Clinical Presentation of T.b. rhodesiense Sleeping Sickness in Second Stage Patients from Tanzania and Uganda

Background A wide spectrum of disease severity has been described for Human African Trypanosomiasis (HAT) due to Trypanosoma brucei rhodesiense (T.b. rhodesiense), ranging from chronic disease patterns in southern countries of East Africa to an increase in virulence towards the north. However, only limited data on the clinical presentation of T.b. rhodesiense HAT is available. From 2006-2009 we conducted the first clinical trial program (Impamel III) in T.b. rhodesiense endemic areas of Tanzania and Uganda in accordance with international standards (ICH-GCP). The primary and secondary outcome measures were safety and efficacy of an abridged melarsoprol schedule for treatment of second stage disease. Based on diagnostic findings and clinical examinations at baseline we describe the clinical presentation of T.b. rhodesiense HAT in second stage patients from two distinct geographical settings in East Africa. Methodology/Principal Findings: 138 second stage patients from Tanzania and Uganda were enrolled. Blood samples were collected for diagnosis and molecular identification of the infective trypanosomes, and T.b. rhodesiense infection was confirmed in all trial subjects. Significant differences in diagnostic parameters and clinical signs and symptoms were observed: the median white blood cell (WBC) count in the cerebrospinal fluid (CSF) was significantly higher in Tanzania (134cells/mm3) than in Uganda (20cells/mm3; p<0.0001). Unspecific signs of infection were more commonly seen in Uganda, whereas neurological signs and symptoms specific for HAT dominated the clinical presentation of the disease in Tanzania. Co-infections with malaria and HIV did not influence the clinical presentation nor treatment outcomes in the Tanzanian study population. Conclusions/Significance We describe a different clinical presentation of second stage T.b. rhodesiense HAT in two distinct geographical settings in East Africa. In the ongoing absence of sensitive diagnostic tools and safe drugs to diagnose and treat second stage T.b. rhodesiense HAT an early identification of the disease is essential. A detailed understanding of the clinical presentation of T.b. rhodesiense HAT among health personnel and affected communities is vital, and awareness of regional characteristics, as well as implications of co-infections, can support decision making and differential diagnosis.


Introduction
Human African Trypanosomiasis (HAT), also known as sleeping sickness, is caused by the protozoan parasites T.b. gambiense (West and Central Africa) and T.b. rhodesiense (East and South Africa). The disease is transmitted by tsetse flies (Glossina ssp.) predominantly in the rural areas of most of sub Saharan Africa. 60 Million people live at risk of infection, but less than 10% are under adequate surveillance [1], reflecting its neglected status. Sleeping sickness caused by either subspecies presents in two disease stages defined as the first, or haemo-lymphatic stage and the second, meningoencephalitic stage. Diagnosis of HAT is made in blood, lymph and the cerebrospinal fluid (CSF). The second stage of the disease is indicated by the presence of trypanosomes and/or an elevated white blood cell (WBC) count ($5WBC/mm 3 ) in the CSF. The disease stage and the causative species of infection direct the choice of treatment. T.b. gambiense infections are treated with pentamidine in the first stage and eflornithine, a combination of eflornithine and nifurtimox, or melarsoprol in the second stage [2][3][4]. T.b. rhodesiense first and second stage infections are treated with suramin and melarsoprol respectively [2]. In the field, the trypanosome subspecies is entirely determined by the geographical location of the patient as the distinction of T.b. gambiense and T.b. rhodesiense is only possible in well equipped laboratories through PCR analysis. The detection of the human serum resistance-associated (SRA) gene unequivocally identifies T.b. rhodesiense trypanosomes [5,6]. In Uganda, the only country where both forms of the disease are present, a potential geographical overlap of the two endemic areas has become likely [7]. This would hamper determination of infective trypanosomes under field conditions and therefore also the identification of the correct treatment.
For first stage infections there are no specific clinical signs and symptoms in both forms of the disease; fever, headache and loss of appetite are common. In T.b. rhodesiense the presence of a chancre at the site of the infective bite may be indicative for a trypanosome infection [8]. Second stage infections show disease-characteristic neuro-psychiatric signs and symptoms: severe endocrinological and mental disturbances and severe motor problems are the main signs [9]. While often considered together, Gambiense and Rhodesiense HAT are clinically and epidemiologically different diseases [10]. T.b. gambiense HAT is a chronic disease, whereas T.b. rhodesiense is characterized by an acute disease progression. If left untreated, both forms of HAT are fatal. The mean time to reach the second stage has been estimated at over one year for T.b. gambiense [11] but only 3 weeks for T.b. rhodesiense HAT [12]. Correspondingly, average times from infection to death are almost 3 years and 6 to 12 months, respectively [11,12].
A diversity of forms of clinical progression from asymptomatic to acute have been reported for T.b. gambiense infections [13][14][15]. This seems to be even more pronounced for T.b. rhodesiense infections; a wide spectrum of disease severity ranging from a chronic disease pattern in southern countries of East Africa with existing reports of asymptomatic carriers [16] to an increase in virulence towards the north had been described [17]. Even though those differences were already described more than 60 years ago [18] the first comparative study was carried out in 2004: on the basis of the SRA gene polymorphism, trypanosomes isolates from Uganda (acute profile) and Malawi (chronic profile) confirmed to be of different genotypes. However, clinical characteristics of the study groups were limited the presence of a chancre and the self-reported duration of illness [19]. Another hypothesis postulates that the differences in disease severity could be attributed to differences in genetic resistance to trypanosomiasis among host populations [18].
From the estimated 509000 to 70900 cases per year [20], over 97% are T.b. gambiense cases and only a few thousand are due to T.b. rhodesiense [1]. Therefore, most literature concentrates on T.b. gambiense HAT. Its clinical picture and related cardiac and endocrinological disorders have been extensively described [21][22][23][24][25][26][27][28]. On the other hand, literature on the clinical aspects of T.b. rhodesiense HAT is scarce. We identified four studies (see table 1) describing its clinical presentation. Only one study in 60 patients infected with T.b. rhodesiense was designed prospectively and used a standardized questionnaire [29].
In this paper we describe the clinical presentation of second stage T.b. rhodesiense HAT in 138 patients from two distinct geographical settings in East Africa. We compare our findings to the existing literature and discuss factors that could explain the differences observed.

