Serum Resistance-associated Gene Research Articles

How Antiparasitic Drugs Work-And Sometimes Stop Working!

How Antiparasitic Drugs Work-And Sometimes Stop Working!

Virulence Profile, Fluoroquinolone and Quinolone Resistance of Uropathogenic Escherichia coli Isolates Recovered from Thabet Hospital-Tulkarm, Palestine

Backcground: Escherichia coli (E. coli) is the most common cause of urinary tract infection (UTI). Virulence factors are mainly responsible for the severity of these emerging infections. Aims: To analyze virulence factors and resistance phenotype to quinolones and fluoroquinolones in a collection of E. coli strains isolated from UTIs with previously known phylogenetic groups. Place and Duration of Study: Department of Biology and Biotechnology, An-Najah N. University, Palestine, during May-December 2012. Methodology: Fifty clinical E. coli isolates were previously recovered from urine specimens obtained from patients suffered from urinary tract infections at Thabet Hospital, Tulkarm-Palestine. Multiplex PCR technique was used to detect the presence of 18 virulence genes and ERIC-PCR Original Research Article Adwan et al.; BMRJ, 5(5): xxx-xxx, 2015; Article no.BMRJ.2015.043 413 was used to detect the heterogeneity of these strains. All E. coli isolates were examined for resistance to different antibiotics using disk diffusion method. Results: It is found that the prevalence of virulence genes ranged from 0% for P-fimbria adhesion variant 1 (pap G I) allele and αhemolysin (hly A) to 86% for Type1 fimbriae adhesion (fim H) and Serum resistance-associated gene (tra T) in strains tested. The results showed that the mean virulence score for group D was 8.2 and ranged from 2 to15, while for group A was 6.2 and ranged from 1 to14 (P=6.2 x 10 -4 ). The mean virulence score for strains resistant to fluoroquinolones and /or quinolones was 7.3, while for strains sensitive to both fluoroquinolones and quinolones was 8.1. Quinolones and/or fluoquinolones sensitive strains related to group D showed an increased prevalence of catecholate siderophore receptor (iron) than resistant strains. It was also found that traT gene was the most common prevalence among strains resistant to nalidixic acid, fluoroquinolones and trimethoprim/sulphamethoxazole and it was 90.1% (30/33), 95.8 (23/24) and 90.6% (29/32), respectively. ERIC-PCR revealed that the 50 isolates were genetically diverse and comprised a heterogeneous population with at total 10 ERIC-PCR profiles at a 50% similarity level. Conclusion: The molecular analysis of strains belonged to groups A and D, results showed that group D had higher mean virulence score than group A. It seems that there is no single virulence factor or virulence profile that is entirely specific to UTI in general.

Outcome of acute East African trypanosomiasis in a Polish traveller treated with pentamidine

BackgroundAfrican trypanosomiasis is a parasitic infection sporadically imported to Europe by tourists or immigrants returning from endemic areas. We present the first and an unusual case of East African trypanosomiasis imported to Poland by a patient returning from a tourist trip to Uganda and Rwanda, which was successfully treated with pentamidine.Case presentationA 61-year-old Polish man was admitted to the Department because of high-grade fever and multi-organ dysfunction after a tourist trip to East Africa. He experienced a single tsetse fly bite during a safari trip to the Queen Elizabeth National Park in Uganda. On admission, his clinical status was severe, with high fever of 41ºC, preceded by chills, bleeding from the gums and oral mucosa, haemorrhages at the sites of venipuncture, numerous ecchymoses, fine-spotted skin rash, tachycardia, hepatosplenomegaly, dehydration, jaundice, dyspnoea, hypoxaemia, generalised oedema and oliguria. There was a typical non-painful trypanosomal chancre with central necrosis and peripheral erythema on his left arm. Laboratory investigations showed leucopenia, thrombocytopenia, haemolytic anaemia, hyperbilirubinaemia, hypoglycaemia, elevated creatinine and urea, high activity of aminotransferases, elevated levels of inflammatory markers, hypoproteinaemia, proteinuria, abnormal clotting and bleeding times, low fibrinogen level, metabolic acidosis, and electrolyte disturbances. A peripheral blood smear showed numerous Trypanosoma brucei trypomastigotes with a massive parasitaemia of 100,000/μl. T. brucei rhodesiense subspecies was finally identified on the basis of the characteristic serum resistance-associated gene using a polymerase chain reaction, and a seroconversion of specific immunoglobulin M and G antibodies in the peripheral blood by enzyme-linked immunosorbent assay. Serological tests for T. brucei gambiense subspecies were negative. A severe clinical course of acute rhodesiense trypanosomiasis with renal failure, respiratory distress, disseminated intravascular coagulation syndrome, haemolysis, liver insufficiency and myocarditis was confirmed. Intensive anti-parasitic and symptomatic treatment was immediately instituted, including intravenous pentamidine, plasmaphereses, oxygen therapy, blood transfusion, catecholamine administration, and fluid infusions, as well as haemostatic, hepatoprotective, anti-inflammatory, antipyretic and diuretic drugs. The final outcome was a full recovery with no late sequelae.ConclusionSleeping sickness should always be considered in the differential diagnosis of fever in people returning from safari trips to the national parks or nature reserves of sub-Saharan Africa.

