Journal of Clinical and Diagnostic Research, ISSN - 0973 - 709X

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On Sep 2018

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"Journal of Clinical and Diagnostic Research is at present a well-known Indian originated scientific journal which started with a humble beginning. I have been associated with this journal since many years. I appreciate the Editor, Dr. Hemant Jain, for his constant effort in bringing up this journal to the present status right from the scratch. The journal is multidisciplinary. It encourages in publishing the scientific articles from postgraduates and also the beginners who start their career. At the same time the journal also caters for the high quality articles from specialty and super-specialty researchers. Hence it provides a platform for the scientist and researchers to publish. The other aspect of it is, the readers get the information regarding the most recent developments in science which can be used for teaching, research, treating patients and to some extent take preventive measures against certain diseases. The journal is contributing immensely to the society at national and international level."

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Sanjay Gandhi institute of trauma and orthopedics,
On Aug 2018

Dr. Mamta Gupta,
"It gives me great pleasure to be associated with JCDR, since last 2-3 years. Since then I have authored, co-authored and reviewed about 25 articles in JCDR. I thank JCDR for giving me an opportunity to improve my own skills as an author and a reviewer.
It 's a multispecialty journal, publishing high quality articles. It gives a platform to the authors to publish their research work which can be available for everyone across the globe to read. The best thing about JCDR is that the full articles of all medical specialties are available as pdf/html for reading free of cost or without institutional subscription, which is not there for other journals. For those who have problem in writing manuscript or do statistical work, JCDR comes for their rescue.
The journal has a monthly publication and the articles are published quite fast. In time compared to other journals. The on-line first publication is also a great advantage and facility to review one's own articles before going to print. The response to any query and permission if required, is quite fast; this is quite commendable. I have a very good experience about seeking quick permission for quoting a photograph (Fig.) from a JCDR article for my chapter authored in an E book. I never thought it would be so easy. No hassles.
Reviewing articles is no less a pain staking process and requires in depth perception, knowledge about the topic for review. It requires time and concentration, yet I enjoy doing it. The JCDR website especially for the reviewers is quite user friendly. My suggestions for improving the journal is, more strict review process, so that only high quality articles are published. I find a a good number of articles in Obst. Gynae, hence, a new journal for this specialty titled JCDR-OG can be started. May be a bimonthly or quarterly publication to begin with. Only selected articles should find a place in it.
An yearly reward for the best article authored can also incentivize the authors. Though the process of finding the best article will be not be very easy. I do not know how reviewing process can be improved. If an article is being reviewed by two reviewers, then opinion of one can be communicated to the other or the final opinion of the editor can be communicated to the reviewer if requested for. This will help one’s reviewing skills.
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Dr. Mamta Gupta
(Ex HOD Obs &Gynae, Hindu Rao Hospital and associated NDMC Medical College, Delhi)
Aug 2018

Dr. Rajendra Kumar Ghritlaharey

"I wish to thank Dr. Hemant Jain, Editor-in-Chief Journal of Clinical and Diagnostic Research (JCDR), for asking me to write up few words.
Writing is the representation of language in a textual medium i e; into the words and sentences on paper. Quality medical manuscript writing in particular, demands not only a high-quality research, but also requires accurate and concise communication of findings and conclusions, with adherence to particular journal guidelines. In medical field whether working in teaching, private, or in corporate institution, everyone wants to excel in his / her own field and get recognised by making manuscripts publication.

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Salient features of the JCDR: It is a biomedical, multidisciplinary (including all medical and dental specialities), e-journal, with wide scope and extensive author support. At the same time, a free text of manuscript is available in HTML and PDF format. There is fast growing authorship and readership with JCDR as this can be judged by the number of articles published in it i e; in Feb 2007 of its first issue, it contained 5 articles only, and now in its recent volume published in April 2011, it contained 67 manuscripts. This e-journal is fulfilling the commitments and objectives sincerely, (as stated by Editor-in-chief in his preface to first edition) i e; to encourage physicians through the internet, especially from the developing countries who witness a spectrum of disease and acquire a wealth of knowledge to publish their experiences to benefit the medical community in patients care. I also feel that many of us have work of substance, newer ideas, adequate clinical materials but poor in medical writing and hesitation to submit the work and need help. JCDR provides authors help in this regards.
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In the era of fast growing newer technologies, and in computer and internet friendly environment the manuscripts preparation, submission, review, revision, etc and all can be done and checked with a click from all corer of the world, at any time. Of course there is always a scope for improvement in every field and none is perfect. To progress, one needs to identify the areas of one's weakness and to strengthen them.
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Thanking you
With sincere regards
Dr. Rajendra Kumar Ghritlaharey, M.S., M. Ch., FAIS
Associate Professor,
Department of Paediatric Surgery, Gandhi Medical College & Associated
Kamla Nehru & Hamidia Hospitals Bhopal, Madhya Pradesh 462 001 (India)
On May 11,2011

