Kapit town is the seat of Kapit Division, an area populated mainly by Iban people who live in traditional longhouses, travel the river by dugout, hunt in the forest, and raise rice and corn in gardens along the forest edges. Plasmodium vivax and P. falciparum are the most commonly reported malarial organisms in Sarawak, with P. malariae third in order, accounting for a small fraction. The blood-borne stages of those three can be distinguished under a microscope, rather quickly and easily, in a smear of blood on a slide—which was how malaria had been diagnosed for decades. But the reported statistics seemed skewed; a large portion of all the P. malariae cases in Sarawak, Singh and Cox-Singh learned, were coming from Kapit. Why? The division had a remarkably high incidence, it seemed, of this particular malaria. Furthermore, most of the Kapit cases were severe enough to require hospital treatment—rather than being mild or scarcely noticeable, as typical for P. malariae. Again, why? And the Kapit victims were mainly adults, who should have been immune because of prior exposure—rather than children, who as nonimmunes were the usual victims of P. malariae. What was going on?
Balbir Singh traveled by boat up to Kapit and took samples from eight patients, pricking the finger of each person and blotting the drop of blood onto a piece of filter paper. Back in Kuching, he and a young research assistant named Anand Radhakrishnan ran the samples through a molecular test using PCR, which was the new standard in malaria diagnostics, as in so many other areas, and a far more precise method of identification than peering at infected blood cells through a microscope.
PCR amplification of DNA fragments, followed by sequencing (reading out the genetic spelling) of those fragments, plumbs far deeper than microscopy. It allows a researcher to see below the level of cellular structure to the letter-by-letter genetic code. That code is written in nucleotides, which are components of the DNA and RNA molecules. Each nucleotide consists of a nitrogenous base linked with a sugar molecule and one or more bits of phosphate. If DNA resembles a spiral staircase supported by two helical strands, those nitrogenous bases are the stair steps connecting the strands. There are four kinds of base in DNA—molecular components known as adenine, cytosine, guanine, and thymine, and abbreviated as A, C, G, and T, little pieces in the great game of genetic Scrabble. You’ve heard this before on the Discovery channel but it’s elemental stuff that bears repeating, because genetic code is one crucial form of evidence by which disease scientists now recognize pathogens. In the RNA molecule, which serves for translating DNA into proteins (and has other roles, as we’ll see), a different piece called uracil substitutes for thymine, and the Scrabble pieces are therefore A, C, G, and U.
Singh and Cox-Singh, with the help of Radhakrishnan, were looking for DNA and RNA fragments characteristic of Plasmodium parasites generally—and they found some. But these fragments hadn’t come from P. malariae, nor from P. vivax nor P. falciparum either. They represented something new—or, anyway, something less expected and familiar.
Further testing and matching showed that five of the eight Kapit patients were infected with Plasmodium knowlesi. And there was no clustering of cases within a single longhouse, another unexpected clue. The absence of clustering meant that these people hadn’t passed the parasite, via mosquitoes, to one another. Each patient seemed to have caught it from a mosquito that had bitten a macaque.
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The Faculty of Medicine and Health Sciences, University of Malaysia Sarawak, resides in a sleek high-rise just ten minutes by cab from the big new hotels and the old market buildings of the Kuching riverfront. I found Balbir Singh there in his office on the eighth floor, a handsome and genial fiftyish man surrounded by books and papers and golf trophies. He wore a dark beard going gray, a purple-black turban, and a pair of reading glasses dangling around his neck. Despite the fact that he and his wife were leaving town the next day, for meetings with health officials elsewhere in Borneo, they had agreed to give me some time. Their discovery of P. knowlesi among the people of Kapit was still rather fresh, with implications for malaria treatment throughout Malaysia and beyond, and they were glad to talk.