The use of Loop-mediated Isothermal Amplification (LAMP) to detect the re-emerging Human African Trypanosomiasis (HAT) in the Luangwa and Zambezi valleys

BackgroundLoop-mediated isothermal amplification (LAMP) is a novel strategy which amplifies DNA with high sensitivity and rapidity under isothermal conditions. In the present study, the performance of the repetitive insertion mobile element (RIME)-LAMP and human serum resistance-associated gene (SRA)-LAMP assays were evaluated using clinical specimens obtained from four male patients from Luangwa and Zambezi valleys in Zambia and Zimbabwe, respectively.FindingsThe cases reported in this preliminary communication were all first diagnosed by microscopy, through passive surveillance, and confirmed by both RIME-LAMP and SRA-LAMP. A good correlation between microscopy and LAMP was observed and contributed to staging and successful treatment of patient. RIME-LAMP and SRA-LAMP complimented each other well in all the cases.ConclusionsBoth RIME-LAMP and SRA-LAMP were able to detect Trypanosoma brucei rhodesiense DNA in patient blood and CSF and hence confirmed HAT in the parasitaemic patients. Our study indicates that the LAMP technique is a potential tool for HAT diagnosis, staging and may be useful for making therapeutic decisions. However, no statistically significant conclusion may be drawn due to the limited sample size used in the present study. It is thus imperative to conduct a detailed study to further evaluate the potential of LAMP as a bedside diagnostic test for HAT.

An expanded family of proteins with BPI/LBP/PLUNC-like domains in trypanosome parasites: an association with pathogenicity?

Trypanosomatids are protozoan parasites that cause human and animal disease. Trypanosoma brucei telomeric ESs (expression sites) contain genes that are critical for parasite survival in the bloodstream, including the VSG (variant surface glycoprotein) genes, used for antigenic variation, and the SRA (serum-resistance-associated) gene, which confers resistance to lysis by human serum. In addition, ESs contain ESAGs (expression-site-associated genes), whose functions, with few exceptions, have remained elusive. A bioinformatic analysis of the ESAG5 gene of T. brucei showed that it encodes a protein with two BPI (bactericidal/permeability-increasing protein)/LBP (lipopolysaccharide-binding protein)/PLUNC (palate, lung and nasal epithelium clone)-like domains and that it belongs to a multigene family termed (GR)ESAG5 (gene related to ESAG5). Members of this family are found with various copy number in different members of the Trypanosomatidae family. T. brucei has an expanded repertoire, with multiple ESAG5 copies and at least five GRESAG5 genes. In contrast, the parasites of the genus Leishmania, which are intracellular parasites, have only a single GRESAG5 gene. Although the amino acid sequence identity between the (GR)ESAG5 gene products between species is as low as 15-25%, the BPI/LBP/PLUNC-like domain organization and the length of the proteins are highly conserved, and the proteins are predicted to be membrane-anchored or secreted. Current work focuses on the elucidation of possible roles for this gene family in infection. This is likely to provide novel insights into the evolution of the BPI/LBP/PLUNC-like domains.

