borrelia immunogenic protein A. Transmission frequencies were also determined by interrupting the bloodmeal 15 seconds after tick attachment. Scanning electron microscopy (SEM) was performed on infected salivary glands to detect spirochetes within acini lumen and excretory ducts. Furthermore, spirochete colonization and dissemination from the bite site was investigated by feeding infected O. turicata on the ears of mice, removing the attachment site after engorment, and evaluating murine infection.
Conclusion/Significance
Our findings demonstrated that three ticks provided a sufficient infectious dose to infect nearly all animals, and B. turicatae was transmitted within seconds of tick attachment. Spirochetes were also detected in acini lumen of salivary glands by SEM. Upon host entry, B. turicatae did not require colonization of the bite site to establish murine infection. These results suggest that once B. turicatae colonizes the salivary glands the spirochetes are preadapted for rapid entry into the mammal.
Author Summary
Relapsing fever spirochetes cause recurrent febrile episodes, rigors, nausea, vomiting, malaise, and pregnancy complications, and are a leading cause of hospital admissions in regions of Africa. Routes of pathogen transmission include crushed human body lice and feces, or through bites by Ornithodoros spp. of ticks. The life cycle of Ornithodoros turicatae, the vector of Borrelia turicatae, includes over six nymphal stages, upwards of a ten year life span, and a bloodmeal that is completed within an hour. We investigated B. turicatae transmission from the tick vector and assessed the rapidity of spirochete entry into the mammal and dissemination in the blood. Salivary glands from infected ticks were also evaluated to visualize B. turicatae within the tissues to determine spirochete localization. We conclude that given the transmission dynamics of B. turicatae, it may be important to target conserved surface proteins that relapsing fever spirochetes produce in the salivary glands in order to develop preventative measures against the pathogens.
doi:10.1371/journal.pntd.0002767
PMCID: PMC3974661  PMID: 24699275
4.  Chagas Disease Vector Control in a Hyperendemic Setting: The First 11 Years of Intervention in Cochabamba, Bolivia 
Background
Chagas disease has historically been hyperendemic in the Bolivian Department of Cochabamba. In the early 2000s, an extensive vector control program was implemented; 1.34 million dwelling inspections were conducted to ascertain infestation (2000–2001/2003–2011), with blanket insecticide spraying in 2003–2005 and subsequent survey-spraying cycles targeting residual infestation foci. Here, we assess the effects of this program on dwelling infestation rates (DIRs).
Methodology/Principal Findings
Program records were used to calculate annual, municipality-level aggregate DIRs (39 municipalities); very high values in 2000–2001 (median: 0.77–0.69) dropped to ∼0.03 from 2004 on. A linear mixed model (with municipality as a random factor) suggested that infestation odds decreased, on average, by ∼28% (95% confidence interval [CI95] 6–44%) with each 10-fold increase in control effort. A second, better-fitting mixed model including year as an ordinal predictor disclosed large DIR reductions in 2001–2003 (odds ratio [OR] 0.11, CI95 0.06–0.19) and 2003–2004 (OR 0.22, CI95 0.14–0.34). Except for a moderate decrease in 2005–2006, no significant changes were detected afterwards. In both models, municipality-level DIRs correlated positively with previous-year DIRs and with the extent of municipal territory originally covered by montane dry forests.
Conclusions/Significance
Insecticide-spraying campaigns had very strong, long-lasting effects on DIRs in Cochabamba. However, post-intervention surveys consistently detected infestation in ∼3% of dwellings, underscoring the need for continuous surveillance; higher DIRs were recorded in the capital city and, more generally, in municipalities dominated by montane dry forest – an eco-region where wild Triatoma infestans are widespread. Traditional strategies combining insecticide spraying and longitudinal surveillance are thus confirmed as very effective means for area-wide Chagas disease vector control; they will be particularly beneficial in highly-endemic settings, but should also be implemented or maintained in other parts of Latin America where domestic infestation by triatomines is still commonplace.
Author Summary
Chagas disease is among the most serious public health problems in Latin America; the highest prevalence of infection by its causative agent, the parasite Trypanosoma cruzi, has historically been recorded in some parts of Bolivia. In the early 2000s, a massive insecticide-spraying program was set up to control dwelling infestation by the blood-sucking bugs that transmit the disease. Here we provide a detailed assessment of the effects of this program in the Department of Cochabamba, one of the most highly-endemic settings worldwide. Our analyses show that municipality-level dwelling infestation rates plummeted from over 70–80% in 2001–2003 to about 2–3% in 2004–2011. This residual infestation was higher in the capital city and, more generally, in municipalities where montane dry forests dominate – probably because wild populations of the main vector, Triatoma infestans, are common in that eco-region. Despite the impressive early achievements of the program, with about 0.5 million people protected from contagion, sustained disease control will require fully operational long-term surveillance systems.
doi:10.1371/journal.pntd.0002782
PMCID: PMC3974664  PMID: 24699407
5.  Expansion of Parasite-Specific CD4+ and CD8+ T Cells Expressing IL-10 Superfamily Cytokine Members and Their Regulation in Human Lymphatic Filariasis 
Background
Lymphatic filariasis (LF) is known to be associated with an increased production of IL-10. The role of the other IL-10 family members in the pathogenesis of infection and/or disease is not known.
Methodology/Principal Findings
We examined the expression patterns of IL-10 family members – IL-19, IL-24 and IL-26 in LF. We demonstrate that both CD4+ and CD8+ T cells express IL-19, IL-24 and IL-26 and that the frequency of CD4+ T cells expressing IL-19 and IL-24 (as well as IL-10) is significantly increased at baseline and following filarial antigen stimulation in patients with LF in comparison to individuals with filarial lymphedema and uninfected individuals. This CD4+ T cell expression pattern was associated with increased production of IL-19 and IL-24 by filarial – antigen stimulated PBMC. Moreover, the frequency of CD4+ and CD8+ T cells expressing IL-26 was significantly increased following filarial antigen stimulation in filarial lymphedema individuals. Interestingly, IL-10 blockade resulted in diminished frequencies of IL-19+ and IL-24+ T cells, whereas the addition of recombinant IL-10 resulted in significantly increased frequency of IL-19+ and IL-24+ T cells as well as significantly up regulated IL-19 and IL-24 gene expression, suggesting that IL-10 regulates IL-19 and IL-24 expression in T cells. In addition, IL-1β and IL-23 blockade also induced a diminution in the frequency of IL-19+ and IL-24+ T cells, indicating a novel role for these cytokines in the induction of IL-19 and IL-24 expressing T cells. Finally, elimination of infection resulted in significantly decreased frequencies of antigen – specific CD4+ T cells expressing IL-10, IL-19 and IL-24.
Conclusions
Our findings, therefore, suggest that IL-19 and IL-24 are associated with the regulation of immune responses in active filarial infection and potentially with protection against development of pathology, while IL-26 is predominantly associated with pathology in LF.
