A recent case of Zika virus infection in Pune has renewed concerns about India’s preparedness for diagnosing emerging infectious diseases. After experiencing symptoms like fever and rashes, the 46-year-old doctor was hospitalised and diagnosed with Zika virus infection after his samples were sent for testing to the apex virology institute. Subsequent testing of his family members revealed that his 15-year-old daughter was also infected. This is not the first time Zika has been identified in India. Cases have been identified from multiple States in India in the past, with larger outbreaks occurring in Kerala and Uttar Pradesh as recently as 2021.

Zika virus is a mosquito-borne pathogen belonging to the flavivirus family of viruses which also includes dengue. Clinical symptoms of Zika infection in many cases could be mild and indistinguishable from other infectious diseases including dengue. However, for pregnant women, the Zika virus poses a significant risk as it can be transmitted from mother to child, potentially leading to microcephaly in the offspring.

Due to climate change, it is not surprising that multiple dengue outbreaks are making headlines. The same vectors that spread dengue could also spread Zika. However, India’s lack of significant Zika surveillance means we might never fully understand its spread. In March 2023, CDSCO, India’s apex organisation for diagnostic approvals, confirmed that there is no approved diagnostic test for Zika. This limitation hinders our ability to diagnose Zika, relying only on classical symptoms and high clinical suspicion, making it complex as we see a concurrent upsurge in dengue cases across the country. Surveillance by ICMR on Aedes mosquitoes showed Zika virus positivity following human cases, indicating that many cases are likely being missed.

Zika is not an isolated case. A case of avian influenza A/H5N1 was recently reported from Australia in a child who had traveled to India, hinting at more undetected infections. Despite multiple avian influenza outbreaks in India this year affecting poultry, and an ongoing outbreak in Kerala, human testing and surveillance have remained limited. This is partially compounded by the lack of widely available diagnostic tests and over-reliance on a few apex institutes.

Consider the case of the Nipah virus, which has seen multiple outbreaks in Kerala. India has experienced several Nipah virus outbreaks, notably in West Bengal (2001 and 2007) and Kerala (2018, 2021, and 2023). In Kerala, identification of the virus relied heavily on clinical suspicion. Some cases were indeed missed during initial admissions due to the lack of routine testing, largely because the diagnostic facilities are not readily available apart from apex national institutes, adding to the complexity of testing, delays, and consequent loss of valuable time to institute countermeasures. Rapid identification and isolation of cases, contact tracing, and targeted screening of contacts are key to the effective containment of Nipah outbreaks.

For the rapid development of widely available and accessible diagnostics, the primary requirement is the quick availability of whole genome sequences in the public domain from outbreaks apart from an independent, time-bound accreditation process, especially in outbreaks. While many countries grant emergency approvals for diagnostics based on synthetic genomic material, India requires validation on clinical samples, which are not readily accessible. These limitations hamper rapid development. Although there have been multiple publications on Zika and Nipah outbreaks over many years now, the genomes from these outbreaks are still not rapidly released in public repositories. For example, the Nipah virus genome from the 2023 outbreak in Kerala was only released last month. Even with ongoing avian influenza outbreaks in multiple States, we do not yet have the whole genome sequences available in GISAID, the primary repository for influenza sequences, hampering our understanding of the disease and spread and more importantly, our ability to rapidly develop and deploy diagnostics.

During the COVID-19 pandemic, India swiftly expanded its testing infrastructure by rapidly decentralising and leveraging the existing network of hospitals, medical colleges, and private laboratories nationwide and tapping into the industry with a systematic approach for approvals of diagnostic tests making diagnostics widely available and accessible. The experience gained from the COVID-19 pandemic can be a stepping stone for improving testing capacities for other emerging diseases.

By decentralising testing facilities particularly at the district and sub-district levels, developing accessible and affordable diagnostic tests for Zika, Nipah, avian influenza, and many more emerging infectious diseases, India can ensure a more effective response to future outbreaks. There has never been a better time to swiftly establish a decentralised system for diagnostics, genomic surveillance, and data sharing to enable preparedness and public health response to emerging infectious diseases.

