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1.  Antimony Resistant Leishmania donovani but Not Sensitive Ones Drives Greater Frequency of Potent T-Regulatory Cells upon Interaction with Human PBMCs: Role of IL-10 and TGF-β in Early Immune Response 
In India the sand fly, Phlebotomus argentipes, transmitted parasitic disease termed kala-azar is caused by Leishmania donovani (LD) in humans. These immune-evading parasites have increasingly developed resistance to the drug sodium antimony gluconate in endemic regions.
Lack of early diagnosis methods for the disease limits the information available regarding the early interactions of this parasite with either human tissues or cell lineages. We reasoned that peripheral blood mononuclear cells (PBMCs) from healthy human beings could help compare some of their immune signatures once they were exposed for up to 8 days, to either pentavalent antimony sensitive (SbS-LD) or resistant (SbR-LD) Leishmania donovani isolates.
At day 2, PBMC cultures exposed to SbS-LD and SbR-LD stationary phase promastigotes had four and seven fold higher frequency of IL-10 secreting monocyte-macrophage respectively, compared to cultures unexposed to parasites. Contrasting with the CD4+CD25−CD127− type-1 T-regulatory (Tr1) cell population that displayed similar features whatever the culture conditions, there was a pronounced increase in the IL-10 producing CD4+CD25+CD127low/− inducible T-regulatory cells (iTregs) in the PBMC cultures sampled at day 8 post addition of SbR-LD.
Sorted iTregs from different cultures on day 8 were added to anti-CD3/CD28 induced naïve PBMCs to assess their suppressive ability. We observed that iTregs from SbR-LD exposed PBMCs had more pronounced suppressive ability compared to SbS-LD counterpart on a per cell basis and is dependent on both IL-10 and TGF-β, whereas IL-10 being the major factor contributing to the suppressive ability of iTregs sorted from PBMC cultures exposed to SbS–LD. Of note, iTreg population frequency value remained at the basal level after addition of genetically modified SbR-LD lacking unique terminal sugar in surface glycan.
Even with limitations of this artificial in vitro model of L. donovani-human PBMC interactions, the present findings suggest that SbR-LD have higher immunomodulatory capacity which may favour aggressive pathology.
Author Summary
The disease Kala-azar is caused by Leishmania donovani (LD). The disease is characterized by the depression of cellular immune response. In the Indian subcontinent LD parasites are mostly resistant to commonly used antileishmanial drug, like sodium antimony gluconate (SAG). It is known that infection with pentavalent antimony (Sb)-resistant parasites induces aggressive pathology- the cause is still not known. Sb-resistant parasites endowed with unique glycan which may also play an important role in the pathogenesis as following removal of terminal sugar of glycan these parasites behave like sensitive parasites. The diagnosis of the disease is possible after the disease sets in and therefore limited information is available on the host-parasite interaction at the onset of disease. As a surrogate of in vivo scenario we studied the interaction between normal human PBMC with Sb-sensitive and Sb-resistant parasites. The Sb-resistant parasites upon interaction with human peripheral blood mononuclear cells (PBMC) in vitro produced two distinct inhibitory cytokines, IL-10 and TGF-β. Similar experiment with Sb-sensitive LD induced much less amount of above cytokines. Thus aggressive pathology induced by Sb-resistant LD, may be, in part attributed to production of dual inhibitory cytokines where surface glycan of the parasite may play a decisive role.
doi:10.1371/journal.pntd.0002995
PMCID: PMC4102415  PMID: 25032977
3.  Detection of Circulating Parasite-Derived MicroRNAs in Filarial Infections 
Filarial nematodes cause chronic and profoundly debilitating diseases in both humans and animals. Applications of novel technology are providing unprecedented opportunities to improve diagnosis and our understanding of the molecular basis for host-parasite interactions. As a first step, we investigated the presence of circulating miRNAs released by filarial nematodes into the host bloodstream. miRNA deep-sequencing combined with bioinformatics revealed over 200 mature miRNA sequences of potential nematode origin in Dirofilaria immitis-infected dog plasma in two independent analyses, and 21 in Onchocerca volvulus-infected human serum. Total RNA obtained from D. immitis-infected dog plasma was subjected to stem-loop RT-qPCR assays targeting two detected miRNA candidates, miR-71 and miR-34. Additionally, Brugia pahangi-infected dog samples were included in the analysis, as these miRNAs were previously detected in extracts prepared from this species. The presence of miR-71 and miR-34 discriminated infected samples (both species) from uninfected samples, in which no specific miRNA amplification occurred. However, absolute miRNA copy numbers were not significantly correlated with microfilaraemia for either parasite. This may be due to the imprecision of mf counts to estimate infection intensity or to miRNA contributions from the unknown number of adult worms present. Nonetheless, parasite-derived circulating miRNAs are found in plasma or serum even for those species that do not live in the bloodstream.
Author Summary
Filarial parasites commonly infect humans and animals, especially in tropical settings. The strongly debilitating panel of diseases they cause in humans contributes to an entrenched cycle of poverty. For efficient treatment strategies, reliable diagnostic tests are necessary. We investigated the potential of parasite-derived microRNAs (miRNAs; short non-coding RNA molecules present in eukaryotes) as biomarkers of infection. Using deep-sequencing technologies and bioinformatics, we identified over two-hundred mature miRNA candidates of nematode origin in plasma from Dirofilaria immitis-infected dogs. Similarly, we discovered twenty-one miRNA candidates predicted to be released by Onchocerca volvulus in infected human sera. We developed two RT-qPCR assays for the detection of D. immitis miR-71 and miR-34 in dog plasma that discriminated infected from uninfected samples. We demonstrated the presence of filarial miRNAs in host blood, regardless of localization in their respective hosts, and suggest that they are suitable targets for detection by RT-qPCR.
doi:10.1371/journal.pntd.0002971
PMCID: PMC4102413  PMID: 25033073
4.  Transcriptome Sequencing and Developmental Regulation of Gene Expression in Anopheles aquasalis 
Background
Anopheles aquasalis is a major malaria vector in coastal areas of South and Central America where it breeds preferentially in brackish water. This species is very susceptible to Plasmodium vivax and it has been already incriminated as responsible vector in malaria outbreaks. There has been no high-throughput investigation into the sequencing of An. aquasalis genes, transcripts and proteins despite its epidemiological relevance. Here we describe the sequencing, assembly and annotation of the An. aquasalis transcriptome.
Methodology/Principal Findings
A total of 419 thousand cDNA sequence reads, encompassing 164 million nucleotides, were assembled in 7544 contigs of ≥2 sequences, and 1999 singletons. The majority of the An. aquasalis transcripts encode proteins with their closest counterparts in another neotropical malaria vector, An. darlingi. Several analyses in different protein databases were used to annotate and predict the putative functions of the deduced An. aquasalis proteins. Larval and adult-specific transcripts were represented by 121 and 424 contig sequences, respectively. Fifty-one transcripts were only detected in blood-fed females. The data also reveal a list of transcripts up- or down-regulated in adult females after a blood meal. Transcripts associated with immunity, signaling networks and blood feeding and digestion are discussed.
