CCMB finds cheaper more effective RT-PCR method to test COVID-19 Samples
The procedure would be useful in the face of increasing need for testing across the country
Increased tracing, testing and isolation of people with novel coronavirus infection is an effective way to contain the virus spread. Currently, the preferred protocol for testing uses the RT-PCR (Reverse Transcription-Polymerase Chain Reaction) test. This protocol does, however, take time and is expensive.
With the steeply rising number of infected persons, there is a need for a reliable test that would give results quickly and also be less expensive. Researchers from the Centre for Cellular and Molecular Biology (CCMB), Hyderabad, have studied such a method, also using RT-PCR but with dry swabs, bypassing the RNA isolation stage, which they find consumes less time and is less expensive.
They also suggest a variant method which apparently shows a higher efficiency that the conventional one. The results have been posted in bioRxiv and medRxiv preprint servers. Preprints are yet to be peer-reviewed and published in scientific journals.
In the usual method of testing, nasal swabs collected from a person are placed in a viral transport medium (VTM). From this, a part of the liquid is taken, the viral RNA is extracted and RT-PCR test is carried out. The remainder is stored. It is the step of isolating the RNA that takes time and is expensive. So, the authors have proposed an alternative method.
Dry swabs
Instead of placing the nasal swabs in the VTM, they are put in a Tris-EDTA (TE) buffer solution, protected by ice. “Virus in dry swabs can stay for several days at 4 degree [ice temperature]. For longer storage, it can be kept in minus 80 degree,... it [dry swab] is much more suitable than VTM, and testing can be delayed, if necessary,” says Rakesh Mishra from CCMB, and one of the authors of the preprint. He adds that handling and transporting dry swabs is safer and more convenient.
A small part of the dry swab-TE extract was taken in a new vial and heated to 98 degree C. This destroyed the protective wall of the virus particles, releasing its RNA and this was sent for the RT-PCR test. In all, 40 patients were put through both testing protocols (heated TE without RNA extraction and current standard method). While 22 tested positive and 18 negative in the new method, the standard method yielded 23 positives and 17 negatives. The researchers found that the new protocol of using dry swab-TE extract for RT-PCR was at par with the standard method.
However, the standard method is known to have a problem of false negatives. To address this, the researchers took the dry swabs-TE extract, extracted RNA from it and studied the samples. In this variant method, they found that a few samples that were consistently negative in both methods now showed a positive result. So out of 40 samples they now had 28 positives and 12 negatives. This result was reproduced on testing multiple times. They surmise that this is because of low viral load, which was picked up in the new variant method.
Handy improvisation
“This is an improvised method, makes the COVID-19 testing rapid and less expensive. Both are welcome considering limited availability of reagents for VTM and RNA isolation,” says L.S. Shashidhara, from Ashoka University, Delhi, who was not involved in the research. “This is a good development as we need to increase the number of tests considerably across India. More and more new labs are being enrolled as testing centres and large numbers of people are being trained in RT-PCR-based testing. Fewer the number of steps, fewer would be the errors,” he adds.
Further study is needed to see if the samples collected and stored in TE can be used for culturing of the virus for any future studies and full sequencing of the viral genomes.
- In the usual method of testing, nasal swabs collected from a person are placed in a viral transport medium (VTM).
- From this, a part of the liquid is taken, the viral RNA is extracted and RT-PCR test is carried out. The remainder is stored. It is the step of isolating the RNA that takes time and is expensive. So, the authors have proposed an alternative method.
- Instead of placing the nasal swabs in the VTM, they are put in a TrisEDTA (TE) buffer solution, protected by ice
- "Virus in dry swabs can stay for several days at 4 degree (ice temperature)
- For longer storage, it can be kept in minus 80 degree.....it (dry swabs)
- is much more sutaible than VTM and testing can be delayed , if necessary."
Dry swabs
- The researchers from the Center for Cellular and Molecular Biology (CCMB), Hydrabad have studied such a method,also using RTPCR but with dry swabs bypassing thr RNA isolation stage, which they find consumes less time and is less expensive.
- Instead of placing the nasal swabs in the VTM, they are put in a Tris-EDTA (TE) buffer solution, protected by ice. “Virus in dry swabs can stay for several days at 4 degree [ice temperature]. For longer storage, it can be kept in minus 80 degree,... it [dry swab] is much more suitable than VTM, and testing can be delayed, if necessary,” says Rakesh Mishra from CCMB, and one of the authors of the preprint. He adds that handling and transporting dry swabs is safer and more convenient.
