Detection of Group 1 Trypanosoma brucei gambiense by loop-mediated isothermal amplification.

Trypanosoma brucei gambiense group 1 is the major causative agent of the Gambian human African trypanosomiasis (HAT). Accurate diagnosis of Gambian HAT is still challenged by lack of precise diagnostic methods, low and fluctuating parasitemia, and generally poor services in the areas of endemicity. In this study, we designed a rapid loop-mediated isothermal amplification (LAMP) test for T. b. gambiense based on the 3' end of the T. b. gambiense-specific glycoprotein (TgsGP) gene. The test is specific and amplifies DNA from T. b. gambiense isolates and clinical samples at 62°C within 40 min using a normal water bath. The analytical sensitivity of the TgsGP LAMP was equivalent to 10 trypanosomes/ml using purified DNA and ∼1 trypanosome/ml using supernatant prepared from boiled blood, while those of classical PCR tests ranged from 10 to 10(3) trypanosomes/ml. There was 100% agreement in the detection of the LAMP product by real-time gel electrophoresis and the DNA-intercalating dye SYBR green I. The LAMP amplicons were unequivocally confirmed through sequencing and analysis of melting curves. The assay was able to amplify parasite DNA from native cerebrospinal fluid (CSF) and double-centrifuged supernatant prepared from boiled buffy coat and bone marrow aspirate. The robustness, superior sensitivity, and ability to inspect results visually through color change indicate the potential of TgsGP LAMP as a future point-of-care test.

African trypanosomiasis: sensitive and rapid detection of the sub-genus Trypanozoon by loop-mediated isothermal amplification (LAMP) of parasite DNA.

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.

Structure of some East African Glossina fuscipes fuscipes populations.

Glossina fuscipes fuscipes Newstead 1910 (Diptera: Glossinidae) is the primary vector of human sleeping sickness in Kenya and Uganda. This is the first report on its population structure. A total of 688 nucleotides of mitochondrial ribosomal 16S2 and cytochrome oxidase I genes were sequenced. Twenty-one variants were scored in 79 flies from three geographically diverse natural populations. Four haplotypes were shared among populations, eight were private and nine were singletons. The mean haplotype and nucleotide diversities were 0.84 and 0.009, respectively. All populations were genetically differentiated and were at demographic equilibrium. In addition, a longstanding laboratory culture originating from the Central African Republic (CAR-lab) in 1986 (or before) was examined. Haplotype and nucleotide diversities in this culture were 0.95 and 0.012, respectively. None of its 27 haplotypes were shared with the East African populations. A first approximation of relative effective population sizes was Uganda > CAR-lab > Kenya. It was concluded that the structure of G. f. fuscipes populations in East Africa is localized.

First recorded outbreak of yellow fever in Kenya, 1992-1993. I. Epidemiologic investigations.

Outbreaks of yellow fever (YF) have never been recorded in Kenya. However, in September 1992, cases of hemorrhagic fever (HF) were reported in the Kerio Valley to the Kenya Ministry of Health. Early in 1993, the disease was confirmed as YF and a mass vaccination campaign was initiated. Cases of suspected YF were identified through medical record review and hospital-based disease surveillance by using a clinical case definition. Case-patients were confirmed serologically and virologically. We documented 55 persons with HF from three districts of the Rift Valley Province in the period of September 10, 1992 through March 11, 1993 (attack rate = 27.4/100,000 population). Twenty-six (47%) of the 55 persons had serologic evidence of recent YF infection, and three of these persons were also confirmed by YF virus isolation. No serum was available from the other 29 HF cases. In addition, YF virus was isolated from a person from the epidemic area who had a nonspecific febrile illness but did not meet the case definition. Five patients with confirmed cases of YF died, a case-fatality rate of 19%. Women with confirmed cases of YF were 10.9 times more likely to die than men (P = 0.010, by Fisher's exact test). Of the 26 patients with serologic or virologic evidence of YF, and for whom definite age was known, 21 (81%) were between 10 and 39 years of age, and 19 (73%) were males. All patients with confirmed YF infection lived in rural areas. There was only one instance of multiple cases within a single family, and this was associated with bush-clearing activity. This was the first documented outbreak of YF in Kenya, a classic example of a sylvatic transmission cycle. Surveillance in rural and urban areas outside the vaccination area should be intensified.

First recorded outbreak of yellow fever in Kenya, 1992-1993. II. Entomologic investigations.

