A study on viruses and bacteria with particular interest on Mycoplasma pneumoniae in children with exacerbation of asthma from a tertiary care hospital in Sri Lanka
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Asthma is a significant public health concern, particularly in children with severe symptoms. Exacerbation of asthma (EOA) is life-threatening and respiratory infections (RIs) play a crucial role. Though viruses play a significant role in EOA, patients are empirically treated with antibiotics which contribute to the development of antibiotic resistance. Although there are widely reported association of EOA with viral or M. pneumoniae infections, there are no published data in Sri Lanka. The present study aimed to identify the association of common respiratory viruses, typical respiratory bacterial pathogens, and M. pneumoniae in children with EOA and relate them with the compatibility of antimicrobial use. A case-control study was conducted in the pediatric unit of North Colombo Teaching Hospital, Sri Lanka, involving two groups of children between 5-15 years of age. Group-1: children with EOA, Group-2: children with stable asthma (SA). Each group consisted of 100 children. Sputum/throat swabs were tested for common respiratory viruses using virus specific FITC-labelled monoclonal antibodies (MAbs), bacteria by routine culture and M. pneumoniae by RT-PCR. Macrolide-resistance in M. pneumoniae was detected using conventional PCR and sequencing specific genetic mutations in the 23S rRNA gene. M. pneumoniae was genotyped using nested multilocus sequence typing (MLST), which targeted eight housekeeping genes (ppa, pgm, gyrB, gmk, glyA, atpA, arcC, adk). There was no significant difference in age, gender, demographic or geographical locations between the two groups. In children with EOA, antibiotics were used in 66% (66/100) and macrolides in 42% (42/100) in children with EOA. Samples consisted of 78% (78/100) sputum and 22% (22/100) throat swabs. Adenovirus was the most common virus identified, and it was significantly higher in children with EOA compared to those with SA, but no significant difference in typical bacteria findings between the two groups. M. pneumoniae was detected in one patient with EOA, with none detected in the SA group. The M. pneumoniae was macrolide sensitive, and it was ST14 by Multi Locus Sequence Typing. This study showed that the empiric use of antibiotics in children with asthma may be better targeted with prior pathogen screening to inform appropriate treatment to minimize antibiotic resistance.
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Thank you for responding to all comments.
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Thank you for responding to the reviewer comments. I notice the gels in Fig 3 are still labelled as Fig 4 after the removal of the previously named Fig 3. Please could this be amended and the manuscript checking for figure numbering consistency; for example, the text at line 255/256 no longer makes sense after the figure removal.
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Thank you for submitting a revised manuscript that answers the majority of the reviewer queries. Before this is accepted for publication, please could you address the following points: 1. The abbreviations for the viruses and bacteria detected using the DFA test (lines 198-202) should be included in the legend for Table 1. 2. Reviewer 2 - "To clarify is there a link between which pathogens were detected together? was the patient who had M. pneumoniae infected with other pathogens? how were the statistics measured?" - please could your response to this be included in the manuscript as it is an interesting point of note. 3. Thank you for your clarification regarding Fig 3. Given the sequencing did not find resistance mutations and the point raised regarding the clarity of the gel, I recommend this figure be removed. 4. Fig 4A - There are …
Thank you for submitting a revised manuscript that answers the majority of the reviewer queries. Before this is accepted for publication, please could you address the following points: 1. The abbreviations for the viruses and bacteria detected using the DFA test (lines 198-202) should be included in the legend for Table 1. 2. Reviewer 2 - "To clarify is there a link between which pathogens were detected together? was the patient who had M. pneumoniae infected with other pathogens? how were the statistics measured?" - please could your response to this be included in the manuscript as it is an interesting point of note. 3. Thank you for your clarification regarding Fig 3. Given the sequencing did not find resistance mutations and the point raised regarding the clarity of the gel, I recommend this figure be removed. 4. Fig 4A - There are arrows missing from this figure. Please include them to indicate the sizes of the bands in the ladder. 5. Fig 4B - The reviewer raised a query about the other bands in the gel, please could you provide an explanation for those. 6. Please could all positive and negative controls be defined clearly as per reviewer request.
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The reviewers have highlighted areas of concern that need to be addressed. Please pay particular attention to the points raised about the figures and related context, and consider a title that is more reflective of the manuscript. In addition, please consider an alternative way to present Figure 2 that is more appropriate for the data being presented.
