Clinical and Diagnostic Virology 10 (1998) 17–23

Acute lower respiratory infections due to respiratory syncytialvirus and adenovirus among hospitalized children from

Argentina

Cristina Videla a, Guadalupe Carballal a,*, Alicia Misirlian b, Marıa Aguilar b

a Centro de Educacion Medica e In6estigaciones Clınicas ‘Norberto Quirno’ (CEMIC), Departamento de Reumatologıa,Inmunologıa y Virologıa, Billinghurst 2447 (1425), Buenos Aires, Argentina

b Hospital de Pediatrıa ‘P. de Elizalde’, Buenos Aires, Argentina

Received 26 August 1997; received in revised form 12 February 1998; accepted 17 February 1998

Abstract

Background: Acute lower respiratory infection (ALRI) is one of the main causes of morbidity and mortality insmall children. Objecti6e: The aim of this study was to determine the frequency, seasonality and association withclinical entities of respiratory syncytial virus (RSV) and adenoviruses in children with ALRI. Study design: During 2consecutive years (1991–1992), 168 children under 2 years of age hospitalized due to ALRI in a public pediatrichospital of Buenos Aires, Argentina, were studied. RSV and adenoviruses were investigated on nasopharyngealaspirates (NPA) by indirect immunofluorescence (IIF). HEp-2 cells were used for adenovirus isolation. Results: RSVwas detected in 36.3% and adenoviruses in 14.3% of the cases (PB0.0001). All adenoviruses detected by IIF were alsoisolated in culture. Out of 61 RSV cases, 57% corresponded to bronchiolitis and 43% to pneumonia. Ninety-two percent of children with RSV were less than 1 year old and 70% were less than 5 months. The highest number of RSVcases were observed during winter, with a clear peak in July. Seventy-one per cent of adenovirus cases were associatedwith pneumonia and only 24% with bronchiolitis (PB0.02), and predominated in children older than 5 months of age(PB0.0001). Adenoviruses were detected in almost all months of the year with a small peak at the end of winter andbeginning of spring. No significant differences in clinical features at admission, breast feeding or malnutrition wereobserved among children with RSV or adenovirus diagnosis versus those with no viral etiology. The overall fatalityrate was 2.4%. In all fatal cases adenovirus was detected in NPA. Thus, fatality rate among patients with adenovirusesreached 16.7%. Conclusions: Our findings show the importance of RSV and adenoviruses associated with ALRI inhospitalized children under 2 years of age and the different epidemiological patterns of the two viruses in BuenosAires, Argentina. © 1998 Elsevier Science B.V. All rights reserved.

Keywords: Acute lower respiratory infections; Respiratory syncytial virus; Adenovirus; Hospitalized children under 2years of age

* Corresponding author. Fax: 54 1 8053233.

0928-0197/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved.

PII S0928-0197(98)00017-8

C. Videla et al. / Clinical and Diagnostic Virology 10 (1998) 17–2318

1. Introduction

Acute lower respiratory infection (ALRI) isone of the main causes of morbidity and mortal-ity in children all around the world. However,due to social, nutritional and environmental fac-tors the risk of death due to ALRI is 30 timeshigher in developing countries than in the north-ern hemisphere (Organizacion Panamericana dela Salud, 1995).

In Argentina, ALRI is the third most frequentcause of death in children under 1 year of age,after perinatal and congenital pathology, ac-counting for almost 10% of deaths in this agegroup. In 1991, 2000 deaths due to ALRI wererecorded in children under 5 years of age al-though no etiologic diagnosis was available (Mi-celli, I., Ministerio de Salud Publica y AccionSocial, personal communication). Research onthe etiology and epidemiology of ALRI in Ar-gentina is scarce due to difficulties in diagnosingall possible pathogens involved. Furthermore,there is undernotification to public health au-thorities.

From 1984 to 1988, the first study on theetiology and epidemiology of ALRI in 1003 chil-dren under 5 years of age was carried out inArgentina with the support of the Board of Sci-ence and Technology for International Develop-ment (BOSTID), National Academy of Sciences,USA. For 3 consecutive years, patients attendingpublic hospitals of Buenos Aires were studied.Eighty per cent of the cases were inpatients. Eti-ological diagnosis was obtained for 44.3% ofcases and the highest frequency was due toviruses (30%), followed by bacteria (11%) andmixed viral-bacterial infections (3.3%) (Weis-senbacher et al., 1990).

