Kim Picozzi

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

BACKGROUND 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. METHODS 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. FINDINGS 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. INTERPRETATION 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.

Sleeping sickness in Uganda: a thin line between two fatal diseases

Abstract Objective To determine, through the use of molecular diagnostic tools, whether the two species of parasite that cause human African trypanosomiasis have become sympatric. Design Blood sampling of all available patients between June 2001 and June 2005 in central Uganda and between July and September 2003 in northwest Uganda and analysis of subcounty sleeping sickness records in Uganda between 1985 and 2005. Setting Sleeping sickness treatment centres in central and northwest Uganda and in south Sudan. Participants Patients presenting at the treatment centres and diagnosed as having sleeping sickness. Main outcome measure Classification of parasites from patients from each disease focus as either Trypanosoma brucei rhodesiense (acute form) or T b gambiense (chronic form). Results Blood from 231 patients with sleeping sickness in central Uganda and from 91 patients with sleeping sickness in northwest Uganda and south Sudan were screened for T b rhodesiense (detection of SRA gene) and T b gambiense (detection of TgsGP gene). All samples from central Uganda were classified as T b rhodesiense, and all samples from northwest Uganda and south Sudan were identified as T b gambiense. Conclusions The two focuses of human African trypanosomiasis remain discrete, but the area of Uganda affected by the acute form of human sleeping sickness has increased 2.5-fold since 1985, spreading to three new districts within the past five years through movement of infected livestock. Without preventive action targeted at the livestock reservoir of this zoonotic disease, it is likely that the two disease focuses will converge. This will have a major impact on diagnosis and treatment of this neglected disease. Real time monitoring is recommended, using molecular diagnostic tools (at a regional surveillance centre, for example) targeted at both livestock and human patients.

A burgeoning epidemic of sleeping sickness in Uganda

The epidemic of Trypanosoma brucei rhodesiense sleeping sickness in eastern Uganda, which began in 1998 as a result of movements of the livestock reservoir of the parasite, has continued to spread. An additional 133 000 people have been put at risk of infection in Kaberamaido, another newly affected district. The few resources committed to control interventions in Soroti district have failed to contain the epidemic. The high prevalence of the parasite in cattle presents a significant risk for transmission to human beings and further spread of this neglected zoonotic disease. Targeted interventions are urgently needed to control epidemics and reduce the high mortality resulting from sleeping sickness.

Human african trypanosomiasis : Epidemiology and control

Human African trypanosomiasis (HAT), or sleeping sickness, describes not one but two discrete diseases: that caused by Trypanosoma brucei rhodesiense and that caused by T. b. gambiense. The Gambian form is currently a major public health problem over vast areas of central and western Africa, while the zoonotic, Rhodesian form continues to present a serious health risk in eastern and southern Africa. The two parasites cause distinct clinical manifestations, and there are significant differences in the epidemiology of the diseases caused. We discuss the differences between the diseases caused by the two parasites, with an emphasis on disease burden, reservoir hosts, transmission, diagnosis, treatment and control. We analyse how these differences impacted on historical disease control trends and how they can inform contemporary treatment and control options. We consider the optimal ways in which to devise HAT control policies in light of the differing biology and epidemiology of the parasites, and emphasise, in particular, the wider aspects of control policy, outlining the responsibilities of individuals, governments and international organisations in control programmes.

LAMP for human African trypanosomiasis: a comparative study of detection formats.

Loop-mediated isothermal amplification (LAMP) is at the forefront of the search for innovative diagnostics for human African trypanosomiasis (HAT). Several simple endpoint detection methods have been developed for LAMP and here we compare four of these: (i) visualization of turbidity; (ii) addition of hydroxynaphthol blue before incubation; (iii) addition of calcein with MnCl2 before incubation and (iv) addition of Quant-iT PicoGreen after incubation. These four methods were applied to four LAMP assays for the detection of human African trypanosomiasis, including two Trypanozoon specific and two Trypanosoma brucei rhodesiense specific reactions using DNA extracted from cryo-preserved procyclic form T. b. rhodesiense. A multi-observer study was performed to assess inter-observer reliability of two of these methods: hydroxynapthol blue and calcein with MnCl2, using DNA prepared from blood samples stored on Whatman FTA cards. Results showed that hydroxynaphthol blue was the best of the compared methods for easy, inexpensive, accurate and reliable interpretation of LAMP assays for HAT. Hydroxynapthol blue generates a violet to sky blue colour change that was easy to see and was consistently interpreted by independent observers. Visible turbidity detection is not possible for all currently available HAT LAMP reactions; Quant-iT PicoGreen is expensive and addition of calcein with MnCl2 adversely affects reaction sensitivity and was unpopular with several observers.