(Bani Jolly is a senior scientist at Karkinos Healthcare, and Vinod Scaria is a senior consultant at Vishwanath Cancer Care Foundation and Adjunct Professor at IIT Kanpur and DY Patil Vidyapeeth)

Bat populations in many Indian States harbour serological evidence of exposure to Nipah virus,” the Nature article points out. 
| Photo Credit: CraigRJD

Many places in Southeast Asia, such as Myanmar, Thailand, Laos, southern China, Bhutan, Nepal, Sri Lanka and some Indian States, have Nipah virus reservoirs and could experience cross-species transmission to humans, says an article published in the recent issue of Nature journal.

The article in the ‘World view’ section, titled ‘a personal take on science and society’, is authored by T.S. Anish, one of the leaders of the Nipah surveillance team in Kerala, and epidemiology professor at Government Medical College, Manjeri, Malappuram.

Based on his experience during the 2018 and 2023 outbreaks in the State, Dr. Anish calls for more scientific and policy work on the infection.

As a key first step, all countries likely to have Nipah virus reservoirs should have early detection systems. “The strain in Kerala’s outbreaks originated from Bangladesh in 2001. Health systems have missed this strain because its mortality rate is so high that it often causes small outbreaks or single cases,” he says.

“To reach Kerala, the virus must have spread undetected over more than 2,000 km, from Bangladesh or the neighbouring Indian State of West Bengal. It is highly probable that many places in Southeast Asia have Nipah virus reservoirs and could experience spillovers. Myanmar, Thailand, Laos, southern China, Bhutan, Nepal, Sri Lanka and many Indian States all lie in a similar distance from Bangladesh and are home to fruit bats. Bat populations in many Indian States harbour serological evidence of exposure to Nipah virus,” the article points out.

Dr. Anish says that lack of treatment options for Nipah is a concern as drug trials are difficult because outbreaks typically last for only a few days. “Kerala’s experience is that using antivirals not specific to Nipah might have helped some people infected near the end of the outbreaks in 2018 and 2023. More research is needed, as is wider distribution of general antivirals,” he says.

The development of monoclonal antibodies from Nipah survivors in Kerala is also a priority. These will be specific to the local variant and could be given to people with early symptoms and to high-risk contacts, such as frontline healthcare workers, to save lives. The International Centre for Diarrhoeal Disease Research, Dhaka, Bangladesh, is already studying about 50 survivors of Nipah.

According to Gavi, the vaccine alliance, several candidate vaccines for Nipah are in clinical trials, including one based on messenger RNA, one based on a viral vector, and one containing the protein subunit of Hendra virus, which closely resembles Nipah.

Because Nipah is an RNA virus that is prone to mutate, studying the virological factors contributing to severity is important for monitoring its pandemic potential. It is also crucial to study variation in immunological mechanisms known to affect people’s susceptibility to Nipah, the article adds.

On September 11, samples sent to Calicut Medical College for testing were confirmed as Nipah virus. But only on September 20, after 323 samples were tested for the virus, did ICMR permit Kerala to use Truenat for Nipah testing. Even the belated permission came only after Kerala “strongly demanded” for Truenat during discussions with ICMR, Health Minister Veena George said during a press conference.

Truenat for Nipah virus testing was granted an emergency use authorisation (EAU) by the Indian regulator in September 2021 days after the third Nipah outbreak in Kerala. The EAU was based on validation of the Truenat test to detect the virus and the Trueprep AUTO lysis buffer (to inactivate the virus) by NIV Pune in 2018 in Kerala and external validation at the Institute of Epidemiology Disease Control and Research, Dhaka, Bangladesh. Both human and bat samples from Kerala were used for validating the test in 2018. In 2019, NIV installed Truenat at the Government Medical College, Ernakulam during the Nipah outbreak, and at two field sites in Punjab as part of the Nipah virus surveillance in bats. Of the 120 human clinical specimens and 25 samples from bats tested in 2019, Truenat correctly diagnosed all the positive and negative samples, except for one positive human sample.