Dr. Shankar P.R.

"On looking back through my Gmail archives after being requested by the journal to write a short editorial about my experiences of publishing with the Journal of Clinical and Diagnostic Research (JCDR), I came across an e-mail from Dr. Hemant Jain, Editor, in March 2007, which introduced the new electronic journal. The main features of the journal which were outlined in the e-mail were extensive author support, cash rewards, the peer review process, and other salient features of the journal.
Over a span of over four years, we (I and my colleagues) have published around 25 articles in the journal. In this editorial, I plan to briefly discuss my experiences of publishing with JCDR and the strengths of the journal and to finally address the areas for improvement.
My experiences of publishing with JCDR: Overall, my experiences of publishing withJCDR have been positive. The best point about the journal is that it responds to queries from the author. This may seem to be simple and not too much to ask for, but unfortunately, many journals in the subcontinent and from many developing countries do not respond or they respond with a long delay to the queries from the authors 1. The reasons could be many, including lack of optimal secretarial and other support. Another problem with many journals is the slowness of the review process. Editorial processing and peer review can take anywhere between a year to two years with some journals. Also, some journals do not keep the contributors informed about the progress of the review process. Due to the long review process, the articles can lose their relevance and topicality. A major benefit with JCDR is the timeliness and promptness of its response. In Dr Jain's e-mail which was sent to me in 2007, before the introduction of the Pre-publishing system, he had stated that he had received my submission and that he would get back to me within seven days and he did!
Most of the manuscripts are published within 3 to 4 months of their submission if they are found to be suitable after the review process. JCDR is published bimonthly and the accepted articles were usually published in the next issue. Recently, due to the increased volume of the submissions, the review process has become slower and it ?? Section can take from 4 to 6 months for the articles to be reviewed. The journal has an extensive author support system and it has recently introduced a paid expedited review process. The journal also mentions the average time for processing the manuscript under different submission systems - regular submission and expedited review.
Strengths of the journal: The journal has an online first facility in which the accepted manuscripts may be published on the website before being included in a regular issue of the journal. This cuts down the time between their acceptance and the publication. The journal is indexed in many databases, though not in PubMed. The editorial board should now take steps to index the journal in PubMed. The journal has a system of notifying readers through e-mail when a new issue is released. Also, the articles are available in both the HTML and the PDF formats. I especially like the new and colorful page format of the journal. Also, the access statistics of the articles are available. The prepublication and the manuscript tracking system are also helpful for the authors.
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Overall, the publishing process with JCDR has been smooth, quick and relatively hassle free and I can recommend other authors to consider the journal as an outlet for their work."

Dr. P. Ravi Shankar
KIST Medical College, P.O. Box 14142, Kathmandu, Nepal.
On April 2011

Dear team JCDR, I would like to thank you for the very professional and polite service provided by everyone at JCDR. While i have been in the field of writing and editing for sometime, this has been my first attempt in publishing a scientific paper.Thank you for hand-holding me through the process.

Dr. Anuradha
On Jan 2020

Important Notice

Original article / research
Year : 2021 | Month : August | Volume : 15 | Issue : 8 | Page : DC01 - DC06 Full Version

Phenotypic and Genotypic Study of Klebsiella species with Reference to Extended Spectrum Beta-Lactamase

Published: August 1, 2021 | DOI:
Rakesh Prasad Sah, Rakesh Kumar Mukhia, AD Urhekar, Kshitija Rane

1. Assistant Professor, Department of Microbiology, Saraswati Medical College, Unnao, Uttar Pradesh, India. 2. Assistant Professor, Department of Microbiology, Hind Medical College, Sitapur, Uttar Pradesh, India. 3. Professor, Department of Microbiology, MGM Medical College, Navi Mumbai, Maharashtra, India. 4. Research Associate, Department of Medical Biotechnology, MGM Medical College, Navi Mumbai, Maharashtra, India.