Author Summary
Lymphatic filariasis afflicts over 120 million people worldwide. While the infection is mostly clinically asymptomatic, approximately 40 million people suffer from overt, morbid clinical pathology, characterized by swelling of the scrotal area and lower limbs (hydrocele and lymphedema). Host immunologic factors that influence the pathogenesis of disease in these individuals are not completely understood. CD4+ and CD8+ T cells are known to play a role in promoting pathogenesis through the secretion of pro-inflammatory cytokines, while IL-10 is known to play an important role in dampening inflammation. IL-10 belongs to a family of cytokines that include IL-19, IL-24 and IL-26, known as the IL-10 superfamily. We investigated whether these cytokines have a function similar to IL-10 in individuals with asymptomatic infection and no clinical pathology and those with overt, clinical pathology. We first identify that CD4+ and CD8+ T cells produce these cytokines. We next identify a significant association of IL-19 and IL-24 secreting T cells with asymptomatic infection. IL-26 secreting T cells, in contrast, appear to be significantly associated with the presence of lymphatic pathology in filarial infection. Therefore, we have uncovered a potentially new regulatory pathway involving the IL-10 superfamily cytokines in filarial infections.
doi:10.1371/journal.pntd.0002762
PMCID: PMC3974669  PMID: 24699268
6.  Trichuris suis and Oesophagostomum dentatum Show Different Sensitivity and Accumulation of Fenbendazole, Albendazole and Levamisole In Vitro 
Background
The single-dose benzimidazoles used against Trichuris trichiura infections in humans are not satisfactory. Likewise, the benzimidazole, fenbendazole, has varied efficacy against Trichuris suis whereas Oesophagostomum dentatum is highly sensitive to the drug. The reasons for low treatment efficacy of Trichuris spp. infections are not known.
Methodology
We studied the effect of fenbendazole, albendazole and levamisole on the motility of T. suis and O. dentatum and measured concentrations of the parent drug compounds and metabolites of the benzimidazoles within worms in vitro. The motility and concentrations of drug compounds within worms were compared between species and the maximum specific binding capacity (Bmax) of T. suis and O. dentatum towards the benzimidazoles was estimated. Comparisons of drug uptake in living and killed worms were made for both species.
Principal findings
The motility of T. suis was generally less decreased than the motility of O. dentatum when incubated in benzimidazoles, but was more decreased when incubated in levamisole. The Bmax were significantly lower for T. suis (106.6, and 612.7 pmol/mg dry worm tissue) than O. dentatum (395.2, 958.1 pmol/mg dry worm tissue) when incubated for 72 hours in fenbendazole and albendazole respectively. The total drug concentrations (pmol/mg dry worm tissue) were significantly lower within T. suis than O. dentatum whether killed or alive when incubated in all tested drugs (except in living worms exposed to fenbendazole). Relatively high proportions of the anthelmintic inactive metabolite fenbendazole sulphone was measured within T. suis (6–17.2%) as compared to O. dentatum (0.8–0.9%).
Conclusion/Significance
The general lower sensitivity of T. suis towards BZs in vitro seems to be related to a lower drug uptake. Furthermore, the relatively high occurrence of fenbendazole sulphone suggests a higher detoxifying capacity of T. suis as compared to O. dentatum.
Author Summary
The human whipworm Trichuris trichiura is together with the roundworm Ascaris lumbricoides and the hookworms Ancylostoma duodenale and Necator Americanus the most common intestinal worms worldwide. Together they place more than 5 billion people at risk of infection. The current global control strategy against these worms is regular administration of anthelmintic drugs, mostly albendazole and mebendazole, both belonging to the drug-class benzimidazoles. Both drugs have a low effect against T. trichiura infections, but the reasons for this are not known. We evaluated the in vitro effect of two benzimidazoles; i.e., albendazole, fenbendazole, and another type of anthelmintic, levamisole, on the whipworm (T. suis) and the nodular worm (Oesophagostomum dentatum) of the pig. Oesophagostomum dentatum is highly sensitive towards benzimidazoles in comparison to T. suis. We measured and compared the drug uptake in both species in both living and killed worms. Our results suggest that the reason for the difference in sensitivity is due to a lower drug uptake into T. suis as compared to O. dentatum. Furthermore, T. suis was able to metabolise fenbendazole into an inactive metabolite to a much larger extent than O. dentatum, suggesting a higher detoxifying capacity of T. suis as compared to O. dentatum.
doi:10.1371/journal.pntd.0002752
PMCID: PMC3974671  PMID: 24699263
7.  Characterization of Guinea Pig Antibody Responses to Salivary Proteins of Triatoma infestans for the Development of a Triatomine Exposure Marker 
Background
Salivary proteins of Triatoma infestans elicit humoral immune responses in their vertebrate hosts. These immune responses indicate exposure to triatomines and thus can be a useful epidemiological tool to estimate triatomine infestation. In the present study, we analyzed antibody responses of guinea pigs to salivary antigens of different developmental stages of four T. infestans strains originating from domestic and/or peridomestic habitats in Argentina, Bolivia, Chile and Peru. We aimed to identify developmental stage- and strain-specific salivary antigens as potential markers of T. infestans exposure.
Methodology and Principal Findings
In SDS-PAGE analysis of salivary proteins of T. infestans the banding pattern differed between developmental stages and strains of triatomines. Phenograms constructed from the salivary profiles separated nymphal instars, especially the 5th instar, from adults. To analyze the influence of stage- and strain-specific differences in T. infestans saliva on the antibody response of guinea pigs, twenty-one guinea pigs were exposed to 5th instar nymphs and/or adults of different T. infestans strains. Western blot analyses using sera of exposed guinea pigs revealed stage- and strain-specific variations in the humoral response of animals. In total, 27 and 17 different salivary proteins reacted with guinea pig sera using IgG and IgM antibodies, respectively. Despite all variations of recognized salivary antigens, an antigen of 35 kDa reacted with sera of almost all challenged guinea pigs.
Conclusion
Salivary antigens are increasingly considered as an epidemiological tool to measure exposure to hematophagous arthropods, but developmental stage- and strain-specific variations in the saliva composition and the respective differences of immunogenicity are often neglected. Thus, the development of a triatomine exposure marker for surveillance studies after triatomine control campaigns requires detailed investigations. Our study resulted in the identification of a potential antigen as useful marker of T. infestans exposure.
Author Summary
Chagas disease is caused by the protozoan parasite Trypanosoma cruzi, and currently affects approximately 8 million people in Latin American countries. Although vector control campaigns against the most effective Chagas disease vector, Triatoma infestans, have been highly successful, T. infestans is re-establishing in once-endemic regions. To monitor re-establishing triatomines, new epidemiological tools are needed. Antibody responses of hosts to triatomine salivary proteins represent a promising tool to detect biting bugs, and highly immunogenic salivary antigens may be used as markers of triatomine exposure. Therefore, we analyzed the antibody response of guinea pigs, common peridomestic hosts of T. infestans, to salivary proteins of nymphs and adults of four different T. infestans strains from Argentina, Bolivia, Chile and Peru. Developmental stage- and strain-specific proteins in the saliva of T. infestans influenced the antibody response of guinea pigs, and different salivary antigens were recognized by guinea pig sera. Despite the variations of immunogenic salivary antigens, a 35 kDa antigen was recognized by almost all guinea pig sera and this antigen may be a useful marker of T. infestans exposure.
doi:10.1371/journal.pntd.0002783
PMCID: PMC3974673  PMID: 24699441
8.  Challenges in Dengue Fever in the Elderly: Atypical Presentation and Risk of Severe Dengue and Hospita-Acquired Infection 
Background/methods
To better understand dengue fever in the elderly, we compared clinical features, World Health Organization (WHO) dengue classification and outcomes between adult (<60) and elderly (≥60) dengue patients. We explored the impact of co-morbidity and hospital-acquired infection (HAI) on clinical outcomes in the elderly. All patients managed at the Communicable Disease Centre, Singapore, between 2005 and 2008 with positive dengue polymerase chain reaction (PCR) or who fulfilled WHO 1997 or 2009 probable dengue criteria with positive dengue IgM were included.
