Indian researchers reverse multidrug resistance in E. coli

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(From left) Dr. Saurabh Mishra, Dr. Amit Singh and Prashant Shukla of IISc have been able to make drug-resistant E. coli become sensitive to antibiotics by inhibiting hydrogen sulphide synthesis.

Indian researchers have unravelled the mechanism by which hydrogen sulphide (H2S) gas produced by bacteria protects them from antibiotics and plays a key role in helping bacteria develop drug resistance. And by blocking/disabling the enzyme that triggers the biosynthesis of hydrogen sulphide in bacteria, the researchers from Bengaluru’s Indian Institute of Science (IISc) and Indian Institute of Science Education and Research (IISER) Pune have been able to reverse antibiotic resistance in E. coli bacteria; E. coli bacteria were isolated from patients suffering from urinary tract infection. The results were published in the journal Chemical Science.

Antibiotics kill by increasing the levels of reactive oxygen species (oxidative stress) inside bacterial cells. So any mechanism that detoxifies or counters reactive oxygen species generated by antibiotics will reduce the efficacy of antibiotics. “Hydrogen sulphide does this to nullify the effect of antibiotics,” says Dr. Amit Singh from the Department of Microbiology and Cell Biology at IISc and one of the corresponding authors of the paper. “When bacteria face reactive oxygen species a protective mechanism in the bacteria kicks in and more hydrogen sulphide is produced.” Hydrogen sulphide successfully counters reactive oxygen species and reduces the efficacy of antibiotics.

There was nearly 50% reduction in drug-resistance when hydrogen sulphide production was blocked.The researchers carried out simple experiments to establish this. They first ascertained that regardless of the mode of action of antibiotics, the drugs uniformly induce reactive oxygen species formation inside E. coli bacteria. Then to test if increased levels of hydrogen sulphide gas inside bacteria counter reactive oxygen species produced upon treatment with antibiotics, a small molecule that produces hydrogen sulphide in a controlled manner inside the bacteria was used. “Hydrogen sulphide  released by the molecule was able to counter reactive oxygen species and reduce the ability of antibiotics to kill bacteria,” says Dr. Singh.

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Prof. Harinath Chakrapani’s team at IISER Pune synthesised the small molecule.

The small molecule was synthesised by a team led by Prof. Harinath Chakrapani from the Department of Chemistry, IISER, Pune; he is one of the corresponding authors of the paper. “We designed the small molecule keeping in mind that synthesis should be easy, efficiency in producing hydrogen sulfide should be high and the molecule should release hydrogen sulfide only inside bacteria and not mammalian cells,” says Vinayak S. Khodade from the Department of Chemistry, IISER, Pune and one of the authors of the paper who contributed equally like the first author. The researchers were able to selectively increase hydrogen sulphide levels inside a wide variety of bacteria.

To reconfirm hydrogen sulphide’s role in countering reactive oxygen species, the team took multidrug-resistant, pathogenic strains of E. coli from patients suffering from urinary tract infection and measured the hydrogen sulphide levels in these strains. “We found the drug-resistant strains were naturally producing more hydrogen sulphide compared with drug-sensitive E. coli,” says Prashant Shukla from the Department of Microbiology and Cell Biology at IISc and the first author of the paper. So the team used a chemical compound that inhibits an enzyme responsible for hydrogen sulphide production. “There was nearly 50% reduction in drug-resistance when hydrogen sulphide production was blocked,” Dr. Singh says.

“Bacteria that are genetically resistant to antibiotics actually become sensitive to antibiotics when hydrogen sulphide synthesis is inhibited,” says Prof. Chakrapani. The multidrug-resistant E. coli regained its ability to survive antibiotics when hydrogen sulphide was once again supplied by introducing the small molecule synthesised by Prof. Chakrapani.

“As a result of our study, we have a found new mechanism to develop a new class of drug candidates that specifically target multidrug-resistant bacteria,” says Prof. Chakrapani. The researchers already have a few inhibitors that seem capable of blocking hydrogen sulfide production. But efforts are on to develop a library of inhibitors to increase the chances of success.

