Keywords
Carbapenem resistance, Enterobacteriaceae, Klebsiella pneumoniae, Escherichia coli, Modified Carbapenem Inactivation Method (mCIM), Antibiotic susceptibility testing
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Kunjalwar R and Mudey G. A cross sectional study on endemicity of VIM, NDM, KPC, IPM & OXA-48 genes in Carbapenemase producing Klebsiella pneumoniae and Escherichia coli from a tertiary hospital using mCIM, eCIM, and PCR in Central India [version 1; peer review: 1 approved with reservations]. F1000Research 2024, 13:636 (https://doi.org/10.12688/f1000research.147644.1)
NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article.
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Study Protocol
A cross sectional study on endemicity of VIM, NDM, KPC, IPM & OXA-48 genes in Carbapenemase producing Klebsiella pneumoniae and Escherichia coli from a tertiary hospital using mCIM, eCIM, and PCR in Central India
[version 1; peer review: 1 approved with reservations]
PUBLISHED 14 Jun 2024
OPEN PEER REVIEW
REVIEWER STATUS
This article is included in the Datta Meghe Institute of Higher Education and Research collection.
Abstract
Corresponding author:
Radha Kunjalwar
Competing interests:
No competing interests were disclosed.
Grant information:
ICMR Financial support for the ICMR MD thesis for the June 2022 batch. Ref No. MD22JUN-0259
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Copyright:
© 2024 Kunjalwar R and Mudey G.
This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
How to cite: Kunjalwar R and Mudey G. A cross sectional study on endemicity of VIM, NDM, KPC, IPM & OXA-48 genes in Carbapenemase producing Klebsiella pneumoniae and Escherichia coli from a tertiary hospital using mCIM, eCIM, and PCR in Central India [version 1; peer review: 1 approved with reservations]. F1000Research 2024, 13:636 (https://doi.org/10.12688/f1000research.147644.1)
First published: 14 Jun 2024, 13:636 (https://doi.org/10.12688/f1000research.147644.1)
Latest published: 14 Jun 2024, 13:636 (https://doi.org/10.12688/f1000research.147644.1)
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Introduction
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.1,2
Several studies have reported an alarming increase in the prevalence of Carbapenemase-producing strains, highlighting the need for robust surveillance and detection methods.3,4 Carbapenemase enzymes, encoded by genes such as blaVIM, blaNDM, blaKPC, blaIPM, and blaOXA-48, contribute to resistance and are associated with difficult-to-treat infections.5,6
In India, where antimicrobial resistance is a growing concern, understanding the local epidemiology of CP-CRE becomes crucial for guiding effective treatment strategies.7 Previous research has underscored the importance of combining phenotypic and genotypic methods for accurately detecting and characterizing Carbapenemase production.8,9
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.
Aim
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.
Objectives
1. Isolation and identification: To employ conventional microbiological methods for isolating and identifying Escherichia coli and Klebsiella pneumoniae from various clinical samples.
2. Carbapenemase production testing: To determine Carbapenemase production in isolated strains using the Modified Carbapenem Inactivation Method (mCIM) and EDTA-Modified Carbapenem Inactivation Method (eCIM) based on CLSI Guidelines 2022.
3. Genotypic identification: To identify Carbapenemase production through conventional PCR and gel electrophoresis for specific genes (blaVIM, blaNDM, blaKPC, blaIPM, & blaOXA-48).
4. Antibiotic-sensitivity testing: To perform antibiotic-sensitivity testing using the Kirby-Bauer Disk Diffusion method according to CLSI Guidelines 2022 to understand the resistance patterns.
Method
Study design
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)
Study population
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.
Place of study
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.
Inclusion and exclusion criteria
Inclusion criteria:
1. Clinical isolates: Isolates of Escherichia coli and Klebsiella pneumoniae obtained from clinical samples.
2. Specimens: Isolates recovered from various clinical specimens indicate the presence of these bacteria.
3. Timeframe: Isolates collected during the specified study duration from July 2022 to July 2023.
4. Study location: Isolates obtained from patients under “Acharya Vinoba Bhave Rural Hospital,” Sawangi (M) Wardha.
Exclusion criteria:
1. Non-clinical isolates: Isolates not obtained from clinical samples, excluding those recovered from routine environmental monitoring or non-human sources.
2. Specimens from outside the specified timeframe: Isolates collected outside the defined study duration (before July 2022 or after July 2023).
3. Isolates from locations outside the designated hospital: Not obtained from patients under “Acharya Vinoba Bhave Rural Hospital,” Sawangi (M) Wardha.
