Journal of Medical Virology 78:820–824 (2006)

Prevalence of Antibodies Against A and B InfluenzaViruses in South-Western Papua New Guinea

Simona Puzelli,1 Stefano Boros,1 Chiara Affinito,1 Laura Calzoletti,1 Marzia Facchini,1

Robert T. Danaya,2 Ifor L. Owen,3 Edoardo Pozio,1 Giovanni Rezza,1 and Isabella Donatelli1*1Department of Infectious, Parasitic and Immunomediated Diseases, Istituto Superiore di Sanita, Rome, Italy2Port Moresby General Hospital, Port Moresby, Papua New Guinea3National Veterinary Laboratory, National Agriculture Quarantine and Inspection Authority,Port Moresby, Papua New Guinea

Influenza viruses remain a major cause ofrespiratory disease in both developed and devel-oping countries. Nevertheless, there is littleinformation on the prevalence of this respiratoryinfection in many developing countries, such asPapua New Guinea, since most of the availabledata originate from studies carried out in indus-trialized countries. In the present study, a ser-osurvey among residents of 47 remote villages ofPapua New Guinea was conducted to evaluatethe intensity of exposure to human influenza Aand B viruses. The data obtained confirm the co-circulation of these antigenic variants, reportedhaving circulated in the Southern Hemisphere.Variations in seroprevalence of influenza A and Bvirus infection were found within the study area.J. Med. Virol. 78:820–824, 2006.� 2006 Wiley-Liss, Inc.

KEY WORDS: human influenza viruses; ser-oepidemiologic study; melane-sian population; developingcountries; human transmis-sion; seroprevalence rates

INTRODUCTION

Acute respiratory infections are a major cause ofmorbidity and mortality throughout the world. Themorbidity from acute respiratory infections is roughlythe same indevelopingas indeveloped countries, but theassociatedmortality is many times higher in developingcountries [WHO, 1987a]. In tropical countries, a highproportion of hospital admissions and death are dueto acute respiratory infections [Karaivanova, 1995].Although information on the etiological agents of theseinfections is rather scanty in the tropical area, availabledata show that about one-third of the cases of respira-tory tract infections are due to viruses [Karaivanova,1995]; among these, influenza, respiratory syncytial

virus, parainfluenza, and adenoviruses play the mostimportant role [Karaivanova, 1995; Weber et al., 1998].

Influenza appears to occur frequently in tropicalcountries, where the prevalence of individual virusvariants is not substantially different from that foundin the temperate zone [WHO, 1987b]. However, thereare still several issues, such as those concerning theseasonal occurrence and the extent to which influenzamay affect partially isolated populations such as thoseliving in some tropical islands. In the present study, theresults of a serosurvey conducted in remote villages ofPapua New Guinea are reported, with the aim ofassessing prevalence of exposure to specific influenzavirus variants and identifying factors associated toseropositivity.

PATIENTS AND METHODS

Area of Investigation and Collectionof Serum Samples

Study participants were Melanesian living in 47remote villages of the Morehead District, WesternProvince of Papua New Guinea (Fig. 1). The villages(total population 11,504 inhabitants, 0.5 person per sqkm in the 2,000 census) are located in a flat, rural zone,sited in the south west of the country, as describedpreviously [Owen et al., 2001, 2005].

Essential demographic information (i.e., age, gender,and village of residence) andat least 5ml of venous bloodwere collected fromvolunteers of at least 15 years of age,after having obtained informed consent, during three

The authors declare that they do not have any commercial orother associations that might pose a conflict of interest.

*Correspondence to: Dr. Isabella Donatelli, Istituto Superioredi Sanita, Viale Regina Elena 299, 00161 Rome Italy.E-mail: donatell@iss.it

Accepted 14 February 2006

DOI 10.1002/jmv.20629

Published online in Wiley InterScience(www.interscience.wiley.com)

� 2006 WILEY-LISS, INC.

surveys conducted in November 2001, and in April andNovember 2002. Serum samples were separated andtreated as described elsewhere [Owen et al., 2001,2005]. Approval for the study was given by theMedical Research Advisory Committee of Papua NewGuinea.

