Neural Damage in Experimental Trypanosoma brucei gambiense Infection: Hypothalamic Peptidergic Sleep and Wake-Regulatory Neurons.

Neuron populations of the lateral hypothalamus which synthesize the orexin (OX)/hypocretin or melanin-concentrating hormone (MCH) peptides play crucial, reciprocal roles in regulating wake stability and sleep. The disease human African trypanosomiasis (HAT), also called sleeping sickness, caused by extracellular Trypanosoma brucei (T. b.) parasites, leads to characteristic sleep-wake cycle disruption and narcoleptic-like alterations of the sleep structure. Previous studies have revealed damage of OX and MCH neurons during systemic infection of laboratory rodents with the non-human pathogenic T. b. brucei subspecies. No information is available, however, on these peptidergic neurons after systemic infection with T. b. gambiense, the etiological agent of 97% of HAT cases. The present study was aimed at the investigation of immunohistochemically characterized OX and MCH neurons after T. b. gambiense or T. b. brucei infection of a susceptible rodent, the multimammate mouse, Mastomys natalensis. Cell counts and evaluation of OX fiber density were performed at 4 and 8 weeks post-infection, when parasites had entered the brain parenchyma from the periphery. A significant decrease of OX neurons (about 44% reduction) and MCH neurons (about 54% reduction) was found in the lateral hypothalamus and perifornical area at 8 weeks in T. b. gambiense-infected M. natalensis. A moderate decrease (21% and 24% reduction, respectively), which did not reach statistical significance, was found after T. b. brucei infection. In two key targets of diencephalic orexinergic innervation, the peri-suprachiasmatic nucleus (SCN) region and the thalamic paraventricular nucleus (PVT), densitometric analyses showed a significant progressive decrease in the density of orexinergic fibers in both infection paradigms, and especially during T. b. gambiense infection. Altogether the findings provide novel information showing that OX and MCH neurons are highly vulnerable to chronic neuroinflammatory signaling caused by the infection of human-pathogenic African trypanosomes

SNPs in IL4 and IFNG show no protective associations with human African trypanosomiasis in the Democratic Republic of the Congo: a case-control study

Background: Human African trypanosomiasis (HAT) is a protozoal disease transmitted by tsetse flies. Infection with trypanosomes can lead directly to active HAT or latent infection with no detectable parasites, which may progress to active HAT or to spontaneous self- cure. Genetic variation could explain these differences in the outcome of infection. To test this hypothesis, polymorphisms in 17 candidate genes were tested (APOL1 [G1 and G2], CFH, HLA-A, HPR, HP, IL1B, IL12B, IL12RB1, IL10, IL4R, MIF, TNFA, IL6, IL4, IL8, IFNG, and HLA-G). Methods: Samples were collected in Democratic Republic of the Congo. 233 samples were genotyped: 100 active HAT cases, 33 from subjects with latent infections and 100 negative controls. Commercial service providers genotyped polymorphisms at 96 single nucleotide polymorphisms (SNPs) on 17 genes. Data were analyzed using Plink V1.9 software and R. Loci, with suggestive associations (uncorrected p < 0.05) validated using an additional 594 individuals, including 164 cases and 430 controls. Results: After quality control, 87 SNPs remained in the analysis. Two SNPs in IL4 and two in IFNG were suggestively associated (uncorrected p<0.05) with a differential risk of developing a Trypanosoma brucei gambiense infection in the Congolese population. The IFNG minor allele (rs2430561, rs2069718) SNPs were protective in comparison between latent infections and controls. Carriers of the rs2243258_T and rs2243279_A alleles of IL4 and the rs2069728_T allele of IFNG had a reduced risk of developing illness or latent infection, respectively. None of these associations were significant after Bonferroni correction for multiple testing. A validation study using more samples was run to determine if the absence of significant association was due to lack of power. Conclusions: This study showed no evidence of an association of HAT with IL4 and IFNG SNPs or with APOL1 G1 and G2 alleles, which have been found to be protective in other studies

A CATT Negative Result after Treatment for Human African Trypanosomiasis Is No Indication for Cure

