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Abstract Konzo is a neglected paralytic neurological disease associated with food (cassava) poisoning that affects the world’s poorest children and women of childbearing ages across regions of sub-Saharan Africa. Despite understanding the dietary factors that lead to konzo, the molecular markers and mechanisms that trigger this disease remain unknown. To identify potential protein biomarkers associated with a disease status, plasma was collected from two independent Congolese cohorts, a discovery cohort (n = 60) and validation cohort (n = 204), sampled 10 years apart and subjected to multiple high-throughput assays. We identified that Glutathione Peroxidase 3 (GPx3), a critical plasma-based antioxidant enzyme, was the sole protein examined that was both significantly and differentially abundant between affected and non-affected participants in both cohorts, with large reductions observed in those affected with konzo. Our findings raise the notion that reductions in key antioxidant mechanisms may be the biological risk factor for the development of konzo, particularly those mediated through pathways involving the glutathione peroxidase family.

Abstract Background Aedes albopictus has been reported in several Central African countries, including the Democratic Republic of the Congo (DRC). The establishment of this mosquito species poses a serious threat as a vector of various infectious diseases. Although Ae. albopictus has been reported in the western region of the DRC, information about its distribution is still scarce in the country. The aim of this study was to investigate the current nationwide distribution of the invasive Ae. albopictus, as well as other native Aedes mosquitoes, in the DRC and to identify suitable areas for its future expansion. Methods Two entomological surveys were conducted in 2017–2019 and 2022. Based on the occurrence sites of Ae. albopictus, important environmental variables were identified. Then, geographical areas suitable for Ae. albopictus establishment were determined using the maximum entropy model. The distribution and abundance of Ae. albopictus were also compared with those of the major native Aedes species. Results Aedes albopictus was found in the western, northern, central, and eastern regions of the DRC, but it was not found in the southeastern region. The maximum entropy model predicted that most parts of the DRC are suitable for the establishment of this mosquito. The unsuitable areas encompassed the eastern highlands, known for their low temperatures, and the southeastern highlands, which experience both low temperatures and a long dry season. The native Aedes species found were Aedes aegypti, Aedes simpsoni, Aedes africanus, and Aedes vittatus. Aedes albopictus dominated in the western and northern regions, while Ae. aegypti was more prevalent in other regions. Conclusions Aedes albopictus has been well established in the western and northern regions of the DRC. This mosquito is expanding its distribution while replacing the native Aedes species. Most of the country is suitable for the establishment of this mosquito species, except the highlands of the eastern and the southeastern regions. Graphical Abstract

Abstract African trypanosomes colonise the skin to ensure parasite transmission. However, how the skin responds to trypanosome infection remains unresolved. Here, we investigate the local immune response of the skin in a murine model of infection using spatial and single cell transcriptomics. We detect expansion of dermal IL-17A-producing Vγ6+ cells during infection, which occurs in the subcutaneous adipose tissue. In silico cell-cell communication analysis suggests that subcutaneous interstitial preadipocytes trigger T cell activation via Cd40 and Tnfsf18 signalling, amongst others. In vivo, we observe that female mice deficient for IL-17A-producing Vγ6+ cells show extensive inflammation and limit subcutaneous adipose tissue wasting, independently of parasite burden. Based on these observations, we propose that subcutaneous adipocytes and Vγ6+ cells act in concert to limit skin inflammation and adipose tissue wasting. These studies provide new insights into the role of γδ T cell and subcutaneous adipocytes as homeostatic regulators of skin immunity during chronic infection.

Abstract Background Copy number variation is an important class of genomic variation that has been reported in 75% of the human genome. However, it is underreported in African populations. Copy number variants (CNVs) could have important impacts on disease susceptibility and environmental adaptation. To describe CNVs and their possible impacts in Africans, we sequenced genomes of 232 individuals from three major African ethno-linguistic groups: (1) Niger Congo A from Guinea and Côte d’Ivoire, (2) Niger Congo B from Uganda and the Democratic Republic of Congo and (3) Nilo-Saharans from Uganda. We used GenomeSTRiP and cn.MOPS to identify copy number variant regions (CNVRs). Results We detected 7608 CNVRs, of which 2172 were only deletions, 2384 were only insertions and 3052 had both. We detected 224 previously un-described CNVRs. The majority of novel CNVRs were present at low frequency and were not shared between populations. We tested for evidence of selection associated with CNVs and also for population structure. Signatures of selection identified previously, using SNPs from the same populations, were overrepresented in CNVRs. When CNVs were tagged with SNP haplotypes to identify SNPs that could predict the presence of CNVs, we identified haplotypes tagging 3096 CNVRs, 372 CNVRs had SNPs with evidence of selection (iHS > 3) and 222 CNVRs had both. This was more than expected (p

Background: Approximately 25% of the risk of Schistosoma mansoni is associated with host genetic variation. We will test 24 candidate genes, mainly in the Th2 and Th17 pathways, for association with S. mansoni infection intensity in four African countries, using family based and case-control approaches. Methods: Children aged 5-15 years will be recruited in S. mansoni endemic areas of Ivory Coast, Cameroon, Uganda and the Democratic Republic of Congo (DRC). We will use family based (study 1) and case-control (study 2) designs. Study 1 will take place in Ivory Coast, Cameroon, Uganda and the DRC. We aim to recruit 100 high worm burden families from each country except Uganda, where a previous study recruited at least 40 families. For phenotyping, cases will be defined as the 20% of children in each community with heaviest worm burdens as measured by the circulating cathodic antigen (CCA) assay. Study 2 will take place in Uganda. We will recruit 500 children in a highly endemic community. For phenotyping, cases will be defined as the 20% of children with heaviest worm burdens as measured by the CAA assay, while controls will be the 20% of infected children with the lightest worm burdens. Deoxyribonucleic acid (DNA) will be genotyped on the Illumina H3Africa SNP (single nucleotide polymorphisms) chip and genotypes will be converted to sets of haplotypes that span the gene region for analysis. We have selected 24 genes for genotyping that are mainly in the Th2 and Th17 pathways and that have variants that have been demonstrated to be or could be associated with Schistosoma infection intensity. Analysis: In the family-based design, we will identify SNP haplotypes disproportionately transmitted to children with high worm burden. Case-control analysis will detect overrepresentation of haplotypes in extreme phenotypes with correction for relatedness by using whole genome principal components.

