Transmission dynamics of Q fever in French Guiana: A population-based cross-sectional study.

Background: Q fever is a zoonosis caused by Coxiella burnetii which is among the major agents of community-acquired pneumonia in French Guiana. Despite its relatively high incidence, its epidemiology in French Guiana remains unclear, and all previous studies have considered transmission from livestock unlikely, suggesting that a wild reservoir is responsible for transmission. Methods: A country-wide seroprevalence survey of 2697 participants from French Guiana was conducted. Serum samples were tested for phase II IgG antibodies by ELISA and indirect immunofluorescence assays (IFAs). Factors associated with Q fever were investigated, and a serocatalytic model was used to reconstruct the annual force of infection. Findings: The overall weighted seroprevalence was estimated at 9.6% (95% confidence interval (CI): 8.2%-11.0%). The model revealed constant, low-level circulation across French Guiana, particularly affecting middle-aged males (odds ratio (OR): 3.0, 95% credible interval (CrI): 1.7-5.8) and individuals living close to sheep farms (OR: 4, 95% CrI: 1.5-12). The overall annual number of cases was estimated at 579 (95% CrI: 492-670). In the region around Cayenne, the main urban municipality, the high seroprevalence was explained by an outbreak that may have occurred between 1996 and 2003 and that infected 10% (95% CrI: 6.9%-14%) of the population and males and females alike. Interpretation: This study reveals for the first time Q fever dynamics of transmission and the role of domestic livestock in transmission in French Guiana and highlights the urgent need to reinforce Q fever surveillance in livestocks of the entire Guianese territory. Funding: This study was supported by the "European Regional Development Fund" under EPI-ARBO grant agreement (GY0008695), the "Regional Health Agency of French Guiana" and the "National Center of Spatial Studies". The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Neopterin and CXCL-13 in Diagnosis and Follow-Up of Trypanosoma brucei gambiense Sleeping Sickness: Lessons from the Field in Angola.

Human African Trypanosomiasis may become manageable in the next decade with fexinidazole. However, currently stage diagnosis remains difficult to implement in the field and requires a lumbar puncture. Our study of an Angolan cohort of T. b. gambiense-infected patients used other staging criteria than those recommended by the WHO. We compared WHO criteria (cell count and parasite identification in the CSF) with two biomarkers (neopterin and CXCL-13) which have proven potential to diagnose disease stage or relapse. Biological, clinical, and neurological data were analysed from a cohort of 83 patients. A neopterin concentration below 15.5 nmol/L in the CSF denoted patients with stage 1 disease, and a concentration above 60.31 nmol/L characterized patients with advanced stage 2 (trypanosomes in CSF and/or cytorachia higher than 20 cells) disease. CXCL-13 levels below 91.208 pg/mL denoted patients with stage 1 disease, and levels of CXCL-13 above 395.45 pg/mL denoted patients with advanced stage 2 disease. Values between these cut-offs may represent patients with intermediate stage disease. Our work supports the existence of an intermediate stage in HAT, and CXCL-13 and neopterin levels may help to characterize it.

Using detergent-enhanced LAMP for African trypanosome detection in human cerebrospinal fluid and implications for disease staging.

