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Biochemistry

Mysterious illness kills children season after season in northern India, leaving researchers frustrated

A toxin in lychees, combined with malnourishment, is the culprit, scientists say. Yet not everyone agrees

by Dinsa Sachan, special to C&EN
August 25, 2019 | APPEARED IN VOLUME 97, ISSUE 33

 

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Credit: DIPTENDU DUTTA/AFP/Getty Images
Some think lychees are being blamed unfairly for an illness that breaks out among children in the north India state of Bihar each year.

Each summer in Muzaffarpur, in the north Indian state of Bihar, children suddenly wake up vomiting and delirious or they start having seizures. Some slip into a coma, and some die within hours of being wheeled into a hospital.

This phenomenon has been recurring for more than two decades. It generally begins in April and ends in June, when temperatures peak in this part of the country, soaring as high as 44 °C. This year, the outbreak claimed the lives of 116 children in the state.

Some doctors thought it might be heat stroke. Others initially linked the recurring epidemic to acute encephalitis, an inflammation of the brain that can be caused in a variety of ways, including by bacterial or viral infection. They call it acute encephalitis syndrome (AES). But the situation in Bihar has been particularly puzzling: the symptoms come on too fast for encephalitis, and they’re accompanied by a drop in blood sugar.

So the search has continued. And within the past decade, researchers have identified a different culprit—a controversial one—lurking in fruit orchards.

Molecular detective work

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In 2012, the Bihar government invited T. Jacob John, a retired pediatrician and virologist who had worked at Christian Medical College in Tamil Nadu, to investigate the deaths in Muzaffarpur. “Until my visit, there were two main ideas. One was viral encephalitis due to an unknown virus, and the second was heat-related disease,” John says. Acute encephalitis was an option because it’s widespread in other parts of India: more than 3,300 cases of AES have been reported in the country so far this year. And heat-related disease was on the table, John says, because of the extreme temperatures and their ability to affect the brain and lead to death.

Encephalitis typically occurs in two stages. The first, called the prodromal phase, usually takes place over 2–3 days and resembles a regular infection. Patients develop fever and headaches, and they are lethargic. When the infection takes hold of the brain, the second stage begins. During this period, patients experience seizures, delirium, and loss of consciousness. “No case in Muzaffarpur had a prodromal phase,” John explains. “And all the children fell ill suddenly overnight,” which doesn’t occur with most cases of encephalitis.

The evidence that clinched the case against encephalitis was tests of the patients’ cerebrospinal fluid (CSF). Increased numbers of immune cells in CSF is “a hallmark of brain inflammation,” John says. In viral encephalitis, he says, doctors can find up to 100 immune cells per mm3 of CSF. But this wasn’t the case for the sick children in Muzaffarpur.

Heat stroke was also eventually ruled out because the children who fell ill began having symptoms early in the morning, “not the usual time when signs of heat-related disease would arise,” John says. Moreover, heat stroke also affects adults, while only very young children were affected by these outbreaks.

In the end, John categorized the illness he was seeing as acute encephalopathy, a broad term for brain disease caused by multiple factors and linked with a variety of symptoms, including seizures and loss of consciousness. Not only do immune-cell counts stay low in CSF samples from patients with encephalopathy, but symptoms also tend to start suddenly.

“This was my first aha moment in this investigation,” John says. Looking for other, similar cases, John and his colleague Mukul Das, a toxicologist then at the Indian Institute of Toxicology Research, sifted through the scientific literature. There, they learned about Jamaican vomiting sickness, also a type of acute encephalopathy, which is caused by consuming an unripe fruit, the ackee. This find was their second breakthrough.

Ackee is grown extensively across Caribbean countries. Importantly for John and Das, it is also a cousin to the lychee (also spelled litchi), a white-fleshed fruit popular in India. In Jamaica, residents prepare a local delicacy by cooking salted codfish with ackee and vegetables. In the 19th and 20th centuries, outbreaks of Jamaican vomiting sickness were common in the Caribbean. Scientists made the connection between the disease and ackee in the early 1900s.

Later, scientists found that unripened ackee contains a toxin called hypoglycin A that can cause a person’s blood glucose levels to drop dramatically, leading to hypoglycemia. In malnourished children who already have low blood sugar levels, the toxin can trigger Jamaican vomiting sickness.

Ever since that connection was made, fewer outbreaks in the Caribbean have occurred.

Upon learning of ackee poisoning, John and Das took a fresh look at the situation in Muzaffarpur.

