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Authors are the souls of any journal, and deserve much respect. To publish a journal manuscripts are needed from authors. Authors have a great responsibility for producing facts of their work in terms of number and results truthfully and an individual honesty is expected from authors in this regards. Both ways its true "No authors-No manuscripts-No journals" and "No journals�No manuscripts�No authors". Reviewing a manuscript is also a very responsible and important task of any peer-reviewed journal and to be taken seriously. It needs knowledge on the subject, sincerity, honesty and determination. Although the process of reviewing a manuscript is a time consuming task butit is expected to give one's best remarks within the time frame of the journal.
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.
Timely publication of journal: Publication of manuscripts and bringing out the issue in time is one of the positive aspects of JCDR and is possible with strong support team in terms of peer reviewers, proof reading, language check, computer operators, etc. This is one of the great reasons for authors to submit their work with JCDR. Another best part of JCDR is "Online first Publications" facilities available for the authors. This facility not only provides the prompt publications of the manuscripts but at the same time also early availability of the manuscripts for the readers.
Indexation and online availability: Indexation transforms the journal in some sense from its local ownership to the worldwide professional community and to the public.JCDR is indexed with Embase & EMbiology, Google Scholar, Index Copernicus, Chemical Abstracts Service, Journal seek Database, Indian Science Abstracts, to name few of them. Manuscriptspublished in JCDR are available on major search engines ie; google, yahoo, msn.
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.
It is well said that "happy beginning is half done" and it fits perfectly with JCDR. It has grown considerably and I feel it has already grown up from its infancy to adolescence, achieving the status of standard online e-journal form Indian continent since its inception in Feb 2007. This had been made possible due to the efforts and the hard work put in it. The way the JCDR is improving with every new volume, with good quality original manuscripts, makes it a quality journal for readers. I must thank and congratulate Dr Hemant Jain, Editor-in-Chief JCDR and his team for their sincere efforts, dedication, and determination for making JCDR a fast growing journal.
Every one of us: authors, reviewers, editors, and publisher are responsible for enhancing the stature of the journal. I wish for a great success for JCDR."
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)
E-mail: drrajendrak1@rediffmail.com
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.
Areas for improvement: In certain cases, I felt that the peer review process of the manuscripts was not up to international standards and that it should be strengthened. Also, the number of manuscripts in an issue is high and it may be difficult for readers to go through all of them. The journal can consider tightening of the peer review process and increasing the quality standards for the acceptance of the manuscripts. I faced occasional problems with the online manuscript submission (Pre-publishing) system, which have to be addressed.
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.
E-mail: ravi.dr.shankar@gmail.com
On April 2011 Anuradha
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
E-mail: anuradha2nittur@gmail.com
On Jan 2020
Original article / research
Full Version
Comparison of Various Phenotypic Methods and mecA Based PCR for the Detection of MRSA
Correspondence Address :
Pramodhini S.
Assistant Professor
Department of Microbiology,
Mahatma Gandhi Medical College and Research Institute,
Pillaiyarkuppam, Pondicherry – 607 402
Phone: +91 9445216145
E-mail: pramo4@yahoo.co.in
Background: Methicillin resistant Staphylococcus aureus (MRSA) is the most commonly emerging pathogen in community and hospital acquired infections. Hence, an accurate detection is not only important for the control of the infection, but also to control the endemicity of MRSA.
Aim: To evaluate the efficacy of various phenotypic methods with mecA based PCR to detect MRSA. We also studied the resistance pattern of the MRSA isolates. Settings and Design: This was a prospective study which was conducted at a tertiary care hospital.
Materials and Methods: A total of 55 S. aureus strains which were isolated from patients with superficial and deep abscesses were included in this study. Methicillin resistance which was determined by oxacillin disc diffusion, cefoxitin disc diffusion and the oxacillin screen agar test was compared with mecA based PCR.