OBJECTIVE: Where human African trypanosomiasis (HAT) patients are seen, failure to microscopically diagnose infections by Trypanosoma brucei gambiense in blood smears and/or cerebrospinal fluid (CSF) in the critical early stages of the disease is the single most important factor in treatment failure, a result of delayed treatment onset or its absence. We hypothesized that the enhanced sensitivity of detergent-enhanced loop-mediated isothermal amplification (LAMP) will allow for point of care (POC) detection of African trypanosomes in the CSF of HAT patients where the probability for detecting a single parasite or parasite DNA molecule in 1 µL of CSF sample is negligible by current methods. METHODOLOGY: We used LAMP targeting the multicopy pan-T. brucei repetitive insertion mobile element (RIME LAMP) and the Trypanosoma brucei gambiense 5.8S rRNA-internal transcribed spacer 2 gene (TBG1 LAMP). We tested 1 µL out of 20 µL sham or Triton X-100 treated CSFs from 73 stage-1 and 77 stage-2 HAT patients from the Central African Republic and 100 CSF negative controls. RESULTS: Under sham conditions, parasite DNA was detected by RIME and TBG1 LAMP in 1.4% of the stage-1 and stage-2 gambiense HAT CSF samples tested. After sample incubation with detergent, the number of LAMP parasite positive stage-2 CSF's increased to 26%, a value which included the 2 of the 4 CSF samples where trypanosomes were identified microscopically. Unexpected was the 41% increase in parasite positive stage-1 CSF's detected by LAMP. Cohen's kappa coefficients for RIME versus TBG1 LAMP of 0.92 (95%CI: 0.82-1.00) for stage-1 and 0.90 (95%CI: 0.80-1.00) for stage-2 reflected a high level of agreement between the data sets indicating that the results were not due to amplicon contamination, data confirmed in χ2 tests (p<0.001) and Fisher's exact probability test (p = 4.7e-13). CONCLUSION: This study detected genomic trypanosome DNA in the CSF independent of the HAT stage and may be consistent with early CNS entry and other scenarios that identify critical knowledge gaps for future studies. Detergent-enhanced LAMP could be applicable for non-invasive African trypanosome detection in human skin and saliva or as an epidemiologic tool for the determination of human (or animal) African trypanosome prevalence in areas where chronically low parasitemias are present.

Proteome characterization in various biological fluids of Trypanosoma brucei gambiense-infected subjects.

Human African trypanosomiasis (HAT) is a neglected tropical disease that is endemic in sub-Saharan Africa. Control of the disease has been recently improved by better screening and treatment strategies, and the disease is on the WHO list of possible elimination. However, some physiopathological aspects of the disease transmission and progression remain unclear. We propose a new proteomic approach to identify new targets and thus possible new biomarkers of the disease. We also focused our attention on fluids classically associated with HAT (serum and cerebrospinal fluid (CSF)) and on the more easily accessible biological fluids urine and saliva. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) established the proteomic profile of patients with early and late stage disease. The serum, CSF, urine and saliva of 3 uninfected controls, 3 early stage patients and 4 late stage patients were analyzed. Among proteins identified, in CSF, urine and saliva, respectively, 37, 8 and 24 proteins were differentially expressed and showed particular interest with regards to their function. The most promising proteins (Neogenin, Neuroserpin, secretogranin 2 in CSF; moesin in urine and intelectin 2 in saliva) were quantified by enzyme-linked immunosorbent assay in a confirmatory cohort of 14 uninfected controls, 23 patients with early stage disease and 43 patients with late stage disease. The potential of two proteins, neuroserpin and moesin, with the latter present in urine, were further characterized. Our results showed the potential of proteomic analysis to discover new biomarkers and provide the basis of the establishment of a new proteomic catalogue applied to HAT-infected subjects and controls. SIGNIFICANCE: Sleeping sickness, also called Human African Trypanosomiasis (HAT), is a parasitic infection caused by a parasitic protozoan, Trypanosoma brucei gambiense or T. b. rhodesiense which are transmitted via an infected tsetse fly: Glossina. For both, the haemolymphatic stage (or first stage) signs and symptoms are intermittent fever, lymphadenopathy, hepatosplenomegaly, headaches, pruritus, and for T. b. rhodesiense infection a chancre is often formed at the bite site. Meningoencephalitic stage (or second stage) occurs when parasites invade the CNS, it is characterised by neurological signs and symptoms such as altered gait, tremors, neuropathy, somnolence which can lead to coma and death if untreated. first stage of the disease is characterizing by fevers, headaches, itchiness, and joint pains and progressive lethargy corresponding to the second stage with confusion, poor coordination, numbness and trouble sleeping. Actually, diagnosing HAT requires specialized expertise and significant resources such as well-equipped health centers and qualified staff. Such resources are lacking in many endemic areas that are often in rural locales, so many individuals with HAT die before the diagnosis is established. In this study, we analysed by mass spectrometry the entire proteome of serum, CSF, urine and saliva samples from infected and non-infected Angolan individuals to define new biomarkers of the disease. This work of proteomics analysis is a preliminary stage to the characterization of the whole proteome, of these 4 biological fluids, of HAT patients. We have identified 69 new biomarkers. Five of them have been thoroughly investigated by ELISA quantification. Neuroserpine and Moesin are respectively promising new biomarkers in CSF and urine's patient for a better diagnosis.