Results
Of the 6989 cases, 295 (4.4%) were elderly. PCR was positive in 29%. The elderly suffered more severe disease with more dengue haemorrhagic fever (DHF) (29.2% vs. 21.4%) and severe dengue (SD) (20.3% vs. 14.6%) (p<0.05). Classic dengue symptoms were more common in the adult group. The elderly were less likely to fulfill WHO 1997 (93.6% vs. 96.4%) (p = 0.014), but not WHO 2009 probable dengue (75.3% vs. 71.5%). Time to dengue diagnosis was similar. There was no significant difference in the frequency of warning signs between the two groups, but the elderly were more likely to have hepatomegaly (p = 0.006) and malaise/lethargy (p = 0.033) while the adults had significantly more mucosal bleeding (p<0.001). Intensive care admission occurred in 15 and death in three, with no age difference. Notably, the elderly stayed in hospital longer (median 5 vs. 4 days), and suffered more pneumonia (3.8% vs. 0.7%) and urinary infection (1.9% vs. 0.3%) (p = 0.003). Predictors of excess length of stay were age (adjusted odds ratio [aOR] 2.01, 95% confidence interval [CI] 1.37–2.88), critical illness (aOR 5.13, 95%CI 2.59–9.75), HAI (aOR 12.06, 95%CI 7.39–19.9), Charlson score (aOR 6.9, 95%CI 2.02–22.56) and severe dengue (DHF/dengue shock syndrome/SD) (aOR 2.24, 95%CI 1.83–2.74).
Conclusion
Elderly dengue patients present atypically and are at higher risk of DHF, SD and HAI. Aside from dengue severity, age, co-morbidity and HAI were associated with longer hospital stay.
Author Summary
Dengue is a neglected tropical disease that is increasingly affecting elderly patients; however, there is a paucity of data on clinical presentation and outcomes in this group. The limited data suggests that elderly dengue patients have the highest case-fatality rate but the pathogenesis of mortality in elderly dengue patients remains unclear. To better understand dengue fever in the elderly we compared clinical features, WHO dengue classification and outcomes between adult (<60) and elderly (≥60) dengue patients and explored the impact of co-morbidity and HAI on clinical outcomes in the elderly. We found that diagnosis in the elderly may be challenging due to atypical presentation. Elderly patients have worse outcomes compared with their younger counterparts with increased rates of DHF and SD. Elderly patients have higher rates of HAI placing them at risk of infection-related mortality. Aside from dengue severity, age, co-morbidity and HAI were associated with longer hospital stay. This will place further burden on already stretched hospital systems.
doi:10.1371/journal.pntd.0002777
PMCID: PMC3974675  PMID: 24699282
9.  Identification of Differentially Expressed Proteins from Leishmania amazonensis Associated with the Loss of Virulence of the Parasites 
Background
The present study analyzed whether or not the in vitro cultivation for long periods of time of pre-isolated Leishmania amazonensis from lesions of chronically infected BALB/c mice was able to interfere in the parasites' infectivity using in vivo and in vitro experiments. In addition, the proteins that presented a significant decrease or increase in their protein expression content were identified applying a proteomic approach.
Methodology/Principal Findings
Parasites were cultured in vitro for 150 days. Aliquots were collected on the day 0 of culture (R0), as well as after ten (R10; 50 days of culture), twenty (R20; 100 days of culture), and thirty (R30; 150 days of culture) passages, and were used to analyze the parasites' in vitro and in vivo infectivity, as well as to perform the proteomic approach. Approximately 837, 967, 935, and 872 spots were found in 2-DE gels prepared from R0, R10, R20, and R30 samples, respectively. A total of 37 spots presented a significant decrease in their intensity of expression, whereas a significant increase in protein content during cultivation could be observed for 19 proteins (both cases >2.0 folds). Some of these identified proteins can be described, such as diagnosis and/or vaccine candidates, while others are involved in the infectivity of Leishmania. It is interesting to note that six proteins, considered hypothetical in Leishmania, showed a significant decrease in their expression and were also identified.
Conclusions/Significance
The present study contributes to the understanding that the cultivation of parasites over long periods of time may well be related to the possible loss of infectivity of L. amazonensis. The identified proteins that presented a significant decrease in their expression during cultivation, including the hypothetical, may also be related to this loss of parasites' infectivity, and applied in future studies, including vaccine candidates and/or immunotherapeutic targets against leishmaniasis.
Author Summary
Leishmania amazonensis can induce a diversity of clinical manifestations in mammal hosts, including tegumentary and visceral leishmaniasis. The present study evaluated the variation of infectivity of L. amazonensis, which was pre-isolated from lesions of chronically infected mice and in vitro cultured for 150 days, in turn connecting these results with the profile of parasite protein expression using a proteomic approach. Parasites were recovered after the first passage, as well as after 50, 100, and 150 days of axenic cultures, and were subsequently evaluated. A total of 37 proteins presented a significant decrease, whereas 19 proteins presented a significant increase in their protein expression content in the assays (both cases >2.0 fold). Some of the identified proteins have been reported in prior literature, including diagnosis and/or vaccine candidates for leishmaniasis, while others proved to be involved in the infectivity of Leishmania. It is interesting to note that proteins related to the parasites' metabolism were also the majority of the proteins identified in the old cultures of L. amazonensis, suggesting a possible relation between the metabolic state of parasites and their possible loss of infectivity. In conclusion, the proteins identified in this study represent a contribution to the discovery of new vaccine candidates and/or immunotherapeutic targets against leishmaniasis.
doi:10.1371/journal.pntd.0002764
PMCID: PMC3974679  PMID: 24699271
10.  Molecular Analysis of Echinostome Metacercariae from Their Second Intermediate Host Found in a Localised Geographic Region Reveals Genetic Heterogeneity and Possible Cryptic Speciation 
Echinostome metacercariae are the infective stage for humans and animals. The identification of echinostomes has been based until recently on morphology but molecular techniques using sequences of ribosomal RNA and mitochondrial DNA have indicated major clades within the group. In this study we have used the ITS2 region of ribosomal RNA and the ND1 region of mitochondrial DNA to identify metacercariae from snails collected from eight well-separated sites from an area of 4000 km2 in Lamphun Province, Thailand. The derived sequences have been compared to those collected from elsewhere and have been deposited in the nucleotide databases. There were two aims of this study; firstly, to determine the species of echinostome present in an endemic area, and secondly, to assess the intra-specific genetic diversity, as this may be informative with regard to the potential for the development of anthelmintic resistance and with regard to the spread of infection by the definitive hosts. Our results indicate that the most prevalent species are most closely related to E. revolutum, E. trivolvis, E. robustum, E. malayanum and Euparyphium albuferensis. Some sites harbour several species and within a site there could be considerable intra-species genetic diversity. There is no significant geographical structuring within this area. Although the molecular techniques used in this study allowed the assignment of the samples to clades within defined species, however, within these groupings there were significant differences indicating that cryptic speciation may have occurred. The degree of genetic diversity present would suggest the use of targeted regimes designed to minimise the selection of anthelmintic resistance. The apparent lack of geographic structuring is consistent with the transmission of the parasites by the avian hosts.
