[version 1; peer review: 1 approved with reservations]
No competing interests were disclosed.
Carbapenem-resistant Enterobacteriaceae (CRE) represent a growing global health concern, necessitating comprehensive investigations into their prevalence and resistance mechanisms. This study protocol focuses on detecting carbapenemase genes, including blaVIM, blaNDM, blaKPC, blaIPM, and blaOXA-48, in clinical isolates of Klebsiella pneumoniae and Escherichia coli from a tertiary hospital in Eastern India. The rise of carbapenem resistance poses challenges to effective antimicrobial therapy and infection control strategies.
Conducted at the Department of Microbiology, Jawaharlal Nehru Medical College, the study employs a cross-sectional design from July 2022 to December 2023. The sample size calculation follows Daniel’s formula, considering a non-response rate of 10%. Modified Carbapenem Inactivation Method (mCIM) and EDTA-Modified Carbapenem Inactivation Method (eCIM) will be used for phenotypic detection, along with polymerase chain reaction (PCR) for genotypic confirmation. Antibiotic susceptibility testing using the Kirby-Bauer Disk Diffusion method will complement resistance profiling.
Anticipated outcomes include insights into the efficacy of mCIM and eCIM in detecting carbapenem resistance, the prevalence of carbapenemase genes in Klebsiella pneumoniae and Escherichia coli, and the antibiotic resistance pattern of carbapenemase-producing CRE. This study aims to provide valuable data for guiding empirical treatment strategies and reinforcing infection control measures in the region.
Antibiotic resistance, particularly the emergence of Carbapenemase-Producing Carbapenem-Resistant Enterobacteriaceae (CP-CRE), poses a significant global health threat. Carbapenems are considered last-resort antibiotics, and the rise in resistance among critical pathogens such as Escherichia coli and Klebsiella pneumoniae undermines the efficacy of these vital drugs.
Several studies have reported an alarming increase in the prevalence of Carbapenemase-producing strains, highlighting the need for robust surveillance and detection methods.
In India, where antimicrobial resistance is a growing concern, understanding the local epidemiology of CP-CRE becomes crucial for guiding effective treatment strategies.
The proposed study in the Department of Microbiology at Jawaharlal Nehru Medical College aims to address this gap by employing a comprehensive approach to identify CP-CRE in clinical isolates of E. coli and Klebsiella spp. The study will utilize modified Carbapenem Inactivation Methods (mCIM and eCIM), polymerase chain reaction (PCR), and antibiotic susceptibility testing to provide insights into the prevalence and antibiotic resistance patterns of CP-CRE in Eastern India.
This research study aims to detect Carbapenemase-Producing Carbapenem-Resistant Enterobacteriaceae (CP-CRE) among clinical isolates of Escherichia coli and Klebsiella pneumoniae in a tertiary hospital in Eastern India. The primary goal is to understand the prevalence of Carbapenemase genes and assess the antibiotic resistance patterns of CP-CRE in the specified region.
The study design is a cross-sectional study. In a cross-sectional study, data is collected from a population at a specific time to understand the prevalence and distribution of a particular condition or characteristic. In this case, the researchers aim to assess the presence of Carbapenemase-Producing Carbapenem-Resistant Enterobacteriaceae (CP-CRE) in clinical isolates of Escherichia coli and Klebsiella pneumoniae. Microbial DNA kit - Hi purA Bacterial Genetic DNA purification (Code- MB505), Hi PCR Carbapenemase Gene (Multiplex) probe PCR kit (Code- MBPCR132)
The study population includes patients from the OPD (Outpatient Department) and IPD (Inpatient Department) at “Acharya Vinoba Bhave Rural Hospital,” Sawangi (M) Wardha, Eastern India. The inclusion criteria likely involve clinically relevant isolates of Escherichia coli, and Klebsiella pneumoniae recovered from various clinical samples.
The study is conducted at the Department of Microbiology, Jawaharlal Nehru Medical College, Sawangi (Meghe) Wardha, Eastern India. The specific setting within the department needs to be detailed. Still, it is mentioned that routine and conventional methods, as well as automated methods like VITEK-2, will be used to isolate and identify the bacterial strains.
The study aims to detect Carbapenemase-Producing Carbapenem-Resistant Enterobacteriaceae (CP-CRE), focusing on Escherichia coli and Klebsiella pneumoniae in a tertiary hospital in Eastern India. The enrollment process involves the collection and analysis of isolates from clinical samples. All clinically relevant isolates of Escherichia coli and Klebsiella spp. It will be obtained from specimens received at the Department of Microbiology, Jawaharlal Nehru Medical College, Sawangi (Meghe) Wardha. These isolates are expected to be recovered from OPD/IPD patients under “Acharya Vinoba Bhave Rural Hospital,” Sawangi (M) Wardha, during the enrollment period spanning from July 2022 to July 2023.