Comparative Detection of Trypanosomal DNA by Loop-Mediated Isothermal Amplification and PCR from Flinders Technology Associates Cards Spotted with Patient Blood

We analyzed DNA eluted from FTA (Flinders Technology Associates) cards spotted with blood from human African trypanosomiasis (HAT) patients admitted at Lwala Hospital in eastern Uganda and Kaliua Health Centre in northwestern Tanzania. The aims were to evaluate loop-mediated isothermal amplification (LAMP) for detection of trypanosomal DNA in clinical samples and to characterize the infecting trypanosomes to the subspecies level. LAMP targeting the Trypanozoon conserved random inserted mobile element (RIME-LAMP) and that for the serum resistance-associated (SRA) gene (SRA-LAMP) were performed. For comparison, PCRs for the SRA gene specific for Trypanosoma brucei rhodesiense (SRA-PCR) and that to amplify the Trypanosoma brucei gambiense-specific surface glycoprotein (TgSGP-PCR) were done. Out of 128 samples analyzed, SRA-PCR was positive in 101 samples (78.9% sensitivity; 95% confidence interval [CI], 71.1 to 85.1%), SRA-LAMP was positive in 120 (93.8%; 95% CI, 88.2 to 96.8%), while RIME-LAMP revealed signals in 122 (95.3%; 95% CI, 90.2 to 97.8%). RIME-LAMP and SRA-LAMP were each significantly more sensitive than SRA-PCR (P values of 0.000 and 0.001, respectively; Fisher's exact test). There was poor agreement between RIME-LAMP and SRA-LAMP and the SRA-PCR, yielding kappa values of 0.31 and 0.40, respectively. Agreement between SRA-LAMP and RIME-LAMP was almost perfect (kappa value, 0.85; 95% CI, 0.64 to 1). All the 128 field samples were negative by TgSGP-PCR. Blood spots from three T. b. gambiense HAT cases from northwestern Uganda were positive by TgSGP-PCR and RIME-LAMP. PCR took five times longer to execute than LAMP. LAMP may be useful to monitor emerging HAT foci or to test travelers returning from countries where HAT is endemic. It should be evaluated in a case-control study to determine its utility as a HAT diagnostic.

Evolution of the serum resistance-associated SRA gene in African trypanosomes

Serum resistance-associated (SRA) protein, a protein unique for Trypanosoma brucei rhodesiense, is responsible for resistance of this parasite to the lysis by normal human serum (NHS) and is a vital molecular marker to distinguish this species from other African trypanosomes. We cloned and sequenced the SRA basic copy (SRAbc) gene from T. b. rhodesiense and related species and found that this gene is confined to the subgenus Trypanozoon. The average 82% identity among the sequenced SRAbc genes indicates that they may have a common origin and are highly conserved. Since SRAbc coexists in the T. b. rhodesiense genome with SRA, we propose that SRAbc might be the ‘donor VSG’, which after duplication became inserted into the expression site by recombination. Under natural selection, SRAbc could reform into SRA following mosaic formation.

Pathogenicity of bloodstream and cerebrospinal fluid forms of <i>Trypanosoma brucei rhodesiense</i> in Swiss White Mice

Trypanosoma brucei rhodesiense (T.b.r.), the causative agent of the East African form of human African trypanosomiasis (HAT), is capable of crossing the blood brain barrier and invade the central nervous system (CNS). However, it is not clear whether bloodstream forms (BSF) of T.b.rhodesiense differ in biological characteristics from the cerebrospinal fluid (CSF) forms. The present study was carried out to compare the pathogenicity of CSF and BSF of T.b. rhodesiense parasites in Swiss white mice following intraperitoneal inoculation with 106 trypanosomes. The parasites were tested for presence of the serum resistance associated (SRA) gene. Parasitaemia, body weight, packed cell volume (PCV) and survival of the mice was monitored daily until the experiment was terminated. Data was analyzed using general linear model. Both forms of parasite were positive for the SRA gene, and there was no significant difference in progression of parasitaemia, PCV values or survival of the mice. However, the weights of BSF infected mice initially dropped faster than those of CSF infected mice (P

