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

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Dr Mohan Z Mani

"Thank you very much for having published my article in record time.I would like to compliment you and your entire staff for your promptness, courtesy, and willingness to be customer friendly, which is quite unusual.I was given your reference by a colleague in pathology,and was able to directly phone your editorial office for clarifications.I would particularly like to thank the publication managers and the Assistant Editor who were following up my article. I would also like to thank you for adjusting the money I paid initially into payment for my modified article,and refunding the balance.
I wish all success to your journal and look forward to sending you any suitable similar article in future"



Dr Mohan Z Mani,
Professor & Head,
Department of Dermatolgy,
Believers Church Medical College,
Thiruvalla, Kerala
On Sep 2018




Prof. Somashekhar Nimbalkar

"Over the last few years, we have published our research regularly in Journal of Clinical and Diagnostic Research. Having published in more than 20 high impact journals over the last five years including several high impact ones and reviewing articles for even more journals across my fields of interest, we value our published work in JCDR for their high standards in publishing scientific articles. The ease of submission, the rapid reviews in under a month, the high quality of their reviewers and keen attention to the final process of proofs and publication, ensure that there are no mistakes in the final article. We have been asked clarifications on several occasions and have been happy to provide them and it exemplifies the commitment to quality of the team at JCDR."



Prof. Somashekhar Nimbalkar
Head, Department of Pediatrics, Pramukhswami Medical College, Karamsad
Chairman, Research Group, Charutar Arogya Mandal, Karamsad
National Joint Coordinator - Advanced IAP NNF NRP Program
Ex-Member, Governing Body, National Neonatology Forum, New Delhi
Ex-President - National Neonatology Forum Gujarat State Chapter
Department of Pediatrics, Pramukhswami Medical College, Karamsad, Anand, Gujarat.
On Sep 2018




Dr. Kalyani R

"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."



Dr Kalyani R
Professor and Head
Department of Pathology
Sri Devaraj Urs Medical College
Sri Devaraj Urs Academy of Higher Education and Research , Kolar, Karnataka
On Sep 2018




Dr. Saumya Navit

"As a peer-reviewed journal, the Journal of Clinical and Diagnostic Research provides an opportunity to researchers, scientists and budding professionals to explore the developments in the field of medicine and dentistry and their varied specialities, thus extending our view on biological diversities of living species in relation to medicine.
‘Knowledge is treasure of a wise man.’ The free access of this journal provides an immense scope of learning for the both the old and the young in field of medicine and dentistry as well. The multidisciplinary nature of the journal makes it a better platform to absorb all that is being researched and developed. The publication process is systematic and professional. Online submission, publication and peer reviewing makes it a user-friendly journal.
As an experienced dentist and an academician, I proudly recommend this journal to the dental fraternity as a good quality open access platform for rapid communication of their cutting-edge research progress and discovery.
I wish JCDR a great success and I hope that journal will soar higher with the passing time."



Dr Saumya Navit
Professor and Head
Department of Pediatric Dentistry
Saraswati Dental College
Lucknow
On Sep 2018




Dr. Arunava Biswas

"My sincere attachment with JCDR as an author as well as reviewer is a learning experience . Their systematic approach in publication of article in various categories is really praiseworthy.
Their prompt and timely response to review's query and the manner in which they have set the reviewing process helps in extracting the best possible scientific writings for publication.
It's a honour and pride to be a part of the JCDR team. My very best wishes to JCDR and hope it will sparkle up above the sky as a high indexed journal in near future."