The diagnosis of trypanosome infections: applications of novel technology for reducing disease risk

Reliable DNA based methodologies to determine prevalence of trypanosome species in domestic livestock have been available for over 10 years. Despite this, they are rarely used to generate baseline data for control operations for these diseases in the field. Rather, such operations tend to rely on data which can be generated using low technology methods such as direct observation of parasites by light microscopy. Here we show the pitfalls of relying on such low tech methodology which, although simple in its application, can provide inaccurate and inadequate data on which to base control methodologies. Our analysis of 61 cattle selected for trypanosome carrier status by either microscopy, low PCV or poor condition score, showed that 90% were infected with trypanosomes while 84% of the total were infected with T. brucei. Diagnosis by PCR on buffy coat preparations on Whatman® FTA® matrices was the most sensitive methodology relative to the gold standard, whereas microscopy was the least sensitive. (African Journal of Biotechnology: 2002 1(2): 39-45)

Characterisation of the wildlife reservoir community for human and animal trypanosomiasis in the Luangwa Valley, Zambia.

Background Animal and human trypanosomiasis are constraints to both animal and human health in Sub-Saharan Africa, but there is little recent evidence as to how these parasites circulate in wild hosts in natural ecosystems. The Luangwa Valley in Zambia supports high densities of tsetse flies (Glossina species) and is recognised as an historical sleeping sickness focus. The objective of this study was to characterise the nature of the reservoir community for trypanosomiasis in the absence of influence from domesticated hosts. Methodology/Principal Findings A cross-sectional survey of trypanosome prevalence in wildlife hosts was conducted in the Luangwa Valley from 2005 to 2007. Samples were collected from 418 animals and were examined for the presence of Trypanosoma brucei s.l., T. b. rhodesiense, Trypanosoma congolense and Trypanosoma vivax using molecular diagnostic techniques. The overall prevalence of infection in all species was 13.9% (95% confidence interval [CI]: 10.71–17.57%). Infection was significantly more likely to be detected in waterbuck (Kobus ellipsiprymnus) (Odds ratio [OR] = 10.5, 95% CI: 2.36–46.71), lion (Panthera leo) (OR = 5.3, 95% CI: 1.40–19.69), greater kudu (Tragelaphus strepsiceros) (OR = 4.7, 95% CI: 1.41–15.41) and bushbuck (Tragelaphus scriptus) (OR = 4.5, 95% CI: 1.51–13.56). Bushbucks are important hosts for T. brucei s.l. while the Bovidae appear the most important for T. congolense. The epidemiology of T. vivax was less clear, but parasites were detected most frequently in waterbuck. Human infective T. b. rhodesiense were identified for the first time in African buffalo (Syncerus caffer) and T. brucei s.l. in leopard (Panthera pardus). Variation in infection rates was demonstrated at species level rather than at family or sub-family level. A number of significant risk factors interact to influence infection rates in wildlife including taxonomy, habitat and blood meal preference. Conclusion and Significance Trypanosoma parasites circulate within a wide and diverse host community in this bio-diverse ecosystem. Consistent land use patterns over the last century have resulted in epidemiological stability, but this may be threatened by the recent influx of people and domesticated livestock into the mid-Luangwa Valley.

