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Africa contains more human genetic variation than any other continent, but the majority of the population-scale analyses of the African peoples have focused on just two of the four major linguistic groups, the Niger-Congo and Afro-Asiatic, leaving the Nilo-Saharan and Khoisan populations under-represented. In order to assess genetic variation and signatures of selection within a Nilo-Saharan population and between the Nilo-Saharan and Niger-Congo and Afro-Asiatic, we sequenced 50 genomes from the Nilo-Saharan Lugbara population of North-West Uganda and 250 genomes from 6 previously unsequenced Niger-Congo populations. We compared these data to data from a further 16 Eurasian and African populations including the Gumuz, another putative Nilo-Saharan population from Ethiopia. Of the 21 million variants identified in the Nilo-Saharan population, 3.57 million (17%) were not represented in dbSNP and included predicted non-synonymous mutations with possible phenotypic effects. We found greater genetic differentiation between the Nilo-Saharan Lugbara and Gumuz populations than between any two Afro-Asiatic or Niger-Congo populations. F3 tests showed that Gumuz contributed a genetic component to most Niger-Congo B populations whereas Lugabara did not. We scanned the genomes of the Lugbara for evidence of selective sweeps. We found selective sweeps at four loci (SLC24A5, SNX13, TYRP1, and UVRAG) associated with skin pigmentation, three of which already have been reported to be under selection. These selective sweeps point toward adaptations to the intense UV radiation of the Sahel., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

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 < 0.0001) and included loci where CNVs have previously been associated with HIV, Rhesus D and preeclampsia. When integrated with 1000 Genomes CNV data, we replicated their observation of population stratification by continent but no clustering by populations within Africa, despite inclusion of Nilo-Saharans and Niger-Congo populations within our dataset., Conclusions: Novel CNVRs in the current study increase representation of African diversity in the database of genomic variants. Over-representation of CNVRs in SNP signatures of selection and an excess of SNPs that both tag CNVs and are subject to selection show that CNVs may be the actual targets of selection at some loci. However, unlike SNPs, CNVs alone do not resolve African ethno-linguistic groups. Tag haplotypes for CNVs identified may be useful in predicting African CNVs in future studies where only SNP data is available.

Background: Rhodesiense sleeping sickness is caused by infection with T. b rhodesiense parasites resulting in an acute disease that is fatal if not treated in time. The aim of this study was to understand the global impact of active T. b rhodesiense infection on the patient's immune response in the early and late stages of the disease., Methods: RNASeq was carried out on blood and cerebral spinal fluid (CSF) samples obtained from T. b. rhodesiense infected patients. The control samples used were from healthy individuals in the same foci. The Illumina sequenced reads were analysed using the Tuxedo suite pipeline (Tophat, Cufflinks, Cuffmerge, Cuffdiff) and differential expression analysis carried out using the R package DESeq2. The gene enrichment and function annotation analysis were done using the ToppCluster, DAVID and InnateDB algorithms., Results: We previously described the transcriptomes of T. b rhodesiense from infected early stage blood (n = 3) and late stage CSF (n = 3) samples from Eastern Uganda. We here identify human transcripts that were differentially expressed (padj < 0.05) in the early stage blood versus healthy controls (n = 3) and early stage blood versus late stage CSF. Differential expression in infected blood showed an enrichment of innate immune response genes whereas that of the CSF showed enrichment for anti-inflammatory and neuro-degeneration signalling pathways. We also identified genes (C1QC, MARCO, IGHD3-10) that were up-regulated (log 2 FC > 2.5) in both the blood and CSF., Conclusion: The data yields insights into the host's response to T. b rhodesiense parasites in the blood and central nervous system. We identified key pathways and signalling molecules for the predominant innate immune response in the early stage infection; and anti-inflammatory and neuro-degeneration pathways associated with sleep disorders in second stage infection. We further identified potential blood biomarkers that can be used for diagnosis of late stage disease without the need for lumbar puncture.

