Epidemiological Features of Leptospirosis and Identification of Leptospira wolffii as a Persistently Prevailing Species in North–Central Bangladesh

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Author Response

1. Major grammatical errors present, the manuscript needs language editing.

-eg: residents in rural areas, Leptospira (should be in capital), Clinical characteristics,

 

Response: Thank you for careful checking on the manuscript. We carefully checked the sentences in the text with regard to spelling of words and grammar along with usages of general and technical words in English. The pointed portions were also corrected. In case of journals issued by MDPI, English editing will be done for all the manuscripts, which will complete keeping good quality of English.

 

2. Here are the specific comments and suggestions to enhance the clarity, coherence, and overall quality of the manuscript:

Method section needs to be more systematically written with sufficient technical details.

 

Response: Thank you for the comment. Originally, as a brief report format, the manuscript was written briefly and many descriptions of methods were omitted. According to the suggestions, Methods section was revised and more descriptions of sample collection, detailed methods of IgM-LAT, IgM-ELISA and nested PCR were added.

 

3. Use the term Visceral leishmaniasis instead of kala-azar.

 

Response: Thank you for the indication. This was corrected as pointed out.

 

4. Providing a clear inclusion criteria: were samples taken from all patients with febrile illness or only the samples that was tested negative for other diseases were chosen?

such as malaria, kala-azar, dengue etc.: what are the other diseases tested?

What was the exclusion criteria?

 

Response: Thank you for the comments. In the revised manuscript, inclusion criteria and exclusion criteria were added (line 93-102). In this study, we analyzed samples that were tested negative for Malaria, Visceral leishmaniasis, Dengue fever and Typhoid fever. This was written in the revised version.

 

5. 2-10 samples showed positive results by only one or two methods?

“In this study, the 88 samples (47%, 88/186) showing positivity in any of the three methods were judged as those of leptospirosis.” ELISA is not considered a definitive confirmatory test for leptospirosis.

 

Response: Yes, 2-10 samples were positive in only one or two tests. This finding is shown in Figure 1 in the revised manuscript. Fig.1 was supplementary Fig. S1 in original submission, but moved to main manuscript to show the findings to readers more easily.

It is acceptable that ELISA is not considered a definitive confirmatory test for leptospirosis. However, in our present study, solely ELISA-positive sample was only two, while most of ELISA-positive (67 samples among 69) samples were also positive by LAT and/or PCR showing high rate. This fact was added to the revised text. (line 156-157).

 

6. The primers used were published in 2009, would it detect new species of Leptospira effectively due to genetic variations?

 

Response: Yes, these primers could effectively amplify leptospiral DNA in PCR. Because the primers were designed on the conserved region of 16S rRNA, primers could work well in PCR.  

 

7. By the PCR-RFLP analysis for the nested PCR-positive samples, all the 78 samples were assigned to “pathogenic” or “intermediate” species (i.e., not non-pathogenic group), and were all negative by PCR with Lig1/Lig2 primers specific to “pathogenic” Leptospira?

 

Response: Yes, your comment is correct. PCR-RFLP showed that 78 samples belong to “pathogenic” or “intermediate” species, and PCR with Lig1/Lig2 primers showed the 78 samples were not pathogenic leptospira. These findings indicated that the 78 samples belong to “intermediate” leptospira group. This conclusion by PCR-RFLP and Lig1/Lig2-PCR was added to the revised manuscript. (line 163).

 

8. What is the justification for the selection of the 22 samples for 16S rRNA sequencing?

                            

Response: Thank you for pointing out it. Among all the samples, 22 samples that showed the prominent band on gel electrophoresis were selected for sequencing. This was added to the revised version (line 164-166).

 

9. Table 1: the title should clearly describe the content of the table

 

Response: Thank you for checking this point. The title of Table 1 was revised.

 

10. Table 2: inconsistent capitalisation

 

Response: Thank you for pointing out. Capitalisation of words in Table 2 was corrected.

 

11. The discussion was not presented effectively; laboratory findings which is found to be statistically significant is not discussed.

 

Response : Thank you for the comment. Discussion was also revised accordingly. The laboratory findings showing significant difference between leptospirosis group and non-leptospirosis group were discussed in two parts, line 342-345 and 350-355 in the revised manuscript. 

 

12. Are there any latest references for studies in Thailand and Malaysia?

 

Response: Thank you for the suggestion. As latest reference of epidemiological study in Thailand and Malaysia, reference no. 37 and 54 were added and described in line 362-363.  

Reviewer 2 Report

Comments and Suggestions for Authors

Introduction:

What animal species transmit Leptospirosis? How is this disease transmitted?

What about the findings of studies on Leptospirosis in other countries?

What about the symptoms of Leptospirosis, and how are patients treated?

 

Results:

Provide the overlap of positive samples detected by the three methods.

What diseases are non-leptospirosis patients infected with?

Author Response

1. Introduction:

What animal species transmit Leptospirosis? How is this disease transmitted?

 

Response: Thank you for the suggestion. Because of the Brief Report format, Introduction section was written somewhat simply in the original version. According to the suggestion, in the revised manuscript, animal species of reservoir of Leptospira and transmission pathway were written. (Line 43-45, 48-50)

 

2. What about the findings of studies on Leptospirosis in other countries?

 

Response: Thank you for the comment. In Introduction section, to avoid making it too lengthy, only global prevalence of leptospirosis with a higher incidence in the tropics and subtropics was written. Instead, in Discussion section, studies in Asia (Thailand, Malaysia, India) and Africa were cited and briefly explained (line 283-288). In the revised manuscript, two more reference (no.37, 54) of latest paper of Thailand and Malaysia were added in Discussion (line 362-363). 

 

3. What about the symptoms of Leptospirosis, and how are patients treated?

 

Response: Thank you for the suggestion. According to it, descriptions about symptoms and treatment were added to Introduction section of the revised manuscript. (line 51-61).

 

4. Results:

Provide the overlap of positive samples detected by the three methods.

 

Response: Thank you for the suggestion. Overlapping positive samples by three methods were described in Result section (line 155). In addition, numbers of overlapping positive samples by two (three) methods are shown in Figure 1. Although this figure was a supplementary material in original version, this was brought to Figure 1 in the main text of the revised manuscript.

 

5. What diseases are non-leptospirosis patients infected with?

 

Response: Thank you for the important question. Actually, we have not done any analysis to detect etiologic agents of non-leptospirosis febrile disease. Although we can just presume it based on any past evidence, there are some reports of Rickettsiosis (Rickettsial diseases) caused by R. felis and O. tsutsugamushi in the same study site. Such Rickettsiosis (Rickettsial diseases) may be a part of the non-leptospirosis disease. This was briefly described in the revised manuscript with the published references. (line 350-355)