Identification of human African Trypanosomiasis foci using school-going children in post-conflict era in Nwoya District, Northern Uganda: A cross-sectional study

Background: Human African Trypanosomiasis (HAT) is fatal if untreated; the drugs to treat it are toxic making its management difficult and diagnosis complex. Nwoya district has a long history of sleeping-sickness dating back to pre-colonial times. The civil war of 1986-2008 displaced many who upon return complained of cattle and dogs dying of unknown causes alongside increased tsetse flies infestation hence, the needs for the study. Methods: We enrolled local 3,040 pupils and recorded their social-demographic characteristics and access to different domesticated animals/fowls in their homes. Screening for HAT using the card agglutination test for trypanosomiasis (CATT) was performed; positive individuals had their titres determined, followed by microscopy and loop mediated isothermal amplification analysis (LAMP). R was used for analysis where associations were sought between dependent and independent variables. Any factor with P-value <0.05 was taken as statistically significant. Results: HAT serological prevalence of 1.2% (95% CI 0.8-1.6) was obtained, 58.3% being boys while 41.7% were girls with titres ranging from 1:2 - 1:16. Two schools alone, constituted 47% of the CATT positive cases. Pupils who came from homes with dogs were more likely to be CATT/ Trypanosoma brucei gambiense positive; (adjusted odds ratio = 3.12, 95% CI 1.41-6.99 & p=0.005). Conclusions: Though no parasites were detected, with prevalence of CATT positive at 1.2%, active surveillance in the district is still recommended. CATT positive cases needs follow-ups were immune trypanolysis test done to ascertain their exposure.


Introduction
Human African Trypanosomiasis (HAT) commonly known as sleeping sickness is still active in over 30 sub-Saharan African countries 1 , and found within the regions between latitude 14 0 North and 20 0 South in more than 250 active recognized different foci 2 . If left untreated, it may lead to death 3-7 . The etiologic agents are; Trypanosoma brucei rhodesiense, the East African type that runs an acute course, and T. b. gambiense, the West African type that runs a chronic course 4,8 . The two species, unfortunately are morphologically undistinguishable, with characteristically different epidemiological features and drugs of treatment 9,10 . Up to 70 million people are at risk of this debilitating and fatal disease with 30,000 individuals estimated to be infected 11 . Since 2010, T. b. gambiense was still endemic in at least 24 countries in Africa, and accounted for over 95% of HAT reported cases 2,7,12 .
Uganda is the only country that harbours both species 1,13 , with T. b. rhodesiense having afflicted the South, through Eastern into mid-northern Uganda, while T. b. gambiense is endemic in North-West Nile districts and part of the Amuru district 12 .
In Northern Uganda, HAT was the main cause of relocations, a policy of compulsory evacuation of the natives from the affected areas 4,14 , an action that remained in force until the early 1950's 15 . Large swaths of land, especially in Anaka and Purongo sub-counties, had to be bush cleared in an attempt to rid the vectors of suitable habitation (The Monitor Newspaper of 22 nd Nov. 1999). Tsetse Control Camps were permanently maintained in several places that bordered the Murchison National Game Park and East Madi game reserve up to the period just before the northern insurgency in 1986.
Over two decades of civil war  led to mass displacement of people leading to total breakdown of social and physical infrastructures 16 . This same region is reported to be a potential site for a possible overlap of the two pathogenic brucei subspecies estimated to be within 160 km apart, and yet the so called frontier that divides the two seems to be only imaginary 1,2,17 .
Our study was therefore designed to use primary schoolgoing children to ascertain the prevalence of HAT, identify and map the current possible HAT foci, and to determine if there is any association among card agglutination test for trypanosomiasis (CATT) positive individuals and domestic animals/fowls kept at homes.