Loop-Mediated Isothermal Amplification (LAMP) Method for Rapid Detection of Trypanosoma brucei rhodesiense

Loop-mediated isothermal amplification (LAMP) of DNA is a novel technique that rapidly amplifies target DNA under isothermal conditions. In the present study, a LAMP test was designed from the serum resistance-associated (SRA) gene of Trypanosoma brucei rhodesiense, the cause of the acute form of African sleeping sickness, and used to detect parasite DNA from processed and heat-treated infected blood samples. The SRA gene is specific to T. b. rhodesiense and has been shown to confer resistance to lysis by normal human serum. The assay was performed at 62°C for 1 h, using six primers that recognised eight targets. The template was varying concentrations of trypanosome DNA and supernatant from heat-treated infected blood samples. The resulting amplicons were detected using SYTO-9 fluorescence dye in a real-time thermocycler, visual observation after the addition of SYBR Green I, and gel electrophoresis. DNA amplification was detected within 35 min. The SRA LAMP test had an unequivocal detection limit of one pg of purified DNA (equivalent to 10 trypanosomes/ml) and 0.1 pg (1 trypanosome/ml) using heat-treated buffy coat, while the detection limit for conventional SRA PCR was ∼1,000 trypanosomes/ml. The expected LAMP amplicon was confirmed through restriction enzyme RsaI digestion, identical melt curves, and sequence analysis. The reproducibility of the SRA LAMP assay using water bath and heat-processed template, and the ease in results readout show great potential for the diagnosis of T. b. rhodesiense in endemic regions.

Characterisation of the <i>Trypanosoma brucei rhodesiense</i> isolates from Tanzania using serum resistance associated gene as molecular marker

Serum resistance associated (SRA) gene has been found to confer resistance to the innate trypanolytic factor (TLF) found in normal human serum; thus allowing Trypanosoma brucei brucei to survive exposure to normal human serum. This study was carried out to examine the presence of SRA gene and identify the origin of T. b. rhodesiense isolates from three districts in Tanzania, namely Kibondo, Kasulu and Urambo. Twenty-six T. b. rhodesiense isolates and two references T. b. rhodesiense isolates from Kenya were examined for SRA gene using simple Polymerase Chain Reaction technique. The gene was found to be present in all 26 T. b. rhodesiense isolates including the two references isolates from Kenya. The SRA gene was confirmed to be specific to T. b. rhodesiense since it could not be amplified from all other Trypanozoon including T. b. gambiense; and gave an amplified fragment of the expected size (3.9kb), confirming that all these isolates were T. b. rhodesiense of the northern variant. Although the geographic distributions of T. b. gambiense and T. b. rhodesiense are clearly localized to west/central Africa and eastern Africa, respectively, natural movement of people and recent influx of large number of refugees into Tanzania from the Democratic Republic of Congo, could have brought T. b. gambiense in western Tanzania. The overlap in distribution of both of these pathogenic sub-species could result in erroneous diagnoses since both trypanosome sub-species are morphologically identical, and currently serologic methods have low specificity. Both the susceptible and resistant T. b. rhodesiense isolates possessed the SRA gene suggesting that there is no correlation between drug resistance and presence of SRA gene. The use of SRA gene helps to confirm the identity and diversity of some of the isolates resistant to various drugs.

Detection of T.b. rhodesiense Trypanosomes in Humans and Domestic Animals in South East Uganda by Amplification of Serum Resistance‐Associated Gene

