Challenges in the Diagnostic Performance of Parasitological and Molecular Tests in the Detection of African Trypanosomiasis in Cattle in Mambwe District in Eastern Zambia


 Background: Trypanosomiasis is a Neglected Tropical Disease with serious health and economic implications. Disease eradication and control programs rely on active case detection through mass population screening. Screening tools and techniques therefore need to be adequately sensitive, practically quick to perform, and affordable. This study compared the field application of the polymerase chain reaction, the loop mediated isothermal amplification technique, and microscopy, in the detection of trypanosomes in cattle blood in Mambwe district in eastern Zambia. Methods: Blood samples were collected from 227 cattle into three heparinised micro capillary tubes, and tested for trypanosomiasis infection using microscopy, ITS-PCR and RIME-LAMP. The comparative diagnostic performance of each of the methods was evaluated using the chi-square test, kappa test and receive operator curves. Results: Microscopy on buffy coat detected 17 cases (n=227), by thin smears detected 26 cases (n=227), and by thick smears detected 28 cases (n=227). In total, microscopy detected 40 cases (n=227). ITS-PCR- on blood spots stored on filter paper detected 47 cases (n=227), ITS-PCR- on blood spots stored on FTA cards detected 83 cases (n=227) and RIME-LAMP-FTA detected 18 cases (n = 131). Using microscopy as gold standard, sensitivity and specificity of ITS-PCR was compared. ITS-PCR-FTA had a better specificity and sensitivity (SE=77.5%; SP=72.2%; k = 0.35) than ITS-PCR-FP (SP = 88%; SE = 60%; kappa = 0.45). Prevalence of Trypanosoma brucei s.l. was higher on RIME-LAMP-FTA (18/131) than ITS-PCR-FTA (19/227). Conclusion: Our results are not perfect but are a good illustration of the current diagnostic challenges in rural Africa. Findings showed that none of the diagnostic tests could be taken as having performed better than the others and that each of the tests offered some advantages and limitations. In endemic rural areas of Africa, the use of PCR and LAMP requires specialised staff, laboratory supplies and infrastructure which is often not available. For this reason, microscopy remains the most practical option for field diagnosis of trypanosomes but understanding its limitations is critical particularly when applied for surveillance purposes.


Background
Tsetse-transmitted trypanosomiasis, caused by protozoan parasites of the genus Trypanosoma, affects both man and animals. While Trypanosoma congolense, Trypanosoma vivax and Trypanosoma brucei s.l. cause nagana or Animal African Trypanosomiasis (AAT) in livestock, the two sub species of T. brucei s.l.: Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense are responsible for Human African Trypanosomiasis (HAT) commonly known as 'sleeping sickness'. Countries affected by nagana have continued to suffer from economic losses in millions of dollars (1)(2)(3).
Historically, microscopy has been traditionally regarded as the gold standard in detecting the presence of trypanosomes. Microscopic examination of buffy boat and wet blood lms, as well as thin and thick blood smears stained with Giemsa, are the most common methods used in Africa for trypanosome detection. Microscopy is considered a good diagnostic method because it is simple, cheap and can also simultaneously detect other haemoparasites (micro laria and Plasmodium spp.) (4). However, microscopy has very low sensitivity especially in detecting early infections that are associated with low parasitaemia (5)(6)(7).
Molecular techniques such as the polymerase chain reaction (PCR) have signi cantly improved the level of sensitivity and accuracy in trypanosome diagnosis, in comparison to the traditional parasitological methods. However, most remote areas of Africa have not had the resources to facilitate the use of such molecular techniques (8,9). Molecular tests have the ability to facilitate differentiation of trypanosome species and subspecies, e.g. through use of speci c primers (10)(11)(12)(13). Internal Transcribed Spacers (ITS)-PCR can be used for the detection of both AAT and HAT, but its use in the eld is limited by high costs and the need for highly trained personnel (14,15).
The invention of the repetitive insertion mobile element-loop-mediated isothermal ampli cation (RIME-LAMP) method a decade ago has given new impetus to development of point-of-care diagnostic tests, based on ampli cation of pathogen DNA -a technology that has been the precinct of well-developed laboratories (13,16). RIME-LAMP is a simple diagnostic test that ampli es the RIME sequence of the Trypanozoon subgenus group, and is reportedly highly sensitive, and speci c (17)(18)(19). The advantage of RIME-LAMP over ITS-PCR is that it is less expensive and quicker to carry out. Furthermore, the sensitivity of RIME-LAMP is reported to be equal to or higher than that of the ITS-PCR that targets the same gene (16,17).
Understanding the capabilities of each diagnostic technique is key to quick and accurate detection of trypanosomiasis in samples and is critical to disease control and eradication. Unfortunately, in most rural settings of Africa, poor detection of trypanosome infections has occurred due to the poor understanding of the limitations of diagnostic tests used. Against this background, this study compared the diagnostic performance of microscopy, ITS-PCR, and RIME-LAMP in detecting trypanosomes.

