Showing the limitations of available phenotypic assays to detect B. pseudomallei from clinical specimens in Nigeria
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Burkholderia is a genus comprising Gram-negative bacteria that are metabolically complex and versatile, thriving in frequently hostile settings. Burkholderia pseudomallei, the causative agent of melioidosis, is a prominent member of the genus and a clinical pathogen of tropical and sub-tropical regions. This pathogen is well known for its multidrug resistance nature and possible bioweapon potential. There is currently no report of the pathogen from clinical specimens in Nigeria, this might be due to misdiagnosis with phenotypic assays. This study aims to explore the accuracy in the use of phenotypic assays to accurately diagnose B. pseudomallei in Nigeria. Two hundred and seventeen (217) clinical samples and twenty-eight (28) Gram-negative clinical isolates were collected and analysed using Ashdown selective agar and monoclonal antibody-based latex agglutination. The species-level identification was achieved using the Analytical profile index (API) 20NE system. The susceptibility of the isolates to nine different antimicrobial agents was determined using the disk diffusion method. Sixty-seven (74) culture-positive isolates were obtained using Ashdown selective agar. Twenty-two isolates were believed to be B. pseudomallei through the monoclonal antibody-based latex agglutination test and API 20NE system subsequently identified 14 isolates as Burkholderia. The predominant Burkholderia species was B. cepacia with isolation rate of 30.8% (8/26). No isolate was distinctively identified as B. pseudomallei but five isolates were highly suspected to be B. pseudomallei with similarity indices ranging from 81.9 – 91.3%. Other bacterial species with definitive identity include Aeromonas sp., Sphingomonas sp. and Pseudomonas aeruginosa. The antibiotic susceptibility results revealed an overall resistance to amoxicillin-clavullanic acid (71.4%), cefepime (33.3%), trimethoprim-sulfamethoxazole (38.1%), piperacillin-tazobactam (33.3%), imipenem (66.7%), doxycycline (57.1%), and ceftazidime (66.7%). The highest intermediate resistance was observed for cefepime and piperacillin-tazobactam with a value 66.7% each while no intermediate-resistance for gentamicin, colistin and imipenem. Our findings, therefore, show that phenotypic assays alone are not enough in the diagnosis of melioidosis. Additionally, it provides a robust basis for supporting present and future decisions to expand diagnostic capability for melioidosis beyond phenotypic assays in low-resource settings.
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All changes have been made appropriately and the manuscript is now ready for publication.
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The major outstanding issue with this manuscript is the references, which was flagged by reviewer 1 but has not been addressed. The vast majority of the references are either not referencing what you are talking about, or are not the primary sources of the information. For example, one line in the text reads "Subsequently, some species have been detected from poultry birds (15), freshwater fishes (16) and hospital effluents (17)", but reference 17 is about the misidentification of different species of bacteria, and it is in fact reference 8 which talks about detection in hospital effluents. Another example is reference 1 which is a review article discussing immune evasion mechanisms but it is being used as a reference for the statement "The Gram-negative bacteria, Burkholderia, are ecologically diverse genus with over 70 species whose …
The major outstanding issue with this manuscript is the references, which was flagged by reviewer 1 but has not been addressed. The vast majority of the references are either not referencing what you are talking about, or are not the primary sources of the information. For example, one line in the text reads "Subsequently, some species have been detected from poultry birds (15), freshwater fishes (16) and hospital effluents (17)", but reference 17 is about the misidentification of different species of bacteria, and it is in fact reference 8 which talks about detection in hospital effluents. Another example is reference 1 which is a review article discussing immune evasion mechanisms but it is being used as a reference for the statement "The Gram-negative bacteria, Burkholderia, are ecologically diverse genus with over 70 species whose heterogeneousness has led to multiple taxonomic changes over time 1, 2". This is not an appropriate reference for that statement. Please carefully check that all references are backing up the statements being made, and are the appropriate primary sources of information. Other minor changes required; Fig 5 legend- recurrence should be occurrence Fig 3- please rotate the X axis labels to be horizontal Ashdown should be Ashdown's throughout the manuscript Lastly, I think the data provided in the supplementary excel sheet could be made more useful if the phenotypic test results (catalase, oxidase, motility, agglutination) for each of the strains was added to sheet "sample info of identified strains" so the reader could see what combination of test results make up the diagnostic profile of each species.
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Thank you to both reviewers for their thorough reports on this manuscript. Please revise your manuscript in line with these comments, including paying close attention to discrepancies in number of samples reported at different points throughout the manuscript. I agree with both reviewers that a title change would be beneficial to reflect the findings of the manuscript, rather than a description of the work that has been carried out. Reviewer 2's suggestion to add a flow chart describing the number of samples at each diagnostic stage would help to make clearer the testing strategy.