Study sites
The Kaliua Health Centre (KHC), a 50-bed missionary hospital in Tanzania (Urambo District) and the Lwala Hospital, a designated 100-bed district hospital in Uganda (Kaberamaido District) participated in the IMPAMEL III program (improved application of melarsoprol).

Study design and data collection
A proof-of-concept trial (n = 60) followed by a utilization study (n = 78) to assess the safety and efficacy of the abridged, 10-day melarsoprol schedule for the treatment of second stage HAT [30,31] in T.b. rhodesiense patients.
Eligible for enrolment were second stage patients with a minimum age of 6 years and confirmed second stage HAT. Patients with first stage infections, pregnant women and moribund or unconscious patients were excluded. Patients were passively enrolled at the study sites.
Diagnosis of HAT was made in blood and in CSF. Blood was examined using microscopy and/or the haematocrit centrifugation technique [32]. If trypanosomes were present, a lumbar puncture was performed for disease staging. Analysis of the CSF was done by direct microscopy and/or single modified centrifugation technique and white blood cell (WBC) count using counting chambers. Second stage infections were confirmed by the presence of trypanosomes and/or $5 WBC/mm 3 in the CSF. The standard assessment of co-infections included malaria, filariasis and voluntary testing for HIV/AIDS.
The local Principal Investigators filled individual case report forms (CRFs). Data used for describing the clinical presentation of the disease were patient demographics, diagnostic findings, self reported duration of illness and clinical signs and symptoms on admission graded by scale of severity (grade 0, 1, 2).

Ethics statement
Each participant gave written informed consent. For the participation of children and adolescents (below 18 years) the parents, the legal representative or the guardian gave written informed consent. Ethical clearances were obtained from the Ethics Committees in Tanzania (National Institute for Medical Research), Uganda (Ministry of Health) and Switzerland (Ethics Committee of both cantons of Basel). Before first patient enrolment, the IMPAMEL III program was registered in the database of Current Controlled Trials (ISRCTN40537886).

