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Risk of Influenza A (H5N1) Infection among Poultry Workers, Hong Kong, 1997– 1998 Carolyn Buxton Bridges,1,3,a Wilina Lim,4 Jean Hu-Primmer,1 Les Sims,6 Keiji Fukuda,1 K. H. Mak,5 Thomas Rowe,1 William W. Thompson,1,a Laura Conn,2 Xiuhua Lu,1 Nancy J. Cox,1 and Jacqueline M. Katz1
1
Influenza Branch, Division of Viral and Rickettsial Diseases, 2Office of the Director, National Center for Infectious Diseases, and 3Epidemic Intelligence Service, Epidemiology Program Office, Centers for Disease Control and Prevention, Atlanta, Georgia; 4 Government Virus Unit, Queen Mary Hospital, 5Department of Health, and 6Agriculture and Fisheries Department, Hong Kong Special Administrative Region, People’s Republic of China
In 1997, outbreaks of highly pathogenic influenza A (H5N1) among poultry coincided with 18 documented human cases of H5N1 illness. Although exposure to live poultry was associated with human illness, no cases were documented among poultry workers (PWs). To evaluate the potential for avian-to-human transmission of H5N1, a cohort study was conducted among 293 Hong Kong government workers (GWs) who participated in a poultry culling operation and among 1525 PWs. Paired serum samples collected from GWs and single serum samples collected from PWs were considered to be anti– H5 antibody positive if they were positive by both microneutralization and Western blot testing. Among GWs, 3% were seropositive, and 1 seroconversion was documented. Among PWs, 10% had anti– H5 antibody. More-intensive poultry exposure, such as butchering and exposure to ill poultry, was associated with having anti– H5 antibody. These findings suggest an increased risk for avian influenza infection from occupational exposure.
In 1997, 18 human cases of avian influenza A (H5N1) illness were documented among persons living in Hong Kong; all the case patients were hospitalized, and 6 died [1]. The outbreak occurred coincident with outbreaks of highly pathogenic avian influenza (HPAI) H5N1 among poultry on 3 Hong Kong farms from March through May (1 human case) and among poultry in wholesale and retail markets from November through December (17 human cases). This was the first time that an avian influenza A virus of any subtype was documented to cause respiratory infections in humans. The genomes of the avian H5N1 viruses that were isolated from chickens and humans in Hong Kong in 1997 were closely related, on the basis of gene sequence data, and all possessed a multibasic Received 17 October 2001; revised 17 December 2001; electronically published 19 March 2002. Presented in part: Options for the Control of Influenza IV, Hersonissos, Crete, Greece, September 2000 (abstract P2-88). Verbal informed consent was obtained from study participants. Human experimentation guidelines of the US Department of Health and Human Services and those of the Hong Kong Special Administration Region Department of Health were followed in the conduct of the clinical research. a Present affiliations: Influenza Branch, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases (C.B.B.), and Vaccine Safety and Development Activity, Division of Epidemiology and Surveillance, National Immunization Program (W.W.T.), Centers for Disease Control and Prevention, Atlanta, Georgia. Reprints or correspondence: Dr. Carolyn Buxton Bridges, Influenza Branch, Div. of Viral and Rickettsial Diseases, National Center for Infectious Diseases, MS A-32, Centers for Disease Control and Prevention, 1600 Clifton Rd., Atlanta, GA 30333 (
[email protected]). The Journal of Infectious Diseases 2002;185:1005–10 q 2002 by the Infectious Diseases Society of America. All rights reserved. 0022-1899/2002/18508-0002$02.00
