Human African trypanosomiasis (HAT), also known as Sleeping Sickness, is a fatal parasitic disease transmitted by the bite of the tsetse fly. It is distributed in pockets throughout sub-Saharan Africa, placing an estimated 60 million people at risk of infection . There are two forms of this deadly disease: in West Africa, infection with Trypanosoma brucei gambiense gives rise to a chronic disease, mainly affecting humans – Gambian sleeping sickness; in East Africa, infection with Trypanosoma brucei rhodesiense generates acute disease in humans, and also circulates in a relatively unaffected livestock reservoir.
Both forms are characterised by a two-stage disease pathogenesis. The first stage is characterised by an initial skin chancre at the site of the infective bite, swollen lymph glands, waves of fever, enlarged spleen and generalised skeletal muscle atrophy. Specifically with Gambian sleeping sickness the lymph glands of the posterior cervical triangle are enlarged – an important diagnostic feature known as Winterbottom’s sign. In the second stage, parasites enter and damage the central nervous system causing progressive neurological damage, which manifests in a number of ways, effecting mental, sensory and motor processes, as well as disturbances to the circadian rhythm (hence the name of the disease). The Rhodesian form progresses rapidly; more than 80% of deaths occur within six months of the onset of illness while, in contrast, the Gambian form may progress for years.
Active screening is necessary for any disease with an extended asymptomatic pre-clinical period and historically the strategy of active case-finding and treatment has been very successful in controlling Gambian sleeping sickness; huge declines in reported cases were seen between 1930 and 1960. However, in the immediate post-colonial era sleeping sickness control programmes lost their impetus and the gradual decline in incidence showed a dramatic reversal. Thankfully, since 1997, the number of reported cases has once again begun to fall . Active case-finding remains the mainstay of successful disease control and the screening techniques have developed to some extent. Nevertheless, under-reporting is a significant problem  and sleeping sickness has become a truly ‘neglected tropical disease’. Over time HAT has simply not attracted sufficient resources for innovative diagnostic and therapeutic strategies to be developed. This trend is changing and there is now an increasing recognition of the neglected tropical diseases, and real efforts to push diagnosis and treatment up the research agenda .
Sleeping sickness tends to persist in well-established epidemiological foci and while these foci may expand and contract in epidemic waves, new foci are rarely seen. However, in late 2007, polymerase chain reaction (PCR) analysis of human blood samples from a previously ‘silent’ region around Lake Albert in western Uganda generated positive signals for T. b. gambiense. The PCR assay for T. b. gambiense  detects and amplifies the single copy T. b. gambiense-specific glycoprotein gene, which is widely accepted as an appropriate ‘indicator’ gene for this parasite. These PCR findings stimulated the Ugandan Ministry of Health and the University of Edinburgh (UK) with support from World Health Organisation (WHO) to support further investigations. I had the privilege of observing the first active search for T. b. gambiense infections in the area surrounding Lake Albert in September 2008 as a representative of the postgraduate Global Health student body from the College of Medicine and Veterinary Medicine at the University of Edinburgh. The Ugandan Ministry of Health brought with them extensive experience of screening activities from the established Gambian sleeping sickness focus in north-western Uganda.
Several thousand people were screened at central sites in two districts of Uganda, Hoima and Bulisa. Finger prick blood samples were taken for serological testing using the Card Agglutination Test for Trypanosomiasis (CATT). The CATT test detects anti-trypanosomal antibodies in patient blood and was designed for use in field surveys; the test is available in kit form from WHO, and is the mainstay of active screening for T. b. gambiense. However, treatment which is dangerous, complicated, and expensive is only justified when the parasites are seen directly . All CATT whole blood positive individuals therefore provided a second blood sample for CATT on 1/4, 1/8 and 1/16 plasma dilutions and parasite detection by micro-haematocrit centrifugation. CATT whole-blood positive individuals were also examined for clinical signs by cervical lymph node palpitation. Micro-haematocrit centrifugation of blood in capillary tubes concentrates parasites in the white blood cell layer and parasites are then visible under a microscope . In addition small droplets of patient blood were placed on Whatman FTA cards (chemically treated filter paper matrices specially designed to hold DNA for long term storage) for PCR analysis in the UK.
Parasites were not found in the blood of any of the screened individuals by micro-haematocrit centrifugation, nor did any of the individuals examined show enlarged lymph nodes. However these findings disguised a much more complicated situation. Several individuals were serologically positive by the CATT test (down to 1/16 plasma dilution) and several of the blood samples were positive for T. brucei s.l. parasite DNA by PCR . Further investigation was necessary; was the micro-haematocrit centrifugation method simply not sensitive enough to detect low levels of parasites in these people or is the CATT test worryingly non-specific for detecting low levels of parasite?