All of our current knowledge of African trypanosome metabolism is based on results from trypanosomes grown in culture or in rodents. Drugs against sleeping sickness must however treat trypanosomes in humans. We here compare the transcriptomes of Trypanosoma brucei rhodesiense from the blood and cerebrospinal fluid of human patients with those of trypanosomes from culture and rodents. The data were aligned and analysed using new user-friendly applications designed for Kinetoplastid RNA-Seq data. The transcriptomes of trypanosomes from human blood and cerebrospinal fluid did not predict major metabolic differences that might affect drug susceptibility. Usefully, there were relatively few differences between the transcriptomes of trypanosomes from patients and those of similar trypanosomes grown in rats. Transcriptomes of monomorphic laboratory-adapted parasites grown in in vitro culture closely resembled those of the human parasites, but some differences were seen. In poly(A)-selected mRNA transcriptomes, mRNAs encoding some protein kinases and RNA-binding proteins were under-represented relative to mRNA that had not been poly(A) selected; further investigation revealed that the selection tends to result in loss of longer mRNAs.

Background: Human African trypanosomiasis (HAT) manifests as an acute form caused by Trypanosoma brucei rhodesiense (Tbr) and a chronic form caused by Trypanosoma brucei gambiense (Tbg). Previous studies have suggested a host genetic role in infection outcomes, particularly for APOL1. We have undertaken candidate gene association studies (CGAS) in a Ugandan Tbr and a Tbg HAT endemic area, to determine whether polymorphisms in IL10, IL8, IL4, HLAG, TNFA, TNX4LB, IL6, IFNG, MIF, APOL1, HLAA, IL1B, IL4R, IL12B, IL12R, HP, HPR, and CFH have a role in HAT., Methodology and Results: We included 238 and 202 participants from the Busoga Tbr and Northwest Uganda Tbg endemic areas respectively. Single Nucleotide Polymorphism (SNP) genotype data were analysed in the CGAS. The study was powered to find odds ratios > 2 but association testing of the SNPs with HAT yielded no positive associations i.e. none significant after correction for multiple testing. However there was strong evidence for no association with Tbr HAT and APOL1 G2 of the size previously reported in the Kabermaido district of Uganda., Conclusions/significance: A recent study in the Soroti and Kaberamaido focus in Central Uganda found that the APOL1 G2 allele was strongly associated with protection against Tbr HAT (odds ratio = 0.2, 95% CI: 0.07 to 0.48, p = 0.0001). However, in our study no effect of G2 on Tbr HAT was found, despite being well powered to find a similar sized effect (OR = 0.9281, 95% CI: 0.482 to 1.788, p = 0.8035). It is possible that the G2 allele is protective from Tbr in the Soroti/Kabermaido focus but not in the Iganga district of Busoga, which differ in ethnicity and infection history. Mechanisms underlying HAT infection outcome and virulence are complex and might differ between populations, and likely involve several host, parasite or even environmental factors.

African trypanosomes of the sub-genus Trypanozoon) are eukaryotic parasitesthat cause disease in either humans or livestock. The development of genomic resources can be of great use to those interested in studying and controlling the spread of these trypanosomes. Here we present a large comparative analysis of Trypanozoon whole genomes, 83 in total, including human and animal infective African trypanosomes: 21 T. brucei brucei, 22 T. b. gambiense, 35 T. b. rhodesiense and 4 T. evansi strains, of which 21 were from Uganda. We constructed a maximum likelihood phylogeny based on 162,210 single nucleotide polymorphisms (SNPs.) The three Trypanosoma brucei sub-species and Trypanosoma evansi are not monophyletic, confirming earlier studies that indicated high similarity among Trypanosoma "sub-species". We also used discriminant analysis of principal components (DAPC) on the same set of SNPs, identifying seven genetic clusters. These clusters do not correspond well with existing taxonomic classifications, in agreement with the phylogenetic analysis. Geographic origin is reflected in both the phylogeny and clustering analysis. Finally, we used sparse linear discriminant analysis to rank SNPs by their informativeness in differentiating the strains in our data set. As few as 84 SNPs can completely distinguish the strains used in our study, and discriminant analysis was still able to detect genetic structure using as few as 10 SNPs. Our results reinforce earlier results of high genetic similarity between the African Trypanozoon. Despite this, a small subset of SNPs can be used to identify genetic markers that can be used for strain identification or other epidemiological investigations.