Konzo, a disease characterized by sudden, irreversible spastic paraparesis, affecting up to 10% of the population in some regions of Sub-Saharan Africa during outbreaks, is strongly associated with dietary exposure to cyanogenic bitter cassava. The molecular mechanisms underlying the development of konzo remain largely unknown. Here, through an analysis of 16 individuals with konzo and matched healthy controls from the same outbreak zones, we identified 117 differentially methylated loci involved in numerous biological processes that may identify cyanogenic-sensitive regions of the genome, providing the first study of epigenomic alterations associated with a clinical phenotype of konzo.

Human African Trypanosomiasis (HAT) is a major public health problem in the Democratic Republic of the Congo (DRC). Active and passive surveillance for HAT is conducted but may underestimate the true prevalence of the disease. We used ELISA to screen 7,769 leftover dried blood spots from a nationally representative population-based survey, the 2007 Demographic and Health Survey. 26 samples were positive by ELISA. Three of these were also positive by trypanolysis and/or PCR. From these data, we estimate that there were 18,592 people with HAT (95% confidence interval, 4,883-32,302) in the DRC in 2007, slightly more than twice as many as were reported.

ABSTRACT The growing resistance to amoxicillin (AMX)—one of the main antibiotics used in Helicobacter pylori eradication therapy—is an increasing health concern. Several mutations of penicillin-binding protein 1A (PBP1A) are suspected of causing AMX resistance; however, only a limited set of these mutations have been experimentally explored. This study aimed to investigate four PBP1A mutations (i.e., T558S, N562H, T593A, and G595S) carried by strain KIN76, a high-level AMX-resistant clinical H. pylori isolate with an AMX minimal inhibition concentration (MIC) of 2 µg/mL. We transformed a recipient strain 26695 with the DNA containing one to four mutation allele combinations of the pbp1 gene from strain KIN76. Transformants were subjected to genomic exploration and antimicrobial susceptibility testing. The resistance was transformable, and the presence of two to four PBP1A mutations (T558S and N562H, or T593A and G595S), rather than separate single mutations, was necessary to synergistically increase the AMX MIC up to 16-fold compared with the wild-type (WT) strain 26695. An AMX binding assay of PBP1A was performed using these strains, and binding was visualized by chasing Bocillin, a fluorescent penicillin analog. This revealed that all four-mutation allele-transformed strains exhibited decreased affinity to AMX on PBP1A than the WT. Protein structure modeling indicated that functional modifications occur as a result of these amino acid substitutions. This study highlights a new synergistic AMX resistance mechanism and establishes new markers of AMX resistance in H. pylori.IMPORTANCEThe development of resistance to antibiotics, including amoxicillin, is hampering the eradication of Helicobacter pylori infection. The identification of mechanisms driving this resistance is crucial for the development of new therapeutic strategies. We have demonstrated in vitro the synergistic role of novel mutations in the pbp1 gene of H. pylori that is suspected to drive amoxicillin resistance. Also deepening our understanding of amoxicillin resistance mechanisms, this study establishes new molecular markers of amoxicillin resistance that may be useful in molecular-based antibiotic susceptibility testing approaches for clinical practice or epidemiologic investigations.

Objectives: Partial artemisinin resistance, mediated by Plasmodium falciparum K13 (PfK13) mutations, has been confirmed in certain areas of East Africa that are historically associated with high-level antimalarial resistance. The Democratic Republic of Congo (DRC) borders these areas in the East. This study aimed to determine the prevalence of resistance markers in six National Malaria Control Program surveillance sites; Boende, Kabondo, Kapolowe, Kimpese, Mikalayi, and Rutshuru. Methods: The single nucleotide polymorphisms (SNPs) in P. falciparum genes PfK13, Pfdhfr, Pfdhps, Pfmdr1, and Pfcrt were assessed using targeted next-generation sequencing of isolates collected at enrollment in therapeutic efficacy studies. Results: PfK13 SNPs were detected in two samples: in Kabondo (R561H) and in Rutshuru (P441L), both areas near Uganda and Rwanda. The Pfdhps ISGEGA haplotype, associated with reduced sulfadoxine-pyrimethamine chemoprevention efficacy, ranged from 0.8% in Mikalayi (central DRC) to 42.2% in Rutshuru (East DRC). Conclusions: R561H and P441L observed in eastern DRC are a concern, as they are associated with delayed artemisinin-based combination therapies-clearance and candidate marker of resistance, respectively. This is consistent with previous observations of shared drug resistance profiles in parasites of that region with bordering areas of Rwanda and Uganda. The likely circulation of parasites has important implications for the ongoing surveillance of partial artemisinin-resistant P. falciparum and for future efforts to mitigate its dispersal.

Abstract Background Ocular toxoplasmosis (OT) is the leading cause of infectious posterior uveitis in several areas worldwide. The combination of Trimethoprim/Sulfamethoxazole (TMP/SMX) has been presented as an attractive alternative to the “classic’ treatment therapy (Pyrimethamine/Sulfadiazine). Methods A prospective study was carried out between February 2020 and September 2021 in 2 ophthalmic centers in Kinshasa. This study aimed to describe TMP/SMX treatment outcomes for OT in a cohort of immunocompetent Congolese patients. Results 54 patients were included, with a mean age at presentation of 37.5 ± 13.6 years old and a Male-Female ratio of 1.45:1. Three patients (5.6%) presented a recurrence during the follow-up period. At the end of the follow-up, improvement in VA and resolution of inflammation concerned 75.9% and 77.5% of patients, respectively. Cataracts (3.7%), macular scars (3.7%), and vitreous opacities (3.7%) were the principal causes of non-improvement in VA. Treatment-related adverse events were present in 10 patients (18.5%); gastrointestinal (14.8%) and dermatological (3.7%) adverse events were the most frequent. Dermatological adverse events led to discontinuation of treatment. Conclusion TMP/SMX regimen appears to be a safe and effective treatment for OT in Congolese patients. The low cost and the accessibility of the molecules make this regimen an option for treating OT in resource-limited countries.