Metabolomics Identifies Multiple Candidate Biomarkers to Diagnose and Stage Human African Trypanosomiasis.

Treatment for human African trypanosomiasis is dependent on the species of trypanosome causing the disease and the stage of the disease (stage 1 defined by parasites being present in blood and lymphatics whilst for stage 2, parasites are found beyond the blood-brain barrier in the cerebrospinal fluid (CSF)). Currently, staging relies upon detecting the very low number of parasites or elevated white blood cell numbers in CSF. Improved staging is desirable, as is the elimination of the need for lumbar puncture. Here we use metabolomics to probe samples of CSF, plasma and urine from 40 Angolan patients infected with Trypanosoma brucei gambiense, at different disease stages. Urine samples provided no robust markers indicative of infection or stage of infection due to inherent variability in urine concentrations. Biomarkers in CSF were able to distinguish patients at stage 1 or advanced stage 2 with absolute specificity. Eleven metabolites clearly distinguished the stage in most patients and two of these (neopterin and 5-hydroxytryptophan) showed 100% specificity and sensitivity between our stage 1 and advanced stage 2 samples. Neopterin is an inflammatory biomarker previously shown in CSF of stage 2 but not stage 1 patients. 5-hydroxytryptophan is an important metabolite in the serotonin synthetic pathway, the key pathway in determining somnolence, thus offering a possible link to the eponymous symptoms of "sleeping sickness". Plasma also yielded several biomarkers clearly indicative of the presence (87% sensitivity and 95% specificity) and stage of disease (92% sensitivity and 81% specificity). A logistic regression model including these metabolites showed clear separation of patients being either at stage 1 or advanced stage 2 or indeed diseased (both stages) versus control.

Sensitivity and Specificity of a Prototype Rapid Diagnostic Test for the Detection of Trypanosoma brucei gambiense Infection: A Multi-centric Prospective Study.

BACKGROUND: A major challenge in the control of human African trypanosomiasis (HAT) is lack of reliable diagnostic tests that are rapid and easy to use in remote areas where the disease occurs. In Trypanosoma brucei gambiense HAT, the Card Agglutination Test for Trypanosomiasis (CATT) has been the reference screening test since 1978, usually on whole blood, but also in a 1/8 dilution (CATT 1/8) to enhance specificity. However, the CATT is not available in a single format, requires a cold chain for storage, and uses equipment that requires electricity. A solution to these challenges has been provided by rapid diagnostic tests (RDT), which have recently become available. A prototype immunochromatographic test, the SD BIOLINE HAT, based on two native trypanosomal antigens (VSG LiTat 1.3 and VSG LiTat 1.5) has been developed. We carried out a non-inferiority study comparing this prototype to the CATT 1/8 in field settings. METHODOLOGY/PRINCIPAL FINDINGS: The prototype SD BIOLINE HAT, the CATT Whole Blood and CATT 1/8 were systematically applied on fresh blood samples obtained from 14,818 subjects, who were prospectively enrolled through active and passive screening in clinical studies in three endemic countries of central Africa: Angola, the Democratic Republic of the Congo and the Central African Republic. One hundred and forty nine HAT cases were confirmed by parasitology. The sensitivity and specificity of the prototype SD BIOLINE HAT was 89.26% (95% confidence interval (CI) = 83.27-93.28) and 94.58% (95% CI = 94.20-94.94) respectively. The sensitivity and specificity of the CATT on whole blood were 93.96% (95% CI = 88.92-96.79) and 95.91% (95% CI = 95.58-96.22), and of the CATT 1/8 were 89.26% (95% CI = 83.27-93.28) and 98.88% (95% CI = 98.70-99.04) respectively. CONCLUSION/SIGNIFICANCE: After further optimization, the prototype SD BIOLINE HAT could become an alternative to current screening methods in primary healthcare settings in remote, resource-limited regions where HAT typically occurs.

Detection of pathogen-specific antibodies by loop-mediated isothermal amplification.