Author Summary
Infections by food-borne trematodes are estimated to infect over 40 million people worldwide, although infections by echinostomes make up only a portion of these cases, usually in regions where their prevalence is high. In South East Asia and in the far east of Asia, human infection is associated with cultural and dietary factors and the prevalence of infection may reach 50% in parts of Thailand, Cambodia, and Laos. Treatment is generally dependent on the use of praziquantel or benzimidazole drugs but with the occurrence of anthelmintic resistance to these compounds it would be desirable to have an understanding of the diversity present in the echinostome populations within a given locality. This study deals with the systematics of echinostomes and informs various aspects of the epidemiology of echinostomiasis which may aid the development of future control strategies.
doi:10.1371/journal.pntd.0002778
PMCID: PMC3974680  PMID: 24699358
11.  The Mycetoma Knowledge Gap: Identification of Research Priorities 
Mycetoma is a tropical disease which is caused by a taxonomically diverse range of actinomycetes (actinomycetoma) and fungi (eumycetoma). The disease was only recently listed by the World Health Organization (WHO) as a neglected tropical disease (NTD). This recognition is the direct result of a meeting held in Geneva on February 1, 2013, in which experts on the disease from around the world met to identify the key research priorities needed to combat mycetoma. The areas that need to be addressed are highlighted here. The initial priority is to establish the incidence and prevalence of the disease in regions where mycetoma is endemic, prior to determining the primary reservoirs of the predominant causal agents and their mode of transmission to susceptible individuals in order to establish novel interventions that will reduce the impact of the disease on individuals, families, and communities. Critically, economical, reliable, and effective methods are required to achieve early diagnosis of infections and consequential improved therapeutic outcomes. Molecular techniques and serological assays were considered the most promising in the development of novel diagnostic tools to be used in endemic settings. Improved strategies for treating eumycetoma and actinomycetoma are also considered.
doi:10.1371/journal.pntd.0002667
PMCID: PMC3967943  PMID: 24675533
12.  Helminth-Associated Systemic Immune Activation and HIV Co-receptor Expression: Response to Albendazole/Praziquantel Treatment 
Background
It has been hypothesized that helminth infections increase HIV susceptibility by enhancing systemic immune activation and hence contribute to elevated HIV-1 transmission in sub-Saharan Africa.
Objective
To study systemic immune activation and HIV-1 co-receptor expression in relation to different helminth infections and in response to helminth treatment.
Methods
HIV-negative adults with (n = 189) or without (n = 57) different helminth infections, as diagnosed by Kato-Katz, were enrolled in Mbeya, Tanzania. Blinded to helminth infection status, T cell differentiation (CD45RO, CD27), activation (HLA-DR, CD38) and CCR5 expression was determined at baseline and 3 months after Albendazole/Praziquantel treatment. Plasma cytokine levels were compared using a cytometric bead array.
Results
Trichuris and Ascaris infections were linked to increased frequencies of “activated” CD4 and/or CD8 T cells (p<0.05), whereas Hookworm infection was associated with a trend towards decreased HLA-DR+ CD8 T cell frequencies (p = 0.222). In Trichuris infected subjects, there was a linear correlation between HLA-DR+ CD4 T cell frequencies and the cytokines IL-1β and IL-10 (p<0.05). Helminth treatment with Albendazole and Praziquantel significantly decreased eosinophilia for S. mansoni and Hookworm infections (p<0.005) but not for Trichuris infection and only moderately modulated T cell activation. CCR5 surface density on memory CD4 T cells was increased by 1.2-fold during Trichuris infection (p-value: 0.053) and reduced after treatment (p = 0.003).
Conclusions
Increased expression of T cell activation markers was associated with Trichuris and Ascaris infections with relatively little effect of helminth treatment.
Author Summary
Helminth infections are common in sub-Saharan Africa where about half of the population may be infected with one or more helminth species. HIV infection is also highly prevalent in this region. Because of the geographic overlap of helminth and HIV infections, it has been hypothesized that helminth infections may increase susceptibility to HIV by increasing systemic immune activation, which has been linked to increased HIV susceptibility. We therefore investigated the profile of T cell activation in individuals infected with different helminth species before and after helminth treatment within the WHIS cohort in Mbeya, Tanzania. Our study shows that systemic T cell activation differs between infections with different helminths. Particularly Trichuris but also Ascaris and S. mansoni infections were linked to increased frequencies of activated, HLA-DR+ T cells with relatively little effect of helminth treatment. Hookworm infection was associated with a trend towards decreased frequencies of activated, HLA-DR+ CD8+ T cells. Our study supports the concept that helminth infections, which are linked to systemic immune activation, could potentially also contribute to increased HIV transmission.
doi:10.1371/journal.pntd.0002755
PMCID: PMC3967945  PMID: 24675895
13.  RNA-Seq Reveals Infection-Induced Gene Expression Changes in the Snail Intermediate Host of the Carcinogenic Liver Fluke, Opisthorchis viverrini 
Background
Bithynia siamensis goniomphalos is the snail intermediate host of the liver fluke, Opisthorchis viverrini, the leading cause of cholangiocarcinoma (CCA) in the Greater Mekong sub-region of Thailand. Despite the severe public health impact of Opisthorchis-induced CCA, knowledge of the molecular interactions occurring between the parasite and its snail intermediate host is scant. The examination of differences in gene expression profiling between uninfected and O. viverrini-infected B. siamensis goniomphalos could provide clues on fundamental pathways involved in the regulation of snail-parasite interplay.
Methodology/Principal Findings
Using high-throughput (Illumina) sequencing and extensive bioinformatic analyses, we characterized the transcriptomes of uninfected and O. viverrini-infected B. siamensis goniomphalos. Comparative analyses of gene expression profiling allowed the identification of 7,655 differentially expressed genes (DEGs), associated to 43 distinct biological pathways, including pathways associated with immune defense mechanisms against parasites. Amongst the DEGs with immune functions, transcripts encoding distinct proteases displayed the highest down-regulation in Bithynia specimens infected by O. viverrini; conversely, transcription of genes encoding heat-shock proteins and actins was significantly up-regulated in parasite-infected snails when compared to the uninfected counterparts.