Aliquots of samples collected in 2001 were also testedfor antibodies against other viral agents [Rezza et al.,2001]. Serum samples were stored at �808C at theIstituto Superiore di Sanita, Rome.

Serological Tests

Titres of anti-haemagglutinin antibody to threeinfluenza virus strains, that is, A/Moscow/10/99(H3N2), A/New Caledonia/20/99 (H1N1) and B/Guan-dong/120/00, were determined using the Haemaggluti-nation-Inhibition (HI) test, according to standardprocedures [Kendal et al., 1982]. Viruses selected asreference antigens and used in the tests were represen-tative of the three predominant serological groups ofinfluenza viruses circulating at the time of serumcollection, in the Southern Hemisphere [WHO, 2001].

Data Analysis and Statistical Methods

Seroprevalence rates for the two influenza A virussubtypes and for the B type were calculated by studyperiod, considering HI titers�40 as positive. Crude andadjusted odds ratios (OR) were calculated using logisticregression univariate andmultivariate models, in orderto evaluate associations between seropositivity to eachstrain and demographic variables, such as age, gender,and geographical area. To evaluate the effect ofgeographic area, two different criteria were used: (i)villages were grouped in accordance with their locationand intensity of inter-village contact, based on anecdo-tical reports in five areas, ‘‘southern coast,’’ ‘‘south-west,’’ ‘‘central,’’ ‘‘eastern’’ and ‘‘northern;’’ (ii) villageswere stratified in two zones, within and outside a radiusof 40 km from the district headquarter of Morehead(Fig. 1). The multivariate analysis was based on thissecond criterion. The statistical significance of theassociations was evaluated through the w2-test fordichotomic variables or the Mann–Whitney test forcontinuous variables, and the 95% confidence intervalsof the OR.

J. Med. Virol. DOI 10.1002/jmv

Fig. 1. Geographical area of PapuaNewGuineawhere the serosurvey to the three influenza viral strains(A/Mos/10/99, A/NC/20/99,B/Gua/120/00)was conducted. The villages are located in a flat, rural zone, sitedin the South-west of the country. Themarked area shows a radius of 40 km from the district headquarter ofMorehead.

Influenza Antibodies in Residents of Papua New Guinea 821

Overall, serum samples from 754 individuals weretested; of them, 238 were recruited in November 2001,315 in April and 201 inNovember 2002. Themedian ageof the participants was 33 years (range: 15–80), and51.3% of them were male. The main characteristics ofthe study population and seroprevalence rates for eachstrain are shown in Table I.

RESULTS

In general terms, seroprevalence peaked inApril 2002and then tended to decline; gender and geographicalarea were also associated with variations in theproportion of seropositive individuals.The OR of seropositivity to each circulating viral

strain for the main study variables is reported inTable II. Individuals recruited in April 2002, were morelikely than those studied in November 2001, to beseropositive for every single strain at both univariateand multivariate analysis, that is, after adjusting forage, gender, and residence outside 40 km from More-head. We found geographical differences in the risk ofbeing seropositive to each of the three viral strains.Overall, the risk was higher for individuals living in theNorth or in the South and, in any case, for those livingmore than 40 km fromMorehead. After adjusting for theother variables, living outside 40 km from Moreheadremained associated with a two-fold risk of seropositiv-ity to A/New Caledonia/20/99 and B/Guandong/120/00but not to A/Moscow/10/99.

DISCUSSION

This study was undertaken to evaluate the intensityof exposure to two influenzaAvirus subtypes (H3N2andH1N1) and to the B type, which were known to circulateworldwide in the years 2001/02, in a remote area ofSouth-western Papua New Guinea. The prevalenceof antibodies to influenza A/Moscow/10/99 (H3N2), A/New Caledonia/20/99 (H1N1), and B/Guandong/120/00peaked in April, towards the end of the wet season, withhigherprevalence ofA/Moscow/10/99 comparedwith theother strains. A local variation was also observed, withhigher prevalence rates found in peripheral villagesthan in those more close to the district headquarter.