Loop-mediated isothermal amplification (LAMP) is a method for enzymatically replicating DNA that has great utility for clinical diagnosis at the point of care (POC), given its high sensitivity, specificity, speed, and technical requirements (isothermal conditions). Here, we adapted LAMP for measuring protein analytes by creating a protein-DNA fusion (referred to here as a "LAMPole") that attaches oligonucleotides (LAMP templates) to IgG antibodies. This fusion consists of a DNA element covalently bonded to an IgG-binding polypeptide (protein L/G domain). In our platform, LAMP is expected to provide the most suitable means for amplifying LAMPoles for clinical diagnosis at the POC, while quantitative PCR is more suitable for laboratory-based quantification of antigen-specific IgG abundance. As proof of concept, we measured serological responses to a protozoan parasite by quantifying changes in solution turbidity in real time. We observed a >6-log fold difference in signal between sera from vaccinated versus control mice and in a clinical patient sample versus a control. We assert that LAMPoles will be useful for increasing the sensitivity of measuring proteins, whether it be in a clinical laboratory or in a field setting, thereby improving acute diagnosis of a variety of infections.

Neopterin is a cerebrospinal fluid marker for treatment outcome evaluation in patients affected by Trypanosoma brucei gambiense sleeping sickness.

BACKGROUND: Post-therapeutic follow-up is essential to confirm cure and to detect early treatment failures in patients affected by sleeping sickness (HAT). Current methods, based on finding of parasites in blood and cerebrospinal fluid (CSF) and counting of white blood cells (WBC) in CSF, are imperfect. New markers for treatment outcome evaluation are needed. We hypothesized that alternative CSF markers, able to diagnose the meningo-encephalitic stage of the disease, could also be useful for the evaluation of treatment outcome. METHODOLOGY/PRINCIPAL FINDINGS: Cerebrospinal fluid from patients affected by Trypanosoma brucei gambiense HAT and followed for two years after treatment was investigated. The population comprised stage 2 (S2) patients either cured or experiencing treatment failure during the follow-up. IgM, neopterin, B2MG, MMP-9, ICAM-1, VCAM-1, CXCL10 and CXCL13 were first screened on a small number of HAT patients (n = 97). Neopterin and CXCL13 showed the highest accuracy in discriminating between S2 cured and S2 relapsed patients (AUC 99% and 94%, respectively). When verified on a larger cohort (n = 242), neopterin resulted to be the most efficient predictor of outcome. High levels of this molecule before treatment were already associated with an increased risk of treatment failure. At six months after treatment, neopterin discriminated between cured and relapsed S2 patients with 87% specificity and 92% sensitivity, showing a higher accuracy than white blood cell numbers. CONCLUSIONS/SIGNIFICANCE: In the present study, neopterin was highlighted as a useful marker for the evaluation of the post-therapeutic outcome in patients suffering from sleeping sickness. Detectable levels of this marker in the CSF have the potential to shorten the follow-up for HAT patients to six months after the end of the treatment.

New biomarkers for stage determination in Trypanosoma brucei rhodesiense sleeping sickness patients.

Accurate stage determination is crucial in the choice of treatment for patients suffering from sleeping sickness, also known as human African trypanosomiasis (HAT). Current staging methods, based on the counting of white blood cells (WBC) and the detection of parasites in the cerebrospinal fluid (CSF) have limited accuracy. We hypothesized that immune mediators reliable for staging T. b. gambiense HAT could also be used to stratify T. b. rhodesiense patients, the less common form of HAT.A population comprising 85 T. b. rhodesiense patients, 14 stage 1 (S1) and 71 stage 2 (S2) enrolled in Malawi and Uganda, was investigated. The CSF levels of IgM, MMP-9, CXCL13, CXCL10, ICAM-1, VCAM-1, neopterin and B2MG were measured and their staging performances evaluated using receiver operating characteristic (ROC) analyses.IgM, MMP-9 and CXCL13 were the most accurate markers for stage determination (partial AUC 88%, 86% and 85%, respectively). The combination in panels of three molecules comprising CXCL13-CXCL10-MMP-9 or CXCL13-CXCL10-IgM significantly increased their staging ability to partial AUC 94% (p value < 0.01).The present study highlighted new potential markers for stage determination of T. b. rhodesiense patients. Further investigations are needed to better evaluate these molecules, alone or in panels, as alternatives to WBC to make reliable choice of treatment.

Cerebrospinal fluid neopterin as marker of the meningo-encephalitic stage of Trypanosoma brucei gambiense sleeping sickness.

