Trypanosoma cruzi and Trypanosoma rangeli in Acre, Brazilian Amazonia: Coinfection and Notable Genetic Diversity in an Outbreak of Orally Acquired Acute Chagas Disease in a Forest Community, Wild Reservoirs, and Vectors

Round 1

Reviewer 1 Report

Reviewer Evaluation

Trypanosoma cruzi and Trypanosoma rangeli in Acre, Brazilian Amazonia: Coinfection and unprecedented genetic diversity in an outbreak of orally acquired acute Chagas disease in a forest community, wild reservoirs, and vectors.

 

This interesting MS shows a study on an orally transmitted acute Chagas disease outbreak, occurred in a forest community locality at the state of Acre, Brazilian Amazonia, afflicting 13(11 adults and 2 children) members of a family group, shortly after ingesting açai pulp. Patients were clinically, parasitologically and serologically diagnosed in a local health center, and blood samples were taken, for molecular diagnosis and trypanosome genotyping, to a reference center for specific characterization. Additionally, 25 more samples including triatomines (8), bats (6), non-human primates (10) and dog (1) were taken to be molecularly characterized by using FFLB method, as well as detecting the genetic diversity of T. cruzi and T. rangeli, and the coinfection in 8/13 (62%) of individuals suffering ACD. This relevant finding together with the discovery of T. cruzi DTUs and T. rangeli genotypes, in the same sample, make the present MS with merit enough to be published in Parasitologia -MDPI.

However, some minor corrections are necessary for a better presentation and more fluid information to avoid confusion for non-specialists on the subject. These are as follows:

 

Title: The word unprecedent, an adjective that describe something “never having happened or existed in the past”, introduce doubt on the studied phenomenon because it is hard to be proven. It should be change for discovery or remove it because the AA-manifested intention is revealed in the text when say: for the first time.

Abstract: One child and one baby=two children

Introduction: Antecedents on T. cruzi causing ACD in Acre is well documented. However, AA do not mention any background information on T. rangeli neither in Acre, place of study, nor in other states of the Brazilian Amazonia.

Results: Table 2. in accordance with the MS proposed title, the 16 samples from Rondônia, including 10 triatomines, 3 didelphis, 2 bats and 1 dog, and detected T. cruzi-DTUs (6TcI-5TcIV) and T. rangeli-genotypes (5TrA), should be removed to a separate table with the information belong to the state of Rondônia, closed to Acre, which supports the point 3.4. Phylogenetic relationships between T. cruzi and T. rangeli from Brazilian Amazonia and the information in Fig.3.

Pag.7 line 238: TcI alone occurred in four patients, correct four for five (5).

 

Author Response

We would like to thank immensely all reviewers for all the constructive comments and valuable suggestions, and their careful review that very much improved our manuscript.

Point 1: Title: The word unprecedent, an adjective that describe something “never having happened or existed in the past”, introduce doubt on the studied phenomenon because it is hard to be proven. It should be change for discovery or remove it because the AA-manifested intention is revealed in the text when say: for the first time.

Response: Our idea was to emphasize that the genetic diversity we found in a single outbreak is uncommon, never published before. For precision, we changed unprecedented for notable. 

Point 2: Abstract: One child and one baby=two children

Response: Ok, changed.

Point 3: Introduction: Antecedents on T. cruzi causing ACD in Acre is well documented. However, AA do not mention any background information on T. rangeli neither in Acre, place of study, nor in other states of the Brazilian Amazonia.

Response: We agreed and added a sentence about T. rangeli in Brazilian Amazonia to the introduction (Line 86).

Point 4: Results: Table 2. in accordance with the MS proposed title, the 16 samples from Rondônia, including 10 triatomines, 3 didelphis, 2 bats and 1 dog, and detected T. cruzi-DTUs (6TcI-5TcIV) and T. rangeli-genotypes (5TrA), should be removed to a separate table with the information belong to the state of Rondônia, closed to Acre, which supports the point 3.4. Phylogenetic relationships between T. cruzi and T. rangeli from Brazilian Amazonia and the information in Fig.3.