As per a 2021 paper published in the Indian Journal of Medical Research, the sensitivity of Truenat was 97% and specificity was 100%. It was able to correctly diagnose Nipah even when other viruses were present. The efficacy of the Trueprep AUTO lysis buffer for inactivating Nipah virus prior to virus detection was also tested and found to be high. The “inactivation of Nipah virus was evident by the absence of Ct value”, notes the paper. A study was carried out at NIV in April 2020 using SARS-CoV-2 virus to evaluate the virus inactivating efficiency of both the lysis buffer and the Trueprep AUTO transport medium. The study, which is yet to be published but shared with The Hindu, found high virus inactivating efficiency by both the lysis buffer and the transport medium.

“The Truenat test for Nipah virus was already developed and ready as our idea is to work on diseases that have the potential to become a pandemic if not diagnosed early,” says Dr. Chandrasekhar Nair, Director and Chief Technology Officer at Molbio Diagnostics Pvt Limited.

Despite the validation as a point-of-care test by NIV and an EUA granted in September 2021, ICMR did not permit Kerala to use Truenat till September 2023. A senior scientist based in Kerala tells The Hindu that it is the Indian regulator and not the ICMR that is authorised to approve the use of Truenat for Nipah testing. With an EUA granted in 2021, States should have been free to use Truenat without any permission from ICMR, the scientist says.

BSL-2 facilities

 “Truenat will be used for testing Nipah virus at five government medical colleges that have a BSL-2 facility with BSL-3 practices. Private medical colleges that have similar facilities will also be allowed to use Truenat,” says Hanish Mohammad, Principal Secretary (Health), Kerala.

“Testing for Nipah using Truenat is being considered only for hospital-based BSL-2 facilities and not in stand-alone labs,” says Dr. Aravind R, Head of Infectious Diseases, Government Medical College, Thiruvananthapuram. According to him, allowing only hospital-based BSL-2 facilities to test for Nipah is to discourage people from walking in to get tested at stand-alone labs. “The decision to test for Nipah should be taken by doctors, not patients. There should be a pretest probability of a patient being positive for Nipah,” says Dr. Aravind.

“The lysis buffer inactivates the virus and the risk is reduced maximally. There is no need for a BSL-2 lab for Nipah virus testing when Truenat is used,” says Dr. Raman Gangakhedkar, who was the Head Scientist of Epidemiology and Communicable Diseases at ICMR and a co-author of the 2021 paper.  

Explaining why a BSL-2 facility is essential for testing Nipah virus, Dr. Rajiv Bahl, Director-General of ICMR says: “Even though in the diagnostic samples the virus may be inactivated by using lysis buffer, handling the initial samples before the addition to lysis buffer without strict containment measures may pose a risk of exposure to healthcare workers or any accidental environmental release if no proper decontamination is taken care of.”

However, Dr. Nair of Molbio Diagnostics confirms to The Hindu that for Truenat, the virus is inactivated at the site of sample collection and not when the sample reaches the BSL-2 facility. Therefore, the inactivated virus is inert and non-infectious when the sample reaches the lab for testing. “The Trueprep AUTO transport media is a proprietary medium that has reagents to inactivate the virus. The transport media inactivates the virus while the lysis media in Trueprep AUTO completely breaks open the pathogens to release nucleic acids,” he explains. The April 2020 study at NIV found both the lysis buffer and the transport medium highly efficient at inactivating the SARS-CoV-2 virus.  

Virus confirmation

ICMR and NIV are very clear that for an official confirmation, the sample must be tested only in a BSL-3/4 facility, says Mr. Mohammad. The insistence on a BSL-3 facility for virus confirmation is based on the 2021 government of India memorandum, he adds.

However, India’s rules on a BSL-3 facility for Nipah virus confirmation run counter to the WHO’s draft high priority diagnostics for Nipah. The WHO document mentions that Nipah can be confirmed by detection of the virus RNA or viral culture. For a reference laboratory setting, the “diagnostic options for confirmation can include laboratory NAT, NPT/POC NAT assays, virus isolation (if BSL-3/4 available), and serum neutralisation assays”. Of the diagnostic options for Nipah confirmation, WHO has included both near-patient testing (NPT) and point-of-care (POC) NAT testing; Truenat is a POC NAT test. The WHO also clearly mentions that BSL-3/4 facility is needed only when virus isolation is undertaken; isolation of viruses is only for research purposes.