BACKGROUND: Sleeping sickness, or human African trypanosomiasis (HAT), is a protozoan disease that affects rural communities in sub-Saharan Africa. Determination of the disease stage, essential for correct treatment, represents a key issue in the management of patients. In the present study we evaluated the potential of CXCL10, CXCL13, ICAM-1, VCAM-1, MMP-9, B2MG, neopterin and IgM to complement current methods for staging Trypanosoma brucei gambiense patients. METHODS AND FINDINGS: Five hundred and twelve T. b. gambiense HAT patients originated from Angola, Chad and the Democratic Republic of the Congo (D.R.C.). Their classification as stage 2 (S2) was based on the number of white blood cells (WBC) (>5/µL) or presence of parasites in the cerebrospinal fluid (CSF). The CSF concentration of the eight markers was first measured on a training cohort encompassing 100 patients (44 S1 and 56 S2). IgM and neopterin were the best in discriminating between the two stages of disease with 86.4% and 84.1% specificity respectively, at 100% sensitivity. When a validation cohort (412 patients) was tested, neopterin (14.3 nmol/L) correctly classified 88% of S1 and S2 patients, confirming its high staging power. On this second cohort, neopterin also predicted both the presence of parasites, and of neurological signs, with the same ability as IgM and WBC, the current reference for staging. CONCLUSIONS: This study has demonstrated that neopterin is an excellent biomarker for staging T. b. gambiense HAT patients. A rapid diagnostic test for detecting this metabolite in CSF could help in more accurate stage determination.

Hepatitis C virus infection may lead to slower emergence of P. falciparum in blood.

BACKGROUND: Areas endemic for Plasmodium falciparum, hepatitis B virus (HBV) and hepatitis C virus (HCV) overlap in many parts of sub-Saharan Africa. HBV and HCV infections develop in the liver, where takes place the first development stage of P. falciparum before its further spread in blood. The complex mechanisms involved in the development of hepatitis may potentially influence the development of the liver stage of malaria parasites. Understanding the molecular mechanisms of these interactions could provide new pathophysiological insights for treatment strategies in Malaria. METHODOLOGY: We studied a cohort of 319 individuals living in a village where the three infections are prevalent. The patients were initially given a curative antimalarial treatment and were then monitored for the emergence of asexual P. falciparum forms in blood, fortnightly for one year, by microscopy and polymerase chain reaction. PRINCIPAL FINDINGS: At inclusion, 65 (20.4%) subjects had detectable malaria parasites in blood, 36 (11.3%) were HBV chronic carriers, and 61 (18.9%) were HCV chronic carriers. During follow-up, asexual P. falciparum forms were detected in the blood of 203 patients. The median time to P. falciparum emergence in blood was respectively 140 and 120 days in HBV- and HBV+ individuals, and 135 and 224 days in HCV- and HCV+ individuals. HCV carriage was associated with delayed emergence of asexual P. falciparum forms in blood relative to patients without HCV infection. CONCLUSIONS: This pilot study represents first tentative evidence of a potential epidemiological interaction between HBV, HCV and P. falciparum infections. Age is an important confounding factor in this setting however multivariate analysis points to an interaction between P. falciparum and HCV at the hepatic level with a slower emergence of P. falciparum in HCV chronic carriers. More in depth analysis are necessary to unravel the basis of hepatic interactions between these two pathogens, which could help in identifying new therapeutic approaches against malaria.

Immunophenotypic lymphocyte profiles in human african trypanosomiasis.

Human African trypanosomiasis (HAT) is a deadly vector-born disease caused by an extracellular parasite, the trypanosome. Little is known about the cellular immune responses elicited by this parasite in humans. We used multiparameter flow cytometry to characterize leukocyte immunophenotypes in the blood and cerebrospinal fluid (CSF) of 33 HAT patients and 27 healthy controls identified during a screening campaign in Angola and Gabon. We evaluated the subsets and activation markers of B and T lymphocytes. Patients had a higher percentage of CD19+ B lymphocytes and activated B lymphocytes in the blood than did controls, but lacked activated CD4+ T lymphocytes (CD25+). Patients displayed no increase in the percentage of activated CD8+ T cells (HLA-DR+, CD69+ or CD25+), but memory CD8 T-cell levels (CD8+CD45RA2) were significantly lower in patients than in controls, as were effector CD8 T-cell levels (CD8+CD45RA+CD62L2). No relationship was found between these blood immunophenotypes and disease severity (stage 1 vs 2). However, CD19+ B-cell levels in the CSF increased with disease severity. The patterns of T and B cell activation in HAT patients suggest that immunomodulatory mechanisms may operate during infection. Determinations of CD19+ B-cell levels in the CSF could improve disease staging.