In Papua New Guinea, influenza may cause sporadicoutbreaks but also larger epidemics,which occur despitelow overall population density and remoteness of manyvillages [Sungu and Sanders, 1991]. The first well-documented report of influenza activity in Papua NewGuinea date from 1930s. Nevertheless, subsequentreports or information related to human influenza viruscirculation have been rather scanty. An influenza-likeillness, caused by a type B virus, was reported along theEastern cost of Papua New Guinea in 1964 [Sunguand Sanders, 1991]. The disease caused by this virusbecame much more serious as it spread into the high-lands, where it was associated with over 100 deaths.The geographical isolation of these people, at thattime, probably accounted for the severity of the illnessin those mountain areas. Today, very few groups in

J. Med. Virol. DOI 10.1002/jmv

TABLE I. Characteristics of the Study Population by Age, Gender, Time of Sample Collection and Geographical Area andSeroprevalence to Each of the Three Influenza Viral Strains

Virus strain

A/Mos/10/99 A/NC/20/99 B/Gua/120/00

Negative Positive Negative Positive Negative Positive

N N % N N % N N %

Gendera

Female 136 229 62.7 280 85 23.3* 246 119 32.6Male 167 218 56.6 319 66 17.1 265 120 31.2

Age (years)Median(range)

33 (15–78) 33 (12–80) N/A 34 (12–80) 30 (14–80)** N/A 33 (12–80) 33 (14–80) N/A

Calendar monthNovember2001

133 105 44.1** 194 44 18.5** 192 46 19.3**

April 2002 67 248 78.7 225 90 28.6 160 155 49.2November2002

106 95 47.3 183 18 9.0 162 39 19.4

Zone ICenter 101 126 55.5** 195 32 14.1** 191 36 15.9**South 15 67 81.7 63 19 23.2 44 38 46.3North 19 52 73.2 49 22 31.0 26 45 63.4East 33 51 60.7 73 11 13.1 62 22 26.2South-west 138 152 52.4 222 68 23.4 191 99 34.1

Zone II�40 km 154 182 54.2** 294 42 12.5** 266 70 20.8**>40 km 152 266 63.6 308 110 26.3 248 170 40.7

N/A, not applicable.aFour individuals had missing data.*P< 0.05.**P< 0.01.

822 Puzelli et al.

Papua New Guinea live in such isolation and, conse-quently, influenza could now penetrate and spreadmuch easier among the population living in these inlandregions, as also demonstrated by the rapid spread ofthe pandemic disease, known as ‘‘Hong Kong flu’’ andcaused by an A/H3N2 influenza strain, in previouslyremote areas of the highlands in 1969 [Sungu andSanders, 1991].

More recent reports concerning influenza virusescirculation in Papua New Guinea were provided bythe National Influenza Centre, located in Goroka, inthe Eastern highlands of PNG, particularly during the1980s and at the beginning of the 1990s [Sungu andSanders, 1991]. The surveillance activities of thisCentre were mostly limited to the surroundings areasand largely influenced by technical and financialconstraints. The surveillance conducted in the area ofGoroka showed that, although the number of isolateswas small, influenza activity might be detected almostall year round, in both wet and dry seasons, with asignificant increase from June to August (dry season);both influenza A (i.e., H3N2 and H1N1) and B strainswere isolated during outbreaks occurred in the period1988–1990 [Sungu and Sanders, 1991]. Another influ-enza outbreak was detected in the highlands of PapuaNewGuinea in the wet season, between December 1982and March 1983 [Canil et al., 1984]. A large epidemic ofan influenza-like illness was reported in a remotehighland population of Irian Jaya (Indonesia), an areabordering Papua NewGuinea, between November 1995and February 1996. Attack rates were higher amongadults, and the overall case-fatality was as high as 15%[Corwin et al., 1998].

After the reports by the National Influenza Centre in1991 [Sungu andSanders, 1991], no further informationon influenza viruses circulating in Papua New Guineawas available [Alan W. Hampson, WHO ReferenceInfluenza Centre of Melbourne, Australia, personalcommunication].