“Viruses are sometimes cultured [for detection]. This used to be done a lot before nucleic acid-based amplification became available… Used much less now (and viral culture experts and lines are hardly available),” says microbiologist Dr. Gagandeep Kang, former professor at CMC Vellore. Dr. E. Sreekumar, Director of the Institute of Advanced Virology, Thiruvananthapuram says: “All labs in India, including the NIV’s, use only RT-PCR for Nipah diagnosis, be it for initial detection or confirmation. No other methods are used now.” 

Usually, it is the same specimen that has been used for testing that is sent for virus confirmation, says Dr. Aravind. Dr. Sreekumar adds: “At present, ICMR encourages collection of only live samples so that they can do both diagnostic testing and research by virus isolation in the same sample. This [collection of live samples] prevents early detection as local labs are prevented from doing testing.”

But now, by default, all samples sent for Truenat testing are collected in a transport medium, which inactivates the virus at the site of sample collection. Since inactivated viruses are not viable, they cannot infect people; genomes can be sequenced using inactivated viruses. This makes a BSL-3 facility redundant for virus confirmation. Relying on BSL-2 labs for testing and confirmation can speed up the process as many such facilities in Kerala can be roped in.  

Inactivated viruses are not only used for testing and genome sequencing but also for developing diagnostics, such as Truenat. “For any nucleic acid tests (NAT), the U.S. FDA accepts synthetic DNA. That is precisely how the U.S. and other countries developed COVID tests and the U.S. FDA authorised for emergency use even before outbreaks happened in those countries,” says Dr. Vinod Scaria, Senior Consultant at the Vishwanath Cancer Care Foundation, Bengaluru. “Even for drug discovery, live viruses are needed only for screening molecules and understanding the infection and immune processes. Even here, many scientists use pseudoviruses, which are better amenable and easier to use, for studying antibody escape.”

Responding to a question on why ICMR insists on a BSL-3 facility for Nipah virus confirmation despite the virus being already inactivated, Dr. Bahl, ICMR Director-General says: “BSL-3 facilities have specialised equipment and containment protocols to prevent the release of infectious materials. This is essential in case the virus is not fully inactivated or if there are any procedural errors during diagnosis.” But NIV had validated the Trueprep AUTO lysis buffer and the transport medium and found it to be really effective in inactivating the virus. 

New Delhi:
The Nipah alert in Kerala’s Kozhikode after two ‘unnatural’ deaths were reported due to fever has brought back panic and grim memories of the earlier outbreaks that claimed several lives.

Here is a 5-point explainer on Nipah virus

Thiruvananthapuram:

The Kerala Health Department sounded a health alert in Kozhikode district on Monday following two “unnatural” deaths suspected to be due to the Nipah virus infection.

State Health Minister Veena George held a high-level meeting and reviewed the situation, the health department said in a statement on Monday night.

It said two “unnatural” deaths following fever were reported from a private hospital, and it is suspected that these were due to the Nipah virus.

Relatives of one of the deceased are also admitted to the intensive care unit, it said.

Deaths due to Nipah virus infection were reported in Kozhikode district in 2018 and 2021.

The first Nipah virus (NiV) outbreak in south India was reported from Kozhikode on May 19, 2018.

According to the World Health Organization (WHO), Nipah virus infection is a zoonotic illness that is transmitted to people from animals and can also be transmitted through contaminated food or directly from person-to-person.

Among infected people, it causes a range of illnesses, from asymptomatic (subclinical) infection to acute respiratory illness and fatal encephalitis.

The virus can also cause severe disease in animals such as pigs, resulting in significant economic losses for farmers, WHO said.

(Except for the headline, this story has not been edited by NDTV staff and is published from a syndicated feed.)