From the high-rise, Balbir Singh and I walked across the street to a very modest South India café, his favorite, where he bought me a biryani lunch and told me about his Punjabi Sikh grandfather who had emigrated to Malaysia and his own circuit through Liverpool. I heard about P. knowlesi living successfully, asymptomatically, in long-tailed macaques amid the forest canopy. I heard about some surveyor, a spy, out in the Malaysian forest somewhere, but the information was flying and the food was good and I could hardly make sense of that part until later. Back in his office, Singh recounted with great élan the story of Julius Wagner-Juaregg and malaria pyrotherapy for syphilitics, Professor Ciuca’s adaptation of Plasmodium knowlesi for that purpose in Romania, and again the mysterious American surveyor who got infected with this monkey disease in the wild. Singh showed me photos, on his computer screen, of Iban longhouses along the upper Rajang River. Eight different ethnic groups, but mostly Ibans, he said. Here’s a longhouse, accommodating anywhere from five families to fifty. Great for doing blood surveys—you don’t have to travel from house to house. Here’s another typical scene: You see that greenery, you think it’s grass, right? But it’s not grass, it’s hill paddy. Rice. They also grow corn. At harvest time the people stay out at night in huts by their fields, trying to haze off the macaques that come to raid the crops. They don’t shoot the animals, because bullets are too expensive and a long-tailed macaque offers very little meat. Also, in some of the longhouses there’s a taboo: Kill a monkey and its spirit will visit the womb of your pregnant wife, with dreadful effects on the baby. The monkeys are bold and persistent, and they’ve got to be kept off the paddy rice—evidently a matter of arm waving, shouting, clanging of pots. Two nights, three nights in a row the people stay out there. Of course they get bitten by nocturnal forest mosquitoes, including Anopheles latens, the main insect transmitting P. knowlesi hereabouts.
“So control is a problem,” he said. “How are you going to control this?” Both men and women are infected. Their livelihood depends on going into the forest, where the macaques are abundant and so are the mosquitoes.
He showed me blown-up images from microscope slides full of malaria-infected human cells. Circles and dots, to me. Trophozoites, schizonts, gametocytes, to him. He was talking fast. Yes, easy to mistake P. knowlesi for P. malariae if that’s what you’re looking at, I agreed. No wonder the methods of molecular genetics have opened new vistas of discrimination. No wonder this zoonotic malaria was misdiagnosed for so long. Then we went downstairs to visit his wife in the lab.
Janet Cox-Singh is a small woman with short auburn-black hair and fine features, her speech reflecting almost no trace of her Belfast origins. She sat at a lab bench, not far from the PCR machine, before her own large computer monitor, and beneath shelves on which rested boxes filled with filter-paper samples of blood, dried and packed away, a precious archive of raw material from which she and her husband had extracted much of their data. Think of it as DNA jerky. “We developed this PCR method so we could take blood spots on filter paper and do very nice malaria epidemiology from very remote places,” Cox-Singh told me. Kapit Division, Sarawak, is indeed a remote place if anywhere is.
Nearby on the floor rested several large liquid-nitrogen storage tanks for transport of frozen specimens, a more cumbersome method of bringing blood to the laboratory, not quite obsolete but now circumvented, for their purposes, by the filter-paper technique. After the first trip upriver, during which Singh had pricked eight fingers and blotted up eight samples, yielding the first signal of P. knowlesi, he and Cox-Singh continued their data gathering with visits to the Kapit hospital and nearby longhouses. They also expanded their reach by delegating the filter-paper technique. They sent kits of such papers to other parts of Sarawak, in the hands of trained helpers, and got back blood spots, dried but valuable. Using an old-fashioned paper punch (carefully sterilized to avoid contamination), they punched two sma
ll dark dots out of each paper and processed those dots through the PCR machine. Two crusty dots held about twenty microliters of blood, just enough for extracting DNA. Then the DNA had to be selectively amplified so they could work with it. Cox-Singh began describing to me the particular method they used, known as “nested PCR,” diagramming it roughly on the back side of a journal paper as she spoke. Small subunits, fifteen hundred nucleotides, ribosomal RNA. I stared at the squiggles. Once they possessed amplified product, they sent that off to a mainland lab for genetic sequencing. The sequenced results were a longish series of letters, a passage written in genetic code as though to spell a choking expletive (ACCGCAGGAGCGCT . . . !), which could be entered into a vast online database for matching against known referents. That’s how they had identified P. knowlesi in those first samples, she said, and in many more since.