4. Non-relevant isolates: Isolates lacking clinical relevance, such as those from healthy individuals or individuals without signs of infection.
5. Duplicate isolates: Multiple isolates from the same patient for the same infection episode, considering only the first isolate.
6. Unsuitable samples: Isolates with compromised sample quality or integrity that may affect the accuracy of test results.
7. Non-consented isolates: Isolates obtained without proper ethical approval or without following ethical guidelines, particularly if written informed consent is required.
Bias
1. Selection bias: The study may be vulnerable to selection bias if the samples collected do not represent the broader population of patients in the hospital with Escherichia coli and Klebsiella pneumoniae infections. For example, if only certain patient groups or specific types of infections are included, the results may not be generalizable.
2. Sampling bias: If the samples are not collected randomly or there is a bias in the selection of samples, it could lead to sampling bias. For instance, if certain patients are more likely to be tested for CP-CRE, it may not reflect the actual prevalence in the overall population.
3. Detection bias: The methods used for detection, such as PCR and phenotypic tests, may have inherent biases. If the tests are more sensitive or specific to certain strains or genes, it could affect the accuracy of the results.
Enrolment
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.
Data collection process
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-2,10 are employed for isolating and identifying Escherichia coli and Klebsiella spp. Antibiotic-susceptibility testing uses the Kirby-Bauer Disk Diffusion method, adhering to CLSI Guidelines 2022.11 Phenotypic methods, specifically the Modified Carbapenem Inactivation Method (mCIM) and EDTA-Modified Carbapenem Inactivation Method (eCIM) are used for Carbapenemase detection.12 Molecular methods, such as Real-Time PCR and Conventional PCR, are utilized for identifying specific Carbapenemase genes (blaNDM, blaIPM, blaVIM, blaKPC, and blaOXA-48).13 Control strains, like Escherichia coli ATCC 25922, ensure quality control in phenotypic tests.14
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.
Sample size calculation
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.
Statistical method
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.
Discussion
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.15 These methods align with the Clinical and Laboratory Standards Institute (CLSI) guidelines, providing a standardized approach to assess carbapenem resistance.16
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.17 The utilization of real-time PCR and gel electrophoresis aims to enhance the accuracy and efficiency of gene identification. Antibiotic susceptibility testing using the Kirby-Bauer Disk Diffusion method complements the study by providing insights into the overall resistance patterns of carbapenemase-producing isolates. This information is crucial for understanding the challenges in treating infections caused by these strains and guiding empirical treatment strategies.18
Ethics and consent
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.
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how to cite this article
Kunjalwar R and Mudey G. A cross sectional study on endemicity of VIM, NDM, KPC, IPM & OXA-48 genes in Carbapenemase producing Klebsiella pneumoniae and Escherichia coli from a tertiary hospital using mCIM, eCIM, and PCR in Central India [version 1; peer review: 1 approved with reservations]. F1000Research 2024, 13:636 (https://doi.org/10.12688/f1000research.147644.1)
NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article.
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PUBLISHED 14 Jun 2024
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Kumar P and V P. Reviewer Report For: A cross sectional study on endemicity of VIM, NDM, KPC, IPM & OXA-48 genes in Carbapenemase producing Klebsiella pneumoniae and Escherichia coli from a tertiary hospital using mCIM, eCIM, and PCR in Central India [version 1; peer review: 1 approved with reservations]. F1000Research 2024, 13:636 (https://doi.org/10.5256/f1000research.161864.r357085)
The direct URL for this report is:
https://f1000research.com/articles/13-636/v1#referee-response-357085
https://f1000research.com/articles/13-636/v1#referee-response-357085
NOTE: it is important to ensure the information in square brackets after the title is included in this citation.
Reviewer Report 27 Jan 2025
Praveen Kumar,
Department of Zoology, Government College for Women, Thiruvananthapuram, Kerala, India
Parvathi V,
Department of Zoology, Government College for Women, Thiruvananthapuram, Kerala, India
Approved with Reservations
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- 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.
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Kumar P and V P. Reviewer Report For: A cross sectional study on endemicity of VIM, NDM, KPC, IPM & OXA-48 genes in Carbapenemase producing Klebsiella pneumoniae and Escherichia coli from a tertiary hospital using mCIM, eCIM, and PCR in Central India [version 1; peer review: 1 approved with reservations]. F1000Research 2024, 13:636 (https://doi.org/10.5256/f1000research.161864.r357085)
The direct URL for this report is:
https://f1000research.com/articles/13-636/v1#referee-response-357085
https://f1000research.com/articles/13-636/v1#referee-response-357085
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
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