Reduced susceptibility to infectious disease can increase the frequency of otherwise deleterious alleles. In populations of African ancestry, two apolipoprotein-L1 (APOL1 ) variants with a recessive kidney disease risk, named G1 and G2, occur at high frequency. APOL1 is a trypanolytic protein that confers innate resistance to most African trypanosomes, but not Trypanosoma brucei rhodesiense or T.b. gambiense, which cause human African trypanosomiasis. In this case-control study, we test the prevailing hypothesis that these APOL1 variants reduce trypanosomiasis susceptibility, resulting in their positive selection in sub-Saharan Africa. We demonstrate a five-fold dominant protective association for G2 against T.b. rhodesiense infection. Furthermore, we report unpredicted strong opposing associations with T.b. gambiense disease outcome. G2 associates with faster progression of T.b. gambiense trypanosomiasis, while G1 associates with asymptomatic carriage and undetectable parasitemia. These results implicate both forms of human African trypanosomiasis in the selection and persistence of otherwise detrimental APOL1 kidney disease variants.

Background: Trypanosoma brucei rhodesiense is the causative agent of acute human African trypanosomiasis. Identification of T. b. rhodesiense in tsetse populations is essential for understanding transmission dynamics, assessng human disease risk, and monitoring spatiotemporal trends and impact of control interventions. Accurate detection and characterisation of trypanosomes in vectors relies on molecular techniques. For the first time in Malawi, a molecular technique has been used to detect trypanosomes in tsetse flies in Nkhotakota Wildlife Reserve., Methods: A polymerase chain reaction (PCR) technique was used to identify the serum resistance associated ( SRA ) gene of T. b. rhodesiense in tsetse flies. Of 257 tsetse flies that were randomly caught, 42 flies were dissected for microscopic examination. The midguts of 206 flies were positive and were individually put in eppendorf tubes containing phosphate-buffered saline (PBS buffer) for DNA extraction. Internal transcribed spacer (ITS)-PCR was first used to isolate all trypanosome species from the flies. TBR PCR was then used to isolate the Trypanozoon group. T. brucei -positive samples were further evaluated by SRA PCR for the presence of the SRA gene., Results: Of 257 flies caught, 185 (72%) were Glossina morsitans morsitans and 72 (28%) were Glossina pallidipes . Three were tenerals and 242 were mature live flies. Of the 242 flies dissected, 206 were positive, representing an 85.1% infection rate. From 206 infected flies, 106 (51.5%) were positive using ITS-PCR, 68 (33.0%) being mixed infections, 18 (8.7%) T. brucei, 9 (4.4%) Trypanosoma vivax , 4 (1.9%) Trypanosoma godfrey , 3 (1.5%) Trypanosoma congolense savanna , 3 (1.5%) Trypanosoma simae , and 1 (0.4%) Trypanosoma simaetsavo . When subjected to TBR PCR, 107(51.9%) were positive for T. brucei . Of the 107 T. brucei- positive samples, 5 (4.7%) were found to have the SRA gene., Conclusions: These results suggest that wild tsetse flies in Malawi are infected with human-infective trypanosomes that put communities around wildlife reserves at risk of human African trypanosomiasis outbreaks. Further studies need to be done to identify sources of blood meals for the flies and for surveillance of communities around wildlife reserves.

Background: During natural Trypanosoma brucei infections, the parasites differentiate spontaneously into a non-dividing "stumpy" form when a certain level of parasitaemia is attained. This form is metabolically adapted for rapid further differentiation into procyclic forms upon uptake by Tsetse flies., Results: We describe here four central Ugandan isolates of Trypanosoma brucei rhodesiense that have undergone only three rodent passages since isolation from human patients. As expected, SNP analysis shows that these isolates are more closely related to each other than to the commonly used strains Lister 427, Antat1.1, and TREU927. TREU927 generally has smaller copy numbers of repeated genes than the other strains, while Lister 427 trypanosomes with a 30-year history of in vitro culture and cloning have more histone genes than the other isolates. The recently isolated trypanosomes were grown in rats, and their transcriptomes characterised. In comparison with cultured procyclic and bloodstream forms, there were increases in mRNAs encoding the stumpy-form markers ESAG9 and PIP39, with coordinated alterations in the levels of over 600 additional mRNAs. Numerous mRNAs encoding proteins of no known function were either increased or decreased. The products of the mRNAs that were increased in parallel with PIP39 included not only enzymes of procyclic-form metabolism, but also components of the translational and RNA control machineries. Many of the mRNAs that were decreased in cells with elevated PIP39 reflected reduced cell division., Conclusions: These transcriptomes suggest new avenues for research into the regulation of trypanosome differentiation.