Her husband pulled down a box and opened it. “This is our collection of blood spots,” he said with quiet pride. Borneo is off the beaten path and, I suppose, not many science journalists visit. Inside the box was a neat file of plastic envelopes, each one containing a piece of porous paper no bigger than a business card; on each card was a rusty black spot. Near the center of the dark spot, on the card I inspected closely, was a perfectly round little hole. The punched dot, missing there, had already surrendered its secrets to science. DNA confetti.
During their first two years of work on the Kapit population, using filter-paper dots and PCR, the Singh–Cox-Singh team (like all scientists, they have helpers and colleagues) found 120 cases of P. knowlesi. Under earlier diagnostic assumptions and methods, most or all of those people would have been judged to have P. malariae, the benign form, and therefore received little or no medical care. They would have suffered, or worse. Properly diagnosed, and treated aggressively with drugs such as chloroquine, they had recovered. The paper describing those results appeared in an august British journal, The Lancet, delivering solid proof of what the strange case of BW the Surveyor had suggested: that P. knowlesi malaria is a zoonotic disease.
Expanding their search between 2001 and 2006, the team identified hundreds more cases of P. knowlesi, including 266 from Sarawak, 41 from Sabah (the other Malaysian state on the island of Borneo), and 5 from an area of Peninsular Malaysia just northeast of Kuala Lumpur—not far, probably, from where BW caught his case in 1965. They also found P. knowlesi in most of the long-tailed macaques from which they were able to take blood, confirming that those monkeys are a reservoir.
More dramatically, the team detected four human fatalities—four malaria patients, each of whom had gone to a hospital, been misdiagnosed with P. malariae (based on microscopy, the old way), developed severe symptoms, and died. Retrospective analysis of their blood samples by PCR showed that all four had suffered from P. knowlesi. These revelations suggested something more than that P. knowlesi is a zoonotic disease; they suggested that people were dying because doctors and microscopists were unaware of that fact. The paper in which Cox-Singh, Singh, and their colleagues presented the four-fatalities work, she told me, was initially rejected for publication. “Because we were saying that this was—”
Her husband completed the sentence: “—causing deaths.”
“It was causing deaths,” she concurred. “And they didn’t like that.” By “they” she meant anonymous manuscript reviewers for The Lancet. The editors of that journal, who had favored their first paper, declined this one on advice from such reviewers, in part because there was no absolute proof as to the cause of death in the four cases. There was no absolute proof, of course, because Cox-Singh and Singh had been working from archived blood samples, and reconstructing stories from medical files, to understand the illnesses of four people whose bodies were long since unavailable for postmortem. “So we ran into trouble with that one.” But eventually the paper was accepted by another good journal and, published there in early 2008, caused a sizable stir. Its title stated the essence, which was that, far from being rare and innocuous, “Plasmodium knowlesi Malaria in Humans Is Widely Distributed and Potentially Life Threatening.”
Science is a process performed in laboratories and in the field, but it’s also a conversation conducted through the journals. Being part of this conversation is especially important, even in the age of email, if a scientist is separated by distance from most of his or her peers. Within that context, Singh and Cox-Singh had followed the second paper with an article in still another journal, summarizing their discoveries, reviewing previous knowledge, and offering some concrete recommendations. It was labeled “Opinion,” a cautious editorial disclaimer, but it was really much more than that: a deeply informative overview, a thoughtful essay, and a warning. There was no list of coauthors; Cox-Singh and Singh spoke together, alone. The piece appeared in print not long before I met them, and I was carrying a copy.