The study will commence after obtaining approval from the Institutional Ethics Committee, and written informed consent is deemed unnecessary as the focus is on isolates recovered from specimens. The sample size calculation is based on Daniel’s formula, considering the expected prevalence, precision, and confidence level. The study setting is the Department of Microbiology, Jawaharlal Nehru Medical College, Sawangi (Meghe) Wardha.
Quality control strains, including Escherichia coli (ATCC 25922) and specific strains for mCIM and eCIM, will be utilized to ensure the reliability of phenotypic tests. The study incorporates a comprehensive approach involving phenotypic and genotypic methods to address the detection and characterization of Carbapenemase-Producing Enterobacteriaceae. The analysis will include Student’s t-test, Chi-square test, and Fisher’s exact test, if necessary, with a significance level set at P < 0.05.
The study’s expected outcomes include evaluating the utility of mCIM and eCIM in detecting Carbapenem resistance and determining the prevalence of Carbapenemase genes in the studied region. Additionally, understanding the antibiotic resistance pattern of CP-CRE in the area will contribute to instituting appropriate empirical treatment strategies. The study aims to provide valuable insights into the prevalence and antibiotic resistance patterns, enhancing treatment strategies in the specified region.
The study involves a comprehensive approach to collect and analyze data on Carbapenemase-Producing Carbapenem-Resistant Enterobacteriaceae (CP-CRE), with a focus on Escherichia coli and Klebsiella pneumoniae in a tertiary hospital in Eastern India. The enrollment process targets clinically relevant isolates, and the study duration spans from July 2022 to December 2023. Ethical approval from the Institutional Ethics Committee is a prerequisite, with written informed consent not required due to the use of isolates recovered in the Department of Microbiology.
Clinical isolates are obtained from OPD/IPD patients under “Acharya Vinoba Bhave Rural Hospital,” Sawangi (M) Wardha. Following Daniel’s formula, sample size calculation determines that 203 patients are needed for the study, considering a non-response rate of 10%. The study setting is the Department of Microbiology, Jawaharlal Nehru Medical College, Sawangi (Meghe) Wardha.
Data collection involves multiple steps. Conventional microbiological methods, including automated methods like VITEK-2,
Statistical analysis, including Student’s t-test, Chi-square test, and Fisher’s exact test, is applied to analyze the collected data. The expected outcomes include evaluating the utility of mCIM and eCIM in detecting Carbapenem resistance and determining the prevalence of Carbapenemase genes in the studied region. Additionally, the study aims to understand the antibiotic resistance pattern of CP-CRE in the area for informed empirical treatment decisions.
The sample size for the study was determined using a formula based on established statistical principles. With a level of confidence set at 95%, an expected prevalence (P) of 0.38 for OXA-48, like producing Enterobacteriaceae, and a desired precision (d) of 0.07, the formula yielded a sample size (n). To account for potential non-response, a 10% non-response rate was assumed. Consequently, the total sample size was adjusted to 203, ensuring robust statistical power for the investigation. This calculation, referencing the work of Daniel et al. (1977), was performed using R Studio Version 4.3.1, providing a solid foundation for the study’s research design and ensuring adequate representation of the population under investigation. The methodology follows established standards, as evidenced by the study reference to Renru Han et al., and emphasizes the importance of achieving statistical significance in understanding the prevalence and characteristics of Carbapenemase-Producing Carbapenem-Resistant Enterobacteriaceae.
The study employs several essential statistical methods to analyze and interpret data gathered in the investigation of Carbapenemase-Producing Carbapenem-Resistant Enterobacteriaceae (CP-CRE), with a specific focus on Escherichia coli and Klebsiella pneumoniae. The statistical toolkit includes the student’s t-test, which compares means between two groups, aiding in identifying any statistically significant differences in measured variables. The Chi-square test is also applied to analyze categorical data, examining potential associations between variables. Fisher’s exact test, a valuable tool for situations with small sample sizes, ensures accurate assessments of associations between categorical variables.
These statistical analyses play a crucial role in evaluating data collected from various stages of the study, encompassing phenotypic methods (Modified Carbapenem Inactivation Method - mCIM and EDTA-Modified Carbapenem Inactivation Method - eCIM), molecular detection (Real-Time PCR and Conventional PCR), and antibiotic susceptibility testing. The overarching objective is to derive meaningful insights into the prevalence of Carbapenemase genes, assess the efficacy of different detection methods, and delineate the antibiotic resistance patterns of CP-CRE within the specified region.