The seroprevalence peak observed in the presentstudy appears to be consistent with an increasedinfluenza activity during the wet season. However, asa possible limit of this study, it should be mentionedthat different villages were recruited in the threedistinct periods of time; this makes difficult to disen-tangle space effect from calendar time effect. Althoughthe calendar time effect remained at the multivariateanalysis, we cannot completely rule out the possibilitythat the April peak and the subsequent decline ofantibody prevalence was due, at least in part, to arecruitment bias. However, a similar decline after theinfluenza season was also observed in other studiesconducted in industrialized countries [Pyhala andAho, 1981].

With regard to geographical variation observedwithin the study area, this could be attributed, at leastin part, to limited population movement due to the lackof rapid transport modalities. More complete informa-tion on behavior, social networks andmobility is neededin order to better interpret these findings.

J. Med. Virol. DOI 10.1002/jmv

TABLE

II.Cru

deandAdjusted

OR

(andTheir95%CI)

ofSerop

ositivityto

Each

oftheThreeIn

fluen

zaViralStrainsbyAge,

Gen

der

andGeo

graphicalArea

Virusstrain

A/M

os/10/99

A/N

C/20/99

B/G

ua/120/00

Cru

deOR

(95%

CI)

Adjusted

OR

(95%

CI)

Cru

deOR

(95%

CI

Adjusted

OR

(95%

CI)

Cru

deOR

(95%

CI)

Adjusted

OR

(95%

CI)

Gen

der

Fem

ale

1.0

1.0

1.0

1.0

1.0

1.0

Male

0.77(0.58–1.04)

0.81(0.59–1.11)

0.68(0.48–0.98)

0.78(0.53–1.14)

0.94(0.69–1.27)

1.00(0.72–1.40)

Age(yea

rs)

Med

ian(range)

1.00(0.99–1.01)

1.00(0.99–1.01)

0.99(0.97–1.00)

0.99(0.97–1.00)

1.00(0.99–1.01)

1.00(0.99–1.01)

Calendarmon

thNov

ember

2001

1.0

1.0

1.0

1.0

1.0

1.0

April2002

4.69(3.23–6.80)

4.57(3.13–6.68)

1.76(1.17–2.65)

1.56(1.02–2.39)

4.04(2.74–5.97)

3.49(2.34–5.20)

Nov

ember

2002

1.13(0.78–1.65)

1.14(0.78–1.67)

0.43(0.24–0.78)

0.43(0.23–0.78)

1.00(0.62–1.62)

0.95(0.59–1.55)

Zon

eI

Cen

ter

1.0

—1.0

—1.0

—Sou

th3.58(1.93–6.64)

—1.84(0.97–3.47)

—4.58(2.61–8.03)

—North

2.19(1.22–3.95)

—2.74(1.46–5.12)

—9.18(5.04–16.73)

—East

1.24(0.74–2.06)

—0.92(0.44–1.92)

—1.88(1.03–3.44)

—Sou

th-w

est

0.88(0.62–1.25)

—1.87(1.18–2.96)

—2.75(1.79–4.23)

—Zon

eII

�40km

1.0

1.0

1.0

1.0

1.0

1.0

>40km

1.48(1.10–1.98)

1.19(0.87–1.64)

2.50(1.69–3.69)

2.22(1.48–3.33)

2.60(1.88–3.61)

2.21(1.57–3.12)

Influenza Antibodies in Residents of Papua New Guinea 823

Comparisons with other developing countries of Asiaare difficult, due to the lack of serological surveysconducted in the same time period. A serological surveyof influenza in Pakistan showed that 36% of thepopulation had antibodies to influenza B and 89% toinfluenza A during the period 1976–1980 [Ghafour andBurney, 1981].In industrialized countries, where a systematic

virological monitoring is usually carried out, serologicalstudies, although useful in the detection of asympto-matic cases [Fox et al., 1982], represent a delayedmarker of influenza epidemic activity. In addition, thesestudies are of little value in the immediate clinicalmanagement. On the contrary, in developing countries,such as Papua New Guinea, the lack of a nation-widevirological surveillance system, makes the seropreva-lence studies interesting and valuable to provide retro-spective information on the circulation of influenzaviruses in the population.In conclusion, the results of this study confirm the co-

circulation of different influenza strains (i.e., thosereported having circulated in other countries in theSouthern Hemisphere) in remote areas of Papua NewGuinea. Large variations in the distribution of theseroprevalence have been found within the study area.The determinants of such variations are undefined andneed further investigation.

