Dr G Arunkumar, Director of Manipal Institute of Virology, was the scientist at the forefront of the identification and diagnosis of NiV in the 2018 outbreak in Kerala, India. For the first time in India, Arunkumar and his team at Manipal Institute of Virology detected the virus using Real-time PCR. The eminent virologist, who has been part of the myriad of encephalitis outbreak investigations in India talks to Aster Medical Journal.
AMJ: Rapid Detection of Nipah Virus helped to contain the out- break in Kerala. As the first person to detect the Nipah virus in India, could you please tell us what made you test for Nipah?
Dr Arunkumar: For the past few years, we have been conducting a study to understand the etiology of hospitalised febrile illness cases in rural India. So as part of that, we have sentinel sites in 10 states. We had sentinel sites near to Bangladesh, i.e. in Tripura and Assam. In these two places, we were actively looking for Nipah among the febrile illness causes, but none were previously found. Because of that, we had the test ready, and our team was prepared. When the Kerala case came what led us to check for Nipah was a cluster of Encephalitis.
An adult cluster of Encephalitis is the classical epidemiological presentation of Nipah in south-east Asia. The only virus that can cause Encephalitis in a family cluster is Nipah. With other Encephalitis viruses like herpes or Japanese Encephalitis virus, you don’t see family clusters. In Japanese Encephalitis, a mosquito borne disease, typically for every 300 people infected, only one person develops Encephalitis. You may see one case per village. Instead, in the 2018 outbreak of Nipah in Kerala, we had a family cluster.
Moreover, the epidemiological report from Bangladesh clearly showed the family pattern within the adult cluster. This was the reason for the Nipah testing. We used a Real-Time PCR, wherein a sample is inactivated to reduce the risk, to screen the RNA.
AMJ: In most of the previous Nipah outbreaks, detection and diagnosis happened retrospectively. It took months or years to detect the virus. For instance, in Siliguri, it took five years to identify the infectious virus as Nipah, whereas in Kerala the virus was detected and the diagnosis was confirmed while the outbreak was still active.
Dr Arunkumar: Yes. That’s true. Unlike the past outbreak, this is the first time in the history of Nipah that the diagnosis was made in the outbreak country. All the previous diagnoses were made at CDC Atlanta. The issue in Malaysia when Nipah appeared was that it was a totally new virus, meaning it took some time to figure it out, in total six months because the samples were finally taken to the US. The Malaysian team had a good connection with the CDC, so they could send them the sample to identify. The availability of another virus, Hendra virus from Australia really helped. The Malaysian team were able to grow the virus in their laboratory and the antibodies of Hendra were reacting to it, which finally clinched identifying Nipah in Malaysia.
In Bangladesh, again there was unusual deaths which were not explained by the routine Japanese Encephalitis story. There were no pigs involved, but Malaysia, there were pigs involved. So again, it took some time to understand that there was something wrong, and the samples again had to go to the US. It took around three years for them to diagnose it as Nipah.
In 2001, India had the Siliguri outbreak, and only some time into the outbreak was it noticed. Again, clinically it did not fit stories such as it being a modified form of measles, or measles without rashes. Unfortunately, they could not identify it, so again CDC was involved. It took some time to transfer the samples out of the country, therefore the diagnosis was a retrospective one which took five years. Only after five years, it was confirmed that this was Nipah, and in 2006 the team published a report. In 2007 there was another small outbreak in Siliguri, but by 2007 the outbreak reportedly involved five people and all five were dead. That outbreak was confirmed in India, and again was a retrospective confirmation rather than identification in real time.
When Nipah struck in Kerala, two things were different; We had two trained laboratories in India, both of which were ready to do the test, and we were already regularly looking for this virus so that we could analyse it. In fact, we were anticipating that this virus would surface in other places in different ways, but when the cluster of Encephalitis came, we were in a position to quickly test it. This case came from a private hospital, the Baby Memorial Hospital in Kerala, from Dr. Anoop, Critical care Physician.