Conclusions/Significance
This study represents the first large-scale effort to sequence the transcriptome of An. aquasalis. It provides valuable information that will facilitate studies on the biology of this species and may lead to novel strategies to reduce malaria transmission on the South American continent. The An. aquasalis transcriptome is accessible at http://exon.niaid.nih.gov/transcriptome/An_aquasalis/Anaquexcel.xlsx.
Author Summary
The mosquito Anopheles aquasalis is responsible for transmitting malaria parasites to humans in South America coastal areas. An. aquasalis females transmit Plasmodium vivax and Plasmodium falciparum, the two major malaria etiological agents in these endemic sites. Although the vectorial importance of this mosquito has been demonstrated, molecular aspects of its biology have been poorly explored. In this study, we present the transcriptome of An. aquasalis using 454 sequencing followed by automated bioinformatic analyses. Our study identified and annotated more than 9,000 putative proteins based on homology, gene ontology, and/or biochemical pathways, including putative secretory proteins. The comparison of RNAs present in samples extracted from larvae, sugar fed adult females, or blood fed adult females, reveal gene expression regulation during mosquito development. The present dataset provides a useful resource and adds greatly to our understanding of a human malaria vector from developing countries.
doi:10.1371/journal.pntd.0003005
PMCID: PMC4102416  PMID: 25033462
5.  Synergy Testing of FDA-Approved Drugs Identifies Potent Drug Combinations against Trypanosoma cruzi 
An estimated 8 million persons, mainly in Latin America, are infected with Trypanosoma cruzi, the etiologic agent of Chagas disease. Existing antiparasitic drugs for Chagas disease have significant toxicities and suboptimal effectiveness, hence new therapeutic strategies need to be devised to address this neglected tropical disease. Due to the high research and development costs of bringing new chemical entities to the clinic, we and others have investigated the strategy of repurposing existing drugs for Chagas disease. Screens of FDA-approved drugs (described in this paper) have revealed a variety of chemical classes that have growth inhibitory activity against mammalian stage Trypanosoma cruzi parasites. Aside from azole antifungal drugs that have low or sub-nanomolar activity, most of the active compounds revealed in these screens have effective concentrations causing 50% inhibition (EC50's) in the low micromolar or high nanomolar range. For example, we have identified an antihistamine (clemastine, EC50 of 0.4 µM), a selective serotonin reuptake inhibitor (fluoxetine, EC50 of 4.4 µM), and an antifolate drug (pyrimethamine, EC50 of 3.8 µM) and others. When tested alone in the murine model of Trypanosoma cruzi infection, most compounds had insufficient efficacy to lower parasitemia thus we investigated using combinations of compounds for additive or synergistic activity. Twenty-four active compounds were screened in vitro in all possible combinations. Follow up isobologram studies showed at least 8 drug pairs to have synergistic activity on T. cruzi growth. The combination of the calcium channel blocker, amlodipine, plus the antifungal drug, posaconazole, was found to be more effective at lowering parasitemia in mice than either drug alone, as was the combination of clemastine and posaconazole. Using combinations of FDA-approved drugs is a promising strategy for developing new treatments for Chagas disease.
Author Summary
Chronic infection with Trypanosoma cruzi causes progressive damage to the heart and other organs that is fatal in about 30% of cases. Known as Chagas disease, this is a major public health problem in Latin America. The existing medicines were developed over forty years ago and are not widely used because of toxicity and unreliable effectiveness. To discover better treatments, we screened a collection of existing drugs for growth inhibitory activity on Trypanosoma cruzi. Several dozen orally administered drugs were discovered, but when used by themselves they were not strong enough to cure the infection in an animal model. We tested a set of 24 of these drugs in every two-way combination and identified eight synergistic partners. At least two of these combinations were able to substantially lower parasite levels in the mouse model of Trypanosoma cruzi infection. Thus, finding pairs of FDA-approved drugs that can be used in combination may be a pragmatic and effective strategy for designing new therapies for Chagas disease.
doi:10.1371/journal.pntd.0002977
PMCID: PMC4102417  PMID: 25033456
6.  Leishmania amazonensis Amastigotes Highly Express a Tryparedoxin Peroxidase Isoform That Increases Parasite Resistance to Macrophage Antimicrobial Defenses and Fosters Parasite Virulence 
Professional phagocytes generate a myriad of antimicrobial molecules to kill invading microorganisms, of which nitrogen oxides are integral in controlling the obligate intracellular pathogen Leishmania. Although reactive nitrogen species produced by the inducible nitric oxide synthase (iNOS) can promote the clearance of intracellular parasites, some Leishmania species/stages are relatively resistant to iNOS-mediated antimicrobial activity. The underlying mechanism for this resistance remains largely uncharacterized. Here, we show that the amastigote form of L. amazonensis is hyper-resistant to the antimicrobial actions of cytokine-activated murine and human macrophages as compared to its promastigote counterpart. Amastigotes exhibit a marked ability to directly counter the cytotoxicity of peroxynitrite (ONOO−), a leishmanicidal oxidant that is generated during infection through the combined enzymatic activities of NADPH oxidase and iNOS. The enhanced antinitrosative defense of amastigotes correlates with the increased expression of a tryparedoxin peroxidase (TXNPx) isoform that is also upregulated in response to iNOS enzymatic activity within infected macrophages. Accordingly, ectopic over-expression of the TXNPx isoform by L. amazonensis promastigotes significantly enhances parasite resistance against ONOO− cytotoxicity. Moreover, TXNPx-overexpressing parasites exhibit greater intra-macrophage survival, and increased parasite growth and lesion development in a murine model of leishmaniasis. Our investigations indicate that TXNPx isoforms contribute to Leishmania's ability to adapt to and antagonize the hostile microenvironment of cytokine-activated macrophages, and provide a mechanistic explanation for persistent infection in experimental and human leishmaniasis.
Author Summary
Pathogens of the genus Leishmania are the causative agents of leishmaniasis, a neglected tropical disease responsible for significant morbidity and mortality worldwide. Although it is well accepted that host-derived leishmanicidal molecules mediate resolution of Leishmania infection, some Leishmania species/stages are relatively resistant to host cell antimicrobial activity. These intracellular pathogens have developed evasive strategies to subvert host antimicrobials, and promote pathogen survival within the harsh intracellular environment. However, the underlying mechanisms remain largely uncharacterized. Here, we show that L. amazonensis, an agent of persistent infection in humans and non-healing skin lesions in mice, antagonize macrophage antimicrobial activity. The superb ability of the amastigote form to survive within host cells is related to its increased expression of a tryparedoxin peroxidase isoform that confers resistance to the cytotoxicity of host-derived antimicrobial molecules. Parasites induce higher expression of the TXNPx in response to iNOS activity during infection of macrophages, indicating that parasites can “sense” the microenvironment of host cells and regulate the expression of relevant virulence factors accordingly. Our investigations are consistent with a model by which Leishmania amastigotes utilize TXNPx to defend against host-derived molecules thereby promoting their intracellular survival and persistent infection.
doi:10.1371/journal.pntd.0003000
PMCID: PMC4102420  PMID: 25033301
7.  Eosinophil Granule Proteins ECP and EPX as Markers for a Potential Early-Stage Inflammatory Lesion in Female Genital Schistosomiasis (FGS) 
Background
Genital granulomas induced by Schistosoma haematobium eggs can manifest as different lesion types visible by colposcopy; rubbery papules (RP), homogenous sandy patches (HSP) and grainy sandy patches (GSP). Pronounced tissue eosinophilia is a candidate marker for active S. haematobium pathology, as viable schistosome egg granulomas often are eosinophil rich. Here it was investigated whether eosinophil granule proteins ECP (eosinophil cationic protein) and EPX (eosinophil protein-X) in urine and genital lavage can be used as markers for active FGS lesions.