amino acid motif in the hemagglutinin (HA) gene at the cleavage site between HA1 and HA2, a characteristic of HPAI [2, 3]. A case-control study conducted in January 1998 implicated exposure to poultry in retail markets as the primary risk factor for human H5N1 illness [4]. Taken together, the molecular and epidemiologic data suggested that the human H5N1 infections were the result of multiple, independent poultry-to-human transmissions of H5N1 viruses [2, 4]. Because of the potential for further poultry-to-human spread of H5N1 viruses in the poultry markets, the Hong Kong government enlisted government employees from several agencies to assist in the Hong Kong– wide slaughter of chickens and other fowl. This 4-day effort, beginning on 29 December, resulted in the slaughter of 1.5 million chickens and several hundred thousand other domestic fowl. No additional human H5N1 cases were detected after the poultry slaughter [1]. Surveillance in the Hong Kong live bird markets conducted just prior to the depopulation of poultry indicated that 20% of chickens tested from poultry markets were infected with H5N1 viruses [5]. However, no H5N1 case patients were detected among poultry workers (PWs), a group expected to have the highest level of exposure to H5N1-infected birds. To better understand the potential for H5N1 viruses to spread to humans, we evaluated the rates of and risk factors for H5N1 infection among persons exposed to poultry by conducting a retrospective cohort study among PWs and among government workers (GWs) involved in the poultry slaughter. Materials and Methods Enrollment. GWs involved in the poultry slaughter and PWs were invited to visit any 1 of 14 Hong Kong Government outpatient
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clinics and to participate in a study of H5N1 infection among persons exposed to poultry. Participating GWs and PWs were enrolled in the study from 29 December 1997 through 15 January 1998. All participants were asked to complete a self-administered questionnaire written initially in English and translated into Chinese. Participants were asked about their age, sex, the type of work that they did with poultry, exposure to persons with H5N1 illness (the “bird flu”), respiratory illnesses experienced since 1 November 1997, and exposure to persons with known H5N1 illness. PWs also were asked whether they had observed .10% mortality among poultry with which they had worked since 1 November 1997. Blood collection. Because the timing of PW exposure to poultry potentially infected with H5N1 could not be identified accurately, PWs were asked to donate a single serum sample. GWs, whose period of concentrated poultry exposure was defined by the dates of the culling operation, were asked to provide paired serum samples, with the first sample collected 0– 7 days after the end of the culling operations and the second sample collected 2 weeks after the first sample. Serum was separated from blood samples and stored at 2 20 C until tested. Antibody testing. Although the hemagglutination inhibition (HI) assay is commonly used to detect influenza antibody in human serum, the HI test lacks sensitivity for the detection of antibodies to the H5N1 virus in human serum [6]. Therefore, a microneutralization assay followed by confirmation with a Western blot assay was used to detect H5-specific antibody in the present study. The sensitivity and specificity of these assays for the detection of anti– H5 HA antibody (among patients >15 years old) have been described elsewhere [6]. Serum samples were tested by the microneutralization assay by use of a nonpathogenic avian H5N3 virus A/Duck/Singapore/-Q/ F119-3/97 (Duck/Singapore) at the Government Virus Unit, Hong Kong Department of Health, Hong Kong. Serum samples were considered to be positive by microneutralization assay if anti-H5 titers of > 80 were obtained. A confirmatory Western blot assay was performed at Centers for Disease Control and Prevention (CDC) on serum samples that were positive by the microneutralization assay [6]. This assay used a highly purified baculovirus-expressed HA protein from A/Hong Kong/ 156/97 virus (kindly provided by Bethanie Wilkinson, Protein Sciences, Meriden, CT) to detect IgG antibody in serum diluted 1:100. Serum samples that tested positive by both microneutralization assay and Western blot were considered to be positive for anti– H5 antibody [6]. Statistical analysis. Because of limited resources, not all PW serum samples positive by microneutralization assay could be tested by Western blot at CDC. Thus, a random sample of microneutraliza-