By 2009, H1N1 pandemic flu had come to India. As a response, India had to establish influenza testing facilities nationwide. From 2007 onwards there was some activity in preparation for a bird flu outbreak, if bird flu affected humans, the government had plans preparing some laboratories to test bird flu, so we were part of that program. Our labs were prepared for testing influenza, but when pandemic Influenza A H1N1 arrived in India in May 2009, we became the regional laboratory for testing influenza in Kerala, Karnataka and Goa. As part of the acute febrile illness surveillance program under the Global Health Security Agenda (GHSA), CDC shared technical expertise, specific reagents, and training for diagnosis of dangerous viruses including Nipah.
From 2009, because of this testing facility, many of the hospitals were connected to us. So they did send samples for influenza testing. Apart from that, whenever they spotted something unusual, they used to contact us, and we would provide diagnostic services. Slowly from 2009, we had around 10-15 diagnostic tests coming from these peripheral hospitals, and by 2018, it was almost 100 tests. Our capacity had increased. These kinds of relationships between hospital and lab were one of the reasons that Dr. Anoop could alert us about this unusual cluster of Encephalitis and we could identify it quickly.
The case study was described to me by Dr. Anoop, and then samples were collected and sent. That is how things become faster. All of these things contributed to rapid detection. If the clinicians were not alerting us, then there would have been no question of anybody knowing what was happening. Also, in the 2018 outbreak, if each member of the family had gone to three different hospitals, then we may not have even recognized Nipah. Since all members went to the single hospital, to the same doctor, he saw it as something unusual, which was the real indicator.
AMJ: In the 2018 outbreak in Kozhikode, Nipah was spotted in the second case and in 2019 in Kochi, Nipah was diagnosed in the index case itself. The rapid detection of Nipah in patient zero accelerated Kerala’s response to the outbreak. Additionally, there was no transmission from the index case, and all other suspected cases proved Nipah negative. What has changed since the first outbreak in Kerala - is it because of the change in the strain of virus or in pathogenicity?
Dr Arunkumar: We cannot say this strain is different or pathogenically changed based on clinical situations, because death typically occurs when the disease becomes a severe condition and affects the respiratory system or the CNS, but in the current case, only the cerebellum was affected. Last year there was a case with only upper respiratory symptoms, not even pneumonia or any altered sensorium, and the patient recovered. So, we will definitely have a spectrum of clinical presentations, and also if we look at the transmission dynamics, every Nipah case is not going to transmit, but there will be some cases where it will transmit more readily.
In last year’s outbreak, only two infected people survived. One person had only mild illness, and he was picked up only because his wife died of Nipah. If that wasn’t the case, he wouldn’t have been picked up, because he had only upper respiratory illness.
Obviously, we can’t identify every upper respiratory disease as Nipah. The second case was an Encephalitis case, and she was a nursing student. She went through the whole spectrum, but she recovered. She was not given any monoclonal antibody, but she was given supportive therapy and ribavirin. In that case, the patient was not admitted immediately because she was a nursing student with fever and went home and later vomited and was admitted, because initially there was no apparent connection. The connection was later established because she was in the ER to which the index case was brought. Last year we had a case which was very mild, but also other fatal cases although the virus strain was the same.
AMJ: In 2003, the eminent epidemiologist Jonathan Epstein et al. conducted a serological study in north India to check for the presence of Nipah. The investigation was later published in the Journal of Emerging Infectious Disease. Jonathan Epstein et al. captured 41 Pteropus giganteus bats from a colony in Haryana from June 24 to June 30 2003, using mist nets. The bats were tested for antibodies against Nipah and Hendra viruses to determine whether henipaviruses circulate in Pteropid fruit bats (Pteropus giganteus) in northern India. The result proved twenty bats were seropositive for Nipah virus. However, there was no re- ported case of Nipah in this region until now. Does this suggest that while in Kerala, we found Nipah because we tested for Nipah, whilst in other states in India, Nipah may be misdiagnosed or provisionally diagnosed as Japanese Encephalitis?
Dr Arunkumar: Nipah outbreak should be a wake-up call for AES surveillance in India. Our Encephalitis surveillance is primarily based on Japanese Encephalitis (JE) because that is a vaccine-preventable disease and has a vaccine. Hence, the whole national program moves around JE surveillance. It’s true that Nipah is not tested for in most of the Encephalitis cases in India. Wherever fruit bats were tested for Nipah in India, we found it. Jonathan’s study reported antibodies in bats in Haryana, and the National Institute of Virology, Pune found Nipah virus among fruit bats in West Bengal, Assam and in Kerala. These are the only places where they tested, and if they increase the survey and test in other places, you will find the presence of Nipah in many states.