Methods
Uro-genital samples from 118 Malagasy women were analysed for ECP and EPX by standard sandwich avidin/biotin amplified ELISA.
Principal findings
The women with RP lesions had significantly higher levels of ECP and EPX in both lavage and urine. Furthermore, women with RP lesions were significantly younger than those with GSP. This could indicate that RP lesions might be more recently established and thus represent an earlier inflammatory lesion stage.
Conclusion
ECP in genital lavage might be a future tool aiding the identification of FGS pathology at a stage where reversibility remains a possibility following praziquantel treatment.
Author Summary
The blood-dwelling fluke Schistosoma haematobium produce eggs which can inflict lesions both in the urinary and genital tract. Lesions in the female genital tract have been hypothesised to confer higher risk of contraction of HIV and other genital infections. These epithelial genital lesions are visibly different and three types can be observed; rubbery papules, homologous sandy patches and grainy sandy patches. Like other helminths, active S. haematobium infection is associated with eosinophilia. Therefore eosinophil granule proteins might be useful markers for active inflammation related to female genital schistosomiasis lesions. This study identifies the rubbery papules as a potential early-stage inflammatory lesion type associated with high levels of eosinophil granule proteins in vaginal lavage. It may be advantageous to identify female genital lesions relatively early after infection as chronic inflammation stage lesions might not respond to praziquantel treatment.
doi:10.1371/journal.pntd.0002974
PMCID: PMC4102437  PMID: 25033206
8.  Changing Patterns of Human Anthrax in Azerbaijan during the Post-Soviet and Preemptive Livestock Vaccination Eras 
We assessed spatial and temporal changes in the occurrence of human anthrax in Azerbaijan during 1984 through 2010. Data on livestock outbreaks, vaccination efforts, and human anthrax incidence during Soviet governance, post-Soviet governance, preemptive livestock vaccination were analyzed. To evaluate changes in the spatio-temporal distribution of anthrax, we used a combination of spatial analysis, cluster detection, and weighted least squares segmented regression. Results indicated an annual percent change in incidence of +11.95% from 1984 to 1995 followed by declining rate of −35.24% after the initiation of livestock vaccination in 1996. Our findings also revealed geographic variation in the spatial distribution of reporting; cases were primarily concentrated in the west early in the study period and shifted eastward as time progressed. Over twenty years after the dissolution of the Soviet Union, the distribution of human anthrax in Azerbaijan has undergone marked changes. Despite decreases in the incidence of human anthrax, continued control measures in livestock are needed to mitigate its occurrence. The shifting patterns of human anthrax highlight the need for an integrated “One Health” approach that takes into account the changing geographic distribution of the disease.
Author Summary
Zoonotic diseases, such as anthrax, represent a threat to public and veterinary health in many developing parts of the world. Control of anthrax is dependent upon several factors, including proper management of outbreaks and livestock vaccination. Following the dissolution of the Soviet Union, resources for disease management in Azerbaijan were dramatically diminished leading to increases in zoonotic diseases. In this study, our objective was to analyze human anthrax incidence during Soviet governance, post-Soviet governance, and after the implementation of a preemptive livestock vaccination campaign to identify potential changes in the occurrence of the disease. We applied spatial and temporal statistical approaches in a geographic information system to describe changes. Our findings provide evidence of a changing incidence and a shift in the geographic patterns of human anthrax. These findings highlight the importance of proper livestock disease management to mitigate human disease and the need for dynamic surveillance that takes into account changes in the distribution of disease.
doi:10.1371/journal.pntd.0002985
PMCID: PMC4102439  PMID: 25032701
9.  Serology of Paracoccidioidomycosis Due to Paracoccidioides lutzii 
Paracoccidioides lutzii is a new agent of paracoccidioidomycosis (PCM) and has its epicenter localized to the Central-West region of Brazil. Serological diagnosis of PCM caused by P. lutzii has not been established. This study aimed to develop new antigenic preparations from P. lutzii and to apply them in serological techniques to improve the diagnosis of PCM due to P. lutzii. Paracoccidioides lutzii exoantigens, cell free antigen (CFA), and a TCA-precipitated antigen were evaluated in immunodiffusion (ID) tests using a total of 89 patient sera from the Central-West region of Brazil. Seventy-two sera were defined as reactive for P. brasiliensis using traditional antigens (AgPbB339 and gp43). Non-reactive sera for traditional antigens (n = 17) were tested with different P. lutzii preparations and P. lutzii CFA showed 100% reactivity. ELISA was found to be a very useful test to titer anti-P. lutzii antibodies using P. lutzii-CFA preparations. Sera from patients with PCM due to P. lutzii presented with higher antibody titers than PCM due to P. brasiliensis and heterologous sera. In western blot, sera from patients with PCM due to P. lutzii were able to recognize antigenic molecules from the P. lutzii-CFA antigen, but sera from patients with PCM due to P. brasiliensis could not recognize any P. lutzii molecules. Due to the facility of preparing P. lutzii CFA antigens we recommend its use in immunodiffusion tests for the diagnosis of PCM due to P. lutzii. ELISA and western blot can be used as complementary tests.
Author Summary
Tropical diseases, such as paracoccidioidomycosis, are the most common type of neglected diseases. From 1980 to 1995, 3,181 deaths from paracoccidioidomycosis occurred in Brazil, representing the eighth most common cause of death from predominantly chronic or recurrent types of infectious and parasitic diseases, showing considerable magnitude and low visibility. Paracoccidioidomycosis is traditionally assumed to be caused solely by Paracoccidioides brasiliensis, but a new species, Paracoccidioides lutzii, was discovered in the Central-Western region of Brazil. Thus, new antigenic preparations and tests for an accurate differential diagnosis between these two species appear to be needed. This study aimed to develop new antigenic preparations from P. lutzii isolates to improve the diagnosis of paracoccidioidomycosis. We used patient serum samples predominantly from the Central-Western region of Brazil. Various antigenic preparations were tested, and a cell free antigen derived from P. lutzii was an excellent antigen for serological diagnosis and able to diagnose 100% of sera from patients with PCM due to P. lutzii.
doi:10.1371/journal.pntd.0002986
PMCID: PMC4102441  PMID: 25032829
10.  Recombinant Antigens Expressed in Pichia pastoris for the Diagnosis of Sleeping Sickness Caused by Trypanosoma brucei gambiense 
Background
Screening tests for gambiense sleeping sickness, such as the CATT/T. b. gambiense and a recently developed lateral flow tests, are hitherto based on native variant surface glycoproteins (VSGs), namely LiTat 1.3 and LiTat 1.5, purified from highly virulent trypanosome strains grown in rodents.