The most frequent etiological agent was res-piratory syncytial virus (RSV) which was de-tected in 18% of ALRI patients, reaching 60%of the positive viral diagnosis and mainly associ-ated with bronchiolitis. The second most com-mon virus was adenovirus, which was detectedin 2.7% of the cases. Parainfluenza and InfluenzaA and B were found in approximately 1% of thepatients. The fatality rate for this study was3.8%. Pathogens were identified in 65% of the

fatal cases. Bacterial and viral infections eachaccounted for 22.6% of deaths, while mixed bac-terial-viral infections were recorded for 19.4%(Carballal et al., 1990).

A new genomic variant of adenovirus (Ad 7h) was described for the first time in childrenwith ALRI, associated with severe and fre-quently fatal outcome of necrotizing bronchioli-tis or pneumonia (Kajon and Wadell, 1992;Murtagh et al., 1993).

The objective of the present study was to fo-cus over a 2-year period on viral etiology ofALRI in children under 2 years of age hospital-ized due to ALRI in a public pediatric hospitalof Buenos Aires. The frequency of RSV andadenovirus, seasonality and the association withclinical diagnosis were determined.

2. Materials and methods

2.1. Population

Children under 2 years of age (n=168) hospi-talized due to ALRI in one ward of the ‘P. deElizalde’ Hospital from May 1991 to December1992 were studied. Children were selected bytwo pediatricians during the entire study. Chil-dren eligible for the study were those under 2years of age, with clinical diagnosis of ALRIand less than 7 days of evolution at admission.Children with a history of asthma, HIV 1/2 di-agnosis, cystic fibrosis or with nosocomial infec-tions were ineligible.

ALRI was defined as an illness resulting intwo or more of the following signs and symp-toms: tachypnea, cough, rales, chest indrawing,wheezing and stridor. Clinical diagnoses ofpneumonia, bronchiolitis, etc., were made bymeans of both physical examination and chestroentgenographic studies according to the WHOguidelines (Pan American Health Organization,1983). The children studied belonged to a disad-vantaged urban community. At admission, acomplete clinical history, physical examination,chest X-ray and a nasopharyngeal aspirate(NPA) were obtained. An informed consentsigned by the parents was obtained in all cases.

C. Videla et al. / Clinical and Diagnostic Virology 10 (1998) 17–23 19

Table 1Adenovirus and respiratory syncytial virus and clinical diagnosis in children under 2 years with ALRI

TotalClinical diagnosis Virus detected

RSV Adenovirus Negative

n % n % n %

35 45.5* 6Bronchiolitis 7.877 36 46.726 32.1 17Pneumonia 21**81 38 46.90 0 110 10Other ALRI syndromes 9 9061 36.3*** 24 14.3 83Total 49.4168

*Compare to adenovirus frequency in bronchiolitis: x2=4.42, PB0.03; **compare to adenovirus frequency in bronchiolitis andother ALRI syndromes: x2=4.7, PB0.02; ***versus negative and adenovirus cases: x2=14.34, PB0.0001.

2.2. Virologic studies

Methods were in accordance with those in theManual of Laboratory Procedures (Board of Sci-ence and Technology for International Develop-ment, 1986). NPA were obtained with a softsterile catheter. Mucus was collected with a vac-uum pump into a mucus trap (Nunc, Roskilde,Denmark) and immediately sent on ice in trans-port media (Hanks’ supplemented with 2% fetalcalf serum and antibiotics) to the laboratory.

Only RSV and adenovirus were investigated byindirect immunofluorescence (IIF). For RSV, amonoclonal antibody (Mab 858.4, Chemicon,USA) and a fluorescein labelled anti-mouse IgG(Sigma) were employed. For adenovirus, a poly-clonal anti-hexon antiserum prepared in rabbits(generously donated by Dr. M. Grandien, Swe-den) and a fluorescein labelled anti-rabbit IgG(Sigma) were employed. The readings were per-formed with a Zeiss microscope fitted with epifl-uorescent equipment.

Supernatants of NPAs were kept at −70°C forfurther adenovirus isolation. Later, 120 superna-tants were inoculated in duplicate in HEp-2 cells.Tubes were examined every other day for cy-tophatic effect (CPE). Cells exhibiting CPE weretested by IIF for adeno antigen. Viral isolateswere kept at −70°C for further genomic typing.

2.3. Statistical analysis

Data were analyzed by the x2 test or Fisher’sexact test.

