[version 1; peer review: 2 not approved]
No competing interests were disclosed.
Enteric fever, predominantly caused by
The study will employ an Analytical Observational Study design conducted at Acharya Vinoba Bhave Rural Hospital in central India over a two-year period. Participants will include adults above 18 years admitted to the ward or Intensive Care Unit (ICU) with acute febrile illnesses. Inclusion criteria encompass a wide age range, ensuring a diverse study population. Peripheral blood samples will be collected for Salmonella PCR, Widal testing, and blood culture. Dot-PCR for Typhi will be employed, and DNA extraction will follow strict protocols. Data will be systematically recorded, and statistical analyses, including sensitivity, specificity, and comparative assessments, will be conducted to evaluate diagnostic performance.
It is anticipated that Salmonella PCR will exhibit superior sensitivity and specificity compared to traditional diagnostic methods, offering a more rapid and accurate identification of enteric fever cases. The study aims to contribute valuable evidence supporting the incorporation of Salmonella PCR into routine diagnostic algorithms, potentially revolutionizing the approach to enteric fever diagnosis. Moreover, insights gained from the study are expected to inform clinical practices, enhance patient management, and potentially reduce the economic burden associated with misdiagnosis or delayed diagnosis of enteric fever. The outcomes of this research are poised to impact public health strategies, providing a foundation for more effective and efficient diagnostic protocols in the context of enteric fever.
Enteric fever, predominantly caused by
Previous studies have highlighted the diagnostic potential of Salmonella PCR in various clinical settings, demonstrating its ability to complement traditional methods.
The study will be conducted at Acharya Vinoba Bhave Rural Hospital (AVBRH), a tertiary care teaching hospital in central India. The research design, encompassing two years, adheres to ethical guidelines and seeks to enroll a diverse cohort of adult patients admitted with acute febrile illnesses.
Salmonella PCR and its comparison with routine laboratory parameters in cases of enteric fever.
To diagnose enteric fever in patients with acute febrile Illness by Widal and blood culture test. To perform Salmonella PCR in diagnosed cases of enteric fever. To compare Salmonella PCR with typhi dot, Widal and blood culture.
This study will employ an Analytical Observational Study design to assess the diagnostic efficacy of Salmonella Polymerase Chain Reaction (PCR) and its comparison with routine laboratory parameters in cases of enteric fever.
The study will include individuals above the age of 18 years who are admitted to the ward or Intensive Care Unit (ICU) with acute febrile illnesses. The participants will be selected based on predefined inclusion and exclusion criteria.
Individuals aged 18 years and above. Patients admitted to the ward or ICU with acute febrile illnesses.
Individuals below the age of 18 years. Patients admitted with a confirmed diagnosis other than enteric fever (e.g., malaria, dengue). Those who refuse to participate in the study.
The study will be conducted at Acharya Vinoba Bhave Rural Hospital (AVBRH), a tertiary care teaching hospital located in Sawangi, Meghe, in the rural sector of Wardha district, Central India. The outpatient medicine department at AVBRH will be the primary site for participant enrollment and data collection.
This setting has been chosen due to its accessibility, relevance to the study population, and the availability of necessary facilities for conducting the required diagnostic tests. Ethical approval will be obtained from the Institute’s review board before the initiation of the study, ensuring adherence to ethical guidelines and standards. The study will span two years, from March 2023 to March 2025, during which data will be systematically collected and analyzed to fulfill the study objectives.
The research protocol got approval from the Datta Meghe Institute of Higher Education and Research (Deemed to be university) Institutional ethical committee in the meeting held on 11-07-2022 with DMIMS (DU)/IEC/2022/28. All the participants will be educated about the research, and written and verbal informed consent will be obtained from all the participants before the intervention.
In an Analytical Observational Study context, the sample size is calculated using a specific formula. The formula, represented as Z 2 1-a/2 Sens(1-Sens) > 2d X Prev, considers various parameters for precise determination. Alpha (a) is set at 0.05, representing the significance level. The estimated sensitivity (Sens) is designated as 0.99, indicating the test’s ability to identify true positive cases correctly. The prevalence of the disease (Prev) is defined as 0.5, reflecting the anticipated frequency of the condition within the population. Additionally, an estimation error (d) of 0.03 is considered. By applying this formula, the minimum number of diseases required for the study is calculated to be 43, and correspondingly, the minimum total sample size needed is 86. These calculations are pivotal for ensuring the statistical robustness of the analytical observational study, facilitating reliable and meaningful conclusions.
The data collection process for this study involves a systematic series of steps designed to gather relevant information and conduct diagnostic tests among eligible participants. To begin, potential participants meeting the study’s inclusion criteria are identified, and informed consent is obtained from those willing to contribute to the research. A thorough clinical assessment is conducted, encompassing detailed medical history, symptom evaluation, and physical examinations.