Response: Sorry for the puzzling Table 1 and the lack of enough information regarding the phylogenetic analysis. Our purpose in comparing samples from Acre and Rondônia was to infer, for the first time, phylogenetic relationships among trypanosomes isolated from humans from southwestern Amazonia. All samples from this region used in the analysis, obtained in the present study and retrieved from GenBank, were included in Table 1. For clarity, Table I was changed; we separated the samples according to their origin, first Acre and then Rondônia. The title was improved, and a forgotten footnote was now included to explain the samples. The phylogenetic analysis in Fig. 3 showed the relationships between trypanosomes from the southwestern and those from distant areas in eastern (PA, AP) and western (AM) Amazonia; the title improved to make clear our purpose.

Point 5: Pag.7 line 238: TcI alone occurred in four patients, correct four for five (5).

Response: Thank you for your revision. Number corrected.

Reviewer 2 Report

In the manuscript “Trypanosoma cruzi and Trypanosoma rangeli in Acre, Brazilian Amazonia: Coinfection and unprecedented genetic diversity in an outbreak of orally acquired acute Chagas disease in a forest community, wild reservoirs, and vectors” the authors characterize an outbreak of orally acquired Chagas disease in the community Seringal Miraflores in Acre Brazil.

 

It is interesting the clinical characterization of the patients and mainly the genetic diversity detected by FFLB of the infections with T. cruzi and T. rangeli. It is also important that they found this genetic diversity in the digestive tract of some vectors

 

I recommend the authors make the following corrections:

 

2.1 Material and Methods

Correct font size in several phrases and words from lines 123 to 134 and 141

 

It is necessary that the authors discuss the limitations of their research, such as the lack of follow-up protocol to ensure that the patients have been cured.

 

In Material and methods, the authors referred that they did serological tests and even in the results (line 190) they mentioned the detection of IgM, nevertheless they don't show or mention these results in any table of results nor in the supplementary material. The authors must include the results of the IHA and IFA-IgM that they mentioned in Methods section 2.2  lines 153 and 154 and results from 3.1 section line 190.

 

Did the authors search for IgG antibodies? How did the authors discard previous infections in all the patients? If the authors did not discard previous infections they should discuss it as a limitation and that some co-infections could have occurred due to previous exposure.

 

The authors mentioned that the follow-up was only clinical and the diagnostic test only included blood smears, as they mentioned in lines 219 -221 they could not assess for possible therapeutic failures and development of chronic infection. It is a serious limitation and this is possibly beyond the scope of the project presented; nevertheless, the authors should discuss it and suggest including it as part of the future protocols and as a suggestion to the SESACRE.

Author Response

We would like to thank immensely all reviewers for all the constructive comments and valuable suggestions, and their careful review that very much improved our manuscript.

Point 1: "2.1 Material and Methods: Correct font size in several phrases and words from lines 123 to 134 and 141"

Response: Ok, corrected.

Point 2: "It is necessary that the authors discuss the limitations of their research, such as the lack of follow-up protocol to ensure that the patients have been cured".

Response: This is a deplorable issue underlying public health in isolated areas in Amazonia. It is important to mention that the outbreak herein characterized occurred more than six years ago when ACD began to be detected in southwestern Amazonia. At that time, Acre had not implemented the recommendations from the Brazilian Health Ministry. For this reason, serological tests of the outbreak were performed at the IEC in Belem, Pará. After the end of the treatment, serology was not performed because of logistical troubles in sending the sera for serological tests. Nowadays, SESACRE can support the whole protocol for ACD diagnosis, treatment and follow-up post-treatment recommended by the Brazilian Ministry of Health. 

Before answering this question, we gathered all available information about the patient's past and current exams. As mentioned in the manuscript, patients were clinically followed for three years, and those that could be examined during the last year were in good health condition. After the COVID pandemic, they were invited for clinical and serological evaluation. Unfortunately, data about patients obtained outside the Hospital of Feijó and IEC-Belém are unavailable for the present study. We changed the sentence as recommended (Line 227). 

