Since the clinical signs of HAT are non-specific, in most cases the disease is only suspected in geographical areas where it is endemic. Sleeping sickness is endemic in areas where other tropical diseases like malaria exist [
7,
19,
20], making HAT an incidental finding on a blood smear meant for malaria diagnosis. Currently there is an increased bias towards the use of rapid diagnostic tests (RDTs) for the diagnosis of malaria [
21]. Therefore, the advent of RDT’s for malaria will consequentially lead to reduced detection
T. b. rhodesiense HAT as this relies on the detection of trypanosomes on blood smears. Sleeping sickness occurs in rural sub-Saharan Africa necessitating diagnostic techniques that are simple and cheap to perform [
22]. A major constraint in
T. b. rhodesiense HAT diagnosis as compared to
T. b. gambiense HAT is the fact that no suspicion serological tests are yet available thus impairing greatly the detection of cases (both for passive and active detection). Therefore, the most feasible approach for the detection of
T. b. rhodesiense infections is through direct microscopic observation of trypanosomes in blood, lymph node aspirates or in cerebrospinal fluids (CSF) of highly suspected individuals [
23]. Unlike
T. b. gambiense HAT, parasitemia due to
T. b. rhodesiense is in most cases above the threshold for microscopic detection reaching values of up to 10,000 trypanosomes/ml [
24]. Thick blood films prepared from a finger prick have limited sensitivity (detection limit is 5000 trypanosomes/ml) but are easy to perform with quick results [
25]. In cases of low parasitemia, concentrations/enrichment methods have been used to improve sensitivity. The micro-hematocrit centrifugation technique (mHCT) has a detection limit of 500 trypanosomes/ml [
26,
27] while the quantitative buffy coat technique offers an improved detection limit of <500 trypanosomes/ml [
28,
29]. Mini-anion-exchange centrifugation technique [
30] offers an improved sensitivity, detecting <30 trypanosomes/ml while its improvement on buffy coat goes lower than 10 trypanosomes/ml [
31]. Molecular biology techniques that detect parasite nucleic acids with increased sensitivity are becoming more common. The most commonly used PCR technique in research laboratory settings has been the detection of the serum resistance antigen (SRA) to confirm the presence of
T. b. rhodesiense [
32]. The SRA gene is reported to be responsible for
T. b. rhodesiense human serum resistance but is absent in
T. b. gambiense sub species that is also resistant to lysis by human serum [
33]. SRA is able to discriminate
T. b. rhodesiense from other
T. b. brucei sub species with a sensitivity equivalent to 1 trypanosome/ml [
34]. Although PCR based techniques have a sensitivity of up to 96 % [
35], the techniques have limited application in a field setting due to the need for a thermocycler, power supply and a cold chain for reagents. To improve on their applicability, an isothermal DNA amplification technique called loop mediated isothermal amplification (LAMP) has been developed [
36,
37]. LAMP is easier to perform and requires less sophisticated equipment than conventional PCR. However, before its adoption as a diagnostic tool, further clinical validation and standardization on large cohorts is required. Other alternatives like the RNA based real-time nucleic sequence based amplification [
38] and oligochromatography-PCR [
39] have been developed but are yet to undergo clinical evaluation and validation.