A New Disease in Singapore
One afternoon in March 1999 during the third month of my infectious diseases fellowship program in a public hospital in Singapore, an emergency room doctor who sounded very alarmed beeped me. He had just seen two consecutive patients with fever and confusion and was admitting a third, all of whom were abattoir workers. One of them needed to be intubated. The same afternoon saw another two workers from the same abattoir with similar symptoms. Later it was learned that a further six abattoir workers had presented to other hospitals the same week, one of whom died after a rapid neurological deterioration. So what was this outbreak of encephalitis involving abattoir workers?
Fever and altered mental status were the most common findings in all 11 patients . Seven of them had various neurological signs involving the lower motor neuron, brainstem, and cerebellum. Cerebrospinal fluid analysis revealed predominantly lymphocytic meningitis with negative microscopy and culture, complement-fixation tests, and serological tests for bacterial and viral pathogens. There were curious findings on neuroimaging. Even more intriguingly, at least two patients with neurological signs presented concomitantly with atypical pneumoniae . Of note, nine of the patients had recently been vaccinated against Japanese Encephalitis (JE).
Singapore had no pig farms and its two abattoirs slaughtered imported pigs from Malaysia. At that time, Malaysia was experiencing an epidemic of presumed JE among pig farmers. A similar scenario was suspected among local abattoir employees despite a ban on pig imports from affected Malaysian states in early March 1999. However, concerns were mounting that the epidemiology and clinical features of the outbreak were not consistent with JE.
Speculation of a new mystery illness was rife. Many others and I were caught in the middle of an outbreak of a new and deadly disease whose mode of transmission was unknown. Answers were feverishly sought while infection control and quarantine issues were tackled. Parallel events were unfolding in neighboring Malaysia, which culminated in the discovery of a new virus.
The Outbreak in Malaysia
The epidemic began in late September 1998 near the city of Ipoh, in the northern state of Perak in Malaysia (Figure 1). Health authorities were quick to implicate JE, which was endemic in the region especially at that time of the year. Also, antibodies against JE were found in the blood and cerebrospinal fluid of some victims . Despite mass vaccination and the fogging of thousands of pig farms and nearby residences, the outbreak spread to the southern Malaysian State of Negeri Sembilan (Figure 1). From there a batch of pigs was brought to Singapore before the ban in early March 1999 of imports from this Malaysian State (Figure 1).
While children were the usual victims of JE, this epidemic involved adults. Family members of victims who did not have close contact with pigs remained unscathed, unlike a mosquito-borne outbreak. Moreover, pigs serve as amplifying hosts for JE and it did not make sense that they were also were falling sick. Some pigs developed cough and hemoptysis, while others had neurologic features like muscle spasms, tremors, myoclonus, and gait ataxia. Vaccination against JE did not protect some pig farmers from developing this disease. By May 1999 the Malaysia Ministry of Health in association with the Centers for Disease Control (CDC) reported a total of 258 cases of encephalitis in adults with a case fatality rate of almost 40% .
A New Virus
A breakthrough occurred after the expertise of Dr Lam Sai Kit was solicited. He and his team of microbiologists at the University of Malaya in the Malaysian capital Kuala Lumpur isolated a paramyxovirus (a family that JE did not belong to) 5 days after obtaining the first patient samples . The discovery in recent years of two new paramyxoviridae in Australia raised the possibility of a related virus. The Menangle virus was isolated from stillborn piglets with deformities at a large commercial piggery in New South Wales in 1997 . Infection also resulted in a flu-like illness with a rash in at least two human workers. The other new paramyxovirus, Hendra, was discovered in Queensland, Australia in 1994 during an outbreak of a fatal respiratory disease among horses and humans . Humans became infected through the body fluids and excretions of horses. The bat was the common reservoir for both Menangle and Hendra viruses.