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Comments to Author
Dear Authors, I have a few concerns about your manuscript: Pathogen detection in childhood exacerbated asthma in Sri Lanka, role and characteristics of Mycoplasma pneumoniae and antibiotic stewardship. 1. Please add a reference into the Material and Methods section: Detection of macrolide resistance in M. pneumoniae by conventional PCR. 2. In regards to all figures- they tend to appear without any context or referencing text so it can often be hard to follow what the data is, what it is showing/it's importance, why each thing was done. 3. Figure 1 is missing a figure legend? In terms of this data, and it's relation to the manuscript topic/title: which of these antibiotics are macrolides? is there a difference in susceptibility between macrolides and non-macrolides? did they clear infections? which …
Comments to Author
Dear Authors, I have a few concerns about your manuscript: Pathogen detection in childhood exacerbated asthma in Sri Lanka, role and characteristics of Mycoplasma pneumoniae and antibiotic stewardship. 1. Please add a reference into the Material and Methods section: Detection of macrolide resistance in M. pneumoniae by conventional PCR. 2. In regards to all figures- they tend to appear without any context or referencing text so it can often be hard to follow what the data is, what it is showing/it's importance, why each thing was done. 3. Figure 1 is missing a figure legend? In terms of this data, and it's relation to the manuscript topic/title: which of these antibiotics are macrolides? is there a difference in susceptibility between macrolides and non-macrolides? did they clear infections? which were used to treat M. pneumoniae? 4. Figure 2:- what is the FA test? What does it do? to clarify is there a link between which pathogens were detected together? was the patient who had M. pneumoniae infected with other pathogens? how were the statistics measured? 5. Figure3: What is this gel in relation to? what are the samples? Presumably this is the M. pneumoniae sample, but what is the positive control? How does figure 3 relate to the fig legend title: how is this showing sensitivity, what is the size of these bands? what would a 'resistant' isolate look like? Lanes 2 and 3 show the positive control in duplicate, but there is an additional band in lane 3 that doesn't look like it appears in lane 2- what is this? 6. Figure4: Is there data missing here? figure shows an image of a gel labelled lanes 1-10 but legend discusses up to lane 18? 4A ladder is a smear so very hard to definitively know the size of these bands. without a positive control or text to refer back to here, it is hard to know if these are the size we should be expecting? or what the size of bands in the gel actually are? 4b- to clarify is this only showing the results of the pygm PCR? This gel is quite unclear- its quite hard to see the bands, is it possible to have a version without the background black band? there appears to be multiple bands in the patient samples that are not present in the positive control? what are these? why are they there? Again what is the positive sample? Is lane 6 a negative control or is it empty? 7. The title of the manuscript is: Pathogen detection in childhood exacerbated asthma in Sri Lanka, role and characteristics of Mycoplasma pneumoniae and antibiotic stewardship. In this cohort there is only one patient where M. pneumoniae is detectable, can you explain why this is a large enough cohort to for you to be able to explain 'the role and characteristics of Mycoplasma pneumoniae and antibiotic stewardship'? I think the data discussed in the discussion leans more into this, but I think a larger cohort from for your own studies is important to back up this title? In relation to the antibiotic stewardship, and to link back to the aims of your project (lines 55-58) would it be possible to include the susceptibility/resistance profiles to the antibiotics used if this is available?
Please rate the manuscript for methodological rigour
Poor
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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Comments to Author
Dear Authors, Thank you for submitting the manuscript. Unfortunately the paper lacks many significant information which are required for such studies. Some of these information are: 1. The title doesn't reflect what you had actually in the findings. You mentioned in the title (characteristics of Mycoplasma pneumoniae and antibiotic stewardship) however you just had one patient who was positive for M. pneumoniae which doesn't make any sense. Also, you did not focus on antibiotic stewardship in the paper. 2. The introduction lacks many significant background information and doesn't include enough references. 3. For the results you need to include a baseline characteristics table for both groups with data on previous colonisation, lung function, inflammatory markers etc. which is essential for this …
Comments to Author
Dear Authors, Thank you for submitting the manuscript. Unfortunately the paper lacks many significant information which are required for such studies. Some of these information are: 1. The title doesn't reflect what you had actually in the findings. You mentioned in the title (characteristics of Mycoplasma pneumoniae and antibiotic stewardship) however you just had one patient who was positive for M. pneumoniae which doesn't make any sense. Also, you did not focus on antibiotic stewardship in the paper. 2. The introduction lacks many significant background information and doesn't include enough references. 3. For the results you need to include a baseline characteristics table for both groups with data on previous colonisation, lung function, inflammatory markers etc. which is essential for this study. 4. Please consider a better context of this research, in this way, the audience could better appreciate the significance of this study. 4. Please consider add a clear and testable hypothesis of this research. 5. Figures are not clear.
Please rate the manuscript for methodological rigour
Poor
Please rate the quality of the presentation and structure of the manuscript
Poor
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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