How H2S acts

The researchers identified that E. coli has two modes of respiration involving two different enzymes. The hydrogen sulfide gas produced shuts down E. coli’s aerobic respiration by targeting the main enzyme (cytochrome bo oxidase (CyoA)) responsible for it. E. coli then switches over to an alternative mode of respiration by relying on a different enzyme — cytochrome bd oxidase (Cydb). Besides enabling respiration, the Cydb enzyme detoxifies the reactive oxygen species produced by antibiotics and blunts the action of antibiotics.

“So we found that hydrogen sulfide activates the Cydb enzyme, which, in turn, is responsible for increasing resistance towards antibiotics,” says Dr. Singh. “If we have a drug-like molecule(s) that blocks hydrogen sulfide production and inhibits Cydb enzyme activity then the combination will be highly lethal against multidrug-resistant bacteria.” This combination can also be used along with antibiotics to effectively treat difficult-to-cure bacterial infections.

The link between hydrogen sulfide and Cydb enzyme in the emergence of drug resistance is another key finding of the study.

Published in The Hindu on May 6, 2017

IIT Madras team produces white light using pomegranate, turmeric extract

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Dr. Vikram Singh produced white light by irradiating the pomegranate extract and carbon nanoparticles made from pomegranate extract with UV light.

Dr. Vikram Singh, former research scholar in the Department of Chemistry, IIT Madras won the BIRAC Gandhian Young Technological Innovation (GYTI) Award 2017 for his work on producing white light emission using natural extracts.

Dr. Singh and Prof. Ashok Mishra from the Department of Chemistry, IIT Madras used a mixture of two natural extracts — red pomegranate and turmeric — to produce white light emission. The researchers used a simple and environment-friendly procedure to extract dyes from pomegranate and turmeric.

While polyphenols and anthocyanins present in red pomegranate emit at blue and orange-red regions of the wavelength respectively, curcumin from turmeric emits at the green region of the wavelength. White light emission is produced when red, blue and green mix together. This is probably the first time white light emission has been generated using low-cost, edible natural dyes. The results were published in the journal Scientific Reports.

“We had to mix the two extracts in a particular ratio to get white light,” says Dr. Singh, the first author of the paper; he is currently at Lucknow’s CSIR-Central Drug Research Institute (CDRI). By changing the concentration of the two extracts the researchers were able to get different colour temperatures (tunability).

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White light produced by irradiating UV at 380 nm.

“When we mix the two extracts and irradiate it with UV radiation at 380 nm, we observed energy transfer (FRET mechanism) taking place from polyphenols to curcumin to anthocyanins, which helps to get perfect white light emission,” says Dr. Singh. For FRET mechanism to take place there must be spectral overlap between a donor and an acceptor.

In this case, there is a perfect overlap of emission of polyphenols with absorption by curcumin so the energy from polyphenols is transferred to curcumin. Since there is also a perfect overlap of emission of curcumin with absorption by anthocyanin, the energy of curcumin is transferred to anthocyanin.

As a result of this energy transfer from one dye to the other, when the extract is irradiated with UV light at 380 nm (blue region of the wavelength), the polyphenols emit in the blue region of the wavelength and transfers its energy to curcumin. The excited curcumin emits in the green region of the wavelength and transfers its energy to anthocyanin, which emits light in the red region of the wavelength.

“Because of the energy transfer, even if you excite in the blue wavelength we were able to get appropriate intensity distribution across the visual wavelength,” says Prof. Mishra, who is the corresponding author of the paper.

White light sans turmeric  

Taking the work further, the duo produced carbon nanoparticles using pomegranate and to their surprise it was producing fairly green emission. So instead of using turmeric to get green wavelength, the researchers used carbon nanoparticles made from pomegranate extract. “We could get white emission, though it is not as white as when we used turmeric. It’s slightly bluish but well within the white zone,” says Prof. Mishra. “It is attractive to use a single source to create white light emission.”

The principle by which the pomegranate extract and carbon nanoparticles made from the extract is the same as in the case when pomegranate and turmeric extracts were used. The results were published in the Journal of Materials Chemistry C.

Though this natural mixture of dye can be used in a wide variety of applications such as tunable laser, LEDs, white light display, much work needs to be done in terms of photostability and chemical stability before it becomes ready for translation. Biosystems have an inherent tendency to breakdown and so this has to be addressed.