ACKNOWLEDGMENTS

The assistance given by Asmo Pisau, MoreheadHealth Service and the cooperation of the participantsare acknowledged. Access to equipment from theNational Veterinary Laboratory, National AgricultureQuarantine and Inspection Authority, Papua NewGuinea, is acknowledged. The field work was in partsupported by the Australian Aid Health SupportServices Program.

REFERENCES

Canil KA, Pratt RD, Sungu MS, Smith DE, Phillips PA. 1984. Anoutbreak of influenza A in the highlands of Papua New Guinea.Southeast Asian J Trop Med Public Health 15:265–269.

Corwin AL, Simianjuntak CH, Ingkokusumo G, Sukri N, Larasati RP,Subuanto B,MuslimHZ, Burni E, LarasK, PutriMP,Hayes C, CoxN. 1998. Impact of epidemic influenza A-like acute respiratoryillness in a remote jungle highland population in Irian Jaya,Indonesia. Clin Infect Dis 26:880–888.

Fox JP, Hall CE, CooneyMK, FoyMF. 1982. Influenza virus infectionsin Seattle families, 1975–79 I. Study design, methods and theoccurrence of infections by time and age. Am J Epidemiol 116:212–227.

Ghafour A, Burney MI. 1981. Studies on influenza epidemics onRawalpindi and Islamabad. Pak J Med Res 20:36–39.

Karaivanova GM. 1995. Viral respiratory infections and their role aspublic health problem in tropical countries (Review). Afr JMedMedSci 24:1–7.

Kendal AP, Pereira MS, Skehel JJ. 1982. Concepts and procedures forlaboratory-based influenza surveillance. Atlanta, GA: US. Depart-ment of Health and Human Services, Centers for Disease Controland Prevention.

Owen IL, Pozio E, Tamburrini A, Danaya RT, Bruschi F, GomezMorales MA. 2001. Focus of human trichinellosis in Papua NewGuinea. Am J Trop Med Hyg 65:553–557.

Owen IL, Gomez Morales MA, Pezzotti P, Pozio E. 2005. Trichinellainfection in a hunting population of PapuaNewGuinea suggests anancient relationship of Trichinella with human beings. Trans R SocTrop Med Hyg 99:618–624.

Pyhala R, Aho K. 1981. Seroepidemiology of H1N1 influenza: Theinfection and re-infection rate in winter 1978–79. J Hyg (Lond)86:27–33.

Rezza G, Danaya RT, Wagner TM, Sarmati L, Owen IL, Monini P,AndreoniN,Suligoi B,Ensoli B, PozioE. 2001.Humanherpesvirus-8 and other viral infections, Papua New Guinea. Emerg Infect Dis7:893–895.

Sungu M, Sanders R. 1991. Influenza virus activity in Papua NewGuinea. PNG Med J 34:199–203.

Weber MW, Mulholland EK, Greenwood BM. 1998. Respiratorysyncytial virus infection in tropical and developing countries. TrpMed Int J Health 3:268–280.

WHO. 1987a.Globalmortality fromacute respiratory infections amongchildren aged below 5 years. BullWorld Health Organ 65:114–116.

WHO. 1987b. Progress in the development of influenza vaccines:Memorandum from a WHO meeting. Bull World Health Organ65:289–293.

WHO. 2001. Recommended composition of influenza virus vaccines foruse in the 2002 influenza season. Wkly Epidemiol Rec 76:309–316.

J. Med. Virol. DOI 10.1002/jmv

824 Puzelli et al.