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tion-positive serum was selected for testing by Western blot from the following age groups: 15–29, 30–44, and 45–59 years. To assess risk factors for H5 infection, we conducted a nested unmatched casecontrol analysis. Case patients included all PWs who tested positive by both microneutralization and Western blot testing. Control subjects included all PWs who tested negative by microneutralization assay plus those who tested positive by microneutralization assay but negative by Western blot. Not included in the nested case-controls were PWs who tested positive by microneutralization but whose serum samples were not tested by Western blot. We estimated the seroprevalence of H5 among PWs on the basis of the percentage of PWs who tested positive by microneutralization and the percentage of those expected to test positive by Western blot, weighted by age group. For GWs, all microneutralization-positive serum samples were tested by Western blot, and results for this population were analyzed on the basis of a cohort study design. Data were analyzed by use of SAS for Windows (version 8.01; SAS Institute). Persons >60 or < 14 years old and those who did not report their age were excluded from the analysis because the microneutralization and Western blot assays were found to be less specific for persons > 60 and 1 serum sample. The median age was 41 years (range, 22– 58 years), 85% were male, and 22.5% smoked cigarettes. Overall, 78% (229/293) had paired serum samples. Among 8 persons with paired serum samples, both specimens were collected .14 days after the culling operation. Fifty-two (81%) of the 64 persons with a single serum sample had the blood collected ,14 days after the culling operation. Among the 293 GWs, 9 (3%) were both microneutralizationand Western blot–positive on >1 serum samples (table 3). The proportions of seropositive GWs was 0% (0/30) among 22– 29year-olds, 4% (6/166) among 30–44-year-olds, and 3% (3/97) among 45–58-year-olds. Of the 229 GWs that had paired serum samples, 1 person seroconverted. This person did report having a respiratory illness with onset on 27 December; however, no viral cultures were collected. Being a current smoker was associated with having any blood specimen positive for H5 (5/66 vs. 4/ 223; P ¼ :03, Fisher’s exact test). No other risk factors for having anti–H5 antibody were identified.
1145 849 754 172 830 865 695 330
(93.0) (69.0) (61.3) (14.0) (67.4) (70.3) (56.5) (26.8)
Odds ratio (95% confidence interval)
5.8 3.1 2.4 1.2 1.6 1.7 1.6 1.7
(0.9–113.6) (1.6–5.9) (1.4–4.1) (0.6–2.2) (0.9–2.7) (0.9–2.9) (1.0–2.5) (1.1–2.7)
Discussion The results of this study demonstrate that individuals with occupational exposure to domestic poultry were at increased risk of infection with H5N1 virus. Although no H5N1-infected GWs or PWs were identified among the 18 hospitalized case patients, the H5 seroprevalence rates of 3% and 10%, respectively, suggest that a substantial number of mild or asymptomatic infections occurred in these occupationally exposed populations. Occupationally exposed persons may represent important populations for evaluating the risks of human infection with nonhuman influenza viruses. The observed H5 seroprevalence rate of 10% among PWs is high, compared with the other Hong Kong cohorts studied during 1997 by use of similar antibody testing methods. Seroprevalence rates among groups not exposed to infected human case patients and with presumed low levels of poultry exposure were
Figure 1. Percentage of poultry workers seropositive for H5 antibody, on the basis of the cumulative number of types of poultry-related tasks performed.
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Table 3.
Case 1 2 3 4 5 6 7 8 9 NOTE.
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Characteristics of government workers with any H5-positive serum sample.
Age
Sex
Current smoker
39 37 41 43 48 43 53 39 55
F M M M M M M M M
No No Yes Yes No Yes Yes No Yes
Febrile respiratory illness since 1 Nov 1997
Date of illness onset
Sample 1 date, result
Sample 2 date, result
Yes Yes Yes Yes No Yes No Yes No
27 Dec 1997 6 Jan 1998 5 Jan 1998 2 Jan 1998 NA 3 Jan 1998 NA 15 Nov 1997 NA
7 8 7 7 9 6 6 9 7
2 þ þ þ þ þ þ þ þ
21 Jan 1998, þ 22 Jan 1998, þ 21 Jan 1998, þ 21 Jan 1998, 2 23 Jan 1998, 2 20 Jan 1998, 2 20 Jan 1998, 2 Not available Not available
Jan Jan Jan Jan Jan Jan Jan Jan Jan
1999, 1998, 1998, 1998, 1998, 1998, 1998, 1998, 1998,
NA, not applicable; þ, positive; 2 , negative.