The best practice for preparation of samples from FTA®cards for diagnosis of blood borne infections using African trypanosomes as a model system

BackgroundDiagnosis of blood borne infectious diseases relies primarily on the detection of the causative agent in the blood sample. Molecular techniques offer sensitive and specific tools for this although considerable difficulties exist when using these approaches in the field environment. In large scale epidemiological studies, FTA®cards are becoming increasingly popular for the rapid collection and archiving of a large number of samples. However, there are some difficulties in the downstream processing of these cards which is essential for the accurate diagnosis of infection. Here we describe recommendations for the best practice approach for sample processing from FTA®cards for the molecular diagnosis of trypanosomiasis using PCR.ResultsA comparison of five techniques was made. Detection from directly applied whole blood was less sensitive (35.6%) than whole blood which was subsequently eluted from the cards using Chelex®100 (56.4%). Better apparent sensitivity was achieved when blood was lysed prior to application on the FTA cards (73.3%) although this was not significant. This did not improve with subsequent elution using Chelex®100 (73.3%) and was not significantly different from direct DNA extraction from blood in the field (68.3%).ConclusionsBased on these results, the degree of effort required for each of these techniques and the difficulty of DNA extraction under field conditions, we recommend that blood is transferred onto FTA cards whole followed by elution in Chelex®100 as the best approach.

Spatial Predictions of Rhodesian Human African Trypanosomiasis (Sleeping Sickness) Prevalence in Kaberamaido and Dokolo, Two Newly Affected Districts of Uganda

The continued northwards spread of Rhodesian sleeping sickness or Human African Trypanosomiasis (HAT) within Uganda is raising concerns of overlap with the Gambian form of the disease. Disease convergence would result in compromised diagnosis and treatment for HAT. Spatial determinants for HAT are poorly understood across small areas. This study examines the relationships between Rhodesian HAT and several environmental, climatic and social factors in two newly affected districts, Kaberamaido and Dokolo. A one-step logistic regression analysis of HAT prevalence and a two-step logistic regression method permitted separate analysis of both HAT occurrence and HAT prevalence. Both the occurrence and prevalence of HAT were negatively correlated with distance to the closest livestock market in all models. The significance of distance to the closest livestock market strongly indicates that HAT may have been introduced to this previously unaffected area via the movement of infected, untreated livestock from endemic areas. This illustrates the importance of the animal reservoir in disease transmission, and highlights the need for trypanosomiasis control in livestock and the stringent implementation of regulations requiring the treatment of cattle prior to sale at livestock markets to prevent any further spread of Rhodesian HAT within Uganda.

Constraints to estimating the prevalence of trypanosome infections in East African zebu cattle

BackgroundIn East Africa, animal trypanosomiasis is caused by many tsetse transmitted protozoan parasites including Trypanosoma vivax, T. congolense and subspecies of T. brucei s.l. (T. b. brucei and zoonotic human infective T. b. rhodesiense) that may co-circulate in domestic and wild animals. Accurate species-specific prevalence measurements of these parasites in animal populations are complicated by mixed infections of trypanosomes within individual hosts, low parasite densities and difficulties in conducting field studies. Many Polymerase Chain Reaction (PCR) based diagnostic tools are available to characterise and quantify infection in animals. These are important for assessing the contribution of infections in animal reservoirs and the risk posed to humans from zoonotic trypanosome species. New matrices for DNA capture have simplified large scale field PCR analyses but few studies have examined the impact of these techniques on prevalence estimations.ResultsThe Whatman FTA matrix has been evaluated using a random sample of 35 village zebu cattle from a population naturally exposed to trypanosome infection. Using a generic trypanosome-specific PCR, prevalence was systematically evaluated. Multiple PCR samples taken from single FTA cards demonstrated that a single punch from an FTA card is not sufficient to confirm the infectivity status of an individual animal as parasite DNA is unevenly distributed across the card. At low parasite densities in the host, this stochastic sampling effect results in underestimation of prevalence based on single punch PCR testing. Repeated testing increased the estimated prevalence of all Trypanosoma spp. from 9.7% to 86%. Using repeat testing, a very high prevalence of pathogenic trypanosomes was detected in these local village cattle: T. brucei (34.3%), T. congolense (42.9%) and T. vivax (22.9%).ConclusionsThese results show that, despite the convenience of Whatman FTA cards and specific PCR based detection tools, the chronically low parasitaemias in indigenous African zebu cattle make it difficult to establish true prevalence. Although this study specifically applies to FTA cards, a similar effect would be experienced with other approaches using blood samples containing low parasite densities. For example, using blood film microscopy or PCR detection from liquid samples where the probability of detecting a parasite or DNA molecule, in the required number of fields of view or PCR reaction, is less than one.