Background: While Human African Trypanosomiasis (HAT) is in decline on the continent of Africa, the disease still remains a major health problem in Uganda. There are recurrent sporadic outbreaks in the traditionally endemic areas in south-east Uganda, and continued spread to new unaffected areas in central Uganda. We evaluated the evolutionary dynamics underpinning the origin of new foci and the impact of host species on parasite genetic diversity in Uganda. We genotyped 269 Trypanosoma brucei isolates collected from different regions in Uganda and southwestern Kenya at 17 microsatellite loci, and checked for the presence of the SRA gene that confers human infectivity to T. b. rhodesiense., Results: Both Bayesian clustering methods and Discriminant Analysis of Principal Components partition Trypanosoma brucei isolates obtained from Uganda and southwestern Kenya into three distinct genetic clusters. Clusters 1 and 3 include isolates from central and southern Uganda, while cluster 2 contains mostly isolates from southwestern Kenya. These three clusters are not sorted by subspecies designation (T. b. brucei vs T. b. rhodesiense), host or date of collection. The analyses also show evidence of genetic admixture among the three genetic clusters and long-range dispersal, suggesting recent and possibly on-going gene flow between them., Conclusions: Our results show that the expansion of the disease to the new foci in central Uganda occurred from the northward spread of T. b. rhodesiense (Tbr). They also confirm the emergence of the human infective strains (Tbr) from non-infective T. b. brucei (Tbb) strains of different genetic backgrounds, and the importance of cattle as Tbr reservoir, as confounders that shape the epidemiology of sleeping sickness in the region.

Background: The prevalence of Helicobacter pylori infection varies in relation to geography, ethnicity and socioeconomic factors. Available data on the prevalence of Helicobacter pylori infection in Uganda are not representative of the general population. We sought to describe the epidemiology of this infection in pregnant women in Uganda to provide background data for a study into the effect of H. pylori infection during pregnancy on the hematological response to iron supplementation., Methods: Using a cross-sectional design, H. pylori infection was assessed by the stool antigen test among 447 pregnant women attending antenatal care clinics in Apac, Mbale, Mbarara and Rakai Districts which are in different geographical regions in Uganda, and at Kawempe Health Center which serves a low-income densely populated area in Kampala City. Socio-demographic and household data were collected by face-to-face interviews using a questionnaire. Associations between H. pylori infection and socio-demographic and household characteristics were analyzed using logistic regression., Results: The overall prevalence of H. pylori infection was 45.2% but varied by geographical location from 18.2% in Apac District to 60.5% at Kawempe Health Centre. At 18.4%, the Langi ethnic group, who were enrolled exclusively in Apac District, had the lowest prevalence of H. pylori infection while the Gisu had the highest prevalence (58.4%). H. pylori was independently associated with enrollment at clinics not in Apac (adjusted OR = 5.68; 95% CI: 3.02-10.7) and with using water from public wells, boreholes or springs (AOR = 3.20; 95% CI: 1.19-8.61) and from rivers, lakes or streams (AOR = 5.20; 95% CI: 1.58-17.05). Urban residence (AOR = 1.71; 95% CI: 1.13-2.60) and no formal education (AOR = 1.95; 95% CI: 1.03-3.67) were also independently associated with H. pylori infection., Conclusions: The unexpected variation in the prevalence of H. pylori infection in Uganda calls for population-based studies in the region and offers an opportunity to study the transmission dynamics of H. pylori infection. The association between H. pylori infection and surface water sources for household use suggests waterborne transmission of H. pylori infection highlighting the need for concerted efforts in environmental health in communities and at the household level.

