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 T. brucei group and typically die of symptoms similar to those of late stage 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 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.

Is the work clearly and accurately presented and does it cite the current literature?

Yes

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

Is the study design appropriate and is the work technically sound?

Partly

Are the conclusions drawn adequately supported by the results?

No

Are sufficient details of methods and analysis provided to allow replication by others?

Partly

Reviewer Expertise:

Parasitology, HAT surveillance and epidemiology

I confirm that I have read this submission and 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.

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 ¹ . 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.

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 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.

It is not clear if authors want to assess the situation of gambiense HAT or rhodesiense HAT, and it would be important to clarify it.

Is the work clearly and accurately presented and does it cite the current literature?

No

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

Is the study design appropriate and is the work technically sound?

No

Are the conclusions drawn adequately supported by the results?

No

Are sufficient details of methods and analysis provided to allow replication by others?

Partly

Reviewer Expertise:

NA

I confirm that I have read this submission and 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.

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.

Is the work clearly and accurately presented and does it cite the current literature?

Partly

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

Is the study design appropriate and is the work technically sound?

Partly

Are the conclusions drawn adequately supported by the results?

Partly

Are sufficient details of methods and analysis provided to allow replication by others?

Partly

Reviewer Expertise:

NA

I confirm that I have read this submission and 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.

Abstract

Conclusions (under Abstract): Pg 1

“CATT positive cases needs follow-ups were immune trypanolysis test done to ascertain their exposure” This sentence is unclear, thus re-write to correct grammatical mistakes.

Keywords: Pg 1

CATT/T. b. gambiense = Follow rules when writing a scientific name. The genus and species names are always italicized; T. b. gambiense

Introduction: Pg 3

1 ^st paragraph, 3 ^rd sentence- the word ‘runs’ is repeated two times in the same sentence. In the 4 ^th sentence, substitute ‘undistinguishable’ with the more commonly used ‘indistinguishable’

3 ^rd paragraph, 1 ^st sentence; In Northern Uganda… = In northern Uganda…

Methods: Pg 3

Study area

Re-write the second sentence in the 1 ^st paragraph;

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.

Study design: Pg 3

1 ^st sentence on 1 ^st paragraph;

Rephrase the following sentence

Our study was a cross-sectional survey; [revise usage of semi-colon] questionnaire was used to collect socio-demographic characteristics [data] and risk factors[,] while laboratory form was used for registering blood sample [laboratory forms were used to record the details of collected blood samples].

In the 2 ^nd & 3 ^rd sentences, include punctuation marks (e.g. commas) where appropriate [‘In total [insert a comma] we…’], in another case, a comma was wrongly inserted into the sentence (see ‘… and those closer to River Nile, [delete comma] were eligible…’).

Last sentence under study design;

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.

Question;

How did you define maturity? What age bracket is mature? This statement on maturity is subjective.

Data collection: Pg 3

Socio-demographic characteristics.[:] Data on socio-demographic characteristics of [delete to avoid repetition]; past history (e.g. places where they could have lived other than their homes [ insert: within the study sites]), and the different [domestic] animals/fowls kept at home were recorded [ insert: for each pupil].

Blood collection and preparation

Rephrase the following statement;

“After obtaining their assent, they were then enrolled by giving [= assigning] them [= unique] identification numbers before going through the questionnaires to capture demographic information as well as animals/fowls kept at home” [This is repetition, see the above sub-title on socio-demographic characteristics]

2 ^nd sentence

“…whole blood was collected aseptically following vein puncture in the cubital fossa into sterile [insert=4.0 ml] plasma tubes (BD, Franklin Lakes, NJ, USA)…”

CATT test and microscopy

Screening was performed using CATT/ T. b. gambiense in accordance to [= with] the manufacturer’s (ITM, Antwerp, Belgium) manual by diluting blood 1:2 in CATT buffer (ITM, Antwerp, Belgium).

Comment: By using CATT/ T. b. gambiense test alone, the authors seem to have overlooked the possibility of T. b. rhodesiense infections in the study area, even though past history of participants living in other regions was recorded. Yet they clearly state that “the so called frontier that divides the two pathogenic brucei subspecies separated by a distance of about 160km in northern Uganda seems to be only imaginary” This is supported by recent findings by Opiro et al., 2017.

- Does CATT/ T. b. gambiense kit detect T. b. gambiense only?

Based on the findings of LAMP assay used in this study, the authors have raised doubts on its sensitivity to detect T. b. gambiense and T. b. gambiense.