The meningeal space is a critical brain structure providing immunosurveillance for the central nervous system (CNS), but the impact of infections on the meningeal immune landscape is far from being fully understood. The extracellular protozoan parasite Trypanosoma brucei, which causes human African trypanosomiasis (HAT) or sleeping sickness, accumulates in the meningeal spaces, ultimately inducing severe meningitis and resulting in death if left untreated. Thus, sleeping sickness represents an attractive model to study immunological dynamics in the meninges during infection. Here, by combining single-cell transcriptomics and mass cytometry by time-of-flight (CyTOF) with in vivo interventions, we found that chronic T. brucei infection triggers the development of ectopic lymphoid aggregates (ELAs) in the murine meninges. These infection-induced ELAs were defined by the presence of ER-TR7+ fibroblastic reticular cells, CD21/35+ follicular dendritic cells (FDCs), CXCR5+ PD1+ T follicular helper-like phenotype, GL7+ CD95+ GC-like B cells, and plasmablasts/plasma cells. Furthermore, the B cells found in the infected meninges produced high-affinity autoantibodies able to recognise mouse brain antigens, in a process dependent on LTβ signalling. A mid-throughput screening identified several host factors recognised by these autoantibodies, including myelin basic protein (MBP), coinciding with cortical demyelination and brain pathology. In humans, we identified the presence of autoreactive IgG antibodies in the cerebrospinal fluid (CSF) of second stage HAT patients that recognised human brain lysates and MBP, consistent with our findings in experimental infections. Lastly, we found that the pathological B cell responses we observed in the meninges required the presence of T. brucei in the CNS, as suramin treatment before the onset of the CNS stage prevented the accumulation of GL7+ CD95+ GC-like B cells and brain-specific autoantibody deposition. Taken together, our data provide evidence that the meningeal immune response during chronic T. brucei infection results in the acquisition of lymphoid tissue-like properties, broadening our understanding of meningeal immunity in the context of chronic infections. These findings have wider implications for understanding the mechanisms underlying the formation ELAs during chronic inflammation resulting in autoimmunity in mice and humans, as observed in other autoimmune neurodegenerative disorders, including neuropsychiatric lupus and multiple sclerosis.

Objective To identify the risk factors associated with ocular toxoplasmosis (OT) in a cohort of Congolese patients with uveitis.Methods and analysis A cross-sectional study was conducted between March 2020 and July 2021 in two ophthalmic clinics in Kinshasa. Patients with a diagnosis of uveitis were enrolled in the study. Each patient underwent an interview, an ophthalmological examination and serology testing. Logistic regression was performed to identify risk factors for OT.Results 212 patients were included in the study with a mean age at presentation of 42.1±15.9 years (limits: 8–74 years) and a sex ratio of 1.1:1. OT concerned 96 patients (45.3%). The age of the patients below 60 years (p=0.001, OR=9.75 CI 95% 2.51 to37.80)), the consumption of cat meat (p=0.01, OR=2.65 CI 95% 1.18 to 5.96)) and undercooked meat (p=0.044, OR=2.30 CI 95% 1.02 to 5.21)) and living in rural area (p=0.021, OR=11.4 (CI 95% 1.45 to 89.84])) were identified as risk factors for OT.Conclusion OT affects more young people. It is associated with dietary habits. Informing and educating the population is necessary to avoid infection.

ABSTRACT Trypanosoma brucei gambiense is the primary causative agent of human African trypanosomiasis (HAT), a vector-borne disease endemic to West and Central Africa. The extracellular parasite evades antibody recognition within the host bloodstream by altering its variant surface glycoprotein (VSG) coat through a process of antigenic variation. The serological tests that are widely used to screen for HAT use VSG as one of the target antigens. However, the VSGs expressed during human infection have not been characterized. Here, we use VSG sequencing (VSG-seq) to analyze the VSGs expressed in the blood of patients infected with T. b. gambiense and compared them to VSG expression in Trypanosoma brucei rhodesiense infections in humans as well as Trypanosoma brucei brucei infections in mice. The 44 VSGs expressed during T. b. gambiense infection revealed a striking bias toward expression of type B N termini (82% of detected VSGs). This bias is specific to T. b. gambiense, which is unique among T. brucei subspecies in its chronic clinical presentation and anthroponotic nature. The expressed T. b. gambiense VSGs also share very little similarity to sequences from 36 T. b. gambiense whole-genome sequencing data sets, particularly in areas of the VSG protein exposed to host antibodies, suggesting the antigen repertoire is under strong selective pressure to diversify. Overall, this work demonstrates new features of antigenic variation in T. brucei gambiense and highlights the importance of understanding VSG repertoires in nature. IMPORTANCE Human African trypanosomiasis is a neglected tropical disease primarily caused by the extracellular parasite Trypanosoma brucei gambiense. To avoid elimination by the host, these parasites repeatedly replace their variant surface glycoprotein (VSG) coat. Despite the important role of VSGs in prolonging infection, VSG expression during human infections is poorly understood. A better understanding of natural VSG gene expression dynamics can clarify the mechanisms that T. brucei uses to alter its VSG coat. We analyzed the expressed VSGs detected in the blood of patients with trypanosomiasis. Our findings indicate that there are features of antigenic variation unique to human-infective T. brucei subspecies and that natural VSG repertoires may vary more than previously expected.

