The Presence of Nipah Virus in Respiratory Secretions and Urine of Patients during an Outbreak of Nipah Virus Encephalitis in Malaysia

Journal of Infection (2001) 42, 40–43 doi:10.1053/jinf.2000.0782, available online at http://www.idealibrary.com on

The Presence of Nipah Virus in Respiratory Secretions and Urine of Patients during an Outbreak of Nipah Virus Encephalitis in Malaysia K. B. Chua*1, S. K. Lam1, K. J. Goh2, P. S. Hooi1, T. G. Ksiazek3, A. Kamarulzaman2, J. Olson3 and C. T. Tan2 1

Department of Medical Microbiology, 2Department of Medicine, Faculty of Medicine, University of Malaya, Kuala Lumpur; and 3Division of Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, Atlanta, U.S.A. Objectives: To study the excretion of Nipah virus in the upper respiratory secretions and urine of infected patients in relation to other clinical features. Methods: Isolation of Nipah virus from the respiratory secretions and urine was made in Vero cells and identified by indirect immunofluorescence assay using anti-Hendra specific hyperimmune mouse ascitic fluid and FITC-conjugated goat anti-mouse IgG. Results: During the peak outbreak of Nipah virus encephalitis in Malaysia, Nipah virus was isolated from the upper respiratory secretions and urine in eight of 20 patients who were virologically and/or serologically confirmed to be infected with the virus. From these eight patients, Nipah virus was isolated from six throat swab specimens, three urine specimens and only one nasal swab specimen. The positive virus isolation rate was related to the collection of these specimens during the early phase of the illness (P:0.068). The presence of serum anti-Nipah specific IgM appeared to reduce the chance of isolating the virus (P:0.049). There was no significant difference in the isolation rate with respect to the age, gender, ethnic group and clinical features associated with grave prognosis and mortality outcome of the patients. Conclusion: This study shows that it is possible to be infected from secretions of infected patients, but epidemiological survey on close contacts so far did not suggest that human-to-human transmission is common. © 2001 The British Infection Society

Introduction An outbreak of severe febrile encephalitis associated with fatal human cases was reported in peninsular Malaysia beginning in late September 1998.1–4 The outbreak was first recognized in the suburb of Ipoh, a city in the state of Perak, and was initially attributed to Japanese encephalitis (JE). Subsequently, additional foci of pig and human infections were recognized in late December 1998 and February 1999 in the state of Negeri Sembilan (Sikamat and Bukit Pelandok), and by the month of April and May spread to the state of Selangor (Sepang and Sungei Buloh). Infection at these foci appeared to be associated with the movement of a large number of infected pigs from Ipoh

* Please address all correspondence to: K. B. Chua, Department of Medical Microbiology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia. Accepted for publication 28 November 2000. 0163-4453/01/010040;04 $35.00/0

southwards into the states of Negeri Sembilan and Selangor. As the outbreak progressed, some of the epidemiological characteristics of the disease were recognized as being distinct from JE. Finally, in early March 1999, culture of cerebrospinal fluids from three patients who died in Negeri Sembilan resulted in the successful isolation of a virus with characteristics that were unlike JE virus. This virus, presently known as Nipah virus (named after the village of the victim from which the virus was first isolated), has been characterized as a new member of the family Paramyxoviridae, sub-family Paramyxovirinae, with closest relationship to the Hendra virus, another recently discovered paramyxovirus in Australia.5–10 Epidemiological features of this outbreak indicate that infection in man is due to close contact with live infected pigs (unpublished data). There was a strong suspicion that infected patients may excrete the virus in their respiratory secretions and urine. Hence, this study was carried out to determine whether there was any excretion of live Nipah © 2001 The British Infection Society

Nipah Virus Encephalitis Outbreak in Malaysia virus in the secretions of humans infected with the virus, and whether there was any relationship of virus shedding in relation to the duration and severity of disease.

Materials and Methods All febrile patients with a history of contact with live pigs from farms of known outbreak of febrile encephalitis admitted to University Hospital, Kuala Lumpur were taken as cases for study. Only those cases which were subsequently virologically and/or serologically confirmed to have Nipah virus infection were included in the analysis. As soon as the patient was admitted, nasal and throat swabs were taken and placed in 3 ml of viral transport medium prior to starting Ribavirin. At the same time, a sample of patient's urine was collected in a sterile container. These samples were transported in wet ice to the virus diagnostic laboratory within 6 h of collection. The nasal and throat secretions were treated with penicillin (200 IU/ml), streptomycin (200 ␮g/ml) and amphotericin B (100 ␮g/ml), while the pH of urine samples was adjusted with 1 N sodium hydroxide to pH 7.4. The samples were subsequently inoculated into a 24-well culture plate containing 105 Vero (ATCC, CCL-81) cells in 1 ml of Eagle's minimal essential growth medium containing 10% fetal calf serum in each well. Each sample was inoculated into the wells at doubling incremental volume, starting at 10 ␮l up to the maximum of 320 ␮l. The culture plate was carefully sealed and incubated at 37 ⬚C. The culture was examined daily for the presence of characteristic syncytial cytopathic effect (CPE) with formation of multi-nucleated giant cells. The supernatant was stored at 980 ⬚C from cultures which showed CPE. The infected cells were resuspended using phosphate buffered saline (PBS), centrifuged 10 min at 800 g and the supernatant discarded. The pelleted cells were resuspended again and washed twice with PBS. After the final wash, the pelleted cells were resuspended and spotted onto Teflon coated slide. The slide was allowed to dry inside the biosafety cabinet under UV irradiation. The dried slide was then fixed in cold acetone for 10 min and stained by indirect immunofluorescence assay using anti-Hendra hyperimmune mouse ascitic fluid as primary antibody and goat antimouse IgG fluorescein isothiocynate conjugate as secondary antibody (DAKO A/s, Denmark). All cultures with negative CPE after 10 days of cultivation were passaged once and discarded if no syncytial CPE was noted. The method for the serological diagnosis of Nipah virus infection was adapted from the original ELISA method developed by the Division of Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, Atlanta, USA, for the serological testing of Ebola virus. The detection of