Patterns of gene expression in cultured Trypanosoma brucei bloodstream and procyclic forms have been extensively characterized, and some comparisons have been made with trypanosomes grown to high parasitaemias in laboratory rodents. We do not know, however, to what extent these transcriptomes resemble those in infected Tsetse flies - or in humans or cattle, where parasitaemias are substantially lower. For clinical and field samples it is difficult to characterize parasite gene expression because of the large excess of host cell RNA. We have here examined two potential solutions to this problem for bloodstream form trypanosomes, assaying transcriptomes by high throughput cDNA sequencing (RNASeq). We first purified the parasites from blood of infected rats. We found that a red blood cell lysis procedure affected the transcriptome substantially more than purification using a DEAE cellulose column, but that too introduced significant distortions and variability. As an alternative, we specifically amplified parasite sequences from a mixture containing a 1000-fold excess of human RNA. We first purified polyadenylated RNA, then made trypanosome-specific cDNA by priming with a spliced leader primer. Finally, the cDNA was amplified using nested primers. The amplification procedure was able to produce samples in which 20% of sequence reads mapped to the trypanosome genome. Synthesis of the second cDNA strand with a spliced leader primer, followed by amplification, is sufficiently reproducible to allow comparison of different samples so long as they are all treated in the same way. However, SL priming distorted the abundances of the cDNA products and definitely cannot be used, by itself, to measure absolute mRNA levels. The amplification method might be suitable for clinical samples with low parasitaemias, and could also be adapted for other Kinetoplastids and to samples from infected vectors.

This article documents the addition of 153 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Brassica oleracea, Brycon amazonicus, Dimorphandra wilsonii, Eupallasella percnurus, Helleborus foetidus, Ipomoea purpurea, Phrynops geoffroanus, Prochilodus argenteus, Pyura sp., Sylvia atricapilla, Teratosphaeria suttonii, Trialeurodes vaporariorum and Trypanosoma brucei. These loci were cross-tested on the following species: Dimorphandra coccicinea, Dimorphandra cuprea, Dimorphandra gardneriana, Dimorphandra jorgei, Dimorphandra macrostachya, Dimorphandra mollis, Dimorphandra parviflora and Dimorphandra pennigera., (© 2013 Blackwell Publishing Ltd.)

Tsetse flies (Glossina spp.) are the sole vectors of Trypanosoma brucei--the agent of human (HAT) and animal (AAT) trypanosomiasis. Glossina fuscipes fuscipes (Gff) is the main vector species in Uganda--the only country where the two forms of HAT disease (rhodesiense and gambiense) occur, with gambiense limited to the northwest. Gff populations cluster in three genetically distinct groups in northern, southern, and western Uganda, respectively, with a contact zone present in central Uganda. Understanding the dynamics of this contact zone is epidemiologically important as the merger of the two diseases is a major health concern. We used mitochondrial and microsatellite DNA data from Gff samples in the contact zone to understand its spatial extent and temporal stability. We show that this zone is relatively narrow, extending through central Uganda along major rivers with south to north introgression but displaying no sex-biased dispersal. Lack of obvious vicariant barriers suggests that either environmental conditions or reciprocal competitive exclusion could explain the patterns of genetic differentiation observed. Lack of admixture between northern and southern populations may prevent the sympatry of the two forms of HAT disease, although continued control efforts are needed to prevent the recolonization of tsetse-free regions by neighboring populations.