Study area
Nwoya district is located in mid-northern Uganda and lies within 02 0 38N, 32 0 00E covering 4,736.2 km 2 (1,736.2 sq miles) with a population of 54,000 people. Subsistence agriculture and livestock husbandry is the main economic activities besides tourism however; currently, there is steady increase in commercial agriculture according to Uganda Bureaus of Statistics, 2011.
The district is made up of 4 sub-counties (Koch Goma, Alero, Anaka and Purongo) in addition to Nwoya town council. Koch Goma and Purungo sub-counties form part of Murchison National Game Park; and thus are heavily infested with tsetse flies which act as vectors for sleeping sickness. In 2013, there were 44 Government-aided primary schools in the district with 39,632 pupils; 15,428 being boys while 14,436 were girls according to records obtained from district education office.

Study design
Our study was a cross-sectional survey; questionnaire was used to collect socio-demographic characteristics and risk factors while laboratory form was used for registering blood sample. The study was conducted in schools in sub-counties that form parts of the Murchison National Game Park and those closer to River Nile, were eligible for sampling. In total we purposively selected 19 Government-aided and 2 community schools since they are closer to the game park or river Nile which are foci for tsetse flies. In each school, we recruited pupils using consecutive sampling methods in each class. Pupils were drawn from primary three to primary seven, except for Gony-Cogo community school that had the whole school enrolled due to their small numbers and the children generally being more mature with one only who was 4 years old.

Data collection
Socio-demographic characteristics. Data on socio-demographic characteristics of each pupil; past history (e.g. places where they could have lived other than their homes), and the different animals/fowls kept at home were recorded. A Global Positioning System (GPS) was used to record locations of salient features; schools, sub-county headquarters, health units, district headquarter and homes of pupils who were found to be CATT/T.b. gambiense positive for easy follow-ups in the future.
Blood collection and preparation. At each selected schools, ethical procedures were observed e.g. explaining the purpose of the study and why they needed to participate although they would experience some slight pain during sample collection. After obtaining their assent, they were then enrolled by giving them identification numbers before going through the questionnaires to capture demographic information as well as animals/fowls kept at home. Finally, 2-3 ml of whole blood was collected aseptically following vein puncture in the cubital fossa into sterile plasma tubes 4.0 ml (BD, Franklin Lakes, NJ, USA) spray-coated with 60 USP (Units of Sodium Heparin) as an anticoagulant 18 . The blood was gently homogenized with the anticoagulant, 3% Phosphate Saline Glucose (PSG) was added to keep the trypanosomes active for a prolonged period. Samples were kept in cold boxes at temperature not exceeding 20 0 C to avoid exposure to heat and direct sunlight.
CATT test and microscopy. Screening was performed using CATT/T. b. gambiense in accordance to the manufacturer's (ITM, Antwerp, Belgium) manual by diluting blood 1:2 in CATT buffer. Briefly, a drop of whole blood was mixed with a corresponding amount of the reagent and rotated onto a flat orbital rotator for 5 minutes at 60 rpm . Both positive and negative controls were set along test samples and results read as  positive, if there were visible agglutinations with the naked  eye 19,20 . Titres were obtained by making twofold dilutions of 1:4, 1:8, 1:16 and 1:32 plasma in CATT buffer. 25 µl freshly reconstituted CATT/T. b. gambiense reagent (Institute of Tropical Medicine, Antwerp, Belgium) was added to each dilution, mixed and rocked for 5 minutes at 60 rpm. Titre was read as the highest dilution where visible agglutination was observed.
Wet preparations were made, mounted with cover slips 24 × 32 mm and examined using Olympus CX21 microscope under x20 and x40 magnifications and the results recorded. Thick blood smears of CATT positive samples were made, air dried, labeled and packaged into slide folders for eventual staining with Giemsa stain (SIGMA-ALDRICH ® , Catalog No. GS) for laboratory examinations under oil immersion (x100).