Blood sample collection is a critical step with strict adherence to aseptic techniques. Two milliliters of blood are drawn into an EDTA vial for
Subsequently, the Widal agglutination test is performed on the blood samples, utilizing commercially manufactured colored antigens for
Specifically, for Typhi, a dot-PCR is conducted using the appropriate primers, following standard PCR procedures, including DNA extraction, amplification, and gel electrophoresis. Additionally, polymerase chain reaction (PCR) for
Strict ethical considerations are maintained throughout the data collection process, including participant confidentiality, privacy protection, and compliance with institutional ethical review board approvals. All pertinent data, including clinical information, test results, and participant demographics, are meticulously recorded in a structured database. The study spans from March 2023 to March 2025, during which these procedures will be carried out to fulfill the research objectives.
The primary outcome measure of this study is the diagnostic accuracy of Salmonella Polymerase Chain Reaction (PCR) in identifying enteric fever among patients with acute febrile illnesses. The primary focus is assessing the sensitivity, specificity, positive predictive value, and negative predictive value of Salmonella PCR compared to the reference standards of Widal testing and blood culture.
Evaluate and compare the diagnostic performance of Salmonella PCR with routine laboratory parameters, including Widal test and blood culture. Assess the concordance and discrepancies between the results of Salmonella PCR, Widal test, and blood culture.
Explore the practicality and feasibility of implementing Salmonella PCR in routine clinical settings. Examine the turnaround time for Salmonella PCR compared to traditional diagnostic methods.
Investigate the impact of Salmonella PCR results on patient management and clinical decision-making. Assess whether using Salmonella PCR leads to more timely and accurate interventions.
Conduct a preliminary cost-effectiveness analysis comparing the expenses associated with Salmonella PCR and routine laboratory parameters. Evaluate the economic implications of incorporating Salmonella PCR into the diagnostic algorithm for enteric fever.
Explore the diagnostic performance of Salmonella PCR in specific subgroups, such as age categories or severity of illness. Identify potential variations in sensitivity and specificity across different patient characteristics.
Efficient data management is integral to the study’s success, ensuring the collected information’s accuracy, security, and confidentiality. Standardized data collection forms and electronic databases will be employed, accompanied by unique identifiers to protect participant confidentiality. Trained personnel will ensure timely and precise data entry, incorporating validation checks and double-entry verification processes to minimize errors. Electronic data will be securely stored on restricted-access servers, with regular backup procedures to prevent data loss. Physical copies of data, if any, will be stored in locked and secure locations. Adherence to institutional and ethical guidelines for data security will be paramount, including encryption and password protection for electronic databases. Periodic data quality checks, audit trails, and the establishing of a Data Monitoring Committee will contribute to ongoing quality control. The study will use statistical software R Studio 4.3.1 to analyze exploratory data and identify patterns and trends. Reports will be generated for internal use and dissemination, with considerations for anonymized data-sharing aligned with ethical guidelines. A robust data archiving plan will be implemented for long-term storage, ensuring accessibility and compliance with data retention policies. Through these measures, the study aims to uphold the integrity and reliability of the collected data, facilitating accurate analysis and contributing to the overall success of the research.
The statistical analysis for this study will encompass a multifaceted approach to assess the diagnostic accuracy of Salmonella Polymerase Chain Reaction (PCR) and compare its outcomes with routine laboratory parameters in cases of enteric fever. The initial steps involve descriptive statistics to summarize participant demographics and baseline characteristics. Diagnostic accuracy measures will be calculated for Salmonella PCR, Widal test, and blood culture, including sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). Receiver operating characteristic (ROC) curves and the area under the curve (AUC) will be utilized to gauge overall diagnostic performance. Comparative analyses will be conducted using statistical tests like McNemar’s test to discern differences between Salmonella PCR and routine laboratory parameters. Subgroup analyses based on factors such as age, gender, and illness severity will be performed to explore potential variations in diagnostic accuracy. The study will also evaluate the practicality and feasibility of implementing Salmonella PCR in routine clinical settings and assess its impact on patient management. Cost-effectiveness analysis will compare the economic implications of Salmonella PCR with routine parameters. Regression analysis, if applicable, will identify factors influencing diagnostic outcomes. The statistical analyses will be executed using R studio Version 4.3.1, and results will be presented transparently, adhering to predetermined significance levels. Visual representations will be created to illustrate key findings. The study aims to derive comprehensive insights into the diagnostic utility of Salmonella PCR, contributing to evidence-based advancements in clinical practice and diagnostic protocols.
After the completion of the study, we will publish it in an indexed journal or conference.
The study has not yet started after the publication of the protocol; we will start recruitment in the study.
The emergence of enteric fever, driven primarily by Salmonella enterica serotype Typhi, presents a persistent public health challenge globally.
The choice of Salmonella PCR as a diagnostic tool is grounded in its potential to overcome some of the limitations associated with traditional methods. Previous research has indicated that PCR-based assays can provide enhanced sensitivity and specificity, facilitating early and accurate detection of bacterial pathogens.