Dr. Arunava Biswas
MD, DM (Clinical Pharmacology)
Assistant Professor
Department of Pharmacology
Calcutta National Medical College & Hospital , Kolkata




Dr. C.S. Ramesh Babu
" Journal of Clinical and Diagnostic Research (JCDR) is a multi-specialty medical and dental journal publishing high quality research articles in almost all branches of medicine. The quality of printing of figures and tables is excellent and comparable to any International journal. An added advantage is nominal publication charges and monthly issue of the journal and more chances of an article being accepted for publication. Moreover being a multi-specialty journal an article concerning a particular specialty has a wider reach of readers of other related specialties also. As an author and reviewer for several years I find this Journal most suitable and highly recommend this Journal."
Best regards,
C.S. Ramesh Babu,
Associate Professor of Anatomy,
Muzaffarnagar Medical College,
Muzaffarnagar.
On Aug 2018




Dr. Arundhathi. S
"Journal of Clinical and Diagnostic Research (JCDR) is a reputed peer reviewed journal and is constantly involved in publishing high quality research articles related to medicine. Its been a great pleasure to be associated with this esteemed journal as a reviewer and as an author for a couple of years. The editorial board consists of many dedicated and reputed experts as its members and they are doing an appreciable work in guiding budding researchers. JCDR is doing a commendable job in scientific research by promoting excellent quality research & review articles and case reports & series. The reviewers provide appropriate suggestions that improve the quality of articles. I strongly recommend my fraternity to encourage JCDR by contributing their valuable research work in this widely accepted, user friendly journal. I hope my collaboration with JCDR will continue for a long time".



Dr. Arundhathi. S
MBBS, MD (Pathology),
Sanjay Gandhi institute of trauma and orthopedics,
Bengaluru.
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.
My best wishes to Dr. Hemant Jain and all the editorial staff of JCDR for their untiring efforts to bring out this journal. I strongly recommend medical fraternity to publish their valuable research work in this esteemed journal, JCDR".



Dr. Mamta Gupta
Consultant
(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.


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

Important Notice

Original article / research
Year : 2021 | Month : August | Volume : 15 | Issue : 8 | Page : DC20 - DC24 Full Version

A Study on Requirement of Cold Chain Maintenance for Reliable Testing of SARS-CoV-2 Samples


Published: August 1, 2021 | DOI: https://doi.org/10.7860/JCDR/2021/50307.15298
Varunika Vijayvergia, Aruna Vyas, Nazneen Pathan, Rajni Sharma, Snigdha Purohit, Akriti Aggarwal, Neha Sharma, Nitya Vyas

1. Assistant Professor, Department of Microbiology, SMS Medical College, Jaipur, Rajasthan, India. 2. Senior Professor, Department of Microbiology, SMS Medical College, Jaipur, Rajasthan, India. 3. Assistant Professor, Department of Microbiology, SMS Medical College, Jaipur, Rajasthan, India. 4. Senior Professor, Department of Microbiology, SMS Medical College, Jaipur, Rajasthan, India. 5. Senior Resident, Department of Microbiology, SMS Medical College, Jaipur, Rajasthan, India. 6. Resident, Department of Microbiology, SMS Medical College, Jaipur, Rajasthan, India. 7. Resident, Department of Microbiology, SMS Medical College, Jaipur, Rajasthan, India. 8. Senior Professor, Department of Microbiology, Mahatma Gandhi Medical College Jaipur, Rajasthan, India.

Correspondence Address :
Dr. Nitya Vyas,
283, Ganga Vihar Enclave, Behind VIT, Jagatpura, Jaipur, Rajasthan, India.
E-mail: drnityavyas@hotmail.com

Abstract

Introduction: Coronavirus Disease 2019 (COVID-19) has been haunting the world since December 2019 and has grown to pandemic proportions from March 2020. Even after a full year of research and study, the most effective way to control the spread of this infection is early diagnosis and isolation of the cases. Real-time Reverse Transcription Polymerase Chain Reaction (RT-PCR) is considered the standard test all over the world for the diagnosis of Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) infection. All the sample collection guidelines have recommended stringent maintenance of the cold chain for the sample transport. However, it is not possible for the resource constrained developing countries with inadequate infrastructure to comply with these guidelines all the time.