Published in The Hindu on May 6, 2017

Novel molecule synthesised by Indian researchers shows promise in treating cancer

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The efficiency of Disarib in selectively killing cancer cells was found to be high in all possible systems that Supriya Vartak (left) and Prof. Sathees Raghavan experimentally tested.

A novel small molecule designed and synthesised by Indian researchers has shown promise in targeted killing of cancer cells. The molecule (Disarib) binds to a protein BCL2 and inhibits the protein from suppressing cell death in cancer cells. While BCL2 protein is produced in excess in cancer cells, its expression is almost undetectable in normal cells. Hence, Disarib targets and kills only cancer cells while sparing normal cells.

Inside a cell there is always a balance between proteins that promote cell death (apoptosis) and those that suppress cell death. Since the proteins (BAX and BAK) that promote cell death get bound to BCL2, normal cell death is suppressed and cancer cells are able to live longer.

A team led by Prof. Sathees C. Raghavan at the Department of Biochemistry, Indian Institute of Science (IISc), Bengaluru demonstrated that Disarib was able to disrupt the interaction of BCL2 and apoptosis-causing BAK protein and cause the death of cancer cells.

However, expression of BCL2 is low in certain cancer cell lines such as breast cancer, chronic myelogenous leukemia and cervical cancer. So the Disarib molecule will be ineffective in these cancers.

The efficiency of Disarib to cause cell death and tumour regression was far superior compared with the FDA-approved ABT199.Disarib is the culmination of eight years of collaborative research involving 24 researchers from eight different research groups across various labs.

Superior than FDA-approved molecule

Unlike the FDA-approved BCL2 inhibitor ABT199, the small molecule binds predominantly to a different domain (BH1) of BCL2 and showed better efficiency in killing cancer cells. Also, compared with ABT199 inhibitor, the small molecule synthesised by Prof. Raghavan’s team did not cause any side effects. The results were published in the journal Biochemical Pharmacology.

Earlier studies had shown that once Disarib binds to BCL2, the proteins that promote cell death were able to create holes in the mitochondria leading to death of cancer cells.

“We have experimentally tested Disarib in all possible systems and the efficiency of Disarib in selectively killing cancer cells was high,” says Supriya V. Vartak from the Department of Biochemistry, Indian Institute of Science (IISc) and one of the first authors of the study. Studies were carried out on three animal models for three different cancers — lymphoma, breast adenocarcinoma and ovarian cancer. Similarly, studies were carried out using cancer cells lines.

“In every case, both in animal studies and cancer cell lines, the efficiency of Disarib to cause cell death and tumour regression was far superior compared with ABT199 when same dosage of Disarib and ABT199 was used,” says Prof. Raghavan. “This is why the molecule has to be taken up for further investigation.”

The team has carried out quite a lot of toxicity studies already. Next step will be to test the toxicity and efficacy of the molecule in cancer cells taken from patients, and also test it in combination with known cancer drugs. If results from humanised mouse models are also encouraging then the molecule can be taken up for clinical trials in humans.

Published in The Hindu on May 5, 2017

What elephants teach us about cancer prevention

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Elephants express many extra genes derived from the critical tumour suppressor gene TP53. – Photo: Stephen Tan/Flickr

Joshua Schiffman, University of Utah and Lisa Abegglen, University of Utah

Every time a cell divides, there is a chance for a mutation (mistake) to occur in the DNA – the substance that carries genetic information in all living organisms. These mutations can lead to cancer. The Conversation

If all cells have a similar chance of developing cancer-causing mutations, then very large and long-lived animals with more cells undergoing more cell divisions should develop cancer at a higher rate than smaller, short-lived animals with fewer cells dividing over less time.

But in 1977, Sir Richard Peto noted that humans develop cancer at a rate similar to mice. This is despite having 1,000 times as many cells and living 30 times as long. Another example of this phenomenon can be found in elephants. They are 100 times larger than people and can live 60 to 70 years, and yet, their cancer rates are exceedingly low.