 

Point 3: "In Material and methods, the authors referred that they did serological tests and even in the results (line 190) they mentioned the detection of IgM, nevertheless they don't show or mention these results in any table of results nor in the supplementary material. The authors must include the results of the IHA and IFA-IgM that they mentioned in Methods section 2.2 lines 153 and 154 and results from 3.1 section line 190".

Response: We changed sentences about serological tests in Material and Methods (Line 160) and Results (Line 197).

Point 4: "Did the authors search for IgG antibodies? How did the authors discard previous infections in all the patients? If the authors did not discard previous infections they should discuss it as a limitation and that some co-infections could have occurred due to previous exposure".

Response: Sorry for this mistake. Serological tests performed routinely for diagnosis in the IEC at IEC-Belém always include IHA, IFA-IgM and IFA-IgG. We changed sentences about serological tests in Material and Methods (Line 161) and Results (Line 199) to include IFA-IgG.

Point 5: "The authors mentioned that the follow-up was only clinical and the diagnostic test only included blood smears, as they mentioned in lines 219 -221 they could not assess for possible therapeutic failures and development of chronic infection. It is a serious limitation and this is possibly beyond the scope of the project presented; nevertheless, the authors should discuss it and suggest including it as part of the future protocols and as a suggestion to the SESACRE".

Response: We fully agree, and although not within the scope of the present study, we explain everything possible about the patients of the Miraflores outbreak (Results, Line 227). We already mentioned in the manuscript that “Acre adopted the Brazilian National Health Surveillance Agency program to prevent and manage orally transmitted ACD” (Line 141). In the revised manuscript we added the sentence “ Nowadays, SESACRE can support the whole protocol for ACD diagnosis, treatment and follow-up post-treatment recommended by the Brazilian Ministry of Health” (Line 232).

 

Reviewer 3 Report

Firstly, I must congratulate the workers for this big effort in typing and registering the extensive number of hosts and potential vectors of T.cruzi and T.rangeli in the Brazilian Amazonia. The data is well presented, but I have some doubts: e.g. the presence of T.rangeli in blood after an oral infection is an intriguing fact to me. As far as I know, T. rangeli lacks or has fewer of the MASP, Mucins and Transilidase genes repertoire as to wistand the drastic acid environment of a human stomach, perhaps the Authors should elaborate more about this interesting finding. Second, I am not so sure that this is the first direct Trypanosoma typing from blood as the Authors claim; third, although the Authors admit not doing a further follow-up using immunological or molecular technologies after the treatment with  Benznidazol, they should be alert to reemergency of the disease. Finally, I was surprised  by the omission of the extensive experience with oral cases accumulated by Dr. Noya de Alarcon in Venezuela where the main T.cruzi DTU is I. Although as the Authors report their strains groups separate from the Northern Type I T. cruzi there are still many lessons to learn from Dr. Noya´s group

Author Response

We would like to thank immensely all reviewers for all the constructive comments and valuable suggestions, and their careful review that very much improved our manuscript.

Point 1: "the presence of T.rangeli in blood after an oral infection is an intriguing fact to me. As far as I know, T. rangeli lacks or has fewer of the MASP, Mucins and Transilidase genes repertoire as to withstand the drastic acid environment of a human stomach, perhaps the Authors should elaborate more about this interesting finding".

Response: We were also surprised and still intrigued by our discovery. Although T. rangeli had already been reported in 1986 in three individuals from Brazilian Amazonia (now included in the Introduction – Line 86), our data is notable regarding both high prevalence, and possible oral infection. We have been careful to say that oral infection by T. rangeli remains undocumented, that or epidemiological study suggest oral infection (Discussion, Lines 334, 404). Confirmation of oral infections is one possibility that requires future studies. Because the outbreak herein characterized occurred in an area endemic to T. rangeli, and vectors are present in peridomicile palms, we could not discard the possibility of vectorial transmission.  We change the sentence to clarify this point (Line 337).