Aim: To determine necessity of stringent transport criteria and the effect of temperature on the clinical sensitivity of a RT-PCR assay for diagnosis of SARS-CoV-2 infection.

Materials and Methods: In this prospective experimental study conducted in November 2020, 49 positive samples were kept at ambient room temperature and were tested everyday with RT-PCR for the detection of SARS-CoV-2 Ribonucleic Acid (RNA). The samples were also kept under refrigeration at 4°C and were also tested by RT-PCR and the results were compared with their respective counterparts kept at room temperature till nine days. Python Jupiter notebook SciPy and Anaconda software was used for statistical analysis.

Results: It was observed that the positivity of the RT-PCR results were not deteriorated till five days and there was no significant deterioration even after nine days of samples being stored at room temperature suggesting that even if the viral RNA itself is not stable outside strict temperature control but small fragment or target genetic sequences are enough for detection of virus by RT-PCR.

Conclusion: It is possible to keep samples at this ambient temperature for five days without any loss of positivity in RT-PCR.

Keywords

Ambient temperature, Coronavirus disease 2019, Developing countries, Nucleic acid, Reverse transcription polymerase chain reaction, Viral transport medium

COVID-19 pandemic caused by SARS-CoV-2 virus since its emergence in December 2019 in Wuhan (1), China has taken the world with surprise and has been raging war against humanity with unprecedented ferocity. Total 93,698,260 cases have occurred worldwide till 14 January 2021 (2) which has led to a global catastrophises in financial losses and loss of more than 2 million lives directly due to COVID and unaccounted indirect loss of lives due to post COVID complications. Coronaviruses comprise a unique clade under the subgenus Sarbecovirus (3) and consists of a group of SARS-like bat coronaviruses, including both SARS-CoV-1 and SARS-CoV-2. Earlier epidemics like the Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS) had raised awareness regarding timely diagnosis to prevent rapid transmission of such viral infections, but COVID-19 had spread uncontrollably beyond all restrictions. In spite of all understanding we got about this virus in a span of almost one year, best effective tool to control the spread of infection is early diagnosis and isolation of the cases (4). In such a scenario inability to detect the positive samples may lead to missing out an important source of infection in community undiagnosed. Out of various modalities for diagnosis the most reliable and gold standard method for detection of SARS-CoV-2 viral infection is real-time RT-PCR which is the most sensitive and specific assay (5). Diagnosis of COVID-19 disease by real-time RT-PCR mainly requires the preservation of RNA instead of replication competent virus. It utilises the amplification of the viral nucleic acid present in the virus from the patient sample. Coronaviruses have a number of molecular targets like E (Envelope), N (Nucleocapsid), Orf (Open reading frame) and RdRp (RNA dependent RNA polymerase) gene within their positive-sense, single-stranded RNA genome that can be used for PCR assays (6).

Earlier, some studies have been conducted to understand the effect of temperature on virus viability (7),(8), but very little is known about the effect of temperature on the results of RT-PCR for viral RNA detection which does not require viable virus. For screening or early diagnosis of coronavirus infectious disease, a nasopharyngeal or an oropharyngeal swab is the recommended specimen of choice (9),(10). After collection, these swabs are immediately put in Viral Transport Medium (VTM) before sending them to laboratory for testing. All the national (11) and international (12) sample collection and transport guidelines strongly recommend the maintenance of cold chain during transport and storage of samples. The outrageous increase in the number of cases has outreached the capacity of the healthcare system enormously and often it becomes very difficult to comply with these sample collection and storage guidelines especially in resource limited developing countries. Even the developed countries of the world had faced similar problem as none of them had expected and were prepared for such a massive outburst. In such a situation either these samples are rejected or even if tested, it raises a concern about the quality of testing and reliability of results obtained with such samples.