Peto proposed that evolutionary considerations might explain the differences in per-cell cancer incidence across species. When comparing cancer rates in mice and men, he proposed that as humans evolved to grow larger and live longer throughout evolutionary history – with more human cells dividing over a longer period of time – that they also evolved to resist cancer. This surprising cancer resistance found in larger, long-lived animals, like elephants, has become known as Peto’s Paradox.

Our research team provided the first empirical data documenting cancer across species in support of Peto’s Paradox.

We showed that cancer mortality does not increase with body size or life span. Actually, we observed that some larger, longer living animals may develop less cancer. We calculated elephant cancer mortality rates at less than 5%, compared to human cancer mortality rates of 11% to 25%.

Elephants have had 55 million years of development to figure out how to resist cancer, and we hope that we can one day apply these lessons to develop effective treatments for cancer.

Cancer resistance

Our team looked at the genome of the African elephant for changes in oncogenes and tumour suppressor genes. Oncogenes can cause cells to grow out of control while tumour suppressor genes slow down cell division. These are the two main types of genes that play a role in cancer and could help explain potential mechanisms of cancer resistance in elephants.

Our analysis revealed the shocking discovery that elephants express many extra genes derived from the critical tumour suppressor gene TP53.

TP53 is called the “Guardian of the Genome” due to its ability to protect cells from accumulating cancer causing mutations. The TP53 gene responds to DNA damage, or pre-cancer, by stopping the cell from dividing until the DNA can be repaired. If the cell cannot fix the DNA, then TP53 causes the cell to die through a process called apoptosis. Sacrificing damaged cells prevents the propagation of cells with mutations that could lead to cancer.

People with Li-Fraumeni Syndrome have a mutation in one copy of their TP53 genes, with more than 90% lifetime risk to develop cancer. This high rate of cancer associated with TP53 dysfunction illustrates the critical role that TP53 plays in protecting us from cancer.

Naturally cancer resistant

Our lab at the University of Utah studies the broken DNA damage response in people with Li-Fraumeni Syndrome who are missing their TP53 genes and have a very high rate of cancer.

When we learned that elephants were naturally cancer resistant and also had 20 times as much TP53 as humans (40 gene copies total in elephants vs. 2 gene copies in healthy humans), we teamed up with Dr. Carlo Maley, an evolutionary and cancer biologist who helped to make the initial discovery about extra elephant TP53.

We used our clinical and research experience from studying patients with Li-Fraumeni Syndrome to try to understand if elephant TP53 could be playing a role in protecting elephants from cancer. Because we already were measuring TP53 function in people with and without Li-Fraumeni Syndrome, we could use the same laboratory tests to measure how elephant cells responded to DNA damage.

To perform these experiments, we collaborated closely with Utah’s Hogle Zoo (who have African elephants) as well as Ringling Bros. and Barnum Bailey Circus (who have Asian elephants). Both groups routinely draw blood from their elephants to monitor their health, and we received approval to study the blood when it was drawn for these routine elephant health screening procedures.

The blood was sent to our lab where the white blood cells, called lymphocytes, were exposed to ionising radiation to induce DNA breakage. We monitored how quickly broken DNA was repaired in the African and Asian elephant lymphocytes compared to human lymphocytes.

We predicted that elephant cells would repair their DNA faster than human cells, but discovered that the rate of DNA repair was similar between elephant and human cells. But we noticed something interesting about the elephant cells after it was exposed to radiation: more elephant cells than human cells underwent programmed cell death or apoptosis.

We next undertook rigorous experiments to compare the percent of elephant cells vs. human cells vs. Li-Fraumeni Syndrome cells that died from DNA damage, or pre-cancer.

We discovered that the amount of apoptosis correlated with the number of TP53 genes and that this followed the same pattern of lifetime cancer risk – elephants (~5%), humans (~50%), patients with LFS (~90%). This makes sense because more TP53 makes the cell more effective at removing pre-cancer cells that could go on to form cancer.

Learning from elephants to help people

We showed that elephant TP53 helps elephants to more quickly remove pre-cancerous cells with DNA damage and that this possibly contributes to elephant cancer resistance.

Now, we are focusing our research efforts to better understand the specific mechanism of how elephant TP53 works. The ultimate goal of our laboratory work is to help patients who already have cancer, and maybe even those people who could be at risk for cancer in the future.