Conclusions/Significance
The present study lays the foundation for functional studies of genes and gene products potentially involved in immune-molecular mechanisms implicated in the ability of the parasite to successfully colonize its snail intermediate host. The annotated dataset provided herein represents a ready-to-use molecular resource for the discovery of molecular pathways underlying susceptibility and resistance mechanisms of B. siamensis goniomphalos to O. viverrini and for comparative analyses with pulmonate snail intermediate hosts of other platyhelminths including schistosomes.
Author Summary
Despite recent significant advances in knowledge of the fundamental biology of the carcinogenic liver fluke Opisthorchis viverrini, little is known of the complement of molecular interactions occurring between this parasite and its prosobranch snail intermediate host, Bithynia siamensis goniomphalos. The determination of such interactions is a key, necessary component of the development of future integrated control strategies for liver fluke infection and associated bile duct cancer. Here, we use cutting-edge high-throughput sequencing technologies and advanced bioinformatic analyses to characterize, for the first time, qualitative and quantitative differences in gene expression between uninfected and O. viverrini-infected B. siamensis goniomphalos collected from an endemic region of Northeast Thailand. The analyses led to the identification of a number of molecules putatively involved in immune defense pathways against invading O. viverrini, and of key biological mechanisms potentially implicated in the ability of the parasite to successfully colonize its snail intermediate host. We believe that this ready-to-use molecular resource will provide the scientific community with new tools for the development of strategies to control the spread of liver fluke infection and the resulting bile duct cancer.
doi:10.1371/journal.pntd.0002765
PMCID: PMC3967946  PMID: 24676090
15.  Enhanced Protective Efficacy of Nonpathogenic Recombinant Leishmania tarentolae Expressing Cysteine Proteinases Combined with a Sand Fly Salivary Antigen 
Background
Novel vaccination approaches are needed to prevent leishmaniasis. Live attenuated vaccines are the gold standard for protection against intracellular pathogens such as Leishmania and there have been new developments in this field. The nonpathogenic to humans lizard protozoan parasite, Leishmania (L) tarentolae, has been used effectively as a vaccine platform against visceral leishmaniasis in experimental animal models. Correspondingly, pre-exposure to sand fly saliva or immunization with a salivary protein has been shown to protect mice against cutaneous leishmaniasis.
Methodology/Principal Findings
Here, we tested the efficacy of a novel combination of established protective parasite antigens expressed by L. tarentolae together with a sand fly salivary antigen as a vaccine strategy against L. major infection. The immunogenicity and protective efficacy of different DNA/Live and Live/Live prime-boost vaccination modalities with live recombinant L. tarentolae stably expressing cysteine proteinases (type I and II, CPA/CPB) and PpSP15, an immunogenic salivary protein from Phlebotomus papatasi, a natural vector of L. major, were tested both in susceptible BALB/c and resistant C57BL/6 mice. Both humoral and cellular immune responses were assessed before challenge and at 3 and 10 weeks after Leishmania infection. In both strains of mice, the strongest protective effect was observed when priming with PpSP15 DNA and boosting with PpSP15 DNA and live recombinant L. tarentolae stably expressing cysteine proteinase genes.
Conclusion/Significance
The present study is the first to use a combination of recombinant L. tarentolae with a sand fly salivary antigen (PpSP15) and represents a novel promising vaccination approach against leishmaniasis.
Author Summary
More than 98 countries are reported as endemic for leishmaniasis, a vector-borne disease transmitted by sand flies. Drug-resistant forms have emerged and there is an increased need to develop advanced preventive strategies. Live attenuated vaccines are the gold standard for protection against intracellular pathogens such as Leishmania and there have been new developments in this field. The lizard protozoan parasite, L. tarentolae, is nonpathogenic to humans and has been used effectively as a vaccine platform against visceral leishmaniasis in experimental animal models. Correspondingly, pre-exposure to sand fly saliva or immunization with salivary proteins has been shown to protect mice against cutaneous leishmaniasis. Herein, we used DNA/Live and Live/Live prime-boost vaccination strategies against cutaneous leishmaniasis based on recombinant L. tarentolae stably expressing CPA/CPB genes with and without the sand fly salivary antigen PpSP15 in both resistant and susceptible mice models. Assessment of the immune response and parasite burden in vaccinated mice at different time intervals post-challenge demonstrated that combination of recombinant L. tarentolae CPA/CPB with PpSP15 DNA elicits an enhanced protective immune response against cutaneous leishmaniasis in mice. This parasite- and insect vector-derived antigen combination represents an important step forward in the development of new vaccine strategies against Leishmania infections.
doi:10.1371/journal.pntd.0002751
PMCID: PMC3967951  PMID: 24675711
16.  Mycobacterium ulcerans Persistence at a Village Water Source of Buruli Ulcer Patients 
Buruli ulcer (BU), a neglected tropical disease of the skin and subcutaneous tissue, is caused by Mycobacterium ulcerans and is the third most common mycobacterial disease after tuberculosis and leprosy. While there is a strong association of the occurrence of the disease with stagnant or slow flowing water bodies, the exact mode of transmission of BU is not clear. M. ulcerans has emerged from the environmental fish pathogen M. marinum by acquisition of a virulence plasmid encoding the enzymes required for the production of the cytotoxic macrolide toxin mycolactone, which is a key factor in the pathogenesis of BU. Comparative genomic studies have further shown extensive pseudogene formation and downsizing of the M. ulcerans genome, indicative for an adaptation to a more stable ecological niche. This has raised the question whether this pathogen is still present in water-associated environmental reservoirs. Here we show persistence of M. ulcerans specific DNA sequences over a period of more than two years at a water contact location of BU patients in an endemic village of Cameroon. At defined positions in a shallow water hole used by the villagers for washing and bathing, detritus remained consistently positive for M. ulcerans DNA. The observed mean real-time PCR Ct difference of 1.45 between the insertion sequences IS2606 and IS2404 indicated that lineage 3 M. ulcerans, which cause human disease, persisted in this environment after successful treatment of all local patients. Underwater decaying organic matter may therefore represent a reservoir of M. ulcerans for direct infection of skin lesions or vector-associated transmission.