Plasmodium knowlesi malaria, they wrote, is not a new emergent infection of humans. It has been getting into people for some while but it was overlooked. Three kinds of Asian primate serve as its reservoir hosts: the long-tailed macaque, the pig-tailed macaque, and the banded leaf monkey. Other monkeys, still unidentified, might be harboring the parasite too. Transmission from monkey to monkey (and from monkey to human) occurs by way of mosquitoes belonging to one group of closely related species, Anopheles leucosphyrus and its cousins, including Anopheles latens in Borneo. Anopheles latens is a forest-dwelling mosquito accustomed to biting macaques, but it will bite humans too, if presented with the necessity and the opportunity. As humans have increasingly entered the Bornean forests—killing and displacing macaques, cutting timber, setting fires, creating massive oil-palm plantations and small family farm plots, presenting themselves as an alternative host—both the necessity and the opportunity have increased. (Borneo has been deforested at a high rate within recent decades, to the point that its forest coverage is now less than 50 percent; meanwhile the island’s human population has grown to about 16 million. Cox-Singh and Singh didn’t cite these facts but clearly had them in mind.) Given such circumstances, Cox-Singh and Singh wrote, “it is possible that we are setting the stage for a switch of host for P. knowlesi, similar to the one postulated for P. vivax.” A host switch, they meant, from macaques to humans.
They expressed the same concern to me. “Have we created this nice opening for knowlesi to come into?” It was Cox-Singh voicing the question. By “opening” she meant an ecological opportunity. “What’s a mosquito going to do? If we start taking so much of the habitat, will the mosquito adapt then to being in a less-forest environment?”
She let that thought trickle off, paused, and then started again. “I honestly believe we’re at a sort of critical point. And we should be watching. We should be watching the situation very, very carefully,” she said. “And hopefully nothing will happen.” But of course, as she well knew, something always does happen. It’s just a question of what and when.
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Months and years after my conversation with Balbir Singh and Janet Cox-Singh, I was still wondering about Plasmodium knowlesi. I remembered a curious point the two scientists had made: that, unlike other malaria parasites, P. knowlesi is capable of reproducing in several kinds of primate. Its tastes in warm-blooded hosts are eclectic. It infects long-tailed macaques and pig-tailed macaques and banded leaf monkeys without distressing them much. It infects humans, sometimes, causing malaria that can be severe. It infects rhesus macaques—as laboratory experiments have shown—killing them quickly and surely. Further experimental work has revealed that it can infect a wide range of primates, including marmosets from South America, African baboons, and other kinds of Asian macaque. So with regard to hosts for the asexual phase of its life cycle—the sporozoite-to-gametocyte phase, occurring in mammalian blood and livers—it is a generalist. Generalists tend to do well in changing ecological circumstances.
I remembered also a vivid illustration from their overview article. It was a sketched map of the region, showing India, Southeast Asia, and the island realm of which Bor
neo sits at the center. The map showed, at a glance, how widely Anopheles leucosphyrus mosquitoes and long-tailed macaques are distributed. A solid line demarcated the native range of the mosquitoes, encircling southwestern India and Sri Lanka in a small loop, separate to themselves, and then a much larger, irregular loop sprawling over the map like a monstrous continental amoeba. The larger loop encompassed Bhutan and Myanmar and half of Bangladesh; the northeastern Indian states, including Assam; southern China, including Yunnan and Hainan and Taiwan; Thailand and Cambodia and Vietnam and Laos; all of Malaysia, all of the Philippines; and most of Indonesia, stretching eastward beyond Bali and Sulawesi. The area within that line, by my rough calculations, contains about 818 million people—that is, roughly one-eighth of the world’s human population, living within the greater ambit of Anopheles leucosphyrus mosquitoes. The distributional range of the long-tailed macaque was also traced on the map: a line of dashes, encircling almost the same area as the mosquitoes’ range, though not quite so large.
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