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Comments to Author
This article is a good reminder that phenotypic assays are still a unique resource for some countries. Thus, it is important to improve identification tests when resources are limited especially since sequencing is routinely used for isolate identification in the majority of countries. The authors have proven that existing phenotypic assays fail to identify closed-related bacterial species within the Burkholderia genus. 1. Methodological rigour, reproducibility and availability of underlying data The article comprises minor mistakes, such as spelling, missing information, and mistakes in reported numbers between the text and the figures. Most of the supplementary data are missing. Adding them will add strength to the article. I believe that the study does not request any complementary experiments. …
Comments to Author
This article is a good reminder that phenotypic assays are still a unique resource for some countries. Thus, it is important to improve identification tests when resources are limited especially since sequencing is routinely used for isolate identification in the majority of countries. The authors have proven that existing phenotypic assays fail to identify closed-related bacterial species within the Burkholderia genus. 1. Methodological rigour, reproducibility and availability of underlying data The article comprises minor mistakes, such as spelling, missing information, and mistakes in reported numbers between the text and the figures. Most of the supplementary data are missing. Adding them will add strength to the article. I believe that the study does not request any complementary experiments. However, I have noted that the article lacks linking bacterial isolates with their original origin (provided in sheet 1 in Excel supplementary data) and their phenotypic characterization. I believe that the phenotypic assays used in this study are specific for Burkholderia identification. However, the method part is missing information about protocol and references for most phenotypic assays. Without this information, it will be difficult to reproduce the experiments. 2. Presentation of results In my opinion, provided figures are sufficient for the conclusion of the study. I have made some suggestions to improve the figures. 3. How the style and organization of the paper communicates and represents key findings The article follows a logical process to identify bacterial isolates based on phenotypic assays. 4. Literature analysis or discussion The authors have discussed their results with the literature and have identified the limitations of their study including the confirmation of the species of the identified Burkholderia isolates and the lack of repeat experiments due to low resources. I believe that this PCR or sequencing verification and repeating the identification process would have added strength to the study. However, all phenotypic assays direct to the same conclusion: they fail identifying bacterial species within a bacterial genus. 5. Any other relevant comments Please find the comments: Title: I would suggest changing the title to represent the main conclusion of the study; showing the limitations of available phenotypic assays to detect B. pseudomallei in Nigeria. Line 24: misspelling of "pseudomallei" Line 104-106: Please provide more information and references about protocols and replicates used for all phenotypic assays. Could you also provide pictures or data for the colony morphotypes for isolates? Line 132-134: Please provide references to support the antibiotic quantities used for the antimicrobial resistance assay. Line 138: the dot at the end of the sentence is missing. Line 141: There are no heatmaps in the manuscript. Lines 144-146: Sheet 1 from the supplementary data Excel file records 218 patients/clinical specimens, the text says 217. The result of growth on both MacConkey Agar and Ashdown medium should be linked to the clinical specimen in supplementary data. Lines 147 to 149: The origin of samples should be added as supplementary data. Line 149: Please specify how the isolates have been confirmed to be Gram-negative. Line 149-156: Please add the positive/negative results for each performed test in supplementary data. Line 166-168: The numbers of clinical specimens isolated from patients ranked by age do not match with Figure 3. For example, 7 isolates belong to the age 31-40 group in the text while the graph only shows five females + one male if my understanding is correct. Line 173: Please specify the total number of isolates that have been tested by API 20NE and the raw data in supplementary data. I am not sure what is "(99.9% ID,". Line 175: Supplementary data are missing. Line 179: I believe that "Figure 3" should be Figure 4. Line 188: Please justify the choice of the antimicrobials that have been tested. Line 203: Please provide details for the characterization by including the range of zones of inhibition that define Susceptible, Intermediate and Resistance phenotypes. Line 195: I would suggest adding the abbreviation used for each antibiotic in the paragraph. Line 255: "B. cepacia", B is missing italic font. Figure 2: I would suggest adding a flowchart describing the results (from 217/218 clinical specimens to 26 API-tested isolates) with the barplot for each test to prove the results. Figure 3: My first understanding is the graph is a stacked barplot. However, from the description in the text at line 166, it seems that the female columns are in the background of the male columns. I would suggest either separating female and male into two columns side by side with the actual y-axis or using a stacked barplot where one column is split between male/female with a total number of both sexes. Figure 4: I would suggest adding two y-axes for the graph with names. The supplementary data Excel file name is "Burlkhoderia" instead of Burkholderia. The First sheet is not named. Supplementary data should be named by tables and figures and reported by table and figure numbers in the text.
Please rate the manuscript for methodological rigour
Satisfactory
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?
No: N/A, the article does not comprise animal work.