Results: Among the 55 S . aureus isolates, 20 (36.4%) isolates were positive for the mecA gene by PCR. Both the cefoxitin disc diffusion and the oxacillin screen agar test showed 100% sensitivity and 100% specificity, while oxacillin disc diffusion showed 90% sensitivity and 100% specificity. The resistance percentage of the MRSA isolates to erythromycin, ciprofloxacin and amikacin were 80%, 30% and 25%, respectively.
Conclusion: Conventional MRSA detection assays like the cefoxitin disc diffusion test and the oxacillin screen agar test are simple and relatively cheap and can be used as alternatives to PCR for the detection of MRSA in resource constraint settings. Also, most of the MRSA strains in this study showed coresistance to many classes of antibiotics and thus they qualified as multi-drug resistant S. aureus.
MRSA, cefoxitin disc diffusion, oxacillin screen agar, mecA gene
INTRODUCTION
Methicillin-resistant Staphylococcus aureus (MRSA) strains were first identified in 1961, immediately after the introduction of methicillin in the clinical settings. Subsequently, an increase in the resistance to methicillin among the S. aureus isolates has been observed globally (1). MRSA is one of the major pathogens which are associated with serious nosocomial infections, because these strains generally show multiple drug resistance which limits the treatment possibilities. MRSA has become established outside the hospital environment and it is now appearing in community populations without any identifiable risk factors (2).
The methicillin resistance in Staphylococci is due to the acquisition of the mecA gene, which encodes the low-affinity penicillin-binding protein 2a (3). The presence of the mecA gene in S. aureus defines methicillin resistance, while the absence of the gene indicates methicillin susceptibility (4). Methicillin resistance can be difficult to detect, because the mecA-positive strains can differ in their level of expression of resistance. The resistance is typically heterogeneous, with only a few cells (one in 104 or 106) expressing the phenotype.
The mecA gene is highly conserved among the Staphylococcal species and therefore, presently, the detection of this gene by the polymerase chain reaction (PCR) is considered as the “goldstandard” for the detection of methicillin resistance in Staphylococci (5),(6),(7). While PCR is considered as the gold standard assay for the detection of methicillin resistance, it still remains a time-consuming and expensive method; besides, it is not available in most of the routine laboratories.
The objective of the present study was to determine the methicillin resistance in S. aureus by mecA based PCR and to evaluate the usefulness of various phenotypic methods for the detection of MRSA in comparison to mecA based PCR. We also aimed to study the resistance pattern of the MRSA isolates.
Clinical Isolates
A prospective study was conducted over a period of one year from May 2008 to June 2009 at a tertiary care hospital in south India. A total of 55 S. aureus strains which were isolated from patients with superficial and deep abscesses were included in this study. All the isolates were identified as S. aureus by their colony morphology, gram staining and their catalase and coagulase tests (both tube and slide tests). These 55 clinical isolates were tested for methicillin resistance by oxacillin disk diffusion, cefoxitin disc diffusion and the oxacillin screen agar test. From this collection, all the isolates were evaluated further by using mecA based PCR.
All the Staphylococcal isolates were tested for susceptibility to a predetermined battery of antibiotics by the Kirby-Bauer disc diffusion method. The antibiotics which were tested included Penicillin (10u), Ampicillin (10μg), Cefotaxime (30μg), Amikacin (30 μg), Erythromycin (15 μg), Ciprofloxacin (5μg) Oxacillin (1μg) and Vancomycin (30 μg). The zones of inhibition were interpreted according to the Clinical Laboratory Standards Institute (CLSI) guidelines (8).
Phenotypic Detection of MRSA
The Oxacillin D isk Diffusion Method: The Oxacillin disk (1 μg) diffusion method was carried out on Mueller-Hinton agar which was supplemented with 2% NaCl to detect MRSA according to the CLSI guidelines (8). The plates were incubated at 35°C and the results were recorded after 24 hrs of incubation. The isolates were considered as resistant when the diameter of inhibition was ≤10 mm, as intermediately resistant when the diameter was 11-12 mm and as sensitive when the diameter was ≥13 mm (8).
