Evaluation of an SNP-Based Diagnostic Assay for Enteric Fever Detection in Resource-Limited Settings
, Farhana Khanam 1, Fahad Khokhar 2,3, Zoe Dyson 2,4, Derek J. Pickard 2, Gordon Dougan 2, Ankur Mutreja 2,5 and Firdausi Qadri 1,*
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors present a method for the identification and differentiation of Salmonella Typhi (non-H58), Salmonella Typhi (H58), and Salmonella Paratyphi A using simplex PCR assays. In this study, new primers targeting gene SSPA2308 are proposed as a marker for S. Paratyphi A, while primers targeting genes STY0307 and C for S. Typhi and S. Typhi H-58, respectively, were described in the authors’ previous work. The topic is relevant, and the development of rapid molecular tools for distinguishing typhoidal Salmonella serovars is of potential diagnostic value. However, several issues should be addressed to improve the clarity, methodological robustness, and clinical relevance of the study.
Major comments
- Introduction and clinical relevance
The Introduction requires revision and expansion, particularly regarding the clinical differences between typhoidal serovars. While the authors highlight the importance of rapid differentiation (lines 66–68), the clinical benefits of such differentiation are not clearly explained. For instance, it remains unclear whether distinguishing between these serovars leads to different clinical management strategies, such as tailored antibiotic therapy. Although some variation in antimicrobial resistance exists, it is not necessarily serovar-specific but rather associated with the acquisition of resistance genes. From a clinical perspective, the development of rapid diagnostics targeting antimicrobial resistance profiles may be more impactful than assays focused solely on serovar identification. This aspect should be clarified and better justified. - Limit of detection (LOD) and sample type
The limit of detection (LOD) is presented in terms of colony-forming units (CFU), whereas PCR-based assays are typically characterized using DNA quantity or copy number. While CFU-based estimation may be acceptable when working with pure cultures, the proposed method relies on colonies obtained from already isolated and selected cultures grown on solid media. Under such conditions, CFU can be approximated to DNA copy number; however, this does not reflect real clinical samples, which contain mixed DNA populations. Therefore, the method requires validation using non-purified samples. Importantly, the need to isolate pure cultures prior to PCR negates the time advantage typically associated with PCR-based diagnostics. Indeed, the time required for culture isolation is comparable to that needed for performing an antibiogram, thus limiting the practical benefit of the proposed approach. - Specificity and workflow of identification
The proposed identification strategy requires further clarification. If amplification of the STY0307 target yields a positive result for all S. Typhi (H58 and non-H58) strains, simplex PCR alone is insufficient for definitive identification. A positive result for gene STY0307 only excludes S. Paratyphi A from S Thypi. Additional amplification of gene STY2513 is required to differentiate between the two S. Typhi types. Consequently, the method relies on sequential testing, which reduces both efficiency and practicality. To improve reliability and speed, the authors should consider either performing all assays for each sample or developing a multiplex PCR approach. - Quality of electrophoresis images (Figure 1)
The electrophoresis images in Figure 1 require significant improvement. The DNA size marker is poorly visible, with faint and insufficiently resolved bands, making it difficult to estimate the size of PCR products. While the authors may rely on comparison with positive controls, the method is based on the detection of amplicons of defined size, and therefore a clearly visible and well-resolved DNA ladder is essential. Additionally, the figure legend should specify the type of DNA ladder used and the sizes of its bands to allow independent assessment by the reader.
Author Response
The authors present a method for the identification and differentiation of Salmonella Typhi (non-H58), Salmonella Typhi (H58), and Salmonella Paratyphi A using simplex PCR assays. In this study, new primers targeting gene SSPA2308 are proposed as a marker for S. Paratyphi A, while primers targeting genes STY0307 and C for S. Typhi and S. Typhi H-58, respectively, were described in the authors’ previous work. The topic is relevant, and the development of rapid molecular tools for distinguishing typhoidal Salmonella serovars is of potential diagnostic value. However, several issues should be addressed to improve the clarity, methodological robustness, and clinical relevance of the study.
Major comments
- Introduction and clinical relevance
The Introduction requires revision and expansion, particularly regarding the clinical differences between typhoidal serovars. While the authors highlight the importance of rapid differentiation (lines 66–68), the clinical benefits of such differentiation are not clearly explained. For instance, it remains unclear whether distinguishing between these serovars leads to different clinical management strategies, such as tailored antibiotic therapy. Although some variation in antimicrobial resistance exists, it is not necessarily serovar-specific but rather associated with the acquisition of resistance genes. From a clinical perspective, the development of rapid diagnostics targeting antimicrobial resistance profiles may be more impactful than assays focused solely on serovar identification. This aspect should be clarified and better justified.