Increased CXCL-13 levels in human African trypanosomiasis meningo-encephalitis.

OBJECTIVES: To determine the role of the B-cell attracting chemokine CXCL-13, which may initiate B-cell trafficking and IgM production in diagnosing HAT meningo-encephalitis. METHODS: We determined CXCL-13 levels by ELISA on paired sera and CSF of 26 patients from Angola and of 16 controls (six endemic and ten non-endemic). Results were compared to standard stage determination markers and IgM intrathecal synthesis. RESULTS: CXCL-13 levels in patients' sera had a median value of 386.6 pg/ml and increased levels were associated with presence of trypanosomes in the CSF but not with other stage markers. CXCL-13 levels in patients' CSF had a median value of 80.9 pg/ml and increased levels were associated with all standard stage determination markers and IgM intrathecal synthesis. CONCLUSION: CXCL-13 levels in CSF increased significantly during the course of HAT. Hence the value of CXCL-13 for diagnosis, follow-up or as a marker of disease severity should be tested in a well-defined cohort study.

Novel markers for treatment outcome in late-stage Trypanosoma brucei gambiense trypanosomiasis.

BACKGROUND: To date, no biological marker for treatment outcome in human African trypanosomiasis (HAT) has been described. The accuracy of biological markers for prediction of treatment outcome of HAT caused by Trypanosoma brucei gambiense was assessed. METHODS: Cerebrospinal fluid (CSF) white blood cell (WBC) count and immunoglobulin M (IgM), trypanosome-specific antibody, total protein, and interleukin-10 levels were determined before and up to 24 months after treatment of late-stage HAT. RESULTS: Treatment failure was experienced by 48 of 260 patients. Pretreatment CSF WBC counts > or = 102 cells/microL, IL-10 concentrations > or = 37 pg/mL, LATEX/IgM end titers > or = 1:32, LATEX/T. b. gambiense end titers > or = 1:2, and protein concentrations > or = 674 mg/L were associated with treatment failure. Six months after treatment, patients with CSF WBC counts < or = 5 cells/microL were at low risk of HAT recurrence (negative predictive value, >0.93). After 12 months, the combination of CSF WBC count > or = 8 cells/microL and LATEX/IgM end titer > or = 1:4 predicted treatment failure with 97% specificity and 79% sensitivity. Eighteen months after treatment, each marker accurately predicted treatment outcome. The combination of CSF WBC count > or = 8 cells/microL and LATEX/IgM end titer > or = 1:4 was 100% specific for treatment failure after 18 and 24 months. CONCLUSIONS: HAT-affected patients with elevated pretreatment CSF levels of WBC, interleukin-10, IgM, trypanosome-specific antibody, and total protein are at risk of treatment failure. Six months after treatment, patients with CSF WBC counts < or = 5 cells/microL can be considered to be cured. The assessment of a combination of CSF WBC count and LATEX/IgM level allowed accurate prediction of outcome beginning at 12 months after treatment, as did each individual marker at 18 months after treatment.

Hypocretin and human African trypanosomiasis.