Correspondence Address :
Dr. Rakesh Prasad Sah,
K-53, Faculty Residence, Saraswati Medical Collgege, Unnao-209859, Uttar Pradesh, India.


Introduction: Klebsiella species is an important nosocomial pathogen with the emergence of Multi Drug Resistance (MDR). MDR in Klebsiella species is increasing worldwide with the production Extended Spectrum Beta-Lactamase (ESBL). The emergence of ESBL is a critical concern in Klebsiella species due to resistance to ceftazidime and other cephalosporins which compromise the efficacy of life saving antibiotics against these infections.

Aim: To know the factors responsible for antimicrobial drug resistance in Klebsiella species with respect to ESBL and their responsible genes.

Materials and Methods: A prospective and experimental study was carried out over a period of three years (August 2013 to July 2016). Total 200 isolates of Klebsiella species were screened for cefotaxime and ceftazidime. The resistant strains (cefotaxime/ceftazidime) were subjected to ESBL agar, Phenotypic Confirmatory Disc Diffusion Test (PCDDT) and Modified Three Dimensional Test (M3DT). Genetic analysis by Polymerase Chain Reaction (PCR) was done for the detection of beta-lactamase (bla) genes i.e., blaTEM, blaSHV & blaCTX-M in 58 isolates of Klebsiella species. The data was presented using frequency and percentage. The proportion was compared using Z-test for the proportions.

Results: Out of 200 isolates, 135 (67.5%) were found resistant to cefotaxime and 125 (62.5%) were resistant to ceftazidime. Among which 110 (55%), 75 (37.5%) and 95 (47.5%) Klebsiella species were found positive for production of ESBL by ESBL agar, PCDDT and M3DT respectively. PCR analysis in 48 isolates were positive by PCDDT/M3DT or both were also positive for beta-lactamase genes i.e., 43 (89.58%) blaTEM; 44 (91.67%) blaSHV and 48 (100%) blaCTX-M. Ten negative isolates either by PCDDT/M3DT or both were also negative by PCR. Co-existence of (blaTEM+blaSHV +blaCTX-M), (blaTEM+blaSHV), (blaTEM+blaCTX-M) and (blaSHV+blaCTX-M) were found 81.25%, 0%, 8.33% and 10.42%, respectively.

Conclusion: The M3DT is the best phenotypic method for the confirmation of ESBL producer in Klebsiella species which is not included by CLSI while inclusion with PCDDT enhances the detection of ESBL producers. Co-existence of all three genes (blaTEM, blaSHV and blaCTX-M) in a single strain is a serious concern for us. So it is important to include M3DT and PCDDT in routine basis for the detection and management of ESBL producers which will help clinician to prescribe proper antibiotics.


Antimicrobial drug resistance, Modified three dimensional test, Multi drug resistance, Phenotypic confirmatory disc diffusion test

Klebsiella is a gram negative, encapsulated, facultative anaerobic bacterium which is opportunistic pathogen responsible for causing hospital and community acquired infections such as pneumonia, urinary tract infection, septicemia, soft tissue infections, liver abscess, meningitis etc., (1). Klebsiella pneumoniae an emerging concern for the worldwide as it is one of the major causes of drug resistant nosocomial infections which are hard to eradicate using available antibiotics (2),(3),(4). Moreover, extensive use of broad spectrum antibiotics in hospitalised patients has led to both increased carriage of Klebsiella and the development of MDR strains that produces ESBL. MDR strains possessing ESBLs have become an increasing problem worldwide (1) which leads to increased morbidity and mortality in hospitalised patients (4). ESBLs enzymes are encoded by certain specific genes which are predominantly found on plasmid. They are classified into three major groups; TEM (named for a patient called Temoniera), SHV (named for sulfhydryl variable) and CTX-M (named cefotaximase as it has strong hydrolytic activity against cefotaxime; CTX as its acronym and M from Munich) (5),(6),(7). Diagnosis of ESBL is extremely important for the proper management and treatment of infection caused by Klebsiella species. Few studies from Maharashtra showed production of ESBL in Klebsiella species as 8.5%, 24.1%, 27.6%, 28.8%, 31.75% and 90% from Mumbai, Navi Mumbai, Mumbai, Pimpri, Mumbai and Nasik, respectively (8),(9),(10),(11),(12),(13). We have very less data from our region regarding the correlation of phenotypic and genotypic expression of ESBL in Klebsiella species which is one of the leading pathogen causing nosocomial infection (9),(12). So this study was done to know the factors responsible for antimicrobial drug resistance in Klebsiella species with respect to ESBL and their responsible genes.