The Cefoxitin Disc Diffusion Test:
The Cefoxitin disc diffusion method was carried out on Mueller-Hinton agar by using a 30 μg cefoxitin disc. An inhibition zone diameter of ≤ 21 mm was reported as methicillin resistant and a diameter of ≥ 22 mm was considered as methicillin sensitive (8).
The Oxacillin Screen Agar Test:
Muller-Hinton agar plates containing 4% NaCl and 6 μg/ml of oxacillin were prepared. To perform the oxacillin screen test, a swab which was dipped in 0.5 Mc Farland’s suspension of the isolate was deposited as a spot on the agar surface and it was incubated at 35°C for 24 h. The plates were observed carefully in transmitted light for any growth. Any growth after 24 hr was interpreted as oxacillin resistance (9).
The Genotypic Detection of MRSA
Detection of the mecA Gene by Polymerase Chain Reaction: Bacterial DNA was obtained by the rapid cell lysis method as described by Unal et al (10). For the DNA extraction, 0.1 mL of an overnight culture of bacteria in Mueller-Hinton broth washarvested by centrifuging the broth in a microcentrifuge tube at 16,000×g for 30 seconds. The precipitates were resuspended in 50 μL lysostaphin (100 μg/mL; Sigma) and they were incubated at 37°C for 10 minutes. Following the addition of 50 μL proteinase K (100 μg/mL; Sigma Aldrich) and 150 μL 100 mm Tris (pH 7.5), the suspension was incubated for 10 minutes at 37°C and then boiled for 5 minutes. After centrifugation at 13,000×g for 2 minutes, the supernatant which contained the extracted bacterial DNA was used in the PCR assay.
From this suspension, a 5μL volume was directly used as the template for the PCR amplification of the mec A gene fragments. The mec A1 (5´ – GTA GAA ATG ACT GAA CGT CCG ATA A – 3´) and the mec A2 (5´ – CCA ATT CCA CAT TGT TTC GGT CTA A – 3´) primers were used for the amplification of the 310 bp fragment of the methicillin-resistant gene ( mec A) (11). A 50 μl PCR reaction consisted of plus 45 μl of the master mix which contained the PCR buffer (1×), dNTP mix (0.2 mM of each), the primer (0.5 μM), Taq DNA polymerase (0.25 U), and MgCl 2 (1.5 mM) with 5 μL of the template DNA. The cycling conditions were as follows: 4 minutes at 94°C, followed by 30 cycles of denaturation at 94°C for 45 seconds, annealing at 50°C for 45 seconds, extension at 72°C for 1 minute and the final extension step at 72°C for 3 minutes. The PCR products were visualized on an 0.8% agarose gel with ethidium bromide dye under a UV transilluminator. Amplicons of 310 bp were consistent with the mecA gene amplification.
Quality control
The quality control strains – methicillin sensitive S. aureus (MSSA) ATCC 25923 and methicillin resistant S. aureus (MRSA) ATCC 43300 – were used as the negative and positive controls, respectively.
Statistical Analysis
The percentages were calculated for the categorical variables. The sensitivity, specificity and the positive and negative predictive values were calculated by using the GraphPad InStat version 3.00 for Windows 95 and the GraphPad Software (San Diego, CA, USA) for determining the diagnostic value of the various phenotypic methods for detecting methicillin resistance.
Among the 55 S . aureus isolates, 20 (36.4%) were positive for the mecA gene by PCR. Methicillin resistance was detected by oxacillin disc diffusion, cefoxitin disc diffusion and the oxacillin screen agar test in 18, 20 and 20 isolates, respectively. The sensitivity, specificity and the positive and negative predictive values of the various phenotypic methods in comparison to PCR, for the detection of MRSA, are summarized in (Table/Fig 1).
In our study, the resistance percentage of the MRSA isolates were as follows: penicillin- 100%, ampicillin- 85%, cephotaxime and erythromycin- 80%, ciprofloxacin- 30%, amikacin- 25% and all the strains were 100% sensitive to vancomycin.
S. aureus is one of the most common causes of nosocomial as well as community-acquired infections. Methicillin resistance (as a result of the mecA gene which encodes the additional penicillin binding protein, PBP2a) renders S. aureus resistant to all the β-lactam antibiotics which is the most important group of antibiotics in the treatment of Staphylococcal infections.