- A small part of the dry swab-TE extract was taken in a new vial and heated to 98 degree C. This destroyed the protective wall of the virus particles, releasing its RNA and this was sent for the RT-PCR test. In all, 40 patients were put through both testing protocols (heated TE without RNA extraction and current standard method). While 22 tested positive and 18 negative in the new method, the standard method yielded 23 positives and 17 negatives. The researchers found that the new protocol of using dry swab-TE extract for RT-PCR was at par with the standard method.
However, the standard method is known to have a problem of false negatives. To address this, the researchers took the dry swabs-TE extract, extracted RNA from it and studied the samples. In this variant method, they found that a few samples that were consistently negative in both methods now showed a positive result. So out of 40 samples they now had 28 positives and 12 negatives. This result was reproduced on testing multiple times. They surmise that this is because of low viral load, which was picked up in the new variant method.
Handy improvisation
“This is an improvised method, makes the COVID-19 testing rapid and less expensive. Both are welcome considering limited availability of reagents for VTM and RNA isolation,” says L.S. Shashidhara, from Ashoka University, Delhi, who was not involved in the research. “This is a good development as we need to increase the number of tests considerably across India. More and more new labs are being enrolled as testing centres and large numbers of people are being trained in RT-PCR-based testing. Fewer the number of steps, fewer would be the errors,” he adds.
Further study is needed to see if the samples collected and stored in TE can be used for culturing of the virus for any future studies and full sequencing of the viral genomes.
Researchers from the center for Cellular or Molecular Biology (CCMB) Hyderabad have studied such as a method also using RTPCR but with dry swabs bypassing the RNA isolation stage which they find consumes less time and is less expensive. Usual method of testing nasal swab collected from a person in a Viral Transport Medium (VTM).
From this, a part of liquid is taken the viral RNA is extracted from and RTPCR test is carried out.
Instead of placing the nasal swabs in the VTM they are put TrisEDTA (TE) buffer solutions protected by ice
What is trisEDTA ?
- Tris and EDTA are two major components which are used throughout the DNA extraction protocol.
- The major role of TE buffer in DNA extraction is to dissolved in liquid form.
- This is hydroxymethyl aminomethane with the molecules of (HOCH2)3CNH2.
- Theajor role of trust in is to maintain the pH of the solution.
- what is EDTA ?
- EDTA, ethylenediamine tetra acetic acid (EDTA) has wide application in medicine industries and laboratories and in cosmetics
- It is colourless (white as in powder form) water soluble and organic molecules however, it can be dissolved in water at high pH (pH near by 8.0)
- The chemical formula of EDTA is a C10H16N2O8.
- This is an improvised method makes the COVID-19 testing rapid and less expensive.
Serotonin triggers desert locust swarm
Recluses though they are, locusts team up during harvest
During the last 10 days, there has been a host of analytical articles in the press about the latest locust swarming from the Rajasthan/Gujarat desert region, all the way into Madhya Pradesh and Chhattisgarh, causing extensive damage to the crops. These articles have also pointed out how India (and indeed Pakistan as well) has been handling this plague since centuries, indeed even since the Mahabharata times (recall how Karna challenged the Pandava’s army: “we will pounce on you, as — shalabasana — a swarm of locusts). The British colonial government had set up Locust Warning Organizations (LWOs) since the early 1900s at Jodhpur and Karachi in the Indian subcontinent. After Independence, the Union Ministry of Agriculture has continued and improved upon the LWOs, one with administrative affairs at Faridabad, near New Delhi, and another LWO at Jodhpur, Rajasthan, where the technical aspects are handled along with local branches in the region. They use the technique of aerial spray of insecticides (using drones these days), as well as spraying by land-based workers in the field. And they are doing a good job of it.
Locust control
The Agriculture Ministry uses a site called <vikaspedia.in>, which gives considerable details of the problem of locust control and plant protection, and the current methods of handling them. The Directorate of Plant Protection, Quarantine and Storage, at the Ministry has a site, <ppqs.gov.in>, which details the contingency plan for desert locust invasions, outbreaks and upsurges.
The locust problem is not confined to India alone, but most of Africa, West Asia, Iran and even parts of Australia. The Food and Agricultural Organization (FAO, this is a part of the United Nations, and based in Rome, Italy) co-ordinates and helps these nations with advice and funds in combating this plague. The informative document from FAO, called the Locust Environmental Booklet, gives an update on the situation and methods of handling locust swarms. And an excellent update (available online) on ‘locust swarm and its management’ has been published on May 29 by the ICRISAT Development Centre (IDC) of the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), based in Hyderabad.
By and large, “detect the swarm and kill it as it moves” has been the method, and countries across the world are using it. We certainly need better and more innovative methods to fight this plaque and win over it.