Material and Methods

A prospective and experimental study was carried out over a period of three years (August 2013 to July 2016) in microbiology laboratory of MGM Medical College and Hospital Kamothe, Navi Mumbai, Maharashtra, India. The research topic was cleared by Ethical committee for research on Human subjects on 27th October 2014 via letter no. MGM/HIS/RS/2014-15. Informed consents were taken from the patients before collection of the samples.

Inclusion criteria: Sample showing pus cells and bacteria were included in the study.

Exclusion criteria: Sample not showing pus cells and bacteria were excluded from the study.

Sample size formula used:

n=z2P*q / d2


Z=2.58 at 1% level of significance
P=the proportion=0.071
d=absolute margin of Error taken as 1%


Hence, the minimum required sample size was 4390, but 4440 samples were taken (14). Out of which, 1722 showed bacterial growth and 2718 showed no growth.

Out of 4440 samples, a total of 200 isolates of Klebsiella species were isolated from different clinical specimens which were characterised into different species by standard protocols (15). The isolates were tested for their susceptibility to the Third Generation Cephalosporins (3GCs) e.g., ceftazidime (30 μg), and cefotaxime (30 μg) by using the standard disc diffusion method as recommended by the Clinical and Laboratory Standards Institute (CLSI). If a zone diameter of <22 mm for ceftazidime and <27 mm for cefotaxime was seen then the strain was considered to be “suspicious for ESBL production” (16). The isolates which were resistant to one of the above 3GCs were selected for the study and were further processed for the detection of ESBL production.


Each resistant bacterial isolates of Klebsiella spp was cultured on Himedia ESBL agar (Hichrome ESBL agar base and Hichrome ESBL agar supplement catalogue no. M1829 and FD278-1VL, respectively) and incubated aerobically at 37°C for 18-24 hours. Colonies of ESBL producers Klebsiella spp develop metallic blue colouration. Non ESBL producers grow with colourless colonies or not at all on ESBL agar as shown in the (Table/Fig 1).

Phenotypic Confirmatory Disc Diffusion Test (PCDDT) (16): The ceftazidime disc (30 μg) and cefotaxime (30 μg) alone and in combination with clavulanic acid (ceftazidime+clavulanic acid (30/10 μg), cefotaxime+clavulanic acid (30/10 μg) tested on Mueller Hinton Agar plate previously inoculated with the test strain. An increase of ≥5 mm in the zone of inhibition of the combination disc in comparison to the ceftazidime or cefotaxime disc alone is considered to be a marker for ESBL production as shown in the (Table/Fig 2).

Modified three-dimensional enzyme extract method (M3DT) (17): This method was previously used for the detection of AmpC β-lactamase but here certain steps have been modified and applied for the detection of ESBL. Overnight culture from the MacConkey agar plate was transferred into a sterile microcentrifuge tube containing peptone water and incubated at 37°C for 3-4 hours then centrifuged at 3000 rpm for 15 minutes. Afterwards, the pellet was subject to repeated freeze-thawing for several times (minimum 7) in the freezer portion of the ordinary refrigerator and crude ESBL enzyme was extracted. Ceftazidime (30 μg) and Cefotaxime (30 μg) disc were placed on Mueller Hinton agar plates containing lawn of 0.5 McFarland of E.coli ATCC (American Type Culture Collection) 25922 culture. Then linear slits (3 cm) was cut using sterile surgical blade, 3 mm away from antibiotic disc. A well was made using sterile standard bacteriological loop having diameter of 2 mm. A total of 30 μL of enzyme extract was loaded in the slit. The plates were kept upright for 5-10 minutes until the liquid dried and incubated at 37°C for 24 hours. The clear distortion of zone of inhibition of ceftazidime and/or cefotaxime was considered as positive test for the presence of ESBL as shown in the (Table/Fig 3), (Table/Fig 4).