In Kerala, it is definitely because of testing that we were able to detect Nipah. When there was a family clustering in an Encephalitis case we have tested Nipah as a first line of action. Where there was not a familial cluster Nipah would have been tested not as a front line, but as a 3rd or 4th line option.
Many times in hospital-based outbreaks, even in Kozhikode, only because we were able to test and connect the deaths, we were able to make the links. Because in the hospital when a patient comes with Nipah and transmits it to others, these people may be bystanders or other patients, it may not be seen as a cluster. However once one healthcare worker is involved it can raise a very high level of suspicion.AMJ: Rapid Detection of Nipah Virus helped to contain the out- break in Kerala. As the first person to detect the Nipah virus in India, could you please tell us what made you test for Nipah?
AMJ: During a new infectious disease outbreak, the source of infection and the route of transmission is always a mystery. How does this virus directly affect humans? Looking at the past outbreaks, transmission pathways in each country were different. In Malaysia it affected the pigs and was transmitted from pigs to pig farm workers, whereas in Bangladesh, bats contaminated the date palm sap and people drank date palm sap and got infected. In the Philippines, it was from bats to horses. When it comes to Kerala, the transmission pathway is not confirmed. It’s still puzzling- how did the index case get infected from the natural reservoir when, moreover, there was no evidence of other animals involved.
Dr Arunkumar: No, It is not completely clear, but it is assumed that because he came from this community, there was a very high probability that it would have been a direct transmission from a bat. The genome of the virus recovered from the bat in Kerala and the genome of the virus recovered from human cases in Kerala was a 100% match, but they were not the Nipah virus strain which was circulating in Bangladesh. They were of the Bangladesh lineage but not the same strains. This was a Kerala specific strain, and so that clearly shows that the virus came from a bat. How that virus came from bat to humans is difficult to trace because the index case in the Kozhikode outbreak died before we could trace the epidemiology. This year, the index case is alive, so we can get some information.
AMJ: Considering the poor AES surveillance in India, do you subscribe to the point of view that every case of Encephalitis should be investigated for Nipah?
Dr Arunkumar: Not every case of Encephalitis. But you should look at the epidemiology and clinical presentation. I have investigated Gorakhpur Encephalitis and Orissa encephalopathy, it is all children, and because of the Acute Encephalitis Syndrome (AES) surveillance definition, people get confused with JE. To increase the sensitivity of JE case detection, they have provided a broad definition, but unfortunately, once you diagnose AES, which is a combination of syndromes including Encephalitis, meningitis, altered sensorium and seizures, people get confused hence further investigation is blocked.
People in these situations immediately start with a JE vaccination. Many times they check serum antibodies for JE IgM and this creates problems when they give false positive results.
AMJ: They don’t do CSF?
Dr Arunkumar: No, in many of these places they don’t do CSF initially. Because it is done only in higher or tertiary care centres. Of course, there is region specific variation in practice, but still, diagnosis is mostly around JE.
AMJ: There are survivors of Nipah virus disease in many countries. What are the common sequelae of Nipah virus disease and recommended evaluations in the survivors? Are you aware of any studies monitoring the NiV survivors?
Dr Arunkumar: Survivors will develop antibodies against Nipah. Even last year’s survivor in Kozhikode had antibodies. The survivors must be monitored for a longer time to look for potential sequalae. Until now they have had no problems, they are doing fine.
In Malaysia, very rarely they found some cases having recurrent Nipah again after around 11 years, so there is a remote possibility of recurrence. Since the numbers of survivors are not very high in our current Indian situation, it may not be a big problem, but these people have to be monitored for a long time to understand whether something is happening. Generally, the viremia lasts only for 8-10 days post onset of illness and by then, the case will have sufficient antibodies and a good immune response. With the present case in Kochi, they could not detect the virus in the blood, but were able to detect the virus in the urine. That clearly shows that the person would have already developed the antibody, that’s why he survived. The moment that we reach 6-8 days post-onset, the survival chance of the patient significantly increases as the immune response including antibodies will clear the virus. However, we have showed that Nipah virus RNA could be detected in semen of the last years male survivor for up to 26 days. This does not imply that it is infectious virus. Further, it may not be like Ebola or Zika which can persist for up to several months. Only in one case was the semen tested. But definitely, the virus remains in semen for some time, because it is an immunologically privileged site and hence has delayed clearance. Additional studies are required to understand the potential of the sexual mode of transmission of Nipah.