The 2 year follow-up period required after treatment of human African trypanosomiasis (HAT) patients is a major challenge for patients and control programmes alike. The patient should return every 6 months for lumbar puncture and cerebrospinal fluid examination since, so far, no markers for cure have been identified in blood. The Card Agglutination Test for Trypanosomiasis (CATT) is a simple, rapid test for trypanosome-specific antibody detection in blood that is extensively used in endemic areas to screen for HAT. We examined the value of a normalising CATT as a marker for treatment outcome. We observed that CATT titres decreased after treatment both in patients who experienced treatment failure as well as in cured patients. We conclude that CATT, though a good screening test, is unreliable for monitoring treatment outcome. We also showed that the sensitivity of CATT in relapse cases was as low as 78%, and as a consequence some relapse cases might be missed in screening programs if they have no clinical signs yet

Sensitivity and specifi city of HAT Sero-K-SeT, a rapid diagnostic test for serodiagnosis of sleeping sickness caused by Trypanosoma brucei gambiense: a case-control study

Background Human African trypanosomiasis (HAT) is a life-threatening infection aff ecting rural populations in sub- Saharan Africa. Large-scale population screening by antibody detection with the Card Agglutination Test for Trypanosomiasis (CATT)/Trypanosoma brucei (T b) gambiense helped reduce the number of reported cases of gambiense HAT to fewer than 10 000 in 2011. Because low case numbers lead to decreased cost-eff ectiveness of such active screening, we aimed to assess diagnostic accuracy of a rapid serodiagnostic test (HAT Sero-K-SeT) applicable in primary health-care centres. Methods In our case-control study, we assessed participants older than 11 years who presented for HAT Sero-K-SeT and CATT/T b gambiense at primary care centres or to mobile teams (and existing patients with confi rmed disease status at these centres) in Bandundu Province, DR Congo. We defi ned cases as patients with trypanosomes that had been identifi ed in lymph node aspirate, blood, or cerebrospinal fl uid. During screening, we recruited controls without previous history of HAT or detectable trypanosomes in blood or lymph who resided in the same area as the cases. We assessed diagnostic accuracy of three antibody detection tests for gambiense HAT: HAT Sero-K-SeT and CATT/T b gambiense (done with venous blood at the primary care centres) and immune trypanolysis (done with plasma at the Institute of Tropical Medicine, Antwerp, Belgium). Findings Between June 6, 2012, and Feb 25, 2013, we included 134 cases and 356 controls. HAT Sero-K-SeT had a sensitivity of 0·985 (132 true positives, 95% CI 0·947–0·996) and a specifi city of 0·986 (351 true negatives, 0·968–0·994), which did not diff er signifi cantly from CATT/T b gambiense (sensitivity 95% CI 0·955, 95% CI 0·906–0·979 [128 true positives] and specifi city 0·972, 0·949–0·985 [346 true negatives]) or immune trypanolysis (sensitivity 0·985, 0·947–0·996 [132 true positives] and specifi city 0·980, 0·960–0·990 [349 true negatives]). Interpretation The diagnostic accuracy of HAT Sero-K-SeT is adequate for T b gambiense antibody detection in local health centres and could be used for active screening whenever a cold chain and electricity supply are unavailable and CATT/T b gambiense cannot be done

SNPs in IL4 and IFNG show no protective associations with human African trypanosomiasis in the Democratic Republic of the Congo: a case-control study.