This study was undertaken to observe the effect of ambient temperature on the RT-PCR results of SARS-CoV-2 samples. It can serve as a guide to the degree of confidence with which the samples are exposed to the ambient temperature for a prolonged duration of time and can be proceeded for testing and the results generated be relied upon.

Material and Methods

This study was a prospective experimental study conducted within the month of November 2020, on the samples (nasopharyngeal and oropharyngeal swabs) received in Vitromed Healthcare, India (VTM) for diagnosis of the SARS-CoV-2 viral disease at the COVID-19 testing laboratory, Department of Microbiology, SMS Medical College, Jaipur, Rajasthan, India. As the study was conducted on the remanent samples which were received for the routine testing for COVID-19 infection after the implied consent of the patient and the study had no direct or indirect implication on the patient, so the ethical clearance for the study was not required.

Inclusion criteria

• Samples which were collected within the hospital premises.
• Samples which reached the laboratory within four hours of collection and were processed in one hour of receiving.
• Primary Ct (Cycle threshold) value between 16-34.
• Samples coming in same type of VTM.

Exclusion criteria

• Samples received after four hours of collection.
• Non compliance in maintenance of cold chain.

Sample size calculation: Sample size was calculated at 95% confidence level assuming 50% positivity till nine days while not maintaining cold chain (maximum variance sampling). At the relative allowable error of 25%, minimum 62 specimens were required as sample size. So initially, 62 samples were separated for research work but on continuous daily follow-up 13 samples were discarded due to various reasons like sample volume insufficient, leakage or some inconclusive result, so total 49 samples were analysed and presented in this study.

Study Procedure

All the samples were tested as per the routine laboratory testing protocol and RT-PCR test for SARS-CoV-2 detection was conducted for all the samples. All the samples were opened in a class II biosafety cabinet and followed by initial lysis in the lysis buffer (PerkinElmer, Germany). Then all the samples were further processed for nucleic acid extraction in an automated magnetic bead based nucleic acid extraction system (chemagic 360 by PerkinElmer, Germany). The extracted RNA was tested by RT-PCR with primers-probes directed at SARS Cov-2 I#IEI?I gene and I#INI?I gene. It was a triplex PCR assay with Internal Control (IC) added as the amplification control in the master mix (Quantiplus Multiplex COVID-19 Detection Kit V2.0 from Huwel Lifesciences Pvt., Ltd., India). PCR for all the samples was carried out on Biorad CFX 96 platform. Cycling conditions were starting from 53°C for 10 minutes for cDNA synthesis followed by initial denaturation for 15 minutes at 95°C and then repeated cycling at 95°C and 60°C for 15 seconds and 30 seconds respectively for 40 cycles. The cut-off threshold was recorded for all the samples and Ct value of less than 36 was taken as positive as per the manufacturer’s protocol. Real-time RT-PCR cycle threshold (Ct) values represent the number of amplification cycles required for the target gene to exceed a threshold level. Ct values are therefore inversely related to viral load and can provide an indirect method of quantifying the copy number of viral RNA in the sample (13). Samples were reported as positive when both E and N gene were detected or even N gene was detected alone. The results were considered as inconclusive if only E gene was detected. If both target genes were not detected then the sample was reported as negative. The results were obtained in about five hours.

After analysing the results a set of positive samples with Ct values ranging from 17.5-33.5 were separated for the research work. The results of all samples were positive and the first day initial testing was considered as day zero for the study purpose. A 500 μL of each sample was separately aliquoted and kept in the refrigerator (4-8°C) at the beginning of the study as required for the short-term storage. The remaining samples from the study group were kept at the room temperature (15-25°C) for the assessment of the effect of the temperature on the integrity of the viral RNA. The room temperature and the temperature of the refrigerator were monitored on daily basis. The test samples were subjected to the same RT-PCR testing protocol repeatedly with 300 μL of sample on daily basis and the results compared (positive and negative controls included in each run). This study continued till the volume of the samples kept at room temperature got exhausted and was insufficient for further testing. As there was no initial dilution of the VTM samples so the available volume of most samples supported the study for nine days. On the last day of the study the part of the samples kept under refrigeration at the 4°C was also tested by RT-PCR and the results were compared with their respective counterparts kept at room temperature till nine days. Initially the samples were stored at 4-8°C as this temperature was considered adequate for short-term storage of 5-7 days. But longer duration of storage mandates lower temperature of -20°C or less.