Genotypic detection (18),(19),(20): For the Plasmid DNA Extraction, the colonies around cefotaxime disc grown on MHA were taken. Plasmid DNA was isolated by using Himedia plasmid DNA extraction kit (Catalogue no. MB 508-50PR) by using manufacturer instructions. PCR analysis for ESBL genes (blaTEM, blaSHV and blaCTX-M) were carried out using following primers which were customised and procured from Eurofin Banglore (Table/Fig 5).

For PCR amplification, 5 μL of template DNA+12.5 μL of master mixture (readymade mastermix of kapa)+1 μL of forward primer+1 μL of reverse primer+5.5 μL of nuclease free water to make final volume 25 μL. Amplification was carried out according to the following thermal and cycling condition.

For TEM and SHV genes (18),(19)

Initial denaturation at 94°C for 5 minutes 35 cycles
Denaturation at 94°C for 1 min
Annealing at 58°C for 1 min
Extension at 72°C for 1 min
Final extension at 72°C for 10 min

For CTX-M gene (20)

Initial denaturation at 94°C for 5 minutes 35 cycles
Denaturation at 94°C for 1 min
Annealing at 45°C for 1 min
Extension at 72°C for 1 min
Final extension at 72°C for 10 min

The PCR products were analysed after electrophoresis in 1.5% agarose gel to detect specific amplified product by comparing with DNA ladder. After completion of electrophoresis the bands were visualised under UV transilluminator. The different amplicon size of different genes are shown in the (Table/Fig 6).

Statistical Analysis

The data was presented using frequency and percentage. The proportion was compared using Z-test for proportions. Diagnostic tests such as sensitivity and specificity were used. The level of significance was set at 5%. All p-values <0.05 were treated as significant.


Out of 1722 positive bacterial growth, 200 isolates of Klebsiella species were isolated from clinical samples with incidence rate of 11.6%. Among which 112 (56%) and 88 (44%) strains were from male and female respectively with the male female ratio 1.27:1. Out of 200, 24% (n=48) of the cases were from Outatient Department (OPD) while remaining 76% (n=152) from the Inpatient Department (IPD). Out of 200 isolates of Klebsiella, 135 isolates were resistant to cefotaxime and 125 isolates were resistant to ceftazidime. So they were suspicious for ESBL production, and further subjected to ESBL agar, PCDDT by using cefotaxime/clavulanic acid and ceftazidime/clavulanic acid as per CLSI guidelines and M3DT (Table/Fig 7).

The incidence of ESBL producers in Klebseilla species in this study was 51.5% by combination of both methods i.e., PCDDT and M3DT (Table/Fig 8).

Species wise distribution of ESBL producers: Out of 200 isolates; 169 were Klebsiella pneumoniae subsp. pneumoniae, seven were Klebsiella pneumonia ozaenae and 24 were Klebsiella oxytoca. Among 169 Klebsiella pneumoniae subsp. pneumoniae, 80 (47.3%), 60 (35.5%) and 78 (46.1%) isolates were positive by ESBL agar, PCDDT and M3DT, respectively. Among seven Klebsiella pneumoniae subsp. ozaenae, 5 (71.4%), 3 (42.9%) and 4 (57.1%) isolates were positive by ESBL agar, PCDDT and M3DT respectively. Among 24 Klebsiella oxytoca; 19 (79.2%), 12 (50%), 13 (54.17%) isolates were positive by ESBL agar, PCDDT and M3DT, respectively.

PCR: Fifty-eight isolates (48 positive and 10 negative by phenotypic methods) of Klebsiella from different groups were selected for the detection of genes (blaTEM, blaSHV and blaCTX-M) responsible for ESBL production. The isolates from different groups were PCDDT and M3DT positive (20), PCDDT negative and M3DT positive (20), PCDDT positive and M3DT negative (8), PCDDT and M3DT both negative (5), ESBL agar positive and PCDDT and M3DT negative (5). Co-existence of (blaTEM+blaSHV+blaCTX-M), (blaTEM +blaSHV), (blaTEM +blaCTX-M) and (blaSHV +blaCTX-M) were found 81.25%, 0%, 8.33% and 10.42%, respectively (Table/Fig 9), (Table/Fig 10), (Table/Fig 11).