The recent increase in the methicillin-resistant and ultipleresistant strains at large hospitals have started to pose a great difficulty in selecting antimicrobial agents for the management of the infections that they cause. Hence, an accurate and rapid detection of methicillin resistance in Staphylococci is therefore important, not only for choosing the appropriate antibiotic therapy for the individual patient, but also for the control of the endemicity of the MRSA.
Our study revealed that, overall the rate of methicillin-resistance with S. aureus was nearly 36.4%. Similar rates which were reported in other studies from tertiary care centers all over the world (12),(13) supported this high incidence which was found in our study.
In our study, the oxacillin disc diffusion method detected 18 of the 20 cases of MRSA with a sensitivity of 90% and a specificity of 100%, which was in concordance to the results of the previous study (14), which showed 87.5% sensitivity and 100% specificity by the oxacillin disc diffusion method. Although the oxacillin disc diffusion test has high sensitivity and specificity for detecting methicillin resistance, some difficulties have been encountered with it in detecting the hetero-resistant isolates of S. aureus. The accurate determination of methicillin resistance in Staphylococci by the oxacillin disc diffusion method may be affected by various components of medium, temperature, and the duration of incubation (15). Hence, other phenotypic methods like the agar screen method and the cefoxitin disc diffusion method have been evaluated.
MRSA detection by the oxacillin screen agar method identified all the 20 MRSA with 100% sensitivity and 100% specificity. A similar study reported that the sensitivity of this method approached 100% for the detection of MRSA and 95% for the coagulase-negative S. aureus (16).
In this study, the cefoxitin disc diffusion method detected 20 (36.4%) out of the 55 isolates as MRSA, which accounted for 100% sensitivity and specificity as compared to the mecA-based PCR. Cefoxitin, a cephamycin, is a more potent inducer of the mecA regulatory system and an accurate surrogate marker for the detection of MRSA in the routine susceptibility testing. It has been suggested that no special medium or incubation temperature is required for cefoxitin as is required for oxacillin (17). Recent studies have indicated that disc diffusion testing by using the cefoxitin disc is far superior to most of the currently recommended phenotypic methods like oxacillin disc diffusion and oxacillin screen agar testing and that it is now an accepted method for the detection of MRSA by many reference groups including CLSI (18).
In our study which was conducted to determine the resistance pattern of the MRSA isolates, the resistance to erythromycin, amikacin and ciprofloxacin were 85%, 40% and 30% respectively and no strains were found to be resistant to vancomycin, which was similar to the findings of several other studies (19),(20). The MRSA showed a high level of resistance to most of the antibiotics in comparison to the methicillin sensitive S. aureus. Also, most of the MRSA in this study showed co-resistance to many classes ofantibiotics at the same time and thus they qualified as multi-drug resistant S. aureus (MDR-MRSA).
In conclusion, as has been shown in this as well as other studies, the oxacillin disk diffusion method was found to be less sensitive for the detection of MRSA. The results of the cefoxitin disc diffusion test and the oxacillin screen agar test were in concordance with the results of PCR for the mecA gene. Moreover, these conventional MRSA detection assays like the cefoxitin disc diffusion test and the oxacillin screen agar test are simple and relatively cheap and they can be used as an alternative to PCR for the detection of MRSA in resource constraint settings.
‘MRSA’ is a term which is used to describe the Staphylococcus aureus isolates that are resistant to all the available β-lactam antibiotics, including the penicillins and the cephalosporins. n Methicillin resistance in S. aureus is primarily mediated by the mecA gene, which codes for the modified penicillin-binding protein 2a (PBP 2a or PBP 2’) Strains that possess the mecA gene are either heterogeneous or homogeneous in their expression of resistance. The phenotypic expression of the resistance can vary, depending on the growth conditions (e.g. temperature, osmolarity and culture medium supplements such as NaCl or sucrose) The detection of the mecA gene by PCR is considered as the gold standard.
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