Our study showed that prevalence can be under-estimated by single microscopy technique, as compared to combined microscopy methods and combined molecular techniques. Differences and discrepancies in the number of cases detected from the three microscopy tests may be attributed to remote conditions under which these tests were conducted, low parasitaemia of trypanosome species, operator expertise and time during which observations were made. The buffy coat which is considered to be more sensitive than thick and thin smears (26) but in this case detected the least number of trypanosomes. Low case detection on buffy coat is a very common scenario as most field conditions are not favourable to allow for thorough screening of samples as compared to laboratory screening where operators take time to thoroughly screen the samples. Factors that may have negatively affected case detection on buffy coat, could include (among others), poor quality of capillary tubes, and high temperatures prevalent in the study area which could have led to increased prospects for diminished motility and/or death of trypanosomes before examiners could observe trypanosome movement in the buffy coat.
To validate available molecular diagnostic techniques for AAT, ITS-PCR and RIME-LAMP were employed using blood spots that were stored and transported on FTA cards and normal filter paper. ITS-PCR using blood spots stored and transported on normal filter paper (ITS-PCR-FP) detected (47/227; 95% CI: 15.4–26.0) while ITS-PCR using blood spots stored and transported on FTA cards (ITS-PCR-FTA) detected a higher number of trypanosome infections (83/227; 95% CI: 30.3–42.8). This result suggested that blood spots collected and stored on FTA paper are more reliable in determining trypanosomiasis prevalence than blood spots collected and stored on common filter paper (Chi-square p-value < 0.01). Such results may be attributed to the fact that FTA paper, unlike common filter paper has the ability to protect DNA from degradation (21, 26).
Unfortunately, due to costs attached to the use of FTA cards, their use may be limited as they may not be readily available by most researchers in trypanosomiasis endemic areas of Africa. Comparative analysis between the use of FTA and FP for blood sample storage and ITS-PCR analysis further indicated a fair agreement between the two techniques (kappa = 0.27) and greater probability in detecting trypanosomes. Our data show that both techniques could be useful in the detection of African trypanosomiasis considering that transportation of whole blood samples for ITS-PCR analysis may not be feasible under remote field conditions. Our study has demonstrated the convenience on the use of dry blood samples in areas with limited refrigeration facilities. Dry blood samples could be practically collected from selected animals and stored on FTA cards or filter papers on a regular basis for onward analysis at diagnostic centres (6, 27). Both FTA cards and filter paper may however, inhibit ITS-PCR making it less accurate compared to if DNA was extracted directly from whole blood samples which could explain why microscopically positive samples tested negative on PCR (26).
Our results showed a significant improvement in the number of T. brucei s.l. detected on RIME-LAMP (18/131) as compared to ITS-PCR (19/227) supporting the notion that RIME-LAMP is more sensitive and specific at detecting T. brucei s.l. than ITS-PCR. ITS-PCR on the other hand is more useful for studying trypanosomes that causes trypanosomiasis in cattle (26). Our findings also showed that filter paper is not good for transporting blood spots for HAT detection as seen in the one case of T. brucei s.l. detected on ITS-PCR-FP. Comparative analysis with respect to blood sample collection techniques employed in our study, showed poor agreement of ITS-PCR-FP with RIME-LAMP (k = 0.09) in detecting Trypanozoon as compared to ITS-PCR-FTA and RIME-LAMP which showed good agreement (k = 0.65) which is consistent with findings from other studies (19, 28).
When ITS-PCR-FTA was compared to microscopy, results indicated a gradual increase in both sensitivity and specificity with single microscopy tests reporting the lowest sensitivity and specificity: buffy coat (SE = 76.5%; SP = 66.7%; k = 0.15), thin smear (SE = 73.1%; SP = 68.2%; k = 0.21), thick smear (SE = 85.7%; SP = 70.4%; k = 0.30) as compared to combined microscopy tests (SE = 77.5%; SP = 72.2%; k = 0.35) which had a relatively higher sensitivity and specificity. This pattern of gradual increase in the ability of the tests to correctly determine diseased and non-diseased cases was also observed for microscopy and ITS-PCR-FP comparisons. Such results indicate the need for combining buffy coat, thin and thick smear techniques when considering microscopy for trypanosomiasis case detection in remote areas of Africa due to limitations attached to the use of molecular tests.