Data management and statistical analysis
All data were double entered and verified using Epi Data 3.1 software (www.epidata.dk) and analysis was accomplished with the statistical software package STATA Version IC10.0 (STATA, StataCorp, USA). The statistical analysis was performed compar-

Author Summary
Sleeping sickness, or Human African Trypanosomiasis (HAT), caused by Trypanosoma brucei rhodesiense is one of the most neglected tropical diseases. It affects mainly rural, poor East African populations and has very high socio-economic impacts. T.b. rhodesiense HAT is an acute disease; patients quickly progress from the first stage, where trypanosomes are detectable in blood and lymph, to the second stage, where parasites penetrate the central nervous system. If left untreated, T.b. rhodesiense HAT is fatal. Disease control is hampered by the absence of sensitive diagnostic tools and safe drugs. Second stage patients can only be treated with melarsoprol, a highly toxic, arsenical drug. It is more difficult to treat patients successfully at advanced stages of the disease, and late onset of treatment should be avoided. Yet, most patients are treated for other conditions prior to HAT diagnosis. Therefore, it is important that health personnel in T.b. rhodesiense endemic regions have a detailed understanding of the clinical presentation of the disease and consider regional characteristics of T.b. rhodesiense HAT for decision making and differential diagnosis.

Results
The use of the abridged 10-day melarsoprol schedule for the treatment of second stage T.b. rhodesiense HAT was highly satisfactory (detailed safety and efficacy data to be published separately). In this paper we describe the clinical presentation of the disease in 138 second stage patients from Tanzania and Uganda. The majority of patients were passively detected. Nine (9) patients from Uganda (13%) were actively identified during a survey of the National Agricultural Research Organisation (NARO) in the HAT endemic region of the country. There was no significant difference between actively and passively recruited patients for the median WBC count in the CSF (actively detected: median WBC = 27, IQR = 24; passively detected: median WBC = 19, IQR = 43, p = 0.067) and the median self-reported duration of illness (actively detected: median = 3 months, IQR = 2, passively detected: median = 2 months, IQR = 4, p = 0.141). 14 patients (11 in Uganda and 3 in Tanzania) could not be examined per protocol as they died or were in a comatose state upon arrival at the study sites which led to an exclusion of those patients from the IMPAMEL III trials.
By molecular analysis of blood samples, the presence of the SRA gene [33] was demonstrated and confirmed T.b. rhodesiense infection in all trial subjects [34].
Data on the demographic and diagnostic baseline characteristics of the study population are shown in table 2. The proportion of male (57.2%) and female (42.8%) patients was comparable. 18.8% (26/138) trial participants were younger than 16 years whereof 88.5% (23/26) were enrolled in Uganda. There were no countyspecific differences for the presence of trypanosomes in blood and CSF: 99% (68/69) of patients from Tanzania and 91% (63/69) from Uganda had trypanosomes in blood (p = 0.0524) and 70% (55/69) and 86% (59/69) respectively had trypanosomes in the CSF (p = 0.3690). However, there was a significant difference for the median WBC count in the CSF in Tanzania and Uganda (134 vs. 20 WBC/mm 3 , p,0.0001). Also, a body mass index (BMI) below 16.5 was more frequent in patients from Uganda (p,0.0001).
Clinical signs and symptoms reported at baseline and the level of significance (95%) are summarized in table 3. Headache, fever, general body pain and joint pains were common in both study populations. Clinical suspicion for cardiac insufficiency was found in both countries: 5.1% (7/138) of the patients had indication for left heart insufficiency (combination of cough and dyspnoe) and 5.8% (8/138) for right heart insufficiency (combination of oedema and hepatomegaly). Patients in Uganda had a more unspecific presentation of the disease whereas specific signs and symptoms for second stage HAT, namely sleeping disorders and aggressiveness were more common in patients from Tanzania. To look at changes of diagnostic markers and clinical signs and symptoms over time we compared them in patients grouped by self-reported duration of illness (see figure 1). In Tanzania and Uganda 21.7% (15/69) and 36.2% (25/69) respectively were diagnosed with HAT having signs and symptoms for one month or less. 47.8% (33/69) of patients from Tanzania and 31.9% (22/69) of patients from Uganda were diagnosed having signs and symptoms of the disease between 1 and 3 months. Respective percentages for diagnosis of HAT after feeling ill for more than 3 months were 30.4% (21/69) in Tanzania and 31.9% (22/69) in Uganda. In both countries, the presence of trypanosomes in blood and/or CSF and the WBC count in the CSF did not significantly change over time. Also, there was no change over time for most of the clinical signs and symptoms. However, we observed that tremor (p = 0.01), walking difficulties (p = 0.040), sleeping disorders at night (p = 0.029), disturbed appetite (p = 0.044) and aggressiveness (p,0.001) aggravated over time in all patients.
Per protocol, standard assessment of co-infections at baseline included malaria and filariasis. 79.7% (55/69) of the patients from Tanzania and 2.9% (2/69) from Uganda were malaria positive on admission. None were found positive for filariasis. The HIV status was determined on voluntary basis. In Tanzania, 94.2% (65/69) of the patients tested their status and 24.6% (16/65) were found positive. In Uganda, 31.9% (22/69) tested their status and 9.1% (2/22) were found positive. We used the data from Tanzania to study implications of malaria and HIV co-infections on the clinical presentation and treatment outcomes of T.b. rhodesiense HAT. No significant difference either in the clinical appearance or in treatment outcomes for those patients was found. Details are shown in table 4 and 5.