Summary: Background: Detection of spliced leader (SL)-RNA allows sensitive diagnosis of gambiense human African trypanosomiasis (HAT). We investigated its diagnostic performance for treatment outcome assessment. Methods: Blood and cerebrospinal fluid (CSF) from a consecutive series of 97 HAT patients, originating from the Democratic Republic of the Congo, were prospectively collected before treatment with acoziborole, and during 18 months of longitudinal follow-up after treatment. For treatment outcome assessment, SL-RNA detection was compared with microscopic trypanosome detection and CSF white blood cell count. The trial was registered under NCT03112655 in clinicaltrials.gov. Findings: Before treatment, respectively 94.9% (92/97; CI 88.5–97.8%) and 67.7% (65/96; CI 57.8–76.2%) HAT patients were SL-RNA positive in blood or CSF. During follow-up, one patient relapsed with trypanosomes observed at 18 months, and was SL-RNA positive in blood and CSF at 12 months, and CSF positive at 18 months. Among cured patients, one individual tested SL-RNA positive in blood at month 12 (Specificity 98.9%; 90/91; CI 94.0–99.8%) and 18 (Specificity 98.9%; 88/89; CI 93.9–99.8%). Interpretation: SL-RNA detection for HAT treatment outcome assessment shows ≥98.9% specificity in blood and 100% in CSF, and may detect relapses without lumbar puncture. Funding: The DiTECT-HAT project is part of the EDCTP2 programme, supported by Horizon 2020, the European Union Funding for Research and Innovation (grant number DRIA-2014-306-DiTECT-HAT).

Abstract Rapid diagnostic tests (RDTs) detecting histidine-rich protein 2 (HRP2) and HRP3 are widely used throughout sub-Saharan Africa (SSA) to diagnose Plasmodium falciparum malaria. However, multiple SSA countries have reported pfhrp2 and pfhrp3 (pfhrp2/3) gene deletions. Blood samples (n = 1109) collected from patients with P. falciparum infection from six health facilities throughout the Democratic Republic of the Congo (DRC) from March 2017 to January 2018 were evaluated for pfhrp2/3 deletions. Samples were assayed for HRP2, pan-Plasmodium LDH (pLDH) and aldolase (pAldolase) antigens by bead-based multiplex antigen assay. Samples with low HRP2 concentration compared to pLDH and pAldolase antigens were selected for further pfhrp2/3 genotyping PCRs. The majority of blood samples (93.3%, 1035/1109) had high concentrations of the HRP2 antigen. Single deletions of pfhrp2 were identified in 0.27% (3/1109) of screened samples, with one sample from each of the Kapolowe, Mikalayi, and Rutshuru study sites. A pfhrp3 single deletion (0.09%, 1/1109) was found in the Kapolowe site. Dual pfhrp2 and pfhrp3 deletions were not observed. Due to, the low numbers of pfhrp2 deletions and the sporadic locations of these deletions, the use of HRP2-based RDTs appears to still be appropriate for these locations in DRC.

Here, using metagenomic profiling in 180 individuals from the Democratic Republic of the Congo, the authors find associations between the gut microbiome and konzo, a neurodegenerative disease that mostly affects children and is caused by the consumption improperly processed cassava.

BackgroundSpliced Leader (SL) trypanosome RNA is detectable only in the presence of live trypanosomes, is abundant and the Trypanozoon subgenus has a unique sequence. As previously shown in blood from Guinean human African trypanosomiasis (HAT) patients, SL-RNA is an accurate target for diagnosis. Detection of SL-RNA in the cerebrospinal fluid (CSF) has never been attempted. In a large group of Congolese gambiense HAT patients, the present study aims i) to confirm the sensitivity of SL-RNA detection in the blood and; ii) to assess the diagnostic performance of SL-RNA compared to trypanosome detection in CSF.Methodology/principal findingsBlood and CSF from 97 confirmed gambiense HAT patients from the Democratic Republic of Congo were collected using PAXgene blood RNA Tubes. Before RNA extraction, specimens were supplemented with internal extraction control RNA to monitor the extraction, which was performed with a PAXgene Blood RNA Kit. SL-RNA qPCR was carried out with and without reverse transcriptase to monitor DNA contamination. In blood, 92/97 (94.8%) HAT patients tested SL-RNA positive, which was significantly more than combined trypanosome detection in lymph and blood (78/97 positive, 80.4%, p = 0.001). Of 96 CSF RNA specimens, 65 (67.7%) were SL-RNA positive, but there was no significant difference between sensitivity of SL-RNA and trypanosome detection in CSF. The contribution of DNA to the Cq values was negligible. In CSF with normal cell counts, a fraction of SL-RNA might have been lost during extraction as indicated by higher internal extraction control Cq values.Conclusions/significanceDetection of SL-RNA in blood and CSF allows sensitive demonstration of active gambiense HAT infection, even if trypanosomes remain undetectable in blood or lymph. As this condition often occurs in treatment failures, SL-RNA detection in blood and CSF for early detection of relapses after treatment deserves further investigation.Trial registrationThis study was an integral part of the diagnostic trial "New Diagnostic Tools for Elimination of Sleeping Sickness and Clinical Trials: Early tests of Cure" (DiTECT-HAT-WP4, ClinicalTrials.gov Identifier: NCT03112655).

Beside diagnostic uncertainties due to the lack of a perfect gold standard test for Helicobacter pylori infection, the diagnosis and the prevalence estimation for this infection encounter particular challenges in Africa including limited diagnostic tools and specific genetic background. We developed and evaluated the accuracy of an enzyme-linked immunosorbent assay (ELISA) system tailored for H. pylori genetics in Africa (HpAfr-ELISA). Strains belonging to main genetic populations infecting Africans were exploited as sources for whole-cell antigens to establish in-house the ELISA system. A phase II unmatched case-control study explored the diagnostic accuracy of the HpAfr-ELISA using a training set of samples collected from dyspeptic patients from Kinshasa, the Democratic Republic of Congo (DRC) who had been tested with invasive standard tests (i.e., histology, culture, and rapid urease test) in 2017. Then the assay was cross-validated through a community-based survey assessing the prevalence of H. pylori and associated factors in 425 adults from Mbujimayi, DRC in 2018. Bayesian inferences were used to deal with statistical uncertainties of estimates (true prevalence, sensitivity, and specificity) in the study population. At its optimal cut-off-value 20.2 U/mL, the assay achieved an estimated sensitivity of 97.6% (95% credible interval [95%CrI]: 89.2; 99.9%) and specificity of 90.5% (95%CrI: 78.6; 98.5). Consistent outcomes obtained at repeated tests attested the robustness of the assay (negative and positive agreements always > 70%). The true prevalence of H. pylori was estimated 53.8% [95%CrI: 42.8; 62.7%]. Increasing age (adjusted odds ratio [aOR] > 1.0 [95% confidence interval (CI): > 1.0; 1.1]; p