3. Results

A viral pathogen was identified in 50.6% (85) ofthe 168 ALRI cases (Table 1). RSV was detectedin 36.3% (61) and adenovirus in 14.3% (24) (x2=14.34, PB0.0001) while for 49.4% (83) of thepatients no viral diagnosis was achieved. RSV wasdiagnosed by IIF on NPA cells. Adenovirus caseswere detected by both IIF and isolation in HEp-2cells.

Clinical diagnoses at admission were bronchi-olitis (46%), pneumonia (48%) and other ALRIsyndromes (6%) (Table 1). RSV was found in45.5% of patients with bronchiolitis while aden-oviruses were detected in only 7.8% (x2=4.42,PB0.03). In pneumonia cases, RSV was detectedin 32.1% and adenoviruses in 21%. Consideringthe RSV cases, 57% (35/61) were associated withbronchiolitis and 43% (26/61) with pneumonia.On the other hand, 71% (17/24) of adenoviruscases were associated with pneumonia, and only25% (6/24) were associated with bronchiolitis(x2=4.7, PB0.02).

Sixty per cent of children studied were under 5months of age; 23% were 6–11 months and 17%were 12–24 months of age (Fig. 1). RSV wasdetected in 42.6% (43/101), adenoviruses in only5.9% (6/101) of children under 5 months of age(x2=13.56, PB0.0001), and 51.5% did not yielda virus (Table 2). In children 6 months of age orolder, no significant differences were observed infrequencies of RSV and adenoviruses.

Considering the 61 RSV cases, 92% occurred inchildren under 1 year of age and 70% in those

C. Videla et al. / Clinical and Diagnostic Virology 10 (1998) 17–2320

Fig. 1. Distribution in age of children hospitalized due to ALRI with RSV or adenovirus.

under 5 months. Of the 24 adenovirus cases,75% were found in patients 6 months of age orolder. The mean age of children with aden-oviruses was 8.7 months versus 4.3 for thosewith RSV (difference not significant).

Sixty-three per cent of children with ALRIwere males. Both RSV and adenoviruses weremore common in males. The association ofadenoviruses and male gender was significant(PB0.04).

No statistical differences were observed inclinical features at admission, malnutrition orbreast feeding among children with ALRI and adiagnosis of RSV or adenovirus etiology versusno viral etiology.

The overall fatality rate in this study was2.4% (4/168). For the four fatal cases an aden-

ovirus was detected (16.7%). These cases were inpatients with congenital cardiopathy (n=1);malnutrition (n=1) and no underlying condition(n=2).

During the 2 consecutive years, a clear peakof RSV was detected during the cold months ofthe year. Seventy-eight per cent (48/61) of thecases were observed during winter and the be-ginning of spring (Fig. 2). RSV was not detectedduring the warm months (from November toJanuary) of 1991 while only two cases wererecorded in January and February of 1992. Ade-noviruses were detected throughout the year. In1991, the first adenovirus case was recorded inAugust. In 1992, it was observed during almostall months with a small peak at the end of thewinter and spring.

C. Videla et al. / Clinical and Diagnostic Virology 10 (1998) 17–23 21

Table 2Clinical features of children under 2 years of age with ALRI

Etiologic diagnosisFeature

Adenovirus (%) (n=24) Not achieved (%) (n=75)RSV (%) (n=61)

Age (months)0–5 42.6* 5.9 51.56–11 34.2 31.6 34.2

21.417.8 60.812–244.394.5Mean age9S.E. 8.796.4 6.896.7

Sex56.0Male (n=101) 79.0** 64.044.0Female (n=67) 21.0 36.0

60.061.4 57.5Breast feeding22.8Malnutrition 40.0 33.8

85.061.4 65.0Fever (\38°C)68.4Rhinorrea 67.570.080.086.0 78.8Tachypnea

Respiratory rate/min X9S.E. 4092846930 3993175.080.7 73.8Cough

80.6Chest indrawing 65.0 68.842.1Wheezing 60.0 38.8

50.043.9 38.8Roncus75.0Rales 76.382.515.014.0 8.8Apneas

31.6Conjunctivitis 30.0 22.50Fatal outcome 16.7***

*Versus adenovirus cases: x2=13.56, PB0.0001; **versus adenovirus in female: x2=4.03, PB0.04; ***versus RSV and negativecases: Fisher’s test, PB0.0001.