How locusts form swarms
This however raises the important scientific question of how and why locusts collect together by the thousands in order to make a swarm. Insect biologists have long since known that the locust is by nature a recluse and a singleton, not mixing with others in the same group, Yet, when the harvest season arrives, these singletons team up with others as an army of swarms to attack plants for food. What is the mystery? What is the biological mechanism by which this sociological transformation comes about? If we know this mechanism, there can be novel ways of stopping this group rampage.
Stephen Rogers of Cambridge University, U.K. (and University of Sydney, Australia) is an acknowledged world expert in the study of how and why such swarms come about. In one of his papers, way back in 2003, he showed that when solitary locusts happen to come near each other (looking for food) and happen to touch each other, this tactile stimulation, even just in a little area of the back limbs, causes their behaviour to change. This mechanical stimulation affects a couple of nerves in the animal’s body, their behaviour changes, leading to their coming together. And if more locusts come nearby, the crowding starts, and what was once a simple looking insect becomes larger in size and shape, and its colour and morphology changes. In the next paper, his group showed substantial changes in some molecules that modulate the central nervous system of the locust, the most important among them being serotonin, which regulates mood and social behaviour. And putting all these together, they came out with a publication in Science in 2009 <https://science.sciencemag.org/content/323/5914/627>, that serotonin is indeed responsible for swarm formation. In this paper, they did a lab experiment wherein they placed locusts in a container one by one, and as the numbers increased, the coming together triggered mechanical (touch) and neurochemical (serotonin) stimulations to make crowding (‘gregarisation’) occur within a few hours! Interestingly, when they started adding substances that inhibit the production of serotonin (for instance, molecules such as 5HT or AMTP), the crowding was significantly less.
Stopping swarms
Now, here is a potential way of stopping swarms from forming! Can we work with the LWOs in Jodhpur and other places, spray serotonin inhibitor molecules as the swarm begins to form? Rogers had indeed hinted this in his Science paper. Is this possible or a quixotic idea? Let the experts tell us. It is well worth a try.
Finally, the insecticides (mainly malathion) sprayed on the swarms need to be looked at for side-effects. Though many studies have cleared it as not very harmful, we need to work on biopesticides which would be environmentally and animal/human health-friendly, using natural and animal products of India.
LOCUST PROBLEM
- The locust problems is not alone in India, but also in Africa, West Asia Iran, Iran and even Parts of Australia.
- The Food and Agriculture Organization (FAO) this is a part of the the United Nations and based in Rome, Italy.) coordinate and help these nation with advice and funds in combating this plague.
- The Informative document from (FAQ) called the locust Environmental Booklet, gives an update in the the situation and method of handling locust swarm.
- This however raises the important scientific of how and why locust collect together by the thousand in order to make swarm.
- Insects biologist have long since know that the locust is by nature a recluse and singleton not mixing with other in the same group.
- Yet, when harvest session arrives the S3 singleton team with others as an army of swarms to attack plant for food.
- Stephen Rogera of Cambridge University U. K. And (University of Sydney, Australia) is an acknowledge world expert in the study of how and why such swarms comes about.
- In one of this paper way back in 2003 he shows that when solitary locusts each other this textile stimulation even just in little area of back limbs cause their behaviour to change.
- This mechanical stimulation affect a couple of nerves in animals body there behaviour changes leading to there coming together.
- And if more locust come near by the crowding starts and that was once a simple looking insects becomes larger in size and shape and it's colour and morphology changes in the next paper, his group substancial changes in some molecules that modulate the central nervous system of the locust the most important among them being serotonin which regulate mood and social behaviour
- Serotonin which regulates moods and social behaviour
- Serotonin is indeed responsible for swarm formation.
Ventilators can heal but also harm
An evaluation is needed to assess what the patient needs
Ventilators are not easy to understand, this is true even for most doctors. Only a relatively small percentage of people who work in ICUs are able to disentangle, de-mystify and decipher these complex machines.
Now, we find ourselves on our knees before a disease that takes away our fundamental ability to draw breath. The seemingly logical solution appears to be the creation and use of more ventilators. Unfortunately, it is not that simple.
If the breathing process is compromised, it most often leads to the failure of every other organ system, making the ventilator the undisputed hero of the piece. A ventilator is able to force air into the lungs so that breathing in is no longer the person’s responsibility. This sounds deceptively simple.
The nose and the mouth draw in air. The windpipe or the trachea directs the air into passages called the bronchi. These tubes pass through our lungs and divide further into tiny bronchioles that end in balloon-shaped pockets called alveoli, of which the average human has about three million. Blood vessels called capillaries surround the alveoli. It is here that oxygen enters the bloodstream.
By simply drawing in air, we call into being a silent orchestral dance that can be felt if we place our palms upon our heaving chests. Even the best artificial ventilators fall far short of duplicating this rhythm and flow.