0% among adult blood donors [7] and 0.7% among health care workers [8]. These low background rates are in contrast to rates of 3.7% among health care workers who cared for H5N1-infected case patients [8] and 3.8% among persons who traveled with a symptomatic case patient [9], rates similar to that found among GWs. The seroprevalence rate among PWs was similar to the 12% rate found among household contacts of case patients, a group likely to have had similar environmental exposures as the case patients, including contact with infected poultry, in addition to case-patient exposure [9]. Although epidemiologic evidence of human-to-human transmission was found in the study of health care workers [8], the higher H5 seroprevalence rate among PWs, only 1 of whom, a seronegative person, reported knowing a case patient, and evidence from a case-control study [4] indicate that poultry-to-human transmission was the primary means of human infection. Although the initial outbreaks of HPAI among poultry in Hong Kong were first detected on 3 farms in the spring of 1997, having ever lived on a farm or working on a farm were not associated with having anti– H5 antibody. Although the numbers of PWs who reported these exposures were small, this evidence and the fact that work in retail was a risk factor support the suggestion that viral amplification was primarily occurring in the retail and wholesale markets and not on the farms [10]. GWs likely had a lower H5 seroprevalence rate than PWs because PWs had more-prolonged exposures to poultry and, possibly, because most of the GWs participated in depopulation of poultry on farms, not in retail markets, which were more likely to have H5N1-infected poultry (Les Sims, personal communication). Thus, GWs would have been less likely to be exposed to H5-infected birds. GWs also often wore protective clothing, such as gowns, masks, and gloves, when working directly with poultry during the culling operation. Using the single radial hemolysis (SRH) assay to detect antibody to different avian virus subtypes, Shortridge [11] reported human seroprevalence rates for antibody to H5 virus ranging from 0% in an urban Hong Kong population to 2%–7% in rural populations in southern China, where H5 viruses were isolated from 4% of domestic ducks tested. Because the SRH assay may
detect not only subtype-specific antibody to influenza surface glycoproteins but also subtype cross-reactive antibody to the internal nucleoprotein, these rates may have been an overestimate. Another serosurvey of farm families who raised ducks and pigs found no antibody against the HAs of avian viruses (H11N2, H4N4, H7N4, and H3N8) isolated from duck feces on these farms [12]. However, the use of the HI test to detect antibody to the avian viruses in that study may have compromised the sensitivity of detection. The HI test is less sensitive than the microneutralization assay in detecting human antibody against H5N1 viruses [6]. Lu et al. [13] reported a similar finding when attempting to detect antibodies in other mammalian species infected with an avian H2N2 virus. The H5N1 HPAI viruses isolated from both humans and chickens in Hong Kong caused rapidly progressive, fatal illness in experimentally infected chickens [2]. The HPAI H5N1 viruses replicate primarily in vascular endothelial cells, cardiac myocytes, and myeloid inflammatory cells. The pathophysiology of HPAI supports the results of our study, which found an association between having anti– H5 antibody and butchering and preparing poultry for restaurants, tasks that involve very close contact with poultry. A limitation of the PW study was that only a single serum sample was collected. Thus, the timing of infection with H5 virus cannot be known with certainty. It is possible that the anti– H5 antibody detected in at least some PWs may have been a result of prior infection with a related H5 virus. Virus surveillance conducted in retail markets immediately prior to the depopulation of poultry isolated H5N1 viruses from 20% of chickens tested and from 2.4% and 2.5% of ducks and geese, respectively [5]. It remains possible that prior subclinical or unrecognized infection of PWs with a related H5 virus may have actually protected them against subsequent infection or severe illness with the HPAI H5N1 virus during the 1997– 1998 outbreak. The examination of GWs provided a unique opportunity to investigate a population with brief but intense exposure to H5N1 virus–infected birds. Seroconversion was documented in 1 individual. Eight other GWs had first serum samples that tested positive for anti–H5 antibody. The first serum samples were col-