The patients there displayed signs of hypoglycemia as well. Das, now retired, came across some studies in which scientists isolated a compound called methylene cyclopropyl glycine (MCPG) from lychee seeds. When researchers fed this compound to rats, the animals developed hypoglycemia (Biochem. J. 1989, DOI: 10.1042/bj2590921).

Even more important, Das and John noted that hypoglycin A and MCPG are structurally similar amino acids, suggesting that they act similarly in the body. In 2013, the duo reported this possible link between MCPG from lychees and the outbreaks in Muzaffarpur, publishing their findings the following year (Curr. Sci. 2014, 106, 1184).

“Now, the only point which was missing was whether the lychee fruit that is consumed by the population contains MCPG or not,” Das recalls. To find out, John and Das examined MCPG levels in the fruit from local orchards and found the compound in the lychee pulp and seeds, in both ripe and unripe samples.

Uptick

In India's northern state of Bihar, cases of what doctors there are still mistakenly calling AES (acute encephalitis syndrome) had been limited to less than 200 for the past few years. During the first half of 2019, however, more than 500 patients were reported.
Source: National Vector Borne Disease Control Programme, Indian Ministry of Health and Family Welfare.
a Data captured only through June 30.

From molecules to medicine

An international team later confirmed the findings of the duo by studying children admitted to hospitals in Muzaffarpur in 2014. The group, led by Aakash Shrivastava at India’s National Centre for Disease Control, analyzed urine samples from about 70 patients and found molecular fragments left over from the breakdown of hypoglycin A, MCPG, or both in 66% of them. Previous studies had linked only MCPG with lychees. Control patients from the hospitals had none of the fragments. In blood samples, the team found signs of hypoglycemia, and the researchers saw evidence that fatty acid metabolism had been disrupted—a tell-tale sign of hypoglycin A and MCPG activity—in 90% of patients (Lancet Global Health 2017, DOI: 10.1016/S2214-109X(17)30035-9).

Participants in this study were more likely to fall ill if they had eaten lychees within 24 h of developing symptoms and if they hadn’t eaten supper the night before the symptoms started. The study participants came mostly from poor families and had poor nutrition. By conducting surveys, the research team determined that patients who landed in the hospital were more likely to have consumed lychees than control patients.

The hypothesis is that malnourished children have dangerously low levels of a polysaccharide called glycogen—a stored form of glucose—in their livers. So their bodies use a process called gluconeogenesis to create energy, particularly for the brain. MCPG, as well as hypoglycin A, disrupts this process, leading to hypoglycemia. These chemicals also interrupt fatty acid oxidation, another process the body uses to create energy. And if that wasn’t enough, when fatty acid oxidation is disrupted, toxic intermediates accumulate that lead to the neurological symptoms of acute encephalopathy. MCPG and hypoglycin A do not impact well-fed individuals because their livers hold adequate glycogen.

Government data show that almost 50% of Bihar’s children under the age of five have low height and weight for their age. “We don’t want to blame lychees,” Das says, pointing out that the fruit by itself isn’t causing the disease and that malnutrition plays a large role. He and others in India worry that the outbreaks have given the fruit a bad reputation.

The good news is that if caught soon enough, John says, cases of lychee-induced encephalopathy can be reversed. John and a pediatrician local to Muzaffarpur, Arun Shah, published a study in 2014 demonstrating that if they administered a 10% dextrose solution to kids with encephalopathy within 4 h of symptoms kicking in, the children could fully recover. The doctors were able to discharge 58% of kids from the hospital within 48 hours (Curr. Sci. 2014, DOI: 10.1007/s13312-016-0861-9). “If the unconscious children go without treatment for more than 4 h, the neurological damage becomes permanent,” Das explains. He points out that he and John also recommended this 10% dextrose therapy to the Bihar government in 2014 to tackle the recurring epidemic.

One of John’s big concerns now is the continued use of the term AES to describe all the cases in Muzaffarpur and elsewhere. Even though cases of acute encephalitis do occur during this time of the year, the treatment is not the same as for lychee-induced encephalopathy. “AES as a clinical diagnosis is unacceptable for any child with any disease other than true encephalitis,” John says. He worries that doctors who aren’t sufficiently trained or who don’t have access to labs that could make the true diagnosis will treat patients incorrectly. “An AES diagnosis puts the child at risk of medical negligence and consequent brain damage or death,” he says.

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Credit: Shutterstock
Lychees (left) and ackees (right) are related tropical fruits.