Author Summary
Buruli ulcer (BU) is a neglected tropical disease caused by Mycobacterium ulcerans which affects mainly children in West Africa. Although it is commonly believed that the infection originates from an environmental source, both the reservoir of M. ulcerans and the mode of transmission to human patients remain to be elucidated. Previous investigations indicated that transmission likely takes place away from the homes of patients. We therefore screened the farms as well as village and farm water locations of 46 laboratory confirmed BU patients of the Mapé Basin of Cameroon for the presence of M. ulcerans DNA by real-time PCR. In this analysis three positive village water locations were identified. By studying one of these locations in great detail we found that M. ulcerans DNA persists in underwater detritus in one section of the village water location even after all local cases had been treated. The detritus may represent a reservoir of M. ulcerans from where infection could take place through either direct contamination of skin lesions or through contamination or colonization of insect vectors.
doi:10.1371/journal.pntd.0002756
PMCID: PMC3967953  PMID: 24675964
18.  Active Matrix Metalloprotease-9 Is Associated with the Collagen Capsule Surrounding the Madurella mycetomatis Grain in Mycetoma 
Madurella mycetomatis is the main causative organism of eumycetoma, a persistent, progressive granulomatous infection. After subcutaneous inoculation M. mycetomatis organizes itself in grains inside a granuloma with excessive collagen accumulation surrounding it. This could be contributing to treatment failure towards currently used antifungal agents. Due to their pivotal role in tissue remodelling, matrix metalloproteinases-2 (MMP-2) and 9 (MMP-9) or tissue inhibitor of metalloproteinases (TIMP) might be involved in this process. Local MMP-2 and MMP-9 expression was assessed by immunohistochemistry while absolute serum levels of these enzymes were determined in mycetoma patients and healthy controls by performing ELISAs. The presence of active MMP was determined by gelatin zymography. We found that both MMP-2 and MMP-9 are expressed in the mycetoma lesion, but the absolute MMP-2, -9, and TIMP-1 serum levels did not significantly differ between patients and controls. However, active MMP-9 was found in sera of 36% of M. mycetomatis infected subjects, whereas this active form was absent in sera of controls (P<0.0001). MMP-2, MMP-9, and TIMP-1 polymorphisms in mycetoma patients and healthy controls were determined through PCR-RFLP or sequencing. A higher T allele frequency in TIMP-1 (+372) SNP was observed in male M. mycetomatis mycetoma patients compared to controls. The presence of active MMP-9 in mycetoma patients suggest that MMP-9 is activated or synthesized by inflammatory cells upon M. mycetomatis infection. Inhibiting MMP-9 activity with doxycycline could prevent collagen accumulation in mycetoma, which in its turn might make the fungus more accessible to antifungal agents.
Author Summary
Eumycetoma, mainly caused by the fungus Madurella mycetomatis, is a chronic infection which, without treatment, results in deformation of the infected body part. Inside the body, the fungus organises itself in grains which are surrounded by collagen. This collagen could act as a natural barrier for antifungal agents. Since collagen modulation is regulated by matrix metalloproteinase-2 (MMP-2), MMP-9 and tissue inhibitors of metalloproteinases (TIMPs), these enzymes could play a role in the formation of the collagen capsule surrounding the fungal grain. Indeed, we demonstrated that MMPs were found surrounding the mycetoma grain and that measurable levels of both MMPs were found in serum of both mycetoma patients and healthy controls. Only in mycetoma patients the active form MMP-9 was found. The presence of active MMP-9 in the serum of mycetoma-patients was not the result of lower levels TIMP-1 but more likely from differences in allele frequencies in the TIMP-1 gene. In conclusion, our results showed an increased MMP-9 activity in mycetoma patients. We hypothesize that inhibition of MMP-9 activity by doxycycline will result in breakdown of the collagen capsule surrounding the grain, which in turn will make the entrance of antifungal drugs into the grain easier.
doi:10.1371/journal.pntd.0002754
PMCID: PMC3967957  PMID: 24675764
19.  Trypsin- and Chymotrypsin-Like Serine Proteases in Schistosoma mansoni – ‘The Undiscovered Country’ 
Background
Blood flukes (Schistosoma spp.) are parasites that can survive for years or decades in the vasculature of permissive mammalian hosts, including humans. Proteolytic enzymes (proteases) are crucial for successful parasitism, including aspects of invasion, maturation and reproduction. Most attention has focused on the ‘cercarial elastase’ serine proteases that facilitate skin invasion by infective schistosome larvae, and the cysteine and aspartic proteases that worms use to digest the blood meal. Apart from the cercarial elastases, information regarding other S. mansoni serine proteases (SmSPs) is limited. To address this, we investigated SmSPs using genomic, transcriptomic, phylogenetic and functional proteomic approaches.
Methodology/Principal Findings
Genes encoding five distinct SmSPs, termed SmSP1 - SmSP5, some of which comprise disparate protein domains, were retrieved from the S. mansoni genome database and annotated. Reverse transcription quantitative PCR (RT- qPCR) in various schistosome developmental stages indicated complex expression patterns for SmSPs, including their constituent protein domains. SmSP2 stood apart as being massively expressed in schistosomula and adult stages. Phylogenetic analysis segregated SmSPs into diverse clusters of family S1 proteases. SmSP1 to SmSP4 are trypsin-like proteases, whereas SmSP5 is chymotrypsin-like. In agreement, trypsin-like activities were shown to predominate in eggs, schistosomula and adults using peptidyl fluorogenic substrates. SmSP5 is particularly novel in the phylogenetics of family S1 schistosome proteases, as it is part of a cluster of sequences that fill a gap between the highly divergent cercarial elastases and other family S1 proteases.
Conclusions/Significance
Our series of post-genomics analyses clarifies the complexity of schistosome family S1 serine proteases and highlights their interrelationships, including the cercarial elastases and, not least, the identification of a ‘missing-link’ protease cluster, represented by SmSP5. A framework is now in place to guide the characterization of individual proteases, their stage-specific expression and their contributions to parasitism, in particular, their possible modulation of host physiology.
Author Summary
Schistosomes are blood flukes that live in the blood system and cause chronic and debilitating infection in hundreds of millions of people. Proteolytic enzymes (proteases) produced by the parasite allow it to survive and reproduce. We focused on understanding the repertoire of trypsin- and chymotrypsin-like Schistosoma mansoni serine proteases (SmSPs) using a variety of genomic, bioinformatics, RNA- and protein-based techniques. We identified five SmSPs that are produced at different stages of the parasite's development. Based on bioinformatics and cleavage preferences for small peptide substrates, SmSP1 to SmSP4 are trypsin-like, whereas SmSP5 is chymotrypsin-like. Interestingly, SmSP5 forms part of a ‘missing link’ group of enzymes between the specialized chymotrypsin-like ‘cercarial elastases’ that help the parasite invade human skin and the more typical chymotrypsins and trypsins found in the nature. Our findings form a basis for further exploration of the functions of the individual enzymes, including their possible contributions to influencing host physiology.
doi:10.1371/journal.pntd.0002766
PMCID: PMC3967958  PMID: 24676141
20.  Serologic Prevalence of Toxoplasma gondii in Indian Women of Child Bearing Age and Effects of Social and Environmental Factors 
Background
Seroprevalence and incidence of toxoplasmosis in women of child bearing age has remained a contentious issue in the Indian subcontinent. Different laboratories have used different patient recruitment criteria, methods and variable results, making these data difficult to compare.
Aim
To map the point-prevalence and incidence of toxoplasmosis in India.
Material and Methods
In this cross-sectional study, a total of 1464 women of fertile age were recruited from 4 regions using similar recruitment plans. This included women from northern (203), southern (512), eastern (250) and western (501) regions of India. All samples were transported to a central laboratory in Delhi and tested using VIDAS technology. Their age, parity, eating habits and other demographic and clinical details were noted.