The first recorded outbreak of yellow fever in Kenya occurred from mid-1992 through March 1993 in the south Kerio Valley, Rift Valley Province. We conducted entomologic studies in February-March 1993 to identify the likely vectors and determine the potential for transmission in the surrounding rural and urban areas. Mosquitoes were collected by landing capture and processed for virus isolation. Container surveys were conducted around human habitation. Transmission was mainly in woodland of varying density, at altitudes of 1,300-1,800 m. The abundance of Aedes africanus in this biotope, and two isolations of virus from pools of this species, suggest that it was the principal vector in the main period of the outbreak. A third isolate was made from a pool of Ae. keniensis, a little-known species that was collected in the same biotope. Other known yellow fever vectors that were collected in the arid parts of the valley may have been involved at an earlier stage of the epidemic. Vervet monkeys and baboons were present in the outbreak area. Peridomestic mosquito species were absent but abundant at urban sites outside the outbreak area. The entomologic and epidemiologic evidence indicate that this was a sylvatic outbreak in which human cases were directly linked to the epizootic and were independent of other human cases. The region of the Kerio Valley is probably subject to recurrent wandering epizootics of yellow fever, although previous episodes of scattered human infection have gone unrecorded. The risk that the disease could emerge as an urban problem in Kenya should not be ignored.

Comparative sensitivity of dot-ELISA, PCR and dissection method for the detection of trypanosome infections in tsetse flies (Diptera: glossinidae).

A visually read dot-enzyme linked immunosorbent assay (dot-ELISA) developed for the detection of trypanosomes in tsetse flies (Glossina spp.) was evaluated in the laboratory and under field conditions. In the evaluation, the fly dissection method was used as a standard technique and compared to the polymerase chain reaction (PCR). In laboratory studies, 133 and 126 tsetse flies were experimentally infected with different stocks of Trypanosoma brucei and T. congolense, respectively. Twenty-five days after infection, the flies were dissected and tested for the presence of trypanosomes using dot-ELISA and PCR. Dot-ELISA detected 98.4% of T. brucei and 94% of T. congolense infections in tsetse midguts, while PCR detected 97.6% of T. brucei and 96% of T. congolense tsetse midgut samples. For field evaluation of dot-ELISA, 700 tsetse flies were caught and screened for trypanosome infections by dissection. Seven of these (1%) had trypomastigotes in the midgut, 23 (3.3%) in the proboscis and none had trypanosomes in the salivary glands. All the flies with midgut infections also had trypanosomes in their proboscides. Five of the seven flies (71.4%) with midgut infections revealed by dissection, were also positive for T. congolense by the dot-ELISA and PCR techniques. Dot-ELISA detected T. congolense infections in an additional 86 (12.4%) of the 700 flies dissected. Of the 23 infections in the proboscis, 16 were T. vivax. Dot-ELISA detected 13 of the 16 (81%) while PCR detected 15 of 16 (94%) T. vivax infections. No T. brucei infection was detected by any of the methods in all the 700 tsetse flies examined. The results obtained from both the laboratory and field studies indicate that the dot-ELISA and PCR techniques are sensitive and species-specific in revealing trypanosome infections in tsetse flies. While dot-ELISA required a single test to detect T. congolense, several primer pairs were needed for PCR. The potential use of dot-ELISA as a tool for studying the epidemiology of trypanosomosis, while considering its field applicability and relatively lower cost is discussed.

Changes in classical pathway complement activity in dromedary camels experimentally infected with Trypanosoma evansi.

The complement system is known to have important effector functions in immune responses. However, its role in camel trypanosomosis has not been determined. The present study was undertaken to evaluate haemolytic complement activity in Trypanosoma evansi-infected and uninfected camels. Five dromedary camels were experimentally infected with T. evansi and classical pathway haemolytic complement activity was assayed. Parasitaemia and packed cell volume were also monitored. Following infection, classical pathway haemolytic complement showed a slight initial increase (7%) in all the camels. The amounts later dropped as the infection progressed and correlated negatively with parasitaemia. Haemolytic complement recovered following elimination of trypanosomes by treatment with melarsomine. Treatment of uninfected camels had no effect on complement. This study has demonstrated that complement concentration increases in the initial phase of infection followed by a drop as the infection progresses towards chronicity. In addition, the study has shown that activation of the classical complement pathway occurs in camels infected with T. evansi. Complement could therefore be involved in the in vivo control of parasitaemia in dromedary camels infected with T. evansi. Decreased complement levels in this species could lead to immunosuppression, widely reported in animal trypanosomosis.