The Malaysian virus was brought to the CDC in the United States, where it reacted with antibodies to the Hendra virus. By sequencing the viral genome, the CDC showed the new virus to be about 20% different from the Hendra virus . Phylogenetic studies showed that Hendra and this new virus were closely linked and distinct from other genera within the paramyoviridae .
In April 1999, the Malaysian virus was named Nipah after a river in the town where the first victim lived. Subsequently, it became clear that there were several distinguishing features between JE and the illness caused by this new deadly virus (Table 1). The Nipah virus was found in the tissues of humans and affected pigs as well as all eleven abattoir workers in Singapore. An IgM capture enzyme linked immunosorbent assay (ELISA) was developed by the CDC using the prototype Hendra virus antigen. Subsequently, an indirect IgG capture ELISA employing the Nipah antigen was also made available.
Control of the Outbreak
The outbreak in Singapore was halted quickly. With the confirmation of a new virus, Singapore imposed a blanket ban on the import of all Malaysian pigs, horses, dogs, cats, and other mammals. The two abattoirs in the country were closed and thoroughly disinfected . It was apparent that transmission involved close contact with pigs. Therefore, all abattoir workers and their contacts were clinically and serologically screened. Three symptomatic and eight asymptomatic cases were diagnosed .
In Malaysia, the government ordered mass culling of more than a million pigs in the outbreak areas, resulting in successful control of the epidemic. The World Health organization (WHO) declared the outbreak over in May 1999.
Autopsy studies revealed the central pathology of this disease to be vasculitis-induced thrombosis causing ischemia and infarction. Multi-organ necrotizing vasculitis and syncytial formation were characteristic [1,10]. This was especially so in the central nervous system where a diffuse small blood vessel vasculitis and lytic necrosis were evident in the cerebral cortex and brainstem associated with infection of endothelial cells.
Malaysian authorities  studied 94 patients with Nipah virus infection, the majority (93%) of whom had direct contact with pigs 2 weeks before the onset of illness. This implied a short incubation period. Fever, headache, dizziness, and vomiting featured prominently. Reduced levels of consciousness and brainstem dysfunction were present in 52 patients (55%). Segmental myoclonus, areflexia, hypotonia, hypertension, and tachycardia were other features. Antibodies against Nipah virus were detected in serum or cerebrospinal fluid (CSF) in 76% of 83 patients tested. Severe brain-stem involvement resulted in the deaths of 30 patients (32%). Fifty patients (53%) achieved full recovery, while neurological deficits persisted in 14 (15%). Three patients relapsed neurologically after initial mild disease, a finding that is unique to Nipah encephalitis. Apart from the CSF lymphocytic predominance, other laboratory markers were not specific. These included abnormalities like moderate thrombocytopenia and elevation of liver transaminases.
The cases in Singapore manifested similarly except for the finding of abnormal chest radiographs in 8 of the 11 patients. In two of these individuals there was evidence of an atypical pneumonia. Serological tests for mycoplasma, chlamydia, Q fever, and legionella were negative on acute and convalescent sera . Curiously, one of these patients with atypical pneumonia developed a transient episode of visual hallucinations during which he described seeing pigs flying outside his hospital room! One of the three symptomatic patients discovered during the screening of abattoir workers had mild neurological deficit. It turned out that he had been hospitalized for an atypical pneumonia coincidentally during the outbreak (Figure 2). Thus, this disease may present initially as pneumonia without any neurological feature.
Given the long latency of infection in the Hendra virus infection, it was initially feared that new cases might develop long after the control of the outbreak. Fortunately, this has generally been shown to be untrue.
Most of the computed tomography (CT) scans of the brain did not reveal any abnormality. However, MRI abnormalities (Figures 3 and 4) were unique, often showing multiple, small (less than 5 mm), asymmetric focal lesions in the subcortical and deep white matter without surrounding edema [1,11–13]. These were in stark contrast to the bilateral thalamic and basal ganglia involvement in neuroimaging of JE. Even seropositive patients who were asymptomatic had white matter changes on MRI of the brain . Pathology studies indicated that underlying microinfarction might be responsible for these lesions.