Statistical Analysis

Statistical analysis was done by using python Jupiter notebook SciPy and Anaconda software and Chi-square test was applied for finding the independence between the original Ct value and test results.

Results

In this study, it was observed that of the 49 positive samples (22 females and 27 males with mean age of 43 years), that were left at ambient room temperature for nine days, 42 samples remained positive by RT-PCR till the end of the study period. Of these 49 samples one sample became negative on day 6, two samples turned negative on day 7, three on day 8 and one sample turned negative on day 9 as shown in (Table/Fig 1), (Table/Fig 2). So in total 42 out of 49 samples (85.71%) remained positive for SARS-CoV-2 by RT-PCR after nine days of keeping at room temperature. It was observed that all the samples stored outside refrigerator remained RT-PCR positive till five days. There was one sample which gave negative result on fourth day but again gave positive results from next day onwards, which may be due to a false negative result on day four due to some technical issues discussed further. The seven samples which gave negative result at room temperature by the end of nine days were found to show a strong concordance with the original Ct value of the samples as shown in (Table/Fig 3). It was found that six out of seven samples were of Ct value of more than 30. Of these seven negative samples, only one was found negative when the test was conducted with its corresponding part kept in the refrigerator as summarised in (Table/Fig 4). So, out of 49 only one sample gave negative results on keeping them at 4-8°C as compared to seven samples becoming negative on being stored at room temperature. All the results were statistically analysed by using python Jupiter notebook SciPy and Anaconda software and Chi-square test was applied for finding the independence between the original Ct value and test results. The p-value was found to be much below the significance level of 0.05 (0.00000282) suggesting that the negative results were highly dependent on the original Ct value of the sample and were strongly associated with Ct value of more than 30. While comparing the Ct values of the samples on various days it was observed that in most samples Ct value was increasing but there was no orderly trend and many a times Ct value also decreased. It was also noted that all the samples that turned negative by the end of study period were of Ct value of 28 and above.

Discussion

With the sudden upsurge of the coronavirus pandemic all over the world, healthcare system has come under tremendous pressure for establishing infrastructure and maintaining the continuous supply of all the diagnostic and treatment modalities. In such a scenario adherence to all the guidelines proposed by World Health Organisation (WHO) for sample transportation and storage often becomes difficult. This study has clearly shown that SARS-CoV-2 RNA in the samples kept properly in VTM at ambient temperature remains detectable till five days without any drop in detection rate. In this study, it was observed that the majority of the samples (85.71%) remain RT-PCR positive for detection of coronavirus RNA till the end of the study period i.e., day 9 of samples kept on room temperature. Few previous studies (8),(14) of temperature also support the findings of present study and showed that the virus remains viable at room temperature till seven days. The results of this study was in contradiction of the study done by Kirkland PD and Frost MJ from Australia (15) which reported that PCR positivity was adversely affected at room temperature in commercial VTM and no viral RNA was detected after 48 hours. This was attributed to the presence of RNA in the commercial VTM used for sample collection by them. It suggests that composition of various VTM may affect the outcome of the test as it may vary between different manufacturers and individual studies comparing various commercial VTM would give a better insight. In this study, it was seen that such decay and positivity with RT-PCR was completely maintained till five days and fairly maintained at room temperature even after nine days of sample collection. It is to be noted that in this study none of the samples turned negative till first five days of being kept at room temperature so it gives us a sufficient pocket of time if samples are exposed to ambient temperature during transportation. One sample that turned out to be negative on day four also became positive next day onwards pointing out to be a false negative result due to some technical issue. This false negativity may be due to some technical faults like pipetting errors or improper vertexing leading to uneven composition of sample or some sensitivity issues.