Increasing resistance to 3GCs has become a serious concern among pathogens causing nosocomial infections. The prevalence of ESBL is a critical concern to the available beta-lactam antibiotics causing treatment failure which leads to increase hospital stay and treatment costs. Among available wide range of antibiotics, beta-lactam antibiotics are widely used for almost more than 50% of the all systemic bacterial infections (21).

Knowledge of ESBL strains is necessary for proper treatment and management of patients. ESBL production in Klebsiella species was studied by various researchers in which incidence of it ranges from 8.5-90% (8),(13). In present study, 37.5% and 47.5% isolates of Klebsiella species were ESBL producer by PCDDT and M3DT respectively. Some results were similar to present study, where others reported lower or higher values which is shown in the (Table/Fig 12) (22),(23),(24),(25),(26),(27),(28),(29),(30),(31),(32),(33),(34),(35),(36),(37). These differences could be due to various reasons like methodology followed by different researchers (Disc approximation method, combined disc diffusion method, phenotypic disc diffusion test, E-test etc.,), constant use of particular antibiotic can lead to development of drug resistance by ESBL which will depend on the quantum of drug prescribed- can be directly proportional to prescription polices.

The genotypic method using specific PCR amplification of resistance genes seems to have 100% specificity and sensitivity (22). The ESBL producers usually consist of multiple resistant plasmid mediated genes e.g., blaTEM, blaSHV and blaCTX-M. In present study, it was found blaTEM 43 (89.58%), blaSHV 44 (91.67) and blaCTX-M 48 (100%) which was compared with other researchers given in the (Table/Fig 12) (22),(23),(24),(25),(26),(28),(29),(30),(31),(32),(33),(34),(35),(36),(37). In present study, the best phenotypic method for the confirmation of ESBL were shown by M3DT in comparison to PCDDT; as all the negative strains by PCDDT and positive by M3DT were found positive for the ESBL genes by PCR.

The co-existence of ESBL genes in a single isolates were 39 (81.25%) for blaTEM+blaSHV+blaCTX-M, 4 (8.33%) for blaTEM+blaCTX-M and 5 (10.42%) for blaSHV+blaCTX-M. Similar results have been reported by Bora A et al., Chandra V et al., and Khosravi AD et al., (23),(24),(25). The presence of multiple genes in a single isolates like blaTEM+blaSHV+blaCTX-M may be related to complex antimicrobial resistance (22). So it is important to continuously monitor resistance pattern and enhancing the infection control for these strains in healthcare units.


Advance molecular methods for the characterisation of Klebsiella species were not conducted. PCR in all cefotaxime/ceftazidime resistant isolates of Klebsiella for detection of ESBL genes were not conducted due to lack of funding as it was self sponsored.


To conclude this, it is suggested that M3DT is best phenotypic methods for the detection of ESBL than PCDDT using both antibiotics i.e., cefotaxime and ceftazidime. The result seems to have 100% sensitive and specific by PCR. Some isolates positive by ESBL agar and negative by M3DT and PCDDT were also found negative by PCR so present study suggests not to use ESBL agar for the detection of ESBL. BlaCTX-M was a dominant gene in Klebsiella species for ESBL producer. Increasing existence of blaCTX-M and co-existence of all the three responsible ESBL genes (blaTEM+blaSHV+blaCTX-M) are a serious concern for us. So, it is important to continuously monitor resistance pattern and enhancing the infection control for these strains in healthcare units. It is also important to report ESBL producers on routine basis which will help the clinicians to prescribe proper antibiotics to manage infections caused by them.


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Date of Submission: Jun 16, 2021
Date of Peer Review: Jun 29, 2021
Date of Acceptance: Jul 21, 2021
Date of Publishing: Aug 01, 2021

• Financial or Other Competing Interests: None
• Was Ethics Committee Approval obtained for this study? Yes
• Was informed consent obtained from the subjects involved in the study? Yes
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