Tsetse flies are notoriously difficult to observe in nature, particularly when populations densities are low. It is therefore difficult to observe them on their hosts in nature; hence their vertebrate species can very often only be determined indirectly by analysis of their gut contents. This knowledge is a critical component of the information on which control tactics can be developed. The objective of this study was to determine the sources of tsetse bloodmeals, hence investigate their feeding preferences. We used mitochondrial cytochrome c oxidase 1 (COI) and cytochrome b (cytb) gene sequences for identification of tsetse fly blood meals, in order to provide a foundation for rational decisions to guide control of trypanosomiasis, and their vectors. Glossina swynnertoni were sampled from Serengeti (Tanzania) and G. pallidipes from Kenya (Nguruman and Busia), and Uganda. Sequences were used to query public databases, and the percentage identities obtained used to identify hosts. An initial assay showed that the feeds were from single sources. Hosts identified from blood fed flies collected in Serengeti ecosystem, included buffaloes (25/40), giraffes (8/40), warthogs (3/40), elephants (3/40) and one spotted hyena. In Nguruman, where G. pallidipes flies were analyzed, the feeds were from elephants (6/13) and warthogs (5/13), while buffaloes and baboons accounted for one bloodmeal each. Only cattle blood was detected in flies caught in Busia and Uganda. Out of four flies tested in Mbita Point, Suba District in western Kenya, one had fed on cattle, the other three on the Nile monitor lizard. These results demonstrate that cattle will form an integral part of a control strategy for trypanosomiasis in Busia and Uganda, while different approaches are required for Serengeti and Nguruman ecosystems, where wildlife abound and are the major component of the tsetse fly food source.

Human African trypanosomiasis, or sleeping sickness, is a parasitic disease endemic in sub-Saharan Africa, transmitted to humans through the bite of a tsetse fly. The first or hemolymphatic stage of the disease is associated with presence of parasites in the bloodstream, lymphatic system, and body tissues. If patients are left untreated, parasites cross the blood-brain barrier and invade the cerebrospinal fluid and the brain parenchyma, giving rise to the second or meningoencephalitic stage. Stage determination is a crucial step in guiding the choice of treatment, as drugs used for S2 are potentially dangerous. Current staging methods, based on counting white blood cells and demonstrating trypanosomes in cerebrospinal fluid, lack specificity and/or sensitivity. In the present study, we used several proteomic strategies to discover new markers with potential for staging human African trypanosomiasis. Cerebrospinal fluid (CSF) samples were collected from patients infected with Trypanosoma brucei gambiense in the Democratic Republic of Congo. The stage was determined following the guidelines of the national control program. The proteome of the samples was analyzed by two-dimensional gel electrophoresis (n = 9), and by sixplex tandem mass tag (TMT) isobaric labeling (n = 6) quantitative mass spectrometry. Overall, 73 proteins were overexpressed in patients presenting the second stage of the disease. Two of these, osteopontin and β-2-microglobulin, were confirmed to be potential markers for staging human African trypanosomiasis (HAT) by Western blot and ELISA. The two proteins significantly discriminated between S1 and S2 patients with high sensitivity (68% and 78%, respectively) for 100% specificity, and a combination of both improved the sensitivity to 91%. The levels of osteopontin and β-2-microglobulin in CSF of S2 patients (μg/ml range), as well as the fold increased concentration in S2 compared with S1 (3.8 and 5.5 respectively) make the two markers good candidates for the development of a test for staging HAT patients.

Control of human African trypanosomiasis (HAT) is dependent on accurate diagnosis and treatment of infected patients. However, sensitivities of tests in routine use are unsatisfactory, due to the characteristically low parasitaemias in naturally infected individuals. We have identified a conserved sequence in the repetitive insertion mobile element (RIME) of the sub-genus Trypanozoon and used it to design primers for a highly specific loop-mediated isothermal amplification (LAMP) test. The test was used to analyse Trypanozoon isolates and clinical samples from HAT patients. The RIME LAMP assay was performed at 62 degrees C using real-time PCR and a water bath. DNA amplification was detectable within 25min. All positive samples detected by gel electrophoresis or in real-time using SYTO-9 fluorescence dye could also be detected visually by addition of SYBR Green I to the product. The amplicon was unequivocally confirmed through restriction enzyme NdeI digestion, analysis of melt curves and sequencing. The analytical sensitivity of the RIME LAMP assay was equivalent to 0.001 trypanosomes/ml while that of classical PCR tests ranged from 0.1 to 1000 trypanosomes/ml. LAMP detected all 75 Trypanozoon isolates while TBR1 and two primers (specific for sub-genus Trypanozoon) showed a sensitivity of 86.9%. The SRA gene PCR detected 21 out of 40 Trypanosoma brucei rhodesiense isolates while Trypanosoma gambiense-specific glycoprotein primers (TgsGP) detected 11 out of 13 T. b. gambiense isolates. Using clinical samples, the LAMP test detected parasite DNA in 18 out of 20 samples which included using supernatant prepared from boiled blood, CSF and direct native serum. The sensitivity and reproducibility of the LAMP assay coupled with the ability to detect the results visually without the need for sophisticated equipment indicate that the technique has strong potential for detection of HAT in clinical settings. Since the LAMP test shows a high tolerance to different biological substances, determination of the appropriate protocols for processing the template to make it a user-friendly technique, prior to large scale evaluation, is needed.