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a patient's specific anti-Nipah IgM in the serum or cerebrospinal fluid was carried out by IgM-capture ELISA using Hendra virus-infected Vero E6 cells slurry (after treatment with cobalt irradiation) as antigen. The Chi-squared test and Student’s t-test were used for the statistical testing of significant association of the presence of any particular clinical features in relation to isolation rate of Nipah virus from the upper respiratory secretions and urine of infected patients.

Results At the peak of the outbreak of Nipah virus encephalitis, upper respiratory secretions (nasal and throat) and urine samples were collected from 26 febrile patients admitted to University Hospital, Kuala Lumpur, suspected to be infected with the virus from pig farms situated in Bukit Pelandok, Negeri Sembilan, Malaysia. Of these 26 patients, only 20 were virologically and/or serologically confirmed to be acutely infected with the virus. All the infected patients were adults (ages ranged from 14 to 57 years) who were either pig-farm owners tending to their own pigs or pig-farm workers who had direct close contact with live pigs. There were five females and 15 males, of whom 17 were Chinese and three Indian (Table I). None of the patients had obvious nasal discharge or other respiratory symptoms such as cough, sneezing or sore throat. Nipah virus was isolated from six throat swab specimens, three urine samples and only one nasal swab sample of eight patients. One patient (No. 18, Table I) had positive isolation in all three types of samples collected. All isolates were obtained at initial inoculation without the need for passage. Further passage did not increase positive isolation. All positive isolation gave syncytial CPE in Vero cells within 5–10 days post-inoculation. Most of the positive samples had infectious Nipah virus particles in 80 ␮l of the inoculum. However, there was one urine sample which contained infectious virus even at 10 ␮l of inoculum (Table I). There was no significant difference in the virus isolation rate with respect to age (Z:0.0000, P:1), sex (Chi-squared:0.000, df:1, P:1) and ethnic group (Chi-squared:1.027, df:1, P:0.311). However, there was an apparent positive correlation in the virus isolation rate with respect to the samples collected at the early phase of the illness (Chi-squared:3.32, df:1, P:0.068). It appeared that by the seventh day of illness no more infectious virus could be cultivated from the secretions of the infected patients, though the sample size of this study was relatively small (Table I). The presence of serum anti-Nipah virus specific IgM appeared to reduce the isolation rate (Fisher exact, Chi-squared:2.76,

K. B. Chua et al.

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Table I. Clinical profiles and virus isolation from the patients infected with Nipah virus. No.

I.D.

Age

Sex

Race

IgM;

D.I.

01 02 03 04 05 06 07 08 09 10 11 12 13 14 15 16 17 18 19 20

8-F03844 3-F03077 6-F04543 5-F03319 0-F03705 6-F03909 0-F03695 0-F03907 0-F03146 0-F03834 0-F03664 0-F03865 4-F03927 3-F05332 2-F03833 4-813298 9-F03267 6-459555 0-E78586 3-F03130

49 47 20 29 33 57 35 43 52 35 14 22 48 28 55 56 37 48 25 22

M M M M M M M M M F M F M M F M F F M M

C C C C C C C C C C C I C I C C C I C C

Y Y Y Y Y N N Y Y Y Y N Y Y Y Y Y Y Y Y

4 5 2 4 4 3 5 3 4 2 4 1 5 7 7 2 7 2 5 2

T/S

N/S

Urine ;(160 ␮l)

;(160 ␮l)

;(10 ␮l)

;(80 ␮l) ;(80 ␮l)

;(80 ␮l)

;(20 ␮l) ;(40 ␮l) ;(80 ␮l)

;(40 ␮l)

;(80 ␮l) ;(80 ␮l)

JE Vaccine N Y N Y Y Y N N Y Y Y N N N N Y Y Y N Y

I.D.:Identification number; M:male, F:female; C:Chinese, I:Indian; Y:yes, N =no; ;:positive virus isolation, bracket indicates minimal volume of inoculum with isolates; D.I.:days of illness prior to collection of specimens or admission to hospital; T/S:throat swab; N/S:nasal swab.