Charting methods to monitor the operational performance of ELISA method for the detection of antibodies against trypanosomes.

Four indirect enzyme-linked immunosorbent assays (ELISAs) for the detection of antibody against trypanosomes using antigen-precoated plates (Trypanosoma congolense and T. vivax) were used in 15 veterinary diagnostic laboratories in Africa and Europe. The study provided data allowing an evaluation of charting methods with respect to the operational performance of each ELISA. Data from standardised internal quality control (IQC) samples were plotted on charts and used as the assay performance indicators with reference to expected upper and lower control limits. Based on unprocessed (optical density) and normalised absorbance values (calculated as a percentage positivity of a control), dispersion of values from the expected data range was estimated plotting the location and deviation of the values. In addition, assay precision was estimated plotting the distribution of coefficients of variation<10% of the IQCs. Binding ratios of controls were calculated to estimate the assay proficiency with respect to the accuracy of assessing that the IQC samples tested positive or negative in the test proper. The graphical analysis of dispersion of absorbance values in combination with assay precision and proficiency criteria was considered fully satisfactory to evaluate the operational performance of the ELISAs and provided useful decision criteria for plate acceptance and rejection. The establishment of standardised and transparent IQC data charting methods for the indirect ELISAs provided an increased measure of confidence to national laboratories with respect to their reports on disease occurrence. Moreover, the relative assay performances between all laboratories were examined using summary data charts with reference to the performance criteria described. The IQC data were also examined using modified Youden plot analysis demonstrating that indirect ELISA methods can be successfully applied at diagnostic laboratories in the tropics for monitoring trypanosomosis control programmes.

Loop-mediated isothermal amplification test for Trypanosoma vivax based on satellite repeat DNA.

Trypanosoma vivax is major cause of animal trypanosomiasis and responsible for enormous economic burden in Africa and South America animal industry. T. vivax infections mostly run low parasitaemia with no apparent clinical symptoms, making diagnosis a challenge. This work reports the design and evaluation of a loop-mediated isothermal amplification (LAMP) test for detecting T. vivax DNA based on the nuclear satellite repeat sequence. The assay is rapid with results obtained within 35 min. The analytical sensitivity is ∼ 1 trypanosome/ml while that of the classical PCR tests ranged from 10 to 10(3)trypanosomes/ml. The T. vivax LAMP test reported here is simple, robust and has future potential in diagnosis of animal trypanosomiasis in the field.

Patterns of genetic diversity and differentiation in the tsetse fly Glossina morsitans morsitans Westwood populations in East and southern Africa.

Genetic diversity and differentiation within and among nine G. morsitans morsitans populations from East and southern Africa was assessed by examining variation at seven microsatellite loci and a mitochondrial locus, cytochrome oxidase (COI). Mean COI diversity within populations was 0.63+/-0.33 and 0.81 taken over all populations. Diversities averaged over microsatellite loci were high (mean number of alleles/locus>or=7.4; mean HE>or=65%) in all populations. Diversities averaged across populations were greater in East Africa (mean number of alleles=22+/-2.6; mean he=0.773+/-0.033) than in southern Africa (mean number of alleles=18.7+/-4.0; mean he=0.713+/-0.072). Differentiation among all populations was highly significant (RST=0.25, FST=0.132). Nei's Gij statistics were 0.09 and 0.19 within regions for microsatellites and mitochondria, respectively; between regions, Gij was 0.14 for microsatellites and 0.23 for mitochondria. GST among populations was 0.23 for microsatellite loci and 0.40 for mitochondria. The F, G and R statistics indicate highly restricted gene flow among G. m. morsitans populations separated over geographic scales of 12-917 km.

New polymorphic microsatellites in Glossina pallidipes (Diptera: Glossinidae) and their cross-amplification in other tsetse fly taxa.

We report the development and characterization of three new microsatellite markers in the tsetse fly, Glossina pallidipes (Diptera: Glossinidae). Fifty-eight alleles were scored in 192 individuals representing six natural populations. Allelic diversity ranged from 9 to 28 alleles per locus (mean 19.3 +/- 5.5). Averaged across loci, observed heterozygosity was 0.581 +/- 0.209, and expected heterozygosity was 0.619 +/- 0.181. Cross-species amplifications of the G. pallidipes loci in other tsetse fly taxa are reported.