Diagnosis of sleeping sickness (trypanosomiasis) is difficult because of the fluctuating levels of parasitaemia encountered in patients. In the present study we found that the polymerase chain reaction (PCR) demonstrated trypanosome infection in 20 out of 35 (57.1%) blood samples and in 21 out of 34 (61.7%) cerebrospinal fluid (CSF) samples collected from an area endemic for sleeping sickness in north-west Uganda. A total of 14 blood samples and 13 CSF samples that were positive for trypanosomes by double centrifugation were also positive by PCR, demonstrating good concordance between the two methods. However, 6 (28.6%) of the 21 blood samples that were parasitologically negative were positive by PCR, while 8 (38.0%) out of 21 CSF samples that were negative by double centrifugation were positive by PCR. These 14 negative samples could therefore be from sleeping sickness cases even though a positive PCR test is not evidence for the presence of trypanosomes. Furthermore, of these 8 CSF samples, 4 had been designated as early cases, based on the absence of trypanosomes and on a count of < or = 5 white blood cells (WBC) per microliter. This suggests that some late-stage cases could potentially be missed according to the present criteria, and it is therefore important to perform clinical trials to determine whether these cases could be treated successfully with the first-stage drug alone. The remaining four CSF samples had been classified as late-stage cases, based on a count of > 6 WBC per microliter, even though trypanosomes could not be detected in these samples by either double centrifugation or PCR. A cut-off point of 5 WBC per microliter, which is used as a rule of thumb to stage sleeping sickness patients, seems to leave some late-stage cases undetected since trypanosomes were detected in four CSF samples from suspected cases with < 5 WBC per microliter.

We evaluated the treatment failure rate among late-stage human African trypanosomiasis (HAT) patients treated with melarsoprol in Arua, northern Uganda, between September 1995 and August 1996, and identified the risk factors for treatment failure. We conducted a retrospective cohort study in October 1998, and performed a survival analysis. A treatment failure was defined as a late-stage HAT patient fully treated with melarsoprol and classified as an HAT case at any follow-up visit within 2 years after treatment. Among 428 patients treated in the study period, 130 (30.4%) were identified as treatment failure within 2 years after discharge. The multivariate analysis showed that patients who experienced treatment failure after melarsoprol were more likely to have been admitted as a relapsing case (relative hazard, RH = 11.15 [6.34-19.61]), and to have been diagnosed with trypanosomes in the lymph nodes (RH = 3.19 [2.10-4.83]) or in the cerebrospinal fluid (CSF) (RH = 1.66 [1.09-2.53]). The risk of treatment failure also increased with the number of cells in the CSF. The treatment failure rate after melarsoprol observed in Arua is greatly above the expected figures of 3-9%. More research is needed to confirm whether it is related to the variation of melarsoprol pharmacokinetics between individuals, or if it is associated with a reduced susceptibility of the trypanosomes to melarsoprol. The study emphasizes the need for second-line drugs to treat patients that have already received one or several full course(s) of melarsoprol.