Sample preparations & detection of repetitive insertion mobile element (RIME) using loop mediated isothermal amplification (LAMP).
Homogenized whole blood from plasma tubes were sucked in heparinized capillary tubes sealed at one end with plasticine and spun at 800 x g (M24 Hematocrit centrifuge, LW Scientific) for 10 minutes to separate the different blood constituents. Buffy coats are located at the interface of packed cells on the lower end and serum at the top end. Using diamond pencil the tube is cut just above the packed cells and the Buffy coat was applied carefully onto labeled FTA ® Classic Cards (Lot No. 5114552C, Whatman International Ltd, Maidstone, UK) beginning from the centre moving outwards within the circle, air dried, packaged in self-sealing plastic bags containing desiccants, and stored in a lockable cupboard/ drawer for LAMP analysis.
Loopamp TM Trypanosoma brucei detection kit version3.11 was used to carry out the analysis by following the most recently revised standard protocol 21,22 . Test sample was positive if florescence was present indicating the availability of trypanosome DNA and negative if there was no fluorescence 21-23 . Both positive and negative controls were checked to confirm the validity of the test before results were read and recorded.

Data management and analysis
Data were entered in Microsoft Office Excel 2007, exported to R version 3.2.3 converted to comma delimitated (.csv) file, cleaned, edited and exported to STATA version 11 for analysis. Continuous variables were summarized using mean and categorical data were summarized in terms of frequencies and percentages. Univariate analysis was performed for both dependent and independent variables. Prevalence of HAT was obtained by dividing those who were CATT positive with the total number of pupils screened. Bivariate logistic regression was performed to determined association between the independent variables and presence of HAT. We reported Odds Ratio, 95% CI and P-value. Multivariate logistic regression analysis was used to assess for association between presence of HAT and the independent predictors. We run the stepwise logistic regression while adjusting for other independent variables in the model. We also adjusted for clustering of data around schools since the study was a survey. We calculated the adjusted Odds ratio, 95% confidence interval and P-value. Any variable with P-value ≤0.05 was taken as a significant predictor of HAT presence.

Ethical clearance
Ethical clearance (Ref. No. GU/IRC/01/11/11) was sought from Institutional Review Board of Gulu University. Study approval was granted by the Uganda National Council for Science and Technology (UNCST). The school management (Head teacher) was briefed about the study and the pupils were briefed about the study by the investigators. After thorough explanation in Acholi, the local language, the purpose of the study and the procedure involved, the pupils were given an informed consent form to take to their parent and return with it the following day when reporting to school before the study was conducted. The pupils who accepted to participate in the study signed an assent form. Pupils whose parents did not provide consent were excluded from the study and pupils who expressed fear of being pricked with needle even if their parent had consented and had first assented to participate in the study were excluded from the study, because that was a sign of withdrawal of assent. Children who were found to be CATT positive were followed home for a brief of their parents about the results and offer guidance where to seek treatment.
Paraa P/S in Purongo and Lutuk Community School in Koch Goma sub-counties had the highest number of pupils who tested CATT positive with 9 and 8 respectively. Those with titre ¼ (moderate reactions) were 15 while those with strong reactions 1/8 and 1/16 were 12.
School enrolment is at 50-50 for girls and boys although those involved in the study where mainly in the age group 13-18 years (63%). Fowls/animals that are mostly kept at home in the district are chicken, goats, and dogs with pigs and cattle are steadily on the increase over the years (Table 2).
With the use of GPS; villages, schools, sub-counties, district headquarter, hospital/health centres and salient features such as district and sub-county boundaries, road networks and streams/rivers are located on the district map for ease of follow ups especially the CATT positive cases even by someone who has not been involved in the study (Table 3; Figure 1).
Of the 36 CATT/T. b. gambiense positive samples, 34 were subjected to LAMP and all of them were found to be negative. The results were as shown in Figure 2a and 2b.