Study area
The study was undertaken in Mambwe district in the Eastern Province in Zambia, from February to April 2019. The district was purposively selected, considering that most of the district is highly tsetse infested and with a high prevalence of bovine trypanosomiasis (20). Located along the Luangwa river basin, the district covers an area of 4,480 km 2 and is home to the South Luangwa National park. With a human population of 92,445 belonging to 18,489 households, most of the local community rely on tourism and small-scale farming for their livelihoods (Zambia Central Statistical O ce (CSO), 2015).

Study design and sample size
The study compared three diagnostic techniques (RIME-LAMP, ITS-PCR, and microscopy) for the detection of bovine trypanosomiasis under eld conditions. This was done through establishment of prevalence of trypanosome species in 227 cattle among a sample of cattle-owning small-scale farmers in selected parts of Mambwe district -i.e. located in tsetse-infested parts of the district close to the South Luangwa National Park. The cattle farmers were selected largely based on willingness to participate in the study. Oral/written informed consents were provided.

Sample collection
From each animal, blood was drawn into three micro capillary tubes containing heparin (anticoagulant) after puncturing the ear vein of the animal with a blood lancet. One capillary tube was sealed with crista seal for on-site examination by buffy coat. Blood from the second capillary tube was used to make thin and thick smears for analysis on microscopy later at the laboratory (4). About 50 µl of blood from the third capillary tube was applied onto a well labelled After air drying, both lter paper and FTA card samples were separately packed in zip locked storage bags containing silica gel and transported to the laboratory for further processing with ITS-PCR and RIME-LAMP (21).

Application of diagnostic tests
Microscopy: On site microscopic examination was conducted for cattle blood stored in heparinized capillary tubes. The sealed capillary tubes were spun in a microhematocrit centrifuge for ve minutes at 10,000 rpm, after which packed cell volumes (PCVs) were determined using a PCV reader. Buffy coat from each sample was then placed on a microscopic slip with cover slip and examined on site at ⋅400 magni cation for the presence of motile trypanosomes. After staining with 10% of Giemsa solution at the laboratory, thin and thick smears collected from cattle samples were also examined for the presence of trypanosomes (22,23). DNA extraction from Whatman® No. 1 lter paper: DNA from stored blood spots was extracted using the Tris-EDTA buffer technique. Discs of about 6 mm diameter were cut from each blood spot and placed in labelled 1.5 ml sterile tubes. About 66 µl of TE buffer (10 mM Tris-HCl pH 8.0 and 0.1 mM EDTA in distilled water) was added to each tube and incubated at 50 o C for 15 minutes. The discs were then pressed gently at the bottom of the tube using a new rod for each tube and heated at 97 o C for another 15 minutes to eluate the DNA. The tubes were then spun down at 10,000 rpm for 1 minute (24) DNA extraction from FTA cards: DNA was extracted from the stored blood spots using the Chelex method. A disc of about 6 mm diameter from each blood spot was placed in a labelled sterile 1.5 ml sterile tube. About 200 µl of Whatman puri cation reagent was used to wash each disc for 15 minutes, after which the solution was carefully decanted. The discs were then washed twice with 200 µl of 1 ⋅ concentrated TE buffer for 15 minutes after which the solutions were decanted gently. A separate rod for each sample was used during decanting to make sure that the discs did not ow over with the solutions. The discs were then left to air dry for an hour after which 100 µl of 5% (w/v) Chelex-100 (Sigma-Aldrich Japan, Tokyo, Japan) in distilled water solution was added and mixed thoroughly. The discs containing Chelex solution were nally incubated at 90 o C for 30 minutes to elute DNA. The eluted DNA was stored at 4 o C for use within 12 hours and at -20 o C for long-term storage (21) RIME-LAMP: The test was performed for cattle blood spots stored on FTA cards using dried (vitri cated) master mix containing RIME primer for T. brucei s.l. detection. The test tubes were produced in house according to the previously reported procedure (19). About 23 µl of reaction buffer was then added to the tubes followed by 2 µl of DNA eluate from cattle blood spots on FTA cards. The tubes were then carefully closed and turned upside down for 2 minutes and mixed several times to dissolve the reagents. The reaction mixture was then incubated at 63 o C for 45 minutes after which positivity was determined through the con rmation of the uorescent in the reaction mixtures observed by LED detector (17,19).