Objective: To develop a simple field test for diagnosis of Trypanosoma brucei rhodesiense in man., Design: Trypanosomosis Agglutination Card Test (TACT) was developed for the diagnosis of sleeping sickness due to Trypanosoma brucei rhodesiense infection, based on stabilised procyclic forms derived from Utat 4.1. Procyclics were fixed in buffered formalin at 4 degrees for 24 hours and further stabilised in acid/alcohol mixture for 30 minutes. The fixed antigen was stained with Coomassie blue and suspended in 0.1 M PBS/sodium azide buffer pH 7.2 at a concentration of 1 x 10(8) trypanosomes/ml and kept at room temperature. This antigen was used to screen 100 sera from rabbits infected with T. b. rhodesiense, eight from normal rabbits, and 220 only sera 60 of which were from sleeping sickness patients, 50 from normal persons and 110 from other parasitic infections., Setting: Laboratory testing of the antigen types against the rabbit and human sera infected with cloned variable antigen types of T. b. rhodesiense, was routinely carried on test cards under room temperature., Subjects/participants: Serum samples from normal and infected rabbits and human subjects., Results: All sera from infected rabbits and 59 from sleeping sickness patients reacted strongly with the antigen showing agglutination reaction which ranged from 1:4 to 1:1024 serum dilution. There was minimal cross reaction with other parasitic infections as follows: one out of 20 malaria patients none of the 20 hookworm patients, one out of 30 for schistosomiasis patients, none of the 10 amoebiasis patients and one out of 20 for filariasis patients. Agglutination titres from all these non-sleeping sickness patients were below 1:16. Based on rabbit positive and negative sera, TACT gave a sensitivity and specificity of 100% and 80% while for human sera a sensitivity of 98.3% and specificity of 96% were observed., Conclusion: These preliminary results show that TACT could be a promising screening field test for T. b. rhodesiense sleeping sickness.

Although there have been recent molecular biological studies for evidence of possible changes in trypanosome biochemistry, such studies are not yet complemented by parallel clinical studies to determine the possible implications to the sleeping sickness patient. The study of the duration of symptoms and the case fatality of T. b. rhodesiense showed that the disease progressed to the stage of central nervous system involvement between three weeks to two months of infection. Most (> 80%) deaths occurred within six months of illness. The case fatality rate of treated sleeping sickness patients was 6% of which the rate in the late-stage of sleeping sickness was more than two and a half times that in the early stage. The incidence of melarsoprol encephalopathy was 2.5% and case fatality due to this condition was 1.0% and similar to previous findings. Thus it appears the virulence of T. b. rhodesiense circulating in south east Uganda has not changed during the past decades.

For the first time, two clones of Trypanosoma brucei from the same epidemic area in southeastern Uganda were successfully crossed. The cotransmission experiments were as close to natural conditions as possible in that two uncloned isolates from wild-caught Glossina fuscipes fuscipes were directly cotransmitted in an initial recombination experiment. From the first uncloned progeny population, which revealed a majority of recombinants, two clones with different parental phenotypes [characterized by isocitrate dehydrogenase (ICD) analysis] were isolated. These clones were cotransmitted in a second recombination experiment. Nine clones could be isolated from two different progeny populations of the second experiment, and all showed a recombinant phenotype. These nine clones belonged to three different karyotypes with respect to the large chromosomes (1-3 Mb), which were different from those of either parental karyotype or the sum of both parental karyotypes. The results indicate that genetic recombination might well occur between trypanosome populations transmitted within the same epidemic area.

An epidemic of sleeping sickness, which started in 1976 in a focus within the county of Luuka in Central Busoga, has spread to cover the three districts of Busoga and large parts of the neighbouring districts of Tororo and Mukono. Forty-three isolates of the subgenus Trypanozoon from Busoga and Tororo (27 from man, 9 from cows, 2 from pigs and 5 from tsetse flies) were compared by thin-layer starch-gel electrophoresis for seven enzymes. Thirty zymodemes were identified; 17 of them were found circulating in the human population. The zymodemes seen previously in Busoga were still circulating together with several new ones. Of the 16 isolates from cattle, pigs and tsetse flies, only two had the same profile, indicating a high degree of diversity. Two zymodemes from cows and a pig were identical to those found in man, implicating domestic stock in the transmission of human disease in south-east Uganda. A computer analysis of the results produced six main zymodeme groups. One comprised only isolates from man; two were composed of isolates from man, domestic animals and tsetse; and three consisted of stocks from domestic animals only. These groups quite probably indicate the different cycles of transmission involving man, tsetse fly and domestic stock.