Microgeographical breeding structure of the tsetse fly, Glossina pallidipes in south-western Kenya.

The origins of extant Glossina pallidipes Austen (Diptera: Glossinidae) populations in the ecologically well-studied Lambwe and Nguruman valleys in Kenya are controversial because populations have recovered after seemingly effective attempts to achieve high levels of control. The microgeographical breeding structure of the tsetse fly, G. pallidipes, was investigated by analysing spatial and temporal variation at eight microsatellite loci to test hypotheses about endemism and immigration. Samples were obtained at seasonal intervals from trap sites separated by 200 m to 14 km and arranged into blocks. G. pallidipes populations nearest to Lambwe and Nguruman also were sampled. Spatial analysis indicated that genetic differentiation by genetic drift was much less among trapping sites within Lambwe and Nguruman (F(ST) < or = 0.049) than between them (F(ST) = 0.232). F(ST) between Serengeti and Nguruman was 0.16 and F(ST) between Kodera Forest and Lambwe was 0.15. The genetic variance in G. pallidipes explained by dry and wet seasons (0.33%) was about one-fifth the variance among collection dates (1.6%), thereby indicating reasonable temporal stability of genetic variation. Gene frequencies in Kodera and Serengeti differed greatly from Lambwe and Nguruman, thereby falsifying the hypothesis that Lambwe and Nguruman were repopulated by immigrants. Harmonic mean effective (= breeding) population sizes were 180 in Lambwe and 551 in Nguruman. The genetic data suggest that G. pallidipes in Lambwe and Nguruman have been endemic for long intervals.

Glossina swynnertoni (Diptera: Glossinidae): effective population size and breeding structure estimated by mitochondrial diversity.

Nucleotide diversity was examined at mitochondrial COI and r16S2 loci in eight Glossina swynnertoni Austen collections from northern Tanzania and from a culture maintained by the International Atomic Energy Agency. Eighteen composite haplotypes were observed among 149 flies, two of which were common to all samples and 10 were private. Mean haplotype diversity was 0.59 and nucleotide diversity was 0.0013. There were excess singular haplotypes and mutation-drift disequilibrium suggesting that populations had experienced an earlier bottleneck and subsequent expansion. Factorial correspondence analysis showed that haplotype frequencies varied much more temporally (G ST=0.18) than spatially (G ST=0.04). The estimate of effective population size N e in Tarangire was a harmonic mean approximately 50 reproductive flies averaged over approximately 47 generations. The mean rate of gene flow was estimated to be approximately 5+/-1 reproducing females per generation but inflated because of mutation-drift disequilibrium arising from likely earlier bottlenecks.

Macrogeographic population structure of the tsetse fly, Glossina pallidipes (Diptera: Glossinidae).

Tsetse flies are confined to sub-Saharan Africa where they occupy discontinuous habitats. In anticipation of area-wide control programmes, estimates of gene flow among tsetse populations are necessary. Genetic diversities were partitioned at eight microsatellite loci and five mitochondrial loci in 21 Glossina pallidipes Austin populations. At microsatellite loci, Nei's unbiased gene diversity averaged over loci was 0.659 and the total number of alleles was 214, only four of which were shared among all populations. The mean number of alleles per locus was 26.8. Random mating was observed within but not among populations (fixation index FST=0.18) and 81% of the genetic variance was within populations. Thirty-nine mitochondrial variants were detected. Mitochondrial diversities in populations varied from 0 to 0.85 and averaged 0.42, and FST=0.51. High levels of genetic differentiation were characteristic, extending even to subpopulations separated by tens and hundreds of kilometres, and indicating low rates of gene flow.

Characterization of microsatellite markers in the tsetse fly, Glossina pallidipes (Diptera: Glossinidae).

Glossina pallidipes is a vector of African trypanosomiasis. Here we characterize eight new polymorphic microsatellite loci in 288 G. pallidipes sampled from 12 Kenya populations. The number of alleles per locus ranged from four to 36 with a mean of 20.5 +/- 10.1. Expected single locus heterozygosities varied from 0.044 to 0.829. Heterozygosity averaged 0.616 +/- 0.246. No linkage disequilibrium was found. We also report results in eight other tsetse species estimated by using the primers developed in G. pallidipes. The primers worked best in G. swynnertoni and G. austeni and worst in G. m. morsitans and G. m. submorsitans.