The human serum resistance-associated (SRA) gene was identified in 28 (80%) of the 35 T.b. rhodesiense trypanosomes from parasitologically confirmed sleeping sickness cases, using the primers designed by Radwanska and in 27 (77.1%) of the same 35 T.b. rhodesiense trypanosomes using the primers designed by Gibson. However, about 20% of the 35 T.b. rhodesiense trypanosomes could not be detected by SRA-polymerase chain reaction (PCR) even when an aliquot of the first PCR was used in the second PCR, indicating that the gene may be absent in those trypanosomes or the trypanosomes could be having another variant of SRA not detectable by these primers since three variants of SRA genes have so far been identified or the amount of trypanosomal DNA extracted from infected blood was too low to be detected. The trypanosome isolates that are SRA gene negative may indicate the presence of some T.b. rhodesiense trypanosomes with modified or lack SRA genes or simple loss of the SRA gene from the expression site in which it resides during antigenic variation. Analysis of trypanosomes derived from domestic animals showed that 79 (90.8%) of the 87 trypanosomes isolated from cattle were positive by Trypanosoma brucei (TBR)-PCR, indicating that they were Trypanozoon while 8 (9.2%) of the trypanosome isolates which were negative by TBR-PCR could be T. vivax, T. congolense, or T. theileri. When subjected to SRA-PCR, 10 (11.5%) of the 87 trypanosomes isolates derived from cattle were positive, indicating that there could be T.b. rhodesiense circulating in cattle, which is similar to the percentage of T.b. rhodesiense previously obtained in cattle in Serere, Soroti district.

In Vitro Generation of Human High-Density-Lipoprotein-Resistant Trypanosoma brucei brucei

The host range of African trypanosomes is influenced by innate protective molecules in the blood of primates. A subfraction of human high-density lipoprotein (HDL) containing apolipoprotein A-I, apolipoprotein L-I, and haptoglobin-related protein is toxic to Trypanosoma brucei brucei but not the human sleeping sickness parasite Trypanosoma brucei rhodesiense. It is thought that T. b. rhodesiense evolved from a T. b. brucei-like ancestor and expresses a defense protein that ablates the antitrypanosomal activity of human HDL. To directly investigate this possibility, we developed an in vitro selection to generate human HDL-resistant T. b. brucei. Here we show that conversion of T. b. brucei from human HDL sensitive to resistant correlates with changes in the expression of the variant surface glycoprotein (VSG) and abolished uptake of the cytotoxic human HDLs. Complete transcriptome analysis of the HDL-susceptible and -resistant trypanosomes confirmed that VSG switching had occurred but failed to reveal the expression of other genes specifically associated with human HDL resistance, including the serum resistance-associated gene (SRA) of T. b. rhodesiense. In addition, we found that while the original active expression site was still utilized, expression of three expression site-associated genes (ESAG) was altered in the HDL-resistant trypanosomes. These findings demonstrate that resistance to human HDLs can be acquired by T. b. brucei.

Detection of trypanosomes in small ruminants and pigs in western Kenya: important reservoirs in the epidemiology of sleeping sickness?

BackgroundTrypanosomosis is a major impediment to livestock farming in sub-Saharan Africa and limits the full potential of agricultural development in the 36 countries where it is endemic. In man, sleeping sickness is fatal if untreated and causes severe morbidity. This study was undertaken in western Kenya, an area that is endemic for both human and livestock trypanosomosis. While trypanosomosis in livestock is present at high levels of endemicity, sleeping sickness occurs at low levels over long periods, interspersed with epidemics, underscoring the complexity of the disease epidemiology. In this study, we sought to investigate the prevalence of trypanosomes in small ruminants and pigs, and the potential of these livestock as reservoirs of potentially human-infective trypanosomes. The study was undertaken in 5 villages, to address two key questions: i) are small ruminants and pigs important in the transmission dynamics of trypanosomosis? and ii), do they harbour potentially human infective trypanosomes? Answers to these questions are important in developing strategies for the control of both livestock and human trypanosomosis.ResultsEighty-six animals, representing 21.3% of the 402 sampled in the 5 villages, were detected as positive by PCR using a panel of primers that identify trypanosomes to the level of the species and sub-species. These were categorised as 23 (5.7%) infections of T. vivax, 22 (5.5%) of T. simiae, 21 (5.2%) of the T. congolense clade and 20 (5.0%) of T. brucei ssp. The sheep was more susceptible to trypanosome infection as compared to goats and pigs. The 20 T. brucei positive samples were evaluated by PCR for the presence of the Serum Resistance Associated (SRA) gene, which has been linked to human infectivity in T. b. rhodesiense. Three samples (one pig, one sheep and one goat) were found to have the SRA gene. These results suggest that sheep, goats and pigs, which are kept alongside cattle, may harbour human-infective trypanosomes.ConclusionWe conclude that all livestock kept in this T. b. rhodesiense endemic area acquire natural infections of trypanosomes, and are therefore important in the transmission cycle. Sheep, goats and pigs harbour trypanosomes that are potentially infective to man. Hence, the control of trypanosomosis in these livestock is essential to the success of any strategy to control the disease in man and livestock.