OBJECTIVES: To detail clinical and polysomnographic characteristics in patients affected with Trypanosoma brucei gambiense (Tb.g.) human African trypanosomiasis (HAT) at different stages of evolution and to measure and compare cerebrospinal fluid (CSF) levels of hypocretin-1 with narcoleptic patients and neurologic controls. METHODS: Twenty-five untreated patients affected with T.b.g. HAT were included. The patients were evaluated using a standardized clinical evaluation and a specific interview on sleep complaints. Diagnosis of stages I and II and intermediate stage was performed by CSF cell count and/or presence of trypanosomes: 4 patients were classified as stage II, 13 stage I, and 8 "intermediate" stage. Seventeen untreated patients completed continuous 24-hour polysomnography. We measured CSF levels of hypocretin-1 in all patients at different stages and evolutions, and we compared the results with 26 patients with narcolepsy-cataplexy and 53 neurologic controls. RESULTS: CSF hypocretin-1 levels were significantly higher in T.b.g. HAT (423.2 +/- 119.7 pg/mL) than in narcoleptic patients (40.16 +/- 60.18 pg/ mL) but lower than in neurologic controls (517.32 +/- 194.5 pg/mL). One stage I patient had undetectable hypocretin levels and 1 stage II patient showed intermediate levels, both patients (out of three patients) reporting excessive daytime sleepiness but without evidence for an association with narcolepsy. No differences were found in CSF hypocretin levels between patients with HAT stages; however, the presence of major sleep-wake cycle disruptions was significantly associated with lower CSF hypocretin-1 level with a same tendency for the number of sleep-onset rapid eye movement periods. CONCLUSION: The present investigation is not in favor of a unique implication of the hypocretin system in T.b.g. HAT. However, we propose that dysfunction of the hypothalamic hypocretin region may participate in sleep disturbances observed in African trypanosomiasis.

Whole-transcriptome analysis of Plasmodium falciparum field isolates: identification of new pathogenicity factors.

BACKGROUND: Severe malaria and one of its most important pathogenic processes, cerebral malaria, involves the sequestration of parasitized red blood cells (pRBCs) in brain postcapillary venules. Although the pathogenic mechanisms underlying malaria remain poorly characterized, it has been established that adhesion of pRBCs to endothelial cells (ECs) can result in cell apoptosis, which in turn may lead to disruption of the blood-brain barrier. The nature of the parasite molecules involved in the pathogenesis of severe malaria remains elusive. METHODS: Whole-transcriptome profiling of nonapoptogenic versus apoptogenic parasite field isolates obtained from Gabonese children was performed with pan-genomic Plasmodium falciparum DNA microarrays; radiolabeled instead of fluorescent cDNAs were used to improve the sensitivity of signal detection. RESULTS: Our methods allowed the identification of 59 genes putatively associated with the induction of EC apoptosis. Silencing of Plasmodium gene expression with specific double-stranded RNA was performed on 8 selected genes; 5 of these, named "Plasmodium apoptosis-linked pathogenicity factors" (PALPFs), were found to be linked to parasite apoptogenicity. Of these genes, 2 might act via parasite cytoadherence. CONCLUSION: This is the first attempt to identify genes involved in parasite pathogenic mechanisms against human ECs. The finding of PALPFs illuminates perspectives for novel therapeutic strategies against cerebral complications of malaria.

Cytoadherence and genotype of Plasmodium falciparum strains from symptomatic children in Franceville, southeastern Gabon.

BACKGROUND: Plasmodium falciparum causes severe clinical manifestations by sequestering parasitized red blood cells (PRBC) in the microvasculature of major organs such as the brain. This sequestration results from PRBC adherence to vascular endothelial cells via erythrocyte membrane protein 1, a variant parasite surface antigen. OBJECTIVE: To determine whether P. falciparum multiple genotype infection (MGI) is associated with stronger PRBC cytoadherence and greater clinical severity. METHODS: Nested polymerase chain reaction was used to genotype P. falciparum isolates from symptomatic children and to distinguish between single genotype infection (SGI) and MGI. PRBC cytoadhesion was studied with cultured human lung endothelial cells. RESULTS: Analysis of two highly polymorphic regions of the merozoite surface antigen (MSP)-1 and MSP-2 genes and a dimorphic region of the erythrocyte binding antigen-175 gene showed that 21.4% and 78.6% of the 42 children had SGI and MGI, respectively. It also showed that 37 (89%) of the 42 PRBC samples expressed MSP-1 allelic family K1. Cytoadherence values ranged from 58 to 1811 PRBC/mm(2) of human lung endothelial cells monolayer in SGI and from 5 to 5744 PRBC/mm(2) in MGI. MGI was not associated with higher cytoadherence values or with more severe malaria. CONCLUSIONS: These results suggested that infection of the same individual by multiple clones of P. falciparum does not significantly influence PRBC cytoadherence or disease severity and confirmed the predominance of the MSP-1 K1 genotype in southeastern Gabon.