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

BackgroundIn efforts to control malaria infection, the Democratic Republic of Congo has implemented several strategies. Studies assessing their efficiency mainly involved at-risk groups, especially children under five years of age. This study aimed to determine the prevalence and identify the risk factors associated with Plasmodium spp. infection.MethodsFrom October 2014 to March 2015, individuals aged at least 15 years were selected randomly and enrolled in a cross-sectional study conducted throughout the country. Microscopy and polymerase chain reaction (PCR) analysis were used for the detection of Plasmodium ssp.ResultsFrom 2286 individuals recruited, 1870 with valid laboratory results were included in the study for further analysis. The prevalence of Plasmodium spp. infection assessed by microscopy (355/ 1870 (19%) was lower than that estimated by PCR (580/1870 (31%). In addition, the difference between the two results was statistically significant (P < 0.0001). The most prevalent Plasmodium species was P. falciparum, either as mono-infection (96.3%; 95% C.I. 93.9-98.1) or combined with P. malariae (3.7%; 95% C.I. 2.8-5.9). The mean parasite density was 3272739 trophozoites/μL of blood. Women had higher risks of being infected than men (OR 2.03, 95% C.I.: 1.96. 2.62, P = 0.041)].ConclusionIn this study, the molecular detection and species identification of Plasmodium spp. showed that, despite all efforts for malaria control, malaria remains a public health problem in the Democratic Republic of Congo. The high prevalence and parasite density of Plasmodium spp. in adults make this age group a potential parasitic infectious reservoir for the at-risk groups and supports the need to include this age group in further programs for malaria control.

Gambiense human African trypanosomiasis (gHAT), also known as gambiense sleeping sickness, is a parasitic infection caused by Trypanosoma brucei gambiense. During the last decades, gHAT incidence has been brought to an all-time low. Newly developed serological tools and drugs for its diagnosis and treatment put the WHO goal of interruption of transmission by 2030 within reach. However, further research is needed to efficiently adapt these new advances to new control strategies. We assessed the serological evolution of cured gHAT patients over a two-year period using four different tests: the rapid diagnostic test (RDT) HAT Sero K-SeT, ELISA/T.b. gambiense, Trypanosoma brucei gambiense inhibition ELISA (iELISA), and the immune trypanolysis test. High seropositive rates were observed in all the tests, although sero-reversion rates were different in each test: ELISA/T.b. gambiense was the test most likely to become negative two years after treatment, whereas RDT HAT Sero-K-SeT was the least likely. iELISA and trypanolysis showed intermediate and comparable probabilities to become negative. Stage 1 patients were also noted to be more likely to become negative than Stage 2 patients in all four serological tests. Our results confirm previous findings that trypanosome-specific antibody concentrations in blood may persist for up to two years, implying that HAT control programs should continue to take the history of past HAT episodes into consideration.

Abstract Background Worldwide, the highest malaria mortality is due to Plasmodium falciparum infection. However, other species of Plasmodium (Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and Plasmodium knowlesi) can also cause malaria. Therefore, accurate identification of malaria species is crucial for patient management and epidemiological surveillance. This study aimed to determine the different Plasmodium species causing malaria in children under 5 years old in two provinces (Kinshasa and North Kivu) of the Democratic Republic of Congo (DRC). Methods From October to December 2015, a health-facility based cross-sectional study was conducted in General Reference Hospitals in Kinshasa and North Kivu. Four hundred and seven blood samples were collected from febrile children aged ≤ 5 years. Nested polymerase chain reaction assays were performed for Plasmodium species identification. Results Out of 407 children, 142 (34.9%) were infected with Plasmodium spp. and P. falciparum was the most prevalent species (99.2%). Among those infected children, 124 had a mono infection with P. falciparum and one with P. malariae. Mixed infections with P. falciparum/P. malariae and P. falciparum/P. vivax were observed in 6 (1.5%) and 8 (2.0%) children, respectively. The prevalence of infection was higher in females (64.8%) than in males (35.2%), p

Human African trypanosomiasis (HAT) caused by the extracellular protozoon Trypanosoma brucei, is a neglected tropical disease affecting the poorest communities in sub-Saharan Africa. HAT progresses from a hemolymphatic first stage (S1) to a meningo-encephalitic late stage (S2) when parasites reach the central nervous system (CNS), although the existence of an intermediate stage (Int.) has also been proposed. The pathophysiological mechanisms associated with the development of S2 encephalopathy are yet to be fully elucidated. Here we hypothesized that HAT progression toward S2 might be accompanied by an increased release of microvesicles (MVs), sub-micron elements (0.1–1 μm) involved in inflammatory processes and in the determination of the outcome of infections. We studied the morphology of MVs isolated from HAT cerebrospinal fluid (CSF) by transmission electron microscopy (TEM) and used flow cytometry to show that total-MVs and leukocyte derived-CD45+ MVs are significantly increased in concentration in S2 patients' CSF compared to S1 and Int. samples (n = 12 per group). To assess potential biological properties of these MVs, immortalized human astrocytes were exposed, in vitro, to MVs enriched from S1, Int. or S2 CSF. Data-independent acquisition mass spectrometry analyses showed that S2 MVs induced, compared to Int. or S1 MVs, a strong proteome modulation in astrocytes that resembled the one produced by IFN-γ, a key molecule in HAT pathogenesis. Our results indicate that HAT S2 CSF harbors MVs potentially involved in the mechanisms of pathology associated with HAT late stage. Such vesicles might thus represent a new player to consider in future functional studies.