Results
Most women were in the 18–25 years age group (48.3%), followed by 26–30 years (28.2%) and 31–35 years (13.66). Few (45) women older than 35 yr. were included. Overall prevalence of anti-Toxoplasma IgG antibodies was seen in 22.40%, with significantly more in married women (25.8%) as compared to single women (4.3%). Prevalence increased steadily with age: 18.1% in the 18–25 yr. age group to 40.5% in women older than 40 yr. The prevalence was high (66%) in those who resided in mud houses. Region-wise, the highest prevalence was observed in South India (37.3%) and the lowest (8.8%) in West Indian women. This difference was highly significant (P<0.001). Prevalence was 21.2% in East India and 19.7% in North India. The IgM positivity rate ranged from 0.4% to 2.9% in four study centers.
Conclusions
This pan-India study shows a prevalence rate of 22.4% with a wide variation in four geographical regions ranging from as low as 8.8% to as high as 37.3%. The overall IgM positivity rate was 1.43%, indicating that an estimated 56,737–176,882 children per year are born in India with a possible risk of congenital toxoplasmosis.
Author Summary
Toxoplasmosis is a protozoan parasitic disease commonly transmitted and propagated by cats as family pets. Infection acquired during pregnancy can lead to congenital abnormalities in the fetus, still birth or intrauterine death. Seroprevalence and incidence of toxoplasmosis in Indian women of child bearing age has remained a contentious issue. Different laboratories have used different patient recruitment criteria, methods and variable results, making these data unreliable. There is no published pan-India seroprevalence study. Hence, a seroprevalence study was undertaken comprising 1464 women of reproductive age representing four distinct geographical regions of India. This resulted in an estimated prevalence of 22.4% (328); the highest prevalence being in South India (37.3%) followed by East India (21.2%) and North India (19.7%). West Indian women had the lowest seroprevalence (8.8%). This difference was highly significant. In our analysis we determined the possible risk-factors of infection in these women. These included lower socioeconomic status, residing in mud plastered houses, consumption of raw salad, drinking untreated water, owning pets and advanced age. Overall, the incidence rate of toxoplasmosis was 1.43%. Extrapolating the data, we estimate that between 56,737 and 176,882 children a year may be born in India with a possible risk of congenital toxoplasmosis, which can manifest itself in-utero or several years after birth.
doi:10.1371/journal.pntd.0002737
PMCID: PMC3967963  PMID: 24675656
21.  Opportunities for Improved Chagas Disease Vector Control Based on Knowledge, Attitudes and Practices of Communities in the Yucatan Peninsula, Mexico 
Background
Chagas disease is a vector-borne parasitic disease of major public health importance. Current prevention efforts are based on triatomine vector control to reduce transmission to humans. Success of vector control interventions depends on their acceptability and value to affected communities. We aimed to identify opportunities for and barriers to improved vector control strategies in the Yucatan peninsula, Mexico.
Methodology/principal findings
We employed a sequence of qualitative and quantitative research methods to investigate knowledge, attitudes and practices surrounding Chagas disease, triatomines and vector control in three rural communities. Our combined data show that community members are well aware of triatomines and are knowledgeable about their habits. However, most have a limited understanding of the transmission dynamics and clinical manifestations of Chagas disease. While triatomine control is not a priority for community members, they frequently use domestic insecticide products including insecticide spray, mosquito coils and plug-in repellents. Families spend about $32 US per year on these products. Alternative methods such as yard cleaning and window screens are perceived as desirable and potentially more effective. Screens are nonetheless described as unaffordable, in spite of a cost comparable to the average annual spending on insecticide products.
Conclusion/Significance
Further education campaigns and possibly financing schemes may lead families to redirect their current vector control spending from insecticide products to window screens. Also, synergism with mosquito control efforts should be further explored to motivate community involvement and ensure sustainability of Chagas disease vector control.
Author Summary
Chagas disease is an important parasitic disease transmitted by triatomine bugs. Current prevention efforts are based on eliminating triatomines from homes to reduce disease transmission to humans. However, the success of these control interventions depends on their acceptability and value to affected communities. We aimed to identify opportunities for and barriers to triatomine control strategies in the Yucatan peninsula, Mexico. We used a sequence of group discussion, interviews, and a survey to investigate the perception and knowledge of communities on Chagas disease and triatomines in three villages from the Yucatan peninsula, Mexico. Inhabitants are rather familiar with triatomine bugs, but do not associate well these bugs with Chagas disease and its clinical manifestations. Mosquito rather than triatomine control is a common preoccupation, and households frequently use insecticide spray, mosquito coils and plug-in repellents, spending about $32 US per year on these products. Alternative methods such as yard cleaning and window screens are perceived as desirable and potentially more effective. Screens are nonetheless described as unaffordable. The promotion of education campaigns and possibly financing schemes could help families to redirect their current spending from insecticide products to window screens. Also, synergism with mosquito control efforts should be further explored to motivate community involvement and ensure sustainability of Chagas disease vector control.
doi:10.1371/journal.pntd.0002763
PMCID: PMC3967964  PMID: 24676038
22.  Identification of Parasitic Communities within European Ticks Using Next-Generation Sequencing 
Background
Risk assessment of tick-borne and zoonotic disease emergence necessitates sound knowledge of the particular microorganisms circulating within the communities of these major vectors. Assessment of pathogens carried by wild ticks must be performed without a priori, to allow for the detection of new or unexpected agents.
Methodology/Principal Findings
We evaluated the potential of Next-Generation Sequencing techniques (NGS) to produce an inventory of parasites carried by questing ticks. Sequences corresponding to parasites from two distinct genera were recovered in Ixodes ricinus ticks collected in Eastern France: Babesia spp. and Theileria spp. Four Babesia species were identified, three of which were zoonotic: B. divergens, Babesia sp. EU1 and B. microti; and one which infects cattle, B. major. This is the first time that these last two species have been identified in France. This approach also identified new sequences corresponding to as-yet unknown organisms similar to tropical Theileria species.
Conclusions/Significance
Our findings demonstrate the capability of NGS to produce an inventory of live tick-borne parasites, which could potentially be transmitted by the ticks, and uncovers unexpected parasites in Western Europe.
Author Summary
Diseases transmitted by ticks have diverse etiology (viral, bacterial, parasitic) and are responsible for high morbidity and mortality rates around the world, both in humans and animals. The emergence or re-emergence of tick-borne diseases is increasingly becoming a problem as the geographical distribution of several tick species is expanding, as well as the numbers of potential or known tick-borne pathogens are constantly evolving. It is thus necessary to know which microorganisms circulate within communities of this major vector to ensure adequate epidemiological surveillance. In this study, we evaluated the potential of Next-Generation Sequencing techniques (NGS) to produce, without a priori, an inventory of both predicted and non-expected parasites carried by Ixodes ricinus, the most prevalent human biting tick in France. Our findings suggest that NGS strategies could be used to produce an inventory of live parasites residing in ticks from a selected area, thereby expanding our knowledge base of tick-associated parasites.
doi:10.1371/journal.pntd.0002753
PMCID: PMC3967966  PMID: 24675738
23.  Dispersal of Adult Culex Mosquitoes in an Urban West Nile Virus Hotspot: A Mark-Capture Study Incorporating Stable Isotope Enrichment of Natural Larval Habitats 
Dispersal is a critical life history behavior for mosquitoes and is important for the spread of mosquito-borne disease. We implemented the first stable isotope mark-capture study to measure mosquito dispersal, focusing on Culex pipiens in southwest suburban Chicago, Illinois, a hotspot of West Nile virus (WNV) transmission. We enriched nine catch basins in 2010 and 2011 with 15N-potassium nitrate and detected dispersal of enriched adult females emerging from these catch basins using CDC light and gravid traps to distances as far as 3 km. We detected 12 isotopically enriched pools of mosquitoes out of 2,442 tested during the two years and calculated a mean dispersal distance of 1.15 km and maximum flight range of 2.48 km. According to a logistic distribution function, 90% of the female Culex mosquitoes stayed within 3 km of their larval habitat, which corresponds with the distance-limited genetic variation of WNV observed in this study region. This study provides new insights on the dispersal of the most important vector of WNV in the eastern United States and demonstrates the utility of stable isotope enrichment for studying the biology of mosquitoes in other disease systems.