Safety factors
But even if the ventilator is not a completely refined one, isn’t it better than nothing? An artificial ventilator that is not exactly graded or calibrated can become a killing machine. The altered pressure changes can damage the lungs. Ventilators can only drive air into the lungs. They cannot fulfil the equally vital task of transferring oxygen into the blood, should the lungs be too damaged to do so.
In the present situation, first, everyone with a breathing difficulty from COVID-19 does not automatically require a ventilator. Second, they may not even be good candidates for ventilation because, in a large number of people with serious COVID-19, the lungs seem to be too badly damaged to recover. . Third, the general condition of the person determines whether there is a reasonable chance for him or her to come out of it alive and well. This is not to say that ventilation is not a viable option in some cases.
Chances of survival
Unfortunately, however, evidence thus far indicates that in general, the chances of survival after ventilation are not good. A published study based on the experience in 12 New York hospitals showed that only 11.9% of 320 patients survived, in whom ventilators were used and the treatment had come to a logical conclusion (discharge or death of the patient). That result spanned all age groups. In the elderly, particularly among those with specific associated diseases , the chances of survival are even poorer.
Symptoms and their experience vary widely across populations, and all this talk of ventilation is applicable only to the minority – those with severe infection, particularly, the elderly and those with other serious concurrent illnesses.
To understand what is best for a patient an evaluation is necessary. The process is called triage. It is recommended that the attending team and the triage team are separate from one another.
On May 19, the World Health Assembly passed a resolution that palliative care should be made available to all people with COVID19. This would mean being mindful of suffering irrespective of disease status and making a concentrated effort to alleviate it as far as possible. It would involve adequate treatment of pain as well as other distressing symptoms. This would be bolstered by appropriate emotional support. Honest information compassionately given forms the cornerstone of such practice.
Those with reasonable chances of survival are ear-marked to receive care in intensive care units with ventilators when necessary. Such ventilators should not be hastily put-together contraptions, but those that are able to mimic air flow in the lungs as near normal as possible. Enough sedation is given to them so that the ventilator is tolerated and stress minimised.
Those with next-to-no chances of survival and their families would be counselled about the gravity of the situation and assisted in making an informed choice whether to go for aggressive intensive care with a ventilator or to forgo it while continuing to receive comfort care with compassion.
- Triage is a process of determining the priority of patients. Treatment by severityof there condition like hood recovery with and without treatment.
- This ration patient treatment efficiently when resources are insufficient for all to be treated imidiately, influshing the order and priority of emergency treatment emergency transport or transport destination for the patient.
What is Palliative care ?
Palliative care is Ann approach that improves the quality of life that patient and their families facing the problem associate with life threatening illness, through the prevention and the relief of suffering by means of early identification and impeccable assessment and treatment of pain and other problems physical, physiological and spiritual illness and their own bereavement.
- Provide relief from paying and other distressing systomps
- Affirms life and regards and dying as a normal process
- Intenods neither to hasten or postpone death
- Integrates the physiological and spiritual aspects of patients.
- Offers a support a support system to help patient live as actively as possible until death.
Prevalence of diabetes among women high in southern India.
More than one in 10 women aged 35-49 suffer the disease
Researchers have identified a bunch of districts in India that have the maximum prevalence for diabetes among women. At least 50 of the 640 districts studied have high prevalence of diabetes — greater than one in 10 — among women aged 35-49 years. Tamil Nadu, Kerala, Andhra Pradesh and Odisha have districts with the highest prevalence. The results were published in the Journal of Diabetes & Metabolic Disorders.
While Cuttack in Odisha has the highest prevalence of 20%, 14 districts in Tamil Nadu — the maximum among all States — have high prevalence, prompting the researchers to classify them as ‘hotspots’.
- The number of people with diabetes in India increased from 26-0 million 1990 to 65 million in 2016.
- The prevalence of diabetes in adults aged 20 year or older in India increased from 5.5% in 1990 to 7.7% in 2016.
- The prevalence in 2016 was highest in Tamil Nadu and Kerala, according to the Global burden Diseases study 2018.
- The Researchers have identified a bunch of district in India that have the maximum prevalence for diabetes among women at least 50 of the 640 districts studied have high prevalence of the diabetes greater then 1 in 10 among women aged 3549 years.
- Tamil Badu, Kerela, Andhra Pradesh and Pagoda have districts with the prevalence of The results were published in the Journal of Diabetes and metabolic Disordered.
- " Results portray that prevalence of diabetes among women in their late reproductive ages is highest among those with two fewer children ever borne, who are educated belong to economically prosperous household living in urban areas enjoying changing lifestyle increased excess to the energy (refined and processed) food and development.
No comments:
Post a Comment