Antimicrobial susceptibility testing (AST) is increasingly needed to guide the Helicobacter pylori (H. pylori) treatment but remains laborious and unavailable in most African countries. To assess the clinical relevance of bacterial whole genome sequencing (WGS)-based methods for predicting drug susceptibility in African H. pylori, 102 strains isolated from the Democratic Republic of Congo were subjected to the phenotypic AST and next-generation sequencing (NGS). WGS was used to screen for the occurrence of genotypes encoding antimicrobial resistance (AMR). We noted the broad-spectrum AMR of H. pylori (rates from 23.5 to 90.0%). A WGS-based method validated for variant discovery in AMR-related genes (discovery rates of 100%) helped in identifying mutations of key genes statistically related to the phenotypic AMR. These included mutations often reported in Western and Asian populations and, interestingly, several putative AMR-related new genotypes in the pbp1A (e.g., T558S, F366L), gyrA (e.g., A92T, A129T), gyrB (e.g., R579C), and rdxA (e.g., R131_K166del) genes. WGS showed high performance for predicting AST phenotypes, especially for amoxicillin, clarithromycin, and levofloxacin (Youden’s index and Cohen’s Kappa > 0.80). Therefore, WGS is an accurate alternative to the phenotypic AST that provides substantial decision-making information for public health policy makers and clinicians in Africa, while providing insight into AMR mechanisms for researchers.

INTRODUCTION: L'objectif de cette étude était d'élucider le profil socio-émotionnel de l'enfant en milieu Konzo, une paralysie toxico-nutritionnelle sévissant en Afrique sub-saharienne. METHODES: nous avons évalué le profil socio-émotionnel de 210 enfants dont 123 avec konzo et 87 présumés contrôles sains (4-17 ans d'âge) après interview structuré avec les parents lors d'une enquête épidémio-clinique du konzo en 2011 au Congo-Kinshasa. Le profil neurocognitif était documenté par le KABC-II, le BOT-2 et l'indice global des signes neurologiques du Konzo (IGSNK). Les tests associatifs ont été réalisés par le test de Chi-carré, la régression logistique, dans le cas échéant par modèle linéaire généralisé, au seuil de signification de 0,05. RESULTATS: dans l'ensemble, l'irritabilité, la violence physique ou l'inhibition avec ou sans tristesse étaient respectivement retrouvés dans 46,0%, 30,2%, 18,7%; avec un risque accru pour le Konzo (OR = 2,6; IC95%: 1,4 - 4,8; p = 0,001). Le trouble socio-émotionnel était associé à l'insuffisance pondérale (OR: 0,49; IC95%: 0,31 - 0,78; p = 0,002) et à un IGSNK élevé (OR: 1,33; IC 95%: 1,1-1,63; p=0,019); et par ailleurs aggravait les déficits cognitifs dans le Konzo (interaction statut neurologique Ç troubles socio-émotionnels, D = 6,297; p = 0,013). Des performances cognitives élevées étaient observées chez les enfants non-Konzo mais avec troubles socio-émotionnels. La concentration moyenne (écart-type, ET) de thiocyanate urinaire était plus élevé (554,8 , 371,6 ,mol/l) chez les enfants Konzo avec troubles socio-émotionnels. CONCLUSION: l,enfant vivant en milieu Konzo présente des troubles socio-émotionnels. Leur nature psychopathologique et l'impact sur la cognition nécessitent des études approfondies.

We conducted a retrospective study on mortality trends and risk factors in 781 naïve cases of advanced stage-2 sleeping sickness admitted between 1989 and 2012 at the National Reference Center for Human African Trypanosomiasis (HAT), Department of Neurology, Kinshasa University, Democratic Republic of Congo (DRC). Death was the outcome variable whereas age, gender, duration of disease, location of trypanosomes in body fluids, cytorachy, protidorachy, clinical status (assessed on a syndromic and functional basis) on admission, and treatment regimen were predictors in logistic regression models run at the 0.05 significance level. Death proportions were 17.2% in the standard melarsoprol schedule (3-series of intravenous melarsoprol on 3 successive days at 3.6 mg/kg/d, with a one-week interval between the series, ARS 9); 12.1% in the short schedule melarsoprol (10 consecutive days of intravenous melarsoprol at 2.2 mg/kg/d, ARS 10), 5.4% in the first-line eflornithine (14 days of eflornithine at 400 mg/kg/d in 4 infusions a day DFMO B), 9.1% in the NECT treatment regimen (eflornithine for 7 days at 400, mg/kg/d in 2 infusions a day combined with oral nifurtimox for 10 days at 15 mg/kg/d in 3 doses a day); and high (36%) in the group with select severely affected patients given eflornithine because of their clinical status on admission, at the time when this expensive drug was kept for treatment of relapses (14 days at 400 mg/kg/d in 4 infusions a day, DFMO A). After adjusting for treatment, death odds ratios were as follows: 10.40 [(95% CI: 6.55-16.51); p = .000] for clinical dysfunction (severely impaired clinical status) on admission, 2.14 [(95% CI: 1.35-3.39); p = .001] for high protidorachy, 1.99 [(95% CI: 1.18-3.37); p = .010] for the presence of parasites in the CSF and 1.70 [(95% CI: 1.03-2.81); p = .038] for high cytorachy. A multivariable analysis within treatment groups retained clinical status on admission (in ARS 9, ARS 10 and DFMO B groups) and high protidorachy (in ARS 10 and DFMO B groups) as significant predictors of death. The algorithm for initial clinical status assessment used in the present study may serve as the basis for further development of standardized assessment tools relevant to the clinical management of HAT and information exchange in epidemiological reports.