Discussion
A study conducted in Taraba state, Nigeria screened n=400 using CATT/T. b. gambiense obtained a prevalence of 1.8% (7/400). Males tested more positive than their female counterparts 23 and the difference in the number of males and females who were CATT positive was statistically significant, p=0.05. Although this agrees with our study; males (21/36) being more CATT positive than females (15/36), the difference in the sexes was not statistically significance. Due to a small sample size of Taraba study, this could have influenced the prevalence causing a significant difference between the sexes in terms of infectivity. HAT is a disease that is related to behavioural risk factors through complex interactions; environmental and behavioural risk factors, vector and the human host 24 .
Considering the fact that our subjects were school-going children, they were engaged in similar activities in disregard to their sexes and this could have been a key factor of having no statistical difference in the way the two sexes were exposed. In villages where girls go out in search of firewood in the park or forests, as seen in Laworo and Wii Anaka in Purongo subcounty, they got more exposed than the boys, whereas in areas where boys herd animals, go digging or playing in the fields, they were more exposed than girls, as observed in Alwi and Laminomony villages in Koch Goma sub-county. In some cases, pupils who travelled long distances leave schools nearby; Paraa Primary School (P/S)coming from Purongo trading centre, St Kizito Bidati P/S from Ladyema in Alero sub-county, Olwiyo P/S from Aparanga and Koch Goma P/S from Wi-Baka. These pupils were likely to have been exposed along the way as they travelled to school; indicated by the fact pupils who attended schools nearby were CATT negative.
The validity of CATT results has been a subject of controversies in the wake of low endemicity since it's known to work well in endemic areas with prevalence above 5% 25 . In our study however, the distribution of pupils who were CATT positive had a very striking pattern; in most cases, where there were several positive cases arising from a particular school, the pupils tended to come from the same villages as was observed in the villages of Laminomony, Wii Anaka area, Lutuk, Kibar, Koyo and Alwi. In one scenario, 2 cases from Lutuk community school were siblings from the same homestead in Bargunya village. Lutuk had a tsetse control camp in the 1980's due to HAT outbreak then, these results could indicate that it is on the verge of HAT re-emergence once again. While Wii Anaka and Paraa are settlements close to Murchison National Game Park; a place known to be infested with tsetse flies. Like Lutuk, Wii Anaka also had a nearby tsetse control camp at Got Apwoyo that now lies in ruins. Our findings therefore, reconfirms that most of the CATT positive results were certainly not due to errors or cross-reactions as other previous authors have suggested 13 , it's likely that these children could have been exposed, and either the parasites failed to be maintained or got neutralized by their strong immunity due to their long period of stay in this HAT endemic region 26 . It's also likely that over time T. b. gambiense could have under gone a lot of changes while in the human host; a study conducted in northern Uganda had found that 75% of those who were found to be CATT positive where parasitologically positive with careful wet preparations and thick blood smear microscopy 27 . Currently, numerous studies are reporting lots of aparasitaemic cases with gross variations in parasitaemia across foci 24,28 . Humans, like animals, are believed to possess trypanotolerance that protect them from the disease 29 .
Cut-off titres at some points were used to define disease cases; 1:4 required parasitological confirmation whereas 1:16 was regarded as indication of infection that required treatment even without parasites being seen 13 . In our study the following CATT positive cases were detected; 1:2 (n=9), 1:4 (n=15), 1:8 (n=6), and 1:16 (n=6) (see Table 1). Our findings cast doubts on reliability using CATT titres, as high as 1:16 did not reveal presence of parasites or their DNA. In a related study that was done in 2012, a case from Onigo village, Miniki sub-county in Adjumani district that was CATT positive with a titre of 1:4 who was aparasitaemic at the time, turned out to be a stage 2 case after trypanosomes were recovered from his CSF within 2 weeks from the time CATT screening was performed [unpublished study, Luryama Moi K, Anywar D and Madra P].
Though it has now been proven that animals play a yet unclear role in the maintenance of T. b. gambiense even when they have been eliminated from the human population, explaining in parts the reason behind re-emergence of HAT 30 . In a study conducted in West Africa, out of 397 domestic animals sampled, 64% were CATT positive for T. b. gambiense and when PCR analysis was done, 15.4% of sheep, 11.6% of pigs, 3.5% of goats and a low number of dogs where confirmed as infected with HAT 31 . Meanwhile a study carried out in Cameroon did not find any T. b. gambiense infection in dogs; this could have been due to a small number of dogs sampled 32 . In our study however, we did not sample the animals but instead tried looking at possible associations of those pupils who were CATT positive and the domesticated animals. After analysis for possible associations we found that those who had dogs were more than 3 times at increased risk of being CATT positive; Adjusted OR 3.12, (95% CI 1.41-6.99 and p = 0.005). This relationship could be explained in two ways; dogs could be acting as a reservoir of T. b. gambiense, a threat to re-introduction into human population. The other explanation could be due to the fact that dogs are social animals that are routinely used for hunting and herding animals in this region, and it could simply mean that those with dogs spend more time outside and therefore at higher risk of exposure than those without dogs. we also recommend that LAMP may still require more evaluation studies before it's adopted as a gold standard in the diagnosis of T. b. gambiense.