Although microscopy was used as gold standard in this study, there were positive cases that could be missed. Microscopy is more prone to human error because it is a manual test thus its inability to detect most positive cases. If ITS-PCR-FTA is used as gold standard with respect to combined microscopy, we observed a different scenario were positive and negative predictive values of 77.5% and 72.2% respectively became sensitivity and specificity of 37.4% and 93.8% respectively. In this scenario, ITS-PCR considerably improved case detection and demonstrated how its use may impact positively on trypanosomiasis prevalence estimations.
Using the “rule-in” and “rule-out” test as described by Florkwoski (29), results showed that ITS-PCR-FTA (kappa = 0.18) (high NPV and high sensitivity) was a better test for identifying diseased cattle than ITS-PCR-FP (kappa = 0.30). AUC-ROC scores for ITS-PCR-FP (0.7), ITS-PCR-FTA (0.8) and RIME-LAMP-FTA (0.7) were however, within the acceptable range of 0.7 to 0.9 indicating that both ITS-PCR and RIME-LAMP were significant in trypanosome case diagnosis (18, 20). Nevertheless, the robustness, simplicity, and ability to quickly read and visualize results has made RIME-LAMP a more popular diagnostic tool in Africa instead of ITS-PCR which requires a lot of instrumentation and expertise to achieve required amplification (13, 17). Although the technology required to perform RIME-LAMP exist in most developing countries, its use has still been limited due to non-availability and lack of production of dried ready to use master mix containing RIME primers. This study was not immune to this challenge resulting in only 131 out of 227 blood spots being tested using RIME-LAMP. Despite their affordability, RIME-LAMP kits could not be sourced locally or from regional suppliers that supply most molecular reagents. All PCR reagents on the other hand were available from regional suppliers though at an expensive price. Since Zambia does not produce any molecular reagents, importation and transportation costs also posed a challenge. The use of both ITS-PCR and RIME-LAMP is therefore still limited as most rural laboratories have not yet transitioned to the use of molecular techniques for point of care diagnosis of African trypanosomiasis and other zoonotic diseases.
Findings from this study brought out limitations that come with the use of the existing tests for African trypanosomiasis in rural areas of Africa i.e. Microscopy, ITS-PCR and RIME-LAMP, which may have crucial clinical and epidemiological implications (15, 30, 31).
Although previous studies suggested that T. congolense is the main cause of AAT and anaemia in Eastern and Southern Africa (6, 32–34), data from the current study demonstrate that T. vivax (ITS-PCR-FP; 39/227 and ITS-PCR-FTA; 50/227) which is less virulent than T. congolense was responsible for trypanosomiasis in most of the sampled cattle and that anaemia was not an indicator for trypanosome infection. Our mean PCV for trypanosome negative samples was slightly higher mean PCV (35.21) for positive samples. Anaemia in cattle is usually associated with the virulence of circulating trypanosomes (34.21). Secondly, ITS-PCR has previously been reported as being better at detecting T. vivax infections as compared to other trypanosome species (8, 15). High prevalence of T. vivax infections may also suggest that trypanosomiasis transmission within sites included in this study could be mechanical by other blood sucking insects prevalent in the area other rather than by tsetse flies. Finally, the detection of the human infective trypanosomes -T. b. rhodesiense from cattle blood samples analysed in this study 3/227 (1.3%; 95% CI: -0.2-2.8), highlights the risks that communities living in tsetse-infested areas may be facing in contracting sleeping sickness (1). Cattle may be potential sources of sleeping sickness when humans get bitten by tsetse after the fly has taken a blood meal from an infected animal (35–38). Our results support the need for a more holistic approach in the control of trypanosomiasis with a focus on the control of the disease in domestic animal reservoirs.