Methodology/Principal Findings
We have expressed SUMO (small ubiquitin-like modifier) fusion proteins of the immunogenic N-terminal part of these antigens in the yeast Pichia pastoris. The secreted recombinant proteins were affinity purified with yields up to 10 mg per liter cell culture.
Conclusions/Significance
The diagnostic potential of each separate antigen and a mixture of both antigens was confirmed in ELISA on sera from 88 HAT patients and 74 endemic non-HAT controls. Replacement of native antigens in the screening tests for sleeping sickness by recombinant proteins will eliminate both the infection risk for the laboratory staff during antigen production and the need for laboratory animals. Upscaling production of recombinant antigens, e.g. in biofermentors, is straightforward thus leading to improved standardisation of antigen production and reduced production costs, which on their turn will increase the availability and affordability of the diagnostic tests needed for the elimination of gambiense HAT.
Author Summary
Population screening for the chronic form of sleeping sickness or gambiense human African trypanosomiasis (HAT) is still based on an antibody detection test against the native variant surface glycoprotein (VSG) LiTat 1.3. This protein is produced through massive infections of lab animals with highly virulent parasites. We aim to replace this native antigen with recombinant VSGs, both LiTat 1.3 and LiTat 1.5, expressed in the yeast Pichia pastoris. The diagnostic potential of these recombinants was confirmed in ELISA with sera from HAT patients and negative controls. Replacement of the native LiTat 1.3 VSG with these recombinants would prevent the infection and sacrifice of lab animals and the inherent infection risk linked to the production of the screening test. Upscaling production of recombinant antigens, e.g. in biofermentors, is straightforward thus leading to improved standardisation of antigen production and reduced production costs, which on their turn will increase the availability and affordability of the diagnostic tests needed for the elimination of gambiense HAT.
doi:10.1371/journal.pntd.0003006
PMCID: PMC4102443  PMID: 25032684
11.  Neurocognitive Outcome of Children Exposed to Perinatal Mother-to-Child Chikungunya Virus Infection: The CHIMERE Cohort Study on Reunion Island 
Background
Little is known about the neurocognitive outcome in children exposed to perinatal mother-to-child Chikungunya virus (p-CHIKV) infection.
Methods
The CHIMERE ambispective cohort study compared the neurocognitive function of 33 p-CHIKV-infected children (all but one enrolled retrospectively) at around two years of age with 135 uninfected peers (all enrolled prospectively). Psychomotor development was assessed using the revised Brunet-Lezine scale, examiners blinded to infectious status. Development quotients (DQ) with subscores covering movement/posture, coordination, language, sociability skills were calculated. Predictors of global neurodevelopmental delay (GND, DQ≤85), were investigated using multivariate Poisson regression modeling. Neuroradiologic follow-up using magnetic resonance imaging (MRI) scans was proposed for most of the children with severe forms.
Results
The mean DQ score was 86.3 (95%CI: 81.0–91.5) in infected children compared to 100.2 (95%CI: 98.0–102.5) in uninfected peers (P<0.001). Fifty-one percent (n = 17) of infected children had a GND compared to 15% (n = 21) of uninfected children (P<0.001). Specific neurocognitive delays in p-CHIKV-infected children were as follows: coordination and language (57%), sociability (36%), movement/posture (27%). After adjustment for maternal social situation, small for gestational age, and head circumference, p-CHIKV infection was found associated with GND (incidence rate ratio: 2.79, 95%CI: 1.45–5.34). Further adjustments on gestational age or breastfeeding did not change the independent effect of CHIKV infection on neurocognitive outcome. The mean DQ of p-CHIKV-infected children was lower in severe encephalopathic children than in non-severe children (77.6 versus 91.2, P<0.001). Of the 12 cases of CHIKV neonatal encephalopathy, five developed a microcephaly (head circumference <−2 standard deviations) and four matched the definition of cerebral palsy. MRI scans showed severe restrictions of white matter areas, predominant in the frontal lobes in these children.
Conclusions
The neurocognitive outcome of children exposed to perinatal mother-to-child CHIKV infection is poor. Severe CHIKV neonatal encephalopathy is associated with an even poorer outcome.
Author Summary
Chikungunya virus (CHIKV), an alphaviral infection transmitted by day-biting Aedes mosquitoes, is widespread in Asia and in Africa. Usually, CHIKV causes a self-limiting arthritide, except in debilitated people and in neonates, for whom it can lead to severe disease. Mother-to-child perinatal transmission of CHIKV is a rare event that can occur in the setting of large-scale outbreaks when the risk of maternal viremia at the term of pregnancy becomes non-negligible. In that event, CHIKV can give rise to neonatal infection with a probability of 50%, and prostration and encephalopathy, the two leading clinical pictures in the neonate, represent a continuum in the gradation of an undiscovered central nervous system involvement. We have followed-up 33 children infected at birth between June 2005 and April 2006 on the island of La Réunion, Indian Ocean, and 135 uninfected controls, for assessing their neurodevelopmental performance around the age of two years. Fifty-one percent of infected children had a global neurodevelopmental delay compared to 15% of uninfected peers. Multivariate analysis and neuroradiology suggest without irrefutable evidence a causal relationship between CHIKV infection and neurocognitive outcomes. Our findings suggest that CHIKV infection, acquired in the perinatal period, can cause severe disease with lifelong expected disability.
doi:10.1371/journal.pntd.0002996
PMCID: PMC4102444  PMID: 25033077
12.  Long-Term and Seasonal Dynamics of Dengue in Iquitos, Peru 
Introduction
Long-term disease surveillance data provide a basis for studying drivers of pathogen transmission dynamics. Dengue is a mosquito-borne disease caused by four distinct, but related, viruses (DENV-1-4) that potentially affect over half the world's population. Dengue incidence varies seasonally and on longer time scales, presumably driven by the interaction of climate and host susceptibility. Precise understanding of dengue dynamics is constrained, however, by the relative paucity of laboratory-confirmed longitudinal data.
Methods
We studied 10 years (2000–2010) of laboratory-confirmed, clinic-based surveillance data collected in Iquitos, Peru. We characterized inter and intra-annual patterns of dengue dynamics on a weekly time scale using wavelet analysis. We explored the relationships of case counts to climatic variables with cross-correlation maps on annual and trimester bases.