Background: Human African trypanosomiasis (HAT) is a protozoal disease transmitted by tsetse flies. Infection with trypanosomes can lead directly to active HAT or latent infection with no detectable parasites, which may progress to active HAT or to spontaneous self-cure. Genetic variation could explain these differences in the outcome of infection. To test this hypothesis, polymorphisms in 17 candidate genes were tested ( APOL1 [ G1 and G2], CFH, HLA-A, HPR, HP, IL1B, IL12B, IL12RB1, IL10, IL4R, MIF, TNFA , IL6, IL4, IL8, IFNG, and HLA-G). Methods: Samples were collected in Democratic Republic of the Congo. 233 samples were genotyped: 100 active HAT cases, 33 from subjects with latent infections and 100 negative controls. Commercial service providers genotyped polymorphisms at 96 single nucleotide polymorphisms (SNPs) on 17 genes. Data were analyzed using Plink V1.9 software and R. Loci, with suggestive associations (uncorrected p Results: After quality control, 87 SNPs remained in the analysis. Two SNPs in IL4 and two in IFNG were suggestively associated (uncorrected pTrypanosoma brucei gambiense infection in the Congolese population. The IFNG minor allele (rs2430561, rs2069718) SNPs were protective in comparison between latent infections and controls. Carriers of the rs2243258_T and rs2243279_A alleles of IL4 and the rs2069728_T allele of IFNG had a reduced risk of developing illness or latent infection, respectively. None of these associations were significant after Bonferroni correction for multiple testing. A validation study using more samples was run to determine if the absence of significant association was due to lack of power. Conclusions: This study showed no evidence of an association of HAT with IL4 and IFNG SNPs or with APOL1 G1 and G2 alleles, which have been found to be protective in other studies

Diagnostic accuracy of Loopamp Trypanosoma brucei detection kit for diagnosis of human African trypanosomiasis in clinical samples

Background: Molecular methods have great potential for sensitive parasite detection in the diagnosis of human African trypanosomiasis (HAT), but the requirements in terms of laboratory infrastructure limit their use to reference centres. A recently developed assay detects the Trypanozoon repetitive insertion mobile element (RIME) DNA under isothermal amplification conditions and has been transformed into a ready-to-use kit format, the Loopamp Trypanosoma brucei. In this study, we have evaluated the diagnostic performance of the Loopamp Trypanosoma brucei assay (hereafter called LAMP) in confirmed T.b. gambiense HAT patients, HAT suspects and healthy endemic controls from the Democratic Republic of the Congo (DRC). Methodology/Principal findings: 142 T.b. gambiense HAT patients, 111 healthy endemic controls and 97 HAT suspects with unconfirmed status were included in this retrospective evaluation. Reference standard tests were parasite detection in blood, lymph or cerebrospinal fluid. Archived DNA from blood of all study participants was analysed in duplicate with LAMP. Sensitivity of LAMP in parasitologically confirmed cases was 87.3% (95% CI 80.9–91.8%) in the first run and 93.0% (95% CI 87.5–96.1%) in the second run. Specificity in healthy controls was 92.8% (95% CI 86.4–96.3%) in the first run and 96.4% (95% CI 91.1–98.6%) in the second run. Reproducibility was excellent with a kappa value of 0.81. Conclusions/Significance: In this laboratory-based study, the Loopamp Trypanosoma brucei Detection Kit showed good diagnostic accuracy and excellent reproducibility. Further studies are needed to assess the feasibility of its routine use for diagnosis of HAT under field conditions

Expérimentation d’un Nouveau Piège à Glossines dans les Zones Endémiques à la Trypanosomiase Humaine Africaine de la Province du Sankuru en République Démocratique Congo

Trois types de pièges à glossines ont été expérimentés en carrés latins dans trois zones de santé (Dikungu, Tshumbe et Wembo-Nyama) pour lutter contre la Trypanosomiase Humaine Africaine. Au total 468 pièges ont été installés en raison de 234 par saison climatique avec une fréquence cumulée de capture de 466 glossines (Glossina fuscipes fuscipes). Les calculs de la régression logiste et du Ficher avec le logiciel R ont servi pour le traitement des résultats.Il en ressort que les saisons, les zones de santé, le sexe, l’âge et le stade alimentaire des glossines n’ont pas de lien perceptible sur leur capture. Par contre, les sites de capture (villages et points d’eau) et le type de pièges impactent significativement la probabilité de capturer les glossines. Les zones de santé de Dikungu, Tshumbe et Wembo-Nyama sont des zones à risques à cause de la présence de glossines vectrices de la Trypanosomiase humaine africaine. Toutefois, les habitants de la zone de santé de Wembo-Nyama sont à haut risque avec une densité apparente d’au moins deux glossines par piège par jour.Le piège Stop est considéré comme le plus efficace compte tenu de ses résultats de plus de 50 % que BCT et Grémansin combinés. Il serait utile de l’expérimenter dans d’autres zones de santé de la RD Congo ou d’autres pays d’Afrique. La lutte antivectorielle est l’un des piliers pour atteindre l’objectif de l’OMS, éliminer la gTHA en tant que problème de santé publique d’ici 2030.