Regarding the repertoire of mentioned multigene families, T. rangeli has a reduced number of copies and different proteins from the same gene families compared with T. cruzi. As recently confirmed by broad analyses of genomes and proteomes of T. rangeli all these gene families (MASP, Mucins and Transilidase and others) are present in T. rangeli (Watanabe-Costa et al., 2020). To our knowledge, nothing was verified regarding the roles of these proteins for the infection/surviving of T. rangeli in mammalian hosts. Therefore, any comments about this issue will be merely speculative. In addition, recent experimental studies demonstrated that besides gastric mucosa, T. cruzi invasion is highly efficient by the mucosa of oral cavity (Barreto de Albuquerque et al., 2018).

Point 2: "Second, I am not so sure that this is the first direct Trypanosoma typing from blood as the Authors claim".

Response: We mentioned in the line 344 “This is the first time that T. cruzi infecting humans from AC was genotyped, revealing a predominance of TcI and one case of TcI mixed with TcIV”.

Of course, there are countless studies of genotyping T. cruzi in blood samples. On the other hand, this is the first report on genotyping of whole repertoires of trypanosomes directly from blood samples from ACD patients in Acre.

Point 3: "Third, although the Authors admit not doing a further follow-up using immunological or molecular technologies after the treatment with Benznidazol, they should be alert to reemergency of the disease".

Response: We explain everything possible about the patients of the Miraflores outbreak (Results, Line 227). We already mentioned in the manuscript that “Acre adopted the Brazilian National Health Surveillance Agency program to prevent and manage orally transmitted ACD” (Line 141). In the revised manuscript we added the sentence “ Nowadays, SESACRE can support the whole protocol for ACD diagnosis, treatment and follow-up post-treatment recommended by the Brazilian Ministry of Health” (Line 232). Only for information, treatment with Benznidazole according to the protocol from Brazilian Health Ministry has been very successful for hundreds of oral ACD cases in the considerable experience of the IEC-Belém.

Point 4: "Finally, I was surprised by the omission of the extensive experience with oral cases accumulated by Dr. Noya de Alarcon in Venezuela where the main T. cruzi DTU is I. Although as the Authors report their strains groups separate from the Northern Type I T. cruzi there are still many lessons to learn from Dr. Noya´s group"

Response: We regret that we did not have the opportunity within the scope of this manuscript to comment on the various excellent studies of colleagues from Brazil, Venezuela (Dr Noya and Dr Añez), Colombia and others that published excellent studies on oral Chagas disease. It would be necessary to include many articles, and our option was to focus our introduction and discussion (already very long) on the Brazilian Amazon, where more than 80% of all cases reported of oral ACD in South America occurred.

Reviewer 4 Report

Trypanosoma cruzi and Trypanosoma rangeli in Acre, Brazilian Amazonia: Coinfection and unprecedented genetic diversity in an outbreak of orally acquired acute Chagas disease in a forest community, wild reservoirs, and vectors.

José Gabriel Vergara-Meza et al. described an outbreak of oral acute Chagas disease in the Brazilian Amazonia by analyzing the clinical symptoms, microscopy diagnosis, and genetic diversity of the parasites found in the infected patients. They found co-infections of different genotypes of T. cruzi and T. rageli, and were able to correlate their genetic footprint to the possible parasites´ hosts, which is the first time to be reported in that part of the world. The work is relevant to the understanding of the sylvatic transmission cycles of T. cruzi in an endangered area, and its impact on human Chagas disease. There are only minor spelling mistakes to be changed.

Line

123-134, 141- The Font size should be the same.

142- Change “apparatuses” to “devices”

 

192- Italics should be used for Trypanosoma cruzi and Trypanosoma rangeli.

Author Response

We would like to thank immensely all reviewers for all the constructive comments and valuable suggestions, and their careful review that very much improved our manuscript

Point 1: "Line 123-134, 141- The Font size should be the same".

Response:  Corrected

Point 2: 142- Change “apparatuses” to “devices”

Response: Changed

Point 3: "192- Italics should be used for Trypanosoma cruzi and Trypanosoma rangeli".

Response: Corrected