Findings
Transmission was dominated by single serotypes, first DENV-3 (2001–2007) then DENV-4 (2008–2010). After 2003, incidence fluctuated inter-annually with outbreaks usually occurring between October and April. We detected a strong positive autocorrelation in case counts at a lag of ∼70 weeks, indicating a shift in the timing of peak incidence year-to-year. All climatic variables showed modest seasonality and correlated weakly with the number of reported dengue cases across a range of time lags. Cases were reduced after citywide insecticide fumigation if conducted early in the transmission season.
Conclusions
Dengue case counts peaked seasonally despite limited intra-annual variation in climate conditions. Contrary to expectations for this mosquito-borne disease, no climatic variable considered exhibited a strong relationship with transmission. Vector control operations did, however, appear to have a significant impact on transmission some years. Our results indicate that a complicated interplay of factors underlie DENV transmission in contexts such as Iquitos.
Author Summary
Description of long-term temporal patterns in disease occurrence improves our understanding of pathogen transmission dynamics and facilitates predicting new epidemics. Dengue, the most prevalent mosquito-borne, viral disease of humans, typically varies seasonally and on longer, inter-annual time scales. In most studies of these patterns, however, only a fraction of putative dengue cases are confirmed with laboratory diagnostics. Here we analyzed 10 years of fully confirmed dengue cases reported to a sentinel surveillance system in Iquitos, Peru. We describe the inter and intra-annual patterns of weekly case counts and relate these to climate and local vector control efforts. We show that dengue case counts vary seasonally in Iquitos despite very little variation in key climatic conditions, such as temperature and humidity. Overall, transmission correlated poorly with climate regardless of time lag. In seasons when vector control was conducted early, there was an apparent decline in cases later that season. We speculate that the relationships between climatic conditions and transmission of DENV in Iquitos are complex and non-linear, and that other factors, such as herd immunity, virus diversity, and vector control efforts, play key roles determining the timing and intensity of transmission.
doi:10.1371/journal.pntd.0003003
PMCID: PMC4102451  PMID: 25033412
13.  In Vitro and In Vivo Miltefosine Susceptibility of a Leishmania amazonensis Isolate from a Patient with Diffuse Cutaneous Leishmaniasis 
Miltefosine was the first oral compound approved for visceral leishmaniasis chemotherapy, and its efficacy against Leishmania donovani has been well documented. Leishmania amazonensis is the second most prevalent species causing cutaneous leishmaniasis and the main etiological agent of diffuse cutaneous leishmaniasis in Brazil. Driven by the necessity of finding alternative therapeutic strategies for a chronic diffuse cutaneous leishmaniasis patient, we evaluated the susceptibility to miltefosine of the Leishmania amazonensis line isolated from this patient, who had not been previously treated with miltefosine. In vitro tests against promastigotes and intracellular amastigotes showed that this parasite isolate was less susceptible to miltefosine than L. amazonensis type strains. Due to this difference in susceptibility, we evaluated whether genes previously associated with miltefosine resistance were involved. No mutations were found in the miltefosine transporter gene or in the Ros3 or pyridoxal kinase genes. These analyses were conducted in parallel with the characterization of L. amazonensis mutant lines selected for miltefosine resistance using a conventional protocol to select resistance in vitro, i.e., exposure of promastigotes to increasing drug concentrations. In these mutant lines, a single nucleotide mutation G852E was found in the miltefosine transporter gene. In vivo studies were also performed to evaluate the correlation between in vitro susceptibility and in vivo efficacy. Miltefosine was effective in the treatment of BALB/c mice infected with the L. amazonensis type strain and with the diffuse cutaneous leishmaniasis isolate. On the other hand, animals infected with the resistant line bearing the mutated miltefosine transporter gene were completely refractory to miltefosine chemotherapy. These data highlight the difficulties in establishing correlations between in vitro susceptibility determinations and response to chemotherapy in vivo. This study contributed to establish that the miltefosine transporter is essential for drug activity in L. amazonensis and a potential molecular marker of miltefosine unresponsiveness in leishmaniasis patients.
Author Summary
Leishmania amazonensis is the etiological agent of diffuse cutaneous leishmaniasis. The disease is extremely difficult to treat and frequently relapses once the treatment is interrupted. Although not yet approved in Brazil, miltefosine is an attractive alternative for leishmaniasis treatment due to its oral administration and low incidence of side effects. Here, we evaluated the efficacy of miltefosine against a L. amazonensis line that was isolated from a chronic diffuse cutaneous leishmaniasis patient to ascertain whether miltefosine could be considered as a therapeutic option in this case. Parasites isolated from this patient were less susceptible to miltefosine than a reference strain in vitro. The mechanisms underlying this decreased susceptibility were studied in this natural parasite isolate in parallel with mutant strains selected in vitro for miltefosine resistance. A mutation in the gene encoding the miltefosine transporter was identified in the mutants selected in vitro but not in the line isolated from the patient. Notwithstanding the decreased susceptibility in vitro, when used to treat infected mice, miltefosine was equally effective against the isolate from the patient and the type strain, but completely ineffective against the resistant line.
doi:10.1371/journal.pntd.0002999
PMCID: PMC4102453  PMID: 25033218
14.  Identification of sVSG117 as an Immunodiagnostic Antigen and Evaluation of a Dual-Antigen Lateral Flow Test for the Diagnosis of Human African Trypanosomiasis 
Background
The diagnosis of human African trypanosomiasis (HAT) caused by Trypanosoma brucei gambiense relies mainly on the Card Agglutination Test for Trypanosomiasis (CATT). There is no immunodiagnostic for HAT caused by T. b. rhodesiense. Our principle aim was to develop a prototype lateral flow test that might be an improvement on CATT.
Methodology/Principle Findings
Pools of infection and control sera were screened against four different soluble form variant surface glycoproteins (sVSGs) by ELISA and one, sVSG117, showed particularly strong immunoreactivity to pooled infection sera. Using individual sera, sVSG117 was shown to be able to discriminate between T. b. gambiense infection and control sera by both ELISA and lateral flow test. The sVSG117 antigen was subsequently used with a previously described recombinant diagnostic antigen, rISG65, to create a dual-antigen lateral flow test prototype. The latter was used blind in a virtual field trial of 431 randomized infection and control sera from the WHO HAT Specimen Biobank.
Conclusion/Significance
In the virtual field trial, using two positive antigen bands as the criterion for infection, the sVSG117 and rISG65 dual-antigen lateral flow test prototype showed a sensitivity of 97.3% (95% CI: 93.3 to 99.2) and a specificity of 83.3% (95% CI: 76.4 to 88.9) for the detection of T. b. gambiense infections. The device was not as good for detecting T. b. rhodesiense infections using two positive antigen bands as the criterion for infection, with a sensitivity of 58.9% (95% CI: 44.9 to 71.9) and specificity of 97.3% (95% CI: 90.7 to 99.7). However, using one or both positive antigen band(s) as the criterion for T. b. rhodesiense infection improved the sensitivity to 83.9% (95% CI: 71.7 to 92.4) with a specificity of 85.3% (95% CI: 75.3 to 92.4). These results encourage further development of the dual-antigen device for clinical use.