Trypanosoma brucei (T. b.) gambiense is the parasite subspecies responsible for most reported cases of human African trypanosomiasis (HAT) or sleeping sickness. This severe infection leads to characteristic disruption of the sleep-wake cycle, recalling attention on the circadian timing system. Most animal models of the disease have been hitherto based on infection of laboratory rodents with the T. b. brucei subspecies, which is not infectious to humans. In these animal models, functional, rather than structural, alterations of the master circadian pacemaker, the hypothalamic suprachiasmatic nucleus (SCN), have been reported. Information on the SCN after infection with the human pathogenic T. b. gambiense is instead lacking. The present study was aimed at the examination of the SCN after T. b. gambiense infection of a susceptible rodent, the multimammate mouse, Mastomys natalensis, compared with T. b. brucei infection of the same host species. The animals were examined at 4 and 8 weeks post-infection, when parasites (T. b. gambiense or T. b. brucei) were detected in the brain parenchyma, indicating that the disease was in the encephalitic stage. Neuron and astrocyte changes were examined with Nissl staining, immunophenotyping and quantitative analyses. Interestingly, significant neuronal loss (about 30% reduction) was documented in the SCN during the progression of T. b. gambiense infection. No significant neuronal density changes were found in the SCN of T. b. brucei-infected animals. Neuronal cell counts in the hippocampal dentate gyrus of T. b. gambiense-infected M. natalensis did not point out significant changes, indicating that no widespread neuron loss had occurred in the brain. Marked activation of astrocytes was detected in the SCN after both T. b. gambiense and T. b. brucei infections. Altogether the findings reveal that neurons of the biological clock are highly susceptible to the infection caused by human pathogenic African trypanosomes, which have the capacity to cause permanent partial damage of this structure.

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, Mastomysnatalensis. 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.

Dietary cyanogen exposure from ingesting bitter (toxic) cassava as a main source of food in sub-Saharan Africa is related to neurological impairments in sub-Saharan Africa. We explored possible association with early child neurodevelopmental outcomes.We undertook a cross-sectional neurodevelopmental assessment of 12-48 month-old children using the Mullen Scale of Early Learning (MSEL) and the Gensini Gavito Scale (GGS). We used the Hopkins Symptoms Checklist-10 (HSCL-10) and Goldberg Depression Anxiety Scale (GDAS) to screen for symptoms of maternal depression-anxiety. We used the cyanogen content in household cassava flour and urinary thiocyanate (SCN) as biomarkers of dietary cyanogen exposure. We employed multivariable generalized linear models (GLM) with Gamma link function to determine predictors of early child neurodevelopmental outcomes.The mean (SD) and median (IQR) of cyanogen content of cassava household flour were above the WHO cut-off points of 10 ppm (52.18 [32·79]) and 50 (30-50) ppm, respectively. Mean (SD) urinary levels of thiocyanate and median (IQR) were respectively 817·81 (474·59) and 688 (344-1032) μmole/l in mothers, and 617·49 (449·48) and 688 (344-688) μmole/l in children reflecting individual high levels as well as a community-wide cyanogenic exposure. The concentration of cyanide in cassava flour was significantly associated with early child neurodevelopment, motor development and cognitive ability as indicated by univariable linear regression (p < 0.05). After adjusting for biological and socioeconomic predictors at multivariable analyses, fine motor proficiency and child neurodevelopment remained the main predictors associated with the concentration of cyanide in cassava flour: coefficients of -0·08 to -.15 (p < 0·01). We also found a significant association between child linear growth, early child neurodevelopment, cognitive ability and motor development at both univariable and multivariable linear regression analyses coefficients of 1.44 to 7.31 (p < 0·01).Dietary cyanogen exposure is associated with early child neurodevelopment, cognitive abilities and motor development, even in the absence of clinically evident paralysis. There is a need for community-wide interventions for better cassava processing practices for detoxification, improved nutrition, and neuro-rehabilitation, all of which are essential for optimal development in exposed children.

We performed a cohort study to analyze the virulome of Staphylococcus aureus from the Democratic Republic of the Congo using whole genome sequencing and to assess its impact on the course of S. aureus infections. Community-associated S. aureus from nasal colonization (n = 100) and infection (n = 86) were prospectively collected. Phenotypic susceptibility testing and WGS was done for each isolate. WGS data were used to screen for 79 different virulence factors and for genotyping purposes (spa typing, multilocus sequence typing). The majority of the 79 virulence factors were equally distributed among isolates from colonization and infection. Panton-Valentine leukocidin (PVL) and the non-truncated hemolysin β were associated with skin and soft tissue infection (SSTI) and recurrence of disease but did not influence the course of infection (i.e., mortality, surgical intervention). For the first time, we show that not only PVL but also hemolysin β could contribute to the development of SSTI in PVL-endemic areas such as Africa.

The role of mammalian skin in harbouring and transmitting arthropod-borne protozoan parasites has been overlooked for decades as these pathogens have been regarded primarily as blood-dwelling organisms. Intriguingly, infections with low or undetected blood parasites are common, particularly in the case of Human African Trypanosomiasis caused by Trypanosoma brucei gambiense. We hypothesise, therefore, the skin represents an anatomic reservoir of infection. Here we definitively show that substantial quantities of trypanosomes exist within the skin following experimental infection, which can be transmitted to the tsetse vector, even in the absence of detectable parasitaemia. Importantly, we demonstrate the presence of extravascular parasites in human skin biopsies from undiagnosed individuals. The identification of this novel reservoir requires a re-evaluation of current diagnostic methods and control policies. More broadly, our results indicate that transmission is a key evolutionary force driving parasite extravasation that could further result in tissue invasion-dependent pathology.

The present External Quality Assessment (EQA) assessed microscopy of blood parasites among diagnostic laboratories in the Democratic Republic of the Congo. The EQA addressed 445 participants in 10/11 provinces (October 2013-April 2014). Participants were sent a panel of five slides and asked to return a routinely stained slide which was assessed for quality of preparation and staining. Response rate was 89.9% (400/445). For slide 1 (no parasites), 30.6% participants reported malaria, mostly Plasmodium falciparum. Only 11.0% participants reported slide 2 (Plasmodium malariae) correctly, 71.0% reported "malaria" or "Plasmodium falciparum" (considered acceptable). Slide 3 contained Plasmodium falciparum (109/μl) and Trypanosoma brucei brucei trypomastigotes: they were each reported by 32.5% and 16.5% participants respectively, 6.0% reported both. Slide 4 (Trypanosoma) was recognised by 44.9% participants. Slide 5 (Plasmodium ovale) was correctly reported by 6.2% participants, another 68.8% replied "malaria" or "Plasmodium falciparum" (considered acceptable). Only 13.6% of routine slides returned were correctly prepared and stained. The proportion of correct/acceptable scores for at least 4/5 slides was higher among EQA-experienced participants compared to first time participants (40.9% versus 22.4%, p = 0.001) and higher among those being trained < 2 years ago compared to those who were not (42.9% versus 26.3%, p = 0.01). Among diagnostic laboratories in Democratic Republic of the Congo, performance of blood parasite microscopy including non-falciparum species and Trypanosoma was poor. Recent training and previous EQA participation were associated with a better performance.