P :0.049). Recent history of JE vaccination did not influence the virus isolation (Chi-squared:1.02, P:0.196). The clinical features associated with poor prognosis and survival outcome of the 20 patients in relation to the excretion of the virus are shown in Table II. The virus isolation rate was not affected by the presence of various clinical features which were related to bad prognostic outcome of the disease (submitted for publication) such as headache (Chi-squared:0.01, P:0.669), drowsiness at admission (Chi-squared:0.43, P:0.514), lapsed into coma (Chi-squared:0.035, df:1, P:0.852), needed ventilatory support (Chi-squared:0.305, df:1, P:0581), developed segmental myoclonus during the course of the illness (Chi-squared:0.135, df:1, P:0714), presence of hypertension (Chisquared:0.000, df:1, P:1) and tachycardia (Chisquared:0.037, df:1, P:0.848). Neither was the eventual survival of the patients influenced by the excretion of virus in the secretions or urine (Chisquared:0.354, df:1, P:0.552).

Discussion This study confirmed our early clinical suspicion that patients infected with Nipah virus excreted the virus in their respiratory secretions and urine. The excretion rate decreased towards the late phase of the illness, which corresponded to the presence of anti-Nipah virus specific IgM. This finding is in concordance with the pattern of virus shedding in most viral infections.

Unpublished data from the Australia Animal Health Laboratory, Geelong, showed that Nipah virus was found in the respiratory secretions and urine of infected pigs, and that this was the most likely source of transmission among animals housed in close quarters. The result of this study also showed that Nipah virus can be excreted in respiratory secretions and urine of infected patients, and possibly can act as a source of spread to healthcare workers as well as to relatives of patients who helped to look after them. To prevent hospital-acquired infection, it is especially important to nursing personnel and doctors who care for these patients in an intensive care setting where the patients are often intubated and catheterized. In most hospitals in developing countries patients share communal toilets and bathrooms, and ambulatory Nipah virus-infected patients can contaminate the surroundings with infected urine. Although this finding is of interest, epidemiological surveys among patient contact has not indicated that humanto-human transmission is common. This could be due to good clinical care and the practice of standard universal precaution among healthcare workers in the management of infected patients. Another reason for the lack of human-to-human transmission could be due to the lower virus load in human respiratory secretions and urine compared to that in pigs, although this is speculative. The low transmission of Nipah virus is also seen in Hendra virus infection in Australia, where a serological survey among workers in an infected horse stable and contacts of patients revealed that they had not been infected.11,12

Nipah Virus Encephalitis Outbreak in Malaysia

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Table II. Clinical features associated with poor prognosis and mortality outcome of the patients infected with Nipah virus. No.

I.D.

Headache

Admission

Coma

Ventilated

Myoclonus

Hypertension

Tachycardia

Outcome

01 *2 03 04 05 *6 *7 08 09 10 *11 *12 13 14 15 *16 17 *18 19 *20

8-F03844 3-F03077 6-F04543 5-F03319 0-F03705 6-F03909 0-F03695 0-F03907 0-F03146 0-F03834 0-F03664 0-F03865 4-F03927 3-F05332 2-F03833 4-813298 9-F03267 6-459555 0-E78586 3-F03130

N N N N N Y Y N N Y Y Y Y Y Y N Y Y Y N

DROWSY DROWSY NORMAL DROWSY NORMAL NORMAL NORMAL NORMAL NORMAL DROWSY DROWSY NORMAL NORMAL NORMAL NORMAL NORMAL DROWSY NORMAL DROWSY DROWSY

Y Y N Y Y Y Y Y Y Y N N N N N N N Y Y Y

Y Y N Y N Y Y Y Y Y N N N N N N N Y Y Y

Y N N Y N Y Y Y Y N N N N N N N N Y Y Y

Y N N Y N N Y Y Y N N N N N Y Y N Y Y Y

Y N N N N Y Y Y Y N N N N N N N N N Y Y

Alive Alive Alive Alive Alive Dead Dead Dead Dead Alive Alive Alive Alive Alive Alive Alive Alive Alive Dead Dead

*Cases where virus was isolated. I.D.:Identification number; Y:presence of particular clinical feature; N:absence of specific clinical feature.

It is interesting to compare this zoonotic infection with that of Japanese encephalitis, where pigs also act as an amplifying host, and man and many other animals are considered as the dead end host. Perhaps this same situation occurs with Nipah virus, and warrants study. Acknowledgment This work was supported in part by the Malaysian government research grant (IRPA 06-02-03-0307) on emerging diseases.

References 1 CDC, Atlanta. Outbreak of Hendra-like virus – Malaysia and Singapore, 1998–1999. MMWR 199; 48: 265–269. 2 CDC, Atlanta. Outbreak of Nipah virus – Malaysia and Singapore, 1999. MMWR 1999; 48: 335–337. 3 CDC, Atlanta. Outbreak of Hendra-like virus – Malaysia and Singapore, 1998–1999. Report from Center for Disease Control and Prevention, Atlanta. JAMA 1999; 281: 1787–1788.

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