Response: Thank you for this insightful comment. We agree that the clinical relevance of distinguishing between typhoidal serovars needs further clarification. Accordingly, we have revised the Introduction to justify the importance of serovar and lineage-level differentiation targeting the AMR profile. These revisions are now incorporated in the Introduction (Lines 68-76) in the revised manuscript.
- Limit of detection (LOD) and sample type
The limit of detection (LOD) is presented in terms of colony-forming units (CFU), whereas PCR-based assays are typically characterized using DNA quantity or copy number. While CFU-based estimation may be acceptable when working with pure cultures, the proposed method relies on colonies obtained from already isolated and selected cultures grown on solid media. Under such conditions, CFU can be approximated to DNA copy number; however, this does not reflect real clinical samples, which contain mixed DNA populations. Therefore, the method requires validation using non-purified samples. Importantly, the need to isolate pure cultures prior to PCR negates the time advantage typically associated with PCR-based diagnostics. Indeed, the time required for culture isolation is comparable to that needed for performing an antibiogram, thus limiting the practical benefit of the proposed approach.
Response:
We thank the reviewer for this important comment. We agree that LOD in PCR-based assays is often expressed in terms of DNA quantity or genome copy number, particularly for direct clinical sample applications. In our study, we validate the PCR assay from DNA extracted from pure bacterial culture isolates rather than from raw clinical samples (blood, stool). For this reason, we calculated LOD from the CFU-based estimation. We have now clarified this limitation in the revised manuscript (Lines 326-334). Importantly, we have emphasized in the revised manuscript that future work will focus on adapting and optimizing this assay for direct detection from clinical samples using enrichment strategies. This would significantly reduce the turnaround time of culture and enhance its utility as a rapid diagnostic tool.
- Specificity and workflow of identification
The proposed identification strategy requires further clarification. If amplification of the STY0307 target yields a positive result for all S. Typhi (H58 and non-H58) strains, simplex PCR alone is insufficient for definitive identification. A positive result for gene STY0307 only excludes S. Paratyphi A from S Thypi. Additional amplification of gene STY2513 is required to differentiate between the two S. Typhi types. Consequently, the method relies on sequential testing, which reduces both efficiency and practicality. To improve reliability and speed, the authors should consider either performing all assays for each sample or developing a multiplex PCR approach.
Response: We strongly agree with the comments. We acknowledged in our manuscript that optimization of multiplex PCR is underway, which would significantly improve efficiency for simultaneous detection and differentiation of S. Typhi, S. Paratyphi A, and H58 lineage in a single reaction. Regarding this consideration, we already acknowledged this as a limitation and also mentioned that we are working on optimizing the multiplex assay (line 334-338)
- Quality of electrophoresis images (Figure 1)
The electrophoresis images in Figure 1 require significant improvement. The DNA size marker is poorly visible, with faint and insufficiently resolved bands, making it difficult to estimate the size of PCR products. While the authors may rely on comparison with positive controls, the method is based on the detection of amplicons of defined size, and therefore a clearly visible and well-resolved DNA ladder is essential. Additionally, the figure legend should specify the type of DNA ladder used and the sizes of its bands to allow independent assessment by the reader.
Response: We sincerely apologize for the lack of clarity in the electrophoresis images presented in Figure 1 and appreciate the reviewer’s valuable feedback. We have revised Figure 1 to improve the visibility and resolution of the DNA ladder by adjusting image brightness and contrast uniformly. Additionally, we have added the labelling of the DNA ladder and added the information about the DNA ladder in the figure legend (lines 227-228 in the revised manuscript).
Reviewer 2 Report
Comments and Suggestions for AuthorsOverall Assessment
This study evaluates a SNP‑based conventional PCR assay for the detection of Salmonella Typhi, Salmonella Paratyphi A, and the globally dominant, drug‑resistant H58 lineage of Typhi using isolates from blood‑culture‑positive patients in Bangladesh. The assay uses simple crude DNA extraction, achieves 100% sensitivity and specificity on cultured isolates, and has a clear limit of detection (LOD = 5.2 × 10⁴ CFU per reaction). The manuscript provides a useful bridge between genomic surveillance and field‑deployable diagnostics. However, revisions are needed to strengthen novelty, improve rigor in LOD calculation, and address key limitations more transparently.