AMJ: In the last two decades, several zoonotic disease outbreaks have been linked to bat-borne viruses. These include the SARS coronavirus, Hendra virus Nipah virus, Ebola viruses and MERS coronavirus. Bats are potential reservoirs of a large number of known and unknown viruses, many of which could spillover into animal and human populations. However, our knowledge about bat biology and immunology is very minimal, and we have little understanding of major factors contributing to the risk of bat virus spillover events.
Dr Arunkumar: We are increasingly understanding the bats. They may be harbouring much more dangerous viruses also. We can do very limited things with the natural environment, but we can do much more as a community and even in the health- care system. We cannot avoid or disturb the bats’ habitats because they do a lot of good things within the ecosystem such as pollination, but we can create awareness to avoid close contact with bats as well as fruits which have been bitten by bats.
We have a minimal risk when it comes to the direct transmission from the natural reservoir, but we can very effectively control human to human transmission in hospitals. Last year, only one case was from the community, the remaining cases of transmission all happened in the hospital.
If a mask was put on coughing patients and if the nurse and other healthcare workers were using appropriate personal protection, then the transmission wouldn’t have been as high. That is an area where intervention is really possible, along with early detection and increased surveillance i.e., case surveillance and event-based surveillance with laboratory support. So, as lab tests increase, the ability to detect cases in the hospital should also increase.
The first case in Kerala last year came to Kozhikode Medical college and died, although otherwise an apparently healthy patient with two days severe illness. No samples were taken, no investigations were mounted. I think that part also has to improve. We need to increase the surveillance. If that could have been detected early, it would have definitely helped in preparedness, containment, contact tracing etc. All unexplained deaths in otherwise healthy people should be investigated.
AMJ: As we know, the first part of detection comes from the clinical acumen and alertness of doctors to suspect the clinical presentation, and this has played a vital role in both the 2018 and 2019 Nipah outbreaks in Kerala. At the same time, laboratory diagnosis is an essential element of infectious disease surveillance, both for routine confirmation of infections and the rapid identification of the cause of outbreaks. In many instances, only the laboratory can confirm an infectious agent. Do you agree that we could improve detection capability by reducing the waiting time to confirm the diagnosis and also increase the accessibility to advanced laboratories?
Dr Arunkumar: One of the limitations in virus diagnosis is that most of these organisms are classified as risk group 4 pathogens. We are confused with this risk group 4 pathogens and containment level 4 for handling samples. But over the years, technology has changed. We need to rethink the basic concepts of risk groups and laboratory biosafety. We are still talking about culturing the virus, which means increasing the quantity of virus in the laboratory, but currently we are not doing that, we are inactivating the virus in specimen, and we are only amplifying the nucleic acid. This actually reduces the risk and makes it possible to do tests at biosafety level 2, enabling near-patient testing.
When you use serological diagnosis, the antibodies are detectable only after 8-10 days after the onset of illness. Nipah is an acute and fatal disease. Many people may die before they develop the antibody, so we need to use a combination of real-time PCR and antibody detection assays. Hence the accessibility of real-time PCR should be increased.
Increased accessibility of Real-time PCR or even single sample testing devices like point of care tests will help to pick up Nipah cases in tertiary care centers. The disease is not transmissible in the early stage. In last year’s case in Kozhikode, none of the patient’s household had any infection. Only during the last 48 hours did he transmit the virus. His brother and father looked after him in the hospital. No children or other people at home were infected. The transmission happened towards the end of the illness, by which time he was already in the tertiary care centre. So, the major tertiary care centre should have these facilities to test at the point of care. Last year in collaboration with a company, MolBio , we had developed a point of care PCR device for Nipah diagnosis. Currently it is undergoing evaluation and awaiting regulatory approval. I am happy to note that this device was put into use during this year’s Nipah outbreak response in Kochi. Unless we follow these steps and make diagnostics more accessible, we cannot detect these things easily.
Nipah and other zoonotic viruses are here to stay, and we need to learn to live with them to ensure global health security.