Study limitations
This was a one off survey and therefore we cannot account for information on pupils who refuse to participate or were absent from school nor children who have dropout of schools. However, our sample was large enough to be representative of the general pupils' population in the schools in the subcounties which form foci for tsetse fly.
The screening test used only targeted T. b. gambiense and as such making only 36 samples to qualify for testing using LAMP that detects both T. b. gambiense and T. b. rhodesiense. However, the information is good enough for baseline data upon which future research can build on.

Recommendations/conclusion
Pupils who were CATT/T. b. gambiense positive need a follow-up study for repeat CATT test, as well as performance of an Immune trypanolysis test to establish their infectivity status as the latter test seems to be more accurate. There is need to for active screening of populations using superior screening tests that combines both, T. b. gambiense and T. b. rhodesiense for regions that faces the threats of merger. Health workers at lower health facilities need sensitization and skill development in identifying cases with HAT. In conclusion, we confirm that the use of school-going children offers the most efficient means in the identification of HAT foci in places that accessibility is difficult or participation from the community is low.

Data availability
Data underlying the study are available on OSF: http://doi. org/10.17605/OSF.IO/DM7FT 33 Data are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).

Competing interests
No competing interest were disclosed.

Grant information
This research was supported by Training Health Researchers into Vocational Excellence (THRiVE) in East Africa, grant number 087540, funded by the Wellcome Trust.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
The manuscript describes a school-based approach to investigate HAT. This approach (unlike in the case of diseases such as schistosomiasis) is not normally taken in HAT, perhaps because most cases affected are among the middle age-group as a consequence of longer-term exposure via occupational activities that bring them into contact with the Tsetse fly. Nevertheless, the authors have shown that taking a school-based approach, one is able to zero in on microfoci where transmission might be taking place.
In the background part of the abstract, it is indicated that cattle and dogs were dying of undiagnosed disease, in presence of heavy tsetse infestation. It would have been interesting to do a pilot sampling of these to show any evidence for trypanosomes as a basis to suspect possible presence of HAT. But dogs are known to be susceptible to group and typically die of symptoms similar to those of late stage T. brucei HAT, corneal opacity is also a common manifestation of Trypanosomiasis in dogs.
CATT is an antibody test, known to cross react with other parasite antigens. Thus false positives are expected, even when executed outside HAT endemic foci. However, its specificity inside HAT endemic areas is considerable high, making it a useful test to screen for potential HAT cases. The authors should make it clear that they investigated sero-prevalence rather than HAT prevalence since a CATT positive reaction does not exclusively point to HAT presence. There is also need to use generally accepted terminologies e.g. Their CATT 1/2 might be referring to CATT whole blood. Normally in screening we talk about CATT whole blood, CATT ¼, 1/8, and 1/16; 1:4 is not the same as 1/4. Also clarify that the wet smears were read under x20 and x40 objectives; the magnifications were x200 and 400 respectively. Noteworthy is that being an area at the periphery of a T. b. gambiense endemic districts, more sensitive parasitology such as the heamatocrit centrifugation technique (HCT) would have been more appropriate, given the characteristically lower parasiteamia of that subspecies in humans.
For LAMP, the authors give no details of how they processed template before running the test, so it is not clear if it was performed with the best possible template. LAMP is a good molecular test but can not be considered a gold standard. The HAT case definition set by the WHO remains demonstration of the parasite itself in blood, lymph node aspirates, or cerebrospinal fluids; these comprise the so-called "composite reference standard". The authors could also have considered PCR alongside, to show if any "composite reference standard". The authors could also have considered PCR alongside, to show if any signals indicative of T. brucei would have been obtained.
In the discussion, please refer to seropositive individuals as suspects rather than cases.
The major arguments in the discussion regarding trypanotolerance would hold water, however in absence of any evidence for human infective trypanosomes circulating in humans, animal reservoirs or vectors in Nwoya, they remain very highly speculative. The only pointer to HAT is the CATT seropositivity whose results alone remain far from conclusive (it is a screening test that must be follwed by confirmatory parasitology); more techniques need to be deployed in the area in order to make more valid conclusions.
Other minor comments: use either "North west Uganda" or "West-Nile" districts. The words "were" and "where" have been interchangeably used, please rectify. Under recommendations, TL would help establish "infection", not "infectivity" status.