Studies (16),(17) have suggested that corona virus RNA may be degraded if the samples are heated till 56oC, but in this study the temperature was never as high as 56oC so this study recommends that the air conditioning temperature of 15-25oC, temperate climate or the colder months of the tropical countries is good enough to get satisfactory results of RT-PCR for coronavirus testing without any attrition of positivity till five days. When the results were compared with the results of sample part kept in refrigerator the results of most of the samples was very comparable with that of day one with only one sample gave negative result. This also point towards possibility of sample storage at 4-8oC as against recommendation of -70oC for more than five days storage.

This study gives very encouraging results for the testing of COVID-19 samples kept at ambient temperature for long duration especially in pandemic like situation where all resources are on verge of collapsing even in storage and transportation of the samples to the accredited labs for testing. It can serve as a fair guide to analyse the effect of such a breach in compliance with temperature guidelines in COVID-19 testing. It will also help the medical fraternity in preparedness for any future pandemic situations. In few such cases Ct value may increase but detection of positive sample is still possible which makes it highly acceptable with limited resources. As most of the samples which turned out negative in this study were of Ct value 30 and above so this study suggest that there are few chances of missing out such low positive samples on being exposed to higher temperatures but minimal chances of missing out highly infectious samples with low Ct values. In this study, out of 49 samples, 42 were having Ct value less than 30 on day one and seven samples were of Ct value more than 30. Out of those all but one in the Ct value less than 30 turned negative.

Though coronavirus pandemic has astonished the entire world with its rampant course, but the scientific community has come out still stronger against the deadly pathogen making all the developing countries also well equipped with the state of the art advanced molecular diagnostic techniques for its accurate diagnosis. RT-PCR has been taken as the desirable test for diagnosis but the specifications of sample handling required with it points towards the need for the development of some newer diagnostic point of care testing modalities which would obviate the need for transportation of the samples to highly specialised labs under specified conditions and would also decrease the turn around time for earliest diagnosis and isolation of the cases from the suspects. This study also emphasises the fact that for testing of SARS-CoV-2 with RT-PCR, the prerequisite is the intact nucleic acid and not the viable virus so the sample transportation and storage guidelines also need to be revisited after carrying on larger studies.

Limitation(s)

This study has a limitation that the temperature of exposure in this study varied from 15-25oC but more studies need to be done at a much higher temperature ranges as found in many tropical countries. Another limitation of the study was the small sample size of the study so larger studies would be required for better understanding.

Conclusion

In this study, it was observed that the positivity of the RT-PCR results was completely intact till five days of the samples being stored at the ambient room temperature of 15-25°C and 85.71% of the positive samples were also found RT-PCR positive even after nine days of the exposure to ambient temperature of 15-25°C. Most of the samples which gave negative results in study period were of Ct value 30 and above. It was concluded that it is possible to keep samples at this ambient temperature for five days without any loss of positivity in RT-PCR but more rigorous studies with larger sample size at higher temperatures are required to be conducted.

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DOI and Others

10.7860/JCDR/2021/50307.15298

Date of Submission: May 11, 2021
Date of Peer Review: May 28, 2021
Date of Acceptance: Jul 02, 2021
Date of Publishing: Aug 01, 2021

AUTHOR DECLARATION:
• Financial or Other Competing Interests: None
• Was Ethics Committee Approval obtained for this study? No
• Was informed consent obtained from the subjects involved in the study? No
• For any images presented appropriate consent has been obtained from the subjects. No

PLAGIARISM CHECKING METHODS:
• Plagiarism X-checker: May 14, 2021
• Manual Googling: Jul 01, 2021
• iThenticate Software: Jul 30, 2021 (6%)

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