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Comments to Author
Diagnosis is critical to successfully treat Burkholderia pseudomallei, the cause of melioidosis, underlying importance of this study. Here, the authors tested 217 clinical samples and 28 gram-negative purified bacterial isolates in attempt to identify B. pseudomallei. API 20NE and latex agglutination tests which were used by authors are accurate for identification of B. pseudomallei (Accuracy of Burkholderia pseudomallei Identification Using the API 20NE System and a Latex Agglutination Test - PMC (nih.gov)) but cross-reactivity of latex agglutination tests for melioidosis with B. territorii, B. pseudomultivorans, B. multivorans and B. cenocepacia isolates was also reported (Cross-reactivity of latex agglutination assay complicates the identification of Burkholderia pseudomallei from soil - PubMed …
Comments to Author
Diagnosis is critical to successfully treat Burkholderia pseudomallei, the cause of melioidosis, underlying importance of this study. Here, the authors tested 217 clinical samples and 28 gram-negative purified bacterial isolates in attempt to identify B. pseudomallei. API 20NE and latex agglutination tests which were used by authors are accurate for identification of B. pseudomallei (Accuracy of Burkholderia pseudomallei Identification Using the API 20NE System and a Latex Agglutination Test - PMC (nih.gov)) but cross-reactivity of latex agglutination tests for melioidosis with B. territorii, B. pseudomultivorans, B. multivorans and B. cenocepacia isolates was also reported (Cross-reactivity of latex agglutination assay complicates the identification of Burkholderia pseudomallei from soil - PubMed (nih.gov)). The results of identification were inconclusive, maybe due to lack of testing proficiency (e.g. not using correct controls). There were several isolates identified as Burkholderia which maybe were or maybe were not B. pseudomallei. Antimicrobial susceptibility of these isolates was tested by disc diffusion, which is one of EUCAST recommended methods for this bacterium. The high levels of resistance to clinically important antimicrobials for B. pseudomallei were found (ceftazidime, imipenem), but significance of this finding is not clear as the isolates might not be B. pseudomallei. Comments. 1. From the start, the title is grammatically incorrect and misleading: "specimen" should be plural "specimens" ; "Ashdown's" not "Ashdown" is the correct name for the agar, but it is not needed in the title because AST is not be performed on this agar; most importantly, the authors state (lines 37-38) "No isolate was distinctively identified as B. pseudomallei", then why "Burkholderia pseudomallei" is in the title? 2. References do not cite the original work and/or do not correspond to the text at all. The example of completely wrong use is the reference is #6 which should refer to "B. pseudomallei designated a bioterrorism agent" but it is "Adebowale O, Adeyemo O. Characterization of bacterium types isolated from commercial laying hen farms in Ogun State Nigeria. Rev élev méd vét pays trop. 2018; 71(3):137-141." References 8,9 supposed to list endemic areas, but they are susceptibility papers from Nigeria. #14 is not 2011 Salam paper. All references need to be verified. 3. Methods are mainly well written and appropriate. Statistical analysis method needs explanation on which data were analyzed. Was it AST data? Results: 4. not clear what is "n" is in lines 147-149. 5. Line 150: delete "and" 6. For latex agglutination: in the Abstract, 26 isolates were reported positive by latex agglutination (line 34), in Results - 22 isolates were reported positive (line 155). Which one is correct? Which isolates were tested by latex agglutination? From Suppl Material, 45 isolates were tested but not clear, how they were selected for the test. From Abstract, it sounds that 67 isolates strains that grew on Ashdown's from 217 clinical specimens were tested and none of the 28 Gram-negative clinical isolates were tested. This needs verification and explanation. 7. Which isolates were analyzed by API 20NE? Did the authors find 26 or 8 B. cepacia? - line 173. 8. Line 179 - should this be Figure 4? 9. Figures: Legends need more details. The font is too small in the figures. 10. Table 1 and AST results. Is it possible to abbreviate antimicrobial names according to - Abbreviations and Conventions (asm.org) ?
Please rate the manuscript for methodological rigour
Satisfactory
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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My initial checks of this manuscript before sending it for peer review have left me with concerns about the presentation of the data in figures 2, 3, and 4. The use of heat maps for figures 2 and 3 are inappropriate and are not interpretable. The data presented in figure two should be represented by a simple bar chart, table, or even a flow diagram with exact numbers given. The current use of a heat map means that reviewers cannot see from this figure how many samples fall into each category, and are left guessing as to how many samples were positive and negative for each test type. I know the numbers are stated in the text, but the graph needs to be useful in its own right. Similarly for figure 3 a histogram should be used to display this age category data, not a heat map. On figure 4, it is not possible to see the frequency data as …
My initial checks of this manuscript before sending it for peer review have left me with concerns about the presentation of the data in figures 2, 3, and 4. The use of heat maps for figures 2 and 3 are inappropriate and are not interpretable. The data presented in figure two should be represented by a simple bar chart, table, or even a flow diagram with exact numbers given. The current use of a heat map means that reviewers cannot see from this figure how many samples fall into each category, and are left guessing as to how many samples were positive and negative for each test type. I know the numbers are stated in the text, but the graph needs to be useful in its own right. Similarly for figure 3 a histogram should be used to display this age category data, not a heat map. On figure 4, it is not possible to see the frequency data as it is hidden behind the % occurrence bars. Please re-graph the data in the manuscript so that we may consider it for peer review.
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