If applicable, is the statistical analysis and its interpretation appropriate? Yes
Are all the source data underlying the results available to ensure full reproducibility? Yes

Are the conclusions drawn adequately supported by the results? No
No competing interests were disclosed.

Competing Interests:
Reviewer Expertise: Parasitology, HAT surveillance and epidemiology I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. , which permits unrestricted use, distribution, and reproduction in any medium, provided the original Attribution Licence work is properly cited. 1.

Control of Neglected Tropical Diseases, Innovative and Intensified Disease Management, World Health Organization, Geneva, Switzerland
There are important conceptual misunderstandings in the manuscript reviewed that can give a wrong impression about the epidemiological situation of HAT in the area studied.
The CATT is a serological test used for screening and therefore with a specificity limited and well described cross reactions (false positives), as for instance with transient infections by nonhuman trypanosomes . This could be the case in the studied area. In areas of very low or zero HAT prevalence, the predictive positive value of the test is very low and the presence of CATT seropositives does not give any indication about the presence of HAT. No trypanosome was observed in the serological suspects after parasitological test, and therefore with the data presented it is not possible to infer that there is a presence or risk of the disease in the area. To talk than about "HAT serological prevalence" is confusing and wrong (The authors define "Prevalence of HAT" as CATT positive individuals divided by screened population). The statement indicating that these CATT results are not due to errors or cross reactions is not adequately justified.
Nwoya district was affected in the beginning of 20 century by gambiense sleeping sickness . In different outbreaks in the mid-20 century, the distribution of cases did not reach the area of Nwoya (Jonan and Okoro districts) . At the end of the last century (from 80's), an important outbreak of gambiense HAT was described in West Nile , but no cases of gambiense HAT have been diagnosed in the area of Nwoya since that time .
Sporadic cases of rhodesiense have been diagnosed in tourists visiting the neighbouring National Park of Murchinson Falls but these cases are due to (e.g. T. b. rhodesiense http://www.nicd.ac.za/assets/files/NICD-NHLS%20Communicable%20Disease%20Communiqu%C3%A9_DECE ) and linked to the presence of in wild animals. To investigate local populations T.b rhodesiense with gambiense CATT would not have any interest to asses' rhodesiense HAT infections, which could happen in the studied area. At the same time primary school children are not a group at high risk for rhodesiense HAT, usually linked with activities related to wild animals and people entering in the protected areas (rangers, poachers, tourist guides, herders, honey and firewood collectors,…).
It is important to clearly distinguish between gambiense and rhodesiense HAT and also to underline that the presence of tsetse fly and even animal trypanosomiasis does not implies the presence of human African trypanosomiasis.
The presence of animal trypanosomiasis in the area is well-known .
The presence of has been described in different animals but the T.b gambiense epidemiological role that animals can play as reservoir for gambiense HAT is not clear .
There would be an ethical concern as if the authors consider the CATT seropositives as possible cases and no action has been taken to follow them up to confirm the presence or not of a disease that is considered as lethal.
The authors confirm "that the use of school children offers the most efficient means in the identification of HAT foci in places that accessibility is difficult or participation from the community is low". According to the HAT foci in places that accessibility is difficult or participation from the community is low". According to the existing epidemiological data, the district of Nwoya cannot be currently considered as a HAT focus. The data presented in the paper (some children presenting CATT positive reactions but no presence of trypanosomes after parasitological tests) does not allow considering the area as a focus with active transmission of HAT.
The study tries to ascertain the prevalence of HAT using primary school children, and the conclusion should be that just based in some CATT positive results, it is not possible to conclude that there is transmission of HAT in the area. 1.