In April 2009, I was again privileged to take part in the follow-up study. We sought out the CATT whole blood positive cases that had been seen six months earlier. This time the most sensitive parasitological detection method, the mini-anion exchange centrifugation technique (mAECT)  was also used, administered by an expert, Tanoé Miézan. mAECT uses anion chromatography to separate trypanosomes from the venous blood sample in which they are collected, before centrifugation to concentrate the parasites for viewing by microscopy. All parasitological detection was performed indoors, since trypanosome lysis is precipitated by exposure to direct sunlight. Again, no parasite positive cases were found yet again, we saw several CATT and PCR positive results, though these were not in direct agreement. The epidemiological picture became even more peculiar when these CATT and PCR results did not match those seen in the initial screening phase. In this instance cervical lymph node palpitations were not performed.
For me, these experiences highlight our lack of a definitive, field-friendly diagnostic screening tool for Gambian sleeping sickness. The sensitivity of CATT in north-west Uganda has been questioned for failing to detect parasite-positive cases . In contrast to serological techniques, molecular tools offer a direct method of parasite detection but PCR requires equipment, expertise and resources well beyond the scope of an average mobile screening team. Furthermore, PCR has not been properly evaluated against the established serological and direct parasite detection methods. We would expect PCR to be more sensitive and it is difficult to predict what our results mean for the detection of very early or possibly even non-virulent or self-limiting forms of the disease. In my opinion, molecular methods such as PCR need to be validated in a less complicated epidemiological setting. Though classical PCR is perhaps never going to be a field friendly screening tool, other molecular methods such as LAMP (loop-mediated isothermal amplification) may fulfil that role . At the very least, we need to be able to interpret the epidemiological significance of PCR-based research findings.
These results also raise the question of whether we truly understand T. b. gambiense infection? Although it is widely assumed to be pathogenic and ultimately fatal in all instances, the time course of infection is not well understood and a long asymptomatic chronic infection stage may exist before parasites become visible. We rarely know when an individual became infected and do not know how long it takes for individuals to show parasites in their blood. If there are instances of human trypanotolerance, and/or non pathogenic T. b. gambiense strains in circulation, then the current epidemiological paradigm would need to be reworked with major implications for long term planning of disease control programmes .
Finally I had a glimpse of the logistical difficulties faced by active screening teams; affected communities lived in remote villages, frequently only accessible by bicycle/footpaths. At the initial screening it is unlikely that all the individuals in an area were aware of our presence and were willing to travel to our screening post. At follow-up we collected people by four-wheel drive vehicle yet seeking out these people and persuading them to leave their daily tasks to come with us was not easy. Difficulties also arose as the team came together from different organisations with different experiences of T. b. gambiense in different sociological, cultural and environmental settings. It rapidly became apparent that we were working to different case definitions and that not all the necessary laboratory equipment had been sourced. I was continually impressed by the resourcefulness of the screening teams, exemplified when the seal broke on the centrifuge; it was quickly replaced by a bicycle inner tube!
Significant efforts are being made to improve the diagnosis of HAT; for example, the Foundation for Innovative New Diagnostics (FIND), is a public-private partnership established to find novel and improved solutions for the diagnosis of sleeping sickness and other diseases. FIND are pursuing improved fluorescent microscopy and LAMP for molecular diagnosis for sleeping sickness, as well as novel serological antibody and antigen detection tests, see www.finddiagnostics.org.
These experiences have highlighted the need for improving not only the tools we use, but also our fundamental understanding of sleeping sickness biology in order to properly interpret screening results. As diagnostic techniques become more sensitive we must ask ourselves what a positive signal means in terms of disease in the infected individual. In the absence of clinical signs, what response is appropriate in terms of follow-up treatment for that individual and for prevention of transmission to their neighbours. Only when a positive signal definitively equates to ‘infection’ should treatment with the currently available drugs ensue. For the PCR positive ‘cases’ detected in the present study, Ministry of Health, Uganda will continue to monitor their status.
Acknowledgements: Grateful thanks are extended to the Ugandan Ministry of Health, especially Dr Abbas Kakembo, Dr Charles Wamboga, Dr Dimi Okutoi, Julius Asingwire and Albino Louga. I am also extremely grateful to WHO consultant Dr Tanoé Miézan, my PhD supervisors at the University of Edinburgh Professor Sue Welburn and Dr Kim Picozzi and Professor Ian Maudlin for his comments on this manuscript. Finally, I would like to thank the local communities and government workers from Hoima and Bulisa, Uganda.
Sally Wastling is studying for a PhD in infectious diseases at Edinburgh University in the UK
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