Application of a sepsis flow chip (SFC) assay for the molecular diagnosis of paediatric sepsis

  1. Dimas Seto Prasetyo
  2. Mulya Rahma Karyanti
  3. Irene Yuniar
  4. Yulia Rosa Saharman
  5. Livya Holiwono

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Abstract

A delay in detecting sepsis pathogens is a problematic issue for determining definitive antibiotic therapy for the causative pathogens. The gold standard method for sepsis is blood culture but this requires 3 days to detect the definitive pathogen. Molecular methods offer rapid identification of pathogens. We evaluated the use of sepsis flow chip (SFC) assay for identifying pathogens from children with sepsis. Blood samples from children with sepsis were collected and incubated in a culture device. Positive samples were subjected to amplification-hybridization using SFC assay and culture. A total of 94 samples from 47 patients were recovered, from which 25 isolates were recovered, including Klebsiella pneumoniae (11) and Staphylococcus epidermidis (6). From 25 positive blood culture bottles subjected to SFC assay, 24 genus/species and 18 resistance genes were detected. The sensitivity, specificity and conformity was 80, 94.2 and 94.68 % respectively. SFC assay offers promise to identify pathogens from positive blood culture in paediatric patients with sepsis and may support the antimicrobial stewardship programme in hospitals.

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  1. Version published to 10.1099/acmi.0.000474.v4 on Access Microbiology
  3. Version published to 10.1099/acmi.0.000474.v3 on Access Microbiology
  4. Version published to 10.1099/acmi.0.000474.v2 on Access Microbiology
  5. Access Microbiology

    Comments to Author

    Authors have addressed Reviewer 1's comments sufficiently.

    Please rate the manuscript for methodological rigour

    Good

    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

  7. Access Microbiology

    Comments to Author

    1. Methodological rigour, reproducibility and availability of underlying data are appropriate 2. The presentation of the results has been improved in the revised version. 3. The style and organization of the paper communicates and represents key findings very well. 4. The literature analysis and discussion are adequate 5. Minor amendments are needed before publication - Line 57: please correct as: pairs - Line 58: please correct as: bottles - Lines 61-62: please correct as: is a promising assay to identify pathogens from positive blood culture in pediatric patients with sepsis - Line 82: please correct delayed with delay - Line 88: please correct as: assays - Line 100: please correct as: assays - Line 101: please correct as: an amplification - Line 104: please correct as: from positive blood cultures. - Line 105: please replace resistant with resistance - Line 106: please replace with: cephalosporins and carbapenems. - Line 122: please correct: patients - Lines 139-140: please rephrase The resistance gene detection ranged from mecA, to class B carbapenemases (e.g kpc, vim). I would suggest: The resistance gene detection included the most common antibiotic resistance genes, such as: mecA, b-lactamase genes (carbapenemases and ESBLs), vancomycin-restance genes (vanA and vanB). - Table 2. Please clarify: Enterobacteriaceae (other than K. pneumoniae) 2 (8.3) and correct Streptococcus pneumonia as Streptococcus pneumoniae - Table 5 may be omitted, as these data are mentioned in the manuscript (lines 170-172). - Line 186: please correct: as: an amplification-hybridization assay - Line 191: please correct sas: antibiotics - Line 205: please correct as: K. pneumoniae - Line 207: please correct as: A different finding - Line 208: please replace with: patients - Line 210: please replace with assays - Line 211: please replace with microbes - Lines 219-220: please correct as: similar resistance gene such as blaKPC, blaNDM, blaVIM, blaCTX-M, mecA, vanA, and vanB. - Gene names and species should be in italics

    Please rate the manuscript for methodological rigour

    Very good

    Please rate the quality of the presentation and structure of the manuscript

    Very good

    To what extent are the conclusions supported by the data?

    Strongly 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

  9. Access Microbiology

    Comments to Author

    1. Methodological rigour, reproducibility and availability of underlying data Small sample size: large sample size required for proper validation of techniques Study time period not mentioned Statistical analysis not included 2. Presentation of results: Confusing Separately results of Conventional method (Identification and AST) and Molecular method (SFC) presented. No comparison presented between both techniques * How many sample positive in Conventional method but negative in Molecular method (SFC) * Sample to sample comparison by both techniques should be presented * Identification and AST both should be compared with statistical analysis 3. How the style and organization of the paper communicates and represents key findings Need suggested modification 4. Literature analysis or discussion Modified according to result. 5. Any other relevant comments

    Please rate the manuscript for methodological rigour

    Poor

    Please rate the quality of the presentation and structure of the manuscript

    Good

    To what extent are the conclusions supported by the data?

    Not at all

    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?