Author Summary
Human African Trypanosomiasis (HAT) is caused by infection with Trypanosoma brucei gambiense or T. b. rhodesiense. The diagnosis of T. b. gambiense infections currently relies primarily on a Card Agglutination Test for Trypanosomiasis (CATT), which has acknowledged limitations, and there is no simple test for T. b. rhodesiense infection. Our overall aim is to produce a simple lateral flow test device with a similar or better sensitivity and specificity than CATT but with better stability and ease of use at point of care. In this study, we identified a particular variant surface glycoprotein, sVSG117, with good diagnostic potential and combined it with a previously identified recombinant diagnostic antigen, rISG65, to produce a prototype dual-antigen lateral flow test. We performed a virtual field trial by testing the device blind with 431 randomized serum samples provided by the WHO HAT Specimen Biobank. The results show that, although the prototype lateral flow test is un-optimized, it was able to diagnose T. b. gambiense HAT with a sensitivity and specificity of 97.3% and 83.3% and T. b. rhodesiense HAT with a sensitivity and specificity of 83.9% and 85.3%.
doi:10.1371/journal.pntd.0002976
PMCID: PMC4102454  PMID: 25033401
15.  Oroya Fever and Verruga Peruana: Bartonelloses Unique to South America 
Bartonella bacilliformis is the bacterial agent of Carrión's disease and is presumed to be transmitted between humans by phlebotomine sand flies. Carrión's disease is endemic to high-altitude valleys of the South American Andes, and the first reported outbreak (1871) resulted in over 4,000 casualties. Since then, numerous outbreaks have been documented in endemic regions, and over the last two decades, outbreaks have occurred at atypical elevations, strongly suggesting that the area of endemicity is expanding. Approximately 1.7 million South Americans are estimated to be at risk in an area covering roughly 145,000 km2 of Ecuador, Colombia, and Peru. Although disease manifestations vary, two disparate syndromes can occur independently or sequentially. The first, Oroya fever, occurs approximately 60 days following the bite of an infected sand fly, in which infection of nearly all erythrocytes results in an acute hemolytic anemia with attendant symptoms of fever, jaundice, and myalgia. This phase of Carrión's disease often includes secondary infections and is fatal in up to 88% of patients without antimicrobial intervention. The second syndrome, referred to as verruga peruana, describes the endothelialcell-derived, blood-filled tumors that develop on the surface of the skin. Verrugae are rarely fatal, but can bleed and scar the patient. Moreover, these persistently infected humans provide a reservoir for infecting sand flies and thus maintaining B. bacilliformis in nature. Here, we discuss the current state of knowledge regarding this life-threatening, neglected bacterial pathogen and review its host-cell parasitism, molecular pathogenesis, phylogeny, sand fly vectors, diagnostics, and prospects for control.
doi:10.1371/journal.pntd.0002919
PMCID: PMC4102455  PMID: 25032975
17.  Shift in Phenotypic Characteristics of Enterotoxigenic Escherichia coli (ETEC) Isolated from Diarrheal Patients in Bangladesh 
Background
Enterotoxigenic Escherichia coli (ETEC) is one of the most common causes of bacterial diarrhea. Over the last decade, from 1996 to 2012, changes in the virulence antigen properties of ETEC such as heat labile (LT) and heat stable (ST) toxins, colonization factors (CFs), and ‘O’-serogroups have been observed. The aim of this prospective study was to compare changes in antigenic profiles of ETEC strains isolated from a 2% surveillance system at the icddr,b hospital in Dhaka, Bangladesh between 2007–2012 and an earlier time period of 1996–1998 conducted at the same surveillance site.
Methodology
In the surveillance system every 50th patient attending the hospital was screened for major enteric pathogens including ETEC, Vibrio cholerae, Shigella spp. and Salmonella spp. from January 2007 to December 2012.
Principal Findings
Of the 15,152 diarrheal specimens tested between 2007–2012, the overall rate of ETEC isolation was 11%; of these, 43% were LT/ST, 27% LT and 30% ST positive. Isolation rate of ST-ETEC (p<0.009) and LT/ST ETEC (p<0.011) during 2007–2012 period differed significantly compared to those seen between 1996–1998. In comparison to the 1996–1998 period, difference in CF profile of ETEC isolates during 2007–2012 was observed particularly for strains expressing CS7 (12.4%), CS14 (9.5%) and CS17 (10.0%). The predominant CF types were CS5+CS6, CFA/I, CS7, CS17, CS1+CS3, CS6 and CS14. The most common serogroups among the CF positive ETEC isolates were O115, O114, O6, O25 and O8. A strong association was found between CFs and ‘O’ serogroups i.e. between CS5+CS6 and (O115 and O126); CS7 and (O114), CFA/I and (O78 and O126), CS17 and (O8 and O167) and CS1/CS2+CS3 and (O6).
Conclusion
The analyses show a shift in prevalence of antigenic types of ETEC over the study period; the information is important in designing effective ETEC vaccines with broad protective coverage.
Author Summary
Diarrheal diseases constitute a major health problem in Bangladesh, where Vibrio cholerae and enterotoxigenic Escherichia coli (ETEC) are two most important causes of bacterial diarrhea. Prevention through vaccination is helpful to reduce the incidence and severity of diarrheal disease due to ETEC, particularly among children in low-resource settings. In this context, we collected stool and/or rectal swab (RS) specimens from patients with diarrhea between 2007 to 2012 under the 2% systematic routine surveillance system at the icddr,b hospital in Dhaka, Bangladesh and screened for ETEC infection. We tested the specimens for two major virulence factors of ETEC: toxins and colonization factors. In this research article, we have focused on changes in toxin as well as colonization factor profiles of ETEC strains isolated from diarrheal patients seeking care at the icddr,b hospital between 2007–2012 and an earlier time period of 1996–1998. We concluded that, such shift in antigenic profile of ETEC over the study period is important in designing effective ETEC vaccines with broad protective coverage.
doi:10.1371/journal.pntd.0003031
PMCID: PMC4102457  PMID: 25032802
18.  Praziquantel, Mefloquine-Praziquantel, and Mefloquine-Artesunate-Praziquantel against Schistosoma haematobium: A Randomized, Exploratory, Open-Label Trial 
Background
Treatment and morbidity control of schistosomiasis relies on a single drug, praziquantel. Hence, there is a pressing need to develop additional therapeutics against schistosomiasis. The antimalarial drug mefloquine shows antischistosomal activity in animal models and clinical trials, which calls for further investigations.
Methodology
We comparatively assessed the efficacy and tolerability of the following treatments against Schistosoma haematobium in school-aged children in Côte d'Ivoire: (i) praziquantel (40 mg/kg; standard treatment); (ii) mefloquine (25 mg/kg) combined with praziquantel (40 mg/kg); and (iii) mefloquine-artesunate (3× (100 mg artesunate +250 mg mefloquine)) combined with praziquantel (40 mg/kg) (treatments administered on subsequent days). Two urine samples were collected before, and on days 21–22 and 78–79 after the first dosing.