Sleeping sickness caused by Trypanosoma brucei (T.b.) gambiense constitutes a serious health problem in sub-Sahara Africa. In some foci, alarmingly high relapse rates were observed in patients treated with melarsoprol, which used to be the first line treatment for patients in the neurological disease stage. Particularly problematic was the situation in Mbuji-Mayi, East Kasai Province in the Democratic Republic of the Congo with a 57% relapse rate compared to a 5% relapse rate in Masi-Manimba, Bandundu Province. The present study aimed at investigating the mechanisms underlying the high relapse rate in Mbuji-Mayi using an extended collection of recently isolated T.b. gambiense strains from Mbuji-Mayi and from Masi-Manimba.Forty five T.b. gambiense strains were used. Forty one were isolated from patients that were cured or relapsed after melarsoprol treatment in Mbuji-Mayi. In vivo drug sensitivity tests provide evidence of reduced melarsoprol sensitivity in these strains. This reduced melarsoprol sensitivity was not attributable to mutations in TbAT1. However, in all these strains, irrespective of the patient treatment outcome, the two aquaglyceroporin (AQP) 2 and 3 genes are replaced by chimeric AQP2/3 genes that may be associated with resistance to pentamidine and melarsoprol. The 4 T.b. gambiense strains isolated in Masi-Manimba contain both wild-type AQP2 and a different chimeric AQP2/3. These findings suggest that the reduced in vivo melarsoprol sensitivity of the Mbuji-Mayi strains and the high relapse rates in that sleeping sickness focus are caused by mutations in the AQP2/AQP3 locus and not by mutations in TbAT1.We conclude that mutations in the TbAQP2/3 locus of the local T.b. gambiense strains may explain the high melarsoprol relapse rates in the Mbuji-Mayi focus but other factors must also be involved in the treatment outcome of individual patients.

INTRODUCTION: Identifier les facteurs déterminant la persistance du konzo à Kahemba en République Démocratique du Congo. METHODES: Une enquête transversale a été réalisée à Kahemba en 2011 auprès des ménages de 123 enfants avec konzo (critères OMS) et de 87 enfants sans konzo. La récolte des données s'est faite par interviews, enquête socio-économique par le questionnaire HOME; observation, mesures anthropométriques et examen clinique; mesure du taux de cyanure (CN) dans la farine de manioc et thiocyanate (SCN) urinaire; et analyses sérologiques pour exclure les infections rétrovirales HTLV-I/II et HIV-I/II. L'analyse statistique a été faite par ANOVA, test de Chi-carré, et Kruskall-Wallis au seuil de signification de 0.05. RESULTATS: La survenue et la sévérité du konzo étaient associées à la pauvreté des ménages (p=0,05). Les enfants atteints de konzo présentaient une dégradation nutritionnelle avancée (p = ml/l) chez les enfants konzo vs. non-konzo. La population attribuait souvent la maladie à la sorcellerie. CONCLUSION: L'intoxication chronique au manioc amer, la malnutrition, ainsi que les croyances superstitieuses favorisent la persistance du konzo à Kahemba. La pauvreté porte le risque d'apparition et de gravité du konzo. Les épidémies de Kahemba dévoilent le risque transgénérationel associé au konzo. L'antécédent de konzo dans la famille élargie constituait un facteur de risque pour la survenue de la maladie (OR= 1,92 ; p=0,042). Le taux moyen (ET) de cyanure dans la farine de manioc était de 92,2 ( 56,2) ppm pour les ménages testés. Les taux moyens (ET) de SCN urinaire étaient respectivement de 520,4 , 355,7vs. 382,5, 226,3.

OBJECTIVES:Recently, improvements have been made to diagnostics for gambiense sleeping sickness control but their performance remains poorly documented and may depend on specimen processing prior to examination. In a prospective study in the Democratic Republic of the Congo, we compared the diagnostic performance of several parasite detection techniques, immune trypanolysis and of m18S PCR on whole blood stored in a stabilisation buffer or dried on filter paper. METHODS:Individuals with CATT whole blood (WB) titer ≥1∶4 or with clinical signs indicative for sleeping sickness were examined for presence of trypanosomes in lymph node aspirate (LNA) and/or in blood. Blood was examined with Capillary Centrifugation Technique (CTC), mini-Anion Exchange Centrifugation Technique (mAECT) and mAECT on buffy coat (BC). PCR was performed on whole blood (i) stored in guanidine hydrochloride EDTA (GE) stabilisation buffer and (ii) dried on filter paper, and repeatability and reproducibility were assessed. Immune trypanolysis (TL) was performed on plasma. RESULTS:A total of 237 persons were included. Among 143 parasitologically confirmed cases, 85.3% had a CATT-WB titre of ≥1/8, 39.2% were positive in LNA, 47.5% in CTC, 80.4% in mAECT-WB, 90.9% in mAECT-BC, 95.1% in TL and up to 89.5% in PCR on GE-stabilised blood. PCR on GE-stabilised blood showed highest repeatability (87.8%) and inter-laboratory reproducibility (86.9%). Of the 94 non-confirmed suspects, respectively 39.4% and 23.4% were TL or PCR positive. Suboptimal specificity of PCR and TL was also suggested by latent class analysis. CONCLUSION:The combination of LNA examination with mAECT-BC offered excellent diagnostic sensitivity. For PCR, storage of blood in stabilisation buffer is to be preferred over filter paper. TL as well as PCR are useful for remote diagnosis but are not more sensitive than mAECT-BC. For TL and PCR, the specificity, and thus usefulness for management of non-confirmed suspects remain to be determined.