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Influenza A (H5N1) Antibody among Poultry Workers
lected 8–11 days after the first day of the poultry depopulation. Although this may appear to be early for a primary antibody response to a novel virus, seroconversion was detected as early as 7 days after symptom onset in 1 adult H5N1 case patient [9]. Nevertheless, seroconversion > 14 days after symptom onset was more typical. The second serum samples collected 14 days after the first samples were negative for 4 of the 8 GWs. In 1 case, the second sample’s neutralizing antibody titer was ,80, which suggests a weak and transient antibody response to infection. For the other 3 individuals, the microneutralization titers of the second samples were similar to those of the respective first samples, but the second serum samples tested negative by the confirmatory Western blot assay. The microneutralization assay potentially detected IgG, IgM, or IgA antibodies, whereas the Western blot detected only IgG. A subclinical viral infection may result in low levels of antigen expression and induce only a weak and transient IgG response that could not be detected by Western blot in the second serum sample collected 3– 4 weeks after virus exposure. The seroprevalence rate of 3% in the group overall may be an underestimate, because some GWs with a single blood sample did not donate a later convalescent specimen for testing. Smoking was found to be a risk factor for H5 antibody among GWs but not PWs. In other studies of young adults, smoking has been associated with increased rates of both seroconversion and clinical influenza illness [14, 15]. However, smoking appeared to increase the risk only among those without preexisting antibody titers. Thus, the same risk factor may not have been seen among PWs because of preexisting antibody from prior exposures to H5 avian viruses. The results of our study contrast with those of a limited serological survey conducted in individuals exposed to virus or infected poultry in the 1997 HPAI H5N2 outbreak among chickens in Italy [16]. When both a microneutralization assay and the SRH test were used, no anti– H5 antibodies were detected in serum samples from exposed people, and no human cases of H5N2 respiratory disease were identified. Likewise, no human infections were detected during outbreaks of HPAI H5N2 virus, which caused widespread morbidity and mortality in chickens in the northeastern United States during the early 1980s. Although virus was detected in nasal swab samples collected immediately after individuals involved in the depopulation of chickens left the infected chicken houses, no virus was isolated from swabs collected 12 h later, which suggests that humans were not susceptible to this HPAI H5N2 virus [17]. Whether the Hong Kong H5N1 viruses are unique among H5 HPAI viruses in their ability to infect humans remains unknown. It is possible that they possess a unique constellation of genes that permitted limited poultry-to-human transmission of the viruses. In support of this hypothesis, avian H9N2 viruses that possessed internal genes that were highly homologous to those of the H5N1 viruses were isolated from 2 children with respiratory disease in Hong Kong in 1999 [18].
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Surveillance among poultry in Hong Kong identified another outbreak of HPAI H5N1 among poultry during May 2001 that was controlled through culling of poultry. No human cases were detected. This H5 virus is distinct from the H5N1 virus that caused the 1997 outbreak (Yi Guan, personal communication). However, the 2001 outbreak of HPAI H5N1 among poultry and human illness in 1999 from H9N2 virus infection highlight the need for continued surveillance among both animals and humans. The serologic evidence for infections in PWs and GWs presented here further demonstrates the potential of avian influenza viruses to infect humans. These findings highlight the need to conduct additional seroprevalence studies in human populations in Asia and elsewhere that are exposed to domestic poultry in live bird markets. Such studies will improve our understanding of the epizootic potential of avian influenza viruses and may identify candidate avian subtypes with pandemic potential.
Acknowledgments
We thank Anthony Mounts, for assistance with data entry, and Drew Abernathy, for assistance with laboratory testing.
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