2 ^nd sentence

“…corresponding amount of the reagent and rotated onto [= on] a flat orbital rotator for 5 minutes at 60 rpm”

3 ^rd sentence, on page 4

“Both positive and negative controls were set along [=alongside] test samples and results read as positive, if there were visible agglutinations with the naked eye” rephrase the statement to make it succinct, i.e if agglutinations were observed.

Last sentence of 1 ^st paragraph;

“Titre was read as the highest dilution where visible agglutination [Delete the word ‘visible’] was observed”

Next paragraph, 1 ^st sentence; page 4

Wet preparations [of what?] were made, mounted with cover slips 24 × 32 mm and examined using Olympus CX21 microscope under x20 [= 20×] and x40 [= 40×] magnifications and the results recorded.

2 ^nd sentence (page 4)

“…for laboratory examinations under oil immersion (x100)” rephrase as “…for laboratory examinations under oil immersion using 100x objective”

Sample preparations & detection of repetitive insertion mobile element (RIME) using loop mediated isothermal amplification (LAMP)

1 ^st sentence, page 4

“Homogenized whole blood from plasma tubes were sucked in heparinized capillary tubes sealed at one end…”

How was homogenization of blood conducted?

Data management and analysis (Page 4)

1 ^st sentence: “…converted to comma delimitated (.csv) file…” replace the word ‘delimitated’ with the correct one as ‘delimited’

5 ^th sentence: “Bivariate logistic regression was performed to determined association between…” Replace ‘determined’ with ‘determine’

Last sentence: “Any variable with P-value ≤0.05 was taken as a significant predictor of HAT presence” Replace ‘taken’ with ‘considered’

Ethical clearance (Page 4)

3 ^rd sentence: “The school management (Head teacher) was briefed about the study and the pupils were briefed about the study by the investigators” The word ‘briefed’ is repeated two times, replace one with another appropriate word, e.g. ‘informed’

6 ^th sentence: “…had first assented to participate in the study were excluded from the study…” delete “from the study” to avoid repetition of “the study”

Results (Page 4)

3 ^rd paragraph, 1 ^st sentence: “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” Replace ‘where’ with ‘were’.

3 ^rd paragraph, 2 ^nd sentence: “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” Replace “with — pigs and cattle…” with “while — pigs and cattle...”

4 ^th 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 Figure 1. Again Table 3 is not about GPS coordinates, but predictors of being CATT positive — thus remove citation of Table 3 in this sentence.

Table 1 (Page 5):

State the significance of titres at ½, ¼,⅛, 1/16, etc. Briefly, highlight the key message presented in the Table. The GPS coordinates could be provided in this Table that summarizes all surveyed schools.

Table 2 (Page 5):

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?

Figure 2 (Page 7): “(a) Prepared tests samples…” replace “tests” with “test”

Discussion (Page 7)

3 ^rd sentence: “…the difference in the sexes was not statistically significance...” Replace ‘significance’ with ‘significant’

4 ^th sentence: “…sexes in terms of infectivity…” Replace ‘infectivity’ with ‘infection’

3 ^rd paragraph, page 8: “…75% of those who were found to be CATT positive where parasitologically positive with careful wet preparations and thick blood smear microscopy” Replace ‘where — parasitologically positive’ with ‘were — parasitologically positive’

3 ^rd 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 ^th 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, subsection on “ CATT test and microscopy” (page 3).

5 ^th paragraph, 1 ^st sentence, pg 8: “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” Check again the flow of information in this sentence, re-write to make it clear.

5 ^th paragraph, 1 ^st sentence, pg 8: “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” This statement is too speculative and oversimplified since the animals were not screened for trypanosome infection by PCR and gene sequencing. This is very important experiment for determining livestock reservoirs of the HAT. It will be impossible to pinpoint or even propose, without bias, the contributions of dogs in the disease epidemiology if the complex interactions between humans with other domestic, and wild animals are not well understood. In my opinion, the authors should avoid generalizations on the potential animal reservoirs of HAT due to lack of conclusive data.

Study limitations (Page 8)

“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” Rephrase the sentence, replace ‘refuse’ with ‘did not assent’, and also replace ‘dropout’ with ‘dropped out’

Recommendations/conclusion (Page 8)

2 ^nd sentence: “There is need to for active screening of populations…” Delete the word “to”

Is the work clearly and accurately presented and does it cite the current literature?

Yes

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

Is the study design appropriate and is the work technically sound?

Yes

Are the conclusions drawn adequately supported by the results?

Yes

Are sufficient details of methods and analysis provided to allow replication by others?

Yes

Reviewer Expertise:

African trypanosomiasis, molecular parasitology and entomology (the biology of tsetse fly and trypanosomes)

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.