4. Discussion

During a 2-year period, 168 children under 2years of age, hospitalized due to ALRI in onepediatric hospital of Buenos Aires, were studied.For 50.6% of these cases, a positive viral diagnosiswas achieved. RSV was the most frequently de-tected virus (36.3%) followed by adenoviruses(14.5%) (PB0,0001) (Table 1). RSV was morefrequently observed in children under 5 months ofage, predominantly associated with bronchiolitis(Table 1, Fig. 1). The seasonal distribution ofRSV exhibited a peak during cold weather andthe outbreak lasted for 6 months (from May toOctober) (Fig. 2). This finding agreed with previ-ous reports for Argentina (Weissenbacher et al.,1990; Carballal et al., 1993) and Uruguay (Hortalet al., 1990). In the USA, RSV also peaks at theend of autumn, during winter or the beginning ofspring (Kim et al., 1973; Gilchrist et al., 1994).

The RSV frequency (36.3%) detected in our studyis higher than that previously observed for Argen-tinian children under 5 years of age with ALRI.The BOSTID study showed the rate to be 18.2%in inpatients (182/1003) (Weissenbacher et al.,1990) and our second large study has found 25%(312/1234 ALRI cases) to be due to RSV (datanot shown).

The rate of RSV detection (36.3%) in our studyis representative of the American reports whichshowed 32–36% RSV detection in young inpa-tients with ALRI, depending on age, clinical enti-ties and season of the year (Taussig et al., 1989;Gilchrist et al., 1994).

Adenovirus infections exhibited a different pat-tern than RSV. These viruses were more frequentin older children especially in males. Adenoviruseswere found predominantly associated with pneu-monia (71%) rather than with bronchiolitis (7.8%)(PB0.02). Adenoviruses were detected in small

C. Videla et al. / Clinical and Diagnostic Virology 10 (1998) 17–2322

Fig. 2. Monthly distribution of RSV and adenovirus for 168 children under 2 years of age with ALRI hospitalized in a pediatrichospital of Buenos Aires, Argentina during 2 consecutive years.

numbers throughout the year. This seasonal dis-tribution is coincident with previous data forBuenos Aires (Kajon and Wadell, 1992) and forthe South cone of South America (Kajon andWadell, 1994).

The adenovirus frequency detected in our study(14.3%) is significantly higher than that publishedfor Argentinian children. In inpatients under 5years, Weissenbacher et al. (1990) showed only2.7% (27/1003) adenovirus detection; Avila et al.(1989) observed a 3% rate (6/204) in patients withALRI living in an orphanage and Carballal et al.(1996) detected adenoviruses in 2.5% (31/1234) ofinpatients. Adenoviruses were reported in 1996from 34% of inpatients with ALRI in BuenosAires from the R. Gutierrez Hospital(Mistchenko, personal communication). Thishighly specialized hospital receives severely ill pa-tients from peripheral centers but nosocomial in-fections cannot be ruled out.

In developed countries, adenovirus frequency inALRI in civilian populations is lower. In Sweden,adenoviruses are responsible for 5% of ALRI inchildren under 4 years of age who require hospi-talization (Sharp and Wadell, 1995). In the USA,adenoviruses are the third most common viral

agent (3%) after parainfluenza virus (17%)(Glezen et al., 1971; Taussig et al., 1989).

No significant differences in clinical features,breast feeding or malnutrition could be identifiedamong children with RSV or adenoviruses versusthose with negative viral diagnosis (Table 2).However, the high fatality rate among childrenwith adenoviruses (17%) versus 0% for those withRSV or negative viral diagnosis was statisticallysignificant (PB0.0001).

The circulation of a new genomic variant ofadenovirus 7 belonging to the subgenus b, calledAd 7 h, has been reported in Argentina, Chile andUruguay associated with severe and fatal cases ofnecrotizing bronchiolitis or pneumonia (Kajonand Wadell, 1994).

The high mortality observed in our studyamong children with adenoviruses supports thehypothesis that this highly pathogenic genotypemay be involved. Genomic typing by restrictionenzyme analysis is being carried out on the strainsisolated in this study.

Our results show the importance of RSV andadenoviruses associated with bronchiolitis andpneumonia in children under 2 years of age. Thesefindings stress the need for further etiological

C. Videla et al. / Clinical and Diagnostic Virology 10 (1998) 17–23 23

studies in order to determine the impact and themolecular epidemiology of the two main agents ofALRI in Argentina.

Acknowledgements

This study was supported by Grant No. 33-81500/92 from CONICET.The skilful technicalassistance of Cristina Juarez, Carmen Ricarte andBeatriz Ebekian is gratefully acknowledged.

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