Major comments:
The primers for Typhi and H58 were previously published (Khokhar et al., 2022, https://pmc.ncbi.nlm.nih.gov/articles/PMC9307194/), and the Paratyphi A assay is added here.
Please consider adding information which could improve this paper: the independent validation in a Bangladeshi strain panel, direct performance comparison to blood culture as the reference standard, utility for local surveillance and treatment guidance.
Explain why the LOD is reported as 5.2 × 10⁴ CFU per 2 μL template, but this is relatively high for clinical blood samples (which often have <1 CFU/mL), the dilution experiment shows detection down to 10⁻¹ dilution, but CFU counting was done at 10⁻³ and 10⁻⁴.
Table1 use three line table.
Define all abbreviations in the abstract.
Strengthen comparison to other enteric fever PCR assays (sensitivity, cost, ease of use) in the discussion section.
Author Response
This study evaluates a SNP‑based conventional PCR assay for the detection of Salmonella Typhi, Salmonella Paratyphi A, and the globally dominant, drug‑resistant H58 lineage of Typhi using isolates from blood‑culture‑positive patients in Bangladesh. The assay uses simple crude DNA extraction, achieves 100% sensitivity and specificity on cultured isolates, and has a clear limit of detection (LOD = 5.2 × 10⁴ CFU per reaction). The manuscript provides a useful bridge between genomic surveillance and field‑deployable diagnostics. However, revisions are needed to strengthen novelty, improve rigor in LOD calculation, and address key limitations more transparently.
Major comments:
The primers for Typhi and H58 were previously published (Khokhar et al., 2022, https://pmc.ncbi.nlm.nih.gov/articles/PMC9307194/), and the Paratyphi A assay is added here.
Please consider adding information which could improve this paper: the independent validation in a Bangladeshi strain panel, direct performance comparison to blood culture as the reference standard, utility for local surveillance and treatment guidance.
Explain why the LOD is reported as 5.2 × 10⁴ CFU per 2 μL template, but this is relatively high for clinical blood samples (which often have <1 CFU/mL), the dilution experiment shows detection down to 10⁻¹ dilution, but CFU counting was done at 10⁻³ and 10⁻⁴.
Response: We have revised the manuscript to emphasize that the Typhi- and H58-specific primers were originally developed by Khokhar et al. (2022) and independently validated in this study on Bangladeshi clinical isolates (line 90-100 in the revised manuscript). We have emphasized the use of different target regions of Salmonella Paratyphi A for primer and justified the reason behind this (line 302-305 in the revised manuscript).
We have also clarified the comparison with blood culture as the reference standard (line 272-274 in the revised manuscript). We have expanded the discussion to explain the utility of this assay for local surveillance and treatment guidance. In particular, we highlight its potential role in identifying the MDR-associated H58 lineage, which can inform empirical treatment decisions in settings where routine antimicrobial susceptibility testing or genomic analysis is not readily available (line 290-296 in the revised manuscript).
The bacterial load in the blood of typhoid patients is typically very low, which makes the development of PCR-based diagnostic assays for enteric fever from direct clinical samples challenging. In this study, the reported LOD of 5.2 × 10⁴ CFU per 2 μL template was calculated using DNA extracted from cultured bacterial suspensions rather than directly from blood samples. During the blood culture process, bacterial populations are enriched, resulting in a higher detectable CFU for detection. This enrichment likely might be a reason for getting relatively higher LOD for this assay. We have now explained this in line 318-321 in the revised manuscript.
Table1 use three line table.
Response: We have now revised Table 1 as per the suggestion.
Define all abbreviations in the abstract.
Response: We have now added all the abbreviations in the abstract (line 21 and lines 28-31 in the revised manuscript).
Strengthen comparison to other enteric fever PCR assays (sensitivity, cost, ease of use) in the discussion section.
Response: We sincerely appreciate this valuable suggestion. We have now revised the discussion section to include a more comprehensive comparison of our assay with previously reported PCR-based methods for enteric fever detection, highlighting differences in sensitivity, cost, and ease of use. (lines 290-294 and 297-303 and in the revised manuscript)
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsI would like to thank the authors for addressing my comments. I accept the current version of the manuscript and consider it suitable for publication in its present form.
Reviewer 2 Report
Comments and Suggestions for AuthorsI recommend it for publication.