    No: Require major revision

  10. Access Microbiology

    Comments to Author

    In the present study, the authors applied the sepsis flow chip (SFC) assay, a commercial molecular method for in diagnosing pediatric sepsis in Indonesia. As the authors suggest, this paper may be added as valuable data about molecular epidemiology of the causative pathogens of pediatric sepsis in Indonesia, since the genomic surveillances are rarely conducted. The species identification and detection of antibiotic resistance genes results of the method were compared with the results from conventional culture. Moreover, this study is an example for the genomic survellance in any hospital setting. The methodological rigour, reproducibility and availability of underlying data are appropriate, the presentation of results is clear, the style and organization of the paper communicates and represents key findings very well. The Literature analysis is adequate. Major comments: 1. I would suggest an alternative title for the manuscript, such as Application of Sepsis Flow Chip (SFC) assay for the Molecular Diagnosis of Pediatric Sepsis 2.The authors should mention if there were any microorganisms that they would be detected by conventional culture and not by (SFC) assay or vice versa 3. Table 2, Enterobacteriaceae, Klebsiella pneumoniae .Is this the percentage of isolation for K. pneumoniae only? If not, then please provide the percentage for K. pneumoniae only, and then the percentage for other Enterobacteriaceae. Accordingly, define the percentage Enterobacteriaceae 4. There are several grammatical and typographical errors. You would need to improve the grammar and language used. line 32: Delay in dete ''cting sepsis pathogens still become problematic issue. Please rephrase. Problematic issue for what? line 32:please correct as: gold standard method line 34: please correct.Molecular methods offers lines 35-36: Please correct samples line 39:Please use italics for the species names. line 42: Please correct as ''positive blood cultures of pediatric patients with sepsis. lines 57-58: please correct as A total of 94 samples from 47 patients were collected, and 25 isolates were recovered line 61: positive blood cultures in pediatric patients with sepsis line 85: Please replace with antibiotic therapy line 93: please delete ''A study'' and add Pammi M. et al., 2011 line 95: please delete ''Another study'' and add Sinha M. et al. 2018 line 98: please deleteAnother study'' and add Zacharioudakis IM, et al., 2019. lines 104-110:please correct '' is a microarray- based assay which can detect up to 21 genus/species of various species of bacteria and fungi from a positive blood cultures. mention how many genesm conferring resistance to which antibiotic classes??, delete: including mecA, vanA/B, blaCTX, blaSHV, and blaOXA-48 please correct a pediatric setting. lines 139-142: please delete these lines, This information is unnecessary, since it has been stated in the introduction section.

    Please rate the manuscript for methodological rigour

    Very good

    Please rate the quality of the presentation and structure of the manuscript

    Very good

    To what extent are the conclusions supported by the data?

    Strongly 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

  11. Access Microbiology

    Comments to Author

    The manuscript provides evidence about the advantages of the application of molecular methods for pediatric sepsis diagnosis. In that sense, it is relevant and may have a positive impact for clinicians in order to choose the specific antimicrobial therapy according with the identified pathogen. The manuscript shows very good results; nevertheless, authors must clarify some points. Abstract and outcome section: Lane 38 and 56- Authors stated, "There were 24 genus/species and 18 resistance genes detected" This sentence is difficult to understand, it is not clear if they are from the 25 isolated microorganisms or are from the total number of hemocultures. It is necessary to go through all the manuscript to make it clear. Abstract might be clearer to demonstrate the impact of the work. Objective and methods section: Lanes 103-104- the objective of the study was to evaluate the SFC as a diagnostic tool; the study design is prospective cross-sectional (lane 107), but it is necessary to include features of a diagnostic test design. As a good validation tool, author used a 2X2 table, but the calculation of the number of samples to be significant (N) should be determined, or if it was indeed calculated then it should be mentioned. It is not clear if the 94 blood cultures used are statistically significant according to the frequency of sepsis in their hospital or if that number was used as the total of hemocultures performed during a specific period. Lanes 126-128- It is not clear what was the volume of blood culture used for the SFC. Authors wrote 500 ul, but after that it seems that a 1:10 dilution was performed and then 4 ul of the dilution was the real volume used for the molecular technique. Please rewrite the sentence. Lanes 149-150- Sensitivity, specificity and conformity were calculated from 2X2 table. With the same table it is possible to calculate the predictive values (positive and negative PPV, NPV), please justify the reason for not doing that. Results section: Table 1, 2, and 4. There were some differences in species identification between the 2 compared methods, author should explain it. Differences in genus/ species occurred only when one method was positive and the other negative or differences in microbial identification (between methods) were observed using the same positive sample? Discussion section: Lane 183- Hemoculture/Vitek positivity rate was 26%, but positivity obtained using SFC is not mentioned. Is it significant or the value of performing this particular methodology relies in results time? Lanes 22-225- About resistance gene detection. Manuscript mentions the advantage of determining resistance genes to support antimicrobial stewardship programs, but gene identification does not necessary mean that antimicrobial resistance is expressed. Anyway, authors have the results obtained with the Vitek system. Phenotypic resistance (vitek susceptibility test) was related to gene amplification?

    Please rate the manuscript for methodological rigour

    Good

    Please rate the quality of the presentation and structure of the manuscript

    Very good

    To what extent are the conclusions supported by the data?

    Strongly 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

  12. Access Microbiology

    Comments to Author

    1. Methodological rigour, reproducibility and availability of underlying data good 2. Presentation of results good 3. How the style and organization of the paper communicates and represents key findings good 4. Literature analysis or discussion good 5. Any other relevant comments. There are some issues that should be considered. English needs revising. Line: 51 This paper may be added as valuable data about molecular epidemiology of the causative pathogens of pediatric sepsis in Indonesia. --> How a single center with 94 samples is a representative of a high populated county like Indonesia? This sentence should be revised. Line 56 including K. pneumoniae (11), and S. epidermidis (6). --> E. epidermidis and S. saprophyticus are the most reported species as the contaminant of blood cultures. How authors certain about excluding this bias? Line 142 . Of 94 samples, 25 isolates (blood sample positivity was 26.59%) were identified, as shown in table 1. --> Please indicate that this is the results of Vitek-2 Compact in this sentence. Line 167 Table 4 can be presented in a simpler format. Positive, Negative, and Result in the first arrow can be omitted. The table will be easer to understand.

    Please rate the manuscript for methodological rigour

    Good

    Please rate the quality of the presentation and structure of the manuscript

    Good

    To what extent are the conclusions supported by the data?

    Strongly 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

  13. Version published to 10.1099/acmi.0.000474.v1 on Access Microbiology