Discussion
Based on data from the IMPAMEL III trials we describe the clinical presentation of second stage T.b. rhodesiense HAT in Tanzania and Uganda and confirm a wide spectrum of clinical presentation in these two geographically distinct areas in East Africa. In both settings T.b. rhodesiense HAT followed the classical disease pattern, but interestingly the neurological signs and symptoms typical for HAT were seen in a relatively small percentage of patients from Uganda. In patients from Tanzania, however, they were the dominate clinical manifestation. This correlated with the significantly higher reported CSF WBC counts in patients from Tanzania.
Unspecific signs of the disease such as fever, headache, general body pain and joint pains were reported in similar proportions in both study populations. We observed fever ($37.5) in 29.7% (41/ 138) of the trial subjects. In the literature, fever was reported in the range of 31-71% in second stage patients from Zambia [18,29,35]. In the two study populations we saw high fever (.38.5) on admission only in Uganda (5.8%, 4/69) whereof 50% were children. Fever seems to be more common in T.b. rhodesiense than T.b. gambiense second stage patients in which fever was only occasionally reported (16%) and high fever was mostly seen in children [25]. In the two study populations, oedema was reported in Uganda and Tanzania in 20.3% and in 37.7% of the patients,  [18,29,35,36].
The clinical aspects of T.b. gambiense HAT [21,25,26,37] have been systematically studied and show that the hallmark of second stage disease are neurological signs and symptoms [21,25]. Unfortunately, this has never been done for T.b. rhodesiense HAT and hampers comparisons. However, published data report sleeping disorders during daytime hours with 63.3-70.5% of patients being affected [29,35]. We observed sleeping disorders during daytime hours in Uganda and Tanzania in 56.5% and in 95.7% of the patients, respectively (p,0.0001). Similarly, sleeping disorders at night time are reported in the literature in 28.3% of patients [29]. We observed it in 34.8% of the patients from Uganda and in 92.8% of the patients from Tanzania (p,0.0001). Also other neurological signs and symptoms were significantly more frequent in patients from Tanzania; tremor (p = 0.0001), abnormal movements (p,0.0001), inactivity (p = 0.0076) and aggressiveness (p,0.0001). Clearly, the neurological signs and symptoms are more pronounced in Tanzania than in Uganda, and when compared to the literature.
In Uganda, almost 50% of patients were in a poor nutritional status (48% had BMI,16.5) as food security is very poor in this part of the country. This most likely contributes to weakness and, therefore, walking difficulties in the absence of neurological symptoms. Malnutrition is associated with immunodeficiency and higher susceptibility for a wide range of infections such as tuberculosis [38,39] and pneumonia [40], as well as a poorer response to treatment. Another potential consequence of malnutrition in Uganda is an increased number of patients admitted with severe coma indicating a more rapid progression of the disease. Yet, we assume that many HAT cases from T.b. rhodesiense endemic areas in Tanzania die without ever having had contact with the health system due to geographical isolation.
With regards to treatment outcomes, we did not see any differences in the two study populations. In both countries all patients were free of parasites at end of treatment. Also, there was no apparent difference in parasite clearance rates. Time-and treatment-dependant dynamics of CSF WBC counts in the two study populations will be published separately.
Cardiovascular involvement is typical, but rarely of clinical relevance in T.b. gambiense HAT [41,42]. We have limited knowledge of the effects of cardiac involvement in T.b. rhodesiense patients, but there is evidence that perimyocarditis seems to play an important role in the clinical course and fatal outcomes [43,44]. We observed symptoms of cardiac failure such as oedema (swelling of legs) in 29% of the patients. Hepatomegaly occurred in 18%, dyspnoea in 7% and cough in 20% of the patients. However, echocardiography or laboratory testing (i.e. brain natrium peptide) could not be performed to confirm heart failure. Co-infections with malaria and HIV were studied in detail in the patient population from Tanzania as the majority of the patients were malaria-positive on admission (80%) and agreed to voluntary testing of their HIV status (94.2%). Patients that were malaria-positive on admission more often had pruritus (p = 0.025), sleeping disorders during day time hours (p = 0.026) and disturbed appetite (p = 0.01). Also, they exhibited strange behaviour more often (p = 0.001). However, there is insufficient evidence for profound differences in malaria-positive and malaria-negative subjects, possibly due to asymptomatic carriers.