Author Summary
The distance and direction of adult mosquitoes movement on the landscape are important processes in the spread of mosquito-borne diseases, and are critical to understand to the development of effective intervention programs. Here we present a novel approach to study adult mosquito dispersal by using stable isotope enrichment of natural larval habitats. We apply this technique in a focal hotspot of West Nile virus (WNV) transmission in suburban, Chicago, USA to measure dispersal of Culex spp. mosquitoes. We enriched larval mosquitoes in residential catch basins using 15N-potassium nitrate and captured adult mosquitoes in traps surrounding these catch basins. Of 10,817 adult female Culex mosquitoes trapped and tested for stable isotopes, 12 individuals were enriched with 15N, indicating they originated from the catch basins receiving stable isotope amendments. The mean dispersal distance was 1.15 km and maximum flight range was 2.48 km. Ninety percent of the female Culex mosquitoes stayed within 3 km of their larval habitat, which corresponds with the distance-limited genetic variation of WNV observed in this study region. This study provides new insights on the dispersal of the most important vector of WNV in the eastern United States and demonstrates the utility of stable isotope enrichment for studying the biology of mosquitoes in other disease systems.
doi:10.1371/journal.pntd.0002768
PMCID: PMC3967984  PMID: 24676212
24.  Defining the Geographical Range of the Plasmodium knowlesi Reservoir 
Background
The simian malaria parasite, Plasmodium knowlesi, can cause severe and fatal disease in humans yet it is rarely included in routine public health reporting systems for malaria and its geographical range is largely unknown. Because malaria caused by P. knowlesi is a truly neglected tropical disease, there are substantial obstacles to defining the geographical extent and risk of this disease. Information is required on the occurrence of human cases in different locations, on which non-human primates host this parasite and on which vectors are able to transmit it to humans. We undertook a systematic review and ranked the existing evidence, at a subnational spatial scale, to investigate the potential geographical range of the parasite reservoir capable of infecting humans.
Methodology/Principal Findings
After reviewing the published literature we identified potential host and vector species and ranked these based on how informative they are for the presence of an infectious parasite reservoir, based on current evidence. We collated spatial data on parasite occurrence and the ranges of the identified host and vector species. The ranked spatial data allowed us to assign an evidence score to 475 subnational areas in 19 countries and we present the results on a map of the Southeast and South Asia region.
Conclusions/Significance
We have ranked subnational areas within the potential disease range according to evidence for presence of a disease risk to humans, providing geographical evidence to support decisions on prevention, management and prophylaxis. This work also highlights the unknown risk status of large parts of the region. Within this unknown category, our map identifies which areas have most evidence for the potential to support an infectious reservoir and are therefore a priority for further investigation. Furthermore we identify geographical areas where further investigation of putative host and vector species would be highly informative for the region-wide assessment.
Author Summary
Plasmodium knowlesi is a malaria parasite found in monkeys which can infect humans via mosquito bites. People infected with the P. knowlesi parasite can suffer severe disease and death yet this disease has often been misdiagnosed as a different malaria type and its geographical distribution is largely unknown. The lack of data on human infections in much of Southeast Asia means a simple map of reported cases would likely misrepresent the extent of the disease. Instead we evaluated and ranked a range of evidence types according to how informative they are about the presence of an infection risk to humans and we mapped this ranked information. This highlighted those geographical areas where new data on the monkey and mosquito species involved in the infection of humans would add most to our knowledge of the full range of factors involved in disease risk. The resulting map highlights known locations of the parasite, and areas where presence of the disease in humans is unknown but possible.
doi:10.1371/journal.pntd.0002780
PMCID: PMC3967999  PMID: 24676231
25.  Humans and Great Apes Cohabiting the Forest Ecosystem in Central African Republic Harbour the Same Hookworms 
Background
Hookworms are important pathogens of humans. To date, Necator americanus is the sole, known species of the genus Necator infecting humans. In contrast, several Necator species have been described in African great apes and other primates. It has not yet been determined whether primate-originating Necator species are also parasitic in humans.
Methodology/Principal Findings
The infective larvae of Necator spp. were developed using modified Harada-Mori filter-paper cultures from faeces of humans and great apes inhabiting Dzanga-Sangha Protected Areas, Central African Republic. The first and second internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal DNA and partial cytochrome c oxidase subunit 1 (cox1) gene of mtDNA obtained from the hookworm larvae were sequenced and compared. Three sequence types (I–III) were recognized in the ITS region, and 34 cox1 haplotypes represented three phylogenetic groups (A–C). The combinations determined were I-A, II-B, II-C, III-B and III-C. Combination I-A, corresponding to N. americanus, was demonstrated in humans and western lowland gorillas; II-B and II-C were observed in humans, western lowland gorillas and chimpanzees; III-B and III-C were found only in humans. Pairwise nucleotide difference in the cox1 haplotypes between the groups was more than 8%, while the difference within each group was less than 2.1%.
Conclusions/Significance
The distinctness of ITS sequence variants and high number of pairwise nucleotide differences among cox1 variants indicate the possible presence of several species of Necator in both humans and great apes. We conclude that Necator hookworms are shared by humans and great apes co-habiting the same tropical forest ecosystems.
Author Summary
We conducted analyses of DNA sequences obtained from the infective larvae of Necator spp. from humans and great apes inhabiting Dzanga-Sangha Protected Areas, Central African Republic. Three sequence types (I–III) were recognized in the in the ITS region, and 34 cox1 haplotypes represented three phylogenetic groups (A–C). I-A, II-B, II-C, III-B, III-C combinations were determined. Combination I-A, corresponding to Necator americanus, was demonstrated in humans and western lowland gorillas; II-B and II-C were observed in humans (local inhabitants and researchers), western lowland gorillas and chimpanzees; III-B and III-C were found only in humans. Pairwise nucleotide difference in the cox1 haplotypes between the groups was more than 8%, while the difference within each group was less than 2.1%, suggesting that each type represents a distinct species. This is the first molecular evidence that Necator species found in great apes can infect humans and vice versa.
doi:10.1371/journal.pntd.0002715
PMCID: PMC3961186  PMID: 24651493

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