In the context of these statistical analyses, the significance level, often represented by the p-value, holds paramount importance. A p-value less than 0.05 is conventionally considered statistically significant, indicating that observed results are unlikely to occur randomly.
The adoption of SPSS version 27.0 for statistical analysis underscores the study’s commitment to a robust and widely accepted platform for handling and interpreting the collected data. By leveraging these statistical methods, the study aims to draw informed conclusions regarding the prevalence, characteristics, and antibiotic resistance patterns of Carbapenemase-Producing Enterobacteriaceae in the targeted population.
After the completion of the study, we will publish it in an indexed journal or conference.
The study has yet to start. After the publication of the protocol, we will start recruitment in the study.
The study protocol addresses the increasing concern of carbapenem-resistant Enterobacteriaceae (CRE) in a tertiary hospital in Eastern India. By employing a combination of phenotypic and genotypic methods, the study intends to shed light on the prevalence of carbapenemase-producing Klebsiella pneumoniae and Escherichia coli in the region. The choice of phenotypic methods, such as Modified Carbapenem Inactivation Method (mCIM) and EDTA-Modified Carbapenem Inactivation Method (eCIM), is based on their established utility in detecting carbapenemase production, particularly in distinguishing between metallo-beta-lactamases and serine carbapenemases.
Additionally, incorporating genotypic methods, including polymerase chain reaction (PCR) for specific carbapenemase genes, offers a molecular perspective on the genetic diversity and prevalence of resistance determinants.
The Institutional Ethics Committee of Datta Meghe Institute of Higher Education and Research (DU) has granted its approval to the study protocol (Reference number: DMIMS (DU)/IEC/2022/1055. Date: 21-07-2022). Before commencing the study, we will obtain written informed consent from all participants, providing them with a comprehensive explanation of the study’s objectives.
No data are associated with this article.
Ensure that all scientific names are italicized throughout the manuscript. Polymerase Chain Reaction (PCR) should be written in full upon its first mention and abbreviated as PCR thereafter. Ensure such corrections are applied consistently throughout the text. Rewrite “expected outcomes” for the clarity. I suggest to include “designing better diagnostic, therapeutic, and preventive measures against carbapenem-resistant infections” Include “eCIM” in keywords. mCIM and eCIM expand the term on first usage in the text. In introduction “Carbapenems are considered last-resort antibiotics, and the rise in resistance among critical pathogens such as Escherichia coli and Klebsiella pneumoniae undermines the efficacy of these vital drugs” also add “colistin” which is also a last resort drug and give appropriate reference Rewrite the second objective as “Detection of major carbapenemase genes (blaVIM, blaNDM, blaKPC, blaIMP, and blaOXA-48) using PCR and analysis of their expression levels through RT-PCR” The study period mentioned in the abstract (July 2022 to December 2023) does not match the one provided in the methodology (July 2022 to July 2023). Please verify the correct timeframe and ensure consistency across the manuscript. Consider incorporating MIC (Minimum Inhibitory Concentration) testing of carbapenems into the study which is highly relevant. In the study population “The inclusion criteria likely involve clinically relevant isolates of Escherichia coli, and Klebsiella pneumoniae recovered from various clinical samples” Authors must provide details of the clinical samples, such as urine, aspirates, and wound swabs, that will be used in the study In antibiotic sensitivity testing using the Kirby-Bauer Disk Diffusion method, authors should specify the carbapenem antibiotics used, such as meropenem and imipenem. Clearly provide the detailed methodology for the assays (mCIM and eCIM) to ensure reproducibility and transparency. Provide detailed protocols for PCR and RT-PCR, including reaction conditions, primer sequences, and the concentrations of PCR components In the discussion section, emphasize the recent reports of Carbapenem-Resistant Enterobacteriaceae (CRE) from India. In the discussion “The utilization of real-time PCR and gel electrophoresis aims to enhance the accuracy and efficiency of gene identification” replace gel electrophoresis with PCR
Is the study design appropriate for the research question?
Yes
Is the rationale for, and objectives of, the study clearly described?
Partly
Are sufficient details of the methods provided to allow replication by others?
Yes
Are the datasets clearly presented in a useable and accessible format?
Yes
Reviewer Expertise:
Antimicrobial Resistance, Anti-biofilm agents
We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however we have significant reservations, as outlined above.