Principal Findings
Sixty-one children were present on all examination time points and had complete datasets. No difference in efficacy was observed between the three treatment groups on either follow-up. On the 21–22 day posttreatment follow-up, based on available case analysis, cure rates of 33% (95% confidence interval (CI) 11–55%), 29% (95% CI 8–50%), and 26% (95% CI 5–48%) were observed for praziquantel, mefloquine-artesunate-praziquantel, and mefloquine-praziquantel, respectively. The corresponding egg reduction rates were 94% and above. On the second follow-up, observed cure rates ranged from 19% (praziquantel) to 33% (mefloquine-artesunate-praziquantel), and egg reduction rates were above 90%. Praziquantel monotherapy was the best tolerated treatment. In the mefloquine-artesunate-praziquantel group, adverse events were reported by 91% of the participants, and in the mefloquine-praziquantel group, 95% experienced adverse events. With the exception of abdominal pain at moderate severity, adverse events were mild.
Conclusions/Significance
The addition of mefloquine or mefloquine-artesunate does not increase the efficacy of praziquantel against chronic S. haematobium infection. Additional studies are necessary to elucidate the effect of the combinations against acute schistosomiasis.
Author Summary
The antimalarial drug mefloquine shows activity against blood flukes that cause the disease schistosomiasis. In animal studies it has been found that a mefloquine-praziquantel combination kills blood flukes more effectively than praziquantel alone. Combining praziquantel with another drug might therefore increase efficacy, broaden the spectrum of activity, and delay the development of drug resistance. We designed a study in Ivorian school children to assess the efficacy and tolerability of mefloquine and mefloquine-artesunate combined with praziquantel against the blood fluke Schistosoma haematobium. The administration of the antimalarials and praziquantel was spaced by a day. Treatment outcomes were assessed twice, on days 21–22 and 78–79 after the first dosing to determine the effect against adult and juvenile S. haematobium, respectively. At both follow-ups, high reduction in the intensity of infection (egg reduction rates of 94–96%), but low cure rates (26–33%) were observed in the three treatment groups. Adverse events were common, particularly in children treated with mefloquine-praziquantel and mefloquine-artesunate-praziquantel. Our study suggests that the addition of mefloquine and mefloquine-artesunate to praziquantel has no benefit in the treatment of chronic S. haematobium infection. However, further investigations are warranted to evaluate the effect of combination therapy on juvenile flukes and longer-term morbidity profiles.
doi:10.1371/journal.pntd.0002975
PMCID: PMC4102459  PMID: 25033291
20.  Filarial Excretory-Secretory Products Induce Human Monocytes to Produce Lymphangiogenic Mediators 
The nematodes Wuchereria bancrofti and Brugia spp. infect over 120 million people worldwide, causing lymphedema, elephantiasis and hydrocele, collectively known as lymphatic filariasis. Most infected individuals appear to be asymptomatic, but many exhibit sub-clinical manifestations including the lymphangiectasia that likely contributes to the development of lymphedema and elephantiasis. As adult worm excretory-secretory products (ES) do not directly activate lymphatic endothelial cells (LEC), we investigated the role of monocyte/macrophage-derived soluble factors in the development of filarial lymphatic pathology. We analyzed the production of IL-8, IL-6 and VEGF-A by peripheral blood mononuclear cells (PBMC) from naïve donors following stimulation with filarial ES products. ES-stimulated PBMCs produced significantly more IL-8, IL-6 and VEGF-A compared to cells cultured in medium alone; CD14+ monocytes appear to be the primary producers of IL-8 and VEGF-A, but not IL-6. Furthermore, IL-8, IL-6 and VEGF-A induced in vitro tubule formation in LEC Matrigel cultures. Matrigel plugs supplemented with IL-8, IL-6, VEGF-A, or with supernatants from ES-stimulated PBMCs and implanted in vivo stimulated lymphangiogenesis. Collectively, these data support the hypothesis that monocytes/macrophages exposed to filarial ES products may modulate lymphatic function through the secretion of soluble factors that stimulate the vessel growth associated with the pathogenesis of filarial disease.
Author Summary
Lymphatic filariasis is caused by parasitic worms with approximately 120 million people infected worldwide and over 1 billion people at risk. The adult worms reside in host lymphatic vessels (LV) but most infected individuals do not present with overt clinical symptoms. Individuals exhibiting lymphedema, a common form of the disease, are often antigen negative; however, infected individuals, though often asymptomatic, have dilated LVs suggesting that early damage to the lymphatic architecture may lead to lymphedema in these infected individuals. In the LVs, adult worms release excretory-secretory (ES) products. Filarial ES products do not directly activate lymphatic endothelial cells (LEC), so we hypothesized that accessory cells may activate LECs indirectly and contribute to the development of disease. Here, we show that adult filarial ES products induce human blood cells, specifically monocytes, to produce lymphangiogenic factors such as IL-8 and VEGF-A and that these factors induce the formation of LVs in vivo. These results support a role for filarial ES products in altering the lymphatic architecture in filarial-infected individuals and this may contribute to LV pathology and the development of lymphedema.
doi:10.1371/journal.pntd.0002893
PMCID: PMC4091784  PMID: 25010672
21.  The Death and Life of the Resurrection Drug 
doi:10.1371/journal.pntd.0002910
PMCID: PMC4091785  PMID: 25010692
22.  Blood Drain: Soil-Transmitted Helminths and Anemia in Pregnant Women 
doi:10.1371/journal.pntd.0002912
PMCID: PMC4091787  PMID: 25010736
23.  Evidence of Dengue Virus Transmission and Factors Associated with the Presence of Anti-Dengue Virus Antibodies in Humans in Three Major Towns in Cameroon 
Background
Dengue is not well documented in Africa. In Cameroon, data are scarce, but dengue infection has been confirmed in humans. We conducted a study to document risk factors associated with anti-dengue virus Immunoglobulin G seropositivity in humans in three major towns in Cameroon.
Methodology/Principal Findings
A cross sectional survey was conducted in Douala, Garoua and Yaounde, using a random cluster sampling design. Participants underwent a standardized interview and were blood sampled. Environmental and housing characteristics were recorded. Randomized houses were prospected to record all water containers, and immature stages of Aedes mosquitoes were collected. Sera were screened for anti-dengue virus IgG and IgM antibodies. Risk factors of seropositivity were tested using logistic regression methods with random effects.
Anti-dengue IgG were found from 61.4% of sera in Douala (n = 699), 24.2% in Garoua (n = 728) and 9.8% in Yaounde (n = 603). IgM were found from 0.3% of Douala samples, 0.1% of Garoua samples and 0.0% of Yaounde samples. Seroneutralization on randomly selected IgG positive sera showed that 72% (n = 100) in Douala, 80% (n = 94) in Garoua and 77% (n = 66) in Yaounde had antibodies specific for dengue virus serotype 2 (DENV-2).