We want to see if we can translate this fascinating discovery into an effective treatment for cancer, or maybe even potentially as a cancer prevention strategy. In the end, we are working to create a world with more elephants and less cancer.

(Joshua Schiffman, Professor of Pediatrics, University of Utah and Lisa Abegglen, Pediatrics – Visiting Instructor, University of Utah)

This article was originally published on The Conversation. Read the original article.

ISRO asked to remain silent on South Asia Satellite launch

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A terse message about the launch on ISRO’s website.

“We have been asked to [remain] silent on the launch [of the South Asia Satellite from Sriharikota off Andhra Pradesh on May 5 evening],” reads a message sent to me by a senior scientist at the Indian Space Research Organisation (ISRO) when I tried to get details about the satellite launch. Many more attempts to get details about the launch from other scientists also proved futile. Scientists never attended my calls, which is extremely unusual. ISRO scientists have always been very cordial and forthcoming with details whenever I or my colleagues at The Hindu, or for that matter anyone from the media had got in touch with them on other occasions.

Apparently, the scientists have been told not to divulge any details till the satellite is launched. And I wonder what this secrecy is all about. The South Asia Satellite is just a communication satellite and nothing more.

The ISRO website has a terse message about the launch. It says: “GSAT-9 is scheduled to be launched on Friday May 05, 2017 from SDSC SHAR, Sriharikota.” The GSLV-F09 will be launched with a 2,230-kg satellite at around 5.00 pm on May 5.

This is the first time in so many years that I can recall is a launch going to happen without the media being taken to Sriharikota to witness it. According to a report in The Hindu, the last time the media was not taken to Sriharikota for a launch was when ISRO launched TecSAR, Israel’s spy satellite, in January 2008.

But the May 5 launch has nothing to do with any spy satellite. According to an April 30 PTI report, it is just a communication satellite with 12 Ku band transponders that Nepal, Bhutan, Maldives, Afghanistan, Bangladesh and Sri Lanka can use for “telecommunication and broadcasting applications such as television, direct-to-home (DTH), very small aperture terminals (VSATs), tele-education, telemedicine and disaster management support”. Pakistan has stayed away.

“The satellite also has the capability to provide secure hot lines among the participating nations in addition since the region is highly prone to earthquakes, cyclones, floods, tsunamis, it may help in providing critical communication links in times of disasters,” the PTI report says.

India to treat all HIV positive people irrespective of CD4 count

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Lakhs of HIV deaths can now be averted as India follows the WHO’s recommendation.

Two years after the World Health Organisation recommended that antiretroviral therapy (ART) be initiated in people living with HIV irrespective of the CD4 cell (a type of white blood cell) count, India has aligned its policy with the guideline. In a major shift in the HIV treatment guidelines, Union Health Minister J.P. Nadda had recently said that any person who tests positive for HIV will be provided ART “as soon as possible and irrespective of the CD4 count or clinical stage”. By expanding the provision of ART, about 0.45 million deaths can be averted.

It was in 2002 that the WHO first issued its ART guidelines. In the absence of AIDS-defining illnesses, the WHO set CD4 count less than 200 cells per cubic millimetre as the threshold to begin ART treatment. Over time, the WHO changed its guidelines and, in 2013, increased the threshold to CD4 count less than 500 cells per cubic millimetre. But for certain populations — HIV positive people who also have TB disease, pregnant and breastfeeding women, children below five years — ART was to be initiated regardless of the CD4 count.

The recommendation was based on the evidence that earlier initiation of ART will help people with HIV live longer, remain healthier and “substantially reduce” the risk of transmitting the virus to others. The availability of safer, affordable and easy-to-manage medicines that could help to lower the amount of virus in the blood played a key role in WHO’s decision to increase the threshold. Earlier initiation could avert an “additional three million deaths and prevent 3.5 million more new HIV infections between 2013 and 2025,” the WHO noted in 2013.

The biggest challenge will be to identify half a million who have been infected but have not been diagnosed.In 2015, the WHO once again changed its guidelines. Based on evidence from clinical trials and observational studies since 2013, it became clear that earlier use of ART, irrespective of the CD4 count, results in better clinical outcomes compared with delayed treatment. Accordingly, it recommended that ART be initiated in HIV positive people at any CD4 cell count. Early start of treatment has the potential to “significantly reduce the number of people acquiring HIV infection and dying from HIV-related causes and significantly impact global public health” it said.

