Analytical observational study of salmonella PCR and its comparison with routine laboratory parameters in cases of enteric fever in tertiary care hospital in central India

Aim

Salmonella PCR and its comparison with routine laboratory parameters in cases of enteric fever.

Objectives

Study design

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.

Study population

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.

Participants

Inclusion criteria:

Exclusion criteria:

Place of 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.

Ethics and consent

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.

Sample size consideration

In an Analytical Observational Study context, the sample size is calculated using a specific formula. The formula, represented as Z²_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.

Procedure

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 Salmonella PCR, while an additional 3 milliliters are collected in plain tubes for the Widal test. Proper labeling and documentation ensure the integrity of the collected samples.

Subsequently, the Widal agglutination test is performed on the blood samples, utilizing commercially manufactured colored antigens for Salmonella Typhi O and H antigens. Results are interpreted based on observed agglutination reactions and titers. Simultaneously, blood culture procedures are initiated by drawing 5 milliliters of blood and placing it into Brain Heart Infusion (BHI) broth under strict sterile conditions. With all aseptic precautions nearly 30-40 ml of blood was sent for culture and automated mics. Daily monitoring for bacterial growth is undertaken, and blind subculture is implemented without observable progress. Verification of bacterial identity is accomplished through biochemical testing and specialized antisera.

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 Salmonella is executed by extracting total DNA from blood samples using SDS and proteinase K. Real-time PCR techniques are employed for accurate and efficient detection, incorporating positive controls (Salmonella typhi DNA) and negative controls (DNA from E. coli, A. baumannii, and S. pneumoniae) to ensure the reliability of results.

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.

Outcome measures

Primary outcome

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.

Secondary outcomes:

Data management

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.

Statistical methods

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.

Dissemination

After the completion of the study, we will publish it in an indexed journal or conference.

Study status

The study has not yet started after the publication of the protocol; we will start recruitment in the study.