We identified one study that looked at T.b. rhodesiense and HIV coinfections in 25 patients from Kenya. In terms of treatment outcomes no conclusive results were obtained [45]. Our results indicate that the HIV status of the patient does not change the clinical presentation and/or the treatment outcomes of T.b. rhodesiense HAT. For T.b. gambiense HAT, there seems to be no association between HIV and HAT infection rates [46,47] but evidence exists for a negative association with treatment outcomes [47,48]. More research efforts are needed to better understand the complex interactions of coinfections, especially for neglected tropical diseases [49].
Our findings on the different clinical presentation of T.b. rhodesiense HAT in the two study populations could be due to an observation bias, bias in patient selection, or in comparing patients at incongruous time points after infection. Bias due to co-infections or differences in host and/or parasite genetics is also possible.
An observation bias can not be ruled out but is however less likely as the IMPAMEL III program was conducted with a structured case report form (CRF) and one monitoring person. We have seen variability in signs and symptoms with clear definitions (e.g. lymphadenopathy, abnormal movements or tremor) as well as subjective definitions (e.g. insomnia, headache or inactivity). We can not completely rule out a selection bias due to the exclusion of moribund and unconscious patients in which baseline examination per protocol was not possible. However, the number of excluded patients was relatively small (,10%) and the two study populations were similar in regards to self-reported duration of illness. Even though unsuccessful, active case searches were conducted in both countries which reduced a potential selection bias. Central nervous system involvement in T.b. rhodesiense HAT was previously reported within 3 weeks to 2 months of infection [12]. One third of the study population already had clear neurological signs and symptoms within one month of infection which reflects the acuteness of T.b. rhodesiense infections. The WBC count in the CSF as well as most of the clinical signs and symptoms also developed quickly and did not significantly change over time. Disease progression was noticeable by aggravation of tremor, walking difficulties, sleeping disorders at night time, disturbance of appetite and aggressiveness over time, in both study populations. Based on the results shown we rule out a bias of our findings due to co-infections. Previous infections with trypanosomes and/or host genetics might be determinants for the different clinical presentation of the disease in Tanzania and Uganda. There are speculations that apathogenic forms of the disease could influence immune responses to pathogenic infections [50,51] supported by the fact that HAT is more acute in white than in the black populations [52,53]. But we also see a high variability in disease severity among African populations [17,18], a fact that has been related to the descent of people: people of Nilotic descent, who migrated into the East African region from tsetse-free areas during the past 2,000 years may have less tolerance than people of Bantu descent, whose ancestors have been exposed to human trypanosomes for several thousand years [18]. Our findings do not align with this theory as in Tanzania, the majority of the population is of Bantu origin and in Uganda the majority of the population is of Nilotic origin. Different parasite genotypes could be responsible for the observed spectrum of disease severity, a hypothesis has already been raised 60 years ago [16,17,54]. Recent findings on the phylogenetic relationship between different T.b. rhodesiense strains showed that the high variability of the T.b. rhodesiense genome is attributed to multiple and independent evolutions from T.b. brucei [55]. Our data show a clear difference in the clinical presentation of T.b. rhodesiense HAT in Tanzania and Uganda but a detailed assessment of host and parasite genotypes was beyond the scope of this paper.
T.b. rhodesiense HAT is a highly neglected disease and tools for disease control are very limited. There are no sensitive diagnostics at hand and melarsoprol, the only available drug to treat second stage disease, is toxic. An early identification of the disease is vital to prevent late onset of treatment. However, most of the patients are first treated for other conditions such as malaria and pneumonia. A low degree of disease awareness among health personnel is common and aggravated by the low prevalence and the focal distribution of HAT. A detailed understanding of the clinical presentation and regional characteristics of T.b. rhodesiense HAT is important and can support decision making and differential diagnosis at health facility level.