If applicable, is the statistical analysis and its interpretation appropriate? Not applicable
Are all the source data underlying the results available to ensure full reproducibility? Partly

Are the conclusions drawn adequately supported by the results? No
No competing interests were disclosed.

Competing Interests:
I have read this submission. I believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

Mike Brown
North Central London South Hub TB Clinic, Whittington Health, London, UK This is a useful surveillance study covering an important region, from which there have been few data published in the last 15 years. It addresses an important public health issue which is the potential role of domestic livestock in limiting the effectiveness of HAT elimination programmes. The methodology used was good, and the analyses and conclusions fair.
I have the following comments covering sections that would need major revision.
The introduction, while providing some background on HAT in Uganda, is not sufficiently focused on the question of interest which is the role of domestic animals in HAT transmission. It needs to be shortened and re-focused on this topic.

If applicable, is the statistical analysis and its interpretation appropriate? Partly
Are all the source data underlying the results available to ensure full reproducibility?
Yes goats, and dogs with pigs and cattle are steadily on the increase over the years" Replace "with -pigs and cattle…" with "while -pigs and cattle..." 4 paragraph: "With the use of GPS; villages, schools, sub-counties, district headquarter, hospital/health centres and salient features such as district and sub-county boundaries, road networks and streams/rivers are located on the district map for ease of follow ups especially the CATT positive cases even by someone who has not been involved in the study (Table 3; Figure 1)" This long sentence does not provide relevant information because the exact GPS coordinates are not given at all -even under . Again is not about GPS coordinates, but predictors of being CATT positive -thus Figure 1 Table 3 remove citation of in this sentence.  The words kept "at home" are repeated nine (9) times. Remove these words from the Table and retain under the description.
Organize this Table by adding; specific sampling sites, GPS coordinates, number of participants from specific sites, the livestock species kept at home, etc.

Table 3 (Page 6):
The basis for selection of some domestic livestock for inclusion in this study is not highlighted. What's the key message in this Table 3? Why is the row containing 'dogs' highlighted in bold, unlike all others in the Table? "(a) Prepared tests samples…" replace "tests" with "test" Discussion (Page 7) 3 sentence: "…the difference in the sexes was not statistically significance..." Replace 'significance' with 'significant' 4 sentence: "…sexes in terms of infectivity…" Replace 'infectivity' with 'infection' 3 paragraph, page 8: "…75% of those who were found to be CATT positive parasitologically where positive with careful wet preparations and thick blood smear microscopy" Replace 'whereparasitologically positive' with 'were -parasitologically positive' 3 paragraph, last sentence page 8: "Humans, like animals, are believed to possess trypanotolerance that protect them from the disease" This statement is misleading because it generally implies that animals [not clear if domestic or wild] are trypanotolerant. It is better to name specific animals that are known to be trypanotolerant; only some breeds of cattle are trypanotolerant e.g. some West African Taurine cattle, whereas many others are susceptible. 4 paragraph, page 8: "Cut-off titres at some points were used to define disease cases; 1:4 required parasitological confirmation whereas 1:16 was regarded as indication of infection that required treatment even without parasites being seen" This information provides the answer to my earlier question in Table 1 (page 5) on the significance of different titres. Move this to come under Materials and Methods, th rd th rd rd th Reviewer Expertise: African trypanosomiasis, molecular parasitology and entomology (the biology of tsetse fly and trypanosomes) I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.