As there was no effective therapy, supportive care was pivotal during the outbreak and included intensive care and mechanical ventilation. A recently published open-labeled controlled study done during the outbreak reported a 36% reduction in mortality (p = 0.011) with the use of ribavirin . This study used decremental doses of oral or intravenous ribavirin (beginning with 2 g orally or 30 mg/kg iv) over a week to 10 days.
Eating pork did not transmit the infection and all of the reported cases involved pig farmers, handlers, or abattoir workers. In a case control study of risk factors for the acquisition of disease , infected persons were more likely than community-farm controls to report increased numbers of sick or dying pigs on the farm. The former were also more likely than case-farm controls to perform activities requiring direct contact with pigs. Similar results were obtained in a study involving abattoir workers in Singapore . Army personnel who were involved in culling pigs in outbreak-affected states did not appear to be at significant risk. In a cross-sectional survey of 1,412 military personnel involved in culling activities , only six had detectable antibody to the Nipah virus, all of whom had reported direct contact with live pigs.
How did pigs transmit the virus to humans? Infected pigs appeared to have respiratory symptoms and their autopsies demonstrated prominent respiratory tract involvement. Pigs were later found to shed the virus in nasopharyngeal secretions , thereby infecting humans. The abnormal chest radiographs in some humans lend weight to the respiratory route of transmission. A recent study  isolated the virus from the upper respiratory secretions and urine in 8 of 20 infected patients. Despite this, there is no evidence of human-to-human transmission. Moreover, risks to health care workers appear to be negligible. Disease did not develop in a study of 338 exposed health care workers . Despite the negligible risks of nosocomial transmission, the authors advised that standard and droplet infection control practices be maintained in view of the high case-fatality rate of the disease.
The isolation of the virus in respiratory secretions and urine was not associated with poor prognosis . Conversely, there was a significant co-relation between CSF isolation and mortality and clinical features that suggested a poor prognosis . The authors postulated greater viral replication in the central nervous system, resulting in the high mortality.
Tracing the Source
Apart from humans and swine, the Nipah virus showed an extended host range with infections in cats, dogs, horses, and bats. Taking a cue from the Hendra outbreak where bats were found to be the natural hosts, bat colonies were studied in Malaysia . Antibodies that neutralize Nipah virus were found in 21 bats from five species (four species of fruit bat, including two flying fox species and one insectivorous species). However, isolation of the virus in culture and detection of RNA was unsuccessful.
A major breakthrough was achieved by Dr Lam and his team who studied a large bat colony on Tioman island, off the eastern coast of West Malaysia . After collecting over 1,000 bat urine samples, the virus was detected in the urine of a species of flying fox. The virus was also found in a piece of fruit that had been partially eaten by a bat. This implied the presence of the virus in the animal’s saliva. Because many Malaysian pig farms have fruit trees, it is easy enough to imagine how the virus can be transmitted from bats. Pigs probably serve as amplifying hosts after ingesting infected bat urine, saliva, or discarded food items. Dr Lam suggested that pig farmers stop growing or maintaining fruit trees in order to prevent a future outbreak .
During the search for the natural host of the nipah virus, there was an incidental discovery of yet another new paramyxoviridae from the urine of the flying fox bat . The Tioman virus is related phylogenetically to the Menangle virus and has not been associated with any clinical disease.
The cause for the Nipah virus epidemic remains elusive but its story underscores the mysterious and tremendous capacity of unknown viruses in wildlife to cross species and become deadly human pathogens. The discovery of the Tioman virus is just the tip of the iceberg in the search for unknown viruses in wildlife. It is imperative to continue this proactive search to help prevent a future calamity.
The author thanks Dr. Leo Yee Sin for providing the chest radiograph and neuroimaging figures.
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