The Trypanosoma brucei reference strain TREU927/4 contains T. brucei rhodesiense-specific SRA sequences, but displays a distinct phenotype of relative resistance to human serum

The Trypanosoma brucei reference strain TREU927/4 exhibits some resistance to lysis by normal human serum (NHS), but this resistance is never complete even after selection. The genome of this strain contains a minimum of eight sequences related to the T. brucei rhodesiense-specific serum resistance-associated gene (SRA), which encodes a truncated variant surface glycoprotein (VSG) conferring full resistance to lysis by NHS. We selected two sequences showing the highest similarity to SRA and also preceded by a region (“cotransposed region”) present immediately upstream from SRA in the VSG expression site termed R-ES, where SRA is expressed in T. brucei rhodesiense. Whereas one of these sequences appears to be a pseudogene, the other, which is contained within a cluster of VSG basic copies (BCs), encodes a VSG truncated in the C-terminal domain. In the latter gene, an inserted region encoding surface-exposed loops similar to those of the BoTat 1.20 VSG interrupts the full SRA sequence. Therefore, this gene was termed SRA-BC, for the putative VSG basic copy from which SRA was derived. Neither this gene nor other SRA-like sequences appeared to be responsible for the relative resistance of TREU927/4 to NHS, since (i) transfection of SRA-BC in T. brucei brucei did not confer increased resistance; (ii) SRA transcripts could not be detected in this strain, even when focusing the search on the limited SRA sequence necessary to confer resistance and when using strain-specific SRA probes on RNA from cells selected in the presence of NHS.

Detection of Trypanosoma brucei rhodesiense in animals from sleeping sickness foci in East Africa using the serum resistance associated ( SRA) gene

The human serum resistance associated ( SRA) gene has been found exclusively in Trypanosoma brucei rhodesiense, allowing the unequivocal detection of this pathogen in reservoir hosts and the tsetse vector without recourse to laborious strain characterisation procedures. We investigated the presence of the SRA gene in 264 T. brucei ssp. isolates from humans, domestic animals and Glossina pallidipes from foci of human trypanosomiasis in Kenya and Uganda. The SRA gene was present in all isolates that were resistant to human serum, and absent from all serum sensitive isolates tested. Further, the gene was present in all isolates that had previously been shown to be identical to human infective trypanosomes by isoenzyme characterisation. The SRA gene was detected in isolates from cattle, sheep, pigs, dog, reedbuck, hyena and G. pallidipes from sleeping sickness foci, but was not found in Trypanosoma evansi or in Trypanosoma brucei gambiense isolates. The present study indicates that the SRA gene may be invaluable in detecting and differentiating T. brucei rhodesiense from other T. brucei ssp. in reservoir hosts and tsetse.

The Trypanosoma brucei reference strain TREU927/4 contains T. brucei rhodesiense-specific SRA sequences, but displays a distinct phenotype of relative resistance to human serum.

The Trypanosoma brucei reference strain TREU927/4 exhibits some resistance to lysis by normal human serum (NHS), but this resistance is never complete even after selection. The genome of this strain contains a minimum of eight sequences related to the T. brucei rhodesiense-specific serum resistance-associated gene (SRA), which encodes a truncated variant surface glycoprotein (VSG) conferring full resistance to lysis by NHS. We selected two sequences showing the highest similarity to SRA and also preceded by a region ("cotransposed region") present immediately upstream from SRA in the VSG expression site termed R-ES, where SRA is expressed in T. brucei rhodesiense. Whereas one of these sequences appears to be a pseudogene, the other, which is contained within a cluster of VSG basic copies (BCs), encodes a VSG truncated in the C-terminal domain. In the latter gene, an inserted region encoding surface-exposed loops similar to those of the BoTat 1.20 VSG interrupts the full SRA sequence. Therefore, this gene was termed SRA-BC, for the putative VSG basic copy from which SRA was derived. Neither this gene nor other SRA-like sequences appeared to be responsible for the relative resistance of TREU927/4 to NHS, since (i) transfection of SRA-BC in T. brucei brucei did not confer increased resistance; (ii) SRA transcripts could not be detected in this strain, even when focusing the search on the limited SRA sequence necessary to confer resistance and when using strain-specific SRA probes on RNA from cells selected in the presence of NHS.