Including routine laboratory parameters, specifically the Widal test and blood culture, allows for a comprehensive comparison, considering established diagnostic practices. Despite being widely used, Widal testing has faced criticism for its variable sensitivity and specificity.
The study’s analytical observational design aims to provide robust evidence regarding the diagnostic performance of Salmonella PCR in a real-world clinical setting. This research will contribute valuable insights into the potential utility of PCR as a complementary or alternative diagnostic tool for enteric fever. The comprehensive data collection approach, including dot-PCR for Typhi, will further enhance our understanding of the molecular characteristics of Salmonella strains prevalent in the study population. The findings from this study may have implications for clinical practice, guiding healthcare professionals in selecting appropriate diagnostic methods for enteric fever. The potential inclusion of cost-effectiveness analysis will add a practical dimension to the discussion, addressing the economic feasibility of incorporating Salmonella PCR into routine diagnostic algorithms.
While the study’s proposed setting, Acharya Vinoba Bhave Rural Hospital, offers a unique perspective from central India, it is essential to acknowledge potential limitations such as regional variations in bacterial strains and healthcare infrastructure. Additionally, the study duration of two years may not capture long-term trends, necessitating ongoing surveillance.
No data are associated with this article.
This is a protocol document for a study that aims to compare PCR for Salmonella Typhi against other methods like blood culture, Widal and Typhidot.
The rationale for the study is well described in the abstract and the introduction. However, the authors have not described the rationale for using other diagnostic modalities like Widal and Typhidot when it is well documented that this is inferior in sensitivity and specificity to blood culture. The authors have also not described how they would interpret a Widal; if this is a hospital study, paired sampling to observe an increase in titres might not be possible.
The authors have not explained why they have excluded patients less than 18 years. Individuals less than 18 years form a huge part of the burden of typhoid in India and they are also the target of the TCV vaccine.
The sampling frame is not ideal for the study design; patients admitted to the ward would just be a subset of the patients with typhoid and the reviewer would suggest including outpatients as well.
The authors have described the methods for Widal and blood culture, but they have not described satisfactorily the Salmonella PCR method which is the major focus of the study. There is no description of the targets other than “appropriate primers” that they are planning to use. This is problematic because even if they will be developing new primers, that process should be detailed in brief. They have not referenced any article that have examined this work earlier, and there are multiple articles regarding this.
In the data analysis section, they have stated that diagnostic accuracy measures will be carried out in for PCR, Widal and blood culture. In that case, which would be the gold standard method they are comparing this against? If they do not consider blood culture to be the gold standard, they have not stated any plans to do a latent class analysis.
In the discussion, they have stated that “blood culture is constrained by longer turnaround times and potential false negatives, especially in patients receiving prior antibiotic treatment.” If that is so, how would PCR be any better than blood culture considering bacteremia even at the height of Typhoid is ~ 1 bacteria/ml. If they are planning to enrich the blood prior to PCR, this should be described.
Is the study design appropriate for the research question?
No
Is the rationale for, and objectives of, the study clearly described?
Yes
Are sufficient details of the methods provided to allow replication by others?
No
Are the datasets clearly presented in a useable and accessible format?
Not applicable
Reviewer Expertise:
Clinical microbiology, wastewater surveillance, gut microbiome
I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.
This is a protocol paper which details a study about to take place.
The objectives 1 & 2 of the study seem to be inappropriate. From the text, what we understand is that the primary objective of the study is to estimate the diagnostic accuracy of Salmonella PCR as compared to a composite score of Widal + blood culture. Hence the objectives need to be re-framed.
The study design is mentioned as 'analytical observational' study. But it is not clear as to what type of analytical observational study is planned.
In analytical observational studies, researchers try to establish an association between exposure(s) and outcome(s). Depending on the direction of enquiry, these studies can be directed forwards (cohort studies) or backwards (case–control studies). (1)
If it is a diagnostic accuracy (DA) study, it should be explicitly mentioned what type it is- DA case-control, DA cross sectional or DA comparative studies. (2)
Inclusion criteria needs to be more specific. Are you going to do a blood culture, Widal and PCR on all febrile illness cases? Is there any duration of fever cut off? And are these tests going to be done in series or in parallel?
In calculating the sample size, why was a prevalence of 0.5 used?
There is already indexed literature on prevalence of laboratory confirmed typhoid in India. (3)
One of the secondary outcomes expected is mentioned as a cost-effectiveness analysis of Salmonella PCR for typhoid diagnosis. How is this analysis planned? With current methodology in the manuscript, it seems to lack those details.
Enteric fever involves both typhoid and paratyphoid. Are you considering paratyphi also here or are you excluding those?
Is the study design appropriate for the research question?
No
Is the rationale for, and objectives of, the study clearly described?
No
Are sufficient details of the methods provided to allow replication by others?
Partly
Are the datasets clearly presented in a useable and accessible format?
Not applicable
Reviewer Expertise:
Infectious disease epidemiology, operational research
I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.