Treatment failure related to intrathecal immunoglobulin M (IgM) synthesis, cerebrospinal fluid IgM, and interleukin-10 in patients with hemolymphatic-stage sleeping sickness.

Human African trypanosomiasis treatment is stage dependent, but the tests used for staging are controversial. Central nervous system involvement and its relationship with suramin treatment failure were assessed in 60 patients with parasitologically confirmed hemolymphatic-stage Trypanosoma brucei gambiense infection (white blood cell count of or=1.9 mg/liter (OR, 11.7; 95% CI, 2.7 to 50), a CSF end titer by the LATEX/IgM assay of >or=2 (OR, 10.4; 95% CI, 2.5 to 44), and a CSF interleukin-10 concentration of >10 pg/ml (OR, 5; 95% CI, 1.3 to 20). The sensitivities of these markers for treatment failure ranged from 43 to 79%, and the specificities ranged from 74 to 93%. The results show that T. brucei gambiense-infected patients who have signs of neuroinflammation in CSF and who are treated with drugs recommended for use at the hemolymphatic stage are at risk of treatment failure. This highlights the need for the development and the evaluation of accurate point-of-care tests for the staging of human African trypanosomiasis.

Equivalence trial of melarsoprol and nifurtimox monotherapy and combination therapy for the treatment of second-stage Trypanosoma brucei gambiense sleeping sickness.

BACKGROUND: Treatment of second-stage sleeping sickness relies mainly on melarsoprol. Nifurtimox has been successfully used to cure melarsoprol-refractory sleeping sickness caused by Trypanosoma brucei gambiense infection. METHODS: An open, randomized trial was conducted to test for equivalence between the standard melarsoprol regimen and 3 other regimens, as follows: standard melarsoprol therapy (3 series of 3.6 mg/kg/day intravenously [iv] for 3 days, with 7-day breaks between the series); 10-day incremental-dose melarsoprol therapy (0.6 mg/kg iv on day 1, 1.2 mg/kg iv on day 2, and 1.8 mg/kg iv on days 3-10); nifurtimox monotherapy for 14 days (5 mg/kg orally 3 times per day); and consecutive 10-day melarsoprol-nifurtimox combination therapy (0.6 mg/kg iv melarsoprol on day 1, 1.2 mg/kg iv melarsoprol on day 2, and 1.2 mg/kg/day iv melarsoprol combined with oral 7.5 mg/kg nifurtimox twice a day on days 3-10). Primary outcomes were relapse, severe adverse events, and death attributed to treatment. RESULTS: A total of 278 patients were randomized. The frequency of adverse events was similar between the standard melarsoprol regimen and the other regimens. Encephalopathic syndromes occurred in all groups and caused all deaths that were likely due to treatment. Relapses (n=48) were observed only with the 3 monotherapy regimens. CONCLUSION: A consecutive 10-day low-dose melarsoprol-nifurtimox combination is more effective than the standard melarsoprol regimen.

Submicroscopic Plasmodium falciparum infections before and after sulfadoxine-pyrimethamine and artesunate association treatment in Dienga, Southeastern Gabon.

BACKGROUND: It has been shown that Plasmodium falciparum submicroscopic infections (SMI) can contribute to malaria-associated anemia as well as to cerebral malaria. Polymerase chain reaction (PCR) assays are usually used as an alternative to microscopy in detecting subpatently infected individuals. OBJECTIVES: The main objective of this study was to investigate the occurrence of SMI before and after a suppressive antimalarial treatment in the population of the village of Dienga in Gabon. METHODS: Nested PCR was used to detect SMI and to determine genotypes. RESULTS: The prevalence rates of SMI were 13.67% (38/278) at day 0 and 8.99% (25/278) at day 14 after sulfadoxine-pyrimethamine-artesunate treatment. Genotype analysis of two polymorphic regions of the merozoite surface protein (MSP)-1 block 2, MSP-2 and a dimorphic region of the erythrocyte binding antigen (EBA-175) revealed that as many as 88% (22/25) of SMI detected after treatment were completely new alleles, indicating either previously sequestered parasites or newly acquired infections. CONCLUSION: These results demonstrate the usefulness of sulfadoxine-pyrimethamine-artesunate association treatment in the population of Dienga and confirmed early parasite genotype change after a suppressive antimalarial treatment in endemic areas.