Age, temporary house walls materials, having water-storage containers, old tires or toilets in the yard, having no TV, having no air conditioning and having travelled at least once outside the city were independently associated with anti-dengue IgG positivity in Douala. Age, having uncovered water containers, having no TV, not being born in Garoua and not breeding pigs were significant risk factors in Garoua. Recent history of malaria, having banana trees and stagnant water in the yard were independent risk factors in Yaounde.
Conclusion/Significance
In this survey, most identified risk factors of dengue were related to housing conditions. Poverty and underdevelopment are central to the dengue epidemiology in Cameroon.
Author Summary
General awareness of dengue fever in Africa, and particularly in Cameroon, is weak. Many acute febrile illnesses are considered as malaria, although not laboratory confirmed, and the diagnosis of dengue fever is seldom evoked while its laboratory confirmation is even more seldom obtained. On the basis of anti-dengue virus IgG seropositivity in humans, our survey demonstrated that dengue virus transmission occurred in the three main towns of the country. Although the findings varied according to the location, identified risk factors of anti-dengue virus seropositivity were commonly related to housing conditions. Taking into account the risk factors identified in Douala, practical ways to lower the risk of dengue virus transmission are long term development and improvement of sanitation and education. We concluded that poverty and underdevelopment are central to the problem of dengue virus transmission in urban areas in Cameroon.
doi:10.1371/journal.pntd.0002950
PMCID: PMC4091864  PMID: 25009996
24.  Good Quality of Life in Former Buruli Ulcer Patients with Small Lesions: Long-Term Follow-up of the BURULICO Trial 
Background
Buruli Ulcer is a tropical skin disease caused by Mycobacterium ulcerans, which, due to scarring and contractures can lead to stigma and functional limitations. However, recent advances in treatment, combined with increased public health efforts have the potential to significantly improve disease outcome.
Objectives
To study the Quality of Life (QoL) of former Buruli Ulcer patients who, in the context of a randomized controlled trial, reported early with small lesions (cross-sectional diameter <10 cm), and received a full course of antibiotic treatment.
Methods
127 Participants of the BURULICO drug trial in Ghana were revisited. All former patients aged 16 or older completed the Dermatology Life Quality Index (DLQI) and the abbreviated World Health Organization Quality of Life scale (WHOQOL-BREF). The WHOQOL-BREF was also administered to 82 matched healthy controls. Those younger than 16 completed the Childrens' Dermatology Life Quality Index (CDLQI) only.
Results
The median (Inter Quartile Range) score on the DLQI was 0 (0–4), indicating good QoL. 85% of former patients indicated no effect, or only a small effect of the disease on their current life. Former patients also indicated good QoL on the physical and psychological domains of the WHOQOL-BREF, and scored significantly higher than healthy controls on these domains. There was a weak correlation between the DLQI and scar size (ρ = 0.32; p<0.001).
Conclusions
BU patients who report early with small lesions and receive 8 weeks of antimicrobial therapy have a good QoL at long-term follow-up. These findings contrast with the debilitating sequelae often reported in BU, and highlight the importance of early case detection.
Author Summary
Buruli ulcer is an infectious skin disease, mainly occurring in West Africa. It usually starts with a small nodule that over the course of weeks progresses into an ulcer. Effective treatment with antibiotics is available, but patients often report to the hospital late, and there is a serious risk of scarring, contractures and functional limitations of the affected body parts. Although it is often reported that Buruli ulcers can have serious sequelae, the quality of life of former Buruli ulcer patients has not been studied before. In this study we assessed the quality of life of healed Buruli ulcer patients that took part in a drug trial between 2006 and 2009, which only included small and early lesions. On follow-up, we found that most scars were small and functional limitations were rare, and that both general and skin specific Quality of Life was good. Our results demonstrate the potential of the combination of early detection and proper antibiotic treatment of Buruli ulcer and hence stress the importance of public health efforts aimed at diagnosing the disease in its early stage, and providing standardized treatment in endemic areas.
doi:10.1371/journal.pntd.0002964
PMCID: PMC4091870  PMID: 25010061
25.  Effect of Deworming on Physical Fitness of School-Aged Children in Yunnan, China: A Double-Blind, Randomized, Placebo-Controlled Trial 
Background
There is considerable debate on the health impacts of soil-transmitted helminth infections. We assessed effects of deworming on physical fitness and strength of children in an area in Yunnan, People's Republic of China, where soil-transmitted helminthiasis is highly endemic.
Methodology
The double-blind, randomized, placebo-controlled trial was conducted between October 2011 and May 2012. Children, aged 9–12 years, were treated with either triple-dose albendazole or placebo, and monitored for 6 months post-treatment. The Kato-Katz and Baermann techniques were used for the diagnosis of soil-transmitted helminth infections. Physical fitness was assessed with a 20-m shuttle run test, where the maximum aerobic capacity within 1 min of exhaustive exercise (VO2 max estimate) and the number of 20-m laps completed were recorded. Physical strength was determined with grip strength and standing broad jump tests. Body height and weight, the sum of skinfolds, and hemoglobin levels were recorded as secondary outcomes.
Principal Findings
Children receiving triple-dose albendazole scored slightly higher in the primary and secondary outcomes than placebo recipients, but the difference lacked statistical significance. Trichuris trichiura-infected children had 1.6 ml kg−1 min−1 (P = 0.02) less increase in their VO2 max estimate and completed 4.6 (P = 0.04) fewer 20-m laps than at baseline compared to non-infected peers. Similar trends were detected in the VO2 max estimate and grip strength of children infected with hookworm and Ascaris lumbricoides, respectively. In addition, the increase in the VO2 max estimate from baseline was consistently higher in children with low-intensity T. trichiura and hookworm infections than in their peers with high-intensity infections of all soil-transmitted helminths (range: 1.9–2.1 ml kg−1 min−1; all P<0.05).
Conclusions/Significance
We found no strong evidence for significant improvements in physical fitness and anthropometric indicators due to deworming over a 6-month follow-up period. However, the negative effect of T. trichiura infections on physical fitness warrants further investigation.
Author Summary
Children from the developing world are often burdened with intestinal worms due to poor water supply, sanitation, and hygiene. However, the assessment of the burden due to intestinal worms is difficult, and thus, the benefits of deworming are unclear. In this study, we determined the effect of deworming on the physical fitness and strength of 9- to 12-year-old children in Yunnan, China, where intestinal worms are common. Children were treated with triple-dose albendazole or placebo and monitored over a 6-month period. Stool samples were collected for the diagnosis of intestinal worm infections. Physical fitness was estimated with a 20-m shuttle run test and physical strength was assessed with grip strength and standing broad jump tests. Children receiving triple-dose albendazole scored slightly higher values in the primary and secondary outcomes than those children who were given placebo. However, the differences were not significant. We also found that children infected with intestinal worms performed significantly worse in the physical fitness and strength tests than their non-infected counterparts. In particular, the negative impact of whipworm infection on physical fitness warrants further investigation.
doi:10.1371/journal.pntd.0002983
PMCID: PMC4091871  PMID: 25010608

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