As per 2015 estimates, India has 2.1 million HIV positive people, of which only 1.6 million have been diagnosed and about a million are on treatment. But half a million people are not even aware of their HIV status. With the government changing its treatment guidelines, 0.6 million who have been diagnosed but not been on treatment are now eligible for treatment. Of the 0.6 million, about 0.25 million have been enrolled for pre-ART care and can be started on treatment almost immediately. But the biggest challenge will be to identify the 0.5 million who have been infected but have not been diagnosed and about 80,000 people who become infected each year.

Even as efforts are on to expand the 1,600 treatment delivery sites that are currently operational, there should be greater focus now on identifying people with HIV. The government has plans to start community-based testing to bring HIV testing closer to those in need, and target special groups that are more vulnerable to infection such as partners of people who are HIV positive.

Despite the WHO releasing the guidelines for self-testing in December last year to improve access to testing, India has refused to approve it on the grounds that pre- and post-testing counselling will not be possible. Self-testing could have increased the number of people who would have got themselves tested. However, the OraQuick self-test is not highly sensitive and any positive test should be reconfirmed with conventional testing.

Published in The Hindu on May 3, 2017

Number of wild poliovirus cases drop in Pakistan, Afghanistan

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Several vaccine-derived poliovirus cases have been detected in the environment in Nigeria in 2017.

In the last six months, there have been only eight wild-type poliovirus (type 1 and type 3) cases reported in Afghanistan and Pakistan, down from 32 cases during the same period last year. “The overall situation in Afghanistan and Pakistan has significantly improved in common corridors of transmission,” says a summary report of a April meeting of the Strategic Advisory Group of Experts (SAGE) on immunisation. Except for Pishin and Quetta, children 6-11 months old showed 95% ‘seroprotection’ in all districts during a recent ‘serosurvey’ carried out in Pakistan.

Type 1 cases in Nigeria

Between July and August 2016, three cases of wild poliovirus (type 1) were reported from Borno State, Nigeria in children between 2 and 5 years of age; two of them developed acute flaccid paralysis. This is the first time a wild poliovirus was detected in the country since 2014. However, no more wild poliovirus has been reported in Nigeria since then. But with most areas in Borno remaining inaccessible, a huge population of children aged less than five have not been immunised.

A year after 156 countries, including India, made a synchronised global switch from trivalent (containing all three strains of the polovirus — type 1, type 2 and type 3) to bivalent (only type 1 and type 3 strains) oral polio vaccine, several vaccine-derived poliovirus (VDPV) type 2 have been detected from the environment in Bauchi, Gombe and Sokoto in Nigeria in 2017.

SAGE has expressed “concern over the ongoing circulation of VDPV2 in Nigeria”. Since there is increased risk of type 2 outbreak as the immunity levels wane, SAGE had recommended that countries that have circulation of both wild poliovirus (type 1 and/or type 3) and vaccine-derived poliovirus type 2 should give “priority to stopping vaccine-derived polovirus type 2 over wild polio virus elimination”. At least two doses of oral polio vaccine containing only type 2 should be given before the next round of immunisation using bivalent oral polio vaccine, SAGE has recommended.

All the 156 countries that switched to bivalent oral polio vaccine have introduced at least one dose of inactivated polio vaccine (IPV) containing all the three strains in a killed form into their routine immunization programmes.

The last time wild poliovirus type 2 was detected anywhere in the world was in 1999. On 20 September 2015, wild poliovirus type 2 was formally declared as eradicated. But with the continued use of OPV, the live, weakened type 2 strain excreted by an immunised child can, under rare instances, turn virulent and cause vaccine-associated paralytic poliomyelitis in unprotected children. Since its eradication in the wild in 1999, all type 2 cases have been caused only by vaccine-derived polioviruses.

Polio cases, whether caused by wild type or vaccine-derived, can be eradicated only when oral polio vaccine is eventually withdrawn once the transmission of wild polio type 1 and type 3 have been eradicated.

Published in The Hindu on May 1, 2017