Data analysis
Statistical analyses were performed in SPSS version 26 (IBM Corporation., 2019). Trypanosomiasis prevalence determined by microscopy (buffy coat, thin smears, and thick smears) was taken as the gold standard. The prevalence determined by the ITS-PCR were compared against this gold standard and the sensitivity and speci city were calculated on this basis. Chi-square test was used to determine statistical signi cance between the tests. For expected values less than 5, the Fisher's exact test was used. P values less than 0.05 were considered statistically signi cant. Impact of diagnostic test performance was estimated using positive and negative predictive values for each test. The usefulness and bene ts between the tests were measured using the Receive operator curve (ROC) while Kappa coe cient measured agreements and accuracy between tests. Area under the receive operator curve (AUC-ROC) scores were used to distinguish between a perfect and worthless test. AUC scores were classi ed as follows: excellent (0.90-1), good (0.80-0. Microscopic examination of trypanosome infection on buffy coat detected 17/227 cases (7.5%), thin smears detected 26/227 cases (11.5%) while thick smears detected 28/227 cases (12.3%). Combined microscopy using these three microscopic techniques in parallel recorded a total of 40/227 cases (17.6%). (Table 1). RIME-LAMP-FTA detected 18 cases (13.7%, n = 131).  Diagnostic accuracy, sensitivity and speci city of ITS-PCR on blood spots stored on lter paper FP (ITS-PCR-FP) and ITS-PCR on blood spots stored on FTA cards (ITS-PCR-FTA) was determined using Microscopy as the gold standard as shown in Tables 3 and 4 respectively. Agreement between the tests was measured using the Kappa test. The results of comparison of ITS-PCR using FTA and FP as collection methods is shown in Table 5.    For the detection of T. brucei s.l., RIME-LAMP was used to compare trypanosome detection with ITS-PCR (Table 6). Receiver operating characteristic (ROC) curves ( Fig. 1) were used to compare sensitivity and speci city across a range of values and area under the ROC curve was used to measure test performance. The curves show the usefulness of ITS-PCR and its ability to detect trypanosomes when compared with buffy coat (ROC 1), thin smear (ROC 2) thick smear (ROC 3) and combined microscopy (ROC 4) while Fig. 2 illustrates the abilities of ITS-PCR and RIME-LAMP to detect T. brucei s.l.
Area under the receive operator curve (AUC-ROC) scores for ITS-PCR-FP, ITS-PCR-FTA and RIME-LAMP were as shown in Table 7. The higher the AUC score, the better the test at distinguishing diseased from non-diseased individuals.

Discussion
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 eld 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 lter paper. ITS-PCR using blood spots stored and transported on normal lter 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 lter paper (Chi-square p-value < 0.01). Such results may be attributed to the fact that FTA paper, unlike common lter 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 eld 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 lter papers on a regular basis for onward analysis at diagnostic centres (6,27). Both FTA cards and lter 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 signi cant 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 speci c 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 ndings also showed that lter 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 ndings from other studies (19,28). 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 speci city 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.
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 ampli cation (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.
Although previous studies suggested that T. congolense is the main cause of AAT and anaemia in Eastern and Southern Africa (6,(32)(33)(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 ies. 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 y has taken a blood meal from an infected animal (35)(36)(37)(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.

Conclusions
The study illustrates current challenges with AAT diagnosis using molecular and microscopy techniques in rural areas and the need for innovation in the area of diagnostics. However, considering that trypanosomiasis is prevalent in remote rural areas where access to diagnostic facilities is limited, FTA cards and lter papers should be considered for collecting, storing and transportation of blood samples for analysis using ITS-ITS-PCR and RIME-LAMP where collection of whole blood is not feasible. Currently used diagnostic tests have their own advantages and limitations. ITS-PCR is a better screening test of trypanosomes causing Nagana while RIME-LAMP is a better test for the detection of T. brucei s.l. However, their use may be limited and not practical in remote rural areas of Africa were trypanosomiasis is endemic. Microscopy could, therefore, be used for diagnosis but as a combination of the three commonly used techniques; buffy coat, thin and thick smears. Microscopy remains the most practical option for diagnosis of trypanosomes in the eld but understanding its limitations is critical when using it for surveillance purposes.