Abstract

Identification of humaninfective trypanosomes in Ugandan cattle by serum resistance-associated gene, Welburn, S. C., Picozzi, K., Fevre, E. M., Coleman, P. G., Odiit, M., Carrington, M. & Maudlin, I. (2002)...

The human serum resistance associated gene is ubiquitous and conserved in Trypanosoma brucei rhodesiense throughout East Africa

The human serum resistance associated ( SRA) gene isolated from a Ugandan strain of Trypanosoma brucei rhodesiense has been shown to be capable by itself of conferring the trait of human infectivity on T.b. brucei by transfection. This gene has also been identified in several other isolates of T.b. rhodesiense, but not in the other human pathogenic trypanosome in Africa, T.b. gambiense, casting doubt on its ubiquity and function. Here, we show that this gene occurs in T.b. rhodesiense from sleeping sickness foci throughout East Africa (Ethiopia, Uganda, Kenya, Tanzania, Zambia, Botswana), but is not found in T.b. brucei isolates or any other trypanosomes of subgenus Trypanozoon. SRA genes from 10 T.b. rhodesiense isolates from five disease foci were compared and were 97.9–99.7% homologous, with three minor sequence variants. PCR amplification of this gene forms the basis of a new test to identify T.b. rhodesiense. This is the first molecular marker identified for T.b. rhodesiense, despite intensive efforts over the past 20 years. It will be invaluable for identification of animal reservoir hosts and detection of T.b. rhodesiense in its tsetse fly vector. Strain typing using minisatellite markers showed considerable genetic heterogeneity between T.b. rhodesiense isolates, despite the presence of the conserved SRA gene. These results are consistent with the hypothesis that new T.b. rhodesiense strains arise by genetic exchange among T. brucei ssp. spreading the SRA gene and thereby the trait for human serum resistance and human infectivity.

Identification of human-infective trypanosomes in animal reservoir of sleeping sickness in Uganda by means of serum-resistance-associated (SRA) gene

The expansion of sleeping sickness caused by Trypanosoma brucei rhodesiense beyond its traditional focus in southeast Uganda has been linked with large-scale livestock restocking. To assess the risk presented to the human population by domestic livestock, human-infective T b rhodesiense must be distinguished from non-human-infective T brucei brucei, since both parasites can be present in cattle. We investigated the use of a simple genetic marker to characterise parasites collected from cattle in villages within the new sleeping sickness focus in Soroti District, Uganda. 70 T brucei sl samples of known human infectivity status collected from human beings and cattle in Tororo District, Uganda, from 1989 to 1991 were screened for the presence of the human-serum-resistance-associated (SRA) gene by conventional PCR. In 2000-01, blood samples from 200 randomly selected cattle in six villages and two markets in Soroti District were screened for T brucei sl parasites by PCR; positive samples were screened for the presence of the SRA gene. The SRA gene was present in all 29 samples from patients with sleeping sickness in Tororo District. Of the 41 samples collected from cattle at the same time, the SRA gene was present in the eight samples that tested resistant to human serum in vitro, whereas it was absent from all 33 isolates that were sensitive to human serum in vitro. Of the 200 cattle sampled in Soroti District, we estimated that up to 18% (95% CI 12-23) were infected with T b rhodesiense. Detection of the SRA gene could provide the basis for a simple diagnostic test to enable targeted control of T b rhodesiense in the domestic livestock reservoir, thereby reducing the public-health burden of sleeping sickness in east Africa.