The case isn’t closed yet

Still, not everyone is convinced that lychees are to blame for the illness in Muzaffarpur and the surrounding area. Vipin Vashishtha, a pediatrician based in the neighboring state of Uttar Pradesh, says that the epidemics are definitely linked to lychee cultivation but exactly how isn’t clear yet. “There have been problems with the studies that linked the deaths with MCPG,” he says. For example, the 2017 study published in the Lancet Global Health used a control group of patients from the hospitals where the affected patients were being treated. “They should have used controls from the community of the patients, their households or neighboring families,” to make a fair comparison, says Vashishtha, who has investigated similar outbreaks in Uttar Pradesh but determined that children were falling ill after consuming seeds from a plant commonly called coffee senna.

Even the Bihar government hasn’t accepted lychee toxicity as the definite cause of the outbreaks. Vishal Nath, director of the National Research Centre for Litchi, which is run by the Indian agricultural ministry, has rejected the findings associated with MCPG. “Everyone eats lychee in India,” he says. “Why aren’t people in other parts of the country affected?”

Das acknowledges that the restricted location of the outbreaks is still a mystery. The answer, he says, may lie in the size of the lychee cultivation area in Muzaffarpur. Because it’s so large, it makes Bihar the largest producer of the fruit. In 2017, the state generated 300,000 metric tons of lychees. “So it’s possible that other areas don’t see such outbreaks because their lychee cultivation areas are smaller in comparison” and kids therefore have fewer chances to eat lychees from the orchards, he says.

A completely different angle that some scientists have explored is whether the pesticides used on lychee farms could be to blame. An international team of researchers examined an outbreak during the summer of 2012 in northern Bangladesh. Of the 14 cases the team studied, 13 of the children died and did so within 20 hours of the onset of illness. The researchers spoke at length with the families of the 14 affected kids, their neighbors, and lychee orchard farmers in the vicinity to determine what the children could have been exposed to. At least 85% of the affected kids had visited a lychee farm within 3 days of falling ill. Family members of at least eight of the kids were involved in lychee farming in the orchards (Amer. J. Trop. Med. Hygiene 2017, DOI: 10.4269/ajtmh.16-0856).

In particular, the lychee orchard managers revealed that employees sprayed lychee trees with a cocktail of pesticides and other ingredients. In particular, the researchers spotted packets of endosulfan, an organochlorine insecticide banned in many countries, scattered around many of the orchards they visited.

They followed up the interviews with an experiment in which they recruited one set of children from an affected neighborhood and a control set from a nearby village. The kids from the affected neighborhood were 12 times as likely as the control group to have visited a lychee farm within a day of falling sick.

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The Bihar and Bangladesh outbreaks have a baffling similarity: their occurrence coincides with the lychee harvest seasons, and they strike near areas rich with lychee orchards. So why do studies of these epidemics point to different culprits? “We investigated different things. The signs and symptoms to us did suggest a type of poisoning. So we investigated this angle very directly,” says Emily Gurley, an epidemiologist at Johns Hopkins Bloomberg School of Public Health and a coauthor of the Bangladesh study. “When we compared children who had been sick during the outbreak to other healthy children, there were no statistical differences when it came to who had eaten lychees. But there was a difference in recent visits to a lychee garden. So, to me, that suggests it’s not eating the lychee that’s the problem. It’s somehow being in that environment in a garden.”

“Pesticides are toxic chemicals that do not just affect children but adults as well,” Das counters. “Had it been pesticides, we would have seen patients in the adult group as well” when studying cases in Muzaffarpur. Das also points to a 2018 Toxicology Letters study carried out by his team (DOI: 10.1016/j.toxlet.2018.10.033). The researchers gave lychee-seed powder, which contains high levels of MCPG, to young, starved rats. The animals’ weight and glucose levels dropped dramatically compared with control groups. He and his team didn’t find significant levels of pesticides in the fruit samples they analyzed. In addition, Das points out that pesticides don’t cause hypoglycemia, a prominent symptom in patients involved in the outbreaks in India.

Gurley responds that children succumb to poisoning more easily compared with adults because of their low body weight. “Any exposure to chemicals affects them much more than adults in the same doses. They could get sick from a dose that would not make an adult sick,” she explains. She adds that she and her coworkers didn’t investigate the presence of hypoglycemia among their cases because they didn’t have access to blood or urine samples from patients. “Pesticides was the simplest explanation based on our investigation,” Gurley says. “We didn’t have to look elsewhere.”

Meanwhile, the health minister of India, Harsh Vardhan, doesn’t believe the case is closed yet and has announced that a multidisciplinary team of experts will investigate the causes of the Bihar outbreaks.

Frustrated, Das says, “Most of the research on this disease is done and completed. We know its etiology. Now the focus should be on treatment and drugs that can save the lives of these kids.”

Dinsa Sachan is a freelance science writer based in New Delhi, India.

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