Identification par Diagnostic moléculaire des Trypanosomes chez les Glossines dans les Zones endémiques de la Province du Sankuru en République Démocratique Congo

Cette étude a été menée dans la province du Sankuru en République Démocratique du Congo, dans trois zones de santé (Dikungu, Tshumbe et Wembo-Nyama) endémiques à la Trypanosomiase Humaine Africaine (THA). Elle a visé de déterminer les espèces de trypanosomes africains infectant les glossines qui sévissent dans ces trois zones de santé. Au total 446 glossines ont été capturées à l’aide des pièges (BCT, Grémansin et Stop) ; 81 glossines vivantes étaient disséquées pour prélever le proboscis, les glandes salivaires et l’intestin moyen pour les observations microscopiques et le diagnostic moléculaire (qPCR et PCR) des espèces de trypanosomes.Sur l’ensemble d’échantillons positifs, 95,1 %  étaient déterminés par diagnostic moléculaire contre 4,9 % observés exclusivement en microscopie. Les analyses moléculaires impactent significativement (P 0,001<0,05) sur la détection des trypanosomes (sensible et spécifique) par rapport à l’observation microscopique. En effet, elles sont lentes et couteuses.Les groupes taxonomiques de trypanosomes infectant les Glossina fuscipes fuscipes dans des zones étudiées sont Trypanosoma brucei gambiense (14,8 %),  Trypanosoma congolense savane (3,7 %) et Trypanosoma congolense forêt (1,2 %). Il y a coexistence de la trypanosomiase africaine humaine et animale dans ce milieu d’étude. Le développement de nouvelles stratégies intégrant des mesures de lutte contre la trypanosomiase humaine et animale pourrait permettrait l’élimination de la gTHA en tant que problème de santé publique d’ici 2030

Microscopic Characteristics , Chromatographic Profiles and Inhibition of Peroxidase Activity of the Leaves of Manihot esculenta and Manihot glaziovii , Consumed as Traditional Vegetables

peer reviewedMethanolic extracts from the leaves of Manihot esculenta (Two cultivars) and Manihot glaziovii, consumed as traditional vegetables in DR. Congo was chemically characterized by Thin layer Chromatography and High Performance Liquid Chromatography. In vitro biochemical activities of extracts against Radical Oxidative Species (ROS) production were assessed in cellular models, on enzymes, Myeloperoxidase (MPO) and Horseradish Peroxidase (HRP) involved in inflammation. The microscopic analysis of the powder of leaves showed that each species displays specific and discriminating botanical microscopic features. Varieties of M. esculenta had a chemical fingerprint different from M. glaziovii. The majority of compounds were polyphenols, repre- sented mainly by rutin, kaempferol-3-O-rutinoside, amentoflavone, phenolic acids such as gallic acid. All extracts exhibited high cellular antioxidant activity in the range of 0.1 to 10 $\mu$g·mL−1 using lucigenin with neutrophils, but a moderate cellular antioxidant activity ranging between 10 and 100 $\mu$g·mL−1 with DCFDA on HL60 monocytes. Extracts from Manihot leaves showed a pronounced inhibitory effect on the production of extracellular ROS, on HRP and myeloperoxidase activity. Cellular antioxidant activities, the inhibitory effect on HRP of extracts from M. glaziovii, M. esculenta cultivar Mwambu were significantly higher, but their inhibitory effect on the activity of MPO was lower than those of M. esculenta cultivar TEM 419. The biological activities of Manihot esculenta and Manihot glaziovii were well correlated to their phytochemicals